Inhibition of Cytochrome P450 Activities by Propoxazepam: Safety Assessment in Context for Potential Drug Interactions
DOI:
https://doi.org/10.20535/ibb.2025.9.2.309378Keywords:
propoxazepam, CYP3A4, testosterone, midazolam, reversible inhibition, metabolism dependent inhibitionAbstract
Background. Propoxazepam is a new anelgetic agent of the benzodiazepine group, chemically known as 7-bromo-5-(o-chlorophenyl)-3-propyloxy-1,2-dihydro-3H-1,4-benzodiazepine-2-one. Propoxazepam is considered a possible substrate of the CYP system, so its effect of on the CYP3A4 enzyme activity was investigated in vitro using human liver microsomes.
Objective. To evaluate the effects of propoxazepam on CYP3A4 activity in vitro using testosterone and midazolam as markers of metabolic activity for CYP3A4 in human liver microsomes.
Methods. Midazolam (1′-hydroxylation reaction) and testosterone (6β-hydroxylation reaction) were used as markers for CYP3A4-mediated activity. Ketoconazole (0.2 μM) was used as a positive control for reversible inhibition, and troleandomycin (50 μM) for metabolism-dependent inhibition. For reversible inhibition, propoxazepam was added together with the corresponding substrate and cofactor (NADPH), while for metabolism-dependent inhibition, it was incubated with microsomes and cofactor for 30 minutes prior to substrate addition.
Results. Propoxazepam at various concentrations (0 to 100 μM) consistently inhibited CYP3A4 activities for both substrates, showing a similar "concentration–activity inhibition" dependence, with IC50 values of 52.3 ± 4.9 μM for midazolam and 46.1 ± 9.2 μM for testosterone. For metabolism-dependent inhibition, IC50 values were 36.6 ± 8.6 μM for midazolam and 28.3 ± 7.4 μM for testosterone. Given that the binding of propoxazepam to microsomal protein under the experimental conditions, which reflected those in the IC50 experiments, was low, no microsomal binding correction factor was applied to the reported IC50 values.
Conclusions. The highest predicted unbound Cmax plasma concentration of propoxazepam, above which interactions can occur, is between 0.462 and 0.524 μM, or 462 and 524 nM. This corresponds to concentrations of 188 to 214 ng/mL (based on the molecular weight of propoxazepam, 414.73 g/mol). According to pharmacokinetic data, these concentrations are not achievable after a single oral administration. Further studies are required for multiple-dose administration.
References
Cederbaum AI. Molecular mechanisms of the microsomal mixed function oxidases and biological and pathological implications. Redox Biology. 2015;4:60-73. DOI: 10.1016/j.redox.2014.11.008
Sugishima M, Sato H, Higashimoto Y, Harada J, Wada K, Fukuyama K, Noguchi M. Structural basis for the electron transfer from an open form of NADPH-cytochrome P450 oxidoreductase to heme oxygenase. Proceedings of the National Academy of Sciences. 2014;111(7):2524-9. DOI: 10.1073/pnas.1322034111
Rao Gajula SN, Pillai MS, Samanthula G, Sonti R. Cytochrome P450 enzymes: a review on drug metabolizing enzyme inhibition studies in drug discovery and development. Bioanalysis. 2021;13(17):1355-78. DOI: 10.4155/bio-2021-0132
Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich FP. Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals: studies with liver microsomes of 30 Japanese and 30 Caucasians. The Journal of Pharmacology and Experimental Therapeutics. 1994;270(1):414-23. DOI: 10.1016/S0022-3565(25)22379-5
Stringer RA, Strain-Damerell C, Nicklin P, Houston JB. Evaluation of recombinant cytochrome P450 enzymes as an in vitro system for metabolic clearance predictions. Drug Metabolism and Disposition. 2009;37(5):1025-34. DOI: 10.1124/dmd.108.024810
Pelkonen O, Turpeinen M, Hakkola J, Honkakoski P, Hukkanen J, Raunio H. Inhibition and induction of human cytochrome P450 enzymes: current status. Archives of Toxicology. 2008;82(10):667-715. DOI: 10.1007/s00204-008-0332-8
Rendic S, Guengerich FP. Survey of Human Oxidoreductases and Cytochrome P450 Enzymes Involved in the Metabolism of Xenobiotic and Natural Chemicals. Chemical Research in Toxicology. 2015;28(1):38-42. DOI: 10.1021/tx500444e
Guidance for Industry on Drug Interaction Studies - Study Design, Data Analysis, and Implications for Dosing and Labeling. Food and Drug Administration, 2006.
Grimm SW, Einolf HJ, Hall SD, He K, Lim H-K, Ling K-HJ, et al. The conduct of in vitro studies to address time-dependent inhibition of drug-metabolizing enzymes: a perspective of the pharmaceutical research and manufacturers of America. Drug Metabolism and Disposition. 2009;37(7):1355-70. DOI: 10.1124/dmd.109.026716
Golovenko NYa, Larionov VB, Reder AS, Valivodz' IP. An effector analysis of the interaction of propoxazepam with antagonists of GABA and glycine receptors. Neurochemical Journal. 2017;11(4):302-8. DOI: 10.1134/S1819712417040043
Golovenko MYa, Larionov VB, Reder AS, Andronati SA, Valivodz' IP, Yurpalova TO. Pharmacodynamics of Interaction between Propoxazepam and a GABA-Benzodiazepine Receptor-Ionofor Complex. Neurophysiology. 2018;50(1):2-10. DOI: 10.1007/s11062-018-9711-9
Golovenko M, Reder A, Andronati S, Larionov V. Evidence for the involvement of the GABA- ergic pathway in the anticonvulsant and antinociception activity of Propoxazepam in mice and rats. Journal of Pre-Clinical and Clinical Research. 2019;13(3):99-105. DOI: 10.26444/jpccr/110430
Golovenko NYa, Voloshchuk NI, Andronati SA, Taran IV, Reder АS, Pashynska ОS, Larionov VB. Antinociception induced by a novel benzodiazepine receptor agonist and bradykinin receptor antagonist in rodent acute and chronic pain models. European Journal of Biomedical and Pharmaceutical Sciences. 2018;5(12):79-88.
Reder AS. Dispersed substance 7-bromo-5-(o-chlorophenyl)-3-propyloxy-1,2-dihydro-3H-1,4-benzdiazepine-2-one (I) with at least 50% volume fraction of particles less than 30 μm for use as anticonvulsive and analgesic drug. Patent of Ukraine UA118626C2, published 11.02.2019.
Fowler S, Zhang H. In vitro evaluation of reversible and irreversible cytochrome P450 inhibition: current status on methodologies and their utility for predicting drug-drug interactions. The AAPS Journal. 2008;10(2):410-24. DOI: 10.1208/s12248-008-9042-7
Guideline on the investigation of drug interactions. CMP/EWP/560/95/rev. 1. European Medicines Agency, Committee for Human Medicinal Products, 2012.
In Vitro Drug Interaction Studies - Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions. Guidance for Industry. Food and Drug Administration, 2020.
Otani K. [Cytochrome P450 3A4 and Benzodiazepines]. Seishin Shinkeigaku Zasshi. 2003;105(5):631-42.
Greenblatt DJ, Zhao Y, Venkatakrishnan K, Duan SX, Harmatz JS, Parent SJ, et al. Mechanism of cytochrome P450-3A inhibition by ketoconazole. Journal of Pharmacy and Pharmacology. 2011;63(2):214-21. DOI: 10.1111/j.2042-7158.2010.01202.x
Golovenko M, Reder A, Larionov V, Andronati S. Metabolic profile and mechanisms reaction of receptor GABA-targeted propoxazepam in human hepatocytes. Biotechnologia Acta. 2022;15(1):43-51. DOI: 10.15407/biotech15.01.043
Kandel SE, Han LW, Mao Q, Lampe JN. Digging Deeper into CYP3A Testosterone Metabolism: Kinetic, Regioselectivity, and Stereoselectivity Differences between CYP3A4/5 and CYP3A7. Drug Metabolism and Disposition. 2017;45(12):1266-75. DOI: 10.1124/dmd.117.078055
Williams JA, Ring BJ, Cantrell VE, Jones DR, Eckstein J, Ruterbories K, et al. Comparative metabolic capabilities of CYP3A4, CYP3A5, and CYP3A7. Drug Metabolism and Disposition. 2002;30(8):883-91. DOI: 10.1124/dmd.30.8.883
Denisov IG, Grinkova YV, McLean MA, Camp T, Sligar SG. Midazolam as a Probe for Heterotropic Drug-Drug Interactions Mediated by CYP3A4. Biomolecules. 2022;12(6):853. DOI: 10.3390/biom12060853
Houston JB, Kenworthy KE, Galetin A. Typical and atypical enzyme kinetics. In: Drug Metabolizing Enzymes: Cytochrome P450 and Other Enzymes in Drug Discovery and Development. Lausanne, Switzerland: Fontis Media, 2003, pp. 211-54. DOI: 10.1201/9781420028485.ch7
Xie H-G, Wood AJJ, Kim RB, Stein CM, Wilkinson GR. Genetic variability in CYP3A5 and its possible consequences. Pharmacogenomics. 2004;5(3):243-72. DOI: 10.1517/phgs.5.3.243.29833
Deodhar M, Al Rihani SB, Arwood MJ, Darakjian L, Dow P, Turgeon J, Michaud V. Mechanisms of CYP450 Inhibition: Understanding Drug-Drug Interactions Due to Mechanism-Based Inhibition in Clinical Practice. Pharmaceutics. 2020;12(9):846. DOI: 10.3390/pharmaceutics12090846
Golovenko MYa, Reder AS, Larionov VB, Andronati SA, Akisheva AS. Cross-species differential plasma protein binding of Propoxazepam, a novel analgesic agent. Biopolymers and Cell. 2021;37(6):459-68. DOI: 10.7124/bc.000A68
Golovenko M, Reder A, Zupanets I, Bezugla N, Larionov V, Valivodz' I. A Phase I study evaluating the pharmacokinetic profile of a novel oral analgesic propoxazepam. Journal of Pre-Clinical and Clinical Research. 2023;17(3):138-44. DOI: 10.26444/jpccr/169426
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