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

XenoTech’s integrated service capabilities

Enzyme InhibitionEvaluate 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 uptakeBi-directional assay for efflux transporters

Membrane-based vesicles and ATPase assays

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Metabolite characterization/ID Reaction phenotyping (CYP & UGT)

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(human and animal)Ex vivo studies in animals

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XT Consulting Department Expert data review and study consultation

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RI synthesis (radiolabeling), preclinical in vivo PK studies, QWBA, plasma protein binding, humanized chimeric mice (PXB), biomarker analysis, pharmacological receptor assays

BioanalyticalNon-GLP Bioanalysis

GLP and non-GLP in vitro study support

2Products

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Overview

• 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

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Enzymatic biotransformation of drugs

• Cytochrome P450 (CYP) enzymes are responsible for biotransformation of ~70% hepatically-cleared drugs

CYP

UGT

esterase

FMO

NAT

MAO

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

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Drug-drug interactions (DDI)

• 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

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Cytochrome P450 inhibition

• 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 b-blockers and other antihypertensives

FN

NN

H

O O

O

OH

N

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

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• 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

NH

NH O

CH3

CH3 O CH3

OH

O

OH

O

Repaglinide uses

N

NH O

CH3

CH3 O CH3

OH

O

Repaglinide Dicarboxylic acid (M2)

van Heiningen et al. Eur, J. Clin. Pharmacol. Exp. Ther. 1999; 55(7): 521-525.

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• Major biotransformation routes described (Bidstrup et al., 2003)

Repaglinide metabolism

Bidstrup et al. Br. J. Clin. Pharmacol. 2003; 56: 305-314.

CYP2C8 metabolism

M0-OHM4

CYP3A4 metabolism

M1M2M5

CYP2C8 probe

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Repaglinide M4 formation and antibody inhibition

Bidstrup et al. Br. J. Clin. Pharmacol. 2003; 56: 305-314.

Roles for CYP2C8 CYP3A4

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Repaglinide metabolized by CYP3A4/2C8 and UGT1A1

• 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

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Gemfibrozil and repaglinide

• 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.

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Gemfibrozil dosing and pharmacokinetics

• 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.

Parameter 600 mg dose 900 mg dose

Cmax (µg mL-1) 28.8 ± 4.1 40.8 ± 12.6

tmax (h) 1.8 ± 0.8 1.8 ± 0.8

AUC0-8 (µg h mL-1) 80.3 ± 10.3 132.1 ± 35.3

CL (L-1) 7.1 ± 0.9 6.6 ± 1.6

Vd (L-1) 11.6 ± 2.1 12.5 ± 3.4

t1/2 (h) 1.1 ± 0.2 1.3 ± 0.1

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Gemfibrozil metabolism

• 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.

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In vitro experiments with repaglinide

• 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

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• High-resolution LC UV chromatogram (254 nm)HLM-30 min

Time6.00 8.00 10.00 12.00 14.00 16.00

AU

-1.0e-3

0.0

1.0e-3

2.0e-3

3.0e-3

4.0e-3

5.0e-3

6.0e-3

7.0e-3

8.0e-3

9.0e-3

RD117007_MSE_05Apr12_008 Sb (3,40.00 ); Sm (SG, 40x1) 4: Diode Array 254

Range: 1.102e-28.72

17.62

17.24

Repaglinide in human liver microsomes (HLM)

50 mM Repaglinide0.5 mg/mL HLM30 minutes; 37°C; pH 7.4NADPH-generating system

Repaglinide

Hydroxyrepaglinide (M4)

Repaglinide desaturationmetabolites

Repaglinide dicarboxylic acid metabolite (M2)

Unlabeled peaks are not related to repaglinide

Major in vivo metabolite

Probe metabolite

Low abundance High abundance

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Repaglinide human liver microsome metabolite profile

Component tr (min) m/z value

Mass error (ppm)

Mass shift

Proposed biotransformation

M0-OH 5.48 469.2693 -1.9 +15.9940 Hydroxylation

C1 5.94 451.2590 -0.2 -2.0156 Dehydrogenation

C2 6.83 441.2369 4.7 -12.0384 O-deethylation + hydroxylation

M4 7.50 469.2700 -0.4 +15.9947 Hydroxylation

M1 7.95 385.2112 -3.9 -68.0641 N,N-didealkylation

M5 8.26 425.2436 -4.5 -28.0317 O-deethylation

Repaglinide 8.98 453.2738 -3.3 -0.0015 None

C3 10.09 471.2848 -0.9 +18.0095 Hydroxylation+ reduction

M2 10.30 485.2642 -5.1 +31.9889 N-dealkylation + oxidation to the carboxylic acid

C4 16.09 451.2583 -3.1 -2.0170 Dehydrogenation

C5 17.23 451.2591 -1.3 -2.0162 Dehydrogenation

C6 17.52 451.2599 0.4 -2.0154 Dehydrogenation

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0

20

40

60

80

100

Perc

enta

ge o

f max

imum

re

mai

ning

(%)

Recombinant CYP panel for repaglinide substrate loss

• 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 loss10 mM repaglinide10 pmol/inc rCYP20 minutes35°C; pH 7.4

CYP3A4?

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Inhibition experiments for CYP reaction phenotyping

• 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

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Selecting appropriate conditions for inhibition experiments

• Initial-rate conditions desirable

20

0

10

20

30

40

50

60

70

80

90

100

110

C1 (M0-OH) C4 (M4) C5 (M1) C7 (M5) C11 (M2)

Perc

enta

ge o

f co

ntro

l (%

)

Repaglinide metabolite

No inhibitor

Mibefradil

Gemfibrozil glucuronide

Metabolism-dependent CYP2C8 and 3A4 inhibition

3A42C8 Less clear

M0-OH M4 M5 M1 M2

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Correlation data for major metabolites with HLM donor panel

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Exploring the interaction further

• 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?

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In vivo experiments in the rat (Xenometrics/XenoTech)

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Repaglinide PK data in rat (n = 3 per group)

• AUC increase in group 1 animals

• Gemfibrozil concentrations 16 – 125 µg mL-1

Group 1: Gemfibrozil + repaglinide

Group 2: Repaglinide only

Repa

glin

ide

plas

ma

conc

entr

ation

(ng/

mL)

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Repaglinide PK data in rat (n = 3 per group)

• Clear evidence of drug-drug interaction between gemfibrozil and repaglinide in Group 1 animals

Parameter Group 1 Group 2 Fold-change

Cmax (ng mL-1) 284.1 ± 85.2 66.2 ± 6.9 4.3-fold increase

tmax (h) 1.7 ± 0.6 1.2 ± 0.7 1.4-fold increase

AUC0-12 (ng h mL-1) 853.9 ± 344.7 242.8 ± 32.5 3.5-fold increase

AUC0-∞ (ng h mL-1) 837.5 ± 337.0 282.1 ± 43.5 3.0-fold increase

CLobs (L h-1 kg-1) 1299.3 ± 522.9 3622.0 ± 589.6 2.8-fold decrease

Vdobs (L kg-1) 5158.6 ± 3397.4 13154.8 ± 2483.3 2.6-fold decrease

t1/2 (h) 2.6 ± 0.8 2.6 ± 0.6 None

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Repaglinide rat plasma (AUC pool) metabolite profile

Component tr (min) m/z Proposed biotransformation Group 1

Group 2

RP1 3.10 441 O-Deethylation + hydroxylation + +

M0-OH 5.58 469 Hydroxylation + ND

RP2 5.78 469 Oxidation + ND

Repaglinide glucuronide 7.17 629 Glucuronidation + +

M4 7.42 469 Hydroxylation ND ND

M1 7.81 385 N,N-Didealkylation ND ND

RP3 7.87 441 O-Deethylation + hydroxylation + ND

M5 8.30 425 O-deethylation + +

RP4 8.59 451 Dehydrogenation + ND

RP5 8.59 469 Oxidation + NDRepaglinide 8.89 453 None + +

C3 9.74 471 Hydroxylation+ reduction + ND

M2 10.12 485 N-dealkylation + oxidation to the carboxylic acid

+ ND

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Relative abundance of major human metabolites

• Very low abundance metabolites in plasma• Limited plasma sample volume

0%10%20%30%40%50%60%70%80%90%

100%

Perc

enta

ge o

f ob

serv

ed m

axim

um (

%) Plasma

Group 1

Group 2

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Bile metabolite profiles (0-12 h pools)

• 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.

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Exploring the CYP inhibition in bile

• Relative abundance of CYP2C8 (in human) metabolites decreased (~65%) with gemfibrozil dosing

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

M0-OH M4

Perc

enta

ge o

f ob

serv

ed m

axim

um (

%)

Group 1

Group 2

30

Relative abundance of major human metabolites

• All of them decreased with gemfibrozil dosing

• Not characteristic of a specific CYP inhibition interaction

0%10%20%30%40%50%60%70%80%90%

100%

Perc

enta

ge o

f m

axim

um o

bser

ved

(%) Bile

Group 1

Group 2

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Urine metabolite profiles (0-12 h pools)

• Huge differences between the treatment groups– Without gemfibrozil treatment, only 7 metabolites– With gemfibrozil treatment, 27 metabolites

Component tr

(min)m/z Proposed

biotransformationGroup 1 Group 2

M0-OH 5.58 469 Hydroxylation + ND

Repaglinide glucuronide 7.17 629 Glucuronidation + ND

M4 7.42 469 Hydroxylation + ND

M1 7.83 385 N,N-Didealkylation + +

M5 8.30 425 O-Deethylation + ND

Repaglinide 8.89 453 None + +

M2 10.12 485 N-dealkylation + oxidation to the carboxylic acid

+ +

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Metabolite abundance in the urine

• Even the metabolites detected in Group 2 urine are present at relatively low abundance

0%10%20%30%40%50%60%70%80%90%

100%

Perc

enta

ge o

f m

axim

um o

bser

ved

(%) Urine

Group 1

Group 2

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Biliary vs urinary excretion

• Gemfibrozil increases urine and decreases bile excretion

• Why?

0102030405060708090

100

Perc

enta

ge o

f to

tal m

ater

ial (

%)

Group 1

Bile

Urine

0102030405060708090

100

Perc

enta

ge o

f to

tal m

ater

ial (

%) Group 2

Bile

Urine

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Systemic effects of gemfibrozil

• 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.

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Human and rat OATPs

• 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.

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Back to the PK data

• The observed clearance, volume of distribution and t1/2 data do support the transporter hypothesis

Parameter Group 1 Group 2 Fold-change

Cmax (ng mL-1) 284.1 ± 85.2 66.2 ± 6.9 4.3-fold increase

tmax (h) 1.7 ± 0.6 1.2 ± 0.7 1.4-fold increase

AUC0-12 (ng h mL-1) 853.9 ± 344.7 242.8 ± 32.5 3.5-fold increase

AUC0-∞ (ng h mL-1) 837.5 ± 337.0 282.1 ± 43.5 3.0-fold increase

CLobs (L h-1 kg-1) 1299.3 ± 522.9 3622.0 ± 589.6 2.8-fold decrease

Vdobs (L kg-1) 5158.6 ± 3397.4 13154.8 ± 2483.3 2.6-fold decrease

t1/2 (h) 2.6 ± 0.8 2.6 ± 0.6 None

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Next experiments

• 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

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Conclusions

• 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

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Acknowledgements

• XenoTech– Phyllis Yerino– Forrest Stanley– Dr. Sylvie Kandel– Seema Muranjan– Chandra Kollu– Dr. David Buckley– Brian Ogilvie

• Xenometrics– Dr. Kristin Russell– Tom Haymaker

Thank you

Questions?

Joanna Barbara, Ph.D.Division Director, Analytical Services

XenoTech, LLCjbarbara@xenotechllc.com

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