[0016] Pro nerve growth factor (pro-NGF) is a complex of three proteins: alpha NGF, beta NGF, and gamma NGF. Pro-NGF is processed by autoproteolytic cleavage of the N-terminal of the beta subunit, performed by the gamma subunit, to yield native NGF.

[0017] As used herein, “recombinant human nerve growth factor (rhNGF)” refers to cenergermin or human beta-NGF, residues 1-118 (SEQ ID NO: 2) formulated as a non-covalent dimer and produced in E. coli. The full length human beta nerve growth factor amino acid sequence is listed as SEQ ID NO: 1.

[0018] The disclosed compositions may be administered to a subject in need thereof once daily, twice daily, three times daily, four times daily, five times daily, six times daily or more. The disclosed compositions may be administered to a subject in need thereof for about one day, about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about ten days, about eleven days, about tw elve days, about thirteen days about fourteen days, or more as determined by a physician. [0019] The disclosed compositions may be administered by any means known to those skilled in the art, including, but not limited to, oral, topical, intranasal, intraperitoneal, parenteral, intravenous, intramuscular, subcutaneous, intrathecal, transcutaneous, nasopharyngeal, intra-lesional, intradermal, or transmucosal absorption. Thus, the compositions may be formulated as an ingestible, injectable, topical, ophthalmic topical, or suppository formulation. The compositions may also be delivered within a liposomal or time-release vehicle. Administration of the compositions to a subject in accordance with the invention may exhibit beneficial effects in a dose-dependent manner. Thus, within broad limits, administration of larger quantities of the compositions is expected to achieve increased beneficial biological effects than administration of a smaller amount. Moreover, efficacy is also contemplated at dosages below the level at which toxicity is seen.

[0020] It will be appreciated that the specific dosage administered in any given case will be adjusted in accordance with the composition or compositions being administered, the disease to be treated or inhibited, the condition of the subject, and other relevant medical factors that may modify the activity of the compositions or the response of the subject, as is well known by those skilled in the art. For example, the specific dose for a particular subject depends on age, body weight, general state of health, diet, the timing and mode of administration, the rate of excretion, medicaments used in combination, and the severity of the particular disorder to which the therapy is applied. Dosages for a given patient can be determined using conventional considerations, e.g., by customary comparison of the differential activities of the compositions described herein and of a known agent, such as by means of an appropriate conventional pharmacological protocol.

[0021] The maximal dosage for a subject is the highest dosage that does not cause undesirable or intolerable side effects. The number of variables in regard to an individual treatment regimen is large, and a considerable range of doses is expected. The route of administration will also impact the dosage requirements. It is anticipated that dosages of the compositions will improve the condition being treated by at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or more as compared to no treatment.

[0022] The compositions, including ophthalmic formulations, with the disclosed compositions may also include an extended-release vehicle. And an extended-release vehicle may be a biocompatible polymer, dissolved in the carrier or by itself impregnated with the trophic factors to hold the trophic factors and slowly release the drug to the subject, preferably for an extended- release period, e.g., one day, two days, three days, four days, five days, six days, seven days, or more. The biocompatible polymer may be biodegradable or non-biodegradable, depending on desired use and application schedule. Example biocompatible polymers that may be used in the disclosed formulations as an extended-release vehicle include but are not limited to poly-2- hydroxycthylmethacrylate (p-HEMA hydrogels), poly(lactic-co-glycolic) acid (PLGA), polycaprolactone (PCL), hydroxypropyl cellulose, Anecortave acetate (AnA), gelatin, and/or collagen. The inclusion of an extended-release vehicle may, in some cases, allow for less frequent application while still providing effective dosing of the trophic factors.

[0023] The term '‘pharmaceutically acceptable” means that which is useful in preparing a pharmaceutical composition that is generally safe, non-toxic, and neither biologically nor otherwise undesirable, and includes that which is acceptable for use in a subject, including a human subject.

[0024] The terms “pharmaceutically acceptable carrier,” or “excipient” as used herein, mean a nontoxic, inert solid, semi-solid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type. The disclosed pharmaceutical compositions may be formulated for administration by, for example, solid dosing, oral, a topical formulation, injection, inhalation (either through the mouth or the nose), implants, oral, buccal, parenteral, or rectal administration. Techniques and formulations and acceptable pharmaceutically acceptable carriers may generally be found in "Remington's Pharmaceutical Sciences", (Meade Publishing Co., Easton, Pa.). Therapeutic compositions typically are sterile and stable under the conditions of manufacture and storage.

[0025] Suitable pharmaceutically acceptable carriers include, but are not limited to, diluents, preservatives, solubilizers, emulsifiers, liposomes, nanoparticles and adjuvants. Some examples of materials which can serve as pharmaceutically acceptable carriers are sugars such as, but not limited to, lactose, glucose and sucrose; starches such as, but not limited to, com starch and potato starch; cellulose and its derivatives such as, but not limited to, sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; powdered tragacanth; malt; gelatin; talc; excipients such as, but not limited to, cocoa butter and suppository waxes; oils such as, but not limited to, peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, com oil and soybean oil; glycols; such as propylene glycol; esters such as, but not limited to, ethyl oleate and ethyl laurate; agar; buffering agents such as, but not limited to, magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol, and phosphate buffer solutions, as well as other non-toxic compatible lubricants such as, but not limited to, sodium lauryl sulfate and magnesium stearate, as well as coloring agents, releasing agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the composition, according to the judgment of the formulator.

[0026] Additionally, pharmaceutically acceptable carriers may be aqueous or non-aqueous solutions, media, suspensions, and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include isotonic solutions, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.

[0027] In some embodiments, the composition, such as a pharmaceutical composition, is formulated for local delivery, for example topical delivery. Suitable additional components for local or topical delivery’ are known in the art and include creams, gels, and controlled release drug delivery’ materials (for example, but not limited to, e.g., PCNU, PGLA, etc.).

[0028] Compositions of the present disclosure may include liquids, lyophilized, or otherwise dried formulations and may include diluents of various buffer content (e.g., Tris-HCl, acetate, phosphate), pH and ionic strength, additives such as albumin or gelatin to prevent absorption to surfaces, detergents (e. g., Tween 20, Tween 80, Pluronic F68, bile acid salts), solubilizing agents (e.g., glycerol, polyethylene glycerol), anti-oxidants (e.g.. ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimerosal, benzyl alcohol, parabens), bulking substances or tonicity modifiers (e.g., lactose, mannitol), covalent attachment of polymers such as polyethylene glycol to the polypeptide, complexation with metal ions, or incorporation of the material into or onto particulate preparations of polymeric compounds such as polylactic acid, polygly colic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility⁷, stability⁷, rate of in vivo release, and rate of in vivo clearance. Controlled or sustained release compositions include formulation in lipophilic depots (e.g., fatty acids, waxes, oils). [0029] The compositions can be sterilized by conventional, well-known sterilization techniques. The compositions may contain pharmaceutically acceptable additional substances as required to approximate physiological conditions such as a pH adjusting and buffering agent, toxicity adjusting agents, such as, sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, and the like.

[0030] The disclosed trophic factors, or pharmaceutical compositions thereof, described herein may be administered one time or more than one time to the subject to effectively improve the condition being treated, e.g., neurotrophic keratopathy. Suitable dosage ranges are of the order of several hundred micrograms effective ingredient with a range from about 0.01 to 50 mg/kg/day, preferably in the range from about 0. 1 to 1 mg/kg/day. Precise amounts of effective ingredient required to be administered depend on the judgment of the practitioner and may be peculiar to each subject. It will be apparent to those of skill in the art that the therapeutically effective amount of the trophic factors described herein will depend, inter alia, upon the administration schedule, whether the composition is administered in combination with other therapeutic agents, the status and health of the recipient, and the therapeutic activity of the particular composition.

[0031] Methods

[0032] In an aspect of the current disclosure, methods of treating neurotrophic keratopathy in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a p57^NTR inhibitor, e.g., THX-B and/or anti-pro NGF, and a therapeutically effective amount of a TrkA agonist, such as recombinant NGF (rNGF), e.g., recombinant human NGF (rhNGF), or the NGF mimetic Tavilermide (MIMD3), to the subject to treat the neurotrophic keratopathy in the subject. The method may comprise administering an anti-pro-NGF antibody, e.g., a monoclonal antibody, and one or more of rNGF (or rhNGF) and THX-B.

[0033] “Treating neurotrophic keratopathy,” as used herein, may comprise one or more of improvement of comeal healing, improved fluorescein exclusion by the cornea, improved epithelial renewal in the cornea, improved transparency of the cornea, or reduction of opacity in the cornea. Methods of assessing the rate of comeal healing are known in the art and demonstrated in the Examples section and may comprise assaying the cornea with fluorescein for an intact corneal barrier that excludes fluorescence. In contrast, corneas that have not fully healed will appear fluorescent under excitation because they have not excluded fluorescein.

[0034] A subject in need thereof, as used herein, may refer to subject with subject about 50% or less comeal innervation, about 40% or less comeal innervation, about 30% or less comeal innervation, about 20% or less comeal innervation, about 10% or less comeal innervation, or a subject with no comeal innervation. A subject in need thereof may be a subject suffering from comeal anesthesia, e.g., congenital comeal anesthesia.

[0035] The inventors discovered that, surprisingly, administration of a p75^NTR inhibitor, e.g., THX-B or anti-pro NGF, and rNGF (e.g., rhNGF) or the NGF mimetic Tavilermide (MIMD3), results in a complete recovery of the comeal epithelium in a single treatment. See, FIG. 2C and 2E. Furthermore, the inventors discovered that the disclosed methods resulted in a transparent cornea. See, FIG. 2D and 2F.

[0036] In another aspect of the cunent disclosure, methods of improving comeal transparency after comeal injury in a subject in need thereof are provided. As discussed above, the inventors discovered that, surprisingly, administration of the disclosed pharmaceutical compositions resulted in a transparent cornea in animals with denervated corneas, in comparison to animals treated with vehicle control which, though the corneas healed, developed opacity during healing. Methods of measuring the overall opacity of the cornea are known in the art and may be performed by a physician, e.g., by visual assessment.

[0037] Methods of measuring the rate of comeal epithelial renewal are known in the art and may comprise visual assessment, histological assessment of the cornea, or other methods know n in the art. Improving comeal epithelial renewal may improve one or more symptom related to reduced comeal epithelial renewal, which is needed for maintenance of the comeal barrier, and may include, but is not limited to, improved comeal wound closure, improved healing of comeal microtraumas, reduced incidence of infection in the eye, e.g., in the setting of NK.

[0038] As used herein, “comeal innervation” refers to the density and function of sensory nerve endings in the cornea. Methods of measuring comeal innervation are known in the art and may be performed by, e.g., histology.

[0039] As used herein, “comeal anesthesia” refers to a lack of sensation in the cornea. Comeal anesthesia can be a result of a congenital condition, or damage to a branch of the trigeminal nerve which innervates the cornea. Relative sensation in the cornea can be measured by, e.g., Cochet-Bonnet aesthesiometer by one of skill in the art.

[0040] As used herein, a “subject” may be any mammal, suitably a human, domesticated animal such as a dog, cat, horse, cow, pig, or a mouse or rat. A “subject in need thereof’ is. in some embodiments, a subject diagnosed with a neurotrophic keratopathy, or a subject suffering from lack or corneal innervation, or a subject suspected of developing neurotrophic keratopathy in the future. In some embodiments, a subject in need thereof is a subject who has chronically elevated blood sugar who is at risk of developing diabetic keratopathy. In some embodiments, a subject in need thereof has been diagnosed with type 1 diabetes. In some embodiments, a subject in need thereof has been diagnosed with type 2 diabetes.

[0041] In some embodiments, a subject in need thereof is a subject in need of improving comeal healing, e.g., a subject with reduced comeal innervation, which can be detected by assaying comeal sensation in the subject. Relatedly, a subject in need thereof may be a subject in need of improving comeal epithelial renewal.

[0042] In some embodiments, a subject in need thereof is a subject that has suffered a comeal injury' and has a reduced likelihood of the comeal injury healing without the development of opacity in the cornea.

[0043] As used herein the term “effective amount” or “therapeutically effective amount” refers to the amount or dose of the compound, upon single or multiple dose administration to the subject, which provides the desired effect in the subject under diagnosis or treatment. The disclosed methods may include administering an effective amount of the disclosed compounds (e.g. , as present in a pharmaceutical composition) for treating a subject in need thereof, as defined above.

[0044] A “therapeutically effective amount,” also referred to as an “effective amount,” can be readily determined by the attending diagnostician, as one skilled in the art. by the use of known techniques and by observing results obtained under analogous circumstances. In determining the effective amount or dose of compound administered, a number of factors can be considered by the attending diagnostician, such as: the species of the subject; its size, age, and general health; the degree of involvement or the severity of the disease or disorder involved; the response of the individual subject; the particular compound administered; the mode of administration; the bioavailability characteristics of the preparation administered; the dose regimen selected; the use of concomitant medication; and other relevant circumstances. A therapeutically effective amount of THX-B or anti-pro NGF may comprise about 1 mg/ml to about 50 mg/ml or about 20-30 mg/ml administered topically to the eye. In some embodiments, THX-B is administered at about 27 mg/ml topically to the eye.

[0045] A therapeutically effective amount of rNGF, e.g., rhNGF, or Tavilermide may be about 1 mg/ml to about 100 mg/ml or about 50 mg/ml. A therapeutically effective amount of rNGF, e.g., rhNGF, or Tavilermide may be about 1 mg/ml, about 5 mg/ml, about 10 mg/ml, about 15 mg/ml, about 20 mg/ml, about 25 mg/ml, about 30 mg/ml, about 35 mg/ml, about 40 mg/ml, about 45 mg/ml, about 50 mg/ml, about 55 mg/ml, about 60 mg/ml, about 65 mg/ml, about 70 mg/ml, about 75 mg/ml, about 80 mg/ml, about 85 mg/ml, about 90 mg/ml, about 95 mg/ml, or about 100 mg/ml, or greater than 100 mg/ml, as determined by a physician.

[0046] The effective dosage amounts described herein refer to total amounts administered, that is, if more than one composition is administered, the effective dosage amounts correspond to the total amount administered. Treatment, i.e., p75^NTR inhibitor (THX-B or anti-pro NGF) and a TrkA agonist (rNGF or NGF mimetic/Tavilermide). can be administered as a single dose or as divided doses. For example, the treatment may be administered two or more times separated by 4 hours, 6 hours, 8 hours, 12 hours, a day, two days, three days, four days, one week, two weeks, or by three or more weeks. Topical ophthalmic formulations may be administered once daily, twice daily, three times daily, four times daily, five times daily, six times daily or more. The methods may comprise administering a p75^NTR inhibitor (THX-B or anti-pro NGF) and a TrkA agonist (rNGF, e.g., rhNGF, or NGF mimetic/Tavilermide), to a subject in need thereof for about one day, about two days, about three days, about four days, about five days, about six days, about seven days, about eight days, about nine days, about ten days, about eleven days, about twelve days, about thirteen days, about fourteen days, or more as determined by a physician.

[0047] The disclosed methods may comprise administering the p75^NTR inhibitor, e.g., THX-B or anti- pro NGF, and the rNGF, e.g., rhNGF, or NGF mimetic/Tavilermide simultaneously or sequentially. For example, THX-B or anti-pro NGF, and rhNGF or Tavilermide may be administered at the same time, e.g., in a single pharmaceutical composition. Alternatively, a dose of THX-B or anti-pro NGF could be administered first, then rNGF (or rhNGF) or Tavilermide administered subsequently, e g., in the same day, or a dose of rNGF (or rhNGF) or Tavilermide could be administered first, then THX-B or anti-pro NGF administered subsequently, e.g., in the same day.

[0048] Administration may comprise parenteral administration. Administration may comprise topical administration to the eye of a subject in need thereof. Administration may be performed unilaterally or bilaterally, as directed by a physician.

[0049] Administration may be performed for several consecutive days followed by a pause in the administration, whereupon administration may cease entirely or be resumed, as prescribed by a physician.

[0050] Kits

[0051] In an aspect of the current disclosure, kits are provided. In some embodiments, the kits comprise THX-B and/or anti-pro NGF, and one or more of rNGF, rhNGF, and Tavilermide, and instructions for administering the THX-B and/or anti-pro NGF, and one or more of rNGF, rhNGF, and Tavilermide. at least once daily to the eye, e.g., once daily, twice daily, three times daily, four times daily, five times daily, six times daily, or more to the eye. The THX-B and/or anti-pro NGF, and one or more of rNGF, rhNGF, and Tavilermide, may be formulated for topical administration to the eye. The kits may comprise an anti-pro-NGF antibody, e.g., a monoclonal antibody.

[0052] Definitions

[0053] The disclosed subject matter may be further described using definitions and terminology as follows. The definitions and terminology used herein are for the purpose of describing particular embodiments only and are not intended to be limiting.

[0054] As used in this specification and the claims, the singular forms “a,” ‘"an,"’ and “the” include plural forms unless the context clearly dictates otherwise. For example, the term “a substituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0055] As used herein, “about”, “approximately,” “substantially,” and “significantly” will be understood by persons of ordinary skill in the art and will vary’ to some extent on the context in which they are used. If there are uses of the term which are not clear to persons of ordinary skill in the art given the context in which it is used, “about” and “approximately’” will mean up to plus or minus 10% of the particular term and “substantially” and “significantly” will mean more than plus or minus 10% of the particular term.

[0056] As used herein, the terms “include” and “including” have the same meaning as the terms “comprise” and “comprising.” The terms “comprise” and “comprising” should be interpreted as being “open” transitional terms that permit the inclusion of additional components further to those components recited in the claims. The terms “consist” and “consisting of’ should be interpreted as being “closed” transitional terms that do not permit the inclusion of additional components other than the components recited in the claims. The term “consisting essentially of’ should be interpreted to be partially closed and allowing the inclusion only of additional components that do not fundamentally alter the nature of the claimed subject matter.

[0057] The phrase “such as” should be interpreted as “for example, including.” Moreover, the use of any and all exemplary language, including but not limited to “such as”, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed.

[0058] Furthermore, in those instances where a convention analogous to “at least one of A, B and C, etc.” is used, in general such a construction is intended in the sense of one having ordinary skill in the art would understand the convention (e.g, “a system having at least one of A, B and C” would include but not be limited to systems that have A alone, B alone, C alone, A and B together, A and C together, B and C together, and/or A, B, and C together.). It will be further understood by those within the art that virtually any disjunctive word and/or phrase presenting two or more alternative terms, whether in the description or figures, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase “A or B” will be understood to include the possibilities of “A” or ‘B or “A and B.”

[0059] All language such as “up to,” “at least,” “greater than,” “less than,” and the like, include the number recited and refer to ranges which can subsequently be broken down into ranges and subranges. A range includes each individual member. Thus, for example, a group having 1-3 members refers to groups having 1, 2, or 3 members. Similarly, a group having 6 members refers to groups having 1, 2, 3, 4, or 6 members, and so forth. [0060] The modal verb “may” refers to the preferred use or selection of one or more options or choices among the several described embodiments or features contained within the same. Where no options or choices are disclosed regarding a particular embodiment or feature contained in the same, the modal verb “may” refers to an affirmative act regarding how to make or use and aspect of a described embodiment or feature contained in the same, or a definitive decision to use a specific skill regarding a described embodiment or feature contained in the same. In this latter context, the modal verb “may” has the same meaning and connotation as the auxiliary verb “can.”

[0061] Illustrative Embodiments

[0062] The following embodiments are exemplary' only and should not be interpreted to limit the scope of the claimed subject matter.

[0063] Embodiment 1 is a method of treating neurotrophic keratopathy in a subject in need thereof, the method comprising administering a therapeutically effective amount of a p75^NTR inhibitor, such as THX-B and/or anti-proNGF, and a therapeutically effective amount of at least one TrkA agonist selected from recombinant nerve growth factor (rNGF), recombinant human NGF (rhNGF), and NGF mimetic Tavilermide to the subject to treat the neurotrophic keratopathy in the subject.

[0064] Embodiment 2 is the method of embodiment 1, wherein the subject has about 50% or less comeal innervation, about 40% or less comeal innervation, about 30% or less comeal innervation, about 20% or less comeal innervation, about 10% or less comeal innervation, or wherein the subject has no comeal innervation.

[0065] Embodiment 3 is the method of embodiment 1 or 2, wherein the subject suffers from comeal anesthesia.

[0066] Embodiment 4 is the method of embodiment 3, wherein the comeal anesthesia is congenital comeal anesthesia.

[0067] Embodiment 5 is the method of any one of embodiments 1-4, wherein the method results in complete recovery of the comeal epithelium. [0068] Embodiment 6 is the method of any one of embodiments 1-5, wherein the method induces complete comeal re-epithelialization with one treatment of THX-B or anti-proNGF, and one or more of rNGF, rhNGF. and Tavilermide.

[0069] Embodiment 7 is the method of embodiment 5, wherein the method results in a transparent cornea.

[0070] Embodiment 8 is the method of embodiment 6, wherein the method results in a transparent cornea after at least 10 days of administration of THX-B or anti-proNGF, and one or more of rNGF, rhNGF, and Tavilermide.

[0071] Embodiment 9 is the method of any one of embodiments 1-7, wherein administration comprises once daily topical administration.

[0072] Embodiment 10 is the method of any one of embodiments 1-8. wherein the therapeutically effective amount of THX-B or anti-proNGF comprises 20-30 mg/ml and wherein administering comprises topical administration.

[0073] Embodiment 11 is the method of embodiments 1-10, wherein the therapeutically effective amount of THX-B comprises 27 mg/ml.

[0074] Embodiment 12 is a method of improving comeal transparency after comeal injury in a subject in need thereof, the method comprising administering a therapeutically effective amount of THX-B and/or anti-proNGF, and a therapeutically effective amount of one or more of recombinant nerve growth factor (rNGF), recombinant human nerve grow th factor (rhNGF), and Tavilermide to the subject to improve comeal transparency after comeal injury' in the subject.

[0075] Embodiment 13 is the method of embodiment 12, wherein the subject has about 50% or less comeal innervation, about 40% or less comeal innervation, about 30% or less comeal innervation, about 20% or less comeal innervation, about 10% or less comeal innervation, or wherein the subject has no comeal innervation.

[0076] Embodiment 14 is the method of embodiments 12 or 13, wherein the subject suffers from comeal anesthesia. [0077] Embodiment 15 is the method of embodiment 14, wherein the comeal anesthesia is congenital corneal anesthesia.

[0078] Embodiment 16 is the method of any one of embodiments 12-15, wherein the subject is suffering from neurotrophic keratopathy.

[0079] Embodiment 17 is the method of any one of embodiments 12-16, wherein the method results in complete recovery of the corneal epithelium.

[0080] Embodiment 18 is the method of any one of embodiments 12-17, wherein the therapeutically effective amount of THX-B or anti-proNGF comprises 20-30 mg/ml and wherein administering comprises topical administration.

[0081] Embodiment 19 is the method of any one of embodiments 12-18, wherein the therapeutically effective amount of THX-B comprises 27 mg/ml.

[0082] Embodiment 20 is the method of any one of embodiments 12-19, wherein the method results in a transparent cornea.

[0083] Embodiment 21 is the method of embodiment 20, wherein the method results in a transparent cornea after at least 10 days of administration of THX-B and/or anti-proNGF, and one or more of rNGF, rhNGF, and Tavilermide.

[0084] Embodiment 22 is a pharmaceutical composition comprising THX-B and/or anti-proNGF, and one or more of rNGF, rhNGF, and Tavilermide, formulated for once daily administration to the eye of a subject.

[0085] Embodiment 23 is the pharmaceutical composition of embodiment 22, wherein the pharmaceutical composition is formulated for topical administration to the eye of a subject.

[0086] Embodiment 24 is a kit comprising THX-B and/or anti-pro NGF, and one or more of rNGF, rhNGF. and Tavilermide. and instructions for administering the THX-B and rhNGF once daily to the eye. [0087] Embodiment 25 is the kit of Embodiment 24, wherein the THX-B and/or anti-pro NGF, and the one or more of rNGF, rhNGF, and Tavilermide, are formulated for topical administration to the eye.

EXAMPLES

[0088] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.

[0089] Example 1 - Treatment of denervated injured corneas with p75^NTR inhibitor, e.g., THX-B, results in improved rate of corneal healing

[0090] A single stereotactic ablation of the ophthalmic maxillary branch of the trigeminal nerve¹ was performed to induce corneal denervation. On day five after denervation, the comeal epithelium was removed with an Amoils brush and immediately treated with the selective p75^NTR inhibitor THX-B². THX-B was administrated to the wounded cornea at 27mg/ml, starting from the moment of denervation, then every 24h during the experiment. Corneal wound healing in the THX-B-treated group was compared with the vehicle-treated group (two rats per group) daily by fluorescein staining. In agreement with our hypothesis, topical THX-B treatment accelerated recovery of the wounded cornea four days post-denervation compared to the vehicle- treated rats (Figs 2B,C).

[0091] Inhibition of p75^NTR induced near-complete wound healing in 24 hours and complete epithelial healing after 48 hours, similar to the healing that occurs in normally innervated, untreated corneas. To exclude the possibility of any neuronal survival after the denervation procedure, we performed immunostaining for bill tubulin. There was a complete absence of nerves in the THX-B treated corneas (not shown). Initial biochemical analysis of harvested corneas indicated upregulation of JNK1 activity following denervation and its inhibition by THX-B (not shown). Inhibition of p75^NTR in the denervated cornea was associated with an increase in Ki67-positive proliferating cells at the limbus (not shown). Although, yet to be validated with more biological samples, together these initial results indicate that p75^NTR inhibition-mediated corneal w ound healing happens independently of the neuronal trophic support and suggest that p75^NTR negatively regulates the activity of LSC. [0092] The reason for the change in the balance between TrkA and p75^NTR activities, following corneal denervation or SC ablation was investigated. The scRNA-seq analysis indicated that following denervation, MLNCs (that usually do not express detectible NGF) start expressing NGF mRNA in significantly higher levels compared to SC in a healthy cornea. A change in the balance between NGF and pro-NGF expression, in favour of pro-NGF, was reported for neurodegenerative conditions in another system. Following denervation, this NGF of MLNC origin could potentially serve as an axonal attraction signal, as well as an activator of the limbal TrkA. designed to induce comeal re-innervation and maintain the epithelial renewal, respectively. At the same time, pro-NGF, responsible for activation of p75^N1R, may affect activity and/or survival of LSC. This hypothesis is supported by the observation that neutralization of pro-NGF accelerates recovery of acutely denervated & de-epithelialized corneas. It is expected that in nerve-intact deepithelialized corneas, TrkA will be activated and p75^NTR levels and JNK.I activity will be low in the proliferating LSC. Following denervation, p75^NTR levels and phosphorylated JNK will be elevated, with little proliferation of LSC. To further validate and crosscheck the initial results, we will perform immunohistochemical and western blot analyses with anti-TrkA, pTrkA, JNK.1/2, and pJNKl/2 and double stain with LSC markers an EdU in EdU-treated mice, comparing between experimental conditions. A clear phenoty pic similarity is seen between the effects of (i) SC ablation, (ii) inhibition of TrkA and (iii) denervation on comeal w ound healing. Therefore, the effect of p75^NTR inhibition on epithelial renewal in transgenic mice with SC-ablated corneas will also be examined. It is expected that in the absence of trophic factors, including NGF secreted by SC and. possibly. pro-NGF secreted by the MLNCs, p75^NGF activity will be unchecked, while inhibition of p75^NTR activity will critically rescue LSC survival and/or activity, and enhance comeal healing. To genetically validate the results obtained using the biochemical approach, the proposed experiments in an inducible p75^NTR knockout crossed with the Krtl-15^cre/PGR mouse will be repeated, to allow a specific knockout of p75^NTR in LSC of the double mutant mouse. It is anticipated that prolonged LSC survival and enhanced comeal healing following comeal denervation in this mouse model. The initial results suggest that TrkA enhances and p75^NTR suppresses comeal epithelial renewal. Increased p75^NTR activity could cause a complete loss of the LSC pool after prolonged denervation. This observation would explain why NGF treatment is not as efficient in the more severe NK cases. Without wishing to be limited by any theory or mechanism, it is believed that in acutely denervated, de-epithelialized corneas, TrkA mediates the recover^' of the corneas and inhibition of p75^NTR activity accelerates the epithelial recovery.

[0093] Example 2 - NGF Acts Synergistically With p75NTR Inhibitor to Induce Epithelial Healing of Denervated Cornea

[0094] To test the effect of the combination of both nerve growth factor (NGF) or p75^NTR inhibitor, e.g., THX-B as a treatment for neurotrophic keratitis (NK), a single stereotactic ablation of the ophthalmomaxillary branch of the trigeminal nerve was performed to induce comeal denervation, treated the denervated corneas with NGF alone, THX-B alone, or a combination of NGF and THX-B daily beginning five days after the ablation procedure (FIG. 2C). Subjects’ eyes were treated with fluorescein - fluorescent signal indicates a lack of comeal epithelial layer in the images in FIG. 2C.

[0095] It was discovered that administration of either NGF or p75^TR inhibitor, e.g.. THX-B, improved comeal healing in a model of neurotrophic keratitis (NK). Surprisingly, the combination of both NGF and THX-B synergized to improve epithelialization in denervated corneas after a single administration (Fig. 2C, THX-B+NGF 24h vs. either NGF or THX-B alone at 24h). Though each of THX-B and NGF produced improvements in healing of the comeal epithelium, both treatments alone resulted in opacity in the cornea of the subjects (FIG. 2D, 120h, NGF and THX-B alone). Unexpectedly, the combination of THX-B and NGF synergized to produce a transparent cornea by 120h (FIG. 2D, THX-B + NGF, 120h). The opacity of the eyes in experimental and control animals was quantified by blinded observers in FIG. 2F. NGF alone slightly increased the opacity of denervated corneas, while THX-B alone slightly reduced opacity of denervated corneas. Surprisingly, the combination of NGF and THX-B decreased the opacity of denervated injured corneas, as compared to control and significantly decreased the opacity as compared to NGF treatment alone.

[0096] In the foregoing description, it will be readily apparent to one skilled in the art that vary ing substitutions and modifications may be made to the invention disclosed herein without departing from the scope and spirit of the invention. The invention illustratively described herein suitably may be practiced in the absence of any element or elements, limitation or limitations which is not specifically disclosed herein. The terms and expressions which have been employed are used as terms of description and not of limitation, and there is no intention that in the use of such terms and expressions of excluding any equivalents of the features shown and described or portions thereof, but it is recognized that various modifications are possible within the scope of the invention. Thus, it should be understood that although the present invention has been illustrated by specific embodiments and optional features, modification and/or variation of the concepts herein disclosed may be resorted to by those skilled in the art, and that such modifications and variations are considered to be within the scope of this invention.

[0097] Example 3 - TrkA activity promotes corneal epithelial recovery and is antagonized by p75^NTR activity

[0098] Although rhNGF is the only drug cleared by the FDA to treat NK⁶, its mode of action is unknown, and it is not effective for all NK patients³’⁴. There is a knowledge gap in the understanding of NGF’s effects on LSC activity. Therefore, we will use pharmacological and genetic approaches to model different corneal pathological conditions to (a) determine the role of NGF in the behavior of LSC and (b) determine which of the two known NGF receptors, TrkA or p75^NTR, or both, mediate NGF's effect on LSC activity -dependent corneal epithelial renewal. The inventors hypothesize that NGF secreted by SC can both positively and negatively regulate LSC activity and comeal healing by activating its antagonistically acting receptors TrkA and p75^NTR. TrkA is responsible for transducing most of NGF’s known pro-survival, growth and differentiation signaling in neurons⁵. Activated (phosphorylated) TrkA is also thought to stimulate comeal epithelial wound healing⁶’¹⁰. However, direct evidence for TrkA activation in the comeal epithelium and its involvement in comeal epithelial renewal is lacking. Based on previous reports^11,10, our scRNA-seq data (not shown) and immunostaining of harvested corneas, both NGF receptors TrkA and p75^NTR are expressed by limbal cells, including LSC. The initial biochemical analysis demonstrated activation of TrkA upon administration of NGF to rat cornea (Fig ID). Meanwhile, p75^NTR, the other NGF receptor, mainly suppresses the growth of axons and survival of developing and injured neurons, although, in some cases it can enhance TrkA-induced signaling¹²’¹⁴. Unlike TrkA, which is only activated by NGF, p75^NTR is activated by both the mature (NGF) and the often injury -induced precursor (pro-NGF) forms of the ligand⁹⁰. In low-level or absent TrkA activity’, p75^NTR mediates much of its negative effects on growth and survival by activating c-Jun N-terminal kinases (JNK) 1 and 3¹² First whether, and how, local NGF regulates LSC activity will be assessed. It is expected that NGF neutralization causes an early delay in epithelial recovery' via inhibition of LSC activity.

[0099] The recovery' of de-epithelialized corneas in mice will be assessed using genetically-labeled LSC. NGF will be neutralized in vivo (as described above) in de-epithelialized corneas of k!5- EGFP mice, correlating delay in comeal re-epithelialization with proliferation of LSC. The number of LSC will be quantified by counting EGFP-positive cells 24 and 48 hours after initiation of treatment, and their proliferation will be assessed by EdU incorporation. The corneas will be harvested after 48 hours and immunolabeled for the comea-specific epithelial markers K3/K12 and CD44 to compare the number of newly-formed epithelial cells in NGF- neutralizing antibody-treated corneas compared to untreated corneas. This experiment will be repeated using wt mice. Corneas also will be immunolabeled for the phosphorylated activated form of TrkA to confirm that sufficient NGF neutralizing antibody was used to suppress TrkA activity. It is expected that neutralization of endogenous NGF will reduce proliferation of LSC and newly -formed epithelial cells.

[00100] It is expected that TrkA activity’ promotes comeal epithelial recovery and is antagonized by p75^NTR activity.

[00101] Genetic-chemical biological and pharmacological approaches will be used to investigate the role of the two NGF receptors in LSC-dependent epithelial renewal.

[00102] To study the role of TrkA, mice harboring a knock-in mutation for endogenous TrkA (F592A) rendering it susceptible to inhibition by 1NMPP1, a compound that inhibits no other known kinase in mammals¹⁵ will be use. These mice have previously been used to study the role of TrkA in axon degeneration¹⁶. Mice were treated with 1NMPP1 and comeal healing assessed using comeal fluorescein staining one to four days following injury. Inhibition of TrkA resulted in a 50% reduction in comeal epithelial recovery (FIGs. 1B,C). Initial observations indicate that this TrkA inhibition with 1NMPP1 increases p75^NTR immunoreactivity' in de- epithelialized corneas of the treated mice (not shown). To define the role of p75^NTR in comeal recovery, de-epithelialized corneas were topically treated daily for 4 days with a neutralizing antibody to pro-NGF (0.7mg/ml)¹⁷. Pro-NGF is a specific p75^NTR ligand that is often induced upon injury¹⁵. Inhibition of pro-NGF alone had no detectable effect on recovery' (not shown). However, pro-NGF neutralization promoted comeal recovery when TrkA activity was suppressed by 1NMPP1. Neutralization of pro-NGF resulted in an increase in Ki67 immunoreactivity in the limbal area of corneas (not shown), suggesting that p75^NGF may suppress LSC or epithelial cell proliferation under injury conditions. Together, these initial results suggest that following injury (i) NGF-induced TrkA activity' promotes LSC-dependent comeal wound healing, while (ii) p75^NTR activity' is associated with inhibition of the healing process.

[00103] Citations to a number of patent and non-patent references may be made herein. The cited references are incorporated by reference herein in their entireties. In the event that there is an inconsistency between a definition of a term in the specification as compared to a definition of the term in a cited reference, the term should be interpreted based on the definition in the specification.

[00104] References

1. Catapano, J., Antonyshyn, K., Zhang, J. J., Gordon, T. & Borschel, G. H. Comeal Neurotization Improves Ocular Surface Health in a Novel Rat Model of Neurotrophic Keratopathy and Comeal Neurotization. Invest Ophthalmol Vis Sci 59, 4345-4354, doi: 10. 1167/iovs.18-24843 (2018).

2. Bai, Y. et al. Chronic and acute models of retinal neurodegeneration TrkA activity are neuroprotective whereas p75NTR activity is neurotoxic through a paracrine mechanism. J Biol Chem 285, 39392-39400, doi: 10. 1074/jbc.Ml 10.147801 (2010).

3. Pflugfelder, S. C. et al. Topical Recombinant Human Nerve Growth Factor (Cenegermin) for Neurotrophic Keratopathy: A Multicenter Randomized Vehicle-Controlled Pivotal Trial. Ophthalmology’ 127, 14-26, doi: 10.1016/j.ophtha.2019.08.020 (2020).

4. Sheha, H., Tighe, S., Hashem, O. & Hayashida, Y. Update On Cenegermin Eye Drops In The

Treatment Of Neurotrophic Keratitis. Clin Ophthalmol 13, 1973-1980, doi: 10.2147/OPTH.S185184 (2019).

5. Tang, C. et al. Neural Stem Cells Behave as a Functional Niche for the Maturation of Newborn Neurons through the Secretion of PTN. Neuron 101, 32-44. e36, doi: 10. 1016/j. neuron.2018. 10.051 (2019). 6. Garcia-Hirschfeld, J., Lopez-Briones, L. G. & Belmonte, C. Neurotrophic influences on corneal epithelial cells. Exp Eye Res 59, 597-605, doi:10.1006/exer, 1994.1145 (1994).

7. Lambiase, A., Rama, P., Bonini, S., Caprioglio, G. & Aloe, L. Topical treatment with nerve growth factor for comeal neurotrophic ulcers. N Engl J Med 338, 1174-1180, doi: 10. 1056/NEJM199804233381702 (1998).

8. Lambiase, A. et al. Nerve growth factor promotes comeal healing: stmctural, biochemical, and molecular analyses of rat and human corneas. Invest Ophthalmol Vis Sci 41, 1063-1069 (2000).

9. Tan, M. H., Bryars, J. & Moore, J. Use of nerve growth factor to treat congenital neurotrophic comeal ulceration. Cornea 25, 352-355, doi: 10.1097/01.ico.0000176609.42838. df (2006).

10. Touhami, A., Grueterich, M. & Tseng, S. C. The role of NGF signaling in human limbal epithelium expanded by amniotic membrane culture. Invest Ophthalmol Vis Sci 43, 987-994 (2002).

11. Di Girolamo, N. et al. Localization of the low-affinity nerve growth factor receptor p75 in human limbal epithelial cells. J Cell Mol Med 12, 2799-2811, doi: 10. 1111/j. 1582- 4934.2008.00290.x (2008).

12. Kaplan, D. R. & Miller, F. D. Neurotrophin signal transduction in the nervous system. Curr Opin Neurobi ol 10, 381-391 (2000).

13. Reddypalli, S. et al. p75NTR-mediated signaling promotes the survival of myoblasts and influences muscle strength. J Cell Physiol 204, 819-829, doi: 10. 1002/jcp.20330 (2005).

14. Massa, S. M. et al. Small, nonpeptide p75NTR ligands induce survival signaling and inhibit proNGF-induced death. J Neurosci 26, 5288-5300, doi:10.1523/JNEUROSCL3547- 05.2006 (2006).

15. Chen, X. et al. A chemical-genetic approach to studying neurotrophin signaling. Neuron 46, 13-21, doi: 10.1016/j.neuron.2005.03.009 (2005).

16. Barcelona, P. F. et al. The route of administration influences the therapeutic index of an anti-proNGF neutralizing mAb for experimental treatment of Diabetic Retinopathy. PLoS One 13, e0199079, doi:10.1371/joumal.pone.0199079 (2018). [00105] Sequence Listing

Claims

CLAIMS We claim:

1. A method of treating neurotrophic keratopathy in a subject in need thereof, the method comprising administering a therapeutically effective amount of THX-B and/or anti- proNGF, and a therapeutically effective amount of at least one TrkA agonist selected from recombinant nen e growth factor (rNGF), recombinant human NGF (rhNGF), and NGF mimetic Tavilermide to the subject to treat the neurotrophic keratopathy in the subject.

2. The method of claim 1, wherein the subject has about 50% or less comeal innervation, about 40% or less comeal innervation, about 30% or less comeal innervation, about 20% or less comeal innervation, about 10% or less comeal innervation, or wherein the subject has no comeal innervation.

3. The method of claim 1 or 2, wherein the subject suffers from comeal anesthesia.

4. The method of claim 3, wherein the comeal anesthesia is congenital comeal anesthesia.

5. The method of claim 1, wherein the method results in complete recover}' of the comeal epithelium.

6. The method of claim 1, wherein the method induces complete comeal re- epithelialization with one treatment of THX-B and/or anti-proNGF. and one or more of rNGF, rhNGF, and Tavilermide.

7. The method of claim 5. wherein the method results in a transparent cornea.

8. The method of claim 6, wherein the method results in a transparent cornea after at least 10 days of administration of THX-B and/or anti-proNGF. and one or more of rNGF. rhNGF, and Tavilermide.

9. The method of claim 1, wherein administration comprises once daily topical administration.

10. The method of claim 1, wherein the therapeutically effective amount of THX-B and/or anti-proNGF comprises 20-30 mg/ml and wherein administering comprises topical administration.

11. The method of claim 9, wherein the therapeutically effective amount of THX-B comprises 27 mg/ml.

12. A method of improving comeal transparency after comeal injury' in a subject in need thereof, the method comprising administering a therapeutically effective amount of THX-B and/or anti-proNGF, and a therapeutically effective amount of one or more of recombinant nerve growth factor (rNGF), recombinant human nerve growth factor (rhNGF), and Tavilermide to the subject to improve comeal transparency after comeal injury' in the subject.

13. The method of claim 12, wherein the subject has about 50% or less comeal innervation, about 40% or less comeal innervation, about 30% or less comeal innervation, about 20% or less comeal innervation, about 10% or less comeal innervation, or wherein the subject has no comeal innervation.

14. The method of claim 12 or 13, wherein the subject suffers from comeal anesthesia.

15. The method of claim 14, wherein the comeal anesthesia is congenital comeal anesthesia.

16. The method of claim 12. wherein the subject is suffering from neurotrophic keratopathy.

17. The method of claim 12. wherein the method results in complete recovery of the comeal epithelium.

18. The method of claim 12. wherein the therapeutically effective amount of THX-B and/or anti-proNGF comprises 20-30 mg/ml and wherein administering comprises topical administration.

19. The method of claim 17, wherein the therapeutically' effective amount of THX-B comprises 27 mg/ml.

20. The method of claim 12, wherein the method results in a transparent cornea.

21. The method of claim 19, wherein the method results in a transparent cornea after at least 10 days of administration of THX-B and/or anti-proNGF, and one or more of rNGF, rhNGF, and Tavilermide.

22. A pharmaceutical composition comprising THX-B and/or anti-proNGF, and one or more of rNGF, rhNGF, and Tavilermide formulated for once daily administration to the eye of a subject.

23. The pharmaceutical composition of claim 22, wherein the pharmaceutical composition is formulated for topical administration to the eye of a subject.

24. A kit comprising THX-B and/or anti-proNGF, and one or more of rNGF, rhNGF. and Tavilermide, and instructions for administering the THX-B and rhNGF once daily to the eye.

25. The kit of claim 24, wherein the THX-B and/or anti-proNGF, and one or more of rNGF, rhNGF, and Tavilermide, are formulated for topical administration to the eye.