Biotransformation of Daclatasvir In Vitro and in Nonclinical Species: Formation of the Main Metabolite by Pyrrolidine  -Oxidation and Rearrangement

Abstract

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.

Application of in vitro Drug Metabolism Studies in Chemical Structure Optimization for the Treatment of Fibrodysplasia Ossificans Progressiva (FOP)

“…LDN-193189 (10 μM) was incubated with mouse, rat, dog, rabbit, monkey and human liver microsomes (1 mg/mL) with/without cytosol (2 mg/mL) in the presence of an NADPH-regenerating system [(glucose-6-phosphate (3.6 mM), NADP + (1.3 mM), and glucose-6-phosphate dehydrogenase (0.4 units/mL)], MgCl 2 (10 mM), and UDPGA (2 mM) in 0.1 M phosphate buffer (pH 7.4), a procedure similar to previously described MetID workflows (Li et al, 2016; Ahire et al, 2017). Liver microsomal or cytosolic fractions containing appropriate cofactors were also fortified with either KCN (0.1 mM), GSH (2 mM), menadione (1 mM) or allopurinol (0.1 mM).…”

Pencil Graphite Electrode Decorated with Xylenol Orange Flakes for Studying Possible Pharmacokinetic Interaction Between Vardenafil and Daclatasvir

“…The incidence of drug‐drug interaction is increased when there is competition between two or more drugs for oxidation by the same P450 enzyme. Accordingly, VAR dose adjustment may be necessary when administered with DAC as both of them are considered as substrates for CYP3A4 enzyme and moreover, DAC was found to be a weak to moderate inhibitor of CYP3A4 . Therefore, it is essential to evaluate pharmacokinetic drug‐drug interactions by different analytical methods to enhance the efficacy and safety of the treatment and to eliminate the incidence of toxicity.…”

Effect of CYP3A5*3 genetic variant on the metabolism of direct-acting antivirals in vitro: a different effect on asunaprevir versus daclatasvir and beclabuvir

“…Importantly, all the three DAAs; ASV, DCV, and BCV, are considered to be mainly metabolized by CYP3A enzymes (ref. [22][23][24]. However, the differences in the detailed metabolic activities of these three drugs between CYP3A4 and CYP3A5 are ambiguous There are still a few difficult-to-treat patients even with the use of the combination tablet of ASV/DCV/BCV.…”

“…on the previous reports (ref 22,23,. 29), the detection methods for ASV, DCV, and BCV were established with simple modification in the present study.…”

“…The coefficients of variation value (%CV) precision were less than 5%. The mean accuracies calculated as the percentage of the nominal concentration were also less than 5%.Optimization was performed to detect ASV, DCV, and BCV metabolites based on m/z information reported previously (ref 22,23,. 29).…”