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Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Nasal Route for CNS Drug Repurposing and Novel Delivery to the Brain
Prof Joan Z Zuo
School of Pharmacy CUHK
SOLVO Meet the Experts Seoul 2019
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Nasal Delivery Advantages
bull Ability to delivery a wide range of therapeutics(small molecules and larger molecules such as peptides proteins and nucleic acids)
bull Rapid onset of action (critical to some disease states such as pain)
bull Avoidance of first-pass hepatic metabolism
bull Non-invasive compared to injection
bull Potential for direct delivery to the brain
BBB blood-brain barrier BCSFB blood-cerebrospinal fluid barrier
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
What has been done for CNS targeting
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
CNS drugs (small molecules)
bull Phase I and Phase II metabolisms not only detoxify the xenobiotics
but might also contribute to the ADRs by generating toxic metabolites
which have been observed in many CNS drugs
bull Regioselectivity in biotransformation deserves more attention since
selective formation or elimination of the toxic metabolites would play
an important role in intensifying or mitigating the ADRs
bull Structural modifications of drug candidates and alternative routes of
drug delivery are effective approaches to modify metabolite
formation
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Protein drugs
bull Poor penetration across the blood brain barrier (BBB) is the major
limitation of peptides as therapeutic agents for the treatment of CNS
diseases
bull Alternative routes of administrations especially intranasal delivery of
peptides demonstrated its unique advantage to deliver peptide based
CNS drugs into brain leading to improvement in both their
pharmacokinetics and pharmacodynamics
bull Taking advantages of both nanocarriers and intranasal route of
administration have demonstrated significant improvement in brain
uptake of peptide than any other traditional approaches for CNS
targeting delivery of peptides
Commonly used study models for intranasal delivery
bull In-vitro cell model
bull In-situ animal model
bull In-vivo animal model
In vitro cell line models
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Nasal Delivery Advantages
bull Ability to delivery a wide range of therapeutics(small molecules and larger molecules such as peptides proteins and nucleic acids)
bull Rapid onset of action (critical to some disease states such as pain)
bull Avoidance of first-pass hepatic metabolism
bull Non-invasive compared to injection
bull Potential for direct delivery to the brain
BBB blood-brain barrier BCSFB blood-cerebrospinal fluid barrier
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
What has been done for CNS targeting
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
CNS drugs (small molecules)
bull Phase I and Phase II metabolisms not only detoxify the xenobiotics
but might also contribute to the ADRs by generating toxic metabolites
which have been observed in many CNS drugs
bull Regioselectivity in biotransformation deserves more attention since
selective formation or elimination of the toxic metabolites would play
an important role in intensifying or mitigating the ADRs
bull Structural modifications of drug candidates and alternative routes of
drug delivery are effective approaches to modify metabolite
formation
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Protein drugs
bull Poor penetration across the blood brain barrier (BBB) is the major
limitation of peptides as therapeutic agents for the treatment of CNS
diseases
bull Alternative routes of administrations especially intranasal delivery of
peptides demonstrated its unique advantage to deliver peptide based
CNS drugs into brain leading to improvement in both their
pharmacokinetics and pharmacodynamics
bull Taking advantages of both nanocarriers and intranasal route of
administration have demonstrated significant improvement in brain
uptake of peptide than any other traditional approaches for CNS
targeting delivery of peptides
Commonly used study models for intranasal delivery
bull In-vitro cell model
bull In-situ animal model
bull In-vivo animal model
In vitro cell line models
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
BBB blood-brain barrier BCSFB blood-cerebrospinal fluid barrier
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
What has been done for CNS targeting
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
CNS drugs (small molecules)
bull Phase I and Phase II metabolisms not only detoxify the xenobiotics
but might also contribute to the ADRs by generating toxic metabolites
which have been observed in many CNS drugs
bull Regioselectivity in biotransformation deserves more attention since
selective formation or elimination of the toxic metabolites would play
an important role in intensifying or mitigating the ADRs
bull Structural modifications of drug candidates and alternative routes of
drug delivery are effective approaches to modify metabolite
formation
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Protein drugs
bull Poor penetration across the blood brain barrier (BBB) is the major
limitation of peptides as therapeutic agents for the treatment of CNS
diseases
bull Alternative routes of administrations especially intranasal delivery of
peptides demonstrated its unique advantage to deliver peptide based
CNS drugs into brain leading to improvement in both their
pharmacokinetics and pharmacodynamics
bull Taking advantages of both nanocarriers and intranasal route of
administration have demonstrated significant improvement in brain
uptake of peptide than any other traditional approaches for CNS
targeting delivery of peptides
Commonly used study models for intranasal delivery
bull In-vitro cell model
bull In-situ animal model
bull In-vivo animal model
In vitro cell line models
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
What has been done for CNS targeting
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
CNS drugs (small molecules)
bull Phase I and Phase II metabolisms not only detoxify the xenobiotics
but might also contribute to the ADRs by generating toxic metabolites
which have been observed in many CNS drugs
bull Regioselectivity in biotransformation deserves more attention since
selective formation or elimination of the toxic metabolites would play
an important role in intensifying or mitigating the ADRs
bull Structural modifications of drug candidates and alternative routes of
drug delivery are effective approaches to modify metabolite
formation
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Protein drugs
bull Poor penetration across the blood brain barrier (BBB) is the major
limitation of peptides as therapeutic agents for the treatment of CNS
diseases
bull Alternative routes of administrations especially intranasal delivery of
peptides demonstrated its unique advantage to deliver peptide based
CNS drugs into brain leading to improvement in both their
pharmacokinetics and pharmacodynamics
bull Taking advantages of both nanocarriers and intranasal route of
administration have demonstrated significant improvement in brain
uptake of peptide than any other traditional approaches for CNS
targeting delivery of peptides
Commonly used study models for intranasal delivery
bull In-vitro cell model
bull In-situ animal model
bull In-vivo animal model
In vitro cell line models
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
CNS drugs (small molecules)
bull Phase I and Phase II metabolisms not only detoxify the xenobiotics
but might also contribute to the ADRs by generating toxic metabolites
which have been observed in many CNS drugs
bull Regioselectivity in biotransformation deserves more attention since
selective formation or elimination of the toxic metabolites would play
an important role in intensifying or mitigating the ADRs
bull Structural modifications of drug candidates and alternative routes of
drug delivery are effective approaches to modify metabolite
formation
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Protein drugs
bull Poor penetration across the blood brain barrier (BBB) is the major
limitation of peptides as therapeutic agents for the treatment of CNS
diseases
bull Alternative routes of administrations especially intranasal delivery of
peptides demonstrated its unique advantage to deliver peptide based
CNS drugs into brain leading to improvement in both their
pharmacokinetics and pharmacodynamics
bull Taking advantages of both nanocarriers and intranasal route of
administration have demonstrated significant improvement in brain
uptake of peptide than any other traditional approaches for CNS
targeting delivery of peptides
Commonly used study models for intranasal delivery
bull In-vitro cell model
bull In-situ animal model
bull In-vivo animal model
In vitro cell line models
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Protein drugs
bull Poor penetration across the blood brain barrier (BBB) is the major
limitation of peptides as therapeutic agents for the treatment of CNS
diseases
bull Alternative routes of administrations especially intranasal delivery of
peptides demonstrated its unique advantage to deliver peptide based
CNS drugs into brain leading to improvement in both their
pharmacokinetics and pharmacodynamics
bull Taking advantages of both nanocarriers and intranasal route of
administration have demonstrated significant improvement in brain
uptake of peptide than any other traditional approaches for CNS
targeting delivery of peptides
Commonly used study models for intranasal delivery
bull In-vitro cell model
bull In-situ animal model
bull In-vivo animal model
In vitro cell line models
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Commonly used study models for intranasal delivery
bull In-vitro cell model
bull In-situ animal model
bull In-vivo animal model
In vitro cell line models
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
In vitro cell line models
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Choose Appropriate Rat Model
VS
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Intranasal Delivery Targeting
Polyglutamine Diseases
CASE STUDIES
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Background-polyQ diseasesBackground-PolyQ diseases
Huntingtonrsquosdisease (HD)
Spinocerebellar ataxia (SCA) type 1 2 3 6 7
amp17
Dentatorubral -pallidoluysian atrophy
(DRPLA)
Spinobulbarmuscular atrophy
(SBMA)
Polyglutamine (polyQ) diseases
PolyQ diseases are a group of neurodegenerative disorders caused by expanded CAG RNA encoding
a long polyQ tract in brain cells
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Background PolyQ diseases
Movement disorders [1]
Cognitive disorders [1]
No effective clinical treatment based on the pathogenesis of polyQ diseases
[1] Guo F Liu X Cai H et al Brain Pathology 2018 28(1) 3-13
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Background PolyQ diseases
Human neuronal cell line [2] Drosophila disease model [2] R62 Huntingtonrsquos disease mice model [3]
In-vitroIn-vivo
[2] Jackson G R Salecker I Dong X et al Neuron 1998 21(3) 633-642 [3] Luthi-Carter R Hanson S A Strand A D et al Human molecular genetics 2002 11(17) 1911-1926
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
HOW TO DELIVER SMALL MOLECULE DRUG
CANDIDATE
14
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Background DB213
Human neuronal cell line [4] Drosophila disease model [4]
DB213
DB213 is a promising CAG-repeated RNA inhibitor targeting polyQdiseases on its pathogenesis bases [4]
[4] Chan H Y E Ngo J C Chun-Ho W et al US Patent Application 15382380[P] 2017-6-29
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Brain
Therapeutics
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Background Barriers for drug delivery in the brain
Blood-brainbarrier (BBB)
Blood-cerebrospinalfluid barrier
(BCSFB)
Physicalbarrier
Transportbarrier
Metabolicbarrier
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Background Intranasal drug delivery
Brain
Commonly used Routesbull Intravenousbull Intraperitonealbull Oral
Alternative Routebull Intranasal
Blood Brain Barrier
Olfactory bulb
Trigeminal nerve
Therapeutics
Formulation development [5]bull Nanoparticlebull In-situ gel
Intranasally administered in-situ thermosensitive gel is proved to be analternative and noninvasive drug delivery approach which shows a potential to
enhance drug brain uptake
[5] Qian S Wang Q Zuo Z Expert opinion on drug metabolism amp toxicology 2014 10(11) 1491-1508
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
In silico prediction showed DB213 has limited BBB permeability
CompoundPhysical chemical
propertiesBBB permeability predictions
LogPa pKab 1c 2d 3d 4d 5d 6d 7d 8d 9d
DB213 157 980 BBB--094
(BBB-)1491
(BBB+)621
(BBB+)517
(BBB+)-0005(BBB-)
028(BBB+)
083(BBB+)
011(BBB+)
Diazepam 282 341 BBB+782
(BBB+)2318(BBB+)
1085(BBB+)
1439(BBB+)
013(BBB+)
022(BBB+)
056(BBB+)
026(BBB+)
Doxorubicin 127 846 BBB--530(BBB-)
-1603(BBB-)
-1189(BBB-)
-1483(BBB-)
-029(BBB-)
-121(BBB-)
-035(BBB-)
-111(BBB-)
a value predicted by ChemBioDrawb value predicted by MarvinSketchc value predicted by ACDLabsd value predicted by online BBB predictor using different algorithms (AdaBoost and SVM) and fingerprint databases (MACCS OpenbabelMolprint2D and PubChem) 2 (AdaBoost and MACCS) 3(AdaBoost and Openbable) 4(AbaBoost and Molprint2D) 5(AdaBoost and PubChem)6(SVMand MACCS) 7(SVM and Openbable) 8(SVM and Molprint2D) and 9 (SVM and PubChem)
In-silico prediction
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Nasal epithelium permeability
DB213 has limited nasal epithelium permeability
Compound
Loading
concentration
(microM)
Calu-3 Papp(ararrb) (times10-6 cms)
Cell uptake ()
(n=3)
Experimental
value
(n=3)
Reference value [8]
Propranolol 100 1576 plusmn 195 2270 plusmn 550 092
Atenolol 200 013 plusmn 002 01 plusmn 001 198
DB213
10 140 plusmn 021 NA 355 plusmn 114
100 187 plusmn 029 NA 310 plusmn 143
300 198 plusmn 048 NA 435 plusmn 174
[8] Qian S He L Mak M et al International Journal of Pharmaceutics 2014 477(1-2) 442-453
DB213
Calu-3 monolayercell model
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
WT
MDR1
MRP2
MRP3
WT
MDR1
MRP2
MRP3
0
100
200
300
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
MDCK II cell lines
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB21
3
DB21
3
DB21
3
DB21
3
DB21
3+In
DB21
3+In
DB21
3+In
WT MDR1 MRP2 MRP3
DB213 low conc (10 mM) DB213 high conc (100 mM)
Potential transporters
DB213 is identified as substrate of MRP2 and MRP3 efflux transportersW
T
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
WT
OAT3
WT
OAT3
0
50
100
150
200
DB
21
3 C
on
ce
ntr
atio
n
(nM
)
HEK293 cell lines
DB213
DB21
3
DB213
+In
WT OAT3
DB213
DB21
3
DB21
3+In
WT OAT3
DB213 low conc (10 mM) DB213 low conc (100 mM)
20
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Plasma and tissue binding
Binding ()DB213 Spiked
ConcSD rat C57 mice R62 mice
Plasma (n=3)
Incubation Time
2 h 4 h 17 h 2 h 2 h
03 (μgmL) 3 plusmn 3 1 plusmn 4 1 plusmn 4 26 plusmn 10 30 plusmn 5
3 (μgmL) 2 plusmn 3 1 plusmn 5 0 plusmn 3 21 plusmn 5 28 plusmn 1130 (μgmL) 0 plusmn 5 2 plusmn 5 2 plusmn 4 19 plusmn 5 17 plusmn 8
300 (μgmL) 3plusmn7 2 plusmn 3 1 plusmn 2 17 plusmn 9 12 plusmn 5
Brain tissue (n=3)
2 h 4 h 17 h 4 h 4 h50 (ngg) 90 plusmn 05 96plusmn03 96 plusmn 06 89 plusmn 1 89 plusmn 1
500 (ngg) 92 plusmn 03 96plusmn03 95 plusmn 02 87 plusmn 5 90 plusmn 15000 (ngg) 93 plusmn 06 95plusmn04 95 plusmn 02 87 plusmn 1 90 plusmn 1
DB213 demonstrated low protein binding (less than 30) in plasma and ratherhigh protein (greater than 85) in brain homogenate
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Liver metabolism
Phase II metabolismbull lt5 DB213 was metabolized by Phase II glucuronidationbull lt15 DB213 was metabolized by Phase II sulfation
[9] Wager T T Hou X Verhoest P R et al ACS chemical neuroscience 2010 1(6) 435-449
Type of
microsomes
Vmax
(nmolminmg)Km (mM)
CLin
(μLminmg)
RLM 3357 plusmn 130 126 plusmn 14 2664
MLM (C57) 4451 plusmn 136 165 plusmn 11 2698
MLM (R62) 4410 plusmn 104 206 plusmn 77 2141
CL in of other drugs(microLminmg)bull felodipine 500bull propranolol 3120bull imipramine 4500[9]
DB213 demonstrated limited liver metabolism in rats and mice
Phase I metabolism
DB213
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Brain metabolism
DB213 demonstrated limited brain metabolism in rats and mice
bull imipramine 3000microLminmg [9]
[9] Voirol P Jonzier-Perey M Porchet F et al Brain research 2000 855(2) 235-243
Type of
microsome
Vmax
(nmolminmg)Km (mM)
CLin
(mLminmg
)
RBM 1490 plusmn 116 348 plusmn 57 428
MBM (C57) 1517 plusmn 89 256 plusmn 61 592
MBM (R62) 4968 plusmn 102 314 plusmn 94 1582
Phase I metabolism
Phase II metabolismbull lt1 DB213 was metabolized by Phase II glucuronidation
DB213
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
DB213 NEEDS SERIOUS HELP
24
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
IN VIVO EVALUATION IN SD RATS
Which route of administration
25
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Intranasal administration has a potential to enhance brain uptake of DB213
100 200 300 400
-2000
0
2000
4000
6000
8000
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV on short-anesthetized model (N=6)
IP on short-anesthetized model (N=5)
IN on short-anesthetized model (N=8)nnn
0 100 200 300 4000
50
100
150
200
Time(min)
Pla
sm
a C
on
ce
ntr
atio
n
(ng
mL
)
IV (n=5)
IP(n=5)
IN(n=5)
Cmax
(ngmL)49059 plusmn 12256 26012 plusmn 9163 1228 plusmn 688
Tmax (min) NA 125 plusmn 50 350 plusmn 154
t12 (min) 7098 plusmn 161 838 plusmn 449 923 plusmn 387
AUC0-infin
(μgminmL)184 plusmn 42 137 plusmn 200 16 plusmn 11
Bioavailability ()
NA 7419 865
0 30 60 90 120 1500
10
20
30
40
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n
(ng
g)
IN (n=5-6)
IP (n=5)
IV (n=5)
Intranasal as best route for CNS delivery of DB213
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Dose dependency of DB213 Brain
200 400 600
-20
0
20
40
60
80
Time(min)DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n(n
gg
)
IV 1 mgkg (n=5 per time point)
IV 10 mgkg (n=5 per time point)
IV 50 mgkg (n=5 per time point)
60 120 180 240 300
-02
00
02
04
06
Time(min)
0 60 120 180 240 300 360 420 480 540 6000
100
200
300
400
500
Time(min)
DB
21
3 B
rain
Un
bo
un
d C
on
ce
ntr
atio
n
(ng
g)
IN 1 mgkg (n=5 per time point)
IN 10 mgkg (n=5 per time point)
IN 50 mgkg (n=5 per time point)
0 60 120 180 240 30000
05
10
15
Time(min)
IV 1 mgkg
(n=6)
IV 10 mgkg
(n=6)
IV 50 mgkg
(n=6)
IN 1 mgkg
(n=6)
IN 10 mgkg
(n=6)
IN 50 mgkg
(n=6)
Cmax
(μgg)0011plusmn
0004022 plusmn0047
122plusmn 0240022plusmn
00070785plusmn
0127517 plusmn 147
t12 (min) 66 plusmn 24 60 plusmn 13 72 plusmn 24 72 plusmn 27 69 plusmn 22 77 plusmn 11
AUC0-infin
(μgming)078 plusmn
00621plusmn 5 133 plusmn 23 16plusmn 03 649 plusmn 98 428 plusmn 20
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Demonstration of direct nose-to-brain transport by PK modeling
0 30 60 90 120 150 180 210 240 270 300 330 360 3901
10
100
1000
10000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
l)
IN 50 mgkg (n=6)
IN 10 mgkg (n=6)
IN 1 mgkg (n=6)
0 30 60 90 120 150 180 210 240 270 300 330 360 39010
100
1000
10000
100000
1000000
Time(min)
DB
21
3 P
las
ma
Co
nc
en
tra
tio
n(n
gm
L)
IV 50 mgkg (n=6~8)
IV 10 mgkg (n=6)
IV 1 mgkg (n=6)
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Demonstration of direct nose-to-brain transport of DB213
PK modeling suggested that direct nose-to-brain pathway is responsible fordelivering 707 of the absorbed DB213 to the brain[10]
[10] Wang Q Zhang Y Wong C H et al The AAPS journal 2018 20(1) 23
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Brain regional distribution of DB213
Olfactor
y bulb
HippocampusStriatum
CerebellumPosterior cortexMedium cortexFrontal
cortex
Trigemin
al nerve
DB213
Brain
Mid-brainThalamusHypothalamus
Both olfactory bulb and trigeminal nerve could serve as entry point of nose-to-brain delivery of DB213 followed by its diffusion into other brain regions
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
FORMULATION DEVELOPMENT
31
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Design Phase
Exp No Exp Name Run Orderwater(ww)
DB213(ww)
Chitosan(ww)
PF-68(ww)
PF-127(ww)
1 N1 24 078 001 0 001 02
2 N2 5 0655 005 000499999 001 028
3 N3 1 0655 001 0005 003 03
4 N4 21 0775 001 000499999 001 02
5 N5 8 076 001 0 003 02
6 N6 16 0755 001 000499999 003 02
7 N7 14 0715 005 000499999 003 02
8 N8 15 0675 001 0005 001 03
9 N9 3 066 001 0 003 03
10 N10 9 0655 005 0 00166667 0278333
11 N11 10 0655 00216667 0 00233333 03
12 N12 20 0713333 001 0 001 0266667
13 N13 19 0711667 005 0 001 0228333
14 N14 25 0726667 005 0 00233333 02
15 N15 4 0733333 00366667 0 003 02
16 N16 23 0655 005 000166666 003 0263333
17 N17 12 0655 005 000333333 003 0261667
18 N18 7 0655 00233333 000499999 003 0286667
19 N19 11 0655 00333334 000166666 001 03
20 N20 17 0736667 005 000333333 001 02
21 N21 18 07175 001 00025 002 025
22 N22 13 0697222 003 000499999 00188889 0248889
23 N23 6 0696579 00294737 000236842 00194737 0252105
24 N24 2 0696579 00294737 000236842 00194737 0252105
25 N25 22 0696579 00294737 000236842 00194737 0252105
32
DOE for in-situ gel development
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Analysis phase
bull Tsol-gel
Best fit with MLR model
Tsol-gel = 3659+022A2+112A-225B2-131D
bull In vitro release at 1 h
Best fit with PLS model
In vitro release at 1 h=
3742-062A+956B-152AB+69B2+614C2
bull Mucoclearnace time
Best fit with MLR model
Mucoclearance time=2130+113C+568BC+123AC
Factors
A PF-127
B PF-68
C Chitosan
D DB213
DOE for in-situ gel development
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Prediction phase for SD rat
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 1
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2799PF-68 287Chitosan 05DB213 1
PerformanceObservations Tsol-gel 322 plusmn 03 In vitro release at 1 h
71plusmn43 MCT 50plusmn5 min
DOE for in-situ gel development
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Prediction phase for C57 and R62 mice
Target Tsol-gel 31 In vitro release at 1 h gt60 Mucoclearance time (MCT)
gt30 min DB213 amount at 5
Performance predictions Tsol-gel 31 In vitro release at 1 h 75 MCT 54 min
Formulation predictionsPF-127 2499PF-68 207Chitosan 05DB2135
PerformanceObservations Tsol-gel 316 plusmn 04 In vitro release at 1 h
75plusmn53 MCT 60plusmn5 min
Based on DOE approach DB213 in-situ gel with desired performance was developed
Results DOE for in-situ gel development
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120 m
in
240 m
in
0
20
40
60
80
100
Time(min)
DB
21
3 B
ra
in C
on
ce
ntr
atio
n(n
gg
)
Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
15 m
in
30 m
in
60 m
in
90 m
in
120
min
240
min
0
20
40
60
80
100
Time(min)
DB
21
3 B
rain
Co
nc
en
tra
tio
n(n
gg
)Water solution (n=5 per time point)
DB213 in situ gel (n=5 per time point)
plt0001
Brain PK of DB213 delivered by in-situ gel in rats
SD rats
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
30
60 90 120
240
480
960
1200
1440
1560
0
500
1000
1500
2000
Time(min)D
B2
13
Bra
in C
on
ce
ntr
atio
n in
R6
2 m
ice
(ng
g)
DB213 water solution (n=3)
DB213 in situ gel (n=3)
plt005
R62 miceC57 mice
300
600
1200
2400
3600
480
0
6000
12000
1440
0
0
500
1000
1500
2000
Time(min)
DB
213 B
rain
Co
ncen
trati
on
(ng
g)
DB213 water solution (n=3-6)
DB213 in situ gel (n=3)
(b)
plt001
DB213 delivered by in situ thermosensitive gel demonstrated significant increase in its brain uptake in SD rats C57 and R62 mice [17]
Brain PK of DB213 delivered by in-situ gel in mice
[17] Wang Q Wong C H Chan H Y E et al International journal of pharmaceutics 2018 539(1-2) 50-57
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
IN-VIVO EFFICACY EVALUATION
38
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Maximum tolerance dose(MTD) of DB213 delivered by in-situ gel in mice
MTD of daily treatment of DB213 in-situ gel via IN up to 28 days was identifiedat 50 mgkg for C57 mice and 25 mgkg for R62 mice
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Efficacy evaluation of DB213 in-situ gel in mice
Openfield
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Efficacy evaluation of DB213 in-situ gel in mice
Day
0
Wee
k 2
(day
14)
Wee
k 4
(day
28)
0
5000
10000
15000T
ota
l tr
av
ele
d d
ista
nc
e (c
m)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
Openfield
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Wee
k 2
(day
15)
Wee
k 4
(day
29)
0
50
100
150
La
ten
cy
(s
)
C57 DB213 25 mgkg (n=5)
C57 vehicle (n=8)
R62 DB213 25 mgkg (n=5-6)
R62 vehicle (n=5-6)
plt005
Results Efficacy evaluation of DB213 in-situ gel in mice
Rota-rod test
After 29-day treatment at 25 mgkg (once daily) in R62 mice DB213 in-situ thermosensitive gel group showed significant improvement in cognitive function (Open field test) and motor
function (Rota-rod test) in comparison to vehicle group
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
HOW ABOUT BIGGER MOLECULES
43
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Major Challenges
bull Stability (in-vitro and in-vivo)
bull Permeability
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Lipidation of peptide
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Treatment Strategy
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Choice of route of administration and formulation for QBP1
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Evaluation of the efficacy for combination treatment
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Copyright copy 2016 All Rights Reserved Faculty of Medicine The Chinese University of Hong Kong
Take home
message Formulation
Alternative Route of Administration
Chemical modification
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk
Acknowledgments
The Chinese University of Hong Kong
School of Pharmacybull Dr Yanfeng Wang
bull Dr Eric Wong
bull Dr Chores QW Wang
bull Miss Shu Wang
bull Dr SK Wo
bull Dr Mengbi Yang
bull Dr Shuai Qian
bull Dr Yufeng Zhang
Department of Medicine and Therapeutics
Prof Brian Tomlinson
Department of Chemistry
Dr Ho CY
Miss He LS
Department of Psychiatry
bull Prof Wing YK
School of Life Science
bull Prof Edwin Chan amp his team
Hong Kong Polytechnic University
Prof Han YF amp his team
Comprehensive Drug Enterprises Ltd
Hong Kong
bull Dr Benjamin Lee
Western Univ of Health Sciences USA
bull Prof Moses SS Chow
Research Funds from
bull University Grant Council of Hong Kong SAR
bull Innovation Technology Fund HK SAR
joanzuocuhkeduhk