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Role of transport proteins in hepatic drug disposition, hepatotoxicity and drug-drug interactions Pieter Annaert Drug Delivery and Disposition Lab [email protected] 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in any format without prior written consent of the relevant copyright holders (see references) or Pieter Annaert (for unpublished data)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