AU2003227683A1 - Treatment of metabolic syndrome - Google Patents

Description

WO 03/090784 PCT/EP03/04357 Treatment of Metabolic Syndrome The invention relates to the treatment of metabolic syndrome. 5 Metabolic syndrome is a cluster of health problems rather than a single disease. Multiple interrelated abnormalities in glucose and lipid metabolism result in insulin-resistant hyperglycemia, hyperinsulinemia, a high triglyceride (TG) level, a low high-density lipoprotien cholesterol (HDL-C) level and abdominal or visceral obesity. The abnormalities seem to be connected by insulin resistance, that is a reduced 10 sensitivity of the tissues of the body to the action of insulin, caused by visceral obesity. The body compensates for this state by secreting more insulin from the pancreas, hence causing hyperinsulinemia. Type II diabetes can develop when the pancreas is unable to sustain this level of insulin secretion. The high insulin levels in the blood also contribute to abnormalities in blood lipids, that is triglycerides and cholesterol. 15 The consequences of metabolic syndrome are perhaps more important than its causes. The syndrome is characterised by glucose intolerance; abnormal cholesterol and triglyceride levels, in particular there is usually a reduction in HDL-C; and upper body obesity. All of these features are independent high risk factors for cardiac disease. 20 Individually each substantially increases the risk of cardiac disease, but in combination the effect is dramatic. The result is a great increase in the risk of hypertension, atherosclerotic changes and myocardial infarction. As well as cardiac diseases, other resulting conditions include, as mentioned, Type II diabetes which can have such resultant problems as microangiopathy, neuropathy, retinopathy and nephropathy; and 25 Polycystic Ovaryian Syndrome (PCOS) in women. PCOS is characterised by ovarian cysts, high androgen levels, hirsuitism and infertility. Women with PCOS have an increased risk of endometrial cancer. Striking similarities exist between the metabolic syndrome and untreated growth 30 hormone (GH) deficiency due to pituitary or hypothalamic pathologies. The most central findings in both these entities are abdominal obesity and insulin resistance. These similarities indicate that a reduced action of GH may be of importance in the pathogenesis of metabolic aberrations not only in GH deficiency but also in metabolic syndrome. In fact, there is a striking evidence for disturbances in the neuroendocrine 35 regulation of GH secretion in obesity. With an increase in body weight, GH secretion becomes blunted with a decrease in the mass of GH secreted per burst without any WO 03/090784 PCT/EP03/04357 2 major impact on secretory burst frequency. Furthermore, serum levels of IGF-I are inversely related to the percentage of body fat, in particular to the amount of visceral adipose tissue. 5 In patients with GH deficiency, the beneficial effects of replacement therapy with recombinant human GH (rhGH) on most of the clinical features of the adult GH deficiency syndrome are well established. These effects include the reduction of body fat, the improvement in risk factors for cardiovascular disease, and the normalization of other metabolic alterations. Johannsson et al have recently provided first evidence that 10 the beneficial effects of rhGH therapy might also be achieved in abdominally obese patients without evidence of pituitary disease. In this placebo-controlled trial, treatment of viscerally obese males with rhGH over a period of 9 months resulted in a reduction of abdominal fat mass, as well as in an improvement of glucose and lipid metabolism. 15 The use of rhGH in patients with central obesity, however, may be limited by an increase in insulin resistance which is known to occur during the early course of rhGH therapy. This may be of particular importance in metabolic syndrome patients who are characterized by a highly increased risk to develop type 2 diabetes. 20 Present treatment for metabolic syndrome involves lifestyle changes and phannacological treatment with drugs such as metformin. Metformin is a biguanide, which improves glycaemic control by enhancing insulin sensitivity in the liver and in muscles. It does not stimulate insulin secretion. 25 It is clear that metabolic syndrome is an important disorder which can result in serious cardiovarcular disease. Whilst metabolic syndrome may be improved by lifestyle changes, for example to diet and exercise levels, what is required is a pharmacological treatment which can be used to help combat this dangerous syndrome. 30 The inventors have surprisingly found that the combination of growth hormone (GH) and an insulin sensitising agent, for example, metformin provides a beneficial treatment for patients suffering from metabolic syndrome. Although to date the studies have been performed with metformin, as the result depends 35 on the insulin-sentising properties of that drug, similar results will be obtainable with other insulin-sensitising agents.

WO 03/090784 PCT/EP03/04357 3 The invention provides a pharmaceutical composition comprising growth hormone and an insulin sensitising agent, for use in the treatment of metabolic syndrome. Preferably the composition may be in combination with one or more pharmaceutically acceptable carriers. 5 Preferably the GH is recombinant human GH. By analogue we mean a substance having the same biological activity as described here and having at least 65%, preferably 75%, most preferably 85% homology with naturally occurring growth honnone. 10 The insulin sensitising agent is preferably a biguanide, most preferably metformin. Alternative insulin sensitising agents include PPAR gamma insulin sensitising agents and thiazolodeniones, for example the troglitazone and rosiglitazone families. However, this list is not exclusive. 15 Insulin sensitising agents that are known, or are under development include: Troglitazone, Chemical Name: 5-[[4-[3,4-Dihydro-6-hydroxy-2,5,7,8-tetramethyl-2H-]-benzopyran-2-yl) methoxyjphenyl]methyl]-2,4-thiazolidinedione V41 1 (DIABII@, Glaucanin) 20 Pioglitazone (ACTOS, AD 4833, U 72107, U 72107A, U 72107E, ZACTOS@) Chemical Name: 2,4-Thiazolidinedione, 5-[[4-[2-(5-ethyl-2-pyridinyl) ethoxy]phenyl]methyl]-, monohydrochloride, (+/-) ; Rosiglitazone (Avandia@, BRL 49653, BRL 49653C) Chemical Name: 2,4 Thiazolidinedione, 5-[[4-[2-(methyl-2-pyridinylamino)ethoxyphenyl]methyl]; 25 Bexarotene-oral (LGD 1069 oral, Targretin oral, Targretin@, Targretyn oral Targrexin oral) Chemical Name: 4-[1-(3,5,5,8,8-Pentamethyl-5,6,7,8-tetrahydro-2-naphthyl) ethenyl]benzoic acid; ZD 2079, (ICI D 2079) (Chemical Name: R)-N-[2-[4-(Carboxymethyl) 30 phenoxy]ethyl)-N-(2-hydroxy-2-phenethyl) ammonium chloride: Netoglitazone, (Isaglitazone, MCC 555, RWJ 241947) (Chemical Name: 5-[6(2-Fluorobenzyloxy)naphthalen-2-yhnethyl]thiazolidine-2,4-dione); INS 1 (D-chiro-inositol) (Chemical Name: D-1,2,3,4,5,6-Hexahydroxycyclohexane), NN 2344(DRF 2593); Dexlipotam, Chemical Name: 5(R)-(1,2-Dithiolan-3-yl) pentanoic 35 acid; HQL 975, Chemical Name: 3-[4-[2-(5-Methyl-2-phenyloxazol-4-yl) ethoxy]phenyl]-2(S)-(propylamino) propionic acid; WO 03/090784 PCT/EP03/04357 4 YM 268, Chemical Name: 5,5'-Methylene-bis(1,4-phenylene)bismethylenebis (thiazolidine-2,4-dione). PPAR agonists under development include: 5 Reglitazar (JTT 501, PNU 182716, PNU 716) (Chemical Name: Isoxazolidien-3,5-dione, 4-[[4-(2-phenyl-5 -methyl)-1,3-oxazolyl]ethoxyphenyl-4]methyl-, (4RS)); KRP 297, Chemical Name: 10 5-(2,4-Dioxothiazolidin-5-ylmethyl)-2-methoxy-N-(4-(trifluoromethyl) benzyl]benzamide; R 119702 (CI 1037, CS 011) Chemical Name: (+/-)-5-[4-(5-Methoxy-lH benzimidazol-2-ylmethoxy)benzyl]thiazolin-2,4-dione hydrochloride; 15 DRF 2189, Chemical Name: 5-[[4-[2-(l-Indolyl)ethoxy]phenyl]methyl] thiazolidine-2,4-dione A further aspect of the invention provides the use of a growth hormone or analogue thereof and an insulin sensitising agent in the preparation of a pharmaceutical 20 composition to reduce waist circumference. The invention also provides the use of growth hormone or analogue and an insulin sensitising agent in the preparation of pharmaceutical compositions for the treatment of metabolic syndrome. Preferably the insulin sensitising agent reduces the insulin antagonist action of the growth hormone. 25 The invention further provides a method of treatment of metabolic syndrome comprising the step of administering an insulin sensitising agent and growth hormone or analogue thereof to a patient suffering from metabolic syndrome. The insulin sensitising agent and growth hormone may be administered simultaneously, 30 or may be administered separately. Also provided is the use of an insulin sensitising agent in the treatment of metabolic syndrome, in combination with treatment with growth hormone. 35 Further provided is the use of growth hormone in the treatment of metabolic syndrome, in combination with treatment with an insulin sensitising agent.

WO 03/090784 PCT/EP03/04357 5 Preferably metformin is administered orally in the form of metformin hydrochloride tablets. The preferred daily dose is in the range of 1000-2000 mg. The tablets may be given in two doses daily or in extended-release form in one dose. 5 The growth hormone is preferably administered in a subcutaneous injection, the dose being individually regulated according to body weight and IGF-levels. The invention will now be described in detail by way of example with reference to the figures in which: 10 Figure 1 shows the baseline characteristics of 25 men with metabolic syndrome. Figure 2 shows the lipid metabolism characteristics of the same 25 men with metabolic syndrome. 15 Figure 3 Shows the change over 18 months of IGF-I levels in two groups of patients treated either with Metformin+GH or with Metformin+Placebo. The figure gives the results from 14 of the 25 patients who completed the study (Mean±SEM). Analysis of the difference between the two groups by repeated measure analysis of variance (RM 20 ANOVA) included the data from all 25 patients (p=0.001). Figure 4. Shows the changes over 18 months in BMI, total body fat, and waist circumference in the two groups of patients treated either with Metformin+GH or with Metformin+Placebo. The Figure gives the results from the 14 patients who completed 25 the study (mean±SEM). Analysis of the differences between the two groups by repeated measure analysis of variance (RM ANOVA) included the data from all 25 patients and gave significant results only for the change of waist circumference (BMI:p=0.24; total body fat: p=0.91; waist circumference: p=0.048). 30 Figure 5. Shows the change over 18 months in fasting plasma glucose and area under the curve (AUC) of glucose during oGTT in the two groups of patients treated either with Metformin+GH or with Metformin+Placebo. The Figure gives the results from the 14 patients who completed the study (mean±SEM). Analysis of the differences between the two groups by repeated measure analysis of variance (RM ANOVA) included the 35 data from all 25 patients (FPG: p=0.09; AUC glucose: p=052). Figure 6. Shows the percentage change in glucose disposal rate (GDR) in the two groups of patients treated either with Metformin+GH or with Metformin+Placebo. The WO 03/090784 PCT/EP03/04357 6 Figure gives the results from the 14 patients who completed the study (mean+SEM) (difference between the two groups: p=0.07, paired Wilcoxon/Mann/Whitney test). STUDY DESIGN 5 A randomized, double-blind, placebo-controlled trial was used to evaluate the effect of rhGH (recombinant human Growth Hormone) in addition to metformin in patients suffering from metabolic syndrome. 25 non-smoking men with metabolic syndrome were selected to complete the study. The patients had a body mass index (BMI) 10 between 30-40 kg/m2, fasting plasma glucose levels without antidiabetic medication of between 110 mg/dl (6.1 mmol/1) and 145 mg/dl (8.0 mmol/1), and HbAlc <7.5%. Hypertension, if present, was treated by angiotensin converting enzyme inhibitors or angiotensin II receptor antagonists. Antihypertensive drug therapy was kept stable during the study period. Lipid-lowering medication, the use of drugs to reduce body 15 weight such as orlistat or sibutramine, and the use of glucocorticoids was not allowed during the study period. The same was true for any medication influencing growth hormone levels. After providing informed consent, patients attended a pre-inclusion assessment. 20 Growth hormone deficiency was excluded by a combined GHRH/arginine-test. After considering inclusion and exclusion criteria, patients were randomized into the placebo ann or the rhGH arn of the study. In both treatment groups, patients received metformin 850 mg twice daily 25 (Glucophage@, Merck, Darmstadt, Germany) during the whole study period. In the Met+GH group, rhGH (Genotropin@, Phannacia GmbH, Erlangen, Germany) was administered se before bedtime at a daily does of 9.5 g/kg (0.20 rU/kg BW/week) after an initial 4-week dose adjustment period. The rhGH chosen was equivalent to the dose used in the study from Johannsson et al. Patients in the Met+Placebo group 30 administered sc injections of placebo. The placebo vials contained the same vehicle as the rhGH vials, and both preparations were visually indistinguishable. In both treatment groups, the daily dose was reduced by half in the event of side effects. Compliance was assessed by counting the returned empty vials. 35 Before start of the study, the patients were screened by two determinations of FPG levels. At baseline and after 18 months of treatment, all patients were hospitalised. During the study (after 6 weeks, 3 months, 6 months, and 12 months), they were WO 03/090784 PCT/EP03/04357 7 monitored as outpatients. Measurements of blood pressure, ECG, as well as physical and laboratory examinations were performed during all visits. Body weight was measured in the morning to the nearest 0.1 kg wearing indoor clothing, and body height was measured barefoot to the nearest 0.01 m. Waist circumference was measured in the 5 standing position with a flexible plastic tape midway between the lower rib margin and the iliac crest. Systolic and diastolic blood pressure were measured after 10 min supine rest and were repeated after 2 min to calculate mean value. Measurements of body composition were performed at baseline, and after 6, 12 and 18 months. A combined arginine-GHRH-test was performed at baseline, euglycemic hyperinsulinemic clamp 10 tests were performed at baseline and after 18 months. Inclusion Criteria Patients had to fulfil all the following criteria before inclusion in the study: - Age 35 years and 70 year; 15 - Male sex; - Body mass index 25 and 40 kg/n 2 ; - Fasting plasma glucose measured at two different days >110 mg/dl (6.1 mmol/1) and <145 mg/dl (8.0 nimol/1) without antidiabetic medication; - HbAlc <7.5%; 20 - Increase of GH levels to > 5 ng/ml during the GHRH.arginine-test; - The patient must give written informed consent before starting any study-related procedures; - The patient must be willing and able to participate and comply with the requirements of the study. 25 Exclusion Criteria - Serum creatinin >1.2 mg/dl; - Previous cardiovascular or cerebrovascular events; - Evidence of other major cardiac disease; 30 - Reasons for elevated blood glucose concentrations other than type 2 diabetes; - Chronic obstructive pulmonary disease (COPD); - Severe hyperlipidemia requiring treatment with lipid-lowering drugs; - Use of drugs to reduce body weight such as orlistat or sibutramine during the last 3 months before study entry; 35 - Use of glucocorticoids during the last 3 months before study entry; - Present or past therapy with insulin (>1 week); - Oral antidiabetic drugs during the last 6 months before study entry; - Alcoholism or drug abuse; WO 03/090784 PCT/EP03/04357 8 - Elevated liver enzymes (SGOT (AST) >100 U/i; - Any suspicion of malignant disease; - History of a malignant disease with a relapse-free interval of less than 10 years; - State of kidney transplantation; 5 - Hypopituitarism; - Treatment with glucocorticoids; - Participation in any other study up to 30 days before study entry; - Any disease influencing regular participation in the study; - Active smoking or history of smoking during the last 12 months. 10 Criteria for Removal from the Study - Cardiovascular or cerebrovascular events; - Suspicion of malignant disease; 15 - Development of severe diabetes mellitus requiring insulin therapy; - Evidence of impaired renal function (creatinine 1.4 mg/dl.); - Any other circumstance which prevents further therapy and regular assessments; - Decision of the patient to withdraw from the study. 20 RhGH was supplied in vials, each containing I.E. of rhGH by Pharmacia & Upjohn. In accordance with the randomisation list, indistinguishable placebo vials were given to patients from the placebo group as a single subcutaneous injection before bedtime. RhGH and placebo were stored in a secure refrigerator at 2-8"C. 25 PATIENT ASSESSMENTS Baseline Study Assessments (Pre-inclusion (Visit 1)) 30 Before visit 1, patients were screened by two determinations of fasting plasma glucose levels. Baseline study assessments at visit 1 were only performed if both values were >110 mg/dl. (6.1 mmol/1) and <145 mg/dl. (8.0 mmol/1) without antidiabetic medication. Prior to visit 1, the patients fasted overnight. The following procedures were carried out and recorded: 35 - Past medical history and history of concurrent diseases including any allergies; - Current medication; - Family history; WO 03/090784 PCT/EP03/04357 9 - Physical examination including determination of BMI, waist to hip ratio, 2 separate measurements of systolic and diastolic blood pressure in the right arm with the subject seated and after a 10 min. rest, and assessment of heart rate; - 12-lead electrocardiogram (ECG); 5 - Laboratory assessment (see below); - GHRH/arginine-test. Study Assessments (Visits 2, 3, 4, 5, 6, 7) 10 Before each visit, patients fasted for at least 8 hours. Study medication was given to the patient at each visit according to the randomisation list. Empty vials were returned by the patient and were counted to assess compliance with study medication. At each visit the patients had to provide samples of blood before receiving study medication. Each visit included: 15 - Medical history; - Current medication; - Adverse events; - Physical examination including determination of BMI, waist to hip ratio, 2 separate measurements of systolic and diastolic blood pressure in the right arm with the 20 subject seated and after a 10 min. rest, and assessment of heart rate; - 12-leas electrocardiogram (ECG); - Laboratory assessments (see below); - Oral glucose tolerance test; - Assessment of body fat mass (DEXA) (visit 2 (begin), 5 (6 months), 6 (12 months) 25 and 7 (18 months; - Euglycemic-hyperinsulinemic clamp test (visit 2, visit 7). Blood Assessments 30 The following assessments were made: - Haemoglobin; - Haemotocrit; - Platelets; - Red blood cell count; 35 - White blood cell and differential count; - Serum sodium; - Serum potassium; - Serum creatinine; WO 03/090784 PCT/EP03/04357 10 - Serum alanine transaminase and asparetate transaminase; - Total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides; - Lipoprotein (a), apolipoprotein Al, apolipoprotein B; - HbAlc; 5 - Insulin-like growth factor 1, Insulin-like growth factor-binding protein 3; - Growth hormone, growth hormone-linding protein; - Fibrinogen, Plasminogen activator inhibitor (PAI) activity; - Free fatty acids. 10 Pharmacodynamic Assessments Oral Glucose Tolerance Test After an overnight fast, subjects ingested a 75-g oral glucose load over a 3-min. period. 15 Blood samples for plasma glucose and serum insulin were obtained at baseline and after 30, 60, 90 and 120 min. Glucose tolerance status of the subjects was defined by WHO criteria. GHRH/A rginine-Test 20 Recently, application of GHRH and arginine has been reported as a potent, reproducible and age-independent test that is well correlated to the results of growth hormone during insulin induced hypoglycemia and is able to reliably distinguish between growth hormone deficiency and normal adults (14). 25 The test was carried out according to the following protocol: - 1 tg/kg. i.v. Somatorelin (GHR-H Ferring@ at 0 min); - 0.5 g/kg. Arginine i.v. over 30 min. from 0 to 30 min.); - Blood samples for measurement of GH every 15 min. from -15 min. to +90 min. 30 In young, healthy, normal weight subjects, a GH peak>9 pg/1 was regarded as normal. 24-Hour Growth Hormone Profiles 35 The studies were started at 8 a.m. after a 12-h overnight fast. A polyethylene catheter was inserted into an antecubital vein and blood was collected at regular intervals. During the study, the patients received standardised meals.

WO 03/090784 PCT/EP03/04357 11 Measurement of Whole-Body Fat Mass and Fat Free Mass by Dual Energy X-Ray Absorptiometry (DEXA) Whole-body fat mass and fat free mass (FFM) were assessed by dual energy X-ray 5 absorptiometry (DEXA) (model DPX-L, Lunar Corporation, Madison, WI, USA). Analysis was performed using software version 1.31. A typical scan lasted approximately 10 minutes, and the subject received 0.02 mrem of radiation. Euglycenic-Hyperinsulinemnic Insulin-Clamp 10 Euglycemic-hyperinsulinemic clamp test was performed based on the protocol of De Fronzo, et aL. Prior to insulin clamp study, all subjects consumed a diet containing at least 200 g. of carbohydrate per day for 3 days. The studies were performed at 8 a.m. after a 12-h overnight fast. A polyethylene catheter was inserted into an antecubital 15 vein for infusion of insulin and 20% dextrose. A second catheter was inserted into a hand vein and the hand was heated during the study to ensure arterialization of venous blood. All blood samples for glucose and hormone analyses were drawn from the heated hand catheter. Insulin-mediated glucose disposal was determined by the euglycemic insulin clamp technique. Human insulin was administered as a continuous 20 infusion (40 mU/m min) for 180 min. Plasma glucose concentration was measured every 5 min. Infusion of 20% dextrose was not begun until 5 min. after the initiation of insulin infusion and was periodically adjusted to maintain the arterialized plasma glucose concentrations at 95 mg/dl. Blood samples for determination of serum insulin concentrations were collected before insulin infusion was started and after 120 and 180 25 min. It has been previously demonstrated that hepatic glucose production is almost completely suppressed in the hyperinsulinemic state. Under these conditions, peripheral glucose utilisation is equal to the rate of glucose infusion to maintain 30 euglycemia. In this study, the final 60 min. of the infusion period (between 120 and 180 min.) was used for determination of peripheral glucose utilisation. Data are expressed in milligrams per kilogram fat free mass per minute. Assays 35 Serum GH levels were determined by a chemiluminescence immunometric assays (Nichols Institute Diagnostics GmbH , Bad Nauheim, Germany). The assay was calibrated against the WHO 1" international standard (80/505) for human GH. Intra- WO 03/090784 PCT/EP03/04357 12 and interassay coefficients of variation (Cvs) for a low point of the standard curve were 5.4% and 7.9%, respectively. Plasma IGF-I concentrations were measured by an immunoradiometric assay (Nichols Institute Diagnostics GmbH, Bad Nauheim, Germany). The assay was calibrated against the WHO 1 st International Reference 5 Reagent 87/518. Intra- and interassay Cvs for low IGF-I concentrations were 2.4% and 5.2%, respectively. In our laboratory the normal IGF-I ranges were 114-492 pg/L for adults aged 25-39 yr, 90-360 ptg/L for adults aged 40-54 yr, and 71-290 pig/L for adults aged 55 yr. 10 Plasma glucose was determined by the glucose oxidase method (glucose autoanalyzer, EBIO 6666, Eppendorf, Germany). Plasma insulin levels were assessed by radioinmunoassay (Biochem Immunosystems, Freiburg, Germany). HbAlc was determined by DCA 2000 (Bayer, Leverkusen, Germany, upper limit 6.3%). Total testosterone was measured by an automated chemiluminescence immunoassay (ACS 15 180, Bayer diagnostics, Fernwald, Germany). Red blood cell counts, white blood cell counts, serum levels of sodium, potassium, creatinine, liver enzymes, total cholesterol, HDL-cholesterol, LDL-cholesterol, triglycerides, lipoprotein (a) (Lp(a)), apolipoprotein Al, apolipoprotein B, fibrinogen, 20 and prostate specific antigen were determined by routine methods. Statistics The data, if not marked otherwise, represent the mean±standard deviation (SD). 25 Comparisons between baseline values of the two groups were performed using the unpaired Wilcoxon/Mann/Whitney test (WMW). Absolute differences between time points were analyzed per group using the paired Wilcoxon signed rank test (WSR). The areas under the curve of glucose and insulin were determined per times. For those AUC measures as for BMI and total body fat time courses were compared by repeated 30 measure analysis of variance (RM ANOVA). Occurrence of side effects were compared by chi-square test (CHI). All p-values were given unadjusted and therefore interpreted explorative. All tests were conducted at a two-sided significance level at 5%. Univariate analyses were performed with Graph.PadPrism (version 3.00 for Windows, GraphPad Software, San Diego, CA, USA) and RM ANOVA was done in 35 SAS V6.12.

WO 03/090784 PCT/EP03/04357 13 For analysis of the time courses of parameters by RM ANOVA, data from all 25 patients were included. For analyzing the differences between time points, only data from the 14 patients who have completed the study were included. 5 WO 03/090784 PCT/EP03/04357 14 Schedule of Assessment Screening Begin 6 weeks 3 months 6 months 12 months 18 months Visit 1 Visit 2 Visit 3 Visit 4 Visit 5 Visit 6 Visit 7 Informed consent X Physical XXX examination X X X X X X X Medical history X X X X X X X Medication X X X X X X X Adverse events X X X X X Educational programme X Heart rate X X X X X X X Blood pressure X X X X X X X Body mass index X X X X X X X Waist to hip ratio X X X X X X X Haematology X X X X X X X Clinical chemistry ECG X X X X X X X Oral glucose tolerance test X X X X X X Fasting blood glucose X HbAlc X X X X X X Lipid metabolism X X X X X X Lipoproteins X X X X X X IGF-1, IGFBP-3, GHBP X X X X X X GHRH/arginine test X 24-H GH profile X X Insulin clamp test X X Body fat mass (DEXA) X X X X Asservation of whole blood X samples (10ml) Asservation of serum samples X X X X X X (10ml) Study medication X X X X X Return of empty vials X X X X X 5 WO 03/090784 PCT/EP03/04357 15 Results Patients' assignment to the two treatment groups was stratified according to BMI and HbAlc. At baseline, the two groups did not differ significantly in terms of all other 5 characteristics except the systolic blood pressure (Met+GH: 136±10 mmllg vs. Met+Placebo: 126±17 mmGh; WMW p=0.033) (Tables 1,2). Two patients in the Met+GH group and three patients in the Met+Placebo group received treatment for hypertension (Met+GH group: 12.5 mg/d hydrochlothiazide 10 (12.5 mg/die) and 60 mg/d metoprolol, Met+Placebo group: amlodipine 10 gm/d; quinalapril 10 mg plus hydrochlorthiazide 12.5 mg/d and benazepril 10 mg/d). These medications were kept stable during the study period. Side effects 15 Side effects were observed in 7/12 patients of the Met+GH group and in 8/13 patients of the Met+placebo group (CHI p=0.81). The side effects reported in the Met+GH group were arthralgia (5 patients) and peripheral edema (2 patients). These side effects appeared during the first 6 weeks of treatment and subsided spontaneously in 3 patients. 20 in 3 other patients side effects subsided in response to dose reduction. In the Met+Placebo group, side effects reported were headache (2 patients), transient parasthesia (2 patients), diarrhoea (2 patients), muscle stiffness (1 patient) and arthralgia (1 patient). 25 Dropout from the study occurred in 5 patients from the Met+GH group (4 due to non-compliance, 1 due to the requirement for additional antidiabetic drugs). In the Met+Placebo group, dropout occurred in 6 patients (5 due to non-compliance and 1 patient due to a newly diagnosed plasmocytoma). 30 Overall, 14 patients (7 from the Met+GH group, 7 from the Met+Placebo group) completed the study. Results are as shown in Tables 1-21 35 Those include data of the 14 patients who completed the study Visits WO 03/090784 PCT/EP03/04357 16 VI Screening V2 Baseline V3 6 weeks V4 3 months 5 V5 6 months V6 12 months V7 18 months 10 Treatment Patient # 1-7 Treatment with metformin (1700 mg/day) + placebo 15 Patient # 8-14 Treatment with metformin (1700 mg/day) + growth hormone (daily dose 9.5 [ig/kg BW) 20 ARG-GHRH test and IGF-I levels At baseline, mean GH peaks in the ART-GHRH test were reduced in both groups (Met+GH: 6.8±3.7 ng/ml vs. Met+placebo: 3.8±2.9 ng/ml; differences between the two groups: WMW p=0.15). Overall, 22/25 patients (88%) showed a maximum GH 25 response <9 pg/l. The mean IGF-I levels at baseline were in the lower age-adjusted normal range in both groups (Met+GH: 173±58 ng/ml vs. Met+placebo: 144±40 ng/ml; differences between the two groups: WMW p=0.31). In the Met+GH group, IGF-I increased from 30 173±58pig/l to 434±61 Lg/l (WSR p<0.001) after 3 months and remained stable thereafter (differences between the two groups, RM ANOVA p<0.001) (Fig. 1) Body weight and body composition (Tables 1 and 2) 35 Body weight (Met+GH: -2.2±5.7 kg vs. Met+Placebo: -3.8±4.2 kg; differences between the two groups: WMW p=0.

54 ) and BMI (Met+GH: -1.16±1.61 kg/m 2 vs. Met+Placebo: -1.21±1.37 kg/m 2 ; differences between the two groups: M ANOVA WO 03/090784 PCT/EP03/04357 17 p=0.24) slightly decreased in both groups without reaching statistical significance. Mean total body fat significantly decreased by 4.6±5.8 kg (range: 2.2-14.5 kg) in the Met+GH group (WSR p=0.04) and by 3.8±2.1 kg (range: 0.4-6.7 kg) in the Met+Placebo group (WSR p=0.07). The difference between the two groups, however, 5 was not significant (RM ANOVA p=0.

9 1) (Fig 2). Fat free mass did not significantly change in both groups (Met+GH group: 0.5±3.8 kg; WSR p=0.

99 ; Met+Placebo: -1.7±2.7 kg; WSR p=0.69; differences between the two groups: WMW p=0.26). However, waist circumference significantly decreased during the study period only in 10 the Met+GH group (Met+GH: 120±9 cm vs. 114±10 cm; Met+Placebo: 112±9 cm vs. 110±9 cm; differences between the two groups: WMW p=0.048). This is an important result. Glucose metabolism 15 According to the inclusion criteria, baseline FPG levels were slightly elevated in both groups (Met+GH: range 6.2-8.0 mmol/l; Met+Placebo : range 6.2-7.6 nnnol/l; differences between the two groups: WMW p=0.32). During the first 6 months of treatment, FPG and AUC of glucose levels during the oGTT significantly increased in 20 the Met+GH group (FPG: baseline 5.9±0.7, 6 months 6.7±0.4 mmol/l/ WSR p-0.005; AUC glucose: baseline 1005±257 mmol/le 120 min, 6 months 1186 ±203 mmol/l.120 min, WSR p=0.005) (Fig 3). These increases in FPG and AUC of glucose levels at 6 months returned to baseline levels during further treatment (after 18 months: FPG 5.8 ±0.2 mmol/l; AUC glucose 1028 ±202 mmol/1120 min). In the Met+Placebo group, 25 FPG remained stable until 12 months and decreased thereafter (baseline: 6.2 0.3, 18 months: 5.5 ±0.6 mmol/l, WSR p=0.02). No significant differences were seen between both groups with respect to the overall changes of FPG (WSR p=0.09), the AUC of glucose (RM ANOVA p=0.5 2 ), the AUC of insulin (RM ANOVA p- 0

.

5 1), and HbAlc (Met+GH: 5.6 ±0.4% at baseline to 5.6 ±0.3% after 18 months, WSR p=1.0; 30 Met+Placebo: 6.0 ±0.7 to 5.6 ±0.4%, WSR p=0.22; differences between the two groups: WMW p-0.

2 0). At baseline, GDR did not differ significantly between the Met+GH and the Met+Placebo group (Met+GH: 3.9 ±1.8 ing/kgemin; Met+Placebo: 5.7 ±2.1 35 mg/kgemin; differences between the two groups: WMW p=0.12). After 18 months of treatment, mean GDR increased in the Met+GH group (4.6 ±2.4 mg/kgemin) and slightly decreased in the Met+Placebo group (4.8 ±1.4 mg/kgemin). The difference WO 03/090784 PCT/EP03/04357 18 between the two groups, however, did not reach statistical significance (WMW p=0.07) (Fig.6.) Lipid metabolism and fibrinogen 5 At baseline, levels of triglycerides, total cholesterol, Lp (a), apolipoprotein Al and B were not statistically different between the two groups (Table 2). In both groups, triglycerides, total cholesterol, LDL-cholesterol and fibrinogen levels did not change significantly during the 18 months period. HDL-cholesterol significantly increased in 10 both groups (Table 6). In the Met+Placebo group the apolipoprotein Al and B increased significantly, whereas these parameters were unaffected in response to the additional administration of rhGH. A statistically significant increase in Lp (a) was observed in both groups without a significant difference between the two groups. 15 Blood Pressure A slight decrease of systolic and diastolic blood pressure was seen in both treatment groups without statistical difference between the groups (Met+GH: 136 ±11/88 ±10 at baseline to 127 ±13 (WSR p=0.

2 5 ) / 81 ±4 (WSR p=0.

3 8 ) mmHg after 18 months; 20 Met+Placebo: 126 ±16 / 83 ±11 to 118 ±9 (WSR p=0.009)/75 ±8 (WSR p=0.11) mmlg). Total testosterone levels 25 Serum total testosterone levels significantly increased in both groups after 18 months (Met+GH: 8.57 ±1.7 to 10.9 ±3.6 nmol/l, WSR p=0.03; Met+Placebo: 8.9 ±1.6 to 14.2 ±3.5 nmol/l, WSR p=0.02). Despite a higher increase in the Met-Placebo group, the difference between the two groups did not reach statistical significance (WMW p=0.06). 30 WO 03/090784 PCT/EP03/04357 19 Table 1 Body Mass Index (kg/n 2 ) V2 V3 V4 V5V6 V7 1 31,6 30,6 30,4 31,1 31,2 30,5 2 34,1 31,8 33,0 35,1 35,0 34,7 3 31,2 30,1 29,9 28,8 28,9 28,4 4 33,7 32,2 31,6 31,7 33,8 33,3 5 30,5 29,4 29,5 28,7 27,0 27,8 6 34,6 33,8 34,3 32,8 32,8 32,4 7 29,4 29,4 29,3 29,7 29,2 29,5 Min 29,4 29,4 29,3 28,7 27,0 27,8 Max 34,6 33,8 34,3 35,1 35,0 34,7 mean 32,2 31,0 31,1 31,1 31,1 30,9 SD 2,0 1,6 1,9 2,3 2,9 2,6 8 32,7 31,8 31,0 30,3 29,6 30,6 9 41,2 41,2 39,0 38,2 39,7 38,3 10 33,0 32,8 32,6 32,4 31,8 32,1 11 30,7 29,6 29,4 28,9 30,0 30,2 12 33,0 32,8 31,5 30,9 32,2 30,3 13 35,5 35,7 35,6 33,9 35,2 34,7 14 29,6 30,7 31,0 31,4 30,3 31,4 Min 29,6 29,6 29,4 28,9 29,6 30,2 Max 41,2 41,2 39,0 38,2 39,7 38,3 mean 33,7 33,5 32,9 32,3 32,7 32,5 5 D 3,8 3,9 3,3 3,0 3,6 3,0 WO 03/090784 PCT/EP03/04357 20 Table 2 Waist Circumference (cm) 5 # V2 \ V4 V5 V6 V7 1 109,5 109,0 109,5 116,0 112,0 110,0 2 120,0 109,5 107,0 114,5 116,0 116,0 3 106,0 107,0 107,0 104,0 n.d. n.d. 4 119,5 113,0 113,5 113,0 117,0 116,0 5 105,5 103,5 103,0 104,0 94,0 97,0 6 121,0 119,0 118,5 116,5 115,0 117,0 7 100,0 101,0 100,5 104,0 97,0 101,0 Min 100,0 101,0 100,5 104,0 94,0 97,0 Max 121,0 119,0 118,5 116,5 117,0 117,0 mean 111,6 108,9 108,4 110,3 108,5 109,5 SD 8,5 6,0 6,1 6,0 10,3 8,6 8 118,0 109,5 103,5 96,0 104,0 107,0 9 140,0 133,5 126,5 126,5 133,0 128,0 10 117,0 117,0 113,0 113,0 113,0 108,0 11 115,0 104,0 102,0 102,0 97,0 n.d. 12 119,0 112,0 114,0 109,5 112,5 n.d. 13 117,5 121,5 116,0 120,5 127,0 nd. 14 111,0 115,0 114,0 117,0 111,0 111,0 Min 111,0 104,0 102,0 96,0 97,0 107,0 Max 140,0 133,5 126,5 126,5 133,0 128,0 mean 119,6 116,1 112,7 112,1 113,9 113,5 D -9,4 9,5 8,2 10,6 12,5 9,8 10 WO 03/090784 PCT/EP03/04357 21 Table 3 HbAlc (%) 5 # V2 V5 V6 V7 1 6,2 6,1 5,7 5,9 2 7,4 5,3 5,7 5,7 3 5,5 5,2 5,5 5,4 4 5,7 5,8 5,0 5,0 5 5,4 5,7 6,0 6,0 6 5,9 5,4 5,0 5,4 7 5,6 5,1 5,3 5,6 Min 5,4 5,1 5,0 5,0 Max 7,4 6,1 6,0 6,0 mean 6,0 5,5 5,5 5,6 SD 0,7 0,4 0,4 0,3 8 5,4 5,2 5,6 5,4 9 5,3 6,1 5,7 4,9 10 5,4 6,1 5,6 6,3 11 6,3 5,9 5,1 5,4 12 5,4 5,4 5,7 5,9 13 5,4 5,6 5,6 5,6 14 5,8 5,1 5,6 5,4 Min 5,3 5,1 5,1 4,9 Max 6,3 6,1 5,7 6,3 mean 5,6 5,6 5,6 5,6 D 0,4 0,4 0,2 0,4 10 WO 03/090784 PCT/EP03/04357 22 Table 4 Total Cholesterol (mmol/1) 5 V2 V7 1 6,34 6,26 2 4,32 4,91 3 5,56 5,64 4 5,64 5,51 5 3,90 5,53 6 4,55 5,09 7 4,65 5,87 Min 3,90 4,91 Max 6,34 6,26 mean 4,99 5,55 SD 0,86 0,45 8 6,88 5,77 9 6,21 5,82 10 5,28 5,20 11 4,24 4,89 12 5,72 4,19 13 4,29 4,47 14 4,99 n.d. Min 4,24 4,19 Max 6,88 5,82 mean 5,37 5,06 D 0,97 0,67 Storage samples from V3-V6 are available for analysis of lipid parameters 10 WO 03/090784 PCT/EP03/04357 23 Table 5 LDL Cholesterol (mmol/l) 5 0 V2 V7 1 4,53 3,36 2 2,84 2,61 3 3,65 3,62 4 4,09 3,59 5 3,90 3,52 6 3,03 2,95 7 3,08 3,80 Min 2,84 2,61 Max 4,53 3,80 mean 3,59 3,35 SD 0,63 0,42 8 3,75 3,70 9 4,65 3,47 10 3,83 3,41 11 2,84 2,97 12 n.d. 2,25 13 2,07 2,53 14 3,57 n.d. Min 2,07 2,25 Max 4,65 3,70 mean 3,45 3,06 ID 0,89 0,57 10 Storage samples from V3-V6 are available for analysis of lipid parameters WO 03/090784 PCT/EP03/04357 24 Table 6 HDL Cholesterol (mmol/l) 5 # V2 V7 1 1,08 1,42 2 0,88 1,32 3 1,03 1,53 4 0,96 1,50 5 0,96 1,01 6 0,95 1,29 7 0,89 1,45 Min 0,88 1,01 Max 1,08 1,53 mean 0,96 1,36 SD 0,07 0,18 8 0,78 1,01 9 1,01 1,06 10 1,08 1,42 11 0,91 1,32 12 0,88 1,16 13 0,80 0,93 14 0,80 n d. Min 0,78 0,93 Max 1,08 1,42 mean 0,89 1,15 SD 0,12 0,19 Storage samples from V3-V6 are available for analysis of lipid parameters 10 WO 03/090784 PCT/EP03/04357 25 Table 7 Triglycerides (mmol/l) 5 1 2,75 2,46 2 1,35 1,76 3 1,10 1,00 4 1,57 1,15 5 1,72 2,04 6 2,24 1,41 7 2,07 1,74 Min 1,10 1,00 Max 2,75 2,46 mean 1,83 1,65 SD 0,57 0,51 8 2,36 2,28 9 2,21 3,40 10 0,67 0,82 11 1,33 1,25 12 3,59 2,44 13 1,66 2,92 14 1,58 n.d. Min 0,67 0,82 Max 3,59 3,40 mean 1,91 2,19 SD 0,93 0,98 Storage samples from V3-V6 are available for analysis of lipid parameters 10 WO 03/090784 PCT/EP03/04357 26 Table 8 Maximal GH-response in GHRH-arginine stimulation test (mmol/l) 5 # V2 V71) 1 2,75 2,46 2 1,35 1,76 3 1,10 1,00 4 1,57 1,15 5 1,72 2,04 6 2,24 1,41 7 2,07 1,74 Min 1,10 1,00 Max 2,75 2,46 mean 1,83 1,65 SD 0,57 0,51 8 2,36 2,28 9 2,21 3,40 10 0,67 0,82 11 1,33 1,25 12 3,59 2,44 13 1,66 2,92 14 1,58 n.d. Min 0,67 0,82 Max 3,59 3,40 mean 1,91 2,19 SD 0,93 0,98 V7b are data collected 4 weeks after withdrawal of GH / placebo 10 WO 03/090784 PCT/EP03/04357 27 Table 9 IGF-I (ptg/1) 5 U - 3 V4T V5 V6N7 -V'~ 7 1 190 136 127 176 151 162 182 2 97 120 111 134 121 127 162 3 185 153 173 202 202 223 212 4 109 105 102 135 113 113 127 5 159 178 234 244 202 208 212 6 105 90 101 99 124 93 94 7 164 262 210 216 199 192 222 Min 97 90 101 99 113 93 94 Max 190 262 234 244 202 223 222 mean 144 149 151 172 159 160 173 SD 40 58 55 52 41 50 48 8 175 485 489 478 500 508 146 9 71 305 377 458 306 folgt folgt 10 157 349 273 368 416 390 148 11 142 352 352 335 239 190 166 12 210 358 420 431 489 376 155 13 202 372 484 466 419 525 folgt 14 251 433 490 501 430 563 244 Min 71 305 273 335 239 190 146 Max 251 485 490 501 500 563 244 mean 173 379 412 434 400 425 172 D 58 60 83 51 95 138 41 10 V7b are data collected 4 weeks after withdrawal of GH / placebo WO 03/090784 PCT/EP03/04357 28 Table 10 Glucose tolerance test: Glucose 0 min (mmol/l) 5 # V3 ~ V4 5V 1 6,33 5,94 7,33 6,22 6,27 5,61 2 6,27 5,38 5,33 5,83 6,00 4,61 3 6,05 7,05 6,33 6,16 5,83 5,61 4 6,49 5,50 5,88 6,22 6,44 5,88 5 5,77 5,83 6,27 5,38 5,50 4,94 6 6,49 6,38 7,55 6,77 6,22 5,66 7 6,05 6,05 6,77 6,83 6,99 6,33 Min 5,77 5,38 5,33 5,38 5,50 4,61 Max 6,49 7,05 7,55 6,83 6,99 6,33 mean 6,21 6,02 6,49 6,20 6,18 5,52 SD 0,27 0,57 0,78 0,50 0,48 0,58 8 5,94 6,11 6,27 6,99 n.d. 6,00 9 6,77 n.d. 6,33 7,05 6,77 6,11 10 5,44 6,00 6,16 6,44 5,77 5,94 11 6,72 6,77 6,66 7,16 6,38 5,77 12 5,77 6,00 6,44 6,33 6,27 5,77 13 5,88 5,83 5,77 6,27 6,44 5,50 14 4,94 n.d. 6,11 6,49 5,55 5,83 Min 4,94 5,83 5,77 6,27 5,55 5,50 Max 6,77 6,77 6,66 7,16 6,77 6,11 mean 5,92 6,14 6,25 6,68 6,20 5,84 SD 0,66 0,37 0,28 0,38 0,45 0,20 10 WO 03/090784 PCT/EP03/04357 29 Table 11 Glucose tolerance test: Glucose 60 min (mmol/l) 5 # V2 V3 V4 V6 V7 1 11,99 n.d. 10,10 16,71 12,93 14,93 2 8,33 10,05 n.d. 11,99 12,05 10,55 3 9,66 9,71 11,32 10,99 8,55 12,93 4 8,49 9,44 9,88 9,33 9,38 10,88 5 10,27 9,38 10,32 8,10 7,16 5,11 6 12,16 14,49 16,82 12,77 9,71 12,82 7 13,49 7,27 14,99 10,55 13,77 13,38 'Mi ,33 7,27 9,88 8,10 7,16 5,11 Max 13,49 14,49 16,82 16,71 13,77 14,93 mean 10,63 10,06 12,24 11,49 10,51 11,51 SD 1,97 2,38 2,94 2,78 2,44 3,20 8 9,49 10,21 9,38 9,05 n.d. 9,21 9 14,99 13,99 10,05 12,16 16,04 n.d. 10 9,38 9,49 11,21 10,88 11,82 12,27 11 15,71 12,49 13,88 16,38 9,77 15,10 12 12,32 17,37 16,54 14,49 12,16 7,38 13 9,33 9,27 9,88 10,27 8,49 9,05 14 6,61 12,38 11,49 11,38 8,72 12,60 Min 6,61 9,27 9,38 9,05 8,49 7,38 Max 15,71 17,37 16,54 16,38 16,04 15,10 mean 11,12 12,17 11,78 12,09 11,17 10,94 ID 3,34 2,89 2,58 2,54 2,84 2,86 I0 WO 03/090784 PCT/EP03/04357 30 Table 12 Glucose tolerance test: Glucose 120 min (mmol/l) 5 # V2 V3 V4 V5 V6 V7 1 7,27 10,27 7,16 12,10 8,10 12,49 2 13,21 8,60 n.d. 7,99 8,27 8,44 3 6,94 5,33 7,05 6,55 5,50 7,05 4 5,05 5,38 5,38 6,61 7,22 7,60 5 4,83 5,00 8,27 3,33 2,78 3,66 6 6,11 10,66 11,38 7,49 4,11 8,33 7 4,83 13,77 7,22 7,94 6,22 8,49 Min 4,83 5,00 5,38 3,33 2,78 3,66 Max 13,21 13,77 11,38 12,10 8,27 12,49 mean 6,89 8,43 7,74 7,43 6,03 8,01 SD 2,96 3,36 2,01 2,60 2,06 2,60 8 4,44 4,66 5,83 5,16 n.d. 4,05 9 8,94 13,21 11,38 6,16 8,44 8,10 10 5,44 5,88 7,60 10,05 4,94 8,66 11 9,10 6,44 9,94 10,27 7,16 4,88 12 5,22 9,77 6,00 11,55 3,83 3,77 13 10,82 10,88 9,71 10,32 8,66 9,77 14 4,50 7,99 9,33 7,27 7,94 8,44 Min 4,44 4,66 5,83 5,16 3,83 3,77 Max 10,82 13,21 11,38 11,55 8,66 9,77 mean 6,92 8,41 8,54 8,68 6,83 6,81 D 2,62 3,04 2,11 2,45 1,99 2,48 10 WO 03/090784 PCT/EP03/04357 31 Table 13 Glucose tolerance test: Glucose AUC (mmol/l/120 min) 5 V2V3V4 V5 V6 V7 1 856 1041 1552 1207 1439 2 934 1022 1134 1151 1024 3 1107 954 1081 1041 853 1156 4 1136 893 931 944 973 1057 5 881 888 1056 748 678 565 6 1371 1381 1577 1194 893 1189 7 889 1031 1319 1076 1222 1247 Mi 856 888 931 748 678 565 Max 1371 1381 1577 1552 1222 1439 mean 1025 1028 1167 1098 997 1097 SD 189 183 238 247 205 271 8 1417 936 926 908 854 9 1069 1134 1126 1419 10 1061 926 1086 1147 1031 1174 11 679 1146 1331 1505 993 1226 12 1069 1515 1366 1406 1032 729 13 1061 1057 1057 1114 963 1001 14 679 1152 1096 928 1184 Min 679 926 926 908 928 729 Max 1417 1515 1366 1505 1419 1226 mean 1005 1116 1150 1186 1061 1028 ISD 257 241 154 203 180 202 10 WO 03/090784 PCT/EP03/04357 32 Table 14 Glucose tolerance test: Insulin 0 min (pmol/l) 5 V2 V3 V4 V5 V6 V7 1 114,1 179,4 514,4 200,2 124,8 246,1 2 96,1 127,7 169,3 116,2 370,9 175,8 3 70,3 230,3 48,1 65,3 65,3 83,9 4 115,5 76,8 104,8 112,6 148,5 149,2 5 35,9 78,9 86,8 94,7 66,7 87,5 6 66,0 65,3 79,6 66,7 92,6 86,1 7 61,0 62,4 76,1 75,3 40,2 153,5 Min 35,9 62,4 48,1 65,3 40,2 83,9 Max 115,5 230,3 514,4 200,2 370,9 246,1 mean 79,8 117,3 154,2 104,4 129,9 140,3 SD 29,7 65,3 163,3 47,0 112,6 60,0 8 61,0 96,9 76,1 100,5 45,2 83,9 9 223,1 495,1 278,4 231,8 414,7 478,6 10 172,2 264,8 196,6 172,2 132,7 279,1 11 122,0 125,6 108,3 92,6 97,6 99,0 12 295,6 160,0 265,5 171,5 176,5 199,5 13 283,4 260,5 261,2 238,9 252,6 298,5 14 147,1 184,4 142,8 199,5 203,1 290,6 Min 61,0 96,9 76,1 92,6 45,2 83,9 Max 295,6 495,1 278,4 238,9 414,7 478,6 mean 186,3 226,7 189,8 172,4 188,9 247,0 D 85,9 134,1 82,2 58,1 120,8 135,4 10 WO 03/090784 PCT/EP03/04357 33 Table 15 Glucose tolerance test: Insulin 60 min (pmol/l) 5 V2 V3 V4 V5 - V6 VT 1 1021,7 1643,1 1404,9 1626,6 1580,7 1987,5 2 965,8 1239,8 3055,8 2011,2 1054,0 975,8 3 880,4 851,0 1323,1 1435,0 1435,0 1553,4 4 860,3 999,5 910,5 723,2 533,1 1169,5 5 1808,1 922,0 749,1 840,2 1240,6 1043,2 6 695,3 507,3 584,8 355,9 437,7 560,4 7 952,1 462,8 1238,4 481,4 827,3 932,0 Mi 695,3 462,8 584,8 355,9 437,7 560,4 Max 1808,1 1643,1 3055,8 2011,2 1580,7 1987,5 mean 1026,2 946,5 1323,8 1067,6 1015,5 1174,5 SD 360,3 410,4 822,7 626,9 437,7 465,0 8 683,8 1063,3 839,5 1185,3 639,3 901,2 9 2903,7 2874,3 1466,6 2128,1 2227,1 2226,4 10 2196,3 1879,9 1705,5 819,4 2340,5 2062,8 11 1045,4 645,8 602,0 548,9 615,6 1183,2 12 1884,9 2247,9 1624,4 1408,5 1719,8 1153,7 13 2884,4 2382,1 1908,6 1443,6 1262,8 1664,6 14 1991,8 1533,3 1483,1 1435,0 1788,7 2337,6 Min 683,8 645,8 602,0 548,9 615,6 901,2 Max 2903,7 2874,3 1908,6 2128,1 2340,5 2337,6 mean 1941,5 1803,8 1375,7 1281,3 1513,4 1647,1 ID 843,6 779,5 476,2 507,0 700,6 577,4 10 WO 03/090784 PCT/EP03/04357 34 Table 16 Glucose tolerance test: Insulin 120 min (pmol/1) 5 V2 V3 V4 V5 V6 V7 1 791,4 1435,0 726,8 2031,2 1380,5 1881,3 2 1018,1 797,1 1972,4 855,3 863,2 770,6 3 602,7 134,9 277,0 352,3 351,6 657,2 4 284,8 491,5 431,9 647,2 645,8 697,4 5 265,5 413,3 1152,3 122,0 88,3 236,8 6 264,8 495,8 406,8 352,3 94,0 348,0 7 301,4 1139,4 434,8 551,0 191,6 856,0 Min 264,8 134,9 277,0 122,0 88,3 236,8 Max 1018,1 1435,0 1972,4 2031,2 1380,5 1881,3 mean 504,1 701,0 771,7 701,6 516,4 778,2 SD 305,5 452,7 604,9 632,1 479,2 535,8 8 115,5 190,1 336,5 210,2 378, 218,8 9 4734,8 3135,5 3935,5 1188,9 2674,8 1927,2 10 653,6 795,0 1912,9 1686,8 524,5 1826,8 11 731,1 222,4 636,4 375,3 422,6 299,2 12 347,3 852,4 446,3 2019,0 170,0 1772,2 13 3497,1 5237,8 2225,7 1949,4 2446,7 3511,4 14 1693,3 3312,7 3842,9 1435,0 2888,7 2392,1 Min 115,5 190,1 336,5 210,2 170,0 218,8 Max 4734,8 5237,8 3935,5 2019,0 2888,7 3511,4 mean 1681,8 1963,7 1905,2 1266,4 1358,0 1706,8 SD 1770,7 1944,7 1537,2 725,0 1238,4 1153,9 10 WO 03/090784 PCT/EP03/04357 35 Table 17 Glucose tolerance test: Insulin AUC (pmol/1/120 min) 5 V2 V3 V4 V5 V6 V7 1 88468 147016 121530 164537 139999 183070 2 91374 102136 247602 149814 100263 86939 3 73013 62014 89135 98628 98606 115439 4 63628 77016 70731 66189 55814 95571 5 117527 70085 82118 56912 79083 72324 6 51638 47269 49680 33923 31857 46645 7 67997 63822 89630 47678 56589 86208 Min 51638 47269 49680 33923 31857 46645 Max 117527 147016 247602 164537 139999 183070 mean 79092 81337 107204 88240 80316 98028 SD 21867 33497 65601 51290 36073 43021 8 46322 72410 62745 80439 51079 63154 9 322961 281375 214411 170306 226314 205757 10 156551 144583 165613 104934 160146 186945 11 88317 49185 58462 46968 52543 82936 12 132379 165247 118818 150223 113587 128375 13 286476 307872 189119 152268 156745 214174 14 174718 196911 208556 135134 200075 220739 Min 46322 49185 58462 46968 51079 63154 Max 322961 307872 214411 170306 226314 220739 mean 172532 173940 145389 120039 137213 157440 D 100421 97312 66040 44422 68245 65468 10 WO 03/090784 PCT/EP03/04357 36 Table 18 C-peptide (ng/ml) 5 # V2 V3 V4 V5 V6 V7 1 3,6 4,7 9,0 4,6 3,7 4,2 2 5,0 3,4 3,3 3,6 5,7 3,1 3 2,2 7,0 1,8 2,3 2,3 2,6 4 3,5 2,6 3,1 3,1 3,8 3,5 5 3,3 3,0 2,8 3,8 2,7 2,5 6 2,9 2,5 2,9 2,6 2,3 2,3 7 2,4 2,0 2,4 2,1 1,7 4,9 Min 2,2 2,0 1,8 2,1 1,7 2,3 Max 5,0 7,0 9,0 4,6 5,7 4,9 mean 3,3 3,6 3,6 3,2 3,2 3,3 SD 0,9 1,7 2,4 0,9 1,4 1,0 8 1,9 2,4 2,5 2,7 1,9 2,6 9 4,6 7,0 4,5 4,6 5,3 8,5 10 3,6 5,0 4,1 4,3 3,3 4,8 11 3,1 3,0 3,3 2,8 2,9 2,7 12 5,4 7,4 6,5 7,4 6,0 7,4 13 4,5 5,9 5,4 6,1 6,5 7,5 14 5,3 8,5 6,3 7,3 5,9 6,7 Min 1,9 2,4 2,5 2,7 1,9 2,6 Max 5,4 8,5 6,5 7,4 6,5 8,5 mean 4,1 5,6 4,7 5,0 4,5 5,7 SD 1,3 2,3 1,5 2,0 1,8 2,4 10 WO 03/090784 PCT/EP03/04357 37 Table 19 Glucose disposal (mg/kg/min) (euglycemic hyperinsulinemic clamp) 5 V2 V7 1 3,24 2,70 2 4,31 3,27 3 5,71 6,20 4 4,91 5,84 5 7,27 5,81 6 4,58 5,37 7 9,64 4,61 Min 3,24 2,70 Max 9,64 6,20 mean 5,67 4,83 SD 2,15 1,36 8 5,78 7,56 9 1,67 1,96 10 3,14 6,38 11 5,03 6,14 12 4,74 5,55 13 1,36 1,95 14 5,35 2,47 Min 1,36 1,95 Max 5,78 7,56 mean 3,87 4,57 SD 1,81 2,37 10 WO 03/090784 PCT/EP03/04357 38 Table 20 Truncal fat (DEXA) (kg) 5 # V2 V5 V7 1 17,86 16,86 16,32 14,59 2 16,42 13,63 n.d. 13,55 3 13,11 12,72 12,72 n.d. 4 16,49 15,90 15,88 16,84 5 15,43 13,28 17,61 11,28 6 17,25 18,44 17,52 16,85 7 11,29 10,53 10,50 11,79 Min 11,29 10,53 10,50 11,28 Max 17,86 18,44 17,61 16,85 mean 15,40 14,48 15,09 14,15 SD 2,37 2,71 2,87 2,40 8 13,28 10,63 8,59 12,10 9 25,42 17,56 15,01 16,01 10 17,37 16,13 16,48 16,44 11 16,91 13,56 21,64 12,94 12 15,12 12,18 13,49 12,31 13 16,82 16,99 16,89 16,66 14 15,55 16,25 15,21 16,24 Min 13,28 10,63 8,59 12,10 Max 25,42 17,56 21,64 16,66 mean 17,21 14,76 15,33 14,67 SD 3,88 2,65 3,93 2,10 10 WO 03/090784 PCT/EP03/04357 39 Table 21 Total fat (DEXA) (kg) 5 # V2 V5 V6 V7 1 34,65 33,45 33,96 29,16 2 28,99 n.d. n.d. 25,06 3 24,02 22,34 n.d. n.d. 4 41,53 36,73 38,57 41,09 5 26,51 24,49 17,61 19,84 6 40,94 39,70 30,18 37,65 7 21,17 39,70 20,82 18,09 Min 21,17 22,34 17,61 18,09 Max 41,53 39,70 38,57 41,09 mean 31,11 31,34 28,23 28,48 SD 8,09 7,60 8,82 9,36 8 25,66 19,89 16,77 22,88 9 50,09 42,49 35,58 36,60 10 32,91 31,04 32,57 30,58 11 33,08 26,49 n.d. 26,37 12 32,17 25,16 28,18 23,40 13 34,18 35,29 34,92 34,67 14 30,31 33,48 31,45 32,72 Min 25,66 19,89 16,77 22,88 Max 50,09 42,49 35,58 36,60 mean 34,06 30,55 29,91 29,60 D 7,61 7,45 6,96 5,47 10 15 WO 03/090784 PCT/EP03/04357 40 Discussion This randomised, double-blind, placebo-controlled trial has provided first evidence, that rhGH in combination with an insulin sensitising agent such as metformin might be a 5 promising therapeutic approach in high risk patients with metabolic syndrome and that treatment with an insulin-sensitising agent such as metformin might be effective in reducing the insulin antagonistic effects of rhGH. Since the insulin antagonist action of GH has to be regarded as the principal factor for GH induced insulin resistance and since rises in FPG levels during rhGH treatment would primarily be expected in 10 patients with pre-existing insulin resistance, such an effect of metformin is of special interest when treating patients with metabolic syndrome who are characterized by an insulin resistant state and a high risk of developing type 2 diabetes. The study included highly selected male patients presenting with obesity and impaired 15 glucose metabolism at baseline. Consistent with earlier findings of disturbances in the neuroendocrine regulation of GH secretion in obesity, the majority of the patients showed a reduced GH peak in the ARG-GHRH test at baseline. Despite the fact that only patients with impaired FPG levels were included in this study, 20 no sustaining negative effects or rhGH on glucose metabolism were seen and the overall course of FPG and insulin levels did not significantly differ between those patients receiving only metformin and those receiving metformin and rhGH. Consistent to previous studies, we have seen a slight but significant increase in FPG levels and glucose levels during oGTT after 6 months of treatment in the group receiving 25 metformin and rhGH. One patient with a baseline FPG level of 9.0 mmol/l dropped out of the study during this period due to the need for additional antidiabetic drug treatment. The overall increase in FPG and AUC glucose levels, however, was transient with both 30 parameters returning to baseline levels during further rhGH and metformin treatment. Interestingly, after 18 months of treatment, GDR as a measure of insulin sensitivity increased in those patients receiving metformin and rhGH and slightly decreased in the metformin group. This effect, however, did not reach statistical significance due to the small sample size included. Similar effects of rhGH on insulin sensitivity were seen in 35 the study of Johannsson et al., who investigated obese male patients, who, in contrast to the present study, had normal FPG levels and did not receive metformin. The long-term course of FPG levels seen in our study, together with the GDR results after 18 months WO 03/090784 PCT/EP03/04357 41 underlines the need for long-tenn studies when investigating the effects of rhGH on parameters of insulin resistance. The study also showed a significant decrease of total body fat in both treatment groups 5 without a significant difference between the groups. These data are in good accordance with previous studies showing that the administration of rhGH can reduce total body fat and visceral fat in obese patients due to the lipolytic effect of GH. Albeit visceral fat was not directly measured in the present study, waist circumference as a parameter of visceral fat accumulation decreased in those patients receiving a combination of 10 metformin and rhGH, but not in those patients treated only with metformin. This strongly suggests a rhGH specific effect on visceral fat and is of major importance since most of the clinical features of the metabolic syndrome are closely related to the accumulation of visceral fat, which is by itself one of the major determinants of insulin resistance in this syndrome. In consequence, the slight increase in GDR seen in patients 15 treated with metformin and rhGH is most probably due to changes of body composition with a preferential reduction in visceral adipose depots associated with the reduction in visceral adipose depots associated with the reduction of waist circumference. In addition, GDR might have been influenced by the slight increase of skeletal muscle mass seen in the rhGH treated group. 20 Despite the additional effects of rhGH, patients treated with metfonnin alone also experienced positive effects regarding the course of body composition, which is well known from previous studies. In the metformin treated group, FPG levels significantly decreased consistent to previous studies demonstrating a significant improvement of 25 glucose metabolism. The slight decrease of GDR after 18 months may be due to the natural course of disease and due to the fact that metformin has no direct effect on visceral fat mass and muscle mass. Dyslipoproteinemia is one of the major characteristics of the metabolic syndrome and a 30 central finding in patients with growth hormone deficiency. Previous investigations on the importance of GH as a regulating factor for serum lipids have produced conflicting results in terms of the effects on serum cholesterol, LDL-cholesterol, HDL-cholesterol, and apoliprotein B. We have seen no significant 35 changes of total cholesterol, triglycerides and LDL-cholesterol, neither in the metformin-treated group nor in response to the additional administration of rhGH. This may be due to the small sample size as well as the fact that patients with severe hyperlipidemia requiring lipid lowering drug treatment were excluded from the study.

WO 03/090784 PCT/EP03/04357 42 The increase of HDL-cholesterol in response to metformin alone is most probably due to the reduction of total body fat and not due to a direct effect of metformin. In the group treated with metformin and rhGH, the increase of HDL-cholesterol may be influenced by the additional administration of rhGH. As shown for the changes of 5 serum lipids, the increase of lipoproteins were also similar in both groups. This observation might indicate an rhGH independent effect, even though rhGH may increase Lp(a). The increase of lipoproteins in the group treated with metformin alone remains speculative, even though some previous trials could not demonstrate an effect of metformin on lipoprotein levels. 10 Low total testosterone levels were observed in both groups and are most probably the consequence of increased body weight, accumulation of visceral fat, and patients' age. The increase of total testosterone levels in both groups may be related to the effects of a reduced total body fat on testosterone and SHBG levels and most probably are due to 15 direct effects of metformin or rhGH. One shortcoming of the present study is the high number of dropouts. The dropout rate was similar in the two treatment groups and most of the dropouts were due to non-compliance. Since most of the dropouts due to non-compliance occurred after the 20 first 12 months of treatment, the high dropout rate is partly due to the study design including double blind se injections over a period of 18 months. The dose of rhGH used in his study was higher than the dose currently used in adult patients with GH deficiency. This resulted in supraphysiological IFG-I levels in the 25 rhGH treated patients and also is the obvious cause for the high rate of GH related side effects during the early phase of treatment. In consequence, lower doses of rhGH titrated according to individual IGF-I levels are suggested for future studies. In summary, the present study demonstrates that in patients with metabolic syndrome 30 and impaired FPG levels, and 18-month treatment with metformin in combination with rhGH was not associated with sustaining negative effects on glucose metabolism and was even more effective in reducing waist circumference than treatment with metformin alone. Thus, the results provide first evidence that rhGH in combination with an insulin sensitising agent, for example metformin, might be an effective therapeutic 35 approach in high risk patients with central obesity and that treatment with metformin might be effective in reducing the insulin antagonistic effects of rhGH. The transient increase in glucose levels despite the use of metformin as well as the case of one patient with an elevated FPG level at baseline who dropped out due to the need for further WO 03/090784 PCT/EP03/04357 43 antidiabetic drug treatment indicate, that such an approach would be most applicable in insulin resistant patients who show still normal or only slightly elevated FPG levels. Further studies including higher numbers of patients are necessary to investigate the effects of this therapeutic approach with regard to central findings in metabolic 5 syndrome and, most importantly, with regard to its effects on cardiovascular risk.

WO 03/090784 PCT/EP03/04357 44 REFERENCES 1. Johannsson G, Bengtsson BA 1999 Growth hormone and the metabolic 5 syndrome. J Endocrinol Invest 22:41-6. 2. De Fronzo RA, Gunnarsson R, Bjorkman 0, Olsson M, Wahren J 1985 Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type II) diabetes mellitus. J Clin Invest 76:149-55. 10 3. Reaven GM 1988 Banting lecture 1988. Role of insulin resistance in human disease. Diabetes 37:1595-607. 4. Rosen T, Eden S, Larson G, Wilhelmsen L, Bengtsson BA 1993 15 Cardiovascular risk Factors in adult patients with growth hormone deficiency. Acta Endocrinol (Copenh) 129:195-200. 5. Franco C, Bengtsson BA, Johannsson G 2001 Visceral obesity and the role of the somatotropic axis in the development of metabolic complications. Growth 20 Horm IGF Res 11 Suppl A:S97-102. 6. Bjorntorp P, Roamond R 2000 Neuroendocrine abnormalities in visceral obesity. Int J Obes Relat Metab Disord 24 Supp1 2:S80-5. 25 7. Bjorntorp P 1995 Neuroendocrine abnormalities in human obesity Metabolism 44:38-41. 8. Veldhuis JD, Liem AY, South S, Weltman A, Weltman J, Clemnions DA, Abbott R, Mulligan T, Johnson ML, Pincus S, et al 1995 Differential 30 impact of age, sex steroid hormones, and obesity on basal versus pulsatile growth hormone secretion in men as assessed in an ultrasensitive chemiluminescence assay. J Clin Endocrinol Metab 80:3209-22. 9. Veldhuis JD, Iranmanesh A, Weltman A 1997 Elements in the 35 pathophysiology of diminished growth hormone (GH) secretion in aging humans. Endocrine 7:41-8 WO 03/090784 PCT/EP03/04357 45 10. Marin P, Kvist H, Lindstedt G, Sjostrom L, Bjorntorp P 1993 Low concentrations of insulin-like growth factor-I in abdominal obesity. Int J Obes Relat Metab Disord 17:83-9. 5 11. Amato G, Mazziotti G, Di Somma C, Lalli E, De Felice G, Conte M, Rotondi M, Pietrosante M, Lombardi G, Bellastella A, Carella C, Colao A 2000 Recombinant growth hormone (GH) therapy in GH-deficient adults: a long-term controlled study on daily versus thrice weekly injections. J Clin Endocrinol Metab 85:3720-5. 10 12. Feldt-Rasmussen U, Abs R, Bengtsson BA, Bennmarker H, Bramnert M, Hernberg-Stahl E, Monson JP, Westberg B, Wilson P, Wuster C 2002 Growth hormone deficiency and replacement in hypopituitary patients previously treated for acromegaly or Cushing's disease. Eur J Endocrinol 15 146:67-74. 13. Fowelin J, Attvall S, von Schenck H, Smith U, Lager 11995 Characterization of the insulin-antagonistic effect of growth hormone in insulin-dependent diabetes mellitus. Diabet Med 12:990-6. 20 14. DeFronzo RA, Gunnarsson R, Bjorkman 0, Olsson M, Wahren J 1985 Effects of insulin on peripheral and splanchnic glucose metabolism in noninsulin-dependent (type II) diabetes mellitus. J Clin Invest 76:149-55. 25

Claims (19)

1. The use of growth hormone, or an analogue thereof, and an insulin sensitising agent in the preparation of a pharmaceutical composition for the treatment of metabolic 5 syndrome.

2. The use of growth hormone, or an analogue thereof, and an insulin sensitising agent in the preparation of a pharmaceutical composition to reduce waist circumference. 10

3. The use according to either of claims 1 or 2 in which the insulin sensitising agent reduces the insulin antagonist action of the growth hormone.

4. A method of treating metabolic syndrome comprising administering an insulin 15 sensitising agent and growth hormone or an analogue thereof to a patient suffering from metabolic syndrome.

5. A method according to claim 4 in which the insulin sensitising agent and growth hormone are administered separately. 20

6. A method according to claim 4 in which the insulin sensitising agent and growth hormone are administered simultaneously.

7. Use according to any of claims 1 to 3, or a method according to any of claims 4 25 to 6 in which the growth hormone is recombinant human growth hormone.

8. A method according to any of claims 4 to 7 which reduces waist circumference.

9. A method according to any of claims 4 to 8 in which the insulin sensitising agent 30 reduces the insulin antagonist action of the growth hormone.

10. The use of an insulin sensitising agent in the treatment of metabolic syndrome, in combination with treatment with growth hormone. 35

11. The use of growth hormone in the treatment of metabolic syndrome, in combination with treatment with an insulin sensitising agent. WO 03/090784 PCT/EP03/04357 47

12. The use of an insulin sensitising agent to reduce waist circumference, in combination with treatment with growth hormone.

13. The use of growth hormone to reduce waist circumference, in combination with 5 treatment with an insulin sensitising agent.

14. The use according to any of claims I to 3 and 10 to 13, or a method according to any of claims 4 to 9, wherein the insulin sensitising agent is a biguanide. 10

15. Use or method according to claim 14 wherein the biguanide is metformin.

16. A pharmaceutical composition comprising growth hormone or analogue thereof and an insulin sensitising agent, in combination with a pharmaceutically acceptable carrier, for use in the treatment of metabolic syndrome. 15

17. The composition according to claim 16 in which the insulin sensitising agent is a biguanide.

18. The composition according to claim 17 in which the biguanide is metformin. 20

19. The composition according to any of claims 16 to 18 in which the growth hormone is recombinant human growth hormone. 25