Peptides to promote hair follicle neogenesis and prevent / reduce the harmful effects of radiation therapy

Abstract

The disclosed subject matter relates to peptides obtained by treating extracellular matrix (ECM) with collagenase and methods of using the peptides thus obtained, which can be used for applications including, but not limited to, hair follicle neogenesis, promoting pigmentation, and mitigating the adverse effects of radiation therapy.

Description

[Technical field]

[0001] [CROSS-REFERENCE TO RELATED APPLICATIONS] This application claims the benefit of U.S. Provisional Application No. 63 / 363,241, filed April 19, 2022, the contents of which are incorporated herein by reference in their entirety.

[0002] [Statement regarding federally sponsored research or development] This invention was made with Government support under Grants HU0001-21-2-0061, HU0001-21-2-0035, and HU0001-17-2-0009 awarded by the Uniformed Services University of the Health Sciences. The Government has certain rights in this invention.

[0003] The field of the invention relates to peptides obtained by treating extracellular matrix (ECM) with collagenase, and to methods of using the peptides thus obtained. 32 The peptides can be used for applications including, but not limited to, promoting hair follicle neogenesis, preventing skin damage, skin ulcers, hair loss, wound healing, promoting hair growth and coloration, and mitigating the adverse effects of radiation therapy.

[0004] [Reference to Electronic Sequence Listing] The contents of the electronic sequence listing (166118.01300.xml, size: 17,505 bytes, creation date: April 12, 2023) are incorporated herein by reference in their entirety. [Background technology]

[0005] Hair is a distinctive mammalian feature and is important for thermoregulation, non-verbal communication, social acceptance and beautification in animals and humans. Hair morphogenesis occurs primarily in utero, and no new hair follicles are formed during adulthood. The importance of this loss of hair follicle neogenesis is particularly evident following age-related hair loss, hair loss associated with skin diseases, clinical interventions, burns and skin wound healing. Wound healing in humans typically results in scarring and can also lead to deformity of the skin without hair follicles. Hair loss due to wounds or ageing can have adverse psychological sequelae. Current therapies to treat hair loss rely on the presence of existing hair follicles, but currently there are no therapies or a lack of therapies or treatment options to promote hair growth by hair follicle neogenesis, i.e., generating new hair follicles when existing follicles are lost or destroyed by clinical interventions, burns or radiation damage.

[0006] Dermal papilla cells regulate hair follicle development and growth. Methods to promote dermal papilla cells, and in particular the outgrowth of these cells, are paramount to establish hair follicle neogenesis. Unfortunately, maintaining the inductive potential of dermal papilla cells in culture is difficult, as evidenced by the gradual loss of alkaline phosphatase activity in culture. New methods to maintain hair follicle neogenesis of dermal papilla cells are an unmet need across the wound treatment continuum, including hair loss as a result of radiation therapy.

[0007] Radiation therapy is the mainstay of cancer treatment. Unfortunately, nearly all patients who undergo radiation therapy develop skin tissue-related radiation damage or radiation therapy-induced radiation dermatitis. Damage to underlying normal tissues limits the use of radiation therapy in cancer treatment. In all patients, the skin is permanently altered after irradiation and cannot self-repair from wounds, cuts, or surgical interventions. Radiation damage can be painful and debilitating from this treatment, affecting quality of life and treatment outcomes. Radiation can cause lasting damage to the skin, including permanent hair loss and ulceration. Current standard treatments for radiation therapy-induced radiation dermatitis include AQUAPHOR, mineral oil, and lanolin alcohol. Strategies under investigation to prevent or mitigate radiation dermatitis include several clinical trials, treated wound dressings, anti-inflammatory agents, antibiotics, laser treatment, or skin grafts. Despite these attempts to reduce or prevent radiation-induced dermatitis and the associated deleterious effects observed in all patients, there remains a global and urgent unmet clinical need aimed at prevention.

[0008] In view of the foregoing, it would be desirable to provide new methods for safely and efficiently promoting the generation of new hair follicles in subjects exhibiting hair loss due to natural causes, such as age, genetic disease, burns, or radiation damage (e.g., from radiation therapy or other radiation-related damage). It would also be desirable to provide new methods for preventing or mitigating further adverse effects of radiation therapy, such as radiation dermatitis. Summary of the Invention

[0009] Provided herein is a method of treating a subject with bioactive peptides.The disclosed method can be carried out to stimulate hair follicle neogenesis and resulting hair growth in a subject in need thereof.The disclosed method can also be carried out to prevent and / or mitigate and / or treat the adverse effects of radiation therapy.The disclosed method can be used to treat hair loss due to natural causes, such as age and / or genetic disease, burns, or radiation damage (e.g., due to radiation therapy or other radiation-related damage).

[0010] In some embodiments, the disclosed methods can be performed to treat a subject having hair loss, for example, by promoting hair follicle neogenesis in the subject. The method can include administering to the subject an effective amount of a peptide obtained from treating the extracellular matrix (ECM) with a collagenase, such as bacterial collagenase, to treat hair loss in the subject. In some embodiments of the disclosed methods for treating alopecia, the peptide comprises, consists essentially of, or consists of an amino acid sequence of any of SEQ ID NOs: 1-19, or any combination thereof. The subject can have alopecia due to loss or death of hair follicles, such as loss or death of hair follicles due to aging, loss or death of hair follicles due to testosterone levels in the subject (e.g., male pattern baldness), and / or loss or death of hair follicles due to burn and / or radiation damage. The disclosed methods can promote hair follicle neogenesis in subjects treated with the disclosed methods.

[0011] In other embodiments, the disclosed methods may be performed to prevent and / or alleviate adverse effects of radiation therapy in a subject who has undergone radiation therapy or who is scheduled to undergo radiation therapy. Adverse effects of radiation therapy alleviated by the disclosed methods include alleviation of dermatitis and loss or death of hair follicles, or skin ulcers, erythema, fibrosis, or inflammation. The method includes administering to the subject an effective amount of a peptide obtained by treating extracellular matrix (ECM) with a collagenase, such as bacterial collagenase, thereby alleviating adverse effects of radiation therapy in the subject. In some embodiments of the method of alleviating adverse effects of radiation therapy, the peptide comprises, consists essentially of, or consists of an amino acid sequence of any of SEQ ID NOs: 1-19, or combinations thereof. The peptide may be administered to the subject before the subject undergoes radiation therapy, and / or the peptide may be administered to the subject after the subject undergoes radiation therapy. Thus, the disclosed methods may include administering radiation therapy to the subject. The subject may be undergoing radiation therapy to treat cancer, and thus subjects suitable for the disclosed method may include subjects suffering from cancer. The subject may be exposed to radiation accidentally or due to a military conflict involving the deployment of radiation. In the disclosed method, the subject may be administered a radiation dose that causes dermatitis and / or hair follicle loss or death, and the subject may be administered an effective amount of a peptide to treat dermatitis and / or an effective amount of a peptide to treat hair loss, for example by stimulating hair follicle neogenesis. In the disclosed method, the subject may be administered an effective amount of a peptide to promote hair follicle neogenesis, prevent and / or alleviate stem cell aging of the skin, and prevent or alleviate skin damage and / or ulcers.

[0012] The peptides used in the disclosed methods are typically obtained by treating the ECM with collagenases, such as bacterial collagenase. The peptides may be resistant to degradation after being administered to a treatment site of a subject, such as the scalp of a subject, a site of a subject receiving radiation therapy, and / or a wound bed of a subject. Treatment sites such as wound beds are known to contain proteases, and previous attempts to develop advanced protein-based neo-therapeutics to treat wound beds have failed, at least in part, because the proteases present in the wound bed degrade any bioactive proteins delivered to the wound bed. For these reasons, the peptides used in the disclosed methods may be resistant to degradation by proteases, including proteases present in the wound bed.

[0013] Also disclosed herein are compositions comprising the disclosed peptides, which are formulated for use in the methods disclosed herein.The compositions disclosed herein may include compositions formulated for local administration to a treatment site, such as the scalp of a subject, to treat alopecia, and / or for local administration to a treatment site of a subject to prevent and / or mitigate the adverse effects of radiation therapy administered to the treatment site.The disclosed compositions may be formulated, for example, for local administration to a wound bed and / or for local administration to a graft or radiation wound.

[0014] The present technology can be better understood by referring to the following drawings, which are merely exemplary to illustrate certain features that can be used alone or in combination with other features, and the technology is not limited to the embodiments shown. [Brief description of the drawings]

[0015] [Figure 1] Dose response of TSN6 on alkaline phosphatase activity as a marker of hair growth potential for scrambled TSN6 peptide (left) and active TSN6 peptide (right). [Diagram 2]Hair follicle neogenesis in vivo. TSN6 peptide patch assay counting hair follicle numbers in response to 100 μM scrambled peptide (white bars) and TSN6 peptide (black bars). TSN6 increases hair follicle formation 2-fold. One patch corresponds to 250 spheroids (4000 hDP cells / spheroid and ~50,000 mouse epidermal aggregates). N=3 patches for each peptide. Graph shows mean ± SD. [Diagram 3] TSN6 increases human hair follicle formation in transplanted DECs. Immunohistochemical staining for human HLA in TSN6-scramble (far left panel) and TSN6 grafts from two mice showing hair follicle dissection. Mice were euthanized 12 weeks after transplantation and grafts were analyzed for human cells by immunohistochemical staining for human leukocyte antigen (red). [Figure 4] TSN6 stimulates epidermal proliferation in vivo. Ki-67+ cells (cells / mm) in the epidermis of explants treated with 1 μM scrambled (white bars) or active (black bars) TSN6 peptide. P=0.08 [Figure 5AB] Reduction of radiation dermatitis in mice by administration of TSN6 peptide. Female C57BL / 6 mice (12-14 weeks old) were irradiated with 16.5 Gy of chest X-rays. On days 2, 7, and 10 after irradiation, vehicle (saline) or TSN6 peptide (5 μg / day) was injected subcutaneously. Mice were scored for: A) alopecia, B) scaling, C) erythema, and D) ulceration. E) shows a composite score. Data are mean ± SEM, N = 4 mice / group. [Figure 5C-E] Reduction of radiation dermatitis in mice by administration of TSN6 peptide. Female C57BL / 6 mice (12-14 weeks old) were irradiated with 16.5 Gy of chest X-rays. On days 2, 7, and 10 after irradiation, vehicle (saline) or TSN6 peptide (5 μg / day) was injected subcutaneously. Mice were scored for: A) alopecia, B) scaling, C) erythema, and D) ulceration. E) shows a composite score. Data are mean ± SEM, N = 4 mice / group. [Figure 6]Histology of the thoracic dorsal region 83 days after X-ray irradiation. Female C57BL / 6 mice (12-14 weeks old) were exposed to 16.5 Gy of thoracic X-ray irradiation. Mice received sham irradiation treatment (A), irradiation + vehicle (saline) (B), or irradiation + TSN6 peptide (5 μg / day) administered by subcutaneous injection on days 2, 7, and 10 after irradiation (C). Skin sections were harvested after euthanasia on day 83 after irradiation. Skin was formalin fixed, paraffin embedded, and then stained with hematoxylin & eosin. Sections are from cranial to caudal, from left to right. Arrows indicate the irradiated area. Representative images are shown. [Figure 7AB] Alleviation of composite injury by TSN6 peptide administration in mice. Female C57BL / 6 mice (12-14 weeks old) were exposed to 16.5 Gy of chest X-ray irradiation followed by a 9 / 16 inch wound. Mice received either vehicle (saline) or TSN6 peptide (5 μg / day) by subcutaneous injection on days 2, 7, and 10 after irradiation. Mice were scored for A) wound closure, B) hair loss, C) scaling, D) erythema, or E) ulceration at the indicated time points after irradiation. Composite scores are shown in (F). Data represent mean ± SEM, N=4 mice / group. [Figure 7C-F] Alleviation of composite injury by TSN6 peptide administration in mice. Female C57BL / 6 mice (12-14 weeks old) were exposed to 16.5 Gy of chest X-ray irradiation followed by a 9 / 16 inch wound. Mice received either vehicle (saline) or TSN6 peptide (5 μg / day) by subcutaneous injection on days 2, 7, and 10 after irradiation. Mice were scored for A) wound closure, B) hair loss, C) scaling, D) erythema, or E) ulceration at the indicated time points after irradiation. Composite scores are shown in (F). Data represent mean ± SEM, N=4 mice / group. [Figure 8]Histology of the thoracic dorsal region 83 days after combined injury. Female C57BL / 6 mice (12-14 weeks old) were subjected to 16.5 Gy chest X-ray irradiation resulting in a 9 / 16 inch wound. Mice received sham irradiation (A), irradiation and vehicle (saline) (B), or irradiation and TSN6 peptide (5 μg / day) by subcutaneous injection (C) on days 2, 7, and 10 after irradiation. After euthanasia, skin sections were harvested on day 83 after irradiation. The wound site was marked with black ink on the surface and was visible at the top of the section. Skin was formalin fixed, paraffin embedded, and then stained with hematoxylin & eosin. Sections are from left to right from cranial to caudal. Arrows indicate the wound site. Representative images are shown. [Figure 9] HGF administration for radiation dermatitis in mice. Female C57BL / 6 mice (12–14 weeks old) were exposed to 16.5 Gy of chest X-ray irradiation. Mice received either vehicle (saline) or HGF (3.3 μg / day) by subcutaneous injection on days 2, 7, and 10 after irradiation. Mice were scored for (A) alopecia, (B) scaling, (C) erythema, or (D) ulceration at the indicated times after irradiation. Total scores are shown in (E). Data represent the mean ± SEM, N = 4 mice / group. [Figure 10] Histology of the thoracic dorsal region 83 days after X-ray irradiation. Female C57BL / 6 mice (12-14 weeks old) were subjected to thoracic X-ray irradiation at 16.5 Gy (0.77 Gy / min). Mice were sham irradiated (A), irradiated + vehicle (saline) (B), or irradiated + HGF peptide (3.3 μg / day) administered subcutaneously on days 2, 7, and 10 after irradiation (C). Skin sections were obtained after euthanasia on day 83 after irradiation. Skin was formalin fixed, paraffin embedded, and stained with hematoxylin & eosin. Sections are from cranial to caudal, left to right. Arrows indicate the irradiated area. Representative images are shown. [Figure 11AB]Alleviation of composite injury by TSN6 peptide administration in mice. Female C57BL / 6 mice (12-14 weeks old) were exposed to 16.5 Gy (0.77 Gy / min) of chest X-ray irradiation followed by a 9 / 16 inch wound. Mice received either vehicle (saline) or HGF (3.3 μg / day) by subcutaneous injection on days 2, 7, and 10 after irradiation. Mice were scored for (A) wound closure, (B) hair loss, (C) scaling, (D) erythema, or (E) ulceration at the indicated times after irradiation. Composite scores are shown in (F). Data represent mean ± SEM, N=4 mice / group. [Figure 11C-F] Alleviation of composite injury by TSN6 peptide administration in mice. Female C57BL / 6 mice (12-14 weeks old) were exposed to 16.5 Gy (0.77 Gy / min) of chest X-ray irradiation followed by a 9 / 16 inch wound. Mice received either vehicle (saline) or HGF (3.3 μg / day) by subcutaneous injection on days 2, 7, and 10 after irradiation. Mice were scored for (A) wound closure, (B) hair loss, (C) scaling, (D) erythema, or (E) ulceration at the indicated times after irradiation. Composite scores are shown in (F). Data represent mean ± SEM, N=4 mice / group. [Figure 12] Histology of the thoracic dorsal region 83 days after combined injury. Female C57BL / 6 mice (12–14 weeks old) were subjected to 16.5 Gy (0.77 Gy / min) chest X-ray irradiation followed by a 9 / 16-inch wound. Mice received sham irradiation (A), irradiation and vehicle (saline) (B), or irradiation and HGF (3.3 μg / day) by subcutaneous injection (C) on days 2, 7, and 10 after irradiation. Skin sections were obtained after euthanasia on day 83 after irradiation. The wound site was marked with black ink on the skin surface and is visible in most sections. Skin was formalin-fixed, paraffin-embedded, and stained with hematoxylin and eosin. Sections are from left to right from cranial to caudal. Arrows indicate the wound site. Representative images are shown. [Figure 13A]Images of hair regrowth and repair after irradiation. Treatment with TSN6 ameliorates radiation dermatitis and regrows pigmented hair in mice. Images of mice treated with vehicle (top) or TSN6 (bottom) (A) 57 days, (B) 71 days, and (C) 79 days after irradiation. [Figure 13B] Images of hair regrowth and repair after irradiation. Treatment with TSN6 ameliorates radiation dermatitis and regrows pigmented hair in mice. Images of mice treated with vehicle (top) or TSN6 (bottom) (A) 57 days, (B) 71 days, and (C) 79 days after irradiation. [Figure 13C] Images of hair regrowth and repair after irradiation. Treatment with TSN6 ameliorates radiation dermatitis and regrows pigmented hair in mice. Images of mice treated with vehicle (top) or TSN6 (bottom) (A) 57 days, (B) 71 days, and (C) 79 days after irradiation. [Figure 14A] The presence of grafts was evaluated at the end of the experiment (10 weeks) in nude mice transplanted with dermal-epidermal constructs treated with 10 μM active peptide (TSN18) (n=5), 10 μM scrambled peptide (n=5), and vehicle (n=5). [Figure 14B] Graft size was measured at week 10 in groups treated with TSN18 (n=4), scrambled (n=3) peptide 10 μM, and vehicle (n=4). ns: not significant [Figure 15] Injection of the active, but unscrambled, TSN18 peptide reduced the severity of the overall radiodermatitis score in mouse skin by approximately 50% (five mice per group). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0016] [Detailed Description] Other objects, features, and advantages of the technology provided herein will become apparent from the following detailed description. It should be understood, however, that the detailed description and specific examples, while indicating preferred embodiments of the technology provided herein, are intended for illustrative purposes only, since various changes and modifications within the spirit and scope of the technology provided herein will become apparent to those skilled in the art from this detailed description.

[0017] Suitable peptides for use in the disclosed methods can be obtained by treating the extracellular matrix of a tissue with collagenase, which digests proteins present in the ECM to release peptides. Suitable collagenases include bacterial collagenases. Suitable peptides for use in the disclosed methods are described in U.S. Patent No. 10,485,846 and Sheets et al., 2016 PLOS One, the contents of which are incorporated herein by reference in their entirety.

[0018] Peptides suitable for the disclosed methods can include peptides comprising, consisting essentially of, or consisting of any of the following SEQ ID NOs: 1-19, or combinations thereof. TNS1, 14 aa, NFQGVQNRFVFGTP (SEQ ID NO:1) thrombospondin-1 laminin G-like domain; TSN2, 16 aa, MENAELDVPIQSVFTR (SEQ ID NO:2) thrombospondin-1 N-terminal domain; TSN3, 11 aa, NTDNIYPESSC (SEQ ID NO:3) multimerin EGF-like domain; TSN4, 8 aa, PYLGYVFK (SEQ ID NO: 4) multimerin C1q domain; TSN5, 18 aa, MQTVAQLFKTVSSLSLST (SEQ ID NO:5) multimerin-1 coiled-coil domain; TSN6, 19 aa, HSPDIQLQKGLTFEPIQIK (SEQ ID NO:6) multimerin-1 coiled-coil domain; TSN7, 16 aa, STITQPYKTLNNARSP (SEQ ID NO: 7) fibronectin heparin binding domain; TSN8, 16 aa, RPGPSPEGTGQSYNYR (SEQ ID NO:8) fibronectin fibrin-binding 2 domain; TSN9, 16 aa, MENAELDPPYLGYVFK (SEQ ID NO: 9) combination of thrombospondin and multimerin peptides; TSN10, 20 aa, TGQSYNQYSQRPYLGVYVFK (SEQ ID NO: 10) combination of thrombospondin and multimerin peptides TSN11, 10 aa, LYGQTPLETL (SEQ ID NO:11) TGF-β-induced protein: Fas1 / 3 domain TSN12, 11 aa, ELADSPALEIG (SEQ ID NO:12) TGF-β-induced protein: N-terminal domain TSN13, 21 aa, LYGQTPLETLELADSPALEIG (SEQ ID NO: 13) Combination of 11 and 12 TSN14, 14 aa, VSGNTVEYALPTLE (SEQ ID NO: 14) Tenascin-C fibronectin type III domain 14 TSN15, 18 aa, LDSPTAPTVQSTALTWRP (SEQ ID NO:15) Tenascin-C fibronectin III domain 15 TSN16, 17 aa, LDGSAPGPLYTGSALDF (SEQ ID NO:16) Collagen (VI) alpha-3 VWFA domain 3 TSN17, 11 aa, GSEGVRSGRSG (SEQ ID NO:17) Collagen (VI) alpha-3 VWFA domain 6 TSN18, 14 aa, QPQPLPSPGVGGKN (SEQ ID NO: 18) combination of non-helical collagen (VI) alpha-3 chain domains. TSN19, 11aa, KYTLNPVIDAS (SEQ ID NO: 19) Combination of fibronectin type III 14 domains.

[0019] In some embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19, and the peptide comprises 100, 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, or fewer amino acids. Thus, the peptides used in the disclosed methods may comprise 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, It can comprise, consist essentially of, or consist of 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or 100 amino acids.

[0020] In some embodiments, the peptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-19, and the peptide does not include the full-length protein from which the peptide is derived. In some such embodiments, the peptide comprises no more than 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, or 8 consecutive amino acid residues of the protein from which the peptide is derived.

[0021] In some embodiments of the disclosed method, a subject is administered a combinatorial peptide. As used herein, the term "administering" an agent, such as a therapeutic peptide described herein, to an animal or cell refers to distributing, delivering, or applying a substance to an intended target. The term "administering" refers to contacting, distributing, delivering, or applying a therapeutic agent to an object by any suitable route to deliver the therapeutic agent to a desired location in an animal (e.g., a subject).

[0022] A combinatorial peptide is a peptide that includes a combination of amino acid sequences of peptides, for example, a peptide that includes two or more amino acid sequences of the peptides described herein, for example, a fusion of two or more amino acid sequences of the peptides described herein. Combinatorial peptides are not typically naturally occurring and cannot be generated by treating ECM with collagenase. Alternatively, combinatorial peptides may be generated by chemical synthesis or by expression of a recombinant nucleic acid encoding the combinatorial peptide, for example, using an expression vector that includes the recombinant nucleic acid. Non-limiting examples of combinatorial peptides include peptides that include one or more of SEQ ID NOs: 1-8, 11, 12, 14-17. For example, of the peptides disclosed herein, peptides TSN9, TSN10, TSN13, TSN18 and TSN19 (SEQ ID NOs: 9, 10, 13, 18 and 19) are examples of combinatorial peptides (Table 1).

[0023] In some embodiments, the peptides are linked sequentially, with the C-terminus of one peptide linked to the N-terminal amino acid of another peptide via a peptide bond. Additionally or alternatively, in some embodiments, the peptides are linked by one or more linkers that can include one or more amino acids that are not part of the peptide linked to the combinatorial peptide. The peptides can be linked to enzymes, tags, or targeting moieties that change the distribution or localization of the peptide. The peptides can be modified or linked to moieties that change the half-life or stability of the peptide.

[0024] In some embodiments, peptides can be linked to biodegradable scaffolds, i.e., bioinspired microscopic spines that can deliver peptides into or under the skin.

[0025] In some embodiments, the peptides can be combined with a carrier or excipient. The peptides can be administered as a pharmaceutical composition comprising a combination of one or more peptides and one or more pharma- ceutically acceptable carriers or excipients. Such compositions can be aqueous solutions, emulsions, creams, ointments, suspensions, gels, liposomal suspensions, and the like. Suitable carriers (excipients) include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphates, acetates, gelatin, collagen, CARBOPOL™, vegetable oils, and the like. In addition, suitable preservatives, stabilizers, antioxidants, antibacterial agents, buffers, such as BHA, BHT, citric acid, ascorbic acid, tetracycline, and the like, may be included. Cream or ointment bases useful for the formulation include lanolin, SILVADENE™, AQUAPHOR™, and the like. Other topical formulations include aerosols, conditioners, bandages, and other wound dressings. Alternatively, the wound healing peptides can be incorporated or encapsulated in a suitable polymer matrix or membrane to provide a sustained release delivery device suitable for implantation near the site to be locally treated. Other suitable devices for delivering or administering the compositions described herein include devices such as indwelling catheters and ALZET™ mini pumps. Ophthalmic formulations can be formulated using commercially available vehicles such as SORBI-CARE™, NEODECADRON™, LACRILUBE™, or topical formulations such as those described in U.S. Pat. No. 5,124,155 (hereby incorporated by reference).

[0026] The peptides described herein can be combined with additional treatments, therapies or therapeutic devices, for example, provided as a combination therapy. Additional treatments, therapies or therapeutic devices include, but are not limited to, minoxidil, finasteride, baricitinib, spironolactone, dutasteride, corticosteroids, platelet-rich plasma, surgery, laser therapy, microneedles, Follica device, RECELL system, allogeneic cellular scaffold products including StrataGraft, skin scaffolds including natural or bioengineered scaffolds, bioengineered skin substitutes, skin constructs, skin grafts (split-thickness grafts, full-thickness grafts and composite grafts) and cosmetic procedures. In some embodiments, the peptides can be combined with additional treatments or therapies ex vivo. By way of example, and not by way of limitation, an effective amount of the peptide can be combined with a bioengineered skin construct or substitute and a medium and growth factors useful for inducing differentiation, growth and survival of the bioengineered skin construct or substitute.

[0027] In the disclosed methods, an effective amount of peptide is administered to treat, prevent, or alleviate symptoms (e.g., hair loss). In some embodiments, the effective amount administered in the disclosed methods can be at least 1, 10, or 100 μM, or an amount of peptide between 1 nM and 100 μM at the administration site. The effective amount can be administered daily, once or multiple times, or as frequently as needed (e.g., weekly, twice weekly, monthly, etc.).

[0028] In some embodiments, an effective amount of peptide is administered locally. In some embodiments, an effective amount is administered by any effective route, including but not limited to oral, subcutaneous, subdermal, and / or wound bed or lesion site. In some embodiments, an effective amount of peptide is incorporated into topical solutions (including gels, ointments, creams, and suspensions), dressings, patches, follicular units, ex vivo cells, scaffolds, skin equivalents, or transdermal delivery by chemical or physical approaches.

[0029] In some embodiments, an effective amount of the peptide is delivered orally, for example, to prevent and / or alleviate chemotherapy- and / or radiation-induced gastrointestinal damage.

[0030] [Method of Treating Hair Loss in a Subject] The methods disclosed herein can be carried out to treat hair loss, for example by promoting hair follicle neogenesis.The disclosed methods typically include administering peptides obtained by treating ECM with collagenase, such as bacterial collagenase, which releases peptides for use in the method of treating hair loss.Hair follicle neogenesis requires hair-forming dermal papilla cells.The disclosed methods can be carried out to induce hair-forming dermal papilla cells to form hair follicles in a subject in need thereof.

[0031] One aspect of the disclosure provides a method of treating a subject with hair loss, at risk of hair loss or progression of hair loss, or with impaired hair growth or loss of hair or other skin appendage structures, comprising administering to the subject an effective amount of a peptide comprising an amino acid sequence of any of SEQ ID NOs: 1-19, or any combination thereof, to treat hair loss or stimulate the formation, maintenance, or function of hair follicles or other skin appendages. In some embodiments, the peptide comprises the amino acid sequence of SEQ ID NO: 6.

[0032] In the disclosed hair loss treatment method, the peptide is combined with a carrier or excipient or a transdermal delivery system. In some embodiments, the peptide is combined with a follicular unit or matrix before administering the peptide to a subject, optionally via implantation. The follicular unit is a complete anatomical and physiological structure that includes a hair follicle and a small group of neurovascular and arrector pili muscles.

[0033] In the disclosed hair loss treatment method, the peptide may be administered by any suitable route of administration. Suitable routes of administration may include oral administration, topical administration, subcutaneous administration, and / or subcutaneous administration. In some embodiments, the peptide is administered locally to the hair loss site. In some embodiments, the peptide is administered ex vivo to cells, substrates, or artificial skin or tissue before administration to a patient.

[0034] In some embodiments of the method disclosed herein, an effective amount of peptide is delivered to hair loss site to promote hair follicle neogenesis.In some embodiments, an effective amount of peptide is administered to subject to achieve peptide concentration of at least about 1, 10, or 100 μM at administration site.In some embodiments, a therapeutically effective amount of peptide having the amino acid sequence of SEQ ID NO:6 is administered to subject.

[0035] The term "effective amount" as used herein refers to the amount or dosage of a compound that produces a desired effect.In some embodiments, an effective amount is the amount or dosage of a compound that produces a desired effect in a subject under diagnosis or treatment when administered to the subject once or multiple times.Suitably, the desired effect can be treating the subject with alopecia.

[0036] Effective amounts can be easily determined by those skilled in the art, including the attending physician and / or diagnostician, by using known techniques and observing results obtained under similar circumstances. In determining the effective amount or dosage of the compound to be administered, the attending physician can take into account several factors, such as the subject's species, the subject's size, age, and general health, the degree or severity of the disease or disorder involved, the response of the individual subject, the specific compound administered, the method of administration, the bioavailability characteristics of the administered formulation, the selected dosing regimen, the use of concomitant drugs, and other relevant circumstances.

[0037] The terms "treat" or "treating" as used herein refer to alleviating symptoms, eliminating the cause of the resulting symptoms, either temporarily or permanently, and / or preventing or delaying the appearance or reversing the progression or severity of the resulting symptoms of the specified disease or disorder, respectively. In some embodiments, the subject responds to treatment with the peptides disclosed herein, and in some embodiments, includes use in combination with one or more additional therapeutic agents. The term "treat" also includes alleviating one or more symptoms associated with hair loss, such as increasing hair growth, length, density, or stimulating the formation, preservation, or function of hair follicles or other skin appendages.

[0038] In the disclosed method for treating hair loss, peptide can be administered at any suitable frequency.In some embodiments, subject can administer peptide every day or more frequently, or at other intervals, such as every other day, every third day, etc.In some embodiments, subject can administer peptide for 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 4 weeks, or more.

[0039] In the disclosed method for treating hair loss, peptide can be administered alone or as part of a combination or complementary therapy.In some embodiments, an effective amount of peptide and other therapies for treating alopecia are administered to the subject.Other therapies for treating alopecia include, but are not limited to, minoxidil, finasteride, baricitinib, spironolactone, dutasteride, corticosteroids, platelet-rich plasma, hair transplant surgery (e.g., follicular unit transplantation, follicular unit ablation, robotic follicular unit ablation technique), hair follicle cell transplantation, laser therapy, microneedling, or Follica device.

[0040] Subjects suitable for the disclosed alopecia treatment methods may include subjects who have lost hair follicles.Subjects for the disclosed alopecia treatment methods may have lost hair follicles due to age, for example, the subject is at least 20, 30, 40, 50, or 60 years old.

[0041] Subjects suitable for the disclosed method for treating alopecia include those who have lost hair follicles due to the effects of testosterone. Subjects eligible for the disclosed method for treating alopecia may have alopecia due to male pattern baldness.

[0042] Subjects suitable for the disclosed hair loss treatment methods include those who have lost hair follicles due to other conditions. Subjects for the disclosed hair loss treatment methods may have hair loss due to alopecia areata, anagen effluvium, telogen effluvium, tinea capitis, or traction alopecia.

[0043] Subjects suitable for the disclosed hair loss treatment methods may include subjects who have lost hair follicles due to trauma, and optionally physical, thermal, chemical, or radiological trauma, or other forms of trauma. In some embodiments, the trauma results in complete or partial loss of skin and / or a reduction in skin thickness.

[0044] Subjects suitable for the disclosed hair loss treatment methods may include subjects who have lost hair follicles due to burns. Subjects for the disclosed hair loss treatment methods may have alopecia resulting from burns caused by heat, radiation therapy, or a combination thereof.

[0045] Subjects suitable for the disclosed hair loss treatment methods include subjects who have lost hair follicles due to treatments or therapies for other conditions or diseases. In some embodiments, the subject has hair loss due to a treatment that results in the death of hair follicles.

[0046] Subjects suitable for the disclosed hair loss treatment methods include subjects who have lost hair follicles due to radiation therapy. Subjects for the disclosed hair loss treatment methods may have cancer and may have hair loss due to radiation therapy or chemotherapy treatment.

[0047] Subjects suitable for the disclosed methods of treating hair loss may include subjects wishing to prevent or reduce hair loss due to aging, the influence of hormones, including testosterone, disease or other conditions, trauma, burns, therapy, or radiation.

[0048] In some embodiments, efficacy is determined by comparison with one or more untreated control subjects, where the control subjects experience the same or similar symptoms and associated hair loss and are not administered the peptide of the present disclosure. With respect to therapeutic efficacy, in some embodiments, relief is observed within 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or about 4 months after the first therapeutic dose of peptide (e.g., reduced hair loss, or improved hair growth or hair follicle formation). In some embodiments, the peptide may show sustained efficacy for several days, weeks, or longer after administration.

[0049] In the disclosed hair loss reducing method, peptide can be administered at any suitable frequency.In some embodiments, subject can administer peptide daily or more frequently.In some embodiments, subject can administer peptide daily for 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 4 weeks or more.In some embodiments, administration is daily, weekly, biweekly, monthly, or any combination thereof.

[0050] In the disclosed methods, a subject can be treated with the peptide before, during, and / or after a detected disease, treatment, or other condition in which hair loss is observed and / or is a predicted outcome, side effect, or symptom.

[0051] Methods for preventing and / or mitigating the adverse effects of a disease, treatment, or radiation exposure in a subject The methods disclosed herein can be performed to prevent and / or mitigate the effects of disease, chemotherapy, radiation therapy, or other radiation exposure events (e.g., accidental radiation exposure, exposure due to military / combat events, or other radiation-releasing events). In particular, these methods relate to reducing hair loss associated with the aforementioned disease, treatment, or exposure, and / or increasing hair growth following or during such disease, treatment, or exposure. These methods typically involve administering to a subject an effective amount of a peptide comprising any of the amino acid sequences of SEQ ID NOs: 1-19, or any combination thereof, for example to reduce the effects of radiation therapy or other radiation exposure. In some embodiments, the method involves administering an effective amount of a peptide comprising the amino acid sequence of SEQ ID NO: 6 and / or SEQ ID NO: 18.

[0052] In some embodiments, the peptide is administered after radiation exposure, treatment, or disease diagnosis. In some embodiments, the peptide is administered, for example, before treatment, radiation exposure, or disease diagnosis. In some embodiments, the peptide shows sustained efficacy for a period of days, weeks, or more after administration. In some embodiments, the peptide is administered at the time of treatment, radiation exposure, or disease diagnosis. In some embodiments, the peptide shows sustained efficacy for a period of days, weeks, or more after administration. By way of example and not limitation, in some embodiments, the peptide is administered after treatment, radiation exposure, or disease diagnosis, for example, about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 48 ​​hours or more after disease diagnosis, treatment, or exposure, and shows a therapeutic effect (e.g., alleviation or prevention of one or more symptoms, such as hair loss) for a period of days, weeks, or more after the initial administration. By way of example and not limitation, in some embodiments, the peptide is administered prior to diagnosis, treatment, or exposure to a disease, e.g., about 1 hour, 2 hours, 6 hours, 12 hours, 18 hours, 24 hours, 28 hours, 32 hours, 36 hours, 40 hours, 48 ​​hours or more prior to diagnosis, treatment, or exposure to a disease, and exhibits a therapeutic effect (e.g., alleviation or prevention of one or more symptoms, such as hair loss) for days, weeks, or more after radiation exposure. In some embodiments, the peptide is administered before or after radiation exposure, and may have a sustained efficacy for days, weeks, or more after administration. In some embodiments, the peptide is administered before, during, and / or after treatment, radiation exposure, or diagnosis of a disease, and may have a sustained efficacy for days, weeks, or more after administration. Alleviating hair loss includes improving hair growth.

[0053] In some embodiments, efficacy is determined by comparison to one or more untreated control subjects, where the control subjects have experienced the same or similar disease, treatment, or radiation exposure and have not been administered the peptides of the present disclosure. With respect to therapeutic efficacy, in some embodiments, a palliative effect is observed (e.g., reduced hair loss, or improved hair growth or hair follicle formation) within 1 week, 2 weeks, 3 weeks, 1 month, 2 months, 3 months, or about 4 months of the first therapeutic dose of the peptide.

[0054] In some embodiments of the disclosed methods, the subject has cancer and undergoes radiation therapy to treat the cancer. In some embodiments, the subject has cancer and undergoes chemotherapy to treat the cancer. Radiation therapy can result in hair loss at the treatment site. Chemotherapy treatment is systemic and can damage hair follicles, resulting in hair loss throughout the body. The subject can be treated with radiation therapy, chemotherapy, or a combination of the two. The methods disclosed herein can be suitable for reducing or treating hair loss associated with cancer treatment. In some embodiments, the peptides can be suitable for use in the disclosed methods in subjects exposed to radiation therapy and / or chemotherapy.

[0055] In other embodiments, the subject has a disease other than cancer, such as Dupuytren's disease or Lederhodes disease, and the subject receives radiation therapy to treat the other disease. In further embodiments, the subject has a benign tumor, a thyroid disease, or a blood disease, and the subject receives radiation therapy to treat the benign tumor, thyroid disease, or blood disease.

[0056] The subject can undergo radiation therapy and then be administered an effective amount of the peptide after radiation exposure to mitigate the effects of the radiation therapy. In some embodiments, the subject can be administered an effective amount of the peptide prophylactically to counteract the effects of the radiation therapy before the subject undergoes radiation therapy.

[0057] In the method of preventing or reducing the subsequent adverse effects of radiation therapy, the subject can be administered the peptide by any suitable route before receiving radiation therapy. Suitable routes of administration include oral, topical, subcutaneous, and / or subcutaneous administration. In some embodiments, the peptide is administered locally to the site where radiation therapy is administered.

[0058] In the method of reducing the adverse effects of radiation therapy, the peptide can be administered to the subject after radiation therapy by any suitable route. Suitable routes of administration include topical administration, subcutaneous administration, and / or subcutaneous administration. In some embodiments, the peptide is administered locally to the site where radiation therapy is administered. Examples of topical administration include, but are not limited to, gel, ointment, cream, suspension, dressing, biodegradable patch, follicular unit, ex vivo cell, scaffold, skin equivalent, or transdermal delivery by chemical or physical approach.

[0059] In a method for reducing the adverse effects of radiation therapy, a subject may be administered an effective amount of a peptide to treat dermatitis, including erythema, scaling, and ulceration, at the site where radiation therapy is administered. In some embodiments, a subject may be administered an effective amount of a peptide to alleviate stem cell aging in the skin at the site where radiation therapy is administered.

[0060] In the method of mitigating the adverse effects of radiation therapy, a subject may receive a dose of radiation that results in the loss or death of hair follicles at the site where the dose of radiation is administered. The subject may be exposed to radiation accidentally or through military conflict. The subject may be administered an effective amount of peptide before and / or after the subject receives radiation therapy. In this case, the effective amount of peptide is effective to promote neogenesis of hair follicles at the site where the radiation therapy is administered. In some embodiments, the peptide is administered after radiation exposure. In some embodiments, the peptide is administered before, during, and / or after radiation exposure, and may have an effect for several days, weeks, or longer after administration.

[0061] In the disclosed method, preferably, an effective amount of peptide is delivered to the site where radiation therapy is administered to reduce the adverse effects of radiation therapy.In some embodiments, an effective amount of peptide is administered to the subject to achieve a concentration of at least about 1, 10, or 100 μM at the administration site.

[0062] In the disclosed methods for reducing the adverse effects of disease, treatment, or radiation exposure, the peptide may be administered at any suitable frequency. In some embodiments, the subject may be administered the peptide daily or more frequently. In some embodiments, the subject may be administered the peptide daily for 1, 2, 3, 4, 5, 6 days, 1, 2, 3, 4 weeks or more. In some embodiments, administration is daily, weekly, biweekly, monthly, or any combination thereof.

[0063] In some embodiments of the disclosed method, the subject is exposed to non-therapeutic radiation. Non-therapeutic radiation may include any radiation exposure that is not therapeutic. Non-therapeutic radiation includes ionizing radiation or non-ionizing radiation. Examples of ionizing radiation include, but are not limited to, nuclear radiation, X-rays, and gamma rays from radioactive elements. Examples of non-ionizing radiation include, but are not limited to, infrared and ultraviolet radiation (including sunlight, artificial ultraviolet light such as tanning beds and medical devices). In some embodiments, the subject may have been exposed to radiation accidentally or due to a military conflict involving the deployment of radiation. The subject may be administered a therapeutic amount of the peptide of the present disclosure before, during, and / or after such exposure.

[0064] In the disclosed methods for reducing the adverse effects of hair loss in general, or hair loss due to disease, treatment, or radiation exposure, the peptides can be administered alone, in combination with, or in conjunction with other therapies. Other therapies include, but are not limited to, the RECELL™ system, allogeneic cellular scaffold products including StrataGraft™, skin scaffolds including naturally derived or bioengineered scaffolds, bioengineered skin substitutes or skin constructs. The disclosed peptides can also be administered with skin grafts, including split thickness grafts, full thickness grafts, and composite grafts. The disclosed peptides can also be encapsulated for administration or administered via biodegradable microneedles or microneedle array patches.

[0065] [Additional definitions] The technology provided herein is not limited to the particular methodologies, protocols, configurations, formulas, and reagents described, and further includes those known to those of skill in the art. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the technology provided herein.

[0066] The disclosure is not limited to the particular details of the configurations, arrangement of components, or method steps described herein. The compositions and methods disclosed herein can be made, practiced, used, performed, and / or formed in a variety of ways that will be apparent to one of skill in the art in light of the following disclosure. The phraseology and terminology used herein are for descriptive purposes only and should not be construed as limiting the scope of the claims. Ordinal designations such as first, second, third, etc., used to refer to various structures or method steps in the description and claims should not be construed as indicating a particular structure or step, or a particular order or configuration of such structures or steps. All methods described herein can be performed in any suitable order unless otherwise indicated herein or clearly contradicted by context. The use of all examples or illustrative language (such as "etc.") provided herein is intended only to facilitate disclosure and does not limit the scope of the disclosure unless otherwise claimed. No language in the specification or any structure shown in the drawings should be construed as indicating that any unclaimed element is essential to the practice of the disclosed subject matter. As used herein, the terms "including," "comprising," and "having" and variations thereof are meant to encompass the elements listed thereafter, and equivalents thereof, as well as additional elements. Embodiments described as "including," "comprising," or "having" particular elements are also contemplated as "consisting essentially of" and "consisting of" those particular elements.

[0067] Unless otherwise specified or indicated by context, the terms "a", "an", and "the" mean "one or more". For example, "a molecular" is to be interpreted as meaning "one or more molecules". As used herein, "about", "approximately", "substantially", and "significantly" are understood by those of ordinary skill in the art and will vary to some extent depending on the context in which they are used. If there are usages that are not clear to those of ordinary skill in the art from the context in which the terms are used, "about" and "approximately" mean plus or minus ≦10% of the particular term, and "substantially" and "significantly" mean plus or minus >10% of the particular term.

[0068] The description of ranges of values ​​herein is intended only as a shorthand way of referring to each individual value within the range individually, unless otherwise stated herein, and each individual value is incorporated herein as if it were individually stated herein. For example, if a concentration range is described as 1% to 50%, values ​​such as 2% to 40%, 10% to 30%, or 1% to 3%, are to be expressly recited herein. These are only examples of what is specifically intended, and all possible combinations of numerical values ​​between and including the lowest and highest values ​​recited are to be considered to be expressly recited in this disclosure. The use of the word "about" to describe a particular recited amount or range of amounts is intended to indicate that the amount includes values ​​very close to the recited amount, such as values ​​that are naturally taken into account or are taken into account due to manufacturing tolerances, equipment and human error in measuring, etc. All percentages relating to amounts are by weight unless otherwise stated.

[0069] As used herein, the terms "subject" and "patient" are used interchangeably and refer to an individual of interest (e.g., an individual such as a mammal, e.g., a human) who is treated with a peptide of the present disclosure or serves as a "no treatment" control.

[0070] Any references, including non-patent or patent documents, cited herein are not admitted to constitute prior art. In particular, unless otherwise noted, it is understood that reference to any document herein is not an admission that any of these documents constitute part of the general knowledge in the art in the United States or any other country. Any discussion of a reference states the assertions of its author, and applicants reserve the right to challenge the accuracy and pertinence of any of the documents cited herein. Unless expressly noted otherwise, all references cited herein are incorporated by reference in their entirety. In the event of any discrepancy in definitions and / or descriptions of the cited documents, the present disclosure shall control.

[0071] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention or the appended claims.

[0072] 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 the technology provided herein belongs. Methods, materials, and kits similar or equivalent to those described herein can be used in the practice or testing of the technology provided herein.

[0073] Exemplary embodiments Embodiment 1. A method of treating a subject with alopecia, or at risk of developing alopecia or alopecia, or a subject with impaired function or alopecia of hair or other skin appendage structures, comprising administering to the subject an effective amount of a peptide comprising an amino acid sequence of any of SEQ ID NOs: 1-19 to treat alopecia or stimulate the formation, maintenance, or function of hair follicles or other skin appendages.

[0074] <Embodiment 2> The method of embodiment 1, wherein the peptide consists of any one of the amino acid sequences of SEQ ID NOs: 1 to 19.

[0075] Embodiment 3. The method of any of the preceding embodiments, wherein the peptide is 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length, or in a range bounded by any of 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids in length (e.g., between 8 and 100 amino acids in length).

[0076] Embodiment 4. The method of any of the preceding embodiments, wherein the peptide comprises no more than 50, 45, 40, 35, 30, 25, or 20 contiguous amino acid residues of the protein from which the peptide is derived.

[0077] Embodiment 5. The method of any of the preceding embodiments, wherein the peptide consists of a combinatorial peptide comprising one or more of SEQ ID NOs: 1-19 fused to a second peptide or polypeptide.

[0078] Embodiment 6. The method of any of the preceding embodiments, wherein the peptide comprises the amino acid sequence of SEQ ID NO:6.

[0079] Embodiment 7. The method of any of the preceding embodiments, wherein the peptide is combined with a carrier or excipient or transdermal delivery system, and optionally the peptide is combined with a follicular unit or matrix prior to administering the peptide to the subject, optionally via implantation.

[0080] Embodiment 8. The method of any of the preceding embodiments, wherein the peptide is administered locally to a treatment site, and optionally, the peptide is administered ex vivo to a cell, matrix, or artificial skin prior to administration to a patient.

[0081] Embodiment 9. The method of any of the preceding embodiments, wherein the effective amount of the peptide is effective to promote hair follicle neogenesis or preservation or improve hair follicle function.

[0082] Embodiment 10. The method of any of the previous embodiments, wherein an effective amount of the peptide is administered locally.

[0083] Embodiment 11. The method of any of the preceding embodiments, wherein an effective amount of the peptide is administered subcutaneously.

[0084] Embodiment 12. The method of any of the preceding embodiments, wherein an effective amount of the peptide is administered intradermally.

[0085] Embodiment 13. The method of any of the preceding embodiments, wherein the subject is administered an effective amount of the peptide to achieve a concentration of at least about 0.1, 1, 10, 100, or 1000 μM at the site of administration.

[0086] Embodiment 14. The method of any of the preceding embodiments, wherein the subject receives an effective amount daily.

[0087] Embodiment 15. The method of any of the preceding embodiments, wherein the subject has alopecia resulting from loss of hair follicles.

[0088] Embodiment 16. The method of any of the preceding embodiments, wherein the subject is at least 20, 30, 40, 50, or 60 years of age.

[0089] Embodiment 17. The method of any of the preceding embodiments, wherein the subject has hair loss due to androgenetic alopecia.

[0090] Embodiment 18. The method of any of the preceding embodiments, wherein the subject has alopecia resulting from trauma, and optionally physical, thermal, chemical, or radiological trauma, or other forms of trauma.

[0091] Embodiment 19. The method of any of the previous embodiments, wherein the trauma results in complete or partial loss of skin and / or reduced skin thickness.

[0092] Embodiment 20. The method of any of the preceding embodiments, wherein the subject has a scarring type of alopecia selected from central centrifugal cicatricial alopecia, lichen planopilaris, keloid acne nuchal, dissecting cellulitis, traction alopecia, and pseudoalopecia bronchialis.

[0093] Embodiment 21. The method of any of the preceding embodiments, wherein the subject has a non-scarring form of alopecia optionally selected from alopecia areata, anagen effluvium, and telogen effluvium.

[0094] Embodiment 22. The method of any of the preceding embodiments, wherein the subject has alopecia resulting from a burn scar.

[0095] Embodiment 23. The method of any of the preceding embodiments, wherein the subject has alopecia resulting from a treatment that results in the death of hair follicles.

[0096] Embodiment 24. The method of any of the preceding embodiments, wherein the subject has alopecia resulting from radiation or chemotherapy treatment.

[0097] Embodiment 25. The method of any of the preceding embodiments, wherein the subject has cancer and is undergoing radiation or chemotherapy treatment.

[0098] <Embodiment 26> A method for reducing the effects of radiation therapy in a subject undergoing radiation therapy, the method comprising administering to the subject an effective amount of a peptide comprising any one of the amino acid sequences of SEQ ID NOs: 1 to 19, so as to reduce the effects of radiation therapy.

[0099] <Embodiment 27> The method of embodiment 26, wherein the peptide consists of an amino acid sequence of any one of SEQ ID NOs: 1 to 19.

[0100] Embodiment 28. The method of any of embodiments 26 to 27, wherein the peptide has a length of 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids, or a length within a range bounded by any of 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids (e.g., a length of 8 to 100 amino acids).

[0101] Embodiment 29. The method of any of embodiments 26-28, wherein the peptide comprises no more than 50, 45, 40, 35, 30, 25, or 20 contiguous amino acid residues of the protein from which the peptide is derived.

[0102] Embodiment 30. The method of any of embodiments 26-29, wherein the peptide comprises a combinatorial peptide comprising one or more of SEQ ID NOs: 1-19 fused to a second peptide or polypeptide.

[0103] Embodiment 31. The method of any one of embodiments 26 to 30, wherein the peptide comprises the amino acid sequence of SEQ ID NO:6.

[0104] Embodiment 32. The method of any of embodiments 26-31, wherein the peptide is combined with a carrier or excipient or a transdermal delivery system.

[0105] Embodiment 33. The method of any of embodiments 26-32, wherein the peptide is administered locally to the site where radiation therapy is administered.

[0106] Embodiment 34. The method of any one of embodiments 26 to 33, wherein an effective amount of the peptide is administered locally.

[0107] Embodiment 35. The method of any one of embodiments 26 to 34, wherein an effective amount of the peptide is administered subcutaneously.

[0108] Embodiment 36. The method of any of embodiments 26-35, wherein an effective amount of the peptide is administered intradermally.

[0109] Embodiment 37. The method of any of embodiments 26-36, wherein an effective amount of the peptide is effective to treat dermatitis.

[0110] Embodiment 38. The method of any of embodiments 26-37, wherein an effective amount of the peptide is effective to attenuate aging of skin stem cells.

[0111] Embodiment 39. The method of any of embodiments 26-38, wherein an effective amount of the peptide is effective to promote the generation or preservation of hair follicles or promote hair follicle function.

[0112] Embodiment 40. The method of any of embodiments 26-39, wherein the subject is administered an effective amount daily.

[0113] <Embodiment 41> The method of any one of embodiments 26 to 40, wherein the subject is administered a radiation dose that results in the death of hair follicles.

[0114] Embodiment 42. The method of any one of embodiments 26-41, wherein the subject has cancer and the subject is undergoing radiation therapy to treat the cancer.

[0115] <Embodiment 43> The method of any of embodiments 26 to 42, wherein the subject has cancer, the subject is undergoing radiation therapy to treat the cancer, or the subject is undergoing chemotherapy to treat the cancer, and an effective amount of the peptide is administered to the site where radiation therapy is administered. EXAMPLES

[0116] The following examples are included solely for the purpose of illustrating certain embodiments and aspects of the disclosure and are not intended to limit the invention.

[0117] [Example 1: TSN peptide promotes hair follicle regeneration.] The active pharmaceutical ingredient (API) enzyme contained in SANTYL™ collagenase ointment was used to generate peptides from well-defined biosynthetic extracellular matrices obtained from in vitro human epidermal and dermal cell cultures. 7 Fifteen collagenous and collagen-related ECM peptides ranging in length from 8 to 21 amino acids have now been identified, sequenced, and synthesized (TSN peptides). 7 Mass spectrometric identification of the releases revealed an array of cell type-specific peptides, including major peptides derived from the macromolecules (i) thrombospondin, (ii) multimerin, and (iii) fibronectin that bind to the extracellular matrix and associate with collagen. These are sensitive to collagenase and are released from the endothelial ECM. In addition, collagenase generates peptides from (i) TGF-β-induced proteins and (ii) collagen VI, which are derived from the ECM synthesized and organized by human dermal fibroblasts. From these original structures, peptides with hydrophobic and hydrophilic profiles have been synthesized, ensuring that each peptide is soluble in water. 7 .

[0118] In addition to these peptides generated by collagenase degradation of synthetic endothelial or fibroblast matrix, four peptides were "redesigned." These redesigned peptides are not naturally generated by collagenase digestion. The collagenase-generated "parent" peptides were redesigned to optimize and maximize their wound healing potential. 7These include peptides TSN9, TSN10, TSN18, and TSN19, which are identical in amino acid sequence to at least two distinct collagenase-released peptides identified by mass spectrometry (Table 1). The peptides were synthesized at the Tufts University Core Facility and evaluated for biological activity in a series of tests. These tests included (i) the ability to stimulate cell-specific proliferation, (ii) activation of angiogenesis, and (iii) wound healing (in vitro and in vivo). TSN peptides 1-9 are derived from human dermal endothelial cell extracellular matrix. TSN peptides 10-19 are derived from human dermal fibroblast extracellular matrix. 8 .

[0119] [Table 1]

[0120] Dermal papilla cells control hair follicle development and growth, but unfortunately, it is difficult to maintain the inductive potential of dermal papilla cells in culture, as evidenced by the gradual loss of alkaline phosphatase activity in culture. 3 To test whether TSN peptides could enhance alkaline phosphatase activity, keratinocytes and dermal papilla cells were seeded in six replicates in 96-well plates at 4000 cells / well and 2000 cells / well, respectively, and cultured overnight. The medium was replaced and the appropriate treatment (vehicle, scrambled peptide, or active peptide) was added to the wells. The medium was replaced with fresh medium containing peptide 2 days after the first treatment, for a total culture period of 4 days, after which alkaline phosphatase activity was measured (Figure 1). 10 μM active TSN peptide enhanced alkaline phosphatase activity compared to the scrambled peptide.

[0121] We next used a patch assay to test whether TSN peptides could induce hair follicle neogenesis in vivo. Epidermal aggregates were resuspended in DMEM / F12 at 750,000 aggregates / 750 μL (1000 aggregates / μL), and spheroids were resuspended in DMEM / F12 at 1400 spheroids / 140 μL (10 spheroids / μL). Ten million dermal cells were resuspended in 400 μL DMEM / F12 (25,000 cells / μL or 1.25E6 per 50 μL). A positive control (30 μL (30,000) aggregates, 120 μL (3 million) mouse dermal cells, 30 μL DMEM / F12), and a no DP cell control (30 μL (30,000) aggregates, 120 μL (3 million) mouse dermal cells, 30 μL DMEM / F12) were used. Appropriate tubes were prepared for the following solutions: 150 µL), scrambled peptide control (175 µL (175,000 cells) of epidermal aggregates, 70 µL (700 cells) of spheroids, 6.25 µL (4 mg / mL) of scrambled peptide), and active peptide (175 µL (175,000 cells) of epidermal aggregates, 70 µL (700 cells) of spheroids, 6.25 µL (4 mg / mL) of active peptide). The solution was stored on ice until injection into mature 6-10 week old nu / nu mice. A maximum of eight injections are possible per mouse. The cells were injected slowly into the deep dermis without injecting an air bubble or penetrating the entire dermis, ensuring that the cells remained in the limited space within the dermis. The transplanted cells were allowed to develop for 2-4 weeks. Using a dissecting microscope, the number of hair follicles was counted per transplant from the mucosal side of the skin (Figure 2). The TSN peptide increased hair follicle formation by two-fold.

[0122] We next investigated whether TNS peptides could increase human hair follicle formation in transplanted dermal-epidermal composite cultures. Human dermal papilla cells were isolated from temporal scalp dermis and expanded in vitro. Normal foreskin keratinocytes (NFK) were isolated from neonatal foreskins (collected from 5–7 donors) and cultured in serum-free medium supplemented with EGF and bovine pituitary extract. Dermal-epidermal composites (DECs) were prepared with human dermal papilla cells and NFK and transplanted into nude mice. Imaging was performed at 4, 6, 8, 10, and 12 weeks, and hHLA staining was performed to evaluate hair follicle anatomy (Figure 3). TSN peptides increased hair follicle generation (Figure 3) and also increased epidermal proliferation in vivo (Figure 4).

[0123] [method]

[0124] Cell viability assay: Keratinocytes and dermal papilla cells were seeded in six replicates in 96-well plates at 4000 and 2000 cells / well, respectively, and cultured overnight. The medium was changed and the appropriate treatment (vehicle, scrambled peptide, or active peptide) was added to the wells. The medium was replaced with fresh medium containing peptide 2 days after the first treatment, for a total culture period of 4 days. Cell viability was then measured using the CellTiter96™ non-radioactive cell proliferation assay (MTT) (Promega Corporation). Briefly, 15 μL of dye solution was added to each well and incubated at 37°C, 5% CO2 for 4 hours. Then, 100 μL of stop solution was added to the mixture, incubated for 1 hour, and absorbance was recorded at 570 nm using a plate reader.

[0125] Alkaline phosphatase activity: Keratinocytes and dermal papilla cells were seeded in six replicates in 96-well plates at 4000 and 2000 cells / well, respectively, and cultured overnight. The medium was changed and the appropriate treatment (vehicle, scrambled peptide, or active peptide) was added to the wells. The medium was replaced with fresh medium containing peptide 2 days after the first treatment, for a total culture period of 4 days. Alkaline phosphatase activity was measured using the alkaline phosphatase yellow (pNPP) liquid substrate system for ELISA (Sigma Aldrich) according to the manufacturer's instructions.

[0126] [Preparation of spheroids]: Dermal papilla (DP) cells were trypsinized, neutralized, and counted. Cells were centrifuged and resuspended in 30 mL DP cell medium as 6.5x106 cells. 0.9 mL of collagen (3.5 mg / mL stock) was added to form 4285 cells / 20 μL in DP medium containing 0.1 mg / mL collagen. 20 μL of cell droplets (4000 cells / droplet) were pipetted onto the bottom of a 10 cm tissue culture dish lid. Approximately 8-10 mL of PBS was added to the bottom of the dish. The lid was placed on the dish and incubated at 37 °C for 48 h.

[0127] [Preparation of epidermal cell aggregates]: From 12 neonatal foreskins, the epidermis was separated from the dermis by overnight dispase incubation. The epidermis was then minced into a fine paste and incubated with trypsin (0.025% diluted in DPBS at 37 °C) for 20 min with regular agitation. The trypsin was then neutralized with trypsin neutralizing solution and the solution was gravity filtered. The filtrate was washed three times with 10 mL of DMEM. This was then centrifuged at 800 rpm for 5 min. The pelleted aggregates were resuspended in 16 mL of 4.5% Ficoll solution and 8 mL of the 4.5% Ficoll / aggregate suspension was carefully layered onto the 9% Ficoll solution. The tube was centrifuged at 800 rpm for 5 min. The supernatant was aspirated and the pellet was washed twice with 15 mL of DMEM. The final pellet was resuspended in 6 mL of DMEM / F12 (for 12 skins) and the aggregates were counted using a hemocytometer.

[0128] Patch assay: Epidermal aggregates were resuspended in DMEM / F12 at 750,000 aggregates / 750 μL (1000 aggregates / μL), and spheroids were resuspended in DMEM / F12 at 1400 spheroids / 140 μL (10 spheroids / μL). Ten million dermal cells were resuspended in 400 μL DMEM / F12 (25,000 cells / μL or 1.25E6 per 50 μL). Positive control (30 μL (30,000) aggregates, 120 μL (3 million mouse dermal cells), 30 μL DMEM / F12), no DP cell control (30 μL (30,000) aggregates, 120 μL (3 million mouse dermal cells), 30 μL DMEM / F12) Appropriate tubes were prepared for the following injections: scrambled peptide control (175 μL (175,000 cells) of epidermal aggregates, 70 μL (700 cells) of spheroids, 6.25 μL (4 mg / mL) of scrambled peptide), and active peptide (175 μL (175,000 cells) of epidermal aggregates, 70 μL (700 cells) of spheroids, 6.25 μL (4 mg / mL) of active peptide). The solutions were kept on ice until injection. Mature 6-10 week old nu / nu mice were anesthetized and the skin surface of the trunk was cleaned with a 70% alcohol wipe. Excess medium was removed from the cell mixture and the cell pellet was resuspended in 60-80 μL of DMEM / F12 per injection (approximately 150 μL for two injections). A syringe with a 25 gauge needle was inserted into the cell suspension, ensuring that air bubbles were behind the sample and at the bottom of the plunger, but not at the bottom of the plunger. The cells were injected in a manner that did not allow the cells to enter the dermis. Up to eight injections per mouse were possible. The cells were slowly injected deep into the dermis without introducing air bubbles or penetrating the entire dermis, allowing the cells to remain in the limited space within the dermis. The transplanted cells were allowed to develop for 2–4 weeks, as mouse dermal cells and mouse epidermal cells form hair follicles within 12 days after birth. In contrast, human epidermal cells and mouse dermal cells take approximately 17 days to form new hair follicles, and human dermal / mouse epidermal cells take 21–26 days. The transplantation sites were tattooed and labeled with a single needle prick on the peripheral side of the skin at the injection site. At the end of the incubation period, the mice were killed with CO2 anesthesia. The entire skin was harvested and fixed in 10% formalin. The number of hair follicles was counted per transplant from the mucosal side of the skin using a dissecting microscope.

[0129] [Dermal-epidermal complex and transplantation]: Human dermal papilla cells isolated from temporal scalp dermis (Promocell, Heidelberg, Germany) of a female donor (HDP47) were expanded in vitro according to the manufacturer's recommendations. Normal foreskin keratinocytes (NFK) were isolated from neonatal foreskin (pooled from 5–7 donors) and cultured in serum-free medium (Keratinocyte-SFM, Invitrogen) supplemented with EGF and bovine pituitary extract according to the manufacturer's recommendations. Dermal-epidermal complexes (DECs) were prepared with human dermal papilla cells and NFK and transplanted into nude mice. Briefly, dermal equivalents containing isolated human DP cells (passage number 8, 500,000 cells per complex) were suspended in 1 mg / ml rat tail collagen type 1 (BD Biosciences, MA) within a 24 mm insert collagen-coated permeable support (Costar, #3492). These dermal constructs were cultured in epidermalization medium for 3 days, after which 106 human NF-K and accompanying melanocytes (primary (P0), passage 1 (P1), passage 3 (P3), n=8 / group) were added on top. When primary keratinocytes are passaged without freezing, the melanocytes persist for 7 passages in culture. These DECs were cultured submerged in epidermalization medium, then placed at the air-liquid interface using keratinization medium and transplanted into 6-8 week-old NIH(S)-nu / nu female mice. The surgical site was covered with Vaseline gauze and secured with a bandage. The bandage was changed at 2 weeks and removed after 4 weeks. Photographs were taken at 4, 6, 8, 10, and 12 weeks. All animal experiments were performed in accordance with the guidelines of our institution's Animal Care and Use Committee.

[0130] Histology: H&E staining: Unstained slides were deparaffinized in xylene and hydrated in graded alcohols up to water. They were then placed in Carrazzi's hematoxylin, washed in tap water, and placed in 95% ethanol. From there they were placed in eosin-phloxine solution and dehydrated in graded alcohols up to xylene. After xylene, the stained slides were coverslipped using Permount as mounting medium.

[0131] [HLA staining]: Frozen section slides were air-dried for 20 min and fixed in cold acetone at -20°C for 3 min. Slides were then rinsed twice in PBS for 5 min each. Sections were outlined and incubated in blocking solution containing 10% normal goat serum (BTPBS) for 1 h at room temperature. Excess blocking serum was wiped off. Slides were then incubated with 100 μL of diluted specific primary antibody (HLA) or negative control overnight at 4°C in a humidified chamber. The next day, slides were allowed to warm to room temperature. Slides were rinsed three times in PBS for 5 min each, followed by incubation with ABC reagent for 30 min at room temperature. Slides were rinsed three times in PBS for 5 min each. Slides were incubated in alkaline phosphatase substrate solution for 30 min at room temperature, followed by rinsing with tap water. Slides were then incubated in hematoxylin for 5 min and rinsed under running water until the rinse water was colorless. Finally, slides were dehydrated using ethanol and xylene in the following order: 70% ethanol for 30 seconds, 95% ethanol for 30 seconds, 100% ethanol for 30 seconds, and xylene for 5 minutes. Slides were wiped to remove excess xylene, and permanent mounting medium was added and coverslipped.

[0132] Image analysis: Both H&E and HLA slides were scanned with a Zeiss Axioscan scanner and analyzed using Zen Lite (Zeiss) and ImageJ (NIH) software. Slides were scored by a blinded pathologist for the presence or absence of hair follicles, hair follicle density, and total hair follicle area.

[0133] [References Example 1]

[0134] 1. Kurt S Stenn, George Cotsarelis Bioengineering the hair follicle: fringe benefits of stem cell technology. Current Opinion in Biotechnology. Volume 16, Issue 5. 2005, 493-497.

[0135] 2. Marshall, Clement D et al. “Cutaneous Scarring: Basic Science, Current Treatments, and Future Directions.” Advances in wound care vol. 7,2 (2018): 29-45. doi:10.1089 / wound.2016.0696.

[0136] 3. Thangapazham RL, Klover P, Wang JA, Zheng Y, Devine A, Li S, Sperling L, Cotsarelis G, Darling TN. Dissociated human dermal papilla cells induce hair follicle neogenesis in grafted dermal-epidermal composites. J Invest Dermatol. 2014 Feb;134(2):538-540.

[0137] 4. Iacovelli, Nicola Alessandro et al. “Topical treatment of radiation-induced dermatitis: current issues and potential solutions.” Drugs in context vol. 9 2020-4-7. 12 Jun. 2020, doi:10.7573 / dic.2020-4-7.

[0138] 5. https: / / my.clevelandclinic.org / health / diseases / 21995-radiation-burns

[0139] 6. Kawamura, M., Yoshimura, M., Asada, H. et al. A scoring system predicting acute radiation dermatitis in patients with head and neck cancer treated with intensity-modulated radiotherapy. Radiat Oncol 14, 14 (2019). https: / / doi.org / 10.1186 / s13014-019-1215-2.

[0140] 7. Herman, I. (2019). Collagenase-derived peptides promote Tissue Regeneration and Wound Healing. (US 10,485,846 B2). United States Patent Office.

[0141] 8. Herman IM, Castellot JJ Jr. Regulation of vascular smooth muscle cell growth by endothelial-synthesized extracellular matrices. Arteriosclerosis. 1987 Sep-Oct;7(5):463-9.

[0142] 9. Kaur A., ​​et al. Functional Skin Grafts: Where Biomaterials Meet Stem Cells. Stem Cells International. 2019. 1286054.

[0143] [Example 2. TSN peptides in the repair of radiation and combined damage] In humans, radiation exposures as low as 2 Gy can cause moderate radiation dermatitis, but radiation exposures at levels above 20 Gy can cause radiation burns with skin oozing, ulceration, and sloughing.[1-3] Radiation exposures above 25 Gy often damage the muscle layers and bones beneath the affected skin, inhibiting normal repair processes.[2] More than 4 million people in the United States undergo radiation therapy each year for the treatment of cancer, and it is estimated that 95% of them will suffer some level of radiation burns or radiation dermatitis.[1] Most of these cases are mild, but as many as 20% may develop severe radiation dermatitis.[1] Treatment of radiation burns is complex, whether they occur alone or as part of multiorgan damage from accidental high-dose radiation exposure. Repair of severe radiation burns often requires sequential surgical excision and reconstructive surgery, and in severe cases, amputation.[1] Currently, there are no Food and Drug Administration-approved medications to prevent or mitigate radiation-induced skin damage.[1,2,4,5]

[0144] Radiation burns are pathophysiologically distinct from electrical and thermal burns [1,2,6-8]. Radiation, electrical and thermal burns all present with erythema, dry or moist desquamation, ulceration and necrosis [2,9,10]. However, severe radiation burns are characterized by the development of an unpredictable inflammatory cycle that expands the initial injury, leading to an expansion of the affected area in the superficial epidermis and deeper tissues, as well as delayed tissue necrosis and failure to repair [2,11]. Inflammation after exposure to ionizing radiation is known to arise from a variety of sources. Ionizing radiation directly induces acute degranulation of cutaneous mast cells [8,12]. In addition, radiation damage to many other cell types in the skin leads to the release of a wide variety of inflammatory factors and cytokines [1,13,14]. In the skin, dendritic cells, endothelial cells, mast cells, fibroblasts and T cells have been hypothesized to contribute to post-irradiation inflammation [1]. Damage to the endothelial barrier allowing vascular leakage has been demonstrated even after very low doses of radiation, allowing inflammatory cells to invade unhindered into underlying tissues and trigger further inflammatory responses.[15,16] Although direct DNA damage is usually considered to be the primary mechanism of radiation-induced injury, activation of inflammatory pathways can trigger new DNA damage cycles, expanding the area of ​​initial damage and triggering subsequent cascades of damage.[17,18]

[0145] Our research team documented the time course of radiation-induced changes in skin structure and cell density in C57BL / 6 mice exposed to 14–17 (0.77 Gy / min) doses of X-rays

[19] . Exposure to radiation activates various mechanisms that induce loss of cellular replicative capacity, including necrosis, necroptosis, apoptosis, autophagy, and accelerated senescence, also known as stress-induced premature senescence (SIPS) [20–24]. Results from our laboratory and others suggest that accelerated senescence may be the primary response of normal, untransformed, non-immortalized cells to ionizing radiation [20, 24, 25]. Excessive levels of cell death and / or senescence impair normal tissue function. Moreover, although senescent cells are technically still alive, they exhibit changes in their biological activity and interactions with surrounding tissues. For example, (1) abnormal expression of cell cycle regulatory proteins, (2) upregulation of antiapoptotic proteins that inhibit normal clearance, (3) abnormal expression of extracellular matrix proteins, and (4) strong expression of inflammatory cytokines and proteases [23,26,27]. This last feature is called the “senescent secretory phenotype” and makes senescent cells a potent source of persistent inflammation. A recent report demonstrated that in vivo inhibition of radiation-induced stem cell senescence in the submucosa using rapamycin attenuates radiation-induced mucositis after head and neck irradiation

[25] . This suggests a mechanistic link between senescence, inflammation, and loss of normal repair processes.

[0146] Failure to repair radiation-induced skin damage is believed to involve the loss of replicative capacity of adult stem and progenitor cells

[28] . Loss of functional adult skin stem cells (due to cell death or senescence) is also believed to play a key role in the failure of skin grafts after radiation burns. Supporting this hypothesis, the use of autologous mesenchymal stem cells derived from bone marrow as a substitute for adult stem cells has been utilized as a successful strategy to obtain skin grafts for severe radiation burns [29,30]. Furthermore, another report demonstrated that inhibiting radiation-induced keratinocyte stem cell senescence in the submucosa using rapamycin is sufficient to inhibit radiation-induced mucositis

[25] . This suggests that inhibiting stem cell senescence is sufficient to promote tissue repair. Failure to repair radiation skin damage is hypothesized to be due to the loss of adult stem and progenitor cells

[28] . Autologous mesenchymal stem cells derived from bone marrow have been utilized to improve the survival rate of skin grafts for severe radiation burns [29,30].

[0147] [Mouse model and methods]

[0148] Mice of the C57BL / 6 strain have been widely used to study radiation-induced skin damage

[31] . We determined that the minimal dose to induce radiation burns in C57BL / 6 mice that were not repaired within 45 days was 16 Gy

[19] . Mock-irradiated mice without wounds were used as controls for radiation dermatitis and were treated similarly to the irradiated mice. Wounded mice not irradiated were used as controls for combined injury.

[0149] Animals were irradiated with an RS2000 small animal irradiator. The RS2000 provides a cone-shaped radiation field that delivers radiation at distances of 12.39–40.50 cm from the X-ray source. For dosimetry, the following settings were used: 160 kVp, 25 mA, 90 s exposure time, and 0.3 mm Cu beam filter. The approximate HVL provided by the manufacturer was 0.62 mm Cu. The University of Wisconsin Medical Radiological Research Center (UW MRRC) provided eight acrylic mouse phantoms with three (1 × 1 × 1 mm) Harshaw Thermoluminescent Dosimeter (TLD)-100 microcubes (Thermo Electron Corp., Oakwood Village, OH) embedded in each phantom. The cylindrical phantoms had dimensions of 27 mm (D) × 65 mm (L) in diameter and were held in place by a 15 mm (D) × 27 mm (L) cylindrical insert with a 3 mm thick stand. A custom lead shield was placed on the floor of the irradiator, placing the mouse's chest approximately 43.75 cm from the X-ray source. Dose measurements were performed in duplicate to account for setup error and repeatability. TLDs were processed at the UW MRRC using national standards with an expanded uncertainty (k=2) of 5%. Dose rates were reported as absorbed dose rate to water (ADRW, Gy / min) for each aperture position 1-4 and were calculated as the average of two replicate measurements. The average ADRW across positions was 0.775 Gy / min, with a uniformity of 97%, and an expanded uncertainty of ±5.2% when accounting for the uncertainty of the TLD measurements.

[0150] Mice (12–14 weeks old) were anesthetized with inhalation anesthesia (isoflurane) and the hair on the dorsal thorax was clipped. Two days after clipping, mice used for combined injury were orally administered acetaminophen (150 mg / kg in 0.5 ml sterile saline). Animals were then anesthetized with injection anesthesia (ketamine 150 mg / kg / xylazine 18 mg / kg), placed in a Lucite jig, and exposed to 16.5 Gy of thoracic irradiation with an RS2000 X-ray irradiator as previously described

[19] . For combined injury, immediately after thoracic irradiation, while anesthesia was still ongoing, the area of ​​skin was scrubbed with betadine or chlorhexidine, followed by debridement with an alcohol pad. A 1 cm wound was created in the mid-dorsal thorax, located approximately in the center of the radiation field, using a 9 / 16-inch diameter punch biopsy tool. The subcutaneous fat muscle and epithelium are removed. The wound is left exposed to air. The wound size is approximately 15% of the total body skin surface.

[0151] TSN6 peptide (5 μg / day in 50 μl sterile saline) or hepatocyte growth factor (HGF; 3.3 μg / day in 50 μl sterile saline) or vehicle (sterile saline, 50 μl) were injected subcutaneously on days 2, 7, and 10 after irradiation. Injections were made into the irradiated area or caudally adjacent to the irradiated / wounded area.

[0152] Animals were monitored daily for 85 days after irradiation in both the radiation dermatitis and combined injury models. Radiation dermatitis was scored separately for alopecia, erythema, scaling, and ulceration. Scoring of erythema, scaling, and ulceration was as previously described. 0=none, 1=minimal, 2=mild, 3=moderate, 4=marked

[19] . All animals underwent hair clipping prior to irradiation and wounding, so the scoring of alopecia was determined as no hair regrowth in the irradiated area. 0=none, 1=minimal, 2=mild, 3=moderate, 4=marked. Animals were photographed for wound measurement and dermatitis scoring. Animals exhibiting radiation-induced dermatitis were subjected to dermal application of Silvadene cream (silver sulfadiazine), which is approved for human use. It has been found that Silvadene can prevent infection but does not significantly promote radiation dermatitis or wound repair. Body weight was measured as an indicator of overall health.

[0153] [result]

[0154] [Effect of TSN6 peptide on radiation dermatitis in a mouse model] Santyl™ ointment contains a mixture of clostridial collagenase and proteases and is used as an enzymatic wound healing agent [32,33]. Research by Dr. IM Herman has shown that various peptides with biological activity are released from extracellular matrix proteins digested with Santyl™ ointment [32,33]. The isolated peptides were demonstrated to induce cell migration, proliferation, and / or angiogenesis to various degrees [32,33]. TSN6 is a 19 amino acid peptide obtained from multimerin 1 by digesting the extracellular matrix with Santyl™

[32] .

[0155] Treatment of X-ray irradiated mice with TSN6 on days 2, 7, and 10 after irradiation reduced radiation-induced alopecia, scaling, erythema, and dermatitis compared to vehicle treatment (Figure 5A-D). The combined score shown in Figure 5E indicates a significant reduction in radiation skin effects. Hematoxylin and eosin (H&E) stained tissue sections show that radiation caused loss of underlying adipocytes and thickening of the overlying epidermis (Figure 6A,B). The adipocyte layer is replaced by fibrous tissue and inflammatory cells. Localized ulceration is observed along with loss of the epidermal layer. Treatment with TSN6 resulted in the preservation of the adipose layer and reduced thickening of the epidermis (Figure 6C). Furthermore, hair follicle restoration was observed within the irradiated area.

[0156] We investigated the effect of TSN6 on wound healing under radiation (referred to as "combined injury"). Wounds within the radiation field usually show delayed or impaired repair capacity. Wound closure in the absence of radiation was found to be complete within approximately 14 days (Figure 7A). In contrast, radiation delayed complete wound closure until approximately 30 days after irradiation. TSN6 treatment increased the rate of wound closure under radiation until approximately 16 days after irradiation. In the combined injury model, hair loss, scaling, erythema, and ulceration were scored (Figure 7B-F). TSN6 treatment significantly reduced all features of radiation dermatitis, including the combined score. H&E stained tissue sections showed that radiation caused loss of underlying adipocytes accompanied by thickening of the overlying epidermis, which is consistent with the findings in the case of radiation alone (Figure 8A,B). Figures 13A-C show that radiation dermatitis in mice treated with TSN6 was significantly improved on days 57, 71, and 75 after irradiation. At day 79 after pot irradiation, vehicle-treated mice showed severe scaling, erythema, and ulceration and no hair regrowth, whereas TSN6-treated mice showed complete wound closure and hair regrowth (Figure 13C).

[0157] [Effect of hepatocyte growth factor on radiation dermatitis in a mouse model]Hepatocyte growth factor (HGF) is a pluripotency factor that induces cell proliferation, survival, migration, and morphogenesis depending on the recipient cell type

[34] . HGF is required for normal development, whereas in adults, HGF is required for normal tissue repair [35, 36]. HGF is a potent mitogen in human melanocytes

[37] and keratinocytes

[38] . Dermal fibroblasts are the main source of HGF in skin, whereas UV irradiation can induce HGF expression in keratinocytes

[39] . Similarly, maintaining the stability of proteases and inhibitors of the c-Met / HGF pathway is essential for optimal skin repair

[35] . In skin wound repair, HGF has been demonstrated to coordinate and promote re-epithelialization, angiogenesis, and granulation tissue formation [35, 40, 41]. In a study using a diabetic mouse model, Yoshida et al. demonstrated that administration of HGF promoted wound healing with less scarring

[36] and had a therapeutic effect on tissue fibrosis in tight-skin mice, a model of systemic sclerosis

[42] .

[0158] Although HGF has been shown to be effective in a variety of wound models, HGF did not improve any of the indices of radiation dermatitis (Figure 9). Hair loss, scaling, erythema, and ulceration were not significantly different between vehicle and HGF-treated animals. Histological sections showed some improvement in the preservation of the fat layer in HGF-treated animals, but this effect did not appear to be consistent within groups (Figure 10). Furthermore, HGF did not accelerate wound repair in the combined injury model (Figure 11A). In the combined injury model, HGF treatment did not improve any of the indices of radiation dermatitis and appeared to somewhat worsen the ulceration (Figure 11B-F). Histological sections of the combined injuries showed some improvement in the fat layer in some animals, but as with radiation alone, this effect was not consistent within groups (Figure 12).

[0159] Our data indicate that mitigation of radiation dermatitis and combined injury is a unique property of TSN6 peptide administration, and these biological activities are not recapitulated by HGF, a known adult tissue repair factor.

[0160] [Reference Example 2]

[0161] 1. Ryan, J.L., Ionizing radiation: the good, the bad, and the ugly. J Invest Dermatol, 2012. 132(3 Pt 2): p. 985 - 93.

[0162] 2. Benderitter, M., et al., New emerging concepts in the medical management of local radiation injury. Health Phys, 2010. 98(6): p. 851 - 7.

[0163] 3. Giordano, S., Radiation - induced skin injuries during interventional radiography procedures. J Radiol Nurs, 2010. 29: p. 37 - 47.

[0164] 4. Hoashi, T., et al., A case of acute radiation syndrome from the dermatological aspect. Br J Dermatol, 2008. 158(3): p. 597 - 602.

[0165] 5. Ryan Wolf, J., et al., Utility of topical agents for radiation dermatitis and pain: a randomized clinical trial. Support Care Cancer, 2020. 28(7): p. 3303 - 3311.

[0166] 6. Hopewell, J.W., The skin: its structure and response to ionizing radiation. Int J Radiat Biol, 1990. 57(4): p. 751-73.

[0167] 7. Rifkin, L.H., et al., An athymic rat model of cutaneous radiation injury designed to study human tissue-based wound therapy. Radiat Oncol, 2012. 7: p. 68.

[0168] 8. Albrecht, M., et al., Ionizing radiation induces degranulation of human mast cells and release of tryptase. Int J Radiat Biol, 2007. 83(8): p. 535-41.

[0169] 9. Li, Y., et al., Successful Treatment of a Case of Extensive Radiation Burns With Multiple Organ Dysfunction Syndrome. J Burn Care Res, 2013. 34: p. e014-9.

[0170] 10. Koenig, T.R., F.A. Mettler, and L.K. Wagner, Skin injuries from fluoroscopically guided procedures: part 2, review of 73 cases and recommendations for minimizing dose delivered to patient. AJR Am J Roentgenol, 2001. 177(1): p. 13-20.

[0171] 11. Simard, P.F., R.M. Bolton, and N.J. Tarbell, Anti-inflammatory cream reduces skin damage induced by ionizing radiation. Oncologist, 2009. 14(2): p. 197-8.

[0172] 12. Rabenhorst, A., et al., Mast cells play a protumorigenic role in primary cutaneous lymphoma. Blood, 2012.

[0173] 13. Kuilman, T. and D.S. Peeper, Senescence-messaging secretome: SMS-ing cellular stress. Nat Rev Cancer, 2009. 9(2): p. 81-94.

[0174] 14. Liang, L., et al., Celecoxib reduces skin damage after radiation: selective reduction of chemokine and receptor mRNA expression in irradiated skin but not in irradiated mammary tumor. Am J Clin Oncol, 2003. 26(4): p. S114-21.

[0175] 15. Griem, M.L., A. Robotewskyj, and R.H. Nagel, Potential vascular damage from radiation in the space environment. Adv Space Res, 1994. 14(10): p. 555-63.

[0176] 16. Frazier, T.H., et al., Fluoroscopy-induced chronic radiation skin injury: a disease perhaps often overlooked. Arch Dermatol, 2007. 143(5): p. 637-40.

[0177] 17. Hill, R.P., et al., Investigations into the role of inflammation in normal tissue response to irradiation. Radiother Oncol, 2011. 101(1): p. 73-9.

[0178] 18. Calveley, V.L., et al., Partial volume rat lung irradiation: temporal fluctuations of in-field and out-of-field DNA damage and inflammatory cytokines following irradiation. Int J Radiat Biol, 2005. 81(12): p. 887-99.

[0179] 19. McCart, E.A., et al., Accelerated senescence in skin in a murine model of radiation-induced multi-organ injury. J Radiat Res, 2017. 58(5): p. 636-646.

[0180] 20. Panganiban, R.A. and R.M. Day, Inhibition of IGF-1R prevents ionizing radiation-induced primary endothelial cell senescence. PLoS One, 2013. 8(10): p. e78589.

[0181] 21. Panganiban, R.A., A.L. Snow, and R.M. Day, Mechanisms of radiation toxicity in transformed and non-transformed cells. Int J Mol Sci, 2013. 14(8): p. 15931-58.

[0182] 22. Kaliberov, S.A. and D.J. Buchsbaum, Chapter seven--Cancer treatment with gene therapy and radiation therapy. Adv Cancer Res, 2012. 115: p. 221-63.

[0183] 23. Suzuki, M. and D.A. Boothman, Stress-induced premature senescence (SIPS)--influence of SIPS on radiotherapy. J Radiat Res, 2008. 49(2): p. 105-12.

[0184] 24. Panganiban, R.A., O. Mungunsukh, and R.M. Day, X-irradiation induces ER stress, apoptosis, and senescence in pulmonary artery endothelial cells. Int J Radiat Biol, 2013. 89(8): p. 656-67.

[0185] 25. Iglesias-Bartolome, R., et al., mTOR Inhibition Prevents Epithelial Stem Cell Senescence and Protects from Radiation-Induced Mucositis. Cell Stem Cell, 2012. 11(3): p. 401-14.

[0186] 26. Muller, M., Cellular senescence: molecular mechanisms, in vivo significance, and redox considerations. Antioxid Redox Signal, 2009. 11(1): p. 59-98.

[0187] 27. Tchkonia, T., et al., Cellular senescence and the senescent secretory phenotype: therapeutic opportunities. J Clin Invest, 2013. 123(3): p. 966-72.

[0188] 28. Ahmed, E.A., et al., Persistent DNA damage after high dose in vivo gamma exposure of minipig skin. PLoS One, 2012. 7(6): p. e39521.

[0189] 29. Bey, E., et al., Emerging therapy for improving wound repair of severe radiation burns using local bone marrow-derived stem cell administrations. Wound Repair Regen, 2010. 18(1): p. 50-8.

[0190] 30. Agay, D., et al., Multipotent mesenchymal stem cell grafting to treat cutaneous radiation syndrome: development of a new minipig model. Exp Hematol, 2010. 38(10): p. 945-56.

[0191] 31. Haston, C.K., Mouse genetic approaches applied to the normal tissue radiation response. Front Oncol, 2012. 2: p. 94.

[0192] 32. Sheets, A.R., et al., Identification and Characterization of Novel Matrix-Derived Bioactive Peptides: A Role for Collagenase from Santyl(R) Ointment in Post-Debridement Wound Healing? PLoS One, 2016. 11(7): p. e0159598.

[0193] 33. Sheets, A.R., et al., Matrix- and plasma-derived peptides promote tissue-specific injury responses and wound healing in diabetic swine. J Transl Med, 2016. 14(1): p. 197.

[0194] 34. Rubin, J.S., D.P. Bottaro, and S.A. Aaronson, Hepatocyte growth factor / scatter factor and its receptor, the c-met proto-oncogene product. Biochim Biophys Acta, 1993. 1155(3): p. 357-71.

[0195] 35. Buchstein, N., et al., Alternative proteolytic processing of hepatocyte growth factor during wound repair. Am J Pathol, 2009. 174(6): p. 2116-28.

[0196] 36. Yoshida, S., et al., Recombinant hepatocyte growth factor accelerates cutaneous wound healing in a diabetic mouse model. Growth Factors, 2004. 22(2): p. 111-9.

[0197] 37. Matsumoto, K., H. Tajima, and T. Nakamura, Hepatocyte growth factor is a potent stimulator of human melanocyte DNA synthesis and growth. Biochem Biophys Res Commun, 1991. 176(1): p. 45-51.

[0198] 38. Matsumoto, K., et al., Marked stimulation of growth and motility of human keratinocytes by hepatocyte growth factor. Exp Cell Res, 1991. 196(1): p. 114-20.

[0199] 39. Mildner, M., et al., Hepatocyte growth factor establishes autocrine and paracrine feedback loops for the protection of skin cells after UV irradiation. J Invest Dermatol, 2007. 127(11): p. 2637-44.

[0200] 40. Toyoda, M., et al., Overexpression of hepatocyte growth factor / scatter factor promotes vascularization and granulation tissue formation in vivo. FEBS Lett, 2001. 509(1): p. 95-100.

[0201] 41. Bevan, D., et al., Diverse and potent activities of HGF / SF in skin wound repair. J Pathol, 2004. 203(3): p. 831-8.

[0202] 42. Iwasaki, T., et al., Hepatocyte growth factor ameliorates dermal sclerosis in the tight-skin mouse model of scleroderma. Arthritis Res Ther, 2006. 8(6): p. R161

[0203] [Example 3. Use of next-generation bioactive peptides in skin substitutes to promote wound healing and skin regeneration] Introduction: Acute and chronic wounds affect more than 6 million people annually in the United States alone, and full-thickness wound repair and management has been a long-standing challenge. Tissue engineered skin substitutes are being used in the clinic for wound treatment. Dermal-epidermal complexes (DECs), one type of skin substitute, also known as skin equivalents or bilayered living skin constructs, consist of dermal fibroblasts embedded in a matrix such as collagen and covered by keratinocytes. DECs promote wound healing and have been used to model skin development and disease. Potential limitations of skin substitutes include delayed vascularization, contractures, loss of sensation, abnormal pigmentation, and lack of hair follicles (HFs) and sebaceous glands, which can lead to impaired healing and scar formation. The overall goal of our skin research program is to advance our understanding of wound healing and skin regeneration and to develop products that improve skin function and promote scar-free healing. Our quest to develop innovative, advanced therapeutics to treat wounds, accelerate healing, and improve graft outcomes led us to investigate next-generation, protease-resistant, pro-healing peptides that have previously been demonstrated to stimulate granulation tissue formation, neovascularization, and wound re-epithelialization after injury. These bioactive peptides were identified by Dr. Herman at Tufts University as products of cleavage from native human extracellular matrix proteins by agents used to debride necrotic tissue at chronic wound sites and at sites of severe burns. Previous studies demonstrated that TSN18, a peptide generated from the non-helical domain of collagen VI, promotes wound re-epithelialization and closure in vitro and in BALB / c mice. Given these results and the growing evidence that collagen VI has important functions in the stem cell niche and is regulated by cutaneous wounds, we investigated whether TSN18 would promote skin cell survival and improve the durability of grafted DECs in vitro and in vivo.

[0204] Materials and Methods: Cultured dermal papilla (DP) cells, specialized skin fibroblasts isolated from the base of hair follicles, were treated with TSN18 for 4 days. Cell viability was measured using the CellTiter96 non-radioactive cell proliferation assay (MTT) kit. Alkaline phosphatase activity, a marker of hair growth, was measured using the alkaline phosphatase yellow for ELISA (pNPP) liquid substrate system. A pilot study was conducted to determine whether incorporating TSN18 active peptide into DECs would improve graft viability or size. To construct DECs, human DP cells suspended in 1 mg / mL rat tail type I collagen were plated onto inserts placed in 6-well plates and incubated in medium containing TSN18 active peptide or scrambled peptide for 3 days. Human neonatal foreskin keratinocytes were then layered on top and the medium was changed to Epi medium containing TSN18 active or scrambled peptide for 2 days. Fresh Cori medium was added to the bottom to lift the DECs to the air-liquid interface for another 2 days. Cr:NIH(S)-nu / nu mice were grafted by placing DECs into the skin wound, and the mice were bandaged to protect the grafts. Bandages were changed at 2 weeks and removed after 4 weeks, and the grafts were monitored for 10 weeks. As is typical in these experiments, the size of the grafts gradually decreased over time. At the end of the experiment, the histological appearance of the grafts was evaluated to confirm the presence of human stratified squamous epithelium and to assess the presence or absence of human cells by staining for human leukocyte antigen. Graft size was measured by tracing the graft and determining the area using ImageJ (NIH) software.

[0205] Results: TSN18 did not affect cell viability or alkaline phosphatase activity of DP cells in vitro. Transplantation experiments showed that 4 of 5 mice transplanted with DECs without peptide or with TSN18 active peptide retained their grafts up to 10 weeks, whereas 3 of 5 mice transplanted with DECs containing scrambled peptide retained their grafts at 10 weeks (Figure 14). The mean graft sizes, determined using ImageJ (NIH) software, were 15.8+5.5mm2, 16.4+5.4mm2, and 10.9+8.4mm2 in the vehicle, active peptide, and scrambled peptide groups, respectively.

[0206] DISCUSSION:Our data suggest that safe incorporation of bioactive peptides into skin substitutes can promote graft durability and graft size, supporting their use in the development of next-generation skin substitutes to accelerate wound healing.

[0207] [Example 4. Introducing bioactive peptides into bioprinted dermal-epidermal composites to improve skin regeneration] Introduction: Combatants who suffer traumatic blast injuries often suffer from full-thickness skin injuries, resulting in physical, mental, and emotional distress to combatants and their families during and after recovery. Current methods to promote skin repair include the use of autologous skin grafts, which require transplanting existing donor tissue to the wound site. Split-thickness grafts are commonly used but lack the components within the dermal layer necessary to induce hair regrowth and restore more normal skin function. Our goal is to bioengineer a dermal-epidermal complex (DEC) to promote full regeneration of skin without scarring.

[0208] To enhance the proliferative and regenerative phases of wound healing, DECs composed of a collagen matrix embedded with fibroblasts and overlaid with human keratinocytes have been developed. Our laboratory has demonstrated human hair follicle neogenesis in DECs by transplanting adult human dermal papilla cells overlaid with human keratinocytes into immunodeficient mice. Advances in this technology include 1) constructing DECs using 3D bioprinting and 2) incorporating dermal papilla spheroids into collagen. Dermal papilla cells lose their ability to induce hair follicles when grown in monolayer, and their aggregation into spheroids restores their ability to generate hair. In the next phase, we plan to combine these advances with the incorporation of protease-resistant pro-healing peptides developed by Dr. Ira Herman at Tufts University. These peptides have previously been demonstrated to significantly stimulate granulation tissue formation, neovascularization, and wound re-epithelialization after injury, and are expected to promote skin regeneration in DECs. We hypothesized that the bioactive peptides used to form dermal papilla cell spheroids would promote hair follicle formation in bioprinted DECs.

[0209] Materials and Methods: We tested the compatibility of 1, 10, and 50 μM concentrations of active or scrambled peptides with human dermal papilla cells suspended in medium supplemented with 0.1 mg / mL rat tail type 1 collagen during spheroid formation using the hanging drop method. Six DECs were constructed with scrambled peptide, seven with active peptide, and six with medium without peptide. Briefly, human dermal papilla cell spheroids were formed by the hanging drop method in the presence of 10 μM active peptide, scrambled peptide, or medium. Spheroids were added to rat type 1 collagen and bioprinted into transwell inserts of 6-well plates using a CellInk BioX printer. DECs were immersed in wells containing dermal papilla cell medium for 2 days. 1,000,000 human keratinocytes were bioprinted directly onto the DECs and cultured in PRIME AIRLIFT medium for 2 days, after which the DECs were floated at the air-liquid interface for an additional 2 days. One DEC containing the active peptide was constructed with the same components by manual pipetting rather than bioprinting and implanted into a mouse.

[0210] Results: The microscopic appearance of spheroids was maintained in hanging drops when scrambled or active peptide was present at all concentrations. DECs containing 10 μM active or scrambled peptide remained intact after bioprinting, similar to vehicle. Bioprinted DECs were evaluated using an inverted light microscope, showing the formation of a uniformly dense epithelial sheet covering the scattered spheroids within the DEC. Spheroids in DECs maintained their microscopic appearance before and after the addition of keratinocytes. Pipetted DECs containing active peptide were implanted into mice. Histological results showed a thickened epidermal area of ​​human keratinocytes compared to the rest of the mouse epidermis.

[0211] Discussion: Human dermal papilla spheroids formed in the presence of bioactive peptides were bioprinted onto DECs. The microscopic appearance of the spheroids does not appear to be affected by the bioprinting process or the inclusion of peptides. DECs created with a manual pipette and implanted into mice showed graft engraftment and the presence of human cells 4 weeks after implantation. Our plan is to further optimize DECs containing bioactive peptides that can be bioprinted and implanted into mice as an in vivo model of skin regeneration and hair follicle growth. The goal is to further develop DECs that improve wound healing and regeneration of normal skin architecture that can transition from bench to warfighter.

[0212] [Example 5. Development of measures against skin radiation damage and complex damage] Introduction: The Department of Defense (DOD) is prepared to perform military missions following radiological accidents, manage radiological crises associated with terrorist activities, and respond in the event of a nuclear weapon detonation. For example, the U.S. military was one of the first agencies to provide assistance to assist Japan in disaster relief following the 2011 Fukushima nuclear power plant disaster (Operation Tomodachi). Recent studies have shown that military personnel are at increased risk of exposure to ionizing radiation due to increased terrorist activities, such as the use of stolen nuclear weapons, the detonation of improvised nuclear devices (INDs), or large-scale radiological dispersal devices (RDDs). Cutaneous radiation injury (CRI) is a significant cause of morbidity following radiation exposure and is a major concern in nuclear accidents and radiological combat events. Furthermore, studies estimate that in mass-casualty and combat scenarios due to radiation, 65-70% of an individual's radiation exposure will combine with other injuries, including wounds, resulting in a condition called complex injury (CI). Failure to repair CRI and CI has been linked to the development of unpredictable inflammatory cycles, loss of adult skin stem cells, tissue necrosis, and fibrotic remodeling. Unlike burns, skin grafting after CRI is only effective with the concomitant administration of autologous stem cells, making it an unsuitable treatment for mass casualty events. Currently, there are four radiation countermeasures approved by the U.S. Food and Drug Administration for the mitigation of acute radiation syndrome, all of which address the loss of mature white blood cells after high-dose radiation exposure. Agents suitable for mitigating CRI or CI in mass casualty and combat scenarios are needed. To be effective in mass casualty and military combat situations, such agents must be stable, easy to administer, and effective when administered at least 48 hours after injury and radiation exposure. We hypothesized that wound-healing peptides, identified by researchers at Tufts University as protease-resistant fragments of extracellular matrix proteins, are a new class of agents to test for mitigating CRI. Here, we describe the development of robust preclinical mouse models of CRI and CI and promising preliminary results using bioactive peptides as radiation countermeasures.

[0213] Materials and Methods: We used the C57BL / 6 mouse strain, which exhibits both radiation-induced inflammation and fibrotic remodeling, similar to human responses. Mice were exposed to chest X-ray irradiation without an excision wound to induce CRI and were given an experimental excision wound within the irradiated area to induce CI. Animals were scored by an investigator blinded to the treatment groups. Photographic documentation was performed for each experiment. Wound healing rates were assessed from day 1 to day 30 post-irradiation, and dermatitis severity was scored using four variable measures (erythema, scaling, alopecia, and ulceration) in mice from day 14 post-irradiation (dpi) until necropsy. Changes in specific cells and protein expression were determined using Western blotting and immunohistochemistry (IHC). To evaluate the efficacy of bioactive peptides, CI mice were treated with active peptides or scrambled peptides (negative control) by subcutaneous injection near the wound site on days 2, 7, and 10 post-irradiation. Additionally, vehicle control CI animals were injected with the same volume of saline (vehicle for peptides) on the same days.

[0214] Results: In the CRI model, irradiation with 14 Gy (0.77 Gy / min) resulted in mild inflammation without histologically observed alopecia or erythema. Irradiation with 16 or 17 Gy (0.77 Gy / min) resulted in dry desquamation, erythema, and mild ulceration detectable within 14 dpi. Histological evaluation revealed inflammation with mast cell infiltration within 14 dpi at higher doses. Scar formation, i.e., fibrosis, occurred 80 days after 16 or 17 Gy (0.77 Gy / min) and was characterized by collagen deposition, mast cell and neutrophilic dermatitis, and necrotic debris. Using Western blotting and IHC, we identified accelerated senescence at 7 dpi and apoptosis at 30 dpi, biological events preceding inflammation and fibrosis. Importantly, senescence was accelerated in cells at the base of the hair follicle bulb, where the adult stem cells of the skin reside. In the CI model, the presence of radiation increased the time required for wound closure by 2-fold. IHC also confirmed accelerated senescence of keratinocyte and adult stem cell populations in the irradiated field, similar to the CRI model. Furthermore, collagen deposition was significantly altered in CI compared to non-irradiated wounds. A pilot study with 5 mice per group showed that injection of the active peptide, but not the scrambled peptide, reduced the severity of the total radiation dermatitis score in mouse skin by nearly 50% (Figure 15).

[0215] DISCUSSION:Our data show that the C57BL / 6 mouse model of CRI and CI recapitulates many features observed in humans after high-dose radiation injury to the skin, including loss of adult stem cells, activation of several types of inflammatory cells, and excessive fibrotic remodeling. Furthermore, our data show that the combination of radiation in the presence of a wound results in delayed wound healing, as observed in humans with these injuries. Importantly, adult stem cells in the skin are a necessary cell population for normal tissue regeneration, and senescence of this population may underlie repair failure after radiation exposure. Our data also show that Tufts bioactive peptides, when administered over a delayed time course, reduce the development of radiation-induced dermatitis. Thus, these peptides are promising new radiation countermeasures that can be administered after combat or mass casualty events.

[0216] [Incorporated by reference] All publications and patents mentioned in this specification are incorporated herein by reference in their entirety as if each individual publication or patent was specifically and individually indicated to be incorporated by reference. In the case of conflict, the present application, including the definitions herein, will control.

[0217] [equivalent] Although certain embodiments of the present invention are explicitly disclosed herein, the above specification is illustrative and not limiting. Many variations of the present invention will become apparent to those skilled in the art upon review of this specification and the following claims. The full scope of the present invention should be determined by reference to the claims, their full scope of equivalents, and the specification and such variations.

Claims

1. A peptide for treating alopecia, or for stimulating the formation, maintenance, or function of hair follicles or other skin appendages, or for use in combination with radiotherapy and / or chemotherapy, The peptide is a peptide comprising one amino acid sequence from SEQ ID NO: 6, or SEQ ID NOs: 1-5, 7-19.

2. The peptide according to claim 1, wherein the peptide comprises an amino acid sequence of any one of Sequence IDs 1 to 19 or any combination thereof.

3. The peptide is either 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids long, or within a length limited by any of 8, 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acids, or The peptide contains 50, 45, 40, 35, 30, 25, or 20 or fewer consecutive amino acid residues of the protein from which the peptide is derived, or The peptide according to claim 1, wherein the peptide comprises a combinatorial peptide containing one or more sequence numbers 1 to 19 fused to a second peptide or polypeptide.

4. The peptide according to claim 1, wherein the peptide is combined with a carrier or excipient or a transdermal delivery system, and optionally, the peptide is combined with a hair follicle unit or substrate before administering the peptide to the subject.

5. The peptide is administered locally to the treatment site, and optionally, the peptide may be administered extracorporeally to cells, substrates, or artificial skin before being administered to the patient. An effective amount of the peptide is administered locally, or An effective amount of the peptide is administered subcutaneously, or An effective amount of the peptide is administered intradermally, or An effective amount of the peptide is administered to achieve a concentration of at least approximately 0.1, 1, 10, 100, or 1000 μM at the administration site, or The peptide according to claim 1, wherein the peptide is administered locally to the site where radiotherapy is administered.

6. The peptide according to claim 1, wherein the peptide is effective in promoting the regeneration or preservation of hair follicles or improving hair follicle function.

7. The aforementioned alopecia is caused by the loss of hair follicles, or The aforementioned hair loss is caused by male pattern baldness, or The aforementioned alopecia is caused by trauma, and optionally by physical trauma, thermal trauma, chemical trauma, or radiological trauma, or other forms of trauma, and optionally by complete or partial loss of skin and / or a decrease in skin thickness, The aforementioned alopecia is alopecia caused by burns, or The aforementioned alopecia is caused by radiation therapy or chemotherapy, and the peptide is as described in claim 1.

8. For treating scarring alopecia, which is optionally selected from central centrifugal scarring alopecia, lichen planus pilaris, keloid acne of the nape of the neck, dissociative cellulitis, traction alopecia, and pseudoalopecia blockis, or The peptide according to claim 1 for treating non-scarring alopecia, optionally selected from alopecia areata, anagen alopecia, and telogen alopecia.

9. The aforementioned peptide is effective in treating dermatitis, or The peptide is effective in mitigating the aging of skin stem cells, or The peptide according to claim 1, wherein the peptide is effective in the regeneration or preservation of hair follicles or in promoting hair follicle function.

10. The peptide according to claim 1, wherein the subject has cancer, or the subject is undergoing radiation therapy to treat cancer.

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