WO2010046474A2 - Chemoattractants inhibitors - Google Patents

Abstract

The authors of the present invention have found that, surprisingly, ob/ob mice present a dramatic decrease in the resident mesenchymal stem cell pool of several organs, including skeletal muscle, heart, adipose tissue and lung. Moreover, the results herein presented show that this decrease is due to the migration of said MSC cells to the adipose tissue. Evidence is also provided that said migration process is largely explained by the production of high levels of inflammatory cytokines within the adipose tissue.

Description

CHEMOATTRACTANTS INHIBITORS

FIELD OF THE INVENTION

The technical field of the present invention relates to the therapeutic uses of chemoattractants inhibitors in the treatment of obesity and/or obesity-related diseases as well as for the treatment of tissue degenerative conditions.

BACKGROUND OF THE INVENTION

Obesity is a well known risk factor for the development of many very common diseases such as atherosclerosis, hypertension, type 2 diabetes (non-insulin dependent diabetes mellitus (NIDDM)), dyslipidaemia, coronary heart disease, and osteoarthritis and various malignancies. It also causes considerable problems through reduced motility and decreased quality of life.

The term obesity implies an excess of adipose tissue. In this context, obesity is best viewed as any degree of excess adiposity that imparts a health risk. The distinction between normal and obese individuals can only be approximated, but the health risk imparted by obesity is probably a continuum with increasing adiposity.

Even mild obesity increases the risk for premature death, diabetes, hypertension, atherosclerosis, gallbladder disease and certain types of cancer. In the industrialized western world the prevalence of obesity has increased significantly in the past few decades. Because of the high prevalence of obesity and its health consequences, its treatment should be a high public health priority.

Current obesity treatment includes diets. However, although several diets have become quite popular as effective ways of losing weight, none of them have been proven to be vastly superior in rigorously conducted, large-scale clinical trials. Current guidelines recommend drug treatment for individuals, especially those with other obesity-related health conditions, who have failed to respond adequately to dietary and behavioural modifications. A limited number of medications are available for the treatment of obesity. Concerns about side effects have diminished enthusiasm for appetite-suppressant drugs, particularly fenfluramine, which carry serious risks and have been withdrawn from the market. Individuals who have taken either should be evaluated by a physician. Phentermine remains available, but is approved only for short- term use. Sibutramine (acting via serotonergic and noradrenaline mechanisms) is approved for longer-term use, but may cause an increase in blood pressure and should be used with caution and only with regular medical monitoring. Orlistat is a medication that blocks the absorption of dietary fat and is also approved for longer-term use. However, it causes unpleasant side effects (greasy stool), and you also need to supplement your diet with fat-soluble vitamins.

Surgery (such as gastric bypass) is the last resort for the treatment of obesity because such operations can carry significant risks, especially in the post-operative period. Consensus recommendations are to limit surgical therapies to patients with morbid obesity (BMI > 40).

Thus, due to the important effect of obesity as a risk factor in serious (and even fatal) and common diseases, there is still a need for pharmaceutical compounds useful in the treatment of obesity and obesity-related disorders.

Recent advances have implicated the adipocyte in many physiologic and pathologic processes, such as obesity, diabetes, cardiovascular disease and muscular disorders. An active role for the adipocyte in energy metabolism was demonstrated with the discovery of leptin and its role in the pathogenesis of obesity. One increasingly important factor is the generation of additional fat cells, or adipocytes, in response to excess feeding or intake of food, especially food high in fat or comprising fat or fat pre- metabolites, and/or large increases in body fat composition. The generation of new adipocytes is controlled by several adipocyte-specific transcription factors that regulate preadipocyte proliferation and adipogenesis. Generally these adipocyte-specific factors are expressed following the induction of adipogenesis.

In adipose tissue, adipose cells (adipocytes) represent two-thirds of the total cell number. The remaining cells include blood and endothelial cells, along with adipocyte precursors (adipose mesenchymal stem cells). Adipogenesis is a process in which mesenchymal stem cells (MSC) can proliferate in clonal expansion, and at some point, some of these cells can differentiate into preadipocytes or cells committed to fill with lipid (i.e., fat) and then become adipocytes. The adipocyte can continue to enlarge by accumulating additional lipid. The volume of the cell can increase as much as a thousand fold largely because of lipid accumulation.

Most adult tissues harbour a stem cell subpopulation (mesenchymal stem cells) that represent a small proportion of the total cell number and have the potential to differentiate into several cell types within the mesenchymal lineage. In recent years, mesenchymal precursos (MP) or MSC have been increasingly given a role in tissue repair and regeneration. Because of their pluripotency and capacity for self-renewal, stem cells hold great potential to renew tissues that have been damaged by conditions such as type 1 diabetes, Parkinson's disease, heart attacks, and spinal cord injury. In different models of tissue damage, MSC improve the recovery of injured tissues, at least, through two proven mechanisms: by releasing soluble factors with antiinflammatory activity and by promoting and potentiating local regenerative processes.

Proinflammatory cytokines are cytokines that accelerate inflammation and regulate inflammatory reactions either directly or by their ability to induce the synthesis of cellular adhesion molecules or other cytokines in certain cell types. The major proinflammatory cytokines that are responsible for early responses are ILl -alpha, ILl -beta, IL6, and TNF-alpha.

TNF alpha is mainly produced by macrophages, but also by a broad variety of other cell types including lymphoid cells, mast cells, endothelial cells, cardiac myocytes, adipocytes, fibroblasts and neuronal tissue. It has a number of actions on various organ systems, generally together with IL-I and Interleukin-6 (IL-6). It also induces insulin resistance by promoting serine-phosphorylation of insulin receptor substrate- 1 (IRS-I), which impairs insulin signalling. It is a potent chemoattractant for neutrophils, and helps them to stick to the endothelial cells for migration. Tumor necrosis factor promotes the inflammatory response, which in turn causes many of the clinical problems associated with autoimmune disorders. These disorders are sometimes treated by using a TNF inhibitor.

TNF alpha mRNA expression has been reported to be up-regulated in adipose tissue from several rodent models of obesity and diabetes and from obese humans (Hube et al (1999) Eur J Clin Invest, Aug; 29(8): 672-8; Hotamisligil GS, et al J (1995) May; 95(5):2409-15). In the last one, body weight reduction in obese subjects was also associated with a decrease in TNF-alpha mRNA expression in fat tissue. This overexpression in the cytokine is thought to act by stimulating lipolysis and stopping hypertrophy of the fat tissue.

The product of the ob gene is a protein of 16 kDa, leptin, synthesized in adipose tissue and secreted into de bloodstream. The molecule travels to the brain, where it can act as an adipostat signal, i.e. informing the brain of the adipose tissue mass and resulting in the loss of appetite. TNF-α administration results in an increase in circulating leptin concentrations thus, being TNF-α an adipostatic molecule involved in body weight control. SUMMARY OF THE INVENTION The authors of the present invention have found that, surprisingly, ob/ob mice

(leptin deficient homozygous mice) have a dramatic decrease in the resident MSC pool of several organs, including skeletal muscle, heart, adipose tissue and lung. Moreover, the results herein presented show that this decrease is due to the migration of said MSC cells from the organs of residence to the adipose tissue. Once in the adipose tissue, migrant MSCs undergo adipose differentiation, giving rise to new terminally differentiated adipocytes within the adipose abdominal mass. Evidence is also provided that said migration process is largely explained by the production of high levels of inflammatory cytokines within the adipose tissue.

Thus, in a first aspect, the present invention refers to a chemoattractant inhibitor for use as a medicament for the treatment of obesity.

In a second aspect, the invention refers to a chemoattractant inhibitor for use as a medicament for the treatment of a tissue degenerative condition, wherein said inhibitor inhibits the migration of stem cells to the adipose tissue.

In a third aspect, the invention relates to an in vitro method for the identification of compounds for the treatment of obesity and/or for the treatment of a tissue degenerative condition said method comprising i) culturing a population of stem cells in conditions allowing them to migrate toward a chemoattractant; ii) bringing into contact a cell population according to step a) with a test compound; and iii) evaluating the migration capacity of said cell population; wherein if said compound is capable of inhibiting the migration of said stem cells, indicates that the test compound may be used as a medicament for the treatment of obesity and/or for the treatment of a tissue degenerative condition. In a further aspect, the invention refers to a method for the in vivo identification of compounds for the treatment of obesity and/or for the treatment of a tissue degenerative condition said method comprising i) administering a test compound to a non-human animal, wherein said animal is characterized by an enhanced migration of stem cells to the adipose tissue when compared with a control animal; and ii) evaluating the distribution of said cell population in said animal; wherein if said compound is capable of inhibiting the migration of said stem cells to the adipose tissue, indicates that the test compound may be used as a medicament for the treatment of obesity and/or for the treatment of a tissue degenerative condition.

In another aspect, the invention refers to the use of a non-human animal for the identification of compounds for the treatment of a tissue degenerative condition, wherein said animal is characterized by an enhanced migration of stem cells to the adipose tissue when compared with a control animal.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Migration and engraftment of mesenchymal precursors (MP) into adipose tissue. A. Results for epsilon chromosome RT-PCR, 6h or 2 months after i.v. or i..m. male MP injection into female C57 or female ob/ob mice. Note the recruitment of

MP to adipose tissue in ob/ob mice (see red arrows).

Figure 2. Analysis of the migration properties of mesenchymal precursors. A. In vitro transmigration of MP after 6h in the presence or absence of different conditions: mock, SN (600 μl of adypocytes supernatants), recombinant murine TNFα (50ng/ml of anti-TNF (600 μl of adypocytes supernatants in the presence of 10 μg/ml antibodies), and MCPl (20ng/ml). B. In vivo migration into the different tissues of MP population after 6h of C57 or ob/ob i.v. injection, in the presence of anti-TNF-alpha treatment.

Note the reduction of MP in the adipose tissue of Ob/ob mice (red arrow).

DETAILED DESCRIPTION OF THE INVENTION

As mentioned above, the present invention shows that obese leptin deficient homozygous mice have a dramatic decrease in the resident mesenchymal stem cell

(MSC) pool of several organs and that this decrease is due to the migration of said MSC cells from the organs of residence to the adipose tissue. Additionally, the present invention shows that inhibition of inflammatory cytokines, such as TNF alpha, reduces said migration process. As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural references unless the context clearly dictates otherwise.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

Therefore, in a first aspect, the present invention refers to a chemoattractant inhibitor for use as a medicament for the treatment of obesity, wherein said inhibitor inhibits migration of stem cells to the adipose tissue.

The term "chemoattractant" as used herein refers to any inorganic or organic substance possessing chemotaxis inducer effect in motile cells. The term "chemotaxis" as used herein refers to a phenomenon in which cells direct their movements according to certain chemicals and non-chemical substances in their environment. According to the present invention, the term "motile cells" refers to cells that are capable of migrating toward a chemoattractant; in particular, said cells are stem cells. In a particular embodiment of the invention, said stem cells are mesenchymal stem cells.

In another particular embodiment of the invention, said stem cells are endogenous or grafted stem cells. The term "endogenous stem cells" as used herein, refers to the own pool of stem cells that most adult tissues harbour. Accordingly, the term "grafted stem cells" as used herein, refers to externally placed stem cells, i.e. cells coming from an external source. In a particular embodiment of the invention, said cells are autologous stem cells. In another embodiment, said cells are allogeneic stem cells. Preferably grafted cells are autologous cells, i.e. stem cells that are collected from an individual and given back to that same individual.

The term "inhibitor" or "inhibiting", "neutralize" or "neutralizing", "down- regulating" and their cognates as used herein refer to a reduction in the activity of a chemoattractant by an chemoattractant inhibitor, relative to the activity or expression of said chemoattractant in the absence of the same inhibitor. The reduction in activity or expression is preferably at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or higher with respect to reference values as explained below. In a particular embodiment of the invention, said inhibitor is a compound or agent that inhibits activity or expression of the chemoattractant. An agent capable of down-regulating activity or expression of the chemoattractant refers to a molecule such as a chemical, nucleic acid or proteinacious molecule or a combination thereof which is capable of inhibiting activity or expression of the chemoattractant.

Agents capable of down-regulating activity or expression of proteins or mRNA transcripts encoding thereof are well known in the art. In a particular embodiment of the invention, said inhibitor according to the invention is a compound selected from the group consisting of an antibody, an antagonist, a soluble binding protein, a soluble receptor variant, a non- functional derivative, an antisense polinucleotide, a RNA interference oligonucleotide, a DNAzyme, a ribozyme, a triplex forming oligonuclotide (TFO), and a chemical or biological compound. In a particular case, said inhibitor is an antibody. Preferably, the antibody specifically binds to at least one epitope of a protein. As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groups of molecules such as amino acids or carbohydrate side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding to the antigen presented by the macrophages. These functional antibody fragments are defined as follows: (1) Fab, the fragment which contains a monovalent antigen-binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain; (2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule; (3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bridges; (4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single Chain Antibody ("SCA"), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable peptide linker as a genetically fused single chain molecule. Methods of producing polyclonal and monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988). Antibody fragments according to the present invention can be prepared by proteolytic hydrolysis of the antibody or by expression in E. coli or mammalian cells (e.g. Chinese hamster ovary cell culture or other protein expression systems) of DNA encoding the fragment. Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5 S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab<l> monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab<l> fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and 4,331,647, and references contained therein. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent light- heavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

Fv fragments comprise an association of VH and VL chains. This association may be noncovalent. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single peptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula [(1991), Methods 2: 97-105]; Pack et al, [(1993), BioTechnology 11 :1271-77]; and U.S. Pat. No. 4,946,778. Another form of an antibody fragment is a peptide coding for a single complementarity-determining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [(1991) Human Antibodies and Hybridomas, 2:172-189 and U.S. Pat. No. 6,580,016]. Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab<l>, F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non- human residues. Humanized antibodies may also comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non- human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Riechmann et al, Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol, 2:593-596 (1992)]. Methods for humanizing non-human antibodies are well known in the art.

Generally, a humanized antibody has one or more amino acid residues introduced into it from a source that is non- human. These non- human amino acid residues are often referred to as import residues, which are typically taken from an import variable domain. Humanization can be essentially performed following the method well known in the state of the art, by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Accordingly, such humanized antibodies are chimeric antibodies, wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies. Human antibodies can also be produced using various techniques known in the art, including phage display libraries. Similarly, human antibodies can be made by introduction of human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. Alternatively, another proteinaceous agent capable of down-regulating the activity of the chemoattractant can be a non- functional derivative thereof (i.e. dominant negative). Peptides which mimic these non- functional derivatives and others can be synthesized using solid phase peptide synthesis procedures that are well known in the art and further described by John Morrow Stewart and Janis Dillaha Young, [Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984)]. Synthetic peptides can be purified by preparative high performance liquid chromatography and the composition of which can be confirmed by amino acid sequencing.

Alternatively, the agent of this aspect of the present invention may be an agent capable of down regulating the expression of the chemoattractant gene. In this sense, a number of techniques are available in order to modify the expression of a given gene. Gene expression is a process that involves transcription of the DNA code into mRNA, translocation of mRNA to ribosomes, and translation of the RNA message into proteins. Additionally there are other factors that contribute to the great variation in gene expression levels and in the penetrance of gene activity: 1) the mRNA molecule can be more or less stable thus contributing to changes in mRNA levels. 2) there is a precursor of the mature mRNA molecule that can be alternatively spliced, adding complexity to the mechanisms of mRNA expression regulation. 3) mRNAs can be degraded by endogenous cells mechanisms like the existence of RNAses or other more complex systems. 4) translation into proteins can also be regulated at different levels (i.e. initiation of translation, etc.) and, finally, 5) Proteins can be postranslationally modified, thus changing their activity, their molecular stability, etc.

Current methods to suppress a gene include, for example, the use of antisense, co-suppression, and RNA interference. Thus, down-regulation of the chemoattractant can be effected by using an antisense polynucleotide capable of specifically hybridizing with an mRNA transcript encoding said chemoattractant. Design of antisense molecules, which can be used to efficiently down-regulate said gene, must be effected while considering two aspects important to the antisense approach. The first aspect is delivery of the oligonucleotide into the cytoplasm of the appropriate cells, while the second aspect is design of an oligonucleotide that specifically binds the designated mRNA within cells in a way that inhibits translation thereof. The prior art teaches of a number of delivery strategies which can be used to efficiently deliver oligonucleotides into a wide variety of cell types [see, for example, Luft J MoI Med 76: 75-6 (1998); Kronenwett et al, Blood 91 : 852-62 (1998); Rajur et al, Bioconjug Chem 8: 935-40 (1997); Lavigne et al, Biochem Biophys Res Commun 237: 566-71 (1997) and Aoki et al, (1997) Biochem Biophys Res Commun 231 : 540-5 (1997)].

In addition, algorithms for identifying those sequences with the highest predicted binding affinity for their target mRNA based on a thermodynamic cycle that accounts for the energetics of structural alterations in both the target mRNA and the oligonucleotide are also available [see, for example, Walton et al, Biotechnol Bioeng 65: 1-9 (1999)]. Such algorithms have been successfully used to implement an antisense approach in cells. For example, the algorithm developed by Walton et al, enabled scientists to successfully design antisense oligonucleotides for rabbit beta-globin (RBG) and mouse tumor necrosis factor-alpha (TNF alpha) transcripts. The same research group has more recently reported that the antisense activity of rationally selected oligonucleotides against three model target mRNAs (human lactate dehydrogenase A and B and rat gpl30) in cell culture as evaluated by a kinetic PCR technique proved to be effective in almost all cases, including tests against three different targets hi two cell types with phosphodiester and phosphorothioate oligonucleotide chemistries. In addition, several approaches for designing and predicting efficiency of specific oligonucleotides using an in vitro system were also published (Matveeva et ah, Nature Biotechnology 16: 1374 - 1375 (1998)].

Several clinical trials have demonstrated safety, feasibility and activity of antisense oligonucleotides. For example, antisense oligonucleotides suitable for the treatment of cancer have been successfully used [Homlund et ah, Curr Opin MoI Ther 1 :372-85 (1999)], while treatment of hematological malignancies via antisense oligonucleotides targeting c-myb gene, p53 and Bcl-2 had entered clinical trials and had been shown to be tolerated by patients [Gerwitz Curr Opin MoI Ther 1 :297-306 (1999)]. More recently, antisense-mediated suppression of human heparanase gene expression has been reported to inhibit pleural dissemination of human cancer cells in a mouse model [Uno et al, Cancer Res 61 :7855-60 (2001)]. Thus, the current consensus is that recent developments in the field of antisense technology which, as described above, have, led to the generation of highly accurate antisense design algorithms and a wide variety of oligonucleotide delivery systems, enable an ordinarily skilled artisan to design and implement antisense approaches suitable for down-regulating expression of known sequences without having to resort to undue trial and error experimentation.

As used herein, "small interfering RNA" refers to an RNA construct that contains one or more short sequences that are at least partially complementary to and can interact with a polynucleotide sequence of the chemoattractant gene.

Interaction may be in the form of a direct binding between complementary (antisense) sequences of the small interfering RNA and polynucleotide sequences of the target, or in the form of an indirect interaction via enzymatic machinery {e.g., a protein complex) that allows the antisense sequence of the small interfering RNA to recognize the target sequence. In some cases, recognition of the target sequence by the small interfering RNA results in cleavage of chemoattractant sequences within or near the target site that is recognized by the recognition (antisense) sequence of the small interfering RNA. The small interfering RNA can exclusively contain ribonucleotide residues, or the small interfering RNA can contain one or more modified residues, particularly at the ends of the small interfering RNA or on the sense strand of the small interfering RNA. The term "small interfering RNA" as used herein encompasses shRNA and siRNA, both of which are understood and known to those in the art to refer to RNA constructs with particular characteristics and types of configurations. As used herein, "shRNA" refers to an RNA sequence comprising a double-stranded region and a loop region at one end forming a hairpin loop. The double-stranded region is typically about 19 nucleotides to about 29 nucleotides in length on each side of the stem, and the loop region is typically about three to about ten nucleotides in length (and 3'- or 5 '- terminal single-stranded overhanging nucleotides are optional).

As used herein, "siRNA" refers to an RNA molecule comprising a double- stranded region with a 3' overhang of nonhomologous residues at each end. The double- stranded region is typically about 18 to about 30 nucleotides in length, and the overhang may be of any length of nonhomologous residues, for example, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16 or more nucleotides. The siRNA can also comprise two or more segments of 19-30 base pair separated by unpaired regions. Synthesis of RNAi molecules suitable for use with the present invention can be carried out as follows. First, the mRNA sequence target is scanned downstream of the AUG start codon for AA dinucleotide sequences. Occurrence of each AA and the 3' adjacent 19 nucleotides is recorded as potential siRNA target sites. Preferably, siRNA target sites are selected from the open reading frame, as untranslated regions (UTRs) are richer in regulatory protein binding sites. UTR-bin ding proteins and/or translation initiation complexes may interfere with binding of the siRNA endonuclease complex. It will be appreciated though, that siRNAs directed at untranslated regions may also be effective, as demonstrated for GAPDH wherein siRNA directed at the 5' UTR mediated about 90 % decrease in cellular GAPDH mRNA and significantly reduced protein level (wmv.ambiori.cc)m/lcch UMn/9JV9Jjyuml)

Second, potential target sites are compared to an appropriate genomic database (e.g., human, mouse, rat etc.) using any sequence alignment software, such as the BLAST software available from the NCBI server (www.ncbi.nlm.nih.gov/BLAST/). Putative target sites that exhibit significant homology to other coding sequences are filtered out. Qualifying target sequences are selected as template for siRNA synthesis. Preferred sequences are those including low G/C content as these have proven to be more effective in mediating gene silencing as compared to those with G/C content higher than 55 %. Several target sites are preferably selected along the length of the target gene for evaluation. For better evaluation of the selected siRNAs, a negative control is preferably used in conjunction. Negative control siRNA preferably include the same nucleotide composition as the siRNAs but lack significant homology to the genome. Thus, a scrambled nucleotide sequence of the siRNA is preferably used, provided it does not display any significant homology to any other gene.

Another oligonucleotide agent capable of down-regulating said chemoattractant is a DNAzyme molecule capable of specifically cleaving an mRNA transcript or a DNA sequence of the target. DNAzymes are single-stranded polynucleotides which are capable of cleaving both single and double stranded target sequences. A general model (the "10-23" model) for the DNAzyme has been proposed. "10-23" DNAzymes have a catalytic domain of 15 deoxyribonucleotides, flanked by two substrate-recognition domains of seven to nine deoxyribonucleotides each. This type of DNAzyme can effectively cleave its substrate RNA at purine :pyrimidine junctions. Examples of construction and amplification of synthetic, engineered DNAzymes recognizing single and double-stranded target cleavage sites have been disclosed in U.S. Pat. No. 6,326,174.

Another agent capable of down-regulating said chemoattractant is a ribozyme molecule capable of specifically cleaving an mRNA transcript encoding said chemoattractant. Ribozymes are being increasingly used for the sequence- specific inhibition of gene expression by the cleavage of mRNAs encoding proteins of interest. The possibility of designing ribozymes to cleave any specific target RNA has rendered them valuable tools in both basic research and therapeutic applications. In the therapeutics area, ribozymes have been exploited to target viral RNAs in infectious diseases, dominant oncogenes in cancers and specific somatic mutations in genetic disorders. Most notably, several ribozyme gene therapy protocols for HIV patients are already in Phase 1 trials. More recently, ribozymes have been used for transgenic animal research, gene target validation and pathway elucidation. Several ribozymes are in various stages of clinical trials. ANGIOZYME was the first chemically synthesized ribozyme to be studied hi human clinical trials. ANGIOZYME specifically inhibits formation of the VEGF-r (Vascular Endothelial Growth Factor receptor), a key component in the angiogenesis pathway. Ribozyme Pharmaceuticals, Inc., as well as other firms have demonstrated the importance of anti-angiogenesis therapeutics in animal models. HEPTAZYME, a ribozyme designed to selectively destroy Hepatitis C Virus (HCV) RNA, was found effective in decreasing Hepatitis C viral RNA in cell culture assays (Ribozyme Pharmaceuticals, Incorporated-www.rpi.com/index.html).

An additional method of regulating the expression of the chemoattractant gene in cells is via triplex forming oligonuclo tides (TFOs). In the last decade, studies have shown that TFOs can be designed which can recognize and bind to polypurine/polypirimidine regions in double-stranded helical DNA in a sequence- specific manner. These recognition rules are outlined by Maher III, L. J., et al, Science (1989) 245:725-730; Moser, H. E., et al, Science (1987)238:645-630; Beal, P. A., et al, Science (1991) 251 :1360-1363; Cooney, M., et al, Science(1988)241,456-459). Modification of the oligonuclo tides, such as the introduction of intercalators and backbone substitutions, and optimization of binding conditions (pH and cation concentration) have aided in overcoming inherent obstacles to TFO activity such as charge repulsion and instability, and it was recently shown that synthetic oligonucleotides can be targeted to specific sequences. In general, the trip lex- forming oligonucleotide has the sequence correspondence: oligo 3'-A G G T duplex 5'~A G C T duplex 3'~T C G A. However, it has been shown that the A-AT and G-GC triplets have the greatest triple helical stability. Thus, for any given sequence in the regulatory region a triplex forming sequence may be devised. Trip lex- forming oligonucleotides preferably are at least 15, more preferably 25, still more preferably 30 or more nucleotides in length, up to 50 or 100 bp .

Transfection of cells (for example, via cationic liposomes) with TFOs, and formation of the triple helical structure with the target DNA induces steric and functional changes, blocking transcription initiation and elongation, allowing the introduction of desired sequence changes in the endogenous DNA and resulting in the specific downregulation of gene expression. Examples of such suppression of gene expression in cells treated with TFOs include knockout of episomal supFGl and endogenous HPRT genes in mammalian cells, and the sequence- and target-specific downregulation of expression of the Ets2 transcription factor, important in prostate cancer etiology, the pro -inflammatory ICAM-I gene. In addition, Vuyisich and Beal have recently shown that sequence specific TFOs can bind to dsRNA, inhibiting activity of dsRNA-dependent enzymes such as RNA-dependent kinases (Vuyisich and Beal, Nuc. Acids Res (2000); 28:2369-74).

Additionally, TFOs designed according to the abovementioned principles can induce directed mutagenesis capable of effecting DNA repair, thus providing both downregulation and upregulation of expression of endogenous genes. Detailed description of the design, synthesis and administration of effective TFOs can be found in U.S. Patent Application Nos. 2003017068 and 20030096980 and U.S. Pat. No.5,721,138.

It will be appreciated that therapeutic oligonucleotides may further include base and/or backbone modifications, which may increase bioavailability, therapeutic efficacy and reduce cytotoxicity. Such modifications are described in Younes (2002) [Current Pharmaceutical Design 8:1451-1466]. For example, the oligonucleotides of the present invention may comprise heterocylic nucleosides consisting of purines and the pyrimidines bases, bonded in a 3' to 5<1> phosphodiester linkage. Preferably used oligonucleotides are those modified in backbone, internucleoside linkages or bases, as is broadly described herein below.

Specific examples of preferred oligonucleotides useful according to this aspect of the present invention include oligonucleotides containing modified backbones or non-natural internucleoside linkages. Preferred modified oligonucleotide backbones include, for example, phosphorothioates, chiral phosphorothioates, phosphorodithioates, phosphotriesters, aminoalkyl phosphotriesters, methyl and other alkyl phosphonates including 3'- alkylene phosphonates and chiral phosphonates, phosphinates, phosphoramidates including 3 '-amino phosphor ami date s and aminoalkylphosphoramidates, thionophosphoramidates, thionoalkylphosphonates, thionoalkylphosphotriesters, and boranophosphates having normal 3 '-5' linkages, 2 '-5' linked analogs of these, and those having inverted polarity wherein the adjacent pairs of nucleoside units are linked 3'-5' to 5'-3' or 2'-5' to 5'-2'. Various salts, mixed salts and free acid forms can also be used.

Alternatively, modified oligonucleotide backbones that do not include a phosphorus atom therein have backbones that are formed by short chain alkyl or cycloalkyl internucleoside linkages, mixed heteroatom and alkyl or cycloalkyl internucleoside linkages, or one or more short chain heteroatomic or heterocyclic internucleoside linkages. These include those having morpholino linkages (formed in part from the sugar portion of a nucleoside); siloxane backbones; sulfide, sulfoxide and sulfone backbones; formacetyl and thioformacetyl backbones; methylene formacetyl and thioformacetyl backbones; alkene containing backbones; sulfamate backbones; methyleneimino and methylenehydrazino backbones; sulfonate and sulfonamide backbones; amide backbones; and others having mixed N, O, S and CH2 component parts.

Other oligonucleotides which can be used according to the present invention, are those modified in both sugar and the internucleoside linkage, i.e. the backbone, of the nucleotide units are replaced with novel groups. The base units are maintained for complementation with the appropriate polynucleotide target. An example for such an oligonucleotide mimetic includes peptide nucleic acid (PNA). A PNA oligonucleotide refers to an oligonucleotide where the sugar-backbone is replaced with an amide containing backbone, in particular an aminoethylglycine backbone. The bases are retained and are bound directly or indirectly to aza nitrogen atoms of the amide portion of the backbone. United States patents that teach the preparation of PNA compounds include, but are not limited to, U.S. Pat. Nos. 5,539,082; 5,714,331; and 5,719,262.

Oligonucleotides of the present invention may also include base modifications or substitutions. As used herein, "unmodified" or "natural" bases include the purine bases adenine (A) and guanine (G), and the pyrimidine bases thymine (T), cytosine (C) and uracil (U). Modified bases include but are not limited to other synthetic and natural bases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine, hypoxanthine, 2-amino adenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 2- thiouracil, 2- thiothymine and 2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil), 4- thiouracil, 8- halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl and other 8-substituted adenines and guanines, 5 -halo particularly 5-bromo, 5-trifluoromethyl and other 5- substituted uracils and cytosines, 7-methylguanine and 7-methyladenine, 8- azaguanine and 8-azaadenine, 7-deazaguanine and 7-deazaadenine and 3- deazaguanine and 3-deazaadenine. Further bases include those disclosed in U.S. Pat. No: 3,687,808, those disclosed in The Concise Encyclopedia Of Polymer Science And Engineering, pages 858-859, Kroschwitz, J. L, ed. John Wiley & Sons, 1990, those disclosed by Englisch et ah, Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y. S., Chapter 15,

Antisense Research and

Applications, pages 289-302, Crooke, S. T. and Lebleu, B., ed., CRC Press, 1993.

Recombinant agents or oligonucleotide agents of the present invention can be administeretd to the subject employing any suitable mode of administration, described hereinbelow (i.e. in vivo gene therapy). Alternatively, the nucleic acid construct can be introduced into a suitable cell using an appropriate gene delivery vehicle/method (transfection, transduction, etc.) and an appropriate expression system. The modified cells are subsequently expanded in culture and returned to the individual (i.e. ex vivo gene therapy). Examples of suitable constructs include, but are not limited to, pcDNA3, [rho]cDNA3.1 (+/-), pGL3, PzeoSV2 (+/-), pDisplay, pEF/myc/cyto, pCMV/myc/cyto each of which is commercially available from Invitrogen Co. (www.invitrogen.com). Examples of retroviral vector and packaging systems are those sold by Clontech, San Diego, Calif, including Retro-X vectors pLNCX and pLXSN, which permit cloning into multiple cloning sites and transcription of the transgene is directed from the CMV promoter. Vectors derived from Mo-MuLV are also included such as pBabe, where the transgene will be transcribed from the 5'LTR promoter.

It will be appreciated that nucleic acid agents of the present invention can be introduced to the subject using the well known "gene knock-in strategy" which will result in the formation of a non- functional protein [see e.g., Matsuda et aL, Methods MoI Biol. 2004; 259:379-90].

In a particular embodiment of the invention, said chemoattractant inhibitor is a cytokine inhibitor. In a more particular embodiment, said inhibitor is an inflammatory or pro-inflammatory cytokine inhibitor.

In principle, a specific cytokine inhibitor could be an antagonist binding to the same receptor and competing for receptor binding with the genuine cytokine. Another class of cytokine inhibitors according to the present invention comprises specific soluble binding proteins. These binding proteins do not themselves bind to cytokine receptors. However, they prevent the genuine cytokine to interact with its receptor by complexing the cytokine. In another embodiment, said cytokine-binding inhibitors are soluble receptor variants, which are generated either by proteolytic cleavage of membrane-bound receptors or by translation of alternatively spliced receptor RNAs. Many of these soluble cytokine receptors lack a transmembrane and a cytoplasmic domain normally found in membrane-bound forms of the receptor. As a rule cytokine binding proteins show a very high affinity towards the respective cytokines, which may exceed the affinity of cytokine-specific antibodies by a factor of 100-1000. These proteins may be valuable drugs, therefore, that might be used to block, for example, the autocrine activities of some of the cytokines. In addition these physiological inhibitors do not contain structures (such as the Fc portion of antibodies) that would allow interaction with other cells. They should therefore not be recognized by the immune system.

In another particular embodiment, said cytokine inhibitor is an inhibitor of cytokine action and is a non-specific binding protein that does not interfere with binding of the cytokines to their respective receptors. One protein capable of binding unspecifically to several cytokines is Alpha-2-Macroglobulin. Illustrative, non limitative, examples of drugs that also interfere with cytokine actions according to the present invention are, for example, CsA, Cyclosporin A; Pentamidine, and Pentoxifylline.

In a more particular embodiment of the invention, said cytokine is an inflammatory cytokine. Persons skilled in the art are well aware of what is intended by an inflammatory or pro -inflammatory cytokine. For the purpose of this disclosure, it may, however, be further clarified that the expression "inflammatory or pro-inflammatory cytokine" relates to any substance from the cytokine family that posses one or more of the following specific mechanisms of action: 1) increasing vascular permeability, 2) attracting white blood cells (leucotaxia or chemotaxia), 3) activating macrophages, and 4) recruiting macrophages to the site of wound healing. These effects may be assessed for each individual substance by use of the assays disclosed below.

"A substance that inhibits a pro -inflammatory cytokine" as it is used herein thus relates to a substance that may block one or more of the four listed effects in assays such as, for example, the ones disclosed below. However, due to differences between species, one may also translate findings from the experimental setting to the human situation. For instance, if a monoclonal antibody with specificity towards a specific cytokine of a certain species inhibits the action of the cytokine in one of the three ways disclosed below in that specific species, one may assume that a monoclonal antibody, with specificity towards the human version of the cytokine, may inhibit this cytokine in the human situation.

1) Assay for increase of vascular permeability: A golden hamster, weighing 65- 100 g, is anaesthetized. An injection of 0.3 ml of FITC-Dextran (mw 150,000, 25 mg/ml, Sigma, St Louis, USA) is made in the femoral vein for fluorescence vital microscopic observations of macromolecular extravascular leakage. Temperature and humidity is controlled by irrigation of saline at 37° C. An injection of approximately 0.02 ml of a suitable concentration of the substance to be tested is made between the two layers of the cheek-pouch using a thin injection needle (diameter 0.4 mm). The same volume of saline is performed in an adjacent part of the cheek-pouch at a distance from the other injection site sufficient to eliminate the risk of communication between the saline and the tested substance within the cheek-pouch. Microvascular reactions are studied for 60 minutes at various magnifications, using fluorescence microscopic techniques. A pro -inflammatory cytokine as defined according to the present invention induces a leakage of the fluorescent macromolecule FITC-dextran. A similar leakage should not be observed at the site injected by saline.

2) Assay for leucotaxia or chemotaxia: A pig, bodyweight 25-30 kg, is anaesthetized. One ml of a fluid containing a sufficient concentration of the substance to be tested is placed, in a suitable concentration locally in its natural form, in slow-release preparations or by continuous administration by osmotic mini-pumps, in a specially designed titanium-chamber. The chamber is 5 mm high and has a diameter of 15 mm. The chamber, together with one chamber with the same volume of saline, is placed subcutaneously in the lumbar region through separate incisions, with no communication between the chambers. After 7 days the pig is reanaesthetized similar to the first procedure. The chambers are harvested and the content of the chamber is placed in a test-tube together with 1 ml of Hanks' Balanced Salt Solution (Life Technologies, Paisley, Scotland). From this suspension, 100 μl is used to wash out the chamber for remaining cells. This procedure is repeated 5 times. The test-tube is then shaken for 15 seconds. A total of 25 μl of the suspension and 25 μl of Turk's staining medium (Sigma, St Louis, USA) are mixed and placed in a chamber of Bϋrker. The total number of leukocytes in each chamber is determined using light microscopy. The chamber with a pro-inflammatory cytokine as defined according to the present invention then contains significantly more white blood cells than the chamber with only saline. 3) Assay for activation of macrophages: A macrophage cell line is bought and cultured according to the description of the manufacturer. The cells are cultured in multiple-well culture plates. The substance to be tested is applied to the culture-wells in various concentrations. After incubation for 6-72 hours, aliquots of the culture media (25-50 μl) of the culture media are used for assays. Assays of TNF and IL-8 and nitric oxide (NO) are performed using commercially available assays and the results are compared with assays from culture media without the addition of the substance to be tested. A pro -inflammatory cytokine as defined according to the present invention induces significantly higher levels of one or more of TNF, IL-8 or NO in the culture media compared to culture media without the tested substance. 4) Assay for recruitment of macrophages to the site of wound healing: Rats are anaesthetized and the skin on the back is shaved. A 3 cm long midline incision is made in the skin and in the underlying muscle. The substance to be tested is applied in a suitable concentration locally in its natural form, in slow-release preparations or by continuous administration by osmotic mini-pumps. In control experiments, the same amount and administration of saline is executed. The skin is sutured. After 1-4 weeks the rat is re-anaesthetized and the area of wound healing in the skin and in the muscle is harvested and processed for immunohistochemistry. Commercially available antibodies for macrophage specific CD-molecules (e.g., CDwI 7, CD23, CD25, CD26, CD64, CD68, CD69, CD71, CD74, CD 80, CD88, CD91 and CD 105) are used to visualize the presence of macrophages in the healing tissues. The number of macrophages is then found to be significantly higher in the healing tissue when exposed to the tested substance than in tissues exposed to saline control. An inhibitor of a pro-inflammatory cytokine as defined according to the present invention will reduce the effects of the pro -inflammatory cytokine in one or more of the four assays described above, i.e. increase of vascular permeability, leucotaxia and activation or recruitment of macrophages, and/or it will have an inhibitory effect on the recruitment of macrophages in an assay for inhibition of recruitment of macrophages as, for example, the one disclosed below.

5) Assay for inhibition of recruitment of macrophages to site of wound healing: Rats are anaesthetized and the skin on the back is shaved. A 3 cm long midline incision is made in the skin and in the underlying muscle. The skin is sutured. The animal receives treatment by a cytokine inhibitor in a suitable concentration and form of administration. Control animals receive no treatment. After 1-4 weeks the rat is re- anaesthetized and the area of wound healing in the skin and in the muscle is harvested and processed for immunohistochemistry. Commercially available antibodies for macrophage specific CD-molecules (e.g. CDwl7, CD23, CD25, CD26, CD64, CD68, CD69, CD71, CD74, CD 80, CD88, CD91 and CD 105) are used to visualize the presence of macrophages in the healing tissues. The number of macrophages is then found to be significantly lower in the healing tissue after treatment with the cytokine inhibitor than in control animals.

In an even more particular embodiment of the invention, said inflammatory cytokine is selected from the group consisting of IL-I, TNF-alpha, IL-6, IFN-alpha, IFN-gamma, IL-I l, TGF-beta, LIF, IL-8, PF-4, MIP-la/b, MCP-I, MCP-2, MCP-3, Rantes, IL- 12, and Lymphotactin. In a preferred embodiment, said cytokine is TNF alpha. In another preferred embodiment of the invention, said cytokine is MCP-I. There are several different types of inhibitors known in the state of the art of pro-inflammatory cytokines that may be used according to the invention: - Illustrative, non limitative, examples of specific TNF blocking substances, are monoclonal antibodies, such as infliximab, CDP-571 (Humicader™), D2E7, and CDP- 870, soluble cytokine receptors, such as etanercept, lenercept, pegylated TNF-receptor type I, TBP-I, TNF-receptor antagonists, antisense oligonucleotides, such as ISIS- 104838, non-specific TNF blocking substances, such as:

• MMP inhibitors (i.e. matrix metalloproteinase inhibitors, or TACE-inhibitors, i.e., TNF-α Converting Enzyme-inhibitors) such as tetracyclines, for example Doxycycline, Lymecycline, Oxitetracycline, Tetracycline, Minocycline and synthetic tetracycline derivatives, such as CMT, i.e., chemically modified tetracyclines, prinomastat (AG3340), Batimastat, Marimastat, KB-R7785, TIMP-I, TIMP-2, adTIMP-1 (adenoviral delivery of TIMP-I), adTIMP-2 (adenoviral delivery of TIMP-2) • Quinolones, for example Norfloxacin, Levofloxacin, Enoxacin, Sparfloxacin,

Temafloxacin, Moxifloxacin, Gatifloxacin, Gemifloxacin, Grepafloxacin, Trovafloxacin, Ofloxacin, Ciprofloxacin, Pefloxacin, Lomefloxacin and Temafloxacin

• Thalidomide derivates, e.g. SeICID (i.e. Selective Cytokin inhibitors), CC- 1088, CDC-501, CDC-801, and Linomide

• Lazaroids, e.g., non-glucocorticoid 21-aminosteroids such as U-74389G (16- desmethyl tirilazad) and U-74500

• Prostaglandins; iloprost (prostacyclin)

• Cyclosporin • Pentoxifyllin derivates

• Hydroxamic acid derivates

• Napthopyrans

• Phosphodiesterase I, II, III, IV, and V-inhibitors, e.g., CC-1088, Ro 20-1724, rolipram, amrinone, pimobendan, vesnarinone, SB 207499 (Ariflo®) • Melancortin agonists, e.g., HP-228

Illustrative, non limitative, examples of other TNF blocking substances, that can be used according to the present invention, include lactoferrin, and peptides derived from lactoferrin such as those disclosed in U.S. Pat. No. 7,253,143 Bl, CT3, ITF-2357, PD-168787, CLX-1100, M-PGA, NCS-700, PMS-601, RDP-58, TNF-484A, PCM-4, CBP-1011, SR-31747, AGT-I, Solimastat, CH-3697, NR58-3.14.3, RIP-3, Sch-23863, SH-636, etc.

As used herein in the specification and claims section that follows, the terms "treatment" and "treating" refers to preventing, curing, reversing, attenuating, alleviating, minimizing, suppressing or halting the deleterious effects of the disease. Thus, in the context of the present invention the term "treatment" comprises treating obesity or obesity-related condition or disorder to reverse disease's symptoms, more particularly, preventing the development of said disease, as well as managing and/or ameliorating said disease or one or more symptoms thereof. In a preferred embodiment of the invention, the term "treatment" refers to administering a therapeutically effective amount of the inhibitor of the invention to achieve a desired therapeutic effect

Thus, "treatment" as used herein covers any treatment of a disease or condition of a mammal, particularly a human, and includes: (a) preventing the disease or condition from occurring in a subject which may be predisposed to the disease or condition but has not yet been diagnosed as having it; (b) inhibiting the disease or condition, i.e., arresting its development; or (c) relieving the disease or condition, i.e., causing regression of the disease or condition or amelioration of one or more symptoms of the disease or condition. The population of subjects treated by the method includes a subject suffering from the undesirable condition or disease, as well as subjects at risk for development of the condition or disease.

As used herein, said "obesity-related disorder" refers to disorders such as overeating, binge eating, bulimia, diabetes, elevated plasma insulin concentrations, insulin resistance, metabolic syndrome, dyslipidemias, hyperlipidemia, lipodystrophy, osteoarthritis, arthritis deformans, lumbodynia, emmeniopathy, obstructive sleep apnea, cholelithiasis, gallstones, nonalcoholic steato hepatitis, heart disease, abnormal heart rhythms and abnormal heart arrhythmias, myocardial infarction, congestive heart failure, coronary heart disease, coronary artery disease, angina pectoris, hypertension, sudden death, stroke, cerebral infarction, cerebral thrombosis, transient ischemic attack, polycystic ovary disease, craniopharyngioma, Pickwickian syndrome, fatty liver, Prader-Willi Syndrome, Frohlich's syndrome, GH-deficiency, normal variant short stature, Turner's syndrome, pediatric acute lymphoblastic leukemia, infertility, hypogonadism in males, hirsutism in females, gastrointestinal motility disorders, respiratory disorders, cardiovascular disorders, inflammation, arteriosclerosis, hypercholesterolemia, hyperuricaemia, lower back pain, gallbladder disease, gout, endometrial cancer, breast cancer, prostate cancer, colon cancer or kidney cancer.

While it is possible for the active agent, i.e. the inhibitor, to be administered alone, it is preferable to present it as part of a pharmaceutical formulation or composition, comprising as active ingredient an effective amount of an inhibitor according to the invention. The pharmaceutical formulation or composition in the context of the invention is intended to mean a combination of the active agent(s), together or separately, with a pharmaceutically acceptable carrier as well as other additives.

The term "pharmaceutically acceptable carrier" in the context of the present invention denotes any one of inert, non-toxic materials, which do not react with the compound of the invention and which can be added to formulations as diluents, carriers or to give form or consistency to the formulation. The carrier may at times have the effect of the improving the delivery or penetration of the active ingredient to the target tissue, for improving the stability of the drug, for slowing clearance rates, for imparting slow release properties, for reducing undesired side effects etc. The carrier may also be a substance that stabilizes the formulation (e.g. a preservative), for providing the formulation with an edible flavor, etc. For examples of carriers, stabilizers and adjuvants, see E. W. Martin, REMINGTON'S PHARMACEUTICAL SCIENCES, MacK Pub Co (June, 1990).

In addition, the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action. The compounds, i.e. the inhibitor, may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients, such as, for example other agents useful in the treatment of obesity or obesity-related conditions.

Examples of said agents useful in the treatment of obesity according to the present invention, include an insulin sensitizer, an insulin or insulin mimetic, a sulfonylurea, an α-glucosidase inhibitor, a cholesterol lowering agent, a PPARδ agonist, cannabinoid (CB) receptor ligands, anti-obesity serotonergic agents, adrenoceptor agonists, pancreatic lipase inhibitors, apolipoprotein-B secretion/microsomal triglyceride transfer protein (apo-B/MTP) inhibitors, melanin-concentrating hormone (MCH) receptor antagonists, neuropeptide Y (NPY) antagonists, peptide YY (PYY) agonists, orexin antagonists, glucagon-like peptide (GLP)-I agonists, melanocortin (MC) agonists, ghrelin receptor antagonists, leptin agonists, cholecystokinin (CCK) agonists, ciliary neurotrophic factors (CNTF), growth hormone (GH) secretagogues, growth hormone secretagogue receptor modulators, dipeptidyl peptidase IV (DP-IV or DPP-IV) inhibitors, histamine receptor-3 (H3) antagonists/inverse agonists, 5- hydroxytryptamine (5HT) agonists, serotonin transport or serotonin reuptake inhibitors, dopamine agonists, norepinephrine (NE) transport inhibitors, diacylglycerol acyltransferase (DAG) inhibitors, glucose transporter inhibitors, 110-hydroxy steroid dehydrogenase- 1 inhibitors, cholesterol ester transfer protein (CETP) inhibitors, squalene synthase inhibitors, for example, lapaquistat, etc.

The compositions of the present invention are administered and dosed in accordance with good medical practice, taking into account the clinical condition of the individual patient, the site and method of administration, scheduling of administration, patient age, sex, body weight and other factors known to medical practitioners. The choice of carrier will be determined in part by the particular active ingredient, as well as by the particular method used to administer the composition. Accordingly, there is a wide variety of suitable pharmaceutical compositions of the present invention.

In a particular case, said inhibitors are oligonucleotides that recognize and hybridize with the promoter region of the chemoattractant gene thereby inhibiting gene expression. In this case, said oligonucleotides may be delivered using any suitable method. In some embodiments, naked DNA is administered. In other embodiments, lipofection is utilized for the delivery of nucleic acids to a subject. In still further embodiments, oligonucleotides are modified with phosphothiolates for delivery as described, for example, in U.S. Pat. No.6,169,177. In some embodiments, nucleic acids for delivery are compacted to aid in their uptake (See e.g., U.S. Pat. Nos. 6,008,366, 6,383,811). In some embodiments, compacted nucleic acids are targeted to a particular cell type (e.g., adipocytes). In some embodiments, oligonucleotides are conjugated to other compounds to aid in their delivery. For example, in some embodiments, nucleic acids are conjugated to polyethylene glycol to aid in delivery. In yet other embodiments, oligonucleotides are conjugated to protected graft copolymers, which are chargeable drug nano-carriers (Pharmaln). In still further embodiments, the transport of oligonucleotides into cells is facilitated by conjugation to vitamins. In other embodiments, oligonucleotides are conjugated to nanoparticles (e.g., NanoMed Pharmaceuticals; Kalamazoo, Mich.). In other embodiments, oligonucleotides are enclosed in lipids (e.g., liposomes or micelles) to aid in delivery. In still further embodiments, oligonucleotides are complexed with additional polymers to aid in delivery.

Pharmaceutical compositions or medicaments may be administered or coadministered by a wide variety of routes, including for example, orally, parenterally, intraperitoneally, intravenously, intraarterially, transdermally, sublingually, intramuscularly, rectally, transbuccally, intranasally, liposomally, via inhalation, vaginally, intraoccularly, via local delivery (for example by catheter or stent), subcutaneously, intraadipo sally, intraarticularly, or intrathecally. The compositions may also be administered or coadministered in slow release dosage forms. Dosage forms known to those of skill in the art are suitable for delivery of the compounds of the invention. Compositions are provided that contain therapeutically effective amounts of the inhibitor according to the invention. To prepare compositions, one or more inhibitors of the invention are mixed with a suitable pharmaceutically acceptable carrier. Upon mixing or addition of the compound(s), the resulting mixture may be a solution, suspension, emulsion, or the like. Liposomal suspensions may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art. The form of the resulting mixture depends upon a number of factors, including the intended mode of administration and the solubility of the compound in the selected carrier or vehicle. The effective concentration is sufficient for lessening or ameliorating at least one symptom of the disease, disorder, or condition treated and may be empirically determined.

The amount or concentration of active substance in those compositions or preparations is such that a suitable dosage in the range indicated is obtained. The compositions are preferably formulated in a unit dosage form. The term "unit dosage from" refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. In addition, the active materials can also be mixed with other active materials that do not impair the desired action, or with materials that supplement the desired action, or have another action. The compounds, i.e. the inhibitor, may be formulated as the sole pharmaceutically active ingredient in the composition or may be combined with other active ingredients.

Where the compounds normally exhibit insufficient solubility, methods for solubilizing may be used. Such methods are known and include, but are not limited to, using cosolvents such as dimethylsulfoxide (DMSO), using surfactants such as Tween.RTM., and dissolution in aqueous sodium bicarbonate. Derivatives of the compounds, such as salts or prodrugs may also be used in formulating effective pharmaceutical compositions.

The inhibitor of the invention may be prepared with carriers that protect them against rapid elimination from the body, such as time-release formulations or coatings. Such carriers include controlled release formulations, such as, but not limited to, microencapsulated delivery systems. The active compound is included in the pharmaceutically acceptable carrier in an amount sufficient to exert a therapeutically useful effect in the absence of undesirable side effects on the subject treated. Such carriers include controlled release formulations, such as, but not limited to, implants and microencapsulated delivery systems, and biodegradable, biocompatible polymers such as collagen, ethylene vinyl acetate, polyanhydrides, polyglycolic acid, polyorthoesters, polylactic acid, and the like. Methods for preparation of such formulations are known to those skilled in the art.

Compounds of the invention may also be delivered in a nano crystal dispersion formulation. Preparation of such formulations is described, for example, in U.S. Pat. No. 5,145,684. The nano crystalline formulations typically afford greater bioavailability of drug compounds.

The inhibitors and compositions of the invention can be enclosed in multiple or single dose containers. The enclosed compounds and compositions can be provided in kits, for example, including component parts that can be assembled for use. For example, a therapeutic compound in lyophilized form and a suitable diluent may be provided as separated components for combination prior to use. A kit may include an inhibitor according to the present invention and a second therapeutic agent for coadministration. The inhibitor of the invention and second therapeutic agent may be provided as separate component parts. A kit may include a plurality of containers, each container holding one or more unit dose of the inhibitor of the invention. The containers are preferably adapted for the desired mode of administration, including, but not limited to tablets, gel capsules, sustained-release capsules, and the like for oral administration; depot products, pre-fϊlled syringes, ampoules, vials, and the like for parenteral administration; and patches, medipads, creams, and the like for topical administration.

The inhibitor of the invention is administered in amounts which are sufficient to achieve the desired effect, in a preferred embodiment, an anti-obesity effect. As will be appreciated, the amount of the compound will depend on the severity of the disease, the intended therapeutic regiment and the desired therapeutic dose. An amount effective to achieve the desired effect is determined by considerations known in the art. Thus, it is appreciated that the effective amount or concentration depends on a variety of factors including the distribution profile of the compound within the body, a variety of pharmacological parameters such as half life in the body, on undesired side effects, if any, on factors such as age and gender of the subject to be treated, etc. The therapeutically effective amount or concentration may be determined empirically by testing the compounds in known in vitro and in vivo model systems for the treated disorder. The effective amount is typically tested in clinical studies having the aim of finding the effective dose range, the maximal tolerated dose and the optimal dose. The manner of conducting such clinical studies is well known to a person versed in the art of clinical development.

An amount may also at times be determined based on amounts shown to be effective in animals. It is well known that an amount of X mg/Kg administered to rats can be converted to an equivalent amount in another species (notably humans) by the use of one of possible conversions equations well known in the art. The concentration of active compound in the drug composition will depend on absorption, inactivation, and excretion rates of the active compound, the dosage schedule, and amount administered as well as other factors known to those of skill in the art.

The inhibitor may be administered at once, or may be divided into a number of smaller doses to be administered at intervals of time. It is understood that the precise dosage and duration of treatment is a function of the disease being treated and may be determined empirically using known testing protocols or by extrapolation from in vivo or in vitro test data. It is to be noted that concentrations and dosage values may also vary with the severity of the condition to be alleviated. It is to be further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that the concentration ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions.

If oral administration is desired, the inhibitor should be provided in a composition that protects it from the acidic environment of the stomach. For example, the composition can be formulated in an enteric coating that maintains its integrity in the stomach and releases the active compound in the intestine. The composition may also be formulated in combination with an antacid or other such ingredient. Oral compositions will generally include an inert diluent or an edible carrier and may be compressed into tablets or enclosed in gelatin capsules. For the purpose of oral therapeutic administration, the active compound or compounds can be incorporated with excipients and used in the form of tablets, capsules, or troches. Pharmaceutically compatible binding agents and adjuvant materials can be included as part of the composition.

The tablets, pills, capsules, troches, and the like can contain any of the following ingredients or compounds of a similar nature: a binder such as, but not limited to, gum tragacanth, acacia, corn starch, or gelatin; an excipient such as microcrystalline cellulose, starch, or lactose; a disintegrating agent such as, but not limited to, alginic acid and corn starch; a lubricant such as, but not limited to, magnesium stearate; a gildant, such as, but not limited to, colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; and a flavoring agent such as peppermint, methyl salicylate, or fruit flavoring.

When the dosage unit form is a capsule, it can contain, in addition to material of the above type, a liquid carrier such as fatty oil. In addition, dosage unit forms can contain various other materials, which modify the physical form of the dosage unit, for example, coatings of sugar and other enteric agents. The compounds can also be administered as a component of an elixir, suspension, syrup, wafer, chewing gum or the like. A syrup may contain, in addition to the active compounds, sucrose as a sweetening agent and certain preservatives, dyes and colorings, and flavors. The active materials can also be mixed with other active materials for the treatment of the disease that do not impair the desired action, or with materials that supplement the desired action. Thus, in a particular embodiment of the invention, said medicament additionally comprises another compound for the treatment of said disease for simultaneous, separate or sequential use in the treatment of said disease.

In a preferred embodiment of the invention, said other active materials or compounds are other drugs used to treat obesity. In such a combination treatment the other drug and the compound of the invention may be given to patients at the same time or at different times, depending on the dosing schedule of each of the drugs. Solutions or suspensions used for parenteral, intradermal, subcutaneous, or topical application can include any of the following components: a sterile diluent such as water for injection, saline solution, fixed oil, a naturally occurring vegetable oil such as sesame oil, coconut oil, peanut oil, cottonseed oil, and the like, or a synthetic fatty vehicle such as ethyl oleate, and the like, polyethylene glycol, glycerine, propylene glycol, or other synthetic solvent; antimicrobial agents such as benzyl alcohol and methyl parabens; antioxidants such as ascorbic acid and sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates, and phosphates; and agents for the adjustment of tonicity such as sodium chloride and dextrose. Parenteral preparations can be enclosed in ampoules, disposable syringes, or multiple dose vials made of glass, plastic, or other suitable material. Buffers, preservatives, antioxidants, and the like can be incorporated as required.

Where administered intravenously, suitable carriers include physiological saline, phosphate buffered saline (PBS), and solutions containing thickening and solubilizing agents such as glucose, polyethylene glycol, polypropyleneglycol, and mixtures thereof. Liposomal suspensions including tissue-targeted liposomes may also be suitable as pharmaceutically acceptable carriers. These may be prepared according to methods known for example, as described in U.S. Pat. No. 4,522,811.

The compounds of the invention can be administered intranasally. When given by this route, the appropriate dosage forms are a nasal spray or dry powder, as is known to those skilled in the art.

The compounds of the invention can be administered intrathecally. When given by this route the appropriate dosage form can be a parenteral dosage form as is known to those skilled in the art. The compounds of the invention can be administered topically. When given by this route, the appropriate dosage form is a cream, ointment, or patch. The compounds of the invention can be administered rectally by suppository as is known to those skilled in the art. The compounds of the invention can be administered by implants as is known to those skilled in the art. When administering a compound of the invention by implant, the therapeutically effective amount is the amount described above for depot administration.

There is nothing novel about the route of administration or the dosage forms for administering the therapeutic compounds. Given a particular therapeutic compound, and a desired dosage form, one skilled in the art would know how to prepare the appropriate dosage form for the therapeutic compound.

As mentioned above, the inventors have shown the absence of resident mesenchymal precursors (MP) or stem cells (MSC) in several solid tissues of ob/ob mice. Moreover, the present invention shows that this decrease is due to the migration of said cells from the organs of residence (muscle, lung and/or heart) to the adipose tissue.

In recent years, MSC have been increasingly given a role in tissue repair and regeneration. In different models of tissue damage, MSC improve the recovery of injured tissues, at least, through two proven mechanisms: by releasing soluble factors with anti-inflammatory activity and by promoting and potentiating local regenerative processes. Thus, chemoattractants inhibitors could be valuable therapeutic approach to improve repair mechanisms in organs like muscle, lung, heart, skin, kidney, etc. since said inhibitors could inhibit the migration of said stem cells to the adipose tissue.

Therefore, in another aspect, the present invention refers to a chemoattractant inhibitor for use as a medicament for the treatment of a tissue degenerative condition, wherein said inhibitor inhibits the migration of stem cells to the adipose tissue.

The term "tissue degenerative condition" as used herein, refers to tissue which exhibits a pathological condition. Thus, according to the present invention, said inhibitor can be used as a medicament for enhancing the proliferation, regeneration and/or engrafting of stem cells in said tissue. In a particular embodiment of the invention, said tissue degenerative condition is obesity-dependent tissue degeneration. The authors of the present invention have observed that ob/ob muscles and lungs are macro scopically much thinner than their WT counterparts. Thus, the term "obesity-dependent tissue degeneration" as used herein, refers to the enhanced degeneration of solid tissues that occur in an obese subject when compared with that one of a non-obese subject.

In a particular embodiment of the invention, said obesity-dependent tissue degenerative condition is skeletal muscle degeneration, cardiac tissue degeneration, bone tissue degeneration, neural tissue degeneration, lung degeneration, liver degeneration, kidney degeneration or more than one of said tissue degenerative conditions simultaneously.

In a particular embodiment of the invention, said stem cells are endogenous or grafted stem cells. In a particular case, said cells are autologous stem cells. In another embodiment, said cells are allogeneic stem cells. Preferably grafted cells are autologous cells, i.e. stem cells that are collected from an individual and given back to that same individual. In another particular embodiment, said stem cells are mesenchymal stem cells.

In a particular embodiment, said chemoattractant inhibitor is a cytokine inhibitor as mentioned above. In a more particular embodiment, said cytokine is an inflammatory cytokine or pro-inflammatory cytokine. In an even more particular embodiment, said inflammatory cytokine is selected from the group consisting of IL-I, TNF-alpha, IL-6,

IFN-alpha, IFN-gamma, IL-I l, TGF-beta, LIF, IL-8, PF-4, MIP-la/b, MCP-I, MCP-2,

MCP-3, Rantes, IL- 12, and Lymphotactin. In a preferred embodiment, said cytokine is TNF alpha. In another preferred embodiment, said cytokine is MCP-I.

In a particular embodiment of the invention, said inhibitor is a compound selected from the group consisting of an antibody, an antagonist, a soluble binding protein, a soluble receptor variant, a non-functional derivative, an antisense polinucleotide, a RNA interference oligonucleotide, a DNAzyme, a ribozyme, a triplex forming oligonuclotide (TFO), and a chemical or biological compound as mentioned above.

In another aspect, the invention refers to an in vitro method for the identification of compounds for the treatment of obesity and/or for the treatment of a tissue degenerative condition said method comprising i) culturing a population of stem cells in conditions allowing them to migrate toward a chemoattractant; ii) bringing into contact a cell population according to step i) with a test compound; and iii) evaluating the migration capacity of said cell population; wherein if said compound is capable of inhibiting the migration of said stem cells, indicates that the test compound may be used as a medicament for the treatment of obesity and/or a tissue degenerative condition. Methods for evaluating the migration capacity of a cell population in vitro are well known in the art. Illustrative, non limitative, examples of said methods include, the ones described in the Example accompanying the present invention. Additionally, assaying cell migration may be effected by monitoring cell movement per se. Alternatively or additionally, molecular or structural (e.g., pseudopodia formation) determinants which are known to be associated with cell migration may be assayed. Other cell migration assays which may be used in accordance with the present invention include live cell data based on time-lapse movies can be most informative for the quantification of dynamic events in individual cells or cell populations (Dai et al, 2005, Exp Cell Res 311, 272-80), phagokinetic track (PKT) formation on flat surfaces (Kawa et al., 1997, FEBS Lett 420, 196-200; Lin et al., 2005, MoI Cancer 4, 21; Scott et al., 2000, Anal Biochem 287, 343-4).

In a particular embodiment of the invention, said chemoattractant is a cytokine. In a more particular embodiment, said cytokine is an inflammatory cytokine. In a more particular embodiment, said inflammatory cytokine is selected from the group consisting of IL-I, TNF-alpha, IL-6, IFN-alpha, IFN-gamma, IL-11, TGF-beta, LIF, IL- 8, PF-4, MIP-la/b, MCP-I, MCP-2, MCP-3, Rantes, IL-12, and Lymphotactin. In a particular embodiment, said stem cells are mesenchymal stem cells. In a particular embodiment of the invention, said tissue degenerative condition is obesity-dependent tissue degeneration. In another aspect, the invention refers to a method for the in vivo identification of compounds for the treatment of obesity and/or for the treatment of a tissue degenerative condition said method comprising i) administering a test compound to a non-human animal, wherein said animal is characterized by an enhanced migration of stem cells to the adipose tissue when compared with a control animal; and ii) evaluating the distribution of said cell population in said animal; wherein if said compound is capable of inhibiting the migration of said stem cells to the adipose tissue, indicates that the test compound may be used as a medicament for the treatment of obesity and/or a tissue degenerative condition.

The administration of the test compounds can be performed by any suitable route, including, for example, oral, transdermal, intravenous, infusion, intramuscular, etc. administration.

According to the invention, said non-human animal is characterized by an enhanced migration of stem cells to the adipose tissue when compared with a control animal. In a particular embodiment, said animal is an obese animal. In another particular embodiment of the invention, said non-human animal is a genetically modified animal. More particularly, said genetically modified animal which presents an elevated migration rate of stem cells to the adipose tissue when compared with a control animal, is a leptin deficient animal. In a preferred embodiment, said animal is a leptin knock out animal (ob/ob). Thus, said control animal is an animal whose stem cells, either endogenous or grafted, distribute or spread to most adult tissues in said animal, not being the adipose tissue a preferred one.

In a particular embodiment of the invention said non-human mammal is a rodent. In a preferred embodiment, said animal is a mouse.

Methods for evaluating the migration capacity of said endogenous or grafted stem cells in said animal are well know in the state of the art and have already been described in the present document (see Example 1). In a particular case, male stem cells can be administered to a female animal or vice versa and the distribution thereof analysed. Other illustrative, non limitative, methods for evaluating said migration capacity of stem cells to a certain tissue, in particular, to the adipose tissue, are Magnetic resonance imaging (MRI) which allows in vivo monitoring of stem cells after grafting. In another particular case, said grafted stem cells are cells whose migration capacity can be easily evaluated in vivo by means of a label or selectable marker. In this sense, said grafted stem cells can be genetically modified stem cells which express a selectable marker. Usually, the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those precursor cells are then delivered to the animal and their migration capacity and distribution is analyzed. In this embodiment, the desired gene is introduced into a precursor cell prior to administration in vivo of the resulting recombinant cell. Such introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection, infection with a viral or bacteriophage vector containing the gene sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells and may be used in accordance with the present invention, provided that the necessary physiological functions of the recipient cells are not disrupted.

According to the present invention, said animal, characterized by an enhanced migration of stem cells to the adipose tissue, can be treated with the test compound(s), and any change in the distribution, number or other properties of the stem cell as a result of drug treatment, is monitored relative to untreated and/or positive control, where the positive control typically is an animal, treated with a known inhibitor compound, such as, for example, a TNF alpha antibody. In a particular embodiment of the invention, said method comprises the administration of a chemoattractant, which induces migration of said stem cells to the adipose tissue, prior to drug (test compound) treatment, i.e. before step i). In a particular embodiment, said chemoattractant is a cytokine, more particularly, a proinflammatory cytokine, even more particularly, TNF-alpha or MCP-I. In a particular embodiment, said stem cells are mesenchymal stem cells.

Results obtained in this model can then be validated by follow-up pharmacokinetic, toxicological, biochemical and immunologic studies, and ultimately human clinical studies.

In another aspect, the invention refers to the use of a non-human animal for the identification of compounds for the treatment of a tissue degenerative condition, wherein said animal is characterized by an enhanced migration of stem cells to the adipose tissue when compared with a control animal. In a particular embodiment, said stem cells are mesenchymal stem cells.

In another particular embodiment of the invention, said non-human animal according to the method of the invention is a genetically modified animal. More particularly, said genetically modified animal which presents an elevated migration rate of stem cells to the adipose tissue when compared with a control animal, is a leptin deficient animal. In a preferred embodiment, said animal is a leptin knock out animal (ob/ob).

In a particular embodiment of the invention said non-human mammal is a rodent. In a preferred embodiment, said animal is a mouse. The invention has been described in an illustrative manner, and it is to be understood that the terminology which has been used, is intended to be in the nature of words of description rather than of limitation. Obviously, many modifications and variations of the present invention are possible in light of the above teaching. It is therefore, to be understood that within the scope of the appended claims, the invention may be practiced otherwise than as specifically described hereinafter.

The following examples are provided as merely illustrative and are not to be construed as limiting the scope of the invention.

EXAMPLE

Materials and Methods

Mice. C57BL/6 and ob/ob mice were obtained from Charles and River, Co and maintained and used in accordance with the National Institutes of Health Animal Care and Use Committee.

Cells isolation and growing conditions. Three described isolation methods were used. Briefly:

1. Enzimatic digestion (collagenase-dispase mixture) of the different tissues for 30 min at 37° C, followed by centrifugation and collection of supernatants. Cells were replated in DMEM+10% FBS (fetal bovine serum) and clones grown and selected by morphology. (Goodell M.A. et al (2001) Ann. N. Y. Acad. Sci.; 938:208-220).

2. Mecanical disgregation: small pieces of tissues were disgregated using forceps and scrapels and then filtered with medium and collected cells were plated onto plates (Yu X, et al (2008) MoI Reprod Dev. Sep;75(9): 1426-32). 3. Explant technique: collection of small pieces of tissues and place them on gelatin- coated plates. Rounded cells coming out from the explants were collected, cloned and grown to obtain mesenchymal precursors (Minasi, MG., et al. (2002) Development. 129, 2773-2783) Obtained cloned cells were grown in DMEM+10% FBS+glut+pen/step on plates covered with gelatin. Cells were characterized by surface markers and caryotype (data not shown).

Injections. 5x10⁵ MSC were injected into the tail vein or into the muscle fibers with a 0.03 μm needle. Pieces of tissues of muscle, heart, liver and adipose mass were collected after 6 hours or 2 months, RNA extracted by Trizol reagent, and RT-PCR against the mouse epsilon chromosome was performed using the following primers: Yl, fw 5'-GAT GGT GCC TCA TGG AAT CT Yl, Rw 5'-AAA TAT GCC AAG AAGG AGA GCC Conditions: 4 min initial denaturation at 95⁰C. Subsequent cycles were 94⁰C for 45 s, 6O⁰C for 45 s and 72⁰C for 45 s repeated for 30 or 35 cycles and followed by a final extension of 10 min at 72⁰C). Pieces of the same tissues were kept into OCT and frozen to perform immunohistology analysis to localize the injected cells. Statistical analysis. P values were calculated using Student's t-test. Error bars in graphs represent the s.e.m. (standard error of the mean).

Flow cytometry analysis. Mesenchymal precursors isolated from WT C57 mice were analyzed by flow cytometry. The following antibodies were used in order to check de mesenchymal origin of said cells: Sca-1 (Pharmingen), CD31 (ID labs, Inc.), CD34 (BD Biosciences), CD44 (BD Biosciences), CD45 (BD Biosciences). Trasnwell analysis. Adipocytes or macrophages were grown on a p24 well-plate for 2 days. Medium with or without cytokines (10-50 ng/ml) and with or without the corresponding antibodies (10 μg/ml mAb) was added onto the plate. At the same time, 8-μm transwell filters (Corning) were coated with 1% gelatin and placed onto the plate. At the same time, 8-μm transwell filters (Corning) were coated with 1% gelatin and placed onto the plate. 10⁴ Mesenquimal stem cells were then plated in DMEM containing 2% serum on the upper side of transwell chamber. After 6 hours of transmigration, migrated cells on the lower side of the filter were fixed in 4% paraformaldehyde, stained with toloudine blue, and counted using an inverted microscope (five random fields of the lower face of the transwell membrane at 2Ox magnification). The results show migrated cells as a percentage of the total number of input cells. Results

Wt or ob/ob mice (leptin KO) explants (from muscle, heart, lung and adipose mass) were taken after surgery. Three different isolation protocols (see Methods) were assessed for explant processing before choosing the one with highest efficiency (see Table 1). Table 1. Number of MSC isolated with different techniques

Briefly, explants were cleaned in PBS, cut into small pieces and plated on gelatin type I plates. After seven to ten days, rounded bright cells started coming out from the explants and these cells were collected and grown in different mediums on

gelatin-coated plates. Mesenchymal precursors and clones thereof were easily isolated from WT mice (five different clones from adipose or muscle tissues of C57 WT mice were obtained, even with different methods).

However, as shown in Table 1 , explants from ob/ob mice did not yield any mesenchymal precursors at all. This was observed in ob/ob adipose tissue explants as well as in other tissue explants (skeletal muscle, heart and lungs). Only just one clone could be obtained from adipose obese tissue after testing 8 different ob/ob mice and non from the other tissues. Interestingly, ob/ob muscles and lungs were also macroscopically much thinner than their WT counterparts.

Mesenchymal precursors isolated from WT C57 mice can grow with a fast proliferation rate with the sole presence of DMEM + 10% FBS in the culture medium. These cells were analyzed by flow cytometry and were Sca-1+, CD31+, CD34+ CD44+, CD45- (data not shown).

The absence of mesenchymal precursors in ob/ob mice could be explained at least in two ways. Either, for unknown reasons, obese mice could be afflicted by a widespread absence of tissue mesenchymal precursors or, in ob/ob mice there is a general migration of mesenchymal stem cells from distant organs (muscle, lungs, etc.) to the adipose tissue followed by their differentiation into mature adipocytes. To investigate the latter, injected mesenchymal precursors from WT C57 males were injected into the tail vein of WT or Ob females. As shown in Figure 1, a percentage of the injected cells could be detected around different tissues after 6 hours in the control mice. Surprisingly, in obese mice, most of the precursors were found in the adipose abdominal mass. When cells were injected intra-muscularly, 6 hours after injection, most of the cells remained around the muscle in both mice, but, again, we could already visualize some precursors into the adipose mass of Ob mice. A follow-up analysis of those mice was performed two months after the injections. No male cells were detected in the adipose mass of C57 female mice. In addition, some male cells within the muscle tissue, specially in the intra-muscular injection group. In the female obese mice, however, male cells were still abundantly present in the adipose mass two months after both i.v. and i.m. injection. Moreover, these cells, independently of the tissue of origin (fat, muscle or lung), had undergone complete differentiation into mature adipocytes thus contributing to the total adipose mass.

In order to understand the migration mechanism of these cells, their migration properties in vitro was studied. In a transwell assay, mesenchymal precursors were attracted by conditioned media from adipocyte or macrophage cultures. Additionally, the migration induction capacity of several cytokines was analyzed. MCP-I and TNF-α were the best inducers of migration of mesenchymal precursors in vitro.

Blocking the activity of TNF-α with inhibitory antibodies (anti-TNF- α monoclonal antibody, Endogen, Woburn, MA) strongly reduced the number of precursors that migrated across the filter (Figure 2) in the transwell assay. Experiments using an anti-TNF-α antibody as a blocking agent in vivo confirmed the in vitro observation. Indeed, mesenchymal precursors did not migrate to the adipose abdominal area after systemically inhibiting TNF activity. In addition, a reduction in the number of vessels around the adipose mass was observed. If confirmed in other animal models of obesity as well as in human patients, altogether, these experiments suggest that controlling the activity of inflammatory cytokines, such as TNF, could be an approach to reduce the adipose hyperplasia observed in obese patients. The results herein presented have additional implications. Inhibiting mesenchymal stem cell migration with anti-adipotaxis treatment could be of further use in any regenerative therapeutic approach in these patients.

Claims

1. A chemoattractant inhibitor for use as a medicament for the treatment of obesity.

2. Chemoattractant inhibitor according to claim 1, wherein said inhibitor inhibits migration of stem cells to the adipose tissue.

3. Chemoattractant inhibitor according to claim 2, wherein said stem cells are endogenous or grafted stem cells.

4. Chemoattractant inhibitor according to any one of claims 2 or 3, wherein said stem cells are mesenchymal stem cells.

5. Chemoattractant inhibitor according to any one of claims 1 to 4, wherein said chemoattractant inhibitor is a cytokine inhibitor.

6. Chemoattractant inhibitor according to claim 5, wherein said cytokine is an inflammatory cytokine.

7. Chemoattractant inhibitor according to claim 6, wherein said inflammatory cytokine is selected from the group consisting of IL-I, TNF-alpha, IL-6, IFN-alpha, IFN- gamma, IL-I l, TGF-beta, LIF, IL-8, PF-4, MIP-la/b, MCP-I, MCP-2, MCP-3, Rantes, IL- 12, and Lymphotactin.

8. Chemoattractant inhibitor according to claim 7, wherein said cytokine is TNF alpha.

9. Chemoattractant inhibitor according to claim 7, wherein said cytokine is MCP-I .

10. Chemoattractant inhibitor according to any one of claims 1 to 9, wherein said inhibitor is a compound selected from the group consisting of an antibody, an antagonist, a soluble binding protein, a soluble receptor variant, a non-functional derivative, an antisense polinucleotide, a RNA interference oligonucleotide, a DNAzyme, a ribozyme, a triplex forming oligonuclotide (TFO), and a chemical or biological compound.

11. Chemoattractant inhibitor for use as a medicament for the treatment of a tissue degenerative condition, wherein said inhibitor inhibits the migration of stem cells to the adipose tissue.

12. Chemoattractant inhibitor according to claim 11, wherein said tissue degenerative condition is obesity-dependent tissue degeneration.

13. Chemoattractant inhibitor according to any one of claims 11 to 12, wherein said tissue degenerative condition is skeletal muscle degeneration, cardiac tissue degeneration, bone tissue degeneration, neural tissue degeneration, lung degeneration, liver degeneration, kidney degeneration or more than one of said tissue degenerative conditions simultaneously.

14. Chemoattractant inhibitor according to any one of claims 11 to 13, wherein said stem cells are endogenous or grafted stem cells.

15. Chemoattractant inhibitor according to any one of claims 11 to 14, wherein said stem cells are mesenchymal stem cells.

16. Chemoattractant inhibitor according to any one of claims 11 to 15, wherein said chemoattractant inhibitor is a cytokine inhibitor.

17. Chemoattractant inhibitor according to claim 16, wherein said cytokine is an inflammatory cytokine.

18. Chemoattractant inhibitor according to claim 17, wherein said inflammatory cytokine is selected from the group consisting of IL-I, TNF-alpha, IL-6, IFN-alpha,

IFN-gamma, IL-I l, TGF-beta, LIF, IL-8, PF-4, MIP-la/b, MCP-I, MCP-2, MCP-3, Rantes, IL- 12, and Lymphotactin.

19. Chemoattractant inhibitor according to claim 18, wherein said cytokine is TNF alpha.

20. Chemoattractant inhibitor according to claim 18, wherein said cytokine is MCP-I.

21. Chemoattractant inhibitor according to any one of claims 11 to 20, wherein said inhibitor is a compound selected from the group consisting of an antibody, an antagonist, a soluble binding protein, a soluble receptor variant, a non-functional derivative, an antisense polinucleotide, a RNA interference oligonucleotide, a

DNAzyme, a ribozyme, a triplex forming oligonuclotide (TFO), and a chemical or biological compound.

22. An in vitro method for the identification of compounds for the treatment of obesity and/or for the treatment of a tissue degenerative condition said method comprising i) culturing a population of stem cells in conditions allowing them to migrate toward a chemoattractant; ii) bringing into contact a cell population according to step i) with a test compound; and iii) evaluating the migration capacity of said cell population; wherein if said compound is capable of inhibiting the migration of said stem cells, indicates that the test compound may be used as a medicament for the treatment of obesity and/or a tissue degenerative condition.

23. Method according to claim 22, wherein said chemoattractant is a cytokine.

24. Method according to claim 23, wherein said cytokine is an inflammatory cytokine.

25. Method according to claim 24, wherein said inflammatory cytokine selected from the group consisting of IL-I, TNF-alpha, IL-6, IFN-alpha, IFN-gamma, IL-11, TGF- beta, LIF, IL-8, PF-4, MIP-la/b, MCP-I, MCP-2, MCP-3, Rantes, IL-12, and Lymphotactin.

26. Method according to any one of claims 22 to 25, wherein said stem cells are mesenchymal stem cells.

27. Method according to anyone of claims 22 to 26, wherein said tissue degenerative condition is obesity-dependent tissue degeneration.

28. A method for the in vivo identification of compounds for the treatment of obesity and/or for the treatment of a tissue degenerative condition said method comprising i) administering a test compound to a non-human animal, wherein said animal is characterized by an enhanced migration of stem cells to the adipose tissue when compared with a control animal; and ii) evaluating the distribution of said cell population in said animal; wherein if said compound is capable of inhibiting the migration of said stem cells to the adipose tissue, indicates that the test compound may be used as a medicament for the treatment of obesity and/or a tissue degenerative condition.

29. Method according to claim 28, wherein said non-human animal is a genetically modified animal.

30. Method according to any one of claims 28 to 29, wherein said stem cells are mesenchymal stem cells.

31. Use of a non-human animal for the identification of compounds for the treatment of a tissue degenerative condition, wherein said animal is characterized by an enhanced migration of stem cells to the adipose tissue when compared with a control animal.

32. Use according to claim 31, wherein said non-human animal is a genetically modified animal.

33. Use according to any one of claim 31 to 32, wherein said stem cells are mesenchymal stem cells.