Metabolism and excretion of [14C]mobocertinib, a selective covalent inhibitor of EGFR exon 20 insertion mutants, in healthy male subjects.

Mobocertinib (formerly known as TAK-788) is a targeted covalent tyrosine kinase inhibitor of epidermal growth factor receptor with exon 20 insertion mutations. This article describes the metabolism and excretion of mobocertinib in healthy male subjects after a single oral administration of [14C]mobocertinib. Mobocertinib related materials were highly covalently bound to plasma proteins such as human serum albumin. The mean extraction recovery of total radioactivity was only 3.9% for 6 individual Hamilton pooled plasma samples. After extraction, mobocertinib was the most abundant component accounting for 7.7% of total extracted circulating radioactivity (TECRA) in the supernatant. Each of identified metabolites accounted for <10% of TECRA. Mobocertinib underwent extensive first-pass metabolism with the fraction of the dose absorbed estimated to be approximately 91.7%. Fecal excretion of mobocertinib metabolites was the major elimination route. Mobocertinib was mainly eliminated via oxidative metabolism with a fraction of approximately 88% metabolized by CYP3A4/5. The other minor elimination pathways included cysteine conjugation, metabolism by other CYPs, and renal excretion of unchanged mobocertinib. Significance Statement This manuscript describes the metabolism and excretion of a targeted covalent inhibitor mobocertinib in humans after a single oral administration of [14C]mobocertinib. Mobocertinib was highly covalently bound to human plasma proteins. No metabolite accounted for >10% of total extracted circulating radioactivity in human plasma. Mobocertinib was mainly eliminated via CYP3A4/5 mediated oxidative metabolism followed by fecal excretion after approximately 91.7% of the dose was absorbed.

Investigation of the absolute bioavailability, mass balance, metabolism, and excretion of the cholesterol 24-hydroxylase inhibitor soticlestat in healthy volunteers.

AIMS: Our aim was to determine the absolute bioavailability, mass balance, metabolism and excretion of soticlestat (TAK-935). METHODS: An open-label, two-period, single-site, phase 1 study was conducted in six healthy men. In Period 1, a single 300 mg dose of soticlestat was administered orally, followed by a 15-min intravenous infusion of [14 C]soticlestat 50 µg (~1 µCi) 10 min later. In Period 2, a single 300 mg dose (~100 µCi) of [14 C]soticlestat in solution was administered orally. Samples were collected, analysed for radioactivity or unchanged soticlestat, and profiled for metabolites. RESULTS: In Period 1, soticlestat had an absolute bioavailability of 12.6% (90% confidence interval, 7.81-20.23%). In Period 2, there was near-complete recovery of total radioactivity (TRA) following a 300 mg dose of [14 C]soticlestat: urine, 94.8% (standard deviation [SD], 1.35%); faeces, 2.7% (SD, 1.67%). Of TRA, 0.1% (SD, 0.09%) and 0.6% (SD, 0.21%) were recovered as soticlestat and metabolite M-I in urine, respectively. In plasma, soticlestat and M-I reached geometric mean maximum observed concentrations of 1352 ng/mL (geometric percent coefficient of variation [gCV%], 61.3) and 253.2 ng/mL (gCV%, 44.1) after 25 min and declined with mean terminal half-lives (SD) of 5.7 (2.90) and 2.0 (0.15) h, respectively. Soticlestat represented 4.9% of TRA in plasma. Soticlestat was rapidly eliminated primarily via O-glucuronidation to metabolite M3, which was the dominant species in plasma (92.6%) and urine (86%). CONCLUSIONS: This study indicates that soticlestat and its metabolites are rapidly cleared and eliminated, lowering the risk of dose accumulation from repeated dosing and supporting further investigation of soticlestat.

Determination of Acyl-, O-, and N-Glucuronide Using Chemical Derivatization Coupled with Liquid Chromatography-High-Resolution Mass Spectrometry.

Glucuronidation is the most common phase II metabolic pathway to eliminate small molecule drugs from the body. However, determination of glucuronide structure is quite challenging by mass spectrometry due to its inability to generate structure-informative fragments about the site of glucuronidation. In this article, we describe a simple method to differentiate acyl-, O-, and N-glucuronides using chemical derivatization. The idea is that derivatization of acyl-, O-, or N-glucuronides of a molecule results in predictable and different numbers of derivatized functional groups, which can be determined by the mass shift using mass spectrometry. The following two reactions were applied to specifically derivatize carboxyl and hydroxyl groups that are present on the aglycone and its glucuronide metabolite: Carboxyl groups were activated by thionyl chloride followed by esterification with ethanol. Hydroxyl groups were derivatized via silylation by 1-(trimethylsilyl)imidazole. The mass shift per derivatized carboxyl and hydroxyl group was +28.031 Da and +72.040 Da, respectively. This approach was successfully validated using commercial glucuronide standards, including benazepril acyl-glucuronides, raloxifene O-glucuronide, and silodosin O-glucuronide. In addition, this approach was applied to determine the type of glucuronide metabolites that were isolated from liver microsomal incubation, where alvimopan and diclofenac acyl-glucuronides; darunavir, haloperidol, and propranolol O-glucuronides; and darunavir N-glucuronide were identified. Lastly, this approach was successfully used to elucidate the definitive structure of a clinically observed metabolite, soticlestat O-glucuronide. In conclusion, a novel, efficient, and cost-effective approach was developed to determine acyl-, O-, and N-glucuronides using chemical derivatization coupled with liquid chromatography-high resolution mass spectrometry. SIGNIFICANCE STATEMENT: The method described in this study can differentiate acyl-, O-, and N-glucuronides and allow for elucidation of glucuronide structures when multiple glucuronidation possibilities exist. The type of glucuronidation information is particularly useful for a drug candidate containing carboxyl groups, which can form reactive acyl-glucuronides. Additionally, the method can potentially be used for definitive structure elucidation for a glucuronide with its aglycone containing a single carboxyl, hydroxyl, or amino group even when multiple types of functional groups are present for glucuronidation.

Metabolism and Disposition of [14C]Pevonedistat, a First-in-Class NEDD8-Activating Enzyme Inhibitor, after Intravenous Infusion to Patients with Advanced Solid Tumors.

Metabolism and disposition of pevonedistat, an investigational, first-in-class inhibitor of the NEDD8-activating enzyme (NAE), were characterized in patients with advanced solid tumors after intravenous infusion of [14C]pevonedistat at 25 mg/m2 (∼60-85 µCi radioactive dose). More than 94% of the administered dose was recovered, with ∼41% and ∼53% of drug-related material eliminated in urine and feces, respectively. The metabolite profiles of [14C]pevonedistat were established in plasma using an accelerator mass spectrometer and excreta with traditional radiometric analysis. In plasma, unchanged parent drug accounted for approximately 49% of the total drug-related material. Metabolites M1 and M2 were major (>10% of the total drug-related material) circulating metabolites and accounted for approximately 15% and 22% of the drug-related material, respectively. Unchanged [14C]pevonedistat accounted for approximately 4% and 17% of the dose in urine and feces, respectively. Oxidative metabolites M1, M2, and M3 appeared as the most abundant drug-related components in the excreta and represented approximately 27%, 26%, and 15% of the administered dose, respectively. Based on the unbound plasma exposure in cancer patients and in vitro NAE inhibition, the contribution of metabolites M1 and M2 to overall in vivo pharmacological activity is anticipated to be minimal. The exposure to these metabolites was higher at safe and well tolerated doses in rat and dog (the two preclinical species used in toxicology evaluation) plasma than that observed in human plasma. Reaction phenotyping studies revealed that CYP3A4/5 are primary enzymes responsible for the metabolic clearance of pevonedistat. SIGNIFICANCE STATEMENT: This study details the metabolism and clearance mechanisms of pevonedistat, a first-in-class NEDD8-activating enzyme inhibitor, after intravenous administration to patients with cancer. Pevonedistat is biotransformed to two major circulating metabolites with higher exposure in nonclinical toxicological species than in humans. The pharmacological activity contribution of these metabolites is minimal compared to the overall target pharmacological effect of pevonedistat. Renal clearance was not an important route of excretion of unchanged pevonedistat (∼4% of the dose).

Sinusoidal Uptake Determines the Hepatic Clearance of Pevonedistat (TAK-924) as Explained by Extended Clearance Model.

Quantitative assessment of hepatic clearance (CLH) of drugs is critical to accurately predict human dose and drug-drug interaction (DDI) liabilities. This is challenging for drugs that involve complex transporter-enzyme interplay. In this study, we demonstrate this interplay in the CLH and DDI effect in the presence of CYP3A4 perpetrator for pevonedistat using both the conventional clearance model (CCM) and the extended clearance model (ECM). In vitro metabolism and hepatocyte uptake data showed that pevonedistat is actively transported into the liver via multiple uptake transporters and metabolized predominantly by CYP3A4 (88%). The active uptake clearance (CLact,inf) and passive diffusion clearance (CLdiff,inf) were 21 and 8.7 ml/min/kg, respectively. The CLact,inf was underpredicted as Empirical Scaling Factor of 13 was needed to recover the in vivo plasma clearance (CLplasma). Both CCM and ECM predicted CLplasma of pevonedistat reasonably well (predicted CLplasma of 30.8 (CCM) and 32.1 (ECM) versus observed CLplasma of 32.2 ml/min/kg). However, both systemic and liver exposures in the presence of itraconazole were well predicted by ECM but not by CCM (predicted pevonedistat plasma area under the concentration-time curve ratio (AUCR) 2.73 (CCM) and 1.23 (ECM))., The ECM prediction is in accordance with the observed clinical DDI data (observed plasma AUCR of 1.14) that showed CYP3A4 inhibition did not alter pevonedistat exposure systemically, although ECM predicted liver AUCR of 2.85. Collectively, these data indicated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat and are consistent with the lack of systemic clinical DDI with itraconazole. SIGNIFICANCE STATEMENT: In this study, we successfully demonstrated that the hepatic uptake is the rate-determining step in the CLH of pevonedistat. Both the conventional and extended clearance models predict CLplasma of pevonedistat well however, only the ECM accurately predicted DDI effect in the presence of itraconazole, thus providing further evidence for the lack of DDI with CYP3A4 perpetrators for drugs that involve complex transporter-enzyme interplay as there are currently not many examples in the literature except prototypical OATP substrate drugs.

Pharmacokinetics and Catabolism of [3H]TAK-164, a Guanylyl Cyclase C Targeted Antibody-Drug Conjugate.

TAK-164 is an antibody-drug conjugate (ADC) comprising human anti-guanylyl cyclase C (GCC) monoclonal antibody conjugated to indolinobenzodiazepine DNA alkylator IGN-P1 through a cleavable alanine-alanine dipeptide linker. TAK-164 is currently being evaluated for the treatment of gastrointestinal cancers expressing GCC. The catabolism of TAK-164 was studied using 3H-labeled ADC using GCC-expressing HEK-293 (GCC-HEK-293) cells, rat tritosomes, cathepsin B, and tumor-bearing mice. Time- and target-dependent uptake of [3H]TAK-164 was observed in GCC-HEK-293 cells with approximately 12% of radioactivity associated with DNA after 24 hours of incubation. Rat liver tritosomes and cathepsin B yielded IGN-P1 aniline, sulfonated IGN-P1 (s-IGN-P1) aniline, and a lysine conjugate of IGN-P1 (IGN-P1-Lys) aniline as catabolites. In tumor-bearing mice, [3H]TAK-164 exhibited a terminal half-life of approximately 41 and 51 hours in plasma and blood, respectively, with low plasma clearance (0.75 ml/h per kilogram). The extractable radioactivity in plasma and tumor samples revealed the presence of s-IGN-P1 aniline and IGN-P1 aniline as payload-related components. The use of a radiolabeled payload in the ADC in tumor uptake investigations provided direct and quantitative evidence for tumor uptake, DNA binding, and proof of mechanism of action of the payload. SIGNIFICANCE STATEMENT: Since payload-related species are potent cytotoxins, a thorough characterization of released products of ADCs, metabolites, and their drug interaction potential is necessary prior to clinical investigations. This study characterized in vitro and in vivo DNA binding mechanisms and released products of TAK-164. The methodologies described here will be highly useful for characterization of payload-related products of ADCs in general.

Biotransformation Pathways and Metabolite Profiles of Oral [14C]Alisertib (MLN8237), an Investigational Aurora A Kinase Inhibitor, in Patients with Advanced Solid Tumors.

Alisertib (MLN8237) is an investigational, orally available, selective aurora A kinase inhibitor in clinical development for the treatment of solid tumors and hematologic malignancies. This metabolic profiling analysis was conducted as part of a broader phase 1 study evaluating mass balance, pharmacokinetics, metabolism, and routes of excretion of alisertib following a single 35-mg dose of [14C]alisertib oral solution (∼80 µCi) in three patients with advanced malignancies. On average, 87.8% and 2.7% of the administered dose was recovered in feces and urine, respectively, for a total recovery of 90.5% by 14 days postdose. Unchanged [14C]alisertib was the predominant drug-related component in plasma, followed by O-desmethyl alisertib (M2), and alisertib acyl glucuronide (M1), which were present at 47.8%, 34.6%, and 12.0% of total plasma radioactivity. In urine, of the 2.7% of the dose excreted, unchanged [14C]alisertib was a negligible component (trace), with M1 (0.84% of dose) and glucuronide conjugate of hydroxy alisertib (M9; 0.66% of dose) representing the primary drug-related components in urine. Hydroxy alisertib (M3; 20.8% of the dose administered) and unchanged [14C]alisertib (26.3% of the dose administered) were the major drug-related components in feces. In vitro, oxidative metabolism of alisertib was primarily mediated by CYP3A. The acyl glucuronidation of alisertib was primarily mediated by uridine 5'-diphospho-glucuronosyltransferase 1A1, 1A3, and 1A8 and was stable in 0.1 M phosphate buffer and in plasma and urine. Further in vitro evaluation of alisertib and its metabolites M1 and M2 for cytochrome P450-based drug-drug interaction (DDI) showed minimal potential for perpetrating DDI with coadministered drugs. Overall, renal elimination played an insignificant role in the disposition of alisertib, and metabolites resulting from phase 1 oxidative pathways contributed to >58% of the alisertib dose recovered in urine and feces over 192 hours postdose. SIGNIFICANCE STATEMENT: This study describes the primary clearance pathways of alisertib and illustrates the value of timely conduct of human absorption, distribution, metabolism, and excretion studies in providing guidance to the clinical pharmacology development program for oncology drugs, for which a careful understanding of sources of exposure variability is crucial to inform risk management for drug-drug interactions given the generally limited therapeutic window for anticancer drugs and polypharmacy that is common in cancer patients.

Mass balance, routes of excretion, and pharmacokinetics of investigational oral [14C]-alisertib (MLN8237), an Aurora A kinase inhibitor in patients with advanced solid tumors.

Aims This two-part, phase I study evaluated the mass balance, excretion, pharmacokinetics and safety of the investigational aurora A kinase inhibitor, alisertib, in three patients with advanced malignancies. Methods Part A; patients received a single 35-mg dose of [14C]-alisertib oral solution (~80 µCi total radioactivity [TRA]). Serial blood, urine, and fecal samples were collected up to 336 h post-dose for alisertib mass balance and pharmacokinetics in plasma and urine by liquid chromatography-tandem mass spectrometry, and mass balance/recovery of [14C]-radioactivity in urine and feces by liquid scintillation counting. Part B; patients received non-radiolabeled alisertib 50 mg as enteric-coated tablets twice-daily for 7 days in 21-day cycles. Results In part A, absorption was fast (median plasma Tmax, 1 h) for alisertib and TRA. Mean plasma t1/2 for alisertib and TRA were 23.4 and 42.0 h, respectively. Mean plasma alisertib/TRA AUC0-inf ratio was 0.45, indicating presence of alisertib metabolites in circulation. Mean TRA blood/plasma AUC0-last ratio was 0.60, indicating preferential distribution of drug-related material in plasma. On average, 87.8% and 2.7% of administered radioactivity was recovered in feces and urine, respectively (total recovery, 90.5% by 14 days post-dose). In part B, patients received a median 3 cycles of alisertib. The most common any-grade adverse events were fatigue and alopecia. Conclusions Findings suggest that alisertib is eliminated mainly via feces, consistent with hepatic metabolism and biliary excretion of drug-related material. Further investigation of alisertib pharmacokinetics in patients with moderate-severe hepatic impairment is warranted to inform dosing recommendations in these patient populations.

Emerging Kidney Models to Investigate Metabolism, Transport, and Toxicity of Drugs and Xenobiotics.

The kidney is a major clearance organ of the body and is responsible for the elimination of many xenobiotics and prescription drugs. With its multitude of uptake and efflux transporters and metabolizing enzymes, the proximal tubule cell (PTC) in the nephron plays a key role in the disposition of xenobiotics and is also a primary site for toxicity. In this minireview, we first provide an overview of the major transporters and metabolizing enzymes in the PTCs responsible for biotransformation and disposition of drugs. Next, we discuss different cell sources that have been used to model PTCs in vitro, their pros and cons, and their characterization. As current technology is inadequate to evaluate reliably drug disposition and toxicity in the kidney, we then discuss recent advancements in kidney microphysiological systems (MPS) and the need to develop robust in vitro platforms that could be routinely used by pharmaceutical companies to screen compounds. Finally, we discuss the new and exciting field of stem cell-derived kidney models as potential cell sources for future kidney MPS. Given the push from both regulatory agencies and pharmaceutical companies to use more predictive "human-like" in vitro systems in the early stages of drug development to reduce attrition, these emerging models have the potential to be a game changer and may revolutionize how renal disposition and kidney toxicity in drug discovery are evaluated in the future.

A Decade in the MIST: Learnings from Investigations of Drug Metabolites in Drug Development under the "Metabolites in Safety Testing" Regulatory Guidance.

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.

Carbon-carbon bond cleavage and formation reactions in drug metabolism and the role of metabolic enzymes.

Elimination of xenobiotics from the human body is often facilitated by a transformation to highly water soluble and more ionizable molecules. In general, oxidation-reduction, hydrolysis, and conjugation reactions are common biotransformation reactions that are catalyzed by various metabolic enzymes including cytochrome P450s (CYPs), non-CYPs, and conjugative enzymes. Although carbon-carbon (C-C) bond formation and cleavage reactions are known to exist in plant secondary metabolism, these reactions are relatively rare in mammalian metabolism and are considered exceptions. However, various reactions such as demethylation, dealkylation, dearylation, reduction of alkyl chain, ring expansion, ring contraction, oxidative elimination of a nitrile through C-C bond cleavage, and dimerization, and glucuronidation through C-C bond formation have been reported for drug molecules. Carbon-carbon bond cleavage reactions for drug molecules are primarily catalyzed by CYP enzymes, dimerization is mediated by peroxidases, and C-glucuronidation is catalyzed by UGT1A9. This review provides an overview of C-C bond cleavage and formation reactions in drug metabolism and the metabolic enzymes associated with these reactions.

Clinical and Nonclinical Disposition and In Vitro Drug-Drug Interaction Potential of Felcisetrag, a Highly Selective and Potent 5-HT4 Receptor Agonist.

BACKGROUND AND OBJECTIVE: Felcisetrag (previously TAK-954 or TD-8954) is a highly selective and potent 5-HT4 receptor agonist in clinical development for prophylaxis and treatment of postoperative gastrointestinal dysfunction (POGD). The rat, dog, and human absorption, distribution, metabolism, and excretion (ADME) properties of felcisetrag were investigated. METHODS: The metabolism and victim and perpetrator drug interaction potentials towards cytochrome P450s (CYP) and transporters were determined using in vitro models. The excretion, metabolite profile, and pharmacokinetics were determined during unlabeled and radiolabeled ADME studies in rat and dog for comparison with human. Due to a low clinical dose (0.5 mg) and radioactivity (~ 1.5 µCi), a combination of liquid scintillation counting and accelerator mass spectrometry was used for analysis of samples in this study. RESULTS: The ADME properties, including metabolite profile, for felcisetrag are generally conserved across species. Felcisetrag is primarily cleared through renal excretion (0.443) and metabolism in humans (0.420), with intact parent as the predominant species in circulation. There are multiple metabolites, each representing < 10% of the circulating radioactivity, confirming no metabolites in safety testing (MIST) liabilities. Metabolites were also detected in animals. The potential for major CYP- and transporter-based drug-drug interaction (DDI) of felcisetrag as a victim or perpetrator is considered to be low. CONCLUSIONS: Felcisetrag is primarily cleared in humans through renal excretion. Although the metabolism of felcisetrag is primarily through CYP3A, the potential for clinically relevant DDI as a victim is significantly reduced as metabolism plays a minor role in the overall clearance.

Sinusoidal Organic Anion-Transporting Polypeptide 1B1/1B3 and Bile Canalicular Multidrug Resistance-Associated Protein 2 Play an Essential Role in the Hepatobiliary Disposition of a Synthetic Cyclic Dinucleotide (STING Agonist).

The liver is central to the elimination of many drugs from the body involving multiple processes and understanding of these processes is important to quantitively assess hepatic clearance of drugs. The synthetic STING (STimulator of INterferon Genes protein) agonist is a new class of drugs currently being evaluated in clinical trials as a potential anticancer therapy. In this study, we used ML00960317 (synthetic STING agonist) to investigate the hepatobiliary disposition of this novel molecular entity. A bile-duct cannulated (BDC) rat study indicated that biliary excretion is the major route of elimination for ML00960317 (84% of parent dose in bile). The human biliary clearance using in vitro sandwich cultured human hepatocyte model predicted significant biliary excretion of ML00960317 (biliary excretion index (BEI) of 47%). Moreover, the transport studies using transporter expressing cell lines, hepatocytes, and membrane vesicles indicated that ML00960317 is a robust substrate of OATP1B1, OATP1B3, and MRP2. Using relative expression factor approach, the combined contribution of OATP1B1 (fraction transported (ft) = 0.62) and OATP1B3 (ft = 0.31) was found to be 93% of the active uptake clearance of ML00960317 into the liver. Furthermore, OATP1B1 and OATP1B3-mediated uptake of ML00960317 was inhibited by rifampicin with IC50 of 6.5 and 2.3 µM, respectively indicating an in vivo DDI risk (R value of 1.5 and 2.5 for OATP1B1 and OATP1B3, respectively). These results highlighted an important role of OATP1B1, OATP1B3, and MRP2 in the hepatobiliary disposition of ML00960317. These pathways may act as rate-determining steps in the hepatic clearance of ML00960317 thus presenting clinical DDI risk.

Analytical strategies for assessment of human metabolites in preclinical safety testing.

Strategies for the detection, identification, and quantification of in vivo drug metabolites from non-radiolabeled studies are reviewed with an emphasis on the utilization of accurate-mass-based data mining tools. New approaches to the determination of coverage of human drug metabolites in preclinical species without using radiolabeled drugs or synthetic standards are also discussed.

Precision and accuracy in the quantitative analysis of biological samples by accelerator mass spectrometry: application in microdose absolute bioavailability studies.

Determination of the pharmacokinetics and absolute bioavailability of an experimental compound, SCH 900518, following a 89.7 nCi (100 µg) intravenous (iv) dose of (14)C-SCH 900518 2 h post 200 mg oral administration of nonradiolabeled SCH 900518 to six healthy male subjects has been described. The plasma concentration of SCH 900518 was measured using a validated LC-MS/MS system, and accelerator mass spectrometry (AMS) was used for quantitative plasma (14)C-SCH 900518 concentration determination. Calibration standards and quality controls were included for every batch of sample analysis by AMS to ensure acceptable quality of the assay. Plasma (14)C-SCH 900518 concentrations were derived from the regression function established from the calibration standards, rather than directly from isotopic ratios from AMS measurement. The precision and accuracy of quality controls and calibration standards met the requirements of bioanalytical guidance (U.S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research, Center for Veterinary Medicine. Guidance for Industry: Bioanalytical Method Validation (ucm070107), May 2001. http://www.fda.gov/downloads/Drugs/GuidanceCompilanceRegulatoryInformation/Guidances/ucm070107.pdf ). The AMS measurement had a linear response range from 0.0159 to 9.07 dpm/mL for plasma (14)C-SCH 900158 concentrations. The CV and accuracy were 3.4-8.5% and 94-108% (82-119% for the lower limit of quantitation (LLOQ)), respectively, with a correlation coefficient of 0.9998. The absolute bioavailability was calculated from the dose-normalized area under the curve of iv and oral doses after the plasma concentrations were plotted vs the sampling time post oral dose. The mean absolute bioavailability of SCH 900518 was 40.8% (range 16.8-60.6%). The typical accuracy and standard deviation in AMS quantitative analysis of drugs from human plasma samples have been reported for the first time, and the impact of these parameters on quantitative analysis was further assessed using the Z factor. The use of the lowest achievable LLOQ(Z=0) derived from statistical analysis of the control and low-concentration standards for AMS measurements is proposed in future studies.

Identification of human liver cytochrome P450 enzymes involved in the metabolism of SCH 530348 (Vorapaxar), a potent oral thrombin protease-activated receptor 1 antagonist.

Vorapaxar (SCH 530348), a potent oral thrombin protease-activated receptor 1 antagonist, is being developed as an antiplatelet agent for patients with established vascular disease. The objective of this study was to identify the human liver cytochrome P450 (P450) enzyme(s) responsible for the metabolism of SCH 530348. Human liver microsomes metabolized SCH 530348 to M19, an amine metabolite formed via carbamate cleavage, and M20 (monohydroxy-SCH 530348). Recombinant human CYP3A4 exhibited the most activity (11.5% profiled radioactivity) for the formation of M19, followed by markedly less substrate conversion with CYP1A1 and CYP2C19. Trace levels of M19, a major excreted human metabolite, were detected with CYP1A2, CYP3A5, and CYP4F3A. Formation of M19 by human liver microsomes was inhibited 89% by ketoconazole (IC(50), 0.73 µM), 34% by tranylcypromine, and 89% by anti-CYP3A4 monoclonal antibody. There was a significant correlation between the rate of M19 formation and midazolam 1'-hydroxylation (r = 0.75) or M19 formation and testosterone 6ß-hydroxylation (r = 0.92). The results of screening, inhibition, and correlation studies confirmed that CYP3A4 is the major P450 enzyme responsible for M19 formation from SCH 530348. In contrast, formation of M20, a major circulating human metabolite at steady state, was primarily catalyzed by CYP3A4 and CYP2J2. M20 is pharmacologically equipotent to SCH 530348, whereas M19 is an inactive metabolite. Formation of M20 by human liver microsomes was inhibited 89% by ketoconazole, 75% by astemizole (a CYP2J2 inhibitor), and 43% by CYP3A4 monoclonal antibody. These results suggest that CYP3A4 and CYP2J2 are both involved in the formation of M20 metabolite.

Characterization of human liver enzymes involved in the biotransformation of boceprevir, a hepatitis C virus protease inhibitor.

Boceprevir (SCH 503034), a protease inhibitor, is under clinical development for the treatment of human hepatitis C virus infections. In human liver microsomes, formation of oxidative metabolites after incubations with [(14)C]boceprevir was catalyzed by CYP3A4 and CYP3A5. In addition, the highest turnover was observed in recombinant CYP3A4 and CYP3A5. After a single radiolabeled dose to human, boceprevir was subjected to two distinct pathways, namely cytochrome P450-mediated oxidation and ketone reduction. Therefore, attempts were made to identify the enzymes responsible for the formation of carbonyl-reduced metabolites. Human liver S9 and cytosol converted ∼ 28 and ∼ 68% of boceprevir to M28, respectively, in the presence of an NADPH-generating system. Screening of boceprevir with recombinant human aldo-keto reductases (AKRs) revealed that AKR1C2 and AKR1C3 exhibited catalytic activity with respect to the formation of M+2 metabolites (M28 and M31). The formation of M28 was inhibited by 100 µM flufenamic acid (80.3%), 200 µM mefenamic acid (83.7%), and 100 µM phenolphthalein (86.1%), known inhibitors of AKRs, suggesting its formation through carbonyl reduction pathway. Formation of M28 was also inhibited by 100 µM diazepam (75.1%), 1 mM ibuprofen (70%), and 200 µM diflunisal (89.4%). These data demonstrated that CYP3A4 and CYP3A5 are primarily responsible for the formation of oxidative metabolites and the formation of M28 and M31, the keto-reduced metabolites, are most likely mediated by AKR1C2 and AKR1C3. Because the biotransformation and clearance of boceprevir involves two different enzymatic pathways, boceprevir is less likely to be a victim of significant drug-drug interaction with concomitant medication affecting either of these pathways.

Quantitation of parent drug and its unstable metabolites by in situ coulometric oxidation and liquid chromatography-tandem mass spectrometry.

Recent FDA and ICH guidances on safety testing of drug metabolites have challenged the way we traditionally think about quantitative bioanalytical methods. Such assays, in general, require a reference standard for each analyte to construct calibration curves and prepare quality control samples. However, early in the drug development process, metabolite standards may not be readily available, and if they are inherently unstable, they are difficult to synthesize or purify. In this paper, we describe a novel in-line method for producing and then quantifying a very unstable metabolite which is based upon the in situ postcolumn coulometric oxidation of the parent drug. Lacking any metabolite standards, the feasibility of simultaneously quantifying a development drug (compound A) and its unstable hydroxylated metabolites (metabolite B) was investigated. Reference standards for these ostensibly major human metabolites could not be reliably obtained due to rapid degradation upon purification and/or subsequent storage. Following high-performance liquid chromatography (HPLC) separation, parent drug and its [(13)C(3)-(15)N] isotopically labeled internal standard were quantitatively converted to equal amounts of a diastereomeric pair of hydroxylated metabolites using a postcolumn coulometric electrochemical cell before reaching the mass spectrometer. The concentration of the injected parent (which is equal to the total concentration of the in-line generated metabolites since the conversion to metabolite is quantitative) and the tandem mass spectrometry (MS/MS) signals of the electrochemically generated metabolites were used to construct a calibration curve for quantifying both the parent drug and its hydroxylated metabolites. Plasma extracts from humans dosed with compound A contained chromatographically distinct liquid chromatography-mass spectrometry (LC-MS) signals (m/z 538) for in vivo formed hydroxylated metabolites and the electrochemically oxidized parent drug which had been converted in-line into its chemically identical twin. Both peaks in this study sample could be quantified using a single calibration curve obtained under the same coulometric conditions using known amounts of the parent drug. Although no attempt was made to fully validate a bioanalytical method, the practicality of this in situ quantification approach was further confirmed by the preliminary bioanalytical analysis of a selection of plasma samples collected following oral administration (50 mg) of compound A in a clinical study.

Identification of two novel metabolites of SCH 486757, a nociceptin/orphanin FQ peptide receptor agonist, in humans.

The study of human metabolism of endo-8[bis(2-chlorophenyl)methyl]-3-(2-pyrimidinyl)-8-azabicyclo[3.2.1]octan-3-ol (SCH 486757) after a 200-mg oral dose of the drug to healthy volunteers in the first-in-human study is presented. The structural elucidation of two unique metabolites, which were detected in the process of metabolite characterization in human plasma and urine by liquid chromatography-mass spectrometry (LC-MS), is described. These metabolites (M27 and M34) were initially detected in human plasma at high levels (>35% of the LC-MS response of the parent drug). Additional LC-MS experiments (hydrogen/deuterium exchange and accurate mass measurement) were used to determine structures of metabolites. It was found that both metabolites were formed through a loss of the C-C bridge from the tropane moiety with the conversion into a substituted pyridinium compound. This metabolic process has not been reported previously. Because of the apparent high abundance of metabolites based on the LC-MS response, actual circulating amounts of these metabolites relative to the parent drug were determined semiquantitatively to evaluate their coverage in preclinical species. With the use of reference standards, it was shown that the LC-MS response of M27 and M34 in human plasma was much higher than that of the parent compound. Actual amounts of M27 and M34 metabolites were less than 5% of the level of the parent drug; therefore, additional assessment was not required.

Determination of exposure multiples of human metabolites for MIST assessment in preclinical safety species without using reference standards or radiolabeled compounds.

A simple, reliable, and accurate method was developed for quantitative assessment of metabolite coverage in preclinical safety species by mixing equal volumes of human plasma with blank plasma of animal species and vice versa followed by an analysis using high-resolution full-scan accurate mass spectrometry. This approach provided comparable results (within (±15%) to those obtained from regulated bioanalysis and did not require synthetic standards or radiolabeled compounds. In addition, both qualitative and quantitative data were obtained from a single LC-MS analysis on all metabolites and, therefore, the coverage of any metabolite of interest can be obtained.