BACKGROUND OF THE INVENTION

[0002] This invention relates to processes for inducing, preventing, or suppressing activation of major histocompatibility complex (MHC) class I and class II molecules, other molecules involved in antigen presentation and the immune recognition process, molecules controlling the -growth and function of cells, and to the products identified for inhibiting, or enhancing, the processes. This allows manipulation of the immune system, particularly for conditions and diseases characterized as involving abnormal or aberrant regulation of the immune recognition system on normal cells, wherein they are converted to antigen presenting cells (APCs) and cause bystander activation of immune cells. This also allows manipulation of regulation of the immune recognition system on lymphocytes and antigen presenting cells of the host immune defense system. These processes are important for the development of immune response to viruses, bacteria, environmental agents which damage tissues, and oncogene-transformation. They are involved in the immune recognition process developing during gene therapy and vaccinations and are part of a normal host defense system. They coordinately control the growth, apoptosis, and function of cells to maintain the normal homeostatic balance of the cell driving the host defense process.

[0003] An important function of the immune system is to discriminate self from non-self antigens and to eliminate the latter. In addition, tolerance must be achieved so that the immune system does not attack itself or other normal tissues of the body. This recognition by the immune system involves complex cell-cell interactions and depends primarily on lymphocytes (e.g., B and T cells) and antigen-presenting cells (“APC”) (e.g., macrophages and dendritic cells).

[0004] The immune response is mediated by molecules encoded by the MHC which contains polymorphic genetic loci encoding an immune superfamily of structurally- and functionally-related products (D. P. Stites & A. I. Terr (eds), “ Basic and Clinical Immunology ” Appelton and Lange, Norwalk, Connecticut/San Mateo, Calif., (1991)). Recognition by a lymphocyte, through its antigen-MHC receptor of antigen presented in a complex with MHC on the antigen-presenting cell, may then trigger an activation program in the lymphocyte and/or secretion of effector substances by the lymphocyte. The two principal classes of MHC molecules, Class I and Class II, each comprise a heterodimer of glycoproteins expressed on the cell surface. Class I molecules are found on virtually all somatic cell types, although they are expressed at different levels in different cell types. In contrast, Class II molecules are normally expressed only on a few cell types, such as lymphocytes, macrophages, and dendritic cells.

[0005] The Class I molecule is generally comprised of an MHC gene product (e.g., HLA-A, B and C loci encoding the heavy chain of Class I) and β2-microglobulin, which is encoded by a non-MHC gene; the Class II molecule is generally comprised of two MHC gene products (e.g., HLA-DP, DQ and DR loci encoding α and β chains of Class II). Furthermore, non-covalently associated polypeptides (e.g., chaperone proteins and invariant chain) are encoded by non-MHC genes. Determination of the three-dimensional protein structure of MHC molecules by X-ray crystallography shows that although the genetic organizations of Class I and Class II genes are disparate, the protein structures of the different MHC molecules are similar with an antigen-binding pocket lined by polymorphic amino acid residues.

[0006] Antigens together with MHC molecules are presented to the immune system. (J. Klein & E. Gutze, “ Major Histocompatibility Complex ,” Springer Verlag, New York, 1977; E. R. Unanue, Ann. Rev. Immunology 2: 295-428, (1984)). For example, an endogenous antigen or a peptide sequence from a virus infecting a cell and expressing viral genes therein, may bind to the Class I molecule while exogenous antigen, e.g., a peptide sequence from an immunogen taken up by an antigen presenting cell and metabolized therein, may bind to the Class II molecule. The chemical structure of a peptide (e.g., length, amino acid composition, post-translational modification) will determine whether it can be processed and transported by the cell, and bound to the MHC molecule. Processing and transport of Class I related peptides involves, but is not limited to, proteasomes and transporters of antigen peptides (TAP) molecules among other cell organelles and proteins (I. A. York & K. L. Rock, Annu. Rev. Iminunol. 14: 369-96 (1996)). Processing and expression of Class II related peptides involves, but is not limited to, invariant chain and HLA-DM molecules (J. Pieters, Curr. Opin. Immunol. 9: 89-96 (1997)). Controlling the cell-surface expression of an antigen-MHC complex by normal cells or regulating antigen-presenting cells at any point in the pathway producing such complexes (e.g., transcription, translation, post-translational modification, and folding of MHC polypeptides; production of peptide, which are able to bind an MHC molecule, from antigen through intracellular biosynthetic or degradative processes; transport of peptide into an organelle where binding to an “empty” MHC molecule can occur) will affect lymphocyte recruitment, maturation, differentiation, and activation through receptor-mediated recognition of the antigen-MHC complex.

[0007] CD4 is the receptor recognizing the Class II cell-surface molecule and CD4 ⁺ T lymphocytes (usually helper T cells) recognize antigens presented in association with Class II gene products. CD8 is the receptor recognizing the Class I cell-surface molecule and CD8 ⁺ T lymphocytes (usually cytotoxic T cells or CTL) recognize antigens in association with Class I gene products. In addition, co-receptors (e.g., CD28 or CTLA-4 on the lymphocyte, and CD80/B7-1 or CD86/B7-2 on the antigen presenting cell) will affect the activation status of an immune cell recognizing cognate antigen. Signalling through such receptors is integrated within the cell and determines the immune response of the individual cell, such as by secretion of substance that can mediate an immune response. Helper T cells are classified as Th1 or Th2 depending on the types of substances secreted during the immune response; those substances may promote the growth and/or differentiation of the target cell or immune cells recognizing the target cell. Cytotoxic T cells secrete compounds that may form pores in the target cell and degrade its contents. Thus cell-cell communication in the immune system may be accomplished through receptor-ligand interactions by cells in direct contact or at a distance.

[0008] It had been believed that Class I molecules function primarily as the targets of the cellular immune response, whereas Class II molecules regulate both the humoral (antibody mediated) and cellular immune response (J. Klein & E. Gutze, ibid. (1977)). MHC molecules have been the focus of much study with respect to research in autoimmune diseases because of their roles as mediators or initiators of the immune response. Class II molecules have been the primary focus of research in the etiology of autoimmune diseases, whereas Class I molecules have historically been the focus of research in transplantation rejection. But the present invention envisions a role for both classes of MHC molecule in host defense mechanism leading to autoimmunity.

[0009] Numerous experimental animal models for human disease have linked aberrant expression and/or function of MHC Class I and MHC Class II antigens to the autoimmune disease process, for example, insulin-dependent diabetes mellitus (IDDM) (Tisch and McDevitt, Cell 85: 291-297 (1996)), systemic lupus erythematosus (SLE) (Kotzin, Cell 85: 303-306 (1996)), uveoretinitis (Prendergast, et al., Invest. Opthalmol. Vis. Sci. 39: 754-762 (1998)), and Graves' disease (L. D. Kohn, et al., Intern. Rev. Immunol. 9: 135-165 (1992)), L. D. Kohn, et al., in Thyroid Immunity (D. Rayner and B. Champion (Eds.), R.G. Landes Biomedical Publishers, Austin/Georgetown, Tex., pp. 115-170 (1995)).

[0010] The pathological link between MHC Class I and/or Class II expression and disease has been examined in many of these model systems using a variety of biochemical and genetic approaches. One important piece of evidence for aberrant MHC gene function as a mediator of autoimmune disease stems from transgenic animal models in which the MHC genes have been inactivated. Using MHC Class I deficient animals, resistance to the autoimmune disease process and hence the dependence of autoimmunity upon MHC gene expression can be directly demonstrated in animal models for IDDM (Serreze, et al., Diabetes 43: 505-509 (1994)), and SLE (E. Mozes, et al., Science 261: 91-93 (1993)).

[0011] Systemic lupus erythematosus (SLE) is a chronic autoimmune disease that, like Graves' disease, has a relatively high rate of occurrence. SLE affects predominantly women, the incidence being 1 in 700 among women between the ages of 20 and 60 (A. K. Abbus, et al., (eds), “ Cellular and Molecular Immunology, ” W. B. Saunders Company, Philadelphia, pp. 360-370 (1991)). SLE is characterized by the formation of a variety of autoantibodies and by multiple organ system involvement (D. P. Stites & A. I. Terr, ibid, pp. 438-443 (1991)). Current therapies for treating SLE involve the use of corticosteroids and cytotoxic drugs, such as cyclosporin. Immunosuppressive drugs, such as cyclosporin, FK506 or rapamycin suppress the immune system by reducing T cell numbers and function (P.J. Morris, Curr. Opin. in Immun. 3: 748-751 (1991)). While these immunosuppressive therapies alleviate the symptoms of SLE and other autoimmune diseases, they have numerous severe side effects. In fact, extended therapy with these agents may cause greater morbidity than the underlying disease. A link between MHC Class I expression and SLE in animal models has been established. Thus, Class I deficient mice do not develop SLE in the 16/6 ID model (E. Mozes, et al., Science 261: 91-93 (1993)).

[0012] Diabetes Mellitus (DDM) is a disease characterized by relative or absolute insulin deficiency and relative or absolute glucagon excess (D. W. Foster, in J. B. Stanbury, et al., The Metabolic Basis of Inherited Disease , vol. 4, pp. 99-117 (1960)). Type I diabetes appears to require a permissive genetic background and environmental factors. Islet cell antibodies are common in the first months of the disease. They probably arise in part to β cell injury with leakage cell antigens but also represent a primary autoimmune disease. The preeminent metabolic abnormality in Type I diabetes is hyperglycemia and glucosuria. Late complications of diabetes are numerous and include increased atherosclerosis with attendant stroke and heart complications, kidney disease and failure, and neuropathy, which can be totally debilitating. The link to HLA antigens has been known since 1970. Certain HLA alleles are associated with increased frequency of disease, others with decreased frequency. Increased MHC Class I and aberrant MHC Class II expression in islet cells has been described (G. F. Bottazzo, et al., N. Eng. J. Med. 313: 353-360 (1985), Foulis and Farquharson, Diabetes 35: 1215-1224 (1986)). A definitive link to MHC Class I has been made in a genetic animal model of the disease. Thus, MHC Class I deficiency results in resistance to the development of diabetes in the NOD mouse (Serreze, et al., Diabetes 43: 505-509 (1994), L. S. Wicker, et al., Diabetes 43: 500-504 (1994)).

[0013] The dependence of the progressive multifocal inflammatory autoimmune disease phenotype exhibited by TGF-beta deficient transgenic mice (Shull, et al., Nature 359: 693-699 (1992); Kulkarni, et al., Proc. Natl. Acad. Sci. U.S.A. 90: 770-774 (1993); Boivin, et al., Am. J. Pathol. 146: 276-288 (1995)) on MHC Class II expression has recently been demonstrated using MHC Class II deficient animals. Specifically, TGF-beta deficient animals lacking MHC Class II expression are without evidence of inflammatory infiltrates, circulating antibodies, or glomerular immune complex deposits (Letterio, et al., J. Clin. Invest. 98: 2109-2119 (1996)).

[0014] Additional evidence supportive of MHC Class I and Class II antigens on target tissues as critical for the development of autoimmunity in animal models has been demonstrated in over-expression experiments.

[0015] Graves' disease (GD) is a relatively common autoimmune disorder of the thyroid. In Graves' disease, autoantibodies against thyroid antigens, particularly the thyrotropin receptor (TSHR), alter thyroid function and result in hyperthyroidism (D. P. Stites & A. I. Terr (eds), “ Basic and Clinical Immunology ” Appleton and Lang, Norwalk, Conn./San Mateo, Calif., pp. 469-470 (1991)). Thyrocytes from patients with GD have aberrant MHC Class II expression and elevated MHC Class I expression (T. Hanafusa, et al., Lancet 2: 1111-1115 (1983); G. F. Bottazzo, et al., Lancet 2: 1115-1119 (1983); L. D. Kohn, et al., Int. Rev. Immunol. 912: 135-165 (1992)).

[0016] Numerous attempts to develop a GD model by immunizing animals with the extracellular domain of the thyrotropin receptor (TSHR) have largely failed (G. S. Seetharamaiah, et al., Autoimmunity 14: 315-320 (1993); S. Costagliola, et al., J. Mol. Endocrinol. 13: 11-21 (1994); S. Costagliola, et al., Biochem. Biophys. Res. Commun. 199: 1027-1034 (1994); S. Costagliola, et al., Endocrinology 135: 2150-2159 (1994); A. Marion, et al., Cell. Immunol. 158: 329-341 (1994); N. M. Wagle, et al., Autoimmunity 18: 103-108 (1994); G. Carayanniotis, et al., Clin. Exp. Immunol. 99: 294-302 (1995); G. S. Seetharamaiah, et al., Endocrinology 136: 2817-2824 (1995); N. M. Wagle, et al., Endocrinology 136: 3461-3469 (1995); H. Vlase, et al., Endocrinology 136: 4415-4423 (1995)) In most cases antibodies to the TSHR (TSHRAbs) which could inhibit TSH binding were produced and in some cases thyroiditis with a large lymphocytic infiltration developed (G. S. Seetharamaiah, et al., Autoimmunity 14: 315-320 (1993); S. Costagliola, et al., J. Mol. Endocrinol. 13: 11-21 (1994); S. Costagliola, et al., Biochein. Biophys. Res. Commun. 199: 1027-1034 (1994); S. Costagliola, et al., Endocrinology 135: 2150-2159 (1994); A. Marion, et al., Cell. Immunol. 158: 329-341 (1994); N. M. Wagle, et al., Autoimmunity 18: 103-108 (1994); G. Carayanniotis, et al., Clin. Exp. Immunol. 99: 294-302 (1995); G. S. Seetharamaiah, et al., Endocrinology 136: 2817-2824 (1995); N. M. Wagle, et al., Endocrinology 136: 3461-3469 (1995); H. Vlase, et al., Endocrinology 136: 4415-4423 (1995)). However. in no case did the immunization produce thyroid stimulating TSHRAbs which increase thyroid hormone levels, the hallmark of Graves,′ nor were the morphologic or histologic features of the disease induced: glandular enlargement, thyrocyte hypercellularity, and thyrocyte intrusion into the follicular lumen. Further, in most studies (G. S. Seetharamaiah, et al., Autoimmunity 14: 315-320 (1993); S. Costagliola, et al., J. Mol. Endocrinol. 13: 11-21 (1994); S. Costagliola, et al., Biochem. Biophys. Res. Commun. 199: 1027-1034 (1994); S. Costagliola, et al., Endocrinology 135: 2150-2159 (1994); A. Marion, et al., Cell. Immunol. 158: 329-341 (1994); N. M. Wagle, et al., Autoimmunity 18: 103-108 (1994); G. Carayanniotis, et al., Clin. Exp. Immunol. 99: 294-302 (1995); G. S. Seetharamaiah, et al., Endocrinology 136: 2817-2824 (1995); N. M. Wagle, et al., Endocrinology 136: 3461-3469 (1995); H. Vlase, et al., Endocrinology 136: 4415-4423 (1995)) the antibodies that inhibited TSH binding were not shown to inhibit TSH activity mediated specifically by the TSH receptor, a feature characteristic of TSH binding inhibitory immunoglobulins (TBIIs) in GD (P.A. Ealey, et al., J. Clin. Endocrinol. Metab. 58: 909-914 (1984); A. Pinchera, et al., in Autoimmunity and the Thyroid , P. G. Walfish, et al., (Eds), Academic Press, New York, pp. 139-145 (1985); G. F. Fenzi, et al., in Thyroid Autoimmunity , A. Pinchera, et al., (Eds), Plenum Press, New York, pp. 83-90 (1987)).

[0017] These studies depended on the ability of the animal to process the TSHR as an extracellular antigen, rather than as a receptor in a functional state on a cell. Several studies have implicated Class I as an important component in the development of autoimmune thyroid disease and in the action of methimazole (MMI), a drug used to treat GD (M. Saji, et al., J. Clin. Endocrinol. Metab. 75: 871-878 (1992); L. D. Kohn, et al., Intern. Rev. Inmunol. 9: 135-165 (1992); E. Mozes, et al., Science 261: 91-93 (1993); D. S. Singer, et al., J. Immunol. 153: 873-880 (1994); L. D. Kohn, et al., in Thyroid Immunity , D. Rayner and B. Champion (Eds), R. G. Landes Biomedical Publishers, Texas, pp. 115-170 (1995)). In addition, aberrant Class II expression, as well as abnormal expression of Class I molecules, is evident on thyrocytes in autoimmune thyroid diseases (G. F. Bottazzo, et al., Lancet 2: 1115-119 (1983); G. F. Bottazzo, et al., N. Engl. J. Med. 313: 353-360 (1985); I. Todd, et al., Annals N. Y. Acad. Sci. 475: 241-249 (1986)), although the cause and role of aberrant Class II in disease expression was controversial (A. P. Weetman & A. M. McGregor, Endocrinol. Rev. 15: 788-830 (1994)).

[0018] The possibility that abnormal MHC expression, as well as a functional, full-length TSHR, might result in a Graves'-like disease, was tested by transfecting full-length human TSHR (hTSHR) into murine fibroblasts with or without aberrantly expressed Class II antigen (N. Shimojo, et al., Proc. Natl. Acad. Sci. U.S.A 93: 11074-11079 (1996); K. -I. Yamaguchi, et al., J. Clin. Endocrinol. Metab. 82: 4266-4269 (1997); S. Kikuoka, et al., Endocrinology 139: 1891-1898 (1998)). Mice immunized with fibroblasts expressing a Class II molecule and holoTSHR, but not either alone, could develop the major features characteristic of GD: thyroid-stimulating antibodies directed against the TSHR, increased thyroid hormone levels, an enlarged thyroid, and thyrocyte hypercellularity with intrusion into the follicular lumen. The mice additionally develop TBIIs, which inhibit TSH-increased cAMP levels in CHO cells stably transfected with the TSHR and appear to be different from the stimulating TSHR Abs, another feature of the humoral immunity in GD. Thus, by immunizing mice with fibroblasts transfected with the human TSHR and a MHC Class II molecule, but not by either alone, an induced immune hyperthyroidism was induced that has the major humoral and histological features of GD (N. Shimojo, et al., Proc. Natl. Acad. Sci. U.S.A 93: 11074-11079 (1996); K. -I. Yamaguchi, et al., J. Clin. Endocrinol. Metab. 82: 4266-4269 (1997); S. Kikuoka, et al., Endocrinology 139: 1891-1898 (1998)). The articles state that the results indicate that the aberrant expression of MHC Class II molecules on cells that express a native form of the TSHR can result in the induction of functional anti-TSHR antibodies that stimulate the thyroid. They additionally suggest that the acquisition of antigen-presenting ability on a target cell containing the TSHR can activate T and B cells normally present in an animal and induce a disease with the major features of autoimmune Graves'.

[0019] Another source of evidence for the importance of abnormal expression of MHC Class I and Class II in causing autoimmune disease derives from studies with drugs. Thionamide therapy has historically been used to treat GD. The most commonly used thionamides are methimazole, carbimazole and propylthiouracil. These thionamides contain a thiourea group; the most potent are thioureylenes (W. L. Green, in Werner and Ingbar's “ The Thyroid”: A Fundamental Clinical Text, 6th Edition, L. Braverman & R. Utiger (Eds), J. B. Lippincott Co., p. 324 (1991)). The basis for thionamide therapy has, however, not focused on immune suppression. Rather, the basis had been suppression of thyroid hormone formation. Experiments suggesting an effect on immune cells, to inhibit antigen presentation or antibody formation, are largely discounted as nonphysiologic in vitro artifacts of high MMI concentration. MMI activity under those circumstances is suggested to be based on free-radical scavenger activity (D. S. Cooper, in Werner E. Ingbar's “ The Thyroid ”, op. cit., pp. 712-734 (1991)).

[0020] PCT Application WO 92/04033, Faustman, et al., identifies a method for inhibiting rejection of transplanted tissue in a recipient animal by modifying, eliminating, or masking the antigens present on the surface of the transplanted tissue. Specifically, this application suggests modifying, masking or eliminating human leukocyte antigen (HLA) Class I antigens. The preferred masking or modifying drugs are F(ab)′ fragments of antibodies directed against HLA-Class I antigens. However, the effectiveness of such a therapy will be limited by the hosts' immune response to the antibody serving as the masking or modifying agent. In addition, in organ transplantation, this treatment would not affect all of the cells because of the perfusion limitations of the masking antibodies. Faustman, et al., contends that fragments or whole viruses can be transfected into donor cells, prior to transplantation into the host, to suppress HLA Class I expression. However, use of whole or fragments of virus presents potential complications to the recipient of such transplanted tissue since some viruses, SV40 in particular, can increase Class I expression (D. S. Singer & J. Maguire, Crit. Rev. Immunol. 10: 235-237 (1991)).

[0021] British patent 592,453, Durant, et al., identifies isothiourea compositions that may be useful in the treatment of autoimmune diseases in host versus graft (HVG) disease and assays for assessing the immunosuppressive capabilities of these compounds. The British patent does not describe methimazole or the suppression of MHC Class I molecules in the treatment of autoimmune diseases. Additionally, several autoimmune diseases have been treated with methimazole with potential success. In one study, MMI was deemed as good as cyclosporin in treating juvenile diabetes (W. Waldhausl, et al., Akt. Endokrin. Stoffw. 8: 119 (1987). U.S. Pat. No. 5,556,754, Singer et al. (which is equivalent to PCT Application WO 94/28897), issued Sep. 17, 1996, describes a method for treating autoimmune diseases using methimazole, methimazole derivatives and methimazole analogs. U.S. Pat. No. 5,310,742, Elias, issued May 10, 1994, describes the use of thioureylene compounds to treat psoriasis and autoimmune diseases. Propylthiouracil, methimazole, and thiabendazole are the only specific compounds disclosed in the patent.

[0022] It has now been found (L. D. Kohn, et al., Methimazole derivatives and tautomeric cyclic thiones to treat autoimmune diseases . U.S. patent application submitted Aug. 31, 1998, which is herein incorporated by reference in its entirety) that a specific class of methimazole derivatives and tautomeric cyclic thiones are effective in treating autoimmune diseases and suppressing the rejection of transplanted organs, and that these compounds show clear and unexpected benefits over the use of methimazole itself. In particular, these compounds: (a) are more effective in inhibiting basal and IFN-induced Class I RNA expression and in inhibiting γIFN-induced Class II RNA expression than methimazole; (b) inhibit the action of IFN and abnormal MHC expression by acting on the CIITA/Y-box regulatory system; and (c) exhibit therapeutic activities in vivo. Specifically they inhibit development of SLE in the (NZBxNZW)F ₁ mouse model and diabetes in the NOD mouse model, both of which are linked to abnormal expression of MHC genes.

[0023] In sum, the development of tissue-specific autoimmune diseases is associated with abnormal or aberrant expression of MHC molecules, Class I and/or Class II, on the surface of cells in the target tissue (G. F. Bottazzo, et al., Lancet 2: 1115-1119 (1983); I. Todd, et al., Annals N. Y. Acad. Sci. 475: 241-249 (1986); J. Guardiola & A. Maffei, Crit. Rev. Immunol. 13: 247-268 (1993); D. S. Singer, et al., Crit. Rev. Immunol. 17: 463-468 (1997)). Abnormal expression of MHC molecules on these non-immune cells can cause them to mimic antigen presenting cells and present self-antigens to T cells in the normal immune cell repertoire (M. Londei, et al., Nature 312: 639-641 (1984); N. Shimojo, et al., Proc. Natl. Acad. Sci. U.S.A. 93: 11074-11079 (1996)). This leads to a loss in self tolerance and the development of autoimmunity (G. F. Bottazzo, et al., Lancet 2: 1115-1119 (1983); I. Todd, et al., Annals N. Y. Acad Sci 475: 241-249 (1986); J. Guardiola & A. Maffei, Crit. Rev. Immunol. 13: 247-268 (1993); D. S. Singer, et al., Crit. Rev. Immunol. 17: 463-468 (1997); M. Londei, et al., Nature 312: 639-641 (1984); N. Shimojo, et al., Proc. Natl. Acad. Sci. U.S.A. 93: 11074-11079 (1996)). Prior to the present invention, there was, however, no comprehensive explanation as to how abnormal or aberrant MHC expression might develop in the target tissue, or how this might contribute to the ensuing immune cell responses involved in autoimmunity.

[0024] Viral infections can ablate self-tolerance, mimic immune responses to self antigens, and be associated with autoimmune disease (J. Guardiola & A. Maffei, Crit. Rev. Immunol. 13: 247-268 (1993); R. Gianani & N. Sarvetnick, Proc. Natl. Acad. Sci. U.S.A. 93: 2257-2259 (1996); M. S. Horowitz, et al., Nature Med 4: 781-785 (1998); C. Benoist and D. Mathis, Nature 394: 227-228 (1998); H. Wekerle, Nature Med 4: 770-771 (1998)).

[0025] Rheumatoid Arthritis (RA), multiple sclerosis (MS) and insulin-dependent diabetes mellitus (IDDM) are diseases which, at first glance, seem to have little in common. Yet all three are inflammatory disorders that are credited with a common autoimmune etiology. The evidence that autoimmunity is involved in human IDDM, MS and RA is indirect. It relies on the following observations: (1) the character of the lesion, which is largely dominated by mononuclear inflitrates; (2) the underlying genetic susceptibility, which involves major histocompatibility (MHC) genes (and other genes too); and (3) the resemblance of the human disease to animal models where the pathology is known to be autoimmune in origin. A fourth possible line of evidence, namely the efficacy of immunomodulatory or immunosuppressive therapies, is unfortunately much weaker than one would like it to be in these diseases (H. Wekerle, Nature Med 4: 770-771 (1998)).

[0026] Several indirect arguments support the idea that microbial agents influence the occurrence or course of certain autoimmune diseases. For example, there is evidence linking autoimmune thyroid disease to viral and bacterial infections (Y. Tomer & T. Davies, Endocr. Rev. 14: 107-121 (1993)). The mechanism by which this might occur is unknown (Y. Tomer & T. Davies, Endocr. Rev. 14: 107-121 (1993)). It was known that Rous sarcoma virus, adenoviruses 12 and 2, and certain Gross viruses reduced expression of Class I: however, SV40 radiation leukemia virus (RadLV), and Moloney murine leukemia virus (MoMuLV) viruses can increase Class I MHC expression (D. S. Singer & J. E. Maguire CRC Crit. Rev. Immunol. 10: 235-257 (1990)).

[0027] Other indirect evidence includes the fact that migrant populations acquire the disease prevalence of the geographical area to which they move, a prevalence correlated with latitude; that the incidence or frequency of autoimmune diseases has dramatically changed in the last two centuries; and that non-obese-diabetic (NOD) mice are protected from diabetes by bacterial infections. The nature of the agents involved and their mechanism of action remain unclear.

[0028] One mechanism by which a viral infection could ablate self-tolerance is the induction of interferon (IFN) production by an immune cell (I. Todd, et al., Annals N. Y. Acad. Sci. 475: 241-249 (1986); J. Guardiola & A. Maffei, Crit. Rev. Immunol. 13: 247-268 (1993); D. S. Singer, et al., Crit. Rev. Immunol. 17: 463-468 (1997); R. Gianani & N. Sarvetnick, Proc. Natl. Acad. Sci. U.S.A. 93: 2257-2259 (1996)). γIFN can certainly increase MHC gene expression in the target tissue (J. P -Y. Ting & A. S. Baldwin, Curr. Opin. Immunol. 5: 8-16 (1993)).

[0029] A wealth of genetic, biochemical and animal model data support a contributory role of inflammatory cytokines (e.g., IL-12, IL-18; and particularly γIFN) in the autoimmune process (Sarvetnick, J. Clin. Invest. 99: 371-372 (1997)). Studies using non-obese diabetic (NOD) mice, which spontaneously develop auto-immune diabetes reminiscent of Type I human IDDM, are particularly illustrative in demonstrating how γIFN stimulated processes play critical roles in the development of autoimmunity; and how the actions of other pro-inflammatory cytokines are channeled through γIFN stimulated processes—among which are the enhanced expression of MHC Class I and MHC Class II antigens.

[0030] IL-12 and IL-18 (γIFN inducing factor) are known to act synergistically in stimulating production of γIFN in T cells (Micallef, et al., Eur. J. Immunol. 26: 1647-1651 (1996)). In diabetic NOD mice the systemic expression of IL-18 (Roghe, et al., J. Autoininun. 10: 251-256-(1997)) and islet expression of IL-12 are increased (Rabinovitch, et al., J. Autoimmun. 9: 645-651 (1996)). Moreover, additional IL-12 accelerates autoimmune diabetes in NOD mice (Trembleau, et al., J. Exp. Med. 181: 817-821 (1995)). Genetic analysis has determined the IL-18 gene maps to a near a non-MHC IDDM susceptibility gene (Idd2) associated with a genetic susceptibility for autoimmune diabetes (Kothe, et al., J. Clin. Invest. 99: 469-474 (1997)). These reports help to define a critical role for γIFN in the process of autoimmunity.

[0031] The role of γIFN in the autoimmune process is further substantiated by studies where γIFN's signaling capacity was abrogated in some manner. For example, transgenic NOD mice deficient in the cellular receptor for γIFN (Wang, et al., Proc. Natl. Acad. Sci. U.S.A. 94: 13844-13849 (1997)) do not develop autoimmune diabetes. NOD mice treated with a neutralizing antibody for γIFN (Debray-Sachs, et al., J. Autoimmun. 4: 237-248 (1991)) also do not develop autoimmune diabetes. While it is somewhat surprising that the onset of diabetes is only delayed in transgenic NOD mice deficient in IFN-gamma (Hultgren, et al., Diabetes 45: 812-817 (1996)), this observation only further stresses the importance of blocking the γIFN signal and more importantly IFN-gamma stimulated downstream events for the effective prevention of autoimmunity in NOD mice.

[0032] Analogous observations have been made in animal models for SLE. Soluble γIFN receptor blocks disease in the (NZBXNZW)F ₁ spontaneous autoimmune disease model for SLE (Ozmen, et al., Eur. J. Immunol. 25: 6-12 (1995)); uveitis, where the targeted expression of γIFN increases ocular inflammation (Geiger, et al., Invest. Opthanlmol. Vis. Sci. 35: 2667-2681 (1994)); and autoimmune gastritis, where neutralizing γIFN antibody blocks disease (Barret, et al., Eur. J. Immunol. 26: 1652-1655 (1996)). Moreover, in humans treatment with γIFN has been reported to be associated with the development of an SLE-like disease (Graninger, et al., J. Rheumatol. 18: 1621-1622 (1991)).

[0033] It is well recognized that γIFN increases MHC Class I and Class II expression in many tissues and thus is linked to the action of a coregulatory molecule, the Class II transactivator (Mach, et al., Ann. Rev. Immunol. 14: 301-331 (1996); Chang, et al., Immunity 4: 167-178 (1996); Steimle, et al., Science 265: 106-109 (1994); Steimle, et al., Cell 5: 646-651 (1995); Chang, et al., J. Exp. Med. 180: 1367-1374 (1994); Chin, et al. Immunity 1: 687-697 (1994); V. Montani, et al., Endocrinology 139: 280-289 (1998)). It is also known that methimazole (MMI) can inhibit IFN-increased Class I and Class II expression in thyroid (M. Saji, et al., J. Clin. Endocrinol. Metab. 75: 871-878 (1992); V. Montani, et al., Endocrinology 139: 290-302 (1998)). Also, it has been shown that MMI decreases expression of CIITA increased Class II expression and this appears to be related to the action of MMI to enhance Y box protein gene expression; the Y box protein suppresses Class II gene expression (V. Montani, et al., Endocrinology 139: 280-289 (1998)).

[0034] Invoking cytokines or γIFN as a cause of autoimmunity caused by viruses does not, however, address the mechanism by which a tissue or target cell viral infection induces immune cells to produce γIFN; nor is it reasonable that γIFN alone would cause autoimrnunity, since its administration does not induce typical autoimmune disease (F. Schuppert, et al., Thyroid 7: 837-842 (1997)). Moreover, generalized γIFN production by immune cells cannot account for cell-specific autoimmunity, i.e., destruction of pancreatic β but not α cells in insulin-dependent diabetes mellitus or involvement of only thyroid follicular cells, not parafollicular C cells, in autoimmune Graves' disease (G. F. Bottazzo, et al., Lancet 2: 1115-1119 (1983); I. Todd, et al., Annals N. Y. Acad. Sci. 475: 241-249 (1986); N. Shimojo, et al., Proc. Natl. Acad. Sci. U.S.A. 93: 11074-11079 (1996); A. K. Foulis et al Diabelologia 30: 333-343 (1987)).

[0035] Another possibility for autoimmunity caused by viruses is immunological cross-reactivity between anti-pathogen and anti-self responses, i.e., molecular mimicry (H. Wekerle, Nature Med 4: 770-771 (1998); C. Benoist & D. Mathis, Nature 394: 227-228 (1998)).

[0036] The currently fashionable concept of molecular mimicry (M. B. Oldstone, et al., Cell 50: 818-820 (1987)) proposes that pathogens express a stretch of protein that is related in sequence or structure to a particular self-component. This pathogen-encoded epitope can be presented by the major histocompatibility complex and activate self-reactive T cells. Activation could occur because the T cell's antigen receptor has a higher affinity for the pathogen protein than for the self-component, or because T cells are more readily primed in the inflammatory context of an infection. Because primed and amplified T lymphocytes have a lower threshold for activation, they can now attack self-antigens that they previously ignored.

[0037] Still another alternative concept to explain the action of viruses is bystander activation which proposes that pathogens disturb self-tolerance without antigenic specificity coming into play. They can do this by provoking cell death and the release of cellular antigens or increasing their visibility or abundance; thereby attracting and potentiating antigen-presenting cells and by perturbing the cytokine balance through the inflammation associated with infection (C. Benoist & D. Mathis, Nature 394: 227-228 (1998)).

[0038] There is good evidence that molecular mimicry could operate. Relevant homologies between mammalian and pathogen sequences have been found. Experimental support has come from animals immunized with peptides containing such homologous motifs (R. S. Fujinami & M. B. Oldstone, Science 230: 1043-1045 (1985)) and transgenic mice in which a viral epitope is expressed on particular organs (P. Ohashi, et al., Cell 65: 305-317 (1991); M. B. Oldstone, et al., Cell 65: 319-331 (1991).

[0039] Coxsackie B virus, has been linked to autoimmune diabetes (IDDM). Sero-epidemiological evidence for an association is sketchy (P. M. Graves' et al. Diabetes 46: 161-168 (1997)), but attention has been drawn to the homology between determinants of the Coxsackie P2-C protein and glutamate decarboxylase (GAD), one of the autoantigens recognized in IDDM (T. M. Ellis & M. A. Atkinson, Nature Med 2: 148-153 (1996)). It is possible that Coxsackie virus infection could unleash autoreactivity to GAD and thereby provide IDDM.

[0040] If viruses activate pathogenic autoimmunity through molecular mimicry, they should not be able to do so if the immune repertoire is blind to cross-reactive epitopes. M. S. Horwitz et al., ( Nature Med. 4: 781-785 (1998)) tested this possibility and the potential importance of virus-induced bystander activation by studying the BDC2.5 mouse model of diabetes. Most of the T cells in these transgenic mice are reactive against a naturally expressed pancreatic antigen that is distinct from GAD. When carried on the NOD genetic background, BDC2.5 mice show heavy infiltration of the pancreas by T cells; the local lesion is active, as shown by lymphocyte activation. division and programmed cell death, but a balance is somehow maintained such that complete destruction of insulin-producing cells is avoided for a longtime (I. Andre, et al., Proc. Natl. Acad. Sci. U.S.A. 93: 2260-2263 (1996)).

[0041] Horwitz and colleagues found that infection by Coxsackie B4 rapidly provoked diabetes in the transgenic mice, but not in non-transgenic littermates or in NOD animals, which show a less extensive pancreatic infiltration. This effect was at least to some degree virus-specific, because it did not occur after infection by lymphocytic choriomeningitis virus. Coxsackie B4 infects pancreatic cells, so the local inflammation that it provokes probably disturbs the immunoregulatory balance of autoreactive T cells in the vicinity (increased levels of antigen and pro-inflammatory cytokines).

[0042] This interpretation is consistent with a previous analysis from the Zinkemagel group (S. Ehl, et al., J. Exp. Med 185: 1241-1251 (1997)), using another transgenic system. They found that functional cytotoxic T cells could be elicited through bystander activation, but could not home to and destroy the pancreas, unlike T cells activated, in higher numbers, by recognition of cognate viral antigen. The results of Zhao et al. (S. -Z. Zhao, et al., Science 279: 1344-1347 (1998)), although interpreted in the context of molecular mimicry, also underscore the importance of local effects of pathogens. These authors found that T cells activated by a mimic from Herpes simplex virus could not provide corneal keratitis without a local, virus-induced lesion.

[0043] Ultimately, the conclusion is that the suspected connection between Coxsackie B virus and IDDM is linked to viral infection of the pancreas and bystander activation of a pre-existing, but controlled, immune system. Homology to GAD would be a coincidence (C. Benoist & D. Mathis, Nature 394: 227-228 (1998)). Although this could be overstating the case that can be made from the available data, it will be important to keep in mind these demonstrations of viral bystander effects. For example, therapeutic immunointervention focused on cross-reactive epitopes would be misguided if a pathogen's main contribution were bystander activation of dormant autoreactive cells (C. Benoist and D. Mathis, Nature 394: 227-228 (1998)).

[0044] In sum, there is evidence that viral triggering of diverse autoimmune diseases including rheumatoid arthritis, insulin-dependent diabetes, and multiple sclerosis is caused by local viral infection of the tissue not molecular mimicry. It is suggested this involves MHC genes, results in presentation of self-antigens, and induces bystander activation of the T cells; the mechanism for this is obscure, as is its relation to the immune cell cytokine/IFN response (H. Wekerle, Nature Med 4: 770-771 (1998); C. Benoist & D. Mathis, Nature 394: 227-228 (1998)).

[0045] The mammalian immune system also responds to bacterial infection. One means to do this is rapidly initiating an inflammatory reaction that limits the early spread of pathogens and facilitates the emergence of antigen-specific immunity. Microorganisms have evolved to avoid such recognition by altering their expression of protein and lipid products. Yet DNA is an indispensable and highly conserved component of all bacteria. Indeed, the genomes of otherwise diverse bacteria share DNA motifs that are rarely found in higher vertebrates. Recent studies suggest that immune recognition of these motifs may contribute to the host's innate inflammatory response.

[0046] Bacterial, but not mammalian DNA, can boost the lytic activity of NK cells and induce γIFN production, an effect attributed to palindromic sequences present in bacterial DNA (S. Yammamoto, el al., J. Immunol. 148: 4072-4076 (1992)). In addition, other investigators showed that bacterial DNA, especially when complexed to DNA-binding proteins, could induce B cell activation. To better define the size and composition of the relevant immunostimulatory motif(s), Krieg and colleagues examined the activity of a series of synthetic oligodeoxynucleotides (ODNs) (A. M. Krieg, et al., Nature 374: 546-548 (1995)). Optimal stimulation was observed when the ODN contained at least one non-methylated CpG dinucleotide flanked by two 5′ purines (optimally GpA) and two 3′ pyrimidines (optimally TpC or TpT). Immune stimulation persisted despite purine/purine or pyrimidine/pyrimidine replacements, even if these substitutions eliminated a palindromic sequence. Yet if either base pair of the CpG was eliminated, stimulatory activity was lost. Optimizing the flanking region or incorporating two CPGs into a single ODN increased stimulation. The minimal length of a stimulatory ODN was 8 bp. These findings established that immune stimulation was mediated by a six base pair nucleotide motif consisting of an unmethylated CpG dinucleotide flanked by two 5′ purines and two 3′ pyrimidines imbedded in a larger fragment of DNA (A. M. Krieg, et al., Nature 374: 546-548 (1995)). Such motifs are expressed nearly 20 times more frequently in bacterial than vertebrate DNA due to differences in the frequency of utilization and methylation pattern of CpG dinucleotides in prokaryotes versus eukaryotes.

[0047] Evidence suggests that these motifs act directly on cells of the immune system. Cells responsive to CpG ODN include macrophages, B lymphocytes, T lymphocytes, and NK cells. CpG ODN rapidly stimulate B cells to produce IL-6 and IL-12, CD4+ T cells to produce IL-6 and γIFN, and NK cells to produce γIFN both in vivo and in vitro (D. M. Klinman, et al., Proc. Natl. Acad. Sci. U.S.A. 93: 2879-2883 (1996)). This lymphocyte stimulation is polyclonal and antigen non-specific in nature, although specificity is retained with respect to the phenotype of cells activated and the type of cytokine they produced. The finding that NK and T cells as well as B cells are triggered by CpG-containing ODNs suggests that immune recognition of this motif is evolutionarily conserved among multiple types of immunologically active cells. Kinetic studies reveal that CpG ODNs induce cytokine release within four hours of administration, with peak production occurring by 12 hours (D. M. Klinman, et al., Proc. Natl. Acad. Sci. U.S.A. 93: 2879-2883 (1996)). Maximal cytokine production is observed using ODNs at a concentration of 0.10-0.33 ug/ml (D. M. Klinman, et al., Proc. Natl. Acad. Sci. U.S.A. 93: 2879-2883 (1996)). Synthetic ODN expressing stimulatory CpG motifs have been used as adjuvants to boost the immune response to DNA and protein based immunogens. In vivo experiments demonstrate that CpG-containing oligos augment antigen-specific antibody production by up to ten fold, and γIFN production by up to six fold. For example, CpG ODN boost antigen-specific immune responses when co-administered with either protein- or DNA-based vaccines (Y. M. Sato, et al., Science 273: 352-354 (1996); M. E. Roman, et al., Nature Medicine 3: 849-854 (1997); D. M. Klinman, et al., J. Immunol. 158: 3635-3642 (1997)). This activity is present whether the motifs are intrinsic parts of the antigen (as in the backbone of a DNA vaccine), or co-administered along with the antigen (M. E. Roman, et al., Nature Medicine 3: 849-854 (1997)). However, immunogenicity is improved when the CpG oligo is physically linked to the relevant antigen. This is true both in the case of DNA vaccines and protein antigens. These results confirm the intuitive expectation that optimal stimulation occurs when antigen and adjuvant are presented to the immune system in close spatial and temporal sequence. These data suggest that CpG oligos initiate a complex cascade of events in vivo that may have broad application for immune regulation.

[0048] Saji, et al., (Proc. Natl. Acad. Sci. U.S.A. 89: 1944-1948 (1992)) described hormonal regulation of Class I genes in the rat thyroid cell line, FRTL-5. Treatment of the FRTL-5 cell line with thyroid-stimulating hormone (TSH) resulted in decreased transcription of Class I genes and reduced cell surface levels of Class I antigens. Saji, et al., ( J. Clin. Endocrinol. Metab. 75: 871-878 (1992)) demonstrated that agents such as serum, insulin, insulin-like growth factor-I (IGF-1), hydrocortisone, and thyroid-stimulating thyrotropin receptor autoantibodies from Graves' patients decrease Class I gene expression in that FRTL-5 cells. In addition, treatment of the FRTL-5 cells with methimazole (MMI) or high doses of iodide resulted in decreased Class I gene expression. The effect of MMI on reduction of Class I expression was shown to be at the level of transcription and was additive with thyroid stimulating hormone and other hormones which normally suppress Class I in these cells. Saji, et al., ( J. Clin. Endocrinol. Metab. 75: 871-878 (1992)) suggested a mechanism by which MMI may act in the thyroid during treatment of GD; no extrapolation was made to any other autoimmune diseases. The use of MMI as an immunosuppressant has, however, been controversial.

[0049] The U.S.P. Dictionary (US Pharmacopeia, Rockville, Md., 1996) includes methimazole (CAS-60-56-0) and describes it as a thyroid inhibitor. U.S. Patent Re. 24,505, Rimington, et al., reissued Jul. 22, 1958, discloses a group of imidazole compounds useful as anti-thyroid compounds.

[0050] Further, the action of MMI as an immunosuppressant is controversial. Thus, there have been differing reports on the ability of antithyroid drugs to suppress MHC Class II antigen expression in patients with Graves' disease (J. C. Carel, et al., in H. A. Drexhage & W. A. Weirsinga (Eds). The thyroid and autoimmunity . Excerpta Medica, Amsterdam, pp. 145-147 (1986); J. Aguayo, et al., J. Clin Endocrinol. Metab. 66: 903-908 (1988); T. F. Davies et al. Clin Endocrinol. 31: 125-135 (1989)) and concerns were expressed that there was an absence of dose dependencies on immunologic parameters in refractory Graves' patients treated with MMI before surgery (R. Paschke, et al., J. Clin Endocrinol. Metab. 80: 2470-2474 (1995)). D. S. Cooper ( N. Engl. J. Med. 311: 1353-1362 (1984)) concluded that MMI was an effective therapeutic agent because of actions to block thyroid hormone formation and that its activity as an immunosuppressant might be an in vitro artifact.

[0051] Nevertheless, Methimazole has been used to treat autoimmune diseases other than those of the thyroid.

[0052] U.S. Pat. No. 5,310,742, Elias, issued May 10, 1994, describes the use of thioureylene compounds to treat psoriasis and autoimmune diseases. Propylthiouracil, methimazole, and thiabendazole are the only specific compounds disclosed in the patent. Examples show the use of methimazole to treat psoriasis in humans and the use of thioureylene to treat rheumatoid arthritis, lupus and transplant rejection. No methimazole analogs or derivatives are disclosed or discussed. No tautomeric cyclic thiones are disclosed or discussed.

[0053] U.S. Pat. No. 5,556,754, Singer et al. (which is equivalent to PCT Application WO 94/28897), issued Sep. 17, 1996, describes a method for treating autoimmune diseases using methimazole, methimazole derivatives and methimazole analogs. The terms “methimazole derivatives” and “methimazole analog” are not defined or exemplified anywhere in the patent.

[0054] In one study, MMI was deemed as good as cyclosporin in treating juvenile diabetes (W. Waldhausl, et al., Akt. Endokrin. Stoffw. 8: 119 (1987)).

[0055] U.S. Pat. No. 5,051,441, Matsumoto, et al., issued Sep. 24, 1991, discloses diphenyl imidazoline derivatives which are, said to act as immunomodulators, showing efficiency in the treatment of rheumatoid arthritis, multiple, sclerosis, systemic lupus, and rheumatic fever.

[0056] U.S. Pat. No. 5,202,312 Matsumoto, et al., issued Apr. 13, 1993, discloses imidazoline-containing peptides which are said to have immunomodulatory activity.

[0057] Methimazole and methimazole derivatives have, however, been reported to have activities other than as an antithyroid agent or immunosuppressive agent.

[0058] U.S. Pat. No. 4,148,885, Renoux, et al., issued Apr. 10, 1979, describes the use of specific low molecular weight sulfur-containing compounds as immunostimulants. Methimazole, thioguanine and thiouracil are among the compounds specified. No methimazole analogs or derivatives are disclosed or discussed. No tautomeric cyclic thiones are disclosed or discussed.

[0059] U.S. Pat. No. 5,010,092, Elfarra, issued Apr. 23, 1991, describes a method of reducing the nephrotoxicity of certain drugs via the coadministration of methimazole or carbimazole, (which is taught to be the pro-drug of methimazole) together with the nephrotoxic drug. No methimazole analogs or derivatives are discussed in this patent. No tautomeric cyclic thiones are disclosed or discussed.

[0060] U.S. Pat. No. 5,578,645, Askanazi, et al., issued Nov. 26, 1996, describes a method for minimizing the side effects associated with traditional analgesics. This is accomplished via the administration of a mixture of specific branched amino acids together with the analgesic compound. Methimazole is disclosed, in the background section of this patent, as a nonsteroidal anti-inflammatory drug which may provide some of the side effects which this invention is said to address. No tautomeric cyclic thiones are disclosed or discussed.

[0061] U.S. Pat. No. 5,587,369, Daynes, et al., issued Dec. 24, 1996, describes a method for preventing or reducing ischemia following injury. This is accomplished by introducing dehydroepiandrosterone (DHEA), DHEA derivatives, or DHEA congeners to a patient as soon as possible after the injury. The background section of this patent teaches that methimazole is a thromboxane inhibitor which has been shown to prevent vascular changes in burn wounds.

[0062] U.S. Pat. No. 4,073,905, Kummer, et al., issued Feb. 14, 1978, discloses 2-amino-4-phenyl-2-imidazolines, which are said to be useful for treating hypertension.

[0063] U.S. Pat. No. 3,390,150, Henry, issued Jun. 25, 1968, is representative of a group of patents which disclose nitroimidazole derivatives which possess antischistosomal and antitrichomonal activity.

[0064] U.S. Pat. No. 3,505,350, Doebel, et al., issued Apr. 7, 1970, discloses a group of substituted 2-mercaptoimidazole derivatives which are said to be effective as anti-inflammatory agents. Illustrative compounds include 1-(4-fluorophenyl)-5-methyl-2-mercaptoimidazole and 1-methyl-5-phenyl-2-mercaptoimidazole.

[0065] Methimazole, therefore, is known in the art for a variety of pharmaceutical utilities: for the treatment of psoriasis (Elias), as an immunostimulant (Renoux et al.), for the reduction of nephrotoxicity of certain drugs (Elfarra), for the minimization of side effects found with certain analgesics (Oskinasi et al.), as a thyroid inhibitor (U.S.P. Dictionary), and as a thromboxane inhibitor (Daynes et al.). It is also taught in the Singer et al. patent (U.S. Pat. No. 5,556,754), as being useful in the treatment of autoimmune diseases, such as rheumatoid arthritis and systemic lupus. While the Singer et al. patent (U.S. Pat. No. 5,556,754) contains general references to the use of methimazole analogs and derivatives for these therapeutic purposes, no definition of these compounds is given and no specific compounds are suggested.

[0066] It has recently been found (L. D. Kohn, et al., Methimazole derivatives and tautomeric cyclic thiones to treat autoimmune diseases . U.S. patent application submitted Aug. 31, 1998)) that a specific class of methimazole derivatives, tautomeric cyclic thiones, are effective in treating autoimmune diseases and suppressing the rejection of transplanted organs, and that these compounds show clear and unexpected benefits over the use of methimazole itself. In particular, these compounds: (a) are more effective in inhibiting basal and IFN-induced Class I RNA expression and in inhibiting IFN-induced Class II RNA expression than methimazole; (b) inhibit the action of IFN by acting on the CIITA/Y-box regulatory system; (c) may be significantly more soluble than methimazole, leading to significant formulation flexibility and advantages; (d) have less adverse effects on thyroid function than methimazole; (e) have an enhanced ability to bind to targets affected by MMI; and (f) exhibit therapeutic activities in vivo. These properties are unexpected based on the known properties of methimazole and particularly the tautomeric cyclic thiones.

[0067] Cyclic tautomeric thiones have not been described as immunoregulatory agents. Rather Kjellin and Sandstrom, Acta Chemica Scandinavica, 23: 2879-2887 and 2888-2899 (1969), disclosed a series of tautomeric cyclic thiones, i.e., oxazoline, thiazoline, and imidazoline-2-(3)-thiones having methyl and phenyl groups in the 4 and 5 positions. The compounds were used for a study of thione-thiol equilibria. No pharmaceutical, or any other utility, is disclosed or suggested for these compounds.

[0068] U.S. Pat. No. 3,641,049, Sandstrom, et al., issued Feb. 8, 1972, discloses N, N′-dialkyl-4-phenylimidazoline-2-thiones, particularly 1,3-dimethyl-4-phenylimidazoline-2-thione, for use as an antidepressant agent. The dimethyl compound is also said to exhibit antiviral properties against herpes simplex and vaccinia viruses.

[0069] It has been noted that specific viruses or viral promoters operably linked to nucleic acid inserts could increase Class I gene expression in cultured cells (D. S. Singer & J. E. Maguire, CRC Crit. Rev. Immunol. 10, 235-257 (1990)). Whether this might be related to a primary action of the virus on the target tissue to increase Class I and whether this might be the triggering effect on the cascade of events leading to an autoimmune response was determined as disclosed herein.

SUMMARY OF THE INVENTION

[0070] It is demonstrated herein that the introduction of double-stranded nucleic acids into the cytoplasm of mammalian cells results in the increase the expression of immune response recognition molecules. This activation process transforms the affected cell into an APC capable of stimulating an immune response and may be the triggering event in autoimmunity; alternatively, or in addition, it may contribute to the activity of immune and antigen presenting cells normally present in the host. This natural response may also contribute to the pathogenesis of infectious diseases, chronic degenerative diseases and cancer. This discovery of a natural host defense response is exploited for the discovery of drugs and therapies for the treatment of these conditions and for the detection and diagnosis of the same. By artificially mimicking this activation process, systems for drug screening, drug target identification, immunization and diagnostic assays are enabled.

[0071] An object of this invention is the identification of drug compounds which can increase or decrease activation of immune recognition molecules.

[0072] Another object of this invention is to identify foreign or endogenous substances in an organism that induce, prevent, or suppress activation of immune recognition molecules in a target cell or tissue, in immune cells, or in antigen presenting cells.

[0073] Another object is to identify drug compounds and foreign or endogenous substances in an organism that enhance, prevent, or suppress growth and function of host cell or tissue when immune recognition molecules are increased or decreased by the invention disclosed herein.

[0074] Another object is to identify drug compounds and foreign or endogenous substances in an organism that induce, prevent or suppress viral activiation of host cell molecules in a target cell or tissue, in immune cells, or in antigen presenting cells.

[0075] Another object is to identify drug compounds and foreign or endogenous substances in an organism that induce, prevent or suppress bacterial activiation of host cell molecules in a target cell or tissue, in immune cells, or in antigen presenting cells.

[0076] Another object is to identify drug compounds and foreign or endogenous substances in an organism that induce, prevent or suppress activiation of host cell molecules caused by environmental damage to a target cell or tissue, immune cells, or antigen presenting cells.

[0077] Another object is to identify drug compounds and foreign or endogenous substances in an organism that enhance immune recognition by oncogene transformed target cells or tissue, immune cells, or antigen presenting cells.

[0078] Another object is to identify drug compounds and foreign or endogenous substances in an organism that enhance immune recognition by a target cell or tissue within an immunodeficient animal.

[0079] Another object is to identify drug compounds and foreign or endogenous substances in an organism that prevent or suppress oncogene activation of host cell molecules in a target cell or tissue, in immune cells, or in antigen presenting cells.

[0080] Another object is to identify drug compounds and foreign or endogenous substances in an organism that prevent or suppress immune responses associated with gene therapy in a target cell or tissue, in immune cells, or in antigen presenting cells.

[0081] A further object of this invention is the isolation of such compounds and substances. Thus products identified and/or isolated by this invention are also envisioned.

[0082] One additional use could be to prepare comparative cDNA or mRNA expression libraries for identification of differentially expressed genes in order to identify key genes or proteins which participate in the process and may serve as drug targets. The comparison would be between ds polynucleotide treated and untreated cells of various tissue types.

[0083] Another embodiment would be to assess active modulators of the “DNA response” as anti-infectives in in vitro models of viral, bacterial, and parasitic infections, in a two step drug discovery process.

[0084] The invention comprises introduction of a double-stranded polynucleotide into a cell to induce activation of at least one immune recognition molecule in or on the cell. The cell may be derived from any organism with an immune system, preferably a mammal. The cell is preferably a non-immune cell that is converted into a cell capable of presenting antigen to the immune system by the introduction of the double-stranded polynucleotide. The cell may, however, be typical of the immune system (e.g., lymphocytes, “professional” antigen presenting cells).

[0085] Introduction into the cell may be accomplished by, for example, entry of an infectious agent, phagocytosis, transfection, transformation, or leakage from a DNA-containing organelle. Thus the sequence of the polynucleotide is not necessarily related to any of the immune recognition molecules being activated.

[0086] Immune recognition molecules are those involved in antigen presentation such as, for example, MHC Class I and Class II molecules, peptide transporters, proteasome, HLA-DM, invariant chain, immunomodulators, kinases, phosphatases, signal transducers, and activators or coregulators of transcription. If the molecule is expressed on the cell surface, it may be conveniently detected by an antibody reacting to the intact cell or cell membranes. In any case, promoter activity of the gene, RNA transcripts of the molecule, and translation of the protein may be measured to detect expression of the immune recognition molecule. Expression may also be detected indirectly by bioassays that measure presentation of antigen and other processes involved in immune activation (e.g., release of soluble mediators of immunity, expression of receptors for the soluble mediators). Activation may also be measured by the cellular signals (e.g., tyrosine or serine/threonine phosphorylation, ADP ribosylation, proteolytic cleavage) generated during an immune response.

[0087] Increasing the ability of a cell to present antigen and activate the immune system by this invention allows its use as an activated APC. The activated APC may be introduced into an organism, preferably the activated APC is injected or surgically implanted into its own host organism (e.g., a murine cell into a mouse), to initiate an immune response. The immune response may be restricted to the MHC haplotype expressed on the activated APC. Presentation of an autoantigen may lead to development of autoimmunity, a tumor antigen may lead to an immune response against the tumor, or the immune response to a selected antigen presented by the activated APC may be used to immunize or tolerize against that antigen.

[0088] This invention provides a simple system to regulate expression of immune recognition molecules, and allows one to increase or decrease the amount of MHC molecules expressed on the cell surface of professional and nonprofessional antigen-presenting cells. By acting early in the pathway for generating antigen-MHC complexes, this invention can profoundly affect immunization, tolerization, and other biological processes dependent on activation of immune recognition molecules. Also provided are systems for the screening, identification, and isolation of compounds that suppress or enhance activation by decreasing or increasing, respectively, expression of immune recognition molecules.

[0089] The invention can be distinguished from the effects of CpG sequences because methylation does not alter activity whereas methylation eliminates CpG activity. There is no sequence specificity, whereas optimal CpG stimulation depends on sequence, e.g., when the ODN contains at least one non-methylated CpG dinucleotide flanked by two 5′ purines (optimally GpA) and two 3′ pyrimidines (optimally TpC or TpT). Most importantly, CpG motifs act directly only on cells of the immune system, whereas the ds nucleic acids described herein also work on nonimmune cells and convert them to APC.

[0090] The present invention may be used additively or synergistically with synthetic ODN expressing stimulatory CpG motifs, for example as adjuvants to boost the immune response to DNA and protein based immunogens and when coadministered with protein or DNA-based vaccines (Y. M. Sato, et al., Science 273: 352 (1996); M. E. Roman, et al., Nature Medicine 3: 849 (1997); D. M. Klinman, et al., J. Immunol. 158: 3635 (1997)). The one agent (ds nucleic acids) acts on the nonimmune cells to improve immune recognition; the other (CpG motifs) work on the immune cells to activate their responsiveness.

[0091] Examples of autoimmune diseases wherein this invention is relevant include, but are not limited to, rheumatoid arthritis, psoriasis, juvenile or type I diabetes, primary idiopathic myxedema, systemic lupus erythematosus, DeQuervains thyroiditis, thyroiditis, autoimmune asthma, myasthenia gravis, scleroderma, chronic hepatitis, Addison's disease, hypogonadism, pernicious anemia, vitiligo, alopecia areata, Coeliac disease, autoimmune enteropathy syndrome, idiopathic thrombocytopenic purpura, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antispermatazoan antibodies, sudden hearing loss, sensoneural hearing loss, Sjogren's syndrome, polymyositis, autoimmune demyelinating diseases such as multiple sclerosis, transverse myelitis, ataxic sclerosis, pemphigus, progesssive systemic sclerosis, -dermatomyositis, polyarteritis, nodosa, hemolytic anemia, glomerular nephritis and idiopathic facial paralysis. Diseases wherein the autoimmune response is a component of the host defense mechanism and disease process are also relevant to this invention. These include, but are not limited to, athero sclerotic plaque development, transplant rejection, host vs. graft disease, and others yet to be described.