RU2464019C1 - Composition possessing endothelial protective, vasodilating and angioprotective effect - Google Patents

Abstract

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to medicine and pharmaceutical industry, and concerns a composition having a complex effect on the vascular endothelium. The composition contains L-arginine or L-citrulline, and an arginase inhibitor in a solid dosage form. The composition possess an endothelial protective, vasodilating and angioprotective effect. The synergetic effect is provided by combining NO donators and the arginase inhibitors in the retard form.

EFFECT: preparing the composition possessing the complex effect on the vascular endothelium.

7 cl, 5 ex, 54 tbl, 2 dwg

Description

The invention relates to the field of pharmaceuticals, medicine, nutraceuticals and is a composition having a complex effect on the endothelium of blood vessels.

For the first time, the independent role of vascular endothelium in the regulation of vascular tone was announced in an article by Furchgott and Zawadzki, published in the journal Nature in 1980.

Subsequent studies have shown that endothelium is not only a cover acting as a passive barrier between blood and tissues, but is a real regulator of blood flow and tissue homeostasis. For this reason, it was postulated that the vascular endothelium is the largest and most important endocrine gland in the body.

A well-known fact is that the vascular endothelium is an active metabolic system. This system functions or can function as a separate receptor-effector organ, which has the ability to respond to physical or chemical stimuli with the release of appropriate biologically active substances, which helps maintain vasomotor balance and vascular tissue homeostasis (Gomazkov O.A., 2000; Pacher P., 2007, Esper RJ, 2006, Luscher TF, 1997). Endothelium, as it turned out now, is actively involved in modulating vascular tone, secrets a variety of vasoactive substances, one of which turned out to be very significant nitric oxide (NO). Nitric oxide (NO) is an active substance released by endothelial cells during the catabolism of L-arginine with the active participation of NO synthase, which is the most powerful vasodilator currently known (Granik V.G., 2004; Pokrovsky V.I., 2005 ; Forstermaim UT, 2006; Rabelink TJ, 2006).

Endothelial dysfunction - the main link and a critical point in the development of cardiovascular diseases

The mechanism of participation of the endothelium in the emergence and development of various pathological conditions is multifaceted and is associated not only with the regulation of vascular tone, but also with participation in the process of atherogenesis, thrombosis, protection of the integrity of the vascular wall, etc.

In a simplified form, there are three main stimuli that cause a “hormonal” reaction of the endothelial cell:

1) a change in blood flow velocity (an increase in shear stress);

2) platelet mediators (serotonin, ADP, thrombin);

3) circulating and / or "intra-wall" neurohormones (catecholamines, vasopressin, acetylcholine, endothelin, bradykinin, histamine, etc.).

Normally, in response to these stimuli, endothelial cells respond by enhancing the synthesis of substances that relax smooth muscle cells of the vascular wall, and primarily nitric oxide (NO), an endothelium-dependent hyperpolarization factor [Coleman, H.A. TareM., Parkmgton H.C. Endothelialpotassiumchannels, endothelium-dependenthyperpolarizationandtheregulationofvasculartoneinhealthanddisease / H.A. Coleman, T. Mare, N.C. Parkington // Clin. Exp.Pharmacol. Physiol. - 2004 .-- V.31. - No. 9. P.641-649.].

The main functions of the vascular endothelium

1. The release of vasoactive agents (nitric oxide (NO), endothelial hyperpolarizing relaxation factor (EGFR), endothelin, angiotensin I-AI (and possibly angiotensin II-AII), prostacyclin, thromboxane)

2. Obstruction of coagulation (blood coagulation) and participation in fibrinolysis (platelet-resistant surface of the endothelium (the same charge on the surface of the endothelium) and platelets prevents "adhesion" - adhesion - of platelets to the vessel wall, the formation of prostacyclin and NO - natural disaggregants, the formation of t-PA (tissue plasminogen activator), expression on the surface of endothelial cells of thrombomodulin, a protein capable of binding thrombin and heparin-like glycosaminoglycans)

3. Immune functions (presentation of antigens to immunocompetent cells, secretion of interleukin-I (stimulator of T-lymphocytes))

4. Enzymatic activity (expression on the surface of endothelial cells of an angiotensin-converting enzyme - ACE (conversion of AI to AII))

5. Participation in the regulation of growth of smooth muscle cells (MMC) (secretion of endothelial growth factor (EGF), secretion of heparin-like growth inhibitors)

6. Protection of smooth muscle cells from vasoconstrictor influences due to: hypoxia, hemodynamic overload, age-related changes, free radical damage, dyslipoproteinemia (hypercholesterolemia), the action of cytokines, hyperhomocysteinemia, hyperglycemia, pancreatic toxicosis, endogenous toxicity, exogenous intoxication (smoking), etc.

Table 1 presents the main endothelial factors that regulate vascular tone.

Table 1 The main endothelial vasoconstrictor and vasodilating factors Endothelial factors regulating vascular tone Endothelial factors that potentiate vasoconstriction vasodilating vasoconstrictive - prostacyclin - Endothelin-1 - Adhesive molecules - hyperpolarizing - Angiotensin II - Vascular factor relaxation factor - Prostaglandin F2α permeability - Nitrogen oxide - Thromboxane A2 - E-selectin - Endothelial - Free radicals - R-selectin natriuretic peptide - Free fatty acids - von Willebrand factor - Thrombomodulin - Cytokines

With prolonged exposure to various damaging factors, gradual depletion and distortion of the compensatory vasodilating ability of the endothelium occurs, and vasoconstriction and proliferation become the primary response of endothelial cells to conventional stimuli.

Thus, endothelial function can be defined as a balance of oppositely acting principles - relaxing and constrictor factors, anticoagulant and procoagulant factors, growth factors and their inhibitors, and endothelial dysfunction - an imbalance of these oppositely acting principles that violate hemovascular hemostasis. Recently, there has been a narrower idea of endothelial dysfunction as an endothelial condition in which there is insufficient production of nitric oxide [53, 54, 55].

The pathophysiological stages of ED development are represented by a diverse complex of connecting and interdependent mechanisms. However, among them there are the most common, playing a key role in the development of this pathology.

Since nitric oxide (NO) is involved in the most diverse bioregulatory effects of the vascular process, a decrease in its level in endothelial cells (EC), due to various factors, has significant changes in their functions and the circulatory system as a whole. Violations are expressed (Shiffrin E.L., 2001; Egashira K., 2002; Feletou M., 2006; Cooke J.P., 2003, Endemann D.H., 2004, Zotova I.V., 2000; Markov H.M., 2005):

- a decrease in the effectiveness of endothelium-dependent vasodilators and, as a consequence, an increase in vasoconstrictor effects,

- increase in blood pressure,

- violations of local, regional and systemic hemodynamics, impaired cardiac activity,

- an increase in the production of adhesive substances in the endothelium, which leads to platelet aggregation, their adhesion and leukocytes to the vascular wall,

- proliferation and / or migration of smooth muscle cells with the formation of neointima,

- synthesis of extracellular matrix, etc.

Recent studies have convincingly shown that, in various combinations, the noted disorders that make up the endothelial dysfunction as a whole occur in many cardiovascular diseases (arterial hypertension, atherosclerosis, coronary heart disease, heart failure, circulatory disorders in the kidneys, lungs, brain, extremities, intestines (Belousov Yu.B., 2000; Makolkin V.I., 2004; Borovkov NN, 2005; CookeJ.P., 2003; LandmesserU., 2004), as well as their risk factors (hypercholesterolemia, tobacco smoking, diabetes mellitus, insulin resistance, obesity , hypokinesia, aging, hereditary burden) (Shestakova M.V., 2001; Buval'tsev V.I., 2002; Petrishchev N.N., 2003; Vertkin A.A., 2005; Mazurov V.I., 2006; BonettiP.O., 2003; BarbatoJ.E., 2004; Spieker L.E., 2006; GiannottiG., 2007).

Endothelial cells can also appear as the primary etiological factor and target organ in the development of cardiovascular changes associated with a decrease in estrogen levels (Tyurenkov I.N., 2006; KrugE., 2002; ModenaM.G. 2002; TaddeiS., 2006 ) The pathogenetic mechanisms of postmenopausal increase in blood pressure are quite confusing and multifactorial. They are much more complex than just the effect of reduced levels of estradiol in the blood. But it is obvious that one of them is associated with impaired endothelium-dependent vasodilation caused by a decrease in the bioavailability of NO (Karpov, Yu.A., 2001; Hinojosa-LabordeC., 2004; ZanchettiA., 2005; RosanoG.M., 2007; CoylewrightM., 2008) . This may be due to increased plasma renin activity, stimulation of oxidative stress, increased salt sensitivity, obesity, increased sympathetic tone (MajmudarN.G., 2000; SimonT., 2000; DubeyR.K., 2002; ScuteriA., 2003; TeerlinkT., 2003 ; Thompson L.P., 2005; MillerV.M., 2007). Therefore, correction of endothelial dysfunction in the postmenopausal period is considered one of the important aspects of therapy for restoring the balance of regulation of vascular tone, hemostasis, immune response, preventing the migration of blood cells into the vascular wall, reducing the synthesis of inflammatory factors, strengthening the barrier functions of the endothelium (Smetnik V.P., 2001 ; Olbinskaya L.I., 2001; Herrington DM, 1999; Diamanti-KadarakisE., 2001; HarmanS.M., 2005; Min-kinM.J., 2007).

In blood plasma, the level of L-arginine is reduced in many diseases of the cardiovascular system and their risk factors. It was shown that oral and parenteral administration of L-arginine into the body of patients with these diseases restores their reduced level of L-arginine, NO, and endothelial function, giving the corresponding therapeutic effect (Stepanov Yu.S., 2005; Markov Kh.M., 2005 , PiattiP., 2003; PiattiP., 2000; HambrechtR., 2000; LinK.Y., 2002; Tousoulis D. 2007; Bode-Boger S. M., 2003). This suggests that NO deficiency and vascular endothelial dysfunction in such patients may be due to varying degrees of lack of L-arginine as a substrate for eNOS (SchlaichM.P., 2004; NakamuraK., 2003; KawanoH., 2002; BreedersM .A., 2002; PemowJ., 2003; GomikH.L., 2004).

Recently, in studies of the causes of the development and progression of cardiovascular diseases, attention has been focused on asymmetric dimethylarginine (ADMA), called a new marker and risk factor for the development and progression of endothelial dysfunction and atherosclerosis (BogerR. N., 1998, 2004; ZoccaliC., 2001 ; AchanV., 2003; Kielstein J.., 2003).

Based on experimental data, there is an assumption that the enzymatic activity of nitric oxide synthase is regulated by the ratio of the concentrations of endogenous L-arginine and asymmetric dimethylarginine (the so-called ADMA), which is an endogenous inhibitor. The physiological balance is maintained so that in the presence of normal levels of L-arginine, an increase in ADMA leads to a relative deficiency of L-arginine, which affects the optimal activity of nitric oxide synthase. Since ADMA is a competitive inhibitor of nitric oxide synthase, its inhibitory effect can be overcome by increasing the concentration of the enzyme substrate (L-arginine) (LuT.M., 2003; BögerR.H., 2005; 2004, 2007). Therefore, additional administration of L-arginine can help restore the physiological state of nitric oxide synthesis by normalizing the ratio of “L-arginine / ADMA". In addition, estrogens regulate the metabolism and release of asymmetric dimethylarginine (ADMA), an endogenous NO synthase inhibitor (TeerlinkT., 2003; PostM.S., 2003). Research HoldenD.P. etal. (2003) demonstrated a decrease in the level of circulating ADMA after estrogen replacement therapy in postmenopausal women, in addition, estrogens reduce the release of ADMA from endothelial cells and stimulate the activity of the enzyme dimethylarginindimethylaminohydrolase, which metabolizes ADMA in vivo.

Another important factor contributing to an increase in ADMA in postmenopausal women is an increase in salt sensitivity and, as a result, blood pressure, which further aggravates the development of endothelial dysfunction, which is manifested in a decrease in endothelium-dependent vasodilation (ScuteriA., 2003).

The participation of L-arginine in the metabolism of nitric oxide (NO) and NO synthase in violation of the function of the vascular endothelium

Nitric oxide is synthesized in endothelium (isoforms of the enzyme NO synthase: 2 of them are constitutive - endothelial and neuronal and 1 inducible), as a result of the reaction of the amino acid L-arginine, with the participation of molecular oxygen. Catalyst-enzyme NO synthase. The reaction can be represented as follows: L-arginine + O2 → L-citrulline + NO. NO is synthesized. Constitutive endothelial NO synthase (eNOS) implements the regulation of the cardiovascular system. The formation of nitric oxide is carried out in stages with the intermediate formation of N-hydroxy-L-arginine (NHA. [73, 103, 309] (Fig. 1).

The reverse conversion of L-arginine from L-citrulline is regulated by arginine succinate synthase and arginine succinate lyase [Glucocorticoids regulate inducible nitricoxidesynthase by inhibiting tetrahydrobiopterinsynthesis and L-arginine transport / W. W. Simmons, D. Ungurean et. G. etg. // J. Biol. Chem. - 1996. - V.271. - P.23928-23937, Glucocorticoids mediate the enhanced expression of intestinal type II arginase and argininosuccinatelyase in postweaning pigs / N.E. Flynn, C.J. Meininger, K. Kelly et al. // J. Nutr. - 1999. - V.129. - P.799-803.].

Carriers of L-arginine include messengers such as CAT-1 and CAT-2, enzymes that transport cationic amino acids [Guoyao, W.U., Arginine metabolism: nitric oxide and beyond / W.U. Guoyao, S.M. Morris // Biochem. J. - 1998. - V.336. - P.1-17., Glucocorticoids regulate inducible nitric oxide synthase by inhibiting tetrahydrobiopterin synthesis and L-arginine transport / W. W. Simmons, D. Ungureanu-Longrois, G. K. Smith et al. // J. Biol. Chem. - 1996. - V.271. - P.23928-23937]. Among other amino acids, L-arginine is involved in protein synthesis. Unpolymerized L-arginine is involved in hormone uptake, such as insulin, growth hormone, prolactin, glucagon. Arginase (2 isoforms) and NO synthase / NOS (3 isoforms), arginine decarboxylase, as well as other enzymes are involved in the conversion of L-arginine.

U-urea, creatine, L-ornithine, L-citrulline, L-proline, etc. [Guoyao, W.U., Arginine metabolism: nitric oxide and beyond / W.U. Guoyao, S.M. Morris // Biochem. J. - 1998. - V.336. - P.1-17.]. There is a likelihood of something else, the so-called an independent pathway from NO synthase / NOS, the formation of nitric oxide (NO) due to the reaction between arginine and hydrogen peroxide [Antineutrophil cytoplasm antibody-induced neutrophil nitric oxide production is nitric oxide synthase-independent / WYTse, J.Williams, A.Pall et al. // Kidney Int. - 2001 .-- V.59. - P.593-600.].

Endothelial NO synthetase (eNOS) is located in the caveoles (the so-called lacuniform microregions of the endothelial cells of the plasma membrane 50-1000 nm), where it is associated with caveolin. The bound state of eNOS can dramatically decrease enzyme activity [Michel, T. Nitric oxide synthases: which, where, how and why. / T. Michel, O. Feron // J. Clin. Invest. - 1997. - V. - 100. - P.2146-2152., Griffits, P.W. Nitric oxide synthases: properties and catalytic mechanism / P.W. Griffits, D.J. Stuehr // Ann. Rev. Physiol. - 1995. - V.57. - P.707-736.]. Five main factors were noted that affect the rate of NOS-dependent NO synthesis; one of them is the concentration of L-arginine inside NO-synthesizing cells. This factor is post-translational [Pokrovsky, V.I. Nitric oxide, its physiological and pathophysiological properties / V.I. Pokrovsky, N.A. Vinogradov // Therapeutic Archive. - 2005. - No. 1. - S. 82-87].

There is evidence that the ability of L-arginine to separately avoid the destruction process, dysfunction (splitting, "uncoupling) in NOS, arising endothelial dysfunction with pathophysiological factors of various etiologies: an increase in the number of endogenous plasma eNOS inhibitors, oxidized LDL, the oxidative activity of NADPH and et al. [Inducible nitric-oxide synthase generates superoxide from the reductase domain / Y. Xia, LJ Roman, BSS Masters, JLZweier // J. Biol. Chem. - 1998. - Vol 273. - P.22635-22639., Pravastatin attenuates cardiovascular inflammatory and proliferative changes in a rat model of chronic inhibition of nitric oxide synthesis by its cholesterol-lowering independent actions. Egashira K, Ni W, Inoue S, Kataoka C, Kitamoto S, Koyanagi M, et al. Hypertens Res 2000; 23: 353-358., Xia, Y. Inducible nitric-oxide synthase generates superoxide from the reductase domain. / Y. Xia, L.J. Roman, B.S. Masters, J.L. Zweier // J. Biol. Chem. - 1998. - V.273. - P.22635-22639.].

Nitric oxide, after its formation, is released from the endothelial cells and diffuses into the deeper myocytes of the vessel walls. By acting on vascular myocytes, nitric oxide (NO) activates guanylate cyclase, which leads to an increase in the content of 3,5-cyclic guanosine monophosphate, these processes provoke a decrease in calcium concentration and result in relaxation of smooth muscle cells and vasodilation [Cavelin, caveolaeand endothelium cell function. / Frank P. O., Woodman S.E., Park D.S., Lisanii M.P. // ArteriosclerThrombVaseBiol 2003; 23: 1161-1168., Zotova, I.V. Synthesis of nitric oxide and the development of atherosclerosis / I.V. Zotova, D.A. Zateishchikov, B.A. Sidorenko // Cardiology. - 2000. - No. 4. - S. 58-67, Luscher T.F., Endothelial control of vascular tone and growth // Clin. Exp.Hypertens. - No. 12. - 1990.-. P.897-902, Luscher T.F., Endothelium - derived vasoactive factors and regulation of vascular tone in human blood vessels // Lung. - V.168. - 1990. - P.27-34].

It is important to note that endothelial relaxation factors additionally cause systemic effects that prevent pathological processes of thrombosis and prevent damage to the vascular wall. This is blocking platelet aggregation, oxidation of low density lipoproteins (LDL), expression of adhesion molecules, adhesion of monocytes and platelets to the vessel wall, etc. [BarbatoJ.E., TzengE.Nitricoxideandarterialdisease.J VascSurg 2004; 40: 187-193., An association between plasma asymmetric dimethylarginine and membrane fluidity of erythrocytes in hypertensive and normotensive men: an electron paramagnetic resonance investigation. Am J Hypertens, 2005 Sep; 18 (9 Pt 1): 1243-8].

In light of the above, the presence of nitric oxide (NO) in the myocardium becomes important. NO increases the diastolic extensibility of the left ventricle, this process plays a significant role in the development of heart hypertrophy. Nitric oxide is one of the factors in the regulation of inotropic and chronotropic β1-adrenergic effects [Markov Kh.M., Nadirashvili S.A. On the regulation of cardiac activity by the L-arginine-nitric oxide system. Pat Physiol Ex 2003; 4: 9-11., Markov H.M., Nadirashvili S.A. Age-related features of the coronary effects of nitric oxide. Ros.pediat.journ 2004; 4: 17-21].

Endogenous factors of NO deficiency and, as a result, endothelial dysfunction

Slowing down the expression and activity of endogenous NO synthetase (eNOS) is usually associated with endothelial dysfunction. The first among the factors leading to this are endogenous eNOS inhibitors, such as ADMA [Association tanalysis of CA repeat polymorphism of the endothelial nitric synthase gene to essential hypertension in Japanese. NakaymaT., Soma J., Takahashi J. el al. Clin Genet 1997; 51: 26-30., Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, Circulation 99: 1141-1146, 1999, Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Boger R.H., Bode-Boger S.M., SzubaA. et al. Circulation 1998; 98: 1842-1847., Cooke J.P. Does ADMA cause endothelial dysfunction. Arterio-sclerThromb Vase Biol 2002; 20: 2032-2045.].

Asymmetric Dimethylarginine (ADMA)

ENOS activity, NO production, and vascular endothelial function are all interconnected systems that depend on the content of endogenous eNOS inhibitors. One of the selective inhibitors of this enzyme is NG-dimethyl-arginine (asymmetric dimethylarginine, ADMA) and NG-monomethyl-L-arginine (NMMA) [Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Miyazaki PI, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, Circulation 99: 1141-1146, 1999, Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Boger R.H., Bode-Boger S.M., SzubaA. et al. Circulation 1998; 98: 1842-1847., Cooke J.P. Does ADMA cause endothelial dysfunction. Arterio-sclerThromb Vase Biol 2002; 20: 2032-2045.]. In plasma, the content of ADMA is 10 times higher than the level of NMMA, as a result of this ADMA attracts attention to itself.

Two types of enzymes that methylate L-arginine have been identified: protein-arginine-methyltransferase (PRMT-1), which catalyzes the formation of ADMA and NMMA, and PRMT-2, which forms symmetrical dimethylarginine (SDMA) [TaniyamaY., Griendling K.K. Reactive oxygen species in the vasculature: molecular and cellular mechanisms.// Hypertension. - 2003. - Vol.42, N 6. - P.1075-1081.]. The latter is a stereoisomer of ADMA, but does not affect eNOS.

Different types of cells have the ability to produce ADMA, including human EC [Metabolism of methylarginines by human vasculature; implication for the regulation of nitric oxide synthesis. Mac Allister R.J., Pickling S.A., Whilley G.S. et al. Br J Pharmacol 1994; 112: 43-48.].

ADMA is excreted in the urine, but the main pathway of its metabolism is the destruction of dimethylarginine-dimethylaminohydrolase (DDAH), which hydrolyzes ADMA to form dimethylamine and L-citrulline [Regulation ofendothelin-1 release from human endothelial cells by sex steroids and angiotensin-II. Pearson LJ, Yandle TG, Nicholls MG, Evans JJ. Peptides. 2008 Jun; 29 (6): 1057-61. Epub 2008 Feb 13.]. The localization of DDAH and eNOS indicates that the concentration of methylarginine is actively and specifically regulated in NO-generating cells.

It was shown that the concentration of ADMA in the plasma in the range of 2-10 μmol / L significantly inhibits NOS in isolated human blood vessels, in cultured macrophages and EC, reducing the formation of NO. Administration of ADMA to animals causes an increase in mean blood pressure and vascular resistance in the kidneys, intestines, and limbs. Accordingly, salt-sensitive rats with arterial hypertension (Dahl strain) showed an increase in ADMA expression [Asymmetric dimethylarginine, and endogenous nitric oxide inhibitor in experimental hypertension. Matsuoka H., Itoh S., Kimoto M. et al. Hypertension 1997; 29: Pt 2: 242-247]. A similar phenomenon occurs in people with salt-dependent hypertension: high-salt diet increases blood pressure and ADMA in blood plasma, reducing urinary excretion of NO metabolites; low-salt diet eliminates these disorders [Stuhlinger M.C., Abbasi F., Chit J.W. Relationship between insulin resistance and an endogenous nitric oxide synthase inhibitor. JAMA 2002; 287: 11: 1420-1426.]. The same changes in ADMA and NO formation, as well as a decrease in the activity of DDAH (an enzyme that destroys ADMA) were also found in patients with essential [Reduced uterine perfusion pressure during pregnancy in the rat is associated with increases in arterial pressure and changes in renal nitric oxide. Alexander, V.T., Kassab, S.E., Miller, M.T., Abram, S.R., Reckelhoff, J.F., Bennett, W.A. & Granger, J.P. (2001) Hypertension 37: 1191-1195.] And pulmonary [Cooke J.P. A novel mechanism for pulmunary arterial hypcrtensioni. Circulation 2003; 108: 1420-1421., 277] hypertension.

A significant increase in ADMA is observed in atherosclerosis [Asymmetric dimethylarginine plasma concentrations differ in patients with end-stage renal disease: Relationship to treatment method and atherosclerotic disease. Kielstein JT, Boger RH, Bode-Boger SM, Schaffer J, Barbey M, Koch KM, Frolich JC: J Am SocNephrol 10: 594-600, 1999, Nilsson I, Shibuya M, Wennstrom S. Differential activation of vascular genes by hypoxia in primary endothelial cells. Exp Cell Res. 2004 Oct 1; 299 (2): 476-85.]. This increase in ADMA concentration is the cause observed in atherosclerosis of endothelial dysfunction, as evidenced, in particular, by a decrease in endothelium-dependent vasodilation in this disease [Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Boger R.H., Bode-Boger S.M., SzubaA. et al. Circulation 1998; 98: 1842-1847.]. Under these conditions, plasma ADMA levels correlate better with endothelial dysfunction than LIP cholesterol. It was also shown that inhibition of eNOS under the influence of ADMA causes oxidative stress in EC and increases the expression of redox-sensitive genes that encode adhesive endothelial molecules to the same extent as occurs in the early stage of atherosclerosis [Asymmetric dimethylarginine (ADMA): an over risk factor for endothelial dysfunction: itsroleinhypercholesterolemia. Boger RH, Bode-Boger SM, Szuba A, et al. Circulation 1998; 98: 1842-1847., Boger R.IL, Bode-Boger S.M. Is asymmetric dimethylarginine anevcl marker of atherosclerosis. Circulation 2000; 101: 160-162.].

The level of ADMA is inversely proportional to the degree of endothelium-dependent vasodilation in individuals with hypercholesterolemia during administration of L-arginine, a competitive substrate of eNOS for ADMA, normalizing endothelial function [Asymmetric dimethylarginine, blood pressure and renal perfusion in elderly subjects. Kielstein J., Bode-BogerT., Frolich J.C. et al. Circulation 2003; 107: 1891-1895.]. Intravenous administration of ADMA reduces heart rate, cardiac output, and increases blood pressure [Asymmetric dimethylarginine causes hypertension and cardiac dysfunction in animals and is actively metabolized by dimethylargininedimethylaminohydrolase. Achan V., Broadhead M, Malaki M, Whitley G, Leiper J, MacAllister R, Vallance P: Arterioscler Thromb Vase Biol 23: 1455-1459, 2003]. The increased interest in ADMA in recent years is associated not so much with elimination as with the formation of this endogenous substance. ADMA is formed with the participation of the enzyme protein-arginine methyltransferase-1, the amount of which increases with the long-term hemodynamic overload of the conducting arteries (high shear stress), which accordingly leads to an increase in the amounts of ADMA [Effect of shear stress on asymmetric dimethylarginine release from vascular endothelial cells. Osanai T, Saitoh M, Sasaki S, Tomita H, Matsunaga T, Okumura K: Hypertension 42: 985-990, 2003]. It is also observed in hypercholesterolemia [Asymmetric dimethylarginine (ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Boger R.H., Bode-Boger S.M., SzubaA. et al. Circulation 1998; 98: 1842-1847.], In elderly individuals with arterial hypertension [Asymmetric dimethylarginine, blood pressure and renal perfusion in elderly subjects. Kielstein J., Bode-BogerT., Frolich J.C. et al. Circulation 2003; 107: 1891-1895.], I.e. there is a correlation between the plasma content of ADMA and blood pressure and age [Endogenous nitric oxide synthase inhibitor: a novel marker of atherosclerosis. Miyazaki H, Matsuoka H, Cooke JP, Usui M, Ueda S, Okuda S, Circulation 99: 1141-1146,

1999].

Since an increase in ADMA concentration is detected not only with severe renal failure (10 times!), But also long before that (against the background of normal kidney function), the reason for the observed increase in ADMA level should not be associated with a decrease in its excretion, but mainly with a decrease its destruction under the influence of DDAH [Characterization of biochemical markers of vascular endothelial function: development of a model system using cell cultures / V.A. Metelskaya, N.G. Gumanova, O.A. Litinskaya, etc. // Vopr. biol. medicine and farm. chemistry. - 2004. - No. 2. - S.34-39.]. This enzyme is found in the EC of the renal vessels (including at the glomerulus level), as well as in canalicidal cells. It is believed that when the latter are affected in patients with kidney disease, the DDAH level decreases, which leads to an increase in ADMA, a decrease in NO, and vascular endothelial dysfunction [Asymmetrical dimethylarginine (ADMA) in critically ill patients: High plasma ADMA concentration is an independent risk factor ofICU mortality. Nijveldt RJ, Teerlink T, Van Der Hoven B, Siroen MP, Kuik DJ, Rauwerda JA, Van Leeuwen PA: ClinNutr 22: 23-30, 2003, Pieper G.M. Review of alterations in endothelial nitric oxide production in diabetes. Hypertension 1998; 31: 1047-1060.]. Addition of 4124 W (DDAH inhibitor) to the incubation medium of the isolated vascular segment caused its contraction, and L-arginine eliminated this effect [Cooke J.P. Does ADMA cause endothelial dysfunction. Arterioscler Thromb Vase Biol 2002; 20: 2032-2045.].

An increase in ADMA or a decrease in DDAH, accompanied by vascular endothelial dysfunction, has also been established in diabetes mellitus [Impaired nitric oxide synthase pathway in diabetes mellitus: role of asymmetric dimethylarginine dimethylaminohydrolase. Lin K.Y., Ito A., Asagami T. et.al. Circulation 2002; 106: 987-992.], Insulin resistance [Remillard CV, Yuan JX. Cardiac L-arginine transport: the CAT is back. J Physiol. 2007 May 1; 580 (Pt. 3): 699-700.], Alloxan hyperglycemia [Hypercholesterolemia enhances tromboembolism in Arterioles but Not Venules: complete reversal by L-arginine. Breeders M.A. W., Tangelder G.J., SlaafD. W. et al. Arterioscler Thromb Vase Biol 2002; 22: 680-685.], Pregnancy and preeclampsia [Plasma concentration of asymmetrical dimethylarginine and mortality in patients with end-stage renal disease: A prospective study. Zoccali C, Bode-Boger S, Mallamaci F, Benedetto F, Tripepi G, Malatino L, Cataliotti A, Bellanuova I, Fermo I, Frolich J, Boger R: Lancet 358: 2113-2117, 2001], hypercholesterolemia [Asymmetric dimethylarginine ( ADMA): a novel risk factor for endothelial dysfunction: its role in hypercholesterolemia. Boger R.H., Bode-Boger S.M., SzubaA. et al. Circulation 1998; 98: 1842-1847.], Hyperhomocysteinemia [259], heart failure [124, 339], peripheral vascular ischemic diseases [141, Asymmetric dimethylarginine, blood pressure and renal perfusion in elderly subjects. Kielstein J., Bode-BogerT., Frolich J.C. et al. Circulation 2003; 107: 1891-1895.], Hyperthyroidism [Plasma concentration of asymmetric dimethylarginine, a natural inhibitor of nitric oxide synthase, in normal pregnancy and preeclampsia. Holden D., Pickling S., Wliitley G., Nussey S. Am J ObstctGynecol 1998; 178: 551-556.9], obesity [Stoleru, L. Effect of D-nebivolol and L-nebivolol on left ventricular systolic and diastolic function: comparison with DL-nebivolol and atenolol / L. Stoleru, W. Wijns, C.van Eyil // J. Cardiovasc. Pharmacol - 1993. - No. 22. - P.183-190.], Tobacco smoking [L-arginine normalizes coronary vasomolion in long-term smokers. Campisi R., Sudhir K., Sievers R.F. el al. Circulation 1999; 99: 491-497.], Aging [Asymmetric dimethylarginine, blood pressure and renal perfusion in elderly subjects. Kielstein J., Bode-BogerT., Frolich J.C. et al. Circulation 2003; 107: 1891-1895.].

There is also a problem, like a reactive form of oxygen. Excessive production of reactive oxygen species, the so-called oxidative stress, overcomes the protective function of the antioxidant mechanisms of the cell. T.O. oxidative stress becomes a strong pathogenic factor, oxidizing and disrupting the functions of such biological macromolecules as DNA, proteins, carbohydrates, lipids [Glembotski C. C. Endoplasmic Reticulum Stress in the Heart Circ. Res., November 9, 2007; 101 (10): 975-984, Tentolouris C, Tousoulis D, Stefanadis C. L-arginine "paradox" in coronary atherosclerosis. Circulation. 2004 Aug 17; 110 (7): e71, TouyzRM. Oxidative stress and vascular damage in hypertension. Curr Hypertens Rep. 2000 Feb; 2 (1): 98-105.].

An excess of reactive oxygen species, such as superoxide anion, hydroxyl radical, hydroperoxide radical, hydrogen peroxide, also causes other changes in the functions of the vascular endothelium: inhibition of endothelium-dependent vasodilation, increased synthesis of adhesive molecules, adhesion and penetration of monocytes into the vascular wall, and the involvement of pro-inflammatory proteins ; increased platelet aggregation and thrombosis, apoptosis activity, etc., i.e. increased formation of reactive oxygen species in vascular disorders [Lankin V.Z., Tikhase A.K., Belenkov Yu.N.]. Free radical processes in diseases of the cardiovascular system are accompanied by severe dysfunction of the vascular endothelium. [Cardiology 2000; 7: 48-61, CookeJ.P. Flow, NO andatherogenesis. PNAS 2003; 100: 3: 768-770., Ferdinandy P, Schuiz R. Nitric oxide, superoxide, and peroxynitrite in myocardial ischaemia-reperfusion injury and preconditioning. Br J Pharmacol. 2003 Feb; 138 (4): 532-43. Review., 545, Ganz P, VitaJA. Testing endothelial vasomotor function: nitric oxide, a multipotent molecule. Circulation. 2003 Oct 28; 108 (17): 2049-53., Tschudi MR, Luscher TF Nitric oxide: the endogenous nitrate in the cardiovascular system // Herz. - 1996. - V.21. - P.50-60., Tentolouris C, Tousoulis D, Stefanadis C. L-arginine "paradox" in coronary atherosclerosis. Circulation. 2004 Aug 17; 110 (7): e71]. Reactive forms of oxygen, in particular superoxide anion - О ^2- , have the ability to inhibit the expression and reduce the activity of eNOS, as well as to bind and inactivate NO, reduce its content in the cell. A change in the physiological balance between NO and O ² towards the second can lead to the formation of highly toxic peroxynitrite (ONOO ^- ), which damages the cell membrane and DNA, causes mutations, apoptosis, and promotes the development of inflammatory processes and other pathological changes [Ferdinandy P, Schuiz R. Nitric oxide, superoxide, and peroxynitrite in myocardial ischaemia-reperfusion injury and preconditioning. Br J Pharmacol. 2003 Feb; 138 (4): 532-43. Review.].

In experimental modeling of arterial hypertension in animals, an increase in reactive oxygen species leads to endothelial dysfunction, as evidenced by improved endothelium-dependent relaxation with antioxidants [ChenX, Antioxidant effects of vitamins C and E are associated with altered activation of vascular NADPH oxidase and superoxide dismutase in stroke-prone SHR. Touyz R M, Park J B, Schiffrin E L: Hypertension 38: 606-611, 2001].

In clinical practice, there is evidence that an excess of oxygen free radicals in patients with hypertension leads to a decrease in NO production and is directly correlated with the degree of decrease in endothelium-dependent vasodilation and manifestations of cardiovascular diseases [Endothelial dysfunction, oxidative stress, and risk of cardiovascular events in patients with coronary artery disease. Heitzer T, Schlinzig T, Krohn K, Meinertz T, Munzel T Circulation 104: 2673-2678, 2001, Oxygen radicals as second messengers for expression of the monocyte chemoattractant protein, JE / MCP-1, and the monocyte colony-stimulating factor, CSF-1, in response to tumor necrosis factor- and immunoglobulin G: evidence for involvement of reduced nicotinamide adenine dinucleotide phosphate (NADPH) -dependent oxidase. Satriano JA, Shuldiner M, Hora K, Xing Y, ShanZ, SchlondorffD. J Clin Invest. 1993; 92: 1564-1571].

In models of chronic renal failure, it has been shown that increased production of reactive oxygen species leads to a decrease in the bioavailability of NO and manifestations of endothelial dysfunction corrected by prior antioxidant therapy [Endothelial dysfunction and hypertensionin 5/6 nephrectomized rats are mediated by vascular superoxide. Hasdan G, Benchetrit S, Rashid G, et al. Kidney Int 61: 586-590, 2002, Enhanced nitric oxide inactivation and protein nitration by reactive oxygen species in renal insufficiency. Vaziri N D, Ni Z, Oveisi F, Liang K, Pandian R: Hypertension 39: 135-141, 2002].

In type 2 diabetes models, excessive oxygen free radical formation also leads to endothelial dysfunction [FrisbeeJC, SteppDW: Impaired NO-dependent dilation of skeletal muscle arterioles in hypertensive diabetic obese Zuckerrats. Am J Physiol Heart Circ Physiol 281: H1304-H1311, 2001, Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: Role of xanthine-oxidase and extracellular superoxide dismutase. Landmesser U, Spiekermann S, Dikalov S, Tatge H, Wilke R, Kohler C, Harrison DG, Homig B, Drexler H: Circulation 106: 3073-3078, 2002]. Obviously, as follows from the experimental data, reactive oxygen species are involved in the process of exacerbating endothelial damage leading to programmed cell death or apoptosis, or to anoikis, in the form of apoptosis, characterized by cleavage of endothelial cells [Vascular oxidative stress and endothelial dysfunction in patients with chronic heart failure: Role of xanthine-oxidase and extracellular superoxide dismutase. Landmesser U, Spiekermann S, Dikalov S, Tatge H, Wilke R, Kohler C, Harrison DG, Hornig B, Drexler H: Circulation 106: 3073-3078, 2002]. Anoikis is a specific mechanism caused by the loss of cell matrix interactions. Its role in the development of cardiovascular diseases is not fully understood. Eicosapentaenoic acid (polyunsaturated fatty acid found in fish oil) has been shown to protect endothelial cells from anoikis [Eicosapentaenoic acid protects endothelial cells against anoikis through restoration of cFLIP. Suzuki T, Fukuo K, Suhara T. Et al. Hypertension 42: 342-348, 2003], which, in turn, can contribute to the manifestation of antiatherogenic and cardioprotective effects of fish oil.

In recent years, great attention has been paid to L-arginine as a source of NO. There are many works proving the undeniable role of L-arginine in the production of the main vasodilating factor [Durante, W., Arginase: acritical regulator of nitrite oxide synthesis and vascular function / W. Durante, F.R. Jonson, R.A. Jonson // Clin. Exp.Pharmacol. Physiol. - 2007. - No. 34. - P.906-911., Morris JR, S.M. Recent advances in arginine metabolism: roles and regulation of the arginases / S.M. Morris JR // British Journal of Pharmacology. - 2009. - No. 157. - P.922-930].

A known nutritional supplement (US Pat. No. 6,548,087), including L-arginine, ginseng, vitamin PP, dehydroepiandrosterone and zinc, is used to prevent sexual dysfunction and improve blood microcirculation.

Known food dietary supplement (US Patent 6544563), including L-arginine, ginseng, ginkgo biloba, antioxidant vitamins A, C, E, B vitamins and minerals, which helps to increase the resistance of the human body, improve sexual function, function of the cardiovascular system and functions of higher nervous activity.

The use of L-arginine and lysine to increase the level of endothelial NO for inhibiting the development of atherosclerosis, restenosis or thrombosis in the vascular system is known (US patent 5945452). Valine and lysine are known competitive inhibitors of arginase, however, their use may have a cytotoxic effect.

A combination of arginine with amlodipine or indapamide is known (Malykhin, Vitaliy Alekseevich. Study of the endothelioprotective properties of L-arginine and its combinations with amlodipine and indapamide. Abstract of dissertation, 2009, Kursk). It is noted that, obviously, the positive pharmacodynamic interaction of the combined use of L-arginine with antihypertensive drugs is associated with their different points of application of action. And it is also suggested that with prolonged use of arginine, an anti-stress effect is possible. However, prolonged use can lead to side effects.

As the closest analogue, a combination of L-arginine and an arginase inhibitor can be indicated for the treatment of disorders associated with reduced bioavailability of nitric oxide (WO 2004073623). This solution discusses the problem of improving the bioavailability of arginine by combination with any arginase inhibitors. It is noted that the combination allows you to not use large doses of arginine, however, the concentration of arginine in the plasma remains low even in the presence of an arginase inhibitor. To increase bioavailability, magnesium is also proposed. The main tests relate to the treatment of asthma, and the endothelin-protective effect of the combination is not discussed.

The problem solved by the invention is to provide a composition having an enhanced effect, an anti-stress property against vascular endothelium, vasodilating and angioprotective action, including when used to correct erectile dysfunction disorders.

The essence of the invention lies in the fact that the claimed composition is in the form of a solid dosage form, coated with a retarding membrane, contains ingredients that ensure its angioprotective properties - it is L-arginine in the form of L-arginine hydrochloride or L-citrulline and an arginase inhibitor selected from L-norvaline (2-aminopentanoic acid, α-aminovalerianic acid) and nor-NOHA (N-hydroxy-nor-L-arginine, N (omega) -hydroxy-nor-L-arginine) and pharmaceutically acceptable excipients.

The novelty of the invention lies in the previously unknown use of these components, contributing to the normalization of vascular endothelial functions in the human body, in the form of a fixed dosage composition for oral administration.

With the combined use of L-arginine or L-citrulline and an arginase inhibitor, a more pronounced effect of L-arginine on the vessels of a biological organism, in particular a mammal, is manifested. Arginase inhibitors contribute to improved bioavailability of arginine, but not all of them can be used for long-term use and to increase the stress-protective effect. Therefore, for the purposes of this invention, L-norvaline and nor-NOHA are used.

L-arginine, being a metabolic source of nitric oxide in the body, improves the functional state of vascular endothelial cells and immunocompetent cells, which is especially important for endothelial dysfunction or inadequate immune responses.

Moreover, it is taken into account that pure L-arginine is not recommended for pregnant and lactating women, people with cancer and with a diagnosis of schizophrenia. Large doses of L-arginine can also cause or exacerbate an outbreak of herpes.

The active ingredients are used in the following therapeutically effective amounts in mg:

L-arginine hydrochloride or L- citrulline, mg 50-2000 Arginase inhibitor, mg 10-700

Dosage forms of retard are forms that provide a slow release of the drug active principle. Physical and chemical methods are commonly used to produce retard dosage forms. Physical methods include coating crystalline particles, granules, tablets, capsules; mixing drugs with substances that slow down the absorption, biotransformation and excretion; the use of insoluble bases (matrices), etc. The main chemical methods are adsorption on ion exchangers and the formation of complexes. Depending on the production technology, there are distinguished dosage forms of retard of two principal types - reservoir and matrix. Forms of the reservoir type are a core containing a drug and a polymer (membrane) membrane, which determines the rate of release. The reservoir can be a single dosage form (tablet, capsule) or drug microform, many of which form the final form (pellets, microcapsules, etc.). Matrix-type retard forms contain a polymer matrix in which the drug is distributed, and often take the form of a conventional tablet.

For the purposes of the present invention, a retarded release coating is used.

Most preferably, the content of pharmaceutically acceptable excipients is up to 30% of the total weight of the preparation. These substances can be selected from the group of substances: microcrystalline cellulose or lactose, starch, povidone, calcium stearate, talc, aerosil, ethyl cellulose or their mixture in any combinations and ratios that do not impair its biological effect on the body. Preferably, the composition is in the form of a tablet and / or capsule form of the drug and contains excipients in the following amounts, mg:

Microcrystalline cellulose or lactose 80-100 Aerosil 35-50 Talc 110-150 Povidone 40-50 Calcium stearate 20-50

The resulting form is coated with a membrane that provides a retard action (delayed release).

As a shell, compositions based on polyvinyl alcohol, talc, polyethylene glycol, titanium dioxide, acceptable dyes, or as a ready-made mixture of the Opadry II brand can be used.

The shell also gives stability to the composition during storage and improves its appearance and organoleptic properties.

The composition may further comprise succinic acid and / or its pharmaceutically acceptable salts, for example ammonium, potassium, sodium and the like, in an amount of 10-500 mg. Performing a catalytic function with respect to the Krebs cycle, succinic acid reduces the concentration of other intermediates of this cycle in the blood - lactate, pyruvate and citrate, produced in the early stages of hypoxia. The antihypoxic effect of succinic acid is due to its effect on the transport of mediator amino acids, as well as an increase in the content of GABA in the brain. Succinic acid without affecting blood pressure and heart rate. However, the introduction of a "universal metabolite" in the composition enhances its protective function.

The biological properties of the composition were studied.

Example 1

The aim of the study was to study the endothelial effects of a combination of an arginase inhibitor (L-norvaline was taken as this) and a donor of nitric oxide (NO) L-arginine during modeling, the so-called L-NAME-induced endothelial dysfunction.

Materials and methods

Animals - white rats, males of the Wistar line, weight 250-300 g. Groups of animals were formed (n = 10): 1) intact; 2) the introduction of N-nitro-L-arginine methyl ester (L-NAME); 3) the introduction of a combination of L-norvaline at a dose of 10 mg / kg and L-arginine at a dose of 70 mg / kg.

When modeling endothelial dysfunction, an N-nitro-L-arginine methyl ester (L-NAME) endothelial NO synthase blocker was administered, which was administered intraperitoneally once a day, at a dose of 25 mg / kg. The combination of the arginase inhibitor L-norvaline and the donor of nitric oxide (NO) L-arginine was administered daily intragastrically at doses of 10 mg / kg and 70 mg / kg, respectively, for 7 days. A bolus of pharmacological agents was administered into the right femoral vein. On the 8th day from the start of the experiment, under anesthesia (chloral hydrate 300 mg / kg), a catheter was inserted into the left carotid artery to record parameters. Hemodynamic parameters - systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR) were measured continuously. Functional tests were performed: endothelium-dependent vasodilation (ESV) was measured by the intravenous administration of acetylcholine (AX) at a dose of 40 μg / kg; endothelium-independent vasodilation (ENZV) - sodium nitroprusside (NP) was administered intravenously at a dose of 30 μg / kg.

The reliability of the changes in the parameters observed under the action of the investigated substance was determined by the difference method of descriptive statistics with finding the average values of the shifts (M), the average error of the arithmetic mean (± m) and the probability of a possible error (p) calculated using the T-test for groups with different dispersions. Differences were evaluated as significant at p <0.05. Statistical calculations were performed using the Microsoft Office program.

Results

During the experiments, it was found that the blockade of NO synthase caused by the seven-day administration of L-NAME led to arterial hypertension average values: SBP - 191.3 ± 7.1 mm Hg, DBP - 146.0 ± 4.2 mm Hg, SRAD - 159.4 ± 3.9 mm Hg The use of L-norvaline and L-arginine, in combination, blocked a marked increase in blood pressure (table 2). In a group of animals treated with a combination of L-norvaline and L-arginine according to the scheme for 7 days, endothelium-independent vasodilation (ENVD) was evaluated by means of a functional test with nitroprusside (NP) at a dose of 30 μg / kg and was characterized by a decrease in SBP to 87.2 ± 4.1 mmHg, DBP up to 37.3 ± 1.8 mmHg

When performing endothelium-dependent vasodilation of EDVD after administration of acetylcholine AH, hemodynamic parameters were also reduced compared to the group of animals treated with L-NAME.

table 2 The effect of the combined use of L-norvaline and L-arginine on hemodynamic parameters in modeling L-NAME-induced endothelial dysfunction (M ± m) Groups of animals Functional Tests GARDEN, mmHg DBP, mmHg Heart rate, beats in min Intact Source 136.4 ± 4.1 99.9 ± 3.8 417.0 ± 8.1 EZV with AH 82.5 ± 3.9 39.4 ± 3.4 415.1 ± 12.4 ENZV with NP 82.3 ± 2.9 41.8 ± 4.4 416.2 ± 9.1 Receiving L-NAME (25 mg / kg) Source 191.3 ± 7.1 * 146.0 ± 4.2 * 429.3 ± 10.3 EZV with AH 111.2 ± 4.8 * 83.2 ± 5.8 * 424.0 ± 12.9 ENZV with NP 89.2 ± 3.9 52.5 ± 3.8 423.0 ± 12.5 L-NAME (25 mg / kg) + combination of L-norvaline (10 mg / kg) + L-arginine (70 mg / kg) Source 136.4 ± 4.7 ** 109.6 ± 2.9 ** 374.2 ± 8.2 EZV with AH 86.6 ± 2.4 ** 43.7 ± 4.8 ** 341.6 ± 10.9 ENZV with NP 87.2 ± 4.1 ** 37.3 ± 1.8 * 343.1 ± 11 Note * - p <0.05 - in comparison with a group of intact animals; ** - p <0.05 - in comparison with the group. GARDEN - systolic blood pressure, DBP - diastolic blood pressure, S is the area above the curve for the restoration of blood pressure during pharmacological tests.

In order to objectively evaluate the degree of correction of endothelial dysfunction that occurs when modeling nitric oxide deficiency by the introduction of L-NAME, a special coefficient of endothelial dysfunction (QED) was used during the experiments [Pokrovsky, M.V. Methodological approaches for the quantitative assessment of the development of endothelial dysfunction in the L-NAME-induced model of nitric oxide deficiency in the experiment / M.V. Pokrovsky, V.I. Kochkarov, T.G. Pokrovskaya and others // Kuban Scientific Medical Bulletin. Krasnodar, 2006. No. 10. S.72-77.]. It characterizes the degree of endothelial dysfunction and reflects the ratio of the area above the blood pressure reduction reaction in response to the administration of sodium nitroprusside (NP) to the area above the blood pressure reduction reaction in response to the administration of acetylcholine (AX) (Fig. 2).

For each animal in each group, the coefficient of endothelial dysfunction (QED) was calculated. In the group of intact animals, the coefficient of endothelial dysfunction (QED) was 1.1 ± 0.1.

findings

1. Modeling nitric oxide (NO) deficiency by introducing an endothelial NO synthetase (NOS) blocker, N-nitro-L-arginine methyl ester (L-NAME) intraperitoneally daily at a dose of 25 mg / kg for 7 days led to the development of arterial hypertension, which was realized through endothelial dysfunction.

2. The development of endothelial dysfunction (ED) in the experimental model is expressed in a sharp increase in the coefficient of endothelial dysfunction (QED) in comparison with the intact group.

3. The combined use of L-arginine at an average dose of 25 mg / kg and L-norvaline at an average dose of 10 mg / kg has an endothelioprotective effect by increasing the concentration of intracellular L-arginine and increasing the bioavailability of nitric oxide (NO), which is characterized by a decrease in the coefficient of endothelial dysfunction (QED) and its values similar to those in the intact group.

The inventive biologically active composition is obtained by mixing the active agents.

The substance nor-NOHA, which is a selective arginase II inhibitor, was also taken as another arginase inhibitor.

Example 2

The aim of the study was to study the endothelial and cardioprotective effects of combinations of arginase inhibitors (non-selective - L-norvaline and selective, arginase II - nor-NOHA N (omega) -hydroxy-nor-L-arginine) and nitric oxide donor (NO) L-arginine when modeling, the so-called L-NAME-induced endothelial dysfunction.

Materials and methods

Animals - white rats, males of the Wistar line, weight 250-300 g. Groups of animals were formed (n = 10):

1) intact;

2) the introduction of N-nitro-L-arginine methyl ester (L-NAME);

3) L-NAME + L-norvaline at a dose of 10 mg / kg (retard);

4) L-NAME + L-arginine at a dose of 70 mg / kg (retard);

5) L-NAME + L-norvaline at a dose of 10 mg / kg + L-arginine at a dose of 70 mg / kg (retard);

6) L-NAME + L-norvaline at a dose of 100 mg / kg;

7) L-NAME + L-arginine at a dose of 200 mg / kg;

8) L-NAME + L-norvaline at a dose of 100 mg / kg + L-arginine at a dose of 200 mg / kg;

9) L-NAME + nor-NOHA at a dose of 1 mg / kg;

10) L-NAME + nor-NOHA at a dose of 1 mg / kg + L-arginine at a dose of 200 mg / kg.

When modeling endothelial dysfunction, an N-nitro-L-arginine methyl ester (L-NAME) endothelial NOS blocker was administered, which was administered intraperitoneally once a day at a dose of 25 mg / kg. The combination of a non-selective arginase inhibitor L-norvaline, a selective arginase II inhibitor nor-NOHA and a donor of nitric oxide (NO) L-arginine was administered daily intragastrically for 7 days.

On the 8th day from the start of the experiment, under anesthesia (chloral hydrate 300 mg / kg), a catheter was inserted into the left carotid artery to record parameters. Hemodynamic parameters - systolic blood pressure (SBP), diastolic blood pressure (DBP), heart rate (HR) were measured continuously. Functional tests were performed: endothelium-dependent vasodilation (ESV) was measured by the intravenous administration of acetylcholine (AX) at a dose of 40 μg / kg; endothelium independent vasodilation (ENZV) - sodium nitroprusside (NP) was administered intravenously at a dose of 30 μg / kg. To evaluate ecdothelial dysfunction from vascular samples for acetylcholine and nitroprusside, the coefficient of endothelial dysfunction (QED) was calculated (Pokrovsky, M.V. Methodological approaches for quantifying the development of endothelial dysfunction in the L-NAME-induced model of nitric oxide deficiency in the experiment / M.V. .Pokrovsky, V.I. Kochkarov, T.G. Pokrovskaya and others // Kuban Scientific and Medical Bulletin. Krasnodar, 2006. No. 10. P.72-77.)

The reliability of the changes in the parameters observed under the action of the investigated substance was determined by the difference method of descriptive statistics with finding the average values of the shifts (M), the average error of the arithmetic mean (± m) and the probability of a possible error (p) calculated using the T-test for groups with different dispersions. Differences were evaluated as significant at p <0.05. Statistical calculations were performed using the Microsoft Office program.

Results

It was found that the blockade of NO synthase caused by the seven-day administration of L-NAME led to severe arterial hypertension, mean values: SBP -191.3 ± 7.1, DBP - 146.0 ± 4.2 mm Hg, and a 5-fold increase in the coefficient of endothelial dysfunction (Table 3).

Table 3 The effect of the retard form of a fixed combination of L-arginine (70 mg / kg) and L-norvaline (10 mg / kg) on blood pressure and endothelial dysfunction coefficient in modeling L-NAME-induced nitric oxide deficiency in rats (M ± m, n = 10) Groups of animals GARDEN, mmHg DBP, mmHg QED, cu Intact 136.4 ± 4.1 99.9 ± 3.8 1.1 ± 0.1 Receiving L-NAME (25 mg / kg) 7 days 191.3 ± 6.7 * 146.0 ± 3.9 * 5.4 ± 0.06 * Treated with L-NAME + L-Arginine (70 mg / kg) 177.6 ± 9.6 * 140.1 ± 6.4 * # 2.9 ± 0.1 * # Receiving L-NAME + L-Norvaline (10 mg / kg) 186.4 ± 13.1 * 156.8 ± 10.0 * 3.5 ± 0.05 ^{* #} Treated with L-NAME + L-norvaline (10 mg / kg) + L-arginine (70 mg / kg) 136.4 ± 4.7 # 109.6 ± 2.9 # 2.2 ± 0.1 # Note: CAD - systolic blood pressure, DBP - diastolic blood pressure, heart rate - heart rate, * - significant difference with the group of intact animals (p <0.05); # - significance of differences with the group of control animals with L-NAME-induced pathology.

The use of L-arginine and L-norvaline in the form of a retard form led to a significant decrease in QED, respectively, 2.9 ± 0.1 and 3.5 ± 0.05 cu Despite the decrease in QED, blood pressure values did not reach the specified values and were significantly higher than in intact animals (Table 3).

A study of the effect of large doses of L-arginine (200 mg / kg) and L of norvaline (100 mg / kg) and their combination in the form of an uncontrolled dosage form also found a significant increase in the effect, but no further statistically significant decrease in QED and blood pressure was observed (Table 4 )

Table 4 The effect of a fixed combination of L-arginine (200 mg / kg) and L-norvaline (100 mg / kg) on blood pressure and endothelial dysfunction coefficient in modeling L-NAME-induced nitric oxide deficiency in rats (M ± m, n = 10 ) Groups of animals GARDEN, mmHg DBP, mmHg QED, cu Intact 136.4 ± 4.1 99.9 ± 3.8 1.1 ± 0.1 Receiving L-NAME (25 mg / kg) 7 days 191.3 ± 6.7 * 146.0 ± 3.9 * 5.4 ± 0.06 * Receiving L-NAME + L-Arginine (200 mg / kg) 177.6 ± 9.6 * 140.1 ± 6.4 * # 2.5 ± 0.1 * # Receiving L-NAME + L-Norvaline (100 mg / kg) 186.4 ± 13.1 * 156.8 ± 10.0 * 3.5 ± 0.5 * Treated with L-NAME + L-Norvaline (100 mg / kg) + L-Arginine (200 mg / kg) 148.3 ± 9.0 # 105.5 ± 4.8 # 2.0 ± 0.1 # Note: CAD - systolic blood pressure, DBP - diastolic blood pressure, heart rate - heart rate, * - significant difference with the group of intact animals (p <0.05); # - significance of differences with the group of control animals with L-NAME-induced pathology.

The fixed combination of 1-arginine 70 mg / kg and the selective inhibitor of arginase II nor-NONA 1 mg / kg also showed an additive effect on the coefficient of endothelial dysfunction and blood pressure values, however, in general, the combination of arginine norvaline did not achieve the effectiveness (Table 5).

Table 5 The effect of a fixed combination of L-arginine (70 mg / kg) and nor-NOHA (N (omega) -hydroxy-nor-L-arginine) (1 mg / kg) on blood pressure and endothelial dysfunction coefficient in modeling L-NAME- induced nitric oxide deficiency in rats (M ± m, n = 10) Groups of animals GARDEN, mmHg DBP, mmHg QED, cu Intact 136.4 ± 4.1 99.9 ± 3.8 1.1 ± 0.1 Receiving L-NAME (25 mg / kg) 7 days 191.3 ± 6.7 * 146.0 ± 3.9 * 5.4 ± 0.06 * Treated with L-NAME + L-Arginine (70 mg / kg) 183.6 ± 4.7 * 148.2 ± 6.4 * 3.4 ± 0.1 * Receiving L-NAME + nor-NOHA (1 mg / kg) 178.3 ± 9.4 * 144.7 ± 9.3 * 3.6 ± 0.3 * Treated with L-NAME + nor-NOHA (1 mg / kg) + L-arginine (200 mg / kg) 150.3 ± 7.9 * 125.1 ± 7.8 2.9 ± 0.1 # Note: CAD - systolic blood pressure, DBP - diastolic blood pressure, heart rate - heart rate, * - significant difference with the group of intact animals (p <0.05); # - significance of differences with the group of control animals with L-NAME-indupable pathology.

Thus, the results obtained indicate the effectiveness of the combined use of an NO-L-arginine donor and arginase inhibitors, L-norvaline and nor-NONA, for the correction of L-NAME-inducted endothelial dysfunction, which manifests itself in a decrease in QED and blood pressure. The most effective combination was L-arginine 70 mg / kg and L-norvaline 10 mg / kg in the form of a retard form with a release profile of 12 hours. The indicated dosage, taking into account the interspecific conversion of doses from rat to human (according to the formula of Ulanova, Guide to preclinical studies under the editorship of R. Khabriev, 2006), corresponds to 500 mg of L-arginine and 70 mg of L-norvaline, which can be achieved as coated tablet, containing respectively 250 and 35 mg when taken 2 times a day.

The invention is also illustrated by examples of pharmaceutical compositions.

Example 3

The components are mixed in the following amounts:

L-arginine hydrochloride, mg 1000 Arginase inhibitor (L-norvaline), mg 700

The components are mixed in a mixer with the addition of, if necessary, pharmacologically acceptable excipients, ensuring an even distribution of the components, and served to form capsules to fill and cover them with Opadry II.

Example 4

The components are mixed in the following amounts:

L-arginine hydrochloride, mg one hundred Arginase inhibitor (L-norvaline), mg 300

Excipients, mg:

lactose one hundred Aerosil 40 Talc 110 Povidone fifty Calcium stearate twenty

The ingredients and excipients are mixed in a mixer, ensuring uniform distribution of the components, tableted, coated with an “Opadry II” sheath, and packaged in a blister strip or corresponding packaging in the form of cans.

It should be noted that a small group of animals (n = 3) was taken to verify the assumption, i.e. not statistically significant by which L-arginine was replaced with citrulline at a dosage of 100 mg or more. It was noted that the absolute values were slightly lower than the composition with L-arginine, and at the same time, the effect of correction of artificial nitric oxide deficiency was noted.

Example 5

Granule-filled capsule, Opadry II-coated, in mg:

L-citrulline 250 L-norvaline 500 succinic acid 150 Aerosil fifty Povidone fifty Calcium stearate 40

The invention expands the possibility of the amino acid L-arginine or the amino acid L-Citrulline as a biologically active substance that increases the resistance of the endothelium to intravascular stress and normalizes the function of the cardiovascular and reproductive systems in the prevention and complex treatment of diseases of patients.

Claims (7)

1. A composition having endothelioprotective, vasodilating and angioprotective effect, characterized in that it is a solid dosage form, coated for retarid release, contains L-arginine hydrochloride or L-citrulline and an arginase inhibitor selected from L- as active ingredients norvaline and nor-NONA in the following content, mg:
L-Arginine Hydrochloride or L-Citrulline 50-2000 arginase inhibitor 10-700
and pharmaceutically acceptable excipients. 2. The composition according to claim 1, characterized in that it contains pharmaceutically acceptable excipients up to 30% of the total weight of the composition. 3. The composition according to claim 1 or 2, characterized in that the arginase inhibitor is L-norvaline. 4. The composition according to claim 3, characterized in that the pharmaceutically acceptable excipients are selected from the group: microcrystalline cellulose, lactose, starch, povidone, calcium stearate, talc, aerosil, ethyl cellulose or mixtures thereof. 5. The composition according to claim 4, characterized in that it is made in the form of a tablet and / or capsule form of the drug, and contains auxiliary substances in the following amounts, mg:
microcrystalline cellulose or lactose 80-100 aerosil 35-50 talc 110-150 povidone 40-50 calcium stearate 20-50 6. The composition according to claim 1, characterized in that succinic acid and / or its pharmaceutically acceptable salts are introduced into the composition in an amount of 10-500 mg. 7. The composition according to claim 1, characterized in that the composition is used to correct erectile dysfunction disorders.

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