WO2000066149A1 - Medicines useful for treating disorders of regulation of body fatness and diseases related to disorders of leptin production - Google Patents

Abstract

The invention concerns the use of agonists and antagonists of chemiokine receptors for preparing medicines useful for treating disorders of regulation of body fatness, in particular obesity, cachexia, anorexia, diabetes and hyperlipidemia, and for treating diseases related to disorders of leptin production.

Description

 MEDICINES USEFUL FOR TREATMENT

REGULATORY DISORDERS

FAT MASS AND RELATED ILLNESSES

TO THE DISORDERS OF LEPTIN PRODUCTION

The present invention relates to medicaments for the treatment of disorders of the regulation of adipose mass and of diseases associated with disorders of leptin production.

Adipocytes are considered to play a key role in the control of body mass, both in normal cases of this weight control and in pathological cases, where there is a disruption of this control, especially in case of increase in fatty mass (obesity) or in the event of reduction of this fatty mass (cachexia). However, little information exists on the factors that regulate the biology of adipocytes, in particular on those involved in the formation of fat.

Obesity is a chronic disease whose consequences on health are important and whose frequency around the world is expanding (Hill J.O. et al. (1998), Science, 280, 1371-1374). Because obesity is the result of an imbalance in the energy balance and adipocytes are considered to be the main site for storing and mobilizing energy, numerous studies have been carried out to understand the biology of adipocytes.

The cachectic states, characterized by the loss of adipose tissue and skeletal muscle mass, are observed in infections, AIDS and in half of cancer patients. These changes can not ^s' only explain by anorexia observed in these diseases since a nutritional supplement alone is not enough to reverse this weight loss process. An increase in the energy consumption at rest could contribute to weight loss in some cancer patients and may explain the ^increase in fat oxidation. It has been suggested that circulating factors such as cytokines, TNF-α (tumor necrosis factor α), interleukins-1 and -6 and rinterferon γ could be mediators involved in cachexia; however, to date no laboratory or clinical study has shown that these different factors could play a role in cachexia in humans and that the action of cytokines alone could explain the complex mechanism of weight loss , due to the cachexia observed in cancers and AIDS (Argiles J. M et al, (1997). Faseb, 11. 743-751; Tisdale MJ, (1997). J. Natl; Cancer Inst, 89. 1763- 1773). 00/66149

Similarly, no link has been demonstrated between plasma leptin levels and cachectic states while this hormone involved in obesity is secreted by adipocytes (Leroy P. et al. (1996), J. Biol. Chem. 271, 2365-2369) Adipocyte differentiation (or adipogenesis) involves several transcriptional factors. in particular factors from the C / EBP family (CCAAT / enhancer binding proteins) and PPAR (peroxisome proliferator-activated receptors), as well as factors from the HLH (helix-loop-helix) motif family of proteins. The regulation of the activity of these transcriptional factors by certain agents leads to the modulation of adipocyte differentiation. Thus, certain agents promote adipocyte differentiation (adipogenic action), others inhibit adipocyte differentiation (anti-adipogenic action), others, finally, can have both types of action. Among the anti-adipogenic agents, there are the two forms of a cytokine, TNF-α (tumor necrosis factor) which inhibits the expression of C / EBPα and PPARγ2 (Peroxisome Proliferator Activated Receptor γ2) and TNF- β (tumor necrosis factor β) which prevents adipocyte differentiation independently of its mitogenic effect (Loftus TM et al, Curr. Opin. Genêt. Dev. (1997), 7, 603-608).

Studies carried out on adipocytes have also shown that adipocytes secrete TNF-α and another multifunctional cytokine, IL-6, (interleukin-6) which would thus seem to play a role in regulating the entry of fatty acids. in adipose tissue and in their metabolism (Fried SK et al, J. Clin. Endocrinol. Metab. (1998), 83, 847-850).

However, with the exception of TNF and IL-6, which are cytokines with very general action, no study, to date, has shown that other mediators of inflammation intervene in adipocyte differentiation. , or were secreted by adipocytes. Cytokines are synthesized de novo during cell activation and they act by binding to specific receptors, having a single transmembrane region and linked to intracellular kinases. Their action is most often carried out locally (paracrine or autocrine action), but they can also act at a distance (endocrine action). Among the inflammatory mediators are chemotactic cytokines, called c imiokines, which are known to play an important role. 00/66149

in perivascular transmigration and the accumulation of leukocytes at the site of tissue damage.

There are four classes of chemokines according to the distance between the first two of the four cysteine residues involved in the formation of the disulfide bridges, the said classes are:

- gold-chemokines (CXC), such as interleukin-8 (IL-8), SDF1 (stromal cell derivated factor), GRO (melanoma growth-stimulating activity) and IP-10 (interferon-γ inducible 10 KDa protein) ,

- Ê-chemokines (CC), such as RANTES (regulated-upon-activation, normal T-cell expressed and secreted), MlP-lα, MlP-lβ and MIP-2

(macrophage inflammatory proteins) and MCP-1, MCP-2, MCP-3, MCP-4 and MCP-5 (monocyte chemotactic protein),

- chemokines (C), such as lymphotactihe and

- ^ -chemokines (CX C), such as neurotactin. These chemokines also act by binding to receptors which have 7 transmembrane regions and are linked to the G protein.

In addition to their well-established role as mediators of inflammation, it has been shown recently (P. Proost et al, Int. J. Clin. Lab. Res. (1996), 26, 211-223) that chemokines actually had much broader biological functions, such as growth regulator and angiogenesis properties. Likewise, it has been shown that they also play a role in the development of the immune, circulatory and central nervous systems. (R. Horuk, Nature (1998), 393, 524-525). However, no study has yet shown that chemokines can exist in adipocytes and play an important role. However, the inventors have shown, surprisingly, that chemokines, independently of their anti-inflammatory role, could play a role in regulating the biology of adipocytes.

Adipocytes represent 3/4 of the cells of adipose tissue, the other cells being essentially precursor cells, in particular preadipocytes (cells without lipids). Adipose tissue plays a central role in controlling the body's energy balance. The main functions are the storage of energy reserves in the form of triglycerides and their mobilization in the form of free fatty acids; moreover, the excessive development of the adipose mass observed during obesity involves both hypertrophy and cellular hypeφlasia. Although the development of white adipose tissue takes place mainly in the postnatal period, an increase in the adipocyte population, from the recruitment of cellular precursors present in the 00/66149

vascular fraction of the stroma of this tissue, can also occur.

Also, the compounds which could inhibit the proliferation of preadipocytes and / or prevent the accumulation of lipids in adipocytes and / or modify the production of leptin, could be useful for the preparation of medicaments useful in the treatment of disorders of the regulation of body fat.

Consequently, the inventors have given themselves the aim of providing drugs intended for the treatment of disorders of the regulation of adipose mass, in particular in the event of obesity, cachexia, anorexia, diabetes and hyperlipidemia and treatment of diseases associated with leptin production disorders, especially in cases of hypogonadism.

The subject of the present invention is the use of ligands for the α-chemokine and β-chemokine receptors for the preparation of a medicament intended for the treatment of disorders of the regulation of adipose mass, in particular in the case of obesity, cachexia, anorexia, diabetes and hyperlipidemia and the treatment of diseases associated with disorders of leptin production, particularly in cases of hypogonadism.

For the purposes of the present invention, the term “ligand” means chemokine receptors, both natural ligands and synthetic analogs. According to a preferred embodiment of the invention, ligands for IL-8 receptors are used.

According to another preferred embodiment of the invention, ligands chosen from the group consisting of ligands for MCP-1 and MlP-1α receptors are used. During their work, the Inventors showed:

- that primary cultures of human white preadipocytes ^~ e "t of human white adipocytes express the mRNAs of certain chemokines and their receptors and that this expression can be regulated by TNFα,

- that the expression: mRNA of these chemokines, mRNA of their receptors and of the chemokines themselves is modified during adipocyte differentiation,

- that the brown adipose tissue expresses the mRNAs of certain chemokines and their receptors,

- that chemokine receptors are present in human white adipose tissue and that treatment with chemokines leads to a reduction in fat mass,

- that these chemokines induce a decrease in the degree of tyrosine O 00/66149

phosphorylation in differentiated adipocytes and are capable of inhibiting the increase in the degree of tyrosine insulin-induced phosphorylation in these cells,

- that these chemokines decrease the expression of the PPARγ receptor and - that MlP-lα increases the leptin level in human white adipocyte cultures.

Thus, human white preadipocytes in primary culture express the mRNAs of MCP-1, IL-8 and MlP-1α; the expression of MCP-1 mRNA is not modified during the differentiation of preadipocytes, whereas the expression of mRNA of IL-8 and of MlP-1α are reduced in adipocytes, compared to preadipocytes.

TNFa increases mRNA expression of IL-8, MIP-α and MCP-1 in primary cultures of adipocytes while it has no effect on ^the mRNA expression of these chemokines in the preadipocytes. The β3 adrenergic receptor (β3 AR) is specifically expressed in adipocytes where it is involved in the regulation of lipolysis and adipogenesis (Strosberg AD et al. (1996) Trends in Pharmacological Sciences 17, 373-381). In addition, a natural mutation of this receptor has been associated with morbid obesity (Strosberg AD et al. (1997) Trends in Pharmacological Sciences 18, 449-454). Given this role of β3 ARs in regulating the functions of adipocytes, the inventors investigated whether the addition of isoproterenol, an agonist of β3 ARs, modifies the expression of the mRNAs of the chemokines themselves or that of the mRNAs of their receptors. Under these conditions, the expression of the IL-8 mRNA is stimulated by the addition of isoproterenol in the culture medium. The level of expression of these chemokines during the differentiation of adipocytes is correlated with that of their mRNA and the level of expression of MlP-α is lower than that of IL-8 and MCP-1.

In addition, the expression of mRNAs of certain receptors (CXCR1, CXCR2, CCR2, CCR5) increases during adipocyte differentiation. Under these conditions, and unexpectedly, the expression of receptors for different chemokines in adipocytes is closely correlated with the presence of lipid droplets in the cytoplasm.

Incubation of human white adipocytes, in the presence of IL-8, MCP-1 and MlP-1α, leads to a reduction in the lipid content of these adipocytes.

Incubation of differentiated human adipocytes, in the presence of IL-8, MCP-1 and MlP-1 induces a decrease in the degree of tyrosine phosphorylation 00/66149

in these cells, and decreased the ^expression of PPRAγ receptor, which plays a vital role in adipogenesis.

In addition, these chemokines are capable of inhibiting the increase in the degree of insulin-induced tyrosine phosphorylation in differentiated human adipocytes.

Suφrenantly, the inventors have therefore shown that the chemokines IL-8, MCP-1 and MlP-1α are capable of antagonizing the actions of insulin and of inhibiting adipogenesis.

In addition, while the incubation of human white adipocytes, in the presence of IL-8 and MCP-1 does not modify the production of leptin by adipocytes and this whatever the stage of differentiation, the incubation d adipocytes in the presence of MIP-lcc, leads to an increase in the production of leptin.

The present invention also relates to a method for detecting the capacity of a molecule to behave like a ligand with respect to the chemokine receptors carried by preadipocytes and adipocytes, characterized in that it comprises:

- bringing the molecule into contact with preadipocytes or adipocytes carrying chemokine receptors, under conditions which allow the formation of a bond between at least one chemokine receptor and said molecule, as soon as it turns out actually have an affinity for this receptor, and

- detecting the formation of a ligand-receptor type complex by physical or chemical means.

This detection can be carried out by any means conventionally used, in particular by the use of radiolabelled ligands, by immunocytochemistry, by fluorescence, or by measurement of the transduced signal (activated signal transduction pathways).

Such a method allows the selection of specific ligands for the various receptors of the chemokines expressed in the preadipocytes and the adipocytes, said ligands acting in addition on the regulation of adipocyte functions, in particular on adipocyte differentiation, the formation of lipids and the production of leptin. .

The subject of the present invention is also a method for studying the receptors for chemokines expressed by preadipocytes and adipocytes, characterized in that it comprises:

- bringing certain ligands into contact with preadipocytes or adipocytes, and 00/66149

- detecting the formation of a ligand-receptor type complex by physical or chemical means.

This detection can be carried out by any means conventionally used, in particular by the use of radiolabelled ligand, by immunocytochemistry, by fluorescence, or by measurement of the transduction signal (activated signal transduction pathways).

Such a method makes it possible to study the pharmacology of the chemokine receptors involved in the regulation of adipocyte functions, in particular in adipocyte differentiation, the formation of lipids and the production of leptin, to identify ligands having a better affinity for these receptors. and to develop drugs active in the treatment of disorders of the regulation of the adipose mass, in particular in the event of obesity, cachexia, anorexia, diabetes and hyperlipidemia.

The subject of the present invention is also a method for studying the capacity of a ligand for receptors for chemokines expressed by adipocytes, to modify the accumulation of lipids in said adipocytes, characterized in that it comprises:

- bringing the ligand to be studied into contact with adipocytes and

- detecting the formation of lipids in said adipocytes, by physical or chemical means.

This detection can be carried out by any means conventionally used, in particular by marking with OR Red O. according to the method described by Ramirez-Zacarias J.L. et al. (1992), Histochemistry, 97, 493-497).

Such a method makes it possible to select specific ligands for the various chemokine receptors expressed in adipocytes, said ligands acting on the formation of lipids in said adipocytes and to develop drugs which are active in the treatment of disorders of the regulation of adipose mass. , especially in cases of obesity, cachexia, anorexia, diabetes and hyperlipidemia, and active in the treatment of diseases associated with disorders of leptin production, in particular in cases of hypogonadism

The present invention also relates to a method for studying the capacity of a ligand for the receptors of chemokines expressed by adipocytes, to modify the production of leptin by said adipocytes, characterized in that it comprises: in contact with the ligand to be studied with adipocytes and

- detection of leptin production by physical or chemical means. O 00/66149

This detection can be carried out by any means conventionally used, in particular by a radiological-immunological method [RIA (Radio-Immuno-Assay)].

Such a method makes it possible to select specific ligands for the various chemokine receptors expressed in adipocytes, said ligands acting on the production of leptin by said adipocytes and to develop drugs which are active in the treatment of disorders of the regulation of adipose mass. , especially in cases of obesity, cachexia, anorexia, diabetes and hyperlipidemia, and active in the treatment of diseases associated with disorders of leptin production, especially in cases of hypogonadism.

According to the invention, for the implementation of said methods, use is made of preadipocytes and adipocytes originating from white or brown adipose tissue, human or murine or cell lines of preadipocytes, such as for example the cell lines of white preadipocytes of mice 3T3 -L1 [Green H. et al. (1974), Cell, 1, 113-116; ATCC CCL-92J strain, mouse embryo, substrain of 3T3 deposited at the American Type Culture Collection (ATCC)] and 3T3- F442A (Green H. et al. (1976), Cell, 7, 105-113) and the lines cells of human brown preadipocytes PAZ-6 (Zilberfarb V. et al. (1997), J. Cell Se, 110, 801-807; strain 1-1531 deposited at National Collection of Cultures and Microorganisms (CNCM) of l 'Pastor Institute).

Preferably, preadipocytes and adipocytes from human white adipose tissue are used.

A further subject of the present invention is a kit for detecting the possible affinity of a ligand for the receptors for chemokines expressed by preadipocytes and adipocytes, characterized in that it comprises:

- preadipocytes or adipocytes in culture,

- one or more control ligands and

- means for detecting the ligand-receptor complex.

A further subject of the present invention is a kit for detecting the capacity of a ligand for the receptors for chemokines expressed by adipocytes, to modify the formation of lipids in said adipocytes, characterized in that it comprises:

- adipocytes in culture, and

- Means for detecting the lipids formed in said adipocytes.

The present invention further relates to a kit for detecting the capacity of a ligand for the chemokine receptors expressed by adipocytes, O 00/66149

to modify the production of leptin by said adipocytes, characterized in that it comprises:

- adipocytes in culture, and

- Means for detecting the leptin produced by said adipocytes. Other characteristics and advantages of the invention appear in the following description and examples illustrated by the figures in which:

- Figure 1 illustrates the expression of chemokine mRNAs in human preadipocytes and white adipocytes; detection is carried out by RT-PCR according to the procedure described in Example 1. The preadipocytes and the human white adipocytes are cultured in the absence (-) or in the presence (+) of 1 μM isoproterenerol for 18 hours; MlP-lα (macrophage inflammatory protein la); MCP-1 (monocyte chemotactic protein).

- Figure 2 illustrates the expression of mRNAs of chemokine receptors in preadipocytes and human white adipocytes; detection is carried out by RT-PCR according to the procedure described in Example 1. The preadipocytes and the human white adipocytes are cultured in the absence (-) or in the presence (+) of 1 μM isoproterenerol for 18 hours; IL-8 (interleukin 8), MCP-1 (monocyte chemotactic protein), MLP-lo. (macrophage inflammatory protein la).

- Figure 3 shows the expression of chemokine receptors in human white adipocytes detected by immunocytochemistry according to the procedure described in Example 1; CXCR1 and CXCR2 are receptors for IL-8 (interleukin 8), CCR2 is the receptor for MCP-1 (monocyte chemotactic protein) and CCR5 is the receptor for MlP-1 (macrophage inflammatory protein 1)

- Figure 4 shows the effect of chemokines on the accumulation of lipids in human primary white adipocytes. Human white adipocytes are cultured either in the absence of chemokines or in the presence of IL-8 (interleukin 8), MCP-1 (monocyte chemotactin protein) or MIP-1c. (macrophage inflammatory protein la) at 50 nM; lipid formation is measured after 12 days of culture. The results are expressed as the mean ± standard error to the mean (s.e.m.) of 5 or 6 experiments.

- Figure 5 shows the effect of chemokines on the production of leptin by human primary white adipocytes. Human white adipocytes are cultured either in the absence of chemokines (- • -), or in the presence of IL-8 (- - interleukin 8) at 100 nM, or MCP-1 (-_ -monocyte chemotactin protein) at 100 nM or MIP-1α (- -macrophage inflammatory protein la) at 100 nM; leptin production is measured after 4, 8 and 12 days of culture. The results are expressed 00/66149

as the mean ± standard error to the mean (s.e.m.) of an experiment carried out in triplicate.

EXAMPLE 1 Material and methods used to study the expression of chemokines and their receptors in human preadipocytes and white adipocytes.

1. Cell cultures: a) preadip cytes: the omental adipose tissue is obtained from healthy subjects having undergone cosmetic surgery. The tissue is cut into small pieces and digested for 1 hour at 37 ° C. with type 2 collagenase (Worthington) at a concentration of 1 mg / ml. After digestion, the tissue is filtered through a nylon filter, and the preadipocytes are isolated by centrifugation at 200 g for 10 minutes. The pellet, which contains the preadipocytes, is treated with a buffer containing: NFLjCl 154 mM, KHCO 10 mM and EDTA 0J mM in order to eliminate the red blood cells. b) adipocytes: the preadipocytes obtained are cultured to confluence in a mixture (l .J) of Ham's F12 / DMEM (Dubelcco 's modified Eagles medium; Gibco-BRL) supplemented with Hepes 20 mM buffer (Gibco- BRL), 5% decomplemented fetal calf serum (Gibco-BRL) and antibiotics. When the cells have reached confluence, they are transferred to a medium supplemented with biotin (33 μM, Sigma), panthotenate (18 μM, Sigma), triiodothyronine (T3, 1 nM, Sigma), insulin (85 nM, Sigma), dexamethasone (1 μM, Sigma), 3-isobutyl-1-methyl-xanthine (250 nM, Sigma) and naphthiazol (1 μM, PPR- / agonist, don by Dr. J. Duhault, IRIS France). The cells are maintained in this medium for 12 days by changing the medium every 3 days.

2. OR Red O marking and quantification of lipids in adipocyte cells:

The solution of OU Red O. is prepared according to the method described by Ramirez-Zacarias J.L. et al. (1992), Histochemistry, 97, 493-497. Six multi-well plates are washed twice with PB S saline buffer (phosphate buffered saline), fixed for 1 hour with a 10% formalin solution in PBS, washed with water, marked for 2 hours by immersion complete in a solution of OU Red O. and rinsed twice with water. Excess water is evaporated by placing the plates at 37 ° C. The dye is extracted with 200 μl of isopropyl alcohol per well and the absorbance of an aliquot is measured immediately at 492 nm.

The lipids (triglycerides) are quantified using triolein calibration curves (Sigma) according to the method described by Ramirez-Zacarias JL O 00/66149

(Ramirez-Zacarias J.L. et al. (1992), already cited).

3. RT-PCR:

Total RNA is extracted from human primary white adipocytes (passage 1-3) using the isolation agent Trizol RNA (Gibco Life Sciences). 20 μg of total RNA are treated with 6 U of DNAse I without RNase

(RQl DNase Promega) in a medium containing 40 mM Tris-HCl, pH 9, 10 mM NaCl, 6 mM MgCl ₂ , in the presence of 2 U / μl of RNase inhibitor (RNaseOUT, Gibco Life Sciences).

The RNA is then extracted with a mixture of phenol and chloroform, precipitated with ethanol, dried and suspended in water.

The cDNA is prepared from 2 μg of total RNA without DNA using the reverse transcriptase of the Moloney murine leukemia virus (SuperScript ™ II, Gibco Life Sciences) according to the manufacturer's instructions.

Controls without reverse transcriptase are prepared in order to exclude any possibility of DNA contamination.

100 ng of RNA thus transcribed are amplified by 1 U of Taq polymerase (Gibco Life Sciences) in a final volume of 30 μl of buffer containing: 20 mM Tris-HCl, at pH 8.0, 50 mM KC1, MgCl ₂ 1 , 5 mM, dNTP 250 μM and

250 μM of sense and antisense primer sequences specific for a gene in a thermal cycler (GeneAmp PCR System 9600, Perkin-Elmer Cetus).

The amplification products are visualized after electrophoresis on a 1.5% agarose gel with BET (ethidium bromide).

For the semi-quantitative determination of RT-PCRs, the analysis is carried out during the exponential phase of the amplification. The sense and antisense primer sequences are as follows:

- cyclophilin: SEQ ID No. 1 and SEQ ID No. 2;

- interleukin-8: SEQ ID N ° 3 and SEQ ID N ° 4;

- MlP-1α: SEQ ID N ° 5 and SEQ ID N ° 6;

- MCP-1: SEQ ID N ° 7 and SEQ ID N ° 8; - CXCR1: SEQ ID N ° 9 and SEQ ID N ° 10;

- CXCR2: SEQ ID N ° 11 and SEQ ID N ° 12;

- CCR1: SEQ ID N ° 13 and SEQ ID N ° 14

- CCR2: SEQ ID N ° 15 and SEQ ID N ° 16

- CCR4: SEQ ID N ° 17 and SEQ ID N ° 18 - CCR5: SEQ ID N ° 19 and SEQ ID N ° 20

- CCR9 / 10: SEQ ID N ° 21 and SEQ ID N ° 22; 00/66149

4. Characterization of the expression of receptor mRNAs by immunocytochemistry:

The cells are fixed in 4% paraformaldehyde for 15 minutes, treated (quenching) with a 0J M glycine solution in PBS (phosphate buffer saline) and permeabilized with PBS containing 0.2% BSA

(bovine serum albumi) and 0.05% saponin. After several washes, the cells are treated with PBS containing 10% FCS (fetal calf serum).

Then the cells are incubated overnight, either with monoclonal antibodies directed against CXCRl, CXCR2, CXCR4, CCRl, CCR2 and CCR5 (dilution 1 J00, R&D), or with polyclonal antibodies directed against CCR4 (dilution 1 JOO, Santa Cruz ).

Finally, the cells are incubated with a secondary antibody conjugated with a chromophore (fluorescein isothiocyanate (FITC) or Cy3) in the same buffer for 1 hour, then washed thoroughly before being mounted. The controls are prepared by omitting the primary antibodies and replacing them with monoclonal antibodies of the same isotype.

After assembly, the cells are examined under a laser microscope (MRC 1000 Biorad, Hercules, CA, USA).

5. Measurement of leptin production by adipocytes Leptin is measured in the culture medium, by a radioimmunological method, according to the technique described by Ma Z. et al, (1996), Clinical chemistry, 42, 942-946) , using the kit from Linco Research Inc. (St Charles, MO, USA) following the manufacturer's instructions.

The detection limit of the method is 0.5 ng / ml.

EXAMPLE 2 Expression of chemokine mRNAs in human preadipocytes and white adipocytes.

The procedure is that described in Example 1. The results are illustrated in FIG. 1. Cyclophilin is used as a control for the expression of

Total mRNAs.

The expression of MCP-1 is not modified during adipocyte differentiation while the expression of IL-8 and MlP-1α is reduced in adipocytes compared to preadipocytes. Isoproterenol stimulates the expression of IL-8 in adipocytes but has no effect on that of IL-8 in preadipocytes.

Isoproterenol has no effect on the expression of MlP-lα and O 00/66149

MCP-1, both in preadipocytes and in adipocytes.

EXAMPLE 3 Expression of mRNAs of chemokine receptors in human white adipocytes. The operating mode is that described in Example 1.

1. in the preadipocytes: the results are illustrated in FIG. 2 and in Table I below.

The CXCR1 and CCR2 receptors are not expressed in human white preadipocytes. The CXCR2, CCR4 and CCR5 receptors are weakly expressed in human white preadipocytes.

The CCR1 and CCR9 / 10 receptors are strongly expressed in human white preadipocytes.

Isoproterenerol stimulates the expression of the CXCR1, CXCR2, CCR2 and CCR5 receptors in human white preadipocytes, but has no effect on the expression of other receptors.

2. in adipocytes: the results are illustrated in Figures 2 and 3 and in Table I below.

All the CXCR1, CXCR2, CCR1, CCR2, CCR4, CCR5 and CCR10 receptors are strongly expressed in human white adipocytes.

Isoproterener has no effect on the expression of different chemokine receptors in human white adipocytes.

The expression of the CXCR1, CXCR2 and CCR5 receptors is limited to completely differentiated adipocytes since it is linked to the presence of lipid droplets in the cytoplasm (FIG. 3).

The expression of CCR2 receptors, on the other hand, appears in cells without lipid droplets but only after culturing in the differentiation medium.

The expression of the multifunctional receptor CXCR4, receptor of SDF-1 (stromal cell derivated factor), is also limited to completely differentiated adipocytes since it is linked to the presence of lipid droplets in the cytoplasm (Table 1). O 00/66149

TABLE I

RECEPTORS LIGANDS PREADIPOCYTES ADIPOCYTES

CXCRl IL-8, CGP-2 - ++

CXCR2 IL-8, NAP-2, + -H- GROα, ENA-78

CXCR4 SDF-lα + ++

CCRl MlP-lα, RANTES + + MCP-3

CCR2 MCP-1,2,3,4,5 - -H-

CCR4 MEP-lα, RANTES + ++ TARC, MDC

CCR5 MJPJα, β, RANTES + ++

CCR10 MCP-U -H- ++

EXAMPLE 4 Effect of chemokines on the formation of lipids in adipocytes.

The lipids are measured according to the technique described in Example 1.

The results are illustrated in Figure 4.

IL-8 significantly decreases lipid formation in adipocytes after 12 days of culture; inhibition is on average 22% ± 6, with a maximum inhibition rate of 41%.

MlP-1 decreases the formation of lipids in adipocytes after 12 days of culture; inhibition is on average 21% ± 9, with a maximum inhibition rate of 47%.

MCP-1 also significantly decreases lipid formation in adipocytes after 12 days of culture; inhibition is on average 23% ± 7, with a maximum inhibition rate of 47%.

This inhibitory effect appears from the concentration of 10 nM.

In addition, the decrease in the lipid level induced by the chemokines is linked to the state of maturation of the adipocytes; in fact, in cultures which contain numerous cells with large lipid vacuoles, the chemokines induce a reduction of up to 59% for IL-8, 52% for MIP-1α and 53% for MCP-1.

EXAMPLE 5 Production of leptin by differentiated primary white adipocytes in vitro

Leptin is measured according to the procedure described in Example 1. O 00/66149

The results are given in Figure 5.

The production of leptin from adipocytes increases during differentiation and this effect is not changed by ^the addition of IL-8 or MCP-1 in the culture medium, at any stage of differentiation. On the other hand, the addition of MlP-1α increases by approximately 50% and this significantly, the leptin levels, at all stages of differentiation.

These results suggest that chemokines may play an important role in regulating the biology of adipocytes and that chemokine receptor agonists and antagonists may be useful in the preparation of drugs useful for the treatment of disorders of mass regulation. adipose, especially in cases of obesity, cachexia, anorexia, diabetes and hyperlipidemia and in the treatment of diseases associated with disorders of leptin production, in particular in the case of hypogonadism.

Claims

00/66149 1) Use of agonists and antagonists of chemokine receptors for the preparation of medicaments useful in the treatment of disorders of the regulation of adipose mass and in the treatment of diseases associated with disorders of leptin production. 2) Use according to claim 1 characterized in that the agonists and antagonists are chosen from the group consisting of agonists and antagonists of CXCR receptors for α-chemokines. 3) Use according to claim 2, characterized in that agonists and antagonists of IL-8 receptors are used. 4) Use according to claim 1 characterized in that the agonists and antagonists are chosen from the group consisting of agonists and antagonists of CCR receptors for β-chemokines. 5) Use according to claim 4 characterized in that one uses agonists and antagonists chosen from the group consisting of agonists and antagonists of MlP-lα or MCP-1 receptors. 6) Use according to any one of claims 1 to 5, characterized in that the disorder of the regulation of adipose mass is chosen from the group consisting of obesity, cachexia, anorexia, diabetes and hyperlipidemia and the disease associated with leptin production disorders is hypogonadism. 7) Method for detecting the capacity of a molecule to behave like a ligand with respect to chemokine receptors, characterized in that it comprises: - bringing the molecule into contact with preadipocytes or adipocytes carrying chemokine receptors, under conditions which allow the formation of a bond between at least one chemokine receptor and said molecule, as soon as it turns out to actually have an affinity for this receptor, and - detection of the formation of a ligand-receptor complex by physical or chemical means. 8) Method for studying the receptors for chemokines expressed by preadipocytes and adipocytes, characterized in that it comprises: - bringing certain ligands into contact with preadipocytes or adipocytes, and - detecting the formation of a ligand-receptor type complex by physical or chemical means. O 00/66149 9) Method for studying the capacity of a ligand for receptors for chemokines expressed by adipocytes, to modify the accumulation of lipids in said adipocytes, characterized in that it comprises: - bringing the ligand to be studied into contact with adipocytes and - detecting the formation of lipids in said adipocytes, by physical or chemical means. 10) Method for the study of the capacity of a ligand of the chemokine receptors expressed by adipocytes, to modify the production of leptin by said adipocytes, characterized in that it comprises: - bringing the ligand to be studied into contact with adipocytes and - detection of leptin production by physical or chemical means. 1 1) A method according to any one of claims 7 to 10, characterized in that the preadipocytes and adipocytes are from human white adipose tissue. 12) Kit for detecting the possible affinity of a ligand for the chemokine receptors expressed by preadipocytes and adipocytes, characterized in that it comprises: - preadipocytes or adipocytes in culture, - one or more control ligands and - appropriate means for the detection of the ligand-receptor complex. 13) Kit for detecting the capacity of a ligand for the receptors of chemokines expressed by adipocytes, to modify the formation of lipids in said adipocytes, characterized in that it comprises: - adipocytes in culture, and - Means for detecting the lipids formed in said adipocytes. 14) Kit for detecting the capacity of a ligand for the chemokine receptors expressed by adipocytes, to modify the production of leptin by said adipocytes, characterized in that it comprises: - adipocytes in culture, and - Means for detecting the leptin produced by said adipocytes.