(W1130-10-69) Pharmacokinetics, Excretion, and Metabolism of [¹⁴C]Aficamten Following Single Oral Dose Administration to Rats

Purpose: Aficamten (also known as CK-3773274) is a small molecule, allosteric inhibitor of cardiac myosin being developed as a chronic oral treatment for patients with symptomatic obstructive hypertrophic cardiomyopathy (oHCM). The purpose of this study is to determine the pharmacokinetics, distribution, metabolism, and excretion of [¹⁴C]aficamten and to characterize the metabolites present in plasma, urine, bile, and feces in male rats following single oral dose administration. Tissue distribution was investigated by quantitative whole body autoradiography (QWBA) in Long Evans (LE) rats and Sprague Dawley (SD) rats.

Methods: The absorption, distribution, metabolism, and excretion of radioactivity were determined after administration of a single oral dose (8 mg/kg, 100 µCi) of [¹⁴C]aficamten to male intact and bile duct-cannulated (BDC) Sprague Dawley rats. Blood was collected for pharmacokinetic analysis at selected times through 72 hours post-dose. Elimination of radioactivity in urine, feces, and expired air after oral dosing to intact SD rats was determined through 168 hours post-dose. Excretion of radioactivity in bile, urine, and feces through 168 hours was determined in BDC rats after oral administration. Samples were processed and analyzed for total radioactivity by liquid scintillation counting. Profiling and identification of metabolites in plasma, urine, and feces were conducted by liquid chromatography (LC)‑radiometric and LC-tandem mass spectrometric (LC-MS/MS) analyses. LE and SD animal carcasses were processed for QWBA analysis.

Results: After oral dosing to male SD rats, radioactivity was widely distributed to all the tissues in male SD rats by the first collection time point (1 hour post-dose). The majority of the tissues reached the maximum concentration by the 1 to 4 hour time points. The tissues exhibiting the highest maximum concentrations of radioactivity included muscle (semitendinosus), myocardium, muscle, diaphragm, fascia, and liver. Similar to SD rats, radioactivity was also widely distributed to all the tissues in LE rats by the first collection time point (0.5 hour post-dose). Most of the tissues reached maximum concentration by 2 hours post-dose. The tissues exhibiting the highest maximum concentrations of radioactivity included myocardium, muscle (semitendinosus), diaphragm, fascia, muscle, fat (abdominal), eye uveal tract, and liver. Radioactivity was cleared from all the tissues by 72 hours post-dose, with the exception of arterial wall, intervertebral ligament(s), and nasal turbinates. [¹⁴C]Aficamten underwent extensive metabolism in rats following oral administration via hydroxylation followed by Phase II mediated-glucuronidation and sulfonation, as well as oxadiazole ring reduction, and desaturation. [¹⁴C]aficamten and 11 metabolites in total were identified. Metabolites cited in the text below are shown in Figure 1. Unchanged [¹⁴C]aficamten was the major circulating component in the area under the concentration-time curve (AUC)-pooled plasma sample (AUC from 0.25 to 48 hours post-dose) from intact rats and accounted for approximately 80% of the total radioactivity exposure. The hydroxylated metabolite M1 (a mixture of two metabolites M1a and M1b) was a second major circulating component and accounted for approximately 12% of the total radioactivity exposure. Other minor circulating metabolites identified were the desaturated metabolite M2, hydroxylated metabolite M3, M5 a glucuronide metabolite of M1, and M45, a hydroxy‑oxadiazole-ring-opened metabolite contributing < 4% of the total radioactivity exposure. The mean overall recoveries of radioactivity after oral dosing to intact and BDC rats were 100 and 98.1%, respectively. Hepatobiliary excretion was the major route of elimination of radioactivity after oral dosing, with approximately 51% of the administered dose recovered in bile through 72 hours post-dose. Trace levels of unchanged [¹⁴C]aficamten were recovered in bile. Metabolite M5 was the major component in bile and accounted for 35.2% of the dose. Approximately 90% of dose‑related material was recovered in feces from intact rats through 48 hours post-dose after oral dosing. In BDC rats, approximately 37% of the dose was recovered in feces through 48 hours post-dose where no unchanged [¹⁴C]aficamten was recovered. Metabolite M18, an oxadiazole ring-opened diamine derivative, was the major component detected in feces from intact and BDC rats and accounted for 35.3 and 7.12% of the dose, respectively. M18 is proposed to be formed likely from reductive metabolism of metabolite M5 in the gastrointestinal tract (GI tract) by the gut microflora. Renal elimination was a minor route of excretion after oral dosing. Approximately 8% of the dose was recovered in urine from intact and BDC rats through 48 hours post-dose. Trace levels of unchanged [¹⁴C]aficamten were detected in urine. Metabolites M5, M3, and M1 were the abundant metabolites detected in urine.

Conclusion: These results indicated that [¹⁴C]aficamten was eliminated in rats primarily by metabolism and mostly eliminated in feces as metabolite M18 presumably mediated initially by the formation of metabolite M1, followed by glucuronidation to metabolite M5 which then undergoes biliary elimination to the gastrointestinal tract prior to the proposed reductive metabolism by gut microflora.

Figure 1. Predominant biotransformation pathways in rat (*denotes site of the radiolabel).