Prediction of the in vivo OATP1B1-mediated drug–drug interaction potential of an investigational drug against a range of statins
Introduction
OATP1B1 (also known as SLCO1B1 protein and previously known as LST-1, OATP-C, OATP2 and OATP6) is a product of gene SLCO1B1 and represents one of the best-characterized human OATPs (Abe et al., 1999, Hsiang et al., 1999, Konig et al., 2000). It is specifically expressed on the basolateral (sinusoidal) membrane of human hepatocytes and is known to transport a broad range of compounds such as bile acids, conjugated steroids, thyroid hormones, peptides, and drugs including rifampicin and pravastatin (Abe et al., 1999, Konig et al., 2000). Due to its liver-specific expression and its capacity for transporting a large number of structurally different compounds, it has been suggested that OATP1B1 plays an important role in hepatic drug uptake and subsequently elimination. Thus, inhibition of OATP1B1 can alter the pharmacokinetics of OATP1B1 substrate drugs causing clinically relevant drug–drug interactions (DDI), as exemplified between eltrombopag olamine and rosuvastatin (Stasi, 2009, Pharmacped, 0000) and between gemfibrozil and rosuvastatin (Schneck et al., 2004).
HMG-coenzyme A reductase inhibitors (statins) are an important class of drugs widely used in the clinic to treat dyslipidemia (Nawrocki et al., 1995). Based on pharmaceutical sales, the six major statins are simvastatin, atorvastatin, rosuvastatin, pravastatin, pitavastatin and fluvastatin (Business Insights Report, 2007). Active hepatic uptake of these statins by OATP1B1 has been demonstrated previously (Neuvonen et al., 2008). As statins are widely prescribed the likelihood of new drugs being co-administered is high. An increase in the plasma concentration of many of the currently used statins can cause severe side effects such as muscle toxicity or even rhabdomyolysis (Neuvonen et al., 2006). Therefore, the DDI potential with statins is of great clinical importance for the clinical safety of new drug candidates. Except for simvastatin, which is orally administered as an inactive lactone prodrug, all of the other statins mentioned above are given as the active β-hydroxy acid forms to patients. In vivo, lactone and acid forms undergo interconversion (Chen et al., 2005). The acid forms exhibit higher affinities for the OATP1B1 transporter (Chen et al., 2005) and thus any DDI with them, via OATP1B1 inhibition, is likely to impact the systemic concentrations of statin or precipitant (the drug that inhibits transporter is defined as precipitant of DDI). In contrast, the lactone forms are more lipophilic, easily permeate through cell membranes and have been shown to be less affected by inhibition of OATP1B1-mediated uptake (Pasanen et al., 2006, Schneck et al., 2004). Many of the DDIs due to the inhibition of OATP1B1-mediated transport of statins reported in the literature involve the acid form of the statin (Hirano et al., 2004, Neuvonen et al., 2008). Consequently, the acidic forms of statins (simvastatin acid, atorvastatin, pravastatin, pitavastatin, fluvastatin and rosuvastatin) were used in this study.
Estrone 3-sulfate has been extensively used as an OATP1B1 substrate and exhibits characteristic biphasic kinetics (Noe et al., 2007). Estradiol 17β-glucuronide is another commonly used probe substrate for determining OATP1B1 inhibition by drug candidates (Konig et al., 2000, Nakai et al., 2001). However, these probe substrates are differentially affected by the OATP1B1 inhibitor gemfibrozil (Noe et al., 2007) and thus currently it is unclear whether estradiol 17β-glucuronide and estrone 3-sulfate are good surrogate probe substrates to predict OATP1B1-mediated inhibition of statins in vitro.
Recent recommendations of International Transporter Consortium (ITC) (Giacomini et al., 2010) describes systematic approach to determine OATP1B1 based drug–drug interactions due to drugs using IC50 values obtained from in vitro systems. In this approach, the dose, plasma protein binding and systemic concentration of inhibitor are used to determine unbound hepatic plasma concentration (Iin,max) at sinusoidal side of hepatocytes. This Iin,max is then used in conjunction with IC50 or Ki of inhibitor to predict DDI due to inhibition of OATP1B1. Hirano et al. (2006) had previously published successful use OATP1B1 based DDIs using set of marketed drug inhibitors and statins. An investigational drug, AZX is an acidic (pKa = 2.6), hydrophilic (logD7.4 = −0.22), small molecular weight chemical drug (>500) with plasma protein binding of 97%, moderately permeable in Caco-2 assay and might be OATP1B1 inhibitor.
The primary objective of this study was to investigate the OATP1B1-mediated DDI potential of an investigational drug (AZX) against various probe substrates including simvastatin acid, atorvastatin, pravastatin, pitavastatin, fluvastatin, rosuvastatin, estradiol 17β-glucuronide and estrone 3-sulfate using the previously validated in vitro HEK-OATP1B1 test system (Sharma et al., 2010). The inhibitory potentials of the OATP1B1 inhibitors rifamycin SV and gemfibrozil were investigated in parallel. In order to achieve the primary objective, the OATP1B1-mediated transport of the substrates listed above was characterized (by determination of apparent Km) prior to their use as probes in inhibition studies.
The secondary objective was to use the inhibition data generated for AZX to predict the likelihood of a clinical OATP1B1-mediated DDI, using current regulatory authority guidance and recommendations of the International Transporter Consortium, in order to establish if a clinical interaction study is warranted, and further, to identify the statin that would be the most appropriate clinical probe. Utilizing the in vitro data generated in this study for gemfibrozil and existing clinical data in the literature for reported DDIs, predictions were made to establish if they agreed with observed clinical outcome.
Section snippets
Materials
Pravastatin acid sodium salt, estradiol 17β-glucuronide, estrone 3-sulfate, rifamycin SV, gemfibrozil, Dulbecco’s Modified Essential Media (DMEM), Krebs–Henseleit buffer and Triton X-100 were purchased from Sigma–Aldrich (Dorset, UK). [3H]Simvastatin acid sodium salt (specific activity 185 GBq/mmol), [3H]atorvastatin acid calcium salt (specific activity 370 GBq/mmol), [3H]pravastatin acid sodium salt (specific activity 185 GBq/mmol), [3H]fluvastatin acid sodium salt (specific activity 185
Non-specific binding to BD Biocoat™ poly-d-lysine 12-well multiwell plates and chemical stability in Krebs–Henseleit buffer (pH 7.4)
The non-specific binding of [3H]-substrates to the poly-d-lysine Biocoat® BD plates used in the assay was assessed at 37 °C over 10 min in the absence of cells. The results obtained demonstrate that there were no issues with non-specific binding as the mean% recovery (n = 2) at 10 min compared to 0 min ranged between 85% and 103%.
The chemical stabilities of [3H]-substrates were assessed in Krebs–Henseleit buffer over 2 h at 37 °C. Mean percentage recoveries of [3H]-substrate solutions (1 μM, n = 2) from
Discussion
Substrate concentrations in incubation solutions during uptake assays may deplete due to non-specific binding to BD Biocoat™ poly-d-lysine 12-well multiwell plates and/or chemical instability in Krebs–Henseleit buffer solution (pH 7.4). All probe substrates exhibited negligible binding to BD Biocoat™ poly-d-lysine 12-well multiwell plates as determined by LSC analysis and were chemically stable in experimental conditions as determined by radioflow HPLC. The dihydroxy open acid forms of statins
Conclusions
AZX inhibited OATP1B1-mediated uptake of atorvastatin, pravastatin, pitavastatin and rosuvastatin in HEK293-OATP1B1 cells. It also inhibited OATP1B1-mediated uptake estradiol 17β-Glucuronide exhibiting similar IC50 value to that of statins. Estradiol 17β-Glucuronide was identified as a good probe surrogate for statins when assessing OATP1B1 inhibitory potential in vitro. Further assessment of in vitro inhibition data predicts an OATP1B1-mediated DDI between AZX and statins if total plasma
Disclosure statement
All authors were employees of AstraZeneca R&D when the work was performed and work was funded by AstraZeneca.
References (51)
- et al.
Identification of a novel gene family encoding human liver-specific organic anion transporter LST-1
J. Biol. Chem.
(1999) - et al.
A novel human hepatic organic anion transporting polypeptide (OATP2). Identification of a liver-specific human organic anion transporting polypeptide and identification of rat and human hydroxymethylglutaryl-CoA reductase inhibitor transporters
J. Biol. Chem.
(1999) Rate and equilibrium constants for acid-catalyzed lactone hydrolysis of HMG-CoA reductase inhibitors
Int. J. Pharm.
(1990)- et al.
Endogenous gene and protein expression of drug-transporting proteins in cell lines routinely used in drug discovery programs
Drug Metab. Dispos.
(2009) - et al.
Plasma concentrations of active simvastatin acid are increased by gemfibrozil
Clin. Pharmacol. Ther.
(2000) - et al.
Rifampin markedly decreases and gemfibrozil increases the plasma concentrations of atorvastatin and its metabolites
Clin. Pharmacol. Ther.
(2005) - et al.
Rifamycin SV: a review
Arzneimittelforschung
(1965) - et al.
Effect of a single gemfibrozil dose on the pharmacokinetics of rosuvastatin in bile and plasma in healthy volunteers
J. Clin. Pharmacol.
(2010) - Business Insights Report: Jan 2007. The Cardiovascular Market Outlook to 2011, Business Insights, IMS...
- et al.
Differential interaction of 3-hydroxy-3-methylglutaryl-coa reductase inhibitors with ABCB1, ABCC2, and OATP1B1
Drug Metab. Dispos.
(2005)
Therapeuic Drugs
Membrane transporters in drug development
Nat. Rev. Drug Discov.
Goodman & Gilman’s The Pharmacological Basis of Therapeutics
Multiple inhibition mechanisms and prediction of drug–drug interactions: status of metabolism and transporter models as exemplified by gemfibrozil–drug interactions
Pharm. Res.
Contribution of OATP2 (OATP1B1) and OATP8 (OATP1B3) to the hepatic uptake of pitavastatin in humans
J. Pharmacol. Exp. Ther.
Drug–drug interaction between pitavastatin and various drugs via OATP1B1
Drug Metab. Dispos.
Database analyses for the prediction of in vivo drug–drug interactions from in vitro data
Br. J. Clin. Pharmacol.
Prediction of pharmacokinetic alterations caused by drug–drug interactions: metabolic interaction in the liver
Pharmacol. Rev.
Effect of OATP1B transporter inhibition on the pharmacokinetics of atorvastatin in healthy volunteers
Clin. Pharmacol. Ther.
Functional characterization of SLCO1B1 (OATP-C) variants, SLCO1B1∗5, SLCO1B1∗15 and SLCO1B1∗15+C1007G, by using transient expression systems of HeLa and HEK293 cells
Pharmacogenet. Genomics
In Vitro and in silico strategies to identify OATP1B1 inhibitors and predict clinical drug–drug interactions
Pharm. Res.
The interconversion kinetics, equilibrium, and solubilities of the lactone and hydroxyacid forms of the HMG-CoA reductase inhibitor, CI-981
Pharm Res.
Prediction of drug clearance by glucuronidation from in vitro data: use of combined cytochrome P450 and UDP-glucuronosyltransferase cofactors in alamethicin-activated human liver microsomes
Drug Metab. Dispos.
Cited by (45)
Microdosing clinical study to clarify pharmacokinetic and pharmacogenetic characteristics of atorvastatin in Japanese hypercholesterolemic patients
2019, Drug Metabolism and PharmacokineticsCitation Excerpt :Atorvastatin exhibits linear pharmacokinetics within the therapeutic dose range [11,12], but linearity of atorvastatin pharmacokinetics in a wider range of dose (from microdose to therapeutic dose) has not been examined. Atorvastatin is a substrate of P-glycoprotein, BCRP, MRP2 and OATP1B1, OATP1B3, and OATP2B1 [13–19]. Previous study indicated that OATP1B-mediated hepatic uptake of atorvastatin is a rate-determining process of overall hepatic clearance [20] and the healthy subjects with SLCO1B1 c.521T>C showed a significant increase in plasma AUC of atorvastatin at the therapeutic dose [21–24].
Cell cultures in drug discovery and development: The need of reliable in vitro-in vivo extrapolation for pharmacodynamics and pharmacokinetics assessment
2018, Journal of Pharmaceutical and Biomedical AnalysisCitation Excerpt :To characterize the operation of the OATP transporters in in vitro conditions, apart from primary cell cultures of hepatocytes and hepatocyte sandwich cultures, cells containing a selective gene coding transporter expression can be used. Such cells were obtained by transfection of HEK293 cDNA cells (Human Embryonic Kidney) responsible for biosynthesis of the OATP1B1 transporter [90,91], or CHO cDNA cells encoding OATP1B3 or OATP1B1 [92]. Employment of the physiologically-based pharmacokinetics modeling can be considered the most suitable method for prediction of DDI resulting from transport across the cell membrane or hepatic metabolism.
Design, synthesis, in vitro characterization and preliminary imaging studies on fluorinated bile acid derivatives as PET tracers to study hepatic transporters
2017, Bioorganic and Medicinal ChemistryCitation Excerpt :Inhibition of the canalicular efflux transporters can result in the accumulation of toxic metabolites in the hepatocyte, causing drug induced liver disease.5 Among the uptake transporters, the organic anion transporter polypeptide OATP1B1 (SLCO1B1, also called OATP-C) is a particularly important liver specific transporter involved in the hepatic uptake of a wide range of clinically relevant drugs.1,6 According to recent studies on the absolute quantitative determination of the amount of transporters in the human liver, OATP1B1 is the most abundant uptake transporter.7
Modeling Organic Anion-Transporting Polypeptide 1B1 Inhibition to Elucidate Interaction Risks in Early Drug Design
2016, Journal of Pharmaceutical Sciences