COSMECEUTICAL COMPOSITION

Abstract

Cosmeceutical compositions for and methods of improving the appearance of skin and promoting hair growth are provided. Said compositions comprise at least one ROS-scavenging polymer gel, at least MG53, or a combination thereof. A novel boronate ester-based copolymer forms an aqueous hydrogel exhibiting lower viscosity at cooler temperatures and higher viscosity at higher temperatures is provided.

Claims

1) A cosmeceutical composition comprising MG53 and one or more cosmetically acceptable excipients.

2) The cosmeceutical composition of claim 1 further comprising a) one or more antioxidants; b) one or more zinc salts; c) aqueous liquid vehicle; or d) a combination thereof.

3) The cosmeceutical composition of claim 2, wherein a) the molar ratio of MG53 to antioxidant ranges from 0.01:1 to 10:1; b) the molar ratio of MG53 to zinc salt is about 1:2 or more; and/or c) the content of aqueous liquid vehicle is at least 30% wt.

4) The cosmeceutical composition of claim 1, wherein said composition a) is adapted for topical administration, dermal administration, transdermal administration, or subcutaneous administration; and/or b) is a gel, cream, or ointment.

5) The cosmeceutical composition of claim 1, wherein said composition a) is a gel comprising at least one cosmetically acceptable antioxidant polymer, an effective amount of MG53, and at least one liquid vehicle, wherein said gel exhibits a low viscosity at less than about 15° C., and said gel solidifies or semi-solidifies (thicken substantially into a high viscosity mass) after contact with skin, or after contact with a surface of at least about 18° C., at least about 20° C., or at least about 25° C.; b) is a cream comprising a cream base and an effective amount of MG53; c) is a cosmeceutical ointment comprising an ointment base and an effective amount of MG53; or d) is a hydrogel that is a liquid prior to topical application to skin and becomes a gel, semi-solid or solid after topical application to skin.

6) The cosmeceutical composition of claim 5, wherein a) said cream base comprises one or more cosmetically acceptable (and/or pharmaceutically acceptable) excipients; b) said antioxidant polymer comprises ROS-scavenging copolymer; c) said hydrogel comprises boronate ester-based copolymer, said hydrogel exhibiting lower viscosity at about a lower temperature and higher viscosity at a higher temperature; and/or d) said hydrogel i) exhibits lower viscosity at about 4° C. and higher viscosity at about 37° C.; ii) is a liquid at about 4° C. and a gel, semi-solid or solid at about 37° C.; and/or iii) at about 4° C. is flowable by hand through an 18-30 gauge needle engaged to a syringe and at about 37° C. is not flowable by hand through an 18-30 gauge needle engaged to a syringe; e)

7) The cosmeceutical composition of claim 6, wherein said copolymer a) is a terpolymer comprising one or more acrylamide monomer(s), one or more methacrylate monomer(s), and one or more boronate ester acrylate monomer(s); and/or b) excludes an alkyne group-containing monomer.

8) The cosmeceutical composition of claim 7, wherein a) said one or more acrylamide (AM) monomer(s) refers to (N-alkyl) acrylamide (NAAM) or homolog(s) or analog(s) thereof; b) said one or more methacrylate (MAc) monomer(s) refers to (hydroxyalkyl) methacrylate (HAMc) or homolog(s) or analog(s) thereof; c) said one or more boronate ester acrylate (BEAc) monomer(s) refers to 4-(hydroxyalkyl)-phenylboronic acid, pinacol ester) acrylate (HPPE-Ac) or homolog(s) or analog(s) thereof, or d) a combination thereof.

9) The cosmeceutical composition of claim 8, wherein a) said one or more acrylamide (AM) monomer(s) or homolog(s) or analog(s) is defined by the following chemical structure ##STR00006## wherein R is propyl, isopropyl, butyl, isobutyl or other alkyl group of 5 or less carbon atoms in length; b) said one or more methacrylate (MAc) monomer(s) or homolog(s) or analog(s) is defined by the following chemical structure ##STR00007## wherein R is (CH.sub.2).sub.n; and n=2, 3, or 4; and/or c) said one or more boronate ester acrylate ester (BEAc) monomer(s) or homolog(s) or analog(s) is defined by the following chemical structure ##STR00008##

10) The cosmeceutical composition of claim 9, wherein alkyl is independently selected upon each occurrence from the group consisting of (C.sub.1-C.sub.2)-alkyl or (C.sub.3-C.sub.8)-alkyl, which alkyl may be linear, branched, or cyclic.

11) The cosmeceutical composition of claim 6, wherein said terpolymer is defined by the formula
poly((AM).sub.m-co-(MAc).sub.n-co-(BEAc).sub.x),  a) wherein: m is in the range of about 75 to about 85, n is in the range of about 5 to about 20, and x is in the range of about 5 to about 20;
poly((NAAM).sub.m-co-(HAMAc).sub.n-co-(HPPE-Ac).sub.x)  b) wherein: m is in the range of about 75 to about 85, n is in the range of about 5 to about 20, and x is in the range of about 5 to about 20; or
poly((NIPAA).sub.m-co-(HEMA).sub.n-co-(AIPPE)x),  c) wherein NIPAAM is N-isopropylacrylamide, HEMA is hydroxyethyl methacrylate, AHPPE is (4-(hydroxymethyl)-phenylboronic acid, pinacol ester) acrylate (or 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)phenyl]methyl ester).

12) A method of improving the appearance of skin by administering to the skin a cosmetically effective amount of the cosmeceutical composition of claim 1.

13) The method of claim 12, wherein said improving is selected from the group consisting of reducing wrinkles, reducing melanin deposition, reducing irritation, reducing inflammation, reducing redness, reducing scarring, reducing collagen formation, reducing differentiation of stem cells into fibroblasts, reducing the period of time for amelioration of dermal of epidermal condition(s).

14) A method of preventing reactive oxygen species (ROS)-related oxidative damage to skin, said method comprising prophylactically administering to the skin a cosmetically effective amount of the cosmeceutical composition of claim 1 prior to exposure of said skin to the ROS.

15) A method of sequestering, neutralizing or eliminating reactive oxygen species (ROS) in skin comprising administering to the skin a cosmetically effective amount of the cosmeceutical composition of claim 1 after exposure of said skin to the ROS.

16) A method of improving hair growth, the method comprising administering to the skin of a subject in need thereof a cosmetically effective amount of the cosmeceutical composition of claim 1.

17) The method of claim 16, wherein said improving hair growth includes at least one of increasing hair strand thickness, improving hair follicle health, increasing the number of hair follicles per unit area of skin, or increasing rate of hair growth.

18) A method of removing skin blemishes or improving the appearance of skin blemishes, the method comprising administering to the skin of a subject in need thereof a cosmetically effective amount of the cosmeceutical composition of claim 1, wherein said removing or improving the appearance of skin blemishes comprises reducing the appearance or number of wrinkle(s), reducing the appearance or number of blotch(es), reducing melanin deposition, and/or reducing the appearance or number of reddened patch(es).

19) A method of healing injured tissue, the method comprising administering to injured tissue an effective amount of the cosmeceutical composition of claim 1, wherein the skin has not been physically injured, such as by impact force, burning, irradiation, or cutting, and/or the skin is otherwise healthy except for exhibiting cosmetically undesired features.

20) Method of treating a healed wound comprising administering to said healed wound, the cosmeceutical composition of claim 1, whereby said administering results in reduction of collagen formation at the site of said healed wound, and/or increase of follicle density at the site of said healed wound.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0072] The following drawings are part of the present specification and are included to further demonstrate certain aspects of the invention. The invention may be better understood by reference to one or more of these drawings in combination with the detailed description of the specific embodiments presented herein.

[0073] FIG. 1 depicts a chart of the number of hair follicles per unit area of skin for wild-type and MG53 knockout (mg53−/−) mice. Quantitation of hair follicles was conducted according to Example 15.

[0074] FIG. 2 depicts photographs of the mice of FIG. 1 twelve days after being shaved and then being administered saline or saline containing MG53 (dose of 1 mg/kg) Example 16.

[0075] FIG. 3 depicts photographs of rats thirty days after being shaved and then administered bovine serum albumin (BSA, dose of 1 mg/kg) in saline or MG53 (dose of 1 mg/kg) in saline (Example 17).

[0076] FIG. 4 depicts a chart of the number of hair follicles per unit area of skin for rats four days after being administered saline or saline containing MG53 (dose of 1 mg of MG53/kg; Example 17).

[0077] FIG. 5 depicts a chart of the number of apoptotic human hair follicle stem cells (HFSC's) twenty-four hours after treatment with hydrogen peroxide (100 micromolar), MG53 (1 microg/ml), or MG53 and hydrogen peroxide as compared to control (Example 18).

[0078] FIG. 6 depicts a chart of the number of hair follicles per unit area for diabetic mice sixteen days after treatment with cream base or cream containing MG53 (10 mg MG53/oz of cream base) (Example 19).

[0079] FIGS. 7A-7C depict chronological charts of degradation of various grades the copolymer after exposure to H.sub.2O.sub.2(50 mM).

[0080] FIG. 8 depicts a chart demonstrating the synergistic efficacy of the combined MG53 and boronate ester-based copolymer based upon wound size reduction over time.

[0081] FIG. 9 depicts a chart demonstrating the synergistic efficacy of the combined MG53 and boronate ester-based copolymer based upon collagen formation after healing of a wound.

[0082] FIG. 10 depicts a chart demonstrating the synergistic efficacy of the combined MG53 and boronate ester-based copolymer based upon follicle density after healing of a wound.

[0083] FIG. 11A depicts a chart of the relative change in melanin deposition after topical treatment of a subject's facial skin with MG53 containing mask according to Example 26.

[0084] FIG. 11B depicts a chart of the relative change in wrinkles after topical treatment of a subject's facial skin with MG53 containing mask according to Example 26.

DETAILED DESCRIPTION OF THE INVENTION

[0085] As used herein, the term ROS-scavenging polymer also refers to a reactive oxygen species-scavenging polymer, whereby such polymers can be more generally referred to as examples of an antioxidant polymer. ROS (reactive oxygen species) comprise both free radical and non-free radical oxygen intermediates such as hydrogen peroxide (H.sub.2O.sub.2), superoxide (O.sub.2.sup.⋅−), singlet oxygen (.sup.1O.sub.2), and the hydroxyl radical. Accordingly, the ROS-scavenging polymer of the invention is able to scavenge such oxygen intermediates so that they do not damage tissue.

[0086] MG53 protein (also referred to as mitsugumin 53 or TRIM72) is known in the art. Unless specified otherwise, all embodiments of the invention comprising or employing “MG53” include all known forms of MG53. It also refers to recombinant human MG53 (rhMG53). As used herein and unless otherwise specified, the term MG53 (or MG53 protein) refers to the MG53 protein present as the native form, optimized form thereof, mutant thereof, derivative thereof or a combination of any two or more of said forms. Native MG53 contains 477 amino acids that are well conserved in different animal species. Methods of preparing and/or isolating MG53 are known: U.S. Pat. No. 7,981,866, WO2008/054561, WO2009/073808, US2011/0202033, US2011/0287004, US2011/0287015, US2013/0123340, WO2011/142744, WO2012/061793, U.S. Pat. Nos. 8,420,338, 9,139,630, 9,458,465, 9,494,602, US2014/0024594, WO2012/134478, WO2012/135868, US2015/0110778, WO2013/036610, US2012/0213737, WO2016/109638, the entire disclosures of which, including sequence information therein, are hereby incorporated by reference.

[0087] The sequence listing information for native MG53, and variants or various forms thereof, is disclosed in U.S. Pat. Nos. 7,981,866 and 9,139,630, the entire disclosures of which, including sequence information therein, are hereby incorporated by reference. The sequence listing information for a cDNA that encodes optimized native human MG53, or a fragment thereof, is disclosed in U.S. Pat. No. 9,139,630, the entire disclosure of which, including sequence information therein, is hereby incorporated by reference.

[0088] As used herein in reference to MG53, the term “mutant” means a recombinant form of MG53 having an amino acid change (replacement) of one, two, three or more amino acids in the amino acid sequence of native MG53. Mutant forms of MG53 and methods of preparing the same are known: US2015/0361146, EP3118317, WO2015/131728, U.S. Pat. No. 9,139,630, the entire disclosures of which, including sequence information therein, are hereby incorporated by reference.

[0089] As used herein the term “endogenous MG53”, refers to MG53 present in a subject prior to treatment with a composition, cosmeceutical, or method according to the invention. As used herein, exogenous MG53 is nonendogenous MG53.

[0090] The present inventors have unexpectedly discovered that an antioxidant polymer applied to the skin can be used to improve the appearance and health of the skin. A boronate ester-based antioxidant polymer can react with ROS in the skin, and thereby prevent and/or reduce the extent of oxidative damage to the skin that is caused by said ROS. Moreover, addition of a cosmetically effective amount (concentration) of MG53 to said antioxidant polymer further improves the appearance of skin and the growth of hair.

[0091] The ROS-scavenging polymer of the invention is a boronate-ester based copolymer gel. It is prepared by reacting acrylamide (or homolog or analog thereof), methacrylate ester (or homolog or analog thereof), and (boronate ester) acrylate ester (or homolog or analog thereof). The molar ratio of the monomers in the reaction milieu approximates the molar ratio of the monomers in the resulting boronate-ester based copolymer: poly((AM).sub.m-co-(MAc).sub.n-co-(BEAc).sub.x), wherein: m, n and x are as defined herein. The identity of each monomer is independently selected upon each occurrence. Thus, the copolymer can comprise one or more acrylamide monomers, one or more methacrylate ester monomers, and one or more (boronate ester) acrylate ester monomers.

[0092] An exemplary boronate ester monomer was prepared according to Example 1. HPPE was dissolved in water immiscible organic solvent and the amine, in slight molar excess, was added thereto. Acryloyl halide, at about the same molar amount as the amine, was added dropwise over a period of time to the prior mixture. After completion of the reaction, the reaction milieu was washed with water and then brine. The organic layer was then dried over desiccant, separated therefrom, and the solvent removed under vacuum. The boronate ester monomer was obtained in high yield.

[0093] An exemplary antioxidant copolymer of the invention was prepared according to Example 2. The boronate ester monomer prepared according to Example 1 was used as the starting material for preparation of the copolymer. Generally, the monomers were mixed in an organic solvent and repeatedly purged with nitrogen. Initiator was then added to the mixture and the entire reaction milieu heated at 65° C. for 20 hours with continuous stirring. The copolymer was isolated by repeated precipitation-dissolution cycles using a mixture of solvents and recovery of solid by filtration prior to each dissolution and after each precipitation. The copolymer with then dried under vacuum with heat. The copolymer was obtained in high yield.

[0094] The liquid gel, comprising the antioxidant (ROS-scavenging) copolymer and an aqueous vehicle, was obtained by dissolving the copolymer in the aqueous vehicle while stirring overnight at about 4° C. (Example 3). The content of copolymer in the gel was about 6 wt % and the content of aqueous vehicle was about 94%.

[0095] Before solidification, a hydrogel solution of the copolymer (about 6 wt % in aqueous vehicle) has a viscosity of less than about 0.1 Pa.Math.sec. Solidification of the hydrogel solution was performed according to Example 3. A portion of hydrogel solution was placed in a tube which was then placed in a heated water bath at 37° C. The gel solidified within seconds. After solidification, the solidified mass viscosity was high.

[0096] The novel antioxidant polymer reduces oxidative damage caused to the skin (tissue) by ROS. A composition comprising antioxidant polymer and one or more antioxidants provides even greater efficacy. A composition comprising antioxidant polymer, MG53, and one or more antioxidants provides still greater efficacy.

[0097] As part of the synthetic pathway toward the target antioxidant copolymer, we have also invented the novel intermediate (or monomer) (4-(hydroxymethyl)-phenylboronic acid, pinacol ester) acrylate. It should be understood that structural homologs and analogs thereof are contemplated within the scope of the invention. Accordingly, the group of monomers defined herein are contemplated.

[0098] The molar ratio of the three monomers in the copolymer may impact performance. The hydrophilicity of the gel increases as the molar ratio increases for MAc or molar ratio decreases for AM/BEAc. As the hydrophilicity changes, the mechanical properties including flexibility, elastic modulus, complex modulus, tensile strength and Young's modulus will change accordingly. For example, a more hydrophilic structure in the copolymer will cause a decrease in tensile strength and modulus as the gels become softer.

[0099] At about 4° C., the antioxidant polymer exhibits low enough viscosity to be syringeable by hand without mechanical assistance through an 18-30-gauge needle. When the antioxidant polymer is warmed to at least 35° C., it forms a much higher viscosity solid or semi-solid gel mass. For example, when the cooled gel is applied as a layer topically to human skin, which is at about 35-38° C., the gel forms a solid or semi-solid layer.

[0100] The compositions of the invention are suitable for use as wound dressings. The ROS-scavenging copolymer (optionally in admixture with MG53) can be administered to a wound in dry form, whereby it will absorb body fluid and become a hydrogel that then solidifies at the site of the wound. The ROS-scavenging copolymer (optionally in admixture with MG53) can be administered to a wound in hydrogel form, whereby it then solidifies at the site of the wound. The copolymer provides reduced collage formation (FIG. 8), increased follicle density (FIG. 9), and faster wound size reduction (FIG. 7) as compared to an untreated wound. The copolymer in combination with MG53 provides even further reduced collage formation, further increased follicle density, and even faster wound size reduction as compared to an untreated wound.

[0101] The ROS-scavenging hydrogel accelerates wound healing due to its function in downregulating the overproduction of ROS on chronic wounds. An appropriate amount of hydrogel at a concentration of 6 wt % administered to a 5 mm-wound on mice ranges from about 150 μL to about 250 μL.

[0102] Suitable concentrations of MG53 in a dosage form include at least 1 ng of MG53/ml, at least 5 ng of MG53/ml, at least 10 ng of MG53/ml, at least 25 ng of MG53/ml, at least 50 ng of MG53/ml, at least 75 ng of MG53/ml, at least 100 ng of MG53/ml, at least 250 ng of MG53/ml, at least 500 ng of MG53/ml, at least 750 ng of MG53/ml, at least 1 μg of MG53/ml, at least 5 μg of MG53/ml, at least 10 μg of MG53/ml, at least 15 μg of MG53/ml, at least 20 μg of MG53/ml, at least 25 μg of MG53/ml, at least 30 μg of MG53/ml, at least 50 μg of MG53/ml, or at least 100 μg of MG53/ml. Higher concentrations are also acceptable, particularly in view the efficacy dose-response trend observed for MG53. These doses can be administered on a frequency as described herein or as determined to be most effective.

[0103] Suitable doses of MG53 that can be administered to a subject in one or more dosage forms include at least 1 ng of MG53, at least 5 ng of MG53, at least 10 ng of MG53, at least 25 ng of MG53, at least 50 ng of MG53, at least 75 ng of MG53, at least 100 ng of MG53, at least 250 ng of MG53, at least 500 ng of MG53, at least 750 ng of MG53, at least 1 μg of MG53, at least 5 μg of MG53, at least 10 μg of MG53, at least 15 μg of MG53, at least 20 μg of MG53, at least 25 μg of MG53, at least 30 μg of MG53, at least 50 μg of MG53, or at least 100 μg of MG53. Such doses can be on a total body weight basis or a per kg of body weight basis.

[0104] The dose of exogenous MG53 can be as low as about 1 up to about 1000 microg per kg of body weight.

[0105] The efficacy of the cosmeceutical composition was established in vivo. The face of a person having undergone laser ablation was subsequently treated with MG53-containing cream (according to Example 13) on only one side of the face according to Example 20 as compared to the standard of care which is a currently marketed conventional moisturizer. The creams were applied daily for seven days. After four days of daily application, the side of the subject's face treated with MG53 was less swollen and exhibited less redness as compared to the conventional moisturizer cream.

[0106] An elderly person having red blistering of unknown etiology was treated with MG53-containing cream (according to Example 13). After three days of daily application (Example 21), the subject's face exhibited reduced blistering as compared to the conventional moisturizer cream. MG53 cream application has significantly reduced time to healing by at least 1 week. This rate of healing was more rapid with daily application AND coverage with bandage. Therefore, keeping the wound moist with MG53 and covered allowed faster epithelialization of the wounds. Furthermore, areas where it is applied regularly (i.e. wrist) are less prone to tearing and blistering.

[0107] An elderly person with wrinkled skin along the cheeks and neck was treated with MG53-containing cream (according to Example 13). The creams were applied for 28 days. After three weeks of daily application (Example 22), the subject's cheeks and neck exhibited substantially reduced wrinkles (both reduced depth and reduced appearance of wrinkles) as compared to the conventional moisturizer cream.

[0108] An elderly person with wrinkled skin along the lips and eyes was treated with MG53-containing cream (according to Example 13). The creams were applied for 28 days. After three weeks of daily application (Example 22), the subject's lips and eyes exhibited substantially reduced wrinkles (both reduced depth and reduced appearance of wrinkles) as compared to the conventional moisturizer cream.

[0109] An elderly person with a long-term chronic nonhealing wound on the skin was treated with MG53-containing cream (according to Example 13). After three weeks of daily application (Example 23), the skin was completed healed and exhibited no scarring. Conventional moisturizer cream was ineffective.

[0110] A 60-year old male underwent a cryofreeze procedure to remove large moles from the left temple. The cryofrozen sites were treated 2-3 times daily with MG53-containing cream (according to Example 13). After 1 week of treatments, the skin was completed healed and exhibited no scarring.

[0111] The invention also provides an MG53-containing cream comprising water (about 65-75%% w/w), at least one preservative (about 0.1-1% w/w), at least one humectant (about 3-5% w/w), at least one exfoliant about 1-5% w/w), at least one penetration enhancer (about 5-15% w/w), at least one emollient (about 1-7% w/w), at least one protectant (skin conditioning agent; about 0.1-5% w/w), at least one antimicrobial (about 0.1-5% w/w), at least one zinc salt (about 0.1-1% w/w).

[0112] MG53 was also evaluated for use in reducing melanin formation and reducing wrinkles according to Example 26. Subjects were treated two to three times per week, for two to three weeks, with a facial mask saturated with normal saline solution comprising rhMG53 (20 microg/mL). In just two weeks, each subject exhibited a substantial reduction in melanin deposition (FIG. 11A) and wrinkles (FIG. 11B).

[0113] Accordingly, the invention provides a method of reducing skin wrinkles by topical administration of rhMG53. The invention also provides a method of reducing melanin deposition by topical administration of rhMG53.

[0114] The amount of therapeutic compound (MG53) incorporated in each dosage form will be at least one or more unit doses and can be selected according to known principles of pharmacy. An effective amount of therapeutic compound is specifically contemplated. By the term “effective amount”, it is understood that, with respect to, for example, pharmaceuticals, a pharmaceutically (therapeutically) effective amount is contemplated. A pharmaceutically effective amount is the amount or quantity of a drug or pharmaceutically active substance which is sufficient to elicit the required or desired therapeutic response, or in other words, the amount which is sufficient to elicit an appreciable biological response when administered to a patient.

[0115] The term “unit dosage form” is used herein to mean a dosage form containing a quantity of the drug, said quantity being such that one or more predetermined units may be provided as a single therapeutic administration.

[0116] The dosage form is independently selected at each occurrence from the group consisting of liquid solution, suspension, gel, cream, ointment, slab, gel, insert (implant), syringe, and hydrogel.

[0117] Compositions and dosage forms of the invention can further comprise one or more pharmaceutically acceptable and/or cosmetically acceptable excipients. Dosage forms can comprise one or more excipients independently selected at each occurrence from the group consisting of acidic agent, alkaline agent, buffer, tonicity modifier, osmotic agent, water soluble polymer, water-swellable polymer, thickening agent, complexing agent, chelating agent, penetration enhancer. Suitable excipients include U.S.F.D.A. inactive ingredients approved for use in parenteral or oral formulations (dosage forms), such as those listed in the U.S.F.D.A.'s “Inactive Ingredients Database (available on the following website: www.fda.gov/Drugs/InformationOnDrugs/ucm113978.htm; October 2018), the entire disclosure of which is hereby incorporated by reference.

[0118] As used herein, an acidic agent is a compound or combination of compounds that comprises an acidic moiety. Exemplary acidic agents include organic acid, inorganic acid, mineral acid and a combination thereof. Exemplary acids include hydrochloric acid, hydrobromic acid, sulfuric acid, sulfonic acid, sulfamic acid, phosphoric acid, or nitric acid or others known to those of ordinary skill; and the salts prepared from organic acids such as amino acids, acetic acid, propionic acid, succinic acid, glycolic acid, stearic acid, lactic acid, malic acid, tartaric acid, citric acid, ascorbic acid, pamoic acid, maleic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, sulfanilic acid, 2-acetoxybenzoic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, ethane disulfonic acid, oxalic acid, isethionic acid, others acids known to those of ordinary skill in the art, or combinations thereof.

[0119] As used herein, an alkaline agent is a compound or combination of compounds that comprises an alkaline moiety. Exemplary alkaline agents include primary amine, secondary amine, tertiary amine, quaternary amine, hydroxide, alkoxide, and a combination thereof. Exemplary alkaline agents include ammonia solution, ammonium carbonate, diethanolamine, monoethanolamine, potassium hydroxide, sodium borate, sodium carbonate, sodium bicarbonate, sodium hydroxide, triethanolamine, diethanolamine, monobasic phosphate salt, dibasic phosphate salt, organic amine base, alkaline amino acids and trolamine, others known to those of ordinary skill in the art, or combinations thereof.

[0120] Exemplary excipients (inactive ingredients as defined by the U.S.F.D.A.) that can be included in dosage forms of the invention include, by way of example and without limitation, water, benzalkonium chloride, glycerin, sodium hydroxide, hydrochloric acid, boric acid, hydroxyalkylphosphonate, sodium alginate, sodium borate, edetate disodium, propylene glycol, polysorbate 80, citrate, sodium chloride, polyvinylalcohol, povidone, copovidone, carboxymethylcellulose sodium, Dextrose, Dibasic Sodium Phosphate, Monobasic Sodium Phosphate, Potassium Chloride, Sodium Bicarbonate, Sodium Citrate, Calcium Chloride, Magnesium Chloride, stabilized oxychloro complex, Calcium Chloride Dihydrate, Erythritol, Levocarnitine, Magnesium Chloride Hexahydrate, Sodium Borate Decahydrate, Sodium Citrate Dihydrate, Sodium Lactate, Sodium Phosphate (Mono- and Dibasic-), Polyethylene Glycol 400, Hydroxypropyl Guar, Polyquaternium-1, Zinc Chloride, white petrolatum, mineral oil, hyaluronic acid, artificial tear, or combinations thereof.

[0121] One or more antioxidants can be included in a composition of dosage form of the invention. Exemplary antioxidants include SS-31, NAC, glutathione, selenium, vitamin A, vitamin C, vitamin E, co-enzyme Q10, resveratrol, other GRAS antioxidant, or a combination of two or more thereof.

[0122] One or more zinc salts can be included in a composition or dosage form of the invention. Such zinc salt(s) may also be administered to a subject receiving exogenous MG53 or expressed MG53. Pharmaceutically acceptable zinc salts include Zinc gluconate, Zinc acetate, Zinc sulfate, Zinc picolinate, Zinc orotate, Zinc citrate, and other such salts comprising a zinc cation and organic or inorganic anion(s).

[0123] It should be understood that compounds used in the art of pharmaceutical formulations generally serve a variety of functions or purposes. Thus, if a compound named herein is mentioned only once or is used to define more than one term herein, its purpose or function should not be construed as being limited solely to that named purpose(s) or function(s).

[0124] As used herein, “pharmaceutically acceptable salts” refer to derivatives of the disclosed compounds wherein the compound is modified by making an acid or base salt thereof. Examples of pharmaceutically acceptable salts include, but are not limited to, mineral or organic acid salts of basic residues such as amines; alkali or organic salts of acidic residues such as carboxylic acids; and others known to those of ordinary skill. The pharmaceutically acceptable salts can be synthesized from the parent therapeutic compound which contains a basic or acidic moiety by conventional chemical methods. Lists of suitable salts are found in Remington's Pharmaceutical Sciences, 17th ed., Mack Publishing Company, Easton, Pa., 1985, p. 1418, the disclosure of which is hereby incorporated by reference.

[0125] The phrase “pharmaceutically acceptable” is employed herein to refer to those compounds, materials, compositions, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.

[0126] MG53 can be used in cotherapy or adjunctive therapy with one or more other active ingredients. Exemplary suitable active ingredients include, among others, U.S.F.D.A. approved drugs for parenteral or oral dosage forms. Other active ingredients that can be used in cotherapy or adjunctive therapy with MG53 include, by way of example and without limitation, Benzoyl Peroxide, Beta-Hydroxy Acid (BHA), Dihydroxyacetone (DHA), Elastin, Sodium Hyaluronate, Zinc Oxide, Titanium Oxide, Vitamin E, or combinations thereof.

[0127] The therapeutically acceptable dose, maximum tolerated dose (MTD), and minimally effective dose (MED) for each of said active ingredients is well known and set forth in the respective U.S.F.D.A. approved product package insert for each said active ingredients.

[0128] A composition, dosage form or formulation of the invention can include one, two or more active ingredients in combination with MG53. The dose of each said active ingredient in said composition, dosage form or formulation of the invention will be a therapeutically effective dose including and above the MED and including and below the MTD.

[0129] In some embodiments, the combination treatment of MG53 with another active ingredient provides at least additive therapeutic efficacy. In some embodiments, said combination provides synergistic therapeutic efficacy. In some embodiments, MG53 reduces the occurrence of, reduces the level of, or eliminates adverse events caused by the other active ingredient.

[0130] The acceptable concentrations of said excipients are well known in the art and specific concentrations (amounts) thereof are set forth in the package insert or package label of known commercial products containing the same.

[0131] It should be understood that compounds used in the art of pharmaceutics may serve a variety of functions or purposes. Thus, if a compound named herein is mentioned only once or is used to define more than one term herein, its purpose or function should not be construed as being limited solely to that named purpose(s) or function(s).

[0132] In the examples below, ranges are specified for the amount of each ingredient. Ranges including “0” as the lowest value indicate an optional ingredient. The lower limit “>0” indicates the respective material is present.

[0133] As used herein, the terms “about” or “approximately” are taken to mean a variation or standard deviation of 10%, +5%, or ±1% of a specified value. For example, about 20 mg is taken to mean 20 mg±10%, which is equivalent to 18-22 mg.

[0134] As used herein, the term “prodrug” is taken to mean a compound that, after administration, is converted within a subject's body, e.g. by metabolism, hydrolysis, or biodegradation, into a pharmacologically active drug. The prodrug may be pharmacologically active or inactive. For example, a prodrug of MG53 (native or mutant) would be converted to the native form or mutant form, respectively, of MG53. The term “precursor” may also be used instead of the term “prodrug”.

[0135] As used herein, the term “derivative” is taken to mean: a) a chemical substance that is related structurally to a first chemical substance and theoretically derivable from it; b) a compound that is formed from a similar first compound or a compound that can be imagined to arise from another first compound, if one atom of the first compound is replaced with another atom or group of atoms; c) a compound derived or obtained from a parent compound and containing essential elements of the parent compound; or d) a chemical compound that may be produced from first compound of similar structure in one or more steps. For example, a derivative may include a deuterated form, oxidized form, dehydrated, unsaturated, polymer conjugated or glycosilated form thereof or may include an ester, amide, lactone, homolog, ether, thioether, cyano, amino, alkylamino, sulfhydryl, heterocyclic, heterocyclic ring-fused, polymerized, pegylated, benzylidenyl, triazolyl, piperazinyl or deuterated form thereof.

[0136] In the examples below, ranges are specified for the amount of each ingredient. Ranges including “0” as the lowest value indicate an optional ingredient. Compositions with quantities of ingredients falling within the compositional ranges specified herein were made. Compositions of the invention comprising quantities of ingredients falling within the compositional ranges specified herein operate as intended and as claimed.

[0137] In view of the above description and the examples below, one of ordinary skill in the art will be able to practice the invention as claimed without undue experimentation. The foregoing will be better understood with reference to the following examples that detail certain procedures for the preparation and use of compositions according to the present invention. All references made to these examples are for the purposes of illustration. The following examples should not be considered exhaustive, but merely illustrative of only a few of the many embodiments contemplated by the present invention. The methods described herein can be followed to prepare and use compositions of the invention and to practice methods of the invention.

[0138] MG53 was kindly provided by TRIM-edicine, Inc. (1275 Kinnear RD, Columbus Ohio 43212-1155).

Example 1

Preparation of AHPPE Intermediate

[0139] 4-(hydroxymethyl)-phenylboronic acid pinacol ester (HPPE) (11.7 g, 50 mmol) was dissolved in dehydrated dichloromethane (DCM, 60 mL) in a flask, followed by adding TEA (6.1 g, 60 mmol). Then the flask was placed on ice to be pre-cooled, and after cooling to 0° C., acryloyl chloride (5.4 g, 60 mmol) in 5.4 mL anhydrous DCM was added dropwise for 1 h. Then the ice under the flask was removed and the reaction mixture was warmed back to room temperature with continuous stirring. After 10 h reaction, the products were filtered, washed by DI water and brine for 3 times, respectively. Then the products were concentrated using rotary evaporator and dried with MgSO.sub.4. The monomer AHPPE was then obtained as faintly yellow powder and the yield was ˜90%.

Example 2

Preparation of AAHPPE Gel

[0140] Starting with the AHPPE of Example 1, the gel was prepared as follows.

[0141] Poly (NIPAAm-co-HEMA-co-AAcPB) was polymerized by dissolving dehydrated NIPAAM, HEMA and AHPPE in dioxane with 20 min purging of N.sub.2. BPO was added as initiator, followed by another 20 min purging with N.sub.2. The mixture was then placed in a preheated oil bath at 65° C. for 20 hours with continuous stirring. The products were obtained and purified by three times precipitation-redissolution cycle using tetrahydrofuran (THF)/hexane and THF/ethyl ether. The final products were further dried by vacuum oven and lyophilizer. Three different compositions of copolymers AHPPE-10, AHPPE-8 and AIPPE-6 were synthesized with the ratios of 75/15/10, 77/15/8 and 79/15/6 for NIPAAm/IEMA/AHPPE, respectively.

Example 3

AAHPPE Gel Solidification

[0142] Starting with the AAHPPE gel of Example 2, the gel (hydrogel solution) was prepared as follows. The purified copolymer was dissolved in DPBS, with continuous stirring at 4° C. overnight. 200 μL of hydrogel solution was transferred in a 1.5 mL microcentrifuge tube. The tubes were then placed in a 37° C. water bath and the gel solution solidified within seconds.

Example 4

Dermal Dressing Comprising AAHPPE Gel Alone

[0143] The AAHPPE gel of Example 2 was applied directly to the skin. Before application, the gel was fluid with a viscosity of less than about 0.1 Pa sec. An applicator was loaded with the fluid gel and placed on the skin of a subject, whereby the gel solidified within about 1 min to form a dressing.

Example 5

Dermal Dressing Comprising AAHPPE Gel and MG53

[0144] The AAHPPE gel (2 mL, 8 wt. % in aqueous vehicle, 160 mg) of Example 2 was mixed with MG53(2 mg, at a concentration of 1 mg/mL). rhMG53 peptide was dissolved in sterilized DPBS and further sterilized by filtering through 0.22 μm syringe filter for two times. The concentration of hydrogel solution was 8 wt. % and the rhMG53 solution in DPBS was thoroughly mixed with hydrogel solution at 4° C. overnight. The final concentration of rhMG53 was 1 mg/mL. The mixture was then applied directly to the skin, whereby the gel solidified within about 1 min to form a dressing. The gel provided a continuous release of MG53. Greater than about 60% wt of the total amount of MG53 was released in 21 days.

Example 6

AAHPPE Gel with Another Active Ingredient

[0145] The AAHPPE gel of Example 2 is mixed with another active ingredient.

[0146] The mixture is then applied directly to the skin, whereby the gel solidifies in less than five min to form a dressing. The gel provides a continuous release of the active ingredient.

Example 7

Evaluation of Antioxidant Activity of AAHPPE Gel

[0147] The ability of the AAHPPE gel (8 wt. % in DPBS) of Example 2 to sequester or capture reactive oxygen species was evaluated in Dulbecco's phosphate buffered saline (DPBS) in the presence of hydrogen peroxide (50 mM). Three different embodiments of the gel (AAHPPE-10, AAHPPE-8, AAHPPE-6) were evaluated. The loss of weight of gel corresponded to the extent of hydrogen peroxide sequestered.

[0148] AAHPPE-10, AAHPPE-8 and AAHPPE-6 (copolymer content of 8 wt. % in each) hydrogel solutions (200 μL) were transferred to 1.5 mL microcentrifuge tubes (tubes were weighed before use as w4), sitting in a 37° C. water bath for 1 h to complete gelation process. After gelation, the supernatants were removed from each tube and 200 μL of DPBS without H.sub.2O.sub.2 and with 50 mM H.sub.2O.sub.2 was added to the tubes. The sample tubes were taken out at each time point on week-0, week-1, week-2, week-3 and week-4. At each time point, the tubes were weighed after removing supernatants and recorded as w5. Finally, the samples were washed by DI H.sub.2O and solutions were evaporated to dryness using lyophilizer and tubes weighed as w6. During the degradation, the medium (DPBS with/without H.sub.2O.sub.2) was exchanged every other day to guarantee the fresh H.sub.2O.sub.2 for use. Four repeats of samples were used for each condition (n=4). Additionally, the one-week degradation products were obtained, freeze dried and characterized using .sup.1H NMR using DMSO as the solvent. The copolymers AAHPPE6/8/10 before degradation were also dissolved in DMSO for NMR test at the same time.

Example 8

[0149] Evaluation of Wound Healing Activity of AAHPPE gel with and without MG53

[0150] The ability of the AAHPPE gel (8 wt. % in DPBS, 200 μL for each wound) of Examples 2 and 5 to accelerate healing of a puncture wound was evaluated as follows.

[0151] Before wound incision, the mice hair on the back was shaved using an electric clipper. Betadine solution and alcohol prep pads were used to wipe the naked area on the back to avoid infection. Two wounds on left and right were created on each mouse using sterilized 5 mm-diameter biopsy punches (Integra Miltex, Plainboro, N.J.) so that the underlying dorsolateral skeletal muscle can be exposed. Wounds left open with no dressing on db/+ mice (C57BL/6 mice) and db/db mice were considered as control groups. 200 μL of hydrogel solution was injected on the wound subcutaneously.

Example 9

Evaluation of Release of MG53 from AAHPPE Gel

[0152] AAHPPE copolymer grades containing different monomer ratios (75/15/10, 77/15/8 and 79/15/6 for NIPAAm/HEMA/AHPPE) were prepared according to Example 1. The corresponding gels (AAHPPE-10, monomer ratio 75/15/10; AAHPPE-8, monomer ratio 77/15/8) were prepared by placing the copolymers in Dulbecco's Phosphate-Buffered Saline (DPBS) (copolymer concentration: 8 wt. %, 200 μL, 16 mg). The copolymers were solidified with slight heat and the release of MG53 over a period of 21 days was monitored by drawings aliquots and analyzing the supernatant. The concentration of released rhMG53 was measured by the microplate reader (Molecular Device). 280 nm wavelength was used in the absorption read mode. The standard curve was acquired by measuring absorbance of various concentrations of rhMG53 in DPBS (0, 0.25, 0.5, 0.75, 1.0, 1.25, 1.5, 1.75 mg/mL) using the plate reader. The observed standard curve was well fitted by a linear regression model. Therefore, the cumulative released rhMG53 concentration was calculated by dividing the release rhMG53 by the total rhMG53 encapsulated in the hydrogel.

TABLE-US-00003 Content of MG53 mg/ml of gel Name AAHPPE-10 AAHPPE-8 9A 1   0 9B 0.5 0 9C 0   1 9D 0   2

Example 10

Comparison of Antioxidant Activity of AAHPPE Gel and APLA Gel

[0153] APLA (poly (NIPAAm-co-HEMA-co-APLA)) is a non-reactive gel, meaning it is non-responsive to reactive oxygen species (ROS).

[0154] Fenton reaction method. 200 μL of APLA or AHPPE hydrogel solution was mixed with 200 μL FeSO.sub.4 solution (2 mM), 200 μL Safranin 0 (360 μg/mL) and 320 μL H.sub.2O.sub.2(6 wt. %) solution, was incubated for 10 min, followed by heating in a 55° C. water bath for 30 min. 200 μL of DI H.sub.2O was used as blank group. For the control group, DI H.sub.2O was used instead of AHPPE gel sample and H.sub.2O.sub.2 solution. The mixtures were then cooled to room temperature and the absorption was read at 492 nm using a plate reader (Molecular Device). The scavenging capacity of hydroxyl radicals was determined by the equation (1):

[00001]$\begin{array}{cc}\mathrm{Hydroxyl}\mathrm{radical}\mathrm{scavenging}\mathrm{effect}\left(\%\right)=\frac{A_{\mathrm{sample}}-A_{\mathrm{blank}}}{A_{\mathrm{control}}-A_{\mathrm{blank}}}\times 100& \left(1\right)\end{array}$

[0155] Pyrogallol assay method. 500 μL of Tris-HCl (50 mM, pH=8.1) was added into a 200 μL gel solution, followed by the drop-wise addition of 80 μL pyrogallol (3 mM) in the dark. At last, 20 μL HCl (8 M) solution was added into the mixture to stop the reaction. For the control group, DI H.sub.2O was used to substitute the gel solution. The absorption was measured using the plate reader. The capability to scavenge superoxide was then calculated based on the equation (2):

[00002]$\begin{array}{cc}\mathrm{Superoxide}\mathrm{scavenging}\mathrm{effect}\left(\%\right)=\frac{A_{\mathrm{control}}-A_{\mathrm{sample}}}{A_{\mathrm{control}}}\times 100& \left(2\right)\end{array}$

Example 11

Evaluation of Hair Growth Promoting Activity of AAHPPE Gel with and without MG53

[0156] The ability of the AAHPPE gel (8 wt. % in DPBS, 200 μL, 16 mg of copolymer) of Examples 2 and 5 to promote hair growth wound was evaluated as follows. A digital camera (Panasonic, Osaka, Japan), a metric ruler and a digital caliper (CD-6″ CSX, Japan) were used for post-surgery wound measurements at each time point (day 0, 2, 4, 6, 8, 10, 12, 14, 16, 18, 20 and 22 post-surgery). The camera was used to capture the wound along with the ruler inside the field. Image-J was then used to accurately calculate wound size. The area of each wound was recorded.

Example 12

Evaluation of AAHPPE Gel with and without MG53 for Protection Against Oxidative Stress

[0157] The ability of the AAHPPE gel of Examples 2 and 5 to protect tissue against oxidative stress was evaluated as follows. FSCs survival on 2D hydrogels with and without H.sub.2O.sub.2 was monitored for 5 days. MTT assay and live cell images illustrated that ROS responsive AHPPE gel enhanced FSCs survival rate under oxidative stress compared with non-ROS responsive APLA gel.

[0158] Method A: (AHPPE gel and APLA gel without MG53): FSCs used for in vitro experiments were passage 4-7. The culture medium used was the complete media with Serum and 1% penicillin. FSCs were digested using trypsin-EDTA (0.25%) from T-75 culture plates and re-suspended in pre-warmed culture medium without serum. The 96 well plate was pre-coated evenly with 200 μL of the hydrogel solution (8 wt. % AHPPE gel and 8 wt. % APLA gel was used as a control) and balanced in a 37° C., 5% CO.sub.2 and 21% 02 incubator. 200 μL of FSCs suspension were seeded into the well with a density of 10,000 cells/mL. The plate was incubated in 37° C. for 24 h and then the culture medium was replaced with fresh medium (500 μM H.sub.2O.sub.2 or without H.sub.2O.sub.2). The medium was exchanged every other day to guarantee fresh H.sub.2O.sub.2.Three plates were used for different time points at day 1, 3 and 5. At each time point, 20 μL of MTT solution (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, 5 mg/mL) was added to the well and incubated for another 4 h before adding 200 μL DMSO. The intensity of fluorescence was read at 560 nm and background at 670 nm was subtracted. The groups included AHPPE gel without H.sub.2O.sub.2, APLA gel without H.sub.2O.sub.2, AHPPE gel with 500 μM H.sub.2O.sub.2 and APLA gel with 500 μM H.sub.2O.sub.2. In addition, FSCs were stained with CMDil (a fluorescent dye used as live cell tracker, Thermofisher) before seeding on the hydrogel. At each time point, the samples were taken out and the medium were removed. Live cell images were obtained by confocal microscope using Z-stack mode.

[0159] Method B: (FSCs with or without MG53): FSCs were cultured in 96-well plate with a seeding density of 2,000 cells/well. FSCs were treated with 500 μM H.sub.2O.sub.2 24 h after seeding and the groups included without MG53, with 1 μg/mL MG53 and 10 μg/mL MG53. MTT assay was performed 5 days post treatment to evaluate the cell viability with or without different concentrations of MG53. TUNEL assay was also performed using TUNEL assay kit-HRP (abcam) to validate the effect of MG53 on reducing cell apoptosis. FSCs were cultured on glass slide inserted in a 6-well plate and treated with or without MG53/500 μM H.sub.2O.sub.2 for 24 h. FSCs were then washed by cold DPBS twice and resuspended in 4% formaldehyde in DPBS at a density of 1×10.sup.6/mL, and incubated at room temperature for 10 min. The cells were then rehydrated in TBS, specimen permeabilized using proteinase K with 1/100 dilution in dH.sub.2O. Inactivation of endogenous peroxidases was achieved by adding diluted H.sub.2O.sub.2 in methanol (1/10), followed by labeling reaction using TdT labeling reaction mixture, and development using methyl green counterstain solution provided by the kit.

Example 13

MG53-Containing Cream

[0160] Lyophilized MG53 is mixed with cream base (1.5 to 10 mg MG53 per oz of cream base) until homogeneity is reached. The cream base comprises water (about 65-75%% w/w), at least one preservative (about 0.1-1% w/w), at least one humectant (about 3-5% w/w), at least one exfoliant about 1-5% w/w), at least one penetration enhancer (about 5-15% w/w), at least one emollient (about 1-7% w/w), at least one protectant (skin conditioning agent; about 0.1-5% w/w), at least one antimicrobial (about 0.1-5% w/w), at least one zinc salt (about 0.1-1% w/w).

Example 14

Oral Dosage Form of rhMG53 and EUDRAGIT S-100

[0161] rhMG53 is provided by TRIM-edicine, Inc. (Columbus, Ohio). EUDRAGIT S-100 (Poly(methacylic acid-co-methyl methacrylate) 1:2) is provided by EVONIK healthcare.evonik.com/product/health-care/en/. The following procedure is used to prepared beads.

[0162] In a 100 mL beaker, add 35 mL water, and stir. While stirring add Eudragit S-100 powder (1.4 g), then and 12N NH.sub.4OH (0.82 mL).

[0163] Add 2-hydroxypropyl)-β-cyclodextrin (0.24 g) to a 10 mL water (CD: 24 mg/mL).

[0164] Prepare a solution of MG53 (70 mg in ˜15.5 mL PBS) at a pH 8. To this solution, add 10 mL of the CD solution and 10 mL of water for a total volume of 35.55 mL.

[0165] Mix the MG53/CD solution with the Eudragit solution while stirring.

[0166] Spray dry the resulting suspension to form the powdered dosage form containing MG53 (70 mg), EUDRAGIT (1.4 g), salts (130 mg), and CD (0.24 g) for a total solids content of 1.77 g or a MG53-loading of 40 mg/g of solid (4% loading). Spray drying conditions used: nozzle size—0.6 mm; air speed—0.3 m.sup.3/min; air outlet temp: 38 C; room temperature: 24 C; room humidity: 53%.

[0167] The powder can be included in a capsule, caplet, tablet or other oral dosage form.

Example 15

Quantitation of Hair Follicles

[0168] Skin samples dissected from the back of experimental animals were fixed in 10% formalin, then processed and embedded in 4 m thick paraffin sections. skin sections were stained with hematoxylin-eosin (H&E). Quantification of the number of hair follicles was made by count hair follicle numbers in each 1000 μM skin section.

Example 16

Evaluation of MG53 Upon Hair Growth in Mice

[0169] Dorsal hair was shaved using electric clippers and cleared by Nair™ (hair remover lotion). Then the mice were immediately received a subcutaneous injection of either 200 μl saline (as control) or rhMG53 (2 mg/kg), and treated daily for 7 successive days. The images were captured at indicated time points by a digital camera (Panasonic DMC-ZS3, Japan).

Example 17

Evaluation of MG53 Upon Hair Growth in Rats

[0170] Dorsal hair was shaved using electric clippers. Then the rats were immediately received a subcutaneous injection of either BSA (1 mg/kg) or rhMG53 (1 mg/kg), and treated daily for 7 successive days. The images were captured at indicated time points by a digital camera (Panasonic DMC-ZS3, Japan).

[0171] Dorsal hair was shaved using electric clippers and disinfected. Two full thickness dermal wounds were created using sterile ϕ 6 mm biopsy punches (Integra™ Miltex®). And the rats were immediately received a subcutaneous injection of either 200 μl saline (as control) or rhMG53 (1 mg/kg) and treated daily for 7 successive days. Skin samples were stained with Hematoxylin-Eosin (H&E). Quantification of the number of hair follicles revealed improved hair follicle growth in the rats with rhMG53 treatment compared to saline control at day 14 following wound.

Example 18

Evaluation of MG53 Upon Improvement of Survivability of Human Hair Follicle Stem Cells Following Treatment with Hydrogen Peroxide

[0172] FSCs were cultured in 96-well plate with a seeding density of 2,000 cells/well. FSCs were treated with 500 μM H.sub.2O.sub.2 24 h after seeding and the groups included without MG53, with 1 μg/mL MG53 and 10 μg/mL MG53. FSCs were then washed by cold DPBS twice and resuspended in 4% formaldehyde in DPBS at a density of 1×10.sup.6/mL, and incubated at room temperature for 10 min. The specimens were then rehydrated in TBS, and permeabilized using proteinase K with 1/100 dilution in dH2O. Inactivation of endogenous peroxidases was achieved by adding diluted H2O2 in methanol (1/10), followed by labeling reaction using TdT labeling reaction mixture, and development using methyl green counterstain solution provided by the kit. All staining was imaged using a KEYENCE microscope. Apoptotic cells were quantified from the acquired images.

Example 19

Evaluation of MG53-Containing Cream for Improvement of Hair Growth in Diabetic Mice

[0173] A mouse model of chronic excisional dermal wound (4 mm×4 mm Biopsy punch) was applied on the db/db mice. Topical creams containing 10 mg/oz of rhMG53 or control (without rhMG53) were applied every other day. At the day 12, the wound tissue was excised, fixed, embedded. H/E stating was performed to evaluate the hair follicle numbers in wound area.

Example 20

Evaluation of MG53-Containing Cream for Treatment of Skin after Laser Ablation

[0174] On the day of the laser ablation procedure, patients had their photo taken for comparative data. Immediately following the treatment, a marketed conventional moisturizer product was applied to the right side of the patient's face and MG53 cream was applied to the patient's left side of the face. The creams were applied daily for 7 days post-procedure. The picture was taken daily. The figure was shown for the day 4.

Example 21

[0175] Evaluation of MG53-containing cream for treatment of red blemishes on skin

[0176] An elderly white male diagnosed with Bullous pemphygoid-like skin condition. Similar to butterfly skin syndrome. Skin is fragile and breaks easily with slight mechanical stress. Skin also blisters easily before bursting, exposing open wounds that heal slowly due to skin condition. Individual frequently tears skin in his sleep waking up with new tears/wounds and bloodied sheets/clothes. MG53 cream was applied daily to both new and scabbed wounds. MG53 cream application has significantly reduced time to healing by at least 1 week. This rate of healing was more rapid with daily application AND coverage with bandage. Therefore, keeping the wound moist with MG53 and covered allowed faster epithelialization of the wounds. Furthermore, areas where it is applied regularly (i.e. wrist) are less prone to tearing and blistering.

Example 22

Evaluation of MG53-Containing Cream for Reduction of Skin Wrinkles

[0177] On the day of the CO.sub.2 procedure, patients had their photo taken for comparative data. Immediately following the treatment, a conventional commercial cream product was applied to the left side of the patient's face and MG53 cream was applied to the patient's right side of the face. The creams were applied daily for 28 days post-procedure. The picture was taken daily. The figure was shown for week 3 post-procedure.

Example 23

Evaluation of MG53-Containing Cream for Treatment of Chronic Open Wound in Elderly Person

[0178] An elderly male diagnosed with Bullous pemphygoid-like skin condition was in otherwise good health. Skin was fragile and broke easily with slight mechanical stress. The hand wound was treated with MG53-containing cream daily for 3 weeks. The skin became more resistant to tearing by slight mechanical stress.

Example 24

Evaluation of MG53-Containing Cream for Treatment of Cryogenically Treated Tissue

[0179] An elderly male had a mole removed from the face by cryofreeze. The dermatologist stated that at least 1 week would be needed before pain would subside and 2 weeks before visible improvements in skin healing would be observed. Daily application of MG53-containing cream resulted in pain reductions after 1-2 days and rapid healing within 1 week. No bandage was applied. Cream was applied 2-3 times per day.

Example 25

rhMG53 Protein Production and Quality Control

[0180] The following process was used to produce native MG53 protein.

[0181] E. coli fermentation was used to obtain high quality (>97% purity) rhMG53 (recombinant human MG53) protein as described by Zhu et al. (“Polymerase transcriptase release factor (PTRF) anchors MG53 protein to cell injury site for initiation of membrane repair” in The Journal of biological chemistry (2011), 286, 12820-12824) and Weisleder et al. (Recombinant MG53 protein modulates therapeutic cell membrane repair in treatment of muscular dystrophy. Science translational medicine (2012), 4, 139ra185), the entire disclosures of which are hereby incorporated by reference. The membrane protective activity of rhMG53 from each preparation was determined with established micro-glass bead injury assay as described previously (ibid).

Example 26

Treatment of Wrinkles and Melanin Production with rhMG53-Containing Mask

[0182] A MG53-containing mask was prepared by saturating a commercially available disposable dry mask cloth with a normal saline solution containing rhMG53 (20 microg/mL).

[0183] About two to three times per week, an MG53-containing mask was placed on the face of a subject after the skin had been cleaned. The mask was administered at night for about 10-30 min per treatment. The subject was administered the mask for about two to three weeks or more. The melanin content and number of wrinkles were quantified with a skin Cutometer before and after the two-week treatment period. Results are depicted in FIGS. 11A-11B.

[0184] All data are expressed as mean±S.D. Groups were compared by Student's t test and analysis of variance for repeated measures. A value of p<0.05 was considered significant.

[0185] For any range herein, the upper and lower limits thereof are considered as being part of the range. Moreover, all integer and fractional values within said ranges are also considered as being within said range. Accordingly, all integers and fractional values within each specified range are hereby incorporated by reference.

[0186] All values disclosed herein may have standard technical measure error (standard deviation) of ±10%. The term “about” or “approximately” is intended to mean±10%, ±5%, ±2.5% or ±1% relative to a specified value, i.e. “about” 20% means 20±2%, 20±1%, 20±0.5% or 20±0.25%. The term “majority” or “major portion” is intended to mean more than half, when used in the context of two portions, or more than one-third, when used in the context of three portions. The term “minority” or “minor portion” is intended to mean less than half, when used in the context of two portions, or less than one-third, when used in the context of three portions. It should be noted that, unless otherwise specified, values herein concerning pharmacokinetic or dissolution parameters are typically representative of the mean or median values obtained.

[0187] The above is a detailed description of particular embodiments of the invention. It will be appreciated that, although specific embodiments of the invention have been described herein for purposes of illustration, various modifications may be made without departing from the spirit and scope of the invention. Accordingly, the invention is not limited except as by the appended claims. All of the embodiments disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure.