Disposition of 1-[3-(Aminomethyl)phenyl]-N-[3-fluoro-2‘- (methylsulfonyl)-[1,1‘-biphenyl]-4-yl]-3-(trifluoromethyl)- 1H-pyrazole-5-carboxamide (DPC 423) by Novel Metabolic Pathways. Characterization of Unusual Metabolites by Liquid Chromatography/Mass Spectrometry and NMR

Abstract

The in vitro and in vivo disposition of DPC 423 was investigated in mice, rats, dogs and humans and the metabolites characterized by LC/MS, LC/NMR and high field-NMR. The rodents produced several metabolites that included an aldehyde (M1), a carboxylic acid (M2), a benzyl alcohol (M3), glutamate conjugates (M4 and M5), an acyl glucuronide (M6) and its isomers; a carbamyl glucuronide (M7); a phenol (M8) and its glucuronide conjugate (M9), two glutathione adducts (M10 and M11), a sulfamate conjugate (M12), isomers of an oxime metabolite (M13), and an amide (M14). Humans and dogs produced less complex metabolite profiles than rats. While unchanged DPC 423 was the major component in plasma and urine samples, differences in the metabolic disposition of this compound among species were noted. M1 is believed to be rapidly oxidized to the carboxylic acid (M2), which forms the potentially reactive acyl glucuronide (M6). The formation of novel glutamate conjugates (M4 and M5) and their role in depleting endogenous glutathione have been described previously. The carbamyl glucuronide M7, found as the major metabolite in rats and in other species, was considered nonreactive and was easily hydrolyzed to the parent compound in the presence of beta-glucuronidase. The identification of GSH adducts M10 and M11 led us to postulate the existence of at least two reactive intermediates responsible for their formation, an epoxide and possibly a nitrile oxide, respectively. Although the formation of GSH adducts such as M10 from epoxides has been described before, there are no reports to date describing the existence of a GSH adduct (M11) of an oxime. The formation of a sulfamate conjugate (M12) formed by direct coupling of sulfate to the nitrogen of benzylamine is described. A mechanism is proposed for the formation of the oxime (M13) that involves sequential oxidation of the benzylamine to the corresponding hydroxylamine and nitroso intermediate. The rearrangement of the nitroso intermediate is believed to produce the oxime (M13). In vitro studies suggested that both the oxime (M13) and the aldehyde (M1) were precursors to the carboxylic acid (M2). This is the first demonstration of carboxylic acid formation via an oxime intermediate produced from an amine. The stability of DPC423 in plasma obtained from several species was studied. Significant species differences in the plasma stability of DPC 423 were observed. The formation of the aldehyde metabolite (M1) was found to be catalyzed by a semicarbazide-sensitive monoamine oxidase (SSAO) found in plasma of rabbits, dogs, and rhesus monkeys. Rat, chimpanzee, and human plasma did not form M1.

Amino Acid Conjugates: Metabolites of 2-Propylpentanoic Acid (Valproic Acid) in Epileptic Patients

“…Reports in the literature indicate that glycine and to a lesser extent glutamine are the most likely amino acids to be involved in the conjugation reactions of carboxylic acids (Hutt and Caldwell, 1990). It should also be added that recently the conjugation of xenobiotic amines with glutamate has also been reported (Mutlib et al, 2000;2002) In this study, quantitative analysis of the conjugates in human urine, serum, and CSF samples revealed that VPA-GLU was consistently the predominant amino acid conjugate compared with VPA-GLN, and VPA-GLY was a minor conjugate. The more extensive conjugation of VPA with glutamic acid than either glutamine or glycine is also an unprecedented observation for the human species.…”

Metabolism, pharmacokinetics and excretion of the GABAAreceptor partial agonist [14C]CP-409,092 in rats

“…Additionally, several reports have appeared in the literature describing the metabolism of benzylamines and structural analogues and results have revealed that the benzylamine moiety was a metabolic soft spot. Oxidative deamination yielding the aldehyde was demonstrated to be a precursor of the corresponding acid metabolite, and the formation of the aldehyde intermediate and/or metabolite has been attributed to mitochondrial monoamine oxidase (MAO)-B. Acyl glucuronides, a carbamyl glucuronide, oxime, a glutathione adduct and N-acetylation were also reported (Timperio et al 2003;Mutlib et al 2001Mutlib et al , 2002aMutlib et al , 2002bMutlib et al , 2002cShaffer et al 2008). …”

Reaction of Primary and Secondary Amines to Form Carbamic Acid Glucuronides

“…Several methods have been described for the preparation and characterization of carbamoyl glucuronides using in vitro techniques, including both hepatocytes and hepatic microsomes, as well as chemical synthesis. Table 1 shows two examples, a piperazine BVT.2938 [46] and a benzylamine DPC 423 [47], where carbamate glucuronides were formed in hepatocytes from rats, monkeys, and humans. A procedure for forming carbamate glucuronide metabolites using liver microsomes fortified with UDPGA in the presence of CO 2 was developed for carvedilol [16] and has been used for the formation of several other carbamate glucuronides summarized in Table 1.…”