Preparation and xenotransplantation of porcine islets

• US 8,142,769 B2
• Filed: 10/31/2007
• Issued: 03/27/2012
• Est. Priority Date: 01/20/2000
• Status: Expired due to Term

• Current Assignee: Diatranz Otsuka Limited (Otsuka Holdings Company Limited)
• Original Assignee: Diabcell Pty Limited
• Inventors: Elliott, Robert Bartlett, Calafiore, Riccardo, Basta, Gusseppe
• Primary Examiner(s): LI, QIAN JANICE
• Application Number:
  US11/932,538
• Publication Number:
  US 20080279827A1
• Time in Patent Office: 1,609 Days
• Field of Search: None
• US Class Current: 424/93.1
• CPC Class Codes: A61K 2035/128 capsules, e.g. microcapsules
• CPC Class Codes: A61K 35/12 Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells vaccines or medicinal preparations containing antigens or antibodies A61K39/00
• CPC Class Codes: A61P 3/10 for hyperglycaemia, e.g. antidiabetics
• CPC Class Codes: C12N 2500/38 Vitamins
• CPC Class Codes: C12N 2501/998 Proteins not provided for elsewhere
• CPC Class Codes: C12N 2501/999 Small molecules not provided for elsewhere
• CPC Class Codes: C12N 2509/00 Methods for the dissociation of cells, e.g. specific use of enzymes
• CPC Class Codes: C12N 5/0677 Three-dimensional culture, tissue culture or organ culture; Encapsulated cells

Abstract

The invention relates to developments in the treatment of diabetes in mammals. Particularly it relates to a method of preparing a xenotransplantable porcine islet preparation capable upon xenotransplantation of producing porcine insulin in an appropriate recipient mammal, the method including or comprising the steps of:

• • (I) harvesting the pancreas of piglets at or near full term gestation, and
  • (ii) extracting pancreatic β islet cells from the harvested pancreas wherein the islets (at least at some stage in the performance of the method) are exposed to nicotinamide.

Further, the invention relates to a method of encapsulation of a xenotransplantable porcine islet preparation, and transplantation of such a preparation, or a capsule containing such a preparation, into an appropriate recipient mammal.

Claims

1. A method of preparing a xenotransplantable porcine islet preparation capable upon xenotransplantation of producing porcine insulin in an appropriate recipient mammal, the method including or comprising the steps of:

(i) harvesting the pancreas of piglets at or near full-term gestation;

(ii) extracting pancreatic β

-islet cells from the harvested pancreas, wherein said β

-islet cells (at least at some stage in the performance of the method) are exposed to nicotinamide during at least one stage in the performance of said method; and

(iii) contacting the harvested pancreas or the extracted β

-islet cells with an effective amount of a trauma-protecting anesthetic agent comprising lignocaine.

2. The method of claim 1, wherein said method comprises the steps of:

(i) harvesting the pancreas of piglets at or near full-term gestation, and

(ii) preparing a culture of pancreatic β

-islet cells either simultaneously with, or after, step (i);

(iii) extracting pancreatic β

-islet cells from said culture of the harvested pancreas and the islets (at least at some stage in the performance of the method) are exposed to nicotinamide; and

(iv) contacting the harvested pancreas or the extracted β

-islet cells with an effective amount of a trauma-protecting anesthetic agent comprising lignocaine.

3. The method of claim 1, where said pancreas is harvested from piglets that are from −

20 to +10 days full-term gestation.

4. The method of claim 3, wherein said pancreas is harvested from piglets that are from −

7 to +10 days full-term gestation.

5. The method of claim 1, wherein the extraction of said β

-islet cells from said pancreas is performed in the presence of a collagenase.

6. The method of claim 5, wherein said collagenase is human or porcine collagenase.

7. The method of claim 6, wherein said collagenase is human collagenase.

8. The method of claim 2, wherein said culture of pancreatic β

-islet cells comprises a supportive, mammalian albumin that is substantially free of non-human microbiological agents.

9. The method of claim 8, wherein said mammalian albumin is human serum albumin (HSA).

10. The method of claim 1, wherein said pancreatic β

-islet cells are contacted with nicotinamide following their extraction from said harvested pancreas.

11. The method of claim 1, wherein said method further comprises the step of:

contacting said pancreatic β

-islet cells with an effective amount of IgF-1 or the N-terminal tripeptide of IgF-1 (GPE).

12. The method of claim 11, wherein the period of contacting said pancreatic β

-islet cells with an effective amount of IgF-1 or GPE is longer for cells harvested from piglets that are furthest from full-term gestation.

13. The method of claim 11, wherein said pancreatic β

-islet cells are contacted with an effective amount of IgF-1.

14. The method of claim 2, wherein the harvested pancreas is mechanically reduced in the presence of the trauma-protecting anesthetic agent.

15. The method of claim 1, wherein said β

-islet cells are exposed to an antibiotic during at least one stage in the performance of said method.

16. The method of claim 15, wherein said antibiotic is ciproxin.

17. A method of preparing a xenotransplantable porcine pancreatic β

-islet cell preparation that is capable upon xenotransplantation of producing porcine insulin in an appropriate recipient mammal, said method comprising the steps of;

(i) harvesting the pancreas of piglets at or near full-term gestation,

(ii) preparing a culture of the pancreatic β

-islet cells;

(iii) simultaneously with step (ii) or after step (ii) extracting pancreatic β

-islet cells from said culture of harvested pancreas;

(iv) contacting the harvested pancreas or the extracted β

-islet cells with an effective amount of a trauma-protecting anesthetic agent, comprising lignocaine; and

(v) encapsulating the extracted pancreatic β

-islet cells with a biocompatible xenotransplantable material, said material being both glucose- and insulin-porous in vivo,

wherein said islet cells are contacted with nicotinamide at a time prior to encapsulation in said biocompatible xenotransplantable material.

18. The method of claim 17, wherein said pancreas is harvested from piglets that are from about −

20 to +10 days full-term gestation.

19. The method of claim 18, wherein said pancreas is harvested from piglets that are from about −

7 to +10 days full-term gestation.

20. The method of claim 17, wherein the extraction of said β

-islet cells from said pancreas is performed in the presence of a mammalian collagenase.

21. The method of claim 20, wherein said mammalian collagenase is human or porcine collagenase.

22. The method of claim 21, wherein said mammalian collagenase is human collagenase.

23. The method of claim 17, wherein said culture comprises harvested pancreatic tissue in a supportive mammalian albumin that is substantially free of non-human microbiological agents.

24. The method of claim 23, wherein said mammalian albumin is human serum albumin (HSA).

25. The method of claim 17, wherein said pancreatic β

-islet cells are contacted with nicotinamide following their extraction from said harvested pancreas.

26. The method of claim 17, wherein said method further comprises the step of contacting said pancreatic β

-islet cells with an effective amount of IgF-1 or GPE.

27. The method of claim 26, wherein the period of contacting said pancreatic β

-islet cells with an effective amount of IgF-1 or GPE is longer for cells harvested from piglets that are furthest from full-term gestation.

28. The method of claim 26, wherein said pancreatic β

-islet cells are contacted with an effective amount of IgF-1.

29. The method of claim 17, wherein the harvested pancreas is mechanically reduced in the presence of the trauma-protecting anesthetic agent.

30. The method of claim 17, wherein said β

-islet cells are exposed to an antibiotic during at least one stage in the performance of said method.

31. The method of claim 30, wherein said antibiotic is ciproxin.

32. The method of claim 17, wherein said biocompatible xenotransplantable material comprises alginate.

33. The method of claim 32, wherein said biocompatible xenotransplantable material comprises ultra-pure alginate.

34. The method of claim 17, wherein said extracted pancreatic β

-islet cells are encapsulated within a biocompatible alginate that is porous to both insulin and glucose in vivo.

35. The method of claim 34, wherein said biocompatible alginate prevents direct contact between said encapsulated pancreatic β

-islet cells and the mammalian tissue into which said xenotransplantable material is implanted.

36. The method of claim 17, wherein the step of encapsulating involves contacting the extracted pancreatic β

-islet cells with a biocompatible alginate solution that comprises a source of compatible cations thereby to entrap said extracted pancreatic β

-islet cells in a cation-alginate gel.

37. The method of claim 36, wherein said cation-alginate gel comprises a calcium-alginate gel.

38. The method of claim 36, wherein said cation-alginate gel comprises a sodium-alginate gel.

39. The method of claim 38, wherein the islet and sodium alginate solution is 1.6% w/w.

40. The method of claim 36, wherein said source of compatible cations is calcium chloride.

41. The method of claim 36, wherein the cation-alginate gel-entrapped β

-islet cells are subsequently coated with a positively-charged material, and thereafter are provided with an outer coat of alginate.

42. The method of claim 41, wherein said positively-charged material comprises poly-L-ornithine.

43. The method of claim 42, wherein the outer coat of alginate on the encapsulated islet cells is subsequently liquefied.

44. The method of claim 43, wherein said outer coat of alginate is subsequently liquefied by the addition of sodium citrate.

45. The method of claim 17, wherein the step of encapsulating said extracted pancreatic β

-islet cells with a biocompatible xenotransplantable material produces a plurality of capsules.

46. The method of claim 45, wherein said capsules comprise a plurality of β

-islet cells.

47. The method of claim 46, wherein said capsules comprise substantially three β

-islet cells.

48. The method of claim 46, wherein said capsules have a diameter that is substantially from about 300 to about 400 microns.

49. The method of claim 44, further comprising additional steps of washing said encapsulated islet cells, and subsequently coating them with alginate.

50. A method for producing porcine insulin in a human, said method comprising the steps of:

(a) extracting pancreatic β

-islet cells from piglets at or near full-term gestation;

(b) simultaneously with, or after (a), treating said β

-islet cells with nicotinamide;

(c) encapsulating said β

-islet cells in a biocompatible xenotransplantable material that permits in vivo glucose movement into, and insulin movement out of, said encapsulated β

-islet cells; and

(d) injecting or implanting said biocompatible xenotransplantable material comprising said encapsulated β

-islet cells into said human, in an amount effective to produce said porcine insulin in said human,

wherein step (a) and/or (c) occurs in the presence of a trauma-protecting agent comprising lignocaine.

51. The method of claim 50, wherein said method further comprises the step of administering nicotinamide to said human at least subsequent to injection or implantation of said biocompatible xenotransplantable material in said human.

52. The method of claim 50, wherein said method further comprises the step of:

prescribing to said human a casein-free diet either prior to, or following, implantation of said biocompatible xenotransplantable material into said human.

53. The method of claim 50, wherein the extraction of step (a) comprises mechanical treatment of said islets.

54. The method of claim 53, wherein said mechanical treatment follows application of the trauma-protecting agent to said pancreatic tissue.

55. The method of claim 50, wherein the piglets from which said pancreatic β

-islet cells are extracted, are from −

20 to +10 days full-term gestation.

56. The method of claim 55, wherein said piglets are from −

7 to +10 days full-term gestation.

57. The method of claim 50, wherein said biocompatible material comprises alginate.

58. The method of claim 57, wherein said biocompatible material comprises ultra-pure alginate.

59. The method of claim 50, wherein said β

-islet cells are encapsulated in an in vivo insulin- and glucose-porous biocompatible xenotransplantable alginate material.

60. The method of claim 59, wherein the encapsulation provides a surround which prevents, once implanted, direct tissue contact with the encapsulated porcine β

-islet cells.

61. The method of claim 59, wherein said encapsulation involves presenting islets and a suitable alginate solution into a source of compatible cations thereby to entrap the islets in a cation-alginate gel.

62. The method of claim 61, wherein said cation-alginate gel comprises a calcium-alginate gel.

63. The method of claim 61, wherein the alginate used in the solution is sodium alginate, and the islet and sodium alginate solution is presented as a droplet into a bath of suitable cations.

64. The method of claim 63, wherein the islet and sodium-alginate solution is 1.6% w/w.

65. The method of claim 61, wherein said source of compatible ions is calcium chloride.

66. The method of claim 65, wherein the gel-encased islets are coated with a positively-charge material, and thereafter are provided with an outer coat of alginate.

67. The method of claim 66, wherein the positively-charged material comprises poly-L-ornithine.

68. The method of claim 67, wherein the gel entrapping said islets within the outer coating is then liquefied.

69. The method of claim 68, wherein said liquification involves the addition of sodium citrate.

70. The method of claim 50, wherein said encapsulation produces a population of capsules.

71. The method of claim 70, wherein at least one of said population of capsules comprises a plurality of porcine β

-islet cells.

72. The method of claim 71, wherein at least one of said population of capsules comprises substantially three porcine β

-islet cells.

73. The method of claim 72, wherein said capsules have a diameter of substantially from about 300 to 400 microns.

74. The method of claim 73, wherein following liquification of the alginate entrapping the islets, the method further comprises the steps of washing the capsules, and further coating the capsules with alginate.