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siRNA-Mediated Knockdown of P450 Oxidoreductase in Rats: A Tool to Reduce Metabolism by CYPs and Increase Exposure of High Clearance Compounds

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ABSTRACT

Purpose

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.

Methods

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.

Results

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.

Conclusions

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.

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ACKNOWLEDGMENTS AND DISCOLSURES

The authors would like to thank Matthew Shipton and Carolyn Six at BioreclamationIVT for their assistance with the production of custom liver and intestinal microsomes from the treated and control rats. All authors were employed by Merck & Co., Inc. at the time this research was performed.

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Author notes

  1. Rob S. Burke

    Present address: Department of Drug Metabolism and Pharmacokinetics,, Avidity Nanomedicines,, La Jolla,, California, 92037, USA

Authors and Affiliations

  1. Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism,, Merck Research Laboratories, Merck & Co., Inc.,, West Point,, Pennsylvania, 19486, USA

    Rob S. Burke, Inthirai Somasuntharam, Paul Rearden, Roy Eisenhandler, Christopher Gibson, Kenneth A. Koeplinger, Karsten Menzel, Nicole Pudvah, Deanne Jackson Rudd, Thomayant Prueksaritanont, Charles D. Thompson, Jerome H. Hochman & Brian A. Carr

  2. Department of RNA Biology,, Merck Research Laboratories, Merck & Co., Inc.,, West Point,, Pennsylvania, 19486, USA

    Duncan Brown, Martha A. DiPietro, Jillian DiMuzio, Kelly A. Hamilton, Traci Q. Lifsted, Jessica Seitzer & Walter R. Strapps

  3. Department of Pharmacokinetics, Pharmacodynamics, and Drug Metabolism,, Merck Research Laboratories, Merck & Co., Inc.,, Boston,, Massachusetts, 02115, USA

    Sujal V. Deshmukh & Ian Knemeyer

  4. Department of Safety Assessment and Laboratory Animal Research,, Merck Research Laboratories, Merck & Co., Inc.,, West Point,, Pennsylvania, 19486, USA

    Scott E. Fauty & Hae Won Kwon

  5. Department of Pharmaceutical Sciences, RNA Therapeutics,, Merck Research Laboratories, Merck & Co., Inc.,, West Point,, Pennsylvania, 19486, USA

    Marian E. Gindy & Mihir Patel

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  1. Rob S. Burke
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Correspondence to Rob S. Burke.

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Burke, R.S., Somasuntharam, I., Rearden, P. et al. siRNA-Mediated Knockdown of P450 Oxidoreductase in Rats: A Tool to Reduce Metabolism by CYPs and Increase Exposure of High Clearance Compounds. Pharm Res 31, 3445–3460 (2014). https://doi.org/10.1007/s11095-014-1433-0

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  • DOI: https://doi.org/10.1007/s11095-014-1433-0

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