Claims for Patent: 6,682,760

Summary for Patent: 6,682,760

Title:	Cross-linked collagen matrices and methods for their preparation
Abstract:	A method for preparing cross-linked collagen and cross-linked collagen products. The method includes incubating collagen in a solution including water, at least one polar solvent and at least one sugar, to form cross-linked collagen. The solution may include a buffer having a suitable pH and ionic strength. The method may include removing excess unreacted sugar(s) and polar solvent(s) by washing the cross-linked collagen or by other methods. The method may also include dehydrating the cross-linked collagen, and may include subjecting the cross-linked collagen to critical point drying, or subjecting the collagen to drying or freeze-drying prior to cross-linking. The collagen may be prepared from atelopeptide collagen to reduce antigenicity, but may also be prepared from other suitable collagen types. The concentration and type of the polar solvent(s), the concentration and type of the reducing sugar(s), and the incubation duration may be varied to control the degree of cross-linking. The cross-linked collagen product may be in the form of a wet or dry matrix or membrane or may be suspended in a liquid in the form of an injectable preparation. The method may be applied to collagenous proteins and collagen-like peptides.
Inventor(s):	Noff; Matitiau (Tel Aviv, IL), Pitaru; Shahar (Givataim, IL)
Assignee:	Colbar R&D Ltd. (Ramat Hasharon, IL)
Application Number:	09/828,189
Patent Claims:	1. A method for preparing cross-linked collagen having improved resistance to collagenase degradation, the method comprising the step of incubating collagen in a cross-linking solution comprising water, at least one polar solvent different than water and at least one sugar, to form cross-linked collagen. 2. The method according to claim 1 wherein said at least one sugar is a reducing sugar. 3. The method according to claim 1 wherein said at least one polar solvent comprises at least one organic polar solvent. 4. The method according to claim 3 wherein said at least one organic polar solvent is an alcohol. 5. The method according to claim 3 wherein said at least one organic polar solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol acetone, teftahydrofuran, dimethylsulfoxide, and combinations thereof. 6. The method according to claim 1 wherein said at least one polar solvent is miscible in water. 7. The method according to claim 1 wherein said solution is a buffered solution comprising a buffer. 8. The method according to claim 1 wherein said solution comprises phosphate buffered saline. 9. The method according to claim 1 wherein said solution comprises water in the range of 15%-95% (v/v), said at least one polar solvent in the range of 5%-85% (v/v), and a buffer. 10. The method according to claim 1 wherein said collagen is selected from, native collagen, fibrillar collagen, fibrillar atelopeptide collagen, lyophylized collagen, collagen obtained from animal sources, human collagen, recombinant collagen, pepsinized collagen, reconstituted collagen, and combinations thereof. 11. The method according to claim 1 wherein said collagen comprises fibrillar collagen reconstituted from monomolecular atelopeptide collagen. 12. The method according to claim 1 wherein said collagen is obtained by reconstituting monomolecular atelopeptide collagen obtained by proteolytic digestion of native collagen. 13. The method according to claim 1 wherein said at least one sugar is a compound represented by one of the following formulae I or II: ##STR3## wherein: R.sup.1 is H or lower alkyl or alkylene, an amino acid, a peptide, a saccharide, a purine or a pyrimidine base, a phosphorylated purine or pyrimidine base; n is an integer between 2-9, and p and q are each independently an integer between 0-8, and the sum of p and q is at least 2 and not more than 8. 14. The method according to claim 1 wherein said at least one sugar is a naturally occurring reducing sugar. 15. The method according to claim 1 wherein said at least one sugar is selected from a group consisting of a diose, a triose, a tetrose, a pentose, a hexose, a septose, an octose, a nanose, and a decose. 16. The method according to claim 1 wherein said at least one sugar is selected from the group consisting of glycerose, tbreose, erythrose, lyxose, xylose, arabinose, ribose, allose, altrose, glucose, mannose, gulose, idose, galactose and talose. 17. The method according to claim 1 wherein said at least one sugar is a disaccharide. 18. The method according to claim 17 wherein said disaccharide is selected from the group consisting of maltose, lactose, sucrose, cellobiose, gentiobiose, melibiose, turanose, and trehalose. 19. The method according to claim 1 wherein at least one substance is added to the solution in which said step of incubating is performed, said at least one substance becoming immobilized within said matrix. 20. The method according to claim 19 wherein said at least one substance is selected from the group consisting of an antimicrobial agent, an anti-inflammatory agent, a factor having tissue inductive properties, and combinations thereof. 21. The method according to claim 1 wherein said at least one sugar is D(-)ribose, and said at least one polar solvent is ethanol. 22. The method according to claim 21 wherein said solution comprises water in the range of 15%-95% (v/v) and ethanol in the range of 5%-85% (v/v). 23. The method according to claim 21 wherein said solution comprises water in the range of 25%-50% (v/v) and ethanol in the range of 50%-75% (v/v). 24. The method according to claim 21 wherein said solution comprises about 30% water (v/v), and about 70% ethanol (v/v). 25. The method according to claim 21 wherein the concentration of D(-)ribose in said solution is in the range of 0.1%-5% (w/v). 26. The method according to claim 21 wherein the concentration of D(-)ribose in said solution is in the range of 0.5%-3% (w/v). 27. The method according to claim 1 further comprising the step of washing said cross-linked collagen after said step of incubating to remove said at least one polar solvent and excess of said at least one sugar. 28. The method according to claim 1 further comprising the step of dehydrating said cross-linked collagen. 29. The method according to claim 28 further comprising the step of subjecting said cross-linked collagen to critical point drying. 30. The method according to claim 1 further comprising the step of drying or freeze-drying said collagen prior to said step of incubating. 31. The method according to claim 1 further comprising the step of drying or freeze-drying said cross-linked collagen. 32. A cross-linked collagen preparation prepared by the method of claim 1. 33. A method for preparing cross-linked collagen having improved resistance to collagenase degradation, the method comprising the step of incubating collagen in a cross-linking solution comprising water, at least one hydrophilic solvent different than water and at least one sugar to form said cross-linked collagen. 34. The method according to claim 33 wherein said step of incubating comprises setting the duration of said incubating of said collagen to control the degree of cross linking of said cross-linked collagen. 35. The method according to claim 33 wherein said step of incubating comprises setting the concentration of said at least one sugar used in said step of incubating to control the degree of cross linking of said cross-linked collagen. 36. The method according to claim 33 wherein said step of incubating comprises setting the concentration of said at least one hydrophilic solvent used in said step of incubating to control the degree of cross linking of said cross-linked collagen. 37. The method according to claim 33 further comprising the step of removing from said solution at least some of the unreacted amount of said at least one sugar, and removing at least some of said at least one hydrophilic solvent. 38. The method according to claim 33 further comprising the step of washing said cross-linked collagen to remove at least some of the unreacted amount of said at least one sugar and to remove at least some of said at least one hydrophilic solvent. 39. A method for preparing cross-linked collagen having improved resistance to collagenase degradation, the method comprising the step of incubating collagen in a cross-linking solution comprising water, at least one polar solvent different than water and D(-)Ribose. 40. The method according to claim 39 wherein said at least one polar solvent is selected from the group consisting of methanol, ethanol, propanol, isopropanol acetone, tetrahydrofuran, dimethylsulfoxide, and combinations thereof. 41. The method according to claim 39 wherein said collagen is selected from, native collagen, fibrillar collagen, fibrillar atelopeptide collagen, lyophylized collagen, collagen obtained from animal sources, human collagen, recombinant collagen, pepsinized collagen, reconstituted collagen, and combinations thereof. 42. The method according to claim 39 wherein said collagen comprises fibrillar collagen reconstituted from monomolecular atelopeptide collagen. 43. The method according to claim 39 wherein said collagen is atelopeptide fibrillar collagen obtained by reconstituting monomolecular atelopeptide collagen obtained by proteolytic digestion of native collagen. 44. The method according to claim 39 wherein the concentration of D(-)ribose in said solution is in the range of 0.1%-5% (w/v). 45. The method according to claim 39 wherein the concentration of D(-)ribose in said solution is in the range of 0.5%-3% (w/v). 46. The method according to claim 39 wherein said solution comprises water in the range of 15%-95% (v/v) and said at least one polar solvent in the range of 5%-85% (v/v). 47. The method according to claim 39 wherein said solution comprises phosphate buffered saline in the range of 15%-95% (v/v) and said at least one polar solvent in the range of 5%-85% (v/v). 48. The method according to claim 39 wherein said solution is a buffered solution comprising a buffer. 49. The method according to claim 39 wherein said solution comprises phosphate buffered saline. 50. The method according to claim 39 wherein said solution comprises water in the range of 15%-95% (v/v), at least one polar solvent in the range of 5%-85% (v/v), and a buffer. 51. A method for preparing cross-linked collagen having improved resistance to collagenase degradation, the method comprising the step of incubating reconstituted atelopeptide fibrillar collagen in a cross-linking solution comprising water, at least one polar solvent different than water and at least one reducing sugar. 52. The method according to claim 51 wherein said cross-linking solution is a buffered solution. 53. A method for preparing a cross-linked collagen having a desired resistance to degradation, the method comprising the steps of: cross-linking collagen by incubating said collagen in a cross-linking solution comprising water, at least one polar solvent different than water and at least one sugar; and controlling the duration of said incubating to obtain cross-linked collagen having a desired resistance to degradation. 54. A method for preparing a cross-linked collagen having a desired resistance to degradation, the method comprising the steps of: cross-linking collagen by incubating said collagen in a cross-linking solution comprising water, at least one polar solvent different than water and at least one sugar; and selecting the concentration of said at least one polar solvent in said cross-linking solution to obtain cross-linked collagen having a desired resistance to degradation. 55. A method for preparing a cross-linked collagen having a desired resistance to degradation, the method comprising the steps of: cross-linking collagen in a cross-linking solution comprising water, at least one polar solvent different than water and at least one sugar; and selecting the concentration of said at least one sugar in said cross-linking solution to obtain cross-linked collagen having a desired resistance to degradation. 56. An improved cross-linked fibrillar collagen matrix obtained by a process for its preparation from fibrillar collagen, said process comprising the following steps: providing a matrix comprising reconstituted fibrillar collagen; and incubating said matrix in a cross-linking solution comprising water, at least one polar solvent different than water and at least one sugar for cross-linking said fibrillar collagen to form a cross-linked fibrillar collagen matrix. 57. The matrix according to claim 56, in the form of an implantable device. 58. The matrix according to claim 57, wherein said implantable device is a collagen based membrane barrier for guided tissue regeneration. 59. The matrix according to claim 56 wherein said process further includes the step of washing said cross-linked collagen matrix after said step of incubating to remove at least some of said at least one polar solvent and unreacted excess of said at least one sugar. 60. The matrix according to claim 56 wherein said process further includes the step of dehydrating said cross-linked fibrillar collagen matrix after said step of incubating. 61. The matrix according to claim 56 wherein said process further includes the step of subjecting said cross-linked fibrillar collagen matrix to critical point drying. 62. The matrix according to claim 56 wherein said process further includes the step of drying or freeze-drying said cross-linked fibrillar collagen matrix. 63. The matrix according to claim 56 wherein said fibrillar collagen comprises fibrillar collagen reconstituted from monomolecular atelopeptide collagen. 64. The matrix according to claim 56 wherein said fibrillar collagen is prepared by reconstituting monomolecular atelopeptide collagen obtained by proteolytic digestion of native collagen. 65. The method according to claim 33 further comprising the step of removing from said cross-linking solution at least some of the unreacted amount of said at least one sugar, and removing at least some of said at least one polar solvent.

Details for Patent 6,682,760

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2020-04-18