WO2006133477A1 - Peptides corresponding to human growth hormone - Google Patents

Abstract

The present invention relates to peptides corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone, and there use in modulating lipid metabolism and promoting weight reduction when administered at a dose of 0.01 - 2 mg per day. Dosage units comprising the peptide are also provided. The invention also relates to use of the peptides in treating glucose intolerance and improving lipid profiles.

Description

PEPTIDES CORRESPONDING TO HUMAN GROWTH HORMONE

This invention relates to peptides corresponding to human growth hormone (hGH) , and more particularly to peptides corresponding to hGH 177-191 and subfragments and analogues thereof .

BACKGROUND OF THE INVENTION

Obesity is an escalating international health problem, and is associated with increased mortality and morbidity risk. Conventional non-pharmacological treatments, based on diet and exercise, are frequently ineffective in promoting sustained weight loss . Consequently, there is a need for pharmacological therapy as an adjunct to diet and lifestyle changes in order to achieve and maintain weight loss .

In the obese state, serum hGH levels are markedly reduced due to decreased secretion and increased clearance of hGH. Since low hGH levels are implicated in a blunted lipolytic response to fasting caused by reduced beta-adrenergic receptor (β-AR) lipolytic sensitivity in subcutaneous adipose tissue, the reduced hGH levels in obesity may contribute to maintenance of the obese state . Most studies have shown that hGH levels normalize following successful weight loss, suggesting that the reduction in circulating hGH in the obese state is likely to be an adaptive phenomenon. The cause of the reduced hGH levels in the obese state is not yet understood. Administration of human Growth hormone (hGH) in an obese state or in a hGH-deficient state, stimulates catecholamine-induced lipolysis in adipose tissue, reduces- fat mass, and increases lean body mass. Pharmacological doses of hGH (≥ 25 μg/kg/day) have limitations as an obesity treatment as the prolonged use of hGH may be associated with insulin resistance, glucose intolerance, oedema and increases in insulin-like growth factor 1 (IGF- 1) . Prolonged increases in IGF-I may have a range of undesirable effects including an increase in cancer risk. Lower doses of hGH closer to physiological levels (≤ 3 μg/kg/day) have been shown to have beneficial effects in obese patients without clear adverse effects, although treatment at these doses can still be associated with some insulin resistance, increased IGF-I levels, and oedema. Titration to maintain IGF-I levels within the physiological range appears to be required to provide modest fat reduction with no adverse metabolic effects and suggestions of metabolic improvement.

There is strong evidence to suggest that the diverse functions carried out by hGH are mediated by discrete functional domains of the hormone. In Australian patent No. 693478 by Monash University, we described the use of a peptide corresponding to a carboxyl-terminal sequence of human growth hormone, or corresponding regions from growth hormone of other mammalian species, for the control of obesity. This region of growth hormone has the ability to modulate lipid metabolism. In particular, synthetic peptide corresponding to amino acid residues 177-191 of the human growth hormone sequence (hereinafter referred to as hGH 177-191) is effective in reducing body weight gain in obese mice and rats , without the adverse effects on insulin sensitivity seen in similar studies using full-length hGH. The peptide also enhances lipolysis in human, pig, mouse and rat adipose tissue ex vivo, and increases fat oxidation in obese mice in vivo. Additionally, the peptide increases the expression of β₃- ARs in mouse and human cell lines in vitro, and in the adipose tissue of obese mice ex vivo. A subsequent application, PCT/AU98/00724 by Metabolic Pharmaceuticals Ltd, discloses analogues of the hGH177-191 peptide which share this activity. Our application, PCT/AUOO/01362 (WO01/33977) , discloses the surprising oral activity of all such peptides. The entire disclosures of AU693478,

PCT/AU98/00724 and PCT/AUOθ/01362 are incorporated herein by this reference.

Peptides corresponding to hGH 177-191 and analogues and subfragments thereof are currently undergoing studies in humans to determine safety, tolerability, weight loss and fat effects.

It is an aim of a preferred embodiment of the present invention to improve the activity of peptides corresponding to hGH 177-191 or analogues or subfragments thereof in modulating lipid metabolism and therefore treating obesity. It is a further aim of a preferred embodiment of the present invention to determine an indication other than treatment of obesity for peptides corresponding to hGH 177-191 or analogues or subfragments thereof .

SUMMARY

The present invention in a first aspect provides the use of a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone, in a method of modulating lipid metabolism in a human, which method comprises administering the peptide orally to the human at a dose of 0.01-2 mg per day

A non-linear dose response to AOD9604 treatment was expected from in vivo and in vitro animal experiments, which show an effective rodent dose of 0.125 to 0.5 mg/kg at the centre of a bell-shaped dose response curve. A 30 mg dose in a 100 kg human is equivalent to 0.3 mg/kg and this was expected to be the most effective dose in humans for promoting lipid metabolism. Surprisingly, however, human trials involving administering 0 (placebo), 1, 5, 10, 20 and 30 mg of an hGH 177-191 analogue indicate that the best activity of the peptide in promoting weight reduction is 1 mg per day. Additionally, in vitro measurements in our laboratory conducted to understand the activity of the peptide at low dose have revealed a bimodal concentration response, with an unexpected low concentration bell shape equimolar with human growth hormone .

According to a second aspect, the invention provides a dosage unit for use in the first aspect of the invention, the dosage unit comprising 0.01 - 2mg of a peptide corresponding to human growth hormone (hGH) 177- 191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone .

According to a third aspect the invention provides a method of reducing body weight comprising administering to a human in need thereof an effective amount of a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone, wherein the peptide is administered at a dose of 0.01 - 2 mg per day. In a preferred embodiment, the method of the third aspect of the invention is performed simultaneously with calorie restriction, at least for an initial period. This serves to optimise the reduction of body weight provided by the method of the third aspect of the invention, as compared to placebo.

Calorie restriction may be achieved by a lifestyle modification program, or by additional pharmacological intervention with an appetite suppressant or digestion inhibitor.

According to a fourth aspect, the invention provides a kit for optimising the reduction of body weight compared to placebo, the kit comprising a peptide corresponding to hGH 177-191 for administering orally at a dose of 0.01 - 2 mg per day, together with information on lifestyle modification capable of enhancing the effect of the peptide in weight reduction.

According to a fifth aspect, the invention provides the use of a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone, in the treatment of glucose intolerance, or in a method of improving lipid profiles in an animal . According to a sixth aspect, the invention provides a method of treating glucose intolerance in an animal, the method comprising administering to the animal an effective amount of a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone.

In a seventh aspect, the present invention provides a pharmaceutical formulation for use in treating glucose intolerance in an animal, which formulation comprises a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone a pharmaceutically acceptable carrier.

According to an eighth aspect, the invention provides a method of improving lipid profiles in an animal, the method comprising administering to the animal an effective amount of a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone.

In a ninth aspect, the present invention provides a pharmaceutical formulation for use in improving lipid profiles in an animal, which formulation comprises a peptide corresponding to human growth hormone (hGH) 177- 191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone a pharmaceutically acceptable carrier. The use of the fifth aspect, the method of the sixth aspect or the formulation of the seventh aspect may be used to treat or prevent any disorder involving glucose intolerance. Such disorders include diabetes and metabolic syndrome. The use of the fifth aspect, the method of the eighth aspect or the formulation of the ninth aspect may be used to treat or prevent any disorder involving abnormal lipid profiles. Such disorders include hypercholesteraemia and metabolic syndrome.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 shows a graph plotting weight change (- SEM) over 12 weeks of treatment with AOD 9604 (ITT population, no data imputed) . Difference in adjusted means is from the general linear model adjusting for effects of gender, centre, age and weight before active treatment. * P < 0.05 (Dunnett's multiple comparisons procedure) , statistically significant for 1 mg AOD9604 female subgroup. Figure 2 shows a graph plotting mean change (-

SEM) in body weight over treatment period for placebo and 1 mg AOD9604 treatment groups (ITT population, no data imputed) . Difference in adjusted means is from the general linear model adjusting for effects of gender, centre, age and weight before active treatment.

Figure 3 shows a graph plotting changes in lipid profiles in the placebo and AOD9604 treatment groups. (ITT population, no data imputed) Figure 4 shows a graph plotting mean weight change after 12 weeks of treatment for patients who complied or did not comply with diet and lifestyle advice (ITT population, no data imputed) . Compliers were defined as patients who lost > 0 kg in the 2 week placebo run-in period. * P < 0.05 (Dunnett's multiple comparisons procedure) .

Figure 5 shows a graph plotting anthropomorphic measurements of total abdominal fat (mean % change - SEM) after 12 weeks of treatment with AOD9604 (ITT population, no data imputed) . Difference in adjusted means is from the general linear model adjusting for the effects of gender, centre, age and pre-treatment value. * P < 0.05 (Dunnett's multiple comparisons procedure) . Figure 6 is a graph showing subcutaneous and visceral fat loss for all treatment groups after 12 weeks of treatment with AOD9604 (ITT population, no data imputed) . Difference in adjusted means is from the general linear model adjusting for the effects of gender, centre, age and pre-treatment value. * P < 0.01 (Dunnett's multiple comparisons procedure) .

Figure 7 is a graph of the dose response of the lipolytic effect of AOD9604 on 3T3L1 cells after 24 hours of incubation (n=6) . Figure 8 shows the number of patients diagnosed as diabetic after the trial treatment (non were diagnosed as diabetic before the treatment period as it was an exclusion criterion) .

Figure 9 the change in the incidence of IGT or diabetes from prior to trial treatment to the end of the trial treatment . DETAILED DESCRIPTION

In a clinical trial involving oral administration of the peptide Tyr-hGH 177-191, the inventors were expecting a dose of 20-30 mg per day to be most effective in promoting body weight reduction, based on animal experiments. What they actually found was that in both sexes, a 1 mg dose produced abdominal fat loss measured by waistline parameters, significant on the repeated measures of waistline tape. The fat loss comes from both abdominal visceral and subcutaneous compartments, with the increased loss over placebo coming more clearly from the larger subcutaneous compartment , which in the placebo group does not reduce. At the 1 mg dose there is also total body fat loss evidenced by weight loss. As the dose increased, there was evidence of similar abdominal fat loss by waistline measurement in both sexes and fat loss increased from the abdominal subcutaneous compartment. However at the same time there was a counteracting stall in the loss of visceral fat and non-abdominal fat possibly increased. As the non- abdominal fat is a larger compartment in females, higher doses produce much less total fat loss (and therefore weight loss) in females than males.

The trial results led the inventors to propose that dose affects how the peptide mobilises fat depots. This was totally unexpected from the animal data and the prior art .

Most of the animal and in vitro data obtained by the inventors in the past was concentrated around an effective rodent dose of 0.125 to 0.5 mg/kg at the centre of a bell shaped pattern. Ex vivo lipogenesis and lipolysis experiments in rat and mouse fat tissue showed bell shapes peaking typically around 500 nM, consistent with this dose range (Tyr-hGH 177-191 has a molecular weight of 1815) . Human fat tissue had a similar peak about 100-200 nM. A 30 mg dose in a 100 kg human is 0.3 mg per kg, within the same range as the rodent data. A low dose bell shaped dose response around 1 mg in humans suggests there may be a much lower concentration in vitro effects in another bell shape, around the low nM region. In terms of molar amounts, 1 mg of orally dosed Tyr-hGH 177-191 is equivalent to 4 mg of injected human growth hormone, assuming 40% oral availability. This is a typical high level hGH dose in humans, not nowadays given but clearly effective on fat loss . Accordingly the in vitro effect of Tyr-hGH 177-191 which may correspond to this should occur at concentrations roughly equimolar with hGH.

Since the clinical trails results were obtained the inventors have been exploring the 1 nM region and initial experiments have confirmed that around 1 - 10 nM there is a substantial peak in lipolysis from both fat tissue and cultured fat cells, disappearing or flattening at 10 - 100 nM and rising again to 500 - 1000 Nm. The 1 - 10 nM region also corresponds to peaks in relevant intracellular signalling. The inventors therefore propose that 1 mg is near the peak of a bell-shaped effect which is probably the genuine equivalent of hGH effects previously observed in humans. This may involve restoration of adrenaline receptor number in subcutaneous fat tissue, restoring the lipolytic response of that depot to low insulin levels caused by fasting and/or to endogenous adrenaline. 5 mg and 10 mg are well above the top of the peak. Optimal activity in the sub microgram region, being molar equivalent with the highest doses of hGH administered to humans is clearly possible.

The inventors also propose that the high dose effect rising at 20 and 30 mg particularly in males may correspond to the direct higher concentration antilipogenic and lipolytic effects observed in fat and the liver. At these higher doses any beneficial effect in non-abdominal fat tissue may disappear or reverse.

The preferred dose for weight reduction is the peak of the dose response curve between 0.01 and 2.00 mg. This is expected to be in the region of 0.05-1.50 mg, preferably 0.25-1.25 mg, more preferably 0.25-0.75 mg and most preferably 0.25-0.50 mg.

Preferably the dose is 0.01, 0.02, 0.03, 0.04,

0.05, 0.06, 0.07, 0.08, 0.09, 0.10 , 0.11, 0.12, 0 .13,

0.14, 0.15, 0.16, 0.17, 0.18, 0.19 , 0.20, 0.21, 0 .22,

0.23, 0.24, 0.25, 0.26, 0.27, 0.28 , 0.29, 0.30, 0 .31,

0.32, 0.33, 0.34, 0.35, 0.36, 0.37 , 0.38, 0.39, 0 .40,

0.41, 0.42, 0.43, 0.44, 0.45, 0.46 , 0.47, 0.48, 0 • 49,

0.50, 0.51, 0.52, 0.53, 0.54, 0.55 , 0.56, 0.57, 0 .58,

0.59, 0.60, 0.61, 0.62, 0.63, 0.64 , 0.65, 0.66, 0 .67,

0.68, 0.69, 0.70, 0.71, 0.72, 0.73 _/ 0.74, 0.75, 0 .76,

0.77, 0.78, 0.79, 0.80, 0.81, 0.82 , 0.83, 0.84, 0 .85,

0.86, 0.87, 0.88, 0.89, 0.90, 0.91 , 0.92, 0.93, 0 .94,

0.95, 0.96, 0.97, 0.98, 0.99, 1.00 , 1.01, 1.02, 1 .03,

0.04, 0.05, 0.06, 0.07, 0.08, 1.09 , l.io, 1.11, 1 .12,

1.13, 1.14, 1.15, 1.16, 1.17, 1.18 , 1.19, 1.20, 1 .21,

1.22, 1.23, 1.24, 1.25, 1.26, 1.27 , 1.28, 1.29, 1 .30,

1.31, 1.32, 1.33, 1.34, 1.35, 1.36 , 1.37, 1.38, 1 .39,

1.40, 1.41, 1.42, 1.43, 1.44, 1.45 , 1.46, 1.47, 1 .48,

1.49, 1.50, 1.51, 1.52, 1.53, 1.54 , 1.55, 1.56, 1 .57,

1.58, 1.59, 1.60, 1.61, 1.62, 1.63 , 1-64, 1.65, 1 .66,

1.67, 1.68, 1.69, 1.70, 1.71, 1.72 , 1.73, 1.74, 1 .75,

1.76, 1.77, 1.78, 1.79, 1.80, 1.81 , 1.82, 1.83, 1 .84,

1.85, 1.86, 1.87, 1.88, 1.89, 1.90 , 1.91, 1.92, 1 .93,

1.94, 1.95, 1.96, 1.97, 1.98, 1.99 or 2.00 mα.

The dose of 0.01 to 2.00 mg is more effective in reducing body weight than higher doses and is active in both genders. Accordingly such a low dose may be used beneficially in methods of promoting weight loss, or overall reduction of body fat.

The use of the first aspect, the dosage unit of the second aspect, the method of the third aspect or kit of the fourth aspect of the invention maybe used to treat obesity. Such treatment may also be used to alleviate or prevent conditions related to obesity. "Obesity" refers to a condition whereby a mammal has a Body Mass Index (BMI) , which is calculated as weight (kg) per height. sup.2 (meters), of at least 25.9. Conventionally, those persons with normal weight have a BMI of 19.9 to less than 25.9. The obesity herein may be due to any cause, whether genetic or environmental. Examples of disorders that may result in obesity or be the cause of obesity include overeating and bulimia, polycystic ovarian disease, craniopharyngioma, the Prader- Willi Syndrome, Frohlich's syndrome, Type II diabetes, GH- deficient subjects, normal variant short stature, Turner's syndrome, and other pathological conditions showing reduced metabolic activity or a decrease in resting energy expenditure as a percentage of total fat-free mass, e.g., children with acute lymphoblastic leukaemia.

"Conditions related to obesity" refer to conditions which are the result of or which are exasperated by obesity, such as, but not limited to dermatological disorders such as infections, varicose veins, Acanthosis nigricans, and eczema, exercise intolerance, diabetes mellitus, insulin resistance, hypertension, hypercholesterolemia, cholelithiasis, osteoarthritis, orthopedic injury, thromboembolic disease, cancer, and coronary (or cardiovascular) heart disease, particular those cardiovascular conditions associated with high triglycerides and free fatty acids in an individual .

As the low dose has particular effect in reducing abdominal fat and subcutaneous fat the low dose may be used beneficially in methods of promoting loss of abdominal fat and loss of subcutaneous fat and particularly in the treatment of obesity. As higher than normal abdominal fat is a risk factor for cardiovascular disease the low dose may be used to prevent the risk of heart disease in obese animals with increased abdominal fat.

The effect of the peptide in promoting weight loss and modulating lipid metabolism being optimised when the peptide is administered to a subject undergoing calorie restriction, was identified by analysing two subgroups of patients in the clinical trial; those who lost weight in the two week placebo run-in period (where all patients received placebo) (" the lifestyle compilers"), and those who did not ("lifestyle non- compliers") . It was found that all of the drug effect is found in the lifestyle compiler group, where the weight loss at the 1 mg dose was 4.2 kg compared to placebo, a highly significant difference and a large difference compared to existing treatments and compared to the group as a whole (2.0 kg) . This synergistic interaction of the peptide with calorie restriction, preferably by lifestyle modification, was unexpected and is unlike that observed with previous obesity drugs.

With existing obesity drugs which pharmacologically induce calorie restriction, the addition of more calorie restriction with lifestyle changes simply adds to the average effect. Consequently with existing drugs, the additional weight loss effect attributable to • the drug is largely independent of the lifestyle changes attempted by the patient and there is no synergy. The inventors therefore believe that the peptide of the invention enhances the effect of calorie restriction, probably by sensitization of the subcutaneous fat tissue to the lipolytic demands of calorie restriction, and that all means of calorie restriction such as pharmacological intervention are to be expected to also combine with the peptide to provide this synergistic effect. The administration of hGH in an obese state or in a hGH-deficient state has been shown to stimulate catecholamine-induced lipolysis in adipose tissue, reduce fat mass and increase lean body mass . However the prolonged use of hGH may be associated with insulin resistance, glucose intolerance, oedema possibly associated with increases in insulin-like growth factor 1 (IGF-I) . The clinical trial undertaken included measurement of glucose levels during an oral glucose tolerance test to determine if peptides corresponding to the hGH C-terminus had the same effect on glucose control as hGH. The results indicated that treatment with Tyr-hGH

177-191 does not worsen glucose control or induce insulin resistance. In fact, surprisingly administration of Tyr- hGH 177-191 caused a reduction in patients with impaired glucose tolerance (ITT) and reduced the risk of onset of diabetes as compared to the placebo group.

Accordingly the inventors propose that peptides corresponding to human growth hormone (hGH) 177-191 or lipid metabolising subfragments or analogues thereof are capable of controlling glucose metabolism and therefore may be used in the treatment of any disorder involving impaired glucose tolerance, such as metabolic syndrome or diabetes. The inventors also propose that peptides corresponding to human growth hormone (hGH) 177-191 or lipid metabolising subfragments or analogues may be useful in preventing the onset of diabetes.

Impaired glucose tolerance as used herein refers to a condition that is midway between normal and diabetic. Blood sugar levels on certain tests are higher than normal , but not quite high enough to say the person has diabetes. People with this condition are at higher than average risk for developing diabetes, heart attack, and strokes .

There are two forms of impaired glucose metabolism: Impaired Fasting Glucose (IFG) , and Impaired Glucose Tolerance (IGT) . A person has impaired fasting glucose when, after fasting for at least eight hours, their blood sugar level is between 110 and 125 mg/dL. This level is higher than normal but lower than the level used to diagnose diabetes. A person has impaired glucose tolerance when an oral glucose tolerance test shows that they have blood sugar levels that are higher than normal but not high enough to diagnose diabetes . Impaired Glucose Tolerance (IGT) is diagnosed when the glucose level is 141 to 199 mg/dL

Metabolic syndrome is a common condition that goes by many names (dysmetabolic syndrome, syndrome X, insulin resistance syndrome, obesity syndrome, and

Reaven's syndrome) . It is a set of risk factors that includes: abdominal obesity, a decreased ability to process glucose (insulin resistance) , dyslipidemia

(unhealthy lipid levels) , and hypertension. Patients who have this syndrome have been shown to be at an increased risk of developing cardiovascular disease and/or type 2 diabetes .

Metabolic syndrome is commonly defined as involving three or more of the following: * Central/abdominal obesity as measured by waist circumference [Men - Greater than 40 inches (102 cm) ;

Women - Greater than 35 inches (88 cm) ]

* Fasting triglycerides greater than or equal to 150 mg/dL (1.69 mmol/L) * HDL cholesterol [Men - Less than 40 mg/dL (1.04 mmol/L) ; Women - Less than 50 mg/ dL (1.29 mmol/L)]

* Blood pressure greater than or equal to 130/85 mm Hg

* Fasting glucose greater than or equal to 110 mg/dL (6.1 mmol/L)

Also frequently seen with metabolic syndrome are prothrombotic (blood clotting) and proinflammatory tendencies. While these combined criteria and risk factors do not usually cause overt symptoms, they are a warning of an increased likelihood of clogged arteries, heart disease, stroke, diabetes, kidney disease, and even premature death. If left untreated, complications from untreated metabolic syndrome can develop in as few as 15 years. Those patients who have metabolic syndrome and also smoke tend to have an even poorer prognosis.

In the United States it is estimated that 20% of adults (about 47 million) have metabolic syndrome, with the prevalence approaching 50% in the elderly. It can affect anyone at any age, but it is most frequently seen in those who are significantly overweight (with most of their excess fat in the abdominal area) and inactive. The root cause of most cases of metabolic syndrome can be traced back to poor eating habits and a sedentary lifestyle. Some cases occur in those already diagnosed with hypertension and in those with poorly controlled diabetes; a few are thought to be linked to genetic factors that are still being researched.

All of the factors associated with metabolic syndrome are interrelated. Obesity and lack of exercise tend to lead to insulin resistance. Insulin resistance has a negative effect on lipid production, increasing VLDL (very low-density lipoprotein) , LDL (low-density lipoprotein - the "bad" cholesterol) , and triglyceride levels in the bloodstream and decreasing HDL (high-density lipoprotein - the "good" cholesterol) . This can lead to fatty plaque deposits in the arteries which, over time, can lead to cardiovascular disease, blood clots, and strokes. Insulin resistance also leads to increased insulin and glucose levels in the blood. Excess insulin increases sodium retention by the kidneys, which increases blood pressure and can lead to hypertension. Chronically elevated glucose levels in turn damage blood vessels and organs, such as the kidneys, and may lead to diabetes.

The primary "treatments" for metabolic syndrome are for patients to lose excess weight, exercise regularly, and stop smoking. Drug treatment may be necessary to address hypertension and high cholesterol levels. Some doctors also recommend aspirin to decrease the risk of clotting, and a few doctors prescribe medications to increase insulin sensitivity (although there is not widespread agreement on this) . Currently there is no pharmaceutical treatment proposed for metabolic syndrome and the present invention proposes to provide such a pharmaceutical treatment . Diabetes as used herein encompasses type I, type II and gestational diabetes. Diabetes is a medical disorder characterized by varying or persistent hyperglycemia (elevated blood sugar levels) , especially after eating. All types of diabetes mellitus share similar symptoms and complications at advanced stages. Hyperglycemia itself can lead to dehydration and ketoacidosis. Longer-term complications include cardiovascular disease (doubled risk) , chronic renal failure (it is the main cause for dialysis) , retinal damage with eventual blindness, nerve damage and and eventual gangrene with probable loss of toes, feet, and even legs in amputation.

Diabetes is characterized by either: an inability of the pancreas to produce insulin (type 1 or insulin- dependent diabetes mellitus) or an inability of insulin to exert its normal physiological actions (type 2 or non- insulin dependent diabetes) .

Gestational diabetes mellitus (GDM) is defined as any degree of glucose intolerance with onset or first recognition during pregnancy. The definition applies whether insulin or only diet modification is used for treatment and whether or not the condition persists after pregnancy. It does not exclude the possibility that unrecognized glucose intolerance may have antedated or begun concomitantly with the pregnancy.

The peptides used in accordance with the present invention corresponds to the C-terminal amino acid sequence of growth hormone. Such a peptide is termed a "C-terminal growth hormone fragment." For the purposes of this specification, the term "C-terminal growth hormone fragment" is to be understood to mean a peptide fragment from the carboxy-terminal region of the amino acid sequence of a mammalian growth hormone which is able to reduce lipogenic activity; and, or to stimulate lipolysis.

"Peptide" as used herein means any chain of amino acids from 2 to 50 amino acid residues in length, preferably 2 to 20, more preferably about 15 amino acid residues in length. Accordingly the term peptide as used herein also encompasses polypeptides and may be used interchangeably therewith. The only proviso is that any peptide for use in accordance with the present invention does not have the full length sequence of human growth hormone or an analogue thereof from another species and does not modulate IGF-I. Full length growth hormone is capable of modulating lipid metabolism but also modulates IGF-I. The amino acids may include synthetic modifications or the peptide bonds may be modified to enhance stability and/or activity, according to the many methods known in the art .

Preferably the peptides used in accordance with the present invention have the ability to stimulate the activity of hormone-sensitive lipase, a key enzyme in lipolysis, and/or to inhibit acetyl CoA carboxylase, a key enzyme in lipogenesis.

The invention also encompasses the use of peptides which are functional analogues of the native carboxy-terminal sequences of mammalian growth hormones, in that the analogue peptide is capable of modulating lipid metabolism without an appreciable effect on IGF-I. Such analogues may be derived from natural sources, produced by recombinant DNA technology, or synthesised using conventional peptide synthetic methods. Such peptides synthetic methods are to be understood to include combinatorial methods . Such analogues include a disulphide bond which confers a cyclic configuration on the peptide. In particular, all of the active peptides disclosed in AU 693478 and PCT/AU98/00724 are to be understood to be within the scope of this invention, for example :

Ref No. STRUCTURE

9502 Leu Arg He VaI GIn Pen Arg Ser VaI GIu GIy Ser Pen GIy Phe SEQ ID NO: 1 9405 CH3CO- Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:2

9410 H - Leu Arg lie VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:3 9404 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe - CONH₂ SEQ ID NO:4

9407 Leu Arg lie VaI GIn Cys Lys Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:5

9408 Leu Arg lie VaI GIn Cys Lys Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:6

(amide bond) 9604 Tyr Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:7

9605 Lys Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:8

9618 Lys Lys Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:9

9607 Ala Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 10

9606 Leu Lys He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 11

9608 Leu Arg Ala VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:12 9403 Leu Arg Lys VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 13 9609 Leu Arg He Ala GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ K) NO: 14

9610 Leu Arg He VaI Ala Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:15

9612 Leu Arg He VaI GIn Cys Arg Ala VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 16

9613 Leu Arg He VaI GIn Cys Arg Ser Ala GIu GIy Ser Cys GIy Phe SEQ ID NO: 17 9615 Leu Arg He VaI GIn Cys Arg Ser VaI GIu Ala Ser Cys GIy Phe SEQ HD NO: 18 9616 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ala Cys GIy Phe SEQ ID NO: 19 9602 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys Ala Phe SEQ ID NO:20 9501 Leu Arg He VaI GIn Cys Arg Ser VaI GIu D-AIa Ser Cys D-AIa Phe SEQ ID NO:21 9601 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Ala SEQ ID NO:22

wherein the amino acid residue abbreviations used are in accordance with the standard peptide nomenclature:

GIy = Glycine; lie = Isoleucine; GIu = Glutamic Acid; Phe = Phenylalanine;

Cys = Cysteine; Arg = Arginine;Gln = Glutamine; Leu = Leucine; Ser = Serine; VaI = Valine; Lys = Lysine; Ala = Alanine; Asp = Aspartic acid; His = Histidine; Orn = Ornithine; Tyr = Tyrosine; Pen = Penicillamine (p, p ' -Dimethyl -Cysteine).

All amino acids, except for glycine, are of the L-absolute configuration, unless indicated as D-absolute configuration. All the above peptides above have a cyclic disulfide bond between Cys(182) and Cys(189) or Pen (182) and Pen (189) as appropriate.

Preferably the peptides comprises amino acids 182-189 (hGH 182-189) , more preferably amino acids 177-191 of human growth hormone (hGH 177-191) . Even more preferably the peptide is the human growth hormone analogue AOD9604 (Tyr-hGH 177-191) . However, it will be clearly understood that the invention is also applicable to peptides corresponding to the amino acid sequences of growth hormones of other mammalian species, including but not limited to those of domestic mammals such as cattle, sheep, pigs and horses, companion animals such as cats and dogs, and zoo animals including felids, canids, and non- human primates. There is strong conservation of the sequence of this region of growth hormone across species, as set out in PCT/AU98/00724 and references cited therein. Peptides may also be conjugated to a fusion partner to enable easier biosynthesis and/or delivery. They may be incorporated in conventional pharmaceutical compositions, or may be present in a genetically-modified food, such as disclosed in WO 01/33997. The peptides may be administered in pharmaceutical compositions together with a pharmaceutically acceptable carrier for administration.

For the treatment of glucose intolerance or the improvement of lipid profiles the peptides are preferably administered orally at a dose of 0.01 to 60.00 mg.

The animal may be a human, or may be a domestic or companion animal . While it is particularly contemplated that the present invention is used in medical treatment of humans, it is also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as non-human primates, felids, canids, bovids, and ungulates.

Preferably the mammal is a human. The human may be a child or an adult .

Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known in the art, as set out in textbooks such as Remington's Pharmaceutical Sciences, 20th Edition, Williams and Williams, Pennsylvania, USA (2000) .

As used herein, a "pharmaceutically acceptable carrier" is a pharmaceutically acceptable solvent, suspending agent, excipient or vehicle for delivering the growth hormone fragment and/or pharmaceutically-active agent to the subject. The carrier or diluent, and other excipients, will depend on the route of administration, and again the person skilled in the art will readily be able to determine the most suitable formulation for each particular case.

Analogues of the peptides described herein are included within the scope of the invention, provided that they are functionally active. As used herein, the terms "functionally active" and "functional activity" in reference to an analogue means that analogue is capable of (or has ability for) modulating lipid metabolism without having an appreciable effect on IGF-I. Analogues as used herein includes amino acid sequence variants of the peptide amino acid sequences provided. Sequence variants include deletions, insertions or substitutions of amino acid residues within the growth hormone fragment amino acid sequence set out above . Any combination of deletion, insertion, and substitution may be made to arrive at an amino acid sequence variant of the growth hormone fragment, provided that the variant possesses the desired functional characteristics described herein; i.e. ability to modulate lipid metabolism without having an appreciable effect on IGF-I.

If such substitutions do not result in loss of functional activity, then more substantial changes, denoted exemplary substitutions in Table 1, or as further described below in reference to amino acid classes, may be introduced, and the resulting variant growth hormone fragment analyzed for functional activity. A person skilled in the art would be able to determine whether a peptide has the ability to modulate lipid metabolisms but has no appreciable effect on IGF-I by methods of the common general knowledge.

As used herein the term "no appreciable effect" means that the effect on IGF-I is not clinically significant and that if any effect of a peptide is registered in an assay it can be considered negligible.

One way in which a skilled person may determine if a peptide is capable of modulating lipid metabolism is by performing a lipolysis assay as described in Example A. In brief, obese rats are treated and sacrificed, adipose tissue is obtained from treated and control rats and placed in vials and terbutaline added. The vials are incubated at 37°C for 1 hour, gassed with carbon and assayed in a standard glycerol assay, for example using an assay kit such as Sigma GPO-337.

To determine is a peptide has an appreciable effect on IGF-I a person skilled in the art may perform an IGF-I assay on a blood sample (for example from a mouse) . A suitable assay kit is available from R & D Systems, Inc., with catalogue number DY791. This is a sandwich ELISA using hamster anti-mouse IGF-I as capture antibody and goat anti-mouse IGF-I as detection antibody.

Table 1

Original Exemplary Preferred

Residue Substitutions Substitutions Ala (A) val ; leu; ile val

Arg (R) lys; gin; asn lys

Asn (N) gin; his ; lys; arg gin

Asp (D) glu glu

Cys (C) ser ser

GIn (Q) asn asn

GIu (E) asp asp

GIy (G) pro pro

His (H) asn; gin; lys; arg arg

He (D leu; val; met; ala; phe; leu norleucine

Leu (L) norleucine ; ile; val ; ile met ; ala; phe

Lys (K) arg; gin; asn arg

Met (M) leu; phe; ile leu

Phe (F) leu; val; ile; ala leu

Pro (P) gly giy

Ser (S) thr thr

Thr (T) ser ser

Trp (W) tyr tyr

Tyr (Y) trp; phe; thr; ser phe

VaI (V) ile; leu; met ; phe; leu ala; norleucine

As used herein, the terms "therapeutically effective amount" and "therapeutic amount" are synonymous, and mean an amount of a peptide of the present invention effective to yield a desired therapeutic response. The specific therapeutically effective amount will obviously vary with such factors as the particular condition being treated, the type of mammal being treated, the physical condition and clinical history of the mammal, the duration of the treatment, the nature of concurrent therapy (if any) , and the specific formulations employed and the structure of the peptide. Generally, the terms "treating", "treatment" and the like are used herein to mean affecting a subject, tissue or cell to obtain a desired pharmacological and/or physiological effect. The effect may be prophylactic in terms of completely or partially preventing onset of disorders involving impaired glucose or lipid metabolism and/or may be therapeutic in terms of promoting fat loss or correcting impaired glucose metabolism.

"Treating" as used herein covers any method of treatment of, or prevention of disease in a mammal, particularly a human, and includes preventing the disease from occurring in a subject who may be predisposed to the disease, but has not yet been diagnosed as having it; inhibiting the disease, i.e., arresting its development; or relieving or ameliorating the effects of the disease, i.e., cause regression of the effects of the disease.

The first and third aspects of the invention involve oral administration of a peptide. Oral administration may be by the use of tablets, capsules, powders or liquids such as suspensions, solutions, emulsions or syrups . As the inventors have found that the peptides are orally active it may be possible to administer the peptide as a transgenic foodstuff.

When formulated into tablets, conventional excipients (e.g. sodium citrate, lactose, macrocrystalline cellulose, starch, etc.)/ lubricating agents (e.g. anhydrous silicic acid, hydrolysed castor oil, magnesium stearate, sodium lauryl sulphate, talc, etc.) and binding agents (e.g. starch paste, glucose, lactose, gelatin, mannitol, magnesium trisilicate, etc.) may be used. When administered as liquids, conventional liquid carriers can be used.

According to the second aspect of the invention provides a dosage unit for oral administration comprising 0.01 - 2 mg of peptide. In the case of solid preparations, each unit dosage form of the active ingredient can contain from about 5% to about 95% of the same by weight of the entire composition with the remainder comprising conventional pharmaceutical carriers. When the therapeutic agent is used as aqueous solution, i.e. for injection, the dosage unit of the solution may contain 0.05 to about 0.5% of the same by weight of the entire solution.

The dosage unit may be of the sustained release type, for example for once daily administration. A suitable sustained or slow release formulation may be achieved, for example, when the peptide is bound to a suitable polymer.

The seventh and ninth aspect of the invention includes various pharmaceutical compositions useful for controlling glucose tolerance or improving lipid profiles and therefore preventing or treating disorders involving abnormal glucose metabolism or lipid profiles, such as diabetes, hypercholesteraemia and metabolic syndrome. The pharmaceutical compositions according to one embodiment of the invention are prepared by bringing a peptide corresponding to a C-terminal growth hormone fragment, analogue, variant or salts thereof into a form suitable for administration to a subject using carriers, excipients and additives or auxiliaries.

The pharmaceutical compositions are preferably prepared and administered in- dosage units. Solid dosage units include tablets, capsules and suppositories. For treatment of a subject, depending on activity of the compound, manner of administration, nature and severity of the disorder, age and body weight of the subject, different daily doses can be used. Under certain circumstances, however, higher or lower daily doses may be appropriate. The administration of the daily dose can be carried out both by single administration in the form of an individual dose unit or else several smaller dose units and also by multiple administration of subdivided doses at specific intervals. The pharmaceutical compositions may be administered locally or systemically in a therapeutically effective dose. Amounts effective for this use will, of course, depend on the severity of the disease and the weight and general state of the subject. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of the pharmaceutical composition, and animal models may be used to determine effective dosages for treatment of the cytotoxic side effects.

An effective amount of the growth hormone fragment to be employed therapeutically will depend, for example, upon the therapeutic objectives, the route of administration, and the condition of the subject. Accordingly, it will be necessary for the therapist to titrate the dosage and modify the route of administration as required to obtain the optimal therapeutic effect. A typical daily dosage might range from about 1 μg/kg to up to 100 mg/kg or more, depending on the mode of delivery. Dosage levels of the growth hormone fragment used for treating glucose intolerance or improving lipid profiles will usually be of the order of about 0.5mcg to about 20mg per kilogram body weight, with a preferred dosage range between about 0.5mcg to about lOmg per kilogram body weight per day (from about 0.5mg to about 3g per patient per day) . The amount of active ingredient which may be combined with the carrier materials to produce a single dosage will vary, depending upon the host to be treated and the particular mode of administration. For example, a formulation intended for oral administration to humans may contain about 0.5mg to Ig of an active compound with an appropriate and convenient amount of carrier material, which may vary from about 5 to 95 percent of the total composition. Dosage unit forms will generally contain between from about 0.5mg to 500mg of active ingredient. It will be understood, however, that the specific dose level for any particular patient will depend upon a variety of factors including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, route of administration, rate of excretion, drug combination and the severity of the particular disease undergoing therapy.

For the purposes of this specification it will be clearly understood that the word "comprising" means "including but not necessarily limited to" , and that the word "comprises" has a corresponding meaning.

For the purposes of this specification the term "consisting essentially of" is used herein to indicate that the peptide includes a bioactive lipid metabolic domain of the carboxyl-terminal sequence of a growth hormone, optionally together with other portions of the carboxyl-terminal sequence or other amino acid sequences which do not materially affect the characteristics of the peptide in modulating lipid metabolism, but not other bioactive domains of the growth hormone.

It will be apparent to the person skilled in the art that while the invention has been described in some detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

It is to be understood that, if any prior art publication is referred to herein, such reference does not constitute an admission that the publication forms a part

^■ of the common general knowledge in the art, in Australia or any other country.

The invention will now be described by way of reference only to the following non-limiting examples.

EXAMPLES EXAMPLE A - LIPOLYSIS IN 3T3-L1 ADIPOCYTES

This protocol has been optimized for 3T3-L1 cells (Source: ATCC reference CL-173) grown and differentiated in a batch of FBS.

1. Serum starve confluent, full differentiated adipocytes in serum free DMEM media.

2. After overnight serum starvation (approximately 16 hrs) , add AOD9604 at required concentrations in KRB working buffer (KRB with 1% BSA and 5.5mM D- glucose) .

3. Incubate in 37°C incubator (5% CO₂) .

4. At 24 hours remove 50μL of supernatant and assay for lipolysis with free glycerol reagent (see below) .

5. Aspirate remaining supernatant, wash cells gently with PBS and lyse cells in 0.4M NaOH-perform protein assay with BCA kit (Pierce) . LIPOLYSIS REACTION:

1. Add 5μL of appropriate supernatant aliguots into 96 well plate (do in triplicate) . Add 5μL of water for negative control or 5μL of glyercol standard for positive control .

2. Add 200μL of free glycerol reagent to each sample_, and leave at room temperature for 15 minutes

3. Read plates at Absorbance of 540nm.

KREBS RINGER BICARBONATE (KRB) BUFFER:

NaCl: 0.13OmM

KCl 4.7mM

CaCl₂ 1.25mM KH₂PO₄ 1.2mM

MgSO₄.6H₂O 1.2mM

NaHCO₃ 25mM

• Make up to desired volume with MiIIiQ water; • Stir solution with magnetic flea until dissolved;

• Bubble with carbogen (95%O₂, 5%CO₂) for approximately 30 minutes;

• pH to 7.4;

• Add 1% BSA and 5.5mM D-glucose and dissolve;

AOD9604 STOCK SOLUTION: AOD9604 is dissolved in sterile MiIIiQ water at a final concentration of 221μM and stored in aliquots at -80⁰C. Immediately before the start of the experiment, single aliquots of the AOD9604 stock solution are thawed on ice and appropriate dilutions of the AOD9604 stock solution are made in KRB/1% BSA/5.5mM D-glucose and immediately added to 3T30L1 cells for the commencement of the 24 hr incubation period as stated above.

BCA PROTEIN ASSAY KIT:

Product number: 2327 Pierce (supplier Progen)

Use according to manufacturers instructions.

FREE GLYCEROL REAGENT:

Catalogue number: F6428-40ml Sigma GLYCEROL STANDARD:

Catalogue number: G7793-5ml Sigma

RESULTS :

The experiment shows very clearly the usual high- concentration bell-shape (peaking around 50OnM) and a second low dose bell-shape peaking at 10 nM with a sharp cutoff, refer to Figure 7.

EXAMPLE B - AOD9604 12 WEEK WEIGHT LOSS TRIAL

This study determined the safety and effect of oral administration of AOD9604 on body weight, adiposity glucose and lipid metabolism.

Design: This was a 12 week multicentre, double-blind, placebo- controlled, parallel group study. A total of 300 patients were randomized to a 14 week period of daily oral dosing, comprising a 2 week placebo run-in period followed by 12 weeks of either placebo or active study drug (1, 5, 10, 20 or 30 mg daily) .

Patients :

Healthy, obese, adult patients (age: 30 - 65 years, BMI: >

35 kg/m2 , abdominal circumference > 110 cm for males and > 95 cm for females) . All patients were diagnosed as not diabetic prior to treatment.

Measurements :

Body weight and abdominal circumference were measured fortnightly and abdominal fat was assessed by computed tomography (CT) scan at baseline and end of treatment. Abdominal circumference was also assessed with a tape measure. Assessments for safety and tolerability included physical status, adverse event questioning, clinical laboratory tests, oral glucose tolerance test, vital signs and electrocardiograms (ECGs) .

AOD9604 Synthesis and Formulation:

The active pharmaceutical ingredient (API) , AOD9604 HCl, was chemically synthesized by conventional solid-phase synthesis on 4-methyl-benzylhydrylamine resin. Side chain- protected butyloxycarbonyl amino acid derivatives were sequentially added from the C-terminal end to the growing resin-bound peptide chain in the presence of diisopropylcarbodiimide .

Following addition of the last amino acid derivative the resin was treated with hydrofluoric acid to yield resin- free unprotected peptide. Mild oxidation of this peptide resulted in the formation of the intramolecular disulphide bridge of AOD9604 peptide.

Subsequent conventional two-step preparative reverse phase high performance liquid chromatography (HPLC) on C-18 resin employing triethylamine phosphate/acetonitrile followed by acetic acid/acetonitrile as mobile followed by acetic acid/acetonitrile as mobile phases yielded high- purity AOD9604 that was lyophilised as it became available. Bulk AOD9604 HCl was generated from lyophilised AOD9604 by dissolution of the purified, lyophilised peptide into a diluted hydrochloric acid/acetonitrile mixture followed by lyophilisation.

AOD9604 was dry-mixed with excipients (mannitol and PEG3350) as detailed below.

Size-0 capsules of 10 mg AOD9604 containing the following: 3.9% AOD9604 (API) 84.1% Mannitol, USP 12% PEG3350, USP Placebo capsules contain the excipients only, and were indistinguishable from capsules containing active drug.

The capsules were a product of Shionogi Qualicaps Co. Ltd. in Japan and distributed by Shionogi Qualicaps, Inc., Whitsett, NC, USA. They were white in colour and composed of hydroxypropylmethylcellulose (USP-grade) , potassium chloride (USP-grade) , carrageenan (USP-grade) , titanium oxide (USP-grade) , carnauba wax (National Formulary (NF) - grade) on the surface as lubricant.

The capsules were filled at the VA Cooperative Studies Program Clinical Research Pharmacy Coordinating Center, Albuquerque, NM, USA. Quality control and stability- testing was shared between the Center and Formatech, Inc., Andover, MA. , USA. The latter developed the pharmaceutical formulation. Results (see Figures) :

Weight loss over 12 weeks was greater in response to all doses of AOD9604 than placebo, with a non-linear dose response. Surprisingly, the largest effect occurred with the 1 mg dose (mean ± SEM of 2.8 + 0.7 kg with AOD9604 vs 0.8 ± 0.6 kg for placebo, P = 0.1042), this was significant in females (2.8 ± 0.7 kg with AOD9604 vs 0.1 ± 0.8 kg with placebo, P = 0.0379) . Repeated measures analysis of the mean weekly ^• rate of weight ^■ loss over the 12 treatment weeks with AOD9604 at doses of 1 mg (-0.22 kg/week, P < 0.0001), 20 mg (-0.13 kg/week, P = 0.0451), or 30 mg (- 0.15 kg/week, P = 0.0107) was statistically significant compared to placebo (-0.07 kg/week). Repeated measures analysis of the mean weekly rate of reduction in abdominal circumference was similarly statistically significant at all doses of AOD9604 compared to placebo. There were trends to improvements in lipid profiles and a lower incidence of diabetes in the AOD9604 as compared to the placebo group.

Conclusion: Daily oral administration of AOD9604 promotes weight and waistline reduction. The dose response is complex, with the lowest dose of the trial giving the greatest effect .

Claims

CLAIMS :

1. Use of a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone, in a method of modulating lipid metabolism in a human, which method comprises administering the peptide orally to the human at a dose of 0.01-2 mg per day.

2. A method of modulating lipid metabolism comprising administering to a human in need thereof an effective amount of a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone, wherein the peptide is administered at a dose of 0.01 - 2 mg per day

3. A dosage unit for use according to claim 1 or in the method of claim 2, the dosage unit comprising 0.01 - 2mg of a peptide corresponding to human growth hormone (hGH) 177-191 or a lipid metabolising subfragment or analogue thereof containing a disulphide bond, with the proviso that the peptide is not intact, full length growth hormone .

4. A use according to claim 1, method according to claim 2 or dosage unit according to claim 3 in which the dose of peptide is in the region of 0.05-1.50 mg per day.

5. A use according to claim 1, method according to claim 2 or dosage unit according to claim 3 in which the dose of peptide is in the region of 0.25-1.25 mg per day.

6. A use according to claim 1, method according to claim 2 or dosage unit according to claim 3 in which the dose of peptide is in the region of 0.25-0.75 mg per day.

7. A use according to claim 1, method according to claim 2 or dosage unit according to claim 3 in which the dose of peptide 0.25-0.50 mg per day.

8. A use according to claim 1 or method according to claim 2 in which the peptide is administered to a human undergoing calorie restriction, at least for an initial period.

9. A kit comprising a peptide corresponding to amino acid residues 177-191 of the human growth hormone sequence or a functional analogue or variant thereof for administering at a dose of 0.01 to 2.00 mg per day, and information on lifestyle modification capable of enhancing the effect of the peptide in weight reduction.

10. Use according to claim 1 or a method according to claim 2, in which modulation of lipid metabolism cases a reduction in body weight .

11. A use or method according to claim 10, in which the reduction of body weight is for treating obesity or preventing or alleviating conditions related to obesity.

12. A method of treating glucose intolerance in an animal, comprising administering to the animal a therapeutically effective amount of a peptide corresponding to amino acid residues 177-191 of the human growth hormone sequence or a functional analogue or variant thereof .

13. A method of improving lipid profiles in an animal, comprising administering to the animal a therapeutically effective amount of a peptide corresponding to amino acid residues 177-191 of the human growth hormone sequence or a functional analogue or variant thereof.

14. Use of the method according to claim 12 for preventing or treating diabetes or metabolic syndrome.

15. Use of the method according to claim 13 for preventing or treating a disorder involving abnormal lipid profiles .

16. Use according to claim 15, in which the disorder is hypercholesteraemia or metabolic syndrome.

17. A method, use or dosage unit according to any preceding claim, in which the peptide is able to reduce lipogenic activity; and, or able to stimulate lipolysis.

18. A method, use or dosage unit according to any preceding claim, in which the peptide comprises at least the disulphide-bonded loop of a mammalian growth hormone.

19. A method, use or dosage unit according to any preceding claim, in which the peptide is selected from:

RefNo. STRUCTURE 9502 Leu Arg lie VaI GIn Pen Arg Ser VaI GIu GIy Ser Pen GIy Phe

9405 CH3CO- Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe 9410 H - Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe 9404 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe - CONH2 9407 Leu Arg He VaI GIn Cys Lys Ser VaI GIu GIy Ser Cys GIy Phe 9408 Leu Arg He VaI GIn Cys Lys Ser VaI GIu GIy Ser Cys GIy Phe

(amide bond)

9604 Tyr Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe

9605 Lys Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe

9618 Lys Lys Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe 9607 Ala Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe

9606 Leu Lys He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe 9608 Leu Arg Ala VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe 9403 Leu Arg Lys VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe

9609 Leu Arg He Ala GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe

9610 Leu Arg He VaI Ala Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe 9612 Leu Arg lie VaI GIn Cys Arg Ala VaI GIu GIy Ser Cys GIy Phe 9613 Leu Arg He VaI GIn Cys Arg Ser Ala GIu GIy Ser Cys GIy Phe

9615 Leu Arg He VaI GIn Cys Arg Ser VaI GIu Ala Ser Cys GIy Phe

9616 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ala Cys GIy Phe 9602 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys Ala Phe 9501 Leu Arg He VaI GIn Cys Arg Ser VaI GIu D-AIa Ser Cys D-AIa Phe 9601 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Ala

20. A method, use or dosage unit according to claim 18, in which the peptide comprises amino acids 182-189 of human growth hormone (hGH 182-189) .

21. A method, use or dosage unit according to claim 18, in which the peptide comprises amino acids 177-191 of human growth hormone (hGH 177-191) .

22. A method, use or dosage unit according to claim 18, in which the peptide is Tyr-hGH 177-191, and has the sequence Tyr Leu Arg lie VaI Gin Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe .

23. A method or use according to any preceding claim, in which the peptide is conjugated to a fusion partner.

24. A method or use according to any preceding claim, in which the peptide is administered as a pharmaceutical composition.

25. A method or use according to claim 24, in which the pharmaceutical composition comprises a pharmaceutically acceptable carrier.