CA2339090C - Methimazole derivatives and tautomeric cyclic thiones to treat autoimmune diseases - Google Patents

Abstract

The present invention provides methods for treating autoimmune diseases in mammals and for preventing or treating transplantation rejection in a transplant recipient. These methods utilize specifically-defined methimazole derivatives and tautomeric cyclic thione compounds, as well as pharmaceutical compositions containing those compounds.
These compounds and compositions have been found to be at least as effective as methimazole in terms of pharmaceutical activity, while having less of an adverse effect on thyroid function. They are also more soluble in conventional pharmaceutical vehicles than methimazole. An assay for screening the activity of compounds useful against autoimmune diseases (ability to suppress expression of MHC Class I and II molecules) is also taught.

Description

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ntolewlea asr, nonnlty ax}waased o* on a few aalt types, such aA lynpbocywa, rna=VbagGS .aW dcndrltic. WU.

Antigens are presented to the immune system by antigen presenting cells in the context of Class I or Class II cell surface molecules, for example, CD4' helper T-lymphocytes recognize antigens in association with Class II MHC molecules, and CD8+ cytotoxic lymphocytes (CTL) recognize antigens in association with Class I
gene products. It is currently believed that MHC Class I molecules function primarily as the targets of the cellular immune response, while Class II
molecules regulate both the humoral and cellular immune response (Klein, J. and Gutze, E., "Major Histocompatibility Complex", Springeir Verlag, New York, 1977; Unanue, E.R., Ann. Rev. Immunology, 2:295-428, (1984)). MHC Class I and Class II
molecules have been the focus of much study with respect to research in autoimmune diseases because of their roles as inediators or initiators of immune response. MHC Class II antigens have been the primary focus of research in the etiology of autoimmune diseases, whereas MHC Class I antigens have historically been the focus of research in transplantation rejection.
Numerous experimental animal models ifor human disease have linked aberrant expression and/or function of MHC Cilass 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)), and uveoretinitis (Prendergast et al., Invest. Opthalmol. Vis. Sci. 39: 754-762 (1998)).

The pathological link between MHC Claiss 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. However, the strongest 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 (Sei-reze et al., Diabetes 43: 505-509 (1994)), and SLE (Mozes et al., Science 261: 91-93 (1993)).

Moreover, the dependence of the progressive multifocal inflammatory autoimmune disease phenotype exhibited by TGF-betal deficient transgenic mice (Shull et al., Nature 359: 693-699 (1992); Kulkarni et al., Proc. Natl. Acad.
Sci.
90: 770-774 (1993); Boivin et al., Am. J. Pathol. 146: 276-288 (1995)) on MHC
Class 11 expression has recently been demonstrated using MHC Class II
deficient animals. Specifically, TGF-betal 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-(1996)).

In addition to the information supportive of MHC Class I and Class II
antigens as critical for the development of autoi.mmunity in animal models there is equally strong evidence linking autoimmune processes with expression of MHC
Class I and MCH Class II antigens in humans.

Graves' disease is a relatively conunon autoimmune disorder of the thyroid.
In Graves' disease, autoantibodies against thyroid antigens, particularly the thyrotropin receptor (TSHR), alter thyroid function and result in hyperthyroidism (Stites, D.P. and Terr, A.I. (eds), "Basic and Clinical Immunology", Appleton and Lang, Norwalk, Connecticut/San Mateo, California, 1991, pp. 469-470)).
Thyrocytes from patients with Graves' disease liave aberrant MHC Class II
expression and elevated MHC Class I expression (Hanafusa et al., Lancet 2:1111-1115 (1983); Bottazzo et al., Lancet 2:1115-11]l9 (1983); Kohn, et al., in "International Reviews of Immunology," Vol. 912, pp. 135-165, (1992)).
Aberrant expression of MIiC Class II and TSHR on fibroblasts, but not either alone, has recently been shown to induce Graves' disease in mice, i.e., aberrant expression of Class II on target tissue can yield autoimmune disease in animals with normal immune systems. Thionamide therapy has historically been used to treat Graves' disease. 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, 6'h Edition, L. Braverman and R. Utiger (eds), J.B.
Lippincott Co., 1991, p. 324). The basis for thionamide therapy has, however, not focused on immune suppression. Rather, the basis has been suppression of thyroid hormone formation. Experiments suggesting an effect on irnmune 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.
See D.S.
Cooper, in Werner E. Ingbar's "The Thyroid", op. cit., pp. 712-734.

Systemic lupus erythematosus (SLE) is a chronic autoimnnune 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 (Abbus, A.K., Lichtman, A.H., Pober, J.S. (eds), "Cellular and Molecular Immunology", W.B. Saunders Comjpany, Philadelphia, 1991, pp. 360-370). SLE is characterized by the formation oi' a variety of autoantibodies and by multiple organ system involvement (Stites and 'Terr, ibid, pp. 438-443).
Current therapies for treating SLE involve the use of corticosteroids and cytotoxic drugs, such as cyclophosphamide. Immunosuppressive drugs, such as cyclosporin, FK506 or rapamycin suppress the immune system by rieducing T cell numbers and function (Morris, P.J., 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 (Mozes, et al., Science 261: 91-93 (1993)).

Women suffering from SLE who have breast cancer face particular difficulties. These individuals are inununosuppressed as a result of corticosteroid and cytotoxic drug treatment for SLE; radiatioin therapy for the treatment of the cancer, a current treatment of choice, would aclditionally exacerbate the immunosuppressed state. Further, radiation therapy can exacerbate disease expression or induce severe radiation complicaitions. For these individuals, alternative therapies that would allow for simultaneous treatment of SLE and cancer are greatly needed.

Diabetes Mellitus is a disease characterized by relative or absolute insulin deficiency and relative or absolute glucagon excess (Foster, D.W., Diabetes Mellitus. In Stanbury, J.B., et al., The Metabc-lic Basis of Inherited Disease. Ch.
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 (3 cell injury with leakage cell antigens but also represent a primary autoimmuane 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 H:LA antigens has been known since 1970. Certain HLA alleles are associated with ilncreased frequency of disease, others with decreased frequency. Increased MH[C class I and aberrant MHC class II
expression in islet cells has been described (Bottazzo et al., NEJM 313: 353-(1985); Foulis and Farquharson, Diabetes 35: 1215-1224 (1986)). A definitive link to MHC class I has been made in a genetic animial model of the disease. Thus MHC
class I deficiency results in resistance to the development of diabetes in the NOD
mouse (Sereze et al., Diabetes 43: 505-509 (1994); Wicker et al., Diabetes 43:

504 (1994)).

A wealth of genetic, biochemical and animal model data support a contributory role of inflammatory cytokines (e.g., IL-12, IL-18; and particularly IFN-gamma) in the autoinimune 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 IFN-gamma stimulated processes play critical roles in the development of autoimmunity; and how the actions of other pro-inflanunatory cytokines are channeled through IFN-gamma stimulated processes - among which are the enhanced expression of MHC Class I ajid MHC Class II antigens.
IL-12 and. IL-18 (IFN-gamma inducing factor) are known to act synergistically in stimulating production of IFN-gamma in T cells (Micallef et al., Eur. J. Immunol. 26: 1647-1651 (1996)). In diabetic NOD mice the systemic expression of IL-18 (Rothe et al., J. Autoimmun. 10: 251-256 (1997)) and islet expression of IL-12 are increased (Rabinovitch et al., J. Autoimmun. 9: 645-(1996)). Moreover, additional IL-12 accelerates autoinunune 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 chromosomal region (Idd2) associated with a genetic susceptibility for autoimmune diabetes (Kothe et al., J. Clin. Invest.
99:
469-474 (1997)). These reports support help to defme a critical role for IFN-gamma in the process of autoimmunity.

The role of IFN-gamma in the autoimmune process is further substantiated by studies where IFN-gamma's signaling capacity was abrogated in some manner.
For example, transgenic NOD mice deficient in the cellular receptor for IFN-ganuna (Wang et al., Proc. Natl. Acad. Sci. 94: 13844-13849 (1997)) do not develop autoimmune diabetes. NOD mice treated with a neutralizing antibody for IFN-gamma (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 IFN-gamma signal - and more ;importantly IFN-gamma stimulated downstream events - for the effective prevention of autoirnmunity in NOD mice.

Analogous observations have been made in animal models for SLE. Soluble IFN-gamma receptor blocks disease in the NZB/NZW Fl spontaneous autoimmune disease model for SLE (Ozmen et al., Eur. J. Immunol. 25: 6-12 (1995));
uveitis, where the targeted expression of IFN-gamma iiicreases ocular inflanunation (Geiger et al., Invest. Opthanlmol. Vis. Sci. 35: 2667-2681 (1994)); and autoimmune gastritis, where neutralizing IFN-gamma antibcdy blocks disesase (Barret et al., Eur. J. Immunol. 26: 1652-1655 (1996)). Moreover, in humans treatment with IFN-gamma has been reported to be associated with the development of an SLE-like disease (Graninger et al., J. Rheumatol. 18: 1621-1622 (1991)).

It is well recognized that r-IFN 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-(1996); Chang et al., Immunity 4: 167-178 (1996); Steimle et al., Science 265:

109 (1994); Chang et al., J Exp Med 180: 1367-1374 (1994); Chin et al.
Immunity 1: 687-697 (1994); Montani, V. et al., Endocrinology 139: 280-289 (1998)). It is also known that MMI can inhibit IFN-increasedl class I and class II expression in thyroid (Saji et al., J. Clin. Endocrinology. Metab. 75: 871-878 (1992);
Montani et al., Endocrinology. 139: 290-302 (1998)). Firially, 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 (Montani et al., Endocrinology 139:
280-289 (1998)).
As is true for autoimmune diseases, there is a great need for new and different ways of treating or preventing transplantation rejection.
Transplantation rejection occurs as a result of histoincompatibililty between the host and the donor; it is the major obstacle in successful transplantaticin of tissues. Current treatment for WO 00/12175 PCT/[JS99/19862 transplantation rejection, as for autoimmune disease, involves the use of a variety of immunosuppressant drugs and corticosteroid treatment.

Kjellin and Sandstrom, Acta Chemica Scandinavica, 23: 2879-2887 and -2888-2899 (1969), discloses a series of tautomeric cyclic thiones, i.e., oxazoline -, thiazoline-, and imidazoline-2-(3)-thiones, havi;ng 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.
U.S. Patent 3,641,049, Sandstrom et al., issued February 8, 1972, discloses N, N'-dialkyl-4-phenylimidazoline-2-thiones, particularly 1,3-dimethyl-4-phenylimidazoline-2-thione, for use as an antidE:pressant agent. The dimethyl compound is also said to exhibit antiviral properties against herpes simplex and vaccinia viruses.

U.S. Patent Re. 24,505, Rimington et al., reissued July 22, 1958, discloses a group of imidazole compounds useful as anti-thyroid compounds.

U.S. Patent 3,505,350, Doebel et al., issued April 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)-methyl-2-mercaptoimidazole and 1-methyl-5-phenyl-2-mercaptoimidazole.

U.S. Patent 3,390,150, Henry, issued June 25, 1968, is representative of a group of patents which disclose nitroimidazole clerivatives which possess antischistosomal and antitrichomonal activity.

U.S. Patent 5,051,441, Matsumoto et al., issued September 24, 1991, discloses diphenyl imidazoline derivatives which are said to act as WO 00/12175 PCT/t7S99/19862 immunomodulators, showing efficiency in the treatment of rheumatoid arthritis, multiple sclerosis, systemic lupus, and rheumatic fever.

U.S. Patent 4,073,905, Kummer, et al., issued February 14, 1978, discloses 2-amino-4-phenyl-2-imidazolines, which are said to be useful for treating hypertension.

U.S. Patent 5,202,312, Matsumoto et al., issued April 13, 1993, discloses iniidazoline-containing peptides which are said ito have immunomodulatory activity.
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 treatmenit 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 (Singer and Maguire, Crit. Rev. Imnnunol., 10:235-237 (1991), see particularly Table 2).

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. However, this patent does not describe methimazole or the suppression of MHC Class I molecules in the treatment of autoimmune diseases. No tautomeric cyclic thiones are disclosed or discussed.

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), and psoriasis has also been treated with MMI.

U.S. Patent 5,556,754, Singer, et al. (wllich is equivalent to PCT
Application WO 94/28897), issued September 17, 1996, describes a method for treating autoimmune diseases using methimazole, methimazole derivatives and methimazole analogs. The terms "methimazole derivative" and "methimazole analog" are not defmed or exemplified anywhere in the patent.
U.S. Patent 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.

U.S. Patent 4,148,885, Renoux, et al., issued April 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.

U.S. Patent 5,010,092, Elfarra, issued April 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 methirnazole analogs or derivatives are discussed in this patent. No tautomeric cyclic thiones are disclosed or discussed.
U.S. Patent 5,578,645, Askanazi, et al., issued November 26, 1996, describes a method for minimizing the side effects associated with traditional analgesics. This is accomplished via the admir.kistration 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 non-steroidal anti-inflanunatory drug which may provide some of' the side effects which this invention is said to address. No tautomeric cyclic thiones are disclosed or discussed.

U.S. Patent 5,587,369, Daynes, et al., iissued December 24, 1996, describes a method for preventing or reducing ischemia following injury. This is accomplished by introducing dehydroepiandrositerone (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.
The U.S.P. Dictionary (US Pharmacopeia, Rockville, Maryland, 1996) includes methimazole (CAS-60-56-0) and descr:ibes it as a thyroid inhibitor.

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 (USP Dictionary), and as a thromboxane inhibitor (Daynes, et al.).
It is also taught in the Singer, et al. patent as being useful in the treatment of autoimmune diseases, such as rheumatoid arthritis and systemic lupus. While the Singer, et al. patent contains general references to the use of inethimazole analogs and derivatives for these therapeutic purposes, no definition of these compounds is given and no specific compounds are suggested. The pharmacological properties of tautomeric cyclic thiones are not discussed nor related to those of inethunazole derivatives.

It has now been found 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 IFN-induced Class H 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 ttian 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 (#) exhibit therapeutic act;ivities in vrvo. These properties are unexpected based on the known properties of methimazole and particularly the tautomeric cyclic thiones.

Finally, the present invention relates to the method by which these agents inhibit interferon-gamma actions, specifically those related to increase MHC
Class I
and MHC Class II expression and mediation of pro-inflammatory processes, and more specifically those processes related to the induction of autoimmune disease and/or transplant rejection.

SLTMMARY OF THE INVENTION

The present invention relates to pharmaceutical compositions comprising a safe and effective amount of an active compound selected from Rz R2 Y. y 1 `
N\ N N

~ Z LN X X
N EN

Rz wherein Y is H, Cl-C4 alkyl, C1-C4 substituted alkyl, -NO2, or the phenyl moiety R4 *)/
Rt wherein no more than one Y group in said active compound may be the phenyl moiety; R' is selected from H, -OH, halogens (F, Cl, Br or I), C1-C4 alkyl, C1-substituted alkyl, C1-C4 ester or Cf-C4 substituted ester; R2 is selected from H, C1-C4 alkyl or C1-C4 substituted alkyl; R3 is selected from H, C1-C4 alkyl, Cl-Ca substituted alkyl or -CH2Ph (wherein Ph is pher.tyl); R4 is selected from H, Ci C,4 alkyl or C1-C4 substituted alkyl; X is selected from S or 0; Z is selected from -SR3, -OR3, S(O)R3 or C1-C4 alkyl; and wherein at leaist two of the RZ and R3 groups on said compound are Cl-C4 alkyl when Y is not a phenyl moiety, and at least one Y is -NO2 when Z is alkyl; together with a pharmaceutically-acceptable carrier.

Preferred compounds for use in these pharmaceutical compositions have the forumlae RZ
Y
Y.
jR ( NZ 4 , X
I R I

wherein Y is selected from H and C,-C4 alkyl or C,-C4 substituted alkyl; R' is selected from H, -OH, halogens (F, Cl, Br, or I), or C,-C4 alkyl, CI-C4 substituted alkyl, Cl-C4 e.ster or CI-C4 substituted ester; RZ is selected from H or Cl-C4 alkyl or C1-C4 substituted alkyl; R3 is selected from H, C,-C4 alkyl, C,-C4 substituted alkyl, or -CH2Ph; R is selected from H, C,-C-4 alkyl or C,-C4 substituted alkyl; X
is selected from S or 0; and Z is selected from -.>R' or -OW.

Particularly preferred compounds are those which have the formulae N N N
j)_SH j_SCH3 JS
O I I O I

Preferred compounds also include those of the formulae:

' N~--SH I N,~---SCH3 I ~S
a~ l i s CH3 R9 /- oCH3 R9o / CHs R

wherein R9 is selected from -OH, -M and MCHxCOO-; and M is selected from F, Cl, Br and I.
The present invention also relates to the method of treating autoimmune diseases or transplantation rejection in a patient in need of such treatment by the administration of a safe and effective amount of'the active compounds and pharmaceutical compositions described above.
The present invention also relates to in vivo assay methods which permit high efficiency screening of the effects of compounds on the expression of MHC
Class I
and Class II proteins.

Finally, the present invention relates to the method by which the compounds defined herein inhibit gamma interferon actions to increase MHC class I or class II
expression. Gamma interferon has been linked to expression of immune disease.

As used herein, all ratios, fractions and percentages are "by weight", unless otherwise specified.

BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1. Diagrammatic representation of the upstream silencer/enhancer region of the MHC Class I PD1 gene (Panel A), the downstream silencer region of the Class I PD1 gene (Panel B), and the regulatory region of the HLA-DRa MHC Class II promoter (Panel C).

Figure 2. Effect of methimazole and several of the active compounds used herein on formation of protein/DNA silencer complexes with Fragment 140 (- 770 to -636 bp) of the MHC (Class I) 5'-flanicing region, which contains within it the upstream silencerlenhancer which controls c;onstitutive MHC class I
expression in different tissues.

Figure 3. Sequence of MHC Class ;[ gene between -250 bp and the start of translation.
Figure 4. Effect of treating FRTL-5 cells with MMI or a representative compound studied herein, compound 8 (see Table 1), on the formation of protein/DNA complexes with radiolabeled Fragment 127 (see Fig. 1) of the MHC
5'-flanking region, -127 to +1 bp, as a function of time.
Figure 5. Nucleotide sequence of the 176 bp 5'-flanking region of the -176 bp HLA-DRa-CAT construct used in the experiments herein.

Figure 6. Electrophoretic mobility shift analysis (EMSA) of the ability of a 32P-radiolabeled DRa-5'-flanking region probe to form protein/DNA
complexes with extracts from FRTL-5 cells maintained with and without TSH and treated or not with x-IFN.

Figure 7. Effect of MMI on the ability of 'r-IFN to increase the formation of protein/DNA complexes with the 3"'P-radiolabeled DRa-5'-flanking region probe.

Figure 8. Effect of 1 mM MMI or 1 mM concentrations of representative compounds studied herein on thi! ability of x-IFN to increase the formation of protein/DNA complexes with the 32P-radiolabeled DRa-5'-flanking region probe.
Figure 9. Effect of MMI and/or TSH on exogenous class I promoter activity in FRTL-5 cells.

Figure 10. Effect of MMI and TSH on the promoter activity of CAT
chimeras of 5'-deletion mutants of the swine class I promoter in FRTL-5 cells.
Figure 11. Effect of x-interferon (IFN) on class II expression in FRTL-5 thyroid cells measured using the -176 bp HLA-DRa-CAT construct and 5'-deletions thereof (A) or the -176 bp DRa-CAT construct having mutations with the S, X,, X2, and Y boxes (B).

Figure 12. Effect of x-IFN and MMI[ on MHC class II antigen expression in FRTL-5 cells.

Figure 13. Effect of 'r-IFN on class ]fI expression in FRTL-5 thyroid cells as a function of x-IFN concentration (A) and in the presence of MMI (B).

Figure 14. Effect of MMI on x-IFN-increased class II expression in FRTL-5 thyroid cells as a function of MMI concentration.
Figure 15. Effect of MMI, compound 10 (5 phenylmethimazole) and compound 3(2-mercaptoimidazole) on immune complexes in the kidneys of (NZBxNZW)F, mice.

Figure 16. Ability of MMI or compounds 10 (5-phenylmethimazole); 7 (5-methylmethimazole), 8 (N-methylmethimazole) or 11 (1-methyl-2-thiomethyl-5(4) nitroimidazole) to reverse the ability of 't-IFN to reduce Y box RNA levels.

Figure 17. Model of the developmen.t of autoimmune disease and the effect of the MMI, MMI derivatives or tautome;ric cyclic thiones on the development process.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to pharmaceutical compositions which may be used to treat autoimmune diseases and to suppress the rejection of transplanted tissue. The methods of treating autoimmune diseases and of suppressing rejection of transplanted tissue utilizing these pharmaceutical compositions are also covered, as are specific methimazole derivatives and tautomeric cyclic thione compounds which may be used to make these pharmaceutical compositions. As used herein, the following terms shall have the definitions given below.

The phrase "safe and effective amount" means a sufficient amount of pharmaceutically active compound to desirably affect the treatment of autoimmune diseases or to suppress the rejection of transplanited tissue, at a reasonable benefit/risk ratio attendant with any medical treatment. Within the scope of sound medical judgement, the required dosage of a pharmaceutically active agent or of the pharmaceutical composition containing that active agent will vary with the severity of the condition being treated, the duration of the treatment, the nature of adjunct treatment, the age and physical condition of the patient, the specific active compound employed, and like considerations discussed more fully hereinafter.
In this regard it should be noted that the use of MMI at high doses can induce side effects, such as aplastic anemia, agranulocytosis, hepatic dysfunction and dermatitis, in certain patients. In arriving at the "safe and effective amount" for a particular compound, these risks must be taken into consideration, as well as the fact that the compounds described herein provide pharmaceutical activity at lower dosage levels _ than conventional methimazole compounds.
.
"Pharmaceutically-acceptable" shall me;an that the pharmaceutically active compound and other ingredients used in the pharmaceutical compositions and methods defined herein are suitable for use in contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, and the like, conunensurate with a reasonable benefit/risk ratio.

The term "administration" of the pharrnaceutically active compounds and the pharmaceutical compositions defined herein includes systemic use, as by injection (especially parenterally), intravenous infusion, suppositories and oral administration thereof, as well as topical application of the compounds and compositions.
Oral administration is particularly preferred in the present invention.

The term "comprising", as used herein, means that various other compatible drugs and medicaments, as well as inert ingredients, can be conjointly employed in the pharmaceutical compositions and methods of this invention, as long as the defined pharmaceutically active compounds and carriers are used in the manner disclosed. The term "comprising" thus encompasses and includes the more restrictive terms "consisting of" and "consisting essentially of'.

The term "patient", as used herein, is intended to encompass any mammal, animal or human, which may benefit from trealtment with the compounds, compositions and methods of the present invention.

By "compatible" herein is meant that the components of the compositions which comprise the present invention are capable of being comingled without interacting in a manner which would substantially decrease the efficacy of the pharmaceutically active compound under ordinary use conditions.

The pharmaceutical compositions of the present invention comprise specifically-defined methimazole derivatives and tautomeric cyclic thiones, used in a safe and effective amount, together with a pharmaceutically-acceptable carrier.

The methimazole derivatives used in the: compositions of the present invention are those having the following structu,ral formulae:

RZ
Y
O
N` N \ N

N
IZ

In these formulae, Y is selected from H, C,-C4 alkyl, C,-C4 substituted alkyl, -NO2, and the phenyl moiety RI

=

Y is preferably H, the phenyi moiety or -NO2, and is most preferably H or the phenyl moiety In the defmed compounds, no more than one Y group may be the phenyl moiety. R' is selected from H, -OH, halogens (F, Cl, Br aind I), C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4ester and C1-C4 substituted ester; preferably R' is H, -OH, halogen, -OOC CH2M (where M is H or a halogen); and is most preferably H. R2 is selected from H, Ct-C4 alkyl and C1-C4 substituted alkyl; preferably one or both of the groups is methyl. As used herein, "substituted, alkyl" or "substituted ester"
is intended to include alkyl, aryl or ester groups which are substituted in one or more places with hydroxyl or alkoxyl groups, carboxyl groups, halogens, nitro groups, amino or acylamino groups, and mixtures of those moieties. Preferred "substituted alkyl" groups are C1-C4 , hydroxyl or alkoxyl groups, as well as groups substituted with halogens. The R3 groups in the above formulae are selected from H, C1-C4 alkyl, Ci-C,, substituted alkyl and -CH2Ph (wherein Ph is phenyl); in preferred compounds, R3 is H or C1-C4 alkyl; most preferably R3 is C1-C4 alkyl, particularly methyl. R4 is selected from H, CX,, alkyl and. C1-C4 substituted alkyl, and preferably is H. X may be S or 0, and is preferably S. Finally, Z is selected from C1-C4 alkyl, -SR3, -S(O)R3 and -OR3, is preferably -SR3, -OR3, and -S(O)R3;
most preferably -SR3 and -OR3; and particularly -SR'`. In the above form,ulae, at least two of the R2 and R3 groups on the compound must be C2-C4 alkyl when Y is not a phenyl moiety. Further, at least one of the Y groups should be -NO2,, when Z
is Ct-C4 alkyl.

Wp pOJ1317S ~K.T/US4'J/19s62 Amended Page 22 Colapouads vseltW ia tAe present invaASlon innludo ft t+nuomeric c'yclie thiones, disclmd in K,jdlia and Sutbtram ACa Chmica SarWemavica 23: 2879-2887 (1969). ltaving ihe ftaoaslw R Rs Rs Rs wharvln R. R' c CFI3- CH3; Pk H; H. Ph.
It~ = 0, S. NH, NCFI, Pcderred compauade for use In, the connpasitiosu of the pmeat lnvendm Include thwa hav9ng ft farmulae;

N N NGA[5s N N
L
~
[\>cH3 , LCHQfl CN

Another group of preferred compositions include those having the formulae:

N C~
N N
S S ~ I
I
Rzo N Rlo ; R~of\ g-CH3 CH3 CFb Ctb wherein R10 is selected from H, NO2, Ph, 4-HOPh and 4-m-Ph (wherein m is F, Cl, Br, or I).

A particularly preferred subset of the pharmaceutical compounds defined herein are those wherein one of the Y groups is the phenyl moiety defmed above.
These compounds have the following formulae:

Y I
N\
jR Z ~ >==x N R~ N
1 j I

RI RI

In these compounds, Y is selected from H, C!-C:4 alkyl and C,-C4 substituted alkyl, and is preferably H. R' is selected from H, -OH, halogens (F, Cl, Br and I), Cl-C4 alkyl, Cl-C4 substituted alkyl, C,-C4 ester, and C,-C4 substituted ester, and is preferably H, -OH, halogen, -OOCCH2M (where) M is H or a halogen), and is not preferably H. RZ is selected from H, C,-C4 alkyl and Cl-C4 substituted alkyl, and it is preferred that at least one of the R2 groups be methyl. R3 is selected from H, C,-C4 alkyl, Cl-C4 substituted alkyl, and -CH2Ph; preferred R3 moieties are H and methyl. R4 is selected from H, C1-C4 alkyl and C,-C4 substituted alkyl, and is preferably H. X is selected from S and 0, and, is preferably S. Finally, the Z
moiety is selected from -SR' and -OR', and is preferably -SR3. Particularly preferred compounds are those having the structural formulae N N
~-- SH ( ~--- SCH3 l ~=S
N N N

Other preferred compounds include:

N N N
I ~--SH ~--SCH3 >==S
( ~ N`CHs N'CH3 N'CH3 wherein R9 is selected from -OH, -M and -OOCCH2M; and M is selected from F, Cl, Br and I.

Most preferred is the compound having the struicture given below. This compound has demonstrated an unexpectedly high activity in terms of suppressing the expression of MHC Class I and Class II proteins. Further, this compound has shown a different effect on thyroid gene expression, i.e., thyroglobin, when compared to MMI. This suggests that it may be used to treat autoimmune diseases and even Graves' Disease, without requiring thyroid hormone supplementation.
N`
- SH
N
0 +

5-phenylmefl*na2oie WO OuV12175 1wGTtus991tsasz Axnended Page 25 Mixtume of the phairnsa~e+utic~l~y active conVounds defted bereia mtlr aleo be used.

Thc medlhnawlc delvativcs and WutAmeric cyclic thiooes deacr-ed abova can ba eynthesizDd uaing tmbrdqqe: wdl iomarn to thaee 6tiRad in the art. Pvr cwmiple, tha :ynthtsls of mC.ccal wmme& cyclic tbiom is descrlbed iA $Jellin and Sandatom. Acta Chemica Scaaftmvka 23: 2879-2887 (1969).

A rape~aenmEiw met'bfCqavle 8ativadVR tua,y be syatboWmad usft the iollm!ng pioeaba. Appcop:iaoely sabeticuccd amlogs of aoctekMyde art brominated lu the 21osidoa by temcttt wOh biro mine ad UV lWt, lbllowcd by fonnatEon of ft ¾ormpopdiog dicthylacMl taft absolute ctlunal. The brpno}aa Is tea cfiaplacod fiom tld's cadpmnd by Ounw-nt whtt adqdtnns naethybmte, or nrber suitable amine, ia a aealad tndo at about 1ZO for up to about 16 hamn.
Raacdaa of ine rteulting aminneaetal witb potassinaa thiocyaNa in the ptreaeoce of krdrochtnrk acie, at aeam bath tmpcrapum& overnight. prwides the methimeznle.
anaiogs.

Tbc pharmaotnteai cantpoalt[oas of the presoud inrentlaa eompeise asofa vnd effecdve ampaot of uns or mme of the mdb.immlc derlvmdves or tsu0onmeric cyclic tbiode oamdpnunKls ('i.4., the aative oompoanda). Preerrod compoaidm contadn frnm abotst 0.01 % to a-bout 25 96 of the active compounds. with most proletred compooitiom combiniaag ttam about 0,196 to about 10% of tbc activa ooaWauoch. The pbumaccut3Gal oomposittops of ft preaeat invcndm ma0r bo ~ iu.auy way oaava~ionatty kaawa, fo~ exampk~ L~pacitooea!!y.
intravonuus#y, intramvaoularly, or tnpioally. although ural ad.tainbtration is preforrCd. Pt+elaeef oomVtlaitious are in unit dosage farm. i.e., pharmaceudeal ootnpoaitimwbich are available in a preamaaaurcd fom suitable fpr smglc dosaga administration without requiring that the indivicluai dosage be measured out by the user, for example, pills, tablets or ampules.

The pharmaceutical compositions of the present invention additionally include a pharmaceutically-acceptable camer compatible with the methimazole derivatives or tautomeric cyclic thiones described above. In addition to the pharmaceutically-acceptable carrier, the pharmaceutical compositions may contain, at their art accepted levels, additional compatiblle ingredients, such as additional pharmaceutical actives, excipients, formulational aids (e.g., tabletting aids), colorants, flavorants, preservatives, and other nriaterials well known to those skilled in the art.

As used herein, the term "pharmaceutical carrier" denotes a solid or liquid filler, diluent or encapsulating substance. These materials are well known to those skilled in the pharmaceutical arts. Some examples of the substances which can serve as pharmaceutical carriers are sugars, such as lactose, glucose, and sucrose;
starches, such as corn starch and potato starch; cellulose and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose, and cellulose acetate;
powdered tragacanth; malt; gelatin; talc; stearic acid; magnesium stearate; calcium sulfate;
vegetable oils, such as peanut oil, cottonseed oft, sesame oil, olive oil, corn oil and oil of theobroma; polyols, such as propylene glycol, glycerine, sorbitol, mannitol, and polyethylene glycol; agar; alginic acid; pyrogen-free water; isotonic saline; and phosphate buffer solutions, as well as other non=-toxic compatible substances used in pharmaceutical formulations. Wetting agents ar.-d lubricants, such as sodium lauryl sulfate, as well as coloring agents, flavoring agents, tableting agents, and preservatives, can also be present. Formulation of the components into pharmaceutical compositions is done using conventional techniques.

The pharmaceutical carrier employed in conjunction with the pharmaceutical compositions of the present invention is used at a concentration sufficient to provide a practical size-to-dosage relationship. Preferably, the pharmaceutical carrier comprises from about 75% to about 99.99%, preferably from about 90% to about 99.9%, by weight of the total pharmaceutical composition. The methimazole derivatives or tautomeric cyclic thiones defined, in the present application may surprisingly be more soluble than methimazole in conventional carrier materials.
This provides significant benefits in allowing gireater flexibility in the fonnulation of pharnaceutical compositions containing those r.nethimazole derivatives, and may allow the use of significantly lower doses of the active compound.

The present invention also provides a method for treating autoimmune diseases and for preventing or treating rejectioni of tissue in a transplant recipient.
More specifically, this invention relates to methods for administering to a mammal in need of such treatment a drug or drugs, as defined herein, capable of suppressing expression of MHC Class I or Class II molecules.
Examples of autoimmune diseases that can be treated using this method 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, autoinunune enteropathy syndrome, idiopathic thrombocytic purpura, acquired splenic atrophy, idiopathic diabetes insipidus, infertility due to antispermatazoan antibodies, sudden hearing loss, sensoneural hearing loss, Sjogren's syndrerme, polymyositis, autoimmune demyelinating diseases such as multiple sclerosis, transverse myelitis, ataxic sclerosis, pemphigus, progressive systemic sclerosis, dermatomyositis, polyarteritis nodosa, hemolytic anemia, glomerular nephritis and idiopathic facial paralysis. In its broadest aspects, methimazole derivatives of the present invention are administered in a dosage range of from about 0.001 to about 100 milligrams, preferably from about 0.05 to about 50 miliigrams, per day. The pharmaceutical compositions of the present invention are administered such that appropriate levels of pharmaceutical active are achieved in the bloodstream. The precise dosage level required in a given case will depend upon, for example, the particular methimazole derivative used, the nature of the disease being treated, and the size, weight, age and physical cormdition of the patient.

In a preferred embodiment, an active compound of the present invention, for example 5-phenylmethimazole, is administered to a manunal, preferably a human, afflicted with an autoimmune disease. Suitable therapeutic amounts of the methimazole derivatives are in the range of froim about 0.05 to about 20 milligrams per day. A preferred dosage is from about 0.05 to about 10 milligrams per day.
The dosage can be administered daily, in approxirnately equally divided amounts at 8 hour intervals or with breakfast, lunch and dinner. Therapy can be continuous, for example, for periods up to one year or longer. Alternatively, therapy can be tapered, for example, starting at a higher dosage level and tapering to a lower daily dosage level within four to ten weeks. Thyroid, hormone (thyroxin T4 or triiodothyronine T3) or thyroid stimulating hornione (TSH) levels in the individual receiving such treatment may, for some compounds, be an index of therapeutic effectiveness. It is understood by one skilled in the art that the dosage administered to a mammal afflicted with an autoimmune disease may vary depending on the mammal's age, severity of the disease and response to the course of treatment.
One skilled in the art will know the clinical parameters to evaluate to determine proper dosage for an afflicted mammal.

In another preferred embodiment, an active compound described herein is administered to a mammal, preferably a human,, afflicted with systemic lupus erythematosus (SLE). A preferred therapeutic Ennount is in the range of from about 0.05 to about 20 milligrams per day, administered over two to twelve months, but can be administered in discontinuous treatment periods of sunilar length over a five-year period or for as long as necessary. Alternatively, the compositions of the WO 00/12175 PCT(tTS99/19862 present invention may be administered in conjunction with the current therapies for SLE, hydrocortisone and cytotoxic drugs, to su;ppress the disease. SLE
patients with breast cancer cannot be readily treated widh radiation therapy since they are already immunosuppressed by the current conventional treatment for SLE. Also SLE may be associated with unusual sensitivity to radiation complications and therefore radiation therapy exacerbates the disease. It is anticipated, therefore, that the use of the methimazole derivatives and tautomeric cyclic thiones of the present invention to treat SLE individuals with breast cancer will allow radiation therapy to be administered to such individuals without radiation complications or exacerbation of their condition.

In another embodiment, the methimazole derivatives, cyclic tautomeric thiones, and pharmaceutical compositions of the present invention are administered to a mammal, preferably a human, afflicted with type I or juvenile diabetes.
In another embodiment, the methimazole derivatives, tautomeric cyclic thiones, and pharmaceutical compositions of the present invention are administered to. a mammal, preferably a human, afflicted with an autoimmune disease associated with the development of thyroid autoantibodies :in the sera of these animals.
In yet another embodiment, the methimazole derivatives, tautomeric cyclic thiones, and pharmaceutical compositions of the present invention are administered to a mammal, preferably a human, afflicted with an autoimmune disease characterized by the development of receptor autoantibodies. For example, autoimmune asthma is associated with a-adrenergic receptor autoantibodies.
Treatment with the compositions of the present invention will alleviate the disease.
Another example of such an autoimmune disease is myasthenia gravis. Myasthenia gravis is associated with acetylcholine receptor autoantibodies. Individuals afflicted with myasthenia gravis have a higher frequency of thyroid autoimmunity.
Because of the structural and functional relationship betvcreen the TSH and acetylcholine receptors, treatment of an animal, preferably a human, afflicted with myasthenia gravis with a drug able to suppress both MHC Class I and Class II, such as the methimazole derivatives or tautomeric cyclic thiones of the present invention, will -help suppress the disease.
The method of this invention is also suitable for preventing or treating rejection of a transplanted tissue in a recipient mammal, preferably a human.
Examples of tissues which may be transplantedl include, but are not limited to, heart, lung, kidney, bone marrow, skin, pancreatic islet cells, thyroid, liver and all endocrine tissues, neural tissue, muscle, fibroblast, and adipocytes.

As an example of the prevention of rejection of transplanted tissue, pancreatic islet cells are isolated from a donor and treated with a methimaz.ole derivative or tautomeric cyclic thione of the present invention prior to transplantation into a recipient suffering from diabetes. Diabetes is caused by loss of islet cells as a result of autoimmune disease. Transplantation of islet cells will correct such a deficiency. Islet cells may be treated with from about 0.05 to about milligrams of active compound per day, for example, in the form of an aqueous solution for from about 24 to about 72 hours or longer as necessary to suppress 20 expression of MHC Class I molecules on the islet cells. After transplantation, the recipient may be further treated with a methimazole derivative or tautomeric cyclic thione together with hydrocortisone and/or other immunosuppressive agents.

The present invention also relates to an in vitro assay for assessing the ability of a drug to suppress expression of MHC Class I and MHC Class II proteins by measuring the activity of a reporter gene operably linked downstream of a MHC
Class I and MHC Class II promoter and its regulatory sequences. The reporter gene operably linked to an MHC Class I and MHC (:lass II promoter and its regulator sequence is introduced into mammalian cells, said mammalian cells are treated with the candidate drag and the activity of the reporter gene in lysates from treated and untreated mammalian cells is measured. A decrease of activity of the reporter gene in cell lysates from treated versus nontreated cells is predictive of the usefulness of the candidate drug in suppressing MHC Class I and MHC Class II expression and, -in turn, suppressing an autoimmune disease or preventing transplant rejection.
Preferred regulatory sequences that may be operably linked to the =reporter gene are sequences corresponding to the regulatory region of the class I gene, -1 Kb to + 1 bp; upstream silencer/enhancer region cif the MHC Class I, PD 1 gene, -to -673 bp (Figure 1A); the downstream regulatory region of the PD1 gene, -203 or -127 to -lbp; the downstream silencer region of the PD1 gene, -127 to -80 bp (Figure 1B); and the regulatory region of the MHC Class II gene containing the S,Y,X, and X2 boxes, -137 to -50 bp (Figure 1C). These sequences are shown in Figure 1, with their cognate promoters. It will be understood by one skilled in the art that sequentially and functionally homologous regions found in the regulatory and promoter domains of other Class I and Class II genes may also be used.
Examples of reporter genes include, but are not limited to, the chloramphenicol acetyltransferase (CAT) gene, the j3-galactosidase gene, the luciferase gene and human growth hormone (hGH) (Sambrook, J. et al., Molecular Cloning: A
Laboratory Manual, Vol. 2, 2"tl ed.; Cold Sprii:ig Harbor Press, NY (1989);
Ausubel, F. et al. in "Current Protocols in Molecular Biology: Supplement 14, section 9.6 (1987); John Wiley and Sons, New York (1990)). Examples of mammalian cells that can be used in this in vitro assay include, but are not limited to, mammalian cell thyrocytes, hepatocytes, neural tissue, muscle, fibroblasts, adipocytes and HELA cells. The means by which the regulatory sequence operably linked to the reporter gene may be introduced into cells are the same as those described above. In a preferred embodiment the luciferase gene is operably linked to one of the above mentioned PD1 sequences and introduced into FRTL-5 cells.

It is understood by one skilled in the art that the ability of a candidate drug to suppress expression of MHC Class I and MHC Class II molecules can also be assessed by coniparing levels of cellular mRNA in mammalian cells treated with the candidate drug versus cells not treated with the candidate drug. Examples of methods for determining cellular mRNA levels include, but are not limited to, Northern blotting (Alwine, J.C. et al. Proc. Natl. Acad. Sci., 74:5350-5354 (1977)), dot and slot hybridization (Kafatos, F.C. et al. Nucleic Acids Res., 7:1541-1522 (1979)), filter hybridization (Hollander, M.C. et al. Biotechniques;
9:174-179 (1990)), RNase protection (Sambrook, J. et al, in "Molecular Cloning, A
Laboratory Manual", Cold Spring Harbor Press, Plainview, NY (1989)), polymerase chain reaction (Watson, J.D. et al.) in "Recombinant DNA" Second Edition, W.H. Freeman and Company, New Y'ork (1992)) and nuclear run-off assays (Ausubel, F. et al. in "Current Protocols in Molecular Biology"
Supplement 9 (1990); John Wiley and Sons, New York (1990)).

The following examples are intended to illustrate the pharmaceutically active compounds, pharmaceutical compositions and inethods of treatment of the present invention, but are not intended to be limiting thereof.

Preparation of 5-2henylmethimazole The preferred compound 5-phenylmethimazole is synthesized as follows:
Step 1- Synthesis of 2-Bromophenylacetaldehyde diethylacetal The bromination of phenylacetaldehyde: may be performed according to the method of P. Duhamel, L. Duhamel and J-Y `/alnot, Bull. Soc. Chim. Fr. 1973, 1465.
120g (1 mol, 117 mL) of phenylacetald.ehyde is cooled in a three-neck flask to 20 C using dry ice/CCl4. 160g (1 mol) of bromine is added dropwise with vigorous stirring and cooling. Following addition, the reaction solution is transferred to a 2L round bottom flask and approximately 1.5 liters of absolute ethanol added. This solution is stirred overnight at room temperature. The solution is evaporated in vacuo, and extracted with a saturated solution of Na2CO3. The crude product is dried over KzC03 and filtered to yield 145.34g. The fmal product is distilled at 94 C under 501L pressure to yield 86.93g (46%) of pure material.
'H-NMR (CDC13): 51.05 (3H t), 1.30 +(3H t), 3.3-3.9 (5H m), 4.91 (2H
dd), 7.32-7.48 (5H m).

SW 2 - Synthesis of 2-Methylaminophenylacetaldehyde diethylacetal This material may be synthesized using the method of Jones, et al.,.J. Am.
Chem. Soc., 1949, 71:4000.
A 200 mL heavy walled glass reaction vessel is cooled in dry ice/acetone and charged with approximately 25 mL anhydrous inethylamine. To this is added approximately 25 mL (32g) of 2-bromophenylacetaldehyde diethylacetal. The mixture is solidified in liquid nitrogen and the reaction vessel evacuated on a vacuum pump. The reaction vessel is closed and heated in an oil bath at 110 C
overnight. The reaction vessei is cooled in dry ice/acetone followed by liquid nitrogen and the methylamine vented to an aqueous solution. Crude reaction product is taken up in methanol and extracted with 1N aqueous KOH solution and dried over solid KOH. The solution is evaporated Ln vacuo and the crude material is purified by distillation. The product is distilled at 100 C under 255 of pressure.
Yield of material is 89.7%.

'H-NMR (CDC13): 50.95 (3H t), 1.24 (3H t), 2.23 (3H s), 3.23 (m), 3.49- ' 3.54 (m), 3.60 (d), 3.7-3.9 (m), 4.41 (d), 7.26-7.40 (5H m).

Sten 3- Synthesis of 5-Phenylmethimazole This material may be synthesized using the method of R.G. Jones, J. Am.
Chem. Soc., 1949, 71: 383-386.
50g (224 mmol) of 2-methylaminophenylacetaldehyde diethylacetal is dissolved in 100 mL of 50% ethanol/water solution. To this is added, with stirring, 26.123 g (268.8 mmol) of potassium thiocyanate and 22.39 mL (268.8 mmol) of concentrated hydrochloric acid. The mixture is heated on a steam bath for four days in an open beaker. The crude reaction mixture is taken up in ethyl acetate (200 mL) and extracted with water (3x50 mL), saturated solution of Na2CO3 (3x50 ml) and a saturated solution of NaCI (3x50 mL). The solution is dried over Na2SO4, and evaporated in vacuo to yield 47.65g of an oranlge-red oil. The material is taken up in benzene and chromatographed on 450g of sillica gel. The column is initially eluted with benzene followed by 10% ethyl acetate:benzene upon the appearance of product. Fractions containing product are com'bined into three groups.
Fractions A
and C are combined to yield 13.93g and recrysitaIlized from ethanol to yield 2.3g.
Group B is recrystallized to yield 3.9g. Recrystallized yield 14.5 %. Melting point 168-173 C.
`H-NMR (CDC13): 53.59 (3H s), 6.76 (1H s), 7.42 (5H m).

PMparation of 1-methyl-5-phen l'imidazoline-2 3-thione See Kjellin and Sandstrom, Acta Chemica Scandinavica 23: 2879-2887 (1969).

(SH..
~

11 -c~
C--C}i-NIJC, +CViCS (Bt)3No ~1~~
FtOf-I CtoNAs To a 1 liter r.b. flask equipped with a nnechanical stirrer, addition funnel, condenser, and N2 inlet is added 50 g of a-aminoacetophenone hydrochloride, 22.5 mL of methyl isothiocyanate, and 600 mL of absolute ethanol. To this mixture is added 42 mL of triethylamine. The reaction solution is heated at reflux for 1 hr.
The reaction mixture is stripped to near-dryness. To the residue is added 150 mL of water. The resulting suspension is suction filtered and 80 g of yellow solids is collected. This filter cake is slurried in 500 mL of 1N NaOH
solution.
The insolubles are filtered off. To the filtrate is added approximately 100 mL
of 37% HCI. The resulting suspension is suction filtered and the solids recrystallized from 50 mL of a 25 % aqueous ethanol solution.

25.7 g of pale pink solids are collected, m.p. 178-179 . HPLC indicates 99.8% purity.
Elemental analysis is as follows:
Found: C, 62.82; H, 5.21; N, 14.74; S, 16.63 Theory: C, 63.13; H, 5.30; N, 14.73; S, 16.86 IR, carbon NMR, and proton NIYIR. all support the desired structure.

Preparation of 1-methvl-2-methylthio-5-phenyi-imidazole N Sti ( ~
+ CH31 NaOH )0. N
{N I
EtOH cHa ~H3 MW 204.3 To a 1 liter r.b. flask equipped with a mechanical stirrer, condenser, thermometer, and addition funnel are added 19.1 g of 1-methyl-5-phenyl-imidazoline-2(3)-thione (Compound 10) and 335 mL of 95% ethanol. To this is added 4 g of NaOH and the mixture is stirred until a solution is realized.

To this is added 15.6 g of methyl iodidE: in 145 mL of 95 % ethanol over 35 minutes at ambient temperature (28-30 C). The resulting mixture is stirred at ambient for 3 hrs. The resulting mixture is stripped on the rotary evaporator.
Approximately 41 g of orange colored solids aire collected. This is dissolved in approximately 600 mL of methylene chloride and to this is added approximately mL of water. The organic layer is separated. The aqueous layer is extracted twice with methylene chloride, and the organic layers combined, dried over sodium sulfate filtered and stripped on the rotary evaporator, to afford 26.7 g of oily solids.
This is suction filtered and air dried to yield 16.5 g of pale orange solids, m.p. 85-87 C. This is recrystallized (along with 0.8 g and 2.5 g of product from 2 small scale preps) from a total of 2.5 L of heptane. 10.7 g of off-white solids is collected;
m.p. 87 C. HPLC indicates 99.8 % purity.
Elemental analysis:
Found: C, 64.70; H, 5.85; N, 13.70; S, 15.55 Theory: C, 64.66; H, 5.93; N, 13.70; S, 15.72 IR, carbon NMR, and proton NMR all give evidence for the desired product.

Preparation of 1.3-dimethyl-4(5)-phenyl-imidazoline-2(3)-thione cl-h s 0 1-h~anol C-Cli-OH + CFj-NH--ll--Nl-t--C}-h alyy N
MW2Ut.3 Ct,H~i~s To a 100 mL r.b. flask equipped with a Dean-Stark trap, condenser, N2 inlet and magnetic stirring are added 13 g of a-hydroxyacetophenone, 10 g of N,N-dimethylthiourea, and 50 mL of 1-hexanol. Thiis is heated at reflux for approximately 3.5 hrs. Approximately 3 mL of water is collected.

Precipitation occurrs after placing the reaction mixture in the freezer for 1 hr. The resulting mixture is suction filtered. 13.2 g of solids are collected, m.p.
105 C. This (along with 10.4 g of product froin another prep.) is recrystallized from approximately 150 niL of absolute ethanol. 12.5 g of white solids are collected, m.p. 126-127 C.
HPLC indicates 99.8% purity.
Elemental analysis indicates:
Found: C, 64.44; H, 5.81; N, 13.70;. S, 15.55 Theory: C, 64.66; H, 5.93; N, :13.71; S, 15.71 IR, carbon NMR, and proton NMR all support the desired structure.

Preparation of 4-nitro-l,3-dimethylimidazole-2-thi ne While there is no report of the preparation of this compound 6 in the literature, using known nitration reactions conditions provides the desired product in reasonable yield. The starting material for this nitration, Compound 5, can be readily prepared in two steps from commercially available materials using known reaction pathways and conditions.

N N CH3 N "CH3 ~ ~
' + CH31 ---~- \ ~~ r ---.- ~ ~s CHg CHg I

N
S ------ .. S
-N

Assessment of the Effect of MMI-derivatives on MHC Class I and MHC Class II Expression by Gel Shift Assay Materials Purified bovine TSH was from the NIII program (NIDDK-bTSH-I-1, 30U/mg) or was prepared as described previously (Kohn, L.D. and Winand, R.J.
J.
Biol. Chem., 250:6503-6508 (1975)). Insulin, hydrocortisone, human transferrin, somatostatin, and glycyl-L-histidyl-L-lysine acetate were from Sigma Chemical Co.
St. Louis, MO.

Cell Culture FRTL-5 rat thyroid cells (Kohn, L.D., et al., U.S. Patent No. 4,609,622 (1986); Ambesi-Impiombato ES., U.S. Patent No. 4,608,341 (1986)) are grown as described below. These cells do not proliferate in the absence of TSH, yet remain viable for prolonged periods in its absence. Their doubling time was approximately 36 6 hours; and, after 6 days in medium wilth no TSH (511) and 5.0% serum, 1x10'10 mol/L TSH elevated iodide uptake 8-10 fold and thymidine incorporation> 10 fold. Cells were diploid, between their 5' and 25' passage in most experiments, and were routinely grown in Coon's modified F12 medium supplemented with 5% calf serum, I mmol/L iionessential amino acids (GIBCO) and a mixture of 6 hormones (6H medium): TSH (1x10'!0 M), insulin (10 g/ml), hydrocortisone 0.4 ng/ml, human transferrin (5 g/ml), somatostatin (10 ng/ml) and glycyl-L hisfidyl-L-lysine acetate (10 ng/ml) (Kohn, L.D. et al., U.S. Patent No.
4,609,622 (1986); Ambesi-Impiombato, E.S., U.S. Patent No. 4,608,341 (1986)).
They were passaged every 7-10 days and provided fresh media every 2 or 3 days.
In individual experiments, cells were grown to near confluency in 6H medium then, in some experiments, were shifted to medium with no TSH (5H), to medium with neither TSH and or insulin (4H), or to medium, with no TSH, no insulin and no hydrocortisone (3H) plus either 5% or 0.2% serum for 4-7 days before use.

Cell Extracts Cells were grown in 6H medium with !i % calf serum for 6-7 days to 70-80%
confluence, then shifted to 5H medium with 5% calf serum for 5 days. TSH
(1x10"" M), gamma-interferon (100 U/mi), MIVII (5 m1VI), and different concentrations of MMI derivatives or tautomeric cyclic thiones (0.0001 to 10 mM) were added as appropriate for 24-48 hours. Cells were then harvested and extracts were made by a modification of a method of Dignam, J., et al. Methods in Enzymologv, 101:582-598 (1983). In brief, cells were harvested by scraping after being washed twice with cold phosphate-buffer saline (PBS). Subsequently they were pelleted, washed in cold PBS and then pelleted again. The pellet was resuspended in Dignam buffer C (20 mM Hepes buffer at pH 7.9, 1.5 mM MgC12, 0.42 M NaCI, 25% glycerol, 0.5 mM dithiotreitol (DTT), 0.5 mM
phenylmethylsulfonylfluoride (PMSF), 1 g/L leupeptin, 1 g/L pepstatin). The final NaCI concentration was adjusted on the basis of cell pellet volume to 0.42 M
and cells were lysed by repeated cycles of freezing and thawing. Extracts were then centrifuged at 10,000 xg at 4 C for 20 min. The supernatant was recovered, aliquoted and stored at -70 C.

Gel Mobidity Shift Assay Binding reactions were performed in a volume of 20 l for 30 min at room temperature. The typical reaction mixture contained 1.5 frnol of 32P DNA, 3 g of cell extracts, 1 to 3 g of poly (dI-dC) in 10 mM Tris-Cl (pH 7.9), 1 mM
MgC121 1 mM DTT, imNi ethylenediamine tetra acetic acid (EDTA), and 5% glycerol. After incubation, reaction mixtures were subjected to electrophoresis in 4%
polyacrylamide gels for 90-120 min at 160 V in,0.5x TBE (Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2' ed. Cold Spring Harbor Press, NY, 2: 11.23-11.44 (1989)), then dried and autoradiographed. Probes were labeled with [a32P] deoxy CTP by Klenow enzyme (In Vitro labeling kit, Amersham) following the manufacturer's instructions, or by [02P] ATI' using T4 polynucleotide kinase.

Labeled probes were then purified either throiugh G-50 columns (5 prime-43 Prime) or on an 8% polyacrylamide gel.

Cell extracts to perform class II gel sh:ift studies were prepared exactly as for class I studies (Dignam et al., ibid, 582-598 (1983); Giuliani C, et al., J.
Biol.
Chem. 270: 11453-11462 (1995); Saji M, et al., J. Biol. Chem. 272: 20096-20107 (1997); Montani, V., et al., Endocrinology 139: 280-289 (1998a); Montani, V., et al., Endocrinology 139: 290-302 (1998b)), with the exception that the extracts were from 'e-interferon treated cells, usually 100 U/'ml interferon for 24 to 48 hours.

Similarly, electrophoretic mobility shift assays (EMSA) were performed in the same way. Oligonucleotides used for EMSA were synthesized (Operon Technologies, Inc.) or were purified from 2 % agarose gels using either QIAEX (Qiagen, Chatsworth, CA) or Jet Sorb (Genomed) following restriction enzyme treatrnent of chimeric HLA-DRa-CAT constructs (Montani et al., 1998a, b). The oligonucleotides were dephosphorylated with calf intestinal alkaline phosphatase, labeled with [Y-32P]dCTP or with [x 32P]ATP using T4 polynucleotide kinase, then purified on an 8% native polyacrylamide gel (Giuliani C, et al., ibid, 11453-(1995); Saji M, et al., ibid, 20096-20107 (1997); Sambrook J, et al., ibid, 2:11.23-11.44 (1989); Montani, V., et al., ibid, 280-289 (1998a), 290-302 (1998b)).
Binding reactions for class II EMSA used similar conditions as well. They were carried out in a volume of 20 l for 30 min at room temperature. The reaction mixtures contained 1.5 fmol of [32P]DNA, 3 g; cell extract and 1.5 to 3 g poly(dI-dC) in 10 mM Tris-Cl at pH 7.9, 1 mM MgC12, 1 mM DTT, 1 mM EDTA, and 5%
glycerol. Where indicated, unlabeled double- c-r single-stranded oligonucleotides were added to the binding reaction as competitors and incubated with the extract for 20 min at room temperature prior to the addition of labeled DNA. Similarly, in experiments using antiserum, extracts were also incubated in the same buffer containing antiserum or nonmal rabbit serum foir 20 min at room temperature before being processed as above. Following incubations, reaction mixtures were subjected to electrophoresis on 3.5 or 5 % native polyac:rylamide gels at 160 V in 0.5xTBE, at room temperature, for 1.5 to 2 h. Gels were dried and autoradiographed at -80 C
overnight unless otherwise noted.

Other Methigds Protein concentration was determined by Bradford's method (Bio-Rad);
recrystallized bovine serum albumin was the standard. DNA was prepared and purified by CsCI gradient centrifugation (Davis LG, et al., Basic Methods in Molecular Biology, Elsevier, NY, pp 93-98 (1986)). The sequences of all constructs were confirmed by a standard method (Sanger F, et al., Proc. Natl.
Acad. Sci. USA 74:5463-5467 (1977)). Values are the mean SE of these experiments where noted. Significance betweeii experimental values was determined by two-way analysis of variance anci are significant if P values were < 0.05 when data from all experiments were considered.
MMI-Sensitive Elements in MHC C:lass I and Class II Genes MHC Class I
Regulatory elements responsive to MMI have been identified in the promoter of the swine MHC class I gene, PDI. Two regEilatory domains have been identified in the 5' flanking region of the PD1 gene. A downstream regulatory domain is between approximately -203 and -1 bp from the transcriptional start site (Figure 1B). This region contains an interferon response element and a major enhancer (Enhancer A), as well as a site homologous to a cyclic AMP response element (CRE) element. Studies using gel mobility shift assays have demonstrated that MMI-induced or modified proteins interact with this region and can regulate transcription initiation (Saji, et al. ibid., 20096-20107 (1997); Singer et al., U.S.
Patent 5,556,754, issued Sep. 17, 1996) particullarly Y box proteins. Another complex regulatory region, showing overlapping silencer and enhancer activity, has been mapped between -769 and -673 base pairs upstream of the promoter (Weissman, J.D. and Singer, D.S. Mol. Cell. B:iol. 11:4217-4227 (1991)). The enhancer and silencer elements in this upstreani regulatory domain are linked to tissue specific expression and tissue specific levels of the Class I gene (Weissman, J.D. and Singer, D.S., ibid, 4217-4227 (1991)) and involve Sox-4 (Singer, DS
et al., U.S. Patent 5,556,754 (1996)).
Saji, et al. (J. Clin. Endocrinol. Metab. 75: 871-878 (1992b)) showed reduced expression of MHC Class I gene in rat FRTL-5 cells treated with MMI.
This study also showed that the effect of MMI in MHC Class I expression was at the level of transcription. MMI increases the appearance of a normal complex with the downstream regulatory domain and decreases the appearance of the silencer complex of the upstream regulatory domain. These changes result in decreased constitutive control (upstream regulatory domain) and dominant hormonal control (downstream regulatory domain). The MMI action is additive to TSH which acts on the downstream regulatory domain (Saji, et al., ibid, 20096-20107 (1997) and Singer, et al., U.S. Patent 5,556,754 (1996)).

The FRTL-5 thyroid cell system is therefore a good system to identify the regulatory DNA sequence elements and trans-acting factors involved in the MMI
effect. PD1 Gel shift mobility assays were performed using the 5' flanking region of the PD 1 gene and cell extracts from FRTL-5 cells treated or not with x-interferon and treated with MMI and MMI derivatives and tautomeric cyclic thiones (see Table 1).

Figure 1A shows the silencer and enhancer regions of the 140 region oliogonucleotide used to map the region for the MMI derivative or tautomeric cyclic thione activity of the gel shifts. The silencer and enhancer regions of relevance are noted by the arrows. The 140 fragment is derived from the PDl promoter of the PD1 Class I MHC gene (Singer, D.S., et a1. Proc. Natl. Acad. Sci. USA, 79:1403-1407 (1982)).

Figure 2 shows a gel shift using the radio labeled 140 fragment noted in Figure 1 and shows the silencer complex regulated by MMI.

Table H
Compounds Imidazole LF4~!N

#1 1-Methylimidazole-2-thiol (Methimzaole) I
C4H6NA LN~rSH
1-Methyl-2-mercaptoimidazole (MMI) i H2CH2OH
#2 2-Methyl-5-nitro-l-imidazole ethanol 02N CH3 (Metronidazole) y C6H9N3O3; MW: 171.16 CN
H
I
NJ SH
#3 2-Mercaptoimidazole ~
MW: 100.14 õ
N
H SH
#4 2 Mercaptobenzimidazole N
MW: 150.20 Y
N
H SH
#5 2-Mercapto-5-nitrobenzimidazole N
MW: 195.20 ~

H SH
#6 2-Mercapto-5-methylbenzimidazole / N
MW: 164.23 Y
N

#7 S-Methylmethimazole N
CSHgN2s' SCH3 MW: 128.20 B.P. 48 @100u (liq.) N
\

#8 N-Methylmethimazole /CH3 CSH8N2S; N
MW: 128.20 ~S
B.P. 188 -194 N
~

#9 5-Methylmethimazole CSHgN2S;
MW: 128.20 H
B.P. 254 - 255 #10 5-Phenylmethimazole N
C10HioN2S; ~ ~-SH
MW: 190.27 N
B.P. 168 -173 'CH3 #11 1-Methyl-2-Thiometlhyl N
-5(4)nitroimidazole NO [)___SCH3 \CHg - 4? -Lane 2 shows the complex fortned between the silencer region and cell extracts from FRTL-5 rat thyroid cells maintained in the presence of 5H medium (no TSH) plus 5% serum. The effect of the addition of 5 mM MMI to cells for 48 -hours prior to extract preparation from cells maintained in 5H medium is shown in lane 1 of Figure 2. The effect of the addition of 1 mM concentration of representative MMI derivatives to cells maintained in 5H medium for 48 hours prior to extract preparation is shown in lanes 3 to 8 in Figure 2. The structures and names of the representative numbered compounds are in Table 1. The MMI or its derivatives decrease the silencer complex. The decrease can be quantitated by densitometry of autoradiograms or phosphoimalging on a Bas 1500 imager (for example). Quantitation is based on the decrease relative to the control and normalized by the unchanged faster migrating complexes.

The following (Table 2) summarizes results from experiments with the compounds described in Table 1 but modified to include different concentrations of active compounds in the EMSA. Compound 10 is the most effective.

Table 2: EFFECT OF 11 COMPOUNDS ON GEL SHIFTS WITH UPSTREAM

WITH THE NOTED ACTIVE COMPOUNDS.

% INHIBITION OF CONTROL SILENCER COMPLEX FORMATION
Compound 10 M 100 M 1 mM 5 mM
Control 0 0 0 0 1 Methimazole 0 0 20 f 15 75 f 12 2 Metronidazole 0 0 45 t 19 86 ~ 15 3 2-inercaptoimidazole 0 0 0 0 4 2-mercaptobenzimidazole 0 5 75 t 10 Insol 5 2-mercapto-5- 0 0 0 Insol nitrobenzimidazole 6 2-mercapto-5- 0 0 20 18 Insol meth lbenzimidazole 7 S-methylmethimazole 0 10 13 50 9 ND
8 N-methylmethimaiole 0 18 t 18 70 f 14 ND
9 5-methylmthimazole 0 0 15 15 30 20 5-Phenylmethimazole 25 60 10 85 15 ND
11 1-methyl-2-thiomethyl- 0 20 f 12 76 18 ND
5(4)nitroimidazole Values from three separate experiments SD.
ND = Not done Figure 3 denotes the sequence of the downstream region of. the PD 1 promoter. The location of enhancer A (-180 to -170 bp), the interferon response element (IRE; -161 to -150 bp), and the cAMP response element (CRE; -107 to -100 bp) are noted. The downstream silencer is between -127 and -80 bp (Saji, et al., ibid, 20096-20107 (1997)). Using a 127 bp probe, -125 to +1 bp, MMI (lane 4) or TSH (lane 10) induce the formation of a rapidly migrating protein/DNA
complex (Fig. 4) which is clearly evident 24 to 48 h after 5H cells are treated.
MMI derivatives can also increase this complex, as illustrated for compound 8 in Figure 4 (lanes 1 to 3). The time most suitable for measurement is 24 to 48 h (Fig.
4) and the complex can be quantitated by comparison to control (lane 7 or 9) using densitometry or the phosphoimager as noted earlier. In this case, the increase over _ control (set at 1) is measured in arbitrary units relative to the maximal increase induced by 5 mM MMI which is arbitrarily set at 10. The results using all 11 compounds in Table 1, each added to cells at diifferent concentrations 48 hours before extracts were prepared, are summarized in Table 3. Compound 10 clearly is again most effective when several experimental sets using extracts from different batches of cells were evaluated.
TABLE 3: EFFECT OF 11 COMPOUNDS ON GEL SHIFTS WITH

CELLS TREATED WITH THE NOTED ACTIVE COMPOUNDS

% INCREASE OVER CONTROL RELATIVE TO THE MAXIMAL (5 mM) MMI-INCREASED SILENCER COMP'LEX WHICH IS SET AT 10 Compound 10 M 100 M 1 mM 5 mM
Control 0 0 0 0 1 Methimazole 0 0 2 t 1 10 2 Metronidazole 0 0 5 f 1 10 t 2 3 2-mercaptoimidazole 0 0 0 0 4 2-mercaptobenzimidazole 0 3 t 0.5 9 f 1.5 Insol 5 2-mercapto-5- 0 0 0 Insol nitrobenzimidazole 6 2-mercapto-5- 0 0 3 2 Insol methylbenzimidazole 7 S-methylmethimazole 0 2 1 6 0.5 ND
8 N-methylmethimazole 0 3 t 2 9 1 ND
9 5-methylmethimazole 0 0 1.5 t 1.5 2.5 t 1 10 5-Phenylmethimazole 3 f 1.5 7 1 10 f 1 ND
11 1-methyl-2-thiomethyl- 0 2 1 10 f 1 ND
5 4 nitroimidazole Values from three separate experiments SD.
ND = Not done MHC Class II

Major histocompatibility complex (MH(:) class II molecules are heterodimeric transmembrane glycoproteins which are encoded by the HI.A-D
region on chromosome 6 and play a central role in immune function (German RM, et al., Ann. Rev. Immunol. 4:281-315 (1986); ,Schwartz RS, and Datta SK, Autoimmunity and Autoimmune Diseases, in Paul, W.E. (ed), Fundamental Immunolov, Raven Press, NY, pp 819-866 (1989); Benoist C, et al., Ann. Rev.
Immunol. 8:681-715 (1990); Gliuncher LH, et al., Ann. Rev. Immunol. 10:13-49 (1992); Ting JP-Y, et al., Curr. Opin. Immunol. 5:8-16 (1993)). Class II
antigens are usually expressed on antigen presenting cells such as B cells, macrophages or dendritic cells; the class II molecules present anitigenic peptides to CD4 positive T
lymphocytes, causing T cell activation (German RM, et al., ibid, (1986);
Schwartz RS, et al., ibid, (1989); Benoist C, et al., ibid, (1990); Glimcher LH, et al., ibid, (1992); Ting JP-Y, et al., ibid, (1993)). Class I1 expression is normally not evident on epithelial cells, such as thyrocytes, synovial cells, or islet cells;
abnormal or aberrant expression of class II molecules on thyrocytes, synovial cells, or islet cells is associated with autoimmune thyroid diseases, rheumatoid arthrids, and diabetes, respectively (Bottazzo GF, et al., Lancet 2:1115-1119 (1983); Bottazzo GF, et al., N. Engl. J. Med. 313:353-360 (1985); Todd I, et al., Ann. NY Acad. Sci.
475:241-249 (1986); German RM, et al., ibid (1986); Burmester GR, et al., J. Clin.
Invest.
80:594-605 (1987); Piccinini LA, et al., Clin. Endocrinol. (Oxf) 26:253-272 (1987); Schwartz RS, et al., ibid (1989); Benoist: C, et al., ibid (1990);
Glimcher LH, et al., ibid (1992); Ting JP-Y, et al., ibid (1993)). The assumption emerged that aberrant class II expression allowed cells to 'become antigen presenting cells, interact with T-cells, and initiate an immune response (Schwartz RS, et al., ibid (1989); Weetman AP, et al., Endocr. Rev. 15:788-830 (1994)). It was, nevertheless, not clear whether this was a secondary response to cytokines, such as 'e-IFN produced by lymphocytes infiltrating the tissue, or was the result of a more primary insult on the tissue itself. More importantly, there was little direct evidence that class II was critical in the initiation of autoiniinune thyroid disease (Weetman AP, et al., ibid (1994)). However, a recent stuciy makes this hypothesis more attractive (Shimojo N, et al., Proc. Natl. Acad. Sci. USA 93:11074-11079 (1996)).

One form of autoimmune thyroid disease (ATD) is Graves' disease, wherein autoantibodies develop to the TSHR and induce hyperthyroidism by mimicking the action of TSH. Whereas numerous attempts to develop a Graves' disease model by immunizing animals with the extracellular domain of the TSHR have largely failed (see, for example, Seetharamaiah, G.S. et aI., Autoimmunity 14: 315-320 (1993);
Costagliola et al., J. Mol. Endocrinol. 13: 11-21 (1994); Costagliola et al., Biochem. Biophys. Res. Commun. 199: 1027-1034 (1994); Costagliola et al., Endocrinology 135: 2150-2159 (1994); Marion, A., et al., Cell. Immunol. 158:
329-341 (1994); Wagle, NM, et al., Autoimmunity 18: 103-108 (1994)), immunizing mice with fibroblasts transfected with the human TSHR and a MHC
class II molecule, but not either alone, has induced ATD with the major humoral and histological features of Graves' (Shimojo N, et al., ibid, 11074-11079 (1996)):
stimulating TSHR antibodies (TSHRAbs), thyrotropin binding inhibiting immunoglobulins (TBII) which are different fronl stimulating TSHRAbs, increased thyroid hormone levels, thyroid enlargement, anci thyrocyte hypercellularity.
These results suggest that aberrant expression of MHC class II molecules on thyrocytes can result in the induction of functional TSHRAbs which stixnuiate the thyroid.
They suggest that acquisition of antigen-presentiElg ability on a thyroid cell, as a result of aberrant class II expression, can activate; T and B cells normally present in an animal, thereby allowing normal immune toleirance to be broken. By analogy, this is relevant to the development of rheumatoid disease, diabetes, and numerous other autoimmune diseases associated with autoimmunity and aberrant MHC class II
expression. Understanding the basis for aberrant class II expression in thyrocytes is, therefore, a potentially unportant aspect of understanding the pathogenesis not only of autoimmune thyroid disease but many other auxounmune diseases. Developing agents which suppress MHC class 11 aberrant expression is important, therefore, in suppressing the autoimmune state. These agents may additionally suppress MHC
class I.

The ability of x-interferon (W-IFN) to induce class II antigen expression in FRTL-5 thyroid cells and mimic changes in hunlan thyrocytes seen in ATD is well described (Todd I, et al., ibid (1985); Platzer M:, et al., Endocrinology 121:2087-2092 (1987); Misaki T, et al., Endocrinology 1?.3:2849-2855 (1988); Zakarija M, et ai., Mol. Cell. Endocrinol. 58:329-336 (1988)). HLA-DR gene expression in rat FRTL-5 thyroid cells has been studied in the FR'.TL-5 thyroid cell model in order to define elements and factors important for x-IFI`f-induced aberrant expression (Montani V., et al., ibid (1998a, b)) and is, therefore, a reasonable model to define elements and factors that might be important in ATD and other immune diseases.
Using an HLA-DR 5 -flanking region construct from -176 to +45 bp coupled to the chioramphenicol acetyl transferase (CAT) reporter gene, it was shown that, unlike lymphocytes, there is no basal class II gene expression in thyrocytes, but, like some immune cells, x-IFN can induce expression (Montani V., et al, ibid, (1998a, b)).
The ability of x-IFN to induce aberrant HLA-DIRt gene expression in FRTL-5 thyroid cells requires, like antigen presenting cells of the immune system which normally express MHC class II, the highly conserved S, Xi, X2, and Y boxes on its 5'-flanking region, -137 to -65 bp (Benoist C, et al., ibid (1990); Glimcher LH, et al., ibid (1992); Ting JP-Y, et al., ibid (1993); R:eith W, et al., Immunobiology 193:248-253 (1995); Montani V., et al, ibid (1998a, b)). Using gel shift assays and the HLA-DR 5'-flankang region from -176 or -137 to +45 bp as radiolabeled probes, the formation of a major protein/DNA ccimplex and a minor, faster migrating complex with extracts from FRTL-5 cells untreated with x-IFN was observed (Montani V., et al, ibid (1998a, b)). x-IFN-induced aberrant expression is associated with increased formation of a specific and novel protein/DNA
complex containing CBP, a coactivator of cAMP response element binding proteins (Montani V., et al., ibid (1998a, b)).

Two factors known to regulate class II gene expression in immune cells are the class II transactivator (CIITA) and a Y box binding protein (Ting JP-Y, et al., -ibid (1993); Reith W, et al., ibid (1995)). CIl[TA is a non DNA-binding protein transactivator that functions as a molecular switch to control constitutive and inducible MHC class II gene expression in izwmune cells; CIITA expression is induced by x' -IFN and is believed to be involved in its activity (Steimle V, et al., Science 265:106-109 (1994); Reith W, et al., ibid (1995); Chang CH, et al., J.
Exp.
Med. 180:1367-1374 (1996); Mach B, et al., Ann. Rev. Immunol. 14:301-331 (1996)). The human Y box protein, YB-1, was cloned based on its ability to bind to the Y box, an inverted CCAAT box, of the MHC class II gene (Didier DK, et al., Proc. Natl. Acad. Sci. USA 85:7322-7326 (19'88)) and has been shown to suppress HLA-DR gene expression in human glioblastoima cells (Ting J P-Y, et al., J.
Exp.
Med. 179:1605-1611 (1994); MacDonald GH, et al., J. Biol.Chem. 270:3527-3533 (1995)). CIITA can induce the formation of the complex induced by 't-IFN and associated with aberrant class II gene expression in FRTL-5 cells (Montani V., et al., ibid (1998b)); overexpression of the Y box: protein in FRTL-5 cells suppresses the formation of this complex (Montani V., et al., ibid (1978b)). It is reasonable to presume, therefore, that this complex is involved in aberrant class II
expression associated with ATD and is related to aberrant class II expression in other autoimmune diseases. Moreover, the data support the conclusion that the negative regulation of class II, as well as the class I genes, involves common transcription factors, the Y box protein, consistent with the hypothesis (Kohn LD, et al., ibid (1997); Saji M., et al., ibid (1998)) that coordinate negative control of class II, as well as class I genes, by common transcription factors maintains self tolerance during hormone-induced increases in thyroid cell growth and function.
Methimazoie (MMI) is an agent effective in treating Graves' disease and preventing experimentai thyroiditis in rats or mice (Cooper DS, N. Engl. J.
Med.
311:1353-1362 (1984); Davies TF, et al., J. Clin. Invest. 73:397-404 (1984);

Reinhardt W, et al., Endocrinol. Invest. 12:559-563 (1989)). The action of methimazole on MHC class II gene expression in thyrocytes has, however, been 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 (Carel JC, et al., in The Thyroid and Autoimmunity, Drexhage and Weirsinga (eds), Excerpta Medica, Amsterdam, pp. 145-147 (1986); Aguayo J, et al., J.
Clin Endocrinol. Metab. 66: 903-908 (1988); Davies TF, et al., Clin. Endocrinol.
(Oxt) 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 (Paschke R, et al., J. Clin. Endocrinol. Metab. 80:2470-(1995)). Nevertheless, formation of x-IFN or CIITA-induced complex, as well as increased HLA-DR gene expression, was suppressed by methimazole in FRTL-5 thyroid cells (Montani V., et al., ibid (1998a)) as a funetion of time and concentration. Thus, MMI suppression of this complex in FRTL-5 cells can be considered a marker of its ability to suppress aberrant MHC class II
expression and autoimmune disease. The clinical relevance oi" aberrant class II and abnormal class I expression in autoimmune disease has recently been demonstrated by the observation that iodide suppresses both class II and class I expression, which are elevated in Graves' thyroids (Schuppert F, et al., J. Clin. Endocrinol. Metab.
81:3622-3628 (1996)); iodide is an agent which like MMI can be used to treat Graves' patients and has been used extensively to prepare Graves' patients for thyroidectomies (Nagataki S, et al., Autoregulation: effects of iodide. In:
Braverman LE, Utiger RD (Eds) Werner and I:ngbar's The Thyroid: a fundamental and clinical text. Lippencott-Raven, Philadelphia, 241-247 (1996)).
Figure 5 depicts the nucleotide sequence of the 5'-flanking region of the HLA-DR gene from -176 bp. The S, X,, X2, and Y boxes or elements, which are conserved in all class II MHC promoters characterized to date, HLA-DR, -DQ, and -DP, are underlined (bold) and their 5' and 3' termini in DR numbered. The more restricted S box site noted in some reports (Tinlg, JP-Y, et al., ibid (1993);
Benoist C, et al., ibid (1990)) is noted by a dashed line over the more extensive S
box used herein or in other reports (Reimold AM, et al., J. Immunol. 151:4173-4182 (1993)).

Figure 6 depicts the electrophoretic mobility shift analysis of the ability of a 32P-radiolabeled DR a-5'-flanking region probe to form protein/DNA complexes with extracts from FRTL-5 cells maintained ivith and without TSH and treated or not with x-IFN. The probe was excised by restriction enzyme treatment of a -137 to +45 bp DR -CAT chimera (Reimold AM, et al., ibid (1993); Montani V., et al., ibid (1998a, b)) and is diagrammatically represented at the bottom of the Figure.
Extracts were from FRTL-5 rat thyroid cells grown to near confluency in TSH or maintained 6 days in 5H medium; duplicate ciultures of each were treated with U/ml w-IFN for the last 48 h before the experiment was terminated. Cell extracts made from each set of cells were incubated with the 32P-radiolabeled probe containing -137 bp of the DR a5'-flanking region and EMSA performed as described above. In this experiment, the autoradiogram was exposed overnight at -70 C.

Figure 7 depicts the effect of MMI on the ability of x-IFN to increase the formation of protein/DNA complexes with the 32P-radiolabeled DR a5'-flanking region probe. The radiolabeled probe is the same as that used and diagrammatically presented in Figure- 6; it contains -137 bp to -I-45 bp of the 5'-flanking region of DRoc -CAT chimera. Extracts were from FRTL-5 rat thyroid cells grown to near confluency in TSH, maintained 6 days in 5H rnedium, then treated with 100 U/ml x-IFN, 5 mM MMI, or both for the last 48 h before the experiment was terminated.
Cell extracts were incubated and EMSA perfo:rmed as in Figure 6 and as described above. The arrows denote the upper and lowe+r complexes seen in Figure 6. In this experiment, the autoradiograms were exposed 72 h at -70 C. Lane 5 contains extract from control cells; extracts from cells treated with IFN or MMI are noted at the top of the panels. The same results were cibtained if cell extracts were from FRTL-5 rat thyroid cells grown to near confluency in TSH and treated with 100 U/ml tt-IFN for the last 48 h before the experiment was terminated.

Using the same protocol as for MMI (Fig. 7), but different exposure times of autoradiograms, various active compounds (MMI derivatives or tautomeric cyclic thiones) were shown to decrease class II complexes linked to interferon-induced (aberrant) or constitutive expression of class II on the cell as exemplified in Figure 8. Compounds 2, 7, 8 are significantly more effective suppressors than MMI
(lanes 4 in Fig. 8A and 2 and 4 in Fig. 8B vs. lanes 2 in Fig. 8A and 3 in Fig. 8B, respectively). Quantitation of the mean decrease in the IFN-induced complex for each of the compounds is evident in Table 4.

In sum, the effect of different derivatives on aberrant class II expression, measured by the decrease in x-IFN increased complex formation differs from compound to compound and can be quantitated. Compound 10 is most effective as is the case for Class I shifts described above.

Table 4: EFFECT OF 11 COMPOUNDS ON' GEL SHIFTS WITH 137 CLASS
II PROBE USING EXTRACTS FROM x-IFN-TREATED SH CELLS WHICH
ARE ALSO TREATED WITH THE NOTELI ACTIVE COMPOUNDS.

% IlITHIBITION OF IFN-INCREASEI) COMPLEX FORMATION
Compound 10 M 100 M 1 mM 5 mM
Control 0 0 0 0 1 Methimazole 0 0 10 t 5 85 t 12 2 Metronidazole 0 0 55 f 14 95 t 10 3 2-mercaptoimidazole 0 0 0 0 4 2-mercaptabenzimidazole 0 5 65 f 15 Insol 5 2-mercapto-5- 0. 0 45 21 Insol nitrobenzimidazole 6 2-mercapto-5- 0 0 35 18 Insol methylbenzimidazole 7 S-methylmethimazole 0 16 11 74 13 ND
8 N-methylmethimazole 0 21 18 86 14 ND
9 5-methylmethimazole 0 0 25 15 40 20 5-Phenylmethimazole 35 t 15 78 10 95 16 ND
11 1-methyl-2-thiomethyl- 0 Z5 9 86 19 ND
5(4)nitroimidazole Values from three separate experiments f SD.
10 ND = Not done Assessment of the effect of MMI deirivatives or tautomeric cyclic thiones on the Promoter activity of MHC +Class I and Class II using transient -transfection ana! sis and CAT assaYs Plasmid conshmction The full length PD1 promoter, PD1 CAT construct pH (-38), inserted into the multicloning site of pSV3CAT, has been previously described (Erhlich, R.
et al.
Immunogenetics 30:18-26 (1989)). Sequential deletion mutants of the full-length PD1 promoter, inserted into the multicloning site of pSV3CAT, have also been previously described (Singer DS, et al., ibid (1.991); Saji, et al., Proc.
Natl. Acad.
Sci. USA 89:1944-1948 (1992a); Saji, et al., J. Clin. Endocrinol. Metab.
75:871-878 (1992b)). Briefly, a nested series of 5' de;Ietions of the upstream regulator region of the PD 1 gene were generated by Ba13 digestion; the series 5' termini ranged from -1012 base pairs to -68 base pairs, but all had a common 3' boundary at + 15 base pairs. The deletion series was also cloned into the pSV3CAT
reporter construct to assess promoter activities (Weisman JD, et al. ibid (1991);
Maguire, J.
et al., Mol. Cell. Biol., 12:3078-3086 (1992)). For screening the action of MMI, MMI derivatives or tautomeric cyclic thiones on Class I promoter activity, three clones are commonly used: p(-1100) CAT, p(-203) CAT, and p(-127) CAT, preferably p( 203) CAT. The number denotes the most 5' -residue in the 5' -flanking region of the PD1 promoter.

Construction of the HLA-DR promoter constructs has also been described as have been their characteristics (Reimold AM, eit al., ibid (1993)). Additional CAT
constructs were constructed by PCR, using the HLA DRa -CAT chimera containing -176 to +45 of 5'-flanking region as template and various primers (Montani V.
et al., ibid (1998a,b)). For example, a plasmid containing -38 to +45 bp of the 5'-flanking region of HLA-DRa was constructed vvith the following primers: a 5' primer with a 5' Sph I restriction site, II' 5' -ACATGCATGCGGTCAGACTCTATTAC:ACCCCAC-3' and a 3' primer, 5'-CTAGTCTAGTTTGGGAGTCAGTAGAGCTCG-3', with an Xba I restriction site (Montani V., et al., ibid (1998a,b)). The PCR products were purified by phenol-chloroform extraction, digested with Xba I and Sph I, purified from a 2%
agarose.

gel with Jet Sorb (Genomed, Frederick, MD), dephosphorylated with calf intestinal alkaline phosphatase (New England Biolabs, Beverly, MA), and religated (DNA
ligation kit, Takara Biochemical, Inc., Berkeley, CA) into the pCAT-Basic Vector (Promega, Madison, WI) at the Sph I and Xba I sites as described (Reimold AM, et al., ibid (1993); Montani V., et al., ibid (1998a,b)).

The thyroglobulin (TG)-CAT construct, pTG-688-CAT, was derived by substituting the TG promoter insert from a previously described pTG-CAT
chimera (Shimura H, et al., Mol. Endocrinol. 8:1049-1069 (1994)) into the HLA-DRa-CAT
chimera from which the class II promoter insert was excised. The vector containing the CAT reporter gene but no insert was the control.

Cell Culture FRTL-5 rat thyroid cells (Interthyr Research Foundation, Baltimore, MD;
ATCC CRL8305) were the same subclone (F1) used in the gel shift experiments described earlier. They were grown in the following medium: Coon's modified F-12 medium containing 5% heat-treated, mycoplasma-free calf serum, 1 mM
nonessential amino acids, and a mixture of six hormones (6H) containing bovine TSH (1 x 10-1 M), insulin (10 g/ml), cortisol (0.4 ng/ml), transferrin (5 g/ml), glycyl-L-histidyl-L-lysine acetate (10 ng/ml), and somatostatin (10 ng/ml).
Cells were diploid and between their 5' and 25tB passage. Fresh medium was added every 2 or 3 days and cells were passaged every 7-10 days. In some experiments, cells were grown to near confluency in 6H medium then maintained for 5-8 days, before experiments were initiated, in 5H medium with no TSH.

TRANSFECTION AND CAT ASSAYS

To measure the promoter activity of MH:C class I promoter constructs, as reported by CAT activity, two procedures were used. In one, rat FRTL-5 thyroid cells were transfected by the electroporation me'thod described previously (Saji, M., et al., ibid (1992b); Giuliani, C., et al., ibid (1995); Saji, M., et al., ibid (1997)).
In brief, FRTL-5 cells were grown to 80% confluence, put in fresh 6H medium containing 5% calf serum for 12 hours, harvested, washed and suspended at 1.5x10' cells/mi in 0.8 mi electroporation buffer (272 mM sucrose, 7 mM sodium phosphate.
at pH 7.4, and 1 mM MgC12). Twenty g of the full-length CAT construct were added with 5 g pSVGH. Cells were then pulsed (330 volts, capacitance 25 farad), plated (approximately 6x106 cell/dish), and cultured for 12 hours in medium containing 5% calf serum. At that time, cells were placed in 5H medium plus 5% calf serum (control), 5H medium plus 5% calf serum plus 5mM MMI or different concentrations of active compound (MIVII derivative or tautomeric cyclic thione), 6H medium plus 5% calf serum, or 6H medium plus 5% calf serum plus 5 mM MMI or active compound at different concentrations. After 48 hours they were harvested. Cell viability was approximately 80%. Medium was taken for hGH
radioimmunoassay to monitor transfection efficiency (Nichols Institute, San Juan Capistrano, CA) and cells were harvested for CAT assays which used 20-50 g cell lysate in a fmal volume of 130 l. Incubation was at 37 C for 2 or 4 hours;
acetylated chloramphenicoi was separated by thin layer chromatograpy (TLC) and positive spots on TLC plates were cut out and quantitated in a scintillation spectrometer. CAT activity was normalized to GH activity and/or cell protein before data were compared.

Alternatively transient transfections used the same class I-CAT chimeras and a DEAE-dextran procedure (Lopata MA, et al., Nucleic Acids Res. 12:5707-5717 (1984); Giuliani C, et al., ibid (1995)). Cells were grown to near (80%) confluency in 6H medium, shifted to 5H medium for 1 day, washed twice with Dulbecco's modified phosphate buffered saline (DPBS), plJ 7.4, and incubated 1 hour with 5 mI
serum-free 5H medium containing the plasmid DNA plus 250 g DEAE-dextran (5 Prime-3 Prime, Inc.). Cells were then exposed to 10% dimethylsulfoxide in DPBS
for 3 min., washed twice in DPBS, cultured in 5H medium for 24 hours, then maintained therein another 48 hours with or wiithout MMI, MMI derivatives or tautomeric cyclic thiones, as noted. Efficiency of transfection was determined by cotransfection as above or with 5 g pRSVLuc (De Wet JR, et al., Mol. Cell.
Biol.
7:725-737 (1987)).

To measure the promoter activity of MHC class II promoter constructs, transient transfections in FRTL-5 cells were performed, using one of the following procedures (Montani V., et al., ibid (1998a, b);). In one, FRTL-5 cells were cultivated in 6H medium to approximately 80% confluency, harvested, washed, and resuspended (1.5x10' cells/mi) in 0.85 ml electroporation buffer (272 mM
sucrose, 7 mM sodium phosphate buffer pH 7.4, and 1 inM MgCIo. Plasmid DNA was added, 20 g of the CAT chirnera together with 2 g pRSV-luciferase which is used to measure the efficiency of transfection. Cells were pulsed (330 V;
capacitance 25 farad), plated (6x 106 cells/10 cm dish), and ciultured in 6H medium plus 5%
calf serum supplemented or not with x-IFN. At the times noted, cells were harvested for CAT and luciferase assays. The second procedure differed as follows. FRTL-cells were grown to 80% confluency in 6H medium, then maintained 6 days in 5H
medium plus 5% calf serum. Cells were returned to 6H medium for 12 hours, transfected as described above, and maintained in 6H medium plus 5% calf serum for twelve hours. The medium was then changed to fresh 5H medium with 5% calf serum supplemented or not with ir-IFN. Cell viability was approximately 80% in all experiments. CAT activity was measured as described above and values were normalized to luciferase activity measured using the Promega (Madison, WI) assay system.

To test the effect of MMI, MMI derivaitives or tautomeric cyclic thiones on x-IFN-increased class II expression in FRTL-5 thyroid cells, the following procedure was used. Transient transfections with the -137 bp or -176 bp DRa-CAT
chimeras were performed as described and treated with 100 U/ml x-IFN for the noted times starting 12 hours after transfection. In duplicate sets of cells, 5 mM
MMI or the noted concentrations of MMI derivatives or tautomeric cyclic thiones were added simultaneously with the T-IFN and CAT activity was measured 48 hours later. Cell viability was approximately 85 5% in all samples. Results were expressed relative to the vector control in the albsence of x-IFN or MMI, after CAT
activities were corrected both for luciferase actiivity and cell protein.
These corrections in all cases resulted in less than 5% changes in activity. The same results were obtained using the alternative transfection protocol involving cells maintained in medium with no TSH.

Representative examples of the effect of MMI on basal class I promoter activity and its additive action with TSH are presented below. In Figure 9, cells maintained in 5H medium plus 5% calf seirum were transfected with a CAT

chimera containing 1100 bp of 5'-flanking region of the swine class I promoter by electroporation as described. After 12 h the medium was changed to fresh 5H
medium in the presence or absence of 1x10"1QM TSH, 5 mM MMI, or both. Cells were assayed 36 hours later. The value from the transfectant with no TSH or MMI
in the medium was the control and was set at 100%. Values are the mean of 3 experiments; significant increases or decreases at P< 0.05 (*) or P< 0.01 (**) are noted. It is evident that MMI, can decrease class I basal promoter activity and that 5 its action is measurable in cells with or without TSH.

In a second experiment (Fig. 10, Panel A), we tested the effect of MMI and TSH on the promoter activity of CAT chimeras of 5'-deletion mutants of the swine class I promoter in FRTL-5 cells. FRTL-5 cells grown in 6H medium (+TSH) were transfected by electroporation with the difi;erent constructs of the PD1 5'-flanking region. After 12 hours, the medium was changed to fresh 6H medium (+TSH), fresh 6H medium plus 5 mM MMI (-1=TSH/+MMI), or fresh 5H medium with no TSH or MMI; CAT activity was measured 36 hours later. Conversion rates were normalized to luciferase levels and protein; the activity of the -1100 bp construct in cells maintained in 6 H medium (first black bar) was assigned a control value of 100%. Values are the mean SE of three different experiments, each performed in duplicate. Differences in the basal level of expression for the different constructs reflect activity of different regulatory elements, some of which are noted in Panel B. Regulatory elements noted include ihe following: (a) the upstream silencer/enhancer region important in regulating constitutive class I levels in different tissues; (b) serum response element; (c) Enhancer A; (d) the interferon response element; (e) the 38 bp constitutive silencer containing the CRE-like sequence within the constitutive silencer element: (Giuliani C., et al., ibid (1995);
Saji M., et al., ibid (1997)). Also noted are (f) the CCAAT and (g) TATA box important in initiation of transcription. These results suggest that any of the constructs could be used to screen MMI derivatives but that p(-203)CAT might be best because its CAT activity is more easily measured and because the MMI
inhibitory effect is best.

The action of MMI is also readily measured in cells treated with 'e-interferon (e-IFN). As noted elsewhere, the ability of r-IFN to increase class I and induce class II antigen expression in FRTL-5 thyroid cells and mimic changes in human thyrocytes seen in ATD is well described (Todd I, et al., ibid (1985); Platzer M, et al., ibid (1987); Misaki T, et al., ibid (1988); 'Fakarija M, et al., ibid (1988)).
Studies of the effect of MMI or MMI derivatives on x-IFN-increased class I or x-IFN induced aberrant class II expression are, therefore, a reasonable model to show activity that is important in ATD and other immune diseases. Using p(-1100)CAT, p(-203)CAT, and p(-127)CAT class I chimeras as examples, the ability of 'e-IFN to increase class I promoter expression is readily measured and the ability of MMI to decrease this action is also readily nieasured (Table 5).

In this experiment, FRTL-5 cells were grown to near confluency in 6H
medium (plus TSH) and then were maintained in 5H medium (no TSH) for 7 days before being treated with x-IFN or x-IFN plus MMI for 40 hours. Control cells were those maintained in 5H medium for the same 40 hours. CAT activity was measured as described above. The x-IFN treatment increased CAT activity significantly (P < 0.05 or 0.01) in cells transfect.ed with all the CAT
plasmids except the pSVO control. Importantly, the MMI significantly decreased the ability of ir-IFN treatment to increase CAT activity (P < 0.01) in cells transfected with all the CAT plasmids except the pSVO control.

TABLE 5: EFFECT OF 5 MM MMI OR 10t1U/M x-INTERFERON ON THE

OF THE SWINE CLASS I PROMOTER.
CHIMERA NO TREATMENT + -tjFN (100U/ml) +xIFN (100U/ml) CONTROL +MMI (5 mM) (CAT ACTIVITY
RELATIVE TO
p(-1100)CAT) % of' Control % of Control p(-1100) 100 460 40 163 f 48 p(-203) 520 750 25 95 t 26 p(-127) 100 500 27 55 f 4 pSVO 45 48 f 7 38 t 9 Table 6, presents results of the effect of' MMI, multiple MMI derivatives or tautomeric cyclic thiones on basal class I activity; Table 7 presents results of the effect of MMI, multiple MMI derivatives or tautomeric cyclic thiones on IFN-increased class I promoter activity. The action of compound 10 is clearly best in both cases, followed by compounds 11, 7, and 8.

The HLA-DRa-176 bp or -137 bp minhnal promoter class II chinmeras coupled to CAT were used to evaluate the expression of the HLA-class II gene in FRTL-5 thyrocytes. HLA-DRa is not expressec[ in transiently transfected FRTL-5 thyrocytes, by comparison to the vector control, unless the cells are treated with rat recombinant 'e-IFN (Fig. 11, first and second set of open and closed bars in each Panel). The action of rat T-IFN is not duplicated by human x-IFN and is associated with an increase in endogenous class II expression measured by flow cytometry using fluorescent all partner analysis (Fig. 12). Thus, the *e-IFN action is specific and appears to reflect effects on the endogenous class H antigen. Evaluation of progressive 5' deletions of the -176 bp DRa-CAT chimera to -137, -122, -111, -97, and -38 bp showed that, like immune cells, x-IFN- induction is lost once the S
box, -137 to -123 bp is removed (Fig. 11 Panel A). Also like immune cells, Y-IFN-induction requires not only the S box, but also the X,, X2, and Y boxes for activity..

Thus, mutation of each element individually also resulted in the loss (Fig. 11 Panel B, MUT S, MUT X,, and MUT Y) or a significant decrease (Fig. 11B, MUT X2) in the *e-IFN response.

TABLE 6: EFFECT OF DIFFERENT COrTCENTRATIONS OF ACTIVE

CELLS TRANSFECTED WITH THE CLASS I CAT CHIMERA, P(-203)CAT.

% INHIBITION OF BASAL (-203)CAT CLASS I PROMOTER ACTIVITY
Compound 25 M 100 M 1.0 mM 5 mM
1 Methimazole 0 0 32 f 7 74 t 10 2 Metronidazole 0 10 8 37 f 11 74 t 9 3 2-mercaptoimidazole None None None None 4 2-mercaptobenzimidazole 0 0 50 12 90 14 5 2-mercapto-5- ND ND ND 45 t 8 nitrobenzimidazole 6 2-mercapto-5- ND ND ND 57 13 meth lbenzimidazole 7 S-methylmethimazole 0 10 9 51 6 68 8 8 N-methylmethimazole 5 5 48 10 72 6 87 11 9 5-methylmethimazole 0 0 14 10 25 7 10 5-Phenylmethimazole 45 9 71 6 92 12 ND
11E 1-methyl-2-thiomethyl- 11 6 51 8 89 11 ND
5 4 nitroiunidazole Values from three experiments in duplicate, mean SD. ND is not done.
Bold values represent significant inhibition (P <: 0.05 or better).
Experiment in each case was in 6H medium. Treatment was for 36 hours.

TABLE 7: EFFECT OF DIFFERENT CON('.ENTRATIONS OF ACTIVE

CELLS TRANSFECTED WITH THE CLASS I CAT CHIMERA, P(-203)CAT, AND TREATED WITH 100 U/ML x-INTERFERON.

% INHIBITION OF IFN-INCREASED p(-203)CAT CLASS I PROMOTER
ACTIVIT3t Compound 10 M 100 M 1.0 mM 5 mM
I Methimazole 0 0 35 t 8 72 t 12 2 Metronidazole 0 0 45 t 5 84 f 13 3 2-mercaptoimidazole None None None None 4 2-mercaptobenzimidazole 0 0 40 8 76 f 10 5 2-mercapto-5- ND ND ND 43 f 9 nitrobenzimidazole 6 2-mrcapto-5- ND ND ND 49 t 7 methylbenzimidazole 7 S-methylmethiunazole 0 14 11 50 13 58 t 12 8 N-methylmethimazole 0 38 9 69 7 77 t 14 9 5-methylmethimazole 0 0 10 10 29 f 11 5-Phenylmethimazole 35 10 81 14 90 13 ND
11 1-methyl-2-thiomethyl- 12 11 43 7 89 11 ND
5(4)nitroimidazole 10 Values from three experiments in duplicate, mean SD. ND is not done.
Bold values represent significant inhibition (P < 0.05 or better).

Experiment in each case was in 6H medium. Treatments with interferon and MMI, methimazole derivative, or tautomeric cyclic thione were for 40 hours.

In this experiment transient transfections were performed in FRTL-5 cells grown to near confluency in medium with TSH ( 6H medium) and treated with 100 U/ml 'r-IFN for 24 h after transfection. Results are expressed relative to the vector control in the absence of x-IFN (first open bar in each panel), after CAT
activities were corrected both for luciferase activity and cell protein. These corrections in all cases resulted in less than 5% changes .in activity. Results are the mean SD
of 3 separate experiments performed on 3 different batches of cells. A single asterisk (*) denotes a statistically significant increase (P <0.,01) in DRa promoter activity induced by x-IFN; two asterisks (**) denote a statistically significant decrease (P < 0.01) in x-IFN-induced DRa promoter activity when the -176 bp DRa minimal promoter contained mutations in the S, Xi, X2, and Y boxes. The same results were obtained using an alternative protocol involving cells maintained in medium with no TSH. In Figure 11 (top left) is a diagrammatic presentation of the -176 bp DRa-CAT chimera with the locations of the S, X,, X;,, and Y boxes noted by black boxes and the locations of the 5'-termini of the deletions noted; on the top right of the Figure, the mutations made in S, Xi, X2, and Y boxes are presented.

In sum, as is the case in other antigen presenting cells or cells exhibiting aberrant class II expression associated with immune disease, x-IFN can increase HLA-DRa promoter expression in FRTL-5 cells and the action of interferon requires the same highly conserved 5'-flanking region elements, S, X,, X2, and Y, present in all class II genes for this effect (Bottazzo GF, et al., ibid (1983); Todd I, et al., ibid (1985); Bottazzo GF, et al., ibid (1985); Todd I, et al., ibid (1986);
Burmester GR, et al., ibid (1987); Schwartz RS, et al., ibid (1989); Benoist C, et al., ibid (1990); Glimcher LH, et al., ibid (1992); Ting JP-Y, et al., ibid (1993)).
The ability of 'r-IFN to increase class II gene expression is dependent on its concentration, whether the -176 bp (data not shown) or -137 bp (Fig. 13) DRa-CAT
chimera is used in transient transfection assays. The maximal effect was in each case evident at 100 U/ml e-IFN (Fig. 13, Panel A). MMI prevents the ability of a maxim.ally effective concentration of lr-IFN to increase the activity of the -137 bp DRa-CAT chimera as a function of time. Thus, CAT activity induced by 100 U/ml x-IFN was progressively decreased 24 and 48 h after MMI addition (Fig. 13, Panel B). The concentration of MMI in Figure 13, Panel B is 5 mM; however, the effect was evident at lower MMI concentrations and was dependent on the concentration of MMI (Fig. 14). In these experiments, transient transfections were performed with the -137 bp DRa -CAT chimera. FRTL-5 cells were grown to near confluency in 6H medium and treated with 100 U/mi x-IFN for the noted times starting 12 h after transfection. MMI was added to duplicate sets of cells, simultaneously with the r-IFN addition or 24 hours after addition of the x-IFN. CAT activity was measured 48 h after the addition of x-IFN and results are expressed relative to the vector control in the absence of x-IFN after CAT activiities were corrected both for luciferase activity and cell protein. These corrections in all cases resulted in less than 5% changes in activity. Results are the mean t SD of 3 separate experiments performed on 3 different batches of cells. In Figure 13, Panel A, a single asterisk (*) denotes a statistically significant increase (P <:0.01) in DR promoter activity induced by it-IFN. In Figure 13, Panel B, two asterisks (**) denote a statistically significant decrease in w-IFN-induced DRa pronioter activity with 24 or 48 hours of methimazole exposure. In Figure 14, two asterisks (**) denote a statistically significant decrease in 'sIFN-induced DRa pronioter activity caused by MMI.
The same results were obtained using the alternative 'transfection protocol involving cells maintained in medium with no TSH.

TABLE 8: EFFECT OF x-IFN AND 1VIMI C-N THE CAT ACTIVITY OF HLA-DRa AND RAT THYROGLOBULIN-CAT CHIMERAS.

CAT ACTIVITY (% OF CONTROL WITH NO TREATMENT) CHIMERA CONTROL + x-IF'N + x-IFN + MMI +MMI
-167 HLA-DRa-CAT 100 790 30` 210 t 24" 99 5 TG-688-CAT 100 49 1.1+ 160 f 30 ++ 260 12+++
Vector-CAT Control 100 102 7 97 5 100 4 'Significant increase over control (P < 0.01);
"significant decrease in IFN-induced activity (F' < 0.01).
+Significant decrease relative to control (P < 0.05);
++significant reversal of the IFN-induced decrease (P<0.01);
+++significant increase in activity relative to control (P < 0.01).

The action of both x-IFN and MMI were specific; thus, neither effected the control vector and each had opposite effects on a TG-CAT chimera (Table 8): x-IFN decreased TG-CAT activity and MMI reversed the x-IFN-induced decrease in TG-CAT activity. Also, MMI alone increased 'TG-CAT activity but not HLA-DRa CAT activity (Table 8). In this experiment, like previous experiments, transient transfections were performed in FRTL-5 cells grown to 80% confluency in 6H
medium, maintained 6 days in 5H medium, and returned to 6H medium for 12 h before transfection as described. Twelve hours later, the medium was changed to fresh 5H medium, supplemented or not with 10() U/ml 'r-IFN and/or 5 mM MMI.
CAT activity was measured 48 h thereafter. Cell viability was approximately 85 %
in all experiments. Results are expressed relative to the control with no treatment, after activities were corrected both for luciferase activity and cell protein.
These corrections in all cases resulted in less than 5% changes in activity. Results are the mean t SD of 3 separate experiments.

Consistent and coincident with its effect, on x-iFN-induced exogenous class II

gene expression in CAT assays (Figs. 13 and 14), M1VxI decreased endogenous class II antigen expression on the cell surface as determined by flow cytometry (Fig. 12).
In this experiment, FRTL-5 cells were grown to near confluency in TSH, maintained 8 days in 5H medium, then treated with 100 U/ml x-IFN, 5 mM MMI, or both for the last 48 hours, as described in Figures 13 and 14. Cells were stained with a fluorescein isothiocyanate conjugated class II-specific monoclonal antibody to RT1.B (clone OX-6, Sera Labs, UK) for 60 min at 4 C, washed twice with Dulbecco's phosphate buffered saline, and subjected to laser flow cytometry.

Table 9 summarizes results of experiments evaluating the effect of MMI, MMI derivatives or tautomeric cyclic thiones on x-IFN induced class II
activity in FRTL-5 thyrocytes. The effectiveness of inhibition is similax to that of class I
derivatives: compound 10 > compound 11 > cotnpounds 7 and 8 > compound 2 >
MMI.

Measuring CAT activity of the chimeric CAT constructs of the Class I or Class II promoter is, therefore, another way to assay the effect of MMI, MMI
derivatives or tautomeric cyclic thiones on Class I or Class II activity.
These assays can be used for evaluating agents able to mimic MMI in therapeutic actions related to treatment of autoimmune disease or transplantation therapy.

TABLE 9: EFFECT OF DIFFERENT C+DNCENTRATIONS OF ACTIVE

CELLS TRANSFECTED WITH THE CL,ASS II CAT CHIMERA, P(-137) CAT AND TREATED WITH 100 U/ML x-INTERFERON.

% INHIBITION OF IFN-INCREASED p(-137)CAT CLASS II PROMOTER
ACTIVITY
Compound 10 M 100 M 0.5 mM 5 mM
1 Methimazole 0 0 44 6 87 f 9 2 Metronidazole 0 +D 55 10 80 t 4 3 2mercaptoimidazole None None None None 4 2-mercaptobenzimidazole 0 0 29 t 12 73 14 5 2-mercapto-5- ND ND ND 39 13 nitrobenzimidazole 6 2-mercapto-5- ND ND ND 44 t 8 meth lbenzimidazole 7 S-methylmethimazole 0 20 15 75 6 84 15 8 N-methylmethimazole 0 43 9 79 11 88 10 9 5-methylmethimazole 0 0 14 12 44 18 5-Phenylmethimazole 45 17 88 15 95 5 ND
11 1-methyl-2-thiomethyl- 22 f 11 52 10 95 7 ND
5(4)nitroimidazole Values from three experiments in duplicate, meain SD. ND is not done.
10 Bold values represent significant inhibition (P<0.05 or better).
Experiment in each case was in 6H medium.

EXAMPLE', 4 Creation of High Through-Put Assay for Use, in Evaluating MMI Derivatives or Tautomeric Cyslic Thiones and Adivitv off Such Materials in These Assays The previous results describing the actic-n of MMI derivatives or tautomeric cyclic thiones on class I RNA levels, class I anci class II gel shifts, and transient transfections using class I and class II promoter constructs indicated that all assays yielded similar results for all compounds. Thus, the order of effectiveness, i.e.
compounds 10 > 11 > 7 or 8> 2> MMI, prevaibed in all assays. This created the possibility that a single set of assays might be useful for high through put screening of different derivatives. From a speed and quantitation point of view, the possibility existed that creating stable transfectants of class I and class II promoter constructs in FRTL-5 thyroid cells might be a useful approac:h. The following experiments indicate this to be true.
CONSTRUCTION OF MHC CLASS I and Class II
PROMOTER-LUCIFERASE CE[I11MRIC PLASMIDS

The swine PD1 5'-flanking sequence-CAT chinieras (-1100, -203, and -127), as well as two mutants with the CRE site with -107 to -100 bp deleted (-and -127ACRE) were used to create luciferase reporter gene constructs. The inserts were released by restriction enzyme digestion, purified from agarose gel using QIAEX (QIAGEN, Chatsworth, CA) and Iigateci in the NheI-HindIII site of the PGL-2 basic vector (PROMEGA, Madison, WI) using a DNA ligation kit from TAKARA biomedicals (TAKARA SHUZO Co). JM109 bacterial competent cells (PROMEGA, Madison, WI) were transformed vvith the ligation reaction and plated in agar plates for 12 h at 37 C. The colonies wE:re picked and screened with minipreps using QlAprep spin plasmid kit (QIAGEN, Chatsworth, CA) and restriction enzyme digestion. The plasmids were then purified by CsCl gradient centrifugation.

WO 00/12175 PCT/US99/19862, Similarly, the HLA DRa promoter constructs, p(-176)CAT and p(-137)CAT, inserted into the pCAT-Basic vector were released by restriction enzyme digestion, purified from agarose gel usiing QIAEX (QIAGEN, Chatsworth, CA), and used as templates for the construction by PCR of inserts with a 5' flanking M1uI restriction site. The PCR products were purified by phenol-chloroform extraction, digested with Miul and Xbal, purified from agarose gels using QIAEX
(QIAGEN, Chatsworth, CA) and ligated in the Mlul-NheI site of the PGL-2 basic vector (PROMEGA, Madison, WI) using the DNA ligation kit from TAKARA
biomedicals (TAKARA SHUZO Co.). JM1091bacterial competent cells (PROMEGA, Madison, WI) were transformed with the ligation reaction and plated in agar plates for 12 h at 37 C. The colonies were picked and screened with minipreps using QIAprep spin plasmid kit (QIAGEN, Chatsworth, CA) and restriction enzyme digestion. The plasmids were then purified by CsCI gradient centrifugation.

The PD1-PGL-2 basic constructs and the Class II-PGL-2 basic constructs were stably transfected in the FRTL-5 cells using a Lipofectamine (GIBCO, Life Technologies, Inc.) method. Near confluent cells in 6H medium were cotransfected with 20 g of plasmid DNA and 2 g of a pcDNA3neo or a pPUR selection vector (CLONTECH, Palo Alto, CA). After 24-48 hoiurs, 400 g/ml of G418 (GIBCO, Life Technologies, Inc.) or 10 M of puromycui (SIGMA) were added to the medium. After 3 weeks the antibiotic-resistant colonies were cloned by limiting dilution and screened to determine their interferon sensitivity. At least 5 cell lines of each construct were isolated, each of whose activity was increased by adding 100 U/ml x interferon to the medium of the cells. The following data (Tables 10-13) were obtained with one clone of each of the class I or class II-luciferase chimera, but were representative of at least three other clones of each.

LUCIFERASE ASSAY

Luciferase activity was measured using the Luciferase assay system (PROMEGA, Madison, WI). Briefly, treated or untreated cells from a 100 mm culture dish were washed with PBS, scraped and collected in microfuge tubes.
The pellet was dissolved in 100 l lx reporter lysis buffer and incubated at room temperature for 15 minutes. Cells were frozen iia dry ice plus ethanol and thawed in water at room temperature. After vortexing for 10 sec, tubes were centrifuged at 12,000xg for 5 min. Twenty l of the supernatant were mixed with 100 l of Luciferase assay reagent and immediately placed in a luminometer. Light was measured for a period of 10 sec after a 2 sec delay.
EFFECT OF MMI DERIVATIVES AND TAIUTOMERIC CYCLIC THOINES
ON STABLE TRANSFECTANTS
Treatment of cells, maintained either in the presence of TSH or its absence, with 100 U/ml interferon, increased class I and class II promoter activity (Table 10). In this experiment FRTL-5 cells were grown to 80% confluency in 6H

medium with TSH then treated with 100 U/ml x-interferon, 5 mM MMI or both together. Luciferase activity was measured after 40 hours as described.
Several points are notable. First, interferon increases both class I and class II
promoter activity in the stable transfectants. Second, MM]1 inhibits both interferon-increased class I and interferon-increased class II promoter activity. Third, unlike transient transfection assays, the MMI does not significantly decrease basal class I
activity.
Finally, the interferon-induced class I activity requires the presence of the CRE; this is consistent with recent results indicating that int:erferon-induced CIITA is the mediator of the increase in class I as well as class II activity and requires the CRE
for its activity (Saji et al., ibid (1997); Montani `J., et al., ibid (1998a, 1998b);
Balducci-Silano et al., Endocrinology 139: 2300-2313 (1998)).

The activity of different MMI derivatives and tautomeric cyclic thiones on interferon-increased class I activity in p(-203)clas;s I luciferase chimeras is presented in Table 11. The same pattern as evident in transient transfection studies is seen:

WO 00/12175 PCT/US99/19862' compound 10 activity > 11 > 7 or 8 > 2 > MMI. Thus, this assay is consistent with the data in the other studies measuring effects on RNA, gel shifts, and transient transfections of promoters. It has however several major advantages. The cells do not require changing to 5H conditions, therefore cell preparation time is reduced.
No labor intensive transient transfections are needed. The assay is rapid and does not require a second prolonged incubation with radioactive materials. Finally, because this involves treatment of living cells with each compound, cell toxicity and viability are readily noted and quantitated, proviiding a better predictive effect for irc vivo use than measurement of an enzyme activity.
The activity of different MMI derivatives and tautomeric cyclic thiones on basal class I activity in p(-203)class I luciferase chimeras is presented in Table 12.
Of interest, despite the fact that MMI at 1 or 5 mM has no significant effect on the basal activity, it appears that more active derivaitives are effective in this screening assay. Again the activities are measured rapidly and within 7 days of splitting cells, including the 40 hour treatment period.

The activity of different MMI derivatives and tautomeric cyclic thiones on interferon-increased class II activity in p(-137)cl.ass II luciferase chimera stably transfected into FRTL-5 cells is presented in Table 13. The same pattern as evident in transient transfection studies is seen: compowxl 10 activity > 11 > 7 or 8 > 2 > MMI. Thus, this assay is consistent with the data in the other studies measuring effects on class I RNA, class I and II gel shifts, and transient transfections of both class I and class II promoters.

TABLE 10: EFFECT OF x-IFN AND MMI ON THE LUCIFERASE
ACTIVITY OF HLA-DRa- AND SWINE CLASS I C:HINI'ERAS STABLY
TRANSFECTED INTO FRTL-5 CELLS.

LUCIFERASE ACTIVITY (Light Units or % of control) CHIMERA CONTROL .}. d-IFN +r-IFN + 5 mM
(Light Units) (% Control) + 5 mM MMI
1VIMI (% Control) (% Control) p(-203) class I-LUC 24,750 495 30 158 23 105 38 p( 203ACRE) class I- 8,290 139 16 106 6 ND
LUC

p(-127) class I LUC 8,340 180 19' 110 8" 135 17 p(-1270CRE) class I- 3,290 92 10 101 4 ND
LUC

-167 HLA-DRaLUC 37,400 790 45' 175 24" 99 5 -137 HLA-DRa-LUC 31,650 695 42f 150 30 103 6 Vector-LUC Control 2190 102 6 105 5 100 6 Values from three experiments (mean t SD). ND is not done.
`Bold values indicate a significant increase over control (P <0.01);
"Bold and Wci.zed values represent a significant decrease relative to the interferon-increased activity (P <0.01).

TABLE 11: EFFECT OF DIFFERENT CONCENTRATIONS OF ACTIVE

CELLS STABLY TRANSFECTED WITH TIEE CLASS I-LUCIFERASE
CHIlVIERA, p(-203)LUC, AND TREATED WTTH 100 U/ML Y-INTERFERON.
% INHIBITION OF IFN-INCREASED p(-203)LUC CLASS I PROMOTER
ACTIVITY
Compound 10 },LM 100 M 1.0 mM

1 Methimazole 0 0 45 10 2 Metronidazole 0 10 6 57 5 3 2-mercaptoimidazole None None None 4 2-mercaptobenzimidazole 0 0 39 6 5 2-mercapto-5-nitrobenzimidazole 0 0 9 7 6 2-mercapto-5- ND ND 19 10 methylbenzimidazole 7 S-methylmethimazole 0 24 6 62 9 8 N-methylmethiunazole 0 42 14 77 10 9 5-methylmethiunazole 0 0 15 11 5-Phenylmethimazole 45 jL 7 90 17 90 13 11 1-methyl-2-thiomethyl- 14 _-L 8 73 14 92 15 5(4)nitroimidazole Values from three experiments in duplicate, mean SD. ND is not done.
10 Bold values represent significant inhibition (P <0.05 or better).
Experiment was in 6H medium.
Treatments with interferon and MMI derivatives or tautomeric cyclic thiones were for 40 hours.

TABLE 12: EFFECT OF DIFFERENT CONCENTRATIONS OF ACTIVE

CELLS STABLY TRANSFECTED WITH TI-IE CLASS I- LUCIFERASE
CIiIMERA, p(-203)LUC.

% INHIBITION OF BASAL p(-203)LUC CLASS I PROMOTERACTIVITY
Compound 25 M 100 M 1.0 mM

1 Methimazole 0 0 0 2 Metronidazole 0 0 42 + 6 3 2-mercaptoimidazole 0 0 0 4 2-mercaptobenzimidazole 0 0 50 5 5 2-mercapto-5-nitrobenzimidazole 0 0 0 6 2-mercapto-5- 0 0 0 methylbenzimidazole 7 S-methylmethimazole 0 10 6 44 4 8 N-methylmethimazole 9 6 44 10 51 7 9 5-methylmethimazole 0 0 10 10 5-Phenylmethimazole 41 1- 5 51 3 52 8 11 1-methyl-2-thiomethyl- 9 , 6 54 9 48 5 5(4)nitroimidazole Values from three experiments in duplicate, mean SD. ND is not done.
Bold values represent significant inhibition (P < 0.05 or better).
10 Experiment in each case was in 6H medium.
Treatment with MMI, MMI derivatives.or tautomeric cyclic thiones was for 40 hours.

TABLE 13: EFFECT OF DIFFERENT CONCENTRATIONS OF ACTIVE

CELLS STABLY TRANSFECTED WITH THE CLASS II LUCIFERASE
CHIMERA., p(-137)LUC AND TREATED WITH 100 U/ML x-INTERFERON.
% INHIBITION OF IFN-INCREASED p(-137)LUC CLASS II PROMOTER
ACTIVITY' Compound 10 M 100 M 1 mM

1 Methimazole 0 0 37 + 10 2 Metronidazole 0 7+ 6 65 + 9 7 S-methylmethimazole 0 30 7 68 10 8 N-methylmethimazole 0 43 5 84 9 5-Phenylmethimazole 43 20 80 7 92 10 11 1-methyl-2-thiomethyl- 16 15 44 11 94 8 5(4)nitroimidazole Values from two experiments in duplicate, mean t SD.
10 Bold values represent significant inhibition (P <0.05 or better).
Experiment was in 6H medium.
Treatments were for 40 hours.

THE GENERIC HIGH THROUGH-PUT ASSAY FOR USE IN EVALUATING
MMI AND TAUTOMERIC CYCLIC TIHONE DERIVATIVES AND
ACTTVITY OF DERIVIATIVES IN THESE ASSAYS
The previous results describing the action of MMI derivatives and tautomeric cyclic thiones on FRTL-5 thyroid cells stably transfected with MHC class I and class II promoter constructs indicated that the assays yielded similar results as for all other assays. Thus, the order or effectiveness, i.e., compounds 10> 11 > 7 or 8> 2> MMI, prevailed. These data established, moreover, a preferable procedure for high through-put screening of different derivatives. The procedure is as follows.

1. FRTL-5 cells stably transfected vrith MHC class I or class II
promoter constructs, preferably p(-203)MHC-class I-LUC or p(-137)MHC class II-LUC, respectively, are grown in complete 6H medium plus 5% calf serum and 400 g/ml G418 (GIBCO, Life Technologies, Inc.) or 10 M of puromycin (SIGMA), as appropriate, and in either 6, 12, 24 or 96 weill plates, according to the numbers of assays required.
2. When cells are 60-70% confluent, the cells are treated with 100 U/ml 'e-interferon with or without MMI derivatives, lautomeric cyclic thiones, or other compounds at 5 mM or lower concentrations together with fresh medium.
Preferably a range of concentrations is tested from 5 mM to 5 nM, in duplicate.
3. After 36 to 48, preferably 40, hours, cells in 6, 12, or 24 well plates are washed with 1 ml PBS, scraped, collected in microfuge tubes, and centrifuged to pellet the cells.

4. The pellet is dissolved in 20, 50 or 100 l lx reporter lysis buffer respectively, by repetitive micropipetting or vort:exing, and incubated at room temperature for 15 minutes.

5. Cells are frozen in dry ice plus ethanol and thawed in water at room temperature. After vortexing for 10 sec, tubes are centrifuged at 12,000 xg for 5 min.
6. Cells in 96 well plates are directly solubilized by the addition of 100 l lx reporter lysis buffer and repetitive pipetting, and incubated at room temperature for 15 min.

7. Five to 100 i of the supernatant, depending on the plate used, are mixed with 100 to 300 l Luciferase assay reagent and immediately placed in a luminometer.

8. Light is measured for a period of 10 seconds after a 2 second time delay.

Usually there is no significant difference in protein concentration between wells; however, the luciferase raw data may be normalized by measuring protein concentrations in the supernatant solution. To increase assay sensitivity or to adjust time schedules in laboratories, the cells may be shifted to 5H medium (no TSH) for 2 to 7 days after reaching 60% confluency, them returned to 6H medium 12 hours before treatment is started.

Since interferon increases both class I and class II promoter activity in the stable transfectants and the inhibition of interferon-induced activity is most representative of the action of the compounds in patients with autoimmune disease, this procedure is optimal. However, for class I transfectants, tests without the presence of interferon can be made to assess effects on basal promoter activity. To insure that the assays are specific for interferon action, tests may be performed with p(-203ACRE) MHC-class I-LUC transfected ce:lls, which do not respond to interferon.

This assay is consistent with data in othe:r studies measuring effects on RNA, gel shifts, antigen expression, and transient transfections of promoters. It has however several major advantages. The cells do not require changing to 5H
conditions, therefore cell prep time is reduced. No labor intensive transient transfections are needed. The assay is rapid and does not require a second prolonged incubation with radioactive materials.

Assessment of the Effect of MMI Derivatives and Tautomeric Cyclic Thiones on mRNA Levels of MHC Class l[ and MHC Class II.
The ability of the MMI derivatives and lautomeric cyclic thiones to decrease the exogenous MHC class I and MHC class II promoter activity in transiently or stably transfected FRTL-5 cells is paralleled by the ability of the derivatives to similarly decrease MHC class I and class II RNA levels in the same cells.
Thus, the promoter activity measurements reflect phenomena within the cell, despite the use of materials from other species such as human or swine promoters or probes and the different assay technologies measuring changes in exogenous vs endogenous gene expression. The data are also consistent with gel shift changes and MHC.
surface expression (Fig. 12) measuring the protein products regulating gene expression.
Cells and Treatment The FRTL-5 rat thyroid cells were the same fresh subclone (F) used previously in the gel shift and transfection studies and had all properties detailed therein. With TSH, their doubling time was 36 6 hours; in its absence, they did not proliferate. After 6 days in 5H medium with no TSH, 1x10'10 M TSH elevated cAMP levels, iodide uptake, and thymidine incorporation into DNA > 10 fold.
Cells were diploid, between the 5th and 25th passage and were grown in Coon's modified F12 medium supplemented with 5% calf serum, 1 mmol/L nonessential amino acids (GIBCO, Grand Island, NY) and a mixture of 6 hormones (6H) as described: TSH (1X10''0 M), insulin (10 g/ml), hydrocortisone (0.4 ng/ml), human transferrin (5 g/ml), somatostatin (10 ng/ml) and glycyl-L-histidyl-L-lysine acetate (10 ng/ml) (Ambesi-Impiombato FS, US Patent 4,608,341 (1986); Kohn, L. D., et al., U.S. Patent 4,609,622 (1986)). Passage was every 7-10 days; fresh media was added every 2 or 3 days. Cells were shifted to imedium with no TSH (5H) for 4-days before use. Experiments were initiated by adding 1x10"' M TSH, 100 U/ml 'e-interferon, the noted concentrations of the MMI derivatives or tautomeric cyclic thiones, or combinations of these in fresh mediium. Fresh medium alone served as a control. RNA was isolated 40 hours later.

R1VA Isolation and Nor:thern Analysis Total cellular RNA was isolated, Northern analyses performed, and filters sequentially hybridized with the following cDNA probes (0.5-1.0 x 106 cpm/niL.) as described (Isozaki 0, et al., Mol Endocrinol 31681-1692 (1989); Saji, M., et al., Proc. Natl. Acad. Sci. U.S.A., 89: 1944-1948 (1992); Saji, M., et al., J.
Clin.
Endocrinol. Metab., 75: 871-878 (1992)). Three of the probes were those previously described: the 1.0 kb HpaU fragme .t of the swine MHC class I pH7 clone which spans the entire cDNA insert (Saji, M., et al., ibid (1992)); a rat thyroglobulin cDNA which used as a positive c:ontrol (Santisteban P, et al., J
Biol Chem 262:4048-4052 (1987); Isozaki 0, et al.,, ibid (1989)); and 0-actin which was the negative control. The MHC class II DNA probe was a PCR amplified 546 bp product, from between 74 and 619 bp of the class II sequence, which was derived from interferon-treated rat FRTL-5 cell RNA using a sense primer having the nucleotide sequence, 5'-AGCAAGCCAGTCACAGAAGG-3', and an antisense primer with the sequence, 5'-GATTCGACTT(iGAAGATGCC-3', two regions which are highly conserved in the class II nucleotide and protein sequence.
After amplification using Pfu DNA polymerase, the product was purified on Agarose gels and then random prime radiolabeled using [32P]IdCTP and Kienow enzyme.

All experiments were repeated at least 21-times with different batches of cells to evaluate biological variability. Values are the mean SD of these experiments unless otherwise noted. Significance between experimental values was determined using the student t-test or by two-way analysis of variance. Values were significant if P was less than 0.05 when data from all experiments were considered.

Results The MMI derivatives and tautomeric cyclic thiones tested decreased both basal class I and interferon-induced class I and class II RNA levels. The effect of -the MMI derivatives and tautomeric cyclic thioines on basal class I RNA levels in cells maintained without TSH is presented in Table 14. The effect on -'s-interferon-induced class I RNA levels in cells maintained with TSH is presented in Table and on x-interferon-induced class II RNA levels in cells maintained with TSH
in Table 16. In all cases, RNA levels are quantitated by laser densitometry of autoradiograms or by BAS phosphoimaging relative to R-actin. The class I or class II ratio to j3-actin levels in control cells determines the 100% level; the %
inhibition reflects an effect of the MMI derivative or tautomeric cyclic thiones to decrease this ratio.

In general, the effect of the derivatives was similar in potency in all RNA
assays: compound 10 > 11> 7 or 8 > 2> MAdI. More importantly, this order matched the effects of the derivatives, with respect to potency, both on class I and class II gene expression measured in transfection studies testing effects on promoter activity.

Studies with thyroglobulin RNA emphasize the specificity of the derivatives and provide an unexpected result. MMI increases TG RNA levels nearly 2-fold (Table 17) rather than decreasing RNA levels a s is the case for class I and class II
(Tables 14-16). Unexpectedly, this is not the case for most MMI derivatives and tautomeric cyclic thiones where no comparable increase is evident, particularly in the case of the most effective compounds (10, 11, 7 and 8) in decreasing class I and class II RNA levels. This suggests that these de:rivatives/thiones may be effective in the treatment of autoimmune diseases with less adverse effects on thyroid function than methimazole TABLE 14: EFFECT OF DIFFERENT CONCENTRATIONS OF ACTIVE

CELLS

% INHIBITION OF CLASS I RNA EXPRESSION
Compound 100 M i mM 5 mM
1 Methimazole None 25 t 10 42 f 8 2 Metronidazole None 49 t 6 63 t 14 3 2-mercaptoimidazole ND None None 4 2-mercaptobenzimidazole None 34 13 ND
5 2-mercapto-5- None 32 11 ND
nitrobenzimidazole 6 2-mercapto-5- None 23 15 ND
methylbenzimidazole 7 S-methylmethimazole 10 8 46 9 ND
8 N-methylmethimazole 14 12 60 6 ND

9 5-methylmethimazole None 15 14 30 5 5-Phenylmethimazole 60 8 85 16 ND

11 1-methyl-2-thiomethyl- 25 15 75 11 ND (Toxic) 5(4)nitroimidazole Values from three separate experiments (Mean f SD).
10 Bold values are significant decreases from basal levels.
The experiment in each case used two 100 mM plates of L5 cells maintained in medium for 5 days after reaching near confluency - then treatment for 40 hours.
None is no effect; ND is not done.

TABLE 15: EFFECT OF DIFFERENT CONCENTRATIONS OF ACTIVE

MAINTAINED IN TSH AND TREATED WITH 100 U/ML Y-INTERFERON.

% INHIBITION OF CLASS I RNA EXPRESSION
Compound 100 M 1.0 mM 5 mM

1 Methimazole None 29 t 7 52 t 15 2 Metronidazole None 42 t 5 68 t 13 3 2-mercaptoimidazole ND None None 4 2-mercaptobenzimidazole None 37 f 10 ND ' 5 2-mercapto-5-nitrobenzimidazole None 19 t 7 ND

6 2-mercapto-5- None 22 t 16 23 t 11 methylbenzimidazole 7 S-methylmethinlazole 14 11 40 t 15 69 t 6 8 N-methylmethimazole 91: 7 49 t 13 73 f 12 9 5-methylmethimazole None 9 5 24 f 6 5-Phenylmethimazole 42 f 14 60 f 12 ND

11 1-methyl-2-thiomethyl- 19 t 12 59 t 5 ND (Toxic) 5(4)nitroimidazole Values from three experiments in duplicate, mean SD.
ND is not done; None is no effect measurable.
Bold values represent significant inhibition (P <; 0.05 or better).
10 Experiment in each case was in 6H medium.
Treatments with interferon and the methimazole derivatives or tautomeric cyclic thiones were for 40 hours.

ii WO 00/12175 PCT/US99/19$61 TABLE 16: EFFECT OF DIFFERENT CONCENTRATIONS OF ACTIVE

WITH 100 U/ML Y-INTERFERON.

% INIIIBITION OF CLASS II RNA EXPRESSION
Compound 100 M 1 mM

1 Methimazole P1one 29 t 9 2 Metronidazole 1.1 t 10 45 t 13 7 S-methylmethimazole 21,2 t 17 62 t 14 8 N-methylmethimazole 32 t 5 59 t 12 5-Phenylmethimazole 75 t 15 87 t 4 11 1-methyl-2-thiomethyl- 40 f 11 85 t 7 5(4)nitroimidazole Values from two experiments in duplicate, mean SD.
Bold values represent significant inhibition (P < 0.05 or better).
Experiment was in 6H medium.
10 Treatment with interferon and the MMI derivatives or tautomeric cyclic thiones was for 40 hours.

TABLE 17: EFFECT OF ACTIVE COMPOIJNDS ON THYROGLOBIJLIN
RNA LEVELS IN FRTL -5 THYROID CELILS.

% INCREASE IN TG ll'NA EXPRESSION
Compound 1 mM

1 Methimazole 207 t 10 2 Metronidazole (15 t 13) 7 S-methylmethimazole 122 f 14 8 N-methylmethimazole 129 t 7 5-Phenylmethimazole (7 13) 11 1-methyl-2-thiomethyl-5(4)nitroimidazole 118 6 Values from two experiments in duplicate, mean SD.
Bold values represent a significant increase (P <; 0.05 or better).
Bold and italicized values represent a measurable decrease or no increase (P <
0.05 or better).
10 Experiment was in 6H medium.
Treatment with interferon and the MMI derivatives or tautomeric cyclic thiones was for 40 hours.

As pointed out in Figure 12, the ability of MMI to decrease protein/DNA
complexes and promoter activity is associated with the ability of MMI to decrease antigen expression as measured by flow cytomet:ry. The MMI derivatives and tautomeric cyclic thiones also decreased w-IFN-iinduced MHC class I and class II
antigen expression as measured by flow cytomet:ry.

In this experiment 106 cells were incubated with MHC class II or MHC class I-specific antibodies as described (Montani, V., et al., ibid (1998b); Saji, M., et al., ibid (1992a); Balducci-Silano, P.L., et al., Endocrinology 139:2309-2313 (1998)).
After 30 rnin on ice, cells were washed with phosphate buffered saline at pH
7.4 and incubated for 30 min with fluorescein-isothiocyanate (FITC)-conjugated antibodies then analyzed by flow cytometry on a FACScan Cytometer using CellQuest software (Becton Dickinson).

The concentration needed to achieve suppression of the interferon induced class I or class II surface expression was determined by testing different concentrations of the compounds (Table 18). Like all other assays, the order of effectiveness was compound 10 > 11 > 7 or 8 > 2> MMI > 3.
TABLE 18: EFFECT OF DIFFERENT CONCENTRATIONS OF ACTIVE
COMPOUNDS ON CLASS I OR CLASS II ANTIGEN EXPRESSION IN
FRTL-5 CELLS MAINTAINED IN TSH A13D TREATED WITH 100 U/ml x-INTERFERON
Concentration to inhibit IFN-increased antigen expression Compound Class I Class II
1 Methimazole 5 mM 5 mM
2 Metronidazole 01.5 1.4 mM 1.5 1.0 mM
3 2-mercaptoimidazole rro Inhibition No Inhibition 7 S-methylmethimazole 424 31 M 405 15 M
8 N-methylmethimazole 356 70 M 439 93 M
10 5-Phenylmethimazole 40 10 M 54 11 M
11 1-methyl-2-thiomethyl- 110 f 14 M 109 5 M
5(4)nitroimidazole Values from three experiments in duplicate, mean f SD.
Bold values represent significant inhibition (P<0.05 or better).
Experiment in each- case was in 6H medium.
Treatments with interferon and the methimazole derivatives or tautomeric cyclic thiones were for 40 hours.

Ability of MMI, MMI derivatives (2-mereaptoimidazole) or tautomeric cyclic thiones (Compound 10) to prevent proteiinuria in (NZBxNZW)Fl Mice or Diabetes in NQD mLce The objective of these experiments was to determine the effect of MMI and the tautomeric cyclic thiones (compound 10) oin the development of lupus in NZB

mice or diabetes in NOD mice by comparison to a methimazole derivative, 2-mercaptoimidazole. Compound 10 was the mc-st effective agent determined in the multiplicity of in vitro assays to suppress class I and class II gene expression; in contrast, 2-mercaptoimidazole was less effective than MMI and had negligible ability to suppress class I and class II gene expression. These were assays to validate the identification procedures with an i,t vivo correlate.

Female (NZB x NZW)F1 mice spontaneously develop an autoimmune disease resembling human systemic lupus erythematosus (SLE) with age (Mozes et al., Clinical Immunology 18:106-113 (1998)). Similarly, female NOD mice develop a disease resembling type 1 diabetes with age (Makino et al., Exp. Anim. 29:1-13 (1980); Wicker, L.S., et al., Diabetes 35:855-860 (1986)). In the former case, proteinuria is a measure of the onset of disease; in the latter case, glucosuria is a measure of the onset of disease.

Methods Female (NZB x NZW)F, mice were obtained from Jackson Labs. Animals were followed with AMES 2855 Uristix (Miles) to semiquantitatively measure proteinuria. Proteinuria was taken as a measure of development of SLE and MMI
suppression of proteinuria as suppression of renal complexes in kidney (Mozes et al., J. Clin Immunology 18:106-1 i3 (1998)). jkt 7-8 mos. mice develop proteinuria reflecting renal disease. Treatment was from 2 mos to 6 mos of age. Treatment was oral. Each group had 8 animals to start.

Female NOD mice were also obtained from Jackson Labs, along with control mice from which the strain was developed f,Maakino et al., Exp. Anim. 29:1-13 (1980); Wicker, L.S., et al., Diabetes 35:855-860 (1986)). Animals showing urine Tes-Tape positivity greater than 1 + were considered positive and to have diabetes (Wicker, L.S., et al., ibid (1986)). At 8-16 weeks, 10-40% of NOD mice develop glucosuria and diabetes according to the literature (Wicker, L.S., et al., ibid (1986)).

Results In an experiment with Female (NZB x NZW)F, mice (Table 19) all surviving control animals and a112-mercaptoimidazole-treated animals developed significant proteinuria at 7.5 mos. In contrast, both MMI and compound 10 (5-phenylmethimazole), at a fifth the MMI concentration, significantly prevented proteinuria. Animals were sacrificed from each group and kidneys evaluated for immune complexes; data revealed that control animals had significant numbers of immune complexes in their kidneys, whereas this was not true for MMI or compound 10 treated animals (Figure 15). In tllis experiment, immunohistology was performed in a blind fashion on 5 m thick frozen kidney sections which were fixed and stained with FITC-conjugated goat antibodies to immunoglobulin G(x chain specific) as described (Mozes, et al., Science 261:91-93 (1993); Mozes, E., et al., ibid (1998)). Compound 10, a tautomeric cyclic thione, and MMI are effective in preventing SLE in this experimental model.

Death of some animals in this experimerit occurred for technical reasons, i.e.
flooding of cages, handling, etc., in the last 2 nios. However, losses in the control group were srtnilar to experiunental groups. MrviI, 2-mercaptoimidazole, and Compound 10 treatments were administered during the period between 2 and 6.25 months of age.

Table 20 presents results of the ability of Female NOD mice (5 animals were in each group) to develop glucosuria and diabetes when treated with MMI and compound 10 (5-phenylmethimazole) at a fifth the MMI concentration, by comparison to no treatment or treatment with 2-mercaptoimidazole. Mice were from Jackson Labs in this experiment. Animals showing urine Tes-Tape positivity greater than 1 + were considered positive and to have diabetes.

In this experiment all surviving control ainimals and all 2-mercaptoimidazole-treated animals developed diabetes by 12 weeks. In contrast, both MMI and 5-phenylmethimazole, at a fifth the MMI concentration, prevented glucosuria.
Compound 10 and MMI are therefore effective in preventing diabetes in the NOD
mouse example of diabetes. Death of animals for technical reasons, i.e.
flooding of cages, handling, etc., was similar in control and experimental groups and did not effect results.
The results in these two experiments support the conclusion that the inr vitro assays can detect effective drugs to treat autoinvnune disease in vivo based on their ability to suppress class I and class II gene expression in vitro in rat FRTL-5 cells.
Moreover, the ability of compound 10 to do this; at one-fifth the MMI
concentration and to be nontoxic appears to validate the hypothesis that use of the assay protocol in FRTL-5 thyroid cells in culture is reasonably predictive of in yi,vo efficacy.

TABLE 19: ABILITY OF MMI, MMI DEIi:IVATIVES (2-MERCAPTOIMIDAZOLE), AND TAUTOMERIC CYCLIC THIONES
(COMPOUND 10) TO PREVENT PROTEIl'1URIA IN (NZB X NZW)Fl MICE
Animals With Proteinuria Treatment 6 mos (mg/dl proteinuiria) 7.5 mos (mg/dI proteinuria) Neg Trace 30-500 > 500 Neg-Trace 30-500 > 500 None 1 of 8 5 of 8 2 of 8 0 of 6 2 of 6 4 of 6 0.05% MMI 8 of 8 0 of 8 0 of 8 3 of 5 2 of 5 0 of 5 0.01%5- 8 of 8 4of8 0 of 8 5 of 6* 1 of 6 0 of 6 Phenylmethimazole 0.05% 2- 3 of 7 4 of 7 0 of 7 0 of 6 3 of 6 3 of 6 mercaptoimidazole *statistically significant, p < 0.05.

TABLE 20: ABILITY OF MMI, MMI DERIVATIVES (2-MERCAPTOIMIDAZOLE) AND TAUTOIV>rERIC CYCLIC THIONES
(COMPOUND 10) TO PREVENT GLUCOSURIA IN NOD MICE

Animals with Glucosuria Treatment 4 weeks 8 weeks 12 weeks 14 weeks None 0 of 5 2 of 5 4 of 4 2 of 2 0.05 kMMI Oof5 Oof5 lof3* Oof3**
0.01% 5-Phen lmethimazole 0 of 5 0 of 5 0 of 4* 0 of 4**
0.05% 2-merca toimidazole 0 of 5 2 of 5 3 of 3 3 of 3 *statistically significant, p< 0.05; **statistically significant, p< 0.01 Effect of Methimazole and Tautomeric Cylic Thione Derivatives on Interferon Induced Decreases in Y Box Protein Levels One factor known to suppress MHC class I and class II gene expression is a Y box binding protein (Saji, M., et al., ibid (1997); Ting, J. P-Y., et al., J. Exp.
Med. 179:1605-1611 (1994)). The human Y box protein, YB-1, was cloned based on its ability to bind to the Y box of the MHC class II gene, an inverted CCAAT
box (Didier, D.K., et al. Proc. Natl. Acad. Sci. USA 85:7322-7326 (1988)).
Transfection of YB-1 was shown to suppress HLA-DRa gene expression in human glioblastoma cells and FRTL-5 thyrocytes (Ting, J.P-Y., et al., ibid (1994);
MacDonald, G.H., et al., J. Biol. Chem. 270:3527-3533 (1995); Montani, V., et al., ibid (1998a)). The Y box protein in FRTL-5 cells was cloned based on its ability to suppress thyrotropin receptor (TSHR) gene expression (Shimura, H., et al., J. Biol. Chem. 268:24125-24137 (1993); Ohmori, M., et al., Mol.
Endocrinol.
10:76-89 (1996)) and therefore termed TSHR suppressor element binding protein-i (TSEP-1). TSEP-1 is a component of the thyroid autoregulatory system wherein TSH/cAMP decreases TSHR and MHC class I gene expression as FTRL-5 thyroid cells progress through the functional and growtli phases of the cell cycle after TSH
challenge (Shimura, H., et al. ibid (1993); Ohmiori, M., et al., ibid (1996);
Kohn, L.D., et al., ibid (1995)). TSH/cAMP increase Y box gene expression, the Y box is a suppressor of TSHR and class I gene expression, and, as a result, TSHR
and class I gene expression is decreased.

Whereas x-IFN decreases Y box protein RNA levels in FRTL-5 cells, MMI
can reverse this action (Fig. 16). In this experiunent, FRTL-5 cells were maintained in complete 6H medium plus 5% calf serum andl treated with 100 Units/ml r-IFN, an active selected from 5 mM methimazole, 0.1 mM compound 10, 0.25 mM
compound 11, 0.5 mM or 0.25 mM compounds 7, 8 and 9, or 0.5 mM
compound 3, or both IFN and the different actives for 40 hours before total cellular RNA was isolated and northern analysis performed as described (Isozaki, 0., et al., ibid (1989); Saji, M. et al., Endocrinology 130:520-533 (1992); Olunori, M., et al., ibid (1996)). The rat TSEP-1 probe was the clone 31 insert, residues 5 to 1395 of the rat TSEP-1 nucleotide sequence reported (C>hmori, M., et al., ibid (1996)); rat ¾-actin was kindly provided by Dr. B. Paterson (NCI, Bethesda, MD).

Radiolabeling of all probes, hybridization (0.5-1.Ox106cpm/ml), and washing were as described (Isozaki, 0., et al., ibid (1989); Saji, M., et al., ibid (1992);
Ohmori, M., et al., ibid (1996)).

The effect of MMI derivatives and tautomeric cyclic thiones on ir-IFN-decreased TSEP-1 levels is presented in Table 21. The ability of the compounds to reverse the it-IFN-induced decrease in TSEP-1 RNA levels is once again 10 > 11 > 7 or 8 > 2 > MMI > 3. This mimics data in all other assays.

YB-1 is the prototype Y box binding protein. It was cloned using the radiolabeled Y box element of the class II promoter to screen akgti I
expression DNA library (Didier, D.K., et al., ibid (1988)). Direct evidence of the ability of YB-1 to suppress ir-IFN-induced class II gene expression was provided in glioblastoma and FRTL-5 cells (Ting, J.P-Y., et al., ibid (1998a)). Separate studies also showed it suppressed MHC class I gene expression (Saji, M., et aI., ibid (1997)). These data show that tt-IFN decreases Y-box RNA levels. Were Y box protein to also decrease, it is reasonable to presume that decreased Y-box suppression of class I and class II would result, since Y box suppresses class I and class II gene expression. In short, it is reasonable to presume that one means by which 'Y-IFN increases MHC expression is to decrease Y box suppre'ssion of both genes. The data further show that MMI, MMI derivatives and tautomeric cyclic thiones reverse the x-IFN induced decrease in Y box RNA levels coincident with their action to decrease x-IFN-induced'increases in MHC gene expression estimated by gel shifts, RNA levels, or promoter activity. In sum, we suggest that x-IFN

sinwltaneousty reduces class II suppressive action by decreasing TSEP-1 RNA
levels and increases class il expression by increasing CIITA RNA levels. The net result is "aberrant" expression of MHC class II and abnormal class I
expression. -Methimazole reverses this by reversing the efi'ect of v-IFN on TSEP-1 (Y box) RNA levels and eliminating the W-IFN-induceci complexes with the HLA-DRa 5'-flanking region (Montani, V., ibid (1998a)). 'The derivatives shown herein to be more active in suppressing IFN-increased class I and class II gene expression have greater abilities than MMI to reverse the effect of IFN on Y box gene expression.
One effect of these agents is thus to prevent or reverse the action of interferon to alter MHC gene expression and exacerbate the autoimmune response (Fig. 17).
The agents are also likely to decrease the initial or primary insult on the target tissue which initiates the autoimmune process (Fig. 17) as evidenced by decreases in basal class I gene expression.

Of interest, the MMI, MMI derivatives, and tautomeric cyclic thiones have a minimal effect on basal Y box RNA levels. Tl:iis reinforces the possibility that the action of the compounds is selective in its effect on w-IFN-induced changes in Y box RNA levels and will not harm normal physiologic processes controlled by Y box.

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... , ~- 1 V ~>' 1' 4 Cy -4 SUBSME SHEET (RULE 26) EXAMPLl , 8 Preparation of Pharmaceutical Compolitions of the Present Invention Composition Admr'nistration Means of administering active compounds of the invention include, but are ..
not limited to, oral, sublingual, intravenous, intramuscular, intraperitoneal, percutaneous, intranasal, intrathecal, subcutaneous, or enteral. Local administration to the afflicted site may be accomplished through means known in the art, including, but not limited to, topical application, injection., infusion and implantation of a porous device in which the active compound(s) or compositions of the invention are contained. Accordingly, the active compounds of the invention will generally be administered as a pharmaceutical composition c;omprising one or more active compounds of the invention in combination with a pharmaceutically acceptable excipient and other formulational aids.

Formutational Aids Such compositions may be aqueous solutions, emulsions, creams, ointments, suspensions, gels, liposomal suspensions, and Oe like. Suitable excipients include water, saline, Ringer's solution, dextrose solution, and solutions of ethanol, glucose, sucrose, dextran, mannose, mannitol, sorbitol, polyethylene glycol (PEG), phosphate, acetate, gelatin, collagen, CarbopolQO, vegetable oils, and the like. One may additionally include suitable preservatives, stabilizers, antioxidants, antimicrobials, and buffering agents, for example, BHA, BHT, citric acid, ascorbic acid, tetracycline, and the like. Cream or ointment bases useful in formulation include lanolin, Silvadene (Marion), Aquaphor (Duke Laboratories), and the like.
Alternatively, one may incorporate or encapsulate the active compounds of the invention in a suitable polymer matrix or memb:rane, thus providing a sustained-release device suitable for implantation near the site to be treated locally.
Other devices include indwelling catheters and devices, such as the Alzet minipump.
Opthalrnic preparations may be formulated using commercially available vehicles such as Sorbi-care'a (Allergan), Neodecdron (Merck, Sharp & Dohme), WO t1G1t275 IUnencled Page 101) LamubeO. xd the !ltoe. Pnrtllor. om nity ,pravide the adive oom*ouadi of ILe iavt-ma In bu-kmg .pan, for ample lamwt serm Wbami& mcrore, mamatol, are tea aiwe. A ftroagn disomloa of pfi.eaKW&dLy amitable mdplea4s Is evOlabte in RaatEngcan's Pfawnuoeut9orl soienoes I QA4c1c Pub. Co.).

OrcC/Parnits,st Adxtkgsbo"
'i'be aetive compouade of tbe invontion.am be admdalaerod both oraily and ParammUy tn aeooadmm t-ith eemva*ood plocedwms for fhe oroatmaot of aotol=ttp0 di:aoe awd per8onamnCe of orgaa aodJor tbeoa Rmaplu=ioa. The an101mt of aCtive aompoumd reqtllro0 to uta aury pardcvlar aaoolrtmme std/or tsoaplta dboda arili. of ccourae. vary depeodlog upon do aMene and sevorlRy of the disorder. tha age md conditiam of ee talbjeet, gad otbor fYcepes raed*
6e10=foed by one of atdimry sloll In do aR. Aclive coa4ounb ta e adodnizened In doaege rmit6 pretbrably dtrhled dosW oaa., cooadnhg the aetEve oompoun4 altlt a midebb pWxoToglpllly aoceptable coerfer or exdpieat. many of whiah aee well kaown to tlmee In tbo art aad m detexbed a6ovo. The doeasa ttoJtf erom be in tbe form Of a ticPid pcspration, e.g., volbtiona. suepmelons. dbpessiooa, or eaallsinme, or thcy mq be In aDlid toam soeh ae pms, tabkts. aapsnlel or the Hloa.
Camapotitiaoc in ut* donge tbrm. l.a, pbunumemian] cotVaWom wHioh ate avaiiobk ia e pe-mpplned Rxtn auinWc for fingle dora wdmiottstsdva wldkmt reWdft tbat elic iadivfduat dosaV be meawuad ovt by the euer. tlor elcamoe.
PiUe.
tabk.h, capmaet, or anoguN are pwetioolRrly RderraQ mdhode of adndnbMdm of u- aetive eomePonada of tbo wr~mt invaodun.

rr+dend bmamgm Fbr tLe rnatmmc of au0oilmnuna sad ayampbmtatios doordaa pbwrauctudcal oomposWoro in doaage w4it tonm comprise an atnotmt of compo.itiam arLiab prorlda froan about 0.03 to ebout 60 mllllgratar. Pneftably frasn about 0.05 to about 20 mlplgirams. of active compomd per day. To prodoen dosage units for peroral administration, the active compound of the invention or a salt thereof is combined, e.g., with solid powdered carriers such as lactose, sucrose, mannitol; starches such as potato starch, corn starch or amylopectin, as well as _ iaminaria powder and citrus pulp powder; celllulose derivatives of gelatin, also lubricants such as magnesium or calcium sterat.e of polyethylene glycols (carbowaxes) of suitable molecular weights may be added, to form compressed tablets or core tablets for sugar coating. The latter are coated, for example, with concentrated sugar solutions which, e.g., can contain gum arabic, talcum and/or titinium dixoide, or they are coated with a lac+quer dissolved in easily volatile organic solvents or mixture of organic solvents. Dyestuffs can be added to these coatings, for example, to distinguish between different contents of active substance.
Capsules useful herein include, for example, soft gelatin capsules (pearl-shaped closed capsules), geltabs, other capsules whicl2 consist, for example, of a mixture of gelatin and glycerin and contain, e.g., mixtures of the active substances or a suitable salt thereof with solid, powdered carriers sucl:i as, e.g., lactose, sucrose, sorbital, mannitol; starches such as potato starch corn starch or amylopectin, cellulose derivatives or gelatin, as well as magnesium sterate or steric acid.
Suppositories are employed as dosage units for rectal application. These consist of a combination of the active substance or a suitable salt thereof with a neutral fatty base, or also gelatin rectal capsules can be employed which consist of a combination of the active substance or a suitable salt thereof with polyethylene glycols (carbowaxes) of suitable molecular weight.

Ampoules for parenteral, particularly intramuscular administration preferably contain an active compound or a water soluble salt thereof and suitable stabilizing agents, and, if necessary, buffer substances in aqueous solution. Anti-oxidizing agents such as sodium bisulfite, sodium sulfite, ascorbic acid or Rongalit (formaldehyde-sodium bisulfite compound), and the like are suitable as stabilizing agents either alone or combined, in total concentrations between 0.01 and about 0.05 percent of the composition. Because of its ability to form chelates, ascorbic acid has an additional stabilizing effect; in this function it can also be replaced by other chelate-formers. The best suitability of the active ingredient is attained, e.g., by mixtures in suitable ratio of sodium sulfite,, sodium bisulfite and/or ascorbic acid, or by the addition of other buffer substances such as citric acid and/or salts thereof.
In addition, the ampoules can contain a slight amount of a preservative.

Useful pharmaceutical formulations foi- administration of the active compounds of this invention may be illustrateci below. They are made using conventional techniques.

CAPSULES
Active ingredient 0.05 to 20 mg Lactose 20-100 mg Corn Starch U.S.P. 20-100 mg Aerosolized silica gel 2-4 mg Magnesium stearate 1-2 mg TABLETS
Active ingredient 0.05 to 20 mg Microcrystalline cellulose 50 mg Corn Starch U.S.P. 80 mg Lactose U.$.P. 50 mg Magnesium stearate U.S.P. 1-2 mg This tablet can be sugar coated according to conventional art practices.
Colors may be added to the coating.

CHEWABLE TABLETS
Active ingredient 0.05 to 20 mg Mannitol, N.F. 100 mg II

WO 00/12175 PCT/US99/19862' Flavor 1 mg Magnesium stearate U.S.P. 2 mg SUPPOSITORIES
Active ingredient 0.05 to 20 mg Suppository base 1900 mg LIQUID
Active ingredient 2.0 percent Polyethylene glyco1300, N.F. 10.0 percent Glycerin 5.0 percent Sodium bisulfite 0.02 percent Sorbitol solution 70%, U.S.P. 50 percent Methylparaben, U.S.P. 0.1 percent Propylparaben, U.S.P. 0.2 percent Distilled water, U.S.P. (q.s.) 100.0 cc INJECTABLE
Active ingredient 0.05 to 60 mg Polyethylene glyco1600 1.0 cc Sodium bisulfite, U.S.P. 0.4 mg Water for injection, U.S.P. (q.s.) 2.0 cc Treatment of Humans Suffering SIX, an Autoimmune Disease For treating humans suffering from SLE, active compound(s) of the present -invention are administered, preferably orally, but administration may also be parenterally, at a dose of up to 100 mg per day initially. Initial dosing can be followed by a step-wise reduction program, to 50 mg for 20 days, 40 mg for up to.
20 days, 35 mg for up to 30 to 60 days, decreasing progressively to 5 mg-30 mg per day. A maintenance dose of 5 mg-10 mg per day for up to 1 year or longer may also be used. Dosages may be decreased by 50 to 100 fold, at least, for preferred active compounds.

Patients can be monitored for alleviation of clinical signs and symptoms of active disease. Specifically monitored parauneters can include, autoantibodies, particularly DNA antibodies; PBL cell surface markers, leukopenia;
proteinuria;
hyperimmunoglobulinemia; and levels of immixne complexes in the kidney by punch biopsy.

In addition, TSH or T31T4 levels may be monitored to access the therapeutic levels of active compounds of the invention required for control of disease in the SLE patient. When TSH levels increase significantly above the normal range, indicative of the effective action of the active compound, dosage can be decreased to the next dose level; When thyroid hormone levels decrease significantly from the normal range, this also 'can be used as an indication to lower dosage. If patients exhibit a decrease in thyroid hormones or an increase in TSH, they can be treated with thyroid hormone (T3 or T4) plus active compounds to maintain a euthyroid state. The TSH level is a better index. The saime parameters may be assessed in children.

Treatment of Humans Suffering From or at Increased Risk of Developing IDDM. an Autoimmiune Disease Humans discovered to be suffering from juvenile diabetes, Type I diabetes, or determined by those skilled in the art to possess an increased risk of developing IDDM may be treated by administration of active compound(s) of the present invention, preferably orally (although they may also be administered parenterally), at a dose of up to 100 mg per day initially. Initial dosing can be followed by a step-wise reduction program, to 50 mg for 20 daysõ 40 mg for up to 20 days, 35 mg for up to 30 to 60 days, decreasing progressively t:o 5 mg-30 mg per day. A
maintenance dose of 5 mg-10 mg per day for up to 1 year or longer can also be used. Dosages may however be lowered by 50 to 100 fold, at least, for preferred active compounds.

Patients can be monitored for alleviation of clinical signs and symptoms of active disease. Specifically monitored parame'ters can include glucosuria, glucosemia, autoantibodies, particularly antibodies known to those skilled in the art to possess positive correlation to disease progression and/or known to those skiiled in the art to possess predictive value with regard to an individual's predisposition, and hence increased risk, for IDDM disease; PBL cell surface markers;
leukopenia;
and glucosuria.

Claims (31)

What is claimed is:

1. A pharmaceutical composition comprising a safe and effective amount for the treatment of an autoimmune disease in a human suffering from said disease of a compound selected from:
wherein Y is selected from the group consisting of H, C1-C4 alkyl, C1-C4 substituted alkyl, and the phenyl moiety;

and wherein no more than one Y group in said compound can be the phenyl moiety: R1 is selected from the group consisting of H, -OH, C1-C4 alkyl, and C1-C4 substituted alkyl; R2 is selected from the group consisting of H and C1-C4 alkyl: R3 is selected from the group consisting of C1-C4 alkyl, C1-C4 substituted alkyl, and -CH2Ph; R4 is selected from the group consisting of H, C1-C4 alkyl, and C1-C4 substituted alkyl; and Z is selected from -OR3; and wherein at least two of the R2 and R3 groups in said compound are C1-C4 alkyl when Y is not a mono-substituted phenyl moiety; and a pharmaccutically-acceptable carrier.

2. A pharmaceutical composition according to Claim 1 wherein Z is selected from OR3.

3. A pharmaceutical composition according to Claim 2 wherein Y is H.

4. A pharmaceutical composition according to Claim 3 wherein R3 is C1-C4 alkyl.

5. A pharmaceutical composition according to Claim 4 wherein R3 is methyl.

6. A pharmaceutical composition according to Claim 5 wherein R2 is methyl.

7. A pharmaceutical composition according to Claim 3 wherein both R2 and R3 groups are methyl.

8. A compound wherein the compound has the formula

9. A compound according to Claim 2 wherein one of the Y groups is the phenyl moiety.

10. A compound according to Claim 9 wherein R1 and R4 are H.

11. A compound according to Claim 10 wherein R3 is methyl and R2 is methyl.

12. A compound according to Claim 10 wherein R3 is H.

13. A compound wherein the compound is

14. A pharmaceutical composition according to Claim 1 in unit dosage form.

15. A pharmaceutical composition according to Claim 1 which comprises from about 0.01% to about 25% of the compound and from about 75% to about 99.99% of the pharmaceutically-acceptable carrier.

16. A pharmaceutical composition comprising a safe and effective amount for the treatment of an autoimmune disease in a human suffering from said disease of a compound selected from wherein R5 and R6 are selected from the following moiety pairs CH3, CH3; Ph, H
and H, Ph;
R7 is selected from H and CH3; and R8 is selected from O, NH and NCH3 ; and a pharmaceutically-acceptable carrier.

17. Use of a safe and effective amount of the pharmaceutical composition according to Claim 1 in the treatment of autoimmune diseases.

18. A pharmaceutical composition as recited in Claim 1 suitable for intraperitoneal injection, intravenous injection. intramuscular injection, oral ingestion, or topical application.

19. A pharmaceutical composition as recited in Claim 18 suitable for oral ingestion.

20. A pharmaceutical composition as recited in Claim 19 wherein the pharmaceutical composition is in a unit dosage form.

21. A pharmaceutical composition as recited in Claim 18 wherein the unit dosage form is in the range of 0.05 to 50 milligrams per day.

22. Use of a safe and effective amount of the compound according to Claim 8 in the treatment of autoimmune diseases.

23. Use of a safe and effective amount of the compound according to Claim 13 in the treatment of autoimmune diseases.

24. Use of a safe and effective amount of the pharmaceutical composition according to Claim 1 in the treatment of systemic lupus erythromatosus (SLE).

25. A pharmaceutical composition comprising a safe and effective amount for the treatment of an autoimmune disease in a human suffering from said disease of a compound wherein Y is selected form the group consisting of H, C1-C4 alkyl, C1-C4 substituted alkyl, and the phenyl moiety;

and wherein no more than one Y group is said compound can be the phenyl moiety; R1 is selected from the group consisting H, -OH, halogens, C1-C4 alkyl, C1-C4 substituted alkyl, C1-C4 ester and C1-C4 substituted ester; R2 is selected from the group consisting of H and C1-C4 alkyl; R3 is selected from the group consisting of C1-C4 alkyl, C1-C4 substituted alkyl, and -CH2Ph; R4 is selected from the group consisting of H, C1-C4 alkyl, C1-C4 substituted alkyl;

Z is selected from -SR3, -OR3, -S(O)R3, and C1-C4 alkyl: and wherein at least two of the R2 and R3 groups in said compound are C1-C4 alkyl when Y is not a mono-substituted phenyl moiety; and a pharmaceutically-acceptable carrier.

26. A pharmaceutical composition according to Claim 25 wherein the compound comprises wherein R9 is selected from the group consisting of -OH, -M and -OOCCH2M;
wherein M is selected from F, Cl, Br and I.

27. A compound wherein the compound is selected from the group consisting of wherein R10 is selected from the group consisting of H, Ph, 4-HOPh and 4-MPh, wherein M
is selected from F, Cl, Br and I.

28. Use of a safe and effective amount of the pharmaceutical composition according to Claim 25 in the treatment of autoimmune diseases.

29. Use of a safe and effective amount of the pharmaceutical composition according to Claim 26 in the treatment of autoimmune diseases.

30. Use of a safe and effective amount of the compound according to Claim 27 in the treatment of autoimmune diseases.

31. Use of a safe and effective amount of a pharmaceutical composition comprising a compound selected from the group consisting of wherein Y is selected from the group consisting of H, C1-C4 alkyl, C1-C4 substituted alkyl, and the phenyl moiety and wherein no more than one Y group in said compound can he the phenyl moiety; R1 is selected from the group consisting of H, -OH, C1-C4 alkyl, and C1-C4 substituted alkyl, R2 is selected from the group consisting of H and C1-C4 alkyl; R3 is selected from the group consisting of C1-C4 alkyl, C1-C4 substituted alkyl, and -CH2Ph; R4 is selected from the group consisting of H, C1-C4 alkyl, and C1-C4 substituted alkyl, Z is selected from -OR3; and wherein at least two of the R2 and R3 groups in said compound are C1-C4 alkyl when Y is not a mono-substituted phenyl moiety; and a pharmaceutically-acceptable carrier; in the treatment of autoimmune diseases.