WO2007068039A1

WO2007068039A1 - Treatment of weight gain in estrogen deficient mammals

Info

Publication number: WO2007068039A1
Application number: PCT/AU2006/001879
Authority: WO
Inventors: Christopher Ian Belyea
Original assignee: Metabolic Pharmaceuticals Limited
Priority date: 2005-12-12
Filing date: 2006-12-12
Publication date: 2007-06-21

Abstract

The invention provides a method of treating weight gain in an estrogen deficient mammal, comprising administering to the mammal a therapeutically effective amount of a peptide, in which the peptide is amino acid residues 177-191 of the human growth hormone sequence or a functional analogue or variant thereof.

Description

TREATMENT OF WEIGHT GAIN IN ESTROGEN DEFICIENT MAMMALS

This invention relates to methods for the treatment or prevention of weight gain in estrogen deficient mammals. In particular, the invention relates to methods for the treatment or prevention of weight gain in post-menopausal women.

BACKGROUND All references, including any patents or patent applications, cited in this specification are hereby incorporated by reference to enable full understanding of the invention. Nevertheless, such references are not to be read as constituting an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

Menopause occurs when a woman stops ovulating and menstruation ceases. Estrogen is the female sex hormone responsible for ovulation. Estrogen has been found to influence body fat distribution.

The results of a number of medical studies indicate that menopause is associated with a progressive increase in weight, and a redistribution of body fat to the abdominal region. Animal studies have shown that a lack of estrogen leads to excessive weight gain, although the exact mechanisms are not yet understood.

Women of childbearing age tend to store fat in the lower body while men and postmenopausal women store fat around the abdomen. Although being overweight is a risk factor alone, the redistribution of fat tissue further increases the risk of cardiovascular disease and diabetes. Postmenopausal women are at increased risk of coronary heart disease (CHD) , partly because of the decline in estrogen production and concurrent elevations in total and low-density lipoprotein (LDL) cholesterol level. Obesity, weight gain, and adverse changes in body fat distribution and composition are part of this phenomenon. Moreover, the rise in LDL cholesterol levels and onset of other CHD risk factors (e.g. high blood pressure, high total cholesterol and triglyceride levels, insulin resistance) is directly influenced by weight gain. The greatest estrogen deficiencies occur when the ovaries have been removed or compromised by surgery or disease. Accordingly, many of these women experience • significant weight problems. This, appears to be related to the incurred deficiency of both testosterone and estrogen. It is an aim of a preferred embodiment of the present invention to provide a method for- the treatment or prevention of the weight gain associated with estrogen deficiency.

SUMMARY

According to the present invention in a first aspect, there is provided a method of treating weight gain in estrogen deficient mammals, comprising orally administering to the mammal a therapeutically effective amount of a C terminal growth hormone peptide at a dose of 0.5mg/kg/day or less.

In Australian patent No. 693478 (US Pat. No. 5,869,452) by Monash University, we described the use of a peptide derived from the 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, a synthetic peptide corresponding to amino acid residues 177-191 of the human growth hormone sequence (hereinafter referred to as hGH 177-191) was found to reduce body weight gain and adipose tissue mass in a model system for obesity, the C57B1/6J (Ob/Ob) mouse. A subsequent application, PCT/AU98/00724 (US Pat. No. 6,737,407) by Metabolic Pharmaceuticals Ltd, discloses analogues of the hGH177-l9l peptide which share this activity. The entire disclosures of US 5,869,452 and US 6,737,407 are incorporated herein by this reference.

Studies of a particular analogue of hGH177-191 - AOD9604 (Tyr-hgH 177-191) have now been performed in a post-menopausal aged rat model. Whist this peptide is known to affect lipid metabolism in an obesity model it was not expected that the peptide would have an effect on such a model. This model is not an obesity model as the type of weight gain associated with estrogen deficiency is different from obesity models. As stated above, the weight gain in estrogen deficient animals is caused by the lack of estrogen and results in a specific form of weight gain to the abdominal region. Therefore, until tested, it was not proposed or suggested that AOD9604 would prevent weight gain in estrogen deficient animals. Additionally, previous unpublished studies in the same rat model at higher doses of lmg/kg and higher injected subcutaneously had shown no effect on the reduction of weight gain associated with the estrogen deficiency. Surprisingly, it has now been found that, at effective doses in this model, orally administered AOD9604 reduce the weight gain associated with estrogen deficiency.

The inventor has recognized that a particular C- terminal fragment peptide of human growth hormone (AOD9604) has effects on reducing weight gain associated with estrogen deficiency. He therefore proposes that all of the C-terminal fragment peptides of human growth hormone previously shown to have ability to modulate lipid metabolism will, have an effect similar to AOD9604 on estrogen deficiency weight gain. Accordingly the inventor proposes that all of the C-terminal fragment peptides of human growth hormone previously shown in US Pat. No. 6,737,407 and US 5,869,452 to have the ability to modulate lipid metabolism can be used to treat or prevent estrogen deficiency weight gain in accordance with the present invention.

According to a second aspect, the invention provides the use of a _C terminal growth hormone fragment peptide in the manufacture of a medicament for use in treating weight gain in estrogen deficient mammals, wherein the medicament is for administering orally to provide a dose of peptide of 0.5mg/kg/day or less. 5 In a third aspect, the present invention provides a pharmaceutical agent for use in treating weight gain in estrogen deficient mammals, which agent comprises a C terminal growth hormone fragment and a pharmaceutically acceptable carrier and the agent is for administering

10 orally to provide a dose of peptide of 0.5mg/kg/day or less.

The formulation according to the third aspect may, in a preferred embodiment, . further comprise one or more additional weight loss agents.

15 The estrogen deficiencies that may be treated by the method of the first aspect or the medicament of the second aspect include deficiencies resulting from a postmenopausal state or surgery or disease.

Preferably, the weight gain is in post-menopausal

20 women .

BRIEF DESCRIPTION OF THE FIGURES

Figure 1: Experimental design summary. Figure 2: Percentage of weight gained over the 25 12-week treatment period examining the effect of AOD on the OVX model .

DETAILED DESCRIPTION

For the purposes of this specification, the term

30 "C-terminal growth hormone fragment peptide" is to be understood to mean a polypeptide fragment from the carboxy-terminal region of the amino acid sequence of a mammalian growth hormone, which has one or more of the following biological activities:

35. (a) ability to reduce lipogenic activity; and (b) ability to stimulate lipolysis.

Preferably the growth hormone fragment peptide has the ability to stimulate the activity of hormone- sensitive lipase, a key enzyme in lipolysis, and to_, inhibit acetyl CoA carboxylase, a key enzyme in lipogenesis . . Preferably the growth hormone fragment peptide comprises at least the disulphide-bonded loop of a mammalian growth hormone.

The term "growth hormone fragment peptide" also encompasses peptides which are analogues of the native carboxy-terminal sequences of mammalian growth hormones, provided that the analogue retains one or more of the biological activities referred to above. 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. Preferably such analogues include a disulphide bond which confers a cyclic configuration on the peptide. In particular, all of the active peptides disclosed in US Pat. No. 5,869,452 and US Pat. No.

6 ,737 , 407 are to be understood to be within the scope of this invention.

Preferably the C-terminal growth hormone fragment peptide comprises amino acids 182-189 (hGH 182- 189) , more preferably amino acid 177-191 of human growth hormone (hGH 177-191) . Even more preferably the C-terminal . growth hormone fragment peptide is the analogue AOD9604, Tyr-hGH 177-191. However, it will be clearly understood that the invention is also applicable to growth hormone fragment peptides derived from 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. The peptide may be an analogue of the C-terminal growth hormone fragment peptide. The analogue may be derived by elongation, insertion, deletion or substitution of amino acids in, or chemical modification of, or introduction of a cyclic amide bond between the side chains of amino acids of, the native carboxyl sequence. Amino acid insertional analogues include amino and/or carboxylic terminal fusions as well as intra-sequence insertions of single or multiple {for example, up to 10, preferably up to 5) amino acids. Insertional amino acid sequence analogues are those in which one or more amino acid residues are introduced into a predetermined site in the protein although random insertion is also possible with suitable screening of the resulting product. Deletional analogues are characterised by the removal of one or more (for example up to 5, preferably up to 3) amino acids from the sequence. Substitutional amino acid analogues are those in which at least one amino acid residue in the sequence, preferably one or two, has been replaced by another of the twenty standard amino acids or by a non standard amino acid. Chemical modification of the native carboxyl -terminal and/or amidation of the carboxyl -terminus and/or sde chain cyclisation of the native carboxyl-terminal sequence. Analogues of the native carboxyl -terminal sequences of human growth hormone or growth hormone of other animal species which in particular retain the same conformation, structure and charge characteristics as the native carboxyl-terminal sequences can be expected to exhibit the same or similar biological activity as the native sequences, in particular in^* the ability to reduce body weight gain and adipose tissue mass in an obese animal .

Whilst the following detailed description refers specifically to analogues of hGH 177-191, it is to be understood that this invention extends to similar analogues of corresponding peptides of non-human mammalian growth hormone as described above.

Peptides comprising amino acid residues 177-191 of native human growth hormone (hGH 177-191) include the following sequence (Ref No. 9401):

Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Phe (SEQ ID NO: 1)

Such a native peptide may be in cyclic disulfide form, and may comprise an organic or inorganic acid addition salt.

Analogues of the hGH 177-191 peptide may be obtained by deletion or insertion of one or more amino acid residues at any position along the native sequence, with the retention of anti-obesity properties as described above. Preferably, the analogue is in a cyclic configuration.

Alternatively, analogues of the hGH 177-191 peptide may be obtained by substitution of one or more amino acid residues at any position along the native sequence .

Screening for in vitro and in vivo activity using alanine substitution scanning and other methods reported herein has revealed positions and relationships between amino acids in hGH 177-191 which are important in the bioactiyity as described above. Preferred analogues of the current invention include peptide analogues of hGH 177-191 wherein

(i) amino acids at positions 182 and 189 of hGH are joined by a bond to promote a cyclic conformation; and/or

(ii) amino acids at positions 183 and 186 of hGH are joined by a salt bridge or a covalent bond.

The bond between amino acids at 182 and 189 of hGH may be a disulfide bond, in which case the amino acids at positions 182 and 189 of hGH may preferably be L- or D-

Cys or Pen. When the amino acids at positions 183 and 186 of hGH are joined by a salt bridge, these amino acids may preferably be (X and Y) or (Y and X), respectively, where:

X is a positively charged amino acid such as L- or D- Arg, Lys or Orn and

Y is a negatively charged amino acid such as L- or D- Asp or GIu.

When the amino acids at positions 183 and 186 of hGH are. joined by a covalent bond, that bond may be an amide bond in which case these amino acids may preferably be (X and Y) or (Y and X) , respectively, where:

X is selected from the group consisting of L- or D- Lys and Orn and Y is selected from the group consisting of L- or D- Asp and GIu.

The amino acid at position 178 of hGH is preferably a positively charged amino acid such as L- or D- Arg, Lys or Orn. . Analogues may also be obtained by elongation of the native hGH 177-191 peptide sequence at one or both ends of the amino acid residues, for example with one or more hydrophilic amino acids to increase solubility in aqueous solution. Such analogues include the following sequence, preferably in cyclic disulphide form:

X^n-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys- Gly-Phe-X²n (SEQ ID NO: 2)

wherein X¹In and X²m are each is selected from the group consisting of L- or D-Arg, His and Lys, and m and n are each 0, 1, 2 or 3 with the provision that at least m or n is.l. [Throughout this specification, elements which are underlined denote differences from the native hGH 177-191 sequence, and unless otherwise stated, amino acids at positions corresponding to 182 and 189 are joined by a disulfide bond.] One elongation analogue not elongated with a hydrophilic amino acid but nonetheless exhibiting especially enhanced anti-obesity properties is the following (Ref No. 9604) :

Tyr-Leu-Arg-Ile-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys- Gly-Phe (SEQ ID NO: 3)

Analogues may also be obtained by chemical modification of the native hGH 177-191 peptide sequence. Such analogues include the sequence:

γ^α-Leu-Arg-lie-VaI-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-GIy- Phe (SEQ ID NO: 4)

wherein Y¹ is selected from the group consisting of the desamino form (H), acetyl (CH. sub.3CO--) and other acyl groups ; or the sequence :

Leu-Arg-IIe-VaI-GIn-Cys-Arg-Ser-VaI-GIu-GIy-Ser-Cys-GIy- Phe-Y² (SEQ ID NO: 5)

where Y² is selected from the group consisting of -C0NH2 and alkyl amide groups.

Specific hGH 177-191 analogues obtained by substitution of amino acids, by elongation, by chemical modification, or by introduction of a cyclic amide bond between side chains of amino acids, of the native hGH 177- 191 peptide sequence, and which exhibit anti-obesity properties and are expected to reduce weight gain associated with estrogen deficiency include the following:

Ref No. ^' STRUCTURE

9502 Leu Arg He VaI GIn Pen Arg Ser VaI GIu GIy Ser Pen GIy Phe SEQ ID N0:6 9405 CH3CO- Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO.-7

9410 H - Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:8 9404 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe - CONH₂ SEQ ID NO:9

9407 Leu Arg Ue VaI GIn Cys Lys Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 10

9408 Leu Arg He VaI Gin Cys Lys Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NOl 1 (amide bond)

9604 Tyr Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:3

9605 Lys Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:12 9618 Lys Lys Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 13

9607 Ala Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 14

9606 Leu Lys He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 15

9608 Leu Arg Ala VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 16 9403 Leu Arg Lys VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 17

9609 Leu Arg He Ala GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 18

9610 Leu Arg He VaI Ala Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe SEQ ID NO: 19

9612 Leu Arg He VaI GIn Cys Arg Ala VaI GIu GIy Ser Cys GIy Phe SEQ ID NO:20

9613 Leu Arg He VaI GIn Cys Arg Ser Ala GIu GIy Ser Cys GIy Phe SEQ ID N0:21 9615 Leu Arg He VaI GIn Cys Arg Ser VaI GIu Ala Ser Cys GIy Phe SEQ ID NO:22

9616 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ala Cys GIy Phe SEQ ID NO:23 9602 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys Ala Phe SEQ ID NO:24 9501 Leu Arg He VaI GIn Cys Arg Ser VaI GIu D-AIa Ser Cys D-AIa Phe SEQ ID NO:25 9601 Leu Arg He VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Ala SEQ ID NO:26

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

GIy = Glycine ; lie = Isoleucine ;

Glu = 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, p¹ -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.

An amino acid sequence variant of the growth hormone fragment defined above is included within the scope of the invention, provided that it is functionally active. As used herein, the terms "functionally active" and "functional activity" in reference to the growth hormone fragment means, that the growth hormone fragment is able to prevent or weight gain in estrogen deficient, mammals.

Amino acid 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 treat or prevent weight gain in estrogen deficient mammals. Particularly, the test to determine whether a variant is functionally active is whether the variant reduces weight gain in an OVX rat model (see Example 1 for further details) .

If such substitutions do not result in a change in 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. ^■ . 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,- gln; lys; arg arg

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

Leu (L) norleucineϊ; 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) giy 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

Where appropriate, the analogues described above may comprise an organic or inorganic acid addition salt.

The growth hormone fragment may also be conjugated to a fusion partner to enable easier biosynthesis and/or delivery. It may be incorporated in a conventional pharmaceutical composition, or may be present in a genetically-modified food, such as disclosed in WO 01/33997.

The term "effective amount" as used herein means an amount of the peptide sufficient to attain the desired effect in the treatment of estrogen deficiency weight gain in the animal, but not so large an amount as to cause serious side effects or adverse reactions.

Previous unpublished results showed no effect in the same rat model of doses of lmg/kg and higher of AOD 9064 injected subcutaneously . The inventor has studied doses of 0.5 and 0.25mg/kg/day and found^' these to be active at reducing the estrogen deficiency weight gain. It is expected that a dose as low as 0.0001mg/kg/day could also be active in reducing weight gain associated with estrogen deficiency. According the therapeutically effective dose as used herein is a dose of 0. OOlmg/kg/day to 0.5mg/kg/day, 0.01 to 0.5mg/kg/day or 0.1 to 0.5mg/kg/day. The dose is particularly, 0.0001, 0.0002,

0.0003, 0.0004, 0.0005, 0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.002, 0.003, 0.004, 0.005, 0..006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05, 0.06, 0.07, 0.08, 0.09,. 0.1, 0.11, 0.12, 0.13, 0.14, 0.15, 0.16, 0.17, 0.18, 0.19, 0.2, 0.21, 0.22, 0.23, 0.24, 0.25, 0.26, 0.27, 0.28, 0.29, 0.3, 0.31, 0.32, 0.32, 0.34, 0.35, 0.36, 0.37, 0.38, 0.39, 0.4, 0.41, 0.42, 0.43, 0.44, 0.45, 0.46, 0.47, 0.48, 0.49 or 0.5. A preferred dose is 0. lmg/kg/day, 0.25mg/kg/day or 0.5mg/kg/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. lmg to 50mg of the active compound with an appropriate and convenient amount of carrier material , which may vary from about 5 to 99 percent of the total composition.

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, rat;e of excretion, drug combination and the severity of the particular disease undergoing therapy.

As used herein, the terms "therapeutically effective amount" and "therapeutic amount" are synonymous, and mean an amount of a growth hormone fragment 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 growth hormone fragment.

The level of a therapeutically effective amount of peptide will be at the discretion of the attendant physician or veterinarian, and will depend on the nature and state of the condition to be treated, the age and • general state of health of the subject to be treated, the route of administration, and any previous treatment which may have been administered.

Preferably the growth hormone fragment is administered in a pharmaceutical composition together with a pharmaceutically acceptable carrier for administration. The compounds and compositions of the invention may be administered by any suitable oral route, and the person skilled in the art will readily be able to determine the most suitable route and dose for the condition to be treated.

Orally as used herein extends to any administration of delivery to the GI tract and includes administration directly to. the oropharyngeal cavity, and administration by mouth in which the actual absorption of the peptide takes place in the GI tract including the stomach, small intestine or large intestine. Oral administration as used herein encompasses sublingual administration (administration by application under the tongue of the recipient, representing one form of administration via the oropharyngeal cavity) and buccal 5 administration (administration of a dosage form between the teeth and the cheek of the recipient) .

The growth hormone fragment may be administered orally, sublingually, buccally, intranasally, or by ■ inhalation, in dosage unit formulations containing 10 conventional non-toxic pharmaceutically acceptable carriers, adjuvants, and vehicles.

The mammal may be a human, or- may be a domestic or companion animal . While it is particularly contemplated that the compounds of the invention are suitable for use 15 in medical treatment of humans, they are also applicable to veterinary treatment, including treatment of companion animals such as dogs and cats, and domestic animals such as horses, cattle and sheep, or zoo animals such as non- human primates, felids, canids, bovids, and ungulates. 20_. Preferably the mammal is a human. The human may be a child or an adult. Preferably the mammal is a post menopausal female.

Methods and pharmaceutical carriers for preparation of pharmaceutical compositions are well known 25 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. 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 weight gain. "Treating" as used herein covers any 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.

"Estrogen deficiency" as used herein applies to a mammal having less estrogen circulating in their blood than a typical healthy female of the same species at reproductive age. All post menopausal women are deemed to have "estrogen deficiencies" .

Estrogens or one or more estrogen mimicking compounds used in Estrogen Replacement may be included in the formulation. Examples of Estrogen-mimicking compounds are phytoestrogens, like genistein, lignans or coumerans or pharmaceutical preparations Iikel7p-estradiol , esterified estrogens, estrone sulfate, conjugated equine Estrogen, and ethinylestradiol . For the phytoestrogens the amount of these compounds is 5-100mg per daily dose. For the pharmaceutical preparations the active amount is defined by the instructions of the manufactures. The formulations according to the third aspect of the invention can further contain other sources of energy, such as fats and carbohydrates, proteins, vitamins, minerals, fibers, flavors, preservatives, colorants, sweeteners, etc.

Any chemically compatible combination of pharmaceutically-active agents is within the scope of the invention, provided that the combination does not eliminate the activity of the growth hormone fragment of this invention.

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.

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.

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. .

The invention will now be described by way of reference only to the following non-limiting example. EXAMPLE 1

Materials and Methods The purpose of this study is to determine the effect of AOD 9604 (AOD) on the weight gain in ovariectomized rats. The experiment thus considers Sham and OVX models and each model contains control (no dose) , low dose (0.25mg/kg/day) and high dose (0.5mg/kg/day) groups as summarized in Table 1.

After a three-week acclimatization period, ovariectomy was performed. One day following the operation, the 12-week dosing period commenced. Dosages were administered by oral gavage with a metal gavage needle.

The rats were individually weighed one day before the AOD treatment commenced and prior to the ovariectomy operation. All measurements were performed on an electronic scale. The rats were weighed a second time immediately after euthanization. From these two weights, a percent in weight gain over the 12-week treatment period was calculated.

Results The OVX groups subjected to . low and high dose

AOD treatment gained significantly (p<0.001) less weight than the OVX Control group (Fig. 2) .

Conclusions

Body weights, of the Sham and OVX control groups reflect those expected in the ovariectomized rat model.

The OVX Controls gained approximately 20% of their initial body weight where as the Sham Controls gained a negligible amount. Both low and high doses of AOD caused a reduction of approximately 50% of the enhanced weight gain in the ovariectomized model. Discussion

We expected that AOD would not prevent the weight gain resulting from the ovariectomy because a previous study at a higher dose of l.0mg/kg/day had shown no effect in preventing the weight gain. The results of this study suggest our expectation was incorrect and that AOD does have a highly significant effect on reducing the weight gain associated with estrogen deficiency.

Claims

CLAIMS :

1. A method of treating weight gain in an estrogen deficient mammal, comprising orally administering to the mammal a therapeutically effective amount of a peptide at a dose of 0.5mg/kg body weight or less, in which the peptide is amino acid residues 177-191 of the human growth hormone sequence or a functional analogue or variant thereof .

2. A method according to claim 1, in which the analogue has one or more of the following biological activities:

(c) ability to reduce lipogenic activity; or

(d) ability to stimulate lipolysis.

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

4. A method according to any preceding claim, in which the peptide is selected from one of the sequences:

RefNo. STRUCTURE

9502 Leu Arg He 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 lie VaI GIn Cys Lys Ser VaI GIu GIy Ser Cys GIy Phe 9408 Leu Arg He VaI GIn Cys Lvs 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 AIa Cys Arg Ser VaI GIu GIy Ser Cys GIy Phe 9612 Leu Arg He 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 lie Va] GIn Cys Arg Ser VaI GIu Ala Ser Cys GIy Phe

9616 Leu Arg lie VaI GIn Cys Arg Ser VaI GIu GIy Ala Cys GIy Phe 9602 Leu Arg lie 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 lie VaI GIn Cys Arg Ser VaI GIu GIy Ser Cys GIy Ala

5. A method according to claim 1, in which the peptide comprises amino acids 182-189 of human growth hormone (hGH 182-189) .

6. A method according to claim 1, in which the peptide comprises amino acids 177-191 of human growth hormone (hGH 177-191) .

7. A method according to claim 1, 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

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

9. A method according to- any preceding claim, in which the peptide is administered as a pharmaceutical composition.

10. A method according to claim 9, in which the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

11. A method according to claim 9 or claim 10, in which the pharmaceutical composition further comprises a further agent active in treating weight gain.

12. A method according to claim 10, in which the composition further comprises an energy source.

13. A method according to any preceding claim, in which the peptide is administered orally, sublingually, buccally, intranasally, or by inhalation.

14. A method according to any one of claims 1 to 13, in which the peptide is administered orally, in the form of a genetically-modified food.

15. A method according to any preceding claims, in which the estrogen deficient mammal is menopausal, post menopausal, has had an ovariectomy, or has damaged ovaries.

16. A method according to any preceding claim in which the mammal is also growth hormone-deficient.

17. A method according to any preceding claim, in which the mammal is a human.

18. Use of a C terminal growth hormone fragment in the manufacture of a medicament for use in treating or preventing weight gain in an estrogen deficient mammal.

19. Use according to claim 18, in which the medicament also comprises further comprises a further agent active in treating or preventing weight gain in an estrogen deficient mammal.

20. A method according to any preceding claim in which the dose is 0.01 to 0.5mg/kg/day .