Abstract
The cytochrome P450-mediated Phase I in vitro metabolism of CRV431 was studied using selective chemical inhibition and recombinant human enzymes. Additionally, the metabolic profile of CRV431 in human, rat, and monkey liver microsomes was investigated. Liver microsomes were incubated for 0–80 min with CRV431, and the metabolite profile was assessed by electrospray ionization liquid chromatography mass spectrometry (ESI-LCMS). CRV431 was extensively metabolized through oxidation to produce various hydroxylated and demethylated species. Species identified included monohydroxylated CRV431 (two distinct products), dihydroxylated CRV431, demethylated CRV431 (two distinct products), demethylated and hydroxylated CRV431 (two distinct products), didemethylated and hydroxylated CRV431, and didemethylated and dihydroxylated CRV431. The magnitude of metabolism was greatest in monkey, followed by human, followed by rat. Importantly, all of the species identified in human microsomes were correspondingly identified in monkey and/or rat microsomes. Human liver microsome studies using selective chemical inhibition, as well as studies using recombinant human cytochrome P450 enzymes, revealed that the major enzymes involved are cytochromes P450 3A4 and 3A5. Enzymes 1A2, 2B6, 2C8, 2C9, 2C19, and 2D6 are not involved in the in vitro metabolism of CRV431. This information will be useful for the further development of CRV431 both preclinically and clinically.
Highlights
Cyclosporine A (CsA) was isolated from Tolypocladium inflatum in 1971, and has been used clinically since the early 1980s as an immunosuppressive drug to prevent rejection after solid organ transplantation [1]
Cyclophilins play an important role in infectious diseases, including for example, treatment of the hepatitis C virus (HCV) and hepatitis B virus (HBV)
We describe the cytochrome P450-mediated Phase I in vitro metabolism of CRV431 using selective chemical inhibition and recombinant human enzymes to gain a better understanding of the fate of the parent molecule, and understand potential routes of elimination of both parent and metabolite
Summary
Cyclosporine A (CsA) was isolated from Tolypocladium inflatum in 1971, and has been used clinically since the early 1980s as an immunosuppressive drug to prevent rejection after solid organ transplantation [1]. In in vitro [4,5] and in vivo (unpublished data) studies to date, CRV431 has been shown to reduce HBV and DNA, as well as target HBV proteins, including protein X (HBx) and surface antigen (HBsAg), through its ability to abrogate binding of cyclophilins to proteins. It is Pharmaceutics 2017, 9, 51; doi:10.3390/pharmaceutics9040051 www.mdpi.com/journal/pharmaceutics. We describe the cytochrome P450-mediated Phase I in vitro metabolism of CRV431 using selective chemical inhibition and recombinant human enzymes to gain a better understanding of the fate of the parent molecule, and understand potential routes of elimination of both parent and metabolite. We investigate the metabolic profile of CRV431 in human, rat, and monkey liver microsomes, and identify multiple metabolites by liquid chromatography—mass spectrometry (LC-MS)
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