Major biotransformation routes of aficamten

Aficamten is extensively metabolized in humans to 3 major circulating metabolites, M1a, M1b and M5 [1].  Initially aficamten is oxidized by multiple cytochrome P450s (CYPs 2C8, 2C9, 2D6, and 3A4) to hydroxylated metabolites, M1a, M1b and M3. M1a and M1b are circulating diastereomers and together make up nearly half of the total radioactivity exposure. They are formed by ω-1-hydroxylation of the ethyl group of the ethyl-1,2,4-oxadiazol-3-yl moiety and whilst some hydroxylated metabolites of parent drugs may possess on/off target activity, M1a and M1b are pharmacologically inactive. M3 is a much more minor metabolite resulting from hydroxylation of the indane-moiety.

Onwards glucuronidation to a major metabolite

The hydroxyl position of both M1a and M1b are further metabolized, either by glucuronidation (M5 and M6) or sulfonation (M7/M64). Glucuronidation is the more dominant route with the O-glucuronide of M1a (M5) forming just over 10% of the total radioactive AUC [1]. In vitro studies in rat liver microsomes looking at production of M1 revealed a 4.2 fold higher formation of M1a compared to M1b after 60 minutes incubation [2]. It was suggested that M1a may serve as a superior substrate for UGT enzymes, which, if mirrored in humans, would explain the higher flux through the M1a to M5 pathway and onwards to gut metabolism.

Involvement of gut microbes to form a major fecal metabolite

Action of bacteria in the gut results in reductive ring opening and hydrolysis of the 1,2,4-oxadiazole moiety of M5 to a major fecal metabolite M18, accounting for just over 44% of the radioactive dose [1]. Previous studies in rat confirmed the involvement of gut bacteria in this biotransformation [2]. M18 is subsequently hydrolyzed at the imidamide moiety to M9.

Whilst the rat study showed that M18 could be formed from M5, there are two routes possible. First from direct reduction of M5 by gut bacteria, or, the conversion of M5 back to M1a by gut bacteria β-glucuronidase then ring opening of the 1,2,4-oxadiazole moiety. It’s hypothesized that any parent drug might also be degraded in the intestine to M18.

Important aspects of oxadiazole gut metabolism

Oxadiazole ring systems are sensitive to reductive ring opening and hydrolysis in the anaerobic gut environment such as also reported for the 1,2,4- oxadiazole ring systems in DS-8500a and ozanimod. Several marketed drugs contain this ring system, for which oxadiazole ring reductive cleavage products have been reported [3]. The process has been reported to be catalyzed by enzymes in liver and by gut microflora [4]. For the drug ataluren, oxadiazole ring cleavage was proposed as a partly enzyme-mediated reaction since cleavage products were detected in both plasma and urine. However, high levels of cleavage metabolites in feces were most likely formed by the microbial flora in the intestine from unabsorbed parent drug and from ataluren acyl glucuronide after biliary excretion.

Formation of the ring-opened metabolites of DS-8500a were observed in rat bile and multi species hepatocyte incubations and in human liver microsomes fortified with an NADPH-generating system under anaerobic conditions [5]. However, a subsequent in vivo study in humans and monkeys revealed a marked species difference in the reductive ring-opening by intestinal microflora and liver pathways vs rat. In monkeys, oxadiazole ring-cleaved metabolites were mainly detected in feces, and not observed in bile like in rats, suggesting that the reductive ring-opening metabolism occurs in the gastrointestinal tract. Interestingly, in vitro incubation with enterobacterial culture media demonstrated that the reductive cleavage of the DS-8500a oxadiazole ring in humans and monkeys was considerably faster than that in rats [6].

In the case of ozanimod, a major human circulating metabolite RP101124 was formed as a result of oxadiazole reduction and hydrolysis of an intermediary amide, discovered during an in vivo ¹⁴C-ADME study in rats where a lag time was observed before its appearance in circulation. Antibiotic studies revealed the involvement of gut microflora in the formation of a RP101124 under strict anaerobic conditions [7].

Aficamten study outcome

PK and metabolism studies to date have not flagged any safety concerns and since multiple CYPs are involved in the primary metabolism of aficamten, no CYP-based drug-drug interaction liability is predicted [8].