WO2006012707A1 - Method for treating diabetes - Google Patents

Abstract

The present invention provides methods of treating or preventing diabetic conditions, comprising the administration of a polypeptide which has the ability to stimulate pancreatic islet β-cell activity but which does not bind to or activate ErbB1 or ErbB4 receptors. Methods of diagnosing diabetic conditions are also provided, as are uses of the polypeptide, and compositions comprising the polypeptide.

Description

METHOD FOR TREATING DIABETES

This invention relates to the treatment or prevention of a diabetic condition, and in particular to the use of a polypeptide or corresponding nucleic acid molecule in the treatment or prevention, or in delaying the onset, of diabetes mellitus of either Type 1 or Type 2.

BACKGROUND

All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. The discussion of the references states what their authors assert, and the applicants reserve the right to challenge the accuracy and pertinency of the cited documents. It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Diabetes mellitus is the most common of the serious metabolic diseases affecting humans. It may be defined as a state of chronic hyperglycemia, i.e. excess sugar in the blood, consequent upon a relative or absolute lack of insulin action.

Diabetes mellitus is classified into two major forms; Type 1 and Type 2 diabetes. Type 1 diabetes, also referred to as insulin-dependent diabetes (IDDM) , is an autoimmune disease which is associated with almost complete loss of the insulin-producing pancreatic β-cells. This loss of β-cells results in life-long insulin dependence. Type 1 diabetes can occur at any age, and it has been estimated that about 1% of all newborns will develop this disease during their lifetime. Type 2 diabetes or non-insulin dependent diabetes (NIDDM) refers to a group of disorders characterized by high blood levels of glucose (hyperglycemia) and a resistance to insulin, and occurs in patients with impaired pancreatic β-cell function. The absence of insulin in patients with Type 1 and the insulin resistance in Type 2 diabetes results in decreased absorption of sugar from the bloodstream, and hence excess sugar accumulates in the blood. Both types of diabetes are associated with shortened life expectancy, and with significant morbidity, such as vascular disease, blindness and atherosclerosis. A new type of diabetes, designated LADA (Latent Autoimmune Diabetes in Adults) , has been proposed to describe adults diagnosed with type 2 diabetes who also have immunological evidence of type 1 diabetes, in that they are Anti-Gad antibody positive. Diabetes is one of the most prevalent chronic diseases in developed countries, and a leading cause of death. In addition to the clinical morbidity and mortality, the economic cost of diabetes is huge, exceeding $90 billion per year in the United States alone, and the prevalence of diabetes is expected to increase^' more than two-fold by the year 2010, largely as a result of Type 2 diabetes associated with obesity.

People of non-European origin are at a greatly increased risk of developing diabetes. Asians, including South Asians, will have a huge epidemic of diabetes in the near future. These individuals develop diabetes in the absence of severe obesity, and therefore may have insulin deficiency as a major component of their condition. In addition, two types of diabetes which are associated with low insulin levels related to malnutrition, fibrocalculous pancreatic diabetes and protein deficient pancreatic diabetes have been described in India.

Insulin is produced by β-cells in the islets of Langerhans in the pancreas. During pancreatic development, islet precursor cells proliferate and eventually differentiate into one of the four major islet cell types. It is thought that the formation of β-cells, in particular, is regulated by the effects of various growth factors, cytokines and hormones .

The numbers and function of β-cells are important to maintain glucose metabolism, and a severe reduction in the number and/or function of β-cells results in the onset of diabetes. It is now established that β-cell mass is maintained by a balance between loss and neoformation from precursor cells. When insulin demand is significantly increased by insulin resistance, for example due to obesity, β-cell neogenesis is stimulated. This leads to expansion of the β-cell mass, and glucose metabolism is maintained. If this compensatory mechanism is impaired, for example in Type 2 diabetes, glucose metabolism is disrupted. Recent studies have shown that β-cell neogenesis is impaired in animal models of type 2 diabetes. Consequently, factors which stimulate β-cell neogenesis should be effective in preventing the onset of diabetes, and thus represent a potentially useful therapeutic approach. The primary aim of treatment in all forms of diabetes mellitus is the same, namely the reduction of blood glucose levels to as near normal as possible, thereby minimizing both the short- and long-term complications of the disease. Long-term management of diabetes often includes insulin therapy, regardless of whether the patient is classified as type 1 or type 2. In healthy individuals, insulin increases glucose uptake by skeletal muscle and decreases glucose production in the liver; however, in individuals with type 2 diabetes, insulin tends not to do so. Many patients with type 2 diabetes, therefore, do not respond well to insulin therapy, even when it is administered at high doses.

The treatment of Type 1 diabetes necessarily involves the administration of replacement doses of insulin, which is conventionally administered by the parenteral route. In combination with dietary measures and - A -

self-monitoring of blood glucose levels, the majority of Type 1 patients can achieve reasonable control of blood glucose. In Type 2 diabetes, initial therapy is a regimen of optimal diet, weight reduction and exercise. Drug therapy is initiated if and when these measures no longer provide adequate metabolic control. Drugs which promote insulin secretion or which lower glucose levels by other means are commonly prescribed. Hypoglycemic agents, for example sulphonylureas or biguanides, are the principal drugs prescribed to such patients. They stimulate insulin production by directly stimulating β-cells; the effectiveness of such drugs therefore depends on the number of functioning β-cells (the β-cell mass) remaining in the pancreas. Sulphonylureas lose their effectiveness in a high proportion of Type 2 diabetics; in one study 30 percent of newly-diagnosed diabetic patients who were treated with sulphonylureas required insulin within the first six years of therapy. It has recently been reported that up to twelve percent of people with type 2 diabetes have a gene variant, the Arg972 variant in insulin receptor substrate-1, which may predispose them to having these drugs fail, so that they require treatment with insulin. Peptide growth factors are implicated in a wide variety of physiological and pathological processes, including signal transduction, cell survival, differentiation, cell adhesion, cell migration, immune response, hematopoiesis, inflammation, tissue repair, atherosclerosis and cancer. In almost all these processes, peptide growth factors exert their biological effects by interacting with the extracellular domain(s) of transmembrane receptor tyrosine kinases (RTKs) . Most RTKs belong to small groups of highly homologous receptors which bind similar ligands and maintain inter-receptor interactions through ligand-induced homo- and hetero-dimer formation. Upon ligand-induced dimerization, these receptor specific tyrosine residues in the cytoplasmic domains are autophosphorylated. The phosphorylated tyrosine residues serve as high-affinity docking sites for proteins which possess SH2 or phosphotyrosine binding (PTB) domains, such as She, Grb2 and the p85 subunit of phosphoinositide 3' -kinase (PI 3-kinase) . This leads to activation of signalling pathways such as the mitogen-activated protein kinase pathway, resulting in a complex cascade of intracellular signals culminating in specific events for target cells. One of the most thoroughly investigated subfamilies of RTKs is the ErbB family, which encompasses four known receptors: ErbB-1 (also called epidermal growth factor receptor (EGFR) ) , ErbB-2 (also called HER2 or Neu) , ErbB-3 and ErbB-4. These receptors are widely distributed throughout different tissues, and, depending on cell type and physiological conditions, act to mediate growth inhibition or induction of cellular proliferation and differentiation.

Many peptide growth factors are ligands for the ErbB receptor family. This group of factors shares a high degree of ^' sequence similarity, particularly with respect to a common six-cysteine 36-40 amino acid residue epidermal growth factor (EGF) motif. This motif has a spacing of CX₇CX₄CXioCXiCX₈C, and forms three intramolecular disulphide bonds (Ci-C₃, C₂-C₄, C₅-C₆) and a characteristic three-loop structure, including the Ci-C₃ disulphide loop, C₂-C₄ disulphide loop and C₅-C₆ disulphide loop.

The EGF-motifs of growth factor peptides belonging to this family all appear to be encoded by two exons with a precisely-located intervening intron, corresponding to the border separating the first two disulphide loops (A loop, C₁-C₃; B loop, C₂-C₄) from the third loop (C loop, C₅-C₆) . A common feature of these molecules is that they are synthesized as larger transmembrane precursors, which are proteolytically cleaved to release the soluble biologically active form of the growth factor. The consensus EGF-motif is crucial for binding to and activating members of the ErbB receptor tyrosine kinase family. Ligand binding to ErbB receptors induces receptor homo- or hetero-dimerization, autophosphorylation and subsequent activation of downstream signalling pathways, resulting in diverse physiological processes including cell proliferation, differentiation, migration and survival. Mammalian ligands for the ErbB family include EGF, transforming growth factor-α (TGF-α) , heparin-binding EGF-like growth factor (HB-EGF) , epiregulin, amphiregulin, neural- and thymus-derived activator for ErbB kinases (NTAK) , the neuregulin (NRG) subfamily, which includes the products of four genes (NRGl) , NRG2, NRG3 and NRG4, and betacellulin (BTC) . BTC was originally purified from the conditioned medium of a mouse pancreatic β-cell carcinoma (insulinoma) cell line, as a 32 kDa glycoprotein with mitogenic activity for fibroblasts, retinal pigment epithelial cells and smooth muscle cells. Human BTC has been cloned from the human breast cancer cell line MCF-7, and bovine BTC has been cloned from a bovine kidney cell line.

BTC is synthesised as a membrane-anchored precursor protein which can be proteolytically cleaved to release the soluble mature growth factor. Mature BTC binds to and activates the ErbBl and ErbB-4 homodimers, and in addition, binds to and activates all possible ErbB heterodimers, including the highly oncogenic heterodimeric ErbB2-ErbB3 receptor complex.

Numerous studies have highlighted the potential importance of the ErbBl receptor, and of the ligands TGFα, HB-EGF and BTC in particular, in the development and function of the pancreas. The ErbB ligands are abundantly expressed throughout the developing pancreas. ErbBl^" mice, which often die in the first post-natal week of life, display a generalised proliferation defect of the pancreatic epithelia, associated with a delay in the differentiation of islet cells into insulin-producing β-cells and disturbed migration and structure-formation of developing islet endocrine cells. These defects indicate that ErbB-1 mediated signaling is critical to normal β-cell development. The development of the pancreas in ErbB-3^"^^" mice is also severely defective.

Many studies have shown that BTC, but not EGF or TGFα, may have a unique, non-redundant role in pancreas development and function. BTC is strongly expressed in the pancreas, particularly in the islets of Langerhans, and stimulates the proliferation and differentiation of various pancreatic cells in vitro and in vivo. Since both ErbB-1 and ErbB-4 are expressed by the duct cells of the pancreas, it is thought that BTC may bind and activate these receptors to stimulate β-cell neogenesis. A surprisingly large number of membrane proteins undergo alternative splicing which removes the exonic sequence encoding the transmembrane domain, generating soluble forms which function differently from the membrane- bound form. Changes in mRNA structure as a result of alternative splicing can produce protein variants which may be differentially expressed in certain tissues, during different developmental stages and/or in different states of cell activation. Where the alternatively spliced exon encodes a protein domain which is functionally important for catalytic activity or binding interactions, the resulting proteins can often exhibit different or even antagonistic activities.

We recently identified an alternatively spliced mRNA transcript encoding a novel isoform of human BTC (International Patent Application No. PCT/AUOl/00010 by GroPep Limited; Dunbar et al . , 2000) . This isoform, designated BTC-δ4, lacks 147 bp encoding exon 4 of the BTC gene, leading to the generation of an mRNA encoding an unusual BTC precursor in which the C-loop of the EGF domain and the transmembrane domain are deleted, while the remainder of the mature molecule, including loops A and B and the "hinge" valine, is fused in-frame to the truncated C-terminal cytoplasmic tail. The retention of the hydrophobic signal sequence and the absence of the transmembrane domain suggests that BTC-δ4 may be a secreted protein. It was proposed in PCT/AUOl/00010 that this BTC splice variant would be useful in modulating activities mediated by ErbB receptors, and therefore would be useful for the treatment of conditions, such as cancer, associated with overexpression of ErbB oncogenes.

We subsequently confirmed that this splice variant was a secreted protein. However, we found that BTC-δ4 did not bind to or activate ErbBl or ErbB4 receptors, and did not bind to ErbB2 or ErbB3 homodimers; we therefore concluded that BTC-δ4 did not have functional activity, at least with respect to ErbBl and ErbB4 receptors and ErbB2 and ErbB3 homodimers, and that the core EGF motif which is missing from BTC-δ4 is likely to be essential for activity (Dunbar et al . , 2000) .

A isoform lacking the third disulphide loop of the EGF domain has been reported for HB-EGF (Loukianov et al . , 1997) . In this case, a 94 bp insertion between exons III and IV causes a frameshift and premature termination, generating a protein which retains the signal peptide, pro- region, heparin-binding domain and the first two conserved disulphide loops of the EGF motif, while a short nine-amino acid tail replaces the third disulphide loop, transmembrane and cytoplasmic domains.

US Patent No. 6,825,159 discloses a variant of betaceullulin, identified as SEQ ID NO. 38, and suggests that this variant has intact betacellulin activity and reduced EGF activity. SEQ ID NO. 38 differs from the polypeptide of the present invention in that it comprises all six cysteines and is not deficient in the C5-C6 loop. - Si -

SUMMARY

Despite the apparent lack of binding of BTC-δ4 to ErbB receptors, we have now found that BTC-δ4 stimulates β- cell differentiation, increases β-cell mass, decreases the decline of β-cell function and increases insulin secretion by β-cells. BTC-δ4 has higher potency than authentic BTC as a stimulator of β-cell differentiation; however the cell growth-promoting activity of BTC-δ4 is absent or reduced compared to that of authentic BTC.

In a first aspect the invention provides a method of treating, preventing^', or delaying the onset of a diabetic condition in a subject suffering from or at risk of suffering from the condition, comprising administering to the subject a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells and which has absent or reduced cell growth promoting activity compared to that of authentic BTC. In a second aspect the invention provides a method of treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from or at risk of the condition, comprising administering to the subject a nucleic acid which encodes a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC.

In a third aspect the invention provides the use of a polypeptide which has ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, for treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from the condition.

In a fourth aspect the invention provides the use of a nucleic acid which encodes a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, for treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from or at risk of suffering from the condition.

In a fifth aspect the invention provides the use of a polypeptide which has ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, for the preparation of a medicament for treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from or at risk of suffering from the condition.

In a sixth aspect the invention provides the use of a nucleic acid which encodes a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, for the preparation of a medicament for treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from or at risk of suffering from the condition.

Preferably, the ability of the polypeptide to activate ErbBl and ErbB4 receptors in the subject is substantially reduced compared to authentic BTC and differentiation of islet cell progenitors into pancreatic β cells is stimulated without stimulating proliferation of other cell types. Preferably the fibroblast cell growth promoting activity is absent or reduced compared to that of authentic BTC. Preferably the epithelial cell growth promoting activity is absent or reduced compared to that of authentic BTC.

Preferably the diabetic condition is Type 2 diabetes or Type 1 diabetes.

In a seventh aspect the invention provides a composition for treating, preventing, or delaying the onset of a diabetic condition, comprising a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, together with a pharmaceutically-acceptable carrier. In an eighth aspect the invention provides a composition for treating, preventing, or delaying the onset of a diabetic condition, comprising a nucleic acid which encodes a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, together with a pharmaceutically- acceptable carrier.

Preferably the ability of the polypeptide to stimulate the proliferation of cell types is absent or reduced compared to authentic BTC and the ability of the polypeptide to activate ErbBl or ErbB4 receptors is absent or reduced compared to that of authentic BTC. Preferably the fibroblast cell growth promoting activity is absent or reduced compared to that of authentic BTC. Preferably the epithelial cell growth promoting activity is absent or reduced compared to that of authentic BTC.

Preferably the ability of the polypeptide to activate ErbB2 -or ErbB3 homodimers is absent or reduced compared to that of authentic BTC.

The risk of cancer arising in the subject as a side-effect of treatment may be reduced compared to the risk arising from treatment with authentic BTC.

Preferably the polypeptide is substantially homologous to a member of the EGF family. More preferably the polypeptide is substantially homologous to epidermal growth factor, transforming growth factor-α, heparin- binding EGF-like growth factor, epiregulin, amphiregulin, neural and thymus-derived activator for ErbB kinases (NTAK) , members of the neuregulin subfamily (NRGl, NRG2, NRG3 and NRG4) , or betacellulin (BTC) . Even more preferably the polypeptide is substantially homologous to BTC-δ4. Most preferably the polypeptide is BTC-δ4. The BTC-δ4 polypeptide has an amino acid sequence shown in Figure 4 (SEQ ID NOs: 11 to 15) .

The polypeptide may be: (a) a splice variant of BTC, in which the C₅-C₆ disulphide loop, which is normally present in the gene which encodes authentic BTC, is absent; or

(b) a polypeptide which has a sequence substantially homologous to that of the molecule of (a) ,or

(c) an analogue, fragment, mutant, derivative, or allelic variant of the molecule of (a) .

The polypeptide may be:

(a) a splice variant of BTC, in which the C₅-C₆ disulphide loop, normally encoded by a nucleic acid sequence present in the gene which encodes authentic BTC, is absent, and the remainder of the nucleic acid sequence encoding the authentic BTC polypeptide molecule, including loops A and B and the "hinge" valine, is fused in-frame to the nucleic acid sequence encoding the truncated C-terminal cytoplasmic tail;

(b) a polypeptide which has a sequence substantially homologous to the molecule of (a) ; or

(c) a fragment, analogue, variant, or derivative of (a) .

Preferably the polypeptide is capable of acting as a β-cell differentiation factor, increasing β-cell mass, decreasing the decline of β-cell function and/or increasing insulin secretion by β-cells.

The fragment, analogue, variant, or derivative may be one which:

(a) one or more of the amino acid residues is substituted with a conserved or non-conserved amino acid residue, preferably a conserved amino acid residue; such a substituted amino acid residue may or may not be one encoded by the genetic code;

(b) one or more of the amino acid residues includes a substituent group,- (c) the mature polypeptide is fused with another compound to increase the half-life of the polypeptide; or

(d) additional amino acids, fused to the mature polypeptide. The risk of cancer arising in the subject as a side-effect of treatment may be reduced compared to the risk entailed in treatment with BTC.

Preferably the subject has one or more of the following risk factors for diabetes: impaired glucose tolerance, metabolic syndrome, a family history of Type 1 or Type 2 diabetes, obesity, polycystic ovary syndrome, hypertension, or elevated cholesterol levels.

It will be clearly understood that the invention includes within its scope the use of variant forms of other members of the EGF family of peptide growth factors which are capable of acting as a β-cell differentiation factor in vitro and/or in vivo, increasing β-cell mass in vitro and/or in vivo, decreasing the decline of β-cell function and/or increasing insulin secretion by β-cells in vitro and/or in vivo, herein defined as "analogous growth factor variants" . In these variant forms amino acid residues normally present in the authentic polypeptide are absent, in a manner analogous to their absence from BTC-δ4. Preferably the C₅-C₆ disulphide loop normally present in the authentic molecule is absent, in a manner analogous to BTC-δ4. Suitable authentic polypeptides selected from the EGF family include epidermal growth factor, transforming growth factor-α, heparin-binding EGF-like growth factor, epiregulin, amphiregulin, neural and thymus-derived activator for ErbB kinases (NTAK) , and members of the neuregulin subfamily (NRGl, NRG2, NRG3 and NRG4) . It is expected that such variant forms will function in a manner analogous to that found in the BTC-δ4 polypeptides of the invention, provided that they have the functional characteristics defined above.

It will be appreciated that, once knowing the amino acid sequence of the polypeptides disclosed herein, both BTC-δ4 and analogous variants of other growth factors may be synthesised by chemical means such as solid phase polypeptide synthesis or by recombinant methods, all of which are well known in the art. Preferably the polypeptide molecule has an amino acid sequence of human origin. However, the invention also encompasses the use of BTC-δ4 from non-human mammals; the BTC-δ4 polynucleotides of the invention may readily be used as probes for isolation of corresponding polynucleotides from cells of other mammals, using methods which are routine in the art.

Thus the polypeptide of the invention may be a recombinant polypeptide, an isolated naturally-occurring polypeptide or a synthetic polypeptide, and is preferably a recombinant polypeptide.

The method of the invention is useful for the treatment of a Type 2 diabetic condition and the prevention or delaying of the onset of a Type 2 diabetic condition. Furthermore, as Type 1 diabetes is characterised by the loss of β-cells and complete loss of β-cell function, the invention is also useful for the treatment of a Type 1 diabetic condition or for the prevention or delay of the onset of a Type 1 diabetic condition.

The polypeptides of the invention may optionally be administered together with an immunosuppressive agent. Suitable immunosuppressive agents include corticosteroids, TNF-α antibodies such as Remicade and soluble TNF-α receptors such as Enbrel . Such combined treatment may be preferable in the treatment of Type 1 diabetes or LADA, in which any new β cells would still be subject to immune attack unless the immune attack was suppressed. It is envisaged that the method of the invention will entail a reduced risk of carcinogenesis compared to the use of authentic BTC. We have found that BTC-δ4 fails to activate ErbBl and ErbB4 receptors, so ErbB-stimulated cell proliferation is prevented, thus decreasing or avoiding the risk of cancer as a side-effect of treatment.

While it is particularly contemplated that the methods and compositions of the invention are suitable for use in medical treatment of a diabetic condition in humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as non-human primates, felids, canids, bovids, and ungulates.

The polypeptides of the invention will generally be administered in the form of pharmaceutical compositions. Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams & Wilkins, Pennsylvania, USA.

The carrier or diluent, and other excipients, will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case. The compounds and compositions of the invention may be administered by any suitable route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated. Dosage will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure IA shows the detection of authentic human BTC and splice variant human BTC-δ4 in MCF-7 cells (Lane 1) and human breast skin fibroblasts (Lane 2) by RT-PCR. Lane 3 is a control without cDNA template. Figure IB shows the corresponding Southern blot using a cDNA probe encompassing polynucleotides encoding amino acids O²²-Y¹¹¹ of human BTC.

Figure 2A compares the partial nucleotide and deduced amino acid sequence (shown in one letter amino acid code) of authentic human BTC with that of splice variant human BTC-δ4. The RT-PCR products obtained from MCF-7 cDNA in Figure IA were cloned into pBluescript II SK and the inserts sequenced. Sequences surrounding the point of the 147 bp deletion (indicated by a downward arrow) are shown. Figure 2B shows the complete nucleotide sequence and deduced amino acid sequence of BTC-δ4 cDNA.

Figure 3 compares the complete amino acid sequences of authentic human BTC and splice variant human BTC-δ4 in a hypothetical alignment. The horizontal dashes within the BTC-δ4 sequence indicate the sites of the missing amino acids when compared to the authentic hBTC sequence. The schematic below shows the overall structure of authentic human BTC compared to BTC-δ4.

Figure 4 shows the amino acid sequences of the BTC-δ4 polypeptides (BTC-δ4i_-129, BTC-δ4_1-94, BTC-δ4₃₂-₉4, BTC-δ4₃₂-i29, BTC-δ432-in and BTC-δ4₉₅-i₂₉) •

Figure 5 illustrates the constructs used for recombinant expression and purification of BTC and BTC-δ4. Figure 5A is a schematic representation of the constructs which were used for BTC and BTC-δ4 respectively. Figure 5B shows the results of assessment of purity of each BTC or BTC-δ4 preparation by analytical RP-HPLC on a C₄ column. Figure 6 shows that BTC-δ4 is a secreted protein. A: COS-7 cells were transiently transfected with either BTC-FLAG, BTC-64-FLAG or empty vector (pcDNA3.1) . Seventy-two hours post-transfection the culture medium was collected and cell lysates prepared. Culture medium and cell lysates were analysed by Western blot with an anti-FLAG M2 antibody. B: COS-7 cells were transfected as above and culture medium or non-permeabilised fixed cells analysed by ELISA with anti-FLAG M2 antibody. Note, similar results for Western blot and ELISA were also obtained with an anti-BTC ectodomain antibody (R&D Systems) (data not shown) .

Figure 7 shows that BTC but not BTC-δ4 stimulates the proliferation of Balb/c 3T3 fibroblasts and also that BTC but not BTC-δ4 binds to ErbBl and ErbB4 in radioreceptor assays. Balb/c 3T3 fibroblasts were incubated with increasing concentrations of either BTC or BTC-δ4 alone (A) or increasing concentrations of BTC-δ4 in the presence of a fixed concentration of BTC (B) . Competitive binding of [¹²⁵I] -labelled BTC in the presence of increasing concentrations of BTC or BTC-δ4 to either AG2804 cells expressing ErbBl (C, D) or CHO cells transfected with ErbB4 (E, F) .

Figure 8 shows that BTC but not BTC-δ4 induces ErbBl and ErbB4 receptor tyrosine phosphorylation in AG2804 and CHO-ErbB4 cells, respectively. Lysates from untreated (-) or treated (10 nmol/1 BTC, 10 nmol/1 BTC-δ4 or 10 nmol/1 BTC + 10 nmol/1 BTC-δ4) . Cells were immunoprecipitated (IP) with either anti-ErbBl (AG2804) or anti-ErbB4 (CHO-ErbB4) antibodies and then blotted and probed with anti-phosphotyrosine antibody (αPY) . Blots were also stripped and re-probed with anti-ErbBl or anti-ErbB4 antibodies.

Figure 9 shows that BTC and BTC-δ4 induce the differentiation of AR42J Cells. AR42J-B20 cells were incubated for 48 hrs with 2 nM activin A and either InM BTC (panels A and C) or BTC-δ4 (panels B and D) and were then fixed and stained with anti-insulin antibody (arrow "1") and DAPI (arrow "2") to stain nuclei. In cells treated with activin A and BTC-δ4, many cells expressed insulin, but nuclei were fragmented (arrow "3") or lost. Magnification, Panels A and B: x 100; Panels C and D: x 400.

Figure 10 shows that BTC but not BTC-δ4 ameliorates activin-induced apoptosis of AR42J cells. AR42J-B20 cells were treated with 2 nM activin A and either 1 nM BTC (A) or BTC-δ4 (B) for 48 hrs. TUNEL-positive cells are indicated by the arrow.

Figure 11 shows that BTC-δ4 administration to STZ-treated rats reduces the plasma glucose concentration and improves glucose tolerance. A: STZ (85 μg/g) was injected on day 0 (neonate of one day old) and daily injection of BTC or BTC-δ4 (3 pmol/g) was started from day 0 for 5 days and the plasma glucose concentration was measured. Values are the means ± S.E. O, STZ group (n = 5) ; •, STZ/ BTC-δ4 group (n = 5) ; Δ, STZ/BTC group (n = 5) ; *P<0.05 vs. the STZ group. B: Glucose tolerance test was performed on 2-month old-rats. Left panel, changes in the plasma glucose concentration. Right panel, changes in the plasma insulin concentration. Values are the means ± SE. O, STZ group (n = 5) ; •, STZ/ BTC-δ4 group (n = 5) ; Δ, STZ/BTC group (n = 5) , ( ) : normal SD rats

(n = 3) . *P<0.05 vs STZ group, **P<0.01 vs STZ group.

Figure 12 shows the number of PDX-I-positive ductal cells (A) and islet-like cell clusters (ICC) (B) measured on Day 4 of the study described in Figure 11. (A) : PDX-I-positive ductal cell (arrow "1") : PDX-I (arrow "2") : cytokeratin. (B) : ICC (arrow "3") : cytokeratin (arrow "4") : insulin (arrow 5") : DAPI. STZ: STZ-injected rats treated with saline. * p<0.05 vs STZ. ** p<0.01vs STZ. Magnification, A: x400; B: xlOO. DETAILED DESCRIPTION

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any materials and methods similar or equivalent to those described herein can be used to practice or test the present invention, the preferred materials and methods are described.

Definitions

As used herein, the singular forms "a" , "an" , and "the" include the corresponding plural reference unless the context clearly dictates otherwise. Thus, for example, a reference to "an enzyme" includes a plurality of such enzymes, and a reference to "an ammo acid" is a reference to one or more amino acids.

In the description of the invention and in the claims which follow, except where the context requires otherwise due to express language or necessary implication, the word "comprise" or variations such as "comprises" or "comprising" is used in an inclusive sense, i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

The term "subject" as used herein refers to any animal having a diabetic condition which requires treatment with a pharmaceutically-active agent. The subject may be a human, or may be a domestic or companion animal. While it is particularly contemplated that the compounds of the invention are suitable for use in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as non-human primates, felids, canids, bovids, and ungulates.

As used herein, the term "therapeutically effective amount" means an amount of a compound of the present invention effective to yield a desired therapeutic response, for example to prevent or treat a disease which is susceptible to treatment by administration of a pharmaceutically-active agent.

The specific "therapeutically effective amount" will of course vary with such factors as the particular condition being treated, the physical condition and clinical history of the subject, the type of animal being treated, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed and the structure of the compound or its derivatives.

The concentration of polypeptide in the treatment composition is not critical, but should be an amount effective to treat a diabetic condition or to prevent or delay the onset of a diabetic condition. The term "effective amount", as used herein in all methods of use for BTC-δ4 polypeptides, means an amount sufficient to elicit a statistically significant response at a 95% confidence level (i.e. p<0.05 that the effect is due to chance alone) . The amount of polypeptide employed can be determined empirically, on the basis of the response of cells in vitro and response of experimental animals to the polypeptide or formulations containing the polypeptide.

The amount of polypeptide employed should be sufficient to elicit β-cell differentiation in vivo, an increase in β-cell mass in vivo, a decrease in the decline of β-cell function in vivo, and/or an increase in the insulin secretion by β-cells in vivo. On average, the daily dose in a patient weighing approximately 75 kg is at least 0.001 mg/kg, preferably 0.01 mg/kg, to about 20 mg/kg, preferably 1 mg/kg, of body weight.

As used herein, a "pharmaceutical carrier" is a pharmaceutically acceptable solvent, suspending agent, excipient or vehicle for delivering BTC-δ4 and/or other pharmaceutically-active agent to the subject. The carrier may be liquid or solid, and is selected with the planned manner of administration in mind.

The term "diabetic condition" as used herein includes both type 1 diabetes, also known as insulin- dependent diabetes mellitus (IDMM) , and type 2 diabetes, also known as non-insulin-dependent diabetes mellitus (NIDDM) .

The splice variant of the invention is designated BTC-δ4, and polynucleotides encoding BTC-δ4 are referred to herein as BTC-δ4 polynucleotides. The term "authentic" when used with reference to BTC means the full-length or mature soluble mature BTC protein of 178 amino acids.

The term "substantially homologous sequence" refers to a polypeptide which is functionally equivalent to the specific BTC-δ4 sequences disclosed herein, and encompasses substitutions, deletions and insertions in the specifically-disclosed polypeptides sequences.

The terms "fragment", "analogue", "variant" and "derivative" when referring to the polypeptide of the invention mean a molecule which retains essentially the same biological function or activity as this polypeptide. Thus an analogue includes a proprotein which can be activated by cleavage of the proprotein portion to produce an active mature polypeptide. The term "fragment" will be clearly understood to include a portion or domain of any size of a full-length sequence, provided only that the fragment is functionally active. The term "fragment of a sequence" or "part of a sequence" means a truncated sequence of the original sequence referred to. The truncated sequence (nucleic acid or polypeptide sequence) can vary widely in length, the minimum size being a sequence of sufficient size to provide a sequence with at least a comparable function and/or activity or the original sequence referred to, while the maximum size is not critical. In some embodiments, the maximum size usually is not substantially greater than that required to provide the desired activity and/or function(s) of the original sequence. The truncated amino acid sequence will typically be at least about 5 amino acids in length. More typically, however, the sequence will be at least about 50 amino acids in length, preferably at least about 60, 80, 100, 120, 150, 200 or 220 amino acids. The fragment sequences may be flanked by further amino acids, or may be modified according to methods known to those skilled in the art. The modified protein must maintain at least one biological activity- of the native protein. The fragments may be used in either the oxidised or the reduced forms, or in conjunction with conjugated or protective groups. For example, they may be conjugated to carrier molecules.

"Variants" of proteins include homologues of the proteins shown in Figure 4, and proteins having conservative substitutions such that the secondary conformation of the protein remain unchanged. Examples of such conservative substitutions include amino acid residues having substantially the same hydrophobicity, size, and charge as the original amino acid residue. Such substitutions are generally well known to those skilled in the art of protein chemistry. For example, conservative substitutions include proline for glycine and vice versa,- alanine or valine for glycine and vice versa; isoleucine for leucine and vice versa,- histidine for lysine and vice versa; serine for asparagine and vice versa; threonine for cysteine and vice versa; serine or alanine for threonine and vice versa; glutamine for asparagine and vice versa; tryptophan for tyrosine and vice versa; and arginine for glutamate and vice versa. The term protein "derivative" includes proteins with one or several amino acid residues substituted by naturally-occurring or synthetic amino acid homologues of the 20 naturally-occurring amino acids. Examples of such homologues are 4-hydroxyproline, 5-hydroxylysine, 3- methylhistidine, homoserine, β-alanine, 4-aminobutanoic acid, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, γ-amino butyric acid, homoserine, citrulline and the l ike .

As used herein, natural amino acid residues are the 20 amino acid residues commonly found in proteins (i.e. alanine, aspartic acid, asparagine, arginine, cysteine, glycine, glutamine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tyrosine, tryptophan and valine) , and includes both the D- and L- forms of such amino acids. As used herein, synthetic amino acid residues include both D- and L- forms of any other amino acid residues whether found in a protein, found in nature or synthetically produced. Synthetic amino acid residues include, but are not limited to, β-alanine, ornithine, norleucine, norvaline, hydroxyproline, thyroxine, gamma-amino butyric acid, homoserine, citrulline and the like.

Where a range of values is expressed, it will be clearly understood that this range encompasses the upper and lower limits of the range, and all values in between these limits.

It is to be clearly understood that this invention is not limited to the particular materials and methods described herein, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and it is not intended to limit the scope of the present invention, which will be limited only by the appended claims.

Unless otherwise indicated, the present invention employs conventional chemistry, protein chemistry, molecular biological and enzymological techniques within the capacity of those skilled in^' the art. Such techniques are well known to the skilled worker, and are explained fully in the literature. See, for example, Coligan, Dunn, Ploegh, Speicher and Wingfield: "Current protocols in

Protein Science" (1999) Volumes I and II (John Wiley & Sons Inc.) ; Sambrook, Pritsch and Maniatis: "Molecular Cloning: A Laboratory Manual" (2001) ; Shuler, M.L. :Bioprocess Engineering: Basic Concepts (2nd Edition, Prentice-Hall International, 1991) ; Glazer, A.N. , DeLange, R.J., and Sigman, D.S. : Chemical Modification of Proteins (North Holland Publishing Company, Amsterdam, 1975) ; Graves, D.J., Martin, B.L., and Wang, J.H. : Co- and post-translational modification of proteins: chemical principles and biological effects (Oxford University Press, 1994) Lundblad, R.L. (1995) Techniques in protein modification. CRC Press, Inc. Boca Raton, Fl. USA; and Goding, J.W

Monoclonal Antibodies: principles and practice (Academic Press, New York: 3^rd ed. 1996) .

The methods of this invention may involve

(a) the administration of BTC-δ4 prior to, together with, or subsequent to the administration of a second pharmaceutically-active agent for the treatment of diabetes; or

(b) the administration of a combination of BTC-δ4 and a second pharmaceutically-active agent for the treatment of diabetes.

Abbreviations used herein are as follows: bp base pairs

BTC betacellulin

CM culture medium DMEM Dulbecco' s minimal essential medium

EGF epidermal growth factor

EIA enzyme immunoassay

ELISA enzyme-linked immunosorbent assay

IDMM insulin-dependent diabetes mellitus IGT impaired glucose tolerance

LADA Latent Autoimmune Diabetes in Adults

NIDMM non-insulin-dependent diabetes mellitus

PCR polymerase chain reaction

PP pancreatic polypeptide PBS phosphate-buffered saline

RT-PCR reverse transcriptase PCR

SD standard deviation SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis STZ streptozotocin

Pre-diabetes, also known as impaired glucose tolerance (IGT) , affects approximately 41 million Americans and is a precursor to type 2 diabetes and cardiovascular disease. The American Diabetes Association (ADA) defines prediabetes as a condition where blood sugar levels are higher than normal, but not high enough to be diagnosed as type 2 diabetes. In addition to weight loss and increased physical activity, clinical trials are testing the efficacy of nutritional supplementation with chromium picolinate before prediabetes progresses into a chronic disease state. Possible underlying conditions which may cause a diabetic condition or which may be associated with diabetic symptoms include metabolic syndrome (hypertension, obesity/overweight, and high cholesterol) , haemochromatosis, chronic pancreatitis, polycystic ovary syndrome (PCOS) , carcinoid syndrome, surgery or trauma to the pancreas, overactive pituitary, overactive adrenals, pancreatic insufficiency, acromegaly, Cushing' s syndrome, cystic fibrosis, adenocarcinoma, somatostatinoma, aldosteronoma-induced hypokalaemia, phaeochromocytoma, primary aldosteronism, Wolfram's syndrome, leprechaunism, Rabson-Mendenhall syndrome, stiff man syndrome, autoimmune lymphoproliterative syndrome; hyperprolactinemia; hyperthyroidism; POEMS or Crow-Fukase syndrome (polyneuropathy, organomegaly, endocrinopathy, M protein, and skin changes) ; prolactinoma; Kearns-Sayre syndrome; niacin overdose; and polyendocrine deficiency syndrome type 2.

In particular, risk factors for Type 2 diabetes include one or more of IGT, a family history of Type 2 diabetes, obesity, hypertension, elevated cholesterol levels, and sedentary lifestyle. It is known that certain ethnic groups, in particular African, Hispanic and Mexican Americans, Australian Aborigines, and Pacific islanders have a higher incidence of Type 2 diabetes. Factors associated with pregnancy are also involved, since a previous history of gestational diabetes, miscarriage, stillbirth, or giving birth to a large baby or one with a birth defect are all associated with increased incidence of Type 2 diabetes.

It has recently been reported that a mutation of a gene known as small ubiquitin-related modifier (SUMO-4) appears more frequently in individuals with type 1 diabetes than in individuals without the disease, and that this gene is associated with type 1 diabetes in some families (Guo et al, 2004; Bohren et al, 2004) .

Methods for the diagnosis of a diabetic condition are well known to those in the art. Two tests are typically used to diagnose a diabetic condition in a human: (1) fasting blood glucose level: The "gold standard" for diagnosing diabetes is an elevated blood sugar level after an overnight fast (no food intake after midnight) . A value above 140 mg/dl on at least two occasions is regarded as diagnostic of diabetes. Normal subjects have fasting sugar levels between 70-110 mg/dl.

Oral glucose tolerance test (OGTT) : An oral glucose tolerance test may be performed in a doctor's office or a laboratory. Blood glucose levels are measured five times over a period of 3 hours following a glucose challenge. The subject starts the test in a fasting state (having had no food or drink except water for at least 10 hours but not more than 16 hours) . An initial blood sample is taken, and the subject is then given a high glucose drink (75 grams of glucose; 100 grams for pregnant women) . Further blood samples are taken at 30 minutes, 1 hour, 2 hours and 3 hours after drinking the high glucose drink. In a non-diabetic person glucose levels in the blood rise following the glucose challenge, but then rapidly return to normal. In a diabetic subject, glucose levels rise to above normal levels after the glucose challenge, and return to normal levels much more slowly.

Mammalian ligands for the ErbB family include EGF (Savage et al . , 1972) , transforming growth factor-α (TGF- α) (Marquardt et al . , 1984) , heparin-binding EGF-like growth factor (HB-EGF) (Higashiyama et al . , 1991) , epiregulin (Toyoda et al . , 1995) , amphiregulin (Shoyab et al . , 1989) , neural- and thymus-derived activator for ErbB kinases (NTAK) (Higashiyama et al . , 1997) , the neuregulin (NRG) subfamily, which includes the products of four genes (NRGl) (Marchionni et al . , 1993) , NRG2 (Chang et al . , 1997; Carraway et al . , 1997) , NRG3 (Zhang et al . , 1997) and NRG4 (Harari et al . , 1999) , and betacellulin (BTC) (Shing et al. , 1993) .

BTC-δ4 polypeptide and its variants are generally as described in PCT/AUOl/00010, the entire contents of which are incorporated herein by this reference. Suitable methods for producing the polypeptides are widely available in the art. Preferably recombinant DNA techniques are employed. For example, the BTC-δ4 polynucleotide sequence is isolated from MCF-7 cells. It contains an open reading frame encoding a polypeptide of 129 amino acids. The polynucleotide sequence is identical to that of human BTC, except for a 147 bp deletion within the open reading frame (encoding 49 amino acids) resulting in the absence of the C₅-C₆ disulphide loop normally present in the EGF domain (See Figure 2B and 3) . This was generated as a result of alternative mRNA splicing (exon skipping) of one of the exons of the human BTC gene. BTC-δ4 polynucleotides may be obtained from a variety of cell sources which express BTC-64 encoding mRNA. The inventors have identified a number of suitable human cell sources for BTC-δ4 polynucleotides, including but not limited to kidney, liver, pancreas, and a variety of breast carcinoma cell lines, such as MCF-7.

For example, polynucleotides encoding BTC-δ4 polypeptides may be obtained by cDNA cloning from RNA isolated and purified from cell sources. cDNA libraries of clones may be prepared using techniques well known to those in the art, and may be screened for BTC-δ4 encoding DNA with nucleotide probes which are substantially complementary to any portion of the BTC gene. Various PCR cloning techniques may also be used to obtain the BTC-δ4 polynucleotides of the invention.

Thus polynucleotides encoding BTC-δ4 polypeptides of the invention may be obtained by PCR, using oligonucleotide primers comprising polynucleotide sequences encoding portions of the BTC gene. Preferably the primer comprises the extreme 5¹ and 3¹ coding regions. More preferably the oligonucleotide primers have the following sequences, or sequences substantially homologous thereto 5¹ GAGCGGGGTTGATGGACCGG 3' (SEQ ID NO: 1) 5' TTAAGCAATATTTGTCTCTTC 3¹ (SEQ ID NO: 2)

Host cells are transformed or transfected with the vectors of this invention, for example a cloning vector or an expression vector. Various expression vector/host systems may be utilised equally well by those skilled in the art for the recombinant expression of BTC-δ4 polypeptides. Such systems include, but are not limited to micro-organisms such as bacteria transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors comprising the desired BTC-δ4 polynucleotide coding sequence; yeast transformed with recombinant yeast expression vectors comprising the desired BTC-δ4 polynucleotide coding sequence; insect cell systems infected with recombinant virus expression vectors (e.g., baculovirus) comprising the desired BTC-δ4 polynucleotide coding sequence; or animal cell systems transfected with appropriate mammalian expression vectors comprising the desired BTC-δ4 polynucleotide coding sequence.

The appropriate DNA sequence may be inserted into the vector by a variety of procedures. In general, the DNA sequence is inserted into an appropriate restriction endonuclease site(s) by procedures well known to those skilled in the art.

The DNA sequence inserted in the expression vector is operatively linked to an expression control sequence (s) (promoter) to direct mRNA synthesis. Depending on the host/vector system utilised, any suitable transcription/translation elements may be used. For instance, when cloning in prokaryotic cells such as E. coli the trc or T7 promoter may be used; when cloning in mammalian expression systems, promoters isolated from the genome of mammalian cells (e.g., mouse metallothionein promoter) or from viruses that grow in these cells (e.g., human cytomegalovirus immediate-early (CMV) promoter) may be used. Promoters produced by recombinant DNA or synthetic techniques may also be used to provide transcription of the inserted sequences. The expression vector also contains a ribosome binding site for translation initiation and a transcription terminator. Specific initiation signals are also required for efficient translation of inserted coding sequences. These signals include the ATG initiation codon and adjacent sequences.

In cases where the entire BTC-δ4 coding sequence, including its own initiation codon and adjacent sequences, is inserted into the appropriate expression vectors, no additional translational control signals may be needed. However, in cases where only a portion of the BTC-δ4 coding sequence is inserted, exogenous translational control signals, including the ATG initiation codon, must be provided.

Furthermore, the initiation codon must be in phase with the reading frame of the BTC-δ4 coding sequences to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of transcription attenuation sequences, enhancer elements etc.

In addition, it is preferable that the expression vectors comprise one or more selectable marker genes to provide a phenotypic trait for selection of transformed or transfected host cells, such as neomycin (G418) resistance for eukaryotic cells, or ampicillin resistance for prokaryotic cells such as E. coli .

The vector containing a DNA molecule of the invention, as well as an appropriate promoter or control sequence, may be employed to transform or transfect an appropriate host to permit the host to express the protein. Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli; insect cells such as Drosophila and Sf9; and animal cells such as CHO, COS, or 293 cells.

The constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence. Alternatively, the polypeptide of the invention can be synthetically produced by conventional peptide synthesisers.

The polypeptide of the invention, produced in a variety of different vector/host expression systems as described above, can be recovered and purified from recombinant cell cultures by a wide variety of methods, including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxyapatite chromatography, and reverse-phase high performance liquid chromatography (HPLC) . Protein refolding steps can be used, as necessary, in completing the configuration of the desired polypeptide.

The polypeptide may be a purified product, isolated from a tissue or cellular source, a product of chemical synthetic procedures, or produced by recombinant techniques using a prokaryotic or eukaryotic host. Depending upon the host employed in a recombinant production procedure, the polypeptides of the invention may be glycosylated or may be non-glycosylated. Methods are available to enable persons skilled in the art to identify polypeptides which have a sequence substantially homologous to BTC-δ4 or analogues, fragments, mutants, derivatives, or allelic variants of BTC-δ4 for use in accordance with the invention, for example by assessing the ability of the polypeptide to induce differentiation of pancreatic cell lines in vitro, or assessing the ability to reduce the severity of diabetic symptoms in animal models of diabetes. Preferably the analogue, fragment, mutant, derivative, or allelic variant of BTC-δ4 for use in accordance with the invention has the following biological properties in vitro;

(1) the polypeptide does not displace radiolabelled mature BTC from ErbBl or ErbB4 receptors on human lung fibroblasts (AG2804) when utilising the method described in Example 5.

(2) the polypeptide does not induce the tyrosine phosphorylation of ErbBl or ErbB4 receptors on human lung fibroblasts (AG2804) when utilising the method described in Example 5.

(3) the polypeptide, together with activin A, stimulates the differentiation of AR42J-B20 cells, as does authentic BTC, but the polypeptide is much less potent in promoting the survival of the cells compared to authentic mature BTC, when utilising the method described in Example 6.

The compound of the invention may be administered orally, rectally, parenterally or by inhalation in dosage unit formulations containing conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles. The term parenteral as used herein includes subcutaneous, intravenous, intramuscular, intrathecal, intracranial, injection or infusion techniques. The invention also provides suitable topical, oral, aerosol, and parenteral pharmaceutical formulations for use in the novel methods of treatment of the present invention. The compounds of the invention may be administered orally as tablets, aqueous or oily suspensions, lozenges, troches, powders, granules, emulsions, capsules, syrups or elixirs. The composition ^■ for oral use may contain one or more agents selected from the group of sweetening agents, flavouring agents, colouring agents and preserving agents in order to produce pharmaceutically elegant and palatable preparations. The tablets contain the active ingredient in admixture with non-toxic pharmaceutically acceptable excipients which are suitable for the manufacture of tablets.

These excipients may be, for example, inert diluents, such as calcium carbonate, lactose, calcium phosphate or sodium phosphate; granulating and disintegrating agents, such as corn starch or alginic acid; binding agents, such as starch, gelatin or acacia; or lubricating agents, such as magnesium stearate, stearic acid or talc. The tablets may be uncoated, or may be coated by known techniques to delay disintegration and absorption in the gastrointestinal tract and thereby provide a sustained action over a longer period. For example, a time-delay material such as glyceryl monostearate or glyceryl distearate may be employed. Coating may also be performed using techniques described in the U.S. Patent Nos . 4,256,108; 4,160,452; and 4,265,874 to form osmotic therapeutic tablets for controlled release.

The BTC-δ4 as well as the pharmaceutically-active agent useful in the method of the invention can be administered parenterally by injection or by gradual perfusion over time independently or together.

Administration may be intravenous, intra-arterial, intraperitoneal, intramuscular, subcutaneously, intracavity, or transdermal. For in vitro studies the agents may be added or dissolved in an appropriate biologically acceptable buffer and added to a cell or tissue.

Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media. Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as anti-microbials, anti-oxidants, chelating agents, growth factors and inert gases and the like.

Generally, the terms "treating", "treatment" and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing a disease or sign or symptom thereof, and/or may be therapeutic in terms of a partial or complete cure of a disease. "Treating" as used herein covers any treatment of, or prevention of disease in a vertebrate, a mammal, particularly a human, and includes preventing the disease from occurring in a subject who may be predisposed to the disease, but has not yet been diagnosed as having it; inhibiting the disease, ie. arresting its development; or relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease.

The invention includes the use of various pharmaceutical compositions useful for ameliorating disease. The pharmaceutical compositions according to one embodiment of the invention are prepared by bringing BTC-δ4, analogues, derivatives or salts thereof and one or more pharmaceutically-active agents or combinations of BTC-δ4 and one or more pharmaceutically-active agents into a form suitable for administration to a subject using carriers, excipients and additives or auxiliaries.

Frequently used carriers or auxiliaries include magnesium carbonate, titanium dioxide, lactose, mannitol and other sugars, talc, milk protein, gelatin, starch, vitamins, cellulose and its derivatives, animal and vegetable oils, polyethylene glycols and solvents, such as sterile water, alcohols, glycerol and polyhydric alcohols. Intravenous vehicles include fluid and nutrient replenishers. Preservatives include antimicrobial, anti-oxidants, chelating agents and inert gases. Other pharmaceutically acceptable carriers include aqueous solutions, non-toxic excipients, including salts, preservatives, buffers and the like, as described, for instance, in Remington's Pharmaceutical Sciences, 20th ed. Williams & Wilkins (2000) and The British National Formulary 43rd ed. (British Medical Association and Royal Pharmaceutical Society of Great Britain, 2002; http://bnf.rhn.net) , the contents of which are hereby incorporated by reference. The pH and exact concentration of the various components of the pharmaceutical composition are adjusted according to routine skills in the art. See Goodman and Gilman's The Pharmacological Basis for Therapeutics (7th ed., 1985) . The pharmaceutical compositions are preferably prepared and administered in dosage units. Solid dosage units include tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the subject, different daily doses can be used. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals . The pharmaceutical compositions according to the invention may be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject.

Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of the cytotoxic side effects. Various considerations are described, eg., in Langer, Science, 249: 1527, (1990) . Formulations for oral use may be in the form of hard gelatin capsules, in which the active ingredient is mixed with an inert solid diluent, for example, calcium carbonate, calcium phosphate or kaolin. They may also be in the form of soft gelatin capsules, in which the active ingredient is mixed with water or an oil medium, such as peanut oil, liquid paraffin or olive oil.

Aqueous suspensions normally contain the active materials in admixture with excipients suitable for the manufacture of aqueous suspensions. Such excipients may be suspending agents such as sodium carboxymethyl cellulose, methyl cellulose, hydroxypropylmethylcellulose, sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia; dispersing or wetting agents, which may be (a) a naturally occurring phosphatide such as lecithin; (b) a condensation product of an alkylene oxide with a fatty acid, for example, polyoxyethylene stearate; (c) a condensation product of ethylene oxide with a long chain aliphatic alcohol, for example, heptadecaethylenoxycetanol; (d) a condensation product of ethylene oxide with a partial ester derived from a fatty acid and hexitol such as polyoxyethylene sorbitol monooleate, or (e) a condensation product of ethylene oxide with a partial ester derived from fatty acids and hexitol anhydrides, for example polyoxyethylene sorbitan monooleate.

The pharmaceutical compositions may be in the form of a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents such as those mentioned above. The sterile injectable preparation may also a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butanediol . Among the acceptable vehicles and solvents which may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed, including synthetic mono-or diglycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables.

Compounds of the invention may also be administered in the form of liposome delivery systems, such as small unilamellar vesicles, large unilamellar vesicles, and multilamellar vesicles. Liposomes can be formed from a variety of phospholipids, such as cholesterol, stearylamine, or phosphatidylcholines.

Dosage levels of BTC-δ4 will usually be of the order ^'of about 0.5mg to about 20mg per kilogram body weight, with a preferred dosage range between about 0.5mg to about lOmg per kilogram body weight per day (from about 0.5g to about 3g per patient per day) . The amount of active ingredient which may be combined with the carrier materials to produce a single dosage will vary, depending upon the host to be treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 5mg to Ig of an active compound with an appropriate and convenient amount of carrier material, which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 5mg to 500mg of active ingredient . It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

The polypeptides of the invention may additionally be combined with other compounds to provide an operative combination. It is intended to include any chemically compatible combination of pharmaceutically- active agents, as long as the combination does not eliminate the activity of BTC-δ4.

In vitro models for testing the efficacy of BTC-δ4

Differentiation of AR42J-B20 cells

Pancreatic AR42J cells are derived from a chemically induced pancreatic tumor and express both exocrine and neuroendocrine properties (Rosewicz et al . ,

1992) . Upon treatment with Activin A, AR42J-B20 cells stop growing, and their morphology changes significantly by extending neurites. In addition, activin-treated cells express mRNA encoding GLUT2 , ATP-sensitive K⁺ channel, and pancreatic polypeptide (PP) . Thus activin A converts AR42J cells into endocrine cells.

Treating these cells with BTC-δ4 and activin A converts them further into insulin-producing cells. The production of insulin in response to BTC-δ4 can be detected by immunofluoresnce following the staining of the cells with an anti-insulin antibody. Alternatively the AR42J clone AR1898-0192 can be used to measure the differentiation of AR42J cells into insulin-secreting cells quantitatively. This clone contains a secreted alkaline phosphatase (SEAP) gene downstream of the rat insulin II gene promoter. The stimulation of these cells with BTC-δ4 results in the synthesis and secretion of alkaline phosphatase into the culture media which can be measured enzymatically.

Immortalised epithelial cell lines Pancreatic precursor cell lines have been isolated from the pancreatic ducts of transgenic mice ("Immortomice") carrying a temperature-sensitive SV40 T antigen (Sharma et al, 2001) . When cells from these mice are cultured at 33°C, the SV40 T antigen is expressed, and cells become conditionally immortalized; however at 37°C, T antigen expression is shut off, and the cells differentiate appropriately. Thus these cells when grown at 33°C do not display islet/neuroendocrine granules, and fail to express islet markers. However, after culture at 37⁰C for approximately 2 weeks, whilst approximately 50% of the cells die, the remainder begin to differentiate into cells which display primitive islet-like insulin and glucagon granules. When transplanted under the kidney capsule of nude mice, cells grown at either temperature develop duct-like and islet-like phenotypes.

This cell line is used in a rapid and sensitive assay similar to the one described above to measure insulin expression and β-cell differentiation. In this case the rat insulin 1 promoter is used to drive expression of the Green Fluorescent Protein (GFP) reporter. This cell line provides a very sensitive method for identification of jS-cell differentiation factors such as BTC-δ4 which stimulate the expression of /3-cell specific genes such as insulin, as assayed by GFP fluorescence in living cells.

In vivo Models of Type 2 Diabetes

In addition to the Streptozotocin (STZ) model (see example 7) , a number of other animal models of Type 2 diabetes are available for use in testing the in vivo activity of BTC-δ4. These models include surgical generation of Type 2 diabetes, for example by pancreatectomy in rats (Bonner-Weir et al . , 1983) and genetic models based on selective breeding. Genetic models include, the Goto-Kakizaki (GK) rat, the Spontaneously Diabetic Torii (SDT) rat, the Otsuka-Long-Evans-Tokushima fatty (OLETF) rat, the db/db mouse and the NOD/Ltj mouse. The Goto-Kakizaki (GK) rat exhibits similar metabolic, hormonal and vascular disorders to those seen in the human diabetes disease. Unlike many other rodent models of Type II diabetes, the GK rat is non-obese. Characteristics of the Goto-Kakizaki rat include fasting hyperglycemia, impaired secretion of insulin in response to glucose both in vivo and in isolated pancreatic cells, and hepatic and peripheral insulin resistance. Late complications such as retinopathy, microangiopathy, neuropathy and nephropathy have been described in the literature.

In the Spontaneously Diabetic Torii (SDT) rat model, male rats spontaneously develop glucose intolerance with impaired insulin secretion after 14 weeks, and develop diabetes with remarkable hyperglycemia and marked hypoinsulinemia after 20 weeks. Diabetic Long-Evans rats are characterized by mild obesity and late-onset hyperglycemia after 18 weeks of age, with complications related to chronic diabetes. Although multiple loci have been identified by genetic analysis, the cause of diabetes in these rats seems to be a combination of insulin resistance and impaired insulin secretion, resembling human type 2 diabetes. Otsuka-Long-Evans-Tokushima fatty (OLETF) rats are derived from a spontaneous combination of insulin resistance and impaired insulin secretion, resembling human type 2 diabetes. In these rats, insulin sensitivity decreases with aging, i.e., it is normal at 6 weeks and reduced by 40% at 12 weeks and 80% after 18 weeks compared with age-matched control Long-Evans Tokushima Otsuka (LETO) rats. Insulin secretion is impaired at 40 weeks (14) and lipotoxicity to islet β-cells may be involved in the pathogenesis of islet dysfunction in hypertriglyceridemic OLETF rats. In addition, OLETF rats exhibit reduced regenerative capacity of pancreatic β-cells compared with LETO rats (17) . Because of the chronic progressive form of diabetes, OLETF rats are suitable for studying the pathophysiological changes during the prediabetic phase. The db/db mice harbour a point mutation in the leptin receptor gene, and spontaneously develop increased levels of blood glucose and depletion of pancreatic β cells by 8-10 weeks after birth. These mice also become overtly obese by about 4 weeks of age . NOD/Ltj mice are characterized by insulitis, a leukocytic infiltrate of the pancreatic islets. Marked decreases in pancreatic insulin content occur in females at about 12 weeks of age, and several weeks later in males. Onset of diabetes is marked by moderate glycosuria and by a non-fasting plasma glucose higher than 250 mg/dl . Diabetic NOD/Ltj mice are hypoinsulinemic and hyperglucagonemic, indicating a selective destruction of pancreatic islet beta cells. Susceptibility to IDDM in NOD/LtJ mice is polygenic, and environment, including housing conditions, health status, and diet, exerts a strong effect on penetrance. NOD/LtJ females are more widely used than males, because the onset of IDDM symptoms occurs earlier and with a higher incidence (90-100% by 30 weeks of age) . NOD/LtJ males develop IDDM at a frequency of between 40-60% by 30-40 weeks of age. Male mice are useful for certain applications, including pharmaceutical studies, "accelerated transfer" of IDDM, and some in vitro studies. The major component of diabetes susceptibility in NOD mice is the unique MHC haplotype (H2⁹⁷ = K^d, Aa^d, Ab⁹⁷, E^nu11, D^b) . NOD mice also exhibit multiple aberrant immunophenotypes, including defective antigen-presenting cell immunoregulatory functions, defects in the regulation of the T lymphocyte repertoire, defective NK cell function, defective cytokine production from macrophages (Fan et al . , 2004) and impaired wound healing. They also lack the α hemolytic complement component, C5. NOD/LtJ mice are also severely hearing-impaired. A variety of mutations causing immunodeficiencies, targeted mutations in cytokine genes and transgenes affecting immune functions, have been backcrossed into the NOD/Lt inbred strain background. Other suitable methods available to persons skilled in the art to test putative polypeptides of the invention for use in accordance with the invention include the use of the in vivo models of Type-2 diabetes discussed above, the testing of β-cell regeneration, amelioration of glucose intolerance and improved glycemic control (fasting serum glucose) , stimulation of insulin secretion and inhibtion of glucagon secretion when blood glusose levels are elevated, hypoglycemia and measuring improvement in markers of β-cell function such as the intravenous glucose tolerence test, arginine stimulation and hypergylcemic clamp. These assays are also applicable to human clinical trials.

The invention will now be described in detail by way of reference only to the following non-limiting examples, and to the figures.

Example 1 : Detection of BTC-δ4 by RT-PCR and cloning into pBluescript II SK

Total RNA was isolated from 70-80% confluent MCF-7 cells and human breast skin fibroblasts using an RNeasy Mini Kit according to the manufacturer's instructions (Qiagen, Clifton Hill, Vic., Australia) . Normal human breast skin fibroblasts were prepared from a piece of skin obtained during surgery for breast reduction. The skin was cultured as an explant for 5 days in DMEM supplemented with 10% fetal calf serum and penicillin- streptomycin sulphate until the fibroblasts had grown into a monolayer. Total RNA (2 μg) was subsequently reverse transcribed to cDNA with Superscript II enzyme according to the manufacturer's instructions using oligo dT primers (Gibco BRL, Gaithersburg, MD) . cDNA corresponding to human BTC-β was amplified by PCR with sense primer (5'-CTCGTCGACGAGCGGGGTTGATGGACCGG-B') (SEQ ID NO: 3) and antisense primer

(5'-CTCCTGCAGTTAAGCAATATTTGTCTCTTC-3') (SEQ ID NO: 4) . The underlined nucleotides correspond to Sail (sense primer) and Pstl (antisense primer) restriction sites. PCR was carried out in 50μl of 60 mM Tris-SO₄, 18 mM (NH₄) ₂SO₄, 1.5 tnM MgSO₄ (pH 9.1) , 0.2 mM dNTPs, 200 ng each primer,

I U eLONGase (Gibco BRL, Gaithersburg, MD) and 1 μl cDNA. Following an initial incubation at 94°C for 3 min, 35 cycles of amplification were carried out as follows: 94⁰C for 1 min, 5O⁰C for 1 min and 68°C for 1 min followed by a final 4 min extension at 68°C. The 416 bp BTC-δ4 PCR product was separated on a 2% agarose gel and visualized under ultraviolet light following staining with ethidium bromide. PCR Markers (Promega, Madison, WI) were used as the size standard, and the results are shown in Figure IA. Following electrophoresis the gels were Southern transferred to positively-charged nylon membrane (Roche, Castle Hill, NSW, Australia) in 0.4 M NaOH. The blot was incubated in prehybridization buffer (5x SSC,

5x Denhardt's, 0.1% sodium dodecyl sulphate (SDS) , and 50 μg/ml salmon sperm DNA) at 55°C for 2 h and hybridized for 16 h in the same buffer containing ³²P-dCTP-labelled human BTC cDNA probe (D³²-Y¹¹¹) generated by the random priming method using a Gigaprime kit (Geneworks, Adelaide, Australia) . Two washes were performed in 2x SSC, 0.1% SDS for 5 min at room temperature, followed by two more 15 min washes in 0.5x SSC, 0.1% SDS at 55⁰C. The blot was then exposed to Kodak XAR X-ray film at -80⁰C, and the results are shown in Figure IB.

BTC and BTC-δ4 PCR products were cloned and sequenced by separating the products by agarose gel electrophoresis and recovering the fragments following gel extraction with a Concert kit (Gibco BRL, Gaithersburg, MD) . Purified PCR products were then digested with Sail and Pstl and subcloned into Sail/Pstl-digested pBluescript

II SK (Strategene, La Jolla, CA) to generate pBlue-BTC and pBlue-BTC-δ4,and introduced into E. coli JM109. The recombinant plasmids were propagated and sequenced by the dideoxynucleotide chain terminator method, using a Thermosequenase cycle sequencing kit (Amersham-Pharmacia Biotech, Sydney, Australia) with Ml3 T7 and T3 promoter primers. PCR products were sequenced in both directions to confirm the sequence; this was done twice from two independent clones, and the results are illustrated in Figures 2A and B.

Example 2 : E. coli expression and purification of recombinant BTC and BTC-δ4

To characterize the biological activity of BTC-δ4, we cloned the corresponding cDNA (Asp³²-Ala¹²⁹; without the signal peptide) into the pET-32a expression vector and expressed the protein as a thioredoxin fusion in E. coli BL21trxB (DE3) . This strain is deficient in thioredoxin reductase, allowing for disulphide bond formation in the cytoplasm. The constructs used are illustrated schematically in Figure 5A. The mature form of BTC (Asp³²-Tyr^llx) or full length BTC-δ4 (Asp³²-Ala¹²⁹, minus the hydrophobic signal peptide Met^Ala³¹) were expressed as thioredoxin fusion proteins in the bacterial expression vector pET3.2a. pET3.2a-BTC and pET3.2a- BTC-δ4 constructs were transformed into the E. coli strain BL21 (DE3) and proteins expressed following induction with IPTG. Following induction, cells were collected, lysed and the BTC or BTC-δ4 purified using Ni-NTA agarose and RP-HPLC. Following expression, cells were lysed and fusion protein purified using Ni-NTA agarose. The purified thioredoxin fusion protein was cleaved with enterokinase to release BTC-δ4, which was purified to homogeneity by reverse-phase HPLC.

The open reading frame sequence of BTC-δ4^32"129 was amplified by PCR using the primer set

5' -CGTCCATGGCTGATGGGAATTCCACCAGAAGT-3' (SEQ ID NO: 5) (sense) and 5'-CGTCTCGAGTCATTAAGCAATATTTGTCTCTTC-B' (SEQ ID NO: 6) (antisense) and pBlue-BTC-δ4 (Dunbar et al . , 2000) as template. Ncol and Xhol recognition sites were attached to the sense and antisense primers, respectively (underlined) . BTC, constituting amino acids 32-111 (BTC^32"111) , was also expressed and purified using the pET system as a positive control as described previously (Seno et al . , 1996) or produced as a thioredoxin fusion protein with the plasmid pET32a. For this construct, the ORF sequence of BTC^32"111 was amplified by PCR using the primer set

5'-CGTCCATGGCTGATGGGAATTCCACCAGAAGT-3' (SEQ ID NO: 7) (sense) and

5' CGTCTCGAGTCAGTAAAACAAGTCAACTGT-3' (SEQ ID NO: 8) (antisense) and pBlue-BTC-δ4 (Dunbar and Goddard, 2000) as template. Ncol and Xhol recognition sites (underlined) were also attached to the sense and antisense primers, respectively. After amplification the PCR products were digested with Ncol/Xhol and cloned into Ncol/Xhol digested pET32a vector as above. The resultant plasmids, pETBTC-δ4 ^32~129 and pETBTC^32"111, were maintained in E. coli JM109 and for expression transformed into E. coli BL21trxB (DE3) cells. For the expression of BTC or BTC-δ4 in BL21trxB(DE3) cells, a single colony was inoculated into a 50 ml overnight culture grown at 37⁰C in LB medium (supplemented with 50 μg/ml ampicillin and 15 μg/ml kanamycin) with constant shaking. Ten ml of this overnight culture was then used to inoculate 200 ml of fresh LB medium. The cells were grown at 37°C to an OD₆oonm of 0.4-0.5, prior to induction with IPTG (1 mmol/1) and then for a further 3 h before being pelleted by centrifugation (4 000 rpm, 10 min 4°C) and stored at -80⁰C prior to purification.

To purify either BTC or BTC-δ4 expressed as fusion proteins, frozen cell pellets were thawed on ice and resuspended in 18 ml BugBuster Protein Extraction Reagent containing lysozyme (100 μg/ml) and incubated at room temperature with gentle shaking for 10 min. Following incubation, the cell lysate was sonicated (3 x 5 sec bursts) to reduce viscosity and clarified by centrifugation (20 min, 16 000 rpm, 10 min) . Following centrifugation, the clarified cell lysate was adjusted to 10 mmol/1 imadazole and incubated with 6 ml of Ni-NTA agarose, pre-equilibrated in 50 mmol/1 NaH₂PO₄, 0.3 mol/1 NaCl, 10 mmol/1 imidazole (pH 8.0) , for 1 h at 4°C with gentle shaking. Following incubation, the resin was centrifuged (5 min, 15 000 rpm, 4°C) , the supernatant removed and the resin washed by resuspension in 50 mmol/1 NaH₂PO₄,

0.3 mol/1 NaCl, 40 mmol/1 imidazole (pH 8.0) . Following three successive washes as above, the protein was eluted by incubating the resin in 12 ml 50 mmol/1 NaH₂PO₄, 0.3 mol/1 NaCl, 250 mmol/1 imidazole (pH 8.0) for 15 min. The resulting supernatant, containing BTC or BTC-δ4, was subsequently dialyzed overnight (Spectra/Por, 3.5 kDa MWCO, Spectrum Laboratories) against several changes of Enterokinase cleavage buffer (50 mmol/1 Tris-Cl, 1 mmol/1 CaCl₂, 0.1% Tween-20, pH 7.4) . To remove the thioredoxin fusion partner from BTC or BTC-δ4, Enterokinase was added to a final concentration of 0.1 units/20 μg protein and incubated overnight at 37⁰C with gentle mixing. BTC or BTC-δ4 was separated from the thioredoxin fusion partner by further Ni-NTA agarose affinity chromatography as described above. In this case, the cleaved thioredoxin fusion partner was captured on the resin and BTC or BTC-δ4 was collected in the flow through fraction. BTC or BTC-δ4 present in the flow through fraction was further purified by reverse-phase HPLC. Briefly, the Ni-NTA agarose flow through fraction was diluted 1:4 (v/v) with 0.1% TFA and applied to a C4 Prep-Pak reverse-phase HPLC column (25 mm x 100 mm; 300 A, 15μm; Millipore-Waters) at a flow rate of 10 ml/min. The column was washed with 0.1% TFA until OD₂i_4nm returned to baseline and the column then eluted with a gradient of 8-80% (v/v) acetonitrile over 150 min in the presence of 0.08% TFA at a flow rate of 10 ml/min. Twenty ml fractions were collected and aliquots of each (50 μl) were analysed by SDS-PAGE and Western Blotting using a goat anti-human BTC ectodomain (Asp³²-Tyr^11:L) antibody to identify pure BTC or BTC-δ4 containing fractions. Fractions containing BTC or BTC-δ4 were pooled and the purity analyzed by analytical RP-HPLC, electrospray ionization mass spectroscopy and SDS-PAGE. Analytical RP-HPLC was performed using a Brownlee aquapore C4 RP-HPLC column (2.1 x 100 mm) (Alltech) with a linear gradient of 8-80% (v/v) acetonitrile and 0.08% TFA over 40 min at a flow rate of 0.5 ml/min. The results are illustrated in Figure 5B.

The molecular mass of recombinant BTC and BTC-δ4 determined by electrospray ionization mass spectrometry was 9211.35 ± 0.29 Da and 11450.26 ± 0.23 Da, respectively, which is consistent with the calculated theoretical masses of 9249 and 11452 Da (data not shown) . Following SDS-PAGE and silver staining a single band at approximately 9 kDa and 11.5 kDa was obtained for BTC and BTC-δ4 detected under reducing or non-reducing conditions. The purity of both BTC and BTC-δ4 was further confimed by N-terminal sequence analysis (5 cycles) which gave the expecte N-terminal sequence with an approximate purity of >95%.

Alternatively, BTC-δ4^32"129 (containing an initiation methionine residue) was cloned into the expression vector pET3b and the recombinant protein, solubilized, and refolded from inclusion bodies, purified by cation exchange column and gel filtration column chromatography as previously described (Maeda et al, 2002) . All the recombinant proteins prepared in this study were lyophilised and stored at -80°C prior to use.

Example 3 : Construction of a BTC-δ4 expression plasmid for mammalian expression

Full length BTC-δ4 (1-129) was cloned into the vector pcDNA3.1 (Invitrogen) to generate expression vectors for the mammalian production of BTC-β as follows. Briefly, pBlue-BTC-δ4 from Example 1 was digested with Apal and BairiRI and the released insert purified following agarose gel electrophoresis. The digested and purified insert was then cloned into Apal/BarriHI-digested pcDNA3.1, and recombinant clones identified following transformation in E. coli TOPlO cells (Invitrogen) . To generate a "Flag- tagged" pcDNA3. l-BTC-64 construct in which the Flag epitope DYKDDDDK is inserted between amino acids S³⁵-T³⁶, as illustrated in Figure 3, the pBlue-BTC-δ4 was used as a template for PCR, using the primers 5'-CTCGGGAATTCCGACTACAAGGACGACGATGACAAGACCAGAAGTCCTGAA-S'

(SEQ ID NO: 9) (sense; underlined nucleotides correspond to an EcoRI restriction site; double underlined nucleotides encode the FLAG epitope tag) and 5'-CTCCTGCAGTTAAGCAATATTTGTCTCTTC-3' (SEQ ID NO: 10) (antisense; underlined nucleotides correspond to a Pstl restriction site) . The resulting PCR product was purified and digested with EcoRI and Pstl and cloned into EcoRI/Pstl-digested pBlue- BTC-δ4. Subsequently, the resultant Flag-tagged pBlue- BTC-δ4 construct was digested with Apal/BamKl and cloned into Apal/BamHl-digested pcDNA3.1 to generate pcDNA3.1-FLAG-BTC-δ4.

Example 4 Expression and analysis of BTC and BTC-δ4

FLAG-tagged constructs in COS-7 cells Like other members of the EGF family, BTC is synthesized as a transmembrane-anchored precursor protein (pro-BTC) which can be proteolytically cleaved to yield soluble BTC containing the EGF-motif (Asp³²-Tyr^li:L) . Retention of the hydrophobic signal peptide and the absence of the transmembrane domain suggests that BTC-δ4 may be a secreted protein. To test this, the complete open-reading frame of either BTC or BTC-δ4, additionally engineered to incorporate a FLAG epitope between amino acids Ser³⁵ and Thr³⁶ for subsequent detection as described in Example 3, was cloned into the expression vector pcDNA3.1 and secretion monitored following transient transfection in COS-7 cells. Seventy-two hours post transfection, culture supernatant and cell lysates were collected and analysed, using ELISA and Western Blot, for the presence of secreted and cell-associated BTC-FLAG and BTC-64-FLAG.

For transient expression of pcDNA3.1-FLAG-BTC-δ4 and pcDNA3.1-FLAG-BTC, COS-7 cells were plated into 12 well plates at 2xlO⁵ cells/well in DMEM/10% FBS. Following overnight incubation, cells were transfected with 2 μg of construct DNA using Lipofectamine 2000 and Optimem-1 medium (both from Invitrogen) according to the manufacturer's instructions. Twelve hours later, the cells were washed twice and replenished with fresh Optimem-1 medium. Seventy-two hours post-transfection, culture medium (CM) was collected and cell lysates prepared. CM was clarified by centrifugation and the presence of BTC or BTC-δ4 in the media analyzed by ELISA or Western blotting using an anti-FLAG-M2 antibody (Sigma) . BTC or BTC-δ4 present in the cell lysate was analysed by Western blotting with the anti-FLAG-M2 antibody. Cell lysates were prepared by washing the cells twice with PBS following the removal of CM and then lysing the cells directly in SDS-PAGE sample buffer and heating to 95°C for 5 min.

For ELISA analysis of the CM, 90μl of CM was mixed with lOμl of 10 x coating buffer (0.15 mol/1 Na₂CO₃, 0.35 mol/1 NaHCO₃, pH 9.3) and loaded into 96 well immunosorbent plates. Plates were coated overnight at 4⁰C then blocked in 2% BSA in PBS/0. l%Tween (PBS-T) . Plates were washed 4 times with PBS-T and then incubated for 1 h at 37°C with anti-FLAG antibody diluted in PBS-T (2.5 μg/ml) . Following incubation plates were washed as above and incubated with HRP-conjugated sheep anti-mouse antibody (1: 2000) diluted in PBS-T (HRP-conjugated sheep anti-mouse antibody was from Silenus Laboratories) . Plates were then washed 4 times with PBS-T and developed with o-phenylamine diamine (OPD) substrate and stopped with 2 mol/1 H₂SO₄. Absorbance was read at 490nm. Results are expressed as the mean ± standard deviation (SD) of triplicate determinations. To analyze the presence of BTC or BTC-δ4 on the cell surface by ELISA, CM was removed and the cells washed 3 times with PBS. The cells were then fixed with 4% paraformaldehyde and stained with anti-FLAG M2 antibody as described above. For Western blot analysis with the anti-FLAG-M2 antibody, CM or cell lysates were resolved by SDS-PAGE (10-20% Tris-Tricine gels) . Proteins were then transferred to nitrocellulose (Hybond-C extra,- Amersham) and membranes probed with mouse anti-FLAG-M2 antibody (2.5 μg/ml) and then HRP-conjugated sheep anti-mouse antibody (1:10 000) . HRP-labeled proteins were visualized using Supersignal West Dura Extended Duration Substrate (Pierce) .

The results are summarised in Figure 6. As shown in Figure 6A and B, BTC-δ4-FLAG was clearly detected in the culture supernatant by either Western blot (~ 14 kDa) or enzyme immunoassay (EIA) , with very little present in the cell lysate or on the cell-surface (Figure 6A and B) . In contrast, BTC-FLAG was predominantly present in the cell lysate (~ 20 kDa) and on the cell surface (Figure 6A and B) . Therefore, unlike membrane-anchored pro-BTC which is secreted following cell surface ectodomain cleavage, BTC-δ4, which lacks a transmembrane domain, appears to be directly secreted following expression.

Example 5 : ErbB-receptor binding and mitogenic activity of BTC-54

To investigate the biological activity of BTC-δ4, we initially tested its ability to stimulate the proliferation of Balb/c 3T3 mouse fibroblasts which express ErbBl. The mitogenic activity of BTC and BTC-δ4 towards Balb/c 3T3 fibroblasts was determined as previously described (Dunbar et al, 1999) . The Balb/c 3T3 cells were obtained from the American Tissue Type Culture Collection.

The results are shown in Figures 7A and 7B. As expected, BTC stimulated Balb/c 3T3 proliferation in a dose-dependent fashion. In contrast, BTC-δ4 did not stimulate cell proliferation even at concentrations as high as 100 nmol/1. In addition, BTC-δ4 did not antagonise the effects of BTC in this cell line.

The binding affinity of BTC and BTC-δ4 for ErbB receptors was determined by measuring the ability of BTC or BTC-δ4 to competitively displace [¹²⁵I] -labeled recombinant human BTC from ErbBl or ErbB4 receptors present on AG2804 fibroblasts (Dunbar et al . , 1999) or Chinese Hamster Ovary (CHO) cells stably transfected with ErbB4 (CHO-ErbB4 cells,- (Tzahar et al . , 1996) , a kind gift from Professor Yosef Yarden, Weizmann Institute, Israel) , respectively. To do this, AG2804 or CHO-ErbB4 cells were grown to 70-80% confluence in DMEM/10% FBS in 24-well plates. (Human lung fibroblast. The cells were then washed twice with binding buffer (100 mmol/1 Hepes, (pH 7.6) , 120 mmol/1 NaCl, 5 mmol/1 KCl, 1.2 mmol/1 MgSO₄, 8 mmol/1 glucose, 0.1% BSA) and then incubated with [¹²⁵I] -rhBTC (10 000-15 000 cpm, labeled with Na[¹²⁵I] using chloramine-T to a specific activity of approximately 20 μCi/μg and increasing concentrations of unlabelled BTC or BTC-δ4 (0-100 nmol/1 for AG2804 cells and 0-10 nmol/1 for CHO-ErbB4 cells) in binding buffer at 4°C for 18 h. Labelled cells were then washed three times in ice-cold Hank's buffered salt solution and lysed with 1 ml 0.5 τnol/1 NaOH/0.1% (v/v) Triton X-100 for 30 mm. Radioactivities of cell lysates were then determined with a γ-counter (Wallac 1470) . Non-specific binding was determined by performing the bioassays in the presence of a 100-fold excess of unlabeled ligand. Non-specific binding was typically about 5% of total binding. We also compared the ability of BTC and BTC-δ4 to stimulate the tyrosine phosphorylation of either ErbBl or ErbB4. AG2804, a cell line overexpressing ErbBl receptors, or CHO cells overexpressing the ErbB4 receptor (CHO-ErbB4 cells; a kind gift from Professor Yosef Yarden, Weizmann Institute, Israel) were grown to confluence in 10 cm dishes and subsequently incubated for 12-14 h in serum-free medium and the cells then stimulated with 10 nmol/1 BTC, BTC-δ4 or a combination of both for 10 min at room temperature. Following stimulation, cells were washed twice in PBS and then lysed in lysis buffer (0.5 ml) (50 mmol/1 Tris-Cl pH 7.4, 150 mmol/1 NaCl, 1% deoxycholate, 1% Triton X-100, 0.1% SDS, 5 mmol/1 sodium orthovanadate, 10 mmol/1 sodium fluoride, 1 mmol/1 EGTA and complete protease inhibitors™. Cell lysates were cleared by centrifugation (20 min, 15 000 g at 4⁰C) and ErbBl or ErbB4 immunoprecipitated by incubating the lysate with 1 μg rabbit polyclonal anti-ErbBl antibody (1005) , Santa Cruz or rabbit polyclonal anti-ErbB4 antibody, respectively for 2 h at 4°C with gentle shaking.

Following incubation, protein-G Sepharose was added and the mixture incubated for a further 1 h at 4⁰C. Immune complexes were collected by centrifugation, washed three times in lysis buffer and heated (3 min, 95°C) in SDS-PAGE sample buffer. Proteins were separated by SDS-PAGE or 10-20% Tricine gels and transferred to nitrocellulose filters (Hybond C, Amersham) . Membranes were probed firstly with anti-phosphotyrosine monoclonal antibody (PY20) and then with HRP-conjugated sheep anti-mouse antibody. HRP-labeled proteins were visualized using enzyme-linked chemiluminescence (ECL) (Amersham) . To confirm equal loading, blots were stripped and re-probed with rabbit polyclonal anti-ErbBl or rabbit polyclonal anti-ErbB4 antibodies and HRP-conjugated rabbit anti-sheep antibody. Anti-ErbBl (1005), anti-ErbB4 (C-18) and anti-phosphotyrosine (PY20) antibodies were purchased from Santa Cruz and HRP-conjugated rabbit anti-sheep antibody from Zymed Laboratories.

The results are summarised in Figures 7C to 7F and Figure 8. We found that BTC but not BTC-δ4 could displace [¹²⁵I] -BTC from ErbBl receptors present on human lung fibroblasts (AG2804) (Figure 7C and D) and induce ErbBl receptor autophosphorylation (Figure 8) . Similarly, BTC but not BTC-δ4 competed for ErbB4 receptor binding (Figure 7E and F) and induced ErbB4 receptor tyrosine phosphorylation (Figure 8) . These results indicate that in contrast to BTC, BTC-δ4 displays no affinity for either ErbBl or ErbB4. Moreover, BTC-δ4 does not activate ErbBl or ErbB4. These results were completely unexpected.

Because this means that BTC-δ4 does not stimulate cell proliferation, our findings suggest that BTC-δ4 would present a lower risk of inducing cancer than does authentic BTC. Moreover, it appears that BTC-δ4 exerts its effect via an as-yet unidentified receptor, rather than via ErbBl or ErbB4 receptors. Further investigations may be pursued using methods described herein, or using BIAcore assays.

Example 6 : Measurement of differentiation and apoptosis of AR42J Cells

Native BTC is known to stimulate the differentiation of pancreatic _/S-cells (Mashima et al . , 1996; Watada et al . , 1996; Yamamoto et al . , 2000; Li et al., 2001; Li L et al . , 2003; Li et al . , 2004) , and there is some evidence to suggest that this may occur through a unique non-ErbB cell surface receptor (Ishiyama et al . , 1998) .

To examine the effect of BTC-δ4 on the differentiation of pancreatic _/S-cells as assessed by induction of insulin expression, we used the model cell line AR42J-B20, a subclone of AR42J, an amylase-secreting pancreatic tumour cell line. In these cells, BTC acts coordinately with activin A and converts them to insulin- secreting cells (Mashima et al . , 1996) . Thus activin A converts amylase-secreting AR42J-B20 into pancreatic polypeptide (PP) -producing endocrine cells. Activin A also induces apoptosis in these cells and, in the absence of a survival factor, many of the activin-treated cells die by apoptosis after their conversion to PP-producing cells (Furukawa et al . , 1999) .

BTC exerts two effects in AR42J-B20 cells: firstly it inhibits apoptosis induced by activin A, and secondly it converts them to insulin-producing cells (Mashima et al . , 1996; Furukawa et al . , 1999) . AR42J-B20 cells were cultured in DMEM medium containing 10% fetal calf serum as described previously (Mashima et al . , 1996) . To assess differentiation into insulin-producing cells, cells were incubated for 48 hrs with 2 nmol activin A and either 1 nmol BTC or BTC-δ4. Cells were then fixed, stained with anti-insulin antibody as described previously (Mashima et al . , 1996) and the number of insulin-positive cells was counted. Nuclei were stained with DAPI.

Apoptosis was assessed by using the terminal deoxynucleotidyl transferase (TUNEL) technique (Wako Pure Chemicals, Osaka, Japan) . Changes in the number of viable cells were assessed by using 3- [4, 5-dimethylthiazole-2-yl] - 2, 5, -diphenyltetrazolium) bromide (MTT) (Carmichael et al . , 1987) .

As shown in Figure 9A, when printed in colour, AR42J-B20 cells differentiated into insulin-producing cells in response to a combination of activin A and BTC. Quantitatively, 78 ± 6.5% of the cells (mean ± S.E., n = 4) became insulin-positive after 48 hrs. Cells treated with activin A and BTC exhibited extended processes and expressed immunoreactive insulin, as shown in Figure 9C when printed in colour. Cells treated with a combination of activin A and BTC-δ4 also became insulin-positive,- 72 + 4.5% (n = 4) of the cells became insulin-positive after 48 hrs, as illustrated in Figure 9B when printed in colour.

In contrast to cells treated with activin A and BTC, the morphology of cells treated with activin A and BTC-δ4 was quite different. Some of the insulin-positive cells displayed extended processes, but most of them remained circular in appearance. The nuclei of these cells were condensed, and in some instances were either fragmented or absent, as illustrated in Figures 9B and D, when printed in colour, which is characteristic of cell death by apoptosis. Consistent with an apoptotic phenomenon, many of the cells treated with activin A and BTC-δ4 were TUNEL-positive, whereas only a small fraction of cells treated with activin A and BTC became TUNEL- positive, as shown in Figure 10.

To provide additional confirmation of the effect of BTC-δ4 on the survival of AR42J-B20 cells, we measured the changes in the number of viable cells after treatment with activin A and either BTC or BTC-δ4. After 48 hrs, the number of viable cells in cultures treated with activin A and BTC was 78.8 ± 4.2% of the number of cells seeded. In contrast, the number of viable cells in cultures treated with activin A and BTC-δ4 was significantly lower (21.8 ±3.2%) (P<0.005) . Taken together, BTC-δ4 was as effective as BTC in stimulating the differentiation of AR42J-B20 cells; however, BTC-δ4 was much less potent in promoting survival of these cells.

Example 7 : BTC-δ4 administration to STZ-treated rats reduces the plasma glucose concentration and improves glucose tolerance . To investigate the /3-cell differentiating activity of BTC-δ4 in vivo, we administered BTC-δ4 to STZ-treated neonatal rats. The experimental protocol was approved by the Animal Care Committee of Gunma University. One-day-old Sprague-Dawley (SD) rats were injected intraperitoneally (ip) with 85 μg/g streptozotocin (STZ) freshly dissolved in 0.05 mmol/1 citrate buffer (pH 4.5) . The pups were left with their mothers until 4 weeks of age. All neonates were tested one day after the STZ injection (day 1) for blood glucose, using Accu-Chek Active (Roche Diagnostics, Germany) . The animals were included in the study only if the blood glucose concentration was between 200 and 350 mg/dl on day 1. Animals whose blood glucose was >350 mg/dl on day 1 developed severe diabetes, and the blood glucose concentrations in these animals were >600 mg/dl on day 2 or 3. Neonatal STZ-treated rats were injected with 3 pmol/g BTC-δ4, BTC or saline every day for five days starting from day 0. The fasting blood glucose concentration and the body weight were measured daily for the first week, and then once a week for up to 8 weeks. Two months after the STZ-treatment, an ip glucose tolerance test (2 g/kg body weight) was performed after 14 h of fasting.

On day 4 and week 8, the animals were injected ip with 1 ml of bromodeoxyuridine (BrdU) labeling reagent per 100 g of body weight (cell proliferation kit, Amersham Pharmacia Biotech, U.K.) and decapitated after 3 h. The pancreas was excised, weighed, and divided into two parts. One portion from the splenic segment was fixed with 4% paraformaldehyde/PBS overnight at 4⁰C and processed for paraffin embedding. Four series of sections from each pancreas were cut at intervals of 100 μm in neonates and 300 μm in adults for immunostaining and histochemistry. The second portion was homogenized in cold acid-ethanol, heated for 5 min in 70⁰C water bath, centrifuged and the supernatant stored at -20⁰C prior to assaying for insulin. Insulin was measured by time-resolved immunofluorometric assay as described previously (Mashima et al . , 1996) .

Immunohistochemistry was performed as described previously (Li et al . , 2001; Li et al . , 2003) . Sources of the primary antibodies and the dilutions were as follows: Guinea pig anti-porcine insulin, 1: 1000 (provided by Dr. Matozaki of Gunma University) ; rabbit anti-PDX-1, 1:

3000; monoclonal mouse anti-BrdU, 1: 100 (Amersham) ; rabbit anti-bovine cytokeratin for wide spectrum screening (Ckwss) , 1: 1000 (DAKO) . Sources of the secondary antibodies and dilutions were as follows: goat Alexa Flour 568 conjugated anti-guinea pig IgG, 1: 1000; goat Alexa Flour 488 conjugated anti-guinea pig IgG, 1: 500; goat Alexa Flour 568 conjugated anti-mouse IgG, 1: 1000; goat Alexa Flour 488 conjugated anti-mouse IgG, 1: 500; goat Alexa Flour 568 conjugated anti-rabbit IgG, 1: 1000; goat Alexa Flour 488 conjugated anti-rabbit IgG, 1: 500 (Molecular Probes Inc., Eugene, OR) . Quantitation of the /3-cell mass was performed on insulin-stained sections using image analysis software (NIH image) by means of an AX70 Epifluorescence microscope (Olympus, Tokyo, Japan) equipped with a PXL 1400 cooled- charge-coupled device camera system (Photometries, Tucson, AZ) operated with IP Lab Spectrum software (Signal Analysis, Vienna, VA) . At least random 40 fields (magnification x 200) from one section (three sections from different series per block) were measured for the area of insulin-positive cells. The /3-cell mass was calculated as described elsewhere (Li L, Seno M, Yamada H, Kojima I. Promotion of /3-cell regeneration by betacellulin in ninety percent pancreatectomized rats. Endocrinology 142: 5379- 5385, 2001; Li L, Seno M, Yamada H, Kojima I: Betacellulin improves glucose metabolism by promoting conversion of Intraislet precursor cells to β-cells in streptozotocin- treated mice. Am J Physiol 285: E577-E583, 2003) . To quantify /3-cell neogenesis, we quantified the number of islet-like cell clusters (ICC) (less than 5 cells across) . The number of ICCs and islets were counted in the section stained with the anti-insulin antibody, and the area in these sections measured. At least five sections were analyzed per animal. Results were expressed as means + SE, and are shown in Table 1 and Figures 11 and 12 (when Figure 12 is printed in colour) . For comparisons between two groups, the unpaired t-test was used.

TABLE 1.

Characteristics of neonatal STZ-treated rats administered either BTC or BTC-δ4

Normal STZ STZ/BTC54 STZ/BTC body weight (g) 224±12.0 (6] 212±9.9 (5) 219±8.4 (5) 220±8.1 (5) pancreas weight (g) 0.80±0.03 (6) 0.82±0.06 (5) 0.91±0.08 (5) 0.92±0.06 (5) plasma glucose (mg/dl) 131.5±6.0 (6) 160.0±6.2 (5) 139.2±3.4 (5)** 150.1+5.3 (5)* plasma insulin (ng/tnl) 2.8±0.5 (6) 0.99±0.3 (5) 1.92±0.4 (5)* 1.14±0.3 (5) insulin content (μg) 1O5_..9±5.2 (6) 39.0±5.4 (5) 70.3±5.5 (5)** 52.4±5.5 (5)* B-cell mass (mg) 8.06±0.70 (6) 2.40±1.0 (5) 5.41±0.8 (5)** 4.12±0.9 (5)*

Data are mean + S. E (π) . Neonatal STZ-treated rats were treated with BTC, BTC-δ4 or saline and various parameters were measured at 8 weeks. *P<0.05 vs the STZ group. **P<0.01 vs the STZ group.

In STZ-treated neonatal rats, the plasma glucose concentration increased markedly and peaked on day 2 (> 400 mg/dl) (Figure HA) , thereafter declining gradually, although still significantly higher compared to control rats after 2 months (Table 1) . In contrast,

STZ-treated rats administered BTC-δ4, the plasma glucose concentration was significantly lower on day 1 and the peak value was markedly reduced (Figure HA) . The plasma glucose concentration was also reduced thereafter up until 2 months (Table 1) . Compared to the effect of BTC-δ4, BTC was less potent in improving hyperglycemia (Figure HA, Table 1) . At 2 months of age, intraperitoneal (ip) glucose tolerance tests were performed. In STZ-treated rats, the glucose response to ip glucose-loading was markedly impaired, whereas in STZ-treated rats administered BTC-δ4, the glucose response was significantly improved (Figure HB) . BTC-δ4 also increased the insulin response but, compared to normal rats, the insulin response to ip glucose-loading was still delayed. In BTC-treated rats, glucose tolerance was improved, but the effect of BTC was less than that of BTC-δ4 (Figure HB) . Moreover, the insulin content and the /3-cell mass were significantly increased in BTC-δ4-treated rats (Table 1) , and histological analysis of pancreatic tissue on day 4 indicated that BTC-δ4 significantly increased the number of PDX-1-positive ductal cells and ICCs (Figure 12, when printed in colour) .

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification. References cited herein are listed on the following pages, and are incorporated herein by this reference. REFERENCES

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Claims

1. A method of treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from or at risk of suffering from the condition, comprising administering to the subject a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells and which has absent or reduced cell growth promoting activity compared to that of authentic BTC.

2. A method of treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from . or at risk of the condition, comprising administering to the subject a nucleic acid which encodes a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the 1 cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC.

3. Use of a polypeptide which has ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to authentic BTC, for treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from the condition.

4. Use of a nucleic acid which encodes a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, for treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from or at risk of suffering from the condition.

5. Use of a polypeptide which has ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, for the preparation of a medicament for treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from or at risk of suffering from the condition.

6. Use of a nucleic acid which encodes a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, for the preparation of a medicament for treating, preventing, or delaying the onset of a diabetic condition in a subject suffering from or at risk of suffering from the condition.

7. A method or use according to any one of claims 1 to 6, wherein the ability of the polypeptide to activate ErbBl and ErbB4 receptors in the subject is substantially reduced compared to that of authentic BTC.

8. A method or use according to any one of claims 1 to 7, in which differentiation of islet cell progenitors into pancreatic β cells is stimulated without stimulating proliferation of other cell types.

9. A method or use according to any one of claims 1 to 8, in which the diabetic condition is Type 2 diabetes.

10. A method or use according to any one of claims 1 to 8, in which the diabetic condition is Type 1 diabetes.

11. A composition for treating, preventing, or delaying the onset of a diabetic condition, comprising a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, together with a pharmaceutically-acceptable carrier.

12. A composition for treating, preventing, or delaying the onset of a diabetic condition, comprising a nucleic acid which encodes a polypeptide which has the ability to stimulate the differentiation of islet cell progenitors into pancreatic β cells, wherein the cell growth promoting activity of the polypeptide is absent or reduced compared to that of authentic BTC, together with a pharmaceutically-acceptable carrier.

13. A method, use or composition according to any one of claims 1 to 12, wherein the ability of the polypeptide to stimulate the proliferation of cell types is absent or reduced compared to that of authentic BTC.

14. A method, use or composition according to any one of claims 1 to 13, wherein the ability of the polypeptide to activate ErbBl or ErbB4 receptors is absent or reduced compared to that of authentic BTC.

15. A method, use or composition according to any one of claims 1 to 14, wherein the ability of the polypeptide to activate ErbB2 or ErbB3 homodimers is absent or reduced compared to that of authentic BTC.

16. A method, use or composition according to any one of claims 1 to 15, wherein the risk of cancer arising in the subject as a side-effect of treatment is reduced compared to the risk arising from treatment with authentic BTC.

17. A method, use, or composition according to any^¬ one of claims 1 to 16, in which the polypeptide is substantially homologous to a member of the EGF family.

18. A method, use, or composition according to claim 17, in which the polypeptide is substantially homologous to epidermal growth factor, transforming growth factor-α, heparin-binding EGF-like growth factor, epiregulin, amphiregulin, neural and thymus-derived activator for ErbB kinases (NTAK) , members of the neuregulin subfamily (NRGl, NRG2, NRG3 and NRG4) , or betacellulin (BTC) .

19. A method, use, or composition according to claim 18, in which the polypeptide is substantially homologous to

BTC-δ4.

20. A method, use, or composition according to claim

19, in which the polypeptide is BTC-δ4.

21. A method, use, or composition according to claim

20, in which the BTC-δ4 polypeptide has the amino acid sequence shown in Figure 4 (SEQ ID NO: 11) .

22. A method, use, or composition according to any one of claims 1 to 12, in which the polypeptide is:

(a) a splice variant of BTC, in which the C₅-C₆ disulphide loop, which is normally present in the gene which encodes authentic BTC, is absent; or (b) a polypeptide which has a sequence substantially homologous to that of the molecule of (a) , or

(c) an analogue, fragment, mutant, derivative, or allelic variant of the molecule of (a) .

23. A method, use, or composition according to any one of claims 1 to 12, in which the polypeptide is: (a) a splice variant of BTC, in which the C₅-C₆ disulphide loop, normally present in the gene which encodes authentic BTC, is absent, and the remainder of the mature molecule, including loops A and B and the "hinge" valine, is fused in-frame to the truncated C-terminal cytoplasmic tail;

(b) a polypeptide which has a sequence substantially homologous to the molecule of (a) ; or (c) a fragment, analogue, variant, or derivative of (a) .

24. A method, use, or composition according to any one of claims 1 to 23, in which the polypeptide is capable of acting as a β-cell differentiation factor, increasing β-cell mass, decreasing the decline of β-cell function and/or increasing insulin secretion by β-cells.

25. A method, use, or composition according to any one of claims 1 to 24, wherein the risk of cancer arising in the subject as a side-effect of treatment is reduced compared to the risk entailed in treatment with BTC.

26. A method, use, or composition according to any one of claims 1 to 25, wherein the subject has one or more of the following risk factors for diabetes: impaired glucose tolerance, metabolic syndrome, a family history of Type 1 or Type 2 diabetes, obesity, polycystic ovary syndrome, hypertension, or elevated cholesterol levels.