Use of insulin in the manufacture of a medicament for the treatment of dry eye syndrome of lacrimal gland dysfunction

Abstract

The present application provides the application of insulin in the preparation of a medicament for treating dry eye syndrome of lacrimal gland dysfunction. Specifically, the present application provides the application of insulin or its analogues in the preparation of a medicament for treating dry eye syndrome of lacrimal gland dysfunction. The present application also provides pharmaceutical compositions, kits and the like thereof.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceuticals, and more specifically, this invention relates to the use of insulin in the preparation of medicaments for treating lacrimal gland dysfunction-related dry eye syndrome. Background Technology

[0002] Dry eye syndrome is a common, multifactorial ocular surface disease, one of the most prevalent eye diseases worldwide. It can be caused by abnormalities in tear dynamics, quality, and quantity, leading to ocular surface microenvironment disturbances or tear film instability. In the middle and late stages, it can cause ocular surface inflammation, tissue and nerve abnormalities, resulting in eye discomfort. Symptoms of dry eye mainly include dryness, foreign body sensation, and redness. Dry eye syndrome can be broadly classified into two types: one is the aqueous hypotonia type, also known as lacrimal gland dysfunction type dry eye syndrome, which is caused by various factors leading to tear dysfunction, reduced tear secretion, and in later stages, secondary symptoms such as corneal epithelial defects and ocular surface inflammation, thus causing dry eye; the other is the evaporative type, where the oil secreted by the meibomian glands or the mucin produced by conjunctival goblet cells reduces ocular surface tension, causing faster tear evaporation and thus dry eye.

[0003] Currently, clinical medications for dry eye primarily target evaporative dry eye and the secondary ocular surface inflammation that occurs in the later stages of dry eye. These medications fall into two categories: the first category includes artificial tears, eye drops, and ocular serums that protect the ocular mucosa and promote repair; the second category consists of anti-inflammatory drugs for the ocular surface, including tetracyclines, corticosteroids, nonsteroidal anti-inflammatory drugs (NSAIDs), and immunosuppressants. Controlling inflammation and breaking the inflammatory cycle on the ocular surface are crucial aspects of dry eye treatment. Cyclosporine A eye drops have shown good clinical efficacy in blocking T-cell activation and inhibiting ocular surface inflammation. However, cyclosporine A is highly lipophilic and has low solubility, which hinders its sustained effect on the ocular surface. The use of surfactants or excipients to improve cyclosporine solubility requires consideration of ocular tolerance. The LFA-1 antagonist Xiidra, which blocks the interaction between LFA-1 and ICAM-1, can inhibit the secretion of cytokines by T cells. However, in clinical practice, this drug usually requires three months of use to relieve symptoms in patients with dry eye, and up to a quarter of patients experience taste abnormalities and irritation after use. It is evident that the range of drug options for dry eye is limited, treatment is challenging, the treatment period is long, and the burden on patients is heavy.

[0004] Therefore, there is an urgent need in this field to explore therapeutic drugs for lacrimal gland dysfunction-related dry eye syndrome, to break through the limitations of traditional drugs, and to provide new ideas for the development of dry eye drugs. Summary of the Invention

[0005] The purpose of this invention is to provide the use of insulin in the preparation of a medicament for treating lacrimal gland dysfunction-related dry eye syndrome.

[0006] A first aspect of the invention provides the use of insulin or an analogue thereof in the preparation of a medicament for treating lacrimal gland dysfunction-related eye diseases.

[0007] In one or more embodiments, the insulin includes animal insulin and human insulin; preferably, the animal insulin includes human insulin, porcine insulin, and bovine insulin.

[0008] In one or more embodiments, the insulin analogue is selected from: 1) An insulin analogue formed by the substitution, deletion or addition of amino acids at one or more positions selected from A4, A5, A8, A9, A10, A12, A13, A14, A15, A16, A17, A18, A19, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B15, B16, B17, B18, B20, B21, B22, B23, B26, B27, B28, B29 and B30, which has the same or similar biological function as said insulin; 2) Insulin analogues formed by the absence of insulin B1-4 and / or B26-30, which have the same or similar biological functions as said insulin; 3) Insulin analogs formed by directly or through polymeric or non-polymeric linkers conjugating one or more acyl, PEG, or glycosyl groups to insulin, which have the same or similar biological functions as said insulin.

[0009] In one or more embodiments, the insulin analogue is selected from one or more of insulin glargine (e.g., Lantus®), insulin detemir (e.g., Novopharm®), insulin aspart (e.g., insulin aspart 30, more specifically, such as NovoRapid®), insulin lispro (e.g., Humalog®, SusB30®), and insulin DesB30.

[0010] In one or more embodiments, the lacrimal gland dysfunction ophthalmopathy is an ophthalmopathy caused by lacrimal gland dysfunction or lack of fluid.

[0011] In one or more embodiments, the lacrimal gland dysfunction type of ophthalmopathy is lacrimal gland dysfunction type of dry eye syndrome.

[0012] In one or more embodiments, the lacrimal gland dysfunction-related dry eye includes: dry eye syndrome induced by lacrimal gland protein or Freund's adjuvant, local lacrimal gland inflammation, damage repair and regeneration induced by IL-1a injection, and local lacrimal gland inflammation and lacrimal gland dysfunction induced by scopolamine injection combined with a dry environment.

[0013] In one or more embodiments, the lacrimal gland dysfunction-related dry eye syndrome is early-stage lacrimal gland dysfunction-related dry eye syndrome.

[0014] In one or more embodiments, the early lacrimal gland dysfunction type dry eye includes: no significant changes in the conjunctiva and meibomian glands, tear dysfunction leading to reduced tear secretion, and inflammatory reactions such as immune cell infiltration in the lacrimal gland.

[0015] In one or more embodiments, for mouse models, the early stage is within 7 days of the onset of lacrimal gland dysfunction; for human subjects, the early stage is the period when the results of the tear secretion test (Schirmer I test) are ≤5mm / 5min, the non-invasive tear film breakup time (NIBUT) is ≤10 seconds and / or the fluorescein-stained tear film breakup time (FBUT) is ≤5 seconds.

[0016] A second aspect of the present invention provides a pharmaceutical composition for treating lacrimal gland dysfunction-related ophthalmopathy, the pharmaceutical composition comprising a therapeutically effective amount of insulin or an analogue thereof, and a pharmaceutically acceptable carrier.

[0017] In one or more embodiments, the lacrimal gland dysfunction ophthalmopathy is lacrimal gland dysfunction dry eye syndrome as described in any embodiment of the present invention.

[0018] In one or more embodiments, the insulin or its analogues are as described in any embodiment of the present invention.

[0019] In one or more embodiments, the pharmaceutically acceptable carrier includes ophthalmologically acceptable salts.

[0020] In one or more embodiments, the ophthalmologically acceptable salt comprises a salt containing one or more cations selected from sodium, potassium, and ammonium, and one or more anions selected from chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, and bisulfite.

[0021] In one or more embodiments, the pharmaceutical composition is applied topically under the conjunctiva, in the anterior chamber, in the vitreous body, under the eyeball, under the retina, under the choroid, on the choroid, in the conjunctival sac, or on the eyelid.

[0022] In one or more embodiments, the pharmaceutical composition is administered at a frequency of 1 to 2 drops each time, 2 to 5 times a day, with an interval of 3 to 6 hours between each administration.

[0023] In one or more embodiments, the pharmaceutical composition is administered at a frequency of 1 drop each time, 4 times a day, with each drop spaced 4 hours apart.

[0024] A third aspect of the present invention provides a medicine box for treating lacrimal gland dysfunction-type eye disease, characterized in that the medicine box contains insulin or an analogue thereof as described in any embodiment of the present invention, or contains a pharmaceutical composition as described in any embodiment of the present invention.

[0025] In one or more embodiments, the lacrimal gland dysfunction ophthalmopathy is lacrimal gland dysfunction dry eye syndrome as described in any embodiment of the present invention.

[0026] Other aspects of the invention will be apparent to those skilled in the art from the disclosure herein. Attached Figure Description

[0027] Figure 1 The method of administering insulin eye drops to mice was demonstrated.

[0028] Figure 2 The results show the staining of ocular tissues after insulin eye drops were administered to normal mice; (A) shows the corneal fluorescence staining results after solvent eye drops and insulin eye drops; (B) shows the PAS staining results of the conjunctival sac after solvent eye drops and insulin eye drops, with the scale bar in the figure being 100 μm; (C) shows the H&E staining results of the lacrimal gland after solvent eye drops and insulin eye drops, with the scale bar in the figure being 100 μm.

[0029] Figure 3 The results show the changes in tear secretion after insulin eye drops were administered to normal mice; (A) is a diagram of the insulin eye drop administration protocol for mice; (B) is a diagram showing the degree of reddening of the phenol red cotton thread in the solvent eye drop group and the insulin eye drop group; (C) is a statistical graph of tear secretion in the solvent eye drop group and the insulin eye drop group.

[0030] Figure 4 The results show the changes in tear secretion in normal mice after instillation of insulin receptor inhibitor eye drops; (A) is a diagram of the instillation protocol for insulin receptor inhibitor eye drops in mice; (B) is a diagram showing the degree of reddening of phenol red cotton threads in the 0.01% DMSO control group and the insulin receptor inhibitor eye drop group; (C) is a statistical graph of tear secretion in the 0.01% DMSO control group and the insulin receptor inhibitor eye drop group.

[0031] Figure 5 The therapeutic effect of insulin eye drops on dry eye syndrome is shown; (A) is a diagram of the mouse modeling of dry eye syndrome (ESS model) and the treatment with insulin eye drops; (B) is a statistical diagram of tear secretion after eye drop treatment in each group of mice; (C) is a diagram of H&E staining of lacrimal gland sections after eye drop treatment in each group of mice. The scale bar in the figure is 100 μm.

[0032] Figure 6 The therapeutic effect of insulin eye drops on early scopolamine-induced dry eye is shown; (A) is a diagram of the short-term scopolamine mouse model and insulin eye drop treatment protocol; (B) is a statistical diagram of tear secretion after eye drop treatment in each group of mice; (C) is a CD45 immunofluorescence staining image of lacrimal gland sections after eye drop treatment in each group of mice. The scale bar in the figure is 100 μm.

[0033] Figure 7 The therapeutic effect of insulin eye drops on late-stage scopolamine-induced dry eye is shown; (A) is a diagram of the long-term scopolamine mouse model and insulin eye drop treatment protocol; (B) is a statistical diagram of tear secretion after eye drop treatment in each group of mice; (C) is a CD45 immunofluorescence staining image of lacrimal gland sections after eye drop treatment in each group of mice. The scale bar in the figure is 100 μm.

[0034] Figure 8 The therapeutic effect of insulin analog eye drops on early scopolamine-induced dry eye is shown; (A) is a diagram of the scopolamine mouse model and the treatment protocol of insulin analog eye drops; (B) is a statistical diagram of tear secretion after eye drop treatment in each group of mice; (C) is an image of CD45 immunofluorescence staining of lacrimal gland sections after eye drop treatment in each group of mice. The scale bar in the figure is 100 μm. Detailed Implementation

[0035] Through in-depth research, the inventors discovered that insulin can significantly increase tear secretion levels in mice and, in a mouse model of dry eye caused by tear secretion dysfunction, it has the effect of inhibiting lacrimal gland inflammation and promoting tear secretion. The inventors also found in a scopolamine dry eye model that insulin eye drops are more effective in the early stages of the disease than in the later stages. Furthermore, insulin eye drops have a high safety profile. Therefore, this invention provides the application of insulin in the preparation of drugs for treating dry eye caused by lacrimal gland dysfunction, especially in the early stages of this condition.

[0036] the term

[0037] In this invention, the term "lacrimal gland" generally refers to a gland located in the eye that secretes an aqueous fluid as well as specific types of proteins and electrolytes. The aqueous fluid and specific types of proteins and electrolytes secreted by the lacrimal gland are gradually distributed and extended to the surface of the eye through blinking, evaporate from the surface of the eye, and are discharged through the nasolacrimal duct to form tears.

[0038] The term "lacrimal gland dysfunction-related dry eye syndrome" refers to dry eye syndrome caused by tear film dysfunction due to various reasons, also known as aqueous hypoplasia-related dry eye syndrome. For example, lacrimal gland dysfunction-related dry eye syndrome includes, but is not limited to: dry eye syndrome induced by lacrimal gland proteins or Freund's adjuvant; local lacrimal gland inflammation, damage repair and regeneration induced by IL-1a injection; and local lacrimal gland inflammation and lacrimal gland dysfunction induced by scopolamine injection combined with a dry environment.

[0039] In this article, "early-stage lacrimal gland dysfunction-related dry eye" refers to a disease caused by abnormal lacrimal gland secretion, leading to insufficient tear production and an inability to meet the eye's normal moisturizing needs, resulting in symptoms such as lacrimal gland inflammation. In the early stages of lacrimal gland dysfunction-related dry eye, the conjunctiva and meibomian glands have not yet undergone significant changes. Due to the reduced tear secretion caused by lacrimal dysfunction, inflammatory reactions such as immune cell infiltration occur in the lacrimal glands. Early-stage dry eye can be detected through tear secretion tests and tear film breakup time measurements, revealing reduced tear production and decreased tear film stability, but damage to corneal, conjunctival, and other ocular tissues is not obvious. In the middle and late stages of lacrimal gland dysfunction-related dry eye, changes in other ocular structures and functions have occurred, such as worsening meibomian gland function, corneal epithelial defects, and ocular surface inflammation, making treatment impossible by improving tear secretion. Ocular examinations in the middle and late stages of dry eye will reveal more serious lesions such as corneal epithelial defects, significant conjunctival hyperemia, and meibomian gland atrophy.

[0040] The term "early stage" typically refers to the period before symptoms such as conjunctival and meibomian gland changes, corneal epithelial defects, and ocular surface inflammation appear. In mouse models, the early stage can be within 7 days of the onset of lacrimal gland dysfunction, such as within 5 days, 3 days, 1 day, or even earlier. In human subjects, the early stage can be a period where the tear secretion test (Schirmer I test) result is ≤5 mm / 5 min (e.g., ≤4 mm / 5 min, ≤3 mm / 5 min, ≤2 mm / 5 min or lower), the non-invasive tear film breakup time (NIBUT) is ≤10 seconds (e.g., ≤9 seconds, 8 seconds, 7 seconds, 6 seconds or less), and / or the fluorescein-stained tear film breakup time (FBUT) is ≤5 seconds (e.g., ≤4 seconds, 3 seconds, 2 seconds, 1 second or less).

[0041] The terms “subject” or “patient” may refer to a patient or other animal, especially a mammal, such as a human, mouse, rat, dog, monkey, cow, horse, etc., who receives the insulin or its analogues described in this invention, or the pharmaceutical composition described in this invention to treat, prevent, improve and / or alleviate the lacrimal gland dysfunction type of dry eye.

[0042] The term "insulin" includes insulin and its analogues. Insulin includes natural insulin such as human insulin, bovine insulin, and porcine insulin, as well as precursor proteins of natural insulin, such as human insulin, porcine insulin, bovine insulin, aspart insulin, glargine insulin, and lispro insulin, which are precursor proteins that do not form the insulin double chain.

[0043] The term "insulin analogue" refers to a substance that is identical or similar to natural insulin in structure, use, and intended effect. These insulin analogues include substances formed by mutating, deleting, or adding amino acid sites to natural insulin or its precursor proteins. The insulin analogues include, but are not limited to: insulin analogues formed by replacing natural amino acids with amino acids at positions selected from A4, A5, A8, A9, A10, A12, A13, A14, A15, A16, A17, A18, A19, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B15, B16, B17, B18, B20, B21, B22, B23, B26, B27, B28, B29, and B30; insulin analogues formed by omitting any one or all of insulin B1-4 and B26-30; insulin analogues formed directly or through polymeric or non-polymeric linkers with one or more acyl, PEG, or glycosyl groups; or any combination thereof. For example, the insulin analogs include, but are not limited to: insulin glargine (e.g., Lantus®), insulin detemir (e.g., NovoRapid®), insulin aspart (e.g., insulin aspart 30, more specifically, such as NovoRapid®), insulin lispro (e.g., Humalog®, SusB30®), and insulin DesB30.

[0044] The term "recombinant" refers to a protein obtained through genetic engineering techniques using recombinant DNA or recombinant RNA. In some embodiments, the insulin is recombinant insulin or an analogue thereof. In some specific embodiments, the insulin is recombinant human insulin or an analogue thereof. For example, the sequence of recombinant human insulin is shown in SEQ ID NO:1 (MALWMRLLPLLALLALWGPDPAAAFVNQHLCGSHLVEALYLVCGER GFFYTPKTRREAEDLQVGQVELGGGPGAGSLQPLALEGSLQKRGIVEQCCTSICSLYQLENYC).

[0045] The term "medicine" refers to a substance capable of treating, preventing, improving, and / or alleviating lacrimal gland dysfunction-related dry eye. The term "medicine" is used in a broad sense, encompassing various forms such as single-drug, multi-drug, and pharmaceutical compositions.

[0046] Pharmaceutical Composition

[0047] The pharmaceutical composition of the present invention comprises a pharmaceutically acceptable carrier and the insulin or an analogue thereof as described in the present invention.

[0048] As used herein, the term "pharmaceutically acceptable carrier" refers to a pharmaceutically acceptable material, composition, or transporter, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that participates in carrying or transporting a reagent from one organ or part of the body to another organ or part of the body. A carrier must be "acceptable" in terms of its compatibility with other components of the formulation; for example, the carrier should not diminish the therapeutic effect of the reagent. In other words, the carrier is pharmaceutically inert.

[0049] The composition can be formulated according to known methods for preparing pharmaceutical compositions, wherein the compound is mixed with a pharmaceutically acceptable carrier. Phosphate buffer is an example of a pharmaceutically acceptable carrier. Other suitable carriers are well known to those skilled in the art. See, for example, Remington's Pharmaceutical Sciences, 19th edition (1995).

[0050] Pharmaceutical compositions suitable for topical administration can be formulated as solutions, ointments, creams, emulsions, suspensions, lotions, powders, pastes, gels, sprays, mists, aerosols, or oils. In some embodiments, the pharmaceutical composition is a solution, such as an ophthalmic solution. In some embodiments, the ophthalmic solution is administered topically to humans or non-human mammals at a dose of one or more drops per eye daily.

[0051] For ocular treatment, the composition can be applied as a topical ointment or cream. When formulated as an ointment, the active ingredient can be used in combination with a paraffin or water-miscible ointment base. Alternatively, the active ingredient can be formulated as a cream using an oil-in-water or water-in-oil base.

[0052] Pharmaceutical compositions suitable for topical ocular administration include eye drops, wherein the active ingredient is dissolved or suspended in a suitable carrier (especially an aqueous solvent). Formulations for ocular administration have ocularly compatible pH and osmotic pressure. The pharmaceutical compositions of the present invention may contain one or more ophthalmologically acceptable pH adjusters and / or buffers, including acids such as phosphoric acid, acetic acid, boric acid, citric acid, lactic acid, and hydrochloric acid; bases such as sodium hydroxide, sodium phosphate, sodium borate, sodium citrate, sodium acetate, and sodium lactate; and buffers such as phosphates, citrates, dextran, sodium bicarbonate, and ammonium chloride. Such acids, bases, and buffers may be contained in amounts necessary to maintain the pH of the composition within an ophthalmologically acceptable range. Pharmaceutical compositions may contain one or more ophthalmologically acceptable salts in amounts sufficient to maintain the osmotic pressure of the pharmaceutical composition within an ophthalmologically acceptable range. Such salts include salts containing sodium, potassium, or ammonium cations and chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, or bisulfite anions. In some embodiments, the pharmaceutical composition is an eye drop comprising the insulin or an analogue of the present invention and a buffer solution containing phosphate ions.

[0053] The pharmaceutical compositions described herein can be applied topically to the eye via subconjunctival, intraocular, intravitreal, subocular, subretinal, subchoroidal, or suprachoroidal administration, into the conjunctival sac or eyelid, using ocular delivery devices or implants. Such devices or implants can be designed to achieve controlled release of one or more therapeutic agents according to various predetermined release rates, sustained-release kinetics, and osmotic pressures. Controlled release can be achieved by designing a polymer matrix, utilizing different polymers and their varying properties, such as biodegradable / biocorrosive polymers (e.g., poly(ethylene-vinyl) acetate (EVA), superhydrolyzed PVA), hydroxyalkyl cellulose (HPC), methylcellulose (MC), hydroxypropyl methylcellulose (HPMC), polycaprolactone, polyglycolic acid, polylactic acid, polyanhydride), polymer molecular weight, polymer crystallinity, copolymerization ratio, processing conditions, surface finishing, geometry, excipient addition, and polymer coatings, thereby enhancing drug diffusion, dissolution, solubility, and penetration.

[0054] For pharmaceutical compositions delivered using ocular devices or implants, one or more active agents may be combined with adjuvants suitable for the designated route of administration. For example, the active agent may be mixed with any pharmaceutically acceptable excipient, lactose, sucrose, starch powder, cellulose esters, stearic acid, talc, magnesium stearate, magnesium oxide, sodium and calcium salts of phosphoric acid and sulfuric acid, gum arabic, gelatin, sodium alginate, polyvinylpyrrolidone, and / or polyvinyl alcohol, tableted, or encapsulated for routine administration. Alternatively, the compound may be dissolved in polyethylene glycol, propylene glycol, carboxymethyl cellulose colloidal solution, ethanol, corn oil, peanut oil, cottonseed oil, sesame oil, tragacanth gum, and / or various buffer solutions. The compound may also be mixed with compositions comprising biodegradable and / or non-biodegradable polymers, and with a carrier or diluent having delay properties. Representative examples of biodegradable compositions may include albumin, gelatin, starch, cellulose, dextran, polysaccharides, poly(D,L-lactide), poly(D,L-lactide-co-glycolic acid), poly(glycolic acid), poly(hydroxybutyrate), poly(alkyl carbonate), and poly(orthoester), and mixtures thereof. Representative examples of non-biodegradable polymers include EVA copolymers, silicone rubber, and poly(methacrylate), and mixtures thereof.

[0055] Pharmaceutical compositions for ocular delivery also include in-situ gellable aqueous compositions. Such compositions comprise a concentration of gelling agent that promotes gelation upon contact with the eye or tear film. Suitable gelling agents include, but are not limited to, thermosetting polymers. As used herein, the term "in-situ gellable" includes not only low-viscosity liquids that form a gel upon contact with the eye or tear film, but also more viscous liquids, such as semi-fluids and thixotropic gels, whose viscosity or gel hardness increases significantly upon delivery to the eye. See, for example, Ludwig (2005), which is incorporated herein by reference for its teachings on example polymers for ocular drug delivery.

[0056] Methods and Applications

[0057] The present invention provides a method for treating lacrimal gland dysfunction-related dry eye, especially early-stage lacrimal gland dysfunction-related dry eye, comprising administering to a subject a therapeutically effective amount of insulin, its analogues, or a pharmaceutical composition thereof.

[0058] The treatment methods described in this article are typically local administration, such as applying the medication to the eye via the subconjunctival, intraocular, vitreous, subocular, subretinal, subchoroidal, or suprachoroidal routes, or to the conjunctival sac or eyelid.

[0059] As used herein, “treatment” includes any beneficial or desired effect on the symptoms or lesions of a disease or pathological condition, and may include symptoms of a disease or condition under treatment, particularly those of lacrimal gland dysfunction-related dry eye, including but not limited to decreased tear production, damage to lacrimal gland tissue structures (e.g., ducts and acini), lacrimal gland inflammation, and lacrimal gland lymphocyte infiltration. Treatment may optionally include a reduction or relief of symptoms of a disease or condition, or a delay in the progression of a disease or condition. “Treatment” does not necessarily mean the complete eradication or cure of a disease or condition or its associated symptoms. As used herein, “treatment” for a disease in a subject means (1) suppressing or preventing the development of lacrimal gland dysfunction-related dry eye; or (2) improving or causing the resolution of symptoms of lacrimal gland dysfunction-related dry eye. As understood in the art, “treatment” is a method for obtaining a beneficial or desired outcome, including clinical outcomes. Beneficial or desired outcomes may include, but are not limited to, relief or improvement of one or more symptoms, whether detectable or undetectable, reduction of the severity of symptoms (including disease), stabilization (i.e., non-worsening) of symptoms (including disease), delay or slowing of the progression of symptoms (including disease), improvement or relief of symptoms (including disease), status, and remission (whether partial or complete).

[0060] The "subject" or "patient" in the treatment can be an animal, especially a mammal, such as a human, mouse, rat, dog, monkey, cow, horse, etc. Subjects are not limited to a specific species, including non-human animals receiving diagnosis or treatment and those receiving infection or animal models, including but not limited to mouse, rat, ape, dog, or rabbit species, as well as other livestock, locomotor animals, or pets. In some embodiments, the subject or patient is a human.

[0061] The term "effective amount" as used herein is intended to mean an amount sufficient to achieve the desired effect. In the case of therapeutic or preventative applications, the effective amount will depend on the type and severity of the condition in question and the characteristics of the individual subject or patient, such as general health, age, sex, weight, and tolerance to the pharmaceutical composition. In some embodiments, when insulin or its analogues are administered, the effective amount is sufficient to cause partial or complete restoration of function of the lacrimal glands in the subject or patient with dysfunction, or to maintain, prevent progression, reduce, improve, or alleviate symptoms of lacrimal gland dysfunction-related dry eye. In some embodiments, the effective amount also depends on the nature and sensitivity of the target subject and the method of administration. Those skilled in the art will be able to determine the effective amount based on these and other considerations. According to embodiments, the effective amount may include one or more administrations of the pharmaceutical composition, consist substantially of one or more administrations of the pharmaceutical composition, or consist of one or more administrations of the pharmaceutical composition.

[0062] As used herein, the term "administration" is intended to mean the delivery of a substance to a subject or patient, such as an animal or human. Administration can be performed at a single dose, continuously or intermittently, throughout the course of treatment. Methods for determining the most effective manner and dosage of administration are known to those skilled in the art and will vary depending on the pharmaceutical composition used for treatment, the purpose of treatment, and the age, health, or sex of the subject being treated. Administration can be performed as a single or multiple doses, with the dosage level and pattern chosen by the treating physician, or, in the case of pets and other animals, by the treating veterinarian. In some specific embodiments, the administration is ocular surface administration, such as intraconjunctival administration.

[0063] Methods for determining the most effective administration route and dosage are known to those skilled in the art and will vary depending on the pharmaceutical composition used for treatment, the purpose of treatment, and the subject being treated. Single or multiple administrations may be performed, with the dosage level and mode chosen by the treating physician. The dosage may be affected by the route of administration. Suitable dosage formulations and methods of administering the reagents are known in the art. A non-limiting example of such a suitable dosage for mice is an administration of 0.5–2 IU / mL of a pharmaceutical composition containing insulin or its analogues per dose. Converting mouse doses to human doses is readily performed by those skilled in the art, for example, by calculation using the Meeh-Rubner formula: Meeh-Rubner formula: A=k×(W 2 / 3 ) / 10,000.

[0064] In the formula, A is the body surface area, expressed in meters. 2 Calculation; W is body weight, expressed in g; K is a constant that varies depending on the animal species: 9.1 for mice and rats, 9.8 for guinea pigs, 10.1 for rabbits, 9.9 for cats, 11.2 for dogs, 11.8 for monkeys, and 10.6 for humans.

[0065] The insulin, its analogues, or pharmaceutical compositions of the present invention can be administered at a single dose, continuously or intermittently, throughout the course of treatment. In some embodiments, the insulin, its analogues, or pharmaceutical compositions of the present invention are administered via intravitreal instillation onto the ocular surface, for example, intraconjunctival sac of the eyelid. In some embodiments, the insulin, its analogues, or pharmaceutical compositions of the present invention are administered at a frequency of 1-2 drops per administration, 2-5 times daily, with intervals of 3-6 hours. In some preferred embodiments, the insulin, its analogues, or pharmaceutical compositions of the present invention are administered at a frequency of 1 drop per administration, 4 times daily, with intervals of 4 hours.

[0066] The insulin, its analogues, and pharmaceutical compositions of the present invention can also be administered in combination with other known drugs and treatments.

[0067] medicine box

[0068] The present invention also provides a kit for treating lacrimal gland dysfunction-related dry eye, especially early-stage lacrimal gland dysfunction-related dry eye, wherein the kit contains the insulin, its analogues, or pharmaceutical compositions thereof described in the present invention.

[0069] In the pillbox of this invention, the insulin, its analogues, or pharmaceutical compositions thereof can be placed in the same container as a pharmaceutically acceptable carrier, or they can be placed in separate containers. In some embodiments, the pillbox comprises: container 1 containing the insulin or its analogue, and container 2 containing a pharmaceutically acceptable carrier. Before use, the substances in container 1 and container 2 are mixed together to form a pharmaceutical composition, which is then administered to the subject.

[0070] The medicine box of the present invention may also contain other known drugs for treating dry eye syndrome, such as, but not limited to: artificial tears, eye drops, ocular serum preparations, and drugs for treating ocular surface inflammation (including tetracyclines, glucocorticoids, nonsteroidal anti-inflammatory drugs, and immunosuppressants). The drugs may be placed in the same container within the medicine box, or they may be placed in separate containers.

[0071] In addition, the medicine box may also contain some auxiliary medication materials.

[0072] In addition, the medicine box may also contain instructions for use, explaining the treatment of lacrimal gland dysfunction-related dry eye, especially early-stage lacrimal gland dysfunction-related dry eye.

[0073] The present invention will be further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0074] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0075] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.

[0076] The present invention will be described in detail below with reference to embodiments.

[0077] The experimental animals used and their housing conditions are as follows in the following examples: Experimental animals: C57Bl / 6JNifdc mice, male or female, 20-25g, purchased from the Experimental Animal Center of Tianjin Medical University, and housed in the clean-grade animal room of the Experimental Animal Center of Tianjin Medical University, with a temperature of 21℃-25℃, relative humidity of 30%-60%, 12-hour light cycle, and provided with sufficient food and water.

[0078] The recombinant human neutral insulin used in the following embodiments of this application was purchased from Procell (PB180432). The insulin analogues used were insulin glargine injection (Lantus®), purchased from Sanofi (Beijing) Pharmaceutical Co., Ltd.; insulin lispro injection (Humalog®), purchased from Eli Lilly Suzhou Pharmaceutical Co., Ltd.; insulin lispro injection (Susoxel®), Gan & Lee Pharmaceuticals Co., Ltd.; insulin detemir injection (Novo Nordisk®), purchased from Novo Nordisk (China) Pharmaceutical Co., Ltd.; and insulin aspart 30 injection (NovoRapid®), purchased from Novo Nordisk (China) Pharmaceutical Co., Ltd.

[0079] Example 1: Preparation and Use of Insulin Eye Drops

[0080] In this embodiment, an eye drop containing a low concentration of insulin for the treatment of dry eye was prepared, which is composed of recombinant human neutral insulin and phosphate buffer.

[0081] Preparation method of insulin eye drops: Recombinant human neutral insulin is dissolved in phosphate buffer, the pH is adjusted to 7.2-7.4, and the insulin concentration is adjusted to 0.5 IU / mL, 1 IU / mL and 2 IU / mL. The solution is filtered through a 0.22 μm microporous membrane for sterilization to obtain insulin eye drops.

[0082] Preparation method of solvent eye drops: Use phosphate buffer to adjust the pH to 7.2-7.4, filter and sterilize with a 0.22μm microporous membrane to obtain solvent eye drops.

[0083] Insulin eye drops usage instructions: Remove the cap from the insulin eye drops bottle, without touching the opening, and place the bottle with the inside of the cap facing up on a clean table. With your other hand, hold the insulin eye drops bottle and squeeze out one drop of eye drops, then drop it into the conjunctival sac of the mouse's lower eyelid. First, drop into the right eye, then the left eye. Gently close both eyelids of the mouse several times to allow the medication to spread fully. Release the mouse and observe the eye surface for two minutes before returning it to the cage.

[0084] Example 2: Safety assessment of insulin eye drops for ocular surface application

[0085] Grouping and treatment of experimental animals: Eight wild-type male C57Bl / 6JNifdc mice, weighing 20-25 grams, were randomly divided into two groups and subjected to the following treatments: 1. Solvent control group: Equal volume of solvent eye drops, applied to the eyes; 2. Insulin eye drop administration group: The 1 IU / mL insulin eye drops prepared in Example 1 were used, 1 drop each time, 4 times a day, with an interval of 4 hours between each application. The insulin eye drops were prepared fresh on the day of use, and the patients were observed after 7 days of administration.

[0086] After the above treatment, mice in each group were anesthetized by intraperitoneal injection of afodin (0.2 mL / 10 g), and 2 μL of 1% sodium fluorescein (purchased from Solarbio, catalog number F8140) was dripped into the lateral conjunctival sac of the mice. Three minutes later, the corneal epithelial staining was observed under cobalt blue light using a slit-lamp microscope, and photographs were taken using a slit-lamp imaging system.

[0087] Figure 2 Figure A shows the corneal epithelial staining in each group of mice. It indicates that neither group showed any staining of the corneal epithelium, demonstrating intact and unstained corneal epithelium. This result suggests that there was no significant difference in corneal epithelial staining between the insulin eye drop group and the solvent control group.

[0088] Mice were euthanized by cervical dislocation, and the orbits were surgically removed. After fixation in 4% paraformaldehyde for at least 24 hours, the orbitals were embedded in paraffin and sectioned at 4 μm using a paraffin microtome. The sections were dewaxed and stained with a PAS staining kit (Solarbio, catalog number G1281). The morphology of the conjunctival sac and goblet cells was observed and photographed using an optical microscope.

[0089] Figure 2 Figure B shows the PAS staining results of orbital sections of mice in each group. It shows that the conjunctival goblet cells in the insulin eye drop group were stained purple-red and were evenly distributed in the conjunctival sacs of the palpebral and ocular conjunctiva. They were round and plump, and there was no significant change in morphology and number compared with the solvent group.

[0090] Mice were euthanized by cervical dislocation, and their lacrimal glands were harvested and fixed in 4% paraformaldehyde for at least 24 hours. The samples were washed and dehydrated in ethanol solutions of increasing concentrations, and after a final wash with anhydrous ethanol, cleared in xylene for 2 hours. The samples were then immersed in soft paraffin at 59°C and hard paraffin at 63°C, respectively, and finally embedded. Sections were prepared at 5 μm using a paraffin microtome and stained with hematoxylin and eosin (Solarbio, catalog number G1120) for morphological examination. The morphology of the lacrimal glands was observed and photographed using an optical microscope.

[0091] Figure 2 Figure C shows the H&E staining results of lacrimal gland sections from each group of mice. It shows that the acinar and ductal epithelial cells in the insulin eye drop group are intact and regularly arranged, with no obvious inflammatory cell infiltration. Compared with the solvent group, there are no significant changes in structure and morphology.

[0092] The above results indicate that insulin eye drops have no significant toxicity to tissues such as the cornea, conjunctiva, and lacrimal glands, and have a high safety profile when applied to the ocular surface.

[0093] Example 3: The effect of insulin eye drops on tear secretion when applied to the ocular surface

[0094] Grouping and treatment of experimental animals: Sixteen wild-type male C57Bl / 6JNifdc mice, weighing 20-25 grams, were randomly divided into 4 groups and subjected to the following treatments: 1. Solvent control group: treated with an equal amount of solvent as eye drops; 2. Insulin eye drop administration group (including 3 dosage groups: 0.5 IU / mL, 1 IU / mL, and 2 IU / mL): The 0.5 IU / mL, 1 IU / mL, and 2 IU / mL insulin eye drops prepared in Example 1 were used, one drop each time, four times daily, with an interval of one drop every four hours. The insulin eye drops were prepared fresh on the day of use, and observation was conducted after 7 days of administration.

[0095] After the above treatment, the lower eyelids of the mice in each group were gently pulled back, and one end of a phenol red cotton thread was placed in the lateral conjunctival sac of the mouse's eye. The absorption of tears would cause the cotton thread to turn from yellow to red, thus reflecting the amount of tear secretion. After 30 seconds, the cotton thread was removed, and the length of the reddened portion was measured and recorded. This was repeated three times for each eye, and the average value of the measurements from both eyes was taken as the tear secretion flow rate. A higher tear secretion flow rate indicates better lacrimal gland function.

[0096] Figure 3 A represents the experimental protocol for administering insulin eye drops to mice; Figure 3 Figure B is a schematic diagram showing the reddening of the phenol red cotton thread after tear measurement. The longer the reddened cotton thread, the greater the tear secretion. It shows that compared with the mice given solvent, the mice given 0.5 IU / mL, 1 IU / mL and 2 IU / mL insulin eye drops had a longer range of reddened phenol red cotton thread. Figure 3 Figure C is a statistical graph of the results of the phenol red cotton thread experiment, which shows that compared with the solvent control group, the mice in the 0.5 IU / mL, 1 IU / mL and 2 IU / mL insulin eye drop administration groups had significantly increased tear secretion.

[0097] The above results indicate that insulin eye drops can improve tear secretion in mice.

[0098] Example 4: In vivo experiments demonstrated that insulin receptor inhibitor eye drops can inhibit tear secretion in mice.

[0099] Preparation method of insulin receptor inhibitor eye drops: Dissolve insulin receptor inhibitor BMS-754807 powder in DMSO, and adjust the mass percentage concentration of insulin receptor inhibitor to 0.01% using phosphate buffer to obtain insulin receptor inhibitor eye drops.

[0100] Grouping and treatment of experimental animals: Six wild-type male C57Bl / 6JNifdc mice, weighing 20-25 grams, were randomly divided into two groups and subjected to the following treatments: 1. 0.01% DMSO control group: treated with eye drops of 0.01% DMSO prepared with an equal volume of solvent; 2. Insulin receptor inhibitor eye drop administration group: 0.01% insulin receptor inhibitor eye drops dissolved in DMSO were administered, 1 drop each time, 4 times a day, with an interval of 4 hours between drops. The insulin receptor inhibitor eye drops were prepared fresh on the day of use, and the group was observed after 7 days of administration.

[0101] After the above treatment, the lower eyelids of the mice in each group were gently pulled back, and one end of a phenol red cotton thread was placed in the conjunctival sac on the outer side of the mouse's eye. The absorption of tears would cause the cotton thread to turn from yellow to red. After 30 seconds, the cotton thread was removed, and the length of the reddened portion of the phenol red cotton thread was measured and recorded. This was repeated three times for each eye, and the average value of the measurements from both eyes was taken as the tear secretion flow rate. A higher tear secretion flow rate indicates better lacrimal gland function.

[0102] The results are as follows Figure 4 As shown. Figure 4 A represents the experimental protocol for administering insulin receptor inhibitor eye drops to mice. Figure 4 Figure B is a schematic diagram showing the reddening of the cotton thread after tear measurement. The longer the reddened cotton thread, the more tear secretion. It shows that compared with the mice in the group given 0.01% DMSO, the mice in the group given insulin receptor inhibitor eye drops had a shorter range of reddened cotton thread. Figure 4 Figure C is a statistical graph of the phenol red cotton thread experiment results, which shows that compared with the mice given 0.01% DMSO control, the mice in the insulin receptor inhibitor eye drop group had significantly reduced tear secretion.

[0103] Example 5: Evaluation of the efficacy of insulin eye drops on lacrimal gland protein-induced dry eye in Sjögren's syndrome.

[0104] Grouping and treatment of experimental animals: Eleven wild-type C57Bl / 6JNifdc mice, female, weighing 20-25 grams, were divided into 3 groups and subjected to the following treatments: 1. Normal Control Group: Four female C57Bl / 6Jnifdc mice were anesthetized by intraperitoneal injection of afodin (0.2 mL / 10 g) on ​​days 0 and 7. Then, equal volumes of Freund's complete adjuvant and phosphate-buffered saline (PBFS) were mixed and injected subcutaneously at multiple points on the back of the mice. This subcutaneous injection was repeated on day 7. On day 14, equal volumes of Freund's incomplete adjuvant and PBFS were mixed and injected subcutaneously at multiple points on the back of the mice anesthetized by intraperitoneal injection of afodin (0.2 mL / 10 g). On day 21, the mice were treated with eye drops prepared using the method described in Example 1, one drop at a time, four times daily, with drops administered every four hours. After 14 days of treatment, changes in tear secretion and lacrimal gland inflammation were observed.

[0105] 2. Experimental Sjögren's syndrome (ESS) model induced by lacrimal gland protein: Adult wild-type mice were euthanized by cervical dislocation. The skin at the outer canthus of the eye and below the ear was cut open, and the subcutaneous tissue was bluntly dissected to expose and isolate the lacrimal gland. Fat and connective tissue were removed, and the tissue was homogenized in a solvent using a homogenizer. The homogenate was then centrifuged at 12000g for 5 minutes at 4°C. The supernatant was collected to obtain a lacrimal gland homogenate. The protein concentration was determined using Nanodrop, and the homogenate was emulsified with an equal volume of Freund's complete adjuvant to adjust the protein concentration to 10 mg / mL.

[0106] Three female C57Bl / 6Jnifdc mice were anesthetized with 4% chloral hydrate via intraperitoneal injection on days 0 and 7, respectively. Then, 0.1 mL of a lacrimal gland homogenate with a protein concentration of 10 mg / mL was injected subcutaneously at multiple points on the back. On day 14, a booster immunization was performed by emulsifying a mixture of the above lacrimal gland protein at a concentration of 20 mg / mL with an equal volume of Freund's incomplete adjuvant. On day 21, one drop of the solvent eye drops prepared in Example 1 was administered four times daily at 4-hour intervals. Changes in tear secretion and lacrimal gland inflammation were observed after 14 days of treatment. 3. ESS Modeling Combined with Insulin Eye Drops: Four female C57Bl / 6Jnifdc mice were anesthetized with intraperitoneal injection of 4% chloral hydrate on days 0 and 7, respectively. Multiple subcutaneous injections of 0.1 mL of the emulsion used in Example 5 (ESS modeling group) were then administered to the back. On day 14, a booster immunization was performed using a mixture of 20 mg / mL lacrimal gland protein (used in Example 5 (ESS modeling group)) and an equal volume of Freund's incomplete adjuvant. On day 21, one drop of 1 IU / mL insulin eye drops prepared in Example 1 was administered four times daily, with an interval of four hours between each application. The insulin eye drops were prepared fresh on the day of use. Changes in tear secretion and lacrimal gland inflammation were observed 14 days after administration.

[0107] The results are as follows Figure 5 As shown in Figure A, the protocol for administering insulin eye drops to mice with Sjögren's syndrome (ESS) combined with the insulin eye drop treatment is illustrated. Figure B shows the statistical results of the phenol red cotton thread experiment, which indicates that compared with the control mice, ESS mice showed significantly reduced tear secretion, while the ESS model combined with insulin eye drops group showed significantly increased tear secretion compared with ESS mice. Figure C shows the H&E staining results of lacrimal gland sections from each group of mice. The results show that compared with the lacrimal glands of control mice, ESS mice showed extensive lymphocyte infiltration, with dense granular cells clustered around the acinar ducts indicating lymphocyte infiltration foci, accompanied by destruction of the ductal and acinar structures. In contrast, the ESS model combined with insulin eye drops group showed a significant reduction in lacrimal gland lymphocyte infiltration foci.

[0108] The above results indicate that combining insulin eye drops with a mouse model of dry eye syndrome can increase tear secretion and reduce lacrimal gland inflammation, thereby alleviating dry eye symptoms.

[0109] Example 6: Evaluation of the efficacy of insulin eye drops in a dry eye model induced by early scopolamine injection combined with a dry environment.

[0110] Grouping and treatment of experimental animals: Twelve wild-type male C57Bl / 6JNifdc mice, weighing 20-25 grams, were divided into three groups and subjected to the following treatments: 1. Normal control group: Four male C57Bl / 6Jnifdc mice were kept in a dry environment (humidity below 30%). The solvent eye drops prepared in Example 1 were used from the first day, one drop each time, four times a day at 4-hour intervals. The changes in tear secretion were observed on the 1st, 3rd, 5th and 7th days after administration, and the lacrimal gland inflammation was observed on the seventh day.

[0111] 2. Scopolamine model group: Four male C57Bl / 6Jnifdc mice were housed in a dry environment (humidity below 30%). Scopolamine (purchased from GLPBIO, catalog number GN10766) was dissolved in PBS and the concentration was adjusted to 2.5 mg / ml. At three time points each day, 9:00 AM, 3:00 PM, and 9:00 PM, for 7 consecutive days, 200 μL of scopolamine solution was injected subcutaneously into the back of the mice at each time point. At the same time, starting from the first day, the solvent eye drops prepared in Example 1 were used, 1 drop each time, 4 times at 4-hour intervals each day. Changes in tear secretion were observed on days 1, 3, 5, and 7 after administration, and the condition of lacrimal gland inflammation was observed on the seventh day.

[0112] 3. Scopolamine modeling combined with insulin eye drops: Four male C57Bl / 6Jnifdc mice were kept in a dry environment (humidity below 30%). Scopolamine (purchased from GLPBIO, catalog number GN10766) was dissolved in PBS and the concentration was adjusted to 2.5 mg / ml. At three time points each day, 9:00 AM, 3:00 PM, and 9:00 PM, for 7 consecutive days, 200 μL of scopolamine solution was injected subcutaneously into the back of the mice at each time point. At the same time, starting from the first day, 1 IU / mL insulin eye drops prepared in Example 1 were used, 1 drop each time, 4 times at 4-hour intervals each day. Changes in tear secretion were observed on days 1, 3, 5, and 7 after administration, and the condition of lacrimal gland inflammation was observed on the seventh day.

[0113] The results are as follows Figure 6 As shown in Figure A, which illustrates the protocol for scopolamine-induced tear production combined with insulin eye drops, and Figure B, which presents a statistical chart of the phenol red cotton thread experiment results, shows that compared to the control mice, scopolamine-induced tear production was significantly reduced from day one. In contrast, the scopolamine-induced tear production combined with insulin administration group showed an increase in tear production compared to scopolamine-induced tear production from day three onwards, with a significant difference observed from day five and continuing until day seven. Figure C shows the location and relative number of CD45 cells in the lacrimal gland sections of mice in each group, stained with immunofluorescence. (CD45 protein is generally expressed in immune cells and is used to indicate inflammation; that is, the degree of tissue inflammation is positively correlated with the number of CD45-positive cells.) The results showed that compared with the lacrimal glands of control mice, a large number of CD45-positive immune cells infiltrated around the acinar ducts in the scopolamine model group, indicating severe lacrimal gland inflammation. In contrast, the infiltration of CD45-positive immune cells in the lacrimal glands was significantly reduced in the scopolamine model combined with insulin administration group. That is, insulin eye drops alleviated the infiltration of inflammatory cells in the lacrimal glands of dry-eye mice induced by early scopolamine injection combined with a dry environment.

[0114] Example 7: Evaluation of the efficacy of insulin eye drops in a dry eye model induced by late-stage scopolamine injection combined with a dry environment.

[0115] Grouping and treatment of experimental animals: Eleven wild-type male C57Bl / 6JNifdc mice, weighing 20-25 grams, were divided into three groups and subjected to the following treatments: 1. Normal control group: Four male C57Bl / 6Jnifdc mice were kept in a dry environment (humidity below 30%). Starting from the seventh day, they were given the solvent eye drops prepared in Example 1, one drop each time, four times a day at 4-hour intervals. The changes in tear secretion and the condition of lacrimal gland inflammation were observed on the third, seventh, tenth and fourteenth days.

[0116] 2. Scopolamine model group: Four male C57Bl / 6Jnifdc mice were kept in a dry environment (humidity below 30%). Scopolamine (purchased from GLPBIO, catalog number GN10766) was dissolved in PBS and the concentration was adjusted to 2.5 mg / ml. At three time points each day, 9:00 AM, 3:00 PM, and 9:00 PM, for 7 consecutive days, 200 μL of scopolamine solution was injected subcutaneously into the back of the mice at each time point. At the same time, starting from the first day, the solvent eye drops prepared in Example 1 were used, one drop each time, four times a day at 4-hour intervals. Changes in tear secretion and lacrimal gland inflammation were observed on the third, seventh, tenth, and fourteenth days.

[0117] 3. Scopolamine modeling combined with insulin eye drops: Four male C57Bl / 6Jnifdc mice were kept in a dry environment (humidity below 30%). Scopolamine (purchased from GLPBIO, catalog number GN10766) was dissolved in PBS and the concentration was adjusted to 2.5 mg / ml. At three time points each day, 9:00 AM, 3:00 PM, and 9:00 PM, for 7 consecutive days, 200 μL of scopolamine solution was injected subcutaneously into the back of the mice at each time point. At the same time, starting from the first day, 1 IU / mL insulin eye drops prepared in Example 1 were used, 1 drop each time, 4 times at 4-hour intervals each day. Changes in tear secretion and lacrimal gland inflammation were observed on the third, seventh, tenth, and fourteenth days.

[0118] The results are as follows Figure 7 As shown in Figure A, which illustrates the protocol for scopolamine-induced tear production combined with insulin eye drops, and Figure B, which presents a statistical chart of the phenol red cotton thread experiment results, shows that compared to the control mice, the tear secretion of scopolamine-induced mice was significantly reduced from day three until day fourteen. In contrast, the tear secretion of mice in the scopolamine-induced combined insulin group was significantly reduced from day three, and after starting eye drops on day seven, the tear secretion did not increase compared to the scopolamine-induced mice, continuing until day fourteen. Figure C shows the location and relative number of CD45 cells in the lacrimal gland sections of mice in each group, obtained through immunofluorescence staining. (CD45 protein is generally expressed in immune cells and is used to indicate inflammation; that is, the degree of tissue inflammation is positively correlated with the number of CD45-positive cells.) The results indicate that compared with the lacrimal glands of control mice, a large number of CD45-positive immune cells infiltrated around the acinar ducts in the scopolamine model group, indicating severe lacrimal gland inflammation. At the same time, the infiltration of CD45-positive immune cells in the lacrimal glands was not significantly reduced in the scopolamine model combined with insulin administration group. That is, the administration of late-stage insulin eye drops in the scopolamine dry eye model could not alleviate the infiltration of inflammatory cells in the lacrimal glands of diabetic dry eye mice.

[0119] Example 8: Evaluation of the efficacy of insulin analog eye drops in a dry eye model induced by early scopolamine injection combined with a dry environment.

[0120] In this embodiment, an eye drop containing a low concentration of insulin analogue for the treatment of dry eye was prepared, which consists of insulin analogue injection solution and phosphate buffer.

[0121] Preparation method of insulin analog eye drops: Dissolve five insulin analog injection solutions (Lantux®, Humalog®, Susquercetin®, NovoRapid®, and NovoRapid®) in phosphate buffer, adjust the pH to 7.2-7.4, and adjust the insulin concentration to 1 IU / mL. Filter the solution through a 0.22μm microporous membrane for sterilization to obtain insulin eye drops.

[0122] Preparation method of solvent eye drops: Use phosphate buffer to adjust the pH to 7.2-7.4, filter and sterilize with a 0.22μm microporous membrane to obtain solvent eye drops.

[0123] Grouping and treatment of experimental animals: 1228 wild-type male C57Bl / 6JNifdc mice, weighing 20-25 grams, were divided into 7 groups and subjected to the following treatments: 1. Normal control group: Four male C57Bl / 6Jnifdc mice were kept in a dry environment (humidity below 30%). The solvent eye drops prepared in Example 1 were used from the first day, one drop each time, and four times a day at 4-hour intervals. The changes in tear secretion were observed for seven days after administration and the condition of lacrimal gland inflammation was observed on the seventh day.

[0124] 2. Scopolamine model group: Four male C57Bl / 6Jnifdc mice were kept in a dry environment (humidity below 30%). Scopolamine (purchased from GLPBIO, catalog number GN10766) was dissolved in PBS and the concentration was adjusted to 2.5 mg / ml. At three time points each day, 9:00 AM, 3:00 PM, and 9:00 PM, for 7 consecutive days, 200 μL of scopolamine solution was injected subcutaneously into the back of the mice at each time point. At the same time, the solvent eye drops prepared in Example 8 were used from the first day, 1 drop each time, 4 times at 4-hour intervals each day. Changes in tear secretion were observed 7 days after administration and the condition of lacrimal gland inflammation was observed on the seventh day.

[0125] 3. Scopolamine modeling combined with insulin analog eye drops: For each insulin analog eye drops, four male C57Bl / 6Jnifdc mice were kept in a dry environment (humidity below 30%). Scopolamine (purchased from GLPBIO, catalog number GN10766) was dissolved in PBS and the concentration was adjusted to 2.5 mg / ml. At three time points each day, 9:00 AM, 3:00 PM, and 9:00 PM, for seven consecutive days, 200 μL of scopolamine solution was injected subcutaneously into the back of the mice at each time point. At the same time, starting from the first day, the 1 IU / mL insulin analog eye drops prepared in Example 8 were used, one drop each time, four times a day at 4-hour intervals. Changes in tear secretion were observed for seven days after administration, and the condition of lacrimal gland inflammation was observed on the seventh day.

[0126] The results are as follows Figure 8 As shown in Figure A, which illustrates the protocol for scopolamine-induced tear production combined with insulin analog eye drops, and Figure B, which presents a statistical chart of the phenol red cotton thread experiment results, shows that compared to the control mice, the tear secretion of scopolamine-induced mice was significantly reduced on day seven, while the tear secretion of the scopolamine-induced mice combined with insulin analog eye drops group was significantly increased on day seven compared to the scopolamine-induced mice. Figure C shows the location and relative number of CD45 cells in the lacrimal gland sections of mice in each group, stained with immunofluorescence. (CD45 protein is generally expressed in immune cells and is used to indicate inflammation; that is, the degree of tissue inflammation is positively correlated with the number of CD45-positive cells.) The results showed that compared with the lacrimal glands of control mice, a large number of CD45-positive immune cells infiltrated around the acinar ducts in the scopolamine model group, indicating severe lacrimal gland inflammation. The infiltration of CD45-positive immune cells in the lacrimal glands was significantly reduced in the scopolamine model combined with insulin analog administration group. That is, the administration of insulin analog eye drops alleviated the infiltration of inflammatory cells in the lacrimal glands of dry-eye mice induced by early scopolamine injection combined with a dry environment.

[0127] The above results indicate that insulin eye drops can improve lacrimal gland function under physiological conditions, thereby increasing tear secretion in mice. Furthermore, in mouse models of Sjögren's syndrome and early scopolamine dry eye, treatment with insulin eye drops increased tear secretion and reduced lacrimal gland inflammation, thus alleviating dry eye symptoms. Similarly, in the early scopolamine dry eye model, treatment with insulin analog eye drops also increased tear secretion and reduced lacrimal gland inflammation. However, in the late scopolamine dry eye model, insulin eye drops did not increase tear secretion or reduce lacrimal gland inflammation. This suggests that for early-stage lacrimal gland dysfunction-related dry eye, insulin eye drops are more effective in the early stages of the disease than in the late stages.

[0128] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the present invention. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this patent should be determined by the appended claims. Furthermore, all documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference.

Claims

1. The use of insulin or its analogues in the preparation of drugs for treating lacrimal gland dysfunction-related eye diseases.

2. The application as described in claim 1, characterized in that, The insulin includes animal insulin and human insulin; preferably, the animal insulin includes human insulin, porcine insulin, and bovine insulin.

3. The application as described in claim 1, characterized in that, The insulin analogues are selected from: 1) An insulin analogue formed by the substitution, deletion or addition of amino acids at one or more positions selected from A4, A5, A8, A9, A10, A12, A13, A14, A15, A16, A17, A18, A19, A21, B1, B2, B3, B4, B5, B9, B10, B13, B14, B15, B16, B17, B18, B20, B21, B22, B23, B26, B27, B28, B29 and B30, which has the same or similar biological function as said insulin; 2) Insulin analogues formed by the absence of insulin B1-4 and / or B26-30, which have the same or similar biological functions as said insulin; 3) Insulin analogs formed by directly or through polymeric or non-polymeric linkers conjugating one or more acyl, PEG, or glycosyl groups to insulin, which have the same or similar biological functions as said insulin.

4. The application as described in claim 3, characterized in that, The insulin analogue is selected from one or more of insulin glargine, insulin detemir, insulin aspart, insulin lispro, and insulin DesB30.

5. The application as described in any one of claims 1-4, characterized in that, The aforementioned lacrimal gland dysfunction type of eye disease is an eye disease caused by lacrimal gland dysfunction or lack of fluid.

6. The application as described in claim 5, characterized in that, The lacrimal gland dysfunction type of eye disease is lacrimal gland dysfunction type of dry eye syndrome.

7. The application as described in claim 6, characterized in that, The lacrimal gland dysfunction-related dry eye includes: dry eye syndrome induced by lacrimal gland protein or Freund's adjuvant; local lacrimal gland inflammation, damage repair and regeneration induced by IL-1a injection; and local lacrimal gland inflammation and lacrimal gland dysfunction induced by scopolamine injection combined with a dry environment.

8. The application as described in any one of claims 1-4, characterized in that, The lacrimal gland dysfunction type of eye disease mentioned above is early-stage lacrimal gland dysfunction type of dry eye syndrome.

9. The application as described in claim 8, characterized in that, The early lacrimal gland dysfunction type of dry eye includes: no obvious changes in the conjunctiva and meibomian glands, tear dysfunction leading to reduced tear secretion, and inflammatory reactions such as immune cell infiltration in the lacrimal gland.

10. The application as described in claim 8, characterized in that, For mouse models, the early stage is within 7 days of the onset of lacrimal gland dysfunction; for human subjects, the early stage is the period when the tear secretion test result is ≤5mm / 5min, the non-invasive tear film breakup time is ≤10 seconds, and / or the fluorescein-stained tear film breakup time is ≤5 seconds.

11. A pharmaceutical composition for treating lacrimal gland dysfunction-related eye diseases, characterized in that, The pharmaceutical composition contains a therapeutically effective amount of insulin or an analogue thereof, and a pharmaceutically acceptable carrier.

12. The pharmaceutical composition according to claim 11, characterized in that, The lacrimal gland dysfunction ophthalmopathy is defined as in claims 5-10; and / or, the insulin or its analogue is defined as in any one of claims 2-4.

13. The pharmaceutical composition of claim 11, characterized in that, Pharmaceutically acceptable carriers include ophthalmologically acceptable salts.

14. The pharmaceutical composition according to claim 13, characterized in that, The ophthalmologically acceptable salts include salts containing one or more cations selected from sodium, potassium, and ammonium, and one or more anions selected from chloride, citrate, ascorbate, borate, phosphate, bicarbonate, sulfate, thiosulfate, and bisulfite.

15. The pharmaceutical composition of claim 11, characterized in that, The pharmaceutical composition is applied topically under the conjunctiva, in the anterior chamber, in the vitreous body, under the eyeball, under the retina, under the choroid, on the choroid, in the conjunctival sac, or on the eyelid.

16. The pharmaceutical composition according to any one of claims 11-15, characterized in that, The drug composition is administered at a frequency of 1-2 drops each time, 2-5 times a day, with an interval of 3-6 hours between each application.

17. The pharmaceutical composition of claim 16, characterized in that, The drug composition is administered at a frequency of 1 drop each time, 4 times a day, with an interval of 4 hours between each application.

18. A medicine box for treating lacrimal gland dysfunction-related eye diseases, characterized in that, The medicine box contains insulin or an analogue thereof as described in any one of claims 2-4, or contains a pharmaceutical composition as described in any one of claims 11-17.

19. The medicine box as described in claim 18, characterized in that, The lacrimal gland dysfunction type of ophthalmopathy is defined as in claims 5-10.

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