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.

Non-Labeled, Stable Labeled, or Radiolabelled Approaches for Provision of Intravenous Pharmacokinetics in Humans: A Discussion Piece.

A review of the use of microdoses and isotopic microtracers for clinical intravenous pharmacokinetic (i.v. PK) data provision is presented. The extent of application of the varied approaches available and the relative merits of each are highlighted with the aim of assisting practitioners in making informed decisions on the most scientifically appropriate design to adopt for any given new drug in development. It is envisaged that significant efficiencies will be realized as i.v. PK data in humans becomes more routinely available for suitable assets in early development, than has been the case prior to the last decade.

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.

The metabolism and disposition of the oral dipeptidyl peptidase-4 inhibitor, linagliptin, in humans.

The pharmacokinetics and metabolism of linagliptin (BI1356, 8-(3R-amino-piperidin-1-yl)-7-but-2-ynyl-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione) were investigated in healthy volunteers. The 10- and 5-mg (14)C-labeled drug was administered orally or intravenously, respectively. Fecal excretion was the dominant excretion pathway with 84.7% (p.o.) and 58.2% (i.v.) of the dose. Renal excretion accounted for 5.4% (p.o.) and 30.8% (i.v.) of the dose. Unchanged linagliptin was the most abundant radioactive species in all matrices investigated. The exposure (area under the curve 0-24 h) to the parent compound in plasma accounted for 191 nM . h (p.o.) and 356 nM . h (i.v.), respectively. The main metabolite 7-but-2-ynyl-8-(3S-hydroxy-piperidin-1-yl)-3-methyl-1-(4-methyl-quinazolin-2-ylmethyl)-3,7-dihydro-purine-2,6-dione (CD1790) was observed with >10% of parent compound systemic exposure after oral administration. The metabolite was identified as S-3-hydroxypiperidinly derivative of linagliptin. Experiments that included stable-labeled isotope techniques indicated that CD1790 was formed by a two-step mechanism via the ketone 7-but-2-yn-1-yl-3-methyl-1-[(4-methylquinazolin-2-yl)methyl]-8-(3-oxopiperidin-1-yl)-3,7-dihydro-1H-purine-2,6-dione (CD10604). The initial ketone formation was CYP3A4-dependent and rate-limiting for the overall reaction to CD1790. Aldo-keto reductases with minor contribution of carbonyl reductases were involved in the subsequent stereoselective reduction of CD10604 to CD1790. The antipodes of linagliptin and CD1790 were not observed with adequate enantioselective liquid chromatography-tandem mass spectrometry methods. Other minor metabolites were identified by mass spectrometry and NMR investigations. However, it was concluded that the metabolites of linagliptin only play a minor role in the overall disposition and elimination of linagliptin.

Untargeted Metabolomic Analysis Combined With Multivariate Statistics Reveal Distinct Metabolic Changes in GPR40 Agonist-Treated Animals Related to Bile Acid Metabolism.

Metabolomics has been increasingly applied to biomarker discovery, as untargeted metabolic profiling represents a powerful exploratory tool for identifying causal links between biomarkers and disease phenotypes. In the present work, we used untargeted metabolomics to investigate plasma specimens of rats, dogs, and mice treated with small-molecule drugs designed for improved glycemic control of type 2 diabetes mellitus patients via activation of GPR40. The in vivo pharmacology of GPR40 is not yet fully understood. Compounds targeting this receptor have been found to induce drug-induced liver injury (DILI). Metabolomic analysis facilitating an integrated UPLC-TWIMS-HRMS platform was used to detect metabolic differences between treated and non-treated animals within two 4-week toxicity studies in rat and dog, and one 2-week toxicity study in mouse. Multivariate statistics of untargeted metabolomics data subsequently revealed the presence of several significantly upregulated endogenous compounds in the treated animals whose plasma level is known to be affected during DILI. A specific bile acid metabolite useful as endogenous probe for drug-drug interaction studies was identified (chenodeoxycholic acid-24 glucuronide), as well as a metabolic precursor indicative of acidic bile acid biosynthesis (7α-hydroxy-3-oxo-4-cholestenoic acid). These results correlate with typical liver toxicity parameters on the individual level.

The metabolism and disposition of the oral direct thrombin inhibitor, dabigatran, in humans.

The pharmacokinetics and metabolism of the direct thrombin inhibitor dabigatran (BIBR 953 ZW, beta-alanine, N-[[2-[[[4-(aminoiminomethyl)phenyl]amino]methyl]-1-methyl-1H-benzimidazol-5-yl]carbonyl]-N-2-pyridinyl) were studied in 10 healthy males, who received 200 mg of [(14)C]dabigatran etexilate (BIBR 1048 MS, the oral prodrug of dabigatran) or an i.v. infusion of 5 mg of [(14)C]dabigatran. Radioactivity was measured in plasma, urine, and feces over 1 week. The metabolite pattern was analyzed by high-performance liquid chromatography with on-line radioactivity detection, and metabolite structures were elucidated by mass spectrometry. Dabigatran etexilate was rapidly converted to dabigatran, with peak plasma dabigatran concentrations being attained after approximately 1.5 h; the bioavailability of dabigatran after p.o. administration of dabigatran etexilate was 7.2%. Dabigatran was predominantly excreted in the feces after p.o. treatment and in the urine after i.v. treatment. The mean terminal half-life of dabigatran was approximately 8 h. The predominant metabolic reaction was esterase-mediated hydrolysis of dabigatran etexilate to dabigatran. Phase I metabolites accounted for

Best practices for metabolite quantification in drug development: updated recommendation from the European Bioanalysis Forum.

Metabolite quantification and profiling continues to grow in importance in today's drug development. The guidance provided by the 2008 FDA Metabolites in Safety Testing Guidance and the subsequent ICH M3(R2) Guidance (2009) has led to a more streamlined process to assess metabolite exposures in preclinical and clinical studies in industry. In addition, the European Bioanalysis Forum (EBF) identified an opportunity to refine the strategies on metabolite quantification considering the experience to date with their recommendation paper on the subject dating from 2010 and integrating the recent discussions on the tiered approach to bioanalytical method validation with focus on metabolite quantification. The current manuscript summarizes the discussion and recommendations from a recent EBF Focus Workshop into an updated recommendation for metabolite quantification in drug development.