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WO2007099345A1 - Utilisation médicale de bmp-2 et/ou bmp-4 - Google Patents

Utilisation médicale de bmp-2 et/ou bmp-4 Download PDF

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WO2007099345A1
WO2007099345A1 PCT/GB2007/000740 GB2007000740W WO2007099345A1 WO 2007099345 A1 WO2007099345 A1 WO 2007099345A1 GB 2007000740 W GB2007000740 W GB 2007000740W WO 2007099345 A1 WO2007099345 A1 WO 2007099345A1
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bmp
cells
variant
combination product
fusion
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Helena Edlund
Ulf Dahl
Joan Goulley
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Betagenon AB
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Betagenon AB
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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
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1875—Bone morphogenic factor; Osteogenins; Osteogenic factor; Bone-inducing factor
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K67/00—Rearing or breeding animals, not otherwise provided for; New or modified breeds of animals
    • A01K67/027—New or modified breeds of vertebrates
    • A01K67/0275—Genetically modified vertebrates, e.g. transgenic
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/71—Receptors; Cell surface antigens; Cell surface determinants for growth factors; for growth regulators
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/8509—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells for producing genetically modified animals, e.g. transgenic
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2227/00—Animals characterised by species
    • A01K2227/10—Mammal
    • A01K2227/105—Murine
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2267/00—Animals characterised by purpose
    • A01K2267/03—Animal model, e.g. for test or diseases
    • A01K2267/035—Animal model for multifactorial diseases
    • A01K2267/0362—Animal model for lipid/glucose metabolism, e.g. obesity, type-2 diabetes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/40—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation
    • C07K2319/42—Fusion polypeptide containing a tag for immunodetection, or an epitope for immunisation containing a HA(hemagglutinin)-tag
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/008—Vector systems having a special element relevant for transcription cell type or tissue specific enhancer/promoter combination

Definitions

  • the present invention relates to the use of a bone morphogenetic protein (BMP), or a variant, fusion or derivative thereof, in the treatment of ⁇ -cell dysfunction.
  • BMP bone morphogenetic protein
  • the invention provides the use of BMP2 and BMP4, and fragments, variants, fusions and derivatives thereof, in the treatment of diseases and disorders characterised by ⁇ -cell dysfunction, such as type 2 diabetes.
  • Type 2 diabetes is a chronic disease that is characterised by persistently elevated blood glucose levels (hyperglycaemia).
  • pancreatic ⁇ -cells In healthy individuals, blood glucose levels are maintained within a narrow range, primarily through the concerted action of two pancreatic hormones, insulin, produced by the pancreatic ⁇ -cells, and glucagon, produced by the pancreatic ⁇ -cells.
  • ⁇ -Cells have the unique ability to sense blood glucose levels, and to respond by production and secretion of appropriate amounts of insulin.
  • insulin promotes glucose uptake by tissues of the body, thereby restoring blood glucose to the physiological range.
  • glucagon acts reciprocally, increasing blood glucose levels under fasting conditions, primarily by stimulating glucose production in the liver.
  • Type 2 diabetics exhibit a combination of reduced action of insulin in skeletal muscle, adipocytes and hepatocytes (insulin resistance), and most importantly, reduced and impaired insulin secretion from the pancreatic ⁇ -cells.
  • Type 2 diabetics exhibit several major ⁇ -cells defects:
  • Intrinsic factors in the ⁇ -cell that also play an important role in the overall insulin export machinery include Ipfl/PDXl (insulin promoter factor 1), Glut2 (glucose transporter 2), PC2 (prohormone convertase 2), Kir6.2 (inward rectifier K+ channel), SURl (sulfonylurea receptor) and Snap-25 (synaptosome- associated protein of 25,000 dalton).
  • Important intrinsic factors additionally present in the ⁇ -cell include Smadl, Smad4, Smad7, IdI, Id2, Evi-1, GCK, Rab3d, Rab27a, CalpainlO, Nkx ⁇ .l, HNFIa, Hifla, FoxOl, Arntl, GIPr and GLP-Ir.
  • GIP glucose-dependent insulinotropic polypeptide receptor
  • GIP The glucose-dependent insulinotropic polypeptide receptor
  • DPP-IV which also cleaves GLP-I and GLP-2, rapidly inactivates GIP both in vitro and in vivo. Enhancement of GIP receptor expression and signalling is considered to be a viable strategy for the treatment of type 2 diabetes.
  • GIP has been shown to stimulate ⁇ -cell proliferation synergistically with glucose in cell lines.
  • many existing therapies for diabetes such as DPP-IV inhibitors, exenatide, and derivatives thereof, stimulate GIPr signalling via reduced degradation of GIP and GIP analogues or agonists by stimulation of GIPr.
  • enhanced expression of GIPr is considered to be of high therapeutic value.
  • the glucagon-like peptide-1 receptor (GLP-Ir) is thought to be of particular importance in ⁇ -cell function.
  • the biological activities on ⁇ -cells of its ligand, glucagon-like peptide-1 (GLP-I), include stimulation of glucose-dependent insulin secretion and insulin biosynthesis. Also, mounting evidence strongly suggests that GLP-I signalling regulates islet proliferation and islet neogenesis.
  • GLP-Ir stimulates GLP-Ir signalling.
  • DPP-IV inhibitors such as DPP-IV inhibitors, GLP-I analogues, exenatide, and derivatives thereof, stimulate GLP-Ir signalling.
  • enhanced expression of GLP-Ir is also considered be of high therapeutic value.
  • a therapy that restores many or all of the above mentioned ⁇ -cell markers, or reduces inhibitory ⁇ -cell markers, is likely have a high therapeutic value for the treatment of humans suffering from impaired glucose tolerance and type 2 diabetes.
  • type 2 diabetes is a progressive disease, and diabetics characteristically pass through stages of disturbed (i.e. impaired) glucose metabolism.
  • the pre-diabetic states are clear and important predictors of the development of type 2 diabetes, and approximately 5% of these individuals develop type 2 diabetes annually.
  • These pre-diabetic states although usually symptomless, are characterised by defective function of both ⁇ - and ⁇ -cells. Correcting the cellular defects in these individuals would effectively prevent onset of clinical disease, thereby greatly diminishing the extent of the diabetes epidemic and the associated complications.
  • hypoinsulinemia is also present in other forms of diabetes, including type 1 diabetes and atypical diabetes.
  • type 2 diabetes has circulating auto-antibodies either to islet cell cytoplasmic antigens or, more frequently, to glutamic acid decarboxylase (GADab).
  • GIDab glutamic acid decarboxylase
  • This subgroup also referred to as latent autoimmune diabetes in adults (LADA)
  • LADA latent autoimmune diabetes in adults
  • LADA shares features with both type 1 and type 2 diabetes.
  • insulin secretion is better preserved in the slowly progressing form compared to in the rapidly progressing form of autoimmune diabetes, insulin secretion tends to deteriorate with time in LADA patients.
  • MODY Maturity-Onset Diabetes of Young
  • these individuals are not typically obese and have onset of disease at a young age, generally less than 25 years.
  • Inheritance of the diabetes in these individuals is autosomal dominant with up to 85-95% penetrance.
  • the genetic defects cause impaired insulin secretion due to mutations in genes that are important for insulin release.
  • HNF-4 ⁇ -associated MODY (formerly MODY 1) was the first MODY to be described and is a rare genetic defect located on chromosome 20.
  • HNF4 ⁇ is involved in the regulation of genes required for glucose transport and metabolism. Patients with (HNF)4 ⁇ defect have a loss of function in this gene.
  • Glucokinase (GCK)-associated MODY (formerly MODY2) is characterised by a heterozygous mutation on chromosome 7 resulting in defects in the expression of glucokinase.
  • This glycolytic enzyme has a low affinity for glucose and controls the rate-limiting step of glucose metabolism.
  • the clinical disease manifests as mild fasting hyperglycaemia with onset during youth.
  • HNF- l ⁇ - associated MODY (formerly MODY 3) is characterised by a mutation on chromosome 12.
  • HNF- l ⁇ is part of the homeodomain-containing superfamily of transcription factors.
  • HNF- l ⁇ is expressed in the liver, kidney, intestine, and pancreatic islets. Glucosuria is often part of the clinical presentation, and diabetic complications are frequently present.
  • Insulin promoter factor (IPF)-I- associated MODY involves a homeodomain-containing transcription factor that plays a role in the development and expression of insulin, glucokinase, islet amyloid polypeptide and glucose transporter 2.
  • HNF- l ⁇ -associated MODY (formerly MODY 5), is a form of diabetes that is characterised by progressive nondiabetic renal dysfunction of variable severity. This gene is functionally related to HNF- l ⁇ and is also part of the homeodomain-containing superfamily. It can be found in the liver, kidney, intestine, stomach, lung, ovary and pancreatic islets.
  • insulin secretagogues such as sulphonylureas stimulate only the insulin secretion step
  • metformin mainly acts on glucose production from the liver
  • peroxisome proliferator-activated receptor- ⁇ (PPAR- ⁇ ) agonists such as the thiazolidinediones, enhance insulin action
  • Exenatide needs to be administered by subcutaneous injection and also has storage stability shortcomings.
  • insulin secretagogues and insulin injections may cause hypoglycaemia and weight gain. Patients may also become unresponsive to insulin secretagogues over time.
  • Metformin and ⁇ - glucosidase inhibitors often lead to gastrointestinal problems and PPAR- ⁇ agonists tend to cause increased weight gain and oedema.
  • Exenatide is also reported to cause nausea and vomiting.
  • Insulin secretion by ⁇ -cells and glucagon secretion by ⁇ -cells are two critical participants in glucose homeostasis and serve as acute regulators of blood glucose concentration. From a medical perspective, insulin in particular is enormously important. Dysfunctional ⁇ -cells hypersecrete proinsulin and insulin split products, which are secretory products believed to be risk factors for cardiovascular disease.
  • Glucose stimulated insulin secretion in pancreatic ⁇ -cells depends on coordinated glucose uptake, oxidative metabolism, and Ca 2+ triggered insulin exocytosis. Impaired GSIS, and in particular loss of first phase insulin release, is an early sign of type 2 diabetes.
  • the glucose transporter type 2 (Glut2) is essential for glucose sensing and efficient uptake of glucose into the ⁇ -cell, and Glut2 null mutant mice are glucose intolerant and lack first-phase insulin secretion in response to glucose (Guillam et al, 1997).
  • Glut2 expression is also perturbed in several diabetic animal models and in islets exposed to free fatty acids (FFA) (Efrat, 1997; Gremlich et al, 1997).
  • TGF Transforming Growth Factor
  • BMP BMP
  • TGF- ⁇ signalling Several members of the Transforming Growth Factor (TGF) ⁇ /Bone Morphogenetic Protein (BMP) superfamily of secreted signalling molecules are expressed in the developing and adult pancreas. Recent data implicate a role for TGF- ⁇ signalling in pancreatic development and disease (Smart et al, 2006, Rane et ⁇ /., 2006).
  • BMP- signalling in the developing and/or adult pancreas.
  • BMP is descriptive of one particular function (induction of ectopic bone and cartilage formation)
  • BMPs also play important roles in a number of non-osteogenic developmental processes, including cell proliferation, apoptosis, differentiation, morphogenesis and dorsal- ventral patterning.
  • TGF- ⁇ /BMPs are known to signal through a family of heteromeric type I and type II, transmembrane serine-threonine kinase receptors.
  • Receptor complex formation is ligand dependent and is initiated by binding of the ligand to the type II receptor kinase (Heldin et al, 1997; Wrana, 2000).
  • the ligand activated type II receptor then recruits and phosphorylates the type I receptor.
  • the activated type I receptor kinase then in turn phosphorylates the receptor- regulated Smads (R-Smads).
  • the phosphorylated R-Smads are released from the receptor complex and form interactions with the Co-Smad, Smad-4.
  • the resulting Smad complex translocates into the nucleus where it regulates the expression of target genes (Massague et al, 2005). BMP-signalling is preferentially transduced via the R-Smads 1, 5, and 8, whereas activin and TGF- ⁇ signalling is transduced via the R-Smads 2 and 3.
  • a third class of Sraads the inhibitor Smads (I-Smads) 6 and 1, inhibits TGF- ⁇ /BMP-signalling by binding to the activated receptor complex, thereby preventing the phosphorylation of the R-Smads (Heldin et al, 1997; Wrana, 2000).
  • Smad ⁇ preferentially inhibits BMP signalling, whereas Smad7 inhibits both TGF- ⁇ /activin and BMP signalling.
  • the co-repressor, Evi-1 exerts its effects by repressing tiie receptor-activated transcription through Smadl .
  • I-Smads have been suggested to compete with Smad-4 for interaction with the R-Smads (Massague et al, 2005).
  • BMP -signals can also be transduced via the "non-canonical" BMP-MAPK pathway in which activated BMP-receptors signal via the TGF- ⁇ activated kinase 1 (TAK-I) and TAK-I binding protein (TABl) (Chen et al, 2004).
  • BMPRIa bone morphogenetic protein type I receptor
  • BMP4 bone morphogenetic protein type I receptor
  • GSIS glucose stimulated insulin secretion
  • BMP2 may also be used to treat type 2 diabetics and patients with associated conditions.
  • a first aspect of the invention provides the use of a bone morphogenetic protein (BMP) or a fragment, variant, fusion or derivative thereof in the preparation of a medicament for treating ⁇ -cell dysfunction, wherein the bone morphogenetic protein is selected from the group consisting of BMP2 and BMP4.
  • BMP bone morphogenetic protein
  • the invention provides a bone morphogenetic protein (BMP) or a fragment, variant, fusion or derivative thereof as defined herein for treating ⁇ - cell dysfunction.
  • BMP bone morphogenetic protein
  • the invention provides the use of a bone morphogenetic protein (BMP) or a fragment, variant, fusion or derivative thereof as defined herein for treating ⁇ -cell dysfunction.
  • ⁇ -cell we mean the cells located in pancreas (and specifically, in the islets of Langerhans) responsible for the production and secretion of the hormone insulin.
  • islets of Langerhans pancreatic islets
  • the endocrine part accounts for only 2% of the total mass of the pancreas.
  • ⁇ -cells constitute 60-80% of all the cells.
  • proinsulin precursor molecule In ⁇ -cells, insulin is synthesised from the proinsulin precursor molecule by the action of proteolytic enzymes known as prohormone convertases (PC 1/3 and PC2), as well as the exoprotease carboxypeptidase E. These modifications liberate the centre portion of the molecule, or C-peptide, from the C- and N- terminal ends of the proinsulin. The two remaining polypeptides, the B- and A- chains, are held together by disulfide bonds and together constitute 51 amino acids.
  • prohormone convertases PC 1/3 and PC2
  • exoprotease carboxypeptidase E exoprotease carboxypeptidase E
  • ⁇ -cell dysfunction includes the treatment of any condition characterised by dysfunction of ⁇ -cells, including pancreatic islet ⁇ -cells, and may this be understood to include any condition that comprises reduced levels of, or dysfunctional key intrinsic factors in, the ⁇ -cell, including type 2 diabetes, atypical forms of diabetes (such as LADA and MODY), glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions, stroke, cancer and infectious diseases, such as tuberculosis.
  • type 2 diabetes atypical forms of diabetes (such as LADA and MODY)
  • glucose intolerance insulin resistance
  • metabolic syndrome dyslipidemia
  • hypercholesterolemia high blood pressure
  • obesity fatty liver conditions
  • diabetic nephropathy diabetic neuropathy
  • diabetic retinopathy cardiovascular disease
  • atherosclerosis cerebrovascular conditions
  • stroke cancer and
  • Bone morphogenetic protein type I receptor also known as Alk-3 signalling and certain BMPs, including BMP2 and BMP4, is/are involved in the control of glucose stimulated insulin secretion. It is known that BMPRIA signals to the nucleus via receptor regulated Smads. The resulting Smad complex is able to translocate into the nucleus where it regulates the expression of target genes (Massague et al, 2006).
  • ⁇ -cell dysfunction is characterised by abnormal glucose stimulated insulin secretion (GSIS), and in particular a reduction thereof.
  • GSIS glucose stimulated insulin secretion
  • any condition associated with reduced levels or dysfunctional key intrinsic factors in the ⁇ -cell could benefit from medicaments described herein.
  • the treatment of conditions where insufficient plasma levels of correctly processed insulin is present, i.e. hypoinsulinemia, is included within the scope of the invention.
  • Dysfunction of ⁇ -cells may also be associated with abnormal levels or dysfunction of key intrinsic factors in the ⁇ -cell.
  • the medicament of the invention may restore normal levels and/or function of one or more intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP- Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25, Hifla and Evi-1.
  • one or more intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP- Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25, Hifla and Evi-1.
  • the medicament of the invention increases expression and/or function of one or more (and most preferably all) intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25 and Hifla. Additionally, or alternatively, the medicament of the invention may decrease expression and/or function of Evi-1.
  • insulin and proinsulin plasma levels, C-peptide levels in blood and/or GSIS may also be used as markers of ⁇ -cell function and/or dysfunction.
  • the invention provides the use of a bone morphogenetic protein (BMP) or a fragment, variant, fusion or derivative thereof, in the preparation of a medicament for treating ⁇ -cell dysfunction associated with a condition selected from the group consisting of type 2 diabetes, atypical forms of diabetes (such as LADA and MODY), glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions, stroke, cancer and infectious diseases (such as tuberculosis).
  • BMP bone morphogenetic protein
  • the medicament is for treating ⁇ -cell dysfunction associated with type 2 diabetes and/or hypoinsulinemia.
  • the medicament is for treating transplanted ⁇ -cells, for example as a treatment for diabetes.
  • the medicament enhances glucose stimulated insulin secretion from ⁇ -cells.
  • the medicament enhances BMPRIa signalling in ⁇ -cells.
  • the medicament may function either as a BMPRIa agonist, to increase expression of Bmp2 and/or Bmp4, or to increase expression of Bmprla.
  • BMPRIa signalling we include signal transduction through the BMPRIa receptor resulting in improved GSIS, proinsulin processing and/or insulin secretion in ⁇ -cells.
  • the medicament modulates the level and/or function of one or more intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, H2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, Calpainl 0, Snap-25, Hifl a and Evi-1.
  • one or more intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, H2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, Calpainl 0, Snap-25, Hifl a and Evi-1.
  • Intrinsic factors we include the cellular machinery, and specifically proteins acting upstream and/or downstream of the BMPRIa receptor, which mediate BMPRIa signalling and insulin secretion in ⁇ cells.
  • the medicament improves (restores) the expression and/or activity of the intrinsic factors to levels found in normal, healthy ⁇ -cells.
  • the medicament preferably increases expression and/or function (activity) of one or more (and most preferably all) intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25 and Hifla.
  • the medicament may decrease expression and/or function of Evi-1.
  • the BMP may be derived from a number of different sources.
  • the BMP may be a mammalian BMP, such as a human BMP.
  • the medicament comprises or consists of BMP4, or a fragment, variant, fusion or derivative thereof.
  • BMP4 proteins are disclosed in Sequence Database Accession Numbers BAA06410, AAC72278, AAX43389, NP_570912, NP_570911 and NP_570911.
  • the BMP4 comprises or consists of the amino acid sequence of SEQ ID NO:1.
  • the medicament comprises or consists of BMP2, or a fragment, variant, fusion or derivative thereof.
  • BMP2 proteins are disclosed in Sequence Database Accession Numbers AAF21646 and NP 001191.
  • the BMP2 comprises or consists of the amino acid sequence of SEQ ID NO:2.
  • the medicament may comprise or consist of a naturally occurring, full length BMP 2 or 4, as described above.
  • 'amino acid' as used herein includes the standard twenty genetically- encoded amino acids and their corresponding stereoisomers in the 'D' form (as compared to the natural 'L' form), omega-amino acids and other naturally- occurring amino acids, unconventional amino acids (e.g. ⁇ , ⁇ -disubstituted amino acids, N-alkyl amino acids, etc.) and chemically derivatised amino acids (see below).
  • each encoded amino acid residue is represented by a single letter designation, corresponding to the trivial name of the conventional amino acid.
  • the, polypeptides of the invention for use in medicine comprise or consist of L-amino acids.
  • the first aspect of the invention encompasses the use in medicine of fragments, variants, fusions and derivatives of the defined BMP polypeptides, as well as fusions of said fragments, variants or derivatives, provided such fragments, variants, fusions and derivatives retain the ability of full length BMP protein to treat ⁇ -cell dysfunction.
  • the fragment, variant, fusion or derivative retains one or more of the following activities of BMP2 and/or 4:
  • BMPRIa signalling can be improved by a functional fragment or synthetic peptide sequence variant with partial functional activity consisting of at least 25%, 35%, 45%, 55%, 65%, 75%, 85%, 95%, 96%, 97%, 98% or 99% functional activity as compared with fully active human mature BMP2 or 4 (as measured in a functional assay).
  • a functional assay may be the use of embryonic mouse cells stable transfected with an expression construct containing a BMP -responsive element fused to the firefly luciferase reporter gene. The expression construct could result from a multirnerisation of DNA sequence elements from the mouse IdI promoter. Test molecules are contacted with the cell and luciferase activity in cell lysates is measured (Logeart-
  • Another example is intraperitoneal glucose tolerance test on fed C57BL76 mice after an acute intraperitoneal administration of test peptide and measuring insulin concentrations.
  • fragment we include polypeptides which comprise or consist of at least 10 contiguous amino acids from the full length BMP, for example at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250, 260, 270, 280, 290, 300, 310, 320, 330, 340 or 350 contiguous amino acids.
  • Variants may be made using the methods of protein engineering and site- directed mutagenesis well known in the art (see example, see Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press, the relevant disclosures in which document are hereby incorporated by reference).
  • polypeptide fused to any other polypeptide we include a polypeptide fused to any other polypeptide.
  • the said polypeptide may be fused to a polypeptide such as glutathione-S-transferase (GST) or protein A in order to facilitate purification of said polypeptide. Examples of such fusions are well known to those skilled in the art.
  • the said polypeptide may be fused to an oligo- histidine tag such as His6 or to an epitope recognised by an antibody such as the well-known Myc tag epitope. Fusions to any variant or derivative of said polypeptide are also included in the scope of the invention. It will be appreciated that fusions (or variants or derivatives thereof) which retain desirable properties, namely the ability to improve ⁇ -cell function are preferred. It is also particularly preferred if the fusions are ones which are suitable for use in the methods described herein.
  • the fusion may comprise a further portion which confers a desirable feature on the said polypeptide of the invention; for example, the portion may be useful in detecting or isolating the polypeptide, or promoting cellular uptake of the polypeptide.
  • the portion may be, for example, a biotin moiety, a radioactive moiety, a fluorescent moiety, for example a small fluorophore or a green fluorescent protein (GFP) fluorophore, as well known to those skilled in the art.
  • GFP green fluorescent protein
  • the moiety may be an immunogenic tag, for example a Myc tag, as known to those skilled in the art or may be a lipophilic molecule or polypeptide domain that is capable of promoting cellular uptake of the polypeptide, as known to those skilled in the art.
  • the fusion protein comprises a polypeptide selected from the group consisting of albumin and the Fc portion of an IgG molecule, and fragments thereof.
  • a polypeptide selected from the group consisting of albumin and the Fc portion of an IgG molecule, and fragments thereof.
  • variant BMP may a non-naturally occurring variant, for example a variant of human BMP2 and/or BMP4.
  • the variant BMP is a chimaeric BMP, i.e. comprising regions from more than one BMP.
  • the chimaeric BMP may comprise amino acid sequences derived from BMP2 and/or BMP4.
  • polypeptide variant has an amino acid sequence which has at least 45% identity with naturally occurring BMP, or a fragment thereof, for example at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 95%, 96%, 97%, 98%, and most preferably at least 99% identity with one or more of the amino acid sequences specified above.
  • the percent sequence identity between two polypeptides may be determined using suitable computer programs, for example the GAP program of the University of Wisconsin Genetic Computing Group, and it will be appreciated that percent identity is calculated in relation to polypeptides whose sequences have been aligned optimally.
  • the alignment may alternatively be carried out using the Clustal W program (as described in Thompson et al, 1994, Nuc. Acid Res. 22:4673-4680, the relevant disclosures in which document are hereby incorporated by reference).
  • the parameters used may be as follows:
  • Fast pairwise alignment parameters K-tuple(word) size; 1, window size; 5, gap penalty; 3, number of top diagonals; 5. Scoring method: x percent. Multiple alignment parameters: gap open penalty; 10, gap extension penalty; 0.05.
  • the BESTFIT program may be used to determine local sequence alignments.
  • BMP or fragment, variant, fusion or derivative thereof, for use in the first aspect of the invention may comprise one or more amino acids which have been modified or derivatised.
  • Chemical derivatives of one or more amino acids may be achieved by reaction with a functional side group.
  • derivatised molecules include, for example, those molecules in which free amino groups have been derivatised to form amine hydrochlorides, p-toluene sulphonyl groups, carboxybenzoxy groups, t- butyloxycarbonyl groups, chloroacetyl groups or formyl groups.
  • Free carboxyl groups may be derivatised to form salts, methyl and ethyl esters or other types of esters and hydrazides.
  • Free hydroxyl groups may be derivatised to form O-acyl or O-alkyl derivatives.
  • Also included as chemical derivatives are those peptides which contain naturally occurring amino acid derivatives of the twenty standard amino acids.
  • 4-hydroxyproline may be substituted for proline
  • 5- hydroxylysine may be substituted for lysine
  • 3-methylhistidine may be substituted for histidine
  • homoserine may be substituted for serine and ornithine for lysine.
  • Derivatives also include peptides containing one or more additions or deletions as long as the requisite activity is maintained.
  • Other included modifications are amidation, amino terminal acylation ⁇ e.g. acetylation or thioglycolic acid amidation), terminal carboxylamidation ⁇ e.g. with ammonia or methylamine), and the like terminal modifications.
  • the present invention provides polypeptides that are linked to polymers which provide increased stability and/or half-life.
  • polymer molecules ⁇ e.g. polyethylene glycol; PEG
  • PEG polyethylene glycol
  • PEG modification of proteins has been shown to alter the in vivo circulating half-life, antigenicity, solubility, and resistance to proteolysis of the protein (Abuchowski et al, J. Biol. Chem. 1977, 252:3578; Nucci et al, Adv. Drug Delivery Reviews 1991, 6:133; Francis et al., Pharmaceutical Biotechnology Vol. 3 (Borchardt, R. T. ed.); and Stability of Protein Pharmaceuticals: in vivo Pathways of Degradation and Strategies for Protein Stabilization, 1991, ⁇ p235-263, Plenum, NY).
  • Attachment of a PEG polymer to an amino acid residue of a polypeptide may be achieved using several PEG attachment moieties including, but not limited to N- hydroxylsuccinimide (NHS) active ester, succinimidyl propionate (SPA), maleimide (MAL), vinyl sulfone (VS), or thiol.
  • a PEG polymer, or other polymer can be linked to a polypeptide at either a predetermined position, or may be randomly linked to the a polypeptide molecule. It is preferred, however, that the PEG polymer be linked to a polypeptide at a predetermined position.
  • a PEG polymer may be linked to any residue in the a polypeptide, however, it is preferable that the polymer is linked to either a lysine or cysteine, which is either naturally occurring in the polypeptide, or which has been engineered into the polypeptide, for example, by mutagenesis of a naturally occurring residue in the polypeptide to either a cysteine or lysine.
  • PEG-linkage can also be mediated through a peptide linker attached to a polypeptide. That is, the PEG moiety can be attached to a peptide linker fused to a polypeptide, where the linker provides the site, e.g. a free cysteine or lysine, for PEG attachment.
  • polymer refers to a macromolecule made up of repeating monomelic units, and can refer to a synthetic or naturally occurring polymer such as an optionally substituted straight or branched chain polyalkylene, polyalkenylene, or polyoxyalkylene polymer or a branched or unbranched polysaccharide.
  • PEG polyethylene glycol
  • PEG polymer refers to polyethylene glycol, and more specifically can refer to a derivitized form of PEG, including, but not limited to N-hydroxylsuccinimide (NHS) active esters of PEG such as succinimidyl propionate, benzotriazole active esters, PEG derivatized with maleimide, vinyl sulfones, or thiol groups.
  • NHS N-hydroxylsuccinimide
  • Particular PEG formulations can include PEG-O- CH 2 CH 2 CH 2 -CO 2 -NHS; PEG-O-CH 2 -NHS; PEG-O-CH 2 CH 2 -CO 2 -NHS; PEG- S-CH 2 CH 2 -CO-NHS; PEG-O 2 CNH-CH(R)-CO 2 -NHS; PEG-NHCO-CH 2 CH 2 - CO-NHS; and PEG-O-CH 2 -CO 2 -NHS; where R is (CH 2 ) 4 )NHCO 2 (mPEG).
  • PEG polymers useful in the invention may be linear molecules, or may be branched wherein multiple PEG moieties are present in a single polymer.
  • a "sulfhydryl-selective reagent” is a reagent which is useful for the attachment of a PEG polymer to a thiol-containing amino acid. Thiol groups on the amino acid residue cysteine are particularly useful for interaction with a sulfhydryl- selective reagent. Sulfhydryl-selective reagents which are useful for such attachment include, but are not limited to maleimide, vinyl sulfone, and thiol. The use of sulfhydryl-selective reagents for coupling to cysteine residues is known in the art and may be adapted as needed according to the present invention (for example, see Zalipsky, 1995, Bioconjug. Chem. 6:150; Greenwald et al, 2000, Crit. Rev. Then Drug Carrier Syst. 17:101; Herman et ah, 1994, Macromol. Chem. Phys. 195:203).
  • PEG or another agent e.g. HSA
  • "retains activity” refers to a level of activity of a PEG-linked polypeptide which is at least 10% of the level of activity of a non-PEG-linked polypeptide, preferably at least 20%, 30%, 40%, 50%, 60%, 70%, 80% and up to 90%, preferably up to 95%, 98%, and up to 100% of the activity of a non-PEG-linked polypeptide comprising the same antigen-binding domain or domains. More specifically, the activity of a PEG-linked polypeptide compared to a non-PEG linked polypeptide should be determined on an polypeptide molar basis; that is equivalent numbers of moles of each of the PEG-linked and non-PEG-linked polypeptides should be used in each trial. In determining whether a particular PEG-linked polypeptide "retains activity", it is preferred that the activity of a PEG-linked polypeptide be compared with the activity of the same polypeptide in the absence of PEG.
  • in vivo half-life refers to the time taken for the serum concentration of a polypeptide, fusion or derivative of the invention to reduce by
  • the anti polypeptides described herein can be stabilized in vivo and their half-life increased by binding to molecules, such as PEG, which resist degradation and/or clearance or sequestration.
  • the half-life of a polypeptide is increased if its functional activity persists, in vivo, for a longer period than a similar polypeptide which is not linked to a PEG polymer.
  • the half-life of a polypeptide is increased if its functional activity persists, in vivo, for a longer period than a similar polypeptide which is not linked to a PEG polymer.
  • PEGylated polypeptide is increased by 10%, 20%, 30%, 40%, 50% or more relative to a non-PEGylated polypeptide. Increases in the range of 2x, 3x, 4x, 5x, 10x, 2Ox, 30x, 4Ox, 50x or more of the half life are possible. Alternatively, or in addition, increases in the range of up to 30x, 4Ox, 50x, 6Ox, 7Ox, 8Ox, 9Ox,
  • peptidomimetic compounds may also be useful.
  • 'polypeptide' we include peptidomimetic compounds which are capable of treating ⁇ -cell dysfunction.
  • the term 'peptidomimetic' refers to a compound that mimics the conformation and desirable features of a particular peptide as a therapeutic agent.
  • the polypeptides of the invention include not only molecules in which amino acid residues are joined by peptide (-CO-NH-) linkages but also molecules in which the peptide bond is reversed.
  • retro-inverso peptidomimetics may be made using methods known in the art, for example such as those described in Meziere et al. (1997) J Immunol. 159, 3230-3237, the relevant disclosures in which document are hereby incorporated by reference. This approach involves making pseudopeptides containing changes involving the backbone, and not the orientation of side chains.
  • Retro-inverse peptides which contain NH-CO bonds instead of CO-NH peptide bonds, are much more resistant to proteolysis.
  • the polypeptide of the invention may be a peptidomimetic compound wherein one or more of the amino acid residues are linked by a -y(CH 2 NH)- bond in place of the conventional amide linkage.
  • the peptide bond may be dispensed with altogether provided that an appropriate linker moiety which retains the spacing between the carbon atoms of the amino acid residues is used; it is particularly preferred if the linker moiety has substantially the same charge distribution and substantially the same planarity as a peptide bond.
  • polypeptide may conveniently be blocked at its N- or C-terminus so as to help reduce susceptibility to exoproteolytic digestion.
  • a presumed bioactive conformation may be stabilised by a covalent modification, such as cyclisation or by incorporation of lactam or other types of bridges, for example see Veber et ah, 1978, Proc. Natl. Acad. ScL USA 75:2636 and Thursell et ah, 1983, Biochem. Biophys. Res. Comm. 111:166, the relevant disclosures in which documents are hereby incorporated by reference.
  • a common theme among many of the synthetic strategies has been the introduction of some cyclic moiety into a peptide-based framework.
  • the cyclic moiety restricts the conformational space of the peptide structure and this frequently results in an increased affinity of the peptide for a particular biological receptor.
  • An added advantage of this strategy is that the introduction of a cyclic moiety into a peptide may also result in the peptide having a diminished sensitivity to cellular peptidases.
  • preferred polypeptides comprise terminal cysteine amino acids.
  • Such a polypeptide may exist in a heterodetic cyclic form by disulphide bond formation of the mercaptide groups in the terminal cysteine amino acids or in a homodetic form by amide peptide bond formation between the terminal amino acids.
  • cyclising small peptides through disulphide or amide bonds between the N- and C-terminus cysteines may circumvent problems of affinity and half-life sometime observed with linear peptides, by decreasing proteolysis and also increasing the rigidity of the structure, which may yield higher affinity compounds.
  • Polypeptides cyclised by disulphide bonds have free amino and carboxy-termini which still may be susceptible to proteolytic degradation, while peptides cyclised by formation of an amide bond between the N-terminal amine and C-terminal carboxyl and hence no longer contain free amino or carboxy termini.
  • the peptides of the present invention can be linked either by a C- N linkage or a disulphide linkage.
  • heterodetic linkages may include, but are not limited to formation via disulphide, alkylene or sulphide bridges.
  • Methods of synthesis of cyclic homodetic peptides and cyclic heterodetic peptides, including disulphide, sulphide and alkylene bridges, are disclosed in US 5,643,872.
  • Other examples of cyclisation methods are discussed and disclosed in US 6,008,058, the relevant disclosures in which documents are hereby incorporated by reference.
  • RCM ring-closing metathesis
  • terminal modifications are useful, as is well known, to reduce susceptibility by proteinase digestion and therefore to prolong the half-life of the peptides in solutions, particularly in biological fluids where proteases may be present.
  • Polypeptide cyclisation is also a useful modification and is preferred because of the stable structures formed by cyclisation and in view of the biological activities observed for cyclic peptides.
  • the BMP, or fragment, variant, fusion or derivative thereof is cyclic.
  • the BMP, or fragment, variant, fusion or derivative thereof is linear.
  • the present invention also includes the use of medicaments comprising pharmaceutically acceptable acid or base addition salts of the above described BMP polypeptides.
  • the acids which are used to prepare the pharmaceutically acceptable acid addition salts of the aforementioned base compounds useful in this invention are those which form non-toxic acid addition salts, i.e.
  • salts containing pharmacologically acceptable anions such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate, fumarate, gluconate, saccharate, benzoate, methanesulphonate, ethanesulphonate, benzenesulphonate, /j-toluenesulphonate and pamoate [i.e. l,l'-methylene-bis-(2-hydroxy-3 naphthoate)] salts, among others.
  • pharmacologically acceptable anions such as the hydrochloride, hydrobromide, hydroiodide, nitrate, sulphate, bisulphate, phosphate, acid phosphate, acetate, lactate, citrate, acid citrate, tartrate, bitartrate, succinate, maleate,
  • Pharmaceutically acceptable base addition salts may also be used to produce pharmaceutically acceptable salt forms of the BMP polypeptides.
  • the chemical bases that may be used as reagents to prepare pharmaceutically acceptable base salts of the present compounds that are acidic in nature are those that form non-toxic base salts with such compounds.
  • Such non-toxic base salts include, but are not limited to those derived from such pharmacologically acceptable cations such as alkali metal cations (e.g. potassium and sodium) and alkaline earth metal cations (e.g. calcium and magnesium), ammonium or water- soluble amine addition salts such as N-methylglucamine-(meglumine), and the lower alkanolammonium and other base salts of pharmaceutically acceptable organic amines, among others.
  • the BMP, or fragment, variant, fusion or derivative thereof may be lyophilised for storage and reconstituted in a suitable carrier prior to use.
  • Any suitable lyophilisation method e.g. spray drying, cake drying
  • reconstitution techniques can be employed. It will be appreciated by those skilled in the art that lyophilisation and reconstitution can lead to varying degrees of activity loss and that use levels may have to be adjusted upward to compensate.
  • the lyophilised (freeze dried) polypeptide loses no more than about 20%, or no more than about 25%, or no more than about 30%, or no more than about 35%, or no more than about 40%, or no more than about 45%, or no more than about 50% of its activity (prior to lyophilisation) when rehydrated.
  • the BMP, or fragment, variant, fusion or derivative thereof may exist in monomeric form or in the form of a homo-or hetero-multimer thereof (e.g. dimer, trimer, tetramer, pentamer, etc.).
  • active BMP proteins can consist of a dimer of two identical proteins or a heterodimer of two related bone morphogenetic proteins.
  • BMP4 forms heterodimers. Therefore, administrations of mixtures of BMPs, or fragments, variants, fusions or derivatives thereof, may lead to improved BMPRIa signalling.
  • BMP polypeptides or fragment, variant, fusion or derivative thereof, for use in the first aspect of the invention are well known in the art.
  • the BMP is a recombinant polypeptide.
  • nucleic acid molecule or polynucleotide encoding the BMP polypeptide, or fragment, variant, fusion or derivative thereof, may be expressed in a suitable host and the polypeptide obtained therefrom.
  • suitable methods for the production of such recombinant polypeptides are well known in the art (for example, see Sambrook & Russell, 2001, Molecular Cloning, A Laboratory
  • expression vectors may be constructed comprising a nucleic acid molecule which is capable, in an appropriate host, of expressing the BMP polypeptide encoded by the nucleic acid molecule.
  • nucleic acid molecules especially DNA
  • vectors for example, via complementary cohesive termini.
  • complementary homopolymer tracts can be added to the DNA segment to be inserted into the vector DNA.
  • the vector and DNA segment are then joined by hydrogen bonding between the complementary homopolymeric tails to form recombinant DNA molecules.
  • Synthetic linkers containing one or more restriction sites provide an alternative method of joining the DNA segment to vectors.
  • the DNA segment e.g. generated by endonuclease restriction digestion, is treated with bacteriophage T4 DNA polymerase or E. coli DNA polymerase I, enzymes that remove protruding, 3 '-single-stranded termini with their 3'-5'-exonucleolytic activities, and fill in recessed 3 '-ends with their polymerising activities.
  • the combination of these activities therefore generates blunt-ended DNA segments.
  • the blunt-ended segments are then incubated with a larger molar excess of linker molecules in the presence of an enzyme that is able to catalyse the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • an enzyme that is able to catalyse the ligation of blunt-ended DNA molecules, such as bacteriophage T4 DNA ligase.
  • the products of the reaction are DNA segments carrying polymeric linker sequences at their ends.
  • These DNA segments are then cleaved with the appropriate restriction enzyme and ligated to an expression vector that has been cleaved with an enzyme that produces termini compatible with those of the DNA segment.
  • Synthetic linkers containing a variety of restriction endonuclease site are commercially available from a number of sources including International Biotechnologies Inc., New Haven, CN, USA.
  • a desirable way to modify the DNA encoding the polypeptide of the invention is to use PCR.
  • This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.
  • the DNA to be enzymatically amplified is flanked by two specific primers which themselves become incorporated into the amplified DNA.
  • the said specific primers may contain restriction endonuclease recognition sites which can be used for cloning into expression vectors using methods known in the art.
  • the DNA (or in the case of retroviral vectors, RNA) is then expressed in a suitable host to produce a polypeptide comprising the compound of the invention or binding moiety thereof.
  • the DNA encoding the BMP polypeptide may be used in accordance with known techniques, appropriately modified in view of the teachings contained herein, to construct an expression vector, which is then used to transform an appropriate host cell for the expression and production of the compound of the invention or binding moiety thereof.
  • Such techniques include those disclosed in US Patent Nos.
  • the DNA (or in the case or retroviral vectors, RNA) encoding the BMP polypeptide may be joined to a wide variety of other DNA sequences for introduction into an appropriate host.
  • the companion DNA will depend upon the nature of the host, the manner of the introduction of the DNA into the host, and whether episomal maintenance or integration is desired.
  • DNA may be linked to the appropriate transcriptional and translational regulatory control nucleotide sequences recognised by the desired host, although such controls are generally available in the expression vector.
  • the vector is then introduced into the host through standard techniques. Generally, not all of the hosts will be transformed by the vector. Therefore, it will be necessary to select for transformed host cells.
  • One selection technique involves incorporating into the expression vector a DNA sequence, with any necessary control elements, that codes for a selectable trait in the transformed cell, such as antibiotic resistance. Alternatively, the gene for such selectable trait can be on another vector, which is used to co-transform the desired host cell.
  • Host cells that have been transformed by the expression vector are then cultured for a sufficient time and under appropriate conditions known to those skilled in the art in view of the teachings disclosed herein to permit the expression of the polypeptide, which can then be recovered.
  • bacteria for example, E. coli and Bacillus subtilis
  • yeasts for example Saccharomyces cerevisiae
  • filamentous fungi for example Aspergillus
  • plant cells animal cells and insect cells.
  • the vectors typically include a prokaryotic replicon, such as the CoIEl on, for propagation in a prokaryote, even if the vector is to be used for expression in other, non-prokaryotic, cell types.
  • the vectors can also include an appropriate promoter such as a prokaryotic promoter capable of directing the expression (transcription and translation) of the genes in a bacterial host cell, such as E. coli, transformed therewith.
  • a promoter is an expression control element formed by a DNA sequence that permits binding of RNA polymerase and transcription to occur.
  • Promoter sequences compatible with exemplary bacterial hosts are typically provided in plasmid vectors containing convenient restriction sites for insertion of a DNA segment.
  • Typical prokaryotic vector plasmids are pUC18, pUC19, pBR322 and pBR329 available from Biorad Laboratories, (Richmond, CA, USA) and pTrc99A and pKK223-3 available from Pharmacia, Piscataway, NJ, USA.
  • a typical mammalian cell vector plasmid is pSVL available from Pharmacia, Piscataway, NJ, USA. This vector uses the SV40 late promoter to drive expression of cloned genes, the highest level of expression being found in T antigen-producing cells, such as COS-I cells.
  • an inducible mammalian expression vector is pMSG, also available from Pharmacia.
  • This vector uses the glucocorticoid-inducible promoter of the mouse mammary tumour virus long terminal repeat to drive expression of the cloned gene.
  • Useful yeast plasmid vectors are pRS403-406 and pRS413-416 and are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Plasmids pRS403, ⁇ RS404, pRS405 and pRS406 are Yeast Integrating plasmids (Yips) and incorporate the yeast selectable markers HIS3, TRPl, LEU2 and URA3.
  • Plasmids pRS413-416 are Yeast Centromere plasmids (Ycps).
  • vectors and expression systems are well known in the art for use with a variety of host cells.
  • the host cell can be either prokaryotic or eukaryotic.
  • Bacterial cells are preferred prokaryotic host cells and typically are a strain of E. coli such as, for example, the E. coli strains DH5 available from Bethesda Research Laboratories Inc., Bethesda, MD, USA, and RRl available from the American Type Culture Collection (ATCC) of Rockville, MD, USA (No. ATCC 31343).
  • Preferred eukaryotic host cells include yeast, insect and mammalian cells, preferably vertebrate cells such as those from a mouse, rat, monkey or human fibroblastic and kidney cell lines.
  • Yeast host cells include YPH499, YPH500 and YPH501 which are generally available from Stratagene Cloning Systems, La Jolla, CA 92037, USA.
  • Preferred mammalian host cells include Chinese hamster ovary (CHO) cells available from the ATCC as CRL 1658 and 293 cells which are human embryonic kidney cells.
  • Preferred insect cells are Sf9 cells which can be transfected with baculovirus expression vectors.
  • Transformation of appropriate cell hosts with a DNA construct is accomplished by well known methods that typically depend on the type of vector used. With regard to transformation of prokaryotic host cells, see, for example, Cohen et al. (1972) Proc. Natl. Acad. Sd. USA 69, 2110 and Molecular Cloning: a Laboratory Manual, 3rd edition, Sambrook & Russell, 2001, Cold Spring Harbor Laboratory Press. Transformation of yeast cells is described in Sherman et al (1986) Methods In Yeast Genetics, A Laboratory Manual, Cold Spring Harbor, NY. The method of Beggs (1978) Nature 275, 104-109 is also useful.
  • reagents useful in transfecting such cells for example calcium phosphate and D ⁇ A ⁇ -dextran or liposome formulations, are available from Stratagene Cloning Systems, or Life Technologies Inc., Gaithersburg, MD 20877, USA. The relevant disclosures in the above documents are hereby incorporated by reference.
  • Electroporation is also useful for transforming and/or transfecting cells and is well known in the art for transforming yeast cells, bacterial cells, insect cells and vertebrate cells.
  • bacterial species may be transformed by the methods described in Luchansky et al (1988) MoI. Microbiol. 2, 637-646, the relevant disclosures in which document are hereby incorporated by reference.
  • the greatest number of transformants is consistently recovered following electroporation of the DNA-cell mixture suspended in 2.5 PEB using 6250V per cm at 25 ⁇ FD.
  • Successfully transformed cells i.e. cells that contain a DNA construct encoding a BMP polypeptide
  • cells resulting from the introduction of an expression construct of the present invention can be grown to produce the polypeptide of the invention.
  • Cells can be harvested and lysed and their DNA content examined for the presence of the DNA using a method such as that described by Southern (1975) J MoI. Biol. 98, 503 or Berent et al (1985) Biotech. 3, 208, the relevant disclosures in which document are hereby incorporated by reference.
  • the presence of the protein in the supernatant can be detected using antibodies as described below.
  • Samples of cells suspected of being transformed are harvested and assayed for the protein using suitable antibodies.
  • the host cell may be a host cell within a non-human animal body.
  • transgenic non-human animals which express a BMP polypeptide by virtue of the presence of the transgene are included.
  • the transgenic non- human animal is a rodent such as a mouse.
  • Transgenic non-human animals can be made using methods well known in the art (see below).
  • polypeptides of the invention are produced in a eukaryotic system, such as a mammalian cell.
  • BMP polypeptides can also be produced in vitro using a commercially available in vitro translation system, such as rabbit reticulocyte lysate or wheatgerm lysate
  • the translation system is rabbit reticulocyte lysate.
  • the translation system may be coupled to a transcription system, such as the TNT transcription-translation system
  • a pharmaceutical composition comprising a BMP, or fragment, variant, fusion or derivative thereof, and a pharmaceutically acceptable excipient, diluent or carrier.
  • composition means a therapeutically effective formulation for use in the methods of the invention.
  • a 'therapeutically effective amount', or 'effective amount', or 'therapeutically effective', as used herein, refers to that amount which provides a therapeutic effect for a given condition and administration regimen. This is a predetermined quantity of active material calculated to produce a desired therapeutic effect in association with the required additive and diluent, i.e. a carrier or administration vehicle. Further, it is intended to mean an amount sufficient to reduce and most preferably prevent, a clinically significant deficit in the activity, function and response of the host. Alternatively, a therapeutically effective amount is sufficient to cause an improvement in a clinically significant condition in a host. As is appreciated by those skilled in the art, the amount of a compound may vary depending on its specific activity.
  • Suitable dosage amounts may contain a predetermined quantity of active composition calculated to produce the desired therapeutic effect in association with the required diluent.
  • a therapeutically effective amount of the active component is provided.
  • a therapeutically effective amount can be determined by the ordinary skilled medical or veterinary worker based on patient characteristics, such as age, weight, sex, condition, complications, other diseases, etc., as is well known in the art.
  • the BMP polypeptides can be formulated at various concentrations, depending on the efficacy/toxicity of the compound being used.
  • the formulation comprises the active agent at a concentration of between 0.1 ⁇ M and 1 mM, more preferably between 1 ⁇ M and 100 ⁇ M, between 5 ⁇ M and 50 ⁇ M, between 10 ⁇ M and 50 ⁇ M, between 20 ⁇ M and 40 ⁇ M and most preferably about 30 ⁇ M.
  • formulations may comprise a lower concentration of a BMP polypeptide, for example between 0.0025 ⁇ M and 1 ⁇ M.
  • the pharmaceutical formulation may comprise an amount of a BMP, or fragment, variant, fusion or derivative thereof, sufficient to treat ⁇ -cell dysfunction.
  • the medicaments generally be administered in admixture with a suitable pharmaceutical excipient diluent or carrier selected with regard to the intended route of administration and standard pharmaceutical practice (for example, see Remington: The Science and Practice of Pharmacy, 19 th edition, 1995, Ed. Alfonso Gennaro, Mack Publishing Company, Pennsylvania, USA, the relevant disclosures in which document are hereby incorporated by reference).
  • the medicaments and agents can be administered orally, buccally or sublingually in the form of tablets, capsules, ovules, elixirs, solutions or suspensions, which may contain flavouring or colouring agents, for immediate-, delayed- or controlled-release applications.
  • the medicaments and agents may also be administered via intracavernosal injection.
  • Such tablets may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine, disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxyl- propylmethylcellulose (HPMC), hydroxy-propylcellulose (HPC), sucrose, gelatin and acacia. Additionally, lubricating agents such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.
  • excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, dibasic calcium phosphate and glycine
  • disintegrants such as starch (preferably corn, potato or tapioca starch), sodium starch glycollate, croscarmellose sodium and certain complex silicates,
  • Solid compositions of a similar type may also be employed as fillers in gelatin capsules.
  • Preferred excipients in this regard include lactose, starch, cellulose, milk sugar or high molecular weight polyethylene glycols.
  • the compounds of the invention may be combined with various sweetening or flavouring agents, colouring matter or dyes, with emulsifying and/or suspending agents and with diluents such as water, ethanol, propylene glycol and glycerin, and combinations thereof.
  • the medicaments can also be administered parenterally, for example, intravenously, intra-articularly, intra-arterially, intraperitoneally, intra-thecally, intraventricularly, intrasternally, intracranially, intra-muscularly or subcutaneously, or they may be administered by infusion techniques. They are best used in the form of a sterile aqueous solution which may contain other substances, for example, enough salts or glucose to make the solution isotonic with blood.
  • the aqueous solutions should be suitably buffered (preferably to a pH of from 3 to 9), if necessary.
  • the preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.
  • Formulations suitable for parenteral administration include aqueous and nonaqueous sterile injection solutions which may contain anti-oxidants, buffers, bacteriostats and solutes which render the formulation isotonic with the blood of the intended recipient; and aqueous and non-aqueous sterile suspensions which may include suspending agents and thickening agents.
  • the formulations may be presented in unit-dose or multi-dose containers, for example sealed ampoules and vials, and may be stored in a freeze-dried (lyophilised) condition requiring only the addition of the sterile liquid carrier, for example water for injections, immediately prior to use.
  • Extemporaneous injection solutions and suspensions may be prepared from sterile powders, granules and tablets of the kind previously described.
  • the daily dosage level of the medicaments will usually be from 1 to 1000 mg per adult ⁇ i.e. from about 0.015 to 15 mg/kg), administered in single or divided doses.
  • the medicaments can also be administered intranasally or by inhalation and are conveniently delivered in the form of a dry powder inhaler or an aerosol spray presentation from a pressurised container, pump, spray or nebuliser with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorof ⁇ uoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2- tetrafluoroethane (HFA 134A3 or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA3), carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorof ⁇ uoro-methane, dichlorotetrafluoro-ethane, a hydrofluoroalkane such as 1,1,1,2- tetrafluoroethane (HFA
  • the dosage unit may be determined by providing a valve to deliver a metered amount.
  • the pressurised container, pump, spray or nebuliser may contain a solution or suspension of the active compound, e.g. using a mixture of ethanol and the propellant as the solvent, which may additionally contain a lubricant, e.g. sorbitan trioleate.
  • a lubricant e.g. sorbitan trioleate.
  • Capsules and cartridges (made, for example, from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.
  • Aerosol or dry powder formulations are preferably arranged so that each metered dose or 'puff contains at least 1 mg of a compound of the invention for delivery to the patient. It will be appreciated that the overall daily dose with an aerosol will vary from patient to patient, and may be administered in a single dose or, more usually, in divided doses throughout the day.
  • the medicaments can be administered in the form of a suppository or pessary, or they may be applied topically in the form of a lotion, solution, cream, ointment or dusting powder.
  • the compounds of the invention may also be transdermally administered, for example, by the use of a skin patch. They may also be administered by the ocular route.
  • the medicaments and agents can be formulated as a suitable ointment containing the active compound suspended or dissolved in, for example, a mixture with one or more of the following: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water.
  • they can be formulated as a suitable lotion or cream, suspended or dissolved in, for example, a mixture of one or more of the following: mineral oil, sorbitan monostearate, a polyethylene glycol, liquid paraffin, polysorbate 60, ceryl esters wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water.
  • Formulations suitable for topical administration in the mouth include lozenges comprising the active ingredient in a flavoured basis, usually sucrose and acacia or tragacanth; pastilles comprising the active ingredient in an inert basis such as gelatin and glycerin, or sucrose and acacia; and mouth-washes comprising the active ingredient in a suitable liquid carrier.
  • a sustained-release drug delivery system such as a microsphere. These are designed specifically to reduce the frequency of injections.
  • a sustained-release drug delivery system such as a microsphere.
  • Nutropin Depot which encapsulates recombinant human growth hormone (rhGH) in biodegradable microspheres that, once injected, release rhGH slowly over a sustained period.
  • Sustained-release polypeptide compositions also include liposomally entrapped polypeptides.
  • Liposomes containing the polypeptides are prepared by methods known per se. See, for example Epstein et ah, Proc. Natl. Acad. ScL USA 82: 3688-92 (1985); Hwang et al, Proc. Natl. Acad. ScL USA 77: 4030-4 (1980); U.S. Patent Nos. 4,485,045; 4,544, 545; 6,139,869; and 6,027,726, the relevant disclosures in which documents are hereby incorporated by reference. Ordinarily, the liposomes are of the small (about 200 to about 800 Angstroms), unilamellar type in which the lipid content is greater than about 30 mole percent (mol. %) cholesterol; the selected proportion being adjusted for the optimal polypeptide therapy.
  • polypeptide medicaments and agents can be administered by a surgically implanted device that releases the drug directly to the required site.
  • Electroporation therapy (EPT) systems can also be employed for the administration of proteins and polypeptides.
  • EPT Electroporation therapy
  • a device which delivers a pulsed electric field to cells increases the permeability of the cell membranes to the drug, resulting in a significant enhancement of intracellular drug delivery.
  • Proteins and polypeptides can also be delivered by electroincorporation (EI).
  • EI occurs when small particles of up to 30 microns in diameter on the surface of the skin experience electrical pulses identical or similar to those used in electroporation. In EI, these particles are driven through the stratum corneum and into deeper layers of the skin. The particles can be loaded or coated with drugs or genes or can simply act as "bullets" that generate pores in the skin through which the drugs can enter.
  • ReGeI thermo-sensitive ReGeI injectable. Below body temperature, ReGeI is an injectable liquid while at body temperature it immediately forms a gel reservoir that slowly erodes and dissolves into known, safe, biodegradable polymers. The active drug is delivered over time as the biopolymers dissolve.
  • Protein and polypeptide pharmaceuticals can also be delivered orally.
  • One such system employs a natural process for oral uptake of vitamin B12 in the body to co-deliver proteins and polypeptides. By riding the vitamin B 12 uptake system, the protein or polypeptide can move through the intestinal wall.
  • Complexes are produced between vitamin B 12 analogues and the drug that retain both significant affinity for intrinsic factor (IF) in the vitamin B 12 portion of the complex and significant bioactivity of the drug portion of the complex.
  • IF intrinsic factor
  • a method of treatment of ⁇ -cell dysfunction comprises the administration of an effective amount of a bone morphogenetic protein (BMP) or a fragment, variant, fusion or derivative thereof, as defined above, to a patient in need of such treatment.
  • BMP bone morphogenetic protein
  • the method and use according to the invention increase human BMPRIa signalling in ⁇ -cells, thereby improving ⁇ -cell function to enable proper glucose stimulated insulin secretion (GSIS).
  • GSIS glucose stimulated insulin secretion
  • treatment include the therapeutic treatment of patients in need of, as well as the prophylactic treatment and/or diagnosis of patients which are susceptible to, ⁇ -cell dysfunction.
  • Patients include mammalian (including human) patients.
  • the term "effective amount” refers to an amount of a compound, which confers a therapeutic effect on the treated patient.
  • the effect may be objective (i.e. measurable by some test or marker) or subjective (i.e. the subject gives an indication of or feels an effect).
  • the method is for treating ⁇ -cell dysfunction associated with a condition selected from the group consisting of type 2 diabetes, atypical forms of diabetes (such as LADA and MODY), glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions, stroke, cancer and infectious diseases (such as tuberculosis).
  • a condition selected from the group consisting of type 2 diabetes, atypical forms of diabetes (such as LADA and MODY), glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions, stroke, cancer and infectious diseases (such as tuberculosis).
  • the method is for treating ⁇ -cell dysfunction associated with type 2 diabetes and/or hypoinsulinemia.
  • the method is for treating transplanted ⁇ -cells, for example as a treatment for diabetes.
  • the method provides enhanced glucose stimulated insulin secretion from ⁇ -cells.
  • the method provides enhanced BMPRIa signalling in ⁇ -cells.
  • the method modulates the level and/or function of one or more intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25, Hifla and Evi-1.
  • one or more intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25, Hifla and Evi-1.
  • the method preferably increases expression and/or function (activity) of one or more (and most preferably all) intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25 and Hifla.
  • the method may decrease expression of Evi- 1.
  • a combination product comprising:
  • a first agent comprising or consisting of a bone morphogenetic protein (BMP) or a fragment, variant, fusion or derivative thereof, as defined in above; and
  • each of components (a) and (b) is formulated in admixture with a pharmaceutically-acceptable adjuvant, diluent or carrier.
  • Such combination products provide for the administration of a BMP, or a fragment, variant, fusion or derivative thereof, in conjunction with the other therapeutic agent, and may thus be presented either as separate formulations, wherein at least one of those formulations comprises a BMP, or a fragment, variant, fusion or derivative thereof, and at least one comprises the other therapeutic agent, or may be presented (i.e. formulated) as a combined preparation (i.e. presented as a single formulation including a BMP, or a fragment, variant, fusion or derivative thereof, and the other therapeutic agent).
  • a combined preparation i.e. presented as a single formulation including a BMP, or a fragment, variant, fusion or derivative thereof, and the other therapeutic agent.
  • a pharmaceutical formulation including a BMP, or a fragment, variant, fusion or derivative thereof; another therapeutic agent useful in the treatment of ⁇ -cell dysfunction or type 2 diabetes; and a pharmaceutically- acceptable adjuvant, diluent or carrier; and
  • kits which components (a) and (b) are each provided in a form that is suitable for administration in conjunction with the other (optionally, the kit may comprise instructions to use that component in conjunction with the other component).
  • the second/other therapeutic agent is a treatment for diabetes and/or hypoinsulinemia.
  • Suitable additional therapeutic agents may be selected from the group consisting of insulin, insulin secretagogues (such as sulphonylureas), metformin, peroxisome proliferator-activated receptor agonists (PPARs; such as thiazolidinediones), ⁇ -glucosidase inhibitors, GLP-I receptor agonists, DPP-IV inhibitors and inhibitors of 11- ⁇ hydroxysteroid dehydrogenase type 1.
  • PPARs peroxisome proliferator-activated receptor agonists
  • ⁇ -glucosidase inhibitors such as GLP-I receptor agonists
  • DPP-IV inhibitors and inhibitors of 11- ⁇ hydroxysteroid dehydrogenase type 1.
  • agonist we include direct and indirect-acting agonists.
  • the second therapeutic agent is GLP-I or a biologically active fragment, variant, fusion of derivative thereof.
  • the agent is selected from the group consisting of Exendin-4 (Exenatide; Byetta), Exenatide long acting release (LAR), Exenatide derivatives (such as ZPlO developed by Zealand Pharmaceuticals), native GLP-I (such as CS-872 [Sankyo]), human GLP-I derivatives (such as BIM51077 [Ipsen and Roche]), DPP-IV resistant GLP-I analogues (for example LY315902 and LY30761 SR [Lilly]), long acting GLP-I derivatives (such as NN2211 [Novo Nordisk] and LY548806 [Lilly]) and complex proteins (such as the GLP-I -albumin complex CJC-1131, CJCl 134 [ConjuChem] and GSK716155 [GSK]).
  • the combination product may comprise BMP4 together with Exendin-4.
  • the second therapeutic agent is a dipeptidyl peptidase IV (DPP-IV) inhibitor.
  • DPP-IV dipeptidyl peptidase IV
  • the agent may be selected from the group consisting of Vildagliptin (LAF237), MK-0431-Sitagliptin and Saxagliptin.
  • the second therapeutic agent is gastric inhibitory polypeptide (GIP), or a biologically active fragment, variant, fusion of derivative thereof.
  • GIP gastric inhibitory polypeptide
  • GIP glucose-dependent insulinotropic polypeptide
  • DPP-4 enzyme DPP-4, which also cleaves GLP-I and GLP-2, rapidly inactivates GIP both in vitro and in vivo. Hence, it may be desirable to administer GIP in combination with a DPP-4 inhibitor.
  • the second therapeutic agent is a selective inhibitor of 11- ⁇ hydroxysteroid dehydrogenase type 1 (1 l ⁇ -HSDl), an enzyme associated with conversion of cortisone to Cortisol in the liver and adipose tissue.
  • suitable 1 l ⁇ -HSDl inhibitors/antagonists include AMG221 (developed by Amgen) and BVT83370 (developed by Biovitrum).
  • a fourth aspect of the invention provides a method of making a combination product as defined above, which method comprises bringing a component (a), namely a BMP, or a fragment, variant, fusion or derivative thereof, into association with a component (b), the second/other therapeutic agent, thus rendering the two components suitable for administration in conjunction with each other.
  • a combination products of the invention are for use in the treatment of ⁇ -cell dysfunction.
  • the combination product is for treating ⁇ -cell dysfunction associated with a condition selected from the group consisting of type 2 diabetes, atypical forms of diabetes (such as LADA and MODY), glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions, stroke, cancer and infectious diseases (such as tuberculosis).
  • a condition selected from the group consisting of type 2 diabetes, atypical forms of diabetes (such as LADA and MODY), glucose intolerance, insulin resistance, metabolic syndrome, dyslipidemia, hypercholesterolemia, high blood pressure, obesity, fatty liver conditions, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, cardiovascular disease, atherosclerosis, cerebrovascular conditions, stroke, cancer and infectious diseases (such as tuberculosis).
  • the combination product is for treating ⁇ -cell dysfunction associated with type 2 diabetes and/or hypoinsulinemia.
  • the combination product is for treating transplanted ⁇ -cells, for example as a treatment for diabetes.
  • the combination product enhances glucose stimulated insulin secretion from ⁇ -cells.
  • the combination product enhances BMPRIa signalling in ⁇ -cells.
  • the combination product modulates the level and/or function of one or more intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25, Hifla and Evi-1.
  • one or more intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, Nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GIPr, GCK, Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Snap-25, Hifla and Evi-1.
  • the combination product preferably increases expression and/or function (activity) of one or more (and most preferably all) intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GCK 5 Kir6.2, SURl, Rab3d, Rab27a, CalpainlO, Hifla, GIPr and Snap-25.
  • the method may decrease expression and/or function (activity) of one or more (and most preferably all) intrinsic factors selected from the group consisting of BMPRIa, BMPRII, Smadl, Smad4, Smad7, IdI, Id2, Ipfl/PDXl, Glut2, nkx ⁇ .l, HNFIa, PC2, PCl/3, GLP-Ir, GCK 5 Kir6.2, SURl, Rab3d, Rab27a, CalpainlO
  • a method of treatment of ⁇ -cell dysfunction comprises the administration of an effective amount of a combination product as defined above, to a patient in need of such treatment.
  • a further aspect of the invention provides a method for treating dysfunction of ⁇ -cells in vitro comprising contacting the ⁇ -cells with a bone morphogenetic protein (BMP) or a fragment, variant, fusion or derivative thereof, or a combination product as defined above.
  • BMP bone morphogenetic protein
  • the method may be used to maintain ⁇ -cell prior to transplantation of the cells into a patient, for example in the treatment of diabetes.
  • a transgenic non- human animal comprising ⁇ -cells which express abnormal levels of a bone morphogenetic protein (BMP) or receptor therefor, wherein the BMP is selected from the group consisting of BMP2 and/or BMP4.
  • BMP bone morphogenetic protein
  • the transgenic non-human animal comprises ⁇ -cells which express increased levels of a bone morphogenetic protein (BMP), for example BMP2 and/or 4, relative to corresponding non-transgenic non-human animals.
  • BMP bone morphogenetic protein
  • the ⁇ -cells comprise a bmp gene under the control of an Ipfl/Pdxl promoter (see Examples below).
  • the ⁇ -cells express decreased levels of a BMP receptor (preferably BMPRIa) relative to corresponding non-transgenic non- human animals.
  • a BMP receptor preferably BMPRIa
  • the ⁇ -cells may comprise a dominant native, kinase-deficient from of the Bmprla gene under the control of an Ipfl/Pdxl promoter (see Examples below).
  • the transgenic non-human animal is preferably a rodent, such as a mouse.
  • the transgenic non-human animals of the invention may be used to identifying candidate compounds with efficacy in the treatment of ⁇ -cell dysfunction.
  • the invention further provides a method for identifying a candidate compound for the treatment of a ⁇ -cell dysfunction, the method comprising administering a compound to be tested alters to a transgenic non-human animal as defined above and determining the effect of the test compound on ⁇ -cell function.
  • Determination of the effect of the test compound on ⁇ -cell function may comprise assaying one or more of the following:
  • the candidate compound may be a drag-like compound or lead compound for the development of a drug-like compound for each of the above methods of identifying a compound. It will be appreciated that the said methods may be useful as screening assays in the development of pharmaceutical compounds or drags, as well known to those skilled in the art.
  • a drug-like compound may be a molecule that may be synthesised by the techniques of organic chemistry, less preferably by techniques of molecular biology or biochemistry, and is preferably a small molecule, which may be of less than 5000 daltons molecular weight.
  • a drug- like compound may additionally exhibit features of selective interaction with a particular protein or proteins and be bioavailable and/or able to penetrate cellular membranes, but it will be appreciated that these features are not essential.
  • 'lead compound' is similarly well known to those skilled in the art, and may include the meaning that the compound, whilst not itself suitable for use as a drag (for example because it is only weakly potent against its intended target, non-selective in its action, unstable, difficult to synthesize or has poor bioavailability) may provide a starting-point for the design of other compounds that may have more desirable characteristics.
  • reagents and conditions used in the method may be chosen such that the interactions between the said test compound and the ⁇ -cell are substantially the same as would exist between them in vivo.
  • the 'drag-like compounds' and 'lead compounds' identified in the screening assays of the invention are suitably tested in further screens to determine their potential usefulness in treating arthritic diseases, inflammatory conditions, proliferative disorders, etc.
  • the method further comprises the step of mixing the candidate compound thus identified with a pharmaceutically acceptable carrier.
  • a further aspect of the invention provide an in vitro method for making cells capable of producing insulin, comprising contacting stem cells or progenitor cells with a bone morphogenetic protein (BMP) or a fragment, variant, fusion or derivative thereof, or a combination product, as defined above.
  • BMP bone morphogenetic protein
  • the cells capable of producing insulin are ⁇ -cells.
  • the invention provides a method for the functional maturation of in vitro differentiated ⁇ -cells generated from stem or progenitor cells.
  • stem cells and "progenitor cells” are well known in the art.
  • stem cell describes primal cells common to all multi-cellular organisms that retain the ability to renew themselves through cell division and can differentiate into a wide range of specialised cell types.
  • progenitor cell is used in cell biology and developmental biology to refer to immature or undifferentiated cells, typically found in post-natal animals.
  • stem cells and progenitor cells include totipotent, pluripotent, multipotent and unipotent cells.
  • Transplantation of functional islets from heart beating donors represents a potential cure for patients with diabetes.
  • the donor material for islet transplantation is insufficient to meet the clinical demand.
  • Pancreatic endocrine stem cells exist in the developing embryonic pancreas. Isolation, expansion and maturation of pancreatic endocrine stem cells would provide an unlimited source of ⁇ -cells for transplantation and treatment of diabetes.
  • Another alternative option to islet transplantation are embryonic stem (ES) cells.
  • ES cells can be obtained from the inner cell mass of blastocysts and ES cells are pluripotent cells with unlimited self-renewal capacity in vitro. Under appropriate conditions, ES cells have the capacity to differentiate into cells of all three germ layers.
  • ES cells are considered to have the potential to serve as a cell source in the replacement therapy for several different medical applications, including diabetes.
  • the stem cells and/or progenitor cells are mammalian cells (for example, from human or mouse).
  • mammalian stem cells for example, from human or mouse.
  • embryonic stem cells derived from blastocysts
  • adult stem cells which are found in adult tissues
  • cord blood stem cells which are found in the umbilical cord.
  • the stems cells are embryonic stem cells.
  • the stems cells are pancreas stem cells.
  • the cells are progenitor cells.
  • FIG. 1 BMPRIa and BMP4 expression in the developing and neonatal pancreas.
  • Figure 4 Determination of total insulin content in isolated pancreas from Ipfl/BMPRIa, IPF1/BMP4, and wild type controls.
  • A Immunohistochemical analyses of differentiated pancreatic markers in wild type and Ipfl/BMP4 transgenic mice using antibodies against insulin and glucagon in upper panel, and GLUT2 in lower panel.
  • FIG. 6 A to D. Expression of intrinsic factors in Ipfl/BMPRIa and Ipfl/BMP4 mice.
  • the figures show blood insulin concentrations in C57BL76 mice in acute tests (A and B) and after three days of treatment (C and D).
  • FIG. 1 Intraperitoneal glucose tolerance test on IPF1/Pdxl-/+ mice after three days treatment of intraperitoneal administration twice daily with vehicle or BMP4 20 ⁇ g/kg bodyweight.
  • the figure shows glucose concentrations in IPF1/Pdxl-/+ mice. Mice were fasted for 12 hours Vehicle or BMP4 were injected at timepoint -30 min and glucose was injected at timepoint 0 min.
  • INS-I cells (5 x 10 5 cells/6- well plate) were starved for 24 hr in serum free RPMI- 1640 medium. Then, cells were incubated with BMP4 (5 ng/ml) in 0.1% BSA/RPMI-1640 medium for indicated time points.
  • INS-I cells (5 x 10 5 cells/6-well plate) were starved for 24 hr in serum free RPMI- 1640 medium. Then, cells were incubated with BMP2 (75 ng/ml), BMP4 (5 ng/ml) or in combination with Noggin (300 ng/ml) in 0.1% BSA/RPMI-1640 medium for 1 hr.
  • BMP-2 and BMP-4 potentiate glucose stimulated insulin secretion.
  • INS-I cells were stimulated with BMP2 (5ng/ml) or BMP4 (5 ng/ml) in KRBB containing 11 mmol/1 glucose. Insulin secreted in the culture medium was determined by a mouse insulin ELISA kit. Data represent the mean value ⁇ SEM of two independent experiments each in duplicate. Figure 12. Animals treated with BMP4 show improved glucose tolerance and enhanced glucose stimulated insulin secretion in response to Exendin-4.
  • the deletion mutant of BMPR-IA that encode 186 amino acids (aa) for BMPR- IA plus 9 aa for influenza virus hemaglutinin (HA) epitope at the COOH terminus was constructed by RT-PCR using mouse BMPR-IA cDNA as template.
  • the 5' primer was 5'-GCG TGC GAA TCA GAC AAT GA-3' [SEQ ID NO: 3] and the 3' primer was 5'-CTA AGC GTA GTC TGG GAC GTC GTA TGG [SEQ ID NO: 4] / ATA GCA AAA GCA GCT GGA GAA-3' [SEQ ID NO: 5] .
  • the resulting trBMPR-IA PCR product was cloned behind the Ipfl/Pdxl -promoter and used for the generation of transgenic mice (Apelqvist et al, 1997; Hogan et ah, 1994).
  • a mouse BMP4 cDNA fragment (Ericson, et ah, 1998) was cloned behind the Ipfl/Pdxl gene promoter and used for the generation of transgenic mice over-expressing BMP4.
  • the genotypes of all offspring were determined by PCR using genomic DNA isolated from the tail biopsies of 3 weeks-old mice.
  • the primers used for genotyping were as follows: 5'-TAGCGAGGGGAAGAGGAGAT-S ' (Ipfl/Pdxl) [SEQ ID NO: 6] and 5' - CCTCAACTCAAATTCGCGT-3' (BMP4) [SEQ ID NO: 7] and 5'- CTATTGTCCTGCGTAGCTGG-3' (BMPRIa) [SEQ ID NO: 8].
  • transgenic mice The production of transgenic mice is also described in detail in Ahlgren et ah, 1998, Genes Dev. 12(12): 1763-8 and Jonsson et ah, 1994, Nature 371(6498):606-9, the relevant disclosures in which documents are hereby incorporated by reference
  • Immunohistochemical localisation of antigens, double-label immunohistochemistry and confocal microscopy was carried out essentially as previously described in Apelqvist et al., 1997, the relevant disclosures in which document are hereby incorporated by reference.
  • the primary antibodies used were guinea pig anti-insulin (Linco), rabbit anti-glucagon (Euro-Diagnostic ⁇ ), and rabbit anti-Glucose transporter 2 (raised against peptide 512-523 of mouse GLUT2, as described in Thorens et al., 1992, the relevant disclosures in which document are hereby incorporated by reference).
  • the secondary antibodies used were fluorescein anti-guinea pig (Jackson) and Cy3 anti-rabbit (Jackson).
  • In situ hybridisation using DIG-labelled RNA probes for BMPRI and BMP4 was performed essentially as described in Apelqvist et ah, 1997, the relevant disclosures in which document are hereby incorporated by reference.
  • mice Twelve hour fasted mice were injected intraperitoneally with Ig of glucose per kg of body weight. Blood samples were obtained from the tail vein and glucose levels were measured using a Glucometer Elite (Bayer Inc.) immediately before and 30, 60, 120 and 150 min after injection of glucose (lg/kg). Total pancreatic insulin was extracted using acid ethanol (75% EtOH, 0.2M HCl) and measured using Sensitive Rat Insulin RIA Kit (Linco). Pancreatic protein concentration was determined using a Bio Rad protein assay (BIO RAD). Serum insulin levels were measured using ELISA (Crystal Chem. Inc.). Statistical significance was calculated using Students t-test.
  • Human BMP4 (Recombinant Human BMP -4, R&D Systems, catalogue nr 314- BP/CF) was administered to C57BL76 mice or IPF1/Pdxl-/+ mice. BMP4 was dissolved in 4 mM HCl, as vehicle. Intraperitoneal glucose tolerance tests were performed on fasted mice either acutely or after three days treatment with intraperitoneal administration twice daily with vehicle or BMP4 20 ⁇ g/kg bodyweight. Vehicle or BMP4 20 ⁇ g/kg bodyweight were injected at timepoint - 30 min and glucose was injected at timepoint 0 min.
  • BMPRIa and BMP4 are expressed in the developing ⁇ -cells.
  • BMP-signalling components was analysed in the developing mouse pancreas by in situ hybridisation and real time PCR. Bmp4 and Bmprla expression was not observed prior to el 3 (data not shown) but from el 3 and to the neonatal stage, expression of Bmprla and BMP4 was prominent within the epithelial part of the pancreas (Fig.l).
  • BMPRIa endogenous BMPRIa gene was greatly enhanced, >24 fold, in I ⁇ fl/BMP4 islets and reduced by -95% in Ipfl/BMPRIa islets (Fig. 6a), indicating a positive, feedback loop for BMP4-BMPRIa signalling in ⁇ -cells.
  • BMPRIa heterodimer partner, BMPRII, and the transducing BMP-signalling components Smadl and Smad4 was also greatly increased in I ⁇ fl/Bmp4 islets and decreased in Ipfl/BmprIA islets (Fig. 6a).
  • Id genes are known BMP target genes and the expression of Id2 was significantly up-regulated, ⁇ 3-fold, in islets isolated from Ipfl/BMP4 mice and reduced by ⁇ 70% in islets of Ipfl/BmprIA mice (Fig. 6a), whereas the expression of IdI was similar in control, Ipfl/BmprIA and I ⁇ fl/BMP4 islets (Fig. 6a). Id3 expression was somewhat reduced in Ipfl/BMPRIa islets but unchanged in Ipfl/BMP4 islets (data not shown).
  • Smad7 another BMP -target gene
  • Smad7 was also significantly increased, >28-fold, in islets of Ipfl/BMP4 mice and slightly but not significantly decreased in islets of Ipfl/BmprIA mice (Fig. 6a) whereas the expression of Smad6 expression was expressed at very low, barely detectable levels in control islets and in islets of Ipfl/BMP4 and Ipfl/BmprIA mice (data not shown).
  • the expression of several BMP-signalling components is regulated in opposite directions in islet cells with enhanced and impaired BMP-signalling, respectively.
  • the enhanced expression of the BMPRIa and BMPRII receptors in islet cells over-expressing BMP4 in ⁇ -cells provides clear evidence of a positive BMP signalling feedback loop.
  • the expression of the IPF1/PDX1 target gene Nkx6.1 was significantly up-regulated by many fold in Ipfl/BMP4 islets and decreased in Ipfl/BMPRIa islets (Fig. 6b).
  • the MODY3 gene HNFl ⁇ was also significantly up-regulated by many fold in Ipfl/BMP4 islets but not significantly reduced in Ipfl/BMPRIa islets (Fig. 6b).
  • proinsulin processing enzymes prohormone convertase 1/3 and 2 were also increased, > 7-fold and ⁇ 2-fold, in islets of Ipfl/BMP4 mice and reduced, by -50% and -80%, respectively, in islets of Ipfl/nBMPRIa mice (Fig. 6b).
  • the expression of GLP-Ir was increased by > 40 fold in Ipfl/BMP4 islets and reduced, by -50% in Ipfl/BMPRIa islets (Fig. 6d).
  • GIP-Ir was increased by >18 fold in Ipfl/BMP4 islets and reduced by ⁇ 50% in Ipfl /BMPRIa islets (Fig. 6b).
  • Glucokinase (GCK) expression was increased ⁇ 6- fold in Ipfl/BMP4 islets and there was a tendency to reduced expression in Ipfl/BMPRIa islets (Fig. 6c).
  • Foxa2/HNF3 ⁇ , HNF4 ⁇ , Foxol, and HIF l ⁇ ARNT, and uncoupling protein 2 (UCP2) were expressed at equal levels in control, Ipfl/BMP4, and Ipfl/BMPRIa islets (Fig. 6c and data not shown).
  • the expression of Kir 6.2, SURl, Rab3d, Rab27a, CalpainlO, Hifla and SNAP25 was also increased in Ipfl/BMP4 islets and reduced in Ipfl/BMPRIa islets (Fig. 6c).
  • BMP4 improves glucose tolerance and increase insulin levels in wild type mice
  • Mature human BMP4 that includes amino acids 293-408 was administered to C57BL76 mice.
  • Intraperitoneal glucose tolerance tests IPGTT were performed on fasted (12 hours) mice after an intraperitoneal administration with BMP4.
  • BMP4-treated C57BL76 mice responded with improved insulin secretion (Fig. 7).
  • diabetic IPF1/Pdxl-/+ mice also responded with improved glucose tolerance (Fig. 8).
  • INS-I cells were maintained at 37 0 C (95% O 2 /5% CO 2 ) in RPMI-1640 medium containing 11 mmol/1 glucose and supplemented with 5% heat-inactivated fetal bovine serum, 1 mmol/1 sodium pyruvate, 10 mmol/1 HEPES, 2 mmol/1 L- glutamine, 50 ⁇ mol/1 ⁇ -mercaptoethanol, 25 units/ml penicillin and 25 ⁇ g/ml streptomycin. The medium was changed every other day until the cells became confluent.
  • INS-I cells were seeded into 6-well plates at a density of 5 x 10 5 cells/well and cultured for 2 days in complete RPMI 1640 at 37°C.
  • the cells were pre-treated with BMP2 (5 ng/ml) or BMP4 (5 ng/ml) for 6 hours in complete DMEM containing 5.5 mmol/1 glucose.
  • the cells were rinsed once with glucose free DMEM followed by a 1 hour incubation in glucose free DMEM at 37°C.
  • INS-I cells were seeded into 6- well plates at a density of 5 x 10 s cells/well and cultured for 2 days in complete RPMI 1640 at 37°C. Cells were further cultured for an additional 24 hours in serum free RPMI 1640. After incubation in serum free RPMI 1640, cells were cultured for 1 hour in RPMI 1640/0.1% BSA with either BMP2 (75 ng/ml), BMP4 (5 ng/ml) or in combination with Noggin (300 ng/ml).
  • INS-I cells were seeded into 6-well plates at a density of 5 x 10 5 cells/well and cultured for 2 days in complete RPMI 1640 at 37°C. Cells were further cultured for an additional 24 hours in serum free RPMI 1640. After incubation in serum-free RPMI 1640, cells were cultured in RPMI 1640/0.1% BSA in the presence of 5 ng/ml of BMP4 for various (0, 0.5, 1, 2, 4, 6, and 24 hours) time points.
  • the membrane were incubated for 1 hour at room temperature with HRP-conjugated secondary antibody (Jackson Laboratories, INC.) (diluted with 5% BSA in TBST), before adding SuperSignal West Dura
  • BMPs are members of the TGF ⁇ superfamily regulating a large variety of biologic responses in many different cells and tissue during embryonic development and postnatal life. BMPs exert their biologic effects via binding to two types of serine/threonine kinase BMP receptors, activation of which leads to phosphorylation and translocation into the nucleus of intracellular signalling molecules, including Smadl, Smad5 and Smad ⁇ . Once Smadl/5/8 are activated by receptor kinases, they can form a heteromeric complex with the common partner Smad, Smad4, and the complex in then translocated into the nucleus to induce transcriptional activation of BMP specific genes.
  • BMP4 Since BMP signal transduction is mediated by Smad proteins, we first evaluated the effects of BMP4 at different concentrations on the activation of Smad signalling. By using a single antibody that recognizes pSmadl(Ser463/465), pSmad5(Ser463/465) and pSmad8(Ser426/428) we could measure the activity of the BMP receptor kinase mediated pathway. BMP4 caused a strong induction of phospho-Smad 1/5/8 at 1, 5, 10, 25 and 50 ng/ml (data not shown). To determine when BMP4 induced phosphorylation of Smad 1/5/8, we incubated cells with 5 ng/ml of BMP4 for 0, 0.5, 1, 2, 4, 6 and 24 hours. We found that treatment of cells with BMP4 had stimulatory effects on phosphorylation of Smad 1/5/8 at 0.5 hr and the effect was more robust at 1 hr (Fig. 9).
  • BMP4 has an effect on GSIS in INS-I cells.
  • Cells were preincubated with BMP2 (5 ng/ml) or BMP4 (5 ng/ml) for 6 hours and subsequently stimulated with the same concentration of BMPs and glucose (2 and 11 mmol/1) for 1 hour.
  • BMP2 and BMP4 were carried out to study the basic effects of BMP2 and BMP4 on insulin secretion using INS-I monolayers.
  • INS-I showed no significant increase in insulin secretion in response to BMP2 or BMP4 (data not shown), whereas a modest increase in insulin secretion was observed in cells treated with BMP2 and a significant increase in insulin secretion was observed in the presence of 11 mmol/1 glucose in cells incubated with BMP4 (Fig. 11).
  • Carrier-free recombinant human bone morphogenetic protein 4 (BMP-4, catalogue nr 314-BP/CF) was obtained from R&D Systems and a stock solution was prepared according to the manufacturer's recommendations.
  • Synthetic exendin-4 was purchased from Sigma and dissolved in PBS. Effects of BMP4 in combination with Exendin-4 were analyzed in CBA male mice. Animals were dosed with either vehicle control or BMP-4 (20 ⁇ g/kg body weight) for 3 consecutive days of twice daily (at 8 a.m. and 4 p.m.) i.p. administration. Animals were fasted overnight (12 h) and a final i.p.
  • BMP4 protein could enhance glucose stimulated insulin secretion and improve glucose tolerance in response to Exendin-4.
  • Beta-cell-specific inactivation of the mouse Ipfl/Pdxl gene results in loss of the beta-cell phenotype and maturity onset diabetes. Genes & Dev 12:1763-1768, 1998.

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Abstract

La présente invention concerne l'utilisation d'une protéine morphogénétique osseuse (BMP), ou d'un fragment, variant, fusion ou dérivé de celle-ci dans la préparation d'un médicament pour traiter le dysfonctionnement de cellules bêta. La protéine morphogénétique osseuse est choisie parmi le groupe constitué de BMP2 et/ou BMP4. En particulier, l'invention concerne des procédés pour traiter le dysfonctionnement de cellules bêta associé au diabète de type 2. D'autres aspects associés de l'invention concernent (i) la combinaison de produits destinés à être utilisés dans le traitement du dysfonctionnement de cellules bêta et (ii) des animaux non humains transgéniques avec une fonction anormale de cellules bêta.
PCT/GB2007/000740 2006-03-02 2007-03-02 Utilisation médicale de bmp-2 et/ou bmp-4 Ceased WO2007099345A1 (fr)

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CN111214649A (zh) * 2020-02-27 2020-06-02 广州领晟医疗科技有限公司 Bm23肽在制备治疗缺血性脑血管疾病的药物中的应用
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