WO2014014966A1 - Screening methods for therapeutic anti-inflammatory agents - Google Patents

Description

SCREENING METHODS FOR THERAPEUTIC

ANTI-INFLAMMATORY AGENTS

CROSS-REFERENCE TO RELATED APPLICATIONS

[1 ] This non-provisional U.S. patent application claims priority under 35 U.S.C. § 1 19(e) to pending U.S. provisional application serial No. 61/672,162, filed July 16, 2013, which is incorporated herein by reference in its entirety.

FI ELD OF THE INVENTION

[2] The invention relates to methods to identify new drugs or low toxicity therapeutic agents that can be used to modulate inflammation and thus treat, ameliorate, prevent or slow the progression of diseases, conditions or disorders whose etiologies involve chronic non-productive inflammation. Those inflammation-based diseases, conditions and disorders include metabolic disorders, e.g., type 2 diabetes and hyperglycemia, lung inflammation conditions, e.g., cystic fibrosis and chronic obstructive pulmonary disease (COPD), autoimmune disorders, e.g., multiple sclerosis, rheumatoid arthritis and lupus erythematosis, inflammatory bowel conditions, e.g., Crohn's disease and ulcerative colitis, which are also autoimmune disorders, neuroinflammatory diseases, e.g., Alzheimer's disease, Parkinson's disease, epilepsy and chronic pain, and vascular diseases such as atherosclerosis, which is often associated with metabolic disorders. Methods to identity inflammation modulators include measuring biological effects of the therapeutic agents on specific effector biomolecules as described herein.

BACKGROUND

[3] The function and mechanism of many effector biomolecules involved in regulating and/or effecting cellular activities can be complex. This stems in part from the capacity of a given biomolecule to exert one set of activities in one biological context, e.g., when pathological inflammation or glucose dysregulation is present, but then sometimes have a different set of activities in another context, e.g., in clinically normal situations. In addition to those considerations, the biological effects of a given biomolecule on a cell can be direct or indirect and influenced by multi-protein complexes with different protein or other components that are themselves affected in differing ways depending on the physiological context.

[4] Biological signaling pathways can involve multiple effector biomolecules that transmit extracellular signals from the cell membrane to the cytoplasm and often also to the nucleus or they can elicit autocrine, paracrine or endocrine effects. Effector biomolecules that are involved include typically elicit genomic effects as seen by changed target gene expression levels and non-genomic effects as seen by modifications such as phosphorylation or sequestration of biomolecules in the signaling pathway. Some effector biomolecules participate in more than one signaling pathway.

[5] Efficient signal transduction through inflammatory promoting pathways typically requires assembly of the participating biomolecules into protein complexes. Those complexes may be pre-assembled with the effector biomolecule prior to input of the inflammatory promoting stimulus that activates the effector biomolecule or are transiently formed to include the effector biomolecule upon that stimulus through the intermediacy of scaffolding and/or adaptor proteins. Disruption of an activated protein complex that contains the effector molecule by a therapeutic agent or a candidate compound found by the methods of the invention provides for a context-dependent effect that preferentially disrupts signal transduction only when the inflammatory promoting stimulus is present. Therefore, in the absence of a pro-inflammatory cytokine such as TNFa, IL6 or IL1 β or a Toll-like receptor ligand such as LPS, basal cell signaling from non-inflammatory protein complexes, which may involve those same participating proteins, is expected to remain unaffected. Therefore, in contrast to "always-on" therapies where an activity of a target effector molecule is continuously inhibited by direct interaction with a therapeutic agent, a context-dependent therapeutic agent or candidate compound found from the methods of the invention is expected to elicit fewer adverse events.

[6] Due to the nature of the protein complexes and their assembly and/or activation resulting from an inflammatory promoting stimulus, disruption of unwanted signal through such complexes is not dependent on a direct interaction of a therapeutic agent or candidate compound found from the methods of the invention with the effector

biomolecule responsible for transmission or propagation of the pro-inflammatory response. Rather it is the protein complex containing the effector biomolecule that is the target. In contrast to the aforementioned "always-on" therapeutic agents, the presence of the molecular targets of the therapeutic agents found from the methods of the invention are conditional on the inflammatory signaling to be inhibited. Therefore, detection of direct interaction of those therapeutic agents with an effector biomolecule may not be observed in isolation, i.e., in absence of other participants in the signaling protein complex.

Furthermore, due to the conditional composition of the target and the inability to detect direct interaction with an effector biomolecule in absence of the other participants in the signaling protein complex, by the therapeutic agent or candidate compound found by the methods of the invention, modulation of signal transduction mediated by the effector molecule is expected. That translates to a dose response curve for the therapeutic agent or candidate compound that when tested in a suitable test system is expected not to achieve the same maximal effect (i.e., efficacy) as a direct inhibitor of the effector protein. Such a therapeutic agent or candidate compound thus behaves as a partial antagonist and is expected to inherently have a greater therapeutic index than a direct inhibitor that behaves as a full antagonist, since residual signaling through the effector protein will remain irrespective of its administered dose mass.

[7] The mitogen-activated protein kinases (MAPK) are important effector molecules in propagating pro-inflammatory signaling initiated at cell surface receptors. Those kinases include the extracellular signal-regulated kinase (ERK) isoforms Erk-1 (also referred to as Mapk-3 or p44 kinase) and Erk-2 (also referred to as Mapk-1 or p42 kinase), c-Jun amino- terminal kinase (JNK) isoforms Jnk-1 , Jnk-2 and Jnk-3 and the p38 isoforms, ρ38α, ρ38β, ρ38γ and ρ38δ. The MAPKs respond to a variety of stimuli with Erk-1 and Erk-2 preferentially responding to growth factors and phorbol esters while JNK and p38 respond to cytokines and other stress stimuli, e.g., ionizing radiation and osmotic shock.

[8] Although the MAPKs respond to a wide array of stimuli and are involved in a wide range of functions, including phosphorylation of phospho-lipids, transcription factors, cytoskeletal protein and other protein kinase termed MAPK-activated protein kinases (MKs or MAPKAPKs), MAPKs contain similar structural binding domains. These domains include the ATP binding site, a catalytic active site that transfers a phospho-group from bound ATP to a specific serine or threonine of a MAPK substrate, and protein-protein interaction domains. Due to the similarity in MAPKs structures, selective inhibitors of these kinases are lacking. Inhibitors of MAPKs that have been studied typically target the ATP-binding site (i.e., ATP binding site-dependent inhibitors). Compounds that exert their activity through MAPK signaling through the ATP binding site are generally considered to be toxic, particularly to the liver in view of human toxicity that is observed for p38 MAPK inhibitors (Morel, C. et al. J. Biol. Chem. (2005) 280: 21384-21393; Laufer, S.A. et al. C em. Med. Chem. (2006) 1 : 197-207; Kumar, S. et al. Nat. Rev. Drug Discov. (2003) 2: 717-726. That toxicity is the direct result of an "always on" (i.e., context-independent complete) therapeutic agent that can achieve complete inhibition of the target molecule, which the compounds found by the present invention avoids.

[9] The MAPKs are regulated in part through phosphorylation cascades. Activation of MAPK requires the phosphorylation of conserved tyrosine and serine (or threonine) residues in its activation loop by an upstream protein kinase referred to as a MAPKK or MEK. Various isoforms of those upstream kinase exhibits differing levels of selectivity for their MAPK substrates. The MAPKK in turn are regulated by phosphorylation by upstream kinases referred to as MAPKKK, MEKK or MAP3K, which in turn can be activated through interaction with a protein that becomes activated by a pro-inflammatory ligand as a consequence of its interaction with its cognate membrane bound receptor. Those membrane receptors typically include receptor tyrosine kinases (RTKs). MAPK activation can also result from GPCR receptor activation though cross-talk with G protein- dependent and G protein-independent signaling.

[10] Another level of regulation of MAPK signaling is provided by scaffold proteins that pre-assemble some of the components of the protein kinase cascade into sub-cellular compartments in order that the incoming signal into the cascade is properly directed to the appropriate downstream effector proteins or integrated with signaling from other signal transduction pathways. The scaffolding protein may in turn be regulated by proteins that affect its phosphorylation state. Additional regulation is provided by phosphatases that remove activating or regulating phosphate groups from their phospho-protein substrate, thus terminating or modulating signaling through a MAPK signal node, with each phosphatase displaying various levels of selectivity for its phospho-protein substrate. These phosphatases are also regulated by their own phosphorylation states and interactions with scaffolding proteins. Therefore, a tightly regulated network of proteins is required to properly respond to the signaling input and output through each MAPK so that signaling coming into this network results in the appropriate outcome.

[11 ] The phosphorylation cascades in MAPK signaling involve multiple protein kinases that amplifies the initial signal entering into each cascade and appears to be a common feature to the diversity of MAPKs signaling effects. Those common cascades allows for multiple unique points of regulation and integration of signaling events originating at the cell membrane or within the cytoplasm that flow through each MAPK signaling node to their diverse array of downstream effectors. The resulting signaling cross-talk sometimes become aberrant resulting in excessive signaling through one or more of those nodes which may be responsible for initiating or propagating conditions resulting from unwanted inflammation. Therefore a compound, which is found by the methods of the invention, that disrupts aberrant signal transduction to an inappropriately assembled signaling node not normally present under basal conditions of the cell would provide conditional inhibition of pro-inflammatory signaling through that node.

[12] Due to the similarity in MAPKs structures, selective inhibitors of these kinases are lacking. Inhibitors of MAPKs that have been studied typically target the ATP-binding site (i.e., ATP binding site-dependent inhibitors). Compounds that exert their activity through MAPK signaling through the ATP binding site are generally considered to be toxic, particularly to the liver in view of human toxicity that is observed for p38 MAPK inhibitors (Morel, C. et al. J. Biol. Chem. (2005) 280: 21384-21393; Laufer, S.A. et al. Chem. Med. Chem. (2006) 1 : 197-207; Kumar, S. et al. Nat. Rev. Drug Discov. (2003) 2: 717-726. That toxicity is the direct result of an "always on" (i.e., context-independent complete) therapeutic agent that can achieve complete inhibition of the target molecule, which the compounds found by the present invention avoids.

[13] Another important effector molecule in propagating pro-inflammatory signaling initiated at cell surface receptors is NF-κΒ. Lrp1 is a regulator of the tumor necrosis factor receptor-1 (TNFR1 ) and the I KK-NFKB pathway. Lrp1 deficient macrophages have decreased NF-κΒ signaling and MCP-1 expression, due to down-regulation of TNFR1 and inhibition of autocrine TNFR1 -initiated cell signaling (Gaultier, A. et al., Blood (2008) 1 1 1 (1 1 ):5316-5325). Lrp1 binds Apolipoprotein E (ApoE) (Croy, J.E. et al., Biochemistry (2004) 43(23)7328-7335), and ApoE induces the anti-inflammatory M2 phenotype in macrophages (Baitsch, D. et al., Arterioscler. Thromb. Vase. Biol., 31 (5) 1 160-1 168). The intracellular domain of Lrp1 has also been shown to interact with scaffolding and signaling proteins in a phosphorylation-dependent manner (Boucher, P. et al., Biochem. Pharmacol. (2010) 81 (1 ):1 -5).

[14] T2DM is primarily characterized by the condition of insulin resistance, a disorder in which peripheral target cells and tissues do not respond to insulin properly.

However, it also involves abnormalities in the secretion of central hormones that regulate glucose metabolism in the body, namely insulin and glucagon, which are produced in pancreatic islets by β and a cells, respectively. It is thought that as the disease progresses towards insulin resistance with the attendant increased demand for insulin secretion, the extent of β-cell compensation becomes insufficient and can no longer maintain normoglycemia This β-cell impairment is a central element in the natural history of the disease as it results in a state of relative insulin deficiency, thereby marking the transition to overt diabetes.

[15] Metabolic disorders related to diabetes and hyperglycemia conditions can include abnormalities such as hyperinsulemia, obesity or elevated levels of

triglycerides, uric acid, fibrinogen, small dense LDL particles and plasminogen

activator inhibitor 1 (PAI-1 ), and decreased levels of HDL-c. Many patients who have insulin resistance but have not yet developed type 2 diabetes are also at a risk of developing metabolic syndrome, also referred to as syndrome X, insulin resistance syndrome or plurimetabolic syndrome. Coincident with sustained insulin resistance is the more easily determined hyperinsulinemia, which can be measured by accurate determination of circulating plasma insulin concentration in the plasma of subjects.

Hyperinsulinemia can be present as a result of insulin resistance, such as is in obese and/or diabetic (NIDDM) subjects and/or glucose intolerant subjects, or in IDDM subjects, as a consequence of over injection of insulin compared with normal

physiological release of the hormone by the endocrine pancreas. [16] The primary goal of treating diabetes is to ameliorate, prevent or slow the progression of the development of diabetic complications. Diabetes is treated with a variety of therapeutic agents including insulin sensitizers, such as PPAR-γ agonists, such as glitazones; biguanides; protein tyrosine phosphatase- 1 B inhibitors; dipeptidyl peptidase IV inhibitors; insulin ; insulin mimetics; sulfonylureas; meglitinides; oc-glucoside hydrolase inhibitors; and oc-amylase inhibitor. Increasing the plasma level of insulin by administration of sulfonylureas (e.g. tolbutamide and glipizide) or meglitinides, which stimulate the pancreatic β-cells to secrete more insulin, and/or by injection of insulin when sulfonylureas or meglitinides become ineffective, can result in insulin concentrations that are high enough to stimulate insulin-resistant tissues. However, dangerously low levels of plasma glucose can result, and increasing insulin resistance due to the even higher plasma insulin levels can occur. The biguanides increase insulin sensitivity resulting in some correction of hyperglycemia. Metformin monotherapy is often used for treating type 2 diabetic patients who are also obese and/or dyslipidemic. Lack of an appropriate response to metformin is often followed by treatment with sulfonylureas,

thiazolidinediones, insulin, or oc-glucosidase inhibitors. However, the two biguanides, phenformin and metformin, can also induce lactic acidosis and nausea/diarrhea, respectively. Alpha glucosidase inhibitors, such as acarbose, work by delaying absorption of glucose in the intestine. Alpha-amylase inhibitors inhibit the enzymatic degradation of starch or glycogen into maltose, which also reduces the amounts of bioavailable sugars.

[17] The glitazones, also known as thiazolidinediones (i.e. 5-benzylthiazolidine-2,4- diones), are a class of compounds that can ameliorate many symptoms of type 2 diabetes. These agents substantially increase insulin sensitivity in muscle, liver and adipose tissue in several animal models of type 2 diabetes resulting in partial or complete correction of the elevated plasma levels of glucose without occurrence of hypoglycemia. The glitazones that are currently marketed are agonists of the

peroxisome proliferator activated receptor (PPAR) gamma subtype. PPAR-y agonism is generally believed to be responsible for the improved insulin sensitization that is observed with the glitazones. Other PPAR agonists that are being developed for treatment of type 2 diabetes and/or dyslipidemia are agonists of one or more of the

PPAR-oc, PPAR-y and PPAR-δ subtypes.

[18] Treatment of diabetes with PPAR-γ agonists has been associated with cardiac hypertrophy, or an increase in heart weight. Recent labeling revisions for Avandia™ (rosiglitazone maleate), a PPAR-γ agonist, indicate that patients may experience fluid accumulation and volume-related events such as edema and congestive heart failure. Cardiac hypertrophy related to PPAR-γ agonist treatment is typically treated by discontinuing the treatment. Therefore, there is a need for insulin sensitizers that do not promote further insulin resistance with chronic use or have the cardiovascular side effects that further aggravate the cardiovascular risks of the disease that such compounds are intended to treat.

[19] Complex signaling is also involved in the regulation of insulin signaling, glucose metabolism and glucose homeostasis and there is growing evidence pointing to a close relationship between inflammation and insulin resistance. Effector biomolecules participating in those pathways include insulin, insulin receptor, which is localized in the cell membrane, the protein kinase Akt2, phosphoinositol 3 kinase (PI3K), insulin receptor substrate 1 (IRS1 ) and 3-phosphoinositide dependent protein kinase-1 (PDPK1 ). Another effector biomolecule that appears to be involved is Lrp1 (Ceschin, D.G. et al., J. Cellular Biochem. (2009) 106(3): 372-380). Lrp1 is regulated by insulin signaling and affects lipid clearance and glucose tolerance. Lrp1 signaling also results in anti-inflammatory effects. Prior studies had shown that mice having an adipocyte-specific inactivation of Lrp1 displayed delayed postprandial lipid clearance, reduced body weight, smaller fat stores, improved glucose tolerance, and resistance to dietary fat-induced obesity and glucose intolerance. Inactivation of the Lrp1 intracellular NpxYxxL motif enhances post prandial dyslipidemia and atherosclerosis, and increases proapoptotic effects via increased secretion of TNFoc in macrophages (Gordts, P.L. et al., Arterioscler. Thromb. Vase. Biol. (2009) 29(9):1258-1264).

[20] Mice deficient in a natural Erk inhibitor, p62, have a high level of Erk activity and develop mature-onset obesity and insulin resistance (A. Rodriguez, A. et al., Cell Metab. (2006) 3(3): 21 1 -222). Activation of TLR2 and TLR4 with fatty acids (Chung, S. et al. J. Biol. Chem. (2005) 280(46): 38445-38456; Kuo, L.H. et al., Diabetologia (201 1 ) 54(1 ):168- 179) contributes to NF-κΒ activation, increased inflammation, and insulin resistance. Tpl2 kinase is up-regulated in adipose tissue in obese mice and human subjects and is reported to mediate NF-κΒ and TNFoc effects upon Erk activation (Jager, J. et al.,

Diabetes (2010) 59(1 ): 61 -70). Inhibitors of ΙΚΚβ or Tpl2 inhibited TNFoc but not insulin- mediated Erk1 and Erk2 activation and also eliminated IRS1 Ser636 phosphorylation stimulated by TNFoc in 3T3-L1 adipocytes. Therefore a compound that interferes with the pro-inflammatory signaling that underlies the diabetic state through modulation of MAPK and NK-KB activities is expected to be an effective treatment for Type 2 diabetes and related conditions. Furthermore, such compounds, which are found by the methods of the invention, are expected not to have the cardiovascular deficits of glitazone therapy, but are instead expected to provide cardiovascular benefits by virtue of their anti-inflammatory properties. [21 ] Multi-component signaling pathways are also involved in control of cell cycling. Sirt2 is a deacetylase that plays a role in cell cycle regulation (Dryden, S.C. et al., Mol. Cell. Biol. (2003) 23(9): 3173-3185) and can also affect a range of other phenomena such as glucose homeostasis and neurodegeneration associated with ageing (Garske, A.L. et al., ACS Chem. Biol. (2007) 2(8): 529-532, 2007; R. Luthi-Carter, R. et al., Proc. Natl Acad. Sci. (USA) (2010) 107(17): 7927-7932). Enhanced expression or activity of Sirt2 appears to decrease the resistance of some cells to stress associated with reoxygenation after anoxia (Lynn, E.G. et al., FEBS Lett. (2008) 582: 2857-2862). Sirt2 can thus exert a range of effects depending on biological context.

[22] The hydroxysteroid dehydrogenase Hsd17b4 is a bifunctional enzyme that catalyzes oxidation of C-17 hydroxyl of C-19 sterols and is involved in β-oxidation of fatty acids. This enzyme modulates the action of 17-hydroxysterols by oxidizing them to 17-oxo derivatives. Inhibition of Hsd17b4 tends to exert an anti-inflammatory effect in vivo.

SUMMARY OF THE INVENTION [23] One embodiment of the invention provides a method to identify a drug candidate, the method comprising (a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins; (b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of Mapk1/3, Jnk1/2, p38 Mapk, p65 NF-κΒ, ΙΚΚα/β and IRS1 ; and (c) selecting the test compound of step (b) that decreases phospho-activation or level(s) or biological activit(ies) of Mapk1 /3, JNK1 /2, p38 Mapk, p65 NF-κΒ and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s), wherein the selected test compound is identified as a candidate compound for determining

immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

[24] Another embodiment of the invention provides a method to identify a drug candidate, the method comprising (a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins; (b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of Mapk1 /3, Jnk1/2, p38 Mapk, p65 NF-κΒ, ΙΚΚα/β and IRS1 ; (c) determining immunotoxicity of the test compound in an suitable

immunosuppressive test system; and (d) selecting the test compound of steps (b) and (c) that decreases the phospho-activation or biological activitv(ies) of Mapk1/2, JNK1/2, p38 Mapk, p65 NF-κΒ and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s) and has sufficiently low immunotoxicity for administration to a subject for determining efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system wherein the selected test compound is identified as a candidate compound.

[25] Another embodiment of the invention provides a method to identify a drug candidate, the method comprising (a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins; (b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of Mapk1 /3, Jnk1/2, p38 MapK, p65 NF-κΒ, ΙΚΚα/β and IRS1 ; (c) determining test compound binding to or transactivation by ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems and (d) selecting the test compound of steps (b) and (c) that decreases the phospho-activation or biological activity(ies) of Mapk1/3, Jnk1/2, p38 Mapk, p65 NF-κΒ and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s) and has EC50's > 10,000 nM in the NHR test systems, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

[26] Another embodiment of the invention provides a method to identify a drug candidate, the method comprising (a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins; (b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of Mapk1 /3, Jnk1/2, p38 Mapk, p65 NF-κΒ, ΙΚΚα/β and IRS1 (c) determining test compound binding to or transactivation by ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems (d) determining test compound binding to or transactivation by PPARy in a suitable PPARy test system; and (e) selecting the test compound of steps (b)-(d) that decreases the phospho-activation or biological activity of Mapk1/3, Jnk1/2, p38 Mapk, p65 NF-κΒ or ΙΚΚα/β or decreases the phospho- deactivation or IRS-1 relative to suitable control test system(s), and has EC50's > 10,000 nM in the NHR and PPARy test systems, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable

immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system. [27] Another embodiment of the invention provides a method to identify a drug candidate, the method comprising (a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins; (b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of Mapk1 /3, Jnk1/2, p38 MapK, p65 NF-κΒ and ΙΚΚα/β; (c) determining cell numbers for cells expressing regulatory T cell phenotypes in two suitable Treg test systems, wherein one Treg system comprises T cells of a subject with an autoimmune condition and the other Treg system comprises T cells from a healthy subject; and (d) selecting the test compound of steps (b) and (c) that decreases the phospho-activation or biological activity(ies) of Mapk1/3, JNK1/2, p38 Mapk, p65 NF-KB and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s) and provides increased numbers of converted regulatory T cells in the autoimmune Treg test system and negligible or no increase of converted regulatory T cells in the healthy Treg test system, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable immunosuppressive test system or efficacy to treat unwanted inflammation in a suitable unwanted

inflammation test system. BRI EF DESCRI PTION OF THE DRAWINGS

[28] Figure 1 . Compound A (17a-ethynyl-androst-5-ene-33, 7β, 17β-ΐποΙ or HE3286) treatment effect on MPTP-induced motor impairment as measured by the ability of mice to maintain balance on a rotating cylinder using the Rotarod test.

[29] Figure 2. Compound A treatment effects on MPTP-induced expression of the pro- inflammatory mediators iNOS, TNF-a and IL-1 β.

[30] Figure 3. Compound A treatment effects on histological counts of MPTP-induced damage of substantia nigra pars compacta (SNpc) dopaminergic neurons.

[31 ] Figure 4. Compound A treatment effects on MPTP induced TH-positive neuronal loss in the SNpc.

[32] Figure 5. Compound A treatment effects on transactivation of PPARa, PPARy, and PPAR5.

[33] Figure 6. Compound A treatment effects on insulin-resistant ob/ob mice.

[34] Figure 7. Compound A treatment effects on MCP-1 and CCR2 mRNA levels.

[35] Figure 8. Compound A treatment effects on NF-κΒ signaling in LPS-stimulated RAW 264.7 macrophages. [36] Figure 9. Compound A treatment effects on NF-kB activation in LPS-stimulated RAW 264.7 macrophages.

[37] Figure 10. Compound A treatment effect on IRS-1 tyrosine phosphorylation in insulin-stimulated H41 1 E hepatocytes and C2C12 differentiated myotubes and on TNFa- stimulated 3T3-L1 adipocytes.

[38] Figure 1 1 . Compound A treatment effect on progression to hyperglycemia in pre- diabetic db/db mice.

[39] Figure 12. Oral glucose tolerance test profile of Compound A in insulin-resistant db/db mice.

[40] Figure 13. Oral glucose tolerance test profile of Compound A in diet-induced diabetic mice.

[41 ] Figure 14. Compound A treatment effect on MAPK and NF-kB activity in LPS- stimulated murine intraperitoneal macrophages.

[42] Figure 15. Compound A treatment effect on MAPK and NF-kB activity in TNFa- stimulated murine intraperitoneal macrophages.

[43] Figure 16. Compound A treatment effect on mRNA profile in adipose tissue derived from Zucker rat model of diabetes.

[44] Figure 17. Compound A treatment effect on macrophage chemotaxis induced by conditioned media derived from 3T3-L1 adipocytes.

[45] Figure 18. Compound A treatment effects on glucose utilization in Zucker rat model of diabetes.

[46] Figure 19. Compound A treatment effects in hepatic gluconeogenesis in Zucker rat model of diabetes.

[47] Figure 20. Compound A treatment effects on hyperinsulinemic euglycemic clamp studies in Zucker rat model of diabetes.

[48] Figure 21 . Compound A treatment effects on serum lipid levels and related gene expression in liver of Zucker rats.

[49] Figure 22. Compound A treatment effects on SREBP-2 protein levels in Zucker rates

[50] Figure 23. Compound A treatment effect on lipid metabolism in liver, epididymal white adipose tissue and skeletal muscle.

[51 ] Figure 24. Compound A early treatment effect on clinical arthritic score for adjuvant induced arthritic rats.

[52] Figure 25. Compound A late treatment effect on clinical arthritic score for adjuvant induced arthritic rats. [53] Figure 26. Compound A treatment effect on clinical score for collagen antibody- induced arthritic mice.

[54] Figure 27. Compound A treatment effects on IL-6 and TNFa in an adjuvant- induced arthritis test system.

[55] Figure 28. Compound A treatment effects on neutrophil infiltration in rat inflamed joints as measure by a myeloperoxidase test system.

[56] Figure 29. Compound A treatment effects on experimental autoimmune encephalomyelitis induced in SJL mice.

[57] Figure 30. Compound A treatment effects on protein scores in MRL-lpr/lpr mouse systemic lupus erythematosus.

[58] Figure 31 . Compound A treatment effects on progression of disease incidence in NOD mouse model of Type 1 diabetes.

[59] Figure 32. Compound A treatment effects on lymphocyte cytokine levels in NOD mouse model of Type 1 diabetes.

[60] Figure 33. Compound A treatment effects on regulatory T cell numbers in healthy and inflamed subjects.

[61 ] Figure 34. Compound A treatment effects on regulatory T cell in NOD mouse model of type 1 diabetes.

[62] Figure 35. Repeated study of Compound A treatment effects on regulatory T cell in NOD mouse model of type 1 diabetes.

[63] Figure 36. Compound A treatment effects on lymphocyte numbers and cytokine levels in a popliteal lymph node test system for immune suppression.

[64] Figure 37. Compound A treatment effects on lung inflammation in the

carrageenan-induce pleurisy test system.

[65] Figure 38. Compound A treatment effects on LPS-induce lung injury as measured by myeloperoxidase activity.

DESCRI PTION OF THE INVENTION

[66] Definitions.

[67] "Unwanted inflammation" as used herein and the like means an inflammatory response that is not desirable or is suboptimal for the subject's condition. Such unwanted responses can arise from various clinical conditions or diseases or as a result of treatment of such conditions or diseases. Typically, unwanted inflammation is an initiated inflammatory response that fails to appropriately resolve leading to a chronic low-grade inflammation that sometimes may flare into an acute inflammatory state. That

inflammatory response sometimes result from immune dysregulation due to autoimmunity or dysregulation of glucose utilization to an extent that immune dysregulation occurs. Conditions whose etiology include unwanted inflammation include metabolic disorders such as metabolic syndrome, dyslipidemia and type 2 diabetes, lung inflammation conditions such as cystic fibrosis, COPD, asthma and other fibrotic lung conditions, autoimmune diseases such as ulcerative colitis and Crohn's disease (which are inflammatory bowel diseases), rheumatoid arthritis, systemic lupus erythematosus and Type 1 diabetes (which is another metabolic disorder). Cancers, particularly those of endothelial origin, are also initiated or propagated by chronic, nonproductive inflammation and includes cancers of the prostate and breast.

[68] "Phosphorylation status" or "phosphorylation state" as used herein interchangeably refers to the number or pattern of phosphate groups covalently bound to a phospho- protein, such as a phosphorylated protein kinase, which may be membrane bound or in a protein complex. In some embodiments phosphorylation status refers to the overall extent of phosphorylation of a collection of proteins for a specified protein kinase or to the extent to which specified amino acid residue(s) of a specified protein kinase in collection of such proteins that are capable of being phosphorylated in a suitable test system are actually phosphorylated.

[69] "Modulation" of an activity or physical state of a protein as used herein means increasing or decreasing an activity of that protein or a property of the protein's physical state resulting from contacting a test or candidate compound to a suitable test system. The modulation may be relative to another activity or property of a different protein, to the same protein in the basal state or subsequent to external stimulation, including contacting a pro-inflammatory cytokine or Toll-like receptor agonist to the test system prior to contacting of the test compound, or relative to the change in activity or property from contacting the test system with vehicle or reference compound.

[70] With respect to a defined kinase, either intracellular or membrane bound, modulation of an activity includes, for example, increasing or decreasing the capacity of the kinase in a suitable test system to phosphorylate one or more of its downstream effector proteins or substrates of that protein kinase, or to increase or decrease signaling through the signal transduction node, cascade or pathway in which the protein kinase participates, upon contacting a test or candidate compound with any suitable test system relative to one or more other kinases or signal transduction nodes, cascades or pathways within the same test system. Modulation of protein activity may be described relative to an isoform of the same protein in the same test system or to the same protein in a control test system to which is contacted the same test or candidate compound or from contact of the same test system with a test compound that is a reference compound (e.g., vehicle or positive or negative control compound). Relative modulation of kinase activity is stated explicitly by describing the comparator protein or by describing the suitable test and control systems, otherwise relative phosphorylation status is implicitly understood by context.

[71 ] With respect to phosphorylation status of a defined protein modulation includes an effected change in phosphorylation state of a specified protein or collection of such proteins in a suitable test system that is capable of being phosphorylated upon contacting a test compound to the test system. Modulation of phosphorylation status or state may mean increasing or decreasing the number of covalently bound phosphate groups in a protein, changing the phosphorylation pattern within a protein, which may or may not be accompanied by an increase or decrease in the number of covalently bound phosphate groups, or increasing the amount of a phosphorylated protein resulting from contacting a test or candidate compound to a suitable test system. Modulation of phosphorylation status or state, may also mean changing the number or pattern of covalently bound phosphate groups in a protein or the amount of a phosphorylated protein in comparison to an effect a test compound has on a reference protein that is present in the same suitable test system resulting from contacting a test compound to the same suitable test system. Modulation of phosphorylation status may be described relative to an isoform of the same protein in the same test system or to the same protein in a control test system to which is contacted the same test or candidate compound or from contact of the same test system with a test compound that is a reference compound (e.g., vehicle or positive or negative control compound). Relative modulation of phosphorylation status or state is stated explicitly by describing the comparator protein or by describing the suitable test and control systems, otherwise relative phosphorylation status is implicitly understood by context.

[72] With respect to a defined transcription factor, modulation of an activity includes, for example, increasing or decreasing the capacity of the transcription factor to transactivate gene(s) whose protein product(s) engage in pro-inflammatory signal transduction.

Modulation of that transactivation activity may result from modulation of one or more protein kinase activities upstream of the transcription factor.

[73] Modulation may also refer to increasing (i.e., positively modulating) or decreasing (i.e., negatively modulating) an activity of a cell within a suitable in vivo test system, including its paracrine or autocrine signaling through release of pro-inflammatory cytokines, such as MCP-1 , TNFa, IL-6, RANTES and IL1 β, release of cytotoxic compounds, such as hydrogen peroxide and other free radical generating precursors, and migration of inflammation-inducing cells into tissue sites. That migratory behavior includes infiltration of leukocytes such as macrophages and neutrophils into white adipose tissue as a result of dysregulated glucose utilization or damaged tissue or as a result of an autoimmune response, respectively, or migration into other sites experiencing unwanted inflammation. In a feed-forward mechanism further leukocyte infiltration may be mediated by cytokine release from aberrantly activated leukocytes already present in the inflamed tissue.

[74] Modulation may also refer to increasing or decreasing an activity of cells within an organ or tissue of a suitable in vivo test system, including decreasing gluconeogenesis in the liver, increasing glucose uptake into skeletal muscle, increasing insulin secretion from pancreatic β-cells, increasing Treg cell numbers or suppressing activity of autoreactive T cells from lymphatic tissue. Modulation may also refer to decreasing serum concentration levels of a pro-inflammatory cytokine, glucose, insulin, free fatty acids, cholesterol or cholesterol esters or decreasing gene expression leading to one or more of those changes in serum concentrations.

[75] Modulation may also refer to correcting immune dysregulation (i.e., imbalanced immune responses to disease conditions) that leads to unwanted inflammation in a vertebrate or mammalian subjects, e.g., as disclosed herein. Candidate compounds selected for further study will do so without suppression of appropriate innate or acquired immune response(s) to pathogens. Thus, a test compound selected as a candidate compound will reduce an immune response when it becomes too active or abnormally persists and thus results in unwanted inflammation without inducing widespread immune suppression.

[76] "Th1 " or "Th2 immune responses" as used herein, refers in general to immune responses as observed in mammals and not as observed in the murine system, from which the Th1 and Th2 terminology originated. Thus, in humans, Th1 cells are CD4+ T lymphocytes and they usually preferentially display chemokine receptors CXCR3 and CCR5, while Th2 cells are CD4+ T lymphocytes and usually preferentially express the CCR4, CCR8 and/or CXCR4 chemokine receptor molecule(s) and generally a smaller amount of CCR3, see, e.g., Syrbe, U. et al., Springer Semin. Immunopathol. (1999) 21 : 263-285, Sebastiani, S. et al., J. Immunol. (2001 ) 166: 996-1002. Tc1 and Tc2 immune responses are mediated by CD8+ lymphocytes and means to identify these cells and their associated lymphokines, cell specific antigens and biological activities have been described, see, e.g., Faries, M.B. et al., Blood (2001 ) 98: 2489-2497, Chan, W.L et al., J. Immunol. (2001 ) 167:1238-1244, Prezzi, C. et al., Eur. J. Immunol. (2001 ) 31 :894-906, Ochi, H. et al., J. Neuroimmunol. (2001 ) 1 19: 297-305, Fowler, D.H. and Gress, R.E.,

Leukemia and Lymphoma (2000) 38:221 -234. When the subject suffers from a excessive or pathological activity associated with macrophages, dendritic cells or neutrophils as in the establishment, maintenance or progression of a disease, symptom or a condition associated with unwanted inflammation, a test compound selected as a candidate compound will generally detectably reduce the level or a biological activity(ies) of one or more effector molecule associated with or needed for an optimal or more normal response or immune function that is mediated by the macrophages, dendritic cells or neutrophils.

[77] "Suitable test system" as used herein means an in vitro system to which can be contacted a test compound in order to elicit effect(s) on one or more signal transduction pathways, nodes, complexes, kinase proteins or cascades. Other suitable test systems are in vivo test systems to which can be contacted a test compound in order to elicit antiinflammatory effect(s) on one or more signal transduction pathways, nodes, complexes, kinase proteins or cascades or biological effect(s) that is(are) predictive for treating a disease or condition associated with unwanted inflammation. Such in vitro and in vivo test systems are capable of responding to a test compound to be selected as a candidate compound, or a candidate compound to be selected as a further characterized candidate compound, that modulates an effect as described herein in a qualitatively or quantitatively similar manner when contacted with 17a-ethynyl-androst-5-ene-33, 7β, 17β-ίποΙ

(Compound A) or another positive control.

[78] "Control test system" as used herein refers to a suitable test system that is to be sham treated with compound, contacted with vehicle or contacted with a reference compound or composition that, depending on context, may serve as a positive or negative control test compound. When cells are used in a suitable test system those cells may comprise an in vitro or an in vivo test system. Typically, the cells of the control test system are genetically the same as the cells comprising the test system to which test or candidate compound is contacted. Control test systems may also be derived from the suitable test system to which a test or candidate compound is to be contacted by genetic alterations to or external stimulus of signal transductions pathways of the cells comprising the suitable test system. In this context the same test compound may be applied to both suitable test systems (i.e., the control test system and the original test system). Cells within a control test system used in screening of test compounds are sometimes referred to as control test cells.

[79] "Test compound" as used herein means a compound, or a composition comprising the compound (e.g. a formulation), to be evaluated in a suitable test system for the presence of one or more of the activities for 17a-ethynyl-androst-5-ene-33, 7β, 17β-ίηΌΙ (Compound A) described herein. Test compounds also include reference compounds whose effect on a suitable test system is known and which is to be compared to an effect (or lack thereof) provided by contact of another test compound to the same test system (i.e., a test or reference compound is contacted with a control test system). The reference compound may be a positive control compound (e.g., Compound A) or a negative control compound.

[80] Test compounds additionally include test compounds shown to have one or more activities qualitatively or quantitatively similar to Compound A, which are required for consideration or selection as a candidate compound, and which may also serve as a positive control compound for that activity. Those test compounds include antiinflammatory glucocorticoids (e.g., dexamethasone) whose anti-inflammatory effects are compared to the test compound. Other test compounds include insulin sensitizers, including compounds commonly referred to as "glitazones" (e.g., rosiglitazone) whose anti-diabetic effects are compared to the test compound. Test compounds also include candidate compounds to be evaluated for identification as a further characterized candidate compound.

[81 ] Typically, test compounds have a molecular weight of 200-1 ,000 amu or 200-800 amu, and are non-peptidic and is initially screened for a biological response by contacting the test compound with a suitable test system in a concentration range of about 100 μΜ to 0.001 μΜ, preferably in a range including, e.g., about 25 μΜ, 10 μΜ or 1 μΜ concentration (final concentration within the test system). Preferred initial suitable test systems are in vitro test systems that screen test compounds for one or more biological responses qualitatively similar to an activity elicited by Compound A when that reference compound is contacted with a control test system. Other suitable test systems for initial screening are in vitro test systems that screen for test compounds that oppose one or more biological responses elicited by a pro-inflammatory cytokine or Toll-like receptor agonist when that reference compound is contacted to those same test systems. Other initial suitable test systems are in vitro test systems that screen for test compounds that are immunosuppressive or bind to nuclear hormone or PPAR receptors for elimination from further consideration as a candidate compound. A test compound for consideration as a candidate compound provides a desired biological response in a suitable in vitro test system within an EC₅o range of 0.1 μΜ or less, more preferably between about 0.1 μΜ to 0.001 μΜ (i.e., 100 nM to 1 nM) or less or about 0.005 μΜ (i.e., 5 nM) or less in a subsequent screen for that biological response. Other test compounds for consideration as candidate compounds provide a desired biological response in a suitable in vivo test system when administered in an amount of less than 200 mg/Kg, more preferably 100 mg/Kg or less. [82] "Candidate compound" as used herein is a test compound that exhibits one or more of the activities of Compound A or opposes one or more activities of a proinflammatory cytokine or Toll-like receptor ligand in in vitro or in vivo model(s) predictive or indicative of efficacy for treating an unwanted inflammation condition described herein in a mammal or a mammal that is expected to have that condition. Typically, candidate compounds exhibits one or more anti-inflammatory effects of an anti-inflammatory glucocorticoid without exhibiting the immunosuppressive effects of that glucocorticoid. Other candidate compounds do not result in transactivation by nuclear hormone receptors in a suitable in vitro test system while exhibiting one or more anti-inflammatory effects of an anti-inflammatory glucocorticoid in a suitable in vitro or in vivo test system. Other candidate compounds do not result in transactivation by nuclear hormone receptors and PPARy in suitable in vitro test systems at concentrations at or below 10 μΜ while exhibiting one or more of the anti-diabetic effects of a glitazone compound such as rosiglitazone in a suitable in vivo test system. Other candidate compounds decrease pro- inflammatory signal transduction mediated through NF-κΒ, preferably with no detectable direct binding interaction with that transcription factor, without exhibiting

immunosuppressive effects associated with an anti-inflammatory glucocorticoid such as dexamethasone within a suitable test system. Other candidate compounds oppose proinflammatory signal transduction mediated by a pro-inflammatory cytokine such as TNFa, IL1 β or a Toll-like receptor ligand such as LPS without exhibiting immunosuppressive effects associated with an anti-inflammatory glucocorticoid within a suitable test system. Still other candidate compounds decrease pro-inflammatory signal transduction mediated through NF-κΒ, preferably with no detectable direct binding interaction with that transcription factor and interact directly with or indirectly through scaffold proteins with one or more of Mapk-1 , Mapk-3 Lrp1 , Rps6ka3, Sirt2 and Hsd17b4 as determined by a suitable SILAC test system. Other candidate compounds are compounds that interact directly or indirectly with Mapk-1 and Mapk-3 without inhibiting at 10 μΜ or less the kinase activity of either isoform as determined in a suitable cell-free test system that contains no scaffold proteins.

[83] "Reference compound" or "control compound" as used herein is test compound that has one or more of the activities as described herein for Compound A for which comparison is to be made in a suitable test system to another test compound to be screened for that activity (i.e., a positive control). Other reference or control compounds lack one or more of these activities for which comparison is to be made in a suitable test system to another test compound to be screened for that activity (i.e., negative control). Reference compounds include Compound A, androst-5-ene-33, 7β, 17β-ίποΙ (βΑΕΤ), 16α- bromoepiandrosterone (BrEA) and anti-inflammatory glucocorticoids. [84] Detailed description. Unless otherwise contraindicated or implied, e.g., by requiring mutually exclusive elements or options, the terms "a" and "an" mean one or more and the term "or" means and/or.

[85] The methods described herein can be performed using based on assays that are conducted in vitro and/or in vivo. Assays performed in vitro will typically use human or mammalian cell lines or cell extracts to determine the effect of test compounds on biomolecule effectors such as Mapk-1 , Mapk-2, Hsd17b4 or Sirt2. Cell assays can utilize CNS, e.g., microglia or neuron, cell lines to assess effects of test compounds for efficacy in treating or slowing the progression of CNS-related disorders, e.g., Gao, H.-M. et al., J. Neurosc. (201 1 ) 31 (3): 1081 -1092; A.K. Cross, A.K. et al., J. Neurosci. Res. (1999) 55(1 ): 17-23; Noda, M. et al., Nature (1985) 318: 73-75; Stanciu, M. et al., J. Biol. Chem. (2000) 275: 12200-12206).

[86] Similarly, vascular cells, such as vascular endothelial cells, are used to assess effects of test compounds for efficacy in treating or slowing the progression of vascular related disorders, e.g., Ades, E.W. et al., J. Invest. Dermatol. (1992) 99: 683-690;

Buonassisi, V. et al., Proc. Nat'l. Acad. Sci. (USA) )1976) 73(5): 1612-1616; Bouis, D. et al., Angiogenesis (2001 ) 4(2):91 -102.

[87] Exemplary cell lines that can be used for assays or cell sources for cell extracts include the DBTRG-05MG (ATCC number CRL-2020), PFSK-1 (CRL-2060) B35 (CRL- 2754), HCN-2 (CRL-10742), CPAE (CCL-209), HUV-EC-C (CRL-1730), primary endothelial cells (PCS-100-010, PCS-100-01 1 or PCS-100-020), 3T3-L1 , Ob1754, Ob1771 , MC3T3-G2/PA6 and PAZ6 cell lines.

[88] In some embodiments, the drug candidate displays its effects in vivo, e.g., in rodents such as mice or rats, canine, porcine or human species, but not in vitro in cells or cell extracts from those species or taxonomic groups of species. In those cases, the biological activity of the drug candidate is masked by the absence of other cell types that contribute to biological effects related to efficacy or toxicity or to endocrine, paracrine or other effects that require multiple cell types or tissue types are not found in in vitro assays using cell lines. In these embodiments, step (a) and or step (b) described in original claim 1 is preferably performed in vivo and optionally in vitro to asses whether or to what extent endocrine, paracrine or other effects may contribute to effects caused by or associated with a drug candidate.

[89] Signal transduction pathway(s) whose modulation(s) by a test compound that characterizes that compound as a drug candidate of the invention and is responsible for one or more of the desired biological effects of Compound A are described below. [90] One intracellular central mediator of insulin action is insulin receptor substrate-1 (IRS-1 ), which is a major enzymatic substrate for the tyrosine kinase activity of the insulin receptor. IRS-1 acts as a scaffolding protein and contains numerous tyrosine residues that undergo insulin receptor catalyzed phosphorylation. The resulting phosphotyrosyl groups in turn become docking sites for effector proteins by virtue of their Src homology 2 (SH2) domains. The resulting protein complexes are then activated to initiate and propagate the intracellular insulin signal that ultimately causes a specific biochemical event. For example, binding of the enzyme phosphatidylinositol-3-kinase (PI3K) to phosphorylated IRS-1 occurs through its SH2-containing regulatory subunit p85, which then recruits its p1 10 catalytic subunit to the complex. Once the p85/p1 10 dimeric PI3K complex is bound to the IRS-1 molecule, it catalyzes the production of the

polyphosphoinositide mediator phosphatidylinositol (3,4,5,)-triphosphate or

Ptdlns(3,4,5)P3, which is a major requirement for the activation of glucose transport, or the serine/threonine kinase Akt in insulin target cells.

[91 ] In contrast, phosphorylation of IRS-1 on serine/threonine residues is associated with the opposite effect, i.e., substantial inhibition of insulin-stimulated phosphorylation of IRS-1 and thus inhibition of its association with PI3K. Numerous enzymes of the serine/threonine kinase family are known to phosphorylate IRS-1 , including mitogen- activated protein kinase (MAPK/ERK), c-Jun NH3-terminal kinase (JNK), atypical protein kinases (PKC-Θ, ΡΚΟζ) or inhibitor of NF-κΒ kinase β (ΙΚΚβ). Several of these enzymes, i.e., JNK, ΙΚΚβ and PKC-Θ are known to be activated by inflammatory stimuli and therefore, may contribute to the inhibition of the insulin signaling pathway. Therefore, there is significant crosstalk between insulin signaling and mitogen-induced inflammation.

[92] For example, in response to a cytokine challenge or fatty acids, JNK becomes activated and associated with IRS-1 , causing direct phosphorylation of Ser307 and impairing insulin action. Furthermore, in animal models of obesity, JNK activity is increased in various tissues such as liver, skeletal muscle and fat and its modulation impacts hepatic glucose metabolism. Conversely, lack of functional JNK results in decreased adiposity, increased insulin sensitivity and enhanced insulin receptor signaling. On the other hand, infusion of lipids leads to elevated levels of intracellular fatty acid metabolites, such as diacylglycerol (DAG) and fatty acyl CoA, which in turn activate PKC9 and increase Ser307 phosphorylation on IRS-1 , again impairing insulin action.

[93] Activation of ΙΚΚβ not only engages the inflammatory pathway through

phosphorylation of ΙκΒ and consequent activation of NF-κΒ (and proinflammatory cytokines), but it can also phosphorylate IRS-1 directly on serine residues, which inhibits insulin signaling. In fact, the glucose lowering and antidiabetic properties of salicylates when administered at high doses are associated with inhibition of ΙΚΚβ activity.

Accordingly, mice heterozygous for a mutation that disables expression of the ΙΚΚβ gene exhibit markedly reduced ΙΚΚβ activity as expected, a phenotype which is accompanied by reversal of insulin resistance produced by high-fat feeding or present in genetically obese models. Thus, it is clear that activation of enzymes that participate and regulate the inflammatory cascade can also downregulate insulin action and thus place them and the NF-KB pathway at a crossroads with insulin signaling. That supports the concept that chronic inflammation can lead to, and be a mechanism for, insulin resistance.

[94] Recent studies using genetically obese or high-fat induced insulin resistance models have shown that white adipose tissue (WAT) depots from these animals are characterized by a significant increase in the population size of resident macrophages. This infiltration by macrophages is consistent with the observed pattern of increased expression of inflammation and macrophage-specific genes detected in WAT from these animals. In fact, expression analysis comparing the macrophage and non-macrophage populations indicates that macrophages are responsible for virtually all of the TNFa, IL-6 and iNOS detected in WAT, and therefore supports the notion that WAT plays an important role in inflammatory pathways known to be activated in obesity states.

[95] The role of the ΙΚΚβ/NF-KB pathway in macrophages has been addressed using specially engineered knockout models in which ΙΚΚβ has been disabled specifically in liver or in myeloid cells. In these studies, insulin responsiveness specifically in liver is conserved in animals lacking hepatic ΙΚΚβ, but they develop insulin resistance in skeletal muscle and fat when rendered obese with a high fat diet or as they age. In contrast, animals lacking ΙΚΚβ in macrophages (myeloid cells) are protected from systemic insulin resistance and exhibit enhanced insulin sensitivity. These findings not only underscore the key function of hepatocyte ΙΚΚβ in determining liver insulin resistance, but also point to a previously unknown role of macrophage ΙΚΚβ to influence the development of systemic insulin resistance. A reasonable corollary of these studies is the proposal that inhibiting ΙΚΚβ, particularly in macrophages, may be used to treat insulin resistance.

[96] Although the mechanism by which macrophages are recruited to adipose tissue is still unclear, plausible candidates are chemotactic molecules that might be secreted by adipocytes, such as C-C motif chemokine ligand 2 (CCL2), also known as monocyte chemoattractant protein-1 (MCP-1 ). The interaction of MCP-1 with its specific receptor CCR2 is a key event mediating macrophage recruitment in the context of inflammation, and accordingly, MCP-1 is expressed at high levels in adipose tissue from obese subjects and mouse models. Studies with adipocytes in vitro indicate that exposure to MCP-1 inhibits insulin-stimulated glucose uptake, effectively inducing an "insulin-resistant" state. Genetic deficiency of the MCP-1 receptor CCR2 reduces food intake and slows down development of obesity and it is accompanied by a reduction in WAT macrophage content and the inflammatory profile, while enhancing systemic glucose homeostasis and insulin sensitivity. Thus, macrophages that infiltrate adipose tissue appear to play a central role in determining a state of chronic inflammation and the development of insulin resistance.

[97] Transgenic mice overexpressing the MCP-1 gene under the control of the aP2 promoter/enhancer develop insulin resistance and hepatic steatosis, whereas an insulin- sensitive phenotype is observed in homozygous MCP-1 KO mice or animals in which 7ND, a dominant-negative mutant of MCP-1 , is acutely expressed after injection of an expression plasmid (Kanda et al., "MCP-1 contributes to macrophage infiltration into adipose tissue, insulin resistance, and hepatic steatosis in obesity" J. Clin. Invest. (2006) 1 16: 1494-1505). Furthermore, treatment of DIO-C57BL/6J mice with INCB3344, a selective CCR2 antagonist (Brodmerkel et al., "Discovery and pharmacological characterization of a novel rodent-active CCR2 antagonist, INCB3344" J. Immunol. (2005) 175: 5370-5378), results in enhanced glucose disposal and insulin sensitivity (Weisberg et al., "CCR2 modulates inflammatory and metabolic effects of high-fat feeding" J. Clin. Invest. (2006) 1 16: 1 15-124). Therefore, a test compound that attenuates expression and production of MCP-1 can have therapeutic benefit in metabolic disorders such as type 2 diabetes, and is believed to be the result of diminished macrophage infiltration into WAT with attendant improved glucose homeostasis and increased insulin sensitivity.

[98] The ability of a drug candidate to inhibit MCP-1 expression is believed to result from broader anti-inflammatory actions as indicated by the Examples on NF-κΒ function in macrophages. Compound A inhibits LPS-induced NF-κΒ nuclear translocation in cultured RAW264.7 mouse macrophages and also caused inhibition of NF-KB-dependent reporter gene expression and a decrease in LPS-induced p65 phosphorylation. Those three observations strengthen the conclusion that Compound A attenuates NF-κΒ function in macrophages, leading to reduced MCP-1 gene expression.

[99] Compound A inhibits activation and function of NF-κΒ in LPS-stimulated macrophages. Although not bound by theory, the basis for that inhibition may reside upstream of NF-κΒ activation. Thus, prior exposure of macrophages to Compound A resulted in suppression of LPS-induced activation of the IKK/NF- B axis and two major proinflammatory MAPK pathways (JNK and p38). In macrophages, thOse kinase signaling cascades are typically activated by pattern-recognition Toll-like receptors, of which TLR4 is the major target of LPS. Because it has been shown that TLR4 can transduce the proinflammatory signals of fatty acids, which are often elevated in insulin-resistant states, it is believed Compound A may interfere with TLR4 function, which leads to suppression of proinflammatory cascades.

[100] Suitable assays for effects on biomolecule effectors such as Lrp1 , Sirt2 or kinases such as Mapkl include measurement of the level of phosphorylation, mRNA, and/or protein of the effector biomolecule or of upstream or downstream kinase target proteins that can mediate signaling through biomolecule effectors such as Lrp1 , Sirt2 or kinases such as Mapkl or Mapk2. Assays for qualitative and quantitative changes in

phosphorylation of kinase substrates including Lrp1 , Sirt2 and Mapkl 12 include quantitation of levels of phosphorylation using antibodies that detect phosphorylated amino acids such as tyrosine or serine on protein kinase substrates in

immunoprecipitation protocols.

[101 ] in vivo assays may be conducted in humans or mammals having the disease or condition of interest. In vivo protocols, the drug candidates may reduce the biological activity or level of effector biomolecules such as TLR4, RAGE and/or HMGB1 , in healthy or diseased humans or mammals, which effects may or may not be observed in cell line assays in vitro or cell extract assays in vitro. In preferred embodiments, assays for effects on TLR4, RAGE and/or HMGB1 are preferably conducted in vivo.

[102] A brief summary of the binding partners that were identified for 17oc-ethynyl-5- androstene-3 , 7β, 17β-ίηοΙ (Compound A) is shown below in Table 1 . This compound exemplifies one or more of the desired biological effects that characterize the drug candidates of the invention.

[103] Table 1. 17oc-Ethynyl-5-androstene-3p, 7β, 17β-ίποΙ binding partner protein function

catabolism of the branched-chain amino acids leucine, isoleucine, and valine

Cherp Calcium homeostasis, cell growth and proliferation

Member of the CCR4-NOT complex that functions as a general

Cnotl transcription regulation complex. It represses the ligand-dependent transcriptional activation by nuclear receptors

One of three subunits which combine to form cleavage stimulation

Cstfl factor (CSTF), which is involved in the polyadenylation and 3' end cleavage of pre-mRNAs

Catalyzes adenylation of flavin mononucleotide (FMN) to form flavin

Fladl

adenine dinucleotide (FAD) coenzyme

A ssDNA binding protein that regulates the far upstream element

Fubpl (FUSE) of c-myc. May act both as activator and repressor of

transcription.

A lysosomal enzyme involved in the degradation of fucose-containing

Fucal

glycoproteins and glycolipids.

Binds with high affinity to RNA molecules that contain AU-rich elements (AREs) found within the 3'-UTR of many proto-oncogenes

Hnrnpd and cytokine mRNAs. Also binds to double- and single-stranded DNA sequences in a specific manner and functions as a transcription factor.

Acts as a transcriptional regulator that promotes transcription repression. Binds to double- and single-stranded DNA sequences. Binds with high affinity to RNA molecules that contain AU-rich elements (AREs) found within the 3'-UTR of many proto-oncogenes

Hnrpdl

and cytokine mRNAs. Binds both to nuclear and cytoplasmic poly(A) mRNAs. Binds to poly(G) and poly(A), but not to poly(U) or poly(C) RNA homopolymers. Binds to the 5'-ACUAGC-3' RNA consensus sequence.

Bifunctional enzyme that acts on the peroxisomal beta-oxidation pathway for fatty acids. Catalyzes the formation of 3-ketoacyl-CoA

Hsd17b4

intermediates from both straight-chain and 2-methyl-branched-chain fatty acids.

An endocytic receptor involved in processes including intracellular signaling, lipid homeostasis, and clearance of apoptotic cells.

Lrp1 Expression decreases with age. May modulate cellular events, such as APP metabolism, kinase-dependent intracellular signaling, neuronal calcium signaling and neurotransmission. * ^#

Lysophospholipases are enzymes that act on biological membranes tc

Lypla2

regulate the multifunctional lysophospholipids.

A member of the MAP kinase family that acts as an integration point for multiple biochemical signals. Modulates cellular processes such as proliferation, differentiation, transcription regulation and development. Upon activation, this kinase translocates to the nucleus of the stimulated cells, where it phosphorylates nuclear targets. It is involved in both the initiation and regulation of meiosis, mitosis, and postmitotic

Mapkl functions in differentiated cells by phosphorylating a number of (Erk2) transcription factors such as ELK1 . Phosphorylates EIF4EBP1 ;

required for initiation of translation. Phosphorylates microtubule- associated protein 2 (MAP2). Phosphorylates SPZ1 (by similarity). Phosphorylates heat shock factor protein 4 (HSF4) and ARHGEF2. Acts as a transcriptional repressor. Binds to a [GC]AAA[GC] consensus sequence. Represses the expression of interferon γ- induced genes. Binds to the promoter of CCL5, DMP1 , IFIH1 , IFITM1 , IRF7, IRF9, LAMP3, OAS1 , OAS2, OAS3 and ST AT1 . Transcriptional activity is independent of kinase activity. In some contexts, activation of Mapkl is associated with a pro-inflammatory cellular state and inflammation.

Similar to Mapkl in some contexts. Activation of Mapk3 is associated

Mapk3

18 with normalization of pathological inflammation and reestablishment ol (Erk1 )

homeostasis.

This protein may control cell migration by relaying extracellular

19 Memol chemotactic signals to the microtubule cytoskeleton. It is a mediator of

ERBB2 signaling.

Single-stranded nucleic acid binding protein that binds preferentially tc

20 Pcbpl

oligo dC.

The second largest subunit of RNA polymerase II, the polymerase

21 Polr2b

responsible for synthesizing messenger RNA in eukaryotes.

Serine/threonine kinase that may play a role in mediating the growth- factor and stress induced activation of the transcription factor CREB. It phosphorylates various substrates, including members of the

22 Rps6ka3

mitogen-activated kinase (MAPK) signaling pathway. The activity of this protein has been implicated in controlling cell growth and differentiation.

Component of the COPII coat, that covers ER-derived vesicles involved in transport from the endoplasmic reticulum to the Golgi

23 Sec23b apparatus. COPII acts in the cytoplasm to promote the transport of secretory, plasma membrane, and vacuolar proteins from the endoplasmic reticulum to the Golgi complex.

24 Sec24c Similar to Ssec23b and may also play a role in shaping the vesicle.

Cytoplasmic (nuclear in G2-M) NAD-dependent protein deacetylase of 'Lys-40' of oc-tubulin in cytoplasm and histones in G2-M Involved in (i)

25 Sirt2

the control of mitotic exit in the cell cycle, probably through its role in the regulation of cytoskeleton and (ii) resistance to cellular stress. ^#

Part of a post-splicing multiprotein complex involved in both mRNA nuclear export and mRNA surveillance. It is located only in the cytoplasm. Plays a role in replication-dependent histone mRNA

26 Upf1 degradation at the end of phase S. Involved in nonsense-mediated decay as part of the SMG1 C complex, a mRNA surveillance complex that recognizes and degrades mRNAs containing premature translation termination codons.

[104] Numbered Embodiments: The following numbered embodiments further describe the invention and related subject matter, including particular aspects of the invention, and are not meant to limit the invention.

[105] 1 . A method to identify a drug candidate, the method comprising

[106] (a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins; (b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of IRS-1 , JNK1/2, p38, p65 NF-κΒ and ΙΚΚα/β; and (e) selecting the test compound of step (b) that decreases phospho-activation or level(s) or biological activit(ies) of Mapk1 /3, JNK1 /2, p38 Mapk, p65 NF-κΒ and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s), wherein the selected test compound is identified as a candidate compound.

[107] 2. The method of embodiment 2 comprising (a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins; (b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of Mapk1/3, JNK1 /2, p38 Mapk, p65 NF- κΒ, ΙΚΚα/β and IRS-1 ; (c) determining immunotoxicity of the test compound in an suitable immunosuppressive test system; and (e) selecting the test compound of steps (b) and (c) that decreases the phospho-activation or biological activitv(ies) of Mapk1/3, JNK1/2, p38 Mapk, p65 NF-κΒ and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s) and has sufficiently low immunotoxicity for administration to a subject for determining efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system wherein the selected test compound is identified as a candidate compound.

[108] In preferred embodiments the maximal reduction in phospho-activation (or reduction in deactivation) or reduction of pro-inflammatory signaling or modulation of the phosphorylation status of a recited protein within cells of a suitable test system that are activated by a pro-inflammatory cytokine or Toll-like receptor 4 (TLR4) agonist in presence of test compound is between about 20% to about 80% of that of a full antagonist reference compound. In more preferred embodiments the selected test compound that is identified as a candidate compound negatively modulates phosphoactivation of Mapk1/3, JNK1 /2, p38, p65 NF-KB and ΙΚΚα/β. In other more preferred embodiments the selected candidate compound negatively modulates phospho-deactivation of IRS1 by negatively modulating Ser phosphorylation and/or positively modulates phospho-activation or IRS1 by positively modulation Tyr phosphorylation of that scaffold protein. In particularly preferred embodiments the selected test compound affects Mapk1 /3, JNK1/2, p38, p65 NF-KB, ΙΚΚα/β and IRS1 in substantially similar manner to Compound A as a reference compound when tested in the same suitable test system(s).

[109] 3. The method of embodiment 1 or 2 further comprising

[1 10] (d) determining test compound binding to or transactivation by ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems; and (f) further selecting from (e) the test compound of steps (b) and (d) or (b), (c) and (d) has EC50's > 10,000 nM in the NHR test systems, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

[111 ] 4. The method of embodiment 1 , 2 or 3 wherein the test compound of step (b) decreases the phospho-activation of Mapk1/3 and wherein the test compounds binds directly or indirectly to Mapkl and Mapk3 as determined by a suitable SILAC test system.

[112] 5. The method of embodiment 1 , 2, 3 or 4 wherein phospho-activation of Mapkl /3 is decreased in a cell-based suitable kinase test system and wherein the test compound in a suitable cell-free test system has negligible or no effect on phospho-Mapk1/3 phosphorylation of myelin-basic protein or other suitable Mapkl /3 substrate wherein no scaffold proteins are present in the cell-free test system.

[113] 6. The method of embodiment any one of embodiments 1 -5 wherein the suitable test system for determining kinase or ΝΚ-κΒ phosphorylation or activity is cell-based.

[114] 7. The method of embodiment 6 wherein the suitable test system for determining kinase phosphorylation or ΝΚ-κΒ activity determines ΙΚΚα/β phosphorylation state(s).

[115] 8. The method of embodiment 6 wherein the suitable test system for determining kinase or ΝΚ-κΒ activity determines κΒ occupancy or NF-κΒ transactivation of a reporter gene transfected into cells comprising the suitable test system in presence of the test compound when the cells are activated by a pro-inflammatory cytokine or Toll-like receptor 4 (TLR4) agonist, wherein the test compound selected as a candidate compound inhibits κΒ occupancy or NF-kB transactivation of the reporter gene.

[116] In preferred embodiments the selected candidate compound inhibits κΒ occupancy or reporter gene transactivation by NF-κΒ in substantially similar manner to Compound A as a reference compound when tested in the same suitable test system(s).

[117] 9. The method of embodiment 6 wherein the suitable test system for determining kinase or ΝΚ-κΒ activity determines the extent of ΝΚ-κΒ translocation to the nucleus on pro-inflammatory cytokine or LPS challenge wherein the selected candidate compound inhibits translocation to the nucleus induced by pro-inflammatory cytokines or a TLR4 agonist.

[118] In preferred embodiments the selected candidate compound inhibits NF-kB translocation to the nucleus is substantially similar manner to Compound A as a reference compound when tested in the same suitable test system(s).

[119] 10. The method of any one of embodiments 2-9 wherein the suitable

immunosuppressive test system is one or more selected from the group consisting of human mixed lymphocyte response, spleenocytes proliferation, delayed-type

hypersensitivity, popliteal lymph node assay and ovalbumin mouse immunization test system(s) test system. [120] In preferred embodiments the selected candidate compound is not immunosuppressive or lacks immunotoxicity substantially similar to Compound A as a reference compound when tested in at least one of the same suitable immunosuppressive test systems. In more preferred embodiments the selected candidate compound is not immunosuppressive in all of the recited immunosuppressive test systems.

[121 ] 1 1 . The method of any one of embodiments 2-10 wherein the suitable

immunosuppressive test system is an opportunistic infection survival test system.

[122] 12. The method of embodiment 1 1 wherein the opportunistic infection is K.

pneumoniae or P. aeruginosa.

[123] 13. The method of any one of embodiments 1 -12 wherein the test compound binds directly or indirectly to Mapkl , Mapk3, Lrp1 and Sirt2 as determined by a suitable SILAC test system.

[124] 14. The method of any one of claims 2-13 wherein the test compound system selected for determining efficacy to treat unwanted inflammation is sufficiently bioavailable to the CNS as determined by a suitable blood-brain barrier test system.

[125] In preferred embodiments the test compound selected as a candidate compound has CNS bioavailability characterized by a brain :serum concentration ratio from about 0.2 to 1 .0 when serum concentrations are least 1 ng/mL when administered by oral gavage to mice. In more preferred embodiments CNS bioavailability is characterized by a brain :serum concentration ratio from about 0.5 to 1 .0 over serum concentrations between about 1 ng/mL to about 1 ,000 ng/mL or more.

[126] 15. The method of embodiment any one of embodiments 2-14 wherein the suitable unwanted inflammation test system is a neuroinflammation test system.

[127] 16. The method of embodiment 15 wherein the suitable neuroinflammation test system is cell-based.

[128] 17. The method of embodiment 15 wherein the suitable cell-based

neuroinflammation test system for determining efficacy comprises neuronal cells or a neuronal cell line, wherein the cell line is (i) mouse cerebral microvessel endothelial cell line bEnd.3 cultured under a chronic hypoxic and hypoglycemic conditions, (ii) human neuron cells (iii) substantia nigra neuroblastoma hybrid cells such as the or (iv) human brain microvascular endothelial cells or a cell line derived therefrom.

[129] In this embodiment conditions for culturing mouse cerebral microvessel endothelial cell under hypoxic and hypoglycemic condition is given in (Yan, F.L. et al., Acta

Neuropathologica (2008) 1 16(5): 529-535. Human neuronal cells include NT2N cells as described by (Wertkin, A.M. et al, Proc. Natl. Acad. Sci. USA (1993) 90(20): 9513-9517. Substantia nigra/neuroblastoma hybrid cells include MES 23.5 cell line as described by Le, W.D. et al, Brain Research (1995) 686(1 ):49-60. Human brain microvascular endothelial cells include those described in C. Bachmeier, C. et al., Cytotechnology (2010) 62(6): 519-529.

[130] 18. The method of embodiment 15 wherein the suitable neuroinflammation test system test for determining efficacy is MPTP- induced neuroinflammation in a subject.

[131 ] 19. The method of embodiment 18 wherein the subject is a non-human primate.

[132] 20. The method of embodiment 1 , 2 or 3 further comprising

[133] (d) determining test compound binding to or transactivation by nuclear hormone receptors ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems (d') determining test compound binding to or transactivation by PPARy; and (g) further selecting from (e) or (f) the test compound of steps (b), (d) and (d') or (b), (c), (d) and (d') that decreases the phospho-activation or biological activity(ies) of Mapk1/3, JNK1/2, p38 Mapk, p65 NF-κΒ and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s) and has EC50's > 10,000 nM in the NHR and PPARy test systems, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

[134] 21 . The method of any one of embodiments 1 -13 and 20 wherein the suitable cell- based test system for determining kinase or NF-kB phosphorylation state or activity is comprised of RAW264.7 macrophages.

[135] 22. The method of any one of embodiments 2-13, 20, 21 wherein the suitable unwanted inflammation test system for determining efficacy is a suitable insulin-resistant test system.

[136] 23. The method of any one of embodiments 1 -13, 20, 21 and 22 wherein the suitable cell-based test system for determining kinase or NF-kB phosphorylation state or activity are murine intraperitoneal macrophages.

[137] 24. The method of any one of embodiments 2-13 and 20-23 wherein the suitable test system(s) for determining efficacy in a suitable insulin-resistant test system is one or more of db/db mouse, ob/ob mouse, Zucker rat or diet-induce mouse model(s).

[138] 25. The method of any one of embodiments 2-13 wherein the suitable unwanted inflammation test system for determining efficacy is a suitable lung inflammation test system.

[139] 26. The method of any one of embodiments 2-13 and 25 wherein the suitable lung inflammation test system is carrageenan-induced pleurisy.

[140] 27. The method of embodiment 1 , 2, 3 or 20 further comprising further comprising [141 ] (d) determining test compound binding to or transactivation by nuclear hormone receptors ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems; (d") determining cell numbers induced by the test compound for cells expressing regulatory T cell phenotypes in a suitable Treg test system, wherein the Treg system comprises T cells of a subject with an autoimmune condition; and (h) further selecting from (e), (f) or (g) the test compound of steps (b), (d) and (d") or (b), (c), (d) and (d") or (b), (d) (d') and (d") or (b) (c) (d) (d') and (d") that has EC50's > 10,000 nM in the NHR test systems and increased numbers of converted regulatory T cells, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

[142] 28. The method of embodiment 1 , 2, 3 or 20 further comprising further comprising

[143] (d') determining test compound binding to or transactivation by nuclear hormone receptors ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems;

[144] (d^*) determining cell numbers for cells expressing regulatory T cell phenotypes in two suitable Treg test systems induced by the test compound, wherein one Treg system comprises T cells of a subject with an autoimmune condition and the other Treg system comprises T cells from a healthy subject; and (h^*) further selecting (e), (f) or (g) the test compound of (b), (d) and (d^*) or (b), (c), (d) and (d^*) or (b), (d) (d') and (d^*) or (b) (c) (d) (d') and (d^*) having EC50's > 10,000 nM in the NHR test systems and increased numbers of converted regulatory T cells in the autoimmune Treg test system and negligible or no increase of converted regulatory T cells in the healthy Treg test system, wherein the selected test compound is identified as a candidate compound for determining

immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

[145] 29. The method of embodiment 28 wherein the cells of the suitable autoimmune Treg test system are those of NOD mice.

[146] 30. The method of embodiment 27 or 28 wherein the cells of the suitable healthy Treg test system are those of B6.SJL mice.

[147] 31 . The method of any one of embodiments 2-13 and 27-30 wherein the suitable test system for determining efficacy to treat unwanted inflammation is a suitable autoimmune test system

[148] 32. The method of embodiment 32 wherein the suitable autoimmune test system is a suitable adjuvant induced arthritis, collagen antibody-induced arthritis, experimental autoimmune encephalomyelitis, ulcerative colitis, systemic lupus erythematosus or autoimmune diabetes test system. [149] 33. The method of embodiment 1 , 2, 3, 15, 20, 27 or 28 wherein the drug candidate of step (c) (i) decreased the biological activity of Mapk1/3, Jnk1/2, p38Mapk or NF-kB and increased the biological activity of Lrp1 ; (ii) decreased the biological activity of Mapk1/2, Jnk1/2, p38Mapk or NF-kB and increased the biological activity of Lrpl and increased the protein or RNA level, phosphorylation state or biological activity of Rps6ka3; (iii) decreased the biological activity of Mapk1/3, Jnk1/2, p38Mapk or NF-kB and increased the biological activity of Lrp1 and decreased the biological activity of Sirt2; (iv) decreased the biological activity of Mapk1/3, Jnk1/2, p38Mapk or NF-kB and decreased the biological activity of Hsd17b4; and/or (v) decreased the biological activity of Mapk1 /3, Jnk1/2, p38Mapk or NF-kB, increased the biological activity of Lrp1 and decreased the biological activity of Sirt2.

[150] 34. The method of embodiment 1 , 2, 3, 15, 20, 27 or 28 wherein the test compound (i) decreased the biological activity of Mapk-1 , Mapk-3, and Lrp1 and decreased the biological activity of RAGE; (ii) decreased the biological activity of Mapk-1 , Mapk-3, and Lrp1 and decreased the biological activity of RAGE and Sirt2; (iii) decreased the biological activity of Mapk-1 , Mapk-2 and decreased the level, phosphorylation state or biological activity of RAGE and HMGB1 ; (iii) decreased the biological activity of Mapk-1 , Mapk-3, Lrp1 and RAGE and decreased the biological activity of HMGB1 ; or (v) decreased the biological activity of Mapk-1 , Mapk-3 and Lrp1 and decreased the biological activity of Sirt2.

[151 ] 35. The method of embodiment any one of embodiments 1 -34 wherein the suitable test system for determining phosphorylation states or levels of biological activity of Mapk1/3, JNK1/2, p38, p65 NF-κΒ, ΙΚΚα/β or IRS-1 or the suitable tests system for determining efficacy in treating unwanted inflammation is an in vitro or in vivo cell-based test system wherein the cells of the cell-based test system are fibroblasts, CNS neurons, astrocytes, microglia cells, Schwann cells, smooth muscle cells, myocytes, monocytes, macrophages, optionally macrophages derived or obtained from white adipose adipocytes or macrophages derived or obtained from brown adipose adipocytes, white adipose adipocytes, brown adipose adipocytes, Kupffer cells, hepatocytes or vascular endothelial cells (e.g., microvascular endothelial cells in, e.g., brain, liver or white or brown adipose tissue).

[152] 36. The method of embodiment 35 wherein the test system is an in vitro cell- based test system.

[153] 37. The method of embodiment 35 wherein the test system is an in vivo cell- based test system. [154] 38. The method of embodiment any one of embodiment 1 , 2, 3, 15, 20, 27 or 28 wherein the suitable test system for determining phosphorylation states or levels of biological activity of Mapk1/3, Jnk1/2, p38, p65 NF-κΒ or ΙΚΚα/β or I RS-1 or the suitable tests system for determining efficacy in treating unwanted inflammation the test system is an in vitro or in vivo cell-based test system comprising one or more of circulating monocytes or macrophages or tissue or cells from an organ or tissue, optionally liver, white adipose tissue or adipocytes, brown adipose tissue or adipocytes, heart, non-heart muscle, brain, ovary, intestine, cardiovascular cells, optionally microvascular endothelial cells, brain parenchyma, CNS cells, lung tissue or cells or kidney tissue or cells, optionally kidney dendritic interstitial cells.

[155] 39. The method of any one of embodiments 1 -9 wherein the biological activity of NF-KB in vitro or in vivo is reduced by the drug candidate by about 20-80% or by about 30-80%.

[156] 40. The method of embodiment 1 , 2, 3, 15, 20, 27 or 28 wherein the unwanted inflammation disease or condition is one or more of (i) a neurological or

neurodegeneration disease or condition, optionally Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, epilepsy, multiple sclerosis, senile dementia or a stroke; (ii) a trauma, optionally a myocardial infarction, a reperfusion injury or a wound; (iii) a vascular disease or condition, optionally atherosclerosis, arteriosclerosis or cerebral amyloid angiopathy; (iv) an ocular disease or condition, optionally uvietis, macular degeneration, scleritis, keratitis, iritis, retinitis, chorioretinal inflammation, dry eye, blepharitis, conjunctivitis, inflammation associated with an ocular or eye infection, e.g., pink eye, or a retinopathy, e.g., diabetic or hypertensive retinopathy; (v) pain, optionally (i) a CNS neuropathy, a peripheral neuropathy or pain associated with cancer or a trauma, or (ii) pain associated with cancer (e.g., prostate, breast, lung or colon cancer or ) an autoimmune disease (e.g., arthritis, a lupus condition or an inflammatory bowel disease) or an injury, optionally a wound or trauma, organ damage (e.g., liver damage from hepatitis) peripheral or central neuropathy (e.g., chemotherapy-induced peripheral neuropathy), neuritis or an infection (e.g., bacterial or viral conjunctivitis; shingles, postherpetic neuralgia, or skin or mucosa ulcers or sores associated with varicella zoster virus or other herpes viruses), wherein the pain can be mild, moderate or acute; (vi) a CNS or neurodegenerative condition, optionally Parkinson's disease, Alzheimer's disease, epilepsy, amyotrophic lateral sclerosis or Huntington's disease; (vii) a metabolic disease or condition, optionally diabetes, obesity or a hyperlipidemia condition

(hypertriglyceridemia, hypercholesterolemia); or (vii) an autoimmune disease or condition, optionally arthritis, a lupus disease or condition (e.g., lupus erythematosis), ulcerative colitis, Crohn's disease, celiac disease, Hashimoto's thyroiditis, atopic allergy, atopic dermatitis, autoimmune peripheral neuropathy, vasculitis, autoimmune thrombocytopenic purpura or autoimmune hepatitis. In some of these embodiments, the disease or condition is type 2 diabetes. In other embodiments, the disease or condition is type 1 diabetes. In other embodiments, the disease or condition is prediabetic hyperglycemia (as defined herein, a fasting glucose level of about 120 to about 125) or overt hyperglycemia associated type 1 diabetes or type 2 diabetes according to standard medical diagnostic criteria.

[157] 41 . The method of any of embodiments 1 , 2, 3, 15, 20, 27 or 28 wherein the test compound has a saline:octanol partition coefficient of 0.9 to 2.3.

[158] The following examples further exemplify the invention and are not meant to limit it in any way.

[159] Example 1. Protein Interaction Test System

[160] Protein binding partners for the compound 17oc-ethynylandrost-5-ene-3p, 7β, 17β- triol ("Compound A") were determined by the stable isotope labeling with amino acids in cell culture (SILAC) technique (Ong & Mann, Nature Protocols, 1 (6):2650-2660, 2007) combined with affinity enrichment protocols. In brief, the SILAC technique was used to identify proteins that are normally expressed in a murine cell line and bound directly or indirectly but specifically to Compound A and is a suitable test system for identifying new drugs or low toxicity therapeutic agents that can be used to modulate inflammation. A test compound that directly or indirectly but specifically binds to one or more of the target proteins found for Compound A is identified as a candidate compound.

[161 ] Compound A covalently bound to agarose affinity beads was used to capture binding partners. Proteins that directly bound to Compound A on the beads were recovered as single proteins essentially as two molecule complexes (Compound A - directly bound protein). Indirectly bound proteins were recovered as components of multi- protein complexes, i.e., proteins that bound to directly bound proteins essentially as a three or more molecule complex (Compound A -directly bound protein-indirectly bound protein).

[162] SILAC media preparation and cell culture conditions. SILAC media preparation and labeling steps were performed essentially as previously described (Harsha et al, Nature Protocols, 3(3):505-516, 2008) and the affinity enrichment protocol was devised using a previously described protocol (Ong et al, Proc. Nat'l. Acad. Sci. (USA) (2009) 106(12): 4617-4622, including pages 1 -15 of the Supporting Information), both of which are incorporated herein by reference, particularly the protocols described at (i) pages 4617-4618 of Ong et al and at page 1 of the supporting formation for Ong et al and (ii) pages 507-513 of Harsha et al.

[163] L-arginine- ³C₆ ⁵N₄-HCI, L-lysine- ³C₆ ⁵N₂-HCI, 4-iodobenzylamine hydrochloride, 9-fluorenylmethyloxycarbonyl chloride (FMOC-CI), triethylamine, ethanolamine, palladium diacetate, triphenyl phosphine, cuprous iodide, piperidine, urea, iodoacetamide, ammonium bicarbonate, Tris(2-carboxyethyl)phosphine-HCI (TCEP), sodium chloride, formic acid, THF (anhydrous), DMF (anhydrous), acetonitrile (ACN, HPLC grade) and dialyzed fetal bovine serum (FBS) were purchased from Sigma-Aldrich (St. Louis, MO). N- hydroxy-succinimide-activated agarose slurry (cat#26200) and SILAC RPMI media (cat#89984) were purchased from Thermo Scientific (Rockfield, IL). HEPES buffer was purchased from Mediatech (Manassas, VA).

[164] Transformed murine monocyte/macrophage cells (ATCC CRL-2278; RAW 264.7) were grown in RPMI light or heavy labeling media, supplemented with 5% dialyzed FBS, in a humidified atmosphere with 5% C0₂ in air at 37 ^<Ό. Tissue culture media was prepared and divided into two portions and "light" (unlabeled) forms of L-arginine and L- lysine were used for the "light" media, while "heavy" (labeled) L-arginine- ³C₆, ⁵N₄ and L- lysine- ³C₆, ⁵N₄ was added to generate the two SILAC labeling media. Each medium was sterilized by filtration through a sterile 0.22 μΜ filter (Millipore). Cells were grown for at least 6 cell divisions in the light or heavy labeling media before beginning recovery of binding partners.

[165] Separate cultures of RAW 264.7 cells grown either the light or heavy labeling media were lysed in ice-chilled 50 mM pH 8.0 Tris buffer containing protease inhibitors (complete tablets, Roche Applied Science, Indianapolis, IN). Lysates were sonicated on ice for 3 x 10 pulses. Protein concentrations of light and heavy lysates were estimated with the DC Protein Assay (Bio-Rad, Hercules CA) and equalized. Affinity enrichments were performed in 1 .5 mL microcentrifuge tubes. All reagents at each step were well mixed at each step to reduce protocol variability.

[166] Compound A and control affinity beads. The preparation of an amino analog of Compound A for linkage to N-hydroxysuccinimide-activated agarose beads was accomplished using a Sonogashira palladium cross-coupling reaction between the C-17 alkynyl group and a FMOC protected 4-iodobenzylamine essentially as previously described (Li et al, J. Org. Chem. (2005) 70(1 1 ): 4393-4396). The FMOC protecting group was removed after cross-coupling by heating in piperidine. The Compound A-benzyl amine was coupled to the N-hydroxysuccinimide-activated bead according to a standard protocol (Pierce Biotechnology, Rockford, IL, product No. 26200, Pierce^® NHS-Activated Agarose Slurry). For evaluation of non-specific binding of proteins onto the affinity sorbent, control agarose beads were prepared using the same treatments as the

Compound A-agarose beads, but replacing the Compound A -amine derivative with hanolamine.

[168] The SILAC technique for screening of test compounds is conducted similarly to that described for Compound A. In those instances where the test compound has a ethynyl substituent conjugation to agarose affinity beads can use the reaction sequence described for preparing Compound A-agarose. In other instances an alkynyl substituent containing a suitably protected functional group is introduced into the test molecule that upon deprotection presents an amino or carboxylic acid group for conjugation to agarose beads. When the test compound contains a ketone (i.e., =0) substituent, conjugation of that compound to agarose beads can occur through an oxime linker, e.g., using carboxy- methoxyamine to form an 0-(carboxymethyl)-oxime. When the test compound contains a hydroxyl substituent, that substituent can be converted to =0 to permit conjugation through an oxime linker or the hydroxyl may be esterified through condensation with a carboxylic acid anhydride or a suitably protected bi-functional di-carboxylic acid to form a hemi-ester, e.g., a hemi-succinate. The carboxylic acid of the hemi-ester or the carboxylic acid generated from the 0-(carboxymethyl)-oxime is then condensed with amine functionalized agarose beads (e.g., aminoethyl-agarose beads). Additional conjugation methods introduce reactive functionality onto the steroid skeleton which allows covalent attachment of linker groups. Those and other techniques known in the art for

immobilization of steroids to solid supports, as used, for example, in affinity

chromatography, may be used to prepare steroid-agarose bead conjugates for SILAC identification of direct or indirect protein binding partners.

[169] Exemplary techniques for steroid immobilization onto solid supports are described in Suzuki, et al. "Isolation of testosterone-binding globulin from bovine serum by affinity chromatography and its molecular characterization" J. Biochem. (1977) 81 : 1721 -1731 ; Suzuki et al. "Isolation and characterization of sex-steroid-binding protein from rat and rabbit plasma" J. Biochem. (1981 ) 89: 231 -6; Sica et al. "Affinity chromatography and the purification of estrogen receptor" J. Biol. Chem. (1973) 248: 6543-6558; Luden et al. "Criteria for affinity chromatography of steroid binding macromolecules" J. Biol. Chem. (1972) 247: 7533-7538; Bucourt et al. "New biospecific absorbents for the purification of estradiol receptor" J. Biol. Chem. (1978) 253: 8221 -8; Govindan and Manz "Three-step purification of glucocorticoid receptor from rat liver" Eur. J. Biochem. (1980) 108: 47-53; Redeuilh et al. "Properties of biospecific absorbents, obtained by immobilization of oestradiol derivatives, for purification of calf-uterine cytosol oestradiol receptor" Eur. J. Biochem. (1980) 106: 481 -493.

[170] Protein capture. Proteins on beads were reduced in 5 mM Tris(2- carboxyethyl)phosphine-HCI and 1 mM dithiothreitol and then alkylated in 10 mM iodoacetamide. An additional amount of ammonium bicarbonate solution was added to create a 2M urea solution before Trypsin was added and the vials were incubated at 37 °C overnight on a thermomixer. Enzymatic digestion was stopped by the addition of 30 μΙ_ of 100% formic acid solution. The vials were held room temperature for 5 minutes and then spun at 1 ,000 x g to pellet the beads. The supernatant was removed to a separate vial for LC/MS analysis.

[171 ] Protein analysis. LC/MS analysis was accomplished using a multidimensional protein identification protocol essentially as described (Washburn, et al, Anal. Chem.

74(7):1650-1657, 2002), which is incorporated herein by reference, particularly the protein identification protocol described therein. Protein analysis was accomplished by flushing five salt gradients through a capillary column before gradient HPLC to elute peptide fractions into a mass spectrometer for MS and MS/MS spectra collection. Tandem mass spectra were searched using the Sequest algorithm (v 3.0) against the mouse database (ipi.MOUSEv368.fasta) from the European Bioinformatics Institute.

[172] SILAC study design. The following SILAC experimental design was used to identify Compound A protein binding partners in RAW 264.7 cells. Compound A coupled solid phase agarose affinity beads retained candidate binding partner proteins, i.e., Compound A target proteins. Proteins non-specifically bound to Compound A were washed off the beads. Candidate binding partners that remained adsorbed to the solid phase were subsequently released and captured for identification. The resulting mixture could contain target proteins directly bound to Compound A and/or indirect binders, e.g., due to protein-protein interactions such as those involved in scaffold assembly of protein complexes. Control beads were used in each experiment to identify non-specific absorption by the agarose bead itself, and these non-specific absorbed proteins were removed from the candidate list by subtracting the two data sets.

[173] In one protocol (summarized below), soluble Compound A, was also used as a competitor to inhibit binding of candidate target proteins to the affinity beads. Subtracting the soluble competitor data set from a buffer control produced a decreased ratio (> 1.5) for Compound A -specific binding partners in comparison to the binding partners in Table 1. This was also used to identify non-specific binding proteins. The soluble competitor protocol was sensitive to the presence of residual competitor during workup and thorough washing steps were employed to reduce the effects of residual soluble competitor.

[174] 17oc-Ethynyl-5a-androstane-3a, 173-diol ("Compound B") was used as a separate control molecule to reduce non-specific binding of proteins to the agarose beads and to Compound A. Compound B is a molecule having a different biological activity profile than Compound A, e.g., no known effect on NF-κΒ in vitro. Compound B was used to reduce or eliminate proteins that non-specifically bound to the solid phase agarose with low affinity. Compound B was used as a soluble competitor to compete with proteins bound non- specifically to Compound A. Variability in the SILAC protocol was reduced through experimental repetition.

[175] The SILAC protocols are summarized in Table 2 below.

[176] Table 2. Protocol summaries

[177] ^a Compound A was used as a competitor to block non-specific protein binding to the beads. ^b Control - agarose beads not crosslinked to Compound A or Compound B. ^c Competitors were DMSO alone (None), a 50 μΜ Compound A in DMSO solution or a 50 μΜ Compound B in DMSO solution. ^d Compound B was used as a non-specific competitor to reveal non-specific binding.

[178] As indicated in Table 2 above, the expected ratios of light (unlabeled) to heavy (labeled) proteins (L/H ratio) for Compound A binding partners varies with the

experimental condition. In protocol A, light proteins from unlabeled cells were incubated with beads linked to Compound A in the presence of DMSO as a control competitor and heavy proteins from labeled cells were incubated with control beads in the presence of

DMSO. Under those conditions, the L/H ratio for specific binders to Compound A would be > 1 because no specific binding would be expected by heavy proteins in the presence of control beads, which do not contain Compound A.

[179] In protocol B, light proteins from unlabeled cells were incubated with control beads in the presence of DMSO and heavy proteins from labeled cells were incubated with beads linked to Compound A in the presence of DMSO. Under those conditions, the L/H ratio for specific binders to Compound A would be < 1 because no specific binding would be expected by light proteins in the presence of control beads, which do not contain Compound A.

[180] In protocol C, light proteins were incubated as in protocol A but heavy proteins from labeled cells were incubated with Compound A beads in the presence of soluble Compound A. Under those conditions, the LJH ratio for specific binders to Compound A would be > 1 because reduced specific heavy protein binding would be expected in the presence of Compound A beads incubated with soluble Compound A, which would compete for and displace binding partners bound to Compound A on the affinity beads.

[181 ] In protocol D, light proteins from unlabeled cells were incubated with beads linked to Compound A in the presence of soluble Compound B as a control competitor and heavy proteins from labeled cells were incubated with Compound A beads in the presence of DMSO. Under those conditions, the L/H ratio for specific binders to Compound A would be expected to be 1 because specific binding by light proteins to Compound A beads would be unaffected by the presence of the non-specific competitor Compound B compared to binding by heavy proteins to Compound A beads in the presence of DMSO, which is also a non-specific competitor of such binding.

[182] In forward bead control protocols (protocols A, C and D above), 2 mg of protein of heavy labeled RAW 264.7 lysate was incubated with 100 μΙ_ of 50% control beads while 2 mg of light lysate was incubated with 100 μΙ_ of 50% affinity beads. Most proteins that bound non-specifically to Compound A were washed off the beads. Candidate binding partners and/or secondary accessory binders that remained adsorbed to the beads were then released and captured for identification. Control beads were used to identify nonspecific absorption by the agarose beads alone. These non-specific absorbed proteins were removed from the candidate binding partner list by subtracting the two data sets. This was the "forward" label protocol. In the "reverse" label protocol (protocol B), the lysates were swapped for each bead type. In a "forward" soluble competitor experiment, the appropriate amount of Compound A or Compound B in DMSO was added to 2 mg of protein of heavy RAW 264.7 lysate. An equal volume of DMSO was then added to 2 mg of light RAW 264.7 protein as a control. 100 μΙ_ of 50% of Compound A -bead was added to both light and heavy lysates in soluble control experiments. [183] Affinity enrichments were incubated overnight (16 h) on an end-over-end rotator at 4 °C. After incubation, the tubes were spun at 1000 x g to pellet the beads. The supernatant was aspirated without disturbing the beads. In forward bead control experiments, beads were combined at the first wash for subsequent washing steps. For soluble control experiments, each tube in a set was washed with 50mM Tris pH8 buffer at least twice to remove excess soluble small molecule competitor. Beads from the two tubes were then combined for later washing steps. After the third wash, the beads were collected by centrifugation at 1000 x g and the wash was aspirated leaving -25 μΙ_ of buffer in the tube.

[184] The four SILAC variations were forward (A) and reverse (B) conditions, which used a solid-phase Compound A -bead compared to non-sterol modified agarose-beads as a control. The third variation, referred to as forward (C) conditions, used solid-phase Compound A -bead in both light and heavy cell preparations where the heavy cell preparation was pre-incubated with 50 μΜ Compound A as a soluble competitor. The fourth variation, forward (D) conditions, used solid-phase Compound A -beads with 50 μΜ Compound B, pre-incubated in the heavy labeled cells as a soluble competitor, which was compared to binding in DMSO buffer using solid-phase Compound A -beads. The results are summarized in Table 3.

[185] Table 3. Comparison of the number of proteins identified and the number of protein ratios > 1 .5 in representative experiments

+ (Light + DMSO + Compound A Bead)

[186] ^*L/H: (light isotope label/heavy isotope label) is the expected outcome ratio for each set of conditions for Compound A binding partners

[187] The number of proteins found for each experiment and the number identified with a ratio > 1 .5 are shown above. A target decoy database of reverse sequences was used to assure a false positive rate of <1 %. This allowed a comparison of different MS database search algorithms for protein identification (Mann, Nature Methods, 6(10):717- 719, 2009). Search results found with the Sequest database followed by DTAselect processing were compared with the !Xtandem database, followed by Peptide Prophet and Protein Prophet comparisons. In general, the total number of identified proteins was similar. In experiment 2A, 874 proteins were found using !Xtandem, which was very close to the 878 discovered using Sequest. From this, 365 coordinate identifications were established. The alternative !Xtandem methodology identified 577 proteins with a ratio of >1 .5 compared to 372 using the Sequest/DTA analysis. The Sequest/DTA algorithms were used for comparison of separate experiments to reduce false positives. Within each experiment MS/MS data was evaluated for each hit to validate the ratios used in the analysis.

[188] The initial data analysis identified 44 candidate binding partners for Compound A. These 44 were additionally constrained by censoring against known promiscuous binding partners that have been identified through a retrospective analysis of several SI LAC experiments essentially as previously described (Trinkle-Mulcahy et al, J. Cell Biol., 183(2):223-239, 2008). This more stringent analysis identified twenty-six Compound A binding partners that exceeded the binding ratios listed in Table 4. The ratio for the 1 D experiment was set at > 1 .5 for the Compound B control experiment to reveal proteins that non-specifically bind to sterols.

[189] Table 4. Compound A binding gene product enrichment ratios in RAW 264.7 monocyte/macrophage cells

[190] The twenty-six specific Compound A binding partners included low abundance proteins including signal transduction-related proteins and transcription factors. The existence of multiple Compound A target proteins is consistent with a biological mechanism operating through a biosystem network instead of primarily acting on a single target biomolecule. Functional analyses to establish contributions of the twenty-six binding partners to the observed biological phenomena was carried out to define the most pertinent targets associated with Compound A biological activities. [191 ] Correlation of the SILAC data with biological and molecular activities observed in humans or animals treated with Compound A:

[192] In addition to the anti-inflammatory activities found in previously reported preclinical models of autoimmunity and inflammation, Compound A decreases chronic inflammation associated with insulin resistance in rodent models of diabetes and in impaired glucose tolerant and type 2 diabetes mellitus human subjects. Compound A had also been reported to attenuate NF-κΒ and TNFoc signaling and associated proinflammatory cytokines and chemokines in these settings.

[193] Analysis of the correlates identified three important nodes, Lrp1 , Mapkl and Mapk3 that are consistent with a mechanism of action for the previously described antiinflammatory and insulin-sensitizing activities of Compound A. Lrp1 was the only surface receptor that binds to Compound A on RAW 264.7 cells.

[194] In addition to a cell-surface interaction with Lrp1 , Compound A also binds Mapkl and 3, and this modulates scaffold interactions and allows cross-talk between Ras-Erk and Lrp1 signaling. Mapkl and Mapk3 (Erk2, Erk1 respectively) are integral mediators of inflammatory signal transduction that are central to obesity.

[195] Addition of Compound A in vitro to Mapkl in the presence of activated Mek1 does not affect phosphorylation activity of Mapkl nor does its addition to phosphorylated Mapkl effect phosphorylation of a peptide substrate. This finding shows that Compound A binding perturbs scaffolding of Mapk within protein complexes and attenuates Ras-Erk signal transduction instead of acting through direct inhibition of the kinase's catalytic domain. This mechanism is important to the safety of this molecular class as direct inhibition of kinase catalytic activity by ATP site-dependent inhibitors often leads to undesirable toxicities. Based on these data Compound A is believed to be a soft drug that modulates signaling and not a high affinity molecular entity that fully neutralizes protein action.

[196] Analysis of all available data shows that Lrp1 , Mapkl , Mapk3 and Sirt2 are the primary effectors of the biological phenomena observed for Compound A, with the combination of (i) Mapkl , Mapk 2 and Lrp1 , (ii) Mapkl , Mapk 2 and Sirt2 and (iii) Mapkl , Mapk 2, Lrp1 and Sirt2 being the key effectors in the context of metabolic disorders accompanied by inflammation. Other binding partners that also significantly contribute to these effects are enzymes central to glucose metabolism (Agl, glycogen degradation) and fatty acid metabolism (Acadsd, short/branched chain acyl-CoA dehydrogenase: Acox3, acyl-CoA oxidase 3; Hsd17b4, fatty acid β-oxidase; Lypla2, lysophospholipase).

[197] Conclusions. The SILAC experiment generated a list of 26 target proteins specific to Compound A. This result of multiple target proteins is consistent with our expectations given the diverse biological activities for Compound A that have been observed in separate in vitro and in vivo models and the involvement of the identified binding partners to these activities . Future additional functional activity assays will serve to detail the contributions of each binding partner to the broad anti-inflammatory, insulin-sensitizing, and lipid regulating activities of Compound A. Experiments with Lrp1 macrophage knockout mice should further establish the role of Compound A acting upon this binding partner. The connections of the identified Compound A binding targets to inflammation, lipid and insulin pathways are clear. KEGG maps for the adipocytokine, toll-like receptor, chemokine and insulin signaling pathways reveal multiple nodes of interaction for these binding partners that are consistent with the observed biological activities of Compound A.

[198] Example 2. Neuroinflammation Test System.

[199] Drugs that are used to treat neurological trauma, pain and CNS

neurodegeneration conditions generally must cross the blood-brain barrier to be active in CNS tissue. A compound with the capacity to modulate Mapkl , Mapk2 and bind to Lrp1 and Sirt2, 17oc-ethynylandrost-5-ene-3p, 7β, 17β-ίποΙ (Compound A), was shown to cross the blood brain barrier as described below. MPTP was purchased from Sigma Aldrich (Milan, Italy). Compound A was used as a 10 mg/mL suspension in vehicle (0.1 % carboxymethyl cellulose + 0.9% saline, 2% Tween 80 and 0.05% phenol).

[200] Pharmacokinetic studies: Male (6 to 8 weeks old) CD-1 mice (n = 18; Charles River Laboratories, Wilmington, MA) received a single oral gavage (4 mlJkg) of

Compound A (80 mg/kg) in vehicle. An additional group of 3 mice went untreated to provide baseline (time = 0) samples. After dosing, cohorts of three mice were sacrificed at 0.5, 1 , 2, 3, 4, 8, and 24 hours by cardiac puncture under C02 anesthesia. Blood was processed to serum and stored frozen until analyzed. After blood collection, the brain from each mouse was collected, snap frozen, and stored frozen until analyzed. Serum and brain samples were analyzed for Compound A by LC-MS/MS.

[201 ] Animals: Male 8-10 week-old C57/BI6 mice were purchased from Harlan

Laboratories s.r.l. (Udine, Italy). The mice were kept under standard laboratory conditions with free access to food and water and they were allowed to adapt one week to their environment before starting the study.

[202] Experimental study plan: Mice received four injections of 20 mg/kg 1 -methyl-4- phenyl-1 , 2, 3, 6-tetrahydropyridine (MPTP) two hours apart. Compound A (40 mg/kg), vehicle or saline was administered per os twice-daily beginning 1 hour after the last MPTP injection for four consecutive days. [203] Assessment of hypokinesia-like symptoms: Four days after MPTP injection, hypokinesia-like symptoms were assessed by a constant speed Rotarod test. Before MPTP injection, mice were trained at several different speeds (measured in rpm), and the overall rod performance was calculated as the latency to fall from the rotating rod (Ugo Basile, Comerio, Italy). To record a baseline of motor function for each mouse, mice were tested 24 h after training was complete and 24 h before saline or MPTP injection. Mice were tested at 20 rpm speed. Testing consisted of three trials (180 s) with an interval of 30 min. Each individual mouse performance was determined as the mean of the three trials.

[204] Semi-quantitative PCR: Brain sections were stored in RNAIater™ solution (Applied Biosystems, Foster City, CA, USA ) at 4^<C until total RNA was extracted using Trizol™ reagent (Invitrogen, Grand Island, NY, USA), according to manufacturer's instructions. RNA quality was evaluated by measuring the 260/280nm absorbance ratio (> 1 .8) and by electrophoresis. cDNA was synthesized using 1 μg of total RNA using the TaqMan™ retrotranscription reagents as described by the manufacturer (Applied Biosystems, Foster City, CA, USA). PCR was carried out in a 30 μΙ_ final volume containing 200 nM forward and 200 nM reverse primers and 20 ng of cDNA. The following primer pairs were used: iNOS Forward: AATCTTGGAGGGAGTTGTGG [SEQ ID 1 ]; iNOS Reverse:

CAGGAAGTAGGTGAGGGTTTG [SEQ ID 2]; TNFoc Forward:

AGCCCACGTCGTAGCAAACCACCA [SEQ ID 3]; TNFoc Reverse:

ACACCCATTCCCTTCACAGAGCAA [SEQ ID 4]; IL-1 β Forward: ACACTCCTTCGT- CCTCGGCCA [SEQ ID 5]; IL-1 β Reverse: CCATCAGAGGCAAGGAGGAA [SEQ ID 6]; GAPDH Forward: CTAGAGAGCTGACAGTGGGTAT [SEQ ID 7]; GAPDH Reverse:

AGACGACCAATGCGTCCAAA [SEQ ID 8]. The amplified fragments were run in a 1 % agarose gel and densitometric analysis was performed using ImageJ™ software. Gene expression data are presented as the ratio between target gene expression and GAPDH control gene expression.

[205] Histology: Brain portions were fixed in 4% paraformaldehyde for 1 week, embedded in paraffin and sliced in δμηΉΐι^ sections. All sections were stained with hematoxylin-eosin (H&E) to visualize cell bodies. The number of damaged neuronal cells was obtained as an average of cells counted per field. The general criteria to score damaged cells included hyperchromatic nuclei and cytoplasmic vacuolation. The number of damaged neurons was visually estimated on three sections from four animals for each experimental group.

[206] Immunohistochemistry: Four days after MPTP injection, mice were anesthetized and transcardially perfused with paraformaldehyde (4% in 0.1 M phosphate buffer, pH 7.4) for immunohistochemistry. Sections from the substantia nigra pars compacta (SNpc) were coronally cut on a vibratome and immunoreacted with Th antibodies (polyclonal rabbit anti-Th, 1 :1000, Abeam, Cambridge, UK) and secondary antibodies (goat anti-rabbit IgG for Th, Abeam). For visualization, the avidin/biotin-peroxidase protocol (ABC, Vector, UK) was applied, using 3,3-diaminobenzidine (Sigma, Milan Italy) as chromogen. Loss of Th positive neurons was determined on three serial sections per animal. Th-labeled neurons were scored as positive only if their cell-body image included well-defined nuclear counterstaining. The number of Th positive neurons was determined in blinded fashion by an independent pathologist.

[207] Statistical evaluation: Data were expressed as the mean ± standard deviation of the mean. Statistical significance was evaluated by applying a two-tailed Student's t test. Data was considered significant at the p < 0.05 level. All statistical tests were carried out using GraphPad Prism 5 (GraphPad Software Inc., San Diego, CA).

[208] Compound A efficiently penetrated the mouse blood brain barrier. The brain :serum ratio (0.57) was exceptionally constant over a broad range of compound A serum concentrations (3.6 - 1 ,230 ng/mL). The semi-log slopes of the clearance from serum and brain were parallel, indicating the same rate of clearance from each tissue, and that these tissues were in rapid equilibrium. The compound A serum concentration vs. time profile and total AUC in was similar to the PK data previously acquired in mice.

[209] Beneficial effects of compound A treatment on MPTP- induced motor impairment were observed. The day following MPTP-injection, the mortality observed was 25% in both the MPTP and the MPTP + Compound A groups and 12.5% in the MPTP + vehicle group. To verify the ability of Compound A to influence MPTP-induced impairment of motor coordination, the ability of mice to maintain balance on a rotating cylinder using the Rotarod test was used (Jones, B.J. and Roberts, D.J. "The quantitative measurement of motor co-ordination in naive mice using an accelerating rotarod" J. Pharm. Pharmacol. (1968) 20(4):302-4). The baseline of mean latency to fall for all the mice before MPTP injection was >180 sec. At day 4 post-MPTP injection, the mean latency to fall was 57.1 and 59.6 sec in the MPTP and MPTP+vehicle group, respectively (shown in Figure 1 ).

[210] Treatment with 40 mg/kg of compound A significantly ameliorated motor impairment because the mean latency to fall was 98.4 sec (p = 0.042 vs. MPTP group and p = 0.047 vs. MPTP + vehicle group). In a second experiment, treatment with 40 mg/kg Compound A again significantly ameliorated motor impairment as judged by a significantly improved mean latency to fall (87.0 sec; p = 0.003) vs. the vehicle-treated group. A similar result was observed for the mice treated with L-DOPA only, which showed a mean latency to fall of 84.5 sec (p = 0.006 vs. vehicle-treated group). Neither a synergistic nor an additive effect was observed for combination of L-DOPA and Compound A, which exhibited a mean latency to fall of 83.4 sec (p = 0.009 vs. the vehicle-treated group).

[211 ] MPTP- induced neuro-inflammation: To elucidate the therapeutic effects of compound A, glial activation and neuronal damage were assessed in control and treated animals by PCR and histology. As expected, na^'ive control mice showed little or no expression of the pro-inflammatory mediators iNOS, TNF-a and I L-1 β (shown in Figure 2).

[212] On the other hand, vehicle-injected mice expressed high mRNA levels of those mediators. A significant reduction was observed in the compound A treated animals for iNOS (p = 0.002 vs. vehicle, two-tailed Student's t test), TNF-oc (p = 0.045) and IL-1 β (p = 0.042) as measured by reverse-transcriptase polymerase chain reaction. Histological count of damaged SNpc dopaminergic neurons revealed a significant (p = 0.01 vs.

vehicle) reduction of brain damage in the mice treated with Compound A compared to vehicle treated animals (shown in Figure 3).

[213] MPTP induced TH-positive neuronal loss in the SNpc: The day following MPTP intoxication, 3 out of 12 and 2 out of 12 mice were found dead in the vehicle- and compound A- treated groups, respectively. MPTP treatment induced a partial

dopaminergic neuron degeneration in the SNpc. The MPTP induced dopaminergic neuron loss was statistically significant (p < 0.0001 ) when compared to healthy control mice group. Treatment with compound A, significantly attenuated Th-positive neuron loss in SNpc (p = 0.006 vs. vehicle)(shown in Figure 4).

[214] The sparing of neurons by 17oc-ethynylandrost-5-ene-3p, 7β, 17β-ίηοΙ showed that the compound spares neurons in CNS neuroinflammation and it can be used to treat neurodegenerative disorders including Parkinson's disease, Alzheimer's disease, amyotrophic lateral sclerosis, Huntington's disease, epilepsy, multiple sclerosis, neurodegeneration or neuron loss associated with aging or the preclinical or early stages of any of those disorders. Such treatment may be manifested as, e.g., slowing the progression of Parkinson's disease or Alzheimer's disease. Such treatment may be manifested as reducing the frequency or intensity of flares or episodes, e.g., in conditions such as multiple sclerosis or epilepsy. Such treatment may be manifested as

improvements in or at least transient stabilization of, e.g., cognitive or motor function in amyotrophic lateral sclerosis, Huntington's disease or neurodegeneration associated with aging.

[215] Example 3. Nuclear Receptor Binding and Transactivation Test Systems.

[216] cDNA expression constructs: Full-length cDNA fragments encoding GR and ER3 were cloned by PCR using double-stranded PCR-ready cDNA templates (QUICK-Clone™: Clontech, Mountain View, CA) from human adipose tissue (GR) or prostate and ovary (ΕΡιβ). Appropriate PCR primers were designed to obtain blunt-end, full-length target amplicons by using AccuPrime™ Taq polymerase (Invitrogen), thereby making them compatible with topoisomerase-mediated directional Gateway cloning (pENTR-TOPO™; Invitrogen). Primers used were: GR, 5'-CACCTGATATTC-ACTGATGGACTC-3' [SEQ ID 9] (forward), 5'-GGCAGTCACTTTTGATGAAAC-3' [SEQ ID 10] (reverse); and ER3, 5'- CACCTCTCAAGACATGGATATAAA-3' [SEQID1 1 ] (forward), 5'- TCACTGAGACTGTGGGTTCT-3' [SEQID 12] (reverse). Recombinant inserts were subsequently transferred to a pDEST40 expression vector by LR recombination

(Gateway-LR Clonase™, Invitrogen). The identity of the inserts was verified by full bidirectional sequencing, and their functional competency was assessed by transient cotransfection of HEK293 fibroblasts, using appropriate GRE or ERE-reporter plasmids (pGRE-SEAP or pERE-TA-SEAP, Clontech; or ERE-/GRE-luciferase constructs generated in pGL3 vectors, Promega, Madison, Wl). Cells were exposed to increasing concentrations of reference test compounds (dexamethasone for GR and 173-estradiol for ER3), and reporter enzyme activity was determined in whole-cell extracts. These titration experiments revealed concentration-dependent increases in reporter activity for each reference ligand and cognate receptor (EC₅₀ =

0.06 nM for 173-estradiol in ER3-transfected cells; EC₅₀ = 8 nM for dexamethasone in GR-transfected cells (see Table 5).

[217] Nuclear Receptor Binding: Assessment of binding activity for various nuclear receptors was performed by homogeneous competition assays using the PolarScreen™ fluorescence polarization system (Invitrogen). In brief, serial dilutions of Compound A (or test compound) were incubated on 384-well plates for 2 h at room temperature in the presence of an appropriate fluorescent ligand (Fluormone™, Invitrogen) and a nuclear receptor of recombinant origin (AR, kit P3018; GR, kit P2816; ERa, kit P2614; ER3, kit P2615; PR, kit P2895) in a total volume of 30 μΙ_, essentially following the manufacturer's protocols. Fluorescence polarization in each well was determined with a GENios Pro™ reader (Tecan), and based on the extent of fluorescence polarization suppression detected IC₅₀ competition values were derived by using GraphPad Prism™ software (GraphPad Software Inc., San Diego, CA). Most nuclear receptors used in these assays were full-length recombinant proteins of human origin, with the exception of AR

(His/glutathione S-transferase-tagged rat AR ligand binding domain) and PR (glutathione S-transferase-tagged human PR ligand binding domain). Nuclear receptor binding activities of Compound A and reference test compounds used for each receptor are shown in Table 5.

[218] Transactivation activity of sex steroid or corticosteroid receptors was assessed primarily in established stably transfected human cancer cell lines expressing nuclear receptor-sensitive luciferase reporter genes. For AR and GR, the cell line MDA-kb2 (American Type Culture Collection CRL-2713) harboring a mouse mammary tumor virus/luciferase cassette was used (Wilson et al. "A novel cell line, MDA-kb2 that stably expresses an androgen- and glucocorticoid-responsive reporter for detection of hormone receptor agonists and antagonists" Toxicol. Sci. (2002) 66: 69-81 ). For ERa and ER3, the cell line T47D-kBluc (American Type Culture Collection 2865) stably transfected with a synthetic plasmid containing three copies of an estrogen response element (ERE) fused upstream of a luciferase gene was used (Wilson et al. "Development and characterization of a cell line that stably expresses an estrogen-responsive luciferase reporter for the detection of estrogen receptor agonists and antagonists" Toxicol. Sci. (2004) 81 : 69-77). In brief, cells were plated at 20,000 cells/well/100 μΙ_ in 96-well clear-bottom white assay microtiter plates (Corning Life Sciences, Lowell, MA) and kept in phenol red-free RPMI 1640 medium supplemented with 4 mM L-glutamine and 10% charcoal-stripped FBS (CHAR-DEX™; Invitrogen). Cells were exposed to the various compound dilutions as needed, and after an overnight incubation at 37 ^<C, media were aspirated, cells were lysed, and luciferase activity was then determined. In some cases, transactivation of ER3 and GR was also performed by transient transfection of HEK293 fibroblasts using expression plasmids encoding full-length human GR or ER3 and appropriate luciferase reporter vectors (see above). For transactivation activity of the human PPARs PPARy, PPAR5 and PPARa, the fluorimetric Gene Blazer™ β-lactamase assay system

(Invitrogen) was used following the manufacturer's instructions. Assays were conducted on 384-well plates using a fluorogenic substrate (CCF4-AM; Invitrogen). Reference test compounds (Tocris Bioscience, Ellisville, MO) used for each receptor were: rosiglitazone and GW1929 for PPARy (EC₅₀ = 1 .3 nM); L165,041 for PPAR5 (EC₅₀ = 1 .1 nM); and GW7647 for PPARa (EC₅₀ = 0.3 nM). Induction of transactivation activity of sex steroid and corticosteroid receptors by Compound A and reference test compounds is shown in Table 5.

[219] Table 5. Nuclear receptor competition binding and transactivation profile of Compound A

R ii Liquid

AE 10,000 IS ± 4,2 PUT: APJGE'- 10,000 0,06 ± ··.· · ; ι ΐ ϋΠ^':

ERu 10.000 * ± 5.4 CE-! ERu.'EEfi" 25 0.002 .Ε·/·

ER >io,ooo ΐ i 1.6 ίΕ,/:· ERfi" 364» ± 540 0.06 ± 0.02 <¾)

PR 10,000 15 ± 2 1 GB' 10,00ti . f D^ )

GR 10,000 8 ± 0 8 .Dex

[220] E2, 173-estradiol; P, progesterone; Dex, dexamethasone. ^a Data are expressed as IC₅₀ values (mean ± S.E.M. in nM units; n = 3^1). Reference ligands are indicated in parentheses. ^b Data are expressed as EC₅o values (mean ± S.E.M. in nM units; n = 4-9). Reference ligands are indicated in parentheses. ^c MDA-kb2: cells stably transfected with a sex steroid receptor-sensitive promoter/reporter construct (mouse mammary tumor virus promoter) fused upstream of a luciferase gene. These cells endogenously express both AR and GR. ^d T47D-kBluc: cells stably transfected with an estrogen-sensitive synthetic promoter/reporter construct (ERE) fused upstream of a luciferase gene. These cells express endogenously both ERa and ER3. ^e ER3-HEK293: HEK293 fibroblasts transiently co-transfected with an estrogen-sensitive promoter/reporter construct and a cDNA expression vector encoding the full-length human ER3. These cells exhibit virtually undetectable levels of endogenous sex steroid receptors. ^f GR-HEK293: HEK293 fibroblasts transiently co-transfected with a glucocorticoid-sensitive promoter/reporter construct and a cDNA expression vector encoding the full-length human GR.

[221 ] As shown in Table 5, Compound A does not bind to the major sex steroid or corticosteroid receptors, AR, ERa, ER3, PR, and GR, as indicated by IC₅o values of > 10,000 nM in competition binding assays. In addition, transactivation assays revealed no activity of Compound A in MDA-kb2 cells co-expressing AR and GR (EC₅₀ > 10,000 nM), and essentially no transactivation in transiently transfected HEK293 fibroblasts expressing ER3 (EC₅₀ > 3600 nM). In contrast, evidence for weak activity (EC₅₀ = 268 nM) was found in T47D-kBluc cells co-expressing ERa and ER3 (Table 5), suggesting preferential, but weak, potential transactivation of ERa. However, under the conditions of these assays, the natural ER ligand 173-estradiol transact! vates ERa / ER3 (T47D-kBluc cells) with an EC₅o of 0.002 nM. The low transactivation activity observed in cells co-expressing ERa and ER3, but not ER3 alone, suggests a weak potential transactivation of ERa, albeit this activity is virtually negligible when compared with 173-estradiol. Furthermore, other results indicate that Compound A inhibits macrophage NF-κΒ function in the presence of ERa - selective antagonists and its beneficial effects on glucose homeostasis in vivo are preserved in Era deficient mice.

[222] Finally, as shown in Figure 5, Compound A did not cause transactivation of PPARa, PPARy, or PPAR5. Transactivation activity for each of the PPARs [ PPARy (Fig. 5A), PPAR5 (Fig. 5B), and PPARa (Fig. 5C)] was determined by adding the indicated concentrations of Compound or reference test compounds onto HEK293 cells stably transfected with isoform-specific PPAR-LBD/Gal4-DBD chimeric proteins and also harboring a PPAR-sensitive β-lactamase reporter gene (Gene-Blazer™). Cells were loaded with the CCF4-AM - β-lactamase substrate, and after a 24-h incubation period with test compound, the resulting activity was determined by fluorescence. Data are shown as relative transactivation normalized with respect to the maximal response observed in each case after subtracting background activity (symbols represent mean ± S.D.). Assuming that the transactivation experiments described here are equivalent in other cell types, the combined results indicates that the observed therapeutic effects of Compound A to improve glucose metabolism in vivo do not result from transactivation of sex steroid receptors, corticosteroid receptors, or PPARs.

[223] The insulin-sensitizing effects of thiazolidinediones (TZDs) such as rosiglitazone involve at least in part PPARy-mediated anti-inflammatory responses caused by trans- repression of inflammatory mediators. Moreover, deletion of the PPARy gene from macrophages results in impaired glucose tolerance, insulin resistance, and increased expression of inflammatory effectors, indicating that macrophage PPARy is required for the full insulin-sensitizing effects of TZDs. The fact that Compound A fails to transactivate PPARy indicates that its anti-inflammatory and antidiabetic effects occur through a PPAR- independent pathway.

[224] Example 4. Inflammation and Insulin Resistance Test Systems-db/db and ob/ob mice.

[225] Animals: Male BKS.Cg-m +/+ Lept^/J (db/db) mice (5 or 7 weeks old) and male B6.V-Z.ep^oi,/J (ob/ob) mice (6-7 weeks old) were housed in an environmentally controlled room under a 12-h light/dark cycle with free access to a standard mouse diet and water. After a 7-day acclimation period, blood samples were collected by tail nick for glucose measurements, and a baseline oral glucose tolerance test (OGTT) was performed, after which mice were randomly assigned to treatment groups according to equivalence of body weight and non-fasting and fasting blood glucose levels. Age-matched db/+ or ob/+ heterozygous animals were used for lean controls. For studies with diet-induced obese (DIO) mice, male C57BL/6J mice (5 weeks old) were first fed a high-fat diet (20% kcal protein, 60% kcal fat, 20% kcal carbohydrate; ResearchDiets, New Brunswick, NJ) for 8 weeks, until they reached a target body weight of > 30 g. Blood glucose level tests and a baseline OGTT were performed, and animals were randomly assigned to groups as described above. All test articles for animal dosing consisted of soluble formulations of Compound A prepared shortly before each study in a cyclodextrin-based vehicle consisting of 30% (w/v) sulfobutyl-3-cyclodextrin (Captisol™; CyDex, Lenexa, KS) in water at 10 mg/ml and adjusted with NaOH or HCI to a final pH of 6.5 ± 1 . Stability of the test articles was verified by high-performance liquid chromatography analysis at least for equivalent periods of time to the duration of the efficacy studies.

[226] Real-Time Quantitative Reverse Transcription-Polvmerase Chain Reaction:

[227] Epidydymal adipose tissue was dissected and immediately placed in RNA/ater™ solution (Ambion, Austin, TX) until processed. Total RNA was extracted with a RiboPure™ RNA purification kit (Ambion), following the manufacturer's instructions. The quality and integrity of RNA was confirmed by OD₂6o OD₂8o ratios > 1 .9 and denaturing agarose gel electrophoresis. First-strand cDNA synthesis was accomplished with 100 ng of RNA and the iScript cDNA Synthesis™ kit (Bio-Rad, Hercules, CA), and polymerase chain reaction (PCR) amplification of the resulting reverse transcription (RT) products was performed in the presence of iQSYBR Green I Supermix™ dye (Bio-Rad) and target-specific primer pairs. The mouse acidic ribosomal protein P0 (RPLPO) was used as a reference housekeeping gene in the same PCRs to normalize expression of target genes to a suitable endogenous standard. PCR primers used were as follows: MCP-1 , 5'- ACTCACCTGCTGCTACTC-ATTCAC-3' [SEQID 13] (forward), 5'-CTTCTTTGG- GACACCTGCTGCT-3' (reverse) [SEQID 14]; tumor necrosis factor a, 5'-CTTGTC- TACTCCCAGGTTCTCTT-3' [SEQID 15] (forward), 5'-GATAGCAAATCGGCTGA-CGG-3' [SEQID 16] (reverse); CCR2, 5'-GAGCCTGATCCTGCCTCTACTTG-3' [SEQID 17] (forward), 5'-CTCTTCTTCTCATTCCTACAGCGA-3' [SEQID 18] (reverse); and RPLPO, 5'-CTGAGATTCGGGATATGCTGTTG-3' [SEQID 19] (forward), 5'-GTCCTAGACCAG- TGTTCTGAGC-3' [SEQID 20] (reverse). Thermocycling conditions included an initial 3- min denaturing step at 95 °C followed by 40 successive cycles of denaturation at 95^ for 10 s, annealing at 60 'C for 30 s, and extension at 72 °C for 20 s. Each PCR amplification was routinely followed by a 15-min melting curve program (95°C for 1 min, 55 °C for 1 min and ramping from 55 °C to 94 °C in 0.5^ increments in 80 cycles of 10 s) and a final cooling step to 4^<C. Real-time detection of PCR amplification products was determined by fluorescence with an iCycler iQ Multicolor Detection System™ (Bio-Rad). Single T_m peak melting curves showed no evidence of primer-dimer formation. Relative quantification of target gene expression was calculated based on real-time PCR efficiency of amplification and the relative difference in threshold crossing points between a sample and a control. Results are expressed as a ratio in comparison to the reference gene.

[228] Serum MCP-1 and insulin levels: Insulin levels were measured in serum by enzyme-linked immunosorbent assay using 96-well microtiter plates coated with mouse- specific anti-insulin monoclonal antibodies [Insulin (Mouse) Ultrasensitive EIA; Alpco Diagnostics, Salem, NH]. Serum MCP-1 levels were determined by enzyme-linked immunosorbent assay with an affinity-purified anti-MCP-1 polyclonal antibody (Quantikine Mouse CCL2/JE/MCP-1 Immunoassay; R&D Systems, Minneapolis, MN). Assays were conducted following the manufacturer's protocol.

[229] Insulin-resistant ob/ob mice were treated with 80 mg/kg Compound A b.i.d. for 4 weeks and serum levels of MCP-1 (Fig. 6A) and insulin (Fig. 6B B) were measured. For Fig. 6C, expression of mRNA MCP-1 in epidydymal white adipose tissue (WAT) was determined by RT-PCR. Data are expressed as mean ± S.E.M. (N = 5). ^*, P < 0.05; ^**, P < 0.01 relative to vehicle). The anti-inflammatory activity of Compound A was evident in obese insulin-resistant ob/ob mice treated with Compound A, in which serum MCP-1 protein levels decreased (P < 0.01 ; Fig. 6A). In addition, the marked increase in serum insulin levels observed in obese ob/ob mice relative to lean ob/+ mice was significantly abrogated in animals treated with Compound A (P <0.05) but not with vehicle (Fig. 6B), indicating that insulin resistance was ameliorated. These effects were accompanied by markedly reduced expression of MCP-1 mRNA expression (P < 0.05) in WAT (Fig. 6C). Thus, amelioration of insulin resistance and improved glucose utilization by Compound A treatment is associated with decreased MCP-1 production in WAT.

[230] MCP-1 and CCR2 mRNA levels: Levels of MCP-1 and CCR2 mRNA were determined by RT-PCR in total RNA prepared after a 6-h fast from epididymal adipose tissue from diabetic db/db mice treated with vehicle, 40 mg/kg Compound A, or 25 mg/kg rosiglitazone b.i.d. for 4 weeks (N = 6 for each group). Lean db/+ mice are shown for comparison. Relative mRNA expression is normalized with respect to vehicle. B, serum levels of MCP-1 in db/db mice after 4 weeks of indicated treatments are shown. Data are shown as mean ± S.E.M. (N = 6-9). ^**, P < 0.01 ; ^*, P < 0.05 relative to vehicle.

Compound A-induced inhibition of MCP-1 expression in adipose tissue was accompanied by reduced serum levels of MCP-1 (P < 0.05) in the same animals, but not rosiglitazone (Fig. 7B). Thus, mRNA levels of MCP-1 and CCR2 in adipose tissue of diabetic db/db mice receiving vehicle only was markedly increased relative to nondiabetic lean db/+ littermates. However, MCP-1 and CCR2 expression was significantly reduced in db/db mice treated with Compound A or rosiglitazone (P < 0.05) relative to vehicle-treated animals (Fig. 7A).

[231 ] Macrophages test systems: Murine RAW264.7 macrophages [American Type Culture Collection (Manassas, VA) TIB-71 ] were maintained in Dulbecco's modified Eagle medium (DMEM; Mediatech, Herndon, VA), with 10% fetal bovine serum (FBS; Invitrogen, Carlsbad, CA) in a humidified incubator at 37°C. For transient transfections, cells were seeded and transfected after 24 h by using the Lipofectamine™ reagent (Invitrogen) with pNF-κΒ luciferase plasmid (Stratagene, La Jolla, CA) and pRLTK Renilla luciferase control plasmid (Promega, Madison, Wl). After 24 h, cells were exposed to the

compounds indicated for 1 h and then stimulated with 100 ng/mL of LPS for 6 h. Cell lysates were prepared, and both firefly and Renilla luciferase activity were determined sequentially by using the Dual- Luciferase reporter assay system (Promega).

Luminescence was measured with a GENios™ Pro plate reader (Tecan, Durham, NC). Results were corrected for well-to-well relative transfection efficiency with respect to Renilla luciferase activity.

[232] For macrophage immunostaining and immunoblot analysis, RAW264.7

macrophages were cultured on glass coverslips and treated with 100 nM Compound A, vehicle or test compound [0.1 % dimethyl sulfoxide (DMSO)] for 1 h, followed by 100 ng/ml of LPS for 15 min. Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline, washed, and permeabilized in 0.1 % Triton X-100 (Sigma-Aldrich). Cells were washed again and blocked with 1 % normal goat serum (Santa Cruz Biotechnology, Inc., Santa Cruz, CA) for 1 h, followed by the addition of an anti-NF-KB/p65 monoclonal antibody (lgG1 ; Santa Cruz Biotechnology, Inc.) and staining with fluorescein

isothiocyanate-conjugated goat-anti-mouse lgG1 (Santa Cruz Biotechnology, Inc.). The resulting immunofluorescence was visualized and captured at 40X with a fluorescence microscope.

[233] Murine intraperitoneal macrophages were elicited by thioglycolate and isolated as described (Welch et al. "PPARy and PPAR5 negatively regulate specific subsets of lipopolysaccharide and INF-γ target genes in macrophages" Proc. Nat'l. Acad. Sci. (USA) (2003) 100: 6712-7). In brief, cells were seeded in six-well plates (7 X 10⁶/10-cm plate) and maintained in DMEM with 15 mM glucose and 10% FBS for 3 days. Media were changed every 24 h, and cells were then serum-starved in DMEM (15 mM glucose) with 0.5% FBS overnight. For experiments with RAW264.7 mouse macrophages, cells were cultured as described above and serum-starved (0.5% FBS) overnight. In both cases, cells were pretreated with DMSO control (0.01 % final) or 100 nM Compound A or test compound for 2 h (intraperitoneal macrophages) or overnight (RAW264.7 macrophages), followed by 100 ng/ml of LPS stimulation for various times. Cell lysates were collected in RIPA buffer [20 mM Tris-HCI (pH 7.4), 0.15 M NaCI, 1 mM EDTA, 1 %Triton X-100], and a cocktail of protease (Roche Diagnostics, Indianapolis, IN) and phosphatase (Sigma- Aldrich) inhibitors]. Proteins in cell lysates were resolved by 10% SDS-polyacrylamide gel electrophoresis and then transferred onto polyvinylidene difluoride membranes.

Membranes were blocked with 5% nonfat dry milk in TBST [10 mM Tris-HCI (pH 8), 0.15 M NaCI, 0.05% Tween 20] and developed for specific proteins by using the appropriate primary antibodies (all from Cell Signaling Technology Inc., Danvers, MA): phospho-IKKa- Ser180/IKK3-Ser181 , phospho-SAPK/JNK-Thr183/Tyr185, phospho-p38 mitogen- activated protein kinase (MAPK)-Thr180/Tyr182, and phospho-NF-KB-p65/Ser536. For loading controls, blots were stripped and developed with anti-a-tubulin rabbit polyclonal antibody (Cell Signaling Technology, Inc.). Immune complexes were detected by enhanced chemiluminescence. Relative band intensities were quantified by densitometry scanning and normalized to untreated controls.

[234] NF-KB test system: Because genes encoding MCP-1 and other proinflammatory effectors are under the control of the key transcription factor NF-κΒ, the effect of

Compound A on this central regulator of the inflammatory response was determined. LPS stimulation of cultured RAW 264.7 mouse macrophages pretreated with Compound A displayed markedly reduced p65 nuclear staining, indicating decreased nuclear translocation of NF-κΒ (Fig. 8A). The effect of Compound A on NF-κΒ activation was also demonstrated in time course experiments using freshly isolated mouse peritoneal macrophages, which revealed that the extent of NF-KB/p65 serine phosphorylation was decreased by Compound A (Fig. 8B). To confirm that these effects translate into a functional change in NF-KB-driven gene expression, experiments were conducted with RAW264.7 cells transiently transfected with NF-KB-sensitive promoter/reporter constructs. Prior treatment of RAW264.7 cells with Compound A decreased subsequent activation of NF-KB-driven reporter gene expression in response to LPS stimulation by 50 to 60% (Fig. 8C). As expected, NF-KB-dependent luciferase expression was also inhibited by dexamethasone or the MAPK inhibitor PD98059. For Figure 8A, cultured RAW264.7 mouse macrophages were grown on glass coverslips and left untreated (1 ), treated with 100 ng/ml LPS for 15 min (2), or treated with 100 nM Compound A for 1 h followed by LPS for 15 min (3). Cells were then incubated with an anti-p65 monoclonal antibody (lgG1 ), stained with a fluorescein isothiocyanate-conjugated goat-anti-mouse lgG1 , and visualized by immunofluorescence (40X). For Fig. 8B, freshly isolated, thioglycolate- elicited murine intraperitoneal macrophages were seeded and allowed to recover for 3 days in culture. Cells were then pretreated with 0.01 % DMSO or 100 nM Compound A for 2 h followed by LPS stimulation for the indicated times. Untreated cells served as the control (lane C). Cell lysates were prepared and proteins were immunoblotted with a phospho-NFK-B p65 (Ser536) antibody. For Figure 8C, cultured RAW264.7 macrophages transiently transfected with an NF-KB/luciferase reporter vector were left untreated (basal) or exposed to 1 , 10, and 100 nM Compound A, 10 nM PD98059, or 10 nM

dexamethasone for 1 h, followed by stimulation with 100 ng/ml of LPS for 6 h. The resulting activation of NF-κΒ was assessed by luciferase activity in cell lysates. Data are shown as mean ± S.E.M and normalized with respect to LPS-treated cells.

[235] Thus, treatment of RAW264.7 murine macrophages with Compound A inhibits LPS-stimulated nuclear translocation of NF-κΒ, and treatment of mouse peritoneal macrophages in vitro with Compound A inhibits NF-κΒ p65 phosphorylation. Furthermore, Compound A inhibits NF-κΒ transcription activity in RAW264.7 murine macrophages as indicated by decreased reporter signaling. Taken together, these observations show that Compound A limits activation of NF-κΒ in macrophages in response to LPS and that this anti-inflammatory action contributes to the observed amelioration of glucose intolerance in vivo.

[236] Pro-inflammatory kinase test systems: Macrophage stimulation with LPS leads to increased phosphorylation of IKK and NF-kB/p65 and two major proinflammatory MAPK signaling cascades, JNK and p38. However, prior exposure to Compound A resulted in marked suppression in the extent of LPS-induced phosphorylation of these proteins (P < 0.05 for all kinases and NF-kB/p65 at least after 60 min of LPS stimulation) (Fig. 9). For Figure 9, murine RAW264.7 macrophages were pretreated with DMSO or 100 nM

Compound A overnight and then challenged with 100 ng/ml of LPS for the times indicated. Total lysates were prepared, and the phosphorylation status of specific proinflammatory kinases was visualized by immunoblotting (Fig. 9A). Equivalent protein loading was determined by immunoblotting with anti-a- tubulin antibodies. Relative band intensities in two independent experiments were quantified by densitometry and normalized with respect to the extent of phosphorylation at time 0 in DMSO-treated cells. Data are expressed as mean ± S.D. ^*, P < 0.05 (Fig. 9B).

[237] Although it was not possible to distinguish between IKKa and ΙΚΚβ in these experiments given the specificity of the antibodies used (phospho-IKKa/3-Ser 180/181 ), it is of interest to note that ΙΚΚβ has been implicated in down-regulation of insulin receptor substrate-1 signaling through increased phosphorylation on serine residues (Arkan et al., 2005). Those findings indicate that Compound A causes a broad anti-inflammatory effect characterized by impaired LPS-induced upstream activation of IKK and attendant suppression of NF-κΒ activation, as well as reduced activation of other TLR4-sensitive proinflammatory signaling kinase cascades. Inhibition of JNK and p38 not only attenuates their impact in the pro-inflammatory response but also should abrogate down regulation of insulin signaling, which is known to be compromised by activation of IKK, JNK and p38. In fact, experiments with cultured hepatocyte and myocyte cell lines treated with proinflammatory cytokines (IL-1 β, IL-6 and TNFa) indicates that Compound A prevents the cytokine-induced inhibition of an important insulin signaling event, i.e., IRS-1 phosphorylation due to insulin receptor (IR) activation.

[238] The insulin receptor (IR) is composed of two a and two P subunits in a P-a-a-P tetrameric configuration held together by disulfide bonds. While the a subunit is completely extracellular and contains the insulin binding domain, the P subunit contains a transmembrane portion and a cytoplasmic domain, which functions as a protein tyrosine kinase. Upon binding to its receptor, insulin causes immediate activation of the p subunit's tyrosine kinase function, which results in cross-autophosphorylation of the P subunits. As a consequence, the inherent tyrosine kinase activity of the p subunit is greatly activated, resulting in phosphorylation of cellular protein substrates. A major IR substrate in target cells is the insulin receptor substrate-1 (IRS-1 ) protein, which by virtue of its SH2 domains (phosphotyrosine binding sites) avidly binds to tyrosine phosphorylated IR, thereby presenting itself as a substrate and undergoing IR-catalyzed extensive multisite tyrosine phosphorylation. This event then enables IRS-1 to rapidly interact with a number of other SH2-containing proteins which initiate signaling through major pathways including the phosphatidylinositol kinase (P13K) pathway (involved in metabolic actions, e.g. glucose transport) and the mitogen-activated protein kinase (MAPK) pathway (involved in cell growth-related actions). While tyrosine phosphorylation of IRS-1 is required to properly mediate insulin action, phosphorylation of IRS-1 on serine and threonine residues is generally associated with inhibition of IRS-1 's signaling properties and therefore, insulin action. Several protein kinases involved in proinflammatory pathways are known to directly phosphorylate IRS-1 on serine residues, thereby interfering with its normal function to carry through the insulin signal. These include protein kinases such as the atypical protein kinase 5 (PKCC), c-Jun NH2-terminal kinase (JNK1 ) and 1KB kinases (IKK), which are responsible for activation of NF-κΒ and the inflammation response. Thus, serine phosphorylation of IRS-1 by the action of these kinases generally causes downregulation of insulin action in target cells. Since Compound A inhibits activation of kinases involved in the stress and inflammation response, i.e., IKK and JNK1 , then a test compound selected as a candidate compound should enhance tyrosine and/or decreased serine IRS-1 phosphorylation. As a result, IRS-1 -dependent signaling due to action of the test compound in a suitable test system is predicted to increase in order to provide an improvement in insulin sensitivity and action.

[239] To screen for IRS-1 -dependent signaling three different suitable test systems are used: H41 1 E liver hepatoma cells (rat), C2C12 myotubes (mouse) and 3T3-L1 adipocytes (mouse). H41 1 E hepatoma cells (ATCC, CRL-1548) are plated at a density of 3 x 10⁶ in 10-cm plates and maintained in Eagle's Minimum Essential Medium (EMEM)

supplemented with penicillin/streptomycin and 10% FBS and kept in culture in a humidified C0₂ incubator at 37 °C until reaching confluence. Confluent monolayers are washed 2X with PBS and then covered with serum-free medium (supplemented with 1 % BSA) for 24 hr. Cells are exposed to treatments as indicated below for 6 hr and then challenged (or not) with 10 nM insulin for 10 min. Plates are removed from incubator and monolayers were then washed 2X and scraped into ice-cold PBS supplemented with a protease inhibitor cocktail. Cells were centrifuged for 5 min at 300 x g and pellets were recovered and resuspended into 300 μΙ_ of cell lysis buffer (50 mM Tris-HCI pH 7.5, 150 mM NaCI, 2 mM EGTA, 10 mM Na₃V0₄, 100 mM NaF, 10 mM Na4P207, 50 nM okadaic acid, 1 mM PMSF, 10 μg/mL aprotinin and 10 μg/mL leupeptin) with repeated pipetting. Cell suspensions were put on ice and on a shaker for 20 min and then centrifuged at 16,000 x g for 25 min. The supernatant fraction was recovered as the total cell lysate. Protein concentration was determined with the Coomassiem Plus reagent (Pierce) and lysates were then subjected to immunoprecipitation and immunoblotting for IRS-1 as described below. [240] C2C12 myoblasts (ATCC) are plated at a density of 3 x 10⁶ in 10-cm plates and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% FBS and kept in culture in a humidified C0₂ incubator at 37°C for 24 hr. Medium in

subconfluent monolayers are then replaced with DMEM + 2% horse serum to induce differentiation and cell-cell fusion into myotubes. Approximately 5 days later, cells are exposed to treatments indicated below for 1 hr and then challenged (or not) with 100 nM insulin for 3 min. Plates are removed from the incubator and monolayers are then washed 2X and scraped into ice-cold PBS supplemented with a protease inhibitor cocktail. Cells are centrifuged for 5 min at 300 x g and pellets are recovered and resuspended into cell lysis buffer, as described above. Cell suspensions are put on ice and on a shaker for 20 min and then centrifuged at 16,000 x g for 25 min. Total cell lysates (supernatant fractions) were recovered, protein concentration was measured and then subjected to immunoprecipitation and immunoblotting as described below.

[241 ] Mouse 3T3-L1 fibroblasts (preadipocytes) are plated at a density of 2 x 10⁵ in 10- cm plates and maintained in Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% calf serum and kept in culture in a humidified C0₂ incubator at 37 °C for several days until reaching confluence. Differentiation of confluent monolayers into adipocytes was induced by adding DMEM + 10% FBS, supplemented with 0.5 mM

isobutylmethylxanthine, 1 μg/mL insulin and 1 μΜ dexamethasone. Cells were maintained under these conditions for 2 days and then medium changed without supplements, except for FBS and insulin. After 2 days in culture, medium is changed to regular DMEM + 10% FBS. Cells will acquire the morphological appearance of fully differentiated adipocytes typically after 7-9 days in culture following the induction of differentiation.

[242] For experiments to determine changes in IRS-1 phospho-serine content, cells are exposed to 100 nM of test compound or 30 min followed by a challenge with 20 ng/mL

TNFa for the indicated periods of time up to 180 min. Plates are removed from incubation and monolayers are then washed 2X and scraped into ice-cold PBS supplemented with a protease inhibitor cocktail. Cells are centrifuged for 5 min at 300 x g and pellets were recovered and resuspended into cell lysis buffer, as described above. Cell suspensions were put on ice and on a shaker for 20 min and then centrifuged at 16,000 x g for 25 min. Total cell lysates (supernatant fractions) are recovered, protein concentration is measured (see above) and then subjected to immunoprecipitation and immunoblotting (see below).

[243] Immunoprecipitation of IRS-1 is accomplished by adding 1 .7 μg of immunoaffinity- purified rabbit polyclonal anti-IRS1 IgG (Upstate) to 20 μg lysate (300 μΙ_), followed by overnight incubation at 4°C. Immune complexes were recovered by adding 20 μΙ_ of Protein A/G PLUS™-Agarose beads (Santa Cruz) and mixing for 1 hr. Beads were pelleted by brief centrifugation and washed 5X with 50 mM Tris-HCI pH 7.5, 1 % NP-40, 150 mM NaCI. Supernatants were discarded and beads resuspended in 35 μΙ_ of SDS- PAGE sample loading buffer, boiled for 3 min and then applied to an SDS-polyacrylamide gel for electrophoresis, Immunoblotting was accomplished by electrotransfer of separated proteins in gels onto polyvinylidene difluoride (PVDF) membranes. Membranes were probed with anti-phosphotyrosine 4G10 mouse monoclonal antibody (1 :1000; Upstate) or anti-phospho IRS-1 (Ser307) rabbit polyclonal antibody (Upstate), followed by HRP- conjugated goat anti-mouse IgG (Jackson ImmunoResearch). Immune complexes are visualized by enhanced chemiluminescence (ECL).

[244] Treatment of H41 1 E hepatocytes with insulin caused the expected increase in

IRS-1 tyrosine phosphorylation. As exemplified for Compound A, treatment with that test compound does not appear to affect that phosphorylation. Exposure of these cells to IL-1 β or IL-6 led to a marked decrease in the extent of IRS-1 tyrosine phosphorylation in response to brief stimulation with insulin (Fig. 10A, immunoblotted with anti- phosphotyrosine antibody (upper rows) or anti-IRS-1 antibody (lower rows). Controls without cytokine treatment are shown on the left panel). Importantly, this observed cytokine- induced decrease in insulin-stimulated IRS-1 tyrosine phosphorylation was completely prevented in the presence of 100 nM Compound A. These changes generally occurred without appreciable or parallel variations in the total amount of IRS-1 protein. In a similar fashion, treatment of C2C12 differentiated myotubes with insulin also caused an immediate increase in IRS-1 tyrosine phosphorylation (Fig. 10B, immunoblotted with anti- phosphotyrosine antibody (upper rows) or anti-IRS-1 antibody (lower rows). Controls with DMSO (D) and medium (M) but without insulin or cytokine treatment are shown on the left side). However, prior exposure to 10 or 20 ng/mL TNFa substantially reduced pTyr-IRS-1 content in response to insulin stimulation. That reduction in the normal response to insulin was completely restored in the presence of 100 nM Compound A. As noted above, these changes also occurred without parallel variations in the total amount of IRS-1 protein. Treatment of differentiated 3T3-L1 adipocytes with TNFa was associated with a rapid increase in serine (Ser307) phosphorylation of IRS-1 (Fig. 10C, immunoblotted with anti- phosphoserine (Ser307) antibody (upper rows) or anti-IRS-1 antibody (lower rows).

Control without cytokine treatment is shown on the first lane (Ctrl)). Importantly, prior exposure (30 min) to 100 nM Compound A was sufficient to abrogate this effect, particularly after 15 min of cytokine challenge. Although the observed decrease in Ser307 IRS-1 phosphorylation in the presence of Compound A was quite robust at this time point, the effect appeared to be transient.

[245] Oral glucose tolerance tests: Glucose tolerance is assessed by standard OGTT after an overnight fast. Mice receive a bolus of glucose (2 g/kg on days 0 and 14 or 1 g/kg on days 28 or longer) by oral gavage, and blood samples were collected by tail nick 15, 30, 60, and 120 min thereafter. A blood sample for baseline glucose (time 0) was also collected before initiating the OGTT. Blood glucose levels were measured with a glucometer (OneTouch Ultra Meter™; LifeScan, Milpitas, CA), but samples that were > 600 mg/dl were collected separately with heparin-coated microcapillary tubes and processed by a standard enzymatic method (Sigma-Aldrich, St. Louis, MO).

[246] Glucose-lowering activity of a test compound is initially evaluated in two studies using C57BLKs/J-m Lep^ (db/db) mice, which is exemplified for Compound A as follows. Compound A was orally administered by gavage in doses ranging from 20 to 80 mg/kg b.i.d. For the first study, 8-week-old diabetic db/db mice were treated with Compound A (40 or 80 mg/kg b.i.d. for 28 days) by oral gavage twice daily for 28 days. Nonfasting blood glucose levels and daily body weight are shown in Fig. 1 1 A and 1 1 B, respectively,) for animals (N = 10 per group) treated with vehicle (open circle), 40 mg/kg Compound A (open square), 80 mg/kg Compound A (filled square), or 25 mg/kg rosiglitazone (open triangle). In the first study, treatment with Compound A (40 or 80 mg/kg b.i.d) significantly reduced progression of hyperglycemia (P < 0.01 ) at days 10 and 21 . In contrast, vehicle- treated animals showed a steady increase in blood glucose levels, reaching 350 to 400 mg/dl (Fig. 1 1 A). In the second study younger (6 weeks old or younger), "prediabetic" db/db mice are treated with Compound A (20 or 40 mg/kg b.i.d. for 28 days) by oral gavage twice daily for 28 days. Nonfasting blood glucose levels and daily body weight are shown in Fig. 10C and 10D, respectively, for animals (N = 9 per group) treated with vehicle (open circle), 20 mg/kg Compound A (filled circle), 40 mg/kg Compound A (open square) or 25 mg/kg rosiglitazone (open triangle). Arrows in 1 1 D indicate the day of OGTT studies summarized in Figure 12. Data in Figure 1 1 are shown as mean ± S.E.M. ^*, P < 0.05; ^**, P < 0.01 ; ^***, P < 0.001 with respect to vehicle. As expected, animals treated with rosiglitazone (25 mg/kg b.i.d.) showed normal glucose levels throughout the second study (Fig. 1 1 C). In contrast, vehicle-treated mice became hyperglycemic with glucose levels exceeding 450 mg/dl after 32 days. The glucose-lowering effects of Compound A in both studies occurred in the absence of significant changes in body weight because its rate of accretion was virtually the same among all groups (Fig. 1 1 B and 1 1 D).

[247] The OGTT profile in the second study are shown in Fig. 12A for animals (N = 9 per group) before dosing or after 14 and 28 days of treatment with vehicle (open circle), 20 mg/kg Compound A (filled circle), 40 mg/kg Compound A (open square) or 25 mg/kg rosiglitazone (open triangle). Compound A (20 or 40 mg/kg b.i.d.) maintained blood glucose levels below 200 mg/dl at all times, comparable with lean db/+ control animals (Fig. 12C; P < 0.001 versus vehicle). In addition, OGTTs indicated that Compound A, like rosiglitazone, markedly enhanced glucose clearance in db/db mice after 14 days (P > 0.05) or 28 days (P > 0.01 ) of treatment (Fig. 12A and 1 1 B). Fig. 12B shows blood glucose AUC values during OGTTs performed at the indicated days of treatment. Data are expressed as area (mean ± S.E.M.) under each of the OGTT curves for animals treated with vehicle (V), 20 mg/kg Compound A (20), 40 mg/kg Compound A (40), or 25 mg/kg rosiglitazone (R). To determine whether those effects may have resulted from

amelioration of insulin resistance, serum insulin levels were also measured (Fig. 12C). Figure 12C shows serum insulin levels after 4 weeks of treatment with Compound A. For comparison, insulin levels in db/+ lean animals are also shown. Data are shown as mean ± S.E.M. ^*, P < 0.05; ^**, P < 0.01 ; ^***, P < 0.001 with respect to vehicle. Treatment with Compound A for 4 weeks, like rosiglitazone, caused a significant reduction in insulin levels compared with vehicle (P < 0.01 -0.001 ).

[248] Glucose utilization was assessed by OGTT in male C57BL/6J DIO-mice and is given by Fig. 13A and 13B and for genetically obese ob/ob mice by Fig. 13C and 13D). Shown in Figures 13A and 13C are OGTT profiles from DIO (Fig 13A) or ob/ob (Fig. 12C) mice treated with vehicle (open circle, N = 7), 80 mg/kg Compound A (filled square, N = 7), or 10 mg/kg rosiglitazone (open triangle, N = 9) twice daily for 4 weeks. Shown in Figures 13B and 13D are blood glucose AUC values during OGTTs performed after treatment for DIO (Fig. 13B) or ob/ob mice (Fig. 13D). Data are shown as mean ± S.E.M. ^*, P < 0.05; ^**, P < 0.01 ; ^***, P < 0.001 with respect to vehicle. As shown in Fig. 13A and 13B, treatment of DIO mice with Compound A significantly enhanced glucose clearance (P < 0.05-0.001 ). Moreover, a reduction of basal blood glucose levels was already apparent in the Compound A-treated mice before gavaging the glucose load (see Fig. 13A, time 0).

[249] OGTT studies thus indicate that Compound A delays progression to

hyperglycemia in db/db mice and enhances glucose tolerance in db/db and ob/ob mice in a similar fashion as rosiglitazone, and also markedly reduces serum insulin levels.

[250] Example 5. Inflammation and Insulin Resistance Test Systems-Zucker rats.

[251 ] Macrophage pro-inflammatory kinase activity inhibition: Murine primary

macrophages were elicited by intraperitoneal injection of thioglycolate (3 mL/mouse) in C57BL/6J mice. Macrophages were obtained from intraperitoneal lavage and washed twice. Cells were cultured in RPMI supplemented with 10% fetal bovine serum (FBS) for 3 days and then starved in RPMI supplemented with 0.5% FBS for overnight before the treatment. RAW 264.7 cells and 3T3-L1 cells were cultured as described previously in Nguyen, M.T. et al. "A subpopulation of macrophages infiltrates hypertrophic adipose tissue and is activated by free fatty acids via Toll-like receptors 2 and 4 and JNK- dependent pathways" J. Biol. Chem. (2007) 282: 35279-35292 and Yoshizaki, T. et al. "SIRT1 exerts anti-inflammatory effects and improves insulin sensitivity in adipocytes. Mol. Cell Biol. (2009) 29: 1363-1374.

[252] Treatment of primary murine intraperitoneal macrophages with 100 ng/mL of the Toll-like receptor 4 ligand LPS broadly activates proinflammatory signaling cascades, including phosphorylation of IKK and MAPKs such as JNK, p38, and extracellular signal- regulated kinases (ERK) as shown in Figure 14. Pretreatment with 100 nM Compound A in comparison to vehicle (DMSO) partially, but significantly, blocked the activation of IKK, JNK, p38, and ERK (Fig. 14A). Although ΙκΒ phosphorylation and degradation were not influenced by Compound A treatment, NF-κΒ phosphorylation was attenuated. Consistent with that chromatin immunoprecipitation studies showed that Compound A treatment led to decreased NF-κΒ occupancy of the κΒ site in the IL-6 promoter. When macrophages were treated with Compound A alone, no activation of phospho-JNK was detected demonstrating that the inhibition of LPS-induced inflammation by Compound A is unlikely due to macrophage tolerance caused by preconditioning.

[253] TNFa stimulates inflammation and activates NF-κΒ, Akt, and MAPKs by binding to TNF receptor 1 and 2. Of note, LPS-activated macrophages secrete TNFa, which then stimulates macrophages in an autocrine or paracrine fashion. When TNFa was

immunodepleted in the culture media, the effect of LPS to stimulate inflammatory signaling was reduced significantly. At the same time, when TNFa was neutralized, no additional inhibition of phospho-JNK and phospho-NF-κΒ by Compound A treatment was observed. Inhibition of signal transduction TNFa Compound A is indicated by no observed alteration in LPS-induced TNFa secretion. That is further shown in Figure 15 by decreased phosphorylation of IKK, NF-κΒ, and p38 but not JNK in murine macrophages when pretreated 100 nM Compound A, followed by stimulation with 10 ng/mL TNFa in comparison to vehicle treatment. Although changes in phosphorylation or degradation of IKB with Compound A treatment were not observed, the recovery of ΙκΒ appeared to be enhanced (cf. DMSO and Compound A-treated cells at 30 and 60 min in Fig. 15A).

[254] For Western blotting, Cells were serum starved and pretreated with DMSO or Compound A (100 nM) overnight before LPS (100 ng/ml) or TNFa (10 ng/ml) stimulation for the indicated times. Cells were lysed, and total cell lysates were subjected to Western blotting, as described previously in Lu, M. "Adiponectin activates adenosine

monophosphate-activated protein kinase and decreases luteinizing hormone secretion in LbetaT2 gonadotropes" Mol. Endocrinol. (2008) 22: 760-771 . TNFa and IL-1 β were measured by ELISA assays (Biosource).

[255] Gene expression analyses: Total RNA was extracted from tissue using the

Purelink™ total RNA purification system (Invitrogen, Calsbad, CA). PCR was carried out on an MJ Research Chromo4 real-time PCR system (Bio-Rad Laboratories, Hercules, CA). The mRNA expression of all genes reported was normalized to multiple

housekeeping genes (34B4, RNA polymerase II, and cyclophilin A), and comparable results were observed. For quantitative nuclease protection assay, cells were lysed in lysis buffer after various treatments. The quantitative nuclease protection assay

ArrayPlate™ assays were performed by High Throughput Genomics Macrophage chemotaxis assay. Differentiated 3T3-L1 adipocytes (day 1 1 post-differentiation) were incubated for 24 h with compounds (10 ng/ml TNFa with or without 100 nM Compound A) or vehicle in DMEM with 0.2% FFA- and endotoxin-free BSA.

[256] Consistent with its effect on NF-kB transactivation, Compound A negatively modulates LPS-induced transcription of various inflammatory genes such as IL1 β, IL6, IL12, Tnfa, Nos2, CxcM O, and CxcM as shown in Fig. 14C. Consistent with its effects on attenuating TNFa signaling Compound A negatively modulates expression of IL1 b, CxcM O, Vcaml , Mmp9, and Ccl2 induced by TNFa as shown in Fig. 15C, thus indicating that Compound A ameliorates intracellular inflammatory responses elicited by Toll-like receptor 4 signaling. In accordance with activation of proinflammatory signaling cascades by LPS and TNFa, Compound A negatively modulates phosphorylation of IKK and MAPKs such as JNK, p38, and extracellular signal-regulated kinases (ERK) as shown in Fig. 14 (A and B) and Fig. 15 (A and B), respectively. Therefore, it appears that Compound A causes inhibition of the macrophage inflammatory program primarily by inhibiting TNFa action and NF-kB transactivation. The negative modulation on Erk activation is further supported by the SI LAC test system described herein showing binding (directly or indirectly) to Mapkl and Mapk3, although in a cell- and scaffold free suitable test system there is no inhibition of substrate phosphorylation by phospho-Erk.

[257] The decrease in adipose tissue macrophages as shown herein for Zucker rats is accompanied by decreased F4/80 mRNA expression in Compound A- and rosiglitazone- treated animals (Fig. 16A). Moreover, there was a striking decrease in a variety of inflammatory markers, including TNFa and MCP-1 , in adipose tissue from both treated groups. M1 macrophage markers (NOS2, CXCL1 , and IL-1 ) and M2 markers (arginase-1 and IL-10) were comparably reduced by Compound A treatment, and the ratio of M1 /M2 markers remained the same. In contrast to these broad changes in adipose tissue inflammatory markers, expression of genes such as glucose transporter 4, adiponectin, adipose triglyceride lipase, and hormone-sensitive lipase was not altered by Compound A treatment. In contrast, rosiglitazone treatment led to an increase in glucose transporter 4 and adipose triglyceride lipase expression as well as very large increases in the lipogenic program, as measured by fatty acid synthase and acetyl-CoA carboxylase expression. Consistent with the decrease in inflammatory gene expression profile in the adipose tissue, circulating TNFa and IL-1 β levels were also markedly reduced in the treated rats (Fig. 16B). In contrast to the effects of rosiglitazone treatment, which deceases adipocyte size, there was no significant difference in adipocyte size after Compound A treatment that would account for the observed changes in gene expression. To determine whether Compound A treatment also produces anti-inflammatory effects in the liver, the hepatic expression of inflammatory cytokines such as TNFa, IL-1 β, IL-6, CXCL1 , and MCP-1 was determined. As in adipose tissue, Compound A led to suppression of inflammatory programs (Fig. 16C). Rosiglitazone treatment led to qualitatively similar, but quantitatively less marked, effects. In Fig. 16 statistical significance vs. vehicle-treated rats is indicated by ^*P < 0.05,†P < 0.01 , or†P < 0.001 .

[258] Macrophage chemotaxis test system: Increased macrophage infiltration and accumulation in adipose tissue occurs in obesity. To determine the effect of Compound A in regulating macrophage chemotaxis, conditioned media (CM) from 3T3-L1 adipocytes was used to induce chemotaxis of RAW 264.7 monocyte/macrophages. Chemotaxis assay was performed as described previously in Patsouris, D. et al. "Glucocorticoids and thiazolidinediones interfere with adipocyte-mediated macrophage chemotaxis and recruitment" J. Biol. C em. (2009) 284: 31223-31235.

[259] Figure 17A shows that CM from TNFa -treated (10 ng/mL) adipocytes markedly stimulated macrophage migration, which was reduced by 30% (P < 0.05) when adipocytes were pretreated with Compound A (100 nM). In addition, adipocyte secretion as measured by ELISA of inflammatory cytokines, such as MCP-1/CCL2 and chemokine (C-C motif) ligand 5 (CCL5/regulated upon activation, normal T cell expressed and secreted or RANTES), was augmented in TNFa -treated adipocyte CM and was significantly impaired by Compound A-pretreatment of the adipocytes (Fig. 16, B and C, respectively)

[260] Animals: The study was staggered into six cohorts conducted on different days. Forty-two male ZDF rats and six Zucker fatty rats at 7 wk of age were housed individually on a 12:12-h light-dark cycle with the lights on at 0600 and were fed ad libitum except during the experiments. After 1 wk of acclimation, the ZDF rats began daily oral treatment for 32-35 days with vehicle (n = 18), 100 mg/kg/day Compound A (n = 15), or 10 mg/kg/day rosiglitazone (n = 9). The ZDF rat, a model of obesity, insulin resistance, and diabetes, is known to develop hyperinsulinemia at 8-9 wk of age and hyperglycemia after 9-10 wk of age. The treatment was initiated at 8 wk of age so that the progression of diabetes in the animals could be evaluated.

[261 ] Insulin and glucose tolerance test systems: Effects of Compound A on insulin secretion and glucose utilization were studied using the insulin tolerance test (ITT) and the oral glucose tolerance test (OGTT). Glucose, insulin, and pyruvate tolerance tests were performed on 6-h-fasted rats. For glucose tolerance test (GTT), animals were orally gavaged with glucose (1 g/kg), whereas for insulin tolerance test (ITT), 0.35 U/kg insulin was injected intraperitoneally. Eight-week-old male ZDF rats were administered orally with vehicle (n = 12), Compound A (100 mg/kg/-day; n = 1 1 ), or rosiglitazone (Rosi; 10 mg/kg/day; n = 8) for 30 days. On day 7, glucose (Fig. 18A) and insulin levels (Fig. 18B) were measured at fed, 6-h fasting, and re-fed state (6-h re-feeding after overnight fasting). Compound A treatment completely normalized fasting and fed glucose levels throughout the study. Thus, 1 wk of treatment with Compound A was sufficient to normalize fasting and fed glucose levels as well as plasma insulin levels. At 10 wk of age (day 14 during treatment), vehicle-treated ZDF rats began to exhibit fasting hyperglycemia, whereas Compound A-treated animals had reduced glycemia both in the fasting state and after glucose administration (Fig. 18C). Glucose-induced insulin levels were also reduced. In those studies, the effects of Compound A were comparable with those of rosiglitazone administration (Fig. 18, A and C). ITTs were performed on a subset of the treated rats on day 21 . Upon insulin injection, both Compound A- and rosiglitazone-treated rats showed enhanced glucose clearance (Fig. 18D, data shown are the percentages compared with glucose levels at 0 min), consistent with improved insulin sensitivity. Rosiglitazone, a known insulin sensitizer, was used as a positive control and had the expected effects to ameliorate hyperglycemia and hyperinsulinemia. However, rosiglitazone treatment also caused increased weight gain, whereas body weight of the vehicle- and Compound A- treated rats was comparable.

[262] Hepatic qluconeoqenesis test systems: Since it has been suggested that fasting glucose level and the area under the curve for the first 30 min during a OGTT largely represent insulin effects on the liver, Compound A should also regulate hepatic glucose production (HGP) by reducing gluconeogenic substrate levels. In ZDF rats, glycerol is a key gluconeogenic substrate for increased HGP, whereas other substrates such as lactate and pyruvate also contribute. To address that issue, plasma levels of glycerol, lactate, and pyruvate were measured on day 14. Fasting and fed glycerol levels were both markedly reduced by Compound A treatment (Fig. 18E). Likewise, lactate and pyruvate levels were also decreased (Fig. 19A). Rosiglitazone also effectively reduced the level of

gluconeogenic substrates. In addition, the ability of insulin to suppress HGP was enhanced in the treated rats (Fig. 19B). Glycerol levels were measured using an assay kit from Sigma. Lactate and pyruvate levels were measured by colorimetric assay kits obtained from Biovision. Statistical significance vs. vehicle-treated rats is indicated by an asterisk (p < 0.05) , a single dagger (p < 0.01 ) or double dagger (p < 0.001 ).

[263] Glucose clamp test systems: Rat hyperinsulinemic euglycemic clamp studies were performed as described previously in Satoh, H. et al. "Adenovirus-mediated adiponectin expression augments skeletal muscle insulin sensitivity in male Wistar rats" Diabetes (2005) 54: 1304-1313 with modifications. Briefly, dual jugular venous cannulae and one carotid arterial cannula were implanted in rats. After 4-5 days of recovery, the

hyperinsulinemic euglycemic clamp experiments were begun with a priming injection (7.5 μϋί/0.2 ml_) and constant infusion (0.25 μΟίΛηίη) of D-[3-³H]-glucose. After 60 min of tracer equilibration and basal sampling at t = -10 and 0 min, glucose (50% dextrose, variable infusion) and tracer (0.25 μΟίΛηίη) plus insulin (20 mU/kg/min) were infused into the jugular vein. The achievement of steady-state conditions (100 mg/dl-5 mg/dl) was confirmed at the end of the clamp by measuring blood glucose every 10 min and ensuring that steady state for glucose infusion and plasma glucose levels was maintained for a minimum of 30 min. Blood samples were taken at t = -60 (start of experiment), -10, 0 (basal), 1 10, and 120 min (end of experiment) to determine glucose-specific activity and insulin and free fatty acid ( ) levels. All blood samples were immediately centrifuged, and plasma was stored at -80 'C for subsequent analysis. Following a 3-day recovery after clamp, rats were fasted for 6 h and euthanized. Tissues were harvested at basal state or acute insulin-stimulated state (5 U/kg). Glucose, insulin, and FFA levels were measured as described previously in Patsouris, D. et al. "Ablation of CD1 1 c-positive cells normalizes insulin sensitivity in obese insulin resistant animals. Cell. Metab. (2008) 8: 301-309.

[264] Euglycemic hyperinsulinemic clamp studies provide a quantitative measurement of in vivo insulin sensitivity. Thus, ZDF rats treated with vehicle (n = 14), Compound A (n =1 1 ), or Rosiglitazone (n = 8) for 4 wk were subjected to euglycemic hyperinsulinemic clamps. Glucose infusion rate (GIR; Fig. 20A), insulin-stimulated glucose disposal rate (GDR; Fig 19B), basal hepatic glucose production (HGP; Fig. 20C), and HGP suppression by insulin (Figure 19D) are shown as means ± SE. Statistical significance vs. vehicle- treated rats is indicated by ^*P < 0.05,†P < 0.01 , or†P < 0.001 .

[265] As summarized in Fig. 20, after 4 wk of treatment, both Compound A and rosiglitazone treatment led to an increase in the glucose infusion rate and insulin- stimulated glucose disposal rate (IS-GDR), with the effects of rosiglitazone being more robust (Fig. 20, A and B). Since 70-80% of IS-GDR is attributable to skeletal muscle, this implies that Compound A treatment improves skeletal muscle insulin action. In support of this, Akt phosphorylation was enhanced in insulin-stimulated skeletal muscle and liver (Fig. 20E). Importantly, basal rates of HGP were markedly and equally reduced by Compound A and rosiglitazone treatment (Fig. 20C). Since basal HGP is the major contributor to basal hyperglycemia, those results are fully consistent with the marked reduction in basal glucose levels. [266] In humans, 10 obese, insulin-resistant subjects with impaired glucose tolerance (according to standard American Diabetes Association OGTT criteria) were treated with Compound A (5 mg BID, n = 5, or 10 mg BID, n = 5) for 4 weeks. Hyperinsulinemic euglycemic glucose clamp studies were performed on the day prior to treatment and on day 28 of treatment on 4 male and 6 female subjects of 49.9 ± 8.9 years of age with body mass index (BMI) = 33.6 + 3.0 Kg/m², fasting plasma glucose (FPG) = 102 ± 10.1 mg/dL, and fasting plasma insulin = 25.1 ± 12.2 μΙΙ/mL The results show a significant 34% increase (P < 0.0304) in the glucose infusion rate (M value) in these patients. Statistical significance was calculated using a paired (2-tailed) t test. The clamp study data in insulin- resistant subjects are fully consistent with data from the macrophage in vitro and ZDF rat test systems. Furthermore, results from the gluconeogenesis and glucose clamp test systems support the conclusion that Compound A treatment leads to robust effects on hepatic glucose metabolism, which result in a marked reduction in gluconeogenic flux, and near normalization of hyperglycemia and enhanced hepatic insulin sensitivity.

[267] Quantitative Lipidomics: In addition to glycerol, lactate and pyruvate, FFAs can contribute indirectly to gluconeogenesis by interfering with insulin suppression on HGP and by providing an energy source for ATP generation. Dyslipidemia usually coexists with insulin resistance/diabetes, and increased tissue lipid accumulation has been

demonstrated in several animal models of obesity and diabetes, including ZDF rats.

Therefore, lipid profiles were determined in three key insulin-responsive tissues: liver, fat, and skeletal muscle.

[268] Quantitative lipidomic studies were performed by Lipomics Technologies, as described previously in Cao, H. et al. "Identification of a lipokine, a lipid hormone linking adipose tissue to systemic metabolism" Cell (2008) 134: 933-944. Briefly, lipids from serum and tissues were extracted in the presence of internal standards with chloroform- methanol (2:1 vol/vol) as described in Folch, J. et al. "A simple method for the isolation and purification of total lipids from animal tissues" J. Biol. Chem. (1957) 226: 497-509, 1957. Individual lipid classes were separated by high-performance liquid chromatography. Each lipid class fraction was transesterified in 1 % sulfuric acid in methanol in a sealed vial under nitrogen at l OO'C for 45 min. The fatty acid methyl esters were extracted from the mixture with hexane containing 0.05% butylated hydroxytoluene and prepared for gas chromatography under nitrogen. Fatty acid methyl esters were then separated and quantified by capillary gas chromatography equipped with a 30-m DB-88MS capillary column and a flame ionization detector.

[269] Figure 21 A shows that Compound A treatment led to a marked decrease in intracellular triacylglycerol content in livers, as did rosiglitazone. Compound A treatment significantly reduced hepatic cholesteryl ester (CE) levels by 73%, and as a result, total cholesterol content in the liver was reduced by 15%. Interestingly, Compound A treatment led to a marked reduction in serum CEs and total cholesterol (CT) levels (by 59 and 25%, respectively) despite an increase in serum free cholesterol (FC) levels (Fig. 21 B). The opposite is true for rosiglitazone treatment. Gene expression measurements

demonstrated enhanced low-density lipoprotein receptor (LDLR) and HMG-CoA reductase expression with Compound A treatment (Fig. 16C). In Figures 21 A and 21 B abbreviations used are TAG, triacylglycerol; PC, phosphatidylcholine; PE, phosphatidylethanolamine; CE, cholesteryl ester; FC, free cholesterol; TC, total cholesterol. On the basis of those results it appear that hepatic LDLR and HMG-CoA reductase expression induced by Compound A resulted from activation of sterol regulatory element-binding protein-2 (SREBP-2).

[270] Since the mRNA level of SREBP-2, a key transcription factor regulating both HMG CoA reductase and LDLR, remained unchanged (Fig. 16C), we believe without being bound by theory that Compound A treatment leads to inhibition of cholesterol synthesis with proteolytic activation of SREBP-2 as a feedback response to low intrahepatic cholesterol. In support of that belief protein levels of SREBP-2 were in treated rats were measured. Figure 22A shows protein levels of (P) and mature form (M) of SREBP-2 by immunoblotting in Compound A treated rats. As positive control for blotting culture cells were infected with adenovirus expressing SREBP-2 NH2 terminus (Ad-2N). Thus,

Compound A treatment enhanced the level of nuclear SREBP-2 (nuclear form) in the liver, indicating an increase in SREBP-2 protein cleavage and activation.

[271 ] To further understand the effect of Compound A and rosiglitazone on lipid metabolism, tissue fatty acid composition was examined. Figure 23A demonstrates that Compound A decreased the levels of palmitate (16:0), palmitoleate (16:1 n7), and linolenate (18:3n3) in the liver, whereas arachidonic acid (20:4n6) and docosahexaenoic acid (22:6n3) were increased. Interestingly, treatment with rosiglitazone increased only the level of eicosapentaenoic acid (20:5n3) but not docosahexaenoic acid. Figure 23B summarizes the fatty acid levels in epididymal white adipose tissue. It has been suggested that palmitoleate (16:1 n7) is a lipokine that can promote insulin sensitivity, and an elevation of palmitoleate levels was found in both Compound A- and rosiglitazone- treated animals. Since 16:1 n7 is an indicator of de novo lipogenesis, these results imply enhanced adipocyte lipogenesis in the treated rats.

[272] Since fatty acids released by adipose tissue are the dominant source of circulating fatty acids, one would expect serum FFA patterns to largely mirror adipose tissue. Thus, an increase in palmitoleate levels in FFAs in Compound A- and rosiglitazone- treated rats (Fig. 23C) was observed, reflecting the corresponding changes in adipose tissue. De novo fatty acid synthesis in muscle was negligible, and therefore, the fatty composition of muscle was determined largely by circulating FFAs and VLDL. Indeed, an increase in palmitoleate levels in several lipid classes in skeletal muscle was found (Fig. 23D). In Fig. 23 statistical significance vs. vehicle-treated rats is indicated by ^*P < 0.05,†P < 0.01 , or †P < 0.001 and abbreviations used are CL, cardiolipin; DAG, diacylglycerol; FFA, free fatty acid; LYPC, lysophosphatidylcholine; PS, phosphatidylserine.

[273] Immunohistochemistry: Immunohistochemistry studies were conducted as described previously in Patsouris, D (2008), op cit. Paraffin-embedded epididymal adipose tissue sections were incubated with Mac-2 antibody with a 1 :100 dilution overnight at 4° C. Increased adipose tissue macrophage (ATM) content is the central component of the chronic tissue inflammatory state in obesity. Thus, modification of macrophage-mediated inflammation in vivo was determined by histological analysis of adipose tissue sections from control and treated rats. As seen in Fig. 22B, there was a marked reduction in ATM content, as measured by staining for the macrophage-specific marker Mac-2 in the treated rats. That directly demonstrates decreased ATM

accumulation by Compound A treatment.

[274] Example 6. Inflammation and Autoimmunity Test Systems

[275] Adjuvant induced arthritis (AIA) test system: Rat AIA study was performed according to the procedures described in Offner, H. et al. "An orally bioavailable synthetic analog of an active dehydroepiandrosterone metabolite reduces established disease in rodent models of rheumatoid arthritis. J. Pharmacol. Exp. Ther. (2009) 329: 1 100-1 109. A total of 40 male Lewis rats ranging in weight from 162 to 215 grams, were used for the study. The rats were housed in a controlled environment (non-specific pathogen free) and provided with standard rodent chow and water. AIA was induced in Lewis rats by a single s.c. injection (0.1 mL) of heat-killed Mycobacterium tuberculosis H37Ra (0.3 mg) in Freund's incomplete adjuvant into the base of the tail. Rats were divided randomly into two groups and treated orally (gavage) with either Compound A (25 mg/kg) suspended in vehicle (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water) or with vehicle alone to a final volume of 0.125 mL. Two experimental regimens were investigated. One regimen occurs early in the disease process, and the second occurs late in the process. For the early regimen, the rats were treated on day 8 post-immunization and in the late regimen, the rats were treated on day 15 post-immunization. In both cases treatments was given twice daily for 15 consecutive days. On the first day of treatment the rats were randomly allocated to the different experimental groups. Post-randomization analysis showed no significant differences in the mean arthritic score or for paw edema between the different groups. Rats were evaluated daily for arthritis using a macroscopic scoring system: 0, no signs of arthritis; 1 , swelling and/or redness of the paw or one digit; 2, two joints involved; 3, more than two joints involved; and 4, severe arthritis of the entire paw and digits. Arthritic index for each rat was calculated by adding the 4 scores of individual paws. Rats were weighed daily, beginning from the day of immunization. Clinical disease severity was determined using plethysmography. The change in left hind paw volume was determined on a weekly basis.

[276] Results from the early and late regimens are presented in Figures 23 and 24, respectively. The data in Fig. 24A and 24B demonstrated a time-dependent increase in both left hind paw volume (mL) and clinical arthritic score that were induced by adjuvant in vehicle-treated rats. The arthritis incidence in these animals was 100% (10/10). The rats treated with Compound A early in the disease process exhibited significantly lower paw edema than vehicle-treated rats from day 22 to the end of the study on day 30 (Fig. 24A). Compound A also significantly reduced the arthritic score of the treated rats as compared to vehicle from days 18-25 (Fig. 24B). Upon late therapeutic treatment Compound A exhibited powerful anti-arthritogenic effects and favorably influenced the course of rat AIA. In fact, relative to vehicle-treated controls, the rats treated with Compound A exhibited significantly lower paw edema from day 19 until the end of the study on day 30 (Fig. 25A) with exception of non-significant reductions vs. on days 23 and 24. Compound A also significantly reduced the arthritic score of the treated rats as compared to vehicle from days 19-30 (Fig. 25B) with exception on day 22 where the reduction of the arthritic score was close to, but did not reach statistical significance. The fluctuations in body weight seen in Compound A-treated control rats were comparable to those observed in the vehicle-treated animals.

[277] Collagen antibody induced arthritis (CAIA) test system:

[278] DBA/1 Lac/J mice are used for in vivo rheumatoid arthritis test systems. To induce CIA, 8-week-old male mice are immunized with 200 μg of Bovine type II collagen (bCII) (Chondrex, Inc., Redmond, WA, USA) emulsified 1 :1 with CFA containing 100 μg

Mycobacterium tuberculosis (100 μΙ_; Difco, Detroit, Ml, USA) intradermal^ at the base of the tail. Animals are monitored for onset and progression of disease 3 to 7 weeks post immunization. The arthritic severity of mice is evaluated with a grading system for each paw according to the following scale: 0 = no redness or swelling; 0.5 = groove still visible (mouse is beginning to show signs of disease); 1 = slight swelling in ankle or redness in foot; 2 = progressed swelling/inflammation and redness from ankle to mid foot; 3 = swelling/inflammation of entire foot; 4 = swelling and inflammation of entire foot, including toes. [279] Compound A as an exemplary test compound was dissolved in vehicle (30% β- cyclodextrin sulfobutyl ether sodium salt (w/v) in water) and administered by oral gavage to 12 animals daily starting with disease onset on day 22-29 (in a final volume of 50 μΙ_ 25 mg/kg) and 100 μΙ_ (50 mg/kg) on day 30-49. Control mice were treated with an equal volume of vehicle. The average score at onset of disease was similar for the vehicle and treatment groups, 1 .3 ± 2.1 and 1 .8 ± 3.1 , respectively. Within 10 days of Compound A treatment, mice displayed a significant decrease in CIA score (2.3 per paw in vehicle group vs. 1 .0 in the Compound A-treated mice, P < 0.05). Average score per mouse on day 21 post onset was 6.5 for the vehicle-treated group and 2 for the Compound A-treated group. On day 50, animals were euthanized along with vehicle controls.

[280] Relevant tissue for histological examination is harvested for analysis in the following manner. Fixed tibiotarsal joints are decalcified in formic acid and processed for paraffin embedding. Tissue sections (5 μηι) are stained with hematoxylin and eosin for histopathologic scoring. Joints are scored for synovial proliferation as follows: grade 0: proliferation was absent; grade 1 : mild proliferation with 2-4 layers of reactive

synoviocytes; grade 2: moderate proliferation with 4 plus layers of reactive synoviocytes, increased mitotic activity, and mild or absent synovial cell invasion of adjacent bone and connective tissue; and grade 3: severe proliferation characterized by invasion and effacement of joint space and adjacent cartilage, bone, and connective tissue. Articular inflammation was scored as follows: grade 0: inflammation lacked significant leukocyte infiltrates or aggregates; grade 1 : inflammation was mild with one aggregate or minimal diffuse leukocyte infiltrates; grade 2: inflammation was moderate with two or more aggregates of leukocytes; and grade 3: inflammation was severe with significant coalescing to diffuse infiltrates of leukocytes.

[281 ] For histological examination, four-color (fluorescein isothiocyanate, phycoerythrin, propidium iodide, and allophycocyanin) fluorescence flow cytometry analyses were performed to determine the phenotypes of CD4+CD25+FOXp3+ CD127-spleenocytes (Treg cells) following standard monoclonal antibody staining procedures. After staining, cells were washed with staining medium and analyzed immediately with a FACSCalibur™ using CellQuest™ (Becton-Dickinson, Mountain View, CA, USA) software. Nearly a doubling in the numbers of Treg cells in the Compound A-treated animals compared to the vehicle-treated group was observed.

[282] For example, joints harvested from Compound A-treated mice had fewer infiltrating cells compared to vehicle controls, which showed severe inflammation and synovial hyperplasia with several layers of reactive synovial tissue. The tissue also showed increased mitotic activity and mild synovial invasion of adjacent bone and connective tissue. Diffuse infiltrates of leukocytes, which were absent in the joints of Compound A- treated mice, were also observed in the vehicle controls. The Compound A-treated tissue also revealed reduced synovial proliferation and erosion compared to vehicle controls.

[283] Active suppression of autoreactive T cells by Treg cells is important in the maintenance of self-tolerance, and Treg cells are implicated in a broad range of medical conditions, such as autoimmune disease, graft-versus-host disease, allograft rejection, sterilizing immunity to infectious agents, allergy, and cancer. It has been observed that inhibition of NF-κΒ and oxidative pathways in human dendritic cells generates Treg cells (Tan, P.H. et al. 2005. Inhibition of NF-kappa B and oxidative pathways in human dendritic cells by antioxidative vitamins generates regulatory T cells. J. Immunol. (2005) 174: 7633- 7644. Without being bound by theory it is believed that the ability of Compound A to modulate NF-κΒ activation plays a role in the activation of Treg cells leading to the clinical benefit that has been observed in the Compound A-treated mice with CIA.

[284] For cytokine analysis spleens are cultured at 4 ^χ 10⁶ cells/well in a 24-well flat- bottom culture plate in stimulation medium with 25 μg/mL BCTII (bovine collagen type II) for 48 h. Supernatants are then harvested and stored at -70 °C until tested for cytokines. The Bio-Rad ^"l Oplex Luminex™ kit is used to detect cytokines. Briefly, 50 μΙ_ of sample is incubated with 50 μΙ_ of the mixed capture beads. The beads are washed and left for a short time in the presence of a detection antibody and then strep-avidin HRP. The beads are then resuspended in 125 μΙ_ of the assay buffer before acquisition on the Bio-Plex 200 System. The data are analyzed using the Luminex™ software (Bio-Rad Hercules, CA, USA). Standard curves are generated for each cytokine using the mixed bead standard provided in the kit, and the concentration of cytokine in the cell supernatant is determined by interpolation from the appropriate standard curve. Averages, standard errors, and median and inter-quartile range limits are used for description. Statistical analyses is based on the linear mixed model, with scores at different times as the dependent variables, and treatment time from CAIA onset (as a classification variable), real time to CAIA onset, and the interaction between time from onset and treatment as independent variables.

The ensuing covariance structure to model the time dependence within each individual is assumed to follow a first-order autoregressive moving average process. Results were then confirmed by means of monotonic modeling (rank transformation of the CAIA scores).

[285] In another study with Compound A, DBA/1 Lac/J male mice (5 per group) are treated by gavage daily with either 0.1 ml_ of vehicle (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water) or Compound A (1 , 10 or 40 mg/kg), beginning on day 1 . Animals were treated for 14 days. To induce arthritis, mice were administered 1 mg of an anti-bCII antibody cocktail (Chondrex, Redmond, WA) intravenously on day -2. On day 0, animals were treated intraperitoneal^ (IP) with LPS (12.5 μg) as above. The arthritic severity of the mice was evaluated as in the AIA test system.

[286] In the CAIA model, daily treatment with Compound A (40 mg/kg) produced benefit when compared to animals receiving either vehicle or 1 or 10 mg/kg Compound as shown in Figure 26. In this study, CAIA began to develop by day 3 (score < 4) in both vehicle- and Compound A-treated animals. Disease peaked in all groups on days 7-8, where average scores were between 9 and 12 in both the vehicle-treated and lower dose (1 and 10 mg/kg) Compound A-treated groups. In contrast, the high dose (40 mg/kg) became statistically lower on day 4 (p < 0.031 ) and remained lower for the remainder of the study (day 14). Dose response trends were clearly detectable and statistically significant starting at day 4.

[287] IL-6 and TNF-g determinations: Circulating levels of IL-6 and TNF-a are measured by specific solid phase ELISA kits in undiluted plasma samples from the different groups of rats used in the rheumatoid arthritis test systems on day 0 (prior to immunization), 15th (at the beginning of the treatment) and at the end of the study. Inter- and intra-assay of variations are typically below 10 and 5%, respectively. The assays are carried out according to the manufacturer's instructions (Bender MedSystems, Prodotti Gianni, Milan, Italy). The lower limit of sensitivity is 31 pg/mL for IL-6 and 1 1 .2 pg/mL for TNF-a. To allow evaluation of statistical analysis, those samples with levels below the limit of sensitivity are assigned a value corresponding to the lower limit of sensitivity.

[288] As exemplified for Compound A, late therapeutic treatment in the AIA test system reduced blood levels of IL-6 and TNF-a that were found to be elevated in vehicle-treated controls. IL-6 and TNF-a were undetectable in most of these rats before immunization and increased thereafter with progression of the disease. A fluctuation similar to that seen in the vehicle-treated rats was also seen in the group of rats assigned to Compound A treatment. However, when re-measured at the end of the study after 15 consecutive days of treatment with Compound A the circulating levels of these cytokines were significantly lower in the treated rats as compared to those receiving the vehicle (Figs. 27A and 27B).

[289] Myeloperoxidase activity test system: Neutrophil infiltration into the inflamed joints of rats used in the rheumatoid arthritis test systems are indirectly quantified using an MPO assay in both groups of rats treated with test compound or vehicle at the end of the treatment (day 30). Briefly, the left hind-paw tissue was removed and snap frozen in liquid nitrogen. Upon thawing, the tissue (0.1 g of tissue per 1 .9 mL buffer) was homogenized in buffer (0.1 M NaCI, 0.2 M NaP04 and 0.015 M NaEDTA; pH 4.7), centrifuged at 3000 x g for 10 min and the pellet subjected to hypotonic lysis (1 .5 mL of 0.2% NaCI solution followed 30 sec later by addition of an equal volume of a solution containing 1 .6% NaCI and 5% glucose). After further centrifugation, the pellet was resuspended in 0.05 M NaP04 buffer (pH 5.4) containing 0.5% hexadecyltrimethylammonium bromide (HTAB) and rehomogenized. Aliquots (1 mL) of the suspension were transferred into 1 .5-mL Eppendorf tubes followed by three freeze-thaw cycles using liquid nitrogen. The aliquots were then centrifuged for 15 min at 3000 x g, the pellet was resuspended to a volume of 1 mL and samples of hind paw were assayed. Myeloperoxidase activity in the resuspended pellet was assayed by measuring the change in optical density at 450 nm using tetramethylbenzidine (1 .6 mM) and H₂0₂ (0.5 mM). Effects of late therapeutic treatment with Compound A on reduction of MPO activity in the AIA rat test system is shown by Figure 28.

[290] Experimental autoimmune encephalomyelitis test system: Experimental autoimmune encephalomyelitis (EAE) is a model for the human inflammatory

demyelinating disease, multiple sclerosis (MS). EAE is characterized by inflammation, demyelination, axonal loss and gliosis, and thus recapitulates the pathological features of MS. Resolution of inflammation and remyelination also occur in EAE. Thus, EAE serves as a model for those processes as well (Constantinescu, C.S. et al. "Experimental autoimmune encephalomyelitis (EAE) as a model of multiple sclerosis (MS)" Br. J.

Pharmacol. (201 1 ) 164: 1079-1 106).

[291 ] For the EAE test system SJL mice (10 per group) are treated by gavage with 0.4, 4, and 40 mg/Kg test compound (in 100 μί of vehicle) or with vehicle (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water) alone, as when Compound A is the test compound, starting at onset of EAE to day 34 (post-immunization with 150 μg PLPI200 mg CFA to induce EAE). Some groups of animals also receive ICI 182,780 compound (10 mg/kg/day), which is and ERa inhibitor, given subcutaneously in 10% EtOH and 90% corn oil, beginning one week prior to induction of EAE. The mice are randomized to each experimental group before immunization to induce EAE as described by St. Louis, J. et al. "Tolerance induction by acylated peptides: suppression of EAE in the mouse with palmitoylated PLP peptides" J. Neuroimmunol. (2001 ) 1 15(1 -2):79-90). Briefly, the mice are immunized with PLP emulsified in CFA with 6 mg/mL Mycobacterium tuberculosis H37RA to make a 1 :1 emulsion. Each mouse receives subcutaneous injections of 200 μί emulsion divided among four sites draining into the auxiliary and inguinal lymph nodes. Pertussis toxin is used as a co-adjuvant and is administered IP at the dose of 200 ng/mouse on day 0 and 2 post immunization. The mice are observed every day by measuring clinical signs of EAE until 34 days after immunization. The clinical grading is carried out by an observer unaware of the treatment and are the following: 0 = no illness, 1 = flaccid tail, 2 = moderate paraparesis, 3 = severe paraparesis, 4 = moribund state, 5 = death. For significant differences on clinical scores performed by ANOVA for unpaired data and to the non-parametric Mann-Whitney test a p value < 0.05 is considered to be statistically significant.

[292] As exemplified for Compound A, classical signs of EAE developed in all of the vehicle-treated mice within 26 days post immunization to induce EAE (see Figure 28). The mean day of onset was 18.7 ± 4.5. In this group of animals the duration of the disease was 9.6 ± 5.3 days. The mean cumulative score from day 1 to 34 was 24.5 ±10.9. A more severe course of EAE was observed in mice treated with the ICI compound alone. In that group of animals, the classical signs of EAE also developed in all of animals. The mean day of onset was similar to the vehicle-treated group (day 18.2 ± 3.7). Thus, the duration of the disease was 10.2 ± 5.5 days and the mean cumulative score from day 1 to 34 was 25.7 ±13.9. However, there was a higher peak of disease (see Fig. 29) and higher mortality in the ICI-treated group (3 in the ICI-treated group versus 1 animal in the vehicle- treated group). In contrast, the mice treated with all doses of Compound A, except the lowest dose (0.4 mg/kg), exhibited an improved course of EAE as compared to the vehicle-treated mice, as judged by reductions in incidence, mortality, duration of disease and mean cumulative scores (1 3.7 ± 12.8 for the 40 mg/kg dose [p = 0.045], 15.7 ± 1 1 .0 for the 4 mg/kg group [p = 0.074] and 21 .4 ± 18.5 for the 0.4 mg/kg group [p = 0.662]). Those benefits were observed even though animals in all the Compound A-treated groups had earlier onset of EAE compared to the vehicle-treated group. The observations suggest that the minimally effective dose in this model is approximately 4 mg/kg. The benefit of Compound A treatment was unaffected by the addition of the ICI compound. Thus, mice receiving both Compound A (40 mg/kg) and ICI (10 mg/Kg) treatment had indistinguishable courses of EAE compared to the group that received Compound A alone (mean cumulative scores of 12.5 ± 14.8 [p = 0.041 ] versus 13.7 ± 12.8 [p = 0.045], respectively).

[293] Ulcerative colitis test system: Male Wistar rats (8-10 weeks old, 180 ± 20 g) in 5 groups (10 per group) are administered by oral gavage 30 mg/Kg or 10 mg/Kg test compound (in 125 μΙ_ vehicle), vehicle alone (0.1 % carboxymethylcellulose, 0.9% NaCI, 2% Polysorbate 80, 0.05% phenol), as when Compound A is the test compound, saline (sham treatment) or 300 mg/Kg sulfasalazine (SZ, as positive control) in 2% Tween (10 mL/Kg) daily for 7 days. Distal colitis is induced by intra-colonic instillation of 0.5 ml_ of an ethanolic solution of 2,4-dinitrobenzene sulfonic acid (DNBS; 60 mg/mL, 30% EtOH in saline). The first administration occurs after DNBS challenge. Animals in the sham treated are not challenged with DNBS. Before starting treatment and before sacrifice, the animals are individually weighed and the body weights are recorded. At 24 hours following the last dose animals are euthanized by ether inhalation and the distal colons (10 cm length) are isolated, carefully cleaned from mesenterium, vessels and fat, rinsed with saline, weighed and opened longitudinally by button scissors for macroscopic evaluation. Endpoints include daily clinical signs, body weight, distal colon weight, intestinal adhesion and presence of ulcers at 24 hrs after the last treatment. Microscopic evaluation of the small and large intestines are performed on tissues previously fixed in 10% neutral buffered formalin, embedded in paraffin, sectioned at approximately 3-5 mm and stained with hematoxylin-eosin. The sections are evaluated by light microscopy based upon ulceration and inflammation within the intestinal mucosa. Mucosal Damage Area (MDA) is measured macroscopically and expressed as mm²/rat. Macroscopic Damage Score (MDS) was scored on a 0 to 6 scale according to the following criteria: 0 = no damage; 1 = localized hyperemia and/or edema; 2 = linear ulcer < half of the width of the colon; 3 = linear ulcer > half the width of the colon; 4 = circular ulcer < 1 cm; 5 = circular ulcer between 1 and 2 cm; 6 = circular ulcer > 2 cm. Statistical analysis for significant differences on clinical parameter were performed by Student's t test. A p value < 0.05 was considered to be statistically significant.

[294] As exemplified for Compound A as the test compound, DNBS induced colitis and mucosal necrosis in the colons of all challenged animals. Vehicle and 30-mg/kg

Compound A treatment had similar mean areas of necrosis, 675 ± 304 and 624 ± 275 mm², respectively. In rats treated with 10 mg/kg Compound A or SZ, the area of mucosal damage was significantly reduced (P = 0.023 and 0.037, respectively) compared to the vehicle-treated group (mean necrotic areas of 319 ± 294 and 391 ± 202 mm²,

respectively). There was no necrosis in sham-treated animals. All DNBS-challenged animals had reduced body weight relative to the sham group. Body weights were decreased in the vehicle- (209 ± 17.9 g) and Compound A-treated groups (191 ± 22.8 and 203 ± 26.0 g for the 30 and 10 mg/kg, respectively) but not in the SZ-treated group (223 ± 16.0 g) compared to sham (275 ± 8.1 g). Compound A- (10 mg/kg) and SZ-treated groups also showed reductions in colon weight (2.5 ± 1 .0 g and 3.6 ± 1 .5 g, respectively) compared to the vehicle-treated group (4.9 ± 2.0 g) but only reached statistical significance in the Compound A-treated group (P = 0.005).

[295] The data demonstrate that when administered orally at the dose of 10 mg/kg for 7 consecutive days, starting 30 minutes after rectal application of DNBS, Compound A favorably influenced the development of colitis in that the treated rats exhibited reduced necrotic areas of the colon and lower increase in colon weights as compared to vehicle- treated control rats. The effects of 10 mg/kg Compound A on the development of DNBS- induced colitis were comparable to that of the positive control drug sulfasalazine. [296] Systemic lupus erythematosus (SLE) test system: Systemic lupus erythematosus (SLE) is considered an autoimmune disease (Perry, D., et al. "Murine models of systemic lupus erythematosus" J. Biomed. Biotechnol. (201 1 ) 201 1 :271694. Similar to other autoimmune conditions, its etiology is multifactorial entailing genetic, environmental, hormonal, and immunologic factors. The MRL-lpr/lpr mouse is an important model in the study of lupus, and serves as a unique prototype for research concerning T-cell dysfunction and autoimmune disease. The link between a single gene abnormality and the autoimmune phenotype also increases the value of this particular strain. Important features include lymphoproliferation and infiltration of lymphoid tissues by T cells of the double negative phenotype. Clinically, major manifestations are immune

glomerulonephritis and massive enlargement of the lymph nodes and spleen. Another attractive clinical characteristic of the MRL strain, although not universally present, is rheumatoid arthritis-like manifestations, with production of rheumatoid factor. Parallels to human disease include lymphadenopathy and kidney disease, and specific anti-Sm and anti-ribosomal P antibody production, together with the anti-DNA response. The incidence of lethality usually reaches 50% by the 25th week of age in female mice. Although the exact defects that give rise to disease in this rodent model are unknown, they are thought to include a mutation in FAS ligand, which regulates cellular apoptosis. It is interesting to note that MRL-lpr/lpr mice also develop a synovial inflammation that has been thought of as a model for rheumatoid arthritis.

[297] Male mice 12 weeks of age are divided into 4 groups of 10 animal each. The animals are maintained under standard laboratory conditions (non-specific pathogen free) with free access to food and water. The animal groups are treated with 20 mg/Kg test compound in 100 μί vehicle, vehicle alone (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water as when Compound A is the test compound), cyclosporin (as positive control) or saline (sham treated). Animals are first dosed with test compound as they develop clinical signs of SLE as assessed by the appearance of proteinuria at week 12 of age. This is a spontaneous model in that MRL-lpr/lpr mice naturally develop disease without any challenge. Mice are treated daily with test compound from the age of 12 until the age of 25 weeks. Development of proteinuria is tested to monitor renal disease by using urine test paper and is clinically scored as follows: negative (0-30 mg/dL), 1 + (30- 100 mg/dL), 2+ (100-300 mg/dL), and 3+ (300-1000 mg/dL). Parameters are subjected to ANOVA with Duncan's new multiple-range post hoc testing between groups. Differences are deemed significant when p < 0.05. Mice are checked for proteinuria weekly through the study period. [298] As exemplified for Compound A as the test compound, urine protein scores of all groups were between 0.94 and 1 .8 at the beginning of treatment (Figure 30A). In the untreated and vehicle treated groups, protein scores rose and fell during the course of the study, peaking at about 2.75 on the 1 1 th week of the study post SLE appearance. In contrast, protein score for the Compound A treated group never exceeded 1 .5, and peaked at 1 .2. That was similar to the cyclosporin treated group (positive control), which exceeded a protein score of 1 .5 (i.e., 1 .56) for only for one time point. Furthermore, treatment with Compound A, like treatment with cyclosporin, was associated with a survival advantage in that treated animals appeared to succumb to disease more slowly than did untreated animals, or animals treated with vehicle alone (Figure 30B).

[299] Autoimmune diabetes test system: The female non-obese diabetic (NOD) mouse is a good model for T1 DM, which resembles the human disease by sharing predisposing genetic factors and characteristics of disease initiation and progression. Diabetes in that model develops spontaneously as a result of pathogenic autoreactive T cells of the Th1 and Th17 phenotype recognizing pancreatic islet β-cell autoantigens, such as portions of insulin and glutamic acid decarboxylase (GAD)65, which promotes insulitis, the initial stage of the disease in which inflammatory leukocytes infiltrate the pancreas and are responsible for lesions within islets and the destruction of insulin-producing β-cells. That occurs from NOD mice having a mutation in exon 2 of the CTLA-4 gene, which causes it to be spliced incorrectly. Since CTLA-4 plays a major role in suppressing the T-cell immune response, the mutation in that gene results in T-cell attack of the insulin producing cells, which leads to Type 1 diabetes (Brode, S. et al. "Cyclophosphamide- induced Type-1 diabetes in the NOD mouse Is associated with a reduction of

CD4+CD25+Eoxp3+ regulatory T cells" J. Immunol. (2006) 177: 6603-6612; Kikutani, H. and Makinom, S. "The murine autoimmune diabetes model: NOD and related strains" Adv. Immunol. (1992) 51 : 285-322). Therefore, therapies designed to intervene in the insulitis from leukocyte infiltration of the pancreatic islets could provide benefit to T1 DM patients.

[300] Female NOD mice (N = 12 per group) are treated by oral gavage with 80 mg/kg test compound A in 100 μΙ_ vehicle or vehicle alone (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water), as when Compound A is the test compound, or with 0.25 mg/Kg dexamethasone (Dex) IP (as positive control) once daily from 15 to 24 weeks of age. Glycosuria and fasting glycemia were measured twice weekly. Mice were defined as diabetic when positive for glycosuria and with fasting glycemia above 1 1 .8 mmol for two consecutive days. The animals are housed under pathogen-free conditions and

acclimated for at least one week prior to initiation of dosing. [301 ] As exemplified for Compound A as the test compound, 25% of vehicle-treated and 8.6% of Compound A-treated mice developed diabetes (Figure 31 A) by week 19. None of the Dex-treated animals had developed diabetes at that time point. By the end of the study (week 24), 66.7% of the vehicle-treated animals had become diabetic. In contrast, only 25% of the Compound A-treated animals had developed disease, as compared to 8.3% of the Dex-treated animals.

[302] In another study with Compound A six-week old female NOD mice (approximately 20-25 grams) were randomized into 5 per cage upon receipt and cages were

subsequently randomized into 4 dosing groups (N = 15 per group). Female NOD mice were treated once daily with vehicle, Compound A (20 or 80 mg/kg in 100 μΙ_, p.o.) or dexamethasone (0.25 mg/kg, 100 μΙ_, i.p.) from 12 to 26 weeks of age (9 days after the first incidence of diabetes). Blood glucose was monitored using glucometers (ENCORE™ Glucometer, Bayer Corp., Elkhart, IN) at weekly intervals, beginning at 10 weeks of age. Mice with blood glucose levels > 200 mg/dL on two consecutive occasions were considered diabetic. Data are given as percentage of animals with diabetes over the course of the experiment.

[303] For histological assessment mice were sacrificed via C0₂ euthanasia at the time of disease diagnosis or at the end of the 26-week study and pancreata were immediately isolated and placed in 10% buffered formalin at room temperature overnight, and then embedded in paraffin. After de-paraffinizing, pancreas samples were rehydrated and 5 μηι sections were prepared and fixed on glass slides. Some sections were stained with hematoxylin and eosin (H&E) while others were stained with anti-insulin antibody as follows; prepared sections were immersed in Tris-Buffered Saline (TBS: 10 mM Tris buffer, pH 7.4, 0.15 M NaCI) containing 1 % of bovine serum albumin (BSA) and 5% normal goat serum for 2 h at room temperature followed by incubation with 2 μg/mL rat anti-insulin lgG2a monoclonal antibody (MAB1417; R&D Systems, Minneapolis, MN) in TBS/1 % BSA overnight at 4°C in a humidified chamber. After washing with TBS/0.5 % Tween-20, sections were covered with biotinylated anti-rat IgG secondary antibody (BD biosciences, 1 :1 ,000) for 1 h at room temperature, followed by washing with TBS/Tween- 20 and incubation with streptavidin-horseradish peroxidase for 30 min. Sections were then incubated with chromogen substrate, DAB (BD Biosciences), for 5 min and counterstained with hematoxylin. The degree of insulitis (leukocyte infiltration per islet; H&E) and functional β-cell content per islet (insulin production) was scored using a scale of 0 to 3 in which 0 = none, 1 = few, 2 = moderate, 3 = high content of inflammatory cells or functional p-cells. Pancreata that were scored were obtained from a randomly-selected portion of mice from each cohort and scores were reported as the mean ± SEM for each cohort. [304] Cytokine levels in lymphocyte cultures and serum were determined by aseptically isolating the inguinal and popliteal lymph nodes and spleens from mice sacrificed at the time of disease diagnosis or at the end of the 26-week study. Organs were aseptically crushed to yield single-cell suspensions in culture medium that consisted of RPMI-1640 medium supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 1 mM sodium pyruvate, 100 units/mL penicillin, and 5 μg/mL streptomycin. After centrifuging spleen cell suspensions at 300 x g for 10 min, red blood cells were lysed with 3 ml_ of chilled Red Blood Cell lysis buffer (Sigma) on ice for 5 min and then washed three times with chilled culture medium via centrifugation. Lymphocytes from pooled lymph nodes (5 x 10⁵/well) or a spleen (10⁶/well) were cultured in duplicate with or without Concanavalin A (Con A; 2.5 g/mL) in 1 ml_ of culture medium for 48 h, and conditioned medium was collected and stored at - 80 °C until ELISA analysis of IFN-γ (BD Biosciences, San Diego, CA) and IL-17 (BioLegend, San Diego, CA) levels. The mean values of duplicate cultures from each mouse were used to calculate the mean ± SEM values from several mice per cohort. Serum samples were obtained from blood samples (cardiac puncture method) collected immediately after C0₂ euthanasia and assessed for cytokine levels via ELISA.

[305] As shown in Figure 31 B the 80 mg/kg dose of Compound A suppressed the progressive increase in disease incidence observed in the vehicle-treated cohorts. There was a significantly (p = 0.009, Fischer's Exact test) response between 20 and 80 mg/kg of Compound A in which 20 mg/kg administration was completely ineffective. The mean ± SEM body weights of each cohort at the end of the study (i.e., Week 26) were 21 .0 ± 0.5g, 24.7 ± 0.3g, 24.6 ± 0.8 g, and 21 .3 ± 0.3g, for the vehicle- 80 mg/kg-, 20 mg/kg-, and dexamethasone-treated cohorts, respectively. Because Compound A improves peripheral insulin resistance in the type 2 diabetes test system by suppressing inflammatory cell activity in adipose tissue it is believed the efficacy of Compound A in the autoimmune test system for type 1 diabetes is a result of modulation of the anti-inflammatory process in the NOD mouse pancreas (i.e., preservation of insulin secretion in the T1 D test system rather than an improvement in insulin sensitivity as was observed in the T2D test system).

Histological examination after Compound A treatment was conducted on pancreata randomly isolated from 6 to 9 mice per cohort for quantitative histological analysis of islet (H&E) and functional β-cell numbers (i.e., immunohistological staining for insulin) and of the degree of leukocyte infiltration of islets. Compound A administered at 80 mg/kg, but not at 20 mg/kg, led to a significant preservation of islet number per pancreas and β-cell number per islet, and a significant reduction in the degree of inflammatory leukocyte infiltrate of islets (Table 6). Those results support the belief that efficacy of Compound A in suppressing autoimmune diabetes in the NOD mouse is a result of down-regulating destructive inflammatory cell infiltration into the pancreas, thus preserving functional p- cells.

[306] Table 6. Effects of Compound A on pancreas pathology in NOD mice

No. islets Insulins $-CeI!

Disease pancreas Score/Islet Score/Islet

Incidence H&E) _.(N)

Vehicle 0.63 ± 0,18 (8) 2.4 + 0.6 (8) 2.7 ± 0.3 (6) 1.0 ± 0.3 (6)

Trioles (20 mg/kg) 0.78 ± 0.15 (9) 2,4 ± 0.9 (9) 2.5 ± 0.3 (6) L5 ± 0.4 (6)

Trioiex (80 mg/kg) 0.17 ± 0.57* (6) 8.0 * 3.1* (6) 0.8 ± 0.4* (5) 2.6 ± 0.4* (6)

Dexamethasofie 0.20 ± 0.20 (5) 8.2 ± 6.3 fS) 2.0 ± 0.6 (3) 2.3 ± 0,3* (3)

[307] Because autoimmune diabetes in the NOD mouse is driven by pathogenic T cells of the Th1 and Th17 phenotypes the status of these cellular phenotypes in peripheral lymphoid organs was determined. Thus, pooled inguinal and popliteal lymph nodes and spleens were isolated from 6 to 9 randomly-selected mice per cohort for cytokine analysis. Lymphocytes were cultured with or without Con A for 48 h and conditioned medium was analyzed for levels of IFN-γ (Th1 phenotype) and IL-17 (Th17 phenotype). While there was a substantial increase in IFN-γ production from T lymphocytes from mice with hyperglycemia (disease), vs. those with normoglycemia (no disease), in the vehicle- treated group, Compound A administered at 80 mg/kg, but not at 20 mg/kg, led to a significant reduction in T lymphocyte IFN-γ production (Fig. 32A and 32B). While there was only a trend in increased IL-17 production in those mice with disease vs. those without disease in the vehicle-treated group, treatment with 80 mg/kg of Compound A also led to a significant reduction in IL-17 production by spleenocytes cultures (Fig. 32C). L-17 levels were below the level of detection in lymph node cultures. Cytokine levels were < 0.5 pg/mL in all cultures containing medium only. That suppressive effect of Compound A on lymphocyte IL-17 production was also apparent with serum IL-17 levels (Fig. 32D). The status of the Th2 phenotype could not be evaluated because IL-4 and IL-10 levels were not detectable in the culture conditioned medium. The above results demonstrate that the efficacy of Compound A is associated with down-modulation of the destructive Th1 and Th17 autoimmune response, suggesting an immunoregulatory function of that test compound.

[308] Regulatory T cell (Treq) test systems: In one Treg test system ten million ultra- purified CD4+CD25- (depleted of regulatory T cells) cells from congenic B6.SJL mice (CD45.1 ) are adoptively transferred into six B6 Mice (CD45.2). Animals differ at the CD45 loci, allowing distinction between transferred and endogenous cells in recipient animals. Vehicle (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water as when

Compound A is the test compound) or test compound (1 mg/mouse/day when Compound A is the test compound [40 mg/kg] in 100 μΙ_ vehicle) was injected IP just before (< 10 minutes) transfer of cells and then daily for 14 days. Thymus, lymph nodes (LN) and spleen are collected at day 15, cells labeled with CD45.1 , CD45.2, CD4, CD25, CD103 and/or Foxp3 antibodies, and analyzed by flow cytometry. The remainder of cells from LN and spleen of each treatment group are pooled and pre-enriched for CD4+CD25+ cells. Converted (CD45.1 +) and endogenous (CD45.2+) CD4+CD25+ cells are then sorted using a cell sorter. To test for regulatory function, graded numbers of purified converted or endogenous CD4+CD25+ cells are co-cultured with CD4+CD25- responder cells, irradiated spleen cells as antigen presenting cells (APC), and anti-CD3 (to induce proliferation). Fresh CD4+CD25+ cells from untreated mice were used as controls.

Proliferation was determined by measurement of H³-thymidine uptake 4 days after initiation of culture.

[309] In another test system, ten million ultra-purified CD4+CD25- T cells from young (6 week old) NOD mice are labeled with CFSE and adoptively transferred into six 12-week- old NOD mice. Vehicle or test compound (40 mg/kg in 100 μΙ_ vehicle when Compound A is the test compound) is administered (gavage) to mice 10 minutes before transfer of cells and then daily for 14 days. Thymus, LN and spleen were collected at day 15, and cells labeled with CD4, CD25, CD103 or Foxp3 antibodies then analyzed by flow cytometry. The remainder of cells from LN and spleen of each treatment group were pooled, pre- enriched for CD4+CD25+ cells then converted (CFSE+) and endogenous (CFSE-)

CD4+/C D25+ cells sorted by cell sorter. To test for regulatory function, graded numbers of purified converted or endogenous CD4+/CD25+ cells were co-cultured with

CD4+CD25- responder cells, irradiated spleen cells as APC, and anti-CD3. Fresh

CD4+CD25+ cells from untreated mice were used as controls.

[310] In healthy (i.e., non-inflamed) B6.SJL mice there were no significant differences in either Foxp3 or CD25 expression by converted (CD45.1 +) CD4+ cells found in any organ of Compound A- versus vehicle-treated recipient mice nor was there any change observed in the numbers of converted CD4+CD25+ cells expressing Foxp3. However, there were significant changes in CD25 expression by endogenous cells where

Compound A treatment significantly increased it in spleen but decreased it in lymph nodes (Fig. 33A). Significant increases were also observed in CD103 expression in both lymph nodes and spleen of Compound A-treated animals compared to those treated with vehicle (Fig. 33B). In functional studies, when purified converted (CD45.1 +) CD4+CD25+ cells from Compound A-treated or vehicle-treated mice were co-cultured with CD4+CD25- responder cells, less suppression was observed using cells from the Compound A-treated mice compared to cells from the vehicle-treated group. That difference produced a strong trend (p = 0.054) and only narrowly missed statistical significance. However, significantly less suppression of proliferation was observed when purified endogenous (CD45.2) CD4+CD25+ cells from Compound A-treated mice were co-cultured with CD4+CD25- responder cells compared to cells from the vehicle-treated animals (p < 0.05) (Fig 33C).

[311 ] In the second Treg test system, NOD mice, which provides a model of

spontaneous type-1 diabetes known to involve both a derangement of regulatory T cell function and a systemic inflammation that presages disease by several weeks, a significant (p < 0.02) increase in converted (phenotypically regulatory T) cells was observed in mesenteric lymph nodes (MLNs) where the percentage of stained (converted) CD4+ Foxp3+ cells averaged 2.2% in the Compound A-treated group compared to 0.6% in the vehicle-treated group (Figure 34A). No other differences were observed in either the converted or endogenous population.

[312] In a repeat of the NOD mice study, although similar effects were observed, differences achieved statistical significance only in spleen, where the percentage of stained CD4+ Foxp3+ cells averaged 9.5% in the Compound A-treated group compared to 1 .7% in the vehicle-treated group (p < 0.04; Fig. 34B). In MLN, an average of 9.6% of cells from the Compound A-treated group were stained CD4+ Foxp3+ compared to only 1 .9% in the vehicle treated group, but failed to achieve statistical significance (p = 0.14). There was also a significant increase in unstained (endogenous) CD4+ Foxp3+ cells in spleen, which had not been observed in the previous NOD mice experiment, where the percentage of stained CD4+ Foxp3+ cells was 15.3% in the Compound A-treated group compared to 9.8% in the vehicle-treated group (p = 0.01 ; Figure 34C). No other differences were observed in either the converted or endogenous population. Meta analysis of the two NOD mice studies revealed significant (p = 0.0056) increased regulatory T cell phenotype in MLN and a similar trend in spleen.

[313] From the first NOD mice experiment enough viable cells to perform functional studies were collected. Significantly less suppression of proliferation was observed when purified endogenous (CD45.2) CD4+CD25+ cells from Compound A-treated mice were co-cultured with CD4+CD25- responder cells compared to cells from the vehicle-treated animals (p < 0.05). As expected, endogenous CD4+CD25+ cells from vehicle-treated animals induced significant suppression compared to the control p = 0.01 ). Proliferation was not suppressed when purified endogenous (unstained) CD4+CD25+ cells from Compound A-treated mice were co-cultured with CD4+CD25- responder cells.

[314] Compound A treatment was associated with increased numbers of cells expressing phenotypes thought to represent regulatory T cells in NOD mice but were unchanged in B6 mice. Thus, the effect on regulatory T cells appears to be dependent on the inflammatory or disease state of the recipient animals. The functional regulatory activity correlated well with the phenotypic observations. Changes in B6 mice with respect to increases in CD25 and CD103 expression (markers of cell activation and motility) suggest effects on cell migration. The observations in B6 animals confirm results of a previous study where healthy female B6 mice (6 per group) were given Compound A (50 mg/kg) or vehicle once daily by gavage for 7 days. In that study there was no effect of Compound A treatment on the regulatory T cell phenotype (Foxp3 and PD1 expression) while treatment with estradiol (positive control) nearly tripled it. However, in the present NOD studies, significant increases (and/or trends in that direction) in converted regulatory cells were observed in spleen and MLN of recipient animals treated with Compound A as compared to those treated with vehicle alone. Whether or not these changes achieved statistical significance in either MLN or spleen in any particular study probably relates to the small number (n = 6) of recipient animals in each group.

[315] In the functional study associated with first NOD mice experiment, proliferation was suppressed when purified converted (stained) CD4+CD25+ cells from Compound A- treated (but not vehicle-treated) mice were co-cultured with CD4+CD25- responder cells, indicating the functional activity of the Compound A- converted CD4+ Foxp3+ cells. That suggests treatment of recipient animals with Compound A, but not vehicle, converted cells functionally to regulatory T cells. The observations are consistent with the hypothesis that treatment with Compound A induces conversion of T cells from responder to regulatory function in NOD mice. Combined with observations in healthy B6 animals, the

observations also suggest that Compound A functions as an immune modulator on regulatory T cells where its activity highly dependent on context.

[316] Example 7. Immune suppressive test systems.

[317] Human mixed lymphocyte response (MLR) test system: Human blood samples are obtained from 3 healthy, fasting male volunteers, ranging from 23-31 years of age, who gave informed consent. The subjects are not to have used immune modulating, antiallergic drugs or antibiotics in the three months prior to the study. Samples are drawn between 9 and 10 AM to avoid fluctuations in the circulating levels of hormones or cytokines. Peripheral blood mononuclear cells (PBMC) are isolated by centrifugation on Ficoll-Hypaque gradients (density 1 .077) and resuspended in culture medium (RPMI-1640 supplemented with 2 mM L-glutamine, penicillin (100 U/mL) and streptomycin (100 μg/mL. Autologous (responder) inactivated plasma are used as vehicle at 10% concentration. Five hundred thousand responder PBMC (PBMCr) and 500,000 allogeneic irradiated (30 Gy) stimulator PBMC (PBMCs) are mixed (ratio 1 :1 in 200 μί medium) and cultured for 6 days in a flat bottom 96-well plate. Test compound is dissolved in ethanol and then diluted to test concentrations (0.3 or 0.03 μΜ for Compound A as the test compound) with culture medium leading to a final solution containing 0.01 % ethanol. Controls include vehicle, PBMCr and PBMCs cultured separately in the same vehicle at the same concentration. During the final 8 h, the PBMC are pulsed with 1 μΟϊ/ννθΙΙ [³H]-thymidine. Cells are harvested and incorporation of radioactivity into DNA measured with a beta cell counter. The mean cpm of quadruplicate wells are calculated. The hypothesis of non-inferiority is typically not analyzed formally due to the small sample size (three subjects). Instead, an exploratory three-step procedure is typically executed as follows: First, an exact 95% Hodges-Lehman confidence interval is constructed for the difference in population medians for each subject, regarding the replicates within that subject as independent. That step is then confirmed by means of the significance of non-inferiority of an active compound relative to a control via an exact one-sided Mann- Whitney test, per subject as above, as described in Conover, W.J. Practical Nonparametric Statistics. 3rd edition, John Wiley & Sons Inc., New York, NY, pp 271 -364 (1999). Second, a decision is to be made as to whether each subject meets the non-inferiority criteria. Finally, the percentage of subjects adjudicated as cases of non-inferiority is calculated, with its attendant exact Blyth-Still-Casella confidence interval for the total fraction of subjects that, under similar experimental conditions, would show non-inferiority as described in Casella, G. "Refining binomial confidence intervals". Can. J. Stat. (1986) 14: 1 13-129). That is a measure of the strength of the evidence at the population level inferred from the three cases.

[318] Results from the MLR test system are exemplified for Compound A as follows. Minimal proliferative responses (>2000 CPM) were observed at the end of the culture period in individual cultures of control responder and stimulator PBMCs (obtained from each of three donors R1 , R2 and R3). In contrast, a marked proliferative response was observed when PBMCr and PBMCs were cultured together with or without vehicle.

Overall, there was no profound suppressive influence of any Compound A concentration tested in the mixed lymphocyte responses, which is a classical measurement of cell- mediated (i.e., Th1 ) immunity.

[319] Spleenocvtes proliferation test system: Spleens are obtained from 6-8 week old C57BU6 mice (or other standard laboratory strain) by aseptic techniques. The spleens are teased and the suspension filtered to obtain single cells. Cells (10⁶ mononuclear cells/mL RPMI-1640 medium/10% fetal bovine serum/50 μΜ 2-mercaptoethanol) are incubated in 96-well plates, 0.2 mL per well, for three days with no stimulus, or with approximately 5 μg/mL Concanavalin A (Con A), 2 μg/ml phytohemmaglutinin (PHA), or 1 :100 dilution of pokeweed mitogen (PWM). Parallel cultures contained test compound at 1000,100 and 10 ng/mL. Test compounds are dissolved in DMSO at 10 mg/mL and then diluted to the desired concentration with culture medium leading to a maximal final solution containing 0.01 % of DMSO. Dexamethasone at 40nM and 1000 nM serves as the positive control. Cells are then incubated overnight with approximately 1 pCi/ml ³H-thymidinaen and proliferation is determined by incorporation of label into cell DNA following filtration, washing, and liquid scintillography. Five individual mice are used, with each spleen cell preparation incubated in triplicate for each condition. Means and the effect of IRH

(stimulation or inhibition) are determined, with standard deviations. Statistical analysis was performed by two-tailed Student's t test with equal variance.

[320] The spleenocytes proliferation test is exemplified for Compound A with the following results. Compound A did not suppress proliferation when used alone. On the contrary, Compound A appeared to increase PHA, Con A, but not PWM induced proliferation. In contrast dexamethasone was powerfully immune suppressive

(approximately 60 to 80%) at both doses in all proliferation studies, regardless of stimulant. The study demonstrates that Compound A does not suppress mitogen-induced proliferation or suppress or enhance proliferation when used alone, without stimuli.

[321 ] Delayed-type hypersensitivity (DTH) test system: The purpose of the DHT test system of this work is to evaluate the ability of a test compound, in the presence or absence of treatment with dexamethasone, to affect the T cell-dependent anamnestic immune responses in the DTH assay. Animals are treated with test compound after sensitization between the induction and effector phases of the response. Test compound is not on board (or may be only at very low levels) during the inflammatory process. Thus, the DTH protocol specifically determines the effect test compound treatment may have on the generation of immunological memory rather than on acute inflammation.

[322] The DTH test is exemplified for Compound A as follows. On the day prior to the start of the experiment, male CD 1 mice (8 per group) were shaved on their right flank. On day 0, mice were painted (sensitized) on the shaved flank with 50 μΙ_ of 2.5% oxazolone solution (in 95% EtOH). Mice were then (beginning on the same day) treated by oral gavage with 40 mg/Kg Compound A in vehicle, 3 mg/Kg dexamethasone (Dex, as positive control) in vehicle given IP or with 40 mg/Kg Compound A + 3 mg/kg Dex or with vehicle alone (approx 100 μΙ_ 30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water) once daily on days 1 -4. After the final treatment, mice were challenged 24 hours later on the right dorsal ear surface with 25 μΙ_ 0.25% oxazolone solution. The right and left pinnae thickness were both measured 2, 6, 24, 48, and 72 hours later using a micrometer caliper. Statistical significance between the control and experimental groups was determined using the Student's t-test. P values < 0.05 are considered significant.

[323] Mice treated with either vehicle or with saline (PBS) exhibited robust DTH responses between 2 and 72 h after challenge with oxazolone solution (Figure 35).

Treatment with Compound did not appear to effect the generation of immunological memory in this model in that there was no significant difference in ear swelling between the Compound A-treated animals and those treated with saline or vehicle. In contrast, mice treated with dexamethasone, a well known highly immune suppressive steroid, had significantly reduced ear swelling when compared to either the Compound A-, saline- or vehicle-treated groups, indicating that dexamethasone powerfully suppressed the DTH memory response. Treatment with Compound A did not appear to counteract or enhance the dexamethasone mediated suppression of DTH memory responses in that the group receiving both Compound A and dexamethasone was not significantly different from the group receiving dexamethasone alone.

[324] Popliteal lymph node (PLN) test system: The purpose of the PLN test system is to determine the anti-GC activity of a test compound. The immune suppressive activities of GC include (1 ) suppression in numbers of total lymphocytes, antigen specific IgM, lgG1 and lgG2a antibody secreting cells (ASC) (ELISPOT assay); (2) cell surface marker (CD4, CD8, CD19, F480, CD80, CD86) expression (flow cytometry of living cells in suspension); (3) IL-4, TNF alpha, and IFN gamma production by cultured lymphocytes (ELISA).

[325] Specific pathogen-free BALB/C mice (n=5 per group) are injected in to the right hind foot pad with 50 μί of a freshly prepared sensitizing dose of TNP-OVA. Dex

(dexamethasone sodium phosphate) is given intraperitoneal^ (200 μΙ_) on five constitutive days starting immediately following sensitization with TNP-OVA (5 μg dose chosen based on previous studies to he minimally suppressive). Compound A compound is given immediately afterwards by gavage, also for five days (100 μΙ_ in vehicle). Five days after injection of TNP-OVA, mice are killed by cervical dislocation and popliteal lymph nodes are removed and separated from adherent fatty tissue. Single cell suspensions are prepared, resuspended in 1 ml_ PBS-BSA (1 %) and counted. For flow cytometric analysis, 1 x 10⁵ cells in PBS/BSA are centrifuged, resuspended, and incubated with predetermined dilutions of FITC-, PE-, CY- conjugated mAbs in 96-well plates (30 min in darkness at 4 °C). Cells are washed, resuspended, stored in formalin (0.1 %) and analyzed within 18 h on a FACscan™with standard FACsflow™ using CellQuest software (BD Biosciences, Franklin Lakes, NJ). For cytokine measurements, cell suspensions (1 x 10⁵ cells in 100 μί complete RPMI 1640 from supplemented with 10% FCS, 50 μΜ B-ME, and 200 mM L- glutamine) are incubated with 50 μί Con A (15 μg mL) in 96 well plates overnight in 5% C0₂ in air. After centrifugation for 10 min at 1000 rpm, supernatant is collected and stored at -70 °C until analysis. Cytokine levels were determined by sandwich ELISA.

[326] For Compound A as the test compound the groups of BALB/C mice used in the PLN test system were TNP-OVA + Vehicle (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water), TNP-OVA + Dex (10 μg/mouse/day), TNP-OVA + Compound A (1 mg/mouse/day) oral, TNP-OVA + Compound A (0.1 mg/mouse/day) oral, TNP-OVA + Dex + Compound A (1 mg/mouse/day) oral, and TNP-OVA + Dex + Compound A (0.1 mg/mouse/day) oral. Statistical analysis was performed by two-tailed Student's t test with equal variance. When pathogen-free BALB/C mice were injected in to the right hind foot pad with 50 μΙ_ of a freshly prepared sensitizing dose of TNP-OVA and treated (gavage) with Compound A daily for five days (beginning immediately following sensitization with TNP-OVA numbers of lymphocytes detected in PLN on day 5 were unchanged or increased at 1 and 0.1 mg dose compared to vehicle controls (Figure 36A). In contrast, treatment with dexamethasone, a well known immune suppressive steroid, reduced numbers of lymphocytes detected on day 5 compared to vehicle controls. Compound A did not appear to interfere with the dexamethasone-induced suppression in that numbers of lymphocytes were still suppressed in animals treated with both dexamethasone and Compound A compared to the vehicle control. This was true with Compound A at both doses when compared to the vehicle control. Compound A treatment also appeared to increase, or not change, the production of IL-4 (Fig. 36B), IL-5 (Figure 36C) and IFN gamma (Figure 36D). In no case did Compound A suppress cytokine production on par with dexamethasone. Only in the case of IL-5 did low dose did Compound A appear to reverse dexamethasone induced inhibition. Therefore, it appears that Compound A is not immune suppressive since it clearly did not significantly reduce cytokine production, but on the contrary, appeared to enhance it, which is an effect not observed when compounds were given to dexamethasone-treated animals (with the one exception noted above). Compound A at the low dose did not appeared to alter T/B cell ratios (Figure 36E), however, when used at the high dose, an increase in CD4+Tcells and a decrease in CD19+ cells was observed. Dexamethasone treatment induced several significant changes in cell surface marker expression including increases in the percentages of all T cells and a decrease in the percentages of B cells.

[327] Ovalbumin mouse immunization test system: Female BALB/c mice (5 per group) were sensitized by intraperitoneal injection (total volume 0.2 mL) on days 1 and 8 with 100 μg ovalbumin (endotoxin-free) precipitated with aluminum hydroxide in saline. Mice were treated by gavage daily with Compound A (40 mg/kg) or vehicle (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water) on days 0-20. On day 20, 2 h after the final treatment, blood was drawn by terminal cardiac puncture, serum prepared and tested by ELISA for antibody titers against OVA. Briefly, OVA was coated overnight (4^<C) on 96 well plates in carbonate buffer (pH 9.6), and then blocked with PBS-Tween 20/3% milk powder for 1 h at 37 ^<C. Serum diluted in PBS Tween 20 (0.5%) was incubated in the wells for 1 h, followed with incubation (1 h, 37^<C) with alkaline phosphatase-conjugated anti-lgG1 antibodies. Subsequently, 1 mg/mL p-nitrophenylphosphate in diethanolamine buffer was used for the color reaction, which was stopped with an EDTA solution. Absorbance at 450 nm was measured using an ELISA reader.

[328] For OVA immunization studies, analysis was performed using the SAS™ system, (version 9.1 ) with certain exact tests implemented by use of the StatXac™ (version 7) software package. Results show no profound immune suppression in the murine ovalbumin immunization model, which is a classical approach to induce antibody (i.e.,Th2) biased immune responses. However, a small (-25%) but statistically significant (p < 0.05) reduction in OVA specific antibody production was observed in mice treated with

Compound A. The statistical analysis shows that, in

terms of derived lgG1 absorbance, Compound A is inferior to its vehicle (p = 0.008). The exact confidence interval for the difference in median absorbance is negative, indicating that the distribution of Compound A optical density is unlikely to be on a par with that of its vehicle.

[329] Example 8. Lung Inflammation Test Systems

[330] Carraqeenan (CAR)-induced pleurisy test system: Six to 8 week old CD1 male mice were housed in a controlled environment and provided with standard rodent chow and water. All animals weighed approximately 25-30 grams each and were acclimated for at least 3 days prior to the start of the experiment. Mice (n = 10 per group) were allocated into one of the following groups: (1 ) Sham (saline) treated animals; (2) CAR only (CAR group); (3) CAR and vehicle (0.1 % carboxymethylcellulose, 0.9% NaCI, 2% Polysorbate 80, 0.05% phenol) by subcutaneous (SC) injection; (4 and 5) CAR and Compound A (40 or 4 mg/kg in vehicle) by SC injection, and (6) CAR and rabbit anti-mouse polyclonal anti- TNFa antibody (200 μg in saline, IP injection). All treatments were given 24 h and 1 h prior to CAR in a final volume of 0.1 ml_.

[331 ] Mice were anaesthetized with isoflurane and the skin was incised at the level of the left sixth intercostal space. The underlying muscle was dissected and saline (sham) or saline containing 2% λ-CAR was injected into the pleural cavity. The skin incision was closed with a suture and the animals were allowed to recover. At 4 h after the injection of CAR, the animals were sacrificed by C0₂ asphyxiation. The chest was carefully opened and the pleural cavity rinsed with 1 ml_ of saline solution containing heparin (5 U/mL) and indomethacin (10 g/mL). The exudate and washing solution were removed by aspiration and the total volume measured. Any exudate, which was contaminated with blood, was discarded. The amount of exudate was calculated by subtracting the volume injected (1 ml_) from the total volume recovered. The leukocytes in the exudate were suspended in phosphate buffer saline (PBS) and counted with an optical microscope in a Burker's chamber after vital Trypan Blue staining. Cells at this time point are predominantly neutrophils. Data are expressed as mL exudate volume or millions of neutrophils per mouse +/- standard deviation.

[332] Parameters of interest were subjected to ANOVA with Duncan's new multiple- range post hoc testing between groups with results shown in Figure 37. When mice were challenged with 0.1 mL of 2% carrageenan in the pleural cavity, high leukocyte numbers (-1 .9 x10⁶ per mouse) were observed in the pleural exudate. Substantially lower leukocytes numbers (-2.8 χ 10⁵ per mouse) were observed in animals undergoing a sham procedure and challenged with saline. When mice were pre-treated with Compound A (40 mg/kg) by SC injection, significantly (p < 0.05) reduced numbers of carrageenan-induced neutrophils (-5.7 χ 10⁵) were observed in pleural exudates compared to those observed in animals given vehicle alone (-1 .8 χ 10⁶). Treatment with the higher dose of Compound A was as effective as treatment with polyclonal anti-mouse TNFa antibody that was used as a positive control. Treatment with Compound A also reduced pleural exudate volumes (compared to vehicle), in a dose-dependent fashion.

[333] LPS -induced lung injury test system: Six to 8-week old C57 black/6 male mice (approximately 25-30 grams) were used in these studies (at least 4-8 animals per group). The animals were housed in a controlled environment and provided with standard rodent chow and water and acclimated for at least 3 days prior to the start of the experiment. Animals were treated with Compound A or vehicle (0.1 % carboxymethylcellulose, 0.9% NaCI, 2% Polysorbate 80, 0.05% phenol) via a single gavage administration (0.1 mL) 24 h and 1 h before LPS challenge. LPS challenge was performed by lightly anesthetizing the mice with isoflurane, and then directly administering the LPS (5 mg/kg, 50 μί of 1 mg diluted in 1 mL sterile saline) into the trachea under direct observation with a gel loading pipette through a medical otoscope. The mice were placed in a vertical position and rotated for 0.5 - 1 min to distribute the instillate evenly within the lungs. At 48 h after the LPS challenge, animals were sacrificed and bronchoalveolar lavage (BAL) samples taken (BAL performed 3χ using sterile PBS; 1 .3 mL were typically recovered). Cells were counted using a hemocytometer and cytokine levels were measured by ELISA.

[334] A meta-analysis of two independent studies revealed that when mice that were pre-treated with 40 mg/kg Compound A by oral gavage were challenged with 50 mg of

LPS, levels of myeloperoxidase (MPO) activity in lungs at 48 hours were significantly (p < 0.025) reduced (-30%) compared to vehicle-treated animals (Figure 38). MPO activity was determined as described in Krawisz, J.E. et al. "Quantitative assay for acute intestinal inflammation based on myeloperoxidase activity. Assessment of inflammation in rat and hamster models" Gastroenterology (1984) 87:1344-50. Data were analyzed by two-sided Student's t test. [335] Opportunistic infection survival test systems: Female BALB/c mice (approximately 25-30 grams) were acclimated for at least 3 days prior to the start of the experiment. Animals were randomized by weight into 4 groups. Group 1 (n = 10) received daily 0.1 ml_ administrations by gavage of 80 mg/kg Compound A in vehicle (0.1 %

carboxymethylcellulose, 0.9% NaCI, 2% Polysorbate 80, 0.05% phenol). Group 2 (n = 10) received equal volumes of vehicle alone. Group 3 (n = 10) received daily intraperitoneal (IP) administrations of dexamethasone (dex; 0.4 mg/kg in 0.1 ml_ saline. Group 4 (n = 8) was untreated. Body weights were measured daily. After 14 days of treatment, infection was induced by subcutaneous inoculation of 107 colony-forming units (LD₅₀ at 72 hours) of Klebsiella pneumoniae (strain AFRRI7). Once daily treatments were given until death. All animals were monitored twice-daily for health status until the end of the study. For K. pneumoniae survival studies, comparison of survival curves (Logrank test for trends) was performed using Prism™ software

[336] K. pneumoniae is an opportunistic infection commonly observed in immune suppressed mice. When animals were challenged with 10⁷ cfu of K. pneumoniae, no significant differences in survival kinetics were found. Therefore, K. pneumoniae challenge to animals conditioned with Compound A resulted in no promotion of death, indicating that the small but significant suppression of OVA specific antibody production described above has no clinical relevance.

[337] In a second survival test system the slow growing mucoid clinical strain P.

aeruginosa M57-15 was used in CFTR-/- mice. P. aeruginosa is another opportunistic bacterial pathogen that is commonly found resident in lungs of patients with cystic fibrosis. For the study CFTR-/- mice were bred, housed and used as previously described in van Heeckeren, A.M. et al. "Role of Cftr genotype in the response to chronic Pseudomonas aeruginosa lung infection in mice" Am. J. Physiol. Lung. Cell. Mol. Physiol. (2004) 287: L944-52 and Van Heeckeren, A.M. "Delivery of CFTR by adenoviral vector to cystic fibrosis mouse lung in a model of chronic Pseudomonas aeruginosa lung infection" Am. J. Physiol. Lung. Cell. Mol. Physiol. (2004) 286: L717-26. Male mice (9 per group) 6-8 weeks of age, body weight at least 16 g, were used in these experiments and bred and housed under standard laboratory conditions. P. aeruginosa-\ader agarose beads were made and used, as essentially described in van Heeckeren, A.M. and Schluchter, M.D. "Murine models of chronic Pseudomonas aeruginosa lung infection" Lab. Anim. (2002) 36:291 - 312. Mice were inoculated with a 1 :35 dilution of the beads (LD₅₀ dose). Compound A, 16a-bromo-epiandrosterone (Compound C) or vehicle (30% β-cyclodextrin sulfobutyl ether sodium salt (w/v) in water) was given by oral gavage 24 h before and 1 h after bacterial challenge. Measurements of bacterial burden in the lungs were performed as in van Heeckeren, A.M. "Effect of Pseudomonas infection on weight loss, lung mechanics, and cytokines in mice" Am. J. Respir. Crit. Care. Med. (2000) 161 : 271 -9.

[338] Neither Compound A nor Compound C (positive control) treatment induced frank toxicity in the CFTR-/- mouse and there was no significant (ANOVA) difference between groups (vehicle versus drug-treated) with respect to body weight or bronchoalveolar lavage cell counts at 24 hours after bacterial challenge. There was significantly greater numbers of bacteria in vehicle-treated mice compared to 40 mg/kg Compound A (p < 0.03) as was found in a previous study Nicoletti, F. et al "16alpha-Bromoepiandrosterone (HE2000) limits non-productive inflammation and stimulates immunity in lungs" Clin. Exp. Immunol. (2009) In contrast, there was no significance with respect to a reduction of bacteria in Compound A- compared to vehicle-treated mice.

[339] All references cited herein are incorporated herein by reference. To the extent not already indicated, it will be understood by those of ordinary skill in the art that any of the various specific embodiments, analysis methods, compounds or compositions described herein may be modified to incorporate other appropriate features, e.g., where one or more protocol steps in a disclosed embodiment is added to or combined with any other compatible protocol step, method or embodiment described herein.

Claims

CLAIMS What is claimed is:

1 . A method to identify a drug candidate, the method comprising

(a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins;

(b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of Mapk1/3, Jnk1/2, p38 MapK, p65 NF-κΒ, ΙΚΚα/β and IRS1 ; and

(e) selecting the test compound of step (b) that decreases phospho-activation or level(s) or biological activit(ies) of Mapk1/3, JNK1/2, p38 Mapk, p65 NF-κΒ and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s), wherein the selected test compound is identified as a candidate compound for determining immunotoxicity of the test compound in an suitable immunosuppressive test system and/or determining efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

2. The method of claim 2 comprising

(a) contacting a test compound with one or more suitable test systems for determining phosphorylation states or levels of biological activities of one or more proteins;

(b) determining phosphorylation states or levels of biological activities for one or more of the proteins of step (a) wherein the proteins are selected from the group consisting of Mapk1/3, Jnk1/2, p38 Mapk, p65 NF-κΒ, ΙΚΚα/β and IRS1 ;

(c) determining immunotoxicity of the test compound in an suitable

immunosuppressive test system; and

(e) selecting the test compound of steps (b) and (c) that decreases the phospho- activation or biological activitv(ies) of Mapk1/3, Jnk1/2, p38 Mapk, p65 NF-κΒ and/or ΙΚΚα/β and/or decreases the phospho-deactivation or IRS-1 relative to suitable control test system(s) and has sufficiently low immunotoxicity for administration to a subject for determining efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system wherein the selected test compound is identified as a candidate compound.

3. The method of claim 1 further comprising

(d) determining test compound binding to or transactivation by ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems; and

(f) further selecting from (e) the test compound of steps (b), (c) and (d) that has

EC50's > 10,000 nM in the NHR test systems, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity of the test compound in an suitable immunosuppressive test system and/or determining efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

4. The method of claim 1 wherein the test compound of step (b) decreases the phospho-activation of Mapk1/3 and wherein the test compound binds directly or indirectly to Mapkl and Mapk3 as determined by a suitable SILAC test system.

5. The method of claim 4 wherein phospho-activation of Mapkl /3 is decreased in a cell-based suitable kinase test system and wherein the test compound in a suitable cell- free test system has negligible or no effect on phospho-Mapk1 /3 phosphorylation of myelin-basic protein or other suitable Mapkl /3 substrate wherein no scaffold proteins are present in the cell-free test system.

6. The method of claim 1 wherein the suitable test system for determining kinase or NK-KB phosphorylation or activity is cell-based.

7. The method of claim 6 wherein the suitable test system for determining kinase phosphorylation or NK-KB activity determines ΙΚΚα/β phosphorylation state(s).

8. The method of claim 6 wherein the suitable test system for determining kinase or NK-KB activity determines κΒ occupancy or NK-KB transactivation of a reporter gene.

9. The method of claim 6 wherein the suitable test system for determining kinase or NK-KB activity determines the extent of NK-KB translocation to the nucleus on cytokine or LPS challenge.

10. The method of claim 2 wherein the suitable immunosuppressive test system is one or more selected from the group consisting of human mixed lymphocyte response, spleenocytes proliferation, delayed-type hypersensitivity, popliteal lymph node assay and ovalbumin mouse immunization test system.

1 1 . The method of claim 2 wherein the suitable immunosuppressive test system is an opportunistic infection survival test system.

12. The method of claim 1 1 wherein the opportunistic infection is K. pneumoniae or P. aeruginosa.

13. The method of any one claim 4 wherein the test compound binds directly or indirectly to Mapkl , Mapk3, Lrp1 and Sirt2 as determined by a suitable SILAC test system.

14. The method of claim 1 wherein the test compound system selected for determining efficacy to treat unwanted inflammation is sufficiently bioavailable to the CNS as determined by a suitable blood-brain barrier test system.

15. The method of claim 14 wherein the suitable unwanted inflammation test system is a neuroinflammation test system.

16. The method of claim 15 wherein the suitable neuroinflammation test system is cell-based.

17. The method of claim 15 wherein the suitable neuroinflammation test system test for determining efficacy is MPTP-induced neuroinflammation in a subject.

18. The method of claim 17 wherein the subject is a non-human primate.

19. The method of embodiment 1 further comprising

(d) determining test compound binding to or transactivation by nuclear hormone receptors ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems;

(d') determining test compound binding to or transactivation by PPARy; and (g) further selecting from (e) the test compound of steps (b), (d) and (d') that has

EC50's > 10,000 nM in the NHR and PPARy test systems, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

21 . The method of claim 6 wherein the suitable cell-based test system for determining kinase or NF-kB phosphorylation state or activity is comprised of RAW 264.7 macrophages.

22. The method of claim 1 wherein the suitable unwanted inflammation test system for determining efficacy is a suitable insulin-resistant test system.

23. The method of claim 6 wherein the suitable cell-based test system for determining kinase or NF-kB phosphorylation state or activity are murine intraperitoneal macrophages.

24. The method of claim 1 wherein the suitable test system(s) for determining efficacy in a suitable insulin-resistant test system is one or more of db/db mouse, ob/ob mouse, Zucker rat or diet-induce mouse model(s).

25. The method of any claim 1 wherein the suitable unwanted inflammation test system for determining efficacy is a suitable lung inflammation test system.

26. The method of any one of claim 1 wherein the suitable lung inflammation test system is carrageenan-induced pleurisy.

27. The method of embodiment 1 further comprising

(d) determining test compound binding to or transactivation by nuclear hormone receptors ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems;

(d") determining cell numbers induced by the test compound for cells expressing regulatory T cell phenotypes in a suitable Treg test system, wherein the Treg system comprises T cells of a subject with an autoimmune condition; and

(h) further selecting from (e) the test compound of steps (b), (d) and (d") that has EC50's > 10,000 nM in the NHR test systems and increased numbers of converted regulatory T cells, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

28. The method of claim 1 further comprising further comprising

(d') determining test compound binding to or transactivation by nuclear hormone receptors ER, PR, GR and AR in suitable nuclear hormone receptor (NHR) test systems;

(d^*) determining cell numbers for cells expressing regulatory T cell phenotypes in two suitable Treg test systems induced by the test compound, wherein one Treg system comprises T cells of a subject with an autoimmune condition and the other Treg system comprises T cells from a healthy subject; and

(h^*) further selecting (e) the test compound of (b), (d) and (d^*) having EC50's > 10,000 nM in the NHR test systems and increased numbers of converted regulatory T cells in the autoimmune Treg test system and negligible or no increase of converted regulatory T cells in the healthy Treg test system, wherein the selected test compound is identified as a candidate compound for determining immunotoxicity in an suitable immunosuppressive test system and/or efficacy to treat unwanted inflammation in a suitable unwanted inflammation test system.

29. The method of embodiment 28 wherein the cells of the suitable autoimmune Treg test system are those of NOD mice.

30. The method of embodiment 28 wherein the cells of the suitable healthy Treg test system are those of B6.SJL mice.

31 . The method of any one claim 1 wherein the suitable test system for determining efficacy to treat unwanted inflammation is a suitable autoimmune test system 32. The method of embodiment 31 wherein the suitable autoimmune test system is a suitable adjuvant induced arthritis, collagen antibody-induced arthritis, experimental autoimmune encephalomyelitis, ulcerative colitis, systemic lupus erythematosus or autoimmune diabetes test system.