Biopolym. Cell. 2021; 37(6):459-468.
Cross-species differential plasma protein binding of Propoxazepam, a novel analgesic agent.
1Golovenko M. Ya., 2Reder A. S., 1Larionov V. B., 1Andronati S. A., 3Akisheva A. S.
  1. O. V. Bogatsky's Physico-chemical Institute, NAS of Ukraine
    86, Lustdorfskaya dor., Odessa, Ukraine, 65080
    86, Lustdorfskaya dor., Odessa, Ukraine, 65080
  3. Odessa I. I. Mechnikov National University
    Dvoryanskaya Str., 2, Odessa, Ukraine, 65082


Aim. To characterize the Propoxazepam properties of bin-ding to the human, dog and rat plasma proteins and to evaluate potential interspecies differences in binding affinity. Methods. The studies on Plasma protein binding were conducted using the Rapid Equilibrium Dialysis system with subsequent LC MS/MS analysis. The albumin binding of the substances was characterized by the percentage of bound drug. Results. Propoxazepam was highly bound to the plasma proteins in rats, dogs and humans. The extent of Propoxazepam binding in human plasma was similar to that observed in rat plasma whereas it was slightly lower in dog plasma, following dialysis for 8 hours. Conclusions. No concentration dependent effects on plasma protein binding were observed in the species investigated for Propoxazepam. Overall mean free fractions of Propoxazepam were 1.60 %, 2.90 % and 1.96 % in rats, dogs and humans, respectively. The results obtained in this work gave insights on the role of HSA in distributing Propoxazepam to target tissues and in determining their pharmacological properties. The results are crucial for the development of benzodizepines based therapeutics drugs.
Keywords: Propoxazepam, plasma protein, interspecies binding


[1] Golovenko N, Voloshchuk N, Andronati S, Taran I, Reder A, Pashynska O, Larionov V. Antinociception induced by a novel benzodiazepine receptor agonist and bradykinin receptor antagonist in rodent acute and chronic pain models. EJBPS. 2018; 5(12): 79-88.
[2] Golovenko N, Larionov V, Reder A, Valivodz I. An effector analysis of the interaction of propoxazepam with antagonists of GABA and glycine receptors. Neurochemical Journal. 2017; 11(4): 302-9.
[3] Golovenko N, Larionov V, Andronati S, Valivodz' I, Yurpalova T. Pharmacodynamic analysis of propoxazepam interaction with GABA-benzodiazepine-receptor-ionophore complex. Neurophysiology. 2018; 50(1): 2-11.
[4] Voloshchuk N, Reder A, Golovenko M, Taran I, Pashinska O. Pharmacological analysis of neurochemical antino-ciceptive mechanisms of propoxazepam action. Pharmacol Drug Toxicol. 2017; 1(52): 3-11.
[5] Lohman JJ, Merkus WH, Rahn KH. Plasma protein binding of drugs. 1986. Pharm Weekblad; 8: 302-4.
[6] Buscher B, Laakso S, Mascher H, Pusecker K, Doig M, Dillen L, Wagner-Redeker W, Pfeifer T, Delrat P, Tim-merman P. Bioanalysis for plasma protein binding studies in drug discovery and drug development: views and recommendations of the European Bioanalysis Forum. Bioanalysis. 2014; 6:673-82.
[7] Howard ML, Hill JJ, Galluppi GR, Mclean MA. Plasma protein binding in drug discovery and development. Comb Chem High Throughput Screen. 2010; 13:170-87.
[8] Jia HY, Yang GL, Li ZW, Xin PY, Zhao Y, Chen Y. Micellar liquid chromatography with dodecyl dimethyl betaine as an in vitro method for prediction of protein-drug binding. J Chromatogr A. 2007; 1143: 88-97.
[9] Bohnert T, Gan LS. Plasma protein binding: from discovery to development. J Pharm Sci. 2013; 102(9): 2953-94.
[10] Colclough N, Ruston L, Wood J, MacFaul P. Species differences in drug plasma protein binding. Med Chem Commun. 2014; 5: 963-67.
[11] Reder A. Dispersed substance 7-bromo-5-(o-chlorophenyl)-3-propiloxy-1,2-dihydro-3H-1,4-benzodiazepine-2-one (I) with at least 50 % volume fraction of particles less than 30μm for use as anticonvulsive and analgesic drug. Patent UA 118626.
[12] Andrade EL, Bento AF, Cavalli J, Oliveira SK, Schwanke RC, Siqueira JM, Freitas CS, Marcon R, Calixto JB. Non-clinical studies in the process of new drug development - Part II: Good laboratory practice, metabolism, pharmacokinetics, safety and dose translation to clinical studies. Braz J Med Biol Res. 2016; 49(12): e5646.
[13] Trainor GL. The importance of plasma protein binding in drug discovery. Expert Opin Drug Discov. 2007; 2(1): 51-64.
[14] European Medicines Agency, Committee for Human Medicinal Products. Guideline on the Investigation of Drug Interactions, Final. CPMP/EWP/560/95/Rev. 1 Corr.*. European Medicines Agency, London, UK. 2012.
[15] Tesseromatis C, Alevizou A. The role of the protein-binding on the mode of drug action as well the interactions with other drugs. Eur J Drug Metab Pharmacokinet. 2008; 33: 225-30.
[16] Phuangsawai O, Hannongbua S, Gleeson, M. Elucidating the origin of the esterase activity of human serum albumin using QM/MM calculations. J Phys Chem. B 2014, 118(41): 11886-89.
[17] Ghuman J, Zunszain P, Petitpas I, Bhattacharya A, Otagiri M, Curry S. Structural basis of the drug-binding specificity of human serum albumin. J Mol Biol. 2005; 353(1): 38-52.
[18] Fasano M, Curry E, Terreno M, Galliano G, Fanali P, Narciso S, Ascenzi N. The extraordinary ligand binding properties of human serum albumin. IUBMB Life. 2005; 57 (12): 787-96.
[19] Jaworski T, Tomasik M, Borkowski L, Buksowicz A, Danevych B, Hulyma Yu, Grudinin A, Khoma S, Delikatnyi A, Stetsenko T, Sitcs M, Talents T, Bi-ders A. Report on conformity of the process of state registration of medicinal products in Ukraine with the EU law and standards. Advice on Regulatory Improvements in Ukraine's Pharmaceutical Sector. 2010. 178p.