1. siRNA-Mediated Knockdown of P450 Oxidoreductase in Rats: A Tool to Reduce Metabolism by CYPs and Increase Exposure of High Clearance Compounds
- Author
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Brian A. Carr, Inthirai Somasuntharam, Traci Q. Lifsted, Martha A. DiPietro, Christopher R. Gibson, Nicole T. Pudvah, Jerome Hochman, Scott Fauty, Marian E. Gindy, Sujal V. Deshmukh, Jillian DiMuzio, Thomayant Prueksaritanont, Hae Won Kwon, Ian Knemeyer, Paul Rearden, Deanne Jackson Rudd, Walter Strapps, Kelly Hamilton, Mihir Patel, Charles D. Thompson, Karsten Menzel, Roy Eisenhandler, Kenneth A. Koeplinger, Rob S. Burke, Duncan Brown, and Jessica Seitzer
- Subjects
Male ,Diclofenac ,Chemistry, Pharmaceutical ,Midazolam ,Pharmacology toxicology ,Pharmaceutical Science ,In Vitro Techniques ,Biology ,Pharmacology ,Cytochrome P-450 Enzyme System ,In vivo ,Microsomes ,Animals ,Pharmacology (medical) ,RNA, Small Interfering ,Gene knockdown ,Organic Chemistry ,Metabolism ,Small molecule ,Rats ,P450 oxidoreductase ,Biochemistry ,Plasma exposure ,Gene Knockdown Techniques ,Microsomes, Liver ,Nanoparticles ,Molecular Medicine ,Protein Binding ,Biotechnology - Abstract
To develop a tool based on siRNA-mediated knockdown of hepatic P450 oxidoreductase (POR) to decrease the CYP-mediated metabolism of small molecule drugs that suffer from rapid metabolism in vivo, with the aim of improving plasma exposure of these drugs.siRNA against the POR gene was delivered using lipid nanoparticles (LNPs) into rats. The time course of POR mRNA knockdown, POR protein knockdown, and loss of POR enzyme activity was monitored. The rat livers were harvested to produce microsomes to determine the impact of POR knockdown on the metabolism of several probe substrates. Midazolam (a CYP3A substrate with high intrinsic clearance) was administered into LNP-treated rats to determine the impact of POR knockdown on midazolam pharmacokinetics.Hepatic POR mRNA and protein levels were significantly reduced by administering siRNA and the maximum POR enzyme activity reduction (~85%) occurred 2 weeks post-dose. In vitro analysis showed significant reductions in metabolism of probe substrates due to POR knockdown in liver, and in vivo POR knockdown resulted in greater than 10-fold increases in midazolam plasma concentrations following oral dosing.Anti-POR siRNA can be used to significantly reduce hepatic metabolism by various CYPs as well as greatly increase the bioavailability of high clearance compounds following an oral dose, thus enabling it to be used as a tool to increase drug exposure in vivo.
- Published
- 2014
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