Background : Carboxylic acids constitute a large and heterogeneous class of both endogenous and xenobiotic compounds. A number of carboxylic acid drugs have been associated with adverse reactions, linked to the metabolic activation of the carboxylic acid moiety of the compounds, i.e., formation of acyl-glucuronides and acyl-CoA thioesters. Objective : The objective is to give an overview of the current knowledge on metabolic activation of carboxylic acids and how such metabolites may play a role in adverse reactions and toxicity. Methods : Literature concerning the formation and disposition of acyl glucuronides and acyl-CoA thioesters was searched. Also included were papers on the chemical reactivity of acyl glutathionethioesters, and literature concerning possible links between metabolic activation of carboxylic acids and reported cellular and clinical effects. Results/conclusion : This review demonstrates that metabolites of carboxylic acid drugs must be considered chemically reactive, and that the current knowledge about metabolic activation of this compound class can be a good starting-point for further studies on the consequences of chemically reactive metabolites.

This paper is available online at http://dmd.aspetjournals.org ABSTRACT:Clofibric acid (p-chlorophenoxyisobutyric acid) is metabolized in vivo to a thioester-linked glutathione conjugate, S-(p-chlorophenoxyisobutyryl)glutathione (CA-SG). The formation of this metabolite is presumed to occur via transacylation reactions between glutathione (GSH) and reactive acyl-linked metabolite(s) of the drug. The present study examines the chemical reactivity of clofibryl-S-acyl-CoA (CA-SCoA), an acyl-CoA thioester intermediary metabolite of clofibric acid, with GSH to form the CA-SG in vitro. Incubations of CA-SCoA (1 mM) with GSH (5 mM) were carried out at pH 7.5 and 37°C, with analysis of the formed reaction products by isocratic reverse-phase high-performance liquid chromatography (HPLC). Results showed a time-dependent and linear formation of CA-SG up to 4 h (50 M CA-SG formed/h), and after a 1-day incubation, the reaction mixture contained 0.7 mM CA-SG. The identity of CA-SG was confirmed by analysis of HPLC-purified material by tandem mass spectrometry. The rate of CA-SG formation was found to be increased 3-fold in incubations containing rat liver glutathione S-transferases (4 mg/ml). Analysis of the chemical stability of CA-SCoA in buffer at 37°C and varying pH showed the derivative to be stable under mildly acidic and basic aqueous conditions but to hydrolyze at pH values greater than 10 after a 1-day incubation (t 1/2 ‫؍‬ ϳ1 day at pH 10.5). Results from these studies show that CA-SCoA is a reactive thioester derivative of clofibric acid and is able to acylate GSH and other thiol-containing nucleophiles in vitro and, therefore, may be able to acylate protein thiols in vivo, which could contribute to the toxic side effects of the drug.

Chemically reactive species formed from the metabolism of carboxylic acid-containing compounds have been proposed as mediators of their toxic side-effects. Two alternative metabolic pathways known to be involved in the generation of reactive acylating metabolites of carboxylic acids are acyl glucuronidation and acyl-CoA formation. Here, we present studies with 2-phenylpropionic acid focused on evaluating the relative abilities of acyl glucuronides versus acyl-CoA derivatives to transacylate the nucleophilic cysteinyl-thiol of glutathione. Thus, synthetic 2-phenylpropionyl-S-acyl-CoA (2-PPA-SCoA) and biosynthetic 2-phenylpropionyl-1-O-acyl glucuronide (2-PPA-1-O-G) were incubated separately, and at varying concentrations (15.6-500 nM as well as at 0.1 mM), with GSH (1, 5, and 10 mM) in buffer (pH 7.4, 37 degrees C), and formation of the transacylation product, 2-phenylpropionyl-S-acyl-glutathione (2-PPA-SG), was quantified by reverse-phase HPLC and LC-MS. HPLC analysis of the products from both the reaction of 2-PPA-SCoA and 2-PPA-1-O-G with GSH showed the presence of 2-PPA-SG, which was confirmed by coelution with authentic 2-PPA-SG as well as by its LC/MS mass spectrum. The formation of 2-PPA-SG was time- and concentration-dependent with a formation rate constant of (1.9 +/- 0.2) x 10(-2) M(-1) x s(-1) from reactions of GSH with 2-PPA-SCoA, and (2.7 +/- 0.4) x 10(-4) M(-1) x s(-1) from reactions of GSH with 2-PPA-1-O-G. Therefore, the reactivity of 2-PPA-SCoA with GSH was 70 times greater than the reactivity of GSH with 2-PPA-1-O-G, which was found to acyl-migrate to less reactive isomers. Analysis of the in vitro stability of 2-PPA-SCoA and 2-PPA-1-O-G in the absence of GSH showed the CoA esters to be completely stable after 24 h, whereas the acyl glucuronides decomposed by 50% in 1.3 and 2.4 h of incubation at pH 7.4 and 37 degrees C for (R)- and (S)-2-PPA-1-O-G, respectively. In addition, studies of the reactivity of 2-PPA-SCoA with bovine serum albumin showed time- and pH-dependent covalent binding to the protein in vitro. These results support the hypothesis that xenobiotic acyl-CoA thioesters are reactive acylating species that, in addition to acyl glucuronides, may contribute to xenobiotic acid-protein adduct formation in vivo.

Mavacamten is a small molecule modulator of cardiac myosin designed as an orally administered drug for the treatment of patients with hypertrophic cardiomyopathy. The current study objectives were to assess the preclinical pharmacokinetics of mavacamten for the prediction of human dosing and to establish the potential need for clinical pharmacokinetic studies characterizing drug-drug interaction potential. Mavacamten does not inhibit CYP enzymes, but at high concentrations relative to anticipated therapeutic concentrations induces CYP2B6 and CYP3A4 enzymes in vitro. Mavacamten showed high permeability and low efflux transport across Caco-2 cell membranes. In human hepatocytes, mavacamten was not a substrate for drug transporters OATP, OCT and NTCP. Mavacamten was determined to have minimal drug-drug interaction risk. In vitro mavacamten metabolite profiles included phase I- and phase II-mediated metabolism cross-species. Major pathways included aromatic hydroxylation (M1), aliphatic hydroxylation (M2); N-dealkylation (M6), and glucuronidation of the M1-metabolite (M4). Reaction phenotyping revealed CYPs 2C19 and 3A4/3A5 predominating. Mavacamten demonstrated low clearance, high volume of distribution, long terminal elimination half-life and excellent oral bioavailability cross-species. Simple four-species allometric scaling led to predicted plasma clearance, volume of distribution and half-life of 0.51 mL/min/kg, 9.5 L/kg and 9 days, respectively, in human.

Cannabidiol (CBD) is a major constituent of marijuana and a potent inhibitor of P450-mediated hepatic drug metabolism. Mouse P450 3A11 metabolism of [14C]CBD resulted in the formation of radiolabeled P450, which after digestion with lysyl endopeptidase C (Lys-C) and HPLC resolution of peptides, revealed one major broadly eluting peak of radioactivity. Electrophoresis/autoradiography of this peak identified several peptide bands, one of which was predominantly radiolabeled and had an apparent molecular mass of approximately 6 kDa. Amino-terminal sequence determination of this band revealed the presence of two peptides whose sequences identified them as Ala344-Lys379 and Gly426-Lys454. To characterize the reactive species that may be generated during P450 3A11-catalyzed CBD metabolism, reduced glutathione (GSH) was used as a trapping agent for possible electrophilic metabolites. Incubation of P450 3A11 in the presence of cofactors NADPH, CBD, and [3H]GSH resulted in the formation of a radiolabeled product which was absent in incubations lacking any of the cofactors. The UV absorption spectra of this compound indicated absorbances at approximately 220, 275, and 350 nm, and mass spectral analysis revealed prominent ions at m/z 634, 599, 505, 402, and 359, ions consistent with that of a GSH adduct of CBD-hydroxyquinone. A synthetic CBD-hydroxyquinone-GSH adduct was also prepared and had UV absorption and mass spectra nearly identical to that of the P450-mediated CBD-GSH adduct. CBD-hydroxyquinone formation may be the penultimate oxidative step involved in CBD-mediated modification and inactivation of P450 3A11.