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WO2006136374A2 - Utilisation du gip et/ou de la vitamine d3 et d'analogues correspondants pour ameliorer la differenciation de cellules souches ou progenitrices en cellules productrices d'insuline - Google Patents

Utilisation du gip et/ou de la vitamine d3 et d'analogues correspondants pour ameliorer la differenciation de cellules souches ou progenitrices en cellules productrices d'insuline Download PDF

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WO2006136374A2
WO2006136374A2 PCT/EP2006/005912 EP2006005912W WO2006136374A2 WO 2006136374 A2 WO2006136374 A2 WO 2006136374A2 EP 2006005912 W EP2006005912 W EP 2006005912W WO 2006136374 A2 WO2006136374 A2 WO 2006136374A2
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cells
gip
insulin
analogue
vitamin
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WO2006136374A3 (fr
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Matthias Austen
Ulrike Burk
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Develogen AG
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Develogen AG
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/59—Compounds containing 9, 10- seco- cyclopenta[a]hydrophenanthrene ring systems
    • A61K31/593—9,10-Secocholestane derivatives, e.g. cholecalciferol, i.e. vitamin D3
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
    • A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics

Definitions

  • GIP and/or vitamin D 3 analogues thereof for enhancing stem or progenitor cell differentiation into insulin producing cells
  • This invention relates to the use of GIP (Gastric Inhibitory Polypeptide or Glucose Dependent lnsulinotropic Polypeptide) or of protease resistant GIP analogues and/or of vitamin D 3 (cholecalciferol or 5,7-cholestadien-3- ⁇ -ol) and analogues thereof, to promote differentiation of stem or progenitor cells into insulin producing cells.
  • GIP Global Inhibitory Polypeptide or Glucose Dependent lnsulinotropic Polypeptide
  • vitamin D 3 cholecalciferol or 5,7-cholestadien-3- ⁇ -ol
  • Pancreatic beta-cells secrete insulin in response to elevated blood glucose levels. Insulin amongst other hormones plays a key role in the regulation of the fuel metabolism. For example, it leads to the storage of glycogen and triglycerides and to the synthesis of proteins. Furthermore, the entry of glucose into muscles and adipose cells is stimulated by insulin. In patients who suffer from diabetes mellitus type I or LADA (latent autoimmue diabetes in adults, Pozzilli & Di Mario, 2001 , Diabetes Care. 8:1460-67) beta cells are being destroyed due to autoimmune attack. Consequently, the amount of insulin produced by the remaining pancreatic islets is too low, resulting in elevated blood glucose levels (hyperglycemia).
  • diabetes mellitus type I or LADA latent autoimmue diabetes in adults, Pozzilli & Di Mario, 2001 , Diabetes Care. 8:1460-67
  • beta cells are being destroyed by autoimmune attack
  • treatments have been devised which modulate the immune system and may be able to stop or strongly reduce islet destruction (Raz et al., 2001 , Lancet 358: 1749-1753; Chatenoud et al., 2003, Nat Rev Immunol. 3: 123-132; Homann et al., Immunity. 2002, 3:403-15).
  • islet destruction due to the relatively slow regeneration of human beta cells such treatments can be enhanced if they are combined with agents that can stimulate beta cell regeneration.
  • Diabetes is a very disabling disease, because today's forms of insulin therapy and common anti-diabetic drugs do not control blood sugar levels well enough to completely prevent the occurrence of high and low blood sugar levels. Frequently elevated blood sugar levels are toxic and cause long-term complications like for example nephropathy, retinopathy, neuropathy and peripheral vascular disease. Extensive loss of beta cells also leads to deregulation of glucagon secretion from pancreatic alpha cells which contributes to an increased risk of dangerous hypoglycemic episodes. There is also a host of related conditions, such as obesity, hypertension, heart disease and hyperlipidemia, for which persons with diabetes are substantially at risk.
  • the technical problem underlying the present invention was to provide for means and methods for treating insulin-dependent diabetes such as type I diabetes or LADA but also diabetes mellitus type Il (particularly late stages).
  • the solution to said technical problems is achieved by providing the embodiments characterized in the claims.
  • beta cells from embryonic stem cells can serve as a model for the stem cell-based regeneration of the same cell type in the adult 5 organism. Being able to generate cells with beta cell character from stem cells also opens possibilities for improved cell based therapies of diabetes, e.g. to generate beta cell replacement material which could be transplanted into diabetic patients.
  • Beta cell replication has recently been described as the major mechanism of beta cell formation after birth in mice (Dor et al., Nature. 2004 429(6987):41- 6). However, in similar experiments, very different results have been obtained by another group (Seymour et al., J Anat. 2004 204(2): 103-16). Beta cell replication appears to be significantly less frequent in humans than 5 in rodents, even in situations of increased need for beta cells such as obesity (Butler et al., Diabetes. 2003 Jan;52(1 ):102-10). A number of mechanisms of beta cell formation not based on beta cell replication has been described in the literature (Lechner and Habener, Am J Physiol Endocrinol Metab. 2003 284(2):E259-66; Bonner-Weir and Sharma, J 0 Pathol. 2002 197(4):519-26).
  • beta cells can be generated from progenitor cells, e.g. from embryonic stem (ES) cells, upon administration of GIP (Glucose-dependent lnsulinotropic Polypeptide) or a GIP analogue.
  • GIP Glucose-dependent lnsulinotropic Polypeptide
  • the differentiated beta cells are useful for improved cell based therapies of diabetes, in particular e.g. for generating replacement material for destroyed beta cells of pancreatic patients which could be transplanted into these patients.
  • vitamin D 3 to stimulate the formation or regeneration of insulin producing beta cells and thus, a use in the treatment and prevention of diabetes.
  • GIP Gastric inhibitory peptide or Glucose-dependent lnsulinotropic Polypeptide
  • GIP GIP
  • GIP dose-dependently stimulates the replication of lns-1 rat insulinoma cells under both low and high glucose conditions at GIP concentrations > 1OnM (Tr ⁇ mper et al., MoI Endocrinol.2001 15:1559-70; Tr ⁇ mper et al., J Endocrinol. 2002 174:233-46; WO 03/082998).
  • GIP has also been described as having a suppressive effect on beta cell death (Wideman and Kieffer, Horm Metab res. 2004 36:782-6).
  • GIP suppresses cell death in lns-1 insulinoma cells (Tr ⁇ mper et al., 2002; supra, WO 03/082898), in dispersed primary beta cells from C57BI/6 mice and Vancouver diabetic fatty (VDF) rats in vivo (Kim et al., J Biol. Chem. 2005 280:22297-307).
  • VDF Vancouver diabetic fatty
  • Beta cell formation from stem or progenitor cells has been previously proposed as an important mechanism of postnatal beta cell regeneration in humans (Butler et al., Diabetes. 2003 52:102-10; Bonner-Weir et al., Pediatr Diabetes 2004; 5 Suppl 2:16-22; Lechner and Habener, Am J Physiol Endocrinol Metab. 2003 284:E259-66).
  • Native GIP has a very short serum half life of 5 minutes in humans, and is therefore not preferred for therapeutic use (Meier and Nauck, Horm Metab Res. 2004 36:859-66).
  • the ubiquitously expressed protease DPP-IV cleaves the peptide bond between amino acids two and three of GIP, resulting in a truncated peptide which can no longer activate the receptor. However, the remaining fragment can still bind to the receptor and thus can act as an antagonist to full length GIP (Deacon, Horm Metab Res. 2004 36(11-12): 761-5; Meier and Nauck, Horm Metab Res. 2004 36:859-66).
  • Modified variants of GIP have been designed which are less prone to DPP-IV- mediated degradation. Examples for such modifications are alterations of amino acids two and three in native GIP or N-terminal modifications of GIP.
  • the present invention relates to the use of GIP and analogues thereof, to stimulate or induce differentiation of stem or progenitor cells into insulin producing cells, which can then be used for the treatment of diabetes, more preferably for the treatment of type I diabetes or LADA or insulin-dependent type Il diabetes.
  • the present invention also relates to new methods for stimulating and/or inducing the differentiation of progenitor cells, e.g. stem cells into insulin- producing cells using vitamin D 3 .
  • Vitamin D a fat soluble vitamin
  • Vitamin D is well-known for its role in mineral (calcium and phosphorus) homeostasis and maintenance of normal skeletal architecture. It is now becoming increasingly clear that vitamin D is involved in a diverse range of cellular functions including cell differentiation, cell proliferation, immune function etc.
  • Vitamin D the biological inert moiety, is converted into the hormonally active form, 1 ⁇ ,25-dihydroxy-vitamin D 3 , (referred to as 1 ,25(OH) 2 D 3 , by successive hydroxylation reactions in liver and kidney.
  • This activated form of vitamin D 3 not only plays a central role in bone and calcium metabolism, but also modulates the immune response via specific receptors expressed in APCs and activated T-cells (Bouillon, R., et.
  • Vitamin D 3 has been shown to preserve the function of in vitro cultured pancreatic islet cells for significantly longer periods of time than control cells that have not been treated with vitamin D 3 . Based on these results, it is suggested to use vitamin D 3 for the treatment of diabetes (Luca G. et al., Diabetes, nutrition and metabolism, 2000 Dec, 13(6), 301-7). The use of vitamin D 3 analogues for the treatment of autoimmune diabetes is also disclosed in WO 02/094247 and in a publication of Nagpal, S. et al. (Vitamin D analogs: Mechanism of action and therapeutic applications, Current Medicinal Chemistry 2001 , 8, 1661-1679).
  • NOD mouse In a prevention study of autoimmune diabetes in NOD mice, 1 ,25(OH) 2 D 3 reduced the incidence of disease by greater than 90% (Mathieu, C. et al.; Diabetologica, 1994, 37, 552).
  • NOD mouse is an animal model for human autoimmune diabetes, characterized by destruction of the beta-Langerhans cells of the pancreas.
  • a synthetic analog of vitamin D, 20-epi-22-oxa- 24,26,27-trishomo-1 ⁇ ,25-dihydroxy-vitamin D 3 (kh 1060) also prevented the onset of type I diabetes in NOD mice at non-hypercalcemic doses (Mathieu, C.
  • EP 0 707 566 describes the use of vitamin D 3 analogues for the preparation of a medicament for therapy and/or prevention of immune disorders, e.g. diabetes mellitus type I, optionally in combination with an immune system interferring drug. Further, the use of said medicament for induction of cell differentiation or any induction of cell proliferation is mentioned. There is, however, no disclosure that vitamin D 3 analogues may stimulate beta cell formation from stem cells.
  • EP 0 972 762 discloses the use of vitamin D 3 analogues for the preparation of a medicament for inhibiting cell proliferation and/or inducing cell differentiation.
  • Said medicament is intended for treatment and/or prevention of immune disorders such as diabetes mellitus type I, among others. Further, said medicament can be used in combination with other drugs known to interfere with the immune system.
  • treatment with vitamin D 3 analogues stimulates beta cell formation from stem cells.
  • US Patent 5,856,189 relates to a method of treating cells by administration of vitamin D 3 analogues and an extracellular matrix. However, there is no hint relating that treatment with vitamin D 3 analogues stimulates beta cell formation from stem cells neither to the treatment of autoimmune or pancreatic disorders by using this method.
  • WO 2004/044146 describes a method for the in vitro induction of cells produced or modified into trans-differentiated organ-specific cells, for example pancreatic islet ⁇ -cells, wherein the administration of vitamin D 3 is mentioned.
  • vitamin D 3 or vitamin D 3 analogue for the manufacture of a medicament for the prevention and/or treatment of pancreatic and/or autoimmune disorders is not mentioned.
  • the present invention also relates to the use of vitamin D 3 or analogues thereof, to stimulate or induce differentiation of stem or progenitor cells into insulin producing cells, which can then be used for the treatment of diabetes, more preferably for the treatment of type I diabetes or LADA or insulin- dependent type Il diabetes.
  • the term ,GIP analogue preferably relates to linear or cyclic peptidic compounds which comprise at least the 14 N-terminal amino acids of GIP, i.e. Tyr-Ala-Glu-Gly-Thr-Phe-lle-Ser-Asp-Tyr-Ser-lle- Ala-Met having at least one modification compared to the native sequence.
  • the modifications are selected from amino acid substitutions, N- terminal modifications, C-terminal modifications, cyclizations, peptide bond modifications and combinations thereof.
  • Amino acid substitutions preferably comprise substitutions of at least one amino acid in the N-terminal amino acid sequence of GIP as indicated above by a D-amino acid, and/or by a naturally occurring or non-naturally occurring amino acid having a different side chain, and/or by a non-amino acid chemical moiety which preserves or enhances activity.
  • Especially preferred amino acid substitutions are on positions 1 , 2, and/or 3 of GIP.
  • the GIP analogue contains 1-4 amino acid substitutions.
  • N-terminal and C-terminal modifications preferably comprise modifications of the N- and C-terminal amino group with a modifying group, e.g. an alkyl, acyl, carbohydrate, cholesteryl, lipid or vitamin group, particularly with a hydrophobic, hydrophilic and/or bulky group.
  • a modifying group e.g. an alkyl, acyl, carbohydrate, cholesteryl, lipid or vitamin group, particularly with a hydrophobic, hydrophilic and/or bulky group.
  • N-terminal modifications can also include the following: alkylation, sulphonylation, glycation, homoserine formation, pyroglutamic acid formation, disulphide bond formation, deamidation of asparagine or glutamine residues, methylation, t-butylation, t-butyloxycarbonylation, 4-methylbenzylation, thioanysilation, thiocresylation, benzyloxymethylation, 4-nitrophenylation, benzyloxycarbonylation, 2- nitrobenzoylation, 2-nitrosulphenylation, 4-toluenesulphonylation, pentafluorophenylation, diphenylmethylation, 2- chlorobenzyloxycarbonylation, 2,4,5-trichlorophenylation, 2- bromobzyloxycarbonylation, 9-fluorenylmethyloxycarbonylation, triphenylmethylation, 2,2,5,7, 8-pentamethylchroman-6-sulphonylation, hydroxylation
  • N- and C-terminal modifications may also comprise the addition of hydrophilic groups, basic groups and preferably amino acids and analogues thereof.
  • the amino acids and analogues are hydrophilic, preferably basic.
  • Particularly preferred is the amino acid lysine.
  • One or more such groups may be added, preferably 1 to 5, preferably 1 to 4, preferably 1 to 3, preferably 2 to 3, preferably 3.
  • the addition of three lysine residues is particularly preferred.
  • N- and C-terminal modifications include steroids and steroid analogues, e.g. lithocholic acid.
  • Peptide bond modifications preferably comprise introduction of reduced peptide bonds and/or other modifications, e.g. alkylation of the N-atom that is part of the peptide bond.
  • Peptide bond modifications preferably comprise the formation of a carbamate bond, preferably between any two adjacent amino acid positions of GIP(I -14) or between any other two adjacent amino acid positions in the GIP analogue of the invention, preferably any two adjacent amino acid positions 2-5, more preferably between amino acid positions 2 and 3.
  • the number of such modified peptide bonds, in particular carbamate bonds is preferably from 1-5, more preferably 1-4, more preferably 1-3, more preferably 2, and most preferably 1.
  • C-terminal modifications comprise any of the modifying groups mentioned above for N-terminal modifications.
  • C-terminal modifications comprise the addition of at least one heterologous amino acid at the C- terminus.
  • a heterologous amino acid is one which is not present at the corresponding position in native GIP.
  • the number of heterologous amino acids or analogues thereof at the C-terminal end is from 1-5, preferably 1-4, more preferably 1-3, in particular 2-3 and most preferably 3.
  • basic and hydrophilic amino acids are preferred, in particular lysine.
  • 3 lysine residues are preferred.
  • the peptide contains no more than 14 native amino acids from the N-terminus of native GIP.
  • the C-terminal modifications are added to the C-terminal position 14 amino acid residue of the GIP analogue.
  • GIP(I -14)-KKK-NH 2 SEQ ID NO: 1
  • Ala2- carbamate-GIP(1-14)-NH 2 SEQ ID NO: 2.
  • the GIP analogues have improved resistance against degradation by proteases, particularly against degradation by dipeptidyl- peptidase IV (DP IV).
  • DP IV dipeptidyl- peptidase IV
  • the therapeutic agent of the present invention may be a preparation of insulin producing cells generated in the presence of vitamin D 3 or an analogue thereof.
  • suitable vitamin D 3 analogues are e.g. described by Nagpal, S. et al. (Vitamin D analogs: Mechanism of action and therapeutic applications, Current Medicinal Chemistry 2001 , 8, 1661-1679), EP 0 707 566, US Patent No. 5,856,189, WO 04/098522, WO 04/098507, by Qiao, G. et al. (Analogs of 1 alpha, 25-dihydroxyvitamin D 3 as novel inhibitors of renin biosynthesis, J. Steroid. Biochem. MoI. Biol.
  • Vitamin D 3 analogues according to the invention are capable of binding the vitamin D receptor (VDR) (Nagpal, S. et al., Current Medicinal Chemistry 2001 , 8, 1661-1679) and have vitamin D 3 agonistic activity, e.g. as determined by binding to the vitamin D 3 nuclear hormone receptor and eliciting a transcriptional response corresponding to a partial or full agonistic activity regarding this receptor.
  • VDR vitamin D receptor
  • vitamin D 3 and analogues thereof are capable of promoting the differentiation of insulin-producing cells, particularly pancreatic beta cells.
  • the agent could be used in stem cell differentiation protocols aiming to the production of beta cell-like cells in culture.
  • the agent can act as a maturation factor promoting the differentiation of stem cells towards the pancreatic lineage or promoting the growth of differentiated cells.
  • the present invention provides methods for treating patients suffering from a pancreatic autoimmune disease caused by, associated with, and/or accompanied by functionally impaired and/or reduced numbers of pancreatic islet cells, particularly insulin producing beta cells, by administering a therapeutically effective amount of differentiated cells as indicated above.
  • Functional impairment or loss of pancreatic islet cells may be due to e.g. autoimmune attack such as in diabetes type I or LADA and/or due to cell degeneration such as in progressed diabetes type II.
  • progenitor cells relates to undifferentiated cells capable of being differentiated into insulin producing cells.
  • the term particularly includes stem cells, i.e. undifferentiated or immature embryonic, adult, or somatic cells that can give rise to various specialized cell types.
  • stem cells can include embryonic stem cells (ES) and primordial germ (EG) cells of mammalian, e.g. human or animal origin. Isolation and culture of such cells is well known to those skilled in the art (see, for example, Thomson et al., (1998) Science 282: 1145-1147; Shamblott et al., (1998) Proc. Natl. Acad. Sci.
  • Embryonic stem cells can be isolated from the inner cell mass of pre- implantation embryos (ES cells) or from the primordial germ cells found in the genital ridges of post-implanted embryos (EG cells). When grown under special culture conditions such as static or rotating culture or hanging drops, both ES and EG cells aggregate to form embryoid bodies (EB). EBs are composed of various cell types similar to those present during embryogenesis. When cultured in appropriate media, EB can be used to generate in vitro differentiated phenotypes, such as extraembryonic endoderm, hematopoietic cells, neurons, cardiomyocytes, skeletal muscle, and vascular cells.
  • beta cell regeneration refers to an at least partial restoration of normal beta cell function by increasing the number of functional insulin secreting beta cells and/or by restoring normal function in functionally impaired beta cells.
  • compositions of the invention are useful in diagnostic and therapeutic applications implicated, for example, but not limited to, pancreatic autoimmune disorders.
  • diagnostic and therapeutic uses for the compositions of the invention are, for example but not limited to, the following: (i) tissue regeneration in vitro (regeneration for all these tissues and cell types composing these tissues and cell types derived from these tissues), and (ii) research tools.
  • composition may be administered via implantation of treated cells.
  • compositions of the invention may be administered alone or in combination with another medicament useful to prevent or treat pancreatic disorders or metabolic syndrome, particularly beta cell degeneration.
  • factors that could be used in a combination therapy are: hormones, growth factors or antioxidants such as GLP-1 and stabilized forms of GLP-1 , GLP-1 analogues, DPP-IV inhibitors, nicotinamide, vitamin C, INGAP pepide, TGF-alpha, gastrin, prolactin, members of the EGF-family, or immune modulating agents such as anti-CD3 antibodies, DiaPep277 or anti-inflammatory agents such as Cox2 inhibitors, acetyl-salicylic acid, or acetaminophen.
  • hormones, growth factors or antioxidants such as GLP-1 and stabilized forms of GLP-1 , GLP-1 analogues, DPP-IV inhibitors, nicotinamide, vitamin C, INGAP pepide, TGF-alpha, gastrin, prolactin, members of the
  • compositions may be administered in combination with the beta cell regenerating proteins, nucleic acids and effectors/modulators thereof described in PCT/EP2004/007917, e.g. pleiotrophin and agonists thereof, or in PCT/EP2004/013175, PCT/EP2004/013535, PCT/EP 2005/000545, PCT/EP 2005/0017111 and EP 04018751.0, which are herein incorporated by reference.
  • compositions may be administered together with factors with beneficial effects on beta cells such as GLP-1 or derivatives thereof, e.g. GLP-1 (7-36 amide), exendin-4, prolactin or a neurotrophins such as NGF.
  • compositions are preferably administered together with pharmaceutical agents which have an immunosuppressive activity, e.g. antibodies, polypeptides and/or peptidic or non-peptidic low molecular weight substances.
  • immunosuppressive agents are listed in the following Table 1.
  • Table 1 Exemplary agents for immune suppression
  • Preferred immunosuppressive agents are DiaPep-277, anti-CD3-antibodies such as hOKT31 gamma (Ala-Ala) and GAD peptides such as DiaMyd GAD peptides if stem or progenitor cells not subject of allograft rejection are used (e.g. autologous cells or cells with reduced visibility to the recipients immue system) in patients with autoimmune diabetes.
  • Use of cells derived from non-autologous stem or progenitor cells preferably requires the use of more potent immunosuppressive agents such as mycophenolate mofetil or tacrolimus or other agents appropriate for a use in an organ transplant setting (see table 1) both in autoimmune diabetes or type Il diabetes patients. No additional immune suppression or immune modulation is needed if differentiated cells derived from autologous stem or progenitor cells or cells with reduced visibility to the recipients' immune system are administered in patients witzh type Il diabetes.
  • the combination therapy may comprise coadministration of the medicaments during the treatment period and/or separate administration of single medicaments during different time intervals in the treatment period.
  • the present invention also relates to methods for differentiating progenitor cells into insulin-producing cells in vitro comprising
  • pancreatic genes activating one or more pancreatic genes in a progenitor, e.g. stem cell
  • embryoid bodies optionally aggregating said cells to form embryoid bodies (optional step, particularly if embryonic stem cells are used)
  • embryoid bodies optionally aggregating said cells to form embryoid bodies (optional step, particularly if embryonic stem cells are used)
  • embryoid bodies optionally cultivating embryoid bodies or cultivating adult stem cells (e.g., duct cells, duct-associated cells, nestin-positive cells) in specific differentiation media containing a composition as indicated above under conditions wherein beta cell differentiation is significantly enhanced, and (d) identifying and selecting insulin-producing cells.
  • adult stem cells e.g., duct cells, duct-associated cells, nestin-positive cells
  • pancreatic genes may comprise transfection of a cell with pancreatic gene operatively linked to an expression control sequence, e.g. on a suitable transfection vector, as described in WO 03/023018, which is herein incorporated by reference.
  • suitable transfection vector e.g. on a suitable transfection vector, as described in WO 03/023018, which is herein incorporated by reference.
  • pancreatic genes are Pdx1 , Pax4, Pax6, neurogenin 3 (Ngn3), Nkx 6.1 , Nkx 6.2, Nkx 2.2, HB 9, BETA2/Neuro D, IsI 1 , HNF1 -alpha, HNF1-beta and HNF3 of human or animal origin.
  • Each gene can be used individually or in combination with at least one other gene.
  • Pax4 is especially preferred.
  • compositions are useful for the modulation, e.g. stimulation, of pancreatic development and/or for the regeneration of pancreatic cells or tissues, e.g. cells having exocrine functions such as acinar cells, centroacinar cells and/or ductal cells, and/or cells having endocrine functions, particularly cells in Langerhans islets such as alpha-, beta-, delta- and/or PP-cells, more particularly beta cells.
  • the invention relates to a cell preparation comprising differentiated progenitor cells, e.g. stem cells exhibiting insulin production, particularly an insulin-producing cell line obtainable by the method described above.
  • the insulin-producing cells may exhibit a stable or a transient expression of at least one pancreatic gene involved in beta cell differentiation.
  • the cells are preferably human cells that are derived from human stem cells. For therapeutic applications the production of autologous human cells from adult stem cells of a patient is especially preferred. However, the insulin producing cells may also be derived from non-autologous ceils. If necessary, undesired immune reactions may be avoided by encapsulation, immunosuppression and/or modulation or due to non-immunogenic properties of the cells.
  • the insulin producing cells of the invention preferably exhibit characteristics that closely resemble naturally occurring beta cells. Further, the cells of the invention preferably are capable of a fast response to glucose. After addition of 27.7 mM glucose, the insulin production is enhanced by a factor of at least 2, preferably by a factor of at least 3. Further, the cells of the invention are capable of normalizing blood glucose levels after transplantation into mice.
  • the invention further encompasses functional pancreatic cells obtainable or obtained by the method according to the invention.
  • the cells are preferably of mammalian, e.g. human origin.
  • said cells are pancreatic beta cells, e.g. mature pancreatic beta cells or stem cells differentiated into pancreatic beta cells.
  • pancreatic beta cells preferably secrete insulin in response to glucose.
  • the present invention may provide functional pancreatic cells that secrete glucagon in response to hypoglycemia.
  • a preparation comprising the cells of the invention may additionally contain cells with properties of other endocrine cell types such as delta-cells and/or PP-cells. These cells are preferably human cells.
  • the cell preparation of the invention is preferably a pharmaceutical composition comprising the cells together with pharmacologically acceptable carriers, diluents and/or adjuvants.
  • the pharmaceutical composition is preferably used for the treatment or prevention of pancreatic autoimmune diseases, e.g. diabetes mellitus type I or LADA, or diabetes mellitus type II.
  • the functional insulin producing cells treated with compositions of the invention may be transplanted preferably intrahepatically, directly into the pancreas of an individual in need, or by other methods.
  • such cells may be enclosed into implantable capsules that can be introduced into the body of an individual, at any location, more preferably in the vicinity of the pancreas, or the bladder, or the liver, or under the skin.
  • Methods of introducing cells into individuals are well known to those of skill in the art and include, but are not limited to, injection, intravenous or parenteral administration. Single, multiple, continuous or intermittent administration can be effected.
  • the cells can be introduced into any of several different sites, including but not limited to the pancreas, the abdominal cavity, the kidney, the liver, the celiac artery, the portal vein or the spleen.
  • the cells may also be deposited in the pancreas of the individual.
  • lmmunomodulating medicaments e.g. immunosuppressive drugs, such as the agents listed in Table 1
  • immunosuppressive drugs such as the agents listed in Table 1
  • Allografts using the cells obtained by the methods of the present invention are also useful because a single healthy donor could supply enough cells to regenerate at least partial pancreas function in multiple recipients.
  • the effect of the transplanted insulin producing cells upon the body reverses the condition of diabetes partially or completely.
  • the dosage of insulin administered may be reduced in strength.
  • further administration can be discontinued entirely and the subject continues to produce a normal amount of insulin without further treatment.
  • the subject is thereby not only treated but could be cured entirely of a diabetic condition.
  • even moderate improvements in beta cell mass can lead to a reduced requirement for exogenous insulin, improved glycemic control and a subsequent reduction in diabetic complications.
  • Fig. 1 shows the GIP dependent induction of the differentiation of insulin producing cells.
  • ES cells Mouse embryonic stem (ES) cells were differentiated into insulin producing cells as described previously (patent application PCT/EP02/04362, published as WO 02/086107, which is incorporated herein by reference).
  • the abundance of insulin mRNA (Fig. 1 ) was determined using quantitative RT-PCR in two independent experiments. Levels were normalized using 18S RNA and a cycle number of 36 as references. The numbers on the vertical line refer to the abundance of the indicated transcripts relative to an abundance for which 36 cycles are necessary for detection.
  • fold expression rel. to delta Ct36' refers to expression of insulin; .undiff.
  • the expression of insulin is shown at different time points: EBs day 5 (end of embryoid body formation), EBs day 5+9 (spontaneously differentiated embryoid bodies shortly before dissociation), and differentiated cells at day 5+33.
  • Fig. 2 and Fig. 3 show the vitamin D 3 dependent induction of the differentiation of insulin producing cells.
  • ES cells Mouse embryonic stem (ES) cells were differentiated to insulin producing cells as described previously (patent application PCT/EP02/04362, published as WO 02/086107, which is incorporated herein by reference).
  • insulin mRNA Fig.2
  • beta cell glucose transporter Glut2 mRNA Fig. 3
  • levels were normalized using 18S RNA as control and a cycle number of 36 as reference.
  • the numbers on the vertical line refer to the abundance of the indicated transcripts relative to an abundance for which 36 cycles are necessary for detection.
  • fold expression rel. to delta Ct36' refers to expression of insulin (Fig. 2) and Glut2 (Fig. 3), respectively; .undiff.
  • ES' refers to R1 mouse embryonic stem (ES) cells stably transfected with a CMV-Pax4 expression construct
  • .control' refers to the differentiation protocol as described in Example 2, without any addition of vitamin D 3
  • ,+ VD3' refers to the differentiation protocol as described in Example 5, with the addition of vitamin D 3 to embryoid bodies.
  • the expression of insulin (Fig. 2) and Glut2 (Fig. 3) is shown at different time points: undifferentiated ES cells, EBs day 5 (end of embryoid body formation), EBs day 5+9 (spontaneously differentiated embryoid bodies shortly before dissociation), and differentiated cells at day 5+33.
  • Example 1 Generation of ES cells expressing the Pax4 gene.
  • Mouse R1 ES cells (Nagy et al. (1993) Proc. Natl. Acad. Sci. U S A. 90: 8424-8428) were electroporated with a plasmid expressing the Pax4 gene under the control of the CMV promoter and the neomycin resistance gene under the control of the phosphoglycerate kinase I promoter (pGK-1 ).
  • ES cells were cultured in Dulbecco's modified Eagle's medium containing 4.5 g/l glucose, 10 "4 M beta-mercaptoethanol, 2 nM glutamine, 1% nonessential amino acids, 1 nM Na-pyruvate, 15% fetal calf serum (FCS) and 500 U/ml leukemia inhibitory factor (LIF). Briefly, approximately 10 7 ES cells resuspended in 0.8 ml phosphate buffered saline (PBS) were subjected to electroporation with 25 ⁇ g/ml of linearized expression vector (Joyner, Gene Targeting: A Practical Approach, Oxford University Press, New York, 1993).
  • PBS 0.8 ml phosphate buffered saline
  • ES cells Five minutes after electroporation, ES cells were plated on petri dishes containing fibroblastic feeder cells previously inactivated by treatment with 100 ⁇ g/ml mitomycin C. One day after electroporation, culture medium was changed to medium containing 450 ⁇ g/ml G418. Resistant clones were separately isolated and cultured 14 days after applying the selection medium. Cells were always cultured at 37 0 C, 5% CO 2 . These untreated and undifferentiated ES cells were used as control for the experiment shown in Fig. 1 , 2 and 3 (referred to as , undiff. ES' in Fig. 1 , 2 and 3).
  • Example 2 Differentiation of ES cells into insulin-producing cells (referred to as .control' in Fig. 1, 3 and 4)
  • the ES cell line R1 constitutively expressing Pax4 (Pax4 ES cells) (.undiff. ES' in Fig. 1 , 3 and 4) were cultivated as embryoid bodies (EB) by the hanging drop method, as described in patent application PCT/EP02/04362, published as WO 02/086107 and by Blyszczuk et al., 2004, Int. J. Dev. Biol. 48: 1095-1104, which are incorporated herein by reference, with media as described below and in Table 2.
  • the embryoid bodies were allowed to form in hanging drop cultures for 2 days and then transferred for three days to suspension cultures in petri dishes.
  • EBs were plated separately onto gelatin-coated 6 cm cell culture dishes containing a differentiation medium prepared with a base of Iscove modified Dulbecco's medium. After dissociation and replating at day 14, cells were cultured up to 40 days in the differentiation medium prepared with a base of Dulbecco's modified Eagle medium: Nutrient Mixture F-12 (DMEM/F12).
  • DEM/F12 Nutrient Mixture F-12
  • Example 3 Expression of pancreas specific genes after differentiation of ES cells into insulin-producing cells
  • RNA from 8x10 4 cells growing on 4 cm 2 surface area of a tissue culture dish was extracted using Qiagen RNAeasy kit according to the instructions of the manufacturer (Qiagen) and 2 ⁇ g was converted into cDNA.
  • Primers for insulin and 18S RNA were designed using the Primer Express 1.5 Software from Applied Biosystems and sequences can be obtained upon request.
  • Quantitative real-time PCR was performed using Applied Biosystems SDS 7000 detection system. Amplifications from 2 independent experiments were performed in duplicate for each transcript and mean values were normalized to the mean value of the reference RNA 18S RNA.
  • Example 4 Induction of differentiation of insulin-producing cells by GIP (referred to as ,+ GIP' in Fig. 1)
  • ES mouse embryonic stem
  • CMV cytomegalovirus
  • Embryoid bodies were formed in the presence of 100 nM of GIP. Embryoid bodies were subsequently plated, again GIP was added every other day until day 14 and afterwards enzymatically dissociated, and replated. After dissociation, cells were cultured without GIP in a differentiation medium containing various growth factors (see Table 2 for more detail). GIP was obtained from SIGMA, USA, Order Number G2269. Under such conditions, the expression of insulin was significantly induced by GIP.
  • ES mouse embryonic stem
  • CMV cytomegalovirus
  • Embryoid bodies were formed in the presence of 10 nM of 1 ⁇ -25-Dihydroxycholecalciferol D 3 (vitamin D 3 ) solution in 95% Ethanol. Embryoid bodies were subsequently plated, again vitamin D 3 was added every second day until day 14 and afterwards enzymatically dissociated, and replated.

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Abstract

L'invention concerne l'utilisation du GIP (polypeptide inhibiteur gastrique ou polypeptide insulinotrope glucose-dépendant) et/ou de la vitamine D3 (cholécalciférol ou 5,7-cholestadién-3-β-ol) et d'analogues correspondants pour déclencher ou favoriser la différenciation de cellules souches ou progénitrices en cellules productrices d'insuline. Ces cellules sont utiles pour le traitement d'affections auto-immunes pancréatiques, telles que le diabète de type I ou LADA ou le diabète de type II.
PCT/EP2006/005912 2005-06-20 2006-06-20 Utilisation du gip et/ou de la vitamine d3 et d'analogues correspondants pour ameliorer la differenciation de cellules souches ou progenitrices en cellules productrices d'insuline Ceased WO2006136374A2 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8299084B2 (en) 2009-04-20 2012-10-30 Auspex Pharmaceuticals, Inc. Piperidine inhibitors of Janus kinase 3
WO2013165800A1 (fr) * 2012-05-02 2013-11-07 Wisconsin Alumni Research Foundation Survie et fonction de support de 2α-méthyl-19-nor-(20s)-1α,25-dihydroxyvitamine d3 (2amd) ou de 2-méthylène-19-nor-(20s)-1α,25-dihydroxyvitamine d3 (2md) de cellules d'îlot transplantées dans le diabète de type 1
WO2014153620A1 (fr) * 2013-03-28 2014-10-02 The University Of Western Australia Procédé de génération de cellules matures de type bêta
US9200051B2 (en) 2013-05-28 2015-12-01 Takeda Pharmaceutical Company Limited Peptide compound
KR20160052220A (ko) * 2014-11-04 2016-05-12 가톨릭대학교 산학협력단 타크롤리무스 및 비타민d를 유효성분으로 포함하는 면역질환의 예방 또는 치료용 조성물
US10501516B2 (en) 2016-05-24 2019-12-10 Takeda Pharmaceutical Company Limited Peptide compound

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DE19921537A1 (de) * 1999-05-11 2000-11-23 Dieter Hoersch Verfahren zur Induzierung von Zellwachstum durch Verwendung geeigneter Mittel
US20030232761A1 (en) * 2002-03-28 2003-12-18 Hinke Simon A. Novel analogues of glucose-dependent insulinotropic polypeptide

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8299084B2 (en) 2009-04-20 2012-10-30 Auspex Pharmaceuticals, Inc. Piperidine inhibitors of Janus kinase 3
US8962638B2 (en) 2009-04-20 2015-02-24 Auspex Pharmaceuticals, Inc. Piperidine inhibitors of janus kinase 3
US9493469B2 (en) 2009-04-20 2016-11-15 Auspex Pharmaceuticals, Inc. Piperidine inhibitors of Janus kinase 3
US9856261B2 (en) 2009-04-20 2018-01-02 Auspex Pharmaceuticals, Inc. Piperidine inhibitors of Janus kinase 3
WO2013165800A1 (fr) * 2012-05-02 2013-11-07 Wisconsin Alumni Research Foundation Survie et fonction de support de 2α-méthyl-19-nor-(20s)-1α,25-dihydroxyvitamine d3 (2amd) ou de 2-méthylène-19-nor-(20s)-1α,25-dihydroxyvitamine d3 (2md) de cellules d'îlot transplantées dans le diabète de type 1
WO2014153620A1 (fr) * 2013-03-28 2014-10-02 The University Of Western Australia Procédé de génération de cellules matures de type bêta
US10568913B2 (en) 2013-03-28 2020-02-25 Healthregen Pty Ltd Method for generating mature β-like cells
US9200051B2 (en) 2013-05-28 2015-12-01 Takeda Pharmaceutical Company Limited Peptide compound
US10087229B2 (en) 2013-05-28 2018-10-02 Takeda Pharmaceutical Company Limited Peptide compound
KR20160052220A (ko) * 2014-11-04 2016-05-12 가톨릭대학교 산학협력단 타크롤리무스 및 비타민d를 유효성분으로 포함하는 면역질환의 예방 또는 치료용 조성물
US10501516B2 (en) 2016-05-24 2019-12-10 Takeda Pharmaceutical Company Limited Peptide compound

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