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.

Metabolite Bioanalysis in Drug Development: Recommendations from the IQ Consortium Metabolite Bioanalysis Working Group.

The intent of this perspective is to share the recommendations of the International Consortium for Innovation and Quality in Pharmaceutical Development Metabolite Bioanalysis Working Group on the fit-for-purpose metabolite bioanalysis in support of drug development and registration. This report summarizes the considerations for the trigger, timing, and rigor of bioanalysis in the various assessments to address unique challenges due to metabolites, with respect to efficacy and safety, which may arise during drug development from investigational new drug (IND) enabling studies, and phase I, phase II, and phase III clinical trials to regulatory submission. The recommended approaches ensure that important drug metabolites are identified in a timely manner and properly characterized for efficient drug development.

Absorption, Metabolism, and Excretion of [14C]-Tolebrutinib After Oral Administration in Humans, Contribution of the Metabolites to Pharmacological Activity.

BACKGROUND AND OBJECTIVE: Tolebrutinib is a covalent inhibitor of Bruton's tyrosine kinase, an enzyme expressed in B lymphocytes and myeloid cells including microglia, which are thought to be major drivers of inflammation in multiple sclerosis. This excretion balance and metabolism study evaluated the metabolite profile of tolebrutinib in healthy male volunteers. METHODS: Six healthy volunteers received a 60-mg oral dose of [14C]-tolebrutinib, and metabolite profiling of 14C-labeled metabolites was performed using a combination of liquid chromatography, mass spectrometry, and radioactivity assay methods. RESULTS: Tolebrutinib was rapidly and completely absorbed from the gastrointestinal tract, followed by rapid and extensive metabolism. Excretion via feces was the major elimination pathway of the administered radioactivity (78%). Tolebrutinib was highly metabolized, with 19 metabolites identified in human plasma. Phase 1 biotransformations were primarily responsible for the circulating metabolites in plasma. Seven metabolites that achieved exposure in plasma similar to or higher than the parent compound were characterized biochemically for inhibition of Bruton's tyrosine kinase activity. Metabolite M8 exceeded the exposure threshold of 10% (18%) of the total radioactivity but had little if any pharmacological activity. Metabolite M2 (4% of circulating radioactivity) retained the ability to irreversibly and potently inhibit Bruton's tyrosine kinase in vitro, similar to the parent compound. Tolebrutinib and metabolite M2 had short (3.5-h) half-lives but durable pharmacodynamic effects as expected for an irreversible antagonist. CONCLUSIONS: Tolebrutinib was extensively metabolized to multiple metabolites. The hydroxylated metabolite M2 demonstrated similar inhibitory potency toward Bruton's tyrosine kinase as the parent compound. Both tolebrutinib and metabolite M2 likely contributed to pharmacological activity in vivo.

Considerations for Human ADME Strategy and Design Paradigm Shift(s) - An Industry White Paper.

The human absorption, distribution, metabolism, and excretion (hADME) study is the cornerstone of the clinical pharmacology package for small molecule drugs, providing comprehensive information on the rates and routes of disposition and elimination of drug-related material in humans through the use of 14 C-labeled drug. Significant changes have already been made in the design of the hADME study for many companies, but opportunity exists to continue to re-think both the design and timing of the hADME study in light of the potential offered by newer technologies, that enable flexibility in particular to reducing the magnitude of the radioactive dose used. This paper provides considerations on the variety of current strategies that exist across a number of pharmaceutical companies and on some of the ongoing debates around a potential move to the so called "human first/human only" approach, already adopted by at least one company. The paper also provides a framework for continuing the discussion in the application of further shifts in the paradigm.

Preclinical in vitro and in vivo pharmacokinetic properties of danicamtiv, a new targeted myosin activator for the treatment of dilated cardiomyopathy.

Dilated cardiomyopathy (DCM) is a disease of the myocardium defined by left ventricular enlargement and systolic dysfunction leading to heart failure. Danicamtiv, a new targeted myosin activator designed for the treatment of DCM, was characterised in in vitro and in vivo preclinical studies. Danicamtiv human hepatic clearance was predicted to be 0.5 mL/min/kg from in vitro metabolic stability studies in human hepatocytes. For human, plasma protein binding was moderate with a fraction unbound of 0.16, whole blood-to-plasma partitioning ratio was 0.8, and danicamtiv showed high permeability and no efflux in a Caco-2 cell line. Danicamtiv metabolism pathways in vitro included CYP-mediated amide-cleavage, N-demethylation, as well as isoxazole- and piperidine-ring-opening. Danicamtiv clearance in vivo was low across species with 15.5, 15.3, 1.6, and 5.7 mL/min/kg in mouse, rat, dog, and monkey, respectively. Volume of distribution ranged from 0.24 L/kg in mouse to 1.7 L/kg in rat. Oral bioavailability ranged from 26% in mouse to 108% in dog. Simple allometric scaling prediction of human plasma clearance, volume of distribution, and half-life was 0.64 mL/min/kg, 0.98 L/kg, and 17.7 h, respectively. Danicamtiv preclinical attributes and predicted human pharmacokinetics supported advancement toward clinical development.

Morphological behaviour and metabolic capacity of cryopreserved human primary hepatocytes cultivated in a perfused multiwell device.

1. The quantitative prediction of the pharmacokinetic parameters of a drug from data obtained using human in vitro systems remains a significant challenge i.e. prediction of metabolic clearance in humans and estimation of the relative contribution of enzymes involved in the clearance. This has become particularly problematic for low turnover compounds. 2. Having human hepatocytes with stable cellular function over several days that adequately mimic the complexity of the physiological environment would be a major advance. Thus, we evaluated human hepatocytes, maintained in culture during 7 days in the microfluidic LiverChip™ system, in terms of morphological appearance, relative mRNA expression of phase I and II enzymes and transporters as a function of time, and metabolic capacity using probe substrates. 3. The results showed that mRNA levels of the major genes for enzymes involved in drug metabolism were well-maintained over a 7-day period of culture. Furthermore, after 4 days of culture, in the Liverchip™ device, human hepatocytes exhibited higher or similar CYPs activities compared to 1 day of culture in 2D-static conditions. 4. The functional data were supported by light/electron microscopies and immunohistochemistry showing viable tissue structure and well-differentiated human hepatocytes: presence of cell junctions, glycogen storage, and bile canaliculi.

Identification of metabolic pathways and enzyme systems involved in the in vitro human hepatic metabolism of dronedarone, a potent new oral antiarrhythmic drug.

The in vitro metabolism of dronedarone and its major metabolites has been studied in human liver microsomes and cryopreserved hepatocytes in primary culture through the use of specific or total cytochrome P450 (CYP) and monoamine oxidase (MAO) inhibitors. The identification of the main metabolites and enzymes participating in their metabolism was also elucidated by using rhCYP, rhMAO, flavin monooxygenases (rhFMO) and UDP-glucuronosyltransferases (rhUGT) and liquid chromatography/tandem mass spectrometry (LC/MS-MS) analysis. Dronedarone was extensively metabolized in human hepatocytes with a metabolic clearance being almost completely inhibited (98 ± 2%) by 1-aminobenzotriazole. Ketoconazole also inhibited dronedarone metabolism by 89 ± 7%, demonstrating the crucial role of CYP3A in its metabolism. CYP3A isoforms mostly contributed to N-debutylation while hydroxylation on the butyl-benzofuran moiety was catalyzed by CYP2D6. However, hydroxylation on the dibutylamine moiety did not appear to be CYP-dependent. N-debutyl-dronedarone was less rapidly metabolized than dronedarone, the major metabolic pathway being catalyzed by MAO-A to form propanoic acid-dronedarone and phenol-dronedarone. Propanoic acid-dronedarone was metabolized at a similar rate to that of N-debutyl-dronedarone and was predominantly hydroxylated by CYP2C8 and CYP1A1. Phenol-dronedarone was extensively glucuronidated while C-dealkyl-dronedarone was metabolized at a slow rate. The evaluation of the systemic clearance of each metabolic process together with the identification of both the major metabolites and predominant enzyme systems and isoforms involved in the formation and subsequent metabolism of these metabolites has enhanced the overall understanding of metabolism of dronedarone in humans.