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WO2006108651A2 - Utilisation de produits de l'activine pour prevenir et/ou traiter le diabete et/ou le syndrome metabolique - Google Patents

Utilisation de produits de l'activine pour prevenir et/ou traiter le diabete et/ou le syndrome metabolique Download PDF

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Publication number
WO2006108651A2
WO2006108651A2 PCT/EP2006/003397 EP2006003397W WO2006108651A2 WO 2006108651 A2 WO2006108651 A2 WO 2006108651A2 EP 2006003397 W EP2006003397 W EP 2006003397W WO 2006108651 A2 WO2006108651 A2 WO 2006108651A2
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cells
activin
pancreatic
product
composition
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PCT/EP2006/003397
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WO2006108651A3 (fr
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Rainer Mussmann
Tri Nguyen
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Develogen Aktiengesellschaft
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    • 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
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/185—Nerve growth factor [NGF]; Brain derived neurotrophic factor [BDNF]; Ciliary neurotrophic factor [CNTF]; Glial derived neurotrophic factor [GDNF]; Neurotrophins, e.g. NT-3

Definitions

  • This invention relates to the use of activin proteins, to the use of polynucleotides encoding these, and to the use of modulators/effectors thereof in the prevention, and/or treatment of pancreatic disorders such as diabetes mellitus and/or metabolic syndrome or in the prevention and/or treatment of neurodegenerative disorders. More particularly, this invention relates to the use of activin proteins in combination with neurotrophin proteins, to the use of polynucleotides encoding these, and to the use of modulators/effectors thereof in the prevention, and treatment of pancreatic disorders such as diabetes mellitus and/or metabolic syndrome and in the prevention and/or treatment of neurodegenerative disorders.
  • 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. Insulin leads to the storage of glycogen and triglycerides and to the synthesis of proteins. 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. The amount of insulin produced by the remaining pancreatic islet cells is too low, resulting in elevated blood glucose levels (hyperglycemia).
  • beta-cell neogenesis and replication does not appear to increase in type Il diabetics, thus causing a reduction in total beta-cell mass over time. Eventually the application of exogenous insulin becomes necessary in type Il diabetics.
  • 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 only be successful if they are combined with agents that can stimulate beta-cell regeneration.
  • Diabetes is a very disabling disease, because today's 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 hyperiipidemia, for which persons with diabetes are substantially at risk.
  • diabetes mellitus type I and LADA Apart from the impaired quality of life for the patients, the treatment of diabetes and its long term complications presents an enormous financial burden to our healthcare systems with rising tendency.
  • diabetes mellitus type I and LADA also for diabetes mellitus type Il there is a strong need in the art to identify factors that induce regeneration of pancreatic insulin producing beta-cells. These factors could restore normal function of the endocrine pancreas once its function is impaired or event could prevent the development or progression of diabetes type I, LADA or diabetes type II.
  • 'metabolic syndrome 1 (syndrome x, insulin-resistance syndrome, deadly quartet) was first described 1966 by Camus and reintroduced 1988 by Reaven (Camus JP, 1966, Rev Rhum MaI Osteoartic 33(1): 10-14; Reaven et al. 1988, Diabetes, 37(12): 1595-1607).
  • Today metabolic syndrome is commonly defined as clustering of cardiovascular risk factors like hypertension, abdominal obesity, high blood levels of triglycerides and fasting glucose as well as low blood levels of HDL cholesterol. Insulin resistance greatly increases the risk of developing the metabolic syndrome (Reaven, 2002, Circulation 106(3): 286-288).
  • the metabolic syndrome often precedes the development of type Il diabetes and cardiovascular disease (McCook, 2002, JAMA 288: 2709-2716).
  • the control of blood lipid levels and blood glucose levels is the essential for the treatment of the metabolic syndrome (see, for example, Santomauro A. T. et al., (1999) Diabetes, 48(9): 1836-1841).
  • the technical problem underlying the present invention was to provide for means and methods for treating pancreatic disorders including diabetes.
  • the solution to said technical problems is achieved by providing the embodiments characterized in the claims.
  • the present invention relates to novel functions of activin proteins, particularly in combination with neurotrophic factors, nucleic acids coding therefore and effectors/modulators thereof in the prevention or treatment of pancreatic disorders and metabolic syndrome.
  • the invention refers to proteins encoded by mammalian activin genes, more preferably human and rodent homologous polypeptides or proteins or sequences encoding these proteins.
  • Activins are members of the TGF- ⁇ superfamily. They are disulfide-linked dimeric proteins with a wide range of biological activities including: mesoderm induction, neural cell differentiation, bone remodeling, hematopoiesis and reproductive physiology. Activins are produced as precursor proteins with an amino-terminal propeptide that is cleaved to release the carboxy-terminal bioactive ligand. Several activins are known in mammals, for example activin A, activin B, activin AB, activin C, and activin E. Similarity to other TGF- ⁇ family members, activins exert their biological activities through binding to the heterodimeric complex composed of two membrane spanning serine-threonine kinases which include at least two type I and type Il receptors.
  • receptors are all transmembrane proteins, composed of a ligand - binding extracellular domain, a transmembrane domain, and a cytoplasmic kinase domain with serine/threonine specificity.
  • Two forms of activin receptor type I Act Rl-A and Act Rl-B
  • two forms of activin receptor type Il Act RII-A and Act RII-B
  • Activin binds directly to the activin receptor type Il (Act RII), the complex then associates with Act Rl and initiates signaling.
  • Activin A was found to increase Pax4 gene expression in pancreatic beta cell lines (Ueda (2000), FEBS. Lett. 480: 101-105; Li et al., (2004), Diabetes 53: 608-615; Brun et al. (2004), J.Cell. Biol. 167: 1123-1135).
  • Activin B is known for treatment of erythropoietin disorders, e.g. anemia (US 5,071 ,834). Activin and analogs of activin are also used for neuronal rescue, e.g. for the treatment of neurological diseases such as Alzheimer and Parkinson (WO 99/15192). Activin antagonists are used for the treatment and/or prophylaxis of diseases associated with fibrosis (WO 03/006057). Activin can also inhibit the maturation of follicles in the ovary of a female mammal (WO 91/10446) and for increasing fertility in a male mammal (WO 91/10444).
  • Activins have been used as effectors for differentiating stem cells into insulin- producing cells (WO 02/086107; US 2003/0138948; US 2002/0182728; WO 03/033697; US 2002/0164307; US 2002/0072115; WO 03/100026). These documents discuss the use of activin A, however, do not contain any specific disclosure for activin B.
  • EP-A-0862451 discloses compositions for improving pancreatic function which may comprise the peptide factor betacellulin in combination with activin, e.g. activin A 1 activin B and activin AB, wherein activin A is preferred (see also Li et al, 2004, supra).
  • activin A was suggested to be a part of a composition comprising the beta-cellulin protein, and Activin A was shown to be effective only in the presence of beta -cellulin.
  • Evidence for the activity of activin B is not provided nor was a combination of activin B and other factors such as neurotrophic factors disclosed.
  • activin A and B stimulate the differentiation of pancreas cells, in particular beta cells, via different mechanisms.
  • Activin B activates pancreatic beta cells via activin receptor-like protein kinase ALK7
  • activin A stimulates beta cells via a different activin receptor-like protein kinase ALK4
  • a patent application discloses a medicament for the treatment and/or prophylaxis of diabetes comprising a protein selected from activin AB, activin B, and protein kinase ALK7 (see JP2003/3113111). This suggestion was based on the analysis of the transcriptional activity in ALK7 positive pancreatic beta cells, and insulin secretion from mouse pancreatic beta cell tumor cells was measured after activin addition.
  • the present invention is based on the finding that activin B and activin AB stimulate the transcription of Pax4 in insulinoma INS-1E cells in vitro.
  • activin B and activin AB are considered to be mitogens capable of promoting the protection, survival and/or regulation of insulin producing cells, particularly pancreatic beta cells.
  • activin B and activin AB may suppress apoptotic events in beta cells thereby preventing beta cell loss.
  • Activin B and/or activin AB may be administered alone or in combination with other medicaments, e.g. known beta cell mitogens such as GLP-1 , prolactin or NGF. Further, activin B and/or activin AB administration may be combined with activin A administration, since their beta cell mitogen activities are surprisingly based on different modes of action and thus the 5 combined administration leads to further increase in activity.
  • medicaments e.g. known beta cell mitogens such as GLP-1 , prolactin or NGF.
  • Activin B and/or activin AB treatment preserves beta cell mass and/or leads to a net increase in beta cell mass. Therefore, activin B and/or activin AB may be used for the prevention, amelioration and/or treatment of pancreatic o disorders, that are associated with beta cell loss.
  • activin B and/or activin AB Treatment in a medical setting could mean the direct application of activin B and/or activin AB to patients for instance by injection.
  • activin B and/or activin AB may be used to promote survival s and growth of donor islets in culture prior to their transfer into recipients.
  • Another use of activin B and/or activin AB is in stem cell differentiation protocols aiming to the production of beta cell-like cells in culture.
  • Activin B and/or activin AB can act as a maturation factor promoting the differentiation of stem cells towards the pancreatic lineage or promoting the growth of o differentiated cells.
  • the present invention is based on the finding that combinations of activins, e.g. activin A, activin B and/or activin AB, and neurotrophins are capable of synergistically activating pancreatic beta cell functions.
  • activins e.g. activin A, activin B and/or activin AB
  • neurotrophins are capable of synergistically activating pancreatic beta cell functions.
  • 5 activins may be used in combinations with neurotrophins capable of binding the P75 neurotrophin receptor (p75 NTR) such as NGF and related molecules.
  • p75 NTR P75 neurotrophin receptor
  • activins may regulate the sensitivity of beta cells to neurotrophins and the exposure of beta cells to activins may enhance the trophic effect of NGF and related molecules. Consequently, a combined administration of activins o and NGF or another neutrophin including a proneurotrophin in regenerative therapies for beta cells is superior to treatment with NGF alone.
  • Activin proteins and nucleic acid molecules coding therefore are obtainable from insect or vertebrate species, e.g. mammals or birds. Particularly preferred are nucleic acids encoding the human activins and the protein encoded thereby.
  • the term "activin” as used in the present application encompasses activin A, activin B, activin AB, activin C or activin E, if not indicated differently.
  • the term "activin product” encompasses activin protein products and activin nucleic acid, e.g. DNA or RNA, products.
  • the invention particularly relates to a nucleic acid molecule encoding a polypeptide contributing to regulating the energy homeostasis and the metabolism of triglycerides and glycogen and regeneration processes, wherein said nucleic acid molecule comprises
  • activin genes particularly a mammalian, e.g. human activin genes and/or sequences complementary thereto,
  • (f) a partial sequence of any of the nucleotide sequences of (a) to (e) having a length of 15-25 bases, preferably 25-35 bases, more preferably 35-50 bases and most preferably at least 50 bases.
  • the human activin nucleic acid and protein sequences are disclosed in the NCBI Genbank.
  • the NCBI accession numbers for human activins are as follows: NM_002193 for activin B, NM_002192 for activin A, NM_005538 for activin C, and AF412024 for activin E.
  • the present invention also relates to modulators/effectors of activins and nucleic acid molecules coding therefore, e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides.
  • modulators/effectors of activins and nucleic acid molecules coding e.g. antibodies, biologically active nucleic acids, such as antisense molecules, RNAi molecules or ribozymes, aptamers, peptides or low-molecular weight organic compounds recognizing said polynucleotides or polypeptides.
  • a first aspect of the present invention relates to compositions for modulating, e.g. stimulating pancreatic development and/or for the regeneration of pancreatic cells or tissues, e.g. cells having exocrinous functions, such as acinar cells, centroacinar cells and/or ductal cells, and/or cells having endocrinous functions, particularly cells in Langerhans islets such as alpha-, beta-, delta-, and/or PP-cells, more particularly beta-cells.
  • exocrinous functions such as acinar cells, centroacinar cells and/or ductal cells
  • endocrinous functions particularly cells in Langerhans islets such as alpha-, beta-, delta-, and/or PP-cells, more particularly beta-cells.
  • compositions comprise as an active ingredient a combination of (a) at least one activin product or a modulator/effector thereof and (b) a neurotrophin product or a modulator/effector thereof. In a still more preferred embodiment, the compositions comprise (a) at least one activin product and/or (b) at least one neurotrophin product.
  • neurotrophin as used in the present application encompasses neurotrophins binding to the p75 NTR surface receptor and precursors such as pro-forms or pre-pro-forms thereof, e.g. nerve growth factor (NGF), proNGF, neurotrophin (NT) -3, pro-NT3, NT-4/5, pro-NT-4/5, brain-derived neurotrophic factor (BDNF), pro-BDNF, and pan-neurotrophin-1.
  • neurotrophin product encompasses neurotrophin protein products and neurotrophic nucleic acid, e.g. DNA or RNA, products.
  • compositions comprise as an active ingredient a combination of (a) at least one activin B and/or activin AB product or a modulator/effector thereof and (b) at least one activin A product or a modulator/effector thereof.
  • a further aspect of the invention relates to the use of compositions comprising an activin B product and/or an activin AB product or a modulator/effector thereof for promoting the protection, survival and/or regeneration of insulin- producing cells and/or for stimulating and/or inducing the differentiation of insulin-producing cells from progenitor cells.
  • the present invention provides methods for treating patients suffering from a 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 compositions 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.
  • the methods of the present invention may also be used to treat patients at risk to develop degeneration of insulin producing beta-cells to prevent the start or progress of such process.
  • compositions may be administered as such e.g. as a protein or a nucleic acid, via implantation of in vitro treated and/or genetically modified cells and/or via gene therapy.
  • the invention relates to cell preparations comprising in vitro treated insulin producing cells or genetically modified insulin-producing cells.
  • 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 cells (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 in special culture conditions such as spinner 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 cells, 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 disorders and/or metabolic syndrome including diabetes mellitus.
  • diagnostic and therapeutic uses for the compositions of the invention of the invention are, for example but not limited to, the following: (i) tissue regeneration in vitro and in vivo (regeneration for all these tissues and cell types composing these tissues and cell types derived from these tissues), (ii) small molecule drug target, (iii) antibody target (therapeutic, diagnostic, drug targeting/cytotoxic antibody), (iv) diagnostic and/or prognostic marker, (v) protein therapy, (vi) gene therapy (gene delivery/gene ablation), and (vii) research tools.
  • the composition may be administered i) as a pharmaceutical composition e.g. enterally, parenterally or topically, preferably directly to the pancreas, ii) via implantation of activin treated and/or activin expressing cells, and/or iii) via gene therapy.
  • a pharmaceutical composition e.g. enterally, parenterally or topically, preferably directly to the pancreas, ii) via implantation of activin treated and/or activin expressing cells, and/or iii) via gene therapy.
  • the activin expression level in a patient might be influenced by an activin modulator/effector administered i) as a pharmaceutical composition e.g. enterally, parenterally or topically, preferably directly to the pancreas, ii) via cell based therapy, and/or iii) via gene therapy.
  • an activin modulator/effector administered i) as a pharmaceutical composition e.g. enterally, parenterally or topically, preferably directly to the pancreas, ii) via cell based therapy, and/or iii) via gene therapy.
  • compositions as indicated above preferably refer to compositions comprising an activin product and a neurotrophin product, compositions comprising an activin B and/or AB product and an activin A product or compositions comprising an activin B product and/or an activin AB product.
  • compositions may be administered alone or in combination with another pharmaceutical composition useful to prevent or treat pancreatic disorders or metabolic syndrome, particularly beta-cell degeneration, for example 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 EGF-family, or immune modulating agents such as anti
  • 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/EP2004/007917, PCT/EP2004/013175, PCT/EP2004/0013535, PCT/EP 2005/000545, PCT/EP 2005/0017111 and EP 04018751.0, which are herein incorporated by reference.
  • compositions may be administered together with beta cell mitogens such as GLP-1 or derivatives thereof such as GLP-1 or derivatives thereof, e.g. GLP-1 (7-36 amide), Exendin-4, prolactin or a neurotrophin such as NGF.
  • beta cell mitogens such as GLP-1 or derivatives thereof such as GLP-1 or derivatives thereof, e.g. GLP-1 (7-36 amide), Exendin-4, prolactin or a neurotrophin such as NGF.
  • compositions may also be 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
  • 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 compositions may be administered in patients suffering from a disease going along with impaired beta-cell function, for example but not limited to one of the diseases for which an anti-apoptotic/pro-survival effect on pancreatic beta cells would be beneficial:
  • Type I diabetes new onset, established, prevention in high-risk patients (identified e.g. via screening for multiple autoantibodies)
  • LADA new onset and established - Type Il diabetes: before loss of beta cell mass has occurred
  • compositions may be administered in indications in which an increased neurotrophic effect would be beneficial: Neurodegenerative diseases (Alzheimers, ALS, Multiple sclerosis, Huntingtons disease, Parkinson, stroke/ischemia etc.) Neuropathy (e.g. diabetic, HIV-linked)
  • Neurodegenerative diseases Alzheimers, ALS, Multiple sclerosis, Huntingtons disease, Parkinson, stroke/ischemia etc.
  • Neuropathy e.g. diabetic, HIV-linked
  • compositions may be administered in diabetes type I, LADA or prognosed diabetes type II, but also preventively to patients at risk to develop complete beta-cell degeneration, like for example but not limited to patients suffering from diabetes type Il or LADA and type I diabetes in early stages, or other types of diseases as indicated above.
  • the compositions may also be used to prevent or ameliorate diabetes in patients at risk for type I diabetes or LADA (identified e.g. by screening for autoantibodies, genetic predisposition, impaired glucose tolerance or combinations thereof).
  • a variety of pharmaceutical formulations and different delivery techniques are described in further detail below.
  • 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
  • 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.
  • the active ingredients of the compositions are preferably produced via recombinant techniques because such methods are capable of achieving high amounts of protein at a great purity, but are not limited to products expressed in bacterial, plant, mammalian, or insect cell systems.
  • 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 exocrinous functions such as acinar cells, centroacinar cells and/or ductal cells, and/or cells having endocrinous functions, particularly cells in Langerhans islets such as alpha-, beta-, delta- and/or PP-cells, more particularly beta-cells.
  • compositions of the invention are useful in therapeutic applications implicated in various applications as described below.
  • cDNAs encoding the proteins of the invention may be useful in gene therapy, and the proteins of the invention may be useful when administered to a subject in need thereof.
  • the compositions of the present invention will have efficacy for treatment of patients suffering from, for example, but not limited to, in metabolic disorders as described above.
  • Cell therapy e.g. pancreatic implantation of cells producing an activin protein product, e.g. an activin B product, is also contemplated.
  • This embodiment would involve implanting cells capable of synthesizing and secreting a biologically active form of activin protein product into patients.
  • Such activin protein product-producing cells may be cells that are natural producers of activin protein product or may be cells that are modified to express the protein.
  • modified cells include recombinant cells whose ability to produce a activin protein product has been augmented by transformation with a gene encoding the desired activin protein product in a vector suitable for promoting its expression and secretion.
  • the cells producing activin protein product be of human origin and produce human activin protein product.
  • the recombinant cells producing activin protein product be transformed with an expression vector containing a gene encoding a human activin protein product.
  • Implanted cells may be encapsulated to avoid infiltration of surrounding tissue.
  • Human or nonhuman animal cells may be implanted in patients in biocompatible, semipermeable polymeric enclosures or membranes that allow release of activin protein product, but that prevent destruction of the cells by the patient's immune system or by other detrimental factors from the surrounding tissue.
  • activin protein product secreting cells may be introduced into a patient in need intraportally via a percutaneous transhepatic approach using local anaesthesia. Between 3000 and 100 000 equivalent differentiated insulin-producing cells per kilogram body weight are preferably administered.
  • Such surgical techniques are well known in the art and can be applied without any undue experimentation, see Pyzdrowski et al, 1992, New England J. Medicine 327:220-226; Hering et al., Transplantation Proc. 26:570-571 , 1993; Shapiro et al., New England J. Medicine 343:230-238, 2000.
  • activin protein product e.g. activin B and/or AB product
  • progenitor e.g. stem cells
  • Preparation, production and purification of such proteins from bacteria, yeast or eukaryotic cells are well known by persons skilled in the art.
  • activin may be added preferably at concentrations between 0.02 nM and 2500 nM, more preferably between 0.2 nM and 2 nM, e.g. at about 1 nM.
  • the present invention also relates to gene therapy. Many methods for introducing nucleic acids into cells or tissues are available and equally suitable for use in vivo, in vitro, and ex vivo.
  • nucleic acids may be introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient. Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any of the therapeutic methods described above may be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • 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 cells. 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 quick 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 diseases, e.g. diabetes.
  • the functional insulin producing cells treated with compositions of the invention may be transplanted preferably intrahepatic, 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.
  • gene therapy ex vivo i.e. the patient's own cells may be transformed ex vivo to produce an activin, e.g. an activin B protein product or a protein stimulating activin, e.g. activin B expression and would be directly reimplanted.
  • an activin e.g. an activin B protein product or a protein stimulating activin, e.g. activin B expression
  • cells retrieved from the patient may be cultured and transformed with an appropriate vector. After an optional propagation/expansion phase, the cells can be transplanted back into the same patient's body, particularly the pancreas, where they would produce and release the desired activin, e.g. activin B protein product. Delivery by transfection and by liposome injections may be achieved using methods, which are well known in the art. Any of the therapeutic methods described above may be applied to any suitable subject including, for example, mammals such as dogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.
  • Activin in particular activin B gene therapy in vivo is also envisioned, by introducing genes coding for an activin protein product into targeted pancreas cells via local injection of a nucleic acid construct or other appropriate delivery methods (Hefti, J. Neurobiol., 25:1418-1435, 1994).
  • a nucleic acid sequence encoding an activin protein product may be contained in an adeno-associated virus vector or adenovirus vector for delivery to the pancreas cells.
  • Alternative viral vectors include, but are not limited to, retrovirus, herpes simplex virus and papilloma virus vectors.
  • Physical transfer may also be achieved by liposome-mediated transfer, direct injection (naked DNA), receptor-mediated transfer (ligand-DNA complex), electroporation, calcium 5 phosphate precipitation or microparticle bombardment (gene gun).
  • liposome-mediated transfer direct injection (naked DNA), receptor-mediated transfer (ligand-DNA complex), electroporation, calcium 5 phosphate precipitation or microparticle bombardment (gene gun).
  • lmmunomodulating medicaments e.g. immunosuppressive drugs, such as cyclosporin
  • immunosuppressive drugs such as cyclosporin
  • Allografts using the cells obtained by the methods o 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.
  • compositions to a subject in need s thereof, particularly a human patient, leads to an at least partial regeneration of pancreatic cells.
  • these cells are insulin producing beta-cells that will contribute to the improvement of a diabetic state.
  • an increase in beta-cell mass can be achieved. This effect upon the body reverses the condition of o diabetes partially or completely.
  • the dosage 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 5 condition.
  • compositions of the present invention will also have efficacy for treatment of patients with other pancreatic diseases such as pancreatic cancer, dysplasia, o or pancreatitis, if beta-cells are to be regenerated.
  • a subject in need thereof particularly a human patient
  • a subject in need thereof leads to an at least partial regeneration of neuronal cells such as cortical neurons, peripheral/sensory neurons, midbrain dopaminergic neurons or cerebellar neurons or spinal cord neurons after injury.
  • neuronal cells such as cortical neurons, peripheral/sensory neurons, midbrain dopaminergic neurons or cerebellar neurons or spinal cord neurons after injury.
  • the combinations are also suitable for the prevention or treatment of neurodegenerative disorders.
  • compositions of the invention are intended for pharmaceutical applications and may comprise with a pharmaceutically acceptable carrier, for any of the therapeutic effects discussed above.
  • Such pharmaceutical compositions may consist of the nucleic acids and the proteins of the invention, antibodies to the proteins of the invention, mimetics, agonists, antagonists or inhibitors of the proteins of the invention.
  • the compositions may be administered alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water.
  • the compositions may be administered to a patient alone or in combination with other agents, drugs or hormones.
  • compositions utilized in this invention may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active compounds into preparations, which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing Co., Easton, Pa.).
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of pancreatic cells or in animal models, usually mice, rabbits, dogs or pigs.
  • the animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example the nucleic acids or the proteins of the invention or fragments thereof or antibodies, which is sufficient for treating a specific condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50/ED50.
  • Pharmaceutical compositions, which exhibit large therapeutic indices, are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage from employed, sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors, which may be taken into account, include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy.
  • Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week or once every two weeks depending on half-life and clearance rate of the particular formulation. Normal dosage amounts may vary from 0.1 to 100,000 ⁇ g, up to a total dose of about 1 g, depending upon the route of administration.
  • the nucleic acids encoding the protein of the invention can be used to generate transgenic animals or site-specific gene modifications in cell lines. These transgenic non-human animals are useful in the study of the function and regulation of the protein of the invention in vivo.
  • Transgenic animals particularly mammalian transgenic animals, can serve as a model system for the investigation of many developmental and cellular processes common to humans.
  • a variety of non-human models of metabolic disorders can be used to test effectors/modulators of the protein of the invention.
  • Misexpression for example, overexpression or lack of expression
  • such assays use mouse models of insulin resistance and/or diabetes, such as mice carrying gene knockouts in the leptin pathway (for example, ob (leptin) or db (leptin receptor) mice).
  • leptin pathway for example, ob (leptin) or db (leptin receptor) mice.
  • Such mice develop typical symptoms of diabetes, show hepatic lipid accumulation and frequently have increased plasma lipid levels (see Bruning J. C. et al., 1998, supra).
  • Susceptible wild type mice for example C57BI/6) show similiar symptoms if fed a high fat diet.
  • mice could be used to test whether administration of a candidate effector/modulator alters for example lipid accumulation in the liver, in plasma, or adipose tissues using standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests and others.
  • standard assays well known in the art, such as FPLC, colorimetric assays, blood glucose level tests, insulin tolerance tests and others.
  • the invention also relates to a kit comprising individual components of the compositions in separate containers.
  • the kit may further contain user instructions.
  • the Figures show:
  • Fig. 1 shows the Pax4 RNA expression level in insulinoma INS-1E cells without treatment (CO) or after treatment with activin A, TGF-beta, activin B, activin AB, BMP-4 and BMP-7 in concentrations as indicated. Pax4 expression levels were quantitatively determined by real time RT-PCR and are indicated in relative amounts.
  • Fig. 2 shows the relative p75 NTR RNA expression level in rat insulinoma INS- IE cells (Fig. 2A) or in mouse insulinoma NIT-1 cells (Fig. 2B) without treatment (Co) or after treatment with 1 nM activin A for different period of time.
  • Fig. 3 shows the p75 NTR protein expression in INS-1 E cells without treatment (Co.) or treated for 8 hours with 1 nM activin A or B. Equal loading of lanes was confirmed by checking the relative expression levels of ⁇ tubulin.
  • Figure 4 shows the p75NTR RNA expression level in isolated mouse (strain NMRI) islets (Fig. 4A) or human islets (Fig. 4B) without treatment (CO) or after treatment with 0.5 nM activin-A or 1 nM TGF-beta.
  • p75NTR expression levels were quantitatively determined by real time RT-PCR and are indicated in relative amounts.
  • Figure 5 shows changes in blood glucose concentrations on STZ-treated neonatal rats.
  • STZ was injected on day 0 and daily injection of the indicated factors (NGF, Activin B, NGF + Activin B) was started a day later until day 6.
  • NGF indicated factors
  • Proteins were dissolved in PBS before the subcutaneous injection. Random fed blood glucose was measured once a day. The average blood glucose values of at least 8 animals per group and the standard deviation are shown.
  • Example 1 Activin B increases Pax4 transcription
  • INS-1E The response of the Pax4 gene to mitogens was investigated in the rat insulinoma cell line INS-1E.
  • INS-1E cells are known to express Pax4 and to upregulate Pax4 levels in response to the treatment with activin-A and betacellulin (Ueda (2000), supra, Li et al. (2004) supra, Bru ⁇ et al. (2004), supra).
  • activin-A and betacellulin Ueda (2000), supra, Li et al. (2004) supra, Bru ⁇ et al. (2004), supra.
  • Activin B an activin AB were found to be almost equally potent in stimulating Pax4 transcription as activin A.
  • TGF-beta family members such as BMP 4 and 7 which are known to recognize the activin-receptor type Il subunit of the heterodimeric activin receptor hardly induced Pax4 gene transcription. Maximal Pax4 induction was observed with 1 nM activin B that induced about a 7.5-fold increase in Pax4 levels. The Pax4 RNA expression level was normalized to this of 18S RNA. The level of the unrelated gene RNA polymerase Il largest subunit (RPB1) was unaffected by activin B treatment.
  • RPB1 RNA polymerase Il largest subunit
  • Fig.1 illustrates a representative experiment in which the relative Pax4 levels were quantified using quantitative real time RT-PCR.
  • activin B and activin AB induce Pax4 gene transcription in INS-1E cells.
  • Quantitative real-time RT-PCR was done with RNA isolated from INS-1E cells cultured under conditions as described below. Low levels of Pax4 are expressed in INS-1E cells.
  • Data are presented as relative levels to the basal Pax4 expression level in untreated INS-1 E cells (Co.).
  • the values for untreated INS-1 E (Co.) and activin-B are averages of three experiments enabling the determination of standard deviations; the other values are averages of two experiments.
  • 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 pax4, 18S RNA, and rat RNA polymerase Il largest subunit (RPB1) 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.
  • INS-1E cells were cultured as described (Merglen, (2004) Endocrinology; 145: 667-678). Cells were seeded at a density of 2x10 4 cells per cm 2 6 to 8 days before the treatment with chemicals. During the growth period the medium was changed once. The cells were incubated for different periods of time with chemicals under serum-free conditions. The cells were harvested in Qiagen RNAeasy cell lysate buffer and immediately transferred to dry ice. The samples were stored at -20 degree until RNA isolation was carried out.
  • NIT-1 cells were grown as indicated in the ATCC product information sheet for CRL-2055. The cells were treated with activins in the presence of 10% serum.
  • lysate aliquots containing 15 ⁇ g protein were mixed with Laemmeli sample buffer and heated to 95 degree Celsius for 5 min before separation on 4 to 12% SDS-polyacrylamide gels (BioRad). Blotting to nitrocellulose was done using standard methods well known in the art.
  • Anti- p75 NTR antibodies (Santa Cruz) were diluted 1 :200 in blotting buffer before immunodetection using secondary anti-goat Ig HRP-labelled antibodies (DAKO).
  • Anti- ⁇ -tubulin antibodies (Sigma) were diluted 1 : 10.000 in blotting buffer before immunodetection using secondary rabbit anti-mouse Ig HRP- labelled antibodies (Pierce). Blots were developed using the SuperSignal WestDura assay (Pierce).
  • Example 2 Synergistic action of activin and neurotrophins on pancreatic beta cell functions
  • Activin A is a mitogen and survival factor of pancreatic beta cells. Activin A stimulates expression of Pax4, a transcription factor that is instrumental for the differentiation of beta cell precursor and the growth of mature pancreatic beta cells.
  • a gene expression profile of INS1-E cells treated for 3 hours with activin A was established using oligonucleotide microarray technology (Affymetrix).
  • expression level of the p75 neurotrophin receptor (p75NTR) went up 14-fold.
  • the differential expression of the p75NTR receptor was verified by quantitative real-time RT-PCR (Fig. 2) and western blotting (Fig. 3).
  • p75NTR binds the nerve growth factor (NGF) and other neurotrophins either alone or as a heterodimer with the so-called TRK receptors.
  • NGF nerve growth factor
  • Fig.2 illustrates representative experiments in which the relative p75 NTR levels in two rodent insulinoma cell lines were quantified using quantitative real time RT-PCR.
  • INS-1 E or NIT-1 cells express p75 NTR at intermediate levels compared to genes such as Pax4 of which only a small number of transcripts are detectable in both cell lines. Data are presented as relative levels to the basal p75 NTR expression level in untreated INS-1 E or NIT-1 cells (Co.).
  • Fig. 2 shows that activin A increases severalfold the p75 NTR RNA levels in INS-1 E or NIT-1 cells. After 48 hours activin treatment INS-1 E cells still express about 2-fold more p75 NTR than untreated cells. The values given are averages of at least three experiments.
  • Fig. 3 illustrates a representative experiment demonstrating that activin A or B induce p75 NTR protein production in INS-1E cells.
  • Figure 4 demonstrates that treatment with activin, e.g. activin A induces p75NTR expression also in mouse or human islets and thus confirms the data obtained in insulinoma cells.
  • activin e.g. activin A induces p75NTR expression also in mouse or human islets and thus confirms the data obtained in insulinoma cells.
  • Figure 5 demonstrates that the administration of a combination of NGF and activin B to STZ-treated neonatal rats improves blood glucose control compared to a treatment with either activin B or NGF alone.
  • p75NTR is expressed on beta cells and NGF has pleiotrophic effects on beta cells such as increasing insulin secretion and preventing apoptosis. Stimulated by these findings it has been suggested to treat diabetic patients with NGF or related molecules to preserve beta cell function.
  • activins can be regulators of the sensitivity of pancreatic beta cells to NGF and the exposure of beta cells to activin enhances the trophic effects of neutrophins, for example NGF 1 e.g. towards pancreatic or neuronal cells.
  • a combination therapy comprising the administration of an activin product together with a neurotrophin product provides an unexpected benefit in treatment of pancreatic or neuronal disorders, particularly in the treatment of diabetes.

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Abstract

La présente invention se rapporte à l'utilisation de protéines d'activine, à l'utilisation de polynucléotides les codant, et à l'utilisation de modulateurs/effecteurs de celles-ci pour prévenir et/ou traiter des troubles pancréatiques tels que le diabète sucré et/ou le syndrome métabolique, ou pour prévenir et/ou traiter des troubles neurodégénératifs. Plus particulièrement, l'invention a trait à l'utilisation de protéines d'activine en combinaison avec une protéine de neurotrophine, à l'utilisation de polynucléotides les codant, et à l'utilisation de modulateurs/effecteurs de celles-ci pour prévenir et/ou traiter des troubles pancréatiques tels que le diabète sucré et/ou le syndrome métabolique, ou pour prévenir et/ou traiter des troubles neurodégénératifs.
PCT/EP2006/003397 2005-04-12 2006-04-12 Utilisation de produits de l'activine pour prevenir et/ou traiter le diabete et/ou le syndrome metabolique WO2006108651A2 (fr)

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN104353059A (zh) * 2014-10-15 2015-02-18 南方医科大学 Activin A在制备治疗精神兴奋类药物成瘾后戒断症状的药物中的应用
CN104353060A (zh) * 2014-10-15 2015-02-18 南方医科大学 Activin B在制备治疗神经退行性疾病的药物中的应用
US20160220639A1 (en) * 2013-09-11 2016-08-04 New York University Methods and compositions for treating bone diseases

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WO1991003569A1 (fr) * 1989-08-30 1991-03-21 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Neurotrophine-3, un nouveau facteur neurotrophique relatif a la croissance des nerfs et facteur neurotrophique derive du cerveau
WO1999015192A1 (fr) * 1997-09-19 1999-04-01 Auckland Uniservices Limited Agent de sauvetage neuronal
EP1010432A1 (fr) * 1997-01-23 2000-06-21 Sumitomo Pharmaceuticals Company, Limited Remedes contre le diabete
WO2002086107A2 (fr) * 2001-04-19 2002-10-31 DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung Procede pour differencier des cellules embryonnaires dans des cellules produisant de l'insuline
WO2003100026A2 (fr) * 2002-05-28 2003-12-04 Novocell, Inc. Procedes, compositions et facteurs de croissance et de differenciation pour cellules productrices d'insuline

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WO1991003569A1 (fr) * 1989-08-30 1991-03-21 Max-Planck-Gesellschaft Zur Forderung Der Wissenschaften E.V. Neurotrophine-3, un nouveau facteur neurotrophique relatif a la croissance des nerfs et facteur neurotrophique derive du cerveau
EP1010432A1 (fr) * 1997-01-23 2000-06-21 Sumitomo Pharmaceuticals Company, Limited Remedes contre le diabete
WO1999015192A1 (fr) * 1997-09-19 1999-04-01 Auckland Uniservices Limited Agent de sauvetage neuronal
WO2002086107A2 (fr) * 2001-04-19 2002-10-31 DeveloGen Aktiengesellschaft für entwicklungsbiologische Forschung Procede pour differencier des cellules embryonnaires dans des cellules produisant de l'insuline
WO2003100026A2 (fr) * 2002-05-28 2003-12-04 Novocell, Inc. Procedes, compositions et facteurs de croissance et de differenciation pour cellules productrices d'insuline

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20160220639A1 (en) * 2013-09-11 2016-08-04 New York University Methods and compositions for treating bone diseases
CN104353059A (zh) * 2014-10-15 2015-02-18 南方医科大学 Activin A在制备治疗精神兴奋类药物成瘾后戒断症状的药物中的应用
CN104353060A (zh) * 2014-10-15 2015-02-18 南方医科大学 Activin B在制备治疗神经退行性疾病的药物中的应用

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