Michael A. Mohutsky

Predictions of the In Vivo Clearance of Drugs from Rate of Loss Using Human Liver Microsomes for Phase I and Phase II Biotransformations

PurposeThe utility of in vitro metabolism to accurately predict the clearance of hepatically metabolized drugs was evaluated. Three major goals were: (1) to optimize substrate concentration for the accurate prediction of clearance by comparing to Km value, (2) to prove that clearance of drugs by both oxidation and glucuronidation may be predicted by this method, and (3) to determine the effects of nonspecific microsomal binding and plasma protein binding.MethodsThe apparent Km values for five compounds along with scaled intrinsic clearances and predicted hepatic clearances for eight compounds were determined using a substrate loss method. Nonspecific binding to both plasma and microsomal matrices were also examined in the clearance calculations.ResultsThe Km values were well within the 2-fold variability expected for between laboratory comparisons. Using both phase I and/or phase II glucuronidation incubation conditions, the predictions of in vivo clearance using the substrate loss method were shown to correlate with published human clearance values. Of particular interest, for highly bound drugs (>95% plasma protein bound), the addition of a plasma protein binding term increased the accuracy of the prediction of in vivo clearance.ConclusionsThe substrate loss method may be used to accurately predict hepatic clearance of drugs.

Prediction of CYP3A Mediated Drug-Drug Interactions Using Human Hepatocytes Suspended in Human Plasma

Cryopreserved human hepatocytes suspended in human plasma (HHSHP) represent an integrated metabolic environment for predicting drug-drug interactions (DDIs). In this study, 13 CYP3A reversible and/or time-dependent inhibitors (TDIs) were incubated with HHSHP for 20 min over a range of concentrations after which midazolam 1′-hydroxylation was used to measure CYP3A activity. This single incubation time method yielded IC50 values for the 13 inhibitors. For each CYP3A inhibitor-victim drug pair, the IC50 value was combined with total average plasma concentration of the inhibitor in humans, fraction of the victim drug cleared by CYP3A, and intestinal availability of the victim drug to predict the ratio of plasma area under the curve of the victim drug in the presence and absence of inhibitor. Of 52 clinical DDI studies using these 13 inhibitors identified in the literature, 85% were predicted by this method within 2-fold of the observed change, and all were predicted within 3-fold. Subsequent studies to determine mechanism (reversible and time-dependent inhibitors) were performed by using a range of incubation periods and inhibitor concentrations. This system differentiated among reversible inhibitors, TDIs, and the combination of both. When the reversible and inactivation parameters were incorporated into predictive models, 65% of 52 clinical DDIs were predicted within 2-fold of the observed changes and 88% were within 3-fold. Thus, HHSHP produced accurate DDI predictions with a simple IC50 determined at a single incubation time regardless of the inhibition mechanism; further if needed, the mechanism(s) of inhibition can be identified.

Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1α,25-dihydroxyvitamin D3 conjugation

The biological effects of 1alpha,25-dihydroxyvitamin D3 (1,25(OH)2D3) are terminated primarily by P450-dependent hydroxylation reactions. However, the hormone is also conjugated in the liver and a metabolite, presumably a glucuronide, undergoes enterohepatic cycling. In this study, the identity of human enzymes capable of catalyzing the 1,25(OH)2D3 glucuronidation reaction was investigated in order to better understand environmental and endogenous factors affecting the disposition and biological effects of vitamin D3. Among 12 different UGT isozymes tested, only UGT1A4 >> 2B4 and 2B7 supported the reaction. Two different 1,25(OH)2D3 monoglucuronide metabolites were generated by recombinant UGT1A4 and human liver microsomes. The most abundant product was identified by mass spectral and NMR analyses as the 25-O-glucuronide isomer. The formation of 25-O-glucuronide by UGT1A4 Supersomes and human liver microsomes followed simple hyperbolic kinetics, yielding respective Km and Vmax values of 7.3 and 11.2 microM and 33.7 +/- 1.4 and 32.9 +/- 1.9 pmol/min/mg protein. The calculated intrinsic 25-O-glucuronide M1 formation clearance for UGT1A4 was 14-fold higher than the next best isozyme, UGT2B7. There was only limited (four-fold) inter-liver variability in the 25-O-glucuronidation rate, but it was highly correlated with the relative rate of formation of the second, minor metabolite. In addition, formation of both metabolites was inhibited >80% by the selective UGT1A4 inhibitor, hecogenin. If enterohepatic recycling of 1,25(OH)2D3 represents a significant component of intestinal and systemic 1,25(OH)2D3 disposition, formation of monoglucuronides by hepatic UGT1A4 constitutes an important initial step.

Predictions of Cytochrome P450-Mediated Drug-Drug Interactions Using Cryopreserved Human Hepatocytes: Comparison of Plasma and Protein-Free Media Incubation Conditions

Cryopreserved human hepatocytes suspended in human plasma (HHSHP) have previously provided accurate CYP3A drug-drug interaction (DDI) predictions from a single IC50 that captures both reversible and time-dependent inhibition. The goal of this study was to compare the accuracy of DDI predictions by a protein-free human hepatocyte system combined with the fraction unbound in plasma for inhibitor(s) with those obtained with protein-containing incubations. Seventeen CYP3A, CYP2C9, or CYP2D6 inhibitors were incubated with hepatocytes in human plasma or hepatocyte maintenance medium (HMM) for 20 min over a range of concentrations after which midazolam 1′-hydroxylation, diclofenac 4′-hydroxylation or (R)-bufuralol 1′-hydroxylation were used to quantify the corresponding cytochrome P450 (P450) catalytic activities. Two methods were used to predict the human exposure ratio of the victim drug in the presence and absence of inhibitor. The HMM Ki, app values were combined with the free average systemic plasma concentration (“free [I] with HMM Ki, app”) and the plasma Ki, app values were combined with the total average systemic plasma concentration (“total [I] with plasma Ki, app”). Of 63 clinical DDI studies, the total [I] with plasma Ki, app method predicted 89% of cases within 2-fold of the reported interaction whereas the free [I] with HMM Ki, app method predicted only 59%. There was a general underprediction by the free [I] with HMM Ki, app method, which is consistent with an underestimation of in vitro inhibition potency in this system. In conclusion, the HHSHP system proved to be a simple, accurate predictor of DDIs for three major P450s and superior to the protein-free approach.

Potentially increasing the metabolic stability of drug candidates via computational site of metabolism prediction by CYP2C9: The utility of incorporating protein flexibility via an ensemble of structures.

Cytochrome P450 enzymes are responsible for metabolizing many endogenous and xenobiotic molecules encountered by the human body. It has been estimated that 75% of all drugs are metabolized by cytochrome P450 enzymes. Thus, predicting a compounds potential sites of metabolism (SOM) is highly advantageous early in the drug development process. We have combined molecular dynamics, AutoDock Vina docking, the neighboring atom type (NAT) reactivity model, and a solvent-accessible surface-area term to form a reactivity-accessibility model capable of predicting SOM for cytochrome P450 2C9 substrates. To investigate the importance of protein flexibility during the ligand-binding process, the results of SOM prediction using a static protein structure for docking were compared to SOM prediction using multiple protein structures in ensemble docking. The results reported here indicate that ensemble docking increases the number of ligands that can be docked in a bioactive conformation (ensemble: 96%, static: 85%) but only leads to a slight improvement (49% vs. 44%) in predicting an experimentally known SOM in the top-1 position for a ligand library of 75 CYP2C9 substrates. Using ensemble docking, the reactivity-accessibility model accurately predicts SOM in the top-1 ranked position for 49% of the ligand library and considering the top-3 predicted sites increases the prediction success rate to approximately 70% of the ligand library. Further classifying the substrate library according to K(m) values leads to an improvement in SOM prediction for substrates with low K(m) values (57% at top-1). While the current predictive power of the reactivity-accessibility model still leaves significant room for improvement, the results illustrate the usefulness of this method to identify key protein-ligand interactions and guide structural modifications of the ligand to increase its metabolic stability.

Apparent High CYP3A5 Expression Is Required for Significant Metabolism of Vincristine by Human Cryopreserved Hepatocytes

Vincristine is metabolized to one primary metabolite, M1, by cDNA-expressed CYP3A4 and CYP3A5 and by CYP3A enzymes in human liver microsomes. For both systems, CYP3A5 is predicted to mediate approximately 80% of the CYP3A metabolism for individuals with high CYP3A5 expression (at least one CYP3A5*1 allele). In the current study, the role of CYP3A5 was quantified in the metabolism of vincristine with human cryopreserved hepatocytes. The hepatocytes were genotyped for common CYP3A5 allelic variants (CYP3A5*3, CYP3A5*6, and CYP3A5*7) to predict CYP3A5 expression. For each hepatocyte preparation, the rates of vincristine depletion and metabolite formation were quantified. Whereas human hepatocytes with predicted low CYP3A5 expression did not detectably metabolize vincristine, human hepatocytes with predicted high CYP3A5 expression metabolized vincristine to one primary metabolite, M1. In paired experiments using cryopreserved hepatocytes from the same donor, vincristine was incubated with intact cells and cell lysates supplemented with NADPH. The rates of M1 formation were 4 to 69-fold higher for the cell lysates compared with the intact cells. For one representative donor, the intact cells had a 3-fold higher Km value and a 3-fold lower Vmax value for M1 formation compared with the cell lysates. Thus, the rate of M1 formation in the hepatocytes may be influenced by the rate of vincristine translocation across the plasma membrane. We conclude that genetically determined CYP3A5 expression in human cryopreserved hepatocytes plays a major role in vincristine metabolism.

Hepatic Drug-Metabolizing Enzyme Induction and Implications for Preclinical and Clinical Risk Assessment

Hepatic drug metabolizing enzyme (DME) induction complicates the development of new drugs owing to altered efficacy of concomitant treatments, reduction in exposure resulting from autoinduction, and potential generation of toxic metabolites. Risk assessment of DME induction during clinical evaluation is confounded by several uncertainties pertaining to hazard identification and dose response analysis. Hepatic DME induction rarely leads to clinical evidence of altered metabolism and toxicity in the patient, which typically occur only if the DME induction is relatively severe. High drug doses are associated with a greater likelihood of hepatic DME induction and downstream effects; therefore, drugs of low potency requiring higher dosing tend to lead to a greater risk of drug–drug interactions. Vigilance in clinical trials for increased or diminished drug effect and, specifically, pharmacokinetic studies in the presence of other drugs and concomitant diseases are necessary for a drug risk assessment profile. Efforts to remove hepatic DME-inducing drugs from development can be facilitated with current in vitro and in vivo assessments and will improve with the development of newer technologies. A carefully tailored case-by-case approach will lead to the development of efficacious drugs with an acceptable risk/benefit profile available to patients.

Metabolic-intermediate complex formation with cytochrome P450: theoretical studies in elucidating the reaction pathway for the generation of reactive nitroso intermediate.

Mechanism‐based inhibition (MBI) of cytochrome P450 (CYP) can lead to drug–drug interactions and often to toxicity. Some aliphatic and aromatic amines can undergo biotransformation reactions to form reactive metabolites such as nitrosoalkanes, leading to MBI of CYPs. It has been proposed that the nitrosoalkanes coordinate with the heme iron, forming metabolic‐intermediate complex (MIC), resulting in the quasi‐irreversible inhibition of CYPs. Limited mechanistic details regarding the formation of reactive nitroso intermediate and its coordination with heme‐iron have been reported. A quantum chemical analysis was performed to elucidate potential reaction pathways for the generation of nitroso intermediate and the formation of MIC. Elucidation of the energy profile along the reaction path, identification of three‐dimensional structures of reactive intermediates and transition states, as well as charge and spin density analyses, were performed using the density functional B3LYP method. The study was performed using Cpd I [iron (IV‐oxo] heme porphine with SH− as the axial ligand) to represent the catalytic domain of CYP, simulating the biotransformation process. Three pathways: (i) N‐oxidation followed by proton shuttle, (ii) N‐oxidation followed by 1,2‐H shift, and (iii) H‐abstraction followed by rebound mechanism, were studied. It was observed that the proton shuttle pathway was more favorable over the whole reaction leading to reactive nitroso intermediate. This study revealed that the MIC formation from a primary amine is a favorable exothermic process, involving eight different steps and preferably takes place on the doublet spin surface of Cpd I. The rate‐determining step was identified to be the first N‐oxidation of primary amine.

Investigational Small-Molecule Drug Selectively Suppresses Constitutive CYP2B6 Activity at the Gene Transcription Level: Physiologically Based Pharmacokinetic Model Assessment of Clinical Drug Interaction Risk

The glycogen synthase kinase-3 inhibitor LY2090314 specifically impaired CYP2B6 activity during in vitro evaluation of cytochrome P450 (P450) enzyme induction in human hepatocytes. CYP2B6 catalytic activity was significantly decreased following 3-day incubation with 0.1–10 μM LY2090314, on average by 64.3% ± 5.0% at 10 μM. These levels of LY2090314 exposure were not cytotoxic to hepatocytes and did not reduce CYP1A2 and CYP3A activities. LY2090314 was not a time-dependent CYP2B6 inhibitor, did not otherwise inhibit enzyme activity at concentrations ≤10 μM, and was not metabolized by CYP2B6. Thus, mechanism-based inactivation or other direct interaction with the enzyme could not explain the observed reduction in CYP2B6 activity. Instead, LY2090314 significantly reduced CYP2B6 mRNA levels (Imax = 61.9% ± 1.4%; IC50 = 0.049 ± 0.043 μM), which were significantly correlated with catalytic activity (r2 = 0.87, slope = 0.77; Imax = 57.0% ± 10.8%, IC50 = 0.057 ± 0.027 μM). Direct inhibition of constitutive androstane receptor by LY2090314 is conceptually consistent with the observed CYP2B6 transcriptional suppression (Imax = 100.0% ± 10.8% and 57.1% ± 2.4%; IC50 = 2.5 ± 1.2 and 2.1 ± 0.4 μM for isoforms 1 and 3, respectively) and may be sufficiently extensive to overcome the weak but potent activation of pregnane X receptor by ≤10 μM LY2090314 (19.3% ± 2.2% of maximal rifampin response, apparent EC50 = 1.2 ± 1.1 nM). The clinical relevance of these findings was evaluated through physiologically based pharmacokinetic model simulations. CYP2B6 suppression by LY2090314 is not expected clinically, with a projected <1% decrease in hepatic enzyme activity and <1% decrease in hydroxybupropion exposure following bupropion coadministration. However, simulations showed that observed CYP2B6 suppression could be clinically relevant for a drug with different pharmacokinetic properties from LY2090314.

Introduction to Hepatic Drug Metabolizing Enzyme Induction in Drug Safety Evaluation Studies

The following three articles represent the output of a combined effort initiated by the Scientific Regulatory Policy Committee of the Society of Toxicologic Pathology to provide a unified review of current scientific practices and relevant literature and provide suggestions regarding the recognition, interpretation, and risk assessment of hepatic drug metabolizing enzyme (DME) induction studies. The core objective was to provide a review that the scientific community including pathologists, regulatory scientists, toxicologists, investigative scientists, and others would find valuable for managing, designing, and interpreting toxicity studies supporting regulatory filings. Three working groups composed of scientists from industry, academia, and regulatory agencies were convened to review the available literature on important aspects of the interpretation and risk assessment of hepatic microsomal DME enzyme induction in three publications. The three reviews are as follows: “Effects of Hepatic Drug Metabolizing Enzyme Induction on Clinical Pathology Parameters in Animals and Man,” Toxicol Pathol “Hepatic Drug Metabolizing Enzyme Induction: Microscopic and Ultrastructural Appearance,” Toxicol Pathol “Hepatic Drug Metabolizing Enzyme Induction and Implications for Preclinical and Clinical Risk Assessment,” Toxicol Pathol The purpose of this introduction is not to summarize the articles but rather to frame the series and to provide a common mechanistic introduction.