Chitosan nitrate for use as a nitric oxide donor (NO donor)

Abstract

The present invention relates to chitosan nitrate for use as a nitric oxide donor, particularly for use in the prevention or treatment of conditions associated with NO deficiency in mammals, preferably humans. Suitable diseases to be treated with chitosan nitrate include skin diseases, sexual dysfunction, diseases requiring pain treatment, eye, nasal and sinus diseases, respiratory tract diseases, digestive diseases, and metabolic disorders. These diseases have been successfully treated or alleviated by the application of the chitosan nitrate of the present invention. The present invention also relates to non-therapeutic or cosmetic methods for skin protection and / or healthy aging.

Description

[Technical field]

[0001] The present invention relates to chitosan nitrate for use as a medicament, chitosan nitrate for use as a nitric oxide donor (NO donor), particularly for use as a nitric oxide (NO) generator, chitosan nitrate for use in the prevention or treatment of conditions associated with NO deficiency in mammals, particularly humans. The present invention further relates to chitosan nitrate for use in the prevention or treatment of skin disease conditions, wherein said conditions are preferably skin diseases. The present invention also relates to chitosan nitrate for use in the prevention or treatment of sexual dysfunction. The present invention further relates to chitosan nitrate for use in the prevention or treatment of diseases requiring pain treatment. The present invention further relates to chitosan nitrate for use in the prevention or treatment of eye, nose and sinus diseases. The present invention further relates to chitosan nitrate for use in the prevention or treatment of airway diseases. The present invention further relates to chitosan nitrate for use in the prevention or treatment of digestive diseases. The present invention also relates to chitosan nitrate for use in the prevention or treatment of metabolic disorders. The present invention also relates to a cosmetic method comprising administering chitosan nitrate, wherein chitosan nitrate is preferably used as NO donor.The present invention also relates to a pharmaceutical or cosmetic composition comprising chitosan nitrate, wherein the composition is a topical preparation, such as cream, emulsion, lotion, transdermal patch, spray, lozenge, chewing gum, dentifrice, nasal spray, nasal and sinus aerosol, eye drops or eye ointment.The pharmaceutical composition comprising chitosan nitrate can be a tablet, capsule, suppository, or pulmonary aerosol.The nasal and sinus aerosol or pulmonary aerosol is preferably administered via a nebulizer. [Background technology]

[0002] The role of nitrate and nitrite, inorganic anions previously thought to be inactive end products of endogenous nitric oxide (NO) metabolism, has become more widely recognized in recent years. Recent studies have shown that NO can be formed in vivo from nitrate and nitrite anions by the nitrate-nitrite-nitric oxide pathway (Lundberg et al., U.S. Pat. No. 10,406,118). This pathway produces nitric oxide by reduction of dietary nitrate, e.g., from nitrate-rich vegetables such as leafy greens, beetroot, and spinach. Humans cannot reduce nitrate to nitrite, a reduction that is only performed by bacteria present in the mouth, intestine, skin, and mucous membranes, i.e., the so-called nitrate-responsive bacteria.

[0003] Several studies have shown that NO plays important biological roles, such as vascular homeostasis, neurotransmission, redox signaling, cellular respiration, and host defense, as a critical regulator. The cardioprotective effect of a vegetable-rich diet may in fact be due to NO. Given its beneficial effects, nitrate supplementation has been widely investigated. However, nitrate supplementation presents challenges, such as providing nitrate to nitrate-responsive bacteria that can convert nitrate to nitrite. Nitrite is biologically active, whereas nitrate is not. Nitrate in nitrate-rich vegetables is efficiently absorbed in the first part of the digestive tract. Most nitrate does not reach the nitrate-reducing bacteria in the colon. 25% of blood nitrate is returned to the mouth by the gut-saliva circulation, where a portion of it is converted to nitrite. Only after this gut-saliva recirculation does nitrate become biologically active, allowing it to be converted to nitrite by nitrate-responsive bacteria on the tongue. Ultimately, only about 5% of ingested dietary nitrate is converted to biologically active nitrite, which, after further reduction, is converted to nitric oxide (derivatives).

[0004] So far, all known NO donors have limitations. Direct administration of NO is impractical because NO is a gas and requires the use of gas cylinders that may not be exposed to acid. Diazeniumdiolates (NONOates) are potentially toxic and form carcinogenic secondary nitrosamines with adverse biological effects from metabolic by-products, e.g., causing oxidative stress that damages tissues. S-nitrosothiols are not stable enough to be used for local delivery, and the potential advantages and disadvantages of NO hybrid drugs are unknown.

[0005] In the field of medicine and cosmetics, the skilled person identifies and distinguishes between various NO donors (Liang et al., Future Sci. OA1(1)2015, FSO54). There are "NO releasers" based on (synthetic) carrier molecules to which active NO is bound. Depending on the carrier selected, NO is released under appropriate physiological or biochemical conditions. Examples of NO releasers are N-diazeniumdiolates, nitrosothiols, nitrosohydroxylamines, metal nitrosyl complexes, sodium nitroprusside, NO-bound diethylamine, V-Pyrro / NO, spermine / NO. In addition, "NO generators" do not release NO from the bound form by cellular metabolic pathways or the nitrate-nitrite NO pathway, but generate NO, i.e., biosynthesize NO. NO generators can be further distinguished into organic NO generators and inorganic NO generators. Both NO generators contain a nitrite or nitrate moiety. NO generators are classified as NO prodrugs or NO precursors, and are therefore called NO prodrugs or NO precursors.

[0006] Organic nitrates and nitrites are NO donor drugs used in the treatment of coronary artery disease. Nitroglycerin (glyceryl trinitrate), isosorbide dinitrate, isosorbide mononitrate, and isoamyl nitrite, isopentyl nitrite, and isopropyl nitrite are examples of organic NO generators. NO is generated via certain endogenous enzymes. Long-term use of nitrate vasodilators not only induces tolerance to nitrates leading to tachyphylaxis, but also leads to strong side effects including increased oxidative stress, endothelial dysfunction, and cardiac autonomic dysfunction. Among organic nitrates, nitroglycerin and its derivatives have been used as vasodilators for a long time in medicine, especially in the field of cardiology. Various side effects are known, such as headaches, tolerance with reduced effectiveness, and oxidative stress that adversely affects blood vessels. Plasters or ointments containing nitroglycerin are often used as NO generators for topical application or, in the case of plasters, for increasing NO systemically. Organic nitrates utilize enzymes and are generally fast-acting, but have the disadvantages of the possibility of overdose and various side effects. It is currently generally believed that bacteria are not involved in the metabolism of organic nitrates.

[0007] US Patent No. 6,261,594 and US Patent Application Publication No. 2008 / 311163 disclose NO releasers, EP Patent Application Publication No. 3120840 discloses NO releasers, namely S-nitrosoglutathione, diazeniumdiolates and iron nitrosyl complexes, and organic nitrates which form and belong to the group of NO generators.

[0008] US Patent No. 6,261,594 discloses chitosan-based polymeric NO donor compositions comprising modified chitosan polymers and nitric oxide dimers [NO], providing "site-specific delivery" and "controlled release" of NO in physiological conditions. Diazeniumdiolates are the bioactive forms of NO that are attached to the chitosan backbone. NO is released directly from chitosan in aqueous media depending on temperature and pH value. Therefore, the chitosan-based polymeric NO donor compositions disclosed in US Patent No. 6,261,594 are classified as NO releasers.

[0009] US 2008 / 311163 discloses certain cosmetic treatment methods and their devices. The disclosed cosmetic disorders are caused by age, environmental factors, changes in the physiological functions of the skin (e.g. psoriasis, dermatitis, acne, cellulite) and viral and / or bacterial attacks. The disclosed devices comprise a nitric oxide (NO) eluting polymer placed in contact with the area to be cosmetically treated, whereby a cosmetic dose of NO is eluted from the NO eluting polymer to said area. The NO eluting polymer is integrated with a carrier material, which adjusts and controls the elution of the cosmetic dose of NO during use. Suitable NO releasing polymers are selected from the group of (among others) amine functionalized chitosans. Chitosan is thus a carrier bound to an activated form of NO, i.e. diazeniumdiolate or S- or O-nitrosylated groups. NO is released depending on the carrier and physiological conditions.

[0010] European Patent Application Publication No. 3120840 relates to a nitric oxide releasing wound treatment film and its preparation method. The nitric oxide releasing film contained S-nitroglutathione, a nitric oxide donor that is naturally formed in the human body. The film can be applied to the human body, where nitric oxide is gradually released to inhibit pathogens that are the main cause of wound infection. Thus, wound healing is promoted. Chitosan may be used as a carrier of S-nitroglutathione, as it has a wound healing effect of absorbing wound secretions and maintaining moisture around the wound surface.

[0011] There remains a general need to provide compositions for use as NO donors, in particular for the delayed release of NO at different sites in the mammalian body, for use in the prevention or treatment of conditions associated with NO deficiency in mammals, and there remains a general need to provide non-therapeutic and / or cosmetic methods aimed at the protection and / or healthy aging of the skin. Summary of the Invention

[0012] The present invention is based on the discovery that inorganic nitrates can be activated by nitrate-responsive bacteria present in the body, such as the skin, colon, tongue and mucous membranes, when bound to a carrier. Chitosan nitrate produces a bioactivatable form of such inorganic nitrates, of which only nitrates in saliva were previously known. By bioactivatable nitrates we mean nitrates that can be converted to nitrites by nitrate-responsive bacteria according to the nitrate-nitrite-NO pathway described by Lundberg et al. (US Pat. No. 10,406,118). Chitosan is suitable for biotechnologically binding different groups to create new molecules. Secondly, the salt form of chitosan with nitric acid can be used to bind and deliver nitrates to the site of action, while the positively charged amine groups of chitosan interact well with negatively charged coatings, such as the pores. The salt form provides an ionic bond, rather than a covalent bond, between chitosan and nitric acid, allowing the nitrate to be bioactivated and delivered to anionic sites where nitrate-responsive bacteria reside. These sites include mucous membranes, gastrointestinal tract, and pores, which are negatively charged. The nitrate anion itself is also negatively charged and does not reach nitrate-responsive bacteria under normal conditions. Chitosan has cationic amine groups, some of which bind to nitric acid. However, the remaining cationic sites still interact with mucous membranes and deliver nitrate to these sites. Cationic chitosan directs the nitrate anion to the negatively charged environment of mucous membranes and pores where nitrate-reducing bacteria reside. In this way, the nitric acid in chitosan nitrate is bioactivated.

[0013] Thus, we have established chitosan nitrate as an inorganic NO generator and its metabolism by bacteria in the colonic mucosa and on the skin. Until now, only the gut-saliva route was known for the nitrate-nitrite-NO pathway. However, chitosan nitrate opens the door to a unique alternative route where nitrate is provided in a novel saliva-independent bio-activatable (SIBA) form.

[0014] The use of chitosan nitrate provides a simple and inexpensive production and a medically safe and effective NO delivery drug. Facultative anaerobic commensal bacteria present on the skin and in the mammalian body provide a controlled and sustained production of NO from nitrate via a nitrite intermediate. The slow, continuous and long-term conversion of nitrate to NO provides a medically safe low concentration of NO.

[0015] The present inventors have developed a chitosan nitrate-based drug, particularly for preventing or treating conditions associated with NO deficiency in mammals, preferably humans. The present inventors have surprisingly found that chitosan nitrate is suitable as an NO donor, more specifically as an NO generator. Without being bound by theory, it is believed that chitosan nitrate is converted to nitrite by anaerobic bacteria present, for example, on the skin, mucous membranes, or digestive tract of a subject. Nitrite is further converted to nitric oxide (NO) by various pathways known in the art. Thus, application of chitosan nitrate to these areas where anaerobic bacteria are present results in the formation of NO, which is absorbed by the body, leading to the prevention or treatment of conditions associated with NO deficiency. It is believed that the chitosan nitrate of the present invention provides a controlled generation and release of NO after activation by bacteria. For example, local NO acts mainly at the site where NO is generated. In the digestive tract, NO not only acts locally, but can also diffuse through the intestinal wall and enter the bloodstream, at least in part. This effect may be limited to a few millimeters. When reoxidized to nitrite and nitrate, allowing further movement or diffusion in the human or animal body, NO can be available at distant locations and can act systemically.As demonstrated in the examples, some conditions related to NO deficiency, such as skin diseases, sexual dysfunction, diseases requiring pain treatment, eye, nose and sinus diseases, airway diseases, digestive diseases, and metabolic disorders, have been successfully treated or alleviated by applying the chitosan nitrate of the present invention.The inventors have also developed a non-therapeutic or cosmetic method that includes administering chitosan nitrate, specifically a method for skin protection, preferably skin protection against environmental effects and / or healthy aging.

[0016] Thus, the present invention relates to chitosan nitrate for use as a medicament. The present invention also relates to chitosan nitrate for use as an NO donor, in particular for use as an agent for donating NO. The use can also be expressed as the use of chitosan nitrate as an NO donor or as a method for donating NO to a subject, comprising administering chitosan nitrate to a subject. In the uses and methods of the present invention, chitosan nitrate may be referred to as an active ingredient. The present invention also relates to chitosan nitrate for use in the prevention or treatment of conditions associated with NO deficiency in mammals. The present invention also relates to non-therapeutic and / or cosmetic methods, particularly methods for protecting the skin, preferably against environmental effects and / or for healthy aging, comprising administering chitosan nitrate. The present invention also relates to pharmaceutical compositions comprising chitosan nitrate, wherein the composition is a topical formulation, such as a cream, emulsion, lotion, transdermal patch, spray, lozenge, chewing gum, dentifrice, nasal spray, nasal and sinus aerosol, eye drops or eye ointment. Alternatively, the composition is an oral formulation, such as a tablet, capsule or suppository.Otherwise, the composition is a pulmonary aerosol.This formulation is ideal for administering chitosan nitrate to the site of human or mammal where anaerobic bacteria exist and can generate NO.Nasal and sinus aerosol or pulmonary aerosol is preferably administered via a nebulizer. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] The present invention resides in the discovery that chitosan nitrate is an efficient NO donor. Thus, chitosan nitrate acts as a nitric oxide prodrug and is metabolized via the nitrate-nitrite-NO pathway after bacterial activation. The present invention is further conceived based on the insight that almost all human systems rely on NO and its direct or indirect effects for homeostasis.

[0018] Chitosan Nitrate Chitosan is a polymer of D-glucosamine and N-acetyl-D-glucosamine monomer residues randomly distributed in β-(1→4) linkages. Chitosan nitrate is also known as CTS-HNO3. Chitosan can be obtained by treating chitin from mushrooms or fungi obtained from Aspergillus niger or chitin shells from shrimp and other crustaceans with alkaline substances, such as sodium hydroxide. Chitosan has a wide range of uses, including biomedical applications. Chitosan can be used in agriculture as a seed treatment agent, a biopesticide, and a fungicide. Medical applications include use in bandages to reduce bleeding, as an antibacterial agent, and as a drug delivery vehicle.

[0019] Luo and Wang reported recent developments in chitosan-based polyelectrolyte complexes with natural polysaccharides for drug delivery (Int J Biol Macromol. 64, 2014, 353-67). Chitosan, a positively charged polysaccharide, is a popular biopolymer for the development of drug delivery systems for various applications due to its promising properties, such as high biocompatibility, excellent biodegradability, and low toxicity, as well as abundant availability and low production costs. Delivery systems made from natural biopolymer-based polyelectrolyte complexes (PECs), which are formed by electrostatic interactions between two biopolymers with opposite charges, have attracted general interest. To suit specific applications of chitosan-based PEC drug delivery systems, various morphologies have been developed, including nanoparticles, microparticles, beads, tablets, gels, and films and membranes. Drug delivery applications of PECs based on chitosan, including other natural polysaccharides, examples of which include alginate, hyaluronic acid, pectin, carrageenan, xanthan gum, gellan gum, gum arabic, and carboxymethylcellulose, have been developed.

[0020] In chitosan nitrate, the primary amine moieties of the D-glucosamine monomers are protonated to form ammonium cations, which have nitric acid as a counterion. Chitosan nitrate is a solid and easily crystallizes. Thus, in one embodiment, chitosan nitrate is in the form of a crystal or powder. The chitosan nitrate of the present invention is composed of nitrate anions and chitosan cations, wherein the weight ratio of nitrate anions to chitosan is 1:1 to 1:10, more preferably 1:1 to 1:5, even more preferably 1:2 to 1:5, even more preferably 1:2 to 1:4, even more preferably 1:2.5 to :3.5. In the most preferred embodiment, the weight ratio of nitrate anions to chitosan is about 1:3. In a preferred embodiment, at least 75 mol%, more preferably at least 90 mol%, and most preferably substantially all of the primary amine moieties of the D-glucosamine monomers of chitosan are protonated and functionalized with nitric acid.

[0021] Chitosan preferably has a weight average molecular weight of 20-500 kDa, more preferably 150-250 kDa, and most preferably 190-210 kDa. In the most preferred embodiment, the molecular weight of chitosan is about 200 kDa. The inventors have found that in these molecular weight ranges, especially about 200 kDa, chitosan nitrate provides optimal results in terms of alleviating conditions associated with NO deficiency. Furthermore, such polymer lengths, when included in topical formulations, provide optimal results in skin applications. At these molecular weights, optimal acceptance into the skin pores inhabited by the relevant anaerobic bacteria is achieved, and the inorganic nitrate is activated by the bacteria, allowing NO to be readily formed and taken up by the body.

[0022] Chitosan nitrate is typically obtained by treating chitosan with nitric acid. In a preferred embodiment, chitosan nitrate can be obtained by a process comprising: (a) obtaining chitin from a natural source, such as crustaceans, fungi, such as mushrooms and / or Aspergillus niger; (b) optionally treating the chitin with an acid, such as an HCl solution, and then with a base, such as a KOH solution, to obtain an intermediate product comprising chitosan, which may be optionally separated; and (c) subsequently contacting the (intermediate) chitosan product with nitric acid (HNO3) to obtain chitosan nitrate. The resulting chitosan nitrate is a salt that readily crystallizes from solution. The chitosan nitrate may then be washed and dried, if desired.

[0023] In an alternative embodiment, chitosan nitrate can be obtained by a process involving the enzymatic treatment of chitin with chitinase, which is commercially available.

[0024] Alternatively, chitosan nitrate can be synthesized as described in Huo, L. et al, Electrorheological properties of chitosan nitrate suspension, Colloids and Surfaces A: Physicochem. Eng. Aspects 316 (2008) 125-130. 3 of nitric acid (16 mol / L) in 20 cm of acetone 3 The chitosan nitrate particles were then crushed and dried in a vacuum oven at 50°C for 2 days. Chitosan nitrate was thus obtained.

[0025] In a preferred embodiment of the above synthesis, an excess of nitric acid is used to protonate substantially all of the primary amines of the D-glucosamine monomers of the resulting chitosan nitrate to the nitrate salt.

[0026] Pharmaceutical Compositions In one aspect, the present invention relates to a pharmaceutical composition comprising chitosan nitrate. The pharmaceutical composition comprising chitosan nitrate can be prepared according to the general procedures known in the art. The pharmaceutical composition of the present invention can take any form, preferably an oral composition, such as a tablet, capsule or suppository, a pulmonary aerosol, and a topical preparation, such as a cream, emulsion, lotion, transdermal patch, spray, lozenge, chewing gum, dentifrice, nasal spray, nasal and sinus aerosol, eye drops or eye ointment. When the composition takes the form of an emulsion, a water-in-oil emulsion is preferred, since it provides optimal results in terms of treating conditions related to NO deficiency. Alternatively, an oil-in-water emulsion also provides good therapeutic results. The nasal and sinus aerosol or pulmonary aerosol is preferably administered via a nebulizer.

[0027] The pharmaceutical composition of the present invention contains the above-mentioned chitosan nitrate. In a preferred embodiment of the present invention, the pharmaceutical composition contains chitosan nitrate having a molecular weight of 20 to 500 kDa, more preferably 150 to 250 kDa, and most preferably 190 to 210 kDa.

[0028] The composition of the present invention preferably contains vitamin C (ascorbic acid). When present, vitamin C is preferably contained in the composition in an amount of 0.5 to 50% by weight, more preferably 1 to 20% by weight, and most preferably 2.5 to 10% by weight, relative to the chitosan nitrate. In a particularly preferred embodiment, the composition contains vitamin C and a phosphate, as described below.

[0029] In one embodiment of the invention, the pharmaceutical composition is a topical formulation, such as a cream. In one embodiment, the cream comprises a water-in-oil (W / O) emulsion. In another embodiment, the cream comprises a water-in-oil-in-water (W1 / O / W2) double emulsion. In yet another embodiment, the cream comprises an oil-in-water (O / W) emulsion. In a W / O emulsion, the aqueous phase comprises chitosan nitrate, usually in combination with a pharma- ceutically acceptable salt, such as NaCl and / or a phosphate-containing salt. Preferred phosphate salts are tetrasodium pyrophosphate (TSPP; Na4P2O7) and / or disodium phosphate (DSP; Na2HPO4). Most preferably, DSP is used. The oil phase preferably comprises petrolatum / paraffin. Petrolatum and paraffin are preferably present in a weight ratio of 2:1 to 1:2, more preferably in approximately equal parts by weight. In a preferred embodiment, the oil phase further comprises lanolin. The cream may be prepared by slowly adding the aqueous phase to the oil phase with stirring. The DSP serves to stabilize the chitosan nitrate particles and is preferably used in a weight percentage of 3-10% by weight, preferably 4-7% by weight, and most preferably about 5% by weight relative to the chitosan nitrate.

[0030] The W / O emulsion or W1 / O / W2 double emulsion preferably contains 50-70% by weight of petrolatum / paraffin, 5-20% by weight of lanolin, 20-50% by weight of distilled water, 0.1-5% by weight of chitosan nitrate, 0.1-1.0% by weight of NaCl, and 3-10% by weight of DSP, based on chitosan nitrate. More preferably, the W / O emulsion or W1 / O / W2 double emulsion contains 55-65% by weight of petrolatum / paraffin, 8-15% by weight of lanolin, 25-35% by weight of distilled water, 0.5-2% by weight of chitosan nitrate, 0.2-0.5% by weight of NaCl, and 4-6% by weight of DSP, based on chitosan nitrate. In a most preferred embodiment, the W / O emulsion or W1 / O / W2 double emulsion comprises about 60% by weight petrolatum / paraffin, about 10% by weight lanolin, about 28.1% by weight distilled water, about 1% by weight chitosan (about 200 kDa) nitrate, about 0.3% by weight NaCl, and about 5% by weight DSP relative to the chitosan nitrate.

[0031] In another embodiment of the present invention, the pharmaceutical composition is a tablet or capsule or a colon-targeted capsule. The tablet or capsule preferably contains 50-2000 mg of chitosan nitrate, more preferably 100-1000 mg of chitosan nitrate, even more preferably 200-800 mg of chitosan nitrate, and most preferably 400-600 mg of chitosan nitrate. In a preferred embodiment, the prepared tablet or capsule contains about 480 mg of chitosan nitrate, consisting of about 120 mg of nitrate anion and about 360 mg of chitosan. In a further preferred embodiment, the chitosan nitrate can be stabilized by adding a phosphate, preferably dihydrogen phosphate (DSP), in an amount of up to 10% by weight relative to the chitosan nitrate.

[0032] In yet another embodiment of the present invention, the pharmaceutical composition is a nasal spray or a nasal aerosol. The nasal spray contains sterile water, a pharma- ceutically acceptable salt, such as NaCl, and preferably 0.05-5% by weight of chitosan nitrate, more preferably 0.1-2% by weight of chitosan nitrate, even more preferably 0.3-1% by weight of chitosan nitrate. In a preferred embodiment, the nasal spray contains sterile water, about 0.5% by weight of chitosan (about 200 kDa) nitrate, and about 0.9% by weight of NaCl. The nasal spray or aerosol is preferably administered via a nebulizer.

[0033] The following embodiments of the invention have been prepared and tested in patients with a variety of medical and medical-like disorders. The compositions described below are the most preferred pharmaceutical compositions in the context of the present invention.

[0034] No.3 - Chitosan nitrate capsules were prepared containing 480 mg of chitosan nitrate consisting of 120 mg of anion and 360 mg of 200 kDa chitosan. In a further preferred embodiment, the chitosan nitrate can be stabilized by adding a phosphate, preferably dihydrogen phosphate (DSP), up to 10% by weight relative to the chitosan nitrate.

[0035] Creams were prepared containing either a water-in-oil (W / O) emulsion or a water-in-oil-in-water (W1 / O / W2) double emulsion. In the W / O emulsion, the aqueous phase contains chitosan (200 kDa) nitrate and NaCl and / or DSP, and the oil phase contains equal parts by weight of petrolatum / paraffin and lanolin. The creams are prepared by slowly adding the aqueous phase to the oil phase with stirring. The emulsions contain 60% by weight petrolatum / paraffin, 10% by weight lanolin, 28.1% by weight distilled water, 1% by weight chitosan (200 kDa) nitrate, 0.3% by weight NaCl, and 0.05% by weight DSP. Another cream based on an O / W emulsion contains 60% by weight distilled water, 2% by weight sorbitol crystal solution, 0.15% by weight sorbic acid, 0.5% by weight chitosan nitrate, 0.025% by weight DSP, 2% by weight D-panthenol, 7% by weight glycerin, about 0.05% by weight citric acid to adjust the pH to about 4.5, 7.5% by weight cetomacrogol wax, 10% by weight decyl oleate, 7% by weight coconut oil, 2% by weight shea butter, and 2% by weight BTMS-25.

[0036] A nasal spray was prepared containing sterile water, 0.5% by weight of chitosan (200 kDa) nitrate, and 0.9% by weight of NaCl. An additional nasal spray was also prepared containing sterile water, 0.25% by weight of chitosan (200 kDa) nitrate, 2.00% by weight of NaCl, 0.00125% by weight of Na2HPO4, 0.05% by weight of eucalyptus oil, and 0.0125% by weight of benzalkonium chloride (used as a preservative).

[0037] Cosmetic composition In one aspect, the present invention relates to a cosmetic composition comprising chitosan nitrate. Apart from not being intended for therapeutic applications, the cosmetic composition may be similar or identical to a pharmaceutical composition. Thus, all preferred aspects for the pharmaceutical composition of the present invention apply equally to the cosmetic composition of the present invention. The cosmetic composition may be referred to as a non-therapeutic composition. The cosmetic composition comprising chitosan nitrate may be prepared in the same manner as the pharmaceutical composition described above. The cosmetic composition of the present invention may take any form, for example, an oral composition or a (nutritional) supplement. Typically, the cosmetic composition takes the form of a tablet, capsule or topical preparation, for example a cream, emulsion or lotion. When the composition takes the form of an emulsion, a water-in-oil emulsion is preferred, as this gives optimal results. However, oil-in-water emulsions may also be used.

[0038] The non-therapeutic composition and / or cosmetic composition of the present invention comprises the above-mentioned chitosan nitrate. In a preferred embodiment of the present invention, the pharmaceutical composition comprises chitosan nitrate having a molecular weight of 20-500 kDa, more preferably 150-250 kDa, and most preferably 190-210 kDa.

[0039] In one embodiment of the present invention, the non-therapeutic composition and / or the cosmetic composition is a topical preparation, such as a cream. In one embodiment, the cream comprises a water-in-oil (W / O) emulsion. In another embodiment, the cream comprises a water-in-oil-in-water (W1 / O / W2) double emulsion. The W / O emulsion or the W1 / O / W2 double emulsion can be prepared in the same manner as described above for the pharmaceutical composition. An oil-in-water (O / W) emulsion can also be used.

[0040] In another embodiment of the present invention, the non-therapeutic composition and / or the cosmetic composition is a tablet or capsule. The tablet or capsule preferably contains 50-2000 mg of chitosan nitrate, more preferably 100-1000 mg of chitosan nitrate, even more preferably 200-800 mg of chitosan nitrate, and most preferably 400-600 mg of chitosan nitrate. In a preferred embodiment, the prepared tablet or capsule contains about 480 mg of chitosan nitrate, consisting of about 120 mg of nitrate anion and about 360 mg of chitosan.

[0041] The non-therapeutic composition and / or cosmetic composition of the present invention may further contain DSP, preferably in an amount of 5 to 10% by weight based on the total weight of the cosmetic composition.

[0042] Chitosan nitrate for use as a drug and as an NO donor The present invention relates to chitosan nitrate for use as a medicament. In a related aspect, the present invention relates to chitosan nitrate for use as a NO donor. In another related aspect, the present invention relates to chitosan nitrate for use in the prevention or treatment of conditions related to NO deficiency. According to the present invention, chitosan nitrate is preferably administered as a pharmaceutical composition comprising chitosan nitrate, where the pharmaceutical composition is either a topical formulation, a tablet, a capsule, or a nasal spray or a pulmonary aerosol. The nasal spray or a pulmonary aerosol is preferably administered via a nebulizer.

[0043] In a preferred embodiment, chitosan nitrate capsules containing 300-600 mg, preferably about 480 mg, of chitosan nitrate are administered twice daily, preferably one capsule in the morning and one capsule in the afternoon, or two capsules in the morning, to maintain or achieve desired NO concentrations. In an alternative embodiment, chitosan nitrate capsules containing 300-600 mg, preferably about 480 mg, of chitosan nitrate are administered at a dose of 6 capsules per day for up to 3 days, most preferably with administration evenly distributed throughout the day, to maintain or achieve higher NO concentrations in the short term.

[0044] In another preferred embodiment, topical application, e.g., using a cream, is initially prescribed / administered twice daily to maintain or achieve the desired NO concentration, but once the desired NO concentration is achieved, once daily administration is sufficient.

[0045] In another preferred embodiment, the nasal spray is administered 1-2 sprays into each nostril 4 times per day initially, and once symptoms are relieved, 1-2 sprays into each nostril once per day to maintain or achieve the desired NO concentration.

[0046] In one aspect, the present invention relates to chitosan nitrate for use in the prevention or treatment of conditions associated with NO deficiency, selected from skin diseases, sexual dysfunction, diseases requiring pain treatment, diseases related to the eyes, nose and sinuses, respiratory tract diseases, digestive diseases, and metabolic disorders. Skin diseases are, for example, psoriasis, atopic dermatitis, acne vulgaris, herpes simplex and / or shingles, the latter also known as zoster or herpes zoster. Alternatively, skin diseases are vascular disorders exhibiting Raynaud's symptoms, in particular scleroderma and / or chilblains. Skin diseases are also caused by ultraviolet oxidative stress (photodamage) and / or skin aging, in particular premature skin aging. In the case of sexual dysfunction, topical application of chitosan nitrate to the genital area may be required, preferably to treat erectile dysfunction and to treat female sexual dysfunction (FSD). Alternatively, the condition may be one for which pain treatment is required, preferably tendonitis, lateral epicondylitis, arthritis, anal fissure, post-traumatic pain and / or swelling, or diabetic neuropathy, carpal tunnel syndrome and / or migraine, which require neuromodulation therapy as part of pain treatment. The eye, nose and sinus disease is preferably allergic conjunctivitis, dry eye, glaucoma, allergic rhinitis, non-allergic rhinitis and / or sinusitis. The airway disease is preferably asthma, chronic obstructive pulmonary disease (COPD) and / or pulmonary arterial hypertension. The digestive disease is preferably leaky gut, reduced gut microbiota diversity, reduced Akkermansia Muciniphila, dysbiosis and / or irritable bowel syndrome (IBS). The metabolic disorder is preferably hypertension, type 2 diabetes and / or atherosclerosis.

[0047] In a preferred embodiment, chitosan nitrate for use in the prevention or treatment of the above diseases is administered as a pharmaceutical composition comprising chitosan nitrate, wherein the pharmaceutical composition is a topical formulation or tablet. In a preferred embodiment, chitosan nitrate for use in the prevention or treatment of the above conditions is administered as a topical formulation, wherein the topical formulation, e.g., a cream, preferably comprises 50-70% petrolatum / paraffin by weight, 5-20% lanolin by weight, 20-50% distilled water by weight, 0.1-5% chitosan nitrate by weight, 0.1-1.0% NaCl by weight, and 3-10% DSP by weight, preferably comprising a water-in-oil (W / O) emulsion or a water-in-oil-in-water (W1 / O / W2) double emulsion, relative to chitosan. More preferably, the W / O emulsion or W1 / O / W2 double emulsion contains 55-65% by weight of petrolatum / paraffin, 8-15% by weight of lanolin, 25-35% by weight of distilled water, 0.5-2% by weight of chitosan nitrate, 0.2-0.5% by weight of NaCl, and 4-7% by weight of DSP relative to chitosan. In yet another preferred embodiment, the cream based on an O / W emulsion contains 60% by weight of distilled water, 2% by weight of sorbitol crystal solution, 0.15% by weight of sorbic acid, 0.5% by weight of chitosan nitrate, 0.025% by weight of DSP, 2% by weight of D-panthenol, 7% by weight of glycerin, about 0.05% by weight of citric acid to adjust the pH to about 4.5, 7.5% by weight of cetomacrogol wax, 10% by weight of decyl oleate; 7% by weight of coconut oil; 2% by weight of shea butter; and 2% by weight of BTMS-25.

[0048] In a further preferred embodiment, the present invention relates to chitosan nitrate for use in the prevention or treatment of the above diseases, wherein the chitosan nitrate is administered as a tablet or capsule, the tablet or capsule preferably containing 50-2000 mg of chitosan nitrate, more preferably 100-1000 mg of chitosan nitrate, even more preferably 200-800 mg of chitosan nitrate, and most preferably 400-600 mg of chitosan nitrate. In a preferred embodiment, the prepared tablet or capsule contains about 480 mg of chitosan nitrate consisting of about 120 mg of nitrate anion and about 360 mg of chitosan (about 200 kDa).

[0049] In a further preferred embodiment, the present invention relates to chitosan nitrate for use in the prevention or treatment of airway diseases, wherein the diseases are preferably asthma, chronic obstructive pulmonary disease (COPD) and / or pulmonary arterial hypertension. In a preferred embodiment, chitosan nitrate for use in the prevention or treatment of airway diseases is administered as a pulmonary aerosol, wherein the pulmonary aerosol comprises sterile water, a pharma- ceutically acceptable salt, such as NaCl, and preferably 0.05-5 wt. % chitosan nitrate, more preferably 0.1-2 wt. % chitosan nitrate, even more preferably 0.3-1 wt. % chitosan nitrate. In a preferred embodiment, the pulmonary aerosol comprises sterile water, about 0.5 wt. % chitosan (about 200 kDa) nitrate, and about 0.9 wt. % NaCl. The pulmonary aerosol is preferably administered via a nebulizer.

[0050] In a further preferred embodiment, the present invention relates to chitosan nitrate for use in the prevention or treatment of a disease requiring pain treatment, preferably for tendonitis, lateral epicondylitis, arthritis, anal fissure, post-traumatic pain and / or swelling, or alternatively for diabetic neuropathy, carpal tunnel syndrome and / or migraine, where neuromodulation therapy is required as part of the pain treatment. In a preferred embodiment, chitosan nitrate for use in the prevention or treatment of the above diseases is administered as a tablet or capsule, wherein the tablet or capsule preferably contains 50-2000 mg of chitosan nitrate, more preferably 100-1000 mg of chitosan nitrate, even more preferably 200-800 mg of chitosan nitrate, most preferably 400-600 mg of chitosan nitrate. In a preferred embodiment, the prepared tablet or capsule contains about 480 mg of chitosan nitrate consisting of about 120 mg of nitrate anion and about 360 mg of chitosan (about 200 kDa). In a more preferred embodiment, chitosan nitrate for use in the prevention or treatment of the above diseases is administered as a topical formulation, wherein the topical formulation, e.g., a cream, preferably comprises 50-70% petrolatum / paraffin, 5-20% lanolin, 20-50% distilled water, 0.1-5% chitosan nitrate, 0.1-1.0% NaCl, and 3-10% DSP by weight relative to chitosan, and preferably comprises a water-in-oil (W / O) emulsion or a water-in-oil-in-water (W1 / O / W2) double emulsion. More preferably, the W / O emulsion or W1 / O / W2 double emulsion contains 55-65% petrolatum / paraffin, 8-15% lanolin, 25-35% distilled water, 0.5-2% chitosan nitrate, 0.2-0.5% NaCl, and 4-7% DSP by weight relative to chitosan. Alternatively, an oil-in-water (O / W) emulsion can be administered.The cream based on an O / W emulsion contains 60% by weight of distilled water, 2% by weight of sorbitol crystal solution, 0.15% by weight of sorbic acid, 0.5% by weight of chitosan nitrate; 0.025% by weight of DSP, 2% by weight of D-panthenol, 7% by weight of glycerin, about 0.05% by weight of citric acid to adjust the pH to about 4.5, 7.5% by weight of cetomacrogol wax, 10% by weight of decyl oleate, 7% by weight of coconut oil, 2% by weight of shea butter and 2% by weight of BTMS-25.

[0051] In a further preferred embodiment, the present invention relates to chitosan nitrate for use in the prevention or treatment of eye, nose and sinus diseases, wherein the diseases are preferably allergic conjunctivitis, dry eye, glaucoma, allergic rhinitis, non-allergic rhinitis and / or sinusitis. In a preferred embodiment, chitosan nitrate for use in the prevention or treatment of nose and sinus diseases is administered as a nasal spray, wherein the nasal spray comprises sterile water, a pharma- ceutically acceptable salt, such as NaCl, and preferably 0.05-5% by weight of chitosan nitrate, more preferably 0.1-2% by weight of chitosan nitrate, even more preferably 0.3-1% by weight of chitosan nitrate. In a preferred embodiment, the nasal spray comprises sterile water, about 0.5% by weight of chitosan (about 200 kDa) nitrate, and about 0.9% by weight of NaCl. In a preferred embodiment, chitosan nitrate for use in the prevention or treatment of eye diseases is administered as eye drops, where the eye drops contain sterile water, a pharma- ceutically acceptable salt, such as NaCl, and preferably 0.05-5% by weight of chitosan nitrate, more preferably 0.1-2% by weight of chitosan nitrate, even more preferably 0.3-1% by weight of chitosan nitrate. In a preferred embodiment, the eye drops contain sterile water, about 0.5% by weight of chitosan (about 200 kDa) nitrate, and about 0.9% by weight of NaCl. A nasal spray was prepared containing sterile water, 0.25% by weight of chitosan (200 kDa) nitrate, 2.00% by weight of NaCl, 0.00125% by weight of Na2HPO4, 0.05% by weight of eucalyptus oil, and 0.0125% by weight of benzalkonium chloride (used as a preservative).

[0052] In a preferred embodiment, chitosan nitrate for use in the prevention or treatment of allergic rhinitis, non-allergic rhinitis and / or sinusitis is administered as a nasal spray, wherein the nasal spray comprises sterile water, a pharma- ceutically acceptable salt, such as NaCl, and preferably 0.05-5% by weight of chitosan nitrate, more preferably 0.1-2% by weight of chitosan nitrate, even more preferably 0.3-1% by weight of chitosan nitrate. In a preferred embodiment, the nasal spray comprises sterile water, about 0.5% by weight of chitosan (about 200 kDa) nitrate, and about 0.9% by weight of NaCl. A nasal spray was prepared containing sterile water, 0.25 wt% chitosan (200 kDa) nitrate, 2.00 wt% NaCl, 0.00125 wt% Na2HPO4, 0.05 wt% eucalyptus oil, and 0.0125 wt% benzalkonium chloride (used as a preservative).

[0053] Non-therapeutic and / or cosmetic methods involving administration of chitosan nitrate In another embodiment, the present invention relates to a cosmetic method comprising administering chitosan nitrate, wherein chitosan nitrate is preferably used as an NO donor.The cosmetic method may be referred to as a non-therapeutic method.In a preferred embodiment, the cosmetic method is aimed at protecting the skin, preferably protecting the skin against environmental influences and / or healthy aging.Furthermore, in the cosmetic method, the lack of nitric oxide is due to aging.The cosmetic method of the present invention comprises administering the cosmetic composition of the present invention described above.

[0054] In a preferred embodiment, the cosmetic method comprises administering a W / O emulsion or a W1 / O / W2 double emulsion, preferably comprising 50-70% by weight of petrolatum / paraffin, 5-20% by weight of lanolin, 20-50% by weight of distilled water, 0.1-5% by weight of chitosan nitrate, 0.1-1.0% by weight of NaCl, and 3-10% by weight of DSP, relative to chitosan. More preferably, the W / O emulsion or the W1 / O / W2 double emulsion comprises 55-65% by weight of petrolatum / paraffin, 8-15% by weight of lanolin, 25-35% by weight of distilled water, 0.5-2% by weight of chitosan nitrate, 0.2-0.5% by weight of NaCl, and 4-7% by weight of DSP, relative to chitosan. In a most preferred embodiment, the W / O emulsion or W1 / O / W2 double emulsion contains about 60% petrolatum / paraffin, about 10% lanolin, about 28.1% distilled water, about 1% chitosan (about 200 kDa) nitrate, about 0.3% NaCl, and about 5% DSP by weight relative to chitosan. Alternatively, an oil-in-water (O / W) emulsion can be administered. The cream based on the O / W emulsion contains 60% by weight distilled water, 2% by weight sorbitol crystal solution, 0.15% by weight sorbic acid, 0.5% by weight chitosan nitrate; 0.025% by weight DSP, 2% by weight D-panthenol, 7% by weight glycerin, about 0.05% by weight citric acid to adjust the pH to about 4.5, 7.5% by weight cetomacrogol wax, 10% by weight decyl oleate; 7% by weight coconut oil, 2% by weight shea butter, and 2% by weight BTMS-25.

[0055] Water-in-oil emulsions for topical application Water-in-oil emulsions are widely used for topical application. There are three routes for intradermal or transdermal drug delivery: the skin appendages (or adnexa) route, also known as the appendageal route, where the drug enters through skin-associated structures such as pores, hair follicles, and sweat ducts; the transcellular route; and the intracellular route. Chitosan nitrate is primarily directed to the appendageal route, and thus W / O emulsions are preferred. Skin as a whole is hydrophobic, whereas the appendages are hydrophilic, so the hydrophilic moiety easily penetrates the appendages. These appendages contain bacteria that can reduce chitosan nitrate via the nitrate-nitrite-NO pathway, also referred to as microbial nitrate bioactivation. The hydrophobic moiety has an affinity with the remaining part of the skin, which is lipophilic. It is therefore recognized that W / O emulsions allow chitosan nitrate to enter the appendages. However, since oil covers the skin except for the hydrophilic pores of the appendages, O / W emulsions or creams based thereon are also suitable. Chitosan nitrate dissolves in the aqueous phase of the cream.

[0056] The stratum corneum, the outermost layer of the epidermis, is lipophilic, so water cannot penetrate the skin. Pores, however, are hydrophilic. Chitosan nitrate is hydrophilic. The W / O emulsion allows for the uptake of chitosan nitrate via the appendage pathway. In pores, there are facultative anaerobes that reduce nitrate to nitrite. This is generally considered to be the first step of the nitrate-nitrite-nitric oxide pathway. In the pores, ducts and hair follicles, bacterial lysozyme and bacterial chitosanase break down the chitosan portion of chitosan nitrate.

[0057] The present inventors have found that oligochitosan (10 kDa) nitrate, oligochitosan (10 kDa) with added nitrate, and oligochitosan (10 kDa) with added nitrite are prone to skin irritation in some individuals. Typical areas of skin affected are the inside of the elbow, the back of the knee, and the back of the neck. It is the applicant's opinion that this may be due to accumulation of nitrite and / or chitosan between the stratum corneum cells. Chitosan (200 kDa) nitrate in a W / O or W / O / W emulsion in the embodiment described herein appears to avoid these problems. Chitosan (200 kDa) nitrate in an O / W emulsion in the embodiment described herein also avoids these problems. It has been found that the addition of 5% by weight of DSP to chitosan nitrate makes the W / O and O / W emulsions even more effective. Nitrate by itself without chitosan in a W / O or O / W emulsion provided very little, if any, improvement. All embodiments with nitrous acid but without chitosan caused itching and irritation of the skin. EXAMPLES

[0058] Example 1: High Blood Pressure (Hypertension) A 65-year-old man was taking antihypertensive drugs. His blood pressure without medication was 145mmHg systolic and 100mmHg diastolic. He was administered one capsule of chitosan nitrate twice daily for one year.

[0059] Chitosan nitrate capsules contain NO3 - Anion 120 mg and 200 kDa (weight average molecular weight M W ) and contains 480 mg of chitosan nitrate, consisting of 360 mg of chitosan.

[0060] The patient's blood pressure returned to normal (systolic 130 / diastolic 85) within three months. There were no side effects.

[0061] Fasting salivary nitrate and nitrite measurements reflect plasma nitrate and nitrite levels. -One capsule of chitosan nitrate containing 120 mg of anion was taken at 11:00 pm. Fasting saliva values ​​measured at 7:30 am the following morning were nitrate (NO3 - ) is >500mg / L, and salivary nitrite (NO2 - ) was >80 mg / L.

[0062] No.3 - The equivalent amount of KNO3 salt (180 mg) was taken at 11 p.m. The fasting value the next morning was NO3 - is 80mg / L, NO2 - was 4.7 mg / L.

[0063] Tests were also carried out using 10 kDa oligochitosan nitrate. - The equivalent of 120 mg was taken at 11 p.m. The fasting values ​​at 7:30 the next morning were NO3 - 144mg / L, NO2 - The value was 14.4 mg / L. Oligochitosan was absorbed before reaching the colon, and NO3 - will also be absorbed.

[0064] Quantofix Nitrate and Nitrite Test Strips (Macherey-Nagel, Germany) were used to measure NO3 - and NO2 - These test strips allow semi-quantitative measurements. The test strips were read using a Quantofix Relax reader.

[0065] In the case of KNO3, it is absorbed immediately and 75% is excreted in the urine, so NO3 - In the case of chitosan (200 kDa) nitrate, the NO3 in saliva was measured 8.5 hours after administration. - and NO2 -The amount of nitrates was higher compared to other studies. This means that at least some of the nitrates reach the colon and are converted to the biologically active NO form. NO is known to increase blood flow to the intestinal wall. Increased blood flow can increase mucus formation in the intestinal wall, which in turn favorably regulates the microflora and supports the growth of bacteria such as Akkermansia muciniphila. Increased mucus formation strengthens the intestinal barrier and prevents disease, as toxic substances cannot enter the bloodstream.

[0066] The daily dose was taken in the morning, and 24 hours later, saliva nitrate and nitrite levels were measured. Two chitosan nitrate capsules contained 240 mg of NO3 - The amount of chitosan in saliva was equivalent to 100 mg of KNO3. The mean value of the fasting saliva was 100 mg of KNO3 for chitosan nitrate. - was 447 mg / l, and NO2 - is 45mg / l, and in KNO3, NO3 - was 274 mg / l, and NO2 - was 15.1 mg / l. The literature suggests that salivary nitrite levels are the best indicator of plasma NO status. Salivary nitrite levels collected 24 hours after chitosan nitrate administration were three times higher than those after administration of an equivalent amount of nitrate anion in the form of KNO3.

[0067] The 16s rRNA of the microbiota was sequenced at the start of treatment and one year later. The abundance of Akkermansia muciniphila increased by 166%. A. muciniphila plays an important role in overweight, obesity and diabetes.

[0068] In conclusion, chitosan nitrate has the effect of maintaining high blood nitrate and (bioactive) nitrite concentrations compared to KNO3 and 10 kDa oligochitosan nitrate, and chitosan nitrate positively regulates the gut microbiota.

[0069] Example 2: Eczema (atopic dermatitis) A 5-year-old girl had long suffered from an extremely itchy rash on her face, neck, arms, and legs. She had been given many corticosteroids to control her condition. Her medication was changed. A cream containing chitosan nitrate was applied to the skin twice a day for one year. After this treatment, a cream containing 0.1% triamcinolone was used approximately twice a month.

[0070] The creams are either water-in-oil (W / O) emulsions or W1 / O / W2 double emulsions. In W / O emulsions, the aqueous phase contains chitosan (200 kDa) nitrate, NaCl and DPS, and the oil phase contains equal amounts of petrolatum and paraffin, as well as lanolin. The creams are prepared by slowly adding the aqueous phase to the oil phase with stirring.

[0071] The emulsion contains 60% by weight petrolatum / paraffin, 10% by weight lanolin, 28.1% by weight distilled water, 1% by weight chitosan (200 kDa) nitrate, and 0.9% by weight NaCl. If DSP is added, the emulsion contains 0.05% by weight DSP and 28.05% by weight distilled water.

[0072] Water-in-oil (W / O) or water-in-oil-in-water (W / O / W) emulsions containing various additional ingredients and emulsifiers can be prepared by standard techniques in the art. Alternatively, oil-in-water (O / W) emulsions containing various additional ingredients and emulsifiers can be prepared by standard techniques in the art. The O / W emulsion cream contains 60% by weight distilled water, 2% by weight sorbitol crystal solution, 0.15% by weight sorbic acid, 0.5% by weight chitosan nitrate, 0.025% by weight DSP, 2% by weight D-panthenol, 7% by weight glycerin, about 0.05% by weight citric acid to adjust the pH to about 4.5, 7.5% by weight cetomacrogol wax, 10% by weight decyl oleate, 7% by weight coconut oil, 2% by weight shea butter, and 2% by weight BTMS-25.

[0073] Example 3: Cold, stiff and painful hands in patients with scleroderma A 50-year-old man with scleroderma frequently complained of Raynaud's syndrome in cold climates. The cold, stiffness and pain in his hands were due to poor circulation. After one year of continuous application of a W / O / W emulsion containing 1% by weight of chitosan (200 kDa) nitrate three times a day (less frequently in summer), the patient felt a marked improvement: he suffered less pain, less stiffness, his hands felt warmer and he felt more comfortable overall.

[0074] Example 4: Diabetic Polyneuropathy A 66-year-old man with diabetes and severe polyneuropathy of the legs for many years. The patient was unable to sleep at night due to pain in his legs. After applying the W / O / W emulsion twice a day to his legs for one month, the patient noticed a significant improvement in his condition, i.e., a reduction in pain. Nitric oxide is thought to regulate the small nerve fibers involved in pain generation. Polyneuropathy is a chronic disease, so lifelong treatment is required.

[0075] Example 5: Migraine; Trigeminal Neuromodulation A 35-year-old woman suffered from migraine with aura approximately 6 days per month. Initially, treatment consisted of topical application of a water-in-oil emulsion containing 1% by weight of chitosan (200 kDa) nitrate to the skin of the forehead and eyebrows. During migraine attacks, the emulsion was applied up to 4 times a day, and between attacks, the emulsion was applied once a day. This treatment is believed to have a positive effect on the ophthalmic branch of the trigeminal nerve. Subsequent treatment included the addition of a nasal spray containing 0.5% by weight of chitosan (200 kDa) nitrate and 0.9% by weight of NaCl, twice a day during migraine attacks. This treatment is believed to have a positive effect on the maxillary branch of the trigeminal nerve. By modulating the two nerve branches of the trigeminal nerve, the number of days the patient suffered from (migraine) headaches was reduced by more than 50%.

[0076] In another patient suffering from both tension headaches and migraines, the same W / O emulsion was applied locally to the occipital triangle, which affects the cervico-trigeminal pathway, in addition to the above treatment. There were no side effects, and the number of days the patient suffered from migraines and tension headaches was reduced. Specifically, the W / O emulsion was applied locally to the so-called occipital triangle. In addition, the topical application of the W / O emulsion to the forehead was continued. In the later stages of treatment, an O / W emulsion containing the same amount of chitosan nitrate was applied, with similar results.

[0077] Example 6: Carpal tunnel syndrome A 70-year-old woman suffered from carpal tunnel syndrome affecting the wrist and hand, which caused sleep deprivation. Nerve decompression was suggested by the neurosurgeon but was not performed. The patient was administered topical application of a water-in-oil emulsion containing 1% by weight of chitosan (200 kDa) nitrate twice daily for the first month and once daily thereafter for six months. The patient then no longer complained of symptoms due to carpal tunnel syndrome.

[0078] Example 7: Rhinitis Several men and women aged 25-65 years with allergic rhinitis complained of nasal itching, sneezing, stuffy nose and runny nose. A nasal spray containing sterile water, chitosan (200 kDa) nitrate 0.5% by weight and NaCl 0.9% by weight was sprayed once or twice into both nostrils twice a day, and good improvement was achieved. That is, nasal itching, sneezing, stuffy nose and runny nose were relieved. Patients were allowed to spray more if they felt it was necessary.

[0079] Patients with non-allergic rhinitis were also administered the nasal spray, and improvements such as reduced snoring and better sleep were observed with the above treatment. Relief of nasal congestion and regulation of the nasal branch of the trigeminal nerve are thought to be part of the mechanism of action. In allergic rhinitis, stabilization of mast cells may also contribute. In allergic and non-allergic rhinitis, reduction of oxidative stress by nitric oxide is thought to play an important role in reducing symptoms due to rhinitis. Since the mechanism of action is different from that of general nasal sprays for rhinitis, chitosan nitrate-containing sprays may be used in combination with other sprays if necessary.

[0080] In the later stages of treatment of allergic and non-allergic rhinitis, the following nasal spray composition was applied twice a day with good results: A nasal spray was prepared containing sterile water, 0.25% by weight of chitosan (200 kDa) nitrate, 2.00% by weight of NaCl, 0.00125% by weight of Na2HPO4, 0.05% by weight of eucalyptus oil, and 0.0125% by weight of benzalkonium chloride (used as a preservative).

[0081] Example 8: Anal fissure A 56-year-old man suffering from pain due to anal fissure. The patient had already been prescribed anal pain by his family doctor. The ointment prescribed was 1% isosorbide dinitrate cream. The patient stopped using this cream due to serious side effects such as severe headache and flushing. Isosorbide dinitrate is an organic nitrate like nitroglycerin, which is quite different from inorganic nitrates like chitosan nitrate. Both nitrate compounds are prodrugs of nitric oxide, but inorganic nitrates are understood to act only locally without causing headache or flushing. The patient switched to administering a W / O emulsion containing 1% by weight of chitosan (200 kDa) nitrate three times a day. There were no side effects during this treatment, and the fissure healed within a few weeks, after which the treatment could be terminated.

[0082] Example 9: Sun protection; promoting skin tanning Topical application of a W / O / W emulsion containing 1% by weight of chitosan (200 kDa) nitrate stores sufficient nitrite in the skin to cause slight sunless tanning through the production of nitric oxide. UV radiation converts nitrite to nitric oxide. Although W / O / W emulsions are not considered sunscreens, application of the W / O / W emulsion twice daily for 3-4 days before sun exposure and again immediately after sun exposure has been shown to reduce solar damage to skin cells.

[0083] Example 10: Tennis elbow (lateral epicondylitis) A 42-year-old man suffered from right armpit and elbow pain for 4 months and was diagnosed with tennis elbow. The patient underwent physical therapy and one injection of a combination of 1ml Kenacort A10 and 1ml Xylocaine into the tendon attachment of the lateral epicondyle. The patient was initially started on twice-daily topical application of a W / O emulsion containing 1% by weight chitosan (200 kDa) nitrate. After 2 weeks of application, the patient felt relief from the pain and continued application of the emulsion once daily for one month.

[0084] Example 11: Erectile Dysfunction A 57-year-old man suffering from obesity, cardiovascular disorders, type 2 diabetes and erectile dysfunction for several years. Erectile dysfunction is a disorder in which the erection is insufficient during penetration. The patient was using organic nitrates such as nitroglycerin, so sildenafil (Viagra®) tablets were contraindicated. In contrast to sildenafil, which is a PDE-5 inhibitor, inorganic nitrates such as chitosan nitrate are permitted since they do not interact with PDE-5. A W / O / W emulsion containing 1% by weight of chitosan nitrate and 0.05% DSP was applied to the glans and penis 30 minutes before sexual activity. This treatment resulted in satisfactory intercourse in 80% of attempts.

[0085] Example 12: Allergic conjunctivitis A 45-year-old woman with hay fever. The patient is taking desloratadine 5 mg 1 tablet daily and levocabastine eye drops, 1 drop in each eye, 3 times daily. The addition of chitosan nitrate eye drops, containing 0.5% by weight chitosan and 0.9% by weight NaCl in sterile water, 1 drop in each eye, 2 times daily, has provided strong relief of the hay fever.

[0086] Example 13: Exercise-induced asthma A 25-year-old man with house dust mite allergy. The patient had been suffering from shortness of breath at the start of running for one year. This symptom lasted only for 5 minutes and then subsided, and he was diagnosed with exercise-induced asthma. The patient started using a Pariboy SX nebulizer. A sterile aqueous solution containing 0.5% by weight of chitosan nitrate and 0.9% by weight of NaCl was prepared. Approximately 10 minutes before starting to run, 1 ml of this solution was inhaled with the Pariboy SX nebulizer. This treatment prevented exercise-induced asthma.

[0087] Example 14: Herpes (Herpes Simplex Virus Infection) A 65-year-old woman suffered from recurrent herpes on the lips and under the nose. Herpes usually recurred when she was exposed to sunlight or had a cold. The patient sensed that a herpes rash was about to appear due to a sudden tingling sensation on her upper lip. Immediately after recognizing this symptom, she started applying the O / W cream. The O / W cream had the following composition: water 60% by weight; sorbitol crystal solution 2% by weight; sorbic acid 0.15% by weight; chitosan nitrate (CTS-HNO3) 0.5% by weight; DSP 0.025% by weight; D-panthenol 2% by weight; glycerin 7% by weight; citric acid 0.05% by weight (adjusted to a pH of about 4.5); cetomacrogol wax 7.5% by weight; decyl oleate 10% by weight; coconut oil 7% by weight; shea butter 2% by weight; BTMS-25 2% by weight. The O / W cream was applied six times a day for the first three days. The cream prevented herpes blisters from forming, so that no crusts formed and the patient experienced little pain. After three days of treatment, the frequency of cream application was reduced, and after another three days treatment was terminated. The herpes simplex virus depends on oxidative stress to grow. The therapeutic effect obtained is thought to be mediated by nitric oxide, a powerful antioxidant derived from chitosan nitrate. The following claims as originally filed of this application are given as embodiments. [1] Chitosan nitrate for use as a medicine. [2] Chitosan nitrate for use as an NO donor. [3] Chitosan nitrate for use in the prevention or treatment of conditions associated with NO deficiency in a mammal, preferably a human. [4] The chitosan nitrate for use according to [3], wherein the condition is a skin disease. [5] The chitosan nitrate for use according to [4], wherein the disease is a skin disease. [6] The chitosan nitrate for use according to [5], wherein the skin disease is psoriasis, atopic dermatitis and / or acne vulgaris. [7] The chitosan nitrate for use according to [4], wherein the skin disease is a vascular disorder exhibiting Raynaud's phenomenon, specifically scleroderma and / or chilblains. [8] The chitosan nitrate for use according to [4], wherein the skin disease is caused by oxidative stress due to ultraviolet rays and / or skin aging, and the skin disease is preferably caused by oxidative stress due to herpes simplex. [9] The chitosan nitrate for use according to [3], wherein the condition is sexual dysfunction.

[10] Chitosan nitrate for use as described in [9], where local application of chitosan nitrate to the genital area is required for the treatment of erectile dysfunction and for the treatment of female sexual dysfunction (FSD).

[11] Chitosan nitrate for use as described in [3], where treatment of pain is required.

[12] The chitosan nitrate for use according to

[11] , wherein the condition is tendonitis, lateral epicondylitis, arthritis, anal fissure, post-traumatic pain and / or swelling.

[13] Chitosan nitrate for use as described in

[11] , where neuromodulation therapy is indicated as part of the treatment of pain in diabetic neuropathy, carpal tunnel syndrome and / or migraine.

[14] The chitosan nitrate for use according to [3], wherein the condition is a disease relating to the eyes, nose and sinuses.

[15] The chitosan nitrate for use according to

[14] , wherein the disease is allergic conjunctivitis, dry eye, glaucoma, allergic rhinitis, non-allergic rhinitis and / or sinusitis.

[16] The chitosan nitrate for use according to [3], wherein the condition is an airway disease.

[17] The chitosan nitrate for use according to

[16] , wherein the disease is asthma, chronic obstructive pulmonary disease (COPD) and / or pulmonary arterial hypertension.

[18] The chitosan nitrate for use according to [3], wherein the condition is a gastrointestinal disease.

[19] The chitosan nitrate for use according to

[18] , wherein the digestive disorder is leaky gut, reduced gut microbiota diversity, reduced Akkermansia muciniphila, dysbiosis and / or irritable bowel syndrome (IBS).

[20] The chitosan nitrate for use according to [3], wherein the condition is a metabolic disorder.

[21] The chitosan nitrate for use according to

[20] , wherein the metabolic disorder is hypertension, type 2 diabetes, obesity and / or atherosclerosis.

[22] The chitosan nitrate for use according to any one of [1] to

[21] , wherein the molecular weight of chitosan is 20 to 500 kDa, more preferably 150 to 250 kDa.

[23] A cosmetic method comprising administering chitosan nitrate, preferably wherein chitosan nitrate is used as an NO donor.

[24] A cosmetic method according to

[23] for protecting the skin and / or promoting healthy aging.

[25] The cosmetic method according to

[24] for protecting the skin, preferably for protecting the skin selected from protection against environmental influences and inhibition of skin aging.

[26] A cosmetic method according to

[24] for promoting healthy aging.

[27] The cosmetic method according to any one of

[23] to

[26] , wherein the deficiency of nitric oxide is caused by aging.

[28] A pharmaceutical composition comprising chitosan nitrate.

[29] The pharmaceutical composition according to

[28] , wherein the molecular weight of chitosan is 20 to 500 kDa, more preferably 150 to 250 kDa.

[30] The pharmaceutical composition of

[28] or

[29] , wherein the composition is a topical formulation, such as a cream, emulsion, lotion, transdermal patch, spray, troche, chewing gum, dentifrice, nasal spray, nasal and sinus aerosol, eye drops, or eye ointment.

[31] The pharmaceutical composition of

[28] or

[29] , wherein the composition is an oral formulation, such as a tablet, capsule or suppository.

[32] The pharmaceutical composition described in

[28] or

[29] , wherein the composition is a pulmonary aerosol.

[33] A cosmetic composition comprising chitosan nitrate, wherein the molecular weight of chitosan is 20 to 500 kDa, more preferably 150 to 250 kDa.

[34] The composition described in

[33] , wherein the composition is a topical formulation or an oral formulation.

[35] The composition according to

[34] , wherein the composition is a topical formulation, preferably selected from a cream, an emulsion, and a lotion.

[36] The composition described in

[34] , wherein the composition is preferably an oral formulation selected from the group consisting of a supplement, more preferably a tablet or capsule form.

Claims

1. A pharmaceutical composition comprising chitosan nitrate, wherein the molecular weight of the chitosan nitrate is 20k-500kDa.

2. 2. A pharmaceutical composition according to claim 1 for use in the prevention or treatment of a condition associated with NO deficiency in a mammal.

3. The pharmaceutical composition described in claim 2, wherein the mammal is a human.

4. The pharmaceutical composition of claim 2, wherein the condition is a skin disease.

5. The pharmaceutical composition according to claim 4, wherein the disease is a skin disease.

6. The pharmaceutical composition according to claim 5 , wherein the skin disease is psoriasis, atopic dermatitis and / or acne vulgaris.

7. The pharmaceutical composition according to claim 4, wherein the skin disease is a vascular disorder exhibiting Raynaud's phenomenon, specifically scleroderma and / or chilblains.

8. The pharmaceutical composition according to claim 4 , wherein the skin disease is caused by oxidative stress due to ultraviolet rays and / or skin aging.

9. The pharmaceutical composition described in claim 8, wherein the skin disease is caused by oxidative stress due to herpes simplex.

10. The method of claim 2, wherein the condition is sexual dysfunction.

11. 11. The pharmaceutical composition of claim 10, wherein topical application of chitosan nitrate to the genital area is indicated for the treatment of erectile dysfunction and for the treatment of female sexual dysfunction (FSD).

12. 3. The pharmaceutical composition of claim 2, wherein pain treatment is indicated.

13. The pharmaceutical composition of claim 12, wherein the condition is tendonitis, lateral epicondylitis, arthritis, anal fissures, post-traumatic pain and / or swelling.

14. 13. The pharmaceutical composition of claim 12, wherein neuromodulation therapy is indicated as part of the treatment of pain in diabetic neuropathy, carpal tunnel syndrome and / or migraine.

15. 3. The pharmaceutical composition of claim 2, wherein the condition is a disease involving the eyes, nose and sinuses.

16. The pharmaceutical composition according to claim 15, wherein the disease is allergic conjunctivitis, dry eye, glaucoma, allergic rhinitis, non-allergic rhinitis and / or sinusitis.

17. The pharmaceutical composition of claim 2, wherein the condition is a respiratory tract disease.

18. 18. The pharmaceutical composition of claim 17, wherein the disease is asthma, chronic obstructive pulmonary disease (COPD) and / or pulmonary arterial hypertension.

19. The pharmaceutical composition of claim 2, wherein the condition is a gastrointestinal disorder.

20. 20. The pharmaceutical composition of claim 19, wherein the digestive disorder is leaky gut, reduced gut microbiota diversity, reduced Akkermansia Muciniphila, dysbiosis and / or irritable bowel syndrome (IBS).

21. The method of claim 2, wherein the condition is a metabolic disorder.

22. 22. The pharmaceutical composition of claim 21, wherein the metabolic disorder is hypertension, type 2 diabetes, obesity and / or atherosclerosis.

23. The pharmaceutical composition according to any one of claims 1 to 22, wherein the molecular weight of chitosan is from 150k to 250kDa.

24. A cosmetic method comprising administering chitosan nitrate, wherein the molecular weight of the chitosan nitrate is 20 kDa to 500 kDa.

25. The cosmetic method described in claim 24, wherein chitosan nitrate is used as an NO donor.

26. 25. The cosmetic method according to claim 24 for protecting the skin and / or promoting healthy aging.

27. The cosmetic method according to claim 26 for protecting the skin.

28. The cosmetic method according to claim 27, selected from protection against environmental influences and protection against skin aging.

29. 27. The cosmetic method according to claim 26 for promoting healthy aging.

30. The cosmetic method according to any one of claims 24 to 29, wherein the deficiency of nitric oxide is caused by aging.

31. The pharmaceutical composition of claim 1 , wherein the composition is a topical formulation.

32. The pharmaceutical composition of claim 31, wherein the topical formulation is selected from a cream, emulsion, lotion, transdermal patch, spray, troche, chewing gum, dentifrice, nasal spray, nasal and sinus aerosol, eye drops or eye ointment.

33. The pharmaceutical composition of claim 1 , wherein the composition is an oral formulation.

34. The pharmaceutical composition of claim 33, wherein the oral formulation is selected from a tablet, capsule or suppository.

35. The pharmaceutical composition of claim 1 , wherein the composition is a pulmonary aerosol.

36. A cosmetic composition comprising chitosan nitrate, wherein the molecular weight of the chitosan is 20 kDa to 500 kDa.

37. The composition of claim 36, wherein the molecular weight of chitosan is between 150k and 250kDa.

38. 37. The composition of claim 36, wherein the composition is a topical or oral formulation.

39. 40. The composition of claim 38, wherein the composition is a topical formulation.

40. The composition of claim 39, wherein the topical formulation is a topical formulation selected from a cream, an emulsion, and a lotion.

41. 40. The composition of claim 38, wherein the composition is an oral formulation.

42. The composition described in claim 41, wherein the oral formulation is a supplement.

43. The composition of claim 41 or 42, wherein the oral formulation is selected from the form of a tablet or capsule.