Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/13—Amines
  • A61K31/135—Amines having aromatic rings, e.g. ketamine, nortriptyline
• • 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
• • 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
  • A61K31/28—Compounds containing heavy metals
• • 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
  • A61K31/28—Compounds containing heavy metals
  • A61K31/295—Iron group metal compounds
• • 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
  • A61K31/40—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with one nitrogen as the only ring hetero atom, e.g. sulpiride, succinimide, tolmetin, buflomedil
• • 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
  • A61K31/44—Non condensed pyridines; Hydrogenated derivatives thereof
• • 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
  • A61K31/555—Heterocyclic compounds containing heavy metals, e.g. hemin, hematin, melarsoprol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P11/00—Drugs for disorders of the respiratory system
  • A61P11/16—Central respiratory analeptics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
  • A61P21/02—Muscle relaxants, e.g. for tetanus or cramps
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • 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
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
• • 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
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/08—Vasodilators for multiple indications
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P9/00—Drugs for disorders of the cardiovascular system
  • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

• the present invention is for methods of use for the decomposition of peroxynitrite by metal complexes, novel pharmaceutical compositions, and methods of use therefor.
• the present invention now provides a method for treating selected diseases comprising the decomposition of peroxynitrite with the use of a compound which is a metal complex.
• This decomposition preferably produces benign agents preventing formation of deleterious decomposition products such as oxygen radicals and which also further prevents inactivation of superoxide dismutase (SOD) by the presence of peroxynitrite.
• SOD superoxide dismutase
• the method of use for selected metal complexes of the present invention, as well as novel pharmaceutical compositions for such use is for the treatment of diseases advantageously affected by treatment comprising decomposition of peroxynitrite at a rate accelerated over a natural background rate of decay which comprises administration of an rate- accelerating effective amount of the metal complex in unit dosage form.
• the methods of treatment and novel compositions of this invention provide a twofold benefit in the treatment of diseases (1) accelerated rate of catalytic decomposition of peroxynitrite and (2) protection of SOD against inactivation by peroxynitrite.
• the present invention provides for a method of treatment of human diseases advantageously affected by such decomposition by protection from the deleterious effects resulting from the presence of peroxynitrite in the human body not heretofore known.
• such decomposition offers protection against diseases associated with the overproduction of superoxide.
• ischemic reperfusion injuries such as stroke, head trauma and myocardial ischemia, sepsis, chronic or acute inflammation (such as arthritis and inflammatory bowel disease and the like), adult respiratory distress syndrome, cancer, bronchopulmonary dysplasia, side effects from drug treatment of cancer, cardiovascular diseases, diabetes (not included for treatment by vanadium porphyrin complexes), multiple sclerosis, parkinson's disease, familial amyotrophic lateral sclerosis, and colitis and specific 5 neuronal disorders, preferably ischemic reperfusion, inflammation, sepsis, multiple sclersis, parkinson's disease and stroke.
• ischemic reperfusion injuries such as stroke, head trauma and myocardial ischemia, sepsis, chronic or acute inflammation (such as arthritis and inflammatory bowel disease and the like), adult respiratory distress syndrome, cancer, bronchopulmonary dysplasia, side effects from drug treatment of cancer, cardiovascular diseases, diabetes (not included for treatment by vanadium porphyrin complexes), multiple s
• Nitric oxide is known for its dual physiological role as helpful messenger and harmful intermediate. Nitric oxide is shown to
• ID be generated in numerous cell types including macrophages, neutrophils, hepatocytes and endothelial cells. See Hibbs et al, Science. 1987, 235, 473-476; Rimele et al, J. Pharmacol. Exp. Ther.. 1988, 245, 102-111; Curran et al, J. Exp. Med.. 1989, 170, 1769-1774; and Plainer et al, Nature. 1987, 327, 524-526; respectively.
• NOS nitric oxide synthases
• peroxynitrite Bactericidal Activity" by L. Brunnelli and J.S. Beckman). Instead a more reactive species, peroxynitrite, produced by the reaction of superoxide and NO, is found to play a role in the cytotoxicity observed with the over-production of NO. Peroxynitrite is known to decompose 0 via a process which is first order in protons. The rate of proton catalyzed decomposition of peroxynitrite (hereinafter "the natural background rate of decay”) is understood from its study over a variety of pH ranges (see;Keith et al. J Chem Soc (A), p.90, 1969).
• peroxynitrite decomposes to give the hydroxyl radical and nitrogen dioxide, a potent nitrating agent. Both of these species are potent oxidants shown to react with lipid membrane and sulfhydryl moieties (See Radi et al "Peroxynitrite Oxidation of Sulfhydryls" in The Journal of Biological Chemistry. Vol.
• the present invention also provides enhancement of known physiological benefits of superoxide dismutase in the treatment of diseases based on such benefits.
• SOD and its mimics have been shown to be useful in the treatment of diseases for the inhibition of an overproduction of superoxide and nitric oxide.
• the present invention relates to the known treatment for diseases by SOD and SOD mimics.
• the Beckman et al PCT application also teaches that SODs catalyze the dismutation of the oxygen radical superoxide and provides references which show SOD and variants thereof have been commonly utilized to prevent or reduce oxidation injury in the treatment of stroke and head trauma, myocardial ischemia, abdominal vascular occlusion, cystitis, and a variety of inflammatory conditions. Beckman et al PCT application also recognizes the presence of peroxynitrite in these same disease conditions associated with O2- without indicating the further improvements of the present invention. Further teachings to the diseases known to be associated with treatment by SOD or its mimics are found in EP Publication No. 0524161 (EP Appl. No. 92870097) which is incorporated by reference therefor.
• Porphyrin complexes are disclosed in U.S. Patent No. 5,284,674 as valuable diagnostic and therapeutic agents, non-peptide phaeophorbide analogs are disclosed in Japanese Patent Publication Hei 5-331063 as endocerine receptor antagonists, carotenoporphyrins are disclosed in U.S. Patent 5,286,474 to be valuable for locating and visualizing mammalian tumor tissue and similar nitrogen containing macrocycles without a complexed metal are disclosed as cytotoxic agents in U.S. Patent No. 5,283,255. No metal complexes and their usefulness are shown as now found in the present invention.
• Metal complexes are, however, shown to be useful compounds in Derwent Abstract as intermediates in JP05277377-A and MRI agents in U.S. Patent No. 5,284,944; cyan pigments in U.S. Patent No. 5,286,592; photoconductive phthalocyanine compositions in U.S. Patent No. 5,283,146; a recording layer in an optical recording medium in U.S. Patent No. 5,284,943 and near infrared absorbers and display/recording materials in an abstract for U.S. 5,296,1632.
• Iron hemoprotein is disclosed to be an effective agent to bind or oxidize nitric oxide which has a deleterious physiological effect when induced by a cytokine or by endotoxin for the treatment of diseases such as septic shock in PCT application No. PCT US93/01288 (Publication No. WO 93/16721).
• nitrogen containing selected macrocycles are shown in JPO5331063 as endothelin receptor antagonists for treating and preventing hypertension, acute renal failure, cardiomyopathy and myocardial infarction.
• the present invention is a method of treating a disease which is advantageously affected by decomposition of peroxynitrite which is accelerated over, ie above or more than, a natural background rate of decay in humans suffering from the disease comprising administering a compound or compound which is a metal complex whereby the peroxynitrite is decomposed.
• peroxynitrite is decomposed to a benign species.
• the compound is a ligand structure providing a complexed metal, such as one of the transition metals, such as Mn, Fe, Ni and V.
• Preferred ligands are macrocyclic ligands, such as porphyrins, aza macrocycles and the like.
• the present invention is a novel method of treating a disease in mammals, including humans, advantageously affected by the absence of peroxynitrite comprising administration of an accelerated- decomposition effective amount of a compound of the formula Structure I
• R 3 , Re, R9 or R 1 2 are independently selected a group consisting of H, alkyl, alkenyl, CH 2 COOH, phenyl, pyridinyl, and N-alkylpyridyl such that phenyl, pyridinyl and N-alkylpyridyl are
• phenyl is optionally substituted by halogen, alkyl, aryl, benzyl, COOH, CONH 2 , SO3H, N0 2 , NH 2 , N(R) 3 +, wherein R is hydrogen, alkyl, or alkylaryl; pyridinyl is optionally substituted by halogen, alkyl, aryl, benzyl, COOH CONH 2 , SO3H, N0 2 , NH 2 , N(R) 3 + or NHCOR' wherein R is as defined above and R' is alkyl; and N-alkylpyridine ring is optionally substituted by halogen, alkyl, aryl, benzyl, COOH, C0NH 2 , SO3H, N0 2 , NH 2 , N(RV or NHCOR' wherein R and R' are as defined above; Ri, R 2 , R 4 , R 5 , R 7 ,
• R' is CH or N
• Rn Ri2» Ri3» Ri4» R15, and Ri ⁇ are independently selected from a group consisting of H, SO 3 H, COOH,
• Ri, R5, Rg, and R 13 are independently a direct bond or CH 2 ; R2, R2 * , R4, R4 * , Re, Re', Re, Re', Rio, Rio', R12, R14, R14',
• R 1 6, Ri ⁇ ' are independently H, or alkyl
• R3, R7, R 11 , R ⁇ 5 are independently H or alkyl; X, Y, Z and M are as defined above;
• Ri, R5, Re, and 12 are independently a direct bond or CH 2;
• R2, R2 ,R4, R4 ,R6, Re', R7, R9, R9', R11, Rn'» R13, R13 1 , R14 are independently H or alkyl;
• R 3 and Rio are independently H or alkyl; X , Y, Z and M are as defined above; wherein Ri, R 4 , Re, R 12 are independently a direct bond or CH 2 ;
• R2, R2', R3, R5, R5', R7, R9, R9', R11, Rii', R13, Ri3', R14 are independently H or alkyl; Rio is H or alkyl;
• X, Y, Z and M are as defined above;
• Ri, R 4 , R 7 and Rio are independently a direct bond or CH 2 ;
• R2, R2', R3, R5, R5', Re, Rs, Re', R9, R11, R11' and R12 are independently H or alkyl;
• X, Y, Z and M are as defined above; wherein Ri, R 4 , Re and Rn are independently a direct bond or CH2;
• R2, R3, R3', R5. R5', » R7', R9, Rio, Rio', R12, R12' and R13 are independently H or alkyl;
• R ⁇ is hydrogen and alkyl; X, Y, Z and M are as defined above;
• Ri, R 4 , R 7 and Rio are independently H or alkyl
• R2, R3, R3', R ⁇ » R5', R ⁇ , R ⁇ , R9, R9', R11, R11' and R12 are independently H or alkyl;
• X, Y, Z and M are as defined above;
• Ri, R3, R 4 and Re are independently H or alkyl;
• R 2 and R5 are independently selected from the group consisting of H, alkyl, SO 3 H, N0 2 , NH 2 , halogen, COOH, and N(R) 3 + wherein R is as defined above;
• X, Y, Z and M are as defined above;
• Ri, R 2 , R3, R 4 are independently selected from the group consisting of H, alkyl, SO3H, N0 2 , NH 2 , halogen, COOH and N(R) 3 + wherein R is as defined above;
• RI, RI', R2, R2 ⁇ R3, R3', R4, R4', R5, R5', R6, R6', R7 and R7' are independently selected from a group consisting of H, alkyl, alkoxy, NO 2 , aryl, halogen, NH 2 , SO3H, and Re, Re', R7 and R7' may each be taken together with one other of Re, R ⁇ ', R7 and R7' to form a cyclic group, preferably a 6 carbon cycloalkyl group;
• Mi is Fe, Ni or V; X, Y and Z are as defined above together with a pharmaceutically acceptable carrier, preferably in unit dosage form.
• the present invention is also a pharmaceutical composition for the treatment of a disease in humans advantageously affected by accelerated decomposition over the natural background rate of decay of peroxynitrite comprising an amount effective for the accelerated decomposition of peroxynitrite in humans of a compound of the formula I, II, IIIA, IIIB, IIIC, HID, IIIE, IIIF, IIIG, IIIH as defined above with a pharmaceutically acceptable carrier in unit dosage form, preferably oral unit dosage form.
• X, Y and Z are each a pharmaceutically acceptable anion or cation.
• FIGURE 1 Plot of & 0 bs vs catalysts concentration for Fe(III)TMPS and Fe(III)TPPS illustrating catalytic nature of decomposition of peroxynitrite by metal complexes.
• FIGURE 2 Plot illustrating the inactivation of CuZnSOD by peroxynitrite.
• FIGURE 3 Plot illustrating the concentration dependant protection of CuZnSOD against inactivation by peroxynitrite using peroxynitrite decomposition catalysts Fe(III)TMPyP.
• FiGURE 4 Plot illustrating the concentration dependant protection of CuZnSOD against inactivation by peroxynitrite using peroxynitrite decomposition catalyst Fe(III)TMPS.
• FIGURE 5 Peroxynitrite-mediated human microvascular endothelial cell injury. Authentic peroxynitrite was overlaid directly onto to 51 Cr- labeled HMDE cells grown in 96-well cell culture plates. After 45 min, the amount of specific cell injury was determined and correlated to peroxynitrite concentration by least squares regression line. Values represent the average of three replicates +/- SEM.
• FIGURE 6 Peroxynitrite catalysts, Fe(TMPyP) (triangle) and Ni(II)dienoN 4 )PF ⁇ ( circle ) were added to HOME cells in the cell injury assay immediately before the addition of authentic peroxynitrite. After 45 min, the amount of specific cell injury was assessed by the amount of radiolabel released into the medium. Values represent the average of three replicas +/- SEM. *p ⁇ 0.01 vs. 0 uM control by Dunnett's t Test.
• FIGURE 7 Inhibition of neutrophil-mediated injury to human aortic endothelial cells by Fe(TMPyP).
• Peroxynitrite catalyst, Fe(TMPyP) was added to neutrophils in the cell injury assay immediately before activation by TNF/C5a. After 2 h, the amount of specific cell injury was assessed by the amount of radiolabel released into the medium. Values represent the average of three replicas +/- SEM. *p ⁇ 0.01 vs. 0 uM control by Dunnett's t Test.
• FIGURE 8 Comparison of Ni and Fe Catalyst Protection of RAW Cells from PN(peroxynitrite)-mediated Injury.
• RAW 264.7 cells were plated at approximately 2xl0 5 per well of a 96-well plate.
• PN(360 micromolar) was added to every well of cells in the presence of increasing concentrations of Ni catalyst or FeTMPyP resulting in total protection from PN-mediated injury as determined by the ability of cells to metabolize Alamar Blue to a fluorescent product. Each condition represents the mean of 4 wells ⁇ sem.
• FIGURE 9 Protection from PN-mediated RAW Cell Injury by Fe Catalysts.
• Cells were treated with 500 micromolar PN in the presence or the absence of the following catalysts: FeTMPyP, FeTMPS, FeTPPS.
• Cell viability was monitored as described in the text and figure legends 1, 2 and 3. Values represent the mean of 4 determinations ⁇ sem.
• FIGURE 10 Effects of FeTMPS, FeTMPyP or ZnTMPyP (30 mg/kg, i.v bolus) administered 3 h after challenge with E. coli lipopolysaccharide (LPS, 3 mg/kg, i.v bolus) on the increase in leakage of radiolabelled albumin (plasma extravasation, ⁇ l/g tissue) observed 1 h later (e.g 4 h after LPS challenge) in the rat jejunum. Results are shown as mean ⁇ s.e.m of 4-8 rats.
• alkyl alone or in combination, means a straight-chain or branched-chain alkyl radical containing from 1 to about 22 carbon atoms, preferably from about 1 to about 18 carbon atoms, and most preferably from about 1 to about 12 carbon atoms.
• radicals include, but are not limited to methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, sec-butyl, tert-butyl, pentyl, iso-amyl, hexyl, octyl, nonyl, decyl, dodecyl, tetradecyl, hexadecyl, octadecyl and eicosyl.
• aryl alone or in combination, means a phenyl or naphthyl radical which optionally carries one or more substituents selected from alkyl, cycloalkyl, cycloalkenyl, aryl, heterocycle, alkoxyaryl, alkaryl, alkoxy, halogen, hydroxy, amine, cyano, nitro, alkylthio, phenoxy, ether, trifluoromethyl and the like, such as phenyl, p-tolyl, 4-methoxy-phenyl, 4-(tert-butoxy)phenyl, 4-fluorophenyl, 4-chlorophenyl, 4-hydroxyphenyl, 1-naphthyl, 2-naphthyl, and the like.
• aralkyl alone or in combination, means an alkyl or cycloalkyl radical as defined herein in which one hydrogen atom is replaced by an aryl radical as defined herein, such as benzyl, 2-phenylethyl, and the like.
• heterocyclic means ring structures containing at least one other kind of atom, in addition to carbon, in the ring. The most common of the other kinds of atoms include nitrogen, oxygen and sulfur.
• Examples of 5 heterocyclics include, but are not limited to, pyrrolidinyl, piperidyl, imidazolidinyl, tetrahydrofuryl, tetrahydrothienyl, furyl, thienyl, pyridyl, quinolyl, isoquinolyl, pyridazinyl, pyrazinyl, indolyl, imidazolyl, oxazolyl, thiazolyl, pyrazolyl, pyridinyl, benzoxadiazolyl, benzothiadiazolyl, triazolyl and tetrazolyl groups.
• cycloalkyl alone or in combination means a cycloalkyl radical containing from 3 to about 10, preferably from 3 to about 8, and most preferably from 3 to about 6 carbon atoms.
• cycloalkyl radicals include, but are not limited to, cyclopropyl, cyclobutyl, cyclophetyl, cyclohexyl, cycloheptyl, cyclooctyl, and perhydronaphthyl.
• cycloalkenyl alone or in combination, means a cycloalkyl radical having one or more double bonds.
• examples of cycloalkenyl radicals include, but are not limited to cyclopentenyl, cyclohexenyl, cycloooctenyl, cyclopentadienyl, cyclohexadienyl, and cyclooctadienyl.
• the macrocyclic ligands useful in the present invention wherein the formula is Structure III can be prepared according to the general synthetic methods known in the art for preparation of certain ligands. See, for example, DGoedken, V. L.; Molin-Case, J.; Whang, Y-A; J.C.S.Chem.Comm.
• the macrocyclic ligands useful in the present invention wherein the formula is Structure IV can be prepared according to the general synthetic methods known in the art for preparation of certain ligands. See, for example,
• the compounds of the present invention can possess one or more asymmetric carbon atoms and are thus capable of existing in the form of optical isomers as well as in the form of racemic or nonracemic mixtures thereof.
• the optical isomers can be obtained by resolution of the racemic mixtures according to conventional processes, for example by formation of diastereoisomeric salts by treatment with an optically active acid.
• appropriate acids are tartaric, diacetyltartaric, dibenzoyltartaric, ditoluoyltartaric and camphorsulfonic acid and then separation of the mixture of diastereoisomers by crystallization followed by liberation of the optically active bases from these salts.
• a different process for separation of optical isomers involves the use of a chiral chromatography column optimally chosen to maximize the separation of the enantiomers.
• Still another available method involves synthesis of covalent diastereoisomeric molecules by reacting one or more secondary amine group( s) of the compounds of the invention with an optically pure acid in an activated form or an optically pure isocyanate.
• the synthesized diastereoisomers can be separated by conventional means such as chromatography, distillation, crystallization or sublimation, and then hydrolyzed to deliver the enantiomerically pure ligand.
• the optically active compounds of the invention can likewise be obtained by utilizing optically active starting materials, such as natural amino acids.
• peroxynitrite is prepared and isolated as its sodium salt by the reaction of acidic hydrogen peroxide with sodium nitrite followed by rapid quenching with NaOH as set out by Halfpenny and Robinson, in J. Chem. Soc. 1952, 928-938.
• Peroxynitrite has an absorbance maximum at 302 nm with an extinction coefficient of 1670 M-icm-i. Therefore, it is possible to directly observe the decomposition of peroxynitrite by stop-flow spectrophotometric analysis by monitoring the decomposition of the absorbance at 302 nm. That is, such observation of the decomposition of peroxynitrite at a rate accelerated over the natural decomposition rate with the addition of the metal complex identifies a compound of the present invention.
• peroxynitrite inactivates CuZnSOD enzyme in a concentration dependant manner. Since it is known peroxynitrite also inactivates MnSOD (See “Peroxynitrite- Mediated Tyrosine Nitration Catalyzed by Superoxide Dismutase” by Ischiropoulos et al in Archives of Biochemistry and Biophysics. Vol. 298, No. 2, November 1, pp. 431-437, 1992), the present invention provides a compound which protects CuZnSOD from inactivation by peroxynitrite.
• the compound of the present invention is shown to be useful in treating a disease in a human advantageously affected by the presence of the SOD enzyme. That is, the treatment of the present invention is for a disease state either caused by the presence of a peroxynitrite of caused by the lack of the protective presence of the SOD enzyme such as in a myocardial infarct, stroke or an autoimmune disease. These latter diseases are also shown to be associated with the presence of peroxynitrite.
• Contemplated equivalents of the general formulas set forth above for the compounds and derivatives as well as the intermediates are compounds otherwise corresponding thereto and having the same general properties such as tautomers of the compounds and such as wherein one or more of the various R groups are simple variations of the substituents as defined therein, e.g., wherein substituents which are a higher alkyl group than that indicated, or where the tosyl groups are other nitrogen or oxygen protecting groups or wherein the O-tosyl is a halide.
• anions having a charge other than 1 will result in a slight modification of the general formula for the complex set forth above.
• a substituent is designated as, or can be, a hydrogen
• the exact chemical nature of a substituent which is other than hydrogen at that position e.g., a hydrocarbyl radical or a halogen, hydroxy, amino and the like functional group, is not critical so long as it does not adversely affect the overall activity and/or synthesis procedure.
• the MeOH was removed by evaporation and the solid was taken up again in a minimal amount of MeOH.
• the mixture was concentrated under vacuum to a total volume 5 of ⁇ 20 mL at which point the unreacted Fe(OAc)2 precipitates.
• the solid was separated by centrifugation and the mother liquor is chromatographed on a Sephadex LH-20 column (2 x 30 cm) using MeOH as eluent.
• the initial colored band was collected and Fe(III)TMPyP(OAc) was isolated by precipitation after evaporation of 10 solvent and trituration with ether to give 85 mg ( 26%) of the desired product as confirmed by mass spectral analysis.
• H 2 TMPS ( 0.2 g, 0.125 mmole) and Mn(OAc) 2 (0.296 g, 1.71 mmole) was dissolved in 38 mL of water and was heated to 85° C for 1 h.
• the reaction was monitored by visible spectroscopy and was determined to be complete when the Soret band ( 416 nm) of the free base porphyrin was replaced by a new band at 468 nm characteristic of Mn(III) porphyrin species.
• the reaction was reduced in volume under vacuum to 10 mL and was chromatographed on a Dowex 50WX-8 cation exchange resin (H+ form) to remove excess Mn(OAc) 2 .
• Example 4 Synthesis of Acetato-5.10.15.20-tetrakis(3.5- disulfonatomesitvDporphvrin Iron (IIP octasodium salt H 2 TMPS ( 0.2 g, 0.125 mmole) and Fe(OAc) 2 ( 0.300 gl.72 mmole) was dissolved in 38 mL of water. The reaction mixture was brought to reflux and was monitored by visible spectroscopy to determine complete metallation. Upon completion the reaction was filtered and reduced in volume to 10 mL. The orange-brown reaction mixture was passed through a Dowex 50WX-8 cation exchange column (H+ from) to remove excess Fe(OAc) 2 .
• IIP octasodium salt H 2 TMPS 0.2 g, 0.125 mmole
• Fe(OAc) 2 0.300 gl.72 mmole
• H 2 TMPS ( 0.1 g, 0.063 mmole) and Ni(OAc) 2 ( 0.156 g, 0.63 mmole) was dissolved in 20 mL of water and was refluxed for 3 h.
• the reaction mixture was orange in color indicative of the Ni porphyrin.
• the completion of the reaction was confirmed by Vis spectroscopy.
• the reaction was reduced in volume to 5 mL and chromatographed on a Dowex 50 WX-8 ion exchange column (H+ form) to remove excess Ni(OAc) 2 .
• Example 6 Synthesis of N.N'-ethvlenebisfS ⁇ 'dimethoxvsalicvlideneamine) ligand.
• a modification of the procedure of Coleman was used (Coleman et al. Inorg. Chem , 20, 700, [1981]).
• a 100 mL round bottom flask equipped with a stir bar was charged with 25 mL of absolute EtOH and 3-methoxysalicyladehyde ( 3.04 g, 0.02 mol).
• a 20 mL solution of absolute EtOH and ethylenediamine ( .601 g, 0.01 mol) was freshly prepared and was added in one portion to the salicylaldehyde.
• the reaction was refluxed for 1 h during which time a yellow-orange precipitate appeared.
• the product was collected by filtration, washed with 100 mL of hot ethanol, and dried under vacuum to give 4.4 g (98%) of the desired product.
• Ni(II)([14]dienoN 4 )PF ⁇ was prepared by the method of Martin and Cummings ( Martin, J.G.; Cummings, S.C. Inorg. Chem.. 12, 1477-1482, [1973]). The compound was characterized by mass spectral analysis and was shown to be consistent with the desired structure.
• Ni(II)([14]dieneN4)(PF ⁇ )2 was prepared from Ni(II)([14]dienoN 4 )PF ⁇ by the method of Martin and Cummings (
• Ni(II)[14]12eneN 4 was prepared by the method of Goendken et. al. (Goendken et. al. J.C.S. Chem.Comm.. 337-338, [1973]). The complex was characterized by mass spectral analysis and which was consistent with the desired structure.
• Example 11 This example describes the preparation of peroxynitrite stock solutions used in these studies. A modified version of the procedure described by Hughs was used (Hughs, M. N.; Nicklin, H. G. J. Chem. Soc. (A), 450-452, [1968]).
• the catalytic rate constant (kc a t -i sec- !) for the complexes tested was determined by varying the complex concentration and plotting kobs vs [complex] Table 1.
• the kobs were obtained from averages of three stopped flow analysis at each catalyst concentration. Data representative of this analysis for a variety of compounds is shown in Figure 1.
• the simple di and trivalent chloride salts of Mn, Fe, Co, Cu, and Ni showed no catalytic peroxynitrite decomposition activity at concentration of 0.050 mM and below.
• Example 13 This example illustrates the inactivation of CuZn-superoxide dismutase (CuZnSOD) by peroxynitrite and that peroxynitrite decomposition catalyst shown to be active in Example 12 protect
• To these assay solution was added various amounts of peroxynitrite ( 25 mM stock solution) such that the final concentration of peroxynitrite in the assay varied from 0, 25, 50, 75 and 100 uM.
• 100 uL of a 2.5 mM stock EDTA solution was added to each assay solution such that the final concentration of EDTA was 250 uM.
• Assay solutions were prepared as described above except for the addition of various of peroxynitrite decomposition catalyst. The final solution volume was maintained at 10 mL. Thus, to the assay solutions Fe(III)TMPyP (0.5 and 1.0 uM final concentration) and Fe(III)TMPS (1.0 and 5.0 uM final concentration) was added. The solution were then treated with various amounts of peroxynitrite such that the final concentrations of 0,25, 50, 75 and 100 uM were achieved. Following treatment with peroxynitrite EDTA was added to a final concentration of 250 uM. The solutions were then assayed for SOD activity.
• Fe(III)TMPyP 0.5 and 1.0 uM final concentration
• Fe(III)TMPS 1.0 and 5.0 uM final concentration
• Example 15 In Vitro Evaluation: Materials: Human recombinant tumor necrosis factor-alpha (TNF-a) was obtained from Genzyme Corporation, Cambridge, MA. Human recombinant complement C5a and L-arginine (L-arg) was purchased from Sigma Chemical Company, St. Louis, MO. Authentic peroxynitrite in 50 mM NaOH was prepared as described above. Isolation of Endothelial Cells: Human dermal microvascular endothelial cells (HDME cells) from neonatal foreskin were prepared as previously described (Marks, R.M., Czerniecki, M., and Penny, R. In Vit. Cell. Devel. Biol.. 21, 627-635 [1985]).
• HDME cells Human dermal microvascular endothelial cells
• neonatal foreskin tissue from several donors was washed in 70% ethanol, cut into small pieces, then emersed in trypsin (0.6 %; Irvine Scientific, Santa Ana, CA) and EDTA (1%; Sigma Chemical Company, St. Louis, MO) for 7-9 minutes.
• trypsin 0.6 %; Irvine Scientific, Santa Ana, CA
• EDTA 1%; Sigma Chemical Company, St. Louis, MO
• the endothelial cells were removed by pressing the unkeratinized surface of the tissue with a scalpel blade.
• the cells were centrifuged through a 35% Percoll density gradient (Sigma Chemical Company, St. Louis, MO). After centrifugation at 250 x g for 10 min, cells corresponding to a density of less than 1.048 g/ml were collected and plated onto gelatin coated tissue culture dishes (0.1%; Sigma Chemical Company, St. Louis, MO).
• Contaminating cells were weeded daily using a 25 gauge needle mounted onto a tuberculin syringe.
• Purified endothelial cells were grown to passage 5 (-8 population doublings) in MCDB 131 (Endothelial basal medium; Clonetics Corporation) supplemented with 30% human serum (BioWittaker, Inc., Walkersville, MD), 10 ng/ml EGF (Collaborative Biomedical Products, Bedford, MA), 2 mM L-glutamine (Irvine Scientific, Santa Ana, CA), and 250 ⁇ g/ml dibutyryl cAMP, 1 ⁇ g/ml hydrocortisone (Sigma Chemical Company, St. Louis, MO).
• Preparations contained >95% neutrophils and were >95% viable by trypan blue (GIBCO Laboratories, Grand Island NY) exclusion.
• Purified neutrophils were suspended in HBSS supplemented with 0.01% BSA (Miles, Inc., Kankakee, IL) and 300 uM L-arg (HBSSBA) at a concentration of 5 x 106 cells/ml.
• Endothelial Cell Iniurv Assays The cytotoxic effects of stimulated neutrophils or peroxynitrite on endothelial cells was determined using a 5iCr-release assay as described by Moldow ( Moldow et. al. Methods Enzvmol.. 105, 378-385, [1984]).
• HDME cells were grown to a density of -1-2 x 104 cells/cm2 (-90 % confluence) in 96 well microtiter plates and labeled for 18 h with 10 uCi/ml sodium [SiCrjchromate (Amersham Corporation, Arlington Heights, IL).
• the HDME cells were cytokine-activated for 4 h with 100 U/ml human recombinant tumor necrosis factor-alpha (TNF-a; Genzyme Corporation, Cambridge, MA), then washed twice with HBSSBA. Suspensions of neutrophils were added at a concentration of 2.5 x 105/well and allowed to settle for 15 min.
• TNF-a tumor necrosis factor-alpha
• the neutrophils were activated by priming with 25 U/ml TNF-a for 10 min followed by activation with 3 ⁇ g ml complement component C5a (Sigma Chemical Company, St. Louis, MO). Incubations were continued for 2 h at 37° C . When authentic peroxynitrite was used, it was added in the absence of neutrophils. Peroxynitrite was added directly to the HDME cell layer from a 25 mM stock in 50 mM NaOH giving a final concentration from 0-800 uM. All inhibitors were made fresh immediately prior to the assay in HBSSBA and added as 1/10 of the well volume before peroxynitrite addition or neutrophil activation..
• Example 16 Protocol for Cell Protection Assays using Peroxynitrite Decomposition Catalysts A cell viability assay was established to rapidly assess the efficacy of peroxynitrite(PN) catalysts in protecting cells from PN- mediated injury and death.
• the peroxynitrite challenge consisted of a pulse of synthetic PN added exogenously to cells.
• a quantity of peroxynitrite(in 50mM NaOH) determined to cause maximal injury(100%) was added as an exogenous pulse to each well of cells in the presence or absence of catalyst.
• the NaOH vehicle was not toxic by itself.
• RAW 264.7 cells or P815 mastocytoma cells were plated to confluence on 96- well tissue culture plates. Each well is washed twice with Dulbecco's phosphate buffered saline(DPBS; GIBCO BRL, Grand Island, NY) to remove protein and other serum components which might react with the exogenous peroxynitrite. To each well is then added 200 ⁇ l of DPBS. PN is next placed into separate wells at increasing concentrations and cell viability monitored. The dose at which maximal cell death is attained is then utilized for the catalyst protection assessment.
• DPBS Dulbecco's phosphate buffered saline
• Phosphate-buffered saline (200 uL) containing increasing concentrations of catalyst is next placed into individual wells of cells.
• the maximal dose of PN is subsequently administered to all wells of cells.
• the medium is removed from each well and the cells are either allowed to recover overnight in Earles minimum essential medium without phenol red and supplemented with 10% fetal bovine serum or alternatively the plate of cells is assayed that day for mitochondrial integrity using the Alamar Blue viability assay(Alamar Biosciences, Inc.; Sacramento, CA.). In either case, cells are incubated at 37° C in 5% C0 2 .
• Cell injury is measured as follows. Briefly, 10% Alamar Blue viability assay(Alamar Biosciences, Inc.; Sacramento, CA.).
• Rats were given a bolus i.v. injection of active or inactive peroxynitrite catalysts 1 hour after the intraplantar injection of carrageenan; paw swelling was assessed thereafter every hour for up to 6 h.
• the relative % inhibition obtained with these agents is summarized in Table 2.
• the inactive peroxynitrite catalysts H 2 TMPS, ZnTMPyP or MnTPPS (all given at 30 mg/kg) or FeCl ⁇ (5 mg/kg, n 6) failed to inhibit edema formation.
• MOFS Multiple organ failure syndrome
• the "motor" of MOFS is the gastrointestinal tract, in particular the small intestine.
• Extensive ischaemia may be found in the intestinal mucosa due to profound vasoconstriction. Ischaemia and hypoxia result in mucous lesions, found both in animals (rat, cat, dogs) and humans.
• the origin of the mucous lesion is hypoxia.
• reperfusion e.g, after the initial severe vasoconstriction
• O 2 - may be liberated and play an important role in the pathogensis of mucous lesions in the GI tract.
• Intestinal vascular permeability was determined as the leakage into the jejunal tissue of [ ⁇ P-labelled bovine serum albumin ([125I]-BSA) administered intravenously (0.5 ml; 0.5 ⁇ Ci) together with either LPS (3 mg kg, serotype Olll:B4) or isotonic saline.
• LPS bovine serum albumin
• segments of jejunal tissue were ligated and removed.
• the intestinal tissues were rapidly washed, blotted dry and weighed. Blood (0.5 ml) was collected into tubes containing tri-sodium citrate (0.318% final concentration) and plasma prepared by centrifugation (10,000 g x 10 min).
• the content in segments of whole tissue and in aliquots of plasma was determined in a gamma counter.
• the total content of plasma in the intestinal tissues was expressed as ⁇ l g tissue.
• Changes in intravascular volume in the intestinal tissue was determined in an additional group of rats by administering ([i25I]-BSA) intravenously 2 min before removal of the jejunum.
• the tissue and plasma content of radiolabel was determined and intravascular volume expressed as ⁇ l/g tissue. This value was substracted from that obtained in the plasma leakage studies to obtain a measure of the intestinal plasma albumin leakage.
• LPS When compared to saline treated rats, LPS evoked profound jejunal damage with severe disruption of plicae and villi. LPS-induced damage was less severe in jejunums taken from rats treated with FeTMPS or FeTMPyP (30 mg/kg, i.v.).
• the compounds which are compounds or complexes of the present invention are novel and can be utilized to treat numerous inflammatory disease states and disorders.
• reperfusion injury to an ischemic organ e.g., reperfusion injury to the ischemic myocardium, inflammatory bowel disease, rheumatoid arthritis, osteoarthritis, hypertension, psoriasis, organ transplant rejections, organ preservation, impotence, radiation-induced injury, asthma, atherosclerosis, thrombosis, platelet aggregation, side effects from drug treatment of cancer metastasis, influenza, stroke, burns, trauma, acute pancreatitis, pyelonephritis, hepatitis, autoimmune diseases, insulin-dependent diabetes mellitus, disseminated intravascular coagulation, fatty embolism, adult and infantile respiratory distress, and hemorrhages in neonates.
• IL-2 induces a complex network of cytokines that include tumor necrosis factor, interleukin 1 and 6. Therefore, IL-2-treated patients resemble patients with endotoxemia (hypotension, elevated TNF levels, elevated cytokine levels etc). Some of these induce release of free radicals as well as inducing iNOS with subsequent release of NO.
• endotoxemia hypertension, elevated TNF levels, elevated cytokine levels etc.
• the present invention is for the methods and compositions for the treatment of a disease or condition advantageously affected by decomposition of peroxynitrite which is accelerated over a natural background rate of decay, preferably in humans suffering from such disease or condition, which comprises administering a metal complex, in dosage unit form, of accelerated-rate-effective amounts for decomposing peroxynitrite preferably wherein the metal complex is as defined above.
• Such methods or compositions accomplish the treatment of these diseases without disadvantageous ⁇ affecting normal biologically advantageous mechanisms.
• Total daily dose administered to a host in single or divided doses may be in amounts, for example, from about 1 to about 100 mg/kg body weight daily and more usually about 3 to 30 mg/kg.
• Dosage unit compositions may contain such amounts of submultiples thereof to make up the daily dose.
• the number of submultiples is preferably about one to three times per day of about 30 mg/kg per unit dosage form.
• the serum concentrations of the doses are about 15 ⁇ M to 1.5 mM with preferred ranges of 3 to 300 ⁇ M.
• the amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated and the particular mode of administration.
• the dosage regimen for treating a disease condition with the compounds and/or compositions of this invention is selected in accordance with a variety. of factors, including the type, age, weight, sex, diet and medical condition of the patient, the severity of the disease, the route of administration, pharmacological considerations such as the activity, efficacy, pharmacokinetic and toxicology profiles of the particular compound employed, whether a drug delivery system is utilized and whether the compound is administered as part of a drug combination.
• the dosage regimen actually employed may vary widely and therefore may deviate from the preferred dosage regimen set forth above.
• the compounds of the present invention may be administered orally, parenterally, by inhalation spray, rectally, or topically in dosage unit formulations containing conventional nontoxic pharmaceutically acceptable carriers, adjuvants, and vehicles as desired. Topical administration may also involve the use of transdermal administration such as transdermal patches or iontophoresis devices.
• parenteral as used herein includes subcutaneous injections, intravenous, intramuscular, intrasternal injection, or infusion techniques.
• injectable preparations for example, sterile injectable aqueous or oleaginous suspensions may be formulated according to the known art using suitable dispersing or wetting agents and suspending agents.
• the sterile injectable preparation may also be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent, for example, as a solution in 1,3-butanediol.
• a nontoxic parenterally acceptable diluent or solvent for example, as a solution in 1,3-butanediol.
• acceptable vehicles and solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution.
• sterile, fixed oils are conventionally employed as a solvent or suspending medium.
• any bland fixed oil may be employed including synthetic mono- or diglycerides.
• fatty acids such as oleic acid find use in the preparation of injectables.
• Suppositories for rectal administration of the drug can be prepared by mixing the drug with a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
• a suitable nonirritating excipient such as cocoa butter and polyethylene glycols which are solid at ordinary temperatures but liquid at the rectal temperature and will therefore melt in the rectum and release the drug.
• Solid dosage forms for oral administration may include capsules, tablets, pills, powders, granules and gels.
• the active compound may be admixed with at least one inert diluent such as sucrose lactose or starch.
• Such dosage forms may also comprise, as in normal practice, additional substances other than inert diluents, e.g., lubricating agents such as magnesium stearate.
• the dosage forms may also comprise buffering agents. Tablets and pills can additionally be prepared with enteric coatings.
• Liquid dosage forms for oral administration may include pharmaceutically acceptable emulsions, solutions, suspensions, syrups, and elixirs containing inert diluents commonly used in the art, such as water.
• Such compositions may also comprise adjuvants, such as wetting agents, emulsifying and suspending agents, and sweetening, flavoring, and perfuming agents.
• compounds of the invention can be administered as the sole active pharmaceutical agent, they can also be used in combination with one or more compounds of the present invention or with one or more compounds which are known to be effective against the specific disease state that one is targeting for treatment.

Landscapes

• Health & Medical Sciences (AREA)
• Medicinal Chemistry (AREA)
• Veterinary Medicine (AREA)
• Public Health (AREA)
• General Health & Medical Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Life Sciences & Earth Sciences (AREA)
• Pharmacology & Pharmacy (AREA)
• Chemical & Material Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Organic Chemistry (AREA)
• General Chemical & Material Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Engineering & Computer Science (AREA)
• Epidemiology (AREA)
• Heart & Thoracic Surgery (AREA)
• Cardiology (AREA)
• Neurology (AREA)
• Pulmonology (AREA)
• Pain & Pain Management (AREA)
• Urology & Nephrology (AREA)
• Vascular Medicine (AREA)
• Communicable Diseases (AREA)
• Oncology (AREA)
• Biomedical Technology (AREA)
• Rheumatology (AREA)
• Immunology (AREA)
• Neurosurgery (AREA)
• Orthopedic Medicine & Surgery (AREA)
• Physical Education & Sports Medicine (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
• Catalysts (AREA)
• Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
• Organic Low-Molecular-Weight Compounds And Preparation Thereof (AREA)

Priority Applications (9)

Applications Claiming Priority (2)

Publications (1)

Family

ID=22915009

Family Applications (1)

Country Status (13)

Cited By (25)

Families Citing this family (2)

Citations (15)

• 1995
  • 1995-05-09 BR BR9507643A patent/BR9507643A/pt not_active Application Discontinuation
  • 1995-05-09 JP JP7529755A patent/JPH10500671A/ja not_active Ceased
  • 1995-05-09 EP EP95919143A patent/EP0758892A1/en not_active Withdrawn
  • 1995-05-09 CZ CZ963234A patent/CZ323496A3/cs unknown
  • 1995-05-09 NZ NZ285648A patent/NZ285648A/xx unknown
  • 1995-05-09 WO PCT/US1995/005886 patent/WO1995031197A1/en not_active Application Discontinuation
  • 1995-05-09 CA CA002189528A patent/CA2189528A1/en not_active Abandoned
  • 1995-05-09 CN CNB951940759A patent/CN1222286C/zh not_active Expired - Fee Related
  • 1995-05-09 AU AU25120/95A patent/AU709553B2/en not_active Ceased
  • 1995-05-09 PL PL95317192A patent/PL317192A1/xx unknown
  • 1995-05-09 HU HU9603140A patent/HUT76327A/hu not_active Application Discontinuation
• 1996
  • 1996-11-12 FI FI964537A patent/FI964537A/fi not_active IP Right Cessation
  • 1996-11-12 NO NO964793A patent/NO964793L/no not_active Application Discontinuation

Patent Citations (15)

Non-Patent Citations (3)

Also Published As

Similar Documents

Legal Events