Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/575—Hormones
  • C07K14/61—Growth hormone [GH], i.e. somatotropin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P3/00—Drugs for disorders of the metabolism
  • A61P3/04—Anorexiants; Antiobesity agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/06—Drugs for disorders of the endocrine system of the anterior pituitary hormones, e.g. TSH, ACTH, FSH, LH, PRL, GH
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K38/00—Medicinal preparations containing peptides

Definitions

• This invention relates to the treatment of obesity in animals.
• the invention relates to the treatment of obesity in humans, although it is to be understood that the present invention also extends to the treatment of obesity in non- human mammals, for example, for the improvement of meat qualities in farm animals used in food production.
• hGH human growth hormone
• hGH globular protein with a molecular weight of 22,000 daltons (22-KD) and consists of 191 amino acid residues in a single-chain, folded by 2 disulphide bonds with a small loop at the carboxyl terminus between residues 182 and 189.
• Recent crystallographic studies also show that the hGH molecule contains four anti-parallel ⁇ -helices which are arranged in a left-twisted, tightly-packed helical bundle 1 .
• the concept that there are discrete functional domains within the hGH molecule responsible for specific metabolic actions of the hormone is generally accepted.
• the amino-terminus has been identified as the functional domain responsible for the insulin-like actions of the hGH molecule 23 .
• the results of the latter effect may include both a stimulation of lipolysis and an inhibition of lipogenesis.
• the anti-lipogenic effect of hGH has been substantiated with the demonstrations of the decrease of the expression of glucose transporter GLUT 4 in adipocytes 18 , the inhibition of the activity of acetyl-CoA carboxylase in adipose tissues 19,20 and the reduction of glucose incorporation into lipid in both isolated cells and tissues 21,22 .
• the present invention provides a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone.
• the peptide may comprise an analogue of the carboxyl-terminal sequence of human growth hormone or the growth hormone of a non-human mammalian species.
• the carboxyl- terminal sequence of growth hormone includes a bioactive lipid metabolic domain.
• the peptide comprises an analogue of the carboxyl-terminal sequence of human growth hormone containing amino acid residues 177-191 or a corresponding sequence of a non-human mammalian growth hormone.
• the analogue may be obtained by insertion, deletion or substitution of amino acids in, or chemical modification of, the native carboxyl-terminal sequence of human growth hormone or the growth hormone of a non-human mammalian species.
• the present invention provides a method for treating obesity comprising administering an effective amount of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone, as described above.
• the treatment may be administered to any animal, including humans.
• the present invention also provides a pharmaceutical composition for use in the treatment of obesity comprising an effective amount of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone as described above, together with one or more pharmaceutically acceptable carriers and/or diluents.
• the present invention provides use of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone as describe dabove, in the manufacture of a pharmaceutical composition for the treatment of obesity in an animal.
• a method for the treatment of obesity in an animal which comprises administering to the animal an effective amount of a peptide which comprises an analogue of the carboxyl- terminal sequence of a growth hormone.
• the animal is a human although the invention also extends to the treatment of non-human mammals.
• the peptide comprises an analogue of the carboxyl-terminal sequence of human growth hormone containing amino acid residues 177-191 (hereinafter referred to as hGH 177-191).
• the peptide may comprise an analogue of the carboxyl-terminal sequence of the growth hormone of other non-human mammalian species, such as bovine, porcine, ovine, equine, feline or canine growth hormone, corresponding to the hGH 177-191 peptide.
• the term "obesity" is used to include both excess body weight and excess adipose tissue mass in the animal, and correspondingly the references to treatment of obesity include both reduction of body weight gain and reduction of adipose tissue mass of the obese animal.
• the expected outcome of any treatment of obesity is the reduction of body weight, body adipose tissue mass in particular.
• the reduction of body adipose tissue mass is directly regulated by two biochemical processes - lipogenesis (fat-production) and lipolysis (fat-reduction) - and it is generally understood that these biochemical processes are controlled by key metabolic enzymes, specifically the fat-reducing key enzyme (hormone-sensitive lipase) and the fat-producing key enzyme (acetyl CoA carboxylase).
• hGH 177-191 is effective in stimulating the fat-reducing key enzyme, hormone-sensitive lipase, and in inhibiting the fat-producing key enzyme, acetyl CoA carboxylase. This is further supported by data showing that in the presence of hGH 177-191, fat utilization is accelerated while fat production is reduced, as measured by metabolic end-products in vitro as well as in vivo. In addition, the mechanism of these molecular actions has been established as resulting from the activation of the production of the cellular second-messenger, diacylglycerol.
• the present invention extends to the use of peptides which are analogues of longer amino acid sequences than the particular sequence 177-191 of growth hormone, for example analogues of the sequence 172- 191 of human growth hormone or the corresponding sequence of growth hormone of other non-human mammalian species.
• the concept of correspondence in amino acid sequences between species is well known in the biological sciences and is determined by aligning comparable sequences (including if necessary theoretical deletions) to match isofunctional or isostereo amino acids thereby maximising homology.
• the published corresponding sequences of the C-terminus region of the growth hormone of selected mammals are tabulated below 26 , using standard single letter notation:
• FRKDMDKVETFLRIVQCR human FRKDMDKVETFLRIVQCR.
• SVEGSCGF human variant FRKDMDKVETFLRIVQCR SVEGSCGF human CS FRKDMDKVETFLRMVQCR.
• the present invention extends to the use of peptides which are analogues of the native carboxyl-terminal sequences of human growth hormone or growth hormone of other animal species, and which are derived from natural or synthetic (including recombinant) sources, provided always that the resulting peptide retains the biological activity of the native carboxyl-terminal sequence described herein, namely the ability to reduce body weight gain and adipose tissue mass in an obese animal.
• these analogues may exhibit a cyclic configuration, which may be induced by a disulfide bond.
• the analogues of the present invention 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-terminal 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 primary protein amino acids, or by a non- protein amino acid.
• Chemical modifications of the native carboxyl-terminal sequence include the acetylation of the amino-terminus and/or amidation of the carboxyl- terminus and/or side 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.
• Peptides comprising amino acid residues 177-191 of native human growth hormone include the following sequence (Ref No. 9401): Leu-Arg-lle-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe
• 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.
• the analogue is in a cyclic configuration.
• 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.
• analogue of the current invention include peptide analogues of hGH 177-191 wherein
• amino acids at positions 182 and 189 of hGH are joined by a bond to promote a cyclic conformation
• 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.
• 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 Glu.
• 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 Glu.
• 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: Xim-Leu-Arg-lle-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe-Xfri (SEQ ID NO: 2) wherein X 1 and X 2 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 1. [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
• One elongation analogue not elongated with a hydrophilic amino acid but nonetheless exhibiting especially enhanced anti-obesity properties is the following (Ref No. 9604): l r-Leu-Arg-lle-Val-Gln-Cys-Arg-Ser-Val-Glu-Gly-Ser-Cys-Gly-Phe.
• Analogues may also be obtained by chemical modification of the native hGH 177-191 peptide sequence.
• Such analogues include the sequence:
• Y 1 is selected from the group consisting of the desamino form (H), acetyl (CH 3 CO-) and other acyl groups; or the sequence:
• 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, include the following:
• Gly Glycine He Isoleucine; Glu Glutamic Acid; Phe Phenylalanine; Cys Cysteine; Arg Arginine; Gin Glutamine; Leu Leucine; Ser Serine; Val Valine; Lys Lysine; Ala Alanine; Asp Aspartic acid; His Histidine; Orn Ornithine; Tyr Tyrosine; Pen Penicillamine ( ⁇ , ⁇ '-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.
• analogues described above may comprise an organic or inorganic acid addition salt.
• an effective amount means an amount of the peptide sufficient to attain the desired effect in the treatment of obesity in the animal, but not so large an amount as to cause serious side effects or adverse reactions.
• the present invention provides the use of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone as described above, in the treatment of obesity in an animal or in the manufacture of a pharmaceutical composition for the treatment of obesity in an animal.
• the present invention provides a pharmaceutical composition for use in the treatment of obesity in an animal, comprising an effective amount of a peptide which comprises an analogue of the carboxyl-terminal sequence of a growth hormone as described above, together with one or more pharmaceutically acceptable carriers and/or diluents.
• the peptide which is the active ingredient of the pharmaceutical composition of this aspect of the invention exhibits advantageous therapeutic activity in the treatment of obesity in an animal when administered in an amount appropriate to the particular case. For example, from about 0.5 ⁇ g to about 20 mg per kilogram of body weight per day may be administered. Dosage regimens may be adjusted to provide the optimum prophylactic or therapeutic response. For example, one or more divided doses may be administered daily, weekly, monthly or in other suitable time intervals or the dose may be proportionally reduced as indicated by the exigencies of the clinical situation.
• the active ingredient may be administered in any convenient manner such as by the oral, parenteral (including intraperitoneal, intravenous, subcutaneous, intramuscular and intramedullary injection), intranasal, intradermal or suppository routes or by implanting (eg using slow release devices).
• oral administration is preferred, however parenteral administration is also quite convenient.
• the active ingredient may be required to be coated in a material that protects said ingredient from the action of enzymes, acids and other natural conditions which may inactivate the said ingredient.
• low lipophilicity of the ingredient may allow it to be destroyed in the gastrointestinal tract by enzymes capable of cleaving peptide bonds and in the stomach by acid hydrolysis.
• the active ingredient may be coated by, or administered with, a material to prevent its inactivation.
• the active ingredient may also be administered in dispersions prepared in glycerol, liquid polyethylene glycols, and/or mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations will usually contain a preservative to prevent the growth of microorganisms.
• the pharmaceutical forms suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and vegetable oils.
• the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
• the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thiomorosal, and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by, for example, the use in the compositions of agents delaying absorption.
• Sterile injectable solutions are prepared by incorporating the active ingredient in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by filtered sterilisation.
• dispersions are prepared by incorporating the sterilised active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
• the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from previously sterile- filtered solution thereof.
• the composition may be orally administered, for example, with an inert diluent or with an assimilable edible carrier, or it may be enclosed in hard or soft shell gelatin capsule, or it may be compressed into tablets, or it may be incorporated directly with the food of the diet.
• the active ingredient may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like.
• Such compositions and preparations should contain at least 0.01% by weight and more preferably at least 0.1-1% by weight of active ingredient.
• compositions and preparations may, of course, be varied and may conveniently be between about 5 to about 80% of the weight of the unit.
• the amount of active ingredient in the pharmaceutical compositions is such that a suitable dosage will be obtained.
• Preferred compositions or preparations according to the present invention may, for example, be prepared so that an oral dosage unit form contains between about 0.5 ⁇ g and 200 mg and more preferably 10 ⁇ g and 20 mg of active ingredient.
• the tablets, troches, pills, capsules and the like may also contain the following: a binder such as gum tragacanth, acacia, corn starch or gelatin; excipients such as dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; a lubricant such as magnesium stearate; and a sweetening agent such a sucrose, lactose or saccharin may be added or a flavouring agent such as peppermint, oil of wintergreen, or cherry flavouring.
• a binder such as gum tragacanth, acacia, corn starch or gelatin
• excipients such as dicalcium phosphate
• a disintegrating agent such as corn starch, potato starch, alginic acid and the like
• a lubricant such as magnesium stearate
• a sweetening agent such as sucrose, lactose or saccharin may be added or a flavouring agent such as pepper
• tablets, pills, or capsules may be coated with shellac, sugar or both.
• a syrup or elixir may contain the active compound, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and flavouring such as cherry or orange flavour.
• any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
• the active ingredient may be incorporated into sustained-release preparations and formulations.
• pharmaceutically acceptable carriers and diluents include any and all solvents, dispersion media, aqueous solutions, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like.
• the use of such media and agents for pharmaceutically active substances is well known in the art, and it is described, by way of example, in Remington's Pharmaceutical Sciences, 18th Edition, Mack Publishing Company, Pennsylvania, USA. Except insofar as any conventional media or agent is incompatible with the active ingredient, use thereof in the pharmaceutical compositions of the present invention is contemplated. Supplementary active ingredients can also be incorporated into the compositions.
• Dosage unit form refers to physically discrete units suited as unitary dosages for the human subjects to be treated; each unit containing a predetermined quantity of active ingredient calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier and/or diluent.
• the specifications for the novel dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active ingredient and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active ingredient for the treatment of obesity.
• Figures 1A & 1B show the effect of hGH 177-191 peptide on cumulative body weight gains in male (1 A) and female (1 B) C57BL/6J (ob/ob) mice during the
• Figures 2A & 2B show the average daily food consumption (g/mouse/day) of C57BL/6J (ob/ob) mice during an 18-day treatment period with hGH 177-191.
• the treatment for the four groups of animals was as described in figures 1 A & 1 B. Each point represents the mean + SEM of 6 animals. No significance between the groups was observed at all times.
• Figures 3A & 3B demonstrate the effect of hGH 177-191 peptide on body weight gain of 14-15 week old male Zucker fatty (fa/fa) rats during the 20 or 27-days treatment period.
• Animals were given a daily intraperitoneal injection of either saline or the peptide (500 ⁇ g/kg body weight) (3A) or implanted intradermally with a slow-release tablet (500 ⁇ g/day/kg body weight) in the lower quadrant of the abdomen of the animals.
• the control group was implanted with a placebo tablet in the same manner. Each point represents the mean + SEM of 6 animals.
• Figures 4A & 4B demonstrate the average daily food consumption (g/rat/day) of Zucker fatty rats during the treatment period with hGH 177-191 peptide.
• the treatment for the four groups of animals was as described in Figures
• Figures 5A & 5B depict the ex vivo effect on lipogenesis in adipose tissues of the C57BU6J (ob/ob) male mice (5A) and female mice (5B) after 18-day treatment with hGH 177-191.
• Data indicate the rate of [C 14 ]-lipid synthesis and are expressed as [C 1 ]-glucose incorporated into lipid (pmol/mg tissue/min) Values are mean ⁇ SEM of 12 determinations from 6 animals of each group. The differences between the hGH 177-191 treated and saline control groups were statistically significant.
• Figure 6A & 6B depict the ex vivo effect on lipolysis in adipose tissue of the C57BL/6J (ob/ob) male mice (6A) and female mice (6B) after 18-days treatment with hGH 177-191.
• Data indicate the rate of glycerol release from adipose tissues (pmol/mg tissue/min). Values are mean ⁇ SEM of 12 determinations from 6 animals of each group. The differences between the hGH 177-191 treated and saline control groups were statistically significant.
• Figure 7 illustrates the in vitro effect of hGH 177-191 on fatty acid oxidation in isolated adipose tissues of C57BL/6J (ob/ob) mice with the determination of the rate of [C ]O 2 production from [C 14 ]-palmitic acid.
• the rate of [C 14 ]-palmitic acid oxidation was expressed as ⁇ mol/g tissue/hr.
• Figure 8 illustrates the in vitro effect of hGH 177-191 on the release of diacylglycerol from isolated adipocytes of normal rats over an incubation period of 40 min. Diacylglycerol was quantitated using radioenzymic assay and the results were expressed as % increase over the basal levels.
• Figure 9 shows the in vitro effect of hGH 177-191 on hormone-sensitive lipase activity in isolated adipocytes of male Zucker fatty (fa/fa) rats was determined by the amount of hydrolyzed [C 14 ]-oleic acid from [C 14 ]-triolein.
• the enzyme was expressed as U/mg protein, where the release of 1 nmole of oleic acid per hour was considered as 1 Unit of enzyme activity.
• Figures 10A & 10B shows the in vitro effect of hGH 177-191 on acetyl-CoA carboxylase in the isolated adipocytes (10A) and hepatocytes (10B) of normal rats was determined by [C 14 ]-bicarbonate fixation reaction and expressed as mU/g cell dry weight, where 1 Unit of acetyl-CoA carboxylase was defined as the carboxylation of 1 ⁇ mole acetyl-CoA per minute.
• Figure 11 A demonstrates the effect of analogue Ref. No. 9403 (SEQ ID NO: 9403
• mice on body weight gain of 26-week-old C57BL/6J (ob/ob) mice during the 18 days treatment period. Animals were given a daily intraperitoneal injection of either saline (as control) or peptide analogue (500 ⁇ g/kg body weight). Each point represents the mean ⁇ SEM of 6 animals.
• Figure 11B shows the average daily food consumption (g/mouse/day) of 26-week-old C57BL/6J (ob/ob) mice during the treatment period with analogue Ref No. 9403 (SEQ ID NO: 6).
• the treatment for the two groups of animals was as described in Figure 11 A. Each point represents the mean ⁇ SEM of 6 animals. No significance between the test group and the control was observed at all times.
• Figure 12 shows the effect on body weight gain of chronic treatment of 16-week old C57BL/6J(ob/ob) mice with analogues Ref Nos. 9604 (SEQ ID NO: 19) and 9605 (SEQ ID NO: 20).
• Figure 13 shows the effect of long-term oral administration of analogue Ref No. 9604 to ob/ob mice.
• Obese C57BU6J (ob/ob) mice and fatty Zucker (fa/fa) rats were used to demonstrate the biological effects of the synthetic hGH 177-191 and analogues.
• the animals of the same age and same sex were randomly divided into two groups, housed 6 per cage and maintained on a normal 12-hr light/dark cycle at a constant room temperature of 25°C in the animal house of the Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia. Animals were fed ad libitum on pre-determined quantity of animal pellets (Clark King, Melbourne, Australia) and allowed free access to water at all times.
• the animals were given a daily intraperitoneal (i.p) injection of 0.1 ml of either the synthetic peptide (200-500 ⁇ g/kg body weight) or equivalent volume of physiological saline (0.9% sodium chloride) for appropriate number of days.
• the i.p. injection was administered with a 30G x 1 /_" (0.31 x 13 mm) needle on a 1-ml disposable tuberculin syringes, and the site of injection was the lower left quadrant of the abdomen of the animals.
• slow-release peptide-pellets in a diameter of 3 mm were implanted intradermally in the abdominal region of Zucker rats under anaesthesia. The body weight and food intake were monitored for the periods of time as indicated.
• the peptides of the present invention were prepared by using standard 9- fluorenylmethyloxycarbonyl(Fmoc) solid-phase techniques.
• the solid-phase synthesis could be commenced from the C-terminal end of the peptide using an ⁇ -amino protected amino acid.
• a suitable starting materials could be prepared, for instance, by attaching the required ⁇ -amino acid to a Wang resin (4- alkoxybenzyl alcohol resin), or Rink amide resin (2,4-dimethoxy-4'- [carboxymethyoxyj-benzhydrylamine linked to amino methyl resin) or PAM resin (4- hydroxymethylphenyl- acetic acid resin). Resins were commercially available from Auspep Pty. Ltd., Parkville, Victoria, Australia.
• a protected amino acid was coupled to a resin with the aid of a coupling agent.
• the ⁇ -amino protecting group was removed by piperidine in organic solvents at room temperature.
• the remaining protected amino acids were coupled stepwise in the desired order.
• a 4-fold excess of each protected amino acid was generally used in the reaction with an appropriate carboxyl group activator such as diisopropylcarbodiimide (DIC) and 1-hydroxybenzotriazole (HOBt), in methylene chloride (DCM)-N,N-dimethylformamide (DMF) mixtures.
• DIC diisopropylcarbodiimide
• HOBt 1-hydroxybenzotriazole
• the peptide was then cleaved from the resin support by treatment with a reagent such as trifluoroacetic acid (TFA) or trifluormethanesulfonic acid (TFMSA) which cleaved the peptide from the resin, as well as all side-chain protecting groups, except for Cys(Acm).
• a reagent such as trifluoroacetic acid (TFA) or trifluormethanesulfonic acid (TFMSA) which cleaved the peptide from the resin, as well as all side-chain protecting groups, except for Cys(Acm).
• TFA treatment resulted in the formation of the free peptide acids.
• Rink amide resin was used
• TFA treatment resulted in the formation of the free peptide amides.
• TFMSA treatment resulted in the formation of the free peptide acids.
• the target peptides might exist in cyclic disulfide form which requires post-synthesis modification.
• Step 5 DMF (8 ml) was added to the reaction vessel. The resulting solution was stirred for 2 minutes. The solution was drained from the resin in the reaction vessel. This washing procedure was repeated twice. DMF (2 ml) was added to the reaction vessel to keep the resin swollen.
• Step 7 DMF (8 ml) was added to the reaction vessel. The resulting solution was vigorously stirred for 2 minutes. The solution was then drained from the reaction vessel. The washing procedure was repeated twice.
• the reaction vessel containing the peptide-resin was then placed in a desiccator and dried overnight under vacuum.
• the yield of peptide-resin was 0.635 g.
• the dried peptide- resin was removed from the reaction vessel and placed in a 50 ml round-bottom flask containing a magnetic stirring bar.
• the cleavage of the peptide from the resin with TFA was carried out with the following procedure: A scavenger solution, containing 0.75 g phenol, 0.5 ml H 2 O, 0.5 ml thioanisole, and 0.25 ml ethanedithio, was added to the round-bottom flask. The resulting mixture was stirred for 5 minutes. 10 ml TFA was added drop by drop into the flask while kept stirring vigorously. The resulting mixture was stirred for 2.5 hours at room temperature.
• the mixture was filtered through a medium-porosity filter, fritted glass funnel.
• the TFA-peptide solution was sucked into another 500 ml round-bottom flask containing 200 mL cold diethyl ether by applying vacuum. Peptide was allowed to be precipitated in the ether solution at 4°C overnight, then collected by filtering the mixture through a fine-porosity, fritted glass funnel. The peptide pellet on the filter was washed with cold ether (10 ml x 3) to remove the scavenger. The peptide pellet was then dissolved with 25% aqueous acetic acid and then lyophilized to yield the crude peptide (about 400 mg dry weight, purity -80%).
• the crude peptide was purified by reversed-phase high performance liquid chromatography (RP-HPLC). Purification was carried out on a preparative 21.2 x 250 mm Supelcosil PLC-18 (octadecyl, C 18 ) column(120 Angstrom pore size, 12 ⁇ m particle size, 190 m 2 /g surface area; Supelco, Bellefonte, PA, U.S.A.) at 5.0 ml/min flow rate at room temperature. A linear gradient program was utilized , where solvent A was water with 0.1% TFA, and solvent B was acetonitrile-water (50/50: v/v, containing 0.1 % TFA). The gradient was developed from 20 to 100% over 80 min.
• RP-HPLC reversed-phase high performance liquid chromatography
• EXAMPLE A The procedure set forth in EXAMPLE A was employed. After completion of the synthesis of the desired deblocked peptide-resin, 5 ml solution of 20% acetic anhydride in DMF was added. After 5 minutes, 71 ⁇ l (0.4 mmols) of diisopropylethylamine (DIEA) was added to neutralize the protons that were generated. Acetylation of the peptide was performed at room temperature for 30 minutes. The peptide-resin was washed twice with DMF and twice with DCM and the N-acetyl peptide resin was ready for TFA cleavage as shown in EXAMPLE A.
• DIEA diisopropylethylamine
• Fmoc-glycine (Fmoc-Gly, 0.238 g, 0.8 mmol), HOBt (108 mg; 0.8 mmol) and DIC (128 ⁇ l; 0.8 mmol) were added to a 10 ml-test tube containing 2 ml DMF. The mixture was stirred for 10 minutes to activate the amino acid. The solution was then added to the resin in the reaction vessel. The resulting mixture was stirred for 1.5 hours or until a negative ninhydrin test was obtained. The solution was then drained from the reaction vessel. Step 4. DMF (8 ml) was added to the reaction vessel. The resulting solution was vigorously stirred for 2 minutes, followed by the removal of the supernatant. The washing procedure was repeated twice. Step 5.
• Step 7 Steps 3 and 4 were repeated to couple Fmoc-Lys(Boc) to Ser in the position 184. After completion of coupling, the reaction vessel containing the peptide-resin was then placed in a desiccator and dried overnight under vacuum, the peptide-resin was then transferred to a 10ml-reaction vessel. DCM (4 ml) was added to the reaction vessel. The resin was washed with vigorous stirring for 2 minutes. The DCM solution was then drained from the reaction vessel. This washing was repeated once.
• Step 8 was used to remove the Boc group and the t-Bu group from the side chain of lysine and glutamic acid, respectively.
• Step 9 1 ml of 1.5% DIEA/DMF was added to the reaction vessel.
• Benzotriazo-1-yl-oxy-tris-(dimethylamino)phosphoniumhexafluorophosphate(BOP) 400 mg; 0.90 mmol
• HOBt 122 mg, 0.90 mmol
• DIEA 400 ⁇ l, 2.25 mmol
• Step 10 Step 10
• Steps 5 through 6 were then repeated to remove the Fmoc group from the ⁇ -amino group of the lysine residue in the peptide-resin.
• Steps 3 through 6 were then repeated with the following order of amino acids: Fmoc-Cys(Acm) Fmoc-GIn Fmoc-Val Fmoc-lle
• Step 4 was then repeated to remove the Fmoc group from Leu and get the deblocked peptide resin.
• the reaction vessel containing the peptide resin was then placed in a desiccator and dried overnight under vacuum. 604 mg peptide-resin was yielded.
• the dried peptide resin was removed from the reaction vessel and placed in a 25 ml round- bottom flask containing a magnetic stirring bar.
• Trifluoromethanesulfonic acid (TFMSA)/TFA cleavage protocol was used to cleave peptide from the PAM resin:
• a scavenger solution containing 500 ⁇ l thioanisole, and 250 ⁇ l ethanedithio, was added to the flask. The resulting mixture was stirred for 10 minutes at room temperature. 5 ml of TFA was added drop by drop into the flask while kept stirring vigorously. The resulting mixture was stirred at room temperature for 15 minutes. Place the flask in an ice-bath and then 500 ml of TFMSA was slowed added while kept stirring vigorously. The resulting mixture was stirred in the ice-bath for 10 minutes and at room temperature another 15 minutes.
• Example A The procedure set forth in Example A was employed, modified by the addition of a further repetition of the steps 4 to 7 using Fmoc-Tyr(t-Bu).
• Cumulative weight gain and food consumption were determined at 3-day intervals by the measurements of body weight and uneaten food remaining in the cages.
• the animals were placed in a covered chamber to minimise movement during the weighing procedure.
• the food consumption data were obtained by subtracting the amount of uneaten food remaining in the cages from the original provision.
• the reagents are based on either a modified glycerol phosphate oxidase (GPO)- Trinder's type colour reaction 24 or a cholesterol oxidase-4-aminoantipyrine method 25 . All assays were performed with the CentrifiChem System 400 (Union Carbide) containing an automated pipetter, a centrifugal analyser and a recording spectrophotometer. Seronorm Lipid (Nycomed Pharma Co., Oslo, Norway) was used as the calibrator.
• GPO modified glycerol phosphate oxidase
• adipose tissue weight The procedure for the isolation and measurement of intact epididymal fat pads was established in previous studies of epididymal growth of GH-deficient (lit/lit) mice. In the present study, white adipose tissues, either whole epididymal or parametrical fat pads, were excised with the identical techniques as previously described 22 from the mice immediately after sacrifice. The tissues were washed in cold physiological saline, blotted and weighed. For ex vivo lipogenic assays, the portions of adipose tissues without blood vessels were used.
• HSL Hormone-sensitive lipase
• HSL Hormone-sensitive lipase activity of isolated adipocytes was used as a model to evaluate the lipolytic effect of hGH 177-191 peptide and analogues.
• [C 14 ]-triolein was used as a substrate by HSL.
• the amount of hydrolyzed [C 1 ]-oleic acid was determined and used as an index of HSL activity.
• the adipocytes were prepared from the epididymal fat pads of male Zucker fatty (fa/fa) rats by collagenase digestion. Fat pads (5 g) were finely-cut into small cells
• the digestion medium contained microbial collagenase (Type II) at a concentration of 1 mg/ml in Krebs-Ringer phosphate buffer (pH 7.4) at half Ca 2+ strength, at 2% (w/v) of bovine serum albumin (BSA Fraction V).
• BSA Fraction V bovine serum albumin
• the adipocytes released from tissue were then filtered through nylon chiffon into a siliconised glass tube and washed twice with 6 ml of collagen free albumin buffer.
• the isolated adipocytes were resuspended with 10 ml collagen-free buffer and the concentration of adipocytes was estimated by counting an aliquot of a pre-determined volume of cells on a microscope slide. It normally gave approximate 10 9 cells/ml of adipocytes in Krebs-Ringer phosphate buffer (pH 7.4).
• the HSL activity was measured at 37 °C for 1 hour in a final volume of 200 ⁇ l, containing 10 ⁇ moles phosphate buffer (pH 7.0), 15 ⁇ moles of emulsified [C 14 ]- triolein, and 10 8 cells.
• the substrate, [C 14 ]-triolein was pre-emulsified with un- labelled triolein to provide the final emulsion contains 15 ⁇ moles of triolein and 375,000 cpm in 0.1 ml..
• Different concentrations of hGH 177-191 peptide or analogues were added to evaluate their effects on the HSL activities.
• the reaction was stopped by adding 1 ml of the fatty acid extraction mixture of choloform- methanol-benzene 2:2:4:1 containing 50 ⁇ g oleic acid, followed by adding 67 ⁇ l of 0.5N NaOH.
• samples were vortexed for 20 seconds and then centrifuged at 1 ,000 x g for 5 minutes.
• a 200 ⁇ l portion of the alkaline aqueous upper phase containing fatty acids was transferred to scitillation vials.
• the [C 14 ]-radioactivity was measured by a liquid scintillation counter.
• the remaining cell suspension was assayed for protein content..
• the HSL activity was expressed as U/mg protein, where the release of 1 nmole of oleic acid per hour was defined as 1 U of enzyme activity.
• Acetyl-CoA carboxylase catalyzes the critical step in fatty acid synthesis.
• the acetyl-CoA carboxylase activities of both isolated adipocytes and hepatocytes in the presence of hGH 177-191 peptide or analogues were measured for the evaluation of the anti-lipogenic effect of the peptides.
• the acetyl-CoA carboxylase activity was determined by the [C 14 ]-bicarbonate fixation reaction - the rate of incorporation of acetyl-CoA dependent H[C 14 ]O 3 into [C 14 ]-malonyl-CoA.
• Adipocytes were prepared with the method described in the HSL assay.
• Hepatocytes were prepared from the livers of male Wistar rats by collagenase digestion.
• the liver was finely-cut with scissors and transferred to a 250 ml Erlenmeyer flask containing 30 ml of digestion medium.
• the digestion medium contained microbial collagenase (Type IV) at a concentration of 30 mg/ml in calcium-free Krebs-Ringer phosphate buffer (pH 7.4) and glucose (5 mM).
• microbial collagenase Type IV
• the digestion medium contained microbial collagenase (Type IV) at a concentration of 30 mg/ml in calcium-free Krebs-Ringer phosphate buffer (pH 7.4) and glucose (5 mM).
• After digestion for 15 minutes at 37°C under an atmosphere of 95% O 2 /5% CO 2 the hepatocytes liberated from the tissue were then filtered through nylon chiffon into a siliconised glass tube and washed twice with fresh collagen-free digestion medium.
• the isolated cells were resuspended in 45 ml of medium containing extra EDTA (0.45 mmoles), gelatine (0.7 ml), 2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethane sulphonic acid (TES) (0.9 mmoles), and gassed with 95% OJ % CO 2 prior to use.
• extra EDTA 0.45 mmoles
• gelatine 0.45 ml
• TES 2- ⁇ [tris(hydroxymethyl)methyl]amino ⁇ ethane sulphonic acid
• the isolated cells were first preincubated at 37°C for 30 minutes in a mixture containing 50 mM Tris-HCI buffer (pH 7.5), 10 mM potassium citrate, 10 mM MgCI 2 , 1 mM dithiothreitol (DTT), and BSA (0.8 mg/ml).
• the reaction was then initiated by the adding an aliquot of the preincubated cells to an assay mixture (final volume, 500 ⁇ l) containing 50 mM Tris-HCI buffer (pH 7.5), 10 mM potassium citrate, 10 mM MgCI 2 , 1 mM dithiothreitol (DTT), BSA (0.8 mg/ml), 3.75 mM ATP, 0.125 mM acetyl- CoA, and 12.5 mM NaH[C 14 ]O 3 (0.44 ⁇ Ci/ ⁇ mol). After incubation at 37° C for 10 minutes, the reaction was terminated with 0.1 ml of 6M HCI.
• reaction mixture was then allowed to stand in a vacuum desiccator for 30 minutes to remove the unreacted NaH[C 14 ]O 3 and followed by centrifugation at 1500 g for 10 minutes to eliminate the insoluble material. A 0.5 ml aliquot of the supernatant was taken and transferred to scintillation vials. The [C 14 ] radioactivity was measured with a liquid scintillation counter. The remaining cell suspention was assayed for protein content. Specific activity of the enzyme was expressed as mU/g cell dry weight, where 1 U of acetyl-CoA carboxylase was defined as that amount which catalyzed the carboxylation of 1 ⁇ mole acetyl-CoA per minute. Assay for lipolytic activity
• hGH 177-191 and analogues on the isolated adipose tissues was demonstrated by the release of glycerol and free fatty acid (FFA) into the medium during incubation at 37°C.
• FFA free fatty acid
• Adipose tissues were removed from animals and sliced into segments of approximately 200 mg each. Then the tissues were pre-incubated in 25 ml vials containing 2 ml of Krebs-Ringer bicarbonate (KRB) buffer, 4% defatted BSA and 5.5 mM glucose under an atmosphere of carbogen (95%O2/ 5%CO2) at 37°C for 1 hour. Tissues were then transferred to another vials with fresh medium and the incubation was initiated by adding hGH 177-191 peptide or analogues into the vials. The mixtures were then incubated at 37°C for 90 minutes.
• KRB Krebs-Ringer bicarbonate
• glycerol or free fatty acid FFA
• enzyme assay glycerol kinase
• colorimetric copper-dye
• Adipose tissues removed from laboratory animals were sliced into segments of approximately 200 mg each.
• the tissues in 25 ml vials containing 2 ml of Krebs- Ringer phosphate (KRP) buffer, and 2% defatted bovine serum albumin (BSA) were pre-incubated at 37°C for 30 minutes under an atmosphere of carbogen (95%O 2 / 5%CO 2 ) Then the tissues were transferred to Konte flasks with fresh incubation medium with 0.15 mM sodium [C 14 ]-palmitate (final [C 14 ]-specific activity, 0.20 ⁇ Ci/ ⁇ mol) and hGH 177-191 peptide or analogues (1 - 1000 nM).
• KRP Krebs- Ringer phosphate
• BSA defatted bovine serum albumin
• a filter paper roll was placed in a well inside of flask and then the flask was seal with rubber septum stopper. The incubation at 37°C was lasted for 1 hour under an atmosphere of carbogen and was then terminated by injecting 250 ⁇ l of 4.5 M H 2 SO 4 with a needle through the rubber septum into the medium of a flask and 250 ⁇ l of hyamine hydroxide was injected to the filter paper roll in the centre well. Flasks were incubated for a further 1 hour to complete the absorption of [C 14 ]O 2 by hyamine hydroxide. The filter paper rolls were then removed and transferred to scintillation vials. The [C 14 ] radioactivity was measured with a liquid scintillation counter. The rate of [C 14 ]-palmitic acid oxidation to [C 14 ]O 2 was calculated and expressed as ⁇ mol/g tissue/hr.
• Adipose tissues were sliced into segments of approximately 200 mg each and then placed in Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) containing 2% defatted BSA, and glucose (0.1 mg/ml) and gassed with 95% O2 - 5% CO2 at 37 °C. After 1 hr preincubation, the tissues were transferred to another 2 ml of fresh media containing [C 14 ]-glucose (final specific activity 0.05 ⁇ Ci/ ⁇ mol), and 0.3 ⁇ M of hGH 177-191 in the presence or absence of insulin (0.1 mU/ml) for a further 90 min incubation (conditions as above).
• KRB Krebs-Ringer bicarbonate
• Diacylglycerol released from isolated adipose tissue or adipocytes was quantitated using a radioenzymic assay, employing E coli DAG kinase and defined mixed micelle conditions to solubilize DAG and allow its quantitative conversion to [ 33 p] phosphatidic acid in the presence of [ 33 p]- ⁇ -ATP. Following a number of extraction steps to remove unreacted [ 33 p]- ⁇ -ATP, separation of [ 33 p] phosphatidic acid was achieved with the use of 1 ml Am-PrepTM minicolumns.
• mice significantly reduced their adipose tissue weights up to 20% in the males and 12% in the females as compared with the controls of the same sex (Table 1).
• Lipogenesis is subject to the supply of precursor metabolites such as glucose and acetate.
• the effect of hGH 177-191 or analogues was therefore determined by measuring the incorporation of [ 1 C]-glucose into lipid in isolated adipose tissues.
• the peptide hGH 177-191 and analogues (Table 5) reduced lipogenic activity in vitro more than 25% as compared with the control in isolated tissues from fatty Zucker rats.
• Table 2 depicts the effect of the hGH 177-191 treatment on the profiles of circulating levels of triglyceride and cholesterol.
• the total cholesterol in plasma was significantly reduced from 4.44 + 0.56 to 3.52 + 0.39 mmol/l in the male animals but the plasma levels of cholesterol in the treated female animals were only slightly lower than those of the control ones.
• hGH 177-191 did not influence the plasma levels of triglyceride in both sexes.
• the oxidation of fatty acids ( Figure 7) and the release of glycerol (Table 4) in adipose tissues isolated from obese animals were enhanced.
• Tables 6 and 7 show the in vitro antilipogenic and lipolytic activity of hGH 177-191 and two representative analogues (Ref Nos. 9604 and 9605) on human adipose tissue.
• Table 8 shows similar positive lipolytic results on porcine adipose tissue. These results support the expectation that hGH 177-191 and peptide variants thereof shown to be effective in one mammal species will have efficacy in all mammals. Since corresponding sequences of non-human mammals are effectively peptide variants of the human sequence, it is also therefore expected that those corresponding sequences will be effective in other mammals including humans.
• Figure 12 shows cumulative weight gain results for analogue Ref No. 9604 and 9605, showing exceptional efficacy of Ref No. 9604 in particular. This in vivo result is consistent with the enhanced in vitro activity of Ref No. 9604 and 9605 compared with hGH 177-191 (Ref No. 9401) shown in Tables 6, 7 and 8.
• Figure 13 shows the result of long-term oral administration of analogue Ref No. 9604 at 500 ⁇ g/kg daily by oral gavage to ob/ob mice.
• analogue Ref No. 9403 500 ⁇ g/kg body weight

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