MXPA06009373A - Methods of treating obesity or diabetes using nt-4/5 - Google Patents

Abstract

The invention concerns methods for treating obesity, non-insulin dependent diabetes mellitus, metabolic syndrome, and other relates diseases by administering an NT-4/5 polypeptide. The invention also concerns compositions and kits comprising an NT-4/5 polypeptide.

Description

METHODS OF TREATMENT OF OBESITY OR DIABETES USING? T-4/5 Field of the Invention The invention relates to the use of? T-4/5 polypeptides in the treatment and / or prevention of obesity, diabetes mellitus not dependent on insulin (type II), metabolic syndrome or related diseases.

Background of the Invention Obesity is a chronic disorder and a major concern in modern society. About 30% of adults in the United States are obese and about 65% adults are overweight. Obesity is associated not only with social stigma, but with a diminished life interval and numerous health problems including hypertension, type 2 diabetes mellitus, high concentrations of insulin in the plasma, insulin resistance, dyslipidemia, hyperlipidemia, cancer of the endometrium, breast, prostate and colon, osteoarthritis; respiratory complications such as obstructive sleep apnea, cholelithiasis, gallstones, arteriosclerosis, heart disease, abnormal heart rhythm and cardiac arrhythmias. Kopelman, P. G.,? Ature 404, 635-643 (2000). Existing therapies for obesity include standard diets and exercise, very low calorie diets, therapy Ref: 175171 of behavior, pharmacotherapy, suppressors that involve appetite, thermogenic drugs, inhibitors of food absorption, mechanical devices such as wires for the jaws, waist and balloon cords and surgery. Jung and Chong, Clinical Endrocrinology, 35: 11-20 (1991); Bray, Am. J. Clin. Nutr., 55: 538S-544 (1992). Fasting modified by the protein replacement has been reported to be effective in reducing weight in adolescents. Lee et al., Clin. Pediatr., 31: 234-236 (April 1992). However, existing therapies are not very effective for many of the obese patients. For more severe obese patients, surgical intervention may be required. Considering the high frequency of obesity in our society and the serious consequences associated with it, as discussed above, any therapeutic drug potentially useful in reducing weight in obese people could have a profound beneficial effect on their health. There is a need for a drug that reduces the total body weight of the obese subjects towards their ideal total weight without significant adverse side effects and which helps the obese subject maintain the reduced weight level. Patients with diabetes mellitus have a weakened glucose disposition due either to insufficient insulin production of the pancreatic β cells ie insulin-dependent diabetes mellitus (type I, IDDM), or due to the resistance of the target organs , that is, diabetes mellitus not dependent on insulin (type II, NIDDM). More than 90% of patients with diabetes mellitus are patients with type II diabetes. Type II diabetes mellitus is often accompanied by hyperlipidemia and an unusual high level of cholesterol or triglycerides (that is, more than 220 mg / dl of total cholesterol, or more than 150 mg / dl of triglycerides) is a risk factor for arteriosclerosis and myocardial infarction. Type II diabetes mellitus is also often accompanied by obesity. Type II diabetes mellitus, hyperlipidemia and obesity are deeply related to one another, however, in order to improve each condition, multiple medications are needed. Dietary restriction and physical exercise can only improve glucose tolerance in the early stage of the disease, but in the later stages multiple classes of anti-hyperglycemic drugs are often needed to contain the glucose level. However, none of the current anti-hyperglycemic drugs are optimal. Patients often suffer from serious side effects and many eventually require insulin. Neurotrophins are a family of small proteins, homodimeric, which play a crucial role in the development and maintenance of the nervous system. Members of the neurotrophin family include nerve growth factor (NGF), brain-derived neurotrophic factor (BD? F), neurotrophin-3 (? T-3), neurotrophin-4/5 (? T-4 / 5), neurotrophin-6 (? T-6) and neurotrophin-7 (? T-7). Neurotrophins, similar to other polypeptide growth factors, affect their target cells through interactions with cell surface receptors. According to current knowledge, two types of transmembrane glycoprotein serve as receptors for neurotrophins. Neurons with neurotrophin responses have a low molecular weight in common (65-80 kDa), a low affinity receptor (L? GFR), also known as p75? TR or p75, which binds to NGF, BD? F,? T-3 and? T-4/5 with a KD of 2xlO "9M; and a large molecular weight (130-150 kDa), high affinity receptors (KD in the range of IO '^ M), which are members of the trk family of receptor tyrosine kinases. The identified members of the Trk receiver family are TrkA, TrkB and TrkC. Both BDNF and? T-4/5 bind to TrkB and p75NTR receptors with similar affinity. However, the NT-4/5 and BD? F mutant mice show quite contrasting phenotypes. While "T-4/5" / _ mice are viable and fertile only with moderate sensory deficiency, BDNF_ / "mice die during the early stages after birth with severe neuronal deficits and behavioral symptoms. Fan et al., Nat. Neurosci. 3 (4): 350-7, 2000; Liu et al.,? Ature 375: 238-241, 1995; Conover et al.,? Ature 375: 235-238, 1995; Ernfors et al.,? Ature 368: 147-150; 1994; Jones et al., Cell 76: 989-999, 1994. Several publications report that "T-4/5 and BDNF have different biological activities in vivo and suggest that different activities may result partially from the differential activation of the TrkB receptor and its Signaling paths in the downward direction by NT-4/5 and BD? F. Fan et al.,? At. eurosci 3 (4): 350-7, 2000; Minichiello et al.,? Euron. 21: 335-45, 1998; Wirth et al., Development. 130 (23): 5827-38, 2003; López et al., Program? 38.6, 2003 Abstract, Society for? Euroscience. BD? F has been shown to have blood lipid and blood glucose controlling activity and anti-obesity activity in type II diabetic animals, C57db / db mice. Patent of E.U.A. ?or. 6,391,312; Itakura et al., Metabolism 49: 129-33 (2000). It has also been shown that BD? F has anti-obesity activity and activity to lower the resistance to lectin in mice fed a high-fat diet. Publication of E.U.A. ?or. 2003/0036512. It has also been reported that BD? F or? T-4/5 can transiently reverse the eating behavior and obesity in heterozygous BD? F agénic mice in which the expression of BDNF genes is reduced. Kernie et al., EMBO J. 19 (6): 1290-300, 2000. It has been reported that a de novo antisense mutation of the Y722C substitution in human TrkB results in weakened receptor phosphorylation and signaling for the MAP kinase; and this mutation seems to result in a unique human syndrome of hyperphagic obesity. Yeo et al., Nat. Neurosci. 7: 1187-1189 (2004). All patents, patent applications and publications cited herein are therefore incorporated in their entirety.

Brief Description of the Invention The present invention is based on the discovery that NT-4/5 is effective in the treatment of non-insulin dependent diabetes mellitus (type II diabetes) in diabetic animal models and an associated effect of NT treatment. -4/5 is the reduction in body weight, dietary intake and triglyceride levels. The present invention is also based on the discovery that NT-4/5 is effective in the reduction of body weight, control of dietary intake, control of blood glucose level and reduction of triglycerides in normal animals and obese animal models. . In one aspect, the invention provides methods for the treatment of non-insulin dependent diabetes mellitus in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. The invention also provides methods for the treatment of any of one or more of hyperglycemia, low glucose tolerance, insulin resistance, abdominal obesity, lipid disorder, dyslipidemia, hyperlipidemia, hypertriglyceridemia and metabolic syndrome in an individual comprising administering a effective amount of an NT-4/5 polypeptide to the individual. The treatment of any of these disorders may be in association with the treatment of non-insulin-dependent diabetes mellitus. In another aspect, the invention provides methods for the control of blood glucose levels in an individual having non-insulin-dependent diabetes mellitus or at risk of non-insulin-dependent diabetes comprising administering an effective amount of an anti-insulin polypeptide. 4/5 to the individual. In some modalities, the individual is human and has an HbAlc value of around 7%, around 8%, around 9%, around 10%, or higher. In some embodiments, the blood glucose level in the individual is reduced and generally maintained within the normal range. In another aspect, the invention provides methods for the control of triglyceride levels in the blood in an individual having insulin dependent diabetes mellitus or at risk of non-insulin dependent diabetes mellitus comprising administering an effective amount of a polypeptide. NT-4/5 to the individual. In some embodiments, the level of triglycerides in the blood in the individual is reduced and generally maintained within the normal range. In another aspect, the invention provides methods for improving insulin resistance in an individual having diabetes mellitus not dependent on insulin or at risk of non-insulin dependent diabetes mellitus, which comprises administering an effective amount of an NT polypeptide. -4/5 to the individual. In some embodiments, the level of insulin in the individual's blood is reduced and generally maintained within the normal range. In another aspect, the invention provides methods for the control of a body weight in an individual having diabetes mellitus not dependent on insulin or at risk for non-insulin-dependent diabetes mellitus comprising administering an effective amount of an NT-4/5 polypeptide. to the individual The invention also provides methods for maintaining body weight or preventing weight gain in an individual having diabetes mellitus not dependent on insulin, or at risk for non-insulin-dependent diabetes mellitus, which comprises administering an effective amount of a polypeptide. NT-4/5 to the individual. In some modalities, body weight is controlled or maintained due to a loss of fat content in the body. In some modalities, the body weight in the individual is reduced and generally maintained within the normal range. In another aspect, the invention provides methods for controlling the fat content in the body in an individual having diabetes mellitus not dependent on insulin, or at risk of non-insulin dependent diabetes mellitus comprising administering an effective amount of a polypeptide NT-4/5 to the individual. In some modalities, the fat content of the individual is generally maintained during the treatment. In another aspect, the invention provides methods for controlling food intake in an individual having diabetes mellitus not dependent on insulin or at risk for non-insulin-dependent diabetes mellitus comprising administering an effective amount of an NT-4 / polypeptide. 5 to the individual. In another aspect, the invention provides methods for delaying the development of non-insulin dependent diabetes mellitus in an individual, which comprises administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the onset of the disease (eg, onset of diabetes from a pre-diabetes condition) is delayed. In other modalities, the progression of diabetes (for example, development of diabetic complications) is delayed. In some modalities, the individual is at risk for diabetes mellitus not dependent on insulin. In some embodiments, the development of non-insulin-dependent diabetes mellitus in the individual is delayed or prevented. In some embodiments, the development of complications associated with non-insulin-dependent diabetes mellitus in the individual is delayed or prevented. The invention also provides methods for treating obesity (including obesity management) in an individual, comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the individual does not have a genetic deficiency of BDNF. In some modalities, obesity is associated with resistance to leptin. In some modalities, obesity is associated with diabetes mellitus not dependent on insulin. In some modalities, obesity is associated with resistance to? T-4/5. The invention also provides methods for treating any one or more of hyperglycemia, low glucose tolerance, insulin resistance, lipid disorder, dyslipidemia, hyperlipidemia, hypertriglyceridemia, and metabolic syndrome in an individual, which comprises administering an effective amount of a Polypeptide NT-4/5 to the individual. The treatment of any of these trats may be in association with the treatment of obesity. The invention also provides methods for controlling body weight in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. The invention also provides methods for maintaining body weight or preventing weight gain in an individual, which comprises administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the individual is obese. In some modalities, the individual does not have a genetic deficiency of BD? F. In some modalities, the individual is at risk of obesity or is overweight. The invention also provides methods for increasing the metabolic ratio in an individual comprising administering an effective amount of a? T-4/5 polypeptide to the individual. In some modalities, the individual is obese. In some modalities, the individual does not have a genetic deficiency of. BD? F. In some modalities, the individual is at risk of obesity or is overweight. The invention also provides methods for controlling dietary intake in an individual, comprising administering an effective amount of a? T-4/5 polypeptide to the individual. In some modalities, the individual is obese. In some modalities, the individual does not have a genetic deficiency of BDNF. In some modalities, the individual is at risk of obesity or is overweight. The invention also provides methods for controlling levels of triglycerides in the blood in an individual, comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the individual is obese. In some modalities, the individual does not have a genetic deficiency of BDNF. In some modalities, the individual is at risk of obesity or is overweight. The invention also provides methods for controlling the level of glucose in the blood in an individual, which comprises administering an effective amount of a NT-4/5 polypeptide to the individual. In some modalities, the individual is obese. In some modalities, the individual does not have a genetic deficiency of BDNF. In some modalities, the individual is at risk of obesity or is overweight. The invention also provides methods for reducing the body fat content in an individual in need of weight loss which comprises administering an effective amount of an NT-4/5 polypeptide to the individual. The treatment may not result in a significant change in fat content in the individual. In some modalities, the individual is obese. In some modalities, the individual does not have a genetic deficiency of BDNF. In some modalities, the individual is at risk of obesity or is overweight. The invention also provides methods for delaying the development of obesity in an individual, which comprises administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the onset of obesity is delayed. In other modalities, the progress of obesity (for example, development of complications associated with obesity, increase in BMI) is delayed. In some modalities, the development of obesity in the individual is avoided. In some modalities, the development of complications associated with obesity in the individual are avoided. In some modalities, the individual is at risk, obese or overweight. The invention also provides methods for treating metabolic syndrome in an individual, comprising administering an effective amount of a NT-4/5 polypeptide to the individual. The invention also provides methods for delaying the development of metabolic syndrome in an individual - comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the metabolic syndrome is avoided. In some modalities, the individual is at risk of developing metabolic syndrome. The invention also provides methods for preventing bone loss in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the bone density in the individual is generally maintained. In some modalities, the individual has osteoporosis. The invention also provides methods for reducing thyroid hormone in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the individual has hyperthyroidism. In any of the methods described herein, the NT-4/5 polypeptide can be administered in conjunction with an antibody that specifically binds to the NT-4/5 polypeptide. In some embodiments, administration in conjunction with the antibody enhances the therapeutic efficacy of the NT-4/5 polypeptide. In some embodiments, administration in conjunction with the antibody reduces the effective amount of NT-4/5 required for the treatment. In some embodiments, administration in conjunction with the antibody reduces the frequency of administration of NT-4/5. The NT-4/5 polypeptide and the antibody can be administered simultaneously or sequentially, or they can be administered in a co-formulation. In some embodiments, binding of the antibody to the NT-4/5 polypeptide does not significantly interfere with the binding of the NT-4/5 polypeptide to the TrkB receptor. Administration of the NT-4/5 polypeptide described herein may be by any means known in the art, including: parenterally, intravenously, subcutaneously, by inhalation, intraarterially, intramuscularly, intracardially, intraventricularly, intrathecally-, intraperitoneally, and transdermally Any NT-4/5 polypeptide described herein can be used for any of the methods described above. In some embodiments, the NT-4/5 polypeptide is a naturally occurring NT-4/5 (termed interchangeably "? T-4/5" herein). In some modalities, the? T-4/5 polypeptide is a human mature? T-4/5 (SEQ ID? O: l). In some embodiments, the "T-4/5" polypeptide is an allelic variant that occurs naturally from human mature "T-4/5" (SEQ ID NO: 1). In some embodiments, the NT-4/5 polypeptide comprises a? T-4/5 sequence that occurs naturally with one or more amino acid substitutions. In some embodiments, the? T-4/5 polypeptide is linked to one or more PEG molecules. In some embodiments, the NT-4/5 polypeptide is linked to one or more PEG molecules at the N-terminus of the? T-4/5 polypeptide. In some embodiments, the? T-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with the amino acid residue G at position 1 changed to S or T, and wherein the? T-4/5 polypeptide it is linked to a PEG molecule at position 1. In some embodiments, the? T-4/5 polypeptide comprises the amino acid sequence of SEQ ID? 0: 1 with amino acid residue S at position 50 changed to C, and wherein the NT-4/5 polypeptide is linked to a PEG molecule at position 50. In another aspect, the invention provides pharmaceutical compositions comprising an effective amount of an NT-4/5 polypeptide, wherein the polypeptide is T-4/5 is linked to one or more PEG molecules. In some embodiments, the? T-4/5 polypeptide comprises one or more amino acid substitutions compared to the naturally occurring sequence of? T-4/5 and the substituted amino acids are linked to one or more PEG molecules. In some embodiments, the? T-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with amino acid residue G in position 1 changed to S or T, and wherein the? T-4/5 polypeptide is ligated to a PEG molecule at position 1. In some embodiments, the "T-4/5" polypeptide comprises the amino acid sequence of SEQ ID? 0: 1 with an amino acid residue substitution, and wherein the? T-4/5 is linked to a PEG molecule in the substituted position (for example, S at position 50 changed to C). In another aspect, the invention provides compositions comprising an effective amount of a? T-4/5 polypeptide and an antibody that specifically binds to the? T-4/5 polypeptide. In some embodiments, the? T-4/5 polypeptide and the antibody are present in a predetermined ratio. In some embodiments, binding of the antibody to the NT-4/5 polypeptide does not significantly interfere with the binding of the NT-4/5 polypeptide to the TrkB receptor. In another aspect, the invention provides kits comprising an NT-4/5 polypeptide for use in any of the methods described herein. In some embodiments, the kit comprises a container, a composition comprising an NT-4/5 polypeptide described herein, in combination with a pharmaceutically acceptable carrier, and instructions for using the composition in any of the methods described herein. . The kits may further comprise an antibody that specifically binds to the NT-4/5 polypeptide and an instruction to administer the antibody in conjunction with the NT-4/5 polypeptide. The methods described herein are generally for treating an individual in need of treatment.

Brief Description of the Figures Figure 1 is a graph showing the effect of? T-4/5 on blood glucose levels in db / db mice. The glucose levels in the blood are expressed as mean + SEM (nM). "*" indicates a statistically significant difference (p <0.05) compared to the vehicle. The "X" axis corresponds to days since the experiment began and the "Y" axis corresponds to blood glucose concentration (mM). Figure 2 is a graph showing the effects of? T 4/5 on the HbAlc level in db / db mice. The HbAlc values are expressed as mean + • SEM (%) -. "*" indicates a statistically significant difference (p <0.05) compared to the vehicle. The "X" axis corresponds to days since the experiment began and the "Y" axis corresponds to HbAlc (%). Figure 3 is a graph showing the effect of NT-4/5 on insulin resistance in db / db mice. Levels of insulin in the blood are expressed as mean _ + SEM (nM). "*" indicates a statistically significant difference (p <; 0..05) compared to the vehicle. The "X" axis corresponds to days since the experiment began and the "Y" axis corresponds to the insulin level (nM). Figure 4 is a graph showing the effect of NT-4/5 on obesity in db / db mice. The body weight is expressed as mean + SEM (g). , * "indicates a statistically significant difference (p <0.05) compared to the vehicle The" X "axis corresponds to days since the experiment began and the" Y "axis corresponds to body weight '(g). is a graph that shows the effect of NT 4/5 on dietary intake in db / db mice.Food intake is expressed as mean + _ SEM (g / kg / day). "*" indicates a statistically significant difference (p <0.05) compared to the vehicle The "X" axis corresponds to days since the experiment began and the "Y" axis corresponds to food intake (g / kg / day).

Figure 6A are graphs showing the blood glucose levels of db / db mice treated with different doses of NT-4/5 to TO (baseline, left) and T6 (right) . The "X" axis corresponds to doses of NT-4/5 (mg / kg) and the "Y" axis corresponds to blood glucose concentration (mg / dL). Figure 6B are graphs showing triglyceride levels in the blood of db / db mice treated with different doses of NT-4/5 to TO (baseline, left) and T6 (right). The "X" axis corresponds to doses of NT-4/5 (mg / kg) and the nY "axis corresponds to the concentration of triglycerides in the blood (mg / dL) Figure 6C are graphs showing cholesterol levels in the blood of db / db mice treated with different doses of NT-4/5 to TO (baseline, left) and 'T6 (right) .The "X" axis corresponds to doses of NT-4/5 (mg / kg) and the "Y" axis corresponds to blood cholesterol concentration (mg / dL) Figure 7 is a graph showing the response to the dose of? T-4/5 in the dietary intakes of db / mice db. "*" indicates a statistically significant difference (p <0.05) compared to the vehicle. The "X" axis corresponds to days since the experiment began and_ the axis "Y" corresponds to food intake (g). Different doses of NT-4/5 are indicated in the graph.

Figure 8A is a graph showing the effect of different doses of NT-4/5 on the body weights of db / db mice. "*" indicates a statistically significant difference (p <0.05) compared to the vehicle. The "X" axis corresponds to days since the experiment began and the "Y" axis corresponds to body weight (g). Different doses of NT-4/5 are indicated in the graph. Figure 8B is a graph showing the effect of different doses of NT-4/5 on the percentage of changes in body weight in db / db mice. "*" indicates a statistically significant difference (p <0.01) compared to the vehicle. The "X" axis corresponds to days since the experiment began and the "Y" axis corresponds to the percentage change in body weight in mice treated with NT-4/5. Different doses of NT-4/5 are indicated in the graph. Figure 9A is a graph showing the effect of different doses of? T-4/5 on the level of glucose without fasting in the blood in obese and diabetic polygenic mice (? O? C? ZO-10). The black bars on the X axis indicate the treatment period. "*" indicates a statistically significant difference of P < 0.05 compared to the vehicle; "**" indicates a statistically significant difference of P < 0.01 compared to the vehicle; and "***" indicates a statistically significant difference of P < 0.001 compared to the vehicle. A? OVA two-way followed by subsequent Bonferroni tests were used for statistical analysis. Figure 9B is a graph showing the effect of different doses of NT-4/5 at the HbAlc level on day 30 in obese and diabetic polygenic mice (NONcNZO-10). One-way ANOVA (F = 7.249, P = 0.0057), followed by subsequent Dunnett tests were used for statistical analysis. The values of P are relative to the vehicle group as indicated in the graph. Figures 10A and 10B are graphs showing the effect of different doses of NT-4/5 on body weight (Fig. 10A) and dietary intake (Fig. 10B) in obese and diabetic polygenic mice (NONcNZO-10). Black bars on the X axis indicate the treatment period. Figures HA and 11B are graphs showing the effect of different doses of NT-4/5 on body weight (Fig. HA) and dietary intake (Fig. 11B) in obese mice induced by a high-fat diet (DIO mice) ). Figures 12A and 12B are graphs showing the effect of different doses of NT-4/5 on fasting glucose (Fig. 12A) and oral glucose tolerance (Fig. 12B) in obese mice induced by a high-fat diet (DIO mice) which were diabetic. The fasting glucose level and oral glucose tolerance tests were carried out on day 60 after three daily doses of NT-4/5.

Figure 13 is a graph showing phosphorylation of the tyrosine receptor in a cell line expressing TrkB by different concentrations of PEG-NT4-S50C (S50C.2) or NT-4/5. Figures 14A and 14B are graphs showing the effect of a single subcutaneous injection of 1 mg / kg PEG-NT4-S50C on body weight (Fig. 14A) and dietary intake (Fig. 14B) in obese and diabetic polygenic mice (Fig. 14A). NONcNZO-10). The vertical arrow indicates the time of the simple injection of either NT-4/5 or PEG-NT4-S50C. The horizontal bars indicate the period of daily subcutaneous injection for wild-type NT-4/5. Two-way ANOVA followed by Bonferroni's subsequent test were used for statistical analysis. "*" indicates a statistically significant difference of P < 0. 05 compared to the vehicle; "**" indicates a statistically significant difference of P < 0.01 compared to the vehicle; and "***" indicates a statistically significant difference of P < 0.001 compared to the vehicle. Figure 15 is a graph showing phosphorylation of the tyrosine receptor in a cell line expressing TrkB by different concentrations of 1PEG-? T4-G1S (GIS 1PEG), 2PEG-? T4-GIS (GIS 2PEG), or? T -4/5. Figure 16 is a graph showing the serum half-life of 2P? G-? T4-G1S and? T-4/5 (? T4) after a single subcutaneous dose of 4 mg / kg in the db / db mice . Figure 17 is a graph showing the effect of a simple subcutaneous injection of N-4/5, 2PEG-NT4-G1S, and 1PEG-NT4-G1S on the fasting serum glucose levels of db / db mice. Figure 18 is a graph showing changes in body weight after weekly dosing of wild-type NT-4/5 (WT) (referred to as WT NT4) against pegylated Gl S? T-4/5 (al which is referred to as Peg GIS? T4) in DIO mice. The data are expressed in average values with the error bars that represent the standard errors of means (-SEM). The treatment groups differed significantly by the 2-way A? OVA (F = 207.01, P < 0.0001). Figure 19 is a graph showing changes in food intake after weekly dosing of wild-type T-4/5 (referred to as WT? T4) against pegilated Gl S? T-4/5 (to which it is referred to as PEG? T4) in DIO mice. The data are expressed in average values with the error bars representing the standard errors of means (SEM). "*" indicates P < 0.05 and "**" indicates P < 0.01 for the corresponding time point and the type of treatment compared to the vehicle control group. Figure 20 is a graph showing the effect of the daily subcutaneous injection of? T-4/5 (lOmg / kg) to the respiratory quotient (RQ = Vco / Vo2, that is, the production ratio of carbon dioxide over the oxygen consumption measured by CCMS) in DIO mice. The individual data points are expressed (P = 0.0025, Student's t test). Figures 2lA, 21B, 21C and 21D are graphs showing the effect of daily subcutaneous injection of NT-4/5 (10 mg / kg) on body weight (table 21A), dietary intake (table 21B), content of body fat (table 21C), and percentage of fat-free mass in the body (table 21D) in DIO mice. The data are expressed in means with error bars representing SEM. Statistical analysis was done by JANOVA 2-way with pairwise comparisons by subsequent Bonferroni tests (* P < 0.05; ** P < 0.01; *** P < 0.001). Figure 22 is a graph showing the effect of weekly intravenous injection of NT-4/5 (2mg / kg) and BDNF (2mg / kg) on body weight in DIO mice. The data are expressed in means with error bars representing SEM. Statistical analysis was done by A? OVA 2-way with pairwise comparisons by Bonferroni's subsequent tests. "*" indicates P < 0.05; "**" indicates P < 0.01; and "***" indicates P <0.001 for the corresponding treatment point and the type of treatment compared to the vehicle control group Figure 23 is a graph showing the effect of the weekly intravenous injection of? T -4/5 (2 mg / kg) and BD? F (2 mg / kg) on dietary intake in DIO mice.The data are expressed in means with error bars representing SEM.The statistical analysis was made by means of ANOVA of 2 tracks with pairwise comparisons through Bonferroni's subsequent tests. "*" Indicates P <; 0.05; "**" indicates P < 0.01; and "***" indicates P <0.001 for the corresponding time point and type of treatment compared to the vehicle control group • Figure 24 is a graph showing the effect ^ of daily subcutaneous injection of NT-4 / 5 (1, 2, 5 and 10 mg / kg) on body weight in male C57BL / 6 non-fat mice The data are expressed in means with error bars representing SEM .. The statistical analysis was made by means of ANOVA of 2 tracks with pairwise comparisons by subsequent Bonferroni tests of each treatment in relation to vehicle control. "*" Indicates P <0.05;? ** "indicates P < 0.01; and "***" indicates P <0.001 for the corresponding time point and type of treatment compared to the vehicle control group Figure 25 is a graph showing the effect of daily subcutaneous injection of NT-4/5 (1, 2, 5 and lOmg / kg) on dietary intake in C57BL / 6 non-fat male mice.The data are expressed in means with error bars representing SEM.The statistical analysis was made by 2-way ANOVA with comparisons in pairs through subsequent Bonferroni tests of each treatment relating to vehicle control, "*" indicates P <0.05; "**" indicates P <0.01; and "***" indicates P <0.001 for the point of corresponding time and type of treatment compared to the vehicle control group Figure 26 is a graph showing the effect of daily subcutaneous injection of NT-4/5 (1, 2, 5 and 10 mg / kg) on blood glucose without fasting in male C57BL / 6 fat-free mice The data are expressed in bar stockings s of error that represent SEM. Statistical analysis was made by means of 2-way ANOVA with pairwise comparisons by subsequent Bonferroni tests of each treatment relative to vehicle control. "*" indicates P < 0.05; "**" indicates P < 0.01; and "***" indicates P <0.001 for the corresponding time point and type of treatment compared to the vehicle control group Figure 27 is a graph showing the survival percentage of female db / db mice after daily subcutaneous injection of NT-4/5 (20 mg / kg / day) from day 1 to day 26 compared to the vehicle control group Figure 28A, 28B, 28C and 28D are graphs showing the effect of NT-4/5 in the control of glucose in the long term (daily subcutaneous injections from day 1 to day 26) in db / db mice. Figure 28A shows the level of HbAlc on day 45 (15 days after dosing of NT-4/5). Figure 28B shows the glucose tolerance test carried out on day 54 (28 days after dosing of NT-4/5). Figure 28C shows the serum glucose level from day 0 to day 30. Figure 28D shows the level of insulin in the blood from day 0 to day 20. Figure 29A is a graph showing the effect of NT4 / 5 and / or Mab 124? in body weights in db / db mice. Body weights are expressed as mean + SEM (% of baseline). "*" indicates a statistically significant difference (p <0.05) compared to the vehicle, "**" represents p <0.01, "***" represents p < 0.001. The "X" axis corresponds to days since the experiment began and the "Y" axis corresponds to body weight (% of baseline). Figure 29B is a graph showing the effect of? T4 / 5 and / or Mab 1241 on dietary intake in db / db mice. Food intake is expressed as mean + SEM (g). ,? * "indicates a statistically significant difference (p <0.05) compared to the vehicle," ** "represents p <0.01;« *** H represents p <0.001, the "X" axis corresponds to days from which started the experiment and the "Y" axis corresponds to food intake (g) Figure 29C is a graph showing the effect of NT4 / 5 and / or Mab 1241 on blood glucose levels in db / db mice Blood glucose levels are expressed as mean + SEM (mg / dL). "*" Indicates a statistically significant difference (p <0.05) compared to the vehicle, "**" represents p <0.01; "* ** »represents p < 0.001. The UX axis "corresponds to days since the experiment began and the" Y "axis corresponds to food intake (g) The" X "axis corresponds to days since the experiment began and the" Y "axis corresponds to glucose concentration in the blood (mg / dL).

Detailed Description of the Invention The present invention is based on the discovery that administration of a therapeutically effective amount of an NT-4/5 polypeptide can be used to treat obesity and non-insulin-dependent diabetes mellitus.

General Techniques The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill in the art. Such techniques are fully explained in the literature, such as, Molecular Cloning: A Laboratory Manual, second edition (Sambrook, et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (M. J. Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (J. E. Cellis, ed., 1998) Academic Press; Animal Cell Culture (R. I. Freshney, ed., 1987); Introduction to Cell and Tissue Culture (J. P. Mather and P. E. Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, J. B. Griffiths, and D. G. Newell, eds., 1993-8) J. Wiley and Sons; Methods in Enzymology "(Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel, et al., Eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis, et al., Eds., 1994); Current Protocols in Immunology (JE Coligan et al., Eds., 1991 ), Short Protocols in Molecular Biology (Wiley and Sons, 1999), Immunobiology (CA Janeway and P. Travers, 1997), Antibodies (P. Finch, 1997), Antibodies: a practical approach (D. Catty., Ed., IRL Press, 1988-1989), Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000), Using antibodies: a laboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999), The Antibodies (M. Zanetti and JD Capra, eds., Harwood Academic Publishers, 1995).

Definitions As used herein, "treatment" is a method for obtaining clinical, beneficial or desired results. For purposes of this invention, beneficial or desired clinical outcomes include but are not limited to, one or more of the following: improving, decreasing severity, alleviating one or more of the symptoms associated with a disease. For example, non-insulin dependent diabetes mellitus, beneficial or desired clinical outcomes include any of the improvement of blood glucose control, blood lipid control and / or insulin resistance. In some embodiments, beneficial or desired clinical outcomes may include improving, decreasing severity and / or alleviating any of the disorders associated with non-insulin-dependent diabetes mellitus such as hyperglycemia, low glucose tolerance, insulin resistance, hyperinsulinemia. , obesity, lipid disorders, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, metabolic syndrome, diabetic nephropathy, diabetic neuropathy, diabetic retinopathy, diabetic microvascularity and other associated diseases known and described herein. For obesity, clinical, beneficial or desired outcomes include any of the reduction or maintenance of body weight, control of food intake (including reduction) or caloric intake; increase in metabolic rate or inhibition of reduction of metabolic rate and improvement, decrease in severity and / or relief of any of the disorders associated with obesity such as diabetes, diabetes mellitus not dependent on insulin, hyperglycemia low tolerance to glucose, insulin resistance, lipid disorder, dyslipidemia, hyperlipidemia, hypertriglyceridemia, hypercholesterolemia, abdominal obesity, eating disorders, metabolic syndrome, hypertension, osteoarthritis, myocardial infarction, stroke, and other associated diseases known and described in the I presented; increase in the quality of life of those who suffer from obesity and / or prolongation of the life interval. An "effective amount" is an amount sufficient to effect beneficial or desired clinical outcomes including the improvement, alleviation and / or reduction of one or more of the symptoms associated with a disease. For the treatment of non-insulin dependent diabetes mellitus, an effective amount of NT-4/5 is an amount sufficient to treat or ameliorate one or more of the symptoms associated with non-insulin-dependent diabetes mellitus. An "Effective amount" is an amount sufficient to result in one or more of the following (which may also correspond to various embodiments of the invention): reducing blood glucose levels and / or blood lipid levels; reduce hyperinsulinemia; improve insulin resistance; improve obesity and / or control the dietary intake that accompanies diabetes. To treat obesity, an effective amount of a NT-4/5 polypeptide is an amount sufficient to treat or ameliorate one or more of the symptoms associated with obesity. An "effective amount" is an amount sufficient to result in one or more of the following (which may also correspond to various embodiments of the invention): reduce body weight, control dietary intake, increase metabolic ratio, decrease one or more of the symptoms that result from diseases associated with obesity increase the quality of life of those who suffer from obesity and / or prolong the life interval. An effective amount may or may not be achieved in conjunction with another drug, compound or pharmaceutical composition. Thus, an "effective amount" can be considered in the context of administering one or more therapeutic agents and a single agent can be considered to be given an effective amount if in conjunction with one or more other agents., the desirable result can be achieved. An "effective amount" can be given in one or more administrations. As used herein, the "retardation" of the development of type II diabetes means deferring, concealing, slowing down, retarding, stabilizing and / or postponing the development of the disease. This delay may be of variable lengths of type depending on the history of the disease and / or the individual to be treated. As is evident to one skilled in the art, a sufficient or significant delay may in effect encompass the prevention in which the individual does not develop the disease. The "development" of type II diabetes means the onset and / or progression of the disease with an individual (which may be different embodiments of the invention). The development of type II diabetes can be detected by using standard clinical techniques as described herein. However, development also refers to the progression of a disease in which it may initially be undetectable. For the purposes of this invention, the advance refers to the biological course of the disease state in this case, when determined by the blood glucose tests, lipids, insulin levels, glucose tolerance test, level of Hb-Alc, as well as the onset and / or worsening of diabetic complications such as cardiovascular diseases, nephropathy, retinopathy and / or neuropathy. A variety of these diagnostic tests include, but are not limited to, blood pressure, resting and exercise electrocardiogram (EKG), echocardiogram, angiocardiogram, blood urea nitrogen (BUN), creatinine level, urinalysis, renal biopsy, glomerular filtration ratio (GFR), ophthalmoscopic examination, conduction velocity in the motor and sensory nerves and biopsy in the peripheral nerve. "Development" includes occurrence, recurrence and beginning. As used in the present "beginning" or "occurrence" of type II diabetes, it includes the onset and / or initial recurrence. As used herein, an individual "at risk" of developing type II diabetes may or may not have a detectable disease or may or may not have a detectable disease displayed prior to the described treatment methods. "At risk" denotes an individual who has one or more of the so-called risk factors, which are measured parameters that correlate with the development of type II dependent diabetes. An individual who has one or more of these risk factors is more likely to develop type II diabetes than an individual without these risk factors. These risk factors include but are not limited to age, diet, physical inactivity, metabolic syndrome, obesity, family history of obesity and / or diabetes, ethnicity (eg, Pima Indians, African-Americans and Hispanics are at greater risk), syndromes hereditary (for example, diabetes at the beginning of the maturity of MODY youth), history of previous illness (for example, a history of gestational diabetes for women), presence of precursor disease (that is, pre-diabetes). The term "metabolic syndrome", also known as syndrome X, is defined in the third report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults (ATP-III). ES Ford et al., JAMA, Vol. 287 (3), Jan. 16, 2002, pp 356-359. Briefly, a person is defined as having metabolic syndrome if the person has three or more of the following disorders: abdominal obesity, hypertriglyceridemia, low HDL cholesterol, high blood pressure, and high fasting plasma glucose. The criteria for these are defined in ATP-III as: abdominal obesity when measured by a waist circumference greater than 40 inches (101.6 cm) for men or greater than 35 inches (88.9 cm) for women; fasting blood triglycerides greater than or equal to 150 mg / dL; HDL cholesterol in blood less than 40 mg / dL for men or less than 50 mg / dL for women; blood pressure equal to or greater than 130/85 mmHg; fasting glucose greater than or equal to 110 mg / dL. The treatment of the metabolic syndrome refers to the administration of an NT-4/5 polypeptide described herein to a subject with metabolic syndrome to obtain clinical, beneficial or desired results. For the purposes of this invention, the clinical, beneficial and desired results include but are not limited to one or more of the following: improvement, decrease in severity, relief of one or more of the symptoms associated with the disease. For example, for the treatment of the metabolic syndrome, the beneficial or desired clinical results include the reduction of one or more of abdominal obesity, triglycerides, HDL cholesterol, blood pressure and blood glucose level. As used herein, the "retardation" of the development of the metabolic syndrome means defer, hinder, slow down, retard, stabilize and / or postpone the development of the disease. This delay can be of variable lengths of time, depending on the history of the disease and / or the individual to be treated. As is obvious to one skilled in the art, a sufficient or significant delay may in effect encompass the prevention in which the individual does not develop the disease. "Development" of the metabolic syndrome means the beginning and / or progression of the disease within an individual (which may be different embodiments of the invention). The development of the metabolic syndrome can be detected using standard clinical techniques known in the art and as described herein. However, development also refers to the progression of the disease that may initially be undetectable. For the purposes of this invention, the advance refers to the biological course of the disease state in this case, as determined by measuring abdominal circumference, testing triglycerides, HDL, blood pressure, blood glucose, tolerance test glucose, Hb-Alc level, as well as the onset and / or- worsening of the metabolic syndrome complications such as cardiovascular diseases and diabetes. "Development" includes presence, recurrence and beginning. As used herein, "onset" or "presence" of metabolic syndrome includes the initial onset and / or recurrence. As used herein, an individual "at risk" for developing the metabolic syndrome may or may not have a detectable disease and may or may not have developed a detectable disease prior to the methods of treatment described herein. For example, in an individual it may have two or fewer disorders or no disorders that define the metabolic syndrome. "at risk" means an individual who has one or more of the so-called risk factors, which are measurable parameters that correlate with the development of the metabolic syndrome. An individual who has one or more of these risk factors has a greater likelihood of metabolic syndrome than an individual without these risk factors. As used herein, "control of blood glucose level" or "improvement in blood glucose level" refers to the reduction of the blood glucose level in an individual (as compared to the level before treatment). In some embodiments, the blood glucose level generally remains within the normal range. In some embodiments, the blood glucose level is reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60% or 70% in the individual compared to the level before the treatment. As used herein, "control of the level of blood lipids" or "improvement in the level of blood lipids" refers to the reduction of the level of lipids in the blood (such as the level of triglycerides in the blood). blood, cholesterol level and / or level of non-esterified fatty acids) in an individual (compared to the level before treatment). In some embodiments, the level of blood lipids (such as triglyceride level in the blood, cholesterol level and / or level of non-esterified fatty acids) is generally maintained within the normal range. In some embodiments, the level of triglycerides in the blood is reduced by at least about any of 10%, 20%, 30%, 40%, 50%, 60% or 70% in the individual as compared to the level before the treatment As used herein, "insulin resistance" refers to a condition wherein the level of insulin to be required to show insulin activity at the same level as a healthy person is much greater than that of the healthy person. That is, it means a condition where insulin activity or sensitivity for insulin is reduced. The target organ for insulin activity includes a liver, muscle (skeletal muscle) and adipose tissue. The insulin shows an activity of suppression of gluconeogenesis, an activity of suppression of glucose release, etc. in the liver Insulin shows an activity promoting the absorption of glucose in the muscle (skeletal muscle) and adipose tissues. The translocation of the glucose transport carrier called GLUT4 from the cytoplasm to the surface of the cell membrane participates in the absorption of glucose. These activities can be measured when evaluating insulin resistance. The clinical evaluation of insulin resistance includes, for example, glucose tolerance test, euglycemic hyperinsulinemic clamp method (or "glucose clamp" in short), Homeostasis Model Assessment (HOMA, ref.; Diabetes Care 23: 57-63, 2000), etc. As used herein, "improved insulin resistance" refers to reversing (which may be partial or complete) the reduced sensitivity of cells to insulin (for example, reduced sensitivity manifests as one or more of the reduction of glucose absorption in peripheral tissues, intensification of glycogenolysis, intensification of gluconeogenesis, etc., all of which are observed in type II diabetes mellitus). For such an improvement, the level of insulin in the blood is reduced (and the level of glucose in the blood can be lowered). Improved insulin resistance can be assessed by the glucose tolerance test, glucose clamp or HOMA. As used herein, "body weight in control" or "improvement in body weight" refers to the reduction of body weight in an individual (as compared to the level before treatment). In some modalities, body weight generally remains within the normal range. Body weight can be reduced by reducing caloric intake and / or reducing the accumulation of body fat. In some embodiments, the body weight is reduced by at least about 5%, 10%, 20%, 30%, 40% or 50% in the individual compared to the level before treatment. As used herein, "food intake in control" refers to the reduction of food intake in an individual (as compared to the level before treatment). In some modalities, food intake is generally maintained in the normal range. In some modalities, food intake is reduced by about either 5%, 10%, 20%, 30%, 40%, 50% or 60% in the individual compared to the level before treatment.

As used herein, "obesity" is a condition in which there is an excess of body fat in a subject. Obesity can be due to any cause, whether genetic or environmental. The operative definition of obesity is based on the body mass index (BMI), which is calculated as body weight by height in square meters (kg / m2). "Vobidity" refers to a condition whereby an otherwise healthy subject has a body mass index (BMI) greater than or equal to 30.0 kg / m2, or a condition whereby a subject with at least one subject orbility has a BMI greater than or equal to 27.0 kg / m2.A "obese subject" is an otherwise healthy subject with a body mass index (BMI) greater than or equal to 30.0 kg / m2 or a subject with at least a co-morbidity with a BMI greater than or equal to 27.0 kg / m2.An obese subject may have a BMI of at least about any of 31.0, 32.0, 33.0, 34. 0, 35.0, 36.0, 37.0, 38.0, 39.0 and 40.0. An "overweight subject" is a subject with a BMI of 25.0 to 29.9 kg / m2.

Different countries can define obesity and overweight with a different BMI. The term "obesity" means that it covers the definitions of all countries. For example, the increased risks associated with obesity occur for a lower body mass index (BMI) in Asians. In Asian countries including Japan, obesity refers to a condition whereby a subject with at least one co-morbidity related to obesity or induced with obesity, who requires a reduction in weight or who would be improved by a reduction in weight has a BMI greater than or equal to 25.0 kg / m2 In Asian countries including Japan, an "obese subject" refers to a subject with at least one co-morbidity related to obesity or induced by obesity that requires a reduction in weight or that would be improved by a reduction in weight with a BMI greater than or equal to 25.0 kg / m2. Co-morbidities related to obesity or obesity-induced co-morbidities include but are not limited to diabetes, non-insulin-dependent type II diabetes mellitus, limited glucose tolerance, fasting weakened glucose, insulin resistance syndrome , dyslipidemia, hypertension, hyperuricacidemia, gout, coronary artery disease, myocardial infarction, angina pectoris, sleep apnea syndrome, Pickwickian syndrome, fatty liver, cerebral infarction, cerebral thrombosis, transient ischemic attack, orthopedic disorders, deformed arthritis, lumbodynia, emeniopathy and infertility. In particular, co-morbidities include: hypertension, hyperlipidemia, dyslipidemia, glucose intolerance, cardiovascular disease, sleep apnea, diabetes mellitus, and other conditions related to obesity. An individual at risk of obesity may or may not have a detectable disease, and may or may not have a detectable disease displayed prior to the treatment methods described herein. "At risk" denotes an individual who has one or more of the so-called factors risk factors which are measurable parameters that correlate with the development of obesity.An individual who has one or more of these risk factors is more likely to be obese than an individual without these risk factors.These risk factors include but they are not limited to age, diet, physical inactivity, metabolic syndrome, family history of obesity, ethnicity, hereditary syndromes, history of previous illnesses (for example, eating disorders, metabolic syndrome and obesity), presence of precursor disease ( for example, overweight.) For example, an otherwise healthy individual with a BMI of 25.0 to less than 30.0 kg / m2 or an individual with A co-morbidity with a BMI of 25.0 kg / m2 to less than 27.0 kg / m2 is at risk of obesity. As used herein, "delaying" the development of obesity means deferring, hinder, slow down, slow down, stabilize and / or postpone the development of the disease. This delay can be of variable duration of time depending on the history of the disease and / or the individual to be treated. As is clear to one skilled in the art, a sufficient or significant delay may in effect encompass the prevention in which the individual does not develop the disease. For example, one result of delaying development may be to reduce the body weight of a subject at risk of obesity relative to the body weight of the subject immediately prior to the administration of the polypeptides or the compositions described herein. Another result of delaying development may be avoiding the recovery of body weight previously lost as a result of diet, exercise or pharmacotherapy. Another result of delaying development may be to prevent obesity from occurring if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. Another result of delaying development may be to decrease the presence and / or severity of obesity-related disorders if the treatment is administered prior to the onset of obesity in a subject at risk of obesity. "Development" of obesity means the beginning and / or progression of the disease within an individual (which may be in different embodiments of the invention). The development of obesity may be detectable by using standard clinical techniques as described herein. However, development also refers to the progression of the disease that may be initially undetectable. For purposes of this invention, the advance refers to the biological course of the disease state in this case, as determined by determining the height and weight by the estimated BMI, measuring the circumference of the waist, assessing co-morbidities as well as the beginning and / or worsening of complications of obesity such as atherosclerosis, type II diabetes, polycystic ovarian disease, cardiovascular diseases, osteoarthritis, dermatological disorders, hypertension, insulin resistance, hypercholesterolemia, hypertriglyceridemia and cholelithiasis. A variety of these diagnostic tests are known in the art. "Development" includes presence, recurrence and beginning. As used herein, "the onset" or "occurrence" of obesity includes the initial onset and / or recurrence. An "individual" or "subject" is a mammal, more preferably a human. Mammals include but are not limited to farm animals, sport animals, pets, primates, horses, dogs, cats, mice and rats. An individual having a genetic deficiency of BDNF refers to an individual having a mutation of at least one BDNF site in the individual's genome which results in a lower level of BDNF expression (eg, the BDNF transcripts in the hypothalamus) or biological activity when compared to an average level in the population of the same species that does not have the mutation. A lower level of expression or biological activity of BDNF can be assessed by another phenotype associated with the lower level of expression or biological activity of BDNF such as mental retardation, memory weakness and / or learning deficiency. In some 'modalities, the level of BDNF expression or BDNF activity in the individual having the mutation may be less than about any of 30%, 40%, 45%, 50%, 55%, 60%, 70% of the level or activity of the average of the same species. In some embodiments, the level of expression of BDNF or biological activity is less than about 50%. In some modalities the individual has only one functional BDNF allele. In some modalities, the individual is a mouse. In some modalities the individual is not human. In some modalities the individual has obesity that is caused by the BD? F place mutation in the individual's genome. An "antibody" is an immunoglobulin molecule capable of specifically binding to an objective such as a carbohydrate, polynucleotide, lipid, polypeptide, etc., through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. As used herein, the term encompasses not only intact polyclonal or monoclonal antibodies but also fragments thereof (such as Fab, Fab ', F (ab') 2, Fv), single chain mutants (ScFv) thereof, fusion proteins comprising a portion of antibodies, and any other modified configuration of the immunoglobulin molecule comprising an antigen recognition site. An antibody includes an antibody of any kind such as IgG, IgA or IgM (or subclass thereof) and the antibody - does not need to be of any particular kind. Depending on the amino acid sequence of the constant domain of its heavy chains, immunoglobulins can be assigned to different classes, there are 5 important classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of these can be further divided into subclasses ( isotypes) for example, IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. the heavy chain constant domains corresponding to the different classes of immunoglobulins are termed alpha, delta, epsilon, gamma, and mu, respectively. The subunit structures and the three-dimensional configurations of different classes of immunoglobulins are well known. It should be noted that as used herein, the singular form, "a", "an", and "the" includes plural references unless otherwise indicated.

Methods of the Invention In some embodiments, the invention provides methods for the treatment of non-insulin dependent diabetes mellitus in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual.

Non-insulin dependent diabetes mellitus (also called type 2 diabetes) is related to insulin resistance. Diagnostic methods for non-insulin dependent diabetes mellitus are well known in the art. In some embodiments, the invention provides methods for the treatment of any one or more of hyperglycemia, low glucose tolerance, insulin resistance, abdominal obesity, lipid disorder, dyslipidemia, hyperlipidemia, hypertriglyceridemia, and metabolic syndrome in an individual. which comprises administering an effective amount of a NT-4/5 polypeptide to the individual. The treatment of any of these disorders may be in association with non-insulin dependent diabetes mellitus treatment. In some embodiments, the invention provides methods for controlling blood glucose levels in an individual having diabetes mellitus not dependent on insulin or at risk for non-insulin dependent diabetes mellitus comprising administering an effective amount of an NT-4 polypeptide / 5 to the individual. In some embodiments, the invention provides methods for controlling triglyceride levels in the blood in an individual having non-insulin-dependent diabetes mellitus or at risk for non-insulin-dependent diabetes mellitus comprising administering an effective amount of an NT-4 polypeptide. / 5 to the individual. In some embodiments, the invention provides methods for improving insulin resistance in an individual having non-insulin-dependent diabetes mellitus or at risk for non-insulin-dependent diabetes mellitus comprising administering an effective amount of a TN-4/5 polypeptide to the individual In some embodiments, the invention provides methods of controlling body weight in an individual having non-insulin dependent diabetes mellitus or at risk of non-insulin dependent diabetes mellitus comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some embodiments, the invention provides methods for controlling food intake in an individual having diabetes mellitus not dependent on insulin or at risk for non-insulin dependent diabetes mellitus comprising administering an effective amount of an NT-4/5 polypeptide to the patient. individual. In some embodiments, the invention provides methods for delaying the development of non-insulin dependent diabetes mellitus in an individual, which comprises administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the individual is at risk for diabetes mellitus not dependent on insulin. The individual having normal blood glucose level but abnormally high blood insulin level, or having blood glucose levels in the high normal range or higher glucose levels can be treated with the NT polypeptide. 4/5 to delay the onset of diabetes. Diabetic complications may also be delayed by the administration of the? T-4/5 polypeptide. In some modalities, the development of non-insulin-dependent diabetes mellitus in the individual is avoided. In some modalities, the development of complications associated with diabetes mellitus not dependent on insulin in the individual is avoided. In some embodiments, the invention also provides methods for the treatment of obesity in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the individual is human. In some modalities, the individual does not have a genetic deficiency of BD? F. Consequently, obesity may be due to any cause, whether genetic (except caused by genetic deficiency of BD? F) or environmental. In some modalities, obesity is associated with resistance to leptin. In some modalities, obesity is associated with diabetes mellitus not dependent on insulin. In some modalities, obesity is associated with resistance to NT-4/5. The treatment of obesity includes the treatment of an individual whose body weight is reduced after treatment and is no longer obese. In some embodiments, the invention also provides methods for the treatment of any one or more of hyperglycemia, low glucose tolerance, insulin resistance, abdominal obesity, lipid disorder, dyslipidemia, hyperlipidemia, hypertriglyceridemia, and metabolic syndrome in a an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. The treatment of any of these disorders may be in association with the treatment of obesity. In some embodiments, the invention also provides methods for controlling body weight, controlling food intake, increased metabolic rate, controlling blood glucose level, controlled triglyceride level, or reducing the body fat content in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the individual is obese. In some modalities the individual does not have a genetic deficiency of BDNF. In some modalities, the individual is at risk of obesity or overweight. In some embodiments, the invention also provides methods for delaying the development of obesity in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the onset of obesity is delayed. In other modalities, the progression of obesity (for example, the development of complications associated with obesity, increased BMI) is delayed. In some modalities, the individual is at risk of obesity or overweight. In some modalities, - the development of obesity in the individual is avoided. In some modalities, the development of complications associated with obesity in the individual is avoided. In some embodiments, the invention also provides methods for the treatment of metabolic syndrome in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. The invention also provides methods for retarding the development of the metabolic syndrome in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the metabolic syndrome is avoided. In some modalities, the individual is at risk of developing the metabolic syndrome. The invention also provides methods for preventing bone loss in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the bone density in the individual is generally maintained. In some modalities, the individual has osteoporosis.

The invention also provides methods for reducing thyroid hormone in an individual comprising administering an effective amount of an NT-4/5 polypeptide to the individual. In some modalities, the individual has hyperthyroidism. In any of the methods described herein, the NT-4/5 polypeptide can be administered in conjunction with an antibody that specifically binds to the NT-4/5 polypeptide. In some modalities, the link of. antibody to the NT-4/5 polypeptide does not generally interfere with the binding of the NT-4/5 polypeptide to a TrkB receptor. For example, binding of the NT-4/5 polypeptide to the TrkB receptor in the presence of the antibody can be reduced by less than about 5%, 10%, 20%, 30%, 40%, and 50% of the link in the absence of the antibody. In some embodiments, the antibody is completed with a TrkB receptor to bind to the NT-4/5 polypeptide, but the binding of NT-4/5 to the TrkB receptor does not usually interfere with the antibody. As used herein, administration as a whole also encompasses administration as a co-formulation (ie, the NT-4/5 polypeptide and the antibody-they are presented (combined) in the same composition) and / or administration as separate compositions. The "administration as a whole" is a means encompassing any circumstance wherein an NT-4/5 polypeptide and an antibody that specifically binds the NT-4/5 polypeptide are administered in an effective amount to an individual. This will be understood that the NT-4/5 and the antibody can be administered at different dose frequencies and / or intervals. For example, an NT-4/5 polypeptide can be administered daily until weekly, while the antibody can be administered less frequently. It will be understood that NT-4/5 and the antibody can be administered using the same route of administration or different routes of administration, and that different dose regimens can change during the course of administration (s). Administration may be prior to the onset of obesity, diabetes mellitus not dependent on insulin, and / or other associated diseases. The term "co-administration" as used herein, means that the antibody and the NT-4/5 polypeptide are administered with a time spacing of no more than about 15 minutes, such as no more than about 10 minutes. . When administered simultaneously, the antibody and the NT-4/5 polypeptide can be contained in the same dose (eg, a unit dose form comprising both the neurotrophin antibody and the neurotrophin) or at a different dose (eg, the antibody is contained in one dosage form and the NT-4/5 polypeptide is contained in another dosage form). In one embodiment, the antibody and the NT-4/5 polypeptide are presented in a single molecule. For example, a chimeric fusion protein can be made comprising an antibody portion and a portion of the NT-4/5 polypeptide in a manner that the antibody portion can bind and stabilize the NT-4/5 polypeptide portion of the protein. '5 chimerical. In some embodiments, the antibody and the NT-4/5 polypeptide are administered in a predetermined ratio. Thus, in some embodiments, the weight ratio of the antibody to the NT-4/5 polypeptide will be about 1 to 1. In some embodiments, this ratio may be between about 0.001 to about 1 and about 1 to about 1000. , between about 0.01 to about 1 and about 1 to about 100, or between about 0.1 to about 1 and about 1 to about 5 10. Other relationships are contemplated. The efficacy of the T-4/5 polypeptide treatment may be increased when at least one aspect of the treatment of the T-4/5 polypeptide is improved (as compared to the treatment of the T-4/5 polypeptide without co-administration to the antibody). 0 The improvement may be reflected in reducing the frequency of polypeptide treatment? T-4/5, reduced dosage of polypeptide? T-4/5, or improved relief of one or more symptoms associated with the disease being treated. For example, the treatment or prevention of diabetes by the T-5 4/5 polypeptide is enhanced by co-administration of an anti-NT4 / 5 polypeptide antibody when co-administration of the antibody allows for better relief (e.g. when one or more symptoms of diabetes have better relief by co-administration of the antibody with the NT-4/5 polypeptide than by administration of the NT-4/5 polypeptide alone). The antibody can also reduce one or more side effects caused by the administration of the NT-4/5 polypeptide. In some embodiments, sufficient of the antibody is administered to allow reduction of the normal dose of the NT-4/5 polypeptide by at least about 5%, at least about S10%, at least about 20%, or more. The reduction can be reflected in terms of the amount administered to a given administration and / or amount administered during a given period of time (reduced frequency). In any of the embodiments described above, the NT-4/5 polypeptide can naturally occur as mature NT-4/5, such as a mature human NT-4/5, any fragment of the mature NT-4/5 polypeptide occurs naturally , or modified forms thereof described herein. The various formulations of the NT-4/5 polypeptide can be used for administration. In some embodiments, the NT-4/5 polypeptide can be administered neat. In other embodiments, the? T-4/5 polypeptide and a pharmaceutically acceptable excipient are administered, and may be in various formulations. Pharmaceutically acceptable excipients are known in the art, and are relatively inert substances that facilitate the administration of a pharmacologically effective substance. For example, an excipient can give a shape or consistency, or act as a diluent. Suitable excipients include but are not limited to stabilizing agents, humectants and emulsifying agents, salts for varying osmolarity, encapsulating agents, buffers, and skin penetration enhancers. The excipients as well as the formulations for parenteral and non-parenteral drug release are set forth in Remington, The Science and Practice of Pharmacy 20th Ed. Mack Publishing (2000). Generally, these agents are formulated by administration by injection (e.g., intraperitoneally, intravenously, subcutaneously, intramuscularly, etc.), although other forms of administration (eg, oral, mucosal, transdermal, inhalation, etc.) can also be used. The administration can be systemic, for example, intravenous and intraperitoneal, or localized. Accordingly, the NT-4/5 polypeptide is preferably combined with pharmaceutically acceptable carriers such as saline, Ringer's solution, dextrose solution, and the like. The different methods can be used to facilitate the development of the NT-4/5 polypeptide to the brain. These methods include: (a) neurosurgical base (intraventricular drug infusion, hyperosmotic opening of the blood-brain barrier (BBB)); (b) pharmacological basis (lipidization of peptide, liposomes); and (c) physiological basis (biochemical opening of BBB, chimeric peptides). The chimeric peptides can be formed by the covalent coupling of the NT-4/5 polypeptide to a transport vector to the brain that is subjected to transcytosis mediated by the receptor or mediated by absorption through the BBB. For example, a transport vector to the brain can be a monoclonal antibody to the transferrin receptor. See Pardridge et al.

Pharmacol. Toxicol 71: 3-10 (1992). The particular dose regimen, that is, dose, schedule and repetition, will depend on the individual in particular and the individual's medical history. Generally, any of the following doses may be used: a dose of at least about 50 mg / kg of body weight; at least about 20 mg / kg of body weight; at least about 10 mg / kg of body weight; at least about 5 mg / kg of body weight; at least about 3 mg / kg of body weight; at least about 2 mg / kg of body weight; at least about 1 mg / kg of body weight; at least about 750 μg / kg body weight; at least about 500 μg / kg body weight; at least about 250 μg / kg of body weight; at least about 100 μg / kg of body weight; at least about 50 μg / kg body weight; at least about 10 μg / kg of body weight; at least about 1 μg / kg of body weight, or more, are administered. Empirical considerations, such as half-life, generally contribute to the determination of the dose. For administrations repeated for several days or longer, depending on the condition, the treatment is sustained until a desired suppression of the symptoms of the present disease or until sufficient therapeutic levels are obtained. For example, the dosage form of one to five times per week is contemplated. Other dosage regimens include a regimen of up to once per day, 1 to 5 times per week, or less frequently. In some embodiments, the NT-4/5 polypeptide is administered about once a week, about 1 to 4 times per month. The intermittent dosing regimen with staggered doses spaced 2 days up to 7 days or up to 14 days can be used. In some modalities, the treatment can start with a daily dose and change the dose until weekly or even monthly. The progress of this therapy is easily monitored by conventional techniques and trials. In some individuals, more than one dose may be required. The frequency of administration can be determined and adjusted during the course of therapy. For example, the frequency of administration can be determined or adjusted based on the type and severity of the disease to be treated (such as obesity and non-insulin dependent diabetes mellitus), whether the agent is administered for preventive or therapeutic purposes, previous, the clinical history of the patient and the response to the agent and the discretion of the doctor to be treated. Typically, the physician will administer the NT-4/5 polypeptide until a dose is reached that requires the desired result. In some cases sustained sustained release formulations of the NT-4/5 polypeptide may be appropriate. The various formulations and devices for achieving sustained release are known in the art. For example, the NT-4/5 polypeptide can be administered through a mechanical pump or encased in a matrix bed for slow or sustained release. In one embodiment, doses for the NT-4/5 polypeptide can be determined empirically in individuals who have been given one or more administrations. Individuals are given dose increments of the NT-4/5 polypeptide. To calculate the efficacy of the NT-4/5 polypeptide, markers of disease status can be monitored. Examples of such markers are blood glucose level, blood lipid level (eg, triglycerides or cholesterol), insulin resistance, body weight, and food intake. ' It will be apparent to someone of skill in the art that the dose varies depending on the individual, the state of the disease (such as obesity and diabetes), and past and parallel treatments are used. Administration of the NT-4/5 polypeptide according to the method in the present invention can be continuous or intermittent, depending for example on the recent physiological condition, whether the purpose of the administration is therapeutic or prophylactic and other factors known by doctors with skill in the art. Administration of a NT-4/5 polypeptide can be essentially continuous for a preselected period of time or can be in series of spaced doses. Other formulations include sustained release forms known in the art including, but not limited to, carriers such as liposomes. See, for example, Mahato et al. (1997) Phare. Res. 14: 853-859. Liposomal preparations include, but are not limited to, cytofectins, multilamellar vesicles and unilamellar vesicles. An expression vector can be used for direct expression of the NT-4/5 polypeptide in the brain, for example hypothalamus. One of skill in the art is familiar with the administration of expression vectors to obtain expression of an exogenous protein in vivo. See, for example, US Patents. Nos. 6,436,908; 6,413,942; and 6,376,471. Administration of expression vectors includes systemic or local administration, including injection, oral administration, particle gun or catheterized administration, and topical administration. In another embodiment, the expression vector is administered directly to the skeletal muscle or subdermal space. Targeted delivery of therapeutic compositions containing an expression vector or subgenomic polynucleotides can also be used. DNA-release techniques mediated by the receptor are described in, for example, Findeis et al., Trends Biotechnol. (1993) 11: 202; Chiou et al., Gene Therapeutics: Methods and Applications of Direct Gene Transfer (J.A. Wolff, ed.) (1994); Wu et al., J. Biol. Chem. (1988) 263: 621; Wu et al., J. Biol. Chem. (1994) 269: 542; Zinke et al., Proc. Nati Acad. Sci. USA (1990) 87: 3655; Wu et al., J. Biol. Chem. (1991) 266: 338. Therapeutic compositions containing a polynucleotide are administered in a range of about 100 ng to about 200 mg of DNA for local administration in a therapy protocol of genes. The concentration ranges from about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, and about 20 μg to about 100 μg of DNA can also be used during a gene therapy protocol. Polynucleotides and therapeutic polypeptides can be delivered using gene delivery vehicles. The gene delivery vehicle may be of viral or non-viral origin (See generally, Jolly, Cancer Gene Therapy (1994) 1:51; Kimura, Human Gene Therapy (1994) 5: 845; Connelly, Human Gene Therapy (1995) "1: 185; and Kaplitt, Nature Genetics (1994) 6: 148.) The expression of such coding sequences can be induced using heterologous or endogenous mammalian promoters, The expression of the coding sequence can be either constitutive or regulated. Vectors, viral bases for delivery of a desired polynucleotide and expression in a desired cell are well known in the art Exemplary viral base vehicles include, but are not limited to, recombinant retroviruses (See, for example, PCT Publications Nos. WO 90/07936, WO 94/03622, WO 93/25698, WO 93/25234, WO 93/11230, WO 93/10218, WO 91/02805, US Patent Nos. 5,219,740 and 4,777,127;, GB Patent No. 2,200,651; and EP Patent No. 0,345,242), vectors based on alphaviruses (for example, vec Sindbis viruses, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373, ATCC VR-1246) and equine encephalitis virus from Venezuela (ATCC VR-923, ATCC VR-1250, ATCC VR 1249, ATCC VR-532), and Vectors (AAV) of virus associated with adeno (See, for example, PCT Publication Nos. WO 94/12649, WO 93/03769, WO 93/19191, WO 94/28938, WO 95/11984 and WO 95/00655). Administration of the adenovirus-linked DNA deleted as described in Curiel, Hum. Gene Ther. (1992) 3: 147 can also be used. Non-viral delivery vehicles and methods may also be employed, including but not limited to, polycationic condensed ligated DNA or unbound to deleted adenovirus alone (See, for example, Curiel, Hum Gene Gene. (1992) 3: 147); Ligand-ligated DNA (See, for example, Wu, J. Biol. Chem. (1989) 264: 16985); cells of eukaryotic cell delivery vehicles (See, for example, U.S. Patent No. 5,814,482, PCT Publication Nos. WO 95/07994, WO 96/17072, WO 95/30763, and WO 97/42338) and neutralization of the nucleic charge or fusion with cell membranes. The discovered DNA can also be used. Methods of introducing the discovered DNA specimens are described in PCT Publication No. WO 90/11092 and US Pat. No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Pat. No. 5,422,120; PCT Publications Nos. WO 95/13796; WO 94/23697; WO 91/14445; and EP 0524968. Additional methodologies are described in Philip, Mol. Cell Biol. (1994) 14: 2411, and in Woffendin, Proc. Nati Acad. Sci. (1994) 91: 1581. The evaluation of the disease is carried out using standard methods known in the art, for example, by monitoring the appropriate markers, such as blood glucose, lipids, insulin levels and body weight. .

Compositions and Methods for Making the Compositions The compositions that are used in methods of the invention comprise an NT-4/5 polypeptide or polynucleotide that encodes an NT-4/5 polypeptide. As used herein, "NT-4/5 polypeptide" includes the naturally occurring mature protein (interchangeably referred to as "NT-4/5") such as mature human NT-4/5 is shown in Table 1 below and figure 1 in the US patent publication request No. 20030203383 and variants of the naturally occurring amino acid sequence of NT-4/5; variants of the amino acid sequence of NT-4/5; fragments of mature NT-4/5 peptides (such as human) and variants of the amino acid sequence; and modified forms of mature NT-4/5 and variants of the amino acid sequence and peptide fragments wherein the polypeptide or peptide is covalently modified by substitution with a portion other than a naturally occurring amino acid, provided that the amino acid sequence variant, peptide fragment, and the modified form thereof show one or more biological activities of the mature NT-4/5 naturally occurring protein. The NT-4/5 polypeptides also include fusion proteins and conjugates comprising any of the embodiments of the NT-4/5 polypeptide described herein, for example, an NT-4/5 polypeptide conjugated or fused to a portion that is extends in the half-life, such as a PEG or a peptide. Variants of the amino acid sequence, peptide fragments (including fragment variants), or modified forms thereof under consideration do not include NGF, BDNF, or NT-3 of any animal species. Variants, peptide fragments, and modified forms of a naturally occurring NT-4/5 are described in the U.S. patent. publication request Nos. 20030203383; 20020045576; patent E.U.A. Nos. 5,702,906; 6,506,728; 6,566,091; 5,830,858; which are incorporated as a reference in its entirety. The NT-4/5 polypeptide includes any of one or more embodiments described herein. For example, the NT-4/5 polypeptide comprises a sequence that occurs naturally with one or more amino acid insertions, deletion, or substitution. In some embodiments, the NT-4/5 polypeptide is a mammalian NT-4/5 polypeptide, which may be a naturally occurring mammalian NT-4/5, or NT-4/5 polypeptide derived from an NT -4/5 of a mammal that occurs naturally and that has a sequence that does not match any part of a non-mammalian NT-4/5 that occurs naturally. In some embodiments, the N T-4/5 polypeptide is a human NT-4/5 polypeptide, which may be a naturally occurring NT-4/5 human, or NT-4/5 polypeptide derived from an NT- 4/5 human that occurs naturally and that has a sequence that does not match any part of a non-human NT-4/5 that occurs naturally.

Table 1. Amino acid sequence of mature human NT-4/5 and the human nucleotide sequence encoding mature human T-4/5 Amino Acid Sequence (SEQ ID NO: l): GVSETZ PASRRGELAVCDAVSGWVTDRRTAVDLRGREVEVLGEVPAAGGSPLRQYFFTR CKAD? AEEGGPGAGGGGCRGVDRKHWVSECKAKQSYVRALTADAQGRVGWRWIRIDTACVC TLLSRTGRA 'Nucleotide Sequence (SEQ ID NO: 2) GGGGTGAGCG AAACTGCACCAGCGAGTCGTCGGGGTGAGCTGGCTGTGTGCGATGCAGTC AGTGGCTGGGTGACAGACCGCCGGACCGCTGTGGACTTGCGTGGGCGCGA GGTGGAGGTGTTGGGCGAGGTGCCTGCAGCTGGCGGCAGTCCCCTCCGCC AGTACTTCTTTGAAACCCGCTGCAAGGCTGATAACGCTGAGGAAGGTGGC CCGGGGGCAGGTGGAGGGGGCTGCCGGGGAGTGGACAGGAGGCACTGGGT ATCTGAGTGCAAGGCCAAGCAGTCCTATGTGCGGGCATTGACCGCTGATG CCCAGGGCCGTGTGGGCTGGCGATGGATTCGAATTGACACTGCCTGCGTC TGCACACTCCTCAGCCGGACTGGCCGGGCCTGAG The T-4/5 polypeptides, which include variants, peptide fragments, modified forms of T-4/5 polypeptides (include a naturally occurring NT-4/5), fusion protein and conjugate of the invention are characterized by any (one or more) of the following features: (a) ability to bind to and activate the TrkB receiver; (b) ability to activate one or more descending trajectories mediated by the signaling function TrkB; (c) treating, preventing or alleviating one or more symptoms of diabetes mellitus not dependent on insulin; Y (d) treating, preventing or alleviating "one or more symptoms of obesity." Thus all NT-4/5 polypeptides (including variants, fragments and modified forms) are functional as described above - The biological activity of the variants may tested in vitro and in vivo using methods known in the art and methods described in the examples .. The T-4/5 polypeptides may have high activity or reduced activity when compared to a naturally occurring NT-4/5 protein. In some embodiments, the functionally equivalent variants have at least about 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% activity when compared to the protein. NT-4/5 native of which the NT-4/5 polypeptide is derived with respect to one or more of the biological assays described above (or known in the art) .In some embodiments, the functionally equivalent variants have an EC5o (half of the maximum effective concentration) less around either 0.01 nM, 0.1 nM, 1 nM, 10 nM, or 100 nM in the TrkB receptor of in vitro activation (eg, assays described in examples 6 and 7). Variants of the amino acid sequence of NT-4/5 include polypeptides having an amino acid sequence which differ from an NT-4/5 which occurs naturally by virtue of insertion, deletion and / or substitution of one or more amino acid residues within the sequence of a naturally occurring NT-4/5 (e.g., the mature human NT-4 shown in Table 1). Variations of the amino acid sequence will generally be at least about 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to either of an NT-4/5 occurring naturally (such as the mature human NT-4/5 shown in SEQ ID N0: 1). In some embodiments, the variant is at least about 70% identical to the amino acid sequence of SEQ ID NO: l. In some embodiments, the variant is at least about 85% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant is at least about 90% identical to the amino acid sequence of SEQ ID NO: 1. In some embodiments, the variant is at least about 95% identical to the amino acid sequence of SEQ ID? 0: 1. The variants of the amino acid sequence of? T-4/5 can be generated by making predetermined mutations in a DNA? T-4/5 previously isolated. The amino acid variants can be designated and generated based on the crystal structure of NT-4/5 and TrkB receptor. Banfield et al., Structure 9: 1191-9 (2001). For example, amino acids that are not directly involved in the interaction between the monomers of NT-4/5 and between NT-4/5 and the TrkB receptor can be mutated, for example, to introduce the binding site to PEG. The methods known in the art can be used to designate variants of the NT-4/5 polypeptide that increase or reduce one or more biological activities when compared to a naturally occurring NT-4/5 protein. There are two main variables to consider when making such predetermined mutations: the location of the mutation site and the nature of the mutation. In general, the location and nature of the mutation are usually indicated depending on the characteristic NT-4/5 to be modified. For example, the candidate NT-4/5 antagonist or super-agonist can initially be selected by locating amino acid residues that are identical or highly conserved between NGF, BDNF, NT-3, and? T-4. Those residues can then be modified in series, for example, by (1) replacing first with conservative selections and then with more radical selections depending on the results obtained, (2) eliminating the objective residue, or (3) inserting residues of the same kind or different adjacent to the localized site, or combinations of options 1-3. One useful technique is called "wing sweep". At the moment, an amino acid residue or group of target residues is identified and replaced by alanine or polyalanine. Those domains that demonstrate functional sensitivity to alanine substitutions are then refined by introducing additional variants or other variants at or for the alanine substitution sites. Obviously, such variants which, for example, convert NT-4/5 into NGF, BDNF, or NT-3 are not included within the scope of this invention. Thus, while the site for introducing a variation in the amino acid sequence is predetermined, the nature of the mutation per se need not be predetermined. For example, to optimize the performance of a mutation at a given site, the "wing" sweep or random mutagenesis is conducted at the target codon or region and the expressed NT-4/5 variants are selected for the desired optimal activity. The deletions of the amino acid sequence are generally in the range from about 1 to 30 residues, more preferably around 1 to 10 residues, and are typically contiguous. The deletions can be introduced in regions of low homology between BDNF, NGF, NT-3, and NT-4/5 to modify the activity of? T-4/5. Removals of? T-4/5 in areas of substantial homology with BDNF, NT-3, and NGF may be more likely to modify the biological activity of? T-4/5 more significantly. The number of consecutive deletions can be selected to preserve the tertiary structures of ΔT-4/5 in the affected domain, for example, beta fold sheet or alpha helix. The insertions of the amino acid sequence include amino and / or carboxyl terminal fusions in the length range from one residue to polypeptides containing one thousand or more residues, as well as intrasequence insertions of single or multiple amino acid residues. The intrasequence inserts (ie, inserts within the mature sequence T-4/5) may be in the range generally from about 1 to 10 residues, more preferably 1 to 5, more preferably 1 to 3. An example of Terminal insertion includes fusion of a terminal signal sequence? heterologo to the terminal? of the? T-4/5 molecule to facilitate the secretion of the mature? T-4/5 from the recombinant host. Such signals will generally be homologous to the intended host cell and include STII or lpp for E. coli, alpha factor for yeast, and viral signals such as herpes gD for mammalian cells. Other insertions include the fusion of a polypeptide to the terminal? or C of NT-4/5. The third group of variants includes those in which at least one amino acid residue in NT-4/5, and preferably only one, is removed and a different residue is inserted in this place. One example is the replacement of arginine and lysine by other amino acids to make NT-4/5 resistant to proteolysis by serine proteases, thus creating a variant of NT-4/5 that is more stable. The sites of greatest interest for substitutional mutagenesis that include sites where amino acids are found in BDNF, NGF, NT-3, and NT-4 are substantially different in terms of side chain volume, charge, or hydrophobicity, but where these also they are of a high degree of homology when selecting the site within various animal analogues of NGF, NT-3, and BDNF (eg, among all animal NGFs, total animal NT-3, and total animal BDNF) . This analysis will highlight the residues that can be involved in the differentiation of the activity of the trophic factors and therefore, the variants to these sites can affect such activities. Examples of such sites in a mature human NT-4/5, numbered from the N-terminus and exemplary substitutions include G77 to K, H, Q or R and R84 to E, F, P, Y or W of NT-4 / 5 of SEQ ID N0: 1, respectively. Other sites of interest are those in which the residues are identical amounts of all animal species BDNF,? GF,? T-3, and? T-4/5, this degree of conformation suggests the importance of achieving the common biological activity in all four factors. For example, the substitution of one or more amino acids includes conservative substitutions. Methods for making conservative substitutions are known in the art. For example, wing (A) can be replaced by val, leu, ile, preferably by val; arg (R) can be replaced by lys, gln, asn, preferably by lys;. asn (N) can be replaced by gln, his, lys, arg, preferably by gln; asp (D) can be replaced by glu; cys (C) can be replaced by being; gln (Q) can be replaced by asn; glu (E) can be replaced by asp; gly (G) can be replaced by pro; his (H) can be replaced by asn, gln, lys, arg; preferably by arg; ile (I) can be replaced by leu, val, met, ala, phe, norleucine, preferably by leu; leu (L) can be replaced by norleucine, ile, val, met; to; phe, preferably by ile; lys (K) can be replaced by arg; gln, asn, preferably by arg; met (M) can be replaced by leu; phe; ile, preferably by leu; phe (F) can be replaced by leu, val, ile, ala, preferably by leu; pro (P) can be replaced by gly; being (S) can be replaced by thr; thr (T) can be replaced by being; trp (W) can be replaced by tyr; tyr (Y) can be replaced by trp, phe, thr, ser, preferably by phe; val (V) can be replaced by ile; leu; met; phe, wing; norleucine, preferably by leu. Particularly suitable sites for conservative substitutions include, enumerated 'from the N terminus of mature human NT-4 (SEQ ID N0: 1), Rll, G12, -E13, V16, D18, W23, V24, D26, V40 , L41, Q54, Y55, F56, E58, T59, G77, R79, G80, H85, W86, A99, L100, T101, W110, RUI, W112, 1113, R114, 1115, D116, and A118. Cysteine residues that are not involved in maintaining the proper conformation of NT-4/5 can also be substituted, generally with serine, in order to improve the oxidative stability of the molecule and prevent aberrant cross-linking. Other sites than those established in this paragraph are suitable for the elimination or insertion studies generally described above. Substantial modifications in function may be completed by selected substitutions that differ significantly in their effect in maintaining (a) the structure of the polypeptide column in the area of the substitution, eg, as a sheet or helix conformation, (b) the load or hydrophobic capacity of the molecule at the target site, or (c) the volume of the side chain. The naturally occurring residues are divided into groups based on common side chain properties (some of these may fall into various functional groups): (1) hydrophobic: norleucine, met, ala, val, leu, ile; (2) neutral hydrophilic: cys, ser, thr; (3) acid: asp, glu; (4) basic: asn, gln, his, lys, arg; (5) residues that influence the orientation of the chain: gly, pro; and (6) aromatic: trp, tyr, phe. Non-conservative substitutions will allow exchanging a member of one of these classes for another. Examples of NT-4 variants include: the SEQ ID? 0: 1 polypeptide with mutation from E67 to S or T (this is added to a glycosylation site linked to?); the polypeptide from the residue of "amino acid R83 to Q94, Gl to C61, Gl to C17, C17 to C61, C17 to C78, C17 to C90, C17 to C119, C17 to C121, R1 to R27, R1 to R34, R34 to R53, C61 to C78, R53 to C61, C61 to C119, C61 to C78, C78 to C119, C61 to C90, R60 to C78, K62 to C119, K61 to K91, R79 to R98, R83 to K93, T101 to RUI, Gl to C121 of SEQ ID? 0: 1; the polypeptide comprises V40-C121 of SEQ ID NO: 1, for example, V40-C121 of SEQ ID? 0: 1 is fused to a N-terminal and / or C-terminal polypeptide; the polypeptide comprises SEQ ID? 0: 1 with removal of C78, C61, Q54-T59, R60-D82, H85-S88, W86-T101 (eliminations of the indicated space of residues, inclusive); SEQ ID? 0: 1 with mutation from R53 to H, from K91 to H, from V108 to F, from R84 to Q, H, N, T, Y or W, and from D 116 to E, N, Q, Y , S or T. Also included are NT-4/5 (SEQ ID N0: 1) wherein position 70 is substituted with an amino acid residue by another G, E, D or P; position 71 different from A, P or M; and / or position 83 different from R, D, S or K; as well as cyclized NT-4 fragments. Two polynucleotide or polypeptide sequences are said to be "identical" if the sequence of nucleotides or amino acids in the two sequences is the same when aligned for maximum correspondence as described below. Comparisons between the two sequences are typically carried out by comparing the sequences during a comparison window to identify and compare the local regions of sequence equality. A "comparison window" as used herein, refers to a segment of about 20 contiguous positions, usually 30 to about 75, 40 to about 50, in which a sequence can be compared to a reference sequence of the same number of contiguous positions after two sequences are optimally aligned. Optimal alignment of the sequences for comparison can be conducted using the Megalign program in the Lasergene suite of bioinformatics software (DNASTAR, Inc.; Madison, Wl), using default parameters. This program covers various alignment schemes described in the following references: Dayhoff, M. O. (1978) A model of evolutionary change in proteins-Matrices for detecting distant relationships. In Dayhoff, M. O. (ed.) Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, Washington DC Vol. 5, Suppl. 3, pp. 345-358; Hein J., 1990, Unified Approach up to Alignment and Phylogenes pp. 626-645 Methods in Enzymology vol. "183, Academic Press, Inc., San Diego, CA, Higgins, DG and Sharp, PM, '1989, CABIOS 5: 151-153, Myers, EW and Muller W., 1988, CABIOS 4: 11-17; Robinson, ED, 1971, Comb. Theor., 11: - 105; Santou, N., Nes, M., 1987, Mol. Biol. Evol. 4: 406-425; Sneath, PHA and Sokal, RR, 1973, Numerical Taxonomy the Principles and Practice of Numerical Taxonomy, Freeman Press, San Francisco, CA; Wilbur, WJ and Lipman, DJ, 1983, Proc. Nati, Acad. Sci. USA 80: 726-730. , the "percent sequence identity" is determined by comparing two sequences optimally aligned during a comparison window of at least 20 positions, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions ( that is, gaps) of 20% or less, usually 5 to 15%, or 10 to 12%, as compared to the sequence of references (which do not include additions or eliminations) to optimally align the two sequences. The percentage is calculated by determining the number of positions in which the identical nucleic acid bases or the amino acid residue occurs in both sequences until the number of matching positions is obtained, dividing the number of matching positions by the total number of positions in the reference sequence (that is, the size of the window) and multiply the result by 100 to provide the percentage of the identical sequence. Variants of the amino acid sequence of NT-4/5 can occur naturally or can be prepared synthetically, such as by introducing appropriate nucleotide changes into a previously isolated NT- / 5 DNA, or by in vitro synthesis of the desired variant polypeptide. As indicated above, such variants may comprise deletions from, or insertions or substitutions of, one or more amino acid residues within the amino acid sequence of mature NT-4/5 (for example, the sequence is shown in Table 1) . Any combination of deletion, insertion and substitution is made to arrive at an amino acid sequence variant of NT-4/5, provided that the polypeptide of the resulting variant possesses a desired characteristic. Changes in the amino acid may also result in additional modifications of NT-4/5 during expression in recombinant hosts, for example, introducing or moving glycosylation sites, or introducing membrane anchor sequences (in accordance with PCT WO 89 / 01041 Published on February 9, 1989). In some embodiments, the NT-4/5 polypeptide comprises an amino acid sequence encoded by a nucleic acid that hybridizes under severe conditions to a nucleic acid sequence (e.g., SEQ ID NO: 2) encoding NT-4/5 mature human. The polynucleotide variants can also or alternatively, be substantially homologous to a native gene, or a portion or complement thereof. Such polynucleotide variants are capable of hybridizing under stringency conditions until the naturally occurring DNA sequence encodes the polypeptide (or a complementary sequence). "Moderate severity conditions" include prewash in a solution of 5 X SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); Hybridize at 50 ° C-65 ° C, 5 X SSC, overnight; followed by washing twice at 65 ° C for 20 minutes with each of 2X, 0.5X and 0.2X SSC containing 0.1% SDS.

As used herein, "highly stringent conditions" or conditions of high stringency are those that: (1) employ a low ionic strength and high temperature for washing eg 0.015 M sodium chloride / 0.0000 M sodium citrate / 0.1% sodium dodecyl sulphate up to 50 ° C; (2) used during the hybridization a denaturing agent such as formamide for example, 50% (v / v) formamide with 0.1% bovine serum albumin 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50mM sodium phosphate buffer solution to a pH of 6.58 with 750 mM sodium chloride, 75 mM sodium citrate up to 42 ° C; or (3) employ 50% formamide, 5x SSC (0.75M NaCl, 0.075M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, Denhardt 5x solution, sonicated salmon sperm DNA ( 50 μg / ml), 0.1% SDS, and 10% dextran sulfate at 42 ° C, washed at 42 ° C in 0.2 x SSC (sodium chloride / sodium citrate) and 50% formamide at 55 ° C followed by a high severity wash consisting of 0.1 x SSC containing EDTA up to 55 ° C. Another severe exemplary hybridization condition in 50% formamide, 5xSSC, 0.1% sodium dodecyl sulfate, 0.1% sodium pyrophosphate, 50mM sodium phosphate pH 6.8, 2x Denhardt solution, and 10% dextran sulfate up to 42 ° C, followed by one wash in O.lxSSC and 0.1% SDS at 42 ° C. The experienced technician will recognize how to adjust the temperature, ionic resistance etc., as necessary to accommodate factors such as the length of the probe and the like. It will be appreciated by those of ordinary skill in the art that as a result of the degeneracy of the genetic code, there are many nucleotide sequences encoding a polypeptide as described herein. Some of these polynucleotides carry minimal homology with the nucleotide sequence of some native gene. However, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention. In addition, alleles of the genes comprising the polynucleotide sequences provided herein are within the scope of the present invention. Alleles are endogenous genes that are altered as a result of one or more mutations such as deletions, additions and / or substitutions of nucleotides. The resulting DNA and protein may have but do not need to have an altered structure or function. Alleles can be identified by using standard techniques (such as hybridization, amplification and / or comparison of database sequences). The ΔT-4/5 variants used in the methods of the invention also include fusion proteins comprising the ΔT-4/5 amino acid sequences (e.g., human ΔT-4/5 shown in table 1) or a functional peptide fragment thereof. The biologically active? T-4/5 polypeptides can be fused with sequences such as sequences that enhance immunological reactivity, facilitate coupling of the polypeptide to a support or carrier, or facilitate retraction and / or purification (eg, sequences that encode epitopes such as Myc, HA derived from the influenza virus hemagglutinin, His-6, FLAG). These sequences can be fused to the NT-4/5 polypeptide at the N-terminus or at the C-terminus end. In addition, the protein or polynucleotide can be fused to others or polypeptides which increase their function or specify their location in the cell such as a secretion sequence. Methods for producing recombinant fusion proteins described above are known in the art. The recombinant fusion protein can be produced, refolded and isolated by methods well known in the art. The NT-4/5 polypeptides described herein can be modified to increase their half-lives in an individual. For example, the NT-4/5 polypeptide can be pegylated to reduce systemic clearance with minimal loss of biological activity. The invention also provides compositions that include pharmaceutical compositions comprising an NT-4/5 polypeptide linked to a PEG molecule. In some modalities, the PEG molecule is linked to the NT-4/5 polypeptide through a reversible ligature. The half-life of a pegylated NT-4/5 polypeptide can be extended by more than about any of 2 times, 5 times, 10 times, 15 times, 20 times, and -30 times the half-life of the NT-4 polypeptide. / 5 not pegylated. Polyethylene glycol (PEG) polymers can be linked to various functional groups of the NT-4/5 polypeptide by using methods known in the art. See, for example, Roberts et al., Advanced Drug Delivery Reviews 54: 459-476 (2002); Sakane et al. Pharm. Res. 14: 1085-91 (1997). PEG can not be linked to the following functional groups on the polypeptide: amino groups, carboxyl groups, natural N termini or modified amine groups and thiol groups. In some embodiments, one or more surface amino acid residues are modified with the PEG molecules. The PEG molecules can be of different size (for example, in the range from about 2 to 40 KDa). The PEG molecules linked to the NT-4/5 polypeptide can have a molecular weight around any of 2000, 10,000, 15,000, 20,000, 25,000, 30,000, 35,000, 40,000 Da. The PEG molecule can be a single or branched chain. To bind PEG to the NT-4/5 polypeptide, it was a PEG derivative having a functional group in one or both termini can be used. The functional group is chosen based on the type of reactive group available on the NT-4/5 polypeptide. Methods for linking derivatives for polypeptides are known in the art.

Roberts et al., Advanced Drug Delivery Reviews 54: 459-476 (2002). The ligation between the NT-4/5 polypeptide and PEG can also be such that they can unfold or naturally degrade (reversible or degradable ligation) in an individual which can improve the half-life but minimize the loss of activity. The PEG ligation site in the NT-4/5 polypeptide can also be created by mutating surface residues for an amino acid residue having a PEG reactive group such as a cysteine. For example, the following amino acids of human NT-4/5 8SEQ ID N0: 1) can be mutated for PEG binding. Gl, V2, S3, E4, T5, S9, RIO, T25, D26, R28, T29, V31, E37, E39, L41, E43, A46, A47, G48, G49, S50, R53, D64, N65, A66, E67, E68, G69, D82, R83, R84, H85, A104, Q105, G106, R107, V108, S125, and T127. These can be applied to the corresponding residues in other species. Various pegylated NT-4/5 have been generated and are shown in Examples 6 and 7. The serine residue in position 50 of mature human NT-4/5 can be changed to cysteine to generate NT4-S50C which is then pegylate wherein the PEG is linked to the cysteine at position 50. An example of a specific binding at the N-terminus for PEG is to mutate the residue at position 1 to a serine or threonine, followed then with pegylation where the PEG is binds serine to position 1. The NT-4/5 polypeptide can also be modified to increase its efficiency of transferring through the brain and blood barrier to the brain after peripheral administration. For example, the NT-4/5 polypeptide can be conjugated or ligated to an anti-transferrin receptor antibody. See Pardridge WM, Pharmacol. Toxicol 71: 3-10 (1992). The NT-4/5 polypeptide can be conjugated to an anti-transferrin receptor antibody by means of the avidin / biotin interaction or covalently coupled to an anti-transferrin receptor antibody (such as the 0X26 antibody). See, Kang et al., J. Pharmacol. Exp. Trier. 269: 344-50 (1994); Yoshikawa, et al., J. Pharmacol. Exp. Ther. 263: 897-903 (1992); Pardridge WM, Pharmacol. Toxicol 71: 3-10 (1992). The pegylated NT-4/5 polypeptides can also be ligated to an anti-transferrin receptor antibody. See, Wu et al., Proc. Nati Acad. Sci. U.S.A. 96: 254-9 (1999). Delivery of the NT-4/5 polypeptides to the brain can also be achieved using immunoliposomes (liposomes targeted to antibodies). The liposomes carrying the NT-4/5 polypeptides can be conjugated to PEG and then coupled to an anti-transferrin receptor antibody. Huwyler et al. Proc. Nati Acad. Sci. U. S. A. 93: 14164-9 (1996). Other antibodies specifically identified for their ability to cross the blood brain barrier can also be used for any of the above formulations. Muruganandam et al., FASEB J. 16: 240-2 (2002).

The NT-4/5 polypeptide can be produced by recombinant means that is, by the expression of a nucleic acid encoding the NT-4/5 polypeptide. In the culture of recombinant cells and optionally the purification of the variant polypeptide of the cell culture for example, by a bioassay of the activity of the variant or by absorption in an immuno-affinity column comprising rabbit polyclonal NT-4/5 antibodies, (which will bind to at least one immune epitope of the variant that is also present in native NT-4/5). Small fragments of peptides in the order of 40 or less residues are conveniently made by in vitro methods. The DNA encoding the NT-4/5 polypeptide can be cloned into an expression vector to express the protein in a host cell. Examples of nucleic acids encoding the NT-4/5 polypeptide are described in U.S. Patent Application Publication No. 2003/0203383. the DNA encoding the NT-4/5 polypeptide in its mature form can be bound at its amino terminus to a secretion signal. This secretion signal is preferably the NT-4/5 pre-sequence which normally directs the secretion of NT-4/5 from human cells in vivo. Nevertheless, the signals of adequate secretion also include signals from other NT-4/5 of animals, signals from NGF, NT-2, or NT-3, viral signals or signals from secreted polypeptides of the same or related species. Any host cell (such as E. coli) can be used to express the protein or polypeptide. The expressed NT-4/5 polypeptide can be purified. The NT-4/5 polypeptide can be recovered from the culture medium as a secreted protein although it can also be recovered from the host cell lysates when they are expressed directly without a secretory signal. Protein purification methods known in the art can be used. The production methods of the NT-4/5 polypeptide and purification of the expressed NT-4/5 polypeptide are described in U.S. Patent Application Publication No. 2003/0203383, and in U.S. Patent No. 6,284,360. The NT-4/5 polypeptide can also be expressed in E coli and refolded according to methods known in the art. NT-4/5 mature human can also be obtained commercially. (for example, from R &; D Systems, Sigma and Upstate). The invention also provides compositions comprising an NT-4/5 polypeptide and an antibody that specifically binds to the NT-4/5 polypeptide. The antibody and the NT-4/5 polypeptide may be present in a predetermined ratio. Thus in some embodiments, the weight ratio of the antibody to the NT-4/5 polypeptide may be about 1 to 1. In some embodiments, this ratio may be between about 0.001 to about 1 and about 1 to about 1000. , between about 0.01 to about 1, and about 1 to about 100, or between about 0.1 to about 1 and about 1 to about 10. Other relationships are contemplated. In some embodiments, the antibody and the NT-4/5 polypeptide are present in a single dosage unit. The binding affinity of the antibody for the NT-4/5 polypeptide can be around 2 pM to about 5800 nM. The binding affinity may be less than about any of 500 nM, 200 nM, 100. nM, 10 nM, 5 nM, 1 nM, 900 pM, 500 pM, 300 pM, 150 pM, 100 pM, 50 pM, 25 pM, and 10 pM. The composition used in the present invention may further comprise pharmaceutically acceptable carriers, excipients or stabilizers (Remington: The Science and Practice of Pharmacy 20th Ed. (2000) Lippincott Williams and Wiikins, Ed. KE Hoover.), In the form of lyophilized formulation. or aqueous solutions. Acceptable carriers, excipients, or stabilizers are not toxic to receptors in doses and concentrations and may comprise buffer solutions such as phosphate, citrate, and other organic antioxidant acids including ascorbic acid and methionine, preservatives (such as octadecyldimethylbenzyl ammonium chloride hexamethonium chloride; benzalkonium chloride; benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl parabens such as methyl or propyl parabens; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); polypeptides of low molecular weight (less than about 10 residues); proteins, such as serum albumin, gelatin or immunoglobulin, hydrophilic polymers, such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, aspargin, histidine, arginine, or lysine; monosaccharides, disaccharides and other carbohydrates including glucose, mannose, or dextran; chelating agents such as EDTA; sugars such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (eg, Zn complexes and proteins); and / or nonionic surfactants such as TWEEN ™, PLURONICS ™ or polyethylene glycol (PEG). The pharmaceutically acceptable excipients are further described herein. The NT-4/5 polypeptides described herein can be formulated for sustained release. Suitable examples of sustained-release preparations include impermeable matrices of solid hydrophobic polymers containing the NT-4/5 polypeptide whose matrices are in the form of formed articles, eg, films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (e.g., poly (2-hydroxyethyl methacrylate), or poly (vinylalcohol)), polylactides (U.S. Patent No. 3,773,919), glutamic acid copolymer-L and 7 ethyl- L-glutamate, non-degradable vinyl ethylene acetate, lactic acid-degradable glycolic acid copolymer such as LUPRON DEPOT ™ (injectable micro spheres composed of glycolic acid copolymer and lactic acid and leuprolide acetate), sucrose acetate isobutyrate and poly-D- (-) -3-hydroxybutyric acid. Another example of a sustained release drug delivery system that can be used is the ATRIGEL® made by Atrix Laboratories. See, for example, US Patent No. 6,665,874. The ATRIGEL® drug delivery system consists of biodegradable polymers similar to those used in biodegradable sutures dissolved in biocompatible carriers. The NT-4/5 polypeptides can be mixed into this liquid delivery system at the time of manufacture or depending on the product they can be added later by the doctor at the time of use. When the liquid product is injected subcutaneously or intramuscularly through a small-gauge needle, or placed in tissue sites accessible through a cannula, the displacement of the carrier with water in the tissue fluids causes the polymer to precipitate out of the fluid. form a solid film or implant. The NT-4/5 polypeptides encapsulated within the implant are then released in a controlled manner as the polymer matrix biodegrades with time. Depending on the patient's medical needs, the Atrigel system can supply proteins for a period ranging from days to months. Injectable sustained release systems such as ProLease®, Medisorb®, manufactured by Alkermes can also be used. In some embodiments, the invention provides compositions (described herein) for use in any of the methods described herein, either in the context of use as a medicament and / or use for the manufacture of a medicament.

Kits comprising the polypeptide with NT-4/5 for therapeutic purposes. The invention also provides kits for use in current methods. Kits of the invention include one or more containers comprising the purified NT-4/5 polypeptide (including naturally occurring NT-4/5) and instructions for use according to any of the methods of the invention described herein. Generally, these instructions comprise a description of the administration of the NT-4/5 polypeptide to treat a disease such as obesity and non-insulin-dependent diabetes mellitus according to any of the methods described herein. The kit may further comprise a description of selecting an individual suitable for treatment based on the identification of that individual having the disease and the stage of the disease (such as obesity)., and diabetes mellitus not dependent on insulin). In some embodiments, the kit further comprises an antibody that binds specifically to the NT-4/5 polypeptide and instructions for administering the NT-4/5 polypeptide in conjunction with the antibody. The instructions relating to the use of the NT-4/5 polypeptide generally include information regarding the dose, schedule of the dose and route of administration for the intended patient. The containers may be of unit doses, volume packs (e.g., multiple dose packs) or subunit doses. The instructions provided in the kits of the invention are typically instructions written on a label pack insert (for example, a sheet of paper included in the kit), but machine-readable instructions (for example, instructions carried on a storage disk). magnetic or optical) are also acceptable. The label insert or package indicates that the composition is used to treat a disease described herein (such as obesity and diabetes mellitus not dependent on insulin). The instructions may be provided to practice any of the methods described herein. -The kits of this invention are in appropriate packages. Appropriate packaging includes, but is not limited to, vials, bottles, jars, flexible packaging (e.g., sealed plastic or Mylar bags), and the like. Packages are also contemplated to be used in combination with a specific device, such as an inhaler, nasal delivery device (e.g., an atomizer), or an infusion device such as a minipump. The kit may have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopping device penetrable by a hypodermic injection needle). The container may also have a sterile access port (for example, the container may be an intravenous solution bag or a vial having a stopping device penetrable by a hypodermic injection needle). At least one active agent in the composition is an NT-4/5 polypeptide. The container may further comprise a second pharmaceutically active agent. The kits may optionally provide additional components such as buffers and information to interpret. Typically, the kit comprises a container and a package label or inserts in or associated with the container. The following examples are provided to illustrate, but not to limit, the invention.

EXAMPLES For all the examples described below, human NT-4/5 was used for all experiments. Thus, in the examples below, NT-4/5 refers to human NT-4/5.

Example 1: Effect of NT-4/5 on the metabolism of carbohydrates and lipids in db / db mice A. Experimental Protocol Test animals: 24 female db / db mice aged 10-12 weeks (C57BL / Ks J Rj - db / db, Janvier, France), which weigh in the target range of 40-50g, were used in this study. These mice were housed in a room controlled by temperature (19.5-24.5 ° C) and relative humidity (45-65%) with a light / dark cycle of '12 hours, with access ad libitum to filtered tap water and food of laboratory irradiated granules (SAFE, France) throughout the study. Upon receiving in animal facilities, 4 per cage covered with filtered lids were housed and at least an acclimation period of 5 days was observed.

Administration of NT-4/5 and reference substances: Rosiglitazone (3 mg / kg, Sequoia Research Products Ltd., UK), a thiazolidinedione compound for the therapy of type 2 diabetes, was used in the current experiment as a reference. Phosphate buffered saline solution was used as a vehicle in this experiment. NT-4/5 were obtained from Genentech. The mice were divided into 3 groups of 8 mice each (groups 1-3). Each group was treated with vehicle (PBS) subcutaneously (s.c.), 3 mg / kg Rosiglitazone orally (p.o.), or 20 mg / kg NT-4/5 subcutaneously (s.c.). From day 1 to day 5 (T1-T5) and then from day 8 to day 12 (T8-T12), the mice in groups 2 and 3 were dosed once a day for 5 consecutive days, with a volume of 10 ml / kg.

Collection of samples for analysis: One day before starting the treatment (TO), db / db mice without fasting were weighed and the blood samples were collected through the retro-orbital plexus (around 300 μL / mouse) under anesthesia. Isoflurane Similarly, on day 5 (T5), day 12 (T12), day 19 (T19) and day 26 (T26), 2 hours after the last administration, blood samples were collected through the retro-orbital plexus under anesthesia. isoflurane in mice without fasting. Blood samples were subjected to HbAlc determination before being kept at room temperature for 5 to 10 minutes to form a spontaneous clot, then placed on ice until they were centrifuged at 3500 xg for 10-15 minutes at 4 ° C using a centrifuge 2K15 model. (Sigma, France). An aliquot of serum was used to measure glucose levels, the rest was frozen until used.

Analysis of blood samples in db / db mice: Before and after the dose serum levels of glucose, HbAlc, cholesterol and triglycerides, insulin, and non-esterified fatty acids (NEFA), as well as activities of aspirin amino-transferase ( AST) and activities of alanine amino-transferase (ALT), were determined at different time points, as also described herein. At each time point and for each parameter, an average% of the effect was calculated according to the following formula: ((T final-T initial) / T initial) * 100. The statistical analysis was consistent in a one-way analysis of variance followed by multiple comparisons against the vehicle group (Dunnett test) in final values. In the case of the equal variance test, a Kruskall-Wallis analysis of a variance path in ranges is conducted. A difference was considered significant for p < 0.05.

B. Results 1. Controlling activity - NT 4/5 blood glucose in db / db mice Blood glucose levels in mice under treatment were determined using a Glucose kit (ref 442640, Beckman Coulter, France) and a Synchron CX-4 analyzer (Beckman Coulter, France). Table 2 summarizes the results of the analysis. The results - are also presented graphically in Figure 1.

Table 2. Effect of NT-4/5 on blood glucose levels (mM) in mice db / db TO T5 T12 b effect T 19% effect T26 effect T12-T0 T19-T0 effect T5-T0 T26-T0 Day 0 5 12 19 26 Vehicle 37.39 29.74 -19.2 34.89 -5.8 43.46 17.0 46.83 25.5 (s.c.) 1.87 3.13 10.1 4.38 13.5 2.14 11.9 1.58 10.4 Rosigli37.56 22.44 -40.5 22.24 -40.9 33.74 -9.0 43.60 17.5 tazona 1.74 2.80 6.8 2.57 5.8 1.85 6.0 2.44 7.6 (3 mg / kg p.o.) NT-4/5 36.95 15.78 -56.4 13.56 -62.6 12.65 -65.2 22.50 -37.9 (20 1.99 1.18 4.2 1.58 5.2 1.48 5.0 1.63 6.7 mg / kg s.c) Values are expressed as mean ± SEM Statistics: Unidirectional analysis of variance (ANOVA) followed by a Dunnett test. * p < 0.05 compared to vehicle The blood glucose level of vehicle-treated mice is kept high through the study. In mice treated with Rosiglitazone, the blood glucose level decreases to around 22.44 mM to T5, remains low until T12, and went up dramatically later. The blood glucose level in mice treated with NT 4/5 decreases to 15.78 mM to T5, decreases to 13.56 mM to T12, and also decreases to 12.65 mM to T19, reaching at least the normal level of blood glucose ( about 10.95 ± 0.41 mM, determined by measuring 8 normal healthy C57BL / 6J mice). The level of blood glucose in mice treated with NT 4/5 is maintained at least 50% less than that of mice treated with vehicle throughout the rest of the study. HbAlc levels were determined using an HbAlc kit (ref 650262, Beckman Coulter, France) and a Synchron CX4 synchronizer. HbAlc is an indicator for blood glucose control during the last 2-3 months. The normal level of HbAlc in C57BL / 6J mice (determined by measuring 8 normal healthy C57BL / 6J mice) was around 3.5%. Table 3 summarizes the results of the analysis. The results are also presented graphically in Figure 2.

Table 3. Effect of NT-4/5 on HbAlc (%) in db / db mice Values are expressed as mean ± SEM Statistics: Unidirectional analysis of variance (ANOVA) * p < 0.05 compared to vehicle The level of HbAlc in both mice treated with Rosiglitazone and NT 4/5 decreased only slightly at T12 (1% and 0.2% respectively). However, the HbAlc level was significantly suppressed at T26. In mice treated with Rosiglitazone, the HbAlc level decreases 9.6%. In mice treated with NT-4/5, the HbAlc to T26 level decreases 30.4% with respect to T0. The results demonstrate that both short-term and long-term blood glucose levels have been significantly reduced in mice treated with NT-4/5. 2. Activity controlling the blood lipid of NT-4/5 in db / db mice The level of triglycerides in the blood and the level of cholesterol in the whole blood were determined with a triglyceride test kit (ref 445850 , Beckman Coutler, France) and a total cholesterol kit (ref 467825, Beckman Coutler, France), "respectively, using a Synchron CX4 analyzer The results of the experiment are shown in Tables 4 and 5. Table 4. Effect of NT-4/5 in triglyceride levels (mM) Values are expressed as mean ± SEM Statistics: Unidirectional analysis of variance (ANOVA) followed by a Dunnett test or Kruskal-Wallis unidirectional analysis of variance in rows (KW) followed by a Dunn test. * p < 0.05 compared to vehicle Table 5. Effect of NT-4/5 on cholesterol levels (mM) Values are expressed as mean ± SEM Statistics: Unidirectional analysis of variance (ANOVA) followed by a Dunnett test. * p < 0.05 compared to vehicle As shown in Table 4, triglyceride levels in the blood are reduced to a normal low level (1.2 ± 0.08 mM, determined by measuring 8 normal healthy C57BL / 6J mice) in mice treated with either Rosiglitazone or NT-4 /5. As shown in Table 5, the level of blood cholesterol level in mice treated with NT 4/5 also decreased by about 20-30% (non-statistically significant). The levels of non-esterified fatty acids in serum (NEFA) were determined by colorimetric method using a non-esterified fatty acid kit (ref FA115, Randox, France). The result of the test is shown in Table 6. Unlike Rosiglitazone, Nt 4/5 does not appear to have affected the blood level of NEFA significantly under these conditions.

Table 6. Effect of NT / 4/5 on NEFA levels (mM) in db / db mice Values are expressed as mean + SEM Statistics: Unidirectional analysis of variance (ANOVA) followed by a Dunnett test. * p < 0.05 compared to vehicle These results show that? T 4/5 significantly reduces the levels of triglycerides in the blood in these mice. 3. T 4/5 improves insulin resistance in db / db mice The level of insulin in the blood was determined by ELISA using an ELIT Plus kit (ref I? SRAT01-8 ?, Eurobio, France). Table 7 summarizes the results of the analysis. The results are also presented graphically in Figure 3.

Table 7. Effect of NT-4/5 on insulin levels (nM) in db / db mice Values are expressed as mean ± S M Statistics: Unidirectional analysis of variance (ANOVA) followed by a Dunnett test. * p < 0.05 compared to vehicle The level of insulin in the blood of db / db mice treated with vehicle decreases to 4.35 mM to T5, 2.29 mM to T12, 2.58 mM to T19, and 1.96 mM to T26. In mice treated with Rosiglitazone, the level of insulin in the blood decreases to 3.94 mM to T5, 2.85 mM to T12. At T19, the blood insulin level in mice treated with Rosiglitazone returns up to 7.30 mM to T19, and 6.33 mM to 'T26. In mice treated with Nt 4/5, the level of insulin in the blood decreases to 1.84 mM to T5 (statistically significant compared to vehicle), and 0.91 mM to T12. At T19, the blood insulin level in mice treated with NT 4/5 returns to 7.6 mM and further decreases to 11.58 mM to T26. These results demonstrate that the insulin level was decreased in mice treated with NT-4/5. However, the insulin level was not reduced to normal level (around 0.118 + 0.010 nM, determined by measuring 8 normal healthy C57BL / 6J mice) by NT-4/5 treatment under these conditions in these mice. The increase in insulin level after treatment with NT 4/5 was stopped may represent rebound hyperinsulinemia and increases the insulin reserve of pancreatic β cells. 4. Enzyme activities AST and ALT of NT 4/5 in db / db mice The activities of amino-transferase of aspartate (AST) and amino-transferase of alanine (ALT) were determined with an AST kit (ref 442665, Beckman Coulter, France ), an ALT kit (ref 442620, Beckman Coulter, France), using an analyzer Synchron CX4. The results of the experiment are shown in Table 8. The AST / ALT ratios at each time point are shown in Table 9.

Table 8. Effect of NT-4/5 on serum transaminase activity in db / db mice Values are expressed as mean ± SEM Statistics: Unidirectional analysis of variance (7ANOVA) followed by a Dunnett test. * p < 0.05 compared to vehicle Table 9 Effect of NT-4/5 on ALT / AST ratio in db / db mice Values are expressed as mean ± SEM Statistics: Unidirectional analysis of variance (ANOVA) As shown in Tables 8 and 9, NT 4/5 had no significant effect on the levels either AST and / or ALT, or the AST / ALT ratios. This result indicates that treatment with NT-4/5 had no significant liver toxicity.

. Treatment effect NT 4/5 in body weight and food intake in db / db mice Throughout the experiment period (T1-T26), the individual body weights were measured daily per cage as well as the food intake. The result of the measurements are shown in Tables 10-12, and are presented graphically in Figures 4 and 5.

Table 10. Effect of NT-4/5 on body weight of db / db mice Values are expressed as mean ± SEM Statistics: 7Análisis de una variancia (ANOVA) followed by a Dunnett test. * p < 0.05 as compared to the vehicle.

Table 11. Effect of NT-4/5 on body weight (expressed as% of baseline) of db / db mice. The data in Table 10 were expressed as a percentage of body weight on Day 0.

Table 12. Effect of NT-4/5 on dietary intake (g / kg / day) of db / db mice As shown in Figure 4 and Tables 10 and 11, mice treated with NT 4/5 gradually lost weight during treatment. Body weights begin to increase to about 3 days after stopping treatment of NT 4/5, but they remained much lower than those of mice treated with vehicle and mice treated with Rosiglitazone. Similarly, as shown in Figure 5 and Table 12, dietary intakes of mice treated with N / T were dramatically suppressed. The dietary intake begins to increase to about 3 days after stopping treatment of NT 4/5, but they remained much lower than those mice treated with vehicle and mice treated with Rosiglitazone. These data demonstrate that g? T 4/5 significantly suppresses weight gain and dietary intake in db / db mice. 6. Effect of NT 4/5 muscle weight in db / db mice Heart weight and gastrocnemius weight were measured on day 28 per vehicle, Rosiglitazone, and mice treated with NT-4/5. The results are shown in Table 13. As shown in Table 13, treatment with NT-4/5 did not significantly change the weight of gastrocnemius and heart muscle as compared to vehicle treatment. This indicates no muscle wasting or lean muscle loss during treatment with NT-4/5.

Table 13. Effect of NT-4/5 on muscle weight (g) in db / db mice Heart weight (g) Gastrocnemius weight (g) Vehicle (sc) - 0.104 0.074 0.003 0.002 Rosiglitazone 0.114 0.074 (3 mg / kg po) 0.005 0.003 NT-4/5 0.104 0.70 (20 mg / kg sc) 0.02 0.002 Values are expressed in mean ± SEM Statistics: Analysis of a variance route (ANOVA) Example 2: NT-4/5 dose response in db / db mice A. Experimental Protocol Db / db mice maintained as described in Example 1 were divided into 5 groups (groups 1-5). Group 1 (7 mice) were administered with vehicle (PBS). Group 2 (7 mice), group 3 (8 mice), group 4 (8 mice), group 5 (8 mice) each were administered with NT-4/5 at doses of 2 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg, respectively. Both the vehicle and NT-4/5 were administered to each group of mice subcutaneously once a day for 5 days. One day before starting the treatment (TO), blood samples were collected from the mice as previously described for the determination of baseline serum biomarker levels (glucose, triglycerides, and cholesterol). Similarly, on day 6 (T6), blood samples were collected for determination of serum biomarker levels. The blood samples were treated in the same manner as described in Example 1.

B. Results 1. NT-4/5 dose response in serum biomarkers Blood glucose, triglycerides, and cholesterol levels were determined as described in Example 1. Table 14 summarizes the results of the analyzes. The results are also presented graphically in Figure 6.

Table 14. NT-4/5 dose response in serum biomarkers of db / db mice (12 weeks, females).

Díaß Values are expressed as mean +/- SEM Statistics: analysis by one way of variance (ANOVA) followed by Dunnett's test * P < 0.05 ** P < 0.01 *** p < o.OOl.

Baseline blood glucose levels of the mice were around 500-600 mg / dL. The administration of 2 mg / kg of? T-4/5 significantly decreases the blood glucose level to around 400 mg / dL. T-4/5 to 5 mg / kg, 10 mg / kg, and 20 mg / kg further decreases levels to less than 300 mg / dL. Triglyceride levels in the baseline blood of the mice were around 200-300 mg / dL. The administration of 2 mg / kg of NT-4/5 significantly decreases the level of triglycerides in the blood to around 150 mg / dL on day 6. NT-4/5 to 5 mg / kg, 10 mg / kg, and 20 mg / kg further decreases the levels to around 100 mg / dL or less on day 6. In contrast, the blood cholesterol levels of mice treated with NT-4/5 remained at the level of the baseline. In another experiment, db / db mice were injected subcutaneously with vehicle (PBS) or NT-4/5 at 2 mg / kg, 5 mg / kg, 10 mg / kg, or 20 mg / kg daily for 2 weeks. Significant reduction in triglyceride level was observed in all groups treated with NT-4/5 on day 14 compared to the vehicle control group. 2. NT-4/5 dose response in dietary intake and body weight of db / db mice From 2 days before treatment (T-1) to day 6 (T6), the individual food intakes of the mice were measured daily. The results of the measurements are shown in Figure 7 and Table 15. The individual body weights were also measured daily for 6 days before treatment (T-5) for day 7 (T7). The results of the measurements are shown in Figure 8A and Table 16. The percentage of changes in body weight were also calculated and plotted in Figure 8B and summarized in Table 17.

Table 15. Response of doses of NT-4/5 in the dietary intake of db / db mice (12 weeks, females) Values are expressed as mean +/- SEM Statistics: 2-way analysis of variance (ANOVA) followed by a Bonferroni test * P < 0.05 as compared to the vehicle group ** P < 0.01 *** P < 0.001 Table 16. NT-4/5 dose response in body weight of db / db mice (12 weeks, females) Values are expressed as mean SEM Statistics: 2-way analysis of variance (ANOVA) followed by a Bonferroni test * P < 0.05 as compared to the vehicle group ** P < 0.01 *** P < 0.001 Table 17. NT-4/5 dose response in the percentage change in body weight of db / db mice (12 weeks, females) Say Day 3 Day 5 Day? Vehicle 101.73 100.82 10X72 100.53 0.48 0.61 0.32 0.28 NT-4/5 (2mg kg) 101.83 98.27 96.24 94.57 0.24 L21 0.34 0.48 *** NT-4/5 (Smg / kg) 101.51 95.71 93.79 90.15 0.44 0.37 0.51 1.09 NT-4/5 (IGmg kg) 101.07 96.28 93.43"90.14 0.45 0.32 0.39 0.48 *** NT-4/5 (Om kg) 100.31 96.01 92.60 88.38 0.34 0.43 0.28 0.47 Values are expressed as mean +/- SEM Statistics: 2-way analysis of variance (ANOVA) followed by Bonferroni test * P < 0.05 ** P < 0.01 *** P < 0.001 The dietary intakes of mice treated with NT-4/5 have already been significantly reduced on the first day of treatment of NT-4/5, and the reduction continues until the end of the experiment period. Treatment with NT-4/5 at 2 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg significantly reduces dietary intake compared to the vehicle. Similarly, the body weights of the treated mice begin to reduce on day 3 of the treatment, and the reduction continues until the end of the experiment period. As shown in Figure 8A and Figure 8B, treatment with NT-4/5 at 2 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg significantly reduces body weight compared to vehicle. Female db / db mice (12 weeks old) - were divided into four groups and injected subcutaneously with the vehicle (PBS) or NT-4/5 at a dose of 2 mg / kg, 5 mg / kg, or 10 mg / kg daily from day 0 to day 4, and then on day 8, day 11, day 15, day 18, day 22, day 25, and day 29. Administration of NT-4/5 reduces body weight by a form dependent on the dose. In addition, body weights in NT-4/5 in all treated groups continue to decrease during the treatment period. On day 31, about 20% reduction in body weight is observed in the group injected with NT-4/5 of 5 mg / kg and 10 mg / kg. Female db / db mice (12 weeks old) were divided into three groups and injected subcutaneously with vehicle (PBS) or NT-4/5 at a dose of 5 mg / kg, or 20 mg / kg daily from day 1 to day 30. Administration of NT-4/5 reduces the body weight in a dependent manner. the dose.

The dietary intake is reduced to around 20% of the level of the vehicle group after 10 days of administration of NT-4/5 at 20 mg / kg and is maintained at this level until day 30. The dietary intake is returned to around the same level as the group with vehicle around 12 days after stopping treatment.

Example 3: Effect of NT-4/5 on carbohydrate metabolism and body weight homeostasis in polygenic obese mice (NONcNZO-10) A. Experimental Protocol Test animals: 19 polygenic NONcNZO-10 obese and diabetic mice from Jackson Laboratory (see Leiter et al., Diabetes 53 (Suppl 1): S4-S11, 2004) at 9 weeks of age weighing in the range of 28-35 g were used in this study. These mice were housed in a room controlled by temperature (19.5-24.5 ° C) and relative humidity (45-65%) with a light / dark cycle of 12 hours, with ad libitum access to the filtered tap water and feed of laboratory in irradiated granules (PURINE) throughout the study. Upon receipt, in animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed.

Administration of NT-4/5: NT-4/5 were obtained from Genentech Inc. Mice were divided into 3 groups of equivalent body weight and baseline glucose distribution (6-7 mice per group). Each group received, from day 1 to day 5 and then from day 8 to day 11, a daily subcutaneous dose of either the vehicle, 2 mg / kg of NT-4/5 or 10 mg / kg of NT- 4/5 Seven days before starting treatment (day 7), blood samples were collected from the mice as previously described for the determination of blood glucose baseline levels and triglycerides. After the treatment starts, blood samples are collected weekly to determine triglyceride and glucose levels as described in the previous examples. Body weight and dietary intake were observed 5 days a week throughout the entire study.

B. Results As shown in Fig. 9, NT-4/5 reduces the non-fasting blood glucose level (Fig. 9A) and shows an improved long-term glycemic control as indicated by HbAlc levels (Fig. 9B) in a dose-dependent manner in these polygenic obese NONcNZO-10 mice. T-4/5 also reduces body weight (Fig. 10A) and dietary intake (Fig. 10B) of polygenic obese NONcNZO-10 mice in a dose-dependent manner. No rebound of hyperphagia or rebound gain / excess body weight is observed even 30 days after the treatment has ended. The experiment was also performed on the same type of mice by intravenous injection of the vehicle (PBS) and NT-4/5 (1 mg / kg) on day 1 and day 5. About 5% up to about 10% reduction body weight was observed between the group treated with NT-475 and the vehicle group between day 2 and day 15. This experiment shows that a significant body weight reduction can be maintained for up to 10 days after 2 stepped intravenous doses of NT-4/5.

Example 4. Effect of NT-4/5 on lipid metabolism, homeostasis of body weight, dietary intake, water intake, electrolyte balance and endocrine functions in polygenic obese mice (NONcNZO-10) Test animals: 40 mice Polygenic obese NONcNZO-10 males obtained from the Jackson Laboratory at 10 weeks of age weighing in the range of 26-32 g were used in this study. These mice were housed in a room controlled by temperature (19.5-24.5 ° C) and relative humidity (45-65%) with a light / dark cycle of 12 hours, with access ad libitum to water from the filtered tap and food from laboratory in irradiated granules (PURINE) throughout the study. When received in animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed.

Administration of NT-4/5: NT-4/5 occurred in E. coli as described in the US Pat. No. 6,184,360. The mice were divided into 6 groups of equivalent body weight and baseline blood glucose distribution (6-7 mice per group). Each group received from day 1 to day 5 and then from day 8 to day 11 a daily subcutaneous dose of either the vehicle, 3 mg / kg, 1 mg / kg, 0.3 mg / kg or 0.1 mg / kg of NT-4/5. A "peer-to-peer" control group receives an injection daily from the vehicle, but only feeds with the same amount of food from the 3 m / k group consumed the previous day. On the day when the treatment starts (day 1), the baseline blood samples were collected to measure glucose and triglycerides. The level of triglycerides was measured on day 10. At the end of the study (day 12), a terminal blood sample was collected to determine the levels of glucose, triglycerides, electrolytes (Na, K, Cl), testosterone, corticosterone, thyroid hormone ( Total T4) and thyroid stimulating hormone (TSH). The electrolyte, free testosterone, T4 and TSH levels of the serum samples were measured by AniLytics (Gaithesburg, M. D) in accordance with the standard protocols employed by the vendor. Other analytes were measured as described above. Body weight, dietary intake were observed 5 days per week throughout the entire study. Water intake was measured daily from day 5 to day 7.

B. Results As shown in Table 18, daily subcutaneous NT-4/5 injection significantly reduced non-fasting blood triglyceride levels. Remarkably, even the lowest daily subcutaneous dose of NT-4/5 (0.1 mg / kg / day) significantly reduced non-fasting blood triglyceride levels in NONcNZO-10 polygenic obese mice.

Table 18. Effect of different doses of NT-4/5 in non-fasting blood triglyceride levels in obese polygenic mice (NONcNZO-10).

Values are expressed as mean ± SEM Statistics: Bi-directional analysis of variance (ANOVA) followed by subsequent Bonferroni test. * P < 0.05, **. P < 0.01 compared to vehicle.

As shown in Tables 19 and 20, injection NT-4/5 also reduces body weight (Table 19) and dietary intake (Table 20) of polygenic obese NONcNZO-10 mice in a dose-dependent manner, mice treated with? T-4/5 3 mg / kg show about 10% loss of body weight and those treated with? T-4/5 1 mg / kg show about 5% relative loss of body weight at control with vehicle. However, only the group with 3 mg / kg, but not the group of 1 mg / kg, shows a significant reduction in dietary intake. The mice in the paired feeding group generally consume a very similar amount of food as the 3 mg / kg group and still only lose about 5% of body weight relative to vehicle control.

Table 19. Effect of different doses of NT-4/5 on body weight in obese polygenic mice (NONcNZO-10). Values are expressed as mean + SEM Statistics: Bi-directional analysis of variance (ANOVA) followed by subsequent Bonferroni test. * P < 0.01, *** P < 0.001 compared to vehicle. Table 20. Effect of different doses of NT-4/5 on food intake in polygenic obese mice (No? CNZO-10).

The effects of NT-4/5 treatment on water intake, electrolyte balance and endocrine functions were also tested. Water intake was measured from day 5 to day 7. Electrolyte concentrations (ie, Na, K, and Cl) were measured using the terminal blood samples taken on day 12. None of the treatment of NT-4 / 5 (0.1 mg / kg, 0.3 mg / kg, 1 mg / kg, and 3 mg / kg for water intake test, and 1 mg / kg and 3 mg / kg for Na, K, and Cl concentration test ) or paired feeding groups show a statistically significant difference. of the group with vehicle in any of these 'measurements. An ANOVA pathway followed by subsequent Dunnett test was used for statistical analysis.

Total T4 thyroxine was significantly reduced (by about 33%, while remaining in the normal range) in the treated group by 3 mg / kg NT-4/5 daily, but not in the matched fed or group treated with 1 mg / kg (one-way ANOVA, followed by Dunnett's subsequent test). ? or change in the level of thyroid stimulating hormone (TSH) in any of the experimental groups compared to vehicle control (one A? OVA route, followed by Dunnett's subsequent test). "In addition," NT-4/5 treatment (0.1-3 mg / kg / day) does not significantly change corticosterone levels in serum and testosterone levels at the end of the experiment (day 12). However, the corticosterone level of the matched feeding group increases significantly (P <0.05) up to about 3 times compared to the vehicle group. A one-way ANOVA followed by the Dunnett posterior test was used for statistical analysis. This is consistent with the possibility that the matched feeding group, but not the groups treated with NT-4/5, is under constant semi-starvation tension.

Example 5. Effect of NT-4/5 on carbohydrates, lipids and body weight homeostasis in obese mice induced by high-fat diet A. Experimental Protocol Test animals: Diet-induced obesity (DIO) mice from the Jackson Laboratory were used in this studio. El-Haschimi, J. Clin. Invest. 105: 1827-1832 (2000) Steppan,? Ature 409: 307-312 (2001). 23 male C57BL / 6J mice were weaned at 4 weeks of age. Immediately after weaning, they were placed on a high fat diet of around 58% (D1233Ü, Research Diet) throughout the study. DIO mice mice at 13 weeks of age weighing in the range of 28-40 g were used in this study. Mice older than 22-24 weeks of age were also used. These mice were housed in a room controlled by temperature (19.5-24.5 ° C) and relative humidity (45-65%) with a light / dark cycle of 12 hours, with access ad libitum to filtered tap water and high diet in 58% fat (D1233Ü, Research Diet) throughout the study. Upon arrival at animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed. Administration of NT-4/5: The? T-4/5 used in this study was obtained from Genentech. The mice were divided into 3 groups of equivalent baseline body weight and blood glucose distribution (7-8 mice per group). Each group received from day 1 to day 5, from day 8 to day 12, from day 15 to 16 a daily subcutaneous dose of either the vehicle, 2 mg / kg or 10 mg / kg of NT-4/5, respectively. Five days before starting treatment (day-5), - baseline blood samples were collected to measure glucose and triglycerides. After the start of treatment (day 1), blood glucose and triglycerides were measured once a week. Body weight, dietary intake were observed 5 days per week throughout the entire study. From days 57-59, the DIO mice receive a daily injection of NT-4/5 at 2 or 10 mg / kg respectively. They fast at night (for 16 hours) before measuring fasting glucose levels and the oral glucose tolerance test is conducted with a load of 2 grams of glucose / kg body weight.

B. Results Administration of NT-4/5 (2 mg / kg and 10 mg / kg) significantly reduced (P <0.001, two-way ANOVA) body weight (Fig. HA) and dietary intake (Fig. llB) of DIO mice in a dose-dependent manner: administration of? T-4/5 (2 mg / kg and 10 mg / kg) does not affect the fasting glucose levels or triglycerides of the DIO mice in the Younger age (12-16 weeks of age) who are not diabetic. However, as shown in Fig. 12, administration of NT-4/5 (2 mg / kg and 10 mg / kg) significantly reduced fasting glucose level and improved glucose tolerance in DIO mice at older age (22-24 weeks of age) when animals develop overt diabetes. Two-way ANOVA followed by subsequent Bonferroni test were used for statistical analysis. In a separate study, a reduction of about 5% of body weight is also observed after intravenous (iv) injection of NT-4/5 (5 mg / kg) on day 1 and day 4, and reduced body weight it is maintained for at least 10 days after the second dose is given.

Example 6. Effect of a pegylated NT-4/5 cysteine mutant on carbohydrates, lipids and body weight homeostasis in polygenic obese mice (? O? CNZO-10) A. Experimental Protocol Test animals: 30 10-week-old, 10-week-old, polygenic obese male mice, weighing in the range of 27-32 g, were used in this study. These mice were housed in a room controlled by temperature (19.5-24.5 ° C) and relative humidity (45-65%) with a light / dark cycle of 12 hours, with access ad libitum to water from the filtered tap and food from laboratory in irradiated granules (PURINE) throughout the study. Upon arrival at animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed.

Method for making pegylated NT-4/5 (S50C): The native form of NT-4/5 used in this study is produced in accordance with the method described in the U.S. Patent. Do not . 6,184,360. A pegged "T-4/5" directed to the site was also produced. The structure of? T-4/5 in complex with domain 5 of TrkB was examined to find side chains of amino acids that may be available surface and contrary to interfering with natural disulfide bonds and also to the contrary to interfere with any of Interactions between monomer subunits of NT-4/5 or the TrkB domain 5 create a PEG binding site. Based on the review, functional linkage with TrkB in the KIRA assay is found to be possible for the following single cysteine mutants: S50C, Q105C, and E67C. A single amino acid mutation is then introduced a_L to change the serine residue at position 50 of the mature human T-4/5 sequence for cysteine, resulting in a mutant form of? T-4/5 (NT4- S50C); changing the aspartic acid residue at position 105 to cysteine; or by changing the glutamic acid residue at position 67 for cysteine.

The mutant proteins are expressed in E. coli, solubilized, extracted, purified and replicated as native NT-4/5. To pegulate the NT4-S50C, NT4-Q105C, and refolded NT-4-E67C, a gentle reduction is first performed with 0.1 mM DTT (dithiothreitol). Excess DTT is removed by dialysis against 50 mM sodium acetate at pH 6, then a 5 molar excess of PEG-maleimide (MW of 10,000 Da, SunBio, Inc., South Korea) is incubated with the protein at 37 ° C for several days with gentle agitation. The pegylated protein, non-pegylated protein and unreacted PEG are separated by ion exchange or size exclusion chromatography.

TrkB specific activation assays by pegylated NT-4/5: Stable cell lines expressing human TrkA, TrkB or TrkC were used to determine functional activity, ie, phosphorylation of the receptor, NT-4/5 and NT-4 / 5 pegylated as previously described (Sadick et al, Experimental Cell Research 234: 354-361, 1997).

Administration of NT-4/5 and pegylated NT-4/5: Mice were divided into 4 equivalent body weight distribution groups (7-8 mice per group). Each group received the first day (day 1) of study one and the only dose of either the vehicle alone, 1 mg / kg of NT-4/5 or 1 mg / kg of PEG-NT4-SSOC; respectively. A final group receives a daily subcutaneous dose of 1 mg / kg NT-4/5 from day 1 to day 5, then from day 7 to day 9. On day 9 after the start of treatment, blood samples were collected to determine serum triglyceride and glucose levels. Body weight and dietary intake were observed 5 days a week throughout the entire study.

B. Results As shown in Fig. 13,? T-4/5 native and PEG-? T4-SSOC increase tyrosine phosphorylation of the cell line receptor expressing TrkB in a dose-dependent manner. But native NT-4/5 and PEG-? T4-SSOC do not increase the tyrosine phosphorylation of the cell line receptor expressing trkA- or trkC-. As a control for the experiment, cells expressing trkA activated with NGF and cells expressing trkC activated with NT-3. As shown in Fig. 14, a simple subcutaneous injection of 1 mg / kg PEG-? T4-S50C, but not NT-4/5 wild-type (WT? T4), significantly reduced dietary intake and weight bodily. In contrast, the daily injection of 1 mg / kg WT? T4 significantly reduced body weight without affecting daily food intake. However, the simple subcutaneous injection of 1 mg / kg PEG-NT4-S50C and NT-4/5 wild-type (WT NT4) does not significantly reduce the level of blood glucose and the level of triglycerides. The daily dose of 1 mg / kg NT-4/5 wild-type reduced significantly (about 50% on average) of both blood glucose levels (P <0.05) and triglyceride level (P <0.01) . An ANOVA pathway followed by Dunnett's test was used for statistical analysis. The pegylated mutant NT-4/5 (Q105C) retains the TrkB receptor agonist activity in the cell-based assay, however it was inactive in vivo (no effect on body weight, dietary intake, glucose or triglyceride of polygenic obese mice ? O? C? ZO-10 to injection 2 mg / kg / week). Pegylated T-4/5 (E67C) was also activated in the activated TrkB receptor in the cell-based assay.

Example 7. Effect of β-4/5 pegylated on carbohydrates, lipids and body weight homeostasis in obese Hypnogenic db / db mice A. Experimental Protocol Test animals: 30 db / db female mice at 7 weeks of age weighing in the range of 27-34 g were used in this study. These mice were housed in a room controlled by temperature (19.5-24.5 ° C) and relative humidity (45-65%) with a light / dark cycle of 12 hours, with access ad libitum to water from the filtered tap and food from laboratory in irradiated granules (PURINE) throughout the study. Upon arrival at animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed.

Methods for making pegylated NT-4/5: A mutation was introduced to the glycine at position 1 of mature human NT-4/5 sequence for serine, resulting in a mutant form of? T-4/5 (NT4- G1S) in order to generate a PEG link site. The? T4-G1S protein was expressed in E. coli, solubilized, extracted, purified and refolded as described above. The refolded NT4-G1S was dialyzed in 50 mM sodium phosphate buffer solution at pH 6.8 and treated with sodium meta-periodate in 1.5 molar excess for 10 minutes to oxidize the serine residue of the N-terminal (or threonine) to an aldehyde The remaining periodate was removed by dialysis against 30 mM sodium acetate at pH 4.5. The exact concentration of? T4-G1S was determined and a molar excess of 2 to 4 times of 20K-m-PEG-HZ (methoxy polyethylene glycol hydrazide) (20 kDa MW, SunBio, South Korea) was added. The reaction was incubated at 37 ° C for several days with gentle agitation. A conjugated PEG, pegylated? T4-G1S and non-pegylated? T4-G1S were separated by means of ion exchange or size exclusion chromatography.

TrkB specific activation assays by pegylated NT-4/5: Stable cell lines expressing TrkA, TrkB or human TrkC were used to determine functional activity, ie, receptor phosphorylation, NT-4/5 and different forms of NT -4/5 pegylated as previously described (Sadick et al., Experimental Cell Research 234: 354-361, 1997).

Administration of NT-4/5 and NT-4 / S pegylated: NT-4/5 wild-type was obtained from Genentech Inc. Mice were divided into 5 groups with equivalent body weight distribution (6 mice per group). Each group received on the first day (day 1) of the study one and only doses of either vehicle, 5 mg / kg of NT-4/5 produced as described above, 5 mg / kg of 2PEG-? T4-G1S, or 5 mg / kg of 1PEG-? T4-G1S. 2PEG-? T4-G1S represents a PEG molecule was conjugated to each monomer NT-4/5 in an NT-4/5 dimer, and 1PEG-NT4-G1S represents a PEG molecule was conjugated to only one monomer of the NT-4 dimer /5 . 2PEG-NT4-G1S and 1PEG-NT4-G1S were separated by ion exchange. 4 days before starting treatment (day-4), blood samples were collected from the mice as previously described for the determination of serum glucose baseline levels. On day 4 after starting the treatment, blood samples were collected again to determine the serum glucose level. Body weight and dietary intake were observed 5 days a week throughout the entire study.

B. Results As shown in Fig. 15, native NT-4/5, 2PEG-? T4-G1S and 1PEG-? T4-G1S increases the tyrosine phosphorylation of the receptor of the cell line expressing TrkB in a dependent manner of the dose. But native NT-4/5, 2PEG-NT4-G1S and 1PEG-NT4-G1S do not increase the tyrosine phosphorylation of the cell line receptor expressing trkA- or trkC. As a control for the experiment, cells expressing trkA activated with? GF and cells expressing trkC activated with? T-3. In addition, pegylated NT-4/5 has extended half-life in vivo. As shown in Fig. 16, the serum half-life of 2PEG-? T4-G1S was estimated to be 764-960 minutes after a single subcutaneous dose of 4 mg / kg in the db / db mice. The half-life of? T-4/5 native was approximately 41-52 minutes. Absolute protein levels of 2PEG-NT4-G1S were also diverse orders of higher magnitude than those of? T-4/5 native at any given point in time.

As shown in Tables 21 and 22 below, a subcutaneous injection of NT-4/5 and pegylated NT-4/5 (2PEG-? T4-G1S and 1PEG-NT4-G1S) reduces body weight growth and significantly reduces Dietary intake of db / db mice.

There was no significant difference between •? T- 4/5 and? T-4/5 pegylated in this experiment using female db / db mice giving a single 5 mg / kg dose of the proteins.

Table 21. Body weight expressed as the percentage of baseline value after a single injection of the vehicle, NT-4/5 or? T-4/5 pegylated. and express values as me + Statistics - Bi-directional analysis of variance (ANOVA) followed by subsequent Bonferroni test compared to vehicle group * P < 0.05 ** P < 0.01 *** p < 0.001 Table 22. Daily dietary intake (g / day) after a single vehicle injection, NT-4/5 or pegylated NT-4/5.

- Values are expressed as mean +/- SEM Statistics: Bi-directional analysis of variance (ANOVA) followed by subsequent Bonferroni test compared to vehicle group * P < 0.05 ** P < 0.01 *** P < 0.001 As shown in Fig. 17, a subcutaneous injection of NT-4/5 and pegylated NT-4/5 (2PEG-? T4-G1S and 1PEG-NT4-G1S) shows a tendency to reduce serum glucose levels without fasting of db / db mice after 4 days. However, the relative lack of efficacy in glucose reduction can be related to the fact that these mice db / db. they were much younger than those used in previous studies and those were not yet diabetic at the start of this study.

Example 8. Differential effects of pegylated NT-4/5 against NT-4/5 wild-type (WT) in homeostasis of body weight and dietary intake in DIO mice A. Experimental Protocol Test animals: Mice with diet-induced obesity (DIO) were used in this study. 29 male C57BL / 6J mice were weaned at 4 weeks of age. Immediately after weaning, they were put on a 58% high fat diet (D1233Ü, Research Diet) throughout the study. DIO mice at 13 weeks of age weighing in the range of 28-40 g were used in this study. These mice were housed in a room controlled by temperature (19 ^ 5-24.5 ° C) and relative humidity (45-65%) with a light / dark cycle of 12 hours, with ad libitum access to water from the filtered tap and 58% high fat diet (D1233Ü, Research Diet) 'throughout the study. Upon arrival at animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed. Administration of NT-4/5 and pegylated NT-4/5: 'The NT-4/5 was obtained from Genentech Inc. or produced as described in US Patent. 6,184,360. The pegylated? T-4/5 occurs as follows: A mutation was introduced when changing position 1 glycine of sequence? T-4/5 of human mature for serine, resulting in • a mutant form of NT-4/5 (? T4-G1S). The NT4-G1S protein was expressed in E. coli, solubilized, extracted, purified and reproduced as described above.

The refolded? T4-G1S was dialysed in 50 mM sodium phosphate buffer solution at pH 6.8 and treated with 1.5 molar excess of sodium meta-periodate for 10 minutes- to oxidize the N-terminal serine residue (or threonine ) to an aldehyde. The remaining periodate was removed by dialysis against 30 mM sodium acetate at pH 4.5. The exact concentration of NT4-G1S was determined and a 2 to 4-fold molar excess of 20K-m-PEG-HZ (methoxy polyethylene glycol hydrazide) was added. The reaction was incubated at 37 ° C for several days with gentle agitation. Unconjugated PEG, pegylated NT4-G1S and non-pegylated NT4-G1S were separated by ion exchange or size exclusion chromatography. Mice were divided into 3 groups of equivalent body weight and baseline blood glucose distribution (7-8 mice per group). Each group received four intravenous doses weekly (on day 1, 8, 15 and 22) of either vehicle, 2 mg / kg of? T-4/5 or 2 mg / kg of GIS? T-4/5 pegylated respectively. Body weight and dietary intake were observed 5 days per week throughout the entire study.

B. Results: As shown in Figure 18, the body weights of the treatment groups (both wild type? T-4/5 and GIS NT-4/5 pegylated) differed significantly by A? OVA 2-way (F = 207.01, P <0.0001). The subsequent tests of Bonferroni showed important differences in pairs between WT NT-4/5 group with vehicle control group (P <0.001 from day 16 to day 38, P <0.01 on day 43, not important at all other time points ) as well as between GIS NT-4/5 pegylated and vehicle control (P <0.05 in days 2-3, P <0.01 in days 4-5, P <0.01 from day 9 to 12, P <0.01 on day 16-18, P <0.05 on day 21 and 22, P <0.001 on day 23-25, not important at all other time points). The first single dose of pegylated NT-4/5 GIS was more effective in reducing body weight. The recovered body weight occurs rapidly within 3-4 days after each weekly dosage of GIS? T-4/5 pegylated. In contrast, the weekly long-term dosage of wild-type (WT)? T-4/5 was more effective in maintaining lower body weight. As shown in Fig. 19, dietary intake of the treatment groups differed significantly by 2-way ANOVA (F = 11.62, P <0.0001). Subsequent Bonferroni tests showed significant differences in pairs between each treatment group with the vehicle control group (* P <0.05 and ** P <0.01 as indicated in the graph). The first single dose of pegylated NT-4/5 GIS was more effective in reducing dietary intake than WT? T-4/5.

Example 9. Effect of daily subcutaneous dosing of WT NT-4/5 on respiratory coefficient and body composition in DIO mice A. Experimental Protocol Test animals: Mice with diet-induced obesity (DIO) were used in this study. 29 C57BL / 6J male mice were weaned at 4 weeks of age. ' Immediately after weaning, they were placed on 58% high fat diet (D1233Ü, Research Diet) throughout the study. DIO mice of 13 weeks of age weighing in the range of 28-40 g were used in this study. These mice were housed in a room controlled by temperature (19.5- 24.5 ° C) and relative humidity (45-65%) controlled in the room with a light / dark cycle of 12 hours, with ad libitum access to tap water filtered and 58% diet high in fat. (D1233Ü Research Diet) throughout the study. Upon arrival at animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed. Administration of NT-4/5:? T-4/5 were obtained from Genentech Inc. or were produced as described in the patent of E.U.A. Do not . 6,184,360. The mice were divided into 2 groups of equivalents of body weight distribution (8 mice per group). The mice were acclimatized individually in the Comprehensive Cage Monitoring System (CCMS, Columbus Instruments) for 2 days, in preparation for indirect calorimetry, before the start of treatment. Each group received 5 daily subcutaneous doses of either vehicle or 10 mg / kg of NT-4/5, respectively. Body weight and dietary intake were observed 5 days per week throughout the entire study. CCMS monitoring and analysis of oxygen consumption and carbon dioxide production data were carried out in accordance with the publication and commonly accepted protocols as described on the website http://pga.jax.org/protocol012.html from the Jackson laboratory.

The composition of the body, that is, percentage of lean body weight, fat content, and bone density determined the day both before the start of treatment (day 0) and the final day of treatment (day 5) with a mouse densitometer PlXImus Mouse (GE Lunar Medical Systems, Madison, Wl) using a software version 1.46. Brommage et al., Am. J. Physiol. Endocrinol Metab. 285: 454-459 (2003).

B. Results: As shown in Fig. 20, daily subcutaneous injection of NT-4/5 (10 mg / kg) significantly (P = 0.0025, Student's t test) reduces the respiratory coefficient (RQ = Vco2 / Vo2 , that is, the ratio of the production of carbon dioxide during oxygen consumption measured by CCMS). Since the body mainly uses fat (RQ-0.67) and carbohydrate (RQ = 1) for oxidation in the resting state, this reduction in RQ indicates a change in the utilization of increased body fat (fatty acids, triglycerides and cholesterol, etc.) relative to carbohydrate as the best source of energy. As shown in Fig. 21, daily subcutaneous injection of NT-4/5 (10 mg / kg) significantly reduced body weight (panel A), dietary intake (panel B), and body fat content (panel C), but not lean body mass (panel D). Bone density at? T-4/5 (10 mg / kg) treated the group compared to the vehicle group. The bone mineral density of the group treated with? T-4/5 was 0.5235 0.0158 g / cm2 (mean ± SEM) on day 0, and 0.5452 0.0142 g / cm2 (mean ± SEM) on day 5. The mineral density of the group bone with vehicle was 0.5070 ± 0.0210 g / cm2 (mean ± SEM) on day 0, and 0.4698 ± 0.0198 g / cm2 (mean ± SEM) on day 5. The difference in bone density of the group treated with T-4/5 was significantly different from that of the vehicle on day 5 (P <0.05, 2-way ANOVA with Bonferroni test). The reduction of the bone density of the group with the vehicle was probably due to the stress of staying in a small confined space in the CCMS for 5 days, as confirmed by extremely high serum corticosterone levels found in both vehicle-treated groups and? T-4/5 on day 5. This study suggests that the treatment of? T-4/5 prevents stress-induced bone loss in the presence of obesity induced by diet. No change in accumulated energy expenditure estimated from day 1 to day 5 was observed in the NT-4/5 treated group (10 mg / kg) compared to the vehicle control group based on the oxygen consumption range measured by indirect calorimetry (http: // page jax.org/ protocol012.html).

Example 10. Differential potency of NT-4/5 against BDNF in body weight and dietary intake of DIO mice A. Experimental Protocol Test animals: Mice with diet-induced obesity (DIO) were used in this study. 22 male C57BL / 6J mice were weaned at 4 weeks of age. Immediately after weaning, they were placed on a 58% high-fat diet (D1233Ü, Research Diet) through the study. DIO mice at 13 weeks of age weighing in the range of 28-40 g were used in this study. These mice were housed in a room controlled by temperature (19.5-2.5 ° C) and relative humidity (45-65%) with a light / dark cycle of 12 hours, with ad libitum access to the water of the filtered tap and 58% high fat diet (D1233Ü, Research Diet) throughout the study. Upon arrival at animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed. Administration of NT-4/5 and BDNF: The NT-4/5 is produced as described in the patent E.U.A. No. 6,184,360. Recombinant human BDNF is obtained from PeproTech Inc. (Rocky Hill, NJ). The BDNF material was tested by the seller to be 3 active in two biological assays: (a) induction of acetyltransferase choline activity in septal culture of the basal forebrain of the primary rat (ED50 = 25-50 ng / mL) and (b) induction of excretion of day 8 embryonic trigeminal neurons chicken (ED50 = 0.1-1 ng / mL). The mice were divided into 3 groups of equivalent body weight distribution (7-8 mice per group). Each group received 2 weekly intravenous doses of either the vehicle, 2 mg / kg of "NT-4/5 or 2 mg / kg of BD? F, respectively." Body weight and dietary intake are taken the day before starting the treatment (day 0) and are subsequently followed on a daily basis.

B. Results: As shown in Fig. 22, weekly intravenous injection of? T-4/5 (2 mg / kg) reduces body weight more effectively than BD? F (2 mg / kg). For example, on day 2, NT-4/5 at 2 mg / kg reduces body weight significantly, but BD? F at 2 mg / kg does not significantly reduce body weight. As shown in Fig. 23, weekly intravenous injection of NT-4/5 (2 mg / kg) reduced dietary intake more effectively than that of BDNF (2 mg / kg). For example, on day 2, NT-4/5 at 2 mg / kg reduced dietary intake significantly, but BDNF at 2 mg / kg did not significantly reduce dietary intake.

Example 11. Activity of WT NT-4/5 in body weight and dietary intake in normal lean mice A. Experimental Protocol Test animals: Mice with normal diet were used in this study. Forty C57BL / 6J mice weighing 25-31 g at 12 weeks of age were housed in a room controlled by temperature (19.5-24.5 ° C) and relative humidity (45-65%) with a light / dark cycle of 12 hours, with access ad libitum to filtered tap water and regular mouse feed (6% fat) throughout the study. Upon arrival at animal facilities, 1 per cage covered with filtered lids were housed and at least one acclimation period of 5 days was observed.

Administration of NT-4/5: NT-4/5 is produced as described in the US Patent. Do not' . 6,184,360. Mice were divided into 5 groups of equivalent body weight at baseline, food intake and non-fasting blood glucose level distribution (8 mice per group). Each group received 4 subcutaneous doses of vehicle daily, Img / kg, 2mg / kg, 5mg / kg or 10 mg / kg of NT-4/5, respectively, from day 1 to day 4. Body weight and intake food were observed on a daily basis. Non-fasting blood glucose was measured as previously described once a week (day 1 and day 8). .

B. Results: As shown in Fig. 24, daily subcutaneous injection of NT-4/5 (1, 2, 5 and 10 mg / kg) significantly reduced body weight in a dose-dependent manner compared to the control group with vehicle. As shown in Fig. 25, daily subcutaneous injection of NT-4/5 (1, 5 and 10 mg / kg) significantly reduced dietary intake in a dose-dependent manner compared to the vehicle control group. As shown in Fig. 26, daily subcutaneous injection of NT-4/5 (2.5 and 10 mg / kg) significantly reduced fasting blood glucose in a dose-dependent manner compared to the control group with vehicle.

Triglyceride levels in the blood were also measured. Daily subcutaneous injection of NT-4/5 (1, 2, 5 and 10mg / kg) significantly reduced triglycerides in the blood in a dose-dependent manner compared to the vehicle control group. Similar experiments were also performed on female C57BL / 6 mice. Statistically significant weight loss in a dose-dependent manner (about 8% for 5 mg / kg, and about 11% for 10 mg / kg on day 2) compared to the vehicle control group is also observed .

Example 12. Effects of NT-4/5 on survival time in db / db mice A. Experimental Protocol Female db / db mice maintained as described in Example 1 were divided into two groups. Group 1 (8 mice) were administered with vehicle (PBS). Group 2 (6 mice) each were administered with NT-4/5 at a dose of 20 mg / kg. Both the vehicle and NT-4/5 were administered subcutaneously once a day from day 1 to day 26, and are then followed for survival. Mortality is defined by body wasting below 60% of the body weight of onset (ie, from about 40 g down to about 24 g) or natural death, whichever comes first.

B. Results As shown in Fig. 27, the mean duration of survival is 257 days (n = 6) for the group? T-4/5 (20 mg / kg), while it is 192.5 days (n = 8) for the vehicle control group. This difference of 65 days is not statistically significant (Logrank test, square Chi = 2.26, P = 0.1331) probably because the number of animals tested is not large enough.

Example 13. Effect of administration of NT-4/5 on pain 5 To test whether NT- / 5 external pain in animals after administration of NT-4/5, an animal heat / thermal hyperalgesia model was used to test. Adult male Sprague-Dawley rats (Charles River) are injected subcutaneously with either the vehicle (PBS), NGF (2 mg / kg), or NT-10 4/5 (2 mg / kg). Then the latency for removal of the paw in response to a constant radiant heat source was measured. See, Hargreaves et al., Neurology 48: 501-505 (1997); Shu et al., Pain 80: 463-470 (1999). No statistically significant changes in latency were observed for removal of the leg in response to a constant radiant heat source in the vehicle group and the group treated with NT-4/5 at 3, 5, and 24 hours after administration . However, the group treated with NGF shows a reduction (around 40% up to 50%) in reduction of latency to 3, 5, and 24 hours after administration. These data indicate that, unlike NGF, NT-4/5 at 2 mg / kg do not cause pain in these animals.

Example 14. Effects of NT-4/5 on the control of long-term glucose A. Experimental Protocol Db / db mice maintained as described in Example 1 were divided into five groups (groups 1-5). Group 1 was administered with vehicle (PBS). Group 2, group 3, group 4, group 5 each were administered with NT-4/5 at doses of 2 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg , .respectively. Both vehicle and NT-4/5 were administered to each group of mice subcutaneously, once a day from day 1 to day 26. One day before the start of treatment (day 0), blood samples were collected of the mice as previously described for the determination of baseline levels of serum biomarkers (glucose and insulin). On day 45, the HbAlc level was measured. The glucose tolerance test was performed on day 54 (28 days after the last dose of treatment? T-4/5). The intraperitoneal glucose (IP) tolerance test was conducted by fasting the mice overnight (16 hours) before giving them an IP injection of a load of 2 grams of glucose / kg body weight. Glucose in the serum and insulin were also measured. Results As shown in Panel A of Figure 28, HbAlc levels in the groups treated with? T-4/5 (2 mg / kg, 5 mg / kg, 10 mg / kg, and 20 mg / kg) are significantly reduced more than the group with vehicle on day 45 (15 days after the dose) after injections are daily from day 1 to day 26. Panels B of Fig.28 show that treatment with NT-4/5 significantly improves glucose tolerance. As shown in Panel C of the .Fig. 28, the fasting serum glucose level is reduced by more than 50% after treatment with NT-4/5 for all treatment groups and remains low until day 29. Panel D of Fig. 28 shows a significant reduction in insulin levels in the serum by 10 mg / kg / d of the group treated with NT-4/5 (day 6, P <0.05) and 20 mg / kg / d of the group treated with NT-4/5 (day 6 and day 14, P <0.05). These data indicate that "T-4/5 improves long-term glucose control in db / db mice.

Example 15. Effects of co-administration of mouse monoclonal NT4 / 5 Mabl241 with NT4 / 5 on the biological activities of? T4 / 5 in an obese, diabetic mouse model A. Experimental Protocol Test animals: 32 mice db / db leptin receptor mutants (BSK.Cg-m + / + Leprdb) obtained from Jackson Laboratory were used for this study. The mice were divided into four groups of 8 animals each (groups 1-4), with homogenous initial body weight and glycemia values based on a random placement table. The mice were housed one per cage.

Anti-NT4 / 5 and NT4 / 5: Both the monoclonal antibody anti-NT4 / 5 Mab 1241 and the human? T4 / 5 are obtained from Genentech. According to the BIAcore assay, the binding affinity between the monovalent Fab fragment of Mab 1241 and NT4 / 5 was 170nM with kon = 2.2xl05M-ls-l and koff = 0.039s-l. The Mabl241 Fab fragment does not interfere with the ability of NT4 / 5 to bind to the human TrkB receptor in a BIAcore surface plasmon resonance assay, (data not shown).

Administration of Mab 1241 and NT4 / 5: On day one, mice in groups 1-4 were treated with vehicle (PBS) subcutaneously (sc), 5 mg / kg Mab 1241 intraperitoneally (ip), 5 mg / kg Mab 1241 intraperitonally (ip) plus 2mg / kg? T4 / 5 subcutaneously (s.c.), and 2mg / kg? T4 / 5 subcutaneously (s.c.), respectively. On day 2 to day 5, group 1 was treated with a daily vehicle dose (PBS) subcutaneously (s.c.) Groups 3 and 4 were treated with a daily dose of 2mg / kg? T4 / 5 subcutaneously (s.c.). Collection of samples for analysis: one day before the start of treatments (day 0), non-fasted mice were weighed and blood samples were collected through the retro-orbital prexo (around 300 ul / mouse) under isoflurane anesthesia. Blood glucose level of non-fasting baseline was measured by the One-Touch Ultra glucose meter of the tail bleeding.

B. Results Effect on body weight: The body weights of each group of mice are taken every other day starting from day 1, and the results are presented graphically in Figure 27A. As shown in the figure, the treatment of NT4 / 5 2mg / kg daily from day 1 to day 5 produces a small but statistically insignificant reduction in body weight. The injection of Mab 1241 alone has no effect on body weight. In contrast, the co-treatment of 2mg / kg NT4 / 5 daily and a single injection of Mab 1241 on day 1 induces a significant reduction in body weight on day 5 and day 8. A two-way ANOVA with pairwise comparisons Subsequent tests Bonferrioni of each treatment in relation to the control group with vehicle was used for statistical analysis.

Effect on dietary intake: Dietary intakes of each group of mice are taken every other day starting from day 1, and the results are presented graphically in Figure 27B. As shown in the figure, treatment of 2 mg / kg NT4 / 5 daily from day 1 to day 5 significantly reduced dietary intake on day 8 and day 10. However, the Mab co-treatment does not change significantly the effect of dietary intake induced by 2 mg / kg NT4 / 5 alone. A two-way ANOVA with paired comparisons by subsequent Bonferrioni tests of each treatment relative to the vehicle control group was used for statistical analysis.

Effect on glucose level: The blood glucose levels of the mice were evaluated on day 1, day 4 and day 9 using the method described above. The results are represented graphically in Figure 27C. As shown in the figure, the treatment of 2 mg / kg NT4 / 5 daily from day 1 to day 5 reduces the glucose level without fasting only on day 9, but not on day 4. In contrast, the Treatment of 2 mg / kg? T4 / 5 daily and a single injection of Mab 1241 on day 1 reduces glucose level without fasting as previously on day 4. The glucose level of the co-treatment group remains significantly lower than the group with vehicle on day 9. A two-way ANOVA with paired comparisons by subsequent Bonferrioni tests of each treatment relative to the vehicle control group was used for statistical analysis.

C. Conclusion This experiment demonstrates that co-administration of a Mabl241 low affinity NT4 / 5 antibody significantly increases the therapeutic efficacy of the daily dose of NT4 / 5 protein in terms of controlling body weight and blood glucose level in db / db mice. It will be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons of ordinary skill in the art and are included. within the spirit and competence of this request. All publications, patents and patent applications cited therein are hereby incorporated by reference in their entirety for all purposes to the same extent as if each individual publication, patent or patent application is specifically and individually indicated to be incorporated thereby reference. It is noted that with this date, the best method known to the applicant to carry out the practice of said invention, is that which is clear from the present description of the invention.

Claims (36)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property.

1. A method for treating obesity in an individual, the method comprises administering to the individual an effective amount of an NT-4/5 polypeptide, characterized in that the individual has no genetic deficiency of BDNF.

2. The method according to claim 1, characterized in that the individual has resistance to leptin.

3, The method according to claim 1, characterized in that a disorder associated with obesity is treated in the individual, and the disorder is selected from the group consisting of hyperglycemia, low glucose tolerance, insulin resistance, lipids, dyslipidemia, hyperlipidemia, hypertriglyceridemia and metabolic syndrome.

4. The method according to claim 1, characterized in that the individual has a reduction in fat content in the body as a result of the treatment.

5. The method according to claim 1, characterized in that the NT-4/5 polypeptide is a naturally occurring NT-4/5.

6. The method according to claim 1, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1. -

7. The method according to claim 1, characterized in that the? T-4/5 polypeptide comprises the amino acid sequence of SEQ ID? 0: 1 with an amino acid substitution.

8. The method according to claim 1, characterized in that the? T-4/5 polypeptide is linked to a polyethylene glycol (PEG) molecule.

9. The method according to claim 8, characterized in that the? T-4/5 polypeptide comprises the amino acid sequence of SEQ ID? 0: 1 with a G amino acid residue at position 1 changed to S or T, and wherein the NT-4/5 polypeptide is linked to a PEG molecule at position 1.

10. The method according to claim 1, characterized in that the NT-4/5 polypeptide is administered in conjunction with an antibody that specifically binds the? T-4/5 polypeptide.

11. A method for reducing body weight or preventing weight gain in an individual, the method comprises administering to the individual an effective amount of a? T-4/5 polypeptide, characterized in that the individual does not have a genetic defect of BD? F.

12. The method according to claim 11, characterized in that the NT-4/5 polypeptide is a naturally occurring NT-4/5.

13. The method according to claim 11, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

14. The method according to claim 11, characterized in that the? T-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with an amino acid substitution.

15. The method according to claim 11, characterized in that the NT-4/5 polypeptide is linked to a molecule of polyethylene glycol (PEG).

16. The method according to claim 15, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with a G amino acid residue at position 1 changed to S or T, and wherein the polypeptide NT-4/5 binds to a PEG molecule at position 1.

17. The method according to claim 11, characterized in that the NT-4/5 polypeptide is administered in conjunction with an antibody that specifically binds to the NT-4/5 polypeptide.

18. A method for treating insulin-dependent diabetes mellitus in an individual, characterized in that it comprises administering to the individual an effective amount of a? T-4/5 polypeptide.

19. The method according to claim 18, characterized in that a disorder associated with non-insulin-dependent diabetes mellitus in the individual is treated, and wherein the disorder is selected from the group consisting of hyperglycemia, low glucose tolerance, resistance to insulin, abdominal obesity, lipid disorders, dyslipidemia, hyperlipidemia, hypertriglyceridemia, and metabolic syndrome.

20. The method according to claim 18, characterized in that the NT-4/5 polypeptide is a naturally occurring NT-4/5. _twenty-one.

The method according to claim 18, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

22. The method according to claim 18, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with an amino acid substitution.

23. The method according to claim 18, characterized in that the NT-4/5 polypeptide is linked to a polyethylene glycol (PEG) molecule.

The method according to claim 23, characterized in that the? T-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with a G amino acid residue in position 1 changed to S or T, and in where the NT-4/5 polypeptide is linked to a PEG molecule at position 1.

25. The method according to claim 18, characterized in that the NT-4/5 polypeptide is administered in conjunction with an antibody that specifically binds the NT-4/5 polypeptide.

26. A method for treating metabolic syndrome in an individual, the method characterized in that it comprises administering to the individual an effective amount of an NT-4/5 polypeptide.

27. The method according to claim 26, characterized in that the NT-4/5 polypeptide is a naturally occurring NT-4/5.

28. The method according to claim 26, characterized in that the? T-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1.

29. The method according to claim 26, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with an amino acid substitution.

30. The method according to claim 26, characterized in that the NT-4/5 polypeptide is linked to a polyethylene glycol (PEG) molecule.

31. The method according to claim 30, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with a G amino acid residue at position 1 changed to S or T, and wherein the polypeptide NT-4/5 binds to a PEG molecule at position 1.

32. The method according to claim 26, characterized in that the NT-4/5 polypeptide is administered in conjunction with an antibody that specifically binds the NT-4/5 polypeptide.

33. A pharmaceutical composition comprising an NT-4/5 polypeptide and a pharmaceutically acceptable excipient, characterized in that the NT-4/5 polypeptide is linked to a PEG molecule.

34. The pharmaceutical composition according to claim 33, characterized in that the NT-4/5 polypeptide comprises an amino acid sequence of a mature NT-4/5 protein with an amino acid substitution, wherein the substituted amino acid is linked to the PEG molecule.

35. The pharmaceutical composition according to claim 33, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with a G amino acid residue at position 1 changed to S or T, and wherein the NT-4/5 polypeptide is linked to a PEG molecule at position 1.

36. The pharmaceutical composition according to claim 33, characterized in that the NT-4/5 polypeptide comprises the amino acid sequence of SEQ ID NO: 1 with an amino acid residue S at position 50 changed to C, and where the NT polypeptide -4 / 5 is linked to a PEG molecule at position 50.