Structure-Property Relationships and Machine Learning Models for Addressing CYP3A4-Mediated Victim Drug-Drug Interaction Risk in Drug Discovery.

Among the FDA-approved small molecule drugs (2005-2016) that are primarily metabolized by cytochrome P450 (CYP), 64% are primarily metabolized by CYP3A4. As the proportion of an individual drug's fraction metabolized through CYP3A4 increases, the risk for the drug to be a victim of an interaction with CYP3A4 inhibitors or inducers increases. Therefore, it is important to assess the extent of involvement of individual CYP enzymes in the overall clearance for a scaffold early in discovery and mitigate the CYP3A4-mediated victim-drug-drug interaction (DDI) risk, if warranted by the desired clinical profile of the drug. To mitigate the CYP3A4-mediated victim DDI risk in discovery, we analyzed the physicochemical properties of the CYP3A4 substrates and found that molecular weight was the property that provided the best separation of the CYP3A4 substrates from other CYP substrates. In addition, neutral and basic compounds with MW ≥ 360 g/mol tend to be primarily metabolized by CYP3A4, whereas acidic compounds with MW < 360 g/mol are most likely to be primarily metabolized by other CYP enzymes. We then developed Support Vector Machine based on fingerprints (SVM-FP) and Deep-Learning (DL) models to predict if a molecule will be primarily metabolized by CYP3A4. Our models were trained on 2306 compounds, which is the largest training set among published models for this endpoint. Both models showed positive predictive values (PPV) > 80% in predicting a CYP3A4 substrate on a prospective testing set. Given the high PPV of the models, project teams can confidently deprioritize compounds predicted to be CYP3A4 substrates to avoid the potential liability of CYP3A4 victim DDI. Teams can then focus time and resources on synthesizing compounds that are predicted to have a lower dependency on CYP3A4 metabolism and confirm that experimentally. Through such iterative in silico-in vitro learning circles, drug discovery teams can decide if metabolism through non-CYP3A4 pathways could be achieved in the SAR of a chemical series to mitigate the CYP3A4 victim DDI risk.

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.

The Effect of Promiscuous Aggregation on in Vitro Drug Metabolism Assays.

PURPOSE: Many bioactive molecules show a type of solution phase behavior, termed promiscuous aggregation, whereby at micromolar concentrations, colloidal drug-rich aggregates are formed in aqueous solution. These aggregates are known to be a major cause of false positives and false negatives in select enzymatic high-throughput screening assays. The goal of this study was to investigate the impact of drug-rich aggregates on in vitro drug screening metabolism assays. METHODS: Cilnidipine was selected as an aggregate former and its impact on drug metabolism was evaluated against rCYP2D6, rCYP1A2, rCYP2C9 and human liver microsomes. RESULTS: The cilnidipine aggregates were shown to non-specifically inhibit multiple cytochrome P450 enzymes with an IC50 comparable with the IC50 of potent model inhibitors. CONCLUSIONS: This newly demonstrated mode of "promiscuous inhibition" is of great importance as it can lead to false positives during drug metabolism evaluations and thus it needs to be considered in the future to better predict in vivo drug-drug interactions.

The Impact of the Hepatocyte-to-Plasma pH Gradient on the Prediction of Hepatic Clearance and Drug-Drug Interactions for CYP2C9 and CYP3A4 Substrates.

Surrogate assays for drug metabolism and inhibition are traditionally performed in buffer systems at pH 7.4, despite evidence that hepatocyte intracellular pH is 7.0. This pH gradient can result in a pKa-dependent change in intracellular/extracellular concentrations for ionizable drugs that could affect predictions of clearance and P450 inhibition. The effect of microsomal incubation pH on in vitro enzyme kinetic parameters for CYP2C9 (diclofenac, (S)-warfarin) and CYP3A4 (midazolam, dextromethorphan, testosterone) substrates, enzyme specific reversible inhibitors (amiodarone, desethylamiodarone, clozapine, nicardipine, fluconazole, fluvoxamine, itraconazole) and a mechanism-based inhibitor (amiodarone) was investigated. Intrinsic clearance through CYP2C9 significantly increased (25% and 50% for diclofenac and (S)-warfarin respectively) at intracellular pH 7.0 compared with traditional pH 7.4. The CYP3A4 substrate dextromethorphan intrinsic clearance was decreased by 320% at pH 7.0, while midazolam and testosterone remained unchanged. Reversible inhibition of CYP2C9 was less potent at pH 7.0 compared with 7.4, while CYP3A4 inhibition potency was variably affected. Maximum enzyme inactivation rate of amiodarone toward CYP2C9 and CYP3A4 decreased at pH 7.0, while the irreversible inhibition constant remained unchanged for CYP2C9, but decreased for CYP3A4 at pH 7.0. Predictions of clearance and drug-drug interactions made through physiologically based pharmacokinetic models were improved with the inclusion of predicted intracellular concentrations based at pH 7.0 and in vitro parameters determined at pH 7.0. No general conclusion on the impact of pH could be made and therefore a recommendation to change buffer pH to 7.0 cannot be made at this time. It is recommended that the appropriate hepatocyte intracellular pH 7.0 be used for in vitro determinations when in vivo predictions are made.

The Impact of the Hepatocyte-to-Plasma pH Gradient on the Prediction of Hepatic Clearance and Drug-Drug Interactions for CYP2D6 Substrates.

The proton gradient from the intracellular space to plasma creates an unbound drug gradient for weak acids and bases that could modulate apparent drug clearance and drug-drug interactions. Cytochrome P450 intrinsic clearance and inhibitor potency are routinely determined in vitro at the plasma pH of 7.4 rather than the intrahepatocyte pH of 7.0. We determined the impact of pH on in vitro enzyme kinetic parameters and inhibition potency for substrates (bufuralol, dextromethorphan), reversible inhibitors (quinidine, amiodarone, desethylamiodarone, clozapine), and mechanism-based inhibitors (paroxetine, desethylamiodarone) of the major drug metabolizing-enzyme CYP2D6. The lower intracellular pH 7.0 compared with pH 7.4 resulted in a 60 and 50% decrease in intrinsic clearance for the substrates bufuralol and dextromethorphan, respectively. Reversible inhibition constants for three of the four inhibitors tested were unaffected by pH, whereas for the inhibitor quinidine, a 2-fold increase in the inhibition constant was observed at pH 7.0. For time-dependent inhibitors desethylamiodarone and paroxetine, changes in time-dependent inhibition parameters were different for each inhibitor. These results were incorporated into physiologically based pharmacokinetic models indicating that the changes in in vitro parameters determined at pH 7.0 offset the effect of increased unbound intracellular concentrations on apparent clearance and extent of drug-drug interactions. However, this offset between concentration and enzyme activity cannot be generalized for all substrates, inhibitors, and enzymes, as the effect of a lower pH in vitro varied significantly; therefore, it would be prudent to determine in vitro enzyme parameters at the hepatocyte-appropriate pH 7.0.

Difference in the Pharmacokinetics and Hepatic Metabolism of Antidiabetic Drugs in Zucker Diabetic Fatty and Sprague-Dawley Rats.

The Zucker diabetic fatty (ZDF) rat, an inbred strain of obese Zucker fatty rat, develops early onset of insulin resistance and displays hyperglycemia and hyperlipidemia. The phenotypic changes resemble human type 2 diabetes associated with obesity and therefore the strain is used as a pharmacological model for type 2 diabetes. The aim of the current study was to compare the pharmacokinetics and hepatic metabolism in male ZDF and Sprague-Dawley (SD) rats of five antidiabetic drugs that are known to be cleared via various mechanisms. Among the drugs examined, metformin, cleared through renal excretion, and rosiglitazone, metabolized by hepatic cytochrome P450 2C, did not exhibit differences in the plasma clearance in ZDF and SD rats. In contrast, glibenclamide, metabolized by hepatic CYP3A, canagliflozin, metabolized mainly by UDP-glucuronosyltransferases (UGT), and troglitazone, metabolized by sulfotransferase and UGT, exhibited significantly lower plasma clearance in ZDF than in SD rats after a single intravenous administration. To elucidate the mechanisms for the difference in the drug clearance, studies were performed to characterize the activity of hepatic drug-metabolizing enzymes using liver S9 fractions from the two strains. The results revealed that the activity for CYP3A and UGT was decreased in ZDF rats using the probe substrates, and decreased unbound intrinsic clearance in vitro for glibenclamide, canagliflozin, and troglitazone was consistent with lower plasma clearance in vivo. The difference in pharmacokinetics of these two strains may complicate pharmacokinetic/pharmacodynamic correlations, given that ZDF is used as a pharmacological model, and SD rat as the pharmacokinetics and toxicology strain.

In vitro characterization of the bioconversion of pomaglumetad methionil, a novel metabotropic glutamate 2/3 receptor agonist peptide prodrug.

To characterize the hydrolysis of the peptide prodrug pomaglumetad methionil (LY2140023; (1R,4S,5S,6S)-4-(L-methionylamino)-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide), to the active drug LY404039 [(1R,4S,5S,6S)-4-amino-2-thiabicyclo[3.1.0]hexane-4,6-dicarboxylic acid 2,2-dioxide], a series of in vitro studies were performed in various matrices, including human intestinal, liver, kidney homogenate, and human plasma. The studies were performed to determine the tissue(s) and enzyme(s) responsible for the conversion of the prodrug to the active molecule. This could enable an assessment of the risk for drug interactions, an evaluation of pharmacogenomic implications, as well as the development of a Physiologically Based Pharmacokinetic (PBPK) model for formation of the active drug. Of the matrices examined, hydrolysis of pomaglumetad methionil was observed in intestinal and kidney homogenate preparations and plasma, but not in liver homogenate. Clearance values calculated after applying standard scaling factors suggest the intestine and kidney as primary sites of hydrolysis. Studies with peptidase inhibitors were performed in an attempt to identify the enzyme(s) catalyzing the conversion. Near complete inhibition of LY404039 formation was observed in intestinal and kidney homogenate and human plasma with the selective dehydropeptidase1 (DPEP1) inhibitor cilastatin. Human recombinant DPEP1 was expressed and shown to catalyze the hydrolysis, which was completely inhibited by cilastatin. These studies demonstrate pomaglumetad methionil can be converted to LY404039 via one or multiple enzymes completely inhibited by cilastatin, likely DPEP1, in plasma, the intestine, and the kidney, with the plasma and kidney involved in the clearance of the circulating prodrug. These experiments define a strategy for the characterization of enzymes responsible for the metabolism of other peptide-like compounds.

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 (r(2) = 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.

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 IC(50) 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 K(i, app) values were combined with the free average systemic plasma concentration ("free [I] with HMM K(i, app)") and the plasma K(i, app) values were combined with the total average systemic plasma concentration ("total [I] with plasma K(i, app)"). Of 63 clinical DDI studies, the total [I] with plasma K(i, app) method predicted 89% of cases within 2-fold of the reported interaction whereas the free [I] with HMM K(i, app) method predicted only 59%. There was a general underprediction by the free [I] with HMM K(i, 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.

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 IC(50) values for the 13 inhibitors. For each CYP3A inhibitor-victim drug pair, the IC(50) 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 IC(50) determined at a single incubation time regardless of the inhibition mechanism; further if needed, the mechanism(s) of inhibition can be identified.

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.

Enterohepatic circulation of glucuronide metabolites of drugs in dog.

The enterohepatic circulation (EHC) of drugs is often the result of the direct glucuronidation, excretion of the metabolite into bile, followed by hydrolysis to the aglycone by the gut microbiome and finally reabsorption of drug into the systemic circulation. The aim of present study to identify key factors in determining the EHC in dog for canagliflozin and DPTQ, two compounds cleared by UDP-glucuronosyltransferase (UGT) mediated O-alkyl glucuronidation and cytochrome P450 (P450) mediated oxidation. The pharmacokinetic profiles of the drugs were compared between bile duct cannulated (BDC) and intact beagle dogs after a single intravenous administration. A long terminal elimination phase was observed for DPTQ but not for canagliflozin in intact dogs, while this long terminal half-life was not seen in BDC animals, suggesting the EHC of DPTQ. Quantification of parent drugs and glucuronide metabolites in bile, urine and feces indicated low recovery of parent in bile and urine and low recovery of conjugated metabolites in urine for both drugs, while biliary excretion of these glucuronide metabolites in BDC dog were low for canagliflozin but much higher for DPTQ. The increased fecal recovery of parent drug in intact dog and the lack of glucuronide metabolites suggested the hydrolysis of DPTQ-glucuronides by gut microbiome. Subsequent characterization of in vitro hepatic metabolism and permeability properties indicated the hepatic fraction metabolized by UGT, hydrolysis of metabolites, and reabsorption of the aglycone were key factors in determining the EHC of DPTQ.

In Vitro and In Vivo Correlation of Hepatic Fraction of Metabolism by P450 in Dogs.

1-Aminobenzotriazole (ABT) has been widely used as a nonspecific mechanism-based inhibitor of cytochrome P450 (P450) enzymes. It is extensively used in preclinical studies to determine the relative contribution of oxidative metabolism mediated by P450 in vitro and in vivo. The aim of present study was to understand the translation of fraction metabolized by P450 in dog hepatocytes to in vivo using ABT, for canagliflozin, known to be cleared by P450-mediated oxidation and UDP-glucuronosyltransferases-mediated glucuronidation, and 3 drug discovery project compounds mainly cleared by hepatic metabolism. In a dog hepatocyte, intrinsic clearance assay with and without preincubation of ABT, 3 Lilly compounds exhibited a wide range of fraction metabolized by P450. Subsequent metabolite profiling in dog hepatocytes demonstrated a combination of metabolism by P450 and UDP-glucuronosyltransferases. In vivo, dogs were pretreated with 50 mg/kg ABT or vehicle at 2 h before intravenous administration of canagliflozin and Lilly compounds. The areas under the concentration-time curve (AUC) were compared for the ABT-pretreated and vehicle-pretreated groups. The measured AUCABT/AUCveh ratios were correlated to fraction of metabolism by P450 in dog hepatocytes, suggesting that in vitro ABT inhibition in hepatocytes is useful to rank order compounds for in vivo fraction of metabolism assessment.

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 K(m) value and a 3-fold lower V(max) 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.

Monitoring the Phase Behavior of Supersaturated Solutions of Poorly Water-Soluble Drugs Using Fluorescence Techniques.

Phase transformations of poorly water-soluble drugs, in low concentration, supersaturated aqueous solutions are of considerable interest. Herein, fluorescence lifetime and steady-state fluorescence spectroscopy were employed to investigate the fluorescence properties of the autofluorescent compound, felodipine (a 1,4-dihydropyridine calcium channel blocker), when present as free drug in solution, drug-rich aggregates, and crystals. Measurements were also performed in the absence and presence of liver microsomes. To study nonfluorescent drugs, an environment-sensitive fluoroprobe, 6-propionyl-2-dimethylaminonaphthalene, was employed. The lifetime of free felodipine in solution in simple media was found to be ∼0.4 ns, whereas felodipine present in drug-rich aggregates and crystals was characterized by a longer lifetime of ∼2 and ∼9 ns, respectively. In the presence of structures containing lipids, the local environment of felodipine was found to change based on fluorescence characteristics and the concentration where felodipine aggregates formed was greatly increased. The lifetime of 6-propionyl-2-dimethylaminonaphthalene in solutions containing clotrimazole (an imidazole derivative with antimycotic activity) or efavirenz (a non-nucleoside reverse transcriptase inhibitor with antiviral activity) increased on aggregate formation as a result of the change in polarity of the probe local environment. Fluorescence lifetime coupled with steady-state fluorescence spectroscopy was demonstrated to be effective in identifying the concentration where drug aggregates formed, contributing to improved understanding of the phase behavior of poorly water-soluble drugs in biologically relevant media.

Mechanistic understanding of the phase behavior of supersaturated solutions of poorly water-soluble drugs.

Amorphous solid dispersions (ASDs) are a promising formulation strategy to increase both the apparent aqueous solubility and bioavailability of poorly water-soluble drugs. Upon dissolution under nonsink conditions, ASDs can generate highly supersaturated drug solutions which can undergo liquid-liquid phase separation (LLPS) and/or crystallization. In this study, the phase behavior of supersaturated solutions generated by antisolvent addition and upon the dissolution of ASDs was evaluated using fluorescence lifetime measurements and several other orthogonal techniques, including steady-state fluorescence spectroscopy, ultraviolet (UV) extinction and concentration profiles, ultracentrifuge measurements and nanoparticle tracking analysis. Ritonavir and lopinavir were chosen as poorly water-soluble model drugs, and the polymer, Kollidon VA64, was selected to form the dispersions. The fluorescence lifetime of the environment-sensitive fluoroprobe, PRODAN, was monitored to determine the occurrence of LLPS and crystallization. It was found that only the 10% w/w drug loading ASDs dissolved to a concentration in solution higher than the LLPS concentration and this led to an increase in the lifetime of PRODAN due to partitioning of the fluoroprobe into the drug-rich phase. In contrast, the 50% w/w drug loading ASDs did not reach the amorphous solubility, pointing to a dissolution behavior controlled by the low water solubility and high hydrophobicity of the drug. Fluorescence lifetime measurements were demonstrated to be extremely useful for the characterization of the phase behavior of supersaturated solutions of poorly water-soluble drugs.

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 compound's 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.

Identification of human UDP-glucuronosyltransferases catalyzing hepatic 1alpha,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 the in vivo clearance of drugs from rate of loss using human liver microsomes for phase I and phase II biotransformations.

PURPOSE: The 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. METHODS: The 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. RESULTS: The 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. CONCLUSIONS: The substrate loss method may be used to accurately predict hepatic clearance of drugs.

Ginkgo biloba: evaluation of CYP2C9 drug interactions in vitro and in vivo.

Ginkgo biloba extract is one of the most widely used herbal products in the United States. However, bleeding episodes in patients taking Ginkgo biloba and warfarin have been documented. Therefore, in vitro and in vivo inhibition studies were done to ascertain the influence of ginkgo on CYP2C9, the P-450 isozyme responsible for the metabolism of the most potent warfarin enantiomer, (S)-warfarin. Ginkgo extract inhibited human liver microsomal CYP2C9 with an apparent Ki=14.8 microg/mL, and the inhibition was increased by acid hydrolysis (apparent Ki=9.1 microg/mL). Two open-label, crossover pharmacokinetic studies in healthy subjects were performed using tolbutamide and diclofenac as probe CYP2C9 substrates. In contrast to the in vitro inhibition of CYP2C9, no interactions between Ginkgo biloba extract and CYP2C9 probe substrates were observed in vivo as evidenced by the lack of effect on the steady-state pharmacokinetics of diclofenac or on the urinary metabolic ratio of tolbutamide.