Histatin combinations and methods for treating or inhibiting cell loss - Patents.com

Abstract

The present disclosure provides methods and compositions for reducing or preventing loss of keratocytes, keratinocytes, and / or keratoblasts, restoring keratocytes, keratinocytes, and / or keratoblasts, inhibiting apoptosis to prevent cell loss in the eye or skin caused by surgical procedures, injury, or disease, preventing or minimizing refractive regression and subepithelial scarring, treating corneal wounds, treating skin or ophthalmic diseases, disorders, and / or conditions, or storing biological samples.

Description

[Technical field]

[0001] FIELD OF THEINVENTION The present disclosure relates to histatins and analogs thereof, optionally in combination with ciliary neurotrophic factor (CNTF) peptides and analogs thereof, for reducing or preventing loss of keratocytes, keratinocytes, and / or keratoblasts, restoring keratocytes, keratinocytes, and / or keratoblasts, inhibiting apoptosis to prevent cell loss in the eye or skin caused by surgical procedures, injury, or disease, preventing or minimizing refractive regression and subepithelial scarring, treating corneal wounds, treating skin or ophthalmic diseases, disorders, and / or conditions, or storing biological samples. [Background technology]

[0002] 2. Background of the Invention Histatins are naturally occurring oral and lacrimal peptides produced by humans and non-human primates that exhibit direct anti-infective activity, have potent anti-inflammatory properties, and stimulate epithelial wound healing in several tissue and organ culture systems. Techniques have been developed to isolate this natural substance, making it an available topical treatment for wounds.

[0003] Ciliary neurotrophic factor (CNTF) is a cytokine of the interleukin-6 family that is sequestered in adult glia and whose production increases during injury. It is essential in the maintenance of dopaminergic neurons of the substantia nigra and spinal motor neurons; when administered to mice with neuromuscular degeneration, it produces a neuroprotective effect.

[0004] Anti-apoptotic agents are compounds or peptides that inhibit cell death.

[0005] Injury to the cornea, from an abrasion, from a photorefractive or surgical procedure, or from disease, such as keratoconus, can result in loss of stromal keratocytes. Cell loss at this point is thought to be driven by the process of apoptosis. There is a need to develop treatments to prevent or reduce the loss of keratocytes, keratinocytes, and / or keratoblasts following surgery, injury, or disease. Summary of the Invention

[0006] BRIEF SUMMARY OF THE INVETION The present disclosure provides methods for reducing or preventing loss of keratocytes, keratinocytes, and / or keratoblasts, restoring keratocytes, keratinocytes, and / or keratoblasts, inhibiting apoptosis to prevent cell loss in the eye or skin caused by injury or disease, preventing or minimizing refractive regression and subepithelial scarring, recruiting keratocytes, or treating corneal wounds, comprising administering to the eye or skin of a subject in need thereof a therapeutically effective amount of a composition comprising a histatin or an analog thereof and / or a ciliary neurotrophic factor (CNTF) peptide or an analog thereof, and optionally an anti-apoptotic agent.

[0007] In some embodiments, the histatin is histatin-1 (SEQ ID NO:4).

[0008] In some embodiments, the CNTF peptide is peptide 6 (SEQ ID NO:34).

[0009] In some embodiments, the composition comprises a histatin or analog thereof at a concentration of about 0.1 μM to about 150 μM.

[0010] In some embodiments, the composition comprises a histatin or analog thereof at a concentration of about 80 μM.

[0011] In some embodiments, the composition comprises a CNTF peptide or analog thereof at a concentration of about 0.1 μM to about 150 μM.

[0012] In some embodiments, the composition comprises a CNTF peptide or analog thereof at a concentration of about 100 μM.

[0013] In some embodiments, about 1 μL to about 100 μL of the composition is administered to the eye or skin of a subject.

[0014] In some embodiments, about 35 μL of the composition is administered to the eye or skin of a subject.

[0015] In some embodiments, the composition is administered daily for about 1 day to about 180 days.

[0016] In some embodiments, the composition is administered daily for about 1 to about 28 days.

[0017] In some aspects, the composition is administered one, two, three, or four times per day.

[0018] In some aspects, the composition is administered three times per day.

[0019] In some aspects, the composition is administered as an eye drop, a gel, an ointment, or a tissue adhesive.

[0020] In some aspects, the composition is administered in an ocular insert or contact lens.

[0021] In some aspects, the subject is a mammal.

[0022] In some aspects, administration of the composition results in a reduction in loss of keratocytes, keratinocytes, and / or keratoblasts.

[0023] In some aspects, the loss of keratocytes, keratinocytes, and / or keratoblasts is caused by an ocular infection, a corneal injury, an ocular surgery, an ocular injury, an ocular infection, an ocular disease, or an ocular disorder.

[0024] In some aspects, the eye surgery is glaucoma surgery, photorefractive keratectomy (PRK) surgery, or a corneal refractive procedure.

[0025] In some aspects, the eye disease is Fuchs' corneal dystrophy.

[0026] In some aspects, the eye disorder is keratoconus.

[0027] In some aspects, the loss of keratocytes, keratinocytes, and / or keratoblasts is caused by skin photodamage.

[0028] In some aspects, the loss of keratocytes, keratinocytes, and / or keratoblasts is caused by chemotherapy.

[0029] The present disclosure also provides a method of reducing or preventing loss of keratocytes, keratinocytes, and / or keratoblasts, restoring keratocytes, keratinocytes, and / or keratoblasts, inhibiting apoptosis to prevent cell loss in the eye or skin caused by injury or disease, preventing or minimizing refractive regression and subepithelial scarring, recruiting keratocytes, keratinocytes, and / or keratoblasts, or treating corneal wounds, comprising administering to the eye or skin of a subject in need thereof a therapeutically effective amount of a composition comprising a histatin or an analog thereof and a second active agent.

[0030] The present disclosure also provides a method for therapeutically or prophylactically treating a skin or ophthalmic disease, disorder, or condition, comprising administering to the eye or skin of a subject in need thereof a therapeutically effective amount of a composition comprising a histatin or an analog thereof and / or a ciliary neurotrophic factor (CNTF) peptide or an analog thereof.

[0031] In some aspects, the composition further comprises an anti-apoptotic agent.

[0032] In some aspects, the skin disease, disorder, or condition is post-CO2 recovery, burns, decubitus ulcers, autonomic dysreflexia in spinal cord injury, diabetic ischemic ulcers, ulcer prevention, skin graft recovery, trauma resulting in skill loss, post-cryo / electrodesiccation recovery, or post-Mohs / skin cancer wound healing.

[0033] In some aspects, the ophthalmic disease, disorder, or condition is post-photorefractive keratectomy (PRK) surgery, glaucoma surgery, infectious corneal ulcer, pigmentary eye disease, post-collagen crosslinking for keratoconus (CSX), neurotrophic keratitis, Fuchs' corneal dystrophy, contact lens intolerance, dry eye disease, keratitis progression inhibition, posterior polymorphous corneal dystrophy (PPCD), aphakic or pseudophakic bullous keratopathy (ABK / PBK), endothelial dysfunction caused by penetrating or blunt trauma, congenital hereditary endothelial dystrophy (CHED), iridocorneal endothelial (ICE) syndrome, refractory glaucoma, previous failed corneal graft, or herpes simplex virus endotheliitis.

[0034] In some aspects, the ophthalmic disease, disorder, or condition is post-photorefractive keratectomy surgery, glaucoma surgery, or Fuchs corneal dystrophy.

[0035] In some aspects, the composition is administered by intraocular injection, subretinal injection, intravitreal injection, periocular administration, subconjunctival injection, suprachoroidal injection, retrobulbar injection, intracameral injection, or subtenon injection.

[0036] In some aspects, the composition is administered by topical administration.

[0037] The present disclosure also provides a method of storing a biological sample prior to a surgical procedure, comprising contacting the biological sample with a composition comprising a histatin or an analog thereof and / or a CNTF peptide or an analog thereof, and optionally an anti-apoptotic agent, and storing the biological sample at a temperature of about -10°C to about 25°C.

[0038] In some aspects, the biological sample is donor tissue.

[0039] In some aspects, the donor tissue is corneal tissue.

[0040] In some embodiments, the biological sample remains viable for between about 0.5 days and about 90 days.

[0041] In some aspects, the surgical procedure is photorefractive keratectomy (PRK) surgery, Descemet's membrane stripping automated endothelial keratoplasty (DSAEK), or Descemet's membrane endothelial keratoplasty (DMEK).

[0042] The present disclosure also provides a method for reducing or preventing corneal endothelial cell loss and / or restoring corneal endothelial cells, comprising administering to an eye of a subject in need thereof a therapeutically effective amount of a composition comprising a histatin or an analog thereof and, optionally, an anti-apoptotic drug.

[0043] In some embodiments, the anti-apoptotic agent is a Fas inhibitor. In some embodiments, the Fas inhibitor is MET12 (SEQ ID NO: 37). In some embodiments, the anti-apoptotic agent is a MET variant (SEQ ID NO: 41).

[0044] In some embodiments, administration of the composition results in about a 20% to about 80% reduction in corneal endothelial cell loss compared to no treatment.

[0045] In some aspects, the loss of corneal endothelial cells is caused by one or more of Fuchs endothelial dystrophy, posterior polymorphous corneal dystrophy, aphakic or pseudophakic bullous keratopathy, endothelial dysfunction caused by penetrating or blunt trauma, congenital hereditary endothelial dystrophy, iridocorneal endothelial syndrome, refractory glaucoma, a previous failed corneal graft, surgical trauma, or herpes simplex virus endotheliitis.

[0046] In some aspects, the loss of corneal endothelial cells is caused by Fuchs endothelial corneal dystrophy.

[0047] In some aspects, the composition is administered prior to a surgical procedure.

[0048] In some aspects, the composition is administered during a surgical procedure.

[0049] In some aspects, the composition is administered after a surgical procedure.

[0050] In some aspects, the composition is administered before and after the surgical procedure.

[0051] In some aspects, the compositions are administered before, during, and after a surgical procedure.

[0052] In some aspects, the surgical procedure is penetrating keratoplasty (PK), Descemet's membrane stripping automated endothelial keratoplasty (DSAEK), or Descemet's membrane endothelial keratoplasty (DMEK).

[0053] The present disclosure also provides a method for restoring, regenerating, or promoting the regeneration of corneal nerves, comprising administering to an eye of a subject in need thereof a therapeutically effective amount of a composition comprising a histatin or an analog thereof, and / or a ciliary neurotrophic factor (CNTF) peptide or an analog thereof, and optionally an anti-apoptotic drug.

[0054] In some aspects, administration of the composition results in about a 20% to about 80% reduction in corneal nerve loss and / or damage compared to no treatment.

[0055] In some aspects, the corneal nerve loss is caused by one or more of neurotrophic keratitis, dry eye disease, herpetic keratitis, leprosy, diabetes, keratoconjunctivitis sicca, or keratoconus.

[0056] In some aspects, the corneal nerve loss or damage is caused by a surgical procedure. [Brief description of the drawings]

[0057] [Figure 1A]FIG. 1A is a line graph showing corneal keratocyte counts in New Zealand White rabbits after surgical keratectomy at 0, 7, 10, 14, and 21 days with administration of phosphate buffered saline (PBS), histatin-1 (Hst-1), or a combination of histatin-1 and peptide 6 (P6) three times per day.

[0058] [Figure 1B] FIG. 1B is a line graph showing corneal keratocyte counts in New Zealand White rabbits after surgical keratectomy at 0, 7, 10, 14, 21, and 28 days with administration of phosphate buffered saline (PBS), histatin-1 (Hst-1), or a combination of histatin-1 and peptide 6 (P6) three times per day.

[0059] [Diagram 2] FIG. 2 is a series of in vivo confocal microscopy images of rabbit corneas following surgical keratectomy at 0 (baseline), 10, 14, and 21 days with administration of phosphate buffered saline (PBS) three times per day.

[0060] [Diagram 3] FIG. 3 is a series of in vivo confocal microscopy images of rabbit corneas after surgical keratectomy at 0 (baseline), 10, 14, and 21 days with administration of histatin-1 (Hst-1) three times per day.

[0061] [Figure 4] FIG. 4 is a series of in vivo confocal microscopy images of rabbit corneas after surgical keratectomy at 0 (baseline), 10, 14, and 21 days with administration of histatin-1 (Hst-1) and peptide 6 (P6) three times per day.

[0062] [Diagram 5]FIG. 5 is a line graph showing corneal endothelial cell density in New Zealand White rabbits after surgical anterior keratectomy at 0, 7, 10, 14, 21, and 28 days with topical phosphate buffered saline (PBS) or histatin-1 (Hst-1) administered three times per day.

[0063] [Figure 6] FIG. 6 is a bar graph showing corneal nerve regeneration in New Zealand White rabbits as measured by percent area of ​​the initial lesion 28 days after surgical anterior keratectomy with phosphate buffered saline (PBS), histatin-1 (Hst-1), or a combination of histatin-1 and peptide 6 (P6) administered three times per day.

[0064] [Figure 7] FIG. 7 is a bar graph showing the percent area of ​​corneal buttons stained with trypan blue (indicating tissue damage) between corneas stored in standard storage solution and corneas stored in standard storage solution plus histatin-1. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Detailed Description of the Invention definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. In case of conflict, the present application, including definitions, will control. Unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. All publications, patents, and other references mentioned herein are incorporated by reference in their entirety for all purposes as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.

[0066] Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The materials, methods, and examples are illustrative only and are not intended to be limiting. Other features and advantages of the present disclosure will be apparent from the detailed description and claims.

[0067] In order to further define this disclosure, the following terms and definitions are provided.

[0068] "Percent identity" refers to the degree of identity between two sequences (e.g., amino acid sequences or nucleic acid sequences). Percent identity (%) can be determined by aligning two sequences and introducing gaps to maximize identity between the sequences. Alignments can be generated using programs known in the art. For purposes herein, alignment of nucleotide sequences can be performed using the blastn program set with default parameters, and alignment of amino acid sequences can be performed using the blastp program set with default parameters (see National Center for Biotechnology Information (NCBI), ncbi.nlm.nih.gov on the World Wide Web).

[0069] The term "pharmaceutical formulation" refers to a preparation that is in a form that allows the biological activity of the active ingredient to be effective and does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation may be administered. The formulation can be sterile.

[0070] As used herein, the term "pharmaceutical acceptable" refers to those compounds, materials, compositions, formulations, and / or dosage forms that are within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, other problem or complication, commensurate with a reasonable benefit / risk ratio.

[0071] The term "pharmaceutically acceptable salts" is art recognized and includes the relatively non-toxic, inorganic and organic acid addition salts of compounds, and the relatively non-toxic, inorganic and organic base addition salts of compounds.

[0072] Inorganic acids that can be used to prepare pharmaceutically acceptable salts include, but are not limited to, hydrochloric acid, phosphoric acid, sulfuric acid, hydrobromic acid, hydroiodic acid, phosphorous acid, and the like. Organic acids that can be used to prepare pharmaceutically acceptable salts include, but are not limited to, aliphatic mono- and dicarboxylic acids, such as tartaric acid, oxalic acid, carbonic acid, citric acid, succinic acid, phenyl heteroatom-substituted alkanoic acids, aliphatic and aromatic sulfuric acids, and the like. Pharmaceutically acceptable salts prepared from inorganic or organic acids thus include, but are not limited to, hydrochloride, hydrobromide, nitrate, sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, hydroiodide, hydrofluoride, acetate, propionate, formate, oxalate, tartrate, citrate, lactate, p-toluenesulfate, methanesulfonate, and maleate salts. Suitable pharmaceutically acceptable salts can also be formed by reacting active agents with organic bases, such as methylamine, ethylamine, ethanolamine, lysine, ornithine, and the like. Pharmaceutically acceptable salts include salts formed between carboxylic acid or sulfonic acid groups, which can be found in some active agents, and inorganic cations, such as sodium, potassium, ammonium, or calcium, or organic cations, such as isopropylammonium, trimethylammonium, tetramethylammonium, and imidazolium. All of these salts can be prepared by conventional means from active agents, for example, by reacting the appropriate acid or base with the active agent.

[0073] The term "excipient" refers to any substance that is not itself a therapeutic agent, but may be used in a composition for delivery of an active therapeutic agent to a subject, or may be combined with an active therapeutic agent to improve its handling or storage characteristics, or to permit or facilitate the formation of a dosage unit of the composition (e.g., to make a pharmaceutical composition). Excipients include, but are not limited to, solvents, penetration enhancers, humectants, antioxidants, lubricants, emollients, substances added to improve the appearance or texture of the composition, and substances used to form hydrogels. Any such excipients may be used in any dosage form according to the present disclosure. The foregoing classes of excipients are not meant to be exhaustive, but merely illustrative, as one of ordinary skill in the art will recognize that additional types and combinations of excipients may be used to achieve the desired goal for delivery of a drug. Excipients can be inert, inactive, and / or pharma- ceutical inactive substances. Excipients can serve a variety of purposes. Those skilled in the art can select one or more excipients with respect to specific desired properties by routine experimentation and without any undue burden. The amount of each excipient used can vary within conventional ranges in the art. Techniques and excipients that can be used to formulate dosage forms are described in Handbook of Pharmaceutical Excipients, 6th edition, Rowe et al., Eds., American Pharmaceuticals Association and the Pharmaceutical Press, publications department of the Royal Pharmaceutical Society of Great Britain (2009); and Remington: the Science and Practice of Pharmacy, 21st edition, Gennaro, Ed., Lippincott Williams & Wilkins (2005).

[0074] As used herein, the term "unit dosage form" or "unit dose composition" refers to a device containing an amount of therapeutic compound such that one or more predetermined units can be provided as a single therapeutic administration.

[0075] The terms "administer," "administering," "administration," and the like, as used herein, refer to methods that can be used to enable delivery of a drug, such as a histatin, to a desired site of biological action (e.g., to the eye or skin). Administration techniques that can be used with the agents and methods described herein can be found, for example, in Goodman and Gilman, The Pharmacological Basis of Therapeutics, current edition, Pergamon; and Remington's, Pharmaceutical Sciences, current edition, Mack Publishing Co., Easton, Pa.

[0076] As used herein, the terms "subject" and "patient" are used interchangeably. A subject can be an animal. In some aspects, a subject is a mammal, such as a non-human animal (e.g., a cow, pig, horse, cat, dog, rat, mouse, monkey, or other primate, etc.). In some aspects, a subject is a human.

[0077] The term "therapeutically effective amount" refers to an amount of a therapeutic agent, such as a histatin, that is effective in treating a disease or disorder in a subject. In the case of eye damage or injury, a therapeutically effective amount of a therapeutic agent can mobilize keratocytes, prevent or reverse the loss of keratocytes caused by epithelial or stromal injury and disease, inhibit or prevent apoptosis to prevent and reverse the loss of cells in the eye and skin caused by injury or disease, and / or improve symptoms in patients with ophthalmic conditions or disorders, such as eye surgery or disease. A "prophylactically effective amount" refers to an amount that is effective to achieve a desired prophylactic result. Desired prophylactic results include prevention of the total amount of damage to the eye (including corneal damage, nerve damage, cell death, etc.) that would be expected to occur in a subject without administration of the composition of the present invention. Effective prophylactic treatment does not mean that the subject to whom the composition is administered will not have any negative outcomes of eye disease or disorder or damage after the surgical procedure, but will show some benefit in relatively healthy cell and / or neuronal mass (compared to untreated subjects) and / or the treated subject will have reduced symptoms compared to untreated subjects. A prophylactically effective amount applied to storage of a biological sample prior to a surgical procedure means that the biological sample can be effectively stored for a longer period prior to surgery than an untreated sample.

[0078] Terms such as "treating," "treatment," "to treat," "alleviating," and "for alleviate" refer to therapeutic measures that cure, slow, reduce symptoms, and / or halt the progression of a pathological condition or disorder. Thus, those in need of treatment include those already diagnosed with the disorder or those suspected of having the disorder. In certain embodiments, a subject is successfully "treated" for an ophthalmic and / or dermatological disease or disorder in accordance with the methods of the invention if the patient exhibits one or more of the following: keratocyte recruitment, prevention or reversal of stromal keratocyte loss, prevention and reversal of stromal keratocyte loss caused by epithelial or stromal injury and disease, inhibition or prevention of apoptosis, cell recovery in the eye and skin, and / or amelioration of one or more symptoms exhibited by the subject prior to treatment.

[0079] Prophylactic or preventative measures refer to measures that prevent and / or delay the onset of an ophthalmic and / or skin condition or disorder, or that alleviate symptoms associated with ophthalmic damage, for example, from eye surgery, injury, or disease. Thus, those in need of prophylactic or preventative measures include those prone to having an ophthalmic and / or skin condition or disorder, as well as those in whom an ophthalmic and / or skin condition or disorder is to be prevented, or subjects undergoing eye surgery.

[0080] As used herein, "pretreatment or prevention" means the substantial absence of loss of keratocytes, keratinocytes, and / or keratoblasts, or a reduction in loss of keratocytes, keratinocytes, and / or keratoblasts by at least 20%, preferably 50%, and more preferably 80%, when measured over the course of 3 months, 6 months, 9 months, or one year.

[0081] As used in this disclosure and the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise.

[0082] Whenever an aspect is described herein with the phrase "comprising," it is understood that otherwise similar aspects described in terms of "consisting of" and / or "consisting essentially of" are also provided. In this disclosure, "comprises," "comprising," "containing," and "having" and the like can have the meanings ascribed to them in U.S. patent law and can mean "includes," "including," and the like; "consisting essentially of" or "consists essentially" likewise have the meanings ascribed to them in U.S. patent law, and the term is open-ended, permitting the presence of more than those recited, so long as the basic or novel characteristics of the recited items are not altered by the presence of more than those recited, but excluding prior art embodiments.

[0083] Unless specifically stated or clear from the context, the term "or" is understood to be inclusive when used herein. The term "and / or" as used in phrases herein, such as "A and / or B", is intended to include both "A and B", "A or B", "A" and "B". Similarly, the term "and / or" as used in phrases, such as "A, B, and / or C", is intended to include each of the following embodiments: A, B, and C; A, B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (single); B (single); and C (single).

[0084] As used herein, the terms "about" and "approximately," when used to modify a numerical value or numerical range, indicate a deviation of 5% to 10% above and 5% to 10% below the value or range while remaining within the intended meaning of the recited value or range.

[0085] Any composition or method provided herein can be combined with one or more of any of the other compositions and methods provided herein.

[0086] explanation The present disclosure provides methods for reducing or preventing keratocyte loss, restoring keratocytes, inhibiting apoptosis to prevent cell loss in the eye or skin caused by surgical procedures, injury or disease, preventing or minimizing refractive regression and subepithelial scarring, mobilizing keratocytes, or treating corneal wounds, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising: (1) a histatin or analog thereof; (2) a histatin or analog thereof and a ciliary neurotrophic factor (CNTF) peptide or analog thereof; (3) a histatin or analog thereof and an anti-apoptotic agent; or (4) a histatin or analog thereof, a CNTF peptide or analog thereof, and an anti-apoptotic agent.

[0087] The present disclosure also provides a method for reducing or preventing keratinocyte loss, restoring keratinocytes, inhibiting apoptosis to prevent cell loss in the eye or skin caused by surgical procedures, injury or disease, preventing or minimizing refractive regression and subepithelial scarring, recruiting keratinocytes, or treating corneal wounds, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising: (1) a histatin or analog thereof; (2) a histatin or analog thereof and a ciliary neurotrophic factor (CNTF) peptide or analog thereof; (3) a histatin or analog thereof and an anti-apoptotic agent; or (4) a histatin or analog thereof, a CNTF peptide or analog thereof, and an anti-apoptotic agent.

[0088] The present disclosure provides methods for reducing or preventing corneal blast cell loss, restoring corneal blast cells, inhibiting apoptosis to prevent cell loss in the eye or skin caused by surgical procedures, injury or disease, preventing or minimizing refractive regression and subepithelial scarring, recruiting corneal blast cells, or treating corneal wounds, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising: (1) a histatin or analog thereof; (2) a histatin or analog thereof and a ciliary neurotrophic factor (CNTF) peptide or analog thereof; (3) a histatin or analog thereof and an anti-apoptotic agent; or (4) a histatin or analog thereof, a CNTF peptide or analog thereof, and an anti-apoptotic agent.

[0089] The present disclosure also provides a method of treating a skin or ophthalmic disease, disorder, and / or condition, comprising administering to a subject in need thereof a therapeutically effective amount of a composition comprising: (1) a histatin or analog thereof; (2) a histatin or analog thereof and a ciliary neurotrophic factor (CNTF) peptide or analog thereof; (3) a histatin or analog thereof and an anti-apoptotic agent; or (4) a histatin or analog thereof, a CNTF peptide or analog thereof, and an anti-apoptotic agent.

[0090] The present disclosure also provides a method of storing a biological sample prior to a surgical procedure, comprising contacting the biological sample with a composition comprising: (1) a histatin or analog thereof; (2) a histatin or analog thereof and a ciliary neurotrophic factor (CNTF) peptide or analog thereof; (3) a histatin or analog thereof and an anti-apoptotic agent; or (4) a histatin or analog thereof, a CNTF peptide or analog thereof, and an anti-apoptotic agent.

[0091] Without wishing to be bound by any particular theory, the histatin or analog thereof and the CNTF peptide or analog thereof, and optionally the anti-apoptotic agent, prevent the loss of keratocytes, keratinocytes, and / or keratoblasts in the subject through an anti-apoptotic mechanism. Other mechanisms that may explain this effect include the known effect of histatin-1 and the CNTF peptide or analog thereof, and optionally the anti-apoptotic agent as an inhibitor of inflammatory factors, such as matrix metalloproteinases, which degrade extracellular substances. The histatin or analog thereof and the CNTF peptide or analog thereof, and optionally the anti-apoptotic agent, provide a supportive environment in the cornea after injury. The histatin or analog thereof and the CNTF peptide or analog thereof, and optionally the anti-apoptotic agent, can also promote the generation of new keratocytes from limbal stem cells. The CNTF peptide or analog thereof, and optionally the anti-apoptotic agent, promote keratocyte recovery.

[0092] In some embodiments, the histatin or analog thereof is histatin-1, which is represented by SEQ ID NO:4 in Table 1 below. In some embodiments, the histatin or analog thereof is histatin-2, which is represented by SEQ ID NO:5 in Table 1 below. In some embodiments, the histatin or analog thereof is histatin-3, which is represented by SEQ ID NO:6 in Table 1 below. In some embodiments, the histatin or analog thereof is histatin-5, which is represented by SEQ ID NO:30 in Table 1 below. In some embodiments, the histatin or analog thereof is a peptide represented by SEQ ID NO:35.

[0093] In some embodiments, the CNTF peptide is peptide 6, which is represented by SEQ ID NO:34 in Table 1 below. In some embodiments, the CNTF peptide is peptide 6c, which is represented by SEQ ID NO:36.

[0094] In some embodiments, the composition comprises a histatin or analog thereof at a concentration of about 0.001 μM to about 100 mM. In some embodiments, the composition comprises from about 0.001 μM to about 0.01 μM, from about 0.001 μM to about 0.1 μM, from about 0.001 μM to about 1 μM, from about 0.001 μM to about 10 μM, from about 0.001 μM to about 100 μM, from about 0.001 μM to about 1 mM, from about 0.001 μM to about 100 mM, from about 0.01 μM to about 0.1 μM, from about 0.01 μM to about 1 μM, from about 0.01 μM to about 100 μM, from about 0.01 μM to about 1 mM, from about 0.01 μM to about 10 mM, from about 0.01 μM to about 100 mM, from about 0.1 μM to about 1 μM, from about 0.1 μM to about 10 μM, The histatin or analogue thereof is contained at a concentration of about 0.1 μM to about 100 μM, about 0.1 μM to about 1 mM, about 0.1 μM to about 10 mM, about 0.1 μM to about 100 mM, about 1 μM to about 10 μM, about 1 μM to about 100 μM, about 1 μM to about 1 mM, about 1 μM to about 10 mM, about 1 μM to about 100 mM, about 10 μM to about 100 μM, about 10 μM to about 1 mM, about 10 μM to about 10 mM, about 100 μM to about 10 mM, about 100 μM to about 10 mM, about 100 μM to about 100 mM, about 1 mM to about 10 mM, about 1 mM to about 100 mM, or about 10 mM to about 100 mM.

[0095] In some embodiments, the composition comprises a histatin or analogue thereof at a concentration of about 80 μM. In some embodiments, the composition comprises a histatin or analogue thereof at a concentration of about 0.001 μM, about 0.01 μM, about 0.1 μM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, about 10 mM, or about 100 mM.

[0096] In some embodiments, the composition comprises a CNTF peptide or analog thereof at a concentration of about 0.001 μM to about 100 mM. In some embodiments, the composition comprises a concentration of from about 0.001 μM to about 0.01 μM, from about 0.001 μM to about 0.1 μM, from about 0.001 μM to about 1 μM, from about 0.001 μM to about 10 μM, from about 0.001 μM to about 100 μM, from about 0.001 μM to about 1 mM, from about 0.001 μM to about 100 mM, from about 0.01 μM to about 0.1 μM, from about 0.01 μM to about 1 μM, from about 0.01 μM to about 100 μM, from about 0.01 μM to about 1 mM, from about 0.01 μM to about 10 mM, from about 0.01 μM to about 100 mM, from about 0.1 μM to about 1 μM, from about 0.1 μM to about 10 μM, about 100 μM, about 0.1 μM to about 1 mM, about 0.1 μM to about 10 mM, about 0.1 μM to about 100 mM, about 1 μM to about 10 μM, about 1 μM to about 100 μM, about 1 μM to about 1 mM, about 1 μM to about 10 mM, about 1 μM to about 100 mM, about 10 μM to about 100 μM, about 10 μM to about 1 mM, about 10 μM to about 10 mM, about 100 μM to about 1 mM, about 100 μM to about 10 mM, about 100 μM to about 100 mM, about 1 mM to about 10 mM, about 1 mM to about 100 mM, or about 10 mM to about 100 mM.

[0097] In some embodiments, the composition comprises a CNTF peptide or analog thereof at a concentration of about 100 μM. In some embodiments, the composition comprises a CNTF peptide or analog thereof at a concentration of about 0.001 μM, about 0.01 μM, about 0.1 μM, about 1 μM, about 10 μM, about 50 μM, about 500 μM, about 1 mM, about 10 mM, or about 100 mM.

[0098] In some aspects, the composition comprises a therapeutically effective amount of an anti-apoptotic agent.

[0099] In some embodiments, the composition comprises an anti-apoptotic agent at a concentration of about 0.001 μM to about 100 mM. In some embodiments, the composition comprises from about 0.001 μM to about 0.01 μM, from about 0.001 μM to about 0.1 μM, from about 0.001 μM to about 1 μM, from about 0.001 μM to about 10 μM, from about 0.001 μM to about 100 μM, from about 0.001 μM to about 1 mM, from about 0.001 μM to about 100 mM, from about 0.01 μM to about 0.1 μM, from about 0.01 μM to about 1 μM, from about 0.01 μM to about 100 μM, from about 0.01 μM to about 1 mM, from about 0.01 μM to about 10 mM, from about 0.01 μM to about 100 mM, from about 0.1 μM to about 1 μM, from about 0.1 μM to about 10 μM, The anti-apoptotic agent is contained at a concentration of 1 μM to about 100 μM, about 0.1 μM to about 1 mM, about 0.1 μM to about 10 mM, about 0.1 μM to about 100 mM, about 1 μM to about 10 μM, about 1 μM to about 100 μM, about 1 μM to about 1 mM, about 1 μM to about 10 mM, about 1 μM to about 100 mM, about 10 μM to about 100 μM, about 10 μM to about 1 mM, about 10 μM to about 100 mM, about 100 μM to about 1 mM, about 100 μM to about 10 mM, about 100 μM to about 100 mM, about 1 mM to about 10 mM, about 1 mM to about 100 mM, or about 10 mM to about 100 mM.

[0100] In some embodiments, the composition comprises an anti-apoptotic agent at a concentration of about 80 μM. In some embodiments, the composition comprises an anti-apoptotic agent at a concentration of about 0.001 μM, about 0.01 μM, about 0.1 μM, about 1 μM, about 10 μM, about 100 μM, about 1 mM, about 10 mM, or about 100 mM.

[0101] In some embodiments, about 1 μL to about 100 μL of the composition is administered to the subject. In some embodiments, the composition is administered to one or both eyes of the subject. In some embodiments, about 1 μL to about 10 μL, about 1 μL to about 100 μL, about 1 μL to about 500 μL, about 1 μL to about 1000 μL, about 10 μL to about 100 μL, about 10 μL to about 500 μL, about 10 μL to about 1000 μL, about 100 μL to about 500 μL, about 100 μL to about 1000 μL, or about 500 μL to about 1000 μL of the composition is administered to one or both eyes of the subject.

[0102] In some embodiments, about 35 μL of the composition is administered to one or both eyes of the subject. In some embodiments, the composition is administered to the eye or skin of the subject. In some embodiments, about 1 μL, about 10 μL, about 50 μL, about 100 μL, about 500 μL, or about 1000 μL of the composition is administered to one or both eyes of the subject.

[0103] In some embodiments, about 0.1 mL to about 10 mL of the composition is administered to the subject. In some embodiments, the composition is administered to the skin of the subject. In some embodiments, about 0.1 mL to about 0.5 mL, about 0.1 mL to about 1 mL, about 0.1 mL to about 5 mL, about 0.5 mL to about 1 mL, about 0.5 mL to about 5 mL, about 0.5 mL to about 10 mL, about 1 mL to about 5 mL, about 1 mL to about 10 mL, or about 5 mL to about 10 mL of the composition is administered to the skin of the subject.

[0104] In some embodiments, about 1 mL of the composition is administered to the skin of a subject. In some embodiments, about 0.1 mL, about 0.5 mL, about 5 mL, or about 10 mL of the composition is administered to the skin of a subject.

[0105] In some embodiments, the composition is administered to the subject daily for between about 1 day and about 6 months. In some embodiments, the composition may be administered for about 1 day to about 7 days, about 1 day to about 14 days, about 1 day to about 21 days, about 1 day to about 1 month, about 1 day to about 2 months, about 1 day to about 3 months, about 1 day to about 4 months, about 1 day to about 5 months, about 7 days to about 14 days, about 7 days to about 21 days, about 7 days to about 1 month, about 7 days to about 2 months, about 7 days to about 3 months, about 7 days to about 4 months, about 7 days to about 5 months, about 7 days to about 6 months, about 14 days to about 21 days, about 14 days to about 1 month, about 14 days to about 2 months, about 14 days to about 3 months, about 14 days to about 4 months, about 14 days to about 5 months, about 14 days to about 6 months, about 21 days to about 1 month. , for about 21 days to about 2 months, for about 21 days to about 3 months, for about 21 days to about 4 months, for about 21 days to about 5 months, for about 21 days to about 6 months, for about 1 month to about 2 months, for about 1 month to about 3 months, for about 1 month to about 4 months, for about 1 month to about 5 months, for about 1 month to about 6 months, for about 2 months to about 3 months, for about 2 months to about 4 months, for about 2 months to about 5 months, for about 2 months to about 6 months, for about 3 months to about 4 months, for about 3 months to about 5 months, for about 3 months to about 6 months, for about 4 months to about 5 months, for about 4 months to about 6 months, or for about 5 months to about 6 months.

[0106] In some embodiments, the composition is administered to the subject daily for about 7 days, hi some embodiments, the composition is administered to the subject daily for about 1 day, about 14 days, about 21 days, about 1 month, about 2 months, about 3 months, about 4 months, about 5 months, or about 6 months.

[0107] In some aspects, the composition is administered to the subject three times per day, hi some aspects, the composition is administered to the subject one, two, four, five, six, seven, eight, nine, or ten times per day.

[0108] In some embodiments, the composition is administered to the subject at 2 hour intervals. In some embodiments, the composition is administered to the subject at 1 hour, 3 hours, 4 hours, 5 hours, or 6 hours intervals.

[0109] In some embodiments, the second active agent is present in an amount known to be therapeutically (clinically) effective for the treatment of the target condition, disease or disorder, such as those described herein, when a unit dose of the pharmaceutical composition is administered to a subject in need thereof. In some embodiments, the second active agent is a CNTF peptide or an analog thereof. In other embodiments, the second active agent is an anti-apoptotic agent. In some embodiments, more than one additional active agent is present, such as both a CNTF peptide and an anti-apoptotic agent. If the active agents are present in a weight ratio such that a superadditive or synergistic therapeutic effect is observed upon administration to a subject, then the overall administered dose may be reduced to produce fewer undesirable side effects.

[0110] In some embodiments, the pharmaceutical composition includes a combination of a histatin and one or more other active agents, and may include an excess of the histatin, an excess of the one or more other active agents, or an equal amount of the histatin and one or more other active agents. In some embodiments, the one or more active agents are CNTF peptides or analogs thereof. In some embodiments, the one or more active agents are anti-apoptotic agents. The weight ratio of the histatin to the one or more other active agents may range from about 100:1 to 1:100. In some embodiments, the one or more other active agents are administered to the subject sequentially or simultaneously in separate dosage forms such that the subject receives a dose of the histatin in the pharmaceutical composition and a dose of the one or more other active agents in the separate dosage forms. The dosage regimen of the one or more other active agents may be the same as or different from the dosage regimen for the histatin. Sequential administration includes active agents administered within seconds, minutes, hours, or days.

[0111] In some embodiments, the weight ratio of histatin to one or more other active agents is about 80:1 to about 1:100, about 60:1 to about 1:100, about 40:1 to about 1:100, about 20:1 to about 1:100, about 10:1 to about 1:100, about 5:1 to about 1:100, about 2:1 to about 1:100, about 1:1 to about 1:100, about 1:2 to about 1:100, about 1:5 to about 1:100, about 1:10 to about 1:100, about 1:20 to about 1:100, about 1:40 to about 1:100, about 1:60 to about 1:100, about 1:80 to about 1:100, about 100:1 to about 1:80, about 80:1 to about 1:80 , about 60:1 to about 1:80, about 40:1 to about 1:80, about 20:1 to about 1:80, about 10:1 to about 1:80, about 5:1 to about 1:80, about 2:1 to about 1:80, about 1:1 to about 1:80, about 1:2 to about 1:80, about 1:5 to about 1:80, about 1:10 to about 1:80, about 1:20 to about 1 :80, about 1:40 to about 1:80, about 1:60 to about 1:80, about 100:1 to about 1:60, about 80:1 to about 1:60, about 60:1 to about 1:60, about 40:1 to about 1:60, about 20:1 to about 1:60, about 10:1 to about 1:60, about 5:1 to about 1:60, about 2:1 to about 1:60, about 1 :1 to about 1:60, about 1:2 to about 1:60, about 1:5 to about 1:60, about 1:10 to about 1:60, about 1:20 to about 1:60, about 1:40 to about 1:60, about 100:1 to about 1:40, about 80:1 to about 1:40, about 60:1 to about 1:40, about 40:1 to about 1:40, about 20:1 to about 1 :40, about 10:1 to about 1:40, about 5:1 to about 1:40, about 2:1 to about 1:40, about 1:1 to about 1:40, about 1:2 to about 1:40, about 1:5 to about 1:40, about 1:10 to about 1:40, about 1:20 to about 1:40, about 100:1 to about 1:20, about 80:1 to about 1:20, about 60:1 ~ about 1:20, about 40:1 ~ about 1:20, about 20:1 ~ about 1:20, about 10:1 ~ about 1:20, about 5:1 ~ about 1:20, about 2:1 ~ about 1:20, about 1:1 ~ about 1:20, about 1:2 ~ about 1:20, about 1:5 ~ about 1:20, about 1:10 ~ about 1:20, about 100:1 ~ about 1:10, about 8 0:1 to about 1:10, about 60:1 to about 1:10, about 40:1 to about 1:10, about 20:1 to about 1:10, about 10:1 to about 1:10, about 5:1 to about 1:10, about 2:1 to about 1:10, about 1:1 to about 1:10, about 1:2 to about 1:10, about 1:5 to about 1:10, about 100:1 to about 1:5,About 80:1 to about 1:5, about 60:1 to about 1:5, about 40:1 to about 1:5, about 20:1 to about 1:5, about 10:1 to about 1:5, about 5:1 to about 1:5, about 2:1 to about 1:5, about 1:1 to about 1:5, about 1:2 to about 1:5, about 100:1 to about 1:2, about 80:1 to about 1:2, about 60:1 to about 1:2, about 40:1 to about 1:2, about 20:1 to about 1:2, about 10:1 to about 1:2, about 5:1 to about 1:2, about 2 :1 to about 1:2, about 1:1 to about 1:2, about 100:1 to about 1:1, about 80:1 to about 1:1, about 60:1 to about 1:1, about 40:1 to about 1:1, about 20:1 to about 1:1, about 10:1 to about 1:1, about 5:1 to about 1:1, about 2:1 to about 1:1, about 100:1 to about 2:1, about 80:1 to about 2:1, about 60:1 to about 2:1, about 40:1 to about 2:1, about 20:1 to about 2:1, about 10:1 to about 2:1, about 5: 1 to about 2:1, about 100:1 to about 5:1, about 80:1 to about 5:1, about 60:1 to about 5:1, about 40:1 to about 5:1, about 20:1 to about 5:1, about 10:1 to about 5:1, about 5:1 to about 5:1, about 100:1 to about 5:1, about 80:1 to about 5:1, about 60:1 to about 5:1, about 40:1 to about 5:1, about 20:1 to about 5:1, about 10:1 to about 5:1, about 100:1 to about 10:1, about 80:1 to about 10:1 , about 60:1 to about 10:1, about 40:1 to about 10:1, about 20:1 to about 10:1, about 100:1 to about 20:1, about 80:1 to about 20:1, about 60:1 to about 20:1, about 40:1 to about 20:1, about 100:1 to about 40:1, about 80:1 to about 40:1, about 60:1 to about 40:1, about 100:1 to about 60:1, about 80:1 to about 60:1, or about 100:1 to about 80:1.

[0112] In some embodiments, the weight ratio of the histatin to one or more other active agents is about 1: 1. In some embodiments, the weight ratio of the histatin to one or more other active agents is about 100: 1, about 80: 1, about 60: 1, about 40: 1, about 20: 1, about 10: 1, about 5: 1, about 2: 1, about 1: 2, about 1: 5, about 1: 10, about 1: 20, about 1: 40, about 1: 60, about 1: 80, or about 1: 100.

[0113] In some embodiments, the pharmaceutical composition includes a combination of a histatin analog or derivative and one or more other active agents, and may include an excess of the histatin analog or derivative, an excess of the one or more other active agents, or an equal amount of the histatin analog or derivative and one or more other active agents. In some embodiments, the one or more active agents is a CNTF peptide or an analog thereof. In some embodiments, the one or more active agents is an anti-apoptotic agent. The weight ratio of the histatin analog or derivative to the one or more other active agents can range from about 100:1 to 1:100. In some embodiments, the one or more other active agents are administered to the subject sequentially or simultaneously in separate dosage forms such that the subject receives a dose of the histatin analog or derivative in the pharmaceutical composition and a dose of the one or more other active agents in the separate dosage forms. The dosage regimen for the one or more other active agents may thus be the same as or different from the dosage regimen for the histatin analog or derivative. Sequential administration includes active agents administered within a few seconds, minutes, hours, or days.

[0114] In some aspects, the composition is administered in the form of eye drops, gels, ointments, or tissue adhesives, or by incorporating the composition into an ocular device or contact lens worn by the subject.

[0115] In some aspects, the composition comprises one or more pharma- ceutically acceptable excipients.

[0116] In some embodiments, the composition may be formulated in any manner suitable for topical administration to the patient's eye, i.e., as an ophthalmic composition. Many suitable topical ophthalmic drug forms are known (see, e.g., Baranowski et al., The Scientific World Journal, Vol. 2014, Article ID 861904, which is incorporated herein by reference). In various examples, the ophthalmic composition is in the form of a solution, suspension, emulsion, ointment, cream, gel, contact lens, ocular insert, depot, matrix, implant, drug delivery system, pledget, punctal plug, or controlled or sustained release medium or system. In some aspects, the ophthalmic composition is in the form of a sustained release vehicle or system described in U.S. Patent No. 7,897,166, U.S. Patent No. 6,991,802, U.S. Patent No. 7,485,318, U.S. Patent No. 10,149,825, U.S. Patent No. 9,226,893, U.S. Patent Publication 2010 / 0119500, U.S. Patent No. 9,949,922, U.S. Patent No. 10,172,792, and U.S. Patent No. 10,548,843, each of which is hereby incorporated by reference in its entirety. As used herein, "topical administration" includes administration of the ophthalmic composition to the corneal surface and / or the conjunctival surface. Topical administration also includes placement of the ophthalmic composition in the pouch of the eye. Ophthalmic compositions may be aqueous or non-aqueous (e.g., mineral oil-based), but are generally aqueous.

[0117] In some embodiments, the ophthalmic formulation is incorporated into biodegradable silica fibers spun from a silica sol, hi some embodiments, the silica fibers have a dissolution rate in simulated body fluid of about 0.2% by weight / hour to about 20% by weight / hour.

[0118] In some aspects, the ophthalmic formulation is incorporated into a device made of a material suitable for medical use in humans and / or animals, which material is capable of carrying or binding a biologically active agent, which material is multi-layered and formed into a body in the shape of a finished device comprising: (a) a core material formed into a body, optionally into a body having the shape of a finished device; (b) two or more layers of coating material, where a first layer is applied over the core material and an additional layer is applied over the coating material of a preceding layer; and (c) at least one layer of a coating material capable of binding to said biologically active agent.

[0119] In some aspects, the coating material is a biopolymer, a sol-gel produced silica gel, or a biologically active molecule. In some aspects, the layers of coating material are asymmetric such that different layers cover different portions of the core material, such that different layers contain different biologically active agents, or both.

[0120] In some embodiments, the ophthalmic composition includes a biodegradable carrier. In some embodiments, the biodegradable carrier is a silica xerogel made with water and a silane in a water:silane ratio range of 35-75 by using an acid or base as a catalyst. In some embodiments, the silane is tetraethoxysilane. In some embodiments, the biodegradable carrier is in the form of a monolith, fiber, net, cone, multiparticulate, or a suitable coating for the implant material.

[0121] In some aspects, the ophthalmic composition includes a bioabsorbable siloxane prepared from tetraethoxysilane. In some aspects, the bioabsorbable siloxane is a monolith. In some aspects, the bioabsorbable siloxane is a coating. In some aspects, the bioabsorbable siloxane is a particle.

[0122] In some aspects, the ophthalmic composition is in the form of an implant or delivery device comprising a water-soluble, undissolved silica-based material consisting of SiO 2 and / or hydrolyzed SiO 2 .

[0123] In some aspects, the composition is administered in any manner suitable for delivery of an active agent to the eye, commonly referred to as "ophthalmic administration". Ophthalmic administration to the eye includes, but is not limited to, intraocular injection, subretinal injection, intravitreal injection, periocular administration, subconjunctival injection, suprachoroidal injection, retrobulbar injection, intracameral injection (including in the anterior chamber or vitreous cavity), subtenon injection or implant, ophthalmic solution, ophthalmic suspension, ophthalmic ointment, ocular implant and ocular insert, intraocular solution, use of iontophoresis, incorporation in surgical irrigation solutions, and packs (by way of example only, saturated cotton balls inserted in formic).

[0124] In some embodiments, the ophthalmic composition is a sterile liquid, such as a solution or suspension contained within a multi-use or single-use eye drop dispenser bottle or vial. In certain embodiments, the ophthalmic composition is provided as a sterile liquid contained within a multi-use eye drop dispenser bottle or vial. Typically, the eye drop formulation comprises an aqueous buffered saline solution, such as phosphate or borate buffered saline, at about pH 4 to about 8, typically about pH 7.0 to about 8.0, more typically about pH 7.2 to about pH 7.4. The osmolality of the formulation is typically in the range of about 200, 250, or 270 mOsm / kg to about 310, 350, or 400 mOsm / kg. The liquid formulation packaged in a multi-use eye drop dispenser bottle may include an added preservative, such as benzalkonium chloride. However, in some embodiments, the ophthalmic composition may be free of preservatives. As used herein, "preservative-free" means that the composition does not contain a preservative in addition to the histatin or analog thereof, the CNTF peptide or analog thereof, and, optionally, the anti-apoptotic agent.

[0125] In some aspects, the composition may include an excipient that extends the period of time that the dose of the composition remains in contact with the cornea, especially when formulated as a liquid, such as a solution or suspension for dispensing by an eye dropper. For example, the excipient may be a viscosity increasing agent and / or a mucoadhesive. Examples of such excipients include high molecular weight hydrophilic polymers, including, but not limited to, polyvinyl alcohol, polyethylene glycol, carbomer, polycarbophil, polyoxyethylene-polyoxypropylene block copolymers (e.g., poloxamer 407), cellulose derivatives (e.g., hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose, and hydroxyethyl cellulose), natural polysaccharides (e.g., hyaluronic acid, dextran, chondroitin sulfate, gellan gum, xanthan gum, guar gum, trehalose, and tamarind seed polysaccharides). Additional mucoadhesive polymers have been described in the literature (see, e.g., Yadav et al. J. Chem. Pharm. Res., 2010, 2(5):418-432, incorporated herein by reference). Any combination of two or more of the foregoing polymers may be included in the composition. As used herein, a high molecular weight polymer has a molecular weight of at least 100,000 Daltons. In another embodiment, the composition includes a cyclodextrin (e.g., 2-hydroxypropyl-beta-cyclodextrin). In one embodiment, the composition is formulated as a sustained release liquid. For example, the composition may be in the form of an ophthalmic suspension that includes mucoadhesive microspheres that sustain the release of a histatin or analog thereof. The microspheres include a mucoadhesive polymer and a histatin or analog thereof. Methods for making mucoadhesive microspheres for ophthalmic suspensions have been described in the literature (see, e.g., Dandagi et al., Sci Pharm (2013) 81(1):259-280).

[0126] In some embodiments, the composition comprises one or more viscosity agents, non-limiting examples of which include hydroxypropyl methylcellulose (HPMC), hydroxyethyl cellulose, hydroxypropyl cellulose, polyvinylpyrrolidone, carboxymethyl cellulose, polyvinyl alcohol, sodium chondroitin sulfate, and sodium hyaluronate. Other acceptable viscosity agents include, but are not limited to, acacia (gum arabic), agar, magnesium aluminum silicate, sodium alginate, sodium stearate, bladderwrack, bentonite, carbomer, carrageenan, Carbopol, xanthan, cellulose, microcrystalline cellulose (MCC), ceratonia, chitin, carboxymethylated chitosan, chondrus, dextrose, furcellan, gelatin, gum ghatti, guar gum, hectorite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, arayago. Ingredients that may be used include, but are not limited to, gum tragacanth, ethyl cellulose, ethyl hydroxyethyl cellulose, ethyl methyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxypolygelatin, pectin, polygeline, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), silicon dioxide, polyvinylpyrrolidone (PVP: povidone), Splenda® (dextrose, maltodextrin and sucralose) and combinations thereof.

[0127] In some embodiments, the composition comprises from about 0.1% to about 1% by weight of one or more viscosity agents. In some embodiments, the composition comprises from about 0.1% to about 0.25%, from about 0.1% to about 0.5%, from about 0.1% to about 0.75%, from about 0.25% to about 0.5%, from about 0.25% to about 0.75%, from about 0.25% to about 1%, from about 0.5% to about 0.75%, from about 0.5% to about 1%, or from about 0.75% to about 1% by weight of one or more viscosity agents.

[0128] In some embodiments, the viscosity of the composition is from about 10 cP to about 30 cP. In some embodiments, the viscosity of the composition is from about 1 cP to about 5 cP, from about 1 cP to about 10 cP, from about 1 cP to about 15 cP, from about 1 cP to about 20 cP, from about 1 cP to about 30 cP, from about 1 cP to about 40 cP, from about 1 cP to about 50 cP, from about 1 cP to about 60 cP, from about 1 cP to about 80 cP, from about 1 cP to about 100 cP, from about 1 cP to about 125 cP, from about 1 cP to about 150 cP, from about 1 cP to about 175 cP, from about 1 cP to about 200 cP, from about 1 cP to about 400 cP, from about 5 cP to about 10 cP, from about 5 cP to about 15 cP, from about 5 cP to about 20 cP, from about 5 cP to about 30 cP, from about 5 cP to about 40 cP, 5cP to about 50cP, about 5cP to about 60cP, about 5cP to about 80cP, about 5cP to about 100cP, about 5cP to about 125cP, about 5cP ~Approx. 150cP, approx. 5cP ~ approx. 175cP, approx. 5cP ~ approx. 200cP, approx. 5cP ~ approx. 400cP, approx. 10cP ~ approx. 15cP, approx. 10cP ~Approx. 20cP, Approx. 10cP~Approx. 30cP, Approx. 10cP~Approx. 40cP, Approx. 10cP~Approx. 50cP, Approx. 10cP~Approx. 60cP, Approx. 10cP~ Approximately 80cP, approximately 10cP to approximately 100cP, approximately 10cP to approximately 125cP, approximately 10cP to approximately 150cP, approximately 10cP to approximately 175cP, approximately 10 cP ~ about 200cP, about 10cP - about 400cP, about 15cP - about 20cP, about 15cP - about 30cP, about 15cP - about 40cP, about 15cP to about 50cP, about 15cP to about 60cP, about 15cP to about 80cP, about 15cP to about 100cP, about 15cP to about 125cP, Approximately 15cP to approximately 150cP, approximately 15cP to approximately 175cP, approximately 15cP to approximately 200cP, approximately 15cP to approximately 400cP, approximately 20cP to approximately 30 cP, about 20cP to about 40cP, about 20cP to about 50cP, about 20cP to about 60cP, about 20cP to about 80cP, about 20cP to about 100 cP, about 20cP to about 125cP, about 20cP to about 150cP, about 20cP to about 175cP, about 20cP to about 200cP, about 20cP ~400cP, approximately 30cP~40cP, approximately 30cP~50cP, approximately 30cP~60cP, approximately 30cP~80cP, approximately 30cP ~100cP, 30cP~125cP, 30cP~150cP, 30cP~175cP, 30cP~200cP, approx. 30cP~about 400cP, about 40cP~about 50cP, about 40cP~about 60cP, about 40cP~about 80cP, about 40cP~about 100cP,Approximately 40cP to approximately 125cP, approximately 40cP to approximately 150cP, approximately 40cP to approximately 175cP, approximately 40cP to approximately 200cP, approximately 40cP to approximately 400cP, approximately 50 cP ~ approx. 60 cP, approx. 50 cP ~ approx. 80 cP, approx. 50 cP ~ approx. 100 cP, approx. 50 cP ~ approx. 125 cP, approx. 50 cP ~ approx. 150 cP, approx. 50 cP ~ approx. 17 5cP, about 50cP to about 200cP, about 50cP to about 400cP, about 60cP to about 80cP, about 60cP to about 100cP, about 60cP to about 125cP, about 60cP to about 150cP, about 60cP to about 175cP, about 60cP to about 200cP, about 60cP to about 400cP, about 80cP to about 100cP, about 80cP ~125cP, 80cP~150cP, 80cP~175cP, 80cP~200cP, 80cP~400cP, 100cP~100cP 125cP, about 100cP~about 150cP, about 100cP~about 175cP, about 100cP~about 200cP, about 100cP~about 400cP, about 125cP~ about 150 cP, about 125 cP to about 175 cP, about 125 cP to about 200 cP, about 125 cP to about 400 cP, about 150 cP to about 175 cP, about 150 cP to about 200 cP, about 150 cP to about 400 cP, about 175 cP to about 200 cP, about 175 cP to about 400 cP, or about 200 cP to about 400 cP.

[0129] In some embodiments, the composition has a viscosity of about 30 cP. In some embodiments, the composition has a viscosity of about 1 cP, about 5 cP, about 10 cP, about 15 cP, about 20 cP, about 30 cP, about 40 cP, about 50 cP, about 60 cP, about 80 cP, about 100 cP, about 125 cP, about 150 cP, about 175 cP, about 200 cP, or about 400 cP.

[0130] In some embodiments, the composition comprises about 0.1% by weight of one or more viscosity agents, hi some embodiments, the composition comprises about 0.25%, about 0.5%, about 0.75%, or about 1% by weight of one or more viscosity agents.

[0131] In some embodiments, the composition comprises one or more buffers, non-limiting examples of which include acetate buffers, borate buffers, citrate borate buffers, citrate buffers, lactate buffers, phosphate buffers, succinate buffers, borate-polyol complex buffers, carbonate buffers, organic buffers, amino acid buffers, and combinations thereof.

[0132] Organic buffers include, but are not limited to, Good's buffers, such as 2-(N-morpholino)ethanesulfonic acid (MES), N-(2-acetamido)iminodiacetic acid, N-(carbamoylmethyl)iminodiacetic acid (ADA), piperazine-N,N'-bis(2-ethanesulfonic acid (PIPES), N-(2-acetamido)-2-aminoethanesulfonic acid (ACES), β-hydroxy-4-morpholinepropanesulfonic acid, acid, 3-morpholino-2-hydroxypropanesulfonic acid (MOPSO), cholamine chloride, 3-(N-morpholino)propanesulfonic acid (MOPS), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-[(2-hydroxy-1,1-bis(hydroxymethyl)ethyl)amino]ethanesulfonic acid (TES), 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid dihydropyrazine-1,4,-bis(2-hydroxypropanesulfonic acid) (POPSO), 4-(2-hydroxyethyl)piperazine-1-(2-hydroxypropanesulfonic acid) hydrate (HEPPSO), 3-[4-(2-hydroxyethyl)-1-piperazinyl]propanesulfonic acid (HEPPS), tricine, glycinamide, bicine or sodium N-tris(hydroxymethyl)methyl-3-aminopropanesulfonate (TAPS), glycine, diethanolamine (DEA); and combinations thereof.

[0133] Amino acid buffers include, but are not limited to, taurine, aspartic acid and its salts (eg, potassium salts, etc.), ε-aminocaproic acid, and combinations thereof.

[0134] In some embodiments, the pH of the ophthalmic formulation is adjusted by a strong acid or strong base. Examples of strong acids and strong bases are well known in the art and include, but are not limited to, NaOH, KOH, HCl, and H2SO4. In some embodiments, the strong acid or base is HCl or NaOH.

[0135] In some embodiments, the composition comprises from about 0.05% to about 1% by weight of one or more buffer solutions. In some embodiments, the composition comprises from about 0.05% to about 0.1% by weight, from about 0.05% to about 0.25% by weight, from about 0.05% to about 0.5% by weight, from about 0.05% to about 0.75% by weight, from about 0.1% to about 0.25% by weight, from about 0.1% to about 0.5% by weight, from about 0.1% to about 0.75% by weight, from about 0.1% to about 1% by weight, from about 0.25% to about 0.5% by weight, from about 0.25% to about 0.75% by weight, from about 0.25% to about 1% by weight, from about 0.5% to about 0.75% by weight, from about 0.5% to about 1% by weight, or from about 0.75% to about 1% by weight of one or more buffer solutions.

[0136] In some embodiments, the composition comprises about 0.5% by weight of one or more buffers, hi some embodiments, the composition comprises about 0.05%, about 0.1%, about 0.25%, about 0.75%, or about 1% by weight of one or more buffers.

[0137] In some embodiments, the composition has a pH of about 7 to about 7.6. In some embodiments, the composition has a pH of about 6 to about 6.5, about 6 to about 7, about 6 to about 7.2, about 6 to about 7.4, about 6 to about 7.6, about 6 to about 7.8, about 6 to about 8, about 6.5 to about 7, about 6.5 to about 7.2, about 6.5 to about 7.4, about 6.5 to about 7.6, about 6.5 to about 7.8, about 6.5 to about 8, about 7 to about 7.2, about 7 to about 7.4, about 7 to about 7.8, about 7 to about 8, about 7.2 to about 7.4, about 7.2 to about 7.6, about 7.2 to about 7.8, about 7.2 to about 8, about 7.4 to about 7.6, about 7.4 to about 7.8, about 7.4 to about 8, or about 7.6 to about 7.8.

[0138] In some embodiments, the composition has a pH of about 7.4. In some embodiments, the composition has a pH of about 6, about 6.5, about 7, about 7.2, about 7.6, or about 8.

[0139] In some embodiments, the composition comprises a preservative. Non-limiting examples of preservatives include benzalkonium chloride, stabilized oxychloro complexes (Purite®), phenylmercuric acetate, chlorobutanol, benzyl alcohol, parabens, and thimerosal. In some embodiments, the composition comprises benzalkonium chloride.

[0140] In some embodiments, the composition comprises about 0.0075% to about 0.0125% by weight of benzalkonium chloride. In some embodiments, the composition comprises about 0.0025% to about 0.005% by weight, about 0.0025% to about 0.0075% by weight, about 0.0025% to about 0.01% by weight, about 0.0025% to about 0.0125% by weight, about 0.0025% to about 0.02% by weight, about 0.005% to about 0.0075% by weight, about 0.005% to about 0.01% by weight. %, about 0.005% to about 0.0125% by weight, about 0.005% to about 0.02% by weight, about 0.0075% to about 0.01% by weight, about 0.0075% to about 0.02% by weight, about 0.01% to about 0.0125% by weight, about 0.01% to about 0.02% by weight, or about 0.0125% to about 0.02% by weight of benzalkonium chloride.

[0141] In some embodiments, the composition comprises about 0.01% benzalkonium chloride by weight. In some embodiments, the composition comprises about 0.0025%, about 0.005%, about 0.0075%, about 0.0125%, or about 0.02% benzalkonium chloride by weight.

[0142] In some embodiments, the composition is preservative-free.

[0143] In some embodiments, the composition includes one or more stabilizers. The stabilizers include, but are not limited to, fatty acids, fatty alcohols, alcohols, long chain fatty acid esters, long chain ethers, hydrophilic derivatives of fatty acids, polyvinylpyrrolidones, polyvinyl ethers, polyvinyl alcohols, hydrocarbons, hydrophobic polymers, hygroscopic polymers, and combinations thereof. In some embodiments, amide analogs of stabilizers are also used. In some embodiments, the stabilizer selected changes the hydrophobicity of the composition, improves the mixing of various components in the composition, controls the moisture level in the composition, or controls phase mobility.

[0144] In some embodiments, the composition comprises one or more stabilizers in an amount sufficient to inhibit degradation of the active agent. Examples of such stabilizers include, but are not limited to, glycerol, methionine, monothioglycerol, EDTA, ascorbic acid, polysorbate 80, polysorbate 20, arginine, heparin, dextran sulfate, cyclodextrin, pentosan polysulfate and other heparinoids, divalent cations such as magnesium and zinc, or combinations thereof.

[0145] In some embodiments, the solubility of the components of the composition may be enhanced by the inclusion of a surfactant or other suitable co-solvent in the composition. Such co-solvents include polysorbates 20, 60, and 80, Pluronic F68, F-84, and P-103, cyclodextrins, or other agents known to those of skill in the art. In some embodiments, the concentration of the co-solvent is from 0.01% to about 2% by weight.

[0146] In some aspects, the composition includes one or more polyols. As used herein, the term "polyol" includes any compound having at least one hydroxyl group on each of two adjacent carbon atoms that are not in a trans configuration relative to each other. The polyol can be linear or cyclic, substituted or unsubstituted, or a mixture thereof, so long as the resulting complex is water-soluble and pharma- ceutically acceptable. Examples of such compounds include sugars, sugar alcohols, sugar acids, and uronic acids. Preferred polyols are sugars, sugar alcohols, and sugar acids, including, but not limited to, mannitol, glycerin, xylitol, sorbitol, and propylene glycol. It is contemplated that the polyol may be composed of two or more different polyols.

[0147] In some embodiments, the composition includes one or more anti-aggregation additives. Anti-aggregation additives enhance the stability of the composition by reducing the rate of protein aggregation. Anti-aggregation additives include, but are not limited to, urea, guanidinium chloride, simple amino acids such as glycine or arginine, sugars, polyalcohols, polysorbates, polymers such as polyethylene glycol and dextrans, alkyl sugars such as alkyl glycosides, and surfactants.

[0148] In some embodiments, the compositions include one or more antioxidants, including but not limited to ascorbic acid, methionine, sodium thiosulfate, sodium metabisulfate, and combinations thereof. Metal chelators, thiol-containing compounds, and other common stabilizers may be acceptable antioxidants.

[0149] In some embodiments, the compositions include one or more osmolality agents, including but not limited to salts, particularly sodium chloride or potassium chloride, organic compounds such as propylene glycol, mannitol, sorbitol, dextrose, and glycerin.

[0150] In some embodiments, the composition has an osmolality of about 260 to about 365 mOsm / kg. In some embodiments, the composition has an osmolality of about 285 mOsm / kg to about 295 mOsm / kg, about 285 mOsm / kg to about 305 mOsm / kg, about 285 mOsm / kg to about 315 mOsm / kg, about 285 mOsm / kg to about 325 mOsm / kg, about 285 mOsm / kg to about 335 mOsm / kg, about 285 mOsm / kg to about 345 mOsm / kg, about 285 mOsm / kg to about 355 mOsm / kg, about 285 mOsm / kg to about 365 mOsm / kg, about 295 mOsm / kg to about 30 ...05 mOsm / kg, about 295 mOsm / kg / kg~about 315mOsm / kg, about 295mOsm / kg~about 325mOsm / kg, about 295mOsm / kg~about 335mOsm / kg, about 295mOsm / kg~about 345mOsm / kg, about 295mOsm / kg~about 355mOsm / kg, about 295mO sm / kg~about 365mOsm / kg, about 305mOsm / kg~about 315mOsm / kg, about 305mOsm / kg~about 325mOsm / kg, about 305mOsm / kg~about 335mOsm / kg, about 305mOsm / kg~about 345mOsm / kg, about 305m Osm / kg~about 355mOsm / kg, about 305mOsm / kg~about 365mOsm / kg, about 315mOsm / kg~about 325mOsm / kg, about 315mOsm / kg~about 335mOsm / kg, about 315mOsm / kg~about 345mOsm / kg, about 31 5mOsm / kg~about 355mOsm / kg, about 315mOsm / kg~about 365mOsm / kg, about 325mOsm / kg~about 335mOsm / kg, about 325mOsm / kg~about 345mOsm / kg, about 325mOsm / kg~about 355mOsm / kg, about 3 25mOsm / kg~Approx. 365mOsm / kg, Approx. 335mOsm / kg~Approx. 345mOsm / kg, Approx. 335mOsm / kg~Approx. 355mOsm / kg, Approx. 335mOsm / kg~Approx. 365mOsm / kg, Approx. 345mOsm / kg~about 365mOsm / kg, about 355mOsm / kg~about 365mOsm / kg, about 260mOsm / kg~about 265mOsm / kg, about 260mOsm / kg~about 275mOsm / kg, about 260mOsm / kg~about 285mOsm / kg,Approx. 260mOsm / kg~Approx. 295mOsm / kg, Approx. 260mOsm / kg~Approx. 305mOsm / kg, Approx. 260mOsm / kg~Approx. 315mOsm / kg, Approx. 260mOsm / k g ~ approx. 325mOsm / kg, approx. 260mOsm / kg ~ approx. 335mOsm / kg, approx. 260mOsm / kg ~ approx. 345mOsm / kg, approx. 260mOsm / kg ~ approx. 355mOsm / kg, about 260mOsm / kg to about 365mOsm / kg, about 265mOsm / kg to about 275mOsm / kg, about 265mOsm / kg to about 285mOsm / kg, about 265mO sm / kg ~ approx. 295mOsm / kg, approx. 265mOsm / kg ~ approx. 305mOsm / kg, approx. 265mOsm / kg ~ approx. 315mOsm / kg, approx. 265mOsm / kg ~ approx. 325m Osm / kg, about 265mOsm / kg to about 335mOsm / kg, about 265mOsm / kg to about 345mOsm / kg, about 265mOsm / kg to about 355mOsm / kg, about 26 5mOsm / kg ~ approx. 365mOsm / kg, approx. 275mOsm / kg ~ approx. 285mOsm / kg, approx. 275mOsm / kg ~ approx. 295mOsm / kg, approx. 275mOsm / kg ~ approx. 3 05 mOsm / kg, about 275 mOsm / kg to about 315 mOsm / kg, about 275 mOsm / kg to about 325 mOsm / kg, about 275 mOsm / kg to about 335 mOsm / kg, about 275 mOsm / kg to about 345 mOsm / kg, about 275 mOsm / kg to about 355 mOsm / kg, about 275 mOsm / kg to about 365 mOsm / kg.

[0151] In some embodiments, the composition has an osmolality of about 260 mOsm / kg, about 265 mOsm / kg, about 275 mOsm / kg, about 285 mOsm / kg, about 295 mOsm / kg, about 305 mOsm / kg, about 315 mOsm / kg, about 325 mOsm / kg, about 335 mOsm / kg, about 345 mOsm / kg, about 355 mOsm / kg, about 365 mOsm / kg, about 370 mOsm / kg, or about 375 mOsm / kg.

[0152] In some aspects, the composition is isotonic. In some aspects, the composition is hypotonic. In some aspects, the composition is hypertonic.

[0153] In some embodiments, the ophthalmic composition is formulated as a unit dose ocular insert for placement in the eye pouch. Methods for making the ocular insert are described in the literature (see, e.g., U.S. Pat. No. 4,730,013, U.S. Pat. No. 7,749,970, and U.S. Pat. No. 2012 / 0215184, which are incorporated herein by reference). The ocular insert is a solid unit dosage form that includes a biodegradable matrix that includes an active agent, which in the present ophthalmic composition is a histatin or an analog thereof, a CNTF peptide or an analog thereof, and optionally an anti-apoptotic agent. The matrix is ​​typically made of a high molecular weight polymer or a combination of high molecular weight polymers, such as the aforementioned hydrophilic polymers and additional polymers disclosed in the aforementioned patent publications that describe the ocular insert. The ocular insert may additionally include a lubricant to enhance comfort. Upon placement in the eye, the ocular insert dissolves or erodes over a period of several hours to a day, and in some cases, over a period of several days.

[0154] In some aspects, the disclosure provides a method of treating an ocular infection in a patient, comprising topically administering to the patient's eye an ophthalmic composition comprising an effective amount of a histatin or analog thereof and / or a CNTF peptide or analog thereof, and optionally an anti-apoptotic agent. In various specific examples, the patient is a human or veterinary patient, and / or the ocular infection is bacterial conjunctivitis or bacterial keratitis, and / or the ocular infection is caused by MRSA.

[0155] In some aspects, the amount of histatin and / or CNTF peptide or analog thereof, and optionally, anti-apoptotic drug in the ophthalmic composition is effective for the treatment or prevention of ocular surgery, injury, infection, disease, or disorder. In certain examples, the amount of histatin and / or CNTF peptide or analog thereof, and optionally, anti-apoptotic drug in the ophthalmic composition is effective for the treatment of ocular surgery, injury, infection, disease, or disorder. In other examples, the amount of histatin and / or CNTF peptide or analog thereof, and optionally, anti-apoptotic drug in the ophthalmic composition is effective for the pretreatment or prevention of ocular surgery, injury, infection, disease, or disorder. As used herein, "pretreatment or prevention" means that ocular surgery, injury, infection, disease or disorder does not lead to or results in reduced keratocyte loss when an ophthalmic composition is administered to an eye (either before or after the ocular surgery, injury, infection, disease or disorder), whereas an eye not administered the ophthalmic composition (or treated with an otherwise identical control composition lacking a histatin) results in statistically significant keratocyte loss.

[0156] Prevention can be, for example, prevention from infection or injury following surgery, such as photorefractive keratectomy surgery or other ocular surgery, or from injury, infection, disease, or disorder.

[0157] In some embodiments, a composition comprising a histatin or analog thereof and / or a CNTF peptide or analog thereof, and optionally an anti-apoptotic agent, is administered to a subject before the subject undergoes surgery. In some embodiments, the surgery is photorefractive keratectomy (PRK) surgery. In some embodiments, the surgery is penetrating keratoplasty (PK). In some embodiments, the surgery is Descemet's membrane stripping automated endothelial keratoplasty (DSAEK) or Descemet's membrane endothelial keratoplasty (DMEK).

[0158] In some embodiments, the composition comprising a histatin or analog thereof and / or a CNTF peptide or analog thereof, and optionally an anti-apoptotic agent, is administered to a subject after the subject has undergone surgery. In some embodiments, the surgery is photorefractive keratectomy (PRK) surgery. In some embodiments, the surgery is penetrating keratoplasty (PK). In some embodiments, the surgery is Descemet's membrane stripping automated endothelial keratoplasty (DSAEK) or Descemet's membrane endothelial keratoplasty (DMEK).

[0159] In some aspects, the subject is a mammal. In some aspects, the mammal is a rabbit, horse, dog, cat, cow, pig, or sheep. In some aspects, the mammal is a human.

[0160] In some aspects, administration of the composition results in a reduction in keratocyte loss following injury or surgery.

[0161] In some aspects, administration of the composition results in restoration of keratocytes lost following injury or surgery.

[0162] In some aspects, the loss of keratocytes in the subject is caused by photorefractive keratectomy, Fuchs' corneal dystrophy, or keratoconus. In some aspects, the loss of keratocytes in the subject is caused by photodamage to the skin. In some aspects, the loss of keratocytes in the subject is caused by chemotherapy.

[0163] In one embodiment, the histatin is a peptide comprising 8 to 44 amino acids. In some embodiments, the peptide is an L-peptide. In other embodiments, the peptide is a cyclic peptide.

[0164] In some embodiments, the amino acid sequence of the histatin peptide is one or more of SEQ ID NOs: 1-33, and SEQ ID NOs: 38-40, or any combination of these sequences. In some embodiments, one or more of the amino acid sequences has up to three amino acid substitutions, deletions, and / or insertions. In some embodiments, one or more of the amino acid sequences has two or fewer amino acid substitutions, deletions, and / or insertions. In other embodiments, one or more of the amino acid sequences has a single amino acid substitution, deletion, and / or insertion.

[0165] The SEQ ID NO:4 peptide is also known as histatin 1 (Hst-1). In some aspects, the first serine in this amino acid sequence may be phosphoserine. The SEQ ID NO:5 peptide is also known as histatin 2 (Hst-2, also equivalent to amino acids 12-38 of Hst-1). The SEQ ID NO:6 peptide is also known as histatin 3 (Hst-3). The SEQ ID NO:30 peptide is also known as histatin 5 (Hst-5). Portions and fragments of each of these amino acid sequences may be used alone or in combination, including but not limited to SEQ ID NOs:1-3, and 7-29 (for histatin 1, histatin 2, and histatin 3) and SEQ ID NO:32 (for histatin 5), to promote wound closure in the disclosures described herein. While L-stereoisomers of amino acids are preferred for the amino acid sequences described herein, D-stereoisomers may alternatively be used. Alternatively, amino acid sequences containing these histatins and other amino acids may be used, such as SEQ ID NO: 33, which is a sortase cyclized histatin (containing all of histatin 1). Any histatin sequence may be cyclized and used.

[0166] In some embodiments, the histatins described herein can include histatins 1-5. Examples of histatins include, but are not limited to, human histatin-1 having the amino acid sequence set forth in SEQ ID NO:4. In some embodiments, histatin analogs, derivatives, fragments, or fusions are also contemplated. In some embodiments, histatin analogs, derivatives, fragments, or fusions of histatins 1-5 set forth in SEQ ID NO:1-33 or SEQ ID NO:38-40 are also contemplated. In some embodiments, two or more histatin analogs, derivatives, fragments, or fusions of histatins 1-5 set forth in SEQ ID NO:1-33 or SEQ ID NO:38-40 connected by a linker are also contemplated. In some aspects, the histatin can have at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NOs:1-33 or SEQ ID NOs:38-40.

[0167] In some embodiments, the ciliary neurotrophic factor (CNTF) peptide, peptide 6, described herein has an amino acid sequence as set forth in SEQ ID NO: 34. In some embodiments, a ciliary neurotrophic factor (CNTF) peptide, peptide 6 derivative is also contemplated. In some embodiments, a CNTF peptide analog, derivative, fragment, or fusion of the CNTF peptide set forth in SEQ ID NO: 34 is also contemplated. In some embodiments, two or more CNTF peptides or analogs, derivatives, fragments, or fusions of the CNTF peptide set forth in SEQ ID NO: 34 connected by a linker are also contemplated. In some embodiments, a derivative of the ciliary neurotrophic factor (CNTF) peptide, peptide 6, may have at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 34.

[0168] The sequences of histatin and CNTF peptides are described in US2013 / 0310327, WO2021 / 108482, and US2020 / 0108117, which are incorporated by reference in their entireties herein.

[0169] The present disclosure provides the sequences in Table 1 below: (Table 1) TIFF2024528700000001.tif170153TIFF2024528700000002.tif185153

[0170] In some aspects, the disclosure provides a method of therapeutically or prophylactically treating a skin or ophthalmic disease, disorder, or condition, comprising administering to a subject in need thereof a composition comprising a histatin or an analog thereof and / or a CNTF peptide or an analog thereof, and optionally an anti-apoptotic agent.

[0171] In some aspects, the dermatological disease, disorder, or condition is post-CO2 recovery, burns, decubitus ulcers, autonomic dysreflexia in spinal cord injury, diabetic ischemic ulcers, ulcer prophylaxis, skin graft recovery, trauma resulting in skill loss, post-cryo / electrodesiccation recovery, or post-Mohs / skin cancer wound healing. In some aspects, the ophthalmic disease, disorder, or condition is post-photorefractive keratectomy (PRK) surgery, glaucoma surgery, infectious corneal ulcers, pigmentary eye disease, post-collagen crosslinking (CSX) for keratoconus, neurotrophic keratitis, Fuchs corneal dystrophy, contact lens intolerance, dry eye disease, or inhibiting keratitis progression. In some aspects, the ophthalmic disease, disorder, or condition is posterior polymorphous corneal dystrophy (PPCD), aphakic or pseudophakic bullous keratopathy (ABK / PBK), endothelial dysfunction caused by penetrating or blunt trauma, congenital hereditary endothelial dystrophy (CHED), iridocorneal endothelial (ICE) syndrome, refractory glaucoma, previous failed corneal graft, or herpes simplex virus endotheliitis.

[0172] In some embodiments, the present disclosure provides a method of processing, preserving, and / or storing a biological sample prior to a surgical procedure, comprising contacting the biological sample with a composition comprising a histatin or an analog thereof and / or a CNTF peptide or an analog thereof, and optionally an anti-apoptotic drug. In some embodiments, the biological sample is a donor tissue. In some embodiments, the donor tissue is a corneal tissue. In some embodiments, the donor tissue is suitable for transplantation into a mammalian subject. In some embodiments, the donor tissue is suitable for transplantation into a human subject. In some embodiments, the method further comprises storing the biological sample contacted with the composition at a temperature of about -10°C to about 25°C.

[0173] In some aspects, the biological sample remains viable for about 0.5 days, about 1 day, about 2 days, about 3 days, about 7 days, about 14 days, about 21 days, about 30 days, about 60 days, or about 90 days. In some embodiments, the biological sample is eluted for about 0.5 days to about 1 day, about 0.5 days to about 2 days, about 0.5 days to about 3 days, about 0.5 days to about 7 days, about 0.5 days to about 14 days, about 0.5 days to about 21 days, about 0.5 days to about 30 days, about 0.5 days to about 60 days, about 0.5 days to about 90 days, about 1 day to about 2 days, about 1 day to about 3 days, about 1 day to about 7 days, about 1 day to about 14 days, about 1 day to about 21 days, about 1 day to about 30 days, about 1 day to about 60 days, about 1 day to about 90 days, about 2 days to about 3 days, about 2 days to about 7 days, about 2 days to about 14 days, about 2 days to about 21 days, about 2 days to about 30 ... The cells remain viable for about 60 days, about 2 to about 90 days, about 3 to about 7 days, about 3 to about 14 days, about 3 to about 21 days, about 3 to about 30 days, about 3 to about 60 days, about 3 to about 90 days, about 7 to about 14 days, about 7 to about 21 days, about 7 to about 30 days, about 7 to about 60 days, about 7 to about 90 days, about 14 to about 21 days, about 14 to about 30 days, about 14 to about 60 days, about 14 to about 90 days, about 21 to about 30 days, about 21 to about 60 days, about 21 to about 90 days, about 30 to about 60 days, about 30 to about 90 days, and about 60 to about 90 days.

[0174] In some aspects, the surgical procedure is photorefractive keratectomy (PRK) surgery, Descemet's membrane stripping automated endothelial keratoplasty (DSAEK), or Descemet's membrane endothelial keratoplasty (DMEK).

[0175] In some aspects, the present disclosure provides a method for reducing or preventing corneal endothelial cell loss and / or restoring corneal endothelial cells, comprising administering to an eye of a subject in need thereof a therapeutically effective amount of a composition comprising a histatin or an analog thereof and, optionally, an anti-apoptotic drug.

[0176] In some embodiments, the anti-apoptotic agent is MET12 (SEQ ID NO: 37). In some embodiments, the anti-apoptotic agent is a MET variant (SEQ ID NO: 41). In some embodiments, the anti-apoptotic agent may have at least 70%, at least 75%, at least 80%, at least 82%, at least 85%, at least 88%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to SEQ ID NO: 37 or SEQ ID NO: 41. The sequence of MET12 is described in U.S. Patent No. 8,343,931, which is incorporated herein by reference in its entirety.

[0177] In some aspects, the composition is administered by topical administration, intraocular injection, subretinal injection, intravitreal injection, periocular injection, subconjunctival injection, suprachoroidal injection, retrobulbar injection, intracameral injection, or subtenon injection.

[0178] In some aspects, the loss of corneal endothelial cells is caused by Fuchs endothelial dystrophy, posterior polymorphous corneal dystrophy, aphakic or pseudophakic bullous keratopathy, endothelial dysfunction caused by penetrating or blunt trauma, congenital hereditary endothelial dystrophy, iridocorneal endothelial syndrome, refractory glaucoma, a previous failed corneal graft, or herpes simplex virus endotheliitis.

[0179] In some aspects, the loss of corneal endothelial cells is caused by Fuchs endothelial corneal dystrophy.

[0180] In some aspects, the composition is administered before a surgical procedure. In some aspects, the composition is administered during a surgical procedure. In some aspects, the composition is administered after a surgical procedure. In some aspects, the surgical procedure is penetrating keratoplasty (PK), Descemet's membrane stripping automated endothelial keratoplasty (DSAEK), or Descemet's membrane endothelial keratoplasty (DMEK).

[0181] In some embodiments, administration of the composition results in about a 20% reduction in corneal endothelial cell loss compared to no treatment, hi some embodiments, administration of the composition results in about a 1%, about 5%, about 10%, about 15%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% reduction in corneal endothelial cell loss compared to no treatment. In some embodiments, administration of the composition reduces or decreases the rate of corneal endothelial cell loss by about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5 ... ~ about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 15%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 15% to about 20%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, About 15% to about 80%, about 15% to about 90%, about 15% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about A reduction of about 40% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100% is brought about.In some aspects, the reduction in corneal endothelial cell loss is determined by measuring corneal endothelial cell density using corneal tomography.

[0182] In some aspects, the present disclosure also provides a method of restoring, regenerating, or promoting the regeneration of corneal nerves, comprising administering to an eye of a subject in need thereof a therapeutically effective amount of a composition comprising a histatin or an analog thereof, and / or a ciliary neurotrophic factor (CNTF) peptide or an analog thereof, and optionally an anti-apoptotic agent.

[0183] In some aspects, the composition comprises a histatin or an analog thereof.

[0184] In some aspects, the composition comprises a histatin or an analog thereof and a ciliary neurotrophic factor (CNTF) peptide or an analog thereof.

[0185] In some aspects, the composition comprises a histatin or an analog thereof, a ciliary neurotrophic factor (CNTF) peptide or an analog thereof, and an anti-apoptotic agent.

[0186] In some aspects, the composition comprises a histatin or an analog thereof and an anti-apoptotic drug.

[0187] In some aspects, the composition comprises a ciliary neurotrophic factor (CNTF) peptide or an analog thereof and an anti-apoptotic agent.

[0188] In some aspects, the composition comprises a ciliary neurotrophic factor (CNTF) peptide or an analog thereof.

[0189] In some aspects, the composition is administered by topical administration, intraocular injection, subretinal injection, intravitreal injection, periocular injection, subconjunctival injection, suprachoroidal injection, retrobulbar injection, intracameral injection, or subtenon injection.

[0190] In some aspects, the corneal nerve loss is caused by neurotrophic keratitis, dry eye disease, herpetic keratitis, leprosy, diabetes, keratoconjunctivitis sicca, or keratoconus.

[0191] In some embodiments, the corneal nerve loss is caused by a surgical procedure, hi some embodiments, the surgical procedure is penetrating keratoplasty (PK), Descemet's membrane stripping automated endothelial keratoplasty (DSAEK), Descemet's membrane endothelial keratoplasty (DMEK), photorefractive keratectomy (PRK), radial keratotomy, or laser in situ keratomileusis (LASIK).

[0192] In some aspects, the composition is administered before the surgical procedure. In some aspects, the composition is administered during the surgical procedure. In some aspects, the composition is administered after the surgical procedure.

[0193] In some aspects, administration of the composition results in about a 50% reduction in corneal nerve loss compared to no treatment, hi some aspects, administration of the composition results in about a 1%, about 5%, about 10%, about 15%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or about 100% reduction in corneal nerve loss compared to no treatment. In some embodiments, administration of the composition reduces or decreases about 1% to about 5%, about 1% to about 10%, about 1% to about 15%, about 1% to about 20%, about 1% to about 30%, about 1% to about 40%, about 1% to about 50%, about 1% to about 60%, about 1% to about 70%, about 1% to about 80%, about 1% to about 90%, about 1% to about 100%, about 5% to about 10%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5 ...0%, about 5% to about 15%, about 5% to about 20%, about 5% to about 30%, about 5% to about About 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 10% to about 15%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 15% to about 20%, about 15% to about 30%, about 15% to about 40%, about 15% to about 50%, about 15% to about 60%, about 15% to about 70%, about 15% to about 80%, about 15% to about 90%, about 15% to about 100%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 40% to about 50%, about 40% to about 60%, about 4 This results in a reduction of 0% to about 70%, about 40% to about 80%, about 40% to about 90%, about 40% to about 100%, about 50% to about 60%, about 50% to about 70%, about 50% to about 80%, about 50% to about 90%, about 50% to about 100%, about 60% to about 70%, about 60% to about 80%, about 60% to about 90%, about 60% to about 100%, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 80% to about 90%, about 80% to about 100%, or about 90% to about 100%.In some aspects, the reduction in corneal nerve loss is determined by measuring the change in the area of ​​a corneal lesion or wound.

[0194] The present disclosure also provides a method for treating or preventing skin aging, cellulite, dry skin, cracks, or wounds, comprising administering to a subject in need thereof a composition comprising a histatin or an analog thereof and / or a CNTF peptide or an analog thereof, and optionally an anti-apoptotic agent. In some aspects, the wound is an internal wound, an oral wound, a skin wound, an external wound, an ulcer, and / or a bedsore wound. In some aspects, the wound comprises an injury to the eye, for example, an injury to the conjunctiva.

[0195] The present disclosure also provides a method of treating or preventing oral ulcers, oral aphthous lesions, burning mouth syndrome, burning tongue, psoriasis, eczema, and hair loss, comprising administering to a subject in need thereof a composition comprising a histatin or an analog thereof and / or a CNTF peptide or an analog thereof, and optionally an anti-apoptotic agent.

[0196] These types of skin damage and wounds are known to those skilled in the art. An internal wound is a wound present in the body, for example, due to a surgical incision. An oral wound is a wound present in the oral cavity. A skin wound is a wound present in the skin. An external wound should be understood as a wound that is visible and accessible from the outside of the body. An ulcer is a lesion on the surface of the skin or on a mucosal surface. A decubitus wound is a wound or ulceration caused by prolonged pressure on the skin and tissues, such as when lying on the floor, remaining in one position for a long period of time. The bone area of ​​the body is the most frequently affected site, which becomes ischemic under continuous and constant pressure. Skin aging is the change in the appearance of the skin due to time or exposure to the environment or the health of an individual. Cellulite is a definition used in cosmetics that relates to the appearance of bulges or dimples in the skin.

[0197] In some aspects, the methods disclosed herein are used for the treatment of skin wounds. Skin wounds can be wounds in the epidermis or dermis of the skin. There are several types of wounds that skin or tissue may need repair: abrasions, lacerations, incisions, punctures, as well as abrasions and burns. The use of the compositions disclosed herein can improve the general health of the skin. In another aspect, the methods disclosed herein are used in the treatment of oral wounds. Oral wounds are wounds in any part of the oral cavity where the oral mucosa is damaged. In another aspect, the methods described herein are used in the treatment of internal wounds. Internal wounds are wounds where a cell layer of endodermal or mesodermal origin is damaged. Examples are wounds in arteries or veins, peritoneum or pericardium.

[0198] The present disclosure also includes a method of treating ocular surface disease by administering to a subject in need thereof a composition comprising a histatin or analog thereof and / or a CNTF peptide or analog thereof, and optionally an anti-apoptotic drug. Ocular surface disease may include, but is not limited to, dry eye, corneal ulcers and erosions, inflammatory and infectious keratitis and conjunctivitis, surgical intervention, and trauma.

[0199] The following examples are illustrative and do not limit the scope of the claimed embodiments. EXAMPLES

[0200] Example 1 Effect of histatin-1 on stromal keratocyte loss. method animal Twenty-four New Zealand white rabbits, approximately 4-6 months old and weighing 2-3 kg, were divided into three groups of eight animals and treated topically with either 35 μl phosphate buffered saline (PBS), 35 μl 80 μM histatin-1 (Hst-1), or a combination of 35 μl 80 μM histatin-1 + 100 μM peptide 6 (P6) three times per day (TID) for 28 days following a unilateral surgical keratectomy procedure.

[0201] surgical keratectomy Animals were administered buprenorphine (0.01-0.05 mg / kg, SC) for pain management immediately prior to the procedure. A second dose of buprenorphine (0.01-0.05 mg / kg, SC) was administered in the evening and then as needed. Animals were induced for surgery with a ketamine / xylazine cocktail (50 mg / kg ketamine and 10 mg / kg xylazine IM) and anesthesia was maintained with isoflurane delivered in oxygen through a mask as needed. After induction of anesthesia, 5% betadine solution was used on autoclaved sterile gauze to clean the periocular (lid) area of ​​the postoperative eye. Betadine was then used to irrigate the ocular surface and conjunctival fornix, which was then thoroughly irrigated with sterile eyewash. One drop each of 0.5% proparacaine HCl and 10% phenylephrine HCl was then applied to the ocular surface for ocular anesthesia and mydriasis, respectively. The rabbit was positioned under an operating microscope and an eyelid retractor was attached. An adjustable 8 mm corneal vacuum trephine (Moria Surgical, Antony, France) was used to create a central corneal defect of approximately 10-25% stromal depth (approximately 100 µm). The perforated section of cornea was removed, leaving the wound open. A single dose of topical antibiotic (neomycin polymyxin B gramicin sulfate ophthalmic solution) was applied immediately after surgery.

[0202] Corneal Tomography The corneas of anesthetized animals were imaged with a Heidelberg Retinal Tomograph equipped with a Rostock Corneal Module (HRT III RCM). This compact ophthalmic device uses confocal scanning laser microscopy to provide high-resolution surface images of corneal cells and structures, including keratocyte subpopulations and the subbasal corneal plexus. Central corneal confocal scans from epithelium to endothelium were performed, generating 400 μm × 400 μm and 2 μm thick optical stacks that were electronically stored for analysis.

[0203] Sampling protocol and data analysis. A sampling area of ​​the stack of optical scans corresponding to approximately 150 μm above the endothelium was selected. This was done by counting 75 images above the endothelium and selecting five adjacent images to serve as replicates. Keratocytes reflectively presented as separate irregularly shaped objects. Counts from the five images were then averaged, expressed as counts / field of view, and displayed graphically as mean ± standard deviation (SD). Unpaired Student's T-test was used to compare the histatin-1 group with the histatin-1 + peptide 6 group; p < 0.05 was considered statistically significant.

[0204] result FIG. 1A shows the results of the experiment. Surgical keratectomy induced a gradual loss of stromal keratocytes that peaked on day 10 and remained relatively unchanged until day 21. The percent reduction (mean ± SD) from baseline in the PBS group for days 7, 10, 14, and 21 was 35.7 ± 3, 58.6 ± 5.5, 54.6 ± 3.7, and 52 ± 2.6, respectively. The response in the histatin-1 and histatin-1 + peptide 6 combination groups on day 7 was similar to the PBS group, with a percent reduction in keratocytes of 40.2 ± 3.3 and 42.4 ± 14.2, respectively. Both drug groups showed a significant reduction in keratocyte loss on days 10, 14, and 21. The percent reduction for the histatin-1 only group on days 10, 14, and 21 was 37.5±2.4, 32.7±15.3, and 26.5±6.0, respectively; whereas for the combination group, the percent reduction was 32.7±9.5, 28.1±13.7, and 3.7±4.4. Interestingly, comparison of the results on day 21 between the histatin-1 group and the histatin-1+peptide 6 combination group shows a statistically significant (p=0.0009) and meaningful difference indicating that inclusion of peptide 6 in the topical formulation appears to have increased the rate of keratocyte recovery.

[0205] FIG. 1B shows the results of the experiment up to day 28. The percent reduction (mean ± SD) from baseline in the PBS group on day 28 was 39.9 ± 4.2. Both the histatin-1 group and the histatin-1 + peptide 6 combination group showed a significant reduction in keratocyte loss on day 28. The percent reduction for the histatin-1 only group on day 28 was 2.7 ± 13; whereas for the combination group, the percent reduction was −8.8 ± 4.3. These data show complete recovery in the histatin-1 only group by day 28 and in the combination group by day 21, suggesting that the adjunctive use of peptide 6 provided an improvement in the time to recovery to about 7 days.

[0206] Example 2 Effect of histatin-1 on corneal endothelial cell density. method animal Sixteen New Zealand white rabbits, approximately 4-6 months old and weighing 2-3 kg, were divided into two groups of eight animals and administered either 35 μl of phosphate-buffered saline (PBS) or 35 μl of 80 μM histatin-1 topically three times per day (TID) for 28 days after unilateral surgical keratectomy procedure.

[0207] surgical keratectomy Animals were administered buprenorphine (0.01-0.05 mg / kg, SC) for pain management immediately prior to the procedure. A second dose of buprenorphine (0.01-0.05 mg / kg, SC) was administered in the evening and then as needed. Animals were induced for surgery with a ketamine / xylazine cocktail (50 mg / kg ketamine and 10 mg / kg xylazine IM); anesthesia was maintained with isoflurane delivered in oxygen through a mask as needed. After induction of anesthesia, 5% betadine solution was used on autoclaved sterile gauze to clean the periocular (lid) area of ​​the postoperative eye. Betadine was then used to irrigate the ocular surface and conjunctival fornix, which was then thoroughly irrigated with sterile eyewash. One drop each of 0.5% proparacaine HCl and 10% phenylephrine HCl was then applied to the ocular surface for ocular anesthesia and mydriasis, respectively. The rabbit was positioned under an operating microscope and an eyelid retractor was attached. An adjustable 8 mm corneal vacuum trephine (Moria Surgical, Antony, France) was used to create a central corneal defect of approximately 10-25% stromal depth (approximately 100 µm). The perforated section of cornea was removed, leaving the wound open. A single dose of topical antibiotic (neomycin polymyxin B gramicin sulfate ophthalmic solution) was applied immediately after surgery.

[0208] Corneal Tomography Corneas of anesthetized animals were imaged with a Heidelberg Retinal Tomograph equipped with a Rostock Corneal Module (HRT III RCM). This compact ophthalmic device uses confocal scanning laser microscopy to provide high-resolution surface images of corneal cells and structures, including the subbasal corneal plexus, keratocyte subpopulations, and the corneal endothelial cell layer. Central corneal confocal scans from epithelium to endothelium were performed, generating 400 × 400 μm and 2 μm thick optical stacks that were electronically stored for analysis.

[0209] Sampling protocol and data analysis The optical slice with the endothelial layer with the best resolution and greatest coverage was selected. Of the 16 animals in the study, only 14 animals had scans with an endothelial cell layer available at one or more time points, and only a maximum of four available scans at any single time point. HRT III cell counting software was used to adjust image contrast and brightness and to enhance manual cell counting within selected regions of interest (ROIs). ROIs were >0.005 mm 2 The cell counts within the ROI ranged from 118 to 287. Partial cells (>1 / 2) at the edge of the ROI were counted only on two perpendicular sides. The software calculated endothelial cell density as cells / mm based on manual counts in the selected ROI. 2 Data are expressed as mean ± standard deviation cells / mm 2 The results are presented as mean ± SEM. Unpaired Student's T-test was used to compare the PBS and histatin-1 groups at each follow-up time point; p < 0.05 was considered statistically significant.

[0210] result Figure 5 shows the results of the experiment. Surgical anterior keratectomy induced a loss of endothelial cell density that peaked on day 14 and remained suppressed until day 28. The percent reduction (mean ± SD) from baseline in the PBS group for days 7, 10, 14, 21, and 28 was 13.8 ± 2.6, 19.5 ± 9.6, 21.9 ± 3.4, 22.5 ± 8.4, and 20.2 ± 5.0, respectively. The response in the histatin-1 group on day 7 was similar to the PBS group, with a percent reduction in endothelial cell density of 15.1 ± 2.8 (p > 0.05). The percent reduction for the histatin-1 group for days 10, 14, 21, and 28 was 1.1 ± 9.1, -0.2 ± 5.9, 4.5 ± 7.8, and 7.4 ± 2.8, respectively. The histatin-1 response was significantly different from PBS (P<0.05) on days 14, 21, and 28. These data show recovery and apparent normalization in the histatin-1 group beginning by day 10.

[0211] Example 3 Effect of histatin-1 and peptide 6 on corneal nerve regeneration. method animal Twelve New Zealand white rabbits, approximately 4-6 months old and weighing 2-3 kg, were divided into three groups of four animals and administered either 35 μl phosphate buffered saline (PBS), 35 μl 80 μM histatin-1, or 35 μl 80 μM histatin-1 + 100 μM peptide 6 topically three times per day (TID) for 28 days following a unilateral surgical keratectomy procedure.

[0212] surgical anterior keratectomy Animals were administered buprenorphine (0.01-0.05 mg / kg, SC) for pain management immediately prior to the procedure. A second dose of buprenorphine (0.01-0.05 mg / kg, SC) was administered in the evening and then as needed. Animals were induced for surgery with a ketamine / xylazine cocktail (50 mg / kg ketamine and 10 mg / kg xylazine IM); anesthesia was maintained with isoflurane delivered in oxygen through a mask as needed. After induction of anesthesia, 5% betadine solution was used on autoclaved sterile gauze to clean the periocular (lid) area of ​​the postoperative eye. Betadine was then used to irrigate the ocular surface and conjunctival fornix, which was then thoroughly irrigated with sterile eyewash. One drop each of 0.5% proparacaine HCl and 10% phenylephrine HCl was then applied to the ocular surface for ocular anesthesia and mydriasis, respectively. The rabbit was positioned under an operating microscope and an eyelid retractor was attached. An adjustable 8 mm corneal vacuum trephine (Moria Surgical, Antony, France) was used to create a central corneal defect of approximately 10-25% stromal depth (approximately 100 µm). The perforated section of cornea was removed, leaving the wound open. A single dose of topical antibiotic (neomycin polymyxin B gramicin sulfate ophthalmic solution) was applied immediately after surgery.

[0213] Corneal whole-mount immunohistochemistry Preparation of the eye Eyes designated for immunohistochemistry were placed in phosphate-buffered saline (PBS) at room temperature (RT) and the corneas were excised with a 2-3 mm scleral edge using a #10 scalpel to create a small incision at the limbus, and each cornea was separated with curved scissors. The iris and lens were carefully peeled away using two pairs of forceps and discarded. The Descemet's membrane (DM) was removed by creating a 0.5-1 mm incision near the intersection of the clear cornea / sclera and the scalpel. An inverted Terry-Sinskey hook was then used to scrape and free the DM flap. The cornea was then transferred to a dish containing PBS and the DM flap was grasped and peeled away.

[0214] fixed Corneas were fixed for 2 hours in 2% paraformaldehyde formulated in 0.1M phosphate buffer, pH 7.4. After fixation, corneas were incubated in 1 / 1,000 mouse anti-TUJ1 antibody (BioLegend catalog number 801213) and 1 / 300 goat anti-Iba1 in 0.1M PBS containing 1% normal donkey serum and 0.15% Triton x-100 for 48 hours at 4°C. Corneas were then washed 5×5 minutes in PBS and incubated with 1 / 500 donkey anti-mouse Cy2, 1 / 500 donkey anti-goat Cy3, and 1 / 5,000 DAPI in 0.1M PBS containing 1% normal donkey serum and 0.15% Triton x-100 for 15 hours at 4°C, after which corneas were washed 5×5 minutes in PBS and mounted as described below.

[0215] Preparation of slides While the corneas were incubated in their final wash, slides with imaging spacers were prepared: 0.36, 0.6 mm, or a combination of the two adhesive imaging spacers (Grace Biolabs catalog number SA-S-3L; custom cut into 22×40 mm rectangles with an oval window cutout) were adhered to glass slides; spacer thickness was determined by the corneal thickness measured on the day of necropsy for the treated eyes; control corneas utilized a 0.36 mm spacer.

[0216] attachment After the final wash, curved scissors were used to remove nearly all of the scleral rim. Then, using a #10 scalpel blade, 4-6 small incisions were made around the periphery to flatten the cornea. The cornea was then carefully placed in the spacer window, endothelial side down, and 3-4 drops of mounting medium (90% glycerol, 0.5% N-propyl gallate, 20 mM Tris pH 8) were applied using a 2 mL transfer pipette. The slide was then gently coverslipped, taking care to avoid air bubbles. Slides were imaged on an Olympus Bx63 upright fluorescent microscope using a 4X phosphor objective, and images were acquired using Olympus CellSens software. The central corneal area was imaged by tiling to create a composite image of the entire surgical area.

[0217] Data analysis Greyscale Tuj1 images from Olympus CellSens software were imported into Fuji ImageJ software (NIH). The system was calibrated to a mm scale on the image. An 8 mm circle was then created and centered on the image to capture the area with the initial injury. The area outside the 8 mm circle was removed. The image was sharpened to better visualize the nerves and divided into quadrants for ease of analysis. The freehand selection tool was then used to map the area in the injury area that contained the regenerating nerves. The measurement tool was used to measure the area in mm 2 The data was read at 50.3 mm. 2 The results are expressed as a percentage of the initial 8 mm lesion area and presented as the mean ± standard deviation.

[0218] result Figure 6 shows the results of the experiment. Surgical keratectomy resulted in approximately 70% reduction in the area covered by corneal nerves on day 28. The area of ​​nerve regeneration in animals treated with PBS was 16.3 ± 7 mm 2 The area of ​​nerve regeneration in eyes treated with histatin-1 was 26.1 ± 7 mm, which is 32.4 ± 14% of the initial lesion area. 2The adjunctive use of peptide 6 produced the strongest effect: the area of ​​nerve regeneration was 31.4 ± 12 mm, which corresponds to 63.4 ± 11% of the lesion area. 2 These data indicate better nerve regeneration in the lesion area, with the following ranked order of efficacy: histatin-1 + peptide 6 > histatin-1.

[0219] Example 4 Effect of histatin-1 on corneal button preservation. method Life4 o The effect of 1 μM histatin-1 formulated in Bausch & Lomb™ Corneal Storage Medium or Optisol™ Corneal Storage Medium was evaluated on the preservation of endothelial cells in nine pairs of human non-surgical corneal buttons stored under stress conditions. After storage at 4°C for 3 days, tissues were stored individually in 20 mL Bausch & Lomb™ Cornea Tissue Viewing chambers at room temperature (24 ± 2°C) for 6 to 14 days. All donors were White / Caucasian. Median age was 64 years, with a range of 14 to 73. The amount of time from time of death to storage of the cornea ranged from 4 hours 15 minutes to 20 hours 48 minutes. Endothelial cell counts at the time of tissue collection were 2222 ± 240 cells / mm 2Histatin-1 was added daily to the right eye (OD) in a volume of 200 μL in an amount sufficient to maintain a concentration of 1 μM. The left eye (OS) received an equal volume of PBS. Endothelial cell viability was assessed by trypan blue staining as nonviable area expressed as a percentage of 80% of the central cornea. Briefly, corneal buttons were transferred endothelial side up from the storage chamber to an observation well (Baron punch cutting block) and 0.06% trypan blue was applied in a volume sufficient to cover the endothelial layer limbus-to-limbus for 90 seconds, then washed twice with PBS. Uptake of trypan blue stain indicated epithelial cell death, so lesser extent of staining correlates with more viable tissue. Corneas were then imaged with a Zeiss Surgical Scope, model # STMI 508, equipped with a Zeiss eyepiece camera model # AXIOCCM 208. Data were analyzed using Image J (NIH) with "Weka Segmentation" software.

[0220] result The stained areas in the PBS and histatin-1 groups were 22.0±5.8% and 12.1±2.9%, respectively; mean±SEM (see FIG. 7). Thus, the addition of histatin-1 to the corneal buttons resulted in a 45% reduction in the stained area in the endothelial area. These data suggest that human corneal buttons incubated with 1 μM histatin-1 in standard corneal storage medium under stress conditions, such as at room temperature, results in significant preservation of endothelial cell loss and thus enhanced preservation of corneal tissue during storage.

Claims

1. 1. A composition for administration to the eye or skin of a subject in need thereof, comprising a histatin or analog thereof, and / or a ciliary neurotrophic factor (CNTF) peptide or analog thereof, and optionally an anti-apoptotic agent, the composition comprising: A composition for use in a method of reducing or preventing the loss of keratocytes, keratinocytes, and / or keratoblasts, restoring keratocytes, keratinocytes, and / or keratoblasts, inhibiting apoptosis to prevent cell loss in the eye or skin caused by injury or disease, preventing or minimizing refractive regression and subepithelial scarring, recruiting keratocytes, keratinocytes, and / or keratoblasts, or treating corneal wounds.

2. The composition of claim 1, wherein the histatin or analog thereof is histatin-1 (SEQ ID NO: 4).

3. 2. The composition of claim 1, wherein the ciliary neurotrophic factor (CNTF) peptide is peptide 6 (SEQ ID NO: 34).

4. The composition of claim 1 , wherein the composition is administered as an eye drop, a gel, an ointment, or a tissue adhesive.

5. The composition of claim 1 , wherein the composition is administered in an ocular insert or contact lens.

6. The composition of claim 1 , wherein the loss of keratocytes, keratinocytes, and / or keratoblasts is caused by an ocular infection, corneal injury, ocular surgery, ocular injury, ocular disease, or ocular disorder.

7. The composition of claim 1 , wherein the loss of keratocytes, keratinocytes, and / or keratoblasts is caused by skin photodamage.

8. The composition of claim 1 , wherein the loss of keratocytes, keratinocytes, and / or keratoblasts is caused by chemotherapy.

9. A composition for use in a method for treating or prophylactically treating a skin or ophthalmic disease, disorder, or condition, comprising a histatin or analog thereof and / or a ciliary neurotrophic factor (CNTF) peptide or analog thereof for administration to the eye or skin of a subject in need thereof.

10. The composition of claim 9 , wherein the composition further comprises an anti-apoptotic agent.

11. The skin disease, disorder, or condition is ２ 10. The composition of claim 9, wherein the wound is a burn, a decubitus ulcer, autonomic dysreflexia in spinal cord injury, diabetic ischemic ulcer, ulcer prophylaxis, skin graft recovery, trauma resulting in skill loss, post cryo / electrodesiccation recovery, or post Mohs / skin cancer wound healing.

12. 10. The composition of claim 9, wherein the ophthalmic disease, disorder, or condition is post-photorefractive keratectomy (PRK) surgery, glaucoma surgery, infectious corneal ulcer, pigmentary eye disease, post-collagen crosslinking (CSX) for keratoconus, neurotrophic keratitis, Fuchs' corneal dystrophy, contact lens intolerance, dry eye disease, keratitis progression inhibition, posterior polymorphous corneal dystrophy (PPCD), aphakic or pseudophakic bullous keratopathy (ABK / PBK), endothelial dysfunction caused by penetrating or blunt trauma, congenital hereditary endothelial dystrophy (CHED), iridocorneal endothelial (ICE) syndrome, refractory glaucoma, previous failed corneal graft, or herpes simplex virus endotheliitis.

13. 13. The composition of claim 12, wherein the ophthalmic disease, disorder, or condition is post-photorefractive keratectomy, glaucoma surgery, or Fuchs corneal dystrophy.

14. The composition of claim 9, wherein the histatin or analog thereof is histatin-1 (SEQ ID NO: 4).

15. 10. The composition of claim 9, wherein the ciliary neurotrophic factor (CNTF) peptide or analog thereof is peptide 6 (SEQ ID NO: 34).

16. 1. A method of storing a biological sample prior to a surgical procedure, comprising: contacting the biological sample with a composition comprising a histatin or an analog thereof and / or a CNTF peptide or an analog thereof, and optionally an anti-apoptotic agent; and storing said biological sample at a temperature of about −10° C. to about 25° C. A method comprising:

17. 17. The method of claim 16, wherein the biological sample is donor tissue.

18. The method of claim 17 , wherein the donor tissue is corneal tissue.

19. 17. The method of claim 16, wherein the biological sample remains viable for about 0.5 days to about 90 days.

20. 17. The method of claim 16, wherein the surgical procedure is photorefractive keratectomy (PRK) surgery, Descemet's membrane stripping automated endothelial keratoplasty (DSAEK), or Descemet's membrane endothelial keratoplasty (DMEK).

Images