High molecular weight hyaluronic acid used in the treatment of corneal nerve damage or loss

Abstract

The present invention provides a fluid for use in treating or preventing corneal nerve injury or loss in humans or non-human animals. [Solution] The present invention provides a method for treating nerve damage or loss (corneal nerve damage or loss) in the cornea of ​​the eye of a human or non-human animal subject, comprising topically administering a fluid containing high molecular weight hyaluronic acid (HMWHA) to the ocular surface, wherein the hyaluronic acid is at least 2.5 mg 3 This relates to a method having an intrinsic viscosity of / kg.

Description

[Technical Field]

[0001] Cross-reference related applications This application claims priority to U.S. Provisional Application No. 63 / 056,081 filed on 24 July 2020 and U.S. Provisional Application No. 63 / 038,361 filed on 12 June 2020, which are incorporated herein by reference, including figures, tables, nucleic acid sequences, amino acid sequences, and other contents. [Background technology]

[0002] The cornea is a densely nerve-supplied surface tissue that has been studied using histochemical and in vivo confocal microscopy (IVCM) (see Non-Patent Literature 1, particularly the schematic diagram in Figure 2). In addition to sensory function, corneal nerves contribute to the blink reflex, tear production, and the maintenance of functional integrity of the ocular surface through the release of nutrients such as substance P(SP), calcitonin gene-related peptide (CGRP), epidermal growth factor (EGF), nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin (NT-)3 (Non-Patent Literature 2, 3, 4).

[0003] Cell-cell interactions between different cell types play a crucial role in maintaining nerve function and integrity, and these interactions are necessary for repairing damaged tissue to restore normal health. Therefore, regulating these cell-cell interactions is considered a potential regenerative strategy in nerve tissue, aiming to re-establish a functional epithelial and stromal microenvironment, including the repair of corneal nerve interactions with surrounding cells after nerve injury. Numerous scientific documents offer starting points for this (Non-Patent Literature 5).

[0004] Mechanical or chemical trauma, inflammation, refractive surgery, infection, and corneal nerve dysfunction due to other causative factors can lead to corneal diseases. For example, because corneal nerves play a crucial role in the homeostasis of the corneal epithelium, neurotrophic keratopathy (NK) can develop when this homeostasis is impaired after nerve damage or loss (Non-patent Literature 6, 7).

[0005] The diagnosis of corneal nerve injury or loss using in vitro corneal keratomileusin (IVCM) has advanced, enabling direct visualization of the subbasal corneal plexus in vivo, leading to laser in situ keratomileusis (LASIK) (Non-patent documents 8, 9, 10, 11, 12). However, treatment options aimed at restoring damaged or lost nerves are limited and expensive (Non-patent document 13). [Prior art documents] [Patent Documents]

[0006] [Patent Document 1] U.S. Patent No. 5,323,907 [Patent Document 2] U.S. Patent No. 5,052,558 [Patent Document 3] U.S. No. 5,033,252 [Non-patent literature]

[0007] [Non-Patent Document 1] Guthoff RF et al., "Epithelial Innervation of Human Cornea - A Three-Dimensional Study Using Confocal Laser Scanning Fluorescence Microscopy", Cornea, 2005, 24(5): 608-613 [Non-Patent Document 2] Shaheen, BS et al., "Corneal nerves in health and disease," Survey of Ophthalmology, 2014, vol. 59, pp. 263-285 [Non-Patent Document 3] Marfurt CF et al.. "Anatomy of the human corneal innervation," Exp Eye Res, 2010; 4:478-492 [Non-Patent Document 4] You L et al., "Neurotrophic factors in the human cornea," Invest Ophthalmol Vis Sci, 2000 Mar; 41(3):692-702 [Non-Patent Document 5] Kowtharapu BS and Stachs O, Corneal Cells: Fine-tuning Nerve Regeneration, Current Eye Research, 2020, 45(3):291-302 [Non-Patent Document 6] Eguchi H et al., "Corneal Nerve Fiber Structure, Its Role in Corneal Function, and Its Changes in Corneal Diseases," Biomed Res Int., 2017, 2017: 3242649 [Non-Patent Document 7] Mastropasqua L et al., "Understanding the Pathogenesis of Neurotrophic Keratitis: The Role of Corneal Nerves, J. Cell. Physiol., 232: 717-724, 2017 [Non-Patent Document 8] Choi EY et al., "Langerhans cells prevent sub-basal nerve damage and upregulate neurotrophic factors in dry eye disease", PLoS One, 2017; 12(4):e0176153); [Non-Patent Document 9] Tuisku, IS et al., "Alterations in corneal sensitivity and nerve morphology in patients with primary Sjogren's syndrome," Experimental Eye Research, 2008, vol. 86, pp. 879-885

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[0008] The present invention relates to a method for treating nerve damage or loss (corneal nerve damage or loss) in the cornea of ​​a human or non-human animal eye, comprising topically administering a fluid containing high molecular weight hyaluronic acid (HMWHA) to the ocular surface, wherein the hyaluronic acid is at least 2.5 mg 3 / kg(2.5m 3 The present invention relates to a method having an intrinsic viscosity of 1 / kg or more. Corneal nerve injury or loss treated using the method of the present invention is any type of nerve injury that inhibits or impairs normal corneal nerve turnover, orientation, growth, function, or any combination of two or more of the above.

[0009] This patent application includes at least one drawing prepared in color. Copies of this patent or the publication of the patent application containing the color drawing will be provided by the Patent Office upon request and payment of the necessary fees. [Brief explanation of the drawing]

[0010] [Figure 1] Total nerve fiber length was greater in patients treated for 8 weeks with high molecular weight hyaluronic acid eye drops (COMFORT SHIELD® preservative-free sodium hyaluronate eye drops) compared to patients treated with a control lubricant eye drop. [Figure 2A] These are single images from individual subbasal plexus (SNPs). [Figure 2B] This image shows the automatically detected nerve fibers used for quantitative analysis. [Figure 3] Typical SNP images of subjects in the control and test groups, as well as schematic diagrams of detected nerve fibers used to identify SNPs at baseline and after 8 weeks of treatment. [Figure 4] Corneal nerve fiber lengths at baseline and 8 weeks for patients treated with high molecular weight hyaluronic acid (COMFORT SHIELD® preservative-free sodium hyaluronate eye drops) and the control group. Figure 4 corresponds to Figure 1 and includes individual patient data points. [Modes for carrying out the invention]

[0011] In the HYLAN M trial, a performance study of a high molecular weight hyaluronic acid eye drop (COMFORT SHIELD® preservative-free sodium hyaluronate eye drop, i.com Medical, Munich, Germany) in severe dry eye, confocal microscopy was an optional diagnostic method performed by researchers with the necessary equipment and experiments at baseline and 8-week follow-up visits to analyze the subbasal epithelial plexus. Images were collected from 16 patients at baseline and after 8 weeks of treatment. Images were from 8 patients in the control group who continued to use the control lubricating eye drop they were using at the time of inclusion in the HYLAN M trial, and from 8 patients whose ophthalmic lubricating eye drop was replaced with COMFORT SHIELD® eye drop.

[0012] To the inventors' surprise, statistically significant growth (p=0.031) in total nerve fiber length was observed in patients treated with Comfort Shield eye drops (see Example 1 and Figure 1). This neurotrophic activity is an unexpected effect of high molecular weight hyaluronic acid (HMWHA) and is highly relevant to many eye conditions, independent of dry eye disease. Further details regarding the HYLAN M test and the effects of HMWHA fluids, such as COMFORT SHIELD® eye drops, on total nerve fiber length and trophication in the subbasal plexus are found in Non-Patent Documents 14 and 15, which are incorporated herein by reference in their entirety.

[0013] The present invention provides a method for treating nerve damage or loss (corneal nerve damage or loss) in the cornea of ​​a human or non-human subject's eye, comprising topically administering a fluid containing HMWHA to the ocular surface, wherein the hyaluronic acid is present in a quantity of at least 2.5 mg. 3 This invention relates to a method having an intrinsic viscosity of / kg. While not limited to the theory of the mechanism of action, it has been proposed that providing HMWHA to the ocular surface supports and promotes the repair of the functional epithelium and interstitial microenvironment, which facilitates axonal extension and nerve regeneration after corneal nerve injury.

[0014] Hyaluronic acid (HA) is a carbohydrate - glycosaminoglycan and is specifically found in the living body. The biological functions of endogenous HA include maintaining the elastic viscosity of liquid connective tissues such as joint synovial fluid and ocular vitreous humor (Non - Patent Documents 16, 17). Although the specific mechanisms involved in the diverse signal transduction of HA have not yet been fully understood, it is known that HA can regulate multifaceted biological effects that vary depending on the HA size (Non - Patent Document 18).

[0015] Sodium hyaluronate and other viscoelastic agents have been used in intraocular surgery since the 1970s and in the treatment of dry eye since the 1980s (Non - Patent Documents 19, 20), but the biological functions of hyaluronic acid in the epithelium have hardly been focused on so far (Non - Patent Document 21).

[0016] The high - molecular - weight hyaluronic acid or "HMWHA" used in the present invention refers to hyaluronic acid having an intrinsic viscosity of at least 2.5 m 3 / kg (2.5 m 3 / kg or more) as determined by Non - Patent Document 22, "Sodium Hyaluronate". Briefly, the intrinsic viscosity [η] is calculated by linear least - squares regression analysis using the Martin equation: Log 10 (n r -1 / c)=log 10 [η]+κ[η]c. In some embodiments, the high - molecular - weight hyaluronic acid has an intrinsic viscosity of at least 2.9 m 3 / kg (2.9 m 3 / kg or more).

[0017] In some embodiments, the hyaluronic acid has a concentration of <0.2% w / v. In some embodiments, the hyaluronic acid has a concentration of 0.1 - 0.19% w / v. In some embodiments, the hyaluronic acid has a concentration of 0.15% w / v.

[0018] In some embodiments, the fluid has a) pH of 6.8 to 7.6, b) an osmolality of 240 to 330 mosmol / kg, c) an NaCl concentration of 7.6 to 10.5 g / l, and / or d) a phosphate concentration of 1.0 to 1.4 mmol / l.

[0019] In some embodiments, the fluid is a clear, colorless solution free from visible impurities. The fluid is assumed to be sterile.

[0020] In some embodiments, the fluid according to the present invention is COMFORT SHIELD® preservative-free sodium hyaluronate eye drops.

[0021] In some embodiments, HA has a molecular weight of at least 3 million dollar-tonnes, as calculated by Mark Howink's formula. In some embodiments, HA has a molecular weight in the range of 3 million to 4 million dollar-tonnes, as calculated by Mark Howink's formula.

[0022] In some embodiments, the high molecular weight HA is hyaluronan. In some embodiments, the high molecular weight HA is crosslinked. In some embodiments, the high molecular weight HA is uncrosslinked. In some embodiments, the high molecular weight HA is linear. In some embodiments, the high molecular weight HA is non-linear (e.g., branched). In some embodiments, the high molecular weight HA is a derivative of hyaluronan, such as an ester derivative, an amide derivative, or a sulfated derivative, or a combination of two or more of the above.

[0023] The fluid is administered to the ocular surface of one or both eyes of the target patient by any local administration method. For example, the fluid may be administered as one or more drops from an eye drop dispenser or other eye drop dispensing device. The fluid may be administered by the patient themselves or by a third party. The dose administered to the ocular surface as a single or multiple dose varies depending on various factors, including the patient's condition and characteristics, the severity of symptoms, concomitant treatments, the frequency of treatment, and the desired effect. For example, one or more drops (e.g., approximately 30 microliters each) may be administered.

[0024] While administration of 1-3 drops 1-3 times a day may be sufficient under certain circumstances, in most cases of corneal nerve damage or loss, it is likely that topical administration of 1-3 drops more frequently, for example, 4, 5, 6, 7, 8, 9, 10 or more times a day, will be required. In some embodiments, 3 or more drops are administered once or twice a day.

[0025] In some embodiments, the frequency of administration and / or the amount per dose can be reduced over time as corneal nerves regenerate. For example, in some cases, after four weeks, the dose may be reduced and / or the frequency of daily administration may be reduced, or the frequency of administration may be reduced to half a day. However, while eye drops are typically taken by the subject for a frequency and duration "as needed," loss of sensation is due to corneal nerve damage or loss, making the subject's self-assessment of the need for frequency and duration unreliable. Therefore, administration of the fluid should not be discontinued simply on the basis of the absence of discomfort, in order to avoid premature discontinuation of treatment.

[0026] Corneal nerve injury or loss treated with the HMWHA fluid and method of the present invention is any type of nerve injury that inhibits or impairs normal corneal nerve turnover, growth, function, or any combination of two or more of the above, and involves any degree of damage to nerve structure (composition and / or continuity) and, optionally, to surrounding tissue. Nerve injury is any type or stage of injury, such as nerve apraxia, axonal rupture, or nerve rupture (Non-Patent Literature 23). For example, nerve injury or loss may include one or more features of reduced nerve fiber length, reduced nerve fiber curvature, reduced nerve fiber density, and complete or partial nerve fiber transection.

[0027] HMWHA fluid is administered topically to the ocular surface in an amount and duration sufficient to reduce the net loss of corneal nerve from corneal nerve injury or loss compared to the net loss of corneal nerve that occurs in the absence of HMWHA fluid. Thus, sufficient topical administration of HMWHA fluid reduces the net loss of corneal nerve that would not occur without treatment with HMWHA fluid.

[0028] The treatment method includes identifying whether the subject has corneal nerve damage or loss before local administration of the HMWHA fluid. Preferably, the subject is identified using in vivo confocal microscopy (IVCM) of the eye having nerve damage or loss (Non-Patent Documents 10, 24, 25).

[0029] Optionally, subjects will be monitored at least once during and / or after treatment using IVCM, and confocal microscopy images will be compared to previous images to assess the status and progress of corneal nerve healing and growth.

[0030] Optionally, the method includes a step of identifying whether a subject has one or more signs or symptoms of corneal nerve injury or loss before administering the HMWHA fluid. Examples of signs of corneal nerve injury or loss include, but are not limited to, decreased corneal innervation or sensation, decreased number of nerve fibers or bundles innervating the cornea, death of neurons innervating the cornea, decreased or loss of neurotransmitter release, decreased or loss of nerve growth factor release, abnormal tearing reflexes, abnormal blink reflexes, abnormal nerve morphology, appearance of abnormal neurobuds, abnormal curvature, increased bead-like neurogenesis, thinning of nerve fiber bundles, thickening of nerve fiber bundles, decreased length of the inferior corneal nerve ring, decreased density of corneal nerves, decreased length of corneal nerves, decreased branching of corneal nerves, recurrent corneal erosions, delayed healing of corneal epithelial wounds, and decreased tearing rate. Examples of symptoms of corneal nerve damage or loss include, but are not limited to, abnormal tear production or dryness, abnormal blinking, difficulty or loss of the ability to focus, decreased or lost visual acuity, and decreased or lost corneal sensitivity.

[0031] In some embodiments, damage to or loss of corneal nerves is caused by disease, trauma (chemical, mechanical, etc.), congenital defects, or medical procedures.

[0032] In some embodiments, damage to or loss of corneal nerves includes impaired corneal innervation caused by viral infection, drug therapy, chronic contact lens use, surgery, diabetes (type 1, type 2, or pregnancy-related), or multiple sclerosis.

[0033] In some embodiments, the subject has neurotrophic keratopathy (mild, moderate, or severe NK) in the eye at the time of administration, and the HMWHA fluid alleviates one or more signs or symptoms of NK. In some embodiments, the NK is mild NK (also known as stage 1) or moderate NK (also known as stage 2) at the time of administration, and the HMWHA fluid prevents or delays the progression of NK to a severe NK state (also known as stage 3). In some embodiments, the NK is severe NK.

[0034] In some embodiments, the subject does not have NK cells at the time of administration, and the HMWHA fluid prevents or delays the onset of NK cells.

[0035] In some embodiments, the subjects have a disease or disorder of the ocular surface (mild, moderate, or severe). In other embodiments, the subjects do not have a disease or disorder of the ocular surface. Examples of diseases or disorders of the ocular surface and their etiologies are given in Non-Patent Literature 26, which is incorporated herein by reference in its entirety. In some embodiments, the subjects have a dry eye disease (mild, moderate, or severe). In other embodiments, the subjects do not have a dry eye disease. In some embodiments, the subjects have tear film deficiency. In other embodiments, the subjects do not have tear film deficiency.

[0036] In some embodiments, the subject has tear deficiency. In some embodiments, the subject does not have tear deficiency. However, the subject has ocular surface abnormalities (topographic abnormalities) including elevations on the cornea or other locations on the ocular surface that are not covered by a normal tear film (tear film with normal surface tension and viscosity), which constitute frictional areas on the ocular surface (Non-Patent Literature 27).

[0037] Diabetes mellitus is a common metabolic disorder known to cause structural and functional changes in the human cornea, and diabetic ocular complications, including corneal nerve loss, frequently occur. In some embodiments, subjects treated by the method of the present invention have diabetes mellitus (type 1, type 2, or pregnancy-related).

[0038] In some embodiments, the subjects have diabetes mellitus (type 1, type 2, or pregnancy-related) and diabetic peripheral neuropathy.

[0039] In some embodiments, the subject has diabetic corneal neuropathy, and the HMWHA fluid reverses the diabetic corneal neuropathy.

[0040] In some embodiments, the subjects have diabetes mellitus (type 1, type 2, or pregnancy-related) but do not yet have diabetic peripheral neuropathy.

[0041] In some embodiments, subjects have a genetic condition known to coexist with ocular surface damage leading to NK (e.g., Riley-Day syndrome (familial autonomic neuropathy), Goldenhar-Gorlin syndrome, Mobius syndrome, and familial corneal hyperemia).

[0042] In some embodiments, the subject has a systemic condition known to be associated with ocular surface damage leading to NK (diabetes mellitus (type 1, type 2, or pregnancy-related), leprosy, vitamin A deficiency, amyloidosis, and multiple sclerosis, etc.).

[0043] In some embodiments, the subjects have NK (e.g., neoplasms, aneurysms, strokes, degenerative disorders of the central nervous system (CNS) (e.g., Alzheimer's disease and Parkinson's disease), and CNS conditions known to cause ocular surface damage leading to post-neurosurgery procedures (e.g., acoustic neuroma, trigeminal neuralgia, or other surgical injury to the trigeminal nerve).

[0044] In some embodiments, subjects have ocular conditions known to result in NK, such as post-herpetic infection (herpes simplex and herpes zoster), other infections associated with keratin neuritis, chemical or physical burns, abuse of local anesthetics, drug toxicity (e.g., trimolol, betaxolol, diclofenac sodium, sulfacetamide 30%), chronic ocular surface injury or inflammation, eye surgery (e.g., cataract surgery, glaucoma surgery, laser epithelial keratectomy (LASIK), photoreactive keratectomy (PRK), deep lamellar keratocyte transplantation (DALK), collagen crosslinking for keratoconus, vitrectomy for retinal detachment, photocoagulation for treating diabetic retinopathy, postoperative or laser treatment), contact lens wear, orbital neoplasms, or corneal dystrophy (lattice or granules).

[0045] In some embodiments, the method further includes performing one or more additional procedures before, during, or after local administration of HMWHA fluid, selected from recombinant human nerve growth factor (senegermin), matrix metalloproteinase inhibitors, growth factor-rich plasma (PRGF), therapeutic contact lenses, temporary tarsal plate suturing (partial or complete after administration of HMWHA fluid), amniotic membrane transplantation, full-thickness corneal transplantation, corneal transplantation, a combination of senegermin and corneal transplantation, or direct or indirect corneal neuropathy. Optionally, if the additional procedure includes the administration of an active agent, it may be administered in the HMWHA fluid or in a separately administered formulation.

[0046] In some embodiments, the HMWHA fluid is administered directly to the ocular surface as eye drops or as a cleansing agent (e.g., an eye wash).

[0047] In some embodiments, 1 to 3 drops are administered 1 to 3 times a day.

[0048] In some embodiments, 1 to 3 drops are administered 4, 5, 6, 7, 8, 9, or 10 or more times per day.

[0049] In some embodiments, three or more drops are administered at least once a day.

[0050] In some embodiments, the HMWHA fluid is indirectly delivered to the ocular surface by a delivery agent (fluid delivery agent) that is locally delivered to the ocular surface or to other parts of the eye (e.g., particles coated with the fluid and / or secreted onto the ocular surface).

[0051] In some embodiments, hyaluronic acid is 2.6m 3 / kg~2.9m 3 It has an intrinsic viscosity of 1 / kg or more.

[0052] In some embodiments, hyaluronic acid has a molecular weight of at least 3 million dollars tons. In some embodiments, hyaluronic acid has a molecular weight in the range of 3 million to 4 million dollars tons.

[0053] In some embodiments, the HMWHA fluid contains HMWHA having a concentration of <0.2% w / v. In some embodiments, the HMWHA fluid contains HMWHA having a concentration of 0.1 to 0.19% w / v. In some embodiments, the HMWHA fluid contains HMWHA having a concentration of about 0.15% w / v.

[0054] In some embodiments, the HMWHA fluid has the following composition / properties corresponding to COMFORT SHIELD® preservative-free sodium hyaluronate eye drops. a) pH 6.8~7.6 b) Osmolar concentration of 240-330 mOsmol / kg c) NaCl concentration of 7.6-10.5 g / l and / or d) Phosphate concentration of 1.0-1.4 mmol / l.

[0055] In some embodiments, the HMWHA fluid is a clear, colorless solution free from visible impurities.

[0056] In some embodiments, the HMWHA fluid is sterile.

[0057] In some embodiments, the HMWHA fluid is a COMFORT SHIELD® preservative-free sodium hyaluronate eye drop.

[0058] In some embodiments, the HMWHA fluid does not contain other bioactive agents (e.g., hydrophobic active ingredients). In other embodiments, the HMWHA fluid further contains bioactive agents (e.g., hydrophobic active ingredients). As used herein, the term “bioactive agent” means any substance that, when administered in an effective amount that affects tissue, affects human or non-human animal subjects. Bioactive agents are any class of substances, such as drug molecules or biological substances (e.g., polypeptides, carbohydrates, glycoproteins, immunoglobulins, nucleic acids), which are natural products or artificially produced and act by any mechanism, such as pharmacological, immunological, or metabolic. Classes of bioactive agents include substances that modify intraocular pressure (e.g., enzyme inhibitors) and anti-angiogenic agents. Some specific examples of bioactive agents include steroids (e.g., corticosteroids), antibiotics, immunosuppressants, immunomodulators, tacrolimus, plasmin activators, anti-plasmins, and cyclosporine A. In some embodiments, the bioactive agent is a glaucoma drug such as a steroid or antibiotic, prostaglandin analog, beta-blocker, alpha-agonist, or carbonic anhydrase inhibitor, an allergic eye reliever such as a histamine antagonist or nonsteroidal anti-inflammatory drug, or a mydriatic agent for treating or preventing eye infections. Unfortunately, in some cases, the bioactive agent contained in the fluid can be irritating or damaging to the eye (e.g., cyclosporine A). An advantage is that, through its rheological and other properties, high molecular weight HA in the fluid can mitigate and / or protect the eye from the irritating and / or damaging effects of the bioactive agent or multiple drugs in the fluid (i.e., the bioactive agent may be irritating or damaging to the eye if administered without high molecular weight HA).

[0059] In some embodiments, the HMWHA fluid does not contain steroids, antibiotics, or immunomodulators. In some embodiments, the fluid does not contain other bioactive agents (e.g., hydrophobic active ingredients).

[0060] In some embodiments, the HMWHA fluid contains preservatives and / or detergents, preferably those that do not cause damage or irritation to the eyes. In other embodiments, the HMWHA fluid does not contain preservatives or detergents (i.e., the fluid is preservative-free and detergent-free). In some situations, it may be desirable to include one or more preservatives or detergents in the fluid. Often, such preservatives or detergents are irritating or damaging to the eyes. As an advantage, due to its rheological and other properties, the fluid can mitigate the irritating and / or damaging effects of preservatives or detergents in the fluid and / or protect the eyes. Thus, in some embodiments, the fluid further contains preservatives or detergents that are irritating or damaging to the eyes (i.e., preservatives or detergents that are more irritating or damaging to the eyes when administered without high molecular weight HA).

[0061] In some embodiments, the HMWHA fluid comprises cyclosporine A, cetalkonium chloride, tyloxapole, or a combination of two or more of the above.

[0062] In some embodiments, the HMWHA fluid is administered to a subject before, during, and / or after administration of another composition containing a bioactive agent to the subject. In some situations, it may be desirable to include one or more preservatives or detergents in the other composition. As mentioned above, in many cases, such preservatives and detergents are irritating or damaging to the eyes, and some bioactive agents themselves are irritating or damaging to the eyes. To offer an advantage, due to its rheological and other properties, the fluid can mitigate the irritating and / or damaging effects of bioactive agents, preservatives, and / or detergents in the other composition and / or protect the eyes. Thus, the bioactive agents, preservatives, and / or detergents in the other composition would be irritating or damaging to the eyes if administered without the fluid.

[0063] In some embodiments, the other composition comprises one or more antibiotics, immunosuppressants, or immunomodulators.

[0064] In some embodiments, the other composition includes cyclosporine A, cetalkonium chloride, tyloxapol, or a combination of two or more of the above.

[0065] Other compositions administered to the subject may be in any form and may be administered by any route (e.g., topically or systemically). In some embodiments, other compositions are administered to the eye, for example, topically or by injection. In some embodiments, other compositions are administered topically to the ocular surface.

[0066] In some embodiments, the preservative or detergent contained in the HMWHA fluid or other composition is a chemical preservative or an oxidative preservative.

[0067] In some embodiments, the preservative or detergent contained in the HMWHA fluid or other composition kills susceptible microbial cells by disrupting the lipid structure of the microbial cell membrane, thereby increasing microbial cell membrane permeability.

[0068] In some embodiments, preservatives or detergents contained in the HMWHA fluid or other compositions typically cause damage to corneal tissues such as the corneal epithelium, endothelium, interstitium, and membrane interfaces, but the HMWHA fluid mitigates or protects against such damage.

[0069] In some embodiments, the preservatives or detergents contained in the HMWHA fluid or other compositions are selected from the group consisting of quaternary ammonium preservatives (e.g., benzalkonium chloride (BAK) or cetalkonium chloride), chlorobutanol, disodium edetate (EDTA), polyquaternarium-1 (e.g., Polyquad® preservative), stabilized oxidizing agents (e.g., stabilized oxychloro complexes (e.g., Purite® preservative)), ionic buffered preservatives (e.g., sofZia® preservative), polyhexamethylene biguanide (PHMB), sodium perborate (e.g., GenAqua® preservative), tyropaxol, and sorbates.

[0070] In some embodiments, the HMWHA fluid is at least substantially mucin-free, in other words, has a mucin concentration of <0.3% w / v.

[0071] In some embodiments, the HMWHA fluid further comprises glycosaminoglycans (GAGs), i.e., high molecular weight HA, an electrolyte (e.g., sodium chloride), a buffer (e.g., phosphate buffer), or a combination of two or more of the above.

[0072] HMWHA fluid can be used in conjunction with bandage contact lenses. Thus, this method further includes applying the bandage contact lens to the eye before, during, and / or after administering the fluid. For example, the fluid can be administered by placing it on the bandage contact lens before applying the bandage contact lens, after applying the contact lens, and / or before applying the bandage contact lens to the eye. The use of the fluid allows the bandage contact lens to apply pressure to the ocular surface while simultaneously minimizing friction on the ocular surface. An advantage is that the fluid and bandage contact lens can be safely used immediately after eye surgery, such as glaucoma surgery.

[0073] Another aspect of the present invention relates to a kit used to carry out the method of the present invention as described herein, i.e., to treat corneal nerve damage or loss. The kit comprises the HMWHA fluid described herein and one or more bandage contact lenses. The bandage contact lenses may be packaged together with the fluid in the same container (with the bandage contact lenses in contact with the fluid), or the bandage contact lenses may be packaged separately from the fluid in a separate container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. Containers can be made from various materials such as glass or plastic.

[0074] The kit may include a delivery agent (separately or in association with a fluid) that comes into contact with the ocular surface or other parts of the eye. For example, the kit may include particles (e.g., microparticles or nanoparticles) that are coated with a fluid and / or release the fluid onto the ocular surface.

[0075] Optionally, the kit may include a device for dispensing eye drops (e.g., an eye drop dispenser) which may or may not function as a container for the HMWHA fluid inside the kit before touching (e.g., opening) the outer packaging of the kit, i.e., the eye drop dispensing device may function to contain the fluid to be provided inside the untouched (unopened) kit, or to become empty and receive the fluid after touching the kit. Optionally, the kit may include a label or accompanying document having printed or digital instructions for, for example, using the kit, or for, for, carrying out the method of the present invention.

[0076] The kit includes a packaging material partitioned to receive one or more containers, such as vials and tubes, each of which contains one of the distinct elements used in the method described herein. Examples of packaging materials used when packaging pharmaceuticals include those described in Patent Documents 1, 2, and 3. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, pumps, bags, vials, light-sealed containers, syringes, bottles, and any packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0077] The kit may include one or more additional containers, each containing one or more different materials desirable from a commercial and user standpoint for use of the compositions described herein. Such materials include, but are not limited to, buffers, diluents, carriers, packaging, containers, vials and / or tube labels, contents and / or instructions for use, and accompanying documents with instructions for use.

[0078] Labels may be on the container or associated with the container. A label is on the container when the letters, numbers, or other characters forming the label are attached to, molded, or etched onto the container itself; a label is associated with the container when it is present, for example, in a container or carrier that also holds the container, such as as an accompanying document. Labels are used to indicate that the contents should be used for a specific therapeutic purpose. Labels may also indicate instructions for the use of the contents, as described herein.

[0079] In some embodiments of the kit, the HMWHA fluid may be provided in a pack or dispensing device containing one or more unit dosage forms containing the compositions disclosed herein. The pack may include, for example, metal or plastic foil such as a blister pack. The pack or dispensing device may be accompanied by instructions for administration.

[0080] Preparation of HMWHA fluid As mentioned above, the hyaluronic acid fluid is at least 2.5 ml 3 It has an intrinsic viscosity of / kg, preferably less than 0.2% w / v. In some embodiments, hyaluronic acid is at least 2.9m 3 It has an intrinsic viscosity of / kg.

[0081] Viscoelasticity is defined as a fluid property that possesses both viscous and elastic properties. Zero shear viscosity is determined as the steady-state shear plateau viscosity at the disappearance shear rate. For high-viscosity formulations, measurement using a controlled stress rheometer is preferred.

[0082] Molecular weight and m 3 The relationship between intrinsic viscosity [η] at / kg is given by Mark Howink's equation: [η]=k·(Mrm) a Given by the formula, where Mrm is the molecular weight of MDa, and the coefficient k = 1.3327 · 10 -4 , and a = 0.6691 Therefore, it was found that the values ​​of k and a are predictable.

[0083] HMWHA fluid is produced by sterilizing the filling line, adding purified water or water for injection (WFI) to a stainless steel mixing tank, adding salt while mixing, gradually adding HA while mixing until a homogeneous solution / fluid is obtained, adjusting the pH value by adding NaOH or HCl as needed while continuing the mixing process, transferring the solution to a sterile holding tank on a 1 μm pore size filter cartridge, and aseptically filling the solution into sterile primary packaging (single dose or vial) by sterile filtration. For single doses, this may be done by a blow-fill-seal (BFS) process.

[0084] Preferably, the HMWHA fluid is at least substantially mucin-free, in other words, has a mucin concentration of <0.3% w / v. This means that its flow behavior or properties are substantially achieved or regulated by hyaluronan rather than by mucin, which is naturally present in the tears of the subject and is primarily responsible for its flow behavior.

[0085] If viscosity-increasing substances are added, they are preferably added toward, during, or as part of the final step. Mixing is carried out to achieve a homogeneous mixture. Alternatively, or in addition, it is preferable to first provide purified water or water for injection as a base, and then optionally add electrolytes, buffers, and substances that do not increase viscosity to the purified water or water for injection first.

[0086] HA is further described in the monograph Non-Patent Document 22 (Sodium Hyaluronate), the entire contents of which are incorporated herein by reference.

[0087] In one embodiment, the fluid used in the method and kit of the present invention has the characteristics listed in Table 1. [Table 1]

[0088] Exemplary Embodiments Embodiment 1 A method for treating nerve damage or loss (corneal nerve damage or loss) in the cornea of ​​the eye of a human or non-human subject, comprising topically administering a liquid containing high molecular weight hyaluronic acid (HMWHA) to the ocular surface, wherein the hyaluronic acid is at least 2.5 mg 3 A method having an intrinsic viscosity of / kg. Embodiment 2 The method according to Embodiment 1, wherein the nerve damage or loss includes one or more of the following characteristics: a decrease in nerve fiber length, a decrease in nerve fiber curvature, a decrease in nerve fiber density, and nerve fiber transection (complete or partial). Embodiment 3 The method according to Embodiment 1 or 2, wherein the HMWHA fluid reduces the net loss of corneal nerves from damage or loss of corneal nerves compared to the net loss of corneal nerves that occurs in the absence of the HMWHA fluid. Embodiment 4 The method of any of the above embodiments, further comprising identifying whether the subject has corneal nerve damage or loss before administering the HMWHA fluid. Embodiment 5 The method according to Embodiment 4, wherein the identification includes performing in vivo confocal microscopy (IVCM) on the target eye. Embodiment 6 The method according to any one of Embodiments 1 to 3, further comprising identifying whether the subject has signs or symptoms of corneal nerve damage or loss before administering the HMWHA fluid. Embodiment 7 The method according to Embodiment 6, wherein the signs of corneal nerve damage or loss are one or more of the following: decreased corneal innervation or sensation, decreased number of nerve fibers or nerve bundles innervating the cornea, death of neurons innervating the cornea, decreased or loss of neurotransmitter release, decreased or loss of nerve growth factor release, abnormal tearing reflex, abnormal blinking reflex, abnormal nerve morphology, appearance of abnormal neurobuds, abnormal curvature, increased bead-like neurogenesis, thinning of nerve fiber bundles, thickening of nerve fiber bundles, decreased length of the inferior corneal nerve ring, decreased density of corneal nerves, decreased length of corneal nerves, decreased branching of corneal nerves, recurrent corneal erosion, delayed healing of corneal epithelial wounds, and decreased tear rate. Embodiment 8 The method according to Embodiment 6, wherein the symptoms of damage or loss of the corneal nerve are one or more of the following: abnormal tear production or dryness, abnormal blinking, difficulty or loss of the ability to focus, decreased or lost visual acuity, and decreased or lost corneal sensitivity. Embodiment 9 The method according to any of the above embodiments, wherein the damage or loss of the corneal nerve is caused by disease, trauma, congenital defect, or medical procedure. Embodiment 10 The method according to any of the above embodiments, wherein the damage or loss of the corneal nerve includes impaired corneal innervation caused by viral infection, drug therapy, chronic contact lens use, surgery, diabetes (type 1, type 2, or pregnancy-related), or multiple sclerosis. Embodiment 11 The method according to any of the embodiments, wherein the subject has neurotrophic keratopathy (mild, moderate, or severe NK) in the eye at the time of administration, and the HMWHA fluid alleviates one or more signs or symptoms of the NK. Embodiment 12 The method according to Embodiment 11, wherein the NK is mild NK (also known as stage 1) or moderate NK (also known as stage 2) at the time of administration, and the HMWHA fluid prevents or delays the progression of NK to a severe NK state (also known as stage 3). Embodiment 13 The method according to embodiment 11, wherein the NK is severe NK. Embodiment 14 The method according to any one of Embodiments 1 to 10, wherein the subject does not have neurotrophic keratopathy (mild, moderate, or severe NK) at the time of administration, and the HMWHA fluid prevents or delays the onset of NK. Embodiment 15 The subject is a person having an ocular surface disease (mild, moderate, or severe), and the method according to any of the embodiments described above. Embodiment 16 The subject is the method according to any one of Embodiments 1 to 14, provided that the subject does not have an eye surface disease. Embodiment 17 The subject is a method according to any of the preceding embodiments, which involves a patient with tear film deficiency. Embodiment 18 The aforementioned subject is the method according to any one of Embodiments 1 to 16, which does not have tear film deficiency. Embodiment 19 The subject is a person having diabetes mellitus (type 1, type 2, or pregnancy-related) and diabetic peripheral neuropathy, and the method according to any of the embodiments described above. Embodiment 20 The method according to any of the preceding embodiments, wherein the subject has diabetic corneal neuropathy, and the HMWHA fluid reverses the diabetic corneal neuropathy. Embodiment 21 The subject is a person who has diabetes mellitus (type 1, type 2, or pregnancy-related) but does not yet have diabetic peripheral neuropathy, and the method according to any one of Embodiments 1 to 18. Embodiment 22 The subject is an ocular condition known to have ocular surface damage leading to NK, such as post-herpetic infection (herpes simplex and herpes zoster), other infections associated with keratin neuritis, chemical or physical burns, abuse of local anesthetics, drug toxicity (e.g., trimolol, betaxolol, diclofenac sodium, sulfacetamide 30%), chronic ocular surface injury or inflammation, eye surgery (e.g., cataract surgery, glaucoma surgery, laser epithelial keratectomy (LASIK), photoreactive keratectomy (PRK), deep lamellar keratocyte transplantation (DALK), collagen crosslinking for keratoconus, vitrectomy for retinal detachment, photocoagulation for treating diabetic retinopathy, postoperative or laser treatment), contact lens wear, orbital neoplasms, or corneal dystrophy (lattice or granules). Embodiment 23 The method according to any of the preceding embodiments, further comprising performing one or more additional procedures before, during, or after local administration of HMWHA fluid, selected from recombinant human nerve growth factor (senegermin), matrix metalloproteinase inhibitor, growth factor-rich plasma (PRGF), therapeutic contact lenses, temporary tarsal plate suturing (partial or complete after administration of HMWHA fluid), amniotic membrane transplantation, full-thickness corneal transplantation, corneal transplantation, a combination of senegermin and corneal transplantation, or direct or indirect corneal neuropathy. Embodiment 24 The method according to any of the preceding embodiments, wherein the HMWHA fluid is administered directly to the ocular surface as eye drops or as a cleansing agent (e.g., an eye wash). Embodiment 25 The method according to any one of Embodiments 1 to 23, wherein the HMWHA fluid is administered indirectly to the ocular surface by a delivery agent (fluid delivery agent) that is locally delivered to the ocular surface or to another part of the eye (e.g., particles coated with the fluid and / or secreted on the ocular surface). Embodiment 26 The aforementioned hyaluronic acid is 2.6 ml 3 / kg~2.9m 3 The method according to any of the prior embodiments, having an intrinsic viscosity of 1 / kg or more. Embodiment 27 The method according to any of the preceding embodiments, wherein the HMWHA fluid further comprises a preservative. Embodiment 28 The method according to any one of Embodiments 1 to 26, wherein the HMWHA fluid further contains no preservatives (i.e., the fluid is preservative-free). Embodiment 29 The method according to any of the preceding embodiments, wherein the HMWHA fluid further comprises an additional glycosaminoglycan (GAG), an electrolyte (e.g., sodium chloride), a buffer (e.g., phosphate buffer), or a combination of two or more of the above. Embodiment 30 The hyaluronic acid has a molecular weight of at least 3 million dollars tons, according to any of the preceding embodiments. Embodiment 31 The hyaluronic acid has a molecular weight in the range of 3 million to 4 million dollars tons, as described in any of the preceding embodiments. Embodiment 32 The method according to any of the preceding embodiments, wherein the HMWHA fluid comprises HMWHA having a concentration of <0.2% w / v. Embodiment 33 The method according to any of the preceding embodiments, wherein the HMWHA fluid comprises HMWHA having a concentration of 0.1 to 0.19% w / v. Embodiment 34 The method according to any of the preceding embodiments, wherein the HMWHA fluid comprises HMWHA having a concentration of about 0.15% w / v. Embodiment 35 The HMWHA fluid is a) pH 6.8~7.6 b) Osmolar concentration of 240-330 mOsmol / kg c) NaCl concentration of 7.6-10.5 g / l and / or d) Phosphate concentration 1.0-1.4 mmol / l A method according to any of the prior embodiments, having the following characteristics. Embodiment 36 The method according to any of the prior embodiments, wherein the HMWHA fluid is a clear, colorless solution free from visible impurities. Embodiment 37 The method according to any of the prior embodiments, wherein the HMWHA fluid is sterile. Embodiment 38 The method according to any of the prior embodiments, wherein the HMWHA fluid is a COMFORT SHIELD® preservative-free sodium hyaluronate eye drop. Embodiment 39 The method according to any of the preceding embodiments, wherein the HMWHA fluid does not contain other bioactive agents (for example, does not contain hydrophobic active ingredients). Embodiment 40 The method according to any one of Embodiments 1 to 38, wherein the HMWHA fluid further comprises a bioactive agent (e.g., a hydrophobic active ingredient).

[0089] definition In the context of this invention (particularly in the context of the claims), the terms “one,” “it,” and similar terms are to be interpreted as encompassing both singular and plural unless otherwise indicated herein or unless the context clearly contradicts this. Thus, for example, when referring to “cell” or “compound,” unless otherwise indicated or unless the context clearly contradicts this, it is to be interpreted as encompassing both a single cell or a single compound and multiple cells and multiple compounds. Similarly, the term “or” is intended to include “and” unless the context clearly indicates otherwise. The abbreviation “eg” is derived from the Latin “exempli gratia” and is used herein to indicate a non-restrictive example. Thus, the abbreviation “eg” is synonymous with the term “for example.” Furthermore, to the extent that terms including “have,” “possess,” “equip,” or variations thereof are used in either the detailed description and / or the claims, such terms are intended to be encompassed in a manner similar to that of the term “include.” The transitional phrases "to include" and "to include," "to become substantially" and "to be substantially," and "to consist of" and "to be consisting of" (and any grammatical variations thereof) can be used interchangeably.

[0090] In the context of the administered fluid of the present invention, the term “effective amount” means the amount of fluid required to obtain the desired result, for example, the amount required to reduce the net loss of corneal nerves from corneal nerve injury or loss compared to the net loss of corneal nerves that occurs in the absence of the HMWHA fluid.

[0091] The term “isolated” means, when used as a preparation of a composition, that the composition is prepared by human intervention or separated from its naturally occurring in vivo environment. Generally, compositions thus isolated substantially do not contain one or more materials that are normally associated with nature, e.g., one or more proteins, nucleic acids, lipids, carbohydrates, or cell membranes. A “substantially pure” molecule can be combined with one or more other molecules. Thus, the term “substantially pure” does not exclude combinations of compositions. Substantially pure is at least about 60% by mass of the molecule. Purity may also be about 70% or 80% or more, e.g., 90% or more. Purity can be determined by any suitable method, including, for example, UV spectroscopy, chromatography (e.g., HPLC, gas phase), gel electrophoresis (e.g., silver or Coomassi staining), and sequence analysis (for nucleic acids and peptides).

[0092] As used herein, the term "hyaluronic acid" (HA) refers to glycosaminoglycans composed of naturally occurring disaccharide repeats of N-acetylglucosamine and glucuronic acid, also known as hyaluronan (e.g., linear, glycosaminoglycan polymers composed of repeating disaccharide units of [-D-glucuronic acid-b1,3-N-acetyl-D-glucosamine-b1,4-]n), as well as derivatives of hyaluronan having chemical modifications such as esters, amide derivatives, alkylamine derivatives, low-molecular-weight and high-molecular-weight forms of hyaluronan, and crosslinked forms such as hyaluron. Thus, the disaccharide chain may be linear or non-linear. Hyaluronan can be crosslinked by linking with crosslinking agents such as thiols, polymethyl methacrylate, hexadecylamide, and tyramine. Hyaluronan can also be directly crosslinked with formaldehyde and divinyl sulfone. Examples of hiran include, but are not limited to, hiran A, hiran B, and hiran G-F20 (Non-Patent Documents 27, 28, 29, 30, and 31, the full contents of which are incorporated herein by reference).

[0093] The term "hyaluronic acid" or HA includes HA itself and its pharmaceutically acceptable salts. HA can be formulated in pharmaceutically acceptable salt forms. Pharmacopoeia of HA can be prepared using conventional techniques.

[0094] The term "high molecular weight" or "HMW" in the context of the hyaluronic acid of this invention means at least 2.5m as determined by the method of Non-Patent Document 22 "Sodium Hyaluronate" (the entire content of which is incorporated herein by reference). 3 / kg(2.5m 3 This refers to hyaluronic acid with an intrinsic viscosity of (1 / kg or more). In short, intrinsic viscosity [η] is given by the Martin equation: Log 10 (n r -1 / c) = log 10 It is calculated by linear least squares regression analysis using [η] + κ[η]c. In some embodiments, high molecular weight hyaluronic acid is at least 2.9m 3 It has an intrinsic viscosity of / kg.

[0095] The terms “nerve injury or loss” (in the cornea) and “corneal nerve injury or loss” are used herein without distinction and refer to any type of nerve injury that impairs or impairs the normal turnover, orientation, growth, function, or any combination of two or more of the above, and involves any degree of damage to nerve structure (composition and / or continuity) and, if applicable, to surrounding tissue. Nerve injury is any type or stage of injury, such as apraxia, axonal rupture, or nerve rupture (Non-Patent Literature 23). For example, nerve injury or loss includes one or more of the following: reduction in nerve fiber length, reduction in nerve fiber curvature, reduction in nerve fiber density, and complete or partial nerve fiber transection. Nerve injury or loss is any type of effect that reduces nerve function (neurotrophic injury) and / or, for example, in the case of nerve transection, a type of result that causes complete absence of nerve function (neuroparalytic injury).

[0096] The term "neurotrophic keratopathy" or "NK (neurotrophic keratopathy)" refers to a degenerative corneal disease caused by impaired corneal innervation, characterized by decreased or absent corneal sensation, leading to epithelial keratopathy, epithelial defects, interstitial ulceration, and ultimately corneal perforation. Any eye or systemic condition that alters normal corneal innervation results in NK. Potential etiological conditions include, for example, infections such as herpes keratitis (herpes zoster and herpes simplex), addiction to local anesthetics, chemical and physical burns, contact lens abuse (chronic contact lens wear), topical drug toxicity, irradiation of the eye or adnexa, chronic use of topical drugs containing benzalkonium chloride (BAK), laser epithelial keratectomy (LASIK), photorefractive keratectomy (PRK), corneal transplantation (especially penetrating keratoplasty (PK) and deep anterior lamellar keratoplasty (DALK)). Non-corneal ophthalmic procedures include keratoplasty, corneal surgery such as collagen cross-linking for keratoconus, vitrectomy and photocoagulation to treat diabetic retinopathy, indirect laser treatment for proliferative diabetic retinopathy, and non-ophthalmic etiologies such as neurosurgical procedures or trauma affecting the fifth cranial nerve, stroke, aneurysm, multiple sclerosis, intracranial mass, diabetes, leprosy, vitamin A deficiency, drugs (narcolepsy and antipsychotics), and congenital trigeminal malformation.

[0097] As used herein, the term “ocular surface” refers to the cornea and conjunctiva, and the portion thereof including the conjunctiva covering the upper and lower eyelids. The HMWHA fluid is administered topically to one or more portions of the ocular surface, for example, including the entire ocular surface.

[0098] "Pharmacologically acceptable salts" include both acid addition salts and base addition salts. Any pharmaceutically acceptable salt of HA or any other compound described herein is intended to encompass all pharmaceutically acceptable salt forms. Preferred pharmaceutically acceptable salts described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0099] "Pharmacologically acceptable acid addition salts" refer to salts formed by inorganic acids, such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, and phosphorous acid, that retain the biological efficacy and properties of the free base and are not biologically or otherwise undesirable. It also includes salts formed by organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanes, hydroxyalkanoates, alkanedionic acids, aromatic acids, and aliphatic and aromatic sulfonic acids, including, for example, acetic acid, trifluoroacetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, and salicylic acid. Examples of such salts include sulfates, pyrosulfates, bisulfates, sulfites, bisulfites, nitrates, phosphates, monohydrogen phosphates, sodium dihydrogen phosphate, metaphosphates, pyrophosphates, chlorides, bromides, iodides, acetates, trifluoroacetates, propionates, caprylates, isobutyrates, oxalates, malonates, succinates, suberates, sebacates, fumarates, maleates, mandelates, benzoic acid, chlorobenzoic acid, methylbenzoic acid, dinitrobenzoates, phthalates, benzenesulfonates, toluenesulfonates, phenyl acetate, citrates, lactates, maleates, tartrates, methanesulfonates, and the like. Salts of amino acids such as alginates, glucons, and galacturons are also possible (see, for example, Non-Patent Document 32, the entire content of which is incorporated herein by reference). Acid addition salts of basic compounds can be prepared by contacting the free base form with a sufficient amount of the desired acid to produce a salt, according to methods and techniques familiar to those skilled in the art.

[0100] A "pharmaceutically acceptable base addition salt" refers to a salt that retains the biological efficacy and properties of a free acid and is not biologically or otherwise undesirable. These salts are prepared by adding an inorganic or organic base to a free acid. pharmaceutically acceptable base addition salts are formed using metals or amines, such as alkalis and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, salts of sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, and aluminum. Examples of salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, naturally occurring substituted amines, cyclic amines, and basic ion exchange resins, such as isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, diethanolamine, 2-dimethylaminoethanol, 2-diethylaminoethanol, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, N,N-dibenzylethylenediamine, chloroprocaine, hydravamin, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, and polyamine resins. See Non-Patent Document 32 above. In some embodiments, the pharmaceutically acceptable salt is a sodium salt (see Non-Patent Document 22 as incorporated herein by reference).

[0101] As used herein, the terms “subject,” “patient,” and “individual” refer to human or non-human animals. Subject also refers to, for example, primates (e.g., humans), cattle, sheep, goats, horses, dogs, cats, rabbits, rats, mice, fish, birds, etc. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject is a bird or fish. Thus, the method can be carried out in hospitals or veterinary hospitals. Non-human animal subjects are, for example, pets or animal models with or without eye diseases.

[0102] The term "local administration" is used herein in its conventional sense, meaning local delivery to a desired anatomical site, such as the ocular surface. The fluid containing high molecular weight hyaluronic acid is applied directly or indirectly to the ocular surface by any means that allows an effective amount of the fluid to come into contact with the ocular surface. For example, the fluid may be applied directly to the ocular surface by eye drops or eye wash, etc., or indirectly by a delivery agent (i.e., a fluid delivery agent) that comes into contact with the ocular surface or other parts of the eye. An example of a delivery agent is a particle (e.g., a microparticle or nanoparticle) that is coated with the fluid and / or releases the fluid onto the ocular surface. Such particles are composed of various materials, such as natural or synthetic polymers. In some embodiments, the delivery agent is administered as a dropper.

[0103] The terms “to treat” and “treatment” include mitigating, improving, inhibiting, or preventing the progression of a medical condition, such as corneal nerve injury or loss, or one or more symptoms or complications associated with that condition, and mitigating, improving, or eradicating one or more causes of that condition.

[0104] The present invention is illustrated only by the embodiments described in the description and drawings, and is not limited thereto, and includes all variations, modifications, substitutions, and combinations that experts may take from the complete documentation of this application, taking into account their specific knowledge and / or in combination with that specific knowledge.

[0105] All patents, patent applications, provisional applications, and documents mentioned or cited herein, including figures and tables, are incorporated herein by reference in their entirety, provided that they do not conflict with the express teachings herein.

[0106] The following are examples of procedures for carrying out the present invention. These examples are not to be considered limiting. All percentages are by weight, and all mixing ratios are by volume unless otherwise specified.

[0107] Example 1 - Promotion of corneal nerve growth by high molecular weight hyaluronic acid eye drops A. Background The HYLAN M trial is an ongoing randomized clinical trial in patients with severe dry eye (according to the ODISSEY primary criteria). In the HYLAN M trial, patients were randomly divided into two groups: one group continued with their previously identified most effective individual patient treatment, and the other switched to high molecular weight hyaluronic acid eye drops (COMFORT SHIELD® preservative-free sodium hyaluronate eye drops (i.com Medical, Munich, Germany)) corresponding to the embodiments in Table 1 herein.

[0108] These patients were already receiving the best possible treatment available from their ophthalmologists. All patients were receiving “stable” treatment at the time of enrollment in the study; that is, their treatment had not been altered for the period specified prior to enrollment in this study. Patients were randomly divided into two groups: one group continued their current treatment for dry eye syndrome, while the other group was treated with the aforementioned fluid drops (COMFORT SHIELD® eye drops) instead of tear substitutes.

[0109] The objectives of the study include (1) comparing the objective and subjective symptoms of dry eye treated with COMFORT SHIELD® eye drops to those treated with the principal investigator (=previous treatment) in patients with severe dry eye conditions, and (2) observing objective performance, patient subjective acceptance, and adverse events of the eye drops. For each patient, both eyes will be examined, and the eye with a higher corneal fluorescein staining score on baseline will be evaluated.

[0110] In the HYLAN M trial, confocal microscopy is an optional diagnostic method performed at baseline and 8-week follow-up visits to analyze the subbasal epithelial plexus. Further details regarding the HYLAN M trial are as follows:

[0111] Further details regarding the HYLAN M test can be found in Non-Patent Document 33, which is incorporated herein by reference in its entirety.

[0112] B. Overview of Corneal Confocal Microscopy The Heidelberg Gretina Tomograph (HRT) is a confocal laser scanning system for acquiring and analyzing two-dimensional or three-dimensional images of the posterior and anterior regions of the eye. The most important routine clinical applications of the Heidelberg Gretina Tomograph are the detection of glaucomatous damage to the optic nerve head and the follow-up of glaucoma progression using the glaucoma module. This instrument enables quantitative description of optic nerve head topography and time-related changes.

[0113] The addition of the Rostock Corneal Module (RCM) transforms the HRT into a confocal corneal microscope, which allows for the acquisition of two-dimensional and three-dimensional images of different corneal layers (including the corneal nerve plexus), as well as the limbus and conjunctiva.

[0114] To acquire corneal images, laser light is focused onto the cornea and periodically deflected by a vibrating mirror, resulting in sequential scanning of a two-dimensional sector of the cornea. The amount of reflected light at each point is measured by a photosensitive detector. In the HRT's confocal optical system, light can only reach the detector if it is reflected or scattered from a narrow region surrounding a preset focal plane. Light reflected or scattered outside the focal plane is highly suppressed. For this reason, the two-dimensional confocal image is considered an envelope optical region passing through the cornea. The focal plane can be manually moved across the entire cornea. Therefore, images of the following different corneal layers can be acquired.

[0115] The actual position of the focal plane is measured and stored along with each acquired image.

[0116] The laser source of the Heidelberg Retina Tomograph / Rostock Corneal Module is a diode laser with a wavelength of 670 microns. The 2D image consists of 384x384 pixels. It covers a 0.4mm x 0.4mm area of ​​the cornea using a "400FOV" field lens, or a 0.3mm x 0.3mm area using a "300FOV" field lens.

[0117] This device is designed to be operated by professional users with a healthcare background and experience in operating ophthalmic imaging equipment, such as physicians, ophthalmic radiographers, or optometrists.

[0118] C. Corneal nerve plexus evaluation RCM measurement follows a clinical routine. Local anesthetic and artificial tears are applied to both eyes of the subject, positioned on the head and chin rests of the device. The operator applies viscous artificial tears to the concave anterior portion of the HRT-RCM patient interface and attaches the "TomoCap" as the contact element between the HRT-RCM device and the patient's eye. The operator then carefully moves the HRT-RCM forward until the "TomoCap" makes gentle contact with the patient's cornea. The focal plane is adjusted to the subbasal plexus layer, and images of the subbasal plexus are acquired at various locations near the center of each cornea, with particular emphasis on the target region. The subbasal plexus images should be uniformly illuminated, artifact-free, and have the maximum signal-to-noise ratio.

[0119] Data collection in the D.HYLAN M trial Ten subbasal plexus images of the central corneal region were collected for each patient's bilateral eye at baseline and at 8-week follow-up visits. The images were saved in single-image mode on the HRT+RCM device and exported to the ReadCentral for evaluation.

[0120] E. Data Analysis and Results Images were collected from 16 patients at baseline and after 8 weeks of treatment. These 16 patients included 8 patients in the control group who continued to use the control lubricating eye drops they were using at the time of inclusion in the HYLAN M trial, and 8 patients whose ophthalmic lubricating eye drops were replaced with COMFORT SHIELD® eye drops.

[0121] To our surprise, as shown in Figure 1, patients treated with COMFORT SHIELD® eye drops showed a statistically significant (p=0.031) increase in total nerve fiber length (51% growth) over 8 weeks compared to the control group. This neurotrophic activity is an unexpected effect.

[0122] Detailed analysis of the 2-HYLAN M trial As described in Example 1, patients with severe dry eye disease (DED) were randomly divided into two parallel arms. The control group continued their current treatments up to the time of enrollment. In the verum group (COMFORT SHIELD® eye drop group, also referred to herein as the HMWHA group), the individual lubricating eye drops used by each patient up to the time of inclusion were replaced with eye drops containing 0.15% HMWHA (COMFORT SHIELD® eye drops, i.com Medical, Munich, Germany). Concomitant treatments for dry eye, such as cyclosporine eye drops, remained unchanged in both arms.

[0123] Demographic data and medical history were recorded at the baseline visit. Symptoms and signs associated with DED were assessed at the baseline visit, and at follow-up visits at 4 and 8 weeks (see Table 2). [Table 2]

[0124] The study centers were asked to optionally acquire CSLM images at baseline and at the 8-week visit and provide them to masked reading centers for evaluation. Four of the 11 study centers participated in this optional study. These four centers provided CSLM images for all patients following the protocol. Thus, the electronic randomization used throughout the HYLAN M trial was also optionally applied to confocal microscopy studies. The evaluation results of the CSLM images from these four study centers are the subject of this report. Results of other diagnostic tests performed on all 84 protocol patients enrolled in the HYLAN M trial, including the Ocular Surface Disease Index (OSDI), frequency of decline, best corrected visual acuity (BCVA), corneal fluorescein staining (CFS), tear film breakup time (TBUT), Scherrer 1, tear osmolality, intraocular pressure (IOP), eyelid wiper epitheliopathy (LWE), and Yamaguchi score, have been previously reported (Non-Patent Literature 14).

[0125] Patients with underlying conditions and a history of DED for more than 18 years were eligible for inclusion. Patients had to have received stable, unchanging dry eye therapy for at least two months (three months in the case of cyclosporine combination therapy) prior to inclusion. Patients were excluded if they had participated in other clinical trials, had other eye conditions, undergone ophthalmic surgery less than three months prior to enrollment in the trial, used tear plugs, or had a masquerading condition as identified by Karpecki (Non-Patent Literature 36). Masquerading conditions included conjunctival degeneration, recurrent corneal erosion, epithelial basement membrane dystrophy, mucus-fishing syndrome, floppy eyelid syndrome, giant papillary conjunctivitis, Salzmann nodular degeneration, and ocular rosacea.

[0126] The primary criteria by Baudouin et al. were selected as the inclusion criteria for severe dry eye (Non-Patent Literature 37). Dry eye symptoms were evaluated using the Ocular Surface Disease Index (OSDI) questionnaire, and an OSDI score of 33 or higher was required for inclusion (Non-Patent Literature 38). Corneal fluorescein staining (CFS) was selected as a sign of dry eye (Non-Patent Literature 39). For inclusion, at least one eye of the patient had to have CFS Oxford grade 3 or higher, but not confluent CFS. Eyes with high staining scores were defined as test eyes.

[0127] The Heidelberg Cretina Tomograph (HRT3), in combination with the Rostock Corneal Module (Heidelberg Engineering, Heidelberg, Germany), was used for in vivo evaluation of subbasal corneal plexuses (SNPs), as described above (Non-Patent Documents 40, 41). Both eyes were anesthetized with a local anesthetic and covered with artificial tears. To prevent eye movement, patients were asked to fix the eye not being examined in front of a spotlight.

[0128] Five non-overlapping images were acquired in the central region of the cornea, close to the apex and at least 0.5 mm away from the inferior whorl (see Figure 2A for an example image, and Figure 2B for an image after processing by the reading center).

[0129] Image processing and quantitative image analysis were performed using Mathematica (version 11.3, Wolfram Research, Champaign, Illinois, USA) with a reading center, as described above (Non-Patent Literature 42). The following SNP parameters were calculated: Unit area (mm / mm²) 2 Corneal nerve fiber length (CNFL), defined as the total length of all nerve fibers per unit area (n / mm²). 2 Corneal nerve fiber density (CNFD), defined as the number of nerve fibers per unit area (n / mm²), is defined as the number of nerve fibers per unit area. 2 Corneal nerve branch density (CNBD) is defined as the number of branching points per unit area, and reflects variations in nerve fiber direction, with absolute nerve fiber curvature / nerve fiber length (μm). -1Mean weighted corneal nerve fiber curvature (CNFTo), defined as the number of nerve fibers crossing a region boundary (number of connections / mm²), 2 Corneal nerve junction points (CNCPs) are defined as the mean corneal nerve single fiber length (CNSFL), defined as the mean length of a nerve fiber (μm), and mean weighted corneal nerve fiber thickness (CNFTh) is measured as the mean thickness perpendicular to the nerve fiber pathway (μm).

[0130] Statistical analysis was performed using IBM SPSS Statistics (version 22, IBM, Armonk, New York, USA). Descriptive statistics were calculated and box plots were created. The data were examined for normal distribution using the Shapiro-Wilk test. Group comparisons were performed using the Wilcoxon signed-rank test and the Mann-Whitney U test, respectively. The significance level was determined to be p<0.05.

[0131] Table 3 includes sociodemographic characteristics of patients with CSLM assessment of SNPs. [Table 3]

[0132] Five CSLM images were analyzed from eight patients in the control group and eight patients in the HMWHA group, collected at the end of baseline visits and at the end of the 8-week visit (see example in Figure 3).

[0133] A statistically significant difference in CNFL was observed between baseline and the 8-week follow-up visit. In contrast to the control group (p=0.294), the HMWHA group showed a significant difference in CNFL (p=0.030). At baseline, CNFL was comparable between HMWHA and the control group (p=0.793), and a significant difference was observed after 8 weeks (p=0.031). Perhaps due to the small patient sample size, no significant differences were found for other SNP parameters (CNFD, CNBD, CNFTo, CNCP, CNSFL, CNFTh). Furthermore, patients with severe dry eye generally lacked well-developed SNPs, and the presence of a large amount of foreign tissue near the SNPs complicated image analysis. Figure 4 summarizes the findings regarding CNFL in the HMWHA and control groups at baseline and the 8-week follow-up visit. Figure 4 corresponds to Figure 1 in Example 1 and shows individual patient data points.

[0134] Further details regarding the HYLAN M trial and the nutritional effects of COMFORT SHIELD® eye drops on total nerve fiber length and subbasal plexus are provided in Non-Patent Documents 14 and 15, which are incorporated herein by reference in their entirety.

[0135] The examples and embodiments described herein are for illustrative purposes only, and various modifications or changes in light thereof are suggested to those skilled in the art and understood to be included in the spirit, scope and appended claims of this application. Furthermore, any element or limitation of the invention or its embodiments disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (individually or in any combination) or with any other invention or its embodiments, and all such combinations are considered to be within the scope of the present invention without limitation.

Claims

1. A fluid containing high molecular weight sodium hyaluronate (HMWHA) for use in treating or preventing corneal nerve injury or loss in humans or non-human animals, The fluid is intended for local administration to the ocular surface of the target, The hyaluronic acid is at least 2.5 ml ３ It has an intrinsic viscosity of / kg, The subjects mentioned above are individuals with diabetes and diabetic peripheral neuropathy, diabetes without yet having diabetic peripheral neuropathy, diabetic corneal neuropathy, stroke, neurotrophic keratopathy (NK), or corneal nerve damage or loss caused by viral infection, trauma or medical procedure.

2. The fluid according to claim 1, wherein the human or non-human subject has nerve damage or loss comprising one or more of the following characteristics: a decrease in nerve fiber length, a decrease in nerve fiber curvature, a decrease in nerve fiber density, and (complete or partial) nerve fiber transection.

3. The fluid according to claim 1 or 2, wherein the subject is identified to have corneal nerve damage or loss prior to the administration of the fluid.

4. The fluid according to claim 3, wherein the identification includes performing in vivo confocal microscopy (IVCM) on the eye of the subject.

5. The fluid according to claim 1 or 2, further comprising recombinant human nerve growth factor (senegermin), a matrix metalloproteinase inhibitor, or plasma rich in growth factors (PRGF).

6. The fluid according to claim 1 or 2, further comprising a preservative.

7. The fluid according to claim 1 or 2, wherein the fluid further contains no preservative.

8. The fluid according to claim 1 or 2, further comprising additional glycosaminoglycans (GAGs), electrolytes, buffers, or combinations of two or more of the above.

9. The fluid according to claim 1 or 2, wherein the fluid contains HMWHA having a concentration of <0.2% w / v.

10. The fluid according to claim 1 or 2, wherein the fluid contains HMWHA having a concentration of 0.1 to 0.19% w / v.

11. The fluid according to claim 1 or 2, wherein the fluid contains HMWHA having a concentration of about 0.15% w / v.

12. The aforementioned fluid is a) pH 6.8-7.6, b) Osmolar concentration of 240–330 mOsmol / kg, c) NaCl concentration of 7.6 to 10.5 g / l, and / or d) The fluid according to claim 1 or 2, having a phosphate concentration of 1.0 to 1.4 mmol / l.

13. The fluid according to claim 1 or 2, wherein the fluid is a transparent, colorless solution that does not contain any visible impurities.

14. The fluid according to claim 1 or 2, wherein the fluid is sterile.

15. The fluid according to claim 1 or 2, wherein the fluid is COMFORT SHIELD® preservative-free sodium hyaluronate eye drops.

16. The fluid according to claim 1 or 2, wherein the fluid does not contain any other biological agents.

17. The fluid according to claim 1 or 2, wherein the fluid further comprises a bioactive agent.

18. The subject is the fluid according to any one of claims 1 to 17, having diabetes and diabetic peripheral neuropathy.

19. The subject is a person with diabetes who does not yet have diabetic peripheral neuropathy, and the fluid is according to any one of claims 1 to 17.

20. The subject is the fluid according to any one of claims 1 to 17, having diabetic corneal neuropathy.

21. The subject is the fluid according to any one of claims 1 to 17, which is used by a person who has suffered a stroke.

22. The subject is the fluid according to any one of claims 1 to 17, having neurotrophic keratopathy (NK).

23. The fluid according to any one of claims 1 to 17, wherein the subject has corneal nerve damage or loss caused by viral infection, trauma, or medical procedure.

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