MXPA99009972A - Leptin as an inhibitor of tumor cell proliferation - Google Patents

Abstract

The use of leptin and leptin-related molecules in oncology is disclosed.

Description

LEPTINE AS TUMOR CELL PROLIFERATION INHIBITOR FIELD OF THE INVENTION The present invention relates to leptin, a cytokine produced by adipocytes and which affects a cell and tissue vapedad. More particularly, the present invention also relates to novel applications of leptin in the field of oncology.

ANTECEDENTS OF THE 1NVECION Leptin, an adipocyte-derived cytokine that regulates body weight, was identified by positional cloning of the murine ob gene (Zhang et al., 1994) and was shown to affect food interference and thermogenesis (Campfield et al. ., 1995, Collins et al., 1996, Halaas et al., 1995, Pelleymounter et al., 1995, Weigle et al., 1995). The high affinity leptin binding sites were located in the choroid plexus, cloning of cDNA expression from said tissue provided the leptin receptor (OB-R) (Tartaglia et al., 1995). The known activities of leptin were mediated through its receptor in the hypothalamus. The leptin receptors were expressed, however, in additional organs, mainly the kidney, lung and liver (Cioffi et al., 1996, Lee et al., 1996, Tartaglia et al., 1995). In addition, a different repertoire of splice variants of leptin receptor, which differs in its cytoplasmic domain, was expressed in a specific tissue form in the mouse (Lee et al., 1996). Therefore, in addition to controlling the interference of food and body heat, leptin can exert other physiological functions. Although leptin is produced by adipocytes, the recent discovery of a correlation between excess fat and high levels of leptin in the serum was contrary to the notion that leptin reduces food interference and body weight (Considine et al. ., 1996, Frederich et al., 1995, Lonnqvist et al., 1995, Maffei et al., 1995). This correlation and the well-established linkage between obesity and insulin resistance (Felber and Golay, 1995), suggested that leptin can modulate insulin-regulated responses. In fact, it was recently reported that leptin significantly reduced basal and insulin-induced tyrosine phosphorylation of the substrate-1 insulin receptor (IRS-1). This effect of leptin on IRS-1 phosphorylation was specific, since phosphorylation with β-chain tyrosine of the insulin receptor (IR) was not reduced (Cohen et al., 1996). Tyrosine phosphorylation of IRS-1 by IR kinase is a key step in the insulin receptor signaling cascade, leading to several of the known insulin activities (Araki et al., 1994; Cheatham and Kahn, 1995; and others, 1994, Myers et al., 1994, Myers and White, 1993, Rose et al., 1994, Tamemoto et al., 1994, White and Kahn, 1994). Signaling towards the 3 'end of IRS-1 is mediated by several associated proteins, one of which is the associated binding protein 2 of growth factor receptor (GRB2) (Cheatham and Kahn, 1995). The insulin receptor (IR) is considered as a metabolic receptor, which is mediated by the effects of insulin in the homeostasis of glucose. As such, it is expressed in terminally differentiated tissues such as adipose tissue, liver and muscle. However, several studies have shown that IR is a powerful mitogenic receptor in vitro and in vivo when expressed in tumor cells. For example, functional IRs were identified in several breast cancer cell lines, as determined by IR tyrosine phosphorylation in response to insulin treatment. In addition, IR measured a mitogenic response in these cells, as determined by [3H] thymidine incorporation (Milazzo et al., 1997; Millazzo et al., 1992). So far, in general, the use of leptin in the field of oncology has not been described, and in particular, leptin has not been described as being useful for the inhibition of cell proliferation, especially proliferating cancer cells.

BRIEF DESCRIPTION OF THE INVENTION It is an object of the present invention to provide the use of leptin as an inhibitor of cell proliferation, for example, for the use of leptin as an inhibitor of the proliferation of cancer cells. It is another object of the invention to provide the use of leptin alone or in combination with other therapeutic agents for the treatment of various malignancies. It is another object of the present invention to provide the use of leptin, leptin fusion proteins, leptin muteins, leptin receptor agonists, active fragments or fractions of any of the foregoing, active analogues or derivatives of any of them, salts of any of them and mixtures of any of them, for the treatment of various malignancies. It is yet another object of the present invention to provide pharmaceutical compositions containing one or more of leptin, leptin fusion proteins, leptin mutein, leptin receptor agonist, fragments or active fractions of any of the foregoing, active analogues or derivatives of any of them, and salts of any of the above, for the treatment of various malignancies. Other objects of the present invention will be described hereinafter or will be readily apparent from the following description.

The present invention provides the use of leptin as an inhibitor of cell proliferation. Leptin can be useful, er alone or in combination with other therapeutic agents or approaches, for the treatment of various malignancies. A preferred embodiment of the invention is the use of leptin to inhibit the proliferation of human breast carcinoma cells. Proliferation of several types of tumor cells is increased in the presence of several growth factors such as insulin and IGF-I. The stimulatory effect of growth of insulin and IGF-I in cells is mediated, at least in part, by IRS-1 / GRB2 (Myers et al., 1993). Said route is inhibited by leptin. In addition, IRS-1 is a substrate of additional growth factor receptor kinases and cytokines, including IL-7 and IL-9 (Pernis et al., 1995; Yin et al., 1995; Yin et al., 1994). . Therefore, leptin can inhibit the mitogenic responses of some or all of the aforementioned growth factors and cytokines, as well as other growth factors, thus inhibiting the proliferation of a variety of tumor cells. Examples provided include the inhibition of IGF-I-induced proliferation and the insulin-induced proliferation of human breast cancer cell lines T-47D and MCF7. The present invention also provides the use of leptin, leptin fusion proteins, leptin muteins, leptin receptor agonists, or active fragments or fractions of any thereof, and salts of any thereof as well as pharmaceutical compositions containing leptin , leptin fusion proteins, leptin mutein, leptin receptor agonists, active fragments or fractions of any of them, or salts of any of them for the treatment of various malignancies. More specifically, the present invention provides the use of an active agent selected from the group consisting of leptin, leptin fusion proteins, leptin mutein, leptin receptor agonists, fragments or active fractions of any of them, active analogues or derivatives of any of them, salts of any of them, and mixtures of any of them, as an inhibitor of tumor cell proliferation. The embodiments of the prior aspect of the present invention include: (i) The use of the above active agent as inhibitor of cell proliferation for the treatment of malignancies in mammals. (ii) The use of the above active agent as a tumor inhibitor dependent on growth factor. (iii) The use of the above active agent as an inhibitor of proliferation of human breast carcinoma cells. (iv) The use of the above active agent for the treatment of human breast carcinomas. (v) The use of the previous agent as an inhibitor of the insulin-stimulating effect and IGF-I in tumor cells, as measured, at least partially, via substrate-1 (IRS-1) of the insulin receptor / protein-2 binding partner of growth factor receptor (GRB2). (vi) The use of the above active agent as an inhibitor of mitogenic responses in tumor cells to one or more receptor kinases, growth factors and cytokines from the group consisting of IL-4 and IL-9, for which IRS- 1 is a substrate, for the treatment of tumors. (vii) The use of the above active agent as a basal inhibitor of tumor cell proliferation, induced by IGF-1 and induced by insulin for the treatment of human breast cancers. (viii) The use of the above active agent wherein said active ingredient is leptin, and said leptin is used as said inhibitor or for said treatment. Likewise, the present invention also provides an active agent selected from the group consisting of leptin, leptin fusion and protein, leptin mutein, leptin receptor agonists, active fragments or fractions of any of the same active analogues or derivatives of leptin. any of them, salts of any of them, and mixtures of any of them, for use in the preparation of a medicament for the inhibition of the proliferation of tumor cells. The embodiments of said aspect of the invention include: (i) an active agent as above for use in the preparation of a medicament for the treatment of malignancies in mammals. (ii) an active agent as above for use in the preparation of a medicament for the inhibition of growth factor-dependent tumors. (iii) an active agent as above for use in the preparation of a medicament for inhibiting the proliferation of human breast carcinoma cells. (iv) an active agent as above for use in the preparation of a medicament for the treatment of human breast carcinomas. (v) an active agent as above for use in the preparation of a medicament for the inhibition of the growth stimulating effect on IGF-I and insulin in tumor cells, as measured, at least partially, by IRS-1 / GRB2. (vi) an active agent as above for use in the preparation of a medicament for the inhibition of mitogenic responses in tumor cells to one or more receptor kinases, growth factors and cytokines of the group consisting of IGF-I, IL -4 and IL-9, for which IRS-1 is a substrate, for the treatment of tumors. (vii) an active agent as above for use in the preparation of a medicament for the inhibition of basal proliferation of tumor cells, induced by IGF-I and by insulin, for the treatment of human breast cancers. (viii) an active agent as above wherein said active agent is leptin, and said leptin is used for the preparation of said medicament. Similarly, in another aspect, the present invention provides a pharmaceutical composition comprising an active ingredient, an active agent as stated above and a pharmaceutically acceptable carrier, diluent or excipient, for inhibiting the proliferation of tumor cells. The embodiments of said aspect of the invention include: (i) a pharmaceutical composition for the treatment of malignancies in mammals. (ii) a pharmaceutical composition for the inhibition of tumors dependent on growth factor. (iii) a pharmaceutical composition for the inhibition of the proliferation of human breast carcinoma cells and therefore for the treatment of human breast carcinoma. (V) a pharmaceutical composition for the inhibition of the growth stimulating effect of IGF-I and insulin in tumor cells, as measured, at least in part, by the IRS-1 / GRB2 pathway. (v) a pharmaceutical composition for the inhibition of mitogenic responses in tumor cells to one or more receptor kinases, growth factors and cytokines from the group consisting of IL-4 and IL-9, for which IRS-1 is a substrate, and therefore for the treatment of tumors. (vi) a pharmaceutical composition for the inhibition of the basal proliferation of tumor cells, induced by IGF-I and by insulin, and in this way for the treatment of human breast cancers. (vii) a pharmaceutical composition wherein said active ingredient is leptin. The present invention also provides a method for the treatment of tumors in mammals or for inhibiting the proliferation of tumor cells in mammals, which comprises administering to a patient a pharmaceutical composition according to the invention, as stated above, in a appropriate dosage form and by an appropriate route of administration. Said dosage forms and routes of administration are usually determined by professional practitioners after the examination of the patient. Other aspects and embodiments of the present invention are described or will be readily apparent from the following detailed description of the invention.

DESCRIPTION PE THE FIGURES Figure 1 shows the dependence of proliferation of T-47D cells on insulin as determined by MTT staining. Figure 2 shows the dependence of T-47D cell on IGF-I as determined by MTT staining.

Figure 3 shows the inhibition of cell proliferation Insulin-induced T-47D in 10% fetal bovine serum (FBS) by murine leptin as determined by MTT staining. Figure 4 shows the inhibition of insulin-induced T-47D cell proliferation in 2% FBS by murine leptin as determined by crystal violet staining. Figure 5 shows the inhibition of T-47D cell proliferation induced by IGF-I in 10% FBS by murine leptin as determined by MTT staining. Figure 6 shows the inhibition of T-47D cell proliferation induced by IGF-I in 2% FBS by murine leptin as determined by crystal violet staining. Figure 7 shows the inhibition of T-47D cell proliferation induced by IGF-I in 2% FBS by human leptin as determined by crystal violet staining. Figure 8 shows the inhibition of insulin-induced MCF7 cell proliferation in the absence of serum by murine leptin as determined by crystal violet staining. Figure 9 shows the inhibition of MCF7 cell proliferation induced by IGF-I in the absence of serum by murine leptin as determined by crystal violet staining.

DETAILED DESCRIPTION OF THE INVENTION The present invention relates to the use of leptin as an inhibitor of the proliferation of tumor cells. Typically, cell lines of human origin, derived from various tumors, can be grown in a culture in the presence of a growth medium supplemented with fetal bovine serum at a concentration of about 10% by volume. The proliferation of the cell under said conditions is defined below as "basal cell proliferation". Ef growth of several tumor cell lines is significantly boosted when several growth factors such as insulin, epidermal growth factor or insulin-like growth factor-1 (IGF-I) are added to the culture medium supplemented with serum . The inclusion of leptin in growth media at a scale of concentrations of 3 to 600 nanomolar produces basal proliferation of cells and the proliferation of growth factor-dependent cells. The results of the cell culture experiments given in the following examples indicate that leptin is useful for inhibiting the growth of various tumors. Therefore, leptin can be useful for the treatment of several malignancies. In a preferred embodiment of the present invention, leptin is used for the inhibition of breast cancer cell proliferation. When leptin is added to the cultures of human ductal breast carcinoma T-47D cells (American Type Culture Collection, Rockville, MD, strain No. ATCC HTB 133), its extension of proliferation is reduced. Similarly, when leptin is added to cultures of human breast adenocarcinoma MCF7 cells (American Type Culture Collection, Rockville, MD, strain No. ATCC HTB 22), its extension of proliferation is reduced. Leptin inhibits the proliferation induced by IGF-I and the insulin-induced proliferation of T-47D and MCF7 cells. Therefore, leptin may be useful specifically for the treatment of breast carcinomas. The stimulatory effect of insulin growth and IFG-I in the cells is mediated, at least in part, by the tyrosine phosphorylation of IRS-1 and the subsequent association of IRS-1 with GRB2, leading to a mitogenic response. The antimitogenic effect of leptin may result from its ability to reduce insulin-induced tyrosine phosphorylation and induced IGF-I by IRS-1, leading to linkage of GRB2 to IRS-1. In addition, IRS-1 is a substrate of receptor kinases of other growth factors and cytokines, including IL-4 and IL-9 (Pernis et al 1995, Yin et al., 1995, Yin et al., 1994). Therefore leptin can inhibit the mitogenic responses of some or all of the aforementioned growth factors and cytosines, as well as other mitogenes, thus inhibiting the proliferation of a variety of tumor cells. The present invention further relates to leptin derivatives and analogues, including leptin fusion proteins, leptin muteins, leptin receptor agonists, or active fractions thereof, and salts thereof, and pharmaceutical compositions containing leptin, leptin fusion proteins, leptin muteins, leptin receptor agonist, active fractions thereof, or salts of the above for the treatment of various cancers. As used herein, the term "muteins" refers to the analogue of leptin, in which one or more of the amino acid residues is replaced by different amino acid residues, or deleted, or one or more amino acid residues are added to the original leptin sequence without significantly changing the activity of the resulting products as compared to wild-type leptin or its fragments or active fractions. Said muteins are prepared by known synthesis and / or by site-directed mutagenesis techniques, or by another known technique suitable for the foregoing. Any mutein preferably has a sufficiently duplicative amino acid sequence of the leptin to have activity substantially similar to leptin or its fragments or active fractions. In this way, it can be determined if any given mutein has substantially the same activity as leptin by routine experimentation comprising the subjecting of a mutein, for example, to a single cell proliferation test, while the mutein which blocks the proliferation of the cell retains sufficient leptin activity and therefore has at least one of the described utilities of leptin and thus has a substantially similar activity thereof. In a preferred embodiment, any mutein has at least 40% identity or homology with the sequence of one of the leptins. More preferably, it has at least 50%, at least 60%, at least 70%, at least 80%, or more preferably, at least 90% identity or homology thereof. Leptin muteins or their active fragments or fractions that can be used according to the present invention, or nucleic acid coding thereof, include a finite set of substantially corresponding sequences such as substitution peptides or polynucleotides that can be commonly obtained by a expert in the art, without undue experimentation, based on the teachings and guides presented herein. For a detailed description of the chemistry and structure of the protein, see Schuiz, G.E. and others., Principies of Protein Structure, Springer-Verlag, New York, 1978; and Creighton, T.E., Proteins: Structure and Molecular Properties, W.H. Freeman & Co., San Francisco, 1983, which are incorporated herein by reference. For a presentation of nucleotide sequence substitutions, such as codon preferences, see Ausubel et al., Supra, in § § 6.3 and 6.4 (1987, 1992) in Appendices C and D. Preferred changes for muteins in accordance with the present invention are what are known as "conservative" substitutions. Conservative amino acid substitutions of leptin polypeptides or proteins or their fragments or active fractions may include synonymous amino acids within a group having sufficiently similar physicochemical properties, so that substitution between members of a group will retain the biological function of the molecule, Grantham, Science, Vol 185, pp. 862-864 (1974). It is clear that the insertions and deletions of amino acids can be made in the sequences defined above without altering their function, particularly if the insertions or deletions only involve a few amino acids, for example, under thirty and preferably under ten, and do not remove or they displace the amino acids that are critical for a functional conformation, that is, residues of cysteine, Anfinsen, "Principies That Govern The Folding of Protein Chains". Science, Vol. 181. pp. 223-230 (1973). The proteins and muteins produced by said deletions and / or insertions are within the scope of the present invention. Preferably, the synonymous amino acid groups are those defined in Table I. More preferably, the synonymous amino acid groups are those defined in Table II; and more preferably the synonymous amino acid groups are those defined in Table III.

TABLE I Preferred amino acid groups synonyms Amino Acid Group Synonym Ser Ser, Thr, Gly, Asn Arg Arg, Gln, Lys, Glu, His Leu lie, Phe, Tyr, Met, Val, Leu Pro Gly, Wing, Thr, Pro Thr Pro, Ser, Wing, Gly, His, Gln, Thr Ala Gly, Thr, Pro, Val Met Wing, Tyr, Phe, Lie, Leu, Val Gly Wing, Thr, Pro, Ser, Gly 10 lie Met, Tyr, Phe, Val, Leu, lie Phe Trp , Met, Tyr, Lie, Val, Leu, Phe Tyr Trp, Met, Phe, Lie, Val, Leu, Tyr Cys Ser, Thr, Cys His Glu, Lys, Gln, Thr, Arg, His Gln Glu, Lys, Asn , His, Thr, Arg, Gln Asn Gln, Asp, Ser, Asn Lys Glu, Gln, His, Arg, Lys Asp Glu, Asn, Asp Glu Asp, Lys, Asn, Gln, His, Arg, Glu Met Phe, Lie, Val, Leu, Met Trop TABLE II Most preferred amino acid groups Amino Acid Group Synonym Ser Ser Arg His, Lys, Arg Leu Leu, Lie, Phe, Met Pro Ala, Pro Thr Thr Ala Pro, Ala Val Val, Met, Lie Gly Lie, Met, Phe, Val, Leu Phe Met, Tyr, Lie, Leu, Phe Tyr Phe, Tyr Cys Cys, Ser His His, Gln, Arg Gln Glu, Gln, His Asn Asp, Asn Lys Lys, Arg Asp Asp, Asn Glu Glu, Gln Met Met, Phe, Me, Val, Leu Trp Trp PICTURE Most preferred amino acid groups Amino Acid Group Synonym Ser Ser Arg Arg Leu Leu, He, Met Pro Pro Thr Thr Wing Wing Val Val Gly Lie, Met, Leu Phe Phe Tyr Tyr Cys Cys, Ser His His Gln Gln Asn Asn Lys Lys Asp Asp Glu Glu Met Met, Me, Leu Trp Met Examples of production of amino acid substitutions in proteins that can be used to obtain leptin muteins or their active fractions for use in the present invention include any of the steps of the known method, such as is presented in the patents of E.U.A. RE 33,653, 4,959,314, 4,588,585 and 4,737,462 to Mark et al .; 5,166,943 to Koths et al., 4,965,195 to Yam and others, 4,879.1 1 to Chong et al .; and 5,017,691 to Lee and others; and substituted lysine proteins presented in the US patent. No. 4,904,584 (Shaw et al.).

In another preferred embodiment of the present invention, any leptin mutein or its active fractions for use in the present invention has an amino acid sequence that essentially corresponds to that of leptin. The term "essentially corresponding to" is intended to understand the proteins with minor changes to the natural protein sequence that does not affect the basic characteristics of the natural proteins, particularly so far with respect to their ability to inhibit the proliferation of the cell. The type of changes that are generally considered within the language "corresponding essentially to" are those that could result from the conventional mutagenesis techniques of DNA encoding leptin, resulting in some minor modifications, and selecting the desired activity in the form described above. Muteins according to the present invention include proteins encoded by a nucleic acid, such as DNA or RNA, which hybridizes to the DNA or RNA encoding leptin according to the present invention, under severe conditions. Said nucleic acid may be a primary candidate for determining whether it encodes a polypeptide that retains the functional activity of leptin of the present invention. The term "severe conditions" refers to hybridization and subsequent washing conditions to which those skilled in the art conventionally refer to as "severe." See Ausubel et al., Current Protocols in Molecular Bioloqy, supra, Interscience, NY, §§6.3 and 6.4 (1987, 1992), and Sambrook et al., Supra.

Without limitation, examples of severe conditions include wash conditions of 12-20 ° C below the calculated Tm of the hybrid under study in, for example, 2 x SSC and 0.5% SDS for 5 minutes, 2 x SSC and 0.1% of SDS for 15 minutes; 0.1 x SSC and 0.5% SDS at 37 ° C for 30-60 minutes and then 0.1 x SSC and 0.5% SDS at 68 ° C for 30-60 minutes. Those skilled in the art understand that severe conditions also depend on the length of DNA sequences, oligonucleotide probes (such as 10-40 bases) or mixed oligonucleotide probes. If mixed probes are used, it is preferred to use tetramethylammopium chloride (TMAC) instead of SSC. See Ausubel, supra. The term "leptin fusion proteins" or simply "fused protein" refers to a polypeptide comprising leptin or its active fractions or a mutein thereof, fused with another protein which, for example, has a long residence time in body fluids The leptin or its active fractions can be fused in this way to another protein, polypeptide or the like. The term "salts" refers herein to the salts of carboxyl groups and to the acid addition salts of leptin amino groups, their active fractions, muteins, or leptin fusion proteins thereof. The salts of a carboxyl group can be formed by methods known in the art and include inorganic salts, for example, sodium, calcium, ammonium, ferric or zinc salts, and the like, and salts with organic bases such as those formed, for example, with amines, such as triethanolamine, arginine or lysine, piperidine, procaine and the like. Acid addition salts include, for example, salts with mineral acids such as, for example, hydrochloric acid or sulfuric acid, and salts with organic acids such as, for example, acetic acid or oxalic acid. Obviously, any of said salts must have an activity substantially similar to leptin or its active fractions. "Functional derivatives", as used herein, encompass leptin derivatives or their active fragments or fractions and their muteins and leptin fusion proteins, which can be prepared from functional groups that occur as side chains in the residues of N- or C-terminal groups, by methods known in the art, and are included in the invention as long as they remain pharmaceutically acceptable, that is, they do not destroy the activity of the protein which is substantially similar to the activity of leptin, and do not confer toxic properties in the compositions that contain them. Such derivatives can, for example, include polyethylene glycol side chains which can cover antigenic sites and extend the residence of leptin or its active fractions in body fluids. Other derivatives include aliphatic esters of the carboxyl groups, amides of the carboxyl groups by reaction with ammonia or with primary or secondary amines, N-acyl derivatives of the free amino groups of the amino acid residues formed with acyl moieties (i.e. carbocyclic alkanoyl or aroyl) or O-acyl derivatives of the free hydroxyl groups (e.g., seryl or treoniio residues) formed with acyl moieties. As "fragments or active fractions" of leptin, leptin muteins and leptin fusion proteins, the present invention covers any fragment or precursors of the leptin polypeptide chain, or fused proteins containing any leptin fragment, alone or together with associated molecules or residues bound thereto, i.e., sugar or phosphate residues, or aggregates of any of the above derivatives, with the proviso that said fraction has an activity substantially similar to leptin. The present invention also relates to the use of natural and synthetic leptin receptor agonists, which are essentially similar to leptin in their ability to inhibit cell proliferation. Said agonists can be selected from a peptide library, a peptide analogue library or a random library of organic molecules. The selection is made by methods known in the art, essentially by the ability of the agonists selected to bind to the leptin receptor. For example, a library of random peptides can be prepared as prokaryotic expression plasmids carrying a DNA coding for a random peptide, fused to a carrier protein. Another example is a phage display system in which the expression system is a phage containing DNA encoding a random peptide and incorporated into one of the outer phage proteins. The phage encoding the fused peptide agonists or antagonists are isolated from the phage library, for example, by visualizing on surfaces coated with the leptin receptor. The bound phages are then isolated and amplified in bacteria. Several rounds of visualization-amplification are usually required in order to obtain phage expressing fused peptides that have high affinity to the leptin receptor. The isolated phage is then amplified and the DNA sequence encoding the peptide is determined. Alternatively, random peptide libraries or libraries of other molecules are prepared by solid phase synthesis in polymeric beads by methods known in the art. Globules carrying a peptide or other molecule having affinity to the leptin receptor are selected from the library, for example, by ligation of the labeled leptin receptor, ie, fluorescently labeled leptin receptor. The positive globules are then collected in the structure of the peptide or another molecule present in the globule is determined. If the globule carries a peptide, the peptide sequence is determined by the analysis of protein sequences. If the globule is representative of a random library of organic molecules then the molecule is separated from the globule and its structure is determined by methods known in the art, such as mass spectrometry, nuclear magnetic resonance and the like. Candidate peptides identified by their affinity to the leptin receptor are then selected for their ability to inhibit cell proliferation in the aforementioned manner.

Accordingly, leptin, its active fractions, the leptin muteins, the leptin fusion proteins, the leptin receptor agonists and their salts, functional derivatives, and fragments or active fractions thereof are indicated for the treatment of various malignancies. , preferably for tumors dependent on the growth factor and more preferably for breast carcinomas. The present invention further relates to the use of pharmaceutical compositions comprising a pharmaceutically acceptable carrier and leptin of the invention, and their active muteins, functioning proteins, leptin receptor agonists and their salts, functional derivatives or active fractions thereof. The pharmaceutical compositions of the invention are prepared for administration by the mixture of leptin or its derivatives, or leptin receptor agonists with physiologically acceptable carriers and / or stabilizers and / or excipients, and prepared in dosage form, that is, by lyophilization in dose jars. The method of administration can be by any of the accepted modes of administration for similar agents and will depend on the condition to be treated, i.e., intravenously, intramuscularly, subcutaneously, by local injection or. topical application, or continuously by infusion, etc. The amount of active compound to be administered will depend on the route of administration, the disease to be treated and the condition of the patient. Local injection, for example, will require a lower amount of the protein based on body weight that requires intravenous infusion. Typical active amounts of leptin to be injected are 0.1-1000 μm / kg body weight and preferably 1 to 10 μm / kg. The active amounts of leptin derivatives and leptin receptor agonists can be essentially the same as those of leptin on a molar basis. Leptin can be administered to patients with cancer, i.e., by injection, either alone or in combination with other therapeutic agents or in combination with other therapeutic approaches. The invention will now be illustrated by the following non-limiting examples: EXAMPLE 1 Determination of cell proliferation by MTT staining Reagents: Supply material 5 mg / ml bromide of (3- [4,5-dimethylthiazole-of MTT 2-yl] .2,5-diphenyltetrazolium bromide in phosphate buffered saline, stored at 120 ° C until Use: Solvent: Conc HCl (450) microliters in 2-propanol (100 ml) Procedure: Cultivate the cells in 96-well plates in the presence of various growth stimulants and growth inhibitors, at a desired time, add material of storage of MTT (10 microliters) to each cavity, incubate for 2.5-3 hours at 37 ° C. Vacuum the supernatant using a fine calibration needle, add the solvent (100 microliters) and read the absorbance with a reader. microplate using a 570 nM filter with background subtraction at 630 nm.

EXAMPLE 2 Determination of cell proliferation by crystal violet staining Procedure Cultivate the cells in 96-well plates in the presence of various growth stimulants and growth inhibitors. At the desired time add 12.5% glutaraldehyde (40 microliters) to each well. Incubate for 30 minutes at room temperature. The microplate was then washed with water, dried and aqueous crystal violet (0.1%, 0.1 ml) was added to each well. The microplate was further incubated for 30 minutes, washed with water and read at 540 nm with background subtraction at 630 nm.

EXAMPLE 3 Determination of insulin-dependent T-47D cell proliferation Human T-47D cells (American Type Culture Collection, Rockville, MD, strain ATCC HTB 133) were seeded in 96-well plates at 3 x 10 5 cells / ml in DMEM and 10% fetal bovine serum (FBS) ), 0.1 ml per cavity. Human insulin was added to different wells at increasing concentrations, the plates were incubated for 72 hours and the cell number was then determined by MTT staining (Figure 1). The results were an average of 8 replicates. Based on the extent of cell proliferation as shown in Figure 1, a 50 nm concentration of insulin was used for further studies.

EXAMPLE 4 Determination of proliferation of T-47D cells dependent on IGF-I Human T-47D cells were seeded in 96-well plates at 3 x 10 5 cells / ml in DMEM and 10% FBS, 0.1 ml per well. The human IGF-I was added to different wells at increasing concentrations, the plates were incubated for 3 days and the cell number was then determined by MTT staining (Figure 2). The results were an average of 8 replicates. Based on the extent of cell proliferation as shown in Figure 2, a concentration of 50 ng / ml IGF-I was used for further studies.

EXAMPLE 5 Inhibition of insulin-induced T-47D cell proliferation by leptin T-47D cells (3 x 10 5 cells / ml) in DMEM supplemented with 10% FBS were plated in 96-well plates (0.1 ml per well). The cells were treated with insulin (50 nM), with or without the indicated concentrations of murine leptin. Plates were incubated at 37 ° C in 5% CO2 for 48 hours. The cells were then stained with MTT. The data are mean ± standard error (SE n = 8) The results showed that leptin significantly inhibited insulin-induced cell proliferation (figure 3). T-47D cells (3 x 10 5 cells / ml) in DMEM supplemented with 10% FBS were plated in 96-well plates (0.1 ml per well). After one day, the medium was replaced with DMEM supplemented with 2% FBS and after one day the cells were treated with insulin (50 nM), with or without the indicated concentrations of murine leptin in DMEM-2% FBS. Plates were incubated at 37 ° C in 5% CO2 for 48 hours. The cells were then stained with crystal violet. The data are mean ± SE, (n = 8). The results showed that leptin significantly inhibited insulin-induced cell proliferation (Figure 4).

EXAMPLE 6 Inhibition of T-47D cell proliferation induced by leptin 1GF-I T-47D cells (3 x 10 5 cells / ml) in DMEM supplemented with 10% FBS were plated in 96-well plates (0.1 ml per well). The cells were treated with IGF-I (50 ng / ml), with or without the indicated concentrations of murine leptin. Plates were incubated at 37 ° C in 5% CO2 for 48 hours. The cells were then stained with MTT. The data are mean ± standard error (SE n = 8) The results showed that leptin significantly inhibited cell proliferation induced by IGF-I (figure 5). T-47D cells (3 x 10 5 cells / ml) in DMEM supplemented with 10% FBS were plated in 96-well plates (0.1 ml per well). After one day, the medium was replaced with DMEM supplemented with 2% FBS and after one day the cells were treated with IGF-I (50 ng / ml), with or without the indicated concentrations of murine leptin in DMEM- 2% FBS. Plates were incubated at 37 ° C in 5% CO2 for 48 hours. The cells were then stained with crystal violet. The data are mean ± standard error (EE, n = 8). The results showed that leptin significantly inhibited cell proliferation induced by IGF-I (figure 6). T-47D cells (3 x 10 5 cells / ml) in DMEM supplemented with 10% FBS were plated in 96-well plates (0.1 ml per well). After one day, the medium was replaced with DMEM supplemented with 2% FBS and after one day the cells were treated with IGF-I (50 ng / ml), with or without the indicated concentrations of human leptin in DMEM-2 % of FBS. Plates were incubated at 37 ° C in 5% CO2 for 48 hours. The cells were then stained with crystal violet. The data are mean ± standard error (EE, n = 8). The results showed that leptin significantly inhibited cell proliferation induced by IGF-I (figure 7).

EXAMPLE 8 Inhibition of leptin insulin-induced MCF7 cell proliferation MCF7 cells from human breast adenocarcinoma (3 x 10 4 cells / ml, American Type Culture Collection, Rockville, MD.; strain No. ATCC HTB 22), supplemented with 6% FBS were plated in 96-well plates (0.1 ml per well). After one day, the medium was replaced with serum-free DMEM and after one day the cells were treated with insulin (50 nM) with or without the indicated concentrations of murine leptin in a serum-free medium. Plates were incubated at 37 ° C in 5% CO2 for 48 hours. The cells were then stained with crystal violet: The data are mean ± SE, (n = 8). The results showed that leptin significantly inhibited insulin-induced cell proliferation (figure 8).

EXAMPLE 9 Inhibition of MCF7 cell proliferation induced by leptin IGF-i MCF7 human breast adenocarcinoma cells in DMEM supplemented with 10% FBS were plated in 96-well plates (3 x 10 4 cells / ml, 0.1 ml per well). After one day, the medium was replaced with a serum-free medium. After one day the cells were treated with IGF-I (50 ng / ml) with or without the indicated concentrations of murine leptin in a serum-free medium. Plates were incubated at 37 ° C in 5% CO2 for 48 hours. The cells were then stained with crystal violet: The data are mean ± SE, (n = 8). The results showed that leptin significantly inhibited insulin-induced cell proliferation (Figure 9).

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Claims (2)

1. NOVELTY OF THE INVENTION CLAIMS 1. - The use of an active agent selected from the group consisting of leptin, leptin fusion proteins, leptin muteins, leptin receptor agonists, fragments or active fractions of any of them, active analogs or derivatives or any of them, salts of any of them, and mixtures of any of them, for the preparation of a medicament for the inhibition of the proliferation of tumor cells.
2. 2. The use according to claim 1 for the treatment of malignancies in mammals 3. The use according to claim 1 or claim 2 for the inhibition of tumors dependent on the growth factor. 4. The use according to any of claims 1-3 for the inhibition of the proliferation of human breast carcinoma cells. 5. The use according to claim 4 for the treatment of human breast carcinomas. 6. The use according to claim 1 or claim 3 for the inhibition of insulin stimulating effect in tumor cells, as it is mediated, at least partially, by IRS-1 / GRRB2. 7. The use in accordance with claim 1 or claim 3 for the inhibition in mitogenic responses in tumor cells for one or more receptor kinases, growth factors and cytokines of the group consisting of IGF-1, II-4 and IL-9, for which IRS-1 is a substrate , for the treatment of tumors. 8. The use according to any of claims 1-7 for the inhibition of basal proliferation of tumor cells and induced by insulin, for the treatment of human breast cancers. 9. The use according to any of claims 1 -8, wherein said active agent is leptin. 10. A pharmaceutical composition comprising as active ingredient an active agent according to claim 1 and a pharmaceutically acceptable carrier, diluent or excipient, for the inhibition of the proliferation of tumor cells. 1 - A pharmaceutical composition according to claim 10, which is useful for the treatment of malignancies in mammals. 12. A pharmaceutical composition according to claim 10 or 1, which is useful for the inhibition of tumors dependent on the growth factor. 13. - A pharmaceutical composition according to any of claims 10-12, which is useful for the inhibition of the proliferation of human breast carcinoma cells and therefore for the treatment of human breast carcinoma. 14. A pharmaceutical composition according to claim 10 or 12, which is useful for inhibiting the insulin-stimulating effect in tumor cells, as is mediated, at least partially, via IRS-1 / GRB2 . 15. A pharmaceutical composition according to claim 10 or 12, which is useful for the inhibition of mitogenic responses in tumor cells to one or more receptor kinases, growth factors and cytokines of the group consisting of IGF-1 , IL-4 and IL-9, for which IRS-1 is a substrate, and therefore for the treatment of tumors. 16. A pharmaceutical composition according to any of claims 10-15, which is useful for the inhibition of basal proliferation of tumor cells and induced by insulin and therefore for the treatment of human breast cancers. 17. A pharmaceutical composition according to any of claims 10-16, further characterized in that said active ingredient is leptin. 18. The use of a pharmaceutical composition according to any of claims 10-17 for the preparation of a medicament for treating tumors in mammals or for inhibiting the proliferation of tumor cells in mammals.