Highly elastic hyaluronan compositions and methods of use thereof

Abstract

To provide a method for effectively treating dry eye conditions, minimizing skin imperfections using an injectable reinforcement device, and promoting wound healing while relieving pain associated with these conditions.SOLUTION: The present invention provides a method of alleviating pain and discomfort associated with a dry eye condition in a subject in need thereof. The method comprises administering to the eye of the subject a composition (e.g., a pharmaceutical composition) comprising hyaluronan, wherein the hyaluronan is present in the composition at a concentration of greater than about 30mg / mL; the hyaluronan has a mean molecular weight of between about 1 million and about 2,000,000; and the hyaluronan is not crosslinked and / or is substantially free of chemical modification, thereby alleviating pain and discomfort in the subject.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 232,364, filed September 24, 2015, the entire contents of which are incorporated herein by reference. [Background technology]

[0002] Background of the Invention Hyaluronan, or hyaluronic acid (HA), is a high-average molecular weight linear polysaccharide widely distributed throughout connective, epithelial, and neural tissues. HA is primarily found in the extracellular and pericellular matrices, but intracellular presence has also been shown. HA's biological functions include maintaining the elastic-viscous properties of liquid connective tissues (such as the synovial fluid in joints and the vitreous body of the eye), regulating tissue hydration and water transport, supramolecular assembly of proteoglycans within the extracellular matrix, and multiple receptor-mediated roles in cell separation, mitosis, migration, and tumor growth.

[0003] Some known uses of HA include the treatment of dry eye, skin care as a dermal filler, and the promotion of wound healing.Dry eye, skin care / dermatological procedures, and wound healing are often associated with pain and discomfort, which typically require the separate application of an analgesic.The HA formulations currently used to treat the above conditions are not effective in treating local pain.Therefore, there is a need in the art for a method that effectively treats dry eye, minimizes skin defects using an injectable augmentation device, and promotes wound healing while alleviating the pain associated with these conditions. Summary of the Invention [Means for solving the problem]

[0004] Summary of the Invention The present inventors have discovered that compositions containing high concentrations of HA (e.g., compositions having an HA concentration of about 30 mg / mL (about 3% weight / volume) or more) can be effectively used for relieving pain and discomfort associated with dry eye conditions, dermatological procedures, and wound healing. Without wishing to be bound by any particular theory, it is believed that the effectiveness of the HA compositions of the present invention containing high concentrations of HA for treating pain and discomfort is determined by their high elasticity, as evidenced by a high value of the elastic modulus G'. Without wishing to be bound by any particular theory, it is also believed that the effectiveness of the HA compositions of the present invention is determined by the relatively high probability of interaction of the HA molecule with pain transduction channels (such as TRPV1), thereby reducing nociceptor excitability.

[0005] Thus, the present invention provides a method for alleviating pain and discomfort associated with a dry eye condition in a subject in need thereof. The method includes administering to the eye of the subject a composition (e.g., a pharmaceutical composition) comprising hyaluronan, wherein the hyaluronan is present in the composition at a concentration of greater than about 30 mg / mL (e.g., about 35 mg / mL or greater, about 40 mg / mL or greater, about 45 mg / mL or greater, about 50 mg / mL or greater, about 55 mg / mL or greater, about 60 mg / mL or greater, about 65 mg / mL or greater, about 70 mg / mL or greater). or greater, about 75 mg / mL or greater, about 80 mg / mL or greater, about 85 mg / mL or greater, about 90 mg / mL or greater, about 95 mg / mL or greater, or about 100 mg / mL or greater); the hyaluronan has an average molecular weight of between about 1 million and about 2 million; and the hyaluronan is not crosslinked and / or is substantially free of chemical modifications, thereby alleviating pain and discomfort in a subject.

[0006] In another embodiment, the hyaluronan is present in the composition at a concentration of about 40 mg / mL to about 60 mg / mL.

[0007] In some embodiments, the present invention also provides a method of alleviating pain and discomfort associated with a dry ocular condition in a subject in need thereof, comprising administering to the eye of the subject a composition (e.g., a pharmaceutical composition) comprising hyaluronan, wherein the hyaluronan is present in the composition at a concentration of at least about 40 mg / mL; the hyaluronan has an average molecular weight of between about 1 million and about 2 million; and the hyaluronan is not cross-linked and / or is substantially free of chemical modifications, thereby alleviating pain and discomfort in the subject.

[0008] In some embodiments, the composition is substantially free of other pharmaceutically active substances.

[0009] In certain embodiments, the composition is polyglycol-free.

[0010] In some embodiments, the composition further comprises a buffer (eg, phosphate buffered saline (PBS)).

[0011] In some embodiments, the elasticity of the composition is at least about 200 Pascals when measured at a frequency of 0.5 Hz; at least about 1,000 Pascals when measured at a frequency of 0.5 Hz; at least about 2,000 Pascals when measured at a frequency of 0.5 Hz; or at least about 4,000 Pascals when measured at a frequency of 0.5 Hz.

[0012] In some embodiments, the composition is in the form of a gel, ointment, salve, lotion, or cream.

[0013] In some embodiments, the composition is administered to the subject's ocular surface, such as under the eyelid (eg, under the upper or lower eyelid) or at the cornea-eyelid junction.

[0014] In certain embodiments, the composition is administered to the subject immediately prior to resting or going to sleep.

[0015] In some embodiments, the composition is administered to the subject intraocularly without the use of an injection.

[0016] In some embodiments, the composition is administered using a container (eg, a single-dose container such as a flexible plastic bottle, a tube, an airless tube, an eyecup, a dropper, or a cartridge).

[0017] In some embodiments, the dry eye condition is associated with one or more symptoms selected from the group consisting of dryness of the eye; decreased tear production, volume, and flow; abnormalities in tear composition; increased tear osmolarity; keratitis; conjunctival and corneal staining; redness; blurry vision; decreased tear film breakup time; increased conjunctival redness; excess debris in the tear film, ocular grittiness; ocular burning; foreign body sensation in the eye; excessive tear production; photophobia; ocular stinging; refractive impairment; ocular sensitivity; and ocular irritation. In other embodiments, the dry eye condition is associated with a condition selected from the group consisting of autoimmune disorders; ophthalmic surgery; drug intake; dry environmental conditions; prolonged computer use; eye fatigue; corneal hypersensitivity due to prolonged contact lens wear; partial eyelid closure; surface irregularities; eyelid irregularities; and corneal nociceptive pain associated with corneal injury or neuropathic pain.In a further embodiment, the dry eye condition is associated with ophthalmic surgery, and the ophthalmic surgery is selected from the group consisting of photorefractive keratectomy (PRK), cataract surgery, retinal detachment surgery, laser in situ keratomileusis (LASIK), and any corneal surgical procedure involving corneal sensory nerve damage.In a further embodiment, the dry eye condition is associated with a condition associated with neuropathic pain (for example, cataract or retinal detachment or surgery designed to treat cataract or retinal detachment).

[0018] In some embodiments, the composition is administered daily for 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 4 weeks, or 10 weeks. In other embodiments, the subject experiences pain and discomfort relief for an extended period of time (e.g., 8 hours, 12 hours, 24 hours, 1 day, 3 days, 5 days, 7 days, 14 days, or 28 days).

[0019] In some embodiments, the subject is a mammal (eg, a human).

[0020] In yet another aspect, the present invention also provides a method for alleviating pain and discomfort while minimizing at least one skin imperfection in a subject in need thereof. The method comprises administering to the subject a composition comprising hyaluronan, wherein the hyaluronan is present in the composition at a concentration of greater than about 30 mg / mL; the hyaluronan has an average molecular weight of between about 1 million and about 2 million; the hyaluronan is not cross-linked and / or is substantially free of chemical modifications; and the composition is substantially free of other pharmaceutically active substances, thereby alleviating pain and minimizing at least one skin imperfection.

[0021] In some embodiments, the hyaluronan is present at a concentration of about 40 mg / mL to about 60 mg / mL.

[0022] In a further aspect, the present invention also provides a method for alleviating pain and discomfort while minimizing at least one skin imperfection in a subject in need thereof, comprising administering to the subject a composition comprising hyaluronan, wherein the hyaluronan is present in the composition at a concentration of at least about 40 mg / mL; the hyaluronan has an average molecular weight of between about 1 million and about 2 million; the hyaluronan is not cross-linked and / or is substantially free of chemical modifications; and the composition is substantially free of other pharmaceutically active substances, thereby alleviating pain and minimizing at least one skin imperfection.

[0023] In some embodiments, the other pharmaceutically active substance is a local anesthetic (eg, lidocaine or bupivacaine).

[0024] In certain embodiments, the composition also includes a buffer (eg, phosphate buffered saline (PBS)).

[0025] In some embodiments, the elasticity of the composition is at least about 200 Pascals when measured at a frequency of 0.5 Hz; at least about 1,000 Pascals when measured at a frequency of 0.5 Hz; at least about 2,000 Pascals when measured at a frequency of 0.5 Hz; or at least about 4,000 Pascals when measured at a frequency of 0.5 Hz.

[0026] In some embodiments, the composition is sterile.

[0027] In certain embodiments, the composition is administered to the subject by intradermal injection.In further embodiments, the composition is injected into the face of the subject.For example, the composition is injected into the area selected from the group consisting of the nasolabial area, the upper lip area, the forehead, the eye area, and the cheek area.

[0028] In some embodiments, the composition is administered by injection using a pre-filled syringe (e.g., a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL pre-filled syringe). In a further embodiment, the pre-filled syringe is sterile.

[0029] In another aspect, the present invention also provides a method for alleviating pain and discomfort while promoting wound healing in a subject in need thereof. The method comprises administering to the subject a composition (e.g., a pharmaceutical composition) comprising hyaluronan, wherein the hyaluronan is present in the composition at a concentration of greater than about 30 mg / mL; the hyaluronan has an average molecular weight of between about 1 million and about 2 million; and the hyaluronan is not cross-linked and / or is substantially free of chemical modifications, thereby alleviating pain and promoting wound healing.

[0030] In a further embodiment, the hyaluronan is present at a concentration of about 40 mg / mL to about 60 mg / mL.

[0031] In a further aspect, the present invention also provides a method for alleviating pain and discomfort while promoting wound healing in a subject in need thereof, comprising administering to the subject a composition (e.g., a pharmaceutical composition) comprising hyaluronan, wherein the hyaluronan is present in the composition at a concentration of at least about 40 mg / mL; the hyaluronan has an average molecular weight of between about 1 million and about 2 million; and the hyaluronan is not cross-linked and / or is substantially free of chemical modifications, thereby alleviating pain and promoting wound healing.

[0032] In some embodiments, the composition is substantially free of other pharmaceutically active substances, such as local anesthetics (eg, lidocaine or bupivacaine).

[0033] In some embodiments, the composition further comprises a buffer (eg, phosphate buffered saline (PBS)).

[0034] In some embodiments, the elasticity of the composition is at least about 200 Pascals when measured at a frequency of 0.5 Hz; at least about 1,000 Pascals when measured at a frequency of 0.5 Hz; at least about 2,000 Pascals when measured at a frequency of 0.5 Hz; or at least about 4,000 Pascals when measured at a frequency of 0.5 Hz.

[0035] In some embodiments, the composition is sterile.

[0036] In certain embodiments, the composition is administered topically (eg, onto the surface of a wound or scar on the skin). The present invention provides, for example, the following items. (Item 1) 1. A method of alleviating pain and discomfort associated with a dry ocular condition in a subject in need thereof, comprising administering to the eye of the subject a composition comprising hyaluronan, wherein: the hyaluronan is present in the composition at a concentration greater than about 30 mg / mL; the average molecular weight of the hyaluronan is between about 1 million and about 2 million; The method, wherein the hyaluronan is not cross-linked and / or is substantially free of chemical modifications, thereby alleviating the pain and discomfort in the subject. (Item 2) 2. The method of claim 1, wherein the hyaluronan is present in the composition at a concentration of about 40 mg / mL to about 60 mg / mL. (Item 3) 1. A method of alleviating pain and discomfort associated with a dry ocular condition in a subject in need thereof, comprising administering to the eye of the subject a composition comprising hyaluronan, wherein: the hyaluronan is present in the composition at a concentration of at least about 40 mg / mL; the average molecular weight of the hyaluronan is between about 1 million and about 2 million; The method, wherein the hyaluronan is not cross-linked and / or is substantially free of chemical modifications, thereby alleviating the pain and discomfort in the subject. (Item 4) 4. The method of claim 1 or 3, wherein the composition is substantially free of other pharmaceutically active substances. (Item 5) 4. The method of claim 1 or 3, wherein the composition does not contain polyglycols. (Item 6) 4. The method of claim 1 or 3, wherein the composition further comprises a buffer. (Item 7) The method according to item 1 or 3, wherein the buffer is phosphate buffered saline (PBS). 4. The method of claim 1 or 3, wherein the elasticity of the composition is at least about 200 Pascals when measured at a frequency of 0.5 Hz. (Item 9) 4. The method of claim 1 or 3, wherein the elasticity of the composition is at least about 1,000 Pascals when measured at a frequency of 0.5 Hz. (Item 10) 4. The method of claim 1 or 3, wherein the elasticity of the composition is at least about 2,000 Pascals when measured at a frequency of 0.5 Hz. (Item 11) 4. The method of claim 1 or 3, wherein the elasticity of the composition is at least about 4,000 Pascals when measured at a frequency of 0.5 Hz. (Item 12) 4. The method of claim 1 or 3, wherein the composition is sterile. (Item 13) 4. The method of claim 1 or 3, wherein the composition is in the form of a gel, ointment, liniment, lotion, or cream. (Item 14) 4. The method of claim 1 or 3, wherein the composition is administered to the ocular surface. (Item 15) 15. The method of claim 14, wherein the composition is administered under the eyelid of the subject. (Item 16) 15. The method of claim 14, wherein the composition is administered to the corneal-eyelid junction. (Item 17) 15. The method of claim 14, wherein the composition is administered to the subject immediately before resting or going to bed. (Item 18) 4. The method of claim 1 or 3, wherein the composition is administered to the subject intraocularly without injection. (Item 19) 20. The method of claim 18, wherein the composition is administered using a container selected from the group consisting of a single-dose container, a flexible plastic bottle, a tube, an airless tube, an eyecup, a dropper, and a cartridge. (Item 20) 4. The method of claim 1 or 3, wherein the dry eye condition is associated with one or more symptoms selected from the group consisting of dry eye; decreased tear production, volume, and flow; abnormal tear composition; increased tear osmolarity; keratitis; conjunctival and corneal staining; redness; blurred vision; decreased tear film breakup time; increased conjunctival redness; excess debris in the tear film, grittiness in the eye; burning eye; foreign body sensation in the eye; excessive tear production; photophobia; eye irritation; refractive dysfunction; ocular hypersensitivity; eye irritation, and discomfort due to prolonged contact lens wear. (Item 21) 4. The method of claim 1 or 3, wherein the dry eye condition is associated with a condition selected from the group consisting of an autoimmune disorder; eye surgery; drug intake; dry environmental conditions; prolonged computer use; eye fatigue; corneal hypersensitivity due to contact lens wear; partial eyelid closure; surface irregularities; eyelid irregularities; and conditions associated with neuropathic pain. (Item 22) 22. The method of claim 21, wherein the dry eye condition is associated with ophthalmic surgery, and the ophthalmic surgery is selected from the group consisting of photorefractive surgery, cataract surgery, retinal detachment surgery, laser in situ keratomileusis (LASIK), and any corneal surgical procedure involving corneal sensory nerve damage. (Item 23) 22. The method of claim 21, wherein the dry eye condition is associated with a condition associated with neuropathic pain, and the condition is cataract or post-retinal detachment surgery. (Item 24) 15. The method of claim 14, wherein the composition is administered daily for 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 4 weeks, or 10 weeks. (Item 25) 4. The method of item 1 or 3, wherein the subject experiences long-term relief of pain and discomfort. (Item 26) 26. The method of claim 25, wherein the extended period is 8 hours, 12 hours, 24 hours, 1 day, 3 days, 5 days, 7 days, 14 days, or 28 days. (Item 27) 4. The method of item 1 or 3, wherein the subject is a mammal. (Item 28) 28. The method of claim 27, wherein the subject is a human. (Item 29) 1. A method for alleviating pain and discomfort while minimizing at least one skin imperfection in a subject in need thereof, the method comprising administering to the subject a composition comprising hyaluronan, wherein: the hyaluronan is present in the composition at a concentration greater than about 30 mg / mL; the average molecular weight of the hyaluronan is between about 1 million and about 2 million; the hyaluronan is not cross-linked and / or is substantially free of chemical modifications; The method, wherein the composition is substantially free of other pharmaceutically active substances, thereby relieving the pain and minimizing the at least one skin imperfection. (Item 30) 30. The method of item 29, wherein the hyaluronan is present at a concentration of about 40 mg / mL to about 60 mg / mL. (Item 31) 1. A method for alleviating pain and discomfort while minimizing at least one skin imperfection in a subject in need thereof, the method comprising administering to the subject a composition comprising hyaluronan, wherein: the hyaluronan is present in the composition at a concentration of about 40 mg / mL; the average molecular weight of the hyaluronan is between about 1 million and about 2 million; the hyaluronan is not cross-linked and / or is substantially free of chemical modifications; The method, wherein the composition is substantially free of other pharmaceutically active substances, thereby relieving the pain and minimizing the at least one skin imperfection. (Item 32) Item 33. The method according to Item 29 or 31, wherein the other pharmaceutically active substance is a local anesthetic. 33. The method of claim 32, wherein the local anesthetic is lidocaine or bupivacaine. (Item 34) 32. The method of claim 29 or 31, wherein the composition further comprises a buffer. (Item 35) 35. The method of claim 34, wherein the buffer is phosphate buffered saline (PBS). (Item 36) 32. The method of claim 29 or 31, wherein the elasticity of the composition is at least about 200 Pascals when measured at a frequency of 0.5 Hz. (Item 37) 32. The method of claim 29 or 31, wherein the elasticity of the composition is at least about 1,000 Pascals when measured at a frequency of 0.5 Hz. (Item 38) 32. The method of claim 29 or 31, wherein the elasticity of the composition is at least about 2,000 Pascals when measured at a frequency of 0.5 Hz. (Item 39) 32. The method of claim 29 or 31, wherein the elasticity of the composition is at least about 4,000 Pascals when measured at a frequency of 0.5 Hz. (Item 40) 32. The method of claim 29 or 31, wherein the composition is sterile. (Item 41) 32. The method of claim 29 or 31, wherein the composition is administered to the subject by intradermal injection. (Item 42) 42. The method of claim 41, wherein the composition is injected into an area of ​​the subject selected from the group consisting of the face, neck, arms, legs, torso, and chest of the subject. (Item 43) 42. The method of claim 41, wherein the composition is injected into an area selected from the group consisting of the nasolabial area, the upper lip area, the forehead, the eye area, and the cheek area. (Item 44) 42. The method of claim 41, wherein the composition is administered by injection using a prefilled syringe. (Item 45) 45. The method of claim 44, wherein the prefilled syringe is a 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mL prefilled syringe. (Item 46) 46. ​​The method of claim 45, wherein the prefilled syringe is sterile. (Item 47) 1. A method for promoting wound healing while relieving pain and discomfort in a subject in need thereof, the method comprising administering to the subject a composition comprising hyaluronan, wherein: the hyaluronan is present in the composition at a concentration greater than about 30 mg / mL; the average molecular weight of the hyaluronan is between about 1 million and about 2 million; The method wherein the hyaluronan is not cross-linked and / or is substantially free of chemical modifications, thereby alleviating pain and promoting wound healing. (Item 48) 48. The method of item 47, wherein the hyaluronan is present at a concentration of about 40 mg / mL to about 60 mg / mL. (Item 49) 1. A method for promoting wound healing while relieving pain and discomfort in a subject in need thereof, the method comprising administering to the subject a composition comprising hyaluronan, wherein: the hyaluronan is present in the composition at a concentration of about 40 mg / mL; the average molecular weight of the hyaluronan is between about 1 million and about 2 million; The method wherein the hyaluronan is not cross-linked and / or is substantially free of chemical modifications, thereby alleviating pain and promoting wound healing. (Item 50) 50. The method of claim 47 or 49, wherein the composition is substantially free of other pharmaceutically active substances. (Item 51) 50. The method of claim 47 or 49, wherein the composition further comprises a buffer. (Item 52) 52. The method of claim 51, wherein the buffer is phosphate buffered saline (PBS). (Item 53) 50. The method of claim 47 or 49, wherein the elasticity of the composition is at least about 200 Pascals when measured at a frequency of 0.5 Hz. (Item 54) 50. The method of claim 47 or 49, wherein the elasticity of the composition is at least about 1,000 Pascals when measured at a frequency of 0.5 Hz. (Item 55) 50. The method of claim 47 or 49, wherein the elasticity of the composition is at least about 2,000 Pascals when measured at a frequency of 0.5 Hz. (Item 56) 50. The method of claim 47 or 49, wherein the elasticity of the composition is at least about 4,000 Pascals when measured at a frequency of 0.5 Hz. (Item 57) 50. The method of claim 47 or 49, wherein the composition is sterile. (Item 58) 50. The method of claim 47 or 49, wherein the composition is administered topically. (Item 59) 59. The method of claim 58, wherein the composition is administered topically onto the surface of a wound or scar on the skin. [Brief explanation of the drawings]

[0037] [Figure 1]FIG. 1 is a graph showing the pressure required to expel a 4% HA solution through different sized needles. DETAILED DESCRIPTION OF THE INVENTION

[0038] Detailed Description of the Invention The present invention provides methods for relieving pain and discomfort associated with dry ocular conditions; skin care / dermatological treatment; and wound healing. The claimed methods involve administering a HA composition containing a high concentration of hyaluronan (HA) (e.g., about 30 mg / mL or greater). Such compositions have been determined to have high elasticity (e.g., a high elastic modulus G') when measured at frequencies between 0.1 and 10 Hz. HA compositions characterized by high elasticity (e.g., compositions containing high concentrations of HA) are surprisingly effective in relieving pain and discomfort associated with dry ocular conditions; cosmetic treatments; or wound healing. Without wishing to be bound by any particular theory, it is believed that the effectiveness of the HA compositions of the present invention containing high concentrations of HA in treating pain and discomfort is determined by their high elasticity, as evidenced by a high value of the elastic modulus G'. Without wishing to be bound by any particular theory, it is also believed that the effectiveness of the HA compositions of the present invention is determined by a relatively high probability of interaction of HA molecules with pain transduction channels (e.g., TRPV1), thereby reducing nociceptor excitability. The average molecular weight of the HA used in the methods of the present invention can be 2 million or less (eg, about 1 million to 2 million).

[0039] I. Hyaluronan Compositions for Use in the Methods of the Invention The present invention provides methods comprising administering a composition comprising hyaluronan (HA) to a subject in need thereof. In some embodiments, the composition comprises hyaluronan, wherein the hyaluronan is present in the composition at a concentration of greater than about 30 mg / mL (i.e., greater than about 3% weight / volume); the average molecular weight of the hyaluronan is between about 1 million and about 2 million; and the hyaluronan is not cross-linked and / or is substantially free of chemical modifications.

[0040] For example, the hyaluronan concentration in the composition may be about 30 mg / mL (i.e., about 3% w / v), about 35 mg / mL (i.e., about 3.5% w / v), about 40 mg / mL (i.e., about 4% w / v), about 45 mg / mL (i.e., about 4.5% w / v), about 50 mg / mL (i.e., about 5% w / v), about 55 mg / mL (i.e., about 5.5% w / v), about 60 mg / mL (i.e., about 6% w / v), about 65 mg / mL (i.e., about 6.5% w / v), about 70 mg / mL (i.e., about 7% w / v), about 75 mg / mL (i.e., about 7.5% w / v), about 80 mg / mL (i.e., about 8% w / v), about 85 mg / mL (i.e., about 8.5% w / v), about 90 mg / mL (i.e., about 9 % w / v), about 95 mg / mL (i.e., about 9.5% w / v), about 100 mg / mL (i.e., about 10% w / v), about 105 mg / mL (i.e., about 10.5% w / v), about 110 mg / mL (i.e., about 11% w / v), about 115 mg / mL (i.e., about 11.5% w / v), about 120 mg / mL (i.e., about 12% w / v), about 125 mg / mL (i.e., about 12.5% ​​w / v), about 130 mg / mL (i.e., about 13% w / v), about 135 mg / mL (i.e., about 13.5% w / v), about 140 mg / mL (i.e., about 14% w / v), about 145 mg / mL (i.e., about 14.5% w / v), or about 150 mg / mL (i.e., about 15% w / v). In one particular embodiment, HA is present in the composition at a concentration of about 40 mg / mL (ie, about 4% w / v).In other specific embodiments, HA is present in the compositions of the invention at a concentration of about 41 mg / mL (i.e., about 4.1% w / v), about 42 mg / mL (i.e., about 4.2% w / v), about 43 mg / mL (i.e., about 4.3% w / v), about 44 mg / mL (i.e., about 4.4% w / v), about 45 mg / mL (i.e., about 4.5% w / v), about 46 mg / mL (i.e., about 4.6% w / v), about 47 mg / mL (i.e., about 4.7% w / v), about 48 mg / mL (i.e., about 4.8% w / v), about 49 mg / mL (i.e., about 4.9% w / v), about 50 mg / mL (i.e., about 5.0% w / v), about 51 mg / mL (i.e., about 5.1% w / v), about 52 mg / mL (i.e., about 5.2% w / v), about 53 mg / mL (i.e., about 5.3% w / v), about 54 mg / mL (i.e., about 5.4% w / v), about 55 mg / mL (i.e., about 5.5% w / v), about 56 mg / mL (i.e., about 5.6% w / v), about 57 mg / mL (i.e., about 5.7% w / v), about 58 mg / mL (i.e., about 5.8% w / v), 59 mg / mL (i.e., about 5.9% w / v), or about 60 mg / mL (i.e., about 6% w / v).

[0041] In some examples, the hyaluronan concentration in the composition can be greater than about 30 mg / mL (i.e., about 3% w / v) (e.g., greater than about 31 mg / mL (i.e., about 3.1%), greater than about 32 mg / mL (i.e., about 3.2%), greater than about 33 mg / mL (i.e., about 3.3%), greater than about 34 mg / mL (i.e., about 3.4%), greater than about 35 mg / mL (i.e., about 3.5%), greater than about 36 mg / mL (i.e., about 3.6%), greater than about 37 mg / mL (i.e., about 3.7%), greater than about 38 mg / mL (i.e., about 3.8%), or greater than about 39 mg / mL (i.e., about 3.9%).

[0042] In some examples, the hyaluronan concentration in the composition can have an average molecular weight of between about 1 million and about 2 million (e.g., between about 1.1 million and about 2 million, between about 1.2 million and about 2 million, between about 1.3 million and about 2 million, between about 1.4 million and about 2 million, between about 1.5 million and about 2 million, or between about 1.6 million and about 2 million).

[0043] In certain embodiments, the hyaluronan used in the composition is not cross-linked and / or does not contain chemical modifications. For example, the hyaluronan used in the composition does not contain amidation, which may be formed by the reaction between the carboxyl group of HA and the amine group of a derivatizing agent, as described, for example, in European Patent No. 1095064B1. The hyaluronan used in the composition may also be free of chemical modifications and / or cross-linking, which may result from the reaction of hyaluronan with a carbodiimide (such as a monocarbodiimide or a biscarbodiimide), as described, for example, in U.S. Patent No. 8,323,617. In some cases, the hyaluronan used in the composition may also be free of acrylates (e.g., methacrylates), as described, for example, in U.S. Patent Application Publication No. 2010 / 0048755; sulfates, as described, for example, in U.S. Patent Application Publication No. 2013 / 0209531; and deuterium, as described, for example, in U.S. Patent Application Publication No. 2015 / 0148310.

[0044] In some embodiments, the HA compositions of the present invention do not contain other pharmaceutically active substances. As used herein, a "pharmaceutically active substance" is a substance that can exert a biological effect on a subject (e.g., a human or animal subject). The term "pharmaceutically active substance" also includes substances that can modulate the biological effect of an HA composition when the composition is administered to a subject (e.g., relief of pain and discomfort associated with dry eye conditions; skin care / dermatological treatment; or wound healing). In certain embodiments, the pharmaceutically active substance is a protein (e.g., a bone morphogenetic protein (BMP) such as rhGDF-5). In certain embodiments, the pharmaceutically active substance is a glycosaminoglycan (GAG) other than HA (e.g., chondroitin). In some embodiments, the pharmaceutically active substance is hydroxypropyl methylcellulose. In other embodiments, the pharmaceutically active substance is a local anesthetic (e.g., lidocaine or bupivacaine). In some cases, the pharmaceutically active agent is a purinergic receptor agonist (eg, a P2Y2 receptor agonist).

[0045] In certain embodiments, the HA compositions of the invention do not contain molecules that can scavenge free radicals, such as polyols (e.g., sorbitol, maltitol, xylitol, or isomalt). In other embodiments, the HA compositions used in the methods of the invention do not contain molecules that decrease the elasticity of HA (e.g., dextran or sucrose).

[0046] In some cases, the HA compositions used in the methods of the invention do not include polyglycols (eg, polyethylene glycol).

[0047] The HA composition can consist essentially of HA having an average molecular weight of between about 1 million and about 2 million, present in a physiological buffer (e.g., phosphate buffer or bicarbonate buffer) at a concentration of greater than about 30 mg / mL (about 3% w / v) or about 40 mg / mL (about 4% w / v). For example, the HA composition used in the methods of the present invention consists essentially of HA having an average molecular weight of between about 1 million and about 2 million, present at a concentration of about 40 mg / mL (i.e., about 4% w / v).

[0048] The hyaluronan in the compositions used in the methods of the invention may have an elasticity of at least 100 Pascal when measured at a frequency of 0.5 Hz, or at least 400 Pascal when measured at a frequency of 0.5 Hz, or at least 1,000 Pascal when measured at a frequency of 0.5 Hz, or at least 2,000 Pascal when measured at a frequency of 0.5 Hz, or at least 4,000 Pascal when measured at a frequency of 0.5 Hz, or an elasticity between 400 and 5,000 Pascal when measured at a frequency of 0.5 Hz.

[0049] It should be appreciated that various methods are available for measuring the elasticity of biopolymers (such as hyaluronan). In one embodiment, the elasticity of a composition comprising hyaluronan is measured as pressure (expressed in Pascals) at a specific frequency (expressed in Hertz). For example, the frequency that can be used to evaluate the elasticity of the hyaluronan compositions provided herein can be measured at 0.5 Hz, 2.5 Hz, or 5.0 Hz.

[0050] It should be further recognized that elasticity can be exhibited at any relevant frequency. Thus, for example, in one embodiment, a composition that exhibits elasticity at a frequency of 2.5 Hz and comprises highly elastic hyaluronan has an elasticity of at least 200 Pa at a frequency of 2.5 Hz. Similarly, in one embodiment, a composition that exhibits elasticity at a frequency of 5.0 Hz and comprises highly elastic hyaluronan has an elasticity of at least 400 Pa at a frequency of 5.0 Hz.

[0051] In one embodiment, the elasticity of the composition comprising hyaluronan used in the presently claimed methods is at least 100 Pascals when measured at a frequency of 0.5 Hz. In some embodiments, the elasticity of the composition is at least 300 Pascals when measured at a frequency of 2.5 Hz. In some embodiments, the elasticity of the composition is at least 350 Pascals when measured at a frequency of 5.0 Hz.

[0052] In a further embodiment, the elasticity of the composition comprising hyaluronan is at least 400 Pascals when measured at a frequency of 0.5 Hz. In some embodiments, the elasticity of the composition is at least 750 Pascals when measured at a frequency of 2.5 Hz. In some embodiments, the elasticity of the composition is at least 900 Pascals when measured at a frequency of 5.0 Hz.

[0053] In another embodiment, the elasticity of the composition comprising hyaluronan is at least 1000 Pascals when measured at a frequency of 0.5 Hz. In some embodiments, the elasticity of the composition is at least 1600 Pascals when measured at a frequency of 2.5 Hz. In some embodiments, the elasticity of the composition is at least 2000 Pascals when measured at a frequency of 5.0 Hz.

[0054] In yet another embodiment, the elasticity of the composition comprising hyaluronan is at least 2600 Pascals when measured at a frequency of 0.5 Hz. In some embodiments, the elasticity of the composition is at least 4000 Pascals when measured at a frequency of 2.5 Hz. In some embodiments, the elasticity of the composition is at least 4500 Pascals when measured at a frequency of 5.0 Hz.

[0055] In one embodiment, the elasticity of the composition comprising hyaluronan is at least 4000 Pascals when measured at a frequency of 0.5 Hz. In some embodiments, the elasticity of the composition is at least 5000 Pascals when measured at a frequency of 2.5 Hz. In some embodiments, the elasticity of the composition is at least 6000 Pascals when measured at a frequency of 5.0 Hz.

[0056] In some embodiments, the elasticity of the composition is between 100 and 10,000 Pascals when measured at a frequency of 0.5 Hz. In some embodiments, the elasticity of the composition is between 400 and 5,000 Pascals when measured at a frequency of 0.5 Hz. In some embodiments, the elasticity of the composition is between 1,000 and 2,000 Pascals when measured at a frequency of 0.5 Hz.

[0057] In some embodiments, the elasticity of the composition is between 300 and 10,000 Pascals when measured at a frequency of 2.5 Hz. In some embodiments, the elasticity of the composition is between 750 and 6,000 Pascals when measured at a frequency of 2.5 Hz. In some embodiments, the elasticity of the composition is between 1,500 and 4,000 Pascals when measured at a frequency of 2.5 Hz.

[0058] In some embodiments, the elasticity of the composition is between 300 and 10,000 Pascals when measured at a frequency of 5.0 Hz. In some embodiments, the elasticity of the composition is between 900 and 7,000 Pascals when measured at a frequency of 5.0 Hz. In some embodiments, the elasticity of the composition is between 2,000 and 5,000 Pascals when measured at a frequency of 5.0 Hz.

[0059] In some embodiments, elasticity can be measured using a suitable device (e.g., a rheometer). For example, elasticity can be measured using a Stresstech High Resolution Research rheometer (Reologica Instruments AB). Typically, elasticity is determined at ambient temperature and pressure; however, it should be recognized that elasticity can also be measured at non-ambient temperatures and / or non-ambient pressures. It should further be recognized that those skilled in the art will know how to convert the magnitude of elasticity determined at various temperatures and pressures to the magnitude of elasticity at ambient temperature and pressure.

[0060] Highly elastic compositions of hyaluronan can be prepared by increasing the concentration of hyaluronan in the composition. Thus, in one embodiment, the presently claimed methods utilize highly elastic compositions with a high proportion of hyaluronan. For example, the composition may contain at least 3.0% hyaluronan (weight / volume), at least 3.5% hyaluronan (weight / volume), at least 4.0% hyaluronan (weight / volume), at least 4.5% hyaluronan (weight / volume), at least 5.0% hyaluronan (weight / volume), at least 5.5% hyaluronan (weight / volume), at least 6.0% hyaluronan (weight / volume), at least 6.5% hyaluronan (weight / volume), at least 7.0% hyaluronan (weight / volume), at least 7.5% hyaluronan (weight / volume), at least 8. 0% hyaluronan (wt / volume), at least 8.5% hyaluronan (wt / volume), at least 8.9% hyaluronan (wt / volume), at least 9.0% hyaluronan (wt / volume), at least 10.0% hyaluronan (wt / volume), at least 11.0% hyaluronan (wt / volume), at least 12.0% hyaluronan (wt / volume), at least 13.0% hyaluronan (wt / volume), at least 14.0% hyaluronan (wt / volume), or at least 15.0% or more hyaluronan (wt / volume).

[0061] Ranges intermediate to the recited values ​​are also intended to be encompassed in the compositions used in the methods of the invention. For example, the hyaluronan content in the composition can be between about 3% and about 15% (wt / vol), between about 3% and about 10% (wt / vol), between about 3.5% and about 9% (wt / vol), between about 4% and about 8% (wt / vol), or between about 5% and about 7% (wt / vol).

[0062] It should be further recognized that the amount of hyaluronan in a particular volume can be expressed in alternative terms (e.g., grams / liter or moles / liter). Those skilled in the art will know how to convert between different terms for expressing the amount of hyaluronan in a particular volume.

[0063] Hyaluronan compositions having a high concentration of hyaluronan with an average molecular weight of about 1-2 million are particularly effective in treating pain (e.g., pain associated with dry eye conditions, cosmetic treatments, or wound healing). Thus, the average molecular weight of hyaluronan contained in the HA compositions described herein can fall within the range of between 1 million and 2 million, and also less than 2 million, less than 1.9 million, less than 1.8 million, less than 1.7 million, less than 1.6 million, less than 1.5 million, less than 1.4 million, less than 1.3 million, less than 1.2 million, less than 1.1 million, less than 1 million, less than 900,000, less than 800,000, less than 700,000, less than 600,000, or less than 500,000. In other cases, the average molecular weight of the hyaluronan contained in the HA compositions described herein can fall within the range between 1 million and 2 million, and also greater than 100,000, greater than 200,000, greater than 300,000, greater than 400,000, greater than 500,000, greater than 600,000, greater than 700,000, greater than 800,000, greater than 900,000, greater than 1 million, greater than 1.1 million, greater than 1.2 million, greater than 1.3 million, greater than 1.4 million, greater than 1.5 million, greater than 1.6 million, greater than 1.7 million, greater than 1.8 million, or greater than 1.9 million.

[0064] Ranges intermediate to the recited values ​​are also intended to be part of the invention, for example, in the hyaluronan compositions provided herein, the average molecular weight of the hyaluronan is between 1 million and 2 million, between 1 million and 1.5 million, between 0.5 million and 1 million, between 0.5 million and 2 million, or between 0.9 million and 1.4 million.

[0065] In some embodiments of the hyaluronan compositions described herein, the average molecular weight of the majority of the hyaluronan present in the composition falls within the ranges described herein. Thus, for example, in a composition having hyaluronan with an average molecular weight between 200,000 and 2,000,000, at least 95% of the hyaluronan present in the composition falls within the range between 200,000 and 2,000,000. In some embodiments, the average molecular weight of at least 50% of the hyaluronan present in the compositions provided herein falls within the recited range. In some embodiments, the average molecular weight of at least 60% of the hyaluronan present in the compositions provided herein falls within the recited range. In some embodiments, the average molecular weight of at least 70% of the hyaluronan present in the compositions provided herein falls within the recited range. In some embodiments, the average molecular weight of at least 80% of the hyaluronan present in the compositions provided herein falls within the recited range. In some embodiments, the average molecular weight of at least 90% of the hyaluronan present in the compositions provided herein falls within the recited range. In some embodiments, the average molecular weight of at least 95% of the hyaluronan present in the compositions provided herein falls within the recited range. In some embodiments, the average molecular weight of at least 98% of the hyaluronan present in the compositions provided herein falls within the recited range. In some embodiments, the average molecular weight of at least 99% of the hyaluronan present in the compositions provided herein falls within the recited range. In some embodiments, the average molecular weight of at least 99.9% of the hyaluronan present in the compositions provided herein falls within the recited range.

[0066] II. Sources of Hyaluronan The hyaluronan used in the compositions and methods described herein can be obtained from any source. Generally, hyaluronan has the same chemical structure regardless of its source (e.g., chicken or rooster comb, human tissue, or bacterial cell wall). Hyaluronan can be obtained, for example, from chicken or rooster comb, bacterial cell wall, and human tissue (umbilical cord, vitreous body, synovial fluid from joints, etc.). In some embodiments, hyaluronan is isolated from chicken comb. In some embodiments, hyaluronan is isolated from human tissue (e.g., umbilical cord, vitreous body, synovial fluid from joints). In some embodiments, hyaluronan is isolated from cell culture. In some embodiments, hyaluronan is isolated from bacterial cell wall. The isolation of hyaluronan from various sources is known to those skilled in the art. For example, the harvesting and purification of hyaluronan from rooster comb is described in U.S. Patent No. 4,141,973, while the harvesting and purification of hyaluronan from bacterial sources is described in U.S. Patent No. 4,517,295. In some embodiments, hyaluronan is purified and harvested in a 0.15 M NaCl solution at a pH of 6-8. Generally, hyaluronan obtained from various sources is free of proteins and glycosaminoglycans other than hyaluronan.

[0067] In some embodiments, the isolated hyaluronan is further purified to obtain hyaluronan having a desired average molecular weight range (e.g., by column chromatography). Methods for purifying hyaluronan having a desired average molecular weight range are known to those skilled in the art.

[0068] In one aspect, the highly elastic hyaluronan disclosed herein is unmodified hyaluronan. However, it should be recognized that in some embodiments, hyaluronan can be chemically modified. For example, hyaluronan can be chemically modified to increase the elasticity of the hyaluronan.

[0069] III. Sterilization of Hyaluronan Compositions In some embodiments, the HA compositions described herein and used in the methods of the present invention are sterile. As used herein, a "sterile composition" refers to a composition that is safe to administer to a subject (e.g., a human subject). Thus, a sterile composition contains a minimum number of agents that may cause unwanted side effects (such as unwanted tissue response, immune response, inflammation, or infection).

[0070] Methods for sterilizing hyaluronan compositions are known in the art and include, for example, heat sterilization or steam sterilization (e.g., by autoclaving). In some embodiments, the HA compositions of the present invention are sterilized by heating the composition. In some embodiments, the HA compositions of the present invention are sterilized by placing the HA composition in a syringe and autoclaving the syringe containing the HA at 131°C for 2 minutes or 121°C for 15 minutes, followed by immediate cooling.

[0071] IV. Additional Components for Hyaluronan Compositions The HA compositions described herein may contain additional components that can stabilize hyaluronan and / or make the compositions more suitable for administration to a subject. In some embodiments, the HA compositions of the present invention may contain a buffer. The buffer is added to stabilize the pH. Buffers suitable for use in the present invention include phosphate buffers and bicarbonate buffers. In some embodiments, the buffer is Tris-phosphate buffer. In some embodiments, the buffer is present at a concentration between 1 mM and 100 mM, between 2 mM and 50 mM, or between 5 mM and 20 mM. In some embodiments, the buffer concentration is less than 1 mM. In some embodiments, the buffer concentration is greater than 100 mM. In some embodiments, the buffer concentration is 10 mM. It should be recognized that the buffer concentration will depend on the nature of the buffer used. In some embodiments, the pH of the composition is between pH 7 and pH 9 or between pH 7.5 and pH 8.5. In some embodiments, the pH of the composition is 8.0. In some embodiments, the pH of the composition is 7.5. In some embodiments, the pH of the composition is 8.5. If necessary, an acid (such as HCl) or a base (such as NaOH) can be added to the composition to obtain the desired pH.

[0072] For example, the HA composition can include a buffer (eg, a physiologically compatible buffer) but no additional components.

[0073] The HA composition may also include a stabilizing excipient, such as a carboxylic acid or its salt. In some embodiments, the composition includes a monocarboxylic acid and / or a salt thereof. In some embodiments, the composition includes gluconic acid and / or sodium gluconate. In some embodiments, the composition includes a dicarboxylic acid and / or a salt thereof. In some embodiments, the composition includes citric acid, succinic acid, malonic acid, maleic acid, tartaric acid, and / or a salt thereof. In some embodiments, the carboxylic acid is sodium citrate. In some embodiments, the composition includes a tricarboxylic acid. In some embodiments, the composition comprises nitrilotriacetic acid and / or sodium nitrilotriacetate. In some embodiments, the composition comprises a tetracarboxylic acid and / or a salt thereof. In some embodiments, the composition comprises ethylenediaminetetracetic acid (EDTA) and / or sodium EDTA. In some embodiments, the composition comprises a pentacarboxylic acid and / or a salt thereof. In some embodiments, the composition comprises diethylenetriaminepentaacetic acid (DTPA) and / or sodium DTPA. Suitable carboxylic acids include, but are not limited to, citrate compounds (such as sodium citrate); tartrate compounds, succinate compounds, and EDTA. Kaushil et al., Protein Science 1999 8:222-233 and Busby et al., The Journal of Biological Chemistry Volume 256, Number 23, pp. 12140-1210-12147 describes carboxylic acids and their uses. In some embodiments, the concentration of the stabilizing excipient is 50-600 mM, 250-500 mM, or 250-350 mM. In some embodiments, the concentration of the stabilizing excipient is 300 mM. In some embodiments, the concentration of the stabilizing excipient is less than 100 mM. In some embodiments, the concentration of the stabilizing excipient is greater than 600 mM.

[0074] The HA compositions described herein may also contain sugars (e.g., disaccharides). Disaccharides that can be added to the compositions include, but are not limited to, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, dextrose, and dextran. In some cases, the sugar may be present at 0.5-5% (wt / volume). In some cases, the sugar may be present at 1-2% (wt / volume). In one embodiment, the sugar may be present at 1%. In some embodiments, the sugar may be present at less than 1% (wt / volume). In some embodiments, the sugar may be present at more than 5% (wt / volume). In one embodiment, the sugar may be sucrose or trehalose and is present at 1% (wt / volume).

[0075] In some embodiments, the HA compositions described herein may contain salt. Salts that can be used in the compositions include sodium chloride and other physiologically compatible salts. In some embodiments, the concentration of salt present in the HA compositions described herein is between about 10 mM and about 250 mM, between about 25 mM and about 100 mM, between about 30 mM and about 70 mM, between about 45 mM and about 150 mM, between about 125 mM and about 200 mM, between about 150 mM and about 250 mM, or between about 190 mM and about 250 mM. In some embodiments, the salt concentration is 50 mM. In some embodiments, the salt concentration is less than 10 mM. In some embodiments, the salt concentration is greater than 250 mM. In one specific embodiment, the salt concentration is between about 50 mM and about 200 mM.

[0076] In some embodiments, the osmolarity of the HA compositions of the present invention can be normal (e.g., about 310 mOsm / L). In other embodiments, the osmolarity of the HA compositions of the present invention can be less than normal osmolarity (e.g., less than 310 mOsm / L). For example, the osmolarity of the HA compositions of the present invention can be between about 20 mOsm / L and about 500 mOsm / L (e.g., between about 150 mOsm / L and about 310 mOsm / L, between about 150 mOsm / L and about 200 mOsm / L, between about 160 mOsm / L and about 220 mOsm / L, between about 180 mOsm / L and about 250 mOsm / L). The blood glucose level may be between about 200 mOsm / L and about 300 mOsm / L, between about 250 mOsm / L and about 310 mOsm / L, between about 290 mOsm / L and about 310 mOsm / L, between about 250 mOsm / L and about 290 mOsm / L, or between about 270 mOsm / L and about 300 mOsm / L). In some embodiments, the osmolarity of the HA compositions of the present invention is about 150 mOsm / L, about 155 mOsm / L, about 160 mOsm / L, about 165 mOsm / L, about 170 mOsm / L, about 175 mOsm / L, about 180 mOsm / L, about 185 mOsm / L, about 190 mOsm / L, about 195 mOsm / L, about 200 mOsm / L, about 205 mOsm / L, about 210 mOsm / L, about 215 mOsm / L, about 220 mOsm / L, about 225 mOsm / L, about 230 mOsm / L, about 235 mOsm / L, about 240 mOsm / L, about 245 mOsm / L, about 250 mOsm / L, about 255 mOsm / L, about 260 mOsm / L, about 265 mOsm / L, about 270 mOsm / L, about 275 mOsm / L, about 280 mOsm / L, about 285 mOsm / L, about 290 mOsm / L, about 300 mOsm / L, about 310 mOsm / L, about 315 mOsm / L, about 320 mOsm / L, about 325 mOsm / L, about 330 mOsm / L, about 335 mOsm / L, about 340 mOsm / L, about 345 mOsm / L, about 350 mOsm / L, about 355 mOsm / L, about 360 mOsm / L, The blood pressure may be about 230 mOsm / L, about 235 mOsm / L, about 240 mOsm / L, about 245 mOsm / L, about 250 mOsm / L, about 255 mOsm / L, about 260 mOsm / L, about 265 mOsm / L, about 270 mOsm / L, about 275 mOsm / L, about 280 mOsm / L, about 285 mOsm / L, about 290 mOsm / L, about 295 mOsm / L, about 300 mOsm / L, about 305 mOsm / L, or about 310 mOsm / L. In one particular embodiment, the osmolarity of the HA compositions of the present invention is between about 100 mOsm / L and about 400 mOsm / L, e.g., about 100 mOsm / L, about 110 mOsm / L, about 120 mOsm / L, about 130 mOsm / L, about 140 mOsm / L, or about 150 mOsm / L.

[0077] In some embodiments, the HA compositions described herein contain one or more antioxidants. Antioxidants are substances that can inhibit oxidation by scavenging free radicals from a solution. Antioxidants are well known to those skilled in the art and include substances such as ascorbic acid, ascorbic acid derivatives (e.g., ascorbyl palmitate, ascorbyl stearate, sodium ascorbate, or calcium ascorbate), butylated hydroxyanisole, butylated hydroxytoluene, alkyl gallates, sodium metabisulfite, sodium bisulfite, sodium dithionite, sodium thioglycolate, sodium formaldehyde sulfoxylate, tocopherol and its derivatives (d-alpha tocopherol, d-alpha tocopherol acetate, d-alpha tocopherol succinate, beta tocopherol, delta tocopherol, gamma tocopherol, and d-alpha tocopherol polyoxyethylene glycol 1000 succinate), monothioglycerol, and sodium sulfite. Such substances are typically added in the range of 0.01 to 2.0%.

[0078] In some embodiments, the HA composition may include one or more isotonic agents. This term is used interchangeably in the art with isotonic agent and is known as a compound that can be added to a composition to increase osmotic pressure (such as the osmotic pressure of a 0.9% sodium chloride solution that is isotonic with human extracellular fluids (such as plasma)). Preferred isotonic agents that can be used in the HA composition include sodium chloride, mannitol, sorbitol, lactose, dextrose, and glycerol.

[0079] In some embodiments, the HA compositions of the present invention may contain one or more preservatives. Suitable preservatives include, but are not limited to, chlorobutanol (0.3-0.9% w / v), parabens (0.01-5.0%), thimerosal (0.004-0.2%), benzyl alcohol (0.5-5%), and phenol (0.1-1.0%).

[0080] In some embodiments, the HA composition may include components suitable for ophthalmic use.

[0081] In some embodiments, the HA composition may include one or more components that minimize unwanted side effects during injection of the composition.

[0082] V. Hyaluronan Compositions for Ophthalmic Use The present invention provides a method for alleviating pain and discomfort associated with a dry eye condition by administering to the eye of a subject a composition comprising HA.

[0083] Any of a variety of carriers can be used in HA compositions for ophthalmic use, including water, mixtures of water and water-miscible solvents (e.g., C1-C7 alkanols), vegetable or mineral oils containing 0.5-5% non-toxic water-soluble polymers, natural products (e.g., gelatin, alginate, pectin, tragacanth, karaya gum, xanthan gum, carrageenan, agar, and acacia, starch derivatives (e.g., starch acetate and hydroxypropyl starch), and other synthetic products (e.g., polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, and preferably cross-linked polyacrylic acid (e.g., neutral carbopol)), or mixtures of these polymers. The concentration of the carrier is typically 1 to 100,000 times that of the active ingredient.

[0084] Additional components that may be included in HA compositions for ophthalmic use include tonicity enhancing agents, preservatives, solubilizers, non-toxic excipients, emollients, sequestering agents, pH adjusters, co-solvents, and viscosity enhancers.

[0085] Buffers can be particularly useful for adjusting the pH, preferably to physiological pH. The pH of hyaluronan compositions suitable for ocular administration should be maintained within the range of pH 4.0 to 8.0, more preferably about pH 4.0 to 6.0, and more preferably about pH 6.5 to 7.8. Suitable buffers can be added, such as boric acid, sodium borate, potassium citrate, citric acid, sodium bicarbonate, tris(hydroxymethyl)aminomethane (TRIS), and various mixed phosphate buffers (including combinations of NaHPO, NaHPO, and KHPO), and mixtures thereof. Generally, buffers are used in amounts ranging from about 0.05 to 2.5 percent by weight, preferably 0.1 to 1.5 percent.

[0086] Tonicity is typically adjusted, if necessary, using a tonicity enhancer. Such agents can be, for example, ionic and / or non-ionic. Examples of ionic tonicity enhancers include alkali metal halides or alkaline earth metal halides (e.g., CaCl2, KBr, KCl, LiCl, NaI, NaBr or NaCl, Na2SO4, or boric acid). Non-ionic tonicity enhancers include, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose. The osmolality of the hyaluronan composition used in the methods of the present invention can be adjusted to approximately the osmotic pressure of normal tears (equivalent to a 0.9% sodium chloride solution or a 2.5% glycerol solution) using a tonicity agent. In some instances, an osmolality between about 100 mOsm / L and about 150 mOsm / L (e.g., 100 mOsm / L, 110 mOsm / L, 120 mOsm / L, 120 mOsm / L, 130 mOsm / L, 140 mOsm / L, or 150 mOsm / L) is preferred. In other instances, an osmolality between about 225 and 400 mOsm / L (e.g., between about 280 mOsm / L and about 320 mOsm / L, or between about 297 mOsm / L and about 318 mOsm / L) is preferred. In some cases, the average osmolality may vary between about 303.7 mOsm / L and about 306.7 mOsm / L.

[0087] In certain embodiments, the HA composition for ophthalmic use may further comprise a preservative. The preservative can typically be selected from quaternary ammonium compounds (such as benzalkonium chloride (N-benzyl-N-(C8-C18 alkyl)-N,N-dimethylammonium chloride) or benzoxonium chloride). Other examples of suitable preservatives include alkylmercury salts of thiosalicylic acid (e.g., thimerosal); phenylmercuric nitrate, phenylmercuric acetate, or phenylmercuric borate; sodium perborate; sodium chlorite; parabens (e.g., methylparaben or propylparaben); alcohols (e.g., chlorobutanol, benzyl alcohol, or phenylethanol); guanidine derivatives (e.g., chlorhexidine or polyhexamethylene biguanide); imidazolidinyl urea (Germar™), or sorbic acid. Preferred preservatives are quaternary ammonium compounds, particularly benzalkonium chloride or its derivatives (such as Polyquad (see U.S. Pat. No. 4,407,791)), alkyl mercury salts, and parabens. Where appropriate, a sufficient amount of preservative is added to the ophthalmic composition to ensure protection against cross-contamination during use due to bacteria and fungi.

[0088] In another embodiment, the HA composition for ophthalmic use is preservative-free, and such compositions are useful for patients who wear contact lenses, or who use some topical eye drops and / or who have pre-existing ocular surface disorders (e.g., dry eye syndrome), where limiting exposure to preservatives may be more desirable.

[0089] HA compositions for ophthalmic use may further require a solubilizer, especially if the active or inactive ingredients tend to form suspensions or emulsions. Suitable solubilizers for the hyaluronan compositions used in the methods of the present invention include, for example, tyloxapol, fatty acid glycerol polyethylene glycol esters, fatty acid polyethylene glycol esters, polyethylene glycol, glycerol ethers, cyclodextrins (e.g., α-, β-, or γ-cyclodextrins (e.g., alkylated, hydroxyalkylated, carboxyalkylated, or alkyloxycarbonyl-alkylated derivatives), or mono- or diglycosyl-α-, β-, or γ-cyclodextrin, mono- or dimaltosyl-α-, β-, or γ-cyclodextrin, or panosyl-cyclodextrin), polysorbate 20, polysorbate 80, or mixtures of these compounds. A preferred solubilizer may be a reaction product of castor oil with ethylene oxide (e.g., the commercially available products Cremophor EL® or Cremophor RH40®). The reaction product of castor oil with ethylene oxide has proven to be a particularly excellent solubilizer, being very well tolerated by the eye. Another preferred solubilizer may be tyloxapol or cyclodextrin. The concentration used depends, inter alia, on the hyaluronan concentration in the composition. The amount added is typically sufficient to solubilize the hyaluronan in the composition. For example, the solubilizer concentration is 0.1 to 5,000 times the hyaluronan concentration in the composition.

[0090] HA compositions for ophthalmic use may also further include non-toxic excipients such as emulsifiers, humectants, or fillers (e.g., polyethylene glycols designated 200, 300, 400, and 600, or carbowaxes designated 1000, 1500, 4000, 6000, and 10000, etc.) The amount and type of excipient added will depend on the specific requirements and generally range from about 0.0001 to about 90% by weight.

[0091] Other compounds can also be added to the formulations of the present invention to increase the viscosity of the carrier. Examples of viscosity-enhancing agents include, but are not limited to, polysaccharides (such as chondroitin sulfate and its salts, dextran, various polymers of the cellulose family, etc.); vinyl polymers; and acrylic acid polymers.

[0092] In one embodiment, hyaluronan compositions intended for ophthalmic use may contain, in addition to HA, additional components typically found in ophthalmic compositions. Examples of such components may include, but are not limited to, other active ingredients (vasoconstrictors, antiallergic agents, anti-infective agents, steroids, anesthetics, anti-inflammatory agents, analgesics, dry eye agents (e.g., secretagogues, mucus mimetics, polymers, lipids, antioxidants)) or may be administered in conjunction with (simultaneously or sequentially with) compositions containing other active ingredients (vasoconstrictors, antiallergic agents, anti-infective agents, steroids, anesthetics, anti-inflammatory agents, analgesics, or dry eye agents (e.g., secretagogues, mucus mimetics, polymers, lipids, antioxidants)).

[0093] In some embodiments, the hyaluronan compositions of the invention are administered to the ocular surface of a subject, such as under the subject's eyelid (e.g., under the upper or lower eyelid) or at the subject's corneal-eyelid junction. In some embodiments, the hyaluronan compositions of the invention are not suitable for or will not be used as a viscous surgical tool or device during ophthalmic surgery (or are not suitable for or will not be injected intraocularly during ophthalmic surgery).

[0094] In one example, the hyaluronan composition of the invention is administered to a subject immediately prior to resting or going to sleep.

[0095] VI. Hyaluronan Compositions for Skin Care / Dermatological Uses The present invention also provides a method for alleviating pain and discomfort while minimizing at least one skin imperfection in a subject in need thereof by administering a composition comprising HA. Such HA compositions intended for skin care / dermatological use may contain, in addition to HA, additional ingredients typically found in such compositions (e.g., compositions used as dermal fillers). Such ingredients may include, for example, collagen, carnitine, vitamin E, vitamin A, and chondroitin sulfate.

[0096] In some embodiments, the HA composition for skin care / dermatological use is injected into the skin.Therefore, the HA composition can be formulated in the form of a container (for example, a pre-filled syringe) that is filled with the composition.Any pre-filled syringe known to those skilled in the art can be used in combination with the composition of the present invention.Pre-filled syringes that can be used include, for example, those described in PCT International Publication No. 05032627, PCT International Publication No. 08094984, PCT International Publication No. 9945985, PCT International Publication No. 03077976, US Patent No. 6792743, US Patent No. 5607400, US Patent No. 5893842, US Patent No. 7081107, US Patent No. 7041087, US Patent No. 5989 227, 6807797, 6142976, 5899889, U.S. Patent Application Publication Nos. 20070161961A1, 20050075611A1, 20070092487A1, 20040267194A1, or 20060129108A1. The pre-filled syringe can be made from a variety of materials. In one embodiment, the pre-filled syringe is a glass syringe. In another embodiment, the pre-filled syringe is a plastic syringe. Those skilled in the art will understand that the nature and / or quality of the material used to manufacture the syringe can affect the stability of the HA composition stored in the syringe. In one embodiment, the pre-filled syringe contains a silicone-based lubricant. In one embodiment, the pre-filled syringe contains baked-on silicone. In another embodiment, the pre-filled syringe does not contain a silicone-based lubricant. Those skilled in the art also understand that small amounts of contaminants that enter the formulation from the syringe barrel, syringe tip cap, plunger, or stopper can also affect the stability of the composition. For example, it is understood that tungsten introduced during the manufacturing process can adversely affect the stability of the formulation. In one embodiment, the pre-filled syringe can contain tungsten at levels greater than 500 ppb. In another embodiment, the pre-filled syringe is a low-tungsten syringe.In another embodiment, the pre-filled syringe may contain tungsten at a level between about 500 ppb and about 10 ppb, between about 400 ppb and about 10 ppb, between about 300 ppb and about 10 ppb, between about 200 ppb and about 10 ppb, between about 100 ppb and about 10 ppb, between about 50 ppb and about 10 ppb, or between about 25 ppb and about 10 ppb.

[0097] VII. Hyaluronan Compositions for Wound Healing The present invention also provides a method for promoting wound healing while relieving pain and discomfort in a subject in need thereof. Such HA compositions intended for topical use may contain, in addition to HA, additional ingredients typically found in such compositions. Such ingredients may include, for example, 1,3-butylene glycol, glycerin, xanthan gum, sodium chondroitin sulfate, ethanol, methyl p-hydroxybenzoate, polyoxyethylene-polyoxypropylene, decyltetradecyl ether, sodium citrate, sodium edetate, and vitamins (e.g., vitamin A, vitamin B, vitamin C, vitamin D, vitamin E, and vitamin K). Such ingredients may also include additional pharmaceutically active substances. In some cases, these additional pharmaceutically active substances do not include analgesics for pain treatment. In other cases, these pharmaceutically active substances include antibiotics (e.g., antibiotics typically used to treat skin infections and intended for topical use).

[0098] The HA compositions used in the methods disclosed herein also encompass finished, packaged, and labeled pharmaceutical products in which the appropriate unit dosage form is placed in a suitable vessel or container (such as a glass vial, pre-filled syringe, or other sealed container). In one embodiment, the unit dosage form is provided as a sterile, particle-free HA composition suitable for parenteral administration (e.g., intradermal or subcutaneous administration to a subject (e.g., the face, neck, arms, legs, or back of a subject)).

[0099] As with any pharmaceutical product, the packaging materials and containers are designed to protect the stability of the product during storage and transport. Additionally, the products of the present invention include instructions or other informational material that advise a physician, technician, or patient on how to appropriately prevent or treat the disease or disorder of interest, as well as how and how often to administer the HA composition. In other words, the article of manufacture includes instruction means that indicate or suggest a dosing regimen (including, but not limited to, the actual dose, monitoring procedures, and other monitoring information).

[0100] VIII. Methods of Relieving Pain and Discomfort Associated with Dry Eye Disease Dry eye disease is an eye condition that affects approximately 10-20% of the population. It can be associated with a pathological condition (such as cataracts or autoimmune disorders) or can occur in the absence of a pathological condition, under certain circumstances that dry out the ocular surface, such as prolonged eye contact, computer work, dry environments, contact lens use, or exposure to medications.

[0101] Individuals with dry eye disease have impaired blinking reflexes and symptomatic tear secretion. Signs and symptoms of dry eye disease include dry eyes; decreased tear production, volume, and flow; abnormal tear composition; increased tear osmolality; keratitis, conjunctival and corneal discoloration; redness; blurred vision; decreased tear breakup time; increased conjunctival redness; excess debris in the tear film; a gritty feeling in the eye; burning eyes; a foreign body sensation in the eye; excessive tear secretion; photophobia; eye irritation; refractive dysfunction; ocular hypersensitivity; eye irritation and discomfort due to prolonged contact lens wear. Although the excessive tear secretion response may seem counterintuitive, it is a natural reflex response to the irritation and foreign body sensation caused by dry eye disease. Some individuals may also experience itchy eyes due to a combination of ocular allergies and dry eye symptoms.

[0102] There can be many variables that can affect an individual's dry eye symptoms, including blood hormone levels, various autoimmune diseases (e.g., Sjorgren's syndrome and systemic lupus erythematosus), eye surgery (including PRK or LASIK), numerous medications, environmental conditions, visual tasks (such as computer use), eye strain, contact lens wear, and mechanical influences such as corneal sensitivity, partial eyelid closure, surface irregularities (e.g., ptosis, entropion / ectropion, pinguecula), and eyelid abnormalities (e.g., ptosis, entropion / ectropion, pinguecula). Low humidity environments (e.g., environments that cause dehydration) (such as sitting on an airplane or in a car with the defroster on or living in arid climates) can exacerbate or cause dry eye symptoms. Additionally, visual tasks can also exacerbate symptoms. Tasks that can significantly affect symptoms include watching TV or using a computer for long periods of time, which reduces the rate of blinking.

[0103] Compositions containing HA have been used to treat dry eye conditions. HA-containing eye drops are commercially available and typically contain HA at concentrations of less than 1%. Exemplary commercially available HA-containing eye drops include Opticalm eye drops (0.2% HA); Aquify comfort drops (0.1% HA); Blink (0.15% HA); Hyal-drops (0.2% HA); Hycosan (0.1% HA); Oxyal (0.15% HA); and Vismed (0.18% and 0.8% HA).

[0104] Applicants have surprisingly discovered that highly concentrated HA compositions comprising HA at concentrations greater than about 30 mg / mL (i.e., 3% HA), such as HA at a concentration of 40 mg / mL (i.e., 4% HA), can be effective when administered to the eye of a subject for the treatment of dry eye conditions. Such highly concentrated HA compositions have not previously been used to treat dry eye because it was believed that these compositions were too viscous for effective administration to the eye.

[0105] The applicant also surprisingly discovered that, once administered to the eye, the concentrated HA composition is surprisingly effective in treating pain and discomfort associated with dry eye. While not wishing to be bound by any particular theory, it is believed that pain associated with dry eye can be regulated by the TRPV1 channel of nociceptors, and that HA molecules in the highly concentrated HA composition interact with the TRPV1 channel present on the sensory nerves and corneal cells of the eye, thereby reducing the responsiveness of the nociceptors to noxious stimuli. Thus, in some embodiments, pain and discomfort associated with dry eye is regulated by the receptor potential vanilloid subtype 1 (TRPV1) channel. Furthermore, the characteristic viscoelasticity of concentrated HA reduces the amount of solution applied to the ocular surface that is removed by normal tearing and blinking, thereby prolonging the protective effect of the HA solution.

[0106] The present invention provides a method for alleviating pain and discomfort associated with a dry ocular condition in a subject in need thereof, comprising administering to the eye of the subject a composition comprising hyaluronan, wherein the hyaluronan is present in the composition at a concentration of greater than about 30 mg / mL; the hyaluronan has an average molecular weight of between about 1 million and about 2 million; and the hyaluronan is not cross-linked and / or is substantially free of chemical modifications, thereby alleviating pain and discomfort in the subject.

[0107] In some cases, the HA composition intended for ophthalmic use is in the form of a gel, ointment, salve, lotion, or cream. In certain embodiments, the HA composition is not in the form of a lens or microsphere.

[0108] HA compositions intended for ophthalmic use can be administered to the ocular surface (such as under the eyelid (e.g., under the upper or lower eyelid) or at the corneal-eyelid junction). In one embodiment, the HA composition is not injected into the subject's eye. Because the high viscosity of the HA composition can significantly impair the subject's ability to see immediately after administration, these compositions can be administered to the subject's eye just before resting or going to sleep.

[0109] Those skilled in the art can determine the appropriate administration schedule for the ophthalmic HA composition described herein to relieve the pain and discomfort associated with dry eye condition. For example, the HA composition can be administered daily for about 3 days, 4 days, 5 days, 6 days, 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, etc., or any period until the pain and discomfort associated with dry eye condition is relieved. In some embodiments, the pain and discomfort can be relieved for a long period of time (for example, for about 12 hours, about 24 hours, about 1 day, about 3 days, about 5 days, about 7 days, about 14 days, or about 28 days).

[0110] The term "dry eye condition" includes, but is not limited to, dry eye conditions associated with one or more symptoms selected from the group consisting of dry eye; decreased tear film breakup time; decreased tear production, volume, and flow; abnormal tear composition; increased tear osmolality; keratitis; conjunctival and corneal staining; redness; blurred vision; increased conjunctival redness; excess debris in the tear film, grittiness in the eye; burning eye; foreign body sensation in the eye; excessive tear production; photophobia; eye irritation; refractive dysfunction; ocular hypersensitivity; ocular irritation, and discomfort due to prolonged contact lens wear.

[0111] Dry eye condition can also be associated with a condition selected from the group consisting of autoimmune disorders; eye surgery; drug intake; dry environmental conditions; prolonged computer use; eye fatigue; corneal hypersensitivity due to contact lens wear; partial eyelid closure; surface irregularities; eyelid irregularities; and conditions related to neuropathic pain (such as cataract or retinal detachment). In one example, dry eye condition can be associated with ophthalmic surgery (such as photorefractive keratectomy (PRK)), cataract surgery, retinal detachment surgery, laser in situ keratomileusis (LASIK), or any corneal surgical procedure that involves corneal sensory nerve damage).

[0112] IX. Methods for Minimizing Skin Imperfections While Relieving Pain and Discomfort HA is a major component of the extracellular matrix (ECM) and is present in particularly large amounts in soft connective tissues such as skin. In normal skin, HA is primarily synthesized by dermal fibroblasts and epidermal keratinocytes. Because its residues are negatively charged, HA functions as a water pump to maintain skin hydration and elasticity. HA plays a major role in regulating the distribution of food, hormones, vitamins, and inorganic salts in connective tissue and in clearing metabolic waste, which may include inflammatory responses. With aging, the amount of HA and its degree of polymerization decrease, thereby reducing the water retention capacity in connective tissue. Skin then undergoes the aging process, which increases fibrosis and reduces the quality of elastic fibers.

[0113] HA is widely used for cosmetic purposes, including both the use of HA compositions in the form of creams or gels for topical application and the use of HA compositions for injection into the skin as a dermal filler. The latter use involves injecting HA compositions into the top layer of a subject's skin (e.g., the face, neck, arms, legs, trunk, or chest of a subject). This use reduces wrinkles by mechanically filling in the sagging skin caused by wrinkles and by preventing the degradation of ECM, which is essential for skin aging and maintaining the mechanical properties of skin elasticity and firmness.

[0114] Although injecting dermal fillers for soft tissue augmentation is a minimally invasive dermatological procedure, patients often express concern about the pain associated with such procedures. Topical anesthetic creams are often used to alleviate pain during these procedures, and anesthetics are also included in injectable HA compositions.

[0115] The present invention eliminates the need for anesthetics in these procedures. Specifically, the present invention provides a method for alleviating the pain and discomfort associated with dermatological procedures while minimizing at least one skin defect in a subject in need of such a method, by administering a highly concentrated HA composition to the subject. As described above, it has been surprisingly discovered that the highly concentrated HA composition described herein is surprisingly effective in treating the pain and discomfort associated with dermatological procedures, which may include, for example, injection into the skin of a subject. Without wishing to be bound by a particular theory, it is believed that the pain associated with the injection of an HA composition into the skin can be regulated by the TRPV1 channel of nociceptors, and that the HA molecules in the highly concentrated HA composition interact with the TRPV1 channel present in sensory nerves and epithelial and connective tissue cells of the skin and subcutaneous tissue, thereby reducing the responsiveness of nociceptors to noxious stimuli.

[0116] In some embodiments, HA compositions for cosmetic use can be administered to a subject in need thereof by injection (e.g., subcutaneous or intradermal injection) using an injection device (such as a needle, trocar, cannula, or perfusion device). The nominal diameter of an injection device suitable for injecting HA compositions of the present invention can be 2.11 mm or greater (equivalent to a 14G needle or needles greater than 14 gauge). In some embodiments, HA compositions of the present invention may be too viscous for administration using smaller needles (e.g., needles with a nominal diameter of less than 2.11 mm). In other embodiments, HA compositions of the present invention can be administered using smaller injection devices with a nominal diameter of less than 2.11 mm.

[0117] For example, the nominal diameters of devices (such as syringes) suitable for injecting the HA compositions of the present invention are approximately 0.31 mm, 0.34 mm, 0.36 mm, 0.41 mm, 0.474 mm, 0.46 mm, 0.49 mm, 0.515 mm, 0.51 mm, 0.54 mm, 0.57 mm, 0.59 mm, 0.642 mm, 0.64 mm, 0.67 mm, 0.718 mm, 0.72 mm, 0.77 mm, The thickness may be 0.82 mm, 0.87 mm, 0.91 mm, about 0.99 mm, about 1.07 mm, about 1.17 mm, about 1.27 mm, about 1.42 mm, about 1.47 mm, about 1.57 mm, about 1.65 mm, about 1.73 mm, about 1.83 mm, about 1.98 mm, about 2.11 mm, about 2.26 mm, about 2.41 mm, about 2.54 mm, or about 2.77 mm, which are 30, 25, 30, 35, 40, 45, 50, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 102, 103, 104, 105, 106, 107, 108, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 9, 28, 27, 26s, 26, 25.5, 25s, 25, 24.5, 24, 23.5, 23s, 23, 22.5, 22s, 22, 21.5, 21, 20.5, 20, 19.5, 19, 18.5, 18, 17.5, 17, 16.5, 16, 15.5, 15, 14.5, 14, 13.5, 13, 12.5, or 12 gauge (i.e. 30G, 29G, 28G, 27G, 26sG, (This corresponds to a 26G, 25.5G, 25sG, 25G, 24.5G, 24G, 23.5G, 23sG, 23G, 22.5G, 22sG, 22G, 21.5G, 21G, 20.5G, 20G, 19.5G, 19G, 18.5G, 18G, 17.5G, 17G, 16.5G, 16G, 15.5G, 15G, 14.5G, 14G, 13.5G, 13G, 12.5G, or 12G needle.) In one embodiment, the HA compositions of the present invention can be administered using an 18G syringe needle with a nominal diameter of about 1.27 mm. In some embodiments, the HA compositions of the present invention may be too viscous for administration using smaller needles (e.g., needles with a nominal diameter of less than 1.27 mm).

[0118] In some embodiments, the methods of the present invention may also include administering to the subject a highly concentrated HA composition in the form of an injectable implant, particularly a dermal implant.

[0119] The compositions of the present invention are intended for use in humans or animals, in particular in reconstructive or plastic surgery or cosmetic dermatology for filling wrinkles, fine lines, loose skin, and scars, including filling loose skin caused by lipodystrophy or lipoatrophy. The composition may be an implant as defined above.

[0120] X. Methods for Promoting Wound Healing While Relieving Pain and Discomfort Skin provides a mechanical barrier to the external environment and acts to prevent the introduction of infectious agents. Upon injury, the underlying tissue is exposed to infection; therefore, rapid and effective healing is crucial for reestablishing barrier function. Skin wound healing in adults is a complex process, involving multiple stages, including inflammation, granulation tissue formation, re-epithelialization, and remodeling. HA likely plays a multifaceted role in the administration of these cellular and matrix events. To alleviate the pain associated with these wounds, local anesthetics are often administered to the wound.

[0121] The present invention provides a method for effectively treating skin wounds while simultaneously providing pain relief, eliminating the need for local anesthesia. Specifically, the present invention provides a method for promoting wound healing while alleviating pain and discomfort by administering a highly concentrated HA composition to a subject in need thereof. As described above, it has been surprisingly found that the highly concentrated HA composition described herein is surprisingly effective in treating the pain and discomfort associated with wound healing. Without wishing to be bound by any particular theory, it is believed that the pain associated with the injection of an HA composition into the skin can be regulated by the TRPV1 channel of nociceptors, and that the HA molecules in the highly concentrated HA composition interact with the TRPV1 channel present in the sensory nerves innervating the wound area and in the connective tissue cells of this area, thereby reducing the responsiveness of nociceptors to noxious stimuli.

[0122] According to the method of the present invention, a highly concentrated HA composition intended for topical administration to a skin wound can be topically administered onto the surface of a wound or scar on the skin. The composition can be in the form of an aqueous gel or polyol containing a thickening polymer (such as a cellulose derivative or acrylic polymer) along with other commonly used excipients (such as preservatives and fragrances).

[0123] A treatment, prevention, or mitigation effect is manifested when a statistically significant improvement is observed in one or more parameters of a disease state or pathological condition (e.g., pain and discomfort associated with dry eye, skin care / dermatological procedures, or wound healing). A treatment, prevention, or mitigation effect is also manifested by a failure to worsen symptoms or develop unexpected symptoms. By way of example, a favorable change of at least 10%, preferably at least 20%, 30%, 40%, 50% or more, in a measurable parameter of a disease or condition (e.g., pain and discomfort) can indicate effective treatment. The terms "prevent" or "prevention," as used herein, include, for example, the prevention of recurrence of pain and discomfort in a subject who has previously experienced pain.

[0124] In some embodiments, the subject is a human, a mammal (e.g., a companion animal (such as a cat or dog), a livestock animal (such as a cow, sheep, horse, donkey), or a rodent (such as a guinea pig, mouse, or rat)). In one particular embodiment, the subject is a human. In another particular embodiment, the subject is a dog.

[0125] As used herein, the term "relief of at least one symptom" includes the reduction, amelioration, or elimination of at least one symptom associated with pain and discomfort. This term also includes reducing the extent of activation of ion channels (such as TRPV1 channels) involved in pain transmission processes in neurons upon administration of the HA compositions of the present invention. Activation of such channels can be, for example, the reduction of intracellular Ca in neurons following a nociceptive impulse. 2+The reduction in nociceptive activity can be measured by measuring changes in the neuronal activity or by measuring whole-cell currents in neurons upon administration of the HA compositions of the present invention. Furthermore, the term "alleviation of at least one symptom" also includes a decrease in nociceptive firing of neurons upon administration of the HA compositions of the present invention.

[0126] In some embodiments, the method for alleviating pain and discomfort associated with dry eye conditions, skin care / dermatological procedures, or wound healing comprises administering a therapeutically effective amount of the composition of the present invention to a subject in need thereof.The term "therapeutically effective amount," as used herein, is intended to include an amount of the HA composition of the present invention that is sufficient to treat, prevent, reduce, or alleviate pain and discomfort when administered to a subject in need thereof.Those skilled in the art, such as physicians, can easily ascertain the amount of the HA composition that will be therapeutically effective. Generally, a therapeutically effective amount of the composition is from about 0.1 to about 500 mg (e.g., about 0.1 mg, about 1 mg, about 5 mg, about 10 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 110 mg, about 120 mg, about 130 mg, about 140 mg, about 150 mg, about 160 mg, about 170 mg, about 180 mg, about 190 mg, about 200 mg, about 210 mg, about 220 mg, about 230 mg). g, about 240 mg, about 250 mg, about 260 mg, about 270 mg, about 280 mg, about 290 mg, about 300 mg, about 310 mg, about 320 mg, about 330 mg, about 340 mg, about 350 mg, about 360 mg, about 370 mg, about 380 mg, about 390 mg, about 400 mg, about 410 mg, about 420 mg, about 430 mg, about 440 mg, about 450 mg, about 460 mg, about 470 mg, about 480 mg, about 490 mg, or about 500 mg).

[0127] Various treatment regimens are included in the methods disclosed herein.For example, after administering the first dose of the HA composition disclosed herein to a subject, additional doses can be administered.In some embodiments, after administering the first dose, the second dose is administered at a specific interval.In some embodiments, the second dose is administered about 30 days, about 60 days, about 90 days, about 120 days, about 150 days, about 180 days, about 210 days, about 240 days, about 270 days, about 300 days, about 330 days, or about 360 days after the first dose.It should be recognized that the dosage regimen can be adjusted based on the relief of pain and discomfort experienced by the subject. In some embodiments, a subject receives a regular dose once per month, once per two months, once per three months, once per four months, once per five months, once per six months, once per seven months, once per eight months, once per nine months, once per ten months, once per eleven months, or once per twelve months.

[0128] The present invention is further illustrated by the following examples, which should not be construed as further limiting the present invention in any way. The entire contents of all documents cited throughout this application (including literature references, issued patents, published patent applications, and co-pending patent applications) are expressly incorporated herein by reference. [Example]

[0129] Example Unless otherwise specified, the hyaluronan (HA) used in the compositions described herein is obtained from animal, human, or bacterial sources. Unless otherwise specified, the compositions used herein are contained in a physiological buffer.

[0130] Example 1. Reduction of impulse activity in corneal sensory nerve fibers by topical application of 4% sodium hyaluronan to the cornea Nociceptor excitation is the first step in pain sensation generation. For acute pain, the magnitude of pain sensation is highly correlated with the firing rate of nociceptive fibers and the number of fibers recruited by the stimulus (Acosta et al., 2001, J. Physiol. 534(2), 511-525). Furthermore, the persistence of pain after peripheral injury is related to the continued activity of nociceptors (Belmonte et al., 2004, Ocular Surface, 2, 248-253).

[0131] Increased sensory nerve impulse activity in the corneal nerve is the first step in generating ocular discomfort and pain. At the ocular surface, noxious stimuli are detected by polymodal and mechano-nociceptors that innervate the cornea and conjunctiva (Belmonte & Giraldez, 1981, J. Physiol., 437, 709-725; Gallar et al., 1993, J. Physiol., 468, 609-622). During inflammation, polymodal nociceptors become sensitized and fire continuously, eliciting persistent pain sensations. On the other hand, activity of corneal cold thermoreceptors is associated with evaporation of the ocular surface and may also contribute to the conscious sensation of ocular surface dryness (Gallar et al., 1993, J. Physiol., 468, 609-622; Parra et al., 2010, Nature Medicine, 16, 1396-1399; Kovacs et al., 2016, Pain, 157, 399-417).

[0132] The purpose of the following experiments was to test the ability of hyaluronan with an average molecular weight of 1–2 million at a concentration of 4% (4% HA) to modulate spontaneous and stimulus-evoked nerve impulse firing in sensory receptor fibers (mechano-nociceptors, polymodal nociceptors, and cold thermoreceptors) in the guinea pig cornea.

[0133] An effective means of generating chemical stimulation of corneal polymodal nociceptors is the application of 98.5% CO2, which generates stimulation due to acidification caused by the local formation of carbonic acid (Chen et al., 1995, Eur. J. Neurosci. 7, 1154-1163). In all cases, it has been demonstrated that the application of similar CO2 pulses to the human cornea induces an immediate and sharp pain sensation (Chen et al., 1995, Eur. J. Neurosci. 7, 1154-1163; Acosta et al., 2001, J. Physiol. 534(2), 511-525). Due to the rapid formation of protons around the nerve endings, H + The irritating effect of CO2 is likely direct and occurs through activation of proton-gated ion channels, possibly the "capsaicin channel" TRPV1 and / or ASIC channels, which depolarize nerves. Furthermore, a decrease in intracellular pH in corneal epithelial cells exposed to CO2 stimulation can also lead to the formation and release of inflammatory mediators (e.g., prostaglandins), which can sensitize corneal polymodal nociceptors, resulting in background firing and an enhanced impulse response to direct acidic stimulation.

[0134] In the case of corneal cold thermoreceptors, these fibers exhibit background (normal impulse activity at a basal corneal temperature of 34°C) and increase in firing rate with very small temperature decreases (e.g., those that occur during tear evaporation) (Belmonte et al., 2015, Curr. Ophthalmol. Rep. 3, 111-121).

[0135] method Dunkin-Hartley guinea pigs of both sexes, weighing 200–300 g, were used in the experiments. Studies were conducted in accordance with the NIH Guide for the Care and Use of Laboratory Animals, the European Union Directive (2010 / 63 / EU), and the Spanish regulations on the protection of animals used for laboratory experiments. The study was conducted in accordance with the International Research Council (IRRC) and followed a protocol approved and supervised by the Ethics Committee of the Miguel Hernández University.

[0136] Electrophysiological recordings The impulse activity of peripheral corneal receptors of different functional types was recorded. For this purpose, animals were euthanized with an intraperitoneal injection of 100 mg / kg sodium pentobarbitone, and the eyeballs, along with the bulbar and palpebral conjunctivae and the optic and ciliary nerves, were immediately removed and placed in cold saline (4°C).

[0137] Connective tissue and extraocular muscles were carefully removed from the enucleated globe to expose the fundus with the ciliary nerve surrounding the optic nerve. The eye was then placed in a specially designed two-chamber system, which allowed the anterior segment with the conjunctiva to remain separate from the posterior pole and ciliary nerve. In the anterior chamber, the conjunctiva was pinned to a separator wall to isolate both compartments, allowing them to be perfused separately. The anterior compartment was perfused with warmed (34°C) saline, which was continuously instilled onto the superior corneoscleral border. In the posterior compartment of the chamber, filled with warmed mineral oil, a nerve filament was detached from the ciliary nerve and placed on an Ag-AgCl electrode for monopolar recording of single-unit impulse activity using conventional electrophysiological equipment. Electrical signals were recorded with an Ag / AgCl pellet in the posterior compartment. The electrical signals were transferred to a PC with a CED interface and analyzed using appropriate software. Spontaneous activity of selected units was recorded 1 min before any intended stimulation. Mechanical thresholds were then determined using calibrated von Frey hairs (range 0.25–4.00 mN). The receptive fields of corneal afferents were defined using mechanical stimulation with a fine paintbrush and then mapped using suprathreshold von Frey hairs. For chemical stimulation, a gas jet containing 98.5% CO2 was applied to the corneal receptive field for 30 seconds. Thermal stimulation was achieved by cooling the irrigation solution (down to 20°C) with a custom-made Peltier device.

[0138] Data analysis In single-fiber recordings of multimodal fibers, impulse firing of individual units, differentiated according to their stimulation modality, amplitude, and shape, was analyzed. Ongoing impulse activity was expressed as the mean impulse frequency (impulses / s) measured for 30 s from the start of recording and during the inter-impulse period. Responses to CO2 were quantified by measuring the following parameters: latency: the time delay between the onset of the CO2 pulse and the first pulse delivered by the unit; mean firing rate: the mean number of impulses per second (imps / s) throughout the CO2 pulse; and post-firing: the mean firing rate (imps / s) for the 30 s period immediately following the CO2 pulse.

[0139] For cold nerve fiber recordings, the following additional firing pattern parameters were calculated: mean firing rate: the average number of impulses recorded per second (impulses / s); cooling threshold: the temperature during the cooling gradient at which the mean nerve impulse frequency at basal temperature increased by 25%; peak response: the maximum impulse / s value of the impulse frequency during the cooling gradient; peak response temperature: the temperature value (°C) at which the peak frequency was reached.

[0140] Experimental Protocol The following experimental sequence was followed: Identification of polymodal nociceptive fibers The cornea was stimulated to identify filaments containing fibers that responded to mechanical stimulation using a moist fine brush applied to the corneal surface. After division of the neurofilaments, single corneal nociceptive units were localized by mechanical stimulation. For further characterization of each isolated single unit (determination of polymodality), the following routine procedures were performed: 1. Mapping receptive field boundaries and determining mechanical thresholds using von Frey filaments; 2. Measurement of the response to a 30 second pulse of 98.5% CO2. If no response was detected, new fibers were investigated.

[0141] The effect of 4% HA on multimodal nociceptors A 4% HA solution was applied to the corneal surface. To maintain humidity, saline was periodically instilled onto the cornea. After the 4% HA instillation, 30-second CO2 pulses were applied sequentially to the corneal surface (5, 20, 35, 50, 65, and 80 minutes after the 4% HA application). The cornea was then continuously washed with saline for 5 minutes, and the response to CO2 was measured 15 and 20 minutes after the wash. Electrical thresholds were measured at the end of the protocol to confirm the responsiveness of the fibers. The presence of spontaneous activity throughout the entire experimental procedure and the magnitude of the impulse response to CO2 stimulation were measured.

[0142] Identification of cold-sensitive fibers Filaments containing cold thermosensitive fibers innervating the cornea were identified by applying a drop of cold saline onto the cornea. After fiber segmentation, filaments containing single corneal cold units were localized and their receptive fields were mapped by applying an ice-cold metal rod (tip diameter 1 mm) onto the cornea.

[0143] The continuous impulse activity exhibited by a single cold-sensory nerve ending was recorded at 34°C. A cooling gradient from 34°C to 15°C was applied by varying the temperature of the receptive field using a Peltier device.

[0144] Effect of 4% HA on cold thermoreceptor nerve activity 4% HA was applied to the exposed ocular surface in the anterior compartment of the chamber, and the effects on the ongoing activity of cold thermoreceptors and their response to a cooling gradient at 34°C were investigated 5, 20, 35, 50, 65, and 80 minutes after HA application, and 15 and 20 minutes after corneal irrigation.

[0145] The mean sustained activity at a resting temperature of 34°C, the thermal threshold, and the increase in nerve impulse activity during the cooling gradient were determined.

[0146] result In the intact cornea, sensory afferents identified as multimodal nociceptor fibers exhibit corneal receptive fields that typically extend up to 1 mm into the adjacent sclera. Multimodal nociceptor fibers are silent at rest, respond to mechanical stimuli, and respond to 98.5% CO2.

[0147] The effects of 4% HA on ongoing activity and firing responses to CO2 were measured in two single multimodal units and two cool units. The effects of CO2 were analyzed before and after application of 4% HA onto the corneal surface. As shown in Table 1 below, multimodal nociceptors were silent at rest, and no ongoing activity was observed during the 2-minute recording period. Multimodal nociceptors responded for approximately 2.5 seconds to a 30-second CO2 pulse with a mean impulse firing frequency of approximately 6 impulses / second. During the first 35 minutes after application of 4% HA onto the cornea, responses to CO2 significantly decreased and then ceased. Washout 80 minutes after 4% HA treatment gradually restored responses to CO2.

[0148] [Table 1]

[0149] We also recorded impulse activity in cold thermoreceptor fibers, which account for approximately 10% of all corneal sensory afferents. We confirmed their regular, continuous impulse activity at 34°C in intact eyes and the marked increase in frequency due to a 15°C cooling pulse characteristic of ocular cold thermoreceptors (Gallar et al., 1993, J. Physiol., 468, 609-622; Parra et al., 2010, Nature Medicine, 16, 1396-1399).

[0150] As shown in Table 1, 4 h of corneal exposure to 4% HA reduced the background ongoing activity of cold thermoreceptors at 34°C. Although no clear changes in cold thresholds were observed, as also shown in Table 1, responses to the cold gradient were significantly reduced and only partially recovered 20 min after washing, while the peak frequency values ​​elicited by the cold gradient returned to normal during this time.

[0151] In summary, these values ​​indicate that 4% HA attenuates the abnormal impulse activity in polymodal nociceptor fibers caused by acidic corneal stimulation. This is likely the result of the interaction of HA molecules with TRPV1 channels in sensitized nociceptive nerve endings (Caires et al., 2015, Nat. Comm. 6, 8095), demonstrating that high concentrations of HA are highly effective in eliciting this effect. Furthermore, cold thermoreceptor activity was reduced, likely due to a more effective shielding of the corneal surface than lower concentrations of HA solution.

[0152] Example 2. Force required for HA ejection with various needle sizes The pressure required to expel a 4% HA composition from a 3 mL syringe with a needle of different diameters (30 to 18 G) was measured and is shown in Figure 1. A force was applied to one pan of a two-pan balance, acting perpendicular to the syringe embolus. A lighter weight was added to the opposite pan. As evidenced by the results shown in Figure 1, the HA composition of the present invention can be administered to subjects using needles with diameters ranging from 30 to 18 G.

[0153] Doctrine of Equivalents The foregoing specification is believed to be sufficient to enable one skilled in the art to practice the present invention. The present invention should not be limited in scope by the examples provided, as the examples are intended to be an illustration of one aspect of the present invention and other functionally equivalent embodiments are included within the scope of the present invention. Various modifications of the present invention, in addition to the embodiments shown and described herein, will become apparent to those skilled in the art from the foregoing description, and such modifications are included within the scope of the appended claims. The advantages and objectives of the present invention are not necessarily included in each embodiment of the present invention.

Claims

[Claim 1] The method described in the specification.

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