WO2006083798A2

WO2006083798A2 - Methods for treating obesity-related disorders by inhibiting myd88 and methods for identifying myd88 inhibitors

Info

Publication number: WO2006083798A2
Application number: PCT/US2006/003296
Authority: WO
Inventors: Anthony W. Ferrante; Stuart P. Weisberg; Rudolph L. Leibel
Original assignee: Columbia University in the City of New York
Current assignee: Columbia University in the City of New York
Priority date: 2005-01-31
Filing date: 2006-01-31
Publication date: 2006-08-10
Anticipated expiration: 2007-07-31
Also published as: WO2006083798A3

Abstract

The invention is directed to methods for treating an obesity-associated disorder in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88. The invention is also related to methods for lowering body weight in a mammal comprising administering to the mammal in need of losing weight an inhibitor of Myd88 in an effective amount to reduce the weight of the subject.

Description

METHODS FOR TREATING OBESITY-RELATED DISORDERS BY INHIBITING MYD88 AND METHODS FOR IDENTIFYING MYD88 INHIBITORS

[0001] This application claims priority to U.S. Application No. 60/648,644, which was filed on January 31 , 2005, which is hereby incorporated by reference in its entirety.

[0002] The invention disclosed herein was made with U.S. Government support under Nffl Grant No. 1R01DK066525; 1R01DK052431 from the NIDDK. Accordingly, the U.S. Government may have certain rights in this invention.

[0003] This patent disclosure contains material that is subject to copyright protection. The copyright owner has no objection to the facsimile reproduction by anyone of the patent document or the patent disclosure as it appears in the U.S. Patent and Trademark Office patent file or records, but otherwise reserves any and all copyright rights.

[0004] AU patents, patent applications and publications cited herein are hereby incorporated by reference in their entirety. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the invention described herein.

FIELD OF THE INVENTION

[0005] This invention relates to methods for treating obesity-related disorders, methods for losing weight, and methods for identifying inhibitory compositions.

BACKGROUND OF THE INVENTION

[0006] The development of obesity in humans and in mouse models is associated with a proinflammatory response. This inflammatory response is characterized by the overproduction of inflammatory mediators including iNOS, IL-6, MCP-I₅ and TNFα; the overproduction of acute phase proteins such as C-reactive protein, and haptoglobin and increases in white blood cell and monocyte counts. Overproduction of these inflammatory molecules is associated with activation of inflammatory signaling pathways in leukocytes, liver, muscle and fat tissue. These inflammatory responses include increased activity of NF- KB, and JNK, and overproduction of suppressors of cytokine signaling 1 and 3 (SOCSl and S0CS3). AU of these responses can impair insulin signaling at the cellular level.

[0007] Evidence from epidemiological studies in humans and genetic experiments on murine obesity models suggests that this inflammatory response plays an important role in the pathogenesis of type II diabetes mellitus (T2DM). Prospective population studies demonstrate that circulating pro-inflammatory molecules — particularly C-reactive protein and IL-6 — are predictive of future development of T2DM. hi mice, genetic deficiency in TNFα, iNOS and the MCP-I receptor CCR2 confers protection against the development of insulin resistance and hyperglycemia during diet induced obesity. Moreover, disruption of the signaling pathways activated by these inflammatory molecules such as JNK, IKKβ, SOCSl, and S0CS3 confers protection against obesity induced insulin resistance.

[0008] Activation of the innate immune response by exogenous pathogens causes overproduction of many of the same proinflammatory molecules overproduced during obesity albeit at much higher levels. As a result, similar downstream signaling pathways are activated during obesity and sepsis, and both obesity and sepsis lead to insulin resistance.

[0009] It is important to better define the signaling pathways linking obesity to inflammation, because they may play an important role in the pathogenesis of T2DM and possibly other diseases for which obesity and inflammation are important independent risk factors such as myocardial infarction, and stroke.

SUMMARY OF THE INVENTION

[0010] The invention provides for a method for treating an obesity-associated disorder in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88. The invention also provides for a method for preventing an obesity-associated disorder in a human subject, the method comprising administering to the subject an effective amount of an inhibitor of human Myd88. In another aspect, the invention provides for a method for treating insulin resistance in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88. The invention also provides for a method for treating diabetes in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88.

[0011] In accordance with the method of this invention, the inhibitor of Myd88 may be an inhibitor that inhibits Myd88 gene expression, Myd88 protein production, or function of the Myd88 protein. The inhibitor may comprise a polypeptide, a peptidomimetic, an organic molecule, a carbohydrate, a lipid, an antibody or a nucleic acid.

[0012] The subject on which the method is employed may be any mammal, e.g. a human, mouse, cow, pig, dog, cat, or monkey.

[0013] In one aspect of the present invention, the mammalian members of the receptor superfamily for which Myd88 acts as an adapter include Toll-like receptor/Interleukin-1 receptor superfamily (mammalian): Subgroup 1 (Interleukin 1 receptors) - IL-IRl, IL-IRE, B15R, IL-IRAcP, IL-18R, IL-18RAcP, T1/ST2, IL-lRrp2, IL- IRAPL, TIGGIR, SIGGIR, and Subgroup 2 (Toll-like receptors) - TLR-I through TLR-10.

[0014] The administration of the agent may be effected by intralesional, intraperitoneal, intramuscular or intravenous injection; by infusion; or may involve liposome- mediated delivery; or topical, nasal, oral, anal, ocular or otic delivery.

[0015] In the practice of the method, administration of the inhibitor may comprise daily, weekly, monthly or hourly administration, the precise frequency being subject to various variables such as age and condition of the subject, amount to be administered, half- life of the agent in the subject, area of the subject to which administration is desired and the like.

[0016] Li connection with the method of this invention, a therapeutically effective amount of the inhibitor may include dosages which take into account the size and weight of the subject, the age of the subject, the severity of the obesity-related symptoms, the method of delivery of the agent and the history of the symptoms in the subject.

BRIEF DESCRIPTION OF THE FIGURES

[0017] Figures 1A-1B. Graphical representations showing the genotyping of blood cells and weights of mice transplanted with Myd88 -/- bone marrow. Mice lethally irradiated and reconstituted with Myd88 -/- bone marrow are engrafted normally and they grow at the same rate on a high fat diet as control animals that were lethally irradiated and reconstituted with wild type bone marrow cells. Figure IA. Four weeks after bone marrow transplantation, blood cells from the recipient mice were genotyped using qPCR. Figure IB. Weights of mice transplanted with Myd88 -/- bone marrow cells (white circles) and Myd88 +/+ bone marrow cells (black squares) during 20 weeks of being fed a high fat diet (60% kcal from lard). Values are mean ± S. D. [0018] Figures 2A-2D. In obese mice, Myd88 deficiency in hematopoietic cells is associated with decreased levels of inflammatory markers in blood. After 20 weeks on high fat diet (60% kcal from lard) plasma samples were drawn from obese mice that had been transplanted with Myd88 -/- bone marrow (white bars), obese mice that had been transplanted with Myd88 +/+ bone marrow and lean wild type mice. Circulating levels of MCP-I (28.5 ± 15.5 vs. 70.2 ± 28.1 vs. 24 ± 7 pg/ml) were measured (Figure 2A), IL-6 (6.5 ± 1.5 vs. 13.5 ± 4.6 vs. 4.6 ± 0.84 pg/ml) (Figure 2B), and PAI-I (4600 ± 2200 vs. 7000 ± 1100 vs. 2000 ± 800 pg/ml) (Figure 2C). In addition, the circulating monocyte fraction of peripheral blood leukocytes was determined by staining peripheral blood leukocytes for CdI Ib and using flow cytometry to quantify the monocyte population (9 ± 1.7 vs. 11 ± 1.5 vs. 6.9 ± 0.3%) (Figure 2D). * p value < 0.05, ** p value < 0.01. Values are mean ± S.D.

[0019] Figures 3A-3D. Obese animals reconstituted with Myd88 -/- bone marrow have improved insulin sensitivity. Figure 3A. After 20 weeks on high fat diet, obese mice that had been transplanted with Myd88 -/- (white circles) and Myd88 +/+ (black squares) bone marrow were fasted for six hours and given an intraperitoneal injection of insulin (1.5 u/kg). Figure 3B. Blood glucose was measured 0, 15, 30, 45 and 60 minutes after injection. At each time point, mice with Myd88 -/- hematopoietic cells had significantly lower absolute blood glucose levels and a greater percentage reduction in glucose levels compared to mice with Myd88 +/+ hematopoietic cells. Figure 3C. HOMA-IR calculations (4.9 ± 3.0 vs. 12.9 ± 6.3; p value < 0.05) based on B. glucose (100.4 ± 20.5 vs. 127 ± 23 mg/dl; p value < 0.05) and insulin values (0.75 ± 0.31 vs. 1.6 ± 0.65 ng/ml; p value < 0.05) measured after an overnight fast. Figure 3D. After 12 weeks on high fat diet, obese mice with Myd88 -/- hematopoietic cells and obese mice with Myd88 +/+ hematopoietic cells were fasted overnight and given an intraperitoneal injection of glucose (1 g/kg). Compared to obese mice with Myd88 +/+ hematopoietic cells, mice with Myd88 -/- hematopoietic cells had significantly lower blood glucose levels at time points 45, 60, 90 and 120 minutes after injection. * p value < 0.05, ** p value < 0.01. Values are mean ± S.D.

[0020] Figures 4A-4D. Obese animals reconstituted with Myd88 -/- bone marrow have decreased hepatomegaly, hepatic steatosis and decreased hepatic SOCSl expression. After 24 weeks on high fat diet whole livers were removed and weighed. Figure 4A. Liver weights were normalized to body mass (0.0456 ± 0.00880 vs. 0.0686 ± 0.00727; p value < 0.01). Figure 4B. Hepatic steatosis was assessed by measuring total triglyceride content per gram of liver tissue (52.4 ± 17.9 vs. 80.4 ± 27.3 mg/g; p value < 0.05). Figure 4C. RNA was extracted and SOCSl expression was measured using quantitative RT-PCR (93.3 ± 27.5 vs. 206 ± 89.1 arbitrary units; p value < 0.05). Figure 4D. The Pearson correlation coefficient was calculated to examine the relationship between SOCSl expression and HOMA-IR - a quantitative estimate of insulin resistance (r = 0.87; p value < 0.01) - in obese mice that had Myd88 -/- hematopoietic cells (white circles) and obese mice that had Myd88 +/+ hematopoietic cells (black squares). * p value < 0.05, ** p value < 0.01. Values are mean ± S. D.

[0021] Figures 5A-5D. Myd88 -/- (open circles), heterozygous (black triangles) and wild type littermates (black squares) were placed on a high fat diet at five weeks of age and their growth was monitored for two months. Figure 5A. At five weeks of age Myd88 -/- mice weighed significantly less than heterozygous (19.8 +/- 1.8 vs. 24.4 +/- 3.5; n = 13-20; p- value < 0.01) or wild type littermates (19.8 +/- 1.8 vs. 25.0 +/- 3.5; n = 13-20; p-value < 0.01). Figure 5D. During two months of high fat feeding, Myd88 -/- mice continued to weigh less than littermate heterozygotes (one month: 26.0 +/- 3.1 vs. 33.8 +/- 5.9; n = 13-20; p-value < 0.01; two months: 32.0 +/- 4.9 vs. 43.2 +/- 8.3; n = 13-20; p-value < 0.01) or wild types (one month: 26.0 +/- 3.1 vs. 37.1 +/- 4.9; n = 13-20; p-value < 0.01; two months: 32.0 +/- 4.9 vs. 48.1 +/- 2.4; n = 13-20; p-value < 0.01). During high fat feeding the body weights of Myd88 -/- mice and littermate controls were mostly non-overlapping after one month (Figure 5B) and two months (Figure 5C).

[0022] Figures 6A - 6B. cDNA sequence for human Myd88 (SEQ ID NO: 1) as reported in Hardiman, G., Rock, F.L., Balasubramanian, S., Kastelein, R.A. and Bazan, J.F, Molecular characterization and modular analysis of human MyD88, Oncogene 13 (11), 2467- 2475 (1996); Accession No. NM_002468.

[0023] Figure 7. The translation, or protein sequence for human Myd88 (SEQ ID NO:2) based on the nucleotide sequence of SEQ ID NO:1.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The patent and scientific literature referred to herein establishes knowledge that is available to those with skill in the art. The issued patents, applications, and other publications that are cited herein are hereby incorporated by reference to the same extent as if each was specifically and individually indicated to be incorporated by reference. In the case of inconsistencies, the present disclosure will prevail. [0025] The following publications are hereby incorporated by reference in their entireties: U.S. Patent Publication No. US20040038926A1, Antisense modulation of MyD88 expression; U.S. Patent Publication No. US20030186903A1, Antisense modulation of MyD88 expression; U.S. Patent Publication No. US20030148986A1 Methods for treating vascular disease by inhibiting myeloid differentiation factor 88; European Publication No. EP1474444A1, A Novel Splice Variant Of Myd88 And Uses Thereof; and European Publication No. EP1465905A2, Antisense Modulation of Myd88 Expression.

[0026] It is a discovery of the present invention that the expression and function of myeloid differentiation primary response gene (Myd88) in hematopoietic cells links obesity, to inflammation and insulin resistance. The molecule Myd88 plays an important role in the activation of the innate immune response. It serves as an adaptor molecule which associates with the intracellular Toll/Interleukin-1 receptor (TIR) domain of the Interleukin-1 receptor and toll-like receptors (TLRs). Many signaling events downstream of these receptors depend on Myd88 because it is required to recruit signaling mediators such as Interleukin 1 receptor associated kinase (IRAK) to the signaling complex. Several TLR mediated responses are independent of Myd88, however most TLRs require Myd88 in order to stimulate production of cytokines such as TNFα and IL-6.

[0027] This invention provides for the discovery that Myd88 can be used as a target in a drug screening assay to identify drugs that are capable of inhibiting Myd88 activity or function, thereby decreasing insulin resistance.

[0028] The present invention provides that Myd88 plays a role in the overproduction of cytokines during obesity and thereby influences the development of T2DM. To study the effects of genetic Myd88 deficiency on obesity induced inflammation, attempts were made to make Myd88-deficient animals obese using a high fat diet and results showed that Myd88 deficient mice are resistant to developing diet induced obesity. Therefore, a bone marrow transplantation was used to create chimeric animals with Myd88 deficiency only in hematopoietic cells. These animals became as obese as chimeras that were transplanted with wild type hematopoietic cells. As a result, the levels of inflammatory markers in their blood were examined as well as their metabolic phenotype. The results shown for this invention indicate that Myd88 in hematopoietic cells plays an important role in obesity induced inflammation and insulin resistance. [0029] Obesity is associated with a proinflammatory state characterized by the increased accumulation in adipose tissue of macrophages, the increased production of cytokines such as TNF-alpha, IL-6 and MCP-I. However, the mechanisms and functional significance of these pro- inflammatory responses are incompletely characterized. The protein Myd88 plays a central role in activation of the innate immune response by recruiting downstream signaling mediators in response to engagement of pattern recognition receptors. Animals with genetic deficiency of Myd88 exhibit impaired innate immune responses to exogenous pathogens as well as resistance to atherosclerosis. To test whether Myd88- dependent signaling plays a role in obesity-associated inflammation, wild type C57BL/6J mice were lethally irradiated and reconstituted with wild type and Myd88-deficient bone marrow cells. Compared to animals reconstituted with wild type bone marrow, the animals reconstituted with Myd88-deficient bone marrow became equally obese but had reduced circulating concentrations of MCPl and TL6. The decreased production of inflammatory cytokines was associated with decreased SOCSl expression in liver. The obese recipients of MyD88-deficient bone marrow had lower circulating monocyte counts. The decreased activation of inflammatory pathways in these animals was associated with lower circulating glucose concentrations, increased insulin sensitivity as assessed by ITT, GTT and HOMA-IR calculation, and decreased hepatic steatosis. These data show that Myd88-dependent signaling in hematopoietic cells is a critical link between obesity, inflammation and insulin resistance.

[0030] Inhibitors of Myd88 include nucleic acid compounds that inhibit Myd88, such as an antisense Myd88 RNA, a ribozyme designed to cleave Myd88 RNA and double- stranded RNA that is sufficiently homologous to the Myd88 gene product to inhibit the encoding function of Myd88 mRNA (See, U.S. Serial No. 2003 0148986 Al, U.S. Serial No. 2003 0186903 Al). Another inhibitor of Myd88 is a Myd88-specific small interfering RNA (siRNA) which was shown to knockdown expression of Myd88 in a macrophage cell line (Yang et al., J Virol 78(20): 11152-11160 (2004)). Myd88-specific antibodies are available from commercial vendors, for example, Abeam, eBiosciences and ProSci, Inc.

[0031] Inhibitors such as peptides or peptidomimetics can be designed based on structural studies of Myd88, TLRs and IL-IRs (Khan et al., J Biol Chem 279(30):31664- 31670 (2004); Dunne et al, J Biol Chem 278(42): 41443-41451 (2003)). For example, the Toll/interleukin-1 receptor (TIR) domain is required for the interaction of Myd88 with TLRs and IL-IRs. The domain is conserved among the TLRs, IL-IRs and Myd88. Peptide inhibitors or peptidomimetics that share homology with this domain could be used to block the recruitment of Myd88 to the receptors, thus creating a lesion in the signaling pathway and inhibiting downstream events. Additionally, U.S. Patent No. 6,222,019 discloses fragments of Myd88 that function as dominant negative versions of full-length Myd88. For example, a Myd88 fragment comprising amino acid residues 106-296 inhibits IL-lR-induced NF-κB activity.

[0032] The invention provides for a method for treating an obesity-associated disorder in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88. The invention also provides for a method for preventing an obesity-associated disorder in a human subject, the method comprising administering to the subject an effective amount of an inhibitor of human Myd88. In another aspect, the invention provides for a method for treating insulin resistance in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88. The invention also provides for a method for treating diabetes in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88.

[0033] The invention provides for embodiments where the inhibitor comprises an antagonist of Myd88, a Myd88-specific antibody, an antisense oligonucleotide that specifically binds to a region of a nucleic acid that encodes Myd88, an organic molecule, a peptide, or a peptidomimetic. The invention provides for embodiments where the inhibitor comprises a small interfering RNA (siRNA) that specifically binds to a region of a nucleic acid that encodes Myd88, wherein expression of Myd88 is inhibited. The invention provides for embodiments where the inhibitor comprises a peptide that is a fragment of Myd88, a fragment of a Toll-like receptor, or a fragment of an interleukin 1 receptor. The invention provides for embodiments where the inhibitor comprises a peptidomimetic of a peptide fragment of Myd88, a peptide fragment of a Toll-like receptor, or a peptide fragment of an interleukin 1 receptor. The invention provides for embodiments where the inhibitor comprises a peptide fragment, which is of a Toll/interleukin 1 receptor (TIR) domain. The invention provides for embodiments where the obesity-associated disorder comprises diabetes, insulin resistance, hyperinsulinemia, decreased glucose clearance, dyslipidemia, non-alcoholic fatty liver disease, hypertension, inflammation, hepatomegaly, hepatic steatosis, myocardial infarction, asthma, stroke, or any combination thereof.

[0034] The invention provides for embodiments where the administering comprises contacting hematopoietically-derived cells or myeloid cells with the inhibitor. The invention provides for embodiments where the administering comprises contacting bone marrow of the subject with the inhibitor. The invention provides for embodiments where the administering comprises ex vivo treatment of cells or tissue taken from the subject with the inhibitor, and returning the cells or tissues to the subject. The invention provides for embodiments where the administering comprises intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome-mediated delivery; or topical, nasal, oral, ocular, otic delivery, or any combination thereof. The invention provides for embodiments where the subject is a human, mouse, rabbit, monkey, rat, bovine, pig or dog.

[0035] The invention provides for methods for treating an obesity-associated disorder in a subject, the method comprising transplanting Myd88-deflcient bone marrow into the subject. The invention also provides methods for reducing or preventing obesity in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88. The invention provides for methods for lowering body weight in a mammal comprising administering to the mammal in need of losing weight an inhibitor of Myd88 in an effective amount to reduce the weight of the subject, thereby lowering the body weight. The invention provides for embodiments where the administration is systemic administration. The invention provides for embodiments where the subject is suffering from diabetes. The invention provides for embodiments where the mammal is a dog, a mouse, a human, a horse, a monkey, a primate, or a feline.

[0036] The invention also provides for drug screening methods. The invention provides for methods for identifying whether a test compound is capable of inhibiting Myd88 activity, the method comprising: (a) contacting a test compound with a solution containing (i) Myd88, and (ii) either a Toll-like receptor, an interleukin 1 receptor, or both, (b) measuring Myd88 activity in the solution of step (a), and (c) comparing the Myd88 activity in step (b) with Myd88 activity in the absence of the test compound, so as to determine whether or not the test compound is capable of inhibiting Myd88 activity.

[0037] The invention provides for methods for identifying a compound capable of inhibiting Myd88, the method comprising: (a) administering a test compound to a subject, and (b) measuring inhibition of Myd88 activity in the subject as compared to another subject that was not administered the test compound, so as to determine whether or not the test compound inhibits Myd88 activity. The invention also provides for a method for identifying a compound capable of inhibiting Myd88 activity, the method comprising: (a) contacting a test compound with a cell, (b) contacting the cell with a toll-like receptor agonist or interleukin 1 receptor agonist, (c) measuring Myd88 activity in the cell, and (d) comparing the activity measured in (c) with Myd88 activity in a cell in the absence of the test compound, so as to determine whether the test compound is capable of inhibiting Myd88.

[0038] The invention provides for embodiments where the method is carried out in a high-throughput way. The invention provides for embodiments where the activity measured comprises Myd88 gene expression, Myd88 protein production, Myd88 translocation, Myd88 protein function, Myd88 binding activity, cytokine production, immune response, blood glucose concentration, insulin resistance, insulin concentration, NF-κB activity, SOCSl expression, MCP-I production, PAI-I production, IL-6 production, blood monocyte fraction, or any combination thereof. The invention provides for embodiments where Myd88 activity is detected as a complex formed with Myd88 and either Toll-like receptor, interleukin 1 receptor, or both. The invention provides for embodiments where the solution comprises an extract from a cell contacted with a Toll-like receptor agonist or an interleukin 1 receptor agonist. The invention provides for embodiments where the inhibition of Myd88 activity is measured by detecting decreased levels of an inflammatory marker in blood of the subject. The invention provides for embodiments where the inflammatory marker comprises MCP-I, IL-6, PAI-I, monocytes, C-reactive protein or any combination thereof. The invention provides for embodiments where the inhibition of Myd88 activity determined by detecting increased insulin sensitivity, decreased circulating glucose levels, decreased hepatomegaly, decreased hepatic steatosis, decreased hepatic suppressor of cytokine signaling (SOCSl) expression, or any combination thereof.

[0039] The invention provides for embodiments where glucose levels are measured by a glucose tolerance test (GTT) or measuring fasting glucose levels, or a combination thereof. The invention provides for embodiments where insulin sensitivity is measured by an insulin tolerance test (ITT) or a homeostatic model assessment of insulin resistance (HOMA-IR) or an euglycemic hyperinsulinemic clamp, or any combination thereof. The invention provides for embodiments where the cell is a hematopoietic cell, a myeloid cell, a macrophage, or any combination thereof. The invention provides for embodiments where the agonist comprises lipopolysaccharide, interleukin 1, interferon gamma, saturated fatty acids, dsRNA, bacterial cellular components, viral protein, carbohydrate, nucleic acid components, or any combination thereof. The invention provides for embodiments where the inhibition of Myd88 activity is measured as decreased NFKB activity, IRAK activation, IRAK recruitment, decreased production of cytokines, or any combination thereof. The invention provides for embodiments where the cytokine comprises MCP-I₅ EL-6, PAI-I, TNF-a, MCP-3, adiponection, resistin, or any combination thereof.

[0040] In one aspect of the invention, the inhibitor of Myd88 can be combined with a carrier. The term "carrier" is used herein to refer to a pharmaceutically acceptable vehicle for a pharmacologically active agent. The carrier facilitates delivery of the active agent to the target site without terminating the function of the agent. Non-limiting examples of suitable forms of the carrier include solutions, creams, gels, gel emulsions, jellies, pastes, lotions, salves, sprays, ointments, powders, solid admixtures, aerosols, emulsions (e.g., water in oil or oil in water), gel aqueous solutions, aqueous solutions, suspensions, liniments, tinctures, and patches suitable for topical administration.

[0041] The term "about" is used herein to mean approximately, in the region of, roughly, or around. When the term "about" is used in conjunction with a numerical range, it modifies that range by extending the boundaries above and below the numerical values set forth. Li general, the term "about" is used herein to modify a numerical value above and below the stated value by a variance of <20%.

[0042] The term "effective" is used herein to indicate that the inhibitor is administered in an amount and at an interval that results in the desired treatment or improvement in the disorder or condition being treated (e.g., an amount effective to reduce body weight of a subject, or to reduce insulin resistance).

[0043] hi some embodiments, the subject is a mammal. Nonlimiting examples of mammals include: human, primate, mouse, otter, rat, and dog.

[0044] Pharmaceutical formulations include those suitable for oral or parenteral (including intramuscular, subcutaneous and intravenous) administration. Forms suitable for parenteral administration also include forms suitable for administration by inhalation or insufflation or for nasal, or topical (including buccal, rectal, vaginal and sublingual) administration. The formulations may, where appropriate, be conveniently presented in discrete unit dosage forms and may be prepared by any of the methods well known in the art of pharmacy. Such methods include the step of bringing into association the active compound with liquid carriers, solid matrices, semi-solid carriers, finely divided solid carriers or combinations thereof, and then, if necessary, shaping the product into the desired delivery system. [0045] The following examples illustrate the present invention, and are set forth to aid in the understanding of the invention, and should not be construed to limit in any way the scope of the invention as defined in the claims which follow thereafter.

EXAMPLES

Example 1: Body Mass of Myd88 -/- Mice and Bone Marrow Chimeras Is Lower Than Myd88 +/+ Mice

[0046] To determine the effects of Myd88 deficiency on growth and the development of obesity, male Myd88 deficient animals and littermate controls were placed on a high fat diet. Before beginning the high fat diet regimen at 5 weeks of age, the body mass of the Myd88 deficient animals was significantly lower than that of heterozygous and wild type littermates. After one and two months on the diet the body mass of the Myd88 deficient animals was still significantly lower than that of their littermates, and there was very little overlap between the body weights of animals in the two groups (Figures 5A-5D).

[0047] Because fewer Myd88 deficient animals became as obese as controls, it was necessary to create chimeric animals where Myd88 deficiency was restricted to hematopoietic cells in order to determine the role of Myd88 in obesity induced inflammation and insulin resistance. At four weeks of age, wild type animals were lethally irradiated and reconstituted with bone marrow from either Myd88 deficient or wild type littermates. These chimeras were then fed a high fat diet for six months. Animals reconstituted with Myd88 deficient bone marrow grew at the same rate as animals reconstituted with wild type bone marrow (Figure 1). Analysis of body composition using dual x-ray absorbance spectroscopy (DEXA) revealed no significant differences in adiposity or epididymal adipose tissue mass between the two groups of mice (Table 1).

Table 1: Mice that were lethally irradiated and reconstituted with Myd88 -/- bone marrow cells became as obese as those reconstituted with Myd88 +/+ bone marrow cells during high fat diet feeding. After 24 weeks on the high fat diet, dual x-ray absorbance spectroscopy was used to measure the body compositions of mice in the two groups. Organ weights were recorded upon necropsy. ** p value < 0.01. Values are mean ± S.D. [0048] The invention provides for the use of one or more inhibitors of Myd88 to prevent obesity or to reduce body weight of a mammal by systemically administering the Myd88 inhibitor to the mammal (such as a mouse, a primate, or a human). This example shows that the systemic administration of a Myd88 inhibitor in order to reduce Myd88 activity in a subject, will prevent weight gain from the diet of the subject. Systemic inhibitors include siRNA or antisense nucleic acids specifically targeted to affect the expression of Myd88. Other inhibitors include antibodies that specifically bind to Myd88.

[0049] These data link Myd88 - an important molecular component of the innate immune response - to several of the inflammatory responses that are associated with obesity in both mice and humans. Furthermore, the fact that the phenotype of reduced obesity- induced inflammation was observed in animals where Myd88 deficiency was restricted to hematopoietic cells suggests that bone marrow derived cells can serve as 'middlemen' that translate signals associated with obesity and overnutrition into inflammatory signals.

[0050] Many of these inflammatory signals have been implicated in the adverse pathophysiological phenotypes associated with obesity. For example, MCP-I has been linked to the development of atherosclerosis. IL-6 is an important stimulator of c-reactive protein production and both have been linked to progression of coronary artery disease. And the development of obesity induced insulin resistance has been linked to overproduction of iNOS, TNF- α, MCP-I and IL-6.

Example 2: Inhibition of Myd88 in Hematopoietic Cells Results in Decreased Insulin Resistance - A treatment for obesity-induced inflammation

[0051] In humans and mice, increased adiposity is associated with increased circulating concentrations of inflammatory molecules including IL-6, MCP-I and PAI-I. To determine if Myd88 in hematopoietic cells plays a role in stimulating this inflammatory response to obesity, the levels of these molecules was measured in plasma from obese mice, lean mice and obese mice with Myd88 -/- hematopoietic cells.

[0052] As expected, obese mice that had been transplanted with Myd88 +/+ bone marrow cells had elevated plasma concentrations of MCP-I (70.2 ± 28.1 vs. 24 ± 7 pg/mL; p value < 0.01), IL-6 (13.5 ± 4.6 vs. 4.6 ± 0.84 pg/mL; p value < 0.01), and PAI-I (7000 ± 1100 vs. 2000 ± 800 pg/mL; p value < 0.05) compared to lean mice. Myd88 deficiency in hematopoietic cells completely prevented the obesity induced increase in plasma MCP-I levels (28.5 ± 15.5 vs. 70.2 ± 28.1 pg/mL; p value < 0.01) and partially prevented the obesity induced increase in IL-6 (6.5 ± 1.5 vs. 13.5 ± 4.6 pg/mL; p value < 0.01) and PAI-I (4600 ± 2200 vs. 7000 ± 1100 pg/mL; p value < 0.05) levels (Figures 2A-2D).

[0053] It has been reported that obesity is associated with increases in the fraction of circulating monocytes and that increased circulating MCP-I levels may play a role in stimulating this response. Because Myd88 deficiency in hematopoietic cells prevented the obesity induced increase in circulating MCP-I levels, the Myd88 deficiency might also prevent the obesity induced increase in blood monocyte fraction. Therefore, flow cytometry was used to quantify blood monocyte populations in lean mice, obese mice and obese mice with Myd88 -/- hematopoietic cells. As expected, obesity was associated with a significant increase in blood monocyte fraction (11 ± 1.5 vs. 6.9 ± 0.3 %; p value < 0.05) which was attenuated in animals with Myd88 -/- hematopoietic cells (9 ± 1.7 vs. 11 ± 1.5 %; p value < 0.05) (Figures 2A-2D).

Obesity induced insulin resistance

[0054] Evidence from human studies and mouse models suggests that the inflammatory response associated with obesity plays a causal role in the development of insulin resistance and T2DM. Because mice with Myd88 -/- hematopoietic cells became as obese as wild types but had an attenuated inflammatory response, the development of insulin resistance in mice with Myd88 -/- hematopoietic cells might be similarly attenuated.

[0055] Insulin sensitivity was measured in obese mice that had been transplanted with Myd88 -/- bone marrow cells and obese mice that had been transplanted with Myd88 +/+ bone marrow cells. After a six-hour fast, mice with Myd88 -/- hematopoietic cells had lower blood glucose concentrations than wild type controls (166 ± 26.5 vs. 242 ± 41.8 mg/dL; p value < 0.01) and had a much greater hypoglycemic response to an injected bolus of insulin. During the insulin tolerance test (ITT), mice with Myd88 -/- hematopoietic cells had significantly lower blood glucose concentrations at every time point and the percentage decrease in blood glucose concentration was significantly greater at every time point (Figures 3A-3D).

[0056] After an overnight fast, mice with Myd88 -/- hematopoietic cells had significantly lower blood glucose levels (100.4 ± 20.5 vs. 127 ± 23; p value < 0.05) and blood insulin levels that were half that of mice with Myd88 +/+ hematopoietic cells (0.75 ± 0.31 vs. 1.6 ± 0.65; p value < 0.05). Using HOMA-IR values, mice with Myd88 +/+ hematopoietic cells were estimated to be 2.5 times more insulin resistant than mice with Myd88 -/- hematopoietic cells (4.9 ± 3.0 vs. 12.9 ± 6.3; p value < 0.05). Moreover, after injection of a glucose bolus, mice with Myd88 -/- hematopoietic cells cleared the excess glucose faster than mice with Myd88 +/+ hematopoietic cells (Figures 3A-3D). These data suggest that Myd88 deficiency in hematopoietic cells prevents the development of insulin resistance during obesity and thereby prevents hyperglycemia and improves glucose clearance capacity.

Example 3: Hepatomegaly and Hepatic Steatosis Reduced With Inhibition of Myd88

[0057] Enlargement of and increased triglyceride accumulation in the liver is associated with obesity induced hepatic insulin resistance in humans and in mouse models of obesity. To determine whether the improved insulin sensitivity observed in obese mice with Myd88 -/- hematopoietic cells compared to obese mice with Myd88 +/+ hematopoietic cells was associated with decreased hepatomegaly and steatosis liver weight and liver triglyceride content were measured in the two groups of mice.

[0058] Livers from obese mice with Myd88 -/- hematopoietic cells weighed 35% less than those from obese mice with Myd88 -/- hematopoietic cells (1.80 ± 0.534 vs. 2.86 ± 0.434; p value < 0.01), and this difference remained after the liver weights were normalized to body mass (0.0456 ± 0.00880 vs. 0.0686 ± 0.00727; p value < 0.01). Accordingly, the triglyceride content of liver from obese mice with Myd88 -/- hematopoietic cells was 35% less than those from obese mice with Myd88 -/- hematopoietic cells (52.4 ± 17.9 vs. 80.4 ± 27.3; p value < 0.05) (Figures 4A-4D).

[0059] The induction of supressors of cytokine signaling 1 and 3 (SOCSl and SOCS3) plays a role in the pathogenesis of obesity induced hepatic steatosis and insulin resistance. IL-6 is a potent stimulator of SOCSl and SOCS3 expression. Because of the observation that Myd88 in hematopoietic cells is important for stimulating IL-6 production during obesity, Myd88 might thereby regulate hepatic SOCSl and/or SOCS3 expression. Therefore, quantitative RT-PCR was used to measure hepatic expression of SOCSl and SOCS3 in the two groups of mice.

[0060] Expression of SOCS3 was highly variable and no significant differences were observed between the two groups of mice. However, hepatic expression of SOCSl was significantly lower in obese mice with Myd88 -/- hematopoietic cells compared to controls (93.3 ± 27.5 vs. 206 ± 89.1; p value < 0.05) and SOCSl expression level was an excellent predictor of HOMA-IR values in the two groups of mice (r = 0.87; p value < 0.01) (Figures 4A-4D).

[0061] Myd88 in hematopoietic cells plays an important role in the development of several inflammatory responses during diet-induced obesity in mice. Mice that had been transplanted with Myd88 deficient bone marrow cells prior to being placed on a high fat diet became equally obese as controls that had been transplanted with wild type bone marrow cells. But they had lower circulating levels of MCP-I, IL-6, and PAI-I. Consistent with having lower circulating levels of MCP-I, mice with Myd88 -/- hematopoietic cells had a lower fraction of circulating monocytes compared to controls. And consistent with having lower circulating levels of IL-6, mice with Myd88 -/- hematopoietic cells had lower SOCSl expression in liver.

[0062] The data presented here provide direct evidence that Myd88-dependent signaling pathways in hematopoietic cells play a role in causing obesity induced insulin resistance. Compared to controls, obese mice with Myd88 -/- hematopoietic cells had decreased fasting blood glucose levels, increased insulin sensitivity as assessed by HOMA-IR modeling and an ITT, improved ability to clear a glucose load during a GTT as well as decreased hepatic steatosis, hepatomegaly and hepatic SOCSl expression. The most parsimonious explanation for these findings is that inflammatory molecules produced downstream of the Myd88 dependent signaling pathways activated by obesity cause insulin resistance.

[0063] It is unclear how obesity might lead to activation of the innate immune response in a Myd88 dependent manner. Some endogenous molecules including saturated fatty acids and the heat shock protein gp96 can induce inflammatory responses in hematopoietic cells in a TLR and Myd88 dependent manner. Levels of circulating free fatty acids are increased during diet-induced obesity. The endoplasmic reticulum stress response is stimulated during obesity and plays an important role in the pathogenesis of insulin resistance. Without being bound by theory, obesity may, therefore, lead to inflammation by causing increased levels of endogenous pattern recognition receptor ligands to be exposed to their cognate pattern recognition receptors. [0064] While the foregoing invention has been described in some detail for purposes of clarity and understanding, these particular embodiments are to be considered as illustrative and not restrictive. It will be appreciated by one skilled in the art from a reading of this disclosure that various changes in form and detail can be made without departing from the true scope of the invention.

Claims

WHAT IS CLAIMED IS:

1. A method for treating an obesity-associated disorder in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88.

2. A method for preventing an obesity-associated disorder in a human subject, the method comprising administering to the subject an effective amount of an inhibitor of human Myd88.

3. A method for treating insulin resistance in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88.

4. A method for treating diabetes in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88.

5. The method of any of claims 1 to 4, or 22, wherein the inhibitor comprises an antagonist of Myd88, a Myd88-specific antibody, an antisense oligonucleotide that specifically binds to a region of a nucleic acid that encodes Myd88, an organic molecule, a peptide, or a peptidomimetic.

6. The method of any of claims 1 to 4 or 22, wherein the inhibitor comprises a small interfering RNA (siRNA) that specifically binds to a region of a nucleic acid that encodes Myd88, wherein expression of Myd88 is inhibited.

7. The method of any of claims 1 to 4 or 22, wherein the inhibitor comprises a peptide that is a fragment of Myd88, a fragment of a Toll-like receptor, or a fragment of an interleukin 1 receptor.

8. The method of any of claims 1 to 4 or 22, wherein the inhibitor comprises a peptidomimetic of a peptide fragment of Myd88, a peptide fragment of a Toll-like receptor, or a peptide fragment of an interleukin 1 receptor.

9. The method of any of claims 1 to 4 or 22, wherein the inhibitor comprises a peptide fragment, which is of a Toll/interleukin 1 receptor (TIR) domain.

10. The method of any one of claims 1 to 4, wherein the obesity-associated disorder comprises diabetes, insulin resistance, hyperinsulinemia, decreased glucose clearance, dyslipidemia, non-alcoholic fatty liver disease, hypertension, inflammation, hepatomegaly, hepatic steatosis, myocardial infarction, asthma, stroke, or any combination thereof.

11. The method of any one of claims 1 to 4, wherein the administering comprises contacting hematopoietically-derived cells or myeloid cells with the inhibitor.

12. The method of any one of claims 1 to 4, wherein the administering comprises contacting bone marrow of the subject with the inhibitor.

13. The method of any one of claims 1 to 4, wherein the administering comprises ex vivo treatment of cells or tissue taken from the subject with the inhibitor, and returning the cells or tissues to the subject.

14. The method of any one of claims 1 to 4, wherein the administering comprises intralesional, intraperitoneal, intramuscular or intravenous injection; infusion; liposome- mediated delivery; or topical, nasal, oral, ocular, otic delivery, or any combination thereof.

15. The method of any one of claims 1 to 4, wherein the subject is a human, mouse, rabbit, monkey, rat, bovine, pig or dog.

16. A method for treating an obesity-associated disorder in a subject, the method comprising transplanting Myd88-defϊcient bone marrow into the subject.

17. A method for reducing or preventing obesity in a subject, the method comprising administering to the subject an effective amount of an inhibitor of Myd88.

18. A method for lowering body weight in a mammal comprising administering to the mammal in need of losing weight an inhibitor of Myd88 in an effective amount to reduce the weight of the subject, thereby lowering the body weight.

19. The method of claim 17 or 18, wherein the administration is systemic administration.

20. The method of claim 17 or 18, wherein the subject is suffering from diabetes.

21. The method of claim 18, wherein the mammal is a dog, a mouse, a human, a horse, a monkey, a primate, or a feline.

22. A method for identifying whether a test compound is capable of inhibiting Myd88 activity, the method comprising: (a) contacting a test compound with a solution containing (i) Myd88, and (ii) either a Toll-like receptor, an interleukin 1 receptor, or both,

(b) measuring Myd88 activity in the solution of step (a), and

(c) comparing the Myd88 activity in step (b) with Myd88 activity in the absence of the test compound, so as to determine whether or not the test compound is capable of inhibiting Myd88 activity.

23. A method for identifying a compound capable of inhibiting Myd88, the method comprising:

(a) administering a test compound to a subject, and

(b) measuring inhibition of Myd88 activity in the subject as compared to another subject that was not administered the test compound, so as to determine whether or not the test compound inhibits Myd88 activity.

24. A method for identifying a compound capable of inhibiting Myd88 activity, the method comprising:

(a) contacting a test compound with a cell,

(b) contacting the cell with a toll-like receptor agonist or interleukin 1 receptor agonist,

(c) measuring Myd88 activity in the cell, and

(d) comparing the activity measured in (c) with Myd88 activity in a cell in the absence of the test compound, so as to determine whether the test compound is capable of inhibiting Myd88.

25. The method of any of claims 22, 23 or 24, wherein the method is carried out in a high- throughput way.

26. The method of any of claims 22, 23 or 24, wherein the activity measured comprises Myd88 gene expression, Myd88 protein production, Myd88 translocation, Myd88 protein function, Myd88 binding activity, cytokine production, immune response, blood glucose concentration, insulin resistance, insulin concentration, NF-kB activity, SOCSl expression, MCP-I production, PAI-I production, IL-6 production, blood monocyte fraction, or any combination thereof.

27. The method of any of claims 22, 23 or 24, wherein Myd88 activity is detected as a complex formed with Myd88 and either Toll-like receptor, interleukin 1 receptor, or both.

28. The method of claim 22, wherein the solution comprises an extract from a cell contacted with a Toll-like receptor agonist or an interleukin 1 receptor agonist.

29. The method of claim 23, wherein the inhibition of Myd88 activity is measured by detecting decreased levels of an inflammatory marker in blood of the subject.

30. The method of claim 29, wherein the inflammatory marker comprises MCP-I, IL-6, PAI-I, monocytes, C-reactive protein or any combination thereof.

31. The method of any of claims 22, 23 or 24, wherein the inhibition of Myd88 activity determined by detecting increased insulin sensitivity, decreased circulating glucose levels, decreased hepatomegaly, decreased hepatic steatosis, decreased hepatic suppressor of cytokine signaling (SOCSl) expression, or any combination thereof.

32. The method of claim 31 , wherein glucose levels are measured by a glucose tolerance test (GTT) or measuring fasting glucose levels, or a combination thereof.

33. The method of claim 31 , wherein insulin sensitivity is measured by an insulin tolerance test (ITT) or a homeostatic model assessment of insulin resistance (HOMA-IR) or an euglycemic hyperinsulinemic clamp, or any combination thereof.

34. The method of claim 24, wherein the cell is a hematopoietic cell, a myeloid cell, a macrophage, or any combination thereof.

35. The method of claim 24, wherein the agonist comprises lipopolysaccharide, interleukin 1, interferon gamma, saturated fatty acids, dsRNA, bacterial cellular components, viral protein, carbohydrate, nucleic acid components, or any combination thereof.

36. The method of claim 22, 23 or 24, wherein the inhibition of Myd88 activity is measured as decreased NFKB activity, IRAK activation, IRAK recruitment, decreased production of cytokines, or any combination thereof.

37. The method of claim 36, wherein the cytokine comprises MCP-I, IL-6, PAI-I, TNF- a, MCP-3, adiponection, resistin, or any combination thereof.