Bile salt transport profiling of hepatic transporters. Peter Krajcsi

Transcription

1 Bile salt transport profiling of hepatic transporters Peter Krajcsi

2 The human bile Carey and Duane: The liver, 3rd edition

3 Bile acids / salts Cholesterol Hepatocytes Primary bile acids: Chenodeoxycholic acid Cholic acids Intestinal bacteria Secondary bile acids: Lithocholic acid Deoxycholic acid Ursodeoxycholic acid Sulfation, Glucuronidation Sulfated / glucuronated bile acids Amidation: glycine or taurin Monovalent bile salts Sulfation, Glucuronidation Divalent bile salts

4 Inhibition of transport of endobiotics Transport of endobiotics Altered homeostasis Potential toxicity

5 Bile salt transportin hepatocytes BS BSEP deficiency / PFIC2 MRP4 Bile acid in serum Bile acid in bile high low BS OATP1A2 BS Hepatocyte Phospholipid in bile normal BS NTCP BS BSEP MRP2 BS- BS-C Bile / Canaliculus Phase II conjugation BS-C BS OATP1B1 BS BS-C MRP3 Blood / Sinusoidal Space Morgan 2010 Tox Sci BS-C

6 Number of cases Drug induced liver injury cholestatic mixed hepatocellular 5 0 Y. Meier et al., Eur J Clin Pharmacol 61, 135, 2005 ~30% cholestatic or mixed Swedish study: almost 50% of DILI cases were cholestatic or mixed (Björnsson & Olssson 2005 Hepatology)

7 Human BSEP VT a HT screening tool Of particular interest are the compounds with an IC50 value of <25 um...almost all compounds with this level of potency for BSEP interference are associated with liver injury in humans Morgan 2010 Tox Sci

8 Inhibition of transport of endobiotics Transport of endobiotics Indication of transporter function Potential biomarker

9 Plasma levels of sulfates in cynos Thakare 2017 DMD

10 Effect of rifampicin on plasma levels of phase II conjugates of bile acids/salts Takehara 2017 Pharm Res

11 Kinetics of transport sulfates, glucuronides Takehara 2017 Pharm Res

12 Assay systems to study bile salt - transporter interaction BS Inside-out vesicles BS BS ATP Cellular uptake influx / uptake / SLC transporters BS ADP + Pi Vesicular uptake efflux / ABC transporters

13 BSEP BSEP-HAM rbsep rbsep-ham mbsep mbsep-ham BSEP/Bsep vs BSEP/Bsep-HAM species specificity rbsep BSEP mbsep Kis 2009 DMD

14 Cholesterol loading increases activity of BSEP development of high activity membranes (HAM) Kis 2009 DMD

15 BSEP/Bsep vs BSEP/Bsep-HAM Eadie-Hofstee plots BSEP mbsep rbsep Kis 2009 DMD

16 BSEP membranes CsA inhibition Taurocholate accumulation in CPM Inhibition of taurocholate accumulation into BSEP expressing membrane vesicles by Cyclosporin A Cyclosporin A conc. in mm BSEP-Sf9 ATP BSEP-Sf9 AMP BSEP-Hi5 ATP BSEP-Hi5 AMP BSEP-HEK ATP BSEP-HEK AMP Origin Signal/noise (ATP+/ATP-) IC50 for reference inhibitor (mm) SB-BSEP-HEK293 Mammalian SB-BSEP-Sf9 Insect SB-BSEP-Hi5 Insect

17 Bile acids/salts CDC GCDC TCDC 3S-CDC TC 3S-GLC

18 Glycholithocholic acid-sulfate effect on bile flow, morphology Yousef 1981 Gastroenterology

19 Screening strategy Feasibility screen (2 concentrations, 2 timepoints) Time course Kinetics

20 OATP1B1 - feasibility

21 OATP1B1 time courses

22 OATP1B1 - kinetics

23 CL int (ml/mg/min) C L in t (m l/m g /m in ) Bile salt substrate profiling NTCP, OATP1B1 NTCP OATP1B ** ** p= p= * p = * p = p = * GCDC TC 3S-GLC 0 G C D C T C D C T C 3 S -C D C 3 S -G L C

24 HEAT map human transporters Bile Salt / Transporter NTCP OATP1B1 CDC TCDC GCDC TC 3S-CDC 3S-GLC Bile Salt / Transporter BSEP MRP2 MRP3 MRP4 CDC TCDC GCDC TC 3S-CDC 3S-GLC The boxes in red indicate POC folds >4, the boxes in orange indicate POC folds 4>fold>2.5, the boxes in yellow indicate POC folds 2.5>fold>1.5, and the boxes in blue indicate no transport.

25 Bile acids/salts CDC GCDC 3S-CDC 3S-GCDC

26 3 S -C D C u p ta k e (p m o l/m g o f p ro te in ) 3 S -G C D C u p ta k e (pm o l/m g of p rotein ) G C D C u p ta k e (pm o l/m g of p rotein ) G C D C u p ta k e (p m o l/m g o f p ro te in ) BSEP time courses for human and rat G C D C -B S E P G C D C -r B s e p T im e (m in ) T im e (m in ) H E K B S E P H E K M o c k H E K rb s e p H E K M o c k B S E P -3 S -C D C B S E P -3 S -G C D C T im e (m in ) T im e (m in ) H E K B S E P H E K M o c k H E K B S E P H E K M o c k

27 A T P d e p e n d e n t tr a n s p o rt in H E K B S E P (p m o l/m g /m in ) A T P d e p e n d e n t tr a n s p o rt in H E K B S E P (p m o l/m g /m in ) A T P d e p e n d e n t tr a n s p o rt in H E K B S E P (p m o l/m g /m in ) A T P d e p e n d e n t tr a n s p o rt in H E K r B s e p (p m o l/m g /m in ) BSEP kinetics for human and rat B S E P - G C D C ' r B s e p -G C D C C o n c e n tr a tio n (m M ) C o n c e n tr a tio n (m M ) B S E P -3 S -C D C B S E P -3 S -G C D C C o n c e n tr a tio n (m M ) C o n c e n tr a tio n (m M )

28 C L in t (m l/m g /m in ) C L in t (m l/m g /m in ) Bile salt substrate profiling Efflux transporters B S E P ra tb s e p G C D C 3 S -C D C 3 S -G C D C 0 G C D C

29 C L in t (m l/m g /m in ) C L in t (m l/m g /m in ) C L in t (m l/m g /m in ) Bile salt substrate profiling Influx transporters O A T P 1 B 1 O A T P 1 B * p = * p = G C D C 3 S -C D C 0 G C D C 3 S -C D C 3 S -G C D C r a to a tp 1 b * p = G C D C 3 S -C D C 3 S -G C D C

30 C L in t (m l/m g /m in ) C L in t (m l/m g /m in ) Bile salt substrate profiling Influx transporters O A T P 1 A 2 r a to a tp r 1 a G C D C 0 G C D C 3 S -C D C 3 S -G C D C

31 Heat map based on uptake ratios - human and rat transporters CDC GCDC 3S-CDC 3S-GCDC NTCP OATP1A2 OATP1B1 OATP1B3 BSEP rntcp roatp1a1 roatp1a4 roatp1b2 rbsep CDC GCDC 3S-CDC 3S-GCDC The boxes in red indicate an average POC folds >4, the boxes in orange indicate an average POC folds 4>fold>2.5, the boxes in yellow indicate an average POC folds 2.5>fold>1.5, and the boxes in blue indicate no transport

32 Expression of transporters in human hepatocytes Sandwich cultured human hepatocytes Schaefer 2012 DMD Closed squares: n = 2 or 3; open squares n = 1

33 Human hepatic bile salt transport BS + BS-C BS OATP1A2 OSTαβ BS + BS-C NTCP BS Hepatocyte Blood / Sinusoidal Space BS + BS-C OATP1B1 Phase II BS-C BSEP MRP2 BS + BS-C BS-C Bile / Canaliculus BS + BS-C OATP1B3 MRP3 MRP4 BS-C + BS BS-C

34 Acknowledgements Solvo R&D team Beata Toth Marton Jani Erzsebet Beery Nora Szilvasy Zsuzsanna Gáborik Krisztina Herédi-Szabó Emese Kis Production team Zsolt Sáfár Judit Molnár Anita Kurunczi Emese Kis Bioanalytics group Éva Molnár Pádár Petra Plahi Bognar Agata Zoltán Tömöri Collaborators DILI Consortium Anita Dankers (JnJ) Teresa Heslop (GSK) Y Anne Pak (Lilly) Shelby C Hullett (Lilly) Mike Bayliss (U Liverpool) Neil Kitteringham (U Liverpool) Bhavana Jagota (U Liverpool) Kevin B Park (U Liverpool) Richard W Weaver (Servier) Dept Med Chem, U Szeged Prof Gábor Tóth & lab

35 Sulfated conjugates in the focus In fact, conjugates of bile acids represented 4 of the 12 metabolites significantly associated with OATP1B1 genetic variants. These conjugates also increased following administration of CSA. Yee 2016 CPT Morgan 2010 Tox Sci

36 Regulation Alferai 2007 Pharm Res Dawson 2009 JLR

37 FIC1 deficiency

38 Number of cases Drug induced liver injury % cholestatic: mixed: ALT/AP < 2N 2N< ALT/AP < 5N hepatocellular: ALT/AP > 5N (Classification according to CIOMS) Y. Meier et al., Eur J Clin Pharmacol 61, 135, 2005 Swedish study: almost 50% of DILI cases were cholestatic or mixed (Björnsson & Olssson 2005 Hepatology)

39 Dawson 2009 JLR Bile salt transport

40 Bas/BSs in human and rodent liver and serum Garcia-Canaveras 2012 JLR

41 Composition of the BA/BS pool in human and rat liver and serum % Liver % Serum % % 80.00% 80.00% 60.00% 60.00% 40.00% 20.00% human rat 40.00% 20.00% human rat 0.00% 0.00% Human CDCA:CA=1:1 G:T=2-3.7:1 Rat CDCA:CA=1:2 G:T=1:3-6 Garcia-Canaveras 2012 JLR

42 Heat map based on uptake ratios - human and rat transporters NTCP OATP1A2 OATP1B1 OATP1B3 MRP2 MRP3 BSEP CDC CDC GCDC GCDC 3S-CDC 3S-CDC 3S-GCDC 3S-GCDC CDC GCDC 3S-CDC rntcp roatp1a1 roatp1a4 roatp1b2 CDC GCDC 3S-CDC rmrp2 rmrp3 rbsep 3S-GCDC 3S-GCDC The boxes in red indicate an average POC folds >4, the boxes in orange indicate an average POC folds 4>fold>2.5, the boxes in yellow indicate an average POC folds 2.5>fold>1.5, and the boxes in blue indicate no transport

43 Heat map based on Km - human and rat transporters CDC GCDC 3S-CDC 3S- GCDC CDC GCDC 3S-CDC 3S- GCDC NTCP OATP1A2 OATP1B1 OATP1B3 rntcp roatp1a1 roatp1a4 roatp1b2 CDC GCDC 3S-CDC 3S- GCDC CDC GCDC 3S-CDC 3S- GCDC MRP2 MRP3 BSEP rmrp2 rmrp3 rbsep The boxes in red indicate Km < 1 um, the boxes in orange indicate Km 10>Km>1 um, the boxes in yellow indicate Km >10 um, and the boxes in blue indicate no significant transport

44 Inhibition profiling of human rat and monkey OATPs/Oatps OATP 1A2 OATP 1B1 OATP 1B3 roatp 1a1 roatp 1a4 roatp 1b2 cyoatp 1b1 cyoatp 1b3 OATP 1A2 8/8 OATP 1B1 1/8 8/8 OATP 1B3 1*/8 6/8 8/8 roatp 1a1 5*/8 3/8 7*/8 8/8 roatp 1a4 6*/8 2/8 2/8 4/8 8/8 roatp 1b2 1/8 4/8 5/8 6/8 1/8 8/8 cyoatp 1b1 1/8 7/8 5/8 3/8 2/8 5/8 8/8 cyoatp 1b3 2*/8 3/7 5*/8 6*/8 2/8 7*/8 4/8 8/8 Atorvastatin CCK8 Diclofenac NMQ Pravastatin Taxol Telmisartan Troglitazone Substrate: taurocholate