HK1258973B - Sulphated hyaluronic acids as regulator agents of the cytokine activity - Google Patents

Description

Sulfated hyaluronic acid as a modulator of cytokine activity

This application is a divisional application of PCT application PCT/EP2010/003044, filed on May 11, 2010, with the invention title “Sulfated hyaluronic acid as a cytokine activity regulator”. The date on which the PCT application entered the Chinese national phase was November 11, 2011, with the application number 201080020643.2.

Field of the Invention

For many years, scientific/patent literature has studied and described sulfated hyaluronic acid, which is obtained after being suitably sulfated starting from hyaluronic acid (HA) as described in the state of the art (EP0940410B1 and EP0702699B1), due to its anticoagulant effect. HAS can also be obtained (defined as HA-NS) (EP0971961B1) by deacetylation and the subsequent sulfation of the glucosamine of HA, for the preparation of surgical articles and pharmaceutical compositions. Also known are patents EP0754460B1 and EP1385492B1, which describe the purposes of HAS in illness (pathologies) such as ARDS, rheumatism, rheumatoid arthritis and dermatitis.

One object of the present invention relates to the novel and surprising use of HAS as a modulator of cytokine activity, in that the applicants have discovered that HAS has a unique ability to modulate specific cytokines (both pro-inflammatory and anti-inflammatory), have studied its mechanism of action and revealed fundamental differences between the two types of sulfated products (HAS and HA-NS), but in summary the applicants have surprisingly found an unexpectedly high activity against different types and strains of herpes viruses, HIV, cytomegalovirus and vesicular stomatitis virus.

Since 1970, scientists have recognized that selected lymphoid cell populations can produce and release into the circulation molecules that are proteinaceous but not assimilated into antibodies, termed "cytokines." They represent a new type of "hormone" that can act on different cellular targets in various areas of the body.

The progress of scientific knowledge related to the synthesis and biological/biochemical functions of these proteins has changed the "old" view of the immune system (I.S.) in the same scientific world and opened a new stage in the understanding of its multiple functions, thus creating new prospects for the treatment of different local and/or systemic pathologies, including the possibility of new therapies related to the immunotherapy of cancer.

Central ISCs are lymphocytes, representing approximately 20% of all white blood cells. Based on their diverse functions, they are divided into three groups: B lymphocytes, T lymphocytes, and cytotoxic lymphocytes. Many cytokines are soluble proteins produced by lymphocytes and/or monocytes that can act on other cells and tissues, even those located far from the site of cytokine production. They have immune functions and, in fact, play a regulatory role in the synthesis of other cytokines by different ISC cells or target cells involved in the cascade of reactions initiated by ISCs.

A variety of different cytokines have been studied and have a variety of different abbreviations, but those that the applicant has specifically studied are: interleukins 1 and 2, interleukins 6, 7 and 12, hereinafter defined as IL-1, IL-2, IL-6, IL-7 and IL-12, which and TNF are defined as cytokines with pro-inflammatory properties, while interleukin 10 (IL-10) is, in contrast, a cytokine with strong anti-inflammatory properties.

The first cytokine to be identified was IL-1, which exists in both α and β forms and is a potent inducer of local and systemic inflammatory processes. It is primarily produced by B and T lymphocytes and macrophages following bacterial stimulation or stimulation with other agents, including other cytokines; it is also secreted by alveolar and peritoneal macrophages, Kupffer cells, peripheral neutrophils, endothelial, epithelial, and smooth muscle cells, fibroblasts, Langerhans cells of the skin, osteoclasts, synoviocytes, and many other cell types. It is also present in the cerebrospinal fluid, where it is produced and transported. Both forms bind to the same receptors and have very similar, if not identical, biological activities. Many of their proinflammatory functions are 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, interferons, bacterial endotoxins, viruses, and various other antigens. It has been implicated in septic shock, but it should also be noted that recent studies have shown that IL-1 can activate the expression of certain oncogenes and, subsequently, participate in the pathogenesis of tumors. Furthermore, the autocrine control system for the growth of white blood cell blasts present in the circulating layers of patients with leukemia is believed to produce this type of cytokine: these blasts uncontrollably produce IL-1, which in turn stimulates the synthesis of growth factors that enhance the proliferation of these same blasts. In conjunction with other cytokines, IL-1 represents one of the primary regulators of inflammatory processes: it stimulates T cells to produce IL-2 and B cells to produce immunoglobulins. Consequently, it is involved in a variety of conditions such as astrogliosis and neurofibrillary demyelination. It is cytotoxic to insulin-producing Langerhans cells and is involved in bone calculi, activating osteoclasts and inhibiting new bone formation, both of which are involved in the pathology of osteoporosis. By increasing the release of prostaglandins in the hypothalamic center, it can act as an endogenous pyrogen, leading to elevated body temperature. It is also involved in the pathogenesis of rheumatoid arthritis and arthritis: in fact, large amounts of IL-1 are found in the synovial fluid of patients with rheumatoid arthritis and/or osteoarthritis. Finally, it is involved in the development of vascular damage, such as venous thrombosis, and is present in all blood vessels with arterial/atherosclerotic type pathology. For this type of cytokine, receptor antagonists are currently being tested, as blocking the receptor has proven to be an effective method for treating these conditions in which IL-1 plays a major role.

TNF : Necrokines are part of the group of cytokines that promote responses during the acute systemic inflammatory phase. TNF is therefore involved in a wide range of processes, such as cell proliferation, differentiation and apoptosis, carcinogenesis and viral replication.

It is produced primarily by macrophages and a range of other cell types, including mast cells, lymphoid cells, myocytes and endothelial cells, fibroblasts and neurons. Its synthesis can be stimulated by bacterial endotoxins, other cytokines such as IL-2, interferon and IL-1, and it can be inhibited by steroids.

By acting on multiple organs and systems, often together with other cytokines, it participates in the establishment and regulation of many pathogenic processes:

- It regulates the expression of many proteins and important cytokines (such as IL-1 and IL-6), and is therefore involved in many skin disorders such as dermatitis, vitiligo and eczema;

-It stimulates the hypothalamic-pituitary-adrenal axis, increasing the release of certain types of hormones;

-It suppresses appetite;

- It causes fever (it acts as an endogenous pyrogen, inducing the production of prostaglandins in the hypothalamus);

- It stimulates the synthesis of collagenase in synovial cells, and for this reason, large amounts of TNF have been found in the synovial fluid of patients with arthritis and rheumatoid arthritis;

- It activates osteoclasts, thereby inducing bone resorption, characteristic of the osteoporosis condition;

- It is also involved in pathologies of the nervous system, such as astrocytosis and demyelination;

- It strongly attracts neutrophils and helps them to attach to endothelial cells and thus extravasate;

- It stimulates the macrophagic production of molecules with oxidative effects;

- It is particularly involved in the pathology of the cardiocirculatory system, in the pathogenesis of venous thrombosis, arteriosclerosis and vasculitis;

- It increases resistance to insulin, it increases protein catabolism in muscle, but it inhibits lipogenic metabolism in adipose tissue.

High concentrations of TNF can induce shock-like symptoms, but prolonged exposure to low concentrations can lead to cachexia, a syndrome that causes protein and lipid depletion of tissues, particularly muscle and adipose tissue. TNF can itself bind to two receptors, TNF-R1 (receptor for type 1 TNF) and TNF-R2 (receptor for type 2 TNF), which are expressed on all body cells except red blood cells. In short, TNF promotes an inflammatory response, which in turn triggers a variety of conditions of an autoimmune nature, such as rheumatoid arthritis, Crohn's disease, psoriasis, and asthma. Current scientific research attempts to develop "biological" drugs (e.g., monoclonal antibodies) that inhibit the synthesis of TNF and/or block its receptors.

IL-2: This is a potent pro-inflammatory, atherogenic cytokine produced primarily by T lymphocytes, whose synthesis is inhibited by steroids and cyclosporine. Leukemic cells synthesize this cytokine and simultaneously express its receptor, thereby stimulating their growth and generating an autocrine system that exacerbates the leukemic condition. IL-2 plays an important role in regulating the immune response: it stimulates the synthesis of interferon (IFN) in peripheral leukocytes and induces the production of IL-1 and TNF. IL-2 can also disrupt the integrity of the blood-brain barrier and the cerebral vascular endothelium, leading to neuropsychiatric disorders such as disorientation and depression.

Thus, a variety of disorders have been associated with abnormal production of IL-2, such as Hodgkin's lymphoma, organ transplant rejection, multiple sclerosis, rheumatoid arthritis, lupus erythematosus, diabetes, and AIDS.

IL-6: Produced by many cell types in all the above-mentioned I.S., it is, together with TNF, the most important member of the group of chemical regulators of the acute phase of the inflammatory process and is therefore involved in conditions with a strong inflammatory component, such as asthma (wherein it participates in the emergence and maintenance of the inflammatory process), chronic enteritis (Crohn's disease), rheumatoid arthritis and arthropathy. As determined above, in fact, cytokines (such as TNF, IL-1 and IL-6) have been shown to be greatly involved in the process of osteoarthropathy of degenerative joints, since they play an important role in regulating the expression of metalloproteinases (responsible for cartilage degradation), in the production of prostaglandins and in the activation of osteoclasts. For this reason, high cytokine levels are shown in the synovial fluid of patients with arthropathy and rheumatoid arthritis (RA). These findings have inspired the use of inhibitors of the above interleukins and/or receptor antagonists as new therapeutic strategies for arthropathy conditions.

High concentrations of IL-6 are found in the urine of patients undergoing transplantation, and its presence represents one of the early signs of organ rejection. The serum level of this cytokine is also significantly increased in many patients with tumors (such as myeloma, leukemia, cardiac myxoma or in diseases such as lymphadenopathy and cirrhosis), and it can also be used as an indicator for monitoring tumor mass size. Finally, recent studies have associated cancer with life expectancy and revealed how certain tumors are affected by the patient's cytokine protein properties/amount situation: in short, the latest evidence associates the low distribution of IL-10 and the high secretion of IL-6 with the deterioration in patients with tumor diseases who survive clinically, and the genotype that can produce and maintain high levels of IL-10 may be beneficial for survival. Therefore, individuals with high anti-inflammatory cytokine levels and low concentrations of pro-inflammatory cytokines generally have a longer life expectancy (Caruso C. et al., Ann N.Y. Acad. SCI., 2004, 1028: 1-13).

IL-7: A cytokine produced primarily by bone marrow stromal cells and also secreted by the thymus and keratinocytes. IL-7 induces the synthesis of inflammatory cytokines, such as IL-1, IL-6, and TNF, which are involved in the pathogenesis of certain skin diseases (e.g., psoriasis and cutaneous lymphoma) and the osteoarticular system. Indeed, high levels of IL-7 are found in patients with R.A., as IL-1 and TNF (cytokines strongly involved in these conditions) increase interstitial production of IL-7, which in turn stimulates macrophage synthesis of TNF. Finally, IL-7 can induce osteoclast maturation, subsequently increasing bone resorption and thus causing joint degeneration.

IL-12: This protein also plays an important role in regulating I.S. function. It actually acts on lymphocyte differentiation, induces the synthesis of interferon and TNF, and its production can be inhibited by IL-10. Excessive production of this protein is involved in the pathogenesis of autoimmune diseases 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 produced by lymphocytes, it is a cytokine with anti-inflammatory properties that can inhibit the synthesis of IL-2 and interferon produced by lymphocyte T cells. The anti-inflammatory effect of IL-10 is also manifested in its ability to inhibit the synthesis of IL-1, IL-6, IL-8, IL-12 and TNF in macrophages stimulated with bacterial toxins. IL-10 deficiency is associated with diseases such as diabetes and chronic enteritis (such as Crohn's disease). Recent evidence has led to the trial of IL-10 as a new treatment method for lupus erythematosus. Low IL-10 levels are often observed in the skin tissue of patients with diseases such as vitiligo, psoriasis, eczema and dermatitis. It should be noted that during conventional immunosuppressive therapy for the treatment of inflammation and organ rejection, corticosteroids and cyclosporine increase the production and/or release of this type of interleukin from relevant competent cells (Zhou X. et al., Current Drug Targets-Immune, Endocrine & Metabolic Disorders, 2005, 5(465475). Experimental data also confirmed its effectiveness in reducing the release of prostaglandins and cyclooxygenase induced by TNF on human synovial cells in vitro, indicating that IL-10 has the ability to reduce inflammatory processes, including joints affected by osteoarticular degeneration (Alaaeddine N. et al., Arthritis & Rheumatism, 1999, 42:710-718). Recent studies have demonstrated their therapeutic effectiveness against asthma in an experimental animal model of bronchial hyperresponsiveness, demonstrating the high therapeutic potential of this class of cytokines in reducing the inflammation characteristic of the airways of asthmatic patients, where high concentrations of TNF, IL-1, IL-5, IL-6, and IL-8 are found in bronchial washings and/or at the serum and/or tissue levels (Stankiewicz W. et al., Mediators of Inflammation, 2002, 11:307-312). This suggests an important role for these interleukins in maintaining immune homeostasis.

Asthma can be an extremely debilitating disease, affecting approximately 2 billion people worldwide and resulting in over 5,000 deaths annually. It is a condition based on a distorted response of the immune system to environmental factors, and is therefore associated with an increased production of proinflammatory cytokines, which are responsible for the growth and differentiation of mast cells and eosinophils, as well as other types of cells in the immune system. The causes of this imbalanced immune system activity are still not fully understood, but genetic, environmental, viral, and nutritional factors contribute to the development of this condition in different ways. Therefore, the discovery of an effective therapy for its prevention and/or treatment that allows the discontinuation or reduction of steroid use (the conventional treatment method) would represent an effective solution for both more severe forms (because it would in any case reduce steroid use) and less severe cases, since discontinuation of steroid treatment would be total.

Detailed Description of the Invention

An object of the present invention is a new and surprising use of HAS as a modulator of cytokine activity, as the applicants have discovered that it has a unique ability in modulating the activity of particular cytokines, the applicants have studied its mechanism of action and revealed fundamental differences between the various types of sulfated products known in the state of the art, but in summary the applicants have discovered an unexpectedly high activity against different types and strains of herpes viruses, HIV, cytomegalovirus and vesicular stomatitis virus. The sulfated hyaluronic acid suitable for the purposes of the present invention is prepared according to the method described in EP 702699 B1: the sulfation is carried out with the aid of the complex SO ₃ -pyridine and involves the alcoholic hydroxyl groups present in the polysaccharide chain, starting from HA derived from any source (for example from Celosia cristata extraction, fermentation or technology) and having a molecular weight ranging from 400 to 3×10 ⁶ Da, in particular from 1×10 ⁴ Da to 1×10 ⁶ Da, even more particularly from 10,000 to 50,000 Da, from 150,000 to 250,000 Da and from 500,000 to 750,000 Da.

The derivatives obtained retain all the physical properties of the unchanged starting polymer, in particular the molecular weight of the starting HA is not modified by the sulfation process, thereby retaining all the physico-chemical properties of the starting polysaccharide. Sulfation involves the multiple hydroxyl groups of the disaccharide unit and, as is known in the state of the art, it is possible to obtain different degrees of sulfation (0.5 to 3.5) (expressing the number of sulfate groups per disaccharide unit) by varying the amount of SO ₃ -pyridine introduced.

The derivatives used in all the experiments performed generally had 1 or 3 degrees of sulfation and are hereinafter defined as HAS1 and HAS3. All free carboxyl groups in HA can form salts with cations of organic and/or inorganic origin.

Both levels of HSA are soluble in water and they can also be sterilized using conventional techniques known to experts in the field, although sterilization using an autoclave is preferred.

In the experiments described below, the applicants demonstrated that:

HAS can stimulate the production of new mRNA and protein synthesis of anti-inflammatory cytokines (such as IL-10), thereby increasing the immune defense capacity of cells and entire organs. The anti-inflammatory effect of the above cytokines is reflected in the ability to inhibit the synthesis of IL-1, IL-6, IL-8, IL-12 and TNF, all pro-inflammatory proteins.

HAS was effective in reducing new mRNA synthesis and in significantly reducing protein synthesis of IL-2, IL-7 and IL-12 in both cases, both for some synoviocytes (for IL-12) and for cellular components of IS, in both cases without the need for an immune response and, in particular, in events of inflammatory stress, where immune cells respond by producing a cytokine cascade and in this case overall, the emerging data suggest a greater inhibitory effect of HAS.

HAS is effective in inhibiting the binding of TNF, IL-1, and IL-6 to their receptors. These results are very important because they demonstrate that sulfate esters behave exactly like specific monoclonal antibodies against the receptors of these proinflammatory proteins, thereby blocking their function even though they lack this specificity. This receptor blockade represents the most effective way to antagonize the proinflammatory and tumorigenic effects of TNF, IL-1, and IL-6, thus opening a new level of clinical trials and providing an excellent new therapy for the treatment and/or prevention of a wide range of conditions, given the role that TNF, IL-1, and IL-6 play in the onset and progression of numerous diseases.

Therefore, the applicants describe and claim a new use of HAS in the preparation of a medicament:

For the prevention and/or treatment of conditions associated with immunodeficiency, and in particular IL-10 deficiency, by stimulating the synthesis of anti-inflammatory cytokines as a novel topical and systemic therapy for conditions such as vitiligo, eczema, psoriasis and dermatitis;

For the prevention and/or treatment of conditions associated with the elevation/activation of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12 and TNF;

For the prevention and/or treatment of asthma, rheumatoid arthritis and arthritis associated with the activation of IL-1, IL-6 and TNF;

For the prevention and/or treatment of diseases associated with vascular endothelial damage;

For the prevention and/or treatment of skin diseases such as dermatitis and cutaneous lymphoma;

for the prevention and/or treatment of diseases of an autoimmune nature, such as rheumatoid arthritis, Crohn's disease (and all chronic inflammatory bowel diseases), psoriasis and asthma, diabetes, encephalomyelitis, multiple sclerosis, lupus erythematosus, and for the treatment of organ transplant rejection;

For the prevention and/or treatment of neoplasia, such as leukemia and Hodgkin's lymphoma;

For the prevention and/or treatment of astrocyte proliferation, astrocytosis, and nerve fiber demyelination;

For the prevention and/or treatment of conditions associated with osteoclast activation, such as osteoporosis;

For the prevention and/or treatment of vascular disorders of the arterial/atherosclerotic type, venous thrombosis and vasculitis associated with the activation of TNF, IL-1 and IL-6;

For the prevention and/or treatment of fever.

In the experiments described below, the applicants also demonstrated the potent antiviral effect of HAS against different types of viruses:

The results obtained show the effectiveness of HAS (degrees 1 and 3) in inhibiting viral replication of HIV 1 and 2. This experiment also confirms that not all sulfated hyaluronic acids known in the state of the art are effective in viral replication, since only HAS in which the sulfation occurs solely at the hydroxyl group has been shown to be effective, while HA-NS is ineffective in blocking viral cytopathogenicity.

The experimental data confirmed the antiviral effects of HAS1 and HAS3 on herpes simplex virus type 1 and type 2 and on vesicular stomatitis virus (VSV). The first form is very common with characteristic fever blisters, which usually affect the facial skin (lips, nostrils); it is also called herpes simplex labialis . The infection caused by herpes labialis is prone to recurrence because the virus survives inside the cells and is difficult to remove even with effective drugs. The second form is genital infection, also known as genital herpes . Both are caused by physical and sexual contact. Due to the location of the virions in the ganglion, they remain dormant for a long time, and herpes infections have recurring characteristics, corresponding to stress events of the immune system, and usually relapse at the initial site. Vesicular stomatitis virus is an RNA-virus that infects mammals (mainly animals) and is used in the laboratory to study the life cycle development of RNA-viruses. The comparison between HA-NS1 and HAS1 once again shows that not all sulfated hyaluronic acids are equivalent, because HA-NS1 is proven to be ineffective at all, while HAS 1 and 3 show very strong antiviral activity against herpes simplex and VSV. The samples tested did not demonstrate cytotoxicity to host cells; in fact, the minimum cytotoxic concentrations achieved were equivalent to reference drug concentrations commonly used in clinical practice for the treatment of herpes and demonstrated to be on average 100-fold higher than concentrations shown to be effective in inhibiting viral replication.

The experimental data obtained for HAS1 and HAS3 show clear and significant antiviral results against cytomegalovirus : Cytomegalovirus is a special type of virus that enters certain types of cells in our body, where it replicates itself and parasitically causes their death. It belongs to the same family of cold sores and genital herpes, chickenpox and infectious mononucleosis. Epithelial cells, mucosa, and lymph nodes are the sites of multiple primary infections. It remains latent throughout life in peripheral blood, renal tubular epithelium, and salivary gland epithelium. Severe forms are found in individuals with weakened multi-organ immunity (such as those with AIDS and transplant individuals in immunosuppressive therapy): pneumonia, hepatitis, colitis, esophagitis, nephritis. The treatment regimen includes the administration of drugs such as ganciclovir, valganciclovir, and foscarnet (inhibitors of viral DNA synthesis). In this case, it was confirmed that HA-NS1 is ineffective in inhibiting viral proliferation, and the absolute diversity of antiviral ability between the two types of sulfated products was determined.

The applicants therefore describe and claim a new use of HAS in the preparation of medicaments:

For the prevention and/or treatment of HIV;

For the prevention and/or treatment of herpes simplex labialis and genital herpes;

For the prevention and/or treatment of vesicular stomatitis virus;

For the prevention and/or treatment of cytomegalovirus.

Finally, the applicant describes the preparation of various pharmaceutical preparations/compositions comprising HAS as the sole active ingredient or in combination with other pharmacologically and/or biologically active agents, such as steroids, hormones, proteins, nutritional factors, vitamins, non-steroidal anti-inflammatory drugs (FANS), chemotherapeutics, calcium antagonists, antibiotics, antivirals, anticoagulants and/or fibrinolytics, local anesthetics, enzymes such as collagenase and/or hyaluronidase and/or other proteases; which can be formulated together with polymers such as hyaluronic acid and its derivatives, carboxymethylcellulose and/or other polymers of natural (e.g. collagen) or synthetic nature.

The pharmaceutical composition can be administered systemically (intravenously or intraarterially, intramuscularly, intraperitoneally, subcutaneously or orally), it can be used for topical application, absorbed through the skin and/or percutaneously, it can be administered by inhaler/aerosol (particularly for the treatment of asthmatic conditions), intraarticularly or it can be administered directly to the area to be treated by direct injection.

The pharmaceutical composition can be formulated as an ointment, liposome, hydrogel, lipstick, cream, vaginal ovules and bougies, foam, mucosal gel, ophthalmic preparation, douche, mouthwash, skin and/or transdermal absorption patch (especially FANS and hormone patches), solution for inhalation application.

Certain examples of the preparation of HAS degrees 1 and 3, pharmaceutical formulations containing the same, together with the results obtained in in vitro tests are provided for illustrative and non-limiting purposes only.

Example 1

Preparation of tetrabutylammonium chloride (TBC) of hyaluronic acid (HA) with an average molecular weight of 200 kDa (range 150,000 to 250,000 Da) Ammonium salt

5.00 g of fermented sodium salt-derived hyaluronic acid (200 kD) was dissolved in 250 mL of water, and the resulting solution was filtered through a glass column of Dowex resin pre-filled with 100 cm ³ of tetrabutylammonium (TBA) form. The eluted solution of the HA-TBA salt was collected and freeze-dried to obtain 7.50 g of product.

Example 2

The sulfated HA was synthesized starting from HA with an average molecular weight of 200 kD and the degree of sulfation was equal to that of each repeating unit. 3 sulfate groups

Method A

10.0 g of the TBA salt of hyaluronic acid with an average molecular weight of 200 kD, prepared according to Example 1, was dissolved in 300 mL of dimethyl sulfoxide (DMSO). 26.0 g of the complex _SO₃ -pyridine (sulfur trioxide and pyridine, hereinafter abbreviated as _PySO₃ ) was dispersed in 150 mL of DMSO and then added to the HA solution. After mechanical stirring at 21°C for 20 hours, the reaction was terminated by the addition of 0.1 volume of water; the crude reaction product was isolated by precipitation after the addition of 2 volumes of ethanol. The resulting solid was dispersed in 150 mL of water and the pH was adjusted to neutral with 1 M NaOH. The mixture was thoroughly dialyzed against water through a membrane with a molecular weight cutoff of 12-14,000 Da. The dialyzed product was freeze-dried. 9.7 g of product was obtained with a degree of sulfation equal to 3 sulfate groups per repeating unit (yield = 88%).

Method B

32.0 g of the TBA salt of hyaluronic acid with an average molecular weight of 200 kD, prepared according to Example 1, were dissolved in 900 mL of N-methylpyrrolidone (NMP); 100 g of _PySO₃ was dispersed in 600 mL of NMP and then added to the HA solution. After mechanical stirring for 20 hours at 21±1°C, the reaction was stopped by adding 0.5 volumes of water; the pH, initially below 2.5, was adjusted to neutral with NaOH (solution). The crude reaction product was isolated by precipitation after adding 2.5 volumes of methanol and washed with 2 volumes of a methanol/water mixture 8/2. The solid was redissolved and thoroughly dialyzed against water through a membrane with a molecular weight cut-off of 12-14,000 Da. 30.4 g of product was obtained with a degree of sulfation equal to 3 sulfate groups per repeat unit (yield = 86%).

Example 3

The sulfated HA was synthesized starting from HA with an average molecular weight of 200 kD and the degree of sulfation was equal to that of each repeating unit. 1 sulfate group

Using the method described in Example 1, 10.0 g of the TBA salt of HA was prepared and dissolved in 350 mL of DMSO. 10.0 g of the complex PySO ₃ was dispersed in 100 mL of DMSO and then added to the HA solution. After mechanical stirring at 21° C. for 20 hours, the reaction was stopped by adding 0.1 volume of water; the crude reaction product was isolated by precipitation after adding 2.5 volumes of ethanol. The solid obtained was dispersed in 150 mL of water and the pH was adjusted to neutral with NaOH 1 mol/L. The mixture was thoroughly dialyzed against water through a membrane with a molecular weight cutoff of 12-14,000 Da. The dialyzed product was freeze-dried. 7.54 g of product was obtained with a degree of sulfation equal to 1.0 sulfate group per repeat unit (yield = 93%).

Example 4

Sulfated HA was synthesized starting from low molecular weight HA (average MW 10 kD, ranging from 10,000 to 50,000 Da). and the degree of sulfation is equal to 3 sulfate groups per repeating unit

Using the method described in Example 1, 12.4 g of the TBA salt of low molecular weight hyaluronic acid were prepared and dissolved in 300 mL of NMP. 40 g of PySO ₃ were dispersed in 100 mL of NMP and then added to the HA solution. After mechanical stirring at 21° C. for 20 hours, the reaction was stopped by adding 0.5 volumes of water. The pH, initially below 2.5, was adjusted to neutral with NaOH 4M. The crude reaction product was isolated by precipitation after adding 2.5 volumes of methanol and washed with 2 volumes of a methanol/water mixture 8/2. The solid was redissolved and thoroughly dialyzed against water through a membrane with a molecular weight cut-off of 3,500 Da. 12.0 g of product was obtained with a degree of sulfation equal to 3.0 sulfate groups per repeat unit (yield = 85%).

Example 5

The sulfated HA was synthesized starting from HA of low average molecular weight and with a degree of sulfation equal to 1 sulfur per repeating unit. acid ester group

Using the method described in Example 1, 12.4 g of the TBA salt of HA were dissolved in 300 mL of DMSO. 16.0 g of PySO ₃ were dispersed in 100 mL of DMSO and then added to the HA solution. After mechanical stirring at 21° C. for 20 hours, the reaction was stopped by adding 0.1 volume of water; the crude reaction product was isolated by precipitation after adding 2.5 volumes of ethanol. The solid obtained was dispersed in 150 mL of water and the pH was adjusted to neutral with NaOH 1 mol/L. The mixture was thoroughly dialyzed against water through a membrane with a molecular weight cutoff of 3,500 Da. The dialyzed product was freeze-dried. 9.04 g of product was obtained with a degree of sulfation equal to 1.0 sulfate group per repeat unit (yield = 90%).

Example 6

Sulfated HA was synthesized starting from HA with a molecular weight range of 500-730 kDa and a degree of sulfation equal to each Repeating unit 3 sulfate groups

21.0 g of extract-derived hyaluronic acid sodium salt (500-730 kD) was dissolved in 1.5 L of water, and the resulting solution was filtered through a glass column pre-filled with 450 cm ³ of Dowex resin in the form of TBA. The eluted solution of the HA-TBA salt was collected and freeze-dried. 32.0 g of product was obtained and dissolved in 1.35 L of NMP; 100 g of PySO ₃ was dispersed in 650 mL of NMP and then added to the HA solution. After mechanical stirring at 23 ± 1°C for 20 hours, the reaction was terminated by adding 0.5 volumes of water. The pH, initially below 2.5, was adjusted to neutral by adding NaOH (solution, concentration 4 mol/L). The crude reaction product was isolated by precipitation after adding 2.5 volumes of methanol and washed with 3.5 volumes of a methanol/water mixture 8:2. The solid was redissolved and thoroughly dialyzed against water through a membrane with a molecular weight cut-off of 12-14,000 Da. 30.3 g of product are obtained with a degree of sulfation equal to 3 sulfate groups per repeat unit (yield = 83%).

Example 7

Sulfated HA was synthesized starting from HA with a molecular weight range of 500-730 kD and a degree of sulfation equal to that of each repeat Unit 1 sulfate group

21.0 g of extract-derived hyaluronic acid sodium salt (500-730 kD) was dissolved in 1.5 L of water, and the resulting solution was filtered through a glass column pre-filled with 450 cm ³ of Dowex resin in the form of TBA. The eluted solution of the HA-TBA salt was collected and freeze-dried. 32.0 g of product was obtained and dissolved in 1.65 L of NMP; 40 g of PySO ₃ was dispersed in 350 mL of NMP and then added to the HA solution. After mechanical stirring at 25±1°C for 20 hours, the reaction was terminated by adding 0.5 volumes of water. The pH, initially below 2.5, was adjusted to neutral by adding NaOH (solution, concentration 4 mol/L). The crude reaction product was isolated by precipitation after adding 3.5 volumes of methanol and washed with 3.5 volumes of a methanol/water mixture 8:2. The solid was redissolved and thoroughly dialyzed against water through a membrane with a molecular weight cutoff of 12-14,000 Da. 22.5 g of product were obtained with a degree of sulfation equal to 1.0 sulfate group per repeat unit (yield = 87%).

Example 8

Evaluation of the regulatory effects of HAS 1 and 3 on IL-10 and IL-12 gene expression in human synoviocytes

Human synoviocytes previously expanded and maintained incubated in vitro in DMEM culture medium containing 10% FCS at 37°C were seeded at a concentration of 20,000 cells per well. Sulfated HA 1 degree (HAS1) and 3 degrees (HAS3) prepared as described in Example 1-3 were then added to the culture medium at concentrations of 0.1 and 0.5 mg/mL (for both samples), while the control treatment was represented by non-sulfated HA with an average molecular weight (MW) of 200 kD. After treatment for 3 days, real-time PCR was performed to assess the gene expression of IL-10 and IL-12: Cell RNA was extracted using the "Trizol" method according to the supplier's instructions (TRIZOL reagent, LIFE Technologies, GIBCO BRL). In short, cells were lysed by adding 1.0 mL of Trizol, and total RNA was quantified by measuring the absorbance at 260 nm. Application software Primer3 (Roche Molecular Diagnostics, Pleasanton, CA, USA) was used to select suitable primers for amplifying each gene. Gene expression was assessed by real-time PCR using a Rotor-gene TM5500 (Corbettresearch, Sydney, Australia). PCR reactions were performed using primers at 300 nm and SYBR Green (Invitrogen, Carlsbad, CA, USA) for 40 cycles, with amplification conditions of 95°C for 15 seconds and 60°C for 1 minute. The fluorescence threshold (Ct) was automatically determined by the software, and the amplification coefficient of the gene under study was estimated to be between 92 and 110%. For each cDNA sample, the gene expression value was expressed by the ratio of the ct of the housekeeping gene (i.e., the gene for β-actin, which represents a control gene because it is present in every cell and is not affected by HAS) to the ct of the gene of interest (i.e., the genes for IL-10 and IL-12). Therefore, the housekeeping gene ct/gene ct value is represented on the vertical axis, thereby representing the amount of mRNA expressed by the gene under study. The results obtained are shown in Figures 1 and 2:

Figure 1 : Treatment of human synoviocytes with HAS1 and HAS3 results in a significant increase in gene expression of the cytokine IL-10 compared to controls treated with non-sulfated HA.

Figure 2 : In this experiment, two sulfated HSA (1 and 3) were shown to significantly reduce IL-12 gene expression and halve its mRNA synthesis compared to the control treated with non-sulfated HA. Therefore, sulfated hyaluronic acid was shown to have:

It can stimulate the production of new mRNA for the synthesis of anti-inflammatory cytokines, thereby enhancing the defense capacity of cells and the entire organism, in the previously described conditions, in which IL-10 has been shown to be very important for the alleviation and/or improvement of diseases in which IL-10 is involved, such as asthma, rheumatoid arthritis, arthritis and all inflammatory diseases.

• Effectively reduces the synthesis of new mRNA of the highly pro-inflammatory cytokine IL-12, proving to be a potent anti-inflammatory drug, able to interfere with the expression of proteins involved in the pathology of ineffective diseases such as psoriasis, arthritis and all those mentioned above.

Example 9

Inhibition of TNF binding to receptors expressed in monocytic cell lines: Evaluation of the effectiveness of HAS 1 and 3 with different MW values Effectiveness

These assays allow for the evaluation of the effectiveness of the test samples (prepared according to Examples 1-4) in inhibiting the binding of TNF to its receptor expressed in vitro by I.S. cells commonly used in such assays, using iodinated cytokine components for evaluation in radioligand binding assays.

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

Briefly, a human tissue cell line of lymphoma U937 was used, characterized in that monocytes are sensitive to TNF cytotoxicity and express its relevant receptors. The cells were initially incubated with ¹²⁵ I-TNF 0.028 nM (performed in water) and simultaneously with the analyzed samples (at a concentration of 1 mg/mL, which was confirmed to be the lowest concentration that caused maximal inhibition) at 4°C in an incubation buffer containing 50 mM Tris-HCl pH 7.4, 0.5 mM EDTA for 3 hours.

After the incubation period, the cells were centrifuged with dibutyl phthalate/dinonyl phthalate 2/1 and the pellets obtained were counted in a gamma-counter.

The results obtained are shown in Figure 3 :

The results obtained show that HSA of degree 1 and degree 3 and medium and low MW completely (100%) inhibit the effectiveness of TNF binding to its receptor. These results are very important because they confirm that the behavior of the sulfated product is completely similar to that of the monoclonal antibody specific for the TNF receptor, thus blocking its function. Therefore, this receptor blockade represents the most effective method to antagonize the pro-inflammatory and tumorigenic effects of TNF factors.

Example 10

Inhibition of IL-1 cytokine binding to receptors expressed in fibroblast cell lines: Evaluation of HAS with different MW values Effectiveness

These experiments allow the evaluation of the effectiveness of the test samples (prepared according to Examples 1-3 and 4) in inhibiting the binding of IL-1 to its receptor expressed by mouse 3T3 cells commonly used in vitro for this type of assay, using iodinated cytokine components for evaluation in radioligand binding assays.

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

Briefly, the mouse fibroblast 3T3 cell line, which is sensitive to the cytotoxicity of IL-1 and expresses its relevant receptor, was used. The cells were initially incubated with 10 pM of ¹²⁵ I-IL-1 (performed in water) and the sample to be analyzed (at a concentration of 1 mg/mL, which was confirmed to be the minimum concentration that caused maximum inhibition) at 37°C for 2 hours in an incubation buffer containing RPMI 1640 containing 20 mM HEPES pH 7.2 and 1% BSA. After the incubation period, the cells were washed with phosphate buffer, then dissolved in 2.5 M NaOH and counted in a gamma counter.

The results obtained are shown in Figure 4 :

The results obtained show that HAS (medium and low MW) is effective up to 30% in inhibiting the binding of IL-1 to its receptor. These results are very significant because they demonstrate that the behavior of the sulfated product is completely similar to that of monoclonal antibodies specific for the cytokine receptor in question, thus blocking its function. This receptor blockade represents the most effective approach to antagonizing the pro-inflammatory and tumorigenic effects of IL-1, as previously described.

Example 11

Inhibition of IL-6 cytokine binding to receptors expressed on human myeloma cells: evaluation of HAS with different MW values Effectiveness

These experiments allow the evaluation of the effectiveness of the test samples (prepared according to Examples 1-3 and 4) in inhibiting the binding of IL-6 to its receptor expressed by the human myeloma U266 in vitro, which is commonly used in such experiments, using iodinated cytokine components for evaluation in radioligand binding assays.

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

Briefly, the human myeloma U266 cell line, which is sensitive to the cytotoxicity of IL-6 and expresses its relevant receptor, was used. The cells were initially incubated with 0.08 nM ¹²⁵ I-IL-6 (performed in water) and the sample to be analyzed (at a concentration of 1 mg/mL, which was confirmed to be the lowest concentration that caused maximum inhibition) at 4°C for 16 hours in an incubation buffer containing RPMI 1640 containing 25 mM HEPES pH 7.1 and 10% BSA. After the incubation period, the cells were washed with phosphate buffer, centrifuged at 9,000 rpm, and the pellets were counted in a gamma counter.

The results obtained are shown in Figure 5 :

The results obtained show that medium and low MW HAS are 100% effective in inhibiting the binding of IL-6 to its receptor. Therefore, these results confirm that the behavior of the sulfated product in this case is completely similar to that of the monoclonal antibody specific for the cytokine receptor in question, thus blocking its function. This receptor blockade represents the most effective method to block the pro-inflammatory effects of IL-6.

Example 12

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

For these experiments, human peripheral blood mononuclear cells (PBMCs) derived from a number of different hosts were used to assess the effects of HSA on the production of the cytokines listed above using:

- non-sulfated HA (average MW: 200KD),

- HAS1 and HAS3 (prepared as described in Examples 1-3) (average MW: 200 kD).

PBMCs (A., Scand J Clin Lab Invest 21 Suppl, 1968, 97:77-89) were isolated using Ficoll-Paque PLUS (GE Healthcare) according to the manufacturer's protocol. On day 0, 100,000 cells were seeded per well in 200 μL of RPMI 1640 medium supplemented with 10% fetal bovine serum, 10 mM HEPES, 2 mM glutamine, and 100 U/mL of 1% penicillin-streptomycin. The effects of all samples were evaluated in PBMCs that were either untreated with agents that stimulate cytokine synthesis or stimulated with lipopolysaccharide (LPS) (10 μg/mL), a potent proinflammatory agent, or with phytohemagglutinin (PHA) (10 μg/mL), a substance that stimulates lymphocyte autolysis. Cells were then treated with the three compounds at concentrations of 0.1 mg/mL or 1 mg/mL. After incubation at 37°C (5% CO ₂ ) for 24 hours, 100 μL of supernatant was removed from each well and analyzed for IL-2, IL-7, IL-10, and IL-12 production.

Quantification of inflammatory mediators was accomplished using the Custom Human 9-Plex Array plate according to the supplier's protocol specified on the technology card.

The results obtained are shown in Figures 6-9 :

These figures clearly show that HAS 1 and 3 degrees can significantly reduce the synthesis of IL-2, IL-7 and IL-12 on some monocytes when the cells are not stimulated and, on the contrary, when they are stimulated by specific and effective inflammatory factors and/or mitogens. Therefore, HAS proves to be a molecule with clear pharmacological characteristics that can regulate/adjust the synthesis of cytokines with significant anti-inflammatory activity, in the case where the immune response is not stimulated and, in particular, inflammatory stress events (wherein immune cells respond by producing a cytokine cascade), in this case, in short, the data presented show a greater HAS regulatory effect.

On the other hand, Fig. 9 has confirmed that it is the generation of IL-10 and for the obvious stimulation of the cell such as human mononuclear cell (except synoviocyte) belonging to immune system.Therefore, it has been confirmed again that HAS can regulate the synthesis of cytokines, stimulate those cytokines of anti-inflammatory and suppress the synthesis of proinflammatory cytokines.

Example 13

Evaluation of the antiviral effects of HAS 1 and 3 and HA-NS:

HIV-1(III _B ) and HIV-2(ROD) viruses

The activity of the test samples was determined by evaluating the inhibition of cytopathogenicity of HIV-1 virus (obtained from T lymphocytes infected with type III _B virus) and HIV-2 virus (obtained from T lymphocytes infected with type ROD virus) in MT-4 cells. The test method was performed as described in Baba M. et al., ANTIMICROB. AGENTSCHEMOTHER., 1988, 32: 1742-1745.

Briefly, human T lymphocytes (designated MT-4) infected with HIV-1 strain III _B and HIV-2 strain ROD were used in this assay. Different concentrations of test samples were used in this assay, and dextran sulfate, a polysaccharide known to inhibit different types of viruses, was used as a positive control. The test samples were:

1. HS-NS1: Sulfated hyaluronic acid prepared by sulfation of glucosamine followed by deacetylation, 1 degree (EP0971961), compared with the following samples

2. HAS1: Hyaluronic acid sulfated only in its hydroxyl groups, 1 degree, and

3. HAS3: Hyaluronic acid sulfated only in its hydroxyl groups, 3 degrees, prepared as described in Examples 1-3 (average MW of all samples: 200 KD).

The antiviral activity of the test samples is expressed as the minimum concentration required to inhibit the viral cytopathogenicity by 50%: IC50 . Cells were infected with an infectious dose of HIV and immediately treated with various concentrations of the above samples. After 5 days at 37°C, the number of viable cells was determined using the MTT assay: tetrazolium salts undergo redox reactions only through the mitochondrial enzymes of living cells. Infected and untreated cells lose the processive ability to convert tetrazolium salts to formazan. Therefore, if these cells retain this ability after infection and treatment, it means that the treatment inhibited the viral action, thereby blocking the viral pathogenicity (Dezinot F. et al., J Immunol. Methods, 1986, 22(89):271-277).

The results obtained are shown in Figure 10 :

The results obtained demonstrate the effectiveness of HAS (degrees 1 and 3) in inhibiting viral replication of the viruses HIV 1 and 2, an effectiveness that is fully comparable to that of the positive control (represented by the polysaccharide dextran sulfate). The test also demonstrates that not all sulfated hyaluronic acids known to the state of the art have an effective effect on the replication of the virus studied, since the Applicant has demonstrated that only HAS in which the hydroxyl groups are sulfated demonstrates an effective antiviral effect, since HA-NS is ineffective in blocking the viral cytopathogenicity.

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

The activity of the test samples was determined by evaluating the inhibition of cytopathogenicity caused by herpes simplex virus-1 (HSV-1: KOS, F, and McIntyre strains) and herpes simplex virus-2 (HSV-2: G, 196, and Lyons strains) in _E6 SM fibroblasts derived from muscle/embryonic skin tissue. Furthermore, the antiviral activity was tested against _{E6 SM} cells infected with vesicular stomatitis virus (VSV). HSV-1 preferentially infects the oral mucosa, while HSV-2 attacks the genital mucosa. The test method was performed as described in Baba M. et al., ANTIMICROB. AGENTS CHEMOTHER., 1988, 32: 1742-1745.

In short, in the presence of sample HS-NS1 (EP0971961), HAS1 and HAS3 (average MW of all samples: 200KD) prepared as described in Examples 1 and 2, the fused cell culture was exposed to an infectious dose of the above-mentioned virus. After incubation at 37°C for 1 hour, the culture medium was replaced with a fresh culture medium containing only the test sample. The cytopathogenicity of the virus was tested on the 2nd day of incubation. The measurement of the cytopathogenicity of the virus was evaluated by determining the synthesis of DNA and RNA in the "infected" cells, and the above-mentioned treatment was performed: the cells were seeded in micropores containing different concentrations of test samples and culture medium containing 2.5 μCi 3H-thymidine and 3H-uridine per mL. After 16 hours at 37°C, the cells were treated with trichloroacetic acid, washed in ethanol, dried and counted by scintillation in 7.5mL liquid. The antiviral activity of the test sample is expressed as the minimum concentration required to inhibit 50% of the viral cytopathogenicity: IC50. Furthermore, to simultaneously assess the cytotoxicity of the test samples, the minimum concentration required to cause morphological damage to the cells used (observed using light microscopy) was determined. Comparisons with dextran sulfate and the drug acyclovir (both molecules have known antiviral efficacy and were therefore used as positive controls) were also performed.

The results obtained are shown in Figure 11 :

The experimental data confirm the effective antiviral effect observed for HIV, namely HAS1 and HAS3: the comparison between HA-NS1 and HAS1 shows that not all sulfated hyaluronic acids are equal, since HA-NS1 has not been shown to be active, and that this difference in efficacy does not depend on the molecular weight or the degree of sulfation of the hyaluronic acid, and therefore lies entirely in the structure of HA-NS1 and HAS1, namely in the sulfation. In fact, HAS showed an efficacy equivalent to that of dextran sulfate and comparable to that of acyclovir, a reference drug for the treatment of herpes simplex. In addition, it should be noted that acyclovir is inactive against VSV, while HAS 1 and 3 degrees have very strong antiviral activity against VSV.

All tested samples were non-cytotoxic to host cells; in fact, the minimum cytotoxic concentrations achieved were equivalent to those of reference drugs commonly used in clinical practice for the treatment of herpes and demonstrated to be on average 100-fold higher than concentrations proven active in inhibiting viral replication.

Cytomegalovirus

The activity of the test samples was determined by evaluating the inhibition of cytopathogenicity using cytomegalovirus (CMV: AD-169 and Davis strains) using a previous protocol. Antiviral activity was tested on HEL cells (embryonic lung cells) and expressed as the concentration required to inhibit 50% of the number of plaques formed by the above viruses.

The results obtained are shown in Figure 12 :

The table shows that clear and significant positive results were obtained for HAS1 and HAS3, which once again confirms their effectiveness as effective antiviral agents. In this case, HA-NS1 was not shown to be effective in inhibiting viral proliferation, thus confirming the absolute diversity of the two types of 1-degree sulfated products in having antiviral capabilities.

Example 14

Preparation of a formulation in the form of an inhalation solution containing sulfated hyaluronic acid 1

40mg (or 20mg if HAS has a MW of 500-730kD) of low or medium MW sulfated hyaluronic acid was introduced into a 50mL glass flask, followed by the addition of 15mL of sterilized PBS 0.2M pH 7.4. The mixture was stirred for approximately 30 minutes until the powder was completely dissolved. When complete dissolution was achieved, 2mL of propylene glycol and additional sterilized PBS 0.2M pH 7.4 were added until a cumulative volume of 20mL was reached. The solution was maintained under stirring for several minutes.

Example 15

Preparation of a formulation in the form of an inhalation solution containing 3 degrees of sulfated hyaluronic acid

100 mg of sulfated hyaluronic acid obtained from HA 200KD was introduced into a 50 mL glass flask at 3°C, followed by the addition of 15 mL of sterilized PBS 0.2M pH 7.4. The mixture was stirred for approximately 30 minutes until the powder was completely dissolved. Once fully dissolved, 2 mL of propylene glycol and additional sterilized PBS 0.2M pH 7.4 were added until a cumulative volume of 20 mL was reached. The solution was maintained under stirring for several minutes.

Example 16

Preparation of a formulation in the form of an injectable solution for intra-articular application containing sulfated hyaluronic acid 1

500 mg of sulfated hyaluronic acid obtained from HA with a MW of 500-730 kD was introduced into a 50 mL glass flask, and then sterile PBS 0.2 M pH 7.4 was added until the total volume reached 20 mL. The mixture was stirred for about 60 minutes until the powder was completely dissolved.

Example 17

Preparation of a hydrophilic gel formulation containing HAS, HA and CMC

Methylparaben and propylparaben were dissolved in pure water at 80°C. After the solution was cooled to room temperature, sodium hyaluronate was added under stirring until dissolved, followed by HAS1 (or HAS3), and stirring was maintained until completely dissolved. Glycerin and propylene glycol were then added under stirring until completely dissolved. Finally, sodium carboxymethylcellulose (CMC) was added and the mixture was mixed until a colloidal solution was obtained.

Example 18

Preparation of a hydrophilic gel formulation containing HAS and HA for mucosal application (without preservatives)

Sodium hyaluronate and then HAS1 (or HAS3) are dissolved in about 90% of the water set in the formulation under stirring. Propylene glycol, Symdiol 68, and subsequently MP Diol Glycol are added, and the mixture is mixed until the various components are completely dissolved. Carbomer 974P is then added and stirred until the latter is evenly dispersed. Sodium hydroxide beads are dissolved in the remaining 10% water, and this solution is added to the solution obtained above to obtain a gelled aqueous phase.

Example 19

Preparation of a lipstick formulation containing HAS and HA

Place the appropriate amount of liquid paraffin indicated in the production formulation into a suitable container. Heat it to 88-92°C, then add white soft paraffin, hard paraffin, white beeswax, ozokerite, and arlacel while stirring, maintaining stirring until the various components are completely melted. Then add all-rac-α-tocopheryl acetate, allantoin, butylated hydroxytoluene, and propylparaben, and mix the mixture until it is completely dissolved, maintaining the material at 88-92°C.

Pour the set amount of pure water in the preparation into suitable containers respectively, then add sodium hyaluronate and HAS1 (or HAS3) under stirring until they are completely dissolved, and then add disodium edetate and keep stirring until dissolved.

The aqueous phase was transferred to the container containing the molten material under stirring, the system was maintained at 88-92 ° C, and stirred until a clear solution was obtained. The two aromatizers were then added under stirring and the mixture was mixed for 10'. The molten material was poured into a mold and immediately cooled to T < 0 ° C until a solid stick was obtained.

Example 20

Preparation of a vaginal ovule formulation containing HAS and HA

Gelatin is expanded in 70% pure water at 85°C; sodium hyaluronate and HAS1 (or HAS3) are dissolved in the remaining water, and this solution is mixed with glycerol at the same temperature. The glycerol solution is added to the expanded gelatin solution and stirred until the gelatin is completely dissolved. The mass is poured into a mold and cooled to T < 0°C until solid ovules are obtained.

Example 21

Preparation of a hydrophilic cream formulation containing HAS and HA

The oil phase is prepared by melting liquid paraffin, stearic acid and Tefose 1500 at 50° C. under stirring. The aqueous phase is prepared separately by first dissolving methylparaben at 80° C., then cooling to room temperature, and incorporating glycerin, sodium hyaluronate, and then HAS1 (or HAS3) under stirring until the various components are completely dissolved.

The aqueous phase was combined with the oil and emulsification was completed, and the obtained emulsion O/A was cooled to room temperature under stirring.

Example 22

Preparation of ointment-containing formulations containing HAS

The base ointment was prepared by melting light liquid paraffin and white petrolatum with stirring at 70° C. After cooling to room temperature, HAS1 (or HAS3) was incorporated with stirring and the mixture was mixed until a homogeneous suspension was obtained.

Example 23

Preparation of a formulation in the form of a capsule (hard gelatin) containing HAS

HAS1 (HAS3) was mixed with calcium phosphate, magnesium stearate and silicon dioxide by graded dilution and then filled into capsules.

Example 24

Preparation of a tablet formulation containing HAS

HAS1 (or HAS3) was wet granulated with a ligand solution containing water and about 70% of the total amount of sodium CMC in a fluidized bed. The obtained granules were sieved on a 0.8 mm mesh.

The remaining ingredients (calcium sulfate, microcrystalline cellulose, sodium CMC and silicon dioxide) were mixed and sieved on a 0.8 mm mesh.

The granules obtained previously are mixed with a mixture containing the remaining ingredients (additional granules) and finally with magnesium stearate (previously sieved on a 0.8 mm mesh) and subsequently compressed.

Example 25

Preparation of HAS-containing injection solution

After preparing a physiological buffer solution of pH 6.4-7.2 (at room temperature), lactose is dissolved under stirring and finally HAS1 or HAS3 is dissolved. The solution thus obtained is filtered through 0.22 microns.

surface

Hydrophilic gel (Example 17)

Components Amount (mg/1g hydrogel) HAS1(HAS3) 40mg (10mg) CMC 20mg glycerin 100mg Propylene glycol 66.75mg Sodium hyaluronate 2mg Methylparaben 2mg Propylparaben 0.2mg pure water Up to 1g

Hydrophilic gel for mucosal application (Example 18)

Components Amount (mg/1g hydrogel) HAS1(HAS3) 10mg Carbomer 974P 15mg Propylene glycol 100mg Sodium hydroxide 0.33mg Sodium hyaluronate 2mg MP-Diol Glycol 37.5mg SymDiol 68 90mg pure water Up to 1g

Lipstick (Example 19)

Components Amount (mg/1g lipstick) HAS1(HAS3) 30mg (10mg) liquid paraffin 253.2mg White soft paraffin 326.2mg Hard paraffin 144.3mg White beeswax 96mg Ozokerite 28.2mg Arlacel 582 95.8mg Sodium hyaluronate 2mg Allantoin 1.1mg All-rac-α-tocopheryl acetate 1.1mg Propylparaben 0.4mg Butylated hydroxytoluene 0.4mg pure water 19.2mg Edetate disodium 1.1mg Vanilla flavoring 0.5mg Sweet flavoring agents 0.5mg

Vaginal ovules (Example 20)

Components Amount (mg/1g ovule) HAS1(HAS3) 10mg glycerin 580 gelatin 200 Sodium hyaluronate 2 pure water Up to 1g

Hydrophilic cream (emulsion O/A) (Example 21)

Components Amount (mg/1g cream) HAS1(HAS3) 10mg Tefose 1500 110mg glycerin 80mg stearic acid 33mg Sodium hyaluronate 2mg liquid paraffin 40mg Methylparaben 1mg pure water Up to 1g

Ointment (Example 22)

Components Amount (mg/1g ointment) HAS1(HAS3) 20mg Light liquid paraffin 200mg White Vaseline Up to 1g

Capsules (hard gelatin) (Example 23)

Tablet (Example 24)

Components Amount (mg/tablet) HAS-1(HAS-3) 120mg calcium phosphate 200 microcrystalline cellulose 185 Sodium carboxymethyl cellulose 10 magnesium stearate 10 Silicon dioxide 15

Injection preparation (Example 25)

Components Amount (mg/mL solution) HAS-3 50mg lactose 0.93mg Dipotassium hydrogen phosphate 0.36 Potassium dihydrogen phosphate 0.23 Sodium chloride 9

It will be apparent that the instructions and methods thus described may be modified in many ways. These modifications should not be considered as a departure from the spirit and scope of the invention, and all modifications that would be obvious to one skilled in the art are intended to be included within the scope of the following claims.

Claims (3)

1. Use of sulfated hyaluronic acid in the preparation of medicaments for the prevention and/or treatment of HSV-1 herpes simplex labialis, HSV-2 genital herpes simplex, VSV herpestostomosis virus, and CMV cytomegalovirus; wherein the preparation of said sulfated hyaluronic acid (HA) involves an alcohol hydroxyl group present in a polysaccharide chain, starting with HA having a molecular weight range of 150,000 to 250,000 Da, and the degree of sulfated hyaluronic acid (HA) is equal to 3. 2. The use of claim 1, for systemic application. 3. The use of claim 1, for topical or inhalation application.