Topical compositions and methods of using the same

[Related Applications]

This application claims the benefit of and priority to U.S. Provisional Patent Application No. 61/770,615, filed February 28, 2013, the disclosure of which is incorporated herein by reference in its entirety.

The present invention relates generally to compositions and methods of using same.

Skin disorders can be treated topically with a variety of pharmaceutical compositions.

<p>The present invention addresses previous deficiencies in the art by providing topical compositions and methods of using the topical compositions.</p>

<p>A first aspect of the invention includes a composition comprising a first viscosity increasing agent, at least one polyhydric alcohol, at least one buffering agent, at least one preservative, a second viscosity increasing agent, at least one organic solvent, at least one humectant, at least one active pharmaceutical ingredient, and water, buffered to a pH of from about 3 to about 11. In some embodiments, the pH of the composition can be from about 3 to about 8. The composition can be cosmetically elegant.</p><p>A second aspect of the invention includes a composition comprising at least one polyhydric alcohol present in an amount from about 1% to about 30% by weight of the composition, at least one thickening agent present in an amount from about 0.1% to about 5% by weight of the composition, water present in an amount from about 70% to about 99% by weight of the composition, at least one buffering agent present in an amount from about 0.01% to about 2% by weight of the composition, and at least one preservative present in an amount from about 0.01% to about 1% by weight of the composition, buffered to a pH of from about 3 to about 8. The composition can be cosmetically elegant.</p><p>A further aspect of the invention includes a kit comprising a first composition comprising at least one polyhydric alcohol present in an amount of about 1% to about 30% by weight of the composition, at least one thickening agent present in an amount of about 0.1% to about 5% by weight of the composition, and water present in an amount of about 70% to about 99% by weight of the composition, and a second composition that is anhydrous. The composition can be cosmetically elegant.</p><p>Another aspect of the present invention includes a method of increasing nitric oxide release from a topical anhydrous gel containing a nitric oxide-releasing active pharmaceutical ingredient, comprising contacting the topical anhydrous gel with a first composition of the present invention (e.g., a hydrogel of the present invention) having a pH of about 4 to about 6 to form a combined composition. In some embodiments, the combined composition can be applied to the skin of a subject. The combined composition can be cosmetically elegant. The nitric oxide-releasing active pharmaceutical ingredient can be a diazeniumdiolate-modified macromolecule.</p><p>A further aspect of the present invention includes a pharmaceutical composition comprising a topical anhydrous gel comprising a moisture sensitive active pharmaceutical ingredient and a first composition of the present invention (e.g., a hydrogel of the present invention) comprising a means for reducing the pH of the topical anhydrous gel. The moisture sensitive active pharmaceutical ingredient can be a nitric oxide releasing active pharmaceutical ingredient, and the nitric oxide releasing active pharmaceutical ingredient can be a diazeniumdiolate modified polysiloxane molecule.</p><p>Another aspect of the invention includes a method of treating acne vulgaris comprising topically applying to the skin of a subject a composition of the invention. A therapeutically effective amount of the composition may be applied.</p><p>A further aspect of the present invention includes a method of reducing inflammatory and/or non-inflammatory lesions in a subject, comprising topically applying a composition of the present invention to the skin of the subject. A therapeutically effective amount of the composition may be applied. In some embodiments, the composition may include a nitric oxide-releasing active pharmaceutical ingredient in an amount of about 0.5% to about 10% by weight of the composition. The composition may store and/or release nitric oxide in an amount of about 0.05% to about 3% by weight of the composition. The method may reduce inflammatory and/or non-inflammatory lesions by about 10% or more over a given period of time, compared to a subject who has not applied a composition of the present invention over the same period of time. In some embodiments, the method may reduce inflammatory and/or non-inflammatory lesions in a subject over the same period of time, compared to a subject who has not applied a composition including a nitric oxide-releasing active pharmaceutical ingredient. Subjects may experience a reduction in inflammatory and/or non-inflammatory lesions within 12 weeks or less, in some embodiments within 8 weeks or less, and in further embodiments within 4 weeks or less.</p><p>Another aspect of the present invention includes a method of reducing P. acnes counts in a subject, comprising topically applying a composition of the present invention to the skin of the subject. A therapeutically effective amount of the composition may be applied. In some embodiments, the composition may comprise a nitric oxide releasing active pharmaceutical ingredient in an amount of about 0.5% to about 10% by weight of the composition. The composition may store and/or release nitric oxide in an amount of about 0.05% to about 3% by weight of the composition. The method may reduce P. acnes counts by about 10% or more over a given period of time, compared to a subject who has not applied a composition of the present invention over the same period of time. In some embodiments, the method may reduce P. acnes counts in a subject over the same period of time, compared to a subject who has not applied a composition comprising a nitric oxide releasing active pharmaceutical ingredient. The subject may experience a reduction in P. acnes counts within 12 weeks or less, in some embodiments within 8 weeks or less, and in further embodiments within 4 weeks or less.</p><p>The above and other aspects of the present invention will now be described in more detail with reference to other embodiments described herein. It should be understood that the present invention can be embodied in various forms and should not be construed as being limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the present invention to those skilled in the art.</p>

<figref num="1">1 is a graph showing the effect of Nitricil NVN1 topical gel strength, hydrogel pH, and hydrogel to Nitricil NVN1 topical gel ratio on the pH of the admixture.</figref><figref num="2">FIG. 1 is a graph of in vitro pH determination for Nitricil NVN1 topical gel when mixed with unbuffered hydrogel (pH 4) or buffered (0.1% citric acid) hydrogel (pH 4).</figref><figref num="3">1 is a graph of the determination of pH at the skin surface for blends of unbuffered pH 4 hydrogel or buffered pH 4 hydrogel with 8% w/w Nitricil NVN1 topical gel.</figref><figref num="4">Nitricil NVN1 topical gel concentration and hydrogel ratio C_max1 is a graph showing the effect of</figref><figref num="5">1 is a graph showing the effect of Nitricil NVN1 topical gel concentration and hydrogel ratio on cumulative nitric oxide (NO) release.</figref><figref num="6">1 is a graph showing the effect of unbuffered hydrogel pH and ratio on admixture pH in relation to Nitricil NVN1 topical gel concentration.</figref><figref num="7">FIG. 1 is a graph showing the in vitro nitric oxide release profile over time for a 2% Nitricil NVN1 gel formulation.</figref><figref num="8">FIG. 1 is a graph showing in vitro nitric oxide release profiles over time for 2%, 6%, and 12% Nitricil NVN1 gel formulations when mixed with pH 4 hydrogel.</figref><figref num="9">Propionibacterium acnes counts (log/cm) over time based on analysis of covariance (ANCOVA)²) is a graph showing changes in</figref><figref num="10">FIG. 1 is a graph showing the mean percentage reduction over time for non-inflammatory lesions in the on-protocol population.</figref><figref num="11">FIG. 1 is a graph showing the mean percentage reduction in inflammatory lesions over time in the protocol population.</figref><figref num="12">FIG. 1 is a graph showing cumulative nitric oxide release for 2% Nitricil NVN1 gel without hydrogel and 1% Nitricil NVN1 gel with hydrogel (gel to hydrogel ratio was 1:1).</figref>

Next, the present invention will be described in more detail below. However, the present invention can be implemented in various forms and should not be construed as being limited to the embodiments described herein. Rather, by providing these embodiments, the present disclosure will be thorough and complete, and the scope of the present invention will be fully conveyed to those skilled in the art.

The terminology used in the description of the invention herein is intended to describe particular embodiments only and is not intended to limit the invention. As used in the description of the invention and the appended claims, the singular forms "a," "an," and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

Unless otherwise defined, all terms used herein (including technical and scientific terms) have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Furthermore, terms defined in commonly used dictionaries should be interpreted to have a meaning that is not inconsistent with their meaning in the context of this application and the related art, and should not be interpreted in an idealized or overly formal sense unless expressly so defined herein. The terms used in the description of the invention herein are intended to describe only specific embodiments and are not intended to limit the invention. All publications, patent applications, patents, and other references mentioned herein are hereby incorporated by reference in their entirety. In the event of a conflict in terminology, the present specification shall control.

Additionally, as used herein, "and/or" refers to and includes any and all possible combinations of one or more of the associated listed items, as well as no combinations when interpreted in the alternative ("or").

Unless otherwise indicated by context, it is expressly intended that the various features of the invention described herein can be used in any combination. Moreover, the invention also contemplates that in some embodiments of the invention, any feature or combination of features described herein can be excluded or omitted. For example, if the specification states that a complex includes components A, B, and C, it is expressly intended that any of A, B, or C, or any combination thereof, can be excluded and not claimed.

As used herein, the transitional phrase "consisting essentially of" (and grammatical variations) is to be construed to include the recited materials or steps "and materials or steps which do not materially affect the basic and novel characteristic(s) of the presently claimed invention." See In re Herz, 537 F.2d 549, 551-52, 190 USPQ461, 463 (CCPA 1976) (emphasis in original).

See also MPEP §2111.03. Thus, the term "consisting essentially of" as used herein should not be construed as equivalent to "including."

The term "about," when used herein in reference to a measurable value, such as an amount or concentration, is intended to refer not only to the stated value, but also to a variation of up to ±20% of the stated value, such as, but not limited to, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of the stated value. For example, "about X," where X is a measurable value, is intended to include X and a variation of ±20%, ±10%, ±5%, ±1%, ±0.5%, or even ±0.1% of X. For measurable values, the ranges described herein may include any other ranges and/or individual values within those ranges.

According to some embodiments of the present invention, a topical composition is provided. The composition of the present invention can comprise at least two parts. In some embodiments, the composition of the present invention comprises a first part comprising a first composition and a second part comprising a second composition. The second part of the composition of the present invention can comprise a nitric oxide-releasing active pharmaceutical ingredient (NO-releasing API). In some embodiments, the composition of the present invention can comprise a first part comprising a first composition, and the first composition can be in the form of a hydrogel. "Hydrogel", as used herein, refers to a hydrophilic gel comprising a gel matrix and water. In some embodiments, the first composition of the present invention comprises at least one polyhydric alcohol, at least one thickening agent, and water.

In certain embodiments, the composition of the present invention comprises, consists essentially of, or consists of a first composition and a second composition of the present invention, where the first composition and the second composition are compatible with each other such that they can be mixed and/or combined together to form the composition of the present invention. The composition of the present invention may be referred to in some embodiments as a "composite composition." In a further aspect, the composite composition may be suitable for topical application to the skin of a subject.

Exemplary polyhydric alcohols that may be present in the first composition of the present invention include, but are not limited to, glycerol, propylene glycol, polyethylene glycol, polypropylene glycol, triethylene glycol, neopental glycol, butylene glycol, polyethylene glycol, sorbitol, arabitol, erythritol, HSH, isomalt, lactitol, maltitol, mannitol, xylitol, threitol, ribitol, galactitol, fucitol, iditol, inositol, volemitol, and any combination thereof. In some embodiments, the first composition of the present invention includes glycerol, such as, but not limited to, anhydrous glycerol.

The polyhydric alcohol can be present in the first composition of the present invention in an amount of about 1% to about 30% by weight of the first composition or any range and/or individual value therein, for example, but not limited to, about 1% to about 20%, about 1% to about 10%, or about 5% to about 15% by weight of the first composition. In some embodiments, the polyhydric alcohol can be present in the first composition of the present invention in an amount of about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by weight of the first composition or any range and/or individual value therein.

Exemplary thickening agents that may be present in the first composition of the present invention include, but are not limited to, carboxypolymethylene; polyacrylic acid polymers, such as polyacrylic acid, polyacrylate polymers, crosslinked polyacrylate polymers, crosslinked polyacrylic acid, and mixtures thereof; cellulose ethers, such as hydroxyalkyl cellulose polymers, such as hydroxypropylmethylcellulose (HPMC), hydroxypropylcellulose, hyrdoxyethylcellulose, methylcellulose, carboxymethylcellulose, and mixtures thereof; methacrylates; polyvinylpyrollidone; crosslinked polyvinylpyrrolidone; polyvinylpyrrolidone-vinyl acetate copolymers; polyvinyl alcohol; polyethylene oxide; polyethylene glycol; polyvinyl alkyl ether-maleic acid copolymers; carboxyvinyl polymers; polysaccharides; gums, such as sodium alginate, carrageenan, xantham gum, acacia gum, gum arabic, guar gum, pullulan, agar, chitin, chitosan, pectin, karayaga. proteins, such as collagen, whey protein isolate, casein, milk protein, soy protein, gelatin, and mixtures thereof; starches, such as maltodextrin, amylose, high amylose starch, corn starch, potato starch, rice starch, tapioca starch, pea starch, sweet potato starch, barley starch, wheat starch, waxy corn starch, added sugars, and mixtures thereof; Modified starches (e.g., hydroxypropylated high amylose starch), dextrin, levan, elsinan, gluten, and mixtures thereof; bentonite; calcium stearate; calatonia; colloidal silicon dioxide; dextrin; hypromellose; polycarbophil; kaolin; saponite; sorbitan esters; sucrose; sesame oil; tragacanth; potassium alginate; povidone; sodium starch glycolate; phospholipids; and any combination thereof.

In some embodiments, the first composition of the present invention comprises a carboxypolymethylene, such as, but not limited to, those commercially available under the trade name Carbopol® from Lubrizol Corporation of Wickliffe, Ohio. Exemplary Carbopol® polymers that may be present in the first composition of the present invention include, but are not limited to, Carbopol® 974P NF polymers, such as, for example, Type A, Type B and/or Type C homopolymers; Carbopol® Ultrez 10, 20, 21 NF polymers; Carbopol® 971P NF polymers; Carbopol® 980 homopolymer Type C polymers, Carbopol® 980 NF polymers, Carpobol® 980P polymers, Carbopol® ETD 2020 NF polymers, Carbopol® 71G NF polymers, Carbopol® 981P NF polymers, Carbopol® 970P NF polymers, Carbopol® 981P NF polymers, Carbopol® 5984P NF polymers, Carbopol® 934P NF polymers, Carbopol® 940P NF polymers, Carbopol® 941P NF polymers, Carbopol® 13242 NF polymers, Carbopol® AA-1 USP NF polymers, Carbopol® TR1 NF polymers, Carbopol® TR2 NF polymers, Lubrizol Aqua CC polymers and SF-2 polymers, and any combination thereof.

In some embodiments, the thickener present in the first composition of the present invention can be a polymer containing an acidic group, such as, but not limited to, a carboxylic acid group. The acidic group of the polymer can be partially neutralized in the first composition of the present invention. In some embodiments, the thickener present in the first composition of the present invention can be carboxypolymethylene. In some embodiments, the carboxypolymethylene present in the first composition of the present invention can be partially neutralized. The first composition of the present invention can include carboxypolymethylene and have a pH of about 3 to about 7, about 3.5 to about 6.5, about 3.5 to about 6, or about 4 to about 6. In some embodiments, the first composition of the present invention can include carboxypolymethylene and have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, or 7.

A thickening agent can be present in the first composition of the present invention. In some embodiments, the composition of the present invention can include at least two thickening agents, and these thickening agents can be the same or different. In some embodiments, the first thickening agent can be present in the first composition of the present invention in an amount of about 0.01% to about 5% by weight of the first composition or any range and/or individual value therein, for example, but not limited to, about 0.05% to about 3% by weight of the first composition, or about 0.1% to about 1.5% by weight of the first composition. In some embodiments, the first thickening agent is present in the first composition of the present invention in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5% by weight of the first composition, or any range and/or individual value therein.

Water can be present in the first composition of the present invention in an amount of about 70% to about 99% by weight of the first composition or any range and/or individual value therein, such as, but not limited to, about 75% to about 95% by weight of the first composition or about 80% to about 90% by weight of the first composition. In some embodiments, water is present in the first composition of the present invention in an amount of about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% by weight of the first composition or any range and/or individual value therein.

In some embodiments, the first composition of the present invention comprises, consists essentially of, or consists of at least one polyhydric alcohol present in an amount of about 1% to about 30% by weight of the first composition, at least one thickening agent present in an amount of about 0.1% to about 5% by weight of the first composition, and water present in an amount of about 70% to about 99% by weight of the first composition. In some embodiments, the thickening agent can be carboxypolymethylene. The first composition can be a hydrogel.

The first composition of the present invention may include a preservative. The preservative may be present in the first composition of the present invention in an amount of about 0.01% to about 1% by weight of the first composition or any range and/or individual value therein, for example, but not limited to, about 0.01% to about 0.1%, about 0.05% to about 1%, or about 0.1% to about 1% by weight of the first composition. In some embodiments, the preservative is present in the first composition of the present invention in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% by weight of the first composition or any range and/or individual value therein.

Exemplary preservatives that may be present in the first composition of the present invention include, but are not limited to, sorbic acid, benzoic acid, methylparaben, propylparaben, methylchloroisothiazolinone, metholisothiazolinone, diazolidinyl urea, chlorobutanol, triclosan, benzethonium chloride, p-hydroxybenzoate, chlorhexidine, digluconate, hexadecyltrimethylammonium bromide, alcohol, benzalkonium chloride, boric acid, bronopol, butylparaben, butylene calcium acetate, calciumacetate), calcium chloride, calcium lactate, carbon dioxide, cationic preservatives, and bentonite, cetrimide, cetylpyridinium chloride, chlorhexidine, chlorobutanol, chlorocresol, chloroxylenol, citric acid monohydrate, cresol, dimethyl ether, ethylparaben, glycerin, hexetidine, imidurea, isopropyl alcohol, lactic acid, monothioglycerol, pentetic acid, phenol, phenoxyethanol, phenylethyl alcohol, phenylmercuric acetate, phenylmercuric borate, phenylmercuric nitrate, potassium benzoate, potassium metabisulfite, potassium sorbate, propionic acid, propyl gallate, propylene glycol, sodium acetate, sodium benzoate, sodium borate, sodium lactate, sodium sulfite, sodium propionate, sodium metabisulfite, xylitol, sulfur dioxide, carbon dioxide, and any combination thereof.

The first composition of the present invention can include a neutralizing agent. The neutralizing agent can be present in the first composition of the present invention in an amount sufficient to provide a desired pH, such as, but not limited to, from about 3 to about 11, or any range and/or individual value therein, such as, but not limited to, from about 3 to about 8, from about 4 to about 7, or from about 6 to about 7, or from about 6 to 11.

In some embodiments, the neutralizing agent can be present in the first composition in an amount sufficient to bring the first composition to a pH of between about 3 and about 8.

In some embodiments, the neutralizing agent can be present in the first composition of the present invention in an amount sufficient to bring the composition of the present invention to a desired pH when the first composition and the second part (e.g., the second composition) are combined and/or when the first composition and/or a composition comprising the first composition and the second part is administered to the skin of a subject. The neutralizing agent can be present in the first composition of the present invention in an amount sufficient to bring the composition of the present invention (e.g., a composition comprising the first composition and the second composition) to a desired pH, for example, but not limited to, a pH of about 3 to about 11, or any range and/or individual value therein.

The pH of the compositions of the present invention may be determined once a steady state pH has been achieved following contacting, combining and/or mixing of the first and second parts of the compositions of the present invention.

Alternatively or in addition, the pH of the composition of the present invention can be determined after a set period of time, such as, but not limited to, about 5 minutes, about 10 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 60 minutes or more. The pH of the composition of the present invention can be measured in vitro after contacting, combining and/or mixing the first and second parts of the composition of the present invention. Alternatively or in addition, the pH of the composition of the present invention can be measured after administration to a subject, for example, the pH at the skin surface can be measured after administration of the composition of the present invention to the skin of a subject.

In some embodiments, the neutralizing agent adjusts the pH of the first composition and/or composition of the present invention. In some embodiments of the present invention, the neutralizing agent is present in the first composition of the present invention in an amount sufficient to cause the first composition to have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8, or any range and/or individual value therein. In some embodiments of the present invention, the neutralizing agent is present in the composition of the present invention in an amount sufficient to cause the composition to have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11, or any range and/or individual value therein.

Exemplary neutralizing agents that may be present in the first composition of the present invention include, but are not limited to, bases, such as sodium hydroxide, potassium hydroxide, and mixtures thereof; acids, such as hydrochloric acid, citric acid, lactic acid, glycolic acid, acetic acid, and mixtures thereof; sodium carbonate; trolamine; tromethamine; aminomethylpropanol; triisopropanolamine; aminomethylpropanol; tetrahydroxypropylethylenediamine; tetrasodium EDTA; suttocide A; and any combination thereof.

The neutralizing agent may be present in the first composition of the present invention in an amount of about 0.01% to about 1% by weight of the first composition, or any range and/or individual value therein, such as, but not limited to, about 0.01% to about 0.1%, about 0.05% to about 1%, or about 0.1% to about 1% by weight of the first composition. In some embodiments, the neutralizing agent may be present in the first composition of the present invention in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, or 1% by weight of the first composition, or any range and/or individual value therein.

The first composition of the present invention may be unbuffered or buffered. In some embodiments, the first composition of the present invention may be unbuffered. In other embodiments, the first composition of the present invention may be buffered. Exemplary buffers that may be present in the first composition of the present invention include, but are not limited to, acetic acid/acetate buffer; hydrochloric acid/phosphate buffer; citro-phosphate buffer; phosphate buffer; citric acid/citrate buffer; lactate buffer; tartrate buffer; malic acid buffer; glycine/HCl buffer; saline buffer, such as phosphate buffered saline (PBS), Tris buffered saline (TBS), Tris-HCl, NaCl, Tween buffered saline (TNT), phosphate buffered saline, Triton X-100 (PBT) and mixtures thereof; cacodylate buffer; barbital buffer; Tris buffer; and any combination thereof.

In some embodiments, the first composition of the present invention can include a buffering agent.Exemplary buffering agents include, but are not limited to, citric acid, acetic acid, lactic acid, boric acid, succinic acid, malic acid, and any combination thereof.The buffering agent can be present in the first composition of the present invention in an amount of about 0.01% to about 2% by weight of the first composition or any range and/or individual value therein, for example, but not limited to, about 0.01% to about 0.1% by weight of the first composition, about 0.05% to about 1%, about 0.1% to about 0.5%, or about 0.1% to about 2%. In some embodiments, the buffering agent is present in the first composition of the present invention in an amount of about 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, 0.06%, 0.07%, 0.08%, 0.09%, 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, 1.5%, 1.6%, 1.7%, 1.8%, 1.9% or 2% by weight of the first composition or any range and/or individual value therein.

In some embodiments, the buffer and/or buffering agent is present in the first composition of the present invention in an amount sufficient for the first composition to have a pH of about 3 to about 8, or any range and/or individual value therein, such as, but not limited to, about 3 to about 6, about 3 to about 5, about 4 to about 7, about 5 to about 7, or about 6 to about 7. In some embodiments of the present invention, the buffer and/or buffering agent can be present in the first composition of the present invention in an amount sufficient for the first composition to have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, or 8, or any range and/or individual value therein.

In some embodiments, the buffer and/or buffering agent can be present in the first composition of the present invention in an amount sufficient to provide a desired pH to the composition of the present invention comprising the first composition and the second portion (e.g., the second composition). For example, the composition of the present invention can include the second composition and the first composition comprising the buffer and/or buffering agent, where the buffer and/or buffering agent is present in an amount sufficient to provide the composition of the present invention with a pH of about 3 to about 11, for example, but not limited to, about 3 to about 8, about 7 to about 11, about 8 to about 10, about 3 to about 5, about 4 to about 7, about 5 to about 7, or about 6 to about 7. In some embodiments of the present invention, the buffer and/or buffering agent can be present in the first composition of the present invention in an amount sufficient to cause the composition to have a pH of about 3, 3.5, 4, 4.5, 5, 5.5, 6, 6.5, 7, 7.5, 8, 8.5, 9, 9.5, 10, 10.5, or 11, or any range and/or individual value therein. In some embodiments, the buffer and/or buffering agent can be present in the first composition of the present invention in an amount sufficient to provide a desired pH upon administration of the composition of the present invention comprising the first composition and the second portion to the skin of a subject.

In some embodiments, a buffering agent, buffering agent and/or neutralizing agent may be present in the first composition of the present invention in an amount sufficient to bring the composition of the present invention and/or the first composition of the present invention to a desired pH.

In some embodiments, the first composition of the present invention comprises at least one polyhydric alcohol present in an amount of about 1% to about 30% by weight of the first composition, at least one thickening agent present in an amount of about 0.1% to about 5% by weight of the first composition, water present in an amount of about 70% to about 99% by weight of the first composition, and at least one preservative in an amount of about 0.01% to about 1% by weight of the first composition. The first composition may be buffered to have a pH ranging from about 3 to about 8, about 3 to about 6, or about 6 to about 8. The first composition may be a hydrogel.

In some embodiments, the first composition of the present invention can comprise, consist essentially of, or consist of a polyhydric alcohol in an amount of about 1% to about 30% by weight of the first composition, a thickening agent in an amount of about 0.01% to about 5% by weight of the first composition, water in an amount of about 70% to about 99% by weight of the first composition, optionally a buffering agent in an amount of about 0.001% to about 2% by weight of the first composition, optionally a preservative in an amount of about 0.001% to about 1% by weight of the first composition, and optionally a neutralizing agent in an amount of about 0.001% to about 1% by weight of the first composition. The first composition can have a pH ranging from about 3 to about 5, or from about 5 to about 7. In some embodiments, the thickening agent present in the first composition can be carboxypolymethylene. In some embodiments, the first composition can be cosmetically elegant. The first composition can be a hydrogel.

As will be recognized by those skilled in the art in light of the present disclosure, the properties of the first composition of the present invention can impart and/or provide the same and/or similar properties to the composition of the present invention. For example, in some embodiments, the first composition of the present invention can include a preservative present in an amount sufficient to provide antimicrobial activity to the first composition and/or the composition of the present invention. Thus, in some embodiments, the first composition of the present invention and/or the composition of the present invention can be antimicrobial.

The composition of the present invention and/or the first composition can have a viscosity in the range of about 5,000 cP to about 25,000 cP or any range and/or individual value therein, such as, but not limited to, about 5,000 cP to about 20,000 cP or about 7,000 cP to about 15,000 cP. In some embodiments, the composition of the present invention and/or the first composition can have a viscosity of about 5,000, 5,500, 6,000, 6,500, 7,000, 7,500, 8,000, 8,500, 9,000, 9,500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 17,000, 17,500, 18,000, 18,500, 19,000, 20,000, 21,000, 22,000, 23,000, 24,000, 25,000, 26,000, 27,000, 28,000, 29,000, 30,000, 31,000, 32,000, 33,000, 34,000, 35,000, 36,000, 37,000, 38,000, 39,000, 40,000, 41,00 In one embodiment, the viscosity of the composition may be 10,000, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 20,500, 21,000, 21,500, 22,000, 22,500, 23,000, 23,500, 24,000, 24,500 or 25,000 cP or any range and/or individual value therein.

In some embodiments, the composition of the present invention can include a first composition of the present invention having a viscosity that allows it to be mixed and/or combined with a second part of the composition of the present invention. For example, the first composition of the present invention can have a suitable and/or sufficient viscosity to be mixed and/or combined with a second part of the composition of the present invention in a human hand and/or on the skin of a subject. If the viscosity of the first composition is too low, it may run off the skin of a subject before mixing and/or combining. If the viscosity of the first composition is too high, it may be difficult to mix with the second part of the composition of the present invention and/or it may be difficult to spread and/or apply the combined composition on the skin of a subject.

The composition of the present invention and/or the first composition may include an active pharmaceutical ingredient (API). Any suitable API or combination of APIs may be included in the composition of the present invention and/or the first composition. Examples of APIs include, but are not limited to, antibacterial agents, anti-acne agents, anti-inflammatory agents, analgesics, anesthetic agents, antihistamines, antiseptics, immunosuppressants, antihemorrhagic agents, vasodilators, wound healing agents, anti-biofilm agents, and any combination thereof. Exemplary APIs include, but are not limited to, those described in WO2013/006608, which is incorporated herein by reference in its entirety.

In some embodiments, the first composition of the present invention may not include an API.

In some embodiments, the first composition of the present invention does not contain a nitric oxide (NO)-releasing API. In some embodiments, the first composition of the present invention can include at least one API, but the first composition may not include a NO-releasing API.

In some embodiments, the first composition of the present invention can include an API.

The API in the first composition may be a moisture-sensitive API. In some embodiments, the first composition of the present invention comprises an API (e.g., a moisture-sensitive API), and the second part of the composition of the present invention and/or the second composition comprises a second API, where the API in the first composition and the second API in the second part and/or the second composition may not be chemically compatible and/or stable in the composition of the present invention. For example, the API in the first composition and the second API in the second part and/or the second composition may not be chemically compatible and/or stable when stored together in the composition of the present invention.

The first composition of the present invention may be suitable for use and/or compounding with one or more compositions, such as, but not limited to, two, three, four or more compositions, and such one or more compositions may be the same and/or different. In some embodiments, the first composition of the present invention may be suitable for use and/or compounding with one or more pharmaceutical compositions. The first composition of the present invention may be used as a drug delivery system and/or a drug release system. For example, the first composition of the present invention may be configured to modulate the release of an API in the second composition upon contact between the first composition of the present invention (e.g., a hydrogel of the present invention) and the second composition. Alternatively or in addition, the first composition of the present invention may be configured to modulate the pH of the second composition upon contact between the first composition of the present invention (e.g., a hydrogel of the present invention) and the second composition. In some embodiments, a first composition of the present invention may be configured to modulate the pH of a second composition comprising a nitric oxide (NO)-releasing API and/or the release of nitric oxide from the NO-releasing API in the second composition.

"Modulate," "modulating," "modulation," and grammatical variations thereof, as used herein, refer to increasing or decreasing the pH of the second composition and/or the release of an API in the second composition, compared to the pH of the second composition and/or the release of an API in the second composition in the absence of the first composition of the present invention. As used herein, the terms "increase," "increases," "increased," "increasing," and similar terms indicate that the pH and/or release is at least about 5%, 10%, 25%, 50%, 75%, 100%, 150%, 200%, 300%, 400%, 500% or more higher than the pH and/or release in the absence of the first composition of the present invention. As used herein, the terms "reduce," "reduces," "reduced," "reducing," and similar terms refer to a reduction in pH and/or emissions of at least about 5%, 10%, 25%, 35%, 50%, 75%, 80%, 85%, 90%, 95%, 97% or more compared to the pH and/or emissions in the absence of the first composition of the present invention.

"Contacting," as used herein with respect to a first composition (e.g., a hydrogel of the invention) and a second portion and/or second composition, refers to directly and/or indirectly exposing at least one component in the first composition to the second portion and/or second composition. Contacting the first composition with the second portion and/or second composition can be accomplished by any means, including but not limited to, mixing, stirring, blending, dispersing, grinding, homogenizing, combining, applying to the same site or area, and in some embodiments, can optionally form a composite composition of the invention. For example, the first composition may be directly contacted with the second composition, including but not limited to, by mixing and/or combining the first composition with the second composition to form a composite composition of the invention, before, during, and/or after topical application to a subject. Direct contact between the first composition and the second composition may occur by applying the second composition to the subject in one or more layers, and then applying the first composition to the subject in one or more layers, or vice versa, to form a composite composition of the present invention. Indirect contact may occur by applying the second composition to the subject, and then applying the first composition to the subject through a substrate, such as, but not limited to, a cloth, bandage, gauze, etc., or vice versa, optionally forming a composite composition of the present invention.

According to some embodiments of the present invention, upon contacting the first composition of the present invention with the second composition, the first composition of the present invention may be configured to modulate the release of an API, such as, but not limited to, a NO-releasing API, present in the second composition.

In some embodiments, the water and/or proton(s) present in the first composition of the present invention can be contacted with the second composition to modulate the release of the API present in the second composition, such as, but not limited to, a NO-releasing API. Alternatively, or in addition, in some embodiments, the first composition of the present invention can be contacted with the second composition to modulate the pH of the second composition, thereby modulating the release of the API present in the second composition, such as, but not limited to, a NO-releasing API. In some embodiments, the first composition of the present invention is configured to provide water and/or proton(s) to the second composition and/or to modulate the pH of the second composition.

The present inventors have surprisingly found that embodiments of the present invention can significantly increase the amount of nitric oxide released from a composition comprising a NO-releasing API and/or can provide a continuous release of nitric oxide from a composition comprising a NO-releasing API. For example, this can be seen in FIG. 12, which compares a composition not comprising the first composition of the present invention (i.e., 2% Nitricil NVN1 gel) with a composition of the present invention comprising the first composition of the present invention (i.e., 1% Nitricil NVN1 gel). The 2% Nitricil NVN1 gel and the portion of the 1% Nitricil NVN1 gel that comprises a NO-releasing API contained the same amount of NO-releasing API. FIG. 12 demonstrates that the 1% Nitricil NVN1 gel provided a significantly higher release of nitric oxide compared to the 2% Nitricil NVN1 gel, and further provided a continuous release of nitric oxide over a period of time.

Without wishing to be bound by any particular theory, it is believed that the composition of the present invention, which comprises the first composition of the present invention and the NO-releasing API, may provide a proton-donating system that can allow the composition of the present invention to release nitric oxide in large quantities and/or the composition of the present invention to release nitric oxide continuously. Without wishing to be bound by any particular theory, the proton-donating system may be an acid that can be formed by the composition of the present invention (e.g., a composition comprising the first composition of the present invention and the second composition as described herein) and/or the first composition of the present invention (e.g., the hydrogel of the present invention). The composition of the present invention may provide a proton to the NO donor in the NO-releasing API and/or allow the proton to be in close proximity to the NO donor in the NO-releasing API, thereby allowing the release of nitric oxide. The compositions of the present invention can provide protons over an extended period of time and/or allow protons to be in close proximity to the NO donor, thereby providing a continuous release of NO for a period of about 1 hour or more, including but not limited to, for example, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours or more.

The proton donating system can be provided by the first composition of the present invention. For example, the proton donating system can be provided by the first composition of the present invention, which comprises an acidic polymer, such as, but not limited to, carboxypolymethylene. Wherein, the acidic polymer is partially neutralized, and the first composition has a pH of about 3 to about 8 or any range therein, such as, but not limited to, about 3 to about 6, about 3 to about 6, or about 5 to about 7. Without wishing to be bound by any particular theory, the partially neutralized acidic polymer can provide the first composition with a polymer gel matrix having a coiled structure. The coiled structure of the polymer gel matrix can donate and/or donate protons when the protons are in close proximity to the NO donor in the composition of the present invention, and the coiled structure can protect some protons (e.g., inner protons) from reacting with the NO donor in the composition. As the polymer gel matrix uncoils (e.g., as the pH of the composition becomes more basic and/or the polymer gel matrix becomes neutralized), additional protons (e.g., protected protons) can come into proximity with the NO donor, thereby allowing nitric oxide to be released from the composition over a longer period of time and/or increasing the amount of nitric oxide released from the composition.

In certain embodiments, the first composition of the present invention modulates the pH of the second composition such that when the first and second compositions are contacted and/or applied to the skin of a subject, the pH of the second composition and/or combined composition (i.e., the composition of the present invention) is less that about 11, in some embodiments less than about 10, in some embodiments less than about 8.5, in further embodiments less than about 7, and in still further embodiments between about 6 and about 8.

In some embodiments, the pH of the composite composition of the present invention changes when the composite composition is applied to the skin of a subject. In certain embodiments, the pH of the composite composition of the present invention changes when the composite composition is applied to the skin of a subject. In some embodiments, the pH of the composite composition after application of the composite composition of the present invention to the skin of a subject is lower than the pH of the second composition applied to the skin without the first composition. In embodiments where the release kinetics of the API in the second composition varies with pH, the buffering capacity of the skin can be utilized to modulate the release while improving the stability of the composite composition after combination and before application. That is, for example, the pH of the second composition containing the nitric oxide-releasing macromolecule can be greater than 10 before mixing, 9 after mixing, and 8 after application to the skin. With each decrease in pH, the release of nitric oxide from the macromolecule can increase. Thus, taking advantage of the varying pH and the buffering capacity of the skin can allow for increased working time (e.g., mixing and application time) of the composite composition of the present invention.

In some embodiments, the composition of the present invention may include a second composition, which may be an anhydrous composition. "Anhydrous" as used herein means that there is no direct addition of water to the second composition at any time during preparation. However, one of ordinary skill in the art will recognize that water may be physically and/or chemically absorbed by the second composition and/or by one or more ingredients in the second composition (i.e., indirect addition of water to the second composition) at any time during preparation, storage, and/or use of the second composition. In some embodiments, the term "anhydrous" means that the water content of the second composition is less than 5% by weight of the second composition, or any range and/or individual value therein. The water content of the second composition may be less than 5, 4.5, 4, 3.5, 3, 2.5, 2, 1.5, 1, or 0.5% by weight of the second composition, or any range therein. The water content may be measured by methods known to those of ordinary skill in the art, such as, but not limited to, Karl Fischer titration. In some embodiments, upon contact with a second composition, the composition of the present invention adds water to the second composition and/or the second composition absorbs water from the composition of the present invention.

Exemplary second compositions that can be used and/or placed in contact with the first composition of the present invention include, but are not limited to, those described in WO 2013/006608, which is incorporated herein by reference in its entirety. Exemplary second compositions that can be used and/or placed in contact with the first composition of the present invention to form a composition of the present invention (e.g., a composite composition of the present invention) can include an anhydrous composition comprising at least one thickening agent present in the second composition in an amount of about 0.5% to about 30% by weight of the second composition, at least one organic solvent present in the second composition in an amount of about 50% to about 90% by weight of the second composition, and at least one humectant present in the second composition in an amount of about 2% to about 20% by weight of the second composition. The second composition can include at least one water repelling agent, also known as a water repellent. In some embodiments, the second composition can include a composition set forth in Table 11.

Exemplary viscosity agents for use in the second composition include, but are not limited to, copolymers of carboxymethylcellulose and acrylic acid, N-vinylpyrrolidone, polyalkylene glycols (e.g., poly(ethylene glycol)), polyalkylene oxides (e.g., polyethylene oxide), polyvinyl alcohol, polyvinylpyrrolidone, polysiloxanes, poly(vinyl acetate), cellulose, derivatized cellulose, alginates, copolymers thereof, and blends thereof. Specific examples of viscosity agents for the second composition include hydroxypropylcellulose, such as Klucel® Hydroxypropylcellulose (e.g., Klucel® MF). The thickening agent may be present in the second composition in an amount of from about 0.1% to about 30% by weight of the second composition, or any range and/or individual value therein, such as, but not limited to, from about 0.5% to about 20%, from about 1% to about 10%, or from about 1% to about 5% by weight of the second composition. In some embodiments, the thickening agent can be present in the second composition in an amount of about 0.1%, 0.25%, 0.5%, 0.75%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, or 30% by weight of the second composition, or any range and/or individual value therein.

Exemplary organic solvents for use in the second composition include, but are not limited to, acetone, methyl alcohol, ethanol, isopropanol, butyl alcohol, ethyl acetate, dimethyl isosorbide, propylene glycol, glycerol, ethylene glycol, polyethylene glycol, diethylene glycol monoethyl ether, or mixtures thereof. In some embodiments of the present invention, the organic solvent in the second composition can be ethanol and/or isopropyl alcohol. The organic solvent can be present in the second composition in an amount of about 50% to about 90% by weight of the second composition or any range and/or individual value therein, such as, but not limited to, about 60% to about 90%, about 70% to about 90%, or about 75% to about 85% by weight of the second composition. In some embodiments, the organic solvent can be present in the second composition in an amount of about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, or 90% by weight of the second composition, or any range and/or individual value therein.

Exemplary humectants for use in the second composition include, but are not limited to, glycols, such as diethylene glycol monoethyl ether; glycerol; sugar polyols, such as sorbitol, xylitol and maltitol; polyols, such as polydextrose; Quillaja, urea, and blends thereof. In some embodiments, the humectant in the second composition can include alkylene glycols, such as hexylene glycol. The humectant can be present in the second composition in an amount of about 2% to about 20% by weight of the second composition or any range and/or individual value therein, such as, but not limited to, about 2% to about 15%, about 5% to about 15%, or about 15% to about 20% by weight of the second composition.

In some embodiments, the humectant may be present in the second composition in an amount of about 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% by weight of the second composition, or any range and/or individual value therein.

Exemplary water repellents for use in the second composition include, but are not limited to, silicones such as cyclomethicone, dimethicone, simethicone, C26-28 alkyl dimethicone, C26-28 alkyl methicone, polyphenylsesquioxane, trimethylsiloxysilicate, and cyclopentasiloxane and dimethicone/vinyl trimethylsiloxysilicate crosspolymers, and blends thereof. In some embodiments, the second composition can include cyclomethicone. The water repellent can be present in the second composition in an amount of about 0.5% to about 15% by weight of the second composition, or any range and/or individual value therein, such as, but not limited to, about 0.5% to about 10%, about 1% to about 5%, or about 2% to about 5% by weight of the second composition. In some embodiments, the water repellent may be present in the second composition in an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, or 15% by weight of the second composition, or any range and/or individual value therein.

Thus, the compositions of the present invention can include at least one polyhydric alcohol, a first thickening agent, at least one preservative, at least one buffering agent, water, a second thickening agent, at least one organic solvent, at least one humectant, and optionally a water repellent.

The compositions of the present invention (e.g., the first and second compositions of the present invention) may be buffered to a pH of about 3 to about 11, such as, but not limited to, about 3 to about 9.5, or about 3 to about 8. In some embodiments, the compositions of the present invention may have a pH of 9.5 or greater. In some embodiments, the compositions of the present invention include at least one API, such as, but not limited to, a nitric oxide-releasing active pharmaceutical ingredient. In some embodiments, the compositions of the present invention may include a second composition comprising a nitric oxide-releasing active pharmaceutical ingredient.

In some embodiments, the composition of the present invention comprises a second composition, wherein the second composition comprises a moisture-sensitive API. The second composition can stably store the moisture-sensitive API. In some embodiments, the moisture-sensitive API can comprise a NO-releasing API, such as, but not limited to, a diazeniumdiolate-modified macromolecule.

"Nitric oxide-releasing active pharmaceutical ingredient" and "NO-releasing API" as used herein refer to a compound or other composition that provides nitric oxide to the skin of a subject, but not gaseous nitric oxide. In some embodiments, the NO-releasing API includes a nitric oxide-releasing compound, hereinafter referred to as "NO-releasing compound". The NO-releasing compound includes at least one NO donor, which is a functional group that can release nitric oxide under certain conditions. In some embodiments, the at least one NO donor of the NO-releasing compound releases NO upon contact with the composition of the present invention. In some embodiments, the composition of the present invention modulates the amount of NO released from the NO-releasing compound and/or the rate of NO released from the NO-releasing compound. In some embodiments, the composition of the present invention increases the amount of NO released from the NO-releasing compound and/or the rate of NO released from the NO-releasing compound.

Any suitable NO-releasing compound can be used. In some embodiments, the NO-releasing compound includes a small molecule compound containing an NO-donating group. A "small molecule compound" as used herein is defined as a compound having a molecular weight of less than 500 Daltons, including organic and/or inorganic small molecule compounds. In some embodiments, the NO-releasing compound includes a macromolecule containing an NO-donating group. A "macromolecule" is defined herein as any compound having a molecular weight of 500 Daltons or greater. Any suitable macromolecule can be used, including crosslinked or uncrosslinked polymers, dendrimers, metal compounds, organometallic compounds, inorganic compounds, and other macromolecular scaffolds. In some embodiments, the macromolecule has a nominal diameter ranging from about 0.1 nm to about 100 μm, and may be composed of an aggregate of two or more macromolecules. This allows the macromolecular structure to be further modified with an NO-donating group.

In some embodiments, the NO-releasing compound comprises a diazeniumdiolate functional group as the NO donor. The diazeniumdiolate functional group can generate nitric oxide under certain conditions, for example, when exposed to water. As another example, in some embodiments, the NO-releasing compound comprises a nitrosothiol functional group as the NO donor. The NO donor can generate nitric oxide under certain conditions, for example, when exposed to light. Examples of other NO-donor groups include nitrosamines, hydroxylnitrosamines, hydroxylamines, and hydroxyureas. Any suitable combination of NO donors and/or NO-releasing compounds can also be used in the second composition described herein. In addition, the NO donor can be incorporated into or on a small molecule or macromolecule via covalent and/or non-covalent interactions.

The NO-releasing macromolecules may be in the form of NO-releasing particles, such as those described in U.S. Patent Application Publication No. 2009/0214618, the disclosure of which is incorporated herein by reference in its entirety. Other non-limiting examples of NO-releasing compounds include NO-releasing zeolites, as described in U.S. Patent Application Publication No. 2006/0269620 or 2010/0331968; NO-releasing metal-organic frameworks (MOFs), as described in U.S. Patent Application Publication No. 2010/0239512 or 2011/0052650; NO-releasing multi-donor compounds, as described in PCT/US2012/052350, entitled "Tunable Nitric Oxide-Releasing Macromolecules Having Multiple Nitric Oxide Donor Structures." No. 2009/0214618; NO-releasing dendrimer or metallostructures; nitric oxide-releasing coatings; and compounds described in U.S. Patent Publication No. 2010/0098733. The disclosure of each of the references described in this paragraph is hereby incorporated by reference in its entirety. Additionally, NO-releasing macromolecules can be made as described in PCT/US2012/022048, filed Jan. 20, 2012, entitled "Temperature Controlled Sol-Gel Co-Condensation," the disclosure of which is hereby incorporated by reference in its entirety.

By way of example, in some embodiments of the present invention, the nitric oxide releasing active pharmaceutical ingredient can include NO loaded precipitated silica. The NO loaded precipitated silica can be in the form of a co-condensed siloxane network from silane monomers modified with a nitric oxide donor. In one embodiment of the present invention, the nitric oxide donor can be an N-diazeniumdiolate. In some embodiments of the present invention, the nitric oxide releasing active pharmaceutical ingredient can comprise, consist essentially of, or consist of a co-condensed siloxane network containing a diazeniumdiolate (e.g., an N-diazeniumdiolate).

In some embodiments, the nitric oxide donor is administered by a pre-charging method. The co-condensed siloxane network can be formed from aminoalkoxysilanes by the pre-charging method, and the co-condensed siloxane network can be synthesized by condensing a silane mixture containing an alkoxysilane and an aminoalkoxysilane to form a co-condensed siloxane network modified with a nitric oxide donor. As used herein, the "pre-charging method" means that the aminoalkoxysilane is "pre-treated" or "pre-charged" with nitric oxide prior to co-condensation with the alkoxysilane. In some embodiments, the pre-charging of nitric oxide can be accomplished by chemical methods. In another embodiment, the "pre-charging" method may be used to create co-condensed siloxane networks and materials that are more densely functionalized with NO donors. In some embodiments of the present invention, the nitric oxide-releasing active pharmaceutical ingredient can comprise, consist essentially of, or consist of a co-condensed silica network synthesized by condensing a silane mixture containing an alkoxysilane and at least one aminoalkoxysilane that is amine-substituted with a diazeniumdiolate (e.g., N-diazeniumdiolate).

The co-condensed siloxane network may be a uniformly sized silica particle, a collection of silica particles of various sizes, amorphous silica, fumed silica, nanocrystalline silica, ceramic silica, colloidal silica, a silica coating, a silica film, organically modified silica, mesoporous silica, silica gel, bioactive glass, or silica in any suitable form or state.

In some embodiments, the alkoxysilane is a tetraalkoxysilane having the formula Si(OR)4, where R is an alkyl group. The R groups can be the same or different. In some embodiments, the tetraalkoxysilane is selected as tetramethylorthosilicate (TMOS) or tetraethylorthosilicate (TEOS). In some embodiments, the aminoalkoxysilane has the formula R"-(NH-R')n-Si(OR)3, where R is an alkyl group, R' is alkylene, branched alkylene, or aralkylene, n is 1 or 2, and R" is selected from the group consisting of alkyl, cycloalkyl, aryl, and alkylamine.

In some embodiments, the aminoalkoxysilane can be selected from N-(6-aminohexyl)aminopropyltrimethoxysilane (AHAP3), N-(2-aminoethyl)-3-aminopropyltrimethoxysilane (AEAP3), (3-trimethoxysilylpropyl)diethylenetriamine (DET3), (aminoethylaminomethyl)phenethyltrimethoxysilane (AEMP3), [3-(methylamino)propyl]trimethoxysilane (MAP3), N-butylaminopropyltrimethoxysilane (n-BAP3), t-butylaminopropyltrimethoxysilane (t-BAP3), N-ethylaminoisobutyltrimethoxysilane (EAiB3), N-phenylaminopropyltrimethoxysilane (PAP3), and N-cyclohexylaminopropyltrimethoxysilane (cHAP3).

In some embodiments, the aminoalkoxysilane has the formula: NH[R'-Si(OR)], where R is alkyl and R' is alkylene. In some embodiments, the aminoalkoxysilane can be selected from bis(3-triethoxysilylpropyl)amine, bis-[3-(trimethoxysilyl)propyl]amine, and bis-[(3-trimethoxysilyl)propyl]ethylenediamine.

In some embodiments, the aminoalkoxysilane is precharged for NO release and the amino group is substituted with a diazeniumdiolate, as described herein above. Thus, in some embodiments, the aminoalkoxysilane has the formula: R-N(NONO-X+)-R-Si(OR)3, where R is alkyl, R is alkylene or aralkylene, R is alkyl or alkylamine, and X+ is a cation selected from the group consisting of Na+, K+, and Li+.

The composition of the siloxane network (e.g., amount or chemical composition of aminoalkoxysilane) and nitric oxide charging conditions (e.g., solvent and base) can be varied to optimize the amount and duration of nitric oxide release. Thus, in some embodiments, the half-life of NO release from silica particles can be adjusted by modifying the composition of the silica particles.

In another embodiment, the amino group of the aminoalkoxysilane is substituted with a diazeniumdiolate, where the aminoalkoxysilane has the formula: RN(NONO-X+)RSi(OR)3, where R is alkyl, R is alkylene or aralkylene, R is alkyl or alkylamine, and X+ is a cation selected from the group consisting of Na+ and K+.

In some embodiments, the NO-releasing API can comprise a co-condensed silica network comprising diazeniumdiolated aminoethylaminopropyltrimethoxysilane (AEAP3) and tetramethylorthosilicate (TMOS), and/or a co-condensed silica network comprising diazeniumdiolated aminoethylaminopropyltrimethoxysilane (AEAP3) and tetraethylorthosilicate (TEOS). In some embodiments, the NO-releasing API can comprise a co-condensed silica network comprising diazeniumdiolated methylaminopropyltrimethoxysilane (MAP3) and tetramethylorthosilicate (TMOS), and/or a co-condensed silica network comprising diazeniumdiolated methylaminopropyltrimethoxysilane (MAP3) and tetraethylorthosilicate (TEOS).

In some embodiments of the present invention, the particle size of the NO-releasing API may be in the range of about 20 nm to about 20 μm or any range therein, such as, but not limited to, about 100 nm to about 20 μm or about 1 μm to about 20 μm. The particle size can be adjusted to minimize or prevent toxicity and/or to penetrate through the epidermis (or damaged dermis) and into the blood vessels. In certain embodiments, the particle size is distributed around an average particle size of less than 20 μm or any range therein, which allows the particle to enter the hair follicle. In some embodiments, the NO-releasing API can have a particle size distributed around an average particle size of about 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 μm. In further embodiments, the NO-releasing API can have a particle size distribution around an average particle size of less than 10 μm or any range therein, such as, but not limited to, about 2 μm to about 10 μm or about 4 μm to about 8 μm. In other embodiments, the particle size distribution can be around an average particle size of more than 20 μm or any range therein, which can prevent the particles from entering the hair follicle. In still further embodiments, a mixture of particles with average particle sizes distribution around two or more average particle sizes can be provided. The NO-releasing API can be micronized (e.g., ball milled and/or jet milled). Methods for achieving the desired particle size and/or micronization include, but are not limited to, those described in U.S. Patent Application Publication No. 2013/0310533, which is incorporated herein by reference in its entirety.

The nitric oxide-releasing active pharmaceutical ingredient may be present in the compositions of the present invention in an amount of about 0.5% to about 10%, for example, but not limited to, about 1% to about 8%, or about 2% to about 6% by weight of the composition. In some embodiments, the nitric oxide-releasing active pharmaceutical ingredient may be present in the compositions of the present invention in an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The compositions of the present invention may include a nitric oxide-releasing active pharmaceutical composition, which may store and/or release nitric oxide in an amount of about 0.05% to about 3%, for example, but not limited to, about 0.15% to about 2%, about 0.15% to about 1%, about 0.3% to about 1.2% by weight of the composition. In some embodiments, the compositions of the present invention include a nitric oxide-releasing active pharmaceutical composition, which may store and/or release nitric oxide in an amount of about 0.05% to about 3%, for example, but not limited to, about 0.15% to about 2%, about 0.15% to about 1%, about 0.3% to about 1.2% by weight of the composition. and may store and/or release nitric oxide in amounts of about 0.15%, 0.3%, 0.6%, 0.9%, 1%, 1.2%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75%, or 3%.

In some embodiments, the first composition of the present invention can increase the amount of NO released from a composition (e.g., a second composition comprising a NO-releasing API) relative to the amount of NO released from the composition over the same time period in the absence of the first composition of the present invention. In some embodiments, the first composition of the present invention can increase the amount of NO released from a composition of the present invention comprising the first composition and a NO-releasing API by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or more, or any range and/or individual value therein, relative to the amount of NO released over the same time period in the absence of the first composition of the present invention. Thus, a composition of the invention (e.g., a composition comprising a first composition of the invention and a second composition comprising a NO-releasing API) can release from about 1.5 to about 100 times more NO than the amount of NO released over the same period of time in the absence of the first composition of the invention (e.g., the second composition alone), or any range and/or individual value therein, such as, but not limited to, from about 2 to 10 times more NO, or from about 5 to about 50 times more NO.

For example, the effect that the composition of the present invention may have on NO release can be seen in Figures 7 and 8. Figure 7 shows the in vitro nitric oxide release profile over time for 2% Nitricil NVN1 gel with the formulation as described in Table 1. Figure 8 shows the in vitro nitric oxide release profile over time for 2% Nitricil NVN1 gel and 6% and 12% Nitricil NVN1 gels with the formulation as described in Table 1 when mixed with the first composition of the present invention at pH 4 with the formulation as described in Table 2 in a ratio of 1:3 (gel:first composition). As can be seen from Figures 7 and 8, the cumulative NO release for 2% Nitricil NVN1 gel was increased by contact with the first composition of the present invention.

<tables><img file="JP7507204B2_D0001.tif" /></tables>

<tables><img file="JP7507204B2_D0002.tif" /></tables>

The composition of the present invention may comprise a first composition and a second composition of the present invention as described herein. As will be recognized by those skilled in the art, the amount or concentration of each component in the composition of the present invention may vary depending on the amount of the first composition and the second composition present in the composition (e.g., the ratio of the first composition to the second composition present in the composition). In some embodiments, the ratio of the first composition to the second composition of the present invention in the composition of the present invention may be about 5:1 or less, in further embodiments about 4:1 or less, about 3:1 or less, about 2:1 or less, about 1:1 or less, about 0.5:1 or less, or about 0.2:1 or less. In certain embodiments, the ratio may be about 3:1. In further embodiments, the ratio may be about 1:1.

In some embodiments, the compositions of the present invention can comprise, consist essentially of, or consist of a polyhydric alcohol in an amount from about 1% to about 10% by weight of the composition, a first thickening agent in an amount from about 0.01% to about 3% by weight of the composition, water in an amount from about 30% to about 50% by weight of the composition, a second thickening agent in an amount from about 0.01% to about 10% by weight of the composition, an organic solvent in an amount from about 30% to about 50% by weight of the composition, a humectant present in the second composition in an amount from about 2% to about 10% by weight of the composition, a water repellent agent in an amount from about 0.1% to about 10% by weight of the composition, a NO-releasing API in an amount from about 0.5% to about 10% by weight of the composition, optionally a buffering agent in an amount from about 0.001% to about 1% by weight of the composition, optionally a preservative in an amount from about 0.001% to about 1% by weight of the composition, and optionally a neutralizing agent. The neutralizing agent can be present in an amount sufficient to provide a pH of about 3 to about 8 to the first portion of the composition. The composition can have a pH of less than about 11, such as, but not limited to, less than about 9.5, less than about 7, or less than about 6. The first and second thickening agents can be the same and/or different. In some embodiments, the first thickening agent can be a carboxypolymethylene and the second thickening agent can be a cellulose, such as, but not limited to, hydroxypropylcellulose. In some embodiments, the composition can be cosmetically elegant.

In some embodiments, the compositions of the present invention can comprise, consist essentially of, or consist of a polyhydric alcohol in an amount from about 2% to about 7% by weight of the composition, a first thickening agent in an amount from about 0.1% to about 1% by weight of the composition, water in an amount from about 40% to about 45% by weight of the composition, a second thickening agent in an amount from about 0.1% to about 1% by weight of the composition, an organic solvent in an amount from about 35% to about 45% by weight of the composition, a humectant present in the second composition in an amount from about 2% to about 7% by weight of the composition, a water repellent in an amount from about 0.1% to about 5% by weight of the composition, a NO-releasing API in an amount from about 0.5% to about 10% by weight of the composition, optionally a buffering agent in an amount from about 0.01% to about 0.2% by weight of the composition, optionally a preservative in an amount from about 0.01% to about 0.3% by weight of the composition, and optionally a neutralizing agent. The neutralizing agent can be present in an amount sufficient to provide a pH of about 4 or about 6 to the first portion of the composition. The composition can have a pH of less than about 11, such as, but not limited to, less than about 9.5, less than about 7, or less than about 6. The first and second thickening agents can be the same and/or different. In some embodiments, the first thickening agent can be a carboxypolymethylene and the second thickening agent can be a cellulose, such as, but not limited to, hydroxypropylcellulose. In some embodiments, the composition can be cosmetically elegant. In some embodiments, the composition can include a composition as set forth in Table 13.

The composition of the present invention may include at least two different thickening agents. One thickening agent may be present in a first portion of the composition of the present invention, and the other thickening agent may be present in a second portion of the composition. In some embodiments, the composition of the present invention includes carboxypolymethylene and cellulose, such as, but not limited to, hydroxypropylcellulose. Carboxypolymethylene may be present in a first composition of the present invention, and cellulose may be present in a second composition, which may be combined to form the composition of the present invention. The composition of the present invention including at least two different thickening agents may be a cosmetically elegant composition including API, such as, but not limited to, particulate API and/or insoluble API (e.g., aqueous and/or moisture insoluble API, such as benzoyl peroxide).

The composition of the present invention can provide a structure suitable for suspending an API, such as, but not limited to, a particulate API and/or an insoluble API. In some embodiments, the composition of the present invention can encapsulate an API, such as, but not limited to, a particulate API and/or an insoluble API. For example, the composition of the present invention can encapsulate an API in at least one thickening agent and/or provide a structure suitable for encapsulating an API. The composition of the present invention can prevent and/or reduce agglomeration of the API in the composition. Without wishing to be bound by any particular theory, the first composition of the present invention can provide a means for suspending and/or encapsulating an API and/or a means for preventing and/or reducing agglomeration of the API in the composition.

For example, the first composition of the present invention may provide a structure (e.g., a gel matrix) suitable for suspending and/or encapsulating an API in the composition of the present invention and/or suitable for preventing and/or reducing aggregation of the API in the composition of the present invention.

The composition of the present invention can include an API, carboxypolymethylene, and cellulose, and can be a cosmetically elegant composition. The composition can be non-gritty and/or have reduced grittiness compared to the API in the absence of the composition of the present invention. The composition can be non-sticky (i.e., viscous) and/or have reduced greasiness (i.e., stickiness) compared to the API in the absence of the composition of the present invention. The composition can have reduced and/or increased stiffness (i.e., hardness) and/or have increased homogeneity compared to the API in the absence of the composition of the present invention. In some embodiments, the composition of the present invention can include an API and can be a cosmetically elegant homogeneous composition. Without wishing to be bound by any particular theory, the first composition of the present invention can provide a means for making a cosmetically elegant homogeneous composition. For example, the first composition of the present invention can provide a structure (e.g., a gel matrix) suitable for making a cosmetically elegant homogeneous composition.

The first part and/or first composition of the composition of the invention and the second part and/or second composition of the composition of the invention can be contacted (e.g., mixed, stirred, blended, homogenized, etc.) during the process of formulating the composition of the invention. The composition of the invention can then be stored with the first part and/or first composition and the second part and/or second composition combined (e.g., in the same vessel and/or container).

The compositions of the present invention can include APIs that are difficult to formulate, such as, but not limited to, APIs that are difficult to formulate as topical compositions. For example, the API can be a particulate API, an insoluble API (e.g., a water and/or moisture insoluble API), a thermally unstable API, and/or a light sensitive API.

According to an embodiment of the present invention, the composition of the present invention can provide an improved composition by providing two separate parts. The composition of the present invention with at least two parts can provide an API (e.g., a difficult to formulate API) in one part and later combine with the second part to prepare a cosmetically elegant composition and/or can provide a composition with a more stable API compared to the same API prepared with one part of the composition of the present invention and/or in the form of a composition formed in the absence of the composition of the present invention. The API can have a greater activity compared to the activity in the absence of the composition of the present invention and therefore can be more stable and/or the API can be stored for a longer period of time compared to when stored in the absence of the composition of the present invention. In some embodiments, the composition of the present invention including an API (e.g., a difficult to formulate API) can be a more cosmetically elegant composition compared to the API in the composition in the absence of the composition of the present invention. For example, the composition of the present invention including a particulate API and/or an insoluble API can be a less gritty composition and therefore a more cosmetically elegant composition.

According to an embodiment of the present invention, a kit can be provided. In some embodiments, the kit of the present invention can include a first composition and a second composition of the present invention described herein. The first composition can be a hydrogel. The first composition can include at least one polyhydric alcohol present in an amount of about 1% to about 30% by weight of the first composition, at least one thickening agent present in an amount of about 0.1% to about 5% by weight of the first composition, and water present in an amount of about 70% to about 99% by weight of the first composition. The second composition can include an API, such as, but not limited to, a NO-releasing API. In some embodiments, the second composition can include at least one thickening agent present in the second composition in an amount of about 0.5% to about 30% by weight of the second composition, at least one organic solvent present in the second composition in an amount of about 50% to about 90% by weight of the second composition, and at least one moisturizing agent present in the second composition in an amount of about 2% to about 20% by weight of the second composition. In certain embodiments, the second composition may comprise an anhydrous alcohol gel containing nitric oxide-releasing polysiloxane macromolecules as described in International Application Publication No. WO 2013/006608.

In some embodiments, the kits of the invention comprise an aqueous composition, an organic composition, and an API that may be stable and/or soluble in the aqueous composition and/or the organic composition. The kits of the invention may be configured to mix the two compositions at the time of dispensing and/or upon application to a subject, and/or to provide a combination composition in which the performance and/or activity of the API is enhanced compared to the performance and/or activity of the API in the absence of one of the compositions and/or the combination composition.

The kit of the present invention can store the first composition of the present invention and the second composition separately.In some embodiments, the kit of the present invention can contact the first composition and the second composition, for example, but not limited to, by mixing the compositions before applying to a subject.

The first composition of the invention (e.g., a hydrogel of the invention) and the second composition can be mixed together and then applied to the skin of a subject. In other embodiments, the second composition can be applied to the skin of a subject, and then the first composition can be applied over the second composition. Or vice versa. In some embodiments, the ratio of the first composition to the second composition that can be applied to a subject can be about 5:1 or less, in further embodiments about 4:1 or less, about 3:1 or less, about 2:1 or less, or about 1:1. In certain embodiments, the ratio can be about 3:1. In further embodiments, the ratio can be about 1:1.

By providing a first composition and a second composition that are combined at the time of application and/or during application to the skin of a subject, the kit of the present invention can have a longer shelf life than if the compositions were stored in the kit in a mixed state. For example, the API can be formulated and carried in the form of the second composition to provide a stable product with a long shelf life. Thus, for example, the pH and water content of the second composition can be adjusted to reduce or minimize the release of the water-activated API, resulting in a composition that is stable at room temperature. The first composition can then be combined with the second composition to adjust the pH of the combination and provide water to activate the API. The second composition can be combined with the first composition in various ratios to achieve the desired release, pH, and/or dosage for the combined composition. Such an approach can allow a single manufacturing process to be utilized for the production of the more complex and costly second composition, followed by the production of a specific product defined by the composition and/or amount of the first composition with which the second composition is mixed.

As will be appreciated by those of skill in the art in light of the present disclosure, the first composition of the present invention (e.g., the hydrogel of the present invention) can provide a means for adjusting the pH of a pharmaceutical composition as well as a means for activating an API of the pharmaceutical composition. In certain embodiments, the first composition of the present invention can provide a means for reducing the pH of an anhydrous pharmaceutical composition comprising a diazeniumdiolate-modified co-condensed polysiloxane macromolecule. In further embodiments, the first composition of the present invention can provide a means for releasing nitric oxide from an anhydrous pharmaceutical composition comprising a diazeniumdiolate-modified co-condensed polysiloxane macromolecule.

According to some embodiments, the method of the present invention can include administering the composition of the present invention (e.g., the first composition and the second composition of the present invention described herein) and/or the first composition of the present invention (e.g., the hydrogel of the present invention) to the skin of a subject. In some embodiments, the composition of the present invention and/or the first composition can be administered topically. The composition can include at least one API, such as but not limited to, a nitric oxide-releasing active pharmaceutical ingredient; a second thickening agent; at least one organic solvent; at least one moisturizer; at least one polyhydric alcohol; a first thickening agent; at least one preservative, and water. The method of the present invention can include forming an admixture before and/or during administration of the composition of the present invention. The admixture can be prepared by mixing or combining a composition including at least one API, such as but not limited to, a nitric oxide-releasing active pharmaceutical ingredient; a second thickening agent; at least one organic solvent, and at least one moisturizer, with a composition including at least one polyhydric alcohol; a first thickening agent; at least one preservative, and water.

The method of the invention can include topically applying a first composition of the invention to the skin of a subject in combination and/or admixture with a second composition, which can include a nitric oxide-releasing active pharmaceutical ingredient.

In some embodiments, the method of the present invention includes delivering a therapeutically effective amount of the composition of the present invention to the skin of a subject. As used herein, the term "therapeutically effective amount" refers to an amount of the composition of the present invention that elicits a therapeutically useful response in a subject. Those skilled in the art will understand that the therapeutic effect does not have to be complete and curative, as long as some benefit is provided to the subject. In some embodiments, a therapeutically effective amount of the composition of the present invention can include delivering a therapeutically effective amount of the components of the composition, such as, but not limited to, an active pharmaceutical ingredient (e.g., a nitric oxide-releasing API). Thus, a therapeutically effective amount of nitric oxide can be delivered and/or administered by the composition of the present invention.

The present invention has applications in both veterinary and medical applications. Suitable subjects to be treated using embodiments of the method of the present invention include, but are not limited to, avian and mammalian subjects. Mammals of the present invention include, but are not limited to, canines, felines, cows, goats, equines, sheep, pigs, rodents (e.g., rats and mice), rabbits, primates (e.g., apes and humans), non-human primates (e.g., monkeys, baboons, chimpanzees, gorillas), and the like, as well as mammals in utero. Any mammalian subject in need of treatment by the present invention is suitable. Human subjects of both sexes and at any stage of development (i.e., neonate, infant, juvenile, adolescent, adult) may be treated by the present invention. In some embodiments of the present invention, the subject is a mammal, and in some embodiments, the subject is human. Human subjects include both males and females of all ages, including fetal, neonatal, infant, juvenile, adolescent, adult, and geriatric subjects, as well as pregnant subjects. In certain embodiments of the invention, the subject is an adolescent and/or adult human.

Illustrative birds according to the present invention include chickens, ducks, turkeys, geese, quail, pheasants, ratites (e.g., ostriches) and captive birds (e.g., parrots and canaries), as well as birds in ovo.

The methods of the invention may also be carried out on animal subjects, especially mammalian subjects such as mice, rats, dogs, cats, livestock and horses, for veterinary purposes and/or for drug screening and development purposes.

In certain embodiments of the invention, a subject is "in need of" a method of the invention, e.g., the subject has been diagnosed with and/or is at risk for and/or is believed to have a disease or disorder that can be treated using a method of the invention. In some embodiments, the subject has a skin disorder, including but not limited to acne, androgenetic alopecia, atopic dermatitis, seborrheic dermatitis, tinea infection, candida infection, bacterial infection, common warts, and/or psoriasis. In some embodiments of the invention, the subject has an inflammatory skin condition or disorder and/or infection (e.g., impetigo, leishmaniasis, etc.). In some embodiments, the compositions of the invention can be used to treat acne vulgaris. According to some embodiments, the compositions and/or methods of the invention can reduce Propionibacterium acnes counts, inflammatory lesions, and/or non-inflammatory lesions in a subject. In some embodiments, the compositions and/or methods of the present invention can be used to treat any disease, disorder, and/or condition suitable for topical administration of a composition comprising a NO-releasing API and an alcohol additive, including, but not limited to, alopecia (e.g., androgenic alopecia) and/or warts (e.g., common warts, plantar warts, flat warts, filiform warts, genital warts, mosaic warts, and/or periungual warts).

"Treating," "treating," "treatment of" (and grammatical variations thereof), as used herein, refers to any type of treatment that confers a benefit to a subject and can mean that the severity of the subject's condition is reduced, at least partially ameliorated, or improved, and/or some alleviation, reduction, or decrease of at least one clinical symptom is achieved, and/or there is a delay in the progression of the disease, disorder, and/or condition. In certain embodiments, the severity of a skin disorder (e.g., acne) can be reduced in a subject relative to the severity of the skin disorder in the absence of the method of the invention. In other embodiments, the method of the invention can prevent and/or treat an infectious disease.

The compositions of the present invention can be applied topically to any area of the skin of a subject. However, in some embodiments, the face of a subject is treated with the methods described herein. Additionally, in some embodiments, the torso of a subject is treated with the methods described herein. In some embodiments, the hand(s), finger(s), foot(s), toe(s) and/or genitalia(s) of a subject is treated with the methods described herein.

According to some embodiments of the present invention, a method of treating acne vulgaris may be provided, comprising topically applying a composition of the present invention to the skin of a subject. A therapeutically effective amount of the composition may be applied. In some embodiments, the composition may include a nitric oxide releasing active pharmaceutical ingredient in an amount of about 0.5% to about 10%, for example, but not limited to, about 1% to about 8%, or about 2% to about 6% by weight of the composition. In some embodiments, the composition may include a nitric oxide releasing active pharmaceutical ingredient in an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The composition may store and/or release nitric oxide in an amount of about 0.05% to about 3%, for example, but not limited to, about 0.15% to about 2%, about 0.15% to about 1%, about 0.3% to about 1.2% by weight of the composition. In some embodiments, the compositions are capable of storing and/or releasing nitric oxide in amounts of about 0.15%, 0.3%, 0.6%, 0.9%, 1%, 1.2%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75% or 3%.

According to further embodiments of the present invention, a method of reducing inflammatory and/or non-inflammatory lesions in a subject may be provided, comprising topically applying a composition of the present invention to the skin of the subject. A therapeutically effective amount of the composition may be applied. In some embodiments, the composition may include a nitric oxide-releasing active pharmaceutical ingredient in an amount of about 0.5% to about 10%, for example, but not limited to, about 1% to about 8%, or about 2% to about 6% by weight of the composition. In some embodiments, the composition may include a nitric oxide-releasing active pharmaceutical ingredient in an amount of about 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. The composition may store and/or release nitric oxide in an amount of about 0.05% to about 3%, for example, but not limited to, about 0.15% to about 2%, about 0.15% to about 1%, or about 0.3% to about 1.2% by weight of the composition. In some embodiments, the compositions are capable of storing and/or releasing nitric oxide in amounts of about 0.15%, 0.3%, 0.6%, 0.9%, 1%, 1.2%, 1.5%, 1.75%, 2%, 2.25%, 2.5%, 2.75% or 3%.

The method of reducing inflammatory and/or non-inflammatory lesions can include topically applying a combination composition of the present invention. In some embodiments, the method can reduce inflammatory and/or non-inflammatory lesions by about 10% or more, for example, but not limited to, about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, over a given period of time, compared to a subject who has not applied a composition of the present invention over the same period of time. In some embodiments, the method can reduce inflammatory and/or non-inflammatory lesions in a subject, compared to a subject who has not applied a composition comprising a nitric oxide-releasing active pharmaceutical ingredient over the same period of time.

In some embodiments, a method of reducing inflammatory and/or non-inflammatory lesions in a subject may be provided, comprising topically applying a first composition of the present invention to the skin of the subject. The first composition may not include an API, such as, but not limited to, a NO-releasing API. A therapeutically effective amount of the first composition may be applied. The method of reducing inflammatory and/or non-inflammatory lesions comprising topically applying a first composition of the present invention may reduce inflammatory and/or non-inflammatory lesions by about 10% or more, such as, but not limited to, about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, over a given period of time, compared to a subject who has not applied the first composition of the present invention over the same period of time.

In some embodiments, the compositions of the invention can include topically applying a composition of the invention comprising a nitric oxide-releasing active pharmaceutical ingredient, which can reduce inflammatory and/or non-inflammatory lesions by about 10% or more, for example and without limitation, about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, over a defined period of time, relative to a subject receiving a substantially identical composition not comprising a nitric oxide-releasing active pharmaceutical ingredient.

In some embodiments, a subject can reduce inflammatory and/or non-inflammatory lesions within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, or more. In some embodiments, the method can reduce inflammatory and/or non-inflammatory lesions in a subject's skin within 12 weeks or less, in some embodiments within 8 weeks or less, and in further embodiments within 4 weeks or less.

In some embodiments, the method of the present invention can reduce the P. acnes count in a subject who is administered and/or topically applies the composition of the present invention. In some embodiments, the method of the present invention can reduce the P. acnes count by about 10% or more, for example, but not limited to, about 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or more, over a given period of time, compared to a subject who has not applied the composition of the present invention over the same length of time. In some embodiments, the method can reduce the P. acnes count in a subject, compared to a subject who has not applied the composition comprising a nitric oxide-releasing active pharmaceutical ingredient over the same period of time.

In some embodiments, reduction in P. acnes counts can occur within 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 7 weeks, 8 weeks, 9 weeks, 10 weeks, 11 weeks, 12 weeks, 13 weeks, or more. In some embodiments, the methods of the present invention can reduce P. acnes counts on the skin of a subject within 12 weeks or less, in some embodiments within 8 weeks or less, and in further embodiments within 4 weeks or less.

<p>The invention is described in more detail in the following non-limiting experimental examples.</p><p>[Experimental Example 1]</p><p>Unbuffered hydrogel formulations were developed with pH values ranging from 3 to 7. Several pH 6 hydrogel formulations were prepared with varying levels of the carbomer Carbopol® 974P to investigate the effect on consistency and gel rheology. The effect of preservatives such as sorbic acid, benzoic, and parabens on the rheology and consistency of the unbuffered hydrogels was also investigated. The prepared hydrogel formulations were used to determine the pH value of blends with Nitricil NVN1 topical gel formulations containing isopropyl alcohol (IPA) and varying concentrations of Nitricil NVN1 (Example 2) and to determine the in vitro nitric oxide (NO) release kinetics (Example 3). Hydrogels using Carpobol® 980P, buffered at pH 4 with 0.1% w/w citric acid, and Carbopol® ETD 2020NF polymer, buffered at pH 6 with 0.2% w/w 0.1 M phosphate buffer, were also formulated.</p><p>All formulations shown in Tables 3 and 4 were mixed with United States Pharmacopeia (USP) grade water and anhydrous glycerol in either a 0.5 L or 2 L glass beaker at ambient temperature using an IKA overhead mixer. For hydrogel formulations containing preservatives such as sorbic acid, benzoic acid, and methyl and propyl paraben, the preservatives were added to the glycerol-water solution and heated to 70°C using a hot plate to ensure complete dissolution. Once dissolved, the solution was cooled to ambient temperature. The next step in each experiment was to mix the hydrogels in an IKA The Carbopol® polymer was slowly transferred to the beaker while constantly stirring using a combination of overhead stirring and homogenization using a T-18 mixer at speed 3-4 for 20-30 seconds. A clear solution formed after 20 minutes indicating the polymer was completely dissolved. With continued stirring, the pH of the unneutralized mixture was first measured and then the pH was adjusted to the desired value using trolamine as a neutralizing agent in a quantity sufficient (QS) to increase the viscosity of the hydrogel. Finally, once the desired pH was obtained, a final amount of water was added to reach the desired target batch size. For batch lot 112331, titanium dioxide was introduced into the hydrogel as a masking agent.</p><p><tables><img file="JP7507204B2_D0003.tif" /></tables></p><p><tables><img file="JP7507204B2_D0004.tif" /></tables></p><p>The pH of unneutralized mixtures containing the anionic Carbopol® polymer 974P was approximately 2.75-3, with or without preservatives, depending on the polymer concentration. It was found that the viscosity of the pH 3 hydrogel was very low when the pH was adjusted to 3 units by adding only a small amount of trolamine, and thus no thickening effect was observed. A pH adjustment to 6 or 7 with trolamine was necessary to completely neutralize the Carbopol® 974P polymer. However, the viscosity of the resulting unbuffered hydrogel was very high at pH 6 and 7. Without wishing to be bound by any particular theory, it may be necessary to reduce the concentration of Carbopol® 974P polymer from 1% w/w to 0.3-0.5% w/w to reduce the viscosity at pH 6 and 7 for dispensing purposes. Without wishing to be bound by any particular theory, use of Carbopol® 974P polymer at concentrations less than 0.3% w/w may result in a hydrogel that is not viscous enough and may run off the surface of the skin upon application.</p><p>An unbuffered pH 4 hydrogel, when formulated with 0.5% w/w Carbopol®, can have a consistency and rheological properties that are suitable for flowing and dispensing from a pump, as well as not running off the surface of the skin upon application. Table 5 shows viscosity measurements for several unbuffered pH 4 and pH 5 hydrogels containing the preservative(s) listed in Table 4.</p><p><tables><img file="JP7507204B2_D0005.tif" /></tables></p><p>The viscosity of the preservative-containing hydrogels at pH 4 ranged from 7000 to 12500 cP. The addition of preservatives affected the pH and therefore the neutralization (thickening) process with trolamine. Without wishing to be bound by any particular theory, it may be necessary to adjust the Carbopol® polymer level to account for the presence of preservatives and the various concentrations of preservatives, so that a consistent viscosity is obtained after neutralization with base. Increasing the pH of the preservative-containing unbuffered hydrogels to 5 significantly increased the viscosity to approximately 20000 cP.</p><p>Several buffering acids, such as citric, tartaric and lactic acids, were used with Carbopol® 974P polymer to buffer at pH 4, but the hydrogel immediately degraded into water. Carbopol® ETD2020, NF polymer, was found to be unsuitable for use with buffering agents at concentrations of 1-3% w/w.</p><p>A phosphate buffered pH 6 hydrogel was prepared using 0.2% w/w Carbopol® ETD2020 NF polymer in USP water + anhydrous glycerol. A 0.1 M stock of potassium phosphate buffer was added at 0.2% w/w to buffer the hydrogel at 6 pH units. This hydrogel was used with Nitricil NVN1 topical gel containing IPA and various concentrations of Nitricil NVN1 to determine the pH of the in vitro blend (Example 2). The phosphate buffered pH 6 hydrogel had the effect of reducing the pH by 0.5 units at several different Nitricil NVN1 concentrations ranging from 0.2% w/w to 8% w/w.</p><p>A citric acid buffered pH 4 hydrogel containing 0.1% w/w benzoic acid and 0.1% sorbic acid with 1% w/w Carbopol® polymer 980P was successfully formulated at 0.5 kg scale. The composition of the buffered citric acid hydrogel is listed in Table 6. The buffered hydrogel (batch lot: 126335) was measured to have a viscosity of 7285 cP. The prepared citric acid buffered hydrogel was used to determine the pH in vitro and at the skin surface (Example 2), including confirmation of the in vitro nitric oxide release profile (Example 3).</p><p><tables><img file="JP7507204B2_D0006.tif" /></tables></p><p>Hydrogel formulations were prepared covering a range of pH values. The initial hydrogels prepared were unbuffered and formulated with and without preservatives. Preservatives such as benzoic acid, sorbic acid and parabens were used. Parabens were found to react with Nitricil NVN1 IPA topical gel. The pH of the hydrogels was adjusted by varying the amount of trolamine (neutralizer) added. Increasing amounts of neutralizer were added to increase the pH and viscosity. At pH 6 and 7, the viscosity of the unbuffered hydrogels formed without preservatives was high. The Carbopol® polymer concentration was reduced to reduce the viscosity of the hydrogel. The addition of preservatives also affected the initial pH. The amount of polymer and neutralizer were adjusted to reach the desired pH and was not so low as to cause issues with running off the surface of the skin and to allow for the flowability of the product and the dual chamber dispensing system. The goal was to achieve a viscosity that was not so high as to cause problems with pumping from the viscoelastic device. Attempts were made to make a buffered pH 4 hydrogel using Carpobol® 974P and ETD2020 polymers with the buffers citric, lactic, and tartaric acid, but in this case the sudden change in pH resulted in the polymer breaking down into water.</p><p>A pH 4 hydrogel buffered with 0.1% w/w citric acid was successfully formulated using sorbic and benzoic acids by substituting Carbopol® 980P, a homopolymer type C and longer chain polymer. A pH 6 hydrogel buffered with 0.2% w/w 0.1 M phosphate buffer was also successfully formulated using Carbopol® ETD2020 polymer.</p><p>[Experimental Example 2]</p><p>A series of experiments were conducted to determine the pH of the final blends of Nitricil NVN1 topical gels containing IPA and having different concentrations of Nitricil NVN1 ranging from 0.2-12% w/w with unbuffered and buffered hydrogel formulations having pH ranging from 4 to 6 at different ratios of hydrogel to Nitricil NVN1 topical gel ranging from 1:1 to 3:1 (hydrogel to Nitricil NVN1 topical gel). The effect of hydrogel pH, hydrogel to Nitricil NVN1 IPA topical gel mixing ratio, and Nitricil NVN1 concentration on the pH of the final blends was investigated. The purpose of the experiments was to determine whether a final blend pH ranging between 6 and 8 could be obtained.</p><p>For all in vitro pH admixture measurements, an approximately 1 g amount of Nitricil NVN1 IPA topical gel was dispensed into a tared weigh boat either using a 1 mL plastic syringe or by dispensing directly from the aluminum tube.</p><p>Once approximately 1 g portions were dispensed, the weigh boat was re-tared. A predetermined amount of hydrogel ranging from 1 to 3 g was then dispensed into the weigh boat using a 1 mL syringe. The mixture was then mixed using a pH probe (Beckman Φ350 pH meter) until a single steady-state pH reading was recorded. All dispenses were done on a weight basis.</p><p>The pH values of the blends were determined using a phosphate buffered pH 6 hydrogel, an unbuffered pH 4 hydrogel, and an unbuffered pH 6 hydrogel, as shown in Table 7.</p><p><tables><img file="JP7507204B2_D0007.tif" /></tables></p><p>Figure 1 shows the effect of Nitricil NVN1 topical gel concentration, hydrogel pH, and hydrogel to Nitricil NVN1 IPA topical gel ratio on blend pH. The blend pH results demonstrate that Nitricil NVN1 topical gels with 6% Nitricil NVN1 w/w or less can be used in combination with a pH 4 hydrogel in either a 1:1 or 3:1 ratio to achieve a pH of 8 in the final blend. At concentrations of 8% w/w or more, the strong buffering properties of Nitricil NVN1 are evident. The pH value of the hydrogels evaluated was significantly affected by the pH of the blends, for all pH and ratios of hydrogels evaluated. Significant foaming (indicating release of nitric oxide) was observed when Nitricil NVN1 was mixed with both pH 4 and pH 6 hydrogels at concentrations above 4% w/w. This was observed even at blend pH values above 8 when using pH 6 hydrogels. A 1:3 ratio of Nitricil NVN1 topical gel to pH 6 buffered hydrogels allowed the resulting blend pH to remain below pH 8 for Nitricil NVN1 topical gels with concentrations up to 2% w/w of Nitricil NVN1. Buffering the hydrogels at pH 6 with phosphate aided in reducing the pH of the final blends for both 1:1 and 1:3 ratios of hydrogel to Nitricil NVN1 IPA topical gel.</p><p>Further experiments investigated the effect of adding preservatives (e.g., parabens, sorbic acid and benzoic acid) at concentrations ranging between 0.1% and 0.2% w/w to hydrogel formulations adjusted to pH 4 and pH 5. The pH of the admixtures was measured for Nitricil NVN1 topical gels at concentrations of 2% w/w and 8% w/w.</p><p>Table 8 shows the pH values of the blends using both 2% w/w Nitricil NVN1 and 8% w/w Nitricil NVN1. The results in Table 8 show that when 2% w/w Nitricil NVN1 IPA topical gel was used in a 1:1 ratio with pH 4 hydrogel, the resulting blends had a pH between 6 and 7, both with and without a preservative.</p><p><tables><img file="JP7507204B2_D0008.tif" /></tables></p><p>When methylparaben and propylparaben were used as preservatives, the blends turned brown. Without wishing to be bound by any particular theory, this may have been an indication that degradation product(s) were formed. When the pH 5 hydrogel was used with different preservatives, the pH of the blends only increased slightly to about 7.</p><p>The pH of the blends at the skin surface was also measured when 8% w/w Nitricil NVN1 IPA topical gel was used with both the buffered pH 4 hydrogel and the unbuffered pH 4 hydrogel. The buffered pH 4 hydrogel contained Carbopol® 980P NF polymer with 0.1% w/w citric acid. Benzoic acid and sorbic acid at 0.1% w/w were also used as preservatives in the formulated pH 4 citric acid buffered hydrogel.</p><p>Further in vitro pH miscibility measurements using unbuffered pH 4 hydrogel (Carbopol® 974P) and buffered pH 4 hydrogel with 0.1% w/w citric acid (Carbopol® 980P) in a 1:1 ratio confirmed the findings from the experimental results shown in Table 8. Figure 2 shows that when Nitricil NVN1 at concentrations of 6% w/w and 8% w/w was used with unbuffered pH 4 hydrogel, the pH of the blend was around 10, similar to previous results. The use of the 0.1% w/w citric acid buffered pH 4 hydrogel has the effect of reducing the pH of the blend over a range of Nitricil NVN1 concentrations.</p><p>An in vitro skin surface pH determination study was also performed. Figure 3 shows that skin has a pH range between 5 and 6, exerting some buffering capability. When unbuffered pH 4 hydrogel was mixed with 8% w/w Nitricil NVN1 IPA topical gel and applied, the skin pH was slightly below 9. The pH remained constant for more than 30 minutes. However, when pH 4 hydrogel buffered with 0.1% w/w citric acid was used with 8% w/w Nitricil NVN1 IPA topical gel (containing 0.1% w/w benzoic acid and 0.1% w/w sorbic acid as preservatives), the skin surface pH was around 7.5.</p><p>Without wishing to be bound by any particular theory, this experiment demonstrates that using pH 4 buffered and unbuffered hydrogels in 1:1 and 3:1 ratios allows the pH of the final blend to be maintained between 5 and 8 when the concentration of Nitricil NVN1 ranges between 0.2% w/w and 4% w/w. Using the unbuffered pH 4 hydrogel in a 3:1 ratio allows the pH of the blend to be maintained below 8 units when the Nitricil NVN1 concentration is up to 8% w/w. Using the buffered pH 6 hydrogel allows the pH to be maintained below 8 when the Nitricil NVN1 concentration is 2% w/w or less. Nitricil NVN1 at a concentration of 1% w/w or less may be used with the unbuffered pH 6 hydrogel to maintain the pH value of the blend below 8. The use of preservatives in the hydrogel has little to no effect on the pH of the blend. When used in a 1:1 ratio, both unbuffered and buffered pH 4 hydrogels gave pH values above 8 units. However, when applied to the skin, the skin exerts some buffering capacity, reducing the measured pH at the surface. To ensure that the pH of the blend at the skin surface is below 8 units, a pH 4 hydrogel buffered with 0.1% w/w citric acid and containing 0.1% w/w benzoic acid and sorbic acid can be used. However, with the unbuffered pH 4 hydrogel, the pH at the skin surface was above 8.5, compared to the pH of 10 units obtained in the in vitro test.</p><p>[Experimental Example 3]</p><p>In vitro release studies were performed using both single and multi-channel nitric oxide analyzers. An analytical balance was used to weigh Nitricil NVN1 topical gel and hydrogel samples. Approximately 50 mg of Nitricil NVN1 topical gel sample and either about 50 mg or about 150 mg of hydrogel sample were transferred to a single pre-cut weigh boat without the samples touching each other. The two samples were mixed for approximately 5 seconds and then immediately placed into a clean, dry 50 mL NO measurement cell maintained at 37°C. The real-time in vitro release of nitric oxide from the combined Nitricil NVN1 topical gel/hydrogel samples was determined using the following instrument parameters:</p><p>1. Wet nitrogen flow rate: 112-115ml/min 2. Sample temperature: 37°C 3. Detection: Nitric oxide by chemiluminescence 4. Data acquisition frequency: 1Hz, irregular sequential alternating 5. Duration: Time during which the NO release rate is declining linearly (up to 8 hours) 6. Acquisition software: NovanWare v1.05.</p><p>Conversion of NO in parts per billion (PPB) to nitric oxide in moles was accomplished by measuring nitric oxide generated from known amounts of sodium nitrite in potassium iodide solution to obtain a PPB to mole conversion factor. Gaps in the real-time nitric oxide release data resulting from multichannel operation were filled using a linear interpolation program. In cases where nitric oxide was depleted and not measured in some samples, a linear extrapolation of the release data from approximately 5000 seconds immediately preceding the zero release portion was performed. The real-time nitric oxide release data was then integrated to obtain a total nitric oxide accumulation curve. Nitric oxide release parameters, e.g., C_max(i.e., the maximum concentration of NO released), T_max(i.e., C_maxThe cumulative amount of nitric oxide released (i.e., the sum of all data points per unit time), and the time to half of the total amount released (T₅₀) (i.e., the time at which 50% of the cumulative amount of NO was released) was calculated from both the real-time and cumulative nitric oxide release curves. All the above calculations were performed automatically with custom-written data processing software (NovanWare v1.05).</p><p>The results of the in vitro release studies are summarized below in Table 9 along with the respective pH of the blends.</p><p><tables><img file="JP7507204B2_D0009.tif" /></tables></p><p>Figure 4 shows the C_maxThe figures show how the C is affected by the mixture ratio, the pH of the hydrogel, and the concentration of NVN1. Overall, for the pH 4 hydrogel, the C_maxThe effect was more pronounced for the 1:3 mixture of Nitricil NVN1 IPA topical gel to hydrogel. Figure 5 shows that the cumulative release of nitric oxide increases with increasing concentration of Nitricil NVN1.</p><p>At all Nitricil NVN1 concentrations, mixtures containing pH 4 hydrogels generally delivered higher net nitric oxide payloads than those using pH 6 hydrogels._max4) and has a shorter half-life. This effect is more pronounced when comparing mixtures containing pH 4 hydrogels in a 1:3 ratio with mixtures containing pH 6 hydrogels in a 1:1 and 1:3 ratio. FIG. 6 shows the effect of unbuffered hydrogel pH and ratio on the pH of the blend in relation to the concentration of Nitricil NVN1 topical gel. Further in vitro tests were performed using buffered citrate hydrogels at pH 4 with preservative. Measurements were repeated three times. The blends were mixed for 15 seconds before being placed in the measurement cell. The results of the in vitro nitric oxide release tests are shown in Table 10.</p><p><tables><img file="JP7507204B2_D0010.tif" /></tables></p><p>[Experimental Example 4]</p><p>A phase 1, multiple-dose, single-center, observer-blind, randomized, parallel-group, safety and skin tolerability study was conducted in 60 healthy volunteers. The objective of the study was to evaluate the safety and skin tolerability of multiple concentrations of Nitricil NVN1.</p><p>The diagnostic and primary inclusion criteria for the study were healthy male and female volunteers aged 18 years or older with high facial P. acnes counts as demonstrated by a high degree of fluorescence of the facial skin under a Wood's lamp.</p><p>Subjects who met the entry criteria at the screening/baseline (week 0) visit were randomized and assigned in a 1:1:1:1 ratio to receive 2% Nitricil NVN1 topical gel, 4% Nitricil NVN1 topical gel, 8% Nitricil NVN1 topical gel, or topical gel vehicle (Table 11). Subjects returned once daily on weekdays for supervised application of their assigned treatment. Approximately 0.5 g was applied evenly to the entire face (3-4% of total body surface area (TBSA)) after cleansing, avoiding the eyes and mouth. On Saturdays and Sundays, subjects applied the treatment (unsupervised) at home once daily. Approximately 0.5 g doses were applied topically once daily for 4 weeks.</p><p><tables><img file="JP7507204B2_D0011.tif" /></tables></p><p>Assessments included collection of vital signs including skin tolerance, blood chemistry and hematology, methemoglobin analysis, physical examination, urine pregnancy test (UPT), adverse event collection, and blood pressure. Propionobacterium acnes cultures from the central forehead were collected at baseline, week 2, and week 4. Volunteers returned for post-baseline evaluations at week 2 and week 4/early termination (ET).</p><p>Tolerance and safety assessments included skin tolerance assessments, adverse events (AEs), methemoglobin measurements, physical examination including vital signs, and laboratory tests [blood chemistry, hematology, and urine pregnancy test (UPT)].</p><p>Propionibacterium acnes cultures were collected from the central forehead by the swab technique at baseline (BL), 2 weeks, and 4 weeks (Williamson 1965).</p><p>This was a pilot study and was not powered for statistical significance. All statistical procedures were performed using SAS unless otherwise stated. Statistical significance was determined based on a two-tailed test of the null hypothesis with a p value of 0.05 or less. Safety analyses were performed using the safety population.</p><p>Each cutaneous tolerability scale was summarized categorically and continuously by treatment group and visit. For categorical summaries, a modified ridit Cochran-Mantel-Haenszel test was performed for each postbaseline assessment.</p><p>Blood chemistry and hematology values were reported individually at baseline and at Weeks 2 and 4/ET. In addition, changes from baseline were reported at Weeks 2 and 4/ET. Laboratory values (Labs) were reported in SI units.</p><p>Methemoglobin was summarized categorically and continuously by treatment group and visit. In addition, boxplots were presented by treatment group and visit, displaying the mean, median, minimum, maximum, first and third quartiles, and overlaying all observed data points onto the boxplots to allow easy visualization of any outliers.</p><p>All AEs occurring during the study were recorded and classified based on the Medical Dictionary for Regulatory Authorities (MedDRA) glossary. All reported AEs occurring from the baseline date through the last study visit were included in the summary and analysis. If an event occurred before administration of study drug, the event was considered historical and was listed and summarized as such.</p><p>Adverse event summaries included the number and percentage of subjects reporting at least one AE, and the number of events reported by severity, seriousness, and causality to study medication. Adverse event summaries were also presented by system organ class (SOC) and preferred term (PT) according to MEDDRA version 15.1. Subjects were counted only once under each SOC and PT category.</p><p>Adverse events SOCs and PTs were summarized by severity and by causal relationship to the investigational product. When summarizing by severity, subjects were counted only once under the most severe category for a particular SOC or PT. Severity was captured as mild, moderate, severe, or life-threatening. When summarizing by causal relationship to the investigational product, subjects were counted only once under the category for which they were most causally related. Causal relationship was captured as "definite," "probable," "possible," "unlikely," "unrelated," or "not applicable." For causal relationship summaries, "definite," "probable," and "possible" were considered causal, and "unlikely," "unrelated," and "not applicable" were considered non-causal.</p><p>All information on AEs noted during the study was listed by subject, investigator-described detailing verbatim, preferred term, system organ class, date of onset, date of resolution, severity, seriousness, and causal relationship to the investigational product. The occurrence of AEs was also presented (in days) in relation to the date of first randomized dose of the investigational product. A summary of adverse events leading to subject discontinuation of the investigational product was also provided.</p><p>P. acnes counts: Cultures for quantitative bacteriological examination were obtained from the forehead at baseline and at weeks 2 and 4/ET. Samples were obtained according to a modification of the Williamson and Kligman method and cultured for 7 days (Williamson, 1965). Colony forming units (cfu) of P. acnes were counted in dilutions containing between 10 and 100 cfu. The total density of P. acnes was calculated and counted per cm²The results were reported as log10 cfu per sera.</p><p>Descriptive statistics, change from baseline, and percent change from baseline for P. acnes were summarized using mean, median, standard deviation, minimum, maximum, and 95% confidence intervals.</p><p>The percent reduction from baseline at weeks 2 and 4 was calculated using the following formula:<math num="1"><img file="JP7507204B2_D0012.tif" /></math>where X=initial Log value and Y=final Log value.</p><p>Safety and Tolerability: Nitricil NVN1 Topical Gel was demonstrated to be safe and well tolerated in this study. The majority of subjects in the Nitricil NVN1 Topical Gel and Vehicle Gel treatment groups did not experience erythema, scaling, dryness, pruritus, or burning/stinging during the treatment period. Scores other than "none" were generally mild, with one subject reporting moderate erythema.</p><p>A total of 17 AEs were reported by 12 subjects in the Nitricil NVN1 topical gel group and 4 AEs were reported by 4 subjects (27%) in the vehicle gel group. The most common AEs reported were nasal congestion and headache. All AEs were mild or moderate in severity and were deemed by the investigator to be unrelated to study medication and did not require any study medication action. All AEs resolved at the end of the study. None of the AEs were severe.</p><p>For the Nitricil NVN1 topical gel treatment group, methemoglobin levels averaged 0.77-0.91% at baseline, 0.73-0.97% at week 2, and 0.67-0.85% at week 4. For the vehicle gel treatment group, methemoglobin levels averaged 0.77% at baseline, 0.91% at week 2, and 0.83% at week 4. The highest methemoglobin levels observed during the study (1.9%) were in subjects treated with vehicle gel.</p><p>P. acnes counts: No differences in P. acnes counts were observed in subjects treated with Nitricil NVN1 compared to the topical gel vehicle treatment group. The mean reduction in P. acnes from baseline to week 4 was 57% (gel vehicle), 58% (2% Nitricil NVN1), 54% (4% Nitricil NVN1), and 63% (8% Nitricil NVN1).</p><p>Nitricil NVN1 was safe and well tolerated in this study.</p><p>There were no differences between treatment groups in tolerability, reported adverse events, clinical laboratory results including methemoglobin levels, and physical examination including vital signs. There were no differences in percent reduction in P. acnes counts from baseline at weeks 2 and 4 between the Nitricil NVN1 topical gel treatment group and the topical gel vehicle group.</p><p>[Experimental Example 5]</p><p>This was a phase 1, single-center, assessor-blinded, randomized, parallel-group, safety and skin tolerance study conducted in 30 healthy volunteers with elevated Propionobacterium acnes counts.The objective of the study was to evaluate the safety and skin tolerance of Nitricil NVN1 gel.</p><p>Subjects who met the entry criteria at screening and baseline visits were randomized and assigned in a 2:1 ratio to receive 4% Nitricil NVN1 gel or vehicle gel (Table 12). Subjects returned once daily for supervised application of their assigned study treatment. Approximately 1 g was applied twice daily to the entire face (3-4% of total body surface area (TBSA)), avoiding the eyes and mouth.</p><p><tables><img file="JP7507204B2_D0013.tif" /></tables></p><p>Assessments included skin tolerance, methemoglobin and hemoglobin analysis, physical examination including vital signs, and adverse event collection.</p><p>Propionibacterium acnes cultures from the central forehead were collected at baseline and after treatment (weeks 1 and 2). Subjects returned for post-baseline evaluations at weeks 1 and 2/early termination (ET).</p><p>Diagnostic and primary inclusion criteria were healthy male and female volunteers aged 18 years or older with high facial P. acnes burden as demonstrated by high facial skin fluorescence under Wood's light.</p><p>Tolerance and safety assessments included skin tolerance assessments, adverse events (AEs), hemoglobin and methemoglobin measurements, and physical examinations including vital signs.</p><p>Propionibacterium acnes cultures were collected from the central forehead by swab technique at baseline (BL), week 1, and week 2/ET (Williamson 1965).</p><p>This was a pilot study and no emphasis was placed on statistical significance. However, the sample size was large enough to detect a mean score difference of 0.75 between treatment groups in any of the skin tolerance assessments, with 80% power at α=0.05. All statistical procedures were performed using SAS® unless otherwise stated. Statistical significance was determined based on two-sided tests of the null hypothesis, with p values of 0.05 or less, unless otherwise stated. Safety analyses were performed using the safety population.</p><p>Skin tolerance assessments (erythema, scaling, dryness, pruritus, burning/stinging) were summarized using frequency counts and percentages at each assessment for each score category at weeks 1 and 2. Comparison of treatment differences in score distributions was performed using the Cochran-Mantel-Haenszel (CMH) chi-square test with the MODRIDIT option for ordered scores.</p><p>Total hemoglobin was reported, and methemoglobin was reported as a percentage of hemoglobin. Hemoglobin and methemoglobin were summarized descriptively by treatment group at screening and weeks 1 and 2. The summary included n, mean, median, standard deviation, minimum, and maximum. In addition, changes from baseline in hemoglobin and methemoglobin were summarized at weeks 1 and 2. Comparisons of treatment group differences in changes from baseline hemoglobin and methemoglobin were performed using Wilcoxon rank sum tests.</p><p>Cultures for quantitative bacteriological examination were obtained from the forehead at baseline and at weeks 1 and 2. Samples were obtained according to a modification of the Williamson and Kligman method and cultured for 7 days (Williamson, BA; Kligman, AMA new method for the quantitative investigation of cutaneous bacteria. J. Invest. Dermatol. 1965, 45, 498-503). Colony forming units (cfu) of P. acnes were counted in dilutions containing between 10 and 100 cfu. The total density of P. acnes was calculated and counted per cm²Hit log₁₀Reported as cfu.</p><p>Descriptive statistics, change from baseline, and percent change from baseline for P. acnes were summarized using mean, median, standard deviation, minimum, maximum, and 95% confidence intervals.</p><p>The percent reduction from baseline at weeks 1 and 2 was calculated using the following formula:<math num="2"><img file="JP7507204B2_D0014.tif" /></math>where X=initial Log value and Y=final Log value.</p><p>In this study, 4% Nitricil NVN1 Gel was demonstrated to be safe and well tolerated. The majority of subjects in the 4% Nitricil NVN1 Gel and vehicle gel treatment groups did not experience erythema, scaling, dryness, pruritus, or burning/stinging during the treatment period.</p><p>No adverse events were reported in this study. There were no clinically significant changes in blood pressure, pulse, or physical examination findings between baseline and end of treatment. There were no clinically significant changes in methemoglobin and percent hemoglobin concentrations.</p><p>After one week of treatment, the mean log reduction in P. acnes counts was 0.38 in the 4% Nitricil NVN1 gel group versus 0.20 in the vehicle gel group. After two weeks, subjects treated with 4% Nitricil NVN1 gel had a mean reduction in P. acnes counts of 1 cm²0.51 log per₁₀cfu, compared with 1 cm in subjects treated with vehicle gel.²0.26 logs per₁₀cfu. The difference at week 2 was statistically significant using a student's T-test at p=0.04. Post-hoc ANCOVA also demonstrated a statistically significant difference in P. acnes reduction between subjects treated with 4% Nitricil NVN1 gel and those treated with vehicle gel at p=0.03.</p><p>4% Nitricil NVN1 gel was safe and well tolerated in this study. There was a statistically significant difference in the percent reduction in P. acnes counts between the 4% Nitricil NVN1 gel treatment group and the vehicle gel treatment group.</p><p>[Experimental Example 6]</p><p>The primary objective of the study was to evaluate the dermal tolerance of 4% Nitricil NVN1 gel (containing hydrogel) at baseline and at Weeks 1 and 2/ET. A secondary objective was to evaluate the safety profile of Nitricil NVN1 gel. Safety was assessed by comparing adverse events (including clinically meaningful changes in physical examination results and vital signs), change in hemoglobin, and change in percent methemoglobin between groups. Exploratory analyses of efficacy, as measured by reduction of P. acnes as determined by change in culture-derived counts, were performed at the baseline visit, Weeks 1, and 2/ET.</p><p>The study panel consisted of 30 healthy adult subjects, aged 18 years or older, colonized with P. acnes . Subjects were carefully screened to ensure that none had used any prohibited topical or systemic antibiotics within the 4 weeks prior to enrollment. Panelists were instructed not to use any medicated shampoos. Volunteers selected for the study showed high levels of facial skin fluorescence under Wood's light, indicative of the presence of high levels of P. acnes. Baseline P. acnes counts were measured on 1 cm2 facial skin.²There were at least 10,000 colonies per well.</p><p>All subjects met the inclusion/exclusion criteria listed below.</p><p>Inclusion criteria: Signed written informed consent form.</p><p>- Healthy adult male or female volunteers aged 18 or older.</p><p>- Demonstrating high levels of facial skin fluorescence under Wood's light, indicating the presence of high levels of Propionibacterium acnes.</p><p>- No history or current illness of any serious internal organ disease (e.g. cardiovascular disease, pulmonary disease, renal disease, etc.).</p><p>- If women of childbearing potential (WOCBP), have a negative urine pregnancy test (UPT) at baseline.</p><p>- If WOCBP, agree to use effective contraception for the duration of the study and for 30 days after the final study visit. Women taking hormonal contraceptives must have been using the same type of contraceptive for at least 3 months (90 days) prior to study entry and must not change types during the study. Those who have previously used and discontinued hormonal contraceptives must have discontinued use for at least 3 months prior to the start of the study.</p><p>-Agree to refrain from using topical antibacterial products (shampoos, soaps, acne preparations, etc.).</p><p>- You will be able to adhere to all compliance instructions and visit the clinical trial site once a day (Monday to Friday) as instructed for approximately two weeks.</p><p>Exclusion Criteria: Subjects were not enrolled if they met any of the following exclusion criteria: Present with any acute or chronic skin disorder, including psoriasis, eczema, etc.</p><p>- Have experienced significant burning or stinging when applying any facial care product to the face (e.g., makeup, soap, mask, cleanser, sunscreen, etc.)</p><p>- Female subjects who are pregnant, nursing mothers, or planning to become pregnant during the study period.</p><p>-Male subjects who do not agree to sexual abstinence, refrain from sperm donation, and/or use a barrier (male condom) throughout the duration of the study.</p><p>- Have used estrogen (e.g., Depogen, Depo-Testadiol, Gynogen, Valergen, etc.) or oral contraceptives within 90 days prior to the baseline visit, or have discontinued estrogen or oral contraceptive use within 90 days prior to baseline, or are planning to start or discontinue use of this therapy during the treatment period of the study.</p><p>Use of topical or systemic antibiotics known to affect P. acnes populations, such as minocycline, tetracycline, erythromycin, clindamycin, or doxycycline, within the past 4 weeks.</p><p>- Use of other medicines within the past 6 months that may affect the levels of P. acnes on the skin surface (e.g. retinoids).</p><p>- Concomitant use of nitroglycerin or other nitric oxide donors.</p><p>- Clinically significant anemia at baseline (as determined by the principal investigator).</p><p>- Methemoglobin level at screening is 2.0% or higher.</p><p>- Clinically significant anemia at screening, as determined by the investigator.</p><p>- Known allergy to any of the ingredients in the investigational product.</p><p>-Having an intercurrent illness that requires administration of a prohibited antibiotic.</p><p>-Has any condition or situation that the investigator believes would place the subject at significant risk or that may confound the results of the study or significantly prevent the subject's participation in the study.Subjects undergoing endoscopy using local anesthetic should not be enrolled in the study and should be discontinued prior to endoscopy.Subjects who are determined by the investigator to have clinically significant anemia should not be enrolled.</p><p>- Inability to communicate or cooperate with the investigator due to language problems, mental retardation, or brain dysfunction.</p><p>- Use of an investigational drug or investigational device within 30 days of enrollment, or concurrent participation in a different research study.</p><p>Volunteers were free to withdraw consent and discontinue participation in the study at any time.</p><p>Prior to enrollment, candidates were screened for eligibility and, after ensuring eligibility and signing an informed consent, a quantitative baseline measurement of Propionibacterium acnes was obtained (on the forehead) (see "Quantitative Bacteriological Testing" below).</p><p>Treatment plan: Treatment was for 2 weeks. Each weekday morning, under the supervision of a technician at the Skin Study Center, volunteers washed their face and then applied the test product. Approximately 1 g of 4% Nitricil NVN1 gel or gel vehicle (Table 12) was applied evenly to the entire face (4% of TBSA), avoiding the eyes and mouth. The study product was dispensed from a dual-chamber pump by depressing the pump three times. The dispensed material was mixed quickly (3-5 seconds) and applied in a thin layer to the face, avoiding the eyes and mouth. The study product was then gently massaged into the skin for approximately 30 seconds. Subjects washed their hands after applying the test product. This procedure of washing the face, applying the study product, and then washing the hands was performed by panelists at home (unsupervised) in the evening.</p><p>All treatments were recorded in the subject's diary.</p><p>Quantitative Bacteriology: Cultures for quantitative bacteriology were obtained from the study sites at baseline (week 0) and weeks 1 and 2. Samples were obtained according to a modification of the Williamson and Kligman method (Williamson, P. and Kligman, AM: A new method for the quantitative investigation of cutaneous bacteria. Journal of Investigative Dermatology 45:498-503, 1965; Keyworth N., Millar, MR, and Holland, KT: Swab-Wash Method for Quantitation of Cutaneous Microflora. J. Clin. Microbiology, Vol. 28, pp. 941-943, 1990). The forehead was cleaned of surface bacteria by gently wiping the area with sterile gauze saturated with 0.1% Triton-X-100 for 30 seconds to remove surface debris and bacteria. The surface area to be cultured (4 cm2) was then outlined with a sterile plastic template pressed firmly against the skin. A sterile cotton-tipped swab was dipped into 2 ml of irrigation solution (Bacto Letheen Broth, Difco, Sparks, MD, USA). The area was then scrubbed with the cotton swab for 30 seconds. The swab was then placed back into the 2 ml of irrigation solution and squeezed against the side of the tube. The same skin area was then rubbed again for an additional 30 seconds, after which the swab was placed back into the previous 2 ml of wash solution and squeezed against the side of the tube. The swab was then torn off and placed into the previous 2 ml of wash solution. Samples were subsequently processed as described in the Williamson-Kligman method, i.e., 0.05% Wash samples were serially diluted by four ten-fold dilutions in Tween-80 (buffered with 0.075 M phosphate buffer, pH 7.9). Using a micropipettor, 0.05 mL of each dilution was placed in designated areas of an agar plate containing Brucella agar medium supplemented with yeast extract, dextrose, and cysteine. Five drops of dilution were placed per plate. Duplicate plates were prepared for each subject. Plates were dried and incubated anaerobically at 36.5-37.5°C for 7 days in anaerobic jars equipped with BBL Gas Pak Plus anaerobic envelopes. Colony forming units (cfu) of P. acnes were counted in dilutions containing between 10 and 100 cfu. The total density of P. acnes was calculated and counted in 1 cm²Hit log₁₀Reported as cfu.</p><p>Skin Tolerance Assessment: Skin tolerance assessments were performed by a dermatologist prior to treatment at baseline, week 1, and week 2. Skin tolerance endpoints were not reported as AEs unless they reached severity and/or resulted in the subject discontinuing participation in the study. Skin tolerance assessments were performed according to the following scale: Erythema 0: None. No signs of erythema present 1: Mild. Slight pinkness 2: Moderate. Obvious redness 3: Severe. Significant erythema, light red to dull dark red in color Scaling 0: None. No scaling 1: Mild. Barely perceptible fine scales present in limited areas of the face 2: Moderate. Fine scales generalized over the face 3: Scaling and peeling over the face Dryness 0: None. No dryness 1: Mild. Slight but obvious roughness 2: Moderate. Moderate roughness 3: Severe. Significant rash Pruritus 0: None. No itching 1: Mild. Slight itching but not very bothersome 2: Moderate. Moderate itching, some bothersome 3: Severe. Severe itching, obvious discomfort, may interfere with sleep Burning/stinging 0: None. No burning/stinging 1: Mild. Crawling sensation, slight burning/stinging sensation, not very bothersome 2: Moderate. Crawling sensation, burning/stinging sensation obvious and some bothersome 3: Severe. Hot, stinging sensation causing obvious discomfort, may interfere with sleep</p><p>Clinical Laboratory Assessments: Hemoglobin was measured using a Masimo Rainbow® SET® Rad-57 pulse co-oximeter at baseline and at weeks 1 and 2/ET. Hemoglobin (g/dL) was displayed on the pulse co-oximeter and recorded on the CRF.</p><p>Methemoglobin was measured using a Masimo Rainbow® SET® Rad-57 pulse co-oximeter at baseline and at weeks 1 and 2/ET. Percentage of methemoglobin was displayed on the pulse co-oximeter and recorded on the CRF.</p><p>All WOCBP underwent a urine pregnancy test (UPT) at baseline and the final assessment visit (week 2/ET).</p><p>A brief physical examination was performed at baseline and at week 2/ET.</p><p>Systolic and diastolic blood pressure and pulse were collected at baseline and at weeks 1 and 2/ET.</p><p>Data processing: Cultures were plated in duplicate at 10-fold dilutions from 100 to 104. Bacterial counts were obtained from dilutions with significant dispersion of colony forming units (CFU) and then converted to total counts per square centimeter. Raw data and average values for each plate were entered in separate columns. The collation template was based on Excel 2007 spreadsheet software. Collapsed data for each session were tabulated.</p><p>Data analysis included paired t-tests to compare baseline means with those at treatment. Student's t-tests were used to compare means between the two treatment groups. A two-tailed p&lt;0.05 was required for significance.</p><p>Thirty-three panelists were screened for the study, and a total of 30 panelists were enrolled. Twenty-nine panelists completed the study. One panelist withdrew consent to the study due to redness after application. No adverse events were observed.</p><p>No erythema or scaling was noted by the dermatologist during the course of the study. Minimal dryness, pruritus, and burning or stinging were noted as follows:</p><p>Three subjects using 4% Nitricil NVN1 Gel (Study Treatment A) reported dryness at week 2. Two subjects using Study Treatment A reported pruritus at week 1. One subject using Study Treatment A reported a burning/stinging sensation at week 1.</p><p>In this study, the skin tolerance and systemic safety of the 4% Nitricil NVN1 gel formulation were evaluated after 1 and 2 weeks of twice-daily treatment. In addition, an exploratory analysis of in-vivo reduction of Propionibacterium acnes, i.e., the contribution of bacteria to inflammation in acne vulgaris, was performed after 1 and 2 weeks of treatment. The test panel consisted of 100 subjects with P. acnes levels and 100 subjects with P. acnes levels.⁴/cm²The study consisted of 30 healthy volunteers, of which 20 were treated with active drug and 10 with vehicle.</p><p>The test agents and their vehicles were very well tolerated. No objective signs of irritation or subjective symptoms of itching, burning, or stinging were observed, except for moderate pruritus in one panelist and mild pruritus in another at the week 1 visit. Mild burning/stinging was reported by one panelist at week 1. Mild dryness was observed by three panelists at week 2. One panelist withdrew on day 1 due to concerns about physiological vasodilation induced by the test agent. This panelist did not experience any signs of irritation.</p><p>No clinically significant changes were observed in blood pressure, pulse, or physical examination findings between baseline and end of treatment. No clinically significant changes were observed in methemoglobin and percent hemoglobin concentrations.</p><p>After one week of treatment, the mean log reduction was 0.38 for the active group versus 0.20 for the vehicle group. After two weeks, the mean reduction for the active was 0.51 versus 0.26 for the vehicle. The difference at two weeks was statistically significant using a Student's T-test (p=0.04). Responses were mixed for the test agents, with two panelists showing greater than a 1 log reduction after two weeks of treatment and five others showing reductions of 0.5 or greater but less than a 1 log. In the vehicle group, three panelists showed values below the mean reduction.</p><p>Analysis of covariance (ANCOVA) demonstrated that there was a statistically significant difference between P. acnes counts at weeks 1 and 2 (Figure 9).</p><p>The test agents and their vehicles were well tolerated and showed no signs of skin or systemic toxicity. They showed various in vivo antibacterial effects against P. acnes, which may be useful in acne therapy.</p><p>[Experimental Example 7]</p><p>This was a multicenter, randomized, assessor-blinded, vehicle-controlled, parallel-group, three-arm study to compare the efficacy, safety, and tolerability of two strengths of Nitricil NVN1 gel and vehicle gel for 12 weeks of treatment in subjects with acne vulgaris. Subjects who met the study entry criteria were enrolled and randomized to receive topical application of 1% Nitricil NVN1 gel, 4% Nitricil NVN1 gel, or vehicle gel (Table 13). Subjects were randomized in a 1:1:1 ratio to 1% Nitricil NVN1 gel, 4% Nitricil NVN1 gel, or vehicle gel and instructed to administer twice daily (morning and evening) for 12 weeks (84 days). The first application of study medication was administered at the baseline visit at the study site.</p><p><tables><img file="JP7507204B2_D0015.tif" /></tables></p><p>After the baseline visit, study visits occurred approximately every 2 weeks for the first 4 weeks, then every 4 weeks for the next 8 weeks. The study duration was up to 84 days in the treatment phase.</p><p>Efficacy assessments included inflammatory (papules and pustules) and non-inflammatory (open and closed comedones) lesion counts, nodule and cyst counts, and Investigator Global Assessment (IGA), performed at baseline and at weeks 4, 8, and 12/early termination (ET).</p><p>Additional assessments included photography and sebum collection. Photographs were collected at baseline, Week 4, and Week 12/ET. Sebum was collected from the center of the forehead of enrolled subjects using Sebutape® at two study sites at baseline, Week 4, and Week 12/ET.</p><p>Tolerance and safety assessments included skin tolerance assessments, adverse event (AE) collection, physical examination including blood pressure and pulse rate, methemoglobin and hemoglobin measurements, and urine pregnancy test (UPT). Skin tolerance assessments (erythema, scaling, dryness, pruritus, and burning/stinging) were evaluated before and 30 minutes after the first application of study drug at the baseline visit and each visit thereafter. Skin tolerance assessments at visits other than baseline were to be performed at least 30 minutes after study drug application. AE collection began after subjects signed informed consent and completed all study assessments until the final study visit. A brief physical examination was performed at baseline (Visit 1/Day 0) and Week 12/ET. Blood pressure and pulse rate were collected pre-dose at baseline and at Weeks 2, 4, 8, and 12/ET. Methemoglobin and hemoglobin are Masimo software applications that analyze methemoglobin and hemoglobin levels. Measurements were made at baseline and at weeks 2 and 12/ET using a Rainbow® SET® Rad-57 pulse co-oximeter. Subjects with baseline methemoglobin levels greater than 2.0 percent and subjects with baseline clinically significant anemia were ineligible for study participation. All women of childbearing potential (WOCBP) were required to receive UPT at baseline, and a positive result would have excluded the subject from the study. WOCBP were also required to receive UPT at weeks 4, 8, and 12/ET. Subjects who terminated early were required to complete all Week 12/ET assessments at the time of premature discontinuation.</p><p>The number of subjects in the intent to treat (ITT) population was 52 in the vehicle gel group, 51 in the 1% Nitricil NVN1 gel group, and 50 in the 4% Nitricil NVN1 gel group. However, not all subjects completed the study, resulting in 45 subjects in the vehicle gel group, 43 subjects in the 1% Nitricil NVN1 gel group, and 41 subjects in the 4% Nitricil NVN1 gel group completing the study.</p><p>The study enrolled healthy men and women of any race, aged 12 to 40 years (inclusive), with acne vulgaris. Subjects must have at least 20, but no more than about 40, inflammatory lesions (papules and pustules), 25 to 70 noninflammatory lesions (open and closed comedones), no more than 2 nodules, and an Investigator's Global Assessment (IGA) of 2, 3, or 4.</p><p>The 1% Nitricil NVN1 Gel, 4% Nitricil NVN1 Gel, and vehicle gel were administered by applying approximately 1 gram twice daily (morning and evening) evenly to the entire face. The active gel (chamber A) and hydrogel (chamber B) were dispensed simultaneously from a dual-chamber pump and mixed in a 1:1 ratio by the subject immediately prior to application. The vehicle gel (chamber A) and hydrogel (chamber B) were dispensed simultaneously from a dual-chamber pump and mixed by the subject immediately prior to application.</p><p>At baseline and at weeks 4, 8, and 12/ET, the investigator counted the total number of noninflammatory lesions on the subject's face, including the forehead, left and right cheeks, chin, and nose. At baseline and at weeks 4, 8, and 12/ET, the investigator also counted the total number of inflammatory lesions on the subject's face, including the forehead, left and right cheeks, nose, and chin. Nasal lesion counts (inflammatory and noninflammatory) and nodule and cyst counts were reported separately, but for analysis, inflammatory lesion counts included the combined counts of papules, pustules, and nodules/cysts.</p><p>IGAs were performed at baseline and at weeks 4, 8, and 12/ET. IGA scores were determined based on the investigator's assessment of the overall signs and symptoms of acne vulgaris, scored on a scale from 0 (no lesions) to 4 (severe).</p><p>Photographs were collected at baseline, week 4 and week 12/ET.</p><p>Sebum was collected from the central forehead of enrolled subjects using Sebutape® at two investigational sites at baseline, Week 4, and Week 12/ET.</p><p>In addition to assessments at each study visit, the investigator assessed the subject's face before and 30 minutes after the first application of the study product. Skin tolerance assessments at visits other than baseline were to be performed at least 30 minutes after study drug application. Skin tolerance assessments included erythema, scaling, dryness, pruritus, and burning/stinging.</p><p>Methemoglobin and hemoglobin were measured at baseline, week 2, and week 12 using a Masimo Rainbow® SET® Rad-57 pulse co-oximeter to analyze methemoglobin and hemoglobin levels.</p><p>A brief physical examination was performed at baseline (Visit 1/Day 0) and Week 12/ET. Blood pressure and pulse rate were collected pre-dose at baseline and Weeks 2, 4, 8, and 12/ET. Investigators assessed subjects for the occurrence of AEs at each scheduled study visit.</p><p>The primary efficacy outcome measure was absolute change from baseline in noninflammatory lesions on the face, including the nose, at week 12.</p><p>Analysis of the primary endpoint, absolute change in noninflammatory lesions on the face including the nose at week 12, was performed using analysis of covariance (ANCOVA) with treatment and study site as factors and baseline lesion count as a covariate. Linear regression was also performed to determine dose-response, where the slope (β) was estimated for all study treatments (1% and 4%), and vehicle gel was labeled as 0% in the regression. The null test was β=0 against the alternative hypothesis that β=0. The null hypothesis was rejected when β was positive, indicating a dose-response.</p><p>Secondary efficacy outcomes included absolute change from baseline in inflammatory lesion counts at week 12, absolute change from baseline in non-inflammatory lesion counts on the face excluding nose at week 12, and dichotomized IGA at week 12.</p><p>Inflammatory lesion counts included total numbers of papules, pustules, and nodules/cysts.</p><p>Absolute change in inflammatory lesion counts at week 12 was analyzed using the same methods outlined for the primary efficacy analysis of change in non-inflammatory lesion counts, including the nose.</p><p>Analysis of absolute non-inflammatory lesion counts on the face excluding nose at week 12 used the same methodology as outlined for the primary efficacy analysis of change in non-inflammatory lesion counts on the face including nose.</p><p>Dichotomized IGA scores at week 12 were analyzed using a logistic regression model with treatment and study site as independent factors. Dichotomized IGA scores were relabeled as the dependent variable in the logistic regression with 0 for failure and 1 for success. Success was defined as a score of "clear" (0) or "almost clear" (1) with at least a 2-grade improvement from baseline. In addition, treatment groups were compared using the Cochran-Mantel-Haenszel (CMH) test stratified by study site. Pairwise comparisons were computed without accounting for controls for multiplicity.</p><p>Skin tolerance assessments (erythema, scaling, dryness, pruritus, and burning/stinging) were summarized as frequency counts and percentages at each evaluation. Methemoglobin was reported as a percentage of hemoglobin. Total hemoglobin was also reported. Methemoglobin and hemoglobin were summarized descriptively by treatment group at baseline and at weeks 2 and 12. This summary included sample size, mean, median, standard deviation (SD), minimum, and maximum. In addition, changes from baseline in methemoglobin and hemoglobin at weeks 2 and 12 were summarized.</p><p>A summary was presented to describe the characteristics of reported AEs, including the number and percentage of subjects reporting at least one AE, and the number of events reported by severity, seriousness, and causality to study drug.</p><p>Blood pressure and pulse rate at baseline, Week 2, Week 4, Week 8, and Week 12/ET, and changes from baseline at Week 12/ET were summarized by treatment group using mean, SD, minimum and maximum.</p><p>For the ITT population, the least squares mean changes from baseline in absolute non-inflammatory lesion counts (including and excluding nose) for 1% Nitricil NVN1 gel and 4% Nitricil NVN1 gel were statistically significantly greater than the least squares mean changes from baseline in absolute non-inflammatory lesion counts (including and excluding nose) for the corresponding vehicle gels, but no linear dose response was observed. The least squares mean changes from baseline in absolute inflammatory lesion counts for 4% Nitricil NVN1 gel were statistically significantly greater than the least squares mean changes from baseline in absolute inflammatory lesion counts for vehicle gels, but no linear dose response was observed. The least squares mean change from baseline in absolute inflammatory lesion counts in Nitricil NVN1 gel was not statistically different from the least squares mean change from baseline in absolute inflammatory lesion counts in vehicle gel. A linear dose response was observed. No differences were observed between treatment groups for dichotomized IGA. Specifically, the least squares mean absolute change from baseline in noninflammatory lesions (including nose) was -11.8 lesions in the 1% Nitricil NVN1 gel group vs. -0.7 lesions in the vehicle gel group (p=0.022). The least squares mean absolute change from baseline in noninflammatory lesions (including nose) was -11.1 lesions in the 4% Nitricil NVN1 gel group vs. -0.7 lesions in the vehicle gel group (p=0.031). There was no linear dose response between treatment groups (p=0.105). The least squares mean absolute change from baseline in inflammatory lesions was 1% The least squares mean absolute change from baseline in inflammatory lesions was -15.5 lesions in the 4% Nitricil NVN1 gel group versus -9.4 lesions in the vehicle gel group (p=0.018). There was a linear dose response between treatment groups (p=0.033).</p><p>The least squares mean absolute change from baseline in noninflammatory lesions (excluding nose) was -10.9 lesions in the 1% Nitricil NVN1 gel group vs -1.3 lesions in the vehicle gel group (p=0.032). The least squares mean absolute change from baseline in noninflammatory lesions (excluding nose) was -10.6 lesions in the 4% Nitricil NVN1 gel group vs -1.3 lesions in the vehicle gel group (p=0.039). There was no linear dose response between treatment groups (p=0.118).</p><p>- 0.0% (0/51) of subjects in the 1% Nitricil NVN1 gel group were characterized as dichotomized IGA "successful" vs. 1.9% (1/52) of subjects in the vehicle gel group (p&gt;0.317). 2.0% (1/50) of subjects in the 4% Nitricil NVN1 gel group were characterized as dichotomized IGA "successful" vs. 1.9% (1/52) of subjects in the vehicle gel group (p&gt;0.601).</p><p>In the safety population, 1% Nitricil NVN1 Gel and 4% Nitricil NVN1 Gel were safe and generally well tolerated. No serious adverse events were observed, and 1% Nitricil NVN1 Gel and 4% Nitricil NVN1 Gel were well tolerated by the skin. Review of methemoglobin and hemoglobin results revealed no safety signals. Review of vital signs results revealed no safety signals.</p><p>For the PP population, both the 1% Nitricil NVN1 gel and the 4% Nitricil NVN1 gel demonstrated a mean percentage reduction in non-inflammatory lesions over time compared to vehicle gel (Figure 10). Figure 10 shows that divergence from the vehicle gel group occurred at week 4 for both the 1% Nitricil NVN1 gel group and the 4% Nitricil NVN1 gel group.</p><p>At week 4, the vehicle gel group had a mean percentage reduction in non-inflammatory lesions of 6%, compared to the 1% Nitricil NVN1 gel group, which had a mean percentage reduction in non-inflammatory lesions of 14%, and the 4% Nitricil NVN1 gel group, which had a mean percentage reduction in non-inflammatory lesions of 24%. The non-inflammatory lesion counts for the PP population are shown in Table 14.</p><p><tables><img file="JP7507204B2_D0016.tif" /></tables></p><p>In the PP population, both the 1% and 4% Nitricil NVN1 gels showed a mean percentage reduction in non-inflammatory lesions over time compared to vehicle gel (FIG. 11). FIG. 11 shows that divergence occurred from the vehicle gel group at week 4 for both the 1% and 4% Nitricil NVN1 gel groups. At week 4, the vehicle gel group had a mean percentage reduction in inflammatory lesions of 14%, compared to a mean percentage reduction in inflammatory lesions of 26% for the 1% Nitricil NVN1 gel group and a mean percentage reduction in inflammatory lesions of 31% for the 4% Nitricil NVN1 gel group.</p><p>At week 12, the median % reduction in inflammatory lesions for the PP population was 56% for the 1% Nitricil NVN1 gel group, 66% for the 4% Nitricil NVN1 gel group, and 42% for the vehicle gel group. Thus, the median % reduction in inflammatory lesions for the PP population was greater than the mean % reduction in inflammatory lesions for the PP population in each group. For the 4% Nitricil NVN1 gel group, 20 of the 41 patients who completed the study had a reduction in inflammatory lesions of more than 70%. The inflammatory lesion counts for the PP population are shown in Table 15.</p><p><tables><img file="JP7507204B2_D0017.tif" /></tables></p><p>Compared to the vehicle gel group, the 1% Nitricil NVN1 gel group was effective in reducing the number of non-inflammatory and inflammatory lesions (including and excluding the nose), and the 4% Nitricil NVN1 gel group was effective in reducing the number of non-inflammatory lesions (including and excluding the nose) and inflammatory lesions. The 4% Nitricil NVN1 gel group demonstrated a statistically significant difference compared to the vehicle gel group, and the 4% Nitricil NVN1 gel group was different from the vehicle group in the primary and secondary endpoints. Both the 1% Nitricil NVN1 gel and the 4% Nitricil NVN1 gel were safe and well tolerated, and no safety signals were found.</p><p>[Experimental Example 8]</p><p>Three American Board of Dermatology certified dermatologists with experience conducting clinical trials for acne conducted a post-hoc Investigator's Global Assessment (IGA) analysis based on the study described in Example 7. The purpose of this analysis was to have investigators with experience conducting clinical trials for US approval evaluate the results of treatment. A description of the IGA scoring used in Examples 7 and 8 is provided in Table 16. "Success" was defined as a score of "0" (lesions cleared) or "1" (lesions almost cleared) at the end of treatment with a change of at least 2 grades from baseline.</p><p><tables><img file="JP7507204B2_D0018.tif" /></tables></p><p>Three American Board of Dermatology certified dermatologists conducted a blinded review of the subjects' available clinical images at both baseline and week 12 and "scored" the images taken at baseline and week 12. The results are shown in Table 17 below. All three dermatologists determined that Nitricil NVN1 Gel, especially 4% Nitricil NVN1 Gel, was active in treating acne vulgaris.</p><p><tables><img file="JP7507204B2_D0019.tif" /></tables></p><p>[Experimental Example 9]</p><p>Cosmetically elegant compositions containing benzoyl peroxide were prepared. Each composition contained two parts, one part was a benzoyl peroxide gel containing carboxypolymethylene, and the second part was a composition containing cellulose. Each part was filled into one chamber of 2 x 15mL dual chamber pumps manufactured by YonWoo. The benzoyl peroxide gel formulations are shown in Tables 18 and 19, and the cellulose compositions for combining with each of the two benzoyl peroxide gel formulations separately are shown in Table 20.</p><p><tables><img file="JP7507204B2_D0020.tif" /></tables></p><p><tables><img file="JP7507204B2_D0021.tif" /></tables></p><p><tables><img file="JP7507204B2_D0022.tif" /></tables></p><p>The above description is illustrative of the present invention and should not be construed as limiting the present invention. The present invention is defined by the following claims, with equivalents of the claims intended to be included within their scope. All publications, patent applications, patents, patent publications, and other references cited herein are hereby incorporated by reference in their entirety for the teachings relevant to the sentence and/or paragraph in which the reference occurs.</p><p><u style="Single">[Embodiment 1]</u><u style="Single"> A first thickening agent; and</u><u style="Single"> at least one polyhydric alcohol;</u><u style="Single"> at least one buffering agent;</u><u style="Single"> At least one preservative;</u><u style="Single"> A second thickening agent; and</u><u style="Single"> at least one organic solvent;</u><u style="Single"> At least one moisturizer;</u><u style="Single"> At least one active pharmaceutical ingredient</u><u style="Single"> water and</u><u style="Single">Including,</u><u style="Single"> The composition is buffered to a pH of from about 3 to about 8.</u><u style="Single">[Embodiment 2]</u><u style="Single"> The composition of embodiment 1, wherein the at least one active pharmaceutical ingredient, the second thickening agent, the at least one organic solvent, and the at least one humectant are present in the composition in admixture with the at least one polyhydric alcohol, the first thickening agent, the at least one preservative, the at least one buffering agent, and the water.</u><u style="Single">[Embodiment 3]</u><u style="Single"> The composition of embodiment 2, wherein the admixture comprises one part consisting of the at least one nitric oxide-releasing active pharmaceutical ingredient, the second thickening agent, the at least one organic solvent, and the at least one humectant, and a third part consisting of the at least one polyhydric alcohol, the first thickening agent, the at least one preservative, the at least one buffering agent, and the water.</u><u style="Single">[Embodiment 4]</u><u style="Single"> The composition of embodiment 2, wherein the admixture is formed by mixing.</u><u style="Single">[Embodiment 5]</u><u style="Single"> The composition of embodiment 1, wherein the active pharmaceutical ingredient comprises a nitric oxide-releasing active pharmaceutical ingredient.</u><u style="Single">[Embodiment 6]</u><u style="Single"> The composition of embodiment 5, wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a nitric oxide-releasing compound having a diazeniumdiolate functional group.</u><u style="Single">[Embodiment 7]</u><u style="Single"> The composition of embodiment 6, wherein the nitric oxide-releasing compound comprises NO-releasing co-condensed silica particles.</u><u style="Single">[Embodiment 8]</u><u style="Single"> The composition of embodiment 1, further comprising at least one water repellent agent.</u><u style="Single">[Embodiment Item 9]</u><u style="Single"> The composition of embodiment 1, further comprising at least one neutralizing agent.</u><u style="Single">[Embodiment 10]</u><u style="Single"> The composition of embodiment 1, wherein the composition has a pH of from about 3 to about 8 when topically applied to the skin of a subject.</u><u style="Single">[Embodiment 11]</u><u style="Single"> The composition of embodiment 1, which is cosmetically elegant.</u><u style="Single">[Embodiment 12]</u><u style="Single"> The composition of embodiment 5, wherein the at least one nitric oxide-releasing active pharmaceutical ingredient releases nitric oxide in an amount of at least about 50% over a period of about 3 hours or less after topical application of the composition to the skin of a subject.</u><u style="Single">[Embodiment 13]</u><u style="Single"> 1. A composition comprising:</u><u style="Single"> at least one polyhydric alcohol present in an amount from about 1% to about 30% by weight of the composition;</u><u style="Single"> At least one thickening agent present in an amount from about 0.1% to about 5% by weight of the composition;</u><u style="Single"> At least one buffering agent present in an amount from about 0.01% to about 2% by weight of the composition;</u><u style="Single"> at least one preservative present in an amount from about 0.01% to about 1% by weight of the composition;</u><u style="Single"> and water present in an amount from about 70% to about 99% by weight of the composition;</u><u style="Single"> The composition is buffered to a pH of from about 3 to about 8.</u><u style="Single">[Embodiment 14]</u><u style="Single"> 14. The composition of embodiment 13, wherein the at least one polyhydric alcohol is selected from the group consisting of glycerol, propylene glycol, polypropylene glycol, neopental glycol, triethanolamine, diethanolamine, ethanolamione, butylene glycol, polyethylene glycol, n-methyldiethanolamine, isopropanolamine, and any combination thereof.</u><u style="Single">[Embodiment Item 15]</u><u style="Single"> The composition of embodiment 13, wherein the at least one polyhydric alcohol is anhydrous glycerol.</u><u style="Single">[Embodiment 16]</u><u style="Single"> 14. The composition of embodiment 13, wherein the at least one thickening agent is selected from the group consisting of carboxypolymethylene, polyacrylic acid polymers, cellulose ethers, methacrylates, polyvinylpyrrolidones, polysaccharides, gums, proteins, starches, and any combination thereof.</u><u style="Single">[Embodiment 17]</u><u style="Single"> 14. The composition of embodiment 13, wherein the at least one thickening agent is carboxypolymethylene.</u><u style="Single">[Embodiment 18]</u><u style="Single"> The composition of embodiment 13, wherein the preservative is selected from the group consisting of sorbic acid, benzoic acid, methylparaben, propylparaben, and any combination thereof.</u><u style="Single">[Embodiment 19]</u><u style="Single"> The composition of embodiment 13, further comprising a neutralizing agent.</u><u style="Single">[Embodiment 20]</u><u style="Single"> The composition of embodiment 19, wherein the neutralizing agent is present in an amount sufficient to adjust the pH of the composition to a pH of from about 3 to about 8.</u><u style="Single">[Embodiment 21]</u><u style="Single"> The composition of embodiment 19, wherein the neutralizing agent is selected from the group consisting of trolamine, sodium carbonate, tromethamine, aminomethylpropanol, triisopropanolamine, aminomethylpropanol, tetrahydroxypropylethylenediamine, tetrasodium EDTA, suttside A, and any combination thereof.</u><u style="Single">[Embodiment 22]</u><u style="Single"> The composition of embodiment 13, wherein the composition is antibacterial.</u><u style="Single">[Embodiment 23]</u><u style="Single"> The composition of embodiment 13, wherein the composition is in the form of a hydrogel.</u><u style="Single">[Embodiment 24]</u><u style="Single"> The composition of embodiment 13, wherein the composition has a viscosity of about 5,000 cP to about 25,000 cP.</u><u style="Single">[Embodiment 25]</u><u style="Single"> The composition of embodiment 13, wherein the composition is configured to modulate at least one of the pH of the second composition and the release of an active pharmaceutical ingredient present in the second composition.</u><u style="Single">[Embodiment 26]</u><u style="Single"> 26. The composition of embodiment 25, wherein the active pharmaceutical ingredient is a nitric oxide-releasing active pharmaceutical ingredient.</u><u style="Single">[Embodiment 27]</u><u style="Single"> 27. The composition of embodiment 26, wherein the nitric oxide-releasing active pharmaceutical ingredient comprises a nitric oxide-releasing compound having a diazeniumdiolate functional group.</u><u style="Single">[Embodiment 28]</u><u style="Single"> 28. The composition of embodiment 27, wherein said nitric oxide-releasing compound comprises NO-releasing co-condensed silica particles.</u><u style="Single">[Embodiment 29]</u><u style="Single"> 26. The composition of embodiment 25, wherein the second composition is an anhydrous composition.</u><u style="Single">[Embodiment 30]</u><u style="Single"> the second composition is an anhydrous composition;</u><u style="Single"> a second thickening agent present in the second composition in an amount from about 0.5% to about 30% by weight of the composition;</u><u style="Single"> at least one organic solvent present in said second composition in an amount from about 50% to about 90% by weight of said composition;</u><u style="Single"> and at least one moisturizer present in said second composition in an amount from about 2% to about 20% by weight of the composition.</u><u style="Single">[Embodiment 31]</u><u style="Single"> at least one polyhydric alcohol present in an amount from about 1% to about 30% by weight of the composition;</u><u style="Single"> At least one thickening agent present in an amount from about 0.1% to about 5% by weight of the composition; and</u><u style="Single"> a first composition comprising water present in an amount from about 70% to about 99% by weight of the composition;</u><u style="Single"> a second composition which is anhydrous;</u><u style="Single">A kit comprising:</u><u style="Single">[Embodiment 32]</u><u style="Single"> The second composition comprises:</u><u style="Single"> a second thickening agent present in said second composition in an amount from about 0.5% to about 30% by weight of said composition;</u><u style="Single"> at least one organic solvent present in the second composition in an amount from about 50% to about 90% by weight of the composition; and</u><u style="Single"> at least one moisturizing agent present in said second composition in an amount from about 2% to about 20% by weight of said composition;</u><u style="Single">32. The kit of embodiment 31, comprising:</u><u style="Single">[Embodiment 33]</u><u style="Single"> 33. The kit of embodiment 32, wherein said second composition comprises a nitric oxide-releasing active pharmaceutical ingredient.</u><u style="Single">[Embodiment 34]</u><u style="Single"> 34. The kit of embodiment 33, wherein said nitric oxide-releasing active pharmaceutical ingredient comprises a nitric oxide-releasing compound having a diazeniumdiolate functional group.</u><u style="Single">[Embodiment 35]</u><u style="Single"> 35. The kit of embodiment 34, wherein said nitric oxide-releasing compound comprises NO-releasing co-condensed silica particles.</u><u style="Single">[Embodiment 36]</u><u style="Single"> 32. The kit of embodiment 31, wherein said first composition and second composition are stored separately.</u><u style="Single">[Embodiment 37]</u><u style="Single"> 1. A method for enhancing the release of nitric oxide from a topical anhydrous gel containing a diazeniumdiolate-modified macromolecule, comprising:</u><u style="Single"> contacting the topical anhydrous gel with a hydrogel having a pH of from 4 to about 6 to form a composite composition;</u><u style="Single"> applying the composite composition to the skin of a subject;</u><u style="Single">The method includes:</u><u style="Single">[Embodiment 38]</u><u style="Single"> The method of embodiment 37, wherein the contacting step is performed on the skin of the subject.</u><u style="Single">[Embodiment 39]</u><u style="Single"> 38. The method of embodiment 37, wherein said contacting step occurs prior to applying said combination composition to the skin of said subject.</u><u style="Single">[Embodiment 40]</u><u style="Single"> 35. The method of embodiment 34, wherein the hydrogel comprises a pH buffered hydrogel.</u><u style="Single">[Embodiment 41]</u><u style="Single"> a topical anhydrous gel comprising diazeniumdiolate-modified polysiloxane molecules;</u><u style="Single"> a hydrogel comprising a means for reducing the pH of said topical anhydrous gel;</u><u style="Single">13. A pharmaceutical composition comprising:</u><u style="Single">[Embodiment 42]</u><u style="Single"> 42. The pharmaceutical composition of embodiment 41, wherein said hydrogel further comprises a means for releasing nitric oxide from said diazeniumdiolate-modified polysiloxane macromolecule.</u></p>