New therapeutic agents for local administration based on sulphated hyaluronic acid as cytokine activity stimulators or inhibitors

FIELD: medicine, pharmaceutics.

SUBSTANCE: invention refers to pharmaceutical industry and represents using sulphated hyaluronic acid for preparing a therapeutic agent for local administration for treating inflammatory/irritating skin diseases specified in dermatitis, atopic dermatitis, photocontact dermatitis, rash, vitiligo, eczema, psoriasis, all skin irritations related to activation of anti-inflammatory cytokines, such as IL-1, IL-2, IL-7, IL-8, IL-9 and TNF, wherein hyaluronic acid has a molecular weight falling within the ranges of 10000 D to 50000 D, 150000 D to 250000 D and 500000 D to 750000 D, and a sulphatation degree equal to 1.

EFFECT: invention provides stimulating the immune system protein synthesis for eliciting the immune response.

8 cl, 33 ex, 15 dwg, 3 tbl

 

The technical field to which the invention relates

For many years in the scientific/patent literature, published research data sulfated hyaluronic acid, which is derived from hyaluronic acid (HA), as appropriate sulfated in accordance with the method described in the prior art (see patent EP0940410B1 and EP0702699B1), which is credited with anticoagulant effects. HAS (sulfated hyaluronic acid) can also be obtained by deacetylation and subsequent sulfation of glucosamine HA (defined as HA-NS) (EP0971961B1) to obtain surgical products and pharmaceutical compositions. Also known patents EP0754460B1 and EP1385492B1, which describes the application HAS such disorders as, for example, ARDS (severe respiratory failure, respiratory distress syndrome of adults), articular rheumatism and rheumatoid arthritis. The present invention relates to a new and unexpected skin application HAS as agent for regulating cytokine activity, because the applicant has found an exceptional HAS the ability to modulate the activity of specific cytokines (and Pro-and anti-inflammatory), was investigated its mechanism of action and revealed a significant difference between the two types of sulfated product HAS and HA-NS), but moreover, the applicant of neojidan is found unexpectedly high activity against various types and strains of the herpes virus, cytomegalovirus, and vesicular stomatitis virus.

Finally, another object of the present invention relates to the application HAS as a stimulator of absorption into the skin anti-inflammatory medicines and hormonal nature.

Since 1970, scientists realized that the selected population of lymphoid cells can produce and release into the circulatory direction of the molecules of a protein nature, not identified with antibodies defined by the term "cytokines". They represent a new type of "hormone" that can act on different target cells in many areas of the body.

The progress of scientific knowledge related to the synthesis and biological/biochemical functions of these proteins, changed the "old" understanding of the immune system (I. S.) in academic circles and has opened new horizons in the understanding of its many functions, thus creating new perspectives for the treatment of various pathologies, local and/or system, including new therapeutic opportunities associated with the immunotherapy of cancer.

The Central cells of the immune system are lymphocytes, which constitute about 20% of all white cells and, on the basis of their different functions, form 3 groups: B-lymphocytes, T-lymphocytes and lymphocyte-killers. Many cytokines are RA is soluble proteins, produced by lymphocytes and/or monocytes, capable of acting on other cells/tissues, also located very far from the site of their products. They really have immunological functions and regulatory functions in the synthesis of other cytokines in respect of other immune system cells or target cells involved in the cascade of reactions initiated by the immune system.

To date we have researched numerous various cytokines, which also have many different acronyms, but those cytokines, which, in particular, studied by the applicant are: interleukin 1 and 2, interleukin 6, 7 and 12, hereinafter defined in the present description as IL-1, IL-2, IL-6, IL-7 and IL-12, which with TNF (factor tumor necrosis) are defined as inflammatory cytokines nature, whereas interleukin-10 (IL-10), on the contrary, is a cytokine with strong anti-inflammatory properties.

The first cytokine, subject to investigation, was a notably IL-1: it is present in two forms α and β, is a powerful inducer of proinflammatory processes (system and/or skin). It is mainly produced by B-lymphocytes, T-lymphocytes and macrophages after bacterial stimulus or stimulation from other agents, including other cytokines; it is also secreted from the peripheral the ski neutrophils, endothelial, epithelial and smooth muscle cells, fibroblasts, Langerhans cells of the skin, osteoclasts, synoviocytes and many other types of cells. Both forms are associated with the same receptor and have very similar, if not identical, types of biological activity. Many of them Pro-inflammatory functions associated with the stimulation of other cytokines, such as IL-6 and IL-8, and their synthesis can be induced by cytokines, such as TNF, Interferon, bacterial endotoxins, viruses and various other types of antigens. He participates in the development of septic shock, but it should be noted that a recent study demonstrated that IL-1 is able to activate the expression of some oncogenes and, therefore, participate in the pathogenesis of neoplasms. In combination with other cytokines such as IL-1, therefore, represents one of the major mediators of inflammatory processes: it stimulates T-cells, in fact, for the production of IL-2 and B-cells for the production of immunoglobulins. He is also involved in the pathogenesis of rheumatoid arthritis and osteoarthritis: large quantities of IL-1 were in fact detected in the synovial fluid of patients affected with rheumatoid arthritis and/or osteoarthritis. He is also active in numerous pathologies mainly skin nature, such as dermatitis in General, atopic dermatitis is psoriasis. Finally, it participates in the formation of vascular lesions, such as venous thrombosis, and is present in all vessels in pathological conditions, arterio/arteriosclerotic type. Receptor antagonists currently are already in clinical use (and also investigated experimentally for a given cytokine, because it turns out that receptor blockade is an effective way of treatment for these pathologies, in which IL-1 refers to the protagonists.

TNF: Tumor necrosis factor is a part of a group of cytokines, which contributes to the acute phase of systemic inflammation. Therefore, TNF is involved in a very wide number of processes, such as cell proliferation, differentiation and apoptosis, carcinogenesis and viral replication.

It is mainly produced by macrophages and other cell types, including Metacity, lymphoid cells, muscle and endothelial cells, fibroblasts and nerve cells. Its synthesis can be stimulated by bacterial endotoxins, other cytokines, such as IL-2, Interferon and IL-1, and it can also be inhibited by steroids.

By action on numerous organs and systems, in General, together with other cytokines, he participates in the development and regulation of many pathogenic processes:

- it modulates the expression of many proteins and is an important cytokine is in, such as IL-1 and IL-6, thus, leading to participation in skin conditions such as vitiligo, eczema, psoriasis and dermatitis as a whole;

- it stimulates the synthesis of collagenases in synoviocyte, and for this reason large quantities of TNF have been detected in synovial fluids of patients suffering from osteoarthritis and rheumatoid arthritis;

he activates osteoclasts and therefore causes the reabsorption of bone tissue (osteoporosis);

he strongly attracts neutrophils and helps them to attach to endothelial cells for extravasation;

- it stimulates the production of macrophages molecules with the oxidizing effect;

he participates in the development of certain pathological conditions of the cardiovascular system, participating in the formation of venous thrombosis in the pathogenesis of atherosclerosis and vasculitis.

TNF is able to communicate with two receptors, TNF-R1 (TNF receptor type 1) and TNF-R2 (TNF receptor type 2), which is expressed in all somatic cells, except erythrocytes. Briefly, TNF stimulates a systemic and cutaneous inflammatory response, which in turn starts many pathological processes, which also have an autoimmune nature, such as rheumatoid arthritis, Crohn's disease, psoriasis and asthma. To date scientific studies have attempted to improve the "biological the medicines (such as, for example, monoclonal antibodies) that inhibit the synthesis of TNF and/or block the receptor.

IL-2: it has high anti-inflammatory activity, atherogenic cytokine, mainly produced by T-lymphocytes, whose synthesis is inhibited by steroids and cyclosporine. IL-2 plays a Central role in the regulation of immune response: it actually stimulates the synthesis of IFN in peripheral leukocytes and induces the production of IL-1 and TNF. IL-2 can also damage the blood-brain barrier and the integrity of the endothelium of cerebral vessels, causing neuropsychiatric disorders, such as disorientation and depression.

Consequently, there are numerous pathological conditions that have been associated with aberrant production of IL-2, such as Hodgkin's lymphoma, multiple sclerosis, rheumatoid arthritis and lupus erythematosus.

IL-6: is produced by many cell types, in addition to the immune system, he TNF is one of the most important members of the group of chemical mediators of the acute phase of the inflammatory process and therefore involved in the pathological processes with a strong inflammatory component such as asthma (where he is involved in the onset and maintenance of the inflammatory process), chronic inflammation of the intestine (Crohn's disease), rheumatoid arthritis and osteoarthritis. Indeed, as previously UTVA what was expected, cytokines, such as TNF, IL-1 and IL-6, as it turned out, largely involved in articular degenerative process of osteoarthritis, because they play a major role in regulating the expression of metalloproteinases (responsible for the breakdown of cartilage), in the production of prostaglandins and osteoclastic activation and, for this reason, high levels of cytokines were reported in synovial fluids of patients suffering from osteoarthritis and rheumatoid arthritis (R. A.). These data stimulated the use of inhibitors of the above interleukins and/or antagonists of their receptors as a new strategy for the treatment of arthritic pathology.

Finally, recent studies have linked cancer with a life expectancy and identified as tumor effect type/quantity status of cytokine proteins of the patient: in short, recent evidence has linked low profile in the production of IL-10 and high levels of secretion of IL-6 with a decrease in clinical survival of patients affected pathological tumor processes, whereas genotype, and are able to produce and maintain high levels of IL-10 may promote survival (C. Caruso et al., Ann N. Y. Acad. SCI., 2004, 1028:1-13).

IL-7: cytokine, mainly produced by stromal cells in the bone marrow, that he is also secreted by the thymus and keratinocytes. IL-7 induces the synthesis of inflammatory cytokines, such as IL-1, IL-6 and TNF, thus, participating in the pathogenesis of certain skin diseases (such as psoriasis and cutaneous lymphoma) and diseases of the osteoarticular system; indeed, high levels of IL-7 were found in patients suffering from R. A.

IL-12: this protein also plays a Central role in the regulation of immune system functions. He actually acts on the differentiation of cells, it induces the synthesis of Interferon and TNF, and its products can also be inhibited by IL-10. Excessive production of this protein is involved in the pathogenesis of autoimmune diseases nature, such as colitis, arthritis, insulin-dependent diabetes mellitus, encephalomyelitis, psoriasis and multiple sclerosis (Brahmachari, S. et al., Minerva Med., 2008, 99(2):105-118).

IL-10: mainly is produced by lymphocytes, he is an anti-inflammatory cytokine nature, can inhibit the synthesis of IL-2 and Interferon produced by T-lymphocytes. Anti-inflammatory effect of IL-10 are also found in the ability to inhibit the synthesis of IL-1, IL-6, IL-8, IL-12 and TNF in macrophages stimulated with bacterial endotoxin. Deficits IL-10 are associated with such pathological conditions as diabetes and chronic intestinal inflammation, such as Crohn's disease. Recent data has also led to judge the pilot application of IL-10 as a new therapeutic approach to the treatment of systemic lupus erythematosus. Low levels of IL-10 was observed in skin tissues of patients suffering from such pathological conditions such as vitiligo, psoriasis, eczema and dermatitis. It should be noted that corticosteroids, and cyclosporine increase the production and/or release of this interleukin affiliated competent cells during normal immunosuppressive therapy for the treatment of inflammatory conditions and rejection of organs (Zhou X. et al., Current Drug Tar-gets-Immune, Endocrine &Metabolic Disorders, 2005, 5(465-475)). Experimental data also demonstrated efficacy in reducing the release of prostaglandins and cyclooxygenase induced byin vitroTNF, human synoviocyte, thus indicating the ability of IL-10 to reduce the inflammatory processes that involve the joints affected osteoarthritis degeneration (Alaaeddine n, et al., Arthritis & Rheumatism, 1999, 42:710-718). Recent studies have confirmed its therapeutic efficacy in asthma pathology in models of bronchial hyperresponsiveness in experimental animals, indicating that this cytokine has a high therapeutic promise in reducing inflammation that characterizes the Airways with asthma patients, in which high concentrations of TNF, IL-1, IL-5, IL-6 and IL-8 were detected in bronchial wash W is dcosta, and/or serum and/or tissues (Stankiewicz W. et al., Mediators of Inflammation, 2002, 11:307-312). Therefore, for a given interleukin assumed the important role of the regulatory cytokine in maintaining immunological homeostasis.

Asthma can be extremely debilitating disease that affects approximately 200 million people worldwide with a mortality of more than 5,000 cases annually. This pathology, which is based on a perverted immune response to environmental factors, in subsequent associated with increased production of proinflammatory cytokines for growth and differentiation of mast cells and eosinophils with other types of cells of the immune system. The reasons for this unbalanced activity of the immune system is not yet fully known; however, there are genetic, environmental, viral, and nutrition-related factors in different ways contribute to the development of this pathology. Therefore, effective therapy (systemic and/or local therapy) for the prevention and/or treatment, which would provide the ability to stop or reduce the use of steroids (usual medical care), could represent an informed decision about the more severe forms (as it is in any case would provide an opportunity to reduce the use of steroids), and about less severe cases, PQS is LCU could completely discontinue therapy with steroids.

DETAILED description of the INVENTION

The aim of the present invention is a new and unexpected local application HAS as a means of regulating the activity of cytokines, because the applicant has discovered her exceptional ability to modulate the activity of specific cytokines was investigated its mechanism of action and revealed a significant difference between the different types of sulfated products, is famous in this area, but, first of all, the applicant has discovered unexpected activity against various types and strains of the herpes virus, cytomegalovirus, and vesicular stomatitis virus. Finally, another objective of the present invention relates to the application HAS as a stimulator of absorption through the skin medications, mainly anti-inflammatory nature, as fibrinolytic funds, as well as wysokometanowego means to treat all pathological conditions of the skin characterized by dryness, irritation and redness, inflammation and peeling.

Sulfated hyaluronic acid suitable for the purposes of the present invention are in accordance with the method described in patent EP 702699 B1: sulfation is carried out via a set of SO3the pyridine and uses alcoholic hydroxyl present in polysacharide the th chain, on the basis of HA, originating from any source, for example, obtained by extraction from cocks ' combs with or enzymatic, or biotechnological methods and having a molecular weight in the range from 400 to 3×106Yes, in particular from 1×104Yes to 1×106Yes, even more specifically from 10000 to 50000 Yes, from 150000 to 250000 and 500000 to 750000 Yes.

Derived retains all of the physical characteristics of the original unmodified polymer, in particular molecular weight HA source is not reduced by sulfate crystallization, thus providing the ability to save all physico-chemical characteristics of the original polysaccharide. Sulfation affects different hydroxyl group disaccharide glycosides element, and therefore obtain different degrees sulfate crystallization, from 0.5 to 3.5 (referring to the number of sulfate groups per disaccharide glycosides element), by varying the quantity entered, SO3-pyridine, as is known in modern technology.

Derivative used in all performed experiments, in General, sulfate crystallization degree 1 or degree 3 and is further defined in the present description as HAS1 and HAS3. All free carboxyl groups of HA can form a salt with cations of organic and/or inorganic origin.

Both species HAS soluble in water, and that they can the same be sterilized by usual methods, well-known experts in this field, even if the preferred sterilization using autoclave.

The applicant describes and claims a new application HAS to obtain medicines for local use:

• for the prevention and/or treatment of pathological skin conditions associated with immune deficiency and, in particular, deficiency of IL-10, such as vitiligo, eczema, psoriasis and dermatitis in General, by stimulating the synthesis of anti-inflammatory cytokines;

• for the prevention and/or local treatment of asthma, is associated with activation of IL-1, IL-6 and TNF, by inhalation;

• for the prevention and/or treatment of pathological conditions of the skin, associated with damage to the endothelium and/or the walls of the blood vessels due, for example, trauma, vascular hemorrhage superficial nature and/or the average depth to the subsequent formation of blood clots and swelling,

• for the prevention and/or treatment applied to the skin skin related diseases increase/activation of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12 and TNF, such as, for example, dermatitis, atopic dermatitis, psoriasis, vitiligo, photodermatol, hives, all types of skin irritation (and gums) and eczema;

• for the prevention and/or treatment of diseases of the autoimmune nature, such as psoriasis, asthma and cutaneous manifestations of systemic (LES) and discoid in red is lanky;

• for the prevention and/or local treatment of skin tumors, such as basal cell carcinoma, Kaposi's sarcoma, squamous cell carcinoma, cutaneous lymphoma, fungoides mycosis and actin keratosis;

• for the prevention and/or local treatment of vascular pathological conditions, such as vasculitis and scleroderma associated with activation of TNF, IL-1 and IL-6.

The applicant also actually demonstrated in the following experiments, that:

• HAS the capacity to stimulate the production of new mRNA and protein synthesis of anti-inflammatory cytokines nature (such as, for example, IL-10), thus increasing the ability of immune cells and, consequently, of the whole organism. Anti-inflammatory effect of the above cytokines detected by the ability to inhibit the synthesis of IL-1, IL-6, IL-8, IL-12 and TNF, all of which are proteins with high Pro-inflammatory activity involved in numerous pathological conditions of the skin.

• HAS effective in reducing the synthesis of new mRNA and with a significant reduction of protein synthesis, IL-2, IL-7 and IL-12, in situations where not called the immune response, and in specific cases of inflammatory stress, in which cells respond by producing a cascade of cytokines: in particular, in this case, the data presented reveal what HAS a greater effect.

• HAS effective at inhibiting the binding of TNF, IL-1 and IL-6 with their receptors. These data are of fundamental importance, because they prove that the behavior of sulfated product is completely analogous to the behavior of monoclonal antibodies specific receptor above proinflammatory proteins, therefore, is able to block their function, but at the same time with the specificity of this antibody. Blockade of these receptors is the most effective way of countering Pro-inflammatory and neoplastic effects of factor TNF, IL-1 and IL-6, thus opening new horizons for clinical experimentation, providing the opportunity to develop new therapeutic approaches for the treatment and/or prevention of a very large number of pathological conditions, including the role played by TNF, IL-1 and IL-6 in the onset and progression of numerous system and skin diseases.

The applicant also describes and claims a new application HAS to obtain medicines for local use:

• for the prevention and/or treatment of infections of the lips caused by the herpes simplex virus and genital herpes;

• for the prevention and/or treatment of infections caused by vesicular stomatitis virus;

• for the prevention and/or treatment details the functions, caused by cytomegalovirus.

The applicant is demonstrated in the following experiments powerful antiviral action HAS against different virus types:

• Experimental data prove the antiviral effect HAS1 and HAS3 against herpes simplex virus 1 and 2 and against vesicular stomatitis virus (VSV). The first form, is extremely widespread, responsible for the appearance of characteristic accompanied by a febrile reaction of the bubbles, which usually affect the skin of the face (lips, nostrils); it is also calledherpes simplex labialis. Infection caused Hubnik herpes can recur because the virus survives inside the cells and persists even when using effective medicines. The second form is a genital infection, also known asherpesgenitalis. Both forms are transmitted through physical or sexual contact. Due to the localization of virions in the nerve ganglia, where they can remain latent for a long period of time, herpes infection has the characteristics of recurrence in accordance with stresseraser the phenomena that affect the immune system, and usually recurs in the primary plot. Vesicular stomatitis virus is an RNA virus, it is p which expresses mammals and is used in the laboratory for the study of the development lifecycle RNA virus. Comparison of HA-NS1 and HAS1 again shows that not all of the sulfated hyaluronic acid equivalent, because HA-NS1 were inactive, whereas HAS1, and 3 show a very strong antiviral activity against herpes simplex virus, as well as against VSV. None of the tested samples were not cytotoxic against host cell; the resulting minimum cytotoxic concentration was actually equal to the minimum cytotoxic concentration reference drugs commonly used in clinical practice for the treatment of infection by a virus of herpes, and on average it was 100 times higher than the minimum cytotoxic concentration identified as active in the inhibition of viral replication.

• Experimental data obtained for HAS1 and HAS3 revealed a clear and significant antiviral effect in relation to Cytomegalovirus: this specific type of virus that comes in some types of cells, where it reproduces itself parasitically, causing cell death. This virus belongs to the same family, and thatherpes labialisandherpes genitalisthe varicella zoster virus and the virus infectious mononucleosis. Epithelial cells, mucous membranes, lymph nodes are the sites multiple primary infection. The virus is stetsa in latent form for life in the peripheral blood, in the epithelium of the renal tubules and in the epithelium of the salivary glands. Severe forms are found in individuals with impaired immunity (such as individuals infected AIDS, and individuals after organ transplantation, receiving immunosuppressive therapy). Medical therapy consists in the introduction of drugs, such as ganciclovir, valganciclovir and foscarnet (inhibitors of viral DNA synthesis). Also in this case, HA-NS1 was ineffective in the inhibition of proliferation of the virus, confirming the absolute difference in antiviral activity between the two types of sulfated products.

Another objective of the present invention relates to the application HAS as fibrinolytic means for the destruction of fibrin clots that are formed at the level of the skin (surface and/or depth) after the destruction of the endothelium and/or the walls of the capillaries and/or small vessels, due to mechanical injury and/or bleeding moderate/low intensity.

In the following experiments, the applicant really showed:

• what HAS effective as when plasmin fibrinolysis/removing fragments of blood clots and blood clots. The plasmin is an important enzyme belonging to the group of hydrolases that can destroy many blood plasma proteins, and in particular, the fibrin in blood clots and shut the Ah blood. The destruction of fibrin is called fibrinolysis. Failure plasmin can lead to thrombosis, as thrombi are not adequately destroyed. It should be noted a significant difference between the process of anticoagulation and fibrinolytic process: in the first case, an anticoagulant drug is supposed to prevent blood clot formation, and in the latter case, a fibrinolytic agent, on the other hand, should intervene in the situation in which a blood clot is already present and therefore must be destroyed to remove it.

Another objective of the present invention relates to a new use HAS as a stimulator of absorption through the skin medications, such as anti-inflammatory medicines of nature, and, finally, as a means of having a strong dehydrating effect, for the treatment of pathological conditions of the skin characterized by dry, lichenizaciei, irritation, itching and redness of the skin, inflammation and desquamation.

The applicant in fact has demonstrated that:

• sulfation of hyaluronic acid significantly increases absorption through the skin, therefore,

• hydrating power HAS turned out to be much higher than that desulfuromonas HA, and therefore HAS important causes reduction of roughness on Botanik skin surfaces relative to the HA and local control preparative forms thus, identifying its ability to effectively treat and protect the skin, characterized by dryness, irritation, lehenetsia, itching and redness, inflammation and desquamation in all other pathological conditions of the skin that make your skin more sensitive to external tools;

• HAS is a powerful and effective stimulator of skin absorption of drugs. The ability HAS so effectively penetrate through the thickness of the skin is the scientific basis on which to base this unexpected new property, which provides the possibility of its inclusion in the preparative forms with pharmacological means of different nature, such as, for example, nonsteroidal anti-inflammatory drugs (in particular, diclofenac, Ketoprofen and ibuprofen) or the steroid type, hormones, vasodilator, cholinergic remedies, antibiotics and other medicines are prepared in various forms, preferably in the form of gels, creams or patches for the dermal and/or transdermal absorption.

Finally, the applicant describes the various local pharmaceutical preparative forms/compositions containing HAS in view only the beginning of the current, or in Association with other pharmacologically and/or biological the key active means, such as, for example, steroids, hormones, proteins, trophic factors, vitamins, nonsteroidal anti-inflammatory drugs (FANS), such as, for example, diclofenac, Ketoprofen or ibuprofen or their salts, chemotherapeutic drugs for local use, antibiotics, antivirals, local anesthetics, anticoagulants and/or fibrinolytic funds and/or enzymes, such as collagenase and/or hyaluronidase and/or other proteases; it can be prepared in the preparative form with polymers, such as hyaluronic acid and its derivatives, carboxymethylcellulose (CMC) and/or other natural polymers (such as collagen) or synthetic nature.

Consider the pharmaceutical composition may be in the form of ointments, Limoges, hydrogel, lipstick, creams, vaginal suppositories and pessaries, foams, gel mucous membranes, ophthalmic preparations, compositions for vaginal disinfection, rinse the oral cavity, patches for dermal and/or transdermal absorption, in particular FANS and hormones, solutions; therefore, it may be a local application or by inhalation for the treatment of pathological conditions of the respiratory system, such as, for example, asthma.

Special attention is given to compositions containing the m enzymes, such as hyaluronidase, in preparative form of a medicinal product for the treatment of skin bruising, and products containing non-steroidal anti-inflammatory drugs or hormones, in the form of gels, creams and pads for the dermal and/or transdermal absorption of the drug.

Some examples of the preparation HAS 1 and 3 degrees, the pharmaceutical formulation containing it, are intended in a descriptive and non-limiting purposes, together with the results obtained by experimentationin vitro.

Example 1

Getting tetrabutylammonium salt of hyaluronic acid (HA) with an average molecular weight of 200 KD (in the range of 150,000 to 250,000 Da)

to 5.00 g of sodium salt of hyaluronic acid enzymatic origin (200 KD) is dissolved in 250 ml of water and the resulting solution was passed through a glass column, pre-filled 100 cm3resin Dowex in the form of tetrabutylammonium (TBA). Suirvey salt solution HA-TBA collect and sublimate. Get 7.50 g of product.

Example 2

Synthesis of sulfated HA, with an average molecular weight of 200 KD and a degree of sulfate crystallization, equal to 3 sulfate groups on the repeating link

The way A

10.0 g TBA salt of hyaluronic acid having an average molecular weight of 200 KD, obtained according to the accordance with example 1, dissolved in 300 ml of dimethyl sulfoxide (DMSO); 26,0 g at SO3-pyridine (sulfur trioxide and pyridine, hereinafter abbreviated as PySO3) was dispersed in 150 ml of DMSO and then added to a solution of HA. After 20 hours under mechanical stirring at a temperature of 21°C the reaction is interrupted by addition of 0.1 volume of water; the crude reaction product produce precipitation after adding 2 volumes of ethanol. The obtained solid was dispersed in 150 ml of water and the pH adjusted to neutrality 1M NaOH. The mixture is completely deleteroute against water through a membrane with a cutoff 12-14000 Yes. Validirovannyj product is subjected to sublimation. Obtain 9.7 g of product with sulfate crystallization degree equal to 3 sulfate groups on the repeating link (yield = 88%).

Method B

32,0 g TBA salt of hyaluronic acid having an average molecular weight of 200 KD, obtained in accordance with example 1, is dissolved in 900 ml of N-methylpyrrolidone (NMP); 100 g PySO3was dispersed in 600 ml of NMP and then added to a solution of HA. After 20 hours under mechanical stirring at a temperature of 21±1°C the reaction is interrupted by the addition of 0.5 volumes of water. pH, which source was lower than 2.5, adjusted to neutrality by the addition mole of NaOH (solution). The crude reaction product produce precipitation by addition of 2.5 volumes of methanol and washed with 2 volumes of methanol/the ode 8/2. The solid is re-dissolved and fully deleteroute against water using a membrane with a cutoff 12-14000 Yes. Get a 30.4 g of the product with the degree of sulfate crystallization, equal to 3 sulfate groups on the repeating link (yield = 86%).

Example 3

Synthesis of sulfated HA of HA having an average molecular weight of 200 KD and a degree of sulfate crystallization, equal to 1 sulfate group on the duplicate link

Using the procedure illustrated in example 1, to obtain 10.0 g TBA salt of HA, which is dissolved in 350 ml of DMSO. 10.0 g of the complex PySO3was dispersed in 100 ml of DMSO and then added to a solution of HA. After 20 hours under mechanical stirring at a temperature of 21°C the reaction is interrupted by addition of 0.1 volume of water; the crude reaction product produce precipitation after adding 2.5 volumes of ethanol. The obtained solid was dispersed in 150 ml of water and the pH adjusted to neutrality with NaOH 1 mol/L. the Mixture is completely deleteroute against water through a membrane with a cutoff 12-14000 Yes. Validirovannyj product is subjected to sublimation. Receive rate of 7.54 g of the product with the degree of sulfate crystallization 1.0 sulfate group on the duplicate link (yield = 93%).

Example 4

Synthesis of sulfated HA of HA having a low molecular weight (average MW 10 KD in the range from 5000 to 30000 Da) and sulfate crystallization degree equal to 3 sulfate groups on overawes link

Using the procedure illustrated in example 1, to obtain 12.4 g TBA salt of hyaluronic acid with low molecular weight, which is dissolved in 300 ml of NMP. 40 g PySO3was dispersed in 100 ml of NMP and then added to a solution of HA. After 20 hours under mechanical stirring at a temperature of 21°C the reaction is interrupted by the addition of 0.5 volume of water. pH, which source was lower than 2.5, adjusted to neutrality by the addition mole of 4M NaOH. The crude reaction product produce precipitation after adding 2.5 volumes of methanol and washed with 2 volumes of methanol/water 8/2. The solid is re-dissolved and fully deleteroute against water using a membrane with a cutoff of 3500 Da. Obtain 12.0 g of product with a degree of sulfate crystallization, is 3.0 sulfate groups on the repeating link (yield = 85%).

Example 5

Synthesis of sulfated HA of HA having a low molecular weight and degree of sulfate crystallization, equal to 1 sulfate group on the duplicate link

Using the procedure illustrated in example 1, to obtain 12.4 g TBA salt of HA and dissolved in 300 ml DMSO. 16.0 g PySO3was dispersed in 100 ml of DMSO and then added to a solution of HA. After 20 hours under mechanical stirring at a temperature of 21°C the reaction is interrupted by addition of 0.1 volume of water; the crude reaction product produce precipitation after addition of 2.5 on the of yemov ethanol. The obtained solid was dispersed in 150 ml of water and the pH adjusted to neutrality with NaOH 1 mol/L. the Mixture is completely deleteroute against water through a membrane with a cutoff of 3500 Da. Validirovannyj product is subjected to sublimation. Get 9,04 g of the product with the degree of sulfate crystallization 1.0 sulfate group on the duplicate link (yield = 90%).

Example 6

Synthesis of sulfated HA of HA having a molecular weight within the range from 500 to 730 and sulfate crystallization degree equal to 3 sulfate groups on the repeating link

21,0 g of sodium salt of hyaluronic acid extractive origin (500-730 KD) is dissolved in 1.5 liters of water and the resulting solution was passed through a glass column, pre-filled 450 cm3resin Dowex in the form of TBA. Suirvey salt solution HA-TBA collect and sublimate. Get 32,0 g of product which was dissolved in 1.35 l NMP; 100 g PySO3dispersed in 650 ml of NMP and then added to a solution of HA. After 20 hours under mechanical stirring at a temperature of 23±1°C the reaction is interrupted by the addition of 0.5 volume of water. pH, which originally was lower than 2.5, adjusted to neutral by addition of NaOH (in solution at a concentration of 4 mol/l). The crude reaction product produce precipitation by addition of 2.5 volumes of methanol and washed with 3.5 volumes of methanol/water 8/2. Solid westwoodone dissolve and completely deleteroute against water, using the membrane, when the cutoff 12-14000 Yes. Get to 30.3 g of the product with the degree of sulfate crystallization, equal to 3 sulfate groups on the repeating link (yield = 83%).

Example 7

Synthesis of sulfated HA of HA having a molecular weight of from 500 to 730 and the degree of sulfate crystallization, equal to 1 sulfate group on the duplicate link

21,0 g of sodium salt of hyaluronic acid extractive origin (500-730 KD) is dissolved in 1.5 liters of water and the resulting solution was passed through a glass column, pre-filled 450 cm3resin Dowex in the form of TBA. Suirvey salt solution HA-TBA collect and sublimate. Get 32,0 g of product which was dissolved in 1.65 l NMP; 40 g PySO3was dispersed in 350 ml of NMP and then added to a solution of HA. After 20 hours under mechanical stirring at a temperature of 25±1°C the reaction is interrupted by the addition of 0.5 volume of water. pH, which originally was lower than 2.5, adjusted to neutrality by addition of NaOH (in solution at a concentration of 4 mol/l). The crude reaction product produce precipitation with addition of 3.5 volumes of methanol and washed with 3.5 volumes of methanol/water 8/2. The solid is re-dissolved and fully deleteroute against water using a membrane with a cutoff 12-14000 Yes. Get to 22.5 g of the product with the degree of sulfate crystallization 1.0 sulfate group on the duplicate link (yield =87%).

Example 8

Evaluation of regulatory effect HAS with the degree of sulfate crystallization 1 and 3 gene expression of IL-10 and IL-12

Human synoviocyte previously copiedin vitroand maintained in culture at 37°C in DMEM containing 10% FCS (fetal calf serum), were seeded at a concentration of 20,000 cells per well (synoviocyte are cells that can produce different types of cytokines, and therefore are usually used for this type of experimental testing). Then sulfated HA degree 1 (HAS1) and grade 3 (HAS3), obtained as described in examples 1-3, added to culture medium at a concentration of 0.1 and 0.5 mg/ml (for both samples), while the control treatment presents desulfuromonas HA having an average molecular weight (MW) 200 KD. After 3 days of processing carried out PCR (polymerase chain reaction) in real time to assess gene expression of IL-10 and IL-12: cellular RNA extracted using the method "Trizol" and following the instructions of the supplier (TRIZOL reagent, LIFE Techonologies, GIBCO BRL). Briefly, cells are lysed by adding 1.0 ml Trizol and total RNA content was quantitatively determined by measuring its absorbance at 260 nm. Appropriate primers chosen for each gene that is subjected to amplification using the software Primer3 Roche Molecular Diagnostics, Pleasanton, CA, USA). Gene expression assessed by PCR in real time with the help of the device Rotor-gene TM5500 (Corbett research, Sydney, Australia). The PCR reactions performed using primers at 300 nm and dye SYBR Green (Invitroge, Carlsbad, CA, USA) with 40 cycles of 15 s at 95°C and 1 min at 60°C. the Value of "Threshold fluorescence (Ct) is automatically determined by the software, estimating the coefficient of amplification for the studied gene from 92 to 110%. For each cDNA sample, the magnitude of gene expression is expressed from the point of view of the correlation between the ct of the gene of the 'household' (i.e., the gene for the protein beta-actin, which is a control gene, because it is present in every cell and is not affected by the HAS) and ct gene of interest (i.e., the gene for IL-10 and IL-12), therefore, the ratio ct "household"/ct test gene is indicated on the ordinate axis, which therefore indicates the amount of mRNA expressed the analyzed genome. The results obtained are shown in Fig.1 and 2:

Fig.1: processing of human synoviocytes HAS1 and HAS3 caused a significant increase in gene expression of cytokine IL-10 compared with control-treated desulfuromonas HA.

Fig.2: in this experiment, both the degree of sulfate crystallization HAS (degree 1 and degree 3) was also able to significantly reduce gene ek the processes of IL-12, half reducing the synthesis of its mRNA compared to control treated desulfuromonas HA. So it turned out that sulfated hyaluronic acid:

• capable of stimulating the production of new mRNA for the synthesis of anti-inflammatory cytokines, thus increasing the protective ability of the cell and, consequently, of the whole organism, in comparison with the previously described pathological conditions in which IL-10, as it turns out, is fundamental to the resolution and/or improvement in such diseases as asthma, vitiligo and all inflammatory processes that involve IL-10;

• effective in reducing the synthesis of new mRNA is highly proinflammatory cytokine IL-12, being an effective anti-inflammatory agent, which is able to interfere with the expression of proteins involved in the pathogenesis of debilitating diseases, such as psoriasis and all of the above diseases.

Example 9

Inhibition of TNF binding to its receptor, expressed in lines of monocytes: evaluation of the effectiveness HAS degree 1 and degree 3 with different values of molecular weight

These experiments were conducted to evaluate the effectiveness of the tested samples (obtained in accordance with examples 1-4) in terms of their impact on the ability of inhibition swazilan what I TNF to its receptor, expressed by cells of the immune system, usually usedin vitrofor this type of experiment conducted with iodirovannoye cytokine components for evaluation in the analysis of the binding of the radio.

The experimental procedure was performed as described in the publication Baglioni C. et al., J Biol Chem 1985, 260:13395-13397.

In short, used the line of human histiocytes lymphoma U937, with the characteristics of monocytes, which are sensitive to the cytotoxic activity of TNF expressing associated receptor. Cells were initially incubated with 0,028 nm125I-TNF (conducted in water) in conjunction with the underlying analysis of samples (at a concentration of 1 mg/ml, which was the lowest concentration that causes the maximum inhibition) in incubation buffer consisting of 50 mm Tris-HCl pH to 7.4, 0.5 mm EDTA, at 4°C for 3 hours.

At the end of incubation, the cells were centrifuged with dibutyl phthalate/dinonylphenol 2/1 and the precipitate obtained after centrifugation were analyzed γ-counter.

The results obtained are shown in Fig.3.

The obtained results show the efficiency HAS full (100%) inhibition of binding of TNF to its receptor and to the degree 1 and degree 3 HAS medium and low molecular weight. These results are of fundamental importance, because they prove that the behavior of self the target product is completely analogous to the behavior of monoclonal antibodies specific for TNF receptor, and therefore, the sulfated product is able to block its function. Therefore, blockade of this receptor is the most effective way of countering Pro-inflammatory and neoplastic effects of factor TNF.

Example 10

Inhibition of binding of the cytokine IL-1 to its receptor, expressed in lines of fibroblasts: evaluation of the effectiveness HAS degree 3 with different values of molecular weight

These experiments were conducted to evaluate the effectiveness of the tested samples (obtained in accordance with examples 1-3 and 4) in terms of their impact on the ability of inhibiting the binding of IL-1 to its receptor expressed murine 3T3 cells, commonly usedin vitrofor this type of experiment conducted with iodirovannoye cytokine components for evaluation in the analysis of the binding of the radio.

The experimental procedure was performed as described in the publication Chin J, et al., J Exp Med, 1987, 165:70-86.

Briefly, used a line of mouse 3T3 fibroblasts, which are sensitive to the cytotoxic activity of IL-1, expressing the associated receptor. Cells were initially incubated with 10 PM125I-IL-1 (conducted in water) in conjunction with the underlying analysis of samples (at a concentration of 1 mg/ml, which was the lowest concentration, the binding maximum inhibition), in the incubation buffer consisting of medium RPMI 1640 containing 20 mm HEPES pH 7.2 and 1% BSA (bovine serum albumin) at 37°C for 2 hours. At the end of incubation, cells were washed in phosphate buffer, then was dissolved in 2.5 M NaOH and the pulses counted in a γ-counter.

The results obtained are shown in Fig.4.

The obtained results show the efficiency HAS (and medium, and low molecular weight) in the inhibition of binding of IL-1 to its receptor by 30%. These results are extremely important because they prove that the behavior of sulfated product is completely analogous to the behavior of a monoclonal antibody specific for the receptor in question cytokine, and therefore, the sulfated product is able to block its function. Blockade of this receptor is the most effective way of countering Pro-inflammatory and tumor effects of IL-1, as described previously.

Example 11

Inhibition of binding of the cytokine IL-6 to its receptor, expressed in myeloma cells: evaluation of effectiveness HAS degree 3 with different values of molecular weight

These experiments were conducted to evaluate the effectiveness of the tested samples (obtained in accordance with examples 1-3 and 4) in terms of their impact on the ability of inhibition swazilan what I IL-6 to its receptor, expressed in human myeloma U266 commonly usedin vitrofor this type of experiment conducted with iodirovannoye cytokine components for evaluation in the analysis of the binding of the radio.

The experimental procedure was performed as described in the publication Taga, T. et al., J Exp Med, 1987, 166:967-981.

Briefly, used a cell line of human myeloma U266, sensitive to the cytotoxic activity of IL-6 expressing associated receptor. Cells were initially incubated with 0.08 nm125I-IL-6 (conducted in water) in conjunction with the underlying analysis of samples (at a concentration of 1 mg/ml, which was the lowest concentration that causes the maximum inhibition) in incubation buffer consisting of medium RPMI 1640 containing 25 mm HEPES pH 7.1 and 10% BSA at 4°C for 16 hours. At the end of incubation, cells were washed in phosphate buffer, centrifuged at 9000 rpm and the pulses from the precipitate after centrifugation was counted in a γ-counter.

The results obtained are shown in Fig.5.

The results show the effectiveness and HAS medium, and low molecular weight when full (100%) inhibition of binding of IL-6 to its receptor. These results, therefore, show that the behavior of sulfated product in this case is completely analogous to the behavior of mono is analnogo antibodies specific for the receptor in question cytokine, and therefore, the sulfated product is able to block its function. Blockade of this receptor is the most effective way of blocking the proinflammatory effects of IL-6.

Example 12

Evaluation of inhibitory effect HAS degree 1 and degree 3 on the protein synthesis of the cytokines IL-2, IL-7, IL-10 and IL-12 in human PBMC

For these experiments used the mononuclear cells of peripheral blood (PBMC), obtained from different donors, to assess the impact HAS on the products listed above cytokines using:

- desulfuromonas HA (average molecular weight: 200 KD),

- HAS1 and HAS3 (obtained as described in examples 1-3).

The allocation of PBMC (Bøyum, A., Scand J Clin Lab Invest 21 Suppl, 1968, 97:77-89) was performed using the product Ficoll-Paque PLUS (GE Healthcare) according to the Protocol specified by the supplier. In zero day, 100,000 cells were sown per well (using tablets with 96 holes) in 200 μl of RPMI medium 1640, to which was added 10% fetal calf serum, 10 mm HEPES, 2 mm glutamine, 1% penicillin-streptomycin 100 U/ml of the Impact of all samples was assessed in untreated PBMC or PBMC stimulated by lipopolysaccharide LPS (10 μg/ml) (highly Pro-inflammatory) or phytohemagglutinin PHA (10 mg/ml) (a substance capable of stimulating the lymphocyte is for their division), both agent capable of stimulating the synthesis of cytokines. Cells were processed separately by the three compounds at a concentration of 0.1 mg/ml or 1 mg/ml After 24 hours incubation at 37°C (5% CO2) 100 μl of supernatant was taken from each well for analysis of IL-2, IL-7, IL-10 and IL-12.

Quantification of inflammatory mediators was carried out through technology SearchLight® using the tablet Custom Human 9-Plex Array, following the Protocol provided by the supplier in the technical card. The results obtained are shown in Fig.6-9.

These graphs clearly show that HAS degree 1 and degree 3 can significantly reduce the synthesis of IL-2, IL-7 and IL-12 in some of monocytes and when cells are not stimulated, and when, on the contrary, they stimulate specific and potent inflammatory factors and/or mitogens. So HAS is a molecule with the exact pharmacological characteristics, is able to modulate/regulate the synthesis of cytokines with a pronounced anti-inflammatory activity and in situations where the immune response is not stimulated, and under certain phenomena of the inflammatory stress, in which the immune cell responds production of a cascade of cytokines and, above all, in this case, the data reveal a greater modulatory effect HAS.

On the other hand, Fig.9 confirms PTS is a prominent stimulus for IL-10 production also for cells, related to the immune system. Therefore, again confirmed that HAS is able to modulate the synthesis of cytokines, stimulating those that are anti-inflammatory and inhibit the synthesis of proinflammatory cytokines.

Example 13

Evaluation of antiviral action HAS degree 1 and degree 3 in comparison with HA-NS:

Herpes simplex virus-1, herpes simplex virus-2, vesicular stomatitis virus

The activity of the tested samples was determined by evaluating the inhibition of cytopathogenicity caused by herpes simplex virus-1 (HSV-1 strain KOS, F and McIntyre) and herpes simplex virus-2 (HSV-2 strain G, 196 and Lyons) in fibroblasts E6SM originating from muscle/skin embryonic tissue. In addition, the antiviral activity was tested again in the cells E6SM infected with vesicular stomatitis virus (vesicular stomatitis virus: VSV). HSV-1 is a virus that mainly infects the mucous membranes of the mouth, while HSV-2 attacks the mucous membrane of the genital organs. The experimental procedure was in accordance with the description in the publication M. Baba et al., ANTIMICROB. AGENTS CHEMOTHER., 1988, 32:1742-1745.

In short, fused cell cultures were exposed to infectious doses of the viruses listed above, in the presence of samples HS-NS1 (EP0971961), HAS1 and HAS3, obtained as described in the examples 1-3. After a period of incubation for 1 h at 37°C culture medium was replaced with fresh medium containing samples to be tested. Cytopathogenic virus was tested on day 2 of incubation. Measurement of inhibition of virus cytopathogenicity was assessed by determining the inhibition of synthesis of DNA and RNA in infected cells and subjected to processing as described above: cells were sown in microwells in culture medium containing different concentrations to be tested samples with 2.5 µci 3H-thymidine and 3H-uridine in ml After 16 h at 37°C the cells were treated with trichloroacetic acid, washed in ethanol, was left for drying and scintillation counted in 7.5 ml of liquid. Antiviral activity of the tested samples was expressed as the minimum concentration required for inhibition of cytopathogenicity of the virus by 50%: IC50. In addition, to assess the cytotoxicity of the tested samples was determined the minimum concentration needed to call the morphological damage (observed by the optical microscope) used cells. The comparison was carried out with dextran-sulfate (DS) and drug acyclovir (both molecules with known antiviral efficacy, therefore, used as a positive control).

The results of t is established with representation in Fig.10.

Experimental data show potent antiviral activity and HAS1 and HAS3: comparison of HA-NS1 and HAS1 shows that not all of the sulfated hyaluronic acid are equivalent because, as it turned out, HA-NS1 was not active, and this difference in efficiency is not dependent on the molecular weight or degree of sulfate crystallization of hyaluronic acid, and this difference therefore lies in the very structure of HA-NS1 in comparison with HAS1. HAS actually exhibits performance equivalent to dextran sulfate and comparable to the effectiveness of acyclovir, the reference medicinal product for the treatment of infection with herpes simplex virus. In addition, you should indicate that acyclovir is inactive against VSV, whereas HAS degree 1 and 3 has a very powerful antiviral activity against VSV.

All tested samples is non-toxic in relation to the host cell; the resulting minimum cytotoxic concentration is in fact equal to the minimum cytotoxic concentration reference drugs commonly used in clinical practice for the treatment of herpes, and were on average 100 times higher than the concentration found active in the inhibition of viral replication.

Cytomegalovirus

The activity of the tested samples was determined by assessment and is generowania cytopathogenicity, defined by the cytomegalovirus (CMV: strain AD-169 and Davis), using the previous Protocol. Antiviral activity was tested in comparison with HEL cells (embryonic lung cells) and were expressed as the concentration required for inhibition of the number of plaques formed above the virus by 50%. The results obtained are shown in Fig.11:

the table shows a clear and significant positive result obtained for HAS1 and HAS3, which again confirms their efficacy as antiviral agents. Also in this case, HA-NS1 was not active in inhibiting the proliferation of the virus, confirming the absolute difference in antiviral activity between the two types of product with a sulfation degree 1.

Example 14

Assessmentin vitrofibrinolytic properties HAS degree 3 with different values of molecular weight

Evaluation of the fibrinolytic properties of the products tested, compared with recognized fibrinolytic activity of plasmin. In particular, they estimated the rate of dissolution of the fibrin network and the formation of soluble degradation products of fibrin (FDP).

Used plasma samples were obtained from whole blood of healthy individuals who are not receiving any pharmacological treatment.

For testing fibrinolytic efficiency the efficiency shall be tested food samples were divided into different test tubes, which caused the formation of blood clots.

Then the formation of FDP was assessed after adding:

• plasmin as a control treatment;

• HAS3 obtained in accordance with example 1 and 2;

• HAS3 obtained in accordance with example 4.

Experimental study

Fresh samples of blood were divided into different test tubes containing sodium citrate in the ratio of 9:1. The tubes were immediately centrifuged at 3000 rpm for 5 minutes. The resulting plasma was transferred into a new tube and immediately used for the evaluation FDP.

Thrombin (300 IU/ml), preheated to 37°C, was addedthe plasma samples as an activator of fibrinogen by induction of the formation of a clot.

The following materials were added in different cuvettes containing fibrin clot:

the plasmin solution of 0.5 IU 5 IU, 50 IU, 500 IU 1 IU;

- solutions HAS3 in concentrations of 25 mg/ml, 50 mg/ml, 100 mg/ml, 150 mg/ml and 200 mg/ml

The change in absorbance at 405 nm for 60 seconds was assessed spectrophotometrically for each cuvette. The reaction mixture was left to continue the interaction up until there was no complete dissolution of the clot.

Coagulated plasma, the plasmin solution and the solution HAS3 200 KD and HAS3 20 KD were mixed in the ratio 1:1 vol./about. and oderzhivali at the operating temperature of 37°C.

Table 1. Fibrinolytic activity of PLASMIN

The table shows averages mAb/min (average change in absorbance/min), registered in the containing coagulated plasma cells, which was added plasmin in various concentrations. These values indicate the rate at which dissolves fibrin clot, and therefore the speed with which produced FDP. Fibrinolytic activity of plasmin is proportional to its concentration.

Averages mAb/min, registered for each concentration of plasmin, subsequently inflicted on the chart depending on the respective units of the enzyme to establish a mathematical function that correlates the magnitude mAb/min with units of enzyme.

The PLASMIN: fibrinolytic activity (mAb/min)

1 item500 IU50 IU5 IU0.5 IU
190178126681

Table 2. Fibrinolytic activity HAS3 at different values of molecular mass

Expected values mAb/min, the dawn of istrebovanie in containing coagulated plasma cells, which added HAS3 in various concentrations. Values indicate the rate at which dissolved fibrin clot, and therefore the speed with which producirovanie FDP. Fibrinolytic activity HAS3 was proportional to its concentration. In table fibrinolytic activity HAS3 is expressed in units of equivalents of plasmin.

Fibrinolytic activity HAS3 (200 KD) (expressed in IU equivalents plasmin)

200 mg/ml150 mg/ml100 mg/ml50 mg/ml25 mg/ml
1,1 Ed50 IU10 IU3 IU0.5 IU

Fibrinolytic activity HAS3 (10 CD) (expressed in IU equivalents plasmin)

200 mg/ml150 mg/ml100 mg/ml50 mg/ml25 mg/ml
400 IU25 IU2 IU0.3 IUof 0.25 IU

The results:

the factors experiments have shown, what HAS is a powerful fibrinolytic agent with efficacy equal to plasmin. The plasmin is an important enzyme belonging to the group of hydrolases, which destroys many proteins in blood plasma and, in particular, the fibrin of blood clots and blood clots. The destruction of fibrin is called fibrinolysis. Failure plasmin can lead to thrombosis, because the clots dissolved inadequate. Although it HAS not an enzyme, its activity was equivalent enzymatic control, thus providing the possibility of using sulfated product as a new fibrinolytic means having all the advantages of non-enzymatic molecules, such as, for example, the resistance at room temperature at a much longer time periods of storage and ease of inclusion in the preparative form.

Example 15

Evaluation of permeability through the skin HAS1 and HAS3 with different values of molecular weight

Assessment of dermal absorption of diclofenac included in the preparative form, in combination with HAS compared to HA

The skin used for the experiments, were obtained from the abdomen of patients aged 30 to 50 years after surgical treatment surgical reduction of the stomach. Sections of skin full thickness were frozen after surgical interfering is listwa and kept at -20°C until the experiment, when the samples were thawed at room temperature and was carefully separated from the adipose tissue. The skin was divided into square section area of 2.5 cm2, was immersed for one minute in water at 60°C and with the help of special micromycete carefully separated the stratum corneum and the epidermis (SCE) from the underlying tissues. The obtained samples were analyzed by optical microscope and removed, if it was found punctures. SCE collected at the bottom of the Franz cell with the epidermis facing downwards, and the Horny layer in contact with the donor solution, which was located above. The area of penetration was a circular surface and was 0,636 cm2. The lower and upper part of the cell Franz carefully recorded to SCE for separating the donor compartment (amount of donor solution: 0,50 ml) and the acceptor compartment (volume receiving solution of 5.00 ml), the volumes of which have been accurately calibrated. The solvents to be used, degirolami to exclude air bubbles, which should be avoided, especially for receiving the solution, the temperature of which is maintained by a thermostat at 37°C by a thermostatic bath with circulation; in these conditions, the temperature of SCE was 31-33°C. Each experiment was performed in three iterations and the results were represented as the average in the guises in three experiments, expressed in terms of the amount of analyte, which is passed through a unit surface area of skin for 24 hours.

Penetration HA, HAS1 and HAS3 both molecular masses was carried out from solutions with 3% (wt./vol.), determining the amount of penetration of the solution through analysis, respectively glucuronic acid and spectrometry ICP (inductively coupled plasma) on the sulphur.

The penetration of the sodium salt of diclofenac was carried out on the salt in aqueous solution, the salt in the presence of HA 200 KD in the amount of 3% (wt./about.) and salt in the presence of HAS3 obtained in accordance with examples 1 and 2, in the amount of 3% (wt./vol.). In all three cases, the concentration of the sodium salt of diclofenac in the penetrating solution was equal to 1% (wt./vol.). The concentration of diclofenac was determined through analysis of HPLC in the reversed-phase apparatus Agilent 1200 Series and UV detection (254 nm) column C18, eluent acetonitrile/water/acetic acid at a flow of 1.2 ml/min

The results shown in the graph of Fig.12 and Fig.13.

In Fig.12 shows that the sulfation HA significantly increased its penetration through the skin and that this result is especially obvious for the average molecular weight.

In Fig.13, on the other hand, shows how behaves HAS as a stimulator of skin absorption of active diclofenac, doubling E. what about the total penetration number for 24 h in control and Association with HA.

Example 16

Evaluation of hydrating and protective activity obtained using HAS, in comparison with the HA and the main control cream

20 people ranging in age from 18 to 70 years who did not have skin diseases and not treated during the study, pharmacological treatment, daily on the skin at the level of the forearm caused definite and constant number of the products tested. Experimental products were:

• control: consisting of a hydrated basic cream;

• base cream (as a control) containing HA 200 KD of 0.1%;

• base cream (as a control), containing HAS3 of 0.1%, obtained according to examples 1-2.

Cream Foundation: track
DEIONIZED WATER85,10%
DERMOL 88ETHYL HEXYL ETHYLHEXANOATE6,50
NIKKOMULESE 41POLYGLYCERYL-10
PENTASTARCH BEHENYL
ALCOHOL, STEARYLAMINE SODIUM
5,00
SEPIGEL 305POLYACRYLAMIDE, C13-14
ISOPARAFFIN LAURETH 7
2,50
ISOCIDE PFPHENOXYETHANOL,
METHYLPARABEN,
ETHYLPARABEN,
PROPYLPARABEN,
PROPYLENE glycol
0,50
KEMIPURE 100IMIDAZOLIDINYL UREA0,30
DISODIUM EDTADISODIUM EDTA0,10

The way to obtain

Downloaded 90% water and disodium EDTA and Isocide PF. Was heated to 65-70°C in an appropriate container, dissolved Nikkomulese, Dermol 88 by heating to 65-70°C; connected the oily phase with an aqueous phase under the action of the turbine. Was cooled to 30-35°C, was added Kemipure 100 dissolved in the remaining 10% of water, was added Sepigel to adjust the viscosity and cooled to 25°C.

The difference of the values obtained hydration was evaluated by corneometer CM825 on average 3 points and also did profilometry analysis of the skin surface using a video camera Visioscan VC98 at time T0 (the initial value) and T7, after 7 days of using the product.

The obtained results are presented graphically in Fig.14 and 15.

Fig.14 shows more hydrating effect on the skin after treatment HAS3, in comparison with the main cream, and cream containing HA. On the other hand, in Fig.15 demonstrared the significant decrease in the roughness of the skin after 7 days of treatment in relation to controls. These data clearly demonstrate the effectiveness HAS marked improvement index of hydration of the skin, showing effective hydration and protective activity when the effect on the skin by reducing transpiration transepidermal water.

Example 17

Getting preparative form solution for inhalation containing HAS degree 1

40 mg (20 mg, if HAS has a molecular mass of 500-730 KD) sulfated hyaluronic acid grade 1, having a low or medium molecular weight, was introduced in a glass flask with a volume of 50 ml, and then added 15 ml of sterile of 0.2 M PBS (saline phosphate buffer) with a pH of 7.4. The mixture was subjected to stirring for about 30 minutes until complete dissolution of the powder. When the complete dissolution, was added 2 ml of propylene glycol and then with 0.2 M of sterile PBS pH 7.4 to a final volume of 20 ml of stirring the solution was continued for several minutes.

Example 18

Getting preparative form solution for inhalation containing HAS degree 3

100 mg of the sulfated hyaluronic acid grade 3 (HAS3), obtained from the HA 200 KD, was introduced in a glass flask with a volume of 50 ml, and then added 15 ml of sterile of 0.2 M PBS with a pH of 7.4. The mixture was subjected to stirring for about 30 minutes until complete dissolution of the powder. When it was received, what about the complete dissolution, was added 2 ml of propylene glycol and then with 0.2 M of sterile PBS pH 7.4 to a final volume of 20 ml of stirring the solution was continued for several minutes.

Example 19

Getting preparative form of a hydrophilic gel containing HAS, HA and CMC

Methyl and propyl paraben were dissolved in purified water at 80°C. After cooling the solution to room temperature was added sodium hyaluronate under stirring to dissolve, followed by addition of HAS1 (or HAS3), continuing the stirring until complete dissolution. Then under stirring was added glycerin and propylene glycol to dissolve. Finally, added carboxymethylcellulose (CMC) of sodium and the mixture was mixed to obtain a gel-like solution.

Example 20

Obtaining a formulation containing HAS and HA, in the form of a hydrophilic gel (without preservatives) for application to mucous membranes

Sodium hyaluronate, and then HAS1 (or HAS3) was dissolved with stirring in water, which comprised about 90% of the amount stipulated by preparative form. Added propylene glycol, Symdiol 68 with the subsequent addition of MP Diol Glycol, mixing until complete dissolution of the various components. Subsequently added Carbomer 974P and mixing continued until a homogeneous dispersion of the latter. Pellet hydrox is Yes sodium was dissolved in the remaining 10% of water and this solution was slowly added to the previously obtained solution, the mixture was mixed for zastudnevaniju aqueous phase.

Example 21

Getting preparative form of a hydrophilic gel containing HAS and hyaluronidase

Methyl and propyl paraben were dissolved in purified water at 80°C. After cooling the solution to room temperature, was added the enzyme hyaluronidase with stirring followed by the addition HAS3, continuing the stirring until complete dissolution of the two components. Subsequently added Carbomer 974P and mixing continued until a homogeneous dispersion of the latter. Then was added TEA (tetraethylammonium) for zastudnevaniju the aqueous phase. Finally, with stirring, add the glycerin and propylene glycol.

Example 22

Getting preparative form of a hydrophilic gel (emulsion, oil-in-water), containing HAS and hyaluronidase

The oil phase is produced by melting liquid paraffin, stearic acid and Tefose 1500 in conditions of stirring at 50°C. the Aqueous phase separately receive the initial dissolution at 80°C methylparaben and then cooled to room temperature and the final inclusion of glycerin, hyaluronidase and subsequent HAS3 under stirring until complete dissolution of the various components.

The aqueous phase is combined with the oil phase and carry out emulsification; the resulting emulsion oil-in-water on laidout to room temperature with stirring.

Example 23

Obtaining a formulation containing HAS and hyaluronidase, in the form of a foam

Methyl - and propylparaben are dissolved in purified water at 80°C. After cooling the solution to room temperature, add the enzyme hyaluronidase with stirring followed by the addition HAS3, continuing the stirring until complete dissolution. Then add the propylene glycol and the mixture is mixed until dissolved; in subsequent include polyvinylpyrrolidone, mixing until complete dissolution, and, finally, add Polysorbate 80, continuing the stirring until dissolution.

Then carry out the distribution of phases of the obtained solution in the cylinder to increase the pressure of the propellant isobutane, n-butane, propane.

Example 24

Obtaining a formulation containing HAS and hyaluronidase, in the form of ointment

Ointment base is produced by fusion of light liquid paraffin and white vaseline under stirring at 70°C. After cooling to room temperature include hyaluronidase with stirring followed by the addition HAS3, and the mixture is mixed until a homogeneous suspension.

Example 25

Obtaining a formulation containing HAS and hyaluronidase, Limoges

Light liquid paraffin, white vaseline and cetylstearyl alcohol is melted under stirring PR is 90°C. With stirring forming lipoyl agent, gidrirovannoe castor oil to obtain a homogeneous solution and the mixture is then slowly cooled to room temperature. Finally, include hyaluronidase and HAS3 and the mixture is mixed until a homogeneous suspension.

Example 26

Obtaining a formulation containing HAS and HA, in the form of lipstick

The right amount of liquid paraffin specified in visitors the composition is loaded into a suitable container. It is heated up to 88-92°C and then with stirring, add white soft paraffin, paraffin wax, white beeswax, ceresin and arlacel; stirring is continued until complete melting of the various components. Then include racemic mixture of alpha-tocopherol acetate, allantoin, equivalent, propyl-p-hydroxybenzoate and the mixture is mixed until dissolved, maintaining the temperature of the mass at the level of 88-92°C.

The amount of purified water provided in the structure, load separately in a suitable container, then added with stirring sodium hyaluronate, HAS1 (or HAS3) until complete dissolution followed by the addition of disodium edetate, continuing the stirring until dissolution.

The aqueous phase is transferred under stirring in a container containing molten mass, maintaining the temperature of the system at UB is not 88-92°C and continuing the stirring to obtain a clear solution. Then with stirring, add two flavouring agent and the mixture is mixed for 10 minutes Molten mass is poured into molds and immediately cooled down to T<0°C to obtain a solid briquettes.

Example 27

Obtaining a formulation containing HAS and HA, in the form of vaginal suppositories

Gelatino allow to swell in 70% of treated water at 85°C, sodium hyaluronate and HAS1 (or HAS3) dissolved in the remaining amount of water and this solution is mixed with glycerin, brought to the same temperature. A solution of glycerin are added to a solution of the swollen gelatin and continue stirring until the gelatin has dissolved. The mass is poured into molds and cooled to T<0°C to obtain a solid suppositories.

Example 28

Obtaining a formulation containing HAS and HA, in the form of a hydrophilic cream (emulsion, oil-in-water)

The oil phase is produced by melting liquid paraffin, stearic acid and tifosi 1500 under stirring at 50°C. the Aqueous phase separately receive the initial dissolution at 80°C methylparaben and then cooled to room temperature and the inclusion of glycerin, sodium hyaluronate and subsequent HAS1 (or HAS3) under stirring until complete dissolution of the various components.

The aqueous phase is combined with the oil phase and implement e is legirovanie, the resulting emulsion O/A cooled with stirring to room temperature.

Example 29

Obtaining a formulation containing HAS, in the form of ointment

Ointment base is produced by fusion of light liquid paraffin and white vaseline under stirring at 70°C. After cooling to room temperature under stirring include HAS1 (or HAS3) and the mixture is mixed until a homogeneous suspension.

Example 30

Obtaining a formulation containing HAS3, HA and diclofenac, in the form of a hydrophilic gel

Methyl - and propylparaben are dissolved in purified water at 80°C. After cooling the solution to room temperature with stirring diclofenac sodium, sodium hyaluronate and then HAS3, continuing the stirring until complete dissolution of the two components. In subsequent add Carbomer 974P and stirring is continued until a homogeneous dispersion of the latter. Then add TEA to zastudnevaniju the aqueous phase. Finally, under stirring include glycerin and propylene glycol.

Example 31

Obtaining a formulation containing HAS3 and diclofenac, in the form of a hydrophilic cream (emulsion, oil-in-water)

The oil phase is produced by melting liquid paraffin, stearic acid and Tifosi 1500 under stirring at 50°C. the Aqueous phase separately receive the original is owned by dissolving at 80°C methylparaben and then cooled to room temperature and the final inclusion of glycerin, diclofenac sodium and subsequent HAS3 under stirring until complete dissolution of the various components.

The aqueous phase is combined with the oil phase and carry out the emulsification, the resulting emulsion oil-in-water cooled with stirring to room temperature.

Example 32

Obtaining a formulation containing HAS and diclofenac, in the form of a foam

Methyl - and propylparaben are dissolved in purified water at 80°C. After cooling the solution to room temperature with stirring diclofenac sodium and then adding HAS3, continuing the stirring until complete dissolution. Then add the propylene glycol and the mixture is mixed until dissolved; in subsequent include polyvinylpyrrolidone, mixing until complete dissolution, and, finally, Polysorbate 80, continuing the stirring until dissolution.

Then carry out the distribution of phases of the obtained solution in the cylinder to increase the pressure of the propellant isobutane, n-butane, propane.

Example 33

Obtaining a formulation containing HAS and diclofenac, in the form of strips

These experiments relate to the obtaining of a polymeric matrix containing HAS, for controlling the release of drugs for local application, and in this case, non-steroidal anti-inflammatory drug sodium is salt of diclofenac, which improves the dermal and/or transdermal absorption of the contained active due to the stimulating effect HAS. Consider the matrix preferably contains a copolymer of acrylic acid and acrylic and/or methacrylic esters with a glass transition temperature (Tg) lower than room temperature, preferably lower than 0°C, whose free carboxyl groups present in the polymer chain, form salts with organic bases (for example, ammonia, Ethylenediamine, copolymers of ester of acrylic and/or methacrylic acid with cationic ammonium functional group in the alkyl group (preferably EUDRAGIT®E100) or inorganic bases (for example, the hydroxide or carbonate or bicarbonate of alkali, alkaline earth and transition metals), as is well known to specialists in this field. The copolymers typically used in accordance with the present invention, consist of 2 or more monomers different percentage; examples of these monomers are:

• acrylic acid;

• butyl and/or methyl acrylate;

• 2-ethyl hexyl acrylate;

• glycidylmethacrylate;

• vinyl acetate.

On the market there are many copolymers (such as Duro-tak® 280-2416, 280-2516, 87-2620, 87-2852, 380-1054, 87-2051, produced by National andStarch), dissolved in organic solvents, with the percentage of free carboxyl groups is from 0.1 to 15%, which can be converted into salts with organic or inorganic bases, as described above. These copolymers preferably contained in the matrix of the patch in the amount of 30-90% of the mass.

In addition, can be obtained containing HAS a polymeric matrix that includes the following two main components:

- a: polymethacrylates: i.e., the preferred copolymers of ester of acrylic and/or methacrylic acid with cationic ammonium functional group in the alkyl group (EUDRAGIT®E100, EUDRAGIT®RS and EUDRAGIT®RL). These polymers can range from 10 to 40% of the mass. throughout the adhesive matrix after drying, preferably from 10 to 25%.

- b: organic dicarboxylic or tricarboxylic acid (such as, for example, succinic, fumaric, adipic and lauric acid) as the counterion of the cationic component a (in addition, they act as a form secutest agent component (a). Component b can be chosen so that was performed partial or complete neutralization. Other suitable component b is an acid-functional acrylate and methacrylate polymers, such as polyacrylic acid of Carbopol®. Components b to moutsoulas in % of the mass. in the range from 1 to 40 wt%. all adhesive formulation after drying, preferably from 1 to 20%.

The above polymeric matrix inside the final formulation is in the range from 10 to 90% relative to dry weight, preferably from 50 to 90% based on dry weight of the final composition. Included the number of active varies with respect to its nature and desirable dermal or transdermal therapeutic effect. It is usually present in amounts in the range from 0.1 to 30% wt. in relation to the dry weight of the final composition.

Preparative form may also contain excipients, sedatives, softeners, emulsifying means, modulators of adhesion, preservatives, plasticizers, podnikatel/buffers. The number of excipients may vary in large ranges from 0.01 to 30%, depending on their function.

Stimulator absorption through the skin with soothing, antipruritic and antihyperuricemic properties is HAS degree 1 or degree 3, preferably of degree 3, which is present in concentrations in the range from 0.1 to 30% dry weight of the final composition. The above matrix provides an adjustable, continuous penetration of the active product without irritation and adhesion to the skin.

the manual receive

1 kg methacrylate copolymer selected (e.g., Duro-tak® 280-2416, 280-2516 or 87-2852) having a solids content of 30-40% wt./mass., added under mechanical stirring with 300 g of a 30% wt./mass. EUDRAGIT®E100 solution of water-based/solvent; the mixture is left under moderate stirring for 30 minutes Then add active active principle (100 g of sodium salt of diclofenac) and HAS3, pre-dissolved in an aqueous solution. The mixture is left under stirring until complete dissolution. To obtain the matrix layer this mixture was then layered over the film silicone polyester/paper, and drying is carried out by evaporation of residual solvent. Distributed over the film matrix has a dry weight of approximately 60 g/m2. Thus obtained matrix is then combined with non-woven polyester cloth or plastic wrap for the final formation of the patch.

The final composition of each individual trancdermalnoe system contains 140 mg of diclofenac and 40 mg HAS3.

Table 3
Hydrophilic gel (Example 19)
ComponentsQuantity (mg/1 g hydrogel)
HAS1 (HAS3)40 mg (10 mg)
CMC20 mg
Glycerin100 mg
Propylene glycol66,75 mg
Sodium hyaluronate2 mg
Methyl-p-hydroxybenzoate2 mg
Propyl-p-hydroxybenzoate0.2 mg
Purified waterTo 1 g
Hydrophilic gel for application to mucous membranes
(Example 20)
ComponentsQuantity (mg/1 g hydrogel)
HAS1 (HAS3)10 mg
Carbomer 974P15 mg
Propylene glycol100 mg
Sodium hydroxide0.33 mg
Sodium hyaluronate2 mg
MP-Diglycol37.5 mg
SymDiol 6890 mg
Purified waterto 1 g

Hydrophilic gel (Example 21)
ComponentsNumber (IU or mg/1 g hydrogel)
HAS310 mg
Hyaluronidase150 IU
Carbomer 974P15 mg
Glycerin100 mg
Propylene glycol66,75 mg
Triethanolamine13.25 mg
Methyl-p-hydroxybenzoate2 mg
Propyl-p-hydroxybenzoate0.2 mg

Purified waterto 1 g
Hydrophilic cream (Emulsion oil-in-water) (Example 22)
Components Number (IU or mg/1 g cream)
HAS310 mg
Hyaluronidase150 IU
Tifosa 1500110 mg
Glycerin80 mg
Stearic acid33 mg
Liquid paraffin 40 mg40 mg
Methyl-p-hydroxybenzoate1 mg
Purified waterto 1 g
Foam (Example 23)
ComponentsNumber (IU or mg/1 g of solution)
HAS310 mg
Hyaluronidase150 IU
Polysorbate 8040 mg
Propylene glycol40 mg
Polyvinylpyrrolidone30 mg
Methyl-p-hydroxybenzoate
Propyl-p-hydroxybenzoate0.3 mg
Purified waterto 1 g
The cylinder contains approximately 94% of the solution and 6% of the propellant (isobutane, n-butane, propane)
Ointment (Example 24)
ComponentsNumber (IU or mg/1 g ointment)
HAS320 mg
Hyaluronidase200 IU
Light liquid paraffin200 mg
White petrolatumto 1 g
Lipoyl (Example 25)
ComponentsNumber (IU or mg/1 g Limoges)
HAS320 mg

Hyaluronidase200 IU
Gidrirovannoe castor oil10 mg
Cetostearyl the new alcohol 50 mg
White petrolatum365 mg
Light liquid paraffinto 1 g

Lipstick (Example 26)
ComponentsQuantity (mg/1 g lipstick)
HAS1 (HAS3)30 mg (10 mg)
Liquid paraffin253,2 mg
White soft paraffin326,2 mg
Paraffin wax144,3 mg
Beeswax white96 mg
Ceresinof 28.2 mg
Arlacel 58295,8 mg
Sodium hyaluronate2 mg
Allantoin1.1 mg
Racemic mixture of alpha-tocopherol acetate1.1 mg
Propyl-p-hydroxybenzoate/td> 0.4 mg
Equivalent0.4 mg
Purified water19,2 mg
Disodium edetate1.1 mg
Vanilla perfume0.5 mg
Proglacial0.5 mg
Vaginal suppositories (Example 27)
ComponentsQuantity (mg/1 g suppositories)
HAS1 (HAS3)10 mg
Glycerin580
Gelatin200
Sodium hyaluronate2
Purified waterto 1 g
Hydrophilic cream (Emulsion oil-in-water) (Example 28)
ComponentsQuantity (mg/1 g cream)
HAS1 (HAS3)10 mg
Those who Uzzah 1500 110 mg
Glycerin80 mg
Stearic acid33 mg
Sodium hyaluronate2 mg
Liquid paraffin40 mg
Methyl-p-hydroxybenzoate1 mg
Purified waterto 1 g
Ointment (Example 29)
ComponentsQuantity (mg/1 g ointment)
HAS1 (HAS3)20 mg
Light liquid paraffin200 mg
White petrolatumto 1 g

Hydrophilic gel (Example 30)
ComponentsQuantity (mg/1 g hydrogel)
Diclofenac sodium (*)30 mg
Carbomer R15 mg/td>
Glycerin100 mg
HAS320 mg
Laurent sodium2 mg
Propylene glycol66,75 mg
Triethanolamine13.25 mg
Methyl-p-hydroxybenzoate2 mg
Propyl-p-hydroxybenzoate0.2 mg
Purified waterto 1 g
(*): sodium-diclofenac 3%
Hydrophilic cream (Emulsion oil-in-water) (Example 31)
ComponentsQuantity (mg/1 g cream)
Diclofenac sodium (*)10 mg
Tifosa 1500110 mg
Glycerin80 mg
HAS330 mg
Stearic acid33 mg
Liquid paraffin40 mg
Methyl-p-hydroxybenzoate1 mg
Purified waterTo 1 g
(*): sodium diclofenac 1%
Foam (Example 32)
ComponentsQuantity (mg/1 g of solution)
Diclofenac sodium (*)40 mg
Polysorbate 8040 mg
Propylene glycol40 mg
HAS310 mg
Polyvinylpyrrolidone30 mg
Methyl-p-hydroxybenzoate2 mg
Propyl-p-hydroxybenzoate0.3 mg
Purified waterTo 1 g
The cylinder contains approximately 94% of the solution and 6% of the propellant (isobutane, n-butane, propane)
(*): sodium di is lofenac 4%

It is obvious that the methods presented in the present description, can be modified in various ways. These modifications should not be considered as deviating from the essence and prospects of the invention, and all modifications that could clearly offer an expert in this area, included in the scope of the following claims.

1. The use of sulfated hyaluronic acid for the preparation of drugs for topical application for the treatment of inflammatory/cause irritation of skin diseases selected from dermatitis, atopic dermatitis, photodermatitis, rash, vitiligo, eczema, psoriasis, all skin irritations associated with activation of Pro-inflammatory cytokines, such as IL-1, IL-2, IL-6, IL-7, IL-8, IL-12 and TNF, where hyaluronic acid has a molecular weight in the range from 10,000 Da to 50,000 Da, 150,000 Yes up to 250000 and 500000 to 750000 and sulfate crystallization degree equal to 1.

2. The use of sulfated hyaluronic acid for the preparation of medicines for local use as a hydrating agent for the treatment of pathological conditions of the skin characterized by dry, lichenification and desquamation, where hyaluronic acid has a molecular weight in the range from 10,000 Da to 50,000 Da, 150,000 Yes up to 250000 and 500000 to 750000 and the degree sulfa what purpose, equal to 1 or 3.

3. The use of sulfated hyaluronic acid for the preparation of drugs for topical application for the treatment of pathological skin conditions associated with damage to the endothelium and/or the walls of blood vessels, and for processing clots of fibrin and blood clots that form on the skin level, where hyaluronic acid has a molecular weight in the range from 10,000 Da to 50,000 Da, 150,000 Yes up to 250000 and 500000 to 750000 and sulfate crystallization degree equal to 1 or 3.

4. The use of sulfated hyaluronic acid for the preparation of drugs for topical application for the treatment of skin pathological conditions associated with deficiency immune IL-10, selected from vitiligo, eczema and psoriasis, which stimulates the synthesis of inflammatory cytokines, where hyaluronic acid has a molecular weight in the range from 10,000 Da to 50,000 Da, 150,000 Yes up to 250000 and 500000 to 750000 and sulfate crystallization degree equal to 1 or 3.

5. The use of sulfated hyaluronic acid for the preparation of drugs for topical application for the treatment of lymphoma of the skin, where hyaluronic acid has a molecular weight in the range from 10,000 Da to 50,000 Da, 150,000 Yes up to 250000 and 500000 to 750000 and sulfate crystallization degree equal to 1 or 3.

6. Pharmaceutical composition for months the con application under item (1, containing sulfated hyaluronic acid together with diclofenac, and specified sulfated hyaluronic acid is found as a stimulator of skin absorption of medicines in the form of ointments, Limoges, hydrogel, lipstick, creams, patches, vaginal suppositories and pessaries, foams, gel for application to mucous membranes, ophthalmic preparations, compositions for vaginal disinfection, rinse for oral solutions.

7. Pharmaceutical composition for topical application on p. 3, containing sulfated hyaluronic acid together with hyaluronidase in the form of ointments, Limoges, hydrogel, lipstick, creams, patches, vaginal suppositories and pessaries, foams, gel for application to mucous membranes, ophthalmic preparations, compositions for vaginal disinfection, rinse for oral solutions for topical application or inhalation.

8. The pharmaceutical composition according to p. 7, optionally containing polymers, such as hyaluronic acid and its derivatives, carboxymethylcellulose and/or other polymers of natural or synthetic origin.



 

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