KR20070032034A - Treatment of ccr2 mediated diseases or disorders - Google Patents

Abstract

The present invention provides a method of reducing or maintaining the weight and / or body fat of a patient, such as a human and an animal, or by using a CCR2 treatment alone or in combination with a second therapeutic, for example in the treatment of overweight or obese patients. A means for producing fat-free meat in chickens, pigs; Methods of treating diabetes and / or glucose intolerance; A method of treating metabolic disorders in a patient in need thereof, a kit for therapeutic use identified above, and a method of identifying CCR2 therapeutics for treatment with the aforementioned therapeutic uses.

Description

 TREATMENT OF CCR2 MEDIATED DISEASES OR DISORDERS}

The present invention relates to a method of reducing or maintaining body weight and / or body fat by administering a CC chemokine receptor 2 (CCR2) therapeutic. The present invention also relates to a method of treating a metabolic syndrome disorder in a patient by administering a CCR2 therapeutic agent. The present invention also relates to a method of treating diabetes or glucose intolerance in a patient by administering a CCR2 therapeutic.

Obesity is now believed to be prevalent in many parts of the world and is known as a chronic disease that requires treatment to reduce the associated health risks. Weight loss is an important therapeutic outcome in itself, but one of the main purposes of obesity management is to improve cardiovascular and metabolic levels to reduce obesity-related morbidity and mortality. It has been shown that a 5-10% reduction in body weight can substantially improve metabolic and cardiovascular levels such as blood sugar, blood pressure and lipid concentrations. Thus, it is shown that a 5-10% reduction in body weight can reduce morbidity and mortality.

In individuals diagnosed with obesity or overweight, coronary artery disease, stroke, hypertension, type 2 diabetes, dyslipidemia, sleep apnea, osteoarthritis, gallbladder disease, depression and certain forms of cancer (eg, endometrium, breast, prostate and colon) Risk of developing other health symptoms, such as). Obesity has become the second leading cause of preventable death in the United States and has become a major public health concern with serious economic and psychosocial impacts on society (McGinnis and Foege, (1993) JAMA 270). , 2207-2212, and Calle, EE (2003) NEJM 348, 1625-1638).

In addition, prevention of weight and / or body fat gain or maintenance of body weight and / or body fat will also benefit the well being of the individual.

Recently, it has been suggested that some chemokines may play a role in the regulation of adipose tissue or may play a role in the cellular composition of adipose tissue, and may provide a basis for the treatment of obesity, diabetes and cachexia. (Gerhardt, CC et al., (2001) Mol. Cell. Endocrin, 175, 81-92).

Chemokines constitute a diverse group of small secreted basal proteins that regulate the chemotactic migration and activation of numerous cells, including leukocytes, especially with respect to the activation of the immune response during inflammatory symptoms. A new classification scheme for chemokines has recently been proposed (see Zlotnik, A and Yoshie O. (2000) Immunity 12, 121-127).

Examples of cells that appear chemotactically react to and are activated by chemokines are neutrophils, eosinophils, basophils, branch cells, monocytes, phagocytes, as well as B lymphocytes and different types of T lymphocytes (Oppenheim, JJ). (1991) Annu. Rev. Immunol. 9, 617-48; Miller. MD and Krangel, SK (1992) Crit. Rev. Immunol . 12 17-46; and Baggiolini, M. et al . (1994) Adv. Immunol . 55, 97-179).

Chemokines can be classified based on the pattern of cysteine residues that participate in disulfide bond formation in mature proteins. The first group, CXC chemokine or α-chemokine, is characterized by the occurrence of two cysteine residues in the amino-terminal region between the positions of different amino acid residues. CXC chemokine residues, in particular CXC chemokines having the amino acid sequence Glu-Leu-Arg at the amino terminus, mainly act on neutrophils. Examples of CXC chemokines include interleukin-8 (IL-8), GRO-α, -β and -γ, NAP-2, ENA-78 and GCP-2.

The second group, CC chemokines or β-chemokines, is characterized by the generation of two adjacent cysteine residues occurring in the amino-terminal region. CC chemokines act on T and B lymphocytes as well as more diverse white blood cells such as monocytes, phagocytes, eosinophils, basophils. Examples of these include MCP-1, MCP-2, MCP-3, MIP-1α, MIP-1β, Eotaxin, Landes and I-309.

MCP-1, newly classified as CCL2, is produced by monocytes and various tumor cells as well as various tissue cells such as endothelial cells, epithelial cells, fibroblasts, keratinocytes, synovial cells, hemangioblasts, osteoblasts and smooth muscle cells. (See Baggiolini, M et al . (1994) Adv. Immunol . 55, 97-179). MCP-1 is also recently known to be produced by adipocytes (Rollins, BG (1997) Blood 90, 909-928; and Gerhardt, CC et al. (2001) Mol. Cell. Endocrinol . 175, 81-92) Reference).

MCP-1 may play a role in the pathogenesis of atherosclerotic lesions. Supplementation of active monocytes via MCP-1 released from activated endothelial cells has been suggested to play a role in the formation of fat streak and atherosclerotic plaques (Yia-Herttuala, S. et al. (1991) PNAS ). 88, 5252-5256; Schwartz, CJ et al. (1993) Am. J. Cardiol . 71, 9B-14B; and Takeya, M. (1993) Hum. Pathol . 24, 534-9). Hypercholesterolemic mice with gene disruption of MCP-1 or its receptor CCR2 have reduced atheromatous development (see Boring, L. et al. (1998) Nature 394, 894-897).

Expression of MCP-1 appears to be upregulated in white adipose tissue and plasma of obese mice compared to lean controls (Sartipsy, P. and Loskutoff, DJ (2003) PNAS 100, 7265-7270). ] Reference). MCP-1 expression also appears to be elevated in diet-induced obese mice, raising the level of MCP-1 in plasma (Takahashi, K. et al . (2003) J. Bio. Chem . 278, 46654). -46660).

Increasing data suggest an interrelated and potentially workable relationship between inflammation and insulin resistance. It has been pointed out that preinflammatory cytokine TNF-α mediates insulin resistance as a result of obesity in rodent obesity models. Increased TNF-α expression has also been noted in phagocytic cells of adipose tissue from obese individuals (see Weisburg, SP et al. (2003) J. Clin. Invest . 112, 1796-1808).

CCR2-/-knockout (KO) mice have been used as a tool to study the pathogenesis of inflammatory diseases, as well as to determine whether symptoms can be ameliorated or worsened by CCR2 antagonists.

CCR2 KO mice reduced MCP-1 induced leukocyte adhesion to microvascular endothelial and reduced extravasation of leukocytes (see Kuziel, WA (1997), PNAS 94, 12053-12058). In addition, CCR2 KO mice appear to have reduced monocyte supplementation in response to inflammatory agents (see Boring, L. et al ., (1997) J. Clin, Invest . 100, 2252-2261).

Summary of the Invention

The present invention provides methods for reducing or maintaining body weight and / or body fat by administering a CCR2 therapeutic agent (alone or in combination with other therapeutic agents) to a patient, as well as related kits, and screening CCR2 therapeutics for the aforementioned therapeutic uses. Provides a way. The invention also provides a method for treating metabolic syndrome by administering to a patient a CCR2 therapeutic agent (alone or in combination with another therapeutic agent). The present invention also provides a method for treating diabetes or glucose intolerance by administering to a patient a CCR2 therapeutic agent (alone or in combination with another therapeutic agent). CCR2 therapeutics include CCR2 antagonists. Additional CCR2 therapeutics include CCR2 inhibitors and CCR2 ligand inhibitors.

In a first aspect, the present invention provides a method of reducing a body weight and / or body fat, comprising administering a therapeutically effective amount of a CCR2 therapeutic agent to a subject (ie, a patient) in need of treatment for weight and / or body fat loss. Provide a method of treatment. In this embodiment, the object is a human, the object tends to be overweight or obese or obese, and the CCR2 therapeutic agent is a CCR2 antagonist. Additional CCR2 therapeutics include CCR2 inhibitors and CCR2 ligand inhibitors.

In a second aspect, the invention maintains and / or stabilizes body weight and / or body fat by administering a CCR2 therapeutic agent to an object (ie, a patient) in need of maintaining and / or stabilizing body weight and / or body fat. Provide a method of treatment. CCR2 therapeutics include CCR2 antagonists. Additional CCR2 therapeutics include CCR2 inhibitors and CCR2 ligand inhibitors.

In a third aspect, the present invention provides a method of treating diabetes or glucose intolerance comprising administering to a subject in need thereof a therapeutically effective amount of a CCR2 therapeutic agent. Diabetic patients can be type 1 (IDDM) or type 2 (NIDDM) diabetes. In type 1 diabetes, CCR2 therapeutics act to increase insulin sensitivity. CCR2 therapeutics include CCR2 antagonists. Additional CCR2 therapeutics include CCR2 inhibitors and CCR2 ligand inhibitors.

In a fourth aspect, the present invention provides a method of treating metabolic syndrome comprising administering to a subject in need thereof a therapeutically effective amount of a CCR2 therapeutic agent.

In a fifth aspect, the method provides a subject with a second therapeutic agent, preferably an obesity agent such as rimonabant, orlistat, sibutramine, bromocriptine, leptin. ) Or peptide YY _3-36 or an analog thereof.

A second aspect of the present invention is directed to (i) administering a CCR2 therapeutic agent to a test subject, and (ii) the CCR2 therapeutic agent is used to reduce or maintain body fat and / or body weight of a test subject or to treat diabetes, metabolic syndrome or glucose intolerance. A method of identifying agents that can be used to reduce or maintain body fat and / or weight, or to treat diabetes, metabolic syndrome or glucose intolerance, including determining whether or not it is effective. CCR2 therapeutics include CCR2 antagonists. Additional CCR2 therapeutics include CCR2 inhibitors and inhibitors for CCR2 ligands.

As a related aspect, the method may further comprise testing the CCR2 therapeutic in an in vitro test for CCR2 activity prior to administering the CCR2 therapeutic to the test subject.

A third aspect of the present invention is directed to diagnosing Type I or Type II diabetes, comprising (i) administering a CCR2 therapeutic agent to a test subject, and (ii) determining whether the CCR2 therapeutic agent is effective to treat diabetes or glucose intolerance of the test subject. A method of identifying therapeutic agents that can be used to treat them. As a related aspect, the method may further comprise testing the CCR2 therapeutic in an in vitro test for CCR2 activity prior to administering the CCR2 therapeutic to the test subject.

A fourth aspect of the present invention can be used to treat a metabolic syndrome disorder comprising (i) administering a CCR2 therapeutic agent to a test subject, and (ii) determining whether the CCR2 therapeutic agent is effective for treating a metabolic syndrome of the test subject. This is how you identify your treatment. As a related aspect, the method may further comprise testing the CCR2 therapeutic in an in vitro test for CCR2 activity prior to administering the CCR2 therapeutic to the test subject.

In addition, a fifth aspect of the invention features a kit comprising a CCR2 therapeutic agent and instructions for administering a therapeutic agent to the subject to reduce or maintain body fat and / or weight of the subject. Also provided in one aspect of the present invention is a kit comprising CCR2 therapeutics and instructions for administering an antagonist to a subject for treating diabetes or glucose intolerance. In addition, an aspect of the invention features a kit comprising a CCR2 therapeutic agent and instructions for administering a therapeutic agent to a subject for treating a metabolic syndrome disorder. CCR2 therapeutics include CCR2 antagonists. Additional CCR2 therapeutics include CCR2 inhibitors and CCR2 ligand inhibitors. In other embodiments, the kit may further comprise a second therapeutic agent, more preferably an antiobesity agent such as limonabant, orlistat, sibutramine, bromocriptine, leptin or peptide YY _3-36 or an analog thereof.

Those skilled in the art will fully understand the terminology used herein for describing the invention and in the description and the appended claims. Nevertheless, unless stated otherwise herein, the following terms are further described immediately below.

The term "a" means one or more.

The terms “treat”, “treatment” and “treating” include acts that provide prophylaxis, such as prevention and alleviation treatment, or prevention or alleviation treatment.

The term "object" refers to any patient (eg, human or animal) or individual having a beneficial effect from reduced CCR2 activity.

"CCR2-mediated disease or disorder" means any disease, disorder, deleterious condition or condition in which CCR2 is known to play a role or have a certain effect.

The term "therapeutic agent" means a pharmaceutical composition including a compound that can modulate CCR2 or a CCR2 ligand, such as small molecules, biological substances or molecules (natural or synthetic); Such therapeutic agents include CCR2 antagonists or CCR2 inhibitors as defined herein, or inhibitors for CCR2 ligands.

"CCR2 antagonist" or "CCR2 inhibitor" means a therapeutic agent that directly or indirectly reduces or attenuates the biological activity of CCR2 (one or more). The agent may be used to determine the amount of CCR2 present on a protein, such as an anti-CCR2 antibody, nucleic acid, such as CCR2 antisense or RNA interference (RNAi) nucleic acid, amino acids, peptides, carbohydrates, small molecules (organic or inorganic), or cells. Any other compound or composition that effectively reduces or reduces the ability of the CCR2 ligand to interact to reduce the activity of the CCR2 polypeptide. Compounds that are CCR2 antagonists include all solvates, hydrates, pharmaceutically acceptable salts, tautomers, stereoisomers, and prodrugs of the compounds. Preferably, small molecule CCR2 antagonists used in the present invention have an IC ₅₀ of less than 10 μM, more preferably less than 1 μM, even more preferably less than 0.1 μM. Antisense oligonucleotides which inhibit their expression in connection with the CCR2 gene or mRNA are prepared according to standard methods (see Agrawal et al. (1993) Methods in Molecular Biology: Protocols for Oligonucleotides and Analogs, Vol. 20). Similarly, RNA interference molecules that act to reduce CCR2 receptor expression can be used (Hannon, (2002) Nature 418; 244-251, Shi, (2003) Trends in Genetics 19: 9-12, 2003; And Shuey et al., (2002) Drug Discovery Today 7: 1040-1046).

"CCR2 ligand inhibitor" means a therapeutic agent that prevents or reduces the action or interaction of a CCR2 ligand with its receptor.

"Reduced CCR2 activity" is a therapeutic agent that directly or indirectly affects CCR2 activity as a result of gene disruption or manipulation of CCR2 gene function that reduces the level of functional CCR2 polypeptide on the cell, or by interfering with ligand interactions. Means that the total polypeptide activity of CCR2 as a result of the administration of is reduced by manipulation.

The phrase “pharmaceutically acceptable” means that the designated carrier, vehicle, diluent, excipient (s) and / or salt is chemically and / or physically compatible with the formulation, generally containing other ingredients, and the recipient And physiologically compatible.

The term “prodrug” refers to a compound that is a drug precursor that releases the drug in vivo following a chemical or physiological process (eg, at physiological pH or through enzymatic activity) after administration. A review of the synthesis and use of prodrugs is described in Higuchi and Stella, Prodrugs as Novel Delivery Systems , vol. 14 of the ACS Symposium Series, and Bioreversible Carriers in Drug Design , ed. Edward B. Roche, American Pharmaceutical Association and Pergamon Press, 1987.

The terms "salts" and "pharmaceutically acceptable salts" refer to the organic and inorganic salts of the compound, stereoisomer of the compound, or prodrug of the compound.

"Overweight" and more severe "obesity" symptoms refer to cases that are larger than the ideal body weight (more specifically, greater than the ideal body fat) and are generally defined as body mass index (BMI), which is the total body fat and the symptoms of overweight or obesity Correlates with the relative risk of premature death or disability due to disease as a result of the disease. Health risks increase with excessive body fat. BMI is calculated by dividing body weight (in kg) by height (m2) (kg / m2), or alternatively multiplying body weight (in lb) by 703 and dividing by height (in ² units) (lbx703 / in ² ) . “Overweight” typically constitutes a BMI of 25.0 to 29.9. “Obesity” is typically defined as a BMI of 30 or more (eg, National Heart, Lung, and Blood Institute, Clinical Guidelines on the Identification, Evaluation, and Treatment of Overweight and Obesity in Adults, The Evidence Report, Washington, DC (US Department of Health and Human Services, NIH publication no. 98-4083, 1998). In muscular individuals, the correlation between BMI, body fat, and disease risk is weaker than in other individuals. Thus, assessment of whether an individual with muscle is actually overweight or obese can be made more accurately by another measurement, such as a direct measurement of total body fat or an assessment of waist-to-buttock ratio.

National Treatment Panel III (ATP III) as defined herein and National Institutes of Health: Third Report of the National Cholesterol Education Program Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults (Adult Treatment Panel III), Executive Summary; see "Metabolism Syndrome" by Bethesda, MD, National Institutes of Health, National Heart, Lung and Blood Institute, 2001 (NIH pub.no. 01-3670)). Occurs when you have three or more of the failures:

1. Abdominal obesity: waist circumference> 102 cm for men,> 88 cm for women;

2. hyperglyceridemia:> 150 mg / dl (1.695 mmol / l);

3. low HDL cholesterol: <40 mg / dl (1.036 mmol / l) for men, <50 mg / dl (1.295 mmol / l) for women;

4. High blood pressure: ≥130 / 85mmHg;

5. High fasting blood sugar: ≥110 mg / dl (≥6.1 mmol / l); or

According to World Health Organization standards (see Alberti and Zimmet, Diabet. Med. 15: 539-53, 1998), humans have two or more of the following abnormalities in addition to diabetes, impaired glucose tolerance, impaired fasting glucose or insulin resistance Occurs when you have:

1. High blood pressure: ≥160 / 90mmHg;

2. Hyperlipidemia: Triglyceride concentration ≥150 mg / dl (1.695 mmol / l) and / or <35 mg / dl (0.9 mmol / l) for HDL cholesterol and <39 mg / dl (1.0 mmol / l) for females l);

3. Central obesity: waist-to-butt ratio> 0.90 for men,> 0.85 for women and / or BMI> 30 kg / m 2;

4. Microproteinuria: urine albumin release rate ≥ 20 μg / min or albumin-to-creatine ratio ≥ 20 mg / kg

"Therapeutically effective" means a decrease in body fat and / or body weight with respect to appropriate controls; And / or amelioration of one or more signs of diabetes (NIDDM and / or IDDM), metabolic syndrome signs or disorders, and / or glucose intolerance.

Other features and advantages of the present invention will become more apparent from the following detailed description and claims. While the invention has been described in connection with specific embodiments, it is to be understood that other changes and modifications that may be made are also part of the invention and are within the scope of the appended claims. The present application is generally equivalent to any counterpart, modification, use or adaptation of the present invention, which may be found without undue experimentation, generally in accordance with the principles of the present invention, including without departing from the present disclosure which is known in the art or within conventional practice. To deal with it. All publications, including all published patent applications and issued patents, described herein are hereby incorporated by reference in their entirety.

Figure 1 shows the time course of weight change in four experimental groups, namely wild type (C57) mice (C57 LFD) fed low-fat diet; CCR2-/-knockout (KO) mice fed low-fat diet (KO LFD); Wild type mice fed high fat diet (C57 HFD); And the percentage of baseline changes for CCR2 KO mice (KO HFD) fed high fat diet. C57 HFD mice showed weight gain compared to C57 LFD. However, KO HFD mice did not experience similar weight gain compared to KO LFD mice.

Figure 2 is a line graph showing the time course of epididymal fat in weight ratio for four experimental groups. CCR2 KO mice showed reduced fat relative to body weight compared to similar dietary C57 control mice.

3A-3D provide tissue cross-sectional images of adipose tissue from four experimental groups after 12 weeks.

4 is a bar graph showing the time course of food consumption, normalized to weight gain for four experimental groups. Despite the reduced weight gain of KO HFD mice as can be seen in FIG. 1, KO HFD mice consumed the same or slightly higher amounts of HFD as compared to C57 HFD control mice.

5 shows oxygen consumption (VO2) in four experimental groups. No significant change in metabolic rate was found in the four experimental groups.

6 shows resting VO2 in four experimental groups. No significant change was observed among the four experimental groups.

7 is a line graph showing the level of fasting plasma insulin in four experimental groups. CCR2 KO mice had greater insulin sensitivity than C57 control mice in both the HFD and LFD protocols, as can be seen by the lower basal levels of insulin.

8A-8B are line graphs showing the results of oral glucose tolerance test (OGGT) and insulin levels in four experimental groups. CCR2 KO HFD mice showed significantly reduced glucose excursion during OGGT compared to C57 HFD.

9 is a line graph showing the results of the oral glucose tolerance test (OGGT) in CCR2 KO mice and C57 mice on a normal diet. CCR2 KO mice had significantly greater glucose tolerance compared to C57 control mice.

The present invention is in need of treatment by administering a CCR2 antagonist, for example, in the treatment of overweight or obese patients (eg humans or pets) or as a means for producing fat-free meat in meat animals (eg cattle, chickens, pigs). A method of reducing the weight and / or body fat of an animal in a patient.

The invention also relates to a method of treating metabolic syndrome disorder in a patient (human or animal) in need thereof by administering a CCR2 antagonist.

The invention also relates to a method of treating non-insulin dependent diabetes mellitus (insulin dependent and / or non-insulin dependent) and / or glucose intolerance in a patient (human or animal) in need thereof by administering a CCR2 antagonist.

As described in the Examples herein, CCR2-/-knockout mice (KO) relatively block the expression of increased body weight and increased fat. The data indicated that signs of metabolic syndrome decreased after exposure to high fat diet (HFD). Embodiments show that reducing CCR2 activity is an effective way to reduce body weight and / or body fat and can improve the signs of metabolic syndrome, such as being overweight, obese and / or suffering from one or more signs of metabolic syndrome (human and It can be used to treat animals, and it can be effectively used to treat animal breeding animal species to produce fat-free meat.

Exemplary Chemokine Antagonists (Inhibitors)

Chemokine antagonists, in particular CCR2 antagonists, may be useful in the practice of the present invention and may also be identified by the assays mentioned herein, and are described in the patent documents WO 04/050024, WO 04/016769, WO 03/093266, WO 03/093231, WO 03/092586, WO 03/051921, WO 01/51467, WO 00/69815, WO 00/69432, WO 00/46199, WO 98/25617, WO 98/25605, US Publication No. 2003144339, US Publication No. 20030165494, US Publication No. 2003008893 and US Publication No. 2002042370, US Patent No. 6,737,435, US Patent 6,686,353, US Patent 613,760, US Patent 6,569,888, US Patent 6,479,527, US Patent 6,472,410, US Patent 6,451,842, US Patent 6,403,587, US Patent 6,441,004 , U.S. Patent 6,362,177, U.S. Patent 6,387,912, U.S. Patent 6,291,501, U.S. Patent 6,288,103, U.S. Patent 6,140,349, U.S. 6,140,338, U.S. Patent 6,166,037, U.S. Patent 6,136,827, U.S. Patent 6,124,319, U.S. Patent 6,084,075, U.S. Patent 6,013,664, U.S. Patent 5,962,462, and U.S. Patent 5,719,776. can do.

CCR2 antagonist

As used herein, the term “antagonist of CCR2 action” refers to a medicament (eg, molecule, compound) capable of inhibiting the (one or more) action of CCR2. For example, antagonists of CCR2 action can inhibit the binding of one or more ligands (eg, MCP-1, MCP-2, MCP-3, MCP-4 and MCP-5) to CCR2 and / or are mediated by CCR2. Signal transduction (eg, GDP / GTP exchange by CCR2-related G proteins, intercellular calcium flux). Thus, CCR2-mediated processes and cellular responses (eg, proliferation, migration, chemotaxis, secretion or degranulation) can be inhibited by antagonists of CCR2 action. As used herein, “CCR2” refers to naturally occurring CC chemokine receptor 2 (eg, mammalian CCR2 (eg, CCR2 of human (homo sapiens)), and refers to naturally occurring variants such as allelic and splice variants ( Such as CC-chemokine receptor 2a and / or CC-chemokine receptor 2b).

Antagonists of CCR2 action are compounds, for example small organic molecules, natural products, proteins (eg, antibodies, chemokines, cytokines), peptides or pentide derivatives (peptidomimetic).

Organic molecules; Proteins such as antibodies (eg, multibranched serum, monobranched, chimeric, humanized, human) and antigen-binding segments thereof; Chemokine mutants and analogs; And many molecules are known in the art that can antagonize one or more actions of chemokine receptors (eg, CCR2), including peptides.

Antagonists of CCR2 action can be identified using assays or other suitable methods, such as libraries of molecular inventories such as the Chemical Repository of the National Cancer Institute, as described herein.

Another source of antagonist of CCR2 action is a combined library, which may include many structurally different molecular compounds. Combination libraries can be used to identify lead compounds or to optimize previously identified lead compounds. Such libraries can be prepared by well known combinatorial chemistry methods and assayed by suitable methods such as those described herein.

As mentioned previously, other CCR2 antagonists, including CCR2 selective antagonists, can be identified using standard assays known to those skilled in the art. That is, one type of assay for identifying CCR2 selective modulators uses cell lines, including primary cells or infected CCR2 cells. Alternatively, animal models can be used.

Preferably, CCR2 proteins used in assay assays for CCR2 antagonists or inhibitors are human (see US Pat. No. 5,707,815, US Pat. No. 6,132,987 and Charo et al., (1994) PNAS, 91: 2752-2756). to be. Other mammalian species of CCR2 protein are also known and can also be used for assays.

An alternative to the assay of inhibition of CCR2 ligand binding is to assess inhibition of CCR2 action.

Test agents used in assays for CCR2 therapeutics can be individually selected from eg the above-mentioned patent documents or obtained from a compound library. Such agents include combinatorial chemically-derived molecular libraries made of peptides, D- and / or L-form amino acids, modified biological agents including phosphopeptides, antibodies such as modified proteins, and small organic and inorganic compounds. Include. The library is a biological library, a library of natural compounds, a peptoid library (a library of molecules having the function of a peptide but having a novel non-peptide backbone that is bioactive and resistant to enzymatic degradation) (Zuckermann (1994), J Med. Chem . 37; 2678-85), spatially addressable parallel solid or solution phase libraries, synthetic library methods requiring deconvolution, "1-bead, 1-compounds.""Library methods, and synthetic library methods using affinity chromatography selection.

Examples of methods for the synthesis of molecular libraries are described in DeWitt et al. (1993), PNAS. 90; 6909; Erd et al. (1994) PNAS 91: 11422; Zuckermann et al. (1994), J. Med. Chem . 37; 2678; Cho et al. (1995), Science , 261: 1303; And Gallop et al. (1994), J. Med. Chem . 37: 1233.

The compound library may be in solution (see Houghten, (1992) Biotechniques , 13: 412-421), or in beads (Lam, (1991) Nature 354: 82-841), on chip (See Fodor (1993), Nature 364; 555-556), bacteria or spores (see Ladner, US Pat. No. 5,223,409), plasmids (Cull et al. (1992), PNAS 89: 1865-) 1869) or phage (Scott et al. (1990), Science 249: 386-390; Devlin (1990), Science 249: 404-406; Cwirla et al., (1990) PNAS 87: 6378-6382; and Felici ( 1991), J. Mol . Biol . 222: 301-310.

Test method

As mentioned above, the present invention includes assay methods for identifying agents that can be used in the methods of treatment described herein. The method may include determining whether the agent is (directly or indirectly) effective in reducing or maintaining body weight and / or body fat after determining whether it modulates CCR2, ligand binding or action. Confirmation of whether an effective treatment of a disorder or symptom of metabolic syndrome is included. It may also be a confirmation whether it is an effective treatment of insulin dependent or non-insulin dependent diabetes mellitus and / or glucose intolerance. In the case of diabetes, the effectiveness of the therapeutic agent may be determined by glucose tolerance tests. Alternatively, the assay method may include testing a medicament known as a CCR2 therapeutic for efficacy in the method of treatment.

Testing of agents for efficacy of altering CCR2 activity can be carried out using methods well known in the art (see Charo et al. (1994) PNAS 91, 2752-2756).

The therapeutic efficacy of the active compound can be determined by standard therapeutic procedures in cell culture or animal models, for example, to determine the ED 50 (concentration of the compound that produces 50% of the maximum effect). Once the medicament is determined as a CCR2 antagonist or a known CCR2 antagonist is tested, the medicament can then be tested to see if it is effective in the treatment methods described herein. Such testing may be conducted in an appropriate animal model system for the symptoms described herein. For example, genetically obese mice (eg, C57BL (ob / ob), diet-induced obese mice (ie, DIO mice) or rats can be treated with a therapeutic agent and are subject to various factors related to the symptoms described herein). The effect of the medicament on the drug can be compared with that in animals under similar conditions except that it is not treated with a therapeutic agent Factors that can be tested for this purpose include, for example, body weight, body fat, insulin, glucose, triglycerides, Free fatty acids, adiponectin, hemoglobin A1c, cholesterol, leptin and / or fructosamine Some examples of these are provided in the examples below: Therapies found to have a positive effect on these factors are known in the art. It can be selected for testing in other preclinical or clinical studies as can be determined by one of skill in the art.

Characterization of CCR2 Antagonists

CCR2 antagonists found to have a positive impact on factors associated with the methods of treatment reviewed herein can be tested in preclinical or clinical studies as can be determined by one of skill in the art.

Data obtained from cell culture assays and animal models can be used to formulate a range of dosages for use in humans. The dosage may vary depending upon the dosage form employed and the route of administration. For any compound or medicament used in the methods of the invention, the therapeutically effective dosage can be estimated initially from the cell culture assay. Dosages may be formulated in animal models to reach circulating plasma concentrations including IC50. The information can be used to more accurately determine dosages useful for humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

How to treat

Therapeutic agents identified as CCR2 antagonists are sufficient to reduce or maintain or maintain body weight and / or body fat in individuals who wish to reduce the mass of fat depot or maintain body weight and / or body fat, for example in obese patients. Administered in dosage. Such therapeutically effective amounts are determined using conventional optimization techniques, for example, determined by symptoms of the patient (human or animal), route of administration, formulation, judgment of the practitioner, and factors apparent to those skilled in the art in light of the present disclosure. .

CCR2 therapeutic agents suitable for use in accordance with the present invention may be administered alone, but in human treatment are generally administered in admixture with suitable pharmaceutical excipients, diluents or carriers selected with regard to the desired route of administration and standard pharmaceutical practice. do.

For example, CCR2 antagonists or salts or solvates thereof suitable for use in accordance with the present invention may be used orally, intraorally or for buccal, delayed, modified, sustained, dual, controlled release or pulsation delivery applications. Sublingually can be administered in the form of tablets, capsules (including soft gel capsules), composite particles, gels, films, ovules, elixirs, solutions or suspensions, which may contain flavoring or coloring agents. The compounds may also be administered via the belonging or fast dissolving dosage form or in the form of high energy dispersion or in the form of coated particles. Suitable pharmaceutical formulations may be in coated or uncoated form as desired.

Such solid pharmaceutical compositions, such as tablets, may contain excipients such as microcrystalline cellulose, lactose, sodium citrate, calcium carbonate, divalent calcium phosphate, glycine and starch (preferably corn, potato or tapioca starch), disintegrants such as sodium starch Glycolate, croscarmellose sodium and certain complex silicates, and granulation binders such as polyvinylpyrrolidone, hydroxypropylmethyl cellulose (HPMC), hydroxypropyl cellulose (HPC), hydroxypropyl methylcellulose acetate succinate Ate (HPMCAS), sucrose, gelatin and acacia. In addition, lubricants such as magnesium stearate, stearic acid, glyceryl behenate and talc may be included.

Solid compositions of a similar type may also be used as fillers in gelatin capsules or HPMC capsules. Excipients in this regard include lactose, starch, cellulose, lactose, or high molecular weight polyethylene glycols. In the case of aqueous suspensions and / or elixirs, the CCR2 antagonist compounds bind various sweeteners or flavoring agents, coloring substances or pigments, and emulsifiers and / or suspending agents, diluents such as water, ethanol, propylene glycol and glycerin, and Combinations thereof.

The modified release and pulsatile release dosage forms may contain excipients such as those described for immediate release dosage forms with additional excipients acting as release rate modifiers, the excipients being coated on and / or in the body of the device It may be included. Release rate modifiers include HPMC, HPMCAS, methyl cellulose, sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, polyethylene oxide, xanthan gum, carbomer, ammonomethacrylate copolymer, hydrogenated castol oil , Carnauba wax, paraffin wax, cellulose acetate phthalate, hydroxypropylmethyl cellulose phthalate, methacrylic acid copolymers and mixtures thereof, but not limited thereto. Modified release and pulsatile release dosage forms may contain one or a combination of excipients for release rate modification. Excipients for release rate modification can be present in the dosage form, ie on the substrate and / or on the dosage form, for example on the surface or coating.

The belonging or fast dissolving dosage form (FDDF) may contain the following components: aspartame, acesulfame potassium, citric acid, croscarmellose sodium, crospovidone, diascorbic acid, ethyl acrylate, ethyl cellulose, Gelatin, hydroxypropylmethyl cellulose, magnesium stearate, mannitol, methyl methacrylate, mint fragrance, polyethylene glycol, fumed silica, silicon dioxide, sodium starch glycolate, sodium stearyl fumarate, sorbitol, xylitol. As used herein to describe FDDF, the term disintegration or dissolution depends on the availability of the drug substance used, i.e., if the drug substance is insoluble, a belonging dosage form can be prepared and the drug substance is soluble. Fast-soluble dosage forms can be prepared.

CCR2 antagonists suitable for use according to the present invention are also administered parenterally, such as cavernous, intravenous, intraarterial, intraperitoneal, intradural, intraventricular, urethra, intrasternal, intracranial, intramuscularly or subcutaneously. Or may be administered by techniques that do not use infusion or saliva. In the case of parenteral administration they are best used in the form of sterile aqueous solutions which may contain sufficient substances or glucose to make other substances, such as isotonic solutions with the blood. The aqueous solution should suitably be buffered (preferably to a pH of about 3-9) as needed. Preparation of suitable parenteral formulations under sterile conditions is readily accomplished by standard pharmaceutical techniques well known to those skilled in the art.

For oral and parenteral administration to a patient (human or animal), the daily dosage level of a CCR2 antagonist for use in the present invention is generally 1 to 500 mg (single or divided dosages). Dosage ranges from about 1 mg to about 100 mg. The dosage may be by a single dosage, or by a divided dose or multiple daily dosages. Alternatively, continuous dosing can be administered on a daily basis or once in a day or more, such as by a controlled (eg, slow) release dosage form.

Thus, for example, tablets or capsules of CCR2 antagonists suitable for use in accordance with the present invention may suitably contain 1 mg to 250 mg of the active compound for administration alone or in combination of two or more. Preferred tablets or capsules suitably contain from about 1 mg to about 50 mg of the active compound for administration in two or more, alone or simultaneously. In any case, the physician determines the actual dosage, which is best suited for any individual patient and varies with the specific patient's age, weight and response. Of course, there may be respective examples in which larger or smaller dosage ranges are advantageous, which is also within the scope of the present invention.

CCR2 antagonists suitable for use according to the invention may also be administered intranasally or by inhalation, and include suitable propellants such as dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1 Hydrofluoroalkane, carbon dioxide, such as 1,1,2-tetrafluoroethane (HFA 134A ^TM ) or 1,1,1,2,3,3,3-heptafluoropropane (HFA 227EA ^TM ) Conveniently delivered in the form of aerosol spray provision or a dry powder inhaler from a pressurized vessel, pump, spray or nebulizer using other suitable gases. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered amount. Pressurized vessels, pumps, sprays or nebulizers may contain a solution or suspension of the active compound, for example using a mixture of ethanol and propellant as solvent, which may further contain a lubricant such as sorbitan trioleate. Capsules and cartridges (eg, made from gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of a compound of the invention and a suitable powder base such as lactose or starch.

The aerosol or dry powder formulation is preferably adjusted such that each metered dose or "puff" contains 1 to 50 mg of CCR2 antagonist to be delivered to the animal to be treated. The total daily dose in the aerosol ranges from 1 to 50 mg, which may be administered in a single dose or more generally in divided doses throughout the day.

CCR2 antagonists suitable for use in accordance with the present invention may also be formulated for delivery via an atomizer. Formulations for nebulizer devices may contain the following components as solubilizers, emulsifiers or suspending agents: water, ethanol, glycerol, propylene glycol, low molecular weight polyethylene glycols, sodium chloride, fluorocarbons, polyethylene glycol ethers, sorbitan tree Oleate, oleic acid.

Alternatively, CCR2 antagonists suitable for use in accordance with the present invention may be administered in the form of suppositories or pessaries, or may be topically applied in the form of gels, hydrogels, lotions, solutions, creams, ointments or spray powders. CCR2 antagonists suitable for use in accordance with the present invention may also be administered to the dermis or by transdermal, such as using skin patches. They may also be administered by pulmonary or rectal route.

CCR2 antagonists may also be administered by the ocular route. For ocular use, the compounds may be formulated in a microsuspension in isotonic, pH adjusted sterile saline, preferably in solution in isotonic, pH adjusted sterile saline, optionally in combination with a preservative such as benzylalkonium chloride. . Alternatively, they may be formulated in an ointment such as petrolatum.

For topical application to the skin, suitable CCR2 antagonists for use in accordance with the invention are suspended in a mixture with, for example, mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyoxyethylene polyoxypropylene compound, emulsifying wax and water It may be formulated as a suitable ointment containing dissolved active ingredient or medicament. Alternatively, they may be suspended in a mixture with one or more of mineral oil, sorbitan monostearate, polyethylene glycol, liquid paraffin, polysorbate 60, cetyl ester wax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water It may be formulated into a suitable lotion or cream dissolved.

Suitable CCR2 antagonists for use according to the invention may also be used in combination with cyclodextrins. Cyclodextrins are known to form inclusion and non-inclusion complexes with drug molecules. Formation of the drug-cyclodextrin complex can modify the solubility, degradation rate, bioavailability and / or stability of the drug molecule. Drug-cyclodextrin complexes are generally useful for most dosage forms and administration routes. As an alternative to direct complexation with the drug, cyclodextrins can be used as auxiliary additives, such as carriers, diluents or solubilizers. α-, β- and γ-cyclodextrins are some of the most commonly used and suitable examples are described in PCT publications WO 91/11172, WO 94/02518 and WO 98/55148.

In general, oral administration is the preferred route for humans and is often the most convenient. If the recipient experiences a swallowing disorder or a drug absorption disorder following oral administration, the drug can be administered parenterally, sublingually or intranasally. Parenteral administration may be used when the medicament is orally inactive.

For veterinary use, the CCR2 inhibitor is administered in a suitably acceptable formulation in normal veterinary practice, and the veterinarian determines the dosage regimen and route of administration, which is best suited for the specific animal. Such animals include companion animals that are overweight, obese or at risk of becoming overweight or obese. Other animals which can be treated according to the invention are meat animals for the purpose of obtaining meat free of fat than those obtained without treatment according to the invention.

CCR2 antagonists used according to the present invention may also be used with other pharmaceutical agents for the treatment of the diseases, symptoms and / or disorders described herein. The second agent is selected to have a combined beneficial effect on the treatment of the speaker. Thus, there is also provided a method of treatment comprising administering a CCR2 antagonist in combination with other pharmaceutical agents. Suitable pharmaceutical preparations that can be used in combination with the compounds of the invention include apolipoprotein-B secretion / microsomal triglyceride transfer protein (apo-B / MTP) inhibitors, 11β-hydroxy steroid dehydrogenase-1 (11β-HSD 1 Type) inhibitors, peptide YY _3-36 or analogues thereof, MCR-4 agonists, cholecystokinin-A (CCK-A) agonists, monoamine reuptake inhibitors (eg sibutramine), cannabinoid receptor-1 antagonists (eg , Limonavant), sympathetic agonists, β ₃ adrenergic receptor agonists, dopamine agonists (eg bromocriptine), melanocyte-stimulating hormone analogs, 5HT2c agonists, melanin enrichment hormone antagonists, leptin analogs, leptin receptor agonists, galanin Agonists, lipase inhibitors (eg tetrahydrolipstatin such as orlistat), appetite suppressants (eg bombesin agonist), neuropeptide-Y receptor drugs (E.g., NPY Y2 agonist), U.S. Patent 6,566,367, U.S. Patent 6,649,624, U.S. Patent 6,638,942, U.S. Patent 6,605,720, U.S. Patent 6,495,559, U.S. Patent 6,462,053, U.S. Patent 6,388,077 , US Pat. No. 6,335,345, US Pat. No. 6,326,375, US Patent Publication No. 20020151456 and US Patent Publication No. 2003036652, PCT Publications WO 03/010175, WO 03/082190 and WO 02/048152. Selected compounds described, thyroid agents, dihydroepiandrosterone or analogues thereof, selected glycocorticoid receptor agents, orexin receptor antagonists, glucagon-type peptide-1 receptor agonists, ciliary nerve growth factors (e.g., reggae, Terrytown, NY, USA) Axokine ^TM , a inhibitor of human guinea pig-related protein (AGRP), available from Regeneron Pharmaceuticals, Inc., ghrelin Selected antitherapeutic agents, which may include receptor antagonists, histamine 3 receptor antagonists or inverse agonists, neuromedin U receptor agonists, and the like. Other anti-obesity agents, including the preferred agents described below, are well known, or will be readily apparent to the average skilled artisan in the context of the present disclosure.

Orlistat, sibutramine, bromocriptine, leptin, limonabant, peptide YY _3-36 or an analog thereof, and 2-oxo-N- (5-phenylpyrazinyl) spiro- [isobenzofuran-1 (3H), 4 Particularly preferred is an anti-obesity agent selected from the group consisting of '-piperidine] -1'-carboxamide. Preferably, the compounds and combination treatments of the invention are administered in combination with exercise and a sensible diet.

Representative anti-obesity agents for use in the combinations, pharmaceutical compositions, and methods of the present invention can be prepared using methods known to those of ordinary skill in the art, such as for example, when sibutramine is prepared as described in US Pat. No. 4,929,629. Bromocriptine can be prepared as described in US Pat. Nos. 3,752,814 and 3,752,888; Orlistat is US 5,274,143, US 5,420,305, US 5,540,917 and Can be prepared as described in US Pat. No. 5,643,874, and Rimonabant can be prepared as described in US Pat. No. 5,624,941, and PYY _3-36 (including analogues thereof) is disclosed in US Patent Publication No. 20020141985 and PCT Publication WO. 2-oxo-N- (5-phenylpyridinyl) spiro [isobenzofu], which may be prepared as disclosed in US Pat. No. 03/027637, which is an NPY Y5 receptor antagonist. Ran-1 (3H), 4'-piperidine] -1'-carboxamide may be prepared as described in US Patent Publication No. 2002151456.

Kit

The present invention provides a kit or pharmaceutical package comprising a CCR2 antagonist for use in the prevention and treatment of the diseases and symptoms described herein. For example, in addition to one or more CCR2 antagonists in the form of tablets, capsules or lyophilized powders, kits or containers may include instructions for using the antagonists in the prevention or treatment of the diseases and conditions. The antagonist may be provided in a kit or packaging in a bottle or another suitable form (eg, a blister pack). Optionally, the kit or pharmaceutical packaging may also contain other pharmaceutically active agents (e.g., drugs listed above, such as anti-obesity agents) and / or materials used to administer the drug (s), such as diluents, saliva, syringes, applicators ( applicator).

The present invention is based in part on the following experimental results. While the present invention has been described herein in connection with specific spirit, it is to be understood that other changes and modifications that may be made are also part of the invention and are also within the scope of the appended claims. This application is any counterpart, modification, use of the invention that generally conforms to the principles of the invention, including deviations from the disclosure that are known in the art or are within conventional practice and may be readily apparent without unreasonable experimentation. Or to deal with applications.

CCR2 (KO) (-/-) mice were sourced from Dr. Israel Charo of J. David Gladstone Institutes. Homologous recombination was used for embryonic stem cells to produce mice with target disruption of CCR2 (Kuziel, WA et al. (1997) PNAS 94, 12053-12058 and Boring L. et al. (2001) J. Clin. Invest . 100, 2252-2561). Age-adjusted male CCR2 (KO) (-/-) mice (n = 20) and wild-type (+ / +) (C57) litter controls (n = 20) were acclimated for 2 weeks before starting the study and Purina 5001 rodent chow (Furina, Brentwood, Missouri, USA) was freely accessible.

Mice were individually confined and divided into two groups: the first group on diet consisting of less than 10 kg of fat (low fat diet (LFD)) and 45 kcal% fat (high fat diet (HFD)) during the 15-week study period. Second group of composed diets (D12331 Rodent Diets, Research Diet Inc., New Brunswick, NY, USA). Mice had free access to water. Animal intake was measured in a 24 hour, 5 day / week (Monday-Friday) cycle. Body weights were determined on day 0 and every week.

Further studies compared CCR2 KO mice and C57 mice, in which both groups consumed normal feed (Furina 5001 Rodent Chow, Furina, Brentwood, Missouri) but differed in fasting plasma insulin levels.

Energy consumption and oxygen consumption were determined using the Oxymax system (Columbus Instruments, Columbus, Ohio). Mice were locked under standard laboratory conditions and fed on an experimental diet. (1 mouse per chamber). The chamber was placed on an activity monitor. The calorimeter was calibrated before each use and the air flow was adjusted to 1.6 l / min. The system setting and sampling time were set to 60 seconds and 15 seconds, respectively.Oxygen consumption, carbon dioxide production and walking activity were measured every 10 minutes for 4 hours.

The Oral Glucose Tolerance Test (OGGT) was conducted after the end of the 16-week study in mice from four experimental groups, with the first sample taken at about 8:30 am (zero hour) after night fasting. Post-orbital blood samples were given an oral glucose load of 2 g / kg body weight after blood collection at zero time as mentioned. Additional blood samples were drawn at 30, 60, 120 and 180 minutes after glucose administration. 25 [mu] l of blood was added to 100 [mu] l of 0.025% heparin- saline in microvitro (Denville Scientific, Inc., Metuchen, NJ). Test tubes were spun at Microfuge® 12 (Beckman Coulter, Fullerton, Calif.) For 2 minutes at the highest setting.

Body weights were determined on the last morning of the study, and then blood samples were drawn from the orbital sinus for plasma glucose determination. The mice were then killed and approximately 1 ml of blood was passed through a Microtainer® plasma separator test tube (Becton-Dickinson, Inc.), Lithium Heparin, Franklin Lakes, NJ, USA. ), Blood was collected from). The test tubes were spun at a maximum setting for 5 minutes on a Beckman Microfuse 12. Plasma was drawn into 1.5 ml Eppendorf test tubes and frozen in liquid nitrogen. The epididymal fat pad was removed and weighed and frozen in liquid nitrogen. All samples were stored at -80 ° C.

Plasma glucose was measured using a manufacturer's reagent on a commercial machine (Roche / Hitachi 912 Clinical Chemistry Analyzer, Roche Diagnostics Corp., Indiana, USA) Indianapolis).

Plasma insulin was assessed using the Mercodia ELISA insulin kit from APLCO Diagnostics, Wyndham, New Hampshire, according to the manufacturer's instructions.

Example 1 CCR2 Inhibitory Effects on Body Weight, Body Fat and Metabolic Residues of Male Mice Fed High Fat Diet (HFD)

CCR2 gene disruption in CCR2 KO (KO) mice during high fat diet (HFD) gave rise to a robust phenotype that prevented the development of obesity. Wild-type (C57) mice fed HFD showed weight gain compared to low-fat (LFD) C57 mice, while CCR2 KO HFD mice maintained body weight comparable to CCR2 KO LFD mice. The phenotype to prevent obesity in CCR2 KO HFD mice was clearly demonstrated as shown in FIG. 1. In contrast to C57 HFD mice whose body weight increased about 30-35% above baseline body weight in 7 weeks, the weight of CCR2 KO HFD mice increased only 5-10% above baseline body weight, in both CCR2 KO LFD mice and C57 LFD mice. This corresponded to an increase comparable to that observed.

The measurement of epididymal adipose tissue expressed as percentage body weight shown in FIG. 2 proved to be due to increased fat gain in C57 HFD mice. CCR2 KO HFD mice increased body fat during the study, but were significantly lower than C57 mice on the same diet (3.2% versus 5.0% of total body weight, respectively).

Biopsy of adipose tissue, pancreas and liver sections

C57 mice and CCR2 KO mice (n = 5 / group) received LFD or HFD for 12 weeks. At the end of the period animals were necropsied and adipose tissue, pancreas and liver were harvested from mice for histology. White adipose tissue was weighed and the relative ratio to body weight was calculated.

The adipose tissue sections were stained with Mac2, a phagocytic marker, and the amount of staining as well as the fat cell size were determined by morphometric analysis. The final amount of Mac 2 staining was corrected for adipocyte size.

Adipose tissue cross section:

Organ Mass: The epididymal adipose tissue mass increased in both C57 and KO mice fed high fat diet as mentioned in Table 1. This increase was more pronounced in C57 mice (2.6x vs. 2.1x). Adipose tissue mass in KO mice in the low fat (LFD) group was slightly less than in C57 mice.

Microscopy: Adipocytes were larger in both C57 and KO mice fed HFD. 3B and 3D (HFD mice) were compared with FIGS. 3A and 3C (LFD mice). The size of adipocytes taken from C57 mice appeared larger than in KO mice. 3A and 3B (C57 mice) were compared with FIGS. 3C-3D (KO mice). The observation was confirmed by morphometric analysis and consistent with organ mass.

In C57 HFD mice, the number of phagocytes in adipose tissue was increased, which correlates with an increase in adipocyte size. In contrast, KO HFD mice showed a decrease in the number of phagocytes in epididymal adipose tissue. The observation was confirmed by morphometric analysis.

The histochemical data indicate that adipocyte size was increased in both C57 HFD mice and KO HFD mice. The increase was more severe in C57 mice. This increase is associated with phagocytic infiltrates in fat.

The weight difference between C57 HFD mice and KO HFD mice could not be explained by reduced food intake in KO mice. CCR2 KO mice consumed HFD to the same extent as compared to C57 counterparts (FIG. 4).

It was evident that there was no change in metabolic rate in HFD-ingested CCR2 KO mice. Oxygen consumption (VO2), measured by total oxygen consumption (VO2) (FIG. 5) or respiratory exchange rate (FIG. 6), was not significantly different in HFD-ingested KO mice. Constant VO2 values in C57 mice that received both HFD and LFD corresponded to CCR2 KO HFD and LFD mice.

The results demonstrate that a decrease in CCR2 activity is an effective means of losing weight and / or reducing body fat and treating disorders associated with increased fat.

Example 2 Effect of CCR2 on Metabolic Factors in Mice Fed High Fat Diet

CCR2 KO mice had lower basal insulin levels, which means increased insulin sensitivity, such as by decreased glucose locomotion (FIG. 7) and prevents glucose increase during OGTT. In addition, in contrast to C57 HFD mice, CCR2 HFD KO mice did not express hyperinsulinemia and glucose intolerance (FIGS. 8A and 8B).

The results demonstrate that CCR2 therapeutics are effective in treating disorders associated with diabetes mellitus, glucose intolerance or insulin resistance. CCR2 therapeutics are effective in treating disorders associated with metabolic syndrome. In addition, CCR2 therapeutics are effective in reducing or maintaining body weight and / or body fat and also for treating disorders associated with fat gain.

Claims (14)

Subjecting a subject in need of reducing or maintaining body fat and / or weight to reduce or maintain body fat and / or weight, comprising administering a therapeutically effective amount of a CC chemokine 2 (CCR2) therapeutic How to treat it. A method of treating a subject by treating diabetes or glucose intolerance, comprising administering to a subject in need thereof a diabetes or glucose intolerance treatment, a therapeutically effective amount of a CC chemokine 2 (CCR2) therapeutic. A method of treating a subject by treating a metabolic syndrome disorder comprising administering to the subject in need thereof a therapeutically effective amount of a CC chemokine 2 (CCR2) therapeutic. The method according to any one of claims 1 to 3, The CCR2 therapeutic agent is a CCR2 antagonist. The method according to any one of claims 1 to 3, CCR2 therapeutic agent is an inhibitor for CCR2 ligand. (i) administering a CCR2 therapeutic agent to a test subject, and (ii) determining whether the CCR2 therapeutic agent is effective to reduce or maintain body fat and / or weight of the test subject. Confirmation method of drug which there is. A method of identifying a medicament that can be used to treat diabetes or glucose intolerance, comprising (i) administering a CCR2 therapeutic agent to a test subject, and (ii) determining whether the CCR2 therapeutic agent is effective to treat diabetes or glucose intolerance of the test subject. . A method of identifying a medicament that can be used to treat a metabolic syndrome disorder, comprising (i) administering a CCR2 therapeutic agent to a test subject, and (ii) determining whether the CCR2 therapeutic agent is effective for treating a metabolic syndrome disorder in a test subject. The method according to any one of claims 6 to 8, The CCR2 therapeutic agent is a CCR2 antagonist. The method according to any one of claims 6 to 8, CCR2 therapeutic agent is an inhibitor for CCR2 ligand. The method according to any one of claims 1 to 3, Further comprising testing the CCR2 therapeutic agent in an in vitro test for CCR2 therapeutic activity prior to administering the CCR2 antagonist to the test subject. A kit comprising a CCR2 therapeutic agent and instructions for administering a therapeutic agent to a patient to reduce or maintain body fat and / or weight of the subject. A kit comprising a CCR2 therapeutic agent and instructions for administering a therapeutic agent to the patient for treating diabetes and / or glucose intolerance of the patient. A kit comprising a CCR2 therapeutic agent and instructions for administering an antagonist to the patient for treating a metabolic syndrome disorder in the patient.

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