Improved Prediction of the Drug-Drug Interactions of Pemafibrate Caused by Cyclosporine A and Rifampicin via PBPK Modeling: Consideration of the Albumin-Mediated Hepatic Uptake of Pemafibrate and Inhibition Constants With Preincubation Against OATP1B.

Pemafibrate (PMF) is highly albumin-bound (>99.8%) and a substrate for hepatic uptake transporters (OATP1B) and CYP enzymes. Here, we developed a PBPK model of PMF to capture drug-drug interactions (DDI) incurred by cyclosporine (CsA) and rifampicin (RIF), the two OATP1B inhibitors. Initial PBPK modeling of PMF utilized in vitro hepatic uptake clearance (PSinf) obtained in the absence of albumin, but failed in capturing the blood PMF pharmacokinetic (PK) profiles. Based on the results that in vitro PSinf of unbound PMF was enhanced in the presence of albumin, we applied the albumin-facilitated dissociation model and the resulting PSinf parameters improved the prediction of the blood PMF PK profiles. In refining our PBPK model toward improved prediction of the observed DDI data (PMF co-administered with single dosing of CsA or RIF; PMF following multiple RIF dosing), we adjusted the previously obtained in vivo OATP1B inhibition constants (Ki,OATP1B) of CsA or RIF for pitavastatin by correcting for substrate-dependency. We also incorporated the induction of OATP1B and CYP enzymes after multiple RIF dosing. Sensitivity analysis informed that the higher gastrointestinal absorption rate constant could further improve capturing the observed DDI data, suggesting the possible inhibition of intestinal ABC transporter(s) by CsA or RIF.

Rapid Bioavailability and Disposition protocol: A novel higher throughput approach to assess pharmacokinetics and steady-state brain distribution with reduced animal usage.

Besides routine pharmacokinetic (PK) parameters, unbound brain-to-blood concentration ratio (Kp,uu) is an index particularly crucial in drug discovery for central nervous system (CNS) indications. Despite advantages of Kp,uu from steady state after constant intravenous (i.v.) infusion compared with one- or multiple time points after transient dosing, it is seldom obtained for compound optimization in early phase of CNS drug discovery due to requirement of prerequisite PK data to inform the study design. Here, we designed a novel rat in vivo PK protocol, dubbed as Rapid Bioavailability and Disposition (RBD), which combined oral (p.o.) dosing and i.v. infusion to obtain steady-state brain penetration, along with blood clearance, oral exposure and oral bioavailability for each discovery compound, within a 24 hour in-life experiment and only a few (e.g., 3) animals. Protocol validity was verified through simulations with a range of PK parameters in compartmental models as well as data comparison for nine compounds with distinct PK profiles. PK parameters (Kp,brain, CLb and oral AUC) measured from the RBD protocol for all compounds, were within two-fold and/or statistically similar to those derived from conventional i.v./p.o. crossover PK studies. Our data clearly indicates that the RBD protocol offers reliable and reproducible data over a wide range of PK properties, with reduced turnaround time and animal usage.

Correlation between Membrane Protein Expression Levels and Transcellular Transport Activity for Breast Cancer Resistance Protein.

Emerging evidence indicates an important role for the breast cancer resistance protein (BCRP) in limiting brain penetration of substrate drugs. While in vitro transwell assays can provide an indication of BCRP substrate potential, the predictability of these assays in relation to in vivo brain penetration is still under debate. The present study examined the correlation of BCRP membrane protein expression level and transcellular transport activity across Madin-Darby canine kidney (MDCK) II monolayers. We expressed human BCRP or murine BCRP1 in MDCKII wild-type cells using BacMam2 virus transduction. The selective P-glycoprotein (P-gp) inhibitor LY335979 (1 µM) was included in the transport medium to measure BCRP-mediated transcellular transport for P-gp and BCRP cosubstrates. The BCRP levels in membrane extracts from MDCKII-BCRP or MDCKII-Bcrp1 cells were quantified by liquid chromatography-tandem mass spectrometry. The results are summarized as follows: 1) the membrane protein expression levels correlate with the corrected efflux ratios of substrates for human BCRP and murine BCRP1 within the efflux ratios investigated; 2) we demonstrate good concordance in rank order between the BCRP and BCRP1-mediated efflux ratios for 12 drugs; and 3) we propose an approach to contextualize in vitro BCRP transport data of discovery compounds by comparing them to the in vitro and in vivo transport data of the reference drug dantrolene and taking into account interbatch variation in BCRP expression. This approach correctly predicted compromised brain penetration for 25 discovery compounds in rodents, which were BCRP substrates but not P-gp or weak P-gp substrates. These results suggest that BCRP-expressing MDCKII cells are useful in predicting the in vivo role of BCRP in brain penetration.

Role of Hepatic Drug Transporters in Drug Development.

Hepatic drug transporters can play an important role in pharmacokinetics and the disposition of therapeutic drugs and endogenous substances. Altered function of hepatic drug transporters due to drug-drug interactions (DDIs), genetic polymorphisms, and disease states can often result in a change in systemic and/or tissue exposure and subsequent pharmacological/toxicological effects of their substrates. Regulatory agencies including the US Food and Drug Administration, European Medicines Agency, and Japan Pharmaceuticals and Medical Devices Agency have issued guidance for industry on drug interaction studies, which contain comprehensive recommendations on in vitro and in vivo study tools and cutoff values to evaluate the DDI potential of new molecular entities mediated by hepatic drug transporters. In this report we summarize the latest regulatory and scientific progress of hepatic drug transporters in clinical DDIs, pharmacogenetics, drug-induced liver injury (DILI), as well as methods for predicting transporter-mediated pharmacokinetics and DDIs.

Integrating in Silico and in Vitro Approaches To Predict Drug Accessibility to the Central Nervous System.

Estimation of uptake across the blood-brain barrier (BBB) is key to designing central nervous system (CNS) therapeutics. In silico approaches ranging from physicochemical rules to quantitative structure-activity relationship (QSAR) models are utilized to predict potential for CNS penetration of new chemical entities. However, there are still gaps in our knowledge of (1) the relationship between marketed human drug derived CNS-accessible chemical space and preclinical neuropharmacokinetic (neuroPK) data, (2) interpretability of the selected physicochemical descriptors, and (3) correlation of the in vitro human P-glycoprotein (P-gp) efflux ratio (ER) and in vivo rodent unbound brain-to-blood ratio (Kp,uu), as these are assays routinely used to predict clinical CNS exposure, during drug discovery. To close these gaps, we explored the CNS druglike property boundaries of 920 market oral drugs (315 CNS and 605 non-CNS) and 846 compounds (54 CNS drugs and 792 proprietary GlaxoSmithKline compounds) with available rat Kp,uu data. The exact permeability coefficient (Pexact) and P-gp ER were determined for 176 compounds from the rat Kp,uu data set. Receiver operating characteristic curves were performed to evaluate the predictive power of human P-gp ER for rat Kp,uu. Our data demonstrates that simple physicochemical rules (most acidic pKa ≥ 9.5 and TPSA < 100) in combination with P-gp ER < 1.5 provide mechanistic insights for filtering BBB permeable compounds. For comparison, six classification modeling methods were investigated using multiple sets of in silico molecular descriptors. We present a random forest model with excellent predictive power (∼0.75 overall accuracy) using the rat neuroPK data set. We also observed good concordance between the structural interpretation results and physicochemical descriptor importance from the Kp,uu classification QSAR model. In summary, we propose a novel, hybrid in silico/in vitro approach and an in silico screening model for the effective development of chemical series with the potential to achieve optimal CNS exposure.

Solute Carrier Family of the Organic Anion-Transporting Polypeptides 1A2- Madin-Darby Canine Kidney II: A Promising In Vitro System to Understand the Role of Organic Anion-Transporting Polypeptide 1A2 in Blood-Brain Barrier Drug Penetration.

Organic anion-transporting polypeptide (OATP) 1A2 has the potential to be a target for central nervous system drug delivery due to its luminal localization at the human blood-brain barrier and broad substrate specificity. We found OATP1A2 mRNA expression in the human brain to be comparable to breast cancer resistance protein and OATP2B1 and much higher than P-glycoprotein (P-gp), and confirmed greater expression in the brain relative to other tissues. The goal of this study was to establish a model system to explore OATP1A2-mediated transcellular transport of substrate drugs and the interplay with P-gp. In vitro (human embryonic kidney 293 cells stably expressing Oatp1a4, the closest murine isoform) and in vivo (naïve and Oatp1a4 knock-out mice) studies with OATP1A2 substrate triptan drugs demonstrated that these drugs were not Oatp1a4 substrates. This species difference demonstrates that the rodent is not a good model to investigate the active brain uptake of potential OATP1A2 substrates. Thus, we constructed a novel OATP1A2 expressing Madin-Darby canine kidney (MDCK) II wild type and an MDCKII-multidrug resistance protein 1 (MDR1) system using BacMam virus transduction. The spatial expression pattern of OATP1A2 after transduction in MDCKII-MDR1 cells was superimposed to P-gp, confirming apical membrane localization. OATP1A2-mediated uptake of zolmitriptan, rosuvastatin, and fexofenadine across monolayers increased with increasing OATP1A2 protein expression. OATP1A2 counteracted P-gp efflux for cosubstrates zolmitriptan and fexofenadine. A three-compartment model incorporating OATP1A2-mediated influx was used to quantitatively describe the time- and concentration-dependent apical-to-basolateral transcellular transport of rosuvastatin across OATP1A2 expressing the MDCKII monolayer. This novel, simple and versatile experimental system is useful for understanding the contribution of OATP1A2-mediated transcellular transport across barriers, such as the blood-brain barrier.

Temporal expression of transporters and receptors in a rat primary co-culture blood-brain barrier model.

1. The more relevant primary co-cultures of brain microvessel endothelial cells and astrocytes (BMEC) are less utilized for screening of potential CNS uptake when compared to intestinal and renal cell lines. 2. In this study, we characterized the temporal mRNA expression of major CNS transporters and receptors, including the transporter regulators Pxr, Ahr and Car in a rat BMEC co-cultured model. Permeability was compared with the Madin-Darby canine kidney (MDCKII)-MDR1 cell line and rat brain in situ perfusion model. 3. Our data demonstrated differential changes in expression of individual transporters and receptors over the culture period. Expression of ATP-binding cassette transporters was better retained than that of solute carrier transporters. The insulin receptor (IR) was best maintained among investigated receptors. AhR demonstrated high mRNA expression in rat brain capillaries and expression was better retained than Pxr or Car in culture. Mdr1b expression was up-regulated during primary culture, albeit Mdr1a mRNA levels were much higher. P-gp and Bcrp-1 were highly expressed and functional in this in vitro system. 4. Permeability measurements with 18 CNS marketed drugs demonstrated weak correlation between rBMEC model and rat in situ permeability and moderate correlation with MDCKII-MDR1 cells. 5. We have provided appropriate methodologies, as well as detailed and quantitative characterization data to facilitate improved understanding and rational use of this in vitro rat BBB model.

A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes.

Primary human hepatocytes are widely used for metabolic stability evaluations. However, there are limited data directly comparing phase I and phase II drug-metabolizing enzymes in fresh and cryopreserved hepatocytes prepared from the same human donor liver. We evaluated the metabolic competency of human hepatocytes prepared from seven donor tissues before and after cryopreservation. Temporal-dependent enzyme activity in suspension and matched adherent cultures of primary human hepatocytes was also assessed. Cryopreservation of hepatocytes resulted in statistically significant increases in activities of CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A but not CYP2C8, CYP2C19, FMO, UGT, and SULT, relative to fresh hepatocytes. In suspension cultures of hepatocytes, enzyme stabilities were as follows: UGT

Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes.

While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes. In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 µM) was 27.5 (±15.0) µl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17ß-D-glucuronide (E(2)-17ß-G; 1 µM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) µl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 µM) significantly (p<0.01) decreased the accumulation of estrone-3-sulfate and E(2)-17ß-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) µl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 µM) and the OCT3 inhibitor corticosterone (1 µM) resulted in a significant (p<0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation (R(2)=0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.

Hepatocytes as a tool in drug metabolism, transport and safety evaluations in drug discovery.

The liver is the primary site of metabolism for most drugs. Its major roles include detoxification of the systemic and portal blood, and production and secretion of critical blood and biliary components. A number of liver-derived in vitro systems, such as slices, primary and immortalized hepatocytes, microsomes and S9 fractions are used to assess the metabolism and potential toxicity of new chemical entities. Over the past decade, primary hepatocytes have become a standard in vitro tool to evaluate hepatic drug metabolism, cytochrome P450 (P450) induction, and drug interactions affecting hepatic metabolism. While earlier, hepatocytes were used in suspension for metabolic stability evaluations, more recent studies have demonstrated the added value of using these over longer terms in primary culture. Primary hepatocyte cultures are particularly useful in the evaluation of low turn-over compounds. Hepatic transporter studies are recommended for drug candidates that are predominantly eliminated through the bile. An appropriate strategy is to use primary hepatocytes to assess uptake, followed by singly transfected cell lines to identify the specific transporter(s) involved. Primary hepatocytes can also be used to assess biliary clearance to enable improved hepatic clearance predictions. Newer technologies such as siRNA can be used to knock out specific transporters for more predictive evaluations of potential clinically-based drug-drug interactions. In vitro safety (toxicology) studies have historically been conducted using cell lines. There is increasing evidence that co-cultures of primary hepatocytes and Kupffer cells would be more predictive of the in vivo outcome, as this system provides the complete complement of drug metabolizing enzymes, transcription factors and cytokines necessary to get a more in vivo-like toxicological response. In this review, we will discuss standard and novel in vitro approaches for using primary hepatocytes to extrapolate clinical hepatic metabolism, transport and toxicity.

Regulation of cytochrome P450 2C9 expression in primary cultures of human hepatocytes.

Cytochrome P450 2C9 (CYP2C9) expression is regulated by multiple nuclear receptors including the constitutive androstane receptor (CAR) and pregnane X receptor (PXR). We compared coregulation of CYP2C9 with CYP2B6 and CYP3A4, prototypical target genes for human CAR and PXR using human hepatocyte cultures treated for three days with the PXR activators clotrimazole, rifampin, and ritonavir; the CAR/PXR activator phenobarbital (PB); and the CAR-selective agonists CITCO, (6-(4-chlorophenyl)imidazo[2,1-beta][1,3]thiazole-5-carbaldehyde-O-(3,4-dichlorobenzyl)oxime) and phenytoin. Clotrimazole, rifampin, ritonavir, phenytoin, and phenobarbital induced CYP2C9 consistent with previous findings for CYP3A4. We observed EC(50) values of 519 microM (phenobarbital), 11 microM (phenytoin), and 0.75 microM (rifampin), similar to those for CYP3A4 induction. Avasimibe, a potent PXR activator, produced nearly identical concentration-dependent CYP2C9 and CYP3A4 activity profiles and EC(50) values. In 17 donors, rifampin increased mean basal CYP2C9 activity from 59 +/- 43 to 143 +/- 68 pmol/mg protein/min; fold induction ranged from 1.4- to 6.4-fold. Enzyme activity and mRNA measurements after rifampin, CITCO and PB treatment demonstrated potency and efficacy consistent with CYP2C9 regulation being analogous to CYP3A4 rather than CYP2B6. We demonstrate that hepatic CYP2C9 is differentially regulated by agonists of CAR and PXR, and despite sharing common regulatory mechanisms with CYP3A4 and CYP2B6; this enzyme exhibits an induction profile more closely aligned with that of CYP3A4.

The state of in vitro science for use in bioaccumulation assessments for fish.

Through the concerted evaluations of thousands of commercial substances for the qualities of persistence, bioaccumulation, and toxicity as a result of the United Nations Environment Program's Stockholm Convention, it has become apparent that fewer empirical data are available on bioaccumulation than other endpoints and that bioaccumulation models were not designed to accommodate all chemical classes. Due to the number of chemicals that may require further assessment, in vivo testing is cost prohibitive and discouraged due to the large number of animals needed. Although in vitro systems are less developed and characterized for fish, multiple high-throughput in vitro assays have been used to explore the dietary uptake and elimination of pharmaceuticals and other xenobiotics by mammals. While similar processes determine bioaccumulation in mammalian species, a review of methods to measure chemical bioavailability in fish screening systems, such as chemical biotransformation or metabolism in tissue slices, perfused tissues, fish embryos, primary and immortalized cell lines, and subcellular fractions, suggest quantitative and qualitative differences between fish and mammals exist. Using in vitro data in assessments for whole organisms or populations requires certain considerations and assumptions to scale data from a test tube to a fish, and across fish species. Also, different models may incorporate the predominant site of metabolism, such as the liver, and significant presystemic metabolism by the gill or gastrointestinal system to help accurately convert in vitro data into representative whole-animal metabolism and subsequent bioaccumulation potential. The development of animal alternative tests for fish bioaccumulation assessment is framed in the context of in vitro data requirements for regulatory assessments in Europe and Canada.

Current industrial practices in assessing CYP450 enzyme induction: preclinical and clinical.

Induction of drug metabolizing enzymes, such as the cytochromes P450 (CYP) is known to cause drug-drug interactions due to increased elimination of co-administered drugs. This increased elimination may lead to significant reduction or complete loss of efficacy of the co-administered drug. Due to the significance of such drug interactions, many pharmaceutical companies employ screening and characterization models which predict CYP enzyme induction to avoid or attenuate the potential for drug interactions with new drug candidates. The most common mechanism of CYP induction is transcriptional gene activation. Activation is mediated by nuclear receptors, such as AhR, CAR, and PXR that function as transcription factors. Early high throughput screening models utilize these nuclear hormone receptors in ligand binding or cell-based transactivation/reporter assays. In addition, immortalized hepatocyte cell lines can be used to assess enzyme induction of specific drug metabolizing enzymes. Cultured primary human hepatocytes, the best established in vitro model for predicting enzyme induction and most accepted by regulatory agencies, is the predominant assay used to evaluate induction of a wide variety of drug metabolizing enzymes. These in vitro models are able to appropriately predict enzyme induction in patients when compared to clinical drug-drug interactions. Finally, transgenic animal models and the cynomolgus monkey have also been shown to recapitulate human enzyme induction and may be appropriate in vivo animal models for predicting human drug interactions.

Aldehyde oxidase activity and inhibition in hepatocytes and cytosolic fractions from mouse, rat, monkey and human.

Aldehyde oxidase (AO) is a cytosolic enzyme that contributes to the Phase I metabolism of xenobiotics in human and preclinical species. We compared AO activity in cytosol and cryopreserved hepatocytes from human, monkey, rat and mouse livers to assess species differences. We also evaluated possible species differences in drug interactions using seven drugs known to inhibit human cytosolic AO i.e. raloxifene, perphenazine, menadione, maprotiline, ketoconazole, erythromycin, and estradiol. AO activity was measured using the formation of vanillic acid from vanillin. The rate of vanillic acid formation was 2 +/- 0.2 nmol/min/mg in human liver cytosol and 0.79 +/- 0.45 nmol/min/million cells in cryopreserved human hepatocytes. AO activity (V(max,app)) was highest in monkey and lowest in rat. Mouse liver cytosol had the lowest K(m,app) (1.44 +/- 0.16 microM) and highest intrinsic clearance (8.97 ml/min/mg) and rat liver cytosol the highest K(m,app) (10.9 +/- 1.2 microM) and lowest intrinsic clearance (0.47 ml/min/mg). There was a 4.25-fold difference in AO activity between the 5 human hepatocyte preparations. Drug interaction studies with the seven marketed drugs revealed marked species-specific inhibition. Our data indicates major differences in the rate of AO metabolism, and inhibition of AO across species, indicating that results from animal studies cannot be safely extrapolated to humans. Cryopreserved hepatocytes and cytosolic fractions from animals and humans provide qualitatively similar data within the species.

Hepatobiliary disposition in primary cultures of dog and monkey hepatocytes.

Hepatobiliary transporters are a major route for elimination of xenobiotics and endogenous products. In vitro hepatobiliary models have been reported for human and rat, but not for the other preclinical species used in safety evaluation. We have established methodologies for culturing dog and monkey hepatocytes with optimal bile canalicular formation and function, using a sandwich culture comprising rigid collagen substratum and gelled collagen overlay. Hepatic uptake utilizing sinusoidal transporters and biliary excretion through canalicular transporters were assessed using the bile salt taurocholate, salicylate (negative control), and the Bsep inhibitors cyclosporin A (CsA) and glyburide. There was significant taurocholate and salicylate canalicular efflux in dog and monkey hepatocytes, although the amount of salicylate transported was one thousandth that of taurocholate. Species differences were observed, as glyburide significantly inhibited taurocholate uptake in monkey (64% at 10 microM) but not dog hepatocytes, and inhibited taurocholate efflux in dog (100% at 10 microM) but not monkey hepatocytes. CsA did not inhibit bile salt uptake and significantly inhibited canalicular efflux in dog (at 0.1 microM) and monkey (at 1 and 10 microM) hepatocyte cultures. These results suggest that glyburide is a bile salt uptake inhibitor in monkey but not in dog hepatocytes and that CsA inhibits bile salt canalicular efflux but not basolateral uptake in these species. We have established dog and monkey hepatocytes in sandwich culture with intact bile canalicular formation and function. The differences observed in taurocholate transport between dog and monkey hepatocytes may be indicative of in vivo species differences.

Metabolism and transporter-mediated drug-drug interactions of the endothelin-A receptor antagonist CI-1034.

CI-1034, an endothelin-A receptor antagonist was being developed for pulmonary hypertension. Drug-drug interaction studies using human hepatic microsomes were conducted to assess CYP1A2, CYP2C9, CYP2C19, CYP3A4 and CYP2D6 inhibition potential; CYP3A4 induction potential was evaluated using primary human hepatocytes. CI-1034 moderately inhibited CYP2C9 (IC(50) 39.6 microM) and CYP3A4 activity (IC(50) 21.6 microM); CYP3A4 inhibition was metabolism-dependent. In human hepatocytes, no increase in CYP3A4 activity was observed in vitro, while mRNA was induced 15-fold, similar to rifampin, indicating that CI-1034 is both an inhibitor and inducer of CYP3A4. A 2-week clinical study was conducted to assess pharmacokinetics, pharmacodynamics and safety. No significant changes were observed in [formula: see text] between days 1 and 14. However, reversible elevations of serum liver enzymes were observed with a 50mg BID dose and the program was terminated. To further understand the interactions of CI-1034 in the liver and possible mechanisms of the observed hepatotoxicity, we evaluated the effect of CI-1034 on bile acid transport and previously reported that CI-1034 inhibited biliary efflux of taurocholate by 60%, in vitro. This indicated that inhibition of major hepatic transporters could be involved in the observed hepatotoxicity. We next evaluated the in vitro inhibition potential of CI-1034 with the major hepatic transporters OATP1B1, OATP1B3, OATP2B1, MDR1, MRP2 and OCT. CI-1034 inhibited OATP1B1 (K(i) 2 microM), OATP1B3 (K(i) 1.8 microM) and OATP2B1 activity (K(i) 3.3 microM) but not OCT, MDR1 or MRP2 mediated transport. Our data indicates that CI-1034 is an inhibitor of major hepatic transporters and inhibition of bile efflux may have contributed to the observed clinical hepatotoxicity. We recommend that in vitro drug-drug interaction panels include inhibition and induction studies with transporters and drug metabolizing enzymes, to more completely assess potential in vivo interactions or toxicity.

In vitro ADME phenotyping in drug discovery: current challenges and future solutions.

Drug-metabolizing enzymes and drug transporters are key regulators of drug disposition and pharmacodynamics, which are closely linked to drug efficacy and safety. In this article, current challenges and future solutions to predicting their influence on pharmacokinetics and inter-organ distribution in humans, from data generated during the drug discovery decision-making process, are presented. In vitro phenotyping strategies for drug metabolizing enzymes (eg, CYP3A4, UGT1A1) and transporters (eg, OATP1B1) are offered, including perspectives on a selection of in vitro systems, novel in vitro phenotyping reagents and remaining technology gaps, challenges in extrapolating in vitro data to the in vivo situation, in silico models for the prediction of whether compounds are enzyme or transporter substrates, and the impact of pharmacogenomics.

Use of in vitro transporter assays to understand hepatic and renal disposition of new drug candidates.

Hepatic and renal transporters contribute to the uptake, secretion and reabsorption of endogenous compounds, xenobiotics and their metabolites and have been implicated in drug-drug interactions and toxicities. Characterising the renal and hepatic disposition of drug candidates early in development would lead to more rational drug design, as chemotypes with 'ideal' pharmacokinetic characteristics could be identified and further refined. Because transporters are often organ specific, 'custom' transporter panels need to be identified for each major organ and chemotype to be evaluated, and appropriate studies planned. This review outlines the major renal and hepatic transporters and some of the in vitro transporter reagents, assays and processes that can be used to evaluate the renal and hepatic disposition of new chemical entities during drug discovery and development.

Effects of avasimibe on cytochrome P450 2C9 expression in vitro and in vivo.

Avasimibe, an acyl-CoA:cholesterol acyltransferase inhibitor, has been previously shown to be a potent inducer of CYP3A4 and multiple drug resistance protein 1. We have further characterized the drug interaction potential of avasimibe by studying the inductive and inhibitory effect of this compound on major drug-metabolizing enzymes. Enzymes known to be involved in the metabolism of drugs likely to be coadministered with avasimibe, such as CYP1A1/2, CYP2C, and CYP2B6, were evaluated further by microarray analysis, Western immunoblotting, and activity assays, using rifampicin and beta-naphthoflavone as positive controls. No change was observed in CYP1A1/2 mRNA or activity levels after avasimibe treatment. Differential induction of CYP2C9- and CYP2B6-immunoreactive protein and activity was observed depending on drug concentration and donor. Microarray analysis showed a similar increase in CYP2C and CYP2B6 mRNA levels. The inhibition potential of avasimibe on the major drug-metabolizing enzymes was assessed using pooled human liver microsomes. Avasimibe inhibited CYP2C9 (IC50 2.9 microM), CYP1A2 (IC50 13.9 microM), and CYP2C19 (IC50 26.5 microM). A clinical drug interaction study was conducted to determine whether avasimibe might interact with the CYP2C9 substrate warfarin. Volunteers received 750 mg of avasimibe and showed a 54.2% reduction in trough concentrations of S-warfarin and decreased prothrombin times by 12, 15, 19, and 21% on days 6 through 9, respectively. These results demonstrate that avasimibe's inductive spectrum resembles that of rifampin.

Induction of drug metabolism enzymes and MDR1 using a novel human hepatocyte cell line.

Induction of drug-metabolizing enzymes and transporters can cause drug-drug interactions and loss of efficacy. In vitro induction studies traditionally use primary hepatocyte cultures and enzyme activity with selected marker compounds. We investigated the use of a novel human hepatocyte clone, the Fa2N-4 cell line, as an alternative reagent, which is readily available and provides a consistent, reproducible system. We used the Invader assay to monitor gene expression in these cells. This assay is a robust, yet simple, high-throughput system for quantification of mRNA transcripts. CYP1A2, CYP3A4, CYP2C9, UGT1A, and MDR1 transcripts were quantified from total RNA extracts from Fa2N-4 cells treated with a panel of known inducers and compared with vehicle controls. In addition, we used enzyme activity assays to monitor the induction of CYP1A2, CYP2C9, and CYP3A4. The Fa2N-4 cells responded in a similar manner as primary human hepatocytes. Treatment with 10 microM rifampin resulted in increases in CYP3A4 mRNA (17-fold) and activity (6-beta-hydroxytestoterone formation, 9-fold); and in CYP2C9 mRNA (4-fold) and activity (4'-hydroxydiclofenac formation, 2-fold). Treatment with 50 microM beta-naphthoflavone resulted in increases in CYP1A2 mRNA (15-fold) and activity (7-ethoxyresorufin O-dealkylation, 27-fold). UGT1A mRNA was induced by beta-naphthoflavone (2-fold), and MDR1 (P-glycoprotein) mRNA was induced by rifampin (3-fold). These preliminary data using a few prototypical inducers show that Fa2N-4 cells can be a reliable surrogate for primary human hepatocytes, and, when used in conjunction with the Invader technology, could provide a reliable assay for assessment of induction of drug-metabolizing enzymes and transporters.