Title: NITRIC OXIDE AMINO ACID ESTERS FOR SKIN REJUVENATION

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority from US Provisional patent applications No. 61/378,097, filed August 30, 2010, and No. 61/392,998, filed on October 14, 2010, the specifications of which are hereby incorporated by reference.

BACKGROUND

(a) Field

[0002] The subject matter disclosed generally relates to compositions and methods for skin rejuvenation, and more particularly to compositions and methods for skin rejuvenation comprising a nitric oxide amino acid ester compound and a healing promoting agent, and a dermal circulation enhancer.

(b) Related Prior Art

[0003] Skin care can be of particular importance in improving or enhancing the appearance of men and women. Various products and methods can be used to care for skin. For example, exfoliant scrubs, cleansers, and lotions are sometimes used to maintain healthy-looking skin. Exfoliant scrubs can be used to remove dead skin cells from the surface of the skin, which can give the skin an improved tone. Soaps and other cleansers can be used to remove dirt and excess oil from the skin, which can help prevent clogging of pores. Consequently, acne and other types of skin blemishes can be prevented in some cases. Lotions and various other topical ointments can also be used to deliver nutrients and/or moisturizers to the skin in an effort to improve the appearance and/or the health of the skin. Other types of cosmetic products (e.g., creams and lotions) or drug actives are sometimes used in an attempt to eliminate wrinkling and other signs of aging.

[0004] Nitric oxide (NO) is synthesized from L-Arginine through the action of the enzyme NOs (nitric oxide synthetase). NO is synthesized by different NOs enzymes: nNOs (neuronal NOs), which is present in the cytoplasm of the parasympathetic nerves, and eNOs (endothelial NOs), found in the endothelium of the blood vessels and trabecular tissue, which mainly seems to bond to the cell membranes. Numerous experiments have demonstrated that stimulation of the parasympathetic nerves leads to the release of NO as a result of direct action by the nerve endings (reaction catalyzed by nNOs) and indirect action resulting from the effect of Ach, released by the parasympathetic nerves, on the vascular endothelium, with stimulation of eNos.

[0005] Nitric oxide molecules, when combined to a wide variety of drugs are believed to exert a synergistic effect that improves the potency of the coadministered drug. This synergistic effect allows for the use of smaller doses of the drug, and/or improves the potency of the drug to achieve better treatment effects.

[0006] It is thus desirable to provide a composition and method for the skin rejuvenation which contains a Nitric Oxide releasing compound, and does not require any special operational procedures other than the application of a composition onto the tissue that needs to be rejuvenated.

SUMMARY

[0007] The invention features compositions and methods for rejuvenating skin. As used herein, skin rejuvenation includes one or more of the following: reducing the appearance of fine lines and wrinkles; reducing deep wrinkles; enhancing skin tone and color and elasticity; reducing skin blemishes and/or age spots; reducing skin roughness; and producing a younger looking skin. The reduction in nitric oxide levels in the dermis and epidermis has been correlated with skin aging, wrinkles and loss of collagen. Nitric oxide has a well known vasodilator/ function, which may contribute to increase the microcirculation in blood vessels of the epidermis and the dermis.

[0008] In a first embodiment there is disclosed a skin rejuvenation composition comprising:

[0009] · an effective amount of a compound of formula (I):

[0010] [0011] wherein n may be 1 to 10;

[0012] wherein Ri may be an amino acid side chain group (D or L configuration),

[0013] wherein R ₂ may be a hydrogen atom, or an amino acid (D or L configuration) forming a peptide bond;

[0014] or any pharmaceutically acceptable salts thereof;

[0015] · at least one healing promoting agent, and

[0016] · at least one dermal circulation enhancer,

[0017] in association with a pharmaceutically acceptable topical carrier.

[0018] The compound of formula (I) may be (2-nitrooxy)-2-ethylamino-3- methylbutanoate:

[0020] or any pharmaceutically acceptable salts thereof.

[0021] The compound of formula (I) may be L-(2'-nitrooxyethyl)-2-amino-3- methylbutanoate:

[0023] or any pharmaceutically acceptable salts thereof.

[0024] The compound of formula (I) may be D-(2'-nitrooxyethyl-2-amino-3- methylbutanoate:

[0025]

[0026] or any pharmaceutically acceptable salts thereof.

[0027] The compound of formula (I) may be valine butylene glycol nitrate:

[0029] or any pharmaceutically acceptable salts thereof.

[0030] The compound of formula (I) may be 2'-nitrooxy ethyl 2- pentanoate:

[0032] or any pharmaceutically acceptable salts thereof.

[0033] The compound of formula (I) may be L-(2'-nitrooxyethyl)-2-amino- pentanoate:

[0035] or any pharmaceutically acceptable salts thereof. [0036] The compound of formula (I) may be 2'-nitrooxy butyl 2-amino- pentanoate:

[0038] or any pharmaceutically acceptable salts thereof.

[0039] The R ₂ may be a hydrogen atom.

[0040] The Ri may be chosen from:

H ₂

wc CH ₂ CH ₂ vwc CH ₂ C NH ₂

H ₂

vwCH ₂ COOH wc CH ₂ COOH H,N CH,

[0042] wherein when Ri may be « νν , said Ri is also linked to an NH ₂ of said Formula (I) to form a proline or hydroxyproline amino acid side chain.

[0043] The R ₂ may be an amino acid of formula (II) (D or L configuration) and derivatives thereof, forming a peptide bond:

H H O

H N C C O H

[0044] Rx (Π)

[0045] wherein R _x is chosen from:

CH

H

wv*C CH ₃ H ₂ wc C NH ₂

H ₂ II

-SH

OH H

H ₂

- wc CH ₂ CH ₂ vwC CH ₂ C NH ₂

H ₂

wv ^> CH ₂ COOH w -c CH ₂ COOH H,N CH,

[0047] wherein when Ri is said Ri is also linked to an NH ₂ of said Formula (I) to form a proline or hydroxyproline amino acid side chain.

[0048] The at least one healing promoting agent may be chosen from collagen hydrolysate, allantoin, hyaluronic acid, elastin, and ascorbyl palmitate.

[0049] The composition may further comprise a dermal circulation enhancer that may be chosen from gingko biloba, ginger, ethyl alcohol, arginine, cayenne and combinations thereof.

[0050] The composition may be further comprising a blend of amino acid and the blend of amino acid may comprise at least two amino acid chosen from Glycine, Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tyrosine, Tryptophane, Serine, Threonine, Cysteine, Methionine, Proline, Asparagine, Glutamine, Aspartic acid, Glutamic acid, Lysine, Histidine, Arginine, Cystine, Hydroxyproline, ε-Ν-methyllysine, diiodotyrosine, homocysteine, ornithine, Norvaline, selenocysteine, Hypusine, Dehydroalanine, and β-alanine.

[0051] The blend of amino acid may be from about 1 % (w/w) to about 5% (w/w) of said composition.

[0052] The pharmaceutically acceptable carrier may be a topical carrier.

[0053] The pharmaceutically acceptable topical carrier may be chosen from a cream, a gel and a lotion.

[0054] The composition may be a transdermal composition.

[0055] The composition may be further comprising a source of sulfur. [0056] The source of sulfur may comprise at least one of alpha-lipoic acid, chondroitin, glucosamine sulfate, glutathione, methylsulfonylmethane, N- acetylcysteine, S-adenosyl-L-methionine, a garlic extract, and garlic powder.

[0057] The pharmaceutically acceptable topical carrier may be chosen from a water base or an oil, base carrier.

[0058] The composition may be further comprising an anti-oxidants chosen from glutathione, vitamin C, alpha lipoic acid, beta-carotene, alpha-tocopherols, lutein and combinations thereof.

[0059] The composition may be further comprising a moisturizer chosen from stearic acid, myrestyl alcohol, white petrolatum, glycerin, lanolin, hydrogenated polydecene, cetearyl alcohol, panthenol and combinations thereof.

[0060] The composition may be further comprising a humectant chosen from glyceryl triacetate, sorbitol, quillaia, urea, glycerin, lactic acid, aloe vera, propylene glycol and combinations thereof.

[0061] The composition may be further comprising an emollient chosen from butyrospermum parkii oil, licithin, olive oil, glyceryl stearate, stearyl alcohol, cetyl alcohol, behenyl alcohol, limnanthes alba seed oil, palmitic acid and combinations thereof.

[0062] The composition may be further comprising a dermal circulation enhancer selected from the group consisting of gingko biloba, ginger, ethyl alcohol, arginine, cayenne and combinations thereof.

[0063] The composition may be further comprising a vitamin chosen from vitamin A, biotin, vitamin E, vitamin C, vitamin D, nicotinamide, niacin and combinations thereof.

[0064] The composition may be further comprising a mineral chosen from zinc, sodium, potassium, selenium, manganese, copper, calcium and combinations thereof.

[0065] The composition may be further comprising an emulsifier chosen from sodium lauryl sulfate, trideceth-6, pluronic acid F-127, polyacrylate sodium, triethanolamin, hydroxyethylcetearamidopropyl dimonium chloride and combinations thereof.

[0066] The composition may be further comprising a lubricant and the lubricant may be chosen from glycerol, sorbitol, a water soluble cellulose, a polysorbate, a carbomer, a polyethylene glycol (PEG), a polyethylene, polydimethylsiloxane and a thickening agent.

[0067] The water soluble cellulose may be chosen from modified starch, methylcellulose, hydroxypropyl cellulose, hydroxypropyl methylcellulose, methocel® MC, carboxymethyl cellulose, ethyl cellulose, hydroxyl ethyl cellulose, and any combination thereof.

[0068] The polysorbate may be chosen from polyoxyethylene (20) sorbitan monolaurate (polysorbate 20), polyoxyethylene (20) sorbitan monopalmitate (polysorbate 40), polyoxyethylene (20) sorbitan monostearate (polysorban 60), polyoxyethylene (20) sorbitan tristrearate (polysorban 65), and polyoxyethylene (20) sorbitan monooleate (polysorban 80), and any combination thereof.

[0069] The carbomer may be a carbopol® polymer chosen from carbopol® polymer 71 G NF, carbopol® polymer 971 P NF, carbopol® polymer 974P NF, carbopol® polymer 980 NF, carbopol® polymer 981 NF, carbopol® polymer 5984 EP and carbopol® polymer Ultrez 10 NF, and any combination thereof.

[0070] The polyethylene glycol (PEG) may be chosen from PEG 200, PEG 200E, PEG 300, PEG 300E, PEG 400, PEG 400E, PEG 600 and PEG 600E, and any combination thereof.

[0071] The thickening agent may be chosen from alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, locust bean gum, xanthan gum, pectin, and gelatin, and any combination thereof.

[0072] The composition may be further comprising at least one antiseptic agent and the antiseptic agent may be selected from chlorhexidine gluconate, glucono delta-lactone, a paraben compound, benzoic acid, imidazolidinyl urea, a quaternary ammonium compound, and Octenidine dihydrochloride. [0073] The composition may be further comprising a preservative agent.

[0074] The preservative agent may be chosen from EDTA, EGTA, hydroxytoluene butoxide, hydroxyanisol butoxide, sodium hydroxide, calcium propionate, sodium nitrate, sodium nitrite, sulfur dioxide, sodium bisulfite, and potassium hydrogen sulfite.

[0075] The composition may be further comprising an absorption enhancer.

[0076] The absorption enhancer may be chosen from triglycerides of coconut oil, dimethylsulfoxide (DMSO) isopropyl palmitate, isopropyl myristate, laurocapram, glycerol, propylene glycol and derivatives thereof.

[0077] The composition may be a transdermal^ absorbed composition.

[0078] According to another embodiment, there is disclosed a method of skin rejuvenation in a patient which comprises:

[0079] (a) topically treating a patient with a therapeutically effective amount of a compound of formula (I):

[0081] wherein n may be 1 to 10;

[0082] wherein Ri may be an amino acid side chain group (D or L configuration),

[0083] wherein R ₂ may be a hydrogen atom, or an amino acid (D or L configuration) forming a peptide bond, or any pharmaceutically acceptable salts thereof; [0084] The compound of formula (I) may be (2-nitrooxy)-2-ethylamino-3- methylbutanoate:

[0086] or any pharmaceutically acceptable salts thereof.

[0087] The compound of formula (I) may be L-(2'-nitrooxyethyl)-2-amino-3- methylbutanoate:

[0089] or any pharmaceutically acceptable salts thereof.

[0090] The compound of formula (I) may be D-(2'-nitrooxyethyl-2-amino-3- methylbutanoate:

[0091]

[0092] or any pharmaceutically acceptable salts thereof.

[0093] The compound of formula (I) may be 2'-nitrooxyethyl 2-amino- pentanoate:

[0095] or any pharmaceutically acceptable salts thereof.

[0096] The compound of formula (I) may be L-(2'-nitrooxyethyl)-2-amino- pentanoate:

[0098] or any pharmaceutically acceptable salts thereof.

[0099] The R ₂ may be a hydrogen atom.

[00100] The Ri may be chosen from:

NH _{2 ^} w^cH ₂ CH ₂ CH _{3 t} H ₂ _ _{and H;N} [00102] wherein when Ri may be , or , said Ri is also linked to an NH ₂ of said Formula (I) to form a proline or hydroxyproline amino acid side chain.

[00103] The R ₂ may be an amino acid of formula (II) (D or L configuration) forming a peptide bond:

H H O

H N C C— O H

[Q0104] (II)

[00105] wherein R _x may be chosen from

w CH ₂

H ^c

vwC- H ₂ wc C NH ₂

II

v c SH

OH H ₂

H ₂

H ₂ vwc CH ₂ CH ₂ CH ₂ C NH ₂

H ₂

vwCH ₂ COOH w-C CH ₂ COOH H ₂ N CH ₂

NH ₂ _ w^CH ₂ CH ₂ CH _{3 (} H _{2 > anc} j _H;N -

[00107] wherein when Ri may be said Ri is also linked to an NH ₂ of said Formula (I) to form a proline or hydroxyproline amino acid side chain.

[00108] According to another embodiment, there is disclosed a use of a composition according to the present invention for the preparation of a medicament for the rejuvenation of skin.

[00109] According to another embodiment, there is disclosed a use of a therapeutically effective amount of a compound of formula (I):

[00111] wherein n may be 1 to 10;

[00112] wherein R-i may be an amino acid side chain group (D or L configuration),

[00113] wherein R ₂ may be a hydrogen atom, or an amino acid (D or L configuration) forming a peptide bond, or any pharmaceutically acceptable salts thereof; for skin rejuvenation. [00114] According to another embodiment, there is disclosed a use of a therapeutically effective amount of a compound of formula (I):

[00116] wherein n may be 1 to 10;

[00117] wherein Ri may be an amino acid side chain group (D or L configuration),

[00118] wherein R ₂ may be a hydrogen atom, or an amino acid (D or L configuration) forming a peptide bond, or any pharmaceutically acceptable salts thereof; for skin rejuvenation.

[00119] The compound of formula (I) may be (2-nitrooxy)-2-ethylamino-3- methylbutanoate:

[00121] or any pharmaceutically acceptable salts thereof.

[00122] The compound of formula (I) may be L-(2'-nitrooxyethyl)-2-amino-3- methylbutanoate:

[00124] or any pharmaceutically acceptable salts thereof. [00125] The compound of formula (I) may be D-(2'-nitrooxyethyl-2-amino-3- methylbutanoate:

[00126]

[00127] or any pharmaceutically acceptable salts thereof.

[00128] The compound of formula (I) may be 2'-nitrooxyethyl 2-amino- pentanoate:

or any pharmaceutically acceptable salts thereof.

The compound of formula (I) may be L-(2'-nitrooxyethyl)-2-

[00133] or any pharmaceutically acceptable salts thereof.

[00134] The R ₂ may be a hydrogen atom.

[00135] The Ri may be chosen from: File No. P1388PC00

NH ₂ vCH ₂ CH ₂ CH _{3 f 2} _ _anc j

[00137] wherein when R may be said Ri is also linked to an NH ₂ of said Formula (I) to form a proline or hydroxyproline amino acid side chain.

[00138] The R _∑ may be an amino acid of formula (II) (D or L configuration) forming a peptide bond: H H O

H N C— C— O— H

[00139] (II)

[00140] wherein R _x may be chosen from

[00142] wherein when said Ri is also linked to an NH ₂ of said Formula (I) to form a proline or hydroxyproline amino acid side chain. [00143] The pharmaceutically acceptable topical carrier of the composition may be a cream, a gel and a lotion.

[00144] In yet another embodiment, there is disclosed use of a composition for skin rejuvenation. The pharmaceutically acceptable topical carrier used in the composition may be chosen from a cream, a gel and a lotion.

[00145] The following terms are defined below.

[00146] The term "Amino acid ester compound" is intended to mean the condensation product of an amino acid with mononitrated alkane ou aikene diol. As will be evident to those familiar to the art, the condensation reaction could also involve, but not limited to, dipeptides or tripeptides, nitrated alcohols containing aliphatic, alkyl or aromatic moieties, as well as other nitric oxide groups attached to the alkane or aikene diols. Amino acid or dipeptide reactions are preferred as well as the condensation reaction with short chain mononitrated alkane diols such as 1 ,3 propanediol or 1 ,4 butanediol.

[00147] The expression "Therapeutically effective amount" is intended to mean the amount of the compound and/or composition that is effective to achieve its intended purpose.

[00148] The expression "Transdermally absorbed" is intended to mean the delivery of a compound by passage through the skin and into the blood stream.

[00149] The term "Transmucosal" is intended to mean the delivery of a compound by passage of the compound through the mucosal tissue and into the blood stream.

[00150] The terms "Carriers" or "vehicles" are intended to mean carrier materials suitable for compound administration and include any such material known in the art such as, for example, any liquid, lotion, gel, solvent, liquid diluent, solubilizer, or the like, which is non-toxic and which does not interact with any components of the composition in a deleterious manner.

[00151] The term "Nitric oxide adduct" or "NO adduct" is intended to mean compounds and functional groups which, under physiological conditions, can donate, release and/or directly or indirectly transfer any of the three redox forms of nitrogen monoxide (NO ⁺ , NO ^" , NO ^' ), such that the biological activity of the nitrogen monoxide species is expressed at the intended site of action.

[00152] The term "Nitric oxide releasing" or "nitric oxide donating" is intended to mean methods of donating, releasing and/or directly or indirectly transferring any of the three redox forms of nitrogen monoxide (NO+, NO-, NO ^' ), such that the biological activity of the nitrogen monoxide species is expressed at the intended site of action.

[00153] The term "Nitric oxide donor" or "NO donor" is intended to mean compounds that donate, release and/or directly or indirectly transfer a nitrogen monoxide species, and/or stimulate the endogenous production of nitric oxide or endothelium-derived relaxing factor (EDRF) in vivo and/or elevate endogenous levels of nitric oxide or EDRF in vivo and/or are oxidized to produce nitric oxide and/or are substrates for nitric oxide synthase and/or cytochrome P450. "NO donor" also includes compounds that are precursors of L-arginine, inhibitors of the enzyme arginase and nitric oxide mediators.

[00154] The term "pharmaceutical acceptable carrier" is intended to mean a preservative solution, a saline solution, an isotonic (about 0.9%) saline solution, or about a 5% albumin solution, suspension, sterile water, phosphate buffered saline, and the like. Other buffering agents, dispersing agents, and inert non-toxic substances suitable for delivery to a patient may be included in the compositions of the present invention. The compositions may be solutions, suspensions or any appropriate formulation suitable for administration, and are typically sterile and free of undesirable particulate matter. The compositions may be sterilized by conventional sterilization techniques.

[00155] The term "lubricant" is intended to mean a substance (often a liquid) introduced between two moving surfaces to reduce the friction between them, hydrate the surface as well as reducing wear of the body parts.

[00156] The term "rejuvenation" is intended to mean the reversal or mitigation of the aging process, or any other processes (e.g. abrasion caused by a fall, burns, etc.) that may have damaged or caused an accumulation of damage to macromolecules, cells, tissues and organs, including the skin. Rejuvenation is the repair of any of such damage. [00157] Features and advantages of the subject matter hereof will become more apparent in light of the following detailed description of selected embodiments, as illustrated in the accompanying figures. As will be realized, the subject matter disclosed and claimed is capable of modifications in various respects, all without departing from the scope of the claims. Accordingly, the drawings and the description are to be regarded as illustrative in nature, and not as restrictive and the full scope of the subject matter is set forth in the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[00158] In embodiments there is disclosed a skin rejuvenation composition containing a nitric oxide amino acid ester compound, at least one healing promoting factor and at least one dermal enhancer, in association with a pharmaceutically acceptable topical carrier.

[00159] In another embodiment there is disclosed a method of skin rejuvenation in a patient by topically treating said patient with a composition containing a nitric oxide amino acid ester compound and at least one healing promoting factor and at least one dermal enhancer, in association with a pharmaceutically acceptable topical carrier.

[00160] In yet another embodiment, there is disclosed a use of a composition containing a nitric oxide amino acid ester compound and at least one healing promoting factor and at least one dermal enhancer, in association with a pharmaceutically acceptable topical carrier for skin rejuvenation.

[00161] The composition of the present invention contains vasoactive amino acid ester compounds. The compounds may be D- or L-isomers. The D- or L-nitric oxide amino acid ester compounds of the present invention possess many of the required characteristics necessary to fulfill the role of a primary boosting of NO levels. The compounds easily dissociate in water into the amino acid derivative and associated ion forming the pharmaceutical salt. The compounds of the present invention are extremely stable in the form of the salts, and thus possess long shelf lives and stability.

[00162] The nitric oxide releasing groups of the compounds of the present invention are preferably nitro groups (i.e. N0 ₂ ), nitroso groups (i.e. NO) and/or heterocyclic nitric oxide donor groups that are linked to the amino acid ester compounds through one or more sites such as oxygen (hydroxyl condensation), sulfur (sulfhydryl condensation) and/or nitrogen. The heterocyclic nitric oxide donor groups are preferably furoxans, sydnonimines, oxatriazole-5-ones and/or oxatriazole-5-imines.

[00163] Amino acids molecules are actively transported across cell membranes by transporters such as the PEPT1 transporter that mediates their uptake. Without wishing to be bound by theory, it is believed that the fusion of a nitric oxide donating moiety to an amino acid molecule enhances the absorption of the compound through the active transport of the compound as an amino acid, and enhance the bioavailability of such compound. Branched chain amino acids such as leucine, isoleucine and valine are believed to have the most preferred absorption patterns.

[00164] Ester bonds are vulnerable to degradation by carboxylesterase enzymes that are for example abundantly found in the liver. Degradation of the compounds before their absorption may cause release of the active moiety before it reaches systemic circulation, and therefore decrease the amount of actual effective compound present to treat a condition. This represents a net disadvantage. The rate of hydrolysis (degradation) of compounds in vivo may sometime be changed by effecting some manipulation to the stereochemistry of compounds in order to increase or decrease their rate of degradation as may be desired. According to the present invention, using D- or L-isomers of the compounds of the present invention may affect their biological half-life and thus render them better suited for certain treatment, as may be desired.

[00165] As used herein a "slow acting" NO precursor may provide for the release of NO in the tissues over the course of a few hours to several hours after initial exposure to the "slow acting" NO precursor. In contrast, a "fast acting" NO precursor, generally provides almost instantaneous release of detectable levels of NO in the plasma or tissue (e.g., within about a few minutes of exposure to the "fast acting" NO precursor and lasting for several minutes). Such "fast acting" agents may be quickly depleted, however. Thus, a fast acting NO donor may be used to provide a burst of NO, while a slow acting NO donor may be used to provide a more sustained, protracted level of NO release.

[00166] The preferred compounds of the present invention are the valine or norvaline derivatives of the nitric oxide amino acid ester of the present invention. They may be in the D- or L- configuration, depending on the desired release profile and treatment. The most preferred compounds are known as valine nitrooxy ethyl ester (or valine ethylene glycol nitrate), valine nitrooxy butyl ester (or valine butylene glycol nitrate), or any pharmaceutically acceptable salt thereof, which possess many of the required characteristics necessary to fulfill the role of boosting NO levels. The compound easily dissociates in water into the valine derivative valine ethylene or butylene glycol nitrate and the salt forming acid. The compounds are extremely stable in the form of the salt and thus possesses a long shelf life. Another preferred compound is the norvaline derivative of the nitric oxide amino acid ester of the present invention. The most preferred compounds are known as norvaline nitrooxy ethyl ester (or norvaline ethylene glycol nitrate or 2'-nitrooxy ethyl 2-amino- pentanoate), norvaline nitrooxy butyl ester (or norvaline butylene glycol nitrate or 4'- nitrooxybutyl 2-amino-pentanoate), or any pharmaceutically acceptable salts thereof, which possess many of the required characteristics necessary to fulfill the role of boosting NO levels. The compound easily dissociates in water into the norvaline derivative norvaline ethylene or butylene glycol nitrate and the salt forming acid. The compounds are extremely stable in the form of the salt and thus possesses a long shelf life. It has been observed that the preferred compounds of the present invention do not cause hypotension in normotensive or hypotensive individuals. Therefore, upon administration of the preferred compounds of the present invention, an hypertensive individual will experience the vasodilator/ effect caused by the preferred compounds, which will result in a decrease in blood pressure. The decrease in blood pressure may be up to a normotensive blood pressure. Individuals with normal blood pressure will not experience the vasodilator/ effect caused by the preferred compounds, and their blood pressure will remain stable (unchanged). Individuals with lower than normal blood pressure (hypotensive) will not experience a further drop in blood pressure and their blood pressure will remain stable (unchanged). Without wishing to be bound by theory, the compounds of the present invention are believed to be subject to a 2 phase metabolism. The preferred compounds of the present invention have an initial first phase half-life of approximately 1 to about 3 minutes, or from about 1 to about 2.5 minutes, or from about 1 to about 2 minutes, or from about 1 to about 1.5 minutes, or from about 1.5 to about 3 minutes, or from about 1.5 to about 2.5 minutes, or from about 1.5 to about 2 minutes, or from about 2 to about 3 minutes, or from about 2 to about 2.5 minutes, or from about 2.5 to about 3 minutes in the L-configuration, and approximately about 3 to about 6 hours, or from about 3 to about 5 hours, or from about 3 to about 4 hours, or from about 4 to about 6 hours, or from about 4 to about 5 hours, or from about 5 to about 6 in the D-configuration. The half-life of the compounds of the present invention, combined with the half-life of the nitric oxide donating moiety may result in a combined half-life of 9-12 hours or even longer.

[00167] Preferably, a therapeutically effective amount of the D- or L- compounds of the present invention are administered. Therapeutically effective amounts include but are not limited to 0.5 to 200 mg of the compound of the present invention. Preferably, therapeutically effective amounts include 1 to 15 mg, 0.5 to 5 mg, 1 to 5 mg, 5 to 10 mg, 10 to 15 mg, 1 to 15 mg, 1 to 30 mg, 5 to 20 mg, 5 to 15 mg, 5 to 30 mg, 10 to 20 mg, 10 to 30 mg and 15 to 30 mg. Other preferable therapeutically effective amounts also include from about 0.05 mg to about 200 mg, or from about 0.05 mg to about 150 mg, or from about 0.05 mg to about 100 mg, or from about 0.05 mg to about 50 mg, or from about 0.05 mg to about 40 mg, or from about 0.05 to about 30 mg, or from about 0.05 mg to about 20 mg, or from about 0.05 mg to about 15 mg, or from about 0.05 mg to about 10 mg, or from about 0.05 mg to about 5 mg, or from about 0.05 mg to about 1 mg, or from about 0.05 mg to about 0.5 mg, about 0.5 mg to about 200 mg, or from about 0.5 mg to about 150 mg, or from about 0.5 mg to about 100 mg, or from about 0.5 mg to about 50 mg, or from about 0.5 mg to about 40 mg, or from about 0.5 to about 30 mg, or from about 0.5 mg to about 20 mg, or from about 0.5 mg to about 15 mg, or from about 0.5 mg to about 10 mg, or from about 0.5 mg to about 5 mg, or from about 0.5 mg to about 1 mg, about 1 mg to about 200 mg, or from about 1 mg to about 150 mg, or from about 1 mg to about 100 mg, or from about 1 mg to about 50 mg, or from about 1 mg to about 40 mg, or from about 1 to about 30 mg, or from about 1 mg to about 20 mg, or from about 1 mg to about 15 mg, or from about 1 mg to about 10 mg, or from about 1 mg to about 5 mg, about 5 mg to about 200 mg, or from about 5 mg to about 150 mg, or from about 5 mg to about 100 mg, or from about 5 mg to about 50 mg, or from about 5 mg to about 40 mg, or from about 5 to about 30 mg, or from about 5 mg to about 20 mg, or from about 5 mg to about 15 mg, or from about 5 mg to about 10 mg, about 10 mg to about 200 mg, or from about 10 mg to about 150 mg, or from about 10 mg to about 100 mg, or from about 10 mg to about 50 mg, or from about 10 mg to about 40 mg, or from about 10 to about 30 mg, or from about 10 mg to about 20 mg, or from about 10 mg to about 15 mg, about 15 mg to about 200 mg, or from about 15 mg to about 150 mg, or from about 15 mg to about 100 mg, or from about 15 mg to about 50 mg, or from about 15 mg to about 40 mg, or from about 15 to about 30 mg, or from about 15 mg to about 20 mg, about 20 mg to about 200 mg, or from about 20 mg to about 150 mg, or from about 20 mg to about 100 mg, or from about 20 mg to about 50 mg, or from about 20 mg to about 40 mg, or from about 20 to about 30 mg, about 30 mg to about 200 mg, or from about 30 mg to about 150 mg, or from about 30 mg to about 100 mg, or from about 30 mg to about 50 mg, or from about 30 mg to about 40 mg, about 40 mg to about 200 mg, or from about 40 mg to about 150 mg, or from about 40 mg to about 100 mg, or from about 40 mg to about 50 mg, 50 mg to about 200 mg, or from about 50 mg to about 150 mg, or from about 50 mg to about 100 mg, about 100 mg to about 200 mg, or from about 100 mg to about 150 mg, or 150 mg to about 200 mg.

[00168] The compounds and compositions of the invention are described in more detail herein.

[00169] In embodiments, the amino acid ester compounds comprising at least one nitric oxide releasing group of the present invention are D- or L- isomers. The choice of a D- or L- isomer for a given amino acid ester compounds of the present invention will depend on the desired properties of the compound. For example, when an increase in nitric oxide levels is required for a short, rather punctual period of time, a compound with a shorter biological half-life may be desired. On the other hand, when an increase in nitric oxide levels is desired for long periods of time, for example to match the therapeutic duration of an anti-inflammatory compound such as naproxen and modulate its gastric damaging effect, a compound with a longer biological half-life may be desired.

[00170] In embodiments the amino acid ester compounds comprising at least one nitric oxide releasing group, and pharmaceutically acceptable salts thereof, the compounds compound of formula (I) is:

wherein,

n = 1 to 10;

Ri is an amino acid side chain group (D or L configuration), which may be chosen from:

[00171] wherein when R-i is R is also linked to an NH2 of said Formula (I) to form a proline or hydroxyproline amino acid side chain.

[00172] The R ₂ is a hydrogen atom, or an amino acid (D or L configuration) forming a peptide bond, which may be chosen from:

w-c C NH H ₂

wvc CH ₂ C NH ₂

H ₂ II

H vwCH ₂ COOH vw-C CH ₂ COOH

wherein when R ₂ is said R ₂ is also linked to an NH ₂ of said Formula (I) to form a proline or hydroxyproline amino acid side chain. Preferably, the Ri may be chosen from valine, leucine or isoleucine. Preferably, the R ₂ may a hydrogen atom.

[00173] The preferred compounds of formula (I) is:

[00174] L-(2'-nitrooxyethyl-2-amino-3-methylbutanoate:

[00175] or any pharmaceutically acceptable salts thereof.

[00176] Another preferred compound of formula (I) is

[00177] D-(2'-nitrooxyethyl)-2-amino-3-methylbutanoate:

[00178] or any pharmaceutically acceptable salts thereof.

[00179] According to one embodiment, the D-isomers of the compounds of the present invention, depending on the amino acid side chain attached, may exhibit a biological half-life of up to about 6 hours. According to one embodiment, the biological half-life may be from about 3 hours to about 6 hours. According to one embodiment, branched amino acid side chains (e.g. isoleucine, leucine, and valine) may exibit enhanced uptake and biological half-life from about 3 hours to about 6 hours, or from about 4 hours to about 6 hours. The increase in biological half-life of the D-isomers is thought to contribute to a release of the nitric oxide donating moiety over a longer time frame, which is a very desirable property that may result in a more uniform release of nitric oxide over time, and thus a better prophylactic profile. Release over a longer time may also allow preparation of formulations containing the compound that may be only administered twice daily. Longer half-lives are important in certain applications when conditions being treated are persistent ones, such as pulmonary hypertension, hypertension, vascular insufficiencies, and the likes.

[00180] According to some embodiments, the L-isomers of the compounds of the present invention, depending on the amino acid side chain attached, may exhibit a biological half-life of up to about 3 minutes. According to one embodiment, the biological half-life may be from about 1 to about 3 minutes, or from about 1 to about 2.5 minutes, or from about 1 to about 2 minutes, or from about 1 to about 1.5 minutes, or from about 1.5 to about 3 minutes, or from about 1.5 to about 2.5 minutes, or from about 1.5 to about 2 minutes, or from about 2 to about 3 minutes, or from about 2 to about 2.5 minutes, or from about 2.5 to about 3 minutes According to some embodiments, branched amino acid side chains (e.g. isoleucine, leucine, and valine) may exhibit enhanced uptake and biological half-life from about from about 1 minutes to about 3 minutes.

[00181] According to one embodiment, the branched amino acid side chains may exhibit good oral bioavailability.

[00182] According to an embodiment, the compounds of the present invention display very fast onset of nitric oxide release when hard physical activity is performed, and they are capable of long sustained release of nitric oxide for up to 24 hours, with a 40% increase in nitric oxide. Interestingly, the 24 hours sustained release highlights that the molecular mechanism involved in the release of nitric oxide from the D- or L- compounds of the present invention does not involve a mechanism that develops tolerance like other nitric oxide releasing molecules. According to some embodiments, the compounds of the present invention may be administered systemically, for example through oral administration or injection, or locally, for example through transdermal administration. Surprisingly, upon local application the effect of the compounds of the present invention is localized to the tissue where the transdermal formulation has been applied. Without wishing to be bound by theory, it is possible that the mechanism of action of the compounds of the present invention is related to the laminar flow activation of receptors (e.g. GST isoform) in the endothelial cells that metabolize the nitrooxy alcohol to nitric oxide. Thus, the observed localized effect of the compounds suggest that the activation of the compounds of the present invention for release of nitric oxide upon hypertensive conditions may not involve the liver cytosol but rather a localized activation pathway. Furthermore, the sustained release over a period of 24 hours suggests that the nitrooxy alcohol molecules resulting from the breakdown of the compounds of the present invention are being sequestered in the cells of the target tissue(s). These observations also suggest that the physiological effects of the molecule may be related to the two step metabolism of the D- or L- compounds of the present invention. The first step comprises the carboxylesterase cleavage of the D- or L- amino acid moiety from the nitrooxy alcohol, and the second step the metabolism of nitrooxy alcohol in the cytosol of endothelial cells to nitric oxide, which may be glutathione transferase regulated.

[00183] The fast onset of nitric oxide release suggests that the (L-) bond between the amino acid moiety and the nitrooxy alcohol moiety is being quickly cleaved by carboxylesterases enzyme (or carboxylic-ester hydrolase) in the tissue, which have been shown to exist in many tissue types in humans. According to some embodiments of the present invention, the L-isomers of the compounds of the present invention are believed to be more susceptible to carboxylesterase enzymes and to be more rapidly cleaved by these enzymes for fast release of the nitrooxy alcohol moiety in the target tissue.

[00184] According to another embodiment, the D-isomers of the compounds of the present invention are believed to be less susceptible to the carboxylesterase enzymes, affording them greater stability and longer half-lives. The D-isomer compounds, because of their greater stability and longer half-lives may distribute throughout the body into the tissue, to be metabolized to nitrooxy alcohol, which may either be sequestered in the cytosol of the cells for a period of time, or react and be liberated as nitric oxide.

[00185] Compounds of the invention that have one or more asymmetric carbon atoms may exist as the optically pure enantiomers, pure diastereomers, mixtures of enantiomers, mixtures of diastereomers, racemic mixtures of enantiomers, diastereomeric racemates or mixtures of diastereomeric racemates. It is to be understood that the invention anticipates and includes within its scope all such isomers and mixtures thereof.

[00186] Compounds of the invention that have one or more double bounds may exist as a single tautomers or a mixture of tautomers. It is to be understood that the invention anticipates and includes within its scope all such tautomers and mixtures thereof.

[00187] The compounds and compositions of the invention can be formulated as pharmaceutically acceptable salt forms. Pharmaceutically acceptable salts include, for example, alkali metal salts and addition salts of free acids or free bases. The nature of the salt is not critical, provided that it is pharmaceutically-acceptable. Suitable pharmaceutically- acceptable acid addition salts may be prepared from an inorganic acid or from an organic acid. Examples of such inorganic acids include, but are not limited to, hydrochloric, hydrobromic, hydroiodic, nitric, carbonic, sulfuric and phosphoric acid and the like. Appropriate organic acids include, but are not limited to, aliphatic, cycloaliphatic, aromatic, heterocyclic, carboxylic and sulfonic classes of organic acids, such as, for example, formic, acetic, propionic, succinic, glycolic, gluconic, lactic, malic, tartaric, citric, ascorbic, glucuronic, maleic, fumaric, pyruvic, aspartic, glutamic, benzoic, anthranilic, mesylic, salicylic, p-hydroxybenzoic, phenylacetic, mandelic, embonic (pamoic), methanesulfonic, ethanesulfonic, benzenesulfonic, pantothenic, toluenesulfonic, 2-hydroxyethanesulfonic, sulfanilic, stearic, algenic, β-hydroxybutyric, cyclohexylaminosulfonic, galactaric and galacturonic acid and the like. Suitable pharmaceutically-acceptable base addition salts include, but are not limited to, metallic salts made from aluminum, calcium, lithium, magnesium, potassium, sodium and zinc or organic salts made from primary, secondary and tertiary amines, cyclic amines, N, N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine (N- methylglucamine) and procaine and the like. All of these salts may be prepared by conventional means from the corresponding compound by reacting, for example, the appropriate acid or base with the compound. In one embodiment, the pharmaceutically acceptable salts of the compounds of the invention include the nitrate salts. In another embodiment, the pharmaceutically acceptable salts of the compounds of the invention are heterocyclic compounds such as, furoxan, a sydnonimine, an oxatriazole-5-one and/or an oxatriazole-5-imine.

[00188] The compounds of the present invention, because of the small size of the molecule, can be other choices of linkages and/or amino acids or their derivatives. For example, as alternatives to the above choices, propyl, butyl, or longer chains may be linked to any amino acid. Salts such as chloride or hydrochloride salts may be used. Other amino acid derivatives may also be chosen. Derivatives of the base amino acids whether they are in the L or D configuration of these amino acids can be chosen. Non standard amino acids, or synthetic derivative of standard and non-standard amino acids may be elected, such as those containing acetyl groups attached to the amide of the molecule or nor derivatives of the amino acids, when such derivatives can be achieved.

[00189] The amino acid esters compounds may be based on natural, nonstandard or even modified amino acids, with the basic structure as depicted below, where the R _x represents the side chain of the amino acid (wherein R _x may be R-i , R ₂ or R ₃ , as applicable to the specific molecule described herein): _,

H H o

H N C C O H

[00190] ^R x

[00191] Basic amino acid structure File No. P1388PC00

[00192] Natural Amino Acids

^* essential amino acids

[ ino Acids

[00194] The nitric oxide amino acid ester compounds of the present invention are not limited to a single amino acid molecule. The compounds of the present invention may be dipeptide or even tripeptide molecules, with the general formula depicted below and where R _x and R _y independently are any of the amino acid side chains described herein.

[00195] The compounds of the present invention may be formulated in any suitable manner for inclusion in the compositions of the present invention. Preferably, the compounds of the present invention are formulated into liposomes prior to being mixed with the other components of the composition of the present invention.

[00196] The composition containing a compound as defined in the present invention may include a wide variety of additional components, including, for example, one or more of gases, gaseous precursors, liquids, oils, stabilizing materials, pharmaceutical acceptable carriers, photoactive agents.

[00197] The invention provides methods for boosting NO levels for the treatment of skin that needs to be rejuvenated.

[00198] Healing promoting agents

[00199] The composition of the present invention comprises healing promoting agents, which are compounds that promote and enhance the healing or regenerative process of a tissue on the application of a composition according to the present invention. It is thought that the vasodilation caused at the treatment site stimulates the increased absorption of the healing factors comprised in the composition of the present invention. Healing promoting factors include collagen hydrolysate, aldioxa, hyaluronic acid, elastin, ascorbyl palmitate and combinations thereof.

[00200] Dermal circulation enhancers

[00201] Dermal circulation enhancers improve blood circulation into the affected area to enhance the process of natural healing through delivering nutrients carried by the bloodstream to the affected area and contribute to healthy restoration of skin. The dermal circulation enhancers include gingko biloba, ginger, ethyl alcohol, arginine, cayenne and combinations thereof.

[00202] The composition of the present invention is preferably prepared in the form of a cream, a gel or a lotion. The cream form of the present invention may be typical moisturizing creams formulations that are well known in the art, into which the active ingredients of the present invention are incorporated.

[00203] Cream compositions according to the present invention may contain ingredients such as anti-oxidants, moisturizers, humectants, emollients, vitamins, minerals, emulsifiers, and preservatives.

[00204] Blend of amino acids

[00205] According to an embodiment, the composition of the present invention may comprise a blend of amino acid. The blend of amino acid may be incorporated in a composition according to the present invention to favor collagen synthesis or formation. Nitric oxide may enhance the synthesis of collagen of skin fibroblast. The blend of amino acid include at least two of any suitable amino acids. Suitable amino acids include but are not limited to Glycine, Alanine, Valine, Leucine, Isoleucine, Phenylalanine, Tyrosine, Tryptophane, Serine, Threonine, Cysteine, Methionine, Proline, Asparagine, Glutamine, Aspartic acid, Glutamic acid, Lysine, Histidine, Arginine, Cystine, Hydroxyproline, ε-Ν-methyllysine, diiodotyrosine, homocysteine, ornithine, Norvaline, selenocysteine, Hypusine, Dehydroalanine, and β-alanine. Preferably the amino acids in the blends will be L-isomers, although D-isomers may be also incorporated in the blend if desired or as required (e.g. in post-translational modifications). The blend of amino acid may be from about 1% (w/w) to about 5% (w/w), or from about 1 % (w/w) to about 2% (w/w), or from about 1 % (w/w) to about 3% (w/w), or from about 1 % (w/w) to about 4% (w/w), or from about 2% (w/w) to about 5% (w/w), or from about 3% to about 5%, or from about 4% (w/w) to about 5% (w/w) of said composition.

[00206] Source of sulfur

[00207] According to an embodiment, the composition of the present invention may comprise a source of sulfur. The source of sulfur may be incorporated in a composition according to the present invention to favor collagen synthesis or formation. Nitric oxide may enhance the synthesis of collagen of skin fibroblast. The source of sulfur may be at least one of alpha-lipoic acid, chondroitin, glucosamine sulfate, glutathione, methylsulfonylmethane, N-acetylcysteine, S-adenosyl-L- methionine, a garlic extract, and garlic powder.

[00208] Anti-oxidants

[00209] Anti-oxidants include glutathione, vitamin C, alpha lipoic acid, beta- carotene, alpha-tocopherols, lutein and combinations thereof.

[00210] Moisturizer

[00211] Moisturizers include of stearic acid, myrestyl alcohol, white petrolatum, glycerin, lanolin, hydrogenated polydecene, cetearyl alcohol, panthenol and combinations thereof.

[00212] Humectant

[00213] Humectants include glyceryl triacetate, sorbitol, quillaia, urea, glycerin, lactic acid, aloe vera, propylene glycol and combinations thereof.

[00214] Emolient

[00215] The emollients include butyrospermum parkii oil, licithin, olive oil, glyceryl stearate, stearyl alcohol, cetyl alcohol, behenyl alcohol, limnanthes alba seed oil, palmitic acid, dimethiconol and combinations thereof.

[00216] Vitamins

[00217] The vitamins include vitamin A, biotin, vitamin E, vitamin C, vitamin D, nicotinamide, niacin and combinations thereof.

[00218] Minerals

[00219] The minerals include zinc, sodium, potassium, selinium, manganese, copper, calcium and combinations thereof. [00220] Emulsifiers

[00221] The emulsifiers include sodium lauryl sulfate, trideceth-6, pluronic acid F-127, polyacrylate sodium, triethanolamin, hydroxyethylcetearamidopropyl dimonium chloride and combinations thereof.

[00222] Keratolytic agents

[00223] The keratolytic agents include salicylic acid, alcloxa, allantoin, glycolic acid and combinations thereof.

[00224] The composition of the present invention is preferably prepared in the form of a cream, a gel or a lotion, which contains lubricants, such as glycerol, water soluble celluloses, polysorbates, carbomers, polyethylene glycols (PEG), polyethylene, polydimethylsiloxaneand thickening agent.

[00225] Water soluble celluloses

[00226] Celluloses are organic compounds with the general formula (C6H ₁₀ O ₅ )n, a polysaccharide consisting of a linear chain of several hundred to over ten thousands β(1→4) linked D-glucose units. Preferred celluloses include water-soluble celluloses, and modified water-soluble celluloses such as those known in the art and have properties similar to cellulose. Examples are methylcellulose of different viscosity, ethylcellulose, hydroxypropyl cellulose, hydroxymethylcellulose, and hydroxyethylcellulose, hydroxypropyl methylcellulose, methocel® MC, and carboxymethylcellulose. These cellulose compounds, like cellulose itself, are not digestible by humans, and they are not toxic, and not allergenic.

[00227] Polysorbates

[00228] Polysorbates are a class of emulsifiers used in some pharmaceuticals and food preparation. Polysorbates are oily liquids derived from PEG-ylated sorbitan (a derivative of sorbitol) esterified with fatty acids. Polysorbates include but are not limited to polyoxyethylene (20) sorbitan monolaurate (polysorbate 20), polyoxyethylene (20) sorbitan monopalmitate (polysorbate 40), polyoxyethylene (20) sorbitan monostearate (polysorban 60), polyoxyethylene (20) sorbitan tristrearate (polysorban 65), and polyoxyethylene (20) sorbitan monooleate (polysorban 80). [00229] Carbomers

[00230] Carbomer is a generic name for synthetic polymers of acrylic acid used as emulsion stabilizers or thickening agents in pharmaceuticals and cosmetic products. They may be homopolymers of acrylic acid, crosslinked with an allyl ether pentaerythritol, allyl ether of sucrose, or allyl ether of propylene. Carbomers include but are not limited to carbopol® polymer 71 G NF, carbopol® polymer 971 P NF, carbopol® polymer 974P NF, carbopol® polymer 980 NF, carbopol® polymer 981 NF, carbopol® polymer 5984 EP and carbopol® polymer Ultrez 10 NF.

[00231] Polyethylene glycol (PEG)

[00232] PEG refers to an oligomer or polymer of ethylene oxide and are prepared by polymerization of ethylene oxide and are commercially available over a wide range of molecular weights from 300 g/mol to 10,000,000 g/mol. The preferred PEG to be used in the present invention are liquid PEGs including but not limited to PEG 200, PEG 200E, PEG 300, PEG 300E, PEG 400, PEG 400E, PEG 600 and PEG 600E.

[00233] Thickening agents

[00234] Thickening agents are often used in cosmetics and personal hygiene products. They include but are not limited to alginic acid, sodium alginate, potassium alginate, ammonium alginate, calcium alginate, agar, carrageenan, locust bean gum, xanthan gum, pectin, and gelatin.

[00235] Other components

[00236] The composition of the present invention may also be prepared by the addition of an antiseptic agent in order to keep the composition sterile and disinfect the surfaces onto which it is applied during use. The preferred antiseptic agents include but are not limited to chlorhexidine gluconate, glucono delta-lactone, a paraben compound, benzoic acid, imidazolidinyl urea, a quaternary ammonium compound, and Octenidine dihydrochloride.

[00237] The composition of the present invention may also be prepared by the addition of an absorption enhancer. The preferred absorption enhancers include but are not limited to from triglycerides of coconut oil, isopropyl palmitate, isopropyl myristate, laurocapram, glycerol, propylene glycol and derivatives thereof.

[00238] Furthermore, in order to stabilize and keep the composition for extended periods of time, preservative agents may be added to the composition. The preferred preservative agents include but are not limited to EDTA, EGTA, hydroxytoluene butoxide, hydroxyanisol butoxide, sodium hydroxide, calcium propionate, sodium nitrate, sodium nitrite, sulfur dioxide, sodium bisulfite, benzoic acid, caprylyl glycol, Diazolidinyl urea, Phenoxyethanol, Dehydroacetic acid, lodopropynylbutylcarbamate, Sorbic acid, Isopropyl-paraben, Isobutyl-paraben, Butyl-paraben, and potassium hydrogen sulfite.

[00239] The composition of the present invention may also be supplemented with a small quantity of fragrance or perfume.

Alternative embodiments

EXAMPLE I EXAMPLARY COMPOSITION 1

[00240] The compound of the present invention may be added to pharmaceutical compositions in approximately 0.25% to 0.75%, to a maximum of 1%.

EXAMPLE II

EXAMPLARY COMPOSITION 2

EXAMPLE III

Transdermal absorption

[00241] The transdermal absorption of a compound of the present invention (L- Valine Ethyl Ester Nitrate - L-VEEN) is tested /V? vitro across a CaCo-2 cell layer. Permeability across Caco-2 cell monolayers is used to predict human permeability of drug candidates, to perform in-depth mechanistic and absorption studies, to study the effects of transporters on permeability, and transporter-mediated drug-drug interactions, and test transdermal absorption. Briefly, permeability across differentiated monolayers of Caco-2 is measured on fully differentiated cells grown for 3 weeks on permeable filter supports to estimate human intestinal permeability. The integrity of the monolayer is determined by measurement of TEER or by Lucifer Yellow permeability. In a typical experiment, the test agent is applied to the apical (A; "gut" side) or basolateral (B; "blood") side of the monolayer and incubated for 2 h. The amount of test agent on each side is measured by HPLC or LC/MS/MS. Permeability (Papp) is calculated in the apical to basolateral (A→ B) and basolateral to apical (B→ A) directions:

[00242] P _app = - *

[00243] where dQ/dt is the rate of permeation, C ₀ is the initial concentration of tested compound, and A is the area of the monolayer.

[00244] Passively transported compounds show equal permeability in both directions. The role of transporters is demonstrated by asymmetry in the amount of permeability. A high B → A vs. A → B ratio indicates the possibility that the compound is an efflux transporter substrate. The transporter can be identified by performing the permeability assay in the presence of a specific inhibitor on both sides of the monolayer.

[00245] The L-VEEN is formulated as a liposomal formulation of the compound that is incorporated into a gel formulation along with the NSAID naproxen. Diclofenac is formulated as a gel composition (Voltaren™ gel), and is a NSAID gel composition which represents the standard of care for anti-inflammatory gels compositions. Caffeine is dissolved into a phosphate buffer and is used as a permeability control to show that the CaCo-2 cell layers permeable filters are functional. All formulations are applied to the CaCo-2 cell layer. The tests results are:

[00246] The results demonstrate that L-VEEN displays a permeability that is 3,24x greater than Diclofenac (Voltaren™ gel), which is a commonly used and widely commercially available gel formulation for transdermal pain treatment (knee, ankle, feet, wrists, hand, fingers, etc). Naproxen also displays a permeability that is comparable to Diclofenac (1.25x greater). This result is interesting, considering that naproxen may be a NSAID that is difficult to dose and suggesting that the combination of naproxen with L-VEEN may display synergy and improve the naproxen anti-inflammatory effects. The positive control, caffeine, worked according to plan.

[00247] The embodiments and examples presented herein are illustrative of the general nature of the subject matter claimed and are not limiting. It will be understood by those skilled in the art how these embodiments can be readily modified and/or adapted for various applications and in various ways without departing from the spirit and scope of the subject matter disclosed claimed. The claims hereof are to be understood to include without limitation all alternative embodiments and equivalents of the subject matter hereof. Phrases, words and terms employed herein are illustrative and are not limiting. Where permissible by law, all references cited herein are incorporated by reference in their entirety. It will be appreciated that any aspects of the different embodiments disclosed herein may be combined in a range of possible alternative embodiments, and alternative combinations of features, all of which varied combinations of features are to be understood to form a part of the subject matter claimed.