J. B. Houston

Mechanistic pharmacokinetic modeling for the prediction of transporter-mediated disposition in humans from sandwich culture human hepatocyte data

With efforts to reduce cytochrome P450-mediated clearance (CL) during the early stages of drug discovery, transporter-mediated CL mechanisms are becoming more prevalent. However, the prediction of plasma concentration-time profiles for such compounds using physiologically based pharmacokinetic (PBPK) modeling is far less established in comparison with that for compounds with passively mediated pharmacokinetics (PK). In this study, we have assessed the predictability of human PK for seven organic anion-transporting polypeptide (OATP) substrates (pravastatin, cerivastatin, bosentan, fluvastatin, rosuvastatin, valsartan, and repaglinide) for which clinical intravenous data were available. In vitro data generated from the sandwich culture human hepatocyte system were simultaneously fit to estimate parameters describing both uptake and biliary efflux. Use of scaled active uptake, passive distribution, and biliary efflux parameters as inputs into a PBPK model resulted in the overprediction of exposure for all seven drugs investigated, with the exception of pravastatin. Therefore, fitting of in vivo data for each individual drug in the dataset was performed to establish empirical scaling factors to accurately capture their plasma concentration-time profiles. Overall, active uptake and biliary efflux were under- and overpredicted, leading to average empirical scaling factors of 58 and 0.061, respectively; passive diffusion required no scaling factor. This study illustrates the mechanistic and model-driven application of in vitro uptake and efflux data for human PK prediction for OATP substrates. A particular advantage is the ability to capture the multiphasic plasma concentration-time profiles for such compounds using only preclinical data. A prediction strategy for novel OATP substrates is discussed.

Optimizing drug development: Strategies to assess drug metabolism/transporter interaction potential - Toward a consensus

The issue of drug–drug interactions has generated significant concern within the pharmaceutical industry and among regulatory authorities in recent years. This has arisen with respect to early termination of clinical development (e.g. furafylline), refusal of approval (e.g. mibefradil in Sweden), severe prescribing restrictions and withdrawal from the market (e.g. sorivudine, terfenadine, mibefradil, astemizole, cisapride), and threatened litigation. This report summarizes the outcomes of a conference held in Basel in November 2000, held under the auspices of the European Federation of Pharmaceutical Sciences (EUFEPS), the U.S. Food and Drug Administration (FDA) and the American Association of Pharmaceutical Sciences (AAPS). The meeting followed from two previous workshops on drug interactions held in Nuremberg (1997) and Arlington (1999) sponsored by the same groups. Whereas previous conferences had identified the main areas of contention, a specific aim of this meeting was to attempt a consensus on the conduct of in vitro and in vivo studies of metabolic and transport interactions. There were five main conference sessions in which experienced scientists from academia, industry, and regulatory bodies were invited to contribute short presentations formulated, where possible, to address specific questions.

Kinetic characterization of rat hepatic uptake of 16 actively transported drugs

To explore the determinants of hepatic uptake, 16 compounds were investigated with different physicochemical and disposition characteristics, including five statins, three sartans, saquinavir, ritonavir, erythromycin, clarithromycin, nateglinide, repaglinide, fexofenadine, and bosentan. Freshly isolated rat hepatocytes in suspension were used with the oil-spin method to generate kinetic parameters. Clearances, via passive diffusion (Pdiff) and active uptake (CLactive, characterized by maximal uptake rate and Km), were estimated from the initial uptake rate data over a 0.01 to 100 μM concentration range. The Km values had a range of 15-fold, with 10 of the 16 drugs with Km < 10 μM (median 6 μM). Both CLactive and Pdiff ranged over 100-fold (median 188 and 14 μl/min/106 cells). Assessment of the relative contribution of Pdiff and CLactive indicated that, at low concentrations (approximately 0.1 μM), the active process contributes >80% to the overall uptake for 13 drugs. Although high Pdiff values were obtained for ritonavir and repaglinide, active process contributed predominantly to uptake; in contrast, high passive permeability dominates over transporter-mediated uptake for saquinavir over the full concentration range. For bosentan and erythromycin, active and passive processes were equally important. Hepatocyte-to-medium unbound concentration ratio was >10 for 9 of the 16 drugs, ranging from 2 to 494 for bosentan and atorvastatin, respectively. Some drugs showed extensive intracellular binding (fraction unbound range 0.01–0.6), which was not correlated with active uptake. LogD7.4 correlated significantly with Pdiff and the extent of intracellular binding but not with active uptake. This study provides systematic assessment of the role of active uptake relative to the passive process; implications of the findings are discussed.

Physiologically Based Pharmacokinetic Modeling of Intestinal First-Pass Metabolism of CYP3A Substrates with High Intestinal Extraction

Prediction of intestinal availability (FG), in conjunction with hepatic metabolism, is of considerable importance in drug disposition to assess oral clearance and liability to drug-drug interactions. In the current study, FG predictions were performed within a physiologically based pharmacokinetic (PBPK) model using in vitro permeability and clearance data. The prediction success was assessed in comparison with the QGut model. In addition, apparent oral clearance values, predicted using the PBPK model, were compared with in vivo observations from meta-analyses. Finally, unbound intrinsic clearance values (CLuint) were determined for 12 CYP3A substrates in eight individual human jejunal microsome (HJM) samples to assess interindividual variability in intestinal intrinsic clearance and subsequent FG predictions. Overall, the PBPK model improved FG predictions in comparison with the QGut model; this was apparent by a reduced bias and increased precision. In particular, FG predictions of indinavir, saquinavir, and terfenadine were model-dependent. The predicted oral clearance values of the drugs investigated ranged from 8.79 to 6320 l/h for tacrolimus and simvastatin, respectively, and were overall within 3-fold of the observed data with the exception of indinavir, atorvastatin, and buspirone. The individual HJM CLuint values ranged from 17 to 14,000 μl · min−1 · mg−1 for atorvastatin and saquinavir, respectively, and corresponding interindividual variability in CLuint estimates ranged from 41 to 67%. These in vitro data resulted in predicted FG values ranging from 0.03 to 0.94 for simvastatin and indinavir, respectively. The largest interindividual variability of FG was predicted for terfenadine (65%) in contrast with the low variability in the case of indinavir (3%).

Characterization of In Vitro Glucuronidation Clearance of a Range of Drugs in Human Kidney Microsomes: Comparison with Liver and Intestinal Glucuronidation and Impact of Albumin

Previous studies have shown the importance of the addition of albumin for characterization of hepatic glucuronidation in vitro; however, no reports exist on the effects of albumin on renal or intestinal microsomal glucuronidation assays. This study characterized glucuronidation clearance (CLint, UGT) in human kidney, liver, and intestinal microsomes in the presence and absence of bovine serum albumin (BSA) for seven drugs with differential UDP-glucuronosyltransferase (UGT) 1A9 and UGT2B7 specificity, namely, diclofenac, ezetimibe, gemfibrozil, mycophenolic acid, naloxone, propofol, and telmisartan. The impact of renal CLint, UGT on accuracy of in vitro-in vivo extrapolation (IVIVE) of glucuronidation clearance was investigated. Inclusion of 1% BSA for acidic drugs and 2% for bases/neutral drugs in incubations was found to be suitable for characterization of CLint, UGT in different tissues. Although BSA increased CLint, UGT in all tissues, the extent was tissue- and drug-dependent. Scaled CLint, UGT in the presence of BSA ranged from 2.22 to 207, 0.439 to 24.4, and 0.292 to 23.8 ml · min−1 · g tissue−1 in liver, kidney, and intestinal microsomes. Renal CLint, UGT (per gram of tissue) was up to 2-fold higher in comparison with that for liver for UGT1A9 substrates; in contrast, CLint, UGT for UGT2B7 substrates represented approximately one-third of hepatic estimates. Scaled renal CLint, UGT (in the presence of BSA) was up to 30-fold higher than intestinal glucuronidation for the drugs investigated. Use of in vitro data obtained in the presence of BSA and inclusion of renal clearance improved the IVIVE of glucuronidation clearance, with 50% of drugs predicted within 2-fold of observed values. Characterization and consideration of kidney CLint, UGT is particularly important for UGT1A9 substrates.

Reliability of human cryopreserved hepatocytes and liver microsomes as in vitro systems to predict metabolic clearance

A total of 110 drugs, selected to cover a range of physicochemical and pharmacokinetic properties, were used to explore standard approaches to the prediction of in vivo metabolic clearance using drug-depletion profiles from human liver microsomes (HLMs) and cyropreserved hepatocytes. A total of 41 drugs (37% of the compounds tested) showed measurable depletion rates using HLMs (depletion by 20% or more over the time course). The most reliable correlations in terms of bias (average fold error (AFE) = 2.32) and precision (root mean square error (RMSE) = 3501) were observed by comparing in vivo intrinsic clearance (CLint), calculated using the parallel-tube model and incorporating the fraction unbound in blood, with in vitro CLint adjusted for microsomal binding. For these reference drugs, 29% of predictions were within two-fold of the observed values and 66% were within five-fold. Compared with HLMs, clearance predictions with cryopreserved hepatocytes (57 drugs) were of similar precision (RMSE = 3608) but showed more bias (AFE = 5.21) with 18% of predictions within two-fold of the observed values and 46% within five-fold. However, with a broad complement of drug-metabolizing enzymes, hepatocytes catalysed measurable CLint values for a greater proportion (52%) of the reference compounds and were particularly proficient at defining metabolic rates for drugs with predominantly phase 2 metabolic routes.

Evaluation of Recombinant Cytochrome P450 Enzymes as an in Vitro System for Metabolic Clearance Predictions

The aim of this study was to explore the potential of recombinant cytochrome P450 (P450) enzymes for human metabolic clearance prediction. The relative abundance and relative activity approaches were compared as methods to bridge the gap between catalytic activities in recombinant P450 enzymes and human liver microsomes (HLMs). Relative activity factors were measured by determining the intrinsic clearance (CLint) of probe substrates (bufuralol-CYP2D6, diclofenac-CYP2C9, midazolam-CYP3A4, and phenacetin-CYP1A2) in recombinant P450s and 16 HLM donors. Simultaneous determination of drug depletion and metabolite formation profiles has enabled a direct comparison of these methods for CLint determination. Of the 110 drugs tested, 66% were metabolized by one or more P450 enzymes; of these 44% of were metabolized by CYP3A4 (0.3–21 μl/min/pmol of P450), 41% by CYP2D6 (0.6–60 μl/min/pmol of P450), 26% by CYP2C19 (0.4–8.1 μl/min/pmol of P450), 9% by CYP1A2 (0.4–2.5 μl/min/pmol of P450), and 4% by CYP2C9 (0.9–6.4 μl/min/pmol of P450). Recombinant enzymes demonstrated improved prediction reliability relative to HLMs and hepatocytes. The most reliable correlations in terms of lowest bias and highest precision were observed by comparing in vivo CLint, calculated using the parallel-tube model and incorporating fraction unbound in blood, with in vitro CLint determined using relative activity factors and adjusted for nonspecific binding. Predictions were less reliable using the relative abundance approach. For these drugs, recombinant P450 enzymes offer improved assay sensitivity compared with HLMs and cryopreserved hepatocytes for CLint determination using the drug depletion method.

Methodological uncertainty in quantitative prediction of human hepatic clearance from in vitro experimental systems.

Mechanistic prediction of unbound drug clearance from human hepatic microsomes and hepatocytes correlates with in vivo clearance but is both systematically low (10 - 20 % of in vivo clearance) and highly variable, based on detailed assessments of published studies. Metabolic capacity (Vmax) of commercially available human hepatic microsomes and cryopreserved hepatocytes is log-normally distributed within wide (30 - 150-fold) ranges; Km is also log-normally distributed and effectively independent of Vmax, implying considerable variability in intrinsic clearance. Despite wide overlap, average capacity is 2 - 20-fold (dependent on P450 enzyme) greater in microsomes than hepatocytes, when both are normalised (scaled to whole liver). The in vitro ranges contrast with relatively narrow ranges of clearance among clinical studies. The high in vitro variation probably reflects unresolved phenotypical variability among liver donors and practicalities in processing of human liver into in vitro systems. A significant contribution from the latter is supported by evidence of low reproducibility (several fold) of activity in cryopreserved hepatocytes and microsomes prepared from the same cells, between separate occasions of thawing of cells from the same liver. The large uncertainty which exists in human hepatic in vitro systems appears to dominate the overall uncertainty of in vitro-in vivo extrapolation, including uncertainties within scaling, modelling and drug dependent effects. As such, any notion of quantitative prediction of clearance appears severely challenged.

A comprehensive assessment of repaglinide metabolic pathways: impact of choice of in vitro system and relative enzyme contribution to in vitro clearance.

Repaglinide is presently recommended by the U.S. Food and Drug Administration as a clinical CYP2C8 probe, yet current in vitro and clinical data are inconsistent concerning the role of this enzyme in repaglinide elimination. The aim of the current study was to perform a comprehensive investigation of repaglinide metabolic pathways and assess their contribution to the overall clearance. Formation of four repaglinide metabolites was characterized using in vitro systems with differential complexity. Full kinetic profiles for the formation of M1, M2, M4, and repaglinide glucuronide were obtained in pooled cryopreserved human hepatocytes, human liver microsomes, human S9 fractions, and recombinant cytochrome P450 enzymes. Distinct differences in clearance ratios were observed between CYP3A4 and CYP2C8 for M1 and M4 formation, resulting in a 60-fold M1/M4 ratio in recombinant (r) CYP3A4, in contrast to 0.05 in rCYP2C8. Unbound Km values were within 2-fold for each metabolite across all in vitro systems investigated. A major system difference was seen in clearances for the formation of M2, which is suggested to be a main metabolite of repaglinide in vivo. An approximately 7-fold higher unbound intrinsic clearance was observed in hepatocytes and S9 fractions in comparison to microsomes; the involvement of aldehyde dehydrogenase in M2 formation was shown for the first time. This systematic analysis revealed a comparable in vitro contribution from CYP2C8 and CYP3A4 to the metabolism of repaglinide (<50%), whereas the contribution of glucuronidation ranged from 2 to 20%, depending on the in vitro system used. The repaglinide M4 metabolic pathway is proposed as a specific CYP2C8 probe for the assessment of drug-drug interactions.

Metabolite kinetics of ondansetron in rat. Comparison of hepatic microsomes, isolated hepatocytes and liver slices, with in vivo disposition

1. The kinetics of hydroxylation and N-demethylation of ondansetron have been determined in freshly isolated hepatocytes, hepatic microsomes and precision-cut liver slices from the male Sprague-Dawley rat. In vivo studies have also been carried out to characterize the pharmacokinetics of ondansetron and in vitro data have been assessed for their value as predictors of hepatic clearance. 2. In the three in vitro systems, the formation of hydroxylated and demethylated metabolites were characterized as a function of substrate concentration by a high-affinity, low-capacity site and a low-affinity, high-capacity site which was not saturated over the concentration range studied (2.5-500 microM). Slices gave consistently higher Kms (20 and 30 microM for hydroxylation and demethylation respectively) than hepatocytes (3 and 13 microM respectively) and microsomes (2 and 5 microM respectively.) The rank order of Vmax and CL(int) was the same for each system; hydroxylation rates exceeding demethylation rates. Although two hydroxylations (7- and 8-hydroxy metabolites) occurred exclusively in microsomes, these are believed to originate from a common precursor. 3. The high CL(int) of ondansetron (150 ml/min/SRW, where SRW is a standard rat weight of 250g) is well predicted by scaling either microsomal clearance for microsomal protein recovery or hepatocyte clearance for hepatocellularity (212 and 135 ml/min/SRW respectively). In contrast, the use of liver slice data scaled to a whole liver substantially underestimates CL(int) (9 ml/min/SRW).