FIELD OF THE INVENTION

The present invention relates to the field of veterinary and medicine. In particular, the present invention relates to a use of a composition based on a hydrolysate obtained from one or more natural biological resources selected from the group including bivalve mollusks, annelids, and leeches, for the prevention or treatment of endothelial dysfunction (ED) and/or an ED-related disease or condition in a mammal in need of such treatment or prevention. The present invention also relates to methods for the prevention and treatment of ED and/or an ED-related condition or disease using the above composition.

BACKGROUND OF THE INVENTION

Endothelium is a monolayer sheet of squamous cells having mesenchymal origin which lines the inner surface of blood and lymphatic vessels, cardiac cavities. In light of contemporary concepts, the endothelium is an active endocrine organ diffusely distributed throughout all tissues. The endothelium forms the inner lining of blood vessels separating blood flow from the deeper layers of the vascular wall. It is a continuous monolayer of l-6xl0 ¹³ endothelial cells which form a tissue weighting 1.5-2.0 kg and having a total surface area of about 900 m ² in humans. The endothelium continuously produces an enormous number of crucial biologically active substances thus being a giant paracrine organ distributed across the entire area of the human body.

The main functions of endothelial cells include, inter alia, the following: regulation of vascular tone (vasoconstriction and vasodilation); regulation of hemostasis (thrombogenicity and atrombogenicity of the vascular wall); angiogenesis (formation of new blood vessels); participation in immune reactions (activation of leukocyte adhesion, production of cytokines) including an essential barrier immune function.

The endothelium is known to be able to produce biologically active substances (BAS) which maintain the tone of underlying vascular smooth muscle cells (SMC), preserve non-adhesiveness of intima, and influence inflammatory and immune mechanisms in the vascular wall. In addition to a barrier function, endothelial cells perform numerous functions in a tight relationship with SMCs. Therefore, the regulation of vascular tone is considerably associated with BAS synthesis by endothelial cells. Changes in physical, chemical and humoral parameters of the endothelial cell environment prompt them to produce a number of BAS: vasodilators - nitric oxide (NO), prostacyclin (PGI2), bradykinin, endothelium-derived hyperpolarizing factor (EDHF); vasoconstrictors - endothelin-1, prostaglandin H2, superoxide anion, angiotensin II, thromboxane A2. Nitric oxide is the main factor controlling the vascular tone; it can regulate and distribute blood flow in various vascular pools by changing the diameter of both large and small arteries and arterioles via the stimulation of guanylate cyclase and an increase in the intracellular cGMP concentration. Subsequently, cGMP reduces the intracellular Ca ²⁺ concentration resulting in vasodilation. In addition to the regulation of vascular tone, nitric oxide has many additional unique functions what makes it the main factor in antiatherogenesis. NO, as a universal signaling molecule, suppresses platelet adhesion to the endothelium and their aggregation, possesses anti-inflammatory properties, regulates the synthesis and breakdown of the extracellular matrix, prevents cell migration and proliferation, and controls gene transcription. Moreover, NO acts as a secondary messenger for many growth factors, peptides, blood coagulation factors and hormones, and also facilitates transendothelial migration of leukocytes and endothelial cell precursors. Along with this, the endothelium is involved in the regulation of blood coagulation (formation of fibrinolysis activators and inhibitors, pro- and antithrombotic factors) and vascular wall permeability (free radicals, protein kinase C).

It was also found that the endothelium is capable to regulate adhesive properties of the vascular wall (expression of adhesion molecules - ICAMs, YCAMs, as well as E- and P-selectins). P- and E-selectins are involved in leukocyte rolling, whereas ICAMs and VCAMs are responsible for their adhesion through the interaction with relevant ligands for white blood cells.

Since ED is associated with an imbalance between the production of vasodilators and vasoconstrictors, thrombogenic and atrombogenic factors, angiogenic substances and their inhibitors, it is subdivided into vasomotor, thrombophilic, adhesive and angiogenic forms. An increased adhesiveness of the endothelium and uncontrolled stopping of leukocytes underlie the adhesive ED form and play an important role in pathogenesis of inflammation in atherosclerosis and other pathological processes. The development of the thrombophilic ED form is mediated by an impaired ratio between factors which induce platelet adhesion and aggregation, thrombogenesis, suppress fibrinolysis (von Willebrand factor, platelet-activating factor, thromboxane A2, tissue factor, etc.), and atrombogenic substances (NO, prostacyclin, thrombomodulin, t-PA, etc.) with a predominance of the former ones. A significant decrease in vascular thromboresistance has been recorded in atherosclerosis, hypertension, diabetes mellitus, and tumor diseases. The loss of vascular endothelial cells accompanied by the exposure of basement membrane and accumulation of cell lysis products, fibrin, erythrocyte sludges, hemolysis products and other factors enhancing intravascular platelet aggregation lead to the development of chronic disseminated intravascular coagulation (DIC) and other diseases. The angiogenic ED form is associated with pathological angiogenesis - an excessive activity of VEGF-A and other growth factors, and impairment in the system of angiogenesis regulators (angiopoietins, angiostatin, etc.). Finally, the vasomotor ED form is mediated by a defect in endothelial vasoactive substance performance and plays an important role in mechanisms of both a systemic increase in blood pressure and local angiospasm. A change in endothelium-dependent vascular relaxation occurs for many reasons: decreased NO production, enhanced inactivation of vasodilators, weakened diffusion of NO to the underlying smooth muscle cells, reduced availability of L-arginine deposits, a precursor of NO, increased degradation of NO by free oxygen radicals, and increased production of vasoconstrictors.

ED also includes accelerated desquamation of capillary endothelium, weakened intercellular junctions, impaired protein synthesis, as well as impaired expression and formation of adhesive glycoproteins on endothelial cells. This facilitates the attachment of monocytes and leukocytes, as well as their migration across the basement membrane.

Endothelial cells of either large or small vessels are insulin-independent cells. Therefore, under hyperglycemic conditions in diabetes mellitus glucose can freely enter into endothelial cells producing pathological biochemical reactions inside the cells which are an important cause of ED. Hyperglycemia delays the replication of endothelial cells and promotes cell death by increasing oxidative processes and glycation. ED is an integrated syndrome of insulin resistance, with exacerbation thereof, increases vascular reactivity, and provokes cardiovascular disorders.

ED is one of the earliest signs of vascular lesions in patients with DM and could be detected at the initial stages of the disease, even before the appearance of atherosclerotic plaques; endothelial dysfunction remains to be a concern at the later stages of atherosclerotic lesion because impairments of endothelium-dependent vascular relaxation and increased adhesiveness of the endothelial lining could provoke a constriction, and the formation and growth of a plaque followed by its rupture. Accordingly, ED also manifests as the impairment of vascular tone regulation and, as a consequence, central and peripheral hemodynamics. The second consequence is the impairment of substance transport through the vascular wall. Lipid accumulation in the vascular wall and their involvement in atherogenesis should be considered as one of the main manifestations of that.

Various antioxidants were amongst the first drugs used for treating endothelial dysfunction: vitamins C and E, N-acetylcysteine, genistein, and others. Vitamin C was shown to protect the endothelium against superoxide radical effects that prevents lipid oxidation, neutrophil adhesion and ultimately the development of endothelial dysfunction [Taddei S., Virdis A., Ghiadoni L, Magagna A., Salvetti A. Vitamin C improvesendo the lium-dependent vasodilation byrestoring nitricoxide activity in essential hypertension. Circulation. 1998; 97:2222-2229 ]. Vitamin E also has a protective effect on the endothelium in hypercholesterolemia but its use in diabetes mellitus produces an opposite effect [ Economides P.A, Khaodhiar L, Caselli A, Caballero A.E, Keenan H, Bursell S.E., King G.L., Johnstone M.T., Horton E.S., Veves A. The effect of vitamin E on endothelial function of micro- and macrocirculation and left ventricular function in type 1 and type 2 diabetic patients. Diabetes. 2005; 54:204-211\. N-acetyl cysteine influences the production of glutathione which is the main endogenous antioxidant protecting the endothelium against free radicals and the damaging effect of various cytokines, such as tumor necrosis factor alpha [ Bourraindeloup M., Adamy C, Candiani G., Cailleret M., Bourin M.C., Badoual T, Su J.B., Adubeiro S., Roudot-Thoraval F., Dubois-Rande J.L. N-acetylcysteine treatment normalizes serum tumor necrosis factor-alpha level and hinders the progression of cardiac injury in hypertensive rats. Circulation. 2004; 110:2003-2009] Moreover, N-acetylcysteine regulates the expression of metalloproteinases which cleave intercellular junction proteins of the endothelium that significantly contributes to the development of endothelial dysfunction [Moshal K.S., Sen U., Tyagi N., Henderson B., Steed M., Ovechkin A.V., Tyagi S.C. Regulation of homocysteine-induced MMP-9 by ERK1/2 pathway. Am J Physiol Cell Physiol. 2006; 290:C883-C891] The main field of application of substances described above is chronic diseases accompanied by endothelial dysfunction.

Another group of drugs is related to prostacyclin, a substance from the class of prostaglandins. Prostaglandins are endogenous biologically active substances of lipid nature synthesized in almost all cells of the body. While prostaglandin E2 is involved in the development of inflammation, prostacyclin and some other prostaglandins have a vasodilatory effect, and also prevent platelet aggregation with endothelial cells. Prostacyclin and its synthetic analogs (iloprost, belaprost, ventavis, etc.) are successfully being used to reduce the permeability of endothelium in combined treatment of ischemia-reperfusion [H. Schutte, A.Lockinger, W.Seeger, et al. Aerosolized PGE1, PGI2 and nitroprusside protect against vascular leakage in lung ischaemia-reperfusion. Eur. Respir. J., 2001; 18:15-22] In endothelial cells, these substances are thought to induce the adenylate cyclase mechanism which reinforces the endothelial barrier by forming new or strengthening the already existing intercellular adhesion complexes. Therefore, prostacyclin was shown to reduce endothelial permeability in LPS-induced inflammation in mice by activating Rapl protein, one of the last participants in the adenylate cyclase pathway [Birukova A.A., Meng F.,Tian Y., Meliton A., Sarich N, Quilliam LA., Birukov K.G. Prostacyclinpost-treatmentimproves LPS- induce dacutelung in juryandendo the lialbarrier recovery via Rap 1. BiochimBiophysActa. 2015; 852 (5): 778-91] However, under certain conditions, an action of prostacyclin can be blocked by other cytokines leading to opposite effects, i.e., enhanced endothelial dysfunction [Blanco-Rivero J., Cachofeiro V., Lahera V., Aras-Lopez R, -Marquez-Rodas /., Salaices M., Xavier F.E., Ferrer M., Balfagon G. Participation of prostacyclininendo the lialdysfunction induced byaldosterone in normotensive and hypertensiverats. Hypertension 2005; 46:107-112] Angiotensin converting enzyme (ACE) inhibitors and angiotensin receptor (AR) blockers are also antihypertensive agents and exert a protective action on the endothelium. Angiotensin II is a protein hormone synthesized from an inactive precursor angiotensin I by ACE. Angiotensin II has a strong vasoconstrictor effect and for this reason ACE is targeted by many drugs, such as perindopril, fosinopril, losartan, etc. The following positive effects of ACE inhibitors were observed amongst others: increased expression of NO-synthase in ischemia-reperfusion [B.Gemici, R.Tan, G.Ongut, V.N. Izgut-Uysal Expressions of induciblenitricoxidesynthase and cyclooxygenase- 2 ingastricis chemia - reperfusion: role of angiotensinll. J SurgRes, 2010; 161:126-133 ], decreased apoptosis of endothelial cells, decreased concentrations of tumor necrosis factor alpha and other pro-inflammatory cytokines \A.M. Kampoli, D.Tousoulis, C.Tentolouris, C.Stefanadis Novelagentstargetingnitricoxide. CnrrVascPharmacol, 2012; 10:61-76]. ACE inhibitors and angiotensin receptor blockers were also shown to have a beneficial effect on the endothelium in coronary heart disease and hypercholesterolemia [ B.Hornig , U.Landmesser, C.Kohler, D.Ahlersmann, S.Spiekermann, A.Christoph, Comparative effect of aceinhibition and angiotensin II type 1 recept or antagonism on bioavailability of nitricoxidein patients with coronaryarterydisease: role of super oxidedismutase. Circulation, 2001; 103:799-805 ].

Different drugs for the treatment of diseases related to endothelial dysfunction are also widely known from patent literature (see, for example, RU 2464019, RU 2657416, RU 2505290).

Therefore, in veterinary and medicine there is still a need for the development of alternative, preferably more effective, means and methods for preventing or treating ED and/or ED-related diseases and conditions, as well as means which could find application in conjunction with conventional means for preventing or treating ED and/or ED-related diseases and conditions.

An object of the present invention was to provide a more effective method for the prevention and treatment of ED and/or ED-related conditions.

SUMMARY OF THE INVENTION

The present invention is a result of continuing the development and investigation of properties of the composition described in patent RU 2526826, issued in the name of present inventor. In particular, patent RU 2526826 discloses the composition for parenteral administration comprising a hydrolysate based on biological raw materials obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches, and water, and having a broad spectrum of action and properties. Surprisingly, additional studies aimed at assessing the efficacy of treatment of ED induced in laboratory conditions in mice, as well as an increase in the levels of nitric oxide (NO) release in human endothelial cells in vitro, described below in this specification, have demonstrated that said composition is an effective means for treating or preventing ED, as well as an ED-related disease or condition. The disclosure of patent RU 2526826 is entirely incorporated herein by reference.

Therefore, the present disclosure provides a use of a composition based on a hydrolysate obtained from one or more biological resources selected from the group including bivalve mollusks, annelids, and leeches, for the prevention or treatment of ED, as well as diseases or conditions accompanied by or related to ED, in a mammal in need of such prevention or treatment.

Said composition comprises a wide range of essential amino acids, melanoidins acting as antioxidants, regulatory peptides, saturated and unsaturated fatty acids, macro- and microelements in a balanced native natural ratio, and, inter alia, has an immunomodulating property and is employed for therapeutic and/or prophylactic purposes in veterinary and medicine.

More specifically, in a first aspect, the present invention provides a use of a composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, for the prevention or treatment of endothelial dysfunction in a mammal in need of such treatment or prevention.

In a second aspect, the present invention provides a use of a composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, for the prevention or treatment of endothelial dysfunction and/or a disease or condition related to endothelial dysfunction in a mammal in need of such treatment or prevention.

In a third aspect, the present invention provides a use of a composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, for the prevention or treatment of a disease or condition with concomitant endothelial dysfunction in a mammal in need of such treatment or prevention.

In yet another aspect, the present invention relates to the composition for use according to any of applications described above.

In a fourth aspect, the present invention provides a method for the treatment or prevention of endothelial dysfunction in a mammal in need of such treatment or prevention, comprising parenteral administration of the composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, to said mammal. In a fifth aspect, the present invention provides a method for the treatment of endothelial dysfunction and/or a disease or condition related to endothelial dysfunction in a mammal in need of such treatment or prevention, comprising parenteral administration of the composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, to said mammal.

In a sixth aspect, the present invention provides a method for the treatment of a disease or condition with concomitant endothelial dysfunction in a mammal in need of such treatment or prevention, comprising parenteral administration of the composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, to said mammal.

Various embodiments of the present invention are described below in the “Detailed Description” section. Each of said specific embodiments of the invention is to be understood as relating to any of aspects of the invention specified above.

BRIEF DESCRIPTION OF THE DRAWINGS

Fig. 1 shows study data demonstrating the effect of enhancing eNOS activity in human umbilical cord vein endothelial cells (HUVECs) by using the composition according to the present invention.

Fig. 2 shows study data demonstrating the effect of reducing the expression of adhesion molecules VCAM and ICAM in human umbilical cord vein endothelial cells (HUVECs) by using the composition according to the present invention.

Fig. 3 shows study data demonstrating the effect of reducing iNOS activity in the cell culture from peritoneal lavage of mice.

DETAILED DESCRIPTION

A disturbance of endothelium functions is one of universal mechanisms of pathogenesis of many diseases including common ones such as atherosclerosis, hypertension, and diabetes mellitus. Dysfunction of the endothelium or endothelial dysfunction (ED) plays a role in the development of thrombosis, neoangiogenesis, vascular remodeling, and intravascular activation of platelets and leukocytes. The predominant impairment of one or another function of the endothelium depends on the localization of pathological processes, prevalence of one or other of inflammatory mediators, and presence of hemodynamic shifts. An implication of the endothelium in the regulation of vasomotor reactions, hemostasis, angiogenesis, vascular-tissue migration of leukocytes was the most comprehensively studied. The impairment of these particular processes is a nonspecific chain in pathogenesis of many diseases. Endothelial dysfunction is one of the most important pathogenetic mechanisms of many cardiovascular diseases. In particular, endothelial dysfunction could cause vasoconstriction, increased clotting and enhanced adhesion of leukocytes to the endothelium which is accompanied by the impairment of regional blood circulation and microcirculation.

Endothelial dysfunction can be caused by the following factors: genetic features; age-related changes; dyslipoproteinemia (hypercholesterolemia); hypercytokinemia; hyperhomocysteinemia; hyperglycemia; hemodynamic factor (hypertension, ischemia, venous stasis); endogenous intoxications (renal failure, hepatic failure, pancreatitis, etc ); exogenous intoxications (smoking, etc.)

Recent years have led to the understanding that endothelial dysfunction represents a state of the endothelium with insufficient production of nitric oxide (NO). Since nitric oxide is involved in the regulation of almost all functions of the endothelium (regulation of vascular tone, vascular thromboresistance, regulation of leukocyte adhesion and vascular permeability), and additionally is the factor most sensitive to a lesion, this insight into endothelial dysfunction is quite correct, although not entirely comprehensive. The most important factor interfering nitric oxide formation and/or bioavailability is an excessive generation of reactive oxygen species observed in many diseases.

One of the methods for evaluating the severity of ED is the assessment of various substances, which are produced in the endothelium, in the blood. At present, there are techniques for determining in the blood almost all known substances produced in the endothelium; however, not all indicators have the same diagnostic value, since a significant portion of ED markers is produced not only in the endothelium but in other cells as well. By the degree of specificity, the most considerable are, in particular, nitric oxide, prostacyclin, endothelin-1, ICAM-1, VCAM-1, E- selectin, annexin-II, thrombomodulin and others. Some of indicated factors, for example, nitric oxide and prostacyclin, are constantly synthesized in the endothelium, and the disturbance of their synthesis or decrease in bioavailability may indicate the presence of lesions in the endothelium. At the same time, inducible nitric oxide synthase and cyclooxygenase-2 are generated in the endothelium that leads to a significant elevation in the production of NO and prostacyclin. In normal conditions, factors such as endothelin-1, ICAM-1, VCAM-1, and E-selectin are virtually not synthesized; however, their concentrations sharply rise upon activation of the endothelium. Factors such as intracellular proteins (tissue factor, annexin-V) or endothelial membrane receptors (thrombomodulin, protein C receptor) are observed in the blood upon damage of the endothelium or apoptosis.

Since nitric oxide (NO) is involved in very different bioregulatory effects of the vascular process, a decline in its level in endothelial cells (ECs), due to various factors, leads to significant changes in their functions and the circulatory system in general. Impairments are manifested (Shiffrin E.L., 2001; Egashira K., 2002; Feletou M., 2006; Cooke J.P., 2003, Endemann D.H., 2004, Zolotova I.V. (3oToea H.B.), 2000; Markov K.M. (MapxoB X.M.), 2005) by: decreased efficacy of endothelium-dependent vasodilators and, as a consequence, enhanced vasoconstrictive effects, increased blood pressure, impaired local, regional and systemic hemodynamics, disturbance of cardiac activity, elevated production of adhesive substances in the endothelium which leads to the aggregation of platelets, and adhesion of them and leukocytes to the vascular wall, proliferation and/or migration of smooth muscle cells with the formation of neointima, synthesis of the extracellular matrix, etc.

Studies in recent years have convincingly demonstrated that the specified impairments constituting endothelial dysfunction as a whole occur in various combinations during many cardiovascular diseases (hypertension, atherosclerosis, CAD, heart failure, circulatory disorders in kidneys, lungs, brain, limbs, intestines (Belousov Yu.B. (Benoycoe BD.E.), 2000; Makolkin V.I. (MCIKOJIKUH B.H.), 2004; Borovkov N.N. (Eorobkob H.H.), 2005; Cooke J.P., 2003; Landmesser U., 2004), as well as in the presence of risk factors of these diseases (hypercholesterolemia, tobacco smoking, diabetes mellitus, insulin resistance, obesity, hypokinesia, aging of the body, hereditary burden) (Shestakova M.V. ( UlecmaKoea M.B. ), 2001; Buvaltsev V.I. (Eyeaibi/ee B.H.), 2002; Petrishchev N.N. (IJempmi ee H.H.), 2003; Vertkin A. A. (Be pm k an A.A.), 2005; Mazurov V.I. ( Ma3ypoe B.M.), 2006; Bonetti P.O., 2003; Barbato J.E., 2004; Spieker L.E., 2006; Giannotti G., 2007).

Nitric oxide is synthesized in the endothelium (by isoforms of the enzyme NO-synthase: 2 of them are constitutive - endothelial (eNOS) and neuronal and 1 is inducible (iNOS)) through the reaction of amino acid L-arginine, with the involvement of molecular oxygen. A catalyst is the enzyme NO-synthase. The reaction can be schematically depicted as follows: L-arginine + 0 ₂ ®L- citrulline + NO. Constitutive endothelial NO-synthase (eNOS) is involved in the cardiovascular system regulation. Nitric oxide is synthesized stepwise with the formation of intermediate N- hydroxy-L-arginine. Once formed, nitric oxide is released from endothelial cells and diffuses into the deeper myocytes of the vascular walls. By acting on vascular myocytes nitric oxide (NO) activates guanylate cyclase that leads to an increase in the content of 3,5-cyclic guanosine monophosphate, these processes provoke a decrease in the calcium concentration resulting in relaxation of smooth muscle cells and vasodilation [Cavelin, caveolae and endothelium cell function. / Frank P.O., Woodman S.E., Park D.S., Lisanii M.P. // ArteriosclerThrombVaseBiol 2003; 23:1161-1168., Zotova, I.V. (3oTOBa, H.B.) Synthesis of nitric oxide and the development of atherosclerosis / I.V. Zotova (3oTOBa, H.B.), D.A. Zateyshchikov (/f AGaTeiimuKOB), B.A. Sidorenko (B.A.CunopeHKo) // Cardiology. - 2000. - No. 4. - C.58-67, Luscher T.F., Endothelial control of vascular tone and growth // Clin. Exp.Hypertens - JV° 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]

Slowing down the expression and activity of endothelial NO-synthase (eNOS) is typically associated with endothelial dysfunction. Endogenous eNOS inhibitors are the first in a series of factors leading to this, for example, ADMA [Association analysis 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]

By conducting a series of studies, the present inventors have found that the composition obtained on the basis of hydrolysate from biological resources and previously described in patent RU 2526826 may be used to increase eNOS activity and reduce iNOS activity in the body of a mammal in need of prevention or treatment of endothelial dysfunction and/or an ED-related condition or disease, as well as to increase nitric oxide concentrations, decrease adhesion molecules VCAM-1 and ICAM-1 in endothelial cells of said mammal, suggesting that said composition may be effectively employed for treating a number of pathological conditions and diseases related to ED.

In a first aspect, the present invention provides a use of a composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, for the prevention or treatment of ED and/or an ED-related disease or condition in a mammal in need of such treatment or prevention. Said hydrolysate is a product of hydrolysis of biological raw materials. In one embodiment, biological resources are selected from the group including bivalve mollusks, crustaceans, annelids, and leeches. Said biological resources allow for obtaining the hydrolysate with a balanced chemical composition comprising, inter alia, a wide range of amino acids including essential amino acids, saturated and unsaturated fatty acids, melanoidins, carbohydrates, micro- and macroelements, which serves as the basis for manufacturing the composition allowing to achieve the desired therapeutic and prophylactic effect when administered parenterally to a mammal in need of such treatment or prevention.

Preferably, said biological raw materials are bivalve mollusks including swan mussel ( Anodonta ), pearl shell ( Unio ), oysters ( Ostreidae ), mytilids ( Mytilidae ), tridacna ( Tridacna ), pearl oysters ( Pinctada ), scallops (. Pectinidae ), shipworms ( Teredinidae ), goeduck (Panopea abrnpta ), Icelandic cyprine (Arctica Islandica) and other commercial types of mollusks.

By the term “hydrolysate obtained from biological resources” in the context of this specification is understood a liquid solution obtained by enzymatic and/or acidic hydrolysis of the biological resources described above. Various methods for conducting hydrolysis of biological resources are known in the art and can be selected by a person skilled in the art when implementing the present invention. In particular, patents RU 2134523, RU 2374892, RU 5042669, RU 2090084, RU 2066105, RU 2134523, RU 2319409 describe various conditions for conducting the hydrolysis of protein raw materials suitable according to the present invention.

In one embodiment, commercially available hydrolysates from mytilids including but not limiting to MIDEL («MH¾3JI»), MIGI-K («MHGΉ-K»), may be used as an initial hydrolysate for producing the composition.

The dilution factor of solution obtained immediately after aqueous hydrolysis is determined empirically by a person skilled in the art such that the amount of mineral salts, in particular sodium chloride, in the final product (composition) does not exceed the total blood salinity of a mammal to which the composition is to be administered. In one embodiment of the present invention, the amount of hydrolysate in the composition is 2 to 8 wt.%, preferably 3 to 7 wt.%, more preferably 4 to 6%, most preferably about 5 wt.%. In another embodiment, the amount of hydrolysate may be 1 to 4 wt.%, 1 to 3 wt.%, 1 to 2 wt.%, 6 to 9 wt.%, 7 to 9 wt.% and 8 to 9 wt.%. The remaining portion is water and optionally auxiliary ingredients, e.g. glucose.

The amount of hydrolysate does not typically exceed 10 wt.% of the composition, as otherwise the composition will not be suitable for certain types of parenteral administration, in particular, injectional administration, due to the presence of high salt concentration therein. However, the weight fraction of hydrolysate is typically at least 1 wt.% to provide the composition with sufficient amount of components for achieving a desired effect in treating and/or preventing ED and ED-related diseases and conditions.

At the same time, the final dilution factor of hydrolysate, as will be appreciated by a person skilled in the art, will be defined by specific conditions for conducting the hydrolysis of initial biological raw materials and the need for dilution in general, depending on the method of administration of the composition, a species of mammal and other conditions.

In another embodiment, said composition is characterized by the weight fraction of amine nitrogen of at least 0.01 wt.%. Preferably, the weight fraction of amine nitrogen is 0.01 to 0.5 wt.%. More preferably, the weight fraction of amine nitrogen is 0.05 to 0.5 wt%, most preferably 0.02 to 0.1 wt.%. The value of weight fraction of amine nitrogen characterizes the conversion rate for hydrolysis of biological resources and represents a factor which allows for assessing the completeness of this process. This characteristic serves as an indirect indicator of the presence of necessary components within the final composition for parenteral administration when using biological resources in accordance with this specification.

In another embodiment, the provided composition has pH value 4 to 7, preferably 5 to 7, most preferably 5.5 to 6.5. The pH value within the above range indicates a sufficient degree of hydrolysate neutralization, as well as the possibility of using said composition for therapeutic and/or prophylactic purposes in mammals.

In another embodiment, the provided composition has the weight fraction of solids 0.5 to 5 wt.%, preferably 1 to 3 wt.%, most preferably 1.2 to 1.8 wt.%.

By the term “disease or condition related to endothelial dysfunction” is understood the condition and disease which development or pathogenesis is related to the impairment of normal functioning of endothelial cell layer or its damage. In other words, the course of said conditions or diseases is accompanied by the impairment of normal functioning of endothelial cell layer or its damage, or by diagnosed endothelial dysfunction.

In particular, by the disease or condition in the context of this specification is understood one of diseases or conditions selected from the group including: atherosclerosis, hypertension, coronary failure, myocardial infarction, insulin resistance, diabetes mellitus, renal failure, hereditary and acquired metabolic disorders (dyslipidemia, etc.), endocrine age-related disorders, respiratory pulmonary pathologies (asthma), abdominal adhesions, major surgeries, hypothermia, bums (thermal and chemical), extensive blood loss, heart attack, stroke, sepsis, septic shock, cancers, tick-borne diseases, multiple organ failure, vascular dementia,

Alzheimer's disease,

Parkinson's disease, autoimmune diseases, senility, asthma, acute lung injury, acute respiratory distress syndrome,

Middle East respiratory syndrome, chronic obstructive pulmonary disease, viral infections, including infections caused by coronaviruses, including SARS-CoV, SARS- CoV-2, preeclampsy, eclampsy, pregnancy-induced hypertension with significant proteinuria, emphysema, asthma, asthmatic condition, bronchiectasis peripheral vascular diseases, arterial embolism and thrombosis, capillary diseases,

ED of geriatric etiology, chronic renal failure, nephrotic syndrome chronic tubulointerstitial nephritis, glomerulonephritis.

Furthermore, an ED-related condition may be any disease specified in the International Classification of Diseases (ICD 10) in sections C22 (Malignant neoplasm of liver and intrahepatic bile ducts), C26 (Malignant neoplasm of other and ill-defined digestive organs) and T22 (Thermal and chemical body burns), as well as other diseases disclosed in this specification.

It is to be understood that the present invention alternatively provides the use of the composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, for the prevention or treatment of any disease or condition from the group above with concomitant endothelial dysfunction, in a mammal in need of such treatment or prevention.

Furthermore, the present invention provides the use of the composition disclosed in this specification for the prevention or treatment of endothelial dysfunction. This embodiment includes the treatment of endothelial dysfunction, regardless of the presence or absence of any concomitant diseases or conditions specified above.

In one embodiment, the composition is administered “parenterally” which means any methods of administration, excluding entry of the provided composition into the body of a mammal in need thereof through the gastrointestinal tract. In particular, parenteral administration may be injectional, intranasal, subconjunctival administration of the composition in the form of solutions, the use of solutions in the form of gargles, rinsing, soaking treatments, wraps, as well as applying the composition onto affected surface in the form of creams, ointments, gels on a hydrophilic base.

In one specific embodiment, the composition is additionally sterilized and represents the composition for injectional administration. Said injectional administration can be carried out intravenously (i.v.), intradermally, subcutaneously (s.c.), intramuscularly (i.m.) or intraosseously (i.o.), wherein said administration can be carried out in a continuous (permanent) mode using a dropper, various drug dispensers, including osmotic pumps, pumps (similar to insulin pumps), or another suitable dispensing device.

By the term “mammal in need of such treatment or prevention” is understood any animal from the class of “mammals”, including humans, in need of treatment or prevention of ED and/or ED- related conditions and diseases. In one embodiment, the mammal may be an animal or a human. For example, the animal may be a farm animal selected from the group including a cow, buffalo, yak, deer, pig, goat, sheep, rabbit, horse, donkey, camel, lama, sable, fox, mink, ferret, etc., or the animal may be a domestic pet, e.g., such as dog, cat, rat, hamster, guinea pig, etc., or the animal may be a wild animal. In a specific embodiment, the animal is a mouse.

In one embodiment, the composition is administered to said mammal for 1-5 days at a dosage of 0.2 to 6.0 pil · k g/h In particular, this administration may be carried out by continuous parenteral administration, in particular subcutaneous, intravenous or intramuscular injection using a suitable dosing device, for example, dropper, pump (similar to insulin pump), osmotic pump. This dosage was found to be effective in treating/eliminating ED and ED-related diseases and diseases. In this respect, a skilled person in medicine or veterinary will be able to select empirically a necessary specific dosage taking into account characteristics of patient's body, disease severity and other conditions.

In another embodiment, the composition is administered to said mammal at a dosage of 10- 400 pl/h for 1-5 days. In particular, the administration may optionally be carried out by continuous parenteral administration, in particular subcutaneous, intravenous or intramuscular injection using a suitable dosing device, for example, dropper, pump, or osmotic pump.

According to one of the most preferred embodiments, the composition is administered continuously (permanently) into the body of a mammal in need thereof using an osmotic pump, pump or dropper.

In an additional embodiment, the composition is administered to said mammal for 7-10 days.

In one embodiment, the use of said composition may include concurrent administration of said composition and means of conventional combination therapy for the prevention or treatment/elimination of ED in a patient in need thereof.

In a second aspect, the present invention provides a method for the treatment/elimination of ED and/or ED-related diseases and conditions in a mammal in need of such treatment or prevention, comprising parenteral administration of the composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water, to said mammal.

It is to be understood that the present invention alternatively provides a method for the prevention or treatment of the disease or condition from the group of conditions or diseases disclosed in this specification with concomitant endothelial dysfunction, in a mammal in need of such treatment or prevention, using the composition comprising a hydrolysate obtained by acidic and/or enzymatic hydrolysis of one or more biological resources selected from the group including bivalve mollusks, annelids, leeches; and water. Furthermore, the present invention provides a method for treating endothelial dysfunction using the composition disclosed in this specification. This embodiment includes the treatment of endothelial dysfunction, regardless of the presence or absence of any of concomitant diseases or conditions related to endothelial dysfunction.

In one embodiment, the composition is administered to said mammal for 1-5 days at a dosage of 0.2 to 6.0 pl _* kg/h. In particular, this administration may be carried out by continuous parenteral administration, in particular subcutaneous, intravenous or intramuscular injection using a suitable dosing device, e.g., dropper, pump or osmotic pump.

In yet another embodiment, the composition is administered to said mammal at a dosage of 10-400 mΐ/h for 1-5 days In particular, said administration may optionally be carried out by continuous parenteral administration, in particular subcutaneous, intravenous or intramuscular injection using a suitable dosing device, e.g., dropper, pump or osmotic pump.

In an additional embodiment, the provided method of treatment comprises the use of the composition intended for injectional administration which is administered to said mammal for 7-10 days.

In still further embodiment, the provided method of treatment comprises the use of the composition intended for injectional administration and may further comprise the repeated course which is carried out in 1-2 days upon completion of the first one.

In one embodiment, the provided method of treatment comprises the use of the composition intended for injectional administration, and may comprise the concurrent use of said composition and means of conventional combination therapy for the prevention or treatment of ED and/or ED- related diseases and conditions in a patient in need thereof.

Efficiency of using the composition for the treatment and prevention of ED, as well as ED- related diseases and conditions is discussed in more detail below by specific examples of its implementation.

EXAMPLES

EXAMINING THE ACTIVITY OF COMPOSITION IN RELATION TO FACTORS OF ENDOTHELIAL DYSFUNCTION

EXAMPLE 1: Effect of the composition on eNOS activity

For the analysis of eNOS activity human umbilical cord vein endothelial cells (HUVECs) were added to 12-well plates (Sarstedt) and cultured until a confluent monolayer formed. The composition containing 5% wt. hydrolysate from mytilids and water was then added at various dilutions and incubated for 72 h at 37°C in humidified atmosphere with 5% CO2. At the end of incubation period, the DAF-FM DA dye (Invitrogen) was added at a concentration of 1 mM and incubated for 1 h. The monolayer was disintegrated and cells were fixed with 4% formaldehyde solution (Sigma). Sample measurements were conducted using Navios™ flow cytometer (Beckman Coulter).

The study results are shown in Fig. 1. In particular, the composition based on the hydrolysate from mytilids was shown to activate eNOS and increase the production of NO by almost 3 times.

EXAMPLE 2: Effect of the composition on expression of VCAM-1 and ICAM-1

To assess the expression of surface molecules VCAM-1 (CD 106) and ICAM-1 (CD54), HUVECs were added to 24-well plates (Sarstedt) at a concentration of 150,000 cells per ml. The composition containing 5% wt. hydrolysate from mytilids and water was then added for 72 hours at different dilutions. TNFa was added to wells at a concentration of 25 U/ml 24 hours before the end of incubation. The expression of surface molecules was assessed by flow cytometry using anti- CD106PE (cat. #PN A66085) and anti-CD54PE (cat. #PN IM1239U) monoclonal antibodies. Cell suspensions were stained according to manufacturer's recommendation. To assess necrosis level cell suspension was additionally stained with the DNA-binding dye DAPI (Invitrogen) at a concentration of 1 mg/ml. Samples were analyzed using Navios™ flow cytometer (Beckman Coulter).

The study results are shown in Fig. 2. The obtained results indicate that the composition based on the hydrolysate from mytilids reduces the expression of VCAM-1 on HUVECs after induction with TNF-alphaby 3.6 times.

EXAMPLE 3: Effect of the composition on iNOS activity.

To analyze the effect of the preparation on iNOS activity, mouse peritoneal lavage cell culture was used. CBA/BALB (FI) mice from the “Rappolovo” nursery were kept under conditions of unlimited access to food and water in accordance with accepted ethical standards. Peritoneal lavage cells were obtained by washing the abdominal cavity of mice with sterile Hanks solution. Cells were added to 96-well plate (Eppendorf, RF) in the amount of 300,000 in 100 pi of RPMI 1640 medium (Biolot, RF) with 10% fetal calf serum (Flow laboratories, USA) supplemented with 2 mM glutamine (Biolot, RF), 50 pg/ml gentamicin (Biolot, RF), and incubated for 24 hours at temperature of 37°C, 5% CO2.

After the culture medium was replaced to stimulate NO production, LPS (Esherichia coli 055 :B5, Sigma-Aldrich, Germany) at a concentration of 1 pg/ml and the composition containing 5% wt. hydrolysate from mytilids and water were added to wells. After incubation for 24 hours at 37°C and 5% CO2 cells were sedimented by centrifugation for 5 min at 200g. Then 70 mΐ of supernatant was collected, transferred to 96-well plate (Eppendorf, RF), and 70 mΐ of Griss reagent was added. Spectrometric analysis was conducted at a wavelength of 540 nm (Microplate reader, Model 680, Bio-Rad). Nitrite and nitrate concentrations in experimental samples were determined mathematically in accordance with linear fitting by the method of least squares based on the calibration curve constructed using sodium nitrite (NaNCh) solution with known concentration.

Data analysis and processing were performed using STATISTICA 5.0 software using Student's t-test.

The study results are shown in Fig. 3. Incubation of cells with the preparation led to a significant decrease in the level of spontaneous ICAM-1 expression. The preparation based on the composition with dilution of 1:3 (v/v) significantly decreased spontaneous and LPS-induced levels of ICAM-1 expression.