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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.

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in vitro and in vivo metabolism of repaglinide modeling clinically relevant drug drug interactions

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
XenoTech’s integrated service capabilities

XT Consulting Department

Expert data review and study consultation

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

Bioanalytical

Non-GLP Bioanalysis

GLP and non-GLP in vitro study support

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

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

Products

overview
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
enzymatic biotransformation of drugs

Hepatic clearance route by enzyme type

Enzymatic biotransformation of drugs

Esterase

UGT

CYP

Data adapted from: Cassarett and Doull’s Toxicology (2001) C. Klaassen (Ed), New York, NY: McGraw-Hill

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

drug drug interactions ddi
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
cytochrome p450 inhibition
Cytochrome P450 inhibition

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

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

  • CYP inhibition has potential to result in
    • Black box label warnings
    • Withdrawal from market
repaglinide uses
Repaglinide uses

Repaglinide

Dicarboxylic acid (M2)

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

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 Heiningenet. al., 1999)

Other oxidative metabolites and glucuronide conjugate

repaglinide metabolism
Repaglinide metabolism

CYP2C8 metabolismM0-OHM4

CYP2C8 probe

CYP3A4 metabolismM1M2M5

Major biotransformation routes described (Bidstrupet al., 2003)

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

repaglinide m4 formation and antibody inhibition
Repaglinide M4 formation and antibody inhibition

Roles for CYP2C8 CYP3A4

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

repaglinide metabolized by cyp3a4 2c8 and ugt1a1
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
gemfibrozil and repaglinide
Gemfibrozil and repaglinide

Vinik and Colwell Diabetes Care 1993; 16(1): 37-44.

Backmannet al.Drug Metab. Dispos. 2009; 37(12): 2359-66.

  • 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
gemfibrozil dosing and pharmacokinetics
Gemfibrozil dosing and pharmacokinetics

Rouiniet al.Int. J. Pharmacol. 2006; 2: 75-78.

Gemfibrozil usually dosed at 600 mg twice a day or less commonly 900 mg once daily

PK parameters after a single oral dose

gemfibrozil metabolism
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.

in vitro experiments with repaglinide
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

repaglinide in human liver microsomes hlm
Repaglinide in human liver microsomes (HLM)
  • High-resolution LC UV chromatogram (254 nm)

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

Repaglinide

Repaglinide desaturationmetabolites

Major in vivo metabolite

Repaglinide dicarboxylic acid metabolite (M2)

Probemetabolite

Hydroxyrepaglinide (M4)

High abundance

Low abundance

Unlabeled peaks are not related to repaglinide

recombinant cyp panel for repaglinide substrate loss
Recombinant CYP panel for repaglinide substrate loss

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

CYP3A4?

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

inhibition experiments for cyp reaction phenotyping
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
metabolism dependent cyp2c8 and 3a4 inhibition
Metabolism-dependent CYP2C8 and 3A4 inhibition

2C8

3A4

Less clear

M0-OH

M5

M2

M4

M1

exploring the interaction further
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?
repaglinide pk data in rat n 3 per group
Repaglinide PK data in rat (n = 3 per group)

Group 1: Gemfibrozil + repaglinide

Repaglinide plasma concentration(ng/mL)

Group 2: Repaglinide only

AUC increase in group 1 animals

Gemfibrozil concentrations 16 – 125 µg mL-1

repaglinide pk data in rat n 3 per group1
Repaglinide PK data in rat (n = 3 per group)

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

relative abundance of major human metabolites
Relative abundance of major human metabolites

Very low abundance metabolites in plasma

Limited plasma sample volume

bile metabolite profiles 0 12 h pools
Bile metabolite profiles (0-12 h pools)

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

  • 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
exploring the cyp inhibition in bile
Exploring the CYP inhibition in bile

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

relative abundance of major human metabolites1
Relative abundance of major human metabolites

All of them decreased with gemfibrozil dosing

Not characteristic of a specific CYP inhibition interaction

urine metabolite profiles 0 12 h pools
Urine metabolite profiles (0-12 h pools)
  • Huge differences between the treatment groups
    • Without gemfibrozil treatment, only 7 metabolites
    • With gemfibrozil treatment, 27 metabolites
metabolite abundance in the urine
Metabolite abundance in the urine

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

biliary vs urinary excretion
Biliaryvs urinary excretion

Gemfibrozil increases urine and decreases bile excretion

Why?

systemic effects of gemfibrozil
Systemic effects of gemfibrozil

Gan et al. Br. J. Pharmacol. 2010; 70(6): 870-80.

Nakagomi-Hagihara et al. Xenobiotica2007; 37(5): 474-486.

  • 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
human and rat oatps
Human and rat OATPs

Kudo et al. Drug Metab. Dispos. 2012; 41(2): 362-371.

  • Repaglinide PK has been shown to correlate with OATP1B1 polymorphism

Niemi et al. Clin. Pharmacol. Ther. 2005; 77(6): 468-478.

Kallioski et al. Br. J. Clin. Pharmacol. 2008; 66(6): 818-825.

  • 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
back to the pk data
Back to the PK data

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

next experiments
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
conclusions
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
acknowledgements
Acknowledgements
  • XenoTech
    • Phyllis Yerino
    • Forrest Stanley
    • Dr. Sylvie Kandel
    • Seema Muranjan
    • Chandra Kollu
    • Dr. David Buckley
    • Brian Ogilvie
  • Xenometrics
    • Dr. Kristin Russell
    • Tom Haymaker
slide40

Thank youQuestions?Joanna Barbara, Ph.D.Division Director, Analytical ServicesXenoTech, LLCjbarbara@xenotechllc.com