HK1167083B - New medicines for topic use based on sulfated hyaluronic acid as activating or inhibiting agent of the cytokine activity - Google Patents

Description

New topical medicaments based on sulfated hyaluronic acid as activators or inhibitors of cytokine activity

Field of the Invention

For many years, the scientific/patent literature has studied and described sulfated hyaluronic acid, which is obtained by suitable sulfation as described in the prior art (EP0940410B1 and EP0702699B1) using hyaluronic acid (HA) as a raw material, to which the anticoagulant effect is attributed. HAS can also be obtained by deacetylation of the glucosamine of HA followed by sulfation (defined as HA-NS) (EP0971961B1) for the production of surgical articles and pharmaceutical compositions. Patents EP0754460B1 and EP1385492B1 are also known, which describe the use of HAS in pathological conditions such as ARDS (severe respiratory insufficiency), rheumatic arthritis and rheumatoid arthritis. The object of the present invention relates to a new and unexpected skin application of HAS as a modulator of cytokine activity, since the applicant has discovered the unique ability of HAS to modulate the activity of specific cytokines (both pro-inflammatory and anti-inflammatory), it has studied its mechanism of action and revealed the main differences between the two classes of sulfated products (HAS and HA-NS), but most importantly, the applicant has unexpectedly discovered an unexpectedly high activity against different types and strains of herpes viruses, cytomegalovirus and vesicular stomatitis viruses.

Finally, another object of the present invention relates to the use of HAS as a skin absorption enhancer for drugs with anti-inflammatory and hormonal properties.

Since 1970, scientists have understood that selected populations of lymphoid cells can produce and release into the circulatory bed proteinaceous molecules that are not assimilated into antibodies, which are defined by the term "cytokines." They represent a new class of "hormones" that can act on different cellular targets in multiple areas of the body.

The progress in scientific knowledge about 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 has opened new horizons in the understanding of its numerous functions, thus creating new prospects for local and/or systemic treatment of different pathological conditions, including new therapeutic possibilities regarding cancer immunotherapy.

Lymphocytes are central to I.S., accounting for approximately 20% of all white blood cells. They are divided into three functional groups: B lymphocytes, T lymphocytes, and cytotoxic lymphocytes. Many cytokines are soluble proteins produced by lymphocytes and/or monocytes that can act against other cells and tissues distant from their site of production. They possess immunological functions and also play a regulatory role in the synthesis of other cytokines by various I.S. cells or target cells involved in the I.S.-induced reaction cascade.

A large number of different cytokines have been studied to date, which also have a large number 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, along with TNF, are defined as cytokines with inflammatory properties, and interleukin 10 (IL-10), which, on the contrary, is a cytokine with strong anti-inflammatory properties.

The first cytokine to be studied was clearly IL-1 : it exists in two forms, α and β, and is a potent inducer of proinflammatory processes (systemic and/or cutaneous). It is produced primarily by B lymphocytes, T lymphocytes, and macrophages in response to bacterial stimuli or stimulation with other factors, including other cytokines; it is also secreted by peripheral neutrophils, endothelial, epithelial, and smooth muscle cells, fibroblasts, cutaneous Langerhans cells, osteoclasts, synoviocytes, and many other cell types. Both forms bind to the same receptors and possess very similar, if not identical, biological activities. Most of its proinflammatory functions are associated with stimulation by other cytokines, such as IL-6 and IL-8, and its own 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 demonstrated that IL-1 can activate the expression of several oncogenes, thus contributing to the pathogenesis of neoplasia. Therefore, IL-1 represents one of the main mediators of the inflammatory process in combination with other cytokines: it actually stimulates T-cells to produce IL-2 and stimulates B-cells to produce immunoglobulins. It is also involved in the morbidity of rheumatoid arthritis and arthritis: in fact, a large amount of IL-1 has been found in the synovial fluid of patients with rheumatoid arthritis and/or osteoarthritis. It is also active in a large number of pathological conditions with general skin properties, such as common dermatitis, atopic dermatitis and psoriasis. Finally, it participates in the establishment of vascular damage such as venous thrombosis and is present in all blood vessels with arterial/arteriosclerotic type pathological conditions. For this cytokine, receptor antagonists have been used clinically (and also in experiments) at present, because blocking this receptor has been proven to be an effective way to treat these pathological conditions in which IL-1 is one of the protagonists.

TNF : Necrokinin is part of a group of cytokines that promote the acute systemic inflammatory phase. Thus, TNF is 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 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 various pathogenic processes:

- It regulates the expression of many proteins and important cytokines such as IL-1 and IL-6, leading to its involvement in skin pathologies such as vitiligo, eczema, psoriasis and dermatitis in general;

- 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 and thus induces bone resorption (osteoporosis);

-It strongly attracts neutrophils and helps them attach themselves to endothelial cells for extravasation;

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

- It is involved in specific pathological conditions of the cardiocirculatory system participating in the development of venous thrombosis, arteriosclerosis and vasculitis.

TNF is able to bind itself 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 erythrocytes. In short, TNF promotes inflammatory responses both systemically and in the skin, thereby triggering a large number of pathological conditions that are also autoimmune in nature, such as rheumatoid arthritis, Crohn's disease, psoriasis and asthma. To date, scientific research has attempted to perfect "biological" drugs (such as monoclonal antibodies) that inhibit the synthesis of TNF and/or block its receptors.

IL-2 : This is a highly proinflammatory, atherogenic cytokine produced primarily by T lymphocytes, whose production is inhibited by steroids and cyclosporine. IL-2 plays a crucial role in regulating immune responses: it stimulates IFN synthesis in peripheral leukocytes and induces IL-1 and TNF production. 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, there are a number of 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 : Produced by many cell types, especially IS, it is one of the most important members of the group of chemical mediators in the acute phase of inflammatory processes, along with TNF. Therefore, it is involved in pathological conditions with a strong inflammatory component, such as asthma (in which it participates in the development and maintenance of the inflammatory process), chronic enteritis (Crohn's disease), rheumatoid arthritis, and arthropathy. In fact, as previously established, cytokines such as TNF, IL-1, and IL-6 have been shown to be highly involved in the process of degenerative osteoarthritis, as they play a major role in regulating the expression of metalloproteinases (responsible for cartilage degradation), the production of prostaglandins, and osteoclast activation. For this reason, high levels of cytokines have been recorded in the synovial fluid of patients with arthropathy and rheumatoid arthritis (RA). These findings have stimulated the use of inhibitors and/or receptor antagonists of the above-mentioned interleukins as new therapeutic strategies for arthropathy pathologies.

Finally, recent studies have linked cancer to lifespan and revealed how some tumors are affected by the type/amount of cytokine proteins in the patient: in short, recent evidence has linked low IL-10 production characteristics and high IL-6 secretion to worse clinical survival in patients affected by tumor pathology, while a genotype that can produce and maintain high levels of IL-10 can benefit survival (Caruso C. et al., Ann N.Y. Acad. SCI., 2004, 1028: 1-13).

IL-7 : A cytokine produced primarily by bone marrow stromal cells. It is also secreted by the thymus and keratinocytes. IL-7 induces the synthesis of inflammatory cytokines such as IL-1, IL-6, and TNF, thereby participating in the pathogenesis of some skin diseases (such as psoriasis and cutaneous lymphoma) and the osteoarticular system. In fact, high levels of IL-7 have been found in patients with RA.

IL-12 : This protein also plays an important role in regulating IS function. It plays a role in lymphocyte differentiation, induces the synthesis of interferon and TNF, and its production can be inhibited by IL-10. Excessive production of this protein contributes to 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 : Produced primarily by lymphocytes, it is a cytokine with anti-inflammatory properties that can inhibit the synthesis of IL-2 and interferon produced by T lymphocytes. IL-10's anti-inflammatory effects have also been demonstrated by its ability to inhibit the synthesis of IL-1, IL-6, IL-8, IL-12, and TNF in macrophages stimulated with bacterial endotoxins. IL-10 deficiency is associated with pathological conditions such as diabetes and chronic enteritis, such as Crohn's disease. Recent studies have also led to the testing of IL-10 as a new therapeutic approach for lupus erythematosus. Low IL-10 levels have been observed in the skin tissue of patients with pathological conditions such as vitiligo, psoriasis, eczema, and dermatitis. It should be noted that during conventional immunosuppressive therapy for the treatment of inflammation and organ rejection, both corticosteroids and cyclosporine increase the production of this interleukin and/or its release from relevant competent cells (Zhou X. et al., Current Drug Targets-Immune, Endocrine & Metabolic Disorders, 2005, 5(465475). Experimental data have also demonstrated its effectiveness in reducing the release of prostaglandins and cyclooxygenase induced by TNF in vitro on human synoviocytes, thereby demonstrating the ability of IL-10 to alleviate the inflammatory process involved in joints affected by osteoarthrosic degeneration (Alaaeddine N. et al., Arthritis & Rheumatism, 1999, 42: 710-718). Recent studies have demonstrated its therapeutic efficacy against asthma pathology in experimental animal models of bronchial hyperresponsiveness, demonstrating how this cytokine has a high therapeutic potential in reducing the inflammation that characterizes the airways of asthmatic patients, wherein high concentrations of TNF, IL-1, IL-5, IL-6, and IL-8 have been found in bronchial washings and/or at the serum and/or tissue levels (Stankiewicz W. et al., Mediators of Inflammation, 2002, 11: 307-312). Thus, an important role for this interleukin as a mediator cytokine in maintaining immune homeostasis has been postulated.

Asthma can be a debilitating disease that affects approximately 200 million people worldwide, with more than 5,000 deaths each year. It is a pathological condition based on a distorted response of the I.S. to environmental factors, resulting in an increased production of proinflammatory cytokines that promote the growth and differentiation of mast cells, eosinophils, and other cell types of the I.S. The causes of this imbalance in immune system activity are still not fully understood, but there are genetic, environmental, viral, and other nutritional factors that contribute in different ways to the development of this pathology. Therefore, the discovery of an effective treatment (systemic and/or topical) for its prevention and/or treatment that allows the discontinuation or reduction of steroid use (conventional therapeutic regimen) could represent an effective solution for both more severe forms (since it would allow the use of steroids to be reduced) and less severe cases (since steroid treatment could be discontinued altogether).

Detailed Description of the Invention

The object of the present invention is a new and unexpected topical use of HAS as a modulator of cytokine activity, since the applicant has discovered its unique ability to modulate the activity of specific cytokines, it has studied its mechanism of action and revealed the main differences between the various types of sulfated products known in the state of the art, but most importantly the applicant has discovered an unexpected activity against different types and strains of herpes viruses, cytomegalovirus and vesicular stomatitis virus. Finally, another object of the present invention relates to the use of HAS as a dermal absorption accelerator for drugs with general anti-inflammatory properties, as a fibrinolytic agent and as a high hydrating agent for the treatment of all skin pathologies characterized by dryness, irritation and redness, inflammation and peeling.

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 using as starting material HA via the complex SO _3- pyridine, and involving the alcoholic hydroxyl groups present in the polysaccharide chain, said HA coming from any source, for example by fermentation or biotechnological extraction from Celosia cristata, and having a molecular weight of 400-3×10 ⁶ Da, in particular 1×10 ⁴ Da-1×10 ⁶ Da, even more particularly 10,000-50,000 Da, 150,000-250,000 Da and 500,000-750,000 Da.

The resulting derivatives retain all the physical characteristics of the unaltered base polymer. In particular, the molecular weight of the base HA is not reduced by the sulfation process, thus maintaining all the physicochemical characteristics of the base polysaccharide. The sulfation affects each hydroxyl group of the disaccharide unit, so it is possible to obtain different degrees of sulfation (the number of sulfate groups per disaccharide unit) from 0.5 to 3.5 by varying the amount of SO _3- pyridine, as is known in the prior art.

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

Both sulfation degrees of HSA are soluble in water. They can also be sterilized using conventional techniques known to those skilled in the art, although sterilization using an autoclave is preferred.

Applicants describe and claim a novel use of HAS in the preparation of a medicament for topical application which:

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

For the prevention by inhalation and/or topical treatment of asthma associated with activation of IL-1, IL-6, and TNF;

for the prevention and/or treatment of skin pathologies associated with damage to the vascular endothelium and/or vessel wall, due, for example, to trauma, superficial and/or medium-depth vascular bleeding, and subsequent clot and edema formation;

● For the prevention and/or treatment of skin diseases associated with the increase/activation of IL-1, IL-2, IL-6, IL-7, IL-8, IL-12 and TNF, such as dermatitis, atopic dermatitis, psoriasis, vitiligo, solar dermatitis, urticaria, all skin irritations (including gingival irritations) and eczema;

For the prevention and/or treatment of diseases of an autoimmune nature, such as psoriasis, asthma, and cutaneous manifestations of systemic (LES) and discoid lupus erythematosus;

For the prevention and/or topical treatment of skin neoplasia, such as basal cell carcinoma, Kaposi's sarcoma, squamous cell carcinoma, cutaneous lymphoma, mycosis fungoides, and actinic keratosis;

●For the prevention and/or topical treatment of vascular pathologies associated with TNF, IL-1, and IL-6 activation, such as vasculitis and scleroderma;

The applicant has actually also confirmed in the experiments described below:

HAS is able to stimulate the production of new mRNA and protein synthesis of cytokines with anti-inflammatory properties (such as IL-10), thereby increasing the immune defense capacity of cells and thus the entire organism. The anti-inflammatory effect of the above-mentioned cytokines is shown by the ability to inhibit the synthesis of IL-1, IL-6, IL-8, IL-12 and TNF, which are highly pro-inflammatory proteins involved in various skin pathological conditions.

● In situations where no immune response is elicited and in particular in inflammatory stress events where cells respond by producing a cytokine cascade, HAS is effective in reducing the synthesis of new mRNA and significantly reducing protein synthesis of IL-2, IL-7 and IL-12: in this case in particular, the data provided show a higher effect of HAS.

HAS effectively inhibited the binding of TNF, IL-1, and IL-6 to their receptors. These results are of fundamental importance because they demonstrate that the sulfated product behaves exactly like monoclonal antibodies specific for the receptors of these proinflammatory proteins, thus blocking their function without possessing the specificity of these antibodies. This receptor blockade represents the most effective way to antagonize the proinflammatory and tumorigenic effects of TNF, IL-1, and IL-6, thus opening new horizons for clinical trials and potentially enabling the development of new therapeutic approaches for the treatment and/or prevention of a vast array of pathological conditions, given the role that TNF, IL-1, and IL-6 play in the development and progression of numerous systemic and cutaneous diseases.

Applicants also describe and claim a novel use of HAS in the preparation of a medicament for topical application, said medicament:

●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.

The applicant has actually confirmed the potent antiviral effect of HAS against different types of viruses in the experiments described below:

● Experimental data demonstrate 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 (extremely widespread) is responsible for the appearance of characteristic febrile vesicles that usually affect the skin of the face (lips, nostrils); it is also known as herpes simplex labialis . The infection caused by herpes labialis can easily reappear because the virus survives inside the cells and cannot be accurately eliminated with effective drugs. The second form is genital infection, also known as genital herpes . Both forms are infected through physical or sexual contact. Because the virions are located in the ganglia, where they can remain dormant for a long time, herpes infections have recurring characteristics corresponding to stress events of the immune system and usually reappear at the primary site. Vesicular stomatitis virus is an RNA-virus that attacks mammals and is used in the laboratory to study the development of the RNA-virus life cycle. The comparison between HA-NS1 and HAS1 once again shows that not all sulfated hyaluronic acids are equivalent, as HA-NS1 has been shown to be completely inactive, while both HAS1 and 3 have shown very strong antiviral activity against herpes simplex and against VSV. None of the samples tested proved to be cytotoxic to the host cells, in fact, the lowest cytotoxic concentrations obtained were equal to those of the reference drugs commonly used in clinical practice to treat herpes and, on average, proved to be 100 times higher than those shown to be active in inhibiting viral replication.

The experimental data obtained for HAS1 and HAS3 have revealed clear and significant antiviral results against cytomegalovirus : this is a special type of virus that enters some types of cells of our organism, where it parasitically replicates itself, causing them to die. It belongs to the same family as herpes labialis and genital herpes, chickenpox and infectious mononucleosis. Epithelial cells, mucosa, lymph nodes are the sites of multiple primary infections. It remains in peripheral blood, renal tubular epithelium and salivary gland epithelium in a latent state throughout life. Severe forms have been found in immunocompromised patients (such as those affected by AIDS and transplant patients in immunosuppressive therapy). The treatment therapy is to administer drugs such as ganciclovir, valganciclovir and foscarnet (viral DNA synthesis inhibitors). In this case as well, it has been shown that HA-NS1 is inactive in suppressing viral proliferation, thus confirming the absolute diversity of antiviral ability between the two types of sulfated products.

Another object of the present invention relates to the use of HAS as a fibrinolytic agent for degrading fibrin clots that are formed at the skin level (superficial and/or deep) after damage to the endothelium and/or walls of capillaries and/or small blood vessels due to mechanical trauma and/or bleeding of medium/small entities.

In the following experiments, the applicant has actually demonstrated that:

HAS is as effective as plasmin in the fibrinolysis/debridement of clots and thrombi. Plasmin is an important enzyme that belongs to a group of hydrolytic enzymes that are able to degrade many plasma proteins, especially fibrin in thrombi and clots. The degradation of fibrin is called fibrinolysis . A lack of plasmin can lead to thrombosis because the clot is not sufficiently degraded. It should be noted that there is a huge difference between the anticoagulant process and the fibrinolytic process: in the former case, the anticoagulant must prevent the formation of the clot, in the latter case, the fibrinolytic agent, on the other hand, must intervene in a situation where the clot is already present and must therefore be degraded in order to completely eliminate it.

Another object of the present invention relates to the novel use of HAS as a skin absorption promoter for drugs, for example drugs with anti-inflammatory properties, and finally as a high hydration agent for the treatment of all skin pathologies characterized by dryness, lichenification, irritation, itching and redness, inflammation and scaling.

The applicant has actually demonstrated that:

●Sulfation of hyaluronic acid significantly increases skin absorption, therefore,

The hydration capacity of HAS has been shown to be significantly higher than that of non-sulfated HA, thus resulting in a significant reduction in the roughness of the treated skin surface, both with respect to HA and topical control formulations, thereby revealing its ability to effectively treat and protect the skin surface characterized by dryness, irritation, lichenification, itching and redness, inflammation and scaling, as well as all other skin pathologies that make the skin more sensitive to external agents.

HAS is a powerful and effective drug skin absorption promoter. The ability of HAS to penetrate the thickness of the skin so effectively is the scientific basis for the establishment of this surprising and unexpected new property, which enables the preparation of pharmacological substances with different properties such as non-steroidal anti-inflammatory types (especially diclofenac, ketoprofen and ibuprofen) or steroid types, hormones, vasodilators, cholinergic drugs, antibiotics, etc. into various forms, preferably gels, creams or patches, for skin and/or transdermal absorption.

Finally, the applicant describes the preparation of various topical pharmaceutical preparations/compositions containing 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, nonsteroidal anti-inflammatory drugs (FANS) such as diclofenac, ketoprofen or ibuprofen or their salts, topical chemotherapeutics, antibiotics, antivirals, local anesthetics, anticoagulants and/or fibrinolytics and/or enzymes such as collagenase and/or hyaluronidase and/or other proteases; which can be formulated with polymers such as hyaluronic acid and its derivatives, carboxymethylcellulose (CMC) and/or other polymers of natural (e.g. collagen) or synthetic nature.

The pharmaceutical composition can be formulated as an ointment, lipogel, hydrogel, lipstick, cream, vaginal beads and bougies, foams, mucosal gels, ophthalmic preparations, vaginal douches, mouthwashes, patches for cutaneous and/or transdermal absorption (especially of FANS and hormones), solutions, so that it can be administered by topical application or by inhalation to treat respiratory pathologies such as asthma.

Particular attention is paid to compositions containing enzymes such as hyaluronidase and those containing nonsteroidal anti-inflammatory drugs or hormones in the formulations of agents for treating skin hematomas, which are gels, creams and patches for dermal and/or transdermal absorption of drugs.

For purely illustrative and non-limiting purposes, some examples of the preparation of degree 1 and degree 3 HAS and pharmaceutical formulations containing the same are provided, as well as the results obtained through in vitro experiments.

Example 1

Tetrabutylammonium of hyaluronic acid (HA) with an average molecular weight equal to 200 kDa (range 150,000 to 250,000 Da) Salt preparation

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

Example 2

Synthesized from HA with an average molecular weight of 200KD and a sulfation degree equal to 3 sulfate groups per repeating unit. Sulfated HA

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 SO ₃ -pyridine (sulfur trioxide and pyridine, hereinafter abbreviated as PySO ₃ ) 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 adding 0.1 volume of water. The crude reaction product was isolated by precipitation after adding 2 volumes of ethanol. The resulting solid was dispersed in 150 ml of water and the pH was neutralized with 1 M NaOH. The water was thoroughly dialyzed from the mixture using a membrane with a cutoff of 12-14,000 Da. The dialyzed product was freeze-dried. 9.7 g of product with a degree of sulfation equal to 3 sulfate groups per repeating unit was obtained (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, was 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 at 21±1°C for 20 hours, the reaction was terminated by adding 0.5 volumes of water. The pH initially fell below 2.5 and was brought to neutral by adding NaOH (solution). The crude reaction product was isolated by precipitation by adding 2.5 volumes of methanol and washed with 2 volumes of an 8:2 methanol/water mixture. The solid was redissolved and thoroughly dialyzed against water using a membrane with a cutoff of 12-14,000 Da. 30.4 g of product with a degree of sulfation equal to 3 sulfate groups per repeating unit was obtained (yield = 86%).

Example 3

Synthesized from HA with an average molecular weight of 200KD and a sulfation degree equal to 1 sulfate group per repeating unit. Sulfated HA

Using the procedure described in Example 1, 10.0 g of the TBA salt of HA were prepared and dissolved in 350 ml of DMSO. 10.0 g of the complex 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 volumes 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 neutralized with 1 moles/l of NaOH. The water in the mixture was thoroughly dialyzed through a membrane with a cut-off value of 12-14,000 Da. The dialyzed product was freeze-dried. 7.54 g of product with a degree of sulfation equal to 1.0 sulfate group per repeating unit were obtained (yield = 93%).

Example 4

The sulfation degree of each Sulfated HA was synthesized by using HA with 3 sulfate groups as the repeating unit.

Using the procedure described in Example 1, 12.4 g of the TBA salt of low molecular weight HA were prepared and dissolved in 300 ml of NMP. 40 g of _PySO₃ 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 fell below 2.5 and was brought to neutral by adding 4 M NaOH. The crude reaction product was isolated by precipitation by adding 2.5 volumes of methanol and washed with 2 volumes of a methanol/water mixture 8/2. The solid was redissolved and the water was dialyzed thoroughly using a membrane with a cut-off of 3,500 Da. 12.0 g of product with a degree of sulfation equal to 3.0 sulfate groups per repeat unit were obtained (yield = 85%).

Example 5

The sulfated HA was synthesized from HA with low molecular weight and a degree of sulfation equal to 1 sulfate group per repeating unit. HA

Using the procedure 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 neutralized with 1 moles/l of NaOH. The water in the mixture was thoroughly dialyzed through a membrane with a cut-off value of 3,500 Da. The dialyzed product was freeze-dried. 9.04 g of product with a degree of sulfation equal to 1.0 sulfate group per repeating unit were obtained (yield = 90%).

Example 6

Synthesized from HA with a molecular weight of 500-730KD and a sulfation degree equal to 3 sulfate groups per repeating unit. Sulfated HA

21.0 g of extract-derived sodium hyaluronate (500-730 kD) was dissolved in 1.5 liters of water and the resulting solution was diafiltered through a glass column prefilled with 450 ^cm³ of Dowex resin in the form of TBA. The eluted solution of HA-TBA salt was collected and freeze-dried. 32.0 g of product was obtained and dissolved in 1.35 liters of NMP. 100 _g of PySO₃ 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 fell below 2.5 and was brought to neutral by adding NaOH (4 moles/l solution). The crude reaction product was isolated by precipitation by adding 2.5 volumes of methanol and washed with 3.5 volumes of an 8:2 methanol/water mixture. The solid was redissolved and dialyzed thoroughly against water using a membrane with a cutoff of 12-14,000 Da. 30.3 g of product having a degree of sulfation equal to 3 sulfate groups per repeating unit were obtained (yield = 83%).

Example 7

Synthesized from HA with a molecular weight of 500-730 kD and a sulfation degree equal to 1 sulfate group per repeating unit. Sulfated HA

21.0 g of extract-derived sodium hyaluronate (500-730 kD) was dissolved in 1.5 liters of water and the resulting solution was diafiltered through a glass column prefilled with 450 ^cm³ of Dowex resin in the form of TBA. The eluted solution of HA-TBA salt was collected and freeze-dried. 32.0 g of product was obtained and dissolved in 1.65 liters of NMP. 40 _g of PySO₃ 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 fell below 2.5 and was brought to neutral by adding NaOH (4 moles/l solution). The crude reaction product was isolated by precipitation by adding 3.5 volumes of methanol and washed with 3.5 volumes of an 8:2 methanol/water mixture. The solid was redissolved and dialyzed thoroughly against water using a membrane with a cutoff of 12-14,000 Da. 22.5 g of product with a degree of sulfation equal to 1.0 sulfate group per repeating unit were obtained (yield = 87%).

Example 8

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

Human synoviocytes previously expanded in vitro and maintained in culture at 37°C with DMEM medium containing 10% FCS were inoculated at a concentration of 20,000 cells/well (synoviocytes are cells capable of producing different types of cytokines and are therefore commonly used for this type of experimental test). 1-degree sulfated HA (HAS1) and 3-degree sulfated HA (HAS3) prepared as described in Examples 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 unsulfated HA with an average molecular weight (MW) of 200 KD. After 3 days of treatment, real-time PCR was performed to evaluate the gene expression of IL-10 and IL-12: cellular RNA was extracted using the "Trizol" method according to the instructions of the supplier (TRIZOL reagent, LIFETechonologies, GIBCO BRL). Briefly, cells were lysed by adding 1.0 ml of Trizol and total RNA was quantified by measuring its absorbance at 260 nm. Adopt software Primer3 (Roche Molecular Diagnostics, Pleasanton, CA, USA) to select appropriate primers for every kind of gene to be amplified.Gene expression was evaluated by real-time PCR performed by adopting Rotor-gene TM5500 (Corbett research, Sydney, Australia).Adopt primer at 300nm and SYBR Green (Invitroge, Carlsbad, CA, USA) to carry out PCR reaction with 40 cycles after 95 ℃ 15 seconds and 60 ℃ 1 minute.The value of " fluorescence threshold (Ct) " was automatically determined by software, and the amplification coefficient of the studied gene of 92-110% was evaluated. For each cDNA sample, the gene expression value is expressed as 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, which thus represents the amount of mRNA expressed by the gene under investigation. The results 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 relative to controls treated with non-sulfated HA.

Figure 2 :

Still in this experiment, two sulfation degrees of HAS (1 and 3) were shown to significantly reduce IL-12 gene expression, reducing its mRNA synthesis by half compared to the control treated with unsulfated HA. Therefore, sulfated hyaluronic acid was shown to:

● For those pathological conditions previously described in which IL-10 has been shown to be of fundamental importance for the resolution and/or improvement of the disease (such as asthma, vitiligo and all inflammatory conditions in which IL-10 is involved), it is possible to stimulate the production of new mRNA for the synthesis of anti-inflammatory cytokines, thereby increasing the defense capacity of the cells and thus the entire organism.

• Effectively reduces new mRNA synthesis of the highly pro-inflammatory cytokine IL-12, proving to be a potent anti-inflammatory agent capable of intervening in the expression of proteins implicated in the pathogenesis of debilitating diseases such as psoriasis and all those previously described.

Example 9

Inhibition of TNF binding to its receptor expressed in monocytic cell lines: efficacy of 1- and 3-degree HAS of different MW values evaluate

These experiments were performed to evaluate the efficacy of the test samples (prepared according to Examples 1-4) in their ability to inhibit the binding of TNF to its receptor expressed by I.S. cells typically used in vitro for this type of experiment using iodinated cytokine components for evaluation in radioligand binding assays.

The experimental procedures were 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 by the sensitivity of monocytes to the cytotoxic activity of TNF and the expression of its relevant receptors. The cells were initially incubated with 0.028 nM ¹²⁵ I-TNF (carried in water) and the sample to be analyzed (at a concentration of 1 mg/ml, which was shown to be the minimum concentration that caused maximal inhibition) at 4°C for 3 hours in an incubation buffer consisting of 50 mM Tris-HCl pH 7.4 and 0.5 mM EDTA.

At the end of the incubation, the cells were centrifuged with dibutylphthalate/dinonylphthalate 2/1 and the resulting pellets were counted in a gamma-counter.

The results are shown in Figure 3 :

The results show that HAS with medium and low MW for both degree 1 and degree 3 completely (100%) inhibits the binding of TNF to its receptor. These results are of fundamental importance because they demonstrate that the sulfated product behaves exactly like a monoclonal antibody specific for the TNF receptor and is therefore able to block its function. This receptor blockade therefore represents the most effective way to antagonize the pro-inflammatory and tumor effects of TNF factors.

Example 10

Inhibition of the binding of cytokine IL-1 to its receptor expressed in fibroblast cell lines: 3-degree HAS with different MW values Efficacy evaluation

These experiments were performed to evaluate the efficacy of the test samples (prepared according to Examples 1-3 and 4) in their ability to inhibit the binding of IL-1 to its receptor expressed by mouse 3T3 cells typically used in vitro for this type of experiment using iodinated cytokine components for evaluation in radioligand binding assays.

Experimental procedures were 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 cytotoxic activity of IL-1 and expresses its relevant receptor, was used. Initially, the cells were incubated with 10 pM of ¹²⁵ I-IL-1 (carried in water) and the sample to be analyzed (at a concentration of 1 mg/ml, which was shown to be the minimum concentration that caused maximal inhibition) at 37°C for 2 hours in an incubation buffer consisting of RPMI 1640 containing 20 mM HEPES pH 7.2 and 1% BSA. At the end of the incubation period, the cells were washed with phosphate buffer, dissolved in 2.5 M NaOH, and counted in a gamma counter.

The results are shown in Figure 4 :

The results obtained show the efficacy of HAS (with medium and low MW) in inhibiting the binding of IL-1 to its receptor by 30%. These results are extremely important because they demonstrate that the sulfated product behaves exactly like a monoclonal antibody specific for the receptor of the cytokine in question and is therefore able to block its function. This receptor blockade represents the most effective way to antagonize the pro-inflammatory and tumor effects of IL-1, as previously described.

Example 11

Inhibition of IL-6 binding to its receptor expressed in myeloma cells: 3-HSA with different MW values Efficacy evaluation

These experiments were performed to evaluate the efficacy of the test samples (prepared according to Examples 1-3 and 4) in their ability to inhibit the binding of IL-6 to its receptor expressed in human myeloma U266, which is commonly used in vitro for this type of experiment, using iodinated cytokine components for evaluation in radioligand binding assays.

Experimental procedures were 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 cytotoxic activity of IL-6 and expresses its cognate receptor, was used. Initially, the cells were incubated with 0.08 nM ¹²⁵ I-IL-6 (carried in water) and the sample to be analyzed (at a concentration of 1 mg/ml, which was shown to be the minimum concentration that caused maximal inhibition) at 4°C for 16 hours in an incubation buffer consisting of RPMI 1640 containing 25 mM HEPES pH 7.1 and 10% BSA. At the end of the incubation period, the cells were washed with phosphate buffered saline, centrifuged at 9,000 rpm, and the pellet was counted in a gamma counter.

The results are shown in Figure 5 :

The results show that HAS with medium and low MW completely (100%) inhibits the efficacy of IL-6 binding to its receptor. These results therefore prove that the behavior of the sulfated product is also completely similar to that of a monoclonal antibody specific for the receptor of the cytokine in question, and is therefore able to block its function. This receptor blockade represents the most effective way to antagonize the pro-inflammatory effects of IL-6.

Example 12

Inhibition of protein synthesis of cytokines IL-2, IL-7, IL-10 and IL-12 by 1 and 3 degree HAS in human PBMCs Production evaluation

For these experiments, human peripheral blood mononuclear cells (PBMCs) from various donors were used to evaluate the effects of HSA on the production of the cytokines listed above using:

- Unsulfated HA (average MW: 200 kD);

- HAS1 and HAS3 (prepared as described in Examples 1-3).

PBMCs were isolated using Ficoll-Paque PLUS (GE Healthcare) according to the manufacturer's protocol (A., Scand J Clin Lab Invest 21 Suppl, 1968, 97: 77-89). On day 0, 100,000 cells were plated in 200 μl of RPMI 1640 medium per well (96-well plates) supplemented with 10% fetal calf serum, 10 mM HEPES, 2 mM glutamine, and 100 U/ml of 1% penicillin-streptomycin. The effects of all samples were evaluated on PBMCs that had been left untreated or stimulated with lipopolysaccharide (LPS) (10 μg/ml), a highly proinflammatory agent, or with phytohemagglutinin (PHA) (10 μg/ml), a substance that stimulates lymphocyte autolysis; both agents stimulate cytokine synthesis. Cells were treated individually with the three compounds at concentrations of 0.1 mg/ml or 1 mg/ml. After incubation at 37°C (5% _CO2 ) for 24 hours, 100 μl of supernatant was collected from each well to analyze the production of IL-2, IL-7, IL-10, and IL-12.

Inflammatory mediators were quantified by technology using Custom Human 9-Plex Array plates following the supplier's protocol indicated on the technology card.

The results are shown in Figure 6-9 :

These figures clearly show that 1 and 3 degrees of HAS are able to significantly reduce the synthesis of IL-2, IL-7 and IL-12 in terms of monocytes when the cells are not stimulated and when, on the contrary, they are stimulated by specific and potent inflammatory factors and/or mitogens. Thus, HAS has been shown to be a molecule with a precise pharmacological profile, capable of regulating/modulating the synthesis of cytokines with significant anti-inflammatory activity in situations where the immune response is not stimulated and in particular in inflammatory stress events where immune cells respond by producing a cytokine cascade, in particular, in this case, the data presented show a stronger HAS regulatory effect.

On the other hand, Figure 9 demonstrates that a clear stimulus for IL-10 production is also for cells belonging to the immune system. Thus, it is once again demonstrated that HAS is able to regulate cytokine synthesis, stimulate anti-inflammatory cytokines and inhibit the synthesis of pro-inflammatory cytokines.

Example 13

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

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

The activity of the tested samples was determined by evaluating the inhibition of cytopathogenicity caused by herpes simplex virus type 1 (HSV-1: KOS, F, and McIntyre strains) and herpes simplex virus type 2 (HSV _- 2: G, 196, and Lyons strains) in E6SM fibroblasts derived from muscle/skin embryonic tissue. Furthermore, the antiviral activity was again tested against _E6SM cells infected with vesicular stomatitis virus (VSV). HSV-1 is a virus that preferentially infects the oral mucosa, while HSV-2 attacks the genital mucosa. The experimental procedures were performed as described in Baba M. et al., ANTIMICROB. AGENTS CHEMOTHER., 1988, 32: 1742-1745.

In brief, the confluent cell culture was contacted with an infectious dose of the above-listed viruses in the presence of sample HS-NS1 (EP0971961), HAS1 and HAS3 prepared as described in Examples 1-3. After incubation at 37°C for 1 hour, the culture medium was replaced with fresh medium containing only the sample to be tested. The cytopathogenicity of the virus was tested on the second day of incubation. The inhibition of viral cytopathogenicity was evaluated by measuring the inhibition of DNA and RNA synthesis in "infected" cells, and the treatment was performed as described above: the cells were seeded in microwells containing different concentrations of the sample to be tested and 2.5 μCi 3H-thymidine and 3H-uridine/ml culture medium. After 16 h at 37°C, the cells were treated with trichloroacetic acid, washed in ethanol, dried, and counted in 7.5 ml of scintillation fluid. The antiviral activity of the test sample was expressed as the minimum concentration required for 50% inhibition of viral cytopathogenicity: IC50 . Furthermore, to evaluate the cytotoxicity of the test samples, the lowest concentration necessary to cause morphological damage (observable under light microscopy) to the cells used was determined. Comparisons were made with dextran sulfate (DS) and the drug acyclovir (both molecules have known antiviral efficacy and were therefore used as positive controls).

The results are shown in Figure 10 :

Experimental data confirm the potent antiviral effects of both HAS1 and HAS3: comparisons between HA-NS1 and HAS1 show that not all sulfated hyaluronic acids are equivalent, as HA-NS1 has not been shown to be active, and that this difference in efficacy does not depend on the molecular weight or degree of sulfation of the hyaluronic acid, so it depends on the exact structure of HA-NS1 relative to HAS1. In fact, HAS showed efficacy equal to that of dextran sulfate and comparable to that of acyclovir, a reference drug for the treatment of herpes simplex. Furthermore, it should be noted that acyclovir is inactive against VSV, whereas 1- and 3-degree HAS have very potent antiviral activity against VSV.

All tested samples were non-cytotoxic to host cells, the lowest cytotoxic concentrations obtained were practically equal to those of reference drugs commonly used for the treatment of herpes in clinical practice, and were shown to be on average 100-fold higher in inhibition of viral replication than those shown to be active.

Cytomegalovirus

The activity of the test samples was determined by evaluating the inhibition of cytopathogenicity of cytomegalovirus (CMV: AD-169 and Davis strains) using the above protocol. The antiviral activity was tested against HEL cells (lung blast cells) and was expressed as the concentration required for 50% inhibition of the number of plaques formed by the above virus.

The results are shown in Figure 11 :

The table shows clear and significant positive results obtained for HAS1 and HAS3, which again confirms that they are effective antiviral agents. Moreover, in this case, HA-NS1 was not shown to be active in inhibiting viral proliferation, which confirms the absolute diversity in antiviral capacity between the two types of 1-degree sulfated products.

Example 14

In vitro evaluation of the fibrinolytic properties of HAS with different MW values and acidity 3

The evaluation of the fibrinolytic properties of the tested products was compared with the known fibrinolytic activity of plasmin . Specifically, the dissolution rate of the fibrin network and the formation of soluble fibrin degradation products (FDPs) were evaluated.

The plasma samples used were obtained from whole blood of healthy subjects without any ongoing pharmacological treatment.

To test the fibrinolytic efficacy of the product under test, a blood sample was distributed into test tubes in which clot formation was induced.

The formation of FDP was then evaluated after the addition of:

Plasmin as a control treatment

●HAS3 prepared according to Examples 1 and 2

●HAS3 prepared according to Example 4

Experimental research:

Freshly drawn blood samples were divided into test tubes containing sodium citrate in a 9:1 ratio. The tubes were immediately centrifuged at 3,000 rpm for 5 minutes. The resulting plasma was transferred to new test tubes and immediately used for FDP assessment.

Thrombin (300 mU/ml) preheated to 37°C was added to the plasma samples as a fibrinogen activator to induce clot formation.

Add the following components to separate cuvettes containing fibrin clots:

- Plasmin solution 0.5mU, 5mU, 50mU, 500mU and 1U

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

The absorbance change of each cuvette at 405 nm was evaluated by spectrophotometer for 60 seconds.The reaction was allowed to continue until the clot was completely dissolved.

The coagulated plasma, plasmin solution, and HAS3 200KD and HAS3 20KD solutions were mixed at a ratio of 1:1 v/v and maintained at an operating temperature of 37°C.

Table 1. Fibrinolytic activity of plasmin

The table shows the mean mAbs/min recorded in cuvettes containing clotted plasma to which different concentrations of plasmin had been added. The values shown indicate the rate of fibrin clot lysis and, therefore, the rate of FDP production. The fibrinolytic activity of plasmin is directly proportional to its concentration.

The mean mAbs/min values recorded for each plasmin concentration were then plotted against the respective enzyme units to create a mathematical function relating mAbs/min values to enzyme units. Plasmin: Fibrinolytic activity (mAbs/min)

1U 500mU 50mU 5mU 0.5mU 190 178 126 68 1

Table 2. Fibrinolytic activity of HAS3 with different MW values

Calculate the mAbs/min value recorded in a cuvette containing coagulated plasma to which different concentrations of HAS3 have been added. This value shows the rate at which the fibrin clot dissolves and, therefore, the rate at which FDPs are produced. The fibrinolytic activity of HAS3 is directly proportional to its concentration. In the table, the fibrinolytic activity of HAS3 is expressed as a unit equivalent of plasmin.

HAS3 (200KD) fibrinolytic activity (expressed in mU plasmin equivalents)

200mg/ml 150mg/ml 100mg/ml 50mg/ml 25mg/ml 1,1U 50mU 10mU 3mU 0.5mU

HAS3 (10KD) fibrinolytic activity (expressed in mU plasmin equivalents)

200mg/ml 150mg/ml 100mg/ml 50mg/ml 25mg/ml 400mU 25mU 2mU 0.3mU 0.25mU

result:

The experiments performed have shown how HAS is a powerful fibrinolytic agent, with an effect equivalent to that of plasmin. Plasmin is an important enzyme belonging to the hydrolase family that degrades various plasma proteins and, in particular, the fibrin of thrombi and clots. The degradation of fibrin is called fibrinolysis . Plasmin deficiency can lead to thrombosis because thrombi are not sufficiently degraded. Although HAS is not an enzyme, it has been shown to be equivalent to the activity of an enzyme reference, allowing the sulfated product to be used as a new fibrinolytic agent with all the advantages of a non-enzymatic molecule, such as stability at room temperature and a much longer shelf life and ease of formulation.

Example 15

Evaluation of skin permeability of HAS1 and HAS3 with different MW values

Evaluation of the skin absorption of diclofenac formulated with HAS versus HA

Skin for the experiments was obtained from the abdomen of patients aged 30 to 50 years who had undergone surgical tummy tuck surgery. Full-thickness skin sections were frozen after surgery and stored at -20°C until the start of the experiment, at which point the samples were thawed at room temperature and precisely separated from the adipose tissue. The skin was divided into 2.5 ^cm² square sections and, using specialized forceps, immersed in 60°C water for one minute to precisely separate the corneal layer and epidermis (SCE) from the underlying tissue. The resulting samples were analyzed using a light microscope and discarded if perforations were observed. The SCE was placed in the lower half of a Franz cell, epidermis facing downward, with the corneal layer in contact with the donor solution located above. The permeation zone was a 0.636 ^cm² circular area. The lower and upper halves of the Franz cell were precisely fixed to the SCE to separate the donor chamber (donor solution volume: 0.50 ml) and the receptor chamber (receiver solution volume: 5.00 ml), and their volumes were precisely calibrated. The solvents to be used were degassed to eliminate the formation of bubbles, which must be avoided in particular for the receiver solution, which was thermostated at 37° C. by means of a circulating thermostatic bath; under these conditions, the SCE was at a temperature of 31-33° C. Each experiment was performed in triplicate, and the results were assigned as the mean of the three experiments, expressed as the amount of analyte that had passed through the skin surface unit after 24 hours .

HA with two MWs and HAS1 and HAS3 were permeated from 3% (w/v) solutions, and the amount of permeate was determined by glucuronic acid analysis and 1CP (inductively coupled plasma) spectroscopy of sulfur, respectively.

The permeation of diclofenac sodium salt was carried out with the salt in aqueous solution, in the presence of 3% (weight/volume) HA200KD, and in the presence of 3% (weight/volume) HAS3 prepared according to Examples 1-2. In all three cases, the concentration of diclofenac sodium salt in the permeation solution was equal to 1% (weight/volume). The diclofenac concentration was determined by reverse phase HPLC analysis on an Agilent 1200 Series instrument with UV detection (254 nm), a C18 column, and an eluent of acetonitrile/water/acetic acid 50/46/4 at a flow rate of 1.2 ml/min.

The results are shown graphically in Figures 12 and 13:

Figure 12 shows how sulfation of HA significantly increases its penetration through the skin and how this result is particularly pronounced for the mean MW.

FIG13 , on the other hand, shows how HAS acts as a skin absorption enhancer for the active ingredient diclofenac, doubling its total permeate volume over 24 hours relative to the control and in combination with HA.

Example 16

Evaluation of the hydration and protective activity obtained with HAS relative to HA and control base creams

Twenty human subjects aged 18 to 70 years, without skin pathologies and without ongoing pharmacological treatment, were treated once daily at the forearm level with a constant amount of the test product. The test product was:

●Control: Consists of a hydrating base cream

Base cream (as a control) containing HA 200KD 0.1%

Matrix cream (as a control) containing 301% HAS, prepared according to Example 1-2

Cream base: composition

Production method

Add 90% of the water, disodium Edta, and Isocide PF. Heat to 65-70°C. Dissolve Nikkomulese and Dermol 88 in a suitable container by heating to 65-70°C. Blend the lipid phase into the aqueous phase using a turbine. Cool to 30-35°C and add Kemipure 100 dissolved in the remaining 10% of the water. Add Sepigel to adjust viscosity and cool to 25°C.

The difference in hydration values was evaluated by means of a corneometer CM825 from 3-point averages, and a profilometric analysis of the skin surface was performed using a Visioscan VC98 video camera at T0 (baseline value) and T7 (after 7 days of product application).

The results are shown graphically in Figures 14 and 15:

Figure 14 shows that the skin hydration effect after treatment with HAS3 is higher than that of the base cream and the base cream containing HA. Figure 15, on the other hand, shows how the skin roughness index is significantly reduced relative to the control after 7 days of treatment. These data clearly show the efficacy of HAS in significantly improving the skin hydration index, demonstrating its effective hydration and protective activity on the skin by reducing water evaporation through the epidermis.

Example 17

Preparation of a solution for inhalation containing 1% HAS

40 mg (or 20 mg if the MW of the HAS is 500-730 kD) of 1-degree sulfated hyaluronic acid with low or medium MW is added to a 50 ml glass flask, followed by 15 ml of sterile PBS 0.2M pH 7.4. The mixture is stirred for approximately 30 minutes until the powder is completely dissolved. Once complete dissolution has been achieved, 2 ml of propylene glycol and additional sterile PBS 0.2M pH 7.4 are added until the cumulative volume reaches 20 ml. The solution is maintained under stirring for several minutes.

Example 18

Preparation of a solution for inhalation containing 3-degree HAS

100 mg of 3-degree sulfated hyaluronic acid (HAS3) obtained from HA 200KD was added to a 50 ml glass flask, followed by 15 ml of sterile PBS 0.2M pH 7.4. The mixture was stirred for approximately 30 minutes until the powder was completely dissolved. When complete dissolution had been achieved, 2 ml of propylene glycol and additional sterile PBS 0.2M pH 7.4 were added until the cumulative volume reached 20 ml. The solution was maintained under stirring for several minutes.

Example 19

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

Dissolve methylparaben and propylparaben in 80°C purified water. After cooling the solution to room temperature, add sodium hyaluronate and stir until dissolved. Then add HAS1 (or HAS3) and continue stirring until completely dissolved. Then, add glycerin and propylene glycol while stirring until completely dissolved. Finally, add sodium carboxymethylcellulose (CMC) and mix the mixture until a gelled solution is obtained.

Example 20

Preparation of a hydrophilic gel formulation for mucosal application containing HAS and HA (preservative-free)

Sodium hyaluronate and then HAS1 (or HAS3) are dissolved in approximately 90% of the intended amount of water under stirring. Propylene glycol, Symdiol 68, and then MP Diol Glycol are added and mixed until the components are completely dissolved. Carbomer 974P is then added and stirred until the latter is evenly dispersed. Sodium hydroxide granules are dissolved in the remaining 10% of water and this solution is slowly added to the above solution. The mixture is mixed to gel the aqueous phase.

Example 21

Preparation of a hydrophilic gel formulation containing HAS and hyaluronidase

Dissolve methylparaben and propylparaben in 80°C purified water. After cooling the solution to room temperature, add hyaluronidase with stirring, followed by HAS3, and maintain stirring until both components are completely dissolved. Carbomer 974P is then added and maintained stirring until it is evenly dispersed. TEA is then added to gel the aqueous phase. Finally, glycerin and propylene glycol are added with stirring.

Example 22

Preparation of a formulation in the form of a hydrophilic cream (emulsion O/A) containing HAS and hyaluronidase

The oil phase was prepared by melting liquid paraffin, stearic acid and Tefose 1500 at 50° C. with stirring. The aqueous phase was prepared separately by initially dissolving methylparaben at 80° C., then cooling to room temperature, finally incorporating glycerol, hyaluronidase and then HAS3 and stirring until all components were completely dissolved.

The aqueous phase was incorporated into the oil phase and emulsified, and the resulting emulsion O/A was cooled to room temperature while stirring.

Example 23

Preparation of a foam formulation containing HAS and hyaluronidase

Dissolve methylparaben and propylparaben in purified water at 80°C. After cooling the solution to room temperature, add hyaluronidase with stirring, followed by HAS3, and continue stirring until completely dissolved. Propylene glycol is then added, and the mixture is mixed until completely dissolved. Polyvinylpyrrolidone is then added, mixed until completely dissolved, and finally, polysorbate 80 is added and continued stirring until dissolved.

The phases of the resulting solution are then distributed in a cylinder using isobutane, n-butane, or propane propellants under pressure.

Example 24

Preparation of a formulation in the form of an ointment containing HAS3 and hyaluronidase

A base ointment was prepared by melting light liquid paraffin and white petrolatum with stirring at 70° C. After cooling to room temperature, hyaluronidase and then HAS3 were incorporated with stirring, and the mixture was mixed until a homogeneous suspension was obtained.

Example 25

Preparation of liposome gel (LIPOGEL) containing HAS3 and hyaluronidase

Melt light liquid paraffin, white petrolatum, and cetearyl alcohol at 90°C with stirring. Add hydrogenated castor oil, a liposomal gelling agent, and stir until a homogeneous solution is obtained. Slowly cool the mixture to room temperature. Finally, add hyaluronidase and HAS3, and mix the mixture until a homogeneous suspension is obtained.

Example 26

Preparation of a lipstick formulation containing HAS and HA

Add the exact amount of liquid paraffin indicated in the preparation recipe to a suitable container. Heat the mixture to 88-92°C, then add white soft paraffin, hard paraffin, white beeswax, ozokerite, and arlacel while stirring. Maintain stirring until all components are completely melted. Then, add all-rac-α-tocopheryl acetate, allantoin, butylated hydroxytoluene, and propylparaben. Mix the mixture until completely dissolved, maintaining the mass at 88-92°C.

Add the intended amount of purified water in the formula to a suitable container separately, then add sodium hyaluronate and HAS1 (or HAS3) and stir until completely dissolved, then add disodium edetate and keep stirring until dissolved.

The aqueous phase was transferred to the vessel containing the molten mass under stirring, and the system was maintained at 88-92°C and stirred until a clear solution was obtained. The two aromatizing agents were then added under stirring, and the mixture was mixed for 10 minutes. The molten mass was poured into a mold and immediately cooled to T < 0°C until a solid stick was obtained.

Example 27

Preparation of a formulation in the form of vaginal beads containing HAS and HA

The gelatin is swollen in 70% purified water at 85°C; sodium hyaluronate and HAS1 (or HAS3) are dissolved in the remaining water and the solution is mixed with glycerol brought to the same temperature. The glycerol solution is added to the swollen 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 beads are obtained.

Example 28

Preparation of a formulation in the form of a hydrophilic cream (emulsion O/A) containing HAS and HA

The oil phase was prepared by melting liquid paraffin, stearic acid and Tefose 1500 at 50° C. with stirring. The aqueous phase was prepared separately by initially dissolving methylparaben at 80° C., then cooling to room temperature, incorporating glycerol, hyaluronidase and then HAS1 (or HAS3) and stirring until all components were completely dissolved.

The aqueous phase was incorporated into the oil phase and emulsified, and the resulting emulsion O/A was cooled to room temperature while stirring.

Example 29

Preparation of a formulation in the form of an ointment containing HAS

A 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 30

Preparation of a hydrophilic gel formulation containing HAS3, HA and diclofenac

Dissolve methylparaben and propylparaben in 80°C purified water. After cooling the solution to room temperature, add diclofenac sodium and sodium hyaluronate with stirring, followed by HAS3, and continue stirring until both components are completely dissolved. Then, add carbomer 974P and continue stirring until it is evenly dispersed. Then, add TEA to gel the aqueous phase. Finally, add glycerin and propylene glycol with stirring.

Example 31

Preparation of a formulation in the form of a hydrophilic cream (emulsion O/A) containing HAS3 and diclofenac

The oil phase was prepared by melting liquid paraffin, stearic acid and Tefose 1500 at 50° C. with stirring. The aqueous phase was prepared separately by initially dissolving methylparaben at 80° C., then cooling to room temperature, finally incorporating glycerol, diclofenac sodium and then HAS3 and stirring until all components were completely dissolved.

The aqueous phase was incorporated into the oil phase and emulsified, and the resulting emulsion O/A was cooled to room temperature while stirring.

Example 32

Preparation of a formulation in the form of a foam containing HAS and diclofenac

Dissolve methylparaben and propylparaben in purified water at 80°C. After cooling the solution to room temperature, add diclofenac sodium with stirring, followed by HAS3, and continue stirring until completely dissolved. Propylene glycol is then added, and the mixture is mixed until completely dissolved. Polyvinylpyrrolidone is then added, mixed until completely dissolved, and finally, polysorbate 80 is added and continued stirring until dissolved.

The phases of the resulting solution are then distributed in a cylinder using isobutane, n-butane, or propane propellants under pressure.

Example 33

Preparation of a formulation in the form of a skin patch containing HAS and diclofenac

These embodiments relate to the preparation of a polymer matrix containing HAS for the controlled release of topical drugs, in which case the improvement in the skin and/or transdermal absorption of the active ingredient FANS diclofenac sodium salt contained therein is due to the accelerator effect of HAS. The matrix preferably comprises a copolymer of acrylic acid and its acrylates and/or methacrylates having a glass transition temperature (Tg) below room temperature, preferably below 0°C, the free carboxyl groups present along the polymer chain being salified with an organic base (e.g. ammonia, ethylenediamine, an acrylic acid and/or methacrylate copolymer having an ammonium cationic functional group on the alkyl group (preferably E100)) or an inorganic base (e.g. hydroxides or carbonates or bicarbonates of alkali metals, alkaline earth metals and transition metals), as known to those skilled in the art. The copolymers typically used in the present invention are composed of two or more monomers in different percentages; examples of these monomers are:

Acrylic acid

●Butyl and/or methyl acrylate

●2-Ethyl/Hexyl Acrylate

●Glycidyl methacrylate

Vinyl acetate

Many copolymers are commercially available (e.g., 280-2416, 280-2516, 87-2620, 87-2852, 380-1054, 87-2051, National and Starch) that are soluble in organic solvents and have a free carboxyl group percentage of 0.1% to 15%, which can be salted with organic or inorganic bases as described above. These copolymers are preferably included in the skin patch matrix in an amount of 30-90% by weight.

Furthermore, a HAS-containing polymer matrix can be prepared comprising the following two main components:

a: Polymethacrylate: acrylic acid and/or methacrylate copolymers with ammonium cation functional groups on the alkyl groups (E100, RS and RL) are preferred. The polymer may account for 10-40% by weight, preferably 10-25% of the total weight of the adhesive matrix after drying.

b: an organic dicarboxylic acid or tricarboxylic acid (e.g., succinic acid, fumaric acid, adipic acid, and lauric acid) which serves as a counterion to the cationic component a (in addition, it acts as a reticulating agent for component a ). Component b can be adjusted to be partially or completely neutralized. Other suitable components b are acid-functionalized acrylate or methacrylate polymers, such as polyacrylic acid. Component b can be included in an amount of 1-40%, preferably 1-20%, by weight, based on the total weight of the dried adhesive formulation.

The polymer matrix in the final formulation is present in an amount of 10-90%, preferably 50-90%, by dry weight, based on the dry weight of the final composition. The amount of active ingredient incorporated varies depending on its properties and the desired skin or transdermal therapeutic effect. It is generally present in an amount of 0.1-30% by weight of the dry weight of the final composition.

The formulation may also contain excipients, emollients, embellishers, emulsifiers, adhesion regulators, preservatives, plasticizers, acidifiers/buffers. The amount of excipients may vary within a wide range of 0.01-30% depending on their function.

The skin absorption accelerator with moisturizing, anti-itching and anti-redness properties is 1 degree or 3 degree HAS, preferably 3 degree, which is present in a concentration of 0.1-30% of the dry weight of the final composition. The above matrix ensures that the penetration of the active ingredient is controlled and constant, and is non-irritating and non-sticky to the skin.

Preparation method

1 kg of a selected methacrylate copolymer (e.g., 280-2416, 280-2516, or 87-2852) with a solids content of 30-40% w/w and 300 g of a 30% w/w E100 solution based on water/solvent is added under mechanical stirring; the mixture is kept under moderate stirring for 30 minutes. The active ingredient (100 g of diclofenac sodium salt) and HAS3, previously dissolved in aqueous solution, are then added. The mixture is kept under stirring until completely dissolved. For the preparation of the matrix layer, the mixture is then placed on top of a film of silicone polyester/paper and dried by evaporation of the residual solvent. The dry weight of the spread matrix is about 60 g/m ^2. The matrix thus obtained is then combined with a non-woven polyester fabric or a polyethylene film for the final formation of a patch.

The final composition of each individual patch contained 140 mg diclofenac and 40 mg HAS3.

surface

Although the present specification is described in this way, it is obvious that these methods can be changed in various ways. These changes should not be considered as departing from the spirit and scope of the present invention, and all changes that are obvious to those skilled in the art are included in the scope of the following claims.

Claims (5)

1. Use of sulfated hyaluronic acid in the preparation of topical pharmaceutical preparations, said preparations serving as fibrinolytic agents to degrade fibrin clots that form at the skin level after damage to the endothelium and/or walls of capillaries and/or small vessels due to mechanical trauma and/or hemorrhage of medium/small solids, and wherein said fibrin clots are dermal fibrin clots associated with dermatopathological conditions related to damage to vascular endothelium and/or vessel walls, said sulfated hyaluronic acid having a sulfation degree equal to 3 and prepared from hyaluronic acid having a molecular weight of 150,000–250,000 Da, said preparations being in the form of ointments, liposome gels, hydrogels, lipsticks, creams, patches, foams, mucosal gels, mouthwashes, or solutions for topical application. 2. Use of a pharmaceutical composition containing sulfated hyaluronic acid in combination with a pharmacologically active agent in the preparation of a pharmaceutical agent for topical application as defined in claim 1, wherein the sulfated hyaluronic acid has a degree of sulfation equal to 3 and is prepared from hyaluronic acid having a molecular weight of 150,000–250,000 Da. 3. The use of claim 2, wherein the pharmaceutical composition comprises a steroid, a vitamin, a nonsteroidal anti-inflammatory drug, a topical chemotherapeutic agent, an antibiotic, an antiviral agent, and/or a local anesthetic. 4. The use of claim 2, wherein the pharmaceutical composition contains hyaluronidase. 5. The use of claim 2, wherein the pharmaceutical composition contains hyaluronic acid and/or carboxymethyl cellulose.