PEPTIDES FOR HAIR GROWTH

Abstract

A method of activating inactive hair follicles by administering a composition including an effective amount of zinc thymulin to a subject. The method generates new hair growth and may be used to treat or prevent hair loss or, alternatively, to increase the quantity of hair in a subject that does not suffer from hair loss.

Claims

1.-15. (canceled)

16. A method of activating inactive hair follicles comprising administering a composition to a subject, wherein the composition comprises an effective amount of exogenous zinc thymulin.

17. The method of claim 16 wherein the subject has normal hair density.

18. The method of claim 16 wherein the method treats and/or prevents hair loss.

19. The method of claim 18 wherein the subject suffers from hair loss and the hair loss is reversed.

20. The method of claim 16 wherein the number of active hair follicles in at least 1 square centimetre of the subject's skin is increased by 5 to 300%.

21. The method of claim 18 wherein the number of active hair follicles in at least 1 square centimetre of the subject's skin is increased by 20 to 300%.

22. The method of claim 17 wherein the number of active hair follicles in at least 1 square centimetre of the subject's skin is increased by 5 to 100%.

23. The method of claim 20 wherein the increase has occurred at about 4 months of treatment.

24. The method of claim 21 wherein the increase has occurred at about 4 months of treatment.

25. The method of claim 22 wherein the increase has occurred at about 4 months of treatment.

26. The method of claim 16 wherein the subject's hair density is increased.

27. The method of claim 16 wherein the administration is topical or by injection.

28. The method of claim 16 wherein the zinc thymulin is present at an amount of about 0.0001%-0.1% w/v.

29. The method of claim 16 wherein the zinc thymulin is present at an amount of about 0.001%-0.05% w/v.

30. The method of claim 16 wherein the zinc thymulin is present at an amount of about 0.02% w/v.

31. The method of claim 16 wherein LKKTETQ peptide is also administered to the subject.

32. A method of increasing the number of hair follicles producing hair with natural pigment comprising administering an effective amount of zinc thymulin to a subject.

33. A topical composition comprising about 0.0001%-0.1% w/v zinc thymulin and at least one pharmaceutically or cosmetically acceptable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0060] FIG. 1: Broad image of the scalp of subject 4 of the clinical trial described in Example 3 before treatment with 0.0005% zinc thymulin topical spray. Camera: Samsung Galaxy Note 4 camera with settings F2.2, focal length 31 mm, LED flash.

[0061] FIG. 2: Broad image of subject 4 of the clinical trial described in Example 3 after 3 months twice daily treatment with 0.0005% zinc thymulin topical spray. Camera: Samsung Galaxy Note 4 camera with settings F2.2, focal length 31 mm, LED flash.

[0062] FIG. 3: Close-up image of subject 4 of the clinical trial described in Example 3 before treatment before treatment with 0.0005% zinc thymulin topical spray. Camera: Samsung Galaxy Note 4 camera with settings F2.2, focal length 31 mm, LED flash.

[0063] FIG. 4: Close-up image of subject 4 of the clinical trial described in Example 3 after 3 months twice daily treatment with 0.0005% zinc thymulin topical spray. Camera: Samsung Galaxy Note 4 camera with settings F2.2, focal length 31 mm, LED flash.

DETAILED DESCRIPTION OF THE EMBODIMENTS

[0064] Reference will now be made in detail to certain embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that the intention is not to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the scope of the present invention as defined by the claims.

[0065] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention. The present invention is in no way limited to the methods and materials described. It will be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All of these different combinations constitute various alternative aspects of the invention.

[0066] All of the patents and publications referred to herein are incorporated by reference in their entirety.

[0067] For purposes of interpreting this specification, terms used in the singular will also include the plural and vice versa.

[0068] The inventors have identified a novel composition for and method of improving hair growth comprising administration of zinc thymulin. Thymulin is a nonapeptide with the amino-acid sequence pyrGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn.

[0069] As used herein, thymulin includes any peptide with the sequence pyrGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn including synthetic thymulin, isolated thymulin, acetylated thymulin, amidated thymulin, acetylated and amidated thymulin, other forms of thymulin and other derivatives of thymulin (that do not alter the amino acid sequence).

[0070] As used herein, zinc thymulin includes any zinc complex with any form of thymulin including the form of zinc thymulin wherein a single zinc atom is complexed to the two serine amino acids and the asparagine amino acid.

[0071] The phrase effective amount generally refers to either a therapeutically effective amount or a cosmetically effective amount of zinc thymulin.

[0072] The phrase normal hair density generally refers to a density of between 100-300 follicles per square centimetre.

[0073] The words treat or treatment refer to therapeutic treatment wherein the object is to lessen the impact of or reverse a physiological disease or disorder or its symptoms. In the context of this invention, the disease, disorder or symptom thereof may be a form of hair loss.

[0074] The word cosmetic refers to compositions or methods for altering appearance or maintaining the condition of an external part of the body. Altering appearance could include altering visual appearance, feel or odour. In the context of this invention, a cosmetic may alter the appearance or maintain the appearance of hair, for example, hair quantity, volume or density. Improvements in appearance of the hair include visibly detectable improvements.

[0075] The words prevent and prevention generally refer to prophylactic or preventative measures for protecting or precluding an individual not having a given disease or disorder from progressing to that disease or disorder or not having an undesirable cosmetic appearance from progressing to have that undesirable cosmetic appearance.

[0076] Although this invention has been applied in humans, the invention is also useful for therapeutic and/or cosmetic veterinary purposes, for example, to improve hair growth in horses, dogs, cats or other animals.

[0077] The phrases pharmaceutically acceptable and cosmetically acceptable indicate that the substance or composition must be compatible chemically and/or toxicologically, with the other ingredients in the composition or formulation, and/or the mammal being treated therewith.

[0078] Suitable topical vehicles and additional components are well known in the art, and it will be apparent that the choice of a vehicle will depend on the particular physical form and mode of delivery. Topical vehicles include organic solvents such as alcohols (for example, ethanol, iso-propyl alcohol or glycerine), glycols such as butylene, isoprene or propylene glycol, aliphatic alcohols such as lanolin, mixtures of water and organic solvents and mixtures of organic solvents such as alcohol and glycerine, lipid-based materials such as fatty acids, acylglycerols including oils such as mineral oil, and fats of natural or synthetic origin, phosphoglycerides, sphingolipids and waxes, protein-based materials such as collagen and gelatine, silicone-based materials (both nonvolatile and volatile), hydrocarbon-based materials such as microsponges and polymer matrices, polysaccharides such as cellulose and its derivatives, and polyacrylic acids.

[0079] A topical composition optionally includes one or more components adapted to improve the stability or effectiveness of the applied formulation, such as stabilizing agents, suspending agents, emulsifying agents, viscosity adjusters, gelling agents, buffers, preservatives, antioxidants, skin penetration enhancers, moisturizers and sustained release materials. Examples of such components are described in MartindaleThe Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. Formulations may comprise microcapsules, such as hydroxymethylcellulose or gelatine-microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles or nanocapsules.

[0080] A topical formulation is optionally prepared in a variety of physical forms including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays and skin patches. The physical appearance and viscosity of such forms can be governed by the presence and amount of emulsifier(s) and viscosity adjuster(s) present in the formulation. Solids are generally firm and non-pourable and commonly are formulated as bars or sticks, or in particulate form. Creams and lotions are often similar to one another, differing mainly in their viscosity. Both lotions and creams are optionally opaque, translucent or clear and often contain emulsifiers, solvents, and viscosity adjusting agents, as well as moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Gels can be prepared with a range of viscosities, from thick or high viscosity to thin or low viscosity. These formulations, like those of lotions and creams, optionally also contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels, and often do not contain emulsifiers. Liquid topical products often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes/colorants, preservatives and other active ingredients that increase or enhance the final product.

[0081] Emulsifiers for use in topical formulations include, but are not limited to, ionic emulsifiers and non-ionic emulsifiers like cetearyl alcohol, phosphatidylcholine (lecithin), polyoxyethylene oleyl ether, acacia gum, PEG-40 stearate, PEG-100 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, acrylic acid, acryl acrylate and glyceryl stearate.

[0082] Suitable viscosity adjusting agents include, but are not limited to, protective colloids or nonionic gums such as hydroxyethylcellulose, xanthan gum, carrageenan, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate.

[0083] A topical gel composition is optionally formed by the addition of a gelling agent such as chitosan, methyl cellulose, ethyl cellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylceilulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer, ammoniated glycyrrhizinate or other gel/scaffold compounds of alginate, alginic acid, polylactic acid or polyglycolic acid.

[0084] Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic and anionic surfactants. For example, one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate are optionally used within topical formulations.

[0085] Suitable buffers include, but are not limited to, sodium bicarbonate, sodium phosphate, disodium phosphate, citric acid and calcium carbonate. Sodium bicarbonate is a preferred buffer for formulations of zinc thymulin.

[0086] Preservatives include, but are not limited to, antimicrobials such as methylparaben, propylparaben, sorbic acid, benzoic acid, potassium sorbate, sodium benzoate, caprylic acid and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate/ascorbic acid and propyl gallate.

[0087] Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerine, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate and mineral oils.

[0088] Other suitable additional ingredients that are optionally included in a topical formulation include, but are not limited to, abrasives, absorbents, anticaking agents, antifoaming agents, antistatic agents, astringents (such as witch hazel), alcohol and herbal extracts such as chamomile extract, binders/excipients, chelating agents, film forming agents, conditioning agents, propellants, opacifying agents, pH adjusters and protectants.

[0089] Typical modes of delivery for topical compositions include application using the fingers, application using a physical applicator such as a cloth, tissue, swab, stick or brush, spraying including mist, aerosol or foam spraying, dropper application, sprinkling, soaking, and rinsing. Controlled release vehicles can also be used, and compositions are optionally formulated for transdermal administration or for dermal administration, that is, delivery into the skin rather than through the skin.

[0090] Formulations for administration by injection include liquids, dispersions, such as suspensions or emulsions, formulations containing peptide scaffolds, polyglycolic and polylactic threads and matrices, dissolving microneedles, nanoparticles or alginate and alginic acid. These include solvents (in particular physiological saline solvents), chelating agents (such as EDTA and citric acid), solubilisers, preservatives (such as benzyl alcohol, phenol, sorbic acid, parabens and chlorocresol).

[0091] Formulations for administration by injection may be presented as a solid powder or a freeze dried powder ready for reconstitution with a liquid before administration.

[0092] The compositions of the invention optionally include one or more additional active therapeutic or cosmetic ingredients. Suitable ingredients include 3-catenin, LKKTETQ peptide, EEINEINR peptide, RIIEGEEHR peptide, substance P peptide, PACAP peptide, vasoactive intestinal peptide, anti-inflammatory agents, vitamins, antioxidants, amino acids, herbal extracts including polyphenols and flavonoids, hair strengthening agents, temporary hair volumising agents and temporary hair thickening agents.

[0093] -catenin optionally induces hair growth. A peptide with the sequence LKKTETQ (leucine-leucine-lysine-threonine-glutamic acid-threonine-glutamine), which is the actin-binding site with an additional peptide Q, promotes hair growth from existing active hair follicles when topically applied to rodent skin. EEINEINR and RIIEGEEHR are both keratin peptides useful for improving the thickness of the hair shaft. These peptides are optionally used in their native form, a synthetic form or an amidated and/or acetylated form. Anti-inflammatory agents can be used to reduce the inflammation often present during hair loss suitable examples include, but are not limited to, Annexin. Suitable vitamins include, but are not limited to, vitamin B9 and vitamin B3. Suitable herbal extracts include, but are not limited to, Fallopia japonica, epimedium and rubus idaeus. Suitable antioxidants include, but are not limited to, coenzyme Q10. Hair strengthening agents include, but are not limited to, keratin, wheat or oat proteins that reinforce the hair.

Example 1Synthesis of Zinc Thymulln

[0094] A. Preparation of Thymulin

[0095] Thymulin has the amino acid sequence: pyrglutamic acid-alanine-lysine-serine-glutamine-glycine-glycine-serine-asparagine, H-PyrGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-OH. Thymulin peptide was prepared using F-moc peptide protocols.

[0096] Materials: Amino acids (and protecting groups): PyrGlu (OtBu: t-butyl ester), Ala, Lys (Boc: t-butyloxycarbonyl), Ser (tBu: t-butyl ether), Gin (Trt: trityl or triphenylmethyl), Gly, Asn (Trt: trityl or triphenylmethyl) and Asn (Trt: trityl or triphenylmethyl).

[0097] Resin: Rink amide resin.

[0098] Reagents: piperidine, N,N-dimethylformamide (DMF), N-methylpyrrolidone (NMP), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU-coupling agent), N,N-Diisopropylethylamine (DIEA) and dichloromethane (DCM).

[0099] Synthesis for each amino acid cycle (under nitrogen gas): [0100] 1. Swell resin and prewash with DMF for 15 minutes [0101] 2. Deprotection with 20% piperidine in DMF [0102] 3. Coupling with 0.4M HBTU/DMF, 2M DIEA/NMP [0103] 4. Capping with DMF

[0104] Cleavage: removal of DMF from resin with dichloromethane, cleavage cocktail of trifluoroacetic acid 90%, thioanisole 5%, EDT (1,2-ethanedithiol) 3%, anisole 2%; 1 ml per 100 mg resin; incubation for 3 hours. Peptide precipitation (amidated form precipitated from Rink amide resin) with cold diethyl ether and centrifugation, repeated three times.

[0105] B. Modification of Thymulin

[0106] The peptide prepared by the above F-moc protocol is modified by N terminal acetylation and C terminal amidation to improve stability and make the functionality similar to that of the native peptide. Following these modifications, the thymulin has the following structure:

H.sub.3CC(O)-Pyr-Glu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn-NH.sub.2

[0107] The skilled person understands how to make these modifications. The example in A above precipitates the amidated form. One method of acetylation is reconstitution in 10% glacial acetic acid, freeze at minus () 80 degrees C. and lyophilisation; repeated three times. Modified thymulin was stored minus at () 20 degrees C.

[0108] C. Preparation of Zinc Thymulin

[0109] The synthesized amidated and acetylated form of thymulin was reacted with zinc oxide compound to yield zinc thymulin. In the reaction, the ratio is by molar mass: 1M thymulin:3M ZnO or its proportional equivalent of millimolar, micromolar or nanomolar mass in 10% glacial acetic acid at 20 degrees C. Zinc oxide is dissolved in 10% glacial acetic acid at 20 degrees C. and the synthesized thymulin is added. The solution is stirred for 1 hour. Following completion of the reaction the reaction mixture is frozen at minus () 80 degrees C. and lyophilized in a freeze-dryer to yield the final composite product of the active zinc-thymulin (MW 924.23) and zinc acetate (MW 183.48) powder.

Example 2Preparation of Zinc Thymulin for Topical Application

[0110] A topical formulation containing 0.0005% zinc thymulin was prepared as a liquid hair spray was prepared according to Table 1.

TABLE-US-00001 TABLE 1 Ingredient CAS number % concentration Function Zinc-thymulin 63958-90-7 0.0005% Active (to activate and 1314-13-2 hair follicle regeneration) Benzoic acid 65-85-0 0.05% Preservative Sodium 532-32-1 0.08% Preservative benzoate Potassium 24634-61-5 0.12% Preservative sorbate Distilled water To 100% Solvent

[0111] The analytical grade benzoic acid, sodium benzoate and potassium sorbate in table 1 were weighed and then dissolved in distilled water with sonication for 15 minutes at 20 C.

[0112] The zinc thymulin prepared according to the method set out in Example 1, was weighed, added to the formulation and sonicated for 5 minutes. The formulation was decanted into spray bottles at 20 C. Spray bottles were stored at 4 C.

[0113] The above formulation has also been prepared with an additional ingredient, 0.00005% amidated & acetylated LKKTETQ peptide (molecular weight 846.48). Natural form LKKTETQ peptide has been reported to promote growth of hair from active hair follicles but not to activate inactive hair follicles.

[0114] When included in the formulation the LKKTETQ peptide is weighed and added to the formulation at the same time as the zinc thymulin is added and sonicated for 5 minutes along with the zinc thymulin.

Example 3Clinical Trial Using Topical Zinc Thymulin

[0115] Human subjects suffering from hair loss had a marker identified near their hair loss, such as a freckle or mole. The area near the marker affected by hair loss was photographed using a Samsung Galaxy Note 4 camera with settings F2.2, focal length 31 mm, LED flash and the number of active hair follicles in a set area were quantified.

[0116] The aqueous spray formulation prepared in Example 2 was administered topically to the scalp of the trial subjects twice daily to the hair loss region(s) of the scalp. The formulation used for subjects 1, 2 and 3 contained 0.00005% LKKTETQ peptide (molecular weight 846.48). The formulation used by subject 4 did not include LKKTETQ peptide.

[0117] At 16 weeks for subject 1 and 12 weeks for subjects 2, 3 and 4, the number of active hair follicles in the set area was quantified again. Multiple set areas of hair loss were evaluated for trial subjects 1, 2 and 3. These areas are designated a and b in Table 2 and Table 3. Table 1 provides information on each subject before treatment. Table 3 provides information on each subject after treatment. Table 4 provides the results as percentage increase in active hair follicles.

TABLE-US-00002 TABLE 2 Active hair follicles per square centimetre before treatment Number of Number of active hair active hair follicles before Size of subject follicles before treatment per Subject area area (cm.sup.2) treatment cm.sup.2 1a 1.96 31 16 1b 1.69 30 18 2a 0.6 34 57 2b 1.21 217 179 3a 1.26 13 10 3b 3.6 26 7 4 1.04 17 16

TABLE-US-00003 TABLE 3 Active hair follicles per square centimetre after treatment Number of Number of active hair active hair follicles after Size of subject follicles after treatment per Subject area area (cm.sup.2) treatment cm.sup.2 1a 1.96 48 24 1b 1.69 36 21 2a 0.6 76 127 2b 1.21 230 191 3a 1.26 39 31 3b 3.6 32 9 4 1.04 40 38

TABLE-US-00004 TABLE 4 percentage increase in active hair follicles Number of Number of Percentage active hair active hair increase in follicles before follicles after active hair Subject area treatment treatment follicles 1a 31 48 55 1b 30 36 20 2a 34 76 124 2b 217 230 6 3a 13 39 200 3b 26 32 23 4 17 40 135 Range 6-200 Average 80 increase

[0118] Subject 1 was a 48 year old female with 3 years of hair loss. Subject 2 was a 49 year old male with 3 years of hair loss. Subject 3 was a 43 year old male with 6 years of hair loss. Subject 4 was a 60 year old male with 11 years of hair loss.

[0119] Area 2b had an equivalent of 179 active hair follicles per square centimetre prior to treatment. This is within the normal range (ie 100 to 300 hairs per square centimetre) and is an example of an increase in active hair follicles when zinc thymulin is administered to an area of a subject that has a normal quantity of hair.

[0120] Low resolution and high resolution images of Subject 4 before treatment are in FIG. 1 and FIG. 3 respectively and low resolution and high resolution images of Subject 4 after treatment are included in FIG. 2 and FIG. 4, respectively.

Example 4Injectable Formulation of Zinc Thymulin

[0121] 0.0005% zinc thymulin is dissolved in a solvent of normal physiological saline.

[0122] The formulation is optionally buffered, for example with phosphate so that the solvent is phosphate buffered saline. This liquid formulation is suitable for injection into the dermis and/or subdermis at a dosage of 5 to 10 microlitres per square centimetre.

[0123] Additional supporting active peptides and or other active ingredients are optionally added as discussed above.

Example 5Further Clinical Trial Using Topical Zinc Thymulin

[0124] Methods:

[0125] Eighteen consecutive adult subjects were recruited, 17 males and 1 female, age range 35-90 years (mean 55.4, SD 13.3) with a diagnosis of AGA, Norwood classification 2-7, and hair loss duration range of 3-40 years (mean 15.8, SD 9.6). The trial duration for each subject ranged from 4-10 months. The test compound ZT was synthesized by standard Fmoc peptide protocols and administered in water based topical spray to the scalp. Baseline and after treatment images for hair growth were graded by two blinded assessors using two validated scales: 1. numerical visual analog scale (VAS) for global assessment 2. hair growth index (HGI) of images under higher magnification for percentage changes of vellus, intermediate and terminal hair.

[0126] Results:

[0127] ZT demonstrated no adverse systemic effects or local side effects of redness or scalp irritation in any subject over a total of 3,300 treatment days. Three subjects who were concurrently using minoxidil (N=2) and minoxidil/finasteride (N=1) did not report any drug interaction with ZT. VAS hair assessment improvement was significant in subjects who completed 6 months of treatment (P=0.045, t-test). HGI assessment showed a significant increase in the number of newly observed intermediate hairs in previous absent hair regions (P<0.0001) with an average increase of vellus type (32%) and intermediate type (23%) hairs at 6 months. Melanogenesis was observed in several subjects.

[0128] Conclusion:

[0129] Topical applications of ZT demonstrated safety and established efficacy for initiating and maintaining anagen to treat male pattern baldness when applied for >6 months.

[0130] Detail

[0131] Subjects

[0132] Male and female adult subjects with a diagnosis of AGA were invited to participate in the study. Each subject provided written informed consent except the 90 year old subject who gave verbal consent as he was unable to provide written consent due to arthritis of the hand. The study was conducted in compliance with the Declaration of Helsinki, International Conference on Harmonization Good Clinical Practice guidelines. Eighteen consecutive adult subjects were recruited (17 males and 1 female) with an age range 35-90 years (mean 55.4, SD 13.3) with a diagnosis of AGA Norwood-Hamilton classification 2-7, and with a hair loss history ranging from 3-40 years (mean 15.8, SD 9.6). Entry time points of the volunteers occurred over 6 months with the trial completed at the same endpoint resulting in duration of treatment for subjects in the trial ranging from 4-10 months.

[0133] Materials

[0134] Thymulin has an amino acid sequence pyrGlu-Ala-Lys-Ser-Gln-Gly-Gly-Ser-Asn (one letter sequence pEAKSQGGSN) and was synthesized using standard Fmoc protocols on a peptide synthesizer (AAPPTEC Corp. Louisville, Ky., USA). Amino acids, resin, reagents and resin cleavage chemicals were analytical grade (AAPPTEC; Labsupply, Sydney, Australia). Confirmation and purity of thymulin was performed by liquid chromatography coupled to an electrospray mass spectrometer (LCMS). The LCMS was a Shimadzu 2010AD system.

[0135] Purity of the peptide was established at >95% with LC conditions employing reverse phase chromatography with mobile phase A 0.1% acetic acid in MilliQ water and phase B of analytical grade methanol, gradient 0-50% B over 30 minutes, using a 2.1 mm150 mm C8 column (Grace Vydac). The operating conditions of the MS were 1.5 kV detector voltage in positive ion mode, heat block and CDL temperature at 200 C, 1.5 L/min nitrogen nebulizing gas, m/z scan range 200-1000.

[0136] The mass spectra of the eluting peak showed two m/z values of 430 and 375. At the stated MS operating conditions m/z 430 represents the thymulin peptide (mass 858.85) that displayed the [M+2H]2+ state i.e. 4292 (double electron charge)+1 proton from acetic acid=observed mass 859. The m/z 375 is an expected but variable phase between pyruvate and the amino acids glutamic acid and glutamine that occurs uniquely under electrospray kV detector conditions.

[0137] After peptide synthesis the residual trifluoroacetic acid and resin cleavage chemicals were removed by lyophilization with 10% acetic acid, repeated three times. Attachment of the zinc ion was performed by mixing thymulin with zinc oxide (1:3 w/w) in 10% acetic acid and lyophilized to yield ZT. The compound was then dissolved in distilled water with preservatives benzoic acid, sodium benzoate and potassium sorbate.

[0138] Citric acid and sodium bicarbonate were used as buffers that resulted in a colorless and odorless solution at pH 5.4. The product given to subjects was a 100 ml spray bottle containing 0.0005% ZT as the active test agent. Subjects were instructed to spray 1-2 ml of solution, twice daily, and rub into the scalp. The spray bottle solution was to be kept away from direct sunlight and heat. Subjects were resupplied with fresh solutions every 6-8 weeks during the trial.

[0139] Hair Growth Assessment

[0140] Images were captured on a Samsung Galaxy Note 4 phone in high resolution (16 MB image) with flash, F 2.2 and focal length 31 mm. For assessment, baseline and after treatment images were de-identified, paired and randomly allocated with respect to left/right sides. Images were presented for analysis to assessors in Powerpoint format files on a 55 inch high resolution television screen. All images and original assessor scorecards were archived in PDF format for external reviewer confirmation.

[0141] Two validated assessment methods were used to grade the changes:

[0142] 1. Numbered visual analog scale (VAS) where the assessor gives a value between 0-10 of the image (0=total absence of hair, 10=full abundant hair) (Price, 2008).

[0143] 2. Hair Growth Index (HGI) that is a more in-depth scale of analysis where higher magnification of images can identify the four types of hair regrowth that are designated as absent, vellus, intermediate or terminal (Bernado, 2003). For each image these four growth stages are given percentage values that added together give a raw score total of 100%. Each percentage value for absent, vellus, intermediate, and terminal are multiplied by 0,1,2,3 respectively to yield a final adjusted score ranging from a possible minimum score of 0 (total absence of hair) to a maximum score of 300 (full terminal hair). Scores were pooled for paired t-test, and P<0.05 was established for significance.

[0144] Assessment was graded by two independent assessors in a single blind manner. Assessors underwent three training sessions over two weeks prior to the final evaluation. Reliability and validation of the assessors using the scoring methods was conducted by a test/retest at two weeks with Pearson's R. Results showed good intrasubject reliability at two weeks (assessor #1 R=0.69, assessor #2 R=0.71, respectively); and similarly, intersubject reliability had improved to a high level at two weeks (first training session R=0.54, third training session at two weeks R=0.76).

[0145] Results

[0146] Demographic data of the 18 subjects and their individual trial duration of ZT use are shown in Table 5. The VAS analysis showed no significant growth in the total group (P=0.07) at the end of the trial but significant growth was observed in the 11 subjects who had completed at least 6 months of treatment (P=0.045) (Table 6).

TABLE-US-00005 TABLE 5 Demographic data of subjects and trial duration zinc- hair other thymulin sub- age loss Norwood current use ject gender (years) (years) scale treatments (months) 1 male 35 10 2 9 2 female 49 3 2 8 3 male 64 5 4 minoxidil 7 4 male 61 11 3 vertex 4 5 male 53 30 .sup.5A 6 6 male 69 15 .sup.5A 6 1 male 55 15 6 9 8 male 44 15 .sup.2A finasteride & 6 minoxidil 9 male 35 15 .sup.5A 4 10 male 49 10 7 minoxidil 5 11 male 64 5 3 vertex 5 12 male 68 5 6 6 13 male 90 40 7 5 14 male 55 20 7 4 15 male 50 10 .sup.2A 5 16 male 44 20 7 6 17 male 50 30 4 10 18 male 60 15 .sup.5A 6

TABLE-US-00006 TABLE 6 VAS (0-10) of each subject by the two assessors (#1 and #2) [P = 0.07 for poo led results, P = 0.045* (t-test) for subjects using zinc-thymulin for >6 months]. VAS after subject VAS baseline treatment Assessor #1 1 7 8 2 7 8 3 8 6 4 6 8 5 9 8 6 7 8 7 8 6 8 8 9 9 9 8 10 6 7 11 9 8 12 7 8 13 5 8 14 7 6 15 7 8 16 6 7 17 8 9 18 8 9 Assessor #2 1 7 6 2 7 8 3 7 6 4 6 8 5 6 7 6 7 6 7 5 4 8 8 9 9 6 7 10 4 5 11 8 7 12 6 8 13 5 8 14 7 6 15 7 6 16 4 5 17 7 8 18 8 9

[0147] In 10 subjects the images could be magnified with acceptable clarity and definition to perform the HGI analysis. In the raw score analysis (Table 7) there was significantly less absent hair after treatment (P=0.008) and a significant increase of intermediate hair in the baseline/after treatment category (P=0.02). In the HGI adjusted scores (Table 8) there was confirmation of the increase of intermediate hair when compared for the baseline/after treatment category (P=0.03).

[0148] Only one side effect was reported in the entire study from a nonactive constituent. Subject #1 used the topical spray each day for nine months and reported a single transient episode of one hour of redness to the forehead after exposure to the sun for several hours, followed by a facial forehead exfoliative scrub cream and then application of the spray.

[0149] The investigator (ERV, a maxillofacial surgeon) considered that the redness was due to the action of the citric acid buffer on the combined sun-irradiated and abraded skin where it mimicked a low grade cosmetic facial chemical peel effect.

[0150] The subject continued to use the spray with no further recurrence.

TABLE-US-00007 TABLE 7 Hair Growth Index analysis of row percentage scores (N = 10 subjects) by each assessor (A#1 and A#2) The four hair types graded as: A (absent), V (vellus), I (intermediate), T (terminal) before and after trial completion. P values are shown at the bottom of the table. subject % A before % A after % V before % V after % I before % I after % T before % T after A#1 2 50 20 10 25 10 25 30 30 3 30 20 5 10 5 10 60 60 4 50 40 10 25 20 25 20 10 7 50 50 20 10 20 20 10 20 9 30 40 40 20 10 20 20 30 10 50 50 30 20 10 15 10 15 14 50 50 20 10 10 10 20 30 16 50 40 10 25 10 25 30 10 17 50 40 0 15 20 25 30 20 18 30 15 10 5 20 25 40 55 A#2 2 40 20 10 30 30 30 20 20 3 40 30 0 20 10 30 50 20 4 40 10 10 30 30 30 20 30 7 20 30 10 0 40 30 30 40 9 10 10 50 40 20 20 20 30 10 50 40 10 20 20 30 20 10 14 40 50 20 10 20 20 20 20 16 50 40 0 30 20 20 30 10 17 30 30 10 20 40 30 20 20 18 30 10 10 10 10 20 50 60 P= 0.008* 0.17 0.02* 0.86

TABLE-US-00008 TABLE 8 Hair Growth Index analysis of adjusted scores (N = 10 subjects) by each assessor (#1 and #2) The four types graded as: A (absent), V (vellus), I (intermediate), T (terminal) before and after trial completion. P values are shown at the bottom of the table. subject A before A after V before V after I before I after T before T after Assessor #1 2 0 0 10 25 20 50 90 90 3 0 0 5 10 10 20 180 180 4 0 0 10 25 40 50 60 30 7 0 0 20 10 40 40 30 60 9 0 0 40 20 20 20 60 90 10 0 0 30 20 10 30 30 45 14 0 0 20 10 20 20 60 90 16 0 0 10 25 20 50 90 30 17 0 0 0 15 40 50 90 60 18 0 0 10 15 40 50 90 60 Assessor #2 2 0 0 10 30 60 60 60 60 3 0 0 0 20 20 60 150 60 4 0 0 10 30 60 60 60 90 7 0 0 10 0 80 60 90 120 9 0 0 50 40 40 40 60 90 10 0 0 10 20 40 60 60 30 14 0 0 20 10 40 40 60 60 16 0 0 0 30 40 40 90 30 17 0 0 10 20 80 60 60 60 18 0 0 10 10 20 40 150 180 P= 0.18 0.03* 0.52

DISCUSSION

[0151] Safety

[0152] The ZT topical formulation was shown to be very safe with only one side effect from a non-active constituent reported in 18 test subjects over a total of 111 months (3,300 days). Specifically, there was no redness, scalp irritation or deterioration of hair color, quality or quantity. In addition, three subjects were using minoxidil and/or finasteride during the trial with no observable drug interaction suggesting concurrent use of these approved FDA drugs with ZT is possible. The excellent safety profile of ZT demonstrated in this study offers an opportunity for treating people with alopecia where side effects of finasteride and minoxidil may be problematic; depression, suicidal thoughts and persistent sexual problems from finasteride, pruritis and local irritation from minoxidil.

[0153] Anagen Initiation

[0154] In the transition to observed hair development there was a highly significant increased number of intermediate hairs that had progressed from the absent hair stage (P<0.0001) indicating widespread hair follicle activation. To confirm this effect subject #17 (30 year hair loss) underwent a fluorescence spectroscopy procedure of the scalp. The images showed 6 pre-existing terminal hairs under visible white LED light but spectroscopy revealed an additional 59 developing vellus hairs. Further spectroscopic research is required to confirm this effect but this potentially indicates that extended use of ZT may treat people with Norwood classification 6-7. Supporting evidence was seen when comparing the assessor pooled responses of total absent hair where 65% (13/20) of subjects improved.

[0155] Melanogenesis

[0156] Melanogenesis was clearly observed with the higher magnification images in several older subjects who developed numerous intermediate hairs that were dark and resembled the color of hair when they were younger.

CONCLUSION

[0157] Global visual improvement was statistically significant when ZT was used for >6 months. The study also showed that ZT initiated anagen in AGA subjects with Norwood-Hamilton classification 5-7. Moreover, several subjects were concurrently using finasteride and minoxidil with no evidence of adverse drug interactions with ZT. The compound was seen to induce melanogenesis and suggests further cosmetic applications in addition to its anagen stimulating properties.

REFERENCES

[0158] United States patent publication no. 2011/0281802. [0159] Price V H, Hordinsky M K, Olsen E A. Roberts J L, Siegfried E C, et al. (2008) Subcutaneous efalizumab is not effective in the treatment of alopecia areata. J Am Acad Dermatol 58: 395-402. [0160] Bernardo O, Tang L, Lui H, Shapiro J (2003) Topical nitrogen mustard in the treatment of alopecia areata: A bilateral comparison study. Am Acad Dermatol 2: 291-294.