Method for establishing, restoring, and preserving homeostasis of the ocular surface

Abstract

To provide a fluid for the treatment of a subject after ocular surgery, suitable for topical administration to the ocular surface of the eye in a human or non-human animal subject.SOLUTION: The present invention provides a fluid to be used in a method for reducing or delaying the onset of irritation, discomfort, (acute or chronic) inflammation, and / or an immune response at the ocular surface, the fluid comprising high molecular weight hyaluronic acid with an intrinsic viscosity of >2.5 m3 / kg and a concentration of <0.2%w / v.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 62 / 516,911, filed June 8, 2017, and U.S. Provisional Application No. 62 / 408,559, filed October 14, 2016, which are incorporated by reference in their entirety, including any figures, nucleic acid sequence listings, amino acid sequence listings, and drawings.

[0002] The present invention relates to fluids that reduce or delay the onset of, and / or attenuate and provide long-term relief from, irritation, discomfort, inflammation and / or immune response at the ocular surface after onset. [Background technology]

[0003] Dry eye disease is a common ocular condition that significantly impacts vision and quality of life. The term dry eye was recognized in the early 20th century and was primarily used in reference to patients with Sjögren's syndrome. Over the last few decades, the diagnosis of dry eye disease has relied primarily on corneal and conjunctival staining, estimation of tear film breakup time, and Schirmer's test. The currently accepted primary treatment for dry eye is some type of tear substitute to improve symptoms.

[0004] At the 2007 Dry Eye Workshop (DEWS), a consensus document on the current state of dry eye disease was issued by approximately 50 internationally recognized experts. This consensus document has become the international standard for clinical research on dry eye disease. The concepts of aqueous-deficient versus evaporative dry eye, the role of tear osmolarity as a major precipitating factor for ocular inflammation, and the focus on meibomian gland dysfunction are among the DEWS achievements. The lack of predictability of the signs assessed by individual diagnostic test methods with respect to the symptoms experienced by patients is known as the sign-symptom dissociation and is reflected in the published literature. New test methods are becoming available, such as assessment of tear osmolarity and visualization of the meibomian glands.

[0005] Recent clinical dry eye research has focused on inflammatory processes at the cellular and molecular levels. Impression cytology with goblet cell counts, flow cytometry for markers such as HLA-DR, and testing of MMP-9 levels are the results of this focus. New therapeutic developments have explored anti-inflammatory compounds that may interrupt the long-lasting cycle of inflammation. Candidates include cyclosporine, corticosteroids, doxycycline, and essential fatty acids. The goal of treatment is to restore corneal and conjunctival immune homeostasis. In this regard, current clinical research into dry eye disease has expanded to other ocular immune responses, such as allergic keratoconjunctivitis. Summary of the Invention

[0006] The present invention provides a holistic approach to establish, restore and maintain corneal and conjunctival homeostasis. According to the present invention, a fluid composition containing an ultra-high molecular weight form of hyaluronic acid (also called hyaluronan or HA) can be topically administered to the cornea and conjunctiva to reduce or delay the onset of corneal and conjunctival irritation, discomfort (such as itching and eye pain), inflammation (acute or chronic) and / or immune response. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0007] Hyaluronic acid (HA) is a carbohydrate and mucopolysaccharide found in living organisms. The biological functions of endogenous HA include maintaining the viscoelasticity of liquid connective tissues such as joint synovial fluid and ocular vitreous fluid (Necas J et al., "Hyaluronic acid (hyaluronan): a review", Veterinarni Medicina, 2008, 53(8): 397-411; Stern R et al., "hyaluronan fragments: An information-rich system", European Journal of Cell Biology, 2006, 85: 699-715). Although the specific mechanisms involved in the various signaling pathways of HA are still poorly understood, it is known that they can regulate multiple biological effects, which vary depending on the size of HA (Cyphert JM et al., "Size Matters: Molecular Weight Specificity of Hyaluronan Effects in Cell Biology", International Journal of Cell Biology, 2015, Epub 2015 Sep 10, 563818).

[0008] Sodium hyaluronate and other viscoelastic substances have been used in intraocular surgery since the 1970s and in the treatment of dry eye since the 1980s (Higashide T and K Sugiyama, "Use of viscoelastic substance in ophthalmic surgery- focus on sodium hyaluronate," Clinical Ophthalmology, 2008, 2(1): 21-30; Polack FM and MT McNiece, "The treatment of dry eyes with Na hyaluronate (Healon)- preliminary report, 1982, 1(2): 133-136). However, little attention has been paid to the biological functions of hyaluronan in the epithelium (Muller-Lierheim WGK, "Transenersatzlosungen, Neues über Hyaluronsaure," Aktuelle Kontaktologie, April 2015, 17-19).

[0009] An embodiment of the present invention is a method for reducing or delaying the onset of symptoms selected from keratoconjunctival irritation, discomfort (e.g., itching or eye pain), inflammation, immune response, or a combination of two or more thereof, comprising topically administering ultra-high molecular weight hyaluronic acid (HA) to the keratoconjunctival epithelium of a non-human animal subject. Hyaluronic acid is a high molecular weight, >2.5mM. 3 / kg and a concentration of <0.2% w / v. Intrinsic viscosity is determined according to the European Pharmacopoeia 9.0, "Sodium Hyaluronate", p. 3584, the contents of which are incorporated herein by reference. Briefly, the intrinsic viscosity [η] is calculated using the Martin formula Log 10 (n r -1 / c)=log 10 [η] + κ[η]c is calculated by linear least squares regression analysis. In one embodiment, the intrinsic viscosity of hyaluronic acid is 2.9 m 3 / kg.

[0010] In one embodiment, the HA has a molecular weight of at least 3 million daltons as calculated by the Marchouwink formula, hi one embodiment, the HA has a molecular weight of 3-4 million daltons as calculated by the Marchouwink formula.

[0011] In some embodiments, the high molecular weight HA is hyaluronan. In some embodiments, the high molecular weight HA is cross-linked, such as Hylan A and Hylan B. In some embodiments, the high molecular weight HA is non-cross-linked. In some embodiments, the high molecular weight HA is linear. In some embodiments, the high molecular weight HA is not linear. 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 thereof.

[0012] In certain embodiments, the cornea to which the fluid is administered is in homeostasis at the time of administration of the fluid, hi other embodiments, the cornea of ​​the subject's eye is not in homeostasis.

[0013] The administered fluid increases or improves the visual performance of the recipient eye, whether the cornea is homeostatic or not at the time the fluid is administered to the eye. The administered fluid helps stabilize the eye's fluid film (e.g., the cornea and conjunctiva tear film) to optimize vision and visual performance. This is particularly beneficial in extreme conditions such as aviation where blinking is not possible or desirable, space travel, submersion in polluted or poison-inducing fluids, or operation in areas with extreme climates such as cold, heat, and dryness.

[0014] Increased or improved visual performance is defined as an increase or improvement in the speed and / or accuracy of processing visual information. For example, visual performance describes how quickly and accurately an individual can process visual stimuli defined in criteria terms such as adaptation luminance, target contrast, and target size. Methods for assessing visual performance and changes in visual performance are known in the art (see, for example, Toda I et al., "Visual performance after reduced blinking in eyes with soft contact lenses or after LASIK," J Refract. Surg., 2009, Jan., 25(1):69-73; and Rea MS and MJ Quellette, "Relative visual performance: A basis for application," Lighting Res.Technol., 1991, 23(3):135-144, the contents of which are incorporated herein by reference).

[0015] The fluid may be administered topically to the cornea or conjunctiva of one or both eyes of the subject. For example, the fluid may be administered in one or more drops from an eye dropper or other eye drop dispensing device. The fluid may be self-administered or administered by a third party. The amount of the fluid administered to the cornea or conjunctiva in a single or multiple doses may vary depending on a variety of factors, including the patient's condition and characteristics, the extent of symptoms, concurrent treatments, frequency of treatment, and the desired effect. For example, one or more drops (e.g., about 30 microliters each) may be administered. Typically, one to three drops administered one to three times a day is sufficient, especially in acute keratoconjunctivitis. However, more frequent administration may be required in chronic keratoconjunctivitis, especially in the early stages of treatment. For example, one to three drops administered four, five, six, seven, eight, nine, ten or more times a day.

[0016] Advantageously, in some embodiments, once corneal homeostasis is maintained or restored and the cornea stabilizes, the frequency and / or dosage of administration can be reduced over time, for example, in some cases after four weeks, the dosage can be reduced and / or the frequency of daily administration can be reduced to twice daily.

[0017] The fluid may be administered prophylactically to reduce severity and / or delay onset before symptoms appear, i.e., before irritation, discomfort (e.g., eye pain), inflammation, or immune response. Alternatively, the fluid may be administered therapeutically after symptoms appear to reduce severity. Optionally, the fluid is administered prophylactically before symptoms appear or irritation occurs, such as eye trauma (e.g., non-surgical trauma), eye surgery (e.g., glaucoma surgery, strabismus surgery, etc.), or eye infection. In some embodiments, onset is delayed (i.e., prevented) indefinitely. In some embodiments, one or more symptoms are alleviated or eliminated. In some embodiments, all symptoms are alleviated or eliminated.

[0018] The fluid may also be administered prophylactically, particularly to a susceptible subject, to prevent or delay irritation, discomfort, inflammation, immune response, or a combination of two or more thereof. For example, the subject is immunocompromised. The subject's immunocompromise may have one or more causes, such as medical (e.g., radiation therapy, chemotherapy, or other immunosuppressive therapy), environmental exposure (e.g., exposure to radiation), or a genetic defect.

[0019] If the disease is present at the time of fluid administration and the fluid is administered as a treatment, the method may optionally include a step of identifying a subject with the disease prior to administering the fluid.

[0020] In certain embodiments, symptoms include one or more of the following features: leukocyte infiltration of the cornea and tears, cornea and conjunctiva CD44 upregulation, activation of the immune cascade including one or more of IL-1, IL-2, IL-5, IL-6, IL-8, CXCL8, IL-10, IL-12, IL-16, IL-33, MCP1, CCL2, MIP1d (also known as CCL15), ENA-78, CXCL5, sILR1, sIL-6R, sgp sEGFR, sTNFR, I-17A, IL-21, IL-22, CXCL9, MIG, CXCL11, I-TAC, CXCL10, IP-10, MIP-1β, CCL4, RANTES, and CCL5.

[0021] The condition is caused by a variety of stimuli, either external, internal, or both. In some embodiments, the condition is caused by an external stimulus resulting in the disruption of corneal and / or conjunctival smoothness and / or integrity (e.g., medical therapy, ocular surgery, non-surgical trauma, contact lens wear, microbial infection, allergens, haptens, toxins, or irritants).

[0022] The condition is caused by a variety of pharmaceutical therapies, including small molecule drugs and biologics. For example, the condition is caused by "beta blockers," which are drugs that inhibit or block the activity of one or more beta adrenergic receptors. Beta blockers are used to treat, among other conditions, hypertension, stable and unstable angina, arrhythmias, migraine headaches, hemorrhagic esophageal aneurysms, heart failure, and ischemic heart disease. Some beta blockers antagonize certain subtypes of beta adrenergic receptors (e.g., beta-1 selective beta blockers that selectively antagonize beta-1 adrenergic receptors, whereas other beta blockers are nonselective. Some beta blockers can block the effect of ligands, such as noradrenaline and norepinephrine, on one or more beta adrenergic receptors. Thus, the term "beta blocker" refers to beta blockers whether they antagonize one, two, or more beta adrenergic receptors and whether they affect other processes. , refers to all types of antagonists or inhibitors of beta-adrenergic receptors. Examples of beta-blockers include, but are not limited to, acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bopindolol, bucindolol, butaxamine, carteolol, carvedilol, celiprolol, esmolol, labetalol, levobunolol, medroxalol, metipranolol, metoprolol, nadolol, nebivolol, oxyprenolol, penbutolol, pindolol, propafenone, propranolol, sotalol, timolol, and eucommia.

[0023] In some embodiments, the condition is ocular allergy. In some embodiments, the condition is non-infectious keratoconjunctivitis due to trauma, allergic keratoconjunctivitis (such as seasonal allergic keratoconjunctivitis) or infectious keratoconjunctivitis, such as viral, bacterial, fungal and parasitic keratoconjunctivitis. In some embodiments, the condition is caused by an internal stimulus (e.g., hormonal disorders (female and male menopause), rheumatic diseases, epithelial-mesenchymal transition (EMT) or autoimmune diseases).

[0024] The condition may also be due to an injury to the eye. In some embodiments, the injury is due to physical trauma, chemical trauma, or radiation (radiation injury). In some embodiments, the injury is due to eye surgery. Examples of eye surgery include, but are not limited to, natural or artificial corneal transplants, corneal implants (e.g., intracorneal ring replacement (ICR) and keratoprosthesis), glaucoma surgery, cataract surgery (e.g., phacoemulsification, extrapsular cataract surgery, or intracapsular surgery), refractive surgery (e.g., radial keratotomy or refractive keratotomy), retinal surgery, strabismus surgery, corrective laser eye surgery (e.g., laser-assisted keratomileusis (LASIK) or photorefractive keratectomy (PRK)), and cross-linking surgery. Administration of the fluids of the present invention before, during and / or after ocular surgery, such as glaucoma surgery, can improve clinical outcomes, such as enhanced recovery, including restoration of post-operative visual characteristics, reduced scarring, and reduced itching, irritation and other discomfort.

[0025] The fluid is administered to reduce, prevent, or delay the onset of ocular discomfort such as itching and eye pain. Pain can have one or more causes. For example, eye pain is pain associated with mechanical, chemical, or thermal irritation of the ocular surface. Eye pain is associated with acute or chronic inflammation, or immune response. Reduction of pain is due to reduction of secondary neuroinflammatory effects (Belmonte C et al., "TFOS DEWS II pain and sensation report", The Ocular Surface, 15:404-437). Pain can have known or unknown causes.

[0026] Optionally, the fluid further comprises one or more bioactive agents (e.g., hydrophobic active components). As used herein, the term "bioactive agent" refers to any substance that, when administered in an effective amount to affect tissue, has an effect on a human or non-human animal subject. A bioactive agent is any class of substance, such as a drug molecule or a biological entity (e.g., polypeptides, carbohydrates, glycoproteins, immunoglobulins, nucleic acids), which may be a natural product or artificially produced and may act by pharmacological, immunological, or metabolic mechanisms. Types of bioactive agents include intraocular pressure regulators (e.g., enzyme inhibitors) and anti-angiogenic agents. Specific examples of bioactive agents include steroids (e.g., corticosteroids), antibiotics, tacrolimus, plasmin activators, antiplasmin, and cyclosporin A. In some embodiments, the bioactive agent is a steroid or antibiotic to treat or prevent an ocular infection, a glaucoma medication such as a prostaglandin analog, a beta blocker, an alpha agonist or a carbonic anhydrase inhibitor, an allergy eye relief or mydriatic medication such as a histamine antagonist or a nonsteroidal anti-inflammatory drug. Unfortunately, in some cases, the bioactive agent or the agent contained in the fluid can be irritating or damaging to the eye (e.g., cyclosporine A). Through its rheological and other properties, the high molecular weight HA in the fluid can mitigate and / or protect against irritation and / or damage to the eye caused by the bioactive agent or the agent present in the fluid (i.e., the bioactive agent would be more irritating or damaging to the eye if administered without the high molecular weight HA).

[0027] In some embodiments, the fluid does not include a steroid, an antibiotic, or an immunomodulatory agent, hi some embodiments, the fluid does not include a bioactive agent (e.g., does not include a hydrophobic active ingredient).

[0028] In some situations, it may be desirable to include one or more preservatives or detergents in the fluid. Preservatives and detergents are often irritating or damaging to the eye. Through rheological and other properties, the fluid may mitigate irritation and / or damage caused by preservatives or detergents present in the fluid and / or protect the eye. Thus, in some embodiments, the fluid includes a preservative or detergent that is irritating or damaging to the eye (i.e., a preservative or detergent that would be more irritating or damaging to the eye if administered without the high molecular weight HA). In some embodiments, the fluid does not include a preservative or detergent.

[0029] In certain embodiments, the fluid comprises cyclosporine A, cetalkonium chloride, tyloxapol, or two or more thereof.

[0030] In some embodiments, the fluid may be administered to the subject before, during, and / or after the administration of another composition containing a bioactive agent to the subject. In some circumstances, it may be desirable to include one or more preservatives or detergents in the other composition. As discussed above, preservatives and detergents are often irritating or damaging to the eye, and some bioactive agents may themselves be irritating or damaging to the eye. Through rheological and other properties, the fluid may mitigate and / or protect against irritation and / or damage caused by the bioactive agents, preservatives, and / or detergents in the other composition that may be present on the eye. Thus, the bioactive agents, preservatives, and / or detergents in the other composition may be more irritating or damaging to the eye when administered without the fluid.

[0031] In some embodiments, the other compositions administered to the subject are administered in any form and by any route (e.g., topically or systemically). In some embodiments, the other compositions are administered to the eye topically or by injection. In some embodiments, the other compositions are administered topically to the cornea and conjunctiva.

[0032] In certain embodiments, the preservative or detergent included in the fluid or other composition is a chemical preservative or an oxidizing preservative.

[0033] In certain embodiments, the preservatives or detergents contained in the fluid or other composition kill susceptible bacterial cells by disrupting the lipid structure of the bacterial cell membrane, increasing the permeability of the bacterial cell membrane.

[0034] In certain embodiments, preservatives or detergents contained in the fluid or other compositions may cause damage to corneal compositions, such as junctions between the corneal epithelium, endothelium, stroma, and membranes.

[0035] In certain embodiments, the preservative or detergent included in the fluid or other composition is selected from the group consisting of quaternary ammonium preservatives (e.g., benzalkonium chloride (BAK)), chlorobutanol, disodium edetate (EDTA), polyquaternium-1 (e.g., Polyquad® preservatives), stable oxidizing agents (e.g., stable oxychloro complexes (e.g., Purite® preservatives)), ionic buffer preservatives (e.g., sofZia® preservatives), polyhexamethylene biguanide (PHMB), sodium perborate (e.g., GenAqua® preservatives), and sorbates.

[0036] In one embodiment, the fluid is at least essentially mucin-free, ie, has a mucin concentration of <0.3% w / v.

[0037] In some embodiments, the fluid includes a preservative, while in other embodiments, the fluid does not include a preservative (i.e., the fluid is preservative-free).

[0038] In some embodiments, the fluid further comprises a glycosaminoglycan (GAG), 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 thereof.

[0039] When the fluid is administered to the subject's eye, the subject may or may not have dry eye disease (aqueous tear deficient type or qualitative dry eye type). In some embodiments of the therapeutic or prophylactic method, the symptom in the cornea and conjunctiva is irritation, discomfort, inflammation, immune response, or a combination of two or more thereof, and the eye of the subject to which the fluid is administered is not aqueous tear deficient (ATD) (i.e., no ATD) at the time of administration of the fluid. In some embodiments of the therapeutic or prophylactic method, the symptom in the cornea and conjunctiva is irritation, discomfort, inflammation, immune response, or a combination of two or more thereof, and the eye of the subject to which the fluid is administered is not qualitative dry eye (i.e., no aqueous tear deficient or qualitative dry eye) at the time of administration of the fluid.

[0040] In some embodiments of the therapeutic or prophylactic method, the corneal and conjunctival symptoms are irritation, discomfort, inflammation, an immune response, or a combination of two or more thereof, and the subject is not deficient in tear volume, but has corneal and conjunctival abnormalities (location-specific abnormalities) including protuberances on the cornea and other surfaces of the eye that are not covered by a normal tear film (tear film of normal surface tension and viscosity) and that result in areas of friction on the corneal and conjunctival.

[0041] The fluid is used in combination with a bandage contact lens. Thus, the method further comprises applying a bandage contact lens to the eye before, during and / or after administering the fluid. For example, the fluid is administered before applying the bandage contact lens, after the contact lens, and / or before applying the fluid to the bandage contact lens before applying the bandage contact lens to the eye. The use of the fluid allows the bandage contact lens to exert pressure on the cornea and conjunctiva while minimizing friction thereon. The fluid and bandage contact lens can be advantageously used safely immediately after eye surgery, for example glaucoma surgery.

[0042] Another aspect of the invention is a kit for use in carrying out the methods of the invention described herein. The kit includes a fluid as described herein and one or more bandage contact lenses. The bandage contact lenses may be packaged in the same container as the fluid (with the bandage contact lens in contact with the fluid). Alternatively, 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, and the like. The containers may be formed from a variety of materials, including glass and plastic.

[0043] The kit includes a delivery agent (separate from or associated with the fluid) that contacts the cornea or other portion of the eye, for example, the kit includes particles (e.g., microparticles or nanoparticles) that are coated with the fluid and / or release the fluid to the cornea or conjunctiva.

[0044] Optionally, the kit includes an eye drop dispensing device (e.g., an eye dropper) that may or may not function as a container for the fluid in the kit prior to use (opening) of the outer packaging of the kit. That is, the eye drop dispensing device may function to contain the fluid in an unused (unopened) kit, or it may be empty and receive the fluid after the kit is opened. Optionally, the kit may include a label or package with attached printed or digital instructions for using the kit to practice the methods of the invention.

[0045] The kits include packaging material compartmentalized to receive one or more containers, such as vials, tubes, etc., each containing one of the individual components used in the methods described herein. Examples of packaging materials used to package pharmaceutical products include, for example, those described in U.S. Patent Nos. 5,323,907, 5,052,558, and 5,033,252. Examples of pharmaceutical packaging materials include, but are not limited to, blister packs, bottles, tubes, pumps, bags, vials, airtight seals, syringes, bottles, and other packaging materials suitable for the selected formulation and intended mode of administration and treatment.

[0046] The kits may contain one or more additional containers, each containing one or more of a variety of materials desirable from a commercial and user standpoint for use in the compositions described herein, including, but not limited to, buffers, diluents, carriers, packaging, containers, vials, and / or tube labels with contents and / or instructions for use, and packaging containing instructions for use.

[0047] A label can be placed or affixed to a container. A label can be attached to a container by marking, molding or etching letters, numbers or other symbols that form the label onto the container. A label can be affixed to a container, for example, as a package insert, when in a receptacle or carrier that holds the container. A label can be used to indicate the contents to be used for a particular treatment. A label can also provide directions for using the contents as described herein.

[0048] In some embodiments of the kit, the fluid may be presented in a pack or dispenser containing one or more unit dosage forms containing the compositions disclosed herein. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for formulation.

[0049] Acute and chronic keratoconjunctivitis Ocular inflammation takes the form of numerous eye diseases of varying severity, depending on the location of the inflammation. Diseases resulting from ocular inflammation include, for example, uveitis, conjunctivitis, episcleritis, scleritis, optic neuritis, retrobulbar optic neuritis, keratitis, blepharitis, etc. Many of these conditions occur secondary to bacterial or viral infections. Ocular inflammation can also be the result of ocular trauma resulting from ocular surgery or physical damage to the eye.

[0050] Inflammation is a normal component of the immune response and a necessary part of the healing process, but it is judged to be occurring at an inappropriate level or for an inappropriate duration. Acute leukoinflammation is triggered by exposure to a pathogen (e.g., antigen, eye injury, or other trauma) for a few hours and usually represents a time-limited inflammatory response. The end of this phase varies, but the reduction in inflammation is an indirect sign of improvement and a gradual re-establishment of the previous state.

[0051] In contrast to acute ocular inflammation, chronic ocular inflammation is qualitatively different in type, not merely in duration (i.e., prolonged duration). The differences between acute and chronic ocular inflammation are large, similar to the differences in immunological events in acute healing wounds and chronic non-healing wounds (see, e.g., Tarnuzzer RW and Schultz GS, "Biochemical analysis of acute and chronic wound environments," Wound Repair Regen., 1996, Jul-Sep, 4(3):321-325). In particular, these differences in immunological events in the conjunctiva and subconjunctival tissues (vs. the cornea) underlie the diversity of corneal and conjunctival diseases and explain why some patients in a disease or disease population fail effective treatment plans.

[0052] Corneal and conjunctival homeostasis is controlled by resident lymphocytes (CD8+ T cells, gamma delta T cells and natural killer T cells) and CD4+ regulatory T cells, among other factors. These interact with anti-inflammatory factors such as interleukin (IL)-1 receptor antagonists, matrix protease inhibitors such as transforming growth factor (TGF)-beta 2 and inhibitors of metalloproteases (TIMP)-1. Stressors including environmental challenges, infections, endogenous stress, autoimmunity and genetic factors all disrupt the finely tuned homeostatic balance in the corneal and conjunctival tissue and activate acute immune responses. The method of the present invention provides a holistic approach to establish, restore and maintain corneal and conjunctival homeostasis, thereby reducing or delaying the onset of corneal and conjunctival irritation, discomfort, inflammation, immune responses or a combination of two or more of these. In this manner, administration of the fluid before or after an episode can result in attenuation and long-term relief from keratoconjunctival irritation, discomfort, inflammation, immune response, or a combination of two or more of these.

[0053] Epithelial-derived inflammatory cytokines activate immature resident antigen-presenting cells (APCs), mainly dendritic cells, in the cornea and conjunctiva. Mature APCs migrate to regional lymph nodes and initiate adaptive immune responses by priming naive CD4+ T cells, including T helper (Th)1 and Th17. Through activated angiogenesis and lymphangiogenesis, these inflammatory mediators traffic back to the cornea and conjunctiva where Th1-secreting interferon (IFN)-gamma and Th17-secreting IL-17 increase cytokine production, induce epithelial and goblet cell apoptosis, and alter conjunctival homeostasis to perpetuate chronic inflammatory processes. In conditions of goblet cell deficiency, gel-forming mucins are deprived, increasing friction and perpetuating inflammation (Pflugfelder SC et al., "Epithelial-immune cell interaction in dry eye," Cornea, 2008, 27 Suppl 1:S9-11).

[0054] Acute keratoconjunctivitis is characterized by rapid onset and relatively short duration (typically minutes to days), protein exudate, and a cellular infiltrate in the conjunctiva and subconjunctival tissue that is primarily neutrophils, whereas chronic keratoconjunctivitis is characterized by a longer duration (days to years), fewer neutrophils, a cellular infiltrate that is primarily macrophages and lymphocytes, vascular proliferation, fibrosis, and tissue necrosis. Indeed, the primary cells in chronic keratoconjunctivitis are macrophages and lymphocytes in the conjunctiva and subconjunctiva.

[0055] Typically, chronic keratoconjunctivitis can also be identified by keratoconjunctivitis markers including one or more of the following: tear film hyperosmolarity, elevated matrix metalloproteinase-9 (MMP-9) expression by epithelial cells and infiltrating leukocytes in the keratoconjunctiva, increased MMP-9 activity in the keratoconjunctiva (with its MMP-3 and TIMP-1-mediated enzymatic activity), elevated interferon gamma, overexpression of human leukocyte antigen-antigen D-related (HLA-DR) by conjunctival epithelial cells, intracellular adhesion molecule-1 (ICAM-1) by conjunctival epithelial cells, an increased number of activated lymphocytes, and a decreased number of goblet cells.

[0056] Steroids (e.g., corticosteroids) are considered the best standard treatment for ocular inflammation. Dexamethasone, prednisolone, triamcinolone acetonide, fluocinolone acetonide, and loteprednol etabonate are commonly used and act through various mechanisms, such as inhibition of multiple anti-inflammatory cytokines, fibrin deposition, polymorphonuclear leukocyte migration, and angiogenesis by inhibiting the nuclear factor-kappa-B (NF-kB) signaling pathway (Nakao et al., Am J Pathol. 2007;171(3):1058-65).

[0057] The anti-inflammatory effect of steroids is believed to be due to inhibition of arachidonic acid metabolism, i.e., inhibition of phospholipase A2, which releases arachidonic acid from the tissue phospholipid pool. Although steroids are considered effective in treating ocular inflammation, their widespread use is complicated by serious side effects, the most common of which are the development of cataracts, glaucoma, and infectious eye diseases (Kymionis GD et al., "Treatment of chronic dry eye: treatment of cyclosporine," Clin Ophthalmol. 2008 Dec;2(4):829-836; Schultz C, "Safety and efficacy of cyclosporine in the treatment of chronic dry eye," Ophthalmol Eye Dis. 2014;6:37-42). Similarly, immunomodulatory drugs such as cyclosporine and anti-inflammatory drugs such as tetracycline and its derivatives cause eye pain and irritation (e.g., burning and itching) in some patients, which is intolerable in inflamed eyes.

[0058] The topical administration of high molecular weight hyaluronic acid (HA), e.g. 3-4 MDa, as an effective natural immunomodulatory agent is advantageous for restoring corneal and conjunctival immune homeostasis in a group of patients with chronic keratoconjunctivitis or seasonal allergic keratoconjunctivitis without the use of the above-mentioned drugs and their side effects. In particular, the fluid of the present invention can be used to restore corneal and conjunctival immune homeostasis in these patients.

[0059] The influence of the molecular weight of HA molecules in eye drops on their biochemical functions has only recently come to the forefront of ophthalmic research. Due to its function in the extracellular matrix, HA plays a key role in keratinocyte proliferation and differentiation in corneal wound healing and corneal epithelial homeostasis. The prevalence of dry eye in older adults seems to be related to a significant decrease in free HA in the extracellular matrix starting in the 50s. In addition, HA acts as a signaling molecule. On the other hand, high molecular weight HA (3 - 4 MDa) acts as an antiangiogenic and immunosuppressive agent, whereas medium molecular weight HA has proinflammatory, immunostimulatory, and angiogenic properties (Stern R et al., "Hyaluronan fragments: An information-rich system", European Journal of Cell Biology, 2006, 85: 699-715; and Noble PW, "Hyaluronan and its catabolic products in tissue injury and repair," Matrix Biology, 2002, 21: 25-29). Elevated enzyme levels in chronic inflammation lead to increased hydrolysis of HA, shifting it to lower molecular weights. High molecular weight HA in eye drops helps to minimize this inflammation-related shift by reducing friction at the cornea and supporting downregulation of the inflammatory process. Reduction in friction also leads to a reduction in nociceptive nerve stimulation that may trigger or maintain pathways that support neurogenic inflammation.

[0060] It is well known that during acute and chronic inflammation, various putative mediators of inflammation are released from inflamed tissues and leukocytes. The concentration of these mediators and leukocytes is an indicator of the level and severity of inflammation. Similarly, a decrease in the concentration of these mediators and leukocytes is an indicator of the effectiveness of drugs to treat inflammation. Examples of keratoconjunctivitis markers include, but are not limited to, those mentioned above, namely, tear film hyperosmolarity, elevated matrix metalloproteinase-9 (MMP-9) expression by keratoconjunctival epithelial cells and infiltrating leukocytes, elevated keratoconjunctival MMP-9 activity (its MMP-3 and TIMP-1 mediated enzyme activity), elevated interferon-gamma, overexpression of human leukocyte antigen-antigen D-related (HLA-DR) by conjunctival epithelial cells, and intracellular adhesion molecule (ICAM-1) by increased numbers of activated lymphocytes and decreased numbers of goblet cells.

[0061] Optionally, prior to administration of the fluids of the invention, the methods may further comprise identifying the subject's eye as having chronic keratoconjunctivitis by detecting or measuring one or more of these inflammatory markers in the keratoconjunctiva. Optionally, the methods may further comprise detecting or measuring one or more inflammatory markers in the keratoconjunctiva one or more times after administration of the fluids of the invention to monitor the inflammatory state of the eye over time and guide the frequency and duration of further administrations of the fluids of the invention.

[0062] fluid preparation As mentioned above, >2.5 ml of fluid hyaluronic acid 3 / kg and a concentration of <0.2% w / v. In one embodiment, the hyaluronic acid is >2.9 m 3 / kg.

[0063] Viscoelasticity is defined as the characteristic of a fluid that is both viscous and elastic. Zero shear viscosity is defined as the constant steady shear viscosity with decreasing shear rate. For highly viscous formulations, controlled stress rheometer measurements are preferred.

[0064] Molecular weight and intrinsic viscosity [η]m 3The relationship between / k is the Markhouink formula: [η]=k·(Mrm) a where Mrm is the molecular weight in MDa; The coefficient is, k=1.3327 10 -4 and a=0.6691 and the values ​​of k and a were found to be the most predictive.

[0065] The fluid is prepared as follows: Sterilize the filling line. Add purified water or water for injection (WFI) to a stainless steel mixing tank. While mixing, add salt. Slowly add HA and mix until a homogenous solution / fluid is obtained. If necessary, adjust the pH value by adding NaOH or HCl while continuing the mixing process. Transfer the solution through a 1 μm pore size filter cartridge to a sterile holding tank and aseptically fill the solution in sterile direct packaging (monodose or glass bottles) by sterile filtration. In the case of monodose, this is done by a form-fill-while-fill (BFS) process.

[0066] Preferably, the fluid is at least essentially mucin-free, i.e., has a mucin concentration of <0.3% w / v, meaning that the flow behavior or properties are essentially achieved or regulated by hyaluronan, rather than by mucins that are naturally present in the subject's tear fluid and may primarily influence the flow behavior.

[0067] If a viscosity increasing substance is added, it is preferably added toward, during, or as a final step. Mix until a homogeneous mixture is obtained. Alternatively or additionally, it is preferred to initially use purified water or water as the base for injection, and optionally add electrolytes, buffers, and substances that do not increase viscosity to the purified water or water for injection initially.

[0068] HA and its production are further described in the monograph European Pharmacopoeia 9.0, page 3583 (Sodium Hyaluronate).

[0069] In one embodiment, the characteristics of the fluids used in the methods and kits of the present invention are shown in Table 1. [Table 1]

[0070] Embodiment Embodiment 1 A method for reducing or delaying the onset of a symptom selected from irritation, discomfort, inflammation, immune response, or a combination of two or more thereof in the cornea and conjunctiva, comprising administering to the patient a concentration of >2.5 mm. 3 13. A method comprising topically administering a fluid comprising high molecular weight hyaluronic acid having an intrinsic viscosity of 1000 mg / kg and a concentration of <0.2% w / v to the cornea and conjunctiva of the eye of a human or non-human animal subject.

[0023] Embodiment 2. The method of embodiment 1, wherein said fluid is administered prophylactically, prior to the onset of said symptoms. Embodiment 3. The method of embodiment 1, wherein said fluid is administered therapeutically after onset of said symptoms. Embodiment 4 The method of any one of embodiments 1 to 3, wherein the symptoms include one or more of the following features: leukocyte infiltration in the cornea and tears, CD44 upregulation in the cornea and conjunctiva, and activation of the immune cascade, including one or more of IL-1, IL-2, IL-5, IL-6, IL-8, CXCL8, IL-10, IL-12, IL-16, IL-33, MCP1, CCL2, MIP1d (also known as CCL15), ENA-78, CXCL5, sILR1, sIL-6R, sgp sEGFR, sTNFR, I-17A, IL-21, IL-22, CXCL9, MIG, CXCL11, I-TAC, CXCL10, IP-10, MIP-1β, CCL4, RANTES, and CCL5. Embodiment 5 The method of any of the preceding embodiments, wherein the condition is caused by an external stimulus resulting in the disruption of the smoothness and / or integrity of the cornea (e.g., medical therapy, eye surgery, non-surgical trauma, contact lens wear, microbial infection, allergens, haptens, toxins or irritants). Embodiment 6 The method of any one of embodiments 1 to 4, wherein the condition is caused by an internal stimulus (eg, a hormonal disorder, a rheumatic disease, an epithelial-mesenchymal transition (EMT), or an autoimmune disease). Embodiment 7. The method of any of the preceding embodiments, wherein the condition is caused by an injury to the eye. Embodiment 8. The method of embodiment 7, wherein said wound is caused by physical trauma, chemical trauma, or radiation (radiation damage). Embodiment 9. The method of embodiment 7, wherein the wound results from eye surgery. Embodiment 10. The method of embodiment 9, wherein the ophthalmic surgery is selected from natural or artificial corneal transplantation, corneal implants (e.g., intracorneal ring surgery (ICR) and artificial corneas), glaucoma surgery, cataract surgery (e.g., phacoemulsification, extracapsular surgery or intracapsular surgery), refractive surgery (e.g., radial keratotomy or refractive keratotomy), retinal surgery, strabismus surgery, corrective laser eye surgery (e.g., laser-assisted keratomileusis (LASIK) or photorefractive keratectomy (PRK)), and cross-linking surgery. Embodiment 11 The method of any of the preceding embodiments, wherein said inflammation is acute keratoconjunctivitis. Embodiment 12 The method of any one of embodiments 1 to 10, wherein said inflammation is chronic keratoconjunctivitis. Embodiment 13 The method of any one of embodiments 1-5, wherein the condition is an ocular allergy. Embodiment 14 The method of any one of embodiments 49-51, wherein the condition is allergic keratoconjunctivitis (e.g., seasonal or non-seasonal). Embodiment 15 The method of any one of embodiments 1-5, wherein said discomfort is itching or eye pain. Embodiment 16. The method of any of the preceding embodiments, wherein the fluid is administered prophylactically to prevent or delay the onset of symptoms and the subject is immunocompromised. Embodiment 17 The method of any of the preceding embodiments, wherein the fluid does not include other bioactive agents (e.g., does not include a hydrophobic active ingredient). Embodiment 18. The method of any of the preceding embodiments, wherein said fluid does not include other immunomodulatory drugs, immunosuppressants, or antibiotics. Embodiment 19. The method of any one of embodiments 1-14, wherein the fluid further comprises a bioactive agent (eg, a hydrophobic active ingredient). Embodiment 20. The method of embodiment 19, wherein the bioactive agent causes eye irritation or damage. Embodiment 21. The method of any one of embodiments 1-16, 18, 19, or 20, wherein the fluid further comprises a preservative or detergent. Embodiment 22. The fluid of embodiment 21, wherein the preservative or detergent is irritating or damaging to the eyes. Embodiment 23. The method of any of the preceding embodiments, wherein the fluid is administered before, during and / or after administration of a composition comprising a bioactive agent (e.g., a hydrophobic active ingredient), a preservative, a detergent, or a combination of two or more thereof. Embodiment 24. The method of embodiment 23, wherein the bioactive agent, preservative, detergent, or combination thereof is irritating or damaging to the eye. Embodiment 25. The method of any one of embodiments 21 to 24, wherein said preservative or detergent is a chemical preservative or an oxidizing preservative. Embodiment 26. The method of any one of embodiments 21 to 25, wherein the preservative or detergent increases the permeability of bacterial cell membranes by disrupting the lipid structure of the bacterial cell membrane and killing weaker cell membranes. Embodiment 27 The method of any one of embodiments 21-26, wherein the preservative or detergent is damaging to corneal tissue. Embodiment 28. The method of any one of embodiments 21 to 27, wherein the preservative or detergent is selected from the group consisting of quaternary ammonium preservatives (e.g., benzalkonium chloride (BAK)), chlorobutanol, disodium edetate (EDTA), polyquaternium-1 (e.g., Polyquad® preservatives), stable oxidizing agents (e.g., stable oxychloro complexes (e.g., Purite® preservatives)), ionic buffer preservatives (e.g., sofZia® preservatives), polyhexamethylene biguanide (PHMB), sodium perborate (e.g., GenAqua® preservatives), and sorbates. Embodiment 29. The method of any of the preceding embodiments, wherein the fluid is administered directly to the cornea as drops or a wash. Embodiment 30. The method of embodiment 29, wherein 1 to 3 drops are administered 1 to 3 times daily. Embodiment 31 The method of any one of embodiments 1 to 29, wherein the inflammation is chronic keratoconjunctivitis and 1 to 3 drops are administered 4, 5, 6, 7, 8, 9, or 10 or more times per day. Embodiment 32. The method of any one of embodiments 1-28, wherein the fluid is administered indirectly to the cornea by a delivery agent (e.g., particles that are coated with the fluid and / or secrete the fluid into the cornea) that is administered locally to the cornea or other part of the eye. Embodiment 33 The hyaluronic acid is >2.9 ml 3 The method of any of the preceding embodiments, wherein the polymer has an intrinsic viscosity of 100 / kg. Embodiment 34. The method of any one of embodiments 1 to 16, wherein the fluid further comprises a preservative. Embodiment 35. The method of any one of embodiments 1-16, wherein said fluid does not further comprise a preservative (i.e., the fluid is preservative-free). Embodiment 36 The method of any one of embodiments 1 to 16, wherein the fluid comprises additional glycosaminoglycan (GAG), an electrolyte (e.g., sodium chloride), a buffer (e.g., a phosphate buffer), or a combination of two or more thereof. Embodiment 37. The method of any of the preceding embodiments, wherein the subject's eye is not aqueous tear deficient (ATD) (i.e., no ATD) at the time of said administering. Embodiment 38. The method of any of the preceding embodiments, wherein the subject's eye does not have qualitative dry eye (i.e., no qualitative dry eye) at the time of said administering. Embodiment 39. The method of any of the preceding embodiments, wherein the subject's eye does not have dry eye disease (i.e., not aqueous tear deficient or qualitative dry eye type) at the time of administration. Embodiment 40. The method of any of the preceding embodiments, wherein the subject does not suffer from aqueous tear deficiency (ATD), the subject has a corneal or conjunctival abnormality (location-specific abnormality) including a protuberance on the cornea or other surface of the eye that is not covered by a normal tear film (tear film of normal surface tension and viscosity) that results in an area of ​​friction with the cornea and the administered fluid reduces the friction. Embodiment 41 The method of any of the preceding embodiments, wherein the eye of the subject is in corneal and conjunctival homeostasis at the time of said administering. Embodiment 42 The method of any of the preceding embodiments, further comprising applying a bandage contact lens to the eye before, during and / or after said administering. Embodiment 43. The method of embodiment 42, wherein the fluid is administered and the bandage contact lens is applied after eye surgery. Embodiment 44. The method of any of the preceding embodiments, wherein the administered fluid increases the visual performance of the eye. Embodiment 45 The method of embodiment 44, wherein the fluid is administered before, during and / or after eye surgery, and the subject recovers visual performance after the eye surgery (the subject recovers visual performance to a greater extent after eye surgery than in the absence of administration of the fluid). Embodiment 46. The method of any of the preceding embodiments, wherein the molecular weight of the hyaluronic acid is at least 3 million Daltons. Embodiment 47 The method of any of the preceding embodiments, wherein the molecular weight of the hyaluronic acid is between 3 million and 4 million daltons. Embodiment 48. A kit comprising the fluid of any one of embodiments 1 to 35 and one or more bandage contact lenses. The present invention also relates to fluids that are administered topically to the cornea and conjunctiva (topical fluids). Embodiment 49. A fluid for use in reducing or delaying the onset of symptoms selected from keratoconjunctival irritation, discomfort, inflammation, immune response, or a combination of two or more thereof, comprising a fluid having a concentration of >2.5 ml. 3 1. A fluid comprising high molecular weight hyaluronic acid having an intrinsic viscosity of 100 / kg and a concentration of <0.2% w / v, for topical administration to the cornea and conjunctiva of the eye of a human or non-human animal subject. Embodiment 50. The fluid of embodiment 49, wherein said fluid is administered prophylactically, prior to the onset of said symptoms. Embodiment 51. The fluid of embodiment 49, wherein said fluid is administered therapeutically after onset of said symptoms. Embodiment 52 The fluid of any one of embodiments 49 to 51, wherein the symptoms include one or more of the following features: leukocyte infiltration in the cornea and tears, CD44 upregulation in the cornea and conjunctiva, and activation of the immune cascade, including one or more of IL-1, IL-2, IL-5, IL-6, IL-8, CXCL8, IL-10, IL-12, IL-16, IL-33, MCP1, CCL2, MIP1d (also known as CCL15), ENA-78, CXCL5, sILR1, sIL-6R, sgp sEGFR, sTNFR, I-17A, IL-21, IL-22, CXCL9, MIG, CXCL11, I-TAC, CXCL10, IP-10, MIP-1β, CCL4, RANTES, and CCL5. Embodiment 53: The fluid of any one of embodiments 49 to 51, wherein the condition is caused by an external stimulus resulting in the disruption of the smoothness and / or integrity of the cornea and / or conjunctiva (e.g., medical therapy, eye surgery, non-surgical trauma, contact lens wear, microbial infection, allergens, haptens, toxins or irritants). Embodiment 54 The fluid of any one of embodiments 49 to 41, wherein the condition is caused by an internal stimulus (eg, a hormonal disorder, a rheumatic disease, an epithelial-mesenchymal transition (EMT) or an autoimmune disease). Embodiment 55 The fluid of any one of embodiments 49 to 51, wherein the condition is caused by an injury to the eye. Embodiment 56 The fluid of embodiment 55, wherein the wound is caused by physical trauma, chemical trauma or radiation (radiation damage). Embodiment 57 The fluid of embodiment 55, wherein the wound results from eye surgery. The fluid of embodiment 57, wherein the ophthalmic surgery is selected from natural or artificial corneal transplantation, corneal implants (e.g., intracorneal ring surgery (ICR) and artificial corneas), glaucoma surgery, cataract surgery (e.g., phacoemulsification, extracapsular surgery or intracapsular surgery), refractive surgery (e.g., radial keratotomy or refractive keratotomy), retinal surgery, strabismus surgery, corrective laser eye surgery (e.g., laser ablation (LASIK) or photorefractive keratectomy (PRK)), and cross-linking surgery. Embodiment 59 The fluid of any one of embodiments 49 to 51, wherein the inflammation is acute keratoconjunctivitis. Embodiment 60 The fluid of any one of embodiments 49 to 51, wherein the inflammation is chronic keratoconjunctivitis. Embodiment 61 The fluid of any one of embodiments 49 to 51, wherein the condition is an ocular allergy. Embodiment 62 The method of any one of embodiments 49 to 51, wherein the condition is allergic keratoconjunctivitis (eg, seasonal or non-seasonal). Embodiment 63 The fluid of any one of embodiments 49 to 51, wherein said discomfort is itching or eye pain. Embodiment 64 The fluid of any one of embodiments 49-51, wherein the fluid is administered prophylactically to prevent or delay the onset of symptoms and the subject is immunocompromised. Embodiment 65 The fluid of any one of embodiments 49-51, wherein the fluid does not include other bioactive agents (eg, does not include a hydrophobic active ingredient). Embodiment 66. The fluid of any one of embodiments 49 to 51, wherein said fluid does not contain other immunomodulatory drugs, immunosuppressants or antibiotics. Embodiment 67 The fluid of any one of embodiments 49 to 51, wherein the fluid further comprises a bioactive agent (eg, a hydrophobic active ingredient). Embodiment 68. The fluid of embodiment 67, wherein the bioactive agent is irritating or damaging to the eye. Embodiment 69. The fluid of any one of embodiments 49 to 51, wherein said fluid further comprises a preservative or detergent. Embodiment 70. The fluid of embodiment 69, wherein the preservative or detergent is irritating or damaging to the eyes. Embodiment 71 The fluid of any one of embodiments 49 to 51, wherein the fluid is administered before, during and / or after administration of a composition comprising a bioactive agent (e.g., a hydrophobic active ingredient), a preservative, a detergent, or a combination of two or more thereof. Embodiment 72. The fluid of embodiment 71, wherein the bioactive agent, preservative, detergent, or combination thereof is irritating or damaging to the eye. Embodiment 73. The fluid of embodiment 69, wherein said preservative or detergent is a chemical preservative or an oxidizing preservative. Embodiment 74. The fluid of embodiment 69, wherein the preservative or detergent increases the permeability of bacterial cell membranes by disrupting their lipid structure, killing weaker membranes. Embodiment 75 The fluid of embodiment 69, wherein the preservative or detergent is damaging to corneal tissue. Embodiment 76: The fluid of embodiment 69, wherein the preservative or detergent is selected from the group consisting of quaternary ammonium preservatives (e.g., benzalkonium chloride (BAK)), chlorobutanol, disodium edetate (EDTA), polyquaternium-1 (e.g., Polyquad® preservatives), stable oxidizing agents (e.g., stable oxychloro complexes (e.g., Purite® preservatives)), ionic buffer preservatives (e.g., sofZia® preservatives), polyhexamethylene biguanide (PHMB), sodium perborate (e.g., GenAqua® preservatives), and sorbates. Embodiment 77 The fluid of any one of embodiments 49 to 51, wherein the fluid is administered directly to the cornea as drops or a wash. Embodiment 78. The fluid of embodiment 77, wherein 1 to 3 drops are administered 1 to 3 times daily. Embodiment 79 The fluid of any one of embodiments 49 to 51, wherein the inflammation is chronic keratoconjunctivitis, and 1 to 3 drops are administered 4, 5, 6, 7, 8, 9, or 10 or more times per day. Embodiment 80. The fluid of any one of embodiments 49 to 51, wherein the fluid is administered indirectly to the cornea by a delivery agent (e.g., particles coated with the fluid and / or secreting the fluid into the cornea) that is administered locally to the cornea or other part of the eye. Embodiment 81 The hyaluronic acid is >2.9 ml 3 52. The fluid of any one of embodiments 49-51, having an intrinsic viscosity of 1.0 / kg. Embodiment 82. The fluid of any one of embodiments 49 to 51, wherein said fluid further comprises a preservative. Embodiment 83. The fluid of any one of embodiments 49 to 51, wherein said fluid does not further comprise a preservative (i.e., the fluid is preservative-free). Embodiment 84 The fluid of any one of embodiments 49 to 51, wherein the fluid comprises an additional glycosaminoglycan (GAG), an electrolyte (e.g., sodium chloride), a buffer (e.g., a phosphate buffer), or a combination of two or more thereof. Embodiment 85 The fluid of any one of embodiments 49-51, wherein the subject's eye is not aqueous tear deficient (ATD) (i.e., no ATD) at the time of administration. Embodiment 86. The fluid of any one of embodiments 49-51, wherein said subject's eye does not have qualitative dry eye (i.e., no qualitative dry eye) at the time of said administration. Embodiment 87 The fluid of any one of embodiments 49 to 51, wherein the eye of the subject does not have dry eye disease (i.e., not aqueous tear deficient or qualitative dry eye type) at the time of administration. Embodiment 88. The fluid of any one of embodiments 49-51, wherein the subject does not suffer from aqueous tear deficiency (ATD), the subject has a corneal and / or conjunctival abnormality (location-specific abnormality) including a protuberance on the cornea or other surface of the eye that is not covered by a normal tear film (tear film of normal surface tension and viscosity) that results in an area of ​​friction at the cornea and the administered fluid reduces said friction. Embodiment 89. The fluid of any one of embodiments 49 to 51, wherein the subject's eye is in corneal and conjunctival homeostasis at the time of administration. Embodiment 90. The fluid of any one of embodiments 49 to 51, further comprising applying a bandage contact lens to the eye before, during and / or after said administration. Embodiment 91 The fluid of embodiment 90, wherein the fluid is administered and the bandage contact lens is applied after eye surgery. Embodiment 92. The fluid of any one of embodiments 49 to 51, wherein the administered fluid increases the visual performance of the eye. Embodiment 93. The fluid of embodiment 92, wherein the fluid is administered before, during and / or after eye surgery, and the subject recovers visual performance after the eye surgery. Embodiment 94. The fluid of any one of embodiments 49 to 51, wherein the molecular weight of the hyaluronic acid is at least 3 million Daltons. Embodiment 95. The fluid of any one of embodiments 49 to 51, wherein the molecular weight of the hyaluronic acid is 3 million to 4 million daltons.

[0071] definition In the context of the present invention (particularly in the claims), "a," "an," "the," and similar terms are to be construed as including both the singular and the plural, unless otherwise specified or clearly contradicted. For example, a "cell" or a "compound" is to be construed as including both a single cell or a single compound, and a plurality of cells and a plurality of compounds, unless otherwise specified or clearly contradicted. Similarly, "or" is to be construed as including "and," unless otherwise specified. "For example" is an abbreviation of the Latin phrase exempli gratia, and is used herein to indicate a non-limiting example. "For example" is synonymous with "for example."

[0072] The term "isolated," when used as a modifier of a composition, means that the composition is produced by human intervention or is separated from its natural occurrence in an in vivo environment. Typically, a composition isolated in this manner is substantially free of one or more materials with which it is normally associated in nature, such as, for example, one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes, etc. A "substantially pure" molecule can be associated with one or more other molecules. Thus, "substantially pure" does not exclude combinations of compositions. Substantial purity can be at least about 60% or more of the molecule by mass. Purity can also be about 70% or 80% or more, and even, for example, 90% or more. Purity can be determined by any suitable method, including, for example, UV spectroscopy, chromatography (e.g., HPLS, gas phase), gel electrophoresis (e.g., silver or Coomassie staining), and sequence analysis (nucleic acids and peptides).

[0073] As used herein, the term "homeostasis" refers to the ability of a physiological system to maintain internal stability, or the state of stability itself, by the coordinated response of its parts to any situation or stimuli that tend to disturb normal, non-pathological conditions or function.

[0074] The term "hyaluronic acid" (HA) as used herein refers to a glycosaminoglycan composed of repeating disaccharides of N-acetylglucosamine and glucuronic acid found in nature, known as hyaluronan (e.g., a linear glycosaminoglycan composed of repeating units of the disaccharide [-D-glucuronic acid-b1,3-N-acetyl-D-glucosamine-b1,4-]n), as well as derivatives of hyaluronan with chemical modifications such as esters of hyaluronan, amide derivatives, alkyl-amine derivatives, low and high molecular weight forms of hyaluronan, crosslinked forms such as hyalan. Thus, the disaccharide chains may be linear or non-linear. Hyaluronan can be crosslinked by adding crosslinkers such as thiols, methacrylates, hexadecylamides, and tyramine. Hyaluronan can also be crosslinked directly with formaldehyde and divinyl sulfone.Hylan includes, but is not limited to, Hylan A, Hylan A (formaldehyde cross-linked glycosaminoglycan polymer), Hylan B (divinylsulfone cross-linked glycosaminoglycan polymer) and Hylan G-F20 (Cowman MK et al., Carbohydrate Polymers 2000,41:229-235; Takigami S et al., Carbohydrate Polymers, 1993,22:153-160; Balazs EA et al., "Hyaluronan, its cross-linked derivative-Hylan-and their medical applications", in Cellulosics Utilization: Research and Rewards in Cellulosics, Proceedings of Nisshinbo International Conference on Cellulosics Utilization in the Near Future (Eds Inagaki, H and Phillips GO), Elsevier Applied Science (1989), NY, pp.233-241; Koehler L et al., Scientific Reports, 2017, 7, article no. 1210; and Pavan M et al., Carbohydr Polym, 2013, 97(2):321-326, the contents of which are incorporated herein by reference.

[0075] The term "hyaluronic acid" or HA includes HA itself and its pharma- ceutically acceptable salts. HA can be formulated into a pharma- ceutically acceptable salt form. Pharmaceutically acceptable salts of HA can be prepared using conventional techniques.

[0076] The term "high molecular weight" or "HMW" in the context of the hyaluronic acid of the present invention refers to a hyaluronic acid having an intrinsic viscosity of >2.5 m, as determined by the method of the European Pharmacopoeia 9.0, "Sodium Hyaluronate", page 3584, the contents of which are incorporated herein by reference. 3 / kg of hyaluronic acid. Simply put, the intrinsic viscosity [η] is expressed by the Martin formula Log 10 (nr-1 / c)=log 10 [η] + κ[η]c is calculated by linear least squares regression analysis. In one embodiment, the intrinsic viscosity of the high molecular weight hyaluronic acid is 2.9 m 3 / kg.

[0077] The term "immunocompromised" as used herein refers to a subject who is congenitally, acquiredly or induced unable to mount a normal immune response. Thus, an immunocompromised subject has a weakened or damaged immune system compared to a normal subject. A subject with a weakened or damaged immune system is "immunocompromised" or "immunocompromised" which is associated with a primary or secondary nutritional deficiency, induced or not, in one or more components of the normal immune defense system. Immunocompromised states are particularly relevant in cancer treatment or the treatment or prevention of transplant rejection, caused by medical treatment, e.g., radiation therapy, chemotherapy or other immunosuppressive treatments, e.g., steroids, cyclophosphamide, azathioprine, methotrexate, cyclosporine or rapamycin. Whether a subject is immunocompromised can be diagnosed by suitable techniques known to those skilled in the art. A strong indicator that immunocompromised is present is when rare diseases occur or when a subject becomes ill from an organism that does not normally cause disease, especially if the subject is repeatedly infected. Other possibilities are typically recent infections such as HIV, hepatitis, tuberculosis, etc. However, a definitive diagnosis is usually based on clinical testing to determine the exact nature of the immunocompromise. Most tests are performed on a blood sample. Blood contains antibodies, lymphocytes, phagocytes, and complement components, all of which are major immune components that cause immune deficiencies. A blood cell count is used to determine if there are fewer phagocytes or lymphocytes than normal. A lower than normal number of either of these two cell types is associated with immunocompromise. Blood cells are also typically checked for their appearance. A subject may have normal cell counts, but the structure of the cells may be defective. If lymphocyte cell counts are low, further testing is usually performed to determine if there are fewer lymphocytes of a particular type than normal. Lymphocyte proliferation tests may be performed to determine if lymphocytes respond to stimuli. Failure to respond to stimuli is associated with immunocompromise. Antibody and complement levels can be determined to diagnose the presence of immunocompromise. As used herein, the term "corneal conjunctiva" refers to the structures of the eye and its appendages, including the cornea, conjunctiva, eyelids, eyelashes, tear film, major and accessory lacrimal glands, and meibomian glands. Thus, tears are included in the term "corneal conjunctiva" both in terms of the individual components of the production site and as a membrane of the corneal conjunctiva (see Craig JP et al., "TFOS DEWS II Definition and Classification Report", The Ocular Surface, 2017, 15:276-283, the contents of which are incorporated herein by reference). Fluids may be topically administered to one or more portions of the corneal conjunctiva, including, for example, the entire corneal conjunctiva.

[0078] "Pharmaceutically acceptable salts" include both acid and base addition salts. Pharmaceutically acceptable salts of HA or any of the other compounds described herein are intended to include any pharma- ceutically suitable salt form. Preferred pharma-ceutically acceptable salts described herein are pharma-ceutically acceptable acid addition salts and pharma-ceutically acceptable base addition salts.

[0079] "Pharmaceutically acceptable acid addition salts" refers to salts which retain the biological effectiveness and properties of the free base, are free of biological or other undesirable properties, and are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, etc. Also included are salts formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanediacids, aromatic acids, aliphatic and aromatic sulfones, 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, salicylic acid, etc. Exemplary salts include sulfate, pyrosulfate, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate, suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, maleate, tartrate, methanesulfonate, and the like. Also contemplated are the salts of amino acids, such as arginates, gluconates, and galacturonates (see, e.g., Berge SM et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66:1-19 (1997), which are incorporated herein by reference). Acid addition salts of base compounds are prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt according to methods and techniques known to those skilled in the art.

[0080] "Pharmaceutically acceptable base addition salts" refers to salts which retain the biological effectiveness and properties of the free acid, without any biological or other undesirable properties. These salts are prepared by adding an inorganic or organic base to the free acid. Pharmaceutically acceptable base addition salts are formed with metals or amines, such as alkali and alkaline earth metals and organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, manganese, iron, zinc, copper, manganous, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, primary, secondary and tertiary amines, naturally occurring substituted amines, cyclic amines and substituted amines including 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, hydrabamine, choline, betaine, ethylenediamine, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purine, piperazine, piperidine, N-ethylpiperidine, proamine resins, etc. See Berge et al., supra. In some embodiments, the pharmaceutically acceptable salt is a sodium salt (see European Pharmacopoeia 9.0 "Sodium Hyaluronate" p. 3583, the contents of which are incorporated herein by reference).

[0081] As used herein, the terms "subject," "patient," and "individual" refer to a human or non-human animal. A subject can also refer to, for example, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, 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 subject methods are practiced in medical and veterinary settings. A non-human animal subject can be, for example, a pet or an animal model of an ocular or non-ocular disease.

[0082] In certain embodiments, the subject's eye is not aqueous tear deficient (ATD) at the time of administration (ie, no ATD).

[0083] In certain embodiments, the subject's eye does not have qualitative dry eye (ie, is free of qualitative dry eye) at the time of administration.

[0084] In certain embodiments, the subject's eye does not have dry eye disease (ie, neither aqueous tear deficient nor qualitative dry eye) at the time of administration.

[0085] In one embodiment, the subject does not suffer from aqueous tear deficiency (ATD), the subject has a corneal and conjunctival abnormality (location-specific abnormality) that includes a protuberance on the cornea or other surface of the eye that is not covered by a normal tear film (tear film of normal surface tension and viscosity) that results in an area of ​​friction with the cornea and the administered fluid reduces friction.

[0086] In certain embodiments, the subject is immunocompromised, ie, in an immunocompromised state.

[0087] The phrase "topical administration" is used in its conventional sense to mean localized delivery to a desired anatomical site, such as the cornea. The fluid containing high molecular weight hyaluronic acid is applied directly or indirectly to the cornea by a method that allows the cornea to contact an effective amount of the fluid. For example, the fluid can be applied directly to the cornea by eye drops or irrigation, or indirectly by a delivery agent (i.e., a fluid delivery agent) that contacts the cornea or other parts of the eye. One example of a delivery agent is a particle (e.g., microparticle or nanoparticle) that is coated with the fluid and / or releases the fluid to the cornea. Such particles are composed of a variety of materials, such as natural or synthetic polymers. In some embodiments, the delivery agent itself is administered as a droplet.

[0088] The present invention has been described by embodiments in the detailed description and drawings for illustrative purposes only, and the present invention is not limited thereto, but includes all variations, modifications, substitutions and combinations that would occur to a person skilled in the art upon consideration and / or combination of the entire contents of this specification together with his / her knowledge.

[0089] All patents, patent applications, provisional applications and publications mentioned or cited in this specification are incorporated by reference in their entirety, including any figures or diagrams, to the extent not inconsistent with the express teachings of this specification.

[0090] The following are examples illustrating procedures for carrying out the present invention. The examples are not to be construed as limiting. Percentages are by weight and proportions of solvent mixtures are by volume unless otherwise noted. EXAMPLES

[0091] Example 1 - A multi-center, multi-national prospective clinical trial in patients with severe dry eye - The HYLAN M Study A multi-center, multi-national prospective, randomized clinical trial (according to the ODISSEY I criteria) is being conducted in 12 research centers in 9 countries in patients with severe dry eye. In the HYLAN M study, patients were randomized into two groups: one group of patients remained on the most effective treatment recognized previously, and the other group was switched to a high molecular weight hyaluronic acid eye drop (Comfort Shield® preservative-free sodium hyaluronate eye drop (i.com medical GmbH, Munich, Germany)) corresponding to the embodiment in Table 1.

[0092] These patients (192) were already receiving the best treatment their ophthalmologists could provide. All patients were on "stable" treatment at the time of the study, i.e., their treatment had not changed for a defined period of time prior to entering the study. Patients were randomly divided into two groups: the first group continued their current treatment for dry eye disease, and the second group was treated with the fluid droplets described above (Comfort Shield® droplets) instead of tear substitutes.

[0093] The objectives of the study were (1) to compare the objective and subjective symptoms of dry eye treatment with Comfort Shield® drops versus the tear substitute drops that the patient had previously been treated with (=current treatment), and the patient had severe dry eye prior to presenting to the investigators, and (2) to observe the objective outcomes, the patient's subjective acceptance, and adverse events of the eye drops. For each patient, both eyes were examined, and eyes with high corneal fluorescein staining scores at baseline were evaluated.

[0094] At one of the research centers, all commercially available tear substitutes did not provide sufficient relief of signs and symptoms with autologous serum eye drops. There were 11 patients in the study of autologous serum eye drops treatment. Of the 11, 6 were randomized to the Comfort Shield® group, i.e., autologous serum eye drops were used over Comfort Shield® for 8 weeks. Of the 6 patients, 2 dropped out of the study because Comfort Shield® did not provide sufficient relief of symptoms. 2 continued on Comfort Shield® eye drops over the 8 week study, but preferred to return to their original treatment with autologous serum. The remaining 2 patients preferred Comfort Shield® over autologous serum eye drops and continued to use Comfort Shield® eye drops after the study.

[0095] The examples and embodiments described herein are illustrative only, and various modifications and changes will be suggested to those skilled in the art and are set forth in the specification and claims. Furthermore, any element or limitation of any invention or embodiment disclosed herein may be combined (individually or in any combination) with any other element or limitation of any other invention or embodiment disclosed herein, and such combinations are contemplated within the scope of the present invention, without limitation thereto.

Claims

1. Molecular weight of at least 3 million daltons, >2.5m ３ hyaluronic acid having an intrinsic viscosity of 0.10-0.19% w / v / kg and a concentration of 0.10-0.19% w / v; and water; having a pH of 6.8 to 7.6, an osmolality of 240 to 330 mosmol / kg, a NaCl concentration of 7.6 to 10.5 g / l, and a phosphate concentration of 1.0 to 1.4 mmol / l; A fluid for topical administration to the cornea and conjunctiva of the eye of a human or non-human animal subject for treating a subject following ocular surgery.

2. the fluid is a clear, colorless solution, free of visible impurities, and / or the fluid is sterile; The fluid of claim 1 .

3. The fluid is Comfort Shield®, a preservative-free sodium hyaluronate eye drop. A fluid according to claim 1 or 2.

4. The eye surgery is selected from natural or artificial corneal transplantation, corneal implants including intracorneal rings (ICRs) and artificial corneas, glaucoma surgery, cataract surgery including phacoemulsification, extracapsular surgery or intracapsular surgery, refractive surgery including radial keratotomy or refractive keratotomy, retinal surgery, strabismus surgery, corrective laser eye surgery including laser keratomileusis (LASIK) or photorefractive keratectomy (PRK), and cross-linking surgery. Caused by The fluid of claim 1 .

5. The fluid is for direct administration to the cornea as a drop or wash. The fluid of claim 1.

6. The fluid is for indirect administration to the cornea or other part of the eye by a delivery agent that is administered topically to the cornea or other part of the eye, preferably the delivery agent is a particle that is coated with the fluid and / or secretes the fluid to the cornea or other part of the eye. The fluid of claim 1 .

7. The hyaluronic acid is >2.9 m ３ / kg, The fluid of claim 1 .

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