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The propensity for aldehyde oxidase (AO) substrates to be implicated in drug-drug interactions (DDIs) is not well understood due to the dearth of potent inhibitors that elicit in vivo inhibition of AO. Although there is only one reported instance of DDI that has been ascribed to the inhibition of AO to date, the supporting evidence for this clinical interaction is rather tenuous, and its veracity has been called into question. Our group recently reported that the epidermal growth factor receptor inhibitor erlotinib engendered potent time-dependent inhibition of AO with inactivation kinetic constants in the same order of magnitude as its free circulating plasma concentrations. At the same time, it was previously reported that the concomitant administration of erlotinib with the investigational drug OSI-930 culminated in a an approximately twofold increase in its systemic exposure. Although the basis underpinning this interaction remains unclear, the structure of OSI-930 contains a quinoline motif that is amenable to oxidation at the electrophilic carbon adjacent to the nitrogen atom by molybdenum-containing hydroxylases like AO. In this study, we conducted metabolite identification that revealed that OSI-930 undergoes AO metabolism to a mono-oxygenated 2-oxo metabolite and assessed its formation kinetics in human liver cytosol. Additionally, reaction phenotyping in human hepatocytes revealed that AO contributes nearly 50% to the overall metabolism of OSI-930. Finally, modeling the interaction between erlotinib and OSI-930 using a mechanistic static model projected an ∼1.85-fold increase in the systemic exposure of OSI-930, which accurately recapitulated clinical observations. SIGNIFICANCE STATEMENT: This study delineates an aldehyde oxidase (AO) metabolic pathway in the investigational drug OSI-930 for the first time and confirmed that it represented a major route of metabolism through reaction phenotyping in human hepatocytes. Our study provided compelling mechanistic and modeling evidence for the first instance of an AO-mediated clinical drug-drug interaction stemming from the in vivo inhibition of the AO-mediated quinoline 2-oxidation pathway in OSI-930 by erlotinib., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

Aldehyde oxidase (AO) is a molybdenum cofactor-containing cytosolic enzyme that has gained prominence due to its involvement in the developmental failure of several drug candidates in first-in-human trials. Unlike cytochrome P450s (P450) and glucuronosyltransferase, AO substrates have been plagued by poor in vitro to in vivo extrapolation, leading to low systemic exposures and underprediction of human dose. However, apart from measuring a drug's AO clearance rates, it is also important to determine the relative contribution to metabolism by this enzyme (f m,AO ). Although hydralazine is the most well-studied time-dependent inhibitor (TDI) of AO and is frequently employed for AO reaction phenotyping in human hepatocytes to derive f m,AO , multiple studies have expressed concerns pertaining to its utility in providing accurate estimates of f m,AO values due to its propensity to significantly inhibit P450s at the concentrations typically used for reaction phenotyping. In this study, we characterized icotinib, a cyclized analog of erlotinib, as a potent TDI of AO-inactivating human liver cytosolic zoniporide 2-oxidation equipotently with erlotinib with a maximal inactivate rate/inactivator concentration at half maximal inactivation rate ( K I ) ratio of 463 and 501 minute -1 mM -1 , respectively. Moreover, icotinib also exhibits selectivity against P450 and elicits significantly weaker inhibition against human liver microsomal UGT1A1/3 as compared with erlotinib. Finally, we evaluated icotinib as an inhibitor of AO for reaction phenotyping in cryopreserved human hepatocytes and demonstrated that it can yield more accurate prediction of f m,AO compared with hydralazine and induce sustained suppression of AO activity at higher cell densities, which will be important for reaction phenotyping endeavors of low clearance drugs SIGNIFICANCE STATEMENT: In this study, we characterized icotinib as a potent time-dependent inhibitor of AO with ample selectivity margins against the P450s and UGT1A1/3 and demonstrated its utility for reaction phenotyping in human hepatocytes to obtain accurate estimates of fm,AO for victim DDI risk predictions. We envisage the adoption of icotinib in place of hydralazine in AO reaction phenotyping., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

BIIB104 (formerly PF-04958242), N -((3 S ,4 S )-4-(4-(5-cyanothiophen-2-yl)phenoxy)tetrahydrofuran-3-yl)propane-2-sulfonamide, is an α -amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor potentiator investigated for the treatment of cognitive impairment associated with schizophrenia. Preliminary in vitro metabolism studies with non-radiolabeled BIIB104 in rat, dog, and human liver microsomes (RLM, DLM, and HLM) showed O -dealkylation in all three species, tetrahydrofuran hydroxylation dominating in DLM and HLM, and thiophene hydroxylation prevalent in RLM. However, a subsequent rat mass balance study with [nitrile- 14 C]BIIB104 showed incomplete recovery of administered radioactivity (∼80%) from urine and feces over 7 days following an oral dose, and an exceptionally long plasma total radioactivity half-life. Radiochromatographic metabolite profiling and identification, including chemical derivation, revealed that [ 14 C]cyanide was a major metabolite of [nitrile- 14 C]BIIB104 in RLM, but a minor and trace metabolite in DLM and HLM, respectively. Correspondingly in bile duct-cannulated rats, [ 14 C]thiocyanate accounted for ∼53% of total radioactivity excreted over 48 hours postdose and it, as an endogenous substance, explained the exceptionally long plasma radioactivity half-life. The release of [ 14 C]cyanide from the 2-cyanothiophene moiety is postulated to follow an epoxidation-initiated thiophene-opening based on the detection of non-radiolabeled counterpart metabolites in RLM. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate. Additionally, the potential cyanide metabolite of nitrile-containing drug molecules may be detected in liver microsomes with liquid chromatography-mass spectrometry following a chemical derivatization. SIGNIFICANCE STATEMENT: Using [nitrile- 14 C]BIIB104, non-intuitive metabolites of BIIB104 were discovered involving a novel cyanide release from the 2-cyanothiophene motif via a postulated epoxidation-initiated thiophene-opening. This unusual biotransformation serves as a lesson regarding placement of the radioactive label on an aryl nitrile when material will be used for evaluating the metabolism of a new drug candidate., (Copyright © 2024 by The American Society for Pharmacology and Experimental Therapeutics.)

In vitro time-dependent inhibition (TDI) kinetic parameters for cytochrome P450 (CYP) 1A2, 2B6, 2C8, 2C9, 2C19, and 2D6, were determined in pooled human liver microsomes for 19 drugs (and 2 metabolites) for which clinical drug-drug interactions (DDI) are known. In vitro TDI data were incorporated into the projection of the magnitude of DDIs using mechanistic static models and Simcyp®. Results suggest that for the mechanistic static model, use of estimated average unbound exit concentration of the inhibitor from the liver resulted in a successful prediction of observed magnitude of clinical DDIs and was similar to Simcyp®. Overall, predictions of DDI magnitude (i.e., fold increase in AUC of a CYP-specific marker substrate) were within 2-fold of actual values. Geometric mean-fold errors were 1.7 and 1.6 for static and dynamic models, respectively. Projections of DDI from both models were also highly correlated to each other (r2 = 0.92). This investigation demonstrates that DDI can be reliably predicted from in vitro TDI data generated in HLM for several CYP enzymes. Simple mechanistic static model equations as well as more complex dynamic PBPK models can be employed in this process. Significance Statement Cytochrome P450 time-dependent inhibitors (TDI) can cause drug-drug interactions (DDI). An ability to reliably assess the potential for a new drug candidate to cause DDI is essential during drug development. In this report, TDI data for 19 drugs (and 2 metabolites) were measured and used in static and dynamic models to reliably project the magnitude of DDI resulting from inhibition of CYP1A2, 2B6, 2C8, 2C9, 2C19, and 2D6., (Copyright © 2024 American Society for Pharmacology and Experimental Therapeutics.)

The metabolism of lufotrelvir, a novel phosphate prodrug of PF-00835231 for the treatment of COVID-19, was evaluated in healthy human volunteers and clinical trial participants with COVID-19 following intravenous infusion. The prodrug was completely converted to PF-00835231 that was subsequently cleared by hydrolysis, hydroxylation, ketoreduction, epimerization, renal clearance, and secretion into the feces. The main circulating metabolite was a hydrolysis product (M7) that was present at concentrations greater than PF-00835231, and this was consistent between healthy volunteers and participants with COVID-19. On administration of [ 14 C]lufotrelvir, only 63% of the dose was obtained in excreta over 10 days and total drug-related material demonstrated a prolonged terminal phase half-life in plasma. A considerable portion of the labeled material was unextractable from fecal homogenate and plasma. The position of the carbon-14 atom in the labeled material was at a leucine carbonyl, and pronase digestion of the pellet derived from extraction of the fecal homogenate showed that [ 14 C]leucine was released. SIGNIFICANCE STATEMENT: Lufotrelvir is an experimental phosphate prodrug intravenous therapy investigated for the potential treatment of COVID-19 in a hospital setting. The overall metabolism of lufotrelvir was determined in human healthy volunteers and clinical trial participants with COVID-19. Conversion of the phosphate prodrug to the active drug PF-00835231 was complete and the subsequent metabolic clearance of the active drug was largely via amide bond hydrolysis. Substantial drug-related material was not recovered due to loss of the carbon-14 label to endogenous metabolism., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Human absorption, distribution, metabolism, and excretion (hADME) studies represent one of the most important clinical studies in terms of obtaining a comprehensive and quantitative overview of the total disposition of a drug. This article will provide background on the origins of hADME studies as well as provide an overview of technological innovations that have impacted how hADME studies are carried out and analyzed. An overview of the current state of the art for hADME studies will be provided, the impacts of advances in technology and instrumentation on the timing of and approaches to hADME studies will be discussed, and a summary of the parameters and information obtained from these studies will be offered. Additionally, aspects of the ongoing debate over the importance of animal absorption, distribution, metabolism, and excretion studies versus a "human-first, human-only strategy" will be presented. Along with the information above, this manuscript will highlight how, for over 50 years, Drug Metabolism and Disposition has served as an important outlet for the reporting of hADME studies. SIGNIFICANCE STATEMENT: Human absorption, distribution, metabolism, and excretion (hADME) studies have and will continue to be important to the understanding and development of drugs. This manuscript provides a historical perspective on the origins of hADME studies as well as advancements resulting in the current-state-of the art practice for these studies., (Copyright © 2023 by The American Society for Pharmacology and Experimental Therapeutics.)

Cytochrome P450 reaction phenotyping to determine the fraction of metabolism values (f m ) for individual enzymes is a standard study in the evaluation of a new drug. However, there are technical challenges in these studies caused by shortcomings in the selectivity of P450 inhibitors and unreliable scaling procedures for recombinant P450 (rCYP) data. In this investigation, a two-step "qualitative-then-quantitative" approach to P450 reaction phenotyping is described. In the first step, each rCYP is tested qualitatively for potential to generate metabolites. In the second step, selective inhibitors for the P450s identified in step1 are tested for their effects on metabolism using full inhibition curves. Forty-eight drugs were evaluated in step 1 and there were no examples of missing an enzyme important to in vivo clearance. Five drugs (escitalopram, fluvastatin, pioglitazone, propranolol, and risperidone) were selected for full phenotyping in step2 to determine f m values, with findings compared to f m values estimated from single inhibitor concentration data and rCYP with intersystem-extrapolation-factor corrections. The two-step approach yielded f m values for major drug clearing enzymes that are close to those estimated from clinical data: escitalopram and CYP2C19 (0.42 vs 0.36-0.82), fluvastatin and CYP2C9 (0.76 vs 0.76), pioglitazone and CYP2C8 (0.72 vs 0.73), propranolol and CYP2D6 (0.68 vs 0.37-0.56) and risperidone and CYP2D6 (0.60 vs 0.66-0.88). Reaction phenotyping data generated in this fashion should offer better input to physiologically-based pharmacokinetic models for prediction of DDI and impact of genetic polymorphisms on drug clearance. The qualitative-then-quantitative approach is proposed as a replacement to standard reaction phenotyping strategies. Significance Statement P450 reaction phenotyping is important for projecting drug-drug interactions and interpatient variability in drug exposure. However, currently recommended practices can frequently fail to provide reliable estimates of the fractional contributions of specific P450 enzymes (f m ) to drug clearance. In this report, we describe a two-step qualitative-then-quantitative reaction phenotyping approach that yields more accurate estimates of f m .

The utility of chemical inhibitors in cytochrome P450 (CYP) reaction phenotyping is highly dependent on their selectivity and potency for their target CYP isoforms. In the present study, seventeen inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4/5 commonly used in reaction phenotyping were evaluated for their cross-enzyme selectivity in pooled human liver microsomes. The data were evaluated using a statistical desirability analysis to identify (1) inhibitors of superior selectivity for reaction phenotyping and (2) optimal concentrations for each. Among the inhibitors evaluated, α-naphthoflavone, furafylline, sulfaphenazole, tienilic acid, N -benzylnirvanol, and quinidine were most selective, such that their respective target enzymes were inhibited by ~95% without inhibiting any other CYP enzyme by more than 10%. Other commonly employed inhibitors, such as ketoconazole and montelukast, among others, were of insufficient selectivity to yield a concentration that could adequately inhibit their target enzymes without affecting other CYP enzymes. To overcome these shortcomings, an experimental design was developed wherein dose response data from a densely sampled multi-concentration inhibition curve are analyzed by a six-parameter inhibition curve function, allowing accounting of the inhibition of off-target CYP isoforms inhibition and more reliable determination of maximum targeted enzyme inhibition. The approach was exemplified using rosiglitazone N -demethylation, catalyzed by both CYP2C8 and 3A4, and was able to discern the off-target inhibition by ketoconazole and montelukast from the inhibition of the targeted enzyme. This methodology yields more accurate estimates of CYP contributions in reaction phenotyping. Significance Statement Isoform-selective chemical inhibitors are important tools for identifying and quantifying enzyme contributions as part of a CYP reaction phenotyping assessment for projecting drug-drug interactions. However, currently employed practices fail to adequately compensate for shortcomings in inhibitor selectivity and the resulting confounding impact on estimates of the CYP enzyme contribution to drug clearance. In this report, we describe a detailed IC 50 study design with 6-parameter modeling approach that yields more accurate estimates of enzyme contribution.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3C-like protease inhibitor PF-07321332 (nirmatrelvir), in combination with ritonavir (Paxlovid), was recently granted emergency use authorization by multiple regulatory agencies for the treatment of coronavirus disease 2019 (COVID-19) in adults and pediatric patients. Disposition studies on nirmatrelvir in animals and in human reagents, which were used to support clinical studies, are described herein. Plasma clearance was moderate in rats (27.2 ml/min per kg) and monkeys (17.1 ml/min per kg), resulting in half-lives of 5.1 and 0.8 hours, respectively. The corresponding oral bioavailability was moderate in rats (34%-50%) and low in monkeys (8.5%), primarily due to oxidative metabolism along the gastrointestinal tract in this species. Nirmatrelvir demonstrated moderate plasma protein binding in rats, monkeys, and humans with mean unbound fractions ranging from 0.310 to 0.478. The metabolism of nirmatrelvir was qualitatively similar in liver microsomes and hepatocytes from rats, monkeys, and humans; prominent metabolites arose via cytochrome P450 (CYP450)-mediated oxidations on the P1 pyrrolidinone ring, P2 6,6-dimethyl-3-azabicyclo[3.1.0]hexane, and the tertiary-butyl group at the P3 position. Reaction phenotyping studies in human liver microsomes revealed that CYP3A4 was primarily responsible (fraction metabolized = 0.99) for the oxidative metabolism of nirmatrelvir. Minor clearance mechanisms involving renal and biliary excretion of unchanged nirmatrelvir were also noted in animals and in sandwich-cultured human hepatocytes. Nirmatrelvir was a reversible and time-dependent inhibitor as well as inducer of CYP3A activity in vitro. First-in-human pharmacokinetic studies have demonstrated a considerable boost in the oral systemic exposure of nirmatrelvir upon coadministration with the CYP3A4 inhibitor ritonavir, consistent with the predominant role of CYP3A4 in nirmatrelvir metabolism. SIGNIFICANCE STATEMENT: The manuscript describes the preclinical disposition, metabolism, and drug-drug interaction potential of PF-07321332 (nirmatrelvir), an orally active peptidomimetic-based inhibitor of the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) 3CL protease, which has been granted emergency use authorization by multiple regulatory agencies around the globe for the treatment of coronavirus disease 2019 (COVID-19) in COVID-19-positive adults and pediatric patients who are at high risk for progression to severe COVID-19, including hospitalization or death., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

The oxazolidinone antibacterial linezolid has been in clinical use for over 20 years, yet knowledge of the contributions of specific cytochrome (CYP) 450 enzymes to the metabolic clearance of this drug were mostly unknown. In this investigation, it was revealed that three P450 enzymes that had not been previously explored in linezolid metabolism, CYP2J2, CYP4F2, and CYP1B1, catalyzed the 2-hydroxylation and de-ethyleneation of the morpholine moiety of linezolid. The intrinsic clearance for linezolid metabolism in pooled human liver microsomes was low at 0.51 μL/min/mg protein, consistent with its in vivo clearance in humans, and the K M was high (>200 μM). In recombinant human P450 enzymes, a rank order of intrinsic clearance values for linezolid 2-hydroxylation were CYP2J2 ≫ CYP4F2 > CYP2C8 > CYP1B1 ≈ CYP2D6 ≈ CYP3A4 > CYP1A1 > CYP3A5, with nine other P450 enzymes showing no linezolid metabolism. The effect of selective inhibitors for these eight P450 enzymes on linezolid metabolism in pooled human liver microsomes was evaluated to provide estimates of the relative fractional contributions of these enzymes to linezolid metabolism. These experiments suggest that CYP2J2 and CYP4F2 contribute about 50% each to linezolid hepatic metabolism. It is proposed that the oxidative metabolic clearance of linezolid is primarily catalyzed by these two unusual P450 enzymes and that this explains the lack of observation of meaningful effects of common perpetrators of drug interactions on linezolid pharmacokinetics. SIGNIFICANCE STATEMENT: Linezolid is an important antibacterial drug, but the enzymes involved in its oxidative metabolism were unknown. In this study, evidence is shown that supports an important role for two enzymes not frequently associated with the metabolism of drugs: cytochrome P450 2J2 and cytochrome P450 4F2. These observations offer insight to understand the results of clinical drug-drug interaction studies conducted on linezolid., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

The use of intersystem extrapolation factors (ISEF) is required for the quantitative scaling of drug metabolism data generated in individually expressed cytochrome P450 (CYP) enzymes when estimating fractional contribution (f m ) to metabolism by P450 enzymes in vivo. For successful prediction of f m , ISEF values must be universal across all substrates for any individual enzyme. In this study, ISEF values were generated for ten CYP3A4 selective substrates using a common source of recombinant heterologously expressed CYP3A4 (rCYP) and a pool of human liver microsomes. The resulting ISEF values for CYP3A4 were substrate-dependent and ranged 8-fold, with the highest value generated from intrinsic clearance of midazolam depletion (0.36) and the lowest from quinidine depletion (0.044). Application of these ISEF values for estimation of the fractional contribution of CYP3A4 and CYP2C19 to omeprazole clearance yielded values that ranged from 0.21-0.63 and 0.37-0.79, respectively, as compared with back-extrapolated in vivo f m values of 0.27 (CYP3A4) and 0.85 (CYP2C19) from clinical pharmacokinetic data. For risperidone, estimated f m values for CYP3A4 and CYP2D6 ranged from 0.87-0.98 and 0.02-0.13, respectively, as compared with in vivo values of 0.36 (CYP3A4) and 0.63-0.88 (CYP2D6), showing that the importance of CYP3A4 was overestimated, and the importance of CYP2D6 underestimated. Overall, these findings suggest that ISEF values for CYP3A4 can vary with the marker substrate used to derive them, thereby reducing the effectiveness of the approach of using metabolism data from rCYP3A4 with ISEF values for the prediction of fraction metabolized values in vivo. SIGNIFICANCE STATEMENT: Intersystem extrapolation factors are utilized for assigning fractional contributions of individual enzymes to drug clearance (f m ) from drug metabolism data generated in recombinant P450s. The present data shows that intersystem extrapolation factors values for cytochrome P4503A4 vary with the substrate. This can lead to variable and erroneous prediction of f m ., (Copyright © 2022 by The American Society for Pharmacology and Experimental Therapeutics.)

Cytochrome P450 3A (CYP3A) is a frequent target for time-dependent inhibition (TDI) that can give rise to drug-drug interactions (DDI). Yet many drugs that exhibit in vitro TDI for CYP3A do not result in DDI. There were 23 drugs with published clinical DDI evaluated for CYP3A TDI in human liver microsomes (HLM) and hepatocytes (HHEP), and these data were used in static and dynamic models for projecting DDI caused by inactivation of CYP3A in both liver and intestine. TDI parameters measured in HHEP, particularly the maximal rate of enzyme inactivation, were generally lower than those measured in HLM. In static models, the use of estimated average unbound organ exit concentrations offered the most accurate projections of DDI with geometric mean fold errors of 2.0 and 1.7 for HLM and HHEP, respectively. Use of maximum organ entry concentrations yielded marked overestimates of DDI. When evaluated in a binary fashion (i.e., projection of DDI of 1.25-fold or greater), data from HLM offered the greatest sensitivity (100%) and specificity (67%) and yielded no missed DDI when average unbound organ exit concentrations were used. In dynamic physiologically based pharmacokinetic modeling, accurate projections of DDI were obtained with geometric mean fold errors of 1.7 and 1.6 for HLM and HHEP, respectively. Sensitivity and specificity were 100% and 67% when using TDI data generated in HLM and Simcyp modeling. Overall, DDI caused by CYP3A-mediated TDI can be reliably projected using dynamic or static models. For static models, average organ unbound exit concentrations should be used as input values otherwise DDI will be markedly overestimated. SIGNIFICANCE STATEMENT: CYP3A time-dependent inhibitors (TDI) are important in the design and development of new drugs. The prevalence of CYP3A TDI is high among newly synthesized drug candidates, and understanding the potential need for running clinical drug-drug interaction (DDI) studies is essential during drug development. Ability to reliably predict DDI caused by CYP3A TDI has been difficult to achieve. We report a thorough evaluation of CYP3A TDI and demonstrate that DDI can be predicted when using appropriate models and input parameters generated in human liver microsomes or hepatocytes., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

Cytochrome P450 enzymes are responsible for the metabolism of >75% of marketed drugs, making it essential to identify the contributions of individual cytochromes P450 to the total clearance of a new candidate drug. Overreliance on one cytochrome P450 for clearance levies a high risk of drug-drug interactions; and considering that several human cytochrome P450 enzymes are polymorphic, it can also lead to highly variable pharmacokinetics in the clinic. Thus, it would be advantageous to understand the likelihood of new chemical entities to interact with the major cytochrome P450 enzymes at an early stage in the drug discovery process. Typical screening assays using human liver microsomes do not provide sufficient information to distinguish the specific cytochromes P450 responsible for clearance. In this regard, we experimentally assessed the metabolic stability of ∼5000 compounds for the three most prominent xenobiotic metabolizing human cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4, and used the data sets to develop quantitative structure-activity relationship models for the prediction of high-clearance substrates for these enzymes. Screening library included the NCATS Pharmaceutical Collection, comprising clinically approved low-molecular-weight compounds, and an annotated library consisting of drug-like compounds. To identify inhibitors, the library was screened against a luminescence-based cytochrome P450 inhibition assay; and through crossreferencing hits from the two assays, we were able to distinguish substrates and inhibitors of these enzymes. The best substrate and inhibitor models (balanced accuracies ∼0.7), as well as the data used to develop these models, have been made publicly available (https://opendata.ncats.nih.gov/adme) to advance drug discovery across all research groups. SIGNIFICANCE STATEMENT: In drug discovery and development, drug candidates with indiscriminate cytochrome P450 metabolic profiles are considered advantageous, since they provide less risk of potential issues with cytochrome P450 polymorphisms and drug-drug interactions. This study developed robust substrate and inhibitor quantitative structure-activity relationship models for the three major xenobiotic metabolizing cytochromes P450, i.e., CYP2C9, CYP2D6, and CYP3A4. The use of these models early in drug discovery will enable project teams to strategize or pivot when necessary, thereby accelerating drug discovery research., (U.S. Government work not protected by U.S. copyright.)

Time-dependent inhibition (TDI) of CYP3A is an important mechanism underlying numerous drug-drug interactions (DDIs), and assays to measure this are done to support early drug research efforts. However, measuring TDI of CYP3A in human liver microsomes (HLMs) frequently yields overestimations of clinical DDIs and thus can lead to the erroneous elimination of many viable drug candidates from further development. In this investigation, 50 drugs were evaluated for TDI in HLMs and suspended human hepatocytes (HHEPs) to define appropriate boundary lines for the TDI parameter rate constant for inhibition (k obs ) at a concentration of 30 µM. In HLMs, a k obs value of 0.002 minute -1 was statistically distinguishable from control; however, many drugs show k obs greater than this but do not cause DDI. A boundary line defined by the drug with the lowest k obs that causes a DDI (diltiazem) was established at 0.01 minute -1 Even with this boundary, of the 33 drugs above this value, only 61% cause a DDI (true positive rate). A corresponding analysis was done using HHEPs; k obs of 0.0015 minute -1 was statistically distinguishable from control, and the boundary was established at 0.006 minute -1 Values of k obs in HHEPs were almost always lower than those in HLMs. These findings offer a practical guide to the use of TDI data for CYP3A in early drug-discovery research. SIGNIFICANCE STATEMENT: Time-dependent inhibition of CYP3A is responsible for many drug interactions. In vitro assays are employed in early drug research to identify and remove CYP3A time-dependent inhibitors from further consideration. This analysis demonstrates suitable boundaries for inactivation rates to better delineate drug candidates for their potential to cause clinically significant drug interactions., Competing Interests: Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper., (Copyright © 2021 by The American Society for Pharmacology and Experimental Therapeutics.)

( R )-2-(2-methylimidazo[2,1-b]thiazol-6-yl)-1-(2-(5-(6-methylpyrimidin-4-yl)-2,3-dihydro-1 H -inden-1-yl)-2,7-diazaspiro[3.5]nonan-7-yl)ethan-1-one (PF-5190457) was identified as a potent and selective inverse agonist of the ghrelin receptor [growth hormone secretagogue receptor 1a (GHS-R1a)]. The present translational bed-to-bench work characterizes the biotransformation of this compound in vivo and then further explores in vitro metabolism in fractions of human liver and primary hepatocytes. Following oral administration of PF-5190457 in a phase 1b clinical study, hydroxyl metabolites of the compound were observed, including one that had not been observed in previously performed human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation was shown to be on the pyrimidine using nuclear magnetic resonance spectroscopy. The aldehyde oxidase (AO) inhibitor raloxifene and the xanthine oxidase inhibitor febuxostat inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. However, greater inhibition was observed with raloxifene, indicating AO is a dominant enzyme in the biotransformation. The intrinsic clearance of the drug in human liver cytosol was estimated to be 0.002 ml/min per milligram protein. This study provides important novel information at three levels: 1) it provides additional new information on the recently developed novel compound PF-5190457, the first GHS-R1a blocker that has moved to development in humans; 2) it provides an example of a reverse translational approach where a discovery in humans was brought back, validated, and further investigated at the bench level; and 3) it demonstrates the importance of considering the molybdenum-containing oxidases during the development of new drug entities. SIGNIFICANCE STATEMENT: PF-5190457 is a novel ghrelin receptor inverse agonist that is currently undergoing clinical development for treatment of alcohol use disorder. PF-6870961, a major hydroxyl metabolite of the compound, was observed in human plasma, but was absent in human liver microsomal incubations. PF-6870961 was biosynthesized using liver cytosol, and the site of hydroxylation on the pyrimidine ring was characterized. Inhibitors of aldehyde oxidase and xanthine oxidase inhibited the formation of PF-6870961 in human liver cytosol, suggesting both enzymes were involved in the metabolism of the drug. This information is important for patient selection in subsequent clinical studies., Competing Interests: R.S.O. and T.F.R. are active employees at Pfizer Inc., Groton, CT., (U.S. Government work not protected by U.S. copyright.)

This commentary summarizes expert mini-reviews and original research articles that have been assembled in a special issue on novel models of drug metabolism and disposition. The special issue consists of research articles or reviews on novel static or micro-flow based models of the intestine, liver, eye, and kidney. This issue reviews static intestinal systems like mucosal scrapings and cryopreserved intestinal enterocytes, as well as novel bioengineered or chemically engineered intestinal models derived from primary human tissue, iPSCs, enteroids, and crypts. Experts have reviewed hepatic systems like cryopermeabilized Metmax hepatocytes and longer term, hepatocyte coculture system from H µ REL, yielding in vivo-like primary and secondary drug metabolite profiles. Additional liver models, including micropattern hepatocyte coculture, 3D liver spheroids, and microflow systems, applicable to the study of drug disposition and toxicology have also been reviewed. In this commentary, we have outlined expert opinions and current efforts on hepatic- and nephrotoxicity models. Ocular disposition models including corneal permeability models have been included within this special issue. This commentary provides a summary of in vivo mini-reviews of the issue, which have discussed the applications and drawbacks of pig and humanized mice models of P450, UGT, and rat organic anionic transporting polypeptide 1a4. While not extensively reviewed, novel positron emissions tomography imaging-based approaches to study the distribution of xenobiotics have been highlighted. This commentary also outlines in vitro and in vivo models of drug metabolism derived from breakthrough genetic, chromosomal, and tissue engineering techniques. The commentary concludes by providing a futuristic view of the novel models discussed in this issue., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Colon microbiota-based drug metabolism has received little attention thus far in the process of drug development, whereas the role of gut microbiota in clinical safety and efficacy of drugs has become more clear. Many of these studies have been performed using animal studies, but the translational value of these data with respect to drug pharmacokinetics, efficacy, and safety is largely unknown. To investigate human colon microbiota-mediated drug metabolism, we applied a recently developed ex vivo fermentation screening platform, in which human colonic microbiota conditions are simulated. A set of 12 drugs (omeprazole, simvastatin, metronidazole, risperidone, sulfinpyrazone, sulindac, levodopa, dapsone, nizatidine, sulfasalazine, zonisamide, and acetaminophen) was incubated with human colon microbiota under strictly anaerobic conditions, and samples were analyzed using high-performance liquid chromatograph-UV-high-resolution mass spectrometry analysis. The human microbiota in the fermentation assay consisted of bacterial genera regularly encountered in human colon and fecal samples and could be reproducibly cultured in independent experiments over time. In addition, fully anaerobic culture conditions could be maintained for 24 hours of incubation. Five out of the 12 included drugs (sulfasalazine, sulfinpyrazone, sulindac, nizatidine, and risperidone) showed microbiota-based biotransformation after 24 hours of incubation in the ex vivo fermentation assay. We demonstrated that drug metabolites formed by microbial metabolism can be detected in a qualitative manner and that the data are in accordance with those reported earlier for in vivo metabolism. In conclusion, we present a research tool to investigate human colon microbiota-based drug metabolism that may be applied to enable translatability of microbiota-based drug metabolism., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

We report a trend analysis of human intravenous pharmacokinetic data on a data set of 1352 drugs. The aim in building this data set and its detailed analysis was to provide, as in the previous case published in 2008, an extended, robust, and accurate resource that could be applied by drug metabolism, clinical pharmacology, and medicinal chemistry scientists to a variety of scaling approaches. All in vivo data were obtained or derived from original references, either through the literature or regulatory agency reports, exclusively from studies utilizing intravenous administration. Plasma protein binding data were collected from other available sources to supplement these pharmacokinetic data. These parameters were analyzed concurrently with a range of physicochemical properties, and resultant trends and patterns within the data are presented. In addition, the date of first disclosure of each molecule was reported and the potential "temporal" impact on data trends was analyzed. The findings reported here are consistent with earlier described trends between pharmacokinetic behavior and physicochemical properties. Furthermore, the availability of a large data set of pharmacokinetic data in humans will be important to further pursue analyses of physicochemical properties, trends, and modeling efforts and should propel our deeper understanding (especially in terms of clearance) of the absorption, distribution, metabolism, and excretion behavior of drug compounds., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

SAM-760 [(2-methyl-1-(phenylsulfonyl)-4-(piperazin-1-yl)-1H-benzo[d]imidazole)], a 5HT 6 antagonist, was investigated in humans for the treatment of Alzheimer's disease. In liver microsomes and recombinant cytochrome P450 (P450) isozymes, SAM-760 was predominantly metabolized by CYP3A (∼85%). Based on these observations and an expectation of a 5-fold magnitude of interaction with moderate to strong CYP3A inhibitors, a clinical DDI study was performed. In the presence of ketoconazole, the mean C max and area under the plasma concentration-time curve from time zero extrapolated to infinite time values of SAM-760 showed only a modest increase by 30% and 38%, respectively. In vitro investigation of this unexpectedly low interaction was undertaken using [ 14 C]SAM-760. Radiometric profiling in human hepatocytes confirmed all oxidative metabolites previously observed with unlabeled SAM-760; however, the predominant radiometric peak was an unexpected polar metabolite that was insensitive to the pan-P450 inhibitor 1-aminobenzotriazole. In human hepatocytes, radiometric integration attributed 43% of the total metabolism of SAM-760 to this non-P450 pathway. Using an authentic standard, this predominant metabolite was confirmed as benzenesulfinic acid. Additional investigation revealed that the benzenesulfinic acid metabolite may be a novel, nonenzymatic, thiol-mediated reductive cleavage of an aryl sulfonamide group of SAM-760. We also determined the relative contribution of P450 to the metabolism of SAM-760 in human hepatocytes by following the rate of formation of oxidative metabolites in the presence and absence of P450 isoform-specific inhibitors. The P450-mediated oxidative metabolism of SAM-760 was still primarily attributed to CYP3A (33%), with minor contributions from P450 isoforms CYP2C19 and CYP2D6. Thus, the disposition of [ 14 C]SAM-760 in human hepatocytes via novel sulfonamide metabolism and CYP3A verified the lower than expected clinical DDI when SAM-760 was coadministered with ketoconazole., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Since the introduction of metabolites in safety testing (MIST) guidance by the Food and Drug Administration in 2008, major changes have occurred in the experimental methods for the identification and quantification of metabolites, ways to evaluate coverage of metabolites, and the timing of critical clinical and nonclinical studies to generate this information. In this cross-industry review, we discuss how the increased focus on human drug metabolites and their potential contribution to safety and drug-drug interactions has influenced the approaches taken by industry for the identification and quantitation of human drug metabolites. Before the MIST guidance was issued, the method of choice for generating comprehensive metabolite profile was radio chromatography. The MIST guidance increased the focus on human drug metabolites and their potential contribution to safety and drug-drug interactions and led to changes in the practices of drug metabolism scientists. In addition, the guidance suggested that human metabolism studies should also be accelerated, which has led to more frequent determination of human metabolite profiles from multiple ascending-dose clinical studies. Generating a comprehensive and quantitative profile of human metabolites has become a more urgent task. Together with technological advances, these events have led to a general shift of focus toward earlier human metabolism studies using high-resolution mass spectrometry and to a reduction in animal radiolabel absorption/distribution/metabolism/excretion studies. The changes induced by the MIST guidance are highlighted by six case studies included herein, reflecting different stages of implementation of the MIST guidance within the pharmaceutical industry., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Maraviroc (MVC) is a CCR5 coreceptor antagonist indicated in combination with other antiretroviral agents for the treatment of CCR5-tropic human immunodefinciency virus-1 infection. In this study, the metabolism of MVC was investigated in human liver microsomes to delineate the relative roles of CYP3A4 and CYP3A5. MVC is metabolized to five hydroxylated metabolites, all of which were biosynthesized and identified using mass and NMR spectroscopy. The sites of metabolism were the 2- and 3-positions of the 4,4-difluorocyclohexyl moiety and the methyl of the triazole moiety. Absolute configurations were ultimately ascertained by comparison to authentic standards. The biosynthesized metabolites were used for quantitative in vitro experiments in liver microsomes using cyp3cide, a selective inactivator of CYP3A4. (1 S ,2 S )-2-OH-MVC was the main metabolite representing approximately half of the total metabolism, and CYP3A5 contributed approximately 40% to that pathway in microsomes from CYP3A5*1/*1 donors. The other four metabolites were almost exclusively metabolized by CYP3A4. (1 S ,2 S )-2-hydroxylation also correlated to T-5 N -oxidation, a CYP3A5-specific activity. These data are consistent with clinical pharmacokinetic data wherein CYP3A5 extensive metabolizer subjects showed a modestly lower exposure to MVC., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

( R )-4-((4-(((4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)piperidin-1-yl)methyl)tetrahydro-2H-pyran-4-ol (TBPT), a serotonin-4 receptor partial agonist, is metabolized to two metabolites: an N -dealkylation product [( R )-3-(piperidin-4-ylmethoxy)-4-((tetrahydrofuran-3-yl)oxy)benzo[d]isoxazole (M1)] and a cyclized oxazolidine structure [7-(((4-((( R )-tetrahydrofuran-3-yl)oxy)benzo[d]isoxazol-3-yl)oxy)methyl)octahydro-3H (M2)]. After administration of TBPT to humans the exposure to M1 was low and the exposure to M2 was high, relative to the parent drug, despite this being the opposite in vitro. In this study, projection of the plasma metabolite/parent (M/P) ratios for M1 and M2 was attempted using in vitro metabolism, binding, and permeability data in static and dynamic physiologically based pharmacokinetic (PBPK) models. In the static model, the fraction of parent clearance yielding the metabolite (which also required taking into account secondary metabolites of M1 and M2), the clearance of the metabolites and parent, and an estimate of the availability of the metabolites from the liver were combined to yield estimated parent/metabolite ratios of 0.32 and 23 for M1 and M2, respectively. PBPK modeling that used in vitro and physicochemical data input yielded estimates of 0.26 and 20, respectively. The actual values were 0.12 for M1/TBPT and 58 for M2/TBPT. Thus, the ratio for M1 was overpredicted, albeit at values less than unity. The ratio for M2/TBPT was underpredicted, and the high ratio of 58 may exceed a limiting ceiling of the approach. Nevertheless, when considered in the context of determining whether a potential circulating metabolite may be quantitatively important prior to administration of a drug for the first time to humans, the approaches succeeded in highlighting the importance of M2 (M/P ratio >> 1) relative to M1, despite M1 being much greater than M2 in vitro., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Advancement of in silico tools would be enabled by the availability of data for metabolic reaction rates and intrinsic clearance (CLint) of a diverse compound structure data set by specific metabolic enzymes. Our goal is to measure CLint for a large set of compounds with each major human cytochrome P450 (P450) isozyme. To achieve our goal, it is of utmost importance to develop an automated, robust, sensitive, high-throughput metabolic stability assay that can efficiently handle a large volume of compound sets. The substrate depletion method [in vitro half-life (t1/2) method] was chosen to determine CLint The assay (384-well format) consisted of three parts: 1) a robotic system for incubation and sample cleanup; 2) two different integrated, ultraperformance liquid chromatography/mass spectrometry (UPLC/MS) platforms to determine the percent remaining of parent compound, and 3) an automated data analysis system. The CYP3A4 assay was evaluated using two long t1/2 compounds, carbamazepine and antipyrine (t1/2 > 30 minutes); one moderate t1/2 compound, ketoconazole (10 < t1/2 < 30 minutes); and two short t1/2 compounds, loperamide and buspirone (t½ < 10 minutes). Interday and intraday precision and accuracy of the assay were within acceptable range (∼12%) for the linear range observed. Using this assay, CYP3A4 CLint and t1/2 values for more than 3000 compounds were measured. This high-throughput, automated, and robust assay allows for rapid metabolic stability screening of large compound sets and enables advanced computational modeling for individual human P450 isozymes., (U.S. Government work not protected by U.S. copyright.)

Major circulating drug metabolites can be as important as the drugs themselves in efficacy and safety, so establishing methods whereby exposure to major metabolites following administration of parent drug can be predicted is important. In this study, imipramine, a tricyclic antidepressant, and its major metabolite desipramine were selected as a model system to develop metabolite prediction methods. Imipramine undergoes N-demethylation to form the active metabolite desipramine, and both imipramine and desipramine are converted to hydroxylated metabolites by the polymorphic enzyme CYP2D6. The objective of the present study is to determine whether the human pharmacokinetics of desipramine following dosing of imipramine can be predicted using static and dynamic physiologically-based pharmacokinetic (PBPK) models from in vitro input data for CYP2D6 extensive metabolizer (EM) and poor metabolizer (PM) populations. The intrinsic metabolic clearances of parent drug and metabolite were estimated using human liver microsomes (CYP2D6 PM and EM) and hepatocytes. Passive diffusion clearance of desipramine, used in the estimation of availability of the metabolite, was predicted from passive permeability and hepatocyte surface area. The predicted area under the curve (AUCm/AUCp) of desipramine/imipramine was 12- to 20-fold higher in PM compared with EM subjects following i.v. or oral doses of imipramine using the static model. Moreover, the PBPK model was able to recover simultaneously plasma profiles of imipramine and desipramine in populations with different phenotypes of CYP2D6. This example suggested that mechanistic PBPK modeling combined with information obtained from in vitro studies can provide quantitative solutions to predict in vivo pharmacokinetics of drugs and major metabolites in a target human population., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Drug interactions elicited through inhibition of cytochrome P450 (P450) enzymes are important in pharmacotherapy. Recently, greater attention has been focused on not only parent drugs inhibiting P450 enzymes but also on possible inhibition of these enzymes by circulating metabolites. In this report, an ex vivo method whereby the potential for circulating metabolites to be inhibitors of P450 enzymes is described. To test this method, seven drugs and their known plasma metabolites were added to control human plasma at concentrations previously reported to occur in humans after administration of the parent drug. A volume of plasma for each drug based on the known inhibitory potency and time-averaged concentration of the parent drug was extracted and fractionated by high-pressure liquid chromatography-mass spectrometry, and the fractions were tested for inhibition of six human P450 enzyme activities (CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A4). Observation of inhibition in fractions that correspond to the retention times of metabolites indicates that the metabolite has the potential to contribute to P450 inhibition in vivo. Using this approach, norfluoxetine, hydroxyitraconazole, desmethyldiltiazem, desacetyldiltiazem, desethylamiodarone, hydroxybupropion, erythro-dihydrobupropion, and threo-dihydrobupropion were identified as circulating metabolites that inhibit P450 activities at a similar or greater extent as the parent drug. A decision tree is presented outlining how this method can be used to determine when a deeper investigation of the P450 inhibition properties of a drug metabolite is warranted., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Replacement of hydrogen with fluorine is a useful drug design strategy when decreases in cytochrome P450 (P450) metabolic lability are needed. In this paper, a facile two-step method of inserting fluorine into metabolically labile sites of drug molecules is described that utilizes less than 1 mg of starting material and quantitative NMR spectroscopy to ascertain the structures and concentrations of products. In the first step, hydroxyl metabolites are biosynthesized using human P450 enzymes, and in the second step these metabolites are subjected to deoxyfluorination using diethylaminosulfur trifluoride (DAST). The method is demonstrated using midazolam, celecoxib, ramelteon, and risperidone as examples and CYP3A5, 2C9, 1A2, and 2D6 to catalyze the hydroxylations. The drugs and their fluoro analogs were tested for metabolic lability. 9-Fluororisperidone and 4'-fluorocelecoxib were 16 and 4 times more metabolically stable than risperidone and celecoxib, respectively, and 2-fluororamelteon and ramelteon were metabolized at the same rate. 1'-Fluoromidazolam was metabolized at the same rate as midazolam by CYP3A4 but was more stable in CYP3A5 incubations. The P450-catalyzed sites of metabolism of the fluorine-containing analogs were determined. Some of the metabolites arose via metabolism at the fluorine-substituted carbon, wherein the fluorine was lost to yield aldehydes. In summary, this method offers an approach whereby fluorine can be substituted in metabolically labile sites, and the products can be tested to determine whether an enhancement in metabolic stability was obtained., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Methods to predict the pharmacokinetics of drugs in humans from in vitro data have been established, but corresponding methods to predict exposure to circulating metabolites are unproven. The objective of this study was to use in vitro methods combined with static and dynamic physiologically based pharmacokinetic (PBPK) models to predict metabolite exposures, using midazolam and its major metabolites as a test system. Intrinsic clearances (CLint) of formation of individual metabolites were determined using human liver microsomes. Metabolic CLintof hydroxymidazolam metabolites via oxidation and glucuronidation were also determined. Passive diffusion intrinsic clearances of hydroxymidazolam metabolites were determined using sandwich cultured human hepatocytes and the combination of this term along with the metabolic CLint, and liver blood flow was used to estimate the fraction of the metabolite that can enter the systemic circulation after formation in the liver. The metabolite/parent drug area under the plasma concentration-time curve ratio (AUCm/AUCp) was predicted using a static model relating the fraction of midazolam clearance to each metabolite, the clearance rates of midazolam and hydroxymidazolam metabolites, and the availability of the metabolites. Additionally, the human disposition of midazolam metabolites was simulated using a SimCYP PBPK model. Both approaches yielded AUCm/AUCpratios that were in agreement with the in vivo ratios. This study shows that in vivo midazolam metabolite exposure can be predicted from in vitro data and PBPK modeling. This study emphasized the importance of metabolite systemic availability from its tissue of formation, which remains a challenge to quantitative prediction., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Laboratory animal models are the industry standard for preclinical risk assessment of drug candidates. Thus, it is important that these species possess profiles of drug metabolites that are similar to those anticipated in human, since metabolites also could be responsible for biologic activities or unanticipated toxicity. Under most circumstances, preclinical species reflect human in vivo metabolites well; however, there have been several notable exceptions, and understanding and predicting these exceptions with an in vitro system would be very useful. Human micropatterned cocultured (MPCC) hepatocytes have been shown to recapitulate human in vivo qualitative metabolic profiles, but the same demonstration has not been performed yet for laboratory animal species. In this study, we investigated several compounds that are known to produce human-unique metabolites through CYP2C9, UGT1A4, aldehyde oxidase (AO), or N-acetyltransferase that were poorly covered or not detected at all in the selected preclinical species. To perform our investigation we used 24-well MPCC hepatocyte plates having three individual human donors and a single donor each of monkey, dog, and rat to study drug metabolism at four time points per species. Through the use of the multispecies MPCC hepatocyte system, the metabolite profiles of the selected compounds in human donors effectively captured the qualitative in vivo metabolite profile with respect to the human metabolite of interest. Human-unique metabolites that were not detected in vivo in certain preclinical species (normally dog and rat) were also not generated in the corresponding species in vitro, confirming that the MPCC hepatocytes can provide an assessment of preclinical species metabolism. From these results, we conclude that multispecies MPCC hepatocyte plates could be used as an effective in vitro tool for preclinical understanding of species metabolism relative to humans and aid in the choice of appropriate preclinical models., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Cerlapirdine (SAM-531, PF-05212365) is a selective, potent, full antagonist of the 5-hydroxytryptamine 6 (5-HT6) receptor. Cerlapirdine and other 5-HT6 receptor antagonists have been in clinical development for the symptomatic treatment of Alzheimer's disease. A human absorption, distribution, metabolism, and excretion study was conducted to gain further understanding of the metabolism and disposition of cerlapirdine. Because of the low amount of radioactivity administered, total (14)C content and metabolic profiles in plasma, urine, and feces were determined using accelerator mass spectrometry (AMS). After a single, oral 5-mg dose of [(14)C]cerlapirdine (177 nCi), recovery of total (14)C was almost complete, with feces being the major route of elimination of the administered dose, whereas urinary excretion played a lesser role. The extent of absorption was estimated to be at least 70%. Metabolite profiling in pooled plasma samples showed that unchanged cerlapirdine was the major drug-related component in circulation, representing 51% of total (14)C exposure in plasma. One metabolite (M1, desmethylcerlapirdine) was detected in plasma, and represented 9% of the total (14)C exposure. In vitro cytochrome P450 reaction phenotyping studies showed that M1 was formed primarily by CYP2C8 and CYP3A4. In pooled urine samples, three major drug-related peaks were detected, corresponding to cerlapirdine-N-oxide (M3), cerlapirdine, and desmethylcerlapirdine. In feces, cerlapirdine was the major (14)C component excreted, followed by desmethylcerlapirdine. The results of this study demonstrate that the use of the AMS technique enables comprehensive quantitative elucidation of the disposition and metabolic profiles of compounds administered at a low radioactive dose., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

The contribution of drug metabolites to the pharmacologic and toxicologic activity of a drug can be important; however, for a variety of reasons metabolites can frequently be difficult to synthesize. To meet the need of having samples of drug metabolites for further study, we have developed biosynthetic methods coupled with quantitative NMR spectroscopy (qNMR) to generate solutions of metabolites of known structure and concentration. These quantitative samples can be used in a variety of ways when a synthetic sample is unavailable, including pharmacologic assays, standards for in vitro work to help establish clearance pathways, and/or as analytical standards for bioanalytical work to ascertain exposure, among others. We illustrate five examples of metabolite biosynthesis and qNMR. The types of metabolites include one glucuronide and four oxidative products. Concentrations of the isolated metabolite stock solutions ranged from 0.048 to 8.3 mM, with volumes from approximately 0.04 to 0.150 ml in hexadeutarated dimethylsulfoxide. These specific quantified isolates were used as standards in the drug discovery setting as substrates in pharmacology assays, for bioanalytical assays to establish exposure, and in variety of routine absorption, distribution, metabolism, and excretion assays, such as protein binding and determining blood-to-plasma ratios. The methods used to generate these materials are described in detail with the objective that these methods can be generally used for metabolite biosynthesis and isolation., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

Metabolism by cytochrome P4503A (CYP3A) is the most prevalent clearance pathway for drugs. Designation of metabolism by CYP3A commonly refers to the potential contribution by one or both of two enzymes, CYP3A4 and CYP3A5. The metabolic turnover of 32 drugs known to be largely metabolized by CYP3A was examined in human liver microsomes (HLMs) from CYP3A5 expressers (*1/*1 genotype) and nonexpressers (*3/*3 genotype) in the presence and absence of ketoconazole and CYP3cide (a selective CYP3A4 inactivator) to calculate the contribution of CYP3A5 to metabolism. Drugs with the highest contribution of CYP3A5 included atazanavir, vincristine, midazolam, vardenafil, otenabant, verapamil, and tacrolimus, whereas 17 of the 32 tested showed negligible CYP3A5 contribution. For specific reactions in HLMs from *1/*1 donors, CYP3A5 contributes 55% and 44% to midazolam 1'- and 4-hydroxylation, 16% to testosterone 6β-hydroxylation, 56% and 19% to alprazolam 1'- and 4-hydroxylation, 10% to tamoxifen N-demethylation, and 58% to atazanavir p-hydroxylation. Comparison of the in vitro observations to clinical pharmacokinetic data showed only a weak relationship between estimated contribution by CYP3A5 and impact of CYP3A5 genotype on oral clearance, in large part because of the scatter in clinical data and the low numbers of study subjects used in CYP3A5 pharmacogenetics studies. These data should be useful in guiding which drugs should be evaluated for differences in pharmacokinetics and metabolism between subjects expressing CYP3A5 and those who do not express this enzyme., (Copyright © 2014 by The American Society for Pharmacology and Experimental Therapeutics.)

The prediction of human drug metabolites using in vitro experiments containing human-derived reagents is an important approach in modern drug research; however, this can be challenging for drugs that are slowly metabolized. In this report, we describe the use of a recently developed human hepatocyte relay method for the purpose of predicting human drug metabolite profiles. Five compounds for which in vivo human metabolism data were available were selected for the investigation of this method, and the results were compared with data gathered in hepatocyte suspensions as well as previous data from a micropatterned hepatocyte coculture method. The hepatocyte relay method demonstrated an improved performance (generation of 75% of human in vivo metabolites) for those drugs for which previous methods showed a relatively low rate of success (50% of human in vivo metabolites). Metabolites included those arising from both oxidative and conjugative reactions and metabolites that required sequential reactions. Two 4-hour relays were shown to adequately generate metabolites, and no further benefit was derived from more relays. Overall, it can be concluded that the hepatocyte relay assay method can be successfully used in the generation of relevant human metabolites, even for challenging drugs.

Understanding drug-drug interactions (DDIs) is a key component of clinical practice ensuring patient safety and efficacy of medicines. The role of drug metabolites in DDIs is a developing area of science, and has been recently highlighted in a draft regulatory guidance. The guidance states that metabolites representing ≥25% of the parent drug's area under the plasma concentration/time curve and/or >10% of exposure of total drug-related material should trigger in vitro characterization of metabolites for cytochrome P450 inhibition and propensity for DDIs. The relationship between in vitro cytochrome P450 inhibitory potency, systemic exposure, and DDI potential of drug metabolites was examined using the Pfizer development database to identify compounds with pre-existing in vivo biotransformation data, where circulating metabolites were identified in humans. The database yielded 33 structurally diverse compounds with collectively 115 distinct circulating metabolites. Of these, 52% (60/115) achieved exposures >25% of parent drug levels as judged from mass balance/metabolite identification studies. It was noted that 14 metabolite standards for 12 parent drugs had been synthesized, monitored in clinical studies, and examined for cytochrome P450 inhibition. For the 14 metabolite/parent drug pairs, no clinically relevant DDIs were expected to occur against the major human cytochrome P450 isoforms. A review of the literature for parent/metabolite DDI information was also conducted to examine trends using a larger data set. Leveraging the analysis of both internal and literature-based data sets, an algorithm was devised for use in drug discovery/early development to assess cytochrome P450 inhibitory potential of drug metabolites and the propensity to cause a clinically relevant DDI.

Prediction of human pharmacokinetics of new drugs, as well as other disposition attributes, has become a routine practice in drug research and development. Prior to the 1990s, drug disposition science was used in a mostly descriptive manner in the drug development phase. With the advent of in vitro methods and availability of human-derived reagents for in vitro studies, drug-disposition scientists became engaged in the compound design phase of drug discovery to optimize and predict human disposition properties prior to nomination of candidate compounds into the drug development phase. This has reaped benefits in that the attrition rate of new drug candidates in drug development for reasons of unacceptable pharmacokinetics has greatly decreased. Attributes that are predicted include clearance, volume of distribution, half-life, absorption, and drug-drug interactions. In this article, we offer our experience-based perspectives on the tools and methods of predicting human drug disposition using in vitro and animal data.

In vitro-in vivo correlation (IVIVC) of intrinsic clearance in preclinical species of rat and dog was established using the hepatocyte relay method to support high-confidence prediction of human pharmacokinetics for low-clearance compounds. Good IVIVC of intrinsic clearance was observed for most of the compounds, with predicted values within 2-fold of the observed values. The exceptions involved transporter-mediated uptake clearance or metabolizing enzymes with extensive extrahepatic contribution. This is the first assay available to address low clearance challenges in preclinical species for IVIVC in drug discovery. It extends the utility of the hepatocyte relay method in addressing low clearance issues.

Aldehyde oxidase 1 (AOX1) is a cytosolic enzyme highly expressed in liver and plays a key role in metabolizing drugs containing aromatic azaheterocyclic substituents. Rapid metabolism catalyzed by AOX1 can cause a drug to exhibit high clearance, low exposure, and hence decreased efficacy or even increased toxicity (if AOX1 generated metabolites are toxic). There is a need to develop the correlation between AOX1 expression levels and AOX1-substrate clearance. A fast, sensitive, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed to quantify AOX1 in human liver cytosol for the first time. This LC-MS/MS method includes a straightforward ultrafiltration fractionation step and gives great selectivity and wide dynamic range (5.2 pM to 20.7 nM). The AOX1 levels in human liver cytosols of 20 donors were quantified using this method to investigate individual differences in AOX1 expression. No significant individual or gender differences in AOX1 levels were observed, although male donors exhibited a broader distribution than female donors (0.74-2.30 pmol/mg versus 0.74-1.69 pmol/mg, respectively). The AOX1 protein levels measured by LC-MS/MS were consistent with those measured by an enzyme-linked immunosorbent assay. Several donors have a normal AOX1 protein level but low enzyme activity, which might be due to cofactor deficiency, single nucleotide polymorphism, or homodimer dissociation. Cytosols from donors with chronic alcohol consumption had low AOX1-catalyzed carbazeran oxidation activities (<51 µl/min per milligram compared with a median of 455 µl/min per milligram), but preserved similar AOX1 protein expression levels (approximately 15% less than the median value).

Time-dependent inhibition (TDI) of cytochrome P450 (P450) enzymes, especially CYP3A4, is an important attribute of drugs in evaluating the potential for pharmacokinetic drug-drug interactions. The analysis of TDI data for P450 enzymes can be challenging, yet it is important to be able to reliably evaluate whether a drug is a TDI or not, and if so, how best to derive the inactivation kinetic parameters K(I) and k(inact). In the present investigation a two-step statistical evaluation was developed to evaluate CYP3A4 TDI data. In the first step, a two-sided two-sample z-test is used to compare the k(obs) values measured in the absence and presence of the test compound to answer the question of whether the test compound is a TDI or not. In the second step, k(obs) values are plotted versus both [I] and ln[I] to determine whether a significant correlation exists, which can then inform the investigator of whether the inactivation kinetic parameters, K(I) and k(inact), can be reliably estimated. Use of this two-step statistical evaluation is illustrated with the examination of five drugs of varying capabilities to inactivate CYP3A4: ketoconazole, erythromycin, raloxifene, rosiglitazone, and pioglitazone. The use of a set statistical algorithm offers a more robust and objective approach to the analysis of P450 TDI data than frequently employed empirically derived or heuristic approaches.

A novel relay method has been developed using cryopreserved human hepatocytes to measure intrinsic clearance of low-clearance compounds. The relay method involved transferring the supernatant from hepatocyte incubations to freshly thawed hepatocytes at the end of the 4-h incubation to prolong the exposure time to active enzymes in hepatocytes. An accumulative incubation time of 20 h or longer in hepatoctyes can be achieved using the method. The relay method was validated using seven commercial drugs (diazepam, disopyramide, theophylline, timolol, tolbutamide, S-warfarin, and zolmitriptan) that were metabolized by various cytochrome P450s with low human in vivo intrinsic clearance at approximately 2 to 15 ml · min⁻¹ · kg⁻¹. The results showed that the relay method produced excellent predictions of human in vivo clearance. The difference between in vitro and in vivo intrinsic clearance was within 2-fold for most compounds, which is similar to the standard prediction accuracy for moderate to high clearance compounds using hepatocytes. The relay method is a straightforward, relatively low cost, and easy-to-use new tool to address the challenges of low clearance in drug discovery and development.

CYP3cide (PF-4981517; 1-methyl-3-[1-methyl-5-(4-methylphenyl)-1H-pyrazol-4-yl]-4-[(3S)-3-piperidin-1-ylpyrrolidin-1-yl]-1H-pyrazolo[3,4-d]pyrimidine) is a potent, efficient, and specific time-dependent inactivator of human CYP3A4. When investigating its inhibitory properties, an extreme metabolic inactivation efficiency (k(inact)/K(I)) of 3300 to 3800 ml · min⁻¹ · μmol⁻¹ was observed using human liver microsomes from donors of nonfunctioning CYP3A5 (CYP3A5 *3/*3). This observed efficiency equated to an apparent K(I) between 420 and 480 nM with a maximal inactivation rate (k(inact)) equal to 1.6 min⁻¹. Similar results were achieved with testosterone, another CYP3A substrate, and other sources of the CYP3A4 enzyme. To further illustrate the abilities of CYP3cide, its partition ratio of inactivation was determined with recombinant CYP3A4. These studies produced a partition ratio approaching unity, thus underscoring the inactivation capacity of CYP3cide. When CYP3cide was tested at a concentration and preincubation time to completely inhibit CYP3A4 in a library of genotyped polymorphic CYP3A5 microsomes, the correlation of the remaining midazolam 1'-hydroxylase activity to CYP3A5 abundance was significant (R² value equal to 0.51, p value of <0.0001). The work presented here supports these findings by fully characterizing the inhibitory properties and exploring CYP3cide's mechanism of action. To aid the researcher, multiple commercially available sources of CYP3cide were established, and a protocol was developed to quantitatively determine CYP3A4 contribution to the metabolism of an investigational compound. Through the establishment of this protocol and the evidence provided here, we believe that CYP3cide is a very useful tool for understanding the relative roles of CYP3A4 versus CYP3A5 and the impact of CYP3A5 genetic polymorphism on a compound's pharmacokinetics.

Aldehyde oxidase (AO) metabolism could lead to significant underestimation of clearance in prediction of human pharmacokinetics as well as unanticipated exposure to AO-generated metabolites, if not accounted for early in drug research. We report a method using cryopreserved human hepatocytes and the time-dependent AO inhibitor hydralazine (K(I) = 83 ± 27 μM, k(inact) = 0.063 ± 0.007 min(-1)), which estimates the contribution of AO metabolism relative to total hepatic clearance. Using zaleplon as a probe substrate and simultaneously monitoring the AO-catalyzed formation of oxozaleplon and the CYP3A-catalyzed formation of desethyzaleplon in the presence of a range of hydralazine concentrations, it was determined that >90% inhibition of the AO activity with minimal effect on the CYP3A activity could be achieved with 25 to 50 μM hydralazine. This method was used to estimate the fraction metabolized due to AO [f(m(AO))] for six compounds with clearance attributed to AO along with four other drugs not metabolized by AO. The f(m(AO)) values for the AO substrates ranged between 0.49 and 0.83. Differences in estimated f(m(AO)) between two batches of pooled human hepatocytes suggest that sensitivity to hydralazine varies slightly with hepatocyte preparations. Substrates with a CYP2D6 contribution to clearance were affected by hydralazine to a minor extent, because of weak inhibition of this enzyme. Overall, these findings demonstrate that hydralazine, at a concentration of 25 to 50 μM, can be used in human hepatocyte incubations to estimate the contribution of AO to the hepatic clearance of drugs and other compounds.

We previously described a simple liquid chromatography-tandem mass spectrometry (LC-MS/MS) method to determine relative plasma exposures of drug metabolites across species for metabolite safety assessments. It offers time- and resource-sparing advantages to ascertain metabolite exposure comparisons between humans and laboratory animal species for stable metabolites with high confidence. In this study, we tested the limitation of the methodology with compounds possessing six substituents found in unstable metabolites. Stabilization procedures were used, and stabilized samples were compared with untreated samples for structures with established stabilization processes. In most cases, the parent compounds with established stability were used as the intrinsic stability references except in cases in which the metabolite was more stable than the parent compound. Long-term storage stability of the unstable structures was tested by comparing the response ratio of the metabolite to the stability reference compound for multiple independent analyses covering the storage duration. Autosampler stability was tested using the same response ratio of the reinjections of the reconstituted solution overnight over the first injections. The results supported that the possibility that an abbreviated LC-MS/MS peak area ratio comparison can be applied to epoxide, amide, catechol, and acyl glucuronides to determine the relative plasma exposure of drug metabolites across species; but it may not be suitable for iminium ions and esters. Stability of suspected unstable metabolites can be tested using the methodology described above.

The measurement of the effect of new chemical entities on human UDP-glucuronosyltransferase (UGT) marker activities using in vitro experimentation represents an important experimental approach in drug development to guide clinical drug-interaction study designs or support claims that no in vivo interaction will occur. Selective high-performance liquid chromatography-tandem mass spectrometry functional assays of authentic glucuronides for five major hepatic UGT probe substrates were developed: β-estradiol-3-glucuronide (UGT1A1), trifluoperazine-N-glucuronide (UGT1A4), 5-hydroxytryptophol-O-glucuronide (UGT1A6), propofol-O-glucuronide (UGT1A9), and zidovudine-5'-glucuronide (UGT2B7). High analytical sensitivity permitted characterization of enzyme kinetic parameters at low human liver microsomal and recombinant UGT protein concentration (0.025 mg/ml), which led to a new recommended optimal universal alamethicin activation concentration of 10 μg/ml for microsomes. Alamethicin was not required for recombinant UGT incubations. Apparent enzyme kinetic parameters, particularly for UGT1A1 and UGT1A4, were affected by nonspecific binding. Unbound intrinsic clearance for UGT1A9 and UGT2B7 increased significantly after addition of 2% bovine serum albumin, with minimal changes for UGT1A1, UGT1A4, and UGT1A6. Eleven potential UGT and cytochrome P450 inhibitors were evaluated as UGT inhibitors, resulting in observation of nonselective UGT inhibition by chrysin, mefenamic acid, silibinin, tangeretin, ketoconazole, itraconazole, ritonavir, and verapamil. The pan-cytochrome P450 inhibitor, 1-aminobenzotriazole, minimally inhibited UGT activities and may be useful in reaction phenotyping of mixed UGT and cytochrome P450 substrates. These methods should prove useful in the routine assessments of the potential for new drug candidates to elicit pharmacokinetic drug interactions via inhibition of human UGT activities and the identification of UGT enzyme-selective chemical inhibitors.

The pharmacokinetic properties of drugs may be altered by kinetic deuterium isotope effects. With specifically deuterated model substrates and drugs metabolized by aldehyde oxidase, we demonstrate how knowledge of the enzyme's reaction mechanism, species differences in the role played by other enzymes in a drug's metabolic clearance, and differences in systemic clearance mechanisms are critically important for the pharmacokinetic application of deuterium isotope effects. Ex vivo methods to project the in vivo outcome using deuterated carbazeran and zoniporide with hepatic systems demonstrate the importance of establishing the extent to which other metabolic enzymes contribute to the metabolic clearance mechanism. Differences in pharmacokinetic outcomes in guinea pig and rat, with the same metabolic clearance mechanism, show how species differences in the systemic clearance mechanism can affect the in vivo outcome. Overall, to gain from the application of deuteration as a strategy to alter drug pharmacokinetics, these studies demonstrate the importance of understanding the systemic clearance mechanism and knowing the identity of the metabolic enzymes involved, the extent to which they contribute to metabolic clearance, and the extent to which metabolism contributes to the systemic clearance.

Recent regulatory guidance suggests that metabolites identified in human plasma should be present at equal or greater levels in one of the animal species used in safety assessments. In this report, a high-performance liquid chromatography-tandem mass spectrometry method is described whereby quantitative comparisons of exposures to metabolites between species can be obtained in the absence of authentic standards of the metabolites, calibration curves, and other attributes of standard bioanalytical methods. This novel method was tested using six drug-metabolite combinations. Plasma samples from animals are mixed with control plasma from humans and vice versa to remove possible differential effects of matrices. Through multiple ion monitoring-triggered enhanced product ion (EPI) scans, all metabolites were qualitatively confirmed, and daughter ions were selected for the most sensitive mass transitions to trigger EPI scans. Direct comparisons of metabolites in animal versus human plasma were achieved by calculating the peak area ratios of the metabolites versus an internal standard. Linearity of instrument responses was established by serial dilution. A statistical analysis demonstrated that experimentally measured ratios of the parent and metabolites in rat versus human correlated well with the nominal ratios of concentrations using linear regression with an average slope of 0.99 ± 0.08 (r = 0.994 ± 0.005). This analysis showed that if the experimentally determined ratio of mass spectrometer responses is ≥ 2.0, then the actual exposure ratio is unity or greater (p < 0.01). This method offers time- and resource-sparing advantages to ascertaining metabolite exposure comparisons between humans and laboratory animal species. A strategy for application of this approach within standard drug development processes is described.

The metabolism, pharmacokinetics, and excretion of a potent and selective 5-hydroxytryptamine(1B) receptor antagonist elzasonan have been studied in six healthy male human subjects after oral administration of a single 10-mg dose of [(14)C]elzasonan. Total recovery of the administered dose was 79% with approximately 58 and 21% of the administered radioactive dose excreted in feces and urine, respectively. The average t(1/2) for elzasonan was 31.5 h. Elzasonan was extensively metabolized, and excreta and plasma were analyzed using mass spectrometry and NMR spectroscopy to elucidate the structures of metabolites. The major component of drug-related material in the excreta was in the feces and was identified as 5-hydroxyelzasonan (M3), which accounted for approximately 34% of the administered dose. The major human circulating metabolite was identified as the novel cyclized indole metabolite (M6) and accounted for ∼65% of the total radioactivity. A mechanism for the formation of M6 is proposed. Furthermore, metabolism-dependent covalent binding of drug-related material was observed upon incubation of [(14)C]elzasonan with liver microsomes, and data suggest that an indole iminium ion is involved. Overall, the major metabolic pathways of elzasonan were due to aromatic hydroxylation(s) of the benzylidene moiety, N-oxidation at the piperazine ring, N-demethylation, indirect glucuronidation, and oxidation, ring closure, and subsequent rearrangement to form M6.

Metabolism is one of the important determinants of the overall disposition of drugs, and the profile of metabolites can have an impact on efficacy and safety. Predicting which drug metabolites will be quantitatively predominant in humans has become increasingly important in the research and development of new drugs. In this study, a novel micropatterned hepatocyte coculture system was evaluated for its ability to generate human in vivo metabolites. Twenty-seven compounds of diverse chemical structure and subject to a range of drug biotransformation reactions were assessed for metabolite profiles in the micropatterned coculture system using pooled cryopreserved human hepatocytes. The ability of this system to generate metabolites that are >10% of dose in excreta or >10% of total drug-related material in circulation was assessed and compared to previously reported data obtained in human hepatocyte suspensions, liver S-9 fraction, and liver microsomes. The micropatterned coculture system was incubated for up to 7 days without a change in medium, which offered an ability to generate metabolites for slowly metabolized compounds. The micropatterned coculture system generated 82% of the excretory metabolites that exceed 10% of dose and 75% of the circulating metabolites that exceed 10% of total circulating drug-related material, exceeds the performance of hepatocyte suspension incubations and other in vitro systems. Phase 1 and phase 2 metabolites were generated, as well as metabolites that arise via two or more sequential reactions. These results suggest that this in vitro system offers the highest performance among in vitro metabolism systems to predict major human in vivo metabolites.

Induction of cytochrome P450 (P450) activity in the clinic can result in therapeutic failure such as tissue rejection in transplant patients or unwanted pregnancy, among others. CYP3A4 is by far the most abundant isoform and is responsible for the majority of P450-related metabolism of all marketed drugs. However, it is of importance to understand the significance of induction mediated through other P450 enzymes. The objective of this investigation was to evaluate several known inducers in vitro using cryopreserved human hepatocytes, with the aim of assessing the relevant induction of CYP3A4, CYP2B6, CYP2C9, CYP2C19, and CYP3A5, based on mRNA expression. CYP3A4 induction was also assessed based on enzymatic activity in three different lots to investigate whether mRNA expression data have any advantages over enzymatic activity. In general, the mRNA fold-induction data results were more sensitive compared with activity data, and more informative in cases in which the drug is also a P450 inhibitor. The induction of transcription of other drug-metabolizing enzymes including CYP2B6 and CYP2C enzymes occurred every time that CYP3A4 mRNA levels increased, but to a lesser extent, indicating that measurement of CYP3A4 mRNA is a sensitive marker for the induction of these other enzymes. This was the case even for enzymes and inducers that are known to also act via the constitutive androstane receptor pathway. Finally, the utility of in vitro induction measurements in the identification of clinically meaningful inducers was tested by using two simple binary classification approaches: 1) fold-induction versus vehicle control and 2) induction response relative to rifampin. The best classification was observed when the cutoff criteria based on fold induction relative to the vehicle control, using mRNA data are used.

Ramelteon is a melatonin receptor agonist used as a treatment for insomnia. It is subject to a remarkably large drug-drug interaction (DDI) caused by fluvoxamine coadministration, resulting in a more than 100-fold increase in exposure. The objective of this study was to determine whether the DDI could be estimated using in vitro metabolism data. Ramelteon was shown to undergo hydroxylation in human liver microsomes to eight metabolites via six pathways. The main routes of metabolism included hydroxylation on the ethyl side chain and the benzylic position of the cyclopentyl ring, as assessed through enzyme kinetic measurements. Hydroxylation at the other benzylic position was observed in human intestinal microsomes. Ramelteon metabolism was catalyzed by CYP1A2, CYP2C19, and CYP3A4 as shown through the use of recombinant human cytochrome P450 enzymes and specific inhibitors. In liver, CYP1A2, CYP2C19, and CYP3A4 were estimated to contribute 49, 42, and 8.6%, respectively, whereas in intestine only CYP3A4 contributes. The in vitro data were used to estimate the magnitudes of DDI caused by ketoconazole, fluconazole, and fluvoxamine. The DDIs caused by the former were reliably estimated (1.82-fold estimated versus 1.82-fold actual for ketoconazole; 2.99-fold estimated versus 2.36-fold actual for fluconazole), whereas for fluvoxamine it was underestimated (11.4-fold estimated versus 128-fold actual). This suggests that there may be a limit on the magnitude of DDI that can be estimated from in vitro data. Nevertheless, the example of the fluvoxamine-ramelteon DDI offers a unique example wherein one drug can simultaneously inhibit multiple enzymatic pathways of a second drug.

The ability to predict in vivo clearance from in vitro intrinsic clearance for compounds metabolized by aldehyde oxidase has not been demonstrated. To date, there is no established scaling method for predicting aldehyde oxidase-mediated clearance using in vitro or animal data. This challenge is exacerbated by the fact that rats and dogs, two of the laboratory animal species commonly used to develop in vitro-in vivo correlations of clearance, differ from humans with regard to expression of aldehyde oxidase. The objective of this investigation was to develop an in vitro-in vivo correlation of intrinsic clearance for aldehyde oxidase, using 11 drugs known to be metabolized by this enzyme. The set consisted of methotrexate, XK-469, (+/-)-4-(4-cyanoanilino)-5,6-dihydro-7-hydroxy-7H-cyclopenta[d]pyrimidine (RS-8359), zaleplon, 6-deoxypenciclovir, zoniporide, O(6)-benzylguanine, N-[(2'-dimethylamino)ethyl]acridine-4-carboxamide (DACA), carbazeran, PF-4217903, and PF-945863. These compounds were assayed using two in vitro systems (pooled human liver cytosol and liver S-9 fractions) to calculate scaled unbound intrinsic clearance, and they were then compared with calculated in vivo unbound intrinsic clearance. The investigation provided a relative scale that can be used for in vitro-in vivo correlation of aldehyde oxidase clearance and suggests limits as to when a potential new drug candidate that is metabolized by this enzyme will possess acceptable human clearance, or when structural modification is required to reduce aldehyde oxidase catalyzed metabolism.

Excretion and metabolism of zoniporide were investigated in humans after intravenous infusion of [(14)C]zoniporide at an 80-mg dose. Bile was the primary route of excretion because 57% of dose was recovered in the feces after intravenous infusion. Zoniporide was primarily cleared via metabolism in humans. 2-Oxozoniporide (M1) was the major excretory and circulating metabolite in humans and was catalyzed by aldehyde oxidase (K(m) of 3.4 microM and V(max) of 74 pmol/min/mg protein). Metabolites M2 (17% of the dose) and M3 (6.4% of circulating radioactivity), in which the guanidine moiety was hydrolyzed to a carboxylic acid, were also detected in human feces and plasma, respectively, suggesting that hydrolysis was another route of metabolism of zoniporide in humans. The metabolism and excretion of [(14)C]zoniporide in rats and dogs were also evaluated. As in humans, bile was the primary route of excretion of the radiolabeled material in both species, and metabolism was the primary route of clearance. A comparison of plasma metabolites showed that for M3, rats had a higher concentration than human or dog. M1 was absent in dog and present in human and rat plasma at comparable levels, whereas comparison of excreta showed that the total body burden of M1 was greater in rat than that in human. No further evaluation of M2 was considered because it was detected only in the human fecal extracts. Hence, no further toxicological evaluation of the three human metabolites was undertaken.