Compositions comprising neuregulin and methods of using the same to treat or prevent neurotrophic keratitis

Abstract

Disclosed are pharmaceutical compositions comprising the growth factor neuregulin 1 (NRG1). Further disclosed herein are methods of using neuregulin 1, or pharmaceutical compositions comprising NRG1, to treat or prevent neurotrophic keratitis, methods to induce corneal headlining in an eye of a subject that has been denervated, and methods or preventing death of Schwann cells in the cornea of a subject that has suffered a corneal denervation.

Description

COMPOSITIONS COMPRISING NEUREGULIN AND METHODS OF USING THE SAME TO TREAT OR PREVENT NEUROTROPHIC KERATITISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 607.858, filed December 8, 2023. the contents of which is incorporated herein by reference in its entirety.SEQUENCE LISTING

[0002] A Sequence Listing accompanies this application and is submitted as an xml file of the sequence listing named “144578_00423.xml” which is 3,473 bytes in size and was created on November 11, 2024. The sequence listing is electronically submitted via Patent Center and is incorporated herein by reference in its entirety.BACKGROUND

[0003] Comeal epithelial maintenance and wound healing depend on the activity of limbal stem cells (LSCs) residing in the basal epithelium of the limbus. Impaired comeal sensory innervation interferes with the activity of LSC-dependent comeal epithelial renewal, leading to comeal ulceration, opacification, and inevitable blindness. This condition, neurotrophic keratitis (NK). affects 5 / 10,000 people worldwide. Topical recombinant human nerve growth factor (rhNGF) is the only FDA-approved drug for treating NK. However, NGF is not effective in 40% of patients and does not prevent onset of NK. In addition, extremely high dosage of NGF make this approach highly expensive and cumbersome for the patient. Accordingly, there is a need in the art for novel treatments for neurotrophic keratopathy, particularly, for treatments that prevent the onset of NK.SUMMARY

[0004] Provided herein are pharmaceutical compositions comprising neuregulin and methods of using the same to treat or prevent neurotrophic keratitis.

[0005] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise neuregulin 1 (NRG1). In someembodiments, the pharmaceutical compositions are formulated for topical ophthalmic administration. In some embodiments, the pharmaceutical compositions further comprise one or more additional compound. In some embodiments, the NRG1 comprises a sequence with at least 90% identity to one of SEQ ID NOs: 1 or 2. In some embodiments, the NRG1 is SEQ ID NO: 1 or 2.

[0006] In an aspect of the current disclosure, methods of treating neurotrophic keratitis (NK) in a subject in need thereof are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of the pharmaceutical composition comprising neuregulin 1 to the subject to treat the NK. In some embodiments, treating comprises preventing or delaying the onset of neurotrophic keratitis (NK). In some embodiments, the subject has experienced a comeal denervation. In some embodiments, the comeal denervation is a result of a surgery. In some embodiments, the surgery comprises brain surgery. In some embodiments, the subject is suffering from a brain tumor, a tumor of the eye, a tumor of the optic nerve, or a tumor that is near or derived from the trigeminal nerve. In some embodiments, the method prevents the death of Schwann cells in the cornea of the subject.

[0007] In an aspect of the current disclosure, methods of preventing death of Schwann cells in the cornea of a subject that has suffered a comeal denervation are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising neuregulin 1 to the subject. In some embodiments, the comeal denervation is a result of a surgery'. In some embodiments, the surgery comprises brain surgery. In some embodiments, the subject is suffering from a brain tumor, a tumor of the eye, or a tumor of the optic nerve.

[0008] In an aspect of the current disclosure, methods of inducing comeal healing in an eye of a subject that has been denervated are provided. In some embodiments, the methods comprise administering a therapeutically effective amount of a pharmaceutical composition comprising neuregulin 1 to the cornea of a subject to induce comeal healing. In some embodiments, the comeal denervation is a result of a surgery. In some embodiments, the surgery' comprises brain surgery'. In some embodiments, the method reduces comeal opacity' compared to a subject not administered the pharmaceutical composition. In some embodiments, the method results in atransparent cornea. In some embodiments, the method results in complete recovery of the comeal epithelium after one day of treatment.BRIEF DESCRIPTION OF THE FIGURES

[0009] FIGs. 1A, IB, 1C, ID, IE, and IF show NRG1 induces wound healing in denervated corneas by preserving SCs. Live fluorescent images of Thyl-YFP (A) and tamoxifen pre-treated SoxlO-TdT (B) mouse denervated corneas, acquired prior to (Oh) and at the two indicated time points after comeal denervation. At 0 hours healthy YFP-positive axons (in A) are associated with TdT-positive SCs (in B) (arrowheads). The gradual axonal loss, appearing in form of punctate debris by 48 hours (arrows, top center), completely disappear by 72 hours after denervation (A, top row), is associated with a complete loss of SCs, as reported3(B; middle panel) that is prevented by two daily treatments by NRG1 (B; lower panel; arrowheads). Scale bar in A and B: 1mm. C) Post fixation immunohistochemical analysis of SoxlO-TdT-positive innervated (left image), or 72 hours post-denervated vehicle-only- or NRG 1 -treated (central and right images, respectively) corneas, as in A. Indicated zoomed-in areas demonstrate presence or absence of bill-positive (green) epithelial axons in innervated or surgically denervated corneas, respectively (lower panel). The treatment with NRG1 supported survival of TdT-positive SCs in limbus (red; in merged (upper panel) and zoomed-in indicated areas (lower panel)) that, otherwise, degenerated in the vehicle-only treated denervated corneas, as indicated by TdT-positive SC cell debris (lower panel; arrowheads). Epithelial and subepithelial cellular layers are indicated by a dense DAPI-positive cell nuclei (blue; upper panel). Scale bars: upper panel - 200mm, lower panel - 100mm. D) Preliminary quantitative representation of TdT positive areas in limbi of normally innervated (inrv; n=4), denervated NRG1- (n=3) or vehicle-only- (veh; n=4) treated corneas, as in B and C; *p<0.05. ANOVA. E). In vivo photos of deepithelialized rat corneas visualize epithelial healing after denervation compared with normally innervated (inrv) corneas. The corneas were treated either with NRG1 or vehicle-only (veh). Images at “Oh” demonstrates the comeal wound immediately after epithelial removal; fluorescein (green) stains the comeal area lacking epithelium (n =2). F) In vivo photos of rat corneas demonstrate opacification of denervated vehicle-only (veh) treated healed cornea five days after de-epithelialization (arrowheads), compared to clear NRG1 treated corneas.DETAILED DESCRIPTION

[0010] The inventors have discovered that neuregulin 1 type I (NRG1; also known as Heregulin or Neu differentiation factor (NDF)) prevents a loss of corneal Schwann cells follow ing corneal denervation when administrated to the eye (FIGs. IB, 1C, and ID). This finding suggests that the Schwann cells prevent loss and stimulate the activity of comeal epithelial (limbal) stem cells, following denervation. The inventors denervated the corneas of rats and topically administered NRG1 to the corneas daily in concentration of 100 ng / mL which resulted in faster epithelial regeneration and transparent corneas in NRG treated mice as compared to vehicle treated mice (FIGs. IE and IF).Pharmaceutical compositions

[0011] In an aspect of the current disclosure, pharmaceutical compositions are provided. In some embodiments, the pharmaceutical compositions comprise neuregulin 1 ty pe I (NRG1; also known as Heregulin or NDF).

[0012] A variety of different isoforms of neuregulin are derived from the neuregulin gene, which can be distinguished based on differences in their NH2-terminal regions. The different NRG1 isoforms can be categorized into three groups: (i) type I NRG1 includes neu differentiation factor (NDF), the heregulins (HRGs) and acetylcholine receptor inducing activity (ARIA); (ii) type II NRG1 contains the glial growth factors (GGFs); and (iii) type III NRG1 comprises the sensory and motor neuron-derived factor (SMDF). As each of the NRG1, type I isoforms comprise the same active site, they should elicit the same therapeutic benefit.

[0013] As used herein, ‘"neuregulin 1” or “NRG1” refers to neuregulin 1 which encompasses type I, II, and III neuregulins. Human neuregulin 1 may have the amino acid sequence SEQ ID NO: 1 (human NRG1) or 2 (human heregulin-betal, GenBank accession No. AAA58639). The NRG1 may comprise recombinant neuregulin 1, type 1, e.g., recombinant human neuregulin 1 with at least about 80%, at least about 81%, at least about 82%, at least about 83%, at least about 84%, at least about 85%, at least about 86%, at least about 87%, at least about 88%, at least about 9890%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, or more identity to SEQ ID NO: 1 or 2.

[0014] The pharmaceutical compositions may further comprise at least one pharmaceutically acceptable carrier or excipient, examples of which are known in the art. Formulation of pharmaceutical compositions and addition of an appropriate carrier or excipient are considered to be routine activities by one of skill in the art.

[0015] The production of recombinant proteins, e.g., NRG1, by methods known in the art, e.g., expression in a suitable expression system, e.g., a prokaryotic expression system, a eukaryotic expression system, are known in the art. Further, purification of such recombinant proteins, is also considered routine in the art. Thus, one of skill in the art could design a suitable expression and purification systems to generate clinical grade proteins, e.g., NRG1 for the administration to a subject, e.g., a human subject.

[0016] The pharmaceutical compositions may further comprise an additional active compound including, but not limited to, an antibiotic, a steroid, an antihistamine, an anesthetic, an analgesic, etc. The additional active compound should, suitably, not interfere with the actions of NRG1. The disclosed pharmaceutical compositions may also comprise neuronal growth factor (NGF), e.g.. human NGF, which has demonstrated some efficacy in the treatment of neurotrophic keratitis (NK). Neurotrophic keratitis and neurotrophic keratopathy are used interchangeably herein.

[0017] The concentration of NRG1 may be about 100 pg / ml to about 1000 ng / ml, about 1 ng / ml to about 100 ng / ml, about 100 pg / ml to about 100 ng / ml, or about 100 pg / ml to about 50 ng / ml, or any value or subrange inclusive of the endpoints, in the pharmaceutical compositions.

[0018] In an exemplary embodiment, the pharmaceutical composition is formulated for topical ophthalmic administration. The concentrations ofNRGl may be about 100 pg / ml to about 1000 ng / ml, about 1 ng / ml to about 100 ng / ml, about 100 pg / ml to about 100 ng / ml, or about 100 pg / ml to about 50 ng / ml in the pharmaceutical compositions formulated for ophthalmic topical administration.

[0019] The pharmaceutical compositions may comprise about 0.0001%, about 0.0002%, about 0.0003%, 0.0004%, about 0.0005%, about 0.0006%, 0.0007%, about 0.0008%, about 0.0009%, 0.001%, about 0.002%, about 0.003%, 0.004%, about 0.005%, about 0.006%, 0.007%, about 0.008%, about 0.009%, 0.01%, about 0.02%, about 0.03%, 0.04%, about 0.05%, about 0.06%, 0.07%, about 0.08%, about 0.09%, 0.1%, about 0.2%, about 0.3%, 0.4%, about 0.5%, about 0.6%,0.7%, about 0.8%, about 0.9%, 1%, about 2%, about 3%, 4%, about 5%, about 6%, 7%, about 8%. about 9%, or more NRGl.

[0020] The compositions (i.e., formulations comprising NRG1 and pharmaceutical formulations thereof) described herein 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.

[0021] 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 w eight, 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 knowm agent, such as by' means of an appropriate conventional pharmacological protocol.

[0022] 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.

[0023] The pharmaceutical compositions, including ophthalmic formulations, with the disclosed trophic factors 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 -hydroxy ethylmethacrylate (p-HEMA hydrogels), poly (lactic-co-gly colic) 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.

[0024] The terms “pharmaceutically acceptable carrier,” or “excipient” as used herein, mean a non-toxic, 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, e g., ophthalmic 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 pharmaceutical composition is formulated for local delivery. Suitable additional components for topical deliver}' 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, polyglycolic acid, hydrogels, etc., or onto liposomes, microemulsions, micelles, milamellar or multilamellar vesicles, erythrocyte ghosts, or spheroplasts. Such compositions will influence the physical state, solubility, stability, 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 pharmaceutical compositions, 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 keratitis. 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 pharmaceutical compositions 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.Methods

[0031] In an aspect of the current disclosure, methods are provided. In some embodiments, the methods are methods of treating neurotrophic keratitis (NK) in a subject in need thereof and comprise administering a therapeutically effective amount of a pharmaceutical composition comprising neuregulin 1 (NRG1) to the subject to treat the NK.

[0032] As used herein, “treat” and grammatical variations thereof, refers to reducing one or more sign or symptom of the disease or disorder being treated in the subject or preventing, delaying the onset, or reducing the severity of the disease or disorder.

[0033] In some situations, the comeal denervation may be planned or expected including, but not limited to, local cancer removal surgery, e.g., removal of cancer of or near the brain, eye, trigeminal nerve, or optic nerve, and brain cancer surgery with sudden onset of comeal denervation.

[0034] In some embodiments, the methods are methods of preventing death of Schwann cells in the cornea of a subject that has suffered comeal denervation and comprise administering a therapeutically effective amount of a pharmaceutical composition comprising neuregulin 1 (NRG1) to the subject to prevent death of Schwann cells in the cornea of the subject.

[0035] In some embodiments, the methods are methods of inducing comeal healing in the eye of a subject that has been denervated and comprise administering a therapeutically effective amount of a pharmaceutical composition comprising neuregulin 1 (NRG1) to the subject to induce comeal healing in the eye of the subject that has been denervated.

[0036] In some embodiments, the methods reduce comeal opacity in the denervated eye of the subject. As demonstrated in FIG. IE, denervated corneas treated with vehicle heal more slowly than corneas treated with NRG1. Furthermore, vehicle treated corneas develop opacity’, while NRG1 treated corneas do not. In some embodiments, the methods result in a transparent cornea. In some embodiments, the methods result in a complete recovery of the cornea, i.e., the comeal epithelium heals completely, e.g., in about one day of treatment. See FIG. IE.

[0037] 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.

[0038] 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 nen e 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.

[0039] 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 “subj ect in need thereof' is, in some embodiments, a subject diagnosed with a neurotrophic keratitis, or a subject suffering from lack or comeal innervation, or a subject suspected of developing neurotrophic keratitis in the future. NK may result from various common causes, including herpes keratitis (both zoster and simplex), excessive use of contact lenses, toxicity from topical medications, eye irradiation, previous comeal surgery, and jaw fractures. Such subjects are also contemplated as being treated by the disclosed methods.

[0040] 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 disclosedmethods 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.

[0041] 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.

[0042] 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., the disclosed pharmaceutical compositions, 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, or more, and administration may be performed on consecutive days for a time period prescribed by a physician. Alternatively, 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.Definitions

[0043] 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.

[0044] 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 “asubstituent” should be interpreted to mean “one or more substituents,” unless the context clearly dictates otherwise.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.”

[0049] 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.

[0050] 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.”EXAMPLES

[0051] The following Examples are illustrative and should not be interpreted to limit the scope of the claimed subject matter.Example 1 - Neuregulin 1 promotes Schwann cell survival and activity after corneal denervation

[0052] To test the effect of neuregulin 1 type I (NRG1 ; also known as Heregulin or NDF) as a treatment for neurotrophic keratitis (NK), the inventors performed a single stereotactic ablation of the ophthalmomaxillary branch of the trigeminal nerve to induce comeal denervation, prevent and / or treated the denervated corneas with NRG1 (heregulin- 1 beta, SEQ ID NO: 2, purchased from R&D Systems, catalog # 377-HB) daily beginning five days after the ablation procedure (FIG. IE). Subjects’ eyes were treated with fluorescein - fluorescent signal indicates a lack of comeal epithelial layer in the images in FIG. IE. The inventors discovered that administration of NRG1 improved comeal healing in a model of neurotrophic keratitis (NK).Example 2 - Corneal epithelial maintenance and healing after injury depend on SCs

[0053] To further define the role of SCs in corneal wound healing, in a novel gain-of-function experiment and correlate between SC survival and epithelial renewal capacity, the inventors will apply topical NRG1 to surgically-denervated rodent (rat or mouse) corneas in vivo.

[0054] Experimental Design and Preliminary Results: In the peripheral nervous system, neuronal paracrine NRG1 supports survival of SCs. Following sensory denervation, the cornea gradually loses its regenerative capacity, and epithelial instability coincides with withdrawal of SCs. The inventors observed that denervated, de-epithelialized rodent corneas exhibit delayed wound closure, accompanied by opacification (FIG. 1). Previously, the inventors and others used NRG1 to grow primary rodent SCs in vitro. To test the extent to which exogenous NRG1 prevents SC loss following denervation, the inventors used a genetic mouse model of tamoxifen-inducible CreERT2 knocked into the terminally differentiated SC-specific promoter SoxlO and the TdTomato reporter gene SoxlO-iCreERT2 / +;R26-LSL-TdT. This “SoxlO-TdT” strain allows visualization of comeal nerve ensheathing SCs when tamoxifen is applied to the cornea in vivo. Following tamoxifen administration, the left cornea was denervated and, starting from day 0, topically treated with lOOng / ml NRG1 or vehicle-only. Note: all in vivo experiments performed on the left cornea, while the right eye served as an internal positive control. Five days after denervation, the epithelium was removed with an Amoils brush. Comeal epithelial recovery was assessed daily by fluorescein staining for four days.

[0055] To prevent accidental self-wounding by scratching the anesthetic cornea, a tarsorrhaphy (eyelid suture) was maintained. In the inventors’ preliminary study, and consistent with reported results, comeal denervation caused SC withdrawal in 48 hours. The inventors’ initial in vivo fluorescent microscopy observations demonstrate that NRG1 induced survival of SCs in limbus at levels comparable to those of healthy non-denervated corneas (FIGs. IB, 1C, and ID). In turn, this NRGl-mediated survival of SCs induced healing of deepithelialized denervated corneas within 36 hours, comparable to the healing observed in normally innervated corneas in rats (FIGs. IE, IF) and mice (not shown). Consistent with the inventors' previous observations, vehicle-only treated corneas exhibited delayed wound healing and developed comeal opacifications (FIGs. IE and IF). As a control, the inventors used Thyl-YFP transgenic mice, which express YFP in axons, to ensure that NRG1 treatment did not inhibit Wallerian degeneration following comeal denervation (FIG. 1A). These data correlate with immunostaining observations, demonstrating complete axonal loss in the SoxlO-TdT denervated NRGl-treatedmouse corneas (FIG. 1C). To assess changes in SC volume, the inventors measured total TdT- positive limbal areas in fixed experimental corneas, including fluorescent cell debris in denervated vehicle-only treated conditions. This method may overestimate SC survival in the control vehicle-only treatment conditions (FIG. ID).Example 3 - Treatment of neurotrophic keratitis with neuregulin

[0056] In one example, a subject suffering from neurotrophic keratitis (NK), e.g., a human subject, is administered a therapeutically effective amount of a composition comprising neuregulin. The neuregulin may suitably be administered by any route that is indicated by the particular treatment needs of the subject, e.g., topically to the eye. Signs and symptoms of the NK may be reduced by the administration of the neuregulin. Treatment may be administered daily, twice daily, three times daily, or on a schedule as determined by the patient's progress, pursuant to a physician's decision. It is anticipated that the subject will experience an increase in comeal epithelial healing, reduction in comeal injuries, return of sensory perception to the cornea, improvements in comeal opacity or complete reversal of comeal opacity, or other metrics associated with reduction in signs or symptoms of NK, as compared to an untreated subject. Methods of measuring reductions in signs and symptoms of NK are known in the art.

[0057] In the foregoing description, it will be readily apparent to one skilled in the art that varying 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.

[0058] 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 isan 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.Sequences

[0059]

Claims

CLAIMS1. A method of treating neurotrophic keratitis (NK) in a subject in need thereof, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising neuregulin 1 , type I (NRG1 ) to the subj ect to treat NK in the subj ect.

2. The method of claim 1. wherein treating comprises preventing or delaying the onset of neurotrophic keratitis (NK).

3. The method of claim 1, wherein the subject has experienced a comeal denervation.

4. The method of claim 3, wherein the comeal denervation is a result of a surgery.

5. The method of claim 4, wherein the surgery comprises brain surgery.

6. The method of claim 1 , wherein the subj ect is suffering from a brain tumor, a tumor of the eye, a tumor of the optic nerve, or a tumor that is near or derived from the trigeminal nerve.

7. The method of claim 1, wherein the method prevents the death of Schwann cells in the cornea of the subject.

8. A method of preventing death of Schwann cells in a cornea of a subject that has suffered a comeal denervation, the method comprising administering a therapeutically effective amount of a pharmaceutical composition comprising neuregulin 1, type I (NRG1) to the subject to prevent death of Schwann cells in the cornea of the subject.

9. The method of claim 8, wherein the comeal denervation is a result of a surgery.

10. The method of claim 9, wherein the surgery comprises brain surgery'.

11. The method of claim 8, wherein the subject is suffering from a brain tumor, a tumor of the eye, or a tumor of the optic nerve.

12. A method of inducing comeal healing in a denervated eye in a subject, the method comprising administering a therapeutically effective amount of the pharmaceuticalcomposition comprising neuregulin 1, type I (NRG1) to the cornea of a subject to induce comeal healing in the denervated eye.

13. The method of claim 12, wherein the comeal denervation is a result of a surgery.

14. The method of claim 13, wherein the surgery comprises brain surgery.

15. The method of claim 12, wherein the method reduces comeal opacity' compared to a subject not administered the pharmaceutical composition or an eye not administered the pharmaceutical composition.

16. The method of claim 1, 8, or 12, wherein the method results in a transparent cornea.

17. The method of claim 1, 8, or 12. wherein the method results in complete recovery of the comeal epithelium after one day of treatment.

18. A pharmaceutical composition comprising neuregulin 1, type I (NRG1).

19. The pharmaceutical composition of claim 18. wherein the NRG1 comprises a sequence with at least 90% identity' to one of SEQ ID NOs: 1 or 2.

20. The pharmaceutical composition of claim 18, wherein the NRGl is SEQ ID NO: 1 or 2.

21. The pharmaceutical composition of claim 18, wherein the pharmaceutical composition is formulated for topical ophthalmic administration.

22. The pharmaceutical composition of claim 18, further comprising one or more additional compound.

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