NITRIC OXIDE COMPOSITIONS AND TOPICAL USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of priority under 35 U.S.C. §119(e) of U.S. Serial No. 62/843,975, filed May 6, 2019, the entire contents of which is incorporated herein by reference in its entirety.

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION

[0002] The present invention relates generally to nitric oxide (NO) compositions and topical uses thereof, and specifically to the use of NO compositions to increase blood flow and circulation.

BACKGROUND INFORMATION

[0003] Nitric oxide (NO) is a free radical gas produced endogenously by a variety of mammalian cells and is synthesized from arginine by nitric oxide synthase. Physiologically, NO mediates vasodilation, inhibits platelet aggregation, induces disaggregation of aggregated platelets, and inhibits platelet adhesion to the vascular endothelium. Specifically, NO dilates blood vessels and increases blood flow through capillary recruitment.

[0004] Currently, there are known chemical methods to generate physiological and supra- physiological concentrations of nitric oxide. However, NO is an unstable gas which reacts rapidly with oxygen to form nitrogen oxides such as nitrogen dioxide (NO2). NO2 is one of a group of highly reactive gases known as oxides of nitrogen or nitrogen oxides and is a poisonous, noxious gas. Other nitrogen oxides include nitrous acid and nitric acid. NO2 primarily gets in the air from the burning of fuel. NO2 forms from emissions from cars, trucks and buses, power plants, and off road equipment.

[0005] The primary route of exposure to NO2 is by inhalation, but exposure by any route can cause systemic effects. NO2 is irritating to the eyes, skin, mucous membranes, and respiratory tract. Breathing air with a high concentration of NO2 can irritate airways in the human respiratory system. Such exposures over short periods can aggravate respiratory diseases, particularly asthma, leading to respiratory symptoms (such as coughing, wheezing or difficulty breathing), hospital admissions and visits to emergency rooms. Longer exposures to elevated concentrations of NO2 may contribute to the development of asthma and potentially increase susceptibility to respiratory infections. On contact with moisture, NO2 forms a mixture of nitric and nitrous acids, which can cause irritation and inflammation.

SUMMARY OF THE INVENTION

[0006] The present invention is based on the seminal discovery that nitric oxide (NO) can be generated and incorporated into compositions, without the generation of nitric dioxide (NO2), and that NO compositions can be topically applied for the treatment of a variety of diseases, disorders and conditions, as well as for cosmetic, anti-inflammatory and antimicrobial uses.

[0007] In one embodiment, the present invention provides a composition including a mixture of equivalent volumes of a first formulation including sodium nitrite in a viscous hydrogel medium and a second formulation including an electron donor and/or an oxygen independent nitrite reductase in a viscous hydrogel medium.

[0008] In one aspect, the sodium nitrite concentration is about 0.5-5%. In various aspects, the pH of the first formulation is about 6.5-8. In other aspects, the electron donor and/or the oxygen independent nitrite reductase concentration is about 5-25%. In some aspects, the electron donor is ascorbic acid. In many aspects, the pH of the second formulation is about 2.5-3.5. In another aspect, the viscous hydrogel medium is hyaluronic acid (HA), glutaraldehyde, polysorbate 20, hydroxyethylcellulose, d-tocopheryl acetate, dehydroacetic acid, benzyl alcohol, sodium sulfite, sodium metabi sulfite, tetrasodium EDTA, glycol or glycerol. In one aspect, mixing equivalent volumes of the first and second formulations generates nitric oxide (NO) and does not generate nitrogen dioxide (NO2). In some aspects, the mixture generates about 5-30 ppm of NO. In other aspects, the pH of the mixture is about 5-6. In various aspects, the composition further includes one or more agents which may be HA, retinol, kojic acid, acrylates, oxylacrilamide coplymer, silica, cannabidiol (CBD) oil, witch hazel, aloe, glycolic acid, silver ions, silver sulfadiazine, diclofenac diethylamine, triamcinolone acetonide, hydrocortisone, NAD ⁺ , NADH , niacin, glutathione, N-acetyl cysteine, vitamin A, vitamin D or vitamin E.

[0009] In another embodiment, the invention provides a system for the delivery of a nitric oxide (NO) composition for topical administration including a first container comprising sodium nitrite in a viscous hydrogel medium, a second container comprising an electron donor and/or an oxygen independent nitrite reductase in a viscous hydrogel medium, and a means to deliver the composition comprising equivalent volumes of the content of the first and second containers. [0010] In one aspect, the first and the second containers are airless. In another aspect, delivering the composition generates NO and does not generate nitrogen dioxide (NO2).

[0011] In an additional embodiment, the invention provides a method of treating a disease, a disorder or a condition in a subject by topically administering to the subject in need thereof a nitric oxide (NO) composition that improves blood flow and circulation to the administration site.

[0012] In one aspect, administering the NO composition induces an local erythema, and one or more agents that may be hyaluronic acid (HA), retinol, kojic acid, acrylates, oxylacrilamide coplymer, silica, hemp oil, cannabidiol (CBD) oil, witch hazel, aloe, glycolic acid, silver ions, silver sulfadiazine, diclofenac diethylamine, triamcinolone acetonide, hydrocortisone, NAD ⁺ , NADH, niacin, glutathione, N-acetyl cysteine, vitamin A, vitamin D or vitamin E is applied while the erythema induced by the NO composition is visible. In another aspect, the NO composition also contains one or more agents which may be HA, retinol, kojic acid, acrylates, oxylacrilamide coplymer, silica, hemp oil, CBD oil, witch hazel, aloe, glycolic acid, silver ions, silver sulfadiazine, diclofenac diethylamine, triamcinolone acetonide, hydrocortisone, NAD ⁺ , NADH, niacin, glutathione, N-acetyl cysteine, vitamin A, vitamin D or vitamin E. In various aspects, the erythema is visible for about 2-5 minutes. In some aspects, the method also includes administering a nitric oxide oral dissolvable tablet to the subject. In various aspects, the disease, disorder or condition is acne or symptoms thereof, saucerized acne scarring, viral shedding, skin condition, hair loss, wound healing, microvascular disease, incision healing, diabetic ulcer, laser resurfaced tissue, improvement in stretch marks, vaginal dryness, painful intercourse, urinary incontinence, fecal incontinence, skin fungal infection, skin bacterial infection, genital fungal infection, genital bacterial infection, penile dysfunction, bums, pain, swelling, skin inflammation, arthritis, sinusitis, skin irritation, hemorrhoidal irritation, vaginal irritation, Raynauds’ syndrome, multi resistant staphylococcus aureus infection (MRSA) or skin itching.

[0013] In one aspect, the disease or disorder is aging skin, acne, a scar, or wound care. In an aspect, aging skin is characterized by wrinkles;, enlarged pore size; poor skin tone; poor skin texture; pigment variation (i.e. red or brown spots); increased oiliness; and/or vasculature such as telangiectasia or rosacea related erythema. In certain aspects, treating aging skin is determined by improvement in fine lines; wrinkles; pore size; skin tone and texture; coloration and oiliness. In an addition aspect, acne was characterized as mild, moderate or severe based on the number of nodules and pustules. In some aspects, treating acne is determined by pustule and/or nodule reduction. In a further aspect, scars are atrophic scars, striae, acne scars, hypertrophic scars and excised keloid scar beds. In other aspects, treating a scar is determining by a reduction on the visibility of striae; softening of hypertrophic scars; reduction in scar volume; and improved vascularity. In an aspect, wounds are surgical incision scars, surgical local and regional flaps. In another aspect, wounds are non-healing wounds such as diabetic ulcers, burn wounds, and smoking-induced vascular-compromised wounds. In certain aspects, treating wound care is determined by reduction in bruising; reduction in edema; re-epithlialisation, healing of wounds and increased or improved vascularity.

[0014] In a further embodiment, the invention provides a method of treating a disease, a disorder or a condition in a subject by providing a system for topical administration of a nitric oxide (NO) composition and topically administering the NO composition to the subject in need thereof.

[0015] In one aspect, the disease or disorder is aging skin, acne, a scar, or wound care. In an aspect, aging skin is characterized by wrinkles;, enlarged pore size; poor skin tone; poor skin texture; pigment variation (i.e. red or brown spots); increased oiliness; and/or vasculature such as telangiectasia or rosacea related erythema. In certain aspects, treating aging skin is determined by improvement in fine lines; wrinkles; pore size; skin tone and texture; coloration and oiliness. In an addition aspect, acne was characterized as mild, moderate or severe based on the number of nodules and pustules. In some aspects, treating acne is determined by pustule and/or nodule reduction. In a further aspect, scars are atrophic scars, striae, acne scars, hypertrophic scars and excised keloid scar beds. In other aspects, treating a scar is determining by a reduction on the visibility of striae; softening of hypertrophic scars; reduction in scar volume; and improved vascularity. In an aspect, wounds are surgical incision scars, surgical local and regional flaps. In another aspect, wounds are non-healing wounds such as diabetic ulcers, burn wounds, and smoking-induced vascular-compromised wounds. In certain aspects, treating wound care is determined by reduction in bruising; reduction in edema; re-epithlialisation, healing of wounds and increased or improved vascularity.

[0016] In one embodiment, the invention provides a non-toxic method of producing a nitric oxide (NO) composition for topical administration including mixing equivalent volumes of sodium nitrite in a viscous hydrogel medium and electron donor and/or an oxygen independent nitrite reductase in a viscous hydrogel medium, wherein mixing the equivalent volumes generates NO and does not generate nitrogen dioxide (NO2).

[0017] In an additional embodiment, the invention provides a method of increasing vascularity for improved ancillary absorption in a subject including providing a system for topical administration of a nitric oxide (NO) composition and topically administering the NO composition to the subject in need thereof. In many aspects, NO dilates blood vessels and increases blood flow through capillary recruitment.

[0018] In another embodiment, the invention provides a use of a nitric oxide (NO) composition for cosmetic application by providing a system for topical administration of a nitric oxide (NO) composition and topically administering the NO composition to a subject in need thereof.

[0019] In one aspect, the cosmetic application is neocollagenesis, neoellastogenesis; improvement in skin tone, skin quality re-epithelialization, skin healing time, skin moisturization, skin exfoliation, saucerized acne scarring, skin vascularization; lip plumping, time of lipstick application and/or skin cleansing improvement; improvement for acne symptoms, stretch mark appearance, skin pigmentation issues, skin irritation, skin itching, and/or skin inflammation reduction; or hair loss management. In one aspect, the cosmetic application is aging skin, acne, or a scar. In an aspect, aging skin is characterized by wrinkles;, enlarged pore size; poor skin tone; poor skin texture; pigment variation (i.e. red or brown spots); increased oiliness; and/or vasculature such as telangiectasia or rosacea related erythema. In certain aspects, treating aging skin is determined by improvement in fine lines; wrinkles; pore size; skin tone and texture; coloration and oiliness. In an addition aspect, acne was characterized as mild, moderate or severe based on the number of nodules and pustules. In some aspects, treating acne is determined by pustule and/or nodule reduction. In a further aspect, scars are atrophic scars, striae, acne scars, hypertrophic scars and excised keloid scar beds. In other aspects, treating a scar is determining by a reduction on the visibility of striae; softening of hypertrophic scars; reduction in scar volume; and improved vascularity.

[0020] In many aspects, the NO composition is topically administered to the face, neck, declotette, abdomen, back, arm, leg, chest, abdomen, hand and/or lip of the subject.

[0021] In yet another embodiment, the invention provides a method for improving stromal vascular fraction homing, delivery and/or efficacy in a subject in need thereof by administering to said subject a nitric oxide (NO) composition in combination with a stromal vascular fraction, wherein the stromal vascular fraction comprises mesenchymal stem cells. In various aspects, the administration of the stromal vascular fraction is topical or by injection.

[0022] In an additional embodiment, the invention provides a method for improving exosome homing, delivery and/or efficacy in a subject in need thereof by administering to said subject a nitric oxide (NO) composition in combination with an exosome composition.

[0023] In another embodiment, the invention provides a method of enhancing the efficacy of a delivery particle in a subject in need thereof by administering to said subject a nitric oxide (NO) composition in combination with or in conjunction with a delivery particle composition. In various aspects, the delivery particle is a nanoparticle of gold.

[0024] In yet another embodiment, the invention provides a method of dilating a vein in a subject in need of a venipuncture by topically administering to said subject a nitric oxide (NO) composition.

[0025] In one aspect, the NO composition induces an erythema visible for about 2-5 minutes. In another aspect, the NO composition also contains one or more agents which might be lidocaine, bupivacaine, tetracaine or a combination thereof.

[0026] In an additional embodiment, the invention provides a method of dilating a vein in a subject including providing a system for topical administration of a nitric oxide (NO) composition and topically administering the NO composition to the subject in need thereof.

BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure l is a linear analog scale used to determine patient improvement.

[0028] Figures 2A-2B are photographs showing an antecubital vein before (A) and after (B) the application of a NO composition. Dotted line: visible erythema; arrow: dilated antecubital vein.

[0029] Figure 3 is a picture of the NO composition killing pseudomonas using increasing amounts of the serum.

DETAILED DESCRIPTION OF THE INVENTION

[0030] The present invention is based on the seminal discovery that nitric oxide (NO) can be generated and incorporated into compositions, without the generation of nitric dioxide (NO2), and that NO compositions can be topically applied for the treatment of a variety of diseases, disorders and conditions, as well as for cosmetic, anti-inflammatory and antimicrobial uses.

[0031] Before the present compositions and methods are described, it is to be understood that this invention is not limited to particular compositions, methods, and experimental conditions described, as such compositions, methods, and conditions may vary. It is also to be understood that the terminology used herein is for purposes of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only in the appended claims.

[0032] As used in this specification and the appended claims, the singular forms“a”,“an”, and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, references to“the method” includes one or more methods, and/or steps of the type described herein which will become apparent to those persons skilled in the art upon reading this disclosure and so forth.

[0033] All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference.

[0034] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, it will be understood that modifications and variations are encompassed within the spirit and scope of the instant disclosure. The preferred methods and materials are now described.

[0035] In one embodiment, the present invention provides a composition including a mixture of equivalent volumes of a first formulation including sodium nitrite in a viscous hydrogel medium and a second formulation including an electron donor and/or an oxygen independent nitrite reductase in a viscous hydrogel medium.

[0036] As used herein, ‘viscous hydrogel medium’ refers to a semisolid emulsion using a solvent to carry an active ingredient. The solvent might be water or alcohol. For example, the hydrogel of the present invention carries sodium nitrite, an electron donor and/or an oxygen independent nitrite reductase as the active ingredients. Additionally, the hydrogel limits the reaction of NO with oxygen, thereby limiting the formation of NO2. Hydrogels can encapsulate various compounds or elements, and thus can be used as drug delivery systems. In one aspect, the viscous hydrogel medium is hyaluronic acid (HA), glutaraldehyde, polysorbate 20, hydroxyethylcellulose, d-tocopheryl acetate, dehydroacetic acid, benzyl alcohol, sodium sulfite, sodium metabi sulfite, tetrasodium EDTA, glycol or glycerol. [0037] The term‘electron donor’ refers to a chemical entity that donates electrons to another compound. As used herein, it refers to any reducing agent that donates electron and oxidizes itself in the process. Reducing agents undergo permanent chemical alteration through covalent or ionic reaction chemistry. The term‘reducing agent’ refers both to elements and compounds that lose (or donate) electron to another chemical species in a redox chemical reaction. Examples of reducing agents include the earth metals, formic acid, and sulfite compounds. Commons reducing agents include: lithium aluminum hydride (LiAlEE), nascent (atomic) hydrogen, hydrogen, sodium amalgam (Na(Hg)), sodium-lead alloy (Na + Pb), diborane, sodium borohydride (NaBEE), compounds containing the Fe ²⁺ ion, compounds containing the Sn ²⁺ ion, sulfur dioxide (sometimes also used as an oxidizing agent), sulfite compounds, dithionates, thiosulfates, iodides, hydrogen peroxide, hydrazine, diisobutylaluminum hydride, oxalic acid (C2H2O4), formic acid (HCOOH), ascorbic acid (OόEEOό), reducing sugars, phosphites, hypophosphites, and phosphorous acid, dithiothreitol (DTT), carbon monoxide (CO), cyanides, carbon (C), and Tris-2- carboxyethylphosphine hydrochloride (TCEP).

[0038] As used herein,‘oxygen independent nitrite reductase’ refers to any enzyme capable of catalyzing the reduction of nitrite, in an oxygen independent chemical reaction. Examples of oxygen independent nitrite reductases include, but are not limited to, hawthome berry, beet root extract, green tea extract, pine bark, and schizandra.

[0039] In one aspect, the sodium nitrite concentration is about 0.5-1%, 0.5-2%, 0.5-3%, 0.5- 4%, 0.5-5%, 0.5-6% or 0.5-7%. In other aspects, the electron donor and/or the oxygen independent nitrite reductase concentration is about 5-15%, 5-20%, 5-25%, 5-30% or 5-35%.

[0040] In various aspects, the pH of the first formulation is about 6.5-8. In many aspects, the pH of the second formulation is about 2.5-3.5. In other aspects, the pH of the mixture is about 5- 6

[0041] In some aspects, the electron donor is ascorbic acid. Ascorbic acid, or vitamin C, is a weak sugar acid structurally related to glucose. In solutions above pH 5, ascorbic acid is predominantly found in its ionized form, ascorbate. Both ascorbate and ascorbic acid have vitamin C activity and anti-oxidant properties. As a reducing agent, ascorbic acid donates electrons and prevents oxidation.

[0042] In one aspect, mixing equivalent volumes of the first and second formulations generates nitric oxide (NO) and does not generate nitrogen dioxide (NO2). Nitric oxide, nitrogen oxide or nitrogen monoxide is a colorless gas with the formula NO. It is one of the principal oxides of nitrogen. In mammals, including humans, nitric oxide is a signaling molecule in many physiological and pathological pathways. NO is a gaseous signaling molecule also known as an endothelium-derived relaxing factor (EDRF). It is biosynthesized endogenously from L-arginine, oxygen, and NADPH by various nitric oxide synthase (NOS) enzymes, and is a known bioproduct in almost all types of organisms, ranging from bacteria to plants, fungi, and animal cells. Nitric oxide binds to the haem region of target enzymes which leads to its activation, in the presence of iron.

[0043] Nitric oxide is a highly reactive molecule with a short lifetime (a few seconds), which diffuses freely across membranes. Thus, NO can act as a transient paracrine (between adjacent cells) and autocrine (within a single cell) signaling molecule. NO is a potent vasodilator, which induces the endothelium of blood vessels to signal the surrounding smooth muscle to relax, thus resulting in vasodilation and increasing blood flow.

[0044] Nitric oxide should not be confused with nitrogen dioxide (NO2), a brown toxic gas and a major air pollutant. However, nitric oxide can be converted into nitrogen dioxide when exposed to oxygen:

2 NO + O2 2 NO2

[0045] NO2 is a trace gas in the atmosphere of Earth, where it plays a role in absorbing sunlight and regulating the chemistry of the troposphere, especially in determining ozone concentrations. While Mhhas numerous uses, it is a toxic gas, which diffuses into the epithelial lining fluid (ELF) of the respiratory epithelium, dissolves, and chemically reacts with antioxidant and lipid molecules in the ELF. Mh's health effects are caused by the reaction products or their metabolites, which are reactive nitrogen species and reactive oxygen species that can drive bronchoconstriction, inflammation, reduced immune response, and may have effects on the heart.

[0046] The composition of the present invention generates NO, but is prevented from any exposure to O2 and thus from generating toxic NO2. The reaction leading to the production of NO from sodium nitrite is as follow:

NO2- + 2 H ⁺ + e NO + H2O

[0047] In some aspects, the mixture generates about 5-10 ppm, 5-15 ppm, 5-20 ppm, 5-25 ppm, 5-30 ppm, 5-35 ppm or 5-40 ppm of NO. [0048] In various aspects, the composition further includes one or more agents which may be HA, retinol, kojic acid, acrylates, oxylacrilamide coplymer, silica, cannabidiol (CBD) oil, witch hazel, aloe, glycolic acid, silver ions, silver sulfadiazine, diclofenac diethylamine, triamcinolone acetonide, hydrocortisone, NAD ⁺ , NADH, glutathione, niacin, N-acetyl cysteine, vitamin A, vitamin D or vitamin E.

[0049] As used herein, the term‘agent’ refers to any active ingredient that might be included in the NO composition. For example, the term agent can include, but is not limited to, anti inflammatory agents, topical bleaching agents, anti-aging agents, and antimicrobial agents.

[0050] ‘Anti-inflammatory agents’ refers to active ingredients capable of blocking, halting or preventing an inflammatory reaction. In injured tissues, the conversion of arachidonic acid to prostaglandins, mediated by COX2 yields to inflammation. The most common COX2 inhibitors are Celebrex and Vioxx; nonsteroidal anti-inflammatory drugs (NSAIDS), such as acetylsalicylic acid (ASA), Ibuprofen and Naproxen inhibiting both COX1 and COX2 enzymes, which can lead to colon, kidney and liver damage. Examples of topical anti-inflammatory agents include, but are not limited to, turmeric, ginger, kelp, kombu, wakeme, arame, brown algae extract, moui, salmon oil, green tea, papaya, pineapple, blueberry, sweet potato, spinach, rosemary, willow bark, tea tree oil, and cayenne pepper.

[0051] ‘Topical bleaching agents’ refers without any distinction to agent that can be used to lighten, brighten, depigment, and bleach the skin. Skin-lightening (or‘skin-bleaching’) agents are essential tools in the management of disorders of hyperpigmentation, such as melasma and post- inflammatory hyperpigmentation. Examples of topical bleaching agents include, but are not limited to, hydroquinone, kojic acid, alpha-arbutin- from bearberry leaf, Canadian rum ex plant, licorice extract, glycolic acid, retinA, ascorbyl palmitate, indian gooseberry, ginga white, azaelic acid, beta-arbutin, paper mulberry, nicotinamide, aloe, tea tree oil, and grape seed extract.

[0052] ‘Anti-aging agents’ refers to active ingredients capable of blocking, halting or preventing skin aging. Examples of anti-aging agents include, but are not limited to, NAD ⁺ , NADH, glutathione, and N-acetyl cysteine.

[0053] Nicotinamide adenine dinucleotide (NAD) exist in two forms, an oxidized form NAD ⁺ and a reduced form, NADH; both can be included in the NO composition of the present invention.

[0054] ‘Anti-microbial agents’ is meant to include antibacterial, anti-fungal and anti-viral agents, that are capable of blocking, halting or preventing bacterial, fungal and/or viral infection. Examples of topical anti -bacterial agents include, but are not limited to, apple stem cells, salicylic acid powder, pumpkin oil, silver nitrate, mandelic acid, sodium nitrate, cucumber astringent and peach extract.

[0055] As used herein, silver ions is meant to include structured, nanoparticles, and colloidal silver ions.

[0056] In another embodiment, the invention provides a system for the delivery of a nitric oxide (NO) composition for topical administration including a first container comprising sodium nitrite in a viscous hydrogel medium, a second container comprising an electron donor and/or an oxygen independent nitrite reductase in a viscous hydrogel medium, and a means to deliver the composition comprising equivalent volumes of the content of the first and second containers.

[0057] In one aspect, the first and the second containers are airless. In another aspect, delivering the composition generates NO and does not generate nitrogen dioxide (NO2).

[0058] In an additional embodiment, the invention provides a method of treating a disease, a disorder or a condition in a subject by topically administering to the subject in need thereof a nitric oxide (NO) composition that improves blood flow and circulation to the administration site.

[0059] The term‘treating’ is used herein to refer to a therapeutic method and refers to both 1) the application of therapeutic treatments or measures to cure, slow down, lessen symptoms of, and/or halt progression of a diagnosed pathologic conditions or disorder, and 2) the application of prophylactic/ preventative measures. Those in need of treatment may include individuals already having a particular medical disorder as well as those who may ultimately acquire the disorder (z.e., those needing preventive measures).

[0060] The term“subj ect” as used herein refers to any individual or patient to which the subj ect methods are performed. Generally the subject is human, although as will be appreciated by those in the art, the subject may be an animal. Thus other animals, including vertebrate such as rodents (including mice, rats, hamsters and guinea pigs), cats, dogs, rabbits, farm animals including cows, horses, goats, sheep, pigs, chickens, etc., and primates (including monkeys, chimpanzees, orangutans and gorillas) are included within the definition of subject.

[0061] The terms“administration of’ and/or“administering” should be understood to mean providing a pharmaceutical composition in a therapeutically effective amount to the subject in need of treatment. Administration routes can be enteral, topical or parenteral. As such, administration routes include but are not limited to intracutaneous, subcutaneous, intravenous, intraperitoneal, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, transdermal, transtracheal, subcuticular, intraarticulare, subcapsular, subarachnoid, intraspinal and intrasternal, oral, sublingual buccal, rectal, vaginal, nasal ocular administrations, as well infusion, inhalation, and nebulization.

[0062] In one aspect, administering the NO composition induces an local erythema, and one or more agents which may be hyaluronic acid (HA), retinol, kojic acid, acrylates, oxylacrilamide coplymer, silica, cannabidiol (CBD) oil, witch hazel, aloe, glycolic acid, silver ions, silver sulfadiazine, diclofenac diethylamine, triamcinolone acetonide, hydrocortisone, NAD ⁺ , NADH, niacin, glutathione, N-acetyl cysteine, vitamin A, vitamin D or vitamin E is applied while the erythema induced by the NO composition is visible.

[0063] In some aspects the administration of the NO composition can be in combination with one or more additional therapeutic agents. The phrases‘combination therapy’,‘combined with’ and the like refer to the use of more than one medication or treatment simultaneously to increase the response. The composition of the present invention might for example be used in combination with other drugs or treatment in use to treat a disease of interest. Such therapies can be administered prior to, simultaneously with, or following administration of the composition of the present invention.

[0064] As used herein‘erythema’ refers to the redness of the skin caused by the application of the NO composition and visually indicates the transient increased blood flow during which any other active compounds should be applied to the skin.

[0065] In various aspects, the erythema is visible for about 2-5 minutes.

[0066] The erythema generally appears within about 2 seconds following the application of the NO composition, and lasts for up to 5 minutes. In some aspects, the erythema appears within 2 seconds, 5 seconds, 10 seconds, 30 seconds, 45 seconds, or 60 seconds following the application of the NO composition and lasts for up to 5 minutes. In other aspects, the erythema is visible for about 2-3 minutes, 2-4 minutes, or 2-5 minutes.

[0067] In another aspect, the NO composition also contains one or more agents which may be HA, retinol, kojic acid, acrylates, oxylacrilamide coplymer, silica, CBD oil, witch hazel, aloe, glycolic acid, silver ions, silver sulfadiazine, diclofenac diethylamine, triamcinolone acetonide, hydrocortisone, NAD ⁺ , NADH, niacin, glutathione, N-acetyl cysteine, vitamin A, vitamin D or vitamin E. [0068] In order to increase the effects of the NO present in the NO composition, the present invention also includes the complementary systemic administration of NO. The mode of delivery chosen for administration of NO according to the present invention to a subject, such as a human patient or mammalian animal, depends on the formulation of said NO. The actual final dosage for a given route of administration can be easily determined by routine experimentation.

[0069] The NO can be administered in a variety of unit dosage forms depending upon the method of administration. Suitable unit dosage forms, include, but are not limited to powders, tablets, pills, capsules, lozenges, suppositories, patches, nasal sprays, injectables, implantable sustained-release formulations, lipid complexes, etc.

[0070] In some aspects, the method further includes administering a nitric oxide oral dissolvable tablet to the subject.

[0071] In various aspects, the disease, disorder or condition is acne or symptoms thereof, saucerized acne scarring, viral shedding, skin condition, hair loss, wound healing, microvascular disease, incision healing, diabetic ulcer, laser resurfaced tissue, improvement in stretch marks, vaginal dryness, painful intercourse, urinary incontinence, fecal incontinence, skin fungal infection, skin bacterial infection, genital fungal infection, genital bacterial infection, penile dysfunction, burns, pain, swelling, skin inflammation, arthritis, sinusitis, skin irritation, hemorrhoidal irritation, vaginal irritation, Raynauds’ syndrome, multi resistant staphylococcus aureus infection (MRSA) or skin itching.

[0072] In one aspect, the disease or disorder is aging skin, acne, a scar, or wound care. In an aspect, aging skin is characterized by wrinkles;, enlarged pore size; poor skin tone; poor skin texture; pigment variation (i.e. red or brown spots); increased oiliness; and/or vasculature such as telangiectasia or rosacea related erythema. In certain aspects, treating aging skin is determined by improvement in fine lines; wrinkles; pore size; skin tone and texture; coloration and oiliness. In an addition aspect, acne was characterized as mild, moderate or severe based on the number of nodules and pustules. In some aspects, treating acne is determined by pustule and/or nodule reduction. In a further aspect, scars are atrophic scars, striae, acne scars, hypertrophic scars and excised keloid scar beds. In other aspects, treating a scar is determining by a reduction on the visibility of striae; softening of hypertrophic scars; reduction in scar volume; and improved vascularity. In an aspect, wounds are surgical incision scars, surgical local and regional flaps. In another aspect, wounds are non-healing wounds such as diabetic ulcers, burn wounds, and smoking-induced vascular-compromised wounds. In certain aspects, treating wound care is determined by reduction in bruising; reduction in edema; re-epithlialisation, healing of wounds and increased or improved vascularity.

[0073] As used herein, the term“skin conditions” includes, but is not limited to, rosacea, acne, dermatitis, and fungal infections.

[0074] The term“microvascular disease” is meant to refer to diseases such as scleroderma.

[0075] In a further embodiment, the invention provides a method of treating a disease, a disorder or a condition in a subject by providing a system for topical administration of a nitric oxide (NO) composition and topically administering the NO composition to the subject in need thereof.

[0076] In one embodiment, the present invention provides a non-toxic method of producing a nitric oxide (NO) composition for topical administration including mixing equivalent volumes of sodium nitrite in a viscous hydrogel medium and electron donor and/or an oxygen independent nitrite reductase in a viscous hydrogel medium, wherein mixing the equivalent volumes generates NO and does not generate nitrogen dioxide (NO2).

[0077] By‘non-toxic’, it is meant that the method used to generate NO doesn’t generate NO2.

[0078] In an additional embodiment, the invention provides a method of increasing vascularity for improved ancillary absorption in a subject by providing a system for topical administration of a nitric oxide (NO) composition and topically administering the NO composition to the subject in need thereof.

[0079] The term‘vascularity’ generally refers to the number, the ramification, and/or the prominence of superficial veins or capillaries. As used herein,‘increasing vascularity’ means enhancing or improving superficial vein or capillary number, ramification and/or prominence in order to increase ancillary absorption of a compound applied to the skin. It also includes reducing or limiting the distance between said superficial vein or capillary and the skin surface.

[0080] In many aspects, NO dilates blood vessels and increases blood flow through capillary recruitment.

[0081] In another embodiment, the invention provides a use of a nitric oxide (NO) composition for cosmetic application including providing a system for topical administration of a nitric oxide (NO) composition and topically administering the NO composition to a subject in need thereof. [0082] In one aspect, the cosmetic application is neocollagenesis, neoellastogenesis, skin tone, skin quality re-epithelialization, skin healing time, skin moisturization, skin exfoliation, saucerized acne scarring, skin vascularization lip plumping, time of lipstick application and/or skin cleansing improvement; acne symptoms, stretch mark appearance, skin pigmentation issues, skin irritation, skin itching, and/or skin inflammation reduction; or hair loss management.

[0083] The NO composition of the present invention generally improves fibroblast function, which subsequently increases neocollagenesis, neoellastogenesis. Improving fibroblast function has numerous applications associated with skin tone, skin quality and skin healing.

[0084] As used herein‘hair loss management’ refers to both the increase of hair follicle diameter, and the reduction of hair shedding.

[0085] In yet another embodiment, the invention provides a method for improving stromal vascular fraction homing, delivery and/or efficacy in a subject in need thereof by administering to said subject a nitric oxide (NO) composition in combination with a stromal vascular fraction, wherein the stromal vascular fraction comprises mesenchymal stem cells.

[0086] As used herein‘ stromal vascular fraction’ refers to a fraction of an adipose tissue which contains cells including pre-adipocytes, fibroblasts, vascular endothelial cells, mesenchymal stem cells and a variety of immune cells such as adipose tissue macrophages.

[0087] ‘Mesenchymal stem cells’ (MSCs) are spindle-shaped plastic adherent cells that can be isolated from many tissues including bone marrow and adipose tissue. MSCs have multipotent differentiation capacity in vitro and can be treated to generate differentiated cells such as osteoblasts, adipocytes and chondrocytes in vitro, but do not have the capacity to reconstitute an entire organ.

[0088] In various aspects, the administration of the stromal vascular fraction is topical or by injection.

[0089] In an additional embodiment, the invention provides a method for improving exosome homing, delivery and/or efficacy in a subject in need thereof by administering to said subject a nitric oxide (NO) composition in combination with an exosome composition.

[0090] As used herein, ‘exosome’ refers to extracellular vesicles that are produced in the endosomal compartment of eukaryotic cells. Exosomes are a family of small nanoparticles having a diameter comprised between 30 and 100 nm. Exosomes are generated inside multivesicular endosomes or multivesicular bodies (MVBs) and are secreted when these compartments fuse with plasma membrane. Exosome secretion is a general cellular function that plays an important role in the intercellular transfer of information, as such exosomes exert their function through paracrine effects. Exosomes can be found in tissues and can also be found in biological fluids including blood, urine, and cerebrospinal fluid; and are released in vitro by cultured cells into the culture- conditioned medium. These secreted vesicles serve as cell-to-cell messengers that modify cellular function in normal state as well as in disease state. The most intriguing feature of is that they can be isolated from cultured cells and be subcutaneously administered, making exosomes an exciting therapeutic delivery system.

[0091] In another embodiment, the invention provides a method of enhancing the efficacy of a delivery particle in a subject in need thereof by administering to said subject a nitric oxide (NO) composition in combination with or in conjunction with a delivery particle composition.

[0092] In various aspects, the delivery particle is a nanoparticle of gold.

[0093] In yet another embodiment, the invention provides a method of dilating a vein in a subject in need of a venipuncture by topically administering to said subject a nitric oxide (NO) composition.

[0094] As used herein,“venipuncture” refers to the process of obtaining intravenous access for the purpose of intravenous therapy or for blood sampling of venous blood. In subjects presenting “hard-to-fmd” veins, the process of venipuncture can be complicated; the use of the NO composition of the present invention helps dilating the veins, which allows easier access to the veins either for injection or blood withdraw. This also apply to subject that are sensitive or afraid of needles, such as kids.

[0095] In one aspect, the NO composition induces an erythema visible for about 2-5 minutes. In another aspect, the NO composition also contains one or more agents which might be lidocaine, bupivacaine, tetracaine or a combination thereof.

[0096] The NO composition might include one or more additional agents. Combined with numbing or local anesthetic agents, NO dilates veins, which make them more visible and more accessible, and reduces the pain associated with the needle. Examples of local anesthetic agent include, but are not limited to, lidocaine, bupivacaine, tetracaine, and a combination thereof.

[0097] In an additional embodiment, the invention provides a method of dilating a vein in a subject including providing a system for topical administration of a nitric oxide (NO) composition and topically administering the NO composition to the subject in need thereof. [0098] Presented below are examples discussing the non-toxic generation of a NO composition contemplated for the discussed applications. The following examples are provided to further illustrate the embodiments of the present invention, but are not intended to limit the scope of the invention. While they are typical of those that might be used, other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

EXAMPLES

EXAMPLE 1

GENERATION OF A NO COMPOSITION

[0099] By using an airless, dual chamber applicator along with specific carriers that limit oxygen diffusion into the mixture once exposed to outside air, authentic nitric oxide can be generated without the generation of NO2.

[0100] In one chamber 0.5-5% sodium nitrite are added to a viscous hydrogel medium such as water, polysorbate 20, hydroxyethylcellulose, d-tocopheryl acetate, dehydroacetic acid, benzyl alcohol, sodium sulfite, sodium metabisulfite, tetrasodium EDTA, glycol, glycerol, and the like, generating a first composition having a pH of about 6.5-8.0. In another chamber a 5-25% solution of ascorbic acid (or any other electron donor and/or oxygen independent nitrite reductase derived from natural products) is added to a viscous hydrogel medium (as described above), generating a second composition having a pH of about 2.5-3.5.

[0101] Using a dual chamber dispensing applicator a specific volume of about 100-500uL of each composition is dispensed. When mixed together and applied onto the skin, the mixture generates about 5-30ppm of nitric oxide gas on the surface of the skin without the generation of nitrogen dioxide. The resulting pH of the combined mixture is about 5-6. The NO gas diffuses in three dimensions with some being diffused into the space above the skin where it can be detected and quantified. Part of the NO gas diffuses into the dermal layers of the skin to dilate blood vessels and recruits capillaries thereby increasing local blood flow only to the area upon which it is applied to.

EXAMPLE 2

USES OF NO COMPOSITIONS TO INCREASE BLOOD FLOW AND CIRCULATION

[0102] NO compositions, such as a NO serum is applied topically to the skin and has the ability to increase vascularity for increased absorption of ancillary ingredients. [0103] The NO serum by itself, via improving vascularity, improves fibroblast function, and subsequently increases neocollagenesis and neoellastogenesis thereby improving skin tone and quality. The serum is applied on the face, neck, declotette, abdomen, back, arms and/or legs as separate area indications.

[0104] Increasing vascularity through topical NO administration has multiple applications, and is for example useful:

i) where increased blood flow itself is beneficial: for clitoral stimulation, since NO increases blood flow and sensation; for enhancing erection quality, since topical and oral NO improve vascularity;

ii) where an increased vascularity improves the absorption and activity of active compounds, such as CBD oil, witch hazel and aloe (for hemorrhoidal and vaginal irritation relieve for instance), HA (to reduce Raynauds syndrome symptoms), triamcinolone acetonide (at a 0.147 mg/gm dosage in nasal spray to improve symptoms of sinusitis).

[0105] Additionally, NO is useful in vaginal health, reduce vaginal dryness, painful intercourse, as well as for the treatment of urinary and fecal incontinence.

[0106] Moreover, NO composition or serum in conjunction with topical or injectable stromal vascular fraction containing mesenchymal stem cells can improve homing, delivery, and facilitate efficacy; in conjunction with exosomes, NO serum can improve homing, delivery and facilitate delivery; and when combined with or in conjunction with the application of nanoparticles of gold or other delivery substances, NO serum can enhance the efficacy of said particles.

[0107] All mentioned indications are magnified when NO topical serum is coupled with NO oral dissolvable tablets. The additional compounds can either be in the NO composition, or applied while the erythema induced by NO is visible, typically for 2-5 minutes.

EXAMPLE 3

USES OF NO COMPOSISITONS FOR ANTI-MICROBIAL PURPOSES

[0108] When applied at relatively high concentration, nitric oxide has anti-microbial properties; topical application of a NO composition is thus used to treat microbial infection, alone or in combination with other active compounds.

[0109] NO has proven anti -bacterial capability though the effective killing of bacteria without the need for nitrogen dioxide which is also antimicrobial. The NO composition is for example applied with 0.147 mg/gm triamcinolone acetonide in a nasal spray to improve the symptoms of sinusitis.

[0110] NO can also be combined with other topical antibacterial agents such as apple stem cells, salicylic acid powder, pumpkin oil, silver nitrate, mandelic acid, sodium nitrate, cucumber astringent and peach extract.

[0111] Further, NO has proven anti-fungal capability, though the effective killing of fungi, mold spores without the need for nitrogen dioxide which is also antimicrobial. The NO composition can also be complemented by any additional known anti-fungal agent, and thus be useful to treat fungal infection.

[0112] Additionally, NO has proven anti -viral capability, though the effective killing of viruses without the need for nitrogen dioxide which is also antimicrobial. Specifically, NO has proven reduced viral shedding capability.

[0113] The anti-fungal and anti-bacterial activity of NO also accelerates infection healing.

[0114] All the aforementioned indications are magnified when NO topical application is coupled with NO oral dissolvable tablets. The additional compounds can either be in the NO composition, or applied while the erythema induced by NO is visible, typically for 2-5 minutes.

EXAMPLE 4

USES OF NO COMPOSISITONS FOR COSMETIC PURPOSES

[0115] When topically applied, NO increases blood flow and circulation and is thus used for multiple cosmetic applications, when applied to the face, neck, chest abdomen, hands, back or legs.

[0116] NO is used to improve skin health. For instance, NO reduces the appearance of stretch marks (striae); improves vascularization, skin tone and skin quality, especially smoker’s skin tone and quality.

[0117] Combined with hyaluronic acid, NO improves skin moisturization.

[0118] In combination with retinol, or glycolic acid NO improves skin exfoliation and can be used in skin cleansers.

[0119] With kojic acid, NO accelerates the resolution of pigmentation issues.

[0120] With acrylates/oxylacrilamide coplymer/silica, NO enhances lip plumping and lengthens the time of lipstick application.

[0121] With silver sulfadiazine 1% in a hydrophilic water base, NO improves burn care. [0122] With 1% hydrocortisone and vitamin A, D & E, NO reduces skin irritation, itching and inflammation.

[0123] NO is also used in combination with bleaching agents for topical application. For example, the NO composition is combined with hydroquinone 2%, 4%, or 8 %; kojic acid 1-4% (malted rice by-product); alpha-arbutin 1%, isolated from bearberry leaf; Canadian rumex plant, which is lOx more stable and decreases erythema; licorice extract, which contains glabridinin known for its anti-inflammatory properties when applied at 0.5%; glycolic acid; retinA; ascorbyl palmitate, extracted from lemon/orange peel; indian gooseberry from the emblica plant, ginga white, from peppermint, mallow, cowslip; azaelic acid, from pityrosporum ovale, which has cytotoxic properties and thus has great applications for acne; beta-arbutin 1%, from uva ursi; paper mulberry 1%; nicotinamide, which has anti-inflammatory properties; aloe, which contains aleosin; tea tree oil, isolated from camellia plant; or grape seed extract + soy.

[0124] The NO composition (or serum) by itself, via improving vascularity improves fibroblast function, and subsequently increases neocollagenesis and neoellastogenesis thereby improving skin tone and quality. This can be applied on the face, neck, declotette, abdomen, back, arms and legs as separate area indications.

[0125] By having both anti-bacterial and anti-inflammatory properties, NO serum also has proven benefit in improving active acne symptoms and reducing mild to moderate saucerized acne scarring.

[0126] Additionally, the topical application of NO increases follicular diameter and decreases shedding, which provides great application for the treatment and/or management of hair loss.

[0127] All the aforementioned indications are magnified when NO topical application is coupled with NO oral dissolvable tablets. The additional compounds can either be in the NO composition, or applied while the erythema induced by NO is visible, typically for 2-5 minutes.

EXAMPLE 5

USES OF NO COMPOSISITONS FOR ANTI-INFLAMMATORY PURPOSES

[0128] When topically applied, NO has proven anti-inflammatory capabilities, which allows its use for multiple applications.

[0129] Applied with 1% hydrocortisone and vitamin A, D & E, NO reduces skin irritation, itching and inflammation. [0130] With 1% diclofenac diethylamine, NO reduces pain, swelling and inflammation associated with arthritic joints.

[0131] In injured tissues, the conversion of arachidonic acid to prostaglandins, mediated by COX2, yields to inflammation. To enhance NO anti-inflammatory effect, the NO composition is combined with COX2 inhibitors such as Celebrex and Viox; nonsteroidal anti-inflammatory drugs (NSAIDS), such as acetylsalicylic acid (ASA), Ibuprofen and Naproxen; or naturally derived anti inflammatory agents. Naturally derived anti-inflammatory agents include turmeric, which has curcumin, a COX2 inhibitor at 400-600-mg; ginger, which has zingabain, a COX2 inhibitor that blocks arachidonic acid release; kelp, i.e. kombu, wakeme, or arame, which contain fucidin a promoter of collagen synthesis; brown algae extract, which promotes collagen synthesis; moui, a tongan fucidin powerhouse; salmon oil, which contains powerful omega-3, alpha linoleic acid; green tea, containing flavonoids; papaya, which contains papain useful for protein digesting, vitamin C and E; pineapple, whose bromelain has strong NSAID activity; blueberry, which is an anti-oxidant powerhouse; sweet potato, containing beta-carotene, vitamin B6 and C; spinach, containing flavonoids, carotenoids, vitamin A, B2, B6, C, E, K, Ca, folate, and iron; rosemary, which contains tryptophan, Mg, and K; willow bark and tea tree oil, which contain potent natural salicylic acid; and cayenne pepper, which is a strong COX2 inhibitor.

[0132] All the aforementioned indications are magnified when NO topical application is coupled with NO oral dissolvable tablets. The additional compounds can either be in the NO composition, or applied while the erythema induced by NO is visible, typically for 2-5 minutes.

EXAMPLE 6

USES OF NO COMPOSISITONS FOR WOUND HE AT, TNG

[0133] When applied on incisions, diabetic ulcers, laser resurfaced tissues and the like, NO decreases time to re-epithelialization, thereby shortens healing time.

[0134] Combined with NO’s intrinsic anti -fungal and anti -bacterial properties, as well as anti inflammatory properties, the application of a NO composition accelerates infection healing.

[0135] All the aforementioned indications are magnified when NO topical application is coupled with NO oral dissolvable tablets. The additional compounds can either be in the NO composition, or applied while the erythema induced by NO is visible, typically for 2-5 minutes.

EXAMPLE 7 USE OF NO COMPOSITIONS TO DILATE VEINS IN PREPARATION FOR

VENIPUNCTURE

[0136] A NO composition, prepared as disclosed in Example 1 was applied onto the skin, at the antecubital fossa location, to dilate the superficial antecubital vein of a patient, in preparation for venipuncture.

[0137] As illustrated in Figure 2A, prior to the administration of the NO composition, the antecubital vein was merely visible, which rendered the process of venipuncture either for injection or for blood sampling complicated. After the application of the NO composition, and as illustrated in Figure 2B, an erythema quickly appeared and was easy to see (dotted line); and the antecubital vein rapidly appeared dilated which rendered it more visible and more accessible.

[0138] The topical application of the NO composition induced the apparition of an erythema that was visible, for 2-5 minutes.

EXAMPLE 8

CLINICAL SUMMARY

[0139] Methods: One hundred patients were enrolled into a two center, multi-limb study. Ages ranged from 15 to 76 years. The effect of a Nitric Oxide generating topical serum was studied on 100 patients in two Centers between December 2018 and December 2019. There were 82 females and 18 males. The population consisted of 42 Caucasian, 18 Asian, 8 African American, 12 Hispanic, 14 Mediterranean, and 6 Indian patients.

[0140] Patients were enrolled under four categories: 1) Aging Skin; 2) Acne; 3) Scar Therapy; and 4) Wound Care. The Aging skin population utilized NO serum bid after cleansing. Photographs were taken monthly for 4-12 weeks. Linear analog scales were administered examining skin tone, wrinkles, clarity and color variation. No other products were used concomitantly.

[0141] The Active acne patients were divided into mild, moderate and severe states based on nodule and pustule counts. NO serum was applied am and pm after cleansing. Photographs were taken weekly for 4-12 weeks. Pustule and nodule counts were recorded weekly. NO serum was applied bid in the treatment of atrophic scars, striae, acne scars, hypertrophic scars post Needle Radiofrequency, and around the C02 laser excised keloid scar beds. Photographs were taken weekly for 4-12 weeks. Several wound types were examined: Surgical incisions, healing surgical flaps, diabetic ulcers, and smoking induced, vascular compromised skin. NO serum was applied qid daily. Photographs were taken at 4, 8, and 12 weeks.

[0142] Results: The Aging skin population reported improvement in fine lines, wrinkles, pore size, skin tone and texture, coloration and oiliness. A high level of product satisfaction with significant improvement in self-esteem was noted. The Acne population showed a range in pustule and nodule reduction ranging from 20% to 84%. The average reduction was 68%. The Scar therapy population showed positive trends. The atrophic scar patients and those with striae improved the most. Softening of hypertrophic scar tissue was noted. Keloid wound beds healed anecdotally faster than non-treated patients. Improved vascularity is speculated. The Wound healing population without exception showed excellent results. Rhytidectomy, Blepharoplasty, and Rhinoplasty patients displayed less bruising, edema, and more rapid healing. Non-healing ulcers healed within 30-45 days. Revascularization was routinely evident in compromised flaps.

[0143] Conclusion: It is possible to deliver clinically relevant bioactive nitric oxide via a dual chamber NO generating delivery device. This Pilot Study shows valid proof of concept to warrant further IRB approved studies in the areas of aging skin, acne, wound healing and scar therapy.

EXAMPLE 9

CLINICAL RESULTS

[0144] Nitric oxide (NO), is an important biological messenger in human physiology. This multifunctional signaling molecule takes part in the control of vasodilation, and is involved in the inhibition of platelet aggregation and platelet adhesion to vascular endothelium. Several human skin studies have indicated that NO is involved in the proliferation and differentiation of epidermal cells, regulation of innate immune reactions and inflammatory responses, the control of allergic skin manifestations, microbicidal activity, and antigen presentation. NO is also a key molecule in wound healing regulation and tissue regeneration due to its gene regulatory properties, as well as its influence on the proliferation and differentiation of fibroblasts, keratinocytes, monocytes and macrophages.

[0145] The science of NO and the importance of NO in human health have been extensively discussed. By the age of 40, most adults produce only 50% of the NO produced endogenously at the age of 20. Studies have shown that diminished NO can be an early marker of chronic disease.

[0146] Given the importance of NO and its’ natural depletion over time, research was performed yielding the subsequent development of a patented NO lozenge, Neo40 (humanN, Austin TX). Peer-reviewed, placebo- controlled studies demonstrated that the NO lozenge could modify cardiovascular risk factors in patients over age 40, reduce triglycerides, and reduce blood pressure.

[0147] These studies demonstrate the safety and efficacy of restoring NO production in humans.

[0148] The importance of nitric oxide in cellular medicine, regenerative and stem cell therapy has also been highlighted in the literature. The role of NO in stem cell biology is revealing novel strategies for NO repletion prior to, during, and after stem cell deployment, and exosome administration, in order to optimize metabolism for regenerative or healing outcomes.

[0149] NO is a short-lived free radical produced enzymatically by nitric oxide synthase (NOS) endogenously. NOS converts the amino acid precursor L-arginine to NO and L-citrulline with the help of several co-factors such as tetrahydrobiopterin, flavine mononucleotide (FMN), flavine adenine dinucleotide (FAD), nicotinamide-adenine-dinucleotide phosphate (NADHP) and an oxygen molecule. Nitric oxide is synthesized and released by many skin cells including fibroblasts, keratinocytes, melanocytes, endothelial cells, macrophages, neutrophils, and adipocytes. NO is a hydrophobic molecule with a stoke radius of 3-4A that can diffuse across most biological membranes.

[0150] By the age of 40, most adults produce only 50% of the NO produced endogenously at the age of 20. Studies have shown that diminished NO can be an early marker of chronic disease. Given the importance of NO and its’ natural depletion over time, research was performed yielding the subsequent development of a patented NO lozenge, Neo40 (humanN, Austin TX).

[0151] Peer-reviewed, placebo- controlled studies demonstrated that the NO lozenge could modify cardiovascular risk factors in patients over age 40, reduce triglycerides, and reduce blood pressure. These studies demonstrate the safety and efficacy of restoring NO production in humans.

[0152] Given the success of oral repletion, many studies also looked at topically applied NO generating formulations. The diffusion coefficient of NO has been shown to be comparable to other small diatomic molecules such as dioxygen or carbon monoxide. A diffusion distance of 500 microns was estimated for NO in tissues. This is sufficient for NO to penetrate the dermis and reach the microcirculation of the upper horizontal plexus, and the upper dermis after topical application. EPR spectroscopy studies using an acidified - 15N-labelled nitrite confirmed transcutaneous penetration of 15NO up to 3mm into human dermis. The transcutaneous application in vivo of a formula generating NO, was shown to significantly increase circulatory nitrite and S-nitroso-concentrations resulting in improvement of hemodynamic parameters. These included increased blood flow, reduced platelet aggregation, and a decrease in arterial blood pressure.

[0153] In the Aesthetic and Reconstructive patient population, the dermal application of a NO generating serum has a wide range of potential uses. The“aging skin” population would benefit from improved circulation and absorption of fibroblast enhancing ingredients. The“Acne prone” patient would benefit from the anti-bacterial benefits of NO. Other chronic fungal or viral infections could also see benefit. Diabetic ulcers plague millions of patients yearly. NO formulations, improving circulation in this population would prove life-changing. Other “compromised” circulation conditions such as Raynaud’s Syndrome, and Localized Sclerosis have seen benefit. Cutaneous skin cancer at times requires partial thickness grafts, full thickness grafts, local flaps, regional flaps or free flaps for reconstruction.

[0154] The concomitant application of a NO delivery serum would improve the donor site and recipient site prior, during and after graft or flap reconstruction.

[0155] This study looks at the potential application of a novel dual chamber delivery system serum which produces NO in clinically sufficient dosage and decay profile to examine its’ benefit in the Facial Aging, Acne, Wound Healing, and Scar Therapy patient populations.

[0156] Study Population: The effect of a Nitric Oxide generating topical serum was studied on 100 patients in two Centers between December 2018 and December 2019. There were 82 females and 18 males. The population consisted of 42 Caucasian, 18 Asian, 8 African American, 12 Hispanic, 14 Mediterranean, and 6 Indian patients.

[0157] Exclusion criteria in this study included an unwillingness to avoid excessive sunlight or wear protective clothing and sunscreen, unwillingness to forgo any other topical dermatological or drug therapy, the use of topical corticosteroid, alpha and beta-hydroxyacids, retinoids, or vitamin C containing topicals within 30 days before, as well as throughout the course of the study. Washout periods adhered to by subjects in this study included 6 months free from dermabrasion, deep chemical peels, ablative laser treatments, neurotoxin or filler injections: 3 months free from non-ablative laser, light, radiofrequency or ultrasound treatments; and 1 month free from microdermabrasion, light and medium depth peels.

[0158] Methods: A novel NO generating dual chamber mixing serum was examined for preliminary efficacy and proof of concept. Nitric oxide was detected and quantified using an ozone-based gas phase chemiluminescent detector (CLD) (EcoPhysics, Michigan USA).

[0159] Contents of both chambers of the NO generating serums were dispensed and mixed on the skin. The CLD detector was placed within one inch from the skin after contents from both chambers of the product were mixed, nitric oxide gas was released, and detected. Initial nitric oxide levels reached over 15,000 ppb during the mixing period and then exhibited normal decay kinetics. Nitric oxide could still be detected emitting from the skin 30 minutes after the initial application. The efficacy was evaluated by visual and instrumental methods at 4- week intervals over a 12 week period. Visual results will be discussed in this Pilot Study. Instrumental results will be discussed in forthcoming peer reviewed publications.

[0160] One hundred patients were enrolled into a two center multi-limb study. Patients were enrolled under four categories: Aging Skin (40 patients), Active Acne (30 patients), Scar Therapy (15 patients), and Wound Care (15 patients).

[0161] The Aging Skin population used the NO serum morning and evening after cleaning their skin with a gentle cleanser. Photographs were taken at 4- week intervals for 12 weeks. Patient linear analog scales were administered. Tissue attributes examined included wrinkles, pores, skin clarity, quality, and pigmentation.

[0162] Patients were also given a linear analog examining product satisfaction (see Figure 1). Quantification of wrinkle improvement, pore size, oiliness, evenness, red spots and brown spots were calculated using 3-dimentional image analysis of Life Viz App (QuantifiCare, France) and then compared to QuantifiCare’ s reference population database of normal aging skin, adjusted for age, sex, and skin type. The 3-dimentional high resolution micro-imaging of skin surface of those participants were taken using a micro imaging system (QuantifiCare, France).

[0163] The Active Acne population was divided into mild, moderate and severe based upon nodule and pustule counts. The NO serum was applied morning and evening after cleaning their skin with a gentle cleanser. Photographs were taken weekly for 30 days. Pustule and nodule counts were recorded weekly. Linear analog scales were administered. [0164] The Scar Therapy population consisted of atrophic scars, striae, acne scars, hypertrophic scars and excised keloid scar beds. The NO serum was applied twice daily for 12 weeks. Photos were taken at 4 week intervals. Linear analog scales were administered to both patients and physicians to examine scar quality improvement.

[0165] Several Wound Healing scenarios were examined. Surgical incision sites were pre treated and treated after completion. Healing surgical local and regional flaps were treated. Non healing diabetic ulcers, burn wounds, and smoking-induced vascular-compromised wounds were examined. NO serum was applied 4 times daily. Photographs were taken daily for the surgical patients and weekly for 4 weeks for the remainder of the population to examine any benefit.

[0166] Results: There were no hypersensitivity, allergic, or irritant reactions reported by any of the test subjects. Table 1 summarizes the results of the linear analog tests for measured features. Table 1

[0167] The Aging Skin population results were consistently favorable and increasingly positive with greater duration of use. Sixty percent of subjects noticed a decrease in visible wrinkles at 4 weeks, 68% by 8 weeks, and 84% by 12 weeks. Decreased pore size was reported by 45% of patients at 4 weeks, 55% of patients at 8 weeks, and 68% of patients at 12 weeks. An increase in skin tone was perceived by 72% of patients at 4 weeks, 80% by 8 weeks, and 83 % by 12 weeks. Textural improvement was reported by 68% of patients at 4 weeks, 83% at 8 weeks, and 88% at 12 weeks. Unwanted pigment variation was improved in 55% of patients at 4 weeks, 65% by 8 weeks, and by 78% of patients at 12 weeks. A decrease in skin oiliness was perceived by 72% of subjects at 4 weeks, 80% by 8 weeks, and 85% by 12 weeks. Unwanted vasculature such as telangiectasia or rosacea-related erythema was improved in 20% of patients at 4 weeks, 38% by 8 weeks, and 53% by 12 weeks. The Active Acne population showed very promising results for utilizing nitric oxide topically.

[0168] Seventy-five per cent reported a decrease in pustules and nodules by 4 weeks, 83% reported improvement by 8 weeks, and 85% by 12 weeks.

[0169] The Wound Healing study patient population without exception showed excellent results. Rhytidectomy, blepharoplasty, and rhinoplasty patients displayed less bruising, edema, and faster re-epithelialization on the treated side when compared to the contralateral untreated control side. Previously non-healing diabetic ulcers and pressure ulcers healed within 30-45 days. Compromised local and regional flaps showed improved vascularization and viability within 30 days.

[0170] The Scar Therapy population showed positive trends, worthy of further investigation. Atrophic scars and striae, particularly abdominal and breast showed the most improvement with 37% describing less visibility of striae at 4 weeks, 49% at 8 weeks, and 72% by 12 weeks. Softening of hypertrophic scars was noted at 4 weeks, with some scar volume reduction noted by 12 weeks. Improved vascularity in these latter scars is postulated.

[0171] Discussion: The fields of Cellular Medicine, Regenerative and Stem Cell Therapy are growing rapidly. Much has been written on the multiple health benefits of Nitric Oxide. This multifunctional signaling molecule may prove to be the cornerstone of the stem cell regenerative medicine world. NO is recognized as the requisite signal for stem cell mobilization and differentiation into target cell types. Endogenous production is a highly complex, two pronged, regulated process that is significantly reduced by 40 years of age. This makes studies examining exogenous modes of administration intriguing. An oral replacement lozenge is available which has shown tremendous benefit.

[0172] Much has been written on the challenges of formulating a topically applied product which generates sufficient NO to absorb into the dermis, does not decay too rapidly, or produce adverse physiological byproducts.

[0173] There have been many studies demonstrating the metabolic effects, of NO on the maj or components of skin and tissue including: fibroblasts, melanocytes, endothelial cells, keratinocytes, macrophages, neutrophils, and adipocytes. [0174] To our knowledge, this is the first Pilot Study examining a novel, dual chamber, Nitric oxide generating serum for aging skin, acne, wound healing and scar therapy. Our findings indicate that the topical application of nitric oxide, when optimized, has a positive effect on improving vascularity to skin, as well as, the general tone, quality and texture of skin. This fact explains the observed reduction in pore size that was shown. Improvement in unwanted hyper-pigmentation and erythema was also observed.

[0175] Skin care can be likened to working out at a gym. If one goes regularly, the results will be better than if one goes sporadically. If one uses all of the equipment at the gym, the results will be better than if one only uses the biceps machine. A NO generating, topically applied serum is akin to hiring a“tissue personal trainer”. The fact that the majority of skin cells emit NO and are therefor depleted over time suggests their function is compromised over time, hence“aging skin”. The replenishment of NO, via this serum, reinvigorates the cells, allowing for improved function, postulating the positive aesthetic results shown in this study.

[0176] Topically applied Nitric oxide generating serum showed a positive effect in the acne population. The anti-inflammatory and ant-bacterial benefits of NO have been described. As it relates to inflammatory conditions, acne vulgaris and subsequent sequalae, lead in conditions having a negative impact on a patients’ self- esteem.

[0177] Acne vulgaris is a chronic inflammatory disease that affects 40-50 million Americans per year, and hundreds of millions of patients worldwide per year. Acne patients present an excellent model for studying scar pathophysiology. The complex interactions of infection, inflammation, tissue injury, wound healing and the propensity for scar formation allow scientists a unique opportunity to investigate alternate forms of therapy.

[0178] Acne is characterized by comedones, pustules, and papules. Left untreated, saucerized and pitted scars can form. The pathogenesis of acne is multifactorial. Four primary factors play a pivotal role in the formation of acne lesions: excess sebum production, abnormal keritanization, inflammation, and bacterial colonization of Propionibacterium acnes in the pilosebaceous unit. While a general concensus exists on the pathogenetic factors, the developmental sequence of events has been controversial. A traditional belief was that abnormal keritanization results in the microcomedone, the earliest subclinical acne lesion. Sebaceous gland activation by androgens, led to excess sebum production, with a keratin plug being formed. P acnes colonization induced the innate immune system, culminating in the visible inflammation. [0179] It is now felt that subclinical inflammation PRECEDES microcomedone formation. Immunohistochemical studies show significant inflammation around normal follicles of uninvolved skin of acne patients PRIOR to follicular hyperkeritanization. Another study showed inflammatory cell infiltrates without clinically visible inflammatory lesions. This is thought to be via the proinflammatory cytokine ILlalpha, independent of colonization. Given this theory, it is understandable why topical NO has a beneficial effect on acne. The Wound Healing and Scar therapy patient populations also showed positive trends worthy of further study. Examining the concept of“scar-less wound healing, as in the Axolotl Salamander model, one sees a rapid re- epithelialization, and short inflammatory phase with TGFB3 playing a major role.

[0180] The modulatory effect that NO has on skin stem cells and communicating messengers such as exosomes can suggest why a topically applied NO serum would accelerate wound healing and play a vital role in multi-modality scar therapy. Interestingly, as seen in Table 2, no patients were unhappy with NO serum. The majority were satisfied to extremely satisfied. This also had a spillover effect on the question of self-esteem with 55% of study subjects reporting an increase in self-esteem due to their appearance at 4 weeks, 78% at 8 weeks, and 85% at 12 weeks.

Table 2

[0181] Conclusion: It is possible to deliver clinically relevant bioactive nitric oxide via a dual chamber NO generating delivery device. This Pilot Study shows valid proof of concept to warrant further IRB approved studies in the areas of aging skin, acne, wound healing and scar therapy.

EXAMPLE 10

USE OF NO AS AN ANTIBACTERIAL AGENT

[0182] The NO serum was examiner to determined effects on bacteria. Increasing amounts of NO serum was applied to a pseudomonas plate. As shown in Figure 3, the application of the NO serum resulted in clear spots on the plate indicating that the bacteria had been killed. [0183] Although the invention has been described with reference to the above examples, it will be understood that modifications and variations are encompassed within the spirit and scope of the invention. Accordingly, the invention is limited only by the following claims.