WO1995013805A1

WO1995013805A1 - Use of nitric oxide synthase inhibitors in the treatment of autoimmune diseases

Info

Publication number: WO1995013805A1
Application number: PCT/US1994/013239
Authority: WO
Inventors: J. Brice Weinberg
Original assignee: Duke University Medical Center
Priority date: 1993-11-17
Filing date: 1994-11-17
Publication date: 1995-05-26
Also published as: AU1209995A; EP0729356A1; CA2176747A1; JPH09508891A

Abstract

The invention relates to a method of treating or preventing autoimmune diseases, such as rheumatoid arthritis, insulin dependent diabetes mellitus, systemic lupus erythematosus and glomerulonephritis, comprising administering to a patient in need thereof an effective amount of a nitric oxide synthase inhibitor or a nitric oxide scavenger.

Description

USE OF NITRIC OXIDE SYNTHASE INHIBITORS IN THE TREATMENT OF AUTOIMMUNE DISEASES

BACKGROUND OF THE INVENTION

NRL-lpr/lpr mice have been studied as a model for human autoimmune diseases. This strain of mice develops a spontaneous autoimmune disease characterized by lymphadenopathy, autoantibody production and inflammatory manifestations including nephritis, vasculitis, and arthritis (Hang, L. , et al . , J. Exp. Med . 155:1690-1701 (1982); Eisenberg, R.A., et al . , Clin . Exp. heumatol . 7:S35-S40 (1989)). Immune function abnormalities also include enhanced constitutive macrophage class II antigen expression, elevated levels of IFN-γ, TNF, IL-1, and IL-6 in isolated kidney, lymph node, and spleen cells, and an enhanced state of macrophage "activation". These disease manifestations are a result of both a single gene mutation (lpr) of the Fas apoptosis gene on mouse Chromosome 19 and background genes from the MRL strain. Although the MRL genes contributing to disease manifestations have not been identified, two loci contributing to renal disease have been mapped to regions of mouse Chromosomes 7 and 12. NO (nitric oxide) , a multifunctional molecule produced by diverse cell types, results from the conversion of L-arginine to L-citrulline and NO by the action of the enzyme nitric oxide synthase (NOS) . NO has been noted to promote relaxation of smooth muscle, serve as a neurotransmitter, cause stasis and/or lysis of microbes and tumor cells, and modulate function and differentiation of hematopoietic cells.

NO also has potent proinflam atory actions. NO may increase vascular permeability in inflamed tissues. Pain is also an important aspect of inflammation. Several groups have now demonstrated that NO plays a role in the mediation of pain in inflammation. Intradermal injection of solutions containing NO into humans caused a dose-related occurrence of pain in the site (Holthusen, H. , and Arndt, J.O., Neuroscience Letters 165:71-74 (1994) . Thus, pain, a hallmark symptom of inflammation, can be induced by NO.

NO has also been shown to cause increased production of TNF and IL-1 by cells, and to increase the potential of cells to produce hydrogen peroxide. Also, rabbit and human chondrocytes have been shown to produce NO and to express inducible nitric oxide synthase (iNOS) in response to various cytokines and bacterial products. NO is an important mediator in immune complex vasculitis in rats (Mulligan, M.S., et al . , Brit . J. Pharmacol . 107:1159-1162 (1992)). Some researchers have noted a role for NO in inflammatory bowel disease in guinea pigs (Miller, M.J., et al . , J. Pharmacol . Exp. Ther. 264 : 11-16 (1993)), and in induced nephritis in mice or rats (Farrario, R. , et al . , J. Am Soc. Nephrol . 4:1847-1854 (1994)). Research in non- human models of inflammatory diseases has also established that NO participates in experimentally-induced uveitis (Parks, D.J., et al . , Arch . Ophthalmol . 112:544-546 (1994)) .

Studies of humans with arthritis demonstrate further that NO plays an important role in human arthritis. Farrell et al . showed that patients with inflammatory arthritis (rheumatoid arthritis and osteoarthritis) had elevated levels of the NO catabolite nitrite in their synovial fluid and serum (Farrell, A.J., et al . , Ann . Rheum . Dis . 51:1219-1222 (1992)). Patients with rheumatoid arthritis had increased synovial fluid and serum levels of nitrotyrosine (Kaur, H., and Halliwell, B. , Febs Letters 350 : 9-12 (1994)). Cell-derived NO reacts with superoxide (0₂*-) to form peroxynitrite (ONOO-) , which in turn may spontaneously produce hydroxyl radical (HO^•) , a molecule with high potential for cell and tissue injury and destruction. Other investigators have shown in a rat model of inflammation that peroxynitrite is pro-inflammatory (Rachmilewitz, D., et al . , Gastroenterology 105:1681-1688 (1993)) .

Nitrotyrosine is formed by the action of peroxynitrite on tyrosine, and it is felt to be a stable "footprint" or "track" of the presence of NO (Beckman, J.S. et al . , Methods Enzymol . 233:229-240 (1994)). Nitrated proteins have been found associated with macrophages and inflammation (by using an anti-nitrotyrosine antibody) in atheromatous plaques in human vessels (Beckman, J.S., et al., Biol . Chem . Hoppe- Seyler 375:81-88 (1994)), providing further evidence that nitrotyrosine is formed in vivo in humans.

SUMMARY OF THE INVENTION The invention relates to a method of treating or preventing autoimmune diseases, such as rheumatoid arthritis, insulin dependent diabetes mellitus, systemic lupus erythematosus and glomerulonephritis, comprising administering, preferably enterally, to a patient in need thereof an effective amount of a nitric oxide synthase inhibitor or a nitric oxide scavenger.

BRIEF DESCRIPTION OF THE DRAWING

The Figure is a bar graph showing the scores of pathological characteristics in MKL-lpr/lpr mice either treated with N^G-monomethyl-L-arginine (NMMA) or left untreated. DETAILED DESCRIPTION OF THE INVENTION

The level of nitric oxide has now been linked to the manifestation of autoimmune diseases, particularly chronic diseases, such as rheumatoid arthritis, insulin dependent diabetes mellitus, systemic lupus erythematosus and glomerulonephritis. The inhibitors of nitric oxide synthase, known to synthesize nitric oxide in vivo, or nitric oxide scavengers can be useful in the prevention or treatment of autoimmune diseases (Gilkeson et al., Arth. Rheum. 36 (Suppl.) :S219, 1993 (September)).

Inhibitors of nitric oxide synthase which can be used in this invention are those known in the art and include substrate analogs, such as aminoguanidine, N°-amino-L- arginine, N^G-methyl-L-arginine, N^G-nitro-L-arginine, N^G- nitro-L-arginine methyl ester, and N^G-iminoethyl-L- ornithine, flavoprotein binders, such as diphenylene iodonium, iodonium diphenyl and di-2-thienyl iodonium, calmodulin binders, such as calcineurin, trifluoroperazine, N-(4-aminobutyl)-5-chloro-2- naphthalenesulfonamide and N-(6-aminohexyl)-1- naphthalenesulfonamide, heme binders, such as carbon monoxide, depleters and analogs of tetrahydrobiopterin, such as 2,4-diamino-6-hydroxypyrimidine, and induction inhibitors, such as corticosteroids, TGF-0^C-1, -2, 3, interleukin-4, interleukin-10 and macrophage deactivation factor (Nathan, The FASEB Journal, Vol. 6, Sept. 1992, pp. 3051-3064) . Preferred are the substrate analogs of nitric oxide synthase, N^G-amino-L-arginine, N^G-methyl-L-arginine, N°-nitro-L-arginine, N°-nitro-L-arginine methyl ester, and N°-iminoethyl-L-ornithine. Particularly preferred are N^G- amino-L-arginine, N°-methyl-L-arginine, N^G-nitro-L- arginine, and aminoguanidine. Most preferred is N°- methyl-L-arginine. Pharmaceutically acceptable salts may also be administered. Examples of suitable salts include acid salts, such as hydrogen chloride, hydrogen bromide, hydrogen iodide, sulfate and acetate salts, as well as basic salts, such as a ine, ammonium, alkali metal and alkaline earth metal salts.

Scavengers of nitric oxide are compounds which will react with nitric oxide in vivo, such as hemoglobin (Wang et al . , Life Sciences 49:55-60 (1991)) and cobalamins (Rajanayagam, C.G., et al . , Brit . J. Pharmacol . 108:3-5 (1993); Zatarain, J. , et al . , Clin . Res . 41:783A (1993)).

The compounds described above are known in the art and are commercially available.

The compounds of the claimed invention can be administered alone or in a suitable pharmaceutical composition. Modes of administration are those known in the art, such as enteral, parenteral or topical application. Enteral is preferred and oral administration is particularly preferred. Suitable pharmaceutical carriers can be employed and include, but are not limited to water, salt solutions, alcohols, polyethylene glycols, gelatin, carbohydrates such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, hydroxymethylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, flavorants, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds.

For parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants. including suppositories. Ampoules are convenient unit dosages. Oral applications are preferably administered in the forms of capsules, tablets and/or liquid formulations. Unit form dosages are preferred. Topical applications can be administered in the form of a liquid, gel or a cream.

It will be appreciated that the actual amounts of the active compounds in a specific case will vary according to the specific compound being utilized, the particular composition formulated, the mode of administration and the age, weight and condition of the patient, for example.

Dosages for a particular patient can be determined by one of ordinary skill in the art using conventional considerations, (e.g. by means of an appropriate, conventional pharmacological protocol) . The invention is further specifically illustrated by the following exemplification.

EXEMPLIFICATION

Nitrite/nitrate excretion

Mice were housed in metabolic cages (3 per cage) and fed deionized-distilled sterile water and a defined arginine and nitrate-free diet. Urine was collected into isopropanol to inhibit bacterial growth. Urinary nitrite/nitrate concentration was determined spectrophotometrically as described before (Granger, D.L. et al . , J. Immunol . 146:1294 (1991)). Determinations were done in duplicate or triplicate. Urinary protein was measured by the Bradford assay. Total nitrate excretion was then calculated based on the concentration and the urine volume. In animals consuming a nitrate-free diet, urinary excretion of nitrite/nitrate accurately reflects the endogenous production of NO. To determine the production of NO in mice of different strains, we analyzed urine collected daily under basal conditions. MRL-lpr/lpr mice excrete more nitrite/nitrate than do C3H mice, when analyzed over a 10 day period at age 3 months. Likewise, when analyzed over time beginning at age 6 weeks, MRL- lpr/lpr mice excrete higher levels of nitrite/nitrate than do B6 mice as the mice age. Higher nitrate/nitrite excretion begins at approximately age 10 to 12 weeks paralleling that of proteinuria. Oral administration of 50 πM NMMA in water to the MRL-lpr/lpr mice reduced the high level nitrite/nitrate excretion. This signifies that the nitrite/nitrate is a product of NOS, since NMMA is a specific inhibitor of the enzyme. Levels of nitrite/nitrate excretion in 5 month old mice of strains MRL-+/+ (0.8 μmol per mouse per day) and B6-lpr/lpr (1.2 μmol per mouse per day) (3 mice in each group) were normal These results indicate that neither the lpr gene per se nor the MRL genetic background is adequate for the expression of enhanced nitrite excretion, and that both the lpr gene and genetic factors in the MRL background are necessary.

Nitric oxide production in vitro and nitric oxide synthase content

Spleen, liver, kidneys, lymph nodes, and peritoneal cells were collected and quickly frozen in a dry ice-ethanol slurry in a buffer-protease inhibitor cocktail containing 100 μM phenylmethylsulfonyl fluoride, 5 μg/ l aprotinin, 1 μg/ml chymostatin, and 5 μg/ml pepstatin A. The tissue cells were then disrupted with a pestle in repeated freeze-thaw cycles. Cytosol was collected after centrifugation, and assayed for protein and NOS activity using a modification of procedures known previously (Bredt, D.S. and Snyder, S.H. , Proc . Natl . Acad . Sci. USA 86:9030 (1989); Sherman, P.A. , et al . , Biochemistry 32:11600 (1993)). The assay buffer contained 50 mM HEPES (pH 7.5), 200 μM NADPH, 1 mM dithiothreitol, 10 μM FAD, 100 μM tetrahydrobiopterin, and 10 μM L-arginine. We used L-arginine labeled with tritium in the guanidino position (product number NEC453, New England Nuclear, Wilmington, DE) . Thirty microliters of sample were used in a total reaction mixture of 50 microliters. Samples were done in duplicate or triplicate.

Peritoneal macrophages from the normal BALB mice had no enhancement of nitrite/nitrate production when treated with endotoxin or IFN-7 alone, but the combination enhanced the production greatly. In contrast, peritoneal macrophages from MRL-lpr/lpr mice had enhanced responses to treatment with endotoxin and murine IFN-7 alone, as well as with combined endotoxin IFN-γ treatment. To determine if there was enhanced tissue iNOS mRNA expression, RNA was extracted from organs from BALB and MRL-lpr/lpr mice, and then examined by Northern analysis for iNOS mRNA expression. The iNOS mRNA (approximately 4.7 kilobases in size) was noted in tissue from kidney and spleen from MRL-lpr/lpr mice, but not in those tissues from BALB mice. Various tissues and cells from MRL-lpr/lpr and BALB mice were extracted and analyzed for their abilities to convert ¹⁴C-L-arginine (labeled in the guanidino position) to ¹⁴C-L-citrulline. Peritoneal macrophages and spleen extracts from MRL-lpr/lpr mice displayed more NOS activity than did those from BALB mice, while the activity in kidney extracts was not different.

By immunofluorescence analysis using a rabbit anti-mouse iNOS antibody, we noted no NOS antigen expression in spleen, liver, and kidney from BALB mice, and none in liver or kidney from MRL-lpr/lpr mice. However, spleens from MRL-lpr/lpr mice displayed large numbers of cells containing the NOS antigen. Comparable findings were noted when we used a monospecific guinea pig anti-rat inducible NOS antibody. When tissue extracts were analyzed for iNOS antigen by immunoprecipitation and immunoblotting techniques using a rabbit anti-mouse iNOS antibody, we did not detect antigen in extracts from organs of BALB mice, although extracts from spleen and kidney tissues from MRL-lpr/lpr mice had readily detectable antigen.

NMMA treatment

Groups of 8 week old MRL-lpr/lpr mice were given either sterile, distilled deionized water or water containing 50 mM NMMA for ad libitum consumption. NMMA was from CalBiochem (San Diego, CA) and from Dr. Owen Griffith (Milwaukee, WI) . Both groups of mice were maintained on the defined nitrate free diet described above. At weekly intervals, the mice were placed in metabolic cages and 24 hour urine collections were obtained. Urinary nitrite/nitrate was measured as described above, and urinary protein was determined using the Bradford assay (BioRad, Hercules, CA) . After 10 weeks of treatment, the mice were bled and sacrificed with removal of the kidneys and knee joints.

Serum anti-DNA activity was determined by ELISA as previously described. The kidneys were imbedded in paraffin, sectioned and stained with hematoxylin and eosin. Knee joints were decalcified in folic acid, embedded into paraffin, sectioned, and stained. Slides were then read by a pathologist "blinded" as to the group of origin. The amount of kidney and knee joint disease present in each specimen was quantitated as noted before. Briefly, glomeruli were graded for hypercellularity (0-4) , hyperlobularity (0-4) , crescents (0-4) , and necrosis (0-4) . A score was then derived by adding the grading of these features of glomerular disease. Kidneys from normal BALB mice usually have scores from 0-1. Vasculitis was noted when present in medium size vessels in the kidney sections. The synovial score was derived by adding the grading of synovial proliferation (0-3) and subsynovial inflammation (0-3) . Knee joints from normal BALB mice usually have scores from 0-0.5.

Groups of MRL-lpr/lpr mice received either double distilled water (n=10) or water containing 50 M NMMA (n=9) ad libitum beginning at 8 weeks of age. Both groups of mice received the defined nitrate free diet. Mice were treated for a total of 10 weeks. Mice in both groups appeared clinically normal. However, two mice in the NMMA group died during week 3 of treatment leaving 7 mice in the NMMA group for analysis. Extensive autopsies (including careful histological examinations and culturing of serum, urine, and organs) on these two mice and on a comparable mouse that had received NMMA 4 weeks revealed no evidence of microbial infection or other evident cause of death. Administration of NMMA in the drinking water of MRL-lpr/lpr mice effectively blocked nitrite/nitrate excretion (and by inference nitric oxide production) . Also, mice receiving NMMA excreted significantly less protein than did control mice; this difference became apparent at week five of treatment.

Pathologic examination of the kidneys and knee joints of mice from the two groups of mice revealed significantly less disease in the NMMA-treated group. Renal disease as measured by the renal score, and arthritis as measured by the synovial score were both significantly less in the NMMA group as compared to the control group. The observed differences for joint disease and renal disease are statistically different (p<0.05 and p<0.02, respectively, using the Mann-Whitney U test) , while that for anti-DNA antibody level is not. There was minimal to no glomerular proliferation in mice treated with NMMA, while all but one of the control mice had significant glomerular proliferation and hyperlobulation. The chronic interstitial lymphocytic infiltrate seen in the kidneys of all lpr congenic mice (including C3H-lpr/lpr mice that do not develop glomerulonephritis) was present to comparable degrees in both control and NMMA treated mice. Medium vessel vasculitis appears sporadically in the kidneys of untreated MRL-lpr/lpr mice with an overall incidence of 30%. Mild to moderate (1-2+) medium vessel vasculitis was present in 3/10 kidneys from mice in the control group. Mild vasculitis was seen in 1/7 kidneys from the NMMA treated group. There was not a statistical difference in vasculitis between the two groups, but the small numbers of mice with vasculitis makes it difficult to draw firm conclusions regarding the effects of NMMA on this aspect of inflammation. Synovial proliferation was significantly decreased in the mice treated with NMMA. While only 3 of 7 mice in the NMMA-treated group had abnormal knee joints with mild to moderate synovial proliferation and synovial inflammation, all 10 mice in the control water group had some degree of synovial proliferation and inflammation. Levels of serum anti-double stranded DNA measured at age 18 weeks in the two groups were essentially equivalent (see the Figure) .

Formation of nitroso-hemocrlobin NO-Hb forms through an interaction of NO with iron in the heme group of hemoglobin (Huot, A.E., et al . , Biochem . Biophys . Res . Commun . 182 : 151-157 , (1992); Cantilena, L.R.J., et al . , J. Lab . Clin . Med . 120:902-907 (1992)). Whole blood from MRL-lpr/lpr mice at different ages was analyzed for the presence of nitroso-hemoglobin (NO-Hb) . The blood samples were anticoagulated and examined by electron paramagnetic resonance (EPR) at 77°K using a Bruker ESP300 spectrometer (Cha ulitrat, W. et al . , Molec . Pharmacol . 46:391-397 (1994)). Table 1 shows the mean ± SEM EPR units (n=number of animals examined) . An age-dependent increase was observed in the amount of NO-Hb in the blood of the diseased mice. The levels of NO-Hb were higher in MRL-lpr/lpr mice compared to same-age control mice without disease. The differences were statistically significant (p<0.05 at all ages analyzed). The presence of NO-Hb is another important sign that NO is being over-expressed in these mice with autoimmune nephritis and arthritis.

Table 1

Mouse Age 12 weeks Age 16 weeks Age 18 to 20 weeks

MRL-lpr/lpr 1420 ± 130 (n= 16) 2045 ± 226 (n= 10) 4092 ± 711 (n=711)

BALB/c (control) 970 ± 221 (n=5) 1005 ± 160 (n= 160) 811 ± 108 (n=8)

The above studies demonstrated an NO-mediated modification of a protein (hemoglobin) in these mice. To determine if diseased tissue is modified by NO in MRL-lpr/lpr mice, kidneys from normal BALB/c mice (20 weeks old) and MRL-lpr/lpr mice (20 weeks old) were examined by electron paramagnetic resonance (EPR) . For these tissues, the spectrum of the control mouse was subtracted from that of the MRL-lpr/lpr mouse, and the resultant curve showed an easily detectable NO-non-heme iron tyrosyl signal at g=2.04, as well as the typical spectra for NO-heme (presumably due to blood trapped within the MRL-lpr/lpr kidney; see Chamulitrat, W. , et al . , Molec . Pharmacol . 46:391-397 (1994)). It is important to note that control kidney did not have these NO-protein signals, signifying that the nitrosylated non-heme protein might be causally related to the observed severe renal pathology in these mice.

Protein nitration in kidneys from MRL-lpr/lpr mice

As noted above, NO may react with superoxide and form the highly reactive, tissue destructive molecule peroxynitrite. It has been shown previously that cells from MRL-lpr/lpr mice can overproduce reactive oxygen species such as hydrogen peroxide, superoxide (Dang-Vu, A.P. et al . , J. Immunol . 138:1757-1761 (1987)) and nitric oxide (Weinberg, J.B., et al . , J. Exp. Med . 179:651-660 (1994)). A study was done to look for evidence that MRL- lpr/lpr mice also overproduce the destructive molecule peroxynitrite (Beckman, J.S., et al . , Methods Enzymol . 233:229-240 (1994)). Since peroxynitrite causes nitration of tyrosine residues in proteins, a mono-specific, polyclonal anti-tyrosine antibody was used to detect evidence of the presence of peroxynitrite in diseased kidneys of MRL-lpr/lpr mice. Immunoblot analysis was performed on protein extracts from kidneys of 20 week old normal (BALB/c) and MRL-lpr/lpr mice.

Kidney tissue was homogenized with a glass pestle. Proteins in soluble extracts (100 μg per lane) were separated by SDS-polyacrylamide gel electrophoresis and transferred to nitrocellulose (0.45 μm, Novex) . Unbound sites were blocked by incubation with 1% non-fat dry milk in TBS (20 mM Tris, 500 M NaCl, pH 7.5) for 60 min at 25°C. Membranes were incubated overnight at 25°C with a polyclonal anti-nitrotyrosine antibody (0.25 μg/ml) in 1% milk/TBS. Immunoreactivity was visualized by incubation with goat anti-rabbit IgG-HRP conjugate (Bio-Rad) (1:2000) dilution in 1% milk/TBS, followed by enhanced chemiluminescence (ECL) detection (Amersham) . While the immunoblot from kidneys from four individual control mice showed only one protein band reacting with the antibody, the immunoblot from kidneys from MRL-lpr/lpr mice showed this same band plus four other prominent bands. This indicates that in vivo-generated NO in MRL-lpr/lpr mice is converted to peroxynitrite, and that this peroxynitrite subsequently nitrates tyrosine residues in proteins in the kidneys.

Catalase activity in kidneys of MRL-lpr/lpr mice The catalase content in kidneys from 20 week old normal (BALB/c) and MRL-lpr/lpr mice was analyzed. Peroxynitrite or NO can destroy catalase activity. Catalase was measured by the disappearance of hydrogen peroxide noted by absorbance at 240 nm (Beers, R.F., and Sizer, R.W. , J. Biol . Chem . 195:133 (1952)). Values shown in Table 2 are the mean ± SEM of replicate samples expressed in units/mg protein. Table 2 shows that kidneys from the control mice had high levels of catalase while levels of catalase from MRL-lpr/lpr mice were very low.

Table 2

BALB/c mouse MRL-lpr/lpr mouse

Mouse 1 77 ± 6 Mouse 1 12 ±1

Mouse 2 75 ± 2 Mouse 2 20

Mouse 3 67 ± 5

Mouse 4 68 ± 6

In a different type of analysis to determine catalase, we studied protein extracts from kidneys by PAGE, with subsequent visualization of catalase activity by soaking the gels in phosphate buffer containing horseradish peroxidase and hydrogen peroxidase with diaminobenzidine. Soluble extracts (25 μg/lane) were separated by electrophoresis on a 6% native polyacrylamide gel. Bands of catalase activity were visualized by soaking the gels for 45 min at 25°C in 50 mM sodium phosphate, pH 7.0, containing 0.1 mM EDTA and 50 μg/ml horseradish peroxidase. H₂0₂ was added to a final concentration of 5.0 mM and the gel was incubated an additional 15 min. After a brief rinse in water, stain development was initiated by addition of 0.5 mg/ml diaminobenzidine-HCl in 50 mM sodium phosphate, 0.1 mM EDTA, pH 7.0. Results showed clearly that kidneys from 4 different BALB/c control mice contained large amounts of catalase, while those from two MRL-lpr/lpr mice had markedly diminished levels of catalase. However, if the mice had been treated with oral N^G-monomethyl-L-arginine in vivo (See NMMA treatment above) , the catalase level in kidneys from three different MRL-lpr/lpr mice was normal (comparable to that of the control mice) . This signifies that catalase activity (which may be inhibited by the actions of NO or peroxynitrite) is markedly low in MRL-lpr/lpr mice, and that the NO- (or peroxynitrite-) mediated decrease in catalase is blocked by in vivo administration of N^G-monomethyl-L-arginine.

Human investigations

To determine if humans with arthritis have iNOS protein expressed in their synovial tissues, these tissues were studied by immunofluorescence techniques using mouse monoclonal anti-iNOS antibody (purchased from Transduction Laboratories, Inc.). Of six synovial samples removed from patients with advanced arthritis at the time of joint replacement surgery, iNOS antigen was detected in the tissues of two of three rheumatoid arthritis patients and in one of three osteoarthritis patients. These studies demonstrate that patients have synovia that contain iNOS, and that it may be overexpressed in synovia of patients with rheumatoid arthritis.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the invention described specifically herein. Such equivalents are intended to be encompassed in the scope of the following claims.

Claims

1. A method of treating or preventing autoimmune diseases in a patient comprising administering to the patient an effective amount of a nitric oxide synthase inhibitor.

2. A method of claim 1 wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, systemic lupus erythematosus and glomerulonephritis.

3. A method of claim 2 wherein the autoimmune disease is rheumatoid arthritis.

4. A method of claim 3 wherein the mode of administration is enteral.

5. A method of claim 3 wherein the mode of administration is parenteral.

6. A method of claim 4 wherein the nitric oxide synthase inhibitor is selected from a group consisting of N^G- amino-L-arginine, N^G-methyl-L-arginine, N^G-nitro-L- arginine, N^G-nitro-L-arginine methyl ester, N°- iminoethyl-L-ornithine and aminoguanidine.

7. A method of claim 6 wherein the nitric oxide synthase inhibitor is N^G-methyl-L-arginine.

8. A method of treating rheumatoid arthritis in a patient in need of treatment thereof comprising administering to said patient an effective amount of N^G-methyl-L-arginine.

9. A method of treating or preventing autoimmune diseases in a patient comprising administering to the patient an effective amount of a nitric oxide scavenger.

10. A method of claim 9 wherein the autoimmune disease is selected from the group consisting of rheumatoid arthritis, insulin-dependent diabetes mellitus, systemic lupus erythematosus and glomerulonephritis.

11. A method of claim 10 wherein the autoimmune disease is rheumatoid arthritis.

12. A method of treating rheumatoid arthritis in a patient in need of treatment thereof comprising administering to said patient an effective amount of N^G-methyl-L-arginine.

13. A nitric oxide synthase inhibitor for use in therapy, for example for use in treating or preventing an autoimmune disease.