Role of transport proteins in hepatic drug disposition ......Organic Cation Transporter 1 (OCT1) •...

Role of transport proteins in hepatic drug disposition, hepatotoxicity and drug-drug interactions Pieter Annaert Drug Delivery and Disposition Lab pieter.annaert@kuleuven.be Twitter: pannaert71

Nothing in this document may be published in any format without prior written consent of the relevant copyright holders (see references) or Pieter Annaert (for unpublished data)

Liver is a major organ for detoxification of xenobiotics including drugs

CYP

But how do xenobiotics get access to these metabolizing

enzymes ?

Van Herwaarden et al., Trends Pharmacol. Sci. 2009

Histology of the liver

Modified from Albert (1994)

Hepatic drug elimination…

Metabolism Phase-I Phase-II

BILE BLOOD (sinusoid)

Phase-0 Phase-III

Exploring Hepatic Drug Transport...

in 2005...

Ho and Kim, Transporters and drug therapy: Implications for drug disposition and disease, CPT 2005

and in 2011

Niemi et al., Organic Anion Transporting Polypeptide 1B1: a Genetically Polymorphic Transporter of Major Importance for Hepatic Drug Uptake, Pharm. Rev. 2011

Overview of drug transporters in the liver

Hillgren et al., Emerging transporters of clinical importance: an update from the International Transporter Consortium., CPT 2013

Intrahepatic drug exposure is determined by interplay between enzymes and transporters

BILE BLOOD (sinusoid) Metabolism

Renal excretion

Giacomini & HuangClinical Pharmacology & Therapeutics | VOLUME 94 NUMBER 1 | JULY 2013

Clinical relevance of (hepatic) drug transporters ? OCT1 (SLC22A) as an example transporter…

Organic Cation Transporter 1 (OCT1) •  Major facilitator superfamily / SLC22A subfamily

•  Liver: hOCT1 >> hOCT3

•  Clearance of (small) cationic compounds (MW < 500 Da)

•  Rat Oct1 shares 78% AA identity with hOCT1

•  Genetic polymorphisms lead to altered PK of drug substrates

•  OCT1 is the single most abundantly expressed transporter in human liver

•  Electrogenic facilitative transport •  Driven by electrochemical potential •  Bidirectional •  Na+/H+ independent

Koepsell & Keller, Functional properties of OCT1, G. Ciarimboli et al. (eds.), Organic Cation Transporters, DOI 10.1007/978-3-319-23793-0_2

Nies et al., HEPATOLOGY 2009;50:1227-1240

Variability in OCT1 expression… Nies et al., HEPATOLOGY 2009;50:1227-1240

Up to about 100-fold differences in OCT1 expression

Effect of cholestasis/obesity on OCT1 expression

+ increased OCT1 expression in adipose tissue of obese subjects -> increased metformin action in these subjects

Sam et al., J Clin Pharmacol 2017 Nies et al., Hepatology 2009

Typical OCT1 substrates Substrate Km, µM

1-methyl-4-phenyl-pyridinium (MPP+) 15-32

4(4-dimethylaminostyryl)-N-methylpyridinium = ASP+ 2.3

Metformin 1470-2160

Rhodamine 123 0.54 Fenoterol 1.8 Furamidine 6.1 Morphine 3.4 Sulpiride 260 Ganciclovir 516

Sundelin et al., CPT 2017

Role of OCT1 in metformin disposition •  As compared to equal IV doses, oral metformin is much

more efficacious in lowering blood glucose o  Limited hepatic exposure after IV administration

•  Metformin: reduced effect in carriers of M420del and R61C in SLC22A1 o  Based on oral glucose tolerance test

Tzvetkov et al. CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 90 NUMBER 1 | JULY 2011

Tzvetkov et al. CLINICAL PHARMACOLOGY & THERAPEUTICS | VOLUME 90 NUMBER 1 | JULY 2011

Tools to study drug transporters? OCT1 (SLC22A) as an example …

Ultracentrifugation

Homogenizing Centrifugation

S9-fraction

Microsomes

Suspended Hepatocytes

(Fresh & Cryo)

Culturing

Cultured hepatocytes

Isolation

IPRL

Isolation & Perfusion

Vesicles

Transporter Expressing CHO cells

Transfection

Homogenizing Ultracentrifugation

IPRL: Isolated perfused rat liver CHO: Chinese hamster ovary

In vitro drug disappearance in presence of hepatocytes (filled circles: + serum)

Metabolic Cl prediction using hepatocytes

26 test compounds

Blanchard et al., 2006

Suspended hepatocytes: Liver-based in vitro models for hepatic uptake and metabolism of xenobiotics

The“oil-spin”methodtodeterminehepaticuptakeinsuspendedhepatocytes

PRE-INCUBATION cell supsension + buffer/inhibitor

INCUBATION + substrate

oil mixture

NaOH / NaCl

14.000 rpm 2 x 2 min

CENTRIFUGATION STEP to seperate cells from

uptake buffer

QUANTIFICATION tube bottoms were cut and

analysed by liquid scintillation, fluoresence or

mass spectrometry

200 µL SAMPLE

OCT1 activity in human hepatocytes Focus on interindividual variability

Activity profiling for OCT1 in human

Fattah et al., Pharm. Res. 2017

Interindividual variability in hepatic OCT1 activity

Fattah et al., Pharm. Res. 2017

OCT1 mRNA shows poor correlation with OCT1 activity

Fattah et al., Pharm. Res. 2017

Genotype alone does not predict OCT1 activity

Fattah et al., Pharm. Res. 2017

Ontogeny of OCT1 activity in human hepatocytes

PhD thesis Sarinj Fattah

Is transporter expression maintained in suspended hepatocytes ? How does this compare to transporter activity in liver tissue ?

Badée et al., Drug Metab Dispos 43:424–432, April 2015

Suspended hepatocytes are a great tool for determining the role of uptake transporters

•  But what about efflux transporters ?

Metabolism Phase-I Phase-II

BILE BLOOD (sinusoid)

Phase-0 Phase-III

Sandwich-cultured Hepatocytes

© P. Annaert

Sandwich-cultured hepatocytes

light microscopy electron microscopy fluo microscopy

Bile canalicular space in sandwich-cultured rat hepatocytes

De Bruyn T, Chatterjee S, Fattah S, Keemink J, Nicolaï J, Augustijns P, Annaert P. Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol. 2013 May;9(5):589-616.

A

De Bruyn T, Chatterjee S, Fattah S, Keemink J, Nicolaï J, Augustijns P, Annaert P. Sandwich-cultured hepatocytes: utility for in vitro exploration of hepatobiliary drug disposition and drug-induced hepatotoxicity. Expert Opin Drug Metab Toxicol. 2013 May;9(5):589-616.

In vitro biliary excretion of taurocholate

Ansede et al., DMD 2010

Billiary excretion index (BEI) and in vitro biliary clearance (Cl)

Expressing in vitro biliary excretion…

Abe et al., JPET 2008

Prediction of in vivo biliary Cl

Major disadvantage of this method ? •  Cell lysis required after parallel incubations •  Large number of valuable hepatocytes are consumed… •  Consider the use of fluorescent transporter sustrates •  And non-destructive assays… •  Such as confocal microscopy in living hepatocytes •  Question: can we get quantitative information on

biliary excretion and the interference by drugs

Carboxydichlorofluorescein retention in Sandwich-Cultured Hepatocytes

In 2000

…and in 2013…

Exploring MRP2-mediated biliary excretion

Niemi et al., Organic Anion Transporting Polypeptide 1B1: a Genetically Polymorphic Transporter of Major Importance for Hepatic Drug Uptake, Pharm. Rev. 2011

Efflux assay with CDFDA: concept

CDFDA

CDFDA

CDF

CDF

CDFDA: carboxy-dichloro-fluorescein-diacetate (non-fluorescent) CDF: carboxy-dichloro-fluorescein (fluorescent)

passive uptake

esterase

hepatocyte

bile canaliculus

Mrp3

CDF

CDF

Oatp

Holmstock et al., submitted

Biliary excretion assay with CDFDA

•  Q1: Image-based quanitification of CDF efflux? •  Q2: Evaluation of the inhibitory effect of drugs on MRP2?

Holmstock et al., submitted

Visual channel Fluorescent + visual channel Fluorescent channel

Q1: Image-based quantification of CDF efflux?

Holmstock et al., submitted

Q1: How do we quanitify CDF efflux? ImageJ software (NIH)

Manual selection

bile canaliculi only cells+bile

Holmstock et al., submitted

Q2: Can we measure the inhibitory effect of drugs on MRP2?

Aim: Test effect of all HIV protease inhibitors on BEI of CDF Experimental conditions:

o  CDFDA (4, 1 and 0,5 µM) o  CDFDA (4, 1 and 0,5 µM) + HIV PI (10 µM) o  Sandwich-cultured rat hepatocytes (culture day 3)

Holmstock et al., submitted

HIV Protease inhibitors

CDFDA control

Holmstock et al., submitted

With saquinavir

Holmstock et al., submitted

With lopinavir

Holmstock et al., submitted

With tipranavir

Holmstock et al., submitted

Control + saquinavir

+ lopinavir + tipranavir

Image-based Billiary excretion index (BEI) ?

Q1: How do we quanitify CDF efflux?

-> absolute amounts

BEI =Xbile

Xcells+bile

with X = intensity/pixel × area (pixels)

Holmstock et al., submitted

Q2: Can we measure the inhibitory effect of drugs on MRP2?

Holmstock et al., submitted

Conventional assay yields much more variable data…

Ye et al., Biopharm. Drug Dispos. 31: 178–188 (2010)

Conclusions •  Drug transporters play a major role in hepatic

disposition of xenobiotics and in drug-drug interactions

•  Polymorphism in e.g. OCT1 leads to interindividual variability in drug exposure (clinical data)

•  Hepatic uptake clearance can be measured in cryopreserved human hepatocytes (oil-spin) o  Understand interindividual differences in OCT1-

mediated disposition o  OCT1 activity measurements provide useful information

beyond mRNA levels and genotype •  Sandwich-cultured hepatocytes allow studying biliary

excretion processes in vitro (including interactions)

Future perspectives •  Clearance values measured in vitro

o  …can be scaled to in vivo units (IVIVE) o  …and implemented in PBPK models to predict hepatic

disposition of xenobiotics (including concentration-time profiles)

•  Focus on drug disposition mechanisms (e.g. rate-limiting steps)

•  Huge interest in experimental determination of tissue unbound concentrations o  Several methods now available…

•  Former PhD students: o  Tom De Bruyn, PhD o  Sagnik Chatterjee, PhD o  Janneke Keemink, PhD o  Johan Nicolaï, PhD o  Sarinj Fattah, PhD o  Marlies Oorts, PhD

•  Current PhD students: o  Qi Bing o  Tom De Vocht o  Neel Deferm o  Pieter Van Brantegem

•  Prof. Bruno Stieger (University Hospital Zürich)

Dr. Eef Hoeben Dr. Debbie Dewaele Apr. Johan Van Daele

Prof. Lysiane Richert

Hepatic drug disposition and hepatotoxicity research at KU Leuven Drug Delivery and Disposition (‘Liver group’)

Non-clinical Liver models

Human hepatocytes

Bioanalysis

Prediction and Simulation of

drug exposure

Strategies for drug exposure modulation

Transporter-transfected cells Human liver

microsomes

Isolated perfused liver

PBPK modeling

Drug Disposition mechanisms &

kinetics

PK boosting

Applications

Prediction of DILI risk

In vitro drug-induced cholestasis

Focus on transporter

function