WO2017066749A1 - Formulations for increasing gene expression - Google Patents

Abstract

Method of treating a condition, disorder or physiological state which is improved or ameliorated by the increased expression of any one or more of ELOVL4 (Elongation of Very Long Chain Fatty Acids-Like 4), UGCG (Ceramide Glucosyltransferase), IVL (Involucrin), TGMI (Transglutaminase 1), HMOX-1 (heme oxygenase (decycling) 1) and AQP3 (Aquaporin-3) genes in a subject including administering to the subject an effective amount of an Active Agent disclosed herein.

Description

FORMULATIONS FOR INCREASING GENE EXPRESSION

This patent application claims the benefit of U.S. Provisional Application No. 62/243,000, filed October 17, 2015 and hereby incorporated by reference in its entirety.

FIELD OF THE INVENTION Formulations for increasing gene expression, such as the ELOVL4 (Elongation of Very Long

Chain Fatty Acids-Like 4), UGCG (Ceramide Glucosyltransferase), IVL (Involucrin), TGMI

(Transglutaminase 1), HMOX-1 (heme oxygenase (decycling) 1) and AQP3 (Aquaporin-3) genes.

BACKGROUND

The ELOVL4, UGCG, IVL, TGMI and HMOX-1 genes are relevant to a number of conditions, disorders and physiological states. These include, but are not limited to, maintaining the skin barrier. Also, AQP3 is similarly related to a number of conditions, disorders and physiological states, including, but not limited to, water transport and hydration in the human skin epidermis.

There remains a need to increase the expression of these genes in subjects in need thereof, or for promoting or maintaining good health (e.g., healthy skin) in a healthy subject. BRIEF SUMMARY OF THE INVENTION

The present invention provides methods of treating a condition, disorder or physiological state which is improved or ameliorated by the increased expression of any one or more of ELOVL4 (Elongation of Very Long Chain Fatty Acids-Like 4), UGCG (Ceramide Glucosyltransferase), IVL (Involucrin), TGMI (Transglutaminase 1), HMOX-1 (heme oxygenase (decycling) 1) and AQP3 (Aquaporin-3) genes in a subject comprising administering to the subject an effective amount of an Active Agent disclosed herein.

In certain, non-limiting embodiments, the condition, disorder or physiological state that can be treated or ameliorated by the presently disclosed Active Agents can be, for example, dry skin, xeroderma, pruritus, ichthyosis, lamellar ichthyosis, eczema, non-bulous congenital ichthyosiform erythroderma (CIE) disease, age related macular degeneration, and autosomal dominant Stargardt Macular Dystrophy 3. In alternative embodiments, the disclosed Active Agents can be used to reduce the appearance of dark circles under eyes and/or the volume of bags under the eye . Furthermore, the presently disclosed Active Agents can be administered to subjects not afflicted with a disease or condition in order to promote a healthy skin appearance. The presently disclosed Active Agents can also be used as antioxidants in order to inhibit the induction of intracellular reactive oxygen species (ROS) in a subject, and to provide protection against UVB-induced pro-inflammatory cytokines on human keratinocytes. BRIEF DESCRIPTION OF THE DRAWINGS

FIGURE 1 depicts the AQP3 expression after 24 hours of UVB radiation in untreated NHEK cells and NHEK cells treated with the sodium salt of Compound A, analyzed as described in Example 1.

FIGURE 2 depicts the AQP3 expression after the EpiDerm-FT skin model was topically treated with Compound A and Compound B at various concentrations followed by isolation of total RNA. Gene expression was measured by qPCR for AQP3 normalized with GAPDH as endogenous control, as described in Example 1.

FIGURE 3 depicts the results of a radical scavenging antioxidant assay for Compound B (O), Vitamin E (T) and Lipoic Acid (■), as described in Example 2. DETAILED DESCRIPTION OF THE INVENTION

The invention can be more fully appreciated by reference to the following description, including the examples. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described herein. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

For the sake of brevity, all publications, including patent applications, patents, and other citations mentioned herein, are incorporated by reference in their entirety. Citation of any such publication, however, shall not be construed as an admission that it is prior art to the present invention. TERMS AND DEFINITIONS

The use of headings and subheadings, such as "General," "Chemistry," or Formulations," in this section (or any other section of this application) is solely for convenience of reference and not intended to be limiting.

General

As used herein, the term "about" or "approximately" means within an acceptable range for a particular value as determined by one skilled in the art, and may depend in part on how the value is measured or determined, e.g., the limitations of the measurement system or technique. For example, "about" can mean a range of up to 5% on either side of a given value. Alternatively, with respect to biological systems or processes, the term "about" can mean within an order of magnitude, for example, within 2 fold on either side of a value. Numerical quantities given herein are approximate unless stated otherwise, meaning that the term "about" or "approximately" can be inferred when not expressly stated. To provide a more concise description, some of the quantitative expressions given herein are not qualified with the term "about." It is understood that, whether the term "about" is used explicitly or not, every quantity given herein is meant to refer to both the actual given value and the approximation of such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. Whenever a yield is given as a percentage, such yield refers to a mass of the entity for which the yield is given with respect to the maximum amount of the same entity for which that could be obtained under the particular stoichiometric conditions. Concentrations that are given as percentages refer to mass ratios, unless indicated differently.

As used herein, the terms "a," "an," and "the" are to be understood as meaning both singular and plural, unless explicitly stated otherwise. Thus, "a," "an," and "the" (and grammatical variations thereof where appropriate) refer to one or more.

A group of items linked with the conjunction "and" should not be read as requiring that each and every one of those items be present in the grouping, but rather should be read as "and/or" unless expressly stated otherwise. Similarly, a group of items linked with the conjunction "or" should not be read as requiring mutual exclusivity among that group, but rather should also be read as "and/or" unless expressly stated otherwise. Furthermore, although items, elements or components of the invention may be described or claimed in the singular, the plural is contemplated to be within the scope thereof, unless limitation to the singular is explicitly stated.

The terms "comprising" and "including" are used herein in their open, non-limiting sense. Other terms and phrases used in this document, and variations thereof, unless otherwise expressly stated, should be construed as open ended, as opposed to limiting. Thus, the term "example" is used to provide exemplary instances of the item in discussion, not an exhaustive or limiting list thereof. Similarly, adjectives such as "conventional," "traditional," "normal," "criterion," "known," and terms of similar meaning should not be construed as limiting the item described to a given time period or to an item available as of a given time, but they should be read to encompass conventional, traditional, normal, or criterion technologies that may be available or known now or at any time in the future. Likewise, where this document refers to technologies that would be apparent or known to one of ordinary skill in the art, such technologies encompass those apparent or known to the skilled artisan now or at any time in the future.

The presence of broadening words and phrases such as "one or more," "at least," "but not limited to" or other like phrases in some instances shall not be read to mean that the narrower case is intended or required in instances where such broadening phrases may be absent. As will become apparent to one of ordinary skill in the art after reading this document, the illustrated embodiments and their various alternatives may be implemented without confinement to the illustrated examples. Compositions

The term "composition," as in pharmaceutical composition, is intended to encompass a product comprising the active ingredient(s) and the inert ingredient(s) (e.g. , pharmaceutically acceptable excipients) that make up the carrier, as well as any product which results, directly or indirectly, from combination, complexation, or aggregation of any two or more of the ingredients, or from dissociation of one or more of the ingredients, or from other types of reactions or interactions of one or more of the ingredients. Accordingly, the pharmaceutical compositions of the present invention encompass any composition made by admixing an active ingredient or disclosed chemical entity and a pharmaceutically acceptable excipient.

The term "carrier" refers to an adjuvant, vehicle, or excipients, with which the compound is administered. In preferred embodiments of this invention, the carrier is a solid carrier. Suitable pharmaceutical carriers include those described in Remington: The Science and Practice of Pharmacy, 21^st Ed., Lippincott Williams & Wilkins (2005).

The term "dosage form," as used herein, is the form in which the dose is to be administered to the subject or patient. The drug is generally administered as part of a formulation that includes nonmedical agents. The dosage form has unique physical and pharmaceutical characteristics. Dosage forms, for example, can be solid, liquid or gaseous. "Dosage forms" can include, for example, a capsule, tablet, caplet, gel caplet (gelcap), syrup, a liquid composition, a powder, a concentrated powder, a concentrated powder admixed with a liquid, a chewable form, a swallowable form, a dissolvable form, an effervescent, a granulated form, and an oral liquid solution. In a specific embodiment, the dosage form is a solid dosage form, and more specifically, comprises a tablet or capsule.

The term "pharmaceutically acceptable," as used in connection with compositions of the invention, refers to molecular entities and other ingredients of such compositions that are physiologically tolerable and do not typically produce untoward reactions when administered to an animal (e.g., a human). The term "pharmaceutically acceptable" may also mean approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals (e.g. mammals), and more particularly in humans.

A "pharmaceutically acceptable excipient" refers to a substance that is non-toxic, biologically tolerable, and otherwise biologically suitable for administration to a subject, such as an inert substance, added to a pharmacological composition or otherwise used as a vehicle, carrier, or diluents to facilitate administration of an agent and that is compatible therewith. Examples of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, and polyethylene glycols. Suitable pharmaceutical carriers include those described in Remington: The Science and Practice of Pharmacy, 21^st Ed., Lippincott Williams & Wilkins (2005).

A "pharmaceutically acceptable salt" is intended to mean a salt of a free acid or base of a disclosed chemical entity, that is non-toxic, biologically tolerable, or otherwise biologically suitable for administration to the subject. See, generally, G.S. Paulekuhn et al., Trends in Active Pharmaceutical Ingredient Salt Selection based on Analysis of the Orange Book Database, /. Med. Chem. 2007, 50, 6665-6672; Berge et al., Pharmaceutical Salts, J. Pharm. Sci. 1977, 66, 1-19; Stahl and Wermuth (eds), Pharmaceutical Salts; Properties, Selection, and Use: 2nd Revised Edition, Wiley-VCS, Zurich, Switzerland (2011). Examples of pharmaceutically acceptable salts are those that are pharmacologically effective and suitable for contact with the tissues of patients without undue toxicity, irritation, or allergic response. Instantly disclosed compounds, e.g. a compound of Formula (I), may possess a sufficiently acidic group, a sufficiently basic group, or both types of functional groups, and accordingly react with a number of inorganic or organic bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt bases, and inorganic and organic acids, to form a pharmaceutically acceptable salt.

Examples of pharmaceutically acceptable salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, phosphates, monohydrogen-phosphates, dihydrogenphosphates, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, borate, nitrate, nitrite, propionates, decanoates, caprylates, acrylates, formates, isobutyrates, caproates, heptanoates, propiolates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, butyne-l,4-dioates, hexyne-l,6-dioates, benzoates, chlorobenzoates, methylbenzoates, dinitrobenzoates, hydroxybenzoates, methoxybenzoates, phthalates, sulfonates, xylenesulfonates, phenylacetates, phenylpropionates, phenylbutyrates, citrates, lactates, y- hydroxybutyrates, glycolates, tartrates, methane-sulfonates, propanesulfonates, naphthalene- 1 -sulfonates, naphthalene-2-sulfonates, besylate, mesylate, mandelates, sodium, disodium, quaternary ammonium, pyridinium, potassium, magnesium, iron (e.g., ²⁺ and ³⁺), calcium and ammonium. In one embodiment, the pharmaceutically acceptable salt is a disodium salt.

As used herein, the term "inert" refer to any inactive ingredient of a described composition. The definition of "inactive ingredient" as used herein follows that of the U.S. Food and Drug Administration, as defined in 21 C.F.R. 201.3(b)(8), which is any component of a drug product other than the active ingredient.

As used herein, "suitable for oral administration" refers to a sterile, pharmaceutical product produced under good manufacturing practices (GMP) that is prepared and presented in a manner such that the composition is not likely to cause any untoward or deleterious effects when orally administered to a subject (e.g, a human). Unless specified otherwise, all of the compositions disclosed herein are suitable for oral administration. Similarly, "suitable for topical administration" refers to a sterile, pharmaceutical product produced under good manufacturing practices (GMP) that is prepared and presented in a manner such that the composition is not likely to cause any untoward or deleterious effects when administered to the skin of a subject (e.g., a human).

Methods and Uses

As used herein, the term "disorder" is used interchangeably with "disease" or "condition".

As used in the present disclosure, the term "effective amount" is interchangeable with

"therapeutically effective amount" and means an amount or dose of a compound or composition effective in treating the particular disease, condition, or disorder disclosed herein, and thus "treating" includes producing a desired preventative, inhibitory, relieving, or ameliorative effect. In methods of treatment according to the invention, "an effective amount" of at least one compound is administered to a subject (e.g., a mammal). As understood by the person of ordinary skill in this art, "effective amount" will vary, depending on the compound, the disease (and its severity), the treatment desired, age and weight of the subject, etc.

The terms "individual," "subject," and "patient" are used interchangeably herein and can be a vertebrate, in particular, a mammal, more particularly, a primate (including non-human primates and humans) and further includes a laboratory animal in the context of a clinical trial or screening or activity experiment (e.g., dogs, monkeys, rats). Thus, as can be readily understood by one of ordinary skill in the art, the compositions and methods of the present invention are particularly suited to administration to any vertebrate, particularly a mammal, and more particularly, a human.

As used herein, a "control animal" or a "normal animal" is an animal that is of the same species as, and otherwise comparable (e.g., similar age, sex) to the animal that is subjected to the treatment whose efficacy is to be ascertained, but does not undergo such treatment.

Reference will now be made to the embodiments of the present invention, examples of which are illustrated by and described in conjunction with the accompanying drawings and examples. While certain embodiments are described herein, it is understood that the described embodiments are not intended to limit the scope of the invention. On the contrary, the present disclosure is intended to cover alternatives, modifications, and equivalents that can be included within the invention as defined by the appended numbered embodiments.

Active Agents

In certain embodiments, the Active Agent is depicted by Formula I:

R3 S CH2 CH R₂

HN C L R-i

O

wherein:

L is a bivalent, branched or unbranched, saturated or unsaturated, C₂-C hydrocarbon chain wherein one or more methylene units of L is independently replaced by— O— ,— S— ,— NH— ,— C(O)— ,— C=CH₂— , or C3-C6 cycloalkylene, wherein L is optionally substituted by one or more groups selected from halogen, phenyl, an 8-10 membered bicyclic aryl ring, a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, an 8-10 membered bicyclic heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, a 5- to 7- membered monocyclic having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur or a 7-10 membered bicyclic heterocyclyl ring having 1-2 heteroatoms independently selected from nitrogen, oxygen, or sulfur;

Rj is hydrogen, -OH or -OR, wherein each R is independently hydrogen or an optionally substituted group selected from Ci-Ce aliphatic or Ci-Ce heteroaliphatic;

R₂ is -C(0)X, wherein X is independently R, -OR, a hydrogen, aryloxy, amino, alkylamino, dialkylamino, heteroaryloxy, hydrazine, a 6-10 membered aryl ring, a 5-6 membered heteroaryl ring having 1-4 heteroatoms independently selected from nitrogen, oxygen, or sulfur, wherein each R is independently hydrogen or an optionally substituted group selected from Ci_6 aliphatic or Ci_6

heteroaliphatic; and

R₃ is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated, C10-C25 aliphatic,

or a pharmaceutically acceptable salt or ester thereof.

In certain embodiments, the Active Agent includes 4-((l-carboxy-2-(((2E,6E)-3,7,l l- trimethyldodeca-2,6,10-trien-l-yl)thio)ethyl)amino)-4-oxobutanoic acid or a pharmaceutically acceptable salt or ester thereof. In one embodiment the Active Agent includes the disodium salt of 4-((l-carboxy-2- (((2E,6E)-3,7,l l-trimethyldodeca-2,6,10-trien-l-yl)thio)ethyl)amino)-4-oxobutanoic acid. In one embodiment, the Active Agent includes 4-(((R)-l-carboxy-2-(((2E,6E)-3,7,l l-trimethyldodeca-2,6,10- trien-l-yl)thio)ethyl)amino)-4-oxobutanoic acid, or a pharmaceutically acceptable salt or ester thereof. In one embodiment, the Active Agent includes the disodium salt of 4-(((R)-l-carboxy-2-(((2E,6E)- 3,7,1 l-trimethyldodeca-2,6,10-trien-l-yl)thio)ethyl)amino)-4-oxobutanoic acid.

In one embodiment, the Active Agent includes 4-(((S)-l-carboxy-2-(((2E,6E)-3,7,l l- trimethyldodeca-2,6,10-trien-l-yl)thio)ethyl)amino)-4-oxobutanoic acid, or a pharmaceutically acceptable salt or ester thereof. In one embodiment, the Active Agent includes the disodium salt of 4- (((S)-l-carboxy-2-(((2E,6E)-3,7,l l-trimethyldodeca-2,6,10-trien-l-yl)thio)ethyl)amino)-4-oxobutanoic acid.

As used herein, Compound A refers to (2R)-2-(2-acetamido-5-amino-5-oxopentanamido)-3- (((2E,6E)-3,7,l l-trimethyldodeca-2,6,10-trien-l-yl)thio)propanoic acid:

or a pharmaceutically acceptable salt or ester thereof.

In one embodiment, the sodium salt of Compound A, i.e., sodium (2R)-2-(2-acetamido-5-amino- 5-oxopentanamido)-3-(((2E,6E)-3,7,l l-trimethyldodeca-2,6,10-trien-l-yl)thio)propanoate, is employed:

As used herein, compound A refers to 4-(((lR)-l-carboxy-2-(((E)-3,7,l l,15-tetramethylhexadec- 2-en- 1 -yl)thio)ethyl)amino)-4-oxobutanoic acid:

H

or a pharmaceutically acceptable salt or ester thereof.

Other Active Agents, such as any one of those disclosed in U.S. Patent No. 8,372,884, hereby incorporated by reference, can also be employed in the instantly described invention.

Prodru s

The invention also relates to, as active ingredients, prodrugs of the disclosed compounds, and the use of such pharmaceutically acceptable prodrugs in the instantly disclosed methods. A "prodrug" is a drug precursor that is initially inactive or partially active and upon administration in vivo undergoes chemical conversion by metabolic processes into an active pharmacological agent. Prodrugs are often useful because, in some situations, they can be easier to administer than the parent drug. They can, for instance, be bioavailable by oral administration whereas the parent is not. The prodrug can also have improved solubility in pharmaceutical compositions over the parent drug.

Exemplary prodrugs include compounds having an amino acid residue, or a polypeptide chain of two or more (e.g., two, three or four) amino acid residues, covalently joined through an amide or ester bond to a free amino, hydroxy, or carboxylic acid group of the disclosed formulas. Examples of amino acid residues include the twenty naturally occurring amino acids, commonly designated by three letter symbols, as well as 4-hydroxyproline, hydroxylysine, desmosine, isodesmosine, 3-methylhistidine, norvaline, beta-alanine, gamma-aminobutyric acid, citrulline homocysteine, homoserine, ornithine and methionine sulfone.

Additional types of prodrugs can be produced, for instance, by derivatizing free carboxyl groups of structures of the disclosed formulas as amides or alkyl esters. Examples of amides include those derived from ammonia, primary Ci ^alkyl amines and secondary di(Ci_₆alkyl) amines. Secondary amines include 5- or 6-membered heterocycloalkyl or heteroaryl ring moieties. Examples of amides include those that are derived from ammonia, Ci_₃alkyl primary amines, and di(Ci_₂alkyl)amines. Examples of esters of the invention include Ci ^alkyl, Ci ^cycloalkyl, phenyl, and phenyl(Ci_₆alkyl) esters. Preferred esters include methyl esters. Prodrugs can also be prepared by derivatizing free hydroxy groups using groups including hemisuccinates, phosphate esters, dimethylaminoacetates, and phosphoryloxymethyloxycarbonyls, following procedures such as those outlined in Fleisher et al., Adv. Drug Delivery Rev. 1996, 19, 115-130.

Carbamate derivatives of hydroxy and amino groups can also yield prodrugs. Carbonate derivatives, sulfonate esters, and sulfate esters of hydroxy groups can also provide prodrugs. Derivatization of hydroxy groups as (acyloxy)methyl and (acyloxy)ethyl ethers, wherein the acyl group can be an alkyl ester, optionally substituted with one or more ether, amine, or carboxylic acid functionalities, or where the acyl group is an amino acid ester as described above, is also useful to yield prodrugs. Prodrugs of this type can be prepared as described in Robinson et al., /. Med. Chem. 1996, 39, 10-18. Free amines can also be derivatized as amides, sulfonamides or phosphonamides. All of these prodrug moieties can incorporate groups including ether, amine, and carboxylic acid functionalities.

Prodrugs can be determined using routine techniques known or available in the art (e.g., Bundgard (ed.), 1985, Design of prodrugs, Elsevier; Krogsgaard-Larsen et al. (eds.), 1991, Design and Application of Prodrugs, Harwood Academic Publishers).

Metabolites

The present invention also relates to, as the Active Agent, a metabolite of a disclosed compound, as defined herein, and salts thereof. A "metabolite" means a pharmacologically active product of metabolism in the body (i.e., in vivo) of a specified compound. The metabolite is in an isolated form outside the body.

Metabolites of a compound can be determined using routine techniques known or available in the art. For example, isolated metabolites can be enzymatically and synthetically produced (e.g., Bertolini et al, /. Med. Chem. 1997, 40, 2011-2016; Shan et al, /. Pharm. Sci. 1997, 86, 765-767; Bagshawe, Drug Dev. Res. 1995, 34, 220-230; and Bodor, Adv Drug Res. 1984, 13, 224-231).

COMPOSITIONS

In some embodiments instantly disclosed compounds, or derivatives thereof, are used, alone or in combination with one or more additional active agents, to formulate pharmaceutical compositions.

A pharmaceutical composition of the invention can include (a) an effective amount of at least one Active Agent in accordance with the invention; and (b) a pharmaceutically acceptable excipient.

In certain embodiments, a disclosed compound (Active Agent) can be administered to a subject in an amount from about 1 μg/kg to about 1000 mg/kg of body weight of the subject. For example, from about 10 μg/kg to about 100 mg/kg of body weight of the subject of Active Agent can be administered. These amounts can be administered once, twice, thrice, four-times, five-times or six-times per day, or alternatively, these amounts can be administered as needed by the subject to control the symptoms of the indication for which it is administered.

Formulations and Administration

Numerous standard references are available that describe procedures for preparing various formulations suitable for administering the compounds according to the invention. Examples of potential formulations and preparations are contained, for example, in the Handbook of Pharmaceutical Excipients, American Pharmaceutical Association (current edition); Pharmaceutical Dosage Forms: Tablets (Lieberman, Lachman and Schwartz, editors) current edition, published by Marcel Dekker, Inc., as well as Remington's Pharmaceutical Sciences (Osol, ed.),1980, 1553-1593.

Any suitable route of administration can be employed for providing an animal, especially a human, with an effective dosage of a compound of the present invention. For example, oral, rectal, topical, parenteral, ocular, pulmonary, nasal, and the like can be employed. Dosage forms include tablets, troches, dispersions, suspensions, solutions, capsules, creams, ointments, aerosols, and the like. In one embodiment, pharmaceutical compositions comprising an instantly described compound are administered topically. In an alternative embodiment, the pharmaceutical compositions are administered systemically orally.

For topical administration, the present active ingredients can be applied in pure form, i.e. , when they are liquids. However, it will generally be desirable to administer them to the skin as compositions or formulations, in combination with a dermatologically acceptable carrier, which can be a solid or a liquid. Useful solid carriers include finely divided solids such as talc, clay, microcrystalline cellulose, silica, alumina, and the like. Useful liquid carriers include water, alcohols or glycols or water- alcohol/glycol blends, in which the present compounds can be dissolved or dispersed at effective levels, optionally with the aid of non-toxic surfactants. Adjuvants such as fragrances and additional antimicrobial agents can be added to optimize the properties for a given use. The resultant liquid compositions can be applied from absorbent pads, used to impregnate bandages and other dressings, or sprayed onto the affected area using pump-type or aerosol sprayers.

Thickeners such as synthetic polymers, fatty acids, fatty acid salts and esters, fatty alcohols, modified celluloses or modified mineral materials can also be employed with liquid carriers to form spreadable pastes, gels, ointments, soaps, and the like, for application directly to the skin of the user

Suitable carriers, diluents and excipients are well known to those skilled in the art and include materials such as carbohydrates, waxes, water soluble and/or swellable polymers, hydrophilic or hydrophobic materials, gelatin, oils, solvents, water, and the like. The particular carrier, diluent, or excipient used will depend upon the means and purpose for which the compound of the present invention is being applied. Solvents are generally selected based on solvents recognized by persons skilled in the art as safe (GRAS) to be administered to an animal. In general, safe solvents are non-toxic aqueous solvents such as water and other non-toxic solvents that are soluble or miscible in water. Suitable aqueous solvents include water, ethanol, propylene glycol, polyethylene glycols (e.g., PEG400, PEG300), etc. and mixtures thereof. The formulations can also include one or more buffers, stabilizing agents, surfactants, wetting agents, lubricating agents, emulsifiers, suspending agents, preservatives, antioxidants, opaquing agents, glidants, processing aids, colorants, sweeteners, perfuming agents, flavoring agents and other known additives to provide an elegant presentation of the drug (i.e. , a compound of the present invention or pharmaceutical composition thereof) or aid in the manufacturing of the pharmaceutical product (i.e. , medicament).

The formulations can be prepared using conventional dissolution and mixing procedures. For example, the bulk drug substance (i.e. , a compound of the present invention or stabilized form of the compound (e.g., complex with a cyclodextrin derivative or other known complexation agent) is dissolved in a suitable solvent in the presence of one or more of the excipients described above. The compound of the present invention is typically formulated into pharmaceutical dosage forms to provide an easily controllable and appropriate dosage of the drug.

The pharmaceutical composition (or formulation) for application can be packaged in a variety of ways, depending upon the method used to administer the drug. Generally, an article for distribution includes a container having deposited therein the pharmaceutical formulation in an appropriate form. Suitable containers are well-known to those skilled in the art and include materials such as bottles (plastic and glass), sachets, ampoules, plastic bags, metal cylinders, and the like. The container can also include a tamper-proof assemblage to prevent indiscreet access to the contents of the package. In addition, the container has deposited thereon a label that describes the contents of the container. The label may also include appropriate warnings.

The active ingredient can also be administered intravenously or intraperitoneally by infusion or injection. Solutions of the active compound or its salts can be prepared in water, optionally mixed with a nontoxic surfactant. Dispersions can also be prepared in glycerol, liquid polyethylene glycols, triacetin, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.

The present compounds can be systemically administered, e.g., orally, in combination with a pharmaceutically acceptable vehicle such as an inert diluent or an assimilable edible carrier. They can be enclosed in hard or soft shell gelatin capsules, can be compressed into tablets, or can be incorporated directly with the food of the patient's diet. For oral therapeutic administration, the active compound can be combined with one or more excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. Such compositions and preparations should contain at least 0.1% of active compound. The percentage of the compositions and preparations can, of course, be varied and can conveniently be between about 2 to about 60% of the weight of a given unit dosage form. The amount of active compound in such therapeutically useful compositions is such that an effective dosage level will be obtained.

The pharmaceutical dosage forms suitable for injection or infusion can include sterile aqueous solutions or dispersions or sterile powders comprising the active ingredient which are adapted for the extemporaneous preparation of sterile injectable or infusible solutions or dispersions, optionally encapsulated in liposomes. In all cases, the ultimate dosage form should be sterile, fluid, and stable under the conditions of manufacture and storage. The liquid carrier or vehicle can be a solvent or liquid dispersion medium comprising, for example, water, ethanol, a polyol (for example, glycerol, propylene glycol, liquid polyethylene glycols, and the like), vegetable oils, nontoxic glyceryl esters, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the formation of liposomes, by the maintenance of the required particle size in the case of dispersions or by the use of surfactants. The prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars, buffers or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.

Sterile injectable solutions are typically prepared by incorporating the active ingredient in the required amount in the appropriate solvent with a variety of the other ingredients enumerated above, as required, followed by filter sterilization. In the case of sterile powders for the preparation of sterile injectable solutions, common methods of preparation are vacuum drying and the freeze drying techniques, which yield a powder of the active ingredient plus any additional desired ingredient present in the previously sterile-filtered solutions. METHODS AND USES

The present invention provides therapeutic methods using the Active Agents disclosed herein, whether alone or in combination. The instantly disclosed compositions can be used, in certain embodiments, to treat any condition, disorder or physiological state which is improved or ameliorated by the increased expression of any one or more of ELOVL4 (Elongation of Very Long Chain Fatty Acids- Like 4), UGCG (Ceramide Glucosyltransf erase), IVL (Involucrin), TGMI (Transglutaminase 1), HMOX- 1 (heme oxygenase (decycling) 1) and AQP3 (Aquaporin-3) genes.

ELOVL4

As disclosed in U.S. Published Application No. 2005/0262580, the human ELQVL4 gene encodes a putative protein of 314 amino acids with approximately 35% amino acid identity to members of the E1..0 (fatty acid elongation) gene family, which encode components of the membrane-bound fatty acid elongation system. ELO proteins have been identified in yeast (Oh, C. S. et al. J. Biol Chem.

272:17376-17384 (1997)), and rodent (Tvrdik, P. et al. J. Biol. Chem. 272:31738-31746 (1997); Tvrdik, P. et al. /. Cell Biol 149:707-318 (2000)).

Similar to other members of the ELO family, Human ELOVL4 has three features: a hydropathy plot that predicts five transmembrane segments; a single HXXHH motif identified with fatty acid desaturase and other dioxy iron cluster proteins; and a strong signal (diiysine motif with the lysines at positions -3 and -5 relative to the carboxy terminus) shown to be responsible for the retention of transmembrane proteins in the endoplasmic reticulum, the known site of biosynthesis of fatty acids with very long chains (Zhang, . et al. Nature Genetics 27:89-93 (200! )). ELOVL4 has a specific and abundant expression in human retina, as well as lower expressions in brain and testis, in the adult retina, ELOVL4 is expressed exclusively in photoreceptor cells, both rod and cone photoreceptors, (ibid.).

ELOVL4 is believed to play a key role in the synthesis of certain essential fatty acids. Essential fatty acids are polyunsaturated fatty acids that cannot be de novo synthesized by mammals, yet are required for a number of important biochemical processes. Thus, essential fatty acids roust be supplied either directly in the diet, or synthesized from, dietary essential fatty acids, such as iinoleic acid and alpha-linolenic acid (ALA). These two dietary EFAs undergo a number of biosynthetic reactions that convert them into various other EFAs. The reactions include a series of alternating reactions involving the removal of two hydrogens coupled with the insertion of an additional double bond (desaturation) and the lengthening of the fatty acid chain by the addition of two carbons (chain elongation). Through the pathways, docosahexaenoic acid (DB A), an essential fatty acid of omega-3 family, is synthesized from alpha-linolenic acid (ALA), while arachidonic acid (ARA) is synthesized from Iinoleic acid . It is believed that ELOVL4 is involved in one of three elongation steps required for DHA synthesis. See U.S. Published .Application No. 2005/0262580, hereby incorporated by reference.

As disclosed in Agbaga et al., ELO L4 is necessary for the synthesis of very long chain saturated fatty acids and very long chain polyunsaturated fatty acids. Very long chain polyunsaturated fatty acids are uniquely present in retina, sperm, and brain. See Agbaga, M.-P.. Brush, R. S., Mandal, M. N. A., Henry, K., Elliott, M. H.. Anderson, R. E. Role of Stargardt-3 macular dystrophy protein

(ELOVL4) in the biosynthesis of very long chain fatty acids. Proc. Nat Acad. Sei. 1.05: 12843-12848,

2008, hereby incorporated by reference.

Mutations in the ELOVL4 gene has been implicated in, or cause, for example, age related macular degeneration and autosomal dominant Stargardt Macular Dystrophy 3. See, e.g., U.S. Published Application No. 2008/0255000 (macular degeneration); and Agdaba et al. (autosomal dominant Stargardt Macular Dystrophy 3), each of which are hereby incorporated by reference.

Aidahmesh et al discloses that heterozygous mutations in ELOVL4, are known to cause macular degeneration in humans and retinal abnormalities in mice. However, according to Aidahmesh, biallelic ELOVL4 mutations have not been observed in humans, and murine models with homozygous mutations die within hours of birth as a result of a defective epidermal water barrier. Aldahmeh discloses two human individuals with recessive ELOVL4 mutations revealed by a combination of autozygome analysis and exome sequencing who exhibit clinical features of ichthyosis, seizures, mental retardation, and spasticity— a constellation that resembles Sjogren-Larsson syndrome (SLS) but presents a more severe neurologic phenotype. Aldamesh's findings identify recessive mutations in ELOVL4 as the cause of a neuro-ichthyotic disease and emphasize the importance of VLCFA synthesis in brain and cutaneous development. See Adahmesh et al, Am. J. Hum Genet., 2011 Dec 9; 89(6): 745-750, hereby

incorporated by reference.

UGCG

UGCG (also referred to as, inter alia, UDP-glucose eeramide gl ucosy 1 transferase) is a gene that encodes an enzyme that catalyzes the first glycosylation step in the biosynthesis of glycosphingolipids. Glycosphinogolipids, which are membrane components containing lipid and sugar moieties. The product of this reaction is glucosylceramide, which is the core structure of many glycosphingolipids. According to Watanabe et al, GCS is up-regulated at the transcriptional level during keratinocyte differentiation. See Wantabe et al., J. Biol. Chem. 1998 April 17; 273(16):9651-5, hereby incorporated by reference.

IVL

Involucrin is a protein component of human skin and is encoded by the IVL gene. By binding to loricrin, involucrin contributes to the formation of a cell envelope that protects corneocytes in the skin. As disclosed by Green et al., involucrin is a highly reactive, soluble, transglutaminase substrate protein present in keratinocytes of epidermis and other stratified squamous epithelia. See Green et al., Mol. Biol. Evol. 9(6):977-1017, hereby incorporated by reference. According to Eckert et al., involucrin first appears in the cell cytosol, but ultimately becomes cross-linked to membrane proteins by

transglutaminase thus helping in the formation of an insoluble envelope beneath the plasma membrane functioning as a glutamyl donor during assembly of the cornified envelope. See Eckert et al., J. Invest. Dermatol. 100(5):613-17, hereby incorporated by reference. TGMI

The TGMI gene encodes the transglutaminase 1 enzyme. As disclosed in Genetics Home Reference, this enzyme is found in cells that make up the outermost layer of the skin (the epidermis). Transglutaminase 1 is involved in the formation of the cornified cell envelope, which is a structure that surrounds skin cells and helps form a protective barrier between the body and its environment.

Specifically, transglutaminase 1 forms cross-links between the structural proteins that make up the cornified cell envelope. This cross-linking provides strength and stability to the epidermis. See Genetics Home Reference: https://ghr.nlm.nih.gov/gene/TGMl .

More particularly, As disclosed in U.S. Patent No. 8,933,035, the outermost layer of the epidermis, the stratum corneum is formed of keratinocytes in the final stage of their differentiation (corneocytes) which are bonded to one another by an intercellular cement which is both flexible and impermeable. In the stratum corneum, a distinction is thus made between a cellular compartment formed of corneocytes and an extracellular compartment formed primarily of lipids organised in multi-lamellar structures. The corneocytes are surrounded by a specific membrane, called a cornified envelope, which is largely responsible for the strength, insolubility and suppleness of the skin. The cornified envelope is formed of a mixture of structural proteins interconnected by covalent bonds under the action of transglutaminase. The main proteins forming the cornified envelope are envoplakin, periplakin, involucrin, small proline-rich proteins (SPR proteins) and loricrin.

Transglutaminase- 1 (TGI) is expressed in keratinocytes and is present in a form bonded to the membrane. In the epidermis, TGI , TG3 and TG5 are involved in the formation of the cornified envelope (Lorand et al, Nat Rev Mol Cell Biol. February; 4(2), 2003). See U.S. Patent No. 8,933,035, which is hereby incorporated by reference.

As disclosed by Pigg et al., the transglutaminase 1 (TGMI) gene is associated with the clinical subtypes lamellar ichthyosis (LI) and non-bullous congenital ichthyosiform erythroderma (CIE).

Furthermore, expression of TGMI is critical for maturation of mammalian epidermis and occurs during squamous metaplasia. See, Functional API and CRE response elements in the human keratinocyte transglutaminase promoter mediating Whn suppression. Jessen, B.A., Qin, Q., Rice, R.H. Gene (2000) Aug 22; 254(l-2):77-85, which is hereby incorporated by reference. See also, Strong founder effect for a transglutaminase 1 gene mutation in lamellar ichthyosis and congenital ichthyosiform erythroderma from Norway. Pigg, M., Gedde-Dahl, T., Cox, D., Hausser, I., Anton-Lamprecht, I., Dahl, N. Eur. J. Hum. Genet. (1998), which is hereby incorporated by reference in its entirety.

HMOX-1

HMOX-1 is a gene that encodes for the heme oxygenase 1 enzyme, which mediates the first step of heme catabolism, cleaving the heme to form biliverdin. It has been reported that the ability of oxygenase 1 to catabolize free heme and produce carbon monoxide (CO) gives its antiinflammatory properties by up-regulation of interleukin 10 (IL-10) and interleukin 1 receptor antagonist (IL-1RA) expression. See Piantadosi CA, et al. (May 2011). "Heme oxygenase-1 couples activation of mitochondrial biogenesis to anti-inflammatory cytokine expression". J. Biol. Chem. 286 (18): 16374-85, hereby incorporated by reference.

HMOX-1 protects endothelial cells from apoptosis, is involved in blood-vessel relaxation regulating vascular tone, and participates in blood-vessel formation by means of angiogenesis and vasculogenesis. See Lobada A et al., Antioxid Redox Signal, 2008 10(10): 1767-812, hereby incorporated by reference. Agents that increase the expression of HMOX-1 have been disclosed to control vascular tone of skin under the eye, and also to reduce dark circles and puffiness (i.e. the volume of bags) under the eyes. See, e.g., H. Chajara et al. SOFW- Journal, 4-2014, p. 16-31, hereby

incorporated by reference.

AOP3

AQP3 is a gene that encodes the water channel protein aquaporin 3. As disclosed by Li et al., Aquaporin 3 (AQP3) is a protein implicated in skin hydration. AQP3 null mice have relatively dry skin, reduced skin elasticity, and delayed recovery of barrier function after removal of the stratum corneum, a condition which is also present in skin of the older population, as a feature of skin aging is the change in both water content and barrier function of the skin. Li demonstrates that AQP3 decreased with age in both skin and NHEK samples. See Li et al., Australian Journal of Dermatology, Vol. 51, Issue 2, May 2010, pp. 106-112, hereby incorporated by reference.

As disclosed in U.S. Published Patent Application No. 2009/0130223, Aquaporins render possible the quick exchange of larger amounts of water and glycerin through the plasma membrane and intracellular membranes, e.g., in erythrocytes, epithelial cells or growing plant cells. In contrast to uncatalyzed, purely physical diffusion through the lipid layer, in erythrocytes the aquaporin-mediated transport of water through the plasma membrane is characterized by a lower sensitivity to low temperatures and inhibitors, (e.g., HgCl₂). The group of aquaporins includes from a functional standpoint the TIP proteins (TIP=tonoplast intrinsic protein) and PIP proteins (PIP=plasma membrane intrinsic protein) from plant cells and the CHIP proteins (CHIP=channel forming integral protein) from the plasma membrane of animal cells. Through the expression of cDNAs of the TIP, PIP or CHIP genes

in xenopus oocytes (amphibian oocytes, xenopus oocyte expression system) the water exchange through the plasma membranes of these cells is very considerably increased— a strong support for the water transport function of these proteins. From a genetic standpoint, the TIP, PIP and CHIP proteins belong to an evolutionarily old family of channel-forming membrane proteins, the MIP proteins (MIP=major intrinsic protein) and have 6 membrane-spanning domains. They are present in the membrane as tetramers.

For example, and without limitation, the presently disclosed Active Agents can be employed to treat, for example, dry skin, in which the presently disclosed Active Agents improve water transport and hydration in the human skin epidermis. More particularly, in certain embodiments, the presently disclosed Active Agents can provide improved hydration, moisturization, barrier repair, and barrier function. In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to treat and/or prevent ichthyosis (e.g.. khtyosis vulgaris). In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to treat and/or prevent lamellar ichthyosis (LI). In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to treat and/or prevent non-bullous congenital ichthyosiform erythroderma (CIE).

In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to treat and/or prevent the appearance of dark circles under the eyes. In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to reduce the volume of bags under the eyes (i.e. to reduce the puffiness of skin under the eye. In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to improve the vascular tone of skin under the eye.

In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to treat and/or prevent dry skin or otherwise increase the moisture that is present in the skin.

Accordingly, the instantly disclosed Active Agents can be used to treat lamellar ichthyosis and/or non-bulous congenital ichthyosiform erythroderma (CIE) disease by administering to a subject in need thereof a therapeutically effective amount of one or more of the instantly disclosed Active Agents.

In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to treat and/or prevent age related macular degeneration. In certain embodiments, the presently disclosed Active Agents (e.g., Compound A or B) can be used to treat and/or prevent autosomal dominant Stargardt Macular Dystrophy 3. The Active Agents can be applied, for example, systemically or topically to the skin or eye (e.g., eye drops).

Furthermore, as demonstrated here, the presently disclosed Active Agents can be used as antioxidants to reduce the production of reactive oxygen species (ROS) in a subject by administering to the subject a therapeutically effective amount of one or more of the presently disclosed Active Agents.

The methods disclosed herein are illustrative and non-limiting. Indeed the presently disclosed

Active Agents can be used, in certain embodiments, to treat or ameliorate any condition, disorder or physiological state which is improved or ameliorated by the increased expression of any one or more of ELOVL4 (Elongation of Very Long Chain Fatty Acids-Like 4), UGCG (Ceramide Glucosyltransf erase), IVL (Involucrin), TGMI (Transglutaminase 1) and AQP3 (Aquaporin-3) genes by administering (e.g., topically administering) to the subject in need thereof an effective amount of one or more of the instantly disclosed Active Agents.

In one embodiment, a method of modulating an inflammatory response, or treating or preventing inflammation is excluded as a use of the presently claimed compound. In one embodiment, the suppression of an inflammatory response is excluded as a use of the presently claimed compound. In one embodiment, the treatment of inflammatory diseases, such as any one or more of injuries of the cardiovascular, digestive, integumentary, muscular, nervous, reproductive, respiratory and urinary systems, as well as, diseases, disorders, syndromes, conditions and injuries of tissue and cartilage such as atherosclerosis, irritable bowel syndrome, psoriasis, tendonitis, Alzheimer's disease and vascular dementia, multiple sclerosis, diabetes, endometriosis, asthma and kidney failure are excluded as a use of the presently claimed compound. In one embodiment, use of the presently disclosed Active Agents as antioxidants is excluded. In one embodiment, inhibition of helper T-lymphocyte infiltration and accumulation is excluded as a use of the presently claimed compound. In one embodiment, inhibition of oxidative burst response from neutrophils is excluded as a use of the presently claimed compound. In one embodiment, the promotion of healthy skin is excluded as a use of the presently claimed compound. In one embodiment, the treatment of diseases that may benefit from inhibition of infiltration and activation of inflammatory cells (e.g. neutrophils, lymphocytes, monocytes, mast cells), and/or inhibition of expression and activation of cell surface adhesion molecules (e.g. VCAM-1 and ICAM-1) in endothelial and inflammatory cells is excluded as a use of the presently claimed compound. In some embodiments, treating or lessening the severity of inflammatory diseases or disorders selected from inflammation (acute or chronic), inflammation associated with spinal cord injury to promote nerve regeneration, inhibition of rejection of genetically engineered cells by the immune system during in vivo gene therapy, asthma, autoimmune diseases, and chronic obstructive pulmonary disease (COPD) (e.g., emphysema, chronic bronchitis and small airways disease, etc.), inflammatory responses of the immune system, skin diseases (e.g., reducing acute skin irritation for patients suffering from rosacea, atopic dermatitis, seborrheic dermatitis, psoriasis), irritable bowel syndrome (e.g., Chron's disease and ulcerative colitis, etc.), neurodegenerative disorders (e.g., Parkinson's disease, Alzheimer's disease, Huntington's disease, Dementia pugilistica, Pick's disease, Guam Parkinsonism dementia complex, Fronto-temporal dementia, Cortico-basal degeneration, Pallido-pontal-nigral degeneration, Progressive supranuclear palsy, Dementia with Lewy bodies (DLB), and multiple system atrophy (MSA)) are excluded as a use of the presently claimed compound. In certain embodiments, methods for treating or preventing diseases in a subject that may benefit from the modulation of levels of inflammatory mediators such as cytokines are excluded as a use of the presently claimed compound. In certain embodiments, methods for treating or preventing diseases in a subject that may benefit from the inhibition of infiltration and accumulation of helper-T lymphocytes are excluded as a use of the presently claimed compound. In certain embodiments, methods for treating or preventing skin conditions are excluded. In certain embodiments, methods of treating edema, erythema and/or inhibition of myeloperoxidase (MPO) are excluded. In certain embodiments, treating or lessening the severity of one or more diseases in which protein inhibitors that modulate the G-protein signaling cascade are known to play a role are excluded as a use of the presently claimed compound. In alternative embodiments, each of the methods and treatments disclosed in this paragraph are included as a use of the presently claimed compound.

The present disclosure will be further illustrated by the following non-limiting Examples. These Examples are understood to be exemplary only, and they are not to be construed as limiting the scope of the invention as defined by the appended claims. EXAMPLES

Example 1: Increased Gene Expression of ELVOL4. UGCG. IVL TGMI. AOP3 and HMOX-1 in 3D Human Skin Culture Model

Maintaining the skin barrier is a key to promoting healthy, youthful skin. The physical skin barrier is localized in the stratum corneum (SC) and consists of corneocytes and lipid-enriched intercellular domains. The present Example demonstrates that Compound B and the sodium salt of Compound A increase the expression of genes related to the formation of the cell-lipid barrier for the prevention of pathogen invasion and unregulated loss of water and solutes (ELOVL4 - Elongation of Very Long Chain Fatty Acids-Like 4, UGCG - Ceramide Glucosyltransferase, IVL - Involucrin, TGMI - Transglutaminase 1) when applied topically in a 3D human skin culture model (EpidermFT). This was a surprising result as these molecules have been previously characterized as anti-inflammatory and thus unlikely to modulate key genes in enhancing barrier repair and function as well. To date, no isoprenylcysteine compounds to date have been reported to possess this activity in the literature. The present Example also demonstrates that Compound B and the sodium salt of compound A also increases the expression of AQP3, and that the sodium salt of compound A increases HMOX-1 expression.

Gene Expression on Human Keratinocvtes (Barrier Function and Aging Markers)

The EpiDerm-FT™ skin tissues were used from human origin and purchased as preserved inserts from MatTek Corp. (Cat No. EFT-400). Tissues were cultured and acclimated for 18 hours before treatments and dosing procedures and tissue groups were identified using appropriate cell culture 6-well plates. The test materials were single administered by topical application (25μί) using a plastic dosing (dispense) pipette of appropriate size. The test articles were weighted and dissolved with HPLC-grade water to make the sodium salt of Compound A (0.5, 1% w/v) and Compound B (0.25% w/v) and were stored at room temperature. The untreated group did not get any dose. A fixed 25 μΕ/ιΐ88υε volume was administered drop wise topically to each tissue on Day 2 and incubated for 24 hours at 37°C and 5% C0₂. On Day 3, total RNA was extracted from each tissue using the RNAqueous^® kit (Ambion^®; Cat No. 1912) and cDNA was obtained using the High Capacity RNA-to-cDNA kit (Applied Biosystems^®; Cat No. 4387406). Quantitative PCR (qPCR) was performed using the TaqMan^® Fast Advanced Master Mix (Applied Biosystems^®; Cat No. 4444556) and specific TaqMan^®-probes human gene primers (ELOVL4, UGCG, IVL, TGMI, AQP3, GAPDH) to calculate the relative gene fold expression change per treatment. Gene expression analysis was performed using the comparative Ct method (also known as the 2-[delta][delta]Ct ) approach by comparing the Ct values of the treated samples with the untreated samples and normalized to GAPDH gene expression as endogenous housekeeping gene. Results were represented as percent of change in gene expression relative to untreated, and are shown below in Table 1. Table 1. Sodium salt of Compound A and Compound B increase skin barrier-related gene expression in reconstructed human epidermis model.

Gene Expression on human keratinocvtes (UVB and aquaporins)

Normal human epidermal keratinocytes (NHEKs) from neonatal donors were obtained from

Thermo-Fisher (Cat No. C-001-5C). Cells were cultured in 6-well plates for 24 hours before treatments with EpiLife media (Cat No. MEPI500CA) supplemented with keratinocytes growth supplement (Cat No. S0015). On Day 2, media was removed and cells were washed with lx PBS. Cells were irradiated with 25mJ/cm² UVB using an external research irradiator (Daavlin Co.) equipped with broadband UVB lamps (305+12 nm). Later, the test materials were diluted in supplemented media and incubated for 24 hours at 37°C and 5% C0₂. On Day 3, total RNA was extracted from each tissue using the RNAqueous^® kit (Ambion^®; Cat No. 1912) and cDNA was obtained using the High Capacity RNA-to-cDNA kit (Applied Biosystems^®; Cat No. 4387406). Quantitative PCR (qPCR) was performed using the TaqMan^® Fast Advanced Master Mix (Applied Biosystems^®; Cat No. 4444556) and specific TaqMan^®- probes human gene primers (AQP3 and GAPDH) to calculate the relative gene fold expression change per treatment. Gene expression analysis was performed using the comparative Ct method (also known as the 2-[delta][delta]Ct ) approach by comparing the Ct values of the treated samples with the untreated samples and normalized to GAPDH gene expression as endogenous housekeeping gene. Results were represented as percent of change in gene expression relative to untreated-No UVB.

Increased Gene Expression of AQP3 in 3D Human Skin Culture Model

Aquaporin-3 (AQP3) is part of the aquaporin family of transmembrane proteins forming water channels in the basal layer of the epidermis and is responsible for water transport and hydration in the human skin epidermis. The present Example demonstrate that the sodium salt of Compound A dose- dependently increase the expression of aquaporin-3. Compound B was also shown to have activity to increase the expression of aquaporin-3. This was a surprising result for at least two reasons. First, previous literature has demonstrated UVB induced AQP3 down regulation in cultured human keratinocytes. See, e.g., Shan et al. Int J Mol Med. 2012, 29(4):625-9, hereby incorporated by reference. The present results using UVB and cultured human keratinocytes demonstrate that UVB in fact does the opposite, it promotes AQP3 expression, not downregulation (Figure 1). Interestingly, the sodium salt of Compound A was able to further boost AQP3 expression which in of itself was completely unexpected given the previous literature regarding UVB treated keratinocytes and AQP3, These results are shown in Figure 1.

Given these surprising results, the effects of the sodium salt of Compound A was then tested (as well as Compound B) in a human skin 3D model by applying the sodium salt of Compound A topically to investigate whether these compounds could boost AQP3 in the absence of UVB. Results show the sodium salt of Compound A significantly increased AQP3 expression in a dose-dependent manner. These results are shown in Figure 2.

Thus, both in the presence and absence of UVB, the sodium salt of Compound A increased AQP3 expression.

Gene Expression on human keratinocytes (Dark circles marker-HMOX-1)

Normal human epidermal keratinocytes (NHEKs) from neonatal donors were obtained from

Thermo-Fisher (Cat No. C-001-5C). Cells were cultured in 6-well plates for 24 hours before treatments with EpiLife media (Cat No. MEPI500CA) supplemented with keratinocytes growth supplement (Cat No. S0015). On Day 2, media was removed and cells were washed with lx PBS. On Day 3, total RNA was extracted from each tissue using the RNAqueous^® kit (Ambion®; Cat No. 1912) and cDNA was obtained using the High Capacity RNA-to-cDNA kit (Applied Biosystems^®; Cat No. 4387406). Quantitative PCR (qPCR) was performed using the TaqMan^® Fast Advanced Master Mix (Applied Biosystems^®; Cat No. 4444556) and specific TaqMan^®-probes human gene primers (HMOX1, GAPDH) to calculate the relative gene fold expression change per treatment. Gene expression analysis was performed using the comparative Ct method (also known as the 2-[delta][delta]Ct ) approach by comparing the Ct values of the treated samples with the untreated samples and normalized to GAPDH gene expression as endogenous housekeeping gene. Results were represented as percent of change in gene expression relative to untreated cells, and are shown below in Table 2.

Table 2. Sodium salt of Compound A increase HMOX-1 in reconstructed human epidermis model.

Treatment Group HMOX1

Compound A 89+25

(sodium salt) (10

μΜ)

Example 2: Protection Against Reactive Oxygen Species (ROS)

The production of reactive radicals is significantly increased during cell metabolism and dermal tissue aging mechanisms. Antioxidant molecules present in the environment and in our bodies function to counteract ROS. We thus sought to determine Compound B's antioxidant properties by testing its activity as a free radical scavenger and efficacy in inhibiting the induction of intracellular ROS in response to H₂0₂ exposure. Compound B's capacity to scavenge reactive radicals was quantitated using a colorimetric antioxidant assay kit (see below). Results demonstrate that Compound B inhibits hydrogen peroxide-induced free radical reactive oxygen species (IC₅₀ = 68 ± 8μΜ) with similar activity to vitamin E (a-tocopherol; IC₅₀ = 25 ± ΟμΜ) but significantly greater potency than lipoic acid (IC₅₀ = 682 ± 169μΜ); two antioxidants commonly used in skin care products (Figure 3).

The accumulation of intracellular ROS over time has been implicated in cellular aging mechanisms correlated to sun damage, pollution and other environmental factors inducers on skin. Intracellular free radicals and other reactive species are constantly generated by cellular metabolism and exacerbated by environmentally produced oxidative damage to lipids, nucleic acids and proteins. The onset of oxidative stress can be delayed by the introduction of functional antioxidant molecules that can penetrate dermal cells and scavenge these species before their interaction with biomolecules. Therefore, a HDF cell-based assay was used for measuring intracellular reactive oxygen species activity employing the cell-permeable fluorogenic probe 2', 7'-Dichlorodihydrofluorescin diacetate (DCFH-DA). DCFH-DA is diffused into cells pre-treated with potential antioxidants and the fluorescence intensity is proportional to intracellular ROS levels. Compound B's intracellular antioxidant capacity of reactive radical scavenging was determined using this method (see below). Results demonstrate that Compound B inhibits intracellular oxidative stress (37 ± 2%) with similar activity to vitamin E (a-tocopherol; 42 + 3%) and greater potency than lipoic acid (26 + 4%) (Table 3). Table 3. Antioxidant effects of Compound B on hydrogen peroxide induced ROS inside human dermal fibroblasts (HDFs)*

* The data represent the mean+StDev of two independent experiments.

Figure 3 sets forth the results, in which Compound B (O), Vitamin E (T) and Lipoic Acid (■) were tested. The data represent the mean+StDev of three independent experiments. The IC₅₀ value was determined via the four parameter logistic curve fit using SigmaPlot graphical software (San Jose, CA).

These results were unexpected since a previously performed antioxidant assay demonstrated Compound B provided little to no protection against hydrogen peroxide induced oxidation in a different cell based assay. Nevertheless, the sodium salt of Compound A, a different isoprenylcysteine molecule did show antioxidant activity (Table 4).

Table 4. Sodium Salt of Compound A shows antioxidant activity protecting human dermal fibroblasts (HDFs) from hydrogen peroxide induced toxicity.

Radical scavenging antioxidant assay

Radical scavenging antioxidant assay was estimated using the colorimetric antioxidant assay kit obtained from Cayman Chemical Company (Ann Arbor, MI). ABTS (2, 2'-Azino-bis-[3- ehthylbenzthiazoline sulphonate]), was used as the chromogen, which changes into a colored monocation radical form (ABTS*+) by metmyoglobin in the presence of hydrogen peroxide, and monitored by measuring absorption at 750 nm using a plate reader (Envision-PerkinElmer; Waltham, MA). Antioxidant inhibition was calculated based on the discoloration of ABTS by serial concentrations of test compounds added simultaneously with myoglobin and hydrogen peroxide.

Cell-based antioxidant Assay #1

Human dermal fibroblasts (HDF) cells were cultured in DMEM with 10% FBS and seeded into black wall 96-well plates and incubated for 24 hours before treatment. Cells were pre-treated for 3 hours with and without test compounds (0.03-1μΜ final concentrations) and labeled with 50μΜ of dichloro- dihydro-fluorescein diacetate (DCFH-DA). Total fluorescence (Excitation=485nm; Emission=535nm) was measured after cells were co- incubated for 20 minutes with 0.1 mM H₂0₂ and the test compounds. Cell protection against oxidative stress was calculated using the following formula:

Cell protection (%) = [(test compound (%) - hydrogen peroxide-only (%))/ (test compound (%) - untreated control (%))] x 100.

Cell-based antioxidant Assay #2

Human dermal fibroblasts (HDFs) from neonatal donors were obtained from Thermo-Fisher (Cat

No. C-004-5C). Cells were cultured in 96-well plates for 24 hours before treatments with DMEM media (Cat No. 11965-092) supplemented with 10% FBS. Test articles were weighted and dissolved with HPLC-grade water to make lmM stocks and serial dilutions were made in water as vehicle. The untreated group did not get any dose. The test materials were diluted in media with 600μΜ hydrogen peroxide on Day 2 and incubated for 24 hours at 37°C and 5% C0₂. On Day 3, treatment media was removed and cytotoxicity assay was performed using the MTS reagent (Promega; Cat No. G3580) diluted in DMEM-no phenol red medium (Thermo-Fisher; Cat No. 21063-029) supplemented with 10% FBS. After 15 minutes incubation, absorbance at 490nm was determined using a microplate reader. Cell protection analysis was performed by comparing the OD values of the hydrogen peroxide- treated samples with the untreated samples. Results were represented as percent of cell protection relative to hydrogen peroxide- treated cells.

Example 3: Protection Against UVB-induced Pro-inflammatory Cytokines on Human Keratinocvtes

Normal human epidermal keratinocytes (NHEKs) from neonatal donors were obtained from Thermo-Fisher (Cat No. C-001-5C). Cells were cultured in 24-well plates for 24 hours before treatments with EpiLife media (Cat No. MEPI500CA) supplemented with keratinocytes growth supplement (Cat No. S0015). On Day 2, media was removed and replaced with supplement-depleted EpiLife media and incubated for 24 hours at 37°C and 5% C0₂. On Day 3, media was removed and test materials were diluted in supplement-depleted media and incubated for 6 hours. After incubation, cells were washed with lxPBS and irradiated with 25mJ/cm² UVB using an external research irradiator (Daavlin Co) equipped with broadband UVB lamps (305+12 nm) and later incubated for 24 hours with supplement depleted media. On Day 4, media supernatants were harvested and used to measure pro-inflammatory cytokine levels (IL-6, and TNF-alpha) using ELISA kits (BD Biosciences® #555220; R&D Systems® #DY210). Results were represented as percent of inhibition relative to untreated-UVB irradiated cells, and are shown below in Table 5.

Table 5. Sodium Salt of Compound A Protects Against Pro-Inflammatory Cytokines on Human Keratinocytes.

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

While this invention has been particularly shown and described with references to preferred embodiments thereof, it will be understood by those skilled in the art that various changes in form and details can be made therein without departing from the scope of the invention encompassed by the appended claims. Further, all embodiments included herein are given solely for the purpose of illustration and are not to be construed as limitations of the present invention, as many variations thereof are possible without departing from the spirit and scope of the invention.

Claims

1. A method of treating a condition, disorder or physiological state which is improved or ameliorated by the increased expression of any one or more of ELOVL4 (Elongation of Very Long Chain Fatty Acids-Like 4), UGCG (Ceramide Glucosyltransferase), IVL (Involucrin), TGMI (Transglutaminase 1), HMOX-1 (heme oxygenase (decycling) 1) and AQP3 (Aquaporin-3) genes in a subject comprising administering to the subject an effective amount of an Active Agent selected from the group consisting of (a) a compound of the formula:

or a pharmaceutically acceptable salt or ester thereof;

(b) a compound of the formula

or a pharmaceutically acceptable salt or ester thereof; or (c) a combination of (a) and (b).

2. The method of claim 1 , wherein the condition, disorder or physiological state is improved or ameliorated by the increased expression of ELOVL4 (Elongation of Very Long Chain Fatty Acids-Like 4) gene.

3. The method of claim 1, wherein the condition, disorder or physiological state is improved or ameliorated by the increased expression of UGCG (Ceramide Glucosyltransferase) gene.

4. The method of claim 1 , wherein the condition, disorder or physiological state is improved or ameliorated by the increased expression of IVL (Involucrin) gene.

5. The method of claim 1, wherein the condition, disorder or physiological state is improved or ameliorated by the increased expression of TGMI (Transglutaminase 1) gene.

6. The method of claim 1, wherein the condition, disorder or physiological state is improved or ameliorated by the increased expression of HMOX-1 (heme oxygenase (decy cling) 1) gene.

7. The method of claim 1, wherein the condition, disorder or physiological state is improved or ameliorated by the increased expression of AQP3 (Aquaporin-3) gene.

8. The method of claim 1, wherein the condition, disorder or physiological state that is improved or ameliorated is dry skin.

9. The method of claim 1, wherein the condition, disorder or physiological state that is improved is skin hydration.

10. The method of claim 1, wherein the condition, disorder or physiological state that is improved or ameliorated is pruritus.

11. The method of claim 1, wherein the condition, disorder or physiological state that is improved is skin barrier repair.

12. The method of claim 1, wherein the condition, disorder or physiological state that is improved is skin barrier function.

13. The method of claim 1, wherein the condition, disorder or physiological state that is improved or ameliorated is ichthyosis.

14. The method of claim 12, wherein the condition, disorder or physiological state that is improved or ameliorated is ichthyosis vulgaris.

15. The method of claim 12, wherein the condition, disorder or physiological state that is improved or ameliorated is lamellar ichthyosis.

16. The method of claim 12, wherein the condition, disorder or physiological state this is improved or ameliorated is non-bullous congenital ichthyosiform erythroderma.

17. The method of claim 1, wherein the condition, disorder or physiological state that is improved or ameliorated is the appearance of dark circles under the eye.

18. The method of claim 1, wherein the condition, disorder or physiological state that is improved or ameliorated is the volume of bags under the eye.

19. The method of claim 1, wherein the condition, disorder or physiological state that is improved or ameliorated is age related macular degeneration.

20. The method of claim 1 , wherein the condition, disorder or physiological state that is improved or ameliorated is autosomal dominant Stargardt Macular Dystrophy 3.