CN1261278A - Methods for treating inflammatory diseases using padprt inhibitors - Google Patents

Abstract

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

Description

Method with pADPRT inhibitor for treating inflammation and inflammatory diseases

The present invention relates to treat the method for animal or interior inflammation of mammalian body and inflammatory diseases (comprising arthritis).The invention still further relates to treatment and suffer from the Grain-negative that causes because of lipopolysaccharide systemic infection or invasion and attack and the animal or the mammiferous method of Grain-positive endotoxin symptom.These methods relate to the pADPRT inhibition chemical compound that adopts the treatment effective dose.

Background of invention

Reported that pADPRT inhibition chemical compound can be used to treat cancer and viral infection.The example of these methods is in U.S. Patent No. 5,464,871; 5,473,074; 5,482,975; 5,484,951; Describe to some extent in 5,516,941 and 5,583,155, it is for referencial use that these patent contents are all included this paper in.

In the document of publishing, show 5-iodo-6-amino-1,2-benzopyrone (INH recently ₂BP) (a kind of new ribozyme polyadenous glycosides diphosphonic acid ribose polymerase (pADPRT) inhibitor) suppresses oncogenicity effect in the body in the endothelial cell line of Ha-ras transfection; People such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase, 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377.INH ₂The processing of BP also causes topoisomerase I and II and map kinase activity to change; People such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase, 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377.The effect that arrives has according to the observation proposed about INH ₂BP has the hypothesis of potential application in the treatment cancer; People such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase, 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377.

Malignancy and inflammatory process all have the activation of some cell signalling passage, for example map kinase; People such as Kyriakis, 1996, " warning: ", J.BiolChem.271:24313-24316 by stress and the activated protein kinase cascade reaction of inflammation; Ferrell, JE, 1996, " open fabulous switch: how the protein kinase cascade reaction can be transformed into fractionated input the output of similar switch " TIBS 21:460-466.Chronic inflammatory disease causes the oncogenic transformation effect usually, is for example proved in the enteric epithelium example; People such as Kawai, 1993, " lipopolysaccharide-induced inflammation strengthens the rat bladder carcinogenesis " Cancer Res.53:5172-5; People such as Rosin, 1994, " inflammation, chromosome instability and cancer: schistosomicide model " Caner Res.54 (the 7th increases version): 1929s-1933s; People such as Choi, 1994, " similarity of the colorectal carcinoma of Crohn disease and ulcerative colitis: the hint of carcinogenesis and prevention " Gut 35:950-4.According to the association between chronic inflammatory disease and the oncogenic transformation, the purpose of this research is investigation INH ₂Whether BP influences inflammatory process in vitro and in vivo.In our research, (endotoxin LPS) is induced generation to multiple short scorching mediator by bacteria lipopolysaccharide.Known LPS has induced a large amount of cell effects, and has triggered systemic inflammatory reaction.The inductive short scorching mediator of LPS comprises tumor necrosis factor (TNF), interleukin-1, IFN-, and the anti-inflammatory mediator comprises interleukin 10 (IL-10) and interleukin-13; People such as Deltenre, 1995, " gastric cancer: helicobacter pylori trace " Acta Gastroenterol Belg.58:193-200; Beutler, 1995, " TNF, immunity and inflammatory diseases: the lesson in past 10 years " J.Invest.Med.42:277-35; People such as Liles, 1995, " summary: the nomenclature and the biological meaning that participate in the cytokine of inflammation and host immune response " J.Infect.Dis.172:1573-80; Giroir, 1993, " mediator of septicopyemia shock: the method that interrupts endogenous inflammatory cascade reaction " Critical Car.Med.21:780-9.Owing to produced these inflammatory cytokines, LPS causes generation inflammatory free radical (free radical such as nitric oxide [NO] that free radical that oxygen is concentrated such as superoxides and nitrogen are concentrated) and prostaglandin; Nathan, 1992, " nitric oxide is as the secreting type product of mammalian cell " FASEB is J.6:3051-3064; Vane, J.R., The Croonian Lecture 1993, " endothelium: sanguimotor great master (maestro) " Proc.Roy.Soc.Lond B 343:225-246; Szabo, C.; 1995, " variation of NO production in the various forms of cyclical shocks " New Horizons 3:3-32.Produce NO during inflammation and be because the expression of the isotype (iNOS) of a kind of uniqueness of NO synzyme, releasing and activity of inflammatory cytokines then be construed to unique isotype of giving expression to cyclo-oxygenase (cyclo-oxygenase-2, COX-2); Nathan, 1992, " nitric oxide is as the secreting type product of mammalian cell " FASEB is J.6:3051-3064; Vane, J.R., The Croonian Lecture 1993, " endothelium: sanguimotor great master (maestro) " Proc.Roy.Soc.Lond B 343:225-246; Szabo, C.; 1995, " variation of NO production in the various forms of cyclical shocks " New Horizons 3:3-32.INOS, COX-2 and above-mentioned proinflammatory cytokine and free radical play an important role in the inductive inflammatory response of LPS; Nathan, 1992, " nitric oxide is as the secreting type product of mammalian cell " FASEB is J.6:3051-3064; Vane, J.R., The Croonian Lecture 1993, " endothelium: sanguimotor great master (maestro) " Proc.Roy.Soc.Lond B 343:225-246; Szabo, C.; 1995, " variation of NO production in the various forms of cyclical shocks " New Horizons 3:3-32.In addition, existing hint, NO (or its toxic by-products peroxynitrite salt (peroxynitrite)) is to cause inflammatory response to change into the crucial mediator of oncogenic process; People such as Bartsch, 1994, " endogenous forms in the human cancer etiology N-nitroso compound and nitrosylation reagent " Pharmacogenetics 2:272-7; People such as Liu, 1992, " synthetic NO in the alpine marmot hepatitis virus surface antigen inducing hepatocyte: possible effect in hepatocarcinoma forms " Carcinogenesis15:2875-7; People such as Ohshima, 1994, " as the chronic infection and the inflammatory process of the cancer risk factor: nitric oxide possible effect in carcinogenesis " Mutation Res.305:253-64.In present research, we have investigated INH first ₂Whether the processing of BP influences interior generation of body of inflammatory mediator tumor necrosis factor [TNF], interleukin 10, interleukin-6, NO and prostaglandin in the inductive inflammatory model of LPS.

There are many born of the same parents' internal procedures to produce short scorching mediator.The activation of tyrosine kinase; Levitzki, A., 1994, " signal transduction treatment.A kind of new method " Eur.J.Biochem.226:1-13 of treatment disease; People such as Novogrodeky, 1994, " preventing lipopolysaccharide-induced lethal toxicity " Science264U (Wash): 1319-22 by tyrosine kinase inhibitor; People such as Marczin, 1993, " cheese nitronic acid inhibitors of kinases prevents endotoxin and the beta induced NO of IL-1 in the large artery trunks smooth muscle cell to synthesize " Am.J.Physiol.265:H1014-1018; Mitogen activated protein kinase (map kinase); People such as Matsuda, 1994, " the signal conduction pathway of mitogen activated protein (MAP) kinase kinase/map kinase cascade reaction mediation " J.Leukocyte Biol.56:548-53; L ' Allemain, G., 1994, " decoding the map kinase passage " Progr.Growth Factor Res.5:291-334; People such as Cowley, 1994, " the kinase whose activation of map kinase is for PC12 differentiation and transform the NIH3T3 cell fully necessary " Cells 77:841-52; With nuclear factor κ B (NF-κ B) passage; People such as Baeuerle, 1994, " function of the NF-B in the immune system and activation " Ann.Rev.Immunol.12:141-79; People such as Schreck, 1992, " nuclear factor κ B: eukaryotic oxidation stress response transcription factor (summary) " Free Radical Res.Comm 17:221-37; People such as Muller, 1993, " nuclear factor κ B, a kind of mediator " Immunobiol.187:233-56 of lipopolysaccharide effect; They are considered to the important factor in the inflammatory response, and the expression or the generation of inflammatory mediator had effect.Therefore, we have investigated INH ₂Whether BP also influences inductive map kinase of LPS and NF-kB activation.Present result of study shows, INH ₂BP has by regulating the potent antiphlogistic effects that LPS induces a plurality of components of inflammatory response.

Summary of the invention

One aspect of the present invention relates to a kind of method for the treatment of animal or interior inflammation of mammalian body or inflammatory diseases, and this method comprises the step of the pADPRT inhibition chemical compound that gives described animal or mammal effective dose.

The present invention is a kind of method for the treatment of animal or interior inflammation of mammalian body or inflammatory diseases on the other hand, and this method comprises the step of the pADPRT inhibition chemical compound that gives effective dose, and wherein pADPRT inhibition chemical compound is selected from the chemical compound with following formula:

R wherein ₁, R ₂, R ₃, R ₄, R ₅And R ₆Respectively be selected from hydrogen, hydroxyl, amino, alkyl, alkoxyl, cycloalkyl or phenol, these groups can be replaced R arbitrarily by alkyl, alkoxyl, hydroxyl or halogen ₁, R ₂, R ₃, R ₄, R ₅And R ₆In have only one to be amino; Chemical compound with following formula: R wherein ₁, R ₂, R ₃, R ₄And R ₅Be selected from hydrogen, hydroxyl, amino, alkyl, alkoxyl, cycloalkyl or phenol separately, these groups can be replaced R arbitrarily by alkyl, alkoxyl, hydroxyl or halogen ₁, R ₂, R ₃, R ₄, R ₅In have only one to be amino; With the chemical compound with following formula: R wherein ₁, R ₂, R ₃, R ₄And R ₅Be selected from hydrogen, hydroxyl, amino, alkyl, alkoxyl, cycloalkyl or phenol separately, these groups can be replaced R arbitrarily by alkyl, alkoxyl, hydroxyl or halogen ₁, R ₂, R ₃, R ₄And R ₅In have only one to be amino.

Preferable pADPRT chemical compound comprises: 6-amino-1,2-benzopyrone, 3-nitrosobenzene Methanamide, 5-amino-1 (2H)-isoquinolines, 7-amino-1 (2H)-isoquinolines and 8-amino-1 (2H)-isoquinolines.

A further aspect of the invention comprises a kind of method for the treatment of animal or interior Grain-negative of mammalian body and the inductive symptom of Grain-positive, and described method comprises the step of the pADPRT inhibition chemical compound that gives animal or mammal treatment effective dose.

A further aspect of the invention is Grain-negative and the inductive endotoxin symptom of gram positive bacteria in a kind of treatment animal or the mammalian body, this method comprises the pADPRT inhibition chemical compound that gives animal or mammal treatment effective dose, and wherein said chemical compound is selected from above-mentioned Compound I, Compound I I or compound III.

A further aspect of the present invention relates to Grain-negative and the inductive endotoxin symptom of Grain-positive in a kind of treatment animal or the mammalian body, this method comprises the pADPRT inhibition chemical compound that gives animal or mammal treatment effective dose, and wherein said chemical compound has the structural formula shown in above-claimed cpd I, Compound I I or the compound III.

A further aspect of the present invention is a kind of method for the treatment of animal or mammalian body intrinsic articulation inflammation, this method comprises the pADPRT inhibition chemical compound that gives effective dose, and wherein said chemical compound has the structural formula shown in above-claimed cpd I, Compound I I or the compound III.

A further aspect of the present invention is a kind of method for the treatment of Chron disease in animal or the mammalian body, and this method comprises the pADPRT inhibition chemical compound that gives effective dose, and wherein said chemical compound has the structural formula shown in above-claimed cpd I, II or the III.

A further aspect of the present invention is a kind of method for the treatment of Barrett disease in animal or the mammalian body, and this method comprises the pADPRT inhibition chemical compound that gives effective dose, and wherein said chemical compound has the structural formula shown in above-claimed cpd I, II or the III.

PADPRT inhibition chemical compound of the present invention can be used U.S. Patent No. 5,464,871; 5,473,074; 5,482,975; 5,484,951; The method of describing in 5,516,941 and 5,583,155 makes, and it is for referencial use that these patent disclosures are included this paper in.

The preferable chemical compound that is used for the inventive method comprises that halogen group is that iodine, a R group are that amino, a R group can be those chemical compounds of nitroso-group or nitro (as described in above-mentioned patent), but preferably the R group is amino.In addition, have been found that when the iodo part is partly adjoined with amino the active strong demonstration of the inhibition of pADPRT.Under any circumstance, the chemical compound that is used for the inventive method should have pADPRT and suppresses active.

Chemical compound can use separately, or preferably is used in combination with pharmaceutically-acceptable acid addition known in the art or other suitable pharmaceutical carriers.

The accompanying drawing summary

Fig. 1.INH ₂BP is to the generation of LPS in the J774 cell inductive (a) nitrite, the generation of (b) 6-ketone PGF1, generation and (d) influence of preventing of mitochondrial respiratory effect of (c) TNF.TNF is 4 hours mensuration behind LPS, and other all parameters are 24 hours mensuration behind LPS. ^*(comparing with contrast (p＜0.01)) replied LPS and had significant change in representative; ## represents INH ₂BP has remarkable effect (comparing (p＜0.01) with LPS with single) in the presence of LPS; The n=6-12 hole.

Fig. 2.INH ₂The iNOS that BP suppresses in J774 and RAW 264.7 cells expresses.(a) (swimming lane 1), LPS handle back 4 hours (swimming lanes 2) and INH are arranged under collating condition ₂The representational Northern trace of iNOS and 18s mRNA in the cell 4 hours (swimming lane 3) after LPS handles that BP (100 μ M) exists, J774 cell (A) and RAW 264.7 macrophages (B).(b) at collating condition (C and C+INH ₂BP) and LPS handle back 12 hours (LPS and LPS+INH ₂BP), INH ₂BP is to the active influence of iNOS of J774 cell homogenates thing. ^*Represent the remarkable effect (p＜0.01) of LPS compared with the control the time; ## represents INH ₂BP has significant inhibition (p＜0.01); N=4.(c) J774 control cells and LPS handle and had or do not exist INH in back 12 hours ₂Representational iNOS Western trace in the cell of BP.

Fig. 3.(a) give LPS preceding 2 hours, give LPS simultaneously or after giving LPS 2,4 and 6 hours the time, INH ₂BP (100 μ M) suppresses the time-dependent loss of nitrite accumulation.(b) INH ₂BP is to the influence through the J774 cell Central Asia of LPS and IFN combination of stimulation nitrates accumulation; The n=6-12 hole.

Fig. 4.INH ₂BP induces the influence of luciferase activity to LPS in the RAW264.7 cell, and this cell is with total length (about 1592bp) or deletion form (about 367bp) iNOS promoter-luciferase construction transient transfection.In total length or deletion form construction (secret note) cells transfected, the processing of LPS (10 mcg/ml, 4 hours) cause inducing of luciferase activity be control value 10-12 doubly.And INH ₂The common processing of BP has suppressed the increase of the luciferase activity of LPS mediation in total length construction cells transfected, but is not having remarkable influence (grey bar) in the deletion form construction cells transfected of about 367bp.Tables of data is shown as the multiple that luciferase activity increases than control cells, and is corrected into β glucosidase activity separately. ^*Representative is INH when LPS exists ₂BP has significant effect (comparing (p＜0.05) with LPS with single); The transfection that separates for n=4 time.

Fig. 5.INH ₂BP prevents inducing of iNOS in the conscious rat.At preceding 10 minutes (INH of LPS ₂BP+LPS) 2 hours (LPS+INH or behind the LPS ₂BP) time, control rats (c), injected INH ₂Rat (the INH of BP ₂BP), the lung homogenate thing (a) in the rat of LPS (15 mg/kg, intraperitoneal, 6 hours) and the iNOS activity of blood plasma nitrite-nitrate concentrate (b) have been injected; And use INH ₂BP handles the effect of (10 mg/kg, intraperitoneal). ^*The significant compared with the control LPS effect (p＜0.01) of representative; ## represents the remarkable inhibition (p＜0.01) of pADPRT inhibitor; N=4-5.

Fig. 6.Give LPS (4 mg/kg, intraperitoneal) in the time of back 90 minutes, INH ₂The influence that BP replys the inductive TNF of LPS in the mice, IL-10 and IL-6.The significant compared with the control LPS effect (p＜0.01) of ## representative; ## represents INH ₂BP is to replying significant potentiation (p＜0.01); N=4-5.

Fig. 7.INH ₂BP has improved the survival of the mice of standing endotoxin shock: INH ₂BP pretreatment (0.3-10 mg/kg) is to the influence of (120 mg/kg, intraperitoneal) lethal of endotaxin induction in the mice body; Every group has 7-8 animal.

Fig. 8.(a) at 100 μ M PD 98059 or 150 μ M INH ₂BP exists or does not exist down, and carrier or LPS (10 mcg/ml) handle the map kinase activity in the back 24 hours RAW264.7 cell.The numerical value that obtains in the data represented type testing: three different test days have been seen similar result.(b) at 150 μ MINH ₂BP exists or does not exist down, and carrier or LPS handle the interior map kinase test of typical gel in the back 24 hours RAW264.7 cell.Swimming lane 1-4 represents following group respectively: 1: the contrast that vehicle treated is crossed; The 2:LPS processed group; 3:150 μ M INH ₂Vehicle treated group under BP exists; 4:150 μ M INH ₂LPS processed group under BP exists.

Fig. 9.Use INH ₂BP suppresses the nuclear translocation that pADPRT does not change the NF-κ B Western trace of nuclear extract, and described nuclear extract is from contrast J74 cell and at INH ₂There is or does not exist 90 minutes cell after LPS handles down in BP (100 μ M).

Figure 10.INH has been described ₂The inductive beastly pawl edema of BP on Carrageenan (paw edema) Influence and Development.Data show 1-4 hour beastly corpus unguis long-pending (meansigma methods ± SEM, every group has 6 animals) behind the injection carrageenin.From 1 hour, beastly corpus unguis is long-pending remarkable increase (p＜0.01), and INH ₂BP has suppressed the development of beastly pawl edema significantly at 1-4 hour.

Figure 11.INH has been described ₂BP is to the influence of collagen-induced arthritic beginning.There is shown the percent of suffering from the arthritis mice (arthritis clinical score greater than 1 mice).The arrow of locating in 21 days is represented the time of collagen immunization for the second time, represents INH from the 25th day horizontal bar ₂BP (N=6) or carrier (N-10) are handled the time of beginning.

Figure 12.INH has been described ₂BP is to the influence of collagen-induced arthritic seriousness.Arthritis score intermediate value during the collagen-induced arthritis.The arrow of locating in 21 days was represented the time of collagen immunization for the second time, from 25 days horizontal bar representative INH ₂BP (n-6) or carrier (n=10) are handled the time of beginning.From the 26th day, arthritis score obviously increased (Ip＜0.01), INH between 26-35 days ₂BP has significant resistance inhibitor action (#p＜0.05) to arthritis score.

Preferred embodiments of the present invention is described

Definition

As used herein,

" anti-inflammatory " disease refers to that bodily tissue has disease or the illness of inflammation. These diseases for example comprise: Chron Disease, Barrett disease, arthritis, multiple sclerosis, cardiomyopathy, colitis, infectious meningitis, brain Inflammation etc.

" pharmaceutically acceptable acid-addition salts " referred to keep those salt of biological effect and free alkali character, it Can by and inorganic acid (example hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, salicylic acid etc.) instead Should obtain.

" ADPRT " refers to adenosine diphosphate ribose transferase, is also referred to as polyadenous glycosides diphosphonic acid ribose polymerase (EC 2.4.99), it is a kind of eucaryote specific DNA syncaryon albumen of catalysis ADP-ribose polymerisation. Enzymatic processes depends on DNA.

" alkyl " refers to saturated or undersaturated, side chain or straight-chain alkyl. Typical alkyl comprise methyl, ethyl, Propyl group, isopropyl, butyl, isobutyl group, the tert-butyl group, amyl group, hexyl etc.

" alkoxyl " refers to group-O-alkyl. Typical case's alkoxyl is methoxyl group, ethyoxyl, propoxyl group, fourth oxygen Base and amoxy etc.

" cycloalkyl " refers to the saturated monocycle alkyl that contains 3-8 carbon atom, for example cyclopropyl, cyclobutyl, Cyclopenta, cyclohexyl, suberyl, ring octyl group etc.

" phenyl of replacement " refers to all possible isomery phenyl, as with being selected from alkyl, alkoxyl, hydroxyl or halogen Plain substituting group list replaces or disubstituted phenyl.

" halogen " refers to chloro, fluoro, bromo or iodo, preferably iodo.

PADPRT inhibition compound of the present invention (especially above-mentioned compound I, II or III) be potent, Single-minded and atoxic anti-inflammatory compound, it can be used for usually known is the illness of inflammation and disease (joint for example Inflammation, Chron disease, Barrett disease etc.). In addition, these compounds can be used to treatment and Grain-negative and leather The relevant illness of infection that the blue positive is induced especially infects relevant disease with Grain-negative, comprises with fat many The sugar disease disease relevant with pyemia. This compound especially is suitable for, even because it is toxic, and toxicity Also very low.

In practice, compound of the present invention or its pharmaceutically acceptable salt can be enough to suppress animal or lactation The amount of inflammatory disease or illness and/or prevention of inflammation or inflammatory disease gives in the animal body, and can be used for the suitableeest The medicament forms that is used for these purposes.

Reactive compound as herein described and salt can come administration by the therapeutic agent administering mode of accepting.These methods comprise general or topical, for example oral, parenteral, percutaneous (transdermal), subcutaneous or local topical administration mode.The preferable medication of these medicines is oral.In some instances, need give compositions with other parenteral form.

According to the administering mode that is adopted, compositions can be solid, semisolid or liquid dosage form, for example be injectable agent, tablet, suppository, pill, the time release capsule, powder, liquid, suspension etc., preferably unit dosage forms.Compositions can comprise active pADPRT inhibition chemical compound or its pharmaceutically acceptable salt of effective dose, it also can comprise the drug excipient of any routine and other medical science or medicine pharmaceutically or preparation, carrier, adjuvant, diluent etc. in addition, and these all are pharmaceutically conventional.

For solid composite, can adopt these excipient, comprise mannitol, lactose, starch, magnesium stearate, saccharin sodium, Talcum, cellulose, glucose, sucrose, magnesium carbonate of pharmaceutical grade etc.For example utilizing, polyglycols (as propylene glycol) also can be mixed with suppository with above-mentioned active pADPRT inhibition chemical compound as carrier.

Liquid, especially Injectable composition for example can make like this: reactive compound is dissolved, is dispersed in the medical solution (for example water, saline, aqueous glucose, glycerol, ethanol etc.), thereby form injectable solution or suspension.

If desired, treat that the administered agents compositions also can contain a small amount of avirulent auxiliary substance (as wetting agent or emulsifying agent, pH buffer agent), and other material (for example sodium acetate, triethanolamine oleate etc.).

In addition, if desired, treat that the administered agents compositions can contain the liposome prescription, the chemical compound that it comprises phospholipid, electronegative phospholipid and is selected from cholesterol, cholesterol fatty acid ester or unsaturated fatty acid.Typical neutral phospholipid comprises L-a-phosphatidylcholine, L-a-phosphatidylinositols, L-a-phosphatidyl-serine, L-a-phosphatidylinositols, L-a-phosphatidic acid, L-a-phosphatidyl glycerol, L-a-LYSO-PHOSPHATIDYLCHOLINE LYSOPC, sphingomyelins and cardiolipin.

Typical electronegative phospholipid comprises two acyl phosphoric acid or di(2-ethylhexyl)phosphate glyceride, for example two lauroyl phosphoric acid, two myristoyl phosphoric acid, DPPA, distearyl (disteroyl) phosphoric acid.

Typical cholesterol and cholesterol ethers comprise cholesterol, 3S-hydroxyl-5-cholestene, polyoxy ethane cholesterol sebacate, cholesterol-5,6-epoxide, cholesteryl acetate, cholesterol n-butyl ether, cholesterol decanoin, cholesterol dodecylate, cholesterol ether, cholesterol margarate, cholesterol methyl ester.

Typical unsaturated fatty acid comprises arachidonic acid, docosane acid, elaidic acid, erucic acid, linoleic acid, linolenic acid, nervonic acid, oleic acid, palmitoleic acid, petroselic acid.The exercise question that nitrohalogen compound can be submitted to according on February 19th, 1993 is the patent application No.08/020 of " liposome prescription and preparation method thereof and use ", method in 035 is sealed or is assigned in the liposome bilayer of liposome prescription, and it is for referencial use that this patent is included this paper in.

In first example, at first form liposome, add C-amino, nitroso-group or nitro compound then.C-amino, nitroso-group or nitro compound are dispensed into the double-layer of lipoid of (even himself enters) (rather than sealing) liposome.In order to prepare said composition, normally component (for example phosphatidylcholine, diacetyl phosphoric acid and cholesterol) and solvent (as chloroform) are mixed.Remove chloroform after the mixing.Add entry then.After water was added liposome, it had formed multilamellar liposome (be liposome with have multiwalled onion epidermis similar).Next step is that it is carried out cold melting.Be placed on cooling rapidly in the liquid nitrogen.Rapidly the purpose of freeze thawing is the size homogeneous more that makes liposome.This moment, the size of liposome was different, handle one or many, handled usually 5 times again.Be melted in 37 ℃ of water-baths and carry out.Before freeze thawing, the ultrasonication mixture.The combination of ultrasonication and thawing has reduced the quantity of cortex.Purpose is to produce monolayer (unilamellar) system.At this moment, the concentration that adds C-nitroso compound to 10 mM.This concentration can surpass 15 mMs.With regard to this concentration of lipid, for 60 milliliters of batches, the lipid total concentration is 648 milligrams, wherein adds 60 ml waters.Phosphatidylcholine is 500 milligrams, and cholesterol is 36 milligrams; Diacetyl phosphoric acid is 112 grams.

Liposome concentration can make it contain more C-amino, nitroso-group or nitro compound in the increase mixture.For example, it can be condensed into the twice of concentration in the said mixture.For 60 milliliters of batches, quantity is factored two to contain 1000 milligrams of phosphatidylcholines, 224 milligrams of diacetyl phosphoric acid and 72 milligrams of cholesterol.Reduce the amount that concentration has then reduced the C-nitroso compound that enters wherein.For 60 milliliters of batches of supposition, the upper limit of C-amino-compound reaches the C-amino-compound concentration of 15 mMs.For 3-nitrosobenzene Methanamide, be limited to 135 milligrams (60 milliliters of batches) on this.

Next step is hydration again.Then, next step of this process is that (British Columbia Canada) extrudes for LipexBiomembranes, Inc.Vancouver with extruder.

Extrusion has two purposes: the big or small homogeneous that 1) makes liposome; With 2) sterilization.

Extrude and generally include: filter the filter by the j0.1 micron, freeze drying mixt normally then is with mixture lyophilizing (therefrom remove and anhydrate, make it become fine powder).This has improved dissolubility, can set up the solution of about 40 mMs, has concentrated about 3 times before this concentration ratio lyophilization.Lyophilization has produced the mixture of Powdered lipid and Powdered C-amino-compound.Now, make more spissated mixture with regard to the C-amino-compound of available same amount and the lipid of less amount.For example, C-amino, nitroso-group or the nitro compound of available identical weight, but volume has only 1/3rd of original volume.

Also can improve, for example remove such as cryodesiccated step to the step of said process.

This method of first example is not sealed C-amino, nitroso-group or nitro compound significantly.Be not to be that the centre of liposome has chemical compound, but chemical compound this in film.The C-amino, nitroso-group or the nitro compound that are assigned in the liposome membrane will migrate to target cell, and lipid can carry C-amino, nitroso-group or nitro compound and enters cell membrane.

Preferably, this process has been made diameter and has been approximately 0.05-0.45 micron, the better liposome that is about the 0.1-0.2 micron.The single or multiple lift liposome is effective.

Second purpose extruding is that mixture is carried out degerming.For degerming, liposome is made into diameter usually less than 45 microns.Less than 0.05 micron size is active in theory.The method of first example has following advantage, and for example in water, 3NOBA only has the concentration of 0.5 mM.Liposome composition of the present invention has reached the concentration of 15 mMs.

In addition, with only the 3-NOBA in aqueous solution is different, the liposome solutions that contains NOBA has resistant function to ascorbic acid.This makes it can be used for the laboratory mouse test.Solution can contain NOBA monomer or NOBA dimer.

In second example, can make this thin film and pharmaceutical aqueous solution hydration from the thin film of liquid component.So just formed the lipid of catching (sealing) medicine automatically.The chemical compound that can not penetrate liposome membrane can this thing happens.An example of these chemical compounds is U.S. Patent No.s 5,262, those chemical compounds in 564 (submissions on November 16th, 1993), for example the L-cystine sulfinic acid adduct of 3-NOBA.

That the administration of parenteral injectable is generally used for is subcutaneous, intramuscular or intravenous injection and inculcate.The injectable agent can be made into conventionally form, is dissolved in the solid form in the liquid as liquid solution, suspension or before being adapted at injecting.

According to U.S. Patent No. 3,710,795, a kind of parenteral method of invention recently adopts implants a kind of slow release or sustained release system, and this system can guarantee to keep constant dosage level, and it is for referencial use that this patent is included this paper in.

Above-mentioned any pharmaceutical composition can contain 0.1-99%, preferable for the active pADPRT inhibition chemical compound of 1-70% (the especially halo of formula I, II or III-C-amino, nitroso-group or nitro compound) as active component.

Known chronic inflammatory disease can promote the carcinogenecity in the various tissues to transform.A kind of new ribozyme gathers inhibitor (5-iodo-6-amino-1, the 2-benzopyrone (INH of (ADP) ribose polymerase (pADPRT) ₂BP)) be in the news recently the endothelial cell line that can regulate the Ha-ras transfection various cell signalling passages and remove tumorigenicity in the body.One aspect of the present invention has proved that pADPRT inhibition chemical compound is (as INH ₂BP) endotoxin (bacteria lipopolysaccharide (LPS)) is produced in vitro and in vivo the effect of the activation of inflammatory mediator tumor necrosis factor (TNF), interleukin 10 (IL-10) and interleukin (IL-6), nitric oxide (NO) and prostaglandin.In addition, the present invention has shown that also pADPRT inhibition chemical compound is (as INH ₂BP) in the influence of external activation to activated protein kinase of mitosis (map kinase) and nuclear factor κ B (NF-κ B).In J774 that cultivates and RAW264.7 macrophage, LPS induces and produces the prostaglandin metabolism thing, discharges TNF and gives expression to inductible NO-synthase isotype (iNOS).The generation of prostaglandin and NO is by INH ₂BP suppresses in dosage dependence mode, and the release of the short-term of TNF-α is unaffected.In the RAW cell of total length (about 1592bp) mouse macrophage iNOS promoter-luciferase construction transient transfection, INH ₂BP has prevented the luciferase activity of LPS mediation significantly, but does not but prevent luciferase activity in the disappearance construction cells transfected that about 367bp forms.In vivo, INH ₂The pretreatment of BP has suppressed the inducing action of the interior LPS of rat body to iNOS, does not influence inductive TNF of LPS and IL-6 and replys, and produces but strengthened the inductive IL-10 of LPS.INH ₂The pretreatment of BP has significantly improved the mice survival rate in the deadly model of endotoxin shock.These results prove that pADPRT inhibition chemical compound is (as INH ₂BP) has potent antiinflammatory action in vitro and in vivo.

Polyadenous glycosides diphosphonic acid ribose synzyme (PARS) is a kind of by the activated ribozyme of dna single chain interruption.The unworthy circulation that a large amount of PARS activation energies that respond the extensibility dna single chain interruption of hydrogen peroxide, peroxynitrite or ionization radiation induction and produce cause power consumption reaches cell injury.Verified recently, in various forms of inflammation (comprising the inductive beastly pawl edema of arthritis and carrageenin), there is peroxynitrite to produce.The present invention has shown that new, potent PARS inhibitor, pADPRT inhibition chemical compound are (as 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP)) effect in inductive beastly pawl edema rat model of 1-4 hour carrageenin and collagen-induced beastly pawl edema mouse model.At the 1st day and the 21st day injection II Collagen Type VI twice, to induce collagen-induced arthritis in the male DMA/1J mice.INH ₂BP oral medication mice (gram/kilogram every day 0.5) has postponed the development of arthritis clinical symptoms from arthritis outbreak (the 25th day) between 26-35 days.Through INH ₂The animal that BP handled shows the arthritis index and reduces (arthritis score is with the 20-50% that marks seen in the mice of vehicle treated), and has improved histology's situation (measuring in knee joint and foot).These digital proofs, PARS inhibitor INH ₂BP shows antiphlogistic effects in vivo, even give INH ₂The zero-time of BP is later relatively, and it also can postpone collagen-induced arthritic process.Data of the present invention have been supported such viewpoint: i.e. PARS activation is played a role in the arthritis development, and may work in the inflammation of other form and inflammatory diseases.

The following example is used for describing the present invention.These embodiment have been not to be construed as limiting scope of the present invention.

Embodiment 1

Cell culture

Described as following these documents, mouse macrophage J774 and RAW264.7 are cultivated in the improved Eagle culture medium of Dulbecco (DMEM); People such as Szabo, 1996, " macrophage that the activation of DNA chain interruption, polyadenous glycosides diphosphonic acid ribose synzyme and cell energy consumption have participated in contacted nitrite and the cytotoxicity in the smooth muscle cell " Proc.Natl.Acad.Sci.USA.93:1753-1758; People such as Zingarelli, 1996, " in the macrophage that bacteria lipopolysaccharide stimulates, the DNA chain interruption of peroxynitrite mediation has activated polyadenous glycosides diphosphonic acid ribose synzyme and has caused cell energy consumption " J.Immunol.156:350-358.In the research that separates, obtain the intraperitoneal macrophage from male Wistar rat, exist or do not exist and be with or without INH at LPS ₂External cultivation is 24 hours under the BP.Put to death rat, take out intraperitoneal macrophage and cultivation in DMEM.INH at various concentration (1-150mM) ₂BP or other pharmacological inhibitor exist or do not exist down, with Escherichia coli LPS (10 mg/ml) or LPS and INF (50 μ/milliliter) processing cell several.

The map kinase related assays

With PBS washing Raw cell, to collect, per 1,000,000 cells are with 100 milliliters of lysis buffer cracking.(50mMTris-HCl, pH7.4,1%NP-40,0.4M NaCl, 0.1mM NaVO ₃, 50mM KF, 1mM EGTA, 2mM PMSF, 25nM Ao Kadi acid (okadaic acid), presses down each 1 mg/ml of enzyme peptide, amastatin and protease inhibitor at leupeptin).Cracking on ice 20 minutes, centrifugal 14 minutes of 13000rpm in the Eppendorf centrifuge then.Keep supernatant, measure protein content with Bio-Rad dyeing algoscopy.

Map kinase test in the gel

In the 100%SDS-PAGE gel that contains immobilization fringe phospholipid basic protein (MBP, 250 mg/ml gels), protein sample (50 milligrams/swimming lane) is carried out electrophoresis.Behind the electrophoresis, use 50mM TRIS-HCl pH7.7 buffer detergent gel once (25 milliliters, 20 minutes), cultivated twice 30 minutes with the same buffer that contains 25% isopropyl alcohol then.Use Tris-HCl buffer detergent gel then once, and at 50 mM Tris-HClpH7.7, the mM 2 mercapto ethanol soaked 1 hour in the solution of 5M guanidine hydrochloride (50 milliliters), changed the solution of cultivating in the time of 30 minutes.Then in 16 hours changing the 50mM TRIS-HCl pH7.7 of liquid 5 times, cultivate gel in the solution of Mm 2 mercapto ethanol, 0.04%NP-40, cultivate albumen again.Detergent gel twice then, with containing 50mM TRIS-HCl pH7.7,5mM MgCl ₂The solution of 7mm 2 mercapto ethanol is cultivated half an hour in advance.Adding 10mm at last ³²Cultivated 1 hour in the same solution of p-g ATP (50mCi/ test).Cultivating at last, removing unconjugated radioactivity, dry also autoradiography with the 10% acetic acid washing of 3 * 25 milliliters 10%TCA and 3 * 25 milliliters; People such as Sasaki, 1995, " ceramide is to the effect of being received of the cell proliferation of growth factor-induced " Biochem.J.311:829-34.

Map kinase Western trace.

To the 10%SDS-PAGE gel, electrophoresis is transferred on the nitrocellulose filter and carries out immunity and survey with 100 milligrams of cell extract albumen application of samples.First antibody (anti-map kinase) is available from UBI, and second antibody is an alkali phosphatase enzyme mark, available from NEN Biolabs.Detect to adopt and strengthen chemoluminescence method; People such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to lose " Int.J.Oncol.8:239-252.

The preparation of nuclear extract and NF-κ B Western trace.

At INH ₂BP handled cell 90 minutes under existing and not existing.The method described in Anal.Biochem.213:162-7 prepares micro-nuclear extract " to measure the enhanced gel mobility shift assay of dna binding factor " by people such as Hassanain 1993.In brief, scrape cell, centrifugal simply, precipitation is resuspended in 400 milliliters of cold buffer A [Hepes pH7.9 (10mM), KCl (10mM), EDTA (0.1mM), EGTA (0.1mM), DTT (1mM), PMSF (0.5mM), pepstatin A (1 mg/ml), leupeptin (10 mg/ml) and but enzyme peptide (10 mg/ml)] in, in the presence of 25 milliliters of 1%NP-40, placed 15 minutes on ice.Then, the vibration sample, centrifugal 1 minute of 10000g, precipitation is resuspended in 100 milliliters of buffer B [HepespH7.9 (20mM), NaCl (400mM), EDTA (1mM), EGTA (1mM), DTT (1mM), PMSF (0.5mM), pepstatin A (mg/ml), leupeptin (10 mg/ml) and but enzyme peptide (10 mg/ml)] in.On the cage chair 4 ℃ the vibration 15 minutes after, 4 ℃ 15 100, the centrifugal sample of 000g 15 minutes.Handle 70 milliliters of equal portions with 150 milliliters of SDS-PAGE sample buffers then.(CA) (1: 750, join with tween TBS (0.02%)) the Western trace carries out for Santa Cruz Biotechnology, Santa Cruz with the anti-mice NF-of rabbit κ B first antibody as mentioned above.

The mensuration of nitrite or nitrite/nitrate concentration

Stimulate the back 24 hours nitrites in the culture supernatant with following method mensuration; People such as Szabo, 1996, " macrophage that the activation of DNA chain interruption, polyadenous glycosides diphosphonic acid ribose synzyme and cell energy consumption have participated in contacted nitrite and the cytotoxicity in the smooth muscle cell " Proc.Natl.Acad.Sci.USA.93:1753-1758; People such as Zingarelli, 1996, " the DNA chain interruption of peroxynitrite mediation has activated polyadenous glycosides diphosphonic acid ribose synzyme and caused cell energy consumption in the macrophage that bacteria lipopolysaccharide stimulates " J.Immunol.156:350-358; People such as Szabo, 1994, " spermine suppresses NO production in the immunostimulating J774.2 macrophage: the demand of serum factor " Br.J.Pharmacol.112:355-356; In 100 ml sample media, add 100 milliliters of Griess reagent (1% sulfanilamide and 0.1% naphthodiamide, 5% phosphorylated ligand).(Molecular Devices, Sunnyvale CA) measure optical density (OD under the 550nm with Spectramax 250 microplate reader ₅₅₀).In order to measure the total concentration of the nitrite/nitrate in the plasma sample, by nitrate reduction being become nitrite with the nitrate reduction enzyme incubating; People such as Zingarelli, 1996, " in the macrophage that bacteria lipopolysaccharide stimulates, the DNA chain interruption of peroxynitrite mediation has activated polyadenous glycosides diphosphonic acid ribose synzyme and has caused cell energy consumption " J.Immunol.156:350-358.

Measure 6-ketone prostaglandin F ₁₂

Measure LPS with the specificity radioimmunoassay and stimulate 6-ketone prostaglandin F in back 4 hours 100 ml cells culture supernatant samples ₁₂Generation; People such as Szabo, 1994, " spermine suppresses NO production in the immunostimulating J774.2 macrophage: the demand of serum factor " Br.J.Pharmacol.112:355-356.

The mensuration of cytokine

With the cytokine levels in ELISA mensuration blood plasma and the cells and supernatant.Use (Endogen Inc., Boston, ELISA kit measurement IL-10 MA) and the blood plasma level of IL-6 available from Endogen.As people such as Szabo; 1997; described in " isoproterenol regulates tumor necrosis factor, interleukin 10, interleukin-6 and NO production and protection prevents from the vascular reaction deficiency takes place in the endotoxemia " Immunology 90:95-100 like that; use (Genzyme Corp. available from Genzyme; Boston, ELISA kit measurement blood plasma MA) and the TNF-α concentration in the cells and supernatant.

The mensuration of mitochondrial respiratory

Rely on mitochondrion with 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl Thiazolyl blue tetrazolium bromide is reduced into the Jia Za, measures 24 hours mitochondrial respiratory; People such as Szabo, 1996, " macrophage that the activation of DNA chain interruption, polyadenous glycosides diphosphonic acid ribose synzyme and cell energy consumption have participated in contacted nitrite and the cytotoxicity in the smooth muscle cell " Proc.Natl.Acad.Sci.USA.93:1753-1758; People such as Zingarelli, 1996, " in the macrophage that bacteria lipopolysaccharide stimulates, the DNA chain interruption of peroxynitrite mediation has activated polyadenous glycosides diphosphonic acid ribose synzyme and has caused cell energy consumption " J.Immunol.156:350-358.

The Northern trace of iNOS mRNA

At INH ₂BP makes cells contacting LPS after 4 hours under existing or not existing, and uses the TRIZOL extracted total RNA as mentioned above.Make the equal portions that contain 15 milligrams of total RNA contain electrophoresis on 1% agarose gel of 3% formaldehyde.Transfer to the RNA trace on the nylon membrane and carry out the UV self-crosslinking.As people such as Lowenstein, 1993, " the macrophage nitrous oxide synthetase gene: two upstream regions are by interferon gamma and lipopolysaccharide mediate inducing action " Proc.Natl.Acad.Sci.USA 90:9730-9734 is described like that, with guiding method (Pharmacia at random, Piscataway, NJ) make film and [ ³²P] (than living is 3000Ci/mM to dCTP; NEN) the Mus iNOS cDNA probe (10 of labelling ⁶.cpm/ml) 42 ℃ of hybridization are spent the night.The filter membrane of hybridization in 53 ℃ with 2X sodium citrate, sodium chloride, 0.1%SDS and 25mM NAHPO ₄, 1mM EDTA, 0.1%SDS solution continuous washing.After having surveyed iNOS, peel off film with ebullient 5mM EDTA, and and [ ³²P] oligonucleotide probe of radiolabeled 18S ribosomal RNA (as house-keeping gene) hybridizes again.After the washing, spend the night with the exposure of Phosphor Imager screen.

INOS Western trace.

Handled cell 20 hours in the existence of pADPRT inhibitor with not with LPS.Then cell is scraped among the cold PBS centrifugal 30 seconds of 14000g.Remove supernatant, the lysis buffer that add and contain RIPA (500 milliliters), presses down enzyme peptide (10 mg/ml) and PMSF (0.5mM).Make sample pass through 22 gauge needle (gaugeneedle) thus avoid DNA.Measure protein content with Bradford method (Bio-Rad).Cytoplasmic protein (200 milligrams/swimming lane) is added in the SDS-PAGE buffer, seethed with excitement 5 minutes, separate, transfer on the nitrocellulose filter (0.2mm) by the Semi-Dry method with the isotachophoresis buffer system with 7.5%SDS-PAGE.In 3% gelatin after sealing 1 hour and the washing subsequently, make sample at tween Tris buffer salt solution (TTBS) and 0.1% gelatin anti-mice iNOS (the upstate Biotechnology of first rabbit that neutralizes, Lake Placid NY) (is assigned in 0.0%TTBS) immunoblotting 2.5 hours at 1: 1000.With the anti-rabbit iGG of the goat of coupling alkali phosphatase antibody as second antibody.Make conjugated protein colour developing with the nitroblue tetrazolium in the carbonate buffer solution/5-bromo-4-chloro-indole phosphoric acid (NBT/BCIP).

The active mensuration of iNOS

Handled cell 12 hours in the existence of pADPRT inhibitor with not with LPS.As people such as Szabo, 1994, " spermine suppresses NO production in the immunostimulating J774.2 macrophage: the demand of serum factor " Br.J.Pharmacol.112:355-356 is described like that, and the measurement result that the calcium independent form from the 1-arginine to the L-citrulline in J774 cell homogenates or the lung homogenate is changed is as the active index of iNOS.Scrape cell or lung is placed the HCl by 50mM Tris, 0.1mM EDTA is in the homogenate buffer that 0.1mM EGTA and 1mM phenylmethylsulfonyl fluoride (pH7.4) are formed, with Tissue Tearor 985-370 homogenizer (Biospec Products, Racine, WI) homogenate in buffer on ice.Measure then in the homogenate [ ³H]-the L-arginine to [ ³H]-transformation of L-citrulline.[ ³H]-there are following 22 ℃ of cultivation homogenate (30 milliliters) 20 minutes in L-arginine (10mM, 5kBq/ pipe), NADPH (1mM), calmodulin, CaM (30nM), Tetrahydrobiopterin (5mM) and EGTA (5mM).Come cessation reaction with ice-cold HEPES buffer (pH5.5) dilution that contains EGTA (2mM) and EDTA (2mM).Reactant mixture is applied on Dowex 50W (Na+ type) post, with scintillation counting technique measure eluting [ ³H]-L-citrulline activity.

The function test of iNOS promoter

Because under our experimental condition, the J774 cell has resistant function for the transient transfection with calcium phosphate, lipofection and lipofection amine method, so on RAW 264.7 cells, carry out transfection research.With reporter gene construction transient transfection AW264.7 cell, to estimate the iNOS promoter activity.This construction is inserted into 5 ' mouse macrophage iNOS promoter region the upstream of reporter gene luciferase; People such as Lowenstein, 1993, " the macrophage nitrous oxide synthetase gene: two upstream regions are by interferon gamma and lipopolysaccharide mediate inducing action " Proc.Natl.Acad.Sci.USA 90:9730-9734; (by Dr.Charles J.Lowenstein, JohnsHopkins University gifts).Adopt two kinds of constructions: total length promoter construction (1-1592bp) and the deletion form construction of forming by 367bp.Seed cells in 6 well culture plates to 50% and be paved with, (Lipofectin is Giboco) with the iNOS promoter separately-luciferase construction transfection of equimolar amounts to utilize cationic-liposome.In order to control the difference of transfection efficiency, cell pSV40-b-tilactase cotransfection.After the transfection, cellular-restoring is spent the night, list adds INH with medium (contrast), LPS (10 mg/ml) or LPS subsequently ₂BP (100mM) handles.Handle after 4 hours, wash cell once with PBS, cracking in reporter lysis buffer (Promega), and analyze luciferase activity, the increase multiple of proofreading and correct primary separately galactosidase activity and expressing control cells (list medium transfection and processing).

Experiment in the body

Male Wistar rat and male BALB/c mouse available from Charles River Laboratories (Wilmington, MA or Budapest, Hungary).Animal is arbitrarily accepted food and water, and keeps illumination in 12 hours periods., put to death after 6 hours injecting in the rat peritoneum with Escherichia coli LPS (15 mg/kg).Get plasma sample and measure nitrite/nitrate, get lung sample determination iNOS.Other group rat is used INH ₂BP (10 mg/kg, peritoneal injection) handles, and handles preceding 10 minutes of LPS or behind lps injection and carries out in 2 hours.

In the research of measuring the inductive cytokine response of LPS, to injectable drug carrier or INH in the mouse peritoneum ₂BP (10 mg/kg), per 10 gram body weight volumes are 0.1 milliliter.Attack with 4 mg/kg LPS peritoneal injections after half an hour.LPS handles and put to death animal in back 90 minutes, collects blood in the ice-cold Eppendorf pipe that contains EDTA, and 4 ℃ centrifugal 10 minutes.Blood plasma-7 ℃ preservation is extremely measured.

In mice survival research, at 0 o'clock to animal peritoneal injection LPS (120 mg/kg) and the survival in 42 hours after detecting LPS.Other group mice is handled at LPS and accepted carrier or INH in preceding 18 hours, preceding 4 hours, 0 hour, back 6 hours, back 24 hours and back 30 hours ₂BP handles (0.1-10 mg/kg, intraperitoneal).

Material

DMEM, RPM1, TRIZOL and hyclone available from Gibco (Grand Island, NY).[ ³H] NAD+ and [ ³²P] NAD+ available from DuPont NEN (Boston, MA).Ethanol dehydrogenase and ND+ available from BoehringerMannheim (Indianapolis, IN).PD 98059 available from Cal biochem (La Jolla, CA).Other all medicines available from Sigma (St.Louis, MO).

Statistical evaluation

All numerical tabulars are shown as the meansigma methods ± standard error of mean (SEM) of n observation (n＞4) in the figure neutralization literary composition.With the meansigma methods between the not paired t-test in this figure Dent comparable group.Be lower than 0.05 p value and be considered to have statistical significance.

The result

INH ₂BP suppresses the inductive nitric oxide of LPS and prostaglandin in the J774 macrophage but does not suppress the generation of TNF-α

INH ₂The processing of BP causes dose dependent ground to suppress in the J774 macrophage the inductive nitrite of LPS forming that (Fig. 1 a).Similarly, INH ₂BP suppresses the inductive 6-ketone of LPS prostaglandin F ₁₂Generation (Fig. 1 b), but do not suppress the generation (Fig. 1 c) of TNF, and recovered the inductive inhibition of LPS (Fig. 1 d) to the mitochondrial respiratory effect.INH ₂BP has obviously suppressed iNOS mRNA and protein expression (Fig. 2 a-c).When reagent when a few hours give behind LPS, INH ₂BP is greatly reduced the inhibitory action that nitrite produces, and this is opposite with situation before inducing stimulation at iNOS, and (Fig. 3 a).And, when LPS and IFN-(TNF-g 50u/ml) are united when being used for immunostimulation INH ₂BP significantly reduces (Fig. 3 b) to the inhibition effect of iNOS.

Selectivity is regulated INH ₂BP is to the inducing action of iNOS promoter

In order further to study INH ₂BP is to the regulating action of iNOS promoter, and we carry out instantaneous measurement with mouse macrophage iNOS promoter-luciferase construction.We find the transcriptional regulatory of the LPS mediation of mouse macrophage iNOS play an important role (evidence that induces 10-12 luciferase activity doubly from LPS is (Fig. 4) as can be seen), this (people such as Lowenstein that conforms to former result, 1993, " the macrophage nitrous oxide synthetase gene: two upstream regions are by interferon gamma and lipopolysaccharide mediate inducing action " Proc.Natl.Acad.Sci.USA 90:9730-9734).Use INH ₂BP handles jointly through total length (to 1592bp) promoter construction cells transfected, and the result has suppressed the luciferase activity (Fig. 4) of LPS mediation fully.Yet, use INH similarly ₂The common luciferase activity (Fig. 4) that does not but have obviously to influence the LPS-mediation through deletion form construction (to 367bp) cells transfected of handling of BP.

INH ₂Antiphlogistic effects in the body of BP

INH ₂The pretreatment of BP has obviously reduced the active increase of iNOS (Fig. 5) in the increase of blood plasma nitrite-nitrate in the LPS inductive conscious rat body and the lung.When reagent is when LPS stimulates back a few hours just to add cell or give animal, INH ₂BP significantly reduces the inhibition effect that NO produces.With cell transformed be similar, use 100mM INH ₂The generation (n=4) of nitrite through the primary cell (peritoneal macrophages that obtains from rat) that LPS external (10 mg/ml) stimulates that BP has handled obviously minimizing (56 ± 7%, p＜0.01).

With in vitro results similar (Fig. 1 c), INH ₂BP does not have obviously to influence the increase of the inductive Plasma TNF levels of LPS in the mice, and (Fig. 6 a).INH ₂BP does not influence the generation (Fig. 6 C) of the inductive IL-6 of LPS yet.Yet, INH ₂BP causes the inductive IL-10 blood plasma of LPS to reply increase (Fig. 6 b).

INH ₂BP to the pretreatment of mice make be subjected to the LPS fatal dose survival of rats significantly, dose dependent ground improves (Fig. 7).

INH ₂The BP activity has been eliminated the inductive map kinase of LPS and has been activated, but does not change activation and the nuclear translocation of NF-κ B

Many born of the same parents' internal procedures are arranged before inducing of iNOS produces with other inflammatory mediator.The activation of tyrosine kinase; Levitzki, A., 1994, " signal transduction treatment.A kind of new method " Eur.J.Biochem.226:1-13 of treatment disease; People such as Novogrodsky, 1994, " preventing lipopolysaccharide-induced lethal toxicity " Science 264U (Wash): 1319-22 by tyrosine kinase inhibitor; People such as Marczin, 1993, " tyrosine kinase inhibitor prevents endotoxin and the beta induced NO of IL-1 in the large artery trunks smooth muscle cell to synthesize " Am.J.Physiol.265:H1014-1018; Mitogen activated protein kinase (map kinase); People such as Matsuda, 1994, " the signal conduction pathway of mitogen activated protein (MAP) kinase kinase/map kinase cascade reaction mediation " J.Leukocyte Biol.56:548-53; L ' Allemain, G., 1994, " decoding the map kinase passage " Progr.Growth Factor Res.5:291-334; People such as Cowley, 1994, " the kinase whose activation of map kinase is for PC12 differentiation and transform the NIH 3T3 cell fully necessary " Cells 77:841-52; With NF-κ B passage; People such as Baeuerle, 1994, " function and the activation of the NF-κ B in the immune system " Ann.Rev.Immunol.12:141-79; People such as Schreck, 1992, " nuclear factor κ B: eukaryotic oxidation stress response transcription factor (summary) " Free Radical Res.Comm 17:221-37; People such as Muller, 1993, " nuclear factor κ B, a kind of mediator " Immunobiol.187:233-56 of lipopolysaccharide effect; These all are considered to the important factor in the inflammatory mediator.Therefore, we investigate INH ₂Whether BP influences the activation of MPA kinases and NF-κ B response LPS stimulation, and these passages participate in INH in the inflammatory process so that set forth ₂The probability of the depression effect of BP.

In the RAW264.7 macrophage that does not stimulate, there is significant map kinase basis active.LPS handles (10 mg/ml, 24 hours) and induces the active increase of map kinase about 2.5 times (Fig. 8), but does not influence the amount (proving that by the Western trace result does not show) of immunoreactivity map kinase.Use INH ₂BP (150mM) pretreatment cell 3 angel's map kinases basis activity is subjected to about 50% inhibition, and has eliminated the increase (not shown) of the inductive map kinase of LPS.The map kinase basis is active is prevented slightly by the map kinase inhibitors of kinases; People such as Pang, 1995, " the inductive PC-12 cell differentiation of nerve growth factor has been blocked in the kinase whose inhibition of map kinase " J.Biol.Chem.270:13585-8; The activation of PD 98059 (100mM) and the inductive map kinase of LPS also is suppressed (Fig. 8).The generation of the inductive nitrite of LPS is also prevented by PD 98059 and (is prevented 53% under 100mM, n=3), this conforms to nearest data in myocardial cell (people such as Singh, 1996, " the synthetic adjusting of the derivable nitric oxide of cytokine in myocardial cell and the capillary endothelium " J.Biol.Chem.271:1111-1117).

With the nearest observed result in a series of monocytic seriess (people such as Baeuerle, 1994, " function of the NF-B in the immune system and activation " Ann.Rev.Immunol.12:141-79) similar, we find to have in J774 cell and the RAW264.7 cell basic (composing type) nuclear NF-κ B.The LPS stimulation causes NF-κ B nuclear translocation to increase, and INH ₂The inhibition of BP does not influence the nuclear translocation (Fig. 9) of NF-κ B response LPS.

Discuss

Polyadenous glycosides diphosphonic acid ribose synzyme (pADPRT) is abundant modified protein in the nucleus and the enzyme of polymerization ADP of being present in; People such as Ueda, 1985, " ADP ribosylation " Ann.Rev.Biochem.54:73-100.The physiological function of pADPRT has become the theme of many debates.Opposite with initial suggestion (claiming that pADPRT is a kind of DNA repairase), now known that pADPRT does not participate in the reparation of DNA directly; People such as Lindahl, 1995, " post translational modification of the inductive polyadenous glycosides of DNA chain interruption diphosphonic acid ribose polymerase " TrendsBiochem.Sci.20:405-411; The transgenic mice cell that the pADPRT gene has been broken away from has normal DNA and repairs feature; People such as Buki, 1995, " being used for the evaluation of protein binding and self associating polyadenous glycosides diphosphonic acid ribose polymerase domain " J.Biol.Chem.270:3370-3377.Under the physiological condition, pADPRT can be in conjunction with many cell proteins and DNA site, and can bring into play pleiotropy cell regulatory function; People such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase, 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377; People such as Buki, 1995, " being used for the evaluation of protein binding and self associating polyadenous glycosides diphosphonic acid ribose polymerase domain " J.Biol.Chem.270:3370-3377.PADPRT activates also and is pointed out to work in inducing cell death (especially behind radiation damage and oxidation stress); Cochrane, 1991, " mechanism of cellular oxidation damage " Molec.Aspects Med.12:137-147; Berger, 1991, " the inductive cytotoxicity of oxidant: the attack that metabolism is regulated " Am.J.Respir.Cell.Biol.Biol.4:1-3.Inducing action, gene expression and cell differentiation that the important physical sexual function of pARPRT may be a regulatory enzyme; People such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase, 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377; People such as Minaga, 1978, " inducing heart L-ODC Ornithine decarboxylase and adjusted " Eur.J.Biochem.91:577-85 with putrescine with nicotiamide; People such as Griffin, 1984, " Benzoylamide and phenobarbital to liver enzyme-polyadenous glycosides diphosphonic acid ribose polymerase, cytochrome P-450, benzene ethylene oxide hydrolytic enzyme, cholesterol oxide hydrolytic enzyme, glutathione S-transferase and UDP glucuronyl transferase-vivo effect: " Biochem.Biophys.Res.Comm.122:770-5.INH ₂BP to the inducing action of alkali phosphatase (people such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252) be the possible cause of some phosphorylation dependent form enzyme (as map kinase topoisomerase I and topoisomerase II) deactivation.INH in the cattle endotheliocyte of Ha-ras transfection ₂BP has eliminated oncogenicity effect, cellular replication is stopped, topoisomerase I, topoisomerase II and map kinase activity have been improved, reduced dnmt rna and Protein kinase C, and ODC to have increased the proteic phosphorylation of Rb very few, and suppressed the ras expression of gene and do not lost the expression of oncogene, people such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase, 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377.

INH according to nearest description ₂The antitumaous effect of BP (people such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase; 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377) and the contact (especially NO generation aspect) between chronic inflammatory disease and the cancer (see: foreword), we have investigated INH here ₂Whether BP regulates the inductive inflammatory reaction of LPS in vitro and in vivo.We find, have (map kinase, prostaglandin, NO) to be subjected to INH in passage of being studied and the mediator ₂BP prevents, and other (TNF, IL-6, NF-κ B) are uninfluenced, or increases (IL-10) to some extent.In a word, data of the present invention show that pADPRT inhibition chemical compound is (as INH ₂BP) brought into play antiinflammatory action, and the combination of these effects may be the reason that increases through the pretreated animal dis motility rate of this pARPRT inhibitor.

Embodiment 2

INH ₂BP prevents LPS to induce generation iNOS

Know that by background technology the short inflammation of induced isotype (iNOS) response of nitric oxide in various cells (NO) synzyme stimulates and expresses.INOS excessively produce NO the shock and inflammation in play an important role (Nathan, 1992, " nitric oxide is as the secreting type product of mammalian cell " FASEB is J.6:3051-3064; Vane, J.R., The Croonian Lecture 1993, " endothelium: sanguimotor great master (maestro) " Proc.Roy.Soc.Lond B 343:225-246; Szabo, C.; 1995, " variation of NO production in the various forms of cyclical shocks " New Horizons 3:3-32), and may tend to carcinogenecity and transform (people such as Bartsch, 1994, " endogenous forms in the human cancer etiology N-nitroso compound and nitrosylation reagent " Pharmacogenetics 2:272-7; People such as Liu, 1992, " synthetic NO in the alpine marmot hepatitis virus surface antigen inducing hepatocyte: possible effect in hepatocarcinoma forms " Carcinogenesis 15:2875-7; People such as Ohshima, 1994, " as the chronic infection and the inflammatory process of the cancer risk factor: nitric oxide possible effect in carcinogenesis " Mutation Res.305:253-64.Clone now the promoter region of Mus iNOS, and identified the zone that separates of the inducibility of being responsible for replying LPS and IFN.It seems that the inducing action of the iNOS of LPS mediation activity and the nuclear translocation that has participated in NF-κ B combine subsequently with the iNOS promoter.Inducing of iNOS suppressed by tyrosine kinase and the activated medicine inhibitor of NF-κ B also can; Szabo, C.; 1995, " variation of NO production in the various forms of cyclical shocks " New Horizons 3:3-32.

INH ₂The inhibition effect that BP expresses iNOS shows and suppresses nitrite generation, iNOS mRNA expression and iNOS protein expression aspect.Regulating action occurs in the early stage of iNOS inducing action, because along with the after-applied time of the stimulation of inducing iNOS increases INH ₂The effectiveness of BP is lost gradually.INH ₂BP betides in the external and whole animal body the inductive regulating action of iNOS.In addition, our data show that inducing of the generation of the inductive cyclo-oxygenase metabolite of LPS and iNOS is similar, is to be subjected to INH ₂BP regulates.It is owing to induce the process of process to synthesize new mRNA and albumen and express COX-2 by similar with iNOS that pro-inflammatory cytokine produces the cyclo-oxygenase metabolite; People such as Vane, 1995, " to newly seeing clearly of antiinflammatory drug effect mode " Inflamm.Res.44:1-10.Yet the inhibitory action that the inductive inflammatory mediator of LPS is expressed is not to INH ₂Nonspecific reaction of BP is not because LPS is subjected to this agents influence to the inducing action of TNF in the J774 cell.

Interestedly be, when LPS combines when being used for immunostimulation INH with INF ₂BP significantly reduces the inhibition effect of iNOS.This effect may be since the inductive transcription factor of IFN (as interferon regulatory factor, people such as Martin, 1994, " effect of interferon regulatory factor I in inducing nitric oxide synthetase " J.Exp.Med.180:977-84) got around mentioned reagent to the inductive inhibitory action of iNOS.

In vitro study in the past hints that in macrophage, the medicine inhibitor that is subjected to pADPRT of inducing of iNOS is regulated external; People such as Hauschildt, 1992, " tumor necrosis factor induce nitric oxide synthetase is suppressed by the inhibitor of polyadenous glycosides diphosphonic acid ribose polymerase in the L929 cell " Biochem is J.288:255-260; People such as Pellat-Seceunyk, 1994, " nicotiamide suppresses inducing of nitric oxide synthetase mRNA in the activatory macrophage " Biochem.J.297:53-58.Yet, in these researchs, pADPRT inhibitor 30 aminobenzamides of employing and dense (10-30mM) of nicotiamide, they suppress whole albumen and RNA and synthesize, and have other pharmaceutical effect (as removing free radical); People such as Hauschildt, 1992, " tumor necrosis factor induce nitric oxide synthetase is suppressed by the inhibitor of polyadenous glycosides diphosphonic acid ribose polymerase in the L929 cell " Biochem is J.288:255-260.Adopt INH now ₂The experiment of BP hints that further the pADPRT pleiotropy participates in iNOS mRNA transcription.In order to study INH ₂BP carries out the transient transfection test to the regulating action of iNOS promoter with mouse macrophage iNOS promoter luciferase construction.These data indirect suggestions of deletion form construction, INH ₂BP regulated relate to-1592 and-367bp between the behavior of transcribing of Mus iNOS promoter region.The ADP ribosylation of histone and ribozyme may participate in keeping lax chromatin Structure; People such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase, 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377; People such as Ueda, 1985, " ADP ribosylation " Ann.Rev.Biochem.54:73-100.Experimental data in the past (people such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase; 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie 77:347-377) reasonably hints, in these experimental systems, pADPRT inhibition chemical compound is (as INH ₂BP) pretreatment suppresses self polyadenous glycosides diphosphonic acid ribosylation of pADPRT and histone.Known that this effect causes the relaxed type chromatin and changes to dense type chromatin, and the rise effect by utilizing nuclease and other dna structure regulatory enzyme (people such as Bauer, 1995, " usefulness 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252; People such as Bauer, 1995, " with a kind of non-covalent binding partner 5-iodo-6-amino-1 of polyadenous glycosides diphosphonic acid ribose polymerase, 2-benzopyrone reversion of malignant tumor phenotypes " Biochimie77:347-377), it may influence the function of promoter.

Embodiment 3

INH ₂BP is to map kinase and the activated inhibition effect of NF-κ B

These results are verified, INH ₂The processing of BP has suppressed the activation of the inductive map kinase of LPS.In this respect, these data are very similar to the endotheliocyte of conversion; People such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252.To the activated inhibitory action of map kinase may be to pass through INH ₂The pleiotropy cell effect that BP triggers produces.Show that map kinase can be activated in the various cell types that LPS or various proinflammatory cytokine (TNF-α, interleukin-1, nerve growth factor) were handled; People such as Kyriakis, 1996, " warning: ", J.Biol Chem.271:24313-24316 by stress and the activated protein kinase cascade reaction of inflammation; People such as Matsuda, 1994, " the signal conduction pathway of mitogen activated protein (MAP) kinase kinase/map kinase cascade reaction mediation " J.Leukocyte Biol.56:548-53; People such as Cowley, 1994, " the kinase whose activation of map kinase is for PC12 differentiation and transform the NIH 3T3 cell fully necessary " Cells 77:841-52; People such as Pang, 1995, " the inductive PC-12 cell differentiation of nerve growth factor has been blocked in the kinase whose inhibition of map kinase " J.Biol.Chem.270:13585-8; People such as Willis, 1996, " bacteria lipopolysaccharide is to the differentiation-inducing action of the activated protein kinase passage of mitogen in mononuclear cell of cultivating and the spider cell " Biochem.J.313:519-524; People such as Saklatvala, 1993, " interleukin-1 and tumor necrosis factor-alpha activate the activated albumen of mitogen (MAP) kinase kinase in the cultured cell " FEBS Lett.334:189-92.Various intracellular signals converge in map kinase kinases/map kinase cascade reaction by different map kinase kinases-kinases and have caused cell effect time spectrum (vice spectrum); People such as Kyriakis, 1996, " warning: ", J.Biol Chem.271:24313-24316 by stress and the activated protein kinase cascade reaction of inflammation; Ferrell, JE, 1996, " open fabulous switch: how the protein kinase cascade reaction can be transformed into fractionated input the output of similar switch " TIBS21:460-466.The kinase whose blocking-up of map kinase or map kinase has been modified many born of the same parents' internal channels and has been suppressed cell differentiation and propagation; People such as Kyriakis, 1996, " warning: ", J.Biol Chem.271:24313-24316 by stress and the activated protein kinase cascade reaction of inflammation; People such as Matsuda, 1994, " the signal conduction pathway of mitogen activated protein (MAP) kinase kinase/map kinase cascade reaction mediation " J.Leukocyte Biol.56:548-53; People such as Cowley, 1994, " the kinase whose activation of map kinase is for PC12 differentiation and transform the NIH3T3 cell fully necessary " Cells 77:841-52; People such as Pang, 1995, " the inductive PC-12 cell differentiation of nerve growth factor has been blocked in the kinase whose inhibition of map kinase " J.Biol.Chem.270:13585-8; People such as Willis, 1996, " bacteria lipopolysaccharide is to the differentiation-inducing action of the activated protein kinase passage of mitogen in mononuclear cell of cultivating and the spider cell " Biochem.J.313:519-524; People such as Saklatvala, 1993, " interleukin-1 and tumor necrosis factor-alpha activate the activated albumen of mitogen (MAP) kinase kinase in the cultured cell " FEBS Lett.334:189-92.Recently, suppress the map kinase kinases with PD98059 and shown the expression that in endothelial cells cultured and myocardial cell, to prevent iNOS mRNA.People such as Singh, 1996, " the synthetic adjusting of the derivable nitric oxide of cytokine in myocardial cell and the capillary endothelium " J.Biol.Chem.271:1111-1117.This discovery with we observed result (be PD98059 cause LPS in the RAW macrophage the generation of inductive nitrite be subjected to remarkable inhibition) conform to.

Because the activation of NF-κ B is the main thoroughfare in the inflammatory reaction, and it has participated in induce (Szabo, the C. of LPS (rather than INF) to iNOS; 1995, " variation of NO production in the various forms of cyclical shocks " New Horizons 3:3-32; People such as Martin, 1994, " effect of interferon regulatory factor I in inducing nitric oxide synthetase " J.Exp.Med.180:977-84), so we attempt to investigate INH ₂BP is to the potential effect of NF-κ B.Our result proves, INH ₂BP does not change the activated nuclear translocation of NF-κ B, or INH ₂BP may occur in the cell behavior away from NF-κ B nuclear translocation the regulating action (if any) of the cell behavior of NF-κ B mediation.

Embodiment 4

Hint in pathophysiology and the treatment: INH ₂BP is at multiple horizontal adjusted inflammatory reaction

The reduction of the pADPRT inhibitor that short scorching gene iNOS and COX-2 express, and the NO and the prostaglandin of reduction formation thereupon may be favourable (Nathan in various forms of inflammation, 1992, " nitric oxide is as the secreting type product of mammalian cell " FASEB J.6:3051-3064; Vane, J.R., The CroonianLecture 1993, " endothelium: sanguimotor great master (maestro) " Proc.Roy.Soc.Lond B 343:225-246; Szabo, C.; 1995, " variation of NO production in the various forms of cyclical shocks " NewHorizons 3:3-32; People such as Vane, 1995, " to newly seeing clearly of antiinflammatory drug effect mode " Inflamm.Res.44:1-10).In addition, the enhancing of IL-10 release may also have other antiinflammatory action; People such as Liles, 1995, " summary: the nomenclature and the biological meaning that participate in the cytokine of inflammation and host immune response " J.Infect.Dis.172:1573-80; Giroir, 1993, " mediator of septicopyemia shock: the method that interrupts endogenous inflammatory cascade reaction " Critical Car.Med.21:780-9; People such as Szabo, 1997, " isoproterenol regulates tumor necrosis factor, interleukin 10, interleukin-6 and NO production and protection prevents to take place in the endotoxemia vascular reaction deficiency " Immunology 90:95-100.Can imagine that these effects obviously cause pADPRT inhibition chemical compound (as INH ₂BP) raising of the pretreatment and the mice survival rate of attacking through the lethal doses of lipopolysaccharide.Yet, INH be described ₂BP performance to LPS induce effect that various inflammatory mediators express really cutter system also need further detailed research.On the one hand, can believe that pADPRT activity or pADPRT are proteic in conjunction with participating in regulating the generation of inflammatory mediator and/or the expression of inflammatory process component encoding gene.On the other hand, INH ₂BP to the active indirect downward modulation effect of map kinase (people such as Bauer, 1995, " with 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP) handle the growth correlation enzymatic passage modified the cattle endothelial cell line that ras transforms and cause oncogenicity effect obviously to be lost ", Int.J.Oncol.8:239-252) may also cause the observed effect estimated as other institute; People such as Kyriakis, 1996, " warning: ", J.Biol Chem.271:24313-24316 by stress and the activated protein kinase cascade reaction of inflammation; Ferrell, JE, 1996, " open fabulous switch: how the protein kinase cascade reaction can be transformed into fractionated input the output of similar switch " TIBS 21:460-466.This result has proved that pADPRT inhibition chemical compound is (as INH ₂BP) the treatment potentiality are arranged in various inflammatory diseasess.

Embodiment 5

Some cytotoxic effects of nitric oxide (NO) are relevant with the generation of peroxynitrite (a kind of NO and superoxides react the reactive oxidants agent of formation rapidly); People such as Crow, 1995, " effect of peroxynitrite in the toxicity of mediated by nitric oxide ", Current Top Microbiol.Immunol.196:57-73; People such as Pryor, 1995, " chemical property of peroxynitrite: the product of nitric oxide and superoxides reaction ", Am.J.Physiol.L699-L772.The formation of peroxynitrite is confirmed in various inflammatory symptoms, these symptoms comprise the systemic inflammation (people such as Szabo of endotaxin induction, 1995, " variation of NO production in the various forms of cyclical shocks " New Horizons 3:3-32), arthritis (people such as Kaur, " evidence of the oxidative damage of mediated by nitric oxide in the chronic inflammation.Rheumatoid patient's serum and the nitrotyrosine in the synovial fluid " FEBS Lett.1359:9-12) and the inductive beastly pawl edema of carrageenin ( ^*People such as Salvemini, 1996).In fact, reach a conclusion from the pharmaceutical research that utilizes NO synzyme (NOS) inhibitor and superoxide dismutase mimics, peroxynitrite plays important pathogenic effects (Szabo C in the inflammatory process development, 1996, " peroxynitrite reindoctrinates effect in the physiopathology of damage in shock, inflammation and ischemia (schemia) " Shock 6:79-88; ^*People such as Salvemini, 1996; ^*People such as Zingarelli, 1997).And; verified; be used for the treatment of the cleanser that more arthritic reagent are actually peroxynitrite people such as (, 1996 " relying on the tyrosine nitrification and the deactivation of α 1-protease inhibitor of peroxynitrite with some anti-inflammatory drug and tetracycline antibiotic protection opposing " Annals.of the Rheumatic Disease 55:383-7) Whiteman at present.Because recognizing the Cytotoxic pith relevant with NO is owing to form peroxynitrite, therefore just is necessary to center on the formation of peroxynitrite and be used for developing new Therapeutic Method.

Relevant (the people such as Szabo of a kind of born of the same parents' internal channel that peroxynitrite triggers with the activation of dna single chain interruption and polyadenous glycosides diphosphonic acid ribose synzyme (PARS), 1996, " peroxynitrite reindoctrinates effect in the physiopathology of damage in shock, inflammation and ischemia " Shock 6:79-88; ^*Szabo, 1996b).The remarkable activation meeting of PARS consumes the intracellular concentration of its substrate NAD+ rapidly, reduces glycolysis, electron transport speed, thus reduce the formation of ATP and cause cell dysfunction ( ^*Berger, 1991; ^*Cochrane, 1991).Therefore, the inhibitor of PARS is protected in these cases and has been prevented cell injury.This mechanism is called " PARS commit suiside hypothesis ", former H ₂O ₂Once did in damage of inductive oxidant and the radiation damage analysis ( ^*Berger, 1991; ^*Cochrane, 1991), shown that recently it has related to interior toxicogenic shock, apoplexy, ischemia and has reindoctrinated cell injury relevant with NO and peroxynitrite in damage and the diabetes (people such as Szabo, 1996, " peroxynitrite reindoctrinates effect in the physiopathology of damage in shock, inflammation and ischemia " Shock 6:79-88; ^*People such as Zhang, 1994, ^*People such as Heller, 1995).

Kroger and colleague thereof have proposed the latent effect of PARS in arthritis recently.In the inductive model of peroxidating chromic acid potassium, nicotiamide handle cause average arthritis score reduce 25-35% ( ^*People such as Miesel, 1996).Yet this research determine to suppress mechanism yet because its do not indicate nicotiamide remove tangible difference arranged between free radical activity and the PARS depression effect ( ^*People such as Miesel, 1995).In this research, utilize a kind of new potent PARS activity inhibitor (5-iodo-6-amino-1,2-benzopyrone (INH ₂BP)) ( ^*People such as Bauer, 1995a, ^*People such as Bauer, 1995b), we have studied the pharmacology of PARS in carrageenin inductive beastly pawl edema and collagen-induced arthritic course and have suppressed effect.Our result of study has been supported such viewpoint, i.e. the inhibition of PARS has the antiinflammatory probability.

Embodiment 6

The inducing and estimating of the inductive beastly pawl edema of carrageenin

In these researchs, adopt male Wister rat (250-300 gram, Charles River Laboratories, Wilmington, MA).(subplantar) injection contains 0.1 mL of saline of 1%1-carrageenin under the sole of the foot of the right back sufficient pawl of animal.This inflammation reagent that causes is given through INH ₂The animal that BP handles or the animal of vehicle treated.Animal was subjected to INH in preceding 24 hours and 2 hours at the injection carrageenin ₂BP (0.5 gram/kilogram p.o.) handles.As ^*Described in the people such as Sautebin 1995, it is long-pending to use plethysmometry (phlethysmometry) to measure beastly corpus unguis after injection immediately.The interval was read the volume of same beastly pawl in 60 minutes and is compared with initial value subsequently.For these experiments, adopt animal and 6 INH of 6 vehicle treated ₂The animal that BP handles.

Embodiment 7

Collagen-induced arthriticly induce and estimate

The male DBA/1J mice of these research employings (9 weeks, Jackson Laboratory, Bar Harbor, ME).Is that 2 mg/ml 4 ℃ stirrings spend the night make dissolving to concentration with chicken II Collagen Type VI (CII) in 0.01M acetic acid.-70 ℃ of freezing dissolved CII are stand-by.Make Freund adjuvant (CFA) completely by adding Mycobacterium tuberculosis (Mycobacterium tuberculosisH37ra) to 2 mg/ml.Before the injection, with equal-volume CFA emulsifying CII.As people such as Hughes 1994, " with non-mitogenic anti--the CD3 monoclonal antibody induces the t helper cell hyperergy in the autoimmune experimental model " induce collagen-induced arthritis like that described in the J.Immunol.153:3319-3325.At the 1st day, at 100 milliliters of CII of mouse tail root percutaneous injection.The 21st day, injection for the second time gave the CII among the CFA.From the 25th day, every 24 hours with carrier (n=10) or INH ₂BP (n=60 (0.5 gram/kilogram p.o)) handles animal.Be arthritis (the beastly pawl of 1-or toe swelling and/or rubescent of 0 to 4 macroscopic score system evaluation mice every day with rank; 2-relates to two joints; 3-relates to plural joint; 3-relates to plural joint; Whole beastly pawl of 4=and toe have serious arthritis).The arthritis index of each mice is calculated in add up four kinds of scorings of indivedual beastly pawls.In experiment last (the 35th day), animal is put to death in anesthesia, takes off beastly pawl and knee and fixing, to carry out histological examination.Histological examination is undertaken by the investigator who does not know therapeutic scheme.

Data analysis and expression

For the research of the inductive beastly pawl edema of carrageenin, long-pending with the beastly corpus unguis of azygous this figure Dent test comparison process group and untreated fish group animal.For arthritic research, the statistics difference in the arthritis index is tested in usefulness Mann-Whitney U test (two tails, incoherent).Compare intermediate value (rather than meansigma methods) with this nonparametric statistics, because the mensuration scale is according to the order of sequence, and distribution be worth nonnormal distribution normally (people 1994 such as Hughes, " and with non-mitogenic anti--the CD3 monoclonal antibody induces the t helper cell hyperergy in the autoimmune experimental model " J.Immunol.153:3319-3325).

Numerical tabular among Figure 10 is shown as the meansigma methods ± meansigma methods standard error of n observed value, and wherein n represents the number (every group of 6 animals) of rat.Numerical value among Figure 11 is represented sickness rate (%), and the numerical value among Figure 12 is represented intermediate value.Think that the p value is lower than 0.05 meaningful statistically (I '＜0.05; ^*P＜0.02).

Material

As previously mentioned ( ^*People such as Bauer, 1995a; ^*People such as Bauer 1995b), make 5-iodo-6-amino-1,2-benzopyrone (INH ₂BP).Chicken II Collagen Type VI is available from Elastin Products Company, and Inc. (Owensville, MO).Mycobacterium tuberculosis H37Ra available from Difco (Detroit, MI).All other chemical substances all available from Sigma Chemical Co. (St.Louis, MO).The beastly pawl of injection rat causes that beastly corpus unguis is long-pending to be depended on the time and increase under the carrageenin sole of the foot, and maximum is reflected at 3 hours and locates (Figure 10).The inductive beastly pawl edema of this carrageenin is by INH ₂BP handles and obviously reduces (Figure 10).

In the intravital arthritis model of collagen-induced mice, after first time collagen immunization, between 26-35 days, find out that from the evidence (Figure 11-12) that the arthritis sickness rate increases and arthritis score increases the development of joint of animal inflammation increases day by day.INH ₂The processing of BP reduced arthritic sickness rate until the 33rd day, and had reduced severity of disease at whole experimental session.At the 30th day, arthritis score increased to 10, and INH ₂The arthritis score intermediate value still maintains about 5 (Figure 12) in the animal that BP handles.At the 35th day, all animal and great majority through vehicle treated were subjected to INH ₂The animal that BP handles all has arthritis (Figure 11) to a certain degree.Yet, even at the 35th day, INH ₂BP handles and has still significantly reduced arthritis score intermediate value (Figure 12).

At the 35th day, the beastly pawl of the trouble arthritis animal of vehicle treated is carried out Histological evaluation, disclosing has serious suppurative arthritis symptom, and macro-mixing (neutrophilic granulocyte, macrophage and lymphocyte) soaks in bigger ankle joint and the whole toe.In addition, can see that necrosis, hypertrophy and the synovial membrane slough of serious or moderate separates, and inflammation extends to the mucus generation that the musculature of adjoining causes fibrosis and increases.At INH ₂In the animal body that BP handles, arthritic degree significantly reduces.However, in these animal bodies, still have the tangible arthritis of degree, have moderate, mainly be that neutrophil infiltration is in some bigger joints, also with slightly to the necrosis and the synovial hyperplasia of moderate.Similar with the discovery in the beastly pawl, the serious suppurative arthritis symptom of finding in knee is by INH ₂The treatment of BP alleviates (result does not show).

Discuss

The product of peroxynitrite, oxy radical and derivable cyclo-oxygenase has been thought independently that the key factor in various forms of inflammation (the comprising arthritis) pathogeny (sees foreword, other sees: Brahn, 1991, " Animal Model of Rheumatoid Arthritis.Clue " the Clin.Orthop.Rel.Res.265:42-53 of etiology and treatment; People such as Kaur, " the evidence of the oxidative damage of mediated by nitric oxide in the chronic inflammation.Rheumatoid patient's serum and " the FEBS Lett.1359:9-12 of the nitrotyrosine in the synovial fluid; Oyanagui Y, 1994, " nitric oxide and superoxide radical participate in the rat body arthritic initial sum development of internal reaction (envelopment) " Life Sci.54:PL285-9; People such as Miesel, 1994, " allopurinol prevent the effect of mice body intrinsic articulation inflammation and in vitro in mediator's whole blood phagocyte produce oxygen-derived free radicals " Inflammation6:597-612; People such as Whiteman, 1996, " relying on the nitrated and α 1-protease inhibitor deactivation of tyrosine of peroxynitrite with some anti-inflammatory drug and tetracycline antibiotic protection opposing " Annals.of the Rheumatic Disease55:383-7; People such as Anderson, 1996, " selectivity suppresses inflammation and the expression that cyclo-oxygenase (COX)-2 has reversed COX-2 and interleukin-6 in the rat reactive arthritis " J.Clin.Invest.97:2672-2679).Originally studies confirm that INH ₂The antiphlogistic effects of BP in inductive beastly pawl edema model of carrageenin and collagen-induced arthritis model, this has supported such viewpoint, promptly the PARS pharmacology that participates in the progress of inflammatory process and PARS suppress to have antiphlogistic potential may.

INH ₂The primary action pattern of BP may be that cascade reaction is interrupted relevant in the trifling born of the same parents of feature with the DNA damage.The removing of PARS activation, adenosine diphosphate riboseization and NAD+ and ATP is in the various cell types in inflammation joint.With the various inhibitor of PARS (as 3-aminobenzamide, nicotiamide and INH ₂BP) suppress this passage demonstrated can protect various kinds of cell avoid the damage (Berger, 1911; ^*Cochrane, 1991; People such as Szabo, 1996, " peroxynitrite reindoctrinates effect in the physiopathology of damage in shock, inflammation and ischemia (schemia) " Shock 6:79-88; ^*Sazbo, 1996b).

Excessively produce NO in the inflammatory state of an illness and be since derivable NOS isotype (iNOS) prevented (Nathan, 1992, " nitric oxide is as the secreting type product of mammalian cell " FASEB is J.6:3051-3064; Szabo; 1995, " variation of NO production in the various forms of cyclical shocks " New Horizons 3:3-32; People such as Southan, 1996, " the aminoalkyl guanidine is spontaneous be rearranged into mercaptoalkyl guanidine-a class new for inducing isotype nitric oxide synthase inhibitor activity selectively " Br.J.Pharmacol.117:619-632).Several evidences have hinted and iNOS and NO excessively have been created in effect in the arthritis pathogenesis (summary is referring to people such as Stenovic-Racic, 1993, " nitric oxide and arthritis " Arthr.Rhemat.36:1036-1044; ^*People such as Evans, 1995).The first, the verified iNOS of having expresses and NO produces (people such as Haeselmann, 1994, " alginate cultivate in person joint's chondrocyte synthesize nitric oxide and Dan Baijutang " FEBS Lett.352:361-364 in a large number in laboratory animal and people's chondrocyte; People such as Sakurai, 1995, " nitric oxide in the inflammatory arthritis produces and derivable nitric oxide synthetase is expressed " J.Clin.Invest.96:2357-63; People such as Grabowski, 1996, " person joint derive the cell that obtains in produce carbon monoxide " Br.J.Rheumatol.35:207-12; People such as Murrell, 1996, " nitric oxide: important joint free radical " J.Bone Joint Sur.-Am.78:265-74).Second, the cyclical level that has confirmed nitrite/nitrate (catabolite of NO) in arthritic's body increases (people such as Farrell to some extent, 1992, " concentration of nitrite increases synthetic the increasing of nitric oxide in the prompting atrophic diseases in synovial fluid and the blood serum sample " Ann.Rhem.Dis.51:1219-22; People such as Stichtenoth, 1995, " the urine nitrate secretion of patient with rheumatoid arthritis increases, and makes its minimizing by meticortelone (predisolone) " Ann.Rhem.Dis.54:820-4).The 3rd, arthritic development shown by the non-isotype selective depressant of NOS and reduced ( ^*People such as Ialential, 1993; People such as McCartney-Francis, 1993, " inhibitor of nitric oxide synthetase is prevented arthritis " J.Exp.Med.178:749-753; People such as Weinberg, 1994, " the effect of nitric oxide in the pathogenesis of spontaneous Mus autoimmune disease; the expression of NO production and nitric oxide synthetase in the increase MRL-1 pr/1pr mice, and the glomerulonephritis and the arthritis " J.Exp.Med.1979:651-60 that have reduced spontaneous generation by oral NG-monomethyl-L-arginine; People such as Stefanovic-Racic, 1994, " a kind of inhibitor N-monomethylarginin of nitric oxide synthetase is prevented the arthritic development of rat body internal reaction " Arthr.Rheumat.37:1062-9), and it is more recent, by to iNOS selectively inhibitor reduce (people such as Connor, 1995, " selectivity with derivable nitric oxide synthetase suppresses to prevent the inductive arthritis of adjuvant " Eur.J.Pharmacol.273:15-24).In this respect, it should be noted that before immunostimulation and (comprise 3-aminobenzamide, nicotiamide and INH with the PARS inhibitor ₂BP) the pretreatment various kinds of cell shown the mRNA that can prevent iNOS express and reduce the generation of NO ( ^*People such as Hauschildt, 1992, ^*People such as Pellat-Scecunyk, 1994; People such as Zingarelli, 1996, " in the macrophage that bacteria lipopolysaccharide stimulates, the DNA chain interruption of peroxynitrite mediation has activated polyadenous glycosides diphosphonic acid ribose synzyme and has caused cell energy consumption " J.Immunol.156:350-358; ^*People such as Szabo, 1997).Can reach a conclusion from these experimental datas, on behalf of PARS, the expression process that PARS also can regulate iNOS by an also undetermined mechanism, this effect may be suppressed at other favourable binding mode in the multi-form inflammation.Yet, should be careful when explaining above-mentioned discovery.For example, in above-mentioned in vitro study,, need very the PARS inhibitor 3-aminobenzamide and the nicotiamide of high concentration (10-30mM) in order to confirm to prevent iNOS to induce.The high concentration of these reagent has other pharmacotoxicological effect, for example suppress total protein and RNA synthetic and/or remove free radical effect ( ^*Hauschildt, 1992, ^*People such as Pellat-Seceunyk, 1994; People such as Zingarelli, 1996, " in the macrophage that bacteria lipopolysaccharide stimulates, the DNA chain interruption of peroxynitrite mediation has activated polyadenous glycosides diphosphonic acid ribose synzyme and caused cell energy consumption " J.Immunol.156:350-358).On the other hand, even under lower non-cell toxicity concentration (10-300mM), INH ₂BP has prevented the expression of iNOS effectively.Yet, at INH ₂Under the BP situation, will be understood that several binding modes because this reagent is the inducer of alkali phosphatase, have cell effect replenish, the multiple-effect regulating action ( ^*People such as Bauer, 1996; ^*People such as Szabo, 1997).To in the cell of eliminating the PARS gene or animal, experimentize, whether itself prevent iNOS to induce the problem of process so that solve the inhibition of PARS definitely.

In the Ehrlich and the nearest research of working together thereof, show in the rabbit synovial fluid fibroblast of cultivating, the expression of the collagenase activity of cytokine induction prevented by the 3-aminobenzamide ( ^*People such as Ehrlich, 1995).May be able to determine now that the character of its pharmacotoxicological effect (but estimate and will prevent arthritic process), used special inhibitor or it are real relevant with the minimizing of PARS catalytic activity.In this respect, it should be noted that according to the research of pharmacological inhibitor, PARS has participated in the adjusting of range gene, these genes comprise main tissue compatible type complex II genoid ( ^*People such as Hiromatsu, 1992; People such as Taniguchi, 1993), ras c-myc ( ^*People such as Bauer, 1996, ^*People such as Nagao, 1991), the dnmt rna gene ( ^*People such as Bauer, 1996) and the Protein kinase C gene ( ^*People such as Bauer, 1996).

In a word, this research work has confirmed INH ₂BP has improved the development of local inflammatory response and has suppressed collagen-induced arthritic progress.Though reported the effect of PARS in DNA repairs in the past 10 years, nearest observation shows that elimination PARS gene can not damage the reparation of DNA: the animal that PARS knocks out act normally and survive ( ^*People such as Wang, 1995).The antiinflammatory trend of pharmacological inhibitor PARS has been strengthened in this observation.PARS suppresses important antimicrobial effect of (suppressing opposite with iNOS) the impossible NO of interference, because the microorganism of invading does not contain PARS.On the other hand, estimating that PARS suppresses can not only the inductive parts of fine cellular toxicity of inhibited oxidation agent, and more effective when using with other free radical scavenger or other immune co-inhibitor.Result of study of the present invention has been supported such viewpoint, and promptly independent PARS suppresses or represented a kind of promising new antiinflammatory method with the combination of other antiinflammatory.

With similar fashion shown in the foregoing description, with formula II and formula III compounds for treating inflammation or inflammatory diseases, and treatment Grain-negative and Grain-positive infection.

Think that above stated specification is enough to make those skilled in the art to implement the present invention.In fact, it is conspicuous for field of pharmaceutical preparations or various equivalent modifications that the above-mentioned mode that is used to carry out an invention is carried out various modifications, and these are all in the scope of following claim.

Claims (6)

1. method for the treatment of inflammation in animal or the mammalian body or inflammatory diseases, described method comprises the step of the pADPRT inhibition chemical compound that gives described animal or mammal effective dose.

2. method according to claim 1, wherein pADPRT inhibition chemical compound is selected from following compounds: the chemical compound with following formula R wherein ₁, R ₂, R ₃, R ₄, R ₅And R ₆Respectively be selected from hydrogen, hydroxyl, amino, alkyl, alkoxyl, cycloalkyl or phenol, these groups can be replaced R arbitrarily by alkyl, alkoxyl, hydroxyl or halogen ₁, R ₂, R ₃, R ₄, R ₅And R ₆In have only one to be amino; Chemical compound with following formula:

R wherein ₁, R ₂, R ₃, R ₄And R ₅Be selected from hydrogen, hydroxyl, amino, alkyl, alkoxyl, cycloalkyl or phenol separately, these groups can be replaced R arbitrarily by alkyl, alkoxyl, hydroxyl or halogen ₁, R ₂, R ₃, R ₄, R ₅In have only one to be selected from amino, nitroso-group or nitro; With the chemical compound with following formula:

R wherein ₁, R ₂, R ₃, R ₄And R ₅Be selected from hydrogen, hydroxyl, amino, alkyl, alkoxyl, cycloalkyl or phenol separately, these groups can be replaced R arbitrarily by alkyl, alkoxyl, hydroxyl or halogen ₁, R ₂, R ₃, R ₄And R ₅In have only one to be amino.

3. method according to claim 2, wherein said chemical compound is selected from: 6-amino-1,2-benzopyrone, 3-aminobenzamide, 5-amino-1 (2H)-isoquinolines, 7-amino-1 (2H)-isoquinolines and 8-amino-1 (2H)-isoquinolines.

4. method according to claim 1, wherein said chemical compound are 5-iodo-6-amino-1, the 2-benzopyrone.

5. method for the treatment of Grain-negative and the inductive endotoxin symptom of gram positive bacteria in animal or the mammalian body, described method comprises the pADPRT inhibition chemical compound that gives animal or mammal treatment effective dose.

6. method according to claim 5, wherein chemical compound is selected from the chemical compound with following formula.

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