evaluation of transporter abundance variance and its ...evaluation of transporter abundance variance...

Evaluation of transporter abundance variance and its impact on IVIVE and inter-species comparison Buyun Chen June 14th, 2018

2 Transporter abundance is important for PBPK model

Physiological parameter

Compound parameter

PBPK model

3

Scaling transporter data: RAF vs REF

Relative Activity factor

Pitavastatin

CLhep=CLoatp8*RAF1

CLhep=CLoatp2*RAF2

CCK8 E2-Sulfate

Relative Expression Factor

Hepatocyte OATP2/8

Clhep=CLoatp8*REF1

Clhep=CLoatp2*REF2

OATP2 cell OATP8 cell

0

10

20

30

40

50

60

70

80

0

10

20

30

40

50

60

70

80

E2-17βGluc Pitavastatin

Hep

tatic

Cle

arnc

e

Predicted OATP8 CL

Predicted OATP2 CL

Observed Clearance

0

50

100

150

200

250

300

0

50

100

150

200

250

300

E2-17βGluc Pitavastatin

Hep

tatic

Cle

arnc

e

Predicted OATP8 CL

Predicted OATP2 CL

Observed Clearance

Hirano et al, 2004

Lack of selective probe for each transporter.

Over-prediction by REF using western blotting quantitation

4

Challenges in transporter IVIVE

No selective substrate Abundance very low

Welger, et al, Mol Pharm, 2017

5 Huge variation in literature results using LC-MS

Livers from 10 donors tested in 6 labs Variation of OATP1B1 abundance up to 120 fold

Welger, et al, Mol Pharm, 2017

• Significant clearance variation of atorvastatin resulted from

using different ISEF in absolute scaling

6

7 Sources of variation

Extraction

Peptide selection Digestion

8 Impact of peptide selection on quantitation result

Harwood, et al, DMD, 2015

Different peptide selection for PGP and BCRP from BPh and UoM

Different abundance reported for Caco-2 and intestinal abundance

9

Extraction

Digestion

Quantitation

Protein vs peptide based quantitation

Protein Conc Peptide Conc

Pept

ide

sign

al

Pept

ide

sign

al

Sample Standard Sample

Standard

MDR1

10 Peptide screening strategy

FASTA <UniProt>

Hepatocytes/Liver Tissues Membrane Protein Extraction

<Millpore Kit or whole cell lysate>

Protein Denature & Tryptic Digestion <5%DOC, spin column, trypsin>

LC-MS/MS Analysis <Sciex 6500 QTrap>

Trypsin Digestion, 6-16 AA Exist in all isoform No Cys, Met, His No Ragged end No PTM, SNP

No transmembrane region Spike in protein std

Overexpression cell line for low abundance transporters

0

0.2

0.4

0.6

0.8

1

1.2

Selecting peptides with highest digestion efficiency

© 2009, Genentech

0.00

1.00

2.00

3.00

4.00

5.00

6.00

7.00

8.00

9.00

Pept

ide

Con

cent

ratio

n(nM

))

Surrogate Peptides

Pre-Spike

Post Spike

First example from MDR1

Digestion efficiency =Post Spike − Pre Spike peptide concentration

Spiked in protein concentration

8-10 fold higher than commonly used peptide

Chen, B. et al, The AAPS Journal 2017, epub

12 Reasons for different digestion efficiency

EALDESIPPVSFWR 1. “Elbow region” connecting two

halves of homo dimer in lower species

2. Not an integral part of large stretches of tertiary structures

STV and FYD Adjacent to ATP binding sequence, more accessible to digestion enzyme

Jian Payandeh

13

0

1

2

3

4

5

6

7

8

GAA EAL IIG NTT STV FYD VLL VVQ

Con

cent

ratio

n(nM

)

Surrogate Peptide

Male, 37, smokerMale, 61Male, 53Female, 62Female, 54, Alcohol, CocaineFemale, 84

Determination in liver and comparison to literature

Reference Ohtsuki et al, 201214 Prasad et al, 201315 Peng et al, 201512 This study

n= 17 64 141 6 MDR1(NTT) 1.50±0.44 0.4±0.2 0.6±0.2 0.90±0.37 MDR1(IAT) 0.3±0.2 0.77±0.47 MDR1(EAL) 16.78±5.48

Extraction Ultracentrifugation PM Millipore kit PM Millipore kit PM Millipore kit PM

Digestion enhancment procedure

6M Urea Extraction buffer II DOC, dilute/dry DOC, spin column

Chen, et al, AAPS J, 2017

14 Similar correlation for BSEP

Reference Li et al,. 2009 This study

n= 17 Pooled hepatocyte STALQLIQR 3.50±0.60

AAATSIFETIDR 5.41±0.40 VGFFQLFR 7.17±0.02

Extraction Millipore kit Crude membrane Millipore kit Crude membrane

Digestion enhancment procedure

N/A DOC, spin column

15 Extraction procedure effect

Cytosolic

Membrane

Pellet

Whole cell

lysate Hepatocyte

16 Extraction effect: Correction factor possible

• MDR1 Slope>1, not 100% on membrane;

• R2>0.9, a correction factor can be applied

17 Digestion condition effect

Solubilization • Deoxycholate, Urea, or extraction buffer

Detergent removal

• Spin column or MWCO filter

Digestion • Trypsin/LysC

18 Solubilization effect is dependent of peptide selection

Balogh et al., J Proteomics Bioinform 2012, S4

19 Digestion condition effect

• Slope<1: Digestion efficiency less than 5% DOC • R2<0.9, digestion factor not likely applicable

independent of peptide selection

20 Characterizing novel in vitro model

Knock out Canine Mdr1 gene

Overexpression of human MDR1 protein

Comparison with tranditional MDR1 overexpression cell lines

CRISPER-Cas9

Eugene Chen

21 Correlating activity and expression abundance

Human MDR1 with canine background

Human MDR1 without canine background

• Difference in substrate specificity? • Difference in expression abundance?

22 Selecting animal model for OATP DDI in vivo study

• Shared sequence

• Homologous sequence

Peptide seledtion

• Pooled heptoctyes • Comparison of multi-

peptide pairs

Quantitation • Extent of DDI in human and cyno

• Analyze in context of in vitro IC50

In vivo study

23 OATPs: comparison of different species

Literature data

1 B 1 _ H u ma n /C

y n o .ITP

1 B 1 _ H u ma n /C

y n o .NV T

1 B 1 _ H u ma n /C

y n o .YV E

1 B 3 _ H U M A N .NV T

1 B 3 _ H U M A N .IYN

1 B 3 _ H u ma n _ N Q T C

y n o _ S Q T

nM

OATP1B1 OATP1B3

Genentech data

• Hepatocyte o Liver

24 Homologous peptide in cyno and human

Distance to TMD longer

Continuous R/K near cleavage site

Cyno OATP1B3 Human

Cyno OATP1B1 Human

25 monkey as the model of human OATP DDI prediction

Maeda et al, Clinical pharmacology & therapeutics | volume 90 number 4 | october 2011

C o n trol

+ R if

Pra

va

sta

tin

AU

C/d

os

e

In vitro

In vivo

Eugene Chen and Kevin Dement

C o n trol

+ R if

Pra

va

sta

tin

AU

Cla

st

C o n trol

+ R if

Pra

va

sta

tin

AU

Cla

st

0

1

2

3

4

I C 5 0

o f R

i f Human Monkey

26 Conclusion and Future work

Peptide selection could have significant impact on quantitation result

Extraction effect can be corrected by a factor independent of peptide selection

Digestion effect is peptide dependent Interspecies correlation should be conducted with consideration

of homologous peptide selection

Bridging literature data gap and better DOE for meta-analysis Transporter localization/regulation/turn-over

Acknowledgement • Liling Liu • Yuan Chen • Jialin Mao, • Hoangdung Ho • Jian Payandeh • Eugene Chen • Kevin Dement • Edna Choo • Emile Plise • Jonathan Cheung • Laurent Salphati

Alan Deng Brian Dean Marcel Hop