High molecular weight hyaluronic acid for enhancing epithelial survival and reconstitution of body surfaces

Abstract

To provide compositions and methods for use in restoring hyaluronic acid (HA) at an epithelial surface of a human or non-human subject.SOLUTION: The invention concerns a restoration / replenishment method for restoring or replenishing hyaluronic acid at an epithelial surface of a subject, comprising topically administering a composition to the epithelial surface of the subject, wherein the composition comprises high molecular weight hyaluronic acid, high molecular weight hyaluronic acid analogue, or a combination thereof, and wherein the epithelial surface has a deficiency in the amount or function of hyaluronic acid.SELECTED DRAWING: None

Description

[Technical field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 62 / 659,180, filed April 18, 2018, which is incorporated by reference in its entirety, including any figures, tables, nucleic acid sequences, amino acid sequences, or drawings.

[0002] The present invention relates to the use of high molecular weight hyaluronic acid to replenish epithelial surfaces in the body and compensate for deficiencies or dysfunction of naturally occurring polysaccharides. [Background technology]

[0003] Hyaluronic acid (HA) is produced on the surfaces of the human body, especially by epithelial cells. The presence of HA on the surface can greatly contribute to the survival of the organism and the functional integrity of epithelial 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 Regulation of HAS2 Activity in Human Aortic Smooth Muscle Cells", The Journal of Biological Chemistry, 2011, 286(10):7917-7924). Mammalian cells, produced by specific synthases, can produce two specific HA synthases (HAS1 and HAS2) that produce high molecular weight HA (HMW-HA) in the range of millions of Da, while the other isozyme (HAS3) synthesizes HA of low molecular weight HA in the range of thousands of Da.

[0004] Hyaluronan turnover is important for maintaining tissue homeostasis, and approximately 30% of hyaluronan is typically replaced by newly formed hyaluronan within 24 hours (Fox SB et al., "Normal Human Tissues, in Addition to Some Tumors, Express Multiple Different CD44 Isoforms", Cancer Res, 1994, 54:4539-46). Hyaluronan transfer can occur by endocytosis within tissues, particularly in lymph nodes and the liver. The presence of reactive oxygen species (ROS) promotes HA turnover (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 Antioxidative 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).

[0005] The size of HA in tissues at a given time also depends on the specific degradative enzymes (hyaluronidases) that can produce bioactive HA oligosaccharides. Increased levels of HA synthesized in cell plasma membranes have been explained by enhanced growth factor activity and the presence of cytokines (e.g., during tissue regulation and wound healing, as well as during inflammation). HA has a highly reduced chance of immunological reactions due to its unchanged chemical structure, which increases 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, no side effects are expected with exogenously applied HA. In adult tissues, HA synthesis is stimulated by injury, inflammation, and tumors (Tammi RH et al., "Transcriptional and Post-Translational Regulation of Hyaluronan Synthesis", FEBS J, 2011, 278(9):1419-28). There are large differences between different cell types regarding the stimuli they respond to in their synthesis by HAS. On the other hand, HA, despite its simple structure, has a plethora of interaction properties with numerous proteins. HA can elicit several responses by being able to interact with cellular receptors such as CD44 and the receptor for HA-mediated cell motility (RHAMM) (Misra S et al., "Interactions Between Hyaluronan and Its Receptors (CD44, RHAMM) Regulate Activities of Inflammation and Cancer", Front Immunol, 2015, 6:201). The specific action seems to depend on the molecular size.

[0006] It is currently understood that low molecular weight HA (LMWHA) has the most well-known effects. Thus, LMWHA is favored in the dermatology industry. Similarly, HA in ophthalmic formulations for the ocular surface is primarily LMWHA. It has been shown that high molecular weight HA (HMWHA) molecules penetrate the skin and ocular surface less effectively than LMWHA, and therefore HMWHA has not been of major interest for topical application to date. When HMWHA is used, for example, it is used as a lubricant on the ocular surface, taking advantage of its properties as a non-Newtonian fluid. However, in adult tissues, when HA synthesis is stimulated by injury, inflammation, or tumors, increased production of HA contributes directly or indirectly to calm mediators such as cytokines and growth factors that are activated by injury, inflammation, or tumors.

[0007] When tissues are continuously stressed, either by predisposing factors or by constant external challenges, they undergo a continuous and enhanced production of HA. Over time, this can lead to a situation in which the tissue's capacity for regeneration and for HA production is limited and / or exhausted. In such a situation, the epithelium becomes damaged and the regulatory system for HA synthesis becomes unbalanced. In the presence of HA, the relatively stable expression of stimulators of HA expression can lead to their accumulation and secondary effects. One of the secondary effects may be a proinflammatory state, as seen in atopy. Summary of the Invention [Problem to be solved by the invention]

[0008] In healthy subjects and young people, the production of hyaluronic acid (HA) usually meets the demands of normal cell turnover and protection against the environment. However, in other conditions, such as disease, or in elderly subjects, the amount or quality of HA produced is not sufficient to maintain the integrity of tissue function at the epithelial surface of the body. The present inventors propose that topical application of high molecular weight (HMW) HA to the epithelial surface of subjects, such as atopic subjects, can help replenish or restore HA at the epithelial surface and protect the epithelium from the harmful effects associated with HA deficiency or HA impairment at the epithelial surface.

[0009] The average molecular weight of HA in the extracellular matrix (ECM) of healthy epithelia is 3 to 4 MDa and is subject to catabolism (30% to 50% turnover per day, depending on the tissue type). From the fifth decade onwards, the amount of non-cell-bound free HA in the extracellular space of epithelia decreases dramatically, whereas the total amount of HA does not change significantly. This has been reported to be the main reason for the patchy skin observed in elderly patients, and is due, in part, to the loss of repair capacity and water-binding capacity of the skin.

[0010] The average molecular weight of HA in the ECM of the skin may change with age (e.g. because the capacity of cells to synthesize HA decreases with age, leading to a lower average molecular weight). Not all of these effects are directly related to the molecular weight-dependent signaling functions of HA described in the literature. The mechanism of angiogenesis may be controlled by the mechanical stability of epithelial tissues, and possibly other tissues. This succinctly explains why HMWHA is antiangiogenic by simply ensuring a stable ECM, and low molecular weight HA is angiogenic by simply shifting the equilibrium of the molecular weight of HA in the ECM to lower values, thereby reducing the mechanical stability of the tissue. We propose that a stable ECM contributes to downregulating inflammation and preventing it from entering a chronic phase, and furthermore, that this role of HA in the matrix may help to interpret and understand the controversial reports on the role of HA in tumor progression. [Means for solving the problem]

[0011] The present invention relates to compositions and methods for use in restoring HA to epithelial surfaces in human or non-human subjects, the compositions comprising HMWHA, HMWHA analogs, or combinations thereof, and the methods of restoration comprising topically administering the compositions to epithelial surfaces lacking HA quantity or function.

[0012] In various embodiments, the method reduces the severity of or delays the onset of a hypersensitivity reaction on an internal or external epithelial surface of the body of a human or non-human animal subject.By topically administering a composition containing a very high molecular weight form of hyaluronic acid to a body surface, such as the skin or mucosa, the present invention can delay the onset of a hypersensitivity reaction on an epithelial surface.The composition may be topically administered to the epithelial surface before, during, and / or after the onset of a hypersensitivity reaction on the epithelial surface.

[0013] It is proposed that when the composition is administered topically to the surface of the epithelium, the composition can interfere with the binding of proinflammatory cytokines to receptors on the epithelial surface, thereby improving cellular stability and integrity, without being limited by theory of mechanism of action. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hyaluronic acid (HA) is a carbohydrate-mucopolysaccharide that can be found, specifically, in living organisms. The biological functions of endogenous HA include maintaining the elasticity of liquid connective tissues such as joint 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 diverse signaling of HA are not yet fully understood, it is known that HA can regulate pleiotropic biological effects that can 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).

[0015] Viscoelastic substances, including sodium hyaluronate, have been used in intraocular surgery since the 1970s and in dry eye treatment 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 function of hyaluronan in the epithelium (Muller-Lierheim WGK, "Tranenersatzlosungen, Neues über Hyaluronsaure", Aktuelle Kontaktologie, April 2015, 17-19).

[0016] The HA used in the compositions and methods of the present invention is high molecular weight (HMW) HA. In various embodiments, the HMWHA is 2.5mM. 3 / kg. Moreover, when the epithelial surface is an ocular epithelium, the concentration of HMWHA and / or HMWHA analogs is preferably less than 0.2% w / v. In other cases when the epithelial surface is a skin epithelium, for example, the concentration of HMWHA and / or HMWHA analogs is preferably within the range of 0.2% to 3.0% w / v. Intrinsic viscosity may be determined by the method of "Sodium Hyaluronate" at page 3584 of European Pharmacopoeia 9.0, which is incorporated herein by reference in its entirety. Briefly, the intrinsic viscosity [η] is determined according to the Martin formula: Log 10 (n r -1 / c) = log 10 [η] + κ[η]c calculated by linear least squares regression analysis. In various embodiments, the hyaluronic acid is 2.9m3 / kg.

[0017] In various embodiments, the compositions used in the invention have a hyaluronic acid concentration of 0.1 to 0.19% w / v.

[0018] In various embodiments, the compositions used in the present invention have a) a pH of 6.8 to 7.6, b) an osmolality of 240 to 330 mosmol / kg, c) a NaCl concentration of 7.6 to 10.5 g / l, and / or d) a phosphate concentration of 1.0 to 1.4 mmol / l.

[0019] In embodiments, the compositions used in the present invention are clear, colorless solutions free of visible impurities. It is envisioned that the compositions are sterile.

[0020] In embodiments, the composition used in the methods and kits of the invention is Comfort Shield® preservative-free sodium hyaluronate eye drops.

[0021] In embodiments, the HA has a molecular weight of at least 3 million tons as calculated by the Mark-Houwink equation, hi embodiments, the HA has a molecular weight in the range of 3 to 4 million tons as calculated by the Mark-Houwink equation.

[0022] In some embodiments, the HMWHA is hyaluronan. In some embodiments, the HMWHA is cross-linked, such as Hylan A and Hylan B. In some embodiments, the HMWHA is not cross-linked. In some embodiments, the HMWHA is linear. In some embodiments, the HMWHA is non-linear. In some embodiments, the HMWHA 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.

[0023] The composition may be in liquid, solid, or semi-solid form. In embodiments, the composition is a liquid (e.g., fluid, spray, lotion, aerosol). In embodiments, the composition is a solid (e.g., tablet, capsule, granule, powder, sachet, dry powder inhalant, chewable). In embodiments, the composition is a semi-solid (e.g., cream, ointment, gel, jelly, paste, salve, balm, mousse, foam, transdermal patch, suppository).

[0024] In various embodiments, the epithelial surface to which the composition is topically administered lacks an amount or functionality of HA at the time the composition is topically administered, hi other embodiments, the epithelial surface of the subject to which the composition is topically administered is not lacking an amount or functionality of HA at the time the composition is topically administered.

[0025] In embodiments, the epithelial surface to which the composition is topically administered is an epithelial surface of the skin. In embodiments, the epithelial surface to which the composition is topically administered is an epithelial surface of a mucous membrane, such as 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, ureter, urethra, kidney, vas deferens, vulva, vagina, cervix, uterus, fallopian tubes).

[0026] In embodiments, the epithelial surface to which the composition is topically administered is an ocular surface (e.g., the conjunctiva). In embodiments, the epithelial surface to which the composition is topically administered is a non-ocular surface.

[0027] The administered composition can improve the visual performance of the eye to which it is administered, regardless of whether there is a reduced amount of HA or loss of HA function at the ocular surface at the time the composition is administered to the eye. The administered composition stabilizes the fluid film of the eye (e.g., the tear film at the ocular surface) and helps optimize vision and visual performance, which is particularly beneficial in extreme situations such as aviation and other environments where blinking is impossible or undesirable, space travel, diving in polluted or toxic fluids, or operating in areas of extreme climates such as cold, heat, and dryness.

[0028] Improved visual performance can be defined as an increase in the speed and / or accuracy of processing visual information. For example, visual performance can be described as how quickly and accurately an individual can process visual stimuli defined in terms of criteria such as adaptive luminance, target contrast, and target size. Methods for assessing visual performance and changes in visual performance are known (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, which are incorporated herein by reference in their entirety).

[0029] The composition may be administered as a fluid to the ocular surface of one or both eyes of a subject by any topical administration method. For example, the fluid may be administered as one or more eye drops from a device for dispensing eye drops, such as an eye dropper. The fluid may be self-administered or administered by a third party. The dose administered to the ocular surface as a single or multiple doses varies depending on a variety of factors, including the patient's condition and characteristics, the severity of symptoms, concurrent treatments, frequency of treatments, and the desired effect. For example, one or more drops (e.g., about 30 microliters each) may be administered. Typically, one to three drops, one to three times a day, is sufficient, especially for acute ocular surface inflammatory conditions. However, in the case of chronic ocular surface inflammation, more frequent administration may be required, especially in the early stages of treatment, for example, one to three drops, four, five, six, seven, eight, nine, ten or more times a day.

[0030] In embodiments, as HA at the epithelial surface is preserved or restored, the frequency and / or dosage of the composition may be advantageously reduced over time, for example, in some cases after 4 weeks the dosage may be reduced and / or the frequency of daily administration may be reduced, or the frequency of administration may be reduced to twice daily.

[0031] The composition may be administered prophylactically before the abnormality occurs, e.g., before a hypersensitivity reaction occurs, to reduce the severity of the abnormality and / or delay its onset. Alternatively, the composition may be administered therapeutically after the abnormality occurs, such as a hypersensitivity reaction, to reduce the severity of the abnormality. Optionally, the composition is administered prophylactically before the event or stimulus that causes the abnormality occurs, such as trauma (e.g., non-surgical trauma), surgery, infection (e.g., exposure to bacteria, viruses, fungi, protozoa (e.g., amoebas), or exposure to an antigen that causes a hypersensitivity reaction in the subject. In embodiments, the onset of the abnormality is delayed (i.e., prevented) indefinitely. In embodiments, one or more symptoms of the abnormality are reduced or eliminated. In embodiments, all symptoms of the abnormality are reduced or eliminated.

[0032] The compositions may also be administered prophylactically to subjects who are particularly susceptible or susceptible to infection. The subject's immunocompromised condition may have one or more causes, such as a medical treatment (e.g., radiation therapy, chemotherapy, or other immunosuppressant treatment), an environmental exposure (e.g., radiation exposure), or a genetic defect.

[0033] If the abnormality is present at the time of administration of the composition and the composition is administered therapeutically, the method optionally further includes the step of identifying the subject as having the abnormality prior to administering the fluid.

[0034] In embodiments, the abnormality may include the following characteristics: leukocyte infiltration and tears at the ocular surface, CD44 upregulation at the ocular surface, and activity 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, MIG-TAC, CXCL10, IP-10, MIP-1β, CCL4, RANTES, and CCL5.

[0035] The abnormality may be caused by a variety of stimuli - external, internal, or both. In embodiments, the abnormality is caused by an external stimulus (e.g., medical therapy, ocular surgery, non-surgical trauma, contact lens wear, microbial infection, allergens, haptens, toxic substances, or irritants) that results in a disruption of the smoothness and / or integrity of the epithelium of the ocular surface.

[0036] Various medical therapies, such as small molecule pharmaceuticals, radiation, such as UV and radiotherapy, and biologics, can cause abnormalities. For example, abnormalities can be caused by "beta-blockers," which refers to drugs that inhibit or block the activity of one or more beta-adrenergic receptors. Beta-blockers may be used to treat hypertension, stable and unstable angina, arrhythmias, migraines, bleeding esophageal varices, heart failure, and coronary artery disease, among other indications. Some beta-blockers antagonize specific subtypes of beta-adrenergic receptors (e.g., beta-1 selective beta-blockers that selectively antagonize beta-1 adrenergic receptors), while other beta-blockers are nonselective. Some beta-blockers inhibit the effects of ligands, such as noradrenaline and 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-blocker antagonizes one or more beta-adrenergic receptors and whether it affects 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, medroxalol, metopranolol; nadolol, nebivolol, nadolol, oxprenolol, penbutolol, pindolol, propafenone, propranolol, sotalol, timolol, and Yukonia bark.

[0037] In embodiments, the disorder is an ocular allergy. In embodiments, the disorder is a non-infectious keratoconjunctivitis caused by an external injury, an allergic keratoconjunctivitis (such as seasonal allergic keratoconjunctivitis), or an infectious keratoconjunctivitis, such as viral keratoconjunctivitis, bacterial conjunctivitis, fungal keratoconjunctivitis, and parasitic conjunctivitis. In embodiments, the disorder is caused by an internal stimulus, such as a hormonal disorder (such as menopause and andropause), a rheumatic disease, an epithelial-mesenchymal transition (EMT), or an autoimmune disease.

[0038] The defect may also result from injury to the ocular epithelium. In embodiments, the injury is caused by physical trauma, chemical trauma, laser treatment such as excimer, or radiation (radiation injury). In embodiments, the injury is caused by ophthalmic surgery. Examples of ophthalmic surgery include, but are not limited to, natural or artificial corneal transplants, corneal transplants (intracornaring (ICR), artificial corneas), glaucoma surgery, cataract surgery (phacoemulsification, extracapsular cataract surgery, intracapsular surgery, etc.), refractive surgery (such as radial keratotomy or photorefractive keratotomy), retinal surgery, strabismus surgery, corrective laser eye surgery (such as excimer laser in situ keratomileusis (LASIK) or photorefractive keratectomy (PRK)), and cross-sectional surgery. Administration of the fluids of the present invention before, during, and / or after ophthalmic surgery, such as glaucoma surgery, can improve clinical outcomes, for example, by accelerating repair, including restoration of visual ability after surgery, reducing scarring, and reducing itching, irritation, pain, and other discomforts.

[0039] The fluid may be administered to reduce or inhibit or delay the onset of ocular discomfort, such as itching or eye pain. Pain may have one or more causes. For example, eye pain may be pain associated with mechanical, chemical, or thermal irritation of the ocular surface. Eye pain may be associated with acute or chronic inflammation, or an immune response. With the reduction in pain, secondary neuroinflammatory effects are also reduced (Belmonte C et al., "TFOS DEWS II pain and sensation report," The Ocular Surface, 15:404-437). The cause of pain may or may not be known.

[0040] Atopic dermatitis In various embodiments, the subject to whom the HMWHA composition is topically administered has atopy. Normally, the immune system, including antibodies, protects the body from foreign substances called antigens. However, in sensitive individuals, the immune system may overreact when exposed to certain substances (allergens).

[0041] Allergies are the result of hypersensitivity reactions, which 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. It is an individual tendency to produce IgE antibodies in response to various antigens in the individual's environment that leads to an established predisposition to develop immediate hypersensitivity, called atopy. Atopy is not identical to allergy. Although both are associated with inflammation, atopy does not follow the inflammatory cascade and is involved in proinflammatory regulators. Although the connection with allergy exists, atopy is a predisposition to exhibit an excessive immune response (e.g., in the form of a hypersensitivity reaction), whereas allergy is a direct reaction 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 a key role in regulating mast cells and IgE production in atopic dermatitis. Allergic diseases (including atopic diseases) and other hypersensitivity disorders are inappropriate or excessive immune responses to foreign antigens. Inappropriate immune responses include those that are misdirected against intrinsic body components, leading to autoimmune diseases.

[0042] As an interface between the organism and the external environment, the skin plays a vital role in protecting and supporting the life it surrounds. Importantly, in atopic conditions, there is a related dysfunction of the epithelial barrier (Brown S and NJ Reynolds, "atopic and non-atopic eczema", BMJ, 2006, 332:584). On the skin, atopy appears as atopic eczema, which is an itchy, inflammatory skin condition. Atopy is typically associated with poorly cohesive junctions between epithelial cells, which makes the body surface more vulnerable to the environment. Without being limited by theory of mechanism of action, topical administration of a composition comprising HMWHA can contribute to strengthening the ECM and thus help protect the body surface of atopic patients.

[0043] Hypersensitivity reactions are classified into four types according to the Gell and Coombs classification. Hypersensitivity disorders often involve more than one type. Type I reactions (immediate hypersensitivity) are mediated by IgE. Antigen binds to IgE bound to tissue mast cells or blood basophils, resulting in the release of preformed mediators (e.g., histamine, proteases, chemotactic factors) and the synthesis of other mediators (e.g., prostaglandins, leukotrienes, platelet-activating factor, cytokines). These mediators induce vasodilation, increased capillary permeability, mucus hypersecretion, smooth muscle spasm, and the activation of eosinophils, type 2 helper T (T H2) causes tissue infiltration by inflammatory cells, and other inflammatory cells. Type I reactions typically develop within 1 hour after exposure to an antigen. Type I hypersensitivity reactions underlie all atopic diseases (e.g., allergic asthma, rhinitis, conjunctivitis) and many allergic diseases (e.g., anaphylaxis, some angioedema, urticaria, latex and some food allergies). The terms atopy and allergy are often used interchangeably, but as noted above, they are different. Atopy is an excessive immune response mediated by IgE, and all atopic diseases are type I hypersensitivity diseases. Allergy is an excessive immune response to a foreign antigen, regardless of mechanism. Thus, all atopic diseases are considered allergic, but many allergic diseases (such as hypersensitivity pneumonitis) are not atopic. Allergic diseases are the most common diseases among people.

[0044] Atopic diseases most commonly affect the nose, eyes, skin, and lungs. These diseases include conjunctivitis, extrinsic atopic dermatitis, immune-mediated urticaria, immune-mediated angioedema, acute latex allergy, some allergic lung diseases (e.g., allergic asthma, the IgE-mediated component of allergic bronchopulmonary aspergillosis), allergic rhinitis, and allergic reactions to toxic needles.

[0045] The subject to whom the HMWHA composition is administered may have a genetic risk factor associated with atopic or allergic disease, such as a predisposing gene or mutation (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; Bonnelykke, 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 matted mouse gene and is a predisposing gene for atopic dermatitis in human subjects," J Allergy Clin Immunol, 2013, 132(5): 1121-1129).In various embodiments, the subject has a predisposing gene or genetic variation (e.g., polymorphism) in a locus associated with atopy, e.g., C11orf30, STAT6, SLC25A46, HLA-DQB1, IL1RL1 / IL18R1, TLR1 / TLR6 / TLR10, LPP, MYC / PVT1, IL2 / AD1, HLA-B / MICA, Tmem79 / Matt, or a combination of two or more of the above.

[0046] Keratoconus In embodiments, the subject to whom the HMWHA composition is topically administered has keratoconus (KC). Keratoconus is a progressive eye disease in which the normally circular cornea thins and begins to bulge into a cone shape. This cone shape deflects light as it enters the eye on its way to the light-sensitive retina, causing astigmatism. Keratoconus can affect one or both eyes and often begins in the teens or early twenties.

[0047] As the shape of the cornea becomes more irregular, it can result in progressive myopia and irregular astigmatism, creating further problems with astigmatism and blurred vision. Glare and sensitivity to light can also occur.

[0048] Keratoconus patients often experience changes in their eyeglass prescription with each visit to their eye doctor.

[0049] With age, the skin and other surfaces become dry, lose elasticity, and complain of itching. Itching and local irritation are some of the characteristics of atopy. On the eyes, atopy can cause eye rubbing. Again, eye rubbing is a risk factor (Galvin V et al., "Keratoconus: An Inflammatory Disorder?", Eye, 2015, 29:843-859) because it leads to a disturbance in the cytokine balance on the ocular surface, which may be one of the important risk factors for the development 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 is particularly important in human IgE synthesis and may have an important role in controlling mast cells and IgE production in atopic dermatitis. Here, the use of HA may reduce the severity of KC (Kolozsvari BL et al., "Association Between Mediators in the Tear Fluid and the Severity of Keratoconus", Ophthalmic Res., 2014, 51:46-51).The pathology of KC is currently not considered to be directly inflammatory in nature, but pseudoinflammatory, i.e., associated with inflammation (McMonnies CW, "Inflammation and Keratoconus", Optometry and Vision Science, Feb 2015, 92(2):e35-e41), with certain subclinical inflammatory processes (Lema I et al., "Inflammatory Molecules in the Tears of Patients with Keratoconus", Ophthalmology, 2005,112:654-659; and 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 increased binding capacity for 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 an association with inflammation. In the progression of KC, early changes in the corneal stroma may trigger the initiation of contour abnormalities. Increasing abnormalities cause increased stress on the corneal surface.Here, the epithelium plays an important role in maintaining surface cytokine homeostasis, with KC corneas having increased IL-1α and IL-1β expression (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-1a and Tumor Necrosis Factor-a 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). Corneal epithelium has been shown to secrete IL-1 after injury or tissue damage as well as after apoptosis (Wilson SE et al., "Epithelial Injury Induces Keratocyte Apoptosis: Hypothesized Role for the Interleukin-1 System in the Modulation of Corneal Tissue Tissue and Wound Healing", Exp Eye Res, 1996, 62(4):325-327). In general, IL-1α is upregulated by corneal trauma as well as during inflammation (West-Mays JA et al., "Repair Phenotype in Corneal Fibroblasts is Controlled by an Interleukin-1a Autocrine Feedback Loop", Investigative Ophthalmology & Visual Science, June 1997, 38(7):1367-1379).At the same time, fibroblasts from KC patients show increased expression for the IL-1α receptor (Bureau J et al., "Modification of Prostaglandin E2 and Collagen Synthesis in Keratoconus Fibroblasts Associated with an Increase of Interleukin 1 Alpha Receptor Number," CR Acad Sci III, 1993, 316:425-430). Another key factor is IL-17, a proinflammatory cytokine associated with many chronic inflammatory conditions (). Interestingly, Jun et al. (2011) detected elevated levels of IL-17 in tear samples from KC patients. IL-17 has been implicated in pathogenic mechanisms in corneal inflammation by stimulating stromal cells to secrete various proinflammatory cytokines, 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). Maertzdorf J, et al., "IL-17 Expression in Human Herpetic Stromal Keratitis: Modulatory Effects on Chemokine Production by Corneal Fibroblasts", J Immunol, 2002 Nov 15, 169(10):5897-5903).As mentioned above, despite its simple structure, HA has the property of interacting with many proteins (Vignetti 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 Vignetti et al. (2011) state, HMWHA (greater than 1000kD) and CD44 induce cell migration and promote wound healing processes (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). Furthermore, the direction of cell migration is strongly dependent on CD44 expression and HA gradients in the extracellular matrix (ECM) environment (Acharya PS et al., "Fibroblast Migration is Mediated by CD44-Dependent TGF Beta Activation", J Cell Sci, 2008, 121(Pt 9):1393-1402). Other engagements of HA include regulation and binding of growth factors, and regulation of enzyme activity.

[0050] The composition may be administered topically to the ocular surface of atopic patients under the age of 30 as a prophylaxis to prevent or delay the onset of KC, since atopy is a risk factor for the development of KC, and may be administered to all young contact lens wearers, since contact lens wear causes corneal tissue changes similar to those of atopy.

[0051] If desired, the HMWHA may be administered topically to the ocular surface before, during, or after one or more treatments for KC, such as corneal cross-section (CXL), custom soft contact lenses, gas permeable contact lenses, "piggyback" contact lenses, hybrid contact lenses, scleral and hemiscleral lenses, artificial contact lenses, surgically applied corneal inserts (e.g., Intacs products), topography-guided conduction keratoplasty, or corneal transplants, or a combination of two or more of these.

[0052] Additional components 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 has an effect on a human or non-human animal subject when administered in an effective amount that affects tissue. A bioactive agent may be any type of substance, such as a drug molecule or a biological substance (e.g., polypeptides, carbohydrates, glycoproteins, immunoglobulins, nucleic acids), may be a natural product or artificially produced, and may act by any mechanism, such as pharmacological, immunological, or metabolic. Examples of types of bioactive agents include substances that modify intraocular pressure (e.g., enzyme inhibitors) and anti-angiogenic agents. Specific examples of bioactive agents include steroids (e.g., corticosteroids), antibiotics, immunosuppressants, immunomodulators, tacrolimus, plasmin activators, antiplasmins, and cyclosporin A. In various embodiments, the bioactive agent is a steroid or antibiotic for treating or preventing an ocular infection, e.g., a glaucoma drug such as a prostaglandin analog, a beta blocker, an alpha agonist, or a carbonic anhydrase inhibitor; an allergy eye relief drug such as a histamine antagonist or a nonsteroidal anti-inflammatory drug; or a mydriatic. Unfortunately, in some cases, the bioactive agent included in the composition may be irritating or damaging to the eye or epithelial surface (e.g., cyclosporine A). Advantageously, the high molecular weight HA in the composition, by virtue of its rheological and other properties, can reduce and / or protect the epithelium from the irritating and / or damaging effects of the bioactive agent in the composition on epithelia, such as the epithelium of the eye (i.e., the bioactive agent is more irritating or more damaging to the epithelial surface when administered without the high molecular weight HA).

[0053] In various embodiments, the bioactive agent included in the HMWHA composition is an immunomodulatory agent, which can enhance or promote the action of the immunomodulatory agent by making the environment at the epithelial surface more conducive to the activity of the immunomodulatory agent.

[0054] In various embodiments, the composition does not include a steroid, an antibiotic, or an immunomodulatory agent. In various embodiments, the composition does not include other bioactive agents (e.g., does not include a hydrophobic active ingredient).

[0055] In some circumstances, it may be desirable to include one or more preservatives or detergents in the composition. Such preservatives and detergents are often irritating or damaging to epithelia, such as ocular epithelium. The rheological and other properties of the composition may reduce and / or protect the epithelium from the irritating and / or damaging effects of the preservatives or detergents in the composition on the epithelium. Thus, in various embodiments, the composition further includes a preservative or detergent that is irritating or damaging to epithelia, such as ocular epithelium (i.e., a preservative or detergent that is more irritating or more damaging to the epithelium when administered without high molecular weight HA). In various embodiments, the composition does not contain a preservative or detergent.

[0056] In various embodiments, the composition comprises cyclosporine A, cetalkonium chloride, tyloxapol, or a combination of two or more thereof.

[0057] In various embodiments, the composition comprises a bioactive agent that is an immunosuppressant (e.g., a T-cell inhibitor such as cyclosporine A, tacrolimus, or sirolimus; an antimetabolite; an alkylating agent; a TNF inhibitor (e.g., infliximab, etanercept, or azilumab); a lymphocyte inhibitor; or an interleukin inhibitor); a prostaglandin (e.g., latanoprost), a prostaglandin analog, or other agent that reduces intraocular pressure; an antihistamine, and / or a mast cell stabilizer (e.g., ketotifen), or a combination of two or more thereof. The epithelial surface to which the composition is topically administered may be an ocular surface or a non-ocular surface. The composition may further comprise a preservative and / or a detergent, or may be devoid of a preservative or detergent.

[0058] In various embodiments, the composition is selected from the group consisting of glucocorticoids or other steroids (e.g., prednisone, cortisone acetate, prednisolone, methylprednisolone, dexamethasone, betamethasone, triamcinolone, beclomethasone, fludrocortisone acetate, deoxycorticosterone acetate, aldosterone), nonsteroidal anti-inflammatory drugs (e.g., salicylates, arylalkanoic acids, 2-arylpropionic acids, N-arylanthranilic acids, oxicams, coxibs, or sulfonanilides), Cox-2 specific inhibitors (e.g., valdecoxib, celecoxib, , or rofecoxib), leflunomide, aurothioglucose, aurothiomate, aurophin, sulfasalazine, hydroxychloroquine, minocycline, TNF-α binding proteins (e.g., infliximab, etanercept, or adalimumab), abatacept, anakinra, interferon-β, interferon-γ, interleukin-2, allergy vaccines, antihistamines, anti-leukotrienes, beta agonists, theophylline, anticholinergics, antibiotics, tacrolimus, and retinoids.

[0059] In various 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.

[0060] In various embodiments, the composition is administered to the subject before, during, and / or after 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 mentioned above, such preservatives and detergents are often irritating or damaging to the eye, and some bioactive agents may themselves be irritating or damaging to the eye. The composition advantageously, due to its rheological and other properties, can reduce and / or protect the eye from the irritating and / or damaging effects of the bioactive agents, preservatives, and / or detergents in the other composition on the eye. Thus, the bioactive agents, preservatives, and / or detergents in the other composition are more irritating or more damaging to epithelia, such as the epithelium of the eye, when administered without the composition.

[0061] In various embodiments, other compositions administered before, during, or after administration of the HMWHA composition include immunomodulatory agents, which can enhance or promote the action of immunomodulatory agents by making the epithelial surface environment more conducive to their activity.

[0062] In various embodiments, the other composition comprises cyclosporine A, cetalkonium chloride, tyloxapol, or a combination of two or more thereof.

[0063] In embodiments, the epithelial surface is an ocular surface and the other composition is an immunosuppressant (e.g., a T-cell inhibitor such as cyclosporine A, tacrolimus, or sirolimus; an antimetabolite; an alkylating agent; a TNF inhibitor (e.g., infliximab, etanercept, or azilumab); a lymphocyte inhibitor; or an interleukin inhibitor); a prostaglandin (e.g., latanoprost); a prostaglandin analog or other agent that reduces intraocular pressure; an antihistamine and / or a mast cell stabilizer (e.g., ketotifen), or a combination of two or more thereof. The other composition may further comprise a preservative and / or a detergent, or may be devoid of a preservative or detergent.

[0064] The other compositions administered to the subject may be in any form and may be administered by any route (e.g., topically or systemically). In embodiments, the other compositions are administered to the eye, for example, topically or by injection. In embodiments, the other compositions are administered topically to the ocular surface.

[0065] In embodiments, the preservative or cleaning agent included in the composition is a chemical or oxidative preservative.

[0066] In various embodiments, the preservative or detergent contained in the composition kills susceptible microbial cells by disrupting the lipid structure of the microbial cell membrane, thereby increasing the microbial cell membrane permeability.

[0067] In various embodiments, the preservative or cleansing agent included in the composition is one that, in the absence of the administered HMWHA, causes damage to corneal tissues such as the corneal epithelium, endothelium, stroma, and membrane interfaces.

[0068] In various 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) or cetalkonium chloride), chlorobutanol, disodium edetate (EDTA), polyquaternium-1 (e.g., Polyquad™ preservative), stabilizing oxidizing agents (e.g., stabilized oxychloro complexes (e.g., Purite™ preservative)), ionic buffer preservatives (e.g., SofZia™ preservative), polyhexamethylene biguanide (PHMB), sodium perborate (e.g., GenAqua™ preservative), tiropaxol, and sorbates.

[0069] The composition may be a health and beauty product such as one selected from bath and shower products, baby and kid care products, sun care products, shampoos, hair conditions, body lotions, solid or liquid soaps, hair styling products, shaving creams, aftershave products, hand and nail creams, face creams, face cleansing lotions, colognes, mouthwashes, or toothpastes. In embodiments, the composition is a cosmetic product such as a foundation, mascara, eye shadow, eye liner, peel, scrub, face mask, skin tightener, toner, body butter, sun care product, shower gel, eye cream, hand and cuticle cream, or makeup powder.

[0070] The composition may be applied by hand or by an applicator, depending on the epithelial surface. In various embodiments, the composition is administered using an applicator, such as a dropper, swab, cosmetic pad, wipe, wipe stick, towelette, sponge, gauze, puff, wand, adhesive or non-adhesive bandage, or contact lens.

[0071] In various embodiments, the composition is at least essentially mucin-free, ie, has a mucin concentration of less than 0.3% w / v.

[0072] In various embodiments, the composition includes a preservative, hi other embodiments, the fluid does not include a preservative (i.e., the fluid is preservative-free).

[0073] In various embodiments, the composition further comprises a glycosaminoglycan (GAG), i.e., one or more GAGs in addition to high molecular weight HA; an electrolyte (e.g., sodium chloride); a buffer (e.g., phosphate buffer); or a combination of two or more of the above.

[0074] The subject may or may not have dry eye syndrome (aqueous tear deficiency or qualitative dry eye) at the time the composition is administered to the subject's eye. In embodiments of the therapeutic or prophylactic method, the abnormality is irritation, discomfort, inflammation, immune response, or a combination of two or more of the above at the ocular surface, and the subject to whom the fluid is administered does not have aqueous tear deficiency (ATD) at the time the fluid is administered (i.e., in the absence of ATD). In embodiments of the therapeutic or prophylactic method, the abnormality is irritation, discomfort, inflammation, immune response, or a combination of two or more of the above at the ocular surface, and the subject to whom 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 embodiments of the therapeutic or prophylactic method, the abnormality is irritation, discomfort, inflammation, immune response, or a combination of two or more of the above at the ocular surface, and the subject to whom the fluid is administered does not have dry eye syndrome at the time the fluid is administered (i.e., in the absence of aqueous tear deficiency or qualitative dry eye).

[0075] In the embodiments of the treatment or prevention method, the abnormality is irritation, discomfort, inflammation, immune response, or a combination of two or more of the above on the ocular surface, and the subject does not suffer from tear volume deficiency.However, the subject has ocular surface abnormality (topographic abnormality) including protuberances on the cornea or other places on the ocular surface that are not covered by normal tear film (tear film with normal surface tension and viscosity) and cause frictional areas on the ocular surface.

[0076] The fluid may be used with a bandage contact lens. Thus, the method may further include applying a bandage contact lens to the eye before, during, and / or after administering the fluid. For example, the fluid may be administered before applying the bandage contact lens, after applying the bandage contact lens, and / or after placing the fluid on 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 ocular surface, while minimizing friction at the ocular surface. The fluid and bandage contact lens can be safely used immediately after ophthalmic surgery, such as glaucoma surgery, which is advantageous.

[0077] Another aspect of the invention is a kit containing the HMWHA composition for carrying out the method of the invention. Optionally, the kit includes an applicator for applying the composition to the desired epithelial surface.

[0078] The kit further comprises a container that contains the composition, for example a bottle, tube, vase, or pouch.

[0079] The applicator may be pre-treated with the composition or may contain the composition.

[0080] In embodiments, the applicator is a cotton swab, a cosmetic pad, a wipe, a wipe stick, a towelette, a sponge, gauze, a puff, a wand, a brush, a comb, a dropper, or an adhesive or non-adhesive bandage.

[0081] The container of the kit may include a closure selected from a pump, spray, or cap.

[0082] In various embodiments, the kit further comprises instructions for applying the composition to the epithelial surface by topically administering the composition to the epithelial surface, and, if necessary, instructions for replenishing hyaluronic acid to an epithelial surface lacking in amount or functionality of hyaluronic acid.

[0083] In other embodiments, the kit comprises the composition described herein as a fluid, and one or more bandage contact lenses. The bandage contact lenses may be packaged together with the composition in the same container (the bandage contact lenses are in contact with the composition), or the bandage contact lenses may be separated from the fluid and packaged in a separate container. Suitable containers include, for example, bottles, vials, syringes, blister packs, and the like. The containers may be made of various materials, such as glass and plastic.

[0084] The kit may include a delivery agent (either separately or associated with the 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) that are coated with the fluid and / or release the fluid onto the ocular surface.

[0085] Optionally, the kit may include an applicator or device for dispensing eye drops (e.g., eye droppers), which may or may not function as a container for the fluids in the kit prior to accessing (e.g., opening) the outer packaging of the kit, i.e., the eye drop dispensing device may function to contain the fluids provided in an unaccessed (unopened) kit, or may be empty and receive fluids after the kit is accessed. Optionally, the kit may include a label or package insert having printed or digital instructions for using the kit, e.g., for practicing a method of the invention.

[0086] The kits of the present invention can include packaging materials compartmentalized to accommodate one or more containers, such as vials, tubes, etc., each of which contains one of the separate components used in the methods described herein. Examples of packaging materials for use in packaging pharmaceutical products include 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, light-tight seals, syringes, bottles, and any packaging material appropriate for the selected formulation and mode of administration and treatment.

[0087] The kit may contain one or more additional containers, each of which has one or more of various materials that are desirable from a commercial and user perspective for the use of the compositions described herein.Non-limiting examples of such materials include, but are not limited to, buffers, diluents, carriers, packages, containers, vial and / or tube labels that list contents and / or instructions for use, and inserts with instructions for use.

[0088] The label may be on or associated with the container. The label may be present on the container by having letters, numbers, or other characters forming the label attached, formed, or etched onto the container itself. The label may be associated with the container when present in a receptacle or carrier that holds the container, for example, as a package insert. The label may be used to indicate that the contents are to be used for a particular therapeutic application. The label may also indicate directions for use of the contents, such as the methods described herein.

[0089] In various embodiments of the kit, the fluid may be provided in a pack or dispensing device that may contain 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 administration.

[0090] Preparation of fluids As noted above, the HA used in the compositions and methods of the present invention is high molecular weight (HMW) HA. In various embodiments, the fluid hyaluronic acid is 2.5 ml 3 In various embodiments, the hyaluronic acid has an intrinsic viscosity of greater than 2.9 ml / kg. 3 / kg.

[0091] Furthermore, when the epithelial surface is an ocular epithelium, the concentration of HMWHA and / or HMWHA analogs is preferably less than 0.2% w / v. In other cases, when the epithelial surface is a skin epithelium, for example, the concentration of HMWHA and / or HMWHA analogs is preferably within the range of 0.2% to 3.0% w / v.

[0092] Viscoelasticity is defined as the property of a fluid that has both viscous and elastic properties. Zero shear viscosity was determined as the steady shear plateau viscosity at the vanishing shear rate. For highly viscous formulations, controlled stress rheometer measurements are preferred.

[0093] Molecular weight and intrinsic viscosity [η](m 3 The relationship between the mass and the mass of the body (kg) is given by the Mark-Houwink equation: [η] = k (Mrm)a Mrm is the molecular weight in MDa coefficient k = 1.3327 10-4 and a = 0.6691 The values ​​of k and a have been found to be most predictive.

[0094] The fluid can be produced by sterilizing the filling line if desired; adding purified water or water for injection (WFI) to a stainless steel mixing tank; adding salt while mixing; slowly adding HA and mixing until a homogenous solution / fluid is obtained; adjusting the pH value by adding NaOH or HCl while continuing the mixing process, if necessary; transferring the solution to a sterile holding tank on a 1 μm pore size filter cartridge; and aseptically filling the solution into a sterile primary package (single dose or vial) via sterile filtration. For single dose, this may be done by a blow-fill-seal (BFS) process. For application to the eye, it is preferred that the composition is sterile. For application to the skin, for example, the composition may be sterile, but does not necessarily have to be sterile.

[0095] It is preferred that the fluid is at least essentially mucin-free, or in other words has a mucin concentration of less than 0.3% w / v, meaning that the flow behavior or properties are essentially achieved or regulated by hyaluronan and not by mucins that are naturally present in the subject's tears and are primarily responsible for its flow behavior.

[0096] When viscosity-increasing substances are added, they are preferably added toward, during, or as the final step.Mixing is performed to obtain a homogeneous mixture.Instead or in addition, it is preferred to first provide purified water or water for injection as a base, and then, if necessary, electrolytes, buffers, and substances that do not increase viscosity are added to the purified water or water for injection first.

[0097] HA is further described in the European Pharmacopoeia 9.0, monograph at page 3583 (Sodium Hyaluronate), which is incorporated herein by reference in its entirety.

[0098] In one embodiment, the fluids used in the methods and kits of the present invention have the characteristics listed in Table 1.

[0099] [Table 1]

[0100] definition The terms "a", "an", "the" and similar terms used in the context of the present invention (especially in the context of the claims) should be interpreted as including both the singular and the plural, unless otherwise indicated herein or clearly contradicted by the context. Thus, for example, the term "cell" or "compound" should be interpreted as including both a single cell or a single compound and a plurality of cells or compounds, unless otherwise indicated or clearly contradicted by the context. Similarly, the term "or" is intended to include "and", unless otherwise indicated by the context. The abbreviation "eg" is derived from the Latin "exempli gratia" and is used herein to indicate a non-limiting example. Thus, the abbreviation "eg" is synonymous with the term "for example".

[0101] The term "deficiency" refers to a lack of HA (membrane-bound or unbound) in the amount or structure relative to physiological normal in the context of HA at the epithelial surface, or a lack of one or more biological functions of normal HA in the anatomical location relative to the extracellular space within the epithelium and the tissue immediately below. 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, and in particular Chapter 6: Experimental Determination of HA Content and Size, which is incorporated herein by reference in its entirety).

[0102] The phrase "effective amount," in the context of an administered composition, refers to the amount of a composition necessary to obtain a desired result, such as repair (complete or partial) of HA at a desired anatomical site or one or more biological functions of HA. In embodiments, an effective amount may be an amount that can prevent, delay the onset of, treat, or ameliorate a disease or condition, or otherwise provide the intended therapeutic effect.

[0103] The term "epithelial surface" refers to a body surface composed primarily of epithelial cells. Epithelium is a sheet of cells that covers most of the body's surfaces. Epithelia are a group of tissues that perform a wide variety of functions and use different cellular arrangements and structures to perform these functions. Epithelia can be any type of epithelium. Epithelia can be keratinized (keratinized), such as skin, or non-keratinized, such as the mouth, esophagus, and vagina. For example, the epithelium may be simple squamous epithelium (e.g., the lining of the air sacs of the lungs, and the heart, blood vessels, and lymphatic vessels), simple cuboidal epithelium (e.g., the ducts and secretory portions of small glands and the renal tubules), simple columnar epithelium (e.g., the ciliated tissue of the bronchi, uterine ducts, and uterus; the smooth (non-ciliated) tissue of the digestive tract and bladder), pseudostratified columnar epithelium (e.g., the 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 ducts of the ureters and some glands in males), transitional epithelium (e.g., the lining of the bladder, ureters, and urethra), or a combination of two or more of these.

[0104] Epithelial cells are usually characterized by the presence of tight junctions, adherens junctions, and desmosomes. Epithelial cells are typically polarized with an apical surface facing the lumen or external environment, and a basal surface facing the basement membrane. The composition may be administered locally to the apical surface, or to the basal surface if the basal surface is exposed. Alternatively, the composition may be administered locally to the basal surface. The epithelial surface may be undamaged, continuous, or discontinuous, such as may result from trauma.

[0105] The phrase "biological function of hyaluronic acid" refers to one or more functions of biological HA in a tissue of interest (e.g., epithelium). For example, depending on the anatomical location of the epithelium, the functions of HA may include one or more of hydration, water binding of HA in the extracellular matrix (allowing transport of nutrients, catabolites, and gases, thereby nourishing the tissue), lubrication, space-filling capacity, framework for cell migration, tissue viscosity, shock absorption, free radical scavenging, cytokine interaction, and modulation of inflammation, cell migration, proliferation, and differentiation via HA receptors (see, for example, Dicker KT et al., "Hyaluronan: A Simple Polysaccharide with Diverse Biological Functions," Acta Biomater, 2014, 10(4):1558-1570).

[0106] The term "isolated", when used as a modifier of a composition, means that the compositions are made by human intervention or are separated from the in vivo environment in which they naturally occur. Generally, such isolated compositions are substantially free of one or more substances 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 combined with one or more other molecules. Thus, the term "substantially pure" does not exclude combinations of compositions. Substantial purity can be at least about 60% by weight or more of the molecule. Purity can also be about 70% or 80% or more, and even greater, such as 90% or more. Purity can be determined by any suitable method, such as, for example, UV spectroscopy, chromatography (e.g., HPLC, gas phase), gel electrophoresis (e.g., silver or Coomassie staining), and sequence analysis (of nucleic acids and peptides).

[0107] As used herein, the term "homeostasis" refers to the ability of a physiological system to maintain internal stability, or the state of stability itself, due to the coordinated response of its parts to any situation or stimuli that would tend to disrupt normal, non-pathological conditions or function.

[0108] As used herein, the term "hyaluronic acid" or "HA" refers to the glycosaminoglycan, also known as hyaluronan, consisting of disaccharide repeats of N-acetylglucosamine and glucuronic acid found in nature (e.g., a linear glycosaminoglycan polymer consisting 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, amide derivatives, alkyl-amine derivatives, low and high molecular weight forms of hyaluronan, and cross-linked forms (e.g., hylan). Thus, the disaccharide chains may be linear or non-linear. Hyaluronan can be cross-linked by attaching cross-linking agents such as thiols, polymethylmethacrylate, hexadecylamide, and tyramine. Hyaluronan can also be cross-linked directly with formaldehyde and divinylsulfone.Examples of hylan include, but are not limited to, hylan A (a formaldehyde-crosslinked glycosaminoglycan polymer), hylan B (a divinylsulfone-crosslinked 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; incorporated herein by reference in their entireties).

[0109] 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. The pharma- ceutically acceptable salts of HA can be prepared using conventional techniques.

[0110] The term "high molecular weight" or "HMW" in the context of the HA of the present invention means a molecular weight of 2.5 nm or more as determined by the method of "Sodium Hyaluronate" at page 3584 of the European Pharmacopoeia 9.0, which is incorporated herein by reference in its entirety. 3 / kg. In simple terms, 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 various embodiments, the high molecular weight hyaluronic acid is 2.9m 3 / kg.

[0111] The term "immunodeficiency" as used herein refers to a subject who is unable to develop a normal immune response, either congenitally, acquired, or inducibly. Thus, an immunodeficient subject has a weakened or impaired immune system compared to a normal subject. A subject with a weakened or impaired immune system has an "immunodeficiency" or "immunodeficiency state" associated with an induced or uninduced primary or secondary deficiency in one or more components of the normal immune defense system. The immunodeficiency state may be due to medical treatment, e.g., radiation therapy, chemotherapy, or other immunosuppressive treatment, particularly associated with cancer treatment or the treatment or prevention of transplant rejection, such as induced by treatment with steroids, cyclophosphamide, azathioprine, methotrexate, cyclosporine, or rapamycin. The presence of an immunodeficiency state in a subject may be diagnosed by any suitable technique known to those skilled in the art. A strong indicator that an immunodeficiency state may exist is the occurrence of a rare disease or when the subject becomes ill with a microorganism that does not normally cause disease, especially when the subject is repeatedly infected. Other typical possibilities include acquired infections such as HIV, hepatitis, tuberculosis, etc. However, a definitive diagnosis is generally based on laboratory tests that determine the exact nature of the immunodeficiency state. Most tests are performed on blood samples. Blood contains antibodies, lymphocytes, phagocytes, and complement components, all of the major immune components that can cause immunodeficiency. A blood count can be used to determine if the number of phagocytes or lymphocytes is below normal. A lower than normal number of either of these two cell types correlates with an immunodeficiency state. Blood cells are also typically examined for their appearance. A subject may have normal cell numbers, but the cells may be structurally defective. If the lymphocyte count is low, further tests are usually performed to determine if certain types of lymphocytes are less than normal. Lymphocyte proliferation tests may be performed to determine if lymphocytes are able to respond to stimuli. Failure to respond to stimuli correlates with an immunodeficiency state. Antibody and complement levels can also be measured to diagnose the presence of an immunodeficiency state.

[0112] As used herein, the term "ocular surface" refers to the structures of the eye and adnexa, including the cornea, conjunctiva, eyelids, eyelashes, tear film, main and accessory lacrimal glands, and meibomian glands.Thus, tears are included in the term "ocular surface" both in terms of individual components at the site of production and as a film on the ocular surface (see Craig JP et al., "TFOS DEWS II Definition and Classification Report," The Ocular Surface, 2017, 15:276-283, which is incorporated herein by reference in its entirety).The composition may be administered topically to one or more parts of the ocular surface, including, for example, the entire ocular surface.

[0113] "Pharmaceutically acceptable salt" includes both acid and base addition salts.The pharmaceutically acceptable salt of HA or any one of the other compounds described herein is intended to include any and all pharmaceutically appropriate salt forms.Preferred pharmaceutically acceptable salts described herein are pharmaceutically acceptable acid addition salts and pharmaceutically acceptable base addition salts.

[0114] "Pharmaceutically acceptable acid addition salts" refer to salts which retain the biological effectiveness and properties of the free base which are 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, etc. 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, 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, bisulfite, sulfite, bisulfite, nitrate, phosphate, monohydrogen phosphate, dihydrogen phosphate, metaphosphate, pyrophosphate, chloride, bromide, iodide, acetate, trifluoroacetate, propionate, caprylate, isobutyrate, oxalate, malonate, succinate persalt, sebacate, fumarate, maleate, mandelate, benzoate, chlorobenzoate, methylbenzoate, dinitrobenzoate, phthalate, benzenesulfonate, toluenesulfonate, phenylacetate, citrate, lactate, malate, tartrate, methanesulfonate, and the like. Also contemplated are 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), incorporated herein by reference in its entirety). Acid addition salts of basic compounds may be prepared by contacting the free base form with a sufficient amount of the desired acid to produce the salt according to methods and techniques familiar to those of ordinary skill in the art.

[0115] "Pharmaceutically acceptable base addition salts" refer to salts that retain the biological effectiveness and properties of the free acids, which are biologically or otherwise undesirable. These salts are prepared from the addition of an inorganic or organic base to the free acid. Pharmaceutically acceptable base addition salts may 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, ethylenedianiline, N-methylglucamine, glucosamine, methylglucamine, theobromine, purines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, etc. See Berge et al., supra. In various embodiments, the pharma- ceutically acceptable salt is a sodium salt (see European Pharmacopoeia 9.0, p. 3583, "Sodium Hyaluronate," herein incorporated by reference in its entirety).

[0116] As used herein, the terms "subject", "patient" and "individual" refer to a human or non-human animal. A subject also refers to, for example, a primate (e.g., a human), cow, sheep, goat, horse, dog, cat, rabbit, rat, mouse, fish, bird, etc. In embodiments, the subject is a mammal. In embodiments, the subject is a human. In embodiments, the subject is a bird or fish. Thus, the method can be performed in medical and veterinary settings. The non-human animal subject may be, for example, a pet or an animal model of an ocular or non-ocular disease. The subject may be of any age or stage of life. For example, in embodiments, the subject is an infant or an adolescent. In some other embodiments, the subject is an elderly person. In embodiments, the subject is an atopic patient (has atopy).

[0117] In various embodiments, the subject's eye does not have aqueous tear deficiency (ATD) at the time of administration (ie, in the absence of an ATD).

[0118] In various embodiments, the subject's eye does not have qualitative dry eye at the time of said administration (ie, in the absence of qualitative dry eye).

[0119] In various embodiments, the subject's eye does not have dry eye syndrome at the time of said administration (ie, in the absence of aqueous tear deficiency or qualitative dry eye).

[0120] In embodiments, the subject does not suffer from aqueous tear deficiency (ATD), the subject has ocular surface abnormalities (topographical abnormalities) including protuberances on the cornea or elsewhere on the ocular surface that are not covered by a normal tear film (tear film of normal surface tension and viscosity) and result in areas of friction on the ocular surface, and the administered fluid reduces friction.

[0121] In various embodiments, the subject is immunocompromised, ie, in an immunocompromised state.

[0122] The phrase "topical administration" is used herein in its conventional sense to mean local delivery to a desired anatomical site, such as an epithelial surface (the surface of an epithelium). The composition comprising HMWHA may be applied directly or indirectly to an epithelial surface by any method that allows an effective amount of the composition and the epithelial surface to come into contact. For example, the composition may be applied directly to the epithelial surface, such as by eye drop or washing, or may be applied indirectly via a delivery agent that is brought into contact with the epithelial surface. One example of a delivery agent is a particle (e.g., a microparticle or nanoparticle) that is coated with the composition and / or releases the composition onto the ocular surface. Such particles may be composed of various materials, such as natural or synthetic polymers. In various embodiments, the delivery agent itself may be administered as a drop.

[0123] The present invention is described by way of example only by the embodiments in the specification and drawings, and is not limited thereto, but rather includes all variations, modifications, substitutions and combinations that a person skilled in the art may take from the full text of the present application under consideration and / or in combination with the particular knowledge of the person skilled in the art.

[0124] All patents, patent applications, provisional applications, and publications mentioned or cited in this specification are incorporated by reference in their entirety, including any figures and tables, unless they are inconsistent with an express disclosure herein.

[0125] The following are examples illustrating procedures for carrying out the present invention. These examples should not be construed as limiting. All percentages are by weight and all mixture ratios are by volume unless otherwise noted.

[0126] Example 1-A: A multicenter, multinational, prospective clinical trial targeting patients with severe dry eye - HYLAN M study A multicenter, multinational, prospective randomized clinical trial in patients with severe dry eye (according to ODISSEY core criteria) is being conducted at 12 centers in 9 countries. In the HYLAN M trial, patients were randomized into two groups, one remaining on the previously identified most effective individual patient treatment, and the other switching to a high molecular weight hyaluronic acid eye drop (preservative-free sodium hyaluronate eye drop Comfort Shield® (i.com medical GmbH, Munich, Germany)), which corresponds to the embodiment in Table 1 herein.

[0127] These patients (192 enrolled) were already receiving the best treatment their ophthalmologists could provide. All patients were on "stable" therapy at the time of enrollment in the study, i.e., their treatment had not changed for a specified period of time prior to enrollment in the study. Patients were randomized into two groups: patients in the first group continued their current treatment for dry eye symptoms, and patients in the second group were treated with the above drops (Comfort Shield® eye drops) instead of tear substitutes.

[0128] The objectives of the study were (1) to compare the objective and subjective symptoms of dry eye under treatment with Comfort Shield® eye drops to a pre-prescribed tear substitute eye drop (=current treatment) in patients with severe dry eye, and (2) to observe the objective performance, subjective patient acceptance, and adverse events 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.

[0129] Patients at one study site who participated in the HYLA M study had not been able to obtain sufficient relief of signs and symptoms with all commercially available eye drops that had been tested before the doctors decided to treat them with autologous serum eye drops. The study included 11 patients who had been treated with autologous serum eye drops. Of these 11 patients, 6 were randomized to the Comfort Shield® group, i.e., the use of autologous serum eye drops was replaced by Comfort Shield® for 8 weeks. Of these 6 patients, 2 discontinued participation in the study because Comfort Shield® eye drops did not provide sufficient relief of symptoms. Two patients continued their Comfort Shield® eye drop therapy for the 8 weeks of the study, but preferred to return to their original treatment with autologous serum. The remaining two patients preferred Comfort Shield® eye drops over autologous serum eye drops and decided to use Comfort Shield® eye drops beyond the study.

[0130] It should be understood that the examples and aspects described herein are for illustrative purposes only, and that various modifications or changes in light thereof will be suggested to those skilled in the art, and are within the spirit and scope of this application and the scope of the appended claims. Furthermore, any element or limitation of any invention or embodiment thereof disclosed herein can be combined with any and / or all other elements or limitations (individually or in any combination) disclosed herein or with any other invention or embodiment thereof, and all such combinations are contemplated within the scope of the present invention, without limitation thereto.

Claims

1. 1. A fluid for use in the treatment of conjunctivitis, rhinoconjunctivitis, or keratoconus, comprising: an effective amount of said fluid is topically administered to an ocular epithelial surface of a subject; The fluid comprises high molecular weight hyaluronic acid, the high molecular weight hyaluronic acid in the fluid permeates the ocular epithelial surface of the subject, and the high molecular weight hyaluronic acid is 2.5 ml ３ / kg.

2. 2. The fluid of claim 1 , The fluid does not contain other bioactive agents. fluid.

3. 3. A fluid according to claim 1 or 2, The high molecular weight hyaluronic acid is 2.9 m ３ / kg. fluid.

4. A fluid according to any one of claims 1 to 3, A fluid having a concentration of high molecular weight hyaluronic acid of less than 0.2% w / v.

5. A fluid according to any one of claims 1 to 4, The subject has conjunctivitis.

6. A fluid according to any one of claims 1 to 4, The subject has rhinoconjunctivitis.

7. A fluid according to any one of claims 1 to 4, The subject has keratoconus.

Images