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ОРИГИНАЛЬНЫЕ ИССЛЕДОВАНИЯ
© Коллектив авторов, 2017 УДК 615.21.015.44 DOI:10.23888/PAVLOVJ2017137-41
ANALYSIS OF ATTRIBUTION OF NOOPEPT TO SUBSTRATES
AND MODULATORS OF FUNCTIONAL ACTIVITY OF ABCB1-PROTEIN
IN IN VIVO EXPERIMENT
I.V. Chernykh, A.V. Shchulkin, E.N. Yakusheva, M. V. Gatsanoga, N.M. Popova
Ryazan State Medical University named after academician I.P. Pavlov, Vysokovoltnaya str., 9, 390026, Ryazan, Russian Federation
In the article the influence of nootropic drug Noopept (N-phenyl-acetyl-L-prolylglycine ethyl ester) on the functional activity of ABCB1-protein is analyzed and attribution of this drug to the transport protein substrates is studied on male Chinchilla rabbits. Functioning of the transport protein was estimated by the pharmacokinetics of its marker substrate - fexofenadine. Fexofenadine was introduced intragastrically one time in the dose 67,5 mg/kg b.w. before and after 14-day introduction of Noopept in the dose 10 mg/kg b.w. 3 times a day. The quantity of the substance was determined by HPLC method according to the previously developed procedure.
Belonging of Noopept to ABCB1-protein substrates was estimated by comparison of its pharmacokinetic parameters before and after a course of introduction of verapamil (the known inhibitor of the ABCB1-protein) into male rabbits in the dose 20 mg/kg b.w. 3 times a day. The pharmacokinetics of Noopept was studied by the original HPLC method.
It was found that the course of Noopept introduction did not lead to any reliable changes in the pharmacokinetic parameters of the ABCB1-protein marker substrate - fexofenadine, which may evidence preservation of the functional activity of the given transport protein at the initial level. It was also found that the pharmacokinetics of Noopept remained unchanged after a course of introduction of ABCB1-protein inhibitor - verapamil - into the male rabbits, that is, Noopept is not a substrate of the given transport protein.
Keywords: ABCB1 -protein, P-glycoprotein, Noopept, fexofenadine, pharmacokinetics, verapamil, substrate.
ABCB1-protein (P-glycoprotein) is a membrane ATP-dependent efflux transport protein that removes a wide range of endogenous and exogenous substances including medical drugs of different pharmacological and chemical groups, from a cell [1].
ABCB1-protein localizes on the membranes of epithelial cells of small intestinal mucosa and of the proximal tubules of neph-ron, of hepatocytes and endothelial cells of histohematic barriers. The transporter prevents absorption in the intestine of medical drugs that are its substrates, participates in their dis-
tribution and facilitates their excretion into the bile and urine. Thus, the functional activity of this transport protein determines the pharmacokinetics of medical drugs - substrates of ABCB1-protein, and, finally, determines the effectiveness and safety of pharmacotherapy [2]. High activity of ABCB1-protein makes treatment with the substrate substance ineffective, and its low activity leads to overdosage.
Noopept (N-phenyl-acetyl-L-prolylglycine ethyl ester), a dipeptide analog of pyracetam, is a modern original nootropic and neuroprotective drug developed in V.V. Zakusov Research
Institute of Pharmacology. Noopept is used in treatment for chronic ischemic brain damages, schizophrenia, epilepsy [3].
In the scientific literature no research is found on estimation of the attribution of Noo-pept to substrates, inductors and inhibitors of ABCB1-protein. However, based on the chemical structure and physical properties of Noopept it is possible to suggest participation of ABCB1-protein in its pharmacokinetics, or the ability of the drug to modulate its functional activity. Earlier among the modulators of the transporter, substances of peptide nature were found [4]. The mechanism of alteration of the functional activity of ABCB1-protein by substances of peptide nature is described. It consists in their direct interaction with parts of the protein molecule with modification of its spatial structure [5].
The aim of the work is estimation of the influence of Noopept on the functional activity of ABCB1-protein and determination of belonging of the drug to the transport protein substrates.
Materials and methods
The research was conducted on 12 viri-potent male Chinchilla rabbits of 3500-4300 g mass [6]. The work with the animals was conducted according to the good laboratory practice (Supplement to the Order of Ministry of Health and Social Development of Russian Federation of August 23, 2010, 708h).
The animals were divided to 2 series: in the first series (n=6) the influence of Noopept on the functional activity of ABCB1-protein was studied, in the second series (n=6) belonging of Noopept to the transporter substrates was determined.
The animals of the first series received fexofenadine (coated tablets, Telfast, 180 mg, Aventis Pharma, Italy) intragastrically one time in the dose 67,5 mg/kg b.w. [7] before and after 14-day introduction of Noopept (Noopept tablets, 10 mg, ZAO (CJSC) «LEK-KO», Russia) in the dose 10 mg/kg b.w. 3 times a day, and also on the 5th day after its withdrawal [8]. The last introduction of Noo-pept was in the morning 20 min. before introduction of the marker substrate, to evaluate the direct influence of the drug on ABCB1-protein (the period of time was determined by Tmax of Noopept - 15 min.). After that blood was taken from the marginal veins of the rabbits' ears
into heparinized test tubes in the quantity 5 ml with subsequent centrifugation at 3000 rpm for 10 min. to obtain plasma. Concentration of fexofenadine in the blood plasma was determined by HPLC method on «Stayer» chromatograph (Russia) according to the earlier described procedure [9].
The second series animals received the test drug Noopept intragastrically in the dose 50 mg/kg b.w. in the form of suspension prepared on purified water one time before and after introduction of ABCB1-protein inhibitor. Blood samples were taken in 5, 10, 20, 30, 45, 60 and 90 min after introduction of the test drug [10] with the subsequent analysis of its pharmacokinetics by HPLC method. «Stayer» chromatograph was used with UV-spectro-photometric detector with 206 nm wavelength. Composition of the mobile phase: deionized water - acetonitrile - glacial acetic acid (50 -50 - 0.1). Chromatographic column Phenome-nex Synergi 4u Polar-RP 80A (250 x 4.6, 4 |im). The inhibitor to ABCB1-protein was verapamil (coated tablets, 80 mg, OAO (JSC) Valenta Pharmacevtica, Russia) given in the dose 20 mg/kg b.w. 3 times a day [7] in 14-day course. The last introduction of verapamil was conducted in the morning 1 hour before introduction of Noopept (the time is determined by Tmax of verapamil 1-2 h).
Pharmacokinetic parameters of fexofe-nadine and Noopept were calculated by non-compartmental method using Kinetica 5.0 program.
The obtained results were processed using StatSoft Statistica 7.0 program. The statistical significance of the differences between the pharmacokinetic parameters was evaluated on the basis of the concept of the lognormal distribution of data. The test pharmacokinetic parameters were compared using dispersion analysis of repeated measurements (ANOVA) after taking their logarithms. Statistically significant were assumed the differences at the meaning of p<0.05. Additionally, two-tailed 90% confidence interval of the ratio of the mean geometrical values of the pharmacoki-netic parameters with introduction of the test substances (verapamil and Noopept) to the parameters of intact animals was calculated. According to recommendations of Drug Evalua-
tion and Research Center of U.S. Food and Drug Administration [11], the differences between pharmacokinetic parameters are considered significant if the two-tailed 90% confidence interval of their ratio lies beyond the range 0,8-1,25 (80-125%). The obtained results are given in the tables as the geometrical mean and its 95% confidence interval.
Results and Discussion
14-Day introduction of Noopept to the male rabbits did not cause any statistically significant changes in any of the test pharmaco-kinetic parameters of fexofenadine (tabl. 1).
14-Day introduction of ABCB1-protein inhibitor - verapamil - did not lead to any reliable changes in the pharmacokinetics of Noopept in comparison with the initial parameters (tabl. 2).
Table 1
Pharmacokinetic Parameters of Fexofenadine (67,5 mg/kg) before and after Introduction of Noopept (10 mg/kg 3 times a day within 14 days)
Pharmacokinetic parameter of fexofenadine Intact animals (n=6) Animals after introduction of Noopept (n=6) Animals on the 5th day after withdrawal of Noopept (n=6)
Cmax, ng/ml 241,3 (138,2; 421,2) 360,7 (171,8; 757,5) 331,2 (209,0; 524,9)
AUCo-t, ng*h/ml 2520,4 (1012,9; 6271,1) 3023,9 (1682,7; 5433,9) 2799,5 (2318,1; 3380,8)
AUC/t1/2 203,9 (85,6; 486,3) 247,2 (106,2; 575,4) 219,6 (99,3; 485,6)
Cl, l*h-1 *kg-1 15,8 (4,8; 52,3) 13,9 (6,4; 30,3) 15,8 (11,2; 22,3)
Note: the data are presented as the geometrical mean and its 95% confidence interval
Table 2
Pharmacokinetic Parameters of Noopept (50 mg/kg) before and after Introduction of Verapamil (20 mg/kg 3 times a day within 14 Days)
Pharmacokinetic parameter ofNoopept Intact animals (n=6) Animals after introduction of verapamil (n=6)
Cmax, ng/ml 44,43 (25,20; 78,33) 26,16 (11,47; 59,70)
AUC0-6eoK, ng*min/ml 1133,36 (599,19; 2143,74) 932,89 (501,66; 1734,81)
AUC0-t ng*h/ml 1111,39 (596,81; 2069,63) 854,067 (417,11; 1748,77)
Cl, l*min-1kg-1 44,12 (23,32; 83,45) 53,60 (28,82; 99,67)
AUC/t1/2, 80,75 (38,38; 169,90) 62,97 (21,36; 185,68)
Note: the data are presented as the geometrical mean and its 95% confidence interval
Fexofenadine is H1-histamin receptor blocker of the III generation which is recommended by FDA as the marker substrate of ABCB1-protein; it is not metabolized in an organism, and its absorption in the intestine, distribution and elimination depend on the functioning of the transporter. Thus, the changes of the concentration of fexofenadine in blood characterizes the functional activity of ABCB1-protein [7, 11, 12].
Absence of any statistically significant alterations in the pharmacokinetics of fexofena-dine after a course of administration of Noopept into rabbits shows that the rate of absorption of the marker substrate in the intestine and the intensity of its excretion did not change that means
preservation of the functional activity of ABCB1-protein at the initial level.
Earlier in vitro was found that Noopept (10 |iM) increases DNA-binding activity of HIF-1 [13] - transcription factor that enhances expression of ABCB1-protein [14]. Our results do not agree with these data probably because we used Noopept in normal intact animals and studied functioning of the transporter at the level of the whole organism and not in cell culture.
A probable reason for absence of the influence of Noopept on the functional activity of ABCB1-protein at the level of the whole organism found in our research, may be its short halflife period which, according to the literature, in rabbits is 0,3±0,03 h [10] which minimizes the
probability for its direct interaction with the transport protein and for changing its activity. Besides, at the level of the whole organism ABCB1-protein is under the influence of many factors (surrounding cells, hormonal background, etc.) that are absent in vitro.
Belonging of medical drugs to ABCB1-protein substrates is estimated in vivo in the following way. Pharmacokinetics of the test drug is estimated before and after administration of the classic inhibitor and inductor of ABCB1-protein. If after a course of introduction to male rabbits of the transporter inhibitor (verapamil) the concentration of the test substance in plasma increases and its excretion decreases, and after a course of introduction of ABCB1-protein inductor (rifam-picin) the opposite dynamics is observed, the conclusion is made about the drug belonging to the transport protein substrates [15]. In this work the maximal concentration of Noopept in blood plasma after its oral administration into intact animals in the dose 50 mg/kg b.w. was 44,43 (25,20; 78,33) ng/ml. Here, if Noopept belongs to ABCB1-protein substrates, introduction of rifam-picin will further decrease its concentration in plasma that will not permit its detection by the method used by us. Because of this limitation, in our work only the transport protein inhibitor was introduced - verapamil, which increases concentration of the test substrates in blood.
Lack of any reliable differences between pharmacokinetic parameters of Noopept before and after a course of introduction of verapamil into rabbits means that the test drug is not a substrate of ABCB1-protein, and the involvement of the given transporter into the processes of absorption, distribution and excretion of the test substance produces no significant influence on its pharmacokinetics.
Independence of Noopept pharmacokinet-ics of ABCB1-protein functioning, as well as absence of any influence of the drug on the functional activity of the transporter permits to safely administer the drug together with drugs that are substrates of the transport protein (digoxin, da-bigatran etexilate, talinolol, etc.) without correction of its dose with the minimal probability for undesired drug-drug interactions.
Conclusions
1. A course of Noopept introduction in 10 mg/kg b.w. dose 3 times daily to male rabbits within 14 days does not lead to any changes in the functional activity of ABCB1-protein that is estimated by pharmacokinetics of its marker substrate fexofenadine.
2. By the results of in vivo testing, Noo-pept is not a substrate of ABCB1-protein that is confirmed by absence of any significant changes in its concentration after introduction of ABCB1-protein inhibitor - verapamil.
In relation to this article reported no potential conflicts of interest.
References
1. Montanari F, Ecker GF. Prediction of drug-ABC-transporter interaction - Recent advantages and future challenges. Adv. Deliv. Rev. 2015; 86: 17-26.
2. Yakusheva EN, Chernyh IV, Sh-chul'kin AV, Popova NM. Glicoprotein-P: struk-tura, fiziologicheskaya rol' i molekulyarnye mekhanizmy modulyacii funkcional'noj aktiv-nosti [P-Glycoprotein: Structure, Physiological Role and Molecular Mechanisms of Modulation of Functional Activity]. Uspekhi fiziologicheskih nauk [Success of Physiological Sciences]. 2014; 45 (4): 89-98. (in Russian)
3. Ostrovskaya RU, Vahitova YuV, Sa-limgareeva MH, Yamidanov RS, Sadovnikov SV, Kapitsa IG et al. K mekhanizmu dejstviya noopepta: snizhenie aktivnosti stress-induciruemyh proteinkinaz i aktivaciya
ehkspressii nejrotrofinov [On the mechanism of noopept action: decrease in activity of stress-induced kinases and increase in expression of neutrophines]. Ehksperimental'naya i klinicheskaya farmakologiya [Russian Journal of Experimental and Clinical Pharmacology]. 2010; 73 (12): 2-5. (in Russian)
4. Novikov SA. Gidrofil'nye geksapeptidy - novyj klass regulyatorov ATF-zavisimyh transportnyh belkov mnozhestvennoj lekarstven-noj ustojchivosti [Hydrophilic hexapeptides - a new class of regulators of ATP-dependent multidrug resistance transport proteins]: Cand. Diss. (Med. Sci.). Moscow; 2008. (in Russian)
5. Carrigos M, Mir LM, Orlowski S. Competitive and Non-Competitive Inhibition of the Multidrug-Resistance-Associated P-glycoprotein ATPase. Eur. J. Biochem. 1997; 244 (2): 664-673.
6. Gatsanoga MV, Chernykh IV, Sh-chul'kin AV., Yakusheva EN, Popova NM. Mozhno li ocenivat' prinadlezhnost' le-karstvennyh veshchestv k substratam glikopro-teina-R na samkah krolikov porody shinshilla [The method of assessment of drugs belonging to the substrates of P-glycoprotein on female Chinchilla rabbits]. Nauka molodykh (Eruditio Juvenium) [Science of young (Eruditio Juve-nium)]. 2016; 3: 5-10. (in Russian)
7. Kolkhir SV. Klinicheskoe znachenie izucheniya aktivnosti transportera lekarstvennyh sredstv glikoproteina-R dlya optimizacii farmako-terapii [Clinical importance of studying the activity of P-glycoprotein drug transporter for optimization of pharmaco-therapy]: Cand. Diss. (Med. Sci.). Moscow; 2008. (in Russian)
8. Kovalenko LP, Smol'nikova NM, Alekseeva SV, Nemova EP, Sorokina AV, Mi-ramedova MG et al. Doklinicheskoe izuchenie toksichnosti noopepta [Preclinical characterization of noopept toxicity]. Ehksperimental'naya i klinicheskaya farmakologiya [Russian Journal of Experimental and Clinical Pharmacology]. 2002; 65 (1): 62-64. (in Russian)
9. Yakusheva EN, Shchul'kin AV, Cher-nyh IV, Titov DS. Funkcional'naya aktivnost' i ehkspressiya glikoproteina-P pri ehksperimen-tal'nyh manipulyaciyah [Functional activity and expression of P-glycoprotein in experimental manipulations]. Rossijskij mediko-biologicheskij vestnik imeni akademika I.P. Pavlova [I.P. Pavlov Russian Medical Biological Herald]. 2014; 2: 74-77. (in Russian)
10. Bojko SS, Zherdev VP, Gudasheva TA, Ostrovskaya RU, Korotkov SA, Kravcova OYu. Biodostupnost' noopepta - novogo noo-tropnogo preparata dipeptidnoj struktury [Bio-accessibility of the new dipeptide nootropic
drug noopept]. Himiko-farmacevticheskij zhurnal [Pharmaceutical Chemistry Journal]. 2004; 38 (12). 3-5. (in Russian)
11. Guidance for Industry Drug Interaction Studies — Study Design, Data Analysis, Implications for Dosing, and Labeling Recommendations / U.S. Department of Health and Human Services Food and Drug Administration Center for Drug Evaluation and Research (CDER). 2012. 75 p. Available at: http://www.fda.gov/downloads/drugs/guidance complianceregulatoryinforma-tion/guidances/ucm292362.pdf.
12. Ohura K, Nakada Y, Kotani S, Imai T. Design of Fexofenadine Prodrugs Based on Tissue-Specific Esterase Activity and Their Dissimilar Recognition by P-Glycoprotein. J. Pharm. Sci. 2015; 104 (9): 3076-3083.
13. Vakhitova YV, Sadovnikov SV, Bo-risevich SS, Ostrovskaya RU, Gudasheva TA, Seredenin SB. Molecular Mechanism Underlying the Action of Substituted Pro-Gly Dipeptide Noopept. Acta Naturae. 2016; 8 (1): 82-89.
14. Ding Z, Yang L, Xie X, Xie F, Pan F, Li J et al. Expression and significance of hypoxia-inducible factor-1 alpha and MDR1/P-glycoprotein in human colon carcinoma tissue and cells. Cancer Res. Clin. O. 2010; 136 (11): 1697-1707.
15. Yakusheva EN, Shchul'kin AV, Chernykh IV. Ocenka prinadlezhnosti meksi-dola k substratam, ingibitoram ili induktoram glikoproteina-P [Assessment of the Attribution of Mexidol to P-Glycoprotein Substrates, Inhibitors, or Inductors]. Ehksperimental'naya i klinicheskaya farmakologiya [Russian Journal of Experimental and Clinical Pharmacology]. 2015; 78 (5): 19-23. (in Russian)
Chernykh I.V. - Cand. Biol. Sc., Assistant of General and Pharmaceutical Chemistry Department, Ryazan State Medical University named after academician I.P. Pavlov.
Shchulkin A.V. - Cand. Med. Sc., Assistant of Pharmacology Department with Course of Pharmacy of Additional Professional Education Faculty, Ryazan State Medical University named after academician I.P. Pavlov.
Yakusheva E.N. - M.D., Ph.D., D.Sc., Professor, Head of Pharmacology Department with Course of Pharmacy of Additional Professional Education Faculty, Ryazan State Medical University named after academician I.P. Pavlov.
E-mail: e.yakusheva@rzgmu.ru
Gatsanoga M.V. - Postgraduate Student of Pharmacology Department with Course of Pharmacy of Additional Professional Education Faculty, Ryazan State Medical University named after academician I.P. Pavlov.
Popova N.M. - Cand. Med. Sc., Assistant of Pharmacology Department with Course of Pharmacy of Additional Professional Education Faculty, Ryazan State Medical University named after academician I.P. Pavlov.
