CA2176747A1 - Use of nitric oxide synthase inhibitors in the treatment of autoimmune diseases - Google Patents
Use of nitric oxide synthase inhibitors in the treatment of autoimmune diseasesInfo
- Publication number
- CA2176747A1 CA2176747A1 CA002176747A CA2176747A CA2176747A1 CA 2176747 A1 CA2176747 A1 CA 2176747A1 CA 002176747 A CA002176747 A CA 002176747A CA 2176747 A CA2176747 A CA 2176747A CA 2176747 A1 CA2176747 A1 CA 2176747A1
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- lpr
- mice
- nitric oxide
- arginine
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/13—Amines
- A61K31/155—Amidines (), e.g. guanidine (H2N—C(=NH)—NH2), isourea (N=C(OH)—NH2), isothiourea (—N=C(SH)—NH2)
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/185—Acids; Anhydrides, halides or salts thereof, e.g. sulfur acids, imidic, hydrazonic or hydroximic acids
- A61K31/19—Carboxylic acids, e.g. valproic acid
- A61K31/195—Carboxylic acids, e.g. valproic acid having an amino group
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/21—Esters, e.g. nitroglycerine, selenocyanates
- A61K31/215—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids
- A61K31/22—Esters, e.g. nitroglycerine, selenocyanates of carboxylic acids of acyclic acids, e.g. pravastatin
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P13/00—Drugs for disorders of the urinary system
- A61P13/02—Drugs for disorders of the urinary system of urine or of the urinary tract, e.g. urine acidifiers
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P15/00—Drugs for genital or sexual disorders; Contraceptives
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P3/00—Drugs for disorders of the metabolism
- A61P3/08—Drugs for disorders of the metabolism for glucose homeostasis
- A61P3/10—Drugs for disorders of the metabolism for glucose homeostasis for hyperglycaemia, e.g. antidiabetics
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P37/00—Drugs for immunological or allergic disorders
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
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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
W095/13805 ~2 ~ 7 ~ 7 ~ 7 PCIIUS94113239 -USE OF NITRIC OXIDE SYNI~IASE I NH I I~
IN 'rElE lL~l~ l OF AUIY)INMUNE DISEASES
- 8Acr~jK~llJNL) OF THE INVENl~ON
MRL-lpr/lpr mice have been studied as a model for human autoimmune diseases. This strain of mice develops a spontaneous autoimmune disease characterized by S lymrh~ ,yatl~, autoantibody production and inf lammatory manifestations including nephritis, vasculitis, and arthritis (Hang, L., et al., J. Exp. Ned. 155:1690-1701 (1982~ onh~rg~ R.~., et al., Clin. Exp. Rheumatol.
7:S35-S40 (1989) ) . Immune function ~hnr~ l ities also include ~nh~nced constitutive macrophage class II antigen expression, elevated levels of IFN-~y, TNF, IL-1, and IL-6 in isolated kidney, lymph node, and spleen cells, and an ~-nh~nrP~I state of macrophage "activation". These disease manifestations are a result of both a single gene mutation (lpr) of the Fas apoptosis gene on mouse ~1IL~ ~ 19 and background genes from the NRL strain. Although the ~RL
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 n~:uLoLL~lnsmitter, cause stasis and/or lysis of microbes and tumor cells, and modulate function and differentiation of hematopoietic cells.
NO also has potent proinflammatory actions. NO may increase vascular permeability in inf lamed tissues .
Pain is also an important aspect of inflammation. Several groups have now d~ ~L~ted that NO plays a role in the Wo 95/13805 PCrtlJSs4/13239 ~
~:~7~74~ -2-mediation of pain in in~lammation. Intradermal injection of solutions containing N0 into humans caused a dose-related O~ ~ ULL~rlCe of pain in the site tHolthusen, H., and Arndt, J.o., Neuroscience Letters 165:71-74 5 (1994). Thus, pain, a hallmark symptom of inflammation, can be induced by N0.
N0 has also been shown to cause increased production of TNF and IL-1 by cells, and to increase the potential of cells to produce 11YdLUY~ peroxide. Also, rabbit and 10 human chondrocytes have been shown to produce N0 and to express inducible nitric oxide synthase (iNos) in response to various cytokines and bacterial products. N0 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 N0 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-20 human models of inflammatory tli~ c~c has also established that N0 participates in experimentally-induced uveitis (Parks, D.J., et al., Arch. ophthalmol. 112:544-546 (1994) ) .
Studies of humans with arthritis ~ L,~-te further 25 that N0 plays an i ~ Ldl.~ role in human arthritis.
Farrell et al . showed that patients with inf lammatory arthritis (rheumatoid arthritis and osteoarthritis) had elevated levels of the N0 catabolite nitrite in their synovial fluid and serum (Farrell, A.J., et al., Ann.
30 Rheum. Dis. 51:1219-1222 (1992) ) . Patients with rheumatoid arthritis had increased synovial f luid and serum levels of niLLu~y.usine (Kaur, H., and Halliwell, B., Febs Letters 350:9-12 (1994)).
WO 951~3805 ~ ~ ~ 6 7 4 7 PCTIUS941~3239 Cell-derived NO reacts with ,uyerc~ide (2'-) 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 (R~rhm;l~witz, D., et al., Gastroenterology 105:1681-1688 (1993) ) .
NiLL~LyLosine is formed by the action of peroxynitrite on tyrosine, and it is felt to be a ctable "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-niL~ oLyLosine antibody) in atheromatous plaques in human vessels (Beckman, J.S., et al., Biol. Chem. ~oppe-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 d; C~ c-~s, such as rheumatoid arthritis, insulin tl_~ t ~ het~c 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.
13RTPIF DESCRTPTION OF T~F: DRAWING
The Figure is a bar graph showing the scores of pathological characteristics in MRL-lpr/lpr mice either treated with NG-monomethyl-L-arginine (NMMA) or left untreated .
WO95/13805 Pcr/us94ll323s ~
217~
~n DES~ 'K I 1' ' lON OF T~E INVENTION
The level of nitric oxide has now been linked to the manifestation of autoimmune ~;ceAcec~ particularly chronic diseases, such as rheumatoid arthritis, insulin dependent 5 diabetes mellitus, systemic lupus erythematosus and glomer-~1 on~Prhritis. 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 diCPACPC (C.jlkP~on et al., Arth.
10 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 aminogllAn;rlinp~ Na-amino-L-arginine, N'J-methyl-L-arginine, Na-nitro-L-arginine, NG-15 nitro-L-arginine methyl ester, and Na-iminoethyl-L-ornithine, flavoprotein binders, such as diphenylene iodonium, iodonium diphenyl and di-2-thienyl iodonium, CAl-- ' 1 in binders, such as cA1~inpllrin~
trifluoroperazine, N- (4-aminobutyl) -5-chloro-2-20 naphthalenesulfonamide and N- (6-aminohexyl) -1-naphthA1PnPc-l1fonamide, heme binders, such as carbon - ~P, depleters and analogs of tetrahydrobiopterin, such as 2,4-diamino-6-lly~lLo,~y~yLimidine~ and induction inhibitors, such as corticosteroids, TGF-~C-l, -2, 3, 25 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, Na-amino-L-arginine, N~-methyl-L-arginine, Na-nitro-L-arginine, Na-nitro-L-arginine methyl ester, and 30 Na-iminoethyl-L-ornithine. Particularly preferred are NG-amino-L-arginine, Na-methyl-L-arginine, N~-nitro-L-arginine, and aminog~l~nidine. Most preferred is Na-methyl-L-arginine.
7 ~ ~
WO 95/13805 ~ PCrlUS94113139 phAr~-~ce~ltically acceptable salts may also be administered. Examples of suitable salts include acid salts, such as I~YdL~Y~n chloride, llyd~o~ bromide, hydrogen iodide, sulfate and acetate salts, as well as basic salts, such as amine, ammonium, alkali metal and AlkAl inP earth metal salts.
S~;~Vt:IIY~L:~ of nitric oxide are compounds which will react with nitric oxide in vivo, such as hemoglobin (Wang et al., Lffe Sciences 49:55-60 (7991)) and cr~h~lAm;nc (Rajanayagam, C.G., et al., Brit. :r. 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 ~ '~ 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, call-ollydl~tes such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, llydr ~,cy ` ylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired, mixed with i:~llYi 1 i~ry agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsif iers, salts for influencing osmotic pressure, buffers, flavorants, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds .
~or parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, WO 95/13805 ~ PCTIUS94/13239--7 l1 7 i n~ l i n~ suppositories . Ampouleg are convenient unit dosages. Oral applications are preferably administered in the forms of c~rsl~lpc~ tablets and/or liquid formulations.
Unit form dosages are preferred. Topical applications can 5 be administered in the form of a liquid, gel or a cream.
It will be appreciated that the actual amounts of the active ~ in a specif ic case will vary according to the specific Cu~ uuu~-d being utilized, the particular composition f ormulated, the mode of administration and the 10 age, weight and condition of the patient, for example.
Dosages for a particular patient can be det~rm;npd 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.
~FMpLIFIcATIoN
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 det~rmin~
~ue11_Lu~hotometrically as described before (Granger, D.L.
et al., J. Im~nunol. 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 cQn~'lm~n~ 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 ~Wo95/13805 7 6~ 4~ PCrllJS94113239 collected daily under basal conditions. MR~-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-5 lpr/lpr mice excrete higher levels of nitrite/nitrate than do B6 mice as the mice age. Higher nitratelnitrite excretion begin6 at approximately age 10 to 12 weeks paralleling that of proteinuria. Oral administration of 50 mM 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 specif ic inhibitor of the enzyme . Levels of nitrite/nitrate excretion in 5 month old mice of strains MRL-+/+ (0.8 ,~mol per mouse per day) and B6-lpr/lpr (1.2 ,umol 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 ~nhAn~ d nitrite excretion, and that both the lpr gene and genetic f actors in the MRL ba._}.yL vulld are npc~cs;~ry~
Nitric oxide l~rQduction 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 ~M phenylmethylsulfonyl fluoride, 5 ,ug/ml aprotinin, 1 ~g/ml ~:I-y ~ ~tin, and 5 ~Lg/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
Wo 95113805 ! PCTIUS94/13239 ~
IN 'rElE lL~l~ l OF AUIY)INMUNE DISEASES
- 8Acr~jK~llJNL) OF THE INVENl~ON
MRL-lpr/lpr mice have been studied as a model for human autoimmune diseases. This strain of mice develops a spontaneous autoimmune disease characterized by S lymrh~ ,yatl~, autoantibody production and inf lammatory manifestations including nephritis, vasculitis, and arthritis (Hang, L., et al., J. Exp. Ned. 155:1690-1701 (1982~ onh~rg~ R.~., et al., Clin. Exp. Rheumatol.
7:S35-S40 (1989) ) . Immune function ~hnr~ l ities also include ~nh~nced constitutive macrophage class II antigen expression, elevated levels of IFN-~y, TNF, IL-1, and IL-6 in isolated kidney, lymph node, and spleen cells, and an ~-nh~nrP~I state of macrophage "activation". These disease manifestations are a result of both a single gene mutation (lpr) of the Fas apoptosis gene on mouse ~1IL~ ~ 19 and background genes from the NRL strain. Although the ~RL
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 n~:uLoLL~lnsmitter, cause stasis and/or lysis of microbes and tumor cells, and modulate function and differentiation of hematopoietic cells.
NO also has potent proinflammatory actions. NO may increase vascular permeability in inf lamed tissues .
Pain is also an important aspect of inflammation. Several groups have now d~ ~L~ted that NO plays a role in the Wo 95/13805 PCrtlJSs4/13239 ~
~:~7~74~ -2-mediation of pain in in~lammation. Intradermal injection of solutions containing N0 into humans caused a dose-related O~ ~ ULL~rlCe of pain in the site tHolthusen, H., and Arndt, J.o., Neuroscience Letters 165:71-74 5 (1994). Thus, pain, a hallmark symptom of inflammation, can be induced by N0.
N0 has also been shown to cause increased production of TNF and IL-1 by cells, and to increase the potential of cells to produce 11YdLUY~ peroxide. Also, rabbit and 10 human chondrocytes have been shown to produce N0 and to express inducible nitric oxide synthase (iNos) in response to various cytokines and bacterial products. N0 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 N0 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-20 human models of inflammatory tli~ c~c has also established that N0 participates in experimentally-induced uveitis (Parks, D.J., et al., Arch. ophthalmol. 112:544-546 (1994) ) .
Studies of humans with arthritis ~ L,~-te further 25 that N0 plays an i ~ Ldl.~ role in human arthritis.
Farrell et al . showed that patients with inf lammatory arthritis (rheumatoid arthritis and osteoarthritis) had elevated levels of the N0 catabolite nitrite in their synovial fluid and serum (Farrell, A.J., et al., Ann.
30 Rheum. Dis. 51:1219-1222 (1992) ) . Patients with rheumatoid arthritis had increased synovial f luid and serum levels of niLLu~y.usine (Kaur, H., and Halliwell, B., Febs Letters 350:9-12 (1994)).
WO 951~3805 ~ ~ ~ 6 7 4 7 PCTIUS941~3239 Cell-derived NO reacts with ,uyerc~ide (2'-) 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 (R~rhm;l~witz, D., et al., Gastroenterology 105:1681-1688 (1993) ) .
NiLL~LyLosine is formed by the action of peroxynitrite on tyrosine, and it is felt to be a ctable "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-niL~ oLyLosine antibody) in atheromatous plaques in human vessels (Beckman, J.S., et al., Biol. Chem. ~oppe-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 d; C~ c-~s, such as rheumatoid arthritis, insulin tl_~ t ~ het~c 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.
13RTPIF DESCRTPTION OF T~F: DRAWING
The Figure is a bar graph showing the scores of pathological characteristics in MRL-lpr/lpr mice either treated with NG-monomethyl-L-arginine (NMMA) or left untreated .
WO95/13805 Pcr/us94ll323s ~
217~
~n DES~ 'K I 1' ' lON OF T~E INVENTION
The level of nitric oxide has now been linked to the manifestation of autoimmune ~;ceAcec~ particularly chronic diseases, such as rheumatoid arthritis, insulin dependent 5 diabetes mellitus, systemic lupus erythematosus and glomer-~1 on~Prhritis. 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 diCPACPC (C.jlkP~on et al., Arth.
10 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 aminogllAn;rlinp~ Na-amino-L-arginine, N'J-methyl-L-arginine, Na-nitro-L-arginine, NG-15 nitro-L-arginine methyl ester, and Na-iminoethyl-L-ornithine, flavoprotein binders, such as diphenylene iodonium, iodonium diphenyl and di-2-thienyl iodonium, CAl-- ' 1 in binders, such as cA1~inpllrin~
trifluoroperazine, N- (4-aminobutyl) -5-chloro-2-20 naphthalenesulfonamide and N- (6-aminohexyl) -1-naphthA1PnPc-l1fonamide, heme binders, such as carbon - ~P, depleters and analogs of tetrahydrobiopterin, such as 2,4-diamino-6-lly~lLo,~y~yLimidine~ and induction inhibitors, such as corticosteroids, TGF-~C-l, -2, 3, 25 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, Na-amino-L-arginine, N~-methyl-L-arginine, Na-nitro-L-arginine, Na-nitro-L-arginine methyl ester, and 30 Na-iminoethyl-L-ornithine. Particularly preferred are NG-amino-L-arginine, Na-methyl-L-arginine, N~-nitro-L-arginine, and aminog~l~nidine. Most preferred is Na-methyl-L-arginine.
7 ~ ~
WO 95/13805 ~ PCrlUS94113139 phAr~-~ce~ltically acceptable salts may also be administered. Examples of suitable salts include acid salts, such as I~YdL~Y~n chloride, llyd~o~ bromide, hydrogen iodide, sulfate and acetate salts, as well as basic salts, such as amine, ammonium, alkali metal and AlkAl inP earth metal salts.
S~;~Vt:IIY~L:~ of nitric oxide are compounds which will react with nitric oxide in vivo, such as hemoglobin (Wang et al., Lffe Sciences 49:55-60 (7991)) and cr~h~lAm;nc (Rajanayagam, C.G., et al., Brit. :r. 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 ~ '~ 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, call-ollydl~tes such as lactose, amylose or starch, magnesium stearate, talc, silicic acid, viscous paraffin, fatty acid esters, llydr ~,cy ` ylcellulose, polyvinyl pyrrolidone, etc. The pharmaceutical preparations can be sterilized and if desired, mixed with i:~llYi 1 i~ry agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsif iers, salts for influencing osmotic pressure, buffers, flavorants, coloring, and/or aromatic substances and the like which do not deleteriously react with the active compounds .
~or parenteral application, particularly suitable are injectable, sterile solutions, preferably oily or aqueous solutions, as well as suspensions, emulsions, or implants, WO 95/13805 ~ PCTIUS94/13239--7 l1 7 i n~ l i n~ suppositories . Ampouleg are convenient unit dosages. Oral applications are preferably administered in the forms of c~rsl~lpc~ tablets and/or liquid formulations.
Unit form dosages are preferred. Topical applications can 5 be administered in the form of a liquid, gel or a cream.
It will be appreciated that the actual amounts of the active ~ in a specif ic case will vary according to the specific Cu~ uuu~-d being utilized, the particular composition f ormulated, the mode of administration and the 10 age, weight and condition of the patient, for example.
Dosages for a particular patient can be det~rm;npd 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.
~FMpLIFIcATIoN
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 det~rmin~
~ue11_Lu~hotometrically as described before (Granger, D.L.
et al., J. Im~nunol. 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 cQn~'lm~n~ 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 ~Wo95/13805 7 6~ 4~ PCrllJS94113239 collected daily under basal conditions. MR~-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-5 lpr/lpr mice excrete higher levels of nitrite/nitrate than do B6 mice as the mice age. Higher nitratelnitrite excretion begin6 at approximately age 10 to 12 weeks paralleling that of proteinuria. Oral administration of 50 mM 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 specif ic inhibitor of the enzyme . Levels of nitrite/nitrate excretion in 5 month old mice of strains MRL-+/+ (0.8 ,~mol per mouse per day) and B6-lpr/lpr (1.2 ,umol 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 ~nhAn~ d nitrite excretion, and that both the lpr gene and genetic f actors in the MRL ba._}.yL vulld are npc~cs;~ry~
Nitric oxide l~rQduction 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 ~M phenylmethylsulfonyl fluoride, 5 ,ug/ml aprotinin, 1 ~g/ml ~:I-y ~ ~tin, and 5 ~Lg/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
Wo 95113805 ! PCTIUS94/13239 ~
2~
(pH 7.S), 200 ,uM NADPH, l mM dithiothreitol, l0 ~M FAD, 100 ,uM tetrahydrobiopterin, and l0 ~M L-arginine. We used L-arginine labeled with tritium in the gll~ni-l;nn position (product number NEC453, New England Nuclear, Wilmington, 5 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 F~nhAnl ~ of nitrite/nitrate production when treated 10 with endotoxin or IFN-~y alone, but the combination Pnh :~nce~ the production greatly. In contra6t, peritoneal macrophages from MRL-lpr/lpr mice had PnhAnc~-d responses to treatment with endotoxin and murine IFN-~y alone, as well as with -i nPd endotoxin IFN- r treatment. To 15 determine if there wa6 PnhAnred tissue iNos mR~A
expression, RNA was extracted from organs from BALB and MRL-lpr/lpr mice, and then PYAmi n~rl by Northern analysis for iNos mRNA expression. The iNoS mRNA (approximately 4.7 kilobases in size~ was noted in tissue from kidney and 20 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 ~4C-L-arginine ( labeled in the gl1An;rlinn position) to I~C-L-citrulline. Peritoneal 25 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 30 expression in spleen, liver, and kidney from BALB mice, and none in liver or kidney from MRL-lpr/lpr mice.
However, spleens f rom MRL-lpr/lpr mice displayed large numbers of cells containing the NOS antigen. Comparable f indings were noted when we used a monospecif ic guinea pig ~ WO 95113805 ~ 1 7 6 ~ 4 7 PCr/US941~3239 _g_ anti-rat inducible NOS antibody. When ti6sue extracts were analyzed for iNos antigen by; , ~cipitation and lQtting techniques using a rabbit anti-mouse iNos antibody, we did not detect antigen in extracts from 5 organs of BALB mice, although extracts from spleen and kidney tissues from MRL-lpr/lpr mice had readily ~letect Ihle 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 c...,ul~Lion. 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 15 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 det~rm; nPd using the Bradford assay (BioRad, Hercules, CA). After l0 weeks 20 of treatment, the mice were bled and sacrificed with removal of the kidneys and knee joints.
Serum anti-DNA activity was det~ m; n-~cl by ELISA as previously described . The kidneys were ; mh/~ d in paraffin, sectioned and stained with hematoxylin and 25 eosin. Knee joints were decalcified in folic acid, ~mh~ ecl 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 j oint disease present in each specimen was quantitated as noted bef ore .
30 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-l. Vasculitis was WO 95/13805 PCrlUS9~113239 ~
7 4~
noted when present in medium size vessels in the kidney sections. The synovial score was derived by adding the grading of synovial prolif eration ( 0-3 ) and subsynovial inflammation (0-3). Knee joints from normal BALB mice 5 usually have scores from 0-0 . 5 .
Groups of MRJJ-lpr/lpr mice received either double distilled water (n=10) or water containing 50 mM NMMA
(n=9 ) ad li~itum beginning at 8 weeks of age. Both groups of mice received the def ined nitrate f ree diet . Mice were 10 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 ( i nr~ i ng careful histological examinations and culturing 15 of serum, urine, and organs) on these two mice and on a comparable mouse that had received NNMA 4 weeks revealed no evidence of microbial infection or other evident cause of death. Administration of NMMA in the drinking water of MRB-lpr/lpr mice effectively blocked nitrite/nitrate 20 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 f ive of treatment .
Pathologic examination of the kidneys and knee joints 25 of mice from the two groups o~ mice revealed significantly less disease in the NMMA-treated group. Renal disease as mea6ured by the renal score, and arthritis as measured by the synovial score were both signif icantly less in the NMMA group as compared to the control group. The observed 30 differences for ~oint disease and renal disease are statistically different (p<0.05 and p<0.02, respec'cively, 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 35 of the control mice had signif icant glomerular ~ WO 9511380S PCTIUS94113239 2~7~7~7 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 dif f icult to draw f irm conclusions regarding the effects of NMMA on this aspect of ;nfl: -tion. Synovial proliferation was significantly decreased in the mice treated with NMr~ hile 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-hemocrlobi n N0-Hb forms through an interaction of N0 with iron in the heme group of hemoglobin (Huot, A.E., et al., Biochem.
Biopl2ys. 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 ~ A~nin~(l by electron paramagnetic resonance (EPR) at 77l~ using a Bruker ESP300 spectrometer (Chamulitrat, ~. et al., Molec.
Pharmacol . 46:391-397 (1994) ) . Table 1 shows the mean +
WO95/13805 21~ 67 ~7 PCr/US94/13239 ~
SEM EPR units ~ ..u.llbe~ of animals ~YAm;nDcl). An agc d~~ nt increase was observed in the amount of NO-Hb in the blood of the ~:1;P.DA~:D~ mice. The levels of NO-Hb i~
were higher in MRL-lpr/lpr mice compared to s~ - a~c:
5 control mice without disease . The dif f erences were statistically si~n; f; cAnt (p<0 . 05 at all ages analyzed) .
The presence of NO-Hb is another important sign that NO is being over-eYpressed in these mice with autoimmune nephritis and arthritis.
Table l Mouse Age 12 weeks Age 16 weeks Age 18 to 20 weeks MRL-lpr~lpr 1420 ~ 130 (n=16) 2045 i 226 (n=10) 4092 i 711 (n=711) BALB/c (control) 970 i 221 (n=5) 1005 ~ 160 (n=160) 811 ~ 108 (n=8 The above studies .1 Lr ted an NO-mediated 15 modification of a protein (hemoglobin) in these mice. To determine if ~;~eA~l 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 DYAm; nD~l by electron paramagnetic resonance (EPR) . For 20 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 f or NO-heme (presumably due to blood trapped 25 within the MRL-lpr/lpr kidney; see Chamulitrat, W., et al ., IYolec. Ph~rmacol . 46:391-397 (1994) ) . It is important to note that control kidney did not have these NO-protein signals, signifying that the nitrosylated _ W0 95113805 ~ ; 7 ~ 7 PCrtUS94/13239 non-heme protein might be causally related to the observed severe renal pathology in these mice.
, ~
PrgtP;n nitration jn k;rlnpys from MRT-l~r/lDr ~ p As noted above, N0 may react with ~u~eluxide and form 5 the highly reactive, tissue destructive molecule peroxynitrite. It has been shown previously that cells from MRL-lpr/lpr mice can vv~L~Lvduce reactive oxygen species such as hydrogen peroxide, superoxide (Dang-Vu, A.P. et al., .J. Immunol . 138:1757-1761 (1987) ) and nitric 10 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 uv~L~Loduce the destructive molecule peroxynitrite (Beckman, J.S., et al., l~ethods 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 ~l;cp~d kidneys of MRL-lpr/lpr mice. Immunoblot analysis was performed on 20 protein extracts from kidneys of 20 week old normal (BALB/c) and MRL-lpr/lpr mice.
Kidney tissue was homogenized with a glas6 pestle.
Proteins in soluble extracts (100 ,~lg per lane) were separated by SDS-polyacrylamide gel electrophoresis and 25 transferred to nitrocPl lulocP (0.45 ,um, Novex) . Unbound sites were blocked by incubation with 1% non-fat dry milk in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) for 60 min at 2 5 C . Membranes were incubated overnight at 2 5 C with a polyclonal anti-niLLvLyLosine antibody (0.25 ~g/ml) in 30 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 chemil~ i n~c-~n~e (ECL) detection (Amersham) .
Wo 95113805 PcrrUss4r1323s ~
7~7 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 5 other pL, i nPnt 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.
Cat~ P activitY in kidnevs of MRL-lpr/lPr mice The catalase content in kidneys from Z0 week old normal (BALB/c) and MRL-lpr/lpr mice was analyzed.
Peroxynitrite or N0 can destroy catalase activity.
Catalase was measured by the di c:ArpP Irance of hydrogen peroxide noted by absorbance at 240 nm (Beers, R.F., and 15 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.
2 0 Table 2 BALB/c mouse MRl,lpr/lpr mouse Mousi~ 1 77 i 6 Mousc 1 12 il Mouse 2 75 i 2 Mous~ 2 20 Mouse 3 67 i 5 25 Mousc 4 68 i 6 In a different type of analysis to determine catalase, we studied protein extracts from kidneys by ~Iwo 9S/1380S PCrlUS94113239 2~7~7~7 PAGE, with subsequent vi~1~Al;7~A~tion of catala6e activity by soaking the gels in phosphate buffer containing - horseradish peroxidase and IIYdLOY~ peroxidase with ~;Am;nr h~n7idine.
Soluble extracts (25 ,ug/lane) were separated by electrophoresis on a 6% native polyacrylamide gel. Bands of catalase activity were v; !=IIA 1; 7~d by soaking the gels for 45 min at 25C in 50 mM sodium phosphate, pH 7.0, containing 0. l mM EDTA and 50 ,ug/ml horseradish peroxidase. H202 was added to a final concentration of 5 . O
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 rl;Am;nclb~n~idine-HCl in 50 mM sodium phosphate, O. l mM EDTA, pH 7 . O.
Results showed clearly that kidneys from 4 different BALB/c control mice contained large amounts of catalase, while those from two NRL-lpr/lpr mice had markedly ~1; m; n; ~:h~ level5 of catala5e . However, if the mice had been treated with oral NG-- Lhyl-L-arginine in vivo (See NMMA ~t!at ~-)t 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 NRL-lpr/lpr mice, and that the NO- (or peroxynitrite-) mediated decrease in catalase is blocked by in vivo administration of NG ~ y l-L-arginine .
n investiqationS
To determine if humans with arthritis have iNos protein expressed in their synovial tissues, these tissues were studied by immunof luorescence techniques using mouse monoclonal anti-iNOS antibody (purchased from Transduction Laboratories, Inc. ) . of six synovial samples removed from ~17~7~7 patients with advanced arthritis at the time of ; oint rep' ~Ir L 6urgery, iNos antigen was detected in the tissues of two of three rheumatoid arthritis patients and ,~
in one of three osteoarthritis patients. These studies 5 d~ LLc.te that patients have synovia that contain iNos, and that it may be uv~le~Less~d in synovia of patients with rheumatoid arthritis.
EOUIVAT .T~'NTS
Those skilled in the art will rf~ n; ~e, or be able 10 to ascertain using no more than routine experimentation, many equivalents to the specific P~nhsr~ nts of the invention described specif ically herein . Such equivalents are intended to be t~n~ ,-cc~d in the scope of the following claims.
(pH 7.S), 200 ,uM NADPH, l mM dithiothreitol, l0 ~M FAD, 100 ,uM tetrahydrobiopterin, and l0 ~M L-arginine. We used L-arginine labeled with tritium in the gll~ni-l;nn position (product number NEC453, New England Nuclear, Wilmington, 5 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 F~nhAnl ~ of nitrite/nitrate production when treated 10 with endotoxin or IFN-~y alone, but the combination Pnh :~nce~ the production greatly. In contra6t, peritoneal macrophages from MRL-lpr/lpr mice had PnhAnc~-d responses to treatment with endotoxin and murine IFN-~y alone, as well as with -i nPd endotoxin IFN- r treatment. To 15 determine if there wa6 PnhAnred tissue iNos mR~A
expression, RNA was extracted from organs from BALB and MRL-lpr/lpr mice, and then PYAmi n~rl by Northern analysis for iNos mRNA expression. The iNoS mRNA (approximately 4.7 kilobases in size~ was noted in tissue from kidney and 20 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 ~4C-L-arginine ( labeled in the gl1An;rlinn position) to I~C-L-citrulline. Peritoneal 25 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 30 expression in spleen, liver, and kidney from BALB mice, and none in liver or kidney from MRL-lpr/lpr mice.
However, spleens f rom MRL-lpr/lpr mice displayed large numbers of cells containing the NOS antigen. Comparable f indings were noted when we used a monospecif ic guinea pig ~ WO 95113805 ~ 1 7 6 ~ 4 7 PCr/US941~3239 _g_ anti-rat inducible NOS antibody. When ti6sue extracts were analyzed for iNos antigen by; , ~cipitation and lQtting techniques using a rabbit anti-mouse iNos antibody, we did not detect antigen in extracts from 5 organs of BALB mice, although extracts from spleen and kidney tissues from MRL-lpr/lpr mice had readily ~letect Ihle 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 c...,ul~Lion. 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 15 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 det~rm; nPd using the Bradford assay (BioRad, Hercules, CA). After l0 weeks 20 of treatment, the mice were bled and sacrificed with removal of the kidneys and knee joints.
Serum anti-DNA activity was det~ m; n-~cl by ELISA as previously described . The kidneys were ; mh/~ d in paraffin, sectioned and stained with hematoxylin and 25 eosin. Knee joints were decalcified in folic acid, ~mh~ ecl 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 j oint disease present in each specimen was quantitated as noted bef ore .
30 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-l. Vasculitis was WO 95/13805 PCrlUS9~113239 ~
7 4~
noted when present in medium size vessels in the kidney sections. The synovial score was derived by adding the grading of synovial prolif eration ( 0-3 ) and subsynovial inflammation (0-3). Knee joints from normal BALB mice 5 usually have scores from 0-0 . 5 .
Groups of MRJJ-lpr/lpr mice received either double distilled water (n=10) or water containing 50 mM NMMA
(n=9 ) ad li~itum beginning at 8 weeks of age. Both groups of mice received the def ined nitrate f ree diet . Mice were 10 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 ( i nr~ i ng careful histological examinations and culturing 15 of serum, urine, and organs) on these two mice and on a comparable mouse that had received NNMA 4 weeks revealed no evidence of microbial infection or other evident cause of death. Administration of NMMA in the drinking water of MRB-lpr/lpr mice effectively blocked nitrite/nitrate 20 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 f ive of treatment .
Pathologic examination of the kidneys and knee joints 25 of mice from the two groups o~ mice revealed significantly less disease in the NMMA-treated group. Renal disease as mea6ured by the renal score, and arthritis as measured by the synovial score were both signif icantly less in the NMMA group as compared to the control group. The observed 30 differences for ~oint disease and renal disease are statistically different (p<0.05 and p<0.02, respec'cively, 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 35 of the control mice had signif icant glomerular ~ WO 9511380S PCTIUS94113239 2~7~7~7 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 dif f icult to draw f irm conclusions regarding the effects of NMMA on this aspect of ;nfl: -tion. Synovial proliferation was significantly decreased in the mice treated with NMr~ hile 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-hemocrlobi n N0-Hb forms through an interaction of N0 with iron in the heme group of hemoglobin (Huot, A.E., et al., Biochem.
Biopl2ys. 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 ~ A~nin~(l by electron paramagnetic resonance (EPR) at 77l~ using a Bruker ESP300 spectrometer (Chamulitrat, ~. et al., Molec.
Pharmacol . 46:391-397 (1994) ) . Table 1 shows the mean +
WO95/13805 21~ 67 ~7 PCr/US94/13239 ~
SEM EPR units ~ ..u.llbe~ of animals ~YAm;nDcl). An agc d~~ nt increase was observed in the amount of NO-Hb in the blood of the ~:1;P.DA~:D~ mice. The levels of NO-Hb i~
were higher in MRL-lpr/lpr mice compared to s~ - a~c:
5 control mice without disease . The dif f erences were statistically si~n; f; cAnt (p<0 . 05 at all ages analyzed) .
The presence of NO-Hb is another important sign that NO is being over-eYpressed in these mice with autoimmune nephritis and arthritis.
Table l Mouse Age 12 weeks Age 16 weeks Age 18 to 20 weeks MRL-lpr~lpr 1420 ~ 130 (n=16) 2045 i 226 (n=10) 4092 i 711 (n=711) BALB/c (control) 970 i 221 (n=5) 1005 ~ 160 (n=160) 811 ~ 108 (n=8 The above studies .1 Lr ted an NO-mediated 15 modification of a protein (hemoglobin) in these mice. To determine if ~;~eA~l 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 DYAm; nD~l by electron paramagnetic resonance (EPR) . For 20 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 f or NO-heme (presumably due to blood trapped 25 within the MRL-lpr/lpr kidney; see Chamulitrat, W., et al ., IYolec. Ph~rmacol . 46:391-397 (1994) ) . It is important to note that control kidney did not have these NO-protein signals, signifying that the nitrosylated _ W0 95113805 ~ ; 7 ~ 7 PCrtUS94/13239 non-heme protein might be causally related to the observed severe renal pathology in these mice.
, ~
PrgtP;n nitration jn k;rlnpys from MRT-l~r/lDr ~ p As noted above, N0 may react with ~u~eluxide and form 5 the highly reactive, tissue destructive molecule peroxynitrite. It has been shown previously that cells from MRL-lpr/lpr mice can vv~L~Lvduce reactive oxygen species such as hydrogen peroxide, superoxide (Dang-Vu, A.P. et al., .J. Immunol . 138:1757-1761 (1987) ) and nitric 10 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 uv~L~Loduce the destructive molecule peroxynitrite (Beckman, J.S., et al., l~ethods 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 ~l;cp~d kidneys of MRL-lpr/lpr mice. Immunoblot analysis was performed on 20 protein extracts from kidneys of 20 week old normal (BALB/c) and MRL-lpr/lpr mice.
Kidney tissue was homogenized with a glas6 pestle.
Proteins in soluble extracts (100 ,~lg per lane) were separated by SDS-polyacrylamide gel electrophoresis and 25 transferred to nitrocPl lulocP (0.45 ,um, Novex) . Unbound sites were blocked by incubation with 1% non-fat dry milk in TBS (20 mM Tris, 500 mM NaCl, pH 7.5) for 60 min at 2 5 C . Membranes were incubated overnight at 2 5 C with a polyclonal anti-niLLvLyLosine antibody (0.25 ~g/ml) in 30 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 chemil~ i n~c-~n~e (ECL) detection (Amersham) .
Wo 95113805 PcrrUss4r1323s ~
7~7 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 5 other pL, i nPnt 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.
Cat~ P activitY in kidnevs of MRL-lpr/lPr mice The catalase content in kidneys from Z0 week old normal (BALB/c) and MRL-lpr/lpr mice was analyzed.
Peroxynitrite or N0 can destroy catalase activity.
Catalase was measured by the di c:ArpP Irance of hydrogen peroxide noted by absorbance at 240 nm (Beers, R.F., and 15 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.
2 0 Table 2 BALB/c mouse MRl,lpr/lpr mouse Mousi~ 1 77 i 6 Mousc 1 12 il Mouse 2 75 i 2 Mous~ 2 20 Mouse 3 67 i 5 25 Mousc 4 68 i 6 In a different type of analysis to determine catalase, we studied protein extracts from kidneys by ~Iwo 9S/1380S PCrlUS94113239 2~7~7~7 PAGE, with subsequent vi~1~Al;7~A~tion of catala6e activity by soaking the gels in phosphate buffer containing - horseradish peroxidase and IIYdLOY~ peroxidase with ~;Am;nr h~n7idine.
Soluble extracts (25 ,ug/lane) were separated by electrophoresis on a 6% native polyacrylamide gel. Bands of catalase activity were v; !=IIA 1; 7~d by soaking the gels for 45 min at 25C in 50 mM sodium phosphate, pH 7.0, containing 0. l mM EDTA and 50 ,ug/ml horseradish peroxidase. H202 was added to a final concentration of 5 . O
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 rl;Am;nclb~n~idine-HCl in 50 mM sodium phosphate, O. l mM EDTA, pH 7 . O.
Results showed clearly that kidneys from 4 different BALB/c control mice contained large amounts of catalase, while those from two NRL-lpr/lpr mice had markedly ~1; m; n; ~:h~ level5 of catala5e . However, if the mice had been treated with oral NG-- Lhyl-L-arginine in vivo (See NMMA ~t!at ~-)t 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 NRL-lpr/lpr mice, and that the NO- (or peroxynitrite-) mediated decrease in catalase is blocked by in vivo administration of NG ~ y l-L-arginine .
n investiqationS
To determine if humans with arthritis have iNos protein expressed in their synovial tissues, these tissues were studied by immunof luorescence techniques using mouse monoclonal anti-iNOS antibody (purchased from Transduction Laboratories, Inc. ) . of six synovial samples removed from ~17~7~7 patients with advanced arthritis at the time of ; oint rep' ~Ir L 6urgery, iNos antigen was detected in the tissues of two of three rheumatoid arthritis patients and ,~
in one of three osteoarthritis patients. These studies 5 d~ LLc.te that patients have synovia that contain iNos, and that it may be uv~le~Less~d in synovia of patients with rheumatoid arthritis.
EOUIVAT .T~'NTS
Those skilled in the art will rf~ n; ~e, or be able 10 to ascertain using no more than routine experimentation, many equivalents to the specific P~nhsr~ nts of the invention described specif ically herein . Such equivalents are intended to be t~n~ ,-cc~d in the scope of the following claims.
Claims (14)
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-independant 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 NG-amino-L-arginine, NG-methyl-L-arginine, NG-nitro-L-arginine, NG-nitro-L-arginine methyl ester, NG-iminoethyl-L-ornithine and aminoguanidine.
7. A method of claim 6 wherein the nitric oxide synthase inhibitor is NG-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 NG-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 Claim 1 wherein the autoimmune disease is systemic lupus erythematosus.
13. A nitric oxide synthase inhibitor for use in therapy, for example for use in treating or preventing an autoimmune disease.
14. A method of treating systemic lupus erythematosus in a patient in need of treatment thereof comprising administering to said patient an effective amount of NG-methyl-L-arginine.
Applications Claiming Priority (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| US15346993A | 1993-11-17 | 1993-11-17 | |
| US08/153,469 | 1993-11-17 |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CA2176747A1 true CA2176747A1 (en) | 1995-05-26 |
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ID=22547353
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CA002176747A Abandoned CA2176747A1 (en) | 1993-11-17 | 1994-11-17 | Use of nitric oxide synthase inhibitors in the treatment of autoimmune diseases |
Country Status (5)
| Country | Link |
|---|---|
| EP (1) | EP0729356A1 (en) |
| JP (1) | JPH09508891A (en) |
| AU (1) | AU1209995A (en) |
| CA (1) | CA2176747A1 (en) |
| WO (1) | WO1995013805A1 (en) |
Families Citing this family (16)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| FR2730930B1 (en) | 1995-02-27 | 1997-04-04 | Oreal | USE OF NO-SYNTHASE INHIBITORS TO REDUCE THE IRRITANT SKIN EFFECT OF PRODUCTS USED IN THE COSMETIC OR PHARMACEUTICAL FIELD |
| FR2740339B1 (en) * | 1995-10-26 | 1997-12-05 | Oreal | USE OF AT LEAST ONE NO-SYNTHASE INHIBITOR IN THE TREATMENT OF SENSITIVE SKIN |
| DE19549118C2 (en) * | 1995-12-29 | 2000-07-13 | Thomas W Stief | Hemostasis activation inhibitor and method for inhibiting hemostasis activation in blood or other biological fluids |
| AU3128197A (en) * | 1996-05-30 | 1998-01-05 | Children's Hospital Medical Center | Guanidino derivatives as inhibitors of the cytotoxic effect of peroxynitrite |
| US5929055A (en) * | 1997-06-23 | 1999-07-27 | The Research Foundation Of State University Of New York | Therapeutic method for management of diabetes mellitus |
| WO1999011667A1 (en) * | 1997-08-29 | 1999-03-11 | Innogenetics N.V. | METHYLATED, SmD HOMOLOGOUS PEPTIDES, REACTIVE WITH THE ANTIBODIES FROM SERA OF LIVING BEINGS AFFECTED WITH SYSTEMIC LUPUS ERYTHEMATOSUS |
| US6093745A (en) * | 1997-11-25 | 2000-07-25 | Psorx, L.L.C. | Methods and composition for treating skin proliferative diseases |
| EP1112068A2 (en) | 1998-09-08 | 2001-07-04 | Monsanto Company | Methods of treating osteoarthritis with inducible nitric oxide synthase inhibitors |
| US6656925B2 (en) * | 1998-09-09 | 2003-12-02 | Advanced Medical Instruments | Composition and method of treating arthritis |
| WO2002060431A1 (en) * | 2001-01-30 | 2002-08-08 | Ajinomoto Co., Inc. | Remedies/preventives for inflammatory diseases |
| DE10111052A1 (en) * | 2001-03-06 | 2002-09-12 | Beiersdorf Ag | Cosmetic or dermatological preparations for increasing ceramide biosynthesis, e.g. to prevent dryness, containing agents inhibiting the onset of nitrogen monoxide synthase activity |
| DE10111054A1 (en) * | 2001-03-06 | 2002-09-12 | Beiersdorf Ag | Cosmetic or dermatological preparations for reinforcing skin barrier function, e.g. to prevent dryness, containing agents inhibiting the onset of nitrogen monoxide synthase activity |
| DE10111050A1 (en) * | 2001-03-06 | 2002-09-12 | Beiersdorf Ag | Use of substances which prevent the NO-synthase of the warm-blooded organism from exerting its effect, for the preparation of cosmetic or dermatological preparations for the treatment and / or prophylaxis of undesired skin pigmentation |
| DE10111049A1 (en) * | 2001-03-06 | 2002-09-12 | Beiersdorf Ag | Cosmetic or dermatological preparations for combating inflammatory disorders or dryness of the skin, containing agents inhibiting the onset of nitrogen monoxide synthase activity |
| JP2007055900A (en) * | 2003-12-15 | 2007-03-08 | Ajinomoto Co Inc | Medicinal composition for treating and preventing inflammatory disease |
| WO2017139457A1 (en) * | 2016-02-11 | 2017-08-17 | Ohio University | Compositions and methods for inhibiting uvb -irradiation damage by targeting constitutive nitric oxide synthases (cnos) |
Family Cites Families (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US5059712A (en) * | 1989-09-13 | 1991-10-22 | Cornell Research Foundation, Inc. | Isolating aminoarginine and use to block nitric oxide formation in body |
| DE69230928T2 (en) * | 1991-12-16 | 2001-01-11 | Univ St Louis | Use of aminoguamidine in the manufacture of a medicament for suppressing nitrogen oxide formation |
| GB9127376D0 (en) * | 1991-12-24 | 1992-02-19 | Wellcome Found | Amidino derivatives |
-
1994
- 1994-11-17 JP JP7514592A patent/JPH09508891A/en active Pending
- 1994-11-17 AU AU12099/95A patent/AU1209995A/en not_active Abandoned
- 1994-11-17 CA CA002176747A patent/CA2176747A1/en not_active Abandoned
- 1994-11-17 EP EP95903124A patent/EP0729356A1/en not_active Withdrawn
- 1994-11-17 WO PCT/US1994/013239 patent/WO1995013805A1/en not_active Application Discontinuation
Also Published As
| Publication number | Publication date |
|---|---|
| AU1209995A (en) | 1995-06-06 |
| EP0729356A1 (en) | 1996-09-04 |
| JPH09508891A (en) | 1997-09-09 |
| WO1995013805A1 (en) | 1995-05-26 |
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