WO2007100631A2 - Compositions and methods for modulating cytokine production - Google Patents

Abstract

The present invention is directed to methods for identifying compounds that inhibit the production of IL-12p40 and/or increase the production of IL-IO, and methods for treating or preventing diseases or disorders related to IL-12, IL-23, and IL-27 production.

Description

COMPOSITIONS AND METHODS FOR MODULATING CYTOKINE PRODUCTION

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No. 60/775,737, filed on February 22, 2006, the entire teachings of which are incorporated herein by reference.

FIELD OF THE INVENTION

The present invention is directed to methods for identifying compounds that inhibit the production of IL-12p40 and/or increase the production of IL-10, and methods for treating or preventing diseases or disorders related to IL- 12, IL-23, and IL-27 production.

BACKGROUND OF THE INVENTION

The vertebrate immune system protects the body against undesirable foreign matter that enters the body, such as infecting pathogens (e.g., bacteria, viruses, fungi, and parasites) and their by-products. One manner by which this takes place involves the adaptive immune system, through which the body recognizes foreign antigens and generates specific immune responses against them. The induction of adaptive immunity takes time (e.g., 2-3 days post infection), and thus could leave the body vulnerable to the adverse effects of early infection, if it were not for the action of another division of the immune system, the innate immune system.

The innate immune system provides the body with a first line defense against invading pathogens. In an innate immune response, an invading pathogen is recognized by a germline-encoded receptor, the activation of which initiates a signaling cascade that leads to the induction of cytokine expression. Innate immune system receptors have broad specificity, recognizing molecular structures that are highly conserved among different pathogens. These receptors are known as Toll-like receptors (TLRs), due to their homology with receptors that were first identified and named in Drosophila, and are present in cells such as macrophages, dendritic cells, and epithelial cells.

There are at least ten different TLRs in mammals, and ligands and corresponding signaling cascades have been identified for some of these receptors. For example, TLR2 is activated by the lipoprotein of bacteria (e.g., E. coli.), TLR3 is activated by double-stranded RNA, TLR4 is activated by lipopolysaccharide (i.e., LPS or endotoxin) of Gram-negative bacteria (e.g., Salmonella and E. coli O157:H7), TLR5 is activated by flagellin of motile bacteria (e.g., Listeria), and TLR9 is activated by unmethylated CpG sequences of pathogen DNA. The stimulation of each of these receptors leads to activation of NF-κB transcription factors, and other signaling molecules that are involved in regulating the expression of cytokine genes.

NF-κB is a family of transcription factors that play a key role in inflammation, immunity, cell proliferation and apoptosis. NF-κB family members, including c-Rel, ReIA (also referred to as "p65"), ReIB, p50 and p52, exist mainly in the cytoplasm in an inactive form due to association with one or more members of a family of inhibitors known as IKB proteins (IκBα, IκBβ, IκBε, Bcl-3, plOO, plO5). The best characterized of the IkB proteins, IκBα, has a strong nuclear export sequence that keeps complexes of NF-κB: IkB proteins largely in the cytoplasm. Pro-inflammatory cytokines and other stimuli trigger phosphorylation of IκBα by IKB kinase (IKK) which marks it for subsequent ubiquitination and proteasomal degradation. Once liberated from association with IKB proteins, NF-κB proteins can accumulate in the nucleus and form homo- and heterodimers which combine with a coactivator protein (CBP) and activate the transcription of target genes, including those controlling cell proliferation and cell survival (e.g. anti-apoptotic genes). However, activation of NF-κB proteins is usually a transient process because one of the primary target genes of NF-κB is the gene encoding IκBα which can bind to NF-κB proteins and return them to their latent form in the cytoplasm.

When c-Rel is released from the IκB:Nf-κB complex, it is free to enter the nucleus and bind to KB sites on DNA, including the promoter for the p40 subunit of Interleukin-12 (IL- 12). LPS, for example, stimulates the translocation of p50/c-Rel andp50/p65 heterodimers in macrophages from the cytoplasm to the nucleus. Both of these heterodimers bind to the NFKB site in the promoter of p40. However, only c-Rel has been shown to be important for the LPS-induced signaling through TLR4 that leads to the production of p40 in response to numerous pro-inflammatory stimuli in vitro and in vivo.

JL- 12 is a disulfide linked heterodimeric cytokine (p70) which plays key roles in immune responses by bridging innate resistance and antigen-specific adaptive immunity. Trinchieri (1993) Immunol Today 14: 335. For example, it promotes type 1 T helper cell (T_HI) responses and, hence, cell-mediated immunity. Chan et α/. (1991) J Exp Med 173: 869; Seder et a (1993) Proc Natl Acad Sci USA 90: 10188; Manetti et al. (1993) J Exp Med 177: 1199; and Hsieh et al. (1993) Science 260: 547. IL-12 is composed of two independently regulated subunits, p35 and p40. EL-12 is produced by phagocytic cells and antigen presenting cells, in particular, macrophages and dendrite cells, upon stimulation with bacteria, bacterial products such as lipopolysaccharide (LPS), and intracellular parasites. The well-documented biological functions of IL-12 are induction of interferon-γ expression from T and Natural Killer (NK) cells and differentiation toward the T_HI T lymphocyte type. IFN-γ is a strong and selective enhancer of IL-12 production from monocytes and macrophages.

The cytokine IL-23 is a heterodimer composed of a pi 9 subunit and the same p40 subunit of IL-12. IL-23, similarly to IL-12, is involved in type 1 immune defenses and induces IFN-γ secretion from T cells.

IL-27 is formed by the association of EBI3, a polypeptide related to the p40 subunit of IL-12, and p28, a protein related to the p35 subunit of IL-12. IL-27 promotes the growth of T cells and is thought to play a role in the differentiation of T_HI cells. Pflanz et al., Immunity (2002), 16:119-190.

It has been suggested that, particularly in chronic diseases in which there is ongoing production of IFN-γ, IL-12 production is augmented by IFN-γ. It is presumed that after an infective or inflammatory stimulus that provokes IL-12 production, the powerful feedback loop promotes IL-12- and IL-23-induced IFN-γ to further augment IL-12 production, leading to consequent excessive production of pro-inflammatory cytokines. Furthermore, it has been suggested that EL-27 induces the expression of T-bet, a major T_HI -specific transcription factor, and its downstream target IL- 12R β2, independently of IFN-γ. In addition, IL-27 suppresses the expression of GATA-3. GATA-3 inhibits T_HI development and causes loss of IL-12 signaling through suppression of IL-12R β2 and Stat4 expression. Lucas et al., PNAS (2003), /00:15047-15052.

IL-12 plays a critical role in multiple-T_Hl dominant autoimmune diseases including, but not limited to, multiple sclerosis, sepsis, myasthenia gravis, autoimmune neuropathies, Guillain-Barre syndrome, autoimmune uveitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, temporal arteritis, anti-phospholipid syndrome, vasculitides, Wegener's granulomatosis, Behcet's disease, psoriasis, psoriatic arthritis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo, Crohn's disease, ulcerative colitis, interstitial pulmonary fibrosis, myelofibrosis, hepatic fibrosis, myocarditis, thyroditis, primary biliary cirrhosis, autoimmune hepatitis, Type 1 or immune-mediated diabetes mellitus, Grave's disease, Hashimoto's thyroiditis, autoimmune oophoritis and orchitis, autoimmune disease of the adrenal gland; rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, polymyositis, dermatomyositis, spondyloarthropathies, ankylosing spondylitis, common variable immunodeficiency disease (CVID), Sjogren's syndrome and graft-versus-host disease. See, for example, Gately et al. (1998) Annu Rev Immunol. 16: 495; and Abbas et al. (1996) Nature 383: 787.

Stimulation of the immune system, which includes stimulation of either or both innate immunity and adaptive immunity, is a complex phenomenon that can result in either protective or adverse physiologic outcomes for the host. Therefore, compounds that attenuate chronic or inappropriate immune responses, such as overproduction of IL- 12 and other proinflammatory cytokines, would be useful for treating inflammatory disorders.

SUMMARY OF THE ENfVENTION

The present invention relates to methods for identifying compounds that inhibit IL- 12p40 production thereby inhibiting the production of IL-12, IL-23 and IL-27, and methods for treating IL-12, IL-23, or IL-27 production-related diseases or disorders in a subject using one or more of the identified compounds. The experimental evidence herein demonstrates that compounds that induce the phosphorylation of ERK increase the production of antiinflammatory cytokine IL-IO. In addition, without wishing to be bound by any theory, it is believed that compounds which induce the phosphorylation of Ser9 of GSK3β, thereby inhibiting its activity, inhibit the production of IL-12p40 by inhibiting the phosphorylation of CREB341 at serl29 and/or inhibiting the phosphorylation of CREB327 at Serl 15. cAMP response element binding protein (CREB) is a transcription factor that exists mainly as two isoforms CREB341 and CREB327 that arise from alternative splicing of a 42-bp exon coding for a part of the transactivational domain present in CREB341 but not in CREB327 (Muller, et al., Circulation (1995), 92:2041-2043). Phosphorylation of CREB341 at serl 33 and CREB327 at serl 19 is required for recruitment of CREB-binding protein (CBP) and transcriptional activity. However, phosphorylation of CREB341 at serl 33 or CREB327 at serl 19 creates a consensus site for phosphorylation by GSK3β at serl29 or serl 15, respectively. Phosphorylation of CREB by GSK3β has been shown to attenuate the CREB DNA binding activity (Grimes, et al., J. ofNeurochem. (2001), 75:1219-1232). Optimal transcriptional activity of NF-κB family members, such as c-Rel, is mediated through their association with CBP which is present in limited supply (Sheppard, et al., MoI. Cell. Biol. (1999), /9:6367-6378). We believe that phosphorylate CREB341 at serl 29 or CREB327 at Serl 15 by GSK3β decreases its ability to associate with CBP, whereas inhibition of GSK3β allows CREB to compete more successfully with NF-κB family members for CBP and thereby decreases transcription of NF-κB genes, such as IL-12p40 and other cytokine associated with an inflammatory response.

In one embodiment, the present invention relates to a method of identifying a compound that inhibits IL-12p40 production in a cell after proinflammatory stimulation, comprising: a) contacting the cell with one or more candidate compounds; b) measuring the amount of Ser9 phosphorylated GSK3β in cells contacted with the candidate compound and in cells not so contacted; c) measuring the amount of c-Rel in the nucleus of cells contacted with the candidate compound and in cells not so contacted after proinflammatory stimulation; d) comparing the amount of Set9 phosphorylated GSK3β and nuclear c-Rel in cells contacted with the candidate compound with cells not so contacted, wherein an increase in phosphorylated GSK3β and a decrease in nuclear c-Rel in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound inhibits EL-12p40 production.

In another embodiment, the invention relates to a compound that inhibits IL-12p40 production in a cell after proinflammatory stimulation, wherein the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation.

In another embodiment, the invention relates to a method of decreasing the level of IL-12p40 in a subject comprising administering to the subject a compound that increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation.

In another embodiment, the invention relates to a method of treating an IL- 12 overproduction disorder in a subject comprising administering to the subject a compound that increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation.

In another embodiment, the invention relates to a method of identifying a compound that increases IL-12p40 production in cells after proinflammatory stimulation and increases IL-10 production, comprising: a) contacting the cell with one or more candidate compounds; b) measuring the amount of c-Rel in the nucleus of cells contacted with the candidate compound and in cells not so contacted after proinflammatory stimulation; c) measuring the amount of phosphorylated ERK in cells contacted with the candidate compound and in cells not so contacted; d) comparing the amount of nuclear c-Rel and phosphorylated ERK in cells contacted with the candidate compound with cells not so contacted, wherein a decrease in nuclear c-Rel and an increase in phosphorylated ERK in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound decreases the production of IL12-p40 and increase the production ofIL-10.

In another embodiment, the invention relates to a compound that decreases IL-12p40 production in a cell after proinflammatory stimulation and increase IL-10 production, wherein the compound decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation; and increases phosphorylated ERK in cells contacted with the compound relative to cells not so contacted.

In another embodiment, the invention relates to a method of decreasing the level of IL-12p40 and increasing the level of IL-10 in a subject comprising administering to the subject a compound that decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation; and increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted.

In another embodiment, the invention relates to a method of treating an IL- 12 overproduction disorder in a subject comprising administering to the subject a compound that decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation; and increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted.

Compounds identified by the method of the invention are useful for treating chronic inflammatory conditions such as IL- 12 overproduction disorders.

In another aspect, the invention relates to a method of treating common variable immunodeficiency (CVID) in a subject, comprising administering to the subject an effective amount of N-(3-methyl-benzylidene)-N'-[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)- pyrimidin-4-yl] -hydrazine. In one embodiment, the subject is human. BRIEF DESCRIPTION OF THE FIGURES

Figures IA- IB recite the nucleotide and amino acid sequences of human c-Rel (SEQ ID NOS: 1 and 2, respectively).

Figures 2A and 2B are graphs showing the ability of test compounds to inhibit EFN-γ and IFN-γ/LPS induced p40 (Fig. 2A) and p35 (Fig. 2B) expression.

Figure 3 A is a schematic of the different test promoters used and Figure 3B is a graph demonstrating the ability of the various test promoters to respond to IFN-^/LPS stimulation.

Figure 4 is a western blot analysis of THP-I nuclear extracts in stimulated and non- stimulated cells with regard to the presence of NFKB family members c-Rel, p65 or p50; os- tubulin is an internal control.

Figure 5 is a western blot analysis of THP-I nuclear extracts with anti-ICSBP antibody in stimulated and non-stimulated cells.

Figure 6 is a western blot analysis of THP-I nuclear extracts with anti-PU-1 antibody in stimulated and non-stimulated cells.

Figure 7 is an immunoblot that shows the effect of a test compound on NF-kB pSO nuclear accumulation.

Figure 8 graphically presents the results of a densitometry showing the effect of a test compound on p50 nuclear accumulation.

Figure 9 depicts an immunoblot demonstrating the effect of a test compound on NF- kB p65 nuclear accumulation.

Figure 10 graphically presents the results of a densitometry showing the effect of a test compound on p65 nuclear accumulation.

Figure 11 depicts an immunoblot demonstrating the effect of compound 2 on nuclear accumulation of NF-kB members, including c rel.

Figure 12 is an immunofluorescent study indicating that compound 2 can block the accumulation of c-Rel in the nucleus of cells induced by LPS. Figure 13 is an immunofluorescent study indicating that compound 2 does not block the accumulation of p65 in the nucleus of cells induced by LPS.

Figure 14 is an immunoblot showing the amount of phosphorylation of IKKβ in cells treated with Compound 2 and untreated cells 0 min., 5 min., 15 min., 30 min. and 60 min. after stimulation with IFNγ/LPS.

Figure 15 is is an immunoblot showing the amount of phosphorylation of p65 in cells treated with Compound 2 and untreated cells 0 min., 30 min., 1 hour, and 6 hours after stimulation with IFNγ/LPS.

Figure 16 is an immunoblot showing the amount of phosphorylation of p50 in cells treated with Compound 2 and untreated cells 0 min., 30 min., 1 hour, and 6 hours after stimulation with IFNγ/LPS.

Figure 17 is an immunoblot showing that Compound 2 reduces the accumulation of c-Rel in the nucleus of Jurkat T cells after stimulation with PMA + ionomycin but does not significantly reduce the nuclear accumulation of p65 or p50.

Figure 18 is a graph showing the DNA binding activity of c-Rel after stimulation with LPS/IFNγ in treated and untreated cells.

Figure 19 is an immunoblot showing the levels of c-Rel in nuclear extracts and cytosolic extracts in treated and untreated cells after stimulation with LPS/INFγ.

Figure 20 is a graph showing the densitometry measurement of the immunoblot in Figure 21.

Figure 21 is an immunoblot measuring the phosphorylation of GSK3α and GSK3β in RAW cells after stimulation with LPS in the presence and absence of Compound 2. Phosphorylated GSK3β is significantly increased in the presence of Compound 2, whereas phosphorylated GSK3α remains the about the same in the presence or absence of Compound 2.

Figure 22 is an immunoblot showing a time course for phosphorylation of GSK3β in the presence of Compound 2 in RAW cells.

Figure 23 is an immunoblot showing that Compound 2 phosphorylates and inactivates GSK3β in a dose dependent manner in RAW cells.

Figure 24 is an immunoblot showing a time course for phosphorylation of GSK3β in the presence of Compound 2 in human monocytes. Figure 25 is an immunoblot showing that Compound 2 phosphorylates and inactivates GSK3β in a dose dependent manner in human monocytes.

Figure 26 is an immunoblot showing that Compound 2 phosphorylates and inactivates GSK3β in a dose dependent manner in human monocytes even in the absence of a proinflammatory stimulus, such as LPS.

Figure 27 is a series of graphs showing the effect of Compound 2 or GSK3β inhibitors, Azakenpaullone and BIO, on production of IL-12p40 and IL-IO in human monocytes that have been stimulated with LPS.

Figure 28 is a series of graphs showing the effect of Compound 2 on IL-12p40 and IL-6 production in human monocytes that have been stimulated with Flagellin (TLR5), ultrapure LPS (TLR4) and SAC (TLR2).

Figure 29 is a series of graphs showing the effect of Compound 2 on IL-12p40 and IL-6 production in dendritic cells that have been stimulated with Flagellin (TLR5), ultrapure LPS (TLR4) and SAC (JUO).

Figure 30 is a series of graphs showing the effect of Compound 2 on IL-12p40 and IL-6 production in dendritic cells that have been stimulated with FSL-I (TLR2/TLR6), Pam3CSK4 (TLR2/TLR1) and loxoribine (TLR7).

Figure 31 is a series of graphs showing the effect of Compound 2 on IL-IO production in human monocytes stimulated with SAC (TLR2), LPS (TLR4), Flagellin (TLR5), FSL-I (TLR2/6), or PAM3CSK4 (TLR2/1) in the presence of INF-γ.

Figure 32 is a series of graphs showing the effect of Compound 2 on IL-10 production in DCs stimulated with SAC (TLR2), LPS (TLR4), Flagellin (TLR5), FSL-I (TLR2/6), or PAM3CSK4 (TLR2/1) in the presence of INF-γ.

Figure 33 A shows a fluorescent image of RAW267.4 cells treated with DMSO alone. Figure 33B shows a fluorescent image of RAW267.4 cells treated with 100 nM of Compound 2 for 2 hrs. Figure 33C shows a fluorescent image of RAW267.4 cells treated with 100 nM of Compound 2 for 4 hrs. Figure 33D shows a fluorescent image of RAW267.4 cells treated with 100 nM of Compound 2 for 6 hrs. Figures 33A-33D show that treatment with Compound 2 induces accumulation of ERK in the nucleus of RAW267.4 cells. Figure 34 is an immunoblot showing the effects of Compound 2 on the amount of phosphorylated ERK in RAW cells.

Figure 35a (upper panel) is a graph showing that Compound 2 augments IL-IO production in human monocytes stimulated with LPS. Figure 35a (lower panel) is a graph showing that MEK 1/2 inhibitor, UO 126, inhibits IL-10 production in human monocytes stimulated with LPS.

Figure 35b (upper panel) is a graph showing that UO 126 offsets IL-IO augmentation by Compound 2. Figure 35b (lower panel) is a graph showing that UO 126 does not offset IL-12p40 inhibition by Compound 2.

Figure 36 is a series of graphs showing that U0126 offsets IL-10 augmentation by Compound 2 in DCs.

Figure 37 is a graph showing that UO 126 does not offset IL-12p40 inhibition by Compound 2 in DCs.

Figure 38 is an immunoblot showing that treatment with MEK1/2 inhibitor, U0126, does not reduce the level of GSK3β phosphorylation induced by Compound 2 in RAW cells.

Figure 39 is an immunoblot showing that treatment with Compound 2 induces phosphorylation of ERK and that treatment with both UO 126 and Compound 2 reduces the level of phosphorylation of ERK induced by Compound 2.

DETAILED DESCRIPTION OF THE INVENTION DEFINITIONS

As used herein, the term "Nf-kB family members" refers to ReIA (or p65), ReIB, NF-zcBl (or pl05/p50), NF-κB2 (or plOO/p52), and cRel.

As used herein, the term "a IcB site on DNA" refers to one or more sites on DNA that Rel/NF-kB transcription factors bind to as dimers (e.g. 9-10 base pair sites).

As used interchangeably herein, "c-Rel activity," "biological activity of c-Rel," or "activity of c-Rel," include an activity exerted by c-Rel protein on a c-Rel responsive cell or tissue, e.g., a T cell, dendritic cells, NK cells, or on a c-Rel target molecule, e.g., a nucleic acid molecule or protein target molecule, as determined in vivo, or in vitro, according to standard techniques. In one embodiment, c-Rel activity is a direct activity, such as an association with a c-Rel-target molecule. Alternatively, a c-Rel activity is an indirect activity, such as a downstream biological event mediated by interaction of the c-Rel protein with a c-Rel target molecule. The biological activities of c-Rel are described herein and include, but are not limited to: activation and/or repression of expression of genes linked to kB motifs, including the p40 subunit of EL-12 and IL-23.

As used herein, the term "modulate" with respect to c-Rel includes changing the expression, activity or function of c-Rel in such a manner that it differs from the naturally- occurring expression, function or activity of c-Rel under the same conditions. For example, the expression, function or activity can be greater or less than that of naturally occurring c- ReI, e.g., owing to a change in binding specificity, etc. As used herein, the various forms of the term "modulate" include potentiating (e.g., increasing or upregulating a particular response or activity) and inhibition (e.g., decreasing or downregulating a particular response or activity). In one embodiment of the invention, the biological activity of c-Rel is modulated indirectly e.g., by modulating the activity of a molecule that is upstream or downstream of c-Rel in a signal transduction pathway involving c-Rel. In a preferred embodiment, the activity of c-Rel is inhibited.

ERKs (Extracellularly Regulated Kinases) belong to the family of MAP kinases. ERKl and ERK2, proteins of 43 and 41 kDa respectively, are about 85% identical, and both are ubiquitously expressed, although levels vary from tissue to tissue. They are activated by extracellular stimulation by many growth factors, mitogens and cytokines and can be activated through toll-like receptor stimulation. Activation of ERKl and ERK2 occurs when a threonine or tyrosine residue within a Thr-Glu-Tyr motif of the activation loop is phosphorylated by MEKl or MEK2. Both sites must be phosphorylated for maximum activity. As used herein, "ERK" refers either ERKl or ERK2, separately, or to both ERKl and ERK2.

Glycogen synthase kinase-3 (GSK3) is a protein kinase that was originally identified as a regulator of glycogen synthase, a key enzyme in glycogen metabolism. Since then it has been found to be involved in number of additional activities. GSK3 exists in two isoforms, GSK3α and GSK3β which constituitively active but can be inactivated by phosphorylation at the serine 21 (ser21) residue for GSK3α and serine 9 (ser9) for GSK3β.

As used herein, the term "stimulus" means a growth factor, a cytokine, a hormone, a steroid, a lipid, an antigen, a small molecule (e.g., Ca²⁺, cAMP, cGMP), an osmotic shock, a heat or cold shock, a pH change, a change in ionic strength, a mechanical force, a viral or bacterial infection, or an attachment or detachment from a neighboring cell or a surface with or without a coated protein. In one embodiment, the stimulus is a proinflammatory stimulus that activates one or more TLR signaling cascade, such as FSL-I which activates TLR2/6, SAC which activates TLR2/6, Pam3CSK4 which activatesTLR2/l, Flagellin which activates TLR5, Loxoribine which activates TLR7, LPS which activates TLR4, PoIy(LC) which activates TLR3, and CpG which activates TLR9.

As used herein, the term "contacting" (i.e., contacting a cell e.g. a cell, with a compound) includes incubating a compound of the invention and the cell together in vitro (e.g., adding the compound to cells in culture) as well as administering the compound to a subject such that the compound and cells of the subject are contacted in vivo. The term "contacting" does not include exposure of cells to a c-Rel, IL-12p40 or IL-IO modulator that may occur naturally in a subject (i.e., exposure that may occur as a result of a natural physiological process).

As used herein, the terms "subject" , "patient" and "mammal" are used interchangeably. The terms "subject" and "patient" refer to an animal (e.g., a bird such as a chicken, quail or turkey, or a mammal), preferably a mammal including a non-primate (e.g., a cow, pig, horse, sheep, rabbit, guinea pig, rat, cat, dog, and mouse) and a primate (e.g., a monkey, chimpanzee and a human), and more preferably a human. In one embodiment, the subject is a non-human animal such as a farm animal (e.g., a horse, cow, pig or sheep), or a pet (e.g., a dog, cat, guinea pig or rabbit). In a preferred embodiment, the subject is a human.

As used herein, the terms "molecule of the invention," or "compound of the invention" are used interchangeably to refer to a compound such as a small molecule, a peptide, a carbohydrate, a nucleic acid, and the like that has been identified by the screening methods of the invention.

As used herein the term "isolated," when used in reference to a compound of the invention, means that the agent is separated from one or more reagent, precursor or other reaction product. Therefore, an isolated compound is a compound that is free from one or more compounds found in the synthetic reaction or reaction pathway that produces the agent. Also included in the term is a compound that is free from one or more compound that it is found with in nature. An isolated compound also includes a substantially pure compound. The term can include a compound that has been produced by a combinatorial chemistry method and separated from precursors and other products by chemical purification or by binding to second compound with sufficient stability to be co-purified with the second compound. The term can include naturally occurring compounds such as products of biosynthetic reactions or non-naturally occurring compounds. In one embodiment, the compounds of the invention are isolated.

As used herein, the term "substantially pure" when used in reference to the compounds of the invention means that the compounds have been separated from components which naturally accompany it if it is a naturally occurring compound or from other compounds occurring in a reaction mixture if it is a synthetic compounds. Typically, a compound of the invention is substantially pure when it is at least 60%, by weight, free from other compounds. Preferably, a compound of the invention is at least 75%, more preferably at least 90%, and most preferably at least 99%, by weight free of other compounds. In one embodiment, the compounds of the invention are substantially pure.

SCREENING METHODS FOR IDENTIFYING MODULATORS

In some embodiments, the invention provides a method for the identifying a compound that inhibits IL-12p40 production in a cell after stimulation, preferably proinflammatory stimulation, comprising: a) contacting the cell with one or more candidate compounds; b) measuring the amount of Ser9 phosphorylated GSK3β in cells contacted with the candidate compound and in cells not so contacted; c) measuring the amount of c- ReI in the nucleus of cells contacted with the candidate compound and in cells not so contacted after proinflammatory stimulation; d) comparing the amount of Ser9 phosphorylated GSK3β and nuclear c-Rel in cells contacted with the candidate compound with cells not so contacted, wherein an increase in phosphorylated GSK3β and a decrease in nuclear c-Rel in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound inhibits IL-12p40 production. In this embodiment, the amount of Ser9 phosphorylated GSK3β is measured after contact with the candidate compound but may be measured either before or after stimulation, such as proinflammatory stimulation. In addition, cells may be contacted with the candidate compound either before or after stimulation, such as proinflammatory stimulation. In some embodiments, the candidate compound inhibits the production of IL-23. In some embodiments, the candidate compound inhibits the production of IL-12. In some embodiments, the candidate compound inhibits the production of IL-27. In some embodiments, the candidate compound inhibits the production of both IL-12 and IL-23. In some embodiments, the candidate compound inhibits the production of IL-12, IL-23 and IL-27. In some embodiments, the method for identifying a compound that inhibits IL- 12p40 production in a cell after stimulation, preferably proinflammatory stimulation, further comprises: e) measuring the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the candidate compound and in cells not so contacted; and f) comparing the amount of Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the candidate compound with cells not so contacted, wherein a decrease in Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound inhibits IL-12p40 production.

In some embodiments, the method for identifying a compound that inhibits IL- 12p40 production in a cell after stimulation, preferably proinflammatory stimulation, further comprises: e) measuring the amount of phosphorylated ERK in cells contacted with the candidate compound and in cells not so contacted; and f) comparing the amount of phosphorylated ERK in cells contacted with the candidate compound with cells not so contacted, wherein an increase in phosphorylated ERK in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound inhibits IL-12p40 production. In another embodiment, the candidate compound increases the production of IL-10 in cells contacted with the candidate compound relative to cells not so contacted.

In some embodiments, the invention provides a method for identifying a compound that decreases EL-12p40 production in cells after stimulation, preferably proinflammatory stimulation, and increases IL-10 production, comprising: a) contacting the cell with one or more candidate compounds; b) measuring the amount of c-Rel in the nucleus of cells contacted with the candidate compound and in cells not so contacted after proinflammatory stimulation; c) measuring the amount of phosphorylated ERK in cells contacted with the candidate compound and in cells not so contacted; d) comparing the amount of nuclear c- ReI and phosphorylated ERK in cells contacted with the candidate compound with cells not so contacted, wherein a decrease in nuclear c-Rel and an increase in phosphorylated ERK in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound decreases the production of IL12-p40 and increase the production of IL-10. In this embodiment, the amount of phosphorylated ERK is measured after contact with the candidate compound but may be measured either before or after stimulation, such as proinflammatory stimulation. In some embodiments, the candidate compound inhibits the production of IL-23. In some embodiments, the candidate compound inhibits the production of IL- 12. In some embodiments, the candidate compound inhibits the production of IL-27. In some embodiments, the candidate compound inhibits the production of both IL- 12 and IL-23. In some embodiments, the candidate compound inhibits the production of IL-12, IL-23 and IL-27.

In some embodiments, the method for identifying a compound that decreases IL- 12p40 production in cells after stimulation, preferably proinflammatory stimulation, and increases IL-IO production, further comprises: e) measuring the amount of Ser9 phosphorylated GSK3β in cells contacted with the candidate compound and in cells not so contacted; and f) comparing the amount of Ser9 phosphorylated GSK3β in cells contacted with the candidate compound with cells not so contacted, wherein an increase in phosphorylated GSK3β in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound inhibits IL-12p40 production. In one aspect of this embodiment, the method may further comprising the steps of: g) measuring the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the candidate compound and in cells not so contacted; and h) comparing the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the candidate compound with cells not so contacted, wherein a decrease in Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound decrease IL-12p40 production.

Exemplary cells and cell lines useful in the screening methods of the present invention include, but are not limited to, macrophages, dendritic cells, monocytes, peripheral blood mononuclear cells, which preferably are stimulated with a proinflammatory stimulus that activates one or more TLR signaling cascade. In one embodiment, the cells are stimulated with one or more of the following molecules: FSL-I which activates TLR2/6, SAC which activates TLR2/6, Pam3CSK4 which activatesTLR2/l, Flagellin which activates TLR5, Loxoribine which activates TLR7, LPS which activates TLR4, PoIy(LC) which activates TLR3, or CpG which activates TLR9.

Methods that can be used to carry out the foregoing are commonly known in the art and/or those methods disclosed herein. The cells used in the methods of this embodiment of the invention can either endogenously or recombinantly express c-Rel, or a fragment, derivative or analog thereof. Recombinant expression of c-Rel is carried out by introducing c-Rel encoding nucleic acids into expression vectors and subsequently introducing the vectors into a cell to express c-Rel or simply introducing c-Rel encoding nucleic acids into a cell for expression, as described herein or using procedures well known in the art. In a specific embodiment, c-Rel is expressed with a tag for ease of detection but where the tag has no effect on c-Rel activity, or post-translational modification state, phosphorylation state, or subcellular localization thereof. Nucleic acids encoding c-Rel from a number of species have been cloned and sequenced and their expression is well known in the art An illustrative example of a human c-Rel nucleotide and amino acid sequence is set forth in Figure 1 (SEQ ID NOS: 1 and 2). Expression can be from expression vectors or intrachromosomal. In a specific embodiment, standard human cell lines, such as human dendritic cell lines or the human monocyte cell line THP-I, or human peripheral blood mononuclear cells, are employed in the screening assays. In specific aspects, when immune cells are employed, the immune cells are contacted with immunoactivating compounds such as lipopolysaccharide (LPS) or interferon-7(IFN-γ), before, concurrently or after contacting with the one or more candidate compounds.

Any method known to those of skill in the art for the insertion of c-Rel-encoding DNA into a vector may be used to construct expression vectors for expressing c-Rel, including those methods described herein. In addition, a host cell strain may be chosen which modulates the expression of c-Rel, or modifies and processes the gene product in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus, expression of c-Rel protein may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification {e.g., glycosylation, cleavage) of proteins. Appropriate cell lines or host systems can be chosen to ensure the desired modification and processing of the c-Rel protein expressed. Illustrative cell lines are those described in the Examples section.

In specific embodiments of the method claims of this invention (described infra and supra), the cell contacted with the candidate compound(s) is preferably a macrophage, monocyte or dendritic cell.

In specific embodiments of the method claims of this invention (described infra and supra), the cell may be stimulated with IFN-γ, lipopolysaccharide (LPS), or another stimulus prior to, concurrently with or subsequent to contact with the candidate compound(s). In another embodiment, a compound identified in accordance with the methods of the invention as decreasing the level of c-Rel in the nucleus of cells contacted with the compound decreases IL-12p35 transcription.

In certain embodiments the compound is not a compound described in the patents or patent applications listed in Table 2. In certain embodiments the compound is not a compound described in the patents or patent applications listed in Table 1. In certain embodiments the compound is a compound described in the patents or patent applications listed in Table 1. In certain embodiment, the compound is not Compound 2. The patents and patent applications listed in Table 1 and Table 2 are each incorporated by reference herein in their entirety. In one aspect, the compound is identified by any of the screening methods disclosed herein. In another aspect, the compound is purified by techniques known in the art.

Table 1

Table 2

In specific embodiments, the amount of c-Rel bound to DNA in the nucleus of the cell is decreased. In specific embodiments, the cell used is a cultured cell. In other specific embodiments, the candidate compounds are derived from a constrained random peptide library.

Any method known in the art may be used to measure the level of c-Rel accumulated in the nucleus of a cell. For example, the accumulation of c-Rel in the nucleus of a cell may be detected by contacting the cell with an antibody to c-Rel or a binding region of said antibody, and a fluorescently labeled binding partner of said antibody under conditions conducive to immunospecific binding. Alternatively, the accumulation of c-Rel in the nucleus of a cell may be detected by contacting the cell with a fluorescently labeled antibody to c-Rel or a binding region of said antibody under conditions conducive to immunospecific binding. The accumulation of c-Rel in the nucleus of a cell may also be detected by mass spectroscopy sequencing of nuclear proteins isolated from the cell. Further, the accumulation of c-Rel in the nucleus of a cell may be detected by measuring the amount of c-Rel-dependent transcription, e.g., measuring p40 transcription, or total cellular p40 protein levels, or total nuclear p40 protein levels.

Any method known in the art may be used to measure the post-translational modification state or phosphorylation state of c-Rel in a cell. Techniques are well known in the art for analyzing phosphorylation and other post-translational modification states. For example, phosphorylation may be detected by the use of antibodies to phospho-epitopes to detect a phosphorylated polypeptide by Western blot analysis. Detecting shifted molecular weights by mass spectroscopy is another art-recognized example of how phosphorylation can be detected. Another example of an assay that can be used to measure the post- translational modification state is antibody array technology. Another example of an assay that can be used to measure the phosphorylation state of c-Rel is peptide array technology.

Any method known in the art may be used to measure the DNA binding of c-Rel in the nucleus of a cell. For example, DNA binding of c-Rel in the nucleus can be determined by solution UV cross-linking and subsequent SDS-PAGE. Radioactive supershift assays and non-radioactive transfactor assays (e.g. BD Mercury™ TransFactor Profiling Kit from BD Biosciences Clontech) can also be used to measure the DNA binding activity of c-Rel.

Any method known in the art may be used to measure the level of NFKB expression, including, but not limited to, measuring the protein levels of NFKB family members p50, p65 and c-Rel by immunospecific binding or measuring the levels of the encoding mRNA. In a particular embodiment, expression of NFKB refers to the expression of NFKB family members pSO, p65 and c-Rel, as measured, e.g., in a western blot using a whole cell protein extract. Any method known in the art may be used to measure the amount of IKB, including, but not limited to, measuring the total amount of IKB protein or encoding mRNA in the cell, as measured, e.g., in a western blot using either a whole cell or cytoplasmic protein extract, or measuring the level of IKB degradation by, e.g., measuring IKB protein levels in the treated cells as compared to levels in the untreated cells. In the context of NFKB and/or IKB (including IκBα and IκB/3) expression and/or amount, the term "materially inhibiting" as used herein means a greater than 10%, preferably greater than 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 90% or 95% change in the level of expression of NFKB and/or amount of IKB.

DETECTION OF C-REL OR ERK SUBCELLULAR LOCALIZATION

Any method known in the art for detecting the subcellular localization of c-Rel or ERK, i.e., to the nucleus or cytoplasm, can be used in the present invention. For example, and not by way of limitation, one such method of detection is contacting a cell with an antibody specific for c-Rel or ERK and then detecting whether the antibody localizes to the nucleus. A particular method of detecting c-Rel or ERK subcellular localization is to contact a labeled anti-c-Rel or anti-ERK antibody, e.g., labeled with a fluorescent dye, and a labeled anti-DNA antibody, e.g., with a fluorescent dye different from the anti-c-Rel or anti- ERK antibody, to whole cells and then to detect cells having both labels co-localized in the cell by, e.g., laser scanning microscopy.

Thus, detection methods encompassed by the present invention include immunofluorescence or immunoelectron microscopy, for in situ detection of c-Rel or ERK In situ detection may be accomplished by contacting a cell endogenously or recombinantly expressing a c-Rel or ERK with a labeled molecule that binds to c-Rel or ERK and detecting any binding that occurs and that is localized to the nucleus. Alternatively, an unlabeled compound may be used, in combination with a labeled binding partner of the compound. Using such an assay, it is possible to determine not only the presence of c-Rel or ERK, but also its subcellular distribution, i.e., in the nucleus. Alternatively, c-Rel or ERK can be expressed with a detectable moiety, such as a flag tag. An antibody specific for the tag then allows for detection of the recombinant c-Rel or ERK.

Immunoassays for c-Rel or ERK will typically comprise incubating a sample, such as a cell in vivo or in in vitro culture, in the presence of a detectably labeled molecule specific for c-Rel or ERK, e.g., an antibody to c-Rel or ERK, and detecting the bound molecule by any of a number of techniques known in the art.

In a specific embodiment, a biological sample, e.g., freshly obtained cells, may be brought in contact with and immobilized onto a solid phase support or carrier such as nitrocellulose, glass, polystyrene, or other solid support, which is capable of immobilizing cells. The support may then be washed with suitable buffers followed by treatment with the detectably labeled molecule. The solid phase support may then be washed with the buffer a second time to remove unbound molecule. The amount of bound label on solid support may then be detected by conventional means.

The binding activity of a given antibody to a c-Rel or ERK may be determined according to well-known methods. Those skilled in the art will be able to determine operative and optimal assay conditions for each determination by employing routine experimentation.

One of the ways in which an antibody to c-Rel or ERK can be detectably labeled is by linking the same to an enzyme and use in an enzyme immunoassay (EIA) (Voller, A., "The Enzyme Linked Immunosorbent Assay (ELISA)", 1978, Diagnostic Horizons 2:1-7, Microbiological Associates Quarterly Publication, Walkersville, MD); Voller et al., 1978, J. Clin. Pathol., 37:507-520; Butler, 1981, Meth. Enzymol., 73:482-523; Maggio, E. (ed.), 1980, Enzyme Immunoassay, CRC Press, Boca Raton, FL,; Ishikawa etal, (eds.), 1981, Enzyme Immunoassay, Kgaku Shoin, Tokyo)). The enzyme which is bound to the antibody bound to a c-Rel or ERK will react with an appropriate substrate, preferably a chromogenic substrate, in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorimetric or by visual means.

It is also possible to label the antibody with a fluorescent or chemiluminescent or bioluminescent compound or with a radioactive moiety or other label known in the art.

Another method of detecting and/or measuring c-Rel or ERK nuclear localization is to isolate nuclear proteins by any method known in the art and detect whether c-Rel or ERK is present in the pool of nuclear proteins, preferably by mass spectroscopy analysis to identify the proteins in the pool of nuclear proteins. Isolation of nuclear proteins can be accomplished by any method know in the art. After nuclear protein isolation, detection of c-Rel or ERK can be accomplished, e.g., by immunoprecipitating c-Rel or ERK with an anti-c-Rel or anti-ERK antibody or binding to anti-c-Rel or anti-ERK antibody on an immunoaffinity column or immobilized on a plate or in a well, or visualizing the protein by Western blotting. In another embodiment of the invention, c-Rel or ERK localization to the nucleus can be detected and/or measured by isolating and separating nuclear proteins on a SDS-PAGE gel, eluting separated protein from the gel, and subjecting the eluted protein to mass spectroscopy analysis to determine amino acid sequence. Such mass spectroscopy analysis can be carried out by any suitable method of mass spectroscopy known in the art, e.g., as described in Neubauer et al, 1998, Nature Genetics, 20:46-50; Neubauer et al, 1997, Proc. Natl. Acad. ScL USA, 94:385-390; and WiIm et al., 1996, Nature, 579:466-469. By way of example but not limitation, the eluted peptides are dissolved in a 5% methanol/5% formic acid solution and desalted using a capillary column as described in WiIm and Mann, 1996, Anal. Chem., 68:1-8. The peptides are then diluted in one step in a 50% methanol/5% formic acid solution (0.5-2μl) directly into the spraying needle of the nanoelectrospray ion source. A mass spectrum of the peptides is acquired. The peptides are then selected in turn in the first quadrupole. This first part of the mass spectrometer is used as a mass filter, allowing the transmission of a peptide ion species of one m/z value at a time. Each peptide is then fragmented individually by collision-induced dissociation with argon in the collision cell. The resulting peptide fragment ions are separated in the third quadrupole and detected. For tryptic peptides this usually results in a 'nested set' of peptide fragments containing the carboxy-terminus. As the mass difference between two adjacent fragments corresponds with the residue masses of the corresponding amino acid, partial sequence of the peptide from its carboxy to amino terminus can be determined.

The cell in which the localization of c-Rel or ERK is detected and/or measured can be in vitro (e.g., isolated in cell culture) or in vivo. The cell in which c-Rel or ERK subcellular localization is detected can be any cell, e.g., one that endogenously or recombinantly expresses c-Rel or ERK or a fragment or homolog thereof. The cell can be vertebrate, insect (e.g., Drosophila), C. elegans, mammalian, bovine, murine, rat, avian, fish, primate, human, etc. The c-Rel or ERK which is expressed can be vertebrate, insect, C. elegans, mammalian, bovine, murine, rat, avian, fish, primate, human, etc. The cell can be a cell of primary tissue, a cell line, or of an animal containing and expressing a c-Rel or ERK transgene. For example, the transgenic animal can be a Drosophila (e.g., melanogaster) or a C. elegans. In a preferred embodiment, the transgene encodes a human c-Rel or ERK. Transgenic animals can be made by standard methods well known in the art.

In specific embodiments of the invention, antibodies and fragments containing the binding domain thereof, directed against c-Rel or ERK are used to detect c-Rel or ERK in a specific embodiment of the above methods. Accordingly, c-Rel or ERK proteins, fragments or analogs or derivatives thereof, in particular, human c-Rel or human ERK protein or fragments thereof, may be used as immunogens to generate anti-c-Rel or anti-ERK protein antibodies. Such antibodies can be polyclonal, monoclonal, chimeric, single chain, Fab fragments, or from an Fab expression library. Methods for the production of such antibodies are well known in the art, and some of which are described, infra.

The antibodies specific for c-Rel or ERK can be used in methods known in the art, and those methods discussed above, relating to the localization and/or quantification of c- ReI or ERK proteins of the invention, e.g., for imaging these proteins, measuring levels thereof in appropriate physiological samples, in diagnostic methods, etc. This hold true also for a derivative, homolog, or analog of a c-Rel or ERK protein.

DETECTION OF NFKB OR IKB SUBCELLULAR LOCALIZATION

The level of expression of NFKB or amount of I/cB can also be determined by using any method known in the art, including the use of antibodies specific to NFKB family members or any subunit thereof, e.g., p50, p65 or c-Rel or to IKB. For example, using an antibody specific for IKB, the amount of IKB can be determined, for example, by the illustrative method taught in the Examples Section, infra. The levels of expression of NFKB can be determined by measuring the amount of p50, p65 or c-Rel.

Other methods for detection of whether c-Rel, or other NFKB family members, is located in the nucleus can include measuring for the presence of proteins, or their encoding mRNA molecules, that are dependent on c-Rel, or other NFKB family members, for transcriptional activation and whether there is an increase (increased c-Rel, or other NFKB family members, in nucleus) or a decrease in expression (decreased c-Rel, or other NFKB family members, in the nucleus).

ANTIBODY PRODUCTION

Various procedures known in the art may be used for the production of antibodies to c-Rel, ERK, NFKB family members or any subunit thereof, or IKB, or a fragment, derivative, homolog or analog of the protein. Antibodies of the invention include, but are not limited to, synthetic antibodies, monoclonal antibodies, recombinantly produced antibodies, intrabodies, mulrispecific antibodies (including bi-specific antibodies), human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain Fvs (scFv) (including bi-specific scFvs), single chain antibodies Fab fragments, F(ab') fragments, disulfide-linked Fvs (sdFv), and anti-idiotypic (anti-Id) antibodies, and epitope- binding fragments of any of the above. In particular, antibodies of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds to an antigen (e.g., one or more complementarity determining regions (CDRs) of an antibody).

For production of the antibody, various host animals can be immunized by injection with, e.g., a native c-Rel protein or a synthetic version, or a derivative of the foregoing. Such host animals include, but are not limited to, rabbits, mice, rats, etc. Various adjuvants can be used to increase the immunological response, depending on the host species, and include, but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, dinitrophenol, and potentially useful human adjuvants such as bacille Calmette-Guerin (BCG) and Corynebacterium parvum. Although the following refers specifically to c-Rel, any of the methods described herein apply equally to c-Rel, ERK, NFKB family members or subunits thereof, or IKB.

For preparation of monoclonal antibodies directed towards c-Rel or a derivative, fragment, homolog or analog thereof, any technique that provides for the production of antibody molecules by continuous cell lines in culture may be used. Such techniques include, but are not restricted to, the hybridoma technique originally developed by Kohler and Milstein (1975, Nature 256:495-497), the trioma technique (Gustafsson et al, 1991, Hum. Antibodies Hybridomas 2:26-32), the human B-cell hybridoma technique (Kozbor et al, 1983, Immunology Today 4:72), and the EBV hybridoma technique to produce human monoclonal antibodies (Cole et al., 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In an additional embodiment of the invention, monoclonal antibodies can be produced in germ-free animals utilizing recent technology described in International Patent Application PCT/US90/02545.

According to the present invention, human antibodies may be used and can be obtained by using human hybridomas (Cote et al, 1983, Proc. Natl. Acad. Sci. USA 80:2026-2030) or by transforming human B cells with EBV virus in vitro (Cole et al, 1985, In: Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96). In fact, according to the invention, techniques developed for the production of "chimeric antibodies" (Morrison etal, 1984, Proc. Natl. Acad. Sci. USA 81:6851-6855; Neubergere/ al, 1984, Nature 312:604-608; Takeda et al, 1985, Nature 314:452-454) by splicing the genes from a mouse antibody molecule specific for c-Rel together with genes from a human antibody molecule of appropriate biological activity can be used; such antibodies are within the scope of this invention.

According to the present invention, techniques described for the production of single chain antibodies (U.S. Patent 4,946,778) can be adapted to produce c-Rel-specific antibodies. An additional embodiment of the invention utilizes the techniques described for the construction of Fab expression libraries (Huse et al, 1989, Science 246:1275-1281) to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity for c-Rel proteins, derivatives, or analogs thereof. Non-human antibodies can be "humanized" by known methods (e.g., U.S. Patent No. 5,225,539).

Antibody fragments that contain the idiotypes of c-Rel can be generated by techniques known in the art. For example, such fragments include, but are not limited to, the F(ab')2 fragment which can be produced by pepsin digestion of the antibody molecule; the Fab' fragment that can be generated by reducing the disulfide bridges of the F(ab')2 fragment; the Fab fragment that can be generated by treating the antibody molecular with papain and a reducing agent; and Fv fragments. Synthetic antibodies, e.g., antibodies produced by chemical synthesis, are useful in the present invention.

In the production of antibodies, screening for the desired antibody can be accomplished by techniques known in the art, e.g., ELISA (enzyme-linked immunosorbent assay). To select antibodies specific to a particular domain of c-Rel, or a derivative, homolog, or analog thereof, one may assay generated hybridomas for a product that binds to the fragment of the c-Rel protein, or a derivative, homolog, or analog thereof, that contains such a domain.

RECOMBINANT EXPRESSION

Methods for recombinant production of c-Rel and derivatives or fragments or homologs thereof for use in the screening methods of the present invention are well known to those skilled in the art. Nucleic acids encoding c-Rel, derivatives, fragments, and homologs thereof are known in the art. The nucleotide sequence encoding an illustrative human c-Rel molecule is known and is provided in Figure 1 (SEQ ID NO: 1). Nucleic acids encoding c-Rel can be obtained by any method known in the art, e.g., by PCR amplification using synthetic primers hybridizable to the 3¹ and 5' ends of each sequence, and/or by cloning from a cDNA or genomic library using an oligonucleotide specific for each nucleotide sequence.

Homologs (e.g., nucleic acids encoding c-Rel of species other than human) or other related sequences (eg., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe, using methods well known in the art for nucleic acid hybridization and cloning.

The encoded c-Rel protein, which is depicted in Figure 1 (SEQ ID NO:2) can be obtained by methods well known in the art for protein purification and recombinant protein expression. For recombinant expression of one or more of the proteins, the nucleic acid containing all or a portion of the nucleotide sequence encoding the protein can be inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted protein coding sequence. The necessary transcriptional and translational signals can also be supplied by the native promoter of the c- ReI gene, and/or their flanking regions.

A variety of host-vector systems may be utilized to express the protein coding sequence. These include but are not limited to mammalian cell systems infected with virus (e.g., vaccinia virus, adenovirus, etc.); insect cell systems infected with virus (e.g., baculovirus); microorganisms such as yeast containing yeast vectors; or bacteria transformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. The expression elements of vectors vary in their strengths and specificities. Depending on the host-vector system utilized, any one of a number of suitable transcription and translation elements may be used.

In a preferred embodiment, human c-Rel is obtained by expressing the human c-Rel coding sequence. In yet another embodiment, a derivative, fragment or homolog of c-Rel is recombinantly expressed. In one embodiment, the c-Rel protein is expressed as chimeric or fusion protein in which an amino acid sequence different from the c-Rel sequence is linked via a peptide bond to the c-Rel sequence. The different amino acid sequence can be a tag, such as a flag tag, for detection and isolation of the expressed chimeric or fusion protein.

Any method available in the art can be used for the insertion of DNA fragments into a vector to construct expression vectors containing a chimeric gene consisting of appropriate transcriptional/translational control signals and protein coding sequences. These methods may include in vitro recombinant DNA and synthetic techniques and in vivo recombinant techniques (genetic recombination). Expression of nucleic acid sequences encoding c-Rel, or a derivative, fragment or homolog thereof, may be regulated by a second nucleic acid sequence so that the gene or fragment thereof is expressed in a host transformed with the recombinant DNA molecule(s). For example, expression of the proteins may be controlled by any promoter/enhancer known in the art. In a specific embodiment, the promoter is not native to the gene for c-Rel. In another specific embodiment, the promoter is active in immune cells, e.g., peripheral blood mononuclear cells, dendritic cells or monocytes or splenocytes. Promoters that may be used include but are not limited to the SV40 early promoter (Bemoist and Chambon, 1981, Nature, 290:304- 310), the promoter contained in the 3¹ long terminal repeat of Rous sarcoma virus (Yamamoto et al, 1980, Cell, 22:1X1-191), the herpes thymidine kinase promoter (Wagner et al., 1981, Proc. Natl. Acad. Sci. USA, 75:1441-1445), the regulatory sequences of the metallothionein gene (Brinster et al, 1982, Nature, 296:39-42); prokaryotic expression vectors such as the /^-lactamase promoter (Villa-Kamaroff et al., 1978, Proc. Natl. Acad. ScL USA, 75:3727-3731) or the tac promoter (DeBoer et al., 1983, Proc. Natl. Acad. ScL USA, 80:21-25; Gilbert et al., 1980, Scientific American, 242:19-94); plant expression vectors comprising the nopaline synthetase promoter (Herrar-Estrella et al, 1984, Nature, 303:209-213) or the cauliflower mosaic virus 35S RNA promoter (Garder et al, 1981, Nucleic Acids Res., 9:2871), and the promoter of the photosynthetic enzyme ribulose bisphosphate carboxylase (Herrera-Estrella et al, 1984, Nature, 310:115-120); promoter elements from yeast and other fungi such as the Gal4 promoter (Johnston et al, 1987, Microbiol. Rev,. 57:458-476), the alcohol dehydrogenase promoter (Schibler et al, 1987, Annual Review Genetics, 21:231-251), the phosphoglycerol kinase promoter (Struhl et al, 1995, Annual Review Genetics, 29:651-614-251; Guarente 1987, Annual Review Genetics, 27:425-452), the alkaline phosphatase promoter (Struhl et al, 1995, Annual Review Genetics, 29:651-674-257; Guarente 1987, Annual Review Genetics, 27:425-452), and the following animal transcriptional control regions that exhibit tissue specificity and have been utilized in transgenic animals: elastase I gene control region which is active in pancreatic acinar cells (Swift et al, 1984, Cell 38:639-646; Ornitz et al, 1986, Cold Spring Harbor Symp. Quant. Biol. 50:399-409; MacDonald 1987, Hepatology 7:425-515); insulin gene control region which is active in pancreatic beta cells (Hanahan et al, 1985, Nature 315:115-122), immunoglobulin gene control region which is active in lymphoid cells (Grosschedl et al, 1984, Cell 38:647-658; Adams et al, 1985, Nature 318:533-538; Alexander et al, 1987, MoI. Cell Biol. 7:1436-1444), mouse mammary tumor virus control region which is active in testicular, breast, lymphoid and mast cells (Leder et al, 1986, Cell 45:485-495), albumin gene control region which is active in liver (Pinckert et al, 1987, Genes and Devel. 1:268-276), alpha-fetoprotein gene control region which is active in liver (Krumlauf et al, 1985, MoI. Cell. Biol. 5: 1639-1648; Hammer et al, 1987, Science 235:53- 58), alpha-1 antitrypsin gene control region which is active in liver (Kelsey et al, 1987, Genes and Devel. 1:161-171), beta globin gene control region which is active in myeloid cells (Mogram et al, 1985, Nature 315:338-340; Kollias et al, 1986, Cell 46:89-94), myelin basic protein gene control region which is active in oligodendrocyte cells of the brain (Readhead et al., 1987, Cell 48:703-712), myosin light chain-2 gene control region which is active in skeletal muscle (Sard 1985, Nature 314:283-286), and gonadotrophs releasing hormone gene control region which is active in gonadotrophs of the hypothalamus (Mason et al, 1986, Science 234:1372-1378).

In a specific embodiment, a vector is used that comprises a promoter operably linked to the nucleic acid sequence encoding c-Rel, or a fragment, derivative or homolog thereof, one or more origins of replication, and optionally, one or more selectable markers (e.g., an antibiotic resistance gene).

In another specific embodiment, an expression vector containing the coding sequence, or a portion thereof, of c-Rel is made by subcloning the gene sequence into the EcoSl restriction site of each of the three pGEX vectors (glutathione S-transferase expression vectors; Smith and Johnson, 1988, Gene 7:31-40). This allows for the expression of products in the correct reading frame.

Expression vectors containing the sequences of interest can be identified by three general approaches: (a) nucleic acid hybridization, (b) presence or absence of "marker" gene function, and (c) expression of the inserted sequences. In the first approach, c-Rel sequences can be detected by nucleic acid hybridization to probes comprising sequences homologous and complementary to the inserted sequences. In the second approach, the recombinant vector/host system can be identified and selected based upon the presence or absence of certain "marker" functions (e.g., resistance to antibiotics, occlusion body formation in baculovirus, etc.) caused by insertion of the sequences of interest in the vector. For example, if a c-Rel gene, or portion thereof, is inserted within the marker gene sequence of the vector, recombinants containing the c-Rel fragment will be identified by the absence of the marker gene function [e.g., loss of beta-galactosidase activity). In the third approach, recombinant expression vectors can be identified by assaying for the c-Rel expressed by the recombinant vector.

Once recombinant c-Rel molecules are identified and isolated, several methods known in the art can be used to propagate them. Using a suitable host system and growth conditions, recombinant expression vectors can be propagated and amplified in quantity. As previously described, the expression vectors or derivatives which can be used include, but are not limited to, human or animal viruses such as vaccinia virus or adenovirus; insect viruses such as baculovirus, yeast vectors; bacteriophage vectors such as lambda phage; and plasmid and cosmid vectors.

In addition, a host cell strain may be chosen that modulates the expression of the inserted sequences, or modifies or processes the expressed proteins in the specific fashion desired. Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically-engineered c-Rel may be controlled. Furthermore, different host cells have characteristic and specific mechanisms for the translational and post-translational processing and modification (e.g., glycosylation, phosphorylation, etc.) of proteins. Appropriate cell lines or host systems can be chosen to ensure that the desired modification and processing of the foreign protein is achieved. For example, expression in a bacterial system can be used to produce an unglycosylated core protein, while expression in mammalian cells ensures "native" glycosylation of a heterologous protein. Furthermore, different vector/host expression systems may effect processing reactions to different extents.

In other specific embodiments, the c-Rel protein or a fragment, homolog or derivative thereof, may be expressed as fusion or chimeric protein products comprising the protein, fragment, homolog, or derivative joined via a peptide bond to a heterologous protein sequence of a different protein. Such chimeric products can be made by ligating the appropriate nucleic acid sequences encoding the desired amino acids to each other by methods known in the art, in the proper coding frame, and expressing the chimeric products in a suitable host by methods commonly known in the art.

Although the above refers specifically to c-Rel, any of the methods described herein apply equally to recombinant expression of ERK, NFKB family members or subunits thereof, or IKB. CANDIDATE COMPOUNDS

Any compound can be tested for its ability to inhibit the production of IL-12p40 after proinflammatory stimulation and/ or increase IL-IO production according to the method of the invention. Preferred compounds that inhibit IL-12p40 production and/or increase IL-10 production according to the method of the invention include the following compounds:

Compound 1 : N-(lH-indol-3-ylmethylene)-N'-[4-morpholin-4-yl-6-(2-pyridin-2-yl- ethoxy)-[ 1 ,3 ,5]triazin-2-yl]-hydrazine;

Compound 2: N-(3-methyl-benzylidene)-N^>-[6-morpholin-4-yl-2-(2-pyridin-2-yl- ethoxy)-pyrimidin-4-yl]-hydrazine;

Compound 3: N-(lH-indol-3-ylmethylene)-N'-[4-morpholin-4-yl-6-(2-morpholin- 4-yl-ethoxy)-pyridin-2-yl]-hydrazine

Compound 4: N-[3,5-Difluoro-2-morpholin-4-yl-6-(2-morpholin-4-yl-ethoxy)- pyridin-4-yl]-N^l-(3-methyl-benzylidene)-hydrazine;

Compound 5 : N-(3-methyl-benzylidene)-N'-[4-moφholin-4-yl-6-(2-morpholin-4- yl-ethoxy)-pyridin-2-yl]-hydrazine;

Compound 6: N-methyl-N'-(3-methyl-benzylidene)-N-[4-morpholin-4-yl-6-(2- moφholin-4-yl-ethoxy)-pyridin-2-yl]-hydrazine;

Compound 7 : 4-methyl-2- { [4-morpholin-4-yl-6-(2-morpholin-4-yl-ethoxy)-pyridin- 2-yl]-hydrazononomethyl}-phenylamine;

Compound 8: N-(6,7-dimemoxy-2-moφholin-4-yl-quinolin-4-yl)-NX3-methyl- benzylidene)-hydrazine;

Compound 9: N-7-Chloro-2-morpholin-4-yl-quninazolin-4-yl)-N'-(3-methyl- ben2ylidene)-hydrazine;

Compound 10: N-[7-memoxy-2-moφholin-4-yl-6-(2-phenoxy-ethoxy)-quinazolin- 4-yl]-N'-(3-methyl-benzylidene)-hydrazine; .

Compound 11: N-[6-Moφholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimidin-4- ylmethylene]-W-m-tolyl-hydrazine;

Compound 12: //-(3-Chloro-phenyl)- iV-[6-moφholin-4-yl-2-(2-pyridin-2-yl- ethoxy)-pyrimidin-4-ylmethylene]- hydrazine;

Compound 13: iV-(3-Methoxy-phenyl)- ΛT-[6-moφholin-4-yl-2-(2-pyridin-2-yl- ethoxy)-pyrimidin-4-ylmethylene]- hydrazine; and

Compound 14: 7V-(2,5-Dimethyl-phenyl)- iV-[6-moφholin-4-yl-2-(2-pyridin-2-yl- ethoxy)-pyrimidin-4-ylmethylene]- hydrazine. The screening methods of the invention are well suited to screen chemical libraries for compounds which inhibit the production of IL-12p40 and/or increase IL-10. The chemical libraries can be peptide libraries, peptidomimetic libraries, chemically synthesized libraries, recombinant, e.g., phage display libraries, and in vitro translation-based libraries, other non-peptide synthetic organic libraries, etc.

Libraries screened using the methods of the present invention can comprise a variety of types of compounds. Examples of libraries that can be screened in accordance with the methods of the invention include, but are not limited to, peptoids; random biooligomers; diversomers such as hydantoins, benzodiazepines and dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; carbohydrate libraries; and small molecule libraries (preferably, small organic compound libraries). In some embodiments, the compounds in the libraries screened are nucleic acid or peptide molecules. In a non-limiting example, peptide molecules can exist in a phage display library. In other embodiments, the types of compounds include, but are not limited to, peptide analogs including peptides comprising non-naturally occurring amino acids, e.g., D-amino acids, phosphorous analogs of amino acids, such as γ-amino phosphoric acids and γ-amino phosphoric acids, or amino acids having non-peptide linkages, nucleic acid analogs such as phosphorothioates and PNAs, hormones, antigens, synthetic or naturally occurring drugs, opiates, dopamine, serotonin, catecholamines, thrombin, acetylcholine, prostaglandins, organic compounds, pheromones, adenosine, sucrose, glucose, lactose and galactose. Libraries of polypeptides or proteins can also be used in the assays of the invention.

In a preferred embodiment, the combinatorial libraries are small organic compound libraries including, but not limited to, benzodiazepines, isoprenoids, thiazolidinones, metathiazanones, pyrrolidines, morpholino compounds, and benzodiazepines. In another embodiment, the combinatorial libraries comprise peptoids; random bio-oligomers; benzodiazepines; diversomers such as hydantoins, benzodiazepines and dipeptides; vinylogous polypeptides; nonpeptidal peptidomimetics; oligocarbamates; peptidyl phosphonates; peptide nucleic acid libraries; antibody libraries; or carbohydrate libraries. Combinatorial libraries are themselves commercially available (see, e.g., ComGenex, Princeton, New Jersey; Asinex, Moscow, Ru, Tripos, Inc., St. Louis, Missouri; ChemStar, Ltd, Moscow, Russia; 3D Pharmaceuticals, Exton, Pennsylvania; Martek Biosciences, Columbia, Maryland; etc.). In a preferred embodiment, the library is preselected so that compounds of the library are more amenable for cellular uptake. For example, compounds are selected based on specific parameters such as, but not limited to, size, lipophilicity, hydrophilicity, and hydrogen bonding, which enhance the likelihood of compounds getting into the cells. In another embodiment, the compounds are analyzed by three-dimensional or four- dimensional computer computation programs.

The combinatorial compound library for use in accordance with the methods of the present invention may be synthesized. There is a great interest in synthetic methods directed toward the creation of large collections of small organic compounds, or libraries, which could be screened for pharmacological, biological or other activity. The synthetic methods applied to create vast combinatorial libraries are performed in solution or in the solid phase, i.e., on a solid support. Solid-phase synthesis makes it easier to conduct multi- step reactions and to drive reactions to completion with high yields because excess reagents can be easily added and washed away after each reaction step. Solid-phase combinatorial synthesis also tends to improve isolation, purification and screening. However, the more traditional solution phase chemistry supports a wider variety of organic reactions than solid- phase chemistry.

Combinatorial compound libraries may be synthesized using the apparatus described in U.S. Patent No. 6,190,619 to Kilcoin et ah, which is hereby incorporated by reference in its entirety. U.S. Patent No. 6,190,619 discloses a synthesis apparatus capable of holding a plurality of reaction vessels for parallel synthesis of multiple discrete compounds or for combinatorial libraries of compounds.

In one embodiment, the combinatorial compound library can be synthesized in solution. The method disclosed in U.S. Patent No. 6,194,612 to Boger et ah, which is hereby incorporated by reference in its entirety, features compounds useful as templates for solution phase synthesis of combinatorial libraries. The template is designed to permit reaction products to be easily purified from unreacted reactants using liquid/liquid or solid/liquid extractions. The compounds produced by combinatorial synthesis using the template will preferably be small organic compounds. Some compounds in the library may mimic the effects of non-peptides or peptides. In contrast to solid phase synthesize of combinatorial compound libraries, liquid phase synthesis does not require the use of specialized protocols for monitoring the individual steps of a multistep solid phase synthesis (Egner et ah, 1995, J.Org. Chem. 60:2652; Anderson et ah, 1995, J. Org. Chem. 60:2650; Fitch et al, 1994, J. Org. Chem. 59:7955; Look et al, 1994, J. Org. Chem. 49:7588; Metzger et al, 1993, Angew. Chem., Int. Ed. Engl. 32:894; Youngquist et al, 1994, Rapid Commun. Mass Spect. 8:77; Chu et al, 1995, J. Am. Chem. Soc. 117:5419; Brummel et al, 1994, Science 264:399; and Stevanovic et al, 1993, Bioorg. Med. Chem. Lett. 3:431).

Combinatorial compound libraries useful for the methods of the present invention can be synthesized on solid supports. In one embodiment, a split synthesis method, a protocol of separating and mixing solid supports during the synthesis, is used to synthesize a library of compounds on solid supports (see e.g., Lam et al, 1997, Chem. Rev. 97:41-448; Ohlmeyer et al, 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926 and references cited therein). Each solid support in the final library has substantially one type of compound attached to its surface. Other methods for synthesizing combinatorial libraries on solid supports, wherein one product is attached to each support, will be known to those of skill in the art (see, e.g., Nefzi et al, 1997, Chem. Rev. 97:449-472).

As used herein, the term "solid support" is not limited to a specific type of solid support. Rather a large number of supports are available and are known to one skilled in the art. Solid supports include silica gels, resins, derivatized plastic films, glass beads, cotton, plastic beads, polystyrene beads, alumina gels, and polysaccharides. A suitable solid support may be selected on the basis of desired end use and suitability for various synthetic protocols. For example, for peptide synthesis, a solid support can be a resin such as p- methylbenzhydrylamine (pMBHA) resin (Peptides International, Louisville, KY), polystyrenes (e.g., PAM-resin obtained from Bachem Inc., Peninsula Laboratories, etc.), including chloromethylpolystyrene, hydroxymethylpolystyrene and aminomethylpolystyrene, poly (dimethylacrylamide)-grafted styrene co-divinyl-benzene (e.g., POLYHIPE resin, obtained from Aminotech, Canada), polyamide resin (obtained from Peninsula Laboratories), polystyrene resin grafted with polyethylene glycol (e.g., TENTAGEL or ARGOGEL, Bayer, Tubingen, Germany) polydimethylacrylamide resin (obtained from Milligen/Biosearch, California), or Sepharose (Pharmacia, Sweden).

In some embodiments of the present invention, compounds can be attached to solid supports via linkers. Linkers can be integral and part of the solid support, or they may be nonintegral that are either synthesized on the solid support or attached thereto after synthesis. Linkers are useful not only for providing points of compound attachment to the solid support, but also for allowing different groups of compounds to be cleaved from the solid support under different conditions, depending on the nature of the linker. For example, linkers can be, inter alia, electrophilically cleaved, nucleophilically cleaved, photocleavable, enzymatically cleaved, cleaved by metals, cleaved under reductive conditions or cleaved under oxidative conditions. In a preferred embodiment, the compounds are cleaved from the solid support prior to high throughput screening of the compounds.

In certain embodiments of the invention, the compound is a small molecule, such as a molecule having a molecular weight of less than 1000 g/mole, preferably less than 500 g/mole.

Exemplary libraries are commercially available from several sources (ArQuIe, Tripos/PanLabs, ChemDesign, Pharmacopoeia). In some cases, these chemical libraries are generated using combinatorial strategies that encode the identity of each member of the library on a substrate to which the member compound is attached, thus allowing direct and immediate identification of a molecule that is an effective modulator. Thus, in many combinatorial approaches, the position on a plate of a compound specifies that compound's composition. Also, in one example, a single plate position may have from 1-20 chemicals that can be screened by administration to a well containing the interactions of interest. Thus, if modulation is detected, smaller and smaller pools of interacting pairs can be assayed for the modulation activity. By such methods, many candidate compounds can be screened.

Many diversity libraries suitable for use are known in the art and can be used to provide compounds to be tested according to the present invention. Alternatively, libraries can be constructed using standard methods. Chemical (synthetic) libraries, recombinant expression libraries, or polysome-based libraries are exemplary types of libraries that can be used.

The libraries can be constrained or semirigid (having some degree of structural rigidity), or linear or nonconstrained. The library can be a cDNA or genomic expression library, random peptide expression library or a chemically synthesized random peptide library, or non-peptide library. Expression libraries are introduced into the cells in which the assay occurs, where the nucleic acids of the library are expressed to produce their encoded proteins.

In one embodiment, peptide libraries that can be used in the present invention may be libraries that are chemically synthesized in vitro. Examples of such libraries are given in Houghten et al, 1991, Nature 354:84-86, which describes mixtures of free hexapeptides in which the first and second residues in each peptide were individually and specifically defined; Lam et al, 1991, Nature 354:82-84, which describes a "one bead, one peptide" approach in which a solid phase split synthesis scheme produced a library of peptides in which each bead in the collection had immobilized thereon a single, random sequence of amino acid residues; Medynski, 1994, Bio/Technology 12:709-710, which describes split synthesis and T-bag synthesis methods; and Gallop et al, 1994, J. Medicinal Chemistry 37(9):1233-1251. Simply by way of other examples, a combinatorial library may be prepared for use, according to the methods of Ohlmeyer et al, 1993, Proc. Natl. Acad. Sci. USA 90:10922-10926; Erb et al, 1994, Proc. Natl. Acad. Sci. USA 91:11422-11426; Houghten et al, 1992, Biotechniques 13:412; Jayawickreme et al, 1994, Proc. Natl. Acad. Sci. USA 91:1614-1618; or Salmon et al, 1993, Proc. Natl. Acad. Sci. USA 90:11708-11712. PCT Publication No. WO 93/20242 and Brenner and Lemer, 1992, Proc. Natl. Acad. Sci. USA 89:5381-5383 describe "encoded combinatorial chemical libraries," that contain oligonucleotide identifiers for each chemical polymer library member.

In one embodiment, the library screened is a biological expression library that is a random peptide phage display library, where the random peptides are constrained (e.g, by virtue of having disulfide bonding).

Further, more general, structurally constrained, organic diversity (e.g, nonpeptide) libraries, can also be used. By way of example, a benzodiazepine library (see e.g., Bunin et al, 1994, Proc. Natl. Acad. Sci. USA 91:4708-4712) may be used.

Conformationally constrained libraries that can be used include but are not limited to those containing invariant cysteine residues which, in an oxidizing environment, cross-link by disulfide bonds to form cystines, modified peptides (e.g., incorporating fluorine, metals, isotopic labels, are phosphorylated, etc.), peptides containing one or more non-naturally occurring amino acids, non-peptide structures, and peptides containing a significant fraction of γ-carboxyglutamic acid.

Libraries of non-peptides, e.g., peptide derivatives (for example, that contain one or more non-naturally occurring amino acids) can also be used. One example of these is peptide libraries (Simon et al, 1992, Proc. Natl. Acad. Sci. USA 89:9367-9371). Peptoids are polymers of non-natural amino acids that have naturally occurring side chains attached not to the alpha carbon but to the backbone amino nitrogen. Since peptoids are not easily degraded by human digestive enzymes, they are advantageously more easily adaptable to drug use. Another example of a library that can be used, in which the amide functionalities in peptides have been premethylated to generate a chemically transformed combinatorial library, is described by Ostresh et al, 1994, Proc. Natl. Acad. Sci. USA 91:11138-11142).

The members of the peptide libraries that can be screened according to the invention are not limited to containing the 20 naturally occurring amino acids. In particular, chemically synthesized libraries and polysome based libraries allow the use of amino acids in addition to the 20 naturally occurring amino acids (by their inclusion in the precursor pool of amino acids used in library production). In specific embodiments, the library members contain one or more non-natural or non-classical amino acids or cyclic peptides. Non-classical amino acids include but are not limited to the D-isomers of the common amino acids, oamino isobutyric acid, 4-aminobutyric acid, Abu, 2 -amino butyric acid; γ- Abu, f-Ahx, 6-amino hexanoic acid; Aib, 2-amino isobutyric acid; 3-amino propionic acid; ornithine; norleucine; norvaline, hydroxyproline, sarcosine, citrulline, cysteic acid, t- butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine, designer amino acids such as β-methyl amino acids, Cos-methyl amino acids, No-methyl amino acids, fluoro-amino acids and amino acid analogs in general. Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

In another embodiment of the present invention, combinatorial chemistry can be used to identify compounds that inhibit the production of EL-12p40 and/or increase the production of IL-10 according to the screening method of the invention. Combinatorial chemistry is capable of creating libraries containing hundreds of thousands of compounds, many of which may be structurally similar. While high throughput screening programs are capable of screening these vast libraries for affinity for known targets, new approaches have been developed that achieve libraries of smaller dimension but which provide maximum chemical diversity. (See e.g., Matter, 1997, Journal of Medicinal Chemistry 40:1219-1229).

One method of combinatorial chemistry, affinity fingerprinting, has previously been used to test a discrete library of small molecules for binding affinities for a defined panel of proteins. The fingerprints obtained by the screen are used to predict the affinity of the individual library members for other proteins or receptors of interest. The fingerprints are compared with fingerprints obtained from other compounds known to react with the protein of interest to predict whether the library compound might similarly react. (See, e.g., Kauvar et al, 1995, Chemistry and Biology 2:107-118; Kauvar, 1995, Affinity fingerprinting, Pharmaceutical Manufacturing International. 8:25-28; and Kauvar, Toxic-Chemical Detection by Pattern Recognition in New Frontiers in Agrochemical Immunoassay, D. Kurtz. L. Stanker and J.H. Skerritt. Editors, 1995, AOAC: Washington, D.C., 305-312).

Kay et al, 1993, Gene 128:59-65 (Kay) discloses a method of constructing peptide libraries that encode peptides of totally random sequence that are longer than those of any prior conventional libraries. The libraries disclosed in Kay encode totally synthetic random peptides of greater than about 20 amino acids in length. Such libraries can be advantageously screened to identify compounds that inhibit the production of IL-12p40 and/or increase the production of IL-IO according to the method of the invention. (See also U.S. Patent No. 5,498,538 dated March 12, 1996; and PCT Publication No. WO 94/18318 dated August 18, 1994).

Other libraries useful for identify compounds that inhibit the production of IL-12p40 and/or increase the production of IL-IO according to the method of the invention can include antibody libraries and libraries of intrabodies expressed in the cell.

If the library comprises arrays or microarrays of compounds, wherein each compound has an address or identifier, the compound can be deconvoluted, e.g., by cross- referencing the positive sample to original compound list that was applied to the individual test assays.

If the library is a peptide or nucleic acid library, the sequence of the compound can be determined by direct sequencing of the peptide or nucleic acid. Such methods are well known to one of skill in the art.

A comprehensive review of various types of peptide libraries can be found in Gallop etal., 1994, J. Med. Chem. 37:1233-1251.

COMPOUNDS IDENTIFIED IN SCREENING ASSAYS

The present invention is also directed to compounds found by any of the screening methods described herein.

In some embodiments, the invention relates to a compound that inhibits IL-12p40 production in a cell after stimulation, preferably proinflammatory stimulation, wherein the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation. In certain embodiments, the compound is not N-(3-methyl-benzylidene)-N'-[6- morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimidin-4-yl]-hydrazine. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 2. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 1. In some embodiments, the compound decreases the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound increases the production of IL-IO in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound increases the amount of nuclear ERK in cells contacted with the compound relative to cells not so contacted.

In some embodiments, the invention relates to a compound that decreases IL-12p40 production in a cell after stimulation , preferably proinflammatory stimulation, and increase IL-IO production, wherein the compound decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation; and increases phosphorylated ERK in cells contacted with the compound relative to cells not so contacted. In certain embodiments, the compound is not N-(3- methyl-benzylidene)-N'-[6-moφholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimidin-4-yl]- hydrazine. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 2. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 1. In some embodiments, the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound decreases the amount of Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound increases the amount of ERK in the nucleus of cells contacted with the compound relative to cells not so contacted.

The present invention is further directed to compounds identified by the above- described screening assays and to processes for producing such compounds by use of these assays. Compounds indentified by the screening methods of the invention can include, but are not limited to, nucleic acids, antisense nucleic acids, ribozyme, triple helix, antibody, and polypeptide molecules and small inorganic or organic compounds. Accordingly, in one embodiment, the present invention includes a compound obtained by a method comprising the steps of any one of the aforementioned screening assays.

The present invention is further directed to pharmaceutically acceptable salts, prodrugs, solvates and clathrates of compounds identified by the above-described screening assays.

As used herein and unless otherwise indicated, the term "prodrug" means a derivative of a compound that can hydrolyze, oxidize, or otherwise react under biological conditions (in vitro or in vivo) to provide a compound of this invention. Prodrugs may only become active upon such reaction under biological conditions, but they may have activity in their unreacted forms. Examples of prodrugs contemplated in this invention include, but are not limited to, analogs or derivatives of compounds identified by the screening methods of the invention that comprise biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Other examples of prodrugs include derivatives of compounds identified by the screening methods of the invention that comprise -NO, -NO2, -ONO, or -ONO2 moieties. Prodrugs can typically be prepared using well-known methods, such as those described by 1 BURGER'S MEDICINAL CHEMISTRY AND DRUG DISCOVERY (1995) 172-178, 949-982 (Manfred E. Wolff ed., 5^th ed), the entire teachings of which are incorporated herein by reference.

As used herein and unless otherwise indicated, the terms "biohydrolyzable amide", "biohydrolyzable ester", "biohydrolyzable carbamate", "biohydrolyzable carbonate", "biohydrolyzable ureide" and "biohydrolyzable phosphate analogue" mean an amide, ester, carbamate, carbonate, ureide, or phosphate analogue, respectively, that either: 1) does not destroy the biological activity of the compound and confers upon that compound advantageous properties in vivo, such as uptake, duration of action, or onset of action; or 2) is itself biologically inactive but is converted in vivo to a biologically active compound. Examples of biohydrolyzable amides include, but are not limited to, lower alkyl amides, Of-amino acid amides, alkoxyacyl amides, and alkylaminoalkylcarbonyl amides. Examples of biohydrolyzable esters include, but are not limited to, lower alkyl esters, alkoxyacyloxy esters, alkyl acylamino alkyl esters, and choline esters. Examples of biohydrolyzable carbamates include, but are not limited to, lower alkylamines, substituted ethylenediamines, aminoacids, hydroxyalkylamines, heterocyclic and heteroaromatic amines, and polyether amines. As used herein, the term "pharmaceutically acceptable salt," is a salt formed from an acid and a basic group of a compound identified by the screening methods of the invention. Illustrative salts include, but are not limited, to sulfate, citrate, acetate, oxalate, chloride, bromide, iodide, nitrate, bisulfate, phosphate, acid phosphate, isonicotinate, lactate, salicylate, acid citrate, tartrate, oleate, tannate, pantothenate, bitartrate, ascorbate, succinate, maleate, gentisinate, fumarate, gluconate, glucaronate, saccharate, formate, benzoate, glutamate, methanesulfonate, ethanesulfonate, benzenesulfonate,/?-toluenesulfonate, and pamoate (i.e., l,r-methylene-bis-(2-hydroxy-3-naphthoate)) salts. The term "pharmaceutically acceptable salt" also refers to a salt prepared from a compound identified by the screening methods of the invention having an acidic functional group, such as a carboxylic acid functional group, and a pharmaceutically acceptable inorganic or organic base. Suitable bases include, but are not limited to, hydroxides of alkali metals such as sodium, potassium, and lithium; hydroxides of alkaline earth metal such as calcium and magnesium; hydroxides of other metals, such as aluminum and zinc; ammonia, and organic amines, such as unsubstituted or hydroxy-substituted mono-, di-, or trialkylamines; dicyclohexylamine; tributyl amine; pyridine; N-methyl,N-ethylamine; diethylamine; triethylamine; mono-, bis-, or rris-(2-hydroxy-lower alkyl amines), such as mono-, bis-, or tris-(2-hydroxyethyl)- amine, 2-hydroxy-tert-butylamine, or tris-(hydroxymethyl)methylamine, N, N,-di-lower alkyl-N-(hydroxy lower alkyl)-amines, such as N,N-dimethyl-N-(2-hydroxyethyl)- amine, or tri-(2-hydroxyethyl)amine; N-methyl-D-glucamine; and amino acids such as arginine, lysine, and the like. The term "pharmaceutically acceptable salt" also refers to a salt prepared from a compound identified by the screening methods of the invention having a basic functional group, such as an amino functional group, and a pharmaceutically acceptable inorganic or organic acid. Suitable acids include, but are not limited to, hydrogen sulfate, citric acid, acetic acid, oxalic acid, hydrochloric acid, hydrogen bromide, hydrogen iodide, nitric acid, phosphoric acid, isonicotinic acid, lactic acid, salicylic acid, tartaric acid, ascorbic acid, succinic acid, maleic acid, besylic acid, fumaric acid, gluconic acid, glucaronic acid, saccharic acid, formic acid, benzoic acid, glutamic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid,and/τ-toluenesulfonic acid.

As used herein, the term "pharmaceutically acceptable solvate," is a solvate formed from the association of one or more solvent molecules to a molecule identified by the screening methods of the invention. The term solvate includes hydrates (e.g., hemi-hydrate, mono-hydrate, dihydrate, trihydrate, tetrahydrate, and the like). As used herein, the term "clathrate" means a compound of the present invention or a salt thereof in the form of a crystal lattice that contains spaces (e.g., channels) that have a guest molecule (e.g., a solvent or water) trapped within.

Once a test compound has been identified as having an appropriate activity according to the screening methods of the present invention, the test compound can be subject to further testing, for example, in animal models to confirm its activity in the animal, or for potential side effects. The test compound can also be tested against known compounds that inhibit IL-12p40 production and/or increase the production of IL-IO (such as Compounds 1 through 14 disclosed herein), in either cell based or animal assays, to confirm its desired activity. The identified compound can also be tested to determine its toxicity, or side effects that could be associated with administration of such compound. Alternatively, a compound identified as described herein can be used in an animal model to determine the mechanism of action of such a compound.

The present invention also pertains to uses of compounds identified by the above- described screening assays for methods of treatment as described herein. Accordingly, it is within the scope of the present invention to use such compound in the design, formulation, synthesis, manufacture, and/or production of a drug or pharmaceutical composition for use in diagnosis, prognosis, or treatment, as described herein. For example, in one embodiment, the present invention includes a method of synthesizing or producing a drug or pharmaceutical composition by reference to the structure and/or properties of a compound obtainable by one of the above-described screening assays. For example, a drug or pharmaceutical composition can be synthesized based on the structure and/or properties of a compound obtained by the screening methods described supra.

Furthermore, the identified compound, prior to formulation for use in a method for treatment or prophylaxis can be modified using methods known in the art to render the compound more stable, i.e., increase its half-life in the subject, or render the compound more readily absorbed into the tissues of the subject. Such modifications include, but are not limited to, PEGylation, multimerization. Such modifications are performed by a pharmaceutical chemist to make the compound more suitable for administration. Additionally, the identified compound can be modified to allow for passage across the blood-brain barrier.

A compound which display the desired biological activity can be used as lead compound for the development or design of congeners or analogs having useful pharmacological activity. For example, once a lead compound is identified, molecular modeling techniques can be used to design variants of the compound that can be more effective. Examples of molecular modeling systems are the CHARM and QUANTA programs (Polygen Corporation, Waltham, MA). CHARM performs the energy minimization and molecular dynamics functions. QUANTA performs the construction, graphic modeling and analysis of molecular structure. QUANTA allows interactive construction, modification, visualization, and analysis of the behavior of compounds with each other. Exemplary compounds that can be used as lead compounds for the development or design of congeners or analogs having useful pharmacological activity are Compounds 1 through 14 disclosed herein, and those compounds described in the patents and patent applications listed in Table 1, each of which is incorporated by reference herein in its entirety.

A number of articles review computer modeling of drugs interactive with specific proteins, such as Rotivinen et ai, 1988, Acta Pharmaceutical Fennica 97:159-166; Ripka, 1998, New Scientist 54-57; McKinaly & Rossmann, 1989, Annu. Rev. Pharmacol. Toxiciol. 29:111-122; Perry & Davies, OSAR: Quantitative Structure-Activity Relationships in Drug Design pp.189-193 (Alan R. Liss, Inc. 1989); Lewis & Dean, 1989, Proc. R. Soc. Lond. 236:125-140 and 141-162; Askew et al, 1989, J. Am. Chem. Soc. I l l: 1082-1090. Other computer programs that screen and graphically depict chemicals are available from companies such as BioDesign, Inc. (Pasadena, California), Allelix, Inc. (Mississauga, Ontario, Canada), and Hypercube, Inc. (Cambridge, Ontario). Although these are primarily designed for application to drugs specific to particular proteins, they can be adapted to design of drugs specific to any identified region. Alternatively, lead compounds with little or no biologic activity, as ascertained in the screen, can also be used to design analogs and congeners of the compounds that have biologic activity.

PHARMACEUTICAL COMPOSITIONS AND THERAPEUTIC/PROPHYLACTIC ADMINISTRATION

The invention provides methods of treatment (and prophylaxis) by administration to a subject of an effective amount of a therapeutic of the invention, i.e., a compound identified by the screening methods of the present invention. In a preferred aspect, the therapeutic is substantially purified. The subject is preferably an animal including, but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human. In a specific embodiment, a non-human mammal is the subject.

In certain embodiments, the present invention provides a method of decreasing the level of IL-12p40 in a subject comprising administering to the subject a compound that increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after stimulation, preferably proinflammatory stimulation. In certain embodiments, the compound is not N-(3-methyl- benzylidene)-N'-[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimidin-4-yl]-hydrazine. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 2. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 1. In some embodiments, the compound decreases the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound increases the level of IL-IO in the subject.

In certain embodiments, the present invention provides a method of decreasing the level of IL-12p40 and increasing the level of IL-10 in a subject comprising administering to the subject a compound that decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after stimulation, preferably proinflammatory stimulation; and increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted. In certain embodiments, the compound is not N-(3-methyl-benzylidene)-N'-[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimidin-4-yl]- hydrazine. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 2. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 1. In some embodiments, the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound decreases the amount of Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted.

In certain embodiments, the invention relates to a method of treating an IL- 12, IL- 23, or IL-27 production-related diseases or disorders in a subject comprising administering to the subject an effective amount of a compound that increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after stimulation, preferably proinflammatory stimulation. In certain embodiments, the compound is not N-(3-meftyl-benzylidene)-N'-[6-morpholin- 4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimidin-4-yl]-hydrazine. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 2. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 1. In some embodiments, the compound decreases the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound increases the level of IL-IO in the subject. In some embodiments, the IL-12, IL-23, or IL-27 production-related diseases or disorders is selected from the group consisting of multiple sclerosis, sepsis, myasthenia gravis, autoimmune neuropathies, Guillain-Barrέ syndrome, autoimmune uveitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, temporal arteritis, anti-phospholipid syndrome, vasculitides, Wegener's granulomatosis, Behcet's disease, psoriasis, psoriatic arthritis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo, Crohn's disease, ulcerative colitis, interstitial pulmonary fibrosis, myelofibrosis, hepatic fibrosis, myocarditis, thyroditis, primary biliary cirrhosis, autoimmune hepatitis, immune-mediated diabetes mellitus, Grave's disease, Hashimoto's thyroiditis, autoimmune oophoritis and orchitis, autoimmune disease of the adrenal gland, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, common variable immunodeficiency (CVID), polymyositis, dermatomyositis, spondyloarthropathies, ankylosing spondylitis, Sjogren's syndrome and graft-versus-host disease. In some embodiments, the IL-12, IL-23, or IL-27 production-related diseases or disorders is selected from the group consisting of rheumatoid arthritis, sepsis, Crohn's disease, multiple sclerosis, psoriasis, psoriatic arthritis, or immune-mediated diabetes mellitus.

In certain embodiments, the invention relates to a method of treating an IL-12, IL- 23, or IL-27 production-related diseases or disorders in a subject comprising administering to the subject an effective amount of a compound that decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after stimulation, preferably proinflammatory stimulation; and increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted. In certain embodiments, the compound is not N-(3-methyl-benzylidene)-N'-[6-morpholin-4-yl-2-(2- pyridin-2-yl-ethoxy)-pyrimidin-4-yl]-hydrazine. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 2. In certain embodiments, the compound is not a compound disclosed in the patents or patent applications listed in Table 1. In some embodiments, the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted. In some embodiments, the compound decreases the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted. In some embodiments, the IL- 12, IL-23, or IL-27 production-related diseases or disorders is selected from the group consisting of multiple sclerosis, sepsis, myasthenia gravis, autoimmune neuropathies, Guillain-Barrέ syndrome, autoimmune uveitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, temporal arteritis, anti-phospholipid syndrome, vasculitides, Wegener's granulomatosis, Behcet's disease, psoriasis, psoriatic arthritis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo, Crohn's disease, ulcerative colitis, interstitial pulmonary fibrosis, myelofibrosis, hepatic fibrosis, myocarditis, thyroditis, primary biliary cirrhosis, autoimmune hepatitis, immune-mediated diabetes mellitus, Grave's disease, Hashimoto's thyroiditis, autoimmune oophoritis and orchitis, autoimmune disease of the adrenal gland, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, common variable immunodeficiency (CVID), polymyositis, dermatomyositis, spondyloarthropathies, ankylosing spondylitis, Sjogren's syndrome and graft- versus-host disease. In some embodiments, the IL- 12, IL-23, or IL-27 production-related diseases or disorders is selected from the group consisting of rheumatoid arthritis, sepsis, Crohn's disease, multiple sclerosis, psoriasis, psoriatic arthritis, or immune- mediated diabetes mellitus.

In certain embodiments, the present invention is directed to a pharmaceutical composition comprising a compound identified by the screening methods of the invention and a pharmaceutically acceptable carrier or excipient. In specific embodiments, the pharmaceutical composition further comprises a growth hormone or growth hormone secretogogue. In other specific embodiments, the pharmaceutical composition further comprises a TNF agonist.

The present invention is also directed to a method of treating or ameliorating an EL- 12, IL-23, or IL-27 production-related disease or disorder comprising administering a pharmaceutical composition comprising a compound identified by the screening methods of the invention and a pharmaceutically acceptable carrier or excipient to a subject in need thereof in an amount sufficient to treat or ameliorate said disease or disorder.

The compounds and compositions described herein are useful to treat and prevent any IL-12, IL-23, or IL-27 production-related disorders. IL-12, IL-23, or IL-27 production- related disorders include inflammatory disorders, immune diseases, neurological disorders and bone loss diseases.

The term "inflammatory disorders" includes any inflammatory disease, disorder or condition caused, exasperated or mediated by IL-12, IL-23 and/or IL-27 production. Such inflammatory disorders may include, without limitation, asthma, adult respiratory distress syndrome, systemic lupus erythematosus, inflammatory bowel disease (including Crohn's disease and ulcerative colitis), multiple sclerosis, insulin-dependent diabetes mellitus, autoimmune arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, psoriatic arthritis), inflammatory pulmonary syndrome, pemphigus vulgaris, idiopathic thrombocytopenic purpura, autoimmune meningitis, myasthenia gravis, autoimmune thyroiditis, dermatitis (including atopic dermatitis and eczematous dermatitis), psoriasis, Sjogren's Syndrome (including keratoconjunctivitis sicca secondary to Sjogren's Syndrome), alopecia areata, allergic responses due to arthropod bite reactions, aphthous ulcer, iritis, conjunctivitis, keratoconjunctivitis, cutaneous lupus erythematosus, scleroderma, vaginitis, proctitis, drug eruptions (such as Stevens- Johnson syndrome), leprosy reversal reactions, erythema nodosum leprosum, autoimmune uveitis, allergic encephalomyelitis, aplastic anemia, pure red cell anemia, idiopathic thrombocytopenia, polychondritis, CVED, Wegener's granulomatosis, chronic active hepatitis, Graves ophthalmopathy, primary biliary cirrhosis, uveitis posterior and interstitial lung fibrosis.

."Inflammatory disorders" expressly include acute inflammatory disorders. Examples of acute inflammatory disorders include graft versus host disease, transplant rejection, septic shock, endotoxemia, Lyme arthritis, infectious meningitis {e.g., viral, bacterial, Lyme disease-associated), an acute episode of asthma and acute episodes of an autoimmune disease.

"Inflammatory disorders" expressly include chronic inflammatory disorders. Nonlimiting examples of chronic inflammatory disorder include asthma, rubella arthritis, and chronic autoimmune diseases, such as systemic lupus erythematosus, psoriasis, inflammatory bowel disease, including Crohn's disease and ulcerative colitis, multiple sclerosis and rheumatoid arthritis.

The term "immune diseases" includes any immune disease, disorder or condition caused, exasperated or mediated by EL- 12, IL-23 and/or IL-27 production. Such immune diseases may include, without limitation, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, systemic lupus erythematosis, antiphospholipid syndrome, iridocyclitis/uveitis/optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis/Wegener's granulomatosis, sarcoidosis, orchitis/vasectomy reversal procedures, allergic/atopic diseases, asthma, allergic rhinitis, eczema, allergic contact dermatitis, allergic conjunctivitis, hypersensitivity pneumonitis, transplants, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, sepsis syndrome, gram positive sepsis, gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia, trauma/hemorrhage, bums, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatory pathologies, sarcoidosis, Crohn's pathology, sickle cell anemia, diabetes, nephrosis, atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis, pernicious anemia, hemolytic disesease, thrombocytopenia, graft rejection of any organ or tissue, kidney translplant rejection, heart transplant rejection, liver transplant rejection, pancreas transplant rejection, lung transplant rejection, bone marrow transplant (BMT) rejection, skin allograft rejection, cartilage transplant rejection, bone graft rejection, small bowel transplant rejection, fetal thymus implant rejection, parathyroid transplant rejection, xenograft rejection of any organ or tissue, allograft rejection, anti-receptor hypersensitivity reactions, Graves disease, Raynoud's disease, type B insulin-resistant diabetes, asthma, myasthenia gravis, antibody- meditated cytotoxicity, type in hypersensitivity reactions, systemic lupus erythematosus, POEMS syndrome (polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes syndrome), polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, skin changes syndrome, antiphospholipid syndrome, pemphigus, scleroderma, mixed connective tissue disease, idiopathic Addison's disease, diabetes mellitus, chronic active hepatitis, primary billiary cirrhosis, vitiligo, vasculitis, post-MI cardiotomy syndrome, type IV hypersensitivity, contact dermatitis, hypersensitivity pneumonitis, allograft rejection, granulomas due to intracellular organisms, drug sensitivity, metabolic/idiopathic, Wilson's disease, hemachromatosis, alpha-1- antitrypsin deficiency, diabetic retinopathy, hashimoto's thyroiditis, osteoporosis, hypothalamic -pituitary-adrenal axis evaluation, primary biliary cirrhosis, thyroiditis, encephalomyelitis, cachexia, cystic fibrosis, neonatal chronic lung disease, chronic obstructive pulmonary disease (COPD), familial hematophagocytic lymphohistiocytosis, dermatologic conditions, psoriasis, alopecia, nephrotic syndrome, nephritis, glomerular nephritis, acute renal failure, hemodialysis, uremia, toxicity, preeclampsia, okt3 therapy, anti-cd3 therapy, cytokine therapy, chemotherapy, radiation therapy (e.g., including but not limited toasthenia, anemia, cachexia, and the like), chronic salicylate intoxication, and the like. See, e.g., the Merck Manual, 12th-17th Editions, Merck & Company, Rahway, NJ. (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al, eds., Second Edition, Appleton and Lange, Stamford, Conn. (1998, 2000), each entirely incorporated by reference.

The term "neurological disorder" includes any neurological disease, disorder or condition caused, exasperated or mediated by IL- 12, IL-23 and/or IL-27 production. Such neurological disorders may include, without limitation, neurodegenerative diseases, multiple sclerosis, migraine headache, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders' such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; Progressive supranucleo Palsy; structural lesions of the cerebellum; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager, and Machado- Joseph); systemic disorders (Refsum's disease, abetalipoprotemia, ataxia telangiectasia, and mitochondrial multi-system disorder); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; and disorders of the motor unit' such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt- Jakob disease; Subacute sclerosing panencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica, and the like. Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. See, e.g., the Merck Manual, 16, Edition, Merck & Company, Rahway, N.J. (1992).

The term "bone loss disease" includes any bone loss disease, disorder or condition caused, exasperated or mediated by IL- 12, IL-23 and/or IL-27 production e.g., periodontal disease, non-malignant bone disorders (e.g., osteoporosis, Paget's disease of bone, osteogenesis imperfecta, fibrous dysplasia, and primary hyperparathyroidism), estrogen deficiency, inflammatory bone loss, bone malignancy, arthritis, osteopetrosis, and certain cancer-related disorders (e.g., hypercalcemia of malignancy (HCM), osteolytic bone lesions of multiple myeloma and osteolytic bone metastases of breast cancer and other metastatic cancers.

In the case of overlap in these definitions, the disease, condition or disorder may be considered to be a member of any of the above listed classes of IL-12, IL-23, or IL-27 production-related disorders. Specific DL- 12, IL-23, or IL-27 production related diseases include rheumatoid arthritis, sepsis, Crohn's disease, multiple sclerosis, psoriasis, or insulin-dependent diabetes mellitus.

An "effective amount" is the quantity of a compound in which a beneficial clinical outcome is achieved when the compound is administered to a subject with an IL-12, IL-23, or IL-27 overproduction disorder. A "beneficial clinical outcome" includes a reduction in the severity of the symptoms associated with the IL-12, IL-23, or IL-27 overproduction disorder and/or an increase in the longevity of the subject compared with the absence of the treatment. The precise amount of the compound administered to a subject will depend on the type and severity of the disease or condition and on the characteristics of the subject, such as general health, age, sex, body weight and tolerance to drugs. It will also depend on the degree, severity and type of the EL- 12, IL-23, or IL-27 overproduction disorder. The skilled artisan will be able to determine appropriate dosages depending on these and other factors. Effective amounts of the disclosed compounds typically range between about 1 mg/mm² per day and about 10 grams/mm² per day, and preferably between 10 mg/mm² per day and about 5 grams/mm². When co-administered with another agent used to treat the IL- 12, IL-23, or IL-27 overproduction disorder, an "effective amount" of the second agent for treating the IL-12, IL-23, or IL-27 overproduction disorder will depend on the type of drug used. Suitable dosages are known for approved agents for treating IL- 12, IL-23, or IL-27 overproduction disorder and can be adjusted by the skilled artisan according to the condition of the subject, the type of IL-12, IL-23, or IL-27 overproduction disorder being treated and the amount of the compound of the invention being used.

The foregoing and other useful combination therapies will be understood and appreciated by those of skill in the art. Potential advantages of such combination therapies include the ability to use less of each of the individual active ingredients to minimize toxic side effects, synergistic improvements in efficacy, improved ease of administration or use and/or reduced overall expense of compound preparation or formulation.

Formulations and methods of administration that can be employed when the therapeutic comprises a compound identified by the assays described, supra; additional appropriate formulations and routes of administration can be selected from among those described herein below. Moreover, a therapeutic of the invention can be administered in conjunction with any known drug to treat the diseases or disorders of the invention.

Various delivery systems are known and can be used to administer a therapeutic of the invention, e.g., encapsulation in liposomes, microparticles, and microcapsules, use of cells capable of expressing the therapeutic, use of receptor-mediated endocytosis (e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429-4432); construction of a therapeutic nucleic acid as part of a retroviral or other vector, etc. Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes. The compounds may be administered by any convenient route, for example by infusion, by bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral, rectal and intestinal mucosa, etc.), and may be administered together with other biologically active agents. Administration can be systemic or local. In addition, it may be desirable to introduce the pharmaceutical compositions of the invention into the central nervous system by any suitable route, including intraventricular and intrathecal injection; intraventricular injection may be facilitated by an intraventricular catheter, for example, attached to a reservoir, such as an Ommaya reservoir. Pulmonary administration can also be employed, eg., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.

In a preferred embodiment, the therapeutic is formulated for oral administration. These dosage forms include tablets (coated or uncoated), caplets, hard gelatin capsules, soft gelatin capsules, troches, dragees, dispersions, suspensions, solutions, and the like, including sustained release formulations well known in the art. See, e.g., Introduction to Pharmaceutical Dosage Forms, 1985, Ansel, H.C., Lea and Febiger, Philadelphia, PA; Remington's Pharmaceutical Sciences, 1995, Mack Publ. Co., Easton, PA. Because of their ease of administration, tablets and capsules are preferred and represent the most advantageous oral dosage unit form, in which case solid pharmaceutical excipients are employed. If desired, tablets or caplets or capsules may be coated by standard aqueous or non-aqueous techniques.

In a specific embodiment, it may be desirable to administer the pharmaceutical compositions of the invention locally to the area in need of treatment. This may be achieved by, for example, and not by way of limitation, local infusion during surgery, topical application, e.g., in conjunction with a wound dressing after surgery, by injection, by means of a catheter, by means of a suppository, or by means of an implant, said implant being of a porous, non-porous, or gelatinous material, including membranes, such as sialastic membranes, or fibers.

In another embodiment, the therapeutic can be delivered in a vesicle, in particular a liposome (Langer, 1990, Science 249:1527-1533; Treat et al, 1989, In: Liposomes in the Therapy of Infectious Disease and Cancer, Lopez-Berestein and Fidler, eds., Liss, New York, pp. 353-365; Lopez-Berestein, ibid., pp. 317-327; see generally ibid.)

In yet another embodiment, the therapeutic can be delivered via a controlled release system. In one embodiment, a pump may be used (Langer, supra; Sefton, 1987, CRC Crit. Ref. Biomed. Eng. 14:201-240; Buchwald et al., 1980, Surgery 88:507-516; Saudek et al, 1989, N. Engl. J. Med. 321:574-579). In another embodiment, polymeric materials can be used (Medical Applications of Controlled Release, Langer and Wise, eds., CRC Press, Boca Raton, Florida, 1974; Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball, eds., Wiley, New York, 1984; Ranger and Peppas, 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al, 1985, Science 228:190-192; During et al, 1989, Ann. Neurol. 25:351-356; Howard et al, 1989, J. Neurosurg. 71:858- 863). In yet another embodiment, a controlled release system can be placed in proximity of the therapeutic target, i.e., the brain, thus requiring only a fraction of the systemic dose (e.g., Goodson, 1984, In: Medical Applications of Controlled Release, supra, Vol. 2, pp. 115-138). Other controlled release systems are discussed in the review by Langer (1990, Science 249:1527-1533). The present invention also provides pharmaceutical compositions. Such compositions comprise a therapeutically effective amount of a therapeutic, and a pharmaceutically acceptable carrier. In a specific embodiment, the term "pharmaceutically acceptable" means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly, in humans. The term "carrier" refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered. Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, including but not limited to peanut oil, soybean oil, mineral oil, sesame oil and the like. Water can be a preferred carrier when the pharmaceutical composition is administered orally. Saline and aqueous dextrose are preferred carriers when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions are preferably employed as liquid carriers for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol and the like. The composition, if desired, can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents. These compositions can take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, sustained-release formulations and the like. The composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides. Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin. Such compositions will contain a therapeutically effective amount of the therapeutic, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

In a preferred embodiment, the composition is formulated, in accordance with routine procedures, as a pharmaceutical composition adapted for intravenous administration to human beings. Typically, compositions for intravenous administration are solutions in sterile isotonic aqueous buffer. Where necessary, the composition may also include a solubilizing agent and a local anesthetic such as lidocaine to ease pain at the site of the injection. Generally, the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachette indicating the quantity of active agent. Where the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline. Where the composition is administered by injection, an ampoule of sterile water or saline for injection can be provided so that the ingredients may be mixed prior to administration.

The therapeutics of the invention can be formulated as neutral or salt forms.

Preferred pharmaceutical compositions and dosage forms comprise a therapeutic of the invention, or a pharmaceutically acceptable prodrug, salt, solvate, or clathrate thereof, optionally in combination with one or more additional active agents.

The amount of the therapeutic of the invention which will be effective in the treatment of a particular disorder or condition will depend on the nature of the disorder or condition, and can be determined by standard clinical techniques. In addition, in vitro assays may optionally be employed to help identify optimal dosage ranges. The precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. However, suitable dosage ranges for intravenous administration are generally about 1-50 milligrams of active compound per kilogram body weight. Suitable dosage ranges for intranasal administration are generally about 0.1 mg/kg body weight to 50 mg/kg body weight. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems.

Suppositories generally contain active ingredient in the range of 0.5% to 10% by weight; oral formulations preferably contain 10% to 95% active ingredient.

Exemplary doses of a small molecule include milligram or microgram amounts of the small molecule per kilogram of subject or sample weight {e.g., about 1 microgram per kilogram to about 500 milligrams per kilogram, about 100 micrograms per kilogram to about 5 milligrams per kilogram, or about 1 microgram per kilogram to about 50 micrograms per kilogram).

For antibodies, proteins, polypeptides, peptides and fusion proteins encompassed by the invention, the dosage administered to a patient is typically 0.0001 mg/kg to 100 mg/kg of the patient's body weight. Preferably, the dosage administered to a patient is between 0.0001 mg/kg and 20 mg/kg, 0.0001 mg/kg and 10 mg/kg, 0.0001 mg/kg and 5 mg/kg, 0.0001 and 2 mg/kg, 0.0001 and 1 mg/kg, 0.0001 mg/kg and 0.75 mg/kg, 0.0001 mg/kg and 0.5 mg/kg, 0.0001 mg/kg to 0.25 mg/kg, 0.0001 to 0.15 mg/kg, 0.0001 to 0.10 mg/kg, 0.001 to 0.5 mg/kg, 0.01 to 0.25 mg/kg or 0.01 to 0.10 mg/kg of the patient's body weight. Generally, human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible. Further, the dosage and frequency of administration of antibodies of the invention or fragments thereof may be reduced by enhancing uptake and tissue penetration of the antibodies by modifications such as, for example, lipidation.

Moreover, in certain embodiments, since IL-12, IL-23, and/or IL-27 production can be inhibited at a lower drug concentration than that needed to inhibit IL-6 or EFN-γ production, appropriate dosages include those mat selectively inhibit IL-12, IL-23, or IL-27 production but not other cytokines.

The therapeutics of the present invention may also be administered by controlled release means or delivery devices that are well known to those of ordinary skill in the art, such as those described in U.S. Patent Nos. 3,845,770; 3,916,899; 3,536,809; 3,598,123; and 4,008,719, 5,674,533, 5,059,595, 5,591,767, 5,120,548, 5,073,543, 5,639,476, 5,354,556, and 5,733,566. These controlled release compositions can be used to provide slow or controlled-release of one or more of the active ingredients therein using, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination thereof to provide the desired release profile in varying proportions. Suitable controlled-release formulations known to those of ordinary skill in the art may be readily selected for use with the pharmaceutical compositions of the invention.

All controlled-release pharmaceutical products have a common goal of improving drug therapy over that achieved by their non-controlled counterparts. Ideally, the use of an optimally designed controlled-release preparation in medical treatment is characterized by a minimum of drug substance being employed to cure or control the condition in a minimum amount of time. Advantages of controlled-release formulations may include extended activity of the drug, reduced dosage frequency, and/or increased patient compliance.

Most controlled-release formulations are designed to initially release an amount of the therapeutic that promptly produces the desired therapeutic effect, and gradually and continually releases other amounts of the therapeutic to maintain the appropriate level of therapeutic effect over an extended period of time. In order to maintain this constant level of therapeutic in the body, the therapeutic must be released from the composition at a rate that will replace the amount of therapeutic being metabolized and excreted from the body. The controlled-release of the therapeutic may be stimulated by various inducers, for example, pH, temperature, enzymes, water, or other physiological conditions or compounds. Such controlled-release components in the context of the present invention include, but are not limited to, polymers, polymer matrices, gels, permeable membranes, liposomes, microspheres, or the like, or a combination thereof, that facilitates the controlled-release of the active ingredient.

The invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention. Optionally associated with such containers) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.

The methods for treating or preventing an IL- 12, IL-23, and/or IL-27 production related disease or disorders in a patient in need thereof can further comprise administering to the patient being administered a compound of this invention, an effective amount of one or more other therapeutic agents. Such therapeutic agents may include other therapeutic agents such as those conventionally used to prevent or treat disorders associated with IL- 12, IL-23, and/or IL-27 production or symptoms thereof. The other therapeutic agent can be a steroid or a non-steroidal anti-inflammatory agent. Useful non-steroidal anti-inflammatory agents, include, but are not limited to, aspirin, ibuprofen, diclofenac, naproxen, benoxaprofen, flurbiprofen, fenoprofen, flubufen, ketoprofen, indoprofen, piroprofen, carprofen, oxaprozin, pramoprofen, muroprofen, trioxaprofen, suprofen, aminoprofen, tiaprofenic acid, fluprofen, bucloxic acid, indomethacin, sulindac, tolmetin, zomepirac, tiopinac, zidometacin, acemetacin, fentiazac, clidanac, oxpinac, mefenamic acid, meclofenamic acid, flufenamic acid, niflumic acid, tolfenamic acid, diflurisal, flufenisal, piroxicam, sudoxicam, isoxicam; salicylic acid derivatives, including aspirin, sodium salicylate, choline magnesium trisalicylate, salsalate, diflunisal, salicylsalicylic acid, sulfasalazine, and olsalazin; para-aminophennol derivatives including acetaminophen and phenacetin; indole and indene acetic acids, including indomethacin, sulindac, and etodolac; heteroaryl acetic acids, including tolmetin, diclofenac, and ketorolac; anthranilic acids (fenamates), including mefenamic acid, and meclofenamic acid; enolic acids, including oxicams (piroxicam, tenoxicam), and pyrazolidinediones (phenylbutazone, oxyphenthartazone); and alkanones, including nabumetone and pharmaceutically acceptable salts thereof and mixtures thereof. For a more detailed description of the NSAIDs, see Paul A. Insel, Analgesic-Antipyretic and Antiinflammatory Agents and Drugs Employed in the Treatment of Gout, in Goodman & Gilman's The Pharmacological Basis of Therapeutics 617-57 (Perry B. Molinhoff and Raymond W. Ruddon eds., 9^th ed 1996) and Glen R. Hanson, Analgesic, Antipyretic and Anti-Inflammatory Drugs in Remington: The Science and Practice of Pharmacy VoIU \ 196-1221 (A.R. Gennaro ed. 19th ed. 1995) which are hereby incorporated by reference in their entireties.

Other examples of prophylactic and therapeutic agents include, but are not limited to, immunomodulatory agents, anti-inflammatory agents (e.g., adrenocorticoids, corticosteroids (e.g., beclomethasone, budesonide, flunisolide, fluticasone, triamcinolone, methlyprednisolone, prednisolone, prednisone, hydrocortisone), glucocorticoids, steroids, non-steriodal anti-inflammatory drugs {e.g., aspirin, ibuprofen, diclofenac, and COX-2 inhibitors), and leukotreine antagonists {e.g., montelukast, methyl xanthines, zafirlukast, and zileuton), beta2 -agonists (e.g., albuterol, biterol, fenoterol, isoetharie, metaproterenol, pirbuterol, salbutamol, terbutalin formoterol, salmeterol, and salbutamol terbutaline), anticholinergic agents (e.g., ipratropium bromide and oxitropium bromide), sulphasalazine, penicillamine, dapsone, antihistamines, anti-malarial agents (e.g., hydroxychloroquine), anti- viral agents, and antibiotics {e.g., dactinomycin (formerly actinomycin), bleomycin, erythomycin, penicillin, mithramycin, and anthramycin (AMC)).

In combination therapy treatment, both the compounds of this invention and the other drug agent(s) are administered to mammals {e.g., humans, male or female) by conventional methods. The agents may be administered in a single dosage form or in separate dosage forms. Effective amounts of the other therapeutic agents are well known to those skilled in the art. However, it is well within the skilled artisan's purview to determine the other therapeutic agent's optimal effective-amount range. In one embodiment of the invention where another therapeutic agent is administered to an animal, the effective amount of the compound of this invention is less than its effective amount would be where the other therapeutic agent is not administered. In another embodiment, the effective amount of the conventional agent is less than its effective amount would be where the compound of this invention is not administered. In this way, undesired side effects associated with high doses of either agent may be minimized. Other potential advantages (including without limitation improved dosing regimens and/or reduced drug cost) will be apparent to those of skill in the art.

In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, at about 1 hour apart, at about 1 to about 2 hours apart, at about 2 hours to about 3 hours apart, at about 3 hours to about 4 hours apart, at about 4 hours to about 5 hours apart, at about 5 hours to about 6 hours apart, at about 6 hours to about 7 hours apart, at about 7 hours to about 8 hours apart, at about 8 hours to about 9 hours apart, at about 9 hours to about 10 hours apart, at about 10 hours to about 11 hours apart, at about 11 hours to about 12 hours apart, at about 12 hours to 18 hours apart, 18 hours to 24 hours apart, 24 hours to 36 hours apart, 36 hours to 48 hours apart, 48 hours to 52 hours apart, 52 hours to 60 hours apart, 60 hours to 72 hours apart, 72 hours to 84 hours apart, 84 hours to 96 hours apart, or 96 hours to 120 hours part. In preferred embodiments, two or more therapies are administered within the same patent visit.

In certain embodiments, one or more compounds of the invention and one or more other therapies (e.g., prophylactic or therapeutic agents) are cyclically administered. Cycling therapy involves the administration of a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by the administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally, followed by the administration of a third therapy (e.g., prophylactic or therapeutic agent) for a period of time and so forth, and repeating this sequential administration, i.e., the cycle in order to reduce the development of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and/or to improve the efficacy of the therapies.

In certain embodiments, the administration of the same compounds of the invention may be repeated and the administrations may be separated by at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months. In other embodiments, the administration of the same therapy (e.g., prophylactic or therapeutic agent) other than a compound of the invention may be repeated and the administration may be separated by at least at least 1 day, 2 days, 3 days, 5 days, 10 days, 15 days, 30 days, 45 days, 2 months, 75 days, 3 months, or at least 6 months.

Any immunomodulatory agent well-known to one of skill in the art may be used in the co-administration methods and compositions of the invention. Immunomodulatory agents can affect one or more or all aspects of the immune response in a subject. Aspects of the immune response include, but are not limited to, the inflammatory response, the complement cascade, leukocyte and lymphocyte differentiation, proliferation, and/or effector function, monocyte and/or basophil counts, and the cellular communication among cells of the immune system. In certain embodiments of the invention, an immunomodulatory agent modulates one aspect of the immune response. In other embodiments, an immunomodulatory agent modulates more than one aspect of the immune response. In a preferred embodiment of the invention, the administration of an immunomodulatory agent to a subject inhibits or reduces one or more aspects of the subject's immune response capabilities. In a specific embodiment of the invention, the immunomodulatory agent inhibits or suppresses the immune response in a subject.

Examples of immunomodulatory agents include, but are not limited to, proteinaceous agents such as cytokines, peptide mimetics, and antibodies (e.g., human, humanized, chimeric, monoclonal, polyclonal, Fvs, ScF vs, Fab or F(ab)2 fragments or epitope binding fragments), nucleic acid molecules (e.g., antisense nucleic acid molecules and triple helices), small molecules, organic compounds, and inorganic compounds. In particular, immunomodulatory agents include, but are not limited to, methotrexate, leflunomide, cyclophosphamide, Cytoxan, Immuran, cyclosporine A, minocycline, azathioprine, antibiotics (e.g., FK506 (tacrolimus)), methylprednisolone (MP), corticosteroids, steroids, mycophenolate mofetil, rapamycin (sirolimus), mizoribine, deoxyspergualin, brequinar, malononitriloamindes (e.g., leflunamide), T cell receptor modulators, cytokine receptor modulators, and modulators mast cell modulators.

Examples of T cell receptor modulators include, but are not limited to, anti-T cell receptor antibodies (e.g., anti-CD4 antibodies (e.g., cM-T412 (Boeringer), IDEC-CE9.1® (IDEC and SKB), mAB 4162W94, Orthoclone and OKTcdr4a (Janssen-Cilag)), anti-CD3 antibodies (e.g., Nuvion (Product Design Labs), OKT3 (Johnson & Johnson), or Rituxan (IDEC)), anti-CD5 antibodies (e.g., an anti-CD5 ricin-linked immunoconjugate), anti-CD7 antibodies (e.g., CHH-380 (Novartis)), anti-CD8 antibodies, anti-CD40 ligand monoclonal antibodies (e.g., IDEC-131 (IDEC)), anti-CD52 antibodies (e.g., CAMPATH IH (Ilex)), anti-CD2 antibodies (e.g., MEDI-507 (Medlmmune, Inc., International Publication Nos. WO 02/098370 and WO 02/069904), anti-CDl Ia antibodies (e.g., Xanelim (Genentech)), and anti-B7 antibodies (e.g., IDEC-114) (IDEC))), CTLA4-immunoglobulin, and LFA- 3TIP (Biogen, International Publication No. WO 93/08656 and U.S. Patent No. 6,162,432). Examples of cytokine receptor modulators include, but are not limited to, soluble cytokine receptors (e.g., the extracellular domain of a TNF-α receptor or a fragment thereof, the extracellular domain of an EL- Iβ receptor or a fragment thereof, and the extracellular domain of an IL-6 receptor or a fragment thereof), cytokines or fragments thereof (e.g., interleukin IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, EL-9, IL-10, IL-11, IL-12, IL-13, IL-15, IL-23, IL-27, TNF-α, TNF-0, interferon (IFN)-O, IFN-/3, IFN-γ, and GM-CSF), anti- cytokine receptor antibodies {e.g., anti-IFN receptor antibodies, anti-IL-2 receptor antibodies (e.g., Zenapax (Protein Design Labs)), anti-IL-3 receptor antibodies, anti-BL-4 receptor antibodies, anti-IL-6 receptor antibodies, anti-IL-10 receptor antibodies, anti-IL-12 receptor antibodies, anti-IL-13 receptor antibodies, anti-IL-15 receptor antibodies, anti-IL- 23 receptor antibodies, and anti-IL-27 receptor antibodies), anti-cytokine antibodies.

In a specific embodiment, a cytokine receptor modulator is IL-3, IL-4, IL-IO, or a fragment thereof. In another embodiment, a cytokine receptor modulator is the extracellular domain of a TNF-α receptor or a fragment thereof. In certain embodiments, a cytokine receptor modulator is not a TNF-α antagonist.

In one embodiment, a cytokine receptor modulator is a mast cell modulator. In an alternative embodiment, a cytokine receptor modulator is not a mast cell modulator. Examples of mast cell modulators include, but are not limited to stem cell factor (c-kit receptor ligand) inhibitor (e.g., mAb 7H6, mAb 8H7a, pAb 1337, FK506, CsA, dexamthasone, and fluconcinonide), c-kit receptor inhibitor (e.g., STI 571 (formerly known as CGP 57148B)), mast cell protease inhibitor (e.g., GW-45, GW-58, wortmannin, LY 294002, calphostin C, cytochalasin D, genistein, KT5926, staurosproine, and lactoferrin), relaxin ("RLX"), IgE antagonist (e.g., antibodies rhuMAb-E25 omalizumab, HMK-12 and 6HD5, and mAB Hu-901), IL-3 antagonist, IL-4 antagonists, IL-10 antagonists, and TGF- beta.

An immunomodulatory agent may be selected to interfere with the interactions between the T helper subsets (THl or TH2) and B cells to inhibit neutralizing antibody formation. Antibodies that interfere with or block the interactions necessary for the activation of B cells by TH (T helper) cells, and thus block the production of neutralizing antibodies, are useful as immunomodulatory agents in the methods of the invention. For example, B cell activation by T cells requires certain interactions to occur (Durie et ai, Immunol. Today, 15(9):406-410 (1994)), such as the binding of CD40 ligand on the T helper cell to the CD40 antigen on the B cell, and the binding of the CD28 and/or CTLA4 ligands on the T cell to the B7 antigen on the B cell. Without both interactions, the B cell cannot be activated to induce production of the neutralizing antibody.

The CD40 ligand (CD40L)-CD40 interaction is a desirable point to block the immune response because of its broad activity in both T helper cell activation and function as well as the absence of redundancy in its signaling pathway. Thus, in a specific embodiment of the invention, the interaction of CD40L with CD40 is transiently blocked at the time of administration of one or more of the compounds of the invention and immunomodulatory agents. This can be accomplished by treating with an agent which blocks the CD40 ligand on the TH cell and interferes with the normal binding of CD40 ligand on the T helper cell with the CD40 antigen on the B cell. An antibody to CD40 ligand (anti-CD40L) (available from Bristol-Myers Squibb Co; see, e.g., European patent application 555,880, published Aug. 18, 1993) or a soluble CD40 molecule can be selected and used as an immunomodulatory agent in accordance with the methods of the invention.

An immunomodulatory agent may be selected to inhibit the interaction between THl cells and cytotoxic T lymphocytes ("CTLs") to reduce the occurrence of CTL-mediated killing. An immunomodulatory agent may be selected to alter (eg., inhibit or suppress) the proliferation, differentiation, activity and/or function of the CD4+ and/or CD8+ T cells. For example, antibodies specific for T cells can be used as immunomodulatory agents to deplete, or alter the proliferation, differentiation, activity and/or function of CD4+ and/or CD8+ T cells.

In one embodiment of the invention, an immunomodulatory agent that reduces or depletes T cells, preferably memory T cells, is administered to a subject at risk of or with an autoimmune disease, an inflammatory disease or an infection (preferably, a respiratory infection) prior to, subsequent to, or concomitantly with a compound of the invention. See, e.g., U.S. Pat. No.4,658,019. In another embodiment of the invention, an immunomodulatory agent that inactivates CD8+ T cells is administered to a subject at risk of or with an autoimmune disease, an inflammatory disease, a proliferative disease, or an infection (preferably, a respiratory infection) in combination with a compound of the invention. In a specific embodiment, anti-CD8 antibodies are used to reduce or deplete CD8+ T cells.

In another embodiment, an immunomodulatory agent which reduces or inhibits one or more biological activities (e.g., the differentiation, proliferation, and/or effector functions) of THO, THl, and/or TH2 subsets of CD4+ T helper cells is administered to a subject at risk of or with an autoimmune disease, an inflammatory disease or an infection (preferably, a respiratory infection) prior to, subsequent to, or concomitantly with a compound of the invention. One example of such an immunomodulatory agent is IL-4. IL- 4 enhances antigen-specific activity of TH2 cells at the expense of the THl cell function (see, e.g., Yokota et al, 1986 Proc. Natl. Acad. Sci., USA, 83:5894-5898; and U.S. Pat. No. 5,017,691 ). Other examples of immunomodulatory agents that affect the biological activity (e.g., proliferation, differentiation, and/or effector functions) of T-helper cells (in particular, THl and/or TH2 cells) include, but are not limited to, IL-2, IL-4, IL-5, IL-6, EL-IO, IL-13, IL-15, and interferon (DFN)-Y

In another embodiment, an immunomodulatory agent administered to a subject at risk of or with an autoimmune disease, an inflammatory disease or an infection (preferably, a respiratory infection) prior to, subsequent to, or concomitantly with a compound of the invention is a cytokine that prevents antigen presentation. In a specific embodiment, an immunomodulatory agent used in the methods of the invention is EL-IO. IL-10 also reduces or inhibits macrophage action which involves bacterial elimination.

An immunomodulatory agent may be selected to reduce or inhibit the activation, degranulation, proliferation, and/or infiltration of mast cells. In certain embodiments, the immunomodulatory agent interferes with the interactions between mast cells and mast cell activating agents, including, but not limited to stem cell factors (c-kit ligands), IgE, IL-4, environmental irritants, and infectious agents. In a specific embodiment, the immunomodulatory agent reduces or inhibits the response of mast cells to environmental irritants such as, but not limited to pollen, dust mites, tobacco smoke, and/or pet dander. In another specific embodiment, the immunomodulatory agent reduces or inhibits the response of mast cells to infectious agents, such as viruses, bacteria, and fungi. Examples of mast cell modulators that reduce or inhibit the activation, degranulation, proliferation, and/or infiltration of mast cells include, but are not limited to, stem cell factor (c-kit receptor ligand) inhibitors (e.g., mAb 7H6, mAb 8H7a, and pAb 1337 (see Mendiaz et al, 1996, Eur J Biochem 293(3):842-849), FK506 and CsA (Ito et al, 1999 Arch Dermatol Res 291(5):275-283), dexamthasone and fluconcinonide (see Finooto et al. J Clin Invest 1997 99(7): 1721-1728)), c-kit receptor inhibitors {e.g., STI 571 (formerly known as CGP 57148B) (see Heinrich et al, 2000 Blood 96(3):925-932)), mast cell protease inhibitors (e.g., GW-45 and GW-58 (see Temkin et al, 2002 J Immunol 169(5):2662-2669), wortmannin, LY 294002, calphostin C, and cytochalasin D (see Vosseller et al, 1997, MoI Biol Cell 1997:909-922), genistein, KT5926, and staurosproine (see Nagai et al 1995, Biochem Biophys Res Commun 208(2):576-581), and lactoferrin (see He et al, 2003 Biochem Pharmacol 65(6): 1007- 1015)), relaxin ("RLX") (see Bani et al, 2002 Int Immunopharmacol 2(8):1195-1294), ), IgE antagonists (e.g., antibodies rhuMAb-E25 omalizumab (see Finn et al, 2003 J Allergy Clin Immuno 111(2):278-284; Corren et al, 2003 J Allergy Clin Immuno 11 l(l):87-90; Busse and Neaville, 2001 Curr Opin Allergy CHn Immuno 1(1):105-108; and Tang and Powell, 2001, Eur J Pediatr 160(12): 696-704), HMK-12 and 6HD5 (see Miyajima et al, 2202 Int Arch Allergy Immuno 128(l):24-32), and mAB Hu-901 (see van Neerven et al, 2001 Int Arch Allergy Immuno 124(1 -3) :400), IL-3 antagonist, IL-4 antagonists, IL-10 antagonists, and TGF-beta (see Metcalfe et al, 1995, Exp Dermatol 4(4 Pt 2):227-230).

In a preferred embodiment, proteins, polypeptides or peptides (including antibodies) that are utilized as immunomodulatory agents are derived from the same species as the recipient of the proteins, polypeptides or peptides so as to reduce the likelihood of an immune response to those proteins, polypeptides or peptides. In another preferred embodiment, when the subject is a human, the proteins, polypeptides, or peptides that are utilized as immunomodulatory agents are human or humanized.

In accordance with one embodiment of the invention, one or more immunomodulatory agents are administered to a subject at risk of or with an autoimmune disease, an inflammatory disease or an infection (preferably, a respiratory infection) prior to, subsequent to, or concomitantly with a compound of the invention. Preferably, one or more immunomodulatory agents are administered in combination with a compound of the invention to a subject at risk of or with an autoimmune disease, an inflammatory disease or an infection (preferably, a respiratory infection) to reduce or inhibit one or more aspects of the immune response as deemed necessary by one of skill in the art. Any technique well- known to one skilled in the art can be used to measure one or more aspects of the immune response in a particular subject, and thereby determine when it is necessary to administer an immunomodulatory agent to said subject. In a preferred embodiment, a mean absolute lymphocyte count of approximately 500 cells/mm3, preferably 600 cells/mm3, 650 cells/mm3, 700 cells/mm3, 750 cells/mm3, 800 cells/mm3, 900 cells/mm3, 1000 cells/mm3, 1100 cells/mm3, or 1200 cells/mm3 is maintained in a subject. In another preferred embodiment, the subject is not administered a compound of the invention if their absolute lymphocyte count is 500 cells/mm3 or less, 550 cells/mm3 or less, 600 cells/mm3 or less, 650 cells/mm3 or less, 700 cells/mm3 or less, 750 cells/mm3 or less, or 800 cells/mm3 or less.

In a preferred embodiment, one or more immunomodulatory agents are administered prior to, subsequent to, or concomitantly with a compound of the invention so as to transiently reduce or inhibit one or more aspects of the immune response. Such a transient inhibition or reduction of one or more aspects of the immune system can last for hours, days, weeks, or months. Preferably, the transient inhibition or reduction in one or more aspects of the immune response lasts for a few hours (e.g., 2 hours, 4 hours, 6 hours, 8 hours, 12 hours, 14 hours, 16 hours, 18 hours, 24 hours, 36 hours, or 48 hours), a few days (e.g., 3 days, 4 days, 5 days, 6 days, 7 days, or 14 days), or a few weeks (e.g., 3 weeks, 4 weeks, 5 weeks or 6 weeks).

Any anti-inflammatory agent, including agents useful in therapies for inflammatory disorders, well-known to one of skill in the art can be used in the compositions and methods of the invention. Non-limiting examples of anti-inflammatory agents include non-steroidal anti-inflammatory drugs (NSAIDs), steroidal anti-inflammatory drugs, anticholinergics (e.g., atropine sulfate, atropine methylnitrate, and ipratropium bromide (ATROVENT™)), beta2-agonists (e.g., abuterol (VENTOLIN™ and PROVENTIL™), bitolterol (TORNALATE™), levalbuterol (XOPONEX™), metøproterenol (ALUPENT™), pirbuterol (MAXAIR™), terbutlaine (BRETHAIRE™ and BRETHINE™), albuterol (PROVENTIL™, REPETABS™, and VOLMAX™), formoterol (FORADIL AEROLIZER™), and salmeterol (SEREVENT™ and SEREVENT DISKUS™)), and methylxanthines (e.g., theophylline (UNIPHYL™, THEO-DUR™, SLO-BID™, AND TEHO-42™)). Examples of NSAIDs include, but are not limited to, aspirin, ibuprofen, celecoxib (CELEBREX™), diclofenac (VOLTAREN™), etodolac (LODINE™), fenoprofen (NALFON™), indomethacin (INDOCIN™), ketorolac (TORADOL™), oxaprozin (DAYPRO™), nabumentone (RELAFEN™), sulindac (CLINORIL™), tolmentin (TOLECTIN™), rofecoxib (VIOXX™), naproxen (ALEVE™, NAPROSYN™), ketoprofen (ACTRON™) and nabumetone (RELAFEN™). Such NSAIDs function by inhibiting a cyclooxgenase enzyme (e.g., COX-I and/or COX-2). Examples of steroidal anti-inflammatory drugs include, but are not limited to, glucocorticoids, dexamethasone (DECADRON™), corticosteroids (e.g., methylprednisolone (MEDROL™)), cortisone, hydrocortisone, prednisone (PREDNISONE™ and DELTASONE™), prednisolone (PRELONE™ and PEDIAPRED™), triamcinolone, azulfidine, and inhibitors of eicosanoids {e.g., prostaglandins, thromboxanes, and leukotrienes). Anti-inflammatory therapies and their dosages, routes of administration, and recommended usage are known in the art and have been described in such literature as the Physician 's Desk Reference (57th ed., 2003).

For arthritis, inflammation-mediated bone loss and other disorders that have an inflammatory component, preferred conventional treatments for use in combination therapy with compounds and compositions of this invention include (without limitation) naproxen sodium (Anaprox® and Anaprox® DS, Roche), flurbiprofen (Ansaid®; Pharmacia), diclofenac sodium + misoprostol (Arthrotec®, Searle), valdecoxib (Bextra®, Pharmacia), diclofenac potassium (Cataflam® and Voltaren®, Novartis), celecoxib (Celebrex®, Pharmacia), sulindac (Clinoril®, Merck), oxaprozin (Daypro®, Pharmacia), salsalate (Disalcid®, 3M), diflunisal (Dolobid®, Merck), naproxen sodium (EC Naprosyn®, Roche), piroxicam (Feldene®, Pfizer), indomethacin (Indocin® and Indocin SR®, Merck), etodolac (Lodine® and Lodine XL®, Wyeth), meloxicam (Mobic®, Boehringer Ingelheim), ibuprofen (Motrin®, Pharmacia), naproxen (Naprelan®, Elan), naproxen (Naprosyn®, Roche), ketoprofen (Orudis® and Oruvail®, Wyeth), nabumetone (Relafen®, SmithKline), tolmetin sodium (Tolectin®, McNeil), choline magnesium trisalicylate (Trilisate®, Purdue Fredrick), and rofecoxib (Vioxx®, Merck).

In any case where pain in a component of the target disorder, the other therapeutic agent can be an analgesic. Useful analgesics include, but are not limited to, phenacetin, butacetin, acetaminophen, nefopam, acetoamidoquinone, and mixtures thereof.

For use against osteoporosis, Paget's disease and other disorders associated with bone deterioration, preferred conventional agents that may be used in combination with compounds and compositions of this invention include (without limitation) bisphosphonates (such as etidronate (Didronel®, Procter & Gamble), pamidronate (Aredia®, Novartis), and alendronate (Fosamax®, Merck)), tiludronate (Skelid®, Sanofi-Synthelabo, Inc.), risedronate (Actonel®, Procter & Gamble/A ventis), calcitonin (Miacalcin®), estrogens (Climara®, Estrace®, Estraderm®, Estratab®, Ogen®, Ortho-Est®, Vivelle®, Premarin®, and others) estrogens and progestins (Activella™, FemHrt®, Premphase®, Prempro®, and others), parathyroid hormone and portions thereof, such as teriparatide (Forteo®, Eli Lilly and Co.), selective estrogen receptor modulators (SERMs) (such as raloxifene (Evista®)) and treatments currently under investigation (such as other parathyroid hormones, sodium fluoride, vitamin D metabolites, and other bisphosphonates and selective estrogen receptor modulators).

The other therapeutic agent can include bone anti-resorptive agents for example progestins, polyphosphonates, bisphosphonate(s), estrogen agonists/antagonists, estrogen (such as Premarin®), estrogen/progestin combinations, and estrogen derivatives (such as estrone, estriol or 17a, 17/3-ethynyl estradiol). Exemplary progestins are available from commercial sources and include: algestone acetophenide, altrenogest, amadinone acetate, anagestone acetate, chlormadinone acetate, cingestol, clogestone acetate, clomegestone acetate, delmadinone acetate, desogestrel, dimethisterone, dydrogesterone, ethynerone, dthynodiol diacetate, etonogestrel, flurogestone acetate, gestaclone, gestodene, gestonorone caproate, gestrinone, haloprogesterone, hydroxyprogesterone, caproate, levonorgestrel, lynestrenol, medrogestone, medroxyprogesterone acetate, melengestrol acetate, methynodiol diacetate, norethindrone, norethindrone acetate, norethynodrel, norgestimate, norgestomet, norgestrel, oxogestone phenpropionate, progesterone, quingestanol acetate, quingestrone, and tigestol. Preferred progestins are medroxyprogestrone, norethindrone and norethynodrel.

Exemplary bone resorption inhibiting polyphosphonates include polyphosphonates of the type disclosed in U.S. Pat. No. 3,683,080. Preferred polyphosphonates are geminal dipolyphosphonates (also referred to as bis-phosphonates). Tiludronate disodium is an especially preferred polyphosphonate. Ibandronic acid is an especially preferred polyphosphonate. Alendronate is an especially preferred polyphosphonate. Zoledronic acid is an especially preferred polyphosphonate. Other preferred polyphosphonates are 6-amino- 1-hydroxy-hexylidene-biphosphonic acid and l-hydroxy-3(methylpentylamino)- propylidene-bisphosphonic acid. The polyphosphonates may be administered in the form of the acid, or of a soluble alkali metal salt or alkaline earth metal salt. Hydrolyzable esters of the polyphosphonates are likewise included. Specific examples include ethane- 1 -hydroxy 1,1-diphosphonic acid, methane diphosphonic acid, pentane-1 -hydroxy- 1,1-diphosphonic acid, methane dichloro diphosphonic acid, methane hydroxy diphosphonic acid, ethane- 1- amino- 1,1 -diphosphonic acid, ethane-2-amino- 1,1 -diphosphonic acid, propane-3-amino-l- hydroxy-1,1- diphosphonic acid, propane-N,N-dimethyl-3-amino-l -hydroxy- 1,1- diphosphonic acid, propane-3,3-dimethyl-3-amino-l -hydroxy- 1,1 -diphosphonic acid, phenyl amino methane diphosphonic acid, N,N-dimethylamino methane diphosphonic acid, N(2-hydroxyethyl)amino methane diphosphonic acid, butane-4-amino-l- hydroxy- 1,1 - diphosphonic acid, pentane-5-amino-l -hydroxy- 1,1-diphosphonic acid, hexane-6-amino-l- hydroxy-l,l-diphosphonic acid and pharmaceutically acceptable esters and salts thereof.

In particular, the compounds of this invention may be combined with a mammalian estrogen agonist/antagonist. Any estrogen agonist/antagonist may be used for this purpose. The term estrogen agonist/antagonist refers to compounds which bind with the estrogen receptor, inhibit bone turnover and/or prevent bone loss. In particular, estrogen agonists are herein defined as chemical compounds capable of binding to the estrogen receptor sites in mammalian tissue, and mimicking the actions of estrogen in one or more tissue. Estrogen antagonists are herein defined as chemical compounds capable of binding to the estrogen receptor sites in mammalian tissue; and blocking the actions of estrogen in one or more tissues. Such activities are readily determined by those skilled in the art of standard assays including estrogen receptor binding assays, standard bone histomorphometric and densitometer methods, and E. F Eriksen et al, Bone Histomorphometry, Raven Press, New York, pp. 1-74 (1994); S. J. Grier et al, The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol. 31(1): 50-62 (1996); Wahner H. W. and Fogelman L, The Evaluation of Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice., Martin Dunitz Ltd., London, pp. 1-296 (1994)). A variety of these compounds are described and referenced below.

A preferred estrogen agonist/antagonist is droloxifene: (phenol, 3-(l-(4-(2- (dimethyIamino)ethoxy)phenyl)-2-phenyl-l-butenyl)-, (E)-) and related compounds which are disclosed in U.S. Pat No. 5,047,431. Another preferred estrogen agonist/antagonist is 3-(4-(l,2-diphenyl-but-l-enyl)-phenyl)-acrylic acid, which is disclosed in Wilson et al, Endocrinology 138: 3901-11 (1997). Another preferred estrogen agonist/antagonist is tamoxifen: (emanamine,2-(-4-(l,2-diphenyl-l-butenyl)phenoxy)-N,N-dimethyl, (Z)-2-, 2- hydroxy-1 ,2,3-propanetricarboxylate(l : I)) and related compounds which are disclosed in U.S. Pat. No.4,536,516. Another related compound is 4-hydroxy tamoxifen which is disclosed in U.S. PaL No. 4,623,660.

A preferred estrogen agonist/antagonist is raloxifene: (methanone, (6-hydroxy-2-(4- hydroxyphenyl)benzo[b]thien-3-yl)(4-(2-( 1 -piperidinyl)etho xy)phenyl)hydrochloride) which is disclosed in U.S. Pat. No. 4,418,068. Another preferred estrogen agonist/antagonist is toremifene: (ethanamine, 2-(4-(4-chloro-l,2-diphenyl-l- butenyl)phenoxy)-N,N-dimethyl-, (Z)-, 2-hydroxy-l,2,3-propanetricarboxylate (1:1) which is disclosed in U.S. Pat. No. 4,996,225. Another preferred estrogen agonist/antagonist is centchroman: l-(2-((4-(-methoxy-2,2,dimethyl-3-phenyl-chroman-4-yl)-phenoxy)-ethyl)- pyrrolidine, which is disclosed in U.S. Pat. No. 3,822,287. Also preferred is levormeloxifene. Another preferred estrogen agonist/antagonist is idoxifene: (E)-l-{2-(4- (l-(4-iodo-phenyl)-2-phenyl-but-l-enyl)-phenoxy)-ethyl)-pyrrol idinone, which is disclosed in U.S. Pat. No.4,839,155. Another preferred estrogen agonist/antagonist is 2-(4-methoxy- phenyl)-3-[4-(2-piperidin-l-yl-ethoxy)-phenoxy]-benzo[b]thiop hen-6-ol which is disclosed in U.S. Pat. No. 5,488,058. Another preferred estrogen agonist/antagonist is 6-(4-hydroxy- phenyl)-5-(4-(2-piperidin-l-yl-ethoxy)-benzyl)-naphthalen-2-ol which is disclosed in U.S. Pat. No. 5,484,795. Another preferred estrogen agonist/antagonist is (4-(2-(2-aza- bicyclo[2.2.1]hept-2-yl)-ethoxy)-phenyl)-(6-hydroxy-2- (4-hydroxy-phenyl)-benzo[b]thiop hen-3-yl)-methanone which is disclosed, along with methods of preparation, in PCT publication no. WO 95/10513 assigned to Pfizer Inc. Other preferred estrogen agonist/antagonists include compounds as described in U.S. Pat. No. 5,552,412. Especially preferred compounds described therein are: cis-6-(4-fluoro-phenyl)-5-(4-(2-piperidin-l-yl- ethoxy)-phenyl)- 5,6,7,8-tetr ahydro-naphthalene-2-ol; (-)-cis-6-phenyl-5-(4-(2-pyrrolidin- 1 -yl-ethoxy)-phenyl)-5,6,7,8-tetrahydro -naphthalene-2-ol; cis-6-phenyl-5-(4-(2-pyrrolidin- l-yl-ethoxy)-phenyl)-5 ,6,7,8- tetrahydro-nap hthalene-2-ol; cis-l-(6'-pyrrolodinoethoxy-3'- pyridyl)-2-phenyl-6- hydroxy-l,2,3,4-tetrahyd ronaphthalene; l-(4'- pyrrolidinoethoxyphenyl)-2-(4"- fluorophenyl)-6-hydroxy-l ,2,3,4-tetrah ydroisoquinoline; cis-6-(4-hydroxyphenyl)- 5-(4-(2-piperidin- 1 -yl-ethoxy)-phenyl)-5,6,7,8-tetr ahydro- naphthalene-2-ol; and 1 -(4'-pyrrolidinolethoxyphenyl)-2-phenyl-6-hydroxy-l ,2,3,4- tetrahydroisoquinoline. Other estrogen agonist/antagonists are described in U.S. Pat. No. 4,133,814. U.S. Pat. No.4,133,814 discloses derivatives of 2-phenyl-3-aroyl- benzothiophene and 2-phenyl-3-aroylbenzothiophene-l -oxide.

Those skilled in the art will recognize that other bone anabolic agents, also referred to as bone mass augmenting agents, may be used in conjunction with the compounds of this invention. A bone mass augmenting agent is a compound that augments bone mass to a level which is above the bone fracture threshold as detailed in the World Health Organization Study World Health Organization, "Assessment of Fracture Risk and its Application to Screening for Postmenopausal Osteoporosis (1994). Report of a WHO Study Group. World Health Organization Technical Series 843." Any prostaglandin, or prostaglandin agonist/antagonist may be used in combination with the compounds of this invention. Those skilled in the art will recognize that IGF-I, sodium fluoride, parathyroid hormone (PTH), active fragments of parathyroid hormone, growth hormone or growth hormone secretogogues may also be used. The following paragraphs describes in greater detail exemplary compounds that may be administered in combination with compounds of this invention.

Prostaglandins: The term prostaglandin refers to compounds which are analogs of the natural prostaglandins PGDi, PGD₂, PGE₂, PGEj and PGF₂ which are useful in the treatment of osteoporosis and other disorders associated with excessive osteoclastic bone resorption. These compounds bind to the prostaglandins receptors. Such binding is readily determined by those skilled in the art of standard assays (e.g., S. An et al., Cloning and Expression of the EP₂ Subtype of Human Receptors for Prostaglandin E₂ Biochemical and Biophysical Research Communications, 197(1): 263-270 (1993)).

Prostaglandins are alicyclic compounds related to the basic compound prostanoic acid. The carbon atoms of the basic prostaglandin are numbered sequentially from the carboxylic carbon atom through the cyclopentyl ring to the terminal carbon atom on the adjacent side chain. Normally the adjacent side chains are in the trans orientation. The presence of an oxo group at C-9 of the cyclopentyl moiety is indicative of a prostaglandin within the E class while PGE₂ contains a trans unsaturated double bond at the Cn-C_t4 and a cis double bond at the Cs -C^ position.

A variety of prostaglandins are described and referenced below. However, other prostaglandins will be known to those skilled in the art. Exemplary prostaglandins are disclosed in U.S. Pat Nos.4,171,331 and 3,927,197,. Norrdin et al., The Role of Prostaglandins in Bone in Vivo, Prostaglandins Leukotriene Essential Fatty Acids 41: 139- 150 (1990) is a review of bone anabolic prostaglandins. Any prostaglandin agonist/antagonist may be used in combination with the compounds of this invention. The term prostaglandin agonist/antagonist refers to compounds which bind to prostaglandin receptors (e.g.., An S. et al., Cloning and Expression of the EP₂ Subtype of Human Receptors for Prostaglandin E₂, Biochemical and Biophysical Research Communications 197(1): 263-70 (1993)) and mimic the action of prostaglandin in vivo (e.g., stimulate bone formation and increase bone mass). Such actions are readily determined by those skilled in the art of standard assays. Eriksen E. F. et al., Bone Histomorphometry, Raven Press, New York, 1994, pp. 1-74; SJ. Grier et al, The Use of Dual-Energy X-Ray Absorptiometry In Animals, Inv. Radiol. 31(1): 50-62 (1996); H. W. Warmer and I. Fogelman, The Evaluation of Osteoporosis: Dual Energy X-Ray Absorptiometry in Clinical Practice, Martin Dunitz Ltd. London, pp. 1-296 (1994). A number of these compounds are described and reference below. However, other prostaglandin agonists/antagonists will be known to those skilled in the art. Exemplary prostaglandin agonists/antagonists are disclosed as follows. U.S. Pat. No. 3,932,389 discloses 2-descarboxy-2-(tetrazol-5-yl)-l l-desoxy-15-substituted-omega- pentanorpros taglandins useful for bone formation activity. U.S. Pat. No. 4,018,892, discloses 16-aryl-13,14-dihydro-PGE2 p-biphenyl esters useful for bone formation activity. U.S. Pat. No.4,219,483, discloses 2,3,6-substituted-4-pyrones useful for bone formation activity. U.S. Pat. No. 4,132,847, discloses 2,3,6-substituted-4-pyrones useful for bone formation activity. U.S. Pat. No.4,000,309, discloses 16-aryl-13,14-dihydro-PGE₂ p- biphenyl esters useful for bone formation activity. U.S. Pat. No. 3,982,016, discloses 16- aryl-13,14-dihydro-PGE2 p-biphenyl esters useful for bone formation activity. U.S. Pat. No. 4,621,100, discloses substituted cyclopentanes useful for bone formation activity. U.S. Pat. No. 5,216,183, discloses cyclopentanones useful for bone formation activity.

Sodium fluoride may be used in combination with the compounds of this invention. The term sodium fluoride refers to sodium fluoride in all its forms (e.g., slow release sodium fluoride, sustained release sodium fluoride). Sustained release sodium fluoride is disclosed in U.S. Pat No. 4,904,478. The activity of sodium fluoride is readily determined by those skilled in the art of biological protocols.

Bone morphogenetic protein may be used in combination with the compounds of this invention (e.g., see Ono et al., Promotion of the Osteogenetic Activity of Recombinant Human Bone Morphogenetic Protein by Prostaglandin E₁, Bone 19(6): 581-588 (1996)).

Any parathyroid hormone (PTH) may be used in combination with a compound of this invention. The term parathyroid hormone refers to parathyroid hormone, fragments or metabolites thereof and structural analogs thereof which can stimulate bone formation and increase bone mass. Also included are parathyroid hormone related peptides and active fragments and analogs of parathyroid related peptides (see PCT publication No. WO 94/01460). Such bone anabolic functional activity is readily determined by those skilled in the art of standard assays. A variety of these compounds are described and referenced below. However, other parathyroid hormone will be known to those skilled in the art. Exemplary parathyroid hormones are disclosed in the following references. "Human Parathyroid Peptide Treatment of Vertebral Osteoporosis", Osteoporosis Int., 3, (Supp 1 ) : 199-203. "PTH 1 -34 Treatment of Osteoporosis with Added Hormone Replacement Therapy: Biochemical, Kinetic and Histological Responses" Osteoporosis Int. 1: 162-170. Any growth hormone or growth hormone secretogogue may be used in combination with compounds of this invention. The term growth hormone secretagogue refers to a compound which stimulates the release of growth hormone or mimics the action of growth hormone {e.g., increases bone formation leading to increased bone mass). Such actions are readily determined by those skilled in the art of standard assays well known to those of skill in the art. A variety of these compounds are disclosed in the following published PCT patent applications: WO 95/14666; WO 95/13069; WO 94/19367; WO 94/13696; and WO 95/34311. However, other growth hormones or growth hormone secretagogues will be known to those skilled in the art. In particular, a preferred growth hormone secretagogue is N-[l(R)-[l,2-Dihydro-l-methanesulfonylspiro[3H-indole-3,4'-piperidin]-l'-y l)carbonyl]-2- (phenylmethyloxy)ethyl]-2-amino-2-methylpropanamide:MK-667. Other preferred growth hormone secretagogues include 2-amino-N-(2-(3a-(R)-benzyl-2-methyl-3-oxo-2,3,3a,4,6,7- hexahydro-pyrazolo- [4,3-c]pyridin-5-yl)- 1 -(R)-benzyloxymethyl-2-oxo-ethyl)- isobutyramide or its L-tartaric acid salt; 2-amino-N-(l-(R)-benzyloxymethyl-2-(3a-(R)-(4- fluoro-ben2yl)-2-methyl-3-oxo -2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyridin-5-yl)-2-oxo- ethyl)isobutyramide; 2-amino-N-(2-(3a-(R)-benzyl-3-oxo-2,3,3a,4,6,7-hexahydro- pyrazolo[4,3-c]pyridin-5-yl)-l -(R)benzyloxymethyl-2-oxo-ethyl)isobutyramide; and 2- amino-N-(l-(2,4-difluoro-benzyloxymethyl)-2-oxo-2-(3-oxo-3a-pyridin-2-yhn ethyl-2- (2_y2,2-trifluoro-ethyl)-2,3,3a,4,6,7-hexahydro-pyrazolo[4,3-c]pyrid in-5-yl)-ethyl)-2- methyl-propionamide.

Any method of the present invention can comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one compound of this invention to a cell, tissue, organ, animal or patient in need thereof. Such a method can optionally further comprise co-administration or combination therapy for treating an IL- 12, IL-23, or IL-27 production-related disorder, wherein the administering further comprises administering before, concurrently with, and/or after a compound of this invention, at least one additional active agent selected from a TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid antiinflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular blocker, an antimicrobial (e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an ann^'psoriatic, a corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog, domase alpha (Pulmozyme), a cytokine or a cytokine antagonistm. Suitable dosages are well known in the art. See, e.g., Wells et al, eds., Pharmacotherapy Handbook, 2.sup.nd Edition, Appleton and Lange, Stamford, Conn. (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, Calif. (2000), each of which references are entirely incorporated herein by reference.

TNF antagonists suitable for compositions, combination therapy, co-administration, devices and/or methods of the present invention include, but are not limited to, anti-TNF antibodies (such as, Remicade (Infliximab) or Humira (adalimumab)) for example, or , antigen-binding fragments thereof, and receptor molecules which bind specifically to TNF (such as, for example, Enbrel (Etanercept)); compounds which prevent and/or inhibit TNF synthesis, TNF release or its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds which prevent and/or inhibit TNF receptor signaling, such as mitogen activated protein (MAP) kinase inhibitors; compounds which block and/or inhibit membrane TNF cleavage, such as metalloproteinase inhibitors; compounds which block and/or inhibit TNF activity, such as angiotensin converting enzyme (ACE) inhibitors (e.g., captopril); and compounds which block and/or inhibit TNF production and/or synthesis, such as MAP kinase inhibitors.

For clarification, a "tumor necrosis factor antibody," "TNF antibody," "TNF antibody," or fragment and the like decreases, blocks, inhibits, abrogates or interferes with TNF activity in vitro, in situ and/or preferably in vivo. For example, a suitable TNF human antibody of the present invention can bind TNFα and includes anti-TNF antibodies, antigen-binding fragments thereof, and specified mutants or domains thereof that bind specifically to TNFα. A suitable TNF antibody or fragment can also decrease block, abrogate, interfere, prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.

The foregoing and other useful combination therapies will be understood and appreciated by those of skill in the art. Potential advantages of such combination therapies include the ability to use less of each of the individual active ingredients to minimize toxic side effects, synergistic improvements in efficacy, improved ease of administration or use and/or reduced overall expense of compound preparation or formulation. The biological activities of a compound of this invention can be evaluated by a number of cell-based assays. One of such assays can be conducted using cells from human peripheral blood mononuclear cells (PBMC) or human monocytic cell line (THP-I). In one embodiment, the cells are stimulated with a combination of human interferon-γ(IFN-7) and lipopolysaccharide (LPS) or a combination of IFN-γand Staphylococcus aureus Cowan I (SAC) in the presence of a candidate compound. The level of inhibition of IL- 12 production can be measured by, e.g., determining the amount of p70 by using a sandwich ELISA assay with anti-human IL- 12 antibodies. Likewise, the level of inhibition of IL- 12p40 production can be measured using a sandwich ELISA assay with anti-human IL- 12p40 antibodies. IC₅0 of the candidate compound can then be determined. Specifically, PBMC or THP-I cells are incubated with the candidate compound. Cell viability was assessed using the bioreduction of MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3- carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium] (Promega, Madison, WI).

ANIMAL MODELS

Animal models for autoimmune disorders can be used to assess the efficacy of the therapeutics or pharmaceutical compositions of invention. Animal models for autoimmune disorders such as type 1 diabetes, thyroid autoimmunity, systemic lupus eruthematosus, and glomerulonephritis have been developed (Flanders et al., 1999, Autoimmunity 29:235-246; Krogh et al., 1999, Biochimie 81:511-515; Foster, 1999, Semin. Nephrol. 19:12-24).

The following series of examples are presented by way of illustration and not by way of limitation on the scope of the present invention. EXAMPLES

General Experimental Procedures

Cell lines and culture conditions:

THP-I cell line was obtained from American Type Culture Collection (Manassas, VA). The THP-I cells were cultured in RPMI 1640 (ATCC, Manassas, VA), supplemented with 10 % FCS (ATCC, Manassas, VA), and 1% penicillin/Streptomycin (Gibco-BRL, New York, N. Y.). The cells were primed with IFNγ (100 U/ml) followed by 1 μg/ml LPS in the presence of different concentrations of Compound 2. Compound 2 was prepared in DMSO and the final DMSO concentration was adjusted to 0.25% in the cultures, including the compound-free control. Cell-free supernatants were taken 18 h later for the measurement of cytokines.

Isolation of Nuclear extracts:

THP-I cells were suspended in 20 volumes of buffer A containing 10 mM KCl, 10 mM HEPES (pH 7.9), I mM MgCl₂, 1 mM dithiothreitol (DTT), 0.1 % Nonidet p40 (NP- 40), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and homogenized and centrifuged at 10,000 rpm at 4C afor 5 min. Nuclear pellets were then suspended in buffer C containing 400 mM NaCl, 20 mM HEPES 9pH 7.9), 15 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, 25 % glycerol, 1 mM PMSF, and 10 ug of leupeptin, 20 ug of pepstatin, and 10 μg/ml antipain, incubated for 30 min at 4⁰C, and centrifuged at 14,000 rpm for 20 min.. The supernatants were dialyzed against buffer D containing 100 mM NaCl, 20 mM HEPES (pH 7.9), 20% glycerol, 1 mM PMSF, and 1 mM DTT.

Western blot:

10 % SDS Polyacrylamide gels (Inveitrogen) were transferred to Pure Nitrocellulose membrane (BioRed, Hercules, CA). The membranes were blocked with 5% milk in TBST buffer and incubated with anti-c-Rel, anti-p65, anti-p50, anti-ICSBP or anti-PU-1 antibody (all the antibodies were purchased from Santa Cruz) at a dilution of 1 :500 for 1 h at room temperature or overnight at 4⁰C. The membranes were washed and incubated with Horseradish Peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Amersham, England) at a dilution of 1 :2000 at room temperature for 1 h.

Immunoprecipitation : Five hundred mg of the precleared whole cell protein was incubated with 20 μl of the agarose conjugated anti-c-Rel antibody (sc-6955) for overnight at 4⁰C. Immunoprecipitated proteins were washed 3 times with PBS, and eluted with electrophoreses sample buffer. Western blotting of immunoprecipitated protein was performed as described above.

Example 1: Measuring the level of IL-12 p40

Northern blot analysis was performed to examine the mRNA levels of IL-12 p35 and p40. Human PBMC and the human monocyte cell line THP-I cells were stimulated with IFN-V SAC in the presence or absence of Compound 1. Human PBMC were isolated by centrifugation using Ficoll-Paque (Pharmacia Biotech, Uppsala, Sweden) and prepared in RPMI medium supplemented with 10% fetal calf serum (FCS), 100 U/ml penicillin, and 100 μg/ml streptomycin, in a 96-well plate with 5 x 10⁵ cells/well. The cells were then primed with IFN-γ(100 U/ml) and followed by 0.01% SAC or 1 μg/ml LPS, in the presence of different concentrations of Compound 2 or other compounds. The test compounds were prepared in DMSO and the final DMSO concentration was adjusted to 0.25% in all cultures, including the compound-free control. Cell-free supernatants were taken 18 h later for the measurement of cytokines. The THP-I cells were obtained from American Type Culture Collection (Manassas, VA) and were cultured in RPMI 1640 (ATCC, Manassas, VA), supplemented with 10 % FCS (ATCC, Manassas, VA), and 1% penicillin/Streptomycin (Gibco-BRL, New York, N. Y.). Total RNA was isolated and subjected to Northern blot analysis using IL-12 p35 and p40 cDNA probes. We first examined the kinetics of mRNA accumulation in cultures of hPBMC and THP-I cells primed with EFN-γ followed by SAC stimulation in the presence or absence of 1 μM Compound 1.

In hPBMC, both IL-12 p35 and p40 mRNA were detectable by 4 h and peaked at 6 h after the addition of SAC. The expression of p35 mRNA was completely inhibited by Compound 1 at all sampling times, whereas the expression of the mRNA for the p40 subunit was reduced significantly but incompletely. In THP-I cells stimulated with IFN- γ/SAC, IL-12 p35 mRNA was barely visible in compound-free control and was undetectable in the presence of 1 μM Compound 1. In contrast, IL-12 p40 mRNA was readily detectable by 4 h and peaked at 6 h after the addition of SAC. Again, Compound 1 significantly but incompletely reduced the expression of the p40 message. We conducted a dose-response study of the inhibitory effects of Compound 1 on IL- 12 mRNA expression in IFN-ySAC-stimulated hPBMC. Because both IL-12 p35 and p40 mRNA levels were maximal at 6 h after the addition of SAC, this time point was selected for the dose-response analysis. The induction of IL-12 p35 mRNA accumulation by IFN- 7/SAC was completely reversed by 3 nM Compound 1, with an IC₅₀ below 1 nM. In contrast, EL- 12 p40 mRNA accumulation was barely inhibited by 1 nM Compound 1, with maximum, though still incomplete inhibition at 10 nM. This apparent weaker inhibition of p40 relative to p35 could be due to more effective inhibition of the p35 promoter or it simply may be the product of the fact that p40 is produced in vast excess to p35 and its inhibition may require greater concentrations of drug.

Thus, Compound 1 caused a decrease in both p35 and p40 mRNA levels. Subsequent nuclear run-on experiments showed that this effect was at the level of transcription initiation.

Example 2: Effect of Compound 2 on IL-12 p3S and p40 promoter activity

As a result of the Northern blot findings, we undertook a study of the p35 and p40 promoter activities. We transiently transfected the murine macrophage cell line RAW264.7 with DNA constructs in which the p35 and p40 promoters directed expression of the luciferase reporter gene. The RAW264.7 cell line was obtained from American Type Culture Collection (Manassas, VA) and was cultured in DMEM (ATCC, Manassas, VA) supplemented with 10% FCS (ATCC, Manassas, VA), and 1% penicillin/Streptomycin (Gibco-BRL, New York, N. Y.).

Both p35 and p40 promoter-driven luciferase production in response to stimulation were determined in the presence or absence of Compound 1 and Compound 2. To construct the human IL-12 p35 and p40 promoter/luciferase reporter constructs, we generated p35 (- 1.5 kb to +3 bp) and p40 (-1.3 kb to +56 bp) promoter fragments, which contained several sequence motifs of the human IL-12 p35 and p40 genes. The fragments were generated by PCR from genomic DNA obtained from human PBMC using primers as follows: IL-12 p35 1.5 kb-F: 5'-GCAGCATTAGAAGGGGCCTTAGAGA-3'(SEQ IDN0:3) and IL-12 p35 1.5 kb-R: 5'-TnTATAATTGTCCCGAGGCGCG-S' (SEQ IDNO:4); IL-12 p40 -1.3kb-F: 5'-ACGGCGAGGAAAGTTAGCCCG-S' (SEQ ID NO:5) and IL-12 p40 1.3kb-R: 5'- TTGCTCTGGGC AGGACGGAG-3' (SEQ ID NO:6). The deletion in the p40 promoter reporter constructs were generated by PCR with primers as follows: IL-12 p40 -250 bp to +56 bp (p40/-250 bp) F: 5'-CACCCAAAAGTCATTTCCTC-3'(SEQ ID NO:7) and IL-12 p40 -250 bp to +56 bp (p40/-250 bp) R: 5'-TGCTCTGGGCAGGACGGAG-S' (SEQ ID NO:8); IL-12 p40 -150 bp to +56 bp (p40/-150 bp) F: 5'-

AGAGTTGTTTTCAATGTTGCAAC-S' (SEQ ID NO:9) and IL-12 p40 -150 bp to +56 bp (p40/-150 bp) R: 5'-TGCTCTGGGCAGGACGGAG-S '(SEQ ID NO:10). The resulting PCR products were ligated upstream of the luciferase gene in pGL3-Basic vector (Promega). All constructs were verified by DNA sequencing.

RAW267.4 cells were transiently transfected and the cells were then stimulated with murine recombinant IFN-γ(100 ng/ml) for 10 h followed by LPS (1 μg/ml) or SAC (0.025 %) in the presence or absence of Compound 1, Compound 2, or a negative control (a structurally-related inactive compound) at different concentrations for an additional 16 h. Transfection was accomplished using SuperFect Transfection Reagent (Qiagen) by the described protocol. Total amount of transfect DNA was kept constant by including the respective control plasmids without insertions.

Cells were co-transfected with the vector pCMV (BD Biosciences Clontech) in which the constitutively active CMV promoter directs j3-galactosidase expression for the monitoring of transfection efficiency. Luciferase and /3-galactosidase activity were determined in cell extracts prepared according to the Luciferase assay system (Promega) and Luminescent /3-gal Detection system (BD Biosciences Clontech). Luciferase activity was then normalized using the /3-galactosidase value. The luciferase activities were strongly induced in the case of the IL-12 p40 and p35 promoter constructs in RAW264.7 cells after the stimulation with IFN-7/LPS or IFN-7/SAC. This p40 and p35 promoter- driven luciferase expression was suppressed in the presence of Compound 1 and Compound 2, but not the inactive negative control compound. The results are shown in Figures 2A-2B. This result supports a mechanism in which Compound 2 inhibits IL-12 transcription. p35 promoter-driven luciferase expression stimulated by IFN-YLPS was inhibited more effectively by Compound 2 than by Compound 1, while the negative control compound did not suppress the promoter activity at all. The IC₅₀S of Compound 1 , Compound 2, and the negative control compound against IL-12 production in THP-I cells were 40 nM, 10 nM, and greater than 1000 nM, respectively. These results are in agreement with the inhibitory activity against IL-12 protein production evaluated by ELISA, signifying that the inhibition of the p35 promoter activity is a reflection of the inhibitory activity against IL-12. ELISA was performed by the following method. Human IL-12 p70 (heterodimer) was assayed using ELI-PAIR kit from Cell Sciences (Norwood, MA), according to the manufacturer's instructions. Human IL-12 p40 was assayed using ELISA kit from Cell Sciences (Norwood, MA) according to the manufacturer's instructions.

Northern blot analysis to examine the mRNA levels of IL-12 p35 and p40 was used to elucidate the mechanism of action. Compound 1 caused a decrease in the levels of p35 and p40 mRNA. Nuclear run-on experiments showed that this effect was at the level of transcription initiation. When DNA expression plasmids in which the p35 and p40 promoters directed the expression of the luciferase reporter gene were transfected into cells, it was shown that expression of luciferase could be inhibited by Compound 1 and Compound 2. These results confirm a mechanism in which Compound 2 inhibits IL-12 transcription of the p3S and p40 genes.

We then set out to analyze the IL-12 transcriptional promoter elements that played a role in this effect. We performed deletion analyses using the p40 promoter. This promoter, rather than the p35 promoter, was chosen because the transcriptional elements are better defined in the p40 promoter. To identify the IL-12 inhibitor responsive elements involved in the p40 gene transcription activation, three different promoters were constructed and transiently transfected into RAW264.7 cells. As shown in Figures 3 A-3B, the promoters that consisted of the NF-κB through the API element region showed diminished promoter activation, while the promoter that contained the S' flanking region of p40 promoter but had a large promximal deletion displayed significantly decreased promoter activity in response to stimulation with IFN-yLPS. Only the promoter which contained the Ets-2 element along with the PU-I, NF-κB and API elements showed high activity of luciferase in response to IFN-VLPS stimulation, suggesting that the Ets-2 element plays a role in the regulation of IL-12 p40 promoter activity. This IL-12 promoter-driven luciferase activity was significantly suppressed in the presence of Compound 1. These results suggest that the element that is responsive in the suppression of promoter activity lies in the region from the TATA box to -250 bp in the IL-12 p40 promoter.

To assess the role of individual p40 promoter transcription elements in more detail, mutations within many of these elements have been generated. The goal of this work is to assess the effect of mutations which decrease but do not eliminate p40 promoter activity on inhibition by Compound 2. All mutations in the Ets-2 element have completely eliminated the induction of reporter gene expression, emphasizing the importance of this element. Site- directed mutatgenesis of the NFKB element resulted in a p40 promoter having reduced but clearly measurable induction by IFN-γ/LPS. Site-directed mutagenesis was performed with the GeneTailor Site-directed Mutagenesis System (Invitrogen, Carlsbad, CA) according to the manufacturer's instructions. The IL- 12 p40 Mutant primer sequences were as follows: IL-12 p40-Ets2 mut-F: 5'-TATTCCCCACCCAAAAGTCACTTAGTTCATT-S' (SEQ ID NO:11) and IL-12 p40-Ets2 mut-R: 5'-TGACTTTTGGGTGGGGAATAAGGAAGGAGA- 3' (SEQ ID NO: 12); IL-12 p40-AP-l mut-F: 5^*-

TTGTTTTCAATGTTGCAACATTTCTAGTTTA-3' (SEQ ID NO:13) and IL-12 p40-AP- 1 mut-R: 5'-TGTTGCAACATTGAAAACAACTCTCAAAAC-S' (SEQ ID NO: 14); IL-12 p40-Nf-kB mut-F: 5'- CAAACAAAAAAGGAACTTCTCAGAAGGTTTT-3' (SEQ ID NO: 15) and IL-12 p40-Nf-kB mut-R: 5'-AGAAGTTCCTTTTTTGTTTGTCTCTCTCTG- 3' (SEQ IDNO:16); IL-12 p40-PU-l mut-F: 5'-

ACAGAGAGAGACAAACAAAACTTCTTGAAAT-3' (SEQ ID NO:17) and IL-12 p40- PU-I mut-R: 5'- TTTTGTTTGTCTCTCTCTGTGTGTGTATCA-S' (SEQ ID NO:18).

Interestingly, inhibition of expression by Compound 2 was reduced in this mutant construct, indicating a role of NFKB. Since the transcription factor NFKB has been shown to be involved in the regulation of IL-12 p40 gene expression, we examined whether Compound 1 alters the binding of NFKB to its cognate site on the p40 promoter. Nuclear extracts were prepared from IFN-γ-primed THP-I cells that had been treated with or without SAC and incubated in the presence or absence of 1 μM Compound 1 or 10 mM ASA. Isolation of nuclear extracts was accomplished by first suspending THP-I cells in 20 volumes of buffer A containing 10 mM KCl, 10 mM HEPES (pH 7.9), 1 mM MgC12, 1 mM dithiothreitol (DTT), 0.1% Nonidet p40 (NP-40), and 0.5 mM phenylmethylsulfonyl fluoride (PMSF) and then homogenizing and centrifuging at 10,000 rpm at 4°C for 5 min. Nuclear pellets were then suspended in buffer C containing 400 mM NaCl, 20 mM HEPES pH 7.9), 15 mM MgCl₂, 0.2 mM EDTA, 1 mM DTT, 25% glycerol, 1 mM PMSF, and 10 μg of leupeptin, 20 μg of pepstatin, and 10 μg of antipain per ml, incubated for 30 min at 4°C, and centrifuge at 14,000 rpm for 20 min. The supernatants were dialyzed against buffer D containing 100 mM NaCl, 20 mM HEPES (pH 7.9), 20% glycerol, 1 mM PMSF, and 1 mM DTT.

The extracts obtained from this process were used in gel-shift assays using oligonucleotides containing the NFKB target sequence corresponding to the region -121 to - 102 from the transcription initiation site of IL-12 p40 or a mutated NFKB binding site. The binding of NFKB to the probe comprising its cognate sequence from the p40 promoter was strongly induced in IFN-γ/SAC-stimulated THP-I cells. This interaction was specific as it was competed away by an excess of unlabeled probe, but not by a mutated oligonucleotide in which two base-pairs were substituted. Compound 1 did not show any influence on NFKB binding. In contrast, ASA reduced the binding significantly, despite the fact that the percent inhibition of production of IL- 12 p70 protein elicited by 1 /ιM Compound 1 and 10 mM ASA were 97% and 45%, respectively. Combined with the lack of any effect of Compound 2 on IKB, these results show that the strong inhibitory activity of Compound 1 /Compound 2 on IL- 12 production is not due to a gross reduction in total NFKB binding activity.

To understand the action of Compound 2 in NF-κB binding, several NF-κB family members, p50, c-Rel and p6S were investigated using an ELISA based transcriptional factor-DNA binding activity assay system. DNA-transcription factor binding activities assays were performed with EZ-detect transcription Factor kit- NFKB p50 or p65 (Pierce, Rockford, IL), and BD Mercury TransFactor Kits -Nf-kB (BD Biosciences Clontech, Palo Alto, CA) according to the manufacturer's instruction.

The binding activities of p50, c-Rel and p65 were significantly increased in nuclear extracts from THP-I cells 3 hrs after IFN-YLPS stimulation. The binding activity of c-Rel was significantly decreased, and pSO was slightly decreased in the presence of Compound 2 (SOOnm) for 3 hrs. In the case of p65, the increased binding activity was observed in the presence of Compound 2 in response to the IFN-YLPS stimulation. This is a consequence of the lack of binding competition as a result of a decrease in pSO and c-Rel.

Example 3 : NF-kB proteins translocation

Our DNA-protein interaction study showed that the binding activity of c-Rel and p50, which form functional active heterodimers in IL-12, were decreased, and the binding activity of p65 was increased in response to Compound 2 treatment. In order to understand these changes, the subcellular localization of NF-κB proteins was investigated using western blot analysis. The amount of c-Rel and p50 protein in the nucleus were found to be decreased, and the amount of p65 in the nucleus was found significantly increased in cells treated with Compound 2 (50OnM) for 3 hr relative to untreated cells. This finding is in agreement with our DNA-protein interaction study, and indicated that the impaired activity of p50/c-Rel and increased p65 binding activity could cause the imbalance of the NF-κB proteins in the nucleus and affect the binding activity of p50/c-Rel. Example 4: Effect of Compound 2 on c-Rel and ICSBP (measuring the level of both in the nucleus)

Of the transcription factors that have been analyzed, two factors, ICSBP and c-Rel, seem to be affected by Compound I/Compound 2 treatment. ICSBP binds indirectly to the Ets-2 site. The primary NFKB trans-activator for IL- 12 is the c-Rel/p50 heterodimer. Other dimers (p65/p50 and p50/p50) either lack activity or have inhibitory functions. Thus, c-Rel plays a role in IL- 12 transcription as a result of both activation through NFKB and its interaction with ICSBP. Both Western blot analysis and DNA binding studies showed a decrease in nuclear c-Rel levels following Compound 2 treatment. As seen in Figure 4, a western blot assay of THPl nuclear c-Rel, p50 and p65 proteins was carried out by the following method: 10% SDS polyacrylamide gels (Invitrogen) were transferred to a Pure nitrocellulose membrane (BioRed, Hercules, CA). The membranes were blocked with 5% milk in TBST buffer and then incubated with anti-c-Rel, anti-p65, anti-p50, anti-ICSBP or anti-PU-1 antibody (all the antibodies were purchased from Santa Cruz) at a dilution of 1 :500 for 1 h at room temperature or overnight at 4°C. The membranes were washed and incubated with Horseradish Peroxidase-conjugated anti-rabbit IgG or anti-mouse IgG (Amersham, England) at a dilution of 1 :2000 at room temperature for 1 h.

Both IFN-γplus LPS and IFN-γplus SAC treatment strongly increased the amount of nuclear c-Rel, p65 and p50. Compound 2 treatment significantly reduced the levels of c- ReI, with the post-treatment nuclear c-Rel level being equal to or below the non-stimulated level. In contrast, nuclear p65 protein increased following Compound 2 treatment. p50 levels decreased slightly following Compound 2 treatment, but remained above the non- stimulated levels. Thus, it is shown that Compound 2 treatment causes a reduction in the amount of nuclear c-Rel/p50, the primary IL-12 activating NFKB dimer.

ICSBP, whose expression was reduced by Compound 2, was over-expressed using co-transfection with the IL-12 promoter-Luc report system. The over-expression construct of ICSBP was generated by PCR from cDNA of human PBMC using primers as follows: ICSBP-exp-F: 5'-CCGGAATTCAGGATGTGTGACCGGAATGG-S' (SEQ ED NO: 19) and ICSBP-exp-R: 5'-ATATCTAGAATGGATGCAGGACGCAGAC-S' (SEQ ID NO:20), the resulting PCR products was ligated to pCI vector (Promega). ICSBP over- expression increased the level of p40 expression and decreased the inhibition by Compound 2. Example 5: Effect of Compound 2 on IKB

IKB degradation is one of the steps in the signaling pathway of NFKB dependent genes. The activity of Compound 2 in inducible degradation of IκBαand IKBJS was investigated in THP-I cells using Western blot and FACS analysis. The amount of IκBα and IκB/3 in the cytoplasm of THP-I and RAW267.4 cells was significantly reduced at 30 min in response to induction by DFN-7/LPS or IFN-ySAC. However, there was no significant difference observed between the samples which were treated with or without Compound 2 (50OnM) at 30 min and 2 hrs. Similar results were observed from the Compound 2 pre- treatment samples in which Compound 2 was added 30 min before stimulation. These results show that Compound 2 does not induce the degradation of IκBα and IKB/? to allow free NFKB to translocate into the nucleus where it can act as a transcription factor.

Example 6: Measuring the level of Ets2 in the nucleus

The transcription factor ICSBP binds to the Ets-2 element indirectly through binding to PU-I . Nuclear extracts were bound to Ets-2 DNA element beads, the beads isolated to separate bound from free protein, and the proteins analyzed on Western blots using antibodies to either ICSBP or PU-I. Conjugation of Ets-2 DNA to beads was accomplished by the following method. Biotinylated DNA fragment encompassing the IL-12 p40 Ets-2 site (-292 to -196) were synthesized from the 1.3 kb wild-type human IL- 12 p40 reporter by PCR using a biotinylated primer as detailed in The Journal of Immunology, 2000, vol 165. pages 271-279. PCR products were purified by the Qiaquick Kit (Qiagen, Chatsworth, CA). Two μg of biotinylated DNA were conjugated to 100 μl of strptavidin-bound magnetic beads (Dynabeads, M280, Dynal, Lake Success, NY) in buffer containing 10 mM Tris-HCL, pH 8.0, 1 mM EDTA, 0.1 M NaCl. Ten μl of beads conjugated to 2 μg of DNA were equilibrated with TGEDN buffer (120 mM Tris-HCL, pH 8.0, 1 mM EDTA, 0.1 M NaCl, 1 mM DTT, 0.1% Triton X-100, 10% glycerol) and incubated with 500 μg of THP-I cell nuclear extracts and 20 μg of Herring sperm DNA (GibCo) at 4⁰C for 2 h. Beads were washed in TGEDN buffer, and bound materials were eluted in 20 μl of the same buffer supplemented with 0.5% SDS and 1 M NaCl. Eluted materials were separated by 10% SDS-PAGE and detected by immunoblot analysis using anti-ICSBP or anti-PU-1 antibody. Western blot analysis showed a significant reduction in the amount of ICSBP protein in nuclear extracts of THP-I cells treated with Compound 1, see Figure 5. In contrast, the levels of PU-I were unaffected by Compound 1 treatment, see Figure 6.

Of particular interest is the finding that both ICSBP and c-Rel were reduced in the nuclei of Compound 2 treated cells. Since these two transcription factors interact with each other, a decrease in the levels of both factors would be expected to have a profound effect. Compound 2 selectively inhibits expression of genes which are dependent upon the ICSBP- c-Rel interaction for trans-activation.

Example 7: Kinetics of the members of NF-κB nuclear translocation in Compound 2- treated cells

Compound 2 impairs nuclear accumulation of c-Rel and slightly reduces nuclear accumulation of p50. We examined the nuclear translocation kinetics of NF-kB family members in LPS stimulated cells treated with Compound 2. THPl cells were stimulated with LPS in either the presence or absence of 100 nM Compound 2, and the distribution of the NF-κB ReI family members was determined by immunoblotting nuclear (n.p.) extracts collected at 5 min, 15 min, 30 min, Ih, 3h and 6h post-treatment. In response to LPS stimulation, pSO translocated into the nucleus as early as 5 minutes post-stimulation and accumulates as time goes on (Figure 7, immunoblots and Figure 8 densitometry). Treatment of LPS-stimulated cells with Compound 2 had no effect on the kinetics of p50 nuclear entry at 5 minutes to 1 hr post-stimulation, and showed a small decrease in nuclear protein levels at 3 hours. The experiment examining p65 nuclear translocation is shown in Figure 9 (immunoblots) and Figure 10 (densitometry). In LPS stimulated cells, p65 translocated into the nucleus as early as S minutes post-stimulation and accumulated to maximum levels at 15-30 minutes post-stimulation. Treatment of LPS-stimulated cells with Compound 2 had no effect on the kinetics of p65 nuclear entry. The level of nuclear p65 at later times (6 hours) showed a small increase in Compound 2 treated cells relative to untreated cells.

Without wishing to be bound by theory, Compound 2 does not affect the kinetics of p50 and p65 nuclear accumulation in response to LPS stimulation. At later times, Compound 2 impairs nuclear accumulation of p50 (at 3 h time point), and enhances nuclear translocation of p65 (at 6 h time point), indicating a selective effect on the NF-κB family.

Example 8: The effects of Compound 2 on nuclear accumulation of p52 and ReI-B ReI B and p52 are two members of ReI family, which are preferentially complexed with each other. To determine the effect of Compound 2 on p52 and ReI-B nuclear accumulation, THPl cells were stimulated with IFNγ+LPS in either the presence or absence of 100 nM Compound 2, and the distribution of p52 and ReI-B was determined by immunoblotting of nuclear at 6h post-treatment. As shown in Figure 11 , the nuclear ReI-B was slightly increased in the presence of Compound 2. No significant difference was found in p52. This result indicates that Compound 2 specifically inhibits c-Rel and p50 nuclear accumulation, but not other NF-kB p52 and ReI-B nuclear accumulation.

Example 9: Inhibition of IL-23

Compound 2 inhibits the expression of p40 that is a subunit of both IL-12 and IL-23. Therefore, inhibition of BL-23 in addition to IL-12 is expected. In order to confirm the hypothesis, an assay was established to specifically detect EL-23 using polyclonal antibodies recognizing pl9 (R&D Systems, MN), an IL-23 specific subunit. A 96-well plate was coated with the antibodies at lμg/ml, and after washing incubated with the supernatants of human peripheral blood mononuclear cells (PBMC). The culture was stimulated with 1 μg/ml of liposaccharide (LPS) or 0.025% of S. aureus Cowan I (SAC) in the presence of test compound after IFN-γ priming. The captured IL-23 was then detected by a biotinylated goat anti-human p40 antibody that binds to p40 subunit of human IL-12 and IL-23 as a monomer or in the context of the respective heterodimer (Part 840099 of product DY1240 from R&D Systems). The plate was developed by incubation with streptavidin-HRP and then substrate solution (R&D Systems Cat # DY999). Recombinant IL-23 (R&D Systems) was added as standard in the assay. The estimated detection range is from 0.1 to 10ng/ml, and 1 ng/ml recombinant IL-12 heterodimer (Cell Sciences, MA) and p40 monomer (R&D Systems) were under detection limit. To compare with the inhibition of IL-23, the supernatant was also analyzed for IL-12 heterodimer and total p40 proteins using IL-12 specific ELISA kit (Cell Sciences) and p40 ELISA kit (R&D Systems), respectively. IL-23 was significantly induced in EFN- ySAC and IFN- yLPS-stimulated human PBMC, and was inhibited by Compound 2 in a dose-dependent manner. The inhibitory activity of Compound 2 against IL-23 was comparable to that against p40 and slightly lower than that against IL-12.

Example 10: Inhibition of IL-27 (Gene expression of peripheral blood mononuclear cells after treatment with a compound of the invention) Changes in gene expression patterns of peripheral blood mononuclear cells (PBMC) are studied using gene chip microarrays (Affyrnetrix, Inc.). PBMC are stimulated with IFNγ plus SAC, then dosed with 0.1, 1.0, 10, 100, or 1000 nM of a compound of the invention for 3 h. Control PBMC are stimulated with INFγ alone and IFNγ plus SAC. Changes in gene expression patterns between the control samples and the samples dosed with a compound of the invention are compared. In order to know the kinetics in the expression, PBMC with IFNySAC are further studied at different time points (20 min, 1.5 h, 3 h, 6 h, and 16 h) after the addition of the stimulus. In addition, PBMC preparations can be fractionated into T-cell enriched and monocyte-macrophage enriched populations, in order to distinguish the effects of a compound of the invention on these cell populations, following IFNySAC stimulation.

Genes preferentially expressed in monocyte/macrophage cells include first and foremost, those encoding the two subunits of IL- 12, p40 and p35. The kinetics of both subunits of IL- 12 showed the maximum expression at 6 h, which is in agreement with Northern blot analysis (data not shown). The expression of EBD is induced after stimulation with IFNγ/SAC, and dose-dependently inhibited by a compound of the invention. Since IL-27 is a heterodimer formed from subunits EBI3 and p28, its production will be inhibited by a compound of the invention. EBI3 shares 27% amino acid sequence homology with IL-12 p40 and p28 is a protein related to the p35 subunit of IL-12.

Example 11: Compound 2 Blocks accumulation of c-Rel, but not p65, in the nucleus of LPS stimulated cells.

We examined whether compound 2 can block the accumulation of c-Rel in the nucleus of cells induced by LPS (Figure 12). RAW264.7 cells cultured in DMEM with 10% BCS were split and seeded into 4-well chambered slides at 80,000 cells/well density. The cells were then treated with DMSO, Compound 2 (10OnM), LPS (Sigma, 5μg/mL), LPS (5μg/mL) + Compound 2 (100 nM) for 4 hours and fixed with 3% paraformaldehyde solution (1 x PBS) after 1 X quick rinse with 1 x PBS. Fixed cells were permeablized with 0.2% TXlOO and immunostained with anti-cRel antibody (SC70, Santa Cruz, 1 :200 dilution) or anti-NF-κB p65 antibody (SC 109, Santa Cruz, 1:100 dilution), and subsequently stained with Alexa Fluor 488 Goat-anti-Rabbit secondary antibody and DAPI (Molecular Probes, 1.1 μM). Images were obtained with CoolSNAP monochrome CCD camera on a Nikon inverted microscope TE300 using identical imaging parameters and were processed identically with Photoshop CS software. As observed previously, c-Rel localized to the cytoplasm in DMSO-treated cells and to the nucleus in LPS-treated cells. In the absence of LPS, Compound 2 treatment (4h) did not alter the nuclear/cytoplasmic distribution of c-Rel. Treatment of LPS-stimulated cells with Compound 2 inhibited the accumulation of c-Rel in the nucleus resulting in a striking reduction of nuclear c-Rel staining. These data demonstrate that Compound 2 blocks LPS-induced nuclear accumulation of c-Rel. We also examined whether Compound 2 blocks the nuclear accumulation of another NF-kB/Rel family member, p65, in LPS-stimulated RAW cells (Figure 13). As observed for c-Rel, p65 was localized to the cytoplasm in DMSO and Compound 2-treated cells and to the nucleus in LPS-stimulated cells. However, in contrast to c-Rel, p65 nuclear accumulation induced by LPS was not blocked by Compound 2. These data demonstrate the Compound 2 blocks c-Rel but not p65 nuclear translocation in LPS-stimulated cells.

Example 13: Compound 2 does not block phosphorylation of IKKβ

The phosphorylation of IKK is an early step in NF-κB activation. To determine whether Compound 2 inhibits the activation of the IKK complex, the level of phosphorylated IKKβ was investigated in drug-treated, LPS-stimulated cells. Whole cell extracts were prepared from THP-I cells that had been stimulated with IFNγ/LPS for 5min, 15 min 30 min and 1 hr in the either the absence or presence of 500 nM Compound 2. Phosphorylated IKKβ was determined by immunoblot analysis using an anti-phospho IKKβ antibody. As shown in Figure 14, the amount of phosphorylated IKKβ accumulated with time in response to IFNγ/LPS stimulation. Compound 2 treatment had no effect on the induction of phosphorylated IKKβ. These data demonstrate the Compound 2 does not block activation of the IKK complex.

Example 14: Compound 2 does not block LPS-induced phosphorylation of p6S or plO5/p5O NF-κB family members

We have showed that Compound 2 impairs LPS-induced phosphorylation of c-Rel (see Example 12). Next, we examined the effect of Compound 2 on LPS-induced phosphorylation of the NF-κB members p65 and plO5/p5O. THPl monocytic cells were stimulated with IFNγ plus LPS in the presence or absence of 100 nM Compound 2 (30 min, Ih and 3h) and whole-cell extracts were immunoblotted using anti-phospho p65 and plO5/p5O antibodies to detect the phosphorylated forms of these proteins. Figure 15 shows the effect of Compound 2 on p65 phosphorylation. LPS/IFNγ induced phosphorylation of p65 as early as 30 minutes on residues Ser-276, Ser-468 and Ser-927. Compound 2 had no effect on LPS/IFNγ induced phosphorylation at these sites. Figure 16 shows the effect of Compound 2 on pi 05 (the precursor of p50) phosphorylation. LPS/IFNγ induced phosphorylation of plO5 as early as 30 minutes on residues Ser-927 and Ser-933. Compound 2 had no effect on LPS/IFNγ induced phosphorylation at these sites. We conclude that Compound 2 does not interfere with signaling pathways that phosphorylate p65 and plO5/p5O in response to LPS/IFNγ stimulation.

Example 15: Compound 2 inhibits the accumulation of nuclear c-Rel in PMA plus ionomycin stimulated Jurkat T cells

We examined the accumulation of nuclear c-Rel in PMA+ionomycin stimulated Jurkat T cells by immunoblot analysis. As shown in Figure 17, in cells treated with 100 nM of Compound 2, the levels of nuclear c-Rel were reduced. As observed previously with other cell types, the nuclear levels of p50 were slightly reduced whereas nuclear p65 levels remained unchanged. These data demonstrate that Compound 2 is able to reduce nuclear c- ReI accumulation in T cells stimulated with PMA+ionomycin.

Example 16: Compound 2 reduces the DNA binding activity of nuclear c-Rel

In this study, we examined the effect of Compound 2 on the DNA-binding activity of nuclear c-Rel. The BD transfactor assay (a non-radioactive version of a super-shift assay) was used to measure the DNA-binding activity of c-Rel. In this assay, nuclear extracts are added to biotinylated double-stranded oligonucleotides containing the NF-κB binding site bound to a streptavidin 96-well plate. Detection of the transcription factor- DNA complex is performed with a specific primary antibody for c-Rel. The 96-well format allows for simultaneous measurement of multiple conditions and proteins using HRP- conjugated secondary antibodies whose enzymatic product can be measured using a luminometer. The level of c-Rel DNA-binding activity increased 40-fold (relative to DMSO control) in nuclear extracts from RAW cells stimulated with LPS/IFNγ. Compound 2 (1000 nM) treatment resulted in a 40% reduction in the level of c-Rel DNA-binding activity induced by stimulation with LPS/ IFNγ (Figure 18).

Methods: 2O x IO⁶ Raw 264.7 cells were treated with either DMSO, LPS/ IFNγ, or LPS/IFNγ/ Compound 2.(LPS cone. 1 ug/ml f.c. ; mouse IFNγ mouse 100U/ml f.c, Compound 2 1 uM f.c). (LPS: Sigma Cat # L2654. Mouse IFNγ; Cat # R+D 485-MICF). Cells were pre-treated with Compound 2 for 30 min, then LPS/ IFNγ was added. After 3 hrs, nuclear and cytoplasmic extracts were prepared according to the BD™ TransFactor Extraction Kit and user manual. Briefly, cells were washed in PBS, harvested and lysed in hypotonic lysis buffer on ice. Cells were then disrupted by drawing the cell suspension through a No.27 gauge needle 10 times. Next, the cell suspension was centrifuged, and the cytoplasmic extract (supernatant) was collected. The nuclear pellet was then disrupted by resuspension in high salt extraction buffer and was drawn through the needle 10 times. The suspension was centrifuged at high speed, and the nuclear extract was collected.

After measurement of protein concentration using BioRad assay, the nuclear extract was used in a Chemiluminescent NF-κB TransFactor Kit (BD) according to the user manual. Briefly, 2 ug of nuclear extract from either DMSO, LPS/ IFNγ or LPS/ IFNγ/ Compound 2 treated cells was incubated in the wells of a 96 well plate that was coated with biotin labeled NF-κB consensus ds oligos. After washing, kit provided c-Rel specific primary antibody at a 1:500 dilution was incubated in each well. After further washing, kit provided rabbit polyclonal secondary antibody was incubated in each well at a 1:10,000 dilution. Finally, amount of bound antibody to the plate was detected by incubation with chemiluminescent substrate and subsequent detection with a luminometer. The experiment was performed in duplicate.

Western Blot Method: After treatment with Compound 2, nuclear extract and cytoplasmic extract were prepared from 2OxIO⁶ Raw264.7 cells by using Extraction kits from BD Biosciences (Cat.631921), and above for experimental details. 20 μg of each extract was dissolved with 4x sample buffer and run on a 4-12% gradient SDS-PAGE gel, and blotted onto a nitrocellulose membrane by using semi-dry transfer. Non-specific binding to nitrocellulose was blocked with 5% skim milk in TBS with 0.5% Tween at room temperature for 1 hour, then probed with anti-c-Rel(C) mAb (rabbit IgG, SC-71) and anti- beta Actin(I-19) (goat IgG, sc-1616) as a control. HRP-conjugated goat anti-rabbit IgG (H+L) (#7074, Cell Signaling) and HRP-conjugated bovine anti-goat IgG (H+L) (sc-2350) were used as secondary Abs. LumiGLO reagent, 2Ox Peroxide (#7003, Cell Signaling) was used for visualization. Densitometry analysis was performed using Quantity One software from BioRad.

The reduction in c-Rel DNA-binding activity correlated with a 40-50% reduction in the levels of nuclear c-Rel as detected by immunoblot analysis (see immunoblot Figure 19 and densitometry Figure 20). Without wishing to be bound by theory, we believe that Compound 2 reduces the accumulation of c-Rel in the nucleus resulting in a concomitant decrease in c-Rel DNA-binding activity. Example 17: Compound 2 induces phosphorylation and inactivation of GSK3β in RAW cells

GSK3β is a serine-threonine protein kinase that is constitutively active in resting cells but can be inactivated through phosphorylation by other protein kinases. GSK3β regulates a multitude of transcription factors involved in cytokine production including NK- KB, NF-AT and CREB. To determine if Compound 2 regulates GSK3 phosphorylation, we stimulated mouse RAW cells (a mouse monocyte/macrophage-like cell line) with LPS (1 μg/mL (a ligand that activates TLR4) for 18h and measured the level of phosphorylated GSK3α and GSK3β by immunoblot analysis using a phospho-specifϊc antibody that recognizes pSer21 in GSK3α and pSer9 in GSK-3beta (Fig. 21). Stimulation of cells with LPS had no affect on the levels of phosphorylated GSK3α but slightly increased the levels of phosphorylated GSK3β. When the cells were stimulated in the presence of 100 nM Compound 2, the levels of phosphorylated GSK3β were greatly augmented with little or no affect on phosphorylated GSK3α levels. These data indicate that Compound 2 induces the phosphorylation and inactivation of GSK3β.

To determine when Compound 2 induces the phosphorylation of GSK3β after TLR stimulation, we treated RAW cells with 100 nM of Compound 2 in the presence or absence of LPS (1 μg/ml). Whole cell extracts were prepared 3, 6 and 8 hours after treatment, and immunoblotted using specific antibodies for GSK3β and phospho-GSK3β Ser9 (Fig. 22). GSK3β was phosphorylated following LPS treatment, and this effect is augmented by Compound 2 (upper panel). In this experiment, we did not observe a significant effect of Compound 2 or LPS on total levels of GSK3β (lower panel). Therefore, Compound 2 treatment induces the phosphorylation and inactivation of GSK3β at early times following LPS stimulation.

To determine whether Compound 2 alone could increase accumulation of phosphorylated GSK3β, we treated RAW cells with increasing amounts of Compound 2 in the presence or absence of LPS (1 μg/ml). Whole cell extracts were prepared 6 hours after treatment, and immunoblotted using specific antibodies for GSK3β and phospho-GSK3- beta Ser9 (Fig. 23). GSK3β is phosphorylated following LPS treatment, and this effect is augmented by Compound 2 (upper panel). Surprisingly, we also observed a dramatic and dose dependent increase in GSK3β phosphorylation in cells treated with Compound 2 alone. As in the above experiment, we did not observe a significant effect of Compound 2 or LPS on total levels of GSK3β (lower panel). This experiment indicates that Compound 2 treatment alone induces that phosphorylation and inactivation of GSIG β.

Example 18: Compound 2 induces phosphorylation and inactivation of GSK3β in human monocytes

To determine whether Compound 2 treatment similarly induced phosphorylation of GSK3β in a human monocyte-like cell line, we treated THP-I cells with LPS / IFNγ in the presence or absence of Compound 2 (0.5 μM). Whole cell extracts were prepared at 0, 0.5, 1, 3 and 6 hours after stimulation and immunoblotted with antibodies specific for GSK3β and phospho-GSK3β (Fig. 24). The upper panel shows that phospho-GSK3β phosphorylation increases in the presence of both Compound 2 and LPS/IFNγ. This effect begins at 1 hr. The total levels of GSK3β do not significantly change in response to Compound 2.

To determine if the inhibition of GSK3β by Compound 2 and LPS was similar to what was observed in RAW cells (namely that Compound 2 treatment alone can lead to GSK3β phosphorylation) we treated THP-I cells with increasing concentrations of Compound 2 (10 nM, 25 nM, 50 nM and 100 nM) in the presence or absence of LPS. Whole cell extracts were prepared at 6 hours after stimulation and immunobloted with antibodies specific for GSK3β and phospho-GSK3β (Fig. 25). The upper panel shows that there is a significant increase of GSK3β phosphorylation in the presence of Compound 2 even in the absence of LPS stimulation.

Example 19: Compound 2 phosphorylates GSK3β in a dose responsive manner

Compound 2 treatment of both transformed human monocytes and mouse macrophages results in phosphorylation (and thus inactivation) of GSK3β. In addition, we have shown that EL- 12 production is inhibited by as little as 10 nM Compound 2 in primary human monocytes. To determine whether GSK3β is also inhibited at these physiological levels, we performed a dose response experiment with Compound 2. Primary human monocytes were isolated from fresh whole blood, treated with ultrapure LPS and/or increasing doses of Compound 2 (10 nM, 25 nM, 50 nM and 100 nM) and whole cell extracts were prepared at 1 hour after LPS stimulation. These extracts were analyzed using immunoblotting with antibodies specific for total GSK3β, and phosphoGSK3β (Ser 9). In the absence of stimulation, there is a very low level of phosphorylated GSK3β; however, the levels of phosphoGSK3β increased significantly upon treatment with 50 nM Compound 2 treatment alone (see Figure 26). Accordingly, the combination of LPS and Compound 2 treatment results in increased phosphorylation of GSK3β, although Compound 2 alone seems a stronger inducer of GSK3β phosphorylation at this time point.

Example 20: Compound 2 modulates cytokine production in human monocytes

GSK3β has been shown to play an important role in TLR-mediated production of IL-12 and other cytokines. Interestingly, it has been reported that three GSK3β inhibitors, SB216763, azakenpaullone and BIO, inhibited IL-12p40, IL-6, and TNFa production in human monocytes stimulated with LPS, while augmenting the production of IL-10 (see Martin, et al, Nature Immunology (2005), <J(8):777-784). In order to test whether Compound 2 has a similar profile of effects in human monocytes stimulated with LPS alone (in the absence of IFNγ), we analyzed IL-12p40 and IL-10 production from monocytes stimulated with pure LPS (1 μg/ml) in the presence of either Compound 2 (0, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, or 300 nM), azakenpaullone (0, 3 nM, 10 nM, 30 nM, 100 nM, 300 nM, or 1000 nM) or BIO (0, 1 nM, 3 nM, 10 nM, 30 nM, 100 nM, or 300 nM) for 24h. The supernatants were collected analyzed by ELISA (R&D Systems, catalog #DY124O and DY217B). As expected, Compound 2 and each of the GSK3β inhibitors, azakenpaullone and BIO, diminished IL-12p40 production induced by LPS (Fig. 27). The GSK3β inhibitors also had a modest effect in augmenting IL-10 production at higher concentrations. Interestingly, Compound 2 clearly augmented IL-10 production in monocytes stimulated under these conditions.

Example 21: Compound 2 affects signaling through multiple toll-like receptor (TLR) signaling pathways in primary human monocytes

Human monocytes were isolated from peripheral blood through negative selection of other cell populations. To generate dendritic cells, isolated monocytes were cultured for 6 days in the presence of GM-CSF and IL-4. Monocytes or DC were cultured with Compound 2 (0, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM) and the following TLR stimuli in the presence of IFNγ for 24 h: a) FSL-I (TLR2/6) (1 μg/ml) b) SAC (TLR2/6) (0.05%) c) Pam3CSK4 (TLR2/1) (1 μg/ml) d) Flagellin (TLR5) (10 μg/ml) e) Loxoribine (TLR7) (500 μM) f) UPLPS (TLR4) (1 μg/ml)

Supernatants were collected and analyzed for IL-12/23p40 and IL-6 protein levels through ELISA analyses (R&D Systems, catalog #DY206). In human monocytes, three of the stimuli tested, SAC (TLR2), UPLPS (TLR4) and flagellin (TLR5), induced detectable IL-12/23p40. Compound 2 potently inhibited IL-12p40 activated by each of these three stimuli, with IC₅₀ values <2 nM (Fig. 28). In contrast, IL-6 induced by these same three stimuli was only slightly or not inhibited, with IC₅₀ values above 1000 nM.

Similarly, Compound 2 inhibited EL-12/23p40, but not IL-6 production in human DCs activated with SAC (TLR2), UPLPS (TLR4) and flagellin (TLR5) (Fig. 29). In addition, IL-12/23p40 and IL-6 production was detectable when DCs were stimulated with FSL-I (TLR2/TLR6), Pam3CSK4 (TLR2/TLR1) and loxoribine (TLR7) (Fig. 30). In each case, Compound 2 potently inhibited IL-12/23p40 induction. Thus, Compound 2 broadly affects IL-12/23p40 induction in monocytes and DC activated through TLRs. However, in contrast to other GSK3β inhibitors, such as azakenpaullone and BIO, which have been reported to reduce IL-6 production by 60-80% in LPS stimulated human monocytes, Compound 2 has only minor effects on IL-6 production.

Example 22: Compound 2 augments IL-10 production in human monocytes and dendritic cells stimulated with various Toll-like receptor ligands.

We tested the effects of Compound 2 on IL-10 production in human monocytes and monocyte-derived dendritic cells (DC) activated with ligands for TLR2, TLR3, TLR4, TLR5, TLR7, and TLR9 in combination with IFNγ. As described in Example 21, monocytes or DC were cultured with Compound 2 (0, 0.1 nM, 1 nM, 10 nM, 100 nM, or 1000 nM) and the following TLR stimuli in the presence of IFNγ for 24 h: a) FSL-I (TLR2/6) (1 μg/ml) b) SAC (TLR2/6) (0.05%) c) Pam3CSK4 (TLR2/1) (1 μg/ml) d) Flagellin (TLR5) (10 μg/ml) e) Loxoribine (TLR7) (500 μM) f) UPLPS (TLR4) (1 μg/ml) g) Poly(I:C) (TLR3) (25 μg/ml) h) CpG (TLR9) (10 μg/ml) Supematants were collected and analyzed for IL-IO protein levels through ELISA analyses (R&D Systems, catalog # DY217B). Compound 2 augmented IL-IO production in human monocytes stimulated with LPS (TLR4), flagellin (TLR5) and PAM3CSK4 (TLR2/TLR1) (Fig. 31). No augmentation of IL-10 was observed when cells were stimulated with FSL-I (TLR2/TLR6). Loxoribine (TLR7), poly (I:C) (TLR3), and CpG (TLR9), did not induce detectable levels of IL-10 on their own, and Compound 2 did not increase IL-10 production in the presence of these stimuli. In contrast, Compound 2 inhibited IL-10 induced with SAC.

In human monocyte-derived DC, Compound 2 substantially augmented IL-10 induced through most TLR pathways, including TLR4, TLR5, TLR2/TLR6 and TLR2/TLR1 (Fig. 32). The activation of some signaling pathways (TLR7, TLR3, and TLR9) did not induce detectable amounts of IL-10, and Compound 2 did not induce IL-10 production in the presence of these stimuli. As in the case of monocytes, DL-IO induced by one stimulus, SAC, was substantially inhibited by Compound 2.

These findings support an overall pattern wherein Compound 2 augments IL-10 production in the context of TLR signaling, though the magnitude of this effect varies somewhat with the nature of the stimulus and the cell lineage (more robust augmentation of IL-10 was seen in DCs than in monocytes). Interestingly, SAC is the only stimulus tested where Compound 2 had the opposite effect of inhibiting IL-10 production. Unlike the other highly purified and defined TLR ligands used in this study, SAC, as a bacterial cell wall extract, is a complex mixture of stimuli and likely activates multiple signaling pathways. It appears that the mechanism by which SAC stimulates IL-10 is distinct from stimulation through single defined TLR pathways and this obviates the ability of Compound 2 to augment IL-10 production.

Example 23: Compound 2 induces the accumulation of nuclear ERK in RAW cells

A second pathway activated by TLRs that negatively regulates IL- 12 production is the ERK signaling pathway. ERK is mitogen-activated protein (MAP) kinase that is phosphorylated and activated in response to TLR signaling. RAW267.4 cells were seeded and cultured in chambered cover-slides for 24 hrs and subsequently treated with DMSO (1 : 1000 dilution), or 100 nM Compound 2 for 2 hr, 4 hr and 6 hr. Cells were fixed in 3% paraformaldehyde for 10 min, and immunostained with Rabbit anti-ERK antibody followed by staining with secondary FITC conjugated antibody against Rabbit. Fluorescence was imaged under inverted microscope Nikon 2000E (objective 6Ox) with CoolSnap HQ camera and Metamorph software. Images were processed using Metamorph software and Photoshop CS software. As can be seen from Fig. 33, Compound 2 treatment of RAW267.4 cells induced the accumulation of nuclear ERK (see arrows, Fig. 33).

Example 24: Compound 2 induces ERK1/2 phosphorylation in mouse RAW cells.

RAW cells were treated with 100 nM or 500 nM Compound 2 or a control compound, N-(5-methyl-pyridin-3-ylmethyl)-N'-[6-morpholin-4-yl-2-(2-pyridin-2- ylethoxy)-pyrimidin-4-yl]-hydrazine, a structurally similar but functionally inactive derivative of Compound 2. Whole cell extracts were prepared at 0, 0.5, 1, and 3 hours following treatment and analyzed by immunoblotting with antibodies specific for: phosphoERK 1 and 2 and unphosphorylated (total) ERK 1 and 2. The results are shown in Fig. 34. In unstimulated cells, there was a small amount of phosphorylated ERK (upper panel). However, upon treatment with Compound 2, we observed that ERK 1 and 2 are strongly phosphorylated in a time and dose dependent manner, (see 3 hrs, both 0.1 and 0.5 μM). At 3 hours, a strong band was observed for both phosphoERK 1 and 2 (upper panel). This increase in phosphorylation was not observed in cells that were treated with 0.5 μM of the control compound, demonstrating the specificity of the effect. Additionally, we did not observe changes in absolute amounts of unphosphorylated ERK (lower panel), indicating that Compound 2 affects the post-translational modification of ERK

Example 25: Role of MEK1/2 and ERK in effects of Compound 2 on IL-10 and EL- 12p40 production by human monocytes

ERK has been reported to positively regulate IL-10 production upon phosphorylation, raising the possibility that the augmentation of IL-10 by Compound 2 observed in LPS-activated monocytes may be due in part to the increased activation of ERK. We tested the effect of an inhibitor of ERK phosphorylation (a MEK1/2 inhibitor) on IL-10 production in monocytes. The MEK1/2 inhibitor U0126 caused a partial reduction in the amount of IL-10 induced in LPS-stimulated monocytes (Fig. 35a) (LPS lμg/ml; 24 h treatment Compound 2 or U0126 and LPS; ELISA kit, R&D Systems, catalog # DY217B). Under the same conditions, Compound 2 augments IL-10 production. In combination with 100 nM Compound 2, U0126 (lOμM) reduced the levels of IL-10 production to levels observed in monocytes stimulated with LPS alone, suggesting that the augmentation of IL- 10 by Compound 2 may function through its ability to stimulate phosphorylation and thereby activate ERK (Fig. 35b, upper panel). Interestingly, the inhibition of IL-12p40 by Compound 2 does not involve activation of the MEK1/2-ERK pathway, since IL-12p40 levels were inhibited by Compound 2 in the presence or absence of UO 126 (Fig. 35b, lower panel).

Example 26: Inhibition of ERK signaling counteracts the induction of IL-10, but does not reverse the inhibition of IL-12p40 by Compound 2 in human dendritic cells

In two separate experiments (LPS lμg/ml; 24 h treatment Compound 2 or UO 126 and LPS. ELISA kit, R&D Systems, catalog # DY217B), we observed a strong 3-5 fold enhancement of IL-10 production when DC were activated with LPS in the presence of 100 nM Compound 2 (Fig. 36). Strikingly, addition of U0126 (10 μM) reduced IL-10 levels nearly to the levels present in cells activated with LPS alone. Therefore, the mechanism by which Compound 2 increases IL-10 production is mediated at least in part through its ability to activate ERK.

We previously observed that in human monocytes, while UO 126 reverses the modulation of EL-IO by Compound 2, it does not affect the inhibition of DL-12p40 by Compound 2. We tested whether U0126 would have an effect on the inhibition of EL-12p40 production by Compound 2 in DCs. Consistent with our previous findings, at concentrations up to 10 /.M, UO 126 did not relieve the inhibition of IL-12p40 by Compound 2 (Fig. 37) (LPS lμg/ml; 24 h treatment Compound 2 or U0126 and LPS; ELISA kit, R&D Systems, catalog # DY1240). Therefore, the inhibition of IL-12p40 does not appear to be mediated by activation of ERK kinase.

Example 27: MEK1/2 inhibition does not affect Compound 2 induced GSK3β phosphorylation

ERKl/2 has been reported to be linked to phosphorylation of GSK3β. Since we have observed that treatment of human monocytes and DC with Compound 2 also causes phosphorylation of GSK3β, we used a MEK inhibitor, UO 126, to investigate the effect of inhibition of ERK phosphorylation on GSK3β phosphorylation. MEK is a kinase that phosphorylates and activates ERK. RAW cells were treated with Compound 2, UO 126, or both, and whole cell extracts were purified at 3 and 6 hours after treatment. Examination of GSK3β phosphorylation was performed using antibodies specific for phosphoGSK3β(Ser9) (see Figure 38). In the absence of treatment, GSK3β is not phosphorylated, however, following treatment with Compound 2 levels of phospho-GSK3β are dramatically increased in a dose dependent manner. Addition of the MEK inhibitor, UO 126, does not impair this phosphorylation, even at a dose of 10 μM. Therefore, MEK signaling does not appear to play a role in phosphorylation of GSK3β by Compound 2. Using the same whole cell extracts, we examined levels of phosphoERKl/2, to confirm that the MEK inhibitor, UO 126, was functional in these experiments. As in Example 24, we observed that Compound 2 treatment alone results in increased phosphorylation of ERK1/2, and that this was inhibited by U0126. Total ERK1/2 levels were unaffected (see Figure 39). This confirms that ERX is phosphorylated by Compound 2 treatment alone, and that UO 126 blocks this phosphorylation, suggesting that Compound 2 functions through MEK to phosphorylate ERK.

Compounds 3-14 are expected to have similar activity as Compounds 1 and 2 in the procedures described in the above Examples.

Many modifications and variations of this invention can be made without departing from its spirit and scope, as will be apparent to those skilled in the art. The specific embodiments described herein are offered by way of example only, and the invention is to be limited only by the terms of the appended claims, along with the full scope of equivalents to which such claims are entitled. Such modifications are intended to fall within the scope of the appended claims.

All references, patent and non-patent, cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual publication or patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety for all purposes.

All of the features, specific embodiments and particular substituents disclosed herein may be combined in any combination. Each feature, embodiment or substituent disclosed in this specification may be replaced by an alternative feature, embodiment or substituent serving the same, equivalent, or similar purpose. In the case of chemical compounds, specific values can be combined in any combination resulting in a stable structure. Furthermore, specific values (whether preferred or not) for substituents in one type of chemical structure may be combined with values for other substituents (whether preferred or not) in the same or different type of chemical structure. Thus, unless expressly stated otherwise, each feature, embodiment or substituent disclosed is only an example of a generic series of equivalent or similar features feature, embodiments or substituents.

Claims

1. A method of identifying a compound that inhibits IL- 12p40 production in a cell after proinflammatory stimulation, comprising: a) contacting the cell with one or more candidate compounds; b) measuring the amount of Ser9 phosphorylated GSK3β in cells contacted with the candidate compound and in cells not so contacted; c) measuring the amount of c-Rel in the nucleus of cells contacted with the candidate compound and in cells not so contacted after proinflammatory stimulation; d) comparing the amount of Ser9 phosphorylated GSK3β and nuclear c-Rel in cells contacted with the candidate compound with cells not so contacted, wherein an increase in phosphorylated GSK3β and a decrease in nuclear c- ReI in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound inhibits IL-12p40 production.

2. The method of Claim 1, wherein the amount of Ser9 phosphorylated GSK3β is measured before proinflammatory stimulation.

3. The method of Claim 1, wherein the amount of Ser9 phosphorylated GSK3β is measured after proinflammatory stimulation.

4. The method of Claim 1 , wherein the cells are contacted with the candidate compound before proinflammatory stimulation.

5. The method of Claim 1 , wherein the cells are contacted with the candidate compound after proinflammatory stimulation.

6. The method of Claim 1, wherein the candidate compound inhibits IL-23 production.

7. The method of Claim 1, wherein the candidate compound inhibits IL-12 production.

8. The method of Claim 1, further comprising the steps of: e) measuring the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the candidate compound and in cells not so contacted; and f) comparing the amount of Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the candidate compound with cells not so contacted, wherein a decrease in Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound inhibits IL-12p40 production.

9. The method of Claim 1, wherein the candidate compound increase the production of IL-10.

10. The method of Claim 9, further comprising the steps of: e) measuring the amount of phosphorylated ERK in cells contacted with the candidate compound and in cells not so contacted; and f) comparing the amount of phosphorylated ERK in cells contacted with the candidate compound with cells not so contacted, wherein an increase in phosphorylated ERK in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound increase IL- 10 production.

11. A compound that inhibits IL- 12p40 production in a cell after proinflammatory stimulation, wherein the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation, provided that the compound is not a compound disclosed in a patent or patent application listed in Table 1.

12. The compound of Claim 11, the compound decreases the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted.

13. The compound of Claim 11 , wherein the compound increases the production of IL- 10 in cells contacted with the compound relative to cells not so contacted.

14. The compound of Claim 13, wherein the compound increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted.

15. The compound of Claim 13, wherein the compound increases the amount of nuclear ERK in cells contacted with the compound relative to cells not so contacted.

16. A method of decreasing the level of IL-12p40 in a subject comprising administering to the subject a compound that increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation, provided that the compound is not a compound disclosed in a patent or patent application listed in Table 1.

17. The method of Claim 16, wherein the compound decreases the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted.

18. The method of Claim 16, wherein the compound increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted.

19. The method of Claim 18, wherein the compound increases the level of IL-IO in the subject.

20. A method of treating an EL-12, IL-23, or IL-27 production-related disease or disorder in a subject comprising administering to the subject an effective amount of a compound that increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted; and decrease the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation, provided that the compound is not N- (3-methyl-benzylidene)-N'-[6-morpholin-4-yl-2-(2-pvridin-2-yl-ethoxy)-pyrimidin- 4-yl]-hydrazine.

21. The method of Claim 20, wherein the compound decreases the amount of Ser 129 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted.

22. The method of Claim 20, wherein the compound increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted.

23. The method of Claim 22, wherein the compound increases the level of IL- 10 in the subject.

24. The method of Claim 20, wherein the IL- 12, IL-23, or IL-27 production-related diseases or disorders is selected from the group consisting of multiple sclerosis, sepsis, myasthenia gravis, autoimmune neuropathies, Guillain-Barre syndrome, autoimmune uveitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, temporal arteritis, anti-phospholipid syndrome, vasculitides, Wegener's granulomatosis, Behcet's disease, psoriasis, psoriatic arthritis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo, Crohn's disease, ulcerative colitis, interstitial pulmonary fibrosis, myelofibrosis, hepatic fibrosis, myocarditis, thyroditis, primary biliary cirrhosis, autoimmune hepatitis, immune- mediated diabetes mellitus, Grave's disease, Hashimoto's thyroiditis, autoimmune oophoritis and orchitis, autoimmune disease of the adrenal gland, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, common variable immunodeficiency (CVID), polymyositis, dermatomyositis, spondyloarthropathies, ankylosing spondylitis, Sjogren's syndrome and graft-versus- host disease.

25. The method of Claim 20, wherein the IL-12, IL-23, or IL-27 production-related diseases or disorders is selected from the group consisting of rheumatoid arthritis, sepsis, Crohn's disease, multiple sclerosis, psoriasis, psoriatic arthritis, or immune- mediated diabetes mellitus.

26. A method of identifying a compound that decreases IL-12p40 production in cells after proinflammatory stimulation and increases EL-IO production, comprising: a) contacting the cell with one or more candidate compounds; b) measuring the amount of c-Rel in the nucleus of cells contacted with the candidate compound and in cells not so contacted after proinflammatory stimulation; c) measuring the amount of phosphorylated ERK in cells contacted with the candidate compound and in cells not so contacted; d) comparing the amount of nuclear c-Rel and phosphorylated ERK in cells contacted with the candidate compound with cells not so contacted, wherein a decrease in nuclear c-Rel and an increase in phosphorylated ERK in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound decreases the production of IL12-p40 and increase the production of IL-10.

27. The method of Claim 26, wherein the amount of phosphorylated ERK is measured before proinflammatory stimulation.

28. The method of Claim 26, wherein the amount of phosphorylated ERK is measured after proinflammatory stimulation.

29. The method of Claim 26, wherein the cells are contacted with the candidate compound before proinflammatory stimulation.

30. The method of Claim 26, wherein the cells are contacted with the candidate compound after proinflammatory stimulation.

31. The method of Claim 26, wherein the candidate compound inhibits IL-23 production.

32. The method of Claim 26, wherein the candidate compound inhibits IL-12 production.

33. The method of Claim 26, further comprising the steps of: e) measuring the amount of Ser9 phosphorylated GSK3β in cells contacted with the candidate compound and in cells not so contacted; and f) comparing the amount of Ser9 phosphorylated GSK3β in cells contacted with the candidate compound with cells not so contacted, wherein an increase in phosphorylated GSK3β in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound inhibits IL-12p40 production.

34. The method of Claim 33, further comprising the steps of: g) measuring the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the candidate compound and in cells not so contacted; and h) comparing the amount of Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the candidate compound with cells not so contacted, wherein a decrease in Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with a candidate compound relative to cells not so contacted indicates that the candidate compound decrease IL-12p40 production.

35. A compound that decreases IL-12p40 production in a cell after proinflammatory stimulation and increase IL-IO production, wherein the compound decreases the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation; and increases phosphorylated ERK in cells contacted with the compound relative to cells not so contacted, provided that the compound is not a compound disclosed in a patent or patent application listed in Table 1.

36. The compound of Claim 35, wherein the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted.

37. The compound of Claim 36, the compound decreases the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted.

38. The compound of Claim 35, wherein the compound increases the amount of ERK in the nucleus of cells contacted with the compound relative to cells not so contacted.

39. A method of decreasing the level of IL-12p40 and increasing the level of IL-10 in a subject comprising administering to the subject a compound that decreases the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation; and increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted, provided that the compound is not a compound disclosed in a patent or patent application listed in Table 1.

40. The method of Claim 39, wherein the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted.

41. The method of Claim 40, wherein the compound decreases the amount of Serl29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted.

42. A method of treating an IL-12, IL-23, or IL-27 production-related disease or disorder in a subject comprising administering to the subject an effective amount of a compound that decreases the amount of nuclear c-Rel in cells contacted with the compound relative to cells not so contacted after proinflammatory stimulation; and increases the amount of phosphorylated ERK in cells contacted with the compound relative to cells not so contacted, provided that the compound is not N-(3-methyl- benzylidene)-N'-[6-morpholin-4-yl-2-(2-pyridin-2-yl-ethoxy)-pyrimidin-4-yl]- hydrazine.

43. The method of Claim 42, wherein the compound increases the amount of Ser9 phosphorylated GSK3β in cells contacted with the compound relative to cells not so contacted.

44. The method of Claim 43, wherein the compound decreases the amount of Serl 29 phosphorylated CREB341 and/or Serl 15 phosphorylated CREB327 in cells contacted with the compound relative to cells not so contacted.

45. The method of Claim 42, wherein the IL-12, IL-23, or IL-27 production-related diseases or disorders is selected from the group consisting of multiple sclerosis, sepsis, myasthenia gravis, autoimmune neuropathies, Guillain-Barre syndrome, autoimmune uveitis, autoimmune hemolytic anemia, pernicious anemia, autoimmune thrombocytopenia, temporal arteritis, anti-phospholipid syndrome, vasculitides, Wegener's granulomatosis, Behcet's disease, psoriasis, psoriatic arthritis, dermatitis herpetiformis, pemphigus vulgaris, vitiligo, Crohn's disease, ulcerative colitis, interstitial pulmonary fibrosis, myelofibrosis, hepatic fibrosis, myocarditis, thyroditis, primary biliary cirrhosis, autoimmune hepatitis, immune- mediated diabetes mellitus, Grave's disease, Hashimoto's thyroiditis, autoimmune oophoritis and orchitis, autoimmune disease of the adrenal gland, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic lupus erythematosus, scleroderma, common variable immunodeficiency (CVID), polymyositis, dermatomyositis, spondyloarthropathies, ankylosing spondylitis, Sjogren's syndrome and graft-versus- host disease.

46. The method of Claim 42, wherein the IL- 12, IL-23, or IL-27 production-related diseases or disorders is selected from the group consisting of rheumatoid arthritis, sepsis, Crohn's disease, multiple sclerosis, psoriasis, psoriatic arthritis, or immune- mediated diabetes mellitus.