阅读说明：本技术 高弹性透明质酸组合物及其使用方法 (Highly elastic hyaluronic acid compositions and methods of use thereof ) 是由 C·贝尔蒙特 J·L·德林杰 于 2016-09-21 设计创作，主要内容包括：本发明提供了用于减轻与干眼症相关的疼痛和不适的方法；用于减轻疼痛和不适,同时将至少一种皮肤瑕疵最小化的方法；以及用于减轻疼痛和不适,同时促进伤口愈合的方法。该方法包括向需要其的受试者施用包含透明质酸的具有高弹性的组合物。(The present invention provides methods for reducing the pain and discomfort associated with dry eye; methods for reducing pain and discomfort while minimizing at least one skin imperfection; and methods for reducing pain and discomfort while promoting wound healing. The method comprises administering to a subject in need thereof a composition having high elasticity comprising hyaluronic acid.)

1. A method for reducing pain and discomfort associated with a dry eye condition in a subject in need thereof, the method comprising administering to the eye of the subject a composition comprising hyaluronic acid, wherein:

the hyaluronic acid is present in the composition at a concentration of greater than about 30 mg/mL;

the hyaluronic acid has an average molecular weight of between about 100 and about 200 million;

the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications, thereby reducing the pain and discomfort in the subject.

2. The method of claim 1, wherein hyaluronic acid is present in the composition at a concentration of about 40mg/mL to about 60 mg/mL.

3. A method for reducing pain and discomfort associated with a dry eye condition in a subject in need thereof, the method comprising administering to the eye of the subject a composition comprising hyaluronic acid, wherein:

the hyaluronic acid is present in the composition at a concentration of at least about 40 mg/mL;

the hyaluronic acid has an average molecular weight of between about 100 and about 200 million;

the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications, thereby reducing the pain and discomfort in the subject.

4. The method of claim 1 or 3, wherein the composition is substantially free of other pharmaceutically active substances.

5. The method of claim 1 or 3, wherein the composition is free of polyglycol.

6. The method of claim 1 or 3, wherein the composition further comprises a buffering agent.

7. The method of claim 1 or 3, wherein the buffer is Phosphate Buffered Saline (PBS).

8. The method of claim 1 or 3, wherein the composition has an elasticity of at least about 200 pascals when measured at a frequency of 0.5 Hz.

9. The method of claim 1 or 3, wherein the composition has an elasticity of at least about 1,000 pascals when measured at a frequency of 0.5 Hz.

10. The method of claim 1 or 3, wherein the composition has an elasticity of at least about 2,000 pascals when measured at a frequency of 0.5 Hz.

11. The method of claim 1 or 3, wherein the composition has an elasticity of at least about 4,000 pascals when measured at a frequency of 0.5 Hz.

12. The method of claim 1 or 3, wherein the composition is sterile.

13. The method of claim 1 or 3, wherein the composition is in the form of a gel, ointment, liniment, lotion, or cream.

14. The method of claim 1 or 3, wherein the composition is administered to the ocular surface.

15. The method of claim 14, wherein the composition is administered under the eyelid of the subject.

16. The method of claim 14, wherein the composition is administered at the cornea-eyelid interface.

17. The method of claim 14, wherein the composition is administered to the subject immediately prior to rest or sleep.

18. The method of claim 1 or 3, wherein the composition is administered without intraocular injection into the subject.

19. The method of claim 18, wherein the composition is administered using a container selected from the group consisting of: single dose containers, soft plastic bottles, tubes, airless tubes, eye cups, droppers, and kits.

20. 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 eyes; tear production, volume and flow reduction; abnormal tear composition; increased tear osmotic pressure; keratitis; conjunctival and corneal staining; reddening; blurred vision; the tear film rupture time is shortened; exacerbation of conjunctival redness; excessive fragments in the tear film, and gritty feeling of eyes; burning the eye; eye foreign body sensation; excessive lacrimation; photophobia; eye stinging; ametropia; ocular sensitivity; eye irritation and discomfort associated with extended contact lens wear.

21. The method of claim 1 or 3, wherein the dry eye condition is associated with a condition selected from the group consisting of: (ii) an autoimmune disease; eye surgery; taking in the drug; dry ambient conditions; using the computer for a long time; eye fatigue; contact lens wearing, cornea sensitive; partial eyelid closure; the surface is not flat; (iii) eyelid irregularities; and conditions associated with neuropathic pain.

22. The method of claim 21, wherein the dry eye condition is associated with ocular surgery, and wherein the ocular surgery is selected from the group consisting of: refractive surgery, cataract surgery, retinal detachment surgery, laser in situ keratomileusis (LASIK), and any corneal surgical procedure involving damage to the corneal sensory nerve.

23. The method of claim 21, wherein the dry eye is associated with a condition associated with neuropathic pain, and wherein the condition is cataract surgery or surgery following retinal detachment.

24. 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.

25. The method of claim 1 or 3, wherein long-term reduction of the pain and discomfort is achieved in the subject.

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.

27. The method of claim 1 or 3, wherein the subject is a mammal.

28. The method of claim 27, wherein the subject is a human.

29. A method for reducing 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 hyaluronic acid, wherein:

the hyaluronic acid is present in the composition at a concentration of greater than about 30 mg/mL;

the hyaluronic acid has an average molecular weight of between about 100 and about 200 million;

the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications; and

wherein said composition is substantially free of other pharmaceutically active substances, thereby reducing said pain and minimizing said at least one skin imperfection.

30. The method of claim 29, wherein hyaluronic acid is present at a concentration of about 40mg/mL to about 60 mg/mL.

31. A method for reducing 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 hyaluronic acid, wherein:

the hyaluronic acid is present in the composition at a concentration of about 40 mg/mL;

the hyaluronic acid has an average molecular weight of between about 100 and about 200 million;

the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications; and

wherein said composition is substantially free of other pharmaceutically active substances, thereby reducing said pain and minimizing said at least one skin imperfection.

32. The method of claim 29 or 31, wherein the other pharmaceutically active substance is a local anaesthetic.

33. The method of claim 32, wherein the local anesthetic is lidocaine or bupivacaine.

34. The method of claim 29 or 31, wherein the composition further comprises a buffering agent.

35. The method of claim 34, wherein the buffer is Phosphate Buffered Saline (PBS).

36. The method of claim 29 or 31, wherein the composition has an elasticity of at least about 200 pascals when measured at a frequency of 0.5 Hz.

37. The method of claim 29 or 31, wherein the composition has an elasticity of at least about 1,000 pascals when measured at a frequency of 0.5 Hz.

38. The method of claim 29 or 31, wherein the composition has an elasticity of at least about 2,000 pascals when measured at a frequency of 0.5 Hz.

39. The method of claim 29 or 31, wherein the composition has an elasticity of at least about 4,000 pascals when measured at a frequency of 0.5 Hz.

40. The method of claim 29 or 31, wherein the composition is sterile.

41. The method of claim 29 or 31, wherein the composition is administered by injection into the skin of the subject.

42. The method of claim 41, wherein the composition is injected into the subject at a site selected from the group consisting of: the face, neck, arms, legs, torso, and chest of the subject.

43. The method of claim 41, wherein the composition is injected into a site selected from the group consisting of: the nasolabial sulcus region, the upper lip region, the forehead, the eye region, and the cheek region.

44. The method of claim 41, wherein the composition is administered by injection using a prefilled syringe.

45. The method of claim 44, wherein the prefilled syringe is a1, 2,3, 4,5, 6, 7, 8, 9, or 10-mL prefilled syringe.

46. The method of claim 45, wherein the prefilled syringe is sterile.

47. A method for reducing pain and discomfort while promoting wound healing in a subject in need thereof, the method comprising administering to the subject a composition comprising hyaluronic acid, wherein:

the hyaluronic acid is present in the composition at a concentration of greater than about 30 mg/mL;

the hyaluronic acid has an average molecular weight of between about 100 and about 200 million; and

the hyaluronic acid is uncrosslinked and/or substantially free of chemical modification, thereby reducing the pain and promoting the wound healing.

48. The method of claim 47, wherein hyaluronic acid is present at a concentration of about 40mg/mL to about 60 mg/mL.

49. A method for reducing pain and discomfort while promoting wound healing in a subject in need thereof, the method comprising administering to the subject a composition comprising hyaluronic acid, wherein:

the hyaluronic acid is present in the composition at a concentration of about 40 mg/mL;

the hyaluronic acid has an average molecular weight of between about 100 and about 200 million; and

the hyaluronic acid is uncrosslinked and/or substantially free of chemical modification, thereby reducing the pain and promoting the wound healing.

50. The method of claim 47 or 49, wherein the composition is substantially free of other pharmaceutically active substances.

51. The method of claim 47 or 49, wherein the composition further comprises a buffering agent.

52. The method of claim 51, wherein the buffer is Phosphate Buffered Saline (PBS).

53. The method of claim 47 or 49, wherein the composition has an elasticity of at least about 200 pascals when measured at a frequency of 0.5 Hz.

54. The method of claim 47 or 49, wherein the composition has an elasticity of at least about 1,000 pascals when measured at a frequency of 0.5 Hz.

55. The method of claim 47 or 49, wherein the composition has an elasticity of at least about 2,000 pascals when measured at a frequency of 0.5 Hz.

56. The method of claim 47 or 49, wherein the composition has an elasticity of at least about 4,000 pascals when measured at a frequency of 0.5 Hz.

57. The method of claim 47 or 49, wherein the composition is sterile.

58. The method of claim 47 or 49, wherein the composition is administered topically.

59. The method of claim 58, wherein the composition is topically applied to the surface of a wound or scar on the skin.

Background

Hyaluronic acid or Hyaluronic Acid (HA) is a linear polysaccharide with a high average molecular weight, which is widely distributed throughout connective, epithelial and neural tissues. HA is primarily present in the extracellular matrix and the pericellular matrix, but is also present intracellularly. Biological functions of HA include maintaining the elastic viscosity of liquid connective tissues (such as synovial fluid in joints and the vitreous of the eye), controlling tissue hydration and water transport, supramolecular assembly of proteoglycans in the extracellular matrix, and a variety of receptor-mediated effects in cell detachment, mitosis, migration, and tumor progression.

Some known uses of HA include the treatment of dry eye, skin care (e.g., as a dermal filler), and the promotion of wound healing. In general, dry eye, skin care/dermatological procedures, and wound healing are associated with pain and discomfort, which often requires the administration of an analgesic drug alone. HA formulations currently used for the treatment of the above conditions are ineffective in treating local pain. Accordingly, there is a need in the art for methods of effectively treating dry eye, minimizing skin imperfections using injectable augmentation devices, and promoting wound healing while reducing pain associated with these conditions.

Disclosure of Invention

The present inventors have discovered that compositions comprising high concentrations of HA (e.g., compositions having HA concentrations of about 30mg/mL (about 3 wt/vol%) or higher) can be effectively used to reduce the pain and discomfort associated with dry eye, dermatological procedures, and healing wounds. Without wishing to be bound by a particular theory, it is believed that the efficacy of the HA compositions of the present invention comprising high concentrations of HA for treating pain and discomfort is determined by their high elasticity, as evidenced by the high value of the elastic modulus G'. Without wishing to be bound by a particular theory, it is also believed that the efficacy of the HA compositions of the present invention is determined by the relatively high probability of the HA molecule interacting with the pain transduction channel (e.g., TRPV1), thereby reducing nociceptor excitability.

Accordingly, the present invention provides a method for reducing the pain and discomfort associated with dry eye in a subject in need thereof. The method comprises administering to the eye of the subject a composition (e.g., a pharmaceutical composition) comprising hyaluronic acid, wherein: hyaluronic acid is present in the composition at a concentration of greater than about 30mg/mL, e.g., about 35mg/mL or more, about 40mg/mL or more, about 45mg/mL or more, about 50mg/mL or more, about 55mg/mL or more, about 60mg/mL or more, about 65mg/mL or more, about 70mg/mL or more, about 75mg/mL or more, about 80mg/mL or more, about 85mg/mL or more, about 90mg/mL or more, about 95mg/mL or more, or about 100mg/mL or more; hyaluronic acid has an average molecular weight of between about 100 and about 200 million; and the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications; thereby alleviating pain and discomfort in the subject.

In another aspect, the hyaluronic acid is present in the composition at a concentration of about 40mg/mL to about 60 mg/mL.

In some embodiments, the present invention also provides a method for reducing pain and discomfort associated with a dry eye condition in a subject in need thereof, the method comprising administering to the eye of the subject a composition (e.g., a pharmaceutical composition) comprising hyaluronic acid, wherein: hyaluronic acid is present in the composition at a concentration of at least about 40 mg/mL; hyaluronic acid has an average molecular weight of between about 100 and about 200 million; and the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications; thereby alleviating pain and discomfort in the subject.

In some embodiments, the composition is substantially free of other pharmaceutically active agents.

In certain aspects, the composition is free of polyglycol.

In some embodiments, the composition further comprises a buffering agent, such as Phosphate Buffered Saline (PBS).

In some embodiments, the composition has a dielectric constant of 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 an elasticity of at least about 4,000 pascals when measured at a frequency of 0.5 Hz.

In some embodiments, the composition is in the form of a gel, ointment, liniment, lotion, or cream.

In some aspects, the composition is administered to an ocular surface, such as under the eyelid (e.g., under the upper or lower eyelid) or at the cornea-eyelid interface of the subject.

In certain embodiments, the composition is administered to the subject immediately prior to rest or sleep.

In some aspects, the composition is administered without intraocular injection into the subject.

In some aspects, the compositions are administered using containers, e.g., single dose containers, such as soft plastic bottles, tubes, airless tubes (airess tubes), eye cups (eye cups), droppers, or cartridges (cartriges).

In some embodiments, the dry eye is associated with one or more symptoms selected from the group consisting of: dry eyes; tear production, volume and flow reduction; abnormal tear composition; increased tear osmotic pressure; keratitis; conjunctival and corneal staining; reddening; blurred vision; the tear film rupture time is shortened; exacerbation of conjunctival redness; excessive fragments in the tear film, and gritty feeling of eyes; burning the eye; eye foreign body sensation; excessive lacrimation; photophobia; eye stinging; ametropia; ocular sensitivity; and eye irritation. In other embodiments, the dry eye condition is associated with a condition selected from the group consisting of: (ii) an autoimmune disease; eye surgery; taking in the drug; dry ambient conditions; using the computer for a long time; eye fatigue; the cornea is sensitive when the contact lens is worn for a long time; partial eyelid closure; the surface is not flat; (iii) eyelid irregularities; and conditions associated with corneal nociceptive pain associated with corneal injury or conditions associated with neuropathic pain. In another embodiment, the dry eye condition is associated with ocular surgery, and the ocular surgery is selected from the group consisting of: refractive surgery (such as photorefractive keratectomy (PRK)), cataract surgery, retinal detachment surgery, laser in situ keratomileusis (LASIK), and any corneal surgical procedure involving damage to the corneal sensory nerve. In another embodiment, dry eye is associated with a condition associated with neuropathic pain (e.g., cataract or retinal detachment, or surgery designed to treat cataract or retinal detachment).

In some aspects, 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 aspects, long-term (e.g., 8 hours, 12 hours, 24 hours, 1 day, 3 days, 5 days, 7 days, 14 days, or 28 days) reduction in pain and discomfort is achieved in the subject.

In some embodiments, the subject is a mammal, e.g., a human.

In yet another aspect, the present invention also provides a method for reducing 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 hyaluronic acid, wherein: hyaluronic acid is present in the composition at a concentration of greater than about 30 mg/mL; hyaluronic acid has an average molecular weight of between about 100 and about 200 million; the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications; and wherein the composition is substantially free of other pharmaceutically active substances, thereby reducing pain and minimizing at least one skin imperfection.

In some embodiments, the hyaluronic acid is present at a concentration of about 40mg/mL to about 60 mg/mL.

In another aspect, the present invention also provides a method for reducing 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 hyaluronic acid, wherein: hyaluronic acid is present in the composition at a concentration of at least about 40 mg/mL; hyaluronic acid has an average molecular weight of between about 100 and about 200 million; the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications; and wherein the composition is substantially free of other pharmaceutically active substances, thereby reducing pain and minimizing at least one skin imperfection.

In some embodiments, the other pharmaceutically active substance is a local anesthetic, such as lidocaine or bupivacaine.

In certain aspects, the composition further comprises a buffering agent, such as Phosphate Buffered Saline (PBS).

In some embodiments, the composition has a dielectric constant of 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 an elasticity of at least about 4,000 pascals when measured at a frequency of 0.5 Hz.

In some aspects, the composition is sterile.

In certain embodiments, the composition is administered by injection into the skin of the subject. In another embodiment, the composition is injected into the face of the subject. For example, the composition is injected into a region selected from the group consisting of: the nasolabial sulcus region, the upper lip region, the forehead, the eye region, and the cheek region.

In some aspects, the composition is administered by injection using a prefilled syringe (e.g., 1, 2,3, 4,5, 6, 7, 8, 9, or 10-mL prefilled syringe). In another aspect, the prefilled syringe is sterile.

In another aspect, the invention also provides a method for reducing 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 hyaluronic acid, wherein: hyaluronic acid is present in the composition at a concentration of greater than about 30 mg/mL; hyaluronic acid has an average molecular weight of between about 100 and about 200 million; and the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications; thereby alleviating pain and promoting wound healing.

In another embodiment, the hyaluronic acid is present at a concentration of about 40mg/mL to about 60 mg/mL.

In another aspect, the invention also provides a method for reducing 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 hyaluronic acid, wherein: hyaluronic acid is present in the composition at a concentration of at least about 40 mg/mL; hyaluronic acid has an average molecular weight of between about 100 and about 200 million; and the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications; thereby alleviating pain and promoting wound healing.

In some aspects, the composition is substantially free of other pharmaceutically active substances, such as local anesthetics, e.g., lidocaine or bupivacaine.

In some embodiments, the composition further comprises a buffering agent, such as Phosphate Buffered Saline (PBS).

In some aspects, the composition has a dielectric constant of 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 an elasticity of at least about 4,000 pascals when measured at a frequency of 0.5 Hz.

In some embodiments, the composition is sterile.

In certain embodiments, the composition is applied topically, for example, on the surface of a wound or scar on the skin.

Drawings

Figure 1 is a graph depicting the pressure required to eject a 4% HA solution through different gauge needles.

Detailed Description

The present invention provides methods for reducing and ameliorating dry eye; methods of skin care/dermatology treatment of pain and discomfort associated with wound healing. The presently claimed methods include administering an HA composition comprising Hyaluronic Acid (HA) at high concentrations, e.g., above about 30mg/mL or higher. Such compositions are determined to have high elasticity, e.g., high elastic modulus G', when measured at frequencies of 0.1 to 10 Hz. HA compositions characterized by high elasticity (e.g., compositions containing high concentrations of HA) are useful in reducing dry eye syndrome; cosmetic treatment; or pain and discomfort associated with wound healing, are surprisingly effective. Without wishing to be bound by a particular theory, it is believed that the efficacy of the HA compositions of the present invention comprising high concentrations of HA in treating pain and non-moderation is determined by their high elasticity, as evidenced by the high value of elastic modulus G'. Without wishing to be bound by a particular theory, it is also believed that the efficacy of the HA compositions of the present invention is determined by the relatively high probability of the HA molecule interacting with the pain transduction channel (e.g., TRPV1), thereby reducing nociceptor excitability. The average molecular weight of the HA used in the process of the invention may be 200 ten thousand or less, for example between about 100 and 200 ten thousand.

I. Hyaluronic acid compositions for use in the methods of the invention

The present invention provides methods comprising administering to a subject in need thereof a composition comprising Hyaluronic Acid (HA). In some embodiments, the composition comprises hyaluronic acid, wherein hyaluronic acid is present in the composition at a concentration of greater than about 30mg/mL (or greater than about 3 wt/vol%); hyaluronic acid has an average molecular weight of between about 100 and about 200 million; and the hyaluronic acid is uncrosslinked and/or substantially free of chemical modifications.

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

In some examples, the hyaluronic acid concentration in the composition may be greater than about 30mg/mL (or about 3% w/v), such as greater than about 31mg/mL (or about 3.1% w/v), greater than about 32mg/mL (or about 3.2% w/v), greater than about 33mg/mL (or about 3.3% w/v), greater than about 34mg/mL (or about 3.4% w/v), greater than about 35mg/mL (or about 3.5% w/v), greater than about 36mg/mL (or about 3.6% w/v), greater than about 37mg/mL (or about 3.7% w/v), greater than about 38mg/mL (or about 3.8% w/v), or greater than about 39mg/mL (or about 3.9% w/v).

In some examples, the hyaluronic acid in the composition can have an average molecular weight of between about 100 and about 200 million; for example, between about 110 and about 200 tens of thousands, between about 120 and about 200 thousands, between about 130 and about 200 thousands, between about 140 and about 200 thousands, between about 150 and about 200 thousands, or between about 160 and about 200 thousands.

In certain embodiments, the hyaluronic acid used in the composition is uncrosslinked and/or free of chemical modification. For example, hyaluronic acid used in the composition is not amidated, which may be formed by the reaction between the carboxyl groups of HA and the amine groups of the derivatizing agent as described, for example, in EP patent No. 1095064B 1. The hyaluronic acid used in the composition may also be free of chemical modifications and/or uncrosslinked, which may be the result of the reaction of hyaluronic acid 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 hyaluronic acid used in the composition may also be free of acrylates (e.g., methacrylates as described in U.S. publication No. 2010/0048755); sulfates as described, for example, in U.S. publication No. 2013/0209531; and deuterium as described, for example, in U.S. publication No. 2015/0148310.

In some embodiments, the HA compositions of the present invention are free of other pharmaceutically active substances. As used herein, a "pharmaceutically active substance" is a substance that is capable of exerting a biological effect in a subject (e.g., a human or animal subject). The term "pharmaceutically active substance" also includes substances that are capable of modulating the biological effects of the HA composition when the composition is administered to a subject (e.g., reducing the pain and discomfort associated with dry eye; skin care/dermatology treatment or healing wounds). In certain embodiments, the pharmaceutically active substance is a protein, for example a Bone Morphogenetic Protein (BMP) such as rhGDF-5. In certain embodiments, the pharmaceutically active substance is a glycosaminoglycan (GAG), such as chondroitin, other than HA. In some embodiments, the pharmaceutically active substance is hydroxypropyl methylcellulose. In other embodiments, the pharmaceutically active substance is a local anesthetic such as lidocaine or bupivacaine. In some cases, the pharmaceutically active substance is a purinergic receptor agonist, such as P2Y₂A receptor agonist.

In certain embodiments, the HA compositions of the present invention are free of molecules capable of scavenging free radicals, such as polyols, for example sorbitol, maltitol, xylitol or isomalt. In other embodiments, the HA composition used in the methods of the invention is free of molecules that reduce the elasticity of HA, such as dextran or sucrose.

In some cases, the HA compositions used in the methods of the present invention do not contain polyglycols, such as polyethylene glycol.

The HA composition may consist essentially of HA present in a physiological buffer (e.g., phosphate buffer or bicarbonate buffer) at a concentration of greater than about 30mg/mL (about 3% w/v) or about 40mg/mL (about 4% w/v), and the HA HAs an average molecular weight of between about 100 and about 200 million. For example, the HA composition used in the methods of the present invention consists essentially of HA present at a concentration of about 40mg/mL (or about 4% w/v), and the HA HAs an average molecular weight of between about 100 and about 200 million.

The hyaluronic acid in the composition used in the method of the invention may have an elasticity of at least 100 pascals when measured at a frequency of 0.5Hz, or at least 400 pascals when measured at a frequency of 0.5Hz, or at least 1,000 pascals when measured at a frequency of 0.5Hz, or at least 2,000 pascals when measured at a frequency of 0.5Hz, or at least 4,000 pascals when measured at a frequency of 0.5Hz, or between 400 and 5,000 pascals when measured at a frequency of 0.5 Hz.

It will be appreciated that various methods can be used to measure the elasticity of biopolymers such as hyaluronic acid. In one embodiment, the elasticity of the composition comprising hyaluronic acid is measured as pressure (in pascals) at a specific frequency (in hertz). For example, the frequency that can be used to assess the elasticity of the hyaluronic acid compositions provided herein can be measured at 0.5Hz, 2.5Hz, or 5.0 Hz.

It should also be understood that elasticity may be expressed in any relevant frequency. Thus, for example, in one embodiment, elasticity is expressed based on a frequency of 2.5Hz, and a hyaluronic acid-containing composition having high elasticity is a composition having an elasticity of at least 200Pa at a frequency of 2.5 Hz. Similarly, in one embodiment, elasticity is expressed based on a frequency of 5.0Hz, and the hyaluronic acid-containing composition having high elasticity is a composition having elasticity of at least 400Pa at a frequency of 5.0 Hz.

In one embodiment, the composition comprising hyaluronic acid for use in the presently claimed method has an elasticity of at least 100 pascals when measured at a frequency of 0.5 Hz. In some embodiments, the composition has an elasticity of at least 300 pascals when measured at a frequency of 2.5 Hz. In some embodiments, the composition has an elasticity of at least 350 pascals when measured at a frequency of 5.0 Hz.

In another embodiment, the composition comprising hyaluronic acid has an elasticity of at least 400 pascals when measured at a frequency of 0.5 Hz. In some embodiments, the composition has an elasticity of at least 750 pascals when measured at a frequency of 2.5 Hz. In some embodiments, the composition has an elasticity of at least 900 pascals when measured at a frequency of 5.0 Hz.

In another embodiment, the composition comprising hyaluronic acid has an elasticity of at least 1000 pascals when measured at a frequency of 0.5 Hz. In some embodiments, the composition has an elasticity of at least 1600 pascals when measured at a frequency of 2.5 Hz. In some embodiments, the composition has an elasticity of at least 2000 pascals when measured at a frequency of 5.0 Hz.

In yet another embodiment, the composition comprising hyaluronic acid has an elasticity of at least 2600 pascals when measured at a frequency of 0.5 Hz. In some embodiments, the composition has an elasticity of at least 4000 pascals when measured at a frequency of 2.5 Hz. In some embodiments, the composition has an elasticity of at least 4500 pascals when measured at a frequency of 5.0 Hz.

In one embodiment, the composition comprising hyaluronic acid has an elasticity of at least 4000 pascals when measured at a frequency of 0.5 Hz. In some embodiments, the composition has an elasticity of at least 5000 pascals when measured at a frequency of 2.5 Hz. In some embodiments, the composition has an elasticity of at least 6000 pascals when measured at a frequency of 5.0 Hz.

In some embodiments, the composition has an elasticity of between 100 and 10,000 pascals when measured at a frequency of 0.5 Hz. In some embodiments, the composition has an elasticity of between 400 and 5,000 pascals when measured at a frequency of 0.5 Hz. In some embodiments, the composition has an elasticity of between 1,000 and 2,000 pascals when measured at a frequency of 0.5 Hz.

In some embodiments, the composition has an elasticity of between 300 and 10,000 pascals when measured at a frequency of 2.5 Hz. In some embodiments, the composition has an elasticity of between 750 and 6,000 pascals when measured at a frequency of 2.5 Hz. In some embodiments, the composition has an elasticity of between 1,500 and 4,000 pascals when measured at a frequency of 2.5 Hz.

In some embodiments, the composition has an elasticity of between 300 and 10,000 pascals when measured at a frequency of 5.0 Hz. In some embodiments, the composition has an elasticity of between 900 and 7,000 pascals when measured at a frequency of 5.0 Hz. In some embodiments, the composition has an elasticity of between 2,000 and 5,000 pascals when measured at a frequency of 5.0 Hz.

In some embodiments, elasticity can be measured by using a suitable device (e.g., a rheometer). For example, elasticity can be measured by using a Stresstech high resolution research rheometer (reorganic Instruments AB). Typically, elasticity is measured at ambient temperature and pressure; however, it should be understood that elasticity may also be measured at non-ambient temperatures and/or pressures. It should be further understood that one of ordinary skill in the art would know how to convert the elastic measurements taken at different temperatures and pressures to elastic measurements at ambient temperatures and pressures.

Highly elastic compositions of hyaluronic acid can be prepared by increasing the concentration of hyaluronic acid in the composition. Thus, in one aspect, the presently claimed method utilizes a composition having high elasticity, which comprises a high percentage of hyaluronic acid. For example, the composition can comprise at least 3.0% hyaluronic acid (weight by volume), at least 3.5% hyaluronic acid (weight by volume), at least 4.0% hyaluronic acid (weight by volume), at least 4.5% hyaluronic acid (weight by volume), at least 5.0% hyaluronic acid (weight by volume), at least 5.5% hyaluronic acid (weight by volume), at least 6.0% hyaluronic acid (weight by volume), at least 6.5% hyaluronic acid (weight by volume), at least 7.0% hyaluronic acid (weight by volume), at least 7.5% hyaluronic acid (weight by volume), at least 8.0% hyaluronic acid (weight by volume), at least 8.5% hyaluronic acid (weight by volume), at least 8.9% hyaluronic acid (weight by volume), at least 9.0% hyaluronic acid (weight by volume), at least 10.0% hyaluronic acid (weight by volume), At least 11.0% hyaluronic acid (weight by volume), at least 12.0% hyaluronic acid (weight by volume), at least 13.0% hyaluronic acid (weight by volume), at least 14.0% hyaluronic acid (weight by volume), or at least 15.0% or more hyaluronic acid (weight by volume).

Intermediate ranges of the listed values are also intended to be included in the compositions used in the methods of the present invention. For example, the hyaluronic acid may be present in the composition in an amount between about 3% and about 15% (weight/volume), between about 3% and about 10% (weight/volume), between about 3.5% and about 9% (weight/volume), between about 4% and about 8% (weight/volume), or between about 5% and about 7% (weight/volume).

It is further understood that the amount of hyaluronic acid in a particular volume may also be expressed by alternative means (e.g., grams/liter or moles/liter). One of ordinary skill in the art will know how to scale the various ways of expressing the amount of hyaluronic acid in a particular volume.

Hyaluronic acid compositions having high concentrations of hyaluronic acid (even with an average molecular weight of about 100-. Thus, the hyaluronic acid comprised in the HA compositions described herein may have an average molecular weight falling in the range between 100 and 200 million, and it is also less than 200 million, less than 190 million, less than 180 million, less than 170 million, less than 160 million, less than 150 million, less than 140 million, less than 130 million, less than 120 million, less than 110 million, less than 100 million, less than 90 million, less than 80 million, less than 70 million, less than 60 million or less than 50 million. In other instances, the hyaluronic acid included in the HA compositions described herein can have an average molecular weight that falls within a range between 100 and 200 million, and it is also greater than 10 million, greater than 20 million, greater than 30 million, greater than 40 million, greater than 50 million, greater than 60 million, greater than 70 million, greater than 80 million, greater than 90 million, greater than 100 million, greater than 110 million, greater than 120 million, greater than 130 million, greater than 140 million, greater than 150 million, greater than 160 million, greater than 170 million, greater than 180 million, or greater than 190 million.

Intermediate ranges of the listed values are also intended to be part of the present invention. For example, in the hyaluronic acid compositions provided herein, the average molecular weight of the hyaluronic acid is between 100 and 200, between 100 and 150, between 50 and 100, between 50 and 200, or between 90 and 140 million.

In some embodiments of the hyaluronic acid compositions described herein, a majority of the hyaluronic acid present in the composition falls within the range of average molecular weights described herein. Thus, for example, in a composition having an average molecular weight of hyaluronic acid between 20 and 200 million, at least 95% of the hyaluronic acid present in the composition falls within a range between 20 and 2 million. In some embodiments, at least 50% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range. In some embodiments, at least 60% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range. In some embodiments, at least 70% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range. In some embodiments, at least 80% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range. In some embodiments, at least 90% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range. In some embodiments, at least 95% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range. In some embodiments, at least 98% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range. In some embodiments, at least 99% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range. In some embodiments, at least 99.9% of the hyaluronic acid present in the compositions provided herein falls within the average molecular weight range.

Sources of hyaluronic acid

The hyaluronic acid used in the compositions and methods described herein can be obtained from any source. Generally, hyaluronic acid has the same chemical structure regardless of its origin (e.g., chicken comb, human tissue, or bacterial cell wall). Hyaluronic acid can be obtained, for example, from cockscombs, from bacterial cell walls, and from human tissues (umbilical cord, vitreous eye, synovial joint fluid, etc.). In some embodiments, the hyaluronic acid is isolated from rooster combs. In some embodiments, the hyaluronic acid is isolated from human tissue (e.g., umbilical cord, vitreous of the eye, synovial fluid of the joints). In some embodiments, the hyaluronic acid is isolated from a cell culture. In some embodiments, the hyaluronic acid is isolated from a bacterial cell wall. Isolation of hyaluronic acid from various sources is known to those of ordinary skill in the art. For example, the collection and purification of hyaluronic acid from rooster combs is described in U.S. Pat. No. 4,141,973, while the collection and purification of hyaluronic acid from bacterial sources is described in U.S. Pat. No. 4,517.295. In some embodiments, hyaluronic acid is purified and collected into a solution containing 0.15M NaCl at pH 6-8. Typically, hyaluronic acid obtained from different sources will not contain proteins or glycosaminoglycans other than hyaluronic acid.

In some embodiments, the isolated hyaluronic acid is further purified to obtain hyaluronic acid having a desired average molecular weight range (e.g., by column chromatography). Methods for purifying hyaluronic acid having the desired range of average molecular weights are known to those of ordinary skill in the art.

In one aspect, the hyaluronic acid with high elasticity disclosed herein is an unmodified hyaluronic acid. However, it is understood that in some embodiments, the hyaluronic acid may be chemically modified. For example, hyaluronic acid may be chemically modified to increase its elasticity.

Sterilization of hyaluronic acid compositions

In some embodiments, the HA compositions described herein and used in the methods of the invention are sterile. As used herein, "sterile composition" refers to a composition that can be safely administered to a subject (e.g., a human subject). Thus, a sterile composition will have only a minimal number of agents that can cause deleterious side effects (such as a deleterious tissue response, immune response, inflammation, or infection).

Methods for sterilizing hyaluronic acid 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 adding the HA composition to a syringe and autoclaving the HA-containing syringe for 2 minutes at 131 ℃ or 15 minutes at 121 ℃ followed by immediate cooling.

Other Components of hyaluronic acid compositions

The HA compositions described herein may comprise other components that may stabilize the hyaluronic acid and/or make the composition more suitable for administration to a subject. In some embodiments, the HA compositions of the present invention may comprise a buffering agent. A buffer is added to ensure a stable pH. Suitable buffers for use in the present invention include phosphate buffers and bicarbonate buffers. In some embodiments, the buffer is a triphosphate buffer. In some embodiments, the buffer is present at a concentration between 1mM and 100mM, between 2mM and 50mM, or between 5mM and 20 mM. In some embodiments, the concentration of the buffer is less than 1 mM. In some embodiments, the concentration of the buffer is greater than 100 mM. In some embodiments, the concentration of the buffer is 10 mM. It will be appreciated that the concentration of the buffer 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 desired, an acid (e.g., HCL) or base (e.g., NaOH) may be added to the composition to achieve the desired pH.

For example, the HA composition may comprise a buffering agent, such as a physiologically compatible buffering agent, but not comprise any other components.

The HA composition may also comprise a stabilizing excipient, such as a carboxylic acid or salt thereof. In some embodiments, the composition comprises a monocarboxylic acid and/or a salt thereof. In some embodiments, the composition comprises gluconic acid and/or sodium gluconate. In some embodiments, the composition comprises a dicarboxylic acid and/or salt thereof. In some embodiments, the composition comprises citric acid, succinic acid, malonic acid, maleic acid, tartaric acid, and/or salts thereof. In some embodiments, the carboxylic acid is sodium citrate. In some embodiments, the composition comprises a tricarboxylic acid (TCA) and/or a salt thereof. In some embodiments, the composition comprises nitrilotriacetic acid and/or sodium nitrilotriacetate. In some embodiments, the composition comprises a tetracarboxylic acid and/or salt thereof. In some embodiments, the composition comprises ethylenediaminetetraacetic 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 (e.g., sodium citrate); tartrate compounds, succinate compounds and EDTA. Carboxylic acids and their use are described by Kaushil et al in Protein Science 19998: 222-. In some embodiments, the stabilizing excipient has a concentration between 50 to 600mM, between 250 to 500mM, or between 250 to 350 mM. In some embodiments, the concentration of the stabilizing excipient is 300mM, 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.

The HA compositions described herein may also comprise a sugar (e.g., a disaccharide). Disaccharides that may be added to the composition include, but are not limited to, sucrose, lactulose, lactose, maltose, trehalose, cellobiose, dextrose, and dextran. In some cases, the sugar may be present at between 0.5 to 5% (weight/volume). In some cases, the sugar may be present at between 1 and 2% (weight/volume). In one embodiment, the sugar may be present at 1%. In some embodiments, the sugar may be present at less than 1% (weight/volume). In some embodiments, the sugar may be present at greater than 5% (weight/volume). In one embodiment, the sugar may be sucrose or trehalose and is present at 1% (weight/volume).

In some embodiments, the HA compositions described herein may comprise a salt. Salts that may be used in the composition include sodium chloride and other physiologically compatible salts. In some embodiments, the salt is present in an HA composition described herein at a concentration of between about 10mM and about 250mM, between about 25mM and about 100mM, between about 30mM and about 70mM, between about 45mM and about 150mM, between about 125mM and about 200mM, between about 150mM and about 250mM, or between about 190mM 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 a specific embodiment, the salt concentration is between about 50mM and about 200 mM.

In some embodiments, the HA compositions of the present invention may have a normal osmolality, for example, about 310 mOsm/L. In other embodiments, the HA compositions of the present invention may have an osmolality lower than normal, for example, lower than 310 mOsm/L. For example, the HA compositions of the present invention may have an osmolality between about 20 and about 500mOsm/L, e.g., between about 150 and about 310mOsm/L, between about 150 and about 200mOsm/L, between about 160 and about 220mOsm/L, between about 180 and about 250mOsm/L, between about 200 and about 300mOsm/L, between about 250 and about 310mOsm/L, between about 290 and about 310mOsm/L, between about 250 and about 290mOsm/L, or between about 270 and about 300 mOsm/L. In some embodiments, the HA compositions of the invention may have an osmolality of about 150mOsm/L, about 155mOsm/L, about 160mOsm/L, about 165mOsm/L, about 170mOsm/L, about 175mOsm/L, about 180mOsm/L, about 185mOsm/L, about 190mOsm/L, about 195mOsm/L, about 200mOsm/L, about 205mOsm/L, about 210mOsm/L, about 215mOsm/L, about 220mOsm/L, about 225mOsm/L, about 230mOsm/L, about 235mOsm/L, about 240mOsm/L, about 245mOsm/L, about 250mOsm/L, about 255mOsm/L, about 260mOsm/L, about 265mOsm/L, about 275mOsm/L, about 285mOsm/L, about 295mOsm/L, about 220mOsm/L, about 225mOsm/L, about, About 300mOsm/L, about 305mOsm/L, or about 310 mOsm/L. In a specific embodiment, the HA composition of the present invention HAs an osmolality between about 100mOsm/L and about 400mOsm/L, such as about 100mOsm/L, about 110mOsm/L, about 120mOsm/L, about 130mOsm/L, about 140mOsm/L, or about 150 mOsm/L.

In some embodiments, the HA compositions described herein comprise one or more antioxidants. Antioxidants are substances that are capable of inhibiting oxidation by removing free radicals from a solution. Antioxidants are well known to those of ordinary skill in the art and include such materials as ascorbic acid, ascorbic acid derivatives (e.g., ascorbyl palmitate, ascorbyl stearate, sodium ascorbate, or calcium ascorbate), butyl hydroxy anisole, butyl hydroxy toluene, alkyl gallate, sodium metabisulfite, sodium bisulfite, sodium hydrosulfite, 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 polyethylene glycol 1000 succinate), monothioglycerol, and sodium sulfite. Such materials are typically added in the range of 0.01 to 2.0%.

In some embodiments, the HA composition may comprise one or more isotonic agents. The term is used interchangeably in the art with isotonic agents and is known to be compounds that can be added to compositions to increase the osmotic pressure, e.g., the osmotic pressure of a 0.9% sodium chloride solution is isotonic with human extracellular fluids (e.g., plasma). Preferred isotonic agents that may be used in the HA composition include sodium chloride, mannitol, sorbitol, lactose, dextrose, and glycerol.

In some embodiments, the HA compositions of the present invention may comprise 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%), phenol (0.1-1.0%), etc.

In some embodiments, the HA composition may comprise a component suitable for ophthalmic use.

In some embodiments, the HA composition may comprise one or more components that minimize deleterious side effects during injection of the composition.

Hyaluronic acid compositions for ophthalmic use

The present invention provides methods for reducing the pain and discomfort associated with dry eye by administering a composition comprising HA to the eyes of a subject.

Any of a variety of carriers may be used in the ophthalmic HA composition, including water, mixtures of water and water miscible solvents (e.g., C1 to C7 alkanols, vegetable oils, or mineral oils containing 0.5 to 5% non-toxic water soluble polymers), natural products such as gelatin, alginates, pectin, tragacanth, karaya, xanthan gum, carrageenan, agar and gum arabic, starch derivatives such as starch acetate and hydroxypropyl starch, and also other synthetic products such as polyvinyl alcohol, polyvinylpyrrolidone, polyvinyl methyl ether, polyethylene oxide, preferably cross-linked polyacrylic acid such as neutral Carbopol (Carbopol), or mixtures of these polymers. The concentration of the carrier is usually 1 to 100,000 times the concentration of the active ingredient.

Other ingredients that may be included in the ophthalmic HA compositions include tonicity enhancing agents, preservatives, solubilizers, non-toxic excipients, demulcents (demuciclets), chelating agents, pH adjusting agents, co-solvents, and viscosity increasing agents.

For adjusting the pH, preferably to physiological pH, a buffer may be particularly useful. The pH of the hyaluronic acid composition suitable for administration to the eye should be maintained in the range of 4.0 to 8.0, more preferably about 4.0 to 6.0, more preferably about 6.5 to 7.8. Suitable buffering agents such as boric acid, sodium borate, potassium citrate, citric acid, sodium bicarbonate, TRIS (hydroxymethyl) aminomethane (TRIS) and various mixed phosphate buffers including Na may be added₂HPO₄、NaH₂PO₄And KH₂PO₄Combinations of (a) and mixtures thereof. Typically, the amount of buffering agent ranges from about 0.05 to 2.5%, preferably from 0.1 to 1.5% by weight.

If desired, the tonicity is usually adjusted by adding tonicity enhancing agents. Such agents may be, for example, ionic and/or non-ionic. Examples of ionic tonicity enhancing agents include alkali or alkaline earth metal halides (e.g., CaCl)₂KBr, KCl, LiCl, NaI, NaBr or NaCl), Na₂SO₄Or boric acid. Non-ionic tonicity enhancing agents include, for example, urea, glycerol, sorbitol, mannitol, propylene glycol, or dextrose. The osmolality of the hyaluronic acid composition used in the method of the invention can be adjusted to approximate the osmolality of normal tears, which corresponds to the osmolality of normal tears, using isotonic agent adjustment0.9% sodium chloride solution or 2.5% glycerol solution. In some instances, an osmolality between about 100mOsm/L and about 150mOsm/L is preferred, such as 100mOsm/L, 110mOsm/L, 120mOsm/L, 130mOsm/L, 140mOsm/L, or 150 mOsm/L. In other examples, an osmolality of between about 225 and 400mOsm/L is preferred, such as between about 280 and about 320mOsm/L or between about 297 and about 318 mOsm/L. In some cases, the mean osmotic pressure may fluctuate between about 303.7 and about 306.7 mOsm/L.

In certain embodiments, the ophthalmic HA compositions may further comprise a preservative. The preservative may typically be selected from quaternary ammonium compounds such as benzalkonium chloride (N-benzyl-N- (C8-C18 alkyl) -N, N-dimethyl ammonium chloride), benzalkonium chloride and the like. Examples of other suitable preservatives include alkyl mercury salts of thiosalicylic acid, such as thimerosal; phenylmercuric nitrate, phenylmercuric acetate, or phenylmercuric borate; sodium perborate; sodium chlorite; parabens, such as methyl paraben or propyl paraben; alcohols, such as chlorobutanol, benzyl alcohol or phenethyl alcohol; guanidine derivatives, such as chlorhexidine or polyhexamethylene biguanide; imidazolidinyl urea (germa (TM)) or sorbic acid. Preferred preservatives are quaternary ammonium compounds, especially benzalkonium chloride or its derivatives such as Polyquad (see U.S. patent No. 4,407,791), alkyl mercury salts, and parabens. Where appropriate, sufficient preservatives are added to the ophthalmic compositions to ensure the prevention of secondary contamination by bacteria and fungi during use.

In another embodiment, the ophthalmic HA composition does not comprise a preservative. Such compositions would be useful for patients wearing contact lenses, or those using several topical eye drops and/or whose ocular surface has been compromised (e.g., dry eye), where it may be more desirable to limit exposure to preservatives.

Ophthalmic HA compositions may additionally require the presence of a solubilizing agent, particularly if the active or inactive ingredients tend to form a suspension or emulsion. Solubilizers suitable for use in the hyaluronic acid composition used in the method of the invention include, for example, tyloxapol, fatty acid glycerol polyethylene glycol esters, fatty acid polyethylene glycol esters, polyethylene glycols, glycerolsOleyl ether, cyclodextrin (e.g. alpha-, beta-or gamma-cyclodextrin, such as alkylated, hydroxyalkylated, carboxyalkylated or alkoxycarbonyl-alkylated derivatives, or mono-or disaccharide-alpha-, beta-or gamma-cyclodextrin, mono-or dimaltosyl-alpha-, beta-or gamma-cyclodextrin or panosyl (panosyl) -cyclodextrin), polysorbate 20, polysorbate 80 or mixtures of these compounds. Preferred solubilizers may be reaction products of castor oil and ethylene oxide, for example the commercial product CremophorOr CremophorThe reaction product of castor oil and ethylene oxide has proven to be a particularly good solubilizer, to which the eye has particularly good tolerance. Another preferred solubilizer may be tyloxapol or cyclodextrin. The concentration used depends in particular on the concentration of hyaluronic acid in the composition. The amount added is generally sufficient to dissolve the hyaluronic acid in the composition. For example, the concentration of the solubilizing agent is 0.1 to 5000 times the concentration of hyaluronic acid in the composition.

The ophthalmic HA compositions may further comprise non-toxic excipients, such as emulsifiers, wetting agents or fillers, such as polyethylene glycol 200, polyethylene glycol 300, polyethylene glycol 400 and polyethylene glycol 600, or Carbowax (Carbowax)1000, Carbowax 1500, Carbowax 4000, Carbowax 6000 and Carbowax 10000. The amount and type of excipient added is according to particular needs and is typically in the range of from about 0.0001% to about 90% by weight.

Other compounds may 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 salts thereof, dextran, polymers of various cellulose families; a vinyl polymer; and acrylic polymers.

In one embodiment, the hyaluronic acid composition intended for ophthalmic use may comprise, in addition to HA, other ingredients typically present in ophthalmic compositions. Examples of such components may include other active ingredients including, but not limited to, vasoconstrictors, anti-allergic agents, anti-infective agents, steroids, anesthetics, anti-inflammatory agents, analgesics, dry eye agents (e.g., secretagogues, mucoids (mucomimetic), polymers, lipids, antioxidants), or administered in combination (simultaneously or sequentially) with compositions comprising the other active ingredients including, but not limited to, vasoconstrictors, anti-allergic agents, anti-infective agents, steroids, anesthetics, anti-inflammatory agents, analgesics, or dry eye agents (e.g., secretagogues, mucoids, polymers, lipids, antioxidants).

In some embodiments, a hyaluronic acid composition of the invention is administered to an ocular surface of a subject, such as under an eyelid (e.g., under an upper or lower eyelid) of a subject or at a cornea-eyelid interface of a subject. In some embodiments, the hyaluronic acid compositions of the invention are not suitable as or not used as viscoelastic surgical tools or devices during ophthalmic surgery, i.e. they are not suitable for injection or not injected into the eye during ophthalmic surgery.

In one example, a hyaluronic acid composition of the invention is administered to a subject immediately prior to rest or sleep.

Hyaluronic acid compositions for skin care/dermatological use

The present invention also provides methods of reducing 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/dermatology use may comprise, in addition to HA, other ingredients typically present in such compositions (e.g., compositions for use as skin fillers). Such ingredients may include, for example, collagen, carnitine, vitamin E, vitamin a, and chondroitin sulfate.

In some embodiments, the HA composition for skin care/dermatological use is injected into the skin. Thus, it can be formulated in the form of a container filled with the HA composition, for example a prefilled syringe. Any prefilled syringe known to those skilled in the art can be used in conjunction with the compositions of the present invention. Prefilled syringes that can be used are described, for example, in PCT publications WO05032627, WO08094984, WO9945985, WO03077976, US6792743, US5607400, US5893842, US7081107, US7041087, US5989227, US6807797, US6142976, US5899889, US20070161961a1, US20050075611a1, US20070092487a1, US20040267194a1 or US20060129108a 1. The prefilled syringe can be made of various materials. In one embodiment, the prefilled syringe is a glass syringe. In another embodiment, the prefilled syringe is a plastic syringe. One skilled in the art will appreciate that the nature and/or quality of the material used to produce the syringe may affect the stability of the HA composition stored in the syringe. In one embodiment, the prefilled injection contains a silicone-based lubricant. In one embodiment, the prefilled syringe comprises baked-on silicone. In another embodiment, the prefilled syringe is free of silicone-based lubricants. Those skilled in the art will also appreciate that small amounts of contaminating ingredients that are immersed in the formulation from the syringe barrel, syringe tip cap, pintle or stopper may also affect the stability of the composition. For example, it is understood that tungsten introduced during the manufacturing process may adversely affect the stability of the formulation. In one embodiment, the prefilled syringe may contain tungsten at levels above 500 ppb. In another embodiment, the prefilled syringe is a low tungsten syringe. In another embodiment, the prefilled syringe may contain tungsten at a level between about 500ppb and about 10ppb, between about 400ppb and about 10ppb, between about 300ppb and about 10ppb, between about 200ppb and about 10ppb, between about 100ppb and about 10ppb, between about 50ppb and about 10ppb, between about 25ppb and about 10 ppb.

Hyaluronic acid compositions for wound healing

The invention also provides methods for alleviating pain and discomfort while promoting wound healing in a subject in need thereof. Such HA compositions for topical use may contain, in addition to HA, other ingredients commonly used in such compositions. Such ingredients may include, for example, 1, 3-butylene glycol, glycerin, xanthan gum, sodium chondroitin sulfate, ethanol, methylparaben, 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 other pharmaceutically active substances. In some cases, these other pharmaceutically active substances do not include analgesics for treating pain. In other cases, these pharmaceutically active substances include antibiotics, such as those commonly used to treat skin infections and intended for topical use.

HA compositions for use in the methods disclosed herein also include finished, packaged, and labeled pharmaceutical products. The article of manufacture comprises a suitable unit dosage form in a suitable vessel or container, such as a glass vial, prefilled syringe or other container that is sealed. In one embodiment, the unit dosage form is provided in the form of 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).

As with any pharmaceutical product, packaging materials and containers are designed to protect the stability of the product during storage and transport. In addition, the products of the invention include instructions for use or other informational material that advises the physician, technician, or patient as to how to properly prevent or treat the disease or condition under consideration, and how often to administer the HA composition. In other words, the article of manufacture includes instructions indicating or suggesting a dosing regimen, including but not limited to actual dosages, monitoring procedures, and other monitoring information.

Methods for reducing pain and discomfort associated with dry eye

Dry eye is an ocular disease affecting about 10-20% of the population. It may be associated with pathological conditions such as cataracts or autoimmune diseases, or in the absence of pathological conditions, may occur in certain situations such as prolonged eye use, work on computers, in dry environments, use of contact lenses or exposure to drugs that cause dryness of the ocular surface.

In individuals with dry eye, the reflex leading to blinking and secretion of supportive tear material is impaired. Signs and symptoms of dry eye include dry eye; tear production, volume and flow reduction; abnormal tear composition; increased tear osmotic pressure; keratitis; conjunctival and corneal staining; reddening; blurred vision; the tear film rupture time is shortened; exacerbation of conjunctival redness; excessive fragments in the tear film, and gritty feeling of eyes; burning the eye; eye foreign body sensation; excessive lacrimation; photophobia; eye stinging; ametropia; ocular sensitivity; eye irritation and discomfort associated with extended contact lens wear. The excess lachrymal response may appear counterintuitive, but is a natural reflex response to irritation and foreign body sensation caused by dry eye. Some individuals may also develop ocular itching caused by a combination of ocular allergies and dry eye symptoms.

There are also many variables that can affect the symptoms of dry eye in humans, including circulating hormone levels, various autoimmune diseases (e.g., sjogren's syndrome and systemic lupus erythematosus), ocular surgery including PRK or LASIK, various drugs, environmental conditions, eye use (e.g., using a computer), eye fatigue, wearing contact lenses, and mechanical effects (e.g., corneal sensitivity, partial eyelid closure, surface irregularities (e.g., pterygium), and eyelid irregularities (e.g., eyelid droop, lid inversion/lid eversion, conjunctival macula). At this time, the blink rate decreases.

Compositions containing HA have been previously used to treat dry eye. Eye drops containing HA are commercially available and typically contain HA at concentrations below 1%. Exemplary commercially available eye drops containing HA 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).

Applicants have surprisingly found that highly concentrated HA compositions comprising HA at a concentration greater than about 30mg/mL (or 3% HA) (e.g., 40mg/mL (or 4% HA)) can be effectively administered to the eyes of a subject for the treatment of dry eye. Such highly concentrated HA compositions have not been used previously for the treatment of dry eye because these compositions are considered too viscous to be effectively administered to the eye.

Applicants have also surprisingly found that once applied to the eye, the concentrated HA compositions are surprisingly effective in treating the pain and discomfort associated with dry eye. Without wishing to be bound by a particular theory, it is believed that pain associated with dry eye may be modulated by the TRPV1 channel of the nociceptor, while the HA molecule in the highly concentrated HA composition interacts with the TRPV1 channel present on the sensory nerves of the eye and on the corneal cells, thereby reducing the nociceptor response to noxious stimuli. Thus, in some embodiments, pain and discomfort associated with dry eye are modulated by the receptor potential vanilloid 1(TRPV1) channel. Moreover, the characteristic elastoviscous nature of concentrated HA reduces the removal of the solution applied to the ocular surface by normal tearing and blinking, thereby prolonging the protective effect of the HA solution.

The present invention provides a method for reducing pain and discomfort associated with dry eye in a subject in need thereof, comprising administering to the eye of the subject a composition comprising hyaluronic acid, wherein hyaluronic acid is present in the composition at a concentration of greater than about 30 mg/mL; hyaluronic acid has an average molecular weight of between about 100 and about 200 million; and hyaluronic acid is uncrosslinked and/or substantially free of chemical modification, thereby reducing the pain and discomfort in the subject.

In some cases, the HA composition intended for ophthalmic use is in the form of a gel, ointment, liniment, lotion, or cream. In certain aspects, the HA composition is not in the form of lenses or microspheres.

An HA composition intended for ophthalmic use may be administered to the ocular surface, such as under the eyelid (e.g., under the upper or lower eyelid) or at the cornea-eyelid interface. In one aspect, the HA composition is not injected into the eye of the subject. Due to their higher viscosity, the ability of the subject to see clearly immediately after administration of the HA compositions may be affected, and thus these compositions may be administered to the eyes of the subject immediately prior to rest or sleep.

One of ordinary skill in the art will be able to determine suitable administration regimens for the ophthalmic HA compositions described herein to achieve reduction of the pain and discomfort associated with dry eye. For example, the HA composition may 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 for any length of time until relief of the pain and discomfort associated with dry eye is achieved. In some embodiments, such reduction of pain and discomfort can be a long-term reduction, for example, lasting 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.

The term "dry eye" includes, but is not limited to, dry eye associated with one or more symptoms selected from the group consisting of: dry eyes; the tear film rupture time is shortened; tear production, volume and flow reduction; abnormal tear composition; increased tear osmotic pressure; keratitis; conjunctival and corneal staining; reddening; blurred vision; exacerbation of conjunctival redness; excessive fragments in the tear film, and gritty feeling of eyes; burning the eye; eye foreign body sensation; excessive lacrimation; photophobia; eye stinging; ametropia; ocular sensitivity; eye irritation and discomfort associated with extended contact lens wear.

Dry eye may also be associated with a condition selected from the group consisting of: (ii) an autoimmune disease; eye surgery; taking in the drug; dry ambient conditions; using the computer for a long time; eye fatigue; contact lens wearing, cornea sensitive; partial eyelid closure; the surface is not flat; (iii) eyelid irregularities; and conditions associated with neuropathic pain (such as cataracts or retinal detachment). In one example, dry eye may be associated with an eye surgery, such as refractive surgery (e.g., photorefractive keratectomy (PRK)), cataract surgery, retinal detachment surgery, laser in situ keratomileusis (LASIK), or any corneal surgical procedure involving damage to the corneal sensory nerve.

IX. method for reducing pain and discomfort while minimizing skin imperfections

HA is a major component of the extracellular matrix (ECM) and is present in particularly large amounts in soft connective tissue, such as skin. In normal skin, HA is synthesized primarily by dermal fibroblasts and epidermal keratinocytes. With its negatively charged residues, HA acts as a water pump to maintain the hydration and elasticity of the skin. HA HAs a major role in controlling the distribution of food, hormones, microorganisms and inorganic salts in connective tissues and in clearing metabolic wastes that may induce inflammatory reactions. With age, the amount of HA and its degree of aggregation decreases, resulting in a decrease in the amount of water retained in the connective tissue. The skin then undergoes an aging process, resulting in increased fibrosis and a decrease in the quality of the elastic fibers.

HA HAs been widely used in cosmetic applications, including the use of HA compositions in cream or gel form for topical application and the use of HA compositions injected into the skin as a dermal filler. The latter use involves injecting the HA composition into the top skin layer of a subject, such as the face, neck, arms, legs, torso, or chest of a subject. The use allows the reduction of wrinkles, due to a mechanical filling effect on skin depressions caused by wrinkles, and due to a preventive effect on skin aging and degradation of the ECM necessary for maintaining the mechanical properties of skin elasticity and firmness.

Although injection of dermal fillers for soft tissue augmentation is a minimally invasive dermatological procedure, patients often exhibit concerns about the pain associated with such procedures. Local anesthetic creams are commonly used to reduce pain during these procedures, and anesthetic agents may also be included in the HA composition for injection.

The present invention avoids the need to use anesthetic agents during these procedures. In particular, the present invention provides methods for reducing pain and discomfort associated with dermatological procedures while minimizing at least one skin imperfection in a subject in need thereof by administering to the subject a highly concentrated HA composition. As noted above, it HAs been surprisingly found that the highly concentrated HA compositions described herein are surprisingly effective in treating 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 pain associated with injection of the HA composition into the skin may be modulated by the TRPV1 channel of nociceptors, while the HA molecules in the highly concentrated HA composition interact with the TRPV1 channel present in the sensory nerves and epithelial and connective tissue cells of the skin and subcutaneous tissue, thereby reducing nociceptor responses to noxious stimuli.

In some embodiments, the HA composition for cosmetic use may 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 infusion device. An injection device suitable for injecting the HA composition of the invention may have a nominal diameter of 2.11mm or greater (corresponding to a 14G needle, or a 14 gauge or greater). In some embodiments, the HA compositions of the present invention may be too viscous to be administered using a smaller needle (e.g., a needle having a nominal diameter of less than 2.11 mm). In other embodiments, the HA compositions of the present invention may allow for administration using smaller injection devices having a nominal diameter of less than 2.11 mm.

For example, a device (e.g., a syringe) suitable for injecting an HA composition of the invention may have a nominal diameter (e.g., No. 20, No. 5, No. 5.23, No. 5, No. 7, No. 0.23, No. 0.59, No. 0.642, No. 0.64, No. 0.67, No. 0.718, No. 0.72, No. 0.77, No. 0.72mm, No. 0.42 mm, No. 1.42mm, No. 1.47mm, No. 1.42mm, No. 1.47mm, No. 1.42mm, No. 1.47mm, No. 1.7 mm, No. 7mm, No. 1.7 mm, No. 1.73mm, No. 1.7 mm, No. 7mm, No. 1.7 mm, No. 7mm, No. 1.73mm, No. 1.7 mm, No. 7mm, No. 1.7 mm, No. 1.73mm, No. 1.7 mm, No. 7mm, No. 1.73mm, No. 1.7 mm, No. 20, No. 7mm, No. 23, No. 7mm, No. 20, No. 7mm, No. 23, No. 25, No. 7mm, No. 23, No. 7mm, No. 25, No. 23, No. 25, No. 7mm, No. 23.23.23.23.23, no, 28G, 27G, 26sG, 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 composition of the present invention may be administered using an 18G syringe needle having a nominal diameter of about 1.27 mm. In some embodiments, the HA compositions of the present invention may be too viscous to be administered using a smaller needle (e.g., a needle having a nominal diameter of less than 1.27 mm).

In some embodiments, the methods of the invention may further comprise administering the highly concentrated HA composition to the subject in the form of an injectable implant (particularly a dermal implant).

The composition according to the invention is particularly intended for use in the filling of wrinkles, fine lines, skin depressions and scars, including the filling of skin depressions caused by lipodystrophy or lipoatrophy, in reconstructive or plastic surgery or cosmetic dermatology of humans or animals. The composition may be an implant as defined above.

Method for reducing pain and discomfort while promoting wound healing

The skin provides a mechanical barrier to the external environment and acts to prevent the entry of infectious agents. Once injured, the underlying tissue will be exposed to infection; rapid and efficient healing is therefore of crucial importance for reestablishing barrier function. Skin wound healing in adults is a complex process and involves multiple stages, such as inflammation, granulation tissue formation, re-epithelialization and remodeling. HA may play a variety of roles in the mediation of these cellular and matrix events. Typically, local anesthetics are applied to wounds to relieve the pain associated with these wounds.

The present invention provides methods for effectively treating skin wounds while providing pain relief and avoiding the need for the use of local anesthetics. In particular, the present invention provides methods for reducing pain and discomfort while promoting wound healing by administering a highly concentrated HA composition to a subject in need thereof. As noted above, it HAs been surprisingly found that the highly concentrated HA compositions described herein are surprisingly effective in treating pain and discomfort associated with wound healing. Without wishing to be bound by a particular theory, it is believed that pain associated with injection of the HA composition into the skin may be modulated by the TRPV1 channel of nociceptors, while the HA molecules in the highly concentrated HA composition interact with the TRPV1 channel present in the sensory nerves innervating the wounded area and in the connective tissue cells of that area, thereby reducing the nociceptor response to noxious stimuli.

According to the method of the present invention, a highly concentrated HA composition intended for topical application to a skin wound may be topically applied on the surface of a wound or scar on the skin. The composition may be in the form of an aqueous gel or polyol containing a thickening polymer (such as a cellulose derivative or an acrylic polymer) together with other excipients conventionally used (such as preservatives, perfumes, etc.).

The therapeutic, prophylactic or palliative effect is evident when there is a statistically significant improvement in one or more parameters of a disease state or pathological condition (e.g., pain and discomfort associated with dry eye, skin care/dermatology procedures or wound healing). Therapeutic, or palliative or prophylactic effects are also evident if no exacerbations or symptoms are expected to occur or to progress. For example, a favorable change of at least 10% (and preferably at least 20%, 30%, 40%, 50% or more) in a measurable parameter of a disease or condition (e.g., pain and discomfort) may indicate an effective treatment. As used herein, the term "prevention" includes preventing reoccurrence of pain and discomfort, for example, in a subject who has previously experienced pain.

In some embodiments, the subject is a human, a mammal, e.g., a domestic animal (such as a cat or dog), a farm animal (such as a cow, sheep, horse, donkey), or a rodent (such as a guinea pig, mouse, or rat). In a specific embodiment, the subject is a human. In another specific embodiment, the subject is a dog.

As used herein, the term "reducing at least one symptom" includes reducing, alleviating, or eliminating at least one symptom associated with pain and discomfort. The term also includes reducing the extent of activation of an ion channel involved in pain transduction processes in neurons, such as the TRPV1 channel, following administration of the HA compositions of the present invention. Activation of such channels after administration of the HA compositions of the invention can be measured, for example by measuring injuryIntracellular Ca in neurons after sexual stimulation²⁺Or by measuring whole cell current in the neuron. Furthermore, the term "reducing at least one symptom" also includes reducing nociceptive firing of neurons after administration of the HA compositions of the present invention.

In some embodiments, a method of reducing pain and discomfort associated with dry eye, a skin care/dermatological procedure, or wound healing comprises administering to a subject in need thereof a therapeutically effective amount of a composition of the present invention. As used herein, the term "therapeutically effective amount" 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. One of ordinary skill in the art (e.g., a physician) will be readily able to determine the amount of HA composition that will be therapeutically effective. Typically, the therapeutically effective amount of the composition is between about 0.1 to about 500mg, such as about 0.1mg, about 1mg, about 5mg, about 10mg, about 20mg, about 30mg, about 40mg, about 50mg, about 60mg, about 70mg, about 80mg, about 90mg, about 100mg, about 110mg, about 120mg, about 130mg, about 140mg, about 150mg, about 160mg, about 170mg, about 180mg, about 190mg, about 200mg, about 210mg, about 220mg, about 230mg, about 240mg, about 250mg, about 260mg, about 270mg, about 280mg, about 290mg, about 300mg, about 310mg, about 320mg, about 330mg, about 340mg, about 350mg, about 360mg, about 370mg, about 380mg, about 390mg, about 400mg, about 410mg, about 420mg, about 430mg, about 440mg, about 450mg, about 460mg, about 480mg, about 470mg, about 500mg, or about 500 mg.

The methods disclosed herein encompass a variety of treatment regimens. For example, a subject may receive a first dose of an HA composition disclosed herein, followed by another dose. In some embodiments, a first dose is administered followed by a second dose at specific intervals. 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 is understood that the dosing regimen may be adjusted based on the reduction in pain and discomfort experienced by the subject. In some embodiments, the subject will receive a dose every month, every two months, every 3 months, every 4 months, every 5 months, every 6 months, every 7 months, every 8 months, every 9 months, every 10 months, every 11 months, or every 12 months.

The invention is further illustrated by the following examples, which should not be construed as further limiting. All references (including references, issued patents, published patent applications, and co-pending patent applications) cited throughout this application are hereby expressly incorporated by reference in their entirety.

Examples

Unless otherwise specified, Hyaluronic Acid (HA) used in the compositions described herein is obtained from animal, human or bacterial sources. Unless otherwise specified, the compositions used herein are in a physiological buffer.

Example 1: reduction of pulse activity in corneal sensory nerve fibers by topical application of 4% sodium hyaluronate onto the cornea

The excitation of nociceptors is the first step in producing a sensation of pain. For acute pain, the degree of pain perception is closely related to the frequency of firing of the pain fibers and the number of fibers recruited by the stimulus (Acosta et al, 2001, J.Physiol.534(2), 511-. Furthermore, persistent pain following peripheral injury is associated with persistent activity of nociceptors (Belmonte et al, 2004, Ocular Surface,2, 248-.

The increased sensory nerve impulse activity in the corneal nerves is the first step in creating the sensation of ocular discomfort and pain. Nociceptive stimulation is detected in nerves in the ocular surface by polymodal and mechano-nociceptors that innervate the cornea and conjunctiva (Belmonte & Giraldez,1981, J. physiol.,437, 709-. When inflammation occurs, the pleiotropic nociceptors become sensitive and continue to discharge, causing a persistent sensation of pain. On the other hand, activity in corneal thermo-caloric receptors has been found to be associated with ocular surface evaporation, and may also contribute to the perception of ocular surface dryness (Gallar et al, 1993, J.Physiol.,468, 609-.

The experimental aim described below was to examine the capacity of hyaluronic acid, at a concentration of 4% (4% HA) and with an average molecular weight of 100-200 ten thousand, to modify the spontaneous and stimulated discharge of nerve impulses in sensory receptor fibers (mechano-nociceptors, multifactorial nociceptors, thermoreceptors) innervating the guinea pig cornea.

An effective means of generating chemical stimulation of corneal polymorphous nociceptors is the use of 98.5% CO₂Acidification is caused by the local formation of carbonic acid (Chen et al, 1995, Eur. J. Neurosci.7, 1154-1163). It has been demonstrated that similar CO is applied to the human cornea₂Pulses immediately cause acute pain in all cases (Chen et al, 1995, Eur. J. Neurosci.7, 1154-1163; Acosta et al, 2001, J. Physiol.534(2), 511-525). H is due to the rapid formation of protons around nerve endings⁺The stimulation of (a) may be direct and occurs by activation of proton-gated ion channels that depolarize nerves, which may be "capsaicin channels" TRPV1 and/or ASIC channels. And, receiving CO₂The decrease in intracellular pH in stimulated corneal epithelial cells may also lead to the formation and release of inflammatory mediators (e.g., prostaglandins) that may sensitize corneal polymodal nociceptors, cause background discharges and enhance the pulse response to acidic stimuli.

In the case of corneal thermo-thermal receptors, these fibers exhibit background, regular pulsatile activity at a basal corneal temperature of 34 ℃, and increase their discharge frequency with very small temperature decreases (e.g., the temperature decrease that occurs during tear evaporation) (Belmonte et al, 2015, curr. ophthalmol. rep.3, 111-121).

Method

Two sex guinea pigs of the species Goldley (Dunkin Hartley) weighing 200-. The study was conducted according to NIH laboratory animal care and use guidelines and european union directives (2010/63/EU) and spanish regulations regarding animal protection for research, and in accordance with protocols approved and supervised by the ethical committee of the university of miers-ehrlandes.

Electricity generationReason record

The pulse activity of different functional types of peripheral corneal receptors was recorded. To achieve this, animals were euthanized by intraperitoneal injection of 100mg/kg sodium pentobarbital, and both eyes, with bulbar conjunctiva and tarsal conjunctiva and optic nerve and ciliary nerve, were immediately enucleated and placed in cold saline (4 ℃).

Connective tissue and extraocular muscles were carefully removed from the excised eyeball to expose the posterior portion of the eye with the ciliary nerves surrounding the optic nerve. The eye is then placed in a specially designed double chamber to keep the anterior segment of the eyeball with conjunctiva separated from the dorsal pole and ciliary nerves. In the anterior chamber, the conjunctiva is pinned to the septum to isolate the two chambers that are separately perfused. The anterior chamber was perfused with warm (34 ℃) saline and dropped continuously at the upper corneoscleral border. In the posterior chamber of the compartment filled with warm mineral oil, the nerve filaments were separated from the ciliary nerves and placed on an Ag-AgCl electrode to record single unit pulse activity unipolar using conventional electrophysiological equipment. The electrical signal was recorded for Ag/AgCl particles (pellet) in the back chamber. The electrical signals were transferred to a PC with a CED interface and analyzed using appropriate software. The selected units of spontaneous activity were recorded for 1 minute prior to any expected stimulation. The mechanical threshold was then determined using a calibrated von Frey ciliary mechanical stimulator needle (range 0.25-4.00 mN). The receptive field of the corneal afferent fibers was localized with mechanical stimulation using a fine brush and then mapped using a suprathreshold von Frey ciliary mechanical stimulation needle. For chemical stimulation, 98.5% CO was administered in the corneal receptor field₂Jet gas for 30 seconds. The thermal stimulation was performed by cooling (down to 20 ℃) the perfusion solution using a custom-made Peltier device.

Data analysis

In single fiber recordings of multi-modal fibers, the pulse discharges of individual cells, distinguished by their stimulus form, amplitude and shape, are analyzed. The sustained pulse activity is represented as the average pulse frequency (in pulses/second) measured during the 30 seconds and during the stimulation interval at the start of recording. Measuring the following parameter pairs for CO₂The response of (a) was quantified: incubation period: in CO₂Pulse initiation and unit administrationThe time delay between the first pulses of (a); average discharge rate: in the whole CO₂Average number of pulses per second (pulses/second) in the pulses; post-discharge: in CO₂Average discharge frequency (pulse/sec) during 30 seconds immediately after the pulse.

In cold nerve fiber recording, the following additional discharge pattern parameters were calculated: average discharge frequency: the average number of pulses recorded per second (pulses/second); cooling threshold value: a temperature during the cooling ramp to obtain a 25% increase in the average nerve pulse frequency at the base temperature; peak response: maximum pulse/second value of pulse frequency during the cooling ramp; temperature at peak response: temperature value (. degree. C.) at which peak frequency is reached.

Experimental protocol

The following experimental sequence was followed:

identification of multi-sense type pain fiber

The silk-containing fibers that innervate the cornea and respond to mechanical stimulation with a wet thin brush applied to the corneal surface were identified. After the nerve filaments are separated, individual corneal nociceptive units are localized by mechanical stimulation. To further characterize each isolated single unit (determine the polytropic form), the following procedure was performed:

1. drawing a receptive field boundary and determining a mechanical threshold by adopting von Frey wires;

2. measurement on 98.5% CO₂30 seconds pulse response. If no response is detected, the new fiber is examined.

Effect of 4% HA on Multi-nociceptors

A 4% HA solution was applied to the corneal surface. Periodically, 1 drop of saline was applied to the cornea to maintain moisture. After 4% HA instillation, 30 seconds of CO was applied to the corneal surface at successive times (5, 20, 35, 50, 65 and 80 minutes after 4% HA administration)₂And (4) pulse. The cornea was then washed continuously with saline for 5 minutes, and the CO was measured 15 and 20 minutes after washing₂The response of (1). The electrical threshold was measured at the end of the protocol to confirm the responsiveness of the fibers. Measurement of spontaneous Activity and of CO present throughout the course of the experimental work₂The amplitude of the stimulus pulse response.

Identification of Cold-Heat sensitive fibers

The filaments containing the cool-heat sensitive fibers innervating the cornea were identified by applying a drop of cold saline to the cornea. After dividing the fibers, a wire containing a single corneal cold unit was positioned using an ice cold metal rod (tip diameter 1mm) applied to the cornea to map the receptive field.

The sustained pulse activity exhibited by individual cold sensitive nerve endings at 34 ℃ was recorded. A cooling ramp from 34 ℃ to 15 ℃ was performed by changing the temperature of the receptive field using a Peltier device.

Effect of 4% HA on Cold and Heat receptor neural Activity

4% HA was applied to the ocular surface exposed in the anterior chamber of the compartment and the effect on the sustained activity of the cold thermal receptors at 34 ℃ and the response to the cooling ramp was examined 5, 20, 35, 50, 65 and 80 minutes after HA application and 15 and 20 minutes after corneal washing.

The average sustained activity at a resting temperature of 34 ℃, the thermal threshold, and the increase in neural impulse activity during the cooling ramp were determined.

Results

In an intact cornea, sensory afferents identified as polymorphous nociceptor fibers present a corneal receptive field that typically extends up to 1mm into the adjacent sclera. The pleiotropic nociceptor fibers were silent at rest, on mechanical stimulation and on 98.5% CO₂A response is generated.

4% HA was measured for persistent activity and for CO in 2 single multi-sense units and 2 cold sensitivity units₂The effect of the discharge response of (c). CO before and after application of 4% HA to the corneal surface was analyzed₂The function of (1). As shown in table 1 below, the pleiotropic nociceptors were silent at rest, with no sustained activity during the 2 minute recording period. They are for 30 seconds of CO₂The pulses produced a 2.5 second response, with an average pulse discharge frequency of about 6 pulses per second. During the first 35 minutes after 4% HA was applied to the corneaTo CO₂The response of (a) is significantly reduced and then stopped. Washing after 80 minutes of 4% HA treatment gradually restored CO₂The response of (1).

Table 1: continuous action of corneal polymorphous nociceptors, CO₂Response and post-discharge values are expressed in pulses per second; latency is expressed in seconds. For thermal receptors, spontaneous activity, cooling ramp response, and peak frequency values are expressed in pulses per second. The threshold and temperature at peak frequency are expressed in ° c; n.r.: there is no pulsing activity.

Pulse activity in the thermal receptor fibers was also recorded, representing about 10% of all corneal sensory afferents. We demonstrate normal sustained pulse activity at 34 ℃ in the intact eye and a significant frequency increase caused by 15 ℃ cooling pulses, which are characteristic of ocular cold and heat receptors (Gallar et al, 1993, J. Physiol.,468, 609-139622; Parra et al, 2010, Nature Medicine,16, 1396-1399).

As shown in table 1, exposing the cornea to 4% HA for 4 hours at 34 ℃ reduced background persistence of the hot and cold receptors. No significant change in the cold threshold was observed, but as also shown in table 1, the response to the cooling ramp was significantly reduced and only partially recovered 20 minutes after washing, during which the peak frequency value excited by the cooling ramp returned to normal.

Taken together, these results indicate that 4% HA reduces abnormal pulse activity in polymorphous nociceptor fibers caused by acid stimulation of the cornea. This is probably the result of the interaction of the HA molecule with the TRPV1 channel sensitizing the nociceptive nerve endings (Caires et al 2015, nat. comm.6,8095), demonstrating that high concentrations of HA are very effective in inducing this effect. Moreover, the reduced activity in the cold and hot receptors is likely due to the more effective barrier formed at the corneal surface compared to lower concentrations of HA solution.

Example 2: force required to expel HA through different needle sizes

The pressure required to expel the 4% HA composition from a 3mL syringe with different diameter needles (30-18G) was measured and is shown in FIG. 1. One plate, balanced by two plates, applies a force that acts vertically on the pintle of the syringe. An increasing amount of weight was added to the opposite side panel. As evidenced by the results shown in figure 1, the HA compositions of the present invention can be administered to a subject using a needle having a diameter of 30-18G.

Equivalents of

The foregoing written description is considered to be sufficient to enable those skilled in the art to practice the invention. The scope of the invention is not limited by the examples provided, as these examples are intended as single illustrations of one aspect of the invention, and other functionally equivalent embodiments are within the scope of the invention. Various modifications of the invention, in addition to those shown and described herein, will be apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Advantages and objects of the present invention are not necessarily included in every embodiment of the present invention.