This document relates to methods and materials for treating hair loss. For example, compositions containing 4-aminopyridine (4-AP) and/or one or more derivatives of 4-AP can be administered to a mammal having hair loss to treat the mammal.
Hair growth plays a significant role in people's psychosocial lives. Things which negatively affect hair appearance, like hair loss, can be a distressing symptom that can reduce quality of life at any age. In addition to cosmetic issues, loss of hair growth in certain parts of the body can predispose a person to infections.
This document provides methods and materials for treating hair loss. For example, compositions containing 4-AP and/or one or more derivatives of 4-AP can be administered to a mammal (e.g., a human) having hair loss to treat the mammal. In some cases, a composition containing 4-AP and/or one or more derivatives of 4-AP can be administered to a mammal (e.g., a human) having hair loss to promote hair growth on the mammal.
As demonstrated herein, administration of 4-AP can increase hair growth and regeneration. Having the ability to stimulate hair regrowth and regeneration as described herein (e.g., by administering a composition containing 4-AP and/or one or more derivatives of 4-AP) provides a unique way to treat hair loss. For example, 4-AP and/or one or more derivatives of 4-AP can be used to treat hair loss associated with one or more hair loss disorders. For example, 4-AP and/or one or more derivatives of 4-AP can be used to treat hair loss associated with one or more medications and/or medical treatments.
In general, one aspect of this document features methods for treating hair loss. The methods can include, or consist essentially of, (a) identifying a mammal as having hair loss, and (b) administering a composition including 4-AP or one or more derivatives of 4-AP to the mammal. The mammal can be a human. The administering can include a systemic administration. The administering can include a local administration. The composition can be effective to deliver about 0.05 mg/kg to about 1 mg/kg of the 4-AP or the one or more derivatives of 4-AP to the mammal. The mammal can have a disease, disorder, or condition associated with the hair loss. The disease, disorder, or condition associated with the hair loss can be traction alopecia, alopecia areata, trichotillomania, a skin graft, or a scar. The mammal can be administered an additional medical treatment. The additional medical treatment can be radiation therapy, a chemotherapy drug, a hormone therapy, Vitamin A, an acne medication, an antibiotic, an antifungal, an anticoagulant, a cholesterol-lowering drug, an immunosuppressant, an anticonvulsant, a blood pressure medication, an antidepressant, or a weight loss drug.
In another aspect, this document features methods for increasing hair growth. The methods can include, or consist essentially of, administering a composition including 4-AP or one or more derivatives of 4-AP to a mammal identified as being in need of increased hair growth. The method can be effective to increase hair growth on the mammal by at least 1.5 fold. The mammal can be a human. The administering can include a systemic administration. The administering can include a local administration. The composition can be effective to deliver about 0.05 mg/kg to about 1 mg/kg of the 4-AP or the one or more derivatives of 4-AP to the mammal. The mammal can have a disease, disorder, or condition associated with the hair loss. The disease, disorder, or condition associated with the hair loss can be traction alopecia, alopecia areata, trichotillomania, a skin graft, or a scar. The mammal can be administered an additional medical treatment. The additional medical treatment can be radiation therapy, a chemotherapy drug, a hormone therapy, Vitamin A, an acne medication, an antibiotic, an antifungal, an anticoagulant, a cholesterol-lowering drug, an immunosuppressant, an anticonvulsant, a blood pressure medication, an antidepressant, or a weight loss drug.
In another aspect, this document features methods for increasing hair regeneration. The methods can include, or consist essentially of, administering a composition including 4-AP or one or more derivatives of 4-AP to a mammal identified as being in need of increased hair regeneration. The method can be effective to increase a number of hair follicles on the mammal by at least 1.5 fold. The mammal can be a human. The administering can include a systemic administration. The administering can include a local administration. The composition can be effective to deliver about 0.05 mg/kg to about 1 mg/kg of the 4-AP or the one or more derivatives of 4-AP to the mammal. The mammal can have a disease, disorder, or condition associated with the hair loss. The disease, disorder, or condition associated with the hair loss can be traction alopecia, alopecia areata, trichotillomania, a skin graft, or a scar. The mammal can be administered an additional medical treatment. The additional medical treatment can be radiation therapy, a chemotherapy drug, a hormone therapy, Vitamin A, an acne medication, an antibiotic, an antifungal, an anticoagulant, a cholesterol-lowering drug, an immunosuppressant, an anticonvulsant, a blood pressure medication, an antidepressant, and a weight loss drug.
In another aspect, this document features methods for promoting proliferation of a cell, wherein said cell is selected from a keratinocyte, a Schwann cell, and fibroblast. The methods can include, or consist essentially of, administering a composition comprising 4-AP or one or more derivatives of 4-AP to a mammal, wherein said keratinocyte, a Schwann cell, or fibroblast of said mammal proliferates. The method can include identifying said mammal as being in need of proliferation of said cell of said mammal prior to said administering. The mammal can be a human. The administering can be a systemic administration. The administering can be a local administration.
In another aspect, this document features methods for promoting migration of a cell, wherein said cell is selected from the group consisting of a keratinocyte, a Schwann cell, and fibroblast. The methods can include, or consist essentially of, administering a composition comprising 4-AP or one or more derivatives of 4-AP to a mammal, wherein said keratinocyte, a Schwann cell, or fibroblast of said mammal migrates. The method can include identifying said mammal as being in need of migration of said cell in the lung of said mammal prior to said administering. The mammal can be a human. The administering can be a systemic administration. The administering can be a local administration.
In another aspect, this document features methods for promoting re-epithelization of a hair follicle in a mammal. The methods can include, or consist essentially of, administering a composition comprising 4-AP or one or more derivatives of 4-AP to a mammal, wherein said hair follicle of said mammal undergoes re-epithelization. The method can include identifying said mammal as being in need of hair follicle re-epithelization prior to said administering. The mammal can be a human. The administering can be a systemic administration. The administering can be a local administration.
In another aspect, this document features methods for promoting hair follicle formation in a mammal. The methods can include, or consist essentially of, administering a composition comprising 4-AP or one or more derivatives of 4-AP to a mammal, wherein a hair-follicle is formed within said mammal. The method can include identifying said mammal as being in need of hair-follicle formation prior to said administering. The mammal can be a human. The administering can be a systemic administration. The administering can be a local administration.
In another aspect, this document features methods for promoting angiogenesis in a hair follicle of a mammal. The methods can include, or consist essentially of, administering a composition comprising 4-AP or one or more derivatives of 4-AP to a mammal, wherein said hair follicle of said mammal undergoes angiogenesis. The method can include identifying said mammal as being in need of hair follicle angiogenesis prior to said administering. The mammal can be a human. The administering can be a systemic administration. The administering can be a local administration.
In another aspect, this document features methods for promoting reinnervation of a hair follicle in a mammal. The methods can include, or consist essentially of, administering a composition comprising 4-AP or one or more derivatives of 4-AP to a mammal, and wherein said hair follicle of said mammal undergoes reinnervation. The method can include identifying said mammal as being in need of hair follicle reinnervation prior to said administering. The mammal can be a human. The administering can be a systemic administration. The administering can be a local administration.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
This document provides methods and materials for treating hair loss. For example, compositions containing 4-AP and/or one or more derivatives of 4-AP can be administered to a mammal (e.g., a human) having hair loss to treat the mammal. In some cases, a composition containing 4-AP and/or one or more derivatives of 4-AP can be administered to a mammal (e.g., a human) having hair loss to promote hair growth on the mammal.
In some cases, a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having hair loss) to promote hair growth on the mammal. For example, a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be administered to a mammal having hair loss to increase hair growth on the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be administered to a mammal having hair loss to increase hair growth on the mammal by, for example, at least 1.5 fold (e.g., about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 5 fold, about 6 fold, or more).
In some cases, a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having hair loss) to promote hair regeneration on the mammal. For example, a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be administered to a mammal having hair loss to increase the number of hair follicles on the mammal by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent. For example, a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be administered to a mammal having hair loss to increase the number of hair follicles on the mammal by, for example, at least 1.5 fold (e.g., about 1.5 fold, about 2 fold, about 2.5 fold, about 3 fold, about 3.5 fold, about 4 fold, about 5 fold, about 6 fold, or more).
Any appropriate mammal having hair loss can be treated as described herein (e.g., by administering a composition containing 4-AP and/or one or more derivatives of 4-AP). Examples of mammals that can have hair loss and that can be treated as described herein include, without limitation, humans, non-human primates such as monkeys, horses, bovine species, porcine species, dogs, cats, mice, rats, rabbits, and goats. In some cases, a mammal having hair loss can be immunocompromised. In some cases, a human having hair loss can be treated as described herein.
A mammal having any type of hair loss can be treated as described herein (e.g., by administering a composition containing 4-AP and/or one or more derivatives of 4-AP). Hair loss can be permanent hair loss or temporary hair loss. Hair loss can be sudden hair loss or gradual hair loss. In some cases, hair loss can be naturally occurring. Examples of types of naturally occurring hair loss that can be treated as described herein include, without limitation, hereditary hair loss (e.g., androgenic alopecia, male-pattern baldness, and female-pattern baldness), hair loss associated with hormonal changes (e.g., hormonal changes due to pregnancy, childbirth, menopause and thyroid problems), stress related hair loss, and hair loss associated with shaving (e.g., hair loss associated with folliculitis caused by shaving). In some cases, hair loss can be associated with one or more diseases, disorders, and/or conditions. Examples of diseases, disorders, and conditions that can be associated with hair loss that can be treated as described herein include, without limitation, traction alopecia (e.g., hair loss associated with hairstyles that pull hair tight, such as pigtails or cornrows), alopecia areata, trichotillomania, the presence of one or more skin grafts (e.g., one or more surgical skin grafts), and scars (e.g., keloid scars). In some cases, hair loss can be associated with (e.g., can be a side effect of and/or adverse effect of) a medication and/or medical treatment. Examples of medications and medical treatments that can be associated with hair loss that can be treated as described herein include, without limitation, radiation therapy (e.g., radiation therapy to the head), chemotherapy drugs (e.g., carboplatin, cisplatin, docetaxel, doxorubicin, and fluorouracil (5-FU)), hormone therapies (e.g., hormone replacements), Vitamin A, acne medications (e.g., vitamin A-derived acne medications such as isotretinoin (e.g., ACCUTANE®) and tretinoin (e.g., RETIN-A®)), antibiotics, antifungals (e.g., voriconazole), anticoagulants (e.g., heparin and warfarin), cholesterol-lowering drugs (e.g., simvastatin and atorvastatin), immunosuppressants (e.g., methotrexate, leflunomide, cyclophosphamide, and etanercept), anticonvulsants (e.g., valproic acid and trimethadione), blood pressure medications (e.g., beta blockers such as metoprolol, timolol, propranolol, atenolol, and nadolol), ACE inhibitors (e.g., enalapril, lisinopril, and captopril), antidepressants (e.g., paroxetine hydrochloride, sertraline, protriptyline, amitriptyline, and fluoxetine), and weight loss drugs.
Hair loss that can be treated as described herein (e.g., by administering a composition containing 4-AP and/or one or more derivatives of 4-AP) can appear in any manner. For example, hair loss can appear as thinning hair (e.g., thinning hair on the top of the head of a mammal such as a human), receding hair (e.g., receding hair at the hairline on the forehead of a mammal such as a human), a broadening of the part in the hair, and/or patchy (e.g., circular) bald spots.
Hair loss that can be treated as described herein (e.g., by administering a composition containing 4-AP and/or one or more derivatives of 4-AP) can affect any location on a mammal. In some cases, hair loss that can be treated as described herein can be on the scalp (e.g., the top of the head) of a mammal. In some cases, hair loss that can be treated as described herein can be on the face of a mammal. In some cases, hair loss that can be treated as described herein can be on the whole body of a mammal. In some cases, hair loss that can be treated as described herein can be on grafted skin present on a mammal.
In some cases, methods described herein also can include identifying a mammal as having hair loss. Examples of methods for identifying a mammal as having hair loss include, without limitation, physical examinations (e.g., pull tests to see how many hairs come out), blood tests (e.g., to identify medical conditions that can cause hair loss), microscopy (e.g., to examine the hair shafts, hair roots, pigment intensity, and/or hair regrowth length), follicle counts, and biopsy follicle assessments. Once identified as having hair loss, a mammal can be administered or instructed to self-administer a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP).
A composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can include 4-AP and/or any appropriate derivative(s) of 4-AP. Examples of derivatives of 4-AP that can be included in a composition described herein include, without limitation, 3,4-diaminopyridine, 3-hydroxy-4-aminopyridine, N-(4-pyridyl)-t-butyl carbamate, N-(4-pyridyl) ethyl carbamate, N-(4-pyridyl) methyl carbamate, and N-(4-pyridyl) isopropyl carbamate. In some cases, 4-AP and/or one or more derivatives of 4-AP can have a structure according to Formula I.
where R1, R2, R3, R4, and R5 are each independently selected from hydrogen, halogen, amine, hydroxyl, alkoxy, carboxyl, or C1-C6 alkyl. For example, R1, R2, R3, R4, and R5 can all be hydrogen. In some cases, 4-AP or a derivative thereof can be a potassium channel blocker. In some cases, 4-AP or a derivative thereof can be a calcium channel agonist. In some cases, 4-AP or a derivative thereof can be electrically active. In some cases, 4-AP or a derivative thereof can be in the form of a free base. In some cases, 4-AP or a derivative thereof can be in the form of a salt (e.g., pharmaceutically acceptable salt). When 4-AP or a derivative thereof is in the form of a salt, the salt can include any appropriate acid (e.g., an organic acid or an inorganic acid). Examples of acids that can be used to form a salt with 4-AP or a derivative thereof include, without limitation, hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, methanesulfonic acid, ethanesulfonic acid, malic acid, acetic acid, oxalic acid, tartaric acid, citric acid, lactic acid, fumaric acid, succinic acid, maleic acid, salicylic acid, benzoic acid, phenylacetic acid, and mandelic acid.
In some cases, 4-AP and/or one or more derivatives of 4-AP can be as described elsewhere (see, e.g., U.S. Patent Application Publication No. 2018/0271847 and U.S. Pat. No. 9,993,429).
A composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can include any appropriate amount of 4-AP and/or one or more derivatives of 4-AP. In some cases, a composition described herein can include from about 0.5 μM to about 10 μM (e.g., from about 0.5 μM to about 8 μM, from about 0.5 μM to about 6 μM, from about 0.5 μM to about 5 μM, from about 0.5 μM to about 3 μM, from about 0.5 μM to about 2 μM, from about 0.5 μM to about 1 μM, from about 1 μM to about 10 PM, from about 2 μM to about 10 μM, from about 4 μM to about 10 μM, from about 5 μM to about 10 μM, from about 7 μM to about 10 μM, from about 9 μM to about 10 μM, from about 1 μM to about 9 μM, from about 2 μM to about 8 μM, from about 3 μM to about 7 μM, from about 4 μM to about 6 μM, from about 1 μM to about 3 μM, from about 2 μM to about 4 μM, from about 3 μM to about 5 μM, from about 5 μM to about 7 μM, from about 6 μM to about 8 μM, or from about 7 μM to about 9 μM) of 4-AP and/or one or more derivatives of 4-AP. In some cases, a composition described herein can include from about 0.01% to about 99% (e.g., from about 0.01% to about 90%, from about 0.01% to about 80%, from about 0.01% to about 70%, from about 0.01% to about 60%, from about 0.01% to about 50%, from about 0.01% to about 40%, from about 0.01% to about 30%, from about 0.01% to about 20%, from about 0.01% to about 10%, from about 0.01% to about 5%, from about 0.01% to about 1%, from about 1% to about 99%, from about 5% to about 99%, from about 10% to about 99%, from about 20% to about 99%, from about 30% to about 99%, from about 40% to about 99%, from about 50% to about 99%, from about 60% to about 99%, from about 70% to about 99%, from about 80% to about 99%, from about 90% to about 99%, from about 10% to about 90%, from about 20% to about 80%, from about 30% to about 70%, from about 40% to about 60%, from about 10% to about 30%, from about 30% to about 50%, from about 50% to about 70%, or from about 70% to about 90%) of 4-AP and/or one or more derivatives of 4-AP.
In some cases, a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can include one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, saline (e.g., phosphate-buffered saline (PBS)), sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose and cellulose ethers like hydroxypropyl cellulose (HIPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, and lecithin.
A composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be administered to a mammal in need thereof (e.g., a mammal having hair loss) locally or systemically. In some cases, a compositions described herein can be administered locally. For example, a composition described herein can be administered locally by injection directly into, around, and/or near an area of hair loss on a mammal (e.g., a human). For example, a composition described herein can be administered locally by a topical administration onto, around, and/or near an area of hair loss on a mammal (e.g., a human). Compositions suitable for topical administration include, without limitation, creams, foams, gels (e.g., thermogels and cooling gels), balms (e.g., soothing balms), lotions, and ointments. In some cases, a topical composition containing 4-AP and/or one or more derivatives of 4-AP can be applied directly to the skin of a mammal (e.g., a human). In some cases, a topical composition containing 4-AP and/or one or more derivatives of 4-AP can be formulated as an after-shave. For example, 4-AP and/or one or more derivatives of 4-AP can be used to treat hair loss associated with shaving (e.g., hair loss associated with folliculitis caused by shaving).
In some cases, composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can include one or more (e.g., one, two, three, four, five or more) additional agents that can be used together with (e.g., can be synergistic with) 4-AP and/or one or more derivatives of 4-AP to treat hair loss. The one or more additional agents that can be used together with 4-AP and/or one or more derivatives of 4-AP to treat hair loss can include any appropriate agent(s) used to treat hair loss. In some cases, an agent that can be used together with (e.g., can be synergistic with) 4-AP and/or one or more derivatives of 4-AP to treat hair loss can be an anti-inflammatory. In some cases, an agent that can be used together with (e.g., can be synergistic with) 4-AP and/or one or more derivatives of 4-AP to treat hair loss can be a disinfectant. In some cases, an agent that can be used together with (e.g., can be synergistic with) 4-AP and/or one or more derivatives of 4-AP to treat hair loss can be a hydrating agent. In some cases, an agent that can be used together with (e.g., can be synergistic with) 4-AP and/or one or more derivatives of 4-AP to treat hair loss can be an exfoliating agent. In some cases, an agent that can be used together with (e.g., can be synergistic with) 4-AP and/or one or more derivatives of 4-AP to treat hair loss can be a healing agent (e.g., can heal micro-lacerations in the skin).
In some cases, a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be administered systemically. For example, a composition described herein can be designed for oral or parenteral (including intraperitoneal, subcutaneous, intramuscular, intravenous, and intradermal) administration to a mammal having hair loss. Compositions suitable for oral administration include, without limitation, liquids, tablets, capsules, pills, powders, gels, and granules. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain anti-oxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient. In some cases, a composition described herein can be formulated for parenteral administration (e.g., intraperitoneal injection or intravenous injection).
An effective amount (e.g., effective dose) of 4-AP and/or one or more derivatives of 4-AP in a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can vary depending on the severity of the hair loss, the route of administration, the age and general health condition of the subject, excipient usage, the possibility of co-usage with other therapeutic treatments such as use of other agents, and/or the judgment of the treating physician.
An effective amount of 4-AP and/or one or more derivatives of 4-AP in a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be any amount that can treat hair loss on a mammal without producing significant toxicity to the mammal. An effective amount of 4-AP and/or one or more derivatives of 4-AP in a composition described herein can be any appropriate amount. In some cases, an effective amount of 4-AP and/or one or more derivatives of 4-AP in a composition described herein can be from about 0.05 milligrams per kilogram body weight (mg/kg) to about 1 mg/kg (e.g., from about 0.05 mg/kg to about 0.8 mg/kg, from about 0.05 mg/kg to about 0.6 mg/kg, from about 0.05 mg/kg to about 0.5 mg/kg, from about 0.05 mg/kg to about 0.3 mg/kg, from about 0.05 mg/kg to about 0.1 mg/kg, from about 0.1 mg/kg to about 1 mg/kg, from about 0.3 mg/kg to about 1 mg/kg, from about 0.5 mg/kg to about 1 mg/kg, from about 0.8 mg/kg to about 1 mg/kg, from about 0.1 mg/kg to about 0.9 mg/kg, from about 0.2 mg/kg to about 0.8 mg/kg, from about 0.3 mg/kg to about 0.7 mg/kg, from about 0.4 mg/kg to about 0.6 mg/kg, from about 0.1 mg/kg to about 0.3 mg/kg, from about 0.2 mg/kg to about 0.4 mg/kg, from about 0.3 mg/kg to about 0.5 mg/kg, from about 0.5 mg/kg to about 0.7 mg/kg, from about 0.6 mg/kg to about 0.8 mg/kg, or from about 0.7 mg/kg to about 0.9 mg/kg).
The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal's response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and severity of the hair loss may require an increase or decrease in the actual effective amount administered.
The frequency of administration of a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be any frequency that can treat hair loss on a mammal without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a week to about once every two months, from about once every two weeks to about once every six weeks, or from about once every three weeks to about once a month (e.g., once every four weeks). The frequency of administration can remain constant or can be variable during the duration of treatment. A course of treatment with a composition described herein can include rest periods. For example, a composition described herein can be administered once a month over a six-month period followed by a rest period (e.g., a one or two month rest period), and such a regimen can be repeated multiple times. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, route of administration, and severity of the hair loss may require an increase or decrease in administration frequency.
An effective duration for administering a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP) can be any duration that treats hair loss on a mammal without producing significant toxicity to the mammal. For example, the effective duration can vary from several days to several weeks, months, or years. In some cases, the effective duration for the treatment of hair loss can range in duration from about one month to about a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, route of administration, and severity of the hair loss being treated.
In some cases, the methods and materials described herein can be used as the sole active agent used to treat a mammal (e.g., a human) having hair loss. For example, a composition containing 4-AP and/or one or more derivatives of 4-AP can be used as the sole active agent(s) used to treat a mammal having hair loss.
In some cases, methods described herein also can include administering to a mammal (e.g., a human) having hair loss one or more (e.g., one, two, three, four, five or more) additional agents used to treat hair loss in addition to a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP). The one or more additional agents used to treat hair loss can include any appropriate agent(s) used to treat hair loss. Examples of agents that can be used to treat hair loss include, without limitation, minoxidil, finasteride, spironolactone (e.g., CaroSpir® and ALDACTONE®), dutasteride (e.g., AVODART®), and steroids (e.g., cortisone). In cases where a mammal having hair loss is treated with a composition described herein and is treated with one or more additional agents used to treat hair loss, the additional agent(s) used to treat hair loss can be administered at the same time or independently. For example, the additional agent(s) used to treat hair loss can be formulated into a composition containing 4-AP and/or one or more derivatives of 4-AP to form a single composition. In some cases, a composition described herein can be administered first, and the one or more additional agents used to treat hair loss can be administered second, or vice versa.
In some cases, methods described herein also can include can include subjecting a mammal (e.g., a human) having hair loss to one or more (e.g., one, two, three, four, five or more) additional treatments (e.g., therapeutic interventions) that are effective to treat hair loss. Examples of additional treatments that can be used as described herein to treat hair loss include, without limitation, hair transplant surgery, laser therapy (e.g., laser therapy using pulsed-dye lasers), electrical stimulation, scar massage, wearing pressure garments (e.g., compression socks), and silicone gel sheets. In some cases, the one or more additional treatments that are effective to treat hair loss can be performed at the same time as the administration of a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP). In some cases, the one or more additional treatments that are effective to treat hair loss can be performed before and/or after the administration of a composition described herein (e.g., a composition containing 4-AP and/or one or more derivatives of 4-AP).
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
This Example demonstrates that 4-AP can increase hair regrowth and regeneration in mice in vivo.
Wild-type C57BL/6 mice (7-8 weeks old) were placed in the lateral decubitus position on a sterile paper sheet after anesthesia using intraperitoneal injection of ketamine (60 mg/kg) and xylazine (4 mg/kg). Sterile surgical instruments (autoclaved) were used during dorsal hair removal. The skin was prepared for hair removal on the dorso-lateral skin (from neck to tail) by shaving with a mechanical shaver, and then hair removal cream (Nair) was placed on the skin for 30-60 seconds. Hair was removed completely by gently wiping the skin with cotton balls soaked in warm water and disinfected using 70% ethanol wipes and betadine 3 times.
After dorsal hair removal, mice were placed in a clean cage on the heating pad for 30 minutes or until recovery. Immediately after recovery, mice were treated either with 4-AP (systemic, 10-50 μg/daily, intraperitoneal injection) or saline and then were returned to the animal facility.
Functional tests and tissue analyses were performed on mice for dorsal skin hair growth and then 4-AP was administered. 4-AP or saline was delivered systemically (ip) for 14 days after hair removal. All analyses were blinded.
The 4-AP treated and controls mice were subjected to functional analyses by dorsal mouse skin hair photography, and skin tissue was collected at day 14 for histology analysis and biomolecule analysis.
Mice were anesthetized during capturing of individual mouse dorsal photographs on sterile white background surface, and then dorsal area photographs were captured using a digital camera. The day when hair was removed was designated as day 0.
At day 14, the backs of the mice were photographed with a digital camera, to evaluate the darkening of skin color. The hair growth effect in each group was rated on a score ranging from 0 to 100%. In morphological observation (
Systemically administered 4-AP increased hair regrowth and regeneration in a standard model in rodents on the dorsal surface (
Mice were euthanized at day 14 and skin tissue was harvested. The dermal skin samples were fixed in 10% buffered formalin for 24 hours, followed by embedding in paraffin wax using standard techniques. General histology was visualized using hematoxylin and eosin (H&E) staining. The number of hair follicles was increased in the 4-AP treated mice compared with that in the saline treated mice in transverse sections on day 14 (
This Example demonstrates that 4-AP can accelerate skin wound healing by increasing cellular proliferation, migration, and de-differentiation, as well as neuro-mediator production.
The effect of 4-AP in promoting cutaneous wound healing and tissue regeneration was investigated using a full thickness excisional wound model (
Tissue assessments were made on wound tissue harvested 14 days after wounds, when 4-AP treated mice had healed. Wound tissue was analyzed by morphometric analysis (hematoxylin and eosin (H&E) staining), which revealed newly formed epidermis within the healed wound that was significantly thicker in 4-AP treated mice (29.74±1.30 μM) compared to saline treated mice (19.38±0.96 μM) (
During wound healing, fibroblast migration and maturation play a significant role in the contraction, granulation, and proliferation phases. A key marker of fibroblast differentiation is α-smooth muscle actin (α-SMA) which signifies fibroblast differentiation into collagen-producing myofibroblasts. To test whether 4-AP treatment affects fibroblast maturation during wound healing, Masson's Trichrome staining was performed to measure collagen deposition in the healing wound. Staining revealed elevated collagen deposition in dermis from 4-AP treated mice compared to saline (
Immunohistochemistry for vimentin (a fibroblast marker) and α-SMA (
Neo-angiogenesis is necessary to provide nutrients and oxygen to healing wounds. To assess the difference in wound neo-vascularization with 4-AP treatment, immuno-fluorescence staining for the endothelial specific marker CD31 was performed. Larger and more abundant blood-vessel networks were observed in dermal tissue from 4-AP treated mice than saline treated mice (
Re-epithelialization involves keratinocyte proliferation, and is marked by expression of the basal keratinocyte proliferation marker, Keratin 14 (K14). Significant increases in K14+ keratinocytes were observed in the epidermis and in de novo hair follicles in wound beds of 4-AP treated mice (
Whether the role of 4-AP on myelination and regeneration relates to wound healing through improved reinnervation was examined. The proliferation marker Ki-67 was used in conjunction with the neuronal marker NF-H, and significantly increased co-staining was revealed in mice treated with 4-AP treatment compared to saline treated controls. Ki-67+ in hair follicle bulges and epidermis was increased 2-fold (21.87±2.763 cells/mm2 vs. 7.754±1.664% cells/mm2) (
To more completely assess 4-AP's effects on neuropeptides and neuronal modulation in wound healing, 4-AP's effect on protein gene product 9.5 (PGP 9.5), which is also known to promote wound healing, was assessed. Fourteen days post wound, PGP 9.5+ nerve fibres in the healed wounds were twice as abundant in 4-AP treated mice (4096938±713297 integrated density/mm2), than in saline treated mice (2107970±325039 integrated density/mm2) (
Schwann cells (SC) are critical players in wound healing and are associated with axons around hair-follicles in the wound bed. In the setting of injury, SCs de-differentiate to a non-myelinating state and begin to secrete neurotrophins like NGF, a state marked by expression of p75-NTR. Using immunohistochemistry for two different markers of SCs (S100, a pan SC marker and p75-NTR, a marker of SC de-differentiation), it was found that SCs were located within the hypodermis and dermis of the healed wounds (
SC originate from migratory neural crest cells (NCCs) that express SOX10, which is required for myelin production. Elevated SOX10 expression promotes conversion of mesenchymal cells into p75-NTR expressing neural crest stem cells (NCSC) and depletion of SOX10 expression significantly delays wound healing and tissue regeneration. NGF and its receptor, p75-NTR, play significant role in the wound healing process by inducing nerve sprouting from injured nerve endings. NGF also acts on non-neuronal cells to sensitize them to substance-P, which in turns further stimulates more NGF secretion ensuring that keratinocytes, for example can elaborate and respond to neuronal factors along with neurons. Significantly increased SOX10, substance-P, and NGF (NGF 4-fold increase: 2429872±375280 integrated density/mm2 vs. 626967±101856 integrated density/mm2) expression was found in tissue harvested from 4-AP treated mice compared to saline treated mice (
As described above, 4-AP accelerates wound closure and enhances WIHN, angiogenesis and nerve regeneration in healed wound. To further understand the specific effects of 4-AP on skin cells, primary, normal human epidermal keratinocytes (NHEKs), fibroblasts, and dermal SCs were cultured in the presence of 4-AP. The purity and identity of each cell type was confirmed with immunohistochemistry for characteristic markers (
4-AP treatment accelerated scratch closure and keratinocyte migration (
Given that keratinocytes, fibroblasts and SCs cells all interact during wound healing in-vivo, co-culture experiments were conducted with pairs of cell types with and without 4-AP treatment to determine if 4-AP promotes synergistic properties. Co-culturing cells accelerated scratch culture closure rates in general. However, keratinocytes co-cultured with SCs in a ratio mimicking that of actual epidermal skin (10:1 keratinocytes:SCs) closed a scratch within 15 hours with 4-AP treatment, which was significantly faster than scratch closure without treatment, which took 20 hours. At 15 hours, the percentage of wound closure was at 98% in 4-AP treated but only 80% in non-treated scratch cultures (
In co-culture scratch assays of keratinocytes with fibroblasts (ratio 10:1), similar effects on migration and scratch closure were found (
Since 4-AP, NGF, and electrical stimulation are approved for use in various conditions, it was sought to determine if NGF is a factor mediating 4-AP induced scratch closure. Keratinocyte scratch closure cultures were treated with 4-AP, NGF, or a NGF neutralizing antibody, either alone or in combination. Treatment of keratinocyte cultures with either 4-AP or NGF accelerated scratch closure, with 75% vs 56% in control cultures at 12 hours. NGF-neutralizing antibody impaired closure by approximately 8% (67%) in both NGF or 4-AP treated cultures (
Together these results demonstrate that 4-AP can be used for cutaneous wound healing and tissue regeneration. For example, a composition including 4-AP can be administered to a mammal (e.g., a human) to promote cellular proliferation, migration, and de-differentiation.
The primary objective of this study was to investigate the possible therapeutic effect of 4-AP in enhancing skin wound healing and tissue regeneration in C57BL/6 male mice as a form of reinnervation-stimulator neurotrophins and regenerative cells medicine. The therapeutic effect of 4-AP in keratinocytes proliferation, reinneravation, neurotrophic factors expression in mice model and by in-vitro experiments was also investigated. Mice were age-matched and randomized before treatment into different groups. Data were generated by microscopic analysis of immunohistochemistry, immuno-fluorescence on fixed skin sections, and Western blotting of harvested tissue, and human skin primary cells extracts used. Sample sizes were chosen empirically to ensure adequate statistical power and were in the line with field standards for the techniques used in the study. Sample sizes for each experiment are included in the figure legends.
Male C57BL/6 (10 week) mice were purchased from the Jackson Laboratory (Bar Harbor, ME USA). Mice were anesthetized by intraperitoneal injection of ketamine (60 mg/kg) and xylazine (4 mg/kg) body weight, and the dorsal skin hair was removed using mechanical shaver and then, applied the hair removal cream. Skin was disinfected using 70% ethanol and betadine for 3 times. The dorsal skin was folded and raised cranially and caudally at midline using index fingers and thumbs, and place the animal in a lateral position and two 5-mm wounds created in their dorsal skin using sterile biopsy punch. A 5-mm-diameter silicone ring was then placed and sutured around the each wound to restrict contraction. After wound creation and sutured silicone ring, wound site was photographed, and wound surface was covered with a Tegaderm (3M) sterile transparent dressing. After surgery, mice was administered SR Buprenorphine (0.05 mg/kg) as a post-operative analgesia. The mice were randomized into the following two groups: a control group (vehicle control), which received 100 μl of saline, and a 4-AP group. In total 40 g/mouse/daily 4-AP in 90-110 μl of saline intraperitoneally (IP) was administered until day 14 post wound. Digital imaging of the wound was used was determined for each wound healing by quantification of wound area based on digital pictures taken from day 0 to day 14 (days 0, 3, 5, 7, 9, 12 and 14) post-surgery (
At the day 14 wound skin sample were collected after mice were euthanized using CO2 and skin samples were excised, either flash frozen or fixed in a buffered 4% formaldehyde solution at 4° C., overnight for samples to be embedded in paraffin and the wound skin paraffin blocks were processed into serially cut into 5 m sections on a Microtome (Leica RM2235, Germany). The wound skin sections were used for morphometric analysis and immunofluorescence staining. 5 m sectioned wound skin samples of day 14 were sections were deparaffinised using xylene and ethyl alcohol and stained with hematoxylin and eosin (H&E). To evaluate the epidermis thickness, and wound induced hair follicle formation, four randomly fields of each H&E-stained sections were imaged under a light microscope (Olympus BX53, Olympus, Tokyo, Japan) at 1000 magnification. The values of each mice were averaged, and then groups were compared. To measure the wound induced hair follicles, similar to above procedure, the number of hair follicles at dermis were counted and then average value calculated for each group. To understand the collagen formation, maturation and deposition Masson's trichome analysis were performed as per manufacture instructions (Sigma-Aldrich, Catalog No.: HT15-1KT).
Foreskin collection and preparation—Newly born babies foreskin was collected after parental consent and were kept in eppendorff tube containing DMEM basal medium and immediately foreskin transported to cell culture laboratory at 4° C. for cells isolation. The fore skin was rinsed gently with 1×-PBS containing antibiotic. The skin was exposed the dermis and hypodermis. The whole hypodermis and blood vessels were removed. Subsequently, the skin cut into 1-2 mm pieces and the placed in DMEM medium with dispase-I at 4° C. overnight) for 12-18 hours). The dispase-I overnight treatment, the epidermis was separated from the dermis.
Keratinocytes isolation, culture conditions and characterization—The isolated epidermis was placed in a petri dish containing HEPES buffer for 10 minutes at room temperature, then treated with trypsin at 37° C. until the epidermis became loose and medium cloudy due to keratinocytes release. The cloudy medium was collected and trypsin activity neutralized using fetal bovine serum (FBS) in 1:1 ratio. The epidermis suspended keratinocytes were centrifuges at 1500 rpm for 5 minutes. The pellet resuspended in KGM-GOLD keratinocytes medium (Lonza KGM gold and supplements, Catlog No. 00192151 and 00192152). The isolated foreskin cultured on a culture dish and placed at 37° C. in 5% CO2 incubator for 1-2 days to allow keratinocytes to adhere. Adhere keratinocytes were continued incubation in KGM-Gold medium until cells reach about 80% confluent.
Dermal Schwann cells isolation culture conditions and characterization—The separated dermis was minced into small pieces and placed in a petri dish containing collagenase in DMEM basal medium at 37° C. for 2.5 hours. The dermis was completely dissociated and the medium was cloudy. The dissociated medium was collected in tube and centrifuged at 1500 rpm for 5 minutes. The pellet was resuspended in complete DMEM medium, added on poly-L-lysine coated dish and the dish was incubated at 37° C. in 5% CO2 incubator for overnight. Next day, adhered cells were treated with 10 μM cytosine arabinoside containing DMEM complete medium and incubated at 37° C. in 5% CO2 incubator for 24 hours. After, the cells were cultured in Schwann cells culture medium (ScienCell Research, Catlog No. 1701) until the cells reached about 95% confluence.
Dermalfibroblasts isolation culture conditions and characterization—The separated dermis was minced into small pieces and placed in a petri dish containing collagenase containing DMEM basal medium at 37° C. for 2.5 hours. The dermis was completed dissociated and the medium was cloudy. The dissociated medium was collected in tube and centrifuged at 1500 rpm for 5 minutes. The pellet was resuspended in complete fibroblast medium (ScienCell Research, Catlog No. 2331), added on culture dish and the dish was incubated at 37° C. in 5% CO2 incubator for overnight. Next day, adhered cells continued culture complete fibroblast medium, added on culture dish and the dish was incubated at 37° C. in 5% CO2 incubator until cells reach about 95% confluent.
Cell Viability Assay with 4-Aminopyridine
The keratinocytes, Schwann cells and fibroblast were culture on 96-well plates, the wells were pre-coated with respective coating matrix in complete growth medium and incubated cells for 12-18 hours at 37° C. in 5% CO2 incubator. Cells were washed with PBS and added basal Schwann cells/fibroblast/keratinocytes medium to starve, then incubated for 4 hours at 37° C. with 5% CO2. After the incubation period, various concentrations of 4-AP (concentrations ranging from 1 to 10000 μM) in 100 μL containing respective medium's (complete keratinocytes, Schwann cells and fibroblast medium), cells were incubated either 24 hours at +37° C. and 5% CO2 incubator. After completion of incubation time, 10 μL of the MTT labeling reagent (final concentration 0.5 mg/ml) was added to each well and incubate at +37° C. for 4 hours in a humidified atmosphere. Then, 100 μL of the solubilization solution into each well and allow the plate to stand in the incubator overnight or until the formazan crystals had dissolved. Live keratinocytes/Schwann cells/fibroblast were colored with formazan purple-blue, as the dead cells do not turn the purple-blue color. The formazan absorbance was read using wavelength of 550 nm in a plate reader. The color of optical density (OD) is directly propositional to number of live cells. The percentage of live keratinocytes/Schwann cells/fibroblast cells was determined using the following formula.
Cell viability (%)=(OD of 4−AP treated cells at particular concentration−OD of medium)/OD of control cells(No treatment)−OD of medium×100
Keratinocytes/Schwann cells/fibroblasts (7×104 cells/well keratinocytes and 3.5×104 cells/well of SCs and FBs) with their respective medium were seeded on pre-coated either with collagen-I/PLL/no 96-well ImageLock microplate for 6 hours (Incucyte-sartorius plate, Cat log 4379). For the drug treatment cells were pretreated with 4-AP prior to coating for 16-18 hours. Next, the wound scratch was created using IncuCyte automated system (Essen BioScience). After scratches the cells washed with PBS and the respective cells medium was added with or without 1 mM of 4-AP. The plate was incubated in Incucyte automated imaging system, wound healing and cells migration was monitored by time-lapse photography capturing every hour from 0 to 42 hours. The relative area of wound size and cells migration at each time point was analyzed using IncuCyte™ Scratch Wound Cell Migration Software Module (Essen BioScience) and the percent of wound healing was calculated from the area measured after scratching relative to the basal area as expressed in pixels.
Indirect immunofluorescence was used to identify, characterize and to analyze the proliferation after 4-AP treatment. For each cells (keratinocytes/Schwann cells/fibroblasts) treated with 4-AP and untreated cultures were processed in the same experimental session. The equal number of passage-1 cells were seeded on chamber slide. The cells were grown in their respective complete medium in presence and absence of 4-AP for 72 hours. After, the cells were fixed with 4% paraformaldehyde followed by 0.1% tritonX-100 and respective primary antibodies used, such as—cytokeratin-14, keratin-10, keratin-15, S100, p75-NTR, MPZ, vimentin and α-SMA were in 5% BSA containing PBS. Then, incubated with respective secondary antibodies and washed with PBS after incubation. The chamber glass slide mounted using Prolong Gold anti-fade mounting medium with DAPI then carefully coverslip was placed.
Co-Culture of Keratinocytes with Schwann Cells or Fibroblast on Wound Scratch Assay
To determine the influence of Schwann cells and fibroblast on keratinocytes migration and proliferation wound healing scratch assay was conducted. The co-culture experiment performed in Keratinocytes to either Schwann cells or fibroblasts in a ratio of 10:1 with same proposition of their respective medium were seeded on 96-well ImageLock microplate for 6 hours and the wells were pre-coated with collagen-I plate. For the drug treatment cells were pretreated with 4-AP prior to coating for 16-18 hours. Next, the wound scratch was created and treated cells with and without 4-AP in medium. Imaged for every hour until 24 hour and analyzed for percent of closure as described above procedure.
Effect of Combination 4-AP and NGF with without NGF-Antibody on Wound Healing
Keratinocytes (7×104 cells/well) with KC medium were seeded on collagen-I pre-coated 96-well ImageLock microplate for 6 hours. For the drug treatment cells were pretreated with 4-AP prior to coating for 16-18 hours. Next, the wound scratch was created using IncuCyte automated system. After scratches the cells washed with PBS and the respective cells medium was added with individual 1 mM of 4-AP and 200 ng/ml of NGF (MyBioSource, Inc., Catlog No. MBS7114974) in keratinocytes medium and combination of 1 mM of 4-AP & 200 ng/mL NGF with or without 10 μg/ml of NGF-antibody (ThermoFisher Scientific, Catlog No. MA5-32067). The plate was incubated in Incucyte automated imaging system, wound healing and cells migration imaged for every hour until 24 hour and analyzed for percent of closure as described above procedure.
Briefly, immunohistochemical and immunofluorescence analysis were performed on 5 μm thick wound healing mouse skin sections. The sections were deparaffinized and subjected to an antigen retrieval process by using sodium citrate buffer at 95° C. followed by immersed in 0.5% triton X-100. The section slides were blocked using 5% goat serum in 0.1% PBS-T for 1 hour. The following primary antibodies mouse monoclonal [LL002] anti-Cytokeratin14 (catalog No.—ab7800; IF-1:100), rat CD31 antibody (Catalog No.—553370; IF-1:100), mouse S100 antibody (Catalog No.—MA5-12969; IF-1:200), rabbit nerve growth factor receptor—p75-NTR antibody (Catalog No.—AB1554; IF and WB-1:500), Chicken neurofilament Heavy-NFH antibody (Catalog No.—NB300-217; IF-1:500), mouse alpha-smooth muscle actin —α-SMA antibody (Catalog No.—14-9760-82; IF-1:200 and WB-1:500), mouse TGF beta antibody (Catalog No.—27969; IF-1:100 and WB-1:500), rabbit Ki67 antibody (Catalog No.—9129S; IF-1:400), chicken keratin 15 antibody (Catalog No. —833901; IF-1:500), rabbit K17 antibody (gift from Pierre a. coulombe Lab; IF-1:1000), rabbit interleukin-1 beta antibody (Catalog No.—GTX74034; IF-1:100), rat F4/80 antibody (Catalog No.—MCA497GA; IF-1:100), mouse anti-SOX10 antibody (Catalog No.—sc-365692; IF-1:100 and WB-1:200), chicken anti-myelin basic protein antibody (Catalog No.—MBP; IF-1:1000), rabbit vimentin antibody (Catalog No.—10366-1-AP; IF-1:200), rabbit NGF antibody (Catalog No.—MA5-32067; IF-1:100 and WB-1:1000), mouse PGP 9.5 antibody (Catalog No.—PA5-29012; IF-1:200), chicken anti-P-zero myelin protein antibody (Catalog No.—PZO; IF-1:200), and rat substance P antibody (Catalog No. —NB100-65219; IF-1:100) were overnight incubated with 5% BSA in 0.1% PBS-T for IF stain and/or 5% skimmed milk in 0.1% TBS-T for Western blot at 4° C., then incubated with secondary antibodies for 1 hour at room temperature. The ProLong™ Gold Anti-fade Mountant with DAPI (Invitrogen, Caltlog No. P36935) used as nuclear counterstain. The immunofluorescence stained sections were imaged using ZEISS Axio Observer 7—Axiocam 506 mono—Apotome.2 microscope. The image analysis and quantification were formed either using ZEN 2.6 pro (Zeiss) imaging software or ImageJ-1.53e software (National Institutes of Health, USA).
For protein isolation the harvested skin tissue was flash frozen immediately. The frozen skin tissue ground to a fine power using a liquid nitrogen mortar. The harvested cells and/or tissue powder dissolved in RIPA buffer containing Halt™ Phosphatase (Thermo Scientific, Catlog No. 78420,) and Protease Inhibitor Cocktail (Roche complete tablets mini EASYpack, Catlog No. 04694124001). Tissue and cell debris removed by centrifuged at 14000 rpm for 30 minutes at 4° C. The supernatant was collected and the total protein concentration was determined by BCA protein assay (Thermo Scientific™ Pierce™, Catlog No. 23225). The proteins (20-30 μg) of the tissue protein samples were subjected to 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (Bio-Rad mini-PROTEAN TGX Gels, Catlog No. 4561044) and transferred to polyvinylidene fluoride (PVDF) membranes. After the membranes were blocked with 5% skimmed milk in 1×TBS-T for 1 hour, they were incubated with the appropriate primary antibodies (1:200-1:1000) at 4° C. overnight, then incubated with HRP-conjugated secondary antibodies (1:3000) for 1 hour. Immunoreactivity was then detected using chemiluminescent substrate (Thermo Scientific™ SuperSignal™ West Pico PLUS, Catlog No. 34577). The intensities of the bands were quantified using Gel-imaging software. The quantified band intensity were normalized using GAPDH and expressed either normalized intensity or ratios with respect to saline treated mice.
Statistical analysis was performed and graphs created using Prism 7 software (GraphPad Software Inc., San Diego, CA) and data presented as means±SEM. For wound healing multiple time-point comparission study, sidak's test, and two-way analysis of variance (ANOVA) was used for analyses. Unpaired t test was performed to compare two groups, and one-way analysis of variance (ANOVA) was used for analyses. Sample sizes are indicated in the figure legends. P<0.05 was considered statistically significant for all reported analyses, as specified in the corresponding figure and figure legends.
4-AP is incorporated into a formulation designed for local administration.
To apply 4-AP to skin, 4-AP is prepared in either in isopropyl alcohol-propylene glycol-water solution or currently available balms (compatible with skin). The resulting homogenous clear solutions are passed through 2.5 micron polypropylene filters and filled in to Amber colored capacity bottles (4-AP is sensitive to light) and a total dose of about 1 mL.
For localized topical administration, the 4-AP formulated homogeneous solution is applied twice daily to skin after shaving, beginning at the center of the area. The area of application is the size of the affected area. The 4-AP formulated solution is allowed to dry for 2 to 4 hours after application. For application to the scalp, the topical application of 4-AP application continues for 21 days. For application after shaving, the topical application of 4-AP application continues for 2-4 days.
For localized injection, the 4-AP formulated homogeneous solution is injected subcutaneously in small doses under shaved skin. The 4-AP solution is delivered using a PLGA based thermogelling-delivery vehicle.
The 4-AP formulated homogeneous solution is applied locally by both topical administration and subcutaneous injection to skin grafts to induce hair grow on grafted skin.
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.
This application claims the benefit of U.S. Patent Application Ser. No. 63/134,407, filed on Jan. 6, 2021, and U.S. Patent Application Ser. No. 63/242,824, filed on Sep. 10, 2021. The disclosure of the prior applications are considered part of (and are incorporated by reference in) the disclosure of this application.
Filing Document | Filing Date | Country | Kind |
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PCT/US2022/011351 | 1/5/2022 | WO |
Number | Date | Country | |
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63242824 | Sep 2021 | US | |
63134407 | Jan 2021 | US |