WO1996040223A1 - Oxidant scavengers - Google Patents
Oxidant scavengers Download PDFInfo
- Publication number
- WO1996040223A1 WO1996040223A1 PCT/US1996/010497 US9610497W WO9640223A1 WO 1996040223 A1 WO1996040223 A1 WO 1996040223A1 US 9610497 W US9610497 W US 9610497W WO 9640223 A1 WO9640223 A1 WO 9640223A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- sod
- scavenger
- bond
- group
- alkyl group
- Prior art date
Links
- 0 C**(C(C=C1)=C(C(C=C2)=C(C*3C(C(c4ccc(*)cc4)=C4*(I)=C5C=C4)=CC=CC33)C2=C3c2ccc(*)cc2)c(cc2)ccc2O)C1=C5c1ccc(*)cc1 Chemical compound C**(C(C=C1)=C(C(C=C2)=C(C*3C(C(c4ccc(*)cc4)=C4*(I)=C5C=C4)=CC=CC33)C2=C3c2ccc(*)cc2)c(cc2)ccc2O)C1=C5c1ccc(*)cc1 0.000 description 14
- UAEPNZWRGJTJPN-UHFFFAOYSA-N CC1CCCCC1 Chemical compound CC1CCCCC1 UAEPNZWRGJTJPN-UHFFFAOYSA-N 0.000 description 2
- QUWGCJBOMAOKME-UHFFFAOYSA-N CC(CC1)CC[N]1(C)I Chemical compound CC(CC1)CC[N]1(C)I QUWGCJBOMAOKME-UHFFFAOYSA-N 0.000 description 1
- NYMBCFRQTRGTML-UHFFFAOYSA-N CC(CCC1)C1(C)C1(CCC(C)CCC1)C(C(C)=O)=O Chemical compound CC(CCC1)C1(C)C1(CCC(C)CCC1)C(C(C)=O)=O NYMBCFRQTRGTML-UHFFFAOYSA-N 0.000 description 1
- QBSKAJOKDGDQDK-UHFFFAOYSA-N CCCCCCC[N]1(C)CCC(C)CCC1 Chemical compound CCCCCCC[N]1(C)CCC(C)CCC1 QBSKAJOKDGDQDK-UHFFFAOYSA-N 0.000 description 1
- BBXJCPPZZIJGOR-UHFFFAOYSA-N CCCCCN=O Chemical compound CCCCCN=O BBXJCPPZZIJGOR-UHFFFAOYSA-N 0.000 description 1
- AVFZOVWCLRSYKC-UHFFFAOYSA-N CN1CCCC1 Chemical compound CN1CCCC1 AVFZOVWCLRSYKC-UHFFFAOYSA-N 0.000 description 1
Classifications
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
- C07D487/22—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains four or more hetero rings
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
- A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
- A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
- A61K47/54—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic compound
- A61K47/545—Heterocyclic compounds
- A61K47/546—Porphyrines; Porphyrine with an expanded ring system, e.g. texaphyrine
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P39/00—General protective or antinoxious agents
- A61P39/06—Free radical scavengers or antioxidants
-
- 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
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B82—NANOTECHNOLOGY
- B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
- B82Y5/00—Nanobiotechnology or nanomedicine, e.g. protein engineering or drug delivery
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/0004—Oxidoreductases (1.)
- C12N9/0089—Oxidoreductases (1.) acting on superoxide as acceptor (1.15)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
Definitions
- the present invention relates, in general, to a method of modulating physiological and pathological processes and, in particular, to a method of modulating intra- and extracellular levels of oxidants such as superoxide radicals and hydrogen peroxide and thereby processes in which such radicals are a participant.
- the invention also relates to compounds and compositions suitable for use in such methods.
- Oxygen free radicals also play a role in modulating the effects of nitric oxide (NO ⁇ ) . In this context, they contribute to the pathogenesis of vascular disorders, inflammatory diseases and the aging process.
- SODs superoxide dismutases
- Mammals produce three distinct SODs. One is a dimeric copper- and zinc-containing enzyme (CuZn SOD) found in the cytosol of all cells. A second is a tetrameric
- Manganese-containing SOD found within
- E-SOD found in the extracellular fluids and bound to the extracellular matrix.
- Several other important antioxidant enzymes are known to exist within cells, including catalase and glutathione peroxidase. While extracellular fluids and the extracellular matrix contain only small amounts of these enzymes, other extracellular antioxidants are known to exist, including radical scavengers and inhibitors of lipid peroxidation, such as ascorbic acid, uric acid, and ⁇ -tocopherol
- EC-SOD is not thought to function as a bulk scavenger of O 2 -.
- EC-SOD is a tetrameric Cu/Zn-containing
- NO ⁇ nitric oxide
- NO ⁇ can, however, be toxic to neurons in some situations (Dawson et al, Proc. Natl. Acad. Sci. USA 88:6368 (1991)).
- O 2 - is known to inactivate NO ⁇ -induced vasorelaxation (Gryglewski et al, Nature 320:454 (1986); Rubanyi and Vanhoutte, Am. J. Physiol. 250:H822 (1986); Rubanyi and Vanhoutte, Am. J. Physiol. 250:H815 (1986); Bult et al, Br. J. Pharmacol.
- the invention thus relates to methods of manipulating nitric oxide function that involve the use of extracellular antioxidants.
- hydrogen peroxide is an oxidant species that is produced under a wide variety of conditions of oxidant stress.
- the invention thus also provides a method of manipulating hydrogen peroxide levels.
- the methods of the invention find application in various disease and non-disease states in which
- oxidative stress plays a role, including inflammation.
- the invention relates generally to methods of modulating intra- and extracellular processes in which an oxidant such as O 2 - or hydrogen peroxide is a participant.
- the present invention relates to a method of modulating intra- or extracellular levels of oxidants such as superoxide radicals, hydrogen peroxide and peroxynitrite. More particularly, the invention relates to a method of modulating normal or pathological oxidants such as superoxide radicals, hydrogen peroxide and peroxynitrite. More particularly, the invention relates to a method of modulating normal or pathological oxidants such as superoxide radicals, hydrogen peroxide and peroxynitrite. More particularly, the invention relates to a method of modulating normal or pathological
- the present invention relates to an oxidant scavenger, for example, a mimetic of
- superoxide dismutase catalase or peroxidase, comprising a nitrogen-containing macrocyclic moiety and a cell surface or extracellular matrix targeting moiety, or a pharmaceutically acceptable salt thereof.
- the present invention relates to oxidant scavenger of the formula:
- halogen and Y is an alkyl group and wherein
- R 3 is -Y", -OH, -NH 2 , -N + (Y") 3 , -COOH, -COO-,
- R 3 is not Y" (eg -CH 3 ), and wherein, when R 1 is and R 2 is a bond, R 3 is
- the present invention relates to an oxidant scavenger of the formula:
- each R 1 ' is independently a bond
- each R 2 ' is independently a bond, or -(CH 2 ) 2 - wherein n is 1-4, each R 3 ' is independently -Y" , -Y'", -H, -CK, -OY", -NO 2 , -CN, -NH 2 , -COOH, -COY", -COO-, or a heterocyclic group, wherein Y" is as defined above and Y' " is a primary, secondary, tertiary or quaternary amine, '
- the present invention relates to a method of inhibiting damage due to
- oxidation of a substance for example, with the subsequent formation of O 2 -, hydrogen peroxide, or peroxynitrite comprising contacting the substance with an amount of a SOD mimetic sufficient to effect the inhibition.
- the present invention relates to a method of inhibiting xanthine oxidase activity of a cell or tissue comprising contacting the cell or tissue with an amount of an oxidant scavenger sufficient to effect the inhibition.
- the present invention relates to a method of treating a pathological condition of a patient (eg, of the lungs of a patient) resulting from superoxide radical-induced degradation of NO ⁇ .
- the method comprises administering to the patient (eg, to the airways of the patient) an effective amount of a compound having the activity and tissue specificity of SOD (eg EC-SOD) under conditions such that the treatment is effected.
- a compound having the activity and tissue specificity of SOD eg EC-SOD
- the present invention relates to a method of treating an inflammatory
- an effective amount of an oxidant scavenger eg a mimetic of SOD, (eg
- the present invention relates to a method of treating a disorder resulting from aberrant smooth muscle function in a patient in need of such treatment comprising administering to the patient an effective amount of a mimetic of SOD (eg
- the present invention relates to a method of modulating physiologic functions of NO ⁇ in a mammal comprising administering to the mammal an oxidant scavenger in an amount sufficient to effect the modulation.
- Physiologic functions of NO ⁇ include its function as a smooth muscle relaxant, neuro- transmitter and immune modulator.
- the present invention relates to soluble oxidant scavengers, for example, mimetics of SOD, catalase or peroxidase, and to targeted forms thereof, in particular, mimetics of EC-SOD having a GAG binding moiety attached thereto.
- the present invention relates to an isolated EC-SOD gene sequence, or portion thereof.
- FIG. 1 shows the EC-SOD expression vector used to construct transgenic mice.
- Intervening sequence 1 from the human ⁇ -actin promoter Intervening sequence 1 from the human ⁇ -actin promoter.
- FIG. 2 shows the Northern analysis of tissues from transgenic mice. Twenty ⁇ g of total RNA from the tissues of transgenic mice were denatured with glyoxal and electrophoresed through a 1.2% agarose gel and blotted onto nitrocellulose. The filter was probed with the entire human EC-SOD cDNA. The 2.5 Kb band corresponds to mRNA of the human EC-SOD transgene containing the 1 Kb intervening sequence (see Figure 1). The 1.5 Kb band corresponds to the fully processed mRNA of the human EC-SOD transgene.
- Figure 3 shows the percent survival of transgenic and nontransgenic mice exposed to 6 ATA oxygen for
- mice were injected with saline or given 20 mg/kg N-fo-nitro-L-arginine (LNNA) i.p. 10 minutes before compression. 400 mg/kg of diethyldithiocarbamate (DDC) in saline was injected i.p. 55 min before
- Figure 4 shows time to onset of first seizure in transgenic and nontransgenic mice exposed to 6 ATA oxygen. Mice were injected with saline or given
- Figure 6 shows the seizure latency in wild-type mice exposed to 6 ATA oxygen after being treated with saline or 20 mg/kg N-to-nitro-L-arginine (LNNA) cr
- Figure 7 shows the percent survival in wild-type mice exposed to 6 ATA oxygen.
- Mice were given an i.p. injection of normal saline (0.008 cc/g) or 20 mg/kg N-to-nitro-L-arginine (LNNA) (0.008 cc/g) 15 minutes prior to compression.
- Figure 8 shows the survival dose response curve for N- ⁇ -nitro-L-arginine (LNNA). Wild-type mice were given an i.p. injection of normal saline (0.008 cc/g) or 0, 2, 10, 20, or 30 mg/kg LNNA (0.008 cc/g) 15 minutes prior to compression and then exposed to 75 minutes at 6 ATA oxygen. Percent survival was calculated for each treatment group.
- Figure 9 shows the percent survival in wild-type mice pretreated with saline, 20 mg/kg N-Zb-nitro-L- arginine (LNNA), or 20 mg/kg N-fo-nitro-L-arginine plus 50 mg/kg L-arginine (LNNA + L-Arg) and then exposed to 75 minutes of 6 ATA oxygen. *p ⁇ 0.05 tested with a ⁇ - square test with Bonferroni correction.
- Figure 10 shows the percent survival in transgenic and nontransgenic mice exposed to 6 ATA oxygen for
- mice were injected with saline or given 20 mg/kg N-to-nitro-L-arginine (LNNA) i.p. 10 minutes before compression. *p ⁇ 0.05 tested by ⁇ 2 compared to nontransgenic saline treated mice. + p ⁇ 0.05 tested by ⁇ 2 compared to transgenic saline treated mice.
- LNNA N-to-nitro-L-arginine
- Figure 11 shows the comparison of edema formation in EC-SOD transgenic mice to edema formation in
- Figure 12 shows the effect of augmented levels of EC-SOD on vascular permeability changes after cold- induced brain injury.
- Vascular permeability is demonstrated as Evan's blue leakage in the injuried right cerebral hemispheres of nontransgenic (control) and EC- SOD transgenic mice.
- Figure 13 shows a Western blot analysis
- rh-EC-SOD and a human lung homogenate to demonstrate antibody specificity. Proteins were separated on a 10% 0.75 mm SDS-polyacrylamide gel and transferred to nitrocellulose. Proteins were hybridized with the antibody to recombinant human EC-SOD (4.3 ⁇ g/ml) and the antibody was detected by hybridization with 125 I-Protein-A followed by autoradiography.
- the EC-SOD lane contained 0.05 ⁇ g of pure recombinant human type C EC-SOD lane protein.
- the lung lane contained 10 ⁇ g of a 20,000 x g supernatant of a human lung homogenate.
- Figures 14A-14C show the light microscopic
- Figures 15A-15C show the immunohistochemical localization of EC-SOD in human lung. Tissues were labeled using the antibody to recombinant human EC-SOD (5.4 mg/ml; anti-EC-SOD). Antibody was detected using a biotin/streptavidin-horseradish peroxidase labeling technique.
- A Large cartilaginous airway labeled with anti-EC-SOD. Note the intense labeling for EC-SOD in the matrix around smooth muscle cells (short arrow), between the epithelial cells (long arrow), and the lack of labeling on the surface of the epithelial cells (open arrows), and in the matrix of cartilage (asterisk).
- Tissues were labeled using the antibody to recombinant human EC-SOD (40 ⁇ g/ml; anti-EC-SOD) or the same antibody after the anti-EC-SOD IgG was absorbed out using purified recombinant EC-SOD attached to CNBr- sepharose (EC-SOD absorbed).
- Antibody was detected using 10 nm protein-A gold.
- A Vascular collagen labeled with anti-EC-SOD
- B Vascular elastin labeled with anti-EC-SOD.
- C Vascular collagen labeled with
- Figure 17 shows the electron microscopic immunolocalization of EC-SOD around vascular smooth muscle. Tissues were labeled using the antibody to recombinant human EC-SOD (40 ⁇ g/ml). Antibody was detected using 10 nm protein-A gold. There is a high degree of
- Figures 18A-18B show the electron miscropic immunolocalization of EC-SOD on the surface of pulmonary endothelial cells. Tissues were labeled using the antibody to recombinant human EC-SOD (40 ⁇ g/ml).
- Antibody was detected using 10 nm protein-A gold.
- A Endothelial cell from a small muscular pulmonary artery
- B Endothelial cell from a pulmonary capillary. No labeling for EC-SOD was on the surface of the
- EC-SOD endothelial cells
- P plasma
- extracellular matrix proteins beneath the endothelium
- Figure 19 shows the electron microscopic immuno- localization of EC-SOD around bronchial epithelial cells. Tissues were labeled using the antibody to recombinant human EC-SOD (40 ⁇ g/ml). Antibody was detected using 10 nm protein-A gold. EC-SOD was found in the junction between the epithelial cells (arrow) and was also seen to some extent inside the cells.
- Figures 20A-20D show a partial restriction map, sequencing strategy, genomic structure, and protein structure of human EC-SOD Clone #7.
- Fig. 20A a partial restriction map of human EC-SOD genomic clone #7 is shown in the 5' to 3' orientation. A 1 kb size marker is indicated.
- B BamH I; H, Hind III; P, Pst I; S, Sal I; K, Kpn I; E, EcoR I.
- Fig. 20B the subcloning and sequencing strategy is shown.
- Various size is indicated.
- Fig. 20C the exon/intron structure of the human EC-SOD gene is shown. The position of the coding region for preEC-SOD in exon 3 is shown by the dashed lines.
- Fig. 20D the four structural domains of human EC-SOD protein are diagrammed. The signal peptide is indicated by an arrow. This is followed by the mature glycosylated (CHO) amino terminal peptide domain. A third region has very high amino acid sequence homology to human CuZn- SOD.
- FIGS 21A-21B show human multiple tissue Northern blots of EC-SOD.
- Fig. 21A two ⁇ g of poly A(+) mRNA from eight different human tissues were electropnoresed on a denaturing agarose gel, transferred to a charged nylon membrane, and probed with [- 32 P]-labeled antisense human EC-SOD cRNA.
- RNA molecular size markers
- Figures 22A-22B show analysis of the transcription initiation site.
- the 5' rapid amplification of cDNA ends (5' RACE) technique was used to identify the site of transcription initiation for the human EC-SOD gene.
- Fig. 22A a schematic diagram illustrates the
- the dark line represents first -strand reverse transcribed cDNA of human heart poly A(+) mRNA which has been primed with EC7 (an EC-SOD gene specific primer) and poly C tailed using terminal deoxynucleotidyl transferase
- HEC1, HEC2, EC4, and EC7 are 5' human EC-SOD gene specific primers.
- the anchor primer is supplied with the 5' RACE kit (GIBCO BRL) and hybridized to the poly C tail.
- PCR was used to amplify segments of DNA using [anchor + EC4] or [HEC1 + EC7] as primers and either poly C tailed (+TdT, lanes 1 & 4) or non-poly C tailed (-TdT, lanes 2 & 5) cDNA as template.
- Lane 3 includes PCR amplified DNA using [HEC1 + EC7] as primers and a full-length human EC-SOD cDNA as template.
- restriction enzymes shown electropnoresed on a 1% agarose gel and transferred to charged nylon membranes. The blots were proved with a [ 32 P]-labeled EC-SOD partial length cRNA which corresponds to the first approximate 1050 nucleotides and autoradiographsd. The specific restriction endonuclease is shown at the top of each lane. DNA molecular size markers (in kilobases) are shown on the right.
- Figure 24 shows the nucleotide sequence and deduced amino acid sequence of the human EC-SOD gene. The complete nucleotide sequence of the human gene is shown. The deduced amino acid sequence of the signal peptide and mature protein is indicated using the single letter amino acid code.
- Figure 25 shows a Lineweaver-Burk plot
- Figure 26 shows the protection of pulmonary artery endothelial cells from xanthine oxidase-induced injury by MnTBAP.
- Figure 27 shows the protection of lung epithelial cells from paraquat-induced injury of SOD mimetics.
- Figure 28 shows the protection of pulmonary artery endothelial cells from paraquat-induced injury by
- Figure 29 shows the lack of protection of pulmonary artery endothelial cells from paraquat-induced injury by ZnTBAP.
- Figure 30 shows the protection of MnTBAP against paraquat-induced lung injury.
- Figure 31 shows a second order rate constant plot for catalase mimetics.
- Figure 32 shows the effect of MnTBAP on H 2 O 2 -induced endothelial injury.
- Figures 33A and 33B show the reduction by MnTBAP and MnTmPyP of endothelial cell injuries caused by exposure to glucose oxidase-produced hydrogen peroxide.
- Figure 33C shows that ZnTBAP does not reduce hydrogen peroxide-induced endothelial cell injury.
- Figure 33D shows that endothelial cells are not protected from hydrogen peroxide-induced injury by CuZnSOD.
- Figures 34A and 34B show the time course of NMDA- and KA-induced inactivation of aconitase. Fig. 34A.
- Figures 36A and 36B show the blockade of aconitase inactivation and neurotoxicity by MnTBAP. Fig. 36A.
- Cortical cells were treated with 150 ⁇ M PQ ++ for 3 hr. (solid bars), 50 ⁇ M NMDA for 1 hr. (open bars) or 300 ⁇ M KA for 6 hr. (hatched bars) in the presence or absence of 200 ⁇ M MnTBAP (present 15 min. prior to and
- Fig. 36B Cortical cells were treated with 150 ⁇ M PQX (solid bars), 50 ⁇ M NMDA (open bars) or 300 ⁇ M KA (hatched bars) in the presence of varying concentrations of MnTBAP (present 15 min. prior to and throughout the duration of treatment) for 18 hr. and LDH release measured in the medium.
- Asterisk represents a
- Figure 37 shows the inhibition of NMDA toxicity by MnTBAP. Cortical cells were treated with varying concentrations of NMDA in the presence and absence of
- Figures 38A-38C show the differential effect of MnTBAP and ZnTBAP on cell death. Cortical cells were treated with 150 ⁇ M PQ" (Fig. 38A), 50 ⁇ M NMDA (Fig.
- EthD-1 EthD-1 -positive cells were stored and counted in randomly selected fields using a digital image analyzer. Data are expressed as the number of dead cells per field. Each point represents measurements made from 1200-1500 cells.
- Figure 39 shows the effect on learning of removal of the EC SOD gene in a mouse model.
- Figure 40 Effect of Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) (3-100 ⁇ M) on the oxidation of dihydrorhodamine 123 to rhodamine 123 in response to peroxynitrite (5 ⁇ M). Data are expressed as means ⁇ s.e.m. of triplicate determinations.
- Figures 42A-D show synthetic reaction schemes.
- the present invention relates to methods of protecting against the deleterious effects of oxidants, particularly, superoxide radicals, hydrogen peroxide and peroxynitrite, and to methods of preventing and treating disease states that involve or result from oxidant stress.
- oxidants particularly, superoxide radicals, hydrogen peroxide and peroxynitrite
- the invention also relates methods of
- the invention further relates to compounds and compositions, including low molecular weight antioxidants (eg mimetics of scavengers of reactive oxygen species, including mimetics of SODs, catalases and peroxidases) and formulations thereof, suitable for use in such methods.
- low molecular weight antioxidants eg mimetics of scavengers of reactive oxygen species, including mimetics of SODs, catalases and peroxidases
- Mimetics of scavengers of reactive oxygen species appropriate for use m the present methods include manganic derivatives of methine substituted porphines, or pharmaceutically acceptable salts thereof.
- the methine substituents can be selected so as to facilitate electron exchange between the mimetic metal (eg Mn) and the oxygen radical. Substituents that withdraw
- substituents can be selected so as to modulate the redox potential of the porphine.
- Substituents can also be selected so as to render the porphyrin resistant to degradation by hemeoxygenase.
- Hemeoxygenasee a key enzyme in normal porphyrin degradation and an enzyme that plays a role in the regulation of inflammation, attacks at the methine bridge carbons.
- compounds not susceptible to attack eg by introducing substituents at the methine bridge carbons
- the half life of the porphyrin can be increased.
- Such compounds have the further advantage that they do not interfere with normal porphyrin
- substituents can also be made based on a result sought to be achieved. For example, when passage through cell membranes is advantageous in a given treatment regimen, non-polar substituents can be selected so as to render the mimetic lipid soluble.
- Substituents can also be selected so as to render the mimetic capable of binding to cell surface or
- substituents can be selected so as to target the mimetic on the basis of charge, shape, structure, etc. Targeting substituents can be specific, for example, for certain cell surface receptors (eg mannose receptors found on epithelial cells) or for certain sugars or lectins present on the cell surface.
- cell surface receptors eg mannose receptors found on epithelial cells
- sugars or lectins present on the cell surface.
- the mimetics are of the formula:
- Y is an alkyl group and wherein indicates bonding to R 2 at any position and indicates
- n 1 to 8;
- R 3 is a bond, hydrogen, -Y", -OH, -NH-, -N + (Y") 3 , -COO-, -COO-, -SO 3 -, -SO 3 -, -C-PO 3 H- or -C-PO 3 H-, wherein Y" is an alkyl group, and
- R 4 is nothing, hydrogen, a cell surface or extracellular matrix targeting moiety or a linker-cell surface or extracellular matrix targeting moiety
- linker is a moiety that links the mimetic core (porphyrin ring and R 1 , R 2 , R 3 ) to the targeting moiety).
- n is 1 to 4;
- R 3 is a bond, hydrogen, Y", -OH, -NH-, -N + (Y") 3 , -COO-, -COO-, -SO 3 -, -SO 3 -, -C-PO 3 H- or -C-PO 3 H-, wherein Y" is a C 1-4 alkyl group, and
- R 4 is nothing, hydrogen, a cell surface or extra- cellular matrix targeting moiety or a linker-cell surface or extracellular matrix targeting moiety.
- R 4 is nothing, hydrogen, a cell surface or extracellular matrix targeting moiety, or a linker-cell surface or extracellular matrix targeting moiety.
- alkyl more preferably methyl or ethyl
- Y' is hydrogen or alkyl (preferably C 1-4 alkyl, more preferably methyl or ethyl) and wherein n is
- R 3 is a bond, hydrogen, C 1-4 alkyl (preferably methyl or ethyl), -OH, -NH-, -N + (CH 3 ) 3 , -N + (CH 2 CH 3 ) 3 , -COO-, -COO-, -SO 3 -, -SO 3 -, -C-PO 3 H- or -C-PO 3 H-; and R 4 is nothing, hydrogen, a cell surface or extracellular matrix targeting moiety or a linker-cell surface or extracellular matrix targeting moiety.
- one or more of the pyrrole rings of the porphyrin can be substituted (at any or all of carbons 2, 4, 7, 8, 12, 13, 17 or 18) with an electron withdrawing group
- each P being, independently, a NO 2 group, a halogen (eg Cl, Br or F), a nitrile, a vinyl group, or a formyl group.
- a halogen eg Cl, Br or F
- substituents alter the redox potential of the porphyrin and thus enhance its ability to scavenge oxygen
- Each P can, independently, also be hydrogen.
- Specific mimetics suitable for use in the present methods include Mn(III) tetrakis (1-methy-4- pyridyl) porphyrin (MnTMPyP), Mn(III) tetrakis (4- trimethyl-aminophenyl) porphyrin (MnTMAP) and Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), with or without a cell surface or extracellular matrix targeting moiety or a linker-cell surface or extracellular matrix targeting moiety at the R 4 position.
- MnTMPyP Mn(III) tetrakis (1-methy-4- pyridyl) porphyrin
- MnTMAP Mn(III) tetrakis (4- trimethyl-aminophenyl) porphyrin
- MnTBAP Mn(III) tetrakis (4-benzoic acid) porphy
- the metal selected may have various valence states, for example, manganese II, III or V can be used. The change in charge will be dependent upon the acceptance or release of electrons.
- the invention also includes compounds (and targeted forms thereof as described above, including linker-cell surface or extracellular matrix targeted forms) of the formula:
- metal complex thereof wherein the metal is, for example, manganese, copper or iron,
- each R 1 ' is independently a bond
- Y" is an alkyl group (eg C 1 -C 4 ) and
- each R 2 ' is independently a bond, or -(CH 2 ) n - wherein n is 1-4, each R 3 ' is independently -Y", -Y'", -H, -OH, -OY",
- Y is as defined above and Y' " is a primary, secondary, tertiary or quaternary amine
- R 3 ' can represent a heterocyclic moiety.
- Possible heterocyclics include substituted or unsubstituted tetrazoles, furans, thiophenes, indoles, imadazoles, pyridines, oxadiazoles and quinolines.
- Possible substituents on such moieties include halogen (eg Br or Cl), -NO 2 , C 1-4 alkyl, and C 1-4 alkyl alcohol groups.
- one or more of the pyrrole rings of the porphyrin can be substituted (at any or all of carbons 2, 4, 7, 8, 12, 13, 17 or 18) with an electron withdrawing group (designated P), for example, each P being, independently, a NO 2 group, a halogen (eg Cl, Br or F), a nitrile, a vinyl group, or a formyl group.
- P can also be hydrogen.
- R 1 can be selected from the group described above with respect to R 1 or R 1 '
- R 2 can be selected from the group described above with respect to R 2 and R 2 '
- R 3 can be selected from the group described above with respect to R 3 or R 3 '.
- the targeted mimetics can be used, for example, to mimic EC-SOD. Since the sequence of human EC-SOD is known (Hjalmarsson et al, Proc. Natl. Acad. Sci. USA 84:6340 (1987)), the C-terminal oligopeptide can be prepared and attached to the "mimetic core" (eg, a Mn((II)-porphyrin) via, for example, a free amine or carboxy group with a 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDO coupling reaction (Yamada et al, Biochemistry 20:4836 (1981); Davis and Preston, Anal. Biochem. 116:402
- the "mimetic core” eg, a Mn((II)-porphyrin) via, for example, a free amine or carboxy group with a 1-ethyl-3-(3-dimethylaminoprop
- Heparin binding affinity of a mimetic can be assessed by passing the mimetic over a heparin-sepharose CL-6B column (Karlsson et al, Biochem. J. 256:29
- Candidate targeting moieties suitable for attaching to the mimetics of the invention to confer GAG binding properties include the following:
- n is, preferably, in the range of 1 to 12, more preferably, 1 to 8, and, most preferably, 1 to 6, with or without a C-terminal Glu or Ala (Sandstrom et al, J. Biol. Chem. 267:18205 (1992)); and
- polyethyleneimine e.g. (NH-CH 2 -CH 2 -NH) nH, wherein n is 1 to 6.
- targeting moieties also include heparin binding proteins generally and sugars, including mannose and oligosaccharides.
- R' NH 2 -ArgGluHisSerGluArgLysLysArgArgArg
- Mimetics suitable for use in the present methods can be selected by assaying for SOD, catalase and/or peroxidase activity and stability.
- the selective, reversible and SOD-sensitive inactivation of aconitase by known O- 2 generators can be used as a marker of O- 2 generation.
- suitable mimetics can be selected by assaying for the ability to protect aconitase activity.
- SOD activity can be monitored in the presence and absence of EDTA using the method of McCord and Fridovich (J. Biol. Chem. 244:6049 (1969)).
- the efficacy of a mimetic can be determined by measuring the effect of the mimetic on the growth of a SOD null E. coli strain versus a wild type strain.
- wild type E. coli AB1157
- SOD null E. coli . JI132
- M9 medium containing 0.2% casamino acids and 0.2% glucose at pH 7.0 and 37°C
- growth can be monitored in terms of turbidity followed at 700 nm
- rat L2 cells a lung Type II like cell; (Kaighn and Douglas, J. Cell Biol. 59:160a (1973)
- LDH lactate dehydrogenase
- rat L2 cells a lung Type II like cell; (Kaighn and Douglas, J. Cell Biol. 59:160a (1973)
- Ham's F-12 medium with 10% fetal calf serum supplement at pH 7.4 and 37°C
- cells can be seeded at equal densities in 24 well culture dishes and grown to approximately 90% confluence
- SOD mimetics can be added to the cells at log doses (eg micromolar doses in minimal essential medium (MEM) ) and incubated for 24 hours.
- MEM minimal essential medium
- Toxicity can be assessed by morphology and by measuring the release of the cytosolic injury marker, LDH (eg on a thermokinetic plate reader), as described by Vassault (In: Methods of Enzymatic Analysis, Bergmeyer (ed) pp. 118-26 (1983); oxidation of NADH is measured at 340 nm).
- Efficacy of active mimetics can be assessed by determining their ability to protect mammalian cells against methylviologen (paraquat)-induced toxicity.
- rat L2 cells grown as described above and seeded into 24 well dishes can be pre-incubated with various concentrations of the SOD mimetic and then incubated with a concentration of methylviologen
- mice can be randomized into 4 groups of 8 mice each to form a standard 2X2 contingency statistical model. Animals can be treated with either paraquat
- Lung injury can be assessed 48 hours after paraquat treatment by analysis of bronchoalveolar lavage fluid (BALF) damage parameters (LDH, protein and % PMN) as previously described (Hampson et al, Tox.
- BALF bronchoalveolar lavage fluid
- Catalase activity can be monitored by measuring absorbance at 240nm in the presence of hydrogen peroxide (see Beers and Sizer, J. Biol. Chem. 195:133 (1952)) or by measuring oxygen evolution with a Clark oxygen electrode (Del Rio et al, Anal. Biochem. 80:409 (1977)). Peroxidase activity can be measured
- Table IX below provides a summary of the activities of various oxidant scavengers of the invention. The footnote to that Table provides details of the assays used.
- Example XIV includes a detailed description of the synthesis of four specific mimetics.
- porphyrin rings with various methine bridge carbon side groups can be
- Mn(III) (OH) 3 preparation of Mn(III) (OH) 3 by a modification of the Winkler method (Sastry et al, Anal. Chem. 41:857 (1969)) followed by reaction with the porphyrin; (3) stirring MnO 2 with the porphyrin in the presence of NH 2 OH, which serves to reduce the Mn(IV) to Mn(III), which is then trapped by the porphyrin,- or (4) a method modified from Pasternack et al (Biochemistry 22:2406 (1983)) which refluxes excess MnCl 3 with the porphyrin.
- Mn(III)- porphyrin complexes can be precipitated from solution with sodium perchlorate, washed and residue perchlorate removed by strong anionic exchange resin. Formation of the Mn( III)-porphyrin can be followed
- Purification of the mimetics can be effected using art- recognized techniques such as recrystallization, chromatography, etc. Coupling of a binding domain to the "mimetic core" can be carried out as describedabove.
- One embodiment of the present invention results, at least in part, from the realization that EC-SOD
- the invention is based on the realization that EC-SOD is synthesized by epithelial cells and is primarily
- NO ⁇ is an intercellular signal and, as such, NO ⁇ must traverse the extracellular matrix to exert its effects. NO ⁇ , however, is highly sensitive to inactivation mediated by O 2 - present in the extracellular spaces.
- EC-SOD is thus an enzyme ideally suited to increase the bioavailability of NO ⁇ by
- One embodiment of the present invention relates to a method of regulating extracellular NO ⁇ levels using polypeptides having EC-SOD activity.
- the invention also relates to mimetics of EC-SOD that can be targeted to strategic locations and to the use of such mimetics in manipulating extracellular levels of NO ⁇ .
- the invention is not limited to NO ⁇ manipulation as the sole mechanism of action of the compounds, mimetics, etc, of the invention. Rather, the invention relates to oxygen radical, hydrogen peroxide and peroxynitrite scavenging generally.
- the present invention relates, in a further
- the mimetics of the invention are used to inhibit oxidases, such as xanthine oxidase, that are responsible for production of superoxide radicals (see Example VII).
- oxidases such as xanthine oxidase
- the ability of a mimetic to protect mammalian cells from xanthine/xanthine oxidase-induced injury can be used.
- rat L2 cells assessed, for example, by growing rat L2 cells in 24- well dishes.
- Cells can be pre-incubated with various concentrations of a mimetic and then xanthine oxidase (XO) can be added to the culture along with xanthine (X).
- XO/X xanthine oxidase
- the appropriate amount of XO/X used in the study can be pre-determined for each cell line by performing a dose-response curve for injury.
- X/XO can be used in an amount that produces approximately an LC 75 in the culture.
- Efficacy of the mimetic can be correlated with a decrease in XO/X-induced LDH release. The ability of the mimetics to inhibit the production of such radicals makes possible the use the mimetics as therapeutics for the treatment of gout and reperfusion injuries.
- the mimetics of the invention can be used as catalytic scavengers of reactive oxygen species to protect against ischemia reperfusion injuries associated with myocardial infarction, stroke, acute head trauma, organ reperfusion following transplantation, bowel ischemia, pulmonary infarction, surgical occlusion of blood flow, and soft tissue injury.
- the mimetics can further be used to protect against skeletal muscle reperfusion injuries.
- the mimetics can also be used to protect against damage to the eye due to sunlight (and to the skin) as well as glaucoma, and macular
- the mimetics of the invention can be used as catalytic scavengers of
- the invention also provides methods of inhibiting damage due to
- the mimetics of the invention are added to food products, pharmaceuticals, stored blood and the like, in an amount sufficient to inhibit or prevent oxidation damage and thereby to inhibit or prevent the degradation associated with the autoxidation reactions. (For other uses of the mimetics of the invention, see USP 5,227,405).
- the amount of mimetic to be used in a particular treatment or to be associated with a particular substance can be determined by one skilled in the art.
- the present invention relates to diagnostic protocols made possible by the availability of the EC-SOD gene sequence (see Example V and Figure 24).
- a defect in the EC-SOD gene is more likely to occur than a defect in nitric oxide synthase due to the nature and number of physiological functions served by NO ⁇ . Detection of an EC-SOD gene defect would signal the need for measures to be undertaken to elevate levels of functional EC-SOD, or related superoxide scavenging compounds, at strategic locations to correct NO ⁇ imbalances and thus disorders involving oxidative stress.
- molecules (agents) having EC-SOD activity are administered under conditions such that levels of extracellular O 2 - are altered.
- Molecules suitable for use in this method include
- glycosaminoglycans for example, by virtue of the fact that they contain positively charged amino acids near their carboxy terminal end.
- Proteinaceous agents suitable for use in the present method include forms of EC-SOD C described in WO 91/04315, as well as additional polypeptides defined and described therein as having comparable or enhanced binding to heparin as compared to recombinant EC-SOD C (eg, polypeptides G1 and SA216;
- polypeptide SA219 which has the same heparin binding as recombinant EC-SOD C.
- Further proteinaceous agents suitable for use in the present method include chimeric proteins with targeted binding and SOD
- Proteinaceous molecules suitable for use in the present method can be synthesized chemically or
- Non-glycosylated recombinant peptides can be produced, for example, using host cells (ie, E. coli cells) that are incapable of effecting glycosylation, or using DNA sequences encoding functional proteins lacking glycosylation sites.
- molecules suitable for use in the present method include mimetics of EC-SOD (eg targeted mimetics), including those described above.
- mimetics are those that they: (a) be stable enough to retain the ligated metal (eg Cu or Mn) in the presence of the multiple chelating agents present in living systems, (b) be active enough that reasonable doses can serve to significantly augment the total SOD activity in the extracellular spaces, (c) be able to adhere to the surfaces of cells or extracellular matrix elements (eg collagen) when protection against extracellular sources of O 2 - is needed, and d) be of low toxicity.
- suitable mimetics include
- nitrogen-containing macrocyclic ligands effective as catalysts for dismutating superoxide including Mn(III) complexes of porphyrins with bulky cationic substituents on the methine bridge carbons, such as those described above (eg MnTMAP and MnTMPyP).
- MnTMAP and MnTMPyP Such complexes are very active and are stable enough to retain full activity in the presence of excess EDTA or in the presence of tissue extracts.
- compositions suitable for use in the present methods can be formulated into pharmaceutical compositions suitable for use in the present methods.
- Such compositions include the active agent (polypeptide or mimetic) together with a pharmaceutically acceptable carrier, excipient or diluent.
- the composition can be present in dosage unit form for example, tablets, capsules or suppositories.
- the composition can also be in the form of a sterile solution suitable for injection or
- compositions can also be in a form suitable for opthalmic use.
- the invention also includes compositions formulated for topical administration, such compositions taking the form, for example, of a lotion, cream, gel or ointment.
- concentration of active agent to be included in the composition can be selected based on the nature of the agent, the dosage regimen and the result sought.
- composition of the invention to be administered can be determined without undue
- a suitable dosage of protein administered IV can be expected to be in the range of about 10-1000 mg/day.
- For topical treatment it is expected that lower doses will be required (see
- Suitable doses of mimetics will vary, for example, with the mimetic and with the result sought.
- the results of Faulkner et al J. Biol. Chem. 269:23471 (1994))
- Doses that can be used include those in the range of 1 to 50 mg/kg.
- the present invention also includes compositions suitable for use in gene therapy types of protocols.
- the invention includes DNA sequences encoding proteins having EC-SOD activity and formulated so as to be incorporated into cells (eg, lung cells) upon contact therewith (eg, via inhalation).
- the sequence can be present in a vector, eg, a viral vector, and/or the sequence can be present in a delivery
- compositions to be administered can be readily
- diseases of the central nervous system including AIDS dementia, stroke, amyotrophic lateral sclerosis (ALS), Parkinson's disease and Huntington's disease
- diseases of the musculature including diaphramic diseases (eg
- NMDA neuronal calcium concentrations
- NO ⁇ oxygen free radicals and nitric oxide
- Interactions between oxygen free radicals and NO ⁇ have been shown to contribute to neuronal cell death.
- Well-established neuronal cortical culture models of NMDA-toxicity have been developed and used as the basis for drug development. In these same systems, the mimetics of the invention inhibit NMDA induced injury.
- Example XI demonstrate that the formation of O- 2 radicals is an obligate step in the intracellular events culminating in excitotoxic death of cortical neurons and further demonstrate that the mimetics of the invention can be used to scavenge O- 2 radicals and thereby serve as protectants against excitotoxic injury.
- Compound 10303 (see Table IX) decreases NMDA- and kainate-induced excitotoxicity in rat cortical cells in a dose dependent manner with 100% protection against NMDA-induced
- excitotoxicity 50 ⁇ M being achieved at 25 ⁇ M compound 10303, 100 ⁇ M compound 10303 in the case of kainate- induced excitotoxicity (300 ⁇ M).
- the present invention also relates to methods of treating arthritis, systemic hypertension,
- the scavengers of the invention can be used to ameliorate the toxic effects associated with endotoxin, for example, by preserving vascular tone and preventing multi-organ system damage.
- the invention further relates to methods of treating memory disorders. While not wishing to be bound by theory, it is believed that nitric oxide is a neurotransmitter involved in long-term memory
- Inflammations particularly inflammations of the lung, are amenable to treatment using the present invention (note particularly the inflammatory based disorders of asthma, ARDS including oxygen toxicity, pneumonia (especially AIDS-reiated pneumonia), cystic fibrosis, chronic sinusitis and autoimmune diseases (such as rheumatoid arthritis)).
- EC-SOD is localized in the interstitial spaces surrounding airways and
- EC-SOD and O 2 - mediate the antiinflammatory - proinflammatory balance in the alveolar septum. NO ⁇ released by alveolar septal cells acts to suppress inflammation unless it reacts with O 2 - to form ONOO-. By scavenging O 2 -, EC-SOD tips the balance in the alveolar septum against inflammation.
- EC-SOD mimetics such as those described herein, can be used to protect against destruction caused by hyperoxia. Appropriate therapeutic regimens can be readily established by one skilled in the art.
- the present invention relates to diagnostic protocols suitable for use in identifying EC-SOD gene defects. This aspect of the invention is based on the
- the present invention includes within its scope the gene sequence presented in Figure 24 as well as portions of non-coding regions of that sequence of at least 15 bases,
- Positions 1-558 This represents a 5' flanking region which contains transcriptional regulatory
- Mutations here can be expected to lead to deficient levels of EC-SOD or defective enhancement or reduction in EC-SOD levels under conditions which require manipulating the EC-SOD concentration.
- the following regions have been identified as putative regulatory regions. Mutations in these regions can be expected to result in deficient levels of EC-SOD:
- Positions 560-570 Mutations here can be expected to lead to an inability to splice out intron 1. This would result in no EC-SOD production (or much reduced) due to initiation of translation at multiple cryptic ATG sites located in intron 1 which are upstream of the EC-SOD ATG start codon. For example, base pair 653, 656, 720, 743, 748, etc, would potentially initiate translation.
- Intron 1 contains DNA sequence unique to EC-SOD. In addition, there are potential transcription regulatory regions within this DNA stretch which are listed below; mutations in intron 1 would lead to deficient levels of EC-SOD or defective enhancement or reduction in EC-SOD levels under conditions that require manipulating the EC-SOD concentration:
- Positions 71-95 Mutations here can be expected to lead to an inability to splice out intron 1. This would result in no EC-SOD production (or much reduced) due to initiation of translation at multiple cryptic ATG sites located in intron 1 that are upstream of the EC-SOD ATG start codon. For example, base pair 653, 656, 720, 743, 748, etc, would potentially initiate translation.
- processing ie, removal of the signal peptide
- Positions 5403-5405 Mutations here can be expected to result in loss of glycosylation which may result in defective levels due to the synthesis of poorly soluble protein.
- Positions 5424-5720 Mutations here can be expected to result in defective EC-SOD activity. This region is critical for binding to the substrate and catalyzing the dismutation of superoxide anion radical. In addition, this region would also affect any other activities of this enzyme including the reduction of other species such as nitric oxide, etc.
- Positions 5721-5804 Mutations in this region would cause defects in binding of EC-SOD to target tissues such as type I collagen in the extracellular matrix, and around smooth muscle cells in both vessels and bronchi. Such mutations here are highly likely to cause disease.
- the present invention relates not only to the entire EC-SOD gene sequence, but to portions thereof for use, for example, as probes or primers in detecting mutations in regions of the gene including those noted above. Such portions can be expected to be, for
- 18-1500 bases in length preferably 18-22 bases in length, 50-75 bases in length, 100-400 bases in length or 500 to 1500 bases in length. Certain details of the present invention are described in greater detail m the non-limiting Examples that follow.
- the EC-SOD expression vector ( Figure 1) was constructed as follows: The entire human EC-SOD cDNA fragment
- mice 2.5 ⁇ g/ml in 5 mM Tris-HCl, pH 7.4, 0.1 mM EDTA was injected into the pronuclei of fertilized eggs isolated from mice ( (C57BL/6 X C3H)F1 X (C57BL/6 X
- mice carrying the transgene were identified by Southern blot analysis of tail DNA probed with the entire human EC-SOD cDNA. Transgenic founders were found the first litter screened. These mice were bred with (C57BL/6 X C3H)FI to produce offspring for further studies. (In all of the following experiments with the EC-SOD
- transgenic mice the nontransgenic mice refer to
- mice littermates of the transgenic mice that did not contain the transgene for the human EC-SOD.
- wild type (C57BL/6 X C3H)FI were used.
- Homozygous transgenic mice were produced by an F 1 cross of heterozygous transgenic mice. Tail DNA from F 2 mice were isolated and treated with RNAase. 10 ⁇ g of DNA from each mouse was cut with PstI and then
- Homozygous mice were generated by breeding two heterozygous F1 mice. Homozygous mice were detected by differential band intensities found using Southern blot analysis of equal amounts of PstI digested DNA from the offspring. EC-SOD activity in the mice was found to increase in response to the total copies of the EC-SOD transgene (Table I).
- mice were observed continuously for signs of oxygen toxicity from the beginning of the exposure until 4 hours after removal from the chamber. The time to the first
- Treatmen t wi th diethyldi thiocarbamate One hour prior to exposure to 6 ATA oxygen, mice were given either i.p. injections of either 0.008 cc/g saline or 400 mg/kg diethyldithiocarbamate dissolved in normal saline (0.008 cc/g) . The mice were then exposed to 6 ATA oxygen for 25 mintues as described above.
- mice were given diethyldithiocarbamate and sacrificed one hour later. The brains were removed and assayed for EC-SOD and CuZn SOD activity as described above.
- Trea tment wi th ⁇ -mercaptoethanol One hour prior to exposure to 6 ATA oxygen, mice were given either i.p. injections of 0.008 cc/g saline or 180 mg/kg ⁇ - ercaptoethanol (0.008 cc/g). This dose of ⁇ - mercaptoethanol was selected because it contains an equal number of reducing thiols as the dose of
- mice diethyldithiocarbamate. The mice were then exposed to 6 ATA oxygen for 30 minutes as described above. Treatment with N- ⁇ -nitro-L-arginine, an inhibitor of nitric oxide synthase: Ten minutes prior to
- mice were treated with an equimolar amount of reducing thiols in the form of ⁇ -mercaptoethanol and exposed to hyperbaric oxygen.
- Figure 5 shows that ⁇ - mercaptoethanol did not protect against CNS oxygen toxicity.
- nitric oxide is a mediator of CNS oxygen toxicity and EC-SOD is protecting nitric oxide from superoxide mediated
- Figure 7 shows that nitric oxide synthase inhibition also significantly increased survival after exposure to hyperbaric oxygen. Mice given the inhibitor of nitric oxide synthase displayed 50% mortality after exposure to 90 minutes of 6 ATA oxygen and 100%
- Figure 8 shows that the percent survival in hyperbaric oxygen was dependent on the dose of the inhibitor given.
- the protection offered by this competitive inhibitor of nitric oxide synthase could be reversed when an excess of L-arginine was given ( Figure 6 and Figure 9).
- Group 1 edema formation of EC-SOD transgenic mice was compared with that of nontransgenic littermates.
- Group 2 compared edema formation between wild-type (C57BL/6 X C3H)F1 mice treated with saline and (C57BL/6 X C3H)F1 mice treated with 0.33 mg/g deferoxamine (0.51 ⁇ moles/g).
- Group 3 compared (C57BL/6 X C3H)F1 mice treated with saline to (C57BL/6 X C3H)F1 mice treated with 0.51 ⁇ moles/g Fe 3+ - saturated deferoxamine.
- Group 4 consisted of
- Group 6 compared edema formation between nontransgenic mice, EC-SOD transgenic mice treated with saline, and EC-SOD transgenic mice treated with 0.02 mg/g N- ⁇ -nitro- L-arginine methyl ester.
- mice were pretreated with i.p. injections of deferoxamine or saline prior to cold-induced injury.
- Table II shows that pretreatment with deferoxamine resulted in 43% less edema formation compared to mice only given saline.
- Mice were then pretreated with i.p. injections of iron-saturated deferoxamine or saline before cold-induced injury to see if the iron chelating properties of this compound were truly necessary for protection against edema formation.
- Table IV shows that, even when deferoxamine was saturated with iron, it was still capable of protecting against edema formation and resulted in 32-48% less edema than that found in saline treated controls.
- the absolute values for the edema index were found to quite variable, however, repeated experiments consistently show the same
- deferoxamine is capable of protecting against edema formation by a mechanism independent of its ability to scavenge iron. Because deferoxamine is capable of scavenging both the
- deferoxamine that enable it to protect against vasogenic edema.
- Transgenic + LNAME 15 minutes prior to cold-induced injury. Values are presented as mean ⁇ standard error and were compared using analysis of variance with a Fisher PLSD test. No significant difference was seen between transgenic and transgenic + LNAME mice.
- Human lung Five human lung samples were obtained to evaluate the distribution of EC-SOD in human lung tissue. One sample was obtained from a surgical
- the patient was diagnosed with squamous cell carcinoma. Tissue from a region not involved in the carcinoma from this lobe was used in the studies presented here.
- a second lung was obtained from a right upper lobe surgical pathology specimen resected from a 51 year old white male with a 60 pack year smoking history found to have an isolated nodule on X-ray. The patient had no other illness and was diagnosed with squamous cell carcinoma. Lung tissue not involved in the carcinoma from this specimen was used for the localization of EC-SOD.
- a third lung was obtained from a rapid autopsy (tissue obtained 6 hours after death) of a 66 year old white male with dementia, but no history of smoking or lung disease.
- the fourth lung examined was obtained from excess lung tissue of a lung too large for the recipient of a lung transplant.
- the lung was donated from a 45 year old white female with no history of smoking or lung disease.
- the fifth- lung examined in these studies was also from excess lung tissue used for lung transplantation from a 39 year old white male with no history of smoking or lung disease. Notably, no differences in labeling patterns were seen between the surgical pathology specimens, the autopsy tissues from donors for lung transplantation.
- the tissues were fixed in 2% paraformaldehyde/0.2% gluteraldehyde in 0.01 M phosphate buffered saline (PBS; 1.2 g NaH 2 PO 4 , 8 g NaCl, 0.2 g KCl, in 1 liter pH 7.3) for 1 hour followed by overnight fixation in 4%
- PBS phosphate buffered saline
- the tissues were frozen in liquid nitrogen chilled hexane and stored at -70°C until they were sectioned for light microscopic studies.
- lung tissues were processed as in the light microscopic studies up to the equilibration in sucrose. After equilibration in sucrose, the lung tissues were infiltrated with 10% gelatin at 37°C for 10 minutes. The tissue slices, in gelatin, were then solidified on ice, cut into 2 mm/side cubes, and then cryoprotected in 4% polyvinyl alcohol containing 2 M sucrose overnight. These samples were then mounted onto stubs, flash frozen in liquid
- diaminobenzidine (10 mg diamino- benzidine, 50 ml 0.05 M Tris ⁇ Cl, pH 7.6, 100 ⁇ l 3% H 2 O 2 ) and counterstained with 1% methyl green.
- serial sections were separately labeled with either rabbit anti-rh-EC-SOD (EC-SOD), non-immune rabbit IgG, or rabbit anti-rh-EC-SOD from which EC-SOD binding IgG had been absorbed out (EC-SOD absorbed; see above).
- Ultrathin cryo sections (70 nm) of human lung tissue were immunolabeled with rabbit anti rh-EC-SOD and 10-nm protein A-gold as prevsicusly described (Crapo et al, Proc. Natl. Acad. Sci. USA 89:10405 (1992)) (Table VI). Briefly, sections were first incubated three times for five minutes at room temperature in 0.15% glycine in PBS to block aldehyde groups. Nonspecific binding was further blocked by incubation in 1% BSA in PBS for 10 minutes. Primary and secondary antibody dilutions were determined empirically and made in PBS containing 1% BSA.
- Sections were stained with uranyl oxalate and uranyl acetate in methyl cellulose as previously described (Crapo et al, Proc. Natl. Acad. Sci. USA 89:10405 (1992)). Control groups were as described for light microscopy above.
- EC-SOD antibody Characteristic of EC-SOD antibody: The antibody to rh-EC-SOD was characterized by Western blot analysis of rh-EC-SOD and a human lung homogenate. Figure 13 shows that the antibody reacted with both the EC-SOD type C (top band) and the type A (bottom band) subunits
- the antibody to EC-SOD was an IgG polyclonal rabbit antibody which was affinity purified using rh-EC-SOD.
- IgG specific for EC-SOD was absorbed out of the antisera using pure rh-EC-SOD bound to cyanogen bromide
- Nonimmune rabbit IgG was then added to this absorbed antibody in a sufficient amount to replace the absorbed IgG.
- Labeling lung tissues with this preabsorbed antibody preparation resulted in the absence of labeling in all areas of the lung including the pulmonary vasculature ( Figure 14c). Labeling lung tissue with nonimmune IgG alone also resulted in the absence of labeling in all areas of the lung.
- the controls indicate that the labeling observed with the primary antibody is specific for EC-SOD in the lung.
- Electron microscopic immunocytochemistry A summary of the labeling for EC-SOD in the lung found using electron microscopic immunocytochemistry is summarized in Table VII.
- EC-SOD was mainly associated with extracellular matrix proteins in all regions of the lung.
- a high degree of labeling was seen in areas rich in type I collagen (Figure 16) and in association with other unidentified proteoglycans in extracellular matrix (Figure 17).
- no labeling for EC-SOD was seen in elastin-rich areas (Figure 16).
- a high degree of labeling was observed near the surface of smooth muscle cells and in the connective tissue matrix surrounding smooth muscle cells in vessels
- (+) indicates presence of labeling for EC-SOD and (-) indicates no labeling for EC-SOD.
- ( ⁇ ) represents areas where low low amounts of labeling for EC-SOD were inconsistently observed.
- EC-SOD may have an important function in this location.
- Superoxide is known to rapidly react with nitric oxide and inactivate its properties as a smooth muscle relaxant. Therefore, the presence of EC-SOD along the diffusion path of nitric oxide to smooth muscle cells should increase the half life of this short lived intercellular messenger in this particular region and thus increase its potency as a vasodilator.
- the high labeling for EC-SOD seen around vascular and airway smooth muscle cells indicates a function for EC-SOD as a mediator of nitric oxide activity in the maintenance of low pulmonary vascular pressures and airway resistence.
- the superoxide anion may be capable of activating latent collagenases from polymorphonuclear leukocytes (PMN) which can lead to further collagen degradation.
- PMN polymorphonuclear leukocytes
- ATGACCTCCTGCCAGATCTCC-3' following a protocol by GIBCO BRL (5' RACE System).
- the RNA template was degraded by the addition of RNase H at 55°C for 10 minutes.
- the resulting cDNA was isolated using a GlassMAX DNA (GIBCO BRL) isolation spin cartridge.
- the purified cDNA was dC-tailed using terminal deoxynucleotidyl transferase (TdT, 0.5 units/ul). 200 ⁇ M dCTP, 10 mM Tris-HCl (pH 8.4), 25 mM KCl, 1.25 mM MgCl 2 and 50 ⁇ g/ml bovine serum albumin for 10 minutes at 37°C.
- the TdT was heat inactivated for 10 minutes at 70°C.
- the dC-tailed products were PCR amplified using EC7 and HEC1 (a sense- strand EC-SOD gene specific primer,
- the final composition of the reaction included 20 mM Tris-HCl (pH 8.4), 50 mM KCl, 2.5 mM MgCl 2 , 100 ⁇ g/mi bovine serum albumin, 400 nM Anchor primer, 200 nM gene-specific primer, and 200 ⁇ M each of dATP, dCTP, dGTP, dTTP. After incubating the PCR reaction for 5 minutes at 94°C, amplitaq (Perkin
- PCR cycling was performed on a Perkin Elmer 9600 for 35 cycles with melting at 94°C for 45 seconds and annealing at 53°C for 15 seconds and extension at 72°C for 90 seconds.
- the full-length EC-SOD cDNA (6 ng) was used as a positive control in the PCR reaction.
- the PCR products were electrophoresed in a 2% SeaPlaque GTG agarose gel, transferred to charged nylon membranes by the method of Southern (Southern, J. Mol. Biol. 98:503 (1975)) using the alkaline transfer protocol (Reed et al, Nuc. Acids Res. 13:7207 (1985)).
- the DNA was fixed to the membrane by baking at 80°C in a vacuum oven for 2 hours.
- the subsequent blot was hybridized to [ 32 P] end-labeled HEC2 (an internal, nested EC-SOD specific primer, 5'-TCCAGCTCCTCCAAGAGAGC-3') overnight at 37°C.
- the blot was washed at increasing stringencies until background hybridization was removed. This was followed by exposure to XAR-5 film using a Lightening Plus intensifier screen at -70°C.
- Human genomic Southern blot analysis :
- a human adult female leukocyte genomic library constructed in the EMBL-3 vector was obtained from
- Clontech Approximately 1 X 10 6 pfu were screened at a density of -50,000 pfu/plate using [ 32 P]CTP-labeled human EC-SOD cRNA (1 X 10 6 dpm/ml). The primary through tertiary screens identified approximately 7 unique putative positive plaques. Individual plaques were isolated and lambda DNA purified using LambdaSorb phage adsorbent (Promega Biotec). The size of the insert DNA from each clone was assessed by Sal I restriction endonuclease digestion followed by electrophoresis in 0.7% agarose. Selected clones underwent extensive restriction endonuclease mapping. Based on the
- Oligonucleotides derived from this initial sequencing data were synthesized approximately every 250 base pairs until the complete nucleotide sequence was obtained. Sequencing data were obtained from both strands as shown in Figure 203 except at the 3' portion of the gene where DNA sequence was obtained on one strand only.
- the IntelliGenetics Geneworks program (Version 2.2) was used for organizing the DNA sequence data. Homology searching was performed at the NCBI using the BLAST (Altschul et al, J. Mol. Biol. 215:403 (1990)) network service and the non-redundant nucleotide sequence database (GenBank (77.0) + EMBL (35.0) + EMBLUpdate + GBUdate). Transcriptional factor database searching was performed using both the SIGNAL SCAN 3.0 algorithm
- SIGSEQ1 (Folz et al, J. Biol. Chem. 261:14752 (1986)) and SIGSEQ2 were employed (Folz et al, Biochem. Biophys. Res. Commun. 146:870 (1987)). Results :
- poly A(+) mRNA from eight different human tissues was fractionated on a denaturing agarose gel and transferred to a charged nylon membrane. Because a previous paper reported long exposures times in order to identify EC- SOD specific bands during genomic Southern analysis (Hendrickson et al, Genomics 8:736 (1990)), a
- mapping of the site of transcription initiation was attempted using the primer extension method. Using several different end-labeled 5'
- RNA generated by in vi tro oligonucleotides and both human lung and human heart poly A+ mRNA as well as total RNA isolated from human foreskin fibroblasts
- Genomic Southern blot analysis To begin to charcterize the human EC-SOD gene, 10 ⁇ g of the total human genomic DNA was restriction digested and the reaction products electrophoresed on an agarose gel followed by transfer to a nylon membrane. The blot was probed with a [ 32 P]-labeled partial EC-SOD cRNA. An autoradiogram of this blot is shown in Figure 23. As can be seen for each lane, there are unique bands associated with each restriction digest. No shadow bands which might suggest pseudogenes were seen. When a full-length cRNA probe was used for Kpn I
- the Kpn I lane shows a 0.5 kb band which was better seen on other blots.
- This banding pattern was similar to a restriction map of the human EC-SOD clone #7 (see Figure 20A).
- Clone #7 is about 18 to 20 kb and contains at least 5000 bp of 5' flanking DNA and at least 4000 bp of 3' flanking DNA. Restriction mapping of clone #7 is shown in Figure 20A. This map is similar to the results obtained with genomic Southern blot analysis data indicating that clone #7 contains the EC-SOD gene. The strategy for sublconing and sequencing clone #7 is shown in Figure 20B. Various size continguous and overlapping restriction fragments were subcloned into the plasmid vector pGEM32f (+) ( Figure 20B).
- the DNA inserts were sequenced on both strands using a combination of primer walking and vector specific, universal sequencing primers. The 3' half of 7K36 insert was sequenced on one strand only.
- Published sequence data for the human EC-SOD cDNA (Hjalmarsson et al, Proc. Natl. Acad. Sci. USA 84:6340 (1987)) as well as DNA sequence information obtained from an independant cDNA clone which contained additional 5' untranslated data (Hendrickson et al, Genomics 8:736 (1990)) were used to determine the genomic intron/exon structure.
- the human EC-SOD gene was determined to contain three exons and two introns (Figure 20C).
- Exon 1 contains at least 5 base pairs and is probably larger (by about 6 base paris), since the exact start of transcription
- Exon 2 contains 84 bp and is separated from exon 1 by a 572 bp intervening sequence marked as intron 1.
- Exon 3 is separated from exon 2 by intron 2, a 3849 bp segment.
- Exon 3 contains a total of 1336 bp and at 17 bp into this exon starts the beginning of the complete coding sequence for preEC-SOD ( Figure 20D). This includes an 18 amino acid signal peptide that precedes the 222 amino acid mature protein sequence. There are no introns separating the various structural domains of EC-SOD. These domains are shown schematically in Figure 20D and include amino acids 1-95 which contain a glycosylated Asn-89 and show no sequence homology with other
- Resides 96-193 show strong homology with
- the size of the introns and exons are shown in base pairs (bp).
- the uppercase letters indicate exon sequence while the lowercase letters indicate intron sequence.
- the splice junctions shown conform to previously published concensus sequences for splice junctions (Senapathy et al, Methods Enzymol. 183:252 (1990)).
- Figure 24 shows the entire sequence for the human EC-SOD gene. Exonic sequences are shown in boxed uppercase letters while intronic, 5'- and 3'-flanking sequence are shown in lowercase. Exon 3 contains the entire uninterrupted coding region for EC-SOD and the protein sequence is shown using the single letter amino acid code. The 18 amino acid signal peptide and 222 amino acid mature protein sequence are highlighted. The identification of the signal peptide cleavage site is consistent with computer algorithms which predict the site of eukaryotic signal peptide cleavage (Folz et al, Biochem. Biophys. Res. Comm. 146:870 (1987)); Von
- Transcriptional factor database searching was used to putatively identify transcriptional regulatory elements. Although almost all eukaryotic promoters utilize a TATA box element to fix the position of transcription initiation, an obvious TATA box cannot be discerned for the EC-SOD gene. Two CAAT box elements were identified. One is in the reverse orientation and located about 20 bp upstream of the first exon, while the second can be found about 335 bp upstream. The putative signal for polyadenylation is shown and the site of poly A adenylation is indicated.
- adenovirus transcription virus (ATF) element can be found starting at 121 bp (Fink et al, Proc. Natl. Acad. Sci. USA 85:6662 (1988); Sassone-Corsi Proc. Natl. Acad. Sci. USA 85:7192 (1988)).
- ATF adenovirus transcription virus
- GRE glucocorticoid response element
- TGTCCT glucocorticoid response element
- M-CAT skeletal muscle specific trans-activating factor response element
- XRE xenobiotic responsive element
- a metal regulatory element (MRE) (TGCRCYC) is found at position 89 (Culotta et al, Mol. Cell. Biol. 9:1376 (1989)).
- Two putative antioxidant response elements (ARE) (RGTGACNNNGC) are found at position 650 and 5022 (Rushmore et al, J. Biol. Chem. 266:11632 (1991)).
- a sis responsive element (SIF) (CCCGTC) important in the induction of the c-fos proto- oncogene is found in the reverse orientation at position 251 (Wagner et al, EMBO J. 9:4477 (1990)).
- SIF sis responsive element
- CCCGTC important in the induction of the c-fos proto- oncogene is found in the reverse orientation at position 251 (Wagner et al, EMBO J. 9:4477 (1990)).
- TRE TPA responsive element
- CAGCTGTGG can be found at position 171 (Jones et al, Genes Dev. 2:267 (1988)).
- Genomic DNA will be purified utilizing a Qiagen Blood
- PCR Kit One ml of blood containing -10 7 leukocytes/ml will be- collected in sodium citrate from each patient or control subject. The blood is placed into a QIAGEN-spin column and the leukocytes are entrapped in the resin by brief centrifugation, while erythrocytes and hemoglobin are washed through. The leukocytes are lysed by the addition of 0.7 ml of lysis buffer and incubated at room temperature for 30 minutes. DNA that is released, binds to the resin in the tube. Remaining cellular debris is washed away by multiple wash/spin cycles. The DNA is eluted by the addition of 1.2 M KCl, 50 mM MOPS, 15% ethanol, pH 8.3. This typically yields -10 ⁇ g of genomic DNA (Reihsaus et al, Am. J. Respir. Cell. Mol. Biol. 8:334 (1993)).
- Sense and antisense oligonucleotide primers (or use primers already obtained from sequencing the genomic DNA) will be designed containing a 3' GC clamp (Sheffeld et al, Proc. Natl., Acad. Sci. USA 86:232 (1989)). These primers will encode the intronless coding region of the EC-SOD gene. A 172 bp region in the 3' untranslated region has been amplified using DNA sequencing primers and human genomic DNA.
- SSCP analysis has been used to detect single base pair mismatch (Orita et al, Genomics 5:874 (1989)).
- Temperature-gradient gel electrophoresis will be used to detect differences in mobility (Wartell et al, Nuc. Acids Res. 18:2599 (1990)). Samples for TGGE will be prepared by heat denaturing the PCR product at 98°C for 5 min, then renaturing at 50°C for 15 min with the corresponding wild-type DNA derived from PCR of the cloned gene. Electrophoresis will be carried out on a 5% acrylamide, 8 M urea gel over a temperature gradient. The temperature gradient will be optimized for each of the EC-SOD DNA segments. Typical gradients for the detection of ⁇ 2 -adrenergic receptor mutations were between 35°C to 60°C, and required 4 to 6 hours of run time (Rosen, Nature 262:59 (1993)).
- CPA-47 Tissue and Cell
- MnTBAP 50 ⁇ M of MnTBAP in minimum essential medium (MEM) or MEM only. Varing amounts of xanthine oxidase (XO) plus 200 ⁇ M xanthine (X) were added and allowed to incubate for 24 hours. Cell injury was quantitiated by measuring the release of cellular lactate dehydrogenase (LDH) into the medium. The efficacy of MnTBAP is shown in Figure 26 by the decrease in XO/X-induced LDH release.
- Rat pulmonary epithelial cell cultures (L2 (Kaighn and Douglas J. Cell Biol. 59:160a (1973)) were grown to confluence in Ham's F-12K medium with 10% fetal bovine serum at pH 7.4 and 37°C. Cells were then trypsinized and seeded at equal densities in 24 well plates and grown to 90% confluence. Cells were washed and preincubated for 1 hour with 100 ⁇ M of MnTBAP or MnTMPyP in MEM or MEM only. Paraquat (2.5 mM) was added and allowed to incubate for 48 hours. Cell injury was quantitiated by measuring the release of cellular lactate dehydrogenase (LDH) into the medium. Figure 27 shows that MnTPyP (hatched bars) and MnTBAP (grey bars) decrease paraquat-induced LDH release.
- LDH lactate dehydrogenase
- CPA-47 Tissue and Cell
- LDH lactate dehydrogenase
- ZnTBAP does not protect against paraquat-induced injury.
- Calf pulmonary artery endothelial cell cultures (CPA-47) were grown to
- ZnTBAP in MEM or MEM only. Paraquat (2 mM) was added and allowed to incubate for 24 hours. Cell injury was quantitiated by measuring the release of cellular lactate dehydrogenase (LDH) into the medium.
- LDH lactate dehydrogenase
- mice 45 mg/kg, ip) or saline (10 ml/kg, ip) and exposed to MnTBAP (2.5 mg/ml, nebulized into a 2 L chamber at 2 L/min for 30 minutes twice daily for 2 days) or room air. Mice were killed 48 hours after start of treatment and lung injury was assessed by analysis of
- BALF damage markers used were lactate dehydrogenase (LDH, as
- MnTBAP treatment provided partial protection against paraquat-induced lung injury (see Figure 30).
- Catalase activity was measured by means of a Clark oxygen electrode using a modified assay previously described by Del Rio et al, Anal. Biochem. 80:409
- Calf pulmonary endothelial cell (CPA-47) line was grown to near confluence in 12-well plates with F-12K medium containing 10% fetal calf serum.
- CPA-47 cells were loaded with Cr 51 as previously described by Simon et al J. Clin. Invest. 78:1375 (1986) in high glucose minimum essential medium (DMEM) .
- DMEM high glucose minimum essential medium
- Cells were pretreated with MnTBAP (100 ⁇ M) for 1 hour and then exposed to various concentrations of hydrogen peroxide generator, glucose oxidase, for 4 hours. Cell injury was then quantitated as the specific Cr 51 release from CPA-47 cells which has been adjusted for spontaneous Cr 51 release. The results are shown in Figure 32.
- Calf pulmonary endothelial cell (CPA-47) line was grown to near confluence in 12-well plates with F-12K medium containing 10% fetal calf serum.
- CPA-47 cells were loaded with Cr 51 as previously described by Simon et al (J. Clin. Invest. 78:1375 (1986)) in high glucose minimum essential medium (DMEM).
- DMEM high glucose minimum essential medium
- Cells were pretreated with various concentrations of either: (A) MnTBAP; (B) MnTMPyP; (C) ZnTBAP; or (D) CuZnSOD for 1 hour and then exposed to the hydrogen peroxide generator, glucose oxidase (100 Mu/ml), for 4 hours.
- Cell injury was then quantitated as the specific Cr 51 release from CPA-47 cells which has been adjusted for spontaneous Cr 51 release. The results are shown in Figure 33A-33D.
- Tissue culture Mixed neuronal and glial cultures were prepared from embryonic day-18 rat cerebral cortices (Sprague-Dawley, Zivic Miller). Briefly, the cerebral cortices were dissected and enzymatically dissociated by incubation in Ca ++ - , Mg ++ -free Hank's balanced salt solution (HBSS) supplemented with 10 mM HEPES and 0.25 % trypsin for 20 min. at 37°C. The tissue was rinsed and dispersed into single cell suspension by gentle passage through a fire polished Pasteur pipette.
- HBSS Hank's balanced salt solution
- the cell suspension was centrifuged and resuspended in Minimum Essential Media (MEM), containing Earle's salts supplemented with 3 g/L glucose, 5% horse serum and 5% fetal bovine serum (growth media).
- MEM Minimum Essential Media
- the cells were plated in poly-D-lysine-coated multi-well plates: 12-well plates for aconitase measurement, and 24 well plates for toxicity experiments. Cells were maintained at 37°C in a humidified incubator with 5% CO 2 /95% air in growth media. Media was not replaced so as to reduce glial overgrowth and neuronal loss. Mature cells (14-17 days in vi tro) were used for all experiments.
- Cell trea tment The growth media was replaced with MEM supplemented with 25 mM glucose (MEM-g). For both neurotoxicity studies and aconitase measurement, cells were incubated in the designated treatment for the indicated length of time at 37°C. Unless otherwise specified, antagonists or SOD-mimetics were added 15 min. prior to agonists. For measurement of
- NMDA neurotoxicity
- growth media was replaced with HBSS + (Ca ++ -, Mg ++ -free Hank's Balanced Salt Solution supplemented with 2 mM CaCl 2 , 1 mM NaHCO 3 , 10 mM HEPES and 5 ⁇ M glycine), cells were treated with vehicle or 100 ⁇ M NMDA for 15 min. after which HBSS+ was replaced with MEM-g and cells were returned to the incubator for 18 additional hrs.
- HBSS + Ca ++ -, Mg ++ -free Hank's Balanced Salt Solution supplemented with 2 mM CaCl 2 , 1 mM NaHCO 3 , 10 mM HEPES and 5 ⁇ M glycine
- Antagonists or SOD-mimetics were added to the cells 15 min. before NMDA exposure or 15, 30 or 60 min. following final media replacement. For determination of Ca ++ -dependence, cells were incubated in HBSS + with no added Ca ++ . When kainate (KA) was used as an agonist, 100 ⁇ M D-APV was routinely included to block secondary NMDA receptor activation. Catalase (100 U/ml) was always included when xanthine plus xanthine oxidase (X+XO) was used as a treatment to rule out the
- Neurotoxicity was determined by the measurement of LDH released in the supernatant media as previously described (Patel et al, Toxicol. Appl. Pharmacol. 115:124 (1991) and
- LDH was measured by the method of Vassault, Lactate dehydrogenase. In: Methods of Enzymatic Analysis, Bergmeyer HU (ed), Verlag Chemie, Weinheim, pp. 118-126 (1983). Additionally, cell death was confirmed with ethidium- 1 homodimer (EthD-1).
- the cells were washed with HBSS, loaded with 20 ⁇ M EthD-1 for 30 min., rinsed and viewed through fluorescence optics.
- the number of dead cells labeled with EthD-1 were counted in 4-6 randomly selected fields, the numbers averaged to give an estimate of cell death and the experiment was repeated 3 times.
- Aconi tase measurement For the measurement of aconitase, following treatment with agents, media was removed and cells lysed in ice-cold 50 mM Tris/HCl, pH 7.4 containing 0.6 mM MnCl 2 , 1 mM L-cysteine, 1 mM citrate and 0.5% Triton-X 100. The aconitase activity of cell lysates was measured spectrophotometrically by monitoring the formation of cis-aconitate from
- Aconi tase inactivation produced by PQ ++ , NMDA and KA correlate wi th cell dea th : Cortical cells were treated with varying concentrations of KA, NMDA, and PQ ++ for 18 hr. and the media analyzed for LDH activity. Sister cultures were treated with varying concentrations of KA, NMDA, or PQ ++ for 6, 1 or 3 hr., respectively, and cell lysates analyzed for aconitase activity; a shorter time of incubation was assessed for aconitase activity because death of neurons leads to irreversible inactivation of aconitase activity, presumably due to protein degradation.
- KA treatment produced proportionate decreases in aconitase activity and cell death as monitored by LDH release (Fig. 35A).
- PQ ++ treatment produced a proportionate decrease of aconitase activity and the amount of LDH release (Fig. 35 B).
- MnTBAP prevents aconi tase inactivation and cell death produced by PQ, NMDA and KA: To determine whether MnTBAP can prevent aconitase inactivation and cell death produced by NMDA, PQ ++ and KA, cortical cells were incubated with PQ ++ , NMDA and KA in the absence and presence of 200 ⁇ M MnTBAP.
- PQ ++ , NMDA, and KA produced a 70 %, 40 % and 42% decrease in aconitase activity after 3, 1 and 6 hrs., respectively; 200 ⁇ M MnTBAP markedly inhibited the PQ ++ -, NMDA- and KA-mediated decreases of aconitase activity (Fig. 36 A) . Parallel effects were evident on cell death as measured by LDH release.
- MnTBAP (200 ⁇ M) markedly inhibited PQ ++ -, NMDA- and KA- mediated cell death (Fig. 36 B). The effects of MnTBAP on cell death were concentration dependent (Fig. 36 B). MnTBAP (200 ⁇ M) produced a right-ward and down-ward shift in the concentration-response curve for NMDA, providing complete protection at low concentrations of NMDA (Fig 37).
- MnTBAP simply prevented activation of NMDA or AMPA receptors was examined and eliminated.
- NMDA neuroprotective effects
- 100 ⁇ M NMDA was included for 15 min. in HBSS + (Ca ++ -, Mg ++ -free HBSS containing 5.6 mM glucose and supplemented with 2 mM CaCl 2 , 1 mM NaHCO 3 , 10 mM HEPES and 5 ⁇ M glycine).
- This acute exposuru paradigm induced delayed neuronal death (measured 18 hr. later) and allowed determination of the temporal
- MnTBAP neuroprotection.
- the neuroprotective effects of MnTBAP were assessed when it was: 1) present 15 min. prior to and during a 15 min. NMDA application (but not for the 18 hr. period after media change) (condition “pre”); 2) present 15 min. prior to and during a 15 min. NMDA application and for the ensuing 18 hr. (condition “pre + post”); 3) added 15, 30 or 60 min. following a 15 min. incubation with 100 ⁇ M NMDA and left in the media for the ensuing 18 hr.
- Condition "pre” resulted in a 25 % reduction of LDH release measured 18 hr. later; using an identical time course of application of 100 ⁇ M D-APV in condition "pre", a complete blockade of LDH release was observed.
- condition "pre + post” resulted in a 51% reduction of LDH release.
- EC-SOD knocked-out mice were obtained from Dr. Lena Carlsson and Dr. Stephan Marklund, Umea, Sweden.
- mice were derived from (+/+) litter mates bearing normal levels of EC-SOD
- mice were derived from (+/+) litter mates bearing normal levels of EC-SOD
- a count was made of the number of arms the mice went down to recover food before they went down any arm for a second time. Naive animals randomly went down between four and five different arms of the maze before
- nitrate in the culture medium was reduced to nitrite by incubation with nitrate reductase (670 mU/ml) and NADPH (160 ⁇ M) at room temperature for 2h. After 2h, nitrite concentration in the samples was measured by the Griess reaction, by adding 100 ⁇ l of Griess reagent (1% sulfanilamide and 0.1% naphthylethylenediamide in 5% phosphoric acid) to 100 ⁇ l samples of conditioned medium. The optical density at 550 nm (OD 550 ) was measured using a Spectramax 250 microplate reader
- MnTBAP concentrations were calculated by comparison with OD 550 of standard solutions of sodium nitrate prepared in culture medium. All measurements were corrected for the interference of MnTBAP at this wavelength. MnTBAP (up to 300 ⁇ M) did not scavenge nitrite or nitrate and did not interfere with the activity of nitrate reductase.
- Saturated NO solution was prepared by bubbling compressed NO gas through a NaOH trap and then into anaerobic deionized water. Aliquots of the nitric oxide solutions (final concentration: 75-300 nM) were added to the rings (in the presence or absence of 100 ⁇ M MnTBAP) and relaxant responses were recorded.
- MnTBAP is not a scavenger of ni tric oxide : MnTBAP did not inhibit the relaxations in vascular rings in response to authentic NO. Moreover, MnTBAP, at 300 ⁇ M, did not inhibit nitrite/nitrate accumulation in the culture medium in response to the NO donor compound SNAP and caused a slight inhibition in response to DNO.
- MnTBAP dihydrorhodamine 123 to rhodamine 123, which was dose- dependently inhibited by MnTBAP with 50% inhibition at 30 ⁇ M (Fig. 40).
- MnTBAP like cysteine, urate, ascorbate and alpha-tocopherol, inhibits the oxidations caused by peroxynitrite.
- MnTBAP inhibi ts the suppression of mi tochondrial respiration by authentic peroxyni tri te in J774 cells . Mitochondrial respiration was profoundly inhibited by exposure to 1 mM peroxynitrite at lh (Fig. 41A). This effect was partially and dose dependently prevented by MnTBAP.
- MnTBAP inhibits the suppression of mi tochondrial respiration by NO donors in J774 cells : Mitochondrial respiration was also inhibited by exposure to the NO donor compound SNAP (Fig. 4IB) and DNO for 24h
- LPS lipopolysaccharide
- IFN gamma interferon
- MnTBAP caused a dose-dependent inhibition of nitrite/nitrate production in cells stimulated with LPS. However, in cells immunostimulated with the combination of LPS and IFN, MnTBAP caused a less pronounced
- MnTBAP only caused a 25% inhibition of nitrite/nitrate accumulation and pronounced inhibition (61%) was only observed at the highest concentration of MnTBAP tested (300 ⁇ M).
- L-NMA N G -methyl-L-arginine
- MnTBAP had no additional effect on nitrite/nitrate formation in the presence of L-NMA.
- MnTBAP caused a dose-dependent, partial restoration of the immunostimulation- induced suppression of the mitochondrial respiration in both LPS-treated and (less potently) LPS and IFN-treatec cells.
- Inhibition of NOS with L-NMA caused a restoration of the respiration to an extent comparable with that of 300 ⁇ M MnTBAP.
- the combined administration of 300 ⁇ M MnTBAP and 3mM L-NMA caused an additional restoration of the mitochondrial respiration.
- respiration was restored to initial levels in the LPS- stimulated cells, but remained below normal in cells stimulated with LPS and IFN.
- the crude product 2 was dissolved in acetone/H 2 O (200 mL, 83:17) then AgNO 3 (47.25 g, 278.2 mmole) was added. The flask was covered with foil to avoid
- the crude product was usually combined with 2-3 g silica gel to make a mixture which was then divided into 2 batches for 2 separate chromatographic purifications on silica gel (ht of silica: 12-15 cm, diameter 5 cm).
- the first eluent used was ether 1:1 hexanes/Et 2 O or 4:1 hexanes/EtOAc
- Acetonitrile is reacted with hydroxylamine to prepare the amide oxime.
- An excess of the amide oxime is allowed to react with the product described in
- Example XIV section 15, to provide a mixture of 10207 and 10208. Chromatographic purification of the reaction mixture provides pure 10207 and 10208.
Landscapes
- Chemical & Material Sciences (AREA)
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Biotechnology (AREA)
- Genetics & Genomics (AREA)
- Nanotechnology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- General Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Biomedical Technology (AREA)
- Epidemiology (AREA)
- Toxicology (AREA)
- Biophysics (AREA)
- Medical Informatics (AREA)
- Crystallography & Structural Chemistry (AREA)
- Microbiology (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
- Solid-Sorbent Or Filter-Aiding Compositions (AREA)
Abstract
Description
Claims
Priority Applications (6)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
CA2223407A CA2223407C (en) | 1995-06-07 | 1996-06-07 | Oxidant scavengers |
AT96923328T ATE306936T1 (en) | 1995-06-07 | 1996-06-07 | CATCHER FOR OXIDANTS |
EP96923328A EP0831891B1 (en) | 1995-06-07 | 1996-06-07 | Oxidant scavengers |
DE69635304T DE69635304T2 (en) | 1995-06-07 | 1996-06-07 | FAENGER FOR OXIDANTS |
AU63870/96A AU725602B2 (en) | 1995-06-07 | 1996-06-07 | Oxidant scavengers |
JP9502304A JPH11509180A (en) | 1995-06-07 | 1996-06-07 | Oxidant remover |
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US08/476,866 | 1995-06-07 | ||
US08/476,866 US5994339A (en) | 1993-10-15 | 1995-06-07 | Oxidant scavengers |
US61341896A | 1996-03-11 | 1996-03-11 | |
US08/613,418 | 1996-03-11 |
Publications (1)
Publication Number | Publication Date |
---|---|
WO1996040223A1 true WO1996040223A1 (en) | 1996-12-19 |
Family
ID=27045322
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/US1996/010497 WO1996040223A1 (en) | 1995-06-07 | 1996-06-07 | Oxidant scavengers |
Country Status (9)
Country | Link |
---|---|
EP (1) | EP0831891B1 (en) |
JP (1) | JPH11509180A (en) |
AT (1) | ATE306936T1 (en) |
AU (1) | AU725602B2 (en) |
CA (1) | CA2223407C (en) |
DE (1) | DE69635304T2 (en) |
ES (1) | ES2249784T3 (en) |
IL (1) | IL122451A0 (en) |
WO (1) | WO1996040223A1 (en) |
Cited By (22)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP0893951A1 (en) * | 1996-01-26 | 1999-02-03 | The Regents Of The University Of California | METHODS OF MODULATING RADICAL FORMATION BY MUTANT CuZnSOD ENZYMES |
WO2000009111A2 (en) * | 1998-08-11 | 2000-02-24 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Inhibitors of amyloid formation |
EP1045851A1 (en) * | 1997-11-03 | 2000-10-25 | Duke University | Substituted porphyrins |
US6403788B1 (en) | 2000-07-11 | 2002-06-11 | Eukarion, Inc. | Non-genotoxic metalloporphyrins as synthetic catalytic scavengers of reactive oxygen species |
JP2002535332A (en) * | 1999-01-25 | 2002-10-22 | ナショナル・ジュウィッシュ・メディカル・アンド・リサーチ・センター | Substituted porphyrins |
US6479477B1 (en) | 1998-04-24 | 2002-11-12 | Duke University | Substituted porphyrins |
US6583132B1 (en) | 1993-10-15 | 2003-06-24 | Duke University | Oxidant scavengers |
US6630128B1 (en) | 1998-08-28 | 2003-10-07 | Destiny Pharma Limited | Porphyrin derivatives their use in photodynamic therapy and medical devices containing them |
EP1740094A2 (en) * | 2004-03-29 | 2007-01-10 | Inotek Pharmaceuticals Corporation | Pyridyl-substituted porphyrin compounds and methods of use thereof |
WO2008114017A2 (en) * | 2007-03-19 | 2008-09-25 | Imperial Innovations Limited | Compositions and methods relating to influenza infection |
US7642250B2 (en) | 2005-09-28 | 2010-01-05 | Inotek Pharmaceuticals Corporation | N-benzyl substituted pyridyl porphyrin compounds and methods of use thereof |
US7740839B2 (en) * | 2003-10-31 | 2010-06-22 | Tae-Yoon Kim | EC SOD and cell transducing EC SOD and use thereof |
CN102584836A (en) * | 2011-12-31 | 2012-07-18 | 中国科学院化学研究所 | Porphyrin cage compound, and preparation method and application thereof |
US8252595B2 (en) | 2008-05-13 | 2012-08-28 | University Of Kansas | Metal abstraction peptide (MAP) tag and associated methods |
US8318803B2 (en) | 2003-10-31 | 2012-11-27 | Tae-Yoon Kim | EC SOD and use thereof |
US8470808B2 (en) | 1999-01-25 | 2013-06-25 | Jon D. Piganelli | Oxidant scavengers for treatment of type I diabetes or type II diabetes |
US8883858B1 (en) | 2012-12-07 | 2014-11-11 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
US9187735B2 (en) | 2012-06-01 | 2015-11-17 | University Of Kansas | Metal abstraction peptide with superoxide dismutase activity |
CN105399750A (en) * | 2015-12-11 | 2016-03-16 | 西北师范大学 | Tetrazole porphyrin metal complexes, synthesis thereof and applications of the metal complexes as dye sensitizing agents |
EP2759546A4 (en) * | 2011-09-21 | 2016-10-19 | Univ Okayama Nat Univ Corp | Metal porphyrin complex, method for producing same, carbon dioxide immobilization catalyst comprising same, and method for producing cyclic carbonic acid ester. |
CN112694887A (en) * | 2020-12-07 | 2021-04-23 | 黑龙江省农业科学院植物保护研究所 | Light-emitting sensor, construction method thereof and application of light-emitting sensor in detection of salicylic acid content in plants |
US11382895B2 (en) | 2008-05-23 | 2022-07-12 | National Jewish Health | Methods for treating injury associated with exposure to an alkylating species |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
ITFI20020200A1 (en) * | 2002-10-21 | 2004-04-22 | Molteni & C Dei Flii Alitti S P A Societa L | MESO-REPLACED PORPHYRINES. |
JP6444211B2 (en) * | 2015-02-24 | 2018-12-26 | 学校法人同志社 | Porphyrin derivatives and photosensitizers containing the same |
Citations (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5248603A (en) * | 1985-09-03 | 1993-09-28 | Symbicom Aktiebolag | Superoxide dismutase |
US5366729A (en) * | 1989-09-15 | 1994-11-22 | Symbicom Aktiebolag | Non-glycosylated variants of extracellular superoxide dismutase (EC-SOD) |
WO1995010185A1 (en) * | 1993-10-15 | 1995-04-20 | Duke University | Superoxide dismutase and mimetics thereof |
Family Cites Families (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
DE3464252D1 (en) * | 1983-06-03 | 1987-07-23 | Hoffmann La Roche | Labelled molecules for fluorescence immunoassays and processes and intermediates for their preparation |
GB8429845D0 (en) * | 1984-11-26 | 1985-01-03 | Efamol Ltd | Porphyrins & cancer treatment |
US4746735A (en) * | 1986-11-21 | 1988-05-24 | The Dow Chemical Company | Regiospecific aryl nitration of meso-substituted tetraarylporphyrins |
US4892941A (en) * | 1987-04-17 | 1990-01-09 | Dolphin David H | Porphyrins |
US5051337A (en) * | 1987-06-10 | 1991-09-24 | Director-General Of Agency Of Industrial Science And Technology | Optical recording material |
GB8805849D0 (en) * | 1988-03-11 | 1988-04-13 | Efamol Holdings | Porphyrins & cancer treatment |
DE3809671A1 (en) * | 1988-03-18 | 1989-09-28 | Schering Ag | PORPHYRINE COMPLEX COMPOUNDS, METHOD FOR THE PRODUCTION THEREOF AND PHARMACEUTICAL AGENTS CONTAINING THEM |
FR2632187B1 (en) * | 1988-06-02 | 1990-09-14 | Centre Nat Rech Scient | METALLOPORPHYRIN DERIVATIVES, THEIR PREPARATION, THEIR THERAPEUTIC APPLICATION AND THEIR USE FOR THE PREPARATION OF HYBRID MOLECULES |
US5171680A (en) * | 1988-06-14 | 1992-12-15 | Chiron Corporation | Superoxide dismutase analogs having novel binding properties |
US5236915A (en) * | 1990-05-31 | 1993-08-17 | Health Research, Inc. | Meso poly(4-sulfonatophenyl) porphines as MRI image enhancing agents |
FR2676738B1 (en) * | 1991-05-22 | 1995-05-05 | Ir2M | NOVEL ORGANIC TRANSITION METAL WITH PORPHYRINIC STRUCTURE, THERAPEUTIC COMPOSITION CONTAINING SAME, IN PARTICULAR WITH HYPOGLYCEMIC ACTIVITY. |
US5262532A (en) * | 1991-07-22 | 1993-11-16 | E.R. Squibb & Sons, Inc. | Paramagnetic metalloporphyrins as contrast agents for magnetic resonance imaging |
-
1996
- 1996-06-07 EP EP96923328A patent/EP0831891B1/en not_active Expired - Lifetime
- 1996-06-07 IL IL12245196A patent/IL122451A0/en unknown
- 1996-06-07 DE DE69635304T patent/DE69635304T2/en not_active Expired - Lifetime
- 1996-06-07 AT AT96923328T patent/ATE306936T1/en active
- 1996-06-07 WO PCT/US1996/010497 patent/WO1996040223A1/en active IP Right Grant
- 1996-06-07 CA CA2223407A patent/CA2223407C/en not_active Expired - Lifetime
- 1996-06-07 JP JP9502304A patent/JPH11509180A/en active Pending
- 1996-06-07 AU AU63870/96A patent/AU725602B2/en not_active Expired
- 1996-06-07 ES ES96923328T patent/ES2249784T3/en not_active Expired - Lifetime
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5248603A (en) * | 1985-09-03 | 1993-09-28 | Symbicom Aktiebolag | Superoxide dismutase |
US5472691A (en) * | 1985-09-03 | 1995-12-05 | Symbicom Aktiebolag | Superoxide dismutase |
US5366729A (en) * | 1989-09-15 | 1994-11-22 | Symbicom Aktiebolag | Non-glycosylated variants of extracellular superoxide dismutase (EC-SOD) |
WO1995010185A1 (en) * | 1993-10-15 | 1995-04-20 | Duke University | Superoxide dismutase and mimetics thereof |
Non-Patent Citations (3)
Title |
---|
AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY, Volume 8, 1993, CLYDE et al., "Distribution of Manganese Superoxide Dismutase mRNA in Normal and Hyperoxic Rat Lung", pages 530-537. * |
BIOCHEM. J., Volume 298, issued 01 June 1994, STRALIN et al., "Effects of Oxidative Stress on Expression of Extracellular Superoxide Dismutase, CuZn-Superoxide Dismutase and Mn-Superoxide Dismutase in Human Dermal Fibroblast", pages 347-352. * |
GENOMICS, Volume 22, 1994, FOLZ et al., "Extracellular Superoxide Dismutase (SOD3): Tissue-Specific Expression, Genomic Characterization and Computer-Assisted Sequence Analysis of the Human EC SOD Gene", pages 162-171. * |
Cited By (49)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US6583132B1 (en) | 1993-10-15 | 2003-06-24 | Duke University | Oxidant scavengers |
EP0893951A1 (en) * | 1996-01-26 | 1999-02-03 | The Regents Of The University Of California | METHODS OF MODULATING RADICAL FORMATION BY MUTANT CuZnSOD ENZYMES |
EP0893951A4 (en) * | 1996-01-26 | 2003-04-23 | Univ California | METHODS OF MODULATING RADICAL FORMATION BY MUTANT CuZnSOD ENZYMES |
EP1045851A1 (en) * | 1997-11-03 | 2000-10-25 | Duke University | Substituted porphyrins |
EP1045851A4 (en) * | 1997-11-03 | 2001-05-09 | Univ Duke | Substituted porphyrins |
JP2010229145A (en) * | 1997-11-03 | 2010-10-14 | Duke Univ | Substituted porphyrins |
US6479477B1 (en) | 1998-04-24 | 2002-11-12 | Duke University | Substituted porphyrins |
WO2000009111A2 (en) * | 1998-08-11 | 2000-02-24 | The Government Of The United States Of America, Represented By The Secretary, Department Of Health And Human Services | Inhibitors of amyloid formation |
WO2000009111A3 (en) * | 1998-08-11 | 2000-11-23 | Us Gov Health & Human Serv | Inhibitors of amyloid formation |
US6632808B1 (en) | 1998-08-11 | 2003-10-14 | The United States Of America As Represented By The Department Of Health And Human Services | Inhibitors of amyloid formation |
US6630128B1 (en) | 1998-08-28 | 2003-10-07 | Destiny Pharma Limited | Porphyrin derivatives their use in photodynamic therapy and medical devices containing them |
US7189707B2 (en) | 1999-01-25 | 2007-03-13 | National Jewish Medical Research Center | Substituted porphyrins |
JP2002535332A (en) * | 1999-01-25 | 2002-10-22 | ナショナル・ジュウィッシュ・メディカル・アンド・リサーチ・センター | Substituted porphyrins |
US8470808B2 (en) | 1999-01-25 | 2013-06-25 | Jon D. Piganelli | Oxidant scavengers for treatment of type I diabetes or type II diabetes |
US8546562B2 (en) | 1999-01-25 | 2013-10-01 | James D. Crapo | Substituted porphyrins |
US8946202B2 (en) | 1999-01-25 | 2015-02-03 | Aeolus Sciences, Inc. | Substituted porphyrins |
US6544975B1 (en) | 1999-01-25 | 2003-04-08 | National Jewish Medical And Research Center | Substituted porphyrins |
US9289434B2 (en) | 1999-01-25 | 2016-03-22 | Aeolus Sciences, Inc. | Substituted porphyrins |
US8217026B2 (en) | 1999-01-25 | 2012-07-10 | Aeolus Sciences, Inc. | Substituted porphyrins |
US7820644B2 (en) | 1999-01-25 | 2010-10-26 | Aelous Pharmaceuticals, Inc. | Substituted porphyrins |
US6403788B1 (en) | 2000-07-11 | 2002-06-11 | Eukarion, Inc. | Non-genotoxic metalloporphyrins as synthetic catalytic scavengers of reactive oxygen species |
US7740839B2 (en) * | 2003-10-31 | 2010-06-22 | Tae-Yoon Kim | EC SOD and cell transducing EC SOD and use thereof |
US8318803B2 (en) | 2003-10-31 | 2012-11-27 | Tae-Yoon Kim | EC SOD and use thereof |
EP1740094A4 (en) * | 2004-03-29 | 2008-09-03 | Inotek Pharmaceuticals Corp | Pyridyl-substituted porphyrin compounds and methods of use thereof |
EP1740094A2 (en) * | 2004-03-29 | 2007-01-10 | Inotek Pharmaceuticals Corporation | Pyridyl-substituted porphyrin compounds and methods of use thereof |
US7642250B2 (en) | 2005-09-28 | 2010-01-05 | Inotek Pharmaceuticals Corporation | N-benzyl substituted pyridyl porphyrin compounds and methods of use thereof |
WO2008114017A3 (en) * | 2007-03-19 | 2008-11-20 | Imp Innovations Ltd | Compositions and methods relating to influenza infection |
WO2008114017A2 (en) * | 2007-03-19 | 2008-09-25 | Imperial Innovations Limited | Compositions and methods relating to influenza infection |
US8252595B2 (en) | 2008-05-13 | 2012-08-28 | University Of Kansas | Metal abstraction peptide (MAP) tag and associated methods |
US9096652B2 (en) | 2008-05-13 | 2015-08-04 | University Of Kansas | Metal abstraction peptide (MAP) tag and associated methods |
US8278111B2 (en) | 2008-05-13 | 2012-10-02 | University Of Kansas | Metal abstraction peptide (MAP) tag and associated methods |
US8975082B2 (en) | 2008-05-13 | 2015-03-10 | University Of Kansas | Metal abstraction peptide (MAP) tag and associated methods |
US11382895B2 (en) | 2008-05-23 | 2022-07-12 | National Jewish Health | Methods for treating injury associated with exposure to an alkylating species |
EP2759546A4 (en) * | 2011-09-21 | 2016-10-19 | Univ Okayama Nat Univ Corp | Metal porphyrin complex, method for producing same, carbon dioxide immobilization catalyst comprising same, and method for producing cyclic carbonic acid ester. |
CN102584836A (en) * | 2011-12-31 | 2012-07-18 | 中国科学院化学研究所 | Porphyrin cage compound, and preparation method and application thereof |
US9187735B2 (en) | 2012-06-01 | 2015-11-17 | University Of Kansas | Metal abstraction peptide with superoxide dismutase activity |
US8987337B2 (en) | 2012-12-07 | 2015-03-24 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
US9061983B2 (en) | 2012-12-07 | 2015-06-23 | Baylor College Of Medicine | Methods of inhibiting xanthine oxidase activity in a cell |
US8883858B1 (en) | 2012-12-07 | 2014-11-11 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
US8895626B1 (en) | 2012-12-07 | 2014-11-25 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
US9585847B2 (en) | 2012-12-07 | 2017-03-07 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
US9585848B2 (en) | 2012-12-07 | 2017-03-07 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
US9610257B2 (en) | 2012-12-07 | 2017-04-04 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
US9622988B2 (en) | 2012-12-07 | 2017-04-18 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
US8883857B2 (en) | 2012-12-07 | 2014-11-11 | Baylor College Of Medicine | Small molecule xanthine oxidase inhibitors and methods of use |
CN105399750A (en) * | 2015-12-11 | 2016-03-16 | 西北师范大学 | Tetrazole porphyrin metal complexes, synthesis thereof and applications of the metal complexes as dye sensitizing agents |
CN105399750B (en) * | 2015-12-11 | 2018-07-17 | 西北师范大学 | Tetrazole porphyrin metal complex and its synthesis and application as dye sensitizing agent |
CN112694887A (en) * | 2020-12-07 | 2021-04-23 | 黑龙江省农业科学院植物保护研究所 | Light-emitting sensor, construction method thereof and application of light-emitting sensor in detection of salicylic acid content in plants |
CN112694887B (en) * | 2020-12-07 | 2024-04-26 | 黑龙江省农业科学院植物保护研究所 | Luminous sensor, construction method thereof and application thereof in detecting salicylic acid content of plants |
Also Published As
Publication number | Publication date |
---|---|
EP0831891A1 (en) | 1998-04-01 |
ATE306936T1 (en) | 2005-11-15 |
DE69635304D1 (en) | 2006-03-02 |
EP0831891B1 (en) | 2005-10-19 |
ES2249784T3 (en) | 2006-04-01 |
CA2223407C (en) | 2011-02-08 |
AU725602B2 (en) | 2000-10-12 |
EP0831891A4 (en) | 1999-02-03 |
AU6387096A (en) | 1996-12-30 |
JPH11509180A (en) | 1999-08-17 |
CA2223407A1 (en) | 1996-12-19 |
IL122451A0 (en) | 1998-06-15 |
DE69635304T2 (en) | 2006-07-20 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US6583132B1 (en) | Oxidant scavengers | |
CA2223407C (en) | Oxidant scavengers | |
CA2174236C (en) | Superoxide dismutase and mimetics thereof | |
US5994339A (en) | Oxidant scavengers | |
Calcerrada et al. | Nitric oxide-derived oxidants with a focus on peroxynitrite: molecular targets, cellular responses and therapeutic implications | |
AU2003279236B2 (en) | Pharmaceutical use of nitric oxide, heme oxygenase-1 and products of heme degradation | |
Otterbein et al. | Mechanism of hemoglobin-induced protection against endotoxemia in rats: a ferritin-independent pathway | |
JP4160115B2 (en) | Synthetic catalytic free radical scavengers useful as antioxidants for disease prevention and treatment | |
US20080113956A1 (en) | Substituted Porphyrins | |
EP1014980A1 (en) | Use of nitroxides for the treatment of essential hypertension | |
US20060034817A1 (en) | Cloning, overexpression and therapeutic use of bioactive histidine ammonia lyase | |
US6605619B1 (en) | Nitroxides as protectors against oxidatives stress | |
Subramanian et al. | Mitochondrially targeted antioxidants for the treatment of cardiovascular diseases | |
Kinobe et al. | Inhibition of the enzymatic activity of heme oxygenases by azole-based antifungal drugs | |
Chen et al. | Heme oxygenase-2 gene deletion increases astrocyte vulnerability to hemin | |
Sato et al. | Susceptibility of metallothionein-null mice to paraquat | |
Valerio Jr | Mammalian iron metabolism | |
AU769217B2 (en) | Oxidant scavengers | |
AU2004201624A1 (en) | Oxidant Scavengers | |
CA2614621A1 (en) | Superoxide dismutase and mimetics thereof | |
WO2009149201A1 (en) | Methods of treatment for pulmonary fibrosis | |
Kirkpatrick | Redox control as a target for anticancer drug development | |
AU746582B2 (en) | Activating C1-secretion | |
Youn Cho et al. | 1-Benzylimidazole induces rat hepatic microsomal epoxide hydrolase with the elevation of its mRNA levels | |
Chakraborty | Insights into iron trafficking and handling in eukaryotic cells |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AK | Designated states |
Kind code of ref document: A1 Designated state(s): AU CA IL JP |
|
AL | Designated countries for regional patents |
Kind code of ref document: A1 Designated state(s): AT BE CH DE DK ES FI FR GB GR IE IT LU MC NL PT SE |
|
DFPE | Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101) | ||
121 | Ep: the epo has been informed by wipo that ep was designated in this application | ||
ENP | Entry into the national phase |
Ref document number: 2223407 Country of ref document: CA Ref country code: CA Ref document number: 2223407 Kind code of ref document: A Format of ref document f/p: F |
|
ENP | Entry into the national phase |
Ref country code: JP Ref document number: 1997 502304 Kind code of ref document: A Format of ref document f/p: F |
|
WWE | Wipo information: entry into national phase |
Ref document number: 1996923328 Country of ref document: EP |
|
WWP | Wipo information: published in national office |
Ref document number: 1996923328 Country of ref document: EP |
|
WWG | Wipo information: grant in national office |
Ref document number: 1996923328 Country of ref document: EP |