Human Mass Balance Study Research Articles

The practical impact of the FDA draft guidance on human mass balance studies – a year of client discussions

The practical impact of the FDA draft guidance on human mass balance studies – a year of client discussions

A Phase I, Open-Label, Mass Balance Study of [14C]-Iberdomide in Healthy Subjects.

Iberdomide is a novel potent cereblon modulator (CELMoD®) agent, which is currently under clinical development for hematological malignancies. A human mass balance study was conducted to characterize the biotransformation and excretion pathways of iberdomide. After a single dose of radiolabelled [14C]-iberdomide (1 mg) in six healthy subjects. Blood, urine, and fecal samples were collected for pharmacokinetics, mass balance, and clinical laboratory assessments. Results showed that a single oral dose of 1 mg iberdomide was generally well tolerated in healthy subjects. The recovery of [14C]-iberdomide-derived radioactivity in humans was 45.9% in urine and 42.6% in feces. Based on exposure (area under the concentration-time curve [AUC0-24]), iberdomide and M12 (metabolites) accounted for approximately 59% and 14% of circulating total radioactivity (TRA) exposure, respectively. Of the 88.5% TRA excreted, approximately 27% was excreted as unchanged iberdomide and 62% as metabolites, with similar amounts of excreted metabolites in the urine (16%) and feces (11%). Biotransformation of iberdomide in humans included multiple oxidations of the morpholino moiety as well as glutarimide ring hydrolysis of parent and oxidized metabolites and a combination of these pathways. Iberdomide was the predominant component in human plasma, with metabolite M12 being the most prominent circulating metabolite. In excreta, similar iberdomide-derived radioactivity was found in urine and feces. NCT03294603.

Recommendations on the Use of Multiple Labels in Human Mass Balance Studies.

The administration of radiolabeled drug candidates is considered the gold standard in absorption, distribution, metabolism, and excretion studies for small-molecule drugs since it allows facile and accurate quantification of parent drug, metabolites, and total drug-related material independent of the compound structure. The choice of the position of the radiolabel, typically 14C or 3H, is critical to obtain relevant information. Sometimes, a biotransformation reaction may lead to cleavage of a part of the molecule. As a result, only the radiolabeled portion can be followed, and information on the fate of the nonlabeled metabolite may be lost. Synthesis and administration of two or more radiolabeled versions of the parent drug as a mixture or in separate studies may resolve this issue but comes with additional challenges. In this paper, we address the questions that may be considered to help make the right choice whether to use a single or multiple radiolabel approach and discuss the pros and cons of different multiple-labeling strategies that can be taken as well as alternative methods that allow the nonlabeled part of the molecule to be followed. SIGNIFICANCE STATEMENT: Radiolabeled studies are the gold standard in drug metabolism research, but molecules can undergo cleavage with loss of the label. This often results in discussions around potential use of multiple labels, which seem to be occurring with increased frequency since an increasing proportion of the small-molecule drugs are tending towards larger molecular weights. This review provides insight and decision criteria in considering a multiple-label approach as well as pros and cons of different strategies that can be followed.

Absorption, Distribution, Metabolism, and Excretion of [14C]BS1801, a Selenium-Containing Drug Candidate, in Rats.

BS1801 is a selenium-containing drug candidate with potential for treating liver and lung fibrosis. To fully elucidate the biotransformation of BS1801 in animals and provide sufficient preclinical drug metabolism data for human mass balance study, the metabolism of BS1801 in rats was investigated. We used radiolabeling techniques to investigate the mass balance, tissue distribution, and metabolite identification of BS1801 in Sprague-Dawley/Long-Evans rats after a single oral dose of 100 mg/kg (100 μCi/kg) [14C]BS1801: 1. The mean recovery of radioactive substances in urine and feces was 93.39% within 168 h postdose, and feces were the main excretion route. 2. Additionally, less than 1.00% of the dose was recovered from either urine or bile. 3. BS1801-related components were widely distributed throughout the body. 4. Fifteen metabolites were identified in rat plasma, urine, feces, and bile, and BS1801 was detected only in feces. 5. BS1801-M484, the methylation product obtained via a N-Se bond reduction in BS1801, was the most abundant drug-related component in plasma. The main metabolic pathways of BS1801 were reduction, amide hydrolysis, oxidation, and methylation. Overall, BS1801 was distributed throughout the body, and excreted mainly as an intact BS1801 form through feces. No differences were observed between male and female rats in distribution, metabolism, and excretion of BS1801.

Cross-species drug metabolism and impact of metabolic stability testing under anaerobic condition on predicting pharmacokinetics of keto-enol containing compound in humans

After oral administration of [14C]-S-1360 in rats and dogs, [14C]-S-1360 was absorbed rapidly and the bioavailability was 93.7% in rats and 75.1% in dogs. Based on the results in animals, good systemic exposure would be expected in humans. In contrast to the expectation, the exposure was low in healthy volunteers compared to the exposure expected. In addition, human mass balance study using [14C]-S1360 revealed that a large amount of metabolites existed in human plasma. The major metabolites in human plasma were reduced metabolite (HP1) and S-1360 N-glucuronide, and they respectively accounted for approximately 30% of total AUC. Unchanged S-1360 accounted for only 14% of total AUC. The results showed that a significant difference between humans and animals were observed in metabolism of S-1360. Although S-1360 was stable in human hepatocytes under aerobic condition (approximately 84% remaining at 1 h), S-1360 was labile under anaerobic condition (approximately 55% remaining at 1 h). The present study revealed that the reductive metabolism pathways are the key metabolic pathway of S-1360, especially the metabolic stability test under anaerobic condition is important to predict pharmacokinetics of keto-enol containing compound, such as S-1360.

Conference report for the 10th China Bioanalysis Forum annual meeting.

The 10th China Bioanalysis Forum annual conference was held in Suzhou between 9 and 11 June 2023. This year a full range of bioanalytical topics were discussed such as new technology and bioanalytical approaches for biotherapeutics and biomarkers, particularly in the areas of gene and cell therapy. Another research area covered extensively at the conference was drug metabolism, including new drug metabolism and pharmacokinetic methods; absorption, distribution, metabolism and excretion of new modality drugs, recent regulatory guidance such as human mass balance study and preclinical study of antibody-drug conjugates and case studies of drug metabolism support to newly approved drugs.

Evobrutinib pathway to its major metabolite M463-2 and insights from a biotransformation and DDI perspective

Evobrutinib is a highly selective, covalent, central nervous system-penetrant Bruton’s tyrosine kinase (BTK) inhibitor, currently in Phase III trials for the treatment of relapsing multiple sclerosis. One major circulating metabolite of evobrutinib has been previously identified as the racemic dihydro-diol M463-2 (MSC2430422) in a Phase I human mass balance study. Phenotyping experiments were conducted to confirm the metabolic pathway of evobrutinib to M463-2. Ratio of the enantiomers was determined by enantioselective liquid chromatography with tandem mass spectrometry analysis of plasma samples from humans and preclinical species. Drug-drug interaction (DDI) characterisation, evaluation of pharmacological activity on BTK, and off-target screening experiments followed assessing safety of the metabolite. The biotransformation of evobrutinib to M463-2 was determined to be a two-step process with a CYP-mediated oxidation acting to form an epoxide intermediate, which was further hydrolysed by soluble and mitochondrial epoxide hydrolase. Only the (S)-enantiomer was determined to be a major metabolite, the (R)-enantiomer was minor. In vitro studies demonstrated the (S)-enantiomer lacked clinically relevant pharmacological activity, off-target effects and DDIs. The biotransformation of evobrutinib to its major metabolite has been elucidated, with the major (S)-enantiomer being shown to pose no on/off target or DDI risks.

DEVELOPMENT OF PHVS719: AN ORAL EXTENDED-RELEASE BRADYKININ B2 RECEPTOR ANTAGONIST TO PREVENT HEREDITARY ANGIOEDEMA ATTACKS

<h3>Introduction</h3> Hereditary angioedema (HAE) is managed by either of two treatment paradigms: on demand or prophylaxis. PHA121, an orally active bradykinin B2 receptor antagonist, is being developed as two drug products to optimize patient choice. PHVS416, the softgel capsule providing quick absorption for fast onset of symptom relief, is currently in clinical trials for treatment of acute attacks; PHVS719, an extended-release tablet, is being developed for use in prophylaxis. <h3>Methods</h3> To develop a daily oral pill for the prevention of attacks, we developed a PHA121 formulation (PHVS719) providing 24 h plasma exposure. Our strategy relied upon a combination of an optimally designed extended-release formulation and the ability of PHA121 to be absorbed throughout the full gastrointestinal tract. <h3>Results</h3> PHA121 is absorbed following intracolonic administration in rats. A human mass balance study showed only 3% PHA121 excreted in feces and oral bioavailability of 57%. An in vitro dissolution experiment conducted with PHVS719 in conditions mimicking fasted and fed gastrointestinal environments showed a linear dissolution of PHA121 over time. Using PHVS719, a PK study in healthy volunteers demonstrated that a single 40 mg dose provides 24 h plasma exposure above the anticipated therapeutic level. Together these data provide evidence that PHVS719 is optimally suited to serve as a once-daily oral pill. <h3>Conclusion</h3> The active ingredient PHA121 is under clinical investigation in the form of two therapeutic treatment modalities: PHVS416 softgel capsule providing quick absorption for fast symptom relief and PHVS719 once-daily extended-release tablet providing 24 hours coverage to prevent attacks from occurring.

Clinical pharmacology information in regulatory submissions and labeling: A comparative analysis of orphan and non-orphan drugs approved by the FDA.

Clinical pharmacology is an integral discipline supporting the development, regulatory evaluation, and clinical use of drugs for the treatment of both common and rare diseases. Here, we evaluated the recommendations and information available from select clinical pharmacology studies in the therapeutic product labeling of new molecular entities (NMEs) approved from 2017 to 2019 for both common and rare diseases. A total of 151 NMEs, including 72 orphan and 79 non-orphan drugs, were analyzed for recommendations and information available related to food-drug interaction, drug-drug interaction, renal impairment, hepatic impairment, QT assessment, and human radiolabeled mass balance studies using data collected from the original labeling and other regulatory documents. The analysis showed no statistically significant difference in the recommendations between orphan and non-orphan drugs except for renal impairment related recommendations in section 8 of the labeling. Although not significant, fewer hepatic impairment labeling recommendations were available for orphan drugs when compared with non-orphan drugs. At the time of initial approval, 79 postmarketing requirements (PMRs) and postmarketing commitments (PMCs) for 33 orphan drugs and 39 PMRs and PMCs for 19 non-orphan drugs were established; with most difference observed for drug-drug interaction, hepatic impairment, and QT assessment. Overall, although there was a trend for more labeling recommendations and fewer postmarketing studies and clinical trials for non-orphan drugs, there appeared to be no substantial differences in how these select clinical pharmacology studies are leveraged during the development and approval of orphan and non-orphan drugs.

Human radiolabeled mass balance studies supporting the FDA approval of new drugs.

Human radiolabeled mass balance studies are an important component of the clinical pharmacology programs supporting the development of new investigational drugs. These studies allow for understanding of the absorption, distribution, metabolism, and excretion of the parent drug and metabolite(s) in the human body. Understanding the drug's disposition as well as metabolite profiling and abundance via mass balance studies can help inform the overall drug development program. A survey of the US Food and Drug Administration (FDA)-approved new drug applications (NDAs) indicated that about 66% of the drugs had relied on findings from the mass balance studies to help understand the pharmacokinetic characteristics of the drug and to inform the overall drug development program. When such studies were not available in the original NDA, adequate justifications were routinely provided. Of the 104 mass balance studies included in this survey, most of the studies were conducted in healthy volunteers (90%) who were mostly men (>86%). The studies had at least six evaluable participants (66%) and were performed using the final route(s) of administration (98%). Eighty-five percent of the studies utilized a dose within the pharmacokinetic linearity range with 54% of the studies using a dose the same as the approved dose. Nearly all studies were performed as a single-dose (97%) study using a fit-for-purpose radiolabeled formulation. In this analysis, we summarized the current practices for conducting mass balance studies and highlighted the importance of conducting appropriately designed human radiolabeled mass balance studies and the challenges associated with inadequately designed or untimely studies.

CYP2C8-Mediated Formation of a Human Disproportionate Metabolite of the Selective NaV1.7 Inhibitor DS-1971a, a Mixed Cytochrome P450 and Aldehyde Oxidase Substrate.

Predicting human disproportionate metabolites is difficult, especially when drugs undergo species-specific metabolism mediated by cytochrome P450s (P450s) and/or non-P450 enzymes. This study assessed human metabolites of DS-1971a, a potent Nav1.7-selective blocker, by performing human mass balance studies and characterizing DS-1971a metabolites, in accordance with the Metabolites in Safety Testing guidance. In addition, we investigated the mechanism by which the major human disproportionate metabolite (M1) was formed. After oral administration of radiolabeled DS-1971a, the major metabolites in human plasma were P450-mediated monoxidized metabolites M1 and M2 with area under the curve ratios of 27% and 10% of total drug-related exposure, respectively; the minor metabolites were dioxidized metabolites produced by aldehyde oxidase and P450s. By comparing exposure levels of M1 and M2 between humans and safety assessment animals, M1 but not M2 was found to be a human disproportionate metabolite, requiring further characterization under the Metabolites in Safety Testing guidance. Incubation studies with human liver microsomes indicated that CYP2C8 was responsible for the formation of M1. Docking simulation indicated that, in the formation of M1 and M2, there would be hydrogen bonding and/or electrostatic interactions between the pyrimidine and sulfonamide moieties of DS-1971a and amino acid residues Ser100, Ile102, Ile106, Thr107, and Asn217 in CYP2C8, and that the cyclohexane ring of DS-1971a would be located near the heme iron of CYP2C8. These results clearly indicate that M1 is the predominant metabolite in humans and a human disproportionate metabolite due to species-specific differences in metabolism. SIGNIFICANCE STATEMENT: This report is the first to show a human disproportionate metabolite generated by CYP2C8-mediated primary metabolism. We clearly demonstrate that DS-1971a, a mixed aldehyde oxidase and cytochrome P450 substrate, was predominantly metabolized by CYP2C8 to form M1, a human disproportionate metabolite. Species differences in the formation of M1 highlight the regio- and stereoselective metabolism by CYP2C8, and the proposed interaction between DS-1971a and CYP2C8 provides new knowledge of CYP2C8-mediated metabolism of cyclohexane-containing substrates.

Comparison between physiologically based pharmacokinetic and population pharmacokinetic modelling to select paediatric doses of gepotidacin in plague.

AimsTo develop physiologically based pharmacokinetic (PBPK) and population pharmacokinetic (PopPK) models to predict effective doses of gepotidacin in paediatrics for the treatment of pneumonic plague (Yersinia pestis).MethodsA gepotidacin PBPK model was constructed using a population‐based absorption, distribution, metabolism and excretion simulator, Simcyp®, with physicochemical and in vitro data, optimized with clinical data from a dose‐escalation intravenous (IV) study and a human mass balance study. A PopPK model was developed with pooled PK data from phase 1 studies with IV gepotidacin in healthy adults.ResultsFor both the PopPK and PBPK models, body weight was found to be a key covariate affecting gepotidacin clearance. With PBPK, ~90% of the predicted PK for paediatrics fell between the 5th and 95th percentiles of adult values except for subjects weighing ≤5 kg. PopPK‐simulated paediatric means for C max and AUC(0‐τ) were similar to adult exposures across various weight brackets. The proposed dosing regimens were weight‐based for subjects ≤40 kg and fixed‐dose for subjects >40 kg. Comparison of observed and predicted exposures in adults indicated that both PBPK and PopPK models achieved similar AUC and C max for a given dose, but the C max predictions with PopPK were slightly higher than with PBPK. The two models differed on dose predictions in children <3 months old. The PopPK model may be suboptimal for low age groups due to the absence of maturation characterization of drug‐metabolizing enzymes involved with clearance in adults.ConclusionsBoth PBPK and PopPK approaches can reasonably predict gepotidacin exposures in children.

Absorption, distribution, metabolism, and excretion of [14C]NBP (3-n-butylphthalide) in rats

3-n-Butylphthalide (NBP) has a considerable neuroprotective effect and is currently used for the treatment of ischemic stroke. NBP was launched on the market in 2004. However, information on its metabolism in humans and preclinical animal models is insufficient. Although the metabolism of unradiolabeled NBP in humans has been reported, the quantitative metabolite profile, blood-to-plasma radioactivity concentration ratio (B/P), and tissue distribution of this drug remain unclear. We evaluated the pharmacokinetics, tissue distribution, mass balance, and metabolism of NBP in rats after a single oral dose of 60 mg/kg (100 μCi/kg) [14C]NBP to understand the biotransformation of NBP comprehensively and to provide preclinical drug metabolism data prior to human mass balance studies with [14C]NBP in the near future. NBP absorption was rapid (Tmax = 0.75 h) and declined with a terminal half-life of 9.73 h. In rats, the B/P was 0.63 during the 48 h postdose period, indicating that drug-related substances did not tend to be distributed into blood cells. Tissue distribution was determined by using the oxidative combustion method. NBP-related components were widely distributed throughout the body, and high concentrations were detected in the stomach, small intestine, fat, bladder, kidney, liver and ovary. At 168 h after oral administration, the mean cumulative recovered radioactivity was 99.85% of the original dose, and was 85.12% in urine and 14.73% in feces. Metabolite profiles were detected via radiochromatography. A total of 49 metabolites were identified in rat plasma, urine, and feces. The main metabolic pathways were oxidation, glucuronidation, and sulfation. Overall, NBP was absorbed rapidly, distributed throughout the body, and excreted in the form of metabolites. Urine was the main excretion route, and the absorption, distribution, metabolism and excretion of NBP showed no significant gender difference between male and female rats.

Pharmacokinetics and Disposition of Contezolid in Humans: Resolution of a Disproportionate Human Metabolite for Clinical Development.

Contezolid (MRX-I), a novel oxazolidinone antibiotic, was recently approved for the treatment of serious Gram-positive infections. The pharmacokinetics and disposition of [14C]contezolid were investigated in a single-dose human mass balance study. Cross-species comparison of plasma exposure for contezolid and metabolites was performed, and the safety of the disproportionate metabolite in human was evaluated with additional nonclinical studies. After an oral administration of 99.1 μCi/602-mg dose of [14C]contezolid, approximately 91.5% of the radioactivity was recovered in 0 to 168 h postdose, mainly in urine followed by that in feces. The principal metabolic pathway of contezolid in human comprised an oxidative ring opening of the 2,3-dihydropyridin-4-one fragment into polar metabolites MRX445-1 and MRX459, with recovery of approximately 48% and 15% of the dose, respectively, in urine and feces. Contezolid, MRX445-1, and MRX459 accounted for 68.0%, 19.5%, and 4.84% of the plasma exposure of the total radioactivity, respectively. Metabolites MRX445-1 and MRX459 were observed in disproportionately larger amounts in human plasma than in samples from rat or dog, the rodent and nonrodent species, respectively, used for the general nonclinical safety assessment of this molecule. This discrepancy was resolved with additional nonclinical studies, wherein the primary metabolite, MRX445-1, was further characterized. The no-observed-adverse-effect level (NOAEL) of MRX445-1 was determined as 360 mg/kg body weight/day in a 14-day repeat-dose test in pregnant and nonpregnant Sprague Dawley rats. Furthermore, MRX445-1 exhibited no antibacterial activity in vitro. Thus, MRX445-1 is not expected to exert clinically relevant pharmacology and toxicity.

Drug-drug interaction of atazanavir on UGT1A1-mediated glucuronidation of molidustat in human.

Molidustat is an oral inhibitor of hypoxia‐inducible factor (HIF) prolyl‐hydroxylase enhancing the erythropoietin (EPO) response to HIF; it is in clinical development for the treatment of anaemia related to chronic kidney disease. The predominant role of glucuronidation for molidustat clearance (formation of N‐glucuronide metabolite M1) and subsequent renal excretion was confirmed in a human mass balance study, with about 85% of the drug being excreted as M1 in urine. The inhibitory effects of 176 drugs and xenobiotics from various compound classes on the UGT‐mediated glucuronidation of molidustat in human liver microsomes (HLMs) were investigated. Based on preclinical findings, glucuronidation of molidustat was predominantly mediated by the 5'‐diphospho‐glucuronosyltransferase (UGT) isoform UGT1A1. Therefore, atazanavir, which is a potent inhibitor of UGT1A1, was chosen for the evaluation of pharmacokinetics and EPO release following a single oral dose of 25 mg molidustat. Molidustat exposure increased about twofold upon coadministration with atazanavir when considering area under plasma concentration‐time curve from zero to infinity (AUC) and maximum plasma concentration (C max). Baseline‐corrected increase of EPO was 14% and 34% for C max and AUC (calculated over 24 hours), respectively. Coadministration of molidustat and atazanavir was well tolerated.

Physiologically-Based Pharmacokinetic Models as Enablers of Precision Dosing in Drug Development: Pivotal Role of the Human Mass Balance Study.

Physiologically-Based Pharmacokinetic Models as Enablers of Precision Dosing in Drug Development: Pivotal Role of the Human Mass Balance Study.

Metabolism and Pharmacokinetic Drug-Drug Interaction Profile of Vericiguat, A Soluble Guanylate Cyclase Stimulator: Results From Preclinical and Phase I Healthy Volunteer Studies.

BackgroundVericiguat is a stimulator of soluble guanylate cyclase currently under investigation as a first-in-class therapy for worsening chronic heart failure (NCT02861534). Patients with heart failure often require polypharmacy because of comorbidities. Hence, understanding the clearance mechanisms, elimination, and potential for pharmacokinetic drug–drug interactions of vericiguat is important for dose recommendations in this patient population.MethodsBiotransformation and perpetrator properties of vericiguat were characterized in vitro using human hepatocytes, liver microsomes, and recombinant enzymes. This was complemented by a human mass balance study and ten drug–drug interaction studies in healthy volunteers wherein vericiguat was co-administered orally with omeprazole, magnesium/aluminum hydroxide, ketoconazole, rifampicin, mefenamic acid, midazolam, warfarin, digoxin, sacubitril/valsartan, aspirin, or sildenafil.ResultsIn the human mass balance study, mean total radioactivity recovered was 98.3% of the dose administered (53.1% and 45.2% excreted via urine and feces, respectively). The main metabolic pathway of vericiguat is glucuronidation via uridine diphosphate-glucuronosyltransferase 1A9 and 1A1. In vitro studies revealed a low risk of vericiguat acting as a perpetrator by inhibiting cytochrome P450s, uridine diphosphate-glucuronosyltransferase isoforms, or major transport proteins, or by inducing cytochrome P450s. These observations were supported by phase I drug–drug interaction studies. Phase I studies that assessed the propensity of vericiguat as a victim drug showed changes in the range that did not warrant recommendations for dose adjustment in phase III.ConclusionsA low pharmacokinetic interaction potential of vericiguat was estimated from in vitro data and confirmed in vivo. Thus, vericiguat is suitable for a patient population with multiple comorbidities requiring polypharmacy.Electronic supplementary materialThe online version of this article (10.1007/s40262-020-00895-x) contains supplementary material, which is available to authorized users.

The Importance of the Human Mass Balance Study in Regulatory Submissions.

The human mass balance study is a key study in the Clinical Pharmacology package of new drug applications. This study, along with the mass balance studies in toxicology species, provides essential information on the exposure of the parent compound and metabolites. Despite current regulatory guidance and previous publications, a lack of this study, or deficiencies in the study, are still seen in regulatory submissions today. This restricts the assessment of the benefit/risk in all populations and on the potential for drug–drug interactions leading to unnecessary precautions in the label. A review of new drug applications identifies a number of examples of inadequate characterization of circulating drug‐related components or of elimination pathways, with questions raised during the regulatory review. In light of this, new insight is given on what is required from the mass balance study and on how to ensure sufficient information is captured.

Evaluation of cAMS for 14C microtracer ADME studies: opportunities to change the current drug development paradigm.

Although regulatory guidances require human metabolism information of drug candidates early in the development process, the human mass balance study (or hADME study), is performed relatively late. hADME studies typically involve the administration of a 14C-radiolabelled drug where biological samples are measured by conventional scintillation counting analysis. Another approach is the administration of therapeutic doses containing a 14C-microtracer followed by accelerator mass spectrometry (AMS) analysis, enabling hADME studies completion much earlier. Consequently, there is an opportunity to change the current drug development paradigm. To evaluate the applicability of the MICADAS-cAMS method, we successfully performed: the validation of MICADAS-cAMS for radioactivity quantification in biomatrices and, a rat ADME study, where the conventional methodology was assessed against a microtracer MICADAS-cAMS approach. Combustion AMS (cAMS) technology is applicable to microtracer studies. A favorable opinion from EMA to complete the hADME in a Phase I setting was received, opening the possibilities to change drug development.

Pharmacokinetics and Disposition of Momelotinib Revealed a Disproportionate Human Metabolite—Resolution for Clinical Development

Momelotinib (MMB), a small-molecule inhibitor of Janus kinase (JAK)1/2 and of activin A receptor type 1 (ACVR1), is in clinical development for the treatment of myeloproliferative neoplasms. The pharmacokinetics and disposition of [14C]MMB were characterized in a single-dose, human mass-balance study. Metabolism and the pharmacologic activity of key metabolites were elucidated in multiple in vitro and in vivo experiments. MMB was rapidly absorbed following oral dosing with approximately 97% of the radioactivity recovered, primarily in feces with urine as a secondary route. Mean blood-to-plasma [14C] area under the plasma concentration-time curve ratio was 0.72, suggesting low association of MMB and metabolites with blood cells. [14C]MMB-derived radioactivity was detectable in blood for ≤48 hours, suggesting no irreversible binding of MMB or its metabolites. The major circulating human metabolite, M21 (a morpholino lactam), is a potent inhibitor of JAK1/2 and ACVR1 in vitro. Estimation of pharmacological activity index suggests M21 contributes significantly to the pharmacological activity of MMB for the inhibition of both JAK1/2 and ACVR1. M21 was observed in disproportionately higher amounts in human plasma than in rat or dog, the rodent and nonrodent species used for the general nonclinical safety assessment of this molecule. This discrepancy was resolved with additional nonclinical studies wherein the circulating metabolites and drug-drug interactions were further characterized. The human metabolism of MMB was mediated primarily by multiple cytochrome P450 enzymes, whereas M21 formation involved initial P450 oxidation of the morpholine ring followed by metabolism via aldehyde oxidase.