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Pharmacokinetic-Based Design of New CNS-Active Analogs of
Valproic Acid Derivatives with Improved Potency and Low-
Toxicity
Jakob Avi ShimshoniJakob Avi Shimshoni
Supervisors: Prof. Meir Bialer & Prof. Boris YagenSupervisors: Prof. Meir Bialer & Prof. Boris Yagen
OutlineOutline
Common features of epilepsy and bipolar disorder
Part I
Epilepsy and antiepileptic drugsEpilepsy and antiepileptic drugs
Valproic acid and its major side-effectsValproic acid and its major side-effects
Study objectives and resultsStudy objectives and results
Part II
Bipolar disorder and drug treatment thereof Bipolar disorder and drug treatment thereof
Molecular targets of mood-stabilizers and their Molecular targets of mood-stabilizers and their effect on growth cone behavioreffect on growth cone behavior
Study objectives and resultsStudy objectives and results
Common Features of Epilepsy & Bipolar Disorder
Both disorders respond to several identical drugs :
Gradual progression in intensity and frequency of epileptic seizures and manic-depressive episodes
20-40% of bipolar and epileptic patients are resistant to current drug-therapy
Amann et al, Epilepsia, 2005
N
C NH2O
Valproic AcidCarbamazepine Lamotrigine
Common neurological condition occurring in about 1% of the global population
Characterized by periodic and unpredictable occurrence of seizures, due to disordered, synchronous firing of a population of brain neurons
Classification into partial seizure and generalized seizure
Part I: Epilepsy
Antiepileptic Drugs (AEDs)
Old DrugsOld Drugs New DrugsNew Drugs
CarbamazepineCarbamazepine
PhenobarbitalPhenobarbital
PhenytoinPhenytoin
Valproic AcidValproic Acid
FelbamateFelbamate
GabapentinGabapentin
LamotrigineLamotrigine
LevetiracetamLevetiracetam
OxcarbazepineOxcarbazepine
PregabalinPregabalin
RufinamideRufinamide
StiripentolStiripentol
TiagabineTiagabine
TopiramateTopiramate
VigabatrinVigabatrin
ZonisamideZonisamide
Valproic Acid (VPA)
COOH
Efficient in many types of epilepsy
Migraine prophylaxis
Bipolar disorder
Major rare side effects:
hepatotoxicity & teratogenicity
VPA-Induced Fatal HepatotoxicityVPA-Induced Fatal Hepatotoxicity Worldwide till 1999:Worldwide till 1999:
179 cases of fatal 179 cases of fatal hepatotoxicityhepatotoxicity
Risk Factors of Fatal Hepatic FailureRisk Factors of Fatal Hepatic Failure
Children under the age of two
Polytherapy
Metabolic disturbances and
liver disease
Chang et al, Drug Metab Rev, 2006Chang et al, Drug Metab Rev, 2006
COOH COOHCYP2A6CYP2A6CYP2C9CYP2C9
-oxidation-oxidation -oxidation-oxidation
COOHCOOH
VPAVPA
2-ene 2-ene VPAVPA
4-ene VPA4-ene VPA
2,4-diene 2,4-diene VPAVPA
CYP450CYP450
ffmm= 0.5-1%= 0.5-1%
COOH COOH
VPAVPA
2,2,3,3-tetramethylcyclopropane- carboxylic acid (TMCA)
Quaternary Carbons & Acylurea containing compounds & Hepatotoxicity
CONHCONH2
3,3-dimethylbutanoylurea (DBU)
VPA-Induced Teratogenicity
Finnell et al, Epilepsia, 2003; Sankar, Acta Neurol Scan, 2007Finnell et al, Epilepsia, 2003; Sankar, Acta Neurol Scan, 2007
Major malformations associated VPA therapy:
abnormalities of the skeleton, CNS,
cardiovascular and urogenital system
Neural Tube Defects (NTD):
10-20 fold increased risk
Mechanism of VPA-Induced Teratogenicity
Inhibition of folate metabolism
HDAC-inhibition
Inhibition of neuroepithelial proliferation
Formation of cytotoxic, teratogenic metabolites:
2,4-diene-VPA, 4-ene-VPA
VPA Constitutional Isomers, Amide derivatives and Teratogenicity
Radatz et alRadatz et al, Epilepsy Res, 1998; Isoherranen et alIsoherranen et al, Epilepsia, 2002
VPA
ValpromideValpromide
COOHCOOH
Valnoctic Acid
CONH2COOH
Propylisopropyl acetic Acid
AEDs Containing Urea Moiety in Their Structure
NH
HN OO
H3C
O
NH
HN
O
O
N
CO NH2
CH2CONHCONH2 CHCONHCONH2
C2H5
PhenobarbitalPhenobarbital PhenytoinPhenytoin CarbamazepineCarbamazepine
PhenacemidePhenacemide PheneturidePheneturide
CONHCONH2
TMCUTMCU
Study Objectives: Part I
Design and synthesis of urea derivatives of VPA constitutional isomers and homologs
Evaluation of anticonvulsant activity, neurotoxicity and teratogenicity of the above compounds
PK study of the most potent and safe candidate among the urea derivatives
Maximal Electroshock Seizures (MES)
Animal Models of Epilepsy
sc Metrazole (scMet)
Identifies drugs effective against generalized seizures; seizure spread inhibition
Identifies drugs effective against absence seizures; increase in seizure threshold
Mice : Rotated rod Rats : Positional sense test
6Hz psychomotor seizure test
Identifies drugs effective against therapy- resistant epilepsy
Neurotoxicity
Synthesis of Urea Derivatives of VPA Constitutional Isomers
R1 C
O
OHLDA, THF
R1 C
O-
O-
2Li+
R1 C
O
OH
R2
R1 C
O
Cl
R2
UREA, ACN R1 C
O
NHCONH2
R2
R-I
SOCl2
CONHCONH2
VPU
CONHCONH2
VCU
CONHCONH2
DIU
CONHCONH2
PIU
CONHCONH2
R-PIU
CONHCONH2
S-PIU
CONHCONH2
OCU
Shimshoni et al et al, J Med Chem, 2007
*
Shimshoni et alShimshoni et al, J Med Chem, 2007
Anticonvulsant Activity and Toxicity
CONHCONH2
PIU
CONHCONH2
VCU
CONHCONH2
DIU
Significantly different from the corresponding amide (p<0.05); $ significantly different from the corresponding
enantiomer (p<0.05)
CompoundMES-ED50
( mg/kg)
scMet-ED50
(mg/kg)
Neurotoxicity-TD50
(mg/kg)
PI(MES)
PI(scMet)
VPA485646 7841.61.2
VPU 5477 2324.33.0
VCU 2414*9746.9
DIU 33*16*561.73.5
(R,S)-PIU 16*45 955.92.1
(R)-PIU36$22$1243.45.6
(S)-PIU 18*371186.53.2
OCU >300>300>300--
*CONH2
CONH2 CONH2
CompoundED50 at 32mA
(mg/kg)
ED50at 44mA
(mg/kg)
VPA126 (PI=3.2) 310 (PI=1.3)
Levetiracetam191089
VPU 58 (PI<1.7)105 (PI<0)
VCU 21* (PI<4.7)48* (PI<2.1)
DIU 43*(PI<2.3)49* (PI<2)
(R,S)-PIU 42 (PI<2.4)71 (PI<1.4)
(R)-PIU 43 (PI=2.7)56# (PI=2.1)
(S)-PIU 46* (PI<6.5)75 (PI<4)
Anticonvulsant Activity in Mice 6Hz Model
Shimshoni et alShimshoni et al, J Med Chem, 2007
Significantly different from the corresponding amide (p<0.05); $ significantly different from the corresponding enantiomer (p<0.05)
CONHCONH2
VCU
CONHCONH2
DIU
CONHCONH2
S-PIU
CONH2 CONH2
CONH2
DrugMES-ED50
(mg/kg)
scMet- ED50
(mg/kg)
Neurotoxicity-TD50
(mg/kg)
PI(MES)
PI(scMet)
VPA4856467841.61.2
PVU69302283.37.6
IVU>25083300<TD50<500-3.6<PI<6
DBU64261432.25.5
Anticonvulsant Activity and Toxicity of VPU Homologs
Shimshoni et alShimshoni et al, submitted to Epilepsia, 2008
CONHCONH2
CONHCONH2
CONHCONH2
COOH
TestDBU’s ED50 or TD50
(mg/kg)
PI (DBU)VPA’s ED50 or TD50
(mg/kg)
PI (VPA)
6Hz (32mA) 803126 3.2
6Hz (44mA)1331.8310 1.3
Toxicity244-398-
Treatment GroupaDosemg/kg
(mmol/kg)
No. Litters No. of Live Fetuses
Exencephaly%
Control25% CEL151880
VPA 452 (2.7)1314129.1*
VPA301 (1,8)121331.5
VPU261(1.8)108013.7*
VPU167(0.9)101150.9
VCU205 (1.1)10752.7
(R)-PIU335 (1.8)95215.4*#
(R)-PIU205 (1.1)91040
(S)-PIU335 (1.8)101235.7*
(S)-PIU205 (1.1)101240.8
Teratogenicity of Acylurea Isomers of VPU in SWV Mice
Shimshoni et alShimshoni et al, J Med Chem, 2007
Significantly different from control (p<0.05); # significantly different from the corresponding enantiomer (p<0.05)
Treatment GroupDosemg/kg
(mmol/kg)
No. of Litters
No. of Live Fetuses
Exencephaly%
Control25% CEL151880
VPA452 (2.7)1314129.1*
IVU520 (3.6)121350
PVU520 (3.6)131800.6
DBU671 (3.6)10981
DBU502 (2.7)121560.6
Shimshoni et alShimshoni et al, submitted to Epilepsia, 2008
Teratogenicity of Acylurea Homologs of VPU in SWV Mice
Significantly different from control (p<0.05)
PK Profile of DBU in Rats (10mg/kg, i.v.)
CL (L/h•kg) 0.12 0.24 0.36
Vss (L/kg) 0.78 0.87 0.86
t1/2 (h) 4.5 4.0 1.6
MRT (h) 6.5 3.5 2.5
fe (%) 2.4 1.8 6.3
DBU VPA TMCU
Shimshoni et alShimshoni et al, submitted to Epilepsia, 2008
0.0
5.0
10.0
15.0
20.0
25.0
0 5 10 15 20 25
Time (h)
Pla
sm
a C
on
c. (
mg
\L) CONHCONH2
Conclusions: Part I
1. Urea derivatives of VPA constitutional isomers exhibited potent and broad anticonvulsant activity
2. PIU exhibited enantioselective activity (S-PIU was more potent in the MES, whereas R-PIU was more potent in the scMet and 6Hz tests)
3. Even at doses 3 times larger than their ED50 values, PIU, VCU and VPU were non teratogenic
4. PIU enantiomers demonstrated enantioselective teratogenicity (R-PIU was more teratogenic than S-PIU)
5 .Homologs of VPU with 2-3 carbon atoms less, but containing quaternary carbon (DBU and PVU) retain high anticonvulsant activity and are non-teratogenic
6 .Based on the pharmacokinetic study in rats, DBU’s half-life was in a similar magnitude as VPA
7 .VCU, PIU and DBU have the potential to become antiepileptic drugs second generation to VPA
Conclusions: Part I
Part II: Bipolar Disorder (BD)
BD is common, affecting approximately 1-2% of the population
BD is characterized by unpredictable swings in mood from mania to depression
Manic episodes emerge gradually and last as long as several months to years when untreated
Frequent age of onset: 20-30 years
Belmaker, N Engl J Med, 2004
Clinical Features
ManiaDepression
Moodelevated, labiledepressed, miserable
Talkfast, flight of ideasslow, monotonous
Energyexcessive, increased psychomotor activity
lacking energy, apathy
Ideationgrandiose, self confidentguilt, unworthiness
Physicalinsomnia, weight lossfatigue
Behaviordisinhibition, excessive spending
retardation or agitation, poverty of movements
Belmaker, N Engl J Med, 2004
FDA Approved Drug Treatments for BD
ManiaMania MaintenanceMaintenance
AntipsychoticsAntipsychotics
LithiumLithium
CarbamazepineCarbamazepine
Valproic AcidValproic Acid
LithiumLithium
LamotrigineLamotrigine
OlanzapineOlanzapine
AripiprazoleAripiprazole
((atypical antipsychotic)
DepressionDepression
Olanzapine (3mg) Olanzapine (3mg) +
Fluoxetine (25mg) Fluoxetine (25mg)
(Symbyax®)(Symbyax®)
Ketter et al, Psychopharm Bull, 2006
Berridge et al, Cell, 1989; Silverstone et al, Bipolar Disorder, 2005
diacylglycerol
PLC
Inositol
IP IP2
Inositol synthaseIno-1
Glucose-6-phosphate
IMPase
BD & the Inositol Depletion Theory
Mania:
Inositol
Depression:
Inositol
VPA
Li+
Neurites elongate by growth at their distal end called the growth cone
Growth cones integrates external signals and translate them into changes in the rate and direction of growth
The Effect of Mood Stabilizers on Growth Cone Behavior
Harwood et al, Clin Neuroscience Res, 2004
The Effect of Mood Stabilizers on Growth Cone Behavior
VPA, CBZ & Li+
Common mechanism :growth cone spreading via inositol depletion
Antipsychotics and Antidepressant have no growth cone spreading effect
The spread of growth cones provides a cell-based assay that may be utilized as a screening approach for mood stabilizing properties
Williams et al, Nature, 2002
Inositol
Study Objectives: Part II
Evaluate the effect of VPA constitutional isomers, cyclopropyl analogs for mood stabilizing properties
Evaluate the inositol depleting activity of the aforementioned compounds
VPA Constitutional Isomers, Cyclopropyl Analogs & their Corresponding Amide
Shimshoni et al, Mol Pharmacol, 2007
COOH
COOH
COOH COOH COOH
COOH CONH2 CONHCH3 CONHCONH2
COOH
CONH2 CONH2 CONH2 CONH2
VPA
4-ene-VPA 4-yne-VPA
PIA DIA VCA
VPD PID DID VCD
TMCA TMCD MTMCD TMCU
Growth Cone-Based Assay: Dorsal Root Ganglion (DRG)
DRG are composed of several thousand cell bodies of somatosensory neurons
Rat DRG showing neuron outgrowth (x20)
a. Control
0102030
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Spread (um2) Spread (um2) Spread (um2)
Shimshoni et al, Mol Pharmacol, 2007
Growth Cone Spreading Effect of Aliphatic VPA Constitutional Isomers
%
%
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b. 3mM TMCD
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Growth Cone Spreading Effect of VPD Constitutional Isomers, Cyclic VPA Analogs &
their Amides
Shimshoni et al, Mol Pharmacol, 2007
Spread (um2) Spread (um2) Spread (um2)
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Shimshoni et al, Mol Pharmacol, 2007
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trol
VPA
Lith
ium
TMCD
PIA
PID
InsP
3 l
evel
s (%
of
con
tro
l)
* * *
Conclusions : Part II
1. PIA and DIA, the constitutional isomers of VPA, as well as the amide of cyclic VPA analog, MTMCD are more effective than VPA in increasing growth cone spreading via inositol depletion mechanism
2. Anticonvulsant potency and growth cone spreading effect of the above compounds are poorly correlated, suggesting a different mechanisms of their action
3. PIA, DIA and MTMCD have a potential as new antibipolar drugs
1. Shimshoni JA, Dalton EM, Jenkins A, Eyal S, Ewan K, Williams RSB, Pessah N, Yagen B, Harwood AJ, Bialer M. The effects of CNS-active valproic acid constitutional isomers, cyclopropyl analogues and amide derivatives on neuronal growth cone behaviour. Mol Pharmacol, 2007, 71: 884-92
2. Shimshoni JA, Bialer M, Wlodarczyk B, Finnell RH, Yagen B. Potent anticonvulsant urea derivatives of constitutional isomers of valproic acid. J Med Chem, 2007, 50: 6419-6427
3. Shimshoni JA, Bialer M, Yagen B. Synthesis and anticonvulsant activity of aromatic tetramethylcyclopropanecarboxamide aromatic derivatives. Submitted to Bioorg & Med Chem, 2008
4. Shimshoni JA, Yagen B, Pessah N, Wlodarczyk, Finnell, Bialer M. Anticonvulsant profile and teratogenicity of 3,3-dimethylbutanoylurea: a potential for a second generation drug to valproic acid. Submitted to Epilepsia, 2008
List of Publications
Acknowledgements
Prof. Meir Bialer & Prof. Boris YagenProf. Meir Bialer & Prof. Boris Yagen
Prof. Adrian HarwoodProf. Adrian Harwood
Dr Ken EwanDr Ken Ewan
Dr Robin WilliamsDr Robin Williams
Miss Emma DaltonMiss Emma Dalton
Dan KaufmannDan Kaufmann
Dorit MimrodDorit Mimrod
Neta PessahNeta Pessah
Naama HenNaama Hen
Idit AchachIdit Achach
Cardiff University Texas University
Prof. Richard H. FinnellProf. Richard H. Finnell
Dr Bogdan WlodarczykDr Bogdan Wlodarczyk
Lab Members