MXPA99010417A - METHODS FOR TREATING INFLAMMATION AND INFLAMMATORY DISEASES USING pADPRT INHIBITORS - Google Patents

Abstract

The present invention is directed to a method for treating inflammation or inflammatory disease in an animal or mammal, which comprises the steps of administering an effective amount of a pADPRT inhibitory compound to said animal or mammal.

Description

METHODS TO TREAT INFLAMMATION AND INFLAMMATORY DISEASES USING PADPRT INHIBITORS DESCRIPTION OF THE INVENTION The present invention relates to methods for treating inflammation and inflammatory diseases, including arthritis, in animals or mammals. The invention also relates to methods of treating animals or mammals that have both gram-negative and gram-positive endotoxin symptoms that result from systemic infections or that result from lipopolysaccharide infestation. These methods involve the use of therapeutically effective amounts of pADPRT inhibitor compounds.

BACKGROUND OF THE INVENTION The use of pADPRT inhibitor compounds to treat cancer and viral infections has been reported. Examples of these methods are described in U.S. Patent Nos. 5,464,871; 5,473,074; 5,482,975; 5,484,951; 5,516,941 and 5,583,155, the disclosures of which are incorporated herein by reference. In published literature, it has recently been shown that 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a REF .: 32033 novel inhibitor of the nuclear enzyme poly-ADP ribose polymer (pADPRT) inhibits tumorigenicity in vivo in a Ha-ras transfected endothelial cell line; Bauer et al., 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)," Int . J. Oncol. 8: 239-252- Bauer et al. , 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, to non-covalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 77: 347-377. Treatment with INH2BP also results in changes in the activities of I and II and MAP kinase; Bauer et al. , 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (INH ^ BP)," Int. J. Oncol. 8 .: 239-252; Bauer et al. , 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, a non-covalently binding ligand of poly (A-DP-ribose) polymerase," Biochimie 7-7: 347-377. Based on the observed effects, a hypothesis has been presented regarding the potential use of INH2BP in cancer ther Bauer et al. , 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH.BP)." Int. JJ Oncol. 8.239-252. Bauer et al. , 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-l, 2-berizopyrone, to a non-covalently binding ligand of poly (ADP-ribose) polymerase." Biochimie 77: 347-377. Malignant growth and inflammatory processes share the activation of certain cellular signal transduction pathways, for example, MAP kinase; Kyriakis et al, 1996, "Sounding the alarm: protein kinase cascades activated by stress and inflammation," J. Biol Chem. 271: 24313-24316; Ferrell, JE, 1996, "Tripping the fantastic switch: how to protein kinase cascade can convert graded inputs into switc-like outputs," TIBS 21: 460-466. Chronic inflammation often leads to carcinogenic transformation, as demonstrated, for example, in the case of the intestinal epithelium; Kawai et al. , 1993, "Enhancement of rat urinary bladder tumorigenesis by ipopolysaccharide-induced inflammation," Cancer Res .53 .: 5172-5; Rosin et al. 1994, "Inflammation, chromosomal instability, and cancer: the schistosomiasis model," Cancer Res. 54 (7 Suppl): 1929s-1933S; Choi et al. , 1994, "Similarity of colorectal cancer in Crohn's disease and ulcerative colitis. - implications for carcinogenesis and prevention," Gut 35: 950-4. Based on the relationship between chronic inflammation and carcinogenic transformation, the objective of the present study is to investigate whether INH2BP affects the course of inflammatory process in vitro and in vivo. In our study, the production of multiple proinflammatory mediators is induced by bacterial lipopolysaccharides (endotoxins, LPS). It is known that LPS induces many cellular reactions and activates a systemic inflammatory response. The proinflammatory mediators induced by LPS include tumor necrosis factor-a (TNF), interleukin-1, interferon-gamma whereas anti-inflammatory mediators include interleukin-10 (IL-10) and interleukin-13; Deltenre et al, 1995, "Gastric carcinoma: the Helicobacter pylori trail," Acta Gastroenterol Belg. .58: 193-200; Beutler, 1995, "TNF, immunity and inflammatory disease: lessons of the past decade," J. Invest. Med. 42: 227-35; Liles et al, 1995, "Review: nomenclature and biologic significance of cytokines involved in inflammation and the host immune response," J. Infect Dis. 172.-1573-80; Giroir, 1993, "Mediators of septic shock: new approaches to interrupting the endogenous inflammatory cascade," Critical Car. Med. 21: 780-9. As a consequence of the production of these inflammatory cytokines, the LPS initiate the production of inflammatory free radicals (with centered oxygen) such as superoxide and radicals with centered nitrogen, such as nitric oxide [NO]) and prostaglandins; Nathan, 1992, "Nitric oxide as a secretory product of mammalian cells," FASEB J. 6: 3051-3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: master of the blood circulation," Proc. Rov. Soc. Lond B 343: 225-246; Szabo, C., - 1995, "Alterations in the production of nitric oxide in various forms of circulatory shock," New Horizons ¿-32. The production of NO in inflammation is due to the expression of an isoform other than NO synthase (iß OS), while the production of inflammatory cytokines is explained by the expression of a different isoform of cyclooxygenase (cyclooxygenase-2, COX- 2), -? Athan, 1992, "? Itric oxide as a secretory product of mammalian cells," FASEB J..-3051-3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: master of the blood circulation," Proc. Roy. Soc. Lond B 343 .: 225-246; Szabo, C; 1995, "Alterations in the production of nitric oxide in various forms of circulatory shock,"? Ew Horizons 3 .: 3 -32. The RNAs, COX-2, as well as the proinflammatory cytokines mentioned above and the free radicals which may play an important role in the inflammatory response induced by LPS; ? athan, 1992, "? ric oxide as a secretory product of mammalian cells," FASEB J. 6: 3051-3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: master of the blood circulation," Proc. Rov. Soc. Lond B 343: 225-246; Szabo, C., - 1995, "Alterations in the production of nitric oxide in various forms of circulatory shock,"? Ew Horizons 3_: 3-32. In addition, NO (or its toxic byproduct, peroxynitrite) has been implicated as a clear mediator that leads to the transformation of the inflammatory response into a carcinogenic process, - Bartsch et al. , 1994, "Endogenously formed? -nitrous compounds and nitrosating agents in human cancer etiology," Pharmacogenetics 2: 272-7; Liu the al. , 1992, "Woodchuck hepatitis virus surface antigen induces or synthesis in hepatocytes: possible role in hepatocarcinogenesis.," Carcinogenesis 15: 2875-7; Ohshima the al. , 1994, "Chronic infections and inflammatory processes as cancer risk factors.-Possible role of nitric oxide in carcinogenesis," Mutation Res. 305: 253-64. In the current studies, we first investigated whether treatment with INH2BP affects the production of the inflammatory mediators of tumor necrosis factor-alpha [TNF], interleukin-10, interleukin-6, NO, and prostaglandin in vivo, in inflammation models induced by LPS. There are a multitude of intracellular processes which precede the production of proinflammatory mediators. Activation of tyrosine kinases; Levitzki, A., 1994, "Signal-transduction therapy, A novel approach to discase management." Eur. J. Biochem. 226-1-13; Novogrodeky the al. , 1994, "Prevention of lipopolysaccharide induced lethal toxicity by tyrosine kinase inhibitors," Science 264U (Wash): 1319-22; Marczin et al, 1993, "Tyrosine kinase inhibitors suppress endotoxin-and IL-lbeta-induced NO synthesis in aortic smooth muscle cells," Am. J. Physiol. 265: H1014-1018; mitogen-activated protein kinase (MAP kinase); Matsuda et al. , 1994, "Signaling pathways mediated by the mitogen-activated protein (MAP) kinase kinase / MAP kinase cascade," J. Leukocvte Biol. 5.6: 548-53; L'Allemain, G., 1994, "Decip ering the MAP kinase pathway," Progr. Growth Factor Res. 5: 291-334; Cowley et al. , 1994, "Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and transformation of NIH 3T3 cells.," Cells 22 = 841-52 and the nuclear factor kappa B (NF-kB) pathway; Baeuerle et al. , 1994, "Function and activation of NF-B in the immune system," Ann. Re. Immunol. 12: 141-79; Schreck et al, 1992, "Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukaryotic cells (a review)," Free Radical Res. Comm. .17: 221-37; Muller et al, 1993, "Nuclear factor kappa B, a mediator of lipopolysaccharide effects," Immunobiol. 187: 233-56; they are recognized as important factors in the inflammatory response and contribute to the expression or production of inflammatory mediators. Therefore, it has also been investigated whether INH2BP also affects LPS-induced activation of MAP kinase and NF-KB by LPS. The results of the present study demonstrate that INH2BP has potent anti-inflammatory effects by modulating multiple components of the inflammatory response induced by LPS.

BRIEF DESCRIPTION OF THE INVENTION One aspect of the invention is a method for treating inflammation or inflammatory disease in an animal or mammal, which comprises the steps of administering an effective amount of a pADPRT inhibitor compound to the animal or mammal.

Another aspect of the invention is a method for treating inflammation or inflammatory diseases in an animal or mammal, which comprises the steps of administering an effective amount of a pADPRT inhibitor compound wherein the pADPRT inhibitor compound is selected from the group consisting of from: a compound that has the formula: : D wherein R., R2, R3, R4, Rs and R6 are each selected from the group consisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, and one of Rl t R2, R3, R4, Rs and R6 is amino; a compound that has the formula: (II) wherein Rx, R2, R3, R4 and Rs are each selected from the group consisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol optionally substituted with alkyl, alkoxy, hydroxy or halo, and one of Rx, R2, R3, R4 and R5 is amino; and a compound that has the formula: (III) wherein R 1 t R 2, R 3, R 4 and R 5 is each selected from the group consisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, and one of R t R2, R3, R4 and R5 is amino. Preferred pADPRT compounds include: 6-amino-1, 2-benzopyrone, 3-nitrosobenzamide, 5-amino-1 (2H) -isoquinolinone, 7-amino-1 (2H) -isoquinolinone and 8-amino-1 (2H) -isoquinolinone. Yet another aspect of the invention includes a method of treating symptoms induced by both gram-negative and gram-positive symptoms in an animal or mammal, the method comprising the step of administering to a mammal or animal a therapeutically effective amount of a pADPRT inhibitor compound. Yet another aspect of the invention is a method for treating endotoxin-induced gram-negative and gram-positive symptoms in an animal or mammal, which comprises the step of administering to an animal or mammal a therapeutically effective amount of a pADPRT inhibitor compound wherein the The compound is selected from the group consisting of compound I, compound II or compound III as described above. Yet another aspect of the invention is a method for treating endotoxin symptoms induced by both gram-negative and gram-positive in an animal or mammal which comprises the step of administering to an animal or mammal a therapeutically effective amount of a pADPRT inhibitor compound in wherein the compound has the structural formula indicated above as compounds I, II or III. Another additional aspect of the invention is a method for treating arthritis in an animal or mammal, comprising the step of administering an effective amount of, or a pADPRT inhibitor compound, wherein the compound has the structural formula indicated above as compounds I , II or III. Another additional aspect of the invention is a method for treating Crohn's disease in an animal or mammal, comprising the step of administering an effective amount of a pADPRT inhibitor compound, wherein the compound has the structural formula indicated above as compounds I, II or III. Yet another aspect of the invention is a method for treating Barrett's disease in an animal or mammal, comprising the step of administering an effective amount of a pADPRT inhibitor compound, wherein the compound has the structural formula indicated above as compounds I, II or III. The pADPRT inhibitor compounds of the invention can be prepared by the methods described in U.S. Patent Nos. 5,464,871; 5,473,074; 5,482,975; ,484,951; 5,516,941 and 5,583,155, the disclosures of which are incorporated herein by reference. Preferred compounds for use in the methods of the invention include those in which the halo group is iodine, and one of the R groups is amino, one of the R groups can be nitroso or nitro as described in the patents mentioned above, but preferably the R group is amino. In addition, it has been found that the inhibitory activity of pADPRT is strongly shown when the iodine portion is adjacent to the amino moiety. In any case, the compounds to be used in the methods of the invention must have inhibitory activity of pADPRT. The compounds may be used as such, or preferably in combination with a pharmaceutically acceptable acid addition salt or other suitable pharmaceutical carrier known in the art.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. Effect of INH2BP on the production of nitrite induced by LPS (a), production of 6-keto prostaglandin Fl (b), production of TNF (c) and suppression of mitochondrial respiration (d) in Jll cells. TNF is measured at 4 hours, all other parameters at 24 hours after LPS. ** represents a significant change in the response to LPS when compared to the controls (p <0.01) m, ## represents a significant effect on INH2BP in the presence of LPS when compared to LPS alone (p <0.01); n = 6-12 wells. Figure 2. INH2BP inhibits the expression of iNOS in J774 and RAW 264.7 cells. (a) Nortrhern transfers representative of iNOS and 18s mRNA in J774 cells (A) and RAW 264.7 macrophages (B) under control conditions (lane 1), at 4 hours after treatment with LPS (lane 2) and at 4 hours after treatment with LPS in cells in the presence of INH2BP (100 μM) (lane 3). (b) Effect of INH2BP on the activity of iNOS in homogenates of J774 cells under control conditions (C and C + INH2BP) and at 12 hours after treatment with LPS (LSP and LPS + INH2BP). ** represents a significant change of LPS when compared to controls (p <0.01) m, ## represents significant inhibition by INH2BP (p <0.01); n = 4. (c) Representative iNOS Western blot in control J744 cells and in cells 12 hours after LPS in the presence or absence of INH2BP. Figure 3. (a) Time-dependent loss of inhibition of nitrite accumulation by INH2BP (100 μM), when administered at 2 hours before LPS together with LPS, or at 2, 4 and 6 hours after LPS. (b) Effect of INH2BP on the accumulation of nitrite in J774 cells stimulated with the combination of LPS and INF; n = 6-12 wells.

Figure 4. Effect of INH2BP on the induction of luciferase activity by LPS in RAW 264.7 cells transiently transfected with the full-length construct (-1592 bp) or a deletional construct (-367 bp) of the iNOS-luciferase promoter. In cells transfected with either full length or with deletional construction (black bars), treatment with LPS (10 μg / ml), 4 hours) leads to a 10 to 12-fold induction of luciferase activity, with respect to to the control values. Co-treatment with I? H2BP inhibits LPS-mediated increases in luciferase activity in cells transfected with the full-length construct, but has no significant effect on cells transfected with the -367 bp deletional construct (gray bars). The data are expressed as times of increase in luciferase activity on the control cells, and corrected for the respective β-galactosidase activity. * represents a significant effect of I? H2BP in the presence of LPS, when compared to LPS alone (p <0.05); n = 4 separate transfections. Figure 5. I? H2BP suppresses the induction of i? OS in conscious rats. IOS activity in lung homogenates 8a) and nitrite-nitrate plasma concentrations (b) in control rats (c), in rats injected with I? H2BP (I? H2BP); in rats injected with LPS (15 mg / kg ip for 6 hours), -and the effect of treatment with I? H2BP (10 mg / kg, ip), when administered 10 minutes before LPS (INH2BP) + LPS) or 2 hours after LPS (LPS + INH2BP). ** represents a significant effect of LPS when compared to controls (p <0.01); ## represents significant inhibition by the inhibitor pADPRT (p < 0.01); n = 4-5. Figure 6. Effect of INH2BP (10 mg / kg, ip) on the response of TNF, IL-10 and IL-6 induced by LPS in mice, 90 minutes after administration of LPS (4 mg / kg, ip ). ## represents a significant effect of LPS when compared to controls (p <0.01); ## represent a significant increase in the response by INH2BP (p <0.01); n = 4-5. Figure 7. INH2BP improves survival in mice subjected to endotoxic shock: effect of pre-treatment with INH2BP (0.3-10 mg / kg) on mortality induced by endotoxin (120 mg / kg i.p.) in mice, - n = 7-8 animals in each group. Figure 8. (a) MAP kinase activity in RAW 264.7 cells treated with vehicle or with LPS (10 μg / ml) for 24 hours in the presence or absence of 100 μM PD 98059 or 150 μM INH2BP. The data represent values obtained in a typical experiment: Similar results are observed in 3 different experimental days, (b) Representative assay of MAP kinase in gel, in RAW 264.7 cells at 24 hours after treatment with vehicle or with LPS, in presence or absence of INH2BP 150 μM. Lanes 1-4 represent the following groups, respectively: 1: control treated with vehicle, -2: treatment with LPS; 3: vehicle treatment in the presence of 150 μM INH2BP; 4: treatment with LPS in the presence of INH2BP 150 μM. Figure 9. Inhibition of pADPRT with INH2BP does not alter the nuclear translocation of Western blotting of NF-KB from nuclear extracts of control J74 cells and in cells at 90 minutes after treatment with LPS in the presence or absence of INH2BP (100 μM) . Figure 10. The effect of INH2BP on the development of edema in the plant, induced by carrageenan, is described. The data show the plant volumes at 1-4 hours after injection of carrageenan (means + standard error mean, n = 6 animals in each group). There is a significant increase in the volume of the plant of one hour (p <0.01) and there is a significant inhibition of the development of edema in the INH2BP plant at 1-4 hours (** p <0.02). Figure ll. The effect of INH2BP on the access of collagen-induced arthritis is described. The percentage of arthritic mice (mice showing clinical grades of arthritis > 1) is represented. The arrow at 21 days represents the time of the second immunization with collagen, the horizontal bar from day 25 represents the time of the start of treatment with INH2BP (N = 6) or with VEHICLE (N-10).

Figure 12. The effect of INH2BP on the severity of collagen-induced arthritis is described. The median arthritic rating during collagen-induced arthritis. The arrow at 21 days represents the time of the second immunization with collagen, the horizontal bar from day 25 represents the time of the start of treatment with The effect of INH2BP on (n -6) or with vehicle (n = 10). There is a significant increase in the arthritic score on day 26, and there is a significant suppression of the arthritic score for INH2BP between days 26-35 (#p < 0.05).

DESCRIPTION OF THE PREFERRED MODALITIES OF THE INVENTION Definitions As used herein: "Anti-inflammatory" diseases refers to diseases or conditions where there is an inflammation of body tissue. Such diseases include, for example, Crohn's disease, Barrett's disease, arthritis, multiple sclerosis, cardiomyopathic disease, colitis, infectious meningitis, encephalitis and the like. The term "pharmaceutically acceptable acid addition salt" refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, acid nitric acid, phosphoric acid, methanesulfonic acid, salicylic acid and the like. The term "ADPRT" refers to adenosine diphospho-ribose transferase which is also known as poly (ADP-ribose) polymerase (EC 2.4.99), a specific nuclear protein that binds to eukaryotic DNA that catalyzes the polymerization of ADP -bull The enzymatic process is dependent on DNA. The term "alkyl" refers to a branched or straight chain, saturated or unsaturated hydrocarbon radical. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl, and the like. The term "alkoxy" refers to the -O-alkyl radical. Typical alkoxy radicals are methoxy, ethoxy, propoxy, butoxy and pentoxy and the like. The term "cycloalkyl" refers to a saturated monocyclic hydrocarbon radical containing 3-8 carbon atoms such as cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like. The term "substituted phenyl" refers to all possible isomeric phenyl radicals such as mono or disubstituted with a substituent selected from the group consisting of alkyl, alkoxy, hydroxy or halo. The term "halo" refers to chlorine, fluorine, bromine or iodine, and preferably to iodine. The inhibitor compounds of pADPRT of the invention (notably the compounds defined above as compounds I, II or III) are potent, specific and non-toxic anti-inflammatory compounds, which can be used for conditions and diseases typically known by inflammation, such as arthritis, Crohn's disease, Barrett's disease 's and similars. In addition, these compounds are useful in the treatment of conditions associated with gram-negative and gram-positive infections, especially those associated with gram-negative infections and including conditions associated with lipopolysaccharide condition and sepsis. The compounds are especially useful insofar as they have a very low toxicity, if present. In practice, the compounds of the invention or their pharmaceutically acceptable salts will be administered in amounts which will be sufficient to inhibit inflammatory conditions or disease and / or to prevent the development of inflammation or inflammatory diseases in animals or mammals, and are used in the pharmaceutical form more suitable for such purposes.

Administration of the active compounds and salts described herein can be by any of the accepted modes of administration for therapeutic agents. These methods include systemic and local administration such as oral administration modes, parenteral, transdermal, subcutaneous or topical. The preferred method of administration of these medications is oral. In some cases it may be necessary to administer the composition in another parenteral form. Based on the proposed mode, the compositions may be in solid, semisolid or liquid dosage form such as, for example, injectables, tablets, suppositories, pills, sustained release capsules, powders, liquids, suspensions or the like, preferably in dosages unit. The compositions will include an effective amount of active inhibitor compound pADPRT or the pharmaceutically acceptable salt thereof, and may further include any conventional pharmaceutical excipient or other medicaments or medicinal or pharmaceutical agents, carriers, adjuvants, diluents, etc., as is customary in the pharmaceutical sciences. For solid compositions, such excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate and the like, which may be used. The active pADPRT inhibitor compound defined above can also be formulated as suppositories using, for example, polyalkylene glycols, for example propylene glycol as a carrier. Liquid compositions, particularly injectables, can be prepared, for example, by dissolving, dispersing, etc., the active compound in a pharmaceutical solution such as, for example, water, saline, aqueous dextrose, glycerol, ethanol and the like, for This way form the injectable solution or suspension. If desired, the pharmaceutical composition when administered may also contain minor amounts of non-toxic auxiliary substances such as wetting agents or emulsifiers, pH buffering agents and other substances such as, for example, sodium acetate, triethanolamine oleate, etc. In addition, if desired, the pharmaceutical composition to be administered may contain liposomal formulations comprising a phospholipid, a negatively charged phospholipid and a compound selected from cholesterol, a cholesterol fatty acid ester or an unsaturated fatty acid. Typical neutral phospholipids include L-a-phosphatidylcholine, L-a-phosphatidylinosol, L-a-phosphatidyl-serine, L-a-phosphatidylinosol, L-a-phosphatidic acid, L-a-phosphatidylglycerol, L-a-lysophosphatidylcholine, sphingomyceline and cardiolipin. Typical negatively charged phospholipids include diacetyl phosphate or phosphodiglyceride, for example dilauroyl, dimyristoyl phosphate, dipal itoyl phosphate, distereoyl phosphate. Typical cholesterols and cholesterol ethers include cholesterol, 3S-hydroxy-5-cholestene, polyoxyethanescholetheryl sebacate, cholesterol-5, 6-epoxide, cholesteryl acetate, cholesteryl-n-butyl ether, cholesteryl caprate, cholesteryl dodecanoate, cholesterylethylether , cholesteryl heptadecanoate, cholesterylmethyl ester. Typical unsaturated fatty acids include arachidonic acid, docosahexaenoic acid, elaidic acid, erucic acid, linoleic acid, linolenic acid, nervonic acid, oleic acid, palmitoleic acid, petroselinic acid. The halo nitro compounds can be encapsulated or divided into a liposome biolayer of the liposome formulation according to the patent application serial number 08 / 020,035 entitled "Liposomal Formulations and Methods of Making and Using Same "filed on February 19, 1993, which is incorporated herein by reference In the first embodiment, the liposomes are formed first and then the C-amino, nitroso or nitro compounds are added instead of being encapsulated , the divisions of the C-amino, nitroso or nitro compounds (placed in themselves) within the lipid bilayer of the liposome To elaborate this composition, typically, the ingredients, for example phosphatidylcholine, diacetyl phosphate and cholesterol are combined with a solvent, such as chloroform After the combination, the chloroform is extracted Water is added thereto When water is added to the liposomes, a multilamellar liposome is constituted (ie, the liposomes are similar to the covers of an onion which It has many layers.) The next stage is to freeze and reheat them, they are quickly frozen in liquid nitrogen, the purpose of freezing and reheating is to resize the liposomes. more uniform. At this point, the liposomes vary in size and you should try to do this more times, typically about five times. The reheating occurs in a water bath at 37 °. Before freezing and reheating, the mixture is sonicated. The combination of sonication and reheating reduces the number of layers. The objective is to produce a unilamellar system. At this point, the nitrous compound C is added to contain a concentration of 10 millimolar (mM). The concentration can be in excess of 15 millimolar. For this concentration of lipids, for a batch of 60 milliliters, the concentration of total lipids is 648 mg and these 60 milliliters of water are added. Phosphatidylcholine is 500 mg, cholesterol is 36 mg; diacetyl phosphate is 112 mg.

Increasing the liposome concentration of the mixture allows it to contain more C-amino, nitroso or nitro compound. For example, it can be twice as concentrated as it is in the previous mix. For a 60 ml batch, one should duplicate the above numbers to obtain 100 mg of phosphatidylcholine, 224 mg of dicetyl phosphate and 72 mg of cholesterol. By decreasing the concentration decreases the amount of C-nitroso compound that is obtained in it. For the hypothetical 60 milliliter batch, the upper limit of the C amino compound it approaches is a 15 millimolar concentration of C-amino compound. For 3-nitrosobenzamide this is 135 mg for a batch of 60 milliliters. The next stage is to rehydrate. Then, the next stage of the process is extrusion using an extruder device (Lipex Biomembranes, Inc., Vuver, British Columbia, Canada). The extrusion process serves two purposes: 1) standardize the size of the liposomes; and 2) sterilization. Extrusion typically involves filtration through a 0.1 micron filter and is usually followed by freeze-drying the mixture to lyophilize the mixture (extract the water and make it a fine powder). This improves the solubility so that one can place up to about 40 millimolar solution which is about three times as concentrated before lyophilization. The lyophilization processes produce a mixture of powdered lipids and the pulverized C-amino compound. Now one can use the same amount of the C-amino compound and a smaller amount of liquid to make a more concentrated mixture. For example, one can have the same weight of C-amino, nitroso or nitro compound but up to one third of the original volume. One can modify the steps of the above processes by, for example, eliminating steps such as lyophilisate. This process of the first embodiment does not significantly encapsulate the C-amino, nitroso or nitro compound. Instead of having compound in the middle of the liposome, the compound is found in the membrane itself. The C-amino, nitroso or nitro compound divided within the liposome membrane will migrate to the target cells and the lipid will transport the C-amino compound, nitrous or nitro within the cell membranes. Preferably, this process causes the liposomes to be about 0.05-0.45, and more preferably about 0.1-0.2 microns in diameter. The unilamellar and multilamellar liposomes are effective. The second purpose of the extrusion is to sterilize the mixture. To sterilize, liposomes generally become smaller than 45 microns in diameter. Sizes less than 0.05 micrometers theoretically work. The process of the first mode has the advantage that, for example, 3NOBA water only has a concentration of 0.5 millimolar. The present liposomal composition reaches concentrations of 15 millimolar. In addition, unlike 3 NOBA only in aqueous solution, the liposome solution containing NOBA is resistant to ascorbic acid. It becomes useful in laboratory experiments with mice. The solution may contain the NOBA monomer or the NOBA dimer. In a second embodiment one can start with a film of the lipid components, hydrate the film with an aqueous solution of medicine. This automatically forms lipids which trap (encapsulate) the medication. This occurs with compounds which are impervious to the liposome membrane. An example of such compounds are those of U.S. Patent No. 5,262,564 of November 16, 1993, for example, L-cystine sulfinic adducts of 3-NOBA. Parenteral injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions. Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or as solid forms suitable for dissolution in liquid prior to injection.

A more recently designed approach for parenteral administration uses the implementation of slow-release or sustained-release systems, which ensure that a constant level of dosage is maintained, in accordance with the United States patent number 3,710,795, which is incorporated herein by reference. Any of the above pharmaceutical compositions may contain 0.1-99%, preferably 1-70% of the active pADPRT inhibitor compounds, especially the halo-C-amino, nitroso or nitro compounds of the above formulas I, II or III as active ingredients . Chronic inflammation is known to facilitate carcinogenic transformation in various tissues. Recently it has been shown that 5-iodo-6-amino-1,2-benzopyrone (INH2BP), a novel inhibitor of the nuclear enzyme poly-ADP ribose polymerase (pADPRT) regulates a variety of cellular signal transduction pathways and abrogates Tumorigenicity in vivo by a Ha-ras transfected endothelial cell line. As an aspect of the present invention, the effect of inhibitor compounds of pADPRT such as INH2BP on activation by endotoxin (bacterial lipopolysaccharide, LPS) on the production of inflammatory mediators of tumor necrosis factor-alpha (TNF), interleukin is demonstrated. -10 (IL-10) and interleukin-6 (IL-6), nitric oxide (NO) and prostaglandins in vitro and in vivo. In addition, the present invention shows the effect of pADPRT inhibitor compounds such as INH2BP on the activation of nitrogen activated protein kinase (MAP kinase) and nuclear factor kB (NF-kB) in vitro. In cultured J774 and RAW 264.7 macrophages, LPS induces the production of prostaglandin metabolites for TNF release and the expression of the inducible isoform of NO synthase (iNOS). The production of prostaglandins and NO by INH2BP is inhibited in a dose-dependent manner, while the short-term release of TNF-alpha is not affected. INH2BP markedly suppresses LPS-mediated luciferase activity in transiently transfected RAW cells with a murine full length construct (-1592 bp) of luciferase-iNOS macrophage promoter, but not in the deletional construct consisting of -367 bp. In vivo, the pretreatment with INH2BP inhibits the induction of iNOS and by LPS in rats, does not affect the TNF and IL-6 response induced by LPS, but increases the production of IL-10 induced by LPS. Pretreatment with INH2BP markedly improves the survival of mice in a fatal model of endotoxin shock. These results demonstrate that the inhibitor compounds of pADPRT such as INH2BP have a potent anti-inflammatory action in vitro and in vivo. Poly-ADP ribose synthetase (PARS) is a nuclear enzyme activated by single-strand DNA breaks. Massive activation of PARS, in response to single strand breakage of extended DNA induced by hydrogen peroxide-peroxynitrite or ionizing radiation can initiate a futile cycle that eliminates energy, culminating in cell damage. Peroxynitrite production has been recently demonstrated in various forms of inflammation, including arthritis and foot edema induced by carrageenan. The present invention shows the effect of the potent novel inhibitor of PARS, inhibitor compounds of pADPRT such as 5-iodo-6-amino-1,2-benzopyrone (INH2BP) in a rat model of carrageenan induced edema in the paw and in a mouse model of leg edema induced by collagen at 1-4 hours. Collagen-induced arthritis is induced in DMA-1J mice, with two injections of type II collagen on day 1 and on day 21. Oral treatment of mice with INH2BP (0.5 g / kg, daily), beginning at the beginning of arthritis (day 25), suppresses the development of clinical signs of arthritis on days 26-35. Animals treated with INH2BP show a reduced arthritic index (arthritic rating: 20-50% of the rating observed in mice treated with vehicle), and an improved histological state, when examined in the joint or knee and in the leg. These data demonstrate that the PARS inhibitor, INH2BP, shows anti-inflammatory effects of INH2BP in vivo, even with a relatively late onset of administration, which is capable of retarding the course of collagen-induced arthritis. The data of the invention support the concept that activation of PARS plays a role in the development of arthritis and possibly other forms of inflammation and inflammatory diseases. The following examples serve to illustrate the invention. They should not be considered as circumscribing or limiting their scope.

EXAMPLE 1 Cell culture Mouse macrophage cell lines were cultured J774 and RAW 264.7 in the middle of Eagle modified by Dulbecco (DMEM) as described; Szabo et al., 1996, "DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion were involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite," Proc. Nati Acad. Sci. U.S.A. 93: 1753-1758; Zingarelli et al., 1996, "Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccaride," J. Immunol. 156: 350-358. In separate studies, perifonéal macrophages of male Wister rats are obtained and cultured in vitro for 24 hours in the absence or presence of LPS and with or without INH2BP. The rats are sacrificed and the peritoneal macrophages are taken and cultured in DMEM. The cells are treated with LPS of E. coli dO mg / ml), or with LPS and INF (50 μ / ml) for various times, in the presence or absence of various concentrations (1-150 mM) of INH2BP or other pharmacological inhibitors .

Assays related to MAP kinase RAW cells were washed in PBS and harvested and used using 100 ml of lysis buffer per million cells (50 mM Tris-HCl, pH 7.4, 1% NP-40, 0.4 M NaCl, 0.1 mM NaV03, 50 mM KF, 1 mM EGTA, 2 mM PMSF, 25 nM okadaic acid, 1 mg / ml each of leupeptin, aprotinin, artastatin and antipain). The lysis is carried out for 20 minutes on ice, followed by a centrifugation of 14 minutes at 13,000 rpm in an Eppendorf centrifuge. The supernatants are stored and assayed for their protein content using the Bio-Rad dye assay.

MAP kinase gel test Protein samples (50 mg / lane) are subjected to 100% SDS-PAGE gel electrophoresis containing immobilized myelin basic protein (MBP, 250 mg / ml gel). After electrophoresis, the gel is washed once with 50 mM Tris-HCl buffer, pH 7.7 (25 ml, 20 minutes) followed by two 30 minute incubations with the same buffer containing 25% i-propanol. The gel is then washed once with Tris-HCl buffer and rinsed in a solution of 50 mM Tris-HCl, pH 7.7, 2 M mercaptoethanol, 5 M guanidine hydrochloride (50 ml) for one hour, changing the solution of incubation at 30 minutes. The proteins were then renubated by incubating the gene in five changes of a solution of 50 mM Tris-HCl, pH 7.7, 2M-mercaptoethanol,. NP-400.04% during a period of time of 16 hours. The gel is then washed twice and preincubated for half an hour in a solution containing 50 mM Tris-HCl, pH 7.7, 5 mM MgCl2, 7 mM 2-mercaptoethal. The final incubation is carried out in the same solution supplemented with 10 mM 32 P-g] ATP (50 mCi / assay) for one hour. At the end of the incubation, the gel is washed free of unbound radioactivity using 3 x 25 ml of TCA 10% and 3 x 25 ml of 10% acetic acid, dried and subjected to autoradiography; Sasaki et al., 1995, "Permissive effect of ceramide on growth factor-induced cell proliferation", Biochem. J. 311: 829-34.

Western blotting of MAP kinase 100 mg of cell extract proteins are loaded in a 10% SDS-PAG gel, subjected to electrophoresis, transferred to a nitrocellulose membrane and annealed. The first antibody (against MAP kinase) is from UBI, the second antibody is labeled alkaline phosphatase and from NEN Biolabs. Detection is performed by increased chemiluminescence; Bauer et al., 1995, "Modification of gowth related enzymatic pathways and apparent loss of tumorigenicity of ras-transformed bovine andothelial cell line by tratment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)", Int J. Oncol. 8: 239-252.

Preparation of nuclear extracts and transfer (blotting) Wester of NF-kB The cells are treated with LPS in the presence and absence of INH2BP for 90 minutes. Mininuclear extracts are prepared as described, - Hassanain et al., 1993, "Enhanced gel mobility shift assay for DNA-buiding factors," Anal. Biochem. 213 .: 167-7. Briefly, the cells are scraped, centrifuged briefly and the pellet resuspended in 400 ml of cold buffer A (Hepes pH 7.9 (10 mM), KCl (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (1 mM), PMSF (0.5 mM), pepstatin A (1 mg / ml), leupeptin (10 mg / ml) and aprotinin (10 mg / ml)], on ice for 15 minutes, in the presence of 25 ml of NP-40 1%. The samples are then vortexed, centrifuged for 1 minute at 10,000 g and the pellet resuspended with 100 ml of buffer B. { Hepes pH 7.9 (20 mM), NaCl (400 mM), EDTA (1 mM), EGTA (1 mM), DTT (1 mM), PMSF (0.5 mM), pepstatin A (mg / ml), leupeptin (10 mg) / ml) and aprotinin (10 mg / ml)]. After shaking on an oscillatory platform for 15 minutes at 4 ° C, the samples are centrifuged for 15 minutes at 15,000 g at 4 ° C. After the 70 ml aliquots are treated with 150 ml of sample buffer for SDS-PAGE. The Western blot is performed as described above, with rabbit antibody against primary NF-kB from mouse (Santa Cruz Biotechnology, Santa Cruz, CA), 1: 750 in Tween TBS (0.02%).

Measurement of nitrite concentration of nitrite / nitrate The nitrite in the culture supernatants 24 hours after the simulation is measured as described; Szabo et al., 1996, "DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smoot muscle cells exposed to peroxynitrite," Proc. Nati Acad. Sci. U.S.A. 93: 1753-1758; Zingarelli et al. , 1996, "Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion a macrophages stimulated with bacterial lipopolisaccaride," J. Immunol. 156: 350-358; Szabo et al., 1994, "Sperminbe inhibits the production of nitric oxide in immuno-stimulated J774.2 macrophages: requirement of a serum fctor," Br. J. Pharmacol. 112: 355-356, by adding 100 ml of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) to 100 ml samples of medium. The optical density is measured at 550 nM (D0550) using a Spectramax 250 microplate reactor (molecular devices, Sunnyvale, CA). For the determination of total nitrite / nitrate concentrations in plasma samples, nitrate is reduced to nitrite by incubation with nitrate reductase; Zingarelli et al., 1996, "Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccaride," J. Immunol. 156: 350-358.

Measurement of 6-keto prostaglandin Fía- The production of 6-keto prostaglandin Fla is measured at 4 hours after stimulation with LPS in samples of 100 ml of cell culture supernatant using specific radioimmunoassay; Szabo et al., 1994, "Spermine inhibits the production of nitric oxide in immuno-stimulated J774.2 macrophages: requirement of a serum factor," Br. J. Pharmacol. 112: 355-356.

Cytokine measurements Cytokine concentrations in plasma and in cell culture supernatants are determined by ELISA. Plasma concentrations of IL-10 and IL-6 were measured using endogen ELISA kits (Endogen Inc., Boston, MA). Concentrations of TNF-alpha in plasma and in cell culture supernatants were determined using Genzyme ELISA kits (Genzyme Corp., Boston, MA) as described; Szabo et al., 1997, "Isoproterenal regulates tumor necrosis factor, interleukin-10, interleukin-6 and nitric oxide production and protects againts the development of vascular hyporeactivity in emndotoxemia," Immunolocry 90: 95-100.

Measurement of mitochondrial respiration Mitochondrial respiration at 24 hours is determined by mitochondria-dependent reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium bromide to formazan; Szabo et al., 1996, "DNA strand breakage, aation of poly-ADP ribosyl synthetase, and cellular energy depletion were involved in the cytotoxity in macrophages and smooth muscle cells exposed to peroxynitrite," Proc. Nati Acad. Sci. U.S.A. 93: 1753-1758; Zingarelli et al. , 1996, "Peroxynitrite-mediated DNA strand breakage aates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccaride," J. Immunol. 156: 350-358.

Northern blotting for iNOS mRNA After exposing the cells to LPS in the presence or absence of INH2BP for 4 hours, the total RNA is extracted as described using TRIZOL. Aliquots containing 15 mg of total RNA underwent electrophoresis on a 1% agarose gel containing 3% formaldehyde. The RNAs are transferred together to a nylon membrane and UV self-crosslinked is performed. The membranes are then hybridized as described; Lowenstein et al., 1993, "Macrophage nitric oxide synthase gene: two upstream regions mediated induction by interferon gamma and lipopolysaccharide," Proc. Nati Acad. Sci. U.S.A. 90 .: 9730-9734; overnight at 42 ° C with a murine iNOS cDNA probe (106 cpm / ml) labeled with [32 P] dCTP (specific activity, 3,000 Ci / mM; NEN) by random priming (Pharmacia, Piscataway, NJ). The hybridized filters are washed in series at 53 ° C using 2X sodium citrate, sodium chloride, 0.1% SDS and 25 mM NaHP04, 1 mM EDTA, 0.1% SDS solutions. After conducting the probe for iNOS, the membranes are primed with boiling 5 mM EDTA and rehybridized with an oligonucleotide probe radiolabeled with [32 P] for 18S ribosomal RNA as a maintenance gene. After washing, the exposure is carried out overnight using a Phosphor Imager mesh.

INOS Wester transfer Cells are treated with LPS in the presence and absence of pADPRT inhibitor for 20 hours. The cells are then scraped in cold PBS and centrifuged at 14,000 g for 30 seconds. The supernatant is removed and lysis buffer containing RIPA (500 ml), aprotin (10 mg / ml) and PMSF is added. (0.5 mM). The DNA is twisted when passing samples through a 22-gauge needle. The protein content is determined by the Bradford method (BIO-Rad). Cytosolic protein (200 mg / lane) is added to SDS buffer. -PAGE, boiled for 5 minutes, separated with 7.5% SDS-PAGE and transferred to nitrocellulose membranes (0.2 mm) using the semi-dry method with an isotacoforic buffer system. After blocking for 1 hour in 3% gelatin and subsequent washing, the samples are immunoblotted in buffered saline with Tris and Tween (TTBS) and 1% gelatin, with rabbit primary antibody against mouse iNOS (upstate Biotechnology, Lake Placid, NY) 1: 1000 in TTBS (0.0%) for 2.5 hours. A goat antibody, conjugated to alkaline phosphatase, is used against rabbit IgG as a secondary antibody. The antibody binding is visualized by nitroblue tetrazolium / 5-bromo-4-indolyl phosphate (NBT / BCIP) in carbonate buffer (BIO-RAD).

INOS activity measurements Cells are treated with LPS in the presence and absence of pADPRT inhibitor for 12 hours. The measurement of the calcium-independent conversion of l-arginine to L-citrulline in homogenates of J774 cells or in lung homogenates is used as an indicator of iNOS activity as described; Szabo et al., 1994, "Sperminbe inhibits the production of nitric oxide in immuno-stimulated J774.2 macrophages: requirement of a serum factor," Br. J. Pharmacol. 12: 355-356. The cells are scraped or the lungs are placed in homogenization buffer consisting of: 50 mM Tris HCl, 0.1 mM EDTA, 0.1 mM EGTA and 1 mM phenylmethylsulfonyl fluoride (pH 7.4) and homogenized in a buffer on ice using a Tissue Tearor homogenizer 985-370 (Biospec Products, Racine, Wl). The homogenates are then measured by the conversion of [3 H] -L-arginine to [3 H] -L-citrulline. The homogenates (30 ml) are incubated in the presence of [3 H] -L-arginine (10 mM, 5 kBq / tube), NADPH (1 mM), calmodulin (30 nM), tetrahydrobiopterin (5 mM) and 5 mM EGTA) for 20 minutes at 22 ° C. The reactions are stopped by dilution with 0.5 ml of ice-cold Hepes buffer (pH 7.5) containing EGTA (2 mM) and EDTA (2 mM). The reaction mixtures are applied to Dowex 50W columns (Na + form) and the eluted activity of [3 H] -L-citrulline is measured by scintillation counting.

INOS functional test Since under our experimental conditions, J774 cells are resistant to our attempts to transfect them transiently using calcium phosphate, lipofectin and lipofectamine methods, transfection studies were performed on RAW 264.7 cells. The activity of the iNOS promoter was evaluated by transient transfection of AW 264.7 cells with constructs of the reporter gene that incorporate the iNOS promoter region of the 5 'murine macrophage towards the 5' end of the luciferase reporter gene; Lowestein et al., 1993, "Macrophage nitric oxide synthase gene: two upstream regions mediated induction by interferon gamma and lipopolysaccharide," Proc. Nati Acad. Sci. U. S.A. 90: 9730-9734; (kindly provided by Dr. Charles J. Lowenstein, Johns Hopkins University). Two constructs were used: a full length promoter construct (-1592 bp) and a deletional construction of -367 bp. The cells were seeded in plates, in 6-well culture plates at -50% confluence and transfected with the respective construction of iNOS-luciferase promoter in equimolar amounts using cationic liposomes (Lipofectin, Gibco). In order to control the differences in transfection efficiencies, the cells were cotransfected with pSV40-β-galactosidase. After transfection, the cells were allowed to recover overnight, and subsequently treated with medium alone (control), LPS (10 mg / ml), or LPS plus INH2BP (100 mM). After 4 hours of treatment, the cells were washed once in PBS, lysed in indicator lysis buffer (Promega) and analyzed for luciferase activity which is corrected for the respective galactosidase raw activity and is expressed as times of increase over control cells (transfected and treated with medium alone).

In vivo experiments Male Wistar rats and male BALB / c rats are obtained from Charles River Laboratories (Wilmington, MA or Budapest, Hungary). The animals received food and water ad libitum and lighting was maintained in a 12-hour cycle. The i.p. rats were injected. with E. coli LPS (15 mg / kg) and sacrificed at 6 hours. Plasma samples are then taken for nitrite / nitrate determinations and lung samples for iNOS measurements. Separate groups of rats were treated with INH2BP (10 mg / kg, i.p.) 10 minutes before LPS or 2 hours after injection of LPS. Studies for the measurement of the cytokine response induced by LPS, mice injected i.p. either with the drug vehicle or with INH2BP (10 mg / kg) in a volume of 0.1 ml / 10 g of body weight. Half an hour later they were exposed to a mg / kg LPS i.p. The animals are sacrificed at 90 minutes after treatment with LPS, the blood is collected in ice-cold Eppendorf tubes containing EDTA and centrifuged for 10 minutes at 4 ° C. The plasma is stored at -7 ° C until the tests are carried out. In survival studies with mice, they are subjected to animals and i.p. of LPS (120 mg / kg) at time 0 and survival is monitored for 42 hours after administration of LPS. Separate groups of mice received vehicle or treatment with INH2BP (0.1-10 mg / kg, i.p) at times -18 hours, -4 hours, 0 hours, 6 hours, 24 hours and 30 hours in relation to the dose of LPS. materials DMEM, RPMI, TRIZOL and fetal bovine serum are from Gibco (Grand Island, NY). [3H] NAD + and [32P] NAD + are obtained from DuPont NEN (Boston, MA). Alcohol dehydrogenase and ND + is obtained from Boehringer Mannheim (Indianapolis, IN). PD 98059 is obtained from Cal biochem (La Jolla, CA). All other medications are obtained from Sigma (St. Louis, MO).

Statistical evaluation All values in the quantities and in the text are expressed as mean + standard error of the mean (S.E.M.) of n observations (n> 4). Student's unpaired t test is used to compare the means between groups. A p-value less than 0.05 is considered statistically significant.

Results INH2BP suppresses nitric oxide and prostaglandin induced by LPS, but does not produce TNF-a in macrophages J774 Treatment with INH2BP causes a dose-dependent inhibition of LPS-induced nitrite formation in J774 macrophages (figure). Similarly, INH2BP suppresses LPS-induced production of 6-keto prostaglandin Fla (see figure lb), but not TNF production (see figure lc) and restores LPS-induced suppression of mitochondrial respiration (see figure). ld). INH2BP causes marked inhibition of mRNA for iNOS and protein expression (see Figures 2a-c). The inhibition of nitrite production by INH2BP is greatly reduced when the agent is administered several hours of LPS, as opposed to before the stimulation of induction by iNOS (see Figure 3a). In addition, the inhibitory effect of INH2BP on iNOS is greatly reduced when LPS is used in combination with interferon-gamma (INF-g, 50 μ / ml) for immunostimulation (see Figure 3b).

Selective regulation of induction of the iNOS promoter by INH2BP In order to further study the regulation of the iNOS promoter by INH2BP, we performed transient assays using promoter constructs of murine macrophage iNOS-luciferase. Consistent with previous data, -Lowenstein et al., 1993, "Macrophage nitric oxide synthase gene: two upstream regions mediated induction by interferon gamma and lipopolysaccharide," Proc. Nati Acad. Sci. USA 90 .: 9730-9734, we found an important role for the LPS-mediated transcriptional regulation of iNOS from murine macrophage, as evidenced by a 10 to 12 fold increase in luciferase induction by LPS (see Figure 4 ). Co-treatment of cells transfected with the full-length promoter construct (-1592 bp) with INH2BP, completely inhibits LPS-mediated luciferase activity (see Figure 4). However, a similar co-treatment of cells transfected with the -367 bp deletional construct does not significantly affect the luciferase activity mediated by LPS (see Figure 4). In vivo anti-inflammatory effects of INH, BP Pretreatment with INH2BP significantly reduces the LPS-induced increase in plasma nitrite-nitrate and the increase in pulmonary iNOS activity in conscious rats (see Figure 5). The inhibitory effect of INH2BP on NO production is reduced when the agent is added to cells or animals several hours after stimulation with LPS (Figure 5). Similar to the transformed cell lines, treatment with 100 mM INH2BP significantly reduces (by 56 + 7%, p <0.01) the production of nitrile in primary cells (peripheral macrophages obtained from rats) stimulated with LPS (10 mg / ml) in vitro (n = 4). Similar to the results in vi tro (see figure, 1c), INH2BP does not significantly affect the LPS-induced increase in plasma concentrations of TNF in mice (see Figure 6a). INH2BP also does not affect the production of IL-6 induced by LPS (see Figure 6c).

However, INH2BP causes an increase in the plasma response of IL-10 induced by LPS (Figure 6b). Pretreatment of mice by INH2BP causes a significant and dose-dependent improvement in the survival rate subjected to lethal doses of LPS (see Figure 7).

The activity of INH2BP abate the activation induced by LPS of MAP kinase, but does not alter the activation and nuclear translocation of NF-kB There are many intracellular processes which precede the induction if iNOS and the production of other inflammatory mediators. Activation of tyrosine kinases; Levitzki, A., 1994, "Signal-transduction therapy, A novel approach to disease management," Eur. J. Biochem. 226: 1-13; Novogrodsky et al., 1994, "Prevention of lipopolysaccharide-induced lethal toxicity by tyrosine kinase inhibitors," Science 264 (Wash): 1319-22; Marczin et al., 1993, "Tyrosine kinase inhibitors suppress endotoxin-and IL-beta-induced NO synthesis in aortic smooth muscle cells," Am. J. Physiol. 265: H1014-1018, the mitogen-activated protein kinase (MAP kinase), - Matsuda et al., 1994, "Signaling pathways mediated by the mitogen-activated protein (MAP) kinase kinase / MAP kinase cascade," ¡L. Leukocite Biol. 56: 548-53; L'Allemain, G., 1994, "Deciphering the MAP kinase pathway," Progr. Growth Factor Res. 5.:29l-334; Cowley et al., 1994, "Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and transformation of NIH 3T3 cells," Cells 77: 841-52; and the NF-kB pthway, - Baeuerle et al., 1994, "Function and activation of NF-kB in the immune system," Ann. Rev. Immunol. .12: 141-79; Scherck et al., 1992, "Nuclear factor kappa B: an oxidative stress-responsive transcription factor of eukariotic cells (a review)," Free Radical Res. Comm. 12: 221-37; Muller et al., 1993, "Nuclear factor kappa B, a mediator of lipopolysaccharide effects," Immunobiol. 187: 233-56; they are recognized as important factors in inflammatory mediators. Therefore, we investigated whether INH2BP affects the activation of MPA kinase and MF-kB in response to stimulation with LPS in order to elucidate the potential relationship of these pathways in the inhibitory effect by INH2BP of the inflammatory process. There is a significant baseline MAP kinase activity in unstimulated RAW 264.7 macrophages. Treatment with LPS (10 mg / ml, 24 hours) induces an approximately 2.5 fold increase in MAP kinase activity (FIG. 8), without altering the amount of MAP kinase immune-reactive content, as demonstrated by Western blot ( not shown). Pretreatment of the cells for three days with INH2BP (150 mM) suppresses the MAP kinase basal activity by approximately 50% and reduces the LPS-induced increase in MAP kinase (not shown). The MAP kinase basal activity is slightly suppressed by the MAP kinase kinase inhibitor; Pang et al., 1995, "Inhibition of MAP kinase kinase blocks the differentation of PC-12 cells induced by nerve growth factor," J. Biol. Chem. 270: 13585-8; PD 98059 (100 mM), and MAP kinase activation induced by LPS is also inhibited (see Figure 8). According to recent data in cardiac myocytes; Singh et al., 1996, "Regulation of cytoline-inducible nitric oxide synthesis in cardiac myocites and microvascular endothelial cells.," J. Biol Chem 271: 1111-1117; the production of nitrite induced by LPS is also suppressed by PD 98059 (in 53% at 100 mM, n = 3). Similar to recent observations in a range of monocytic cell lines; Baeuerle et al., 1994, "Function and activation of NF-B in the immune system," Ann. Rev.

Immunol. 12: 141-79; we found basal nuclear NF-kB (constitutive) in J774 and RAW 264.7 cells. Stimulation with LPS causes an increase in the nuclear translocation of NF-kB, and the inhibition of INH2BP does not affect the nuclear translocation of NF-kB in response to LPS (see Figure 9).

DISCUSSION Poly (ADP ribose) synthetase (pADPRT) is an ADP protein and polymerizing enzyme that is abundantly present in the nucleus, - Ueda et al. , 1985, "ADP-ribosylation," Ann. Rev. Biochem. 54: 73-100. The physiological function of pADPRT has been subject to much debate. In contrast to the original proposal, which states that pADPRT is a DNA repair enzyme, it is now clear that pADPRT is not directly involved in DNA repair; Lindahl et al. , 1995, "Post-translational modification of poly (ADP-ribose) polymerase induced by DNA strand breaks," Trends Biochem. Sci. 20: 405-411; and that cells from transgenic mice in which the pADPRT gene has been deleted have normal repair characteristics of AD ?; Buki et al, 1995, "Identification of domains of pol (ADP-ribose) polymerase for protein binding and self association" J. Biol. Chem. 220: 3370-3377. Under physiological conditions, pADPRT can bind to numerous cellular proteins and AD sites. and can exert pleyotropic cellular regulatory functions; Bauer et al. , 1995, "Modification of growth related enzymatic atic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-1,2-benzopyrone (I? H2BP)," Int. J. Oncol. .8: 239-252; Bauer et al, 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, to non-covalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 22 = 347-377, Buki et al. , 1995, "Identification of domains of poly (ADP-ribose) polymerase for protein binding and self association," J. Biol.

Chem. 270: 3370-3377. It has also been proposed that the activation of pADPRT serves as a mechanism to induce cell death, in particular after radiation damage and stresses by oxidants; Cochrane, 1991, "Mechanisms of oxidant injury of cells," Molec. Aspects Med. 12: 137-147, - Berger, 1991, "Oxidant-induced cytotoxicity: a challenge for metabolic modulation," Am. J. Respir. Cell. Biol. Biol. 4: 1-3. One of the important physiological functions of pADPRT can be the regulation of enzymatic induction, gene expression and cell differentiation; Bauer et al. , 1995, "Modification of growth related enzymatic pathways and apparent loss of tumonigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)," Int. J. Oncol . 8: 239-252; Bauer eü al. , 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, to non-covalently binding, ligand of poly (ADP-ribose) polymerase," Biochimie 77: 347-377; Minaga et al., 1978, "Induction of cardiac L-ornithine decarboxylase by nicotinamide and its regulation by putrescine," Eur. J. Biochem. 91: 577-85, -Griffin et al. , 1984, "The in vivo effect of benzamide and phenobarbital on the enzymes: poly (ADP-ribose) polymerase, cytochrome P-450, styrene oxide hydrolase, cholesterol oxide hydrolase, cholesterol oxide hydrolase, glutathione S-transferase and UDP-glucuronyl transferase , "Biochem. Biophys. Res. Comm. 122: 770-5. Induction of alkaline phosatase by NIH2B0; Bauer et al. , 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)," Int. J. Oncol. 8.239-252; it is a probable cause of inactivation of certain phosphorylation-dependent enzymes, for example MAP kinase topoisomerase I and topoisomerase II. INH2BP in bovine endothelial cells transfected with ha-ras suppresses tumorigenicity, inhibits cell multiplication, increases toposomerases I, toposomerase II and MAP kinase activity, has a down regulation of DNA-methyltransferase and protein kinase C and increases ODC phosphorylation of Rb protein and inhibits ras gene expression without loss of the oncogene, Bauer et al. , 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone INH2BP)," Int. J. Oncol. ¿239'-752; Bauer et al, 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, to non-covalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 77: 347-377. Based on the recently described anticancer actions of NIH2BP; Bauer et al, 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)," Int. J. Oncol. 8: 239-252; Bauer et al. , 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, to noncovalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 22 = 347-377; and the relationship between chronic inflammation and cancer, with special reference to the production of NO (see introduction), was investigated if INH2BP modulates the inflammatory response induced by LPS in vitro and in vivo. We found that several of the studied pathways and mediators (MAP kinase, prostaglandins, NO) are suppressed by INH2BP, while others (TNF, IL-6, NF kB) are not affected, or are increased (IL-10). Generally, the present invention data show that inhibitor compounds of pADPRT such as INH2BP exert anti-inflammatory actions, and the combination of these effects may underlie the improvement in survival rate in animals or mammals pretreated with this inhibitor of pADPRT.

EXAMPLE 2 INH2BP suppresses induction of iNOS induced by LPS By way of background, the inducible isoform of nitric oxide (NO) synthase (iNOS) is expressed in response to proinflammatory stimuli in many cells. The overproduction of NO by iNOS plays an important role in shock and inflammation; Nathan, 1992, "Nitric oxide as a secretory product of mammalian cells," FASEB J. 6: 3051-3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: master of the blood circulation," Proc. Rov. Soc. Lond B 343: 225-246; Szabo, C., -1995, "Alterations in the production of nitric oxide in various forms of circulatory shock," New-Horizons 3_: 3-32; and may predispose to carcinogenic transformation; Bartsch et al., 1994, "Endogenously formed N-nitroso compounds and nitrosating agents in human cancer etiology," Pharmacogenetics 2.-272-7; Liu et al. , 1992, "Woodchuck hepatitis virus surface antigen induces NO synthesis in hepatocytes: possible role in hepatocarcinogenesis.," Carcinogenesis 15-2875-7; Ohshima et al. , 1994, "Chronic infections and inflammatory processes as cancer risk factors: possible role of nitric oxide in carcinogenesis," Mutation Res. 305: 253-64. The promoter region of the murine iNOS gene has been cloned, and separate regions responsible for the induction capacity have been identified in response to LPS and IFN. Induction mediated by LPS if iNOS appears to involve the mobilization and nuclear translocation of NF-kB, with subsequent binding to the iNOS promoter. Induction of iNOS can also be inhibited by pharmacological inhibitors of tyrosine kinase and inactivation of NF-kB; Szabo, C; 1995, "Alterations in the production of nitric oxide in various-forms of circulatory shock," New Horizons 3: 3-32.

The inhibitory effect of INH2BP on the expression of iNOS is indicated by the inhibition of nitrite production, the expression of mRNA for iNOS and the expression of iNOS proteins. Regulation occurs at the early stage of iNOS production, since INH2BP gradually loses its effectiveness when applied at increasing times after stimulation by induction of iNOS. The regulation of INH2BP of induction of iNOS occurs both in vitro and in whole animals. In addition, our data show that the LPS-induced production of cyclooxygenase metabolites, similar to the induction of i? OS, is modulated by I? H2BP. The production of cyclooxygenase metabolites by proinflammatory cytokines is due to novel ARβ and protein synthesis, and the expression of COX-2 by a process which shares similarities with the induction process of i ?OS; Vane et al. , 1995, "? Ew insights into the mode of action of anti-inflammatory drugs," Inflamm. Res. 44: 1-10. The inhibition of LPS-induced expression of inflammatory mediators, however, is not non-specific "response to I? H2BP, since the induction of T? F by LPS is not affected by this agent in J774 cells. , the inhibitory effect of I? H2BP on i? OS is greatly reduced when LPS is used in combination with I? F for immunostimulation.This effect may be due to the fact that IF? - induced transcription factors such as interferon regulatory factor. Martin et al., 1994, "Role of interferon regulatory factor 1 in induction of nitric oxide synthase," J. Exp. Med. 180: 977-84, derivation of the inhibition of iNOS induction by the agents mentioned above. Previous in vitro studies have suggested that the induction of iNOS is modulated by pharmacological inhibitors of pADPRT in macrophages in vitro, Hauschildt et al., 1992, "Induction of nitric oxide synthase in L929 cells by your necrosis factor alpha is prev ented by inhibitors of poly (ADP-ribose) polymerase, "Biochem. J. 288: 255-260; Pellat-Seceunyk et al. , 1994, "Nicotinamide inhibits nitric oxide synthase mRNA induction in activated macrophages," Biochem. J. 297.-53-58. However, in these studies, the inhibitors of pADPRT 30 aminobenzamide and nicotinamide are used at high concentrations (10-30 mM), which inhibits total protein and RNA synthesis, and may have additional pharmacological actions, such as elimination of free radicals; Hauschildt et al. , 1992, "Induction of nitric oxide synthase in L929 cells by tumor-necrosis factor alpha is prevented by inhibitors of poly (ADP-ribose) polymerase," Biochem. J. 88.:255-260. The present experiments, using INH2BP, also suggest the pleyotropic relationship of pADPRT in the transcription process of the iRNA mRNA. In order to study i? OS promoter regulation by I? H2BP, transient transfection assays were performed using promoter constructs of murine macrophage iNOS-luciferase. These data with the deletional constructs indirectly suggest that INH2BP regulates a transcription event which involves the murine iNOS promoter region between -1592 and -367 bp. The ribosylation with ADP of the histones and nucleases can be involved in the maintenance of a reduced structure of chromatin; Bauer et al. , 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)," Int. J. Oncol . 8.239-252; Bauer et al. , 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, to non-covalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 22 = 347-377; Ueda et al. , 1985, "ADP-ribosylation." Ann. Rev. Biochem. 54: 73-100. Based on our previous experimental data, - Bauer et al. , 1995, "Modification of growth related enzymatie pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)," Int. J. Oncol . 8 ..- 239-252; Bauer et al. , 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1, 2-benzopyrone, to non-covalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 77: 347-377; it is reasonable to suggest that in these experimental systems, pADPRT inhibitor compounds, for example pretreatment with INH2BP, inhibits the ribosylation of ADP poliau omatica from pADPRT and histones. It is known that such action activates the conversion of relaxed chromatin to condensed, and, as an up-regulation of nucleases and other regulatory enzymes of the DNA structure; Bauer et al. , 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)," Int. J. Oncol . 8 .: 239-252; Bauer et al. 1995, "Reversal of malignant phenotype by 5-iodo-6-amino-1,2-benzopyrone, a non-covalently binding ligand of poly (ADP-ribose) polymerase," Biochimie 77: 347-377; It can affect the functions of the promoter.

EXAMPLE 3 Effect of inhibition of I? H2BP on the MPA kinase and activation of? F-kB The results have shown that treatment with INH2BP inhibits the activation of MAP kinase induced by LPS. In this respect, these data are similar to the findings with transformed endothelial cells. Bauer et al, 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH2BP)," Int. J. Oncol. 8: 239-252. It is likely that the inhibition of MAP kinase activation is produced by an activation of pleiotropic cell response by INH2BP. It has been shown that MAP kinase is activated in various types of cells treated with LPS or in various proinflammatory cytokines (T? F-alpha, interleukin-1, nerve growth factor), - Kyriakis et al, 1996, "Sounding the alarm: protein kinase cascades activated by stress and inflammation, "J. Biol Chem. 271: 24313-24316; Matsuda et al, 1994, "Signaling pathways mediated by the mitogen-activated protein (MAP) kinase kinase / MAP kinase cascade," J. Leukocyte Biol. 56 = 548-53; Cowley et al. , 1994, "Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and transformation of IH 3T3 cells.," Cells 77: 841-52; Pang the al. , 1995, "Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor," J. Biol. Chem. 270: 13585-8; Willis et al, 1996, "Differential induction of the mitigen activated protein kinase pathway by bacterial lipopolysaccharide in cultured monocytes and astrocytes," Biochem. J. 313: 519-524; Saklatvala et al, 1993, "Interleukin 1 and tumor necrosis factor-alpha activates the mitogen-activated protein (MAP) kinase kinase in cultured cells," FEBS Lett. 334 = 189-92. A variety of extracellular signals converge in the MPA kinase kinase / MAP kinase cascade through different MAP kinase kinase-kinases and induce a vice-specter of cellular responses; Kyriakis et al. , 1996, "Sounding the alarm: protein kinase cascades activated by stress and inflammation," J. Biol Chem. 271: 24313-24316; Ferrell, JE, 1996, "Tripping the switch fantastic: how to protein kinase cascade can convert graded inputs into switchlike outputs," TIBS 21: 460-466. Blockage of MPA kinase or MAP kinase kinase modifies a multitude of intracellular pathways and inhibits cell differentiation and proliferation; Kyriakis et al. , 1996, "Sounding the alarm: protein kinase cascades activated by stress and inflammation," J. Biol Chem. 271: 24313-24316; Matsuda et al, 1994, "Signaling pathways mediated by the mitogen-activated protein (MAP) kinase kinase / MAP kinase cascade," J. Leukocyte Biol. 56: 548-53; Cowley et al, 1994, "Activation of MAP kinase kinase is necessary and sufficient for PC12 differentiation and transformation of NIH 3T3 cells.," Cells 22 = 841-52; Pang et al. ,. 1995, "Inhibition of MAP kinase blocks the differentiation of PC-12 cells induced by nerve growth factor," J. Biol. Chem. 270: 13585-8; Willis et al, 1996, "Differential induction of the mitigen-activated protein kinase pathway by bacterial lipopolysaccharide in cultured monocytes and astrocytes," Biochem. J. 313: 519-524; Saklatvala et al, 1993, "Interleukin 1 and tumor necrosis factor-alpha activate the mitogen-activated protein (MAP) kinase kinase in cultured cells," FEBS Lett. 334: 189-92. Recently, the inhibition of MAP kinase kinase with PD 98059 has been shown to suppress the expression of mRNA for iNOS in cultured endothelial cells and in cardiac myocytes, - Singh et al. , 1996, "Regulation of cytoline-inducible nitric oxide synthesis in cardiac myocytes and microvascular endothelial cells.," J. Biol. Chem. 271: 111-117. This finding is consistent with our observation that PD 98059 causes a marked suppression of nitrite production by LPS in RAW macrophages. Since the activation of NF-kB is a major pathway in the inflammatory response, and is involved in the induction of iNOS by LPS, but not by INF; Szabo, C; 1995, "Alterations in the production of nitric oxide in various forms of circulatory shock," New Horizons 3: 3-32; Martin et al, 1994, "Role of interferon regulatory factor 1 in induction of nitric oxide synthase,". Exp. Med. 180: 977-84, we seek to investigate the potential effect of INH2BP on NF-kB. Our results demonstrate that INH2BP does not alter the nuclear translocation of NF-kB activation, or the modulation of cellular events mediated by NF-kB, by INH2BP, if found, that can occur at a cellular event distal to the nuclear translocation of NF-kB.EXAMPLE 4 Pathophysiological and therapeutic and therapeutic implications, INH2BP modulate the inflammatory process at multiple levels The reduction by inhibitors of pADPRT of the expression of genes and proinflammatories iNOS and COX-2, and the subsequent reduced formation of NO and prostaglandins can be beneficial in various forms of inflammation; Athan, 1992, "Nitric oxide as a secretory product of mammalian cells," FASEB J. 6: 3051-3064, - Vane, J.R., The Croonian Lecture 1993, "The endothelium: master of the blood circulation," Proc. Roy. Soc. Lond B 343-225-246; Szabo, C. , - 1995, "Alterations in the production of nitric oxide in various forms of circulatory shock," New Horizons 3: 3-32. Vane et al. , 1995, "New insights into the mode of action of anti-inflammatory drugs," Inflamm. Res. 44: 1-10. In addition, the improved release of IL-10 may have additional anti-inflammatory actions; Liles et al. , 1995, "Review: nomenclature and biologic significance of cytokines involved in inflammation and the host immune response," J. InfectDis. 172.-1573-80; Giroir, 1993, "Mediators of septic shock: new approaches to interrupting the endogenous inflammatory cascade," Critical Car. Med. 21: 780-9; Szabo et al. , 1997, "Isoproterenal regulates tumor necrosis factor, interleukin-10, interleukin-6 and nitric oxide production and protects against the development of vascular hyporeactivity in endotoxemia," Immunology £ 0: 95-100. It is conceivable that such effects contribute significantly to the improvement by the pADPRT inhibitor compounds, for example, the pretreatment with INH2BP and the survival rate of mice exposed with fatal doses of endotoxin. However, the establishment of the exact mechanisms by which I? H2BP exerts effects on the LPS-induced expression of the various inflammatory mediators requires further detailed investigations. On the one hand, it is conceivable that the activity of pADPRT or that the binding of the pADPRT protein is involved in the regulation of the production of inflammatory mediators and / or the expression of genes that code for components of the inflammatory process. On the other hand, there is likely to be an indirect down regulation of the activity of MPA kinase by I? H2BP; Bauer et al. , 1995. "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of the transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (I? H2BP)," Int. J. Oncol. 8: 239-252; it can also contribute to the observed effects, as predicted by other studies; Kyriakis et al. . 1996, "Sounding the alarm: protein kinase cascades activated by stress and inflammation," J. Biol Chem. 221 = 24313-24316; Ferrell, JE, 1996, "Tripping the switch fantastic: how to protein kinase cascade can convert graded inputs into switch-like outputs," TIBS .21: 460-466. The present results demonstrate the therapeutic potential of pADPRT inhibitor compounds such as INH2BP in various inflammatory diseases.

EXAMPLE 5 Some of the cytotoxic effects of nitric oxide (NO) are related to the production of peroxynitrite, a reactive oxidant that is formed by rapid reaction of NO and superoxide; Crow et al. , 1995, "The role of peroxynitrite in nitric oxide-mediated toxicity", Current Top Microbiol. Immunol, 196: 57-73. - Pryor et al, 1995, "The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide", Am. J. Phvsiol. L699-L772. Peroxynitrite formation has been demonstrated in various inflammatory conditions, including systemic inflammation induced by entoxin, - Szabo et al. , 1995, "Alterations in nitric oxide production in various forms of circulatory shock" New Horizons 3: 2-32; arthritis, - Kaur et al. , "Evidence for nitric oxide-mediated oxidative damage in chronic inflammation, Nitrotyrosine in serum and synovial fluid from rheumatoid patients", FEBS Lett. 1359: 9-12; and leg edema induced by carrageenan; * Salvemini el al. , 1996. In fact, from pharmacological studies using inhibitors of NO synthase (NOS) and mimics of superoxide dismutase, it is concluded that peroxynitrite plays an important pathogenic role in the development of an inflammatory process; Szabo C, 1996, "The role of peroxynitrite in the pathophysiology of shock, inflammation and schemia-reperfusion injury", Shock 6: 79-88; * Salvemini et al., 1996; * Zingarelli et al, 1997. In addition, it has been shown that some agents currently used in the treatment of arthritis are, in fact, eliminators of peroxynitrite, Whiteman et al., 1996"Protection againts peroxynitrite dependent tyrosine nitration and alpha 1-antiproteinase inactivation by some anti-inf1amatory drugs and by the antibio ic tetracycline "Annals. of the Rheumatic Diseases 55: 383-7. The knowledge that a significant part of the toxicity related to NO is due to the formation of peroxynitrite has required the development of novel therapeutic solutions based on the formation and action of peroxynitrite. One of the intracellular pathways activated by peroxynitrite is related to the breakdown of the single chain of AD? and the activation of poly (ADP-ribose) synthetase (PARS); Szabo et al., 1996, "The role of peroxynitrite in the pathophysiology of shock, inflammation and schemia-reperfusion injury", Shock 6: 79-88; "Szabo, 1996. The pronounced activation of PRAS can rapidly suppress the intracellular concentration of its substrate, NAD +, decreasing the rate of glycolysis, electron transport, and, therefore, the formation of ATP, resulting in cellular dysfunction.; * Berger, 1991; * Cochrane, 1991. Consequently, PARS inhibitors protect against cellular damage under these conditions.This mechanism, known as the "suicide hypothesis of PARS", has previously been characterized in relation to induced oxidant damage. by H202 and in radiation damage, - * Berger, 1991; * Cochrane, 1991; and recently has been implicated in cell damage related to NO and with peroxynitrite in endotoxic shock, attack, ischemia-reperfusion injury and diabetes mellitus; et al., 1996, "The role of peroxynitrite in the pathophysiology of shock, inflammation and schemia-reperfusion injury", Shock 6: 79-88; * Zhang et al., 1994, * Heller, et al., 1995. potential role of PARS in arthritis has recently been established by Kroger et al. In a model induced by potassium peroxochromate, treatment with nicotinamide causes a 25-35% reduction in the average arthritic rating; * Miesel et al., 1996. However, from that study, the mechanism of inhibition remains undefined, since a clear distinction can not be drawn between the free radical scavenging activity and the PARS inhibitory effect of the nicotinamide; * Miesel et al., 1995. In the present study, with the help of 5-iodo-6-amino-1, 2-benzopyrone (INH2BP), a potent novel inhibitor of PARS activity; * Bauer et al., 1995a, * Bauer et al., 1995b; We investigated the effect of pharmacological inhibition of PARS on the development of leg edema induced by carcinogen and collagen-induced arthritis. The results of our study support the concept that the inhibition of PARS is of anti-inflammatory potential.

EXAMPLE 6 Induction and evaluation of leg edema induced by carrageenan Wistar male rats (250-300 g, Charles River Laboratories, Wilmington, MA) were used in these studies. The animals received an injection under the plant of 0.1 ml of saline containing 1-carrageenan 1% in the right hind paw. This flogogenic agent is administered to animals treated with INH2BP or to animals treated with vehicle. Animals were treated with INH2BP (0.5 g / kg p.o.) -24 h and -2 h before injection of carrageenan. The paw volume is measured by phletismometry immediately after injection, as previously described, - * Sautebin, et al., 1995. Subsequent readings of the volume of the same paw are carried out at 60 minute intervals and they are compared with the initial readings. For these experiments, n = 6 animals treated with vehicle and n = 6 animals treated with INH2BP were used.

EXAMPLE 7 Induction and evaluation of collagen-induced arthritis For these studies, male DBA / 1J mice (9 weeks old, Jackson Laboratory, Bar Harbor, ME) were used. Chicken type II collagen (CII) is dissolved in 0.01 M acetic acid at a concentration of 2 mg / ml by shaking overnight at 4 ° C. The dissolved CII is frozen at -70 ° C until used. Freund's complete adjuvant (CFA) is prepared by the addition of Mycobacterium tuberculosis H37ra at a concentration of 2 mg / ml. Before the injection, CII is emulsified with an equal volume of CFA. Collagen-induced arthritis is induced as previously described, - Hughes et al., 1994, "Induction of T helper cell hyporesponsiveness in an experimental model of autoimmunity by using nonmitogenic anti-CD3 monoclonal antibody", J. Immunol. 153: 3319-3325. On day 1, mice are injected intradermally in the tail base with 100 ml of CII). On day 21, a second injection of CII into CFA is administered. The animals are treated with vehicle (n = 10) or with INH2BP (n = 60 (0.5 g / kg po) every 24 hours, starting from day 25. The mice are evaluated daily for arthritis using a macroscopic rating system that varies from 0 to 4 (1 - swelling and / or redness of the paw or finger, - 2 - two joints involved, 3 - more than two joints involved, 3 - more than two joints involved, and 4 = severe arthritis in all the paw and on the fingers.) The arthritic index for each mouse is calculated by adding the four grades of the individual legs.At the end of the experiments (day 35), the animals are sacrificed under anesthesia and the legs and knees are extirpated and are fixed for histological examination.The histological examination is performed by a researcher who does not know the treatment regimen.

Data analysis and presentation For paw edema studies induced by carrageenan, the paw volumes in the groups of treated and untreated animals were compared with the non-stop Student test. For arthritis studies, the Mann-Whitney U test (two-tailed, independent) was used to test statistical differences in arthritic indices. This non-parametric statistic is used to compare medians, instead of averages, because the measurement scale is ordinal, and the distribution values are not normally typically distributed, - Hughes et al., 1994, "Induction of T helper cell hyporesponsiveness in an experimental model of auto-immunity by using nonmitogenic anti-CD3 monoclonal antibody ", J. Immunol. 153: 3319-3325. The values in Figure 10 are expressed as mean ± standard error of the mean of n observations where n represents the number of rats (6 animals for each group). The values in figure 11 represent incidences (%), while the values in figure 12 represent medians. A p-value less than 0.05 is considered statistically significant (l '<0.05; ** p <0.02). materials -iodo-6-amino-1,2-benzopyrone (INH2BP) is prepared as previously described (* Bauer et al., 1995a, - * Bauer et al., 1995b). Chicken type II collagen is from Elastin Products Company, Inc. (Owensville, MO). Mycobacterium tuberculosis H37Ra is from Difco (Detroit, MI). All other chemicals are from Sigma Chemical Co. (St. Louis, MO). Injection under the carrageenan plant in the rat's paw leads to a time-dependent increase in paw volume with a maximum response at 3 h (Figure 10). This foot edema induced by carrageenan was significantly reduced by treatment with INH2BP (Figure 10).

In the model of collagen-induced arthritis in mice, between days 26-35 after the first immunization with collagen, animals progressively develop arthritis, as evidenced by an increase in the incidence of arthritis and an increase in arthritic qualification ( see Figures 11-12). Treatment with INH2BP induces the incidence of arthritis until day 33 and reduces the severity of the disease throughout the experimental period. On day 30, the arthritic rating increases to 10, while the medium arthritic ratings on the animals treated with INH2BP remain at approximately 5 (see Figure 12). On day 35, all animals treated with vehicle and most of the animals treated with INH2BP present a certain degree of arthritis (see Figure 11). However, on day 35, the median arthritic grades decreased significantly by treatment with INH2BP (see Figure 12). On day 35, the histological evaluation of the legs in arthritic animals treated with vehicle shows signs of severe suppurative arthritis, with mixed massive infiltration (neutrophils, macrophages and lymphocytes) in the larger joints of the ankle and in the terminal fingers. In addition, severe or moderate necrosis, hyperplasia and synovial detachment can be observed, along with the extension of inflammation to the adjacent musculature, with fibrosis and increased mucus production. In animals treated with INH2BP, the degree of arthritis is significantly reduced. However, there is still a significant degree of arthritis in these animals, with moderate infiltration, mainly of neutrophils in several of the larger joints, together with medium to moderate necrosis and synovial hyperplasia. Similar to these findings are in the leg, signs of severe suppurative arthritis which are found in the knee, which is reduced by treatment with I? H2BP (not shown).

Discussion Oxygen, peroxynitrite, oxyradicals, and inducible cyclooxygenase products have been independently proposed as important factors in the pathogenesis of various forms of inflammation, including arthritis (see introduction and also Brahn, 1991, "Animal models of rheumatoid arthritis.

Clues to etiology and treatment "Orthop Clinic Res. 265: 42-53.- Kaur et al., 1994," Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. ? itrotyrosine in serum and synovial fluid from rheumatoid patients. FEBS Lett. 1359: 9-12; Oyanagui Y, 1994, "? Itric oxide and superoxide radical are involved in both initiation and envelopment of adjuvant arthritis in rats" Life Sci. 54: PL285-9; Miesel et al., 1994, "Effects of allupironol on in vivo suppression of arthritis in mice and ex vivo modulation of phagocytic production of oxygen roots in whole human blood", Inflammation 6: 597-612; Whiteman et al., 1996"Protection against peroxynitrite dependent tyrosine nitration and alpha 1-antiproteinase inactivation by some anti-inflammatory drugs and by the antibiotic tetracycline" Annals. of the Rheumatic Diseases 55: 383-7; Anderson et al., 1996, "Selective inhibition of cyclooxygenase (COX) -2-reverses inflammation and expression of COX-2 and interleukin 6 in rat adjuvant arthritis", J. Clin. Invest. 97: 2672-2679. The present study, which demonstrates the antiinflammatory effects of INH2BP in the paw edema model induced by carrageenan and in the model of collagen-induced arthritis, supports the concept that PARS is involved in the progress of the inflammatory process and the pharmacological inhibition of PARS is of anti-inflammatory potential. The main mode of action of INH2BP is probably related to the interruption of the futile intracellular cascade characterized by DNA damage. The activation of PARS, the ribosylation of ADP and the suppression of NAD + and ATP in various cell types of inflamed joints. Inhibition of this pathway with various PARS inhibitors, such as 3-aminobenzamide, nicotinamide and INH2BP has been shown to protect different types of multiple cells from damage, -Berger, 1991, * Cochrane, 1991; Szabo et al., 1996, "The role of peroxynitrite in the pathophysiology of shock, inflammation and schemiareperfusion injury", Shock 6: 79-88; * Szabo, 1996b. The production of NO in inflammatory conditions is due to the suppression of the inducible isoform of NOS (iNOS), - Nathan, 1992, "Nitric oxide as a secretory product of mammalian cells", FASEB J. 6: 3051-3064; Szabo, 1995, "Alterations in nitric oxide production in various forms of circulatory shock", New Horizons 3: 2-32; Southan, et al., 1996, "Spontaneous rearrangement of aminoaklyguanidines into mercaptoalkylguadidines - a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform" Br. J. Pharmacol. 117: 619-632. Several lines of evidence suggest a role for OSI and the overproduction of βO in the pathogenesis of arthritis (see for reviews: Stenovic-Racic, et al., 1993, "Itric oxide and arthritis", Arthr. : 1036-1044, - * Evans et al., 1995. Firstly, the expression of i? OS and the production of large amounts of βO have been demonstrated in chondrocytes from experimental and human animals (Haeselmann et al., 1994 , "? itric oxide and proteoglycan synthesis by human articular chondrocytes in alginate culture", FEBS Lett. 352: 361-364, Sakurai et al., 1995, "? itric oxide production and inducible nitric oxide synthase expression in inflammatory arthritis", J Clin.Research 96: 2357-63; Grabowski et al., 1996, "? Ric oxide production in cells derived from the human joint", Br.

J. Rheumatol. 35: 207-12, - Murrell et al., 1996, "Nitric oxide: an important articulate free radical", J. Bone Joint Sur.-Am. 28: 265-74. Secondly, an increase in circulating concentrations of nitrite / nitrate (the products of NO decomposition) has been demonstrated in patients with arthritis (Farrell et al., 1992), "Increased concentrations of nitrite in synovial fluid and serum samples suggest increased nitric oxide synthesis in rheumatic diseases "Ann. Rhem. Dis. 51: 1219-22; Stichtenoth et al., 1995, "Urinary nitrate excretion is increased in patients with rheumatoid arthritis and reduced by predisolone", Ann. Rhem. Dis. 54: 820-4. Third, it has been shown that the development of arthritis is reduced in non-selective inhibitors of the NOS isoform (* Ialential et al, 1993; McCartney-Francis et al., 1993, Suppression of arthritis by an inhibitor of nitric oxide synthase). , J. Exp. Med. 178: 749-753; Weinberg et al., 1994, "The role of nitric oxide in the pathogenesis of spontaneous murine autoimmune disease, increased nitric oxide production and nitric oxide synthase expression in MRL-1 pr / l pr mice, and reduction of spontaneous glomerulonephritis and arthritis by orally administered NG-monomethyl-L-arginine ", J. Exp. Med. 1979: 651-60; Stefanovic-Racic et al., 1994," N-monomethyl arginine, an inhibitor of nitric oxide synthase, suppresses the development of adjuvant arthritis in rats "Arthr .. Rheumat 37: 1062-9: and more preferably, by inhibitors with iNOS selectivity; (Connor et al., 1995, "Suppression of adjuvant-induced arthritis by selective inhibition of inducible nitric oxide synthase," Eur. J. Pharmacol., 273: 15-24.) In this regard, it should be noted that pretreatment with types Multiple cellular with PARS inhibitors (including 3-aminobenzamide, nicotinamide as well as INH2BP) before immunostimulation, have been shown to suppress the expression of mRNA for iNOS and reduce NO production (* Hauschildt et al., 1991, * Pellat-Seceunyk et al., 1994; Zingarelli et al., 1996, "Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide" J. Immunol. 156: 350-358; * Szabo et al 1997. From these experimental data it can be concluded that the PARS pathway is not yet a characterized mechanism, and that it also regulates the expression process of iNOS, and that this effect may represent an additional mode of acc Beneficial ion of PARS inhibition in various forms of inflammation. However, caution should be exercised when interpreting previous findings. For example, in the in vitro studies established above, extremely high concentrations of PARS inhibitors, 3-aminobenzamide and nicotinamide (10-30 mM) are required in order to demonstrate suppression of iNOS induction. These high concentrations of these reagents may have additional pharmacological actions, such as total protein inhibition and RNA synthesis and / or free radical scavenging actions, * * Hauschildt et al., 1992, * Pellat-Seceunyk et al., 1994; Zingarelli et al., 1996, "Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide" J. Immunol. 156: 350-358. On the other hand, INH2BP effectively suppresses the expression of iNOS even at lower non-cytotoxic concentrations (100-300 mM). However, in the case of INH2BP, several modes of action must be considered, since this agent is an inducer of alkaline phosphatases, with secondary pleiotropic modulation of cellular responses, - * Bauer et al. nineteen ninety six; * Szabo et al., 1997). Experiments in cells or animals with deletion of the PARS gene are required to definitively resolve the question of whether PARS inhibition per se suppresses the process of iNOS induction. In a recent study by Ehrlich et al., it has been shown that in cultured rabbit synovial fibroblasts, the cytosine-induced expression of collagenase activity is suppressed by 3-aminobenzamide, - * Ehrlich et al., 1995. It is now possible to determine the pharmacological action (which, However, it would be expected to suppress the course of the arthritis process), the property of the particular inhibitor used, or if, in fact, it is related to a reduction in the catalytic activity of PARS. In this regard, it should be noted that, based on studies with pharmacological inhibitors, PARS has been implicated in the regulation of several genes, which include the gene of the major histocompatibility class II complex (* Hiromatsu et al, 1992, - Taniguchi et al., 1993), ras c-myc (* Bauer et al., 1996, * Nagao et al., 1991), the DNA methyltransferase gene (* Bauer et al, 1996) and protein kinase C (* Bauer et al., 1996). Taken together, the present work demonstrates a decrease in the development of the local inflammatory response and the inhibition of the progress of collagen-induced arthritis by I? H2BP. Although, during the last decade, a role for PARS as a repairman of AD has been proposed, recent observations show that suppression of the gene for PARS does not compromise the repair of AD?; Agénicos animals lacking the gene for PARS appear normal and viable (* Wang et al., 1995). This observation reinforces the anti-inflammatory potential of the pharmacological inhibitors of PARS. It is unlikely that the inhibition of PARS (as opposed to the inhibition of i? OS) interferes with the important antimicrobial effects of? O, since the invading microorganisms do not contain PARS. On the other hand, the inhibition of PARS is not only expected to inhibit part of the oxidant-induced cytotoxicity and thus may be more effective when applied in combination with other scavengers of free radicals or other immunosuppressive agents. The results of the present studies support the concept that the inhibition of PARS, by itself, or in combination with other anti-inflammatory agents, represents a promising novel anti-inflammatory solution. In a manner similar to that demonstrated in the previous examples, the compounds of formulas II and III are used to treat inflammation or inflammatory diseases, as well as in treatment of gram-negative and gram-positive infections. The specification described in the above is considered sufficient to enable a person skilled in the art to carry out the invention. In fact, various modifications of the modes described above for carrying out the invention, which will be obvious to a person familiar in the field of pharmaceutical formulation or related fields, are intended to be within the scope of the following claims. It is noted that in relation to this date, the best method known by the applicant to carry out the aforementioned invention is that which is clear from the present description of the invention.

Claims (9)

CLAIMS Having described the invention as above, the content of the following claims is claimed as property:

1. A method for treating inflammation or inflammatory diseases in an animal or mammal, the method is characterized in that it comprises the steps of administering an effective amount of a pADPRT inhibitor compound to the animal or mammal, wherein the pADPRT inhibitor compound is not aminonobenzamide.

2. The method according to claim 1, characterized in that the pADPRT inhibitor compound is selected from the group consisting of: a compound having the formula: (I) wherein Rlt R2, R3, R < , Rs and R6 are each selected from the group consisting of hydrogen, hydroxy, amino, alkoyl, alkoxy, cycloalkyl or phenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, and one of Rlf R2, R3, R4, RB and R6 is amino; a compound that has the formula: (III) wherein R x, R 2, R 3, R 4 and R 5 are each selected from the group consisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol optionally substituted with alkyl, alkoxy, hydroxy or halo, and one of R 1 t R2, R3, R4 and R5 is an amino moiety.

3. The method according to claim 2, characterized in that the compound ee is selected from the group consisting of: 6-amino-1, 2-benzopyrone, 5-amino-1 (2H) isoguinolinone, 7-amino-1 (2H) - isoquinolinone and 8-amino-1 (2H) isoquinolinone.

4. The method according to claim 1, characterized in that the compound is 5-iodo-6-amino-1,2-benzopyran.

5. A method for treating endotoxin symptoms induced by both gram-negative and gram-positive organisms in an animal or mammal, the method is characterized by comprising the steps of administering to a mammal or animal a therapeutically effective amount of a pADPRT inhibitor compound.

6. The method according to claim 5, characterized in that the compound is selected from the group consisting of: a compound having the structural formula (I) (ID (III) wherein R 1 t R 2, R 3, R 4, R 5 and R 6 are, independently from each other, selected from the group consisting of hydrogen, hydroxy, amino, nitroso, nitro, halogen, Cg alkyl, C 3 -C β alkoxy, cycloalkyl Cj-C7 and phenyl, and pharmaceutically acceptable salts thereof, wherein the last three of the six substituents of Rx, R2, R3, R4, RB and R6 are always hydrogen and at least one of the six substituents R1 t R2 , R3, R4, Rs and Rβ is an amino moiety.

7. The method according to claim 6, characterized by the compound having the structural formula:

8. The method according to claim 6, characterized in that R4 is amino.

9. The method according to claim 8, characterized in that the halogen is iodine.