EP1009404A1 - Procede de traitement de l'inflammation ou de maladies inflammatoires a l'aide d'inhibiteurs de poly-adp ribose polymerase - Google Patents

Procede de traitement de l'inflammation ou de maladies inflammatoires a l'aide d'inhibiteurs de poly-adp ribose polymerase

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Publication number
EP1009404A1
EP1009404A1 EP98922359A EP98922359A EP1009404A1 EP 1009404 A1 EP1009404 A1 EP 1009404A1 EP 98922359 A EP98922359 A EP 98922359A EP 98922359 A EP98922359 A EP 98922359A EP 1009404 A1 EP1009404 A1 EP 1009404A1
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European Patent Office
Prior art keywords
inh
lps
amino
cells
induced
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EP98922359A
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German (de)
English (en)
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EP1009404A4 (fr
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Ernest Kun
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Octamer Inc
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Octamer Inc
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Priority claimed from US08/855,616 external-priority patent/US5908861A/en
Application filed by Octamer Inc filed Critical Octamer Inc
Publication of EP1009404A1 publication Critical patent/EP1009404A1/fr
Publication of EP1009404A4 publication Critical patent/EP1009404A4/fr
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/365—Lactones
    • A61K31/366—Lactones having six-membered rings, e.g. delta-lactones
    • A61K31/37—Coumarins, e.g. psoralen
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/165—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide
    • A61K31/166—Amides, e.g. hydroxamic acids having aromatic rings, e.g. colchicine, atenolol, progabide having the carbon of a carboxamide group directly attached to the aromatic ring, e.g. procainamide, procarbazine, metoclopramide, labetalol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/435—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with one nitrogen as the only ring hetero atom
    • A61K31/47—Quinolines; Isoquinolines
    • A61K31/472—Non-condensed isoquinolines, e.g. papaverine
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to methods for treating inflammation and inflammatory diseases, including arthritis, in animals or mammals.
  • the invention also relates to methods for treating animals or mammals having both gram negative and gram positive endotoxin symptoms resulting from systemic infections or resulting from infestation by lipopolysaccharides. These methods involve the use of therapeutically effective amounts of pADPRT inhibitory compounds.
  • 5-iodo-6-amino-l, 2-benzopyrone a novel inhibitor of the nuclear enzyme poly-ADP ribose polymerase (pADPRT) has recently been shown to inhibit in vivo tumorigencity in a Ha-r ⁇ s transfected endothelial cell line; Bauer et al, 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH,BP)," Int. J. Oncol.
  • LPS-induced pro-inflammatory mediators include tumor necrosis factor alpha (TNF), interleukin-1, interferon-gamma, whereas anti-inflammatory mediators include interleukin-10 (IL-10) and interleukin-13; Deltenre et al, 1995, "Gastric carcinoma: the Helicobacter pylori trail," Acta Gastroenterol Belg. 58:193-200: Beutler, 1995, "TNF, immunity and inflammatory disease: lessons of the past decade," J. Invest. Med. 42:227-35; Liles et al, 1995, “Review: nomenclature and biologic significance of cytokines involved in inflammation and the host immune response.” J. Infect Pis.
  • LPS initiates the production of inflammatory free radicals (oxygen-centered, such as superoxide, and nitrogen-centered radicals, such as nitric oxide [NO]) and of prostaglandins; Nathan, 1992, “Nitric oxide as a secretory product of mammalian cells,” FASEB J. 3:151-3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: maestro of the blood circulation.” Proc. Roy. Soc.
  • the production of NO in inflammation is due to the expression of a distinct isoform of NO synthase (iNOS), while the production of inflammatory cytokines is explained by the expression of a distinct isoform of cyclooxygenase (cyclooxygenase-2, COX-2); Nathan, 1992, "Nitric oxide as a secretory product of mammalian cells," FASEB J. 3:151-3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: maestro of the blood circulation,” Proc. Rov.
  • One aspect of the invention is a method for treating inflammation or inflammatory disease in an animal or mammal, which comprises the steps of administering an effective amount of an pADPRT inhibitory compound to said animal or mammal.
  • Another aspect of the invention is a method for treating inflammation or inflammatory disease in an animal or mammal, which comprises the steps of administering an effective amount of a pADPRT inhibitory compound wherein the pADPRT inhibitory compound is selected from the group consisting of:
  • R contest R 2 , R 3 , R 4 , R 5 and R ⁇ are each selected from the group consisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, and only one of R privilege R 2 , R 3 , R 4 , R 5 and I . is amino; a compound having the formula:
  • R,, R 2 , R 3 , R 4 , and R 5 are each selected from the group consisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, and only one of R perpetrat R 2 , R 3 , R 4 , and-R 5 is amino; and a compound having the formula:
  • R,, R 2 , R 3 , R 4 , and R 5 are each selected from the group consisting of hydrogen, hydroxy, amino, alkyl, alkoxy, cycloalkyl or phenol, optionally substituted with alkyl, alkoxy, hydroxy or halo, and only one of R,, R 2 , R 3 , R 4 , and R 5 is amino.
  • Preferred pADPRT compounds include: 6-amino-l, 2-benzopyrone, 3- nitrosobenzamide, 5-amino- 1 (2H)-isoquinolinone, 7-amino- 1 (2H)-isoquinolinone, and 8-amino-l(2H)-isoquinolinone.
  • Still another aspect of the invention includes a method of treating both gram negative and gram positive induced symptoms in an animal or mammal, said method comprising the step of administering to an animal or mammal a therapeutically effective amount of a pADPRT inhibitory compound.
  • Still another aspect of the invention is a method of treating both gram negative and gram positive induced endotoxin symptoms in an animal or mammal which comprises the step of administering to an animal or mammal a therapeutically effective amount of a pADPRT inhibitory compound wherein the compound is selected from the group consisting of compound I, compound II, or compound III, as described above.
  • Still another aspect of the invention is a method of treating both gram negative and gram positive induced endotoxin symptoms in an animal or mammal which comprises the step of administering to an animal or mammal a therapeutically effective amount of a pADPRT inhibitory compound wherein the compound has the structural formula noted above as compounds I, II or III.
  • Still another aspect of the invention is a method of treating arthritis in an animal or mammal comprising the step of administering an effective amount of or an pADPRT inhibitory compound wherein the compound has the structural formula noted above as compounds I, II or III.
  • Still another aspect of the invention is a method of treating Chron's Disease in an animal or mammal comprising the step of administering an effective amount of an pADPRT inhibitory compound wherein the compound has the structural formula noted above as compounds I, II or III.
  • Still another aspect of the invention is a method of treating Barrett's Disease in an animal or mammal comprising the step of administering an effective amount of an pADPRT inhibitory compound wherein the compound has the structural formula noted above as compounds I, II or III.
  • the pADPRT inhibitory compounds of the invention may be prepared by the methods described in U.S. Patent Nos. 5,464,871; 5,473,074; 5,482,975; 5,484,951; 5,516,941; and 5,583,155, the disclosures of which are incorporated herein by reference.
  • the preferred compounds for use in the methods of the invention include those where the halo group is iodo, and one of the R groups is amino, one of the R groups may be nitroso or nitro as described in the aforementioned patents, but preferably the R group is amino. Also, it has been found that the pADPRT inhibitory activity is strongly exhibited when the iodo moiety is adjacent to the amino moiety. In any event, the compounds to be used in the methods of the invention should have pADPRT inhibitory activity.
  • the compounds may be used as is, or preferably in combination with a pharmaceutically acceptable acid addition salt or other suitable pharmaceutical carrier known in the art.
  • FIG. 2 BP inhibits iNOS expression in J774 and RAW 264.7 cells
  • FIG. 4 Effect of INH 2 BP on the induction of luciferase activity by LPS in RAW 264.7 cells transiently transfected with either a full length (-1592 bp) or a deletional (-367 bp) iNOS promoter-luciferase construct.
  • LPS 10 ⁇ g/ml
  • 4 h led to a 10 to 12-fold induction of luciferase activity, over control values.
  • FIG. 5 INH 2 BP suppresses the induction of iNOS in conscious rats.
  • iNOS activity in lung homogenates (a) and plasma nitrite-nitrate concentrations (b) in control rats (c), in rats injected with INH 2 BP (INH 2 BP); in rats injected with LPS (15 mg/kg i.p. for 6 h); and the effect of treatment with INH 2 BP (10 mg/kg i.p.), when given 10 min. prior to LPS (INH 2 BP + LPS) or at 2 h after LPS (LPS + INH 2 BP). **represents a significant effect of LPS when compared to controls (p ⁇ 0.01); ##represents significant inhibition by the pADPRT inhibitor (p ⁇ O.Ol); n 4-5.
  • FIG. 8 (a) MAP kinase activity in RAW 264.7 cells treated with vehicle or LPS (10 ⁇ g/ml) for 24 h ni presence or absence of 100 ⁇ M PD 98059 or 150 ⁇ M INH 2 BP. Data represent values obtained in a typical experiment: similar results were seen on 3 different experimental days, (b) Representative in gel MAP kinase assay in RAW 264.7 cells at 24 h after vehicle or LPS treatment in the presence or absence of 150 ⁇ M INH 2 BP.
  • Lanes 1-4 represent the following groups, respectively: 1 : vehicle- treated control; 2: LPS treatment; 3: vehicle treatment in the presence of 150 ⁇ M INH 2 BP; 4: LPS treatment in the presence of 150 ⁇ M INH 2 BP.
  • FIG. 10 Describes the effect of INH 2 BP on the development of carrageenan- induced paw edema.
  • FIG. 11 Describes the effect of INH 2 BP on the onset of collagen-induced arthritis.
  • the percentage of arthritic mice (mice showing clinical scores of arthritis >1) are represented.
  • FIG. 12 Describes the effect of INH 2 BP on the severity of collagen-induced arthritis.
  • IpO.Ol There was a significant increase in the arthritic score from day 26 (IpO.Ol), and there was a significant suppression of the arthritic score by INH 2 BP between days 26-35 (#p ⁇ 0.05).
  • Anti-inflammatory diseases refers to diseases or conditions where there is an inflammation of the body tissue. Such disease include for example, Chron's disease, Barrett's disease, arthritis, multiple scelorsis, cardiomyopathic disease, colitis, infectious meningitis, encephalitis, and the like.
  • “Pharmaceutically acceptable acid addition salt” refers to those salts which retain the biological effectiveness and properties of the free bases and which are obtained by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, methanesulfonic acid, salicylic acid and the like.
  • ADPRT refers to adenosinediphosphoribose transferase and is also known as poly(ADP-ribose)polymerase (EC 2.4.99), a specific DNA-binding nuclear protein of eucaryotes that catalyzes the polymerization of ADP-ribose. The enzymatic process is dependent on DNA.
  • Alkyl refers to saturated or unsaturated branched or straight chain hydrocarbon radical. Typical alkyl groups include methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tertiary butyl, pentyl, hexyl and the like.
  • Alkoxy refers to the radical -O-alkyl. Typical alkoxy radicals are methoxy, ethoxy, propoxy, butoxy and pentoxy and the like.
  • Cycloalkyl refers to saturated monocyclic hydrocarbon radical containing 3- 8 carbon atoms such as cycloproply, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl, and the like.
  • Substituted phenyl refers to all possible isomeric phenyl radicals such as mono or disubstituted with a substituent selected from the group consisting of alkyl, alkoxy, hydroxy, or halo.
  • Halo refers to chloro, fluoro, bromo or iodo, and preferably iodo.
  • the pADPRT inhibitory compounds of the invention are potent, specific and non-toxic anti- inflammatory compounds, that can be used for conditions and diseases typically known for inflammation, such as arthritis, Chron's disease, Barrett's disease, and the like. Also, these compounds are useful in the treatment of conditions associated with gram negative and gram positive induced infections, especially those associated with gram negative infections, and including conditions associated with lipopolysaccharide condition and sepis. The compounds are especially useful in that they have very low, if any toxicity.
  • the compounds of the invention or their pharmaceutically acceptable salts will be administered in amounts which will be sufficient to inhibit inflammatory conditions or disease and/or prevent the development of inflammation or inflammatory disease in animals or mammals, and be used in the pharmaceutical form most suitable for such purposes.
  • Administration of the active compounds and salts described herein can be via any of the accepted modes of administration for therapeutic agents. These methods include systemic or local administration such as oral, parenteral, transdermal, subcutaneous, or topical administration modes.
  • systemic or local administration such as oral, parenteral, transdermal, subcutaneous, or topical administration modes.
  • the preferred method of administration of these drugs is oral. In some instances it may be necessary to administer the composition in other parenteral form.
  • compositions may be in the solid, semi- solid or liquid dosage form, such as, for example, injectables, tablets, suppositories, pills, time-release capsules, powders, liquids, suspensions, or the like, preferably in unit dosages.
  • the compositions will include an effective amount of active pADPRT inhibitory compound or the pharmaceutically acceptable salt thereof, and in addition, it may include any conventional pharmaceutical excipients and other medicinal or pharmaceutical drugs or agents, carriers, adjuvants, diluents, etc., as customary in the pharmaceutical sciences.
  • excipients include pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharin, talcum, cellulose, glucose, sucrose, magnesium carbonate, and the like may be used.
  • the active pADPRT inhibitory compound defined above may be also formulated as suppositories using for example, polyalkylene glycols, for example, propylene glycol, as the carrier.
  • Liquid, particularly injectable compositions can, for example, be prepared by dissolving, dispersing, etc. the active compound in a pharmaceutical solution such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • a pharmaceutical solution such as, for example, water, saline, aqueous dextrose, glycerol, ethanol, and the like, to thereby form the injectable solution or suspension.
  • the pharmaceutical composition to be administered may also contain minor amounts of nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
  • nontoxic auxiliary substances such as wetting or emulsifying agents, pH buffering agents, and other substances such as for example, sodium acetate, triethanolamine oleate, etc.
  • the pharmaceutical composition to be administered may contain liposomal formulations comprising a phospholipid, a negatively charged phopholipid and a compound selected from cholesterol, a fatty acid ester of cholesterol or an unsaturated fatty acid.
  • Typical neutral phospolipids include L-a- phophalidycholine, L-a-phosphatidylinosotol, L-a-phosphatidyl-serine, L-a- phosphatidylinosotol, L-a-phosphatidic acid, L-a-phosphatidylglycerol, L-a- lysophosphatidylcholine, sphingomycelin, and cardiolipin.
  • Typical negatively charged phospholipids include diacetyl phosphate or phosphodiglyceride, e.g., dilauroyl, dimyristoyl phosphate, dipalmitoyl phosphate, disteroyl phosphate.
  • Typical cholesterols and cholesterol ethers include cholesterol, 3S-hydroxy-5- cholestene, polyoxyethanylcholesteryl sebacate, cholesterol-5, 6-epoxide, cholesteryl acetate, cholesteryl n-butyl ether, cholesteryl caprate, cholesteryl dodecanoate, cholesteryl ethyl ether, cholesteryl heptadecanoate, cholesteryl methyl ester.
  • Typical unsaturated fatty acids include arachidonic acid, docosahexanoic acid, elaidic acid, erucic acid, linoleic acid, linolenic acid, nervonic acid, oleic acid, palmitoleic acid, petroselinic acid.
  • the halo nitro compounds may be encapsulated or partitioned in a bilayer of liposomes of the liposomal formulation according to patent application Ser. No. 08/020,035 entitled "Liposomal Formulations and Methods of Making and Using Same” filed on February 19, 1993 which is incorporated herein by reference.
  • the liposomes are formed first and then the C-amino, nitroso or nitro compound is added. Rather than be encapsulated, the C-amino, nitroso or nitro compound partitions (locates itself) into the lipid bilayer of the liposome.
  • the ingredients e.g., phosphatidyl choline, dicetyl phosphate and cholesterol are blended with a solvent such as chloroform. After blending the chloroform is driven off. Then water is added to it. When the water is added to the liposomes, it makes a multilamellar liposome (i.e., the liposomes are similar to an onion skin having many layers).
  • the next step is to freeze and thaw them. They are frozen down rapidly in liquid nitrogen. The purpose of the rapid freeze and thaw it to make the liposome size more uniform. The liposomes at this point are varied in size and you treat and that it one or more, typically, five, times. Thawing occurs in a 37 degree water bath. Before the freeze and thaw one sonicares the mixture. The combination of sonication and thawing reduces the number of skins. The goal is to produce a unilamellar system. At this point, the C-nitroso compound is added to get a 10 millimolar (Mn) concentration. The concentration can be in excess of 15 millimolar.
  • Mn millimolar
  • the total lipid concentration is 648 mg and 60- milliliters of water is added to that.
  • the phosphatidyl choline is 500 mg, the cholesterol is 36 mg; the dicetyl phosphate is 112 mg.
  • Increasing the liposome concentration of the mixture permits it to contain more C-amino, nitroso or nitro compound. For example, it could be twice as concentrated as it is in the above mixture. For a 60 mil batch, one could double the numbers above to have 1000 mg of phosphatidyl choline, 224 mg of dicetyl phosphate and 72 mg of cholesterol. Decreasing the concentration decreases the amount of C- nitroso compound to get in there. For the hypothetical 60 milliliter batch, the upper limit of C-amino compound approaches is 15 millimolar concentration of C-amino compound. For 3-Nitrosobenzamide this is 135 mg. for a 60 milliliter batch.
  • the next step is to rehydrate. Then, the next step of the process is extrusion using an extruder device (Lipex Biomembranes, Inc., Vancouver, British Columbia, Canada).
  • the extrusion process serves two purposes; 1) making the size of the liposomes uniform; and 2) sterilization.
  • Extrusion typically involves filtration through a jO.l micron filter and is generally followed by freeze drying the mixture to lyophilize the mixture (takes the water out of it and makes it a fine powder). This improves solubility so that one can put up to about a 40 millimolar solution which is about three times as concentrated as prior to free drying. Freeze drying produces a mixture of powdered lipids and the powdered C-amino compound. Now one can use the same amount of the C-amino compound and a smaller amount of liquid making a more concentrated mixture. For example, one may have the same weight of C-amino, nitroso or nitro compound but have up to one-third of the original volume.
  • steps of the above process could modify steps of the above process by, for example, eliminating steps such as freeze drying.
  • This process of the first embodiment does not significantly encapsulate the C- amino, nitroso or nitro compound. Instead of having the compound in the middle of the liposome the compound resides in the membrane itself. The C-amino, nitroso or nitro compound partitioned within the membrane of the liposome will migrate to the target cells and the lipid will carry the C-amino, nitroso or nitro compound into the cell membranes.
  • this process makes liposomes having about 0.05-0.45, and more preferably about 10.1 -0.2 micron, diameter. Unilamellar or multilamellar liposomes are effective.
  • the second purpose of extrusion is to sterilize the mixture.
  • the liposomes are generally made smaller than 45 microns in diameter. Sizes less than 0.05 microns would theoretically work.
  • the process of the first embodiment has the advantage that, for example, in water 3NOBA only has a 0.5 millimolar concentration.
  • the present liposomal composition achieves concentrations of 15 millimolar.
  • the NOBA-containing liposomal solution is resistant to ascorbic acid. This makes it useful in laboratory mice experiments.
  • the solution may contain the NOBA monomer or NOBA dimer.
  • An example of such compounds are those in U.S. Pat. No. 5,262,564, issued November 16, 1993, e.g., L-cystine sulfmic adducts of 3- NOBA.
  • Parental injectable administration is generally used for subcutaneous, intramuscular or intravenous injections and infusions.
  • Injectables can be prepared in conventional forms, either as liquid solutions or suspensions or solid forms suitable for dissolving in liquid prior to injection.
  • a more recently devised approach for parenteral administration employs the implantation of a slow-release or sustained-released systems, which assures that a constant level of dosage is maintained, according to U.S. Pat. No, 3,710,795, which is incorporated herein by reference.
  • compositions may contain 0.1-99%, preferably 1-70% of the active pADPRT inhibitory compounds, especially the halo- C-amino, nitroso or nitro compounds of the formulae I, II or III, above as active ingredients.
  • 5 -iodo-6-amino-l, 2-benzopyrone (INH 2 BP), a novel inhibitor of the nuclear enzyme poly-ADP ribose polymerase (pADPRT) has recently been shown to regulate a variety of cellular signal transduction pathways and to abrogate in vivo tumorigenicity by a Ha-ras transfected endothelial cell line.
  • the present invention demonstrates the effect of pADPRT inhibitory compounds such as INH 2 BP on the activation by endotoxin (bacterial lipopolysaccharide, LPS) on the production of the inflammatory mediators tumor necrosis factor alpha (TNF), interleukin-lO(IL-lO) and interleukin-6 (IL-6), nitric oxide (NO) and prostaglandins in vitro and in vivo.
  • TNF tumor necrosis factor alpha
  • IL-6 interleukin-6
  • NO nitric oxide
  • the present invention shows the effect of pADPRT inhibitory compounds such as INH 2 BP on the activation of mitogen-activated protein kinase (MAP kinase) and nuclear factor kB (NF-kB) in vitro.
  • MAP kinase mitogen-activated protein kinase
  • NF-kB nuclear factor kB
  • LPS In cultured J774 and RAW 264.7 macrophages, LPS induced the production of prostaglandin metabolites, the release of TNF and the expression of the inducible isoform of NO synthase (iNOS).
  • the production of prostaglandins and of NO were inhibited by INH 2 BP in a dose-dependent manner, while the short-term release of TNF-alpha was unaffected.
  • INH 2 BP markedly suppressed LPS-mediated luciferase activity in RAW cells transiently transfected with a full length (-1592 bp) murine macrophage iNOS promoter-luciferase construct, but not in a deletional construct consisting of -367 bp.
  • INH 2 BP pretreatment inhibited :the induction of iNOS by LPS in rats, did not affect the LPS-induced TNF and IL-6 response, but enhanced LPS-induced IL-10 production.
  • INH 2 BP pretreatment markedly improved the survival of mice in a lethal model of endotoxin shock.
  • Poly-ADP ribose synthetase is a nuclear enzyme activated by DNA single strand breaks. Massive activation of PARS, in response to hydrogen peroxide- peroxynitrite- or ionizing radiation-induced extensive DNA single strand breakage can initiate an energy-depleting futile cycle culminating in cellular injury. The production of peroxynitrite has recently been demonstrated in various forms of inflammation, including arthritis and carrageenan-induced paw edema.
  • the present invention shows the effect of the novel, potent inhibitor of PARS, pADPRT inhibitory compounds such as 5-iodo-6-amino-l,2-benzopyrone (INH 2 BP), in a rat model of carrageenan- induced paw edema and in a mouse model of collagen-induced paw edema at l-4h.
  • Collagen-induced arthritis was induced in male DMA/1 J mice, with two injections of type II collagen at Day 1 and Day 21.
  • Oral treatment of mice with INH 2 BP 0.5 g/kg, daily
  • starting at the onset of arthritis Day 25
  • delayed the development of the clinical signs of arthritis at Days 26-35 delayed the development of the clinical signs of arthritis at Days 26-35.
  • INH 2 BP treated animals exhibited a reduced arthritic index (arthritic score: 20-50%> of the score seen in the vehicle-treated mice), and improved histological status, as examined in the knee and paw.
  • arthritic score 20-50%> of the score seen in the vehicle-treated mice
  • histological status as examined in the knee and paw.
  • mice macrophage cell lines J774 and RAW 264.7 were cultured in Dulbecco's modified Eagle's medium (DMEM) as escribed; Szabo et al, 1996, "DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite, "Proc. Natl. Acad. Sci. U.S.A.
  • DMEM Dulbecco's modified Eagle's medium
  • peritoneal macrophages were obtained from male Wistar rats and cultured in vitro for 24 hours in the absence or presence of LPS and with or without INH 2 BP. Rats were sacrificed and peritoneal macrophages taken and cultured in DMEM. Cells were treated with E. Coli IPS (10 mg/ml) or LPS and INF (50 ⁇ /ML) for various times, in the presence or absence of various concentrations (1-150 mM) INH 2 BP or other pharmacological inhibitors.
  • Raw cells were washed in PBS and collected and lysed using 100 ml of lysis buffer per million cells. (50 mM Tris-HCl pH 7.4, 1% NP-40, 0.4 M NaCl, 0.1 mM NaVO 3 , 50 mM KF, 1 mM EGTA, 2 mM PMSF, 25 nM okadaic acid, 1 mg/mL of each leupeptin, aprotinine, arnastatine and antipaine). Lysis was carried out for 20 minutes on ice followed by a 14 min. centrifugation at 13000 rpm in an Eppendorf centrifuge. Supernatants were saved and their protein content were assayed using the Bio-Rad dye assay.
  • Protein samples (50 mg/lane) were electrophoresed in a 100%) SDS-PAGE gel containing immobilized myelin basic protein (MBP, 250 mg/mL gel). After electrophoresis, the gel was washed once with 50 mM TRIS-HC1 pH 7.7 buffer (25 mL, 20 min.), followed by two 30 min. incubations with the same buffer containing 25%o i-propanol. The gel was then washed once with the Tris-Hcl buffer and soaked into a solution of 50 mM Tris-HCl pH 7.7, mM 2-mercaptoethanol, 5 M guanidine hydrochloride (50 mL) for an hour, changing the incubating solution at 30 min.
  • MBP myelin basic protein
  • the proteins were then renurtured by incubating the gel in five changes of a solution of 50 mM TRIS-HC1 Ph 7.7, Mm 2-mercaptoethanol, 0.04% NP-40 over a 16 hours period of time.
  • the gel was then washed twice and preincubated for half an hour in a solution containing 50 mM TRIS-HC1 pH 7.7, 5 mM MgCl 2 7 mm 2- mercaptoethanol.
  • the final incubation was carried out in the same solution supplemented with 10 mm of 32 p-g] ATP (50 mCi/assay) for an hour.
  • the gel was washed free of unbound radioactivity using 3x25 mL of 10%) TCA and 3x25 ml of 10%> acetic acid, dried and autoradiographed; Sasaki et al, 1995, "Permissive effect of ceramide on growth factor-induced cell proliferation,” Biochem. J. 311 :829-34.
  • cells were scraped, briefly centrifuged and pellets resuspended in 400 ml cold Buffer A [Hepes pH 7.9 (10 mM), KC1 (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (ImM), PMSF (0.5 mM), pepstatin A (1 mg/ml), leupeptin (10 mg/ml), and aprotinin (10 mg/ml)], on ice for 15 minutes, in the presence of 25 ml 1%.
  • cold Buffer A Hepes pH 7.9 (10 mM), KC1 (10 mM), EDTA (0.1 mM), EGTA (0.1 mM), DTT (ImM), PMSF (0.5 mM), pepstatin A (1 mg/ml), leupeptin (10 mg/ml), and aprotinin (10 mg/ml)
  • Nitrite in culture supernatants at 24 hours after stimulation was measured as described; Szabo et al, 1996, "DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite, "Proc. Natl. Acad. Sci. U.S.A. 93:1753-1758; Zingarelli et al, 1996, “Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccaride," J. Immunol. 156:350-358; Szabo et al, 1994, “Spermine inhibits the production of nitric oxide in immuno-stimulated J774.2 macrophages: requirement of a serum factor," Br. J.
  • nitrate was reduced to nitrite by incubation with nitrate reductase; Zingarelli et al, 1996, "Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccaride," J. Immunol. 156:350-358.
  • 6-keto prostaglandin F a production at 4 hours after LPS stimulation was measured in 100 ml samples of cell culture supernatant using a specific radioimmunoassay; Szabo et al, 1994, "Spermine inhibits the production of nitric oxide in immuno-stimulated J774.2 macrophages: requirement of a serum factor," Br. J. Pharmacol. 112:355-356.
  • Cytokine levels in plasma and cell culture supematants were determined by ELISA.
  • Plasma levels of IL-10 and IL-6 were measured using ELISA kits from Endogen (Endogen Inc., Boston, MA).
  • Concentrations of TNF- ⁇ in the plasma and cell culture supematants were determined using ELISA kits from Genzyme (Genzyme Corp., Boston, MA) as described; Szabo et al., 1997, "Isoproterenal regulates tumour necrosis factor, interleukin-10, interleukin-6 and nitric oxide production and protects against the development of vascular hyporeactivity in endotoxemia," Immunology 90:95-100.
  • Mitochondrial respiration at 24 hours was assessed by the mitochondrial-dependent reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan; Szabo et al, 1996, "DNA strand breakage, activation of poly-ADP ribosyl synthetase, and cellular energy depletion are involved in the cytotoxicity in macrophages and smooth muscle cells exposed to peroxynitrite, "Proc. Natl. Acad. Sci. U.S.A.
  • membranes were stripped with boiling 5 mM EDTA and rehybridized with a [ 32 P]-radiolabeled oligonucleotide probe for 18S ribosomal RNA as a housekeeping gene. After washing, exposure was carried out overnight using a Phosphor Imager screen.
  • iNOS promoter activity was evaluated by transient transfection of AW 264.7 cells with reporter gene constructs incorporating the 5 ' murine macrophage iNOS promoter region upstream from the reporter gene luciferase; Lowenstein et al, 1993, "Macrophage nitric oxide synthase gene: two upstream regions mediate induction by interferon gamma and lipopolysaccharide.” Proc. Natl. Acad. Sci. U.S.A.
  • mice Male Wistar rats and Male BALB/c mice were obtained from Charles River Laboratories (Wilmington, MA or Budapest, Hungary). Animals received food and water ad libitum, and lighting was maintained on 12 hour cycle. Rats were injected i.p. with E. coli LPS (15 mg/kg) and sacrificed at 6 hours. Plasma samples were taken for nitrite/nitrate determinations and lung samples for iNOS measurements. Separate groups of rats were treated with INH 2 BP (10 mg/kg i.p.) 10 minutes prior to LPS or 2 hours after LPS injection.
  • INH 2 BP 10 mg/kg i.p.
  • mice were injected i.p. with either drug vehicle, or with INH,BP (10 mg/kg) in a volume of 0.1 ml/10 g body weight. Half an hour later they were challenged with 4 mg/kg of i.p. LPS. The animals were killed at 90 minutes after LPS treatment, blood was collected in ice-cold Eppendorf tubes containing EDTA, and centrifuged for 10 minutes at 4°C. The plasma was stored at -7°C until assayed.
  • mice In survival studies with mice, animals were subjected to i.p. injection of LPS (120 mg/kg) at time 0 and survival was monitored for 42 hours after LPS. Separate groups of mice received vehicle or INH 2 BP treatment (0.1-10 mg/kg i.p.) at times -18 hours, -4 hours, 0 hours, 6 hours, 24 hours and 30 hours relative to LPS.
  • LPS 120 mg/kg
  • INH 2 BP treatment 0.1-10 mg/kg i.p.
  • DMEM, RPM1, TRIZOL and fetal calf serum were from Gibco (Grand Island, NY).
  • [ 3 H]NAD+ and [ 32 P]NAD+ were obtained from DuPont NEN (Boston, MA).
  • Alcohol dehydrogenase and ND+ were obtained from Boehringer Mannheim (Indianapolis, IN).
  • PD 98059 was obtained from Cal biochem (La Jolla, CA). All other drugs were obtained from Sigma (St. Louis, MO).
  • INH j BP suppresses LPS-induced nitric oxide and prostaglandin but no TNF-a production in J774 macrophages
  • INH 2 BP treatment caused a dose-dependent inhibition of LPS-induced nitrite formation in J774 macrophages (Fig. la).
  • INH,BP suppressed LPS-induced production of 6-keto prostaglandin F, a (Fig. lb), but not the production of TNF (Fig. lc), and restored the LPS-induced suppression of mitochondrial respiration (Fig. Id).
  • INH 2 BP caused a marked inhibition of iNOS mRNA and protein expression (Fig. 2a- c).
  • the inhibition of nitrite production by INH 2 BP was greatly diminished when the agent was given several hours LPS, as opposed to prior to the stimulus of iNOS induction (Fig. 3a).
  • the inhibitory effect of INH 2 BP on iNOS was greatly reduced when LPS was used in combination was interferon-gamma (INF-g 50 u/raL) for immunostimulation (Fig. 3b).
  • INH 2 BP pretreatment significantly reduced the LPS-induced increase in plasma nitrite-nitrate and the increase in pulmonary iNOS activity in conscious rats (Fig. 5).
  • the inhibitory effect of INH 2 BP on NO production was reduced when the agent was added to the cells or to the animals several hours after LPS stimulation (Fig. 5).
  • INH 2 BP did not significantly affect the LPS-induced increase in plasma TNF levels in mice (Fig. 6a). Nor did INH 2 BP affect LPS-induced IL-6 production (Fig. 6C). However, INH 2 BP caused an augmentation of the LPS-induced EL- 10 plasma response (Fig. 6b)
  • mice by INH 2 BP had a significant and dose-dependent improvement in the survival rate subjected to lethal doses of LPS (Fig. 7).
  • INH 2 BP activity abolishes LPS-induced activation of MAP kinase but does not alter activation and nuclear translocation of NF-kB.
  • MAP kinase mitogen-activated protein kinase
  • Basal MAP kinase activity was slightly suppressed by the MAP kinase kinase inhibitor; Pang et al, 1995, "Inhibition of MAP kinase kinase blocks the differentiation of PC- 12 cells induced by nerve growth factor," J. Biol. Chem. 270:13585-8; PD 98059 (100 mM), and LPS-induced MAP kinase activation was also inhibited (Fig. 8).
  • pADPRT Poly (ADP-ribose) synthetase
  • pADPRT can bind to numerous cellular protein and DNA site and can exert pleitropic cellular regulatory functions; Bauer et al, 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5 -iodo-6-amino-l, 2-benzopyrone (INH 2 BP)," Int. J. Oncol.
  • pADPRT activation has also been proposed to serve as a mechanism to induce cell death, in particular after radiation injury, and oxidant stress; Cochrane, 1991, “Mechanisms of oxidant injury of cells,” Molec. Aspects Med. 12: 137-147; Berger, 1991, "Oxidant-induced cytotoxicity: a challenge for metabolic modulation,” Am. J. Respir. Cell. Biol. Biol. 4:1-3.
  • pADPRT One of the important physiological functions of pADPRT may be the regulation of enzyme induction, gene expression and cell differentiation; Bauer et al, 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone (INH 2 BP)," Int. J. Oncol.
  • INH 2 BP in bovine endothelial cells transfected with Ha-ras abrogates tumorigenicity, arrests cell multiplication, increases toposomerase I, toposomerase II, and MAP kinase activity, down-regulates DNA- methyl-transferase and protein kinase C, and ODC increases the hypophosphorylation of Rb protein, and inhibits the expression of the ras gene without the loss of the oncogene, Bauer et al, 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5-iodo-6-amino-l, 2-benzopyrone INH,BP)," Int.
  • INH 2 BP suppresses the LPS-induced induction of iNOS.
  • nitric oxide (NO) synthease iNOS
  • NO nitric oxide synthease
  • the promoter region of the murine iNOS gene has been cloned, and separate regions responsible for inducibility in response to LPS and to IFN have been identified.
  • LPS-mediated induction if iNOS appears to involve the mobilization and nuclear translocation of NF-kB, with subsequent binding to the iNOS promoter.
  • the induction of iNOS can also be inhibited by pharmacological inhibitors of tyrosine kinase and NF-kB activation; Szabo, C; 1995, "Alterations in the production of nitric oxide in various-forms of circulatory shock," New Horizons 2:3-32.
  • the inhibitory effect of INH 2 BP on iNOS expression was indicated by the inhibition on nitrite production., iNOS mRNA expression and iNOS protein expression.
  • the regulation occurs in the early stage of iNOS induction, since INH 2 BP gradually loses its effectiveness when applied at increasing times after the stimulus for iNOS induction.
  • the regulation of INH 2 BP of iNOS induction occurs both in vitro and in whole animals.
  • our data show that the LPS-induced production of cyclooxygenase metabolites, similar to the induction of iNOS, is modulated by INH 2 BP.
  • INH 2 BP INF-induced transcription factors
  • the pADPRT inhibitors 30 aminobenzamide and nicotinamide were used at high concentrations (10-30 mM), which inhibited total protein and RNA synthesis, and may have had additional, pharmacological actions, such as free radical scavenging; Hauschildt et al, 1992, "Induction of nitric oxide synthase in L929 cells by tumour-necrosis factor alpha is prevented by inhibitors of poly (ADP-ribose) polymerase," Biochem. J. 288:255- 260.
  • the present experiments, using ⁇ NH 2 BP, further suggest the pleiotropic involvement of pADPRT in the process of iNOS mRNA transcription.
  • ADP-ribosylation of histones and nucleases may be involved in the maintenance of a relaxed chromatin structure; Bauer et al, 1995, "Modification of growth related enzymatic pathways and apparent loss of tumorigenicity of a ras-transformed bovine endothelial cell line by treatment with 5 -iodo-6-amino-l, 2-benzopyrone (INH 2 BP)," Int. J. Oncol.
  • MAP kinase has been shown to be activated in various cell types treated with LPS or various pro- inflammatory cytokines (TNF-alpha, interleukin-1, nerve growth factor); Kyriakis et al, 1996, “Sounding the alarm: protein kinase cascades activated by stress and inflammation," J. Biol Chem. 221:24313-24316; Matsuda et al. , 1994, "Signaling pathways mediated by the mitogen-activated protein (MAP) kinase kinase/MAP kinase ascade," J. Leukocyte Biol.
  • MAP mitogen-activated protein
  • NF-kB Since activation of NF-kB is a major pathway in the inflammatory response, and it is involved in the induction of iNOS by LPS, but not by INF; Szabo, C; 1995, "Alterations in the production of nitric oxide in various forms of circulatory shock," New Horizons 3:3-32: Martin et al, 1994, "Role of interferon regulatory factor 1 in induction of nitric oxide synthase,” J. Exp. Med. 180:977-84. we sought to investigate potential effect of INH 2 BP on NF-kB.
  • INH 2 BP does not alter the nuclear translocation of NF-kB activation, or the modulation of NF-kB- mediated cellular events by INH,BP, if any, may occur at a cellular event distal to nuclear translocation of NF-kB.
  • INH 2 BP modulates the inflammatory process at multiple levels
  • Reduction by pADPRT inhibitors of the expression of pro-inflammatory genes iNOS and COX-2, and the subsequent reduced formation of NO and prostaglandins may be beneficial in various forms of inflammation; Nathan, 1992, “Nitric oxide as a secretory product of mammalian cells,” FASEB J. 3:151-3064; Vane, J.R., The Croonian Lecture 1993, "The endothelium: maestro of the blood circulation,” Proc. Rov. Soc.
  • enhanced release of IL-10 may have additional anti- inflammatory actions; Liles et al, 1995, “Review: nomenclature and biologic significance of cytokines involved in inflammation and the host immune response," J. Infect Pis. 122:1573-80; Giroir, 1993, “Mediators of septic shock: new approaches for interrupting the endogenous inflammatory cascade," Critical Car. Med.
  • nitric oxide a reactive oxidant formed by the rapid reaction of NO and superoxide
  • Crow et al 1995, "The role of peroxynitrite in nitric oxide-mediated toxicity", Current Top Microbiol. Immunol. 196:57-73
  • Pryor et al 1995, "The chemistry of peroxynitrite: a product from the reaction of nitric oxide with superoxide”. Am. J. Physiol. L699- L772.
  • peroxynitrite has been demonstrated in a variety of inflammatory conditions, including systemic inflammation induced by endotoxin; Szabo et al, 1995, "Alterations in nitric oxide production in various forms of circulatory shock” New Horizons 3:2-32; arthritis; Kaur et al, "Evidence for nitric oxide-mediated oxidative damage in chronic inflammation. Nitrotyrosine in serum and synovial fluid from rheumatoid patients", FEBS Lett. 1359:9-12; and carageenan induced paw edema; *Salvemini et al, 1996.
  • SUBST ⁇ UTE SHEET (RULE 26) in the treatment of arthritis are, in fact, scavengers of peroxynitrite; Whiteman et al, 1996 "Protection against peroxynitrite dependent tyrosine nitration and alpha 1- antiproteinase inactivation by some anti-inflammatory drugs and by the antibiotic tetracycline" Annals, of the Rheumatic Diseases 55_:383-7.
  • the realization that a significant part of the NO-related cytotoxicity is due to the formation of peroxynitrite has necessitated the development of novel therapeutic approaches based around the formation and action of peroxynitrite.
  • One of the intracellular pathways triggered by peroxynitrite is related to DNA single strand breakage and activation of poly (ADP-ribose) synthetase (PARS); Szabo et al, 1996, "The role of peroxynitrite in the pathophysiology of shock, inflammation and schemia-reperfusion injury", Shock 6: 79-88; *Szabo, 1996b).
  • Pronounced activation of PARS can rapidly deplete the intracellular concentration if its substrate, NAD+, slowing the rate of glycolysis, electron transport, and, therefore, ATP formation, resulting in cell dysfunction; *Berger, 1991; *Cochrane, 1991. Accordingly, inhibitors of PARS protect against cellular injury under these conditions.
  • the arthritic index for each mouse was calculated by adding the four scores of the individual paws.
  • animals were sacrificed under anesthesia and paws and knees were removed and fixed for histological examination. Histological examination was done by an investigator blinding for the treatment regime.
  • the primary mode of action of INH 2 BP is likely to be related to interruption of the futile intracellular cascade characterized by DNA injury.
  • Inhibition of this pathway with various inhibitors of PARS, such as 3- aminobenzamide, nicotinamide and INH 2 BP has been shown to protect multiple cell types from injury; Berger, 1991; *Cochrane, 1991; Szabo et al, 1996, "The role of peroxynitrite in the pathophysiology of shock, inflammation and schemiareperfusion injury", Shock 6:79-88; *Szabo, 1996b.
  • These high concentrations of these agents may have additional pharmacological actions, such as inhibition of total protein and RNA synthesis, and/or free radical scavenging actions; *Hauschildt et al, 1992, *Pellat-Seceunyk et al, 1994; Zingarelli et al, 1996, "Peroxynitrite-mediated DNA strand breakage activates poly-ADP ribosyl synthetase and causes cellular energy depletion in macrophages stimulated with bacterial lipopolysaccharide" J. Immunol. 156:350- 358.
  • INH 2 BP on the other hand, effectively suppressed the expression of iNOS even at lower, non- cytotoxic concentrations (100-300 mM).
  • INH 2 BP several modes of action should be considered, since this agent is an inducer of alkaline phosphatases, with secondary, pleiotropic modulation of cellular responses; *Bauer et al, 1996; *Szabo et al, 1997).
  • Experiments in cells or animals with ablation of the PARS gene are required to definitely address the question as to whether inhibition of PARS per se suppresses the process of iNOS induction.
  • PARS has been implicated in the regulation of a variety of genes, including the major histocompatibility complex class II gene (*Hiromatsu et al, 1992; Taniguchi et al, 1993), ras c-myc (*Bauer et al, 1996, *Nagao et al. 1991), PNA methyltransferase gene (*Bauer et al, 1996) and protein kinase C (*Bauer et al, 1996).
  • PARS inhibition is not only expected to inhibit part of the oxidant-induced cytotoxicity, and thus may be more effective when applied in combination with other free radical scavengers or other immunosuppressive agents.
  • the results of the present studies support he view that PARS inhibition, alone, or in combination with other anti-inflammatory agents, represents a promising novel anti-inflammatory approach.
  • compounds of formulae II, and III are used to treat inflammation or inflammatory diseases, as well as treating gram negative and gram positive infections.

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Abstract

Procédé de traitement de l'inflammation ou des maladies inflammatoires chez un animal ou un mammifère, qui consiste à administrer à cet animal ou mammifère une quantité efficace d'un composé inhibiteur de poly-ADP ribose polymérase (pADPRT).
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See also references of WO9851308A1

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WO1998051308A1 (fr) 1998-11-19
NZ501650A (en) 2001-11-30
AU7484798A (en) 1998-12-08
WO1998051307A1 (fr) 1998-11-19
CA2289119A1 (fr) 1998-11-19
IL132758A0 (en) 2001-03-19
EP1009404A4 (fr) 2009-07-01
AU745790B2 (en) 2002-03-28
CN1261278A (zh) 2000-07-26
JP2002502367A (ja) 2002-01-22
JP4362638B2 (ja) 2009-11-11
AU7492698A (en) 1998-12-08
CA2289119C (fr) 2011-03-15
CN1198614C (zh) 2005-04-27
BR9809115A (pt) 2002-01-02

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