AU4897100A - A method of treatment and agents useful for same - Google Patents

Description

QA0M\EjhAgst230272 1-apge 1. 214.doc-0 1000 -1- Regulation 3.2

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT

(ORIGINAL)

Name of Applicant: Actual Inventor: Address for Service: The University of Melbourne Geoffrey Charles NICHOLSON DAVIES COLLISON CAVE, Patent Attorneys, Level 3, 303 Coronation Drive, Milton, Queensland, 4064, Australia Invention Title: "A method of treatment and agents useful for same" Details of Associated Provisional Application No.: PQ1999 filed 3 August 1999 The following statement is a full description of this invention, including the best method of performing it known to us:- P: \OPER\Ej H\230272 1. UNIMELB. CAP. DOC 180 -2- A METHOD OF TREATMENT AND AGENTS USEFUL FOR SAME FIELD OF THE INVENTION The present invention relates generally to a method of modulating bone resorption and to agents useful for same. More particularly, the present invention provides for the use of leptin and its derivatives, homologues, analogues, antagonists or agonists to modulate osteoclastogenesis. Even more particularly, the present invention contemplates the treatment of disorders characterised by or associated with excessive bone resorption such as but not limited to osteoporosis and Paget's disease. The present invention further provides for the use of leptin and its derivatives, homologues, analogues, antagonists and agonists in the manufacture of a medicament for the modulation of bone resorption.

BACKGROUND OF THE INVENTION Bibliographic details of the publications referred to by author in this specification are collected at the end of the description. Nucleotide and amino acid sequences are referred to by a sequence identifier, i.e. <400>1, <400>2, etc. A sequence listing is provided after the claims.

Reference to any prior art in this specification is not, and should not be taken as, an acknowledgment or any form of suggestion that this prior art forms part of the common general knowledge in Australia or any other country.

25 The designation of nucleotide residues referred to herein are those recommended by the IUPAC-IUB Biochemical Nomenclature Commission, wherein A represents Adenine, C represents Cytosine, G represents Guanine, T represents thymine, Y represents a pyrimidine residue, R represents a purine residue, M represents Adenine or Cytosine, K represents Guanine or Thymine, S represents Guanine or Cytosine, W represents Adenine or Thymine, H represents a nucleotide other than Guanine, B represents a nucleotide other than Adenine, V represents a nucleotide other than Thymine, D represents a nucleotide other than Cytosine and N represents any nucleotide residue.

P: OPER\EJH\2302721. UNIMELB. CAP. DOC 1 /8/00 -3- The regulation of bone metabolism is a multifaceted process requiring the tight control of bone resorption and bone formation. The latter is the primary function of osteoblasts whereas bone resorption involves osteoclasts.

Osteoclasts are multinucleate cells formed in bone marrow by the fusion of cells from the monocyte/macrophage lineage (Suda et al., 1992; Quinn et al., 1998). A variety of factors play a role in regulating osteoclast formation including growth factors, systemic hormones and cell contact with marrow stroma.

A number of proteins have been identified which are involved in the process of osteoclastogenesis. Osteoprotegerin, also known as osteoclastogenesis inhibitory factor (OPG and OCIF, respectively), is a secreted member of the TNF receptor superfamily that blocks osteoclast differentiation both in vitro and in vivo (Yasuda, et al., 1998; Simonet et 15 al., 1997). The cloning of a membrane bound ligand for OPG (OPG-ligand [OPGL]) resulted in the identification of an essential signal for proliferation and fusion of osteoclast progenitors (Yasuda, et al., 1998). This protein, also called osteoclast differentiation factor (ODF), is expressed on the plasma membrane of osteoblasts/marrow stromal cells and has a membrane bound receptor (in contrast to the soluble receptor, OPG/OCIF) identified as receptor activator of NF-kappa B (RANK).- OPGL/ODF has also been termed TNF-related activation-induced cytokine (TRANCE) and RANK-ligand (RANKL). The combination of M-CSF and a soluble form of recombinant ODF, lacking the transmembrane domain, is necessary and sufficient to stimulate osteoclast generation, in the absence of osteoblast or stromal cells, from either murine spleen cells or human monocytes (Matsuzaki et al., 1998; Quinn et al., 1988).

Leptin, a cytokine produced primarily by mature adipocytes, is linked to food intake and energy expenditure (Friedman and Halaas, 1998) but also has activity in neuroendocrine, metabolic, reproductive and haemopoetic pathways (Auwerz and Staels, 1998).

P:\OPER\EJH\230272 1. UNIMELB.CAP. DOC 1/8/00 -4- In work leading up to the present invention, the inventors investigated the role of leptin in the bone microenvironment. The inventors have now identified leptin as a regulator of osteoclastogenesis. This provides the basis for the development of a range of medicaments for modulating bone resorption.

°e* e .oo P:\OPER\FJHz\230272 1. UNIMELB.CAP. DOC 1/8/00 SUMMARY OF THE INVENTION Throughout this specification, unless the context requires otherwise, the word "comprise", or variations such as "comprises" or "comprising", will be understood to imply the inclusion of a stated element or integer or group of elements or integers but not the exclusion of any other element or integer or group of elements or integers.

One aspect of the present invention contemplates a method of modulating bone resorption in a human or animal, said method comprising administering to said human or animal an effective amount of leptin or a derivative, homologue, analogue, chemical equivalent, antagonist or agonist thereof for a time and under conditions sufficient for the modulation of osteoclastogenesis.

Another aspect of the present invention provides a method for inhibiting, reducing or S 15 otherwise delaying onset or progression of bone resorption in a human or animal, said S. method comprising administering to said human or animal an effective amount of a leptin as hereinbefore defined for a time and under conditions sufficient to inhibit, reduce or otherwise delay onset or progression of osteoclastogenesis.

Yet another aspect of the present invention is directed to the use of leptin as hereinbefore defined in the manufacture of a medicament in the treatment of a disease condition involving excess bone resorption.

Still yet another aspect of the present invention provides a composition useful in the modulation of bone resorption comprising leptin as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents.

P:\OPER\EJH\2302721.UNIMELB.CAP.DOC- 1/8/00 -6- BRIEF DESCRIPTION OF THE FIGURES Figure 1 is a photographic representation showing generation of TRAP +ve multinuclear cells (MNC) and monoclear (Mono) cells. A colour version of this figure is available by request from the Applicant.

Figure 2 is a graphical representation showing the effect of leptin on the generation of TRAP +ve multinuclear (MNCs). TRAP +ve MNCs were generated on bone slices from PBMCs treated with ODF (55 ng/ml) and M-CSF (25 ng/ml) for 21 days. a, b, c denote significant difference, p>0.05, ANOVA, Fischer's multiple comparison.

Figure 3 is a graphical representation showing the effect of leptin on bone resorption by ODF/M-CSF generated human osteoclasts. The percentage of bone surface resorbed by human osteoclast generated from PBMCs was quantified by SEM. a, b, c denotes significant 15 difference, p>0.05, ANOVA, Fischer's multiple comparison.

*Figure 4 shows photographic and graphical representations of the effects of leptin on OPG and RANK mRNA expression. Adherent PBMCs were treated with leptin for 24 hr immediately after settlement. The expression of OPG, RANK and GAPDH mRNA in human PBMCs was quantified using semi-quantitative RT-PCR The net intensity of OPG and RANK product bands of mRNA expression were analysed and corrected for GAPDH Figure 5 is a graphical representation of the effect of leptin on osteoclastogenesis in purified CD14+ cells. CD14+ cells were positively selected from unfractionated PBMCs using anti- CD14 antibody labelled immunomagnetic beads and cultured on bone slices for 3 weeks in the presence of M-CSF (25 ng/ml) and ODF (40 ng/ml) with and without leptin 1.6 Fg/ml.

Figure 6 is a schematic representation of the proposed mechanism of leptin inhibition of osteoclastogenesis. Not shown on the figure is the likely production of leptin by bone marrow adipocytes.

P:\OPER\EJH\2302721.UNIMELB.CAP.DOC 1/8/00 -7- Abbreviations used in the subject specification are defined in Table 1.

TABLE 1

ABBREVIATIONS

9

TNF

OPG (OCIF) OPGL (ODF)

RANKL

RANK

M-CSF

Mono

MNC

TRAP

PBMC

PBS

FCS

TGFB

RT-PCR

CTR

Tumour necrosis factor Osteoprotegerin; Osteoclastogenesis Inhibitory Factor Membrane bound ligand for OPG; osteoclast differentiation factor (same as TRANCE, TNF-related activation-induced cytokine) Ligand for RANK (same as OPGL/ODF/TRANCE) Receptor activator of NP-kappa B Macrophage-colony stimulating factor Mononuclear cell Multinuclear cell Tartrate-resistant acid phosphatase Peripheral blood mononuclear cell Phosphate buffered saline Fetal calf serum Transforming growth factor B Reverse transcriptase polymerase chain reaction Calcitonin receptor P: \QPER\E H\2302721. .UNIMELB.CAP. DOC 1/8/00 -8- DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS The present invention is predicated in part on the identification of leptin as a potent inhibitor of oseoclastogenesis. This provides a means for modulating bone resorption.

Accordingly, one aspect of the present invention contemplates a method of modulating bone resorption in a human or animal, said method comprising administering to said human or animal an effective amount of leptin or a derivative, homologue, analogue, chemical equivalent, antagonist or agonist thereof for a time and under conditions sufficient for the modulation of osteoclastogenesis.

Reference herein to "leptin" includes reference to a polypeptide having the amino acid sequence set forth in <400>7 or an amino acid sequence having at least 60% similarity thereto while retaining leptin activity or antagonist activity as well as a molecule encoded by the nucleotide sequence set forth in <400>8 or a nucleotide sequence having at least about 60% similarity thereto or a nucleotide sequence capable of hybridising to <400>8 under low stringency conditions at 42 0

C.

Reference herein to a low stringency at 42 0 C includes and encompasses from at least about 1% v/v to at least about 15% v/v formamide and from at least about 1 M to at least about 2 M salt for hybridization, and at least about 1 M to at least about 2 M salt for washing conditions. Alternative stringency conditions may be applied where necessary, such as medium stringency, which includes and encompasses from at least about 16% v/v to at least about 30% v/v formamide and from at least about 0.5 M to at least about 0.9 M salt for hybridization, and at least about 0.5 M to at least about 0.9 M salt for washing conditions, or high stringency, which includes and encompasses from at least about 31% v/v to at least about 50% v/v formamide and from at least about 0.01 M to at least about 0.15 M salt for hybridisation, and at least about 0.01 M to at least about 0.15 M salt for washing conditions.

In general, washing is carried out Tm 69.3 0.41 (Marmur and Doty, 1962).

However, the Tm of a duplex DNA decreases by 1°C with every increase of 1% in the number of mismatch base pairs (Bonner and Laskey, 1974).

P:\OPER\EJH\2302721.UNIMELB.CAP. DOC- 1/8/00~ -9- The term "leptin" is defined herein as including all derivatives, homologues, analogues, chemical equivalents, antagonists and agonists thereof.

The term "derivative" and its plural form includes parts, portions, fragments, regions, fusion molecules, mimotopes and mimetics.

Analogues and mimetics include molecules which contain non-naturally occurring amino acids as well as molecules which do not contain amino acids but nevertheless behave functionally the same as leptin. Natural product screening is one useful strategy for identifying analogues and mimetics. Natural product screening involves screening environments such as bacteria, plants, animals, rainforests, riverbeds, seabeds, aquatic environments, coral and antarctic or arctic environments for naturally occurring molecules 4o4* which mimic, agonize or antagonize leptin of the present invention. Analogues of leptin 15 contemplated herein include modifications to side chains, incorporation of unnatural amino acids and/or their derivatives during peptide synthesis and the use of crosslinkers and other .methods which impose conformational constraints on the peptide molecule or their analogues.

Examples of side chain modifications contemplated by the present invention include modifications of amino groups such as by reductive alkylation by reaction with an aldehyde followed by reduction with NaBH 4 amidination with methylacetimidate; acylation with acetic anhydride; carbamoylation of amino groups with cyanate; trinitrobenzylation of amino groups with 2, 4, 6-trinitrobenzene sulphonic acid (TNBS); acylation of amino groups with succinic anhydride and tetrahydrophthalic anhydride; and pyridoxylation of lysine with followed by reduction with NaBH 4 The guanidine group of arginine residues may be modified by the formation of heterocyclic condensation products with reagents such as 2,3-butanedione, phenylglyoxal and glyoxal.

P:\OPER\EJH\2302721.UNIMELB.CAP. DOC The carboxyl group may be modified by carbodimide activation via O-acylisourea formation followed by subsequent derivitization, for example, to a corresponding amide.

Sulphydryl groups may be modified by methods such as carboxymethylation with iodoacetic acid or iodoacetamide; performic acid oxidation to cysteic acid; formation of a mixed disulphides with other thiol compounds; reaction with maleimide, maleic anhydride or other substituted maleimide; formation of mercurial derivatives using 4-chloromercuribenzoate, 4chloromercuriphenylsulphonic acid, phenylmercury chloride, 2-chloromercuri-4-nitrophenol and other mercurials; carbamoylation with cyanate at alkaline pH.

Tryptophan residues may be modified by, for example, oxidation with N-bromosuccinimide or alkylation of the indole ring with 2-hydroxy-5-nitrobenzyl bromide or sulphenyl halides.

Tyrosine residues on the other hand, may be altered by nitration with tetranitromethane to *form a 3-nitrotyrosine derivative.

Modification of the imidazole ring of a histidine residue may be accomplished by alkylation with iodoacetic acid derivatives or N-carbethoxylation with diethylpyrocarbonate.

Examples of incorporating unnatural amino acids and derivatives during peptide synthesis include, but are not limited to, use of norleucine, 4-amino butyric acid, 4-amino-3-hydroxy- 5-phenylpentanoic acid, 6-aminohexanoic acid, t-butylglycine, norvaline, phenylglycine, S'.ornithine, sarcosine, 4-amino-3-hydroxy-6-methylheptanoic acid, 2-thienyl alanine and/or D-isomers of amino acids. A list of unnatural amino acid contemplated herein is shown in Table 2.

P:\OPER\FJH\2302721. UNIMELB. CAP. DOC 118/00 -11 I- TABLE 2 Non-conventional Code Non-conventional Code amino acid amino acid 0 sees a-aminobutyric acid a-amino-oa-methylbutyrate amninocyclopropanecarboxylate amninoisobutyric acid aminonorbomylcarboxylate cyclohexylalanine cyclopentylalanine D-alanine D-arginine D-aspartic acid D-cysteine D-glutamnine D-glutamic acid D-histidine D-isoleucine D-leucine D-lysine D-methionine D-omnithine D-phenylalanine D-proline D-serine D-threonine D-tryptophan D-tyrosine Abu Mgabu Cpro Aib Norb Chexa Cpen Dal Darg Dasp Dcys Dgln Dglu Dhis Dule Dleu Dlys Dmet Dom Dphe Dpro Dser Dthr Dtrp Dtyr L-N-methylalanine L-N-methylarginine L-N-methylasparagine L-N-methylaspartic acid L-N-methylcysteine L-N-methylglutamine L-N-methylglutamic acid L-Nmethylhistidine L-N-methylisolleucine L-N-methylleucine L-N-methyllysine L-N-methylmethionine L-N-methylnorleucine L-N-methylnorvaline L-N-methylomithine L-N-methylphenylalanine L-N-methylproline L-N-methylserine L-N-methylthreonine L-N-methyltryptophan L-N-methyltyrosine L-N-methylvaline L-N-methylethylglycine L-N-methyl-t-butylglycine L-norleucine L-norvaline a-methyl-aminoisobutyrate Nmala Nmarg Nmasn Nmasp Nmcys Nmgln Nmglu Nmnhis Nmile Nmleu Nmlys Nmnmet Nmnle Nmnva Nmom Nmphe Nmpro Nmser Nmthr Nmtrp Nmtyr Nmval Nmetg Nmtbug NMe Nva Maib P:\OPER\EJH\2302721.UNIMELB.CAP.DOC 1/8/00 12

I

I

I

1111 D-valine D-ca-methylalanine D-a-methylarginine D-a-methylasparagine D-a-methylaspartate D-a-methylcysteine D-ca-methylglutamine D-a-methylhistidine D-a-methylisoleucine D-a-methylleucine D-a-methyllysine D-a-methylmethionine D-a-methylomithine D-a-methylphenylalanine D-oa-methylproline D-a-methylserine D-a-methylthreonine D-ca-methyltryptophan D-a-methyltyrosine D-ca-methylvaline D-N-methylalanine D-N-methylarginine D-N-methylasparagine D-N-methylaspartate D-N-methylcysteine D-N-methylglutamine D-N-methylglutamate D-N-methylhistidine D-N-methylisoleucine D-N-methylleucine D-N-methyllysine Dval Dmala Dmarg Dmasn Dmasp Dmcys Dmgln Dmhis Dmile Dmleu Dmlys Dmmet Dmom Dmphe Dmpro Dmser Dmthr Dmtrp Dmty Dmval Dnala Dnmarg Dnasn Dnasp Dnmcys Drngln Dlnglu Dnmhis Dnmile Dnmleu Dnmlys a-methy1--y-aminobutyrate a-methylcyclohexylalanine a-methylcylcopentylalanine a-methy1-oa-napthylalanine a-methylpenicillamine N-(4-aminobutyl)glycine N-(2-aminoethyl)glycine N-(3 -amninopropyl)glycine N-amino-a-methylbutyrate a-napthylalanine N-benzylglycine N-(2-carbamylethyl)glycine N-(carbamylmethyl)glycine N-(2-carboxyethyl)glycine N-(carboxymethyl)glycine N-cyclobutylglycine N-cycloheptylglycine N-cyclohexylglycine N-cyclodecylglycine N-cylcododecylglycine N-cyclooctylglycine N-cyclopropylglycine N-cycloundecylglycine N-(2,2-diphenylethyl)glycine N-(3 ,3-diphenylpropyl)glycine N-(3 -guanidinopropyl)glycine 1 -hydroxyethyl)glycine N-(hydroxyethyl))glycine N-(imidazolylethyl))glycine N-(3-indolylyethyl)glycine N-methy1-,y-aminobutyrate Mgabu Mchexa Mcpen Manap Mpen Nglu Naeg Nom Nmaabu Anap Nphe Ngln Nasn Nglu Nasp Ncbut Nchep Nchex Ncdec Nedod Ncoct Ncpro Ncund Nbhm Nbhe Narg Nthr Nser Nhis Nhtrp Nmgabu P:\OPER\EJ H\2302721. UNIMELB. CAP. DOC 1/8/00 13 N-methylcyclohexylalanine Nmchexa S S

S.

**SS

S

5S55

S

S.

S S

SS

S

S* *SS

S

Se..

D-N-methylornithine N-methylglycine N-methylaminoisobutyrate 1 -methylpropyl)glycine N-(2-methylpropyl)glycine D-N-methyltryptophan D-N-methyltyrosine D-N-methylvaline -y-aminobutyric acid L-t-butylglycine L-ethylglycine L-homnophenylalanine L-a-methylarginine L-a-methylaspartate L-oa-methylcysteine L-ca-methylglutamine L-a-methylhistidine L-a-methylisoleucine L-a-methylleucine L-a-methylmethionine L-a-methylnorvaline L-a-methylphenylalanine L-a-methylserine L-ce-methyltryptophan L-a-methylvaline N-(N-(2,2-diphenylethyl) carbamylmethyl)glycine 1 -carboxy- 1 -(2,2-diphenyl- Dnomn Nala Nmaib Nile Nleu Dntrp Dntyr Dnmval Gabu Tbug Etg Hphe Marg Masp Mcys Mgln Mhis Mile Mleu Mmet Mnva Mphe Mser Mtrp Mval Nnbhm D-N-methylmethionine N-methylcyclopentylalanine D-N-methylphenylalanine D-N-methylproline D-N-methylserine D-N-methylthreonine 1-methylethyl)glycine N-methyla-napthylalanine N-methylpenicillamnine N-(p-hydroxyphenyl)glycine N-(thiomnethyl)glycine penicillammie L-ce-methylalanine L-a-methylasparagine L-a-methyl-t-butylglycine L-methylethylglycine L-ca-methylglutamnate L-ca-methylhomophenylalanine N-(2-methylthioethyl)glycine L-a-methyllysine L-a-methylnorleucine L-a-methylomithine L-ca-methylproline L-ci-methylthreonine L-a-methyltyrosine L-N-methylhomophenylalanine ,3-diphenylpropyl) carbamylmethyl)glycine Dnmmet Nmcpen Dnmphe Dnmpro Dnser Dnmthr Nval Nmanap Nmpen Nhtyr Ncys Pen Mala Masn Mtbug Metg Mglu Mhphe Nmet Mlys Mnle Mom Mpro Mthr Mtyr Nmhphe Nnbhe Nmbc ethylamnino)cyclopropane P:\OPER\EJH\2302721. UNIMELB.CAP. DOC 180 14- Crosslinkers can be used, for example, to stabilize 3D conformations, using homobifunctional crosslinkers such as the bifunctional imido esters having (CH 2 )n spacer groups with n=1 to n=6, glutaraldehyde, N-hydroxysuccinimide esters and heterobifunctional reagents which usually contain an amino-reactive moiety such as Nhydroxysuccinimide and another group specific-reactive moiety such as maleimido or dithio moiety (SH) or carbodiimide (COOH). In addition, peptides can be conformationally constrained by, for example, incorporation of Ca and Na-methylamino acids, introduction of double bonds between Ca and Coj atoms of amino acids and the formation of cyclic peptides or analogues by introducing covalent bonds such as forming an amide bond between the N and C termini, between two side chains or between a side chain and the N or C terminus.

All these types of modifications may be important to stabilize leptin. This may be important if leptin is used, for example, in the manufacture of a therapeutic composition.

The present invention further contemplates chemical equivalents of leptin. Chemical equivalents may not necessarily be derived from leptin itself but may share certain conformational or functional similarities. Alternatively, chemical equivalents may be specifically designed to mimic certain physiochemical properties of the polypeptides.

Chemical equivalents may be chemically synthesized or may be detected following, for example, natural product screening.

9 The term "modulate" means that bone resorption may be stimulated, enhanced or otherwise increased or that it may be inhibited, retarded or otherwise reduced. Reduction in bone resorption is important for disease conditions involving an excess of bone resorption such as osteoporosis or Paget's disease. Preferably, the modulation involves a reduction in bone resorption.

Accordingly, another aspect of the present invention provides a method for inhibiting, reducing or otherwise delaying onset or progression of bone resorption in a human or animal, said method comprising administering to said human or animal an effective P:\OPER\EJH\2302721. UNIMELB. CAP. DOC 1/8/00 15 amount of a leptin as hereinbefore defined for a time and under conditions sufficient to inhibit, reduce or otherwise delay onset or progression of osteoclastogenesis.

Yet another aspect of the present invention is directed to the use of leptin as hereinbefore defined in the manufacture of a medicament in the treatment of a disease condition involving excess bone resorption.

Such conditions include osteoporosis and Paget's disease.

Accordingly, the present invention provides a composition useful in the modulation of bone resorption comprising leptin as hereinbefore defined and one or more pharmaceutically acceptable carriers and/or diluents.

Preferably, the composition inhibits, reduces or otherwise delays onset or progression of osteoclastogenesis.

The preferred form of a composition is as a pharmaceutical composition.

Compositions suitable for injectable use include sterile aqueous solutions (where water 20 soluble) and sterile powders for the extemporaneous preparation of sterile injectable solutions. They are generally 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, polyoil (for example, glycerol, propylene glycol and liquid polyethylene glycol, and the like), suitable mixtures thereof and vegetable oils. The preventions of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thirmerosal and the like. In many cases, it will be preferable to include isotonic agents, for example, sugars or sodium chloride.

P: \OPER\FJ-H\2302721. UNIMELB.CAP. DOC 1/8/003 -16- Sterile injectable solutions are prepared by incorporating leptin as hereinbefore defined in the required amount in the appropriate solvent with various of the other ingredients enumerated above, as required, followed by, for example, filter sterilization by other appropriate means. In the case of sterile powders for the preparation of sterile injectable solutions, a preferred method of preparation includes vacuum drying and freeze-drying which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution.

When leptin is suitable protected, it 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.

The tablets, troches, pills, capsules and the like may also contain the components as listed hereafter: a binder such as gum, acacia, corn starch or gelatin; excipients such as 15 dicalcium phosphate; a disintegrating agent such as corn starch, potato starch, alginic acid and the like; and a lubricant such as magnesium stearate. Any material used in preparing any dosage unit form should be pharmaceutically pure and substantially nontoxic in the amount employed. In addition, the active compound(s) may be incorporated oooo into sustained-release preparations and formulations.

oooo o sees Pharmaceutically acceptable carriers and/or diluents include any and all solvents, o* dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents and the like. The use of such media and agents for pharmaceutically S. active substances is well known in the art. Except insofar as any conventional media or 6ooo S 25 agent is incompatible with the active ingredient, use thereof in the therapeutic compositions is contemplated. Supplementary active ingredients can also be incorporated into the compositions.

Effective amounts of leptin will vary depending on the condition to be treated by may range from 0.001 ng/kg body weight to 100 mg/kg body weight. Leptin may be P:\OPER\FJH\2302721. UNIMELB. CAP. DOC 1/8/00 -17administered every minute or hourly, daily, weekly or monthly. Leptin may be used prophylactically or in the treatment of a disease condition.

The mode of administration may be intravenous, drip, infusion, oral, intraperitoneal, intra-bone, parenteral, inhalation, nasal drip, aerosol or rectal.

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, 17th Edition, Mack Publishing Company, Easton, Pennsylvania, USA.

The present invention further contemplates genetic modulation of endogenous leptin levels to thereby induce modulation of osteoclastogenesis.

Although not intending to limit the present invention to any one theory or mode of 15 action, it is proposed herein that leptin inhibits osteoclastogenesis by antagonism of the osteoclastic effect of ODF by stimulation of OPG and inhibition of RANK expression.

o.o, The preferred subject for treatment is a human. The invention extends, however, to treatment in non-human animals such as primates, livestock animals sheep, cows, 20 pigs, goats, donkeys, horses), laboratory test animals mice, rats, guinea pigs, •hamsters), companion animals dogs, cats) and captive wild animals.

P: \OPER\EJH\2302721. UNIMELB.CAP. DOC 1/8/00 18- EXAMPLE 1 Human Peripheral Blood Monocnuclear Cell (PBMC) Cultures PBMC were isolated from the peripheral blood of haemochromatic patients and healthy volunteers. Whole blood was spun at 700 g and serum discarded. Blood cells were then diluted 1:1 in PBS and layered over ficoll at a volume ratio of 5:3 and spun at 400 g for min. The top layer was discarded and the underlying layer containing the peripheral blood mononuclear cells (PBMCs) collected. PBMCs were washed in PBS to remove ficoll, collected via centrifugation (140 g) and resuspended in eagle's MEM/10% v/v FCS. PBMCs were seeded into 25 cm 3 tissue flasks (20-25 x 106 PBMCs/flask) and 4 x 4 mm cortical bovine bone slices (1 x 10 6 PBMCs/bone slice) and left to adhere for 2 hrs.

Flasks and bone slices were rinsed to remove non-adherent cells and fresh media added.

EXAMPLE 2 15 Action ofLeptin so The inventors proposed that systemic and/or bone marrow-derived leptin acted on o osteoclast precursors (directly and/or indirectly) and regulated osteoclastogenesis. The o inventors cultured adherent human peripheral blood monocytes (hPBMCs) on bone slices 20 for 21 days in the presence of ODF and M-CSF (Quinn, 1998) with and without added leptin at various concentrations. The formation of multinuclear (MNC) osteoclasts was quantified by cytochemical staining for tartrate-resistant acid phosphatase (TRAP), 1251salmon calcitonin (1 25 I-sCT) autoradiography for calcitonin receptor (CTR) and the identification of bone resorption lacunae by SEM.

The inventors found that leptin was a potent inhibitor of ODF-dependent osteoclast (TRAP+ve, CTR+ve MNCs) formation (Figure 1 and Table 3) and that this effect was dose-dependent (Figure Consistent with this, was a corresponding dose-dependent decrease in bone resorption (Figure At the highest concentration used (1.6 ug/ml), leptin reduced the number of osteoclasts and plan area of bone resorption by approximately P: \OPER\EIH\2302721. UNIMELB. CAP. DOC 1/8/00 -19- Although not intending to limit the present invention to any one theory or mode of action, the inventors postulated that the mechanism of the antagonistic effect of leptin on ODF-induced osteoclast generation might be related to stimulation of ODF's decoy receptor, OPG and/or inhibition of its target cell receptor, RANK. To test this hypothesis, PBMCs were treated with increasing concentrations of leptin for 24 hrs and OPG and RANK mRNA expression assessed by RT-PCR. It was found that leptin (0.032-3.2 p.g/ml) increased OPG mRNA and decreased RANK mRNA in a dose-dependent manner (Figure The OPG and RANK PCR products were confirmed by restriction enzyme digest. (The OPG response will be confirmed by RNAse protection assay and/or "realtime" PCR).

The inhibitory effect of leptin on osteoclast generation is also seen in co-cultures of osteoblast-like UMR 106-01 cells and PBMCs, which do not require the addition of ODF 15 since it is produced by the UMR 106-01. Treatment of a number of osteoblast (rat calvarial, UMR 106-01, SAOs-2) and stromal (ST2, giant cell tumour, M3T3-L1) cells with leptin had no consistent effect on expression of OPG, ODF or RANK mRNA.

TABLE 3 Effect of leptin on the generation of calcitonin receptor positive (CTR 20 +ve) cells o Adherent PBMC's cultured on bone slices for 3 weeks in MEM/MCSF (25 ng/ml) ±ODF (30 ng/ml) Leptin (1.6 pg/ml). Bone slices reacted for TRAP activity and calcitonin binding determined autoradiography.

Treatment MNC, CTR+ve Mono Mono, CTR+ve MCSF alone 0 1345 270 0 MCSF+ODF 367 50 55 9 16 4 MCSF+ODF+Leptin 0 1380 360 9 3 P:\OPER\EJH\2302721 .UNIMELB.CAP.DOC 1/8/00 EXAMPLE 3 Gene Expression by Semi-Quantitative RT-PCR Cultured cells were directly lysed in RNAzol B solution and total RNA extracted according to the manufacturer's instructions. For RT and PCR reactions, a Perkin Elmer/Cetus DNA Thermal Cycler was used. Reverse transcription was performed in the presence of 5 mM MgCl 2 1 mM deoxynucleotide mix, 3.2 mg random primers, units RNase inhibitor and 20 units AMV reverse transcriptase. The final mixture was reacted at 25 0 C for 10 min, 42°C for 60 min and 95°C for 5 min to denature the enzyme.

Sense and antisense primers were designed using the MacVector program and synthesised by Gibco BRL, (Gaithersburg, MD). Sequences and sizes are defined in Table 4.

TABLE 4 Gene Forward Primer Backward Primer Product Size GAPDH 5= CAGTCAGCCGCATCTTCTTTTG 3= 5= TGGTTCACACCCATGACGAAC 3= 464 bp OPG 5= GTACGTCAAGCAGGAGTGCAATC 3= 5= TTCTTGTGAGCTGTGTTGCCG 3= 472 bp RANK 5= TTAAGCCAGTGCTTCACGGG 3= 5= ACGTAGACCACGATGATGTCGC 3= 497 bp PCR products were confirmed by restriction enzyme digest and all primer pairs spanned intron-exon splice sites allowing for the detection of mRNA only.

PCR amplification was performed with cycles of denaturation at 95 0 C for 1 min, annealing at 55 0 C for 2 min, and extension at 72°C for 1 min. The reaction mixture contained 40 pmol of each primer, 200 mM dNTPs, 2 ml of 10X reaction buffer, optimised concentrations of MgCl 2 0.75 mM (OPG), and 1.0 mM (GAPDH and Rank), 1U Taq DNA polymerase, and sterile distilled water up to 20 ml. The mixture was then overlayed with paraffin oil. For semi-quantitative RT-PCR analysis, the optimal number P:\OPER\EJH\230272 1. UNIMELB. CAP.DOC 1/8/003 -21 of cyles for each gene was determined as follows: GAPDH, 20 cycles, OPG, 32 cycles and Rank, 30 cycles. PCR products were resolved on a 1.2% w/v agarose gel and visualised using ethidium bromide. The size of the bands were confirmed by a 100 bp DNA ladder (Gibco BRL, Gaithersburg, MD). Complementary DNA from a sample of human giant cell tumour was used a positive control as we found it to express all the genes studied. Band intensities were measured on the Kodak Digital Science T ID Image Analysis Software and expressed as a ratio of GAPDH intensity.

EXAMPLE 4 Osteoclastogenesis assays employing purified CD14+ cells Leptin treatment does not significantly inhibit osteoclastogenesis in assays that use highly purified CD14+ve cells cultured on bone slices for 21 days in the presence of •ODF and M-CSF. CD14+ve cells were positively-selected from PBMCs using anti-CD14 15 antibody-labelled immunomagnetic beads and the VarioMACs system. Purity (90-95%) was confirmed with FACs analysis. CD14+ cells are highly efficient in the production of osteoclasts. PBMC populations depleted of CD14+ve cells CD14-ve) are not able to generate osteoclasts in this assay (Figure 20 Furthermore, leptin does not upregulate expression of OPG mRNA in purified CD14+ve cells, although down-regulation of RANK mRNA is observed.

These results indicate that the mechanism of leptin-induced inhibition of osteoclastogenesis is not via a direct effect of leptin on CD14+ cells, which appear to be the predominant adherent osteoclast precursor present in the PBMC fraction.

Thus, leptin appears to be acting via another cell type(s) present in the PBMC fraction.

At this time the identity of this cell(s) is unknown.

The proposed mechanism of inhibition of osteoclast generation by leptin is shown in Figure 6.

P:\OPER\FJH\2302721. UNIM ELB. CAP. DOC 1/8/00 22 Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to or indicated in this specification, individually or collectively, and any and all combinations of any two or more of said steps or features.

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BIBLIOGRAPHY

Auwerx et al., The LANCET 351: 737-742, 1998.

Bonner and Laskey, Eur. J1 Biochem. 46: 83, 1974.

Friedman et al., Nature 395: 763 -770, 1998.

Marmur and Doty, J1 Mci. Bio. 5: 109, 1962.

Matsuzaki. et al., Biochem. Biophys. Res. Commun. 246: 199-204, 1998.

0 0 *0.0 Simonet et al., Cell 89: 309-3 19, 1997.

92 0000 Suda et al., Endocrine Reviews 13: 6680192 Suda et al., Endocrine Reviews 20: 345-357, 1999.

Yasuda et al., Endocrinology 139: 1329-1337, 1998.

Claims (16)

1. A method of modulating bone resorption in a human or animal, said method comprising administering to said human or animal an effective amount of leptin or a derivative, homologue, analogue, chemical equivalent, antagonist or agonist thereof for a time and under conditions sufficient for the modulation of osteoclastogenesis.

2. A method according to Claim 1 wherein the leptin or its derivative, homologue, antagonist or agonist comprises an amino acid sequence having at least similarity to the amino acid sequence set forth in <400>7 after optimal alignment.

3. A method according to Claim 1 wherein the leptin or its derivative, homologue, antagonist or agonist is encoded by the nucleotide sequence set forth in <400>8 or a nucleotide sequence having at least 60% similarity to <400>8 after optimal alignment or a nucleotide sequence capable of hybridizing to <400>8 or its complementary form under low stringency conditions at 42 0 C.

4. A method according to Claim 1 or 2 or 3 wherein the modulation comprises a reduction in bone resorption.

A method according to Claim 4 for the treatment of osteoporosis or Paget's disease.

6. A method for inhibiting, reducing or otherwise delaying onset or progression of bone resorption in a human or animal, said method comprising administering to said human or animal an effective amount of a leptin or a derivative, homologue, analogue, chemical equivalent or agonist thereof for a time and under conditions sufficient to inhibit, reduce or otherwise delay onset or progression of osteoclastogenesis. PAOp \Ejh\2 30272 1. iedb-~pd.c- 1/08/00 -31

7. A method according to Claim 6 wherein the leptin or its derivative, homologue, antagonist or agonist comprises an amino acid sequence having at least similarity to the amino acid sequence set forth in <400>7 after optimal alignment.

8. A method according to Claim 7 wherein the leptin or its derivative, homologue, antagonist or agonist comprises an amino acid sequence having at least similarity to the amino acid sequence set forth in <400>7 after optimal alignment.

9. A method according to Claim 6 or 7 or 8 for the treatment of osteoporosis or Paget's disease.

10. A composition comprising leptin or a derivative, homologue, analogue, chemical equivalent, antagonist or agonist thereof and one or more pharmaceutically acceptable carriers and/or diluents when used for modulating bone resorption.

11. A composition according to Claim 10 when used for inhibiting bone resorption.

12. A composition according to Claim 11 when used for the treatment of osteoporosis or Paget's disease.

13. A method for inhibiting osteoclastogenesis in a human or animal, said method comprising administering to said human or animal an amount of leptin or a derivative, homologue, analogue, chemical equivalent or agonist thereof effective to antagonize the osteoclastic effect of ODF by stimulation of OPG and/or inhibition of RANK expression.

14. A method according to Claim 13 wherein the leptin or its derivative, homologue, antagonist or agonist comprises an amino acid sequence having at least similarity to the amino acid sequence set forth in <400>7 after optimal alignment.

PAOP \Ejh\230272 L.idb-pd.-O 1/08/00 32 A method according to Claim 13 wherein the leptin or its derivative, homologue, antagonist or agonist is encoded by the nucleotide sequence set forth in <400>8 or a nucleotide sequence having at least 60% similarity to <400>8 after optimal alignment or a nucleotide sequence capable of hybridizing to <400>8 or its complementary form under low stringency conditions at 42 0 C.

16. A method according to Claim 13 or 14 or 15 for the treatment of osteoporosis or Paget's disease. DATED this first day of August 2000. The University of Melbourne by DAVIES COLLISION CAVE Patent Attorneys for the Applicant.