Exploring the Boundaries for In Vitro-In Vivo Extrapolation: Use of Isolated Rat Hepatocytes in Co-culture and Impact of Albumin Binding Properties in the Prediction of Clearance of Various Drug Types.

Prediction of hepatic clearance of drugs (via uptake or metabolism) from in vitro systems continues to be problematic, particularly when plasma protein binding is high. The following work explores simultaneous assessment of both clearance processes, focusing on a commercial hepatocyte-fibroblast co-culture system (HµREL) over a 24-hour period using six probe drugs (ranging in metabolic and transporter clearance and low-to-high plasma protein binding). A rat hepatocyte co-culture assay was established using drug depletion (measuring both medium and total concentrations) and cell uptake kinetic analysis, both in the presence and absence of plasma protein (1% bovine serum albumin). Secretion of endogenous albumin was monitored as a marker of viability, and this reached 0.004% in incubations (at a rate similar to in vivo synthesis). Binding to stromal cells was substantial and required appropriate correction factors. Drug concentration-time courses were analyzed both by conventional methods and a mechanistic cell model prior to in vivo extrapolation. Clearance assayed by drug depletion in conventional suspended rat hepatocytes provided a benchmark to evaluate co-culture value. Addition of albumin appeared to improve predictions for some compounds (where fraction unbound in the medium is less than 0.1); however, for high-binding drugs, albumin significantly limited quantification and thus predictions. Overall, these results highlight ongoing challenges concerning in vitro hepatocyte system complexity and limitations of practical expediency. Considering this, more reliable measurement of hepatically cleared compounds seems possible through judicious use of available hepatocyte systems, including co-culture systems, as described herein; this would include those compounds with low metabolic turnover but high active uptake clearance. SIGNIFICANCE STATEMENT: Co-culture systems offer a more advanced tool than standard hepatocytes, with the ability to be cultured for longer periods of time, yet their potential as an in vitro tool has not been extensively assessed. We evaluate the strengths and limitations of the HµREL system using six drugs representing various metabolic and transporter-mediated clearance pathways with various degrees of albumin binding. Studies in the presence/absence of albumin allow in vitro-in vivo extrapolation and a framework to maximize their utility.

Importance of the Unstirred Water Layer and Hepatocyte Membrane Integrity In Vitro for Quantification of Intrinsic Metabolic Clearance.

Prediction of clearance-a vital component of drug discovery-remains in need of improvement and, in particular, requires more incisive assessment of mechanistic methodology in vitro, according to a number of recent reports. Although isolated hepatocytes have become an irreplaceable standard system for the measurement of intrinsic hepatic clearance mediated by active uptake transport and metabolism, the lack of prediction reliability appears to reflect a lack of methodological validation, especially for highly cleared drugs, as we have previously shown. Here, novel approaches were employed to explore fundamental experimental processes and associated potential limitations of in vitro predictions of clearance. Rat hepatocytes deemed nonviable by trypan blue staining showed undiminished metabolic activity for probe cytochrome P450 (P450) substrates midazolam and propranolol; supplementation with NADPH enhanced these activities. Extensive permeabilization of the plasma membrane using saponin showed either full or minimal P450 activity, depending on the presence or absence of 1 mM NADPH, respectively. The shaking of incubations facilitated P450 metabolic rates up to 5-fold greater than static incubation, depending on intrinsic clearance, indicating the critical influence of the unstirred water layer (UWL). Permeabilization allowed static incubation metabolic rates to approach those of shaking for intact cells, indicating an artificially induced breakdown of the UWL. Permeabilization combined with shaking allowed an increased metabolic rate for saquinavir, resolving the membrane permeability limitation for this drug. These findings advance the interpretation of the rate-limiting processes involved in intrinsic clearance measurements and could be critical for successful in vitro prediction.

Characterization of the comparative drug binding to intra- (liver fatty acid binding protein) and extra- (human serum albumin) cellular proteins.

1. This study compared the extent, affinity, and kinetics of drug binding to human serum albumin (HSA) and liver fatty acid binding protein (LFABP) using ultrafiltration and surface plasmon resonance (SPR). 2. Binding of basic and neutral drugs to both HSA and LFABP was typically negligible. Binding of acidic drugs ranged from minor (fu > 0.8) to extensive (fu < 0.1). Of the compounds screened, the highest binding to both HSA and LFABP was observed for the acidic drugs torsemide and sulfinpyrazone, and for ß-estradiol (a polar, neutral compound). 3. The extent of binding of acidic drugs to HSA was up to 40% greater than binding to LFABP. SPR experiments demonstrated comparable kinetics and affinity for the binding of representative acidic drugs (naproxen, sulfinpyrazone, and torsemide) to HSA and LFABP. 4. Simulations based on in vitro kinetic constants derived from SPR experiments and a rapid equilibrium model were undertaken to examine the impact of binding characteristics on compartmental drug distribution. Simulations provided mechanistic confirmation that equilibration of intracellular unbound drug with the extracellular unbound drug is attained rapidly in the absence of active transport mechanisms for drugs bound moderately or extensively to HSA and LFABP.

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.

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.

Comparison of intrinsic clearances in human liver microsomes and suspended hepatocytes from the same donor livers: clearance-dependent relationship and implications for prediction of in vivo clearance.

Intrinsic clearance (CL(int)) of seven probe cytochrome P450 substrates, across a wide range of clearance, was compared in microsomes and cryopreserved hepatocytes from the same four livers. Previous comparisons have shown system dependence, but using preparations from different donor livers. Four-fold average underprediction of microsomal CL(int) by hepatocytes (scaled to whole liver) for high clearance substrates (midazolam, nifedipine, and diclofenac) was observed with relatively unbiased prediction (within 1.5-fold average) for the low/medium clearance substrates (tolbutamide, alprazolam, bufuralol, and triazolam). CL(int) of midazolam and nifedipine corresponded between livers over a tenfold range, but the absolute ranges were lower for hepatocytes, indicating independence of hepatocyte bias from substrate. In contrast, the absolute ranges of CL(int) for the low clearance CYP3A4 substrate alprazolam were similar between the systems, indicating independence of hepatocyte bias from enzyme. The trend in CL(int) between the systems was similar to that in a dataset of published CL(int) for 46 substrates in microsomes and hepatocytes (unrelated liver sources), supporting a fundamental rate limitation of the hepatocyte system. A tendency of decreasing V(max) in hepatocytes relative to microsomes, with increasing clearance, suggests that a capacity limitation, such as cofactor rate limitation, may be involved in this phenomenon.

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.

Prediction of human metabolic clearance from in vitro systems: retrospective analysis and prospective view.

PURPOSE: To provide a definitive assessment of prediction of in vivo CL (int) from human liver in vitro systems for assessment of typical underprediction. METHODS: A database of published predictions of clearance from human hepatocytes and liver microsomes was compiled, including only intravenous CL (b). The influence of liver model (well-stirred (WS) or parallel tube (PT)), plasma protein binding and clearance level on the relationship between in vitro and in vivo CL (int) was examined. RESULTS: Average prediction bias was about 5- and 4-fold for microsomes and hepatocytes, respectively. Reduced bias using the PT model, in preference to the popular WS model, was only marginal across a wide range of clearance with a consequential minor impact on prediction. Increasing underprediction with decreasing fu (b), or increasing CL (int), was found only for hepatocytes, suggesting fundamental in vitro artefacts rather than failure to model potentially unequilibrated binding during rapid extraction. CONCLUSIONS: In contrast to microsomes, hepatocytes give a disproportionate prediction with increasing clearance suggesting limitations either at the active site, such as cofactor exhaustion, or with intracellular concentration equilibrium, such as rate-limiting cell permeability. A simple log linear empirical relationship can be used to correct hepatocyte predictions.

Utilising Magnetically Isolated Lysosomes for Direct Quantification of Intralysosomal Drug Concentrations by LC-MS/MS Analysis: An Investigatory Study With Imipramine.

Lysosomes are acidic intracellular organelles that can extensively sequester basic lipophilic drugs due to pH and membrane partitioning, and therefore may significantly influence subcellular drug concentrations. Current in vitro methods for lysosomal drug sequestration evaluation typically lack the ability to accurately and sensitively quantify drug concentrations directly within the lysosome. In the current study, magnetic lysosomal isolation was used in the lysosome rich rat NR8383 cell line and combined with LC-MS/MS analysis to quantify intralysosomal concentrations and lysosomal partitioning (KpLysosome) values of imipramine. The purity of the isolated lysosomes was validated by enzymatic and electron microscopy analysis. Lysosomal imipramine accumulation was explored using 2 methods: addition of imipramine to cells followed by lysosomal isolation (Method 1), and direct addition of imipramine to isolated lysosomes (Method 2). This work highlighted that both experimental buffers and ATP influence intralysosomal drug concentrations, and non-specific drug binding and re-distribution limits the use of Method 1. Method 2 may benefit future lysosomal drug accumulation studies, as imipramine demonstrated high KpLysosome values (3500), comparable to in silico predictions. This study reports a novel method for the direct quantification of intralysosomal drug concentrations that has the ability to be adapted to other cell types.

Prediction of in vivo drug-drug interactions from in vitro data : factors affecting prototypic drug-drug interactions involving CYP2C9, CYP2D6 and CYP3A4.

BACKGROUND: Quantitative predictions of in vivo drug-drug interactions (DDIs) resulting from metabolic inhibition are commonly made based upon the inhibitor concentration at the enzyme active site [I] and the in vitro inhibition constant (K(i)). Previous studies have involved the use of various plasma inhibitor concentrations as surrogates for [I] along with K(i) values obtained from published literature. Although this approach has resulted in a high proportion of successful predictions, a number of falsely predicted interactions are also observed. OBJECTIVES: To focus on three issues that may influence the predictive value of the [I]/K(i) ratio approach: (i) the use of unbound K(i) (K(i,u)) values generated from standardised in vitro experiments compared with literature values; (ii) the selection of an appropriate [I]; and (iii) incorporation of the impact of intestinal metabolic inhibition for cytochrome P450 (CYP) 3A4 predictions. To this end we have selected eight inhibitors of CYP2C9, CYP2D6 and CYP3A4 and 18 victim drugs from a previous database analysis to allow prediction of 45 clinical DDI studies. METHODS: In vitro kinetic and inhibition studies were performed in human liver microsomes using prototypic probe substrates of CYP2C9 and CYP2D6, with various inhibitors (miconazole, sulfaphenazole, fluconazole, ketoconazole, quinidine, fluoxetine, fluvoxamine). The K(i) estimates obtained were corrected for non-specific microsomal binding, and the K(i,u) was incorporated into in vivo predictions using various [I] values. Predictions for CYP3A4 were based upon in vitro data obtained from a previous publication within our laboratory, and an assessment of the impact of the interaction in the gut wall is included. Predictions were validated against 45 in vivo studies and those within 2-fold of the in vivo ratio of area under the plasma concentration-time curve of the substrate, in the presence and absence of the inhibitor (AUC(i)/AUC) were considered successful. RESULTS: Predictions based upon the average systemic total plasma drug concentration ([I](av)) [incorporating the effects of parallel drug elimination pathways] and the K(i,u) value resulted in 91% of studies predicted to within 2-fold of the in vivo AUC(i)/AUC. This represents a 35% improvement in prediction accuracy compared with predictions based upon total K(i) values obtained from various published literature sources. A corresponding reduction in bias and an increase in precision were also observed compared with the use of other [I] surrogates (e.g. the total and new unbound maximum hepatic input plasma concentrations). No significant improvement in prediction accuracy was observed by incorporating consideration of gut wall inhibition for CYP3A4. CONCLUSION: DDI predictions based upon the use of K(i,u) data obtained under a set of optimal standardised conditions were significantly improved compared with predictions using in vitro data collated from various sources. The use of [I](av) as the [I] surrogate generated the most successful predictions as judged by several criteria. Incorporation of either plasma protein binding of inhibitor or gut wall CYP3A4 inhibition did not result in a general improvement of DDI predictions.

Evaluation of hepatic clearance prediction using in vitro data: emphasis on fraction unbound in plasma and drug ionisation using a database of 107 drugs.

Underprediction of in vivo intrinsic clearance (CL(int)) of unbound drug from human hepatic in vitro systems using physiological extrapolation methodology is accepted as a common outcome. Poulin et al. (2012. J Pharm Sci 101:838-851) recently proposed an approach involving determination of effective fraction unbound in plasma (fu(p)) based on albumin-facilitated hepatic uptake of acidic/neutral drugs which improved prediction accuracy and precision for 25 drugs highly bound to plasma proteins. This approach includes correction of unbound drug according to the ionisation fraction either side of the plasma membrane based on pH difference. Here, we assessed the proposed method using a larger database of predictions of CL(int) for 107 drugs involving hepatocytes (89 drugs) and microsomes (64 drugs). The proposed method was similarly effective in minimising average prediction bias (to within twofold), unlike the conventional fu(p) correction method. However, precision was similar between methods and there was no evidence in the larger database that prediction bias was associated with fu(p). Prediction bias for hepatocytes was clearance dependent by either method, indicating important sources of bias from in vitro methodology. Therefore, to progress beyond empirical correction of bias, there is further need of mechanistic elucidation to improve prediction methodology.

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.

Saturable uptake of lipophilic amine drugs into isolated hepatocytes: mechanisms and consequences for quantitative clearance prediction.

The hepatic uptake of quinine, fluvoxamine, and fluoxetine (0.1-10 microM) was investigated with freshly isolated rat hepatocytes. The cell-to-medium concentration ratios (K(p)) were concentration-dependent: the mean maximum K(p) values (at 0.1 microM) were 150 (quinine), 500 (fluvoxamine), and 2000 (fluoxetine). There was also a large capacity site that was not saturable over the concentration range used (possibly partition into the phospholipid component of membranes); representing this site, the mean minimum K(p) values (at 10 microM) were 30 (quinine), 200 (fluvoxamine), and 500 (fluoxetine). To eliminate concomitant metabolism, cells were pretreated with the irreversible P450 inhibitor, aminobenzotriazole. The saturable uptake was substantially eliminated after exposure to carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (ATP inhibitor). The difference between the maximum and minimum K(p) for these three amine drugs, as well as for dextromethorphan, propranolol, and imipramine, was within a limited range of 3-fold, indicating a common magnitude of saturable uptake. Basic, permeable drugs are expected to be sequestered into lysosomes, which actively maintain their low internal pH (approximately 5) using ATP, and this process is predictable from the combined effects of pH-driven ion accumulation and unsaturable binding representing partition into membranes. The resultant predicted maximum K(p) correlated strongly with the observed maximum K(p). Thus, at low substrate concentrations, the fraction of drug unbound in the hepatocyte incubation (critical for assessing drug clearance and drug-drug interaction potential) may be dependent upon saturable as well as unsaturable binding, and for lipophilic, basic drugs, this can be readily estimated assuming a common degree of uptake into lysosomes.

Primary hepatocytes: current understanding of the regulation of metabolic enzymes and transporter proteins, and pharmaceutical practice for the use of hepatocytes in metabolism, enzyme induction, transporter, clearance, and hepatotoxicity studies.

This review brings you up-to-date with the hepatocyte research on: 1) in vitro-in vivo correlations of metabolism and clearance; 2) CYP enzyme induction, regulation, and cross-talk using human hepatocytes and hepatocyte-like cell lines; 3) the function and regulation of hepatic transporters and models used to elucidate their role in drug clearance; 4) mechanisms and examples of idiosyncratic and intrinsic hepatotoxicity; and 5) alternative cell systems to primary human hepatocytes. We also report pharmaceutical perspectives of these topics and compare methods and interpretations for the drug development process.

Uptake and intracellular binding of lipophilic amine drugs by isolated rat hepatocytes and implications for prediction of in vivo metabolic clearance.

The hepatic uptake of imipramine and propranolol was investigated after incubation with isolated rat hepatocytes over a wide concentration range (0.04-400 microM). The cell-to-medium concentration ratio (K(p)) was concentration-dependent and could be described using a two-site binding model incorporating a high affinity/low capacity site and a linear component for a site which was apparently not saturated. Maximum (at 0.04 microM) and minimum K(p) values (at 400 microM) were 360 and 280, and 110 and 70 for imipramine and propranolol, respectively. During these incubations, metabolism was inhibited using aminobenzotriazole (an irreversible inhibitor of cytochrome P450). Pretreatment of cells either by freeze-thawing or with saponin (which permeabilizes the plasma membrane) eliminated the saturable process. The saturable uptake process of imipramine was also inhibited by 18 other lipophilic amine drugs (including propranolol). This uptake component may involve membrane transporter(s), whereas the nonsaturable component probably represents partition into the phospholipid component of membranes. Propranolol was further investigated to determine the impact of high K(p) values on hepatocellular clearance. The area under the curve for propranolol concentrations in the total incubate (medium including the cells) from the depletiontime profile was substantially greater than the corresponding area under the curve for the drug concentration in the extracellular medium, and this difference approximated the nonsaturable uptake component. It is concluded that the clearance of propranolol in isolated hepatocytes is not rate-limited by hepatic uptake and is directly proportional to unbound drug concentration, being independent of the higher K(p) value.

CYP3A4 substrate selection and substitution in the prediction of potential drug-drug interactions.

The complexity of in vitro kinetic phenomena observed for CYP3A4 substrates (homo- or heterotropic cooperativity) confounds the prediction of drug-drug interactions, and an evaluation of alternative and/or pragmatic approaches and substrates is needed. The current study focused on the utility of the three most commonly used CYP3A4 in vitro probes for the prediction of 26 reported in vivo interactions with azole inhibitors (increase in area under the curve ranged from 1.2 to 24, 50% in the range of potent inhibition). In addition to midazolam, testosterone, and nifedipine, quinidine was explored as a more "pragmatic" substrate due to its kinetic properties and specificity toward CYP3A4 in comparison with CYP3A5. Ki estimates obtained in human liver microsomes under standardized in vitro conditions for each of the four probes were used to determine the validity of substrate substitution in CYP3A4 drug-drug interaction prediction. Detailed inhibitor-related (microsomal binding, depletion over incubation time) and substrate-related factors (cooperativity, contribution of other metabolic pathways, or renal excretion) were incorporated in the assessment of the interaction potential. All four CYP3A4 probes predicted 69 to 81% of the interactions with azoles within 2-fold of the mean in vivo value. Comparison of simple and multisite mechanistic models and interaction prediction accuracy for each of the in vitro probes indicated that midazolam and quinidine in vitro data provided the best assessment of a potential interaction, with the lowest bias and the highest precision of the prediction. Further investigations with a wider range of inhibitors are required to substantiate these findings.

Impact of parallel pathways of drug elimination and multiple cytochrome P450 involvement on drug-drug interactions: CYP2D6 paradigm.

The success of in vitro derived Ki values for predicting drug-drug interactions in vivo has been mixed. For example, the use of hepatic input concentration of inhibitor has resolved the negative and positive interactions on the qualitative level, eliminating false negative predictions. However, several examples of false positives and a high incidence of over-predictions of true positive interactions indicated a need for incorporation of additional factors. The aim of this study was to investigate the effect of parallel elimination pathways as a possible reason for false positives and over-predictions. Simulation studies indicated that the degree of interaction (assessed by area under the plasma concentration-time curve ratio in the presence and absence of inhibitor) depends largely on the fraction of substrate metabolized by the particular P450 enzyme (fmCYPi) that is inhibited. The current analysis focused on CYP2D6 interactions due to the well documented genetic polymorphism and the ability to estimate fmCYP2D6 readily from in vivo data obtained in extensive and poor metabolizers. Based on either a phenotype study or an alternative regression analysis approach, the fmCYP2D6 values of 0.37 to 0.94 and 0.25 to 0.89, respectively, were obtained for nine substrates. Prediction of 44 drug-drug interaction studies was improved by the combination of parallel pathways of elimination and their susceptibility to inhibition. The overall success of predicting positive and negative interactions was increased from 54% to 84%, and the number of over-predictions was substantially reduced. It is concluded that incorporating parallel pathways provides a valuable step forward in making quantitative predictions of drug-drug interactions from in vitro data.

Prediction of metabolic clearance using cryopreserved human hepatocytes: kinetic characteristics for five benzodiazepines.

Predictions of intrinsic clearance (CL(int)) from human liver microsomes often underestimate in vivo observations. In this study, a series of five benzodiazepines was used as prototypic CYP3A4 substrates to investigate the prediction of clearance from the less studied alternative in vitro system, cryopreserved human hepatocytes. Formation of the two major metabolites from midazolam, triazolam, diazepam, flunitrazepam, and alprazolam was measured in cryopreserved human hepatocytes from five donors; the kinetics were characterized and CL(int) values were determined and scaled to predict CL(int) in vivo. At least one of the two major pathways of metabolic clearance of each benzodiazepine was characterized by autoactivation in hepatocytes; the extent to which this occurred varied depending on substrate and liver (up to 8-fold). Heteroactivation by testosterone of these pathways was also observed (up to 6-fold). The maximum autoactivated clearance was used to predict in vivo CL(int) (1.6-138 ml/min/kg) which closely agreed with values previously obtained using human liver microsomes. Comparison with in vivo CL(int) indicates that cryopreserved human hepatocytes systematically underpredict CL(int). When three previous studies (documenting CL(int) for substrates of various enzymes in cryopreserved human hepatocytes using drug depletion-time profiles) were considered as well, the combined data show a consistent underprediction of 5.6-fold. Collectively it is demonstrated that the predicted hepatic intrinsic clearances from cryopreserved hepatocytes show an excellent rank order with in vivo findings but are systematically underpredicting the in vivo value.

Quantitative prediction of the in vivo inhibition of diazepam metabolism by omeprazole using rat liver microsomes and hepatocytes.

The diazepam (DZ)-omeprazole (OMP) interaction has been selected as a prototype for an important drug-drug interaction involving cytochrome P450 inhibition. The availability of an in vivo K(i) value (unbound K(i), 21 microM) obtained from a series of steady-state inhibitor infusion studies allowed assessment of several in vitro-derived predictions of this inhibition interaction. Studies monitoring substrate depletion with time were used to obtain in vitro K(i) values that were evaluated against the more traditional metabolite formation approach using microsomes and hepatocytes. OMP inhibited the metabolism of DZ to its primary metabolites 4'-hydroxydiazepam, 3-hydroxydiazepam, and nordiazepam to different extents over a range of concentrations (0.3-150 microM), and a competitive inhibition model best fitted the data. The K(i) values observed using the substrate depletion approach (16 +/- 3 microM and 7 +/- 2 microM in microsomes and hepatocytes, respectively) were in good agreement with the overall weighted K(i) values obtained using the standard metabolite formation approach (12 +/- 2 microM and 16 +/- 5 microM in microsomes and hepatocytes, respectively). In vitro binding and cell uptake studies as well as human serum albumin studies in hepatocytes confirmed the importance of both intracellular and extracellular unbound concentrations of inhibitor when considering inhibition predictions. Both kinetic approaches and both in vitro systems predicted the in vivo interaction well and provide a good example of the ability of in vitro inhibition studies to quantitatively predict an in vivo drug-drug interaction successfully.

Predicting P-glycoprotein effects on oral absorption: correlation of transport in Caco-2 with drug pharmacokinetics in wild-type and mdr1a(-/-) mice in vivo.

PURPOSE: Cell-based permeability screens are widely used to identify drug-P-glycoprotein (PGP) interaction in vitro. However, their reliability in predicting the impact of PGP on human drug pharmacokinetics is poorly defined. The aim was to determine whether a quantitative relationship exists between PGP-mediated alterations in Caco-2 permeability and oral pharmacokinetics in mice. METHODS: Two indicators of drug efflux were measured in Caco-2 for a group of 10 compounds, the ratio of A-B and B-A transport (R9B-A/A-B)) and the ratio of A-B transport in the presence and absence of a PGP inhibitor, GF120918 (R(GF)). These data were correlated with ratios of oral plasma levels in either mdr1a(-/-) or mdr1a/1b(-/-) and wild-type mice (R(KO/WT in vivo)) calculated from literature data on these compounds. RESULTS: A significant, positive correlation (r2 = 0.8, p < 0.01) was observed between RGF and R(KO/WT in vivo). In contrast, R(B-A/A-B), a more commonly used in vitro measure, showed a much weaker correlation with in vivo data (r2 = 0.33, p = 0.11). A strong correlation with R(GF) was also observed after correction of in vivo data for PGP effects on IV clearance. CONCLUSION: The increase in A-B drug permeability following inhibition of PGP in Caco-2 allows a reasonable prediction of the likely in vivo impact that PGP will have on plasma drug levels after oral administration.