1. In vitro and in vivo metabolism of repaglinide: Modeling clinically- relevant drug-drug interactions Joanna Barbara, Ph.D. Director of Analytical Services, XenoTech LLC. Pacific Northwest Biosciences Winter Seminar March 3, 2014

2. XenoTech’s integrated service capabilities Enzyme Inhibition Evaluate potential for direct, and metabolism- dependent inhibition(MDI or TDI) Mechanistic studies (direct or MDI) Non-CYP enzymes (e.g., UGT, MAO, AO ) Transporters XenoTech and Sekisui In vitro studies in mono-layer cell lines for uptake Bi-directional assay for efflux transporters Membrane-based vesicles and ATPase assays Drug Metabolism Metabolic stability and species comparison Metabolite characterization/ID Reaction phenotyping (CYP & UGT) Customized services Enzyme Induction In vitro studies in cultured hepatocytes (human and animal) Ex vivo studies in animals Toxicity & mechanistic studies XT Consulting Department Expert data review and study consultation Sekisui Medical (Conducted in Japan) RI synthesis (radiolabeling), preclinical in vivo PK studies, QWBA, plasma protein binding, humanized chimeric mice (PXB), biomarker analysis, pharmacological receptor assays Bioanalytical Non-GLP Bioanalysis GLP and non-GLP in vitro study support 2 Products

3. Overview 3 Enzymatic biotransformation and drug-drug interactions Introduction to repaglinide and project background – Repaglinide as a probe substrate Investigating mechanism of drug-drug interactions – In vitro metabolism Evaluating rat as a preclinical model – In vivo metabolism Conclusions

4. Enzymatic biotransformation of drugs 4 Cytochrome P450 (CYP) enzymes are responsible for biotransformation of ~70% hepatically-cleared drugs Hepatic clearance route by enzyme type Data adapted from: Cassarett and Doull’s Toxicology (2001) C. Klaassen (Ed), New York, NY: McGraw-Hill CYP UGT Esterase

5. Drug-drug interactions (DDI) 5 Altered enzymatic biotransformation can lead to clinically-relevant drug-drug interactions between co- administered drugs, a key safety consideration In preclinical drug development, DDI risk is assessed by evaluating – Major clearance routes (e.g., mass balance, CYP phenotyping) – Enzyme inhibition potential – Enzyme induction potential – Transporter involvement and inhibition potential

6. Cytochrome P450 inhibition 6 CYP inhibition has potential to result in – Black box label warnings – Withdrawal from market Mibefradil: withdrawn 1998 (perpetrator drug) Mibefradil inhibits CYP3A4 and can cause elevated levels of coadministered drugs cleared by these enzymes. Life-threatening interactions can occur with  - blockers and other antihypertensives Terfenadine: withdrawn 1997 (victim drug) Co-administration with CYP3A4 inhibitors (e.g., ketoconazole) reduced clearance of the drug and resulted in cardiotoxicity caused by terfenadine accumulation

7. Repaglinide is an insulin secretagogue used to normalize postprandial hyperglycemia in patients with type 2 diabetes Major human metabolite in vivo is the dicarboxylic acid (van Heiningen et. al., 1999) Other oxidative metabolites and glucuronide conjugate Repaglinide uses 7 RepaglinideDicarboxylic acid (M2) van Heiningen et al. Eur, J. Clin. Pharmacol. Exp. Ther. 1999; 55(7): 521-525.

8. Major biotransformation routes described (Bidstrup et al., 2003) Repaglinide metabolism 8 Bidstrup et al. Br. J. Clin. Pharmacol. 2003; 56: 305-314. CYP2C8 metabolism M0-OH M4 CYP3A4 metabolism M1 M2 M5 CYP2C8 probe

9. Repaglinide M4 formation and antibody inhibition 9 Bidstrup et al. Br. J. Clin. Pharmacol. 2003; 56: 305-314. Roles for CYP2C8 CYP3A4

10. Repaglinide metabolized by CYP3A4/2C8 and UGT1A1 10 Repaglinide therefore has potential for DDIs with other drugs cleared hepatically by CYP3A4 and 2C8 and UGT1A1 According to the University of Washington Drug Interaction Database, repaglinide is known for interactions with 10 drugs – Flucloxacillin and rifampin cause increased CL – Gemfibrozil, clarithromycin, cyclosporine, deferasirox, telithromycin, itraconazole, trimethoprim cause >40% increase in AUC

11. Gemfibrozil and repaglinide 11 Type 2 diabetics have 2-4-fold increased risk of macrovascular disease Gemfibrozil is used to reduce triglycerides (TG) in patients with certain dyslipidemias – Almost 30% TG reduction in diabetics compared to placebo group In patients concommitant administration has resulted in up to 8-fold plasma increase in repaglinide – Reports of severe, prolonged hypoglycemia Backmann et al. Drug Metab. Dispos. 2009; 37(12): 2359-66. Vinik and Colwell Diabetes Care 1993; 16(1): 37-44.

12. Gemfibrozil dosing and pharmacokinetics 12 Gemfibrozil usually dosed at 600 mg twice a day or less commonly 900 mg once daily PK parameters after a single oral dose Rouini et al. Int. J. Pharmacol. 2006; 2: 75-78. Parameter600 mg dose900 mg dose C max (µg mL -1 )28.8 ± 4.140.8 ± 12.6 t max (h)1.8 ± 0.8 AUC 0-8 (µg h mL -1 )80.3 ± 10.3132.1 ± 35.3 CL (L -1 )7.1 ± 0.96.6 ± 1.6 Vd (L -1 )11.6 ± 2.112.5 ± 3.4 t 1/2 (h)1.1 ± 0.21.3 ± 0.1

13. Gemfibrozil metabolism 13 Metabolized in liver to 4 major metabolites but the glucuronide metabolite is a potent CYP2C8 inhibitor Baer et al. Chem. Res. Toxicol. 2009; 22(7): 1298-1309.

14. In vitro experiments with repaglinide 14 Initially worked to establish a simple CYP2C8 assay in vitro to complement the in vivo application of repaglinide Noted discrepancies using reference material potential issues with some of the analytical work described in the literature Subsequently needed to re-establish the specificity of the CYP2C8/CYP3A4 metabolism

15. High-resolution LC UV chromatogram (254 nm) Repaglinide in human liver microsomes (HLM) 15 50  M Repaglinide 0.5 mg/mL HLM 30 minutes; 37°C; pH 7.4 NADPH-generating system Repaglinide Hydroxyrepaglinide (M4) Repaglinide desaturation metabolites Repaglinide dicarboxylic acid metabolite (M2) Unlabeled peaks are not related to repaglinide Major in vivo metabolite Probe metabolite Low abundance High abundance

16. Repaglinide human liver microsome metabolite profile 16 Componentt r (min)m/z value Mass error (ppm) Mass shift Proposed biotransformation M0-OH5.48469.2693 -1.9+15.9940 Hydroxylation C15.94451.2590 -0.2-2.0156 Dehydrogenation C26.83441.2369 4.7-12.0384 O-deethylation + hydroxylation M47.50469.2700 -0.4+15.9947 Hydroxylation M17.95385.2112 -3.9-68.0641 N,N-didealkylation M58.26425.2436 -4.5-28.0317 O-deethylation Repaglinide8.98453.2738 -3.3-0.0015 None C310.09471.2848 -0.9+18.0095 Hydroxylation+ reduction M210.30485.2642 -5.1+31.9889 N-dealkylation + oxidation to the carboxylic acid C416.09451.2583 -3.1-2.0170 Dehydrogenation C517.23451.2591 -1.3-2.0162 Dehydrogenation C617.52451.2599 0.4-2.0154 Dehydrogenation

17. Recombinant CYP panel for repaglinide substrate loss 17 Incubating drug with individual enzymes can help narrow down enzymes involved in metabolism Complicated by involvement of enzymes that would not be involved in a more complete test system Substrate loss 10  M repaglinide 10 pmol/inc rCYP 20 minutes 35°C; pH 7.4 CYP3A4?

18. Inhibition experiments for CYP reaction phenotyping 18 Simple test system to minimize variables – HLM for cytochrome P450-mediated M0-OH, M1, M2, M4 and M5 Use known chemicals (or antibodies) to inhibit specific enzymes – Mibefradil for CYP3A4 (metabolism-dependent) – Gemfibrozil glucuronide for CYP2C8 (metabolism- dependent) Assess the effect of the presence/absence of the inhibitor on formation of the metabolite of interest

19. Selecting appropriate conditions for inhibition experiments 19 Initial-rate conditions desirable

20. Metabolism-dependent CYP2C8 and 3A4 inhibition 20 3A42C8Less clear M0-OH M4 M5 M1 M2

21. Correlation data for major metabolites with HLM donor panel 21

22. Exploring the interaction further 22 Nonclinical species have very limited use in modeling human DDIs One major challenge is species differences in protein expression and function (e.g., enzyme specificity) Rodent studies occur early on for most drugs Rat is not a good model for drugs cleared by CYP3A4 – Ortholog CYP3A1 has limited similarity and little overlap in function The rat ortholog for CYP2C8 is CYP2C22 which has demonstrated some very similar properties Could this DDI be modeled in the rat?

23. In vivo experiments in the rat (Xenometrics/XenoTech) 23

24. Repaglinide PK data in rat (n = 3 per group) 24 AUC increase in group 1 animals Gemfibrozil concentrations 16 – 125 µg mL -1 Group 1: Gemfibrozil + repaglinide Group 2: Repaglinide only Repaglinide plasma concentration (ng/mL)

25. Repaglinide PK data in rat (n = 3 per group) 25 Clear evidence of drug-drug interaction between gemfibrozil and repaglinide in Group 1 animals ParameterGroup 1Group 2Fold-change C max (ng mL -1 )284.1 ± 85.266.2 ± 6.94.3-fold increase t max (h)1.7 ± 0.61.2 ± 0.71.4-fold increase AUC 0-12 (ng h mL -1 )853.9 ± 344.7242.8 ± 32.53.5-fold increase AUC 0-∞ (ng h mL -1 )837.5 ± 337.0282.1 ± 43.53.0-fold increase CL obs (L h -1 kg -1 )1299.3 ± 522.93622.0 ± 589.62.8-fold decrease Vd obs (L kg -1 )5158.6 ± 3397.413154.8 ± 2483.32.6-fold decrease t 1/2 (h)2.6 ± 0.82.6 ± 0.6None

26. Repaglinide rat plasma (AUC pool) metabolite profile 26 Componentt r (min)m/zProposed biotransformationGroup 1 Group 2 RP13.10441 O-Deethylation + hydroxylation ++ M0-OH5.58469 Hydroxylation +ND RP25.78469 Oxidation +ND Repaglinide glucuronide 7.17629 Glucuronidation ++ M47.42469 Hydroxylation ND M17.81385 N,N-DidealkylationND RP37.87441 O-Deethylation + hydroxylation +ND M58.30425 O-deethylation ++ RP48.59451 Dehydrogenation +ND RP58.59469 Oxidation +ND Repaglinide8.89453 None ++ C39.74471 Hydroxylation+ reduction +ND M210.12485 N-dealkylation + oxidation to the carboxylic acid +ND

27. Relative abundance of major human metabolites 27 Very low abundance metabolites in plasma Limited plasma sample volume

28. Bile metabolite profiles (0-12 h pools) 28 Repaglinide predominantly excreted in bile in humans – 90% excreted in feces; 8 % excreted in urine Rat bile profiles contained 49 metabolites across the two groups – Oxidative metabolism – Glucuronidation – Sulfonation Initial focus has to be on metabolites of interest van Heiningen et al. Eur, J. Clin. Pharmacol. Exp. Ther. 1999; 55(7): 521-525.

29. Exploring the CYP inhibition in bile 29 Relative abundance of CYP2C8 (in human) metabolites decreased (~65%) with gemfibrozil dosing

30. Relative abundance of major human metabolites 30 All of them decreased with gemfibrozil dosing Not characteristic of a specific CYP inhibition interaction

31. Urine metabolite profiles (0-12 h pools) 31 Huge differences between the treatment groups – Without gemfibrozil treatment, only 7 metabolites – With gemfibrozil treatment, 27 metabolites Componentt r (min) m/zProposed biotransformation Group 1Group 2 M0-OH5.58469 Hydroxylation +ND Repaglinide glucuronide 7.17629 Glucuronidation +ND M47.42469 Hydroxylation +ND M17.83385 N,N-Didealkylation ++ M58.30425 O-Deethylation +ND Repaglinide8.89453 None ++ M210.12485 N-dealkylation + oxidation to the carboxylic acid ++

32. Metabolite abundance in the urine 32 Even the metabolites detected in Group 2 urine are present at relatively low abundance

33. Biliary vs urinary excretion 33 Gemfibrozil increases urine and decreases bile excretion Why?

34. Systemic effects of gemfibrozil 34 Metabolism-dependent CYP2C8 inhibitor – Does not seem to account for all the metabolic profile changes – As yet, do not have evidence of CYP2C22 inhibition UGT1A1 inhibitor – Repaglinide glucuronidation occurs at least in part through 1A1 mediation – Would not explain other effects OATP1B1 (SLCO1B1) hepatic uptake transporter inhibitor – Would severely reduce abundance of all metabolites in bile – May also account for increased urinary excretion in Group 1 Gan et al. Br. J. Pharmacol. 2010; 70(6): 870-80. Nakagomi-Hagihara et al. Xenobiotica 2007; 37(5): 474-486.

35. Human and rat OATPs 35 Human OATP1B1 inhibition has been described as a confounding factor in the repaglinide/gemfibrozil DDI OATP1B family comprises OATP1B1 and 1B3 Only rodent ortholog for OATP1Bs is Oatp1b2 – Functions similarly to both – Mice deficient in Oatp1b2 have shown some utility as models for OATP1B studies Kudo et al. Drug Metab. Dispos. 2012; 41(2): 362-371. Repaglinide PK has been shown to correlate with OATP1B1 polymorphism Kallioski et al. Br. J. Clin. Pharmacol. 2008; 66(6): 818-825. Niemi et al. Clin. Pharmacol. Ther. 2005; 77(6): 468-478.

36. Back to the PK data 36 The observed clearance, volume of distribution and t 1/2 data do support the transporter hypothesis ParameterGroup 1Group 2Fold-change C max (ng mL -1 )284.1 ± 85.266.2 ± 6.94.3-fold increase t max (h)1.7 ± 0.61.2 ± 0.71.4-fold increase AUC 0-12 (ng h mL -1 )853.9 ± 344.7242.8 ± 32.53.5-fold increase AUC 0-∞ (ng h mL -1 )837.5 ± 337.0282.1 ± 43.53.0-fold increase CL obs (L h -1 kg -1 )1299.3 ± 522.93622.0 ± 589.62.8-fold decrease Vd obs (L kg -1 )5158.6 ± 3397.413154.8 ± 2483.32.6-fold decrease t 1/2 (h)2.6 ± 0.82.6 ± 0.6None

37. Next experiments 37 Still have untapped potential in the liver samples They were flash frozen so cannot do hepatocyte/transporter work Plan to make microsomes and measure CYP/UGT activities to explore the inhibition independently – CYP2C8/CYP3A4 – UGT1A1/1A3 (more complicated) Transporter work will need to be done in vitro – Clear evidence of uptake interactions – Efflux transporter issues may also be involved

38. Conclusions 38 Individual CYP inhibition effects can be modeled well in vitro; repaglinide does seem to have CYP2C8/2C22-specific metabolites but not necessarily as expected More complete systems have both advantages and disadvantages In the case of gemfibrozil and repaglinide, transporter inhibition appeared to be much more involved in PK changes than CYP inhibition – Still some work to be done Rodent utility in transporter studies needs further study

39. Acknowledgements 39 XenoTech – Phyllis Yerino – Forrest Stanley – Dr. Sylvie Kandel – Seema Muranjan – Chandra Kollu – Dr. David Buckley – Brian Ogilvie Xenometrics – Dr. Kristin R ussell – Tom Haymaker

40. Thank you Questions? Joanna Barbara, Ph.D. Division Director, Analytical Services XenoTech, LLC jbarbara@xenotechllc.com 40