WO2012033518A1 - Methods and compositions for treating metabolic disorders - Google Patents

Abstract

The present invention provides methods and compositions for treating insulin resistance related disorders and metabolic symptoms of obesity. The therapeutic methods and compositions of the invention employ compounds that specifically inhibit or suppress the signaling pathway mediated by tissue factor and protease activated receptor 2 (TF-PAR2 signaling pathway).

Description

Methods and Compositions for Treating Metabolic Disorders

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001 ] The subject patent application claims the benefit of priority to U.S. Provisional Patent Application No. 61 /402,984 (filed September 9, 2010). The full disclosure of the priority application is incorporated herein by reference in its entirety and for all purposes.

STATEMENT OF GOVERNMENT SUPPORT

[0002] This invention was made by government support under Grant Nos. HL-71 146, HL77753 and HL-104232 awarded by the National Institutes of Health. The U.S.

Government therefore has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] Imbalance in energy metabolism in the body can lead to various diseased states, e.g., obesity and obesity-induced diabetes. Insulin regulates the concentration of blood sugar and blood lipid through the promotion of glucose and lipid intake into cells and utilization and storage of them. Insulin resistance is a condition in which insulin does not act normally on cells, which causes elevation of the concentration of blood sugar or blood lipid. Insulin resistance in obesity and type 2 diabetes is manifested by decreased insulin-stimulated glucose transport and metabolism in adipocytes and skeletal muscle, and by impaired suppression of hepatic glucose output.

[0004] Obesity is commonly associated with diabetes. Before the development of diabetes, many obese patients develop a peripheral resistance to the actions of insulin.

Elevated level of plasma free-fatty acids contributes to insulin resistance in diabetes and obesity. It was shown that free fatty acids induce insulin resistance in human in a dose dependent fashion. See, e.g., Boden G., Front. Biosci. 3:d 169- 175, 1998; Boden G., Diabetes 46: 3- 10, 1997; and Roden et al., J Clin Invest 97, 2859-65, 1996.

[0005] There is a need in the art for new compounds and methods for treating insulin resistance related disorders and metabolic symptoms of obesity, e.g., insulin resistance and type 2 diabetes. By providing novel methods and compositions for treating insulin resistance, the instant invention addresses this and other needs. SUMMARY OF THE INVENTION

[0006] In one aspect, the invention provides methods for treating subjects suffering from metabolic complications of obesity or disorders associated with or mediated by insulin resistance. The methods entail administering to a subject with one of the noted disorders a therapeutically effective amount of a compound that specifically inhibits or suppresses tissue factor- protease activated receptor 2 (TF-PAR2) signaling. Examples of metabolic complications of obesity or disorders associated with or mediated by insulin resistance include type 2 diabetes, obesity, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome or abnormal weight gain. In some preferred embodiments, the employed compound selectively inhibits TF-PAR2 signaling and does not interfere with hemostatic activity mediated by tissue factor/ factor Vila (TF/VIIa). The compound used in the methods can be either a TF inhibitor or a PAR2 antagonist. Some of the methods use a TF-PAR2 signaling inhibitor that is an antibody or small chemical entity. For example, the methods can employ monoclonal anti-TF antibody 10H 10 produced by the hybridoma with ATCC access number HB9383 or an antibody or an antigen-binding molecule having the same binding specificity of anti-TF antibody 10H 10.

[0007] In a related aspect, the invention specifically provides methods for treating or ameliorating the symptoms of type 2 diabetes in a subject. These methods entail

administering to a subject afflicted with type 2 diabetes a compound that specifically inhibits or suppresses tissue factor- protease activated receptor 2 (TF-PAR2) signaling. Preferably, the employed inhibitor compound selectively inhibits TF-PAR2 signaling and does not interfere with hemostatic activity mediated by tissue factor/ factor Vi la (TF/VIIa). In some specific embodiments, the employed inhibitor compound is monoclonal anti-TF antibody 10H 10 produced by the hybridoma with ATCC access number HB9383 or an antibody or an antigen-binding molecule having the binding specificity of MAb 10H 10.

[0008] In another aspect, the invention provides kits for treating insulin resistance related disorders. The kits typically contain a therapeutically effective amount of a compound that specifically inhibits or suppresses tissue factor- protease activated receptor 2 (TF-PAR2) signaling and a pharmaceutically acceptable carrier. Some of the kits contain a compound that selectively inhibits the TF-PAR2 signaling pathway but does not interfere with TF mediated hemostatic activities. For example, the kits can contain monoclonal anti-TF antibody 10H 10 produced by the hybridoma with ATCC access number HB9383 or an antibody or an antigen-binding molecule having the binding specificity of antibody 10H 10. The kits can additionally contain instructions for use or disposal of the reagents in the kit.

[0009] A further understanding of the nature and advantages of the present invention may be realized by reference to the remaining portions of the specification and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0010] Figures 1 A- 1 E show that TF-PAR2 signaling promotes diet induced obesity (DIO). (a) TF activity in plasma and adipose tissue extracts of male C57BL/6J mice that were fed a low fat ( 10% fat) or high fat (60% fat) diet for 16 weeks, n=6±SD. (b) Comparison of TF and PAR2 mRNA expression in VAD from 22-week old male C57BL/6J mice on a low fat (LFD; 10% fat) or high fat (HFD; 60% fat) diet for 16 weeks, n = 6, mean ± SD. For a and b, *P < 0.05, **P < 0 .01, ***P < 0.001 for LFD versus HFD. (c) HFD-induced weight gain of male TF^ACT, PAR2^''^', TF^ACT PAR2^"'\ or wild type (WT) mice; n = 8-20, mean ± SD. (d) Glucose tolerance test in TF^ACT, PAR2^"A, TF^ACT PAR2^{" "}, or wild type (WT) mice measured after 16 weeks on a HFD; n = 8- 1 5, mean ± SD. For c and d, *P < 0.05, * *P < 0 .01, WT versus PAR-2 ⁷-; *P < 0.05, ^UP < 0 .01 for WT versus TF^ACT PAR2^{" "} ; ⁺P < 0.05, P < 0.01, for WT versus TF^ACT. (e) Oxygen consumption of TF^ACT, ΡΑΡν2^"Λ, TF^ACT/PAR2^"/", or WT mice on a HFD for 16 weeks. Mice were acclimated individually in metabolic cages and l ight-dark cycle normalized V0₂ consumption was averaged over 3 days, n = 4-5, mean ± SD. *P<0.05, **P < 0 .01, ***P<0.001 WT versus knockout.

[0011 ] Figures 2A-2D shows that adipose TF-PAR2 signaling regulates weight gain, (a) TF-VIIa signaling regulates UCP-2 expression by 3T3-L 1 adipocytes. Adipocytes were differentiated and after switch to serum-free conditions for 24 hours stimulated with 25 nM Vila in the absence or presence of inhibitory anti-mouse TF 21 E 10 (50 Dg/ml) for 3 hours, or with 10 nM thrombin, n = 6, mean ± SD. (b) UCP- 2 and leptin mRNA levels in adipocytes isolated from epididymal fat pads of 16 weeks HFD WT, TF^ACT or PAR2^'A mice, n = 6, mean ± SD. (c) Weight gain of WT bone marrow chimeras of WT, TF^ACT or PAR2^{~ ~} mice on a HFD. n = 8 mean ± SD. (d) Oxygen consumption of WT bone marrow chimeras of WT, TF^ACT or PAR2^7' mice on a HFD. n = 5, mean ± SD. (e) Quantification of VAD and SAD CD 1 l b+/CDl l c+ macrophages by flow cytometry of the stromal vascular fraction (SVF) cells from WT, TF^ACT, PAR2^7" and TF^ACT/ PAR2 ^A mice. SVF from 2-3 mice were typically pooled, n = 3, mean ± SD, *P < 0.05, **P < 0 .01, treated versus control (a, b) or WT versus knockouts or WT BMT chimeras into WT versus knock-out (c, d). [0012] Figures 3A-3E show that hematopoietic TF-PAR2 signaling in insulin resistance and adipose inflammation, (a) Weight gains of WT, TF^ACT, or PAR2^"/_ bone marrow chimeras in C57BL/6 WT mice. Pooled data from 2 independent bone marrow transplantation experiments, n = 14, mean ± SD. (b, c) Glucose and insulin tolerance test of TF^ACT or PAR2^'A bone marrow chimeras in comparison with control WT bone marrow transplanted mice after 16 weeks of HFD. n = 10- 14, mean ± SD. (d) Quantification by flow cytometry of macrophage and T cell populations in the VAD SVF from WT, TF^ACT, or PAR2^7' bone marrow chimeras. SVF from 2-3 mice were typically pooled, n = 3, mean ± SD. (e) IL-6 or IL- 10 mR A levels in the VAD (n = 8) or in CD 1 l b-selected SFV cells (n = 4) from WT, TF^ACT, or PAR2^'A bone marrow chimeras; mean ± SD, *P < 0.05, **P < 0.01 for WT versus the corresponding knockout chimeras.

[0013] Figures 4A-4F show that anti-TF antibody therapy improves insulin sensitivity and ameliorates adipose inflammation, (a, b) Glucose and insulin tolerance test in 16 week HFD male WT obese mice 24 hours after intraperitoneal injection of rat anti-mouse TF 21 E 10 (20 mg/kg) or rat IgG control, n = 8, mean ± SD. (c) IL-6 mRNA, IL- 10 mRNA and CD 1 l b+/CDl l c+ macrophage levels in VAD 24 hours after treatment with 21 E 10 or control IgG. For gene expression, n = 8, mean ± SD, for macrophages quantification, n = 3, mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001 control versus antibody treated, (d, e) Glucose and insulin tolerance tests of obese TF I bone marrow chimeras 24 hours after receiving anti- human TF signaling-selective monoclonal antibody 10H 10 (20 mg/kg) were compared to either obese TFKI bone marrow chimeras receiving IgG or control C57BL/6 WT bone marrow chimeras in WT mice receiving 10H 10. The response of both control groups was indistinguishable and data were pooled from these experiments, (f) IL-6 mRNA, IL- 10 mRNA levels of CD 1 1 b-selected adipose tissue macrophages, and CD 1 1 b+/CD 1 1 c+ macrophage levels from 10H 10 treated C57BL/6 WT or TFKI bone marrow chimeras, n = 3- 6, mean ± SD. *P < 0.05, **P < 0.01, control versus treated TFKJ BM chimera.

[0014] Figures 5A-5B show TF expression in obese human subjects, (a) TF gene expression in adipose tissues of obese diabetics compared to non-diabetics; n = 6, mean ± SD. (b) Plasma TF activity in obese diabetics versus non-diabetics; n = 12- 14, mean ± SD. Blood samples and adipose tissue biopsies were obtained in the fasting state. ** P < 0 .01 for diabetics versus non-diabetics.

[0015] Figures 6A-6D show contribution of TF-PAR2 signaling to insulin resistance and metabolism, (a) Insulin resistance of TF^ACT, PAR2^"7", TF^ACT/PAR2^~/~, and wild type (WT) mice measured after 16 weeks on a HFD. Mice were injected with insulin (0.75U/kg body weight) and blood glucose levels were analyzed in venous blood from tail bleeds at the indicated times, n = 8- 15, mean ± SD. (b) Food intake, (c) VC0₂, and (d) total activity of 16 weeks HFD fed WT, TF^ACT or PAR2^7' mice. Mice were acclimated individually in metabolic cages and measurements were taken over 3 days on HFD. Data are averages of light or dark phases from 3 day measurements, n = 4-5, mean ± SD, *P < 0.01, **P < 0.01, ***P < 0.001 knock-out versus the corresponding WT values.

[0016] Figures 7A-7B show TF and PAR2 expression in adipocytes, (a) TF and PAR2 mRNA levels in adipocytes isolated from the epididymal fat pads of male DIO WT mice, n = 6, mean ± SD. (b) TF and PAR2 expression during differentiation of 3T3-L 1 adipocytes with typical cultures shown in the lower panels at the 4 time points. 3T3-L 1 adipocytes were grown and differentiated as previously described in Chavey et al., Cell Metab. 9:339-349, 2009. n = 6 mean ± SD. D-PD: days post differentiation.

[0017] Figures 8A-8B show TF and PAR2 expression in adipose tissue macrophages, (a) Live cell gating using propidium iodide (PI) of a typical preparation of the adipose tissue stromal vascular fraction (SVF). (b) TF expression in CD 1 l b⁺/CD l l c⁺ macrophages from the SVF of the visceral, epididymal (VAD) or subcutaneous (SAD) adipose tissues of WT DIO mice. Typical experiment of 3 independent repeats. TF and PAR2 expression was also confirmed at the mRNA level in CD1 l c-selected cells isolated from the stromal vascular fraction of obese wild-type mice.

[0018] Figures 9A-9B show reconstitution efficiency of bone marrow chimeras. Lethally irradiated WT mice were reconstituted with GFP-tagged bone marrow from WT, TF^ACT or PAR2^{" _} mice. After 6 weeks of recovery, mice were fed a HFD for 16 weeks, (a) Gates of CD 1 l b⁺/CD l l c⁺ macrophages in the SVF fraction from epididymal VAD adipose tissues reconstituted with GFP⁺ BM from WT, TF^ACT or PAR2^_/" mice and quantification of reconstitution with GFP cells. Note that some PAR2 ^A bone marrow donors carried only one copy of the GFP transgene. (b) TF mean fluorescence of CD 1 l b⁺/CDl l c⁺ cells from chimeras with the indicted bone marrow; n = 3, mean ± SD.

[0019] Figures 10A- 10B show control experiments for anti-TF therapy, (a) Insulin tolerance test in TF^ACT mice on a HFD for 16 weeks 24 hours after a single injection of 21 E 10, n = 4, mean ± SD. (b) Weight gain during HFD feeding of chimeric WT mice reconstituted with bone marrow from WT (n = 7) or human TF knock-in (TFKI), n = 12, mean ± SD. [0020] Figures 1 1 A-l I F show contributions of adipocyte TF-PAR2 signaling to the regulation of glucose and lipid metabolism in obesity, (a) Representative Western blot analysis for pAkt and Akt from control, Vila, or Vila + 21 E 10 treated primary adipocyte cultures from WT or TF^ACT mice, (b) Quantification of insulin-induced Akt phosphorylation after 24 hour treatment with or without Vila of primary adipocyte cultures from WT and TF^ACT mice, n = 4-8, mean ± SD. (c) Regulation of gene expression in 3T3-L 1 adipocytes by Vila, n = 6, mean ± SD, * *P < 0.01, * **P < 0 .001 for control versus Vila-treated, (d) Gene expression in adipocytes isolated from VAD of DIO WT or TFKI BM chimeric mice treated overnight with antibody 21 E 10 to mouse TF or antibody 1 OH 10 to human TF; n = 8- 14, mean ± SD, *P < 0.05, **P < 0 .01 for control versus antibody treated, (e) Insulin tolerance tests of obese TFKI BM chimeras 24 hours after receiving antibody 21 E l 0 compared to obese TFKI BM chimeras receiving control IgG. (f) Gene expression in SVF fraction cells isolated from VAD of DIO TFKI BM chimeric mice treated with the murine TF specific antibody 21 E 10. For (e, f) « = 8, mean ± SD, *P < 0.05, **P < 0 .01 for control versus antibody- treated.

DETAILED DESCRIPTION OF THE INVENTION

I. Overview

[0021] The present invention is predicated in part on the discoveries by the present inventors that Tissue factor (TF)-protease activated receptor 2 (PAR2) signaling pathway is crucial for the development in mice of diet-induced obesity and adipose inflammation promoting insulin resistance, and that improvements in glucose homeostasis can be rapidly achieved by selectively targeting the proinflammatory TF-PAR2 signaling pathway. These discoveries underscore a novel approach to selectively and safely treat the metabolic complications of obesity and potentially induce weight loss by independent effects on the regulation of energy expenditure by adipocytes.

[0022] As detailed in the Examples below, the inventors observed that TF expression is increased in adipose tissues from obese humans and mice, that adipocyte TF-PAR2 signaling controls a key regulator of metabolism, and that genetic deletion of the TF cytoplasmic domain or PAR2 in nonhematopoietic cells protects mice from high fat diet (HFD) induced obesity. It was also found that short term pharmacological intervention with a monoclonal antibody that selectively inhibits TF-PAR2 signaling rapidly attenuated HFD-induced adipose tissue inflammation and insulin resistance in the absence of weight loss or adipose tissue macrophage depletion. It was demonstrated that inhibition of TF-PAR2 signaling rapidly improves metabolism and energy expenditure. Additionally, the inventors observed a critical role of TF signaling in regulating activation of AKT phosphorylation and further elucidated the downstream targets for TF signaling in adipocytes.

[0023] Moreover, the present inventors demonstrated that stimulation of TF-PAR2 signaling in adipocytes suppressed uncoupling protein (UCP) 2, a mitochondrial inner membrane protein that promotes energy expenditure, and that blockade of TF-PAR2 signaling prevented suppression of UCP-2. Further, blockade of TF-PAR2 signaling resulted in reduced levels of pro-inflammatory cytokines (e.g., IL-6) and increased mRNA levels of anti-inflammatory cytokine (e.g., IL- 10), alongside with reduced numbers of

proinflammatory macrophages. These discoveries delineated an unexpected signal ing pathway that maintains adipose inflammation and is a pharmacological target for rapid reversal of macrophage-dependent inflammation and altered glucose homeostasis in obesity.

[0024] In accordance with these discoveries, the present invention provides methods for treating insulin resistance related disorders and obesity-induced metabolic complications or pathological conditions. The invention also provides methods for treating or ameliorating inflammation by inhibiting proliferation and function of proinflammatory macrophage. By reverting insulin resistance and promoting energy expenditure, the therapeutic methods and compositions disclosed herein are useful for treating subjects suffering from diseases and disorders associated with abnormal insulin signaling activities and excessive weight gain, e.g., type 2 diabetes and obesity. Subjects suitable for treatment with methods of the invention include ones who have or are at risk of developing any of these diseases. The following sections provide more detailed guidance for practicing the invention.

II. Definitions

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains. The following references provide one of skill with a general definition of many of the terms used in this invention: Academic Press Dictionary of Science and

Technology, Morris (Ed.), Academic Press ( l ^sl ed., 1992); Oxford Dictionary of

Biochemistry and Molecular Biology, Smith et al. (Eds.), Oxford University Press (revised ed., 2000); Encyclopaedic Dictionary of Chemistry, Kumar (Ed.), Anmol Publications Pvt. Ltd. (2002); Dictionary of Microbiology and Molecular Biology, Singleton et al. (Eds.), John Wiley & Sons (3^rd ed., 2002); Dictionary of Chemistry, Hunt (Ed.), Routledge ( l ^sl ed., 1999); Dictionary of Pharmaceutical Medicine, Nahler (Ed.), Springer-Verlag Telos ( 1994);

Dictionary of Organic Chemistry, Kumar and Anandand (Eds.), Anmol Publications Pvt. Ltd. (2002); and A Dictionary of Biology (Oxford Paperback Reference), Martin and Hine (Eds.), Oxford University Press (4^,h ed., 2000). In addition, the following definitions are provided to assist the reader in the practice of the invention.

[0026] Tissue factor (TF) mediated hemostasis (or hemostatic activities) refers to the coagulation cascade triggered by TF-VIIa complex binding to and activation of factor X, which ultimately leads to thrombin-dependent fibrin deposition and platelet activation. TF is a transmembrane glycoprotein expressed by vascular and myeloid cells. It is the primary cellular initiator of blood coagulation. TF exerts its biological activities by forming a catalytic enzyme complex with coagulation factor Vila. The TF-VIIa complex then triggers coagulation by binding and activating factor X, leading to thrombin-dependent fibrin deposition and platelet activation.

[0027] TF-PAR2 signaling (or TF-VIIa-PAR2 signaling) pathway refers to a non- hemostatic signaling cascade mediated by TF. Independent of triggering coagulation in TF- driven thrombin pathways in metastasis, the TF-VIIa protease complex can also promote tumor and developmental angiogenesis through protease-activated receptor-2 (PAR-2) signaling. It was found that in TF cytoplasmic-domain-deleted mice, PAR2 -dependent angiogenesis and tumor growth is enhanced, demonstrating a role for host cell TF signaling. In tumor cells, TF-VIIa complex activates PAR2 and thereby regulates proangiogenic growth factor expression as well as integrins involving crosstalk with the TF cytoplasmic domain.

[0028] "TF-PAR2 signaling inhibitors" or "TF-PAR2 inhibiting compounds" refer to any agents or substances that partially or completely inhibit or suppress the TF-VIIa-PAR2 signaling pathway. Preferably, the compounds selectively inhibit the TF-VIIa-PAR2 signaling while having no substantial (e.g., little or no) effect on the haemostatic activities mediated by TF.

[0029] The phrases "type II diabetes", "type 2 diabetes", "non-insulin dependent diabetes mellitus" and "NIDDM" refer to the same condition. It is a metabolic disorder that is characterized by high blood glucose in the context of insulin resistance and relative insulin deficiency. Testing for type 2 diabetes typically involves drawing blood samples and measuring the glucose (sugar) levels within the blood. During a random glucose test, a sample of blood can be obtained and tested at any time. For human subjects, normal random glucose levels are around 70- 1 10 mg/dl. According to the American Diabetes Association, a random glucose level of greater than about 200 mg/dl is indicative of diabetes. During a fasting glucose test, a sample of blood is obtained following a period of not eating or drinking (except water) for at least 8 hours. According to the American Diabetes Association, a fasting blood glucose level of greater than 125 mg/dl on two occasions is indicative of diabetes.

[0030] Diet-induced obese (DIO) is a mouse model created to study obesity-related diseases such as diabetes. In this model, a mouse is fed a high fat diet, typically for 8 to 20 weeks. As a result it become obese, mildly to moderately hyperglycemic, and develop impaired glucose tolerance. These mice are then used to study the genetic and physiological mechanisms of obesity and type 2 diabetes.

[0031 ] Insulin resistance (IR) is a physiological condition where the natural hormone, insulin, becomes less effective at lowering blood sugars. The resulting increase in blood glucose may raise levels outside the normal range and cause adverse health effects. Certain cell types such as fat and muscle cells require insulin to absorb glucose. When these cells fail to respond adequately to circulating insulin, blood glucose levels rise. The liver helps regulate glucose levels by reducing its secretion of glucose in the presence of insulin. This normal reduction in the liver's glucose production may not occur in people with insulin resistance. Insulin resistance primarily refers to reduced glucose-lowering effects of insulin. However, other functions of insulin can also be affected. For example, insulin resistance in fat cells reduces the normal effects of insulin on lipids and results in reduced uptake of circulating lipids and increased hydrolysis of stored triglycerides. Increased mobilization of stored lipids in these cells elevates free fatty acids in the blood plasma. Elevated blood fatty- acid concentrations (associated with insulin resistance and diabetes mellitus Type 2), reduced muscle glucose uptake, and increased liver glucose production all contribute to elevated blood glucose levels. High plasma levels of insulin and glucose due to insulin resistance are a major component of the metabolic syndrome. If insul in resistance exists, more insulin needs to be secreted by the pancreas. If this compensatory increase does not occur, blood glucose concentrations increase and type 2 diabetes occurs.

[0032] Metabolic syndrome is a combination of medical disorders that increase the risk of developing cardiovascular disease and diabetes. It affects one in five people, and prevalence increases with age. Metabolic syndrome is also referred to as metabolic syndrome X, syndrome X, or insulin resistance syndrome. [0033] Insulin resistance related disorders are disorders that are associated with or mediated by insulin resistance. They encompass those diseases or conditions where the response to insulin is either causative of the disease or has been implicated in the progression or suppression of the disease or condition. Representative examples of insulin related disorders include, without limitation diabetes, diabetic complications, polycystic ovary disease, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome, obesity, inflammatory diseases, diseases of the digestive organs, stenocardia, myocardial infarction, sequelae of stenocardia or myocardial infarction, senile dementia, and cerebrovascular dementia. See, Harrison's Principles of Internal Medicine, 13th Ed., McGraw Hill Companies Inc., New York ( 1994).

[0034) Metabolic complications of obesity refer to a variety of adverse health consequences and conditions that are attributable to obesity. They encompass, e.g., insulin resistance with or without type II diabetes mellitus (DM), hypertension, dyslipidemia, cardiovascular disease, and abnormal or excessive weight gain. The major complications come under the heading of the metabolic syndrome. This syndrome is characterized by plasma lipid disorders (atherogenic dyslipidemia), raised blood pressure, elevated plasma glucose, and a prothrombotic state. The clinical consequences of the metabolic syndrome are coronary heart disease and stroke, type 2 diabetes and its complications, fatty liver, cholesterol gallstones, and possibly some forms of cancer. At the heart of the metabolic syndrome is insulin resistance, which represents a generalized derangement in metabolic processes. Unless otherwise specified, metabolic complications of obesity refer to the same diseases and conditions encompassed by disorders associated with or mediated by insulin resistance.

[0035] The term "agent" includes any substance, molecule, element, compound, entity, or a combination thereof. It includes, but is not limited to, e.g., protein, polypeptide, small organic molecule, polysaccharide, polynucleotide, and the like. It can be a natural product, a synthetic compound, or a chemical compound, or a combination of two or more substances. Unless otherwise specified, the terms "agent", "substance", and "compound" are used interchangeably herein.

[0036] The term "analog" or "derivative" is used herein to refer to a molecule that structurally resembles a reference molecule (e.g., a known anti-TF antibody) but which has been modified in a targeted and controlled manner, by replacing a specific substituent of the reference molecule with an alternate substituent. Compared to the reference molecule, an analog would be expected, by one skilled in the art, to exhibit the same, similar, or improved utility. Synthesis and screening of analogs to identify variants of known compounds having improved traits (such as higher binding affinity for a target molecule) is an approach that is well known in pharmaceutical chemistry.

[0037] "Hemostasis" refers to the arrest of bleeding from an injured blood vessel, requires the combined activity of vascular, platelet, and plasma factors counterbalanced by regulatory mechanisms to limit the accumulation of platelets and fibrin in the area of injury. Hemostatic abnormalities can lead to thrombosis or excessive bleeding.

[0038] As used herein, the term "hyperlipidemia" refers to a disorder manifested by elevated serum concentrations of total cholesterol (>200 mg/dL), LDL cholesterol (> 130 mg dL), or triglycerides (> 150 mg/dL) or decreased HDL cholesterol (<40 mg/dL). Further, as used herein, the term "fat" refers to serum and adipose triglyceride content and

"triglycerides" refers to triacylglyerol esters of fatty acids.

[0039] As used herein, the terms "hyperinsulinemia" and "hyperglycemia" refer to a fasting insulin concentration > \ 7 IU/ml) and fasting glucose > 125 mg/dL.

[0040] The term "treating" or "alleviating" includes the administration of compounds or agents to a subject to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease (e.g., insulin resistance or type 2 diabetes), alleviating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. Subjects in need of treatment include those already suffering from the disease or disorder as well as those being at risk of developing the disorder. Treatment may be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease. In the treatment of a disease or disorder associated with or mediated by insulin resistance, a therapeutic agent may directly decrease the pathology of the disease, or render the disease more susceptible to treatment by other therapeutic agents.

[0041] "In combination with", "combination therapy" and "combination products" refer, in certain embodiments, to the concurrent administration to a subject of a first therapeutic (e.g., a known anti-diabetes drug) and a second therapeutic (e.g., a TF-PAR2 signaling inhibitor as described herein). When administered in combination, each component can be administered at the same time or sequentially in any order at different points in time. Thus, each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect. "Concomitant administration" of a known drug for treating insulin resistance with a pharmaceutical composition of the present invention means administration of the drug and the composition which includes an inhibitor of TF-PAR2 signaling (e.g., antibody or small chemical entity) at such time that both the known drug and the composition of the present invention will have a therapeutic effect. Such concomitant administration may involve concurrent (i.e. at the same time), prior, or subsequent administration of the insulin resistance drug with respect to the administration of a compound of the present invention. A person of ordinary skill in the art, would have no difficulty determining the appropriate timing, sequence and dosages of administration for particular drugs and compositions of the present invention.

[0042] "Dosage unit" refers to physically discrete units suited as unitary dosages for the particular individual to be treated. Each unit can contain a predetermined quantity of active compound(s) calculated to produce the desired therapeutic effect(s) in association with the required pharmaceutical carrier. The specification for the dosage unit forms can be dictated by (a) the unique characteristics of the active compound(s) and the particular therapeutic effect(s) to be achieved, and (b) the limitations inherent in the art of compounding such active compound(s).

[0043] "Pharmaceutically acceptable", "physiologically tolerable" and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.

[0044] A "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to effect treatment for that disease.

[0045] Unless otherwise noted, the terms "patient" or "subject" are used herein interchangeably. The term refers to any animal classified as a mammal, e.g., human and non- human mammals. Examples of non-human animals include dogs, cats, cattle, horses, sheep, pigs, goats, rabbits, and etc. Accordingly, the term "subject" or "patient" as used herein means any mammalian patient or subject to which the compositions of the invention can be administered.

III. Inhibitor compounds that specifically target TF-PAR2 signaling [0046] The therapeutic methods and compositions of the invention typically employ a compound that specifically targets the TF-PAR2 signaling pathway. The methods entail administering to a subject in need of treatment such a compound in an amount effective to inhibit or suppress TF-PAR2 signaling. The inhibitor compound suitable for the invention can be a TF antagonist (e.g., an antibody or a small molecule compound). For example, various TF-specific antibodies can be utilized in the invention to specifically inhibit the TF- PAR2 signaling cascade. Of particular interest are anti-TF antibodies that selectively inhibit TF-PAR2 signaling but do not interfere or significantly affect TF mediated hemostatic activities (e.g., coagulation) in the subject. In addition to TF antagonists, the inhibitor compounds to be used in the invention can also be PAR2 antagonists that inhibit or suppress TF-VIIa complex signaling through PAR2.

[0047] Some therapeutic methods and compositions of the invention employ an antibody or antibody-derived antigen-binding molecule that specifically inhibits TF-PAR2 signaling pathway. The employed antibody can be one that recognizes TF or PAR2. In some embodiments, the antibodies or antibody-derived antigen-binding molecules exhibit a strong monovalent, bivalent or polyvalent binding to a given epitope or epitopes involved in TF- PAR2 signaling (e.g., TF or the specific TF peptide epitope recognized by MAb 10H 10). Unless otherwise noted, antibodies or antigen-binding molecules of the invention can have sequences derived from any vertebrate, camelid, avian or pisces species. They can be generated using any suitable technology, e.g., hybridoma technology, ribosome display, phage display, gene shuffling libraries, semi-synthetic or fully synthetic libraries or combinations thereof. As detailed herein, antibodies or antigen-binding molecules of the invention include intact antibodies, antigen-binding polypeptide chains and other designer antibodies (see, e.g., Serafmi, J. Nucl. Med. 34:533-6, 1993).

[0048] Antibodies or antigen-binding molecules suitable for the invention also include antibody fragments which contain the antigen-binding portions of an intact antibody that retain capacity to bind the cognate antigen (e.g., TF or the specific TF peptide epitope recognized by MAb 10H 10). Examples of such antibody fragments include (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CH I domains; (ii) a F(ab')₂ fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CH I domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward el al.. , Nature 341 :544-546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR). Furthermore, although the two domains of the Fv fragment, VL and VH, are coded for by separate genes, they can be joined, using recombinant methods, by a synthetic linker that enables them to be made as a single protein chain in which the VL and VH regions pair to form monovalent molecules (known as single chain Fv (scFv); See, e.g., Bird et al., Science 242 :423 -426, 1988; and Huston et al., Proc. Natl. Acad. Sci. USA 85:5879-5883, 1988.

[0049] Antibodies or antigen-binding molecules that can be used in the invention further include one or more immunoglobulin chains that are chemically conjugated to, or expressed as, fusion proteins with other proteins. It also includes bispecific antibody. A bispecific or bifunctional antibody is an artificial hybrid antibody having two different heavy/light chain pairs and two different binding sites. Other antigen-binding fragments or antibody portions of the invention include bivalent scFv (diabody), bispecific scFv antibodies where the antibody molecule recognizes two different epitopes, single binding domains (dAbs), and minibodies.

[0050] The various antibodies or antigen-binding fragments described herein can be produced by enzymatic or chemical modification of the intact antibodies, or synthesized de novo using recombinant DNA methodologies (e.g., single chain Fv), or identified using phage display libraries (see, e.g., McCafferty et al., Nature 348:552-554, 1990). For example, minibodies can be generated using methods described in the art, e.g., Vaughan and Sollazzo, Comb Chem High Throughput Screen. 4:417-30, 2001 . Bispecific antibodies can be produced by a variety of methods including fusion of hybridomas or linking of Fab' fragments. See, e.g., Songsivilai & Lachmann, Clin. Exp. Immunol. 79:315-321 , 1990;

ostelny et al., J. Immunol. 148, 1547- 1 553, 1992. Single chain antibodies can be identified using phage display libraries or ribosome display libraries, gene shuffled libraries. Such libraries can be constructed from synthetic, semi-synthetic or nave and immunocompetent sources.

[0051 ] A number of TF- or PAR2-antagonists (e.g., antibodies) known in the art can be useful in the practice of the present invention. For example, monoclonal antibodies specific for human TF are described in Morrissey et al., Thromb. Res. 52:247-61 , 1988; Ruf et al., Biochem. J. 278:729-33, 1991 ; and U.S. Patent Nos. 51 10730, 5437864 and 5622931 . Other types of TF antagonists are also known in the art, e.g., polypeptide inhibitors as described in Paborsky et al., Biochemistry 34: 1 5328-33, 1995; and U.S. Patent No. 6,858,587. Similarly, PAR2 antagonist compounds are also described in the art. See, e.g., Wilson et al., Biochem J. 388:967-72, 2005 (a 5-mer synthetic peptide); Ui et al., Eur. J. Pharmacol. 530: 1 72-8, 2006 (neutralizing antibody and synthetic peptide); ida et al., Infect. Immun. 75: 164-74, 2007 (peptide antagonists); Kelso et al., Arthritis Rheum. 56:765-71 , 2007 (small molecule antagonist "ENMD- 1068"); and European Patent Application 1806141 (organic compound inhibitors). In addition, many PAR-2 specific inhibitors can be obtained from commercial suppliers. For example, anti-human PAR2 monoclonal antibodies from Invitrogen (Carlsbad, CA), LifeSpan Biosciences (Seattle, WA) or US Biological (Swampscott, MA). Similarly, small molecule inhibitors of PAR2 are available from Enzo Life Sciences (Plymouth

Meeting, PA). In the practice of the present invention, the skilled artisans can readily employed and modify as needed any of these known inhibitors and test their abi lity to specifically inhibit TF-PAR2 signaling.

[0052] Some preferred embodiments of the invention employ a specific anti-TF monoclonal antibody or derivatives thereof. This is the murine monoclonal antibody designated 10H 10. As demonstrated in the Examples below, MAb 1 OH 10 is an antibody that acts as an inhibitor of tissue factor signaling without interfering with hemostasis. This antibody has been described in great detail in U.S. Patent Nos. 5,223,427 and 6,001 ,978. Hybridoma secreting this antibody has been deposited pursuant to Budapest Treaty requirements with the American Type Culture Collection (ATCC) (Manassas, VA) on Mar. 27, 1987 with accession number HB9383. In addition to the 10H 10 antibody produced by this hybridoma, any antibody which has the same binding specificity and similar (e.g., the same or better) binding affinity of MAb 10H 10 can also be used in the therapeutic methods of the invention. In addition, the therapeutic methods of the invention can also use any antigen- binding molecule or fragments that are derived from MAb 1 OH 10 or an antibody with the same binding specificity and the same or better binding affinity of MAb 1 OH 10.

[0053] Some of the therapeutic methods of the invention are directed to treating human subjects. In these methods, a humanized antibody, a human antibody, or a chimeric antibody containing human sequences (e.g., in the constant region) is preferred. Compared to an antibody isolated from a non-human animal (e.g., a mouse), such an antibody would have less or no antigenicity when administered to the human subject. A chimeric anti-TF or anti-PAR2 antibody (e.g., one with the same binding specificity as that of MAb 10H 10) can be made up of regions from a non-human anti-TF antibody together with regions of human antibodies. For example, a chimeric H chain can comprise the antigen binding region of the heavy chain variable region of a mouse anti-TF antibody exemplified herein linked to at least a portion of a human heavy chain constant region. This chimeric heavy chain may be combined with a chimeric L chain that comprises the antigen binding region of the light chain variable region of the mouse anti-TF antibody linked to at least a portion of the human light chain constant region.

[0054] Chimeric anti-TF or anti-PAR2 antibodies of the invention can be produced in accordance with methods known in the art. See, e.g., Robinson et al.., International Patent Publication PCT/US86/02269; Akira, et al.., European Patent Application 184, 187;

Taniguchi, M., European Patent Application 171 ,496; Morrison et al.., European Patent Application 173,494; Neuberger e/ al.., International Application WO 86/01533; Cabilly et ο/.. U.S. Patent No. 4,816,567; Cabilly et al.., European Patent Application 125,023; Better et al., Science 240: 1041 - 1043, 1988; Liu et al., PNAS 84:3439-3443, 1987; Liu et al., J.

Immunol. 139:3521 -3526, 1987; Sun et al., PNAS 84:214-218, 1987; Nishimura et al., Cane. Res. 47:999- 1005, 1987; Wood et al., Nature 314:446-449, 1985; Shaw et al., J. Natl. Cancer Inst. 80: 1 553- 1559, 1988.

[0055] Chimeric antibodies which have the entire variable regions from a non-human antibody can be further humanized to reduce antigenicity of the antibody in human. This is typically accomplished by replacing certain sequences or amino acid residues in the Fv variable regions (framework regions or non-CDR regions) with equivalent sequences or amino acid residues from human Fv variable regions. These additionally substituted sequences or amino acid residues are usually not directly involved in antigen binding. More often, humanization of a non-human antibody proceeds by substituting only the CDRs of a non-human antibody (e.g., the mouse anti-TF antibodies exemplified herein) for the CDRs in a human antibody. In some cases, this is followed by replacing some additional residues in the human framework regions with the corresponding residues from the non-human donor antibody. Such additional grafting is often needed to improve binding to the antigen. This is because humanized antibodies which only have CDRs grafted from a non-human antibody can have less than perfect binding activities as compared to that of the non-human donor antibody. Thus, in addition to the CDRs, humanized anti-hTF or anti-PAR2 antibodies of the invention (e.g., one with the same binding specificity as that of MAb 10H 10) can often have some amino acids residues in the human framework region replaced with corresponding residues from the non-human donor antibody (e.g., the mouse antibody exemplified herein). Methods for generating humanized antibodies by CDR substitution, including criteria for selecting framework residues for replacement, are well known in the art. See, e.g., Winter et al., UK Patent Application GB 2188638A ( 1987), U.S. Patent 5,225,539; Jones et al., Nature 321 :552-525, 1986; Verhoeyan et al., Science 239: 1534, 1988; Beidler et al., J. Immunol. 141 :4053-4060, 1988; and WO 94/10332.

[0056] In addition to chimeric or humanized anti-TF or anti-Par2 antibodies, therapeutic methods for treating human subjects can also employ fully human antibodies that exhibit the same binding specificity and comparable or better binding affinity relative to a mouse antibody such as MAb 10H 10. The human anti-TF or anti-PAR2 antibodies can be generated using any of the methods that are well known in the art, e.g., phage display methods using antibody libraries derived from human immunoglobulin sequences. See, e.g., Lonberg and Huszar, Int. Rev. Immunol. 13:65-93 ( 1995), U.S. Pat. Nos. 4,444,887 and 4,716, 1 1 1 ; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO

96/34096, WO 96/33735, and WO 91/10741 .

[0057] Derivative antibodies or antigen-binding molecules which have the same binding specificity and the same or better binding affinity of MAb 10H 10 can be obtained by methods well known in the art and exemplified herein. For example, candidate antibodies or immunoglobulins generated against a tissue factor antigen can be screened for, e.g., an ability to compete with MAb 10H 10 for binding to a tissue factor polypeptide or peptide.

Polypeptide and polynucleotide sequences of human tissue factor are known (see, e.g., Scarpati et al., Biochemistry 26:5234-5238, 1987; and Fisher et al., Thromb. Res. 48:89-99, 1987). Tissue factor polypeptides or peptides suitable for the screening can be generated using methods well known in the art or described herein. In addition, many specific antigenic peptides derived from human tissue factor have been described in the art, e.g., U.S. Patent Nos. 5,223,427 and 6,001 ,978. These patents also disclose the profile of MAb 10H 10 binding to the panel of tissue factor peptides. For example, it was shown that MAb 10H 10 specifically binds to tissue factor peptide with the sequence of

SGTTNTVAAY LTWKST FKTILEWEPKPV (SEQ ID NO: l ) or

ECDLTDEIVKDVKQTY (SEQ ID NO:2) but not several other antigenic peptides derived from human tissue factor. The latter peptides include, e.g.,

TKSGDWKSKCFYTTDTECDLTDEIVKDVKQTY (SEQ ID NO:3) or

LARVFSYPAGNVESTGSAGEPLYENSPEFTPYLC (SEQ ID NO:4). Thus, candidate antibodies (e.g., antibodies generated against a human tissue factor polypeptide) can be screened for ability to block MAb 10H 10 binding to the peptide with the sequence of SEQ ID NO: 1 and/or SEQ ID NO:2. The can also be screened for the same or substantially identical binding profile as that of MAb 10H 10 for binding to the panel of human tissue factor peptides as described in U.S. Patent No 5,223,427. Methods for performing such screening is well known in the art (see, e.g., U.S. Patent Nos. 5,223,427 and 6,001 ,978).

[0058] In addition to in vitro screening assays, in vivo methods can also be used to identify anti-TF or anti-PAR2 antibodies that are suitable for practicing the methods of the present invention. For example, an in vivo method for replacing a nonhuman antibody variable region with a human variable region in an antibody while maintaining the same or providing better binding characteristics has been disclosed in U.S. Patent Application No. 10/778,726 (Publication No. 20050008625). To generate a human antibody with the same binding specificity and the same or better binding affinity as that of mouse MAb 10H 10, this method relies on epitope guided replacement of variable regions of the non-human antibody with a fully human antibody. The resulting human antibody is generally unrelated structurally to the reference nonhuman antibody, but binds to the same epitope on the same antigen as the reference antibody.

[0059] Human antibodies with the same or better affinities for a specific epitope than a starting non-human antibody (e.g., a mouse MAb 10H 10) can also be obtained from companies which customarily produce human antibodies. For example, to generate a desired human antibody, KaloBios, Inc. (Mountain View, CA) employs a human "acceptor" antibody library. A directed or epitope focused library of human antibodies which bind to the identical epitope as the non-human antibody, though with varying affinities, is then generated.

Antibodies in the epitope focused library are then selected for similar or higher affinity than that of the starting non-human antibody. The identified human antibodies are then subject to further analysis for affinity and sequence identity.

[0060] Other than antibodies, some other embodiments of the therapeutic methods and compositions of the invention employ small molecule inhibitor of TF-PAR2 signaling pathway. Preferably, the employed compounds specifically target TF-PAR2 signaling with little or no effect on the hemostatic activities mediated by TF. The employed small molecule compound or small chemical entity can be any chemical or other moiety that can act to affect biological processes, wherein the small chemical entity can act as an inhibitor of tissue factor signaling without interfering with hemostasis in a mammalian subject. They can be any carbon-based compound other than macromolecuies such nucleic acids and polypeptides. Small molecules can include any number of therapeutic agents presently known and used, or can be small molecules synthesized in a library of such molecules for the purpose of screening for biological function(s). Typically, the small molecules of this invention usually have molecular weight less than about 5,000 daltons (Da), preferably less than about 2,500 Da, more preferably less than 1 ,000 Da, most preferably less than about 500 Da. The compounds can be organic compounds, peptidomimetics, antibody mimetics, and conjugates thereof.

[0061] The above-described small molecule inhibitors of TF-PAR2 signaling can be readily employed and modified as necessary in the practice of the methods of the present invention. Small molecule inhibiting compounds suitable for the invention can also be identified by screening test compounds to identify compounds which inhibit or suppress TF- VIla-PAR2 mediated signaling activities but do not interfere with hemostasis in vivo. Such modulators (e.g., small molecule organic compound modulators) can be identified by employing a known compound that possesses such desired properties (e.g., MAb 10H 10) in competitive assay formats. Some of the screening assays are directed to identifying compounds which inhibit TF VIIa signaling but does not block coagulation. For example, these screening assays can entail measuring in the presence or absence of test compounds a binding between (i) an antibody or an antigen-binding molecule having the same binding specificity as that of MAb 10H 10 and (ii) a tissue factor polypeptide, and then detecting an inhibition of the binding in the presence of a test compound relative to the binding in the absence of the test compound. For example, the screening methods can employ the murine MAb 10H 10 produced by the hybridoma with ATCC access number HB9383.

[0062] Some of the screening methods employ test compounds which are preferably small molecule organic compounds, e.g., chemical compounds with a molecular weight of not more than about 5000, and more preferably not more than about 2,500, 1 ,000 or 500. In addition to measuring their ability to compete with MAb 10H 10 for binding to tissue factor, the modulators thus identified can be additionally examined for activity to modulate tissue factor signaling (e.g., inhibiting TF-PAR2 signaling activities while having no significant effect on hemostasis). The compounds can be tested for inhibitory activity on any of the signaling activities that are mediated by TF (e.g., MAP kinase phosphorylation or complex formation with and signaling via protease activated receptor 2). Assays for measuring TF- PAR2 mediated signaling activities are well known in the art. For example, TF-PAR2 mediated signaling activities can be quantitatively measured by a MAP kinase

phosphorylation assay, e.g., assaying by western blot phosphorylation level of a MAP kinase (e.g., ERX kinase) in HUVEC cells or CHO cells stimulated with factors Vila and X. These assays can be readily used in the screening methods described herein for identifying novel modulating compounds (e.g., inhibitors) of TF-PAR2 signalling. A compound is considered a TF signaling inhibitor if the compound can inhibit TF signaling activities by at least 50%, at least 75%, at least 90%, or at least 95% relative to TF signaling in the absence of the compound. The quantitative inhibition can be measured by any of the TF signaling assays well known in the art (see, e.g., Ahamed et al., Blood 105:2384-91 , 2005) or described herein, e.g., a reduction of ERK phosphorylation.

[0063] The identified compounds that inhibit TF-PAR2 signaling can be additionally examined to confirm that they have no significant effect on tissue factor-mediated hemostasis activities (e.g., coagulation). For example, TF mediated coagulation activities can be measured by quantifying factor Xa generation in HaCaT cells by a linked amidolytic assay. A compound does not interfere with or prevent activation of (i.e., having no significant effect on) a TF-mediated hemostasis (e.g., coagulation) if its presence does not lead to more than 5%, more than 10%, more than 1 5%, or more than 25% reduction in the hemostasis activity (e.g., coagulation activity as measured by the Xa generation assay) relative to that in the absence of the compound. In some embodiments, potential blocking activity of a compound on coagulation can be examined by assaying effect of the compound on the binding to tissue factor by an antibody which is known to block tissue factor mediated coagulation. One such antibody is the monoclonal antibody 5G9 produced by the hybridoma with ATCC access number HB9382. Inhibitory activities of this antibody on coagulation and relevant assays are disclosed in great detail in U.S. Patent No. 5,223,427. A lack of significant effect of a compound on MAb 5G5 binding to tissue factor (e.g., a reduction of at least 20%, 30%, 40%, 50%, 75% or more) indicates that the compound is likely not to block tissue factor mediated coagulation.

IV. Treating metabolic complications of obesity or insulin resistance related disorders

[0064] The invention provides methods for treating metabolic complications of obesity and insulin resistance related disorders. By targeting adipose inflammation and insulin resistance, the TF-PAR2 signaling inhibitors described herein can have various therapeutic and prophylactic applications. They can be employed to treat or prevent the development of many diseases or disorders that are caused by or associated with adipose inflammation and/or insulin resistance in subjects (e.g., human subjects). Examples of these diseases or disorders include metabolic syndrome or insulin resistance, diabetes, obesity and cardiovascular diseases. For example, they can be used as the active ingredients in pharmaceutical compositions to treat subjects suffering from diabetes, especially type 2 diabetes. Other examples of diseases or conditions that are associated with insulin resistance include diabetic microangiopathies (diabetic nephropathy, diabetic neuropathy, and diabetic retinopathy), impaired glucose tolerance, hyperinsulinemia, hyperlipemia, arteriosclerosis, hypertension, obesity, ischemic heart diseases, ischemic brain disorders, and peripheral arterial embolism (see, e.g., Teramoto et al., Biomedicine & Therapeutics 29:8-96, 1995; and DeFronzo et al., Cardiomuscular Pharmacol. 20: S I -S I 6, 1992). Particularly suitable for treatment with the TF-PAR2 inhibiting compounds are subjects with obesity induced insulin resistance, e.g., type 2 diabetic patients with obesity.

[0065] The TF-PAR2 signaling inhibitors of the present invention can be directly administered under sterile conditions to the subject to be treated. The modulators can be administered alone or as the active ingredient of a pharmaceutical composition. In some applications, a first inhibitor compound is used in combination with a second inhibitor in order to inhibit TF-PAR2 signaling to a more extensive degree than cannot be achieved when one TF-PAR2 signaling inhibitor is used individually. To treat various insulin resistance related disorders, the therapeutic composition of the present invention can also be combined with or used in association with other therapeutic regimens. For example, a TF-PAR2 inhibiting compound of the present invention may be used in conjunction with other compounds that are known to be able to treat insulin resistance or diabetes, e.g., various oral antihyperglycemic agents. See, e.g., Scheen et al., Drugs 54:355-368, 1997; Scheen et al., Drug Saf. 12:32-45, 1995); Inzucchi et al., JAMA. 287:360-372, 2002); and Gao et al., J. Bio. Chem. 278: 24944-24950, 2003. Thus, many drugs currently on the market for treating diabetes and insulin resistance can be readily employed in the combination therapies of the invention. Examples of such drugs are Actos (pioglitizone, Takeda, Eli Li lly ), Avandia (rosightazone, Smithkline Beacham), Amaryl (glimepiride, Aventis), Glipizide Sulfonlyurea (Generic) or Glucotrol (Pfizer), Glucophage (metformin, Bristol Meyers Squibb), Glucovance (glyburide/metformin, Bristol Meyers Squibb), Glucotrol XL (glipizide extended release, Pfizer), Glyburide (Micronase; Upjohn, Glynase; Upjohn, Diabeta; Aventis), Glyset (miglitol, Pharmacia & Upjohn), Metaglip (glipizide + metformin; fixed combination tablet), Prandin (repaglinide, NOVO), Precose (acarbose, Bayer), Rezulin (troglitazone, Parke Davis), and Starlix (nateglinide, Novartis). In addition to these known anti-diabetes drugs, other example of compounds that are useful in the treatment of insulin resistance are also described in the art, e.g., US Patent Nos. 61 10962, 6,399745, 6521665, 6683 107, and 6765021.

[0066] The TF-PAR2 inhibiting compounds described herein can be used in either prophylactic or therapeutic applications. In prophylactic applications, pharmaceutical compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (i.e. , a diabetic disease) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease. In therapeutic applications, compositions or drugs are administered to a patient suspected of, or already suffering from such a disease in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease (biochemical, histologic and/or behavioral), including its complications and intermediate pathological phenotypes in development of the disease. An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose. In both prophylactic and therapeutic regimes, agents are usually administered in several dosages until a sufficient benefit has been achieved. Typically, the therapeutic benefit is monitored and repeated dosages are given if the benefit starts to wane.

V. Pharmaceutical compositions and kits

[0067] The TF-PAR2 signaling inhibitors (e.g., antibodies with the same binding specificity of antibody 10H 10) and the other therapeutic agents disclosed herein can be administered directly to subjects in need of treatment. However, these therapeutic compounds are preferable administered to the subjects in pharmaceutical compositions which comprise the TF-PAR2 signaling inhibitors and/or other active agents along with a pharmaceutically acceptable carrier, diluent or excipient in unit dosage form. Accordingly, the invention provides pharmaceutical compositions comprising one or more of the TF-PAR2 signaling inhibitors disclosed herein. The invention also provides a use of these TF-PAR2 signaling inhibitors in the preparation of pharmaceutical compositions or medicaments for treating the above described diseases or medical disorders. The pharmaceutical compositions of the invention can be used for either therapeutic or prophylactic applications described herein. [0068] Typically, the pharmaceutical compositions contain as active ingredients compounds that specifically inhibit TF-PAR2 signaling pathway. Some compositions include a combination of multiple (e.g., two or more) TF-PAR2 inhibiting compounds (e.g., antibody or small molecule therapeutics). As described herein, the compositions can additionally contain other therapeutic agents that are suitable for treating or preventing metabolic symptoms of obesity or other insulin resistance related disorders. The active ingredients are typically formulated with one or more pharmaceutically acceptable carrier. Pharmaceutically carriers enhance or stabilize the composition, or to facilitate preparation of the composition. They should also be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the subject. Pharmaceutically acceptable carriers include solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible. The pharmaceutically acceptable carrier employed should be suitable for various routes of administration described herein. For example, the TF-PAR2 inhibiting compound can be complexed with carrier proteins such as ovalbumin or serum albumin prior to their administration in order to enhance stability or pharmacological properties. Additional guidance for selecting appropriate pharmaceutically acceptable carriers is provided in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publ ishing Co., 20^th ed., 2000.

[0069] Pharmaceutical compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^,h ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J.R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions.

Formulations for parenteral administration may, for example, contain excipients, sterile water, or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, or hydrogenated napthalenes. Biocompatible, biodegradable lactide polymer, lactide/glycolide copolymer, or polyoxyethylene-polyoxypropylene copolymers may be used to control the release of the compounds. Other potentially useful parenteral delivery systems for molecules of the invention include ethylene-vinyl acetate copolymer particles, osmotic pumps, implantable infusion systems, and liposomes. Formulations for inhalation may contain excipients, for example, lactose, or may be aqueous solutions containing, e.g., polyoxyethylene-9-lauryl ether, glycocholate and deoxycholate, or may be oily solutions for administration in the form of nasal drops, or as a gel.

(0070] The pharmaceutical compositions can be prepared in various forms, such as granules, tablets, pills, suppositories, capsules, suspensions, salves, lotions and the like. The concentration of therapeutically active compound in the formulation may vary from about 0.1 - 100% by weight. Therapeutic formulations are prepared by any methods well known in the art of pharmacy. The therapeutic formulations can be delivered by any effective means which could be used for treatment. See, e.g., Goodman & Gilman's The Pharmacological Bases of Therapeutics, Hardman et al., eds., McGraw-Hill Professional ( 10¹¹¹ ed., 2001 ); Remington: The Science and Practice of Pharmacy, Gennaro (ed.), Lippincott Williams & Wilkins (20^th ed., 2003); and Pharmaceutical Dosage Forms and Drug Delivery Systems, Ansel et al. (eds.), Lippincott Williams & Wilkins (7^th ed., 1999).

[0071] The TF-PAR2 signaling inhibitors for use in the methods of the invention should be administered to a subject in an amount that is sufficient to achieve the desired therapeutic effect (e.g., eliminating or ameliorating symptoms associated with insulin resistance) in a subject in need thereof. Typically, a therapeutically effective dose or efficacious dose of the TF-PAR2 signaling inhibitor is employed in the pharmaceutical compositions of the invention. Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular subject, composition, and mode of administration, without being toxic to the subject. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, and the rate of excretion of the particular compound being employed. It also depends on the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, gender, weight, condition, general health and prior medical history of the subject being treated, and like factors. Methods for determining optimal dosages are described in the art, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20^lh ed., 2000. Typically, a pharmaceutically effective dosage would be between about 0.001 and 100 mg/kg body weight of the subject to be treated.

[0072] The TF-PA 2 signaling inhibitor compounds and other therapeutic regimens described herein are usually administered to the subjects on multiple occasions. Intervals between single dosages can be daily, weekly, monthly or yearly. Intervals can also be irregular as indicated by measuring blood levels of the TF-PAR2 signaling inhibitor compounds and the other therapeutic agents used in the subject. In some methods, dosage is adjusted to achieve a plasma compound concentration of 1 -1000 μg/ml, and in some methods 25-300 μg/ml or 10- 100 μ¾/ϊη1. Alternatively, the therapeutic agents can be administered as a sustained release formulation, in which case less frequent administration is required. Dosage and frequency vary depending on the half-life of the TF-PAR2 signaling inhibitor compound and the other drugs in the subject. The dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some subjects may continue to receive treatment for the rest of their lives. In therapeutic applications, a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the subject shows partial or complete amelioration of symptoms of disease. Thereafter, the subject can be administered a prophylactic regime.

[0073] The invention also provides kits for carrying out the therapeutic applications disclosed herein. For example, the invention provides therapeutic kits for use in the treatment of insulin resistance or type 2 diabetes subjects in need of treatment. The therapeutic kits of the invention typically comprise as active agent one or more of the described TF-PAR2 signaling inhibitors (e.g., monoclonal antibodies including humanized or human sequence antibodies). The kits can optionally contain suitable pharmaceutically acceptable carriers or excipients for administering the active agents. The pharmaceutically acceptable carrier or excipient suitable for the kits can be coatings, isotonic and absorption delaying agents, binders, adhesives, lubricants, disintergrants, coloring agents, flavoring agents, sweetening agents, absorbants, detergents, and emulsifying agents. Other reagents that can be included in the kits include antioxidants, vitamins, minerals, proteins, fats, and carbohydrates.

[0074] The therapeutic kits can further include packaging material for packaging the reagents and a notification in or on the packaging material. The kits can additionally include appropriate instructions for use and labels indicating the intended use of the contents of the kit. The instructions can be present on any written material or recorded material supplied on or with the kit or which otherwise accompanies the kit.

[0075] The therapeutic kits of the invention can be used alone in the treatment of insulin resistance related disorders. They can also be used in conjunction with other known therapeutic regiments. For example, subjects suffering from insul in resistance can use the therapeutic kit along with antihyperglycem ic agents (e.g., metformin). The therapeutic composition of the invention and other known treatment regimens can be administered to the subjects sequentially or simultaneously. These therapeutic appl ications of the invention can all be indicated on the instructions of the kits.

EXAMPLES

[0076] The following examples demonstrated that mice lacking PAR2 (F2rll) or the cytoplasmic domain of TF (F 3) were shown to be protected from high fat diet (HFD) induced weight gain and insulin resistance. In hematopoietic cells, genetic deletion of TF-PAR2 signaling reduced adipose tissue macrophage inflammation and specific pharmacological inhibition of macrophage TF signal ing rapidly ameliorated insulin resistance. In contrast, non-hematopoietic cell TF-VI Ia-PAR2 signaling specifically promoted obesity.

Mechanistically, adipocyte TF cytoplasm ic domain dependent Vila signaling suppressed Akt phosphorylation with concordant adverse transcriptional changes of key regulators of obesity and metabolism. Pharmacological blockade of adipocyte TF in vivo reversed these effects of TF-VIIa signaling and rapidly improved energy expenditure.

[0077] These Example are provided to further illustrate the invention but not to limit its scope. Other variants of the invention wi ll be readily apparent to one of ordinary ski ll in the art and are encompassed by the appended claims.

Example 1 Subjects and experimental protocols

[0078] Human Subjects. Human subject protocols were approved by the Committee on Human Investigation of the University of Cal ifornia, San Diego. Subjects were recruited from diabetes clinics fol lowing informed written consent and classified as diabetic or non-diabetic by their response to a 75-g oral glucose tolerance test according to the American Diabetes Association criteria and absence or presence of obesity (BMI> 30) as described in Phi llips et al., Diabetes 52, 667-674, 2003. Adipose tissue was obtained by needle biopsy of the lower subcutaneous abdominal depot.

[0079] Mice. All experiments were approved by the animal ethics committee of the Torrey Pines Institute for Molecular Studies and The Scripps Research Institute IACUC. TF^ACT mice, which lack the TF cytoplasm ic domain with the exception of the conserved palm itoylation site, and PAR2^{" '} m ice (aka F2rU~'^' m ice), and TF^ACT/ PAR2^7" m ice were in the C57BL/6 background. Humanized TF knock in mice (TFKl) in the C57BL/6 background were kindly provided by Dr. Mark Anderson (J&J PRD). Male mice were fed either a high fat diet (60% kcal from fat; Research Diets, New Brunswick, NJ) or low fat diet ( 10% kcal from fat) beginning at 6-8 weeks of age. Bone marrow chimeras were generated by injecting 5- 10 x 10⁶ bone marrow cells 4-6 hours after lethal irradiation of mice. Following engraftment under antibiotic prophylaxis for 6 weeks, mice were started on a HFD for 16-20 weeks.

[0080] Indirect Calorimetry/energy balance. Metabolism was evaluated in a computer controlled system (Oxymax, Columbus Instruments) under the regular 12 hour light-dark cycle (see, e.g., Yang et al., Am J Physiol Endocrinol. Metab. 297:E21 1 , 2009. Respiratory chambers equipped with a food tray and connected to a balance and photo beams detected motor activity and measured oxygen consumption (V0₂) and C0₂ production (VC0₂).

Measurements from each metabolic chamber every 15 min for 3 consecutive days were pooled based on light-dark cycles and averaged.

[0081] Metabolic parameters. Glucose tolerance tests were performed on mice fasted for 6 hours, whereas insulin tolerance tests were done on non fasted mice. Mice were injected intraperitoneally with either glucose (2 g per kilogram body weight) or insulin (0.75U/kg, Eli Lilly) and glucose concentration was determined with a Glucometer (Bayer, Elkhart, ΓΝ) in blood samples from tail bleeds at baseline, 15, 30, 60, 90 and 120 minutes.

[0082] Cell culture. 3T3-L 1 mouse embryo fibroblasts were grown and differentiated into adipocytes as described in Samad et al., J. Clin. Invest. 97:37-46, 1996. Differentiated adipocytes were incubated in serum free media in the presence of Vila (25 nM) in the presence or absence of anti-mouse TF antibody 2 1 E 10. Total RNA was extracted 3 hours after treatment for measurement of UCP2 gene expression.

[0083] Analysis of gene expression. Total RNA was extracted from cells or fat tissues using Ultraspec RNA isolation system (Biotecx Laboratories, Houston, TX). For real-time quantitative RT-PCR , cDNAs synthesized from total RNA was analyzed with gene-specific primer sets (Invitrogen) and SYBR Green PCR Master mix (PerkinElmer) in an iCycler (Bio-Rad). The mRNA expression levels of all genes reported were s normalized to the β- actin gene expression.

[0084] Adipose tissue fractionation. Epididymal adipose tissues were washed and minced in DMEM and incubated on a shaking platform for 40 min at 37°C with the same medium containing collagenase (0.5 mg/ml). The mixture was filtered through a nylon filter (pore size, 250 μ^ηι) and the filtrate was centrifuged for 5 min at 200 g at 4°C. The floating cells and the pellet were recovered as the mature adipocyte fraction and the stromal vascular fraction (SVF), respectively.

[0085] Flow cytometry and CD 1 1 b+ selection. Flow cytometry of SVF cells from VAD and SAD were performed by staining in FACS buffer (PBS, 1 % FCS, 1 mM EDTA) at 4°C for 30 min with labeled monoclonal antibodies to CD3, CD4, CD8D, CD 1 l b, CD1 l c, F4/80 (eBioscience), or TF (PhD126) in the presence of Fc receptor blocking antibody CD 16/32 (eBioscience). Propidium iodide (PI, Invitrogen) was employed for live cell gating. After washing, cells were fixed in 1 % formaldehyde and analyzed on a LSR-II cytometer (BD) with data processing using the FlowJo software (Tree Star). CD 1 l b⁺ cells were selected from VAD SVF using anti-CD 1 l b paramagnetic microbeads (Milltenyi) by a single pass of the magnetic columns.

[0086] Statistical analysis. The statistical significance of differences between two groups was determined using the unpaired Student's t-test. Comparisons among several groups were performed by ANOVA followed by the Bonferroni test.

Example 2 TF-PAR2 signaling is involved in glucose metabolism and promotes obesity

[0087] This Example describes results obtained from studies that were designed to examine the effect of TF-PAR2 signaling activities on glucose metabolism and insulin resistance. We found that adipose tissue TF mRNA levels and plasma TF activity were increased in obese diabetic subjects compared to non-diabetics (Figs. 5a and 5b). Diet- induced obesity (DIO) in mice recapitulated human findings of increased TF activity levels in plasma and VAD (Fig. l a) and mRNA levels of TF as well as its signaling receptor PAR2 were significantly up regulated in the VAD of obese relative to lean mice (Fig. l b). To address the role of TF-PAR2 signaling in obesity, mice lacking the cytoplasmic domain of TF (TF^ACT), PAR2, or both (TF^ACT PAR2- ^_) were fed a HFD. In comparison to strain-matched C57BL/6 WT controls, the weight gain was significantly lower in the TF^ACT and PAR2^7" mice (Fig. l c). No additive effects were observed in TF^ACT/PAR2^{' "} mice, indicating that TF and PAR2 are operating on a linked signaling pathway.

[0088] TF^ACT, PAR2^"7", and TF^ACT/PAR2^"A mice on a HFD showed improved glucose metabolism (Fig. I d) and insulin sensitivity (Fig. 6a). The attenuated HFD-induced weight gain of TF^ACT and PAR2^7" mice was associated with improved metabolism. Despite similar food intake by WT, TF^ACT and PAR2^7" mice (Fig. 6b), the knockout strains demonstrated increased oxygen consumption (Fig. l e), C0₂ output and a tendency towards increased ambulatory activity (Figs. 6c and 6d). These results demonstrate that TF-PAR2 signaling plays a pivotal role in the development of obesity and its metabolic complications, including insulin resistance.

[0089] Because TF and PAR2 were expressed by adipocytes isolated from obese mice and also on cultured 3T3-L1 adipocytes throughout the course of adipocyte differentiation in vitro (Figs. 7a and 7b), we characterized TF-VIIa-PAR2 signaling in these cells. Coagulation factor Vila binds TF to cleave PAR2. Stimulation of adipocytes with Vila, but not thrombin, suppressed uncoupling protein (UCP) 2, a mitochondrial inner membrane protein that promotes energy expenditure (Fig. 2a). In addition, a monoclonal antibody (21 E 10) that inhibits Vila binding to mouse TF prevented suppression of UCP-2, demonstrating feasibility to interrupt Vila-dependent signaling in mice. Adipocytes isolated from HFD-fed TF^ACT or PAR2^{" ~} mice had concordantly higher levels of UCP-2 mRNA relative to WT (Fig. 2b), confirming in vivo that TF cytoplasmic domain and PAR2 signaling cooperate to improve energy expenditure. DIO leads to increased leptin levels and resulting leptin resistance in humans and mice. Adipocyte leptin levels were significantly reduced in both knock-outs relative to WT, further supporting improved metabolism. Characterization of the stromal vascular fraction from adipose tissues of obese mice further showed that TF (Fig. 8) and PAR2 mRNA (not shown) were expressed by CD 1 l b⁺/CDl l c⁺ pro-inflammatory

macrophages/dendritic cells specifically in the inflammation prone VAD, but not

subcutaneous adipose tissue (SAD). In order to exclude that loss of inflammatory cell TF- PAR2 signaling was responsible for the observed improvements in obesity, we reconstituted TF-PAR2 signaling of macrophages by wild-type bone marrow transplantation into TF^ACT and PAR2^7' mice. The bone marrow chimeras showed significantly reduced HFD-induced weight gain compared to WT bone marrow chimeric controls (Fig. 2c), with a concordant increase in metabolism as indicated by increased oxygen consumption (Fig. 2d) supporting the proposed regulation of energy expenditure by TF-PAR2 signaling in a non hematopoietic compartment, presumably the adipocyte.

[0090] In subsequent studies, we found that WT BM chimeric TF^ACT and F2rU^''^' were hyperphagic and more active than WT recipient controls, excluding that the reduced weight gain was caused by decreased food intake. Both TF^ACT mice with WT BM and whole body TF^ACT mice showed increased oxygen consumption and C0₂ output in indirect calorimetry, with unchanged or slightly decreased respiratory exchange ratios (RER) relative to WT controls, suggesting that increased metabolism in the absence of major impairments of fat utilization contributed to the reduced body weight in TF^ACT mice.

[0091] To eliminate differences in body weight and activity as variables, we further blocked TF acutely in obese WT mice during metabolic evaluation. Blocking TF with the antibody 21 E l 0 in weight-matched DIO mice caused no changes in activity or food intake relative to control antibody, but significantly increased oxygen consumption and C0₂ output with a modest reduction of the RER indicative of improved fatty acid oxidation. Since TF is not detectable in skeletal muscle cells or hepatocytes by in situ hybridization, the improved energy expenditure and metabolism following anti-TF therapy or genetic TF cytoplasmic domain deletion in the stromal compartment likely originated from alteration of TF signaling in the adipocyte.

Example 3 TF-PAR2 signaling regulates macrophage inflammatory phenotypes

[0092] This Example describes studies that are directed to examining the role that TF- PAR2 signaling plays in proliferation, recruitment, retention or function of proinflammatory macrophage. In obese humans and mice the accumulation of CD l l b⁺/CD l l c⁺ proinflammatory macrophages is linked to the development of insulin resistance. Adipose tissue macrophages are under the reciprocal regulatory control of T cells that either enhance proinflammatory cytokine (e.g. IL-6, TNF-a) production by macrophages to cause insulin resistance, or induce an anti-inflammatory phenotype that produces lL- 10 and is implicated in improved insulin sensitivity. We found that TF^ACT, PAR2^7" and TF^ACT PAR2^{" "} mice on a HFD had reduced numbers of CD1 l b⁺/CDl l c⁺ macrophages relative to WT specifically in the VAD, but not the SAD (Fig. 2e). While the diminished macrophage infiltration may be an indirect effect of reduced obesity in these mice, these data indicated possible roles for TF- PAR2 signaling in inflammatory macrophages.

[0093] We therefore generated bone marrow chimeras with a TF cytoplasmic domain- or PAR2-deficient hematopoietic compartment. Mice were placed on a HFD 6 weeks after the bone marrow transplant and macrophage reconstitution efficiency was assessed in the VAD stromal vascular fraction at sacrifice. Flow cytometry showed that GFP-labeled donor cells accounted for >90% of the VAD macrophages and that macrophage TF expression of knockout bone marrow chimeras was indistinguishable from WT (Figs. 9a and 9b). Deletion of the TF cytoplasmic domain or PAR2 in the hematopoietic compartment did not impair HFD- induced weight gain (Fig. 3a), confirming the crucial role for non-hematopoietic TF-PAR2 signaling in the development of obesity. Remarkably, despite the normal increase of weight under HFD, hematopoietic TF cytoplasmic domain- or PAR2-deficiency caused improved glucose tolerance and insulin sensitivity (Figs. 3b and 3c). TF^ACT and PAR2^7' bone marrow chimeras had reduced numbers of VAD CD1 l b⁺/CDl l c⁺ macrophages, but no appreciable changes in CD4⁺ or CD8⁺ T cell numbers (Fig. 3d). The hematopoietic TF-PAR2 signaling- deficient bone marrow chimeras had also reduced VAD mRNA levels of the proinflammatory cytokine IL-6, and increased mRNA levels of the anti-inflammatory cytokine IL- 10 (Fig. 3e). Concordant changes were seen in CD 1 l b⁺ cells isolated from the VAD stromal vascular fraction (Fig. 3e), indicating that TF-PAR2 signaling directly regulates macrophage inflammatory phenotypes, independent of effects on macrophage recruitment.

Example 4 Treating insulin resistance by inhibition of TF-PAR2 signalinfi

[0094] This Example describes anti-TF antibody therapy for improving insulin sensitivity and ameliorating adipose inflammation. To further substantiate the direct role of TF-VIIa- PAR2 signaling on macrophage inflammatory phenotypes, TF was acutely blocked with monoclonal anti-mouse TF antibody 21 E 10 that inhibits Vila signaling (Fig. 2a). A single intraperitoneal bolus of 20 mg/kg anti-TF antibody 21 E l 0 given to obese WT mice significantly improved glucose tolerance (Fig. 4a) and insulin resistance (Fig. 4b) within 24 hours compared to control IgG treated mice. The normalization of metabolic parameters were transient and reversible upon antibody washout one week later, indicating that the antibody directly inhibited cell signaling, rather than caused cellular toxicity to deplete macrophages. In addition, the antibody effect was specific; anti-TF treatment of TF^ACT mice on a HFD did not further improve insulin resistance in these mice (Fig. 10a). The improvements in glucose homeostasis of obese WT mice in response to anti-TF treatment were associated with markedly decreased levels of VAD pro-inflammatory cytokine IL-6, and an increase in IL- 10 (Fig. 4c). Rapid changes in glucose homeostasis independent of weight loss are not unusual and depletion of proinflammatory CD 1 l c⁺ macrophage populations normalizes insulin resistance in obese mice within 24 hours.

[0095] Although the acute effects of anti-TF antibody closely mimicked the improved adipose inflammation and glucose homeostasis of TF^ACT or PAR2^{_ "} hematopoietic bone marrow chimeras, the antibody may have targeted other cells or reduced TF-VIIa

procoagulant activity to achieve its pharmacological action. We made use of humanized TF knock-in (TFK.I) mice and a unique monoclonal antibody ( 10H 10) to selectively block human TF-PAR2 signaling of the hematopoietic compartment of TF I bone marrow chimeras. Chimeric mice developed obesity indistinguishable from control wild-type bone marrow chimeras (Fig. 10b). Obese chimeric mice expressing human TF in hematopoietic cells were treated with a bolus of the monoclonal antibody 1 OH 10 to specifically inhibit human TF- VIIa-PAR2 signaling. Glucose tolerance (Fig. 4d) and insulin sensitivity (Fig. 4e) were improved in 1 OH 10-treated TFKI bone marrow chimeras, but not in the same chimeric mice treated with control antibody or wild-type bone marrow chimera controls that received 10H 10 (pooled data are shown for these control groups). Basal blood glucose levels were also significantly decreased 24 hours after specific antibody therapy relative to controls (214+ 15 versus 245+ 18 mg/dL). These improvements in glucose homeostasis were associated with decreased IL-6 and increased IL- 10 mRNA levels in CD1 l b-selected VAD macrophages from these mice (Fig. 4f). Notably, treating DIO mice with either the anti-mouse TF antibody or anti-human TF in BMT TFKI mice did not appreciably reduce VAD macrophage counts in HFD fed mice (Figs. 4c and 41), indicating that acute inhibition of TF signaling in established obesity improves glucose hemostasis primarily through inhibition of pro-inflammatory signaling of myeloid cells.

[0096] This study establishes a previously unknown role for adipocyte TF-PAR2 signaling in the development of obesity and demonstrates that adipose inflammation and insulin resistance are sustained by independent macrophage TF-PAR2 signaling.

Example 5 Adipocyte TF-VIIa signaling controls regulators of obesity

[0097] Considering that PAR2 β-arrestin signaling suppresses PI-3 kinase activation and that insulin-induced Akt phosphorylation was not inhibited in obese TF^ACT mice, we asked whether adipocyte Akt activation was directly regulated by TF-VIIa-PAR2 signaling. In in vitro differentiated adipocytes from WT, but not from TF^ACT mice, Vila suppressed basal Akt phosphorylation in a TF-dependent manner (Fig. 1 l a). Insulin-induced Akt phosphorylation was similarly inhibited in Vila-treated WT, but not in TF^ACT adipocytes (Fig. 1 1 b).

Importantly, Akt activation was preserved in insulin-treated WT adipocytes when Vila binding to TF was blocked with antibody 21 E 10 to murine TF. Thus, the data indicate that TF directly regulates adipocyte Akt activation.

[0098] We also found that adipocyte TF-VIIa signaling regulated Akt-dependent target genes implicated in obesity and insulin resistance (Fig. 1 l c). Stimulation of 3T3-L 1 adipocytes for 3 hours with Vila, but not thrombin (not shown), increased in a TF-dependent manner mR A expression of the obesity promoter PAI- 1 that is negatively regulated by Akt in adipocytes. Akt activation supports adiponectin expression in adipocytes. Vila stimulation reduced mRNA of this adipokine that upregulates glucose and lipid metabolism broadly in other insulin-sensitive tissue and thereby prevents obesity. Adiponectin activates AMP and, consistently, Vila suppressed the mRNA expression of targets downstream of AMPK, i.e. UCP-2 and PPAR-a involved in energy expenditure and fatty acid oxidation.

Suppression of AMPK may, in part, also be directly mediated by β-arrestin downstream of TF-VIIa-PAR2 signaling. In addition, TF-VIIa signaling increased adipocyte mRNA synthesis of TNF-a,D a key inflammatory mediator that promotes obesity and insulin resistance. Thus, adipocyte TF-VIIa signaling regulates crucial effectors that contribute to the development of DIO.

[0099] To demonstrate that TF signaling in vivo regulated the same target genes, adipocytes were isolated from the VAD of WT mice 24 hours after treatment with antibody 21 E l 0 to mouse TF. Blockade of TF reduced mRNA levels of PAI- 1 and TNF-a and increased mRNA expression of adiponectin, UCP-2 and PPAR-a (Fig. 1 I d), demonstrating a reversal of TF-VIIa signaling effects observed in cultured adipocytes. These changes were directly caused by TF adipocyte signaling, because the mouse TF-specific antibody 21 E 10 caused the same changes in treated BM chimeric mice with human TF in hematopoietic cells (Fig. 1 I d). Conversely, blocking human TF signaling in the hematopoietic compartment of the chimeric mice with antibody 10H 10 had no effect on the adipocyte expression of the same target genes relative to control. These data establish direct and independent regulatory roles for adipocyte TF signaling in the expression of key mediators of weight gain.

[00100] TF is expressed by neither skeletal muscle nor hepatocytes and restrictions of the blood-brain barrier would exclude central effects of acutely administered antibody. Blocking adipocyte TF in the human/mouse TF chimeric mice ameliorated insulin resistance within 24 hours (Fig. 1 l e). In order to determine whether the improved insulin sensitivity was independent of changes in adipose inflammation, we determined inflammatory cytokines in SVF cells which include macrophages (Fig. 1 I f). As expected, blocking hematopoietic TF signaling with the human TF-specific antibody 10H 10 increased IL- 10 and suppressed IL-6 and TNF-a mRNA levels in SVF cells from these chimeric mice. Blocking TF specifically in non-hematopoietic cells with the murine TF-specific antibody 21 E 10 had no effect on IL- 10 mRNA expression. Thus, macrophage TF signaling specifically regulates IL- 10 expression independent of potential cross talks from adipocytes. Conversely, both antibodies did not change PAI- 1 mRNA expression in SVF cells, confirming adipocyte-specific regulation of PAI- 1 by TF signaling. However, inhibition of non-hematopoietic TF reduced mRNA levels of TNF-a and IL-6 in SVF cells. These data indicated that improved insulin sensitivity in response to TF blockade on adipocytes may, in part, involve reduced adipose tissue inflammation.

* * *

[00101] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it will be readily apparent to one of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[00102] All publications, databases, GenBank sequences, patents, and patent applications cited in this specification are herein incorporated by reference as if each was specifically and individually indicated to be incorporated by reference.

Claims

WHAT IS CLAIMED IS:

1. A method for treating a subject with a metabolic complication of obesity or a disorder associated with or mediated by insulin resistance, comprising administering to the subject a therapeutically effective amount of a compound that specifically inhibits or suppresses tissue factor- protease activated receptor 2 (TF-PAR2) signaling.

2. The method of claim 1 , wherein the metabolic complication of obesity or a disorder associated with or mediated by insulin resistance is type 2 diabetes, obesity, hyperglycemia, dyslipidemia, insulin resistance, metabolic syndrome or abnormal weight gain.

3. The method of claim 1 , wherein the compound selectively inhibits TF- PAR2 signaling and does not interfere with hemostatic activity mediated by tissue factor/ factor Vila (TF/VIIa).

4. The method of claim 1 , wherein the compound is a TF inhibitor or a PAR2 antagonist.

5. T e method of claim 1 , wherein the compound is an antibody or small chemical entity.

6. The method of claim 5, wherein the compound is an antibody or an antigen-binding molecule having the binding specificity of monoclonal antibody 10H 10 produced by the hybridoma with ATCC access number HB9383.

7. The method of claim 5, wherein the compound is a monoclonal antibody 10H 10 produced by the hybridoma with ATCC access number HB9383.

8. A method for treating or ameliorating the symptoms of type 2 diabetes in a subject, comprising administering to a subject afflicted with type 2 diabetes a compound that specifically inhibits or suppresses tissue factor- protease activated receptor 2 (TF-PAR2) signaling.

9. The method of claim 8, wherein the compound selectively inhibits TF- PAR2 signaling and does not interfere with hemostatic activity mediated by tissue factor/ factor Vila (TF/VIIa).

10. The method of claim 8, wherein the compound is an antibody or small chemical entity.

11. The method of claim 8, wherein the compound is an antibody or an antigen-binding molecule having the binding specificity of monoclonal antibody 1 OH 10 produced by the hybridoma with ATCC access number HB9383.

12. The method of claim 8, wherein the compound is a monoclonal antibody 10H 10 produced by the hybridoma with ATCC access number HB9383.

13. A kit for use in the treatment of insulin resistance related disorders in a subject in need thereof, comprising a therapeutically effective amount of a compound that specifically inhibits or suppresses tissue factor- protease activated receptor 2 (TF-PAR2) signaling and a pharmaceutically acceptable carrier.

14. The kit of claim 13, wherein the compound selectively inhibits TF-PAR2 signaling and does not interfere with hemostatic activity mediated by tissue factor/ factor Vila (TF/VIla).

15. The kit of claim 13, wherein the compound is an antibody or small chemical entity.

16. T e kits of claim 13, wherein the compound is an antibody or an antigen- binding molecule having the binding specificity of monoclonal antibody 10H 10 produced by the hybridoma with ATCC access number HB9383.

17. The kit of claim 13, wherein the compound is a monoclonal antibody 10H 10 produced by the hybridoma with ATCC access number HB9383.

18. The kit of claim 13, further comprising instructions for use or disposal of the reagents in the kit.