PREPARATION AND XENOTRANSPLANTATION OF PORCINE ISLETS

• US 20080279827A1
• Filed: 10/31/2007
• Published: 11/13/2008
• Est. Priority Date: 01/20/2000
• Status: Active Grant

• Current Assignee: Diatranz Otsuka Limited (Otsuka Holdings Company Limited)
• Original Assignee: Diabcell Pty Limited
• Inventors: Calafiore, Riccardo, Elliott, Robert Bartlett, Basta, Gusseppe
• Granted Patent: US 8,142,769 B2
• Time in Patent Office: Days
• US Class Current: 424/93.7
• 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; and

(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.

110. 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 (ii); and

(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.

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

20 to +10 days full-term gestation.

112. The method of claim 111, wherein said pancreas is harvested from piglets that are from −

7 to +10 days full-term gestation.

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

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

114. The method of claim 113, wherein said collagenase is human or porcine collagenase.

115. The method of claim 114, wherein said collagenase is human collagenase.

116. The method of claim 1, wherein said culture of pancreatic β

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

117. The method of claim 116, wherein said mammalian albumin is human serum albumin (HSA).

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

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

119. 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).

120. The method of claim 119, 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.

121. The method of claim 119, wherein said pancreatic β

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

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

contacting the harvested pancreas or the extracted β

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

123. The method of claim 122, wherein said trauma-protecting anesthetic agent is lignocaine.

124. The method of claim 110, wherein step (iii) of said method comprises mechanically reducing the harvested pancreas in the presence of a trauma-protecting anesthetic agent.

125. 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.

126. The method of claim 125, wherein said antibiotic is ciproxin.

162. A xenotransplantable porcine islet composition prepared substantially by the method of claim 1.

127. 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, and

(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; and

(iv) 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.

128. The method of claim 127, wherein said pancreas is harvested from piglets that are from about −

20 to +10 days full-term gestation.

129. The method of claim 128, wherein said pancreas is harvested from piglets that are from about −

7 to +10 days full-term gestation.

130. The method of claim 127, wherein the extraction of said β

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

131. The method of claim 130, wherein said mammalian collagenase is human or porcine collagenase.

132. The method of claim 131, wherein said mammalian collagenase is human collagenase.

133. The method of claim 127, wherein said culture comprises harvested pancreatic tissue in a supportive mammalian albumin that is substantially free of non-human microbiological agents.

134. The method of claim 133, wherein said mammalian albumin is human serum albumin (HSA).

135. The method of claim 127, wherein said pancreatic β

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

136. The method of claim 127, wherein said method further comprises the step of:

contacting said pancreatic β

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

137. The method of claim 136, wherein the period of contacting said pancreatic β

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

138. The method of claim 136, wherein said pancreatic β

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

139. The method of claim 127, said method further comprises the step of:

contacting the harvested pancreas or the extracted β

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

140. The method of claim 139, wherein said trauma-protecting anesthetic agent is lignocaine.

141. The method of claim 127, wherein step (iii) of said method comprises mechanically reducing the harvested pancreas in the presence of a trauma-protecting anesthetic agent.

142. The method of claim 127, wherein said β

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

143. The method of claim 142, wherein said antibiotic is ciproxin.

144. The method of claim 127, wherein said biocompatible xenotransplantable material comprises alginate.

145. The method of claim 144, wherein said biocompatible xenotransplantable material comprises ultra-pure alginate.

146. The method of claim 127, wherein said extracted pancreatic β

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

147. The method of claim 146, 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.

148. The method of claim 127, 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.

149. The method of claim 148, wherein said cation-alginate gel comprises a calcium-alginate gel.

150. The method of claim 149, wherein said cation-alginate gel comprises a sodium-alginate gel.

151. The method of claim 150, wherein the islet and sodium alginate solution is 1.6% w/w.

152. The method of claim 148, wherein said source of compatible cations is calcium chloride.

153. The method of claim 148, 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.

157. The method of claim 127, wherein the step of encapsulating said extracted pancreatic β

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

158. The method of claim 157, wherein said capsules comprise a plurality of β

-islet cells.

159. The method of claim 158, wherein said capsules comprise substantially three β

-islet cells.

160. The method of claim 158, wherein said capsules have a diameter that is substantially from about 300 to about 400 microns.

154. The method of claim 154, wherein said positively-charged material comprises poly-L-ornithine.

155. The method of claim 154, wherein the outer coat of alginate on the encapsulated islet cells is subsequently liquefied.

156. The method of claim 155, wherein said outer coat of alginate is subsequently liquefied by the addition of sodium citrate.

161. The method of claim 156, further comprising the additional steps of washing said encapsulated islet cells, and subsequently coating them with alginate.

163. A xenotransplantable porcine pancreatic β

-islet cell composition prepared substantially by the method of claim 19.

164. The xenotransplantable porcine pancreatic β

-islet cell composition of claim 163, comprising at least one viable porcine pancreatic β

-islet cell encapsulated in a biocompatible material that is both glucose- and insulin-porous when implanted in vivo in a recipient mammal.

165. 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.

166. The method of claim 165, 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.

167. The method of claim 165, 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.

168. The method of claim 165, wherein said method further comprises the step of exposing said pancreatic β

-islet cell to IgF-1 or GPE at some stage after extraction from said piglets but prior to encapsulation of said β

-islet cells.

169. The method of claim 165, wherein the extraction of said β

-islet cells occurs in the presence of a trauma-protecting agent.

170. The method of claim 165, wherein the encapsulation of said β

-islet cells occurs in the presence of a trauma-protecting agent.

171. The method of claim 170, wherein said trauma-protecting agent comprises an anesthetic.

172. The method of claim 171, wherein said trauma-protecting agent comprises lignocaine.

173. The method of claim 165, wherein said human prior to, during or after step (d) is provided with a cholesterol-lowering drug regimen.

174. The method of claim 173, wherein said cholesterol-lowering drug is of the “

statin”

family.

175. The method of claim 174, wherein said cholesterol-lowering drug is pravastatin or simvistatin.

176. The method of claim 165, wherein the yield of viable porcine islets obtained from the extraction of step (a) is enhanced by the use of collagenase.

177. The method of claim 176, wherein said collagenase is human or porcine collagenase.

179. The method of claim 165, wherein the extraction of step (a) comprises mechanical treatment of said islets.

180. The method of claim 179, wherein said mechanical treatment follows application of an anesthetic to said pancreatic tissue.

181. The method of claim 180, wherein said anesthetic is lignocaine.

182. The method of claim 165, wherein the piglets from which said pancreatic β

-islet cells are extracted, are from −

20 to +10 days full-term gestation.

184. The method of claim 165, wherein said biocompatible material comprises alginate.

186. The method of claim 165, wherein each islet or grouping of islets is entrapped in an in vivo insulin- and glucose-porous biocompatible xenotransplantable alginate material.

197. The method of claim 165, wherein said encapsulation produces a population of capsules.

198. The method of claim 197, wherein at least one of said population of capsules comprises a plurality of porcine β

-islet cells.

199. The method of claim 198, wherein at least one of said population of capsules comprises substantially three porcine β

-islet cells.

200. The method of claim 199, wherein said capsules have a diameter of substantially from about 300 to 400 microns.

201. The method of claim 200, wherein following liquification of the alginate entrapping the islets there are the further steps of:

washing the capsules further coating the capsules with alginate.

178. The method of claim 178, wherein said collagenase is human collagenase.

183. The method of claim 183, wherein said piglets are from −

7 to +10 days full-term gestation.

185. The method of claim 185, wherein said biocompatible material comprises ultra-pure alginate.

187. The method of claim 187, wherein the encapsulation provides a surround which prevents, once implanted, direct tissue contact with the encapsulated porcine β

-islet cells.

188. The method of claim 187, wherein each 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.

189. The method of claim 188, wherein said cation-alginate gel comprises a calcium-alginate gel.

190. The method of claim 189, 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.

191. The method of claim 190, wherein the islet and sodium-alginate solution is 1.6% w/w.

192. The method of claim 188, wherein said source of compatible ions is calcium chloride.

193. The method of claim 192, wherein the gel-encased islets are coated with a positively-charge material, and thereafter are provided with an outer coat of alginate.

194. The method of claim 193, wherein the positively-charged material comprises poly-L-ornithine.

195. The method of claim 194, wherein the gel entrapping said islets within the outer coating is then liquefied.

196. The method of claim 195, wherein said liquification involves the addition of sodium citrate.

202. A method for the treatment of a mammalian patient suffering from or predisposed to diabetes, said method comprising the steps of:

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

(ii) culturing the harvested pancreas in mammalian albumin that is substantially free of non-human microbiological agents; and

(iii) simultaneously with step (ii) or after step (ii), extracting a population of β

-islet cells from the harvested pancreas using a mammalian collagenase, wherein the islets are exposed to nicotinamide during at least some stage in the performance of step (A);

(B) encapsulating the β

-islet cells prepared in step (A) with a biocompatible encapsulation material that allows both glucose and insulin movement therethrough; and

(C) implanting the encapsulated porcine β

-islet cells prepared in step (B) into said mammalian patient in an amount effective to treat said diabetes in said patient.

203. The method of claim 202, wherein said mammalian collagenase is human or porcine collagenase.

204. The method of claim 203, wherein said mammalian collagenase is human collagenase.

205. The method of claim 202, wherein the extraction in step (A) of said method further comprises mechanical treatment of said harvested pancreas.

206. The method of claim 205, wherein said mechanical treatment follows application of a trauma-protecting anesthetic composition to a tissue of said harvested pancreas.

208. The method of claim 202, wherein said method further comprises the step of:

(D) administering nicotinamide to said mammalian patient prior to, or after, step (C) of said method.

209. The method of claim 202, wherein said method further comprises the step of:

(E) prescribing for said mammalian patient a casein-free diet either prior to, or after, step (C) of said method.

210. The method of claim 202, wherein said method further comprises the step of:

(F) prescribing for said mammalian patient a cholesterol-lowering drug regimen either prior to, or after, step (C) of said method.

211. The method of claim 210, wherein said regimen comprises administering a cholesterol-lowering drug of the “

statin”

family.

212. The method of claim 211, wherein said cholesterol-lowering drug is pravastatin or simvistatin.

207. The method of claim 207, wherein said trauma-protecting anesthetic is lignocaine.

213. A method of treating a mammalian patient suffering from diabetes, the method comprising the steps of:

(i) extracting pancreatic β

-islet cells from a harvested porcine pancreas; and

(ii) encapsulating β

-islet cells obtained from said harvested pancreas with a biocompatible xenotransplantable material, said material being both glucose- and insulin-porous;

(iii) introducing a trauma-protecting agent during or prior to the step of encapsulating; and

(iv) transplanting into the mammalian patient an effective amount of viable islet cells capable of producing insulin in the patient.

214. The method of claim 213, wherein said trauma-protecting agent comprises an anesthetic.

215. The method of claim 214, wherein said trauma-protecting agent is lignocaine.

216. The method of claim 213, further comprising the step of subjecting the patient to cholesterol lowering drug regime prior to, during or after the step of transplanting.

217. The method of claim 216, wherein said drug regimen comprises a member of the “

statin”

family of cholesterol-lowering drugs.

218. The method of claim 217, wherein said drug regimen comprises pravastatin or simvistatin.

219. The method of claim 213, further comprising the step of prescribing to the patient, prior to or after the transplanting step, a casein-free diet.