High molecular weight hyaluronic acid for enhanced epithelial survival and body surface reconstruction

Abstract

The present invention relates to a method for restoring or supplementing hyaluronic acid on an epithelial surface of a subject comprising topically applying to the epithelial surface of the subject a composition wherein the composition comprises high molecular weight hyaluronic acid, a high molecular weight hyaluronic acid analogue, or a combination thereof, and wherein the amount or function of hyaluronic acid on the epithelial surface is insufficient.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 659,180, filed April 18, 2018, which is hereby incorporated by reference in its entirety, including any tables, nucleic acid sequences, and amino acid sequences. Technical Field

[0003] The present invention involves the use of high molecular weight hyaluronic acid to replenish the body's epithelial surfaces and compensate for the absence or dysfunction of natural polysaccharides. Background Art

[0004] Hyaluronic acid (HA) is produced on the surface of the human body by epithelial cells, etc. The presence of HA at the surface can greatly promote the survival of the organism and the functional integrity of the epidermis or other surface structures. HA is an important component of the extracellular matrix (ECM) of most vertebrate tissues (Vigetti D et al., " Hyaluronan Synthesis is Inhibited by Adenosine Monophosphate-activated Protein Kinase Through the Regulation of HAS2 Activity in Human Aortic Smooth Muscle Cells, " The Journal of Biological Chemistry, 2011, 286 (10): 7917-7924). Produced by specific synthases, mammalian cells can produce two specific HA synthases (HAS1 and HAS2), which produce high molecular weight HA (HMW-HA) in the range of millions of Daltons (Da), while the HA synthesized by other isozymes (HAS3) is low molecular weight HA with a molecular weight range of several thousand Da.

[0005] The turnover of HA is important for maintaining tissue homeostasis. Typically, within 24 hours, approximately 30% of HA is replaced by newly formed HA (Fox SB et al., "Normal Human Tissues, in Addition to Some Tumors, Express Multiple Different CD44 Isoforms, Cancer Res, 1994, 54: 4539-46). The clearance of HA can occur by endocytic uptake within tissues, particularly in lymph nodes and liver. The presence of reactive oxygen species (ROS) increases the turnover of HA (Hrabarova E et al., "Pro-oxidative Effect of Peroxynitrite Regarding Biological systems: A Special Focus on High-Molar-Mass Hyaluronan Degradation," Gen Physiol Biophys, 2011, 30: 223-38; Hrabarova E et al., "Free-radical Degradation of High-Molar-Mass Hyaluronan Induced by Ascorbate Plus Cupric Ions: Evaluation of Antioxidant Effect of Cysteine-derived Compounds, Chem Biodivers, 2012, 9: 309-17; and Soltes L et al., "Degradative Action of Reactive Oxygen Species on Hyaluronan Biomacromolecules," 2006, 7: 659–68).

[0006] The size of HA in a given tissue at a given time also depends on the specific degradative enzyme (hyaluronidase) that can produce biologically active HA oligosaccharides. It has been described that the level of HA synthesized in the plasma membrane increases, where there is enhanced growth factor activity and cytokine presence, such as during tissue regulation and wound healing and in inflammatory processes. Due to its unchanged chemical structure, the possibility of HA's immune response is greatly reduced, thereby improving its biocompatibility (Farwick M et al., "Fifty-kDa Hyaluronic Acid Upregulates Some Epidermal Genes Without Changing TNF-α Expression in Reconstituted Epidermis," Skin Pharmacol Physiol, 2011, 24: 210-217). Therefore, it is expected that no adverse reactions will occur to externally applied HA. In adult tissues, HA synthesis is stimulated by damage, inflammation and neoplastic tumors (Tammi RH et al., "Transcriptional and Post-Translational Regulation of Hyaluronan Synthesis," FEBSJ, 2011, 278 (9): 1419-28). In the synthesis carried out by HAS, there are great differences between different cell types in terms of the stimuli to which they respond. On the other hand, although HA has a simple structure, it has too many interaction properties with numerous proteins. Its ability to interact with cell receptors such as CD44 and HA-mediated cell motility receptors (RHAMM) can trigger a variety of reactions (Misra S et al., "Interactions Between Hyaluronan and its Receptors (CD44, RHAMM) Regulate the Activities of Inflammation and Cancer," Front Immunol, 2015, 6: 201). The specific effect seems to depend on the molecular size.

[0007] It is currently understood that low molecular weight HA (LMW HA) has the most well-known effects. Therefore, the dermatology industry prefers LMW HA. Similarly, the HA of ophthalmic preparations used on the surface of the eye is mainly LMW HA. It has been shown that high molecular weight HA (HMW HA) molecules are less efficient than LMW HA in penetrating the surface of the skin and eye. Therefore, to date, HMW HA has not become the main focus of topical application. If used, they are used as lubricants, for example, on the surface of the eye, thereby exerting their properties as non-Newtonian fluids. However, in adult tissues, if HA synthesis is stimulated in injury, inflammation and neoplastic tumors, the increase in HA production should directly or indirectly help to calm those mediators, such as cytokines and growth factors, which are recruited or activated due to injury, inflammation and neoplastic tumors.

[0008] In situations where predisposition or ongoing external challenges constantly stress the tissues, they will continually increase their production of HA. Over time, this can lead to situations where the tissue's regenerative capacity and HA production capacity are exceeded and / or exhausted. In these situations, the epithelium is damaged and the regulatory system for HA synthesis is thrown out of balance. In the presence of HA, the relatively constant expression of stimuli for HA expression may lead to its accumulation and produce secondary effects. One of these secondary effects may be a pro-inflammatory state, such as that found in atopic inflammation. Summary of the Invention

[0009] In healthy subjects and young people, the production of hyaluronic acid (HA) can usually cope with the needs of normal cell turnover and protection from the environment. However, in other cases, such as disease, or in elderly subjects, the amount or quality of HA produced is insufficient to maintain the integrity of human epithelial surface tissue function. The present inventors propose that the topical application of high molecular weight (HMW) HA to the epithelial surface of atopic and other subjects can play a role in replenishing or restoring HA on the epithelial surface, protecting the epithelium from the harmful effects associated with the lack or damage of HA on the epithelial surface.

[0010] The average molecular weight of HA in the extracellular matrix (ECM) of healthy epithelial cells is 3-4 MDa and is catabolized (30%-50% turnover per day, depending on the tissue type). Starting from the fifth decade of life, the amount of free, non-cell-bound HA in the extracellular space of epithelial cells decreases dramatically, but the total amount of HA does not change significantly. This is reported to be the main reason for the skin dehydration (parchy) observed in elderly patients, partly due to the loss of the skin's repair capacity and loss of water binding capacity.

[0011] The average molecular weight of HA in the skin ECM may change with age (for example, because the ability of cells to synthesize HA decreases with age, and thus the average molecular weight becomes lower). All of these effects are not directly related to the molecular weight-dependent signaling functions of HA described in the literature. The mechanism of neovascularization may be controlled by the mechanical stability of epithelial and other tissues. This could well explain why HMW HA exerts an anti-angiogenic effect by simply ensuring a stable ECM, while low molecular weight HA exerts an angiogenic effect by simply shifting the balance of HA molecular weight in the ECM to lower values, thereby reducing the mechanical stability of the tissue. The inventors propose that a stable ECM downregulates inflammation and helps prevent inflammation from entering a chronic phase; furthermore, this role of HA in the matrix may help to explain and understand controversial reports of a role for HA in tumor progression.

[0012] The present invention relates to compositions and methods for restoring HA to epithelial surfaces in human or non-human subjects. The compositions comprise high molecular weight HA, high molecular weight HA analogs, or combinations thereof, and the repair method comprises topically applying the compositions to epithelial surfaces where HA is insufficient in quantity or function.

[0013] In some embodiments, the method reduces the severity of a hypersensitivity reaction on the inside or outside epithelial surface of the health of a human or non-human animal subject or delays its outbreak. By topically administering a composition comprising the hyaluronic acid of a very high molecular weight form to a body surface, such as skin or mucous membrane, the present invention can weaken or delay the generation of a hypersensitivity reaction on an epithelial surface. Can be topically administered to an epithelial surface before, during, and / or after a hypersensitivity reaction on an epithelial surface occurs.

[0014] Without being bound by theory of mechanism of action, it is proposed that when topically applied to an epithelial surface, the composition may interfere with the binding of proinflammatory cytokines to receptors on the epithelial surface, thereby improving cellular stability and integrity. DETAILED DESCRIPTION

[0015] Hyaluronic acid (HA) is a carbohydrate, particularly a mucopolysaccharide, that can be found in living organisms. The biological functions of endogenous HA include maintaining the elastic viscosity of liquid connective tissues (e.g., synovial fluid and vitreous humor) (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 well known that HA can regulate a variety of biological effects, which can vary with 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).

[0016] Sodium hyaluronate and other viscoelastic agents have been used in intraocular surgery since the 1970s and in the treatment of dry eyes since the 1980s (Higashide T and KSugiyama, “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, the biological function of hyaluronate in epithelial cells has received little attention to date (Müller-Lierheim WGK, “ Neuesüber ” Aktuelle Kontaktologie, April 2015, 17-19).

[0017] The HA used in the compositions and methods of the present invention is high molecular weight (HMW). In some embodiments, the HMWHA has a molecular weight greater than 2.5 m 3 / kg of intrinsic viscosity. In addition, in the case where the epithelial surface is the epithelium of the ocular surface, the concentration of HMW HA and / or HMW HA analogs is preferably <0.2% w / v. In other cases, for example, where the epithelial surface is the epithelium of the skin, the concentration of HMW HA and / or HMW HA analogs is preferably in the range of 0.2% to 3.0% w / v. The intrinsic viscosity can be determined by the European Pharmacopoeia, 9.0 edition, "Sodium Hyaluronate", page 3584 (incorporated herein by reference in its entirety). 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, hyaluronic acid has a molecular weight greater than 2.9 m 3 / kg intrinsic viscosity.

[0018] In some embodiments, the hyaluronic acid concentration for use in the compositions of the present invention is 0.1 to 0.19% w / v.

[0019] In some embodiments, the composition for use in the present invention has: a) a pH of 6.8-7.6; b) an osmolarity of 240-330 mosmol / kg; c) a NaCl concentration of 7.6-10.5 g / l; and / or d) a phosphate concentration of 1.0-1.4 mmol / l.

[0020] In some embodiments, the compositions used in the present invention are clear, colorless solutions free of visible impurities. It is contemplated that the compositions are sterile.

[0021] In some embodiments, the compositions used in the methods and kits of the present invention are Preservative-free sodium hyaluronate eye drops.

[0022] In some embodiments, HA has a molecular weight of at least 3 million Daltons as calculated by the Mark-Houwink equation. In some embodiments, HA has a molecular weight in the range of 3 to 4 million Daltons as calculated by the Mark-Houwink equation.

[0023] In some embodiments, the HMW HA is hyaluronic acid. In some embodiments, the HMW HA is cross-linked, such as hylan A and hylan B. In some embodiments, the HMW HA is non-cross-linked. In some embodiments, the HMW HA is linear. In some embodiments, the HMW HA is non-linear. In some embodiments, the HMW HA is a derivative of hyaluronic acid, such as an ester derivative, an amide derivative, or a sulfated derivative, or a combination of two or more thereof.

[0024] The composition can be in liquid, solid or semi-solid form. In some embodiments, the composition is a liquid (e.g., a fluid, a spray, a lotion, an aerosol). In some embodiments, the composition is a solid (e.g., a tablet, a capsule, a granule, a powder, a sachet, a dry powder inhaler, a chewable tablet). In some embodiments, the composition is a semi-solid (e.g., a cream, an ointment, a gel, a jelly, a paste, a salve, a balm, a mousse, a foam, a transdermal patch, a suppository).

[0025] In some embodiments, the epithelial surface to which the composition is topically applied is deficient in the amount of HA or the function of HA at the time the composition is topically applied. In other embodiments, the epithelial surface to which the composition is topically applied is not deficient in the amount of HA or the function of HA at the time the composition is topically applied.

[0026] In some embodiments, the epithelial surface to which the composition is topically applied is the surface of the skin. In some embodiments, the epithelial surface to which the composition is topically applied is the epithelial surface of a mucosa, such as the mucosa of the eye, ear, gastrointestinal tract (e.g., mouth, esophagus, stomach, small intestine, large intestine, colon, cecum, rectum, or anus), respiratory tract (e.g., nose, larynx, trachea, bronchial tree, alveoli), or genitourinary tract (e.g., bladder, ureters, urethra, kidneys, vas deferens, vulva, vagina, cervix, uterus, fallopian tubes).

[0027] In some embodiments, the epithelial surface to which the composition is topically applied is an ocular surface (eg, the conjunctiva). In some embodiments, the epithelial surface to which the composition is topically applied is a non-ocular surface.

[0028] Regardless of whether the HA or HA function of the ocular surface is reduced when the composition is applied to the eye, the applied composition can increase or enhance the visual function of the eye to which it is applied. The applied composition helps stabilize the fluid film of the eye (e.g., the tear film on the ocular surface), optimizing vision and visual function, which is particularly beneficial in extreme conditions such as aviation and other environments where blinking is impossible or undesirable, space travel, diving in contaminated or poisoned liquids, or working in extreme climates such as cold, hot, and dry.

[0029] An increase or enhancement of visual function can be defined as an increase or enhancement in the speed and / or accuracy of processing visual information. For example, visual function can be described as how quickly and accurately a person can process visual stimuli defined based on criteria such as adaptation brightness, target contrast, and target size. Methods for assessing visual function and changes in visual function are known in the art (see, for example, Toda I et al., "Visual performance after reduce dblinking 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 entire contents of which are incorporated herein by reference).

[0030] The composition can be applied to the ocular surface of one or both eyes of the experimenter in a fluid form by any topical application method. For example, the fluid can be applied in the form of one or more drops from a device (such as a dropper) for distributing eye drops. The fluid can be applied by itself or by a third party. The dosage applied to the ocular surface with a single dose or multiple doses will vary according to various factors, including the patient's condition and characteristics, the degree of the symptoms, the treatment carried out simultaneously, the frequency of treatment and the desired effect. For example, one or more drops (each drop is about, for example, 30 microliters) can be applied. Typically, 1 to 3 times a day is administered, and each 1-3 drops is enough, particularly for acute ocular surface inflammation. However, in the case of chronic ocular surface inflammation, more frequent administration may be required, especially in the initial stages of treatment, for example, each 1-3 drops, 4, 5, 6, 7, 8, 9, 10 or more times a day.

[0031] Advantageously, in some embodiments, the frequency of administration of the composition and / or the amount per dose can be reduced over time as HA on the epithelial surface is preserved or restored. For example, in some cases, after four weeks, the amount administered can be reduced and / or the frequency of administration per day can be reduced, or the frequency of administration can be reduced to half a day.

[0032] The composition can be administered prophylactically before the condition exists, for example, before a hypersensitivity reaction occurs, to reduce the severity of the condition and / or delay its onset; or after the condition exists, such as a hypersensitivity reaction, the composition can be administered therapeutically to alleviate the severity of the condition. Optionally, the composition is administered prophylactically before the event or stimulus causing the condition occurs, the event or stimulus causing the symptom, such as trauma (e.g., non-surgical trauma), surgery, infection (e.g., exposure to bacteria, viruses, fungi, protozoa (e.g., amoeba)), or exposure to an antigen that can cause a hypersensitivity reaction in the subject. In some embodiments, the onset of the condition is delayed indefinitely (i.e., prevented). In some embodiments, one or more symptoms of the condition are alleviated or eliminated. In some embodiments, all symptoms of the condition are alleviated or eliminated.

[0033] The composition can also be administered prophylactically to a subject who is particularly susceptible or prone to infection. The subject's immunocompromised condition may have one or more causes, such as medical treatment (e.g., radiotherapy, chemotherapy, or other immunosuppressive therapy), environmental exposure (e.g., radiation exposure), or genetic defects.

[0034] Where the condition is present at the time the composition is administered and the composition is administered therapeutically, the method optionally further comprises the step of determining that the subject has the condition prior to administering the fluid.

[0035] In some embodiments, the disorder can be characterized by one or more of the following: invasion of leukocytes at the ocular surface and tears, upregulation of CD44 at the ocular surface, and activation of an immune cascade including 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.

[0036] The condition may be caused by various stimuli (external, internal, or both). In some embodiments, the condition is caused by an external stimulus that disrupts the smoothness and / or integrity of the epithelium of the ocular surface (e.g., medical treatment, ophthalmic surgery, non-surgical trauma, contact lens wear, microbial infection, allergens, haptens, toxic substances, or irritants).

[0037] Various medical treatments, such as small molecule drugs, radiation (such as ultraviolet light and radiotherapy), and biologics, can cause this condition. For example, the condition can be 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 hypertension, stable and unstable angina, arrhythmias, migraines, esophageal variceal bleeding, heart failure, and coronary artery disease, among other conditions. Some beta-blockers antagonize a specific subtype of beta-adrenergic receptors (e.g., beta-1 selective beta-blockers that selectively antagonize beta-1 adrenergic receptors), while other beta-blockers are non-selective. Some beta-blockers can inhibit the action of ligands such as noradrenaline or norepinephrine on one or more beta-adrenergic receptors. Thus, the term "beta-blocker" refers to all types of antagonists or inhibitors of beta-adrenergic receptors, regardless of whether the beta-blockers antagonize one, two, or more beta-adrenergic receptors, and whether they affect other processes. Examples of beta-blockers include, but are not limited to, acebutolol, alprenolol, atenolol, betaxolol, bisoprolol, bopindolol, bucindolol, butaxamine, carteolol, carvedilol, celiprolol, esmolol, labetalol, levobunolol, mexadolol, metipranolol, metoprolol, nadolol, nebivolol, nadolol, oxprenolol, penbutolol, pindolol, propafenone, propranolol, sotalol, timolol, and eucommia bark.

[0038] In some embodiments, the disease is an eye allergy. In some embodiments, the disease is a non-infectious keratoconjunctivitis caused by external damage, an allergic keratoconjunctivitis (e.g., seasonal allergic keratoconjunctivitis) or an infectious keratoconjunctivitis such as viral keratoconjunctivitis, bacterial conjunctivitis, fungal keratoconjunctivitis, parasitic conjunctivitis. In some embodiments, the disease is caused by internal stimulation (e.g., hormone disorders (e.g., menopause and andropause), rheumatic diseases, epithelial-mesenchymal transition (EMT) or autoimmune diseases).

[0039] The disease may be caused by the wound in the epithelium of the eye. In some embodiments, the wound is caused by physical wound, chemical wound, such as excimer laser treatment or radiation (radiation damage). In some embodiments, the wound is caused by eye surgery. The example of ophthalmic surgery includes but is not limited to natural or artificial cornea transplantation, corneal implantation (such as intracorneal ring (ICR) and artificial cornea), glaucoma surgery, cataract surgery (such as phacoemulsification, cataract extracapsular surgery or intracapsular surgery), refractive surgery (such as, radial keratotomy or refractive corneal incision), retinal surgery, strabismus (eye deviation) surgery, corrective laser eye surgery (such as, laser-assisted in situ keratomileusis (LASIK) or laser optical keratectomy (PRK)) and cross-linking surgery. Before, during and / or after eye surgery such as glaucoma surgery, applying the liquid of the present invention can improve clinical results, such as by accelerating recovery, including recovery of visual function after surgery, reducing scars and alleviating itching, irritation, pain and other discomforts.

[0040] Liquid can be applied to alleviate or prevent or delay the onset of ocular discomfort such as itching or eye pain. The pain may have one or more causes. For example, eye pain may be pain associated with mechanical, chemical or thermal stimulation of the ocular surface. Eye pain may be associated with acute or chronic inflammation or an immune response. As the pain is alleviated, there is a reduction in secondary neuroinflammatory effect (Belmonte C et al., "TFOSDEWS II pain and sensation report", The Ocular Surface, 15: 404-437). The cause of the pain may be known or unknown.

[0041] Atopy

[0042] In some embodiments, the subject to whom the HMW HA composition is topically administered has atopy. Normally, the immune system, including antibodies, protects the body from foreign substances called antigens. However, in susceptible individuals, the immune system can overreact when exposed to certain substances (allergens).

[0043] Allergy is the result of a hypersensitivity reaction that can be immediate or delayed. Classic immunoglobulin E (IgE)-dependent reactions are involved, such as asthma, conjunctivitis, allergic rhinitis, atopic eczema, allergic urticaria, and even anaphylaxis. The tendency of an individual to produce IgE antibodies in response to various antigens in the individual's environment leads to the establishment and enhancement of susceptibility, which develops into an immediate hypersensitivity reaction called atopy. Atopy is different from allergy. Both are associated with inflammation, but atopy does not follow the inflammatory cascade and does involve pro-inflammatory mediators. There is indeed a link with allergy; however, atopy is a tendency to exhibit an excessive immune response (for example, in the form of a hypersensitivity reaction), while allergy is a direct response to an allergen. According to Brown MA and JM Hanifin, "Atopic Dermatitis", Current Opinion in Immunology, 2(4): 531-534, interleukin 4 (IL-4) may be particularly important in human IgE synthesis and may play an important role in controlling mast cell and IgE production in atopic dermatitis. Allergies (including atopy) and other hypersensitivity diseases are inappropriate or exaggerated immune responses to foreign antigens. Inappropriate immune responses include those that are misdirected against inherent body components, resulting in autoimmune diseases.

[0044] The skin, as the interface between the organism and the external environment, plays a vital role in protecting and supporting the life it surrounds. Importantly, in the case of atopy, there is a related dysfunction of the epidermal barrier (Brown S and NJ Reynolds, "Atopic and non-atopic eczema", BMJ, 2006, 332: 584). On the skin, atopy can be seen as atopic eczema, which is an itchy inflammatory skin disorder. Atopy is often associated with loose junctions between epithelial cells, which makes the body surface more susceptible to damage from the environment. Without being limited by the theory of mechanism of action, topical application of a composition comprising HMW HA can help to enhance the ECM, thereby helping to protect the body surface of atopic patients.

[0045] Hypersensitivity reactions are divided into four types according to the Gell and Coombs classification. Hypersensitivity diseases usually involve more than one type. Type I reactions (immediate hypersensitivity reactions) are IgE-mediated. Antigen binds to IgE bound to tissue mast cells and blood basophils, triggering the release of preformed mediators (e.g., histamine, proteases, chemokines) and the synthesis of other mediators (e.g., prostaglandins, leukotrienes, platelet-activating factor, cytokines). These mediators cause vasodilation, increased capillary permeability, excessive mucus secretion, smooth muscle spasm, and tissue infiltration of eosinophils, type 2 helper T (TH2) cells, and other inflammatory cells. Type I reactions usually occur within less than an hour after exposure to the antigen. Type I hypersensitivity reactions are the basis of all atopic diseases (e.g., allergic asthma, rhinitis, conjunctivitis) and many allergic diseases (e.g., anaphylaxis, certain angioedema, urticaria, latex, and certain food allergies). The terms atopy and allergy are often used interchangeably, but as mentioned above, they are different. Atopy is an exaggerated IgE-mediated immune response; all atopic diseases are type I hypersensitivity disorders. Allergy is any exaggerated immune response to a foreign antigen, regardless of the mechanism. Therefore, all atopic diseases are considered allergic, but many allergic diseases (such as hypersensitivity pneumonitis) are not atopic. Allergic diseases are among the most common disorders in the human population.

[0046] Atopic diseases most commonly affect the nose, eyes, skin, and lungs. These disorders include conjunctivitis, extrinsic atopic dermatitis, immune-mediated urticaria, immune-mediated angioedema, acute latex allergy, certain allergic lung diseases (eg, allergic asthma, components of IgE-mediated allergic bronchopulmonary aspergillosis), allergic rhinitis, and anaphylactic reactions to sting venom.

[0047] Subjects administered the HMW HA composition may have genetic risk factors associated with atopy or allergic diseases, such as susceptibility genes or variants (Portelli MA et al., “Genetic risk factors for the development of allergic disease identified by genome-wide association”, Clinical & Experimental Allergy, 2014, 45:21-21; Tamari M et al., “Genome-wide Association Studies of Atopic Dermatitis”, Journal of Dermatology, 2014, 41:213-220; Hinds DA et al., “A Genome-Wide Association Meta-Analysis of Self-reported Allergy Identifies Shared and Allergy-Specific Susceptibility Loci”, Nat Genet, 2013, 45(8):907–911; K et al., "Meta-analysis of genome-wide association studies identifies ten loci influencing allergic sensitization". Nature Genetics, 2013, 45(8):902–906; Saunders SP et al., "Tmem79 / Matt is the mattedmouse gene and is a predisposing gene for atopic dermatitis in human subjects," JAllergy Clin Immunol,2013,132(5):1121-1129). In some embodiments, the subject has a susceptibility gene or genetic variation (e.g., polymorphism) at a locus associated with atopy, such as C11orf30, STAT6, SLC25A46, HLA-DQB1, IL1RL1 / IL18R1, TLR1 / TLR6 / TLR10, LPP, MYC / PVT1, IL2 / ADAD1, HLA-B / MICA, Tmem79 / Matt, or a combination of two or more of the foregoing.

[0048] Keratoconus

[0049] In some embodiments, the subject to whom the HMW HA composition is topically administered has keratoconus (KC). Keratoconus is a progressive eye disease in which the normally round cornea thins and begins to bulge into a cone shape. The cone deflects light rays as they enter the eye and reach the light-sensitive retina, causing distorted vision. Keratoconus can occur in one or both eyes and typically begins in a person's teenage years or 20s.

[0050] As the cornea becomes more irregular in shape, it can lead to progressive myopia and the development of irregular astigmatism, which can cause other problems such as distorted and blurred vision. Glare and light sensitivity may also occur.

[0051] Typically, people with keratoconus experience changes in their eyeglass prescription with each visit to their eye care practitioner.

[0052] On the surface of the skin, as we age, the skin becomes dry, loses elasticity, and is accompanied by itching. Itching and local irritation are one of the hallmarks of atopic diseases. On the eyes, it may cause eye rubbing. Rubbing the eyes again is considered a risk factor (Galvin V et al., "Keratoconus: An Inflammatory Disorder?", Eye, 2015, 29: 843-859), because it can lead to a disorder of the cytokine balance of the ocular surface, which may be a key risk factor for the progression of KC (Balasubramanian SA et al., "Effects of Eye Rubbing on the Levels of Protease, Protease Activity, and Cytokines in Tears: Relevance in Keratoconus," Clin Exp Optom, 2013, 96 (2): 214-218). As mentioned above, IL-4 may be particularly important in human IgE synthesis and may play an important role in controlling the production of mast cells and IgE in atopic dermatitis. Here, the use of HA can reduce the severity of KC (Kolozsvári BL et al., "Association Between Mediators in the Tear Fluid and the Severity of Keratoconus", Ophthalmic Res., 2014, 51:46-51). Currently, the pathophysiology of KC is not considered to be directly inflammatory in nature, but rather para-inflammatory, that is, related or associated with inflammation (McMonnies CW, "Inflammation and Keratoconus", Optometry and Vision Science, Feb 2015, 92(2): e35-e41), involving a specific subclinical inflammatory process (Lema I et al., "Inflammatory Molecules in the Tears of Patients with Keratoconus", Ophthalmology, 2005, 112: 654-659; Lema I et al., "Subclinical Keratoconus and Inflammatory Molecules from Tears", Br J Ophthalmol, 2009; 93: 820–824).This process involves proinflammatory mediators in the tear film, such as IL-6 and MMP-9 (Jun AS et al., "Subnormal Cytokine Profile in the Tear Fluid of Keratoconus Patients," PLoS One, 2011; 6: 1-8). In parallel with corneal KC, fibroblasts show an increased ability to bind to IL-1 (Fabre EJ et al., "Binding Sites for Human Interleukin 1 Alpha, Gamma Interferon and Tumor Necrosis Factor on Cultured Fibroblasts of Normal Cornea and Keratoconus," Curr Eye Res, 1991; 10: 585-592), suggesting a link to inflammation. In the progression of KC, initial changes in the corneal stroma may trigger the development of contour abnormalities. Increased abnormalities can trigger increased pressure on the corneal surface. Here, the epithelium plays a key role in maintaining surface cytokine homeostasis, with KC corneas increasing expression of IL-1α and IL-1β (Zhou L et al., “Expression of Wound Healing and Stress-Related Proteins in Keratoconus Corneas, Curr Eye Res, 1996, 15: 1124–1131; Bosnar D et al., “Influence of Interleukin-1ɑ and Tumor Necrosis Factor-ɑ Production on Corneal Graft Survival”, Croat Med J, 2006, 47(1): 59-66; and Pearson AR et al., “Does Ethnic Origin Influence the Incidence or Severity of Keratoconus?,” Eye (Lond), 2000; 14(Pt 4): 625-628).It has been shown that the corneal epithelium secretes IL-1 after injury or tissue damage and after cell apoptosis (Wilson SE et al., "Epithelial Injury Induces Keratocyte Apoptosis: Hypothesized Role for the Interleukin-1 System in the Modulation of Corneal Tissue Organization and Wound Healing, Exp Eye Res, 1996, 62(4): 325-327). Generally, IL-1α is upregulated not only during inflammation but also in corneal trauma (West-Mays JA et al., "Repair Phenotype in Corneal Fibroblasts is Controlled by an Interleukin-1α Autocrine Feedback Loop", Investigative Ophthalmology & Visual Science, June 1997, 38(7): 1367-1379). At the same time, fibroblasts from KC patients showed increased expression of IL-1α receptors (Bureau J et al., "Modification of Prostaglandin E2 and Collagen Another key factor may be IL-17, a proinflammatory cytokine associated with many chronic inflammatory conditions. Interestingly, Jun et al. (2011) detected elevated IL-17 levels in tear samples from patients with KC.IL-17 is associated with the pathogenesis of corneal inflammation by stimulating stromal cells to secrete various proinflammatory cytokines (Maertzdorf J et al., “IL-17 Expression in Human Herpetic Stromal Keratitis: Modulatory Effects on Chemokine Production by Corneal Fibroblasts,” J Immunol, November 15, 2002, 169(10):5897-5903), including IL-6, IL-8 and intercellular adhesion molecule 1 (ICAM-1) (Gabr MA et al., “Interleukin-17 Synergizes With IFNγ or TNFα to Promote Inflammatory Mediator Release and Intercellular Adhesion Molecule-1 (ICAM-1) Expression in Human Intervertebral Disc Cells,” J Orhop Res, 2011, 29(1):1-7). As mentioned above, despite its simple structure, HA has a large number of interaction properties with various proteins (Vigetti D et al., "Hyaluronan Synthesis is Inhibited by Adenosine Monophosphate-activated Protein Kinase Through the Regulation of HAS2 Activity in Human Aortic Smooth Muscle Cells," Journal of Biological Chemistry, 2011, 286(10):7917-7924).As described by Vignetti et al. (2011), HMW HA (>1000 kD) and CD44 induce cell migration and promote the wound healing process (Tzircotis G et al., "Chemotaxis Towards Hyaluronan is Dependent on CD44 Expression and Modulated by Cell Type Variation in CD44-Hyaluronan Binding", Journal of Cell Science, 2005, 118(21):5119-5128); in addition, the directionality of cell migration is strongly dependent on CD44 expression and the HA gradient in the extracellular matrix (ECM) environment (Acharya PS et al., Fibroblast Migration is Mediated by CD44-Dependent TGFβeta Activation," J Cell Sci, 2008, 121(Pt 9):1393-1402). Other involvements of HA include the regulation and binding of growth factors and the regulation of enzyme activity.

[0053] Since atopy is a risk factor for developing KC, the composition can be topically applied to the ocular surface of atopic patients under 30 years of age as a prophylactic method to prevent or delay the onset of KC, and in all young contact lens wearers because contact lens wear can cause changes in corneal tissue similar to atopy.

[0054] Alternatively, HMW HA can be topically applied to the ocular surface before, during, or after one or more KC treatments, such as corneal cross-linking (CXL), custom-made soft contact lenses, gas permeable contact lenses, "piggy-back" contact lenses, hybrid contact lenses, scleral and semi-scleral lenses, prosthetic contact lenses, surgically applied corneal inserts (e.g., Intacs products), topography-guided conductive keratoplasty, or corneal transplantation, or a combination of two or more of the foregoing.

[0055] Other components

[0056] Optionally, the composition further comprises one or more bioactive agents (e.g., hydrophobic active ingredients). As used herein, the term "bioactive agent" refers to any substance that acts on a human or non-human animal subject when administered in an effective amount to affect tissue. A bioactive agent can be any type of substance, such as a drug molecule or a biological agent (e.g., a polypeptide, a carbohydrate, a glycoprotein, an immunoglobulin, a nucleic acid), can be a natural product or artificially produced, and can act by any mechanism, such as pharmacology, immunology, or metabolism. Examples of bioactive agent categories include substances that change eye 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, antiplasmin, and cyclosporin A. In some embodiments, the bioactive agent is a steroid or antibiotic used to treat or prevent eye infections; a glaucoma medication, such as a prostaglandin analog, a beta blocker, an alpha agonist, or a carbonic anhydrase inhibitor; an allergy eye relief agent, such as a histamine antagonist or a nonsteroidal anti-inflammatory drug; or a mydriatic agent. Unfortunately, in some cases, one or more bioactive agents included in the composition may be irritating or damaging to the eye or epithelial surface (e.g., cyclosporin A). Advantageously, through its rheological and other properties, the high molecular weight HA in the composition can relieve and / or protect the epithelium, such as the epithelium of the eye, from the irritating and / or damaging effects of one or more bioactive agents in the composition (i.e., if administered without the high molecular weight HA, the irritation or damage of the bioactive agent to the epithelial surface will be higher).

[0057] In some embodiments, the bioactive agent included in the HMW HA composition is an immunomodulatory agent. HMW HA can make the environment of the epithelial surface more conducive to the activity of the immunomodulatory agent, thereby enhancing or promoting the effect of the immunomodulatory agent.

[0058] In some embodiments, the composition does not include a steroid, an antibiotic, or an immunomodulator. In some embodiments, the composition does not include other bioactive agents (eg, does not include a hydrophobic active ingredient).

[0059] In some cases, it may be desirable to include one or more preservatives or detergents in the composition. Typically, such preservatives and detergents are irritating or damaging to epithelia, such as ocular epithelia. Advantageously, through their rheological properties and other properties, the composition can alleviate and / or protect the epithelium from the irritating and / or damaging effects of the preservatives or detergents in the composition. Therefore, in some embodiments, the composition further comprises a preservative or detergent that is irritating or damaging to epithelia (e.g., ocular epithelium) (i.e., if administered in the absence of high molecular weight HA, the preservative or detergent would be more irritating or more damaging to the epithelium). In some embodiments, the composition does not comprise a preservative or detergent.

[0060] In some embodiments, the composition comprises cyclosporine A, cetaxel, tyloxapol, or a combination of two or more of the foregoing.

[0061] In some embodiments, the composition comprises a bioactive agent that is an immunosuppressant (e.g., a T cell inhibitor such as cyclosporin A, tacrolimus, or sirolimus; an antimetabolite; an alkylating agent; a TNF inhibitor (e.g., infliximab, etanercept, or adalimumab); a lymphocyte inhibitor; or an interleukin inhibitor); a prostaglandin (e.g., latanoprost), a prostaglandin analog, or other intraocular pressure-lowering drug; an antihistamine and / or a mast cell stabilizer (e.g., ketotifen), or a combination of two or more of the foregoing. The epithelial surface to which the composition is topically applied can be an ocular surface or a non-ocular surface. The composition can further comprise a preservative and / or a detergent, or may not comprise any preservative or detergent.

[0062] In some embodiments, the composition comprises a bioactive agent selected from the group consisting of a glucocorticoid or other steroidal anti-inflammatory agent (e.g., prednisone, cortisone acetate, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone), a nonsteroidal anti-inflammatory drug (e.g., a salicylate, an arylalkanoic acid, a 2-arylpropionic acid, an N-arylanthranilic acid, an oxicams, a coxib, or a sulfonanilide), a Cox-2 specific inhibitor ( For example, valdecoxib, celecoxib, or rofecoxib), leflunomide, gold thioglucosinolate, gold thiomalate, aurofin, sulfasalazine, hydroxychloroquine, minocycline, TNF-alpha binding proteins (such as infliximab, etanercept, or adalimumab), abatacept, anakinra, beta-interferon, gamma-interferon, interleukin-2, allergy vaccines, antihistamines, antileukotrienes, beta-agonists, theophylline or anticholinergics, antibiotics, tacrolimus, or retinoids.

[0063] In some embodiments, the composition comprises one or more of a solvent, a co-solvent, a demulcent, an emollient, a preservative, an antioxidant, a humectant, or a solubilizer.

[0064] In some embodiments, the composition is administered to a subject before, during, and / or after another composition comprising a bioactive agent is administered to the subject. In some cases, it may be desirable to include one or more preservatives or detergents in the other composition. As described above, these preservatives and detergents are often irritating or damaging to the eyes, and some bioactive agents themselves may be irritating or damaging to the eyes. Advantageously, through its rheological and other properties, the composition can mitigate and / or protect the eyes from the irritating and / or damaging effects of the bioactive agents, preservatives, and / or detergents in the other composition. Therefore, if not administered together with the composition, the bioactive agents, preservatives, and / or detergents in the other composition will be more irritating or damaging to epithelia, such as the epithelium of the eye.

[0065] In some embodiments, the other composition administered before, during, or after the HMW HA composition comprises an immunomodulatory agent. HMW HA can make the environment of the epithelial surface more conducive to the activity of the immunomodulatory agent, thereby enhancing or promoting the effect of the immunomodulatory agent.

[0066] In some embodiments, the additional composition comprises cyclosporine A, cetaxel, tyloxapol, or a combination of two or more of the foregoing.

[0067] In some embodiments, the epithelial surface is the ocular surface, and the other composition is an immunosuppressant (e.g., a T cell inhibitor, such as cyclosporin A, tacrolimus, or sirolimus; an antimetabolite; an alkylating agent; a TNF inhibitor (e.g., infliximab, etanercept, or adalimumab); a lymphocyte inhibitor; or an interleukin inhibitor), a prostaglandin (e.g., latanoprost); a prostaglandin analog or other intraocular pressure-lowering drug; an antihistamine and / or a mast cell stabilizer (e.g., ketotifen), or a combination of two or more of the foregoing. Other compositions may further comprise a preservative and / or detergent, or may be free of any preservative or detergent.

[0068] The other composition administered to the subject can be in any form and by any route (e.g., topical or systemic). In some embodiments, the other composition is administered to the eye, for example topically or by injection. In some embodiments, the other composition is administered topically to the ocular surface.

[0069] In some embodiments, the preservative or detergent included in the composition is a chemical preservative or an oxidative preservative.

[0070] In some embodiments, the preservative or detergent included in the composition is an preservative or detergent that kills susceptible microbial cells by destroying the lipid structure of the microbial cell membrane, thereby increasing the permeability of the microbial cell membrane.

[0071] In some embodiments, the preservative or detergent included in the composition is one that causes damage to corneal tissues, such as corneal epithelium, endothelium, stroma, and interfaces such as membranes, in the absence of administration of HMW HA.

[0072] In some embodiments, the preservative or detergent contained in the fluid or other composition is selected from quaternary ammonium preservatives (e.g., benzalkonium chloride (BAK) or cetab chloride), chlorobutanol, disodium ethylenediaminetetraacetic acid (EDTA), polyquaternium-1 (e.g., Polyquaternium-2), polyquaternium-3 (e.g., TM preservatives), stable oxidants (such as stable oxychlorine complexes (such as Purite TM preservatives), ionic buffer preservatives (e.g., sofZia TM preservatives), polyhexamethylene biguanide (PHMB), sodium perborate (e.g. GenAqua TM Preservatives), tylopaxol and sorbate.

[0073] The composition can be a health and beauty product, such as one selected from bath and shower products, baby and child care products, sunscreen products, shampoo, conditioner, body lotion, solid or liquid soap, hair styling products, shaving cream, aftershave products, hand and nail cream, face cream, face cleanser, cologne, mouthwash or toothpaste. In some embodiments, the composition is a cosmetic, such as foundation, mascara, eye shadow, eyeliner, peel, scrub, mask, skin firming agent, toner, body cream, sunscreen products, shower gel, eye cream, hand and cuticle cream or makeup powder.

[0074] Depending on the epithelial surface, the composition can be applied by hand or with an applicator. In some embodiments, the composition is applied using an applicator such as a dropper, cotton swab, cotton pad, wipe, wipe stick, towelette, sponge, gauze, puff, wand, adhesive or non-adhesive bandage, or contact lens.

[0075] In some embodiments, the composition is at least substantially free of mucin; or in other words, the mucin concentration is <0.3% w / v.

[0076] In some embodiments, the composition comprises a preservative. In other embodiments, the fluid does not comprise a preservative (ie, the fluid is preservative-free).

[0077] In some embodiments, the composition further comprises glycosaminoglycans (GAGs), i.e., in addition to high molecular weight HA, one or more GAGs; an electrolyte (e.g., sodium chloride); a buffer (e.g., phosphate buffer); or a combination of two or more of the foregoing.

[0078] At the time the composition is applied to the subject's eye, the subject may or may not be suffering from dry eye syndrome (aqueous-deficient dry eye or qualitative dry eye). In some embodiments of the treatment or prevention methods, the condition is irritation, discomfort, inflammation, immune response at the ocular surface, or a combination of two or more of the foregoing, and the eye of the subject to which the fluid is administered does not have aqueous-deficient dry eye (ATD) at the time the fluid is administered (i.e., in the absence of ATD). In some embodiments of the treatment or prevention methods, the condition is irritation, discomfort, inflammation, immune response at the ocular surface, or a combination of two or more of the foregoing, and the eye of the subject to which the fluid is administered does not have qualitative dry eye at the time the fluid is administered (i.e., in the absence of qualitative dry eye). In some embodiments of the treatment or prevention methods, the condition is irritation, discomfort, inflammation, immune response at the ocular surface, or a combination of two or more of the foregoing, and the eye of the subject to which the fluid is administered does not have dry eye syndrome at the time the fluid is administered (i.e., in the absence of aqueous-deficient dry eye or qualitative dry eye).

[0079] In some embodiments of the methods of treatment or prevention, the condition is irritation, discomfort, inflammation, an immune response, or a combination of two or more of the foregoing on the ocular surface, and the subject does not suffer from insufficient tear volume. However, the subject has an ocular surface abnormality (topographic anomaly) comprising a bulge on the cornea or other part of the ocular surface that is not covered by a normal tear film (tear film of normal surface tension and viscosity), resulting in an area of ​​friction on the ocular surface.

[0080] The fluid can be used in conjunction with a bandage contact lens. Thus, the method can further include placing a bandage contact lens on the eye before, during, and / or after applying the fluid. For example, the fluid can be applied before the bandage contact lens is placed on the eye, after the contact lens is placed on the eye, and / or the fluid can be placed on the bandage contact lens before applying the bandage contact lens. The use of the fluid allows the bandage contact lens to exert pressure on the surface of the eye while minimizing friction on the surface of the eye. Advantageously, the fluid and bandage contact lens can be safely used shortly after ophthalmic surgery, such as glaucoma surgery.

[0081] Another aspect of the invention is a kit comprising a HMW HA composition for practicing the methods of the invention. Optionally, the kit includes an applicator for applying the composition to a desired epithelial surface.

[0082] Optionally, the kit further comprises a container for holding the composition, such as a bottle, test tube, flask or sachet.

[0083] The applicator may be pre-treated with or contain the composition.

[0084] In some embodiments, the applicator is a cotton swab, cotton pad, wipe, wipe stick, towelette, sponge, gauze, puff, stick, brush, comb, dropper, or adhesive or non-adhesive bandage.

[0085] The container of the kit may comprise a closure selected from a pump, a sprayer or a cap.

[0086] In some embodiments, the kit further comprises instructions for applying the composition to the epithelial surface by topically applying the composition to the epithelial surface, and optionally, instructions for restoring hyaluronic acid to an epithelial surface in which the amount or function of hyaluronic acid is insufficient.

[0087] In another embodiment, the kit comprises a composition as described herein as a fluid and one or more bandage contact lenses. The bandage contact lenses can be packaged in the same container as the composition (the bandage contact lenses are in contact with the composition), or the bandage contact lenses can be packaged separately from the fluid in a separate container. Suitable containers include, for example, bottles, vials, syringes, blister packs, etc. The container can be formed from a variety of materials, such as glass or plastic.

[0088] The kit may include a delivery agent (separate or combined with a fluid) to be contacted with the ocular surface or other portion of the eye. For example, the kit may include particles (e.g., microparticles or nanoparticles) coated with a fluid and / or releasing the fluid onto the ocular surface.

[0089] Alternatively, the kit may include an applicator or device (e.g., a dropper) for dispensing eye drops that may or may not serve as a container for the fluid in the kit prior to use (e.g., opening) of the outer packaging of the kit, that is, the eye drop dispensing device may serve to hold the fluid provided in the unused (unopened) kit, or may be empty and receive the fluid after use of the kit. Optionally, the kit may include a label or package insert with printed or digital instructions for use of the kit, e.g., for practicing the methods of the invention.

[0090] The kits of the present invention may include packaging material that is compartmentalized to accommodate one or more containers, such as vials, test tubes, and the like, each container comprising one of the separate components to be used in the methods described herein. By way of example only, packaging materials used to package pharmaceuticals include 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, light-tight sealed containers, syringes, bottles, and any other packaging material suitable for the selected formulation and intended mode of administration and treatment.

[0091] The kit may include one or more additional containers, each containing one or more of various materials required from a commercial and user perspective for using the compositions described herein. Non-limiting examples of such materials include, but are not limited to, buffers, diluents, carriers, packaging, containers, vials, and / or tube labels listing the contents and / or instructions for use, and package inserts with instructions for use.

[0092] The label can be on or associated with the container. A label can be on a container when the letters, numbers, or other characters comprising the label are affixed, molded, or etched onto the container itself; a label can be associated with a container when the label is present in a receptacle or carrier that also holds the container (e.g., as a package insert). The label can be used to indicate that the contents are to be used for a specific therapeutic application. The label can also indicate instructions for use of the contents, such as the methods described herein.

[0093] In some embodiments of the kit, the fluid can be present in a package or dispenser device that can contain one or more unit dosage forms containing the compositions disclosed herein. The package can, for example, comprise metal or plastic foil, such as a blister pack. The package or dispenser device can be accompanied by instructions for administration.

[0094] Fluid preparation

[0095] As described above, the HA used in the compositions and methods of the present invention is high molecular weight (HMW). In some embodiments, the hyaluronic acid of the fluid has a molecular weight greater than 2.5 m 3 In some embodiments, the hyaluronic acid has an intrinsic viscosity greater than 2.9 m 3 / kg intrinsic viscosity.

[0096] Furthermore, in cases where the epithelial surface is the epithelium of the ocular surface, the concentration of HMW HA and / or HMW HA analogs is preferably <0.2% w / v. In other cases, for example, where the epithelial surface is the epithelium of the skin, the concentration of HMW HA and / or HMW HA analogs is preferably in the range of 0.2% to 3.0% w / v.

[0097] Viscoelasticity is defined as the property of a fluid that exhibits both viscous and elastic properties. Zero shear viscosity is determined as the steady shear plateau viscosity at the vanishing shear rate. For high-viscosity formulations, controlled stress rheometers are preferred for measurement.

[0098] Molecular weight and intrinsic viscosity [η] (m 3 The relationship between the two is given by the Mark-Houwink equation:

[0099] [η]=k·(M rm ) a

[0100] Among them, M rm is the molecular weight in MDa, and the coefficient

[0101] k=1.3327·10 -4

[0102] as well as

[0103] a=0.6691

[0104] Among them, the values ​​of k and a were found to be the most predictive.

[0105] The fluid can be produced in the following manner: sterilize the filling line (if necessary); add purified water or water for injection (WFI) to a stainless steel mixing tank; add salt while mixing; slowly add HA and mix until a homogeneous solution / fluid is obtained; if necessary, adjust the pH by adding NaOH or HCl while continuing the mixing process; transfer the solution to a sterile storage tank through a 1 μm pore size filter cartridge; and then aseptically fill the solution into sterile primary packaging (single dose or vial) by sterile filtration. In the case of single doses, this can be accomplished by a blow-fill-seal (BFS) process. For ophthalmic use, the composition is preferably sterile. For applications such as skin, the composition can be sterile, but is not required.

[0106] Preferably, the fluid is at least substantially mucin-free, or in other words, has a mucin concentration < 0.3% w / v. This means that the flow characteristics or properties are achieved or modulated by the hyaluronic acid, rather than by the mucin present in the subject's tears and primarily responsible for their flow behavior.

[0107] Preferably, if viscosity-increasing substances are added, they are added near the end, during the end, or as the last step. Mixing is performed to achieve a homogeneous mixture. Alternatively or additionally, it is preferred to first provide purified water or water for injection as a base and then optionally add electrolytes, buffers, and non-viscosity-increasing substances to the purified water or water for injection.

[0108] HA is further described in European Pharmacopoeia 9.0, page 3583 (sodium hyaluronate), which is incorporated herein by reference in its entirety.

[0109] In one embodiment, the fluids used in the methods and kits of the present invention have the characteristics listed in Table 1:

[0110] Table 1

[0111] feature illustrate Test Method Appearance Clear colorless solution, no visible impurities Ph.Eur. pH 6.8-7.6 Ph.Eur. Osmotic pressure 240-330 mosmol / kg Ph.Eur. HA concentration 0.10-0.19% w / v Ph.Eur. Sodium chloride concentration 7.6-10.5g / l Ph.Eur. Sterility sterile Ph.Eur. Phosphate concentration 1.0-1.4mmol / l Ph.Eur.

[0112] definition

[0113] The terms "a", "an", "the" and similar terms used in the context of the present invention (particularly in the context of the claims) should be interpreted as covering both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Thus, for example, reference to "a cell" or "a compound" should be interpreted to cover a single cell or a single compound as well as multiple cells and multiple compounds, unless otherwise indicated or clearly contradicted by the context. Similarly, the word "or" is intended to include "and" unless the context clearly indicates otherwise. The abbreviation "eg" is derived from the Latin exempligratia and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example".

[0114] In the case of HA at the epithelial surface, the term "deficient" refers to a lack of HA (membrane-bound HA or unbound HA) in amount or structure relative to physiologically normal conditions, or a lack of one or more biological functions of HA that are normal for the extracellular space within the skin and the tissue immediately below it in the anatomical location. Methods for determining HA content and size in vivo are known (Cowman MK, "Hyaluronan and Hyaluronan Fragments", Adv Carbohydr Chem Biochem., 2017, 74: 1-59, in particular Chapter 6: Experimental Determination of HA Content and Size In Vivo, which is incorporated herein by reference in its entirety).

[0115] In the context of an administered composition, the phrase "effective amount" refers to the amount of the composition required to achieve a desired result, such as restoration (in whole or in part) of one or more biological functions of HA or at a desired anatomical site. In some embodiments, an effective amount can be an amount that can prevent a disease or condition, delay the onset of a disease or condition, treat or ameliorate a disease or condition, or otherwise produce a desired therapeutic effect.

[0116] The term "epithelial surface" refers to a body surface composed primarily of epithelial cells. Epithelium is a layer of cells that covers most of the body's surface. Epithelium is a group of tissues that perform a variety of functions and uses different cell arrangements and structures to achieve these functions. Epithelium can be any type of epithelium. It can be cornified (keratinized), such as the skin, or non-keratinized, such as the mouth, esophagus, and vagina. For example, the epithelium can be simple squamous epithelium (e.g., the lining of the alveoli of the lungs and the heart, blood vessels, and lymphatic vessels), simple cuboidal epithelium (e.g., in the ducts and secretory parts of small glands and in the renal tubules), simple columnar epithelium (e.g., ciliated tissue in the bronchi, fallopian tubes, and uterus; smooth (non-ciliated) tissue in the digestive tract and bladder), pseudostratified columnar epithelium (e.g., ciliated tissue lining the trachea and most of the upper respiratory tract), stratified squamous epithelium (e.g., the lining of the esophagus, mouth, and vagina), stratified cuboidal epithelium (e.g., sweat glands, salivary glands, mammary glands), stratified columnar epithelium (e.g., the male urethra and certain glandular ducts), transitional epithelium (e.g., the lining of the bladder, urethra, and ureters), or any combination of two or more of the foregoing.

[0117] Epithelial cells are characterized by the presence of tight junctions, adherens junctions, and desmosomes. Epithelial cells are typically polarized, with the apical surface facing the lumen or external environment and the basal surface facing the basement membrane. The composition can be applied topically to the apical surface or, if the basal surface is exposed, to the basal surface. The epithelial surface can be intact or continuous or discontinuous, for example, due to trauma.

[0118] The phrase "biological function of hyaluronan" refers to one or more functions of biological HA in the tissue in question, such as epithelium. For example, depending on the anatomical site of the epithelium, the functions of HA may include one or more of the following: hydration, water binding of HA in the extracellular matrix (enabling the transport of nutrients, catabolites and gases, thereby nourishing the tissue), lubrication, space filling ability, a framework through which cells migrate, tissue viscosity, shock absorption, free radical scavenging, cytokine interaction, and regulation of one of inflammation, cell migration, proliferation and differentiation through HA receptors (e.g., see Dicker KT et al., "Hyaluronan: A Simple Polysaccharide with Diverse Biological Functions", Acta Biomater, 2014, 10(4): 1558–1570).

[0119] The term "isolated" when used as a modifier for a composition means that the composition is made by human intervention or separated from its naturally occurring in vivo environment. Typically, the composition so separated is substantially free of one or more materials with which they are normally associated in nature, such as one or more proteins, nucleic acids, lipids, carbohydrates, cell membranes. A "substantially pure" molecule can be bound to one or more other molecules. Thus, the term "substantially pure" does not exclude combinations of compositions. Basic purity can be at least about 60% or more of the molecular mass. Purity can also be about 70% or 80% or more, and can be higher, such as 90% or more. Purity can be determined by any appropriate method, including, for example, UV spectroscopy, chromatography (e.g., HPLC, gas phase), gel electrophoresis (e.g., silver or Coomassie staining), and sequence analysis (for nucleic acids and peptides).

[0120] As used herein, the term "homeostasis" refers to the ability of a physiological system to maintain internal stability or its own steady state due to the coordinated response of its parts to any situation or stimulus that might disturb its normal state, non-pathological disease or function.

[0121] As used herein, the term "hyaluronic acid" or "HA" refers to a glycosaminoglycan composed of repeating sequences of disaccharides of N-acetylglucosamine and glucuronic acid found in nature, also known as hyaluronic acid (e.g., a linear glycosaminoglycan polymer composed of repeating units of the disaccharide [-D-glucuronic acid-b1,3-N-acetyl-D-glucosamine-b1,4-]n), and hyaluronic acid derivatives with chemical modifications, such as esters of hyaluronic acid, amide derivatives, alkyl-amine derivatives, low and high molecular weight forms of hyaluronic acid, and cross-linked forms such as hylans. Thus, the disaccharide chains can be linear or nonlinear. Hyaluronic acid can be cross-linked by connecting cross-linking agents such as thiols, methacrylates, hexadecylamide, and tyramine. Hyaluronic acid can also be directly cross-linked with formaldehyde and divinyl sulfone. Examples of hyaluronan include, but are not limited to, hyaluronan A, hyaluronan A (a glycosaminoglycan polymer cross-linked with formaldehyde), hyaluronan B (a glycosaminoglycan polymer cross-linked with divinyl sulfone), and hyaluronan GF 20 (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 et al., Scientific Reports, 2017, 7, article no. 1210; and Pavan M et al., CarbohydrPolym, 2013, 97(2): 321-326; the entire contents of which are incorporated herein by reference).

[0122] The term "hyaluronic acid" or HA includes HA itself and its pharmaceutically acceptable salts. HA can be formulated into pharmaceutically acceptable salt form. Pharmaceutically acceptable salts of HA can be prepared using conventional techniques.

[0123] In the context of the present invention, the term "high molecular weight" or "HMW" refers to a HA having a molecular weight >2.5 mM as determined by the method of European Pharmacopoeia 9.0, page 3584, "Sodium Hyaluronate" (incorporated herein by reference in its entirety). 3 / kg of hyaluronic acid. Briefly, the intrinsic viscosity [η] was 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 a molecular weight greater than 2.9 m 3 / kg intrinsic viscosity.

[0124] As used herein, the term "immunocompromised" refers to a subject who suffers from a congenital, acquired, or induced inability to mount a normal immune response. Thus, an immunocompromised subject's immune system is weakened or impaired relative to that of a normal subject. A subject with a weakened or impaired immune system has an "immunodeficiency" or "immunodeficiency," which is associated with a primary or secondary defect, induced or uninduced, in one or more elements of the normal immune defense system. Immunocompromised conditions may result from medications such as radiation therapy, chemotherapy, or other immunosuppressive therapies, such as steroids, cyclophosphamide, azathioprine, methotrexate, cyclosporine, or rapamycin, particularly in connection with cancer therapy or treatment or prevention of transplant rejection. The presence of immunocompromise in a subject can be diagnosed by any suitable technique known to those skilled in the art. Strong signs of immunocompromise may be present when a rare disease occurs or when a subject becomes ill from an organism that does not normally cause disease, particularly if the subject has had recurrent infections. Other possibilities are often considered, such as recently acquired infections such as HIV, hepatitis, tuberculosis, etc. However, a definitive diagnosis is typically based on laboratory tests that 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 may contribute to immunodeficiency. A blood cell count can be used to determine if the number of phagocytes or lymphocytes is below normal. A lower-than-normal count of either of these cell types is associated with immunosuppression. The appearance of the blood cells is also often examined. Sometimes, a subject may have a normal cell count, but the cells may have structural defects. If the lymphocyte count is low, further testing is usually done to determine if any specific lymphocyte type is below normal. A lymphocyte proliferation test may be performed to determine whether the lymphocytes can respond to a stimulus. Failure to respond to a stimulus is associated with immunosuppression. Antibody and complement levels may also be determined to diagnose the presence of immunosuppression.

[0125] As used herein, the term "ocular surface" refers to the structure of the eye and its accessories, including the cornea, conjunctiva, eyelids, eyelashes, tear film, primary and secondary lacrimal glands, and meibomian glands. Therefore, whether it is a single component at the production site or a thin film formed on the ocular surface, tears are included in the term "ocular surface" (see Craig JP et al. "TFOSDEWS II Definition and Classification Report", The Ocular Surface, 2017, 15: 276-283, the entire contents of which are incorporated herein by reference). The composition can be topically applied to one or more parts of the ocular surface, including, for example, the entire ocular surface.

[0126] "Pharmaceutically acceptable salts" include acid and base addition salts. Pharmaceutically acceptable salts of HA or any other compound described herein are intended to encompass any and all pharmaceutically suitable salt forms. Preferred pharmaceutically acceptable salts described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0127] "Pharmaceutically acceptable acid addition salts" refers to those salts which retain the biological effectiveness and properties of the free bases, which are not biologically or otherwise undesirable, and which are formed with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, hydroiodic acid, hydrofluoric acid, phosphorous acid, and the like. Also included are salts formed with organic acids such as aliphatic mono- and dicarboxylic acids, phenyl-substituted alkanoic acids, hydroxyalkanoic acids, alkanedioic acids, aromatic acids, aliphatic and aromatic sulfonic acids, and the like, 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, salicylic acid, and the like. Thus, exemplary salts include sulfate, pyrosulfate, bisulfate, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, octanoate, isobutyrate, oxalate, malonate, succinate suberate, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, malate, tartrate, methanesulfonate, etc. Salts of amino acids, such as arginate, gluconate, and galacturonate, are also contemplated (see, e.g., Berge SM et al., "Pharmaceutical Salts," Journal of Pharmaceutical Science, 66: 1-19 (1997), incorporated herein by reference in its entirety). Acid addition salts of basic compounds can be prepared by contacting the free base form with a sufficient amount of the desired acid to prepare the salt according to methods and techniques with which the skilled person is familiar.

[0128] "Pharmaceutically acceptable base addition salts" refer to those salts that retain the biological effectiveness and properties of the free acids and are not biologically or otherwise undesirable. These salts are prepared by adding inorganic or organic bases to the free acids. Pharmaceutically acceptable base addition salts can be formed with metals or amines, such as alkali and alkaline earth metals or organic amines. Salts derived from inorganic bases include, but are not limited to, sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum salts, and the like. Salts derived from organic bases include, but are not limited to, salts of primary, secondary, and tertiary amines, substituted amines, including 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, hydrabamine, choline, betaine, ethylenediamine, diaminobibenzyl, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. See Berge et al., supra. In some embodiments, the pharmaceutically acceptable salt is a sodium salt (see European Pharmacopoeia 9.0, page 3583, "Sodium Hyaluronate," which is incorporated herein by reference).

[0129] As used herein, the terms "subject," "patient," and "individual" refer to humans or non-human animals. A subject also refers to, for example, a primate (e.g., a human), a cow, a sheep, a goat, a horse, a dog, a cat, a rabbit, a rat, a mouse, a fish, a 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 a fish. Thus, the method can be performed in both medical and veterinary settings. The non-human animal subject can be, for example, a pet or an animal model of an ocular or non-ocular disease. The subject can be of any age or life stage. For example, in some embodiments, the subject is an infant or a teenager. In some other embodiments, the subject is an elderly person. In some embodiments, the subject is atopic (has atopy).

[0130] In some embodiments, the subject's eye does not have aqueous deficient dry eye (ATD) at the time of the administering (ie, in the absence of ATD).

[0131] In some embodiments, the subject's eye does not have qualitative dry eye at the time of said administering (ie, in the absence of qualitative dry eye).

[0132] In some embodiments, the subject's eye is not suffering from dry eye syndrome at the time of the administering (ie, in the absence of aqueous deficient dry eye or qualitative dry eye).

[0133] In some embodiments, the subject does not have aqueous deficient dry eye (ATD), and wherein the subject has an ocular surface abnormality (topographic abnormality) comprising a bulge on the cornea or elsewhere on the ocular surface that is not covered by a normal tear film (tear film of normal surface tension and viscosity), resulting in an area of ​​friction on the ocular surface, wherein the applied fluid reduces friction.

[0134] In some embodiments, the subject is immunocompromised, ie, immunosuppressed.

[0135] The phrase "topical application" is used herein in its conventional sense to mean local delivery to a desired anatomical site, such as an epithelial surface (epithelial surface). The composition comprising HMW HA can be applied directly or indirectly to the epithelial surface by any means that allows an effective amount of the composition to contact the epithelial surface. For example, the composition can be applied directly to the epithelial surface, for example, by eye drops or lavage fluid, or indirectly by a release agent (delivery agent) that contacts the epithelial surface. An example of a release agent is a particle (e.g., micron or nanoparticle) coated with the composition and / or released onto the ocular surface by the composition. Such particles can be made of various materials, such as natural or synthetic polymers. In some embodiments, the release agent itself can be applied in the form of drops.

[0136] The present invention is described exemplarily only by the embodiments in the description and is not limited thereto, but includes all variants, modifications, substitutions and combinations that experts can obtain from the entire document of this application after considering and / or combining their professional knowledge.

[0137] All patents, patent applications, provisional applications, and publications referred to or cited herein, including all tables, are hereby incorporated by reference in their entirety to the extent they do not contradict the explicit teachings of this specification.

[0138] The following are examples illustrating the steps of implementing the present invention. These examples should not be construed as limiting. Unless otherwise indicated, all percentages are by weight and all solvent mixture ratios are by volume.

[0139] Example 1 - Multicenter, Multinational Prospective Clinical Study in Patients with Severe Dry Eye - Hylan M Study

[0140] A multicenter, multinational, prospective, randomized clinical study of patients with severe dry eye (according to the ODISSEY primary criteria) is ongoing at 12 centers in nine countries. In the HYLAN M study, patients were randomized to receive either the most effective treatment previously determined for each individual patient or a high molecular weight hyaluronic acid eye drop ( Preservative-free sodium hyaluronate eye drops (i.com medical GmbH, Munich, Germany), which correspond to the embodiments of Table 1 herein.

[0141] These patients (192 enrolled) were already receiving the best possible care their ophthalmologists could offer. All patients were on "stable" therapy at the time of study enrollment, meaning their therapy had not changed within a defined period of time prior to enrollment. The patients were randomly divided into two groups: one group continued with their current dry eye treatment, and the other group received drops of the aforementioned fluid ( Eye drops) instead of tear substitutes for treatment.

[0142] The research objectives include: (1) Comparison of the acceptance of Comparison of objective and subjective symptoms of dry eye with eye drop treatment versus tear replacement eye drops that the patient had received before receiving the investigator's treatment (= current treatment); and (2) observation of objective manifestations, subjective patient acceptance, and adverse effects of the eye drops. For each patient, both eyes were examined, and the eye with the higher corneal fluorescein staining score at baseline was evaluated.

[0143] Patients at one of the centers participating in the HYLAN M study had not achieved adequate relief of signs and symptoms with all commercially available eye drops to date, and the physicians decided to proceed with autologous serum eye drops after testing these eye drops. They enrolled 11 patients who were receiving autologous serum eye drops in the study. Of these 11 patients, 6 were randomly assigned to Group, that is, within 8 weeks Of these 6 patients, 2 discontinued the study because Eye drops did not provide adequate relief of symptoms. Two of the patients continued to use the eye drops during the eight weeks of the study. The remaining two patients preferred autologous serum eye drops over autologous serum eye drops. Eye drops and decided to use them outside the scope of the study Eye drops.

[0144] It should be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes thereof will be suggested to those skilled in the art and will be included within the spirit and scope of this application and the scope of the appended claims. In addition, any element or limitation of any invention or embodiment thereof disclosed herein may be combined with any and / or all other elements or limitations disclosed herein (alone or in any combination) or any other invention or embodiment thereof, and all such combinations may be understood to fall within the scope of the present invention, but are not limited thereto.

Claims

1. A composition comprising high molecular weight hyaluronic acid, a high molecular weight hyaluronic acid analogue or a combination thereof and use of said high molecular weight hyaluronic acid, high molecular weight hyaluronic acid analogue or a combination thereof for the manufacture of a medicament for delivering a bioactive agent to an epithelial surface, wherein said epithelial surface is the site of a hypersensitivity reaction in a subject and said composition is topically administered to said epithelial surface after the onset of said hypersensitivity reaction, and wherein said high molecular weight hyaluronic acid and / or high molecular weight hyaluronic acid analogue has a molecular weight greater than 2.5 m 3 / kg intrinsic viscosity.

2. Use according to claim 1, wherein the epithelial surface is the ocular surface.

3. The use according to claim 1, wherein the epithelial surface is a non-ocular surface.

4. The use according to claim 1, wherein the bioactive agent comprises one or more agents selected from the group consisting of immunosuppressants, prostaglandins, prostaglandin analogs, and antihistamines and / or mast cell stabilizers.

5. The use according to claim 1, wherein the bioactive agent comprises an immunomodulator, and wherein the high molecular weight hyaluronic acid makes the environment on the epithelial surface more conducive to the activity of the immunomodulator, thereby enhancing or promoting the effect of the immunomodulator.

6. Use according to claim 4 or 5, wherein the epithelial surface is the ocular surface.

7. The use according to claim 1, wherein the bioactive agent is an anti-inflammatory agent.

8. The method of claim 7, wherein the anti-inflammatory agent is selected from the group consisting of glucocorticoids, steroids, nonsteroidal anti-inflammatory drugs, Cox-2 specific inhibitors, leflunomide, gold thioglucosinate, gold thiomalate, aurofin, sulfasalazine, hydroxychloroquine, minocycline, TNF-α binding protein, abatacept, anakinra, beta-interferon, gamma-interferon, interleukin-2, allergy vaccines, antihistamines, antileukotrienes, beta-agonists, theophylline or anticholinergics, antibiotics, tacrolimus, or retinoids.

9. The method according to claim 1, wherein the high molecular weight hyaluronic acid and / or high molecular weight hyaluronic acid analog has a molecular weight of >2.9 m 3 / kg intrinsic viscosity.

10. The use according to claim 1, wherein the hyaluronic acid has a molecular weight of at least 3 million Daltons.

11. The use according to claim 1, wherein the molecular weight of the hyaluronic acid is in the range of 3 to 4 million Daltons.

12. The use according to claim 1, wherein the hypersensitivity reaction is associated with an atopic disease selected from the group consisting of conjunctivitis, intrinsic atopic dermatitis, extrinsic atopic dermatitis, immune-mediated urticaria, immune-mediated angioedema, acute latex allergy, allergic asthma, IgE-mediated allergic bronchopulmonary aspergillosis, allergic rhinitis, eosinophilic gastroenteritis (EGE) and eosinophilic enterocolitis (ECO).

13. The use according to claim 8, wherein the antihistamine is ketotifen.