Radiolabeled In vitro ADME Studies

After incubation of 10 µM labeled compound [¹⁴C]TA-1 in rat, dog, monkey and human hepatocytes, metabolite profiles were obtained by high performance liquid chromatography coupled with on-line radio-detector detection (see Figure 1). A total of 12 metabolites were detected across all incubation samples. And the radio-profiles of the parent drug and all metabolites in hepatocytes across species were summarized in Figure 1. The results indicated that metabolic rates of the compound in all animal hepatocytes were higher than that in human hepatocytes. Four metabolites found in human hepatocytes were observed in rat and monkey hepatocytes instead of dog hepatocytes. The percentage of total radioactivity of each metabolite is shown in Table 1.

[¹⁴C]TA-2, at the concentration of 10 µM was incubated with cryopreserved hepatocytes in a CO2 incubator with 5% CO2 and saturated humidity at 37°C for 4 hours. The positive control- [¹⁴C]Clozapine, [¹⁴C]Acetaminophen, and [³H]Warfarin at 10 µM, were run in parallel to assess reliability of the test system. Following incubation, samples were extracted. Post-extraction pellets were analyzed for protein concentration and radioactivity to quantify covalent binding levels, while the resulting extracts were analyzed via LC-RAM/HRMS to determine the metabolic turnover rate. The results were used to calculate the net covalent binding (CVBnet), the fraction of metabolism leading to covalent binding (fcvb), and daily CVB burden of the test articles. Detailed data are summarized in Table 2