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  • Atipamezole hydrochloride Fig S an adrenoceptor antagonist i

    2020-11-25

    Atipamezole hydrochloride (Fig. S1), an α2-adrenoceptor antagonist, is commonly used by veterinarians to recover animals from sedation-anesthesia induced by α2-adrenoceptor agonists. Recent studies in animals suggest that atipamezole might have beneficial effects in AGK 2 damage repair, it also has anti-Parkinsonian effects as dopaminergic drug by improving the motor recovery following stroke (Pertovaara et al., 2005; Beltran et al., 2010). In phase 1 clinical trials, atipamezole is well tolerated by human subjects (Karhuvaara et al., 1990). During our recent quantitative evaluation of the inhibitory effects of atipamezole toward major CYP isoforms, the non-specific pan-CYP inhibitory potential of atipamezole was discovered. Hence, the aim of the present study was to investigate the inhibitory effects of atipamezole toward CYPs in vitro and in vivo and to demonstrate it to be a novel pan-CYP inhibitor to be used in drug metabolism studies.
    Materials and methods
    Results
    Discussion Low oral bioavailability is a common concern in drug discovery. Elucidating whether extensive metabolism is the key factor can allow structural modification to improve ADME properties. On the other hand, blocking of P450-mediated drug metabolism offers an approach to either maximize the exposure of highly metabolized compounds or, alternatively, block the formation of metabolites in vivo. Up to date, ABT has been widely used as a non-specific CYP inhibitor because it is known to be a suicidal substrate for CYP isoforms. However, due to its poor inhibition against human CYP2C9 (e.g. diclofenac 4′-hydroxylation) (Emoto et al., 2003), a challenge would rise when using ABT as a pan-P450 inhibitor. As such, non-P450 enzymatic activity (the portions not inhibited by ABT) cannot be accurately assessed. As a time-dependent inhibitor, ABT requires preincubation to inactivate the CYPs. The preincubation procedure could be varying from lab to lab, as well as the effectiveness of the consequent CYP inactivation. To address this concern, a safe, potent and more non-discriminative CYP inhibitor is desired. In the present investigation described above, direct and time-dependent inhibition toward major CYP enzymes in human, dog and rat liver microsomes were compared for atipamezole and ABT. In vivo inhibition effect of CYP2C9 using diclofenac as a substrate was further compared between atipamezole and ABT in rats. In the inhibition study, phenacetin O-deethylation (POD), bupropion hydroxylation (BPOH), amodiaquine N-deethylation (AMND), diclofenac 4′-hydroxylation (DFOH), S-mephenytoin 4′-hydroxylation (SMOH), dextromethorphan O-demethylation (DMOD), and midazolam 1′-hydroxylation (MD1-OH) were selected as the marker reactions for CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6 and 3A4/5 activities in HLM per the FDA guidance (FDA Draft Guidance for Industry, 2017a, FDA Draft Guidance for Industry, 2017b). The concentrations of probe substrates were set at or below their Km values to make the achieved IC50 values closer to their Ki. Despite the existence of species differences in drug metabolism, similar metabolic pathways generated by the human CYP substrates used in this study were observed in rat and dog, like described in literature (Sasaki and Shimoda, 2015). Moreover, the study of drug metabolism in preclinical species plays an important role in drug research and development, especially reviewing the in vitro and in vivo studies together for establishing in vitro-in vivo correlation (IVIVC), which has a great value in pharmacokinetics, pharmacodynamics, and toxicokinetics assessment. Our study demonstrated that atipamezole strongly inhibits the seven-major human CYP isoforms including CYP2C9 (diclofenac 4′-hydroxylation) in HLM, as well as in DLM and RLM. No IC50 shift toward left side was observed indicating the inhibitory effect was in a reversible manner. On the other hand, the depletion of atipamezole caused the slight decreases of IC50 after preincubation with NADPH (Table 1). This type of phenomena also occurred when ketoconazole (a metabolic instable compound) was taken as an inhibitor toward CYP3A4 mediated ritonavir metabolism (Berry et al., 2013).