To be a qualified non specific CYP inhibitor
To be a qualified non-specific CYP inhibitor used as an in vitro or in vivo screening tool to distinguish CYP involvement in the overall metabolism, a complete inhibition of the entire metabolism catalyzed by CYPs in a simple experiment is desired. Based on our results, 100 μM atipamezole can completely inhibit the human, dog, or rat liver microsomal CYP activity in an incubation of 30–60 min (Fig. S3). This approach is far simpler and easier to be applied compared using ABT, in which preincubation is a necessary step. Another distinct advantage over ABT is that the CYP2C9 mediated metabolism can also be well blocked by atipamezole in liver microsomes of human and animals. CYP2C9 plays a critical role in the metabolism of many market drugs including NSAIDs (e.g. diclofenac, celecoxib, ibuprofen etc.), oral hypoglycemic, oral anticoagulants, diuretics and uricosurics, and many others (Zhou et al., 2009). Thus, being able to accurately account for its contribution toward the metabolism of a given drug is critical. To investigate the appropriate dose regimen for an in vivo application of atipamezole, different doses of atipamezole were applied in rat inhibition study by coadministrated CYP2C9 substrate diclofenac as a PK marker. The plasma concentration-time profiles of atipamezole following single oral doses of 3, 10, and 30 mg/kg (n = 5) in fasted male Sprague-Dawley rats are shown in Fig. S4. The results showed that with increasing doses from 3 to 30 mg/kg, atipamezole was well absorbed, the systemic concentration increased as the dose increased. After a single dose of 30 mg/kg atipamezole, the plasma concentration can rapidly reach above ~2000 ng/mL suggesting a rapid Copanlisib in rats. Protein binding of a compound in bio-matrix has impact on its disposition, efficacy, drug-drug interaction and safety as only free drug can act on pharmacological or toxicological target (Kalvass et al., 2018), the in vitro and in vivo free fractions of atipamezole were determined and free concentrations calculated. Given the atipamezole in vitro IC50 of CYP2C9 is about 4.49 μM in RLM, after taking into consideration correction for plasma and microsomal protein binding (Table S1), the 2000 ng/mL in plasma is around the effective concentration to inhibit diclofenac 4′-hydroxylation in rats. Diclofenac, the typical substrate of CYP2C9, is almost completely absorbed after p.o. administration in rats. It is subjected to first-pass metabolism so that only approximately 50% of the drug reaches the systemic circulation in an unchanged form (Kasperek et al., 2015). After co-administrated of a single dose of 30 mg/kg atipamezole with diclofenac (2 mg/kg), the concentration of diclofenac was dramatically increased initially to the same level achieved after ABT(100 mg/kg) co-administration applied according to the routine recommended pretreatment protocol (Boily et al., 2015), then dropped rapidly after 12-hour. As a result, the terminal t1/2 computed between 6 and 12 h increased in as less extend as the Cmax. On the other hand, up on a repeat dosing of atipamezole at 12 h, diclofenac plasma concentration (as well as t1/2) elevated significantly from 12 to 24 h (Fig. S6). This phenomenon further testified that CYP2C9 inhibition by atipamezole is reversible in rats, consistently with our in vitro finding. Given that a poor bioavailability of a well soluble drug mainly attributed to extensive metabolism and/or poor absorption is often affected by P-gp efflux, the interaction of atipamezole with P-gp was evaluated both in vitro and in vivo. Our in vitro study in P-gp transfected cell line and in vivo study in rat both demonstrated that atipamezole is neither a P-gp substrate nor a modulator of P-gp. That agrees with early literature report (Tannergren et al., 2003). Thus, atipamezole can be used as a tool compound to inhibit CYP mediated metabolism without affecting P-gp mediated transport. This can differentiate the metabolic firs-pass effect from the P-gp mediated efflux.