The Critical Role of Passive Permeability in Designing Successful Drugs.

Passive permeability is a key property in drug disposition and delivery. It is critical for gastrointestinal absorption, brain penetration, renal reabsorption, defining clearance mechanisms and drug-drug interactions. Passive diffusion rate is translatable across tissues and animal species, while the extent of absorption is dependent on drug properties, as well as in vivo physiology/pathophysiology. Design principles have been developed to guide medicinal chemistry to enhance absorption, which combine the balance of aqueous solubility, permeability and the sometimes unfavorable compound characteristic demanded by the target. Permeability assays have been implemented that enable rapid development of structure-permeability relationships for absorption improvement. Future advances in assay development to reduce nonspecific binding and improve mass balance will enable more accurately measurement of passive permeability. Design principles that integrate potency, selectivity, passive permeability and other ADMET properties facilitate rapid advancement of successful drug candidates to patients.

Hepatic Organic Anion Transporting Polypeptide-Mediated Clearance in the Beagle Dog: Assessing In Vitro-In Vivo Relationships and Applying Cross-Species Empirical Scaling Factors to Improve Prediction of Human Clearance.

In the present study, the beagle dog was evaluated as a preclinical model to investigate organic anion transporting polypeptide (OATP)-mediated hepatic clearance. In vitro studies were performed with nine OATP substrates in three lots of plated male dog hepatocytes ± OATP inhibitor cocktail to determine total uptake clearance (CLuptake) and total and unbound cell-to-medium concentration ratio (Kpuu). In vivo intrinsic hepatic clearances (CLint,H) were determined following intravenous drug administration (0.1 mg/kg) in male beagle dogs. The in vitro parameters were compared with those previously reported in plated human, monkey, and rat hepatocytes; the ability of cross-species scaling factors to improve prediction of human in vivo clearance was assessed. CLuptake in dog hepatocytes ranged from 9.4 to 135 µl/min/106 cells for fexofenadine and telmisartan, respectively. Active process contributed >75% to CLuptake for 5/9 drugs. Rosuvastatin and valsartan showed Kpuu > 10, whereas cerivastatin, pitavastatin, repaglinide, and telmisartan had Kpuu < 5. The extent of hepatocellular binding in dog was consistent with other preclinical species and humans. The bias (2.73-fold) obtained from comparison of predicted versus in vivo dog CLint,H was applied as an average empirical scaling factor (ESFav) for in vitro-in vivo extrapolation of human CLint,H The ESFav based on dog reduced underprediction of human CLint,H for the same data set (geometric mean fold error = 2.1), highlighting its utility as a preclinical model to investigate OATP-mediated uptake. The ESFav from all preclinical species resulted in comparable improvement of human clearance prediction, in contrast to drug-specific empirical scalars, rationalized by species differences in expression and/or relative contribution of particular transporters to drug hepatic uptake.

Predicting Human Clearance of Organic Anion Transporting Polypeptide Substrates Using Cynomolgus Monkey: In Vitro-In Vivo Scaling of Hepatic Uptake Clearance.

This work explores the utility of the cynomolgus monkey as a preclinical model to predict hepatic uptake clearance mediated by organic anion transporting polypeptide (OATP) transporters. Nine OATP substrates (rosuvastatin, pravastatin, repaglinide, fexofenadine, cerivastatin, telmisartan, pitavastatin, bosentan, and valsartan) were investigated in plated cynomolgus monkey and human hepatocytes. Total uptake clearance and passive diffusion were measured in vitro from initial rates in the absence and presence of the OATP inhibitor rifamycin SV , respectively. Total uptake clearance values in plated hepatocytes ranged over three orders of magnitude in both species, with a similar rank order and good agreement in the relative contribution of active transport to total uptake between cynomolgus monkey and human. In vivo hepatic clearance for these nine drugs was determined in cynomolgus monkey after intravenous dosing. Hepatic clearances showed a range similar to human parameters and good predictions from respective hepatocyte parameters (with 2.7- and 3.8-fold bias on average, respectively). The use of cross-species empirical scaling factors (determined from cynomolgus monkey data either as the data set average or individual drug values) improved prediction (less bias, better concordance) of human hepatic clearance from human hepatocyte data alone. In vitro intracellular binding in hepatocytes also correlated well between species. It is concluded that the minimal species differences observed for the current data set between cynomolgus monkey and human hepatocyte uptake, both in vitro and in vivo, support future use of this preclinical model to delineate drug hepatic uptake and enable prediction of human in vivo intrinsic hepatic clearance.

In Vitro-In Vivo Extrapolation of OATP1B-Mediated Drug-Drug Interactions in Cynomolgus Monkey.

Hepatic organic anion-transporting polypeptides (OATP) 1B1 and 1B3 are clinically relevant transporters associated with significant drug-drug interactions (DDIs) and safety concerns. Given that OATP1Bs in cynomolgus monkey share >90% degree of gene and amino acid sequence homology with human orthologs, we evaluated the in vitro-in vivo translation of OATP1B-mediated DDI risk using this preclinical model. In vitro studies using plated cynomolgus monkey hepatocytes showed active uptake Km values of 2.0 and 3.9 µM for OATP1B probe substrates, pitavastatin and rosuvastatin, respectively. Rifampicin inhibited pitavastatin and rosuvastatin active uptake in monkey hepatocytes with IC50 values of 3.0 and 0.54 µM, respectively, following preincubation with the inhibitor. Intravenous pharmacokinetics of 2H4-pitavastatin and 2H6-rosuvastatin (0.2 mg/kg) and the oral pharmacokinetics of cold probes (2 mg/kg) were studied in cynomolgus monkeys (n = 4) without or with coadministration of single oral ascending doses of rifampicin (1, 3, 10, and 30 mg/kg). A rifampicin dose-dependent reduction in i.v. clearance of statins was observed. Additionally, oral pitavastatin and rosuvastatin plasma exposure increased up to 19- and 15-fold at the highest dose of rifampicin, respectively. Use of in vitro IC50 obtained following 1 hour preincubation with rifampicin (0.54 µM) predicted correctly the change in mean i.v. clearance and oral exposure of statins as a function of mean unbound maximum plasma concentration of rifampicin. This study demonstrates quantitative translation of in vitro OATP1B IC50 to predict DDIs using cynomolgus monkey as a preclinical model and provides further confidence in application of in vitro hepatocyte data for the prediction of clinical OATP1B-mediated DDIs.

Reduced physiologically-based pharmacokinetic model of repaglinide: impact of OATP1B1 and CYP2C8 genotype and source of in vitro data on the prediction of drug-drug interaction risk.

PURPOSE: To investigate the effect of OATP1B1 genotype as a covariate on repaglinide pharmacokinetics and drug-drug interaction (DDIs) risk using a reduced physiologically-based pharmacokinetic (PBPK) model. METHODS: Twenty nine mean plasma concentration-time profiles for SLCO1B1 c.521T>C were used to estimate hepatic uptake clearance (CLuptake) in different genotype groups applying a population approach in NONMEM v.7.2. RESULTS: Estimated repaglinide CLuptake corresponded to 217 and 113 µL/min/10(6) cells for SLCO1B1 c.521TT/TC and CC, respectively. A significant effect of OATP1B1 genotype was seen on CLuptake (48% reduction for CC relative to wild type). Sensitivity analysis highlighted the impact of CLmet and CLdiff uncertainty on the CLuptake optimization using plasma data. Propagation of this uncertainty had a marginal effect on the prediction of repaglinide OATP1B1-mediated DDI with cyclosporine; however, sensitivity of the predicted magnitude of repaglinide metabolic DDI was high. In addition, the reduced PBPK model was used to assess the effect of both CYP2C8*3 and SLCO1B1 c.521T>C on repaglinide exposure by simulations; power calculations were performed to guide prospective DDI and pharmacogenetic studies. CONCLUSIONS: The application of reduced PBPK model for parameter optimization and limitations of this process associated with the use of plasma rather than tissue profiles are illustrated.

Recent advances in 2D and 3D in vitro systems using primary hepatocytes, alternative hepatocyte sources and non-parenchymal liver cells and their use in investigating mechanisms of hepatotoxicity, cell signaling and ADME.

This review encompasses the most important advances in liver functions and hepatotoxicity and analyzes which mechanisms can be studied in vitro. In a complex architecture of nested, zonated lobules, the liver consists of approximately 80 % hepatocytes and 20 % non-parenchymal cells, the latter being involved in a secondary phase that may dramatically aggravate the initial damage. Hepatotoxicity, as well as hepatic metabolism, is controlled by a set of nuclear receptors (including PXR, CAR, HNF-4α, FXR, LXR, SHP, VDR and PPAR) and signaling pathways. When isolating liver cells, some pathways are activated, e.g., the RAS/MEK/ERK pathway, whereas others are silenced (e.g. HNF-4α), resulting in up- and downregulation of hundreds of genes. An understanding of these changes is crucial for a correct interpretation of in vitro data. The possibilities and limitations of the most useful liver in vitro systems are summarized, including three-dimensional culture techniques, co-cultures with non-parenchymal cells, hepatospheres, precision cut liver slices and the isolated perfused liver. Also discussed is how closely hepatoma, stem cell and iPS cell-derived hepatocyte-like-cells resemble real hepatocytes. Finally, a summary is given of the state of the art of liver in vitro and mathematical modeling systems that are currently used in the pharmaceutical industry with an emphasis on drug metabolism, prediction of clearance, drug interaction, transporter studies and hepatotoxicity. One key message is that despite our enthusiasm for in vitro systems, we must never lose sight of the in vivo situation. Although hepatocytes have been isolated for decades, the hunt for relevant alternative systems has only just begun.

Cyclosporine inhibition of hepatic and intestinal CYP3A4, uptake and efflux transporters: application of PBPK modeling in the assessment of drug-drug interaction potential.

PURPOSE: To apply physiologically-based pharmacokinetic (PBPK) modeling to investigate the consequences of reduction in activity of hepatic and intestinal uptake and efflux transporters by cyclosporine and its metabolite AM1. METHODS: Inhibitory potencies of cyclosporine and AM1 against OATP1B1, OATP1B3 and OATP2B1 were investigated in HEK293 cells +/- pre-incubation. Cyclosporine PBPK model implemented in Matlab was used to assess interaction potential (+/- metabolite) against different processes (uptake, efflux and metabolism) in liver and intestine and to predict quantitatively drug-drug interaction with repaglinide. RESULTS: Cyclosporine and AM1 were potent inhibitors of OATP1B1 and OATP1B3, IC(50) ranging from 0.019-0.093 µM following pre-incubation. Cyclosporine PBPK model predicted the highest interaction potential against liver uptake transporters, with a maximal reduction of >70% in OATP1B1 activity; the effect on hepatic efflux and metabolism was minimal. In contrast, 80-97% of intestinal P-gp and CYP3A4 activity was reduced due to the 50-fold higher cyclosporine enterocytic concentrations relative to unbound hepatic inlet. The inclusion of AM1 resulted in a minor increase in the predicted maximal reduction of OATP1B1/1B3 activity. Good predictability of cyclosporine-repaglinide DDI and the impact of dose staggering are illustrated. CONCLUSIONS: This study highlights the application of PBPK modeling for quantitative prediction of transporter-mediated DDIs with concomitant consideration of P450 inhibition.

Use of mechanistic modeling to assess interindividual variability and interspecies differences in active uptake in human and rat hepatocytes.

Interindividual variability in activity of uptake transporters is evident in vivo, yet limited data exist in vitro, confounding in vitro-in vivo extrapolation. The uptake kinetics of seven organic anion-transporting polypeptide substrates was investigated over a concentration range in plated cryopreserved human hepatocytes. Active uptake clearance (CL(active, u)), bidirectional passive diffusion (P(diff)), intracellular binding, and metabolism were estimated for bosentan, pitavastatin, pravastatin, repaglinide, rosuvastatin, telmisartan, and valsartan in HU4122 donor using a mechanistic two-compartment model in Matlab. Full uptake kinetics of rosuvastatin and repaglinide were also characterized in two additional donors, whereas for the remaining drugs CL(active, u) was estimated at a single concentration. The unbound affinity constant (K(m, u)) and P(diff) values were consistent across donors, whereas V(max) was on average up to 2.8-fold greater in donor HU4122. Consistency in K(m, u) values allowed extrapolation of single concentration uptake activity data and assessment of interindividual variability in CL(active) across donors. The maximal contribution of active transport to total uptake differed among donors, for example, 85 to 96% and 68 to 87% for rosuvastatin and repaglinide, respectively; however, in all cases the active process was the major contributor. In vitro-in vivo extrapolation indicated a general underprediction of hepatic intrinsic clearance, an average empirical scaling factor of 17.1 was estimated on the basis of seven drugs investigated in three hepatocyte donors, and donor-specific differences in empirical factors are discussed. Uptake K(m, u) and CL(active, u) were on average 4.3- and 7.1-fold lower in human hepatocytes compared with our previously published rat data. A strategy for the use of rat uptake data to facilitate the experimental design in human hepatocytes is discussed.

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 K(m) 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.

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.

Clearance-dependent underprediction of in vivo intrinsic clearance from human hepatocytes: comparison with permeabilities from artificial membrane (PAMPA) assay, in silico and caco-2 assay, for 65 drugs.

Underprediction of in vivo intrinsic clearance (CLint) from suspended human hepatocytes has recently been shown to be clearance-dependent although the mechanistic basis is currently unknown. One possible explanation is rate limiting transmembrane (passive) permeation into hepatocytes in vitro; evidence to support this has been minor to date, but there has not been a systematic exploration of the impact of passive permeability in vitro. To investigate the relationship between underprediction of in vivo CLint and potentially rate limiting permeability, permeability constants (Px, collated from published studies and determined experimentally in this study), using three alternative methodologies (parallel artificial membrane permeability assay (PAMPA), caco-2 permeability assay and calculated using an empirical model) were compared with CLint from suspended human hepatocytes for 65 drugs from a recently reported database of clearance predictions. Although there was an approximate correspondence between hepatocyte CLint and permeability measured by PAMPA (but not by caco-2 or modelling), prediction accuracy was not dependent on the relative permeability (measured as the ratio of CLint to permeability), indicating the absence of a general rate limitation by passive hepatocyte uptake on metabolic clearance. Further investigation into rate-dependent CLint in hepatocytes is required.

Comparison of cryopreserved HepaRG cells with cryopreserved human hepatocytes for prediction of clearance for 26 drugs.

Prediction of clearance in drug discovery currently relies on human primary hepatocytes, which can vary widely in drug-metabolizing enzyme activity. Potential alternative in vitro models include the HepaRG cell (from immortalized hepatoma cells), which in culture can express drug-metabolizing enzymes to an extent comparable to that of primary hepatocytes. Utility of the HepaRG cell will depend on robust performance, relative to that of primary hepatocytes, in routine high-throughput analysis. In this study, we compared intrinsic clearance (CL(int)) in the recently developed cryopreserved HepaRG cell system with CL(int) in human cryopreserved pooled hepatocytes and with CL(int) in vivo for 26 cytochrome P450 substrate drugs. There was quantitative agreement between CL(int) in HepaRG cells and human hepatocytes, which was linear throughout the range of CL(int) (1-2000 ml · min(-1) · kg(-1)) and not dependent on particular cytochrome P450 involvement. Prediction of CL(int) in HepaRG cells was on average within 2-fold of in vivo CL(int) (using the well stirred liver model), but average fold error was clearance-dependent with greater underprediction (up to at least 5-fold) for the more highly cleared drugs. Recent reporting of this phenomenon in human hepatocytes was therefore confirmed with the hepatocytes used in this study, and hence the HepaRG cell system appears to share an apparently general tendency of clearance-limited CL(int) in cell models. This study shows the cryopreserved HepaRG cell system to be quantitatively comparable to human hepatocytes for prediction of clearance of drug cytochrome P450 substrates and to represent a promising alternative in vitro tool.

Metabolite formation kinetics and intrinsic clearance of phenacetin, tolbutamide, alprazolam, and midazolam in adenoviral cytochrome P450-transfected HepG2 cells and comparison with hepatocytes and in vivo.

Cryopreserved human hepatocytes and other in vitro systems often underpredict in vivo intrinsic clearance (CL(int)). The aim of this study was to explore the potential utility of HepG2 cells transduced with adenovirus vectors expressing a single cytochrome P450 enzyme (Ad-CYP1A2, Ad-CYP2C9, or Ad-CYP3A4) for metabolic clearance predictions. The kinetics of metabolite formation from phenacetin, tolbutamide, and alprazolam and midazolam, selected as substrates probes for CYP1A2, CYP2C9, and CYP3A4, respectively, were characterized in this in vitro system. The magnitude of the K(m) or S(50) values observed in Ad-P450 cells was similar to those found in the literature for other human liver-derived systems. For each substrate, CL(int) (or CL(max)), values from Ad-P450 systems were scaled to human hepatocytes in primary culture using the relative activity factor (RAF) approach. Scaled Ad-P450 CL(int) values were approximately 3- to 6-fold higher (for phenacetin O-deethylation, tolbutamide 4-hydroxylation, and alprazolam 4-hydroxyaltion) or lower (midazolam 1'-hydroxylation) than those reported for human cryopreserved hepatocytes in suspension. Comparison with the in vivo data reveals that Ad-P450 cells provide a favorable prediction of CL(int) for the substrates studied (in a range of 20-200% in vivo observed CL(int)). This is an improvement compared with the consistent underpredictions (<10-50% in in vivo observed CL(int)) found in cryopreserved hepatocyte studies with the same substrates. These results suggest that the Ad-P450 cell is a promising in vitro system for clearance predictions of P450-metabolized drugs.

Multiple inhibition mechanisms and prediction of drug-drug interactions: status of metabolism and transporter models as exemplified by gemfibrozil-drug interactions.

PURPOSE: To assess the consequences of multiple inhibitors and differential inhibition mechanisms on the prediction of 12 gemfibrozil drug-drug interactions (DDIs). In addition, qualitative zoning of transporter-related gemfibrozil and cyclosporine DDIs was investigated. METHODS: The effect of gemfibrozil and its acyl-glucuronide on different enzymes was incorporated into a metabolic prediction model. The impact of CYP2C8 time-dependent inhibition by gemfibrozil acyl-glucuronide was assessed using repaglinide, cerivastatin, loperamide, rosiglitazone and pioglitazone DDIs. Gemfibrozil and cyclosporine inhibition data obtained in human embryonic kidney cells expressing OATP1B1 and hepatic input concentration ([I]in) were used for qualitative zoning of 14 transporter-mediated DDIs. RESULTS: Incorporation of time-dependent inhibition by gemfibrozil glucuronide showed no significant improvement in the prediction, as CYP2C8 contributed <65% to the overall elimination of the victim drugs investigated. Qualitative zoning of OATP1B1 DDIs resulted in no false negative predictions; yet the magnitude of observed interactions was significantly over-predicted. CONCLUSIONS: Time-dependent inhibition by gemfibrozil glucuronide is only important for victim drugs eliminated predominantly (>80%) via CYP2C8. Qualitative zoning of OATP1B1 inhibitors based on [I]in/K (i) is valid in drug screening to avoid false negatives. Refinement of the transporter model by incorporating the fraction of drug transported by a particular transporter is recommended.

Scaling factors for the extrapolation of in vivo metabolic drug clearance from in vitro data: reaching a consensus on values of human microsomal protein and hepatocellularity per gram of liver.

Reported predictions of human in vivo hepatic clearance from in vitro data have used a variety of values for the scaling factors human microsomal protein (MPPGL) and hepatocellularity (HPGL) per gram of liver, generally with no consideration of the extent of their inter-individual variability. We have collated and analysed data from a number of sources, to provide weighted meangeo values of human MPPGL and HPGL of 32 mg g-1 (95% Confidence Interval (CI); 29-34 mg.g-1) and 99x10(6) cells.g-1 (95% CI; 74-131 mg.g-1), respectively. Although inter-individual variability in values of MPPGL and HPGL was statistically significant, gender, smoking or alcohol consumption could not be detected as significant covariates by multiple linear regression. However, there was a weak but statistically significant inverse relationship between age and both MPPGL and HPGL. These findings indicate the importance of considering differences between study populations when forecasting in vivo pharmacokinetic behaviour. Typical clinical pharmacology studies, particularly in early drug development, use young, fit, healthy male subjects of around 30 years of age. In contrast, the average age of patients for many diseases is about 60 years of age. The relationship between age and MPPGL observed in this study estimates values of 40 mg.g-1 for a 30 year old individual and 31 mg.g-1 for a 60 year old individual. Investigators may wish to consider the reported covariates in the selection of scaling factors appropriate for the population in which estimates of clearance are being predicted. Further studies are required to clarify the influence of age (especially in paediatric subjects), donor source and ethnicity on values of MPPGL and HPGL. In the meantime, we recommend that the estimates (and their variances) from the current meta-analysis be used when predicting in vivo kinetic parameters from in vitro data.

Multisite kinetic analysis of interactions between prototypical CYP3A4 subgroup substrates: midazolam, testosterone, and nifedipine.

The potential of substrates and modifiers of CYP3A4 to show differential effects, attributed to the existence of multiple binding sites, confounds the straightforward prediction of in vivo drug-drug interactions from in vitro data. A set of in vitro interaction studies was performed in human lymphoblast-expressed CYP3A4 involving representatives of two CYP3A4 subclasses, midazolam (MDZ) and testosterone (TST); a distinct subgroup, nifedipine (NIF); and its structural analog, felodipine (FEL). Mechanistic insight into the interaction of each pair of substrates was provided by employing a range of multisite kinetic models; most were subtypes of a generic two-site model, but a three-site model was required for TST interactions. The complexity of the inhibition profiles and the selection of the kinetic model with appropriate interaction factors were dependent upon the kinetics of substrates involved (hyperbolic, substrate inhibition, or sigmoidal for MDZ/FEL, NIF, and TST, respectively). In no case was a simple reciprocity seen between pairs of substrates. The interaction profiles observed between TST, MDZ, NIF, and FEL involved several atypical inhibition features (partial, cooperative, concentration-dependent loss of characteristic homotropic behavior) and pathway-differential effects reflecting an 80-fold difference in Ki values and a delta factor (defining the alteration in the binding affinity in the presence of a modifier) ranging from 0.04 to 2.3. The conclusions from the multisite kinetic analysis performed support the hypothesis of distinct binding domains for each substrate subgroup. Furthermore, the analysis of intersubstrate interactions strongly indicates the existence of a mutual binding domain common to each of the three CYP3A4 substrate subclasses.