A methodology for the accurate calculation and mitigation of isotopic interferences in liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS) assays and its application in supporting microdose absolute bioavailability studies are reported for the first time. For simplicity, this calculation methodology and the strategy to minimize the isotopic interference are demonstrated using a simple molecule entity, then applied to actual development drugs. The exact isotopic interferences calculated with this methodology were often much less than the traditionally used, overestimated isotopic interferences simply based on the molecular isotope abundance. One application of the methodology is the selection of a stable isotopically labeled internal standard (SIL-IS) for an LC-MS/MS bioanalytical assay. The second application is the selection of an SIL analogue for use in intravenous (i.v.) microdosing for the determination of absolute bioavailability. In the case of microdosing, the traditional approach of calculating isotopic interferences can result in selecting a labeling scheme that overlabels the i.v.-dosed drug or leads to incorrect conclusions on the feasibility of using an SIL drug and analysis by LC-MS/MS. The methodology presented here can guide the synthesis by accurately calculating the isotopic interferences when labeling at different positions, using different selective reaction monitoring (SRM) transitions or adding more labeling positions. This methodology has been successfully applied to the selection of the labeled i.v.-dosed drugs for use in two microdose absolute bioavailability studies, before initiating the chemical synthesis. With this methodology, significant time and cost saving can be achieved in supporting microdose absolute bioavailability studies with stable labeled drugs.

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A strategy of using selected reaction monitoring (SRM) mass spectrometry for evaluating oral absolute bioavailability with concurrent intravenous (i.v.) microdosing a stable isotopically labeled (SIL) drug was developed and validated. First, the isotopic contribution to SRM (ICSRM) of the proposed SIL drug and SIL internal standard (IS) was theoretically calculated to guide their chemical synthesis. Second, the lack of an isotope effect on drug exposure was evaluated in a monkey study by i.v. dosing a mixture of the SIL and the unlabeled drugs. Third, after the SIL drug (100 μg) was concurrently i.v. dosed to humans, at T(max) of an oral therapeutic dose of the unlabeled drug, both drugs in plasma specimens were simultaneously quantified by a sensitive and accurate SRM assay. This strategy significantly improves bioanalytical data quality and saves time, costs, and resources by avoiding a traditional absolute bioavailability study or the newer approach of microdoses of a radio-microtracer measured by accelerator mass spectrometry.

1. Asunaprevir (ASV, BMS-650032), a highly selective and potent NS3 protease inhibitor, is currently under development for the treatment of chronic hepatic C virus infection. This study describes in vivo biotransformation in humans and the identification of metabolic enzymes of ASV. 2. Following a single oral dose of [(14)C]ASV to humans, the majority of radioactivity (>73% of the dose) was excreted in feces with <1% of the dose recovered in urine. Drug-related radioactivity readily appeared in circulation and the plasma radioactivity was mainly attributed to ASV. A few minor metabolites were observed in human plasma and are not expected to contribute to the pharmacological activity because of low levels. The area under the curve (AUC) values of each circulating metabolite in humans were well below their levels in animals used in the long-term toxicological studies. In bile and feces, intact ASV was a prominent radioactive peak suggesting that both metabolism and direct excretion played important roles in ASV clearance. 3. The primary metabolic pathways of ASV were hydroxylation, sulfonamide hydrolysis and the loss of isoquinoline. In vitro studies with human cDNA expressed CYP enzymes and with human liver microsomes (HLM) in the presence of selective chemical inhibitors demonstrated that ASV was primarily catalyzed by CYP3A4 and CYP3A5.

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GSK3532795, formerly known as BMS-955176 (1), is a potent, orally active, second-generation HIV-1 maturation inhibitor (MI) that advanced through phase IIb clinical trials. The careful design, selection, and evaluation of substituents appended to the C-3 and C-17 positions of the natural product betulinic acid (3) was critical in attaining a molecule with the desired virological and pharmacokinetic profile. Herein, we highlight the key insights made in the discovery program and detail the evolution of the structure-activity relationships (SARs) that led to the design of the specific C-17 amine moiety in 1. These modifications ultimately enabled the discovery of 1 as a second-generation MI that combines broad coverage of polymorphic viruses (EC <15 nM toward a panel of common polymorphisms representative of 96.5% HIV-1 subtype B virus) with a favorable pharmacokinetic profile in preclinical species.

A robust UHPLC-MS/MS assay was developed and validated for CP-I and CP-III in plasma, and is currently applied to clinical studies to confirm suitability of Coproporphyrins as a potential substitute for drug-drug interaction study.

Daclatasvir is a first-in-class, potent, and selective inhibitor of the hepatitis C virus nonstructural protein 5A replication complex. In support of nonclinical studies during discovery and exploratory development, liquid chromatography-tandem mass spectrometry and nuclear magnetic resonance were used in connection with synthetic and radiosynthetic approaches to investigate the biotransformation of daclatasvir in vitro and in cynomolgus monkeys, dogs, mice, and rats. The results of these studies indicated that disposition of daclatasvir was accomplished mainly by the release of unchanged daclatasvir into bile and feces and, secondarily, by oxidative metabolism. Cytochrome P450s were the main enzymes involved in the metabolism of daclatasvir. Oxidative pathways included d-oxidation of the pyrrolidine moiety, resulting in ring opening to an aminoaldehyde intermediate followed by an intramolecular reaction between the aldehyde and the proximal imidazole nitrogen atom. Despite robust formation of the resulting metabolite in multiple systems, rates of covalent binding to protein associated with metabolism of daclatasvir were modest (55.2-67.8 pmol/mg/h) in nicotinamide adenine dinucleotide phosphate (reduced form)-supplemented liver microsomes (human, monkey, rat), suggesting that intramolecular rearrangement was favored over intermolecular binding in the formation of this metabolite. This biotransformation profile supported the continued development of daclatasvir, which is now marketed for the treatment of chronic hepatitis C virus infection.