Oxidant scavengers

ES 2 249 784 T3

DESCRIPTION

Oxidant antioxidants.

Technical field

The present invention relates, in general, to the modulation of physiological and pathological processes and, in particular, to the modulation of concentrations of intra and extracellular oxidants such as superoxide radicals and hydrogen peroxide and consequently processes in which they participate radical sayings. The invention also relates to compounds and compositions suitable for use in such methods.

Background

Oxidants are produced as part of the normal metabolism of all cells but are also an important component in the pathogenesis of many disease processes. Reactive oxygen species, for example, are critical elements in the pathogenesis of diseases of the lung, central nervous system, and skeletal muscle. Free radicals with oxygen 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 aging processes.

A critical balance of defensive enzymes against oxidants is needed to maintain normal cellular and organic function. Superoxide dismutases (SOD) are a family of metalloenzymes that catalyze the intra and extracellular conversion of O₂ in H₂OR₂ more O₂, and represent the first line of defense against the surface effects of superoxide radicals. Mammals produce three different SODs. One is a dimeric copper-zinc-containing enzyme (CuZn SOD) discovered in the cytosol of all cells. A second is a manganese-containing tetrameric SOD (Mn SOD) discovered within the mitochondria and the third is a copper and zinc-containing glycosylated tetrameric enzyme (EC-SOD) discovered in extracellular fluids and bound to the extracellular matrix. Several other important antioxidant enzymes are known to exist inside cells, including catalase and glutathione peroxidase. While extracellular fluids and the extracellular matrix contain only small amounts of these enzymes, other extracellular oxidants are known to exist, including radical antioxidants and lipid peroxidation inhibitors, such as ascorbic acid, uric acid, and α-tocopherol (Halliwell et al. al., Arch. Biochem. Biophys. 280: 1 (1990)). The relative lack of extracellular antioxidant enzymes may reflect the possible role of extracellular reactive oxygen species as bioeffector molecules (Halliwell et al., Arch. Biochem. Biophys. 280: 1 (1990)). The relative insufficiency of these enzymes can also lead to greater sensitivity to extracellular oxidative stresses.

At many extracellular sites the enzyme EC-SOD exists only at low concentrations. Although its physiological role in vivo is yet to be defined, at many extracellular sites, it is believed that EC-SOD does not function as a major antioxidant of O₂ . As noted above, EC-SOD is a Cu / Zn-containing tetrameric glycoprotein with a subunit molecular weight of 30,000 (Marklund, Proc. Natl. Acad. Sci. USA 79: 7634 (1982); Tibell et al., Proc. Natl. Acad. Sci. USA 84: 6634 (1987); see also US Patent No. 5,130,245 and WO 91/04315). Biochemical data suggest that EC-SOD binds to heparan sulfate proteoglycans in endothelial cells, where it has been speculated that it serves as a “protective layer” (Marklund, J. Clin. Invest. 74: 1398 (1984), Karlsson et al., Biochem. J. 25: 223 (1988)) Endothelial cells secrete both O₂ (Halliwell, Free Radical Res. Commun. 5: 315 (1989)) as a relaxing factor derived from the endothelium, supposedly identified as nitric oxide (NO ·) (Noak and Murphy, in Oxidative Stress Oxidants and Antioxidants, eds. Sies, H. (Academic, San Diego), pp. 445-489 (1991)). NO · functions as a vasoregulator and as a neurotransmission regulator (Schuman and Madison, Science 254: 1503 (1991)). NO ·, however, can be toxic to neurons in some situations (Dawson et al., Proc. Natl. Acad. Sci. USA 88: 6368 (1991)). OR₂ is known to inactivate the vasorelaxation produced by NO · (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. 95: 1308 (1988); Nucci et al., Proc. Natl. Acad. Sci. USA 85: 2334 (1998)). For this reason, a possible role of EC SOD is to protect NO released in cells from O2-mediated inactivation.^- .

The O2 reaction ^- with NO is also known because it produces a potentially toxic intermediate product in the form of peroxynitrite anion (ONOO^-) (Beckman et al., Proc. Natl. Acad. Sci. USA 87: 1620 (1990); Mulligan et al., Proc. Natl. Acad. Sci. USA 88: 6338 (1991); Hoog et al., Biochem J. 281: 419 (1992); Matheis et al., Am. J. Physiol. 262: H616 (1992)). Therefore EC-SOD can also work to prevent the formation of ONOO^-.

Surprisingly, it has been found that EC-SOD increases, rather than decreases, the toxicity of O2 in the central nervous system and that this effect of EC-SOD occurs throughout NO · modulation. This result implicates NO · as an important mediator in O2 toxicity. The invention therefore relates to methods of manipulating nitric oxide function involving the use of extracellular antioxidants.

In addition to superoxide radicals, hydrogen peroxide is an important species that occurs under a wide variety of conditions of oxidative stress. The invention therefore also allows the manipulation of hydrogen peroxide concentrations.

ES 2 249 784 T3

The methods of the invention find application in various pathological and non-pathological states in which oxidative insult plays a role, including inflammation.

In a broader sense, the invention relates generally to methods of modulation of inflated and extracellular processes in which an oxidant such as O participates.₂^- or hydrogen peroxide.

Summary of the invention

The present invention relates to the modulation of concentrations of intra and extracellular oxidants such as superoxide radicals, hydrogen peroxide and peroxynitrite. More particularly, the invention relates to the modulation of normal or pathological processes involving superoxide, hydrogen peroxide, nitric oxide or peroxynitrite radicals using low molecular weight antioxidants, eg, SOD, catalase or peroxidase mimetics.

In a first embodiment, the present invention provides an oxidant antioxidant of the following formula:

<img file="ES2249784T3_D0001.tif" />

or a pharmaceutically acceptable salt thereof, or a metal complex thereof, wherein said metal is selected from the group consisting of manganese, copper, or iron, wherein:

each Ri is independently

<img file="ES2249784T3_D0002.tif" />

<img file="ES2249784T3_D0003.tif" />

indicates that it binds to R₂'and the substituent R₁'phenyl in any position; each R₂'is independently a bond or - (CH₂)_nwhere n is 1 to 4, each R₃'is independently - Y ”, -Y”', -H, -OH, -OY ”, -NO₂, -CN, -NH₂, -COOH, -COY ", -COO or a heterocyclic group, where Y" is as defined above and Y "'is a primary, secondary, tertiary or quaternary amine,

ES 2 249 784 T3 in which when Ri 'is

<img file="ES2249784T3_D0004.tif" />

R₃'it's not COOH, COY ”or COO^-, in which when R₁ ' it is

<img file="ES2249784T3_D0005.tif" />

R₃' No means no₂, and in which -R₁ '-R₂-R₃', together, they are not

<img file="ES2249784T3_D0006.tif" />

Preferably, Y "'is a secondary, tertiary, or quaternary amine and each hydrogen substitution group on the amine nitrogen is a C alkyl group.₁-C₄.

The antioxidant can have the following structure:

<img file="ES2249784T3_D0007.tif" />

ES 2 249 784 T3

The antioxidant can have the following structure:

<img file="ES2249784T3_D0008.tif" />

The antioxidant can have the following structure:

<img file="ES2249784T3_D0009.tif" />

The antioxidant can have the following structure:

<img file="ES2249784T3_D0010.tif" />

ES 2 249 784 T3

The present invention also provides a method of protecting plant cells from oxidant toxicity by contacting said cells with a non-toxic amount of an antioxidant of the following formula:

<img file="ES2249784T3_D0011.tif" />

or a pharmaceutically acceptable salt thereof, or a metal complex thereof, wherein said metal is selected from the group consisting of manganese, copper, or iron, wherein:

each Ri 'is independently a bond

<img file="ES2249784T3_D0012.tif" />

where Y ”is an alkyl group, and where

<img file="ES2249784T3_D0013.tif" />

Or u

c-

<img file="ES2249784T3_D0014.tif" />

indicates that it binds to R₂'in any position and

<img file="ES2249784T3_D0015.tif" />

indicates that it binds to R₂'and the substituent R₁'phenyl in any position;

each R₂'is independently a bond or - (CH₂)_n- where n is from 1 to 4, each R₃'is independently - Y ”, -Y”', -H, -OH, -OY ”, -NO₂, -CN, -NH₂, -COOH, -COY ”, -COO^- or a heterocyclic group, where Y "is as defined above and Y" 'is a primary, secondary, tertiary or quaternary amine, where when R1' is

<img file="ES2249784T3_D0016.tif" />

ES 2 249 784 T3

Rs' is not COOH, COY ”or COO^-, and in which when R1 'is

<img file="ES2249784T3_D0017.tif" />

R₃' No means no₂, and in which -R₁'-R₂'-R₃', together, are not -H, sufficient to effect such protection.

The present invention also provides the use of said antioxidant for the preparation of a drug for the protection of mammalian cells from the toxicity caused by the oxidant by contacting said cells with a non-toxic quantity of antioxidant sufficient to effect said protection. .

The present invention also provides the use of an antioxidant of the following formula:

<img file="ES2249784T3_D0018.tif" />

or a pharmaceutically acceptable salt thereof, or a metal complex thereof, wherein said metal is selected from the group consisting of manganese, copper, or iron, wherein:

each R1 'is independently a bond

<img file="ES2249784T3_D0019.tif" />

where Y ”is an alkyl group, and where

<img file="ES2249784T3_D0020.tif" />

ES 2 249 784 T3 indicates that it binds to R₂'in any position and

<img file="ES2249784T3_D0021.tif" />

indicates that it binds to R₂'and to the Rf phenyl substituent in any position; each R₂'is independently a bond or - (CH₂)_n- where n is 1 to 4, each R₃'is independently - Y ”, -Y”', -H, -OH, -OY ”, -NO₂, -CN, -NH₂, -COOH, -COY ”, -COO^- or a heterocyclic group, where Y "is as defined above and Y" 'is a primary, secondary, tertiary or quaternary amine, where when Ri' is

<img file="ES2249784T3_D0022.tif" />

R₃'it's not COOH, COY ”or COO^-, and in which when Ri 'is

<img file="ES2249784T3_D0023.tif" />

R₃' No means no₂, and in which -R₁'-R₂'-R₃', collectively, they are not -H, for the preparation of a medicament for inhibiting the xanthine oxidase activity of a cell or tissue by contacting said cell or tissue with an amount of said antioxidant sufficient to effect said inhibition.

The present invention also provides the use of an antioxidant of the following formula:

<img file="ES2249784T3_D0024.tif" />

or a pharmaceutically acceptable salt thereof, in which:

ES 2 249 784 T3

R1 is a link

<img file="ES2249784T3_D0025.tif" />

where X is a halogen and Y is an alkyl group and where

<img file="ES2249784T3_D0026.tif" />

indicates that it binds to R2 in any position and

<img file="ES2249784T3_D0027.tif" />

indicates that it binds R2 and the substituent at any position; Y

R2 is a bond - (CY'2) n-, - (CY'2-CY '= CY') n, -CY '₂-CY '₂-CH = CH) n-, - (CY '= CY') n- or - (CY'2-COV where Y 'is hydrogen or an alkyl group and where n is 1 to 8; and

R3 is -Y ", -OH, -NH2, -N + (Y") s, -COOH, -COO^-, -SO3H, -SO3^-, CH2-PO3H2 or -CH2-PO3H^-, in which Y "is an alkyl group, optionally complexed with a metal selected from the group consisting of manganese, copper and iron, for the preparation of a drug for modulating the function of NO · as a neurotransmitter in a mammal by contacting said mammal with an amount of antioxidant from the oxidant sufficient to effect said modulation.

The present invention also provides the use of an antioxidant of the following formula:

<img file="ES2249784T3_D0028.tif" />

or of a pharmaceutically acceptable salt thereof, or of a metal complex thereof in which said metal is selected from the group consisting of manganese, copper or iron, in which:

ES 2 249 784 T3 each R1 'is independently a bond

<img file="ES2249784T3_D0029.tif" />

<img file="ES2249784T3_D0030.tif" />

in which Y "is an alkyl group, and in which it indicates that it is attached to R2 'in any position and

<img file="ES2249784T3_D0031.tif" />

indicates that it binds to R₂'and the substituent R₁'phenyl in any position;

each R₂'is independently a bond or - (CH₂)_n- where n is from 1 to 4, each R₃'is independently - Y ”, -Y”', -H, -OH, -OY ”, -NO₂, -CN, -NH₂, -COOH, -COY ”, -COO^- or a heterocyclic group, where Y "is as defined above and Y" 'is a primary, secondary, tertiary or quaternary amine, where when R1' is

<img file="ES2249784T3_D0032.tif" />

R3 'is not COOH, COY ”or COO^-, and in which when R1 'is

<img file="ES2249784T3_D0033.tif" />

R₃' No means no₂, and in which -R₁'-R₂'-R₃', collectively, are not -H, for the preparation of a medicament for modulating the function of NO · as a neurotransmitter in a mammal by contacting said mammal with an amount of said oxidant antioxidant sufficient to effect said modulation.

The present invention also provides a kit comprising the antioxidant of the first embodiment of the invention arranged inside a container.

In a second embodiment, the present invention also provides the use of an antioxidant of the oxidant of the following formula:

ES 2 249 784 T3

<img file="ES2249784T3_D0034.tif" />

or of a pharmaceutically acceptable salt thereof, or of a metal complex thereof in which said metal is selected from the group consisting of manganese, copper or iron, in which:

each Ri 'is independently a bond

<img file="ES2249784T3_D0035.tif" />

in which Y ”is an alkyl group, and in which

<img file="ES2249784T3_D0036.tif" />

indicates that it binds to R₂'in any position and

<img file="ES2249784T3_D0037.tif" />

indicates that it binds to R₂'and the substituent R₁'phenyl in any position;

each R₂'is independently a bond or - (CH₂)_n- where n is from 1 to 4, each R₃'is independently -Y "', -COY" or a heterocyclic group, where Y "is as defined above and Y" 'is a primary, secondary, tertiary or quaternary amine, where when R1' is

<img file="ES2249784T3_D0038.tif" />

R₃'is not COY ”and in which -R₁'-R₂'-R₃', together, they are not

ES 2 249 784 T3

<img file="ES2249784T3_D0039.tif" />

The objects and advantages of the present invention will become apparent from the following description.

Brief description of the drawings

Figure 1 presents the EC-SOD expression vector used to create transgenic mice. Transgenic mice were generated with the ECO-RI-Xbal fragment. IVS1: sequence 1 involved in human jd-actin activator.

Figure 2 presents the Northern analysis of the transgenic mouse tissues. Twenty pg of total RNA from transgenic mouse tissues was denatured with glyoxal and electrophoresis through 1.2% agarose gel and transferred to nitrocellulose. The filter was probed with whole human EC-SOD cDNA. The 2.5 Kb band corresponds to the mRNA of the human EC-SOD transgene that contains the intervening 1 Kb sequence (see Figure 1). The 1.5 Kb band corresponds to the fully processed mRNA of the human EC-SOD transgene.

Figure 3 presents the survival percentage of transgenic and non-transgenic mice exposed to 6 ATAs of oxygen for 25 minutes. Mice were injected with either saline or 20 mg / kg of Nm-nitroL-arginine (LNNA) intraperitoneally 10 minutes before compression. Diethyl dithiocarbamate (DDC) in saline 400 mg / kg was injected intraperitoneally 55 min before compression. * p &lt;0.017 determined by x² with Bonferroni correction, it was compared with the transgenic mice treated with saline solution.

Figure 4 presents the time to the beginning of the first attack in transgenic and non-transgenic mice exposed to 6 ATA of oxygen. Mice were injected with either saline or 20 mg / kg of Nm-nitro-L-arginine (LNNA) intraperitoneally 10 minutes before the start of compression. Diethyl dithiocarbamate (DDC) 400 mg / kg was injected intraperitoneally 55 minutes before compression. Results are expressed as mean ± SD time to first zero time attack taken once on chamber reaching 6 ATA. * p &lt;0.05 determined by analysis of variance with Scheffe's F test was compared with that of non-transgenic mice treated with saline solution.

Figure 5 shows the effect of diethyl dithiocarbamate and ^ -mercaptoethanol on survival in 6 ATA oxygen for 30 minutes. Mice (C57BL / 6 x C3H) F1 were injected intraperitoneally with saline, 180 mg / kg of ^ -mercaptoethanol (2-ME) or with 400 mg / kg of diethyl dithiocarbamate (DDC) in saline solution 55 min. before compression. * p &lt;0.025 determined by χ² Bonferroni correction was compared to saline-treated mice.

Figure 6 presents the latency of attack in wild-type mice exposed to 6 ATA of oxygen after being treated with saline or with 20 mg / kg of Nm-nitro-L-arginine (LNNA) or with 20 mg / kg of Nm- nitro-L-arginine plus 50 mg / kg of L-arginine (LNNA + L-Arg). A Student's t-test for paired data was compared to that of saline-treated mice.

Figure 7 presents the survival percentage in wild type mice exposed to 6 ATAs of oxygen. Mice were given an intraperitoneal injection of normal saline (0.008 cc / g) or 20 mg / kg of Nm-nylroL-arginine (LNNA) (0.008 cc / g) 15 minutes before compression. Mice were exposed to 6 ATAs of oxygen for 20 minutes (n = 10, saline only), 25 minutes (n = 10, both groups), 30 minutes (n = 10, saline only), 50 minutes (n = 6, LNNA only), 75 minutes (n = 12, LNNA only), 90 minutes (n = 14, LNNA only), 105 minutes (n = 6, LNNA only) and 120 minutes (n = 6, LNNA only) and the percentage of survival in each group was measured.

Figure 8 presents the survival dose response curve for Nm-nitro-L-arginine (LNNA). Wild-type mice were given an intraperitoneal injection of normal saline (0.008 cc / g) or 0, 2, 10, 20, or 30 mg / kg of LNNA (0.008 cc / g) 15 minutes before compression and then exposed to 75 minutes of oxygen at 6 ATA. Percentage survival was calculated for each treatment group.

Figure 9 presents the survival percentage in wild-type mice pretreated with saline, 20 mg / kg of Nm-nitro-L-arginine (LNNA) or 20 mg / kg of Nm-nitro-L-arginine plus 50 mg / kg of L-arginine (LNNA + L-Arg) and

ES 2 249 784 T3 were then exposed to 75 minutes of oxygen at 6 ATA. * p &lt;0.05 determined with a-squared test with Bonferroni correction.

Figure 10 presents the survival percentage in transgenic and non-transgenic mice exposed to oxygen at 6 ATA for 75 minutes. Mice were injected with saline or administered 20 mg / kg of N-ωnitro-L-arginine (LNNA) intraperitoneally 10 minutes before compression. * p &lt;0.05 determined by χ²compared to non-transgenic saline-treated mice. f p &lt;0.05 determined by χ² compared to saline-treated transgenic mice.

Figure 11 presents the comparison of edema formation in EC-SOD transgenic mice to edema formation in non-transgenic litters after cold injury to the right brain hemisphere as well as in uninjured mice. Values ​​are presented as mean ± standard error. * p &lt;0.05 compared to the edema index of the respective non-transgenic controls using a Student's t test for paired data.

Figure 12 presents the effect of increased EC-SOD concentrations on vascular permeability changes after cold-induced brain injury. Vascular permeability is demonstrated as the loss of Evan's blue in the injured right cerebral hemispheres of non-transgenic (control) and EC-SOD transgenic mice.

Figure 13 presents a Western blot analysis of rh-EC-SOD and a human lung homogenate to demonstrate the specificity of the antibody. Proteins were separated on 0.75mm SDS-10% polyacrylamide gel and transferred to nitrocellulose. Proteins were hybridized with the antibody to recombinant human EC-SOD (4.3 pg / ml) and the antibody was detected by hybridization with¹²⁵I-protein-A followed by autoradiography. The ECSOD band contained 0.05 pg of pure protein from the human recombinant-type EC-SOD band. The lung band contained 10 pg of a supernatant at 20,000 xg of a human lung homogenate.

Figures 14A to 14C present the light microscopic immunohistochemical localization of EC-SOD in the human lung. Tissues were labeled using the antibody against recombinant human EC-SOD (5.4 mg / ml; anti-EC-SOD) or the same antibody in which anti-EC-SOD IgG was observed using purified recombinant EC-SOD coupled to CNBr-Sepharose (EC-SOD absorbed). The antibody was detected using a biotin / streptavidin-horseradish peroxidase labeling technique. A, Large elastic pulmonary artery labeled with anti-EC-SOD. Note the marking around the smooth muscle cells below the endothelium and below the elastic layer of the vessel (short arrow) and the absence of marking for EC-SOD on the surface of the endothelial cells (open arrow) and on elastin (arrow long). B, muscular pulmonary artery labeled with anti-EC-SOD. Note large amounts of labeling in the connective tissue matrix surrounding the vessel and lymph nodes (long arrow), in the matrix surrounding smooth muscle cells (short arrow), and the absence of labeling on the surface of endothelial cells. (open arrow). C, muscular pulmonary artery labeled with EC-SOD absorbed antiserum. Absorption of anti-EC-SOD IgG suppressed all marking in the muscle vessel. (Bars = 50 pm).

Figures 15A to 15C present the immunohistochemical localization of EC-SOD in the human lung. Tissues were labeled using the human recombinant EC-SOD antibody (5.4 mg / ml; anti-EC-SOD). The antibody was detected using a biotin / streptavidin-horseradish peroxidase labeling technique. A, Large cartilaginous airway labeled with anti-EC-SOD. Note the intense marking for EC-SOD in the matrix around the smooth muscle cells (short arrow), between the epithelial cells (long arrow), and the absence of marking on the surface of the epithelial cells (open arrows) and on the cartilage matrix (asterisk). B, Non-cartilaginous airway labeled with anti-EC-SOD. Note the intense marking for EC-SOD throughout the entire matrix under the epithelium (short arrow) and the absence of marking on the surface of the epithelium (open arrow). C, Lung parenchyma labeled with anti-EC-SOD. EC-SOD labeling is mainly at the tips of the alveolar septum (short arrow) and in the matrix around the small vessels (long arrow). No labeling for ECSOD was observed on the surface of the alveolar epithelial cells (open arrow). (Bars = 50 pm).

Figures 16A to 16C present the immunolocation of EC-SOD in the vascular connective tissue under the electron microscope. Tissues were labeled using the antibody against human recombinant EC-SOD (40 pg / ml; anti-EC-SOD) or the same antibody after absorbing anti-EC-SOD IgG using purified recombinant EC-SOD coupled to CNBr-sepharose (EC -SOD absorbed). Antibody was detected using 10 nm protein-A gold. A, anti-EC-SOD labeled vascular collagen, B, anti-EC-SOD labeled vascular elastin. C, vascular collagen labeled with EC-SOD absorbed antisera. Note the intense EC-SOD labeling along with type I collagen and the absence of elastin-associated labeling (E). Furthermore, absorption of the anti-EC-SOD antibody suppressed all the labeling for EC-SOD associated with type I collagen (Bars = 200 nm).

Figure 17 presents the electron microscope immunolocation of EC-SOD around vascular smooth muscle. Tissues were labeled using the human recombinant EC-SOD antibody (40 pg / ml). Antibody was detected using 10 nm protein-A gold. There is a high degree of labeling in the connective tissue matrix around vascular smooth muscle (S) cells associated with type I collagen (short arrow) and other unidentified matrix elements (long arrow). (Bars = 200 nm).

Figures 18A and 18B show the electron microscope immunolocation of EC-SOD on the surface

ES 2 249 784 T3 from pulmonary endothelial cells. Tissues were labeled using the human recombinant EC-SOD antibody (40 µg / ml). Antibody was detected using 10 nm gold with protein-A. A, endothelial cell from a small muscular pulmonary artery, B, endothelial cell from a pulmonary capillary. There was no label for ECSOD on the surface of the endothelial cells (short arrows). EC-SOD is seen in plasma (P) and is associated with extracellular matrix proteins below the endothelium (long arrows). (Bars = 200 nm).

Figure 19 shows the immunolocation of EC-SOD under the electron microscope around the cells of the bronchial epithelium. Tissues were labeled using the human recombinant EC-SOD antibody (40 µg / ml). Antibody was detected using 10 nm protein-A gold. EC-SOD was observed at the intersection between the epithelial cells (arrow) and was also observed to some extent inside the cells. (Bars = 200 nm).

Figures 20A to 20D (for reference only) present 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 presented in the 5 'to 3' orientation. A marker 1 kb in size is indicated. B, BamH I; H; Hind III; P, Pst I; S, Sal I; K, Kpn I; E, EcoR I. In Fig. 20B, the subcloning and sequencing strategy is presented. Several restriction fragments were subcloned by size overlap into the plasmid vector pGEM3Zf (+) for subsequent DNA sequence analysis. All DNA on both strands was sequenced using Sequenase (USB) and double stranded DNA template, except for ~ 2 kb of the 3 '7K36 fragment where only one orientation was sequenced. In Fig. 20C, the exon / intron structure of the human EC-SOD gene is presented. The position of the coding region for preEC-SOD in exon 3 is shown by dotted lines. In Fig. 20D, the four structural domains of the EC-SOD protein are outlined. The signal peptide is indicated by an arrow. This is followed by the mature glycosylated amino terminal peptide (CHO) domain. A third zone exhibits very high amino acid sequence homology to human CuZn-SOD. The carboxy terminal domain has multiple basic charged residues (+) that are critical for heparin glycosaminoglycan binding.

Figures 21A to 21B (for reference only) present Northern blots of human EC-SOD from multiple tissues. Fig. 21A, transfer of two pg of poly A (+) mRNA from eight different human tissues was performed on a denatured agarose gel, transferred to a loaded nylon membrane and probed with stranded human EC-SOD cRNA Complementary marked with [³²P]. On the right are markers with RNA molecular size (kilobases). Quantitative transfer was monitored by ethidium bromide staining. The results demonstrate a single 1.4 kb mRNA present in all eight tissues examined. Skeletal muscle interestingly demonstrates an mRNA greater than ~ 4.2 kb, while the brain exhibits a faint band of approximately 2.2 kb. In Fig. 21B, the bands corresponding to the EC-SOD mRNA were quantified by laser densitometric scanning, normalized to the 1.4 kb brain band and expressed as relative.

Figures 22A to 22B (for reference only) present the transcription initiation point analysis. The rapid 5 'end extension of the cDNA (5' RACE) technique was used to identify the transcription initiation point for the human EC-SOD gene. In Fig. 22A, a schematic diagram illustrates the hybridization sites for various oligonucleotides. The thick line represents the reverse transcribed cDNA of the first strand poly A (+) mRNA from human heart that has been primed with EC7 (EC-SOD gene specific primer) and poly C tail using terminal deoxynucleotidyl transferase ( TdT). HEC1, HEC2, EC4 and EC7 are specific primers for the 5 'human EC-SOD gene. The anchor primer is supplied with the 5 'RACE kit (GIBCO BRL) and hybridized to the poly C tail. In Fig. 22B, PCR was used to amplify the DNA segments using [anchor + EC4] or [HEC1 + EC7] as primers and poly C tail (+ TdT, bands 1 and 4) or no poly C tail (-TdT, bands 2 and 5) cDNA as template. Lane 3 includes DNA amplified by PCR using [HEC1 + F7] as primers and a complete human EC-SOD cDNA as a template. The resulting amplified DNAs were electrophoresed on a 2% agarose gel, transferred to loaded nylon membranes and probed with HEC2-labeled [³²P], 5 'nested gel specific EC-SOD primer. DNA molecular weight markers were introduced between lanes 2 and 3. The expected size of the PCR-enlarged area in lanes 3, 4 and 5 is 217 bp. Only one is observed in band 1, with a molecular size of approximately 185 to 200 bp.

Figure 23 presents the genomic Southern blot analysis of the human EC-SOD gene. Ten micrograms of human genomic DNA were completely digested with each of the restriction enzymes shown, electrophoresed on a 1% agarose gel, and transferred to loaded nylon membranes. The blots were probed with a [³²P] which corresponds to approximately the first 1050 nucleotides and was autoradiographed. The specific restriction endonuclease is presented at the top of each band. On the right are the DNA molecular size markers (in kilobases).

Figure 24 (for reference only) presents the nucleotide sequence and deduced amino acid sequence of the human EC-SOD gene. The complete nucleotide sequence of the human gene is presented. The deduced amino acid sequence of the signal peptide and the mature protein is indicated using the single letter amino acid code.

Figure 25 presents a Lineweaver-Burk plot demonstrating non-competitive inhibition of xanthine oxidase by MnTBAP.

ES 2 249 784 T3

Figure 26 shows the protection of pulmonary artery endothelial cells against xanthine oxidase injury by MnTBAP. Reference = MnTBAP H.

Figure 27 presents the protection of lung epithelial cells against injury caused by paraquat of SOD mimetics.

Figure 28 shows the protection of pulmonary artery endothelial cells against injury caused by paraquat by MnTBAP.

Figure 29 shows the lack of protection of pulmonary artery endothelial cells against paraquat-induced injury by ZnTBAP.

Figure 30 presents the protection of MnTBAP against paraquat-induced lung injury.

Figure 31 presents a graph of the second order frequency constant for catalase mimetics.

Figure 32 presents the effect of MnTBAP on endothelial injury caused by H₂OR₂.

Figures 33A and 33B show the reduction by MnTBAP and MnTmPyP of endothelial cell lesions produced by exposure to hydrogen peroxide produced by glucose oxidase. Figure 33C demonstrates that ZnTBAP does not reduce endothelial cell injury caused by hydrogen peroxide. Figure 33D demonstrates that endothelial cells are not protected from hydrogen peroxide injury by CuZnSOD.

Figures 34A and 34B show the inactivation of aconitase caused by NMDA and KA over time. Fig. 34A. Cortical cells treated with vehicle or with 50 pM NMDA for 0, 5, 15, 30, 60 and 240 min. and aconitase activity measured in cell lysates. Each point represents the mean ± SEM (n = 6-8). Fig. 34B. Cortical cells treated with vehicle or with 300 pM kainate for 0, 60, 240 and 360 min. and aconitase activity measured in cell lysates. Each point represents the mean ± SEM (n = 4-8).

Figures 35A to 35C depict the correlation of toxicity with aconitase inactivation. The concentration dependence of KA (Fig. 35A), PQ⁺⁺(Fig. 35B) and NMDA (Fig. 35C) to cause toxicity (axes on the left) and activation of aconitase (axes on the right). Values ​​were normalized as percent LDH release or aconitase inhibition and plotted to assess the correlation between LDH release and aconitase inactivation. The black boxes represent LDH and the white boxes represent aconitase. Computer curves were generated using linear regression analysis using the equation: Y = bottom + (top - bottom) / 1 + 10^LogEC50-X (GraphPad Prism). Each point represents the mean value (n = 4-6).

Figures 36A and 36B present the blocking of aconitase inactivity and neurotoxicity by MnTBAP. Fig. 36A. Cortical cells were treated with 150 pM PQ ++ for 3 h. (black bars), 50 pM NMDA for 1 hr. (open bars) or KA 300 pM for 6 h. (shaded bars) in the presence or absence of 200 pM MnTBAP (present 15 min. before and for the duration of treatment) and aconitase activity was measured in the cell lysates. Bars represent mean ± SEM (n = 8-12). The asterisk indicates a difference from the other treatments (p &lt;0.05, one-way ANOVA). Fig. 36B. Cortical cells were treated with 150 pM PQ ++ (black bars), 50 pM NMDA (open bars), or 300 pM KA (shaded bars) in the presence of varying amounts of MnTBAP (present 15 min before and during the duration of treatment for 18 h and the release of LDH in the medium was measured. The asterisk represents a difference in the references (agonist in the absence of MnTBAP; p &lt;0.05 Dunnet's test). Bars represent mean ± SEM (n = 3-6).

Figure 37 presents the inhibition of NMDA toxicity by MnTBAP. Cortical cells were treated with varying concentrations of NMDA in the presence and absence of 200 pM MnTBAP for 18 h and LDH was measured in the medium. Each of the points represents the mean ± SEM, n = 3-4.

Figures 38A to 38C show the differential effect of MnTBAP and ZnTBAP on cell death. Cortical cells were treated with 150 µ / M PQ ++ (Fig. 38A), 50 µ / M NMDA (Fig. 38B), or 300 µ / M KA (Fig. 38C) in the presence or absence of varying concentrations of MnTBAP (open squares) or ZnTBAP (solid squares) and dead cells were stained with EthD-1. Images of EthD-1 positive cells were stored and counted in randomly selected fields using a digital image analyzer. Data is expressed as the number of dead cells per field. Each point represents measurements made on 1,200 to 1,500 cells.

Figure 39 presents the effect on learning of deletion of the EC SOD gene in a mouse model.

Figure 40. Effect of Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) (3 to 100 µ / M) on the oxidation of dihydrorhodamine 123 to rhodamine 123 in response to peroxynitrite (5 juM). Data are expressed as mean ± sem of triplicate determinations.

Figure 41. Suppression by peroxynitrite (Fig. 41A) and by NO S-nitroso-N-acetyl donor compounds15

ES 2 249 784 T3

DL-penicillamine (SNAP, 3 mM) (Fig. 41B) and diethylamine: NO NONOate (DNO) (Fig. 41C) on mitochondrial respiration (expressed as a percentage of unstimulated cell respiration) in J774 macrophages and the protective effect of Mn (III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) (10-300 juM) against this suppression. Data are expressed as means ± sem of n = 12 wells. ** p &lt;0.01 represents the significant effect of SNAP compared to the reference values ​​(C); # '## represent significant protective effects of MnTBAP (p &lt;0.05 and p &lt;0.01, respectively).

Figures 42A to D represent the schemes of the synthesis reaction

Detailed description of the invention

The present invention relates to the provision of protection against the damaging effects of oxidants, particularly superoxide radicals, hydrogen peroxide and peroxynitrite and to the prevention and treatment of pathological states that involve or derive from oxidative aggression. The invention also relates to the modulation of biological processes involving oxidants, including superoxide radicals, hydrogen peroxide, nitric oxide and peroxynitrite. The invention further relates to compounds and compositions, comprising low molecular weight antioxidants, for example antioxidant mimetics of reactive oxygen species, including SOD mimetics, catalases and peroxidases, and formulations thereof, suitable for the above uses. .

Appropriate reactive oxygen species antioxidant mimetics for the aforementioned uses comprise manganic derivatives of methine substituted porphins or the pharmaceutically acceptable salts thereof. Methine substituents can be selected to facilitate electronic exchange between the metal mimetic (eg, Mn) and the oxygen radical. Substituents that give up electrons from the ring help delocalize the charge from the metal and thus increase catalytic activity. Consequently, substituents can be selected in order to modulate the porphine redox potential. Substituents can also be selected in order to render the porphyrin resistant to degradation by heme oxygenase. Heme oxygenase, a key enzyme in the normal breakdown of porphyrin and an enzyme that plays a role in regulating inflammation, attacks the carbons of the metin bridge. By designing compounds not sensitive to attack (eg, by introducing substituents on the methine bridge carbons), the half-life of porphyrin can be increased. Said compounds have the additional advantage that they do not interfere with normal porphyrin metabolism. The selection of the substituents can also be made on the basis of the desired result to be achieved. For example, when passage through cell membranes is advantageous in a given treatment regimen, nonpolar substituents can be selected in order to make the mimetic liquid soluble. Substituents can also be selected to allow the mimetic to be able to bind to the cell surface or extracellular matrix elements. Such substituents can be selected in order to target the mimetic based on charge, shape, structure, etc. The 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.

In one embodiment, the mimetics used according to the invention are those of the following formula:

<img file="ES2249784T3_D0040.tif" />

P

P in which:

R1 is a link

<img file="ES2249784T3_D0041.tif" />

sB^SW’^H

<img file="ES2249784T3_D0042.tif" />

,Y

ES 2 249 784 T3 in which X is a halogen and Y is an alkyl group and in which

<img file="ES2249784T3_D0043.tif" />

indicates that it binds to R2 in any position and indicates that it binds to R₂ and to the substituent at any position; Y

R₂ is a link - (CY '₂)_n-, - (CY '₂-CY '= CY')_n^-, -CY '₂-CY '₂-CH = CH)_n-, - (CY '= CY')_n- or - (CY '₂-CO)_n-, where Y 'is hydrogen or an alkyl group and where n is 1 to 8;

R3 is -Y ", -OH, -NH-, -N + (Y")₃, -COOH, -COO^-, -SO3H, -SO3^-, CH2-PO3H2 or -CH2-PO3H^-, where Y "is an alkyl group.

In a more specific embodiment,

R1 is a link

-O · CT C- · '&gt;^N0’'

S @^SW-- -₀-OR^v where X is Cl or Br and Y is a C alkyl group_1-4;

R₂ is a link - (CY '₂)_n-, - (CY '₂-CY '= CY')_n, -CY '₂-CY '₂-CH = CH)_n-, - (CY '= CY')_n- or - (CY '₂-CO)_n-, where Y 'is hydrogen or an alkyl group and where n is 1 to 4;

R3 is -Y ", -OH, -NH2, -N + (Y") 3, -COOH, -COO^-, -SO3H, -SO3^-, CH2-PO3H- or -CH2-PO3H^-, where Y "is a C1-4 alkyl group.

In a more specific embodiment,

R1 is a link

<img file="ES2249784T3_D0044.tif" />

where X is Cl or Br and Y is methyl or ethyl, and

R2 is a bond - (CY'j), -, - (CY'2-CY '= CY') n-, -CY'2-CY'2-CH = CH) n-, - (CY '= CY ') n- or - (CY'2-CO), - in which Y' is hydrogen, methyl or ethyl and in which n is 1 or 2; Y

R3 is methyl, ethyl, -OH, -NH2, -N + (CH₃)₃, -N + (CH2CH3É, -COOH, -COO^-, -SO3H, -SO3^-, CH2-PO3H- or -CH2PO3H^-.

In another specific embodiment,

R1 is a link

<img file="ES2249784T3_D0045.tif" />

ES 2 249 784 T3 wherein Y is alkyl, preferably C alkyl_1-4, more preferably methyl or ethyl,

R2 is a bond - (CY '2) n-, - (CY' = CY ') n-, or - (CY' 2-CO) n- where Y 'is hydrogen or alkyl (preferably C1-4 alkyl , more preferably methyl or ethyl) and where n is 1 to 4 (preferably 1 or 2); Y

R₃ is C alkyl_1-4 (preferably methyl or ethyl), -OH, -NH₂, -N + (CH₃)₃, -N + (CH₂CH₃)₃, -COOH, -COO^-, SO3H, -SO3^-, -CH2-PO₃H- or -CH₂-PO₃H^-.

In yet another specific embodiment,

R1 'is a link

<img file="ES2249784T3_D0046.tif" />

R₂ is a link - (CY '₂)_n- or - (CY '= CY')_n- where Y 'is hydrogen or alkyl (preferably C_1-4, more preferably methyl or ethyl) and where n is 1 to 4 (preferably 1 or 2);

R₃ is C alkyl_1-4 (preferably methyl or ethyl), -OH, -NH₂, -N + (CH₃)₃, -N + (CH₂CH₃)₃, -COOH, -COO^-, SO3H, -SO3^-, -CH2-PO3H- or -CH2-PO3H^-.

Each P is hydrogen. Specific mimetics suitable for use in the present methods include Mn (III) tetrakis (1-methyl-4-pyridyl) porphyrin (MnTMPyP), Mn (III) tetrakis (4-trimethyl-aminophenyl) porphyrin (MnTMAP) and Mn ( III) tetrakis (4-benzoic acid) porphyrin (MnTBAP).

Although the above mimetics are described as manganese chelates, metals other than manganese can also be used, such as iron (III) and copper (II). The present invention also relates to the metal-free nitrogen-containing macrocyclic ligand. It will be appreciated that the selected metal can have several valence states, for example, manganese II, III or V can be used. The change in charge will depend on the acceptance or release of electrons.

In addition to the above mimetics, the invention also comprises the use of the compounds of the following formula:

<img file="ES2249784T3_D0047.tif" />

or of a pharmaceutically acceptable salt thereof, or of a metal complex thereof in which said metal is selected from the group consisting of manganese, copper or iron, in which:

each R1 'is independently a bond

<img file="ES2249784T3_D0048.tif" />

ES 2 249 784 T3 in which Y "is an alkyl group (eg C₁-C₄) and in which

<img file="ES2249784T3_D0049.tif" />

indicates that it binds to R2 'at any position and

<img file="ES2249784T3_D0050.tif" />

indicates that it binds to R₂'and the substituent R₁'phenyl in any position; each R2 'is independently a bond or - (CH2) n- where n is 1 to 4, each R₃'is independently - Y ”, -Y”', -H, -OH, -OY ”, -NO₂, -CN, -NH₂, -COOH, -COY ”, -COO^- or a heterocyclic group, where Y "is as defined above and Y" 'is a primary, secondary, tertiary or quaternary amine (preferably an alkylamine where the alkyl groups are, for example, C₁-C₅) in which when R₁' it is,

<img file="ES2249784T3_D0051.tif" />

R3 'is not COOH, COY ”or COO^-, in which when R1 'is

<img file="ES2249784T3_D0052.tif" />

R₃' No means no₂, and in which -R₁'-R₂'-R₃', together, they are not -H.

In certain embodiments of the invention, -R₁'-R₂'-R₃', together, they are not

<img file="ES2249784T3_D0053.tif" />

for example, when Y "is a methyl.

As indicated above R3 'may represent a heterocyclic group. Possible heterocyclics include substituted or unsubstituted tetrazoles, furans, thiophenes, indoles, imidazoles, pyridines, oxadiazoles and quinolines. Possible substituents on such groups include halogen (eg Br or Cl), -NO₂, C alkyl_1-4 and C1-4 alkyl alcohol groups.

P is hydrogen.

Where rotational isomers are possible, all isomers of the mimetics described herein (oxidant antioxidants) can be used in accordance with the invention.

The following are specific examples of suitable mimetics for use or for use in the invention:

ES 2 249 784 T3

<img file="ES2249784T3_D0054.tif" />

R '= NH₂-ArgGluHisSerGluArgLysLysArgArgArgGluSerGluCysLysAlaAla-COOH porphyrins of 5,10,15,20-tetra kis [group R] manganese (III)

<img file="ES2249784T3_D0055.tif" />

<img file="ES2249784T3_D0056.tif" />

<img file="ES2249784T3_D0057.tif" />

<img file="ES2249784T3_D0058.tif" />

Suitable mimetics can be selected for use in the present invention by analyzing the activity and stability of SOD, catalase and / or peroxidase. Selective, reversible, and SOD-sensitive inactivation of aconitase by O generators^-two known can be used as a marker for O generation^-two. In this way, suitable mimetics can be selected by testing 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 an E. coli strain without SOD versus a wild type strain. Specifically, wild-type E. coli (AB1157) and E. coli without SOD (JI132) can be cultured in M9 medium containing 0.2% casmino acids and 0.2% glucose at pH 7.0 and 37 ° C; the culture can be monitored as a function of turbidity followed at 700 nm by spectrophotometry. Active mimetics can be tested for toxicity in mammalian cell culture by measuring the release of lactate dehydrogenase (LDH). Specifically, rat L2 cells (type II-like lung cells; (Kaighn and Douglas, J. Cell Biol. 59: 160a (1973)) can be cultured in Ham's F-12 medium enriched with 10% fetal calf serum at pH 7.4 and 37 ° C. Cells can be seeded at equal densities in 24-well culture plates and grown to approximately 90% confluence; SOD mimetics can be added to cells at log doses (eg micromolar doses in minimal essential medium (MEM)) and incubated for 24 hours. The

ES 2 249 784 T3 toxicity can be assessed by morphology and by measuring release of the cytosolic injury marker, LDH (eg, in 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). The efficacy of active mimetics can be assessed by determining their ability to protect mammalian cells against toxicity caused by methylviologen (paraquat). In particular, rat L2 cells grown as described above and seeded in 24-well plates can be pre-incubated with various concentrations of the SOD mimetic and then incubated with a concentration of methylviologen previously shown to produce LC.₇₅ in reference L2 cells. The efficacy of the mimetic can be correlated with a decrease in LDH release caused by methylviologen (St. Clair et al., FEBS Lett. 293: 199 (1991)). The efficacy of SOD mimetics can be evaluated in vivo with mouse and / or rat models using both aerosol administration and parenteral injection. For example, male Balb / c mice can be randomly chosen into 4 groups of 8 mice each to form a 2x2 statistical contingency pattern. Animals can be treated with paraquat (40 mg / kg, ip) or saline and treated with SOD mimetic or reference vehicle. Lung injury can be assessed 48 hours after paraquat treatment by analyzing the parameters (LDH, protein, and% PMN) of the injury with bronchoalveolar lavage fluid (BALF) as previously described (Hampson et al., Tox. Appl. Pharm. 98: 206 (1989); Day et al., J. Pharm. Methods 24: 1 (1990)). The lungs of 2 mice in each group can be fixed by instillation with 4% paraformaldehyde and processed for histopathology at the light microscopic level.

Catalase activity can be monitored by measuring the absorbance at 240 nm in the presence of hydrogen peroxide (see Beers and Sizer, J. Biol. Chem. 195: 133 (1952)) or by measuring the evolution of oxygen with an oxygen electrode of Clark (Del Rio et al., Anal. Biochem. 80: 409 (1997)). Peroxidase activity can be measured spectrophotometrically as previously described by Putter and Becker: Peroxidases. In: Methods of Enzymatic Analysis, HU Bergmeyer (ed.), Verlag Chemie, Weinheim, pp. 286-292 (1983). Aconitase activity can be measured as described in Example XI below.

Table IX below provides a summary of the activities of various oxidant antioxidants of the invention. The footnote to this Table provides details of the analyzes used.

Synthesis of suitable mimetics for use in the present invention can be accomplished using art recognized protocols. Example XIV includes a detailed description of the synthesis of four specific mimetics. In the case of Mn (I II) -porphyrin mimetics, porphyrin rings with various methine-bridged carbon side groups are commercially available and the Mn (III) metal ion can be inserted into the ring of porphyrin by methods comprising the following: (1) mixture of Mn (II) acetate with porphyrin in the presence of oxygen, under which conditions the selective stabilization of Mn (III) by porphyrin produces auto-oxidation of Mn (II);

(2) preparation of Mn (III) (OH) 3 by modification of the Winkler method (Sastry et al., Anal. Chem. 41: 857 (1969)) followed by reaction with porphyrin; (3) agitation of MnO₂ with porphyrin in the presence of NH₂OH, which serves to reduce Mn (IV) to Mn (III), which is then trapped by porphyrin; or (4) a modified method from Pasternack et al. (Biochemistry 22: 2406 (1983)) which refluxes excess MnCl₃ with porphyrin. The Mn (III) -porphyrin complexes can be precipitated from solution with sodium perchlorate, washed, and the perchlorate residue removed by strong anion exchange resin. The formation of the Mn (III) -porphyrin complex can be followed spectrophotometrically by monitoring a characteristic Soret band at 468 nm. The synthesis of compounds bearing electron-donating groups at one or more pyrrolic carbons can be performed as described by Richards et al., Inorg. Chem. 35: 1940 (1996).

Purification of the mimetics can be accomplished using art recognized techniques such as recrystallization, chromatography, etc. Coupling of a binding domain to the "mimetic core" can be performed as described above.

An embodiment of the present invention derives, at least in part, from the embodiment that specifically regulates NO · function with EC-SOD. Furthermore, the invention is based on the realization that EC-SOD is synthesized by epithelial cells and is located mainly in the interstitium, on matrix elements and collagen and around smooth muscle cells (particularly in the pathways pulmonary respiratory systems and in the vascular system). NO · is an intercellular signal and, as such, NO · must cross the extracellular matrix to exert its effects. However, NO is very sensitive to O2-mediated inactivation.^- present in extracellular spaces. EC-SOD is therefore a theoretically suitable enzyme to increase the bioavailability of NO by preventing its degradation by O₂^-.

One embodiment of the present invention relates to the regulation of extracellular NO · concentrations using polypeptides with EC-SOD activity. The invention, however, is not limited to the manipulation of NO · 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 antioxidant generally.

The present invention relates, in a further specific embodiment, to the inhibition of the production of superoxide radicals. In this embodiment, the mimetics of the invention are used to inhibit oxidases, such as xanthine oxidase, which are responsible for the production of superoxide radicals (see Example VII). The ability of a mimetic to protect mammalian cells from xanthine / xanthine oxidase injury

ES 2 249 784 T3 can be evaluated, for example, by culturing rat L2 cells in 24-well plates. Cells can be preincubated with various concentrations of a mimetic and then xanthine oxidase (XO) can be added to the culture along with xanthine (X). The appropriate amount of XO / X used in the study can be predetermined for each cell line by performing a dose-response curve for the lesion. X / XO can be used in an amount that produces approximately one LC₇₅ in cultivation. The efficacy of the mimetic can be correlated with a decrease in LDH release produced by XO / X. The ability of mimetics to inhibit the production of such radicals allows the use of mimetics as therapeutics for the treatment of gout and reperfusion injuries.

The mimetics of the invention can be used as catalytic antioxidants of reactive oxygen species to protect against ischemic reperfusion injuries associated with myocardial infarction, stroke, acute head trauma, organ reperfusion after transplantation, intestinal ischemia, pulmonary infarction, occlusion. Surgical Blood Circulation and Mild Tissue Injury. Mimetics can also be used to protect against skeletal muscle reperfusion injury. Mimetics can also be used to protect against damage to the eyes due to sunlight (and skin) as well as glaucoma and macular degeneration of the eye. Bone diseases are also suitable for treatment with mimetics. Furthermore, connective tissue disorders associated with effects on collagen synthesis or degradation can be expected to be sensitive to treatment with the present mimetics.

In addition to the antioxidation of superoxide, the ability of the mimetics of the invention to antioxidant hydrogen peroxide would protect against possible formation of the highly reactive hydroxyl radical by interfering with Fenton chemistry (Aruoma and Halliwell, Biochem. J. 241: 273 ( 1987), Mello Filho et al., Biochem. J. 218: 273 (1984); Rush and Bielski, J. Phys. Chem. 89: 5062 (1985). These metalloporphyrins have been shown to sequester peroxynitrite as shown indirectly by inhibiting the oxidation of dihydrorhodamine 123 to rhodamine 123 (see Example XIII) and directly by accelerating peroxynitrite degradation by interrupting flow analysis.

In addition to the above, mimetics can be used as catalytic antioxidants or as reactive oxygen species to increase the very limited storage viability of transplanted hearts, kidneys, skin, and other organs and tissues. Mimetics can also be used in methods of inhibition of injury due to autoxidation of substances that produce O formation.₂^- including food products, pharmaceuticals, stored blood, etc. To accomplish this goal, mimetics are added to food products, pharmaceuticals, stored blood, and the like, in an amount sufficient to measure or prevent oxidative damage and thereby inhibit or prevent degradation related to autoxidation reactions. (For other uses of the mimetics of the invention, see US Patent No. 5,227,405). One skilled in the art can determine the amount of mimetic that should be used in a given treatment or that should be related to a given substance.

The availability of mimetics also allows studies of O-mediated processes.₂ , hydrogen peroxide, nitric oxide and peroxynitrite.

To effect modulation of the efficiency of extracellular NO ·, e.g. For example, in smooth muscle relaxation, molecules (agents) with EC-SOD activity are administered under conditions such that concentrations of

Molecules suitable for use in the present invention comprise the EC-SOD mimetics as described above.

The general requirements for such mimetics are that they: (a) be stable enough to keep the metal bound (eg. g., Cu or Mn) in the presence of the multiple chelating agents present in living systems, (b) they are sufficiently active so that reasonable doses can serve to significantly increase the total activity of SOD in the extracellular spaces, ( c) are able to adhere to cell surfaces or extracellular matrix elements (e.g. collagen) when protection against extracellular sources of O is needed₂and (d) are of low toxicity. Examples of suitable mimetics include porphyrin Mn (III) complexes with bulky cationic substituents on the methine bridged carbons, such as those described above (eg, MnTMAP and MnTMPyP). These complexes are very active and are stable enough to retain all activity in the presence of excess EDTA or in the presence of tissue extracts.

The mimetics described above can be formulated into pharmaceutical compositions suitable for use in the present methods. Such compositions include the active agent (mimetic) together with a pharmaceutically acceptable carrier, excipient or diluent. The composition can be present in unit dosage form eg tablets, capsules or suppositories. The composition may also be in the form of a sterile solution suitable for injection or nebulization. The compositions may be in a form suitable for ophthalmic use. The invention also encompasses compositions formulated for topical administration, said compositions taking the form, for example, of a lotion, cream, gel or ointment. The 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 desired result.

The dose of the composition of the invention to be administered can be determined without experimentation.

ES 2 249 784 T3 undue and will depend on several factors including the nature of the active agent, the route of administration, the patient and the desired result to be achieved.

Suitable doses of mimetics will vary, for example, with the mimetic and the desired result. The results of Faulkner et al. (J. Biol. Chem. 269: 23471 (1994)) indicate that the in vivo oxidoreductase activity of the mimetics is such that a pharmaceutically effective dose is low enough to prevent toxicity problems. Doses that can be used include those in the range 1 to 50 mg / kg.

Other examples of diseases or disorders suitable for treatment using the compounds and compositions of the present invention include diseases of the central nervous system (including AIDS dementia, stroke, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Huntington's disease). and musculature diseases (including diaphragm diseases (eg. respiratory fatigue in emphysema, bronchitis and cystic fibrosis), cardiac fatigue from congestive heart failure, muscle weakness syndromes associated with myopathies, ALS and multiple sclerosis). Many neurological disorders (including stroke, Huntington's disease, Parkinson's disease, ALS, Alzheimer's disease, and AIDS dementia) are associated with an overstimulation of the major glutamate receptor subtype, the NMDA (or N-methyl-D-aspartate) subtype. ). In NMDA receptor stimulation, excessive neuronal calcium concentrations contribute to a number of membrane and cytoplasmic events leading to the production of free radicals with oxygen and nitric oxide (NO ·). Interactions between free radicals with oxygen and NO · have been shown to contribute to neuronal cell death. Well-proven 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. The results presented in Example XI demonstrate that radical formation with O^-two It is an obligatory stage in intracellular cases that culminates in the excitotoxic death of cortical neurons and further demonstrates that the mimetics of the invention can be used as antioxidants of O radicals.^-two and thus serve as protectors against excitotoxic injury. Compound 10303 (see Table IX) decreases the excitotoxicity produced by NMDA and kainate in rat cortical cells in a dose-dependent manner with 100% protection against excitotoxicity produced by NMDA (50 juM) achieving compound 10303 at 25 juM, compound 10303 at 100 pM in case of exotoxicity caused by kainate (300 juM).

Also described herein is the use of compounds for the preparation of a medicament for use in the treatment of arthritis, generalized hypertension, atherosclerosis, edema, septic shock, pulmonary hypertension, including primary pulmonary hypertension, impotence, infertility. , endometriosis, premature uterine contractions, microbial infections, gout and for the treatment of type II diabetes mellitus. The antioxidants of the invention can be used to improve the toxic effects associated with endotoxins, for example, by preserving vascular tone and preventing damage to the multi-organ system.

The use of compounds for the preparation of a medicament for use in the treatment of memory disorders is also described. Although not wishing to be bound by theory, nitric oxide is believed to be a neurotransmitter involved in enhancing long-term memory. Using a genetically modified EC-SOC mouse model, it can be shown that impaired learning correlates with the reduction of the antioxidant superoxide in the extracellular spaces of the brain (see Example XII). Reduction of antioxidant produces higher concentrations of O^-two extracellular. It is believed that O^-two it reacts with nitric oxide thus preventing or inhibiting nitric oxide-medicated neurotransmission and therefore long-term memory enhancement. The mimetics of the invention can be used to treat dementias and memory or learning disorders.

One skilled in the art can easily determine the therapeutic regimens, including the mode of administration, appropriate to effect the treatment of the diseases described above.

Inflammations, particularly lung inflammations, are suitable for treatment using the medicaments prepared using the compounds of the present invention (note particularly inflammatory disorders of asthma, ARDS including oxygen toxicity, pneumonia (especially pneumonia related to AIDS), cystic fibrosis, chronic sinusitis and autoimmune diseases such as rheumatoid arthritis)). EC-SOD is located in the interstitial spaces surrounding the airways and smooth muscle cells of the vascular system. EC-SOD and O2^- mediate the anti-inflammatory-pro-inflammatory balance in the alveolar septum. NO released by cells of the alveolar septum acts to suppress inflammation unless it reacts with O2^- to form ONOO^-. By antioxidation with O2^-, EC-SOD shifts the balance in the alveolar septum against inflammation. Significant amounts of ONOO^- will be formed only when ECSOD is insufficient or when there is a large increase in O release₂^-. EC-SOD mimetics, such as those described herein, can be used to protect against destruction caused by hyperoxia. One skilled in the art can easily demonstrate appropriate therapeutic regimens.

Certain aspects of the present invention are described in greater detail in the following non-limiting Examples.

ES 2 249 784 T3

Example I

Preparation and characterization of transgenic mice

Protocols

i) Preparation of transgenic mice

Construction of the human EC-SOD expression vector: The EC-SOD expression vector (Figure 1) was constructed as follows: the complete human EC-SOD cDNA fragment (Hjalmarrson et al., Proc. Natl Acad Sci. USA 84: 6340 (1987); Hendrickson et al., Genomics 8: 736 (1990)) flanked by restriction sites EcoRI was transformed with mung bean nuclease to form blunt ends, ligated to SalI, digested with SalI and then inserted at the SalI site of the expression vector of human ^ -actin pH / lAPr-1. The EcoRI-HindIII fragment was isolated from the resulting plasmid containing the human ^ -actin activator, (provided by Dr. Larry Kedes of the University of Southern California, Los Angeles, California), the intron and the ECSOD cDNA. In addition, the SV40 HpaI site at position 2666 in plasmid pMSG (Pharmacia LKB Biotechnology, Piscataway, NJ) was transformed into the HindIII site by linker ligation and the HindIII-PstI fragment containing the polyadenylation site of the initial zone SV40. These two DNA fragments were then ligated into an EcoRI vector plus PstI digested pKS (Stratagene, La Jolla, California). The EcoRI-XbaI fragment containing the complete expression construct devoid of plasmid sequences was isolated and used to create transgenic mice. All recombinant DNA procedures were performed according to accepted methods (Sambrook et al., Molecular Cloning: A Laboratory Manual 3, Cold Spring Harbor; Cold Spring Harbor Laboratory, 1989).

Development of transgenic mice: Purified DNA was injected at 2.5 pg / ml in 5 mM Tris-HCl, pH 7.4, 0.1 mM EDTA into the pronuclei of fertilized ovules isolated from mice, mice ((C57BL / 6 X C3H) F1 X (C57BL / 6 X C3H) F1) ((C57BL / 6 X C3H) F1 were purchased from Charles River). Microinjection surviving mouse eggs were then implanted into the oviducts of pseudopregnant adoptive mothers (CD1) (CD1 mice were purchased from Charles River) following the procedures described by Hogan et al. (Hogan et al., Manupulating the Mouse Embryo, Cold Spring Harbor; Cold Spring Harbor Laboratory 1986, 32). Mice carrying the transgene were identified by Southern blot analysis of tail DNA probed with full length human EC-SOD cDNA. Transgenic founders were discovered in the first litter examined. These mice were crossed with (C57BL / 6 X C3H) FI to produce offspring for further studies. (In all subsequent experiments with EC-SOD transgenic mice, transgenic mice refer to litters of transgenic mice that do not contain the transgene for human EC-SOD. In the experiments in which EC- transgenic mice were not used SOD, wild-type mice (C57BL / 6 X C3H) FI) were used.

Production of homozygous EC-SOD transgenic mice: Homozygous transgenic mice were produced by crossing F₁ of heterozygous transgenic mice. DNA was isolated from the tail of F mice₂ and treated with RNase. 10 pg of DNA from each mouse was cut with PstI and then electrophoresis was performed through a 1.2% agarose gel. Southern blot analysis of the tail DNA probed with the full length cDNA of human EC-SOD was then performed. The human EC-SOD cDNA did not interact with the mouse EC-SOD gene. The intensity of the band was visually compared to determine which mice were homozygous, heterozygous, or negative for the human EC-SOD transgene.

ii) Characterization of transgenic mice

Northern analysis: Transgenic mice and non-transgenic litters were euthanized with an overdose of pentobarbital. Tissues were rapidly excised and frozen in liquid nitrogen until ready for further treatment. Total RNA was then isolated by the CsCl procedure described in Sambrook et al., Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1989. Twenty pg of total RNA from the tissues of transgenic mice and from non-transgenic litters and one step of RNA were then denatured with glyoxal, electrophoresed through a 1.2% agarose gel and transferred to nitrocellulose. as described (Sambrook et al., Molecular Cloning: A Laboratory Manual. 3. Cold Spring Harbor, Cold Spring Harbor Laboratory, 1989). The blots were then probed with the full length human EC-SOD cDNA.

Separation of SOD isoenzymes by concanavalin A sepharose chromatography: Tissues taken from 3 mice were weighed, then pooled and homogenized in 10 volumes of ice cold 50 mM potassium phosphate, pH 7.4, with KBr 0, 3M, 3mM diethylenetriaminepentaacetic acid and 0.5mM phenylmethylsulfonyl fluoride. Separation of EC-SOD from CuZn SOD and Mn SOD was performed by passing tissue homogenates over a concanavalin A sepharose column as described (Marklund et al., Clin. Chim. Acta 126: 4 (1982)).

SOD Activity: EC-SOD activity and total SOD activity (CuZn SOD and Mn SOD) remaining after EC-SOD extraction were measured by inhibition of cytochrome C reduction at pH 10, as previously described (Crapo et al., Methods Enzymol. 53: 382 (1978)). Total protein was determined by BCA protein analysis (Pierce, Rockford, IL). SOD activities were then expressed in units / mg of total protein.

ES 2 249 784 T3

Results

i) Transgenic mice with EC-SOD

Characterization of transgenic mice: Mice carrying the human EC-SOD transgene were detected by Southern blot analysis. Northern analysis of various tissues from the F1 mouse and a mouse found to carry the transgene is shown in Figure 2. High message concentrations for human EC-SOD were detected in the heart, skeletal muscle, and brain of transgenic mice, with little or no message observed in the lung, liver, and spleen. No messages were detected in the non-transgenic litters.

Homozygous mice were generated by crossing two heterozygous F1 mice. Homozygous mice were detected by the intensities of the differential band observed using Southern blot analysis of equal amounts of PstI-digested DNA in the offspring. EC-SOD activity in mice was found to increase in response to total copies of the EC-SOD transgene (Table I).

TABLE I

EC-SOD activity in non-transgenic, heterozygous transgenic, and homozygous transgenic mouse tissues. Tissues from 3 mice were pooled for each measurement. Activity is expressed in units / g wet weight of tissue

<td>Tissue</td><td>Non-transgenic</td><td>Heterozygous</td><td>Homozygous</td>

<td>Brain</td><td> 18</td><td> 38</td><td> 50</td>

<td>Heart</td><td> 35</td><td> 69</td><td> 102</td>

Example II

Central nervous system oxygen toxicity Protocols

Oxygen exposures: Five mice were exposed at a time for 7 to 8 weeks to hyperbaric oxygen in a small animal chamber (Bethlehem, Pennsylvania). After entrainment in the chamber with pure oxygen, compression was performed at 50 meters (6 ATA) for 5 minutes. The oxygen concentration in the chamber was monitored continuously with a Servomex oxygen analyzer (model 572, Sybron, Norwood, Massachusetts) and kept at&gt; 99%. The carbon dioxide concentration in the intermittent chamber gas samples was analyzed with an IR detector (IR Industries, Santa Barbara, California) and was not allowed to rise by approximately 0.1%. The temperature of the chamber was kept between 25 and 26 ° C, except for the compression of oxygen in the chamber that temporarily reached a temperature between 30 and 32 ° C, except for an environmental control system that restored the normal temperature in the chamber every 3 minutes. The exposures lasted 25 to 75 minutes and were followed by decompression for 5 minutes. Oxygen toxicity symptoms were observed in mice continuously from the beginning of exposure until 4 hours after removal from the chamber. Time to first generalized seizure (attack latency) and time to death were recorded. These exposure conditions are designed to produce CNS oxygen toxicity without appreciable evidence of pulmonary oxygen toxicity.

Diethyl dithiocarbamate treatment: One hour before exposure to 6 ATA oxygen, mice were given ip injections of 0.008 cc / g of saline or 400 mg / kg of diethyl dithiocarbamate dissolved in saline (0.008 cc / g). Mice were then exposed to 6 ATAs of oxygen for 25 minutes as described above.

To determine the extent of the inhibition of EC-SOD and CuZn SOD by diethyl dithiocarbamate, the mice were administered diethyl dithiocarbamate and sacrificed one hour later. Brains were removed and EC-SOD and CuZn SOD activity were analyzed as described above.

Treatment with β-mercaptoethanol: One hour before exposure to 6 ATA of oxygen, mice were given ip injections of 0.008 cc / g of saline or 180 mg / kg of β-mercaptoethanol (0.008 cc / g). This dose of β-mercaptoethanol was selected because it contains an equal number of reducing thiols as the dose of diethyl dithiocarbamate. Mice were then exposed to 6 ATAs of oxygen for 30 minutes as described above.

Treatment with Nm-nitro-L-arginine, nitric oxide synthetase inhibitor: Ten minutes before the start of compression, 0.008 cc / g of saline or 20 mg / kg (0.008 cc / g) of Nm-nitro was administered ip -L-arginine dissolved in stabilized water in transgenic and non-transgenic mice. Mice were then exposed to 6 ATAs of oxygen for 25 or 75 minutes as described above.

ES 2 249 784 T3

Statistical analysis: Student's t test for paired data was used to compare enzyme activities in transgenic and non-transgenic mice. The χ test was used² with Bonferroni correction to assess the significance of differences in survival to hyperbaric exposures. Analysis of variance with a Scheffe's F test was used to compare differences in attack latency in different groups of mice.

Results

Exposures to hyperbaric oxygen: To test the effects of increased EC-SOD concentrations in the brain on CNS oxygen toxicity, both transgenic and non-transgenic mice (see Example I) were exposed to oxygen at 6 ATA for 25 minutes. Transgenic mice were more sensitive (83% mortality) to CNS oxygen toxicity than non-transgenic mice (33% mortality) (Figure 3).

Transgenic and non-transgenic mice were subsequently treated with a CuZn SOD inhibitor, diethyl dithiocarbamate, to confirm that the increased sensitivity of the transgenic mice to CNS oxygen toxicity was the result of increased SOD activity. In both transgenic and non-transgenic mice, treatment with 400 mg / kg of diethyl dithiocarbamate produced 80% inhibition of EC-SOD and 60% inhibition of CuZn SOD in the brain. This is consistent with previous findings (Frank et al., Biochem. Pharmacol. 27: 251 (1978); Heikkila et al., J. Biol. Chem. 251: 2182 (1976)). Diethyl dithiocarbamate treatment conferred increased resistance to CNS oxygen toxicity for both transgenic and non-transgenic mice. Survival increased to 100% in transgenic mice and 93% in non-transgenic mice (Figure 3). The onset of seizures was also delayed four times in mice treated with diethyl dithiocarbamate (Figure 4).

To evaluate whether or not diethyl dithiocarbamate protects against CNS oxygen toxicity by acting as a reducing agent rather than an inhibitor of SOD activity, mice were treated with an equimolar amount of reducing thiols in the form of ^ -mercaptoethanol. and they were exposed to hyperbaric oxygen. Figure 5 demonstrates that ^ -mercaptoethanol did not protect against CNS oxygen toxicity.

One possibility that may explain why EC-SOD worsens CNS oxygen toxicity would be that nitric oxide is a mediator of CNS oxygen toxicity and EC-SOD is nitric oxide that protects against inactivation mediated by superoxide. To demonstrate the hypothesis that nitric oxide contributes to CNS oxygen toxicity, wild-type (C57BL / 6 X C3H) F1 mice were treated with a nitric oxide synthase inhibitor, Nm-nitro-L-arginine. Figure 6 presents the effects of Nm-nitro-L-arginine on attack latency in mice. Nm-nitro-L-arginine pretreatment produced a significant increase in attack latency (13.50 ± 5.6 min) compared to saline-treated mice (2.75 ± 1 min). Figure 7 demonstrates that inhibition of nitric oxide synthase also significantly increased survival after exposure to hyperbaric oxygen. Mice administered nitric oxide synthase inhibitor had 50% mortality after exposure to 90 minutes of 6 ATA oxygen and 100% mortality was not obtained until 2 hours after this challenge. The saline-treated mice, however, exhibited 50% mortality after only 25 minutes of exposure with 100% mortality after only 30 minutes to oxygen at 6 ATA. Figure 8 demonstrates that the percentage of survival in hyperbaric oxygen depended on the dose of inhibitor administered. The protection offered by this competitive nitric oxide synthase inhibitor could be reversed when excess L-arginine is administered (Figure 6 and Figure 9).

The effects of the nitric oxide synthase inhibitor, Nm-nitro-L-arginine, on CNS oxygen toxicity in transgenic mice were then studied. This treatment dramatically reduced CNS oxygen toxicity in both transgenic and non-transgenic mice. Survival after 25 minutes of oxygen exposure to 6 ATA increased to 100% in both groups (Figure 3). Attack latency was also significantly delayed (Figure 4). The exposure time was then increased to 75 minutes to investigate whether the transgenic mice were even more sensitive than non-transgenic mice to hyperbaric oxygen. The results in Figure 10 indicate that Nm-nitro-L-arginine treatment suppressed the difference in sensitivity that was observed between untreated and non-transgenic mice during the 25 minute exposure shown in Figure 3.

Example III

Brain edema caused by cold

Protocols

Injury model: Young mice (6 to 7 weeks old) (see Example I) were anesthetized with 60 mg / kg pentobarbital (Nembutal, Abbot Laboratories, Chicago, Illinois). An incision was then made in the scalp and a steel roller 20 cm long, 3 mm in diameter, equilibrated in liquid nitrogen with an 8 cm bath of 4 cm liquid nitrogen was placed at the end of the roller, in the skull over the right cerebral hemisphere for 30 seconds. The skin incision was then sutured.

ES 2 249 784 T3

Two hours after injury, the mouse was administered an additional dose of pentobarbital. The thoracic cavity was opened, the lungs were excised and the mouse was then perfused with 20 ml of saline via the left ventricle of the heart. The brain was then removed and the cerebellum excised. The right (R) and left (L) cerebral hemispheres were separated and weighed immediately (wet weight, W). Each hemisphere was then dried at 70 ° C for 2 to 3 days in a hot air oven until a constant weight was achieved (dry weight, D). The edema index (I) was then calculated as shown in equation 13.

I = (W / DR - W / DL) / (W / DL) X 100 [13]

This calculation allowed the left hemisphere to serve as an internal reference for the injured right hemisphere in each mouse.

Chemical treatments: Six sets of experiments were conducted to investigate the importance of extracellular superoxide, iron, and nitric oxide in cold-induced brain edema. In all groups, drugs were dissolved in saline and injected at 0.008 cc / g 15 minutes before cold trauma. In group 1, the edema formation of the EC-SOD transgenic mice was compared with that of the non-transgenic litters. Group 2 compared edema formation between wild-type (C57BL / 6 X C3H) F1 mice treated with saline and F1 mice (C57BL / 6 X C3H) treated with 0.33 mg / g deferoxamine (0.51 pmol / g). Group 3 compared saline-treated F1 (C57BL / 6 X C3H) mice with F1 (C57BL / 6 X C3H) mice treated with 0.51 pmol / g Fe-saturated deferoxamine.³+. Group 4 consisted of F1 mice (C57BL / 6 X C3H) treated with saline solution and F1 mice (C57BL / 6 X C3H) treated with 0.02 mg / g of N-ωnitro-L-arginine methyl ester. Group 5 consisted of F1 mice (C57BL / 6 X C3H) treated with saline solution and F1 mice (C57BL / 6 X C3H) treated with 0.02 mg / g of Nw-nitro-L-arginine methyl ester plus 0 .05 mg / g L-arginine. Group 6 compared edema formation between non-transgenic mice, EC-SOD transgenic mice treated with saline, and EC-SOD transgenic mice treated with 0.02 mg / ng of Nw-nitro-Larginine methyl ester.

Iron-saturated deferoxamine was prepared by dissolving equimolar amounts of deferoxamine and then ferric chloride in saline. The saturation of deferoxamine with ferric ion was determined by spectrophotometry measuring the absorbance at 425 nm (e = 2500 M^-1 cm^-1 for Fe³+ -deferoxamine) (Monzyk and Crumbliss, J. Amer. Chem. Soc. 104: 4921 (1982)).

Evan's blue treatment: One hour and 50 minutes after cold trauma, 5 ml / kg of 1% Evan's blue in saline was injected into the femoral artery of transgenic and non-transgenic mice. Mice were sacrificed 10 minutes later. The lungs were then excised and the mice were then prefused with normal saline via the left ventricle until there was no more blue color in the effluent. The brains were then removed and photographed.

Statistical analysis: Student's t-test for paired data was used to compare the significance of the development of edema compared to non-transgenic mice or to saline-treated mice for each of the groups examined. Analysis of variance in a Fisher's PLSD test was used to compare significance.

TABLE III

Effect of inhibition of nitric oxide synthesis on the formation of edema after brain trauma caused by cold. Wild-type F1 (C57BL / 6 X C3H) mice were treated with the competitive inhibitor of nitric oxide synthetase, Nw-nitro-L-arginine methyl ester (LNAME) to determine the effect of nitric oxide on vasogenic edema. Mice were also given Nw-nitro-L-arginine methyl ester plus excess L-arginine (LNAME + L-Arg) to see if the effects seen with LNAME alone could be reversed. Values ​​are presented as mean ± standard error.

<td>Treatment</td><td>n</td><td>Edema index</td>

<td>Saline solution</td><td> 6</td><td> 5,77 ± 0,29</td>

<td>LNAME</td><td> 6</td><td> 3,65 ± 0,51*</td>

<td>Saline solution</td><td> 6</td><td> 6,56 ± 0,21</td>

<td>LNAME + L Arg</td><td> 6</td><td> 6,03 ± 0,71</td>

* p &lt;0.05 compared to the edema index of the controls treated with the respective saline solution using a Student's t test for paired data.

In final experiments, EC-SOD transgenic mice were treated with saline or Nw-nitro-L-arginine methyl ester to determine whether there was an additive effect to prevent edema formation in mice that had increased EC- concentrations. SOD as well as those of the nitric oxide synthetase inhibitor. Table IV

ES 2 249 784 T3 demonstrates that when the nitric oxide synthase inhibitor was administered to transgenic mice with EC-SOD, no protection against edema formation was detected compared to transgenic mice protected only by increased EC concentrations -SOD in the brain.

TABLE IV

Evaluation of the inhibiting effect of nitric oxide synthesis on edema formation in transgenic mice. Comparison of edema formation in non-transgenic mice with edema formation in transgenic mice with high levels of EC-SOD activity in the brain and with edema formation in transgenic mice treated with an inhibitor of nitric oxide synthesis ( 20 mg / kg Nm-nitro-L-arginine; transgenic + LNAME) 15 minutes before cold injury. Values ​​are presented as mean ± standard error and were compared using analysis of variance with a Fisher's PLSD test. No significant difference was observed between transgenic and transgenic mice + LNAME

<td>Treatment</td><td>n</td><td>Edema index</td>

<td>Non-transgenic</td><td> 6</td><td> 7,91 ± 0,67</td>

<td>Transgenic</td><td> 6</td><td> 4,91 ± 0,78*</td>

<td>Transgenic + LNAME</td><td> 6</td><td> 4,30 ± 0,96*</td>

* p &lt;0.05 compared to the edema index of non-transgenic mice

Example IV

EC-SOD immunolocation

Protocols

Human lung: Five human lung samples were obtained to assess the distribution of EC-SOD in human lung tissue. A specimen from a surgical pathology specimen of the right upper lobe excised from a 43-year-old white woman with a history of smoking of 50 packs per year (equivalent to one pack per day for 1 year) and an isolated nodule observed on the chest was obtained. by X-ray. The patient was diagnosed with squamous cell carcinoma. Tissue from an area unaffected by carcinoma of this lobe was used in the studies presented herein. A second lung was obtained from an upper lobe surgical pathology specimen excised from a 51-year-old white man with a history of smoking 60 packs per year, observing that he had a nodule isolated by X-rays. The patient had no other disease and was diagnosed with squamous cell carcinoma. The lung tissue unaffected by carcinoma from this specimen was used for the localization of EC-SOD. A third lung was obtained in a rapid autopsy (tissue obtained 6 hours after death) of a 66-year-old white man 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 a lung transplant recipient. The donated lung came from a 45-year-old white woman 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. No significant differences in marking patterns were observed between surgical pathology specimens and autopsy tissues from lung transplant donors.

Tissues were fixed in 2% paraformaldehyde / 0.2% glutaraldehyde in 0.01 M phosphate buffered saline (PBS; 1.2 g NaH₂PO₄, 8 g NaCl, 0.2 g KCl, in 1 liter pH 7.3) for 1 hour followed by fixation overnight in 4% paraformaldehyde at 4 ° C and then in OCT compound Tissues were frozen in hexane cooled in liquid nitrogen and stored at -70 ° C until sectioned for light microscopic studies.

For electron microscope studies, lung tissues were processed as in light microscope studies until equilibrated in sucrose. After equilibration in sucrose, the lung tissues were infiltrated with 10% gelatin at 37 ° C for 10 minutes. The tissue sections, in gelatin, were then solidified on ice, cut into 2 mm / side cubes and then cold protected in 4% polyvinyl alcohol containing 2M sucrose overnight. These samples were then assembled into chunks, flash frozen in liquid nitrogen, and then stored in liquid nitrogen until sectioned for electron microscope studies.

Characterization of antibodies against human recombinant EC-SOC: human recombinant EC-SOD (provided by SL Marklund, Umea, Sweden; Tibell et al. Proc. Natl. Acad. Sci. USA 84: 6634 (1987)) and the supernatant at 20,000 xg of a human lung homogenate was denatured in the presence of ^ -mercaptoethanol and sodium dodecyl sulfate boiling for 5 minutes and then electrophoresis through a 12% polyacrylamide gel in the presence of sodium dodecyl sulfate. The protein was then transferred to nitrocellulose by electrophoresis. The blot was then incubated with 4.3 jug / ml of an anti-rh-EC-SOD IgG purified fraction

ES 2 249 784 Rabbit T3 provided by SM Marklund, Umea University Hospital, Umea, Sweden affinity purified with rhEC-SOD followed by incubation with ¹²⁵I-protein A and autoradiography.

Anti-EC-SOD IgG uptake: CNBr-activated sepharose was swollen in PBS. The swollen gel was mixed with PBS so that the settled gel occupied 50% of the volume. The gel was suspended and 100 µl was mixed with 100 µg of pure rh-EC-SOD for 2 hours at room temperature with gentle shaking. The gel was then washed 4 times with PBS + 1% bovine serum albumin (BSA) and 100 µl was prepared with PBS + 1% BSA. 100 µl of anti-rh-EC-SOD were then added at twice the concentration used for immunolabeling and mixed for 2 hours with gentle stirring at room temperature. Unimmunized rabbit IgG was then added to the supernatant in a concentration equivalent to the predicted concentration of anti-rh-EC-SOD IgG removed by the procedure. This supernatant was then used for subsequent immunostaining.

Optical microscopic immunohistochemistry: Serial 4 µm sections of OCT-soaked tissue were cut on a cryostat at -20 ° C and placed in poly-L-lysine coated sections (3 sections / section). Sections were stored at -70 ° C until labeling was done. The sections were then marked for EC-SOD using an indirect immunoperoxidase method (Milde et al., J. Histochem. Cytochem. 37: 1609 (1989); Randell et al., Am. J. Resp. Cell. Mol. Biol. 4: 544 (1991)) with a biotinylated goat anti-rabbit IgG and horseradish streptavidin peroxidase (Jackson, ImmunoResearch Laboratories (West Grove, Pennsylvania)) (Table V). To reduce background staining, sections were incubated in H₂OR₂ 1% in methanol to inactivate endogenous peroxidases, 10 mM borohydride to block aldehydes, and non-specific binding was blocked by incubation with 5% normal goat serum (NGS), 5% milk, and 1% BSA in PBS . Optimal primary and secondary antibody dilutions were empirically determined and prepared in PBS with 1% milk plus 1% BSA (milk was not included in the streptavidin solution). Sections were developed using diaminobenzidine (10 mg diaminobenzidine, 50 ml 0.05 M Tris-Cl, pH 7.6, 100 µl H₂OR₂ 3%) and contrast stained with 1% methyl green. As a reference, serial sections were separately labeled with rabbit anti-rh-EC-SOD (ECSOD), non-immunized rabbit IgG or rabbit anti-rh-EC-SOD of which the IgG that binds to EC -SOD had been absorbed (EC-SOD absorbed; see above).

TABLE V

Staining procedures for immunohistochemistry under the light microscope. All incubations were carried out in a humidified chamber at room temperature.

Incubation time

<td>Stage 1</td><td>1% H2O2 in methanol</td><td>30 minutes</td>

<td>Stage 2</td><td>10 mM borohydride in PBS (glutaraldehyde fixed tissue only)</td><td>30 minutes</td>

<td>Stage 3</td><td>5% NGS, 5% milk, 1% BSA / PBS</td><td>30 minutes</td>

<td>Stage 4</td><td>Primary antibody, 1% milk, 1% BSA / PBS (various dilutions)</td><td>1 hour</td>

<td>Stage 5</td><td>Biotin-labeled goat anti-rabbit IgG (1: 6000 in 1% milk, 1% BSA / PBS)</td><td>1 hour</td>

<td>Stage 6</td><td>Streptavidin-horseradish peroxidase (1: 2000 in 1% BSA / PBS)</td><td>1 hour</td>

<td>Stage 7</td><td>diaminobenzidine</td><td>15 minutes</td>

<td>Stage 8</td><td>1% methyl green in water</td><td>15 minutes</td>

Electron microscopic immunocytochemistry: Ultrathin (70 nm) cryogenic sections of human lung tissue were immunostained with rabbit anti rh-EC-SOD and 10-nm protein A-gold as previously described (Crapo et al., Proc. Natl. Acad. Sci. USA 89: 10405 (1992)) (Table VI). Briefly, the sections were first incubated three times for five minutes at room temperature in 0.15% glycine in PBS to block the aldehyde groups. Non-specific binding was further blocked by incubation in 1% BSA in PBS for 10 minutes. The primary and secondary dilutions of antibody were empirically determined and prepared in PBS containing 1% BSA. Sections were stained with uranyl oxalate and uranyl acetate in methylcellulose as previously described (Crapo et al., Proc. Natl. Acad. Sci. USA 89: 10405 (1992)). The reference groups were as described above for light microscopy.

ES 2 249 784 T3

TABLE VI

Staining procedures for electron microscope immunohistochemistry. All incubations were carried out at room temperature

Incubation time

<td>Stage 1</td><td>PBS + 0.15% glycine</td><td>3 x 5 minutes</td>

<td>Stage 2</td><td>1% BSA / PBS</td><td>5 minutes</td>

<td>Stage 3</td><td>Primary antibody in 1% BSA / PBS</td><td>45 minutes</td>

<td>Stage 4</td><td>Protein -A gold</td><td>30 minutes</td>

<td>Stage 5</td><td>uranyl oxalate</td><td>5 minutes</td>

<td>Stage 6</td><td>uranyl acetate / methylcellulose</td><td>10 minutes</td>

Results

EC-SOD Antibody Characteristic: Antibody against rh-EC-SOD was characterized by Western blot analysis of rh-EC-SOD and a human lung homogenate. Figure 13 demonstrates that the antibody reacted with both EC-SOD type C (upper band) and type A (lower band) subunits (Sandstrom et al., Biochem. J. 267: 18205 (1992) in a homogenate from human lung. The type A subunit does not exist in the interstitium of tissues in vivo (Sandstrom et al., Biochem. J. 290: 623 (1993)). The antibody reacted with these three bands in the band containing purified type C rh-EC-SOD. The two lower molecular weight species in Figure 13 are due to partial and sufficient glycosylation of rh-EC-SOD in the overproduction of CHO cells.

Immunohistochemistry under the light microscope: Using an antibody against rh-EC-SOD, this protein was immunolocated in human lungs. The light microscopic immunohistochemistry revealed with EC-SOD is mainly related to the connective tissue matrix around the vessels and airways in the lung (Figure 14a and b, Figure 15a, b and c). EC-SOD was observed in close proximity to airway and vascular smooth muscle (Figure 14a and b, and Figure 15a). EC-SOD was also observed in connective tissue from alveolar septum strips (Figure 15c) suggesting an affinity of EC-SOC with the connective tissue matrix. No marking was observed in relation to vascular endothelial cells in large elastic arteries, medium-sized vessels, or capillaries (Figure 14a and b). EC-SOD was notably absent from airway endothelial cell surfaces (Figure 15a and b) and was also not present in cartilage (Figure 15a).

The antibody against EC-SOD was a polyclonal rabbit IgG antibody that was affinity purified using rh-EC-SOD. To test the specificity of the labeling for EC-SOD, ECSOD-specific IgG was absorbed from the antiserum using pure rh-EC-SOD bound to cyanogen bromide sepharose. Unimmunized rabbit IgG was then added to this absorbed antibody in sufficient quantity to replace the absorbed IgG. Labeling of lung tissues with this pre-absorbed antibody preparation resulted in the absence of labeling in all areas of the lung including the pulmonary vascular system (Figure 14c). Labeling of lung tissue with unimmunized IgG alone also resulted in the absence of labeling in all areas of the lung. The references indicate that the labeling observed with the primary antibody is specific for EC-SOD in the lung.

Electron microscope immunocytochemistry: Table VII summarizes the EC-SOD labeling in the lung observed using electron microscopic immunocytochemistry. EC-SOD was mainly related to extracellular matrix proteins in all areas of the lung. In particular, a high degree of labeling was observed in the areas in type I collagen (Figure 16) and in relation to other unidentified proteoglycans in the extracellular matrix (Figure 17). No noticeable EC-SOD labeling was observed in the 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 around smooth muscle cells in blood vessels (Figure 17) and in the airways. Labeling was markedly absent on the surface of endothelial cells in small, medium, and large vessels (Figures 18a and b). The lack of endothelial cell labeling observed in light microscopic immunochemistry supports electron microscopic observations. EC-SOD labeling was also observed in plasma within the lumen of blood vessels (Figure 18a). Localization of EC-SOD in plasma is to be expected since this protein was first discovered in plasma (Marklund, Acta Physiol. Scand., 5492: 19 (1980)). EC-SOD labeling was observed at intercellular junctions between bronchial epithelial cells (Figure 19), but was absent on the apical surface of these cells. Finally, the EC-SOD labeling was absent from the surface of type I and type II cells. A moderate but uniform amount of intracellular EC-SOD was observed in type II epithelial cells and in bronchial epithelial cells (Figure 19).

ES 2 249 784 T3

TABLE VII

Distribution of EC-SOD in the human lung. (+) indicates the presence of EC-SOD labeling and (-) indicates no labeling for EC-SOD. (±) represents the areas in which very low amounts of labeling for EC-SOD were observed in a non-uniform way

<td>Situation</td><td>EC-SOD</td>

<td>Cell surfaces</td><td></td>

<td>Endothelial</td><td> -</td>

<td>Type I cell</td><td> -</td>

<td>Type II cell</td><td> -</td>

<td>Smooth muscle cell</td><td> +</td>

<td>Fibroblast</td><td> ±</td>

<td>Extracellular matrix</td><td></td>

<td>Type I collagen</td><td> +</td>

<td>Elastin</td><td> -</td>

<td>Cartilage</td><td> -</td>

<td>Unidentified elements of the array</td><td> +</td>

<td>Intracellular</td><td></td>

<td>Endothelial cell</td><td> ±</td>

<td>Type I cell</td><td> -</td>

<td>Type II cell</td><td> +</td>

<td>Bronchial epithelial cell</td><td> +</td>

<td>Smooth muscle cell</td><td> -</td>

<td>Fibroblast</td><td> ±</td>

<td>Blood</td><td></td>

<td>Plasma</td><td> +</td>

<td>Red blood cell</td><td> -</td>

Controls were performed by absorbing EC-SOD-specific antibody outside of the primary antibody and substituting this absorbed antibody for non-immunized mouse IgG produced in the absence of labeling in all areas of the lung including areas rich in type I collagen as observed in Figure 16c. Furthermore, the use of non-immunized rabbit IgG in place of the primary antiserum also produced an absence of labeling in all areas of the lung. The lack of labeling with these controls indicates that the labeling observed with the primary antiserum is specific for EC-SOD in the lung.

The localization of EC-SOD on the surface of smooth muscle cells and in the extracellular matrix around these cells in both blood vessels and airways indicates that EC-SOD may play an important role in this position. Superoxide is known to react rapidly with nitric oxide and inactivate its smooth muscle relaxant properties. Therefore, the presence of EC-SOD throughout the course of nitric oxide dilution in smooth muscle cells should increase the half-life of this brief intracellular messenger active in this particular area and thus increase its potency as a vasodilator. . The elevated marking for EC-SOD observed around airway and vascular smooth muscle cells indicates a role for EC-SOD as a mediator of nitric oxide activity in maintaining low pulmonary vascular pressures and resistance of the respiratory tract.

In addition to labeling EC-SOD in conjunction with smooth muscle cells, EC-SOD also appears to be firmly localized to type I collagen. Collagen has previously been shown to be susceptible to attack by reactive oxygen species such as anion. superoxide. Furthermore, the superoxide anion may be capable of activating latent polymorphonuclear leukocyte (PMN) collagenases which can lead to further degradation of collagen. Because collagen fragments have been shown to chemically attract and activate PMNs, any increased superoxide produced that causes collagen degradation can accelerate inflammatory reactions and tissue destruction through PMN regeneration and activation. Consequently, the association of EC-SOD with collagen may be important in preventing superoxide-mediated degradation of collagen and therefore represents a means of controlling inflammatory responses.

ES 2 249 784 T3

Example V (For reference only)

Human EC-SOD Gene Protocols

Radiochemicals and materials

[α^-35S] dATP (~ 1400 Ci / mmol), | γ ³2P | ATP (3000 Ci / mmol) and [a^-35P] CTP (800 Ci / mmol) were purchased from New England Nuclear. Human genomic DNA, T₇, T₃ and SP6 RNA polymerase, RNasin, and plasmid pGEM3Zf (+) were purchased from Promega Biotec. The Sequenase sequencing kit (V 2.0) was purchased from United States Biochemicals Corporation. Poly A + human RNA was purchased from Clontech. SeaPlaque GTG agarose was from FMC BioProducts. Restriction enzymes were from New England Biolabs. The other reagents used were of molecular biology quality. The oligonucleotides were synthesized using an Applied Biosystems 380B or 392 DNA Synthesis Facility by the Duke University Department of Botany. Nylon filled membranes (GeneScreen Plus) were from Dupont.

Human Northern blot or analysis

Two pg of poly A + RNA were purified from eight different human tissues. These mRNAs were electrophoresed on a 1.2% agarose gel in denatured formaldehyde and transferred to a charged modified nylon membrane followed by fixing by UV irradiation. The membrane was prehybridized in 50% formamide, 0.25 M NaPO4 (pH 7.2), 0.25 M NaCl, 1 mM EDTA, 7% SDS, and 5% polyethylene glycol (8000 molecular weight). The blot was hybridized in the same buffer overnight at 60 ° C with 1 x 10⁶ cpm / ml EC-SOD RNA labeled with [³²P] generated by transcription of the complete cDNA using T3 RNA polymerase in the presence of [α³²P] CTP. The blot was washed in 0.25 M NaPO (pH 7.2), 1% SDS and 1 mM EDTA at 75 ° C followed by a second wash using Na3PO₄ 0.04 M (pH 7.2), 1% SDS and 1 mM EDTA at 75 ° C for 30 minutes. This was followed by exposure to XAR-5 film using a Lightening Plus intensifying screen at -70 ° C. The autoradiogram was scanned using an LKB Ultrascan XL laser densitometer and peaks were quantified by integration using the densitometer's internal digital integrator or by cutting the peak from a printer trace and weighing it.

Rapid amplification at the 5 'ends of the cDNA

Reverse transcription of 0.5 pg of human heart poly A + mRNA was performed using 2 pmoles of EC7, an antisense oligonucleotide specific to the 5 'gene of EC-SOD (5'-ATGACCTCCTGCCAGATCTCC3'), following a GIBCO protocol 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 spinner isolation cartridge (GIBCO BRL). The purified cDNA was tailed to dC using terminal deoxynucleotidyl transferase (TdT, 0.5 units / µl). 200 pM dCTP, 10 mM Tris-HCl (pH 8.4), 25 mM KCl, MgCl₂ 1.25 mM and 50 pg / ml of bovine serum albumin for 10 minutes at 37 ° C. The TdT was thermally inactivated for 10 minutes at 70 ° C.

The products of this reaction were then amplified by PCR using the "docking" primer (GIBCO BRL), which hybridized to the homopolymeric tail and EC4 (nested internal EC-SOD 5 'gene specific antisense oligonucleotide, 5 '-AGGCAGGAACACAGTAGC-3'). Alternatively, the tail products in dC were amplified by PCR using EC7 and HECl, transcribed strand EC-SOD gene specific primer, 5'TGGGTGCAGCTCTCTTTTCAGG-3 '). The final composition of the reaction included 20 mM Tris-HCl (pH 8.4), 50 mM KCl, MgCl₂ 2.5 mM, 100 pg / ml bovine serum albumin, 400 nM anchor primer, 200 nM gene specific primer, and 200 pM each of dATP, dCTP, dGTP, and dTTP. After incubating the PCR reaction for 5 minutes at 94 ° C, amplitaq (Perkin Elmer Cetus) was added to a final concentration of 0.04 units / μl. PCR cycling was performed on a Perkin Elmer 9600 for 35 cycles with fusion at 94 ° C for 45 seconds and hybridization at 53 ° C for 15 seconds and extension at 72 ° C for 90 seconds. Complete EC-SOD cDNA (6 ng) was used as a positive control in the PCR reaction. The PCR products were electrophoresed on a 2% SeaPlaque GTG agarose gel, transferred to loaded nylon membranes by the Southern method (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 heating it at 80 ° C in a vacuum oven for 2 hours. The subsequent blot hybridized to end-labeled HEC2 [³²P] (internal primer, nested EC-SOD specific, 5'-TCCAGCTCCTCCAAGAGAGC-3 ') overnight at 37 ° C. The blot was washed at increasing stringency until background hybridization was removed. This was followed by exposure to XAR-5 film using a Lightening Plus intensifying screen at -70 ° C.

Human genomic analysis by Southern blot

Ten pg of human genomic DNA were digested with restriction endonuclease enzymes BamH I, EcoR I, Kpn I, and Pst I to completion. Next, the electrophoresis of the DNA was carried out on a 1% agarose gel and

ES 2 249 784 T3 was transferred to a nylon membrane loaded by the Southern technique (Southern, J. Mol. Biol. 98: 503 (1975)), after alkaline denaturation (Reed et al., Nuc. Acids Res. 13: 7207 (1985)). The DNA was fixed to the membrane by heating at 80 ° C in a vacuum oven for 2 hours. Human EC-SOd-labeled antisense cRNA was synthesized with [³²P] CTP using the EC-SOD cDNA that had been linearized with Stu I. The blot was hybridized (500 x 10³ cpm / ml) in 50% formamide, NaPO₄ 0.25 M (pH 7.2), 0.25 M NaCl, 1 mM EDTA, 7% SDS and 5% polyethylene glycol (8000 molecular weight) at 50 ° C. After hybridization overnight, they were washed in NaPO₄ 0.25 M (pH 7.2), 2% SDS and 1 mM EDTA followed by NaPO₄ 0.04 M (pH 7.2), 1% SDS and 1 mM EDTA at increasing stringency until background hybridization was minimized. The blot was exposed on XAR-5 film using the Lightening Plus intensifying screen at -70 ° C.

Isolation of the human gene for EC-SOD

An adult female leukocyte library constructed on the EMBL-3 vector was purchased from Clontech. Approximately 1 x 10 were screened⁶ pfu at a density of ~ 50,000 pfu / plate using [³²P] CTP (1 x 10⁶ dpm / ml). Primary to tertiary screens identified approximately 7 unique putative positive plaques. Individual plates were isolated and lambda DNA was purified using LambdaSorb phage adsorbent (Promega Biotec). The insert DNA size of each clone was assessed by restriction endonuclease digestion Sal I followed by 0.7% agarose electrophoresis. Selected clones underwent extensive restriction endonuclease mapping. Based on the results of restriction mapping and asymmetric hybridization using 5 'and 3' hybridizing EC-SOD oligonucleotides, clone # 7 was selected for all subsequent DNA sequence analyzes. Clone # 7 contains a fragment of approximately 18-20 kb.

DNA sequenced from the human EC-SOD gene

The general strategy used for the sequencing of clone # 7 is illustrated in Figure 20. Restriction endonuclease DNA fragments of various sizes from clone # 7 were subcloned into the vector DNA pGEM3Zf (+). The dideoxy sequencing method using double-stranded DNA was used (Ausubel et al., Current Protocols in Molecular Biology, Green Publishing Assoc. and Wiley Interscience, New York (1992)) as template and Sequenase enzyme (United States Biochemicals) (Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463 (1977)). Both Universal and -40 M13 sequencing primers were used to initiate DNA sequencing for each subcloned fragment. The oligonucleotides from these initial sequencing data were synthesized approximately every 250 base pairs until the complete nucleotide sequence was obtained. Sequencing data was obtained from both strands as shown in Figure 20B except at the 3 'position of the gene where the DNA sequence was obtained on one strand only.

Computer-aided sequence analysis and investigation of the transcription database

The IntelliGenetics geneworks program (version 2.2) was used to organize the DNA sequence data. Homology research was performed at NCBI using the BLAST network service (Altschul et al., J. Mol. Biol. 215: 403 (1990)) and the non-redundant nucleotide sequence database (GenBank ( 77.0) + EMBL (35.0) + EMBLUpdate + GBUdate). The investigation of the transcription factor database was carried out using both the SIGNAL SCAn 3.0 algorithm (Prestridge et al., CABIOS 9: 113 (1993)) and the FINDPATTERNS program of the GCG package (V 7.2) using the 6.3 edition of the transcription factor database (Gosh, Nuc. Acids Res. 18: 1749 (1990)). For a prediction of the signal peptide cleavage zone, the SIGSEQ1 programs (Folz et al., J. Biol. Chem. 261: 14752 (1986)) and SIGSEQ2 (Folz et al., Biochem. Biophys. Res. Commun. 146: 870 (1987)).

Results

Tissue-specific expression of human EC-SOD

To investigate the expression of human EC-SOD, poly A (+) mRNA from eight different human tissues was fractionated on a denatured agarose gel and transferred to a loaded nylon membrane. Because a previous article reported long exposure times in order to identify specific EC-SOD bands during genomic Southern analysis (Hendrickson et al., Genomics 8: 736 (1990)), a probe of Radiolabeled antisense cRNA from human EC-SOD full length cDNA (Oury et al., Proc. Natl. Acad. Sci. USA 89: 9715 (1992)). A discrete band of approximately 1.4 kb can be seen in all eight human tissues analyzed (Figure 21A). Furthermore, skeletal muscle contains an approximate 4.2 kb message, not detected in other tissues. By densitometric scanning of the 4.2 and 1.4 kb bands, the largest message can be calculated to prepare approximately 32% of the total skeletal muscle EC-SOD message. In the brain, a highly simulated 2.2 kb band can be seen. This band was too weak for quantification by the laser densitometer. The quantification of these bands was carried out by laser densitometry as well as the integration of the peaks of the autoradiograms obtained in the linear exposure interval (Figure 21B). After normalization in the brain, the heart showed the most expression with 10.1 times that of the brain. This was followed by the placenta, pancreas and lung which gave 13,6,10,2 and 7.5, respectively. Expression in skeletal muscle was 4.7 for the 1.4 kb band or 6.9 for both the 1.4 kb and 4.2 kb messages, while the kidney and liver gave an expression 6.3 and 4.1 times that of the brain. These models of

ES 2 249 784 T3 expressions have been reproduced based on probing an additional independent multiple tissue Northern blot. Bands are specific based on the relatively high stringency of washout and data using a transcribed strand EC-SOD cRNA as a probe that did not hybridize under the given conditions.

Transcription initiation mapping of the area

Initially, mapping of the transcription initiation zone was tested using the primer extension method. Using several different 5 'end-labeled oligonucleotides and poly A + mRNA from both human lung and human heart as well as total RNA isolated from human foreskin fibroblasts, a positive signal was not obtained even after prolonged exposure periods. This did not appear to be due to technique as it was not possible to obtain positive signals using RNA generated by in vitro transcription of the EC-SOD cDNA. It is not clear whether the lack of success using this technique was due to too low an abundance of mRNA encoding EC-SOD or some other problem or problems. Working under the assumption of a low abundance of mRNA, the technique of rapid extension of the ends of the cDNA was tested in order to amplify this signal by PCR. As shown in Figure 22 EC-SOD gene specific primer EC7 was used for hybridization and reverse transcription of human heart poly A + mRNA. Half of this reaction was in the 3 'dC tail using terminal deoxynucleotidyl transferase and the remaining half without using it. These templates were then subjected to PCR amplification using the gene specific HEC1 + EC7 primers as well as the anchor primer + EC4. The products of these reactions were fractionated by agarose electrophoresis, transferred to a nylon membrane, and probed with the nested internal gene specific primer HEC2. An autoradiogram of this experiment is presented in Figure 22A. Using EC-SOD cDNA as a reference template and HEC1 + EC7, a band of 217 bp is to be expected (lane 3 of Figure 22A). As primers HEC1 and EC7 are expected to widen independently of the dC tail, bands of equal intensity are observed in bands 4 and 5, which are also the same size as the EC-SOD reference. Using the docking primer (which hybridizes to the dC tail) and EC4, only a ~ 190 bp band (lane 1) was observed. As the template was not poly C tail, band 2, as expected, does not show any signal. By subtracting 48 bp (anchor primer size), the size of the reverse transcribed DNA would correspond to 5 'of ~ 136 bp of primer EC4. This analysis predicted that there is approximately 6 base pairs of additional 5 'sequence in the cDNA clone and that transcription initiation begins approximately 6 bp upstream of the first intron (indicated by a dotted box). Although the initiation of eukaryotic transcription normally begins at the adenosine moiety, it is expected to begin at a G (Breathnach et al., Ann. Rev. Biochem. 50: 349 (1981)).

Southern blot genomic analysis

To begin characterizing the human EC-SOD gene, 10 pg of total human genomic DNA was restriction digested and reaction products electrophoresed on an agarose gel followed by transfer to a nylon membrane. The blot was probed with a partial EC-SOD cRNA labeled with [³²P]. An autoradiogram of this transfer is shown in Figure 23. As can be seen for each band, there are unique bands associated with each restriction digest. There were no shaded bands that could suggest pseudomonas. When a full length cRNA probe is used for Kpn I digested DNA, an additional band of ~ 4000 bp is observed corresponding to the 3 'end of the gene. Furthermore, the Kpn I band exhibits a 0.5 kb band that was better seen in other blots. This band pattern was similar to a restriction map of human EC-SOD clone # 7 (see Figure 20A).

Isolation and characterization of human EC-SOD by DNA sequencing

Multiple independent positive clones were identified from a human adult lymphocyte library constructed in EMBL-3. These clones underwent extensive restriction endonuclease mapping and were probed with EC-SOD-specific 5 'and 3' oligonucleotides to determine the relative orientation of the inserts. Based on these results, clone # 7 was selected for further analysis. Clone # 7 is approximately 18-20 kb and contains at least 5000 bp of 5 'flanking DNA and at least 4000 bp of 3' flanking DNA. The restriction map of clone # 7 is presented in Figure 20B. This map is similar to results obtained with genomic Southern blot data indicating that clone # 7 contains the EC-SOD gene. The strategy for subcloning and sequencing clone # 7 is presented in Figure 20A. Contiguous and overlapping restriction fragments of various sizes were subcloned into the plasmid vector pGEM32f (+) (Figure 20B). The DNA inserts on both strands were sequenced using a combination of migratory primer and specific vector, universal sequencing primers. The 3 'half of the 7K36 insert was sequenced only in one strand. Published sequence data for human EC-SOD cDNA (Hjalmarsson et al., Proc. Natl. Acad. Sci. USA 84: 6340 (1987)) as well as DNA sequence information obtained from an independent cDNA clone containing additional 5 'untranslated data (Hendrickson et al., Genomics 8: 736 (1990)) was used to determine the genomic structure of the intron / exon. Based on a comparison of these data with the genomic sequence information, the human EC-SOD gene containing three exons and two introns was determined (Figure 20C). Exon 1 contains at least 5 base pairs and is probably larger (by approximately 6 base pairs), as the exact onset of transcription initiation was not determined (note below). Exon 2 contains 84 bp and is separated from exon 1 by a 572 bp intervening sequence labeled 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 in this exon the beginning of the complete coding sequence for preEC-SOD begins (Figure 20D). This comprises a signal peptide of 18

ES 2 249 784 T3 amino acids preceding the mature protein sequence of 222 amino acids. There are no introns that separate the various structural domains of EC-SOD. These domains are presented schematically in Figure 20D and comprise 1 to 95 amino acids that contain a glycosylated Asn-89 and have no sequence homology to other proteins. Residues 96 to 193 show marked homology with the CuZn-SOD protein sequences with preservation of critical amino acids important in the catalysis and structure of the enzyme. Amino acids 194-222 contain multiple charged residues that have been shown to be important in binding to sulfated proteoglycans. In addition, 558 bp of the 5 'flanking zone containing putative regulatory elements and 3675 bp of the 3' flanking zone were sequenced. The exogenous DNA sequence data is in agreement with the published cDNA sequence (Hjalmarsson et al., Proc. Natl. Acad. Sci. USA 84: 6340 (1987)). The intron-exon boundaries and according to the consensus eukaryotic splicing sequence are presented in Table VIII (Senapathy et al., Methods Enzymol. 183: 252 (1990)). Both introns split the sequences in the 5 'untranslated region of the EC-SOD gene.

TABLE VIII

Sequences at the intron / exon splice junctions

The size of the introns and exons is presented in base pairs (bp). The letters in the upper box indicate the sequence of the exon while the letters in the lower box indicate the sequence of the intron. Splicing junctions are presented according to previously published consensus sequences for splicing junctions (Senapathy et al., Methods Enzymol. 183: 252 (1990)).

<td colspan="2">Donor</td><td>Intron size (bp)</td><td colspan="2">Acceptor</td><td>Exon</td>

<td>TGCGGG</td><td>gt ggac</td><td> 572</td><td>gccc ag</td><td>GCTCCA</td><td> 84</td>

<td>GGAAAG</td><td>gt gggt</td><td> 3549</td><td>ccgc ag</td><td>GTGCCC</td><td> 1336</td>

Figure 24 presents the complete sequence for the human EC-SOD gene. The exonic sequences are presented in letters contained in the upper box while the intronic sequence that flanks the 5 'and 3' is presented in the lower box. Exon 3 containing the complete uninterrupted coding region for EC-SOD and the protein sequence are presented using the single letter amino acid code. The 18 amino acid signal peptide and the 222 amino acid mature protein sequence are highlighted. The identification of the signal peptide cleavage point is consistent with computer algorithms that predict the cleavage point of the eukaryotic signal peptide (Folz et al., Biochem. Biophys. Res. Comm. 146: 870 (1987)); Von Heijne, Eur. J. Biochem. 133: 17 (1983)).

Searching the transcription factor database was used to presumably identify transcriptional regulatory elements. Although almost all eukaryotic activators use an element with the TATA sequence to fix the transcription initiation position, an obvious TATA sequence for the EC-SOD gene cannot be discerned. Two elements with CAAT sequence were identified. One is in the opposite orientation and located approximately 20 bp upstream of the first exon, while the second can be approximately 335 bp upstream. The putative signal for polyadenylation is presented and the point of poly A adenylation is indicated. Investigation of the transcription factor database of the 5 'untranslated region and the first intron identified several potential regulatory elements. A cAMP-sensitive element (CREB) (TGACGT) that is similar to the 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)). The middle of the region for the glucocorticoid response element (GRE) (TGTCCT) is located at 370 bp (Karin et al., Nature 308: 513 (1984)). A skeletal muscle-specific transactivation factor (M-CAT) (CATTCCT) response element is found at the beginning of the opposite orientation at position 238 (Mar et al., Mol. Cell. Biol. 10: 4271 (1990) ). Within the first intron at position 1085 bp is a xenobiotic responsive element (XRE) (CACGCW) (Rushmore et al., J. Biol. Chem. 265: 14648 (1990)). At position 89 is a metallic regulatory element (MRE) (TGCRCYC) (Culotta et al., Mol. Cell. Biol. 9: 1376 (1989)). Two putative response antioxidant elements (AREs) (RGTGACNNNGC) are found at position 650 and 5022 (Rushmore et al., J. Biol. Chem. 266: 11632 (1991)). At position 251 in the opposite orientation is a sis-sensitive element (SIF) (CCCGTC) important for the production of the proto-oncogene c-fos (Wagner et al., EMBO J. 9: 4477 (1990)). There is an AP1 binding site or TPA-sensitive element (TRE) (TGACTCA) discovered at position 162 (Risse et al., EMBO J. 8: 3825 (1989)). The AP4 region of the SV40 enhancer (CAGCTGTGG) can be found at position 171 (Jones et al., Dev. Genes 2: 267 (1988)).

ES 2 249 784 T3

Example VI (For reference only)

Identification of patients for gene defects in EC-SOD

Preparation of genomic DNA from patient leukocytes: Normal healthy control patients and patients with asthma, primary pulmonary hypertension, and secondary pulmonary hypertension will be identified. Genomic DNA will be purified using a Qiagen Blood PCR Kit. One ml of blood will be drawn containing ~ 10⁷ leukocytes / ml in sodium citrate from each patient or reference individual. The blood is placed on a Qiagen-spin column and the leukocytes are trapped in the resin by brief centrifugation, while the erythrocytes and hemoglobin are completely washed away. Leukocytes are lysed by adding 0.7 ml of lysis buffer and incubated at room temperature for 30 minutes. The DNA that is released binds to the resin in the tube. The remaining cell debris is washed away by multiple wash / spin cycles. DNA is eluted by elution of 1.2 M KCl, 50 mM MPOS, 15% ethanol, pH 8.3. This normally yields ~ 10 pg of genomic DNA (Reihsaus et al., Am. J. Respir. Cell. Mol. Biol. 8: 334 (1993)).

Primer design and PCR amplification of EC-SOD exonic sequences: Transcribed strand and antisense oligonucleotide primers will be designed (or the use of primers already obtained from genomic DNA sequencing) that contain a 3'GC staple (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 enlarged using human genomic DNA and DNA sequencing primers. The PCR conditions are as described (Reihause et al., Am. J. Respir. Cell. Mol. Biol. 8: 334 (1993); Innis et al. (Eds.) Academic Press San Diego pp. 1-12 (1990)) using Taq polymerase, with the following temperature cycle: initial denaturation at 95 ° C for 5 minutes followed by 35 denaturation cycles at 94 ° C for 20 seconds, hybridization at 57 ° C for 15 seconds and elongation at 72 ° C for 45 s. Due to the GC composition and existing primer sequence, it will be necessary to experimentally optimize the conditions for PCR amplification using each set of primers. Three sets of primers will be used to span the entire coding area.

Identification of mutations with single chain conformational polymorphism (SSCP) analysis: SSCP analysis has been used to detect a single base pair incompatibility (Orita et al., Genomics 5: 874 (1989)). Temperature gradient gel electrophoresis (TGGE) will be used to detect mobility differences (Wartell et al., Nuc. Acids Res. 18: 2699 (1990)). Samples for TGGE will be prepared by thermal denaturation of the product by PCR at 98 ° C for 5 minutes, then renaturation at 50 ° C for 15 minutes with the corresponding natural DNA from the PCR of the cloned gene. Electrophoresis will be performed on an 8M urea gel, 5% acrylamide during a temperature gradient. The temperature gradient will be optimized for each of the EC-SOD DNA segments. Typical Gradients for Detection of β Receptor Mutations₂-adrenergic were between 35 ° C and 60 ° C and required 4 to 6 hours of run time (Rosen, Nature 262: 59 (1993)).

All PCR samples found to be positive for TGGE mutations will be sequenced directly using the dideoxy technique (Sanger et al., Proc. Natl. Acad. Sci. USA 74: 5463 (1977)).

Example VII

Inhibition of xanthine oxidase

In a first study, analyzes were performed in a 1 ml quartz cuvette containing 50 mM carbonate buffer, pH 10, 0.1 mM EDTA, 1 nM xanthine oxidase (Boehringer Mannheim) at 25 ° C. Xanthine oxidase activity was measured spectrophotometrically following the loss of xanthine over time at 295 nm. Four concentrations of xanthine (25, 50,250 and 500 juM) and two concentrations of MnTBAP (5 and 10 juM) were used. The two inhibition constants were obtained after the ordinate at the origin of the curve (Kii = 5.5 juM) and the slopes (Kis = 15 juM). The results presented in Figure 25 demonstrate that MnTBAP inhibits xanthine oxidase in a non-competitive manner.

In a second study, calf pulmonary artery endothelial cell cultures (CPA-47 (Tissue and Cell 10: 535 (1978)) 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 pre-incubated for 1 hour with 50 pM MnTBAP in minimal essential medium (MEM) or MEM only. Varying amounts of xanthine oxidase (XO) plus 200 pM xanthine (X) were added and allowed to incubate for 24 hours. Cell damage was quantified by measuring the release of cellular lactate dehydrogenase (LDH) in the medium. Figure 26 shows the efficacy of MnTBAP by decreasing the release of LDH produced by XO / X.

ES 2 249 784 T3

Example VIII

SOD mimetic provides cellular protection from paraquat injury

Rat lung 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 pre-incubated for 1 hour with 100 pM MnTBAP or MnTMPyP in MEM or MEM only. Paraquat (2.5 mM) was added and allowed to incubate for 48 hours. Cell damage was quantified by measuring the release of cellular lactate dehydrogenase (LDH) in the medium. Figure 27 demonstrates that MnTPyP (hatched bars) and MnTBAP (gray bars) decrease the release of LDH produced by Paraquat.

In another study, calf pulmonary artery endothelial cell cultures (CPA-47 (Tissue and Cell 10: 535 (1987)) 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 pre-incubated for 1 hour with varying concentrations of MnTBAP in MEM or MEM only. Paraquat (2 mM) was added and allowed to incubate for 24 hours. Cell damage was quantified by measuring the release of cellular lactate dehydrogenase (LDH) in the medium. MnTBAP decreases the release of LDH produced by Paraquat as a function of dose (see Figure 28).

In contrast to MnTBAP, ZnTBAP does not protect against injury caused by Paraquat. The endothelial cell cultures of the calf pulmonary artery (CPA-47 were grown until confluence in Ham's F-12K medium with 10% fetal bovine serum at pH 7.4 and 37 ° C. The cells were then treated with Trypsin and seeded at equal densities in 24-well plates and grown to 90% confluence. Cells were washed and pre-incubated for 1 hour with varying concentrations of ZnTBAP in MEM or MEM only. Paraquat (2 mM) was added and allowed to incubate for 24 hours. Cell damage was quantified by measuring the release of cellular lactate dehydrogenase (LDH) in the medium. The results presented in Figure 29 demonstrate that ZnTBAP does not possess SOD-like activity. ZnTBAP can be used as a negative reference to demonstrate that redox metal is important in protecting against paraquat toxicity.

Example IX

MnTBAP Protection Against Paraquat Lung Injury

Mice were treated with Paraquat (PQ, 45 mg / kg, ip) or with saline (10 ml / kg, ip) and exposed to MnTBAP (2.5 mg / ml, nebulized in a chamber of 2 the ratio of 2 l / min for 30 minutes twice a day for 2 days) or in ambient air. Mice were sacrificed 48 hours after the start of treatment and lung injury was assessed by bronchoalveolar lavage fluid (BALF) analysis. The markers of BALF alteration used were lactate dehydrogenase (LDH, in units / l), protein concentration (in mg / dl) and the percentage of mononuclear leukocytes (PMN). Treatment with MnTBAP provided partial protection against Paraquat lung injury (see Figure 30).

Example X

Catalase activity was measured by a Clark oxygen electrode using a modified assay previously described by Del Rio et al., Anal. Biochem. 80: 409 (1977). Briefly, reactions were carried out in a nitrogen degassed phosphate buffer (50 mM, pH 7.8) containing 0.1 mM EDTA at 25 ° C. Three concentrations of hydrogen peroxide (1 to 4 mM) and four concentrations of metalloporphyrin (0.5 to 50 juM) were used to determine second order rate constants. The evolution of the oxygen velocity was followed for 2 minutes. The results are presented in Figure 31.

Calf pulmonary endothelial cell line (CPA-47) was cultured to approximate confluence in 12-well plates with F-12K medium containing 10% fetal calf serum. CPA-47 cells were loaded with Cr⁵¹ as previously described by Simon et al. J. Clin. Invest. 78: 1375 (1986) in high glucose minimal essential medium (DMEM). Cells were pretreated with MnTBAP (100 juM) for 1 hour and then exposed to various concentrations of hydrogen peroxide generator, glucose oxidase, for 4 hours. Cell injury was then quantified as the specific release of Cr⁵¹ of CPA-47 cells that had been adjusted for spontaneous release of Cr⁵¹. The results are presented in Figure 32.

Calf pulmonary endothelial cell line (CPA-47) was cultured to approximate confluence in 12-well plates with F-12K medium containing 10% fetal calf serum. CPA-47 cells were loaded with Cr⁵¹as previously described by Simon et al. J. Clin. Invest. 78: 1375 (1986) in high glucose minimal essential medium (DMEM). Cells were pretreated with various concentrations of: (A) MnTBAP; (B) MnTMPyP; (C) ZnTBAP; or (D) CuZnSOD for 1 hour and then glucose oxidase (100 Mu / ml) was exposed to the hydrogen peroxide generator for 4 hours. Cell injury was then quantified as the specific release of Cr⁵¹ of CPA-47 cells that had been adjusted for spontaneous release of Cr⁵¹. The results are presented in Figures 33A to 33D.

ES 2 249 784 T3

Example XI

Mimetics as protectors against excitotoxic cell death

Experimental procedures

Tissue culture: Mixed glial and neuronal cultures were prepared from 18 day old embryonic rat brain cortices (Sprague-Dawley, Zivic Miller). Briefly, the cerebral cortices were dissected and enzymatically dissociated by incubation in Hank's balanced salt solution free of Ca ++ and mg ++ (HBSS) enriched 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 heat sterilized Pasteur pipette. The cell suspension was centrifuged and resuspended in minimal essential medium (MEM), containing Earle's salts enriched with 3 g / l glucose, 5% horse serum and 5% fetal calf serum (medium cultivation). Cells were placed in poly-D-lysine coated multi-well plates: 12-well plates for aconitase measurement and 24-well plates for toxicity experiments. Cells were kept at 37 ° C in a CO humidified incubator.₂ 5% / 95% air in growth medium. The medium was not substituted in order to reduce glial overgrowth and neuronal loss. Mature cells (14 to 17 days in vitro) were used for all experiments.

Cell treatment: The culture medium was replaced with MEM enriched with 25 mM glucose (MEMg). For both neurotoxicity and aconitase measurement studies, cells were incubated in the designed treatment for the indicated duration at 37 ° C. Unless otherwise specified, SOD antagonists or mimetics were added 15 min. before agonists. For the measurement of neurotoxicity, cells were incubated with treatments for 18 h at 37 ° C in MEM-g. To assess the ability of antagonists to inhibit acute NMDA toxicity or rescue cells from ongoing NMDA insult, an alternative NMDA treatment paradigm was used in addition to that described above. In this paradigm, the culture medium was replaced by HBSS^- (Hank's balanced salt solution free of Ca^-- and mg^-- enriched with 2 mM CaCl2, 1 mM NaHCO3, 10 mM HEPES and 5 juM glycine), the cells were treated with the vehicle or with 100 pM NMDA for 15 min. after which HBSS + was replaced by MEM-g and the cells were returned to the incubator for a further 18 h. SOD antagonists or mimetics were added to cells 15 min. before exposure to NMDA or 15, 30 or 60 min. after the final middle substitution. For the determination of dependence on Ca^--, cells were incubated in HBSS^- without adding Ca^--. When kainate (KA) was used as an agonist, 100 pM 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 regulate the contribution of hydrogen peroxide.

Neurotoxicity studies: Neurotoxicity was determined by treating the LDH released in the supernatant medium as previously described (Patel et al., Toxicol. Appl. Pharmacol. 115: 124 (1991) and Neurotoxicology 14:35 (1992) ). LDH was measured by the Vassault lactate dehydrogenase method. In: Methods of Enzymatic Analysis, Bergmeyer HU (ed), Verlag Chemie, Weinheim, pp. 118-126 (1983). Furthermore, cell death was confirmed with ethidium-1 homodimer (EthD-1). Briefly, cells were washed with HBSS, loaded with 20 pM EthD-1 for 30 min, rinsed, and viewed through fluorescence lenses. The number of dead cells labeled with EthD-1 were counted in 4 to 6 randomly selected fields, the numbers were averaged to give an estimate of cell death, and the experiment was repeated 3 times.

Aconitase measurement: For aconitase measurement, following treatment with agents, the medium was removed and cells were lysed in ice cold 50 mM Tris / HCl, pH 7.4 containing MnCl₂ 0.6 mM, 1 mM L-cysteine, 1 mM citrate, and 0.5% Triton-X 100. Aconitase activity of cell lysates was measured by spectrophotometry by monitoring the formation of cis-aconitate in isocitrate at 240 nm in 50 mM Tris / HCl, pH 7.4, containing MnCl₂ 0.6 mM and 20 mM isocitrate at 25 ° C (Kreb and Holzach, Biochem. J. 52: 527 (1952); Gardner and Fridovich, J. Biol. Chem. 267: 8757 (1992)). The inactive aconitase was reactivated by a 30 min incubation. of cortical cell lysates (90 µl) with 0.5 M DTT (10 Jul), Na₂S 20 mM (1 µl) and 20 mM ferrous ammonium sulfate (1 //.l) in 50 mM Tris / HCl, pH 8.0 at 37 ° C. Aconitase activity in cortical cell lysates was inhibited by 0.1 mM potassium ferricyanide or by boiling the sample.

Statistical analysis: One-way analysis of variance (ANOVA) was used to compare three or more treatments and Dunnet's test to compare multiple treatment groups with a reference group. The Tukey-Kramer multiple comparison test was used to detect differences between treatments. Student's t test was used to compare two treatments.

Results

Validation of aconitase activity as a marker of O2 radical formation^- in cortical cell culture: With the use of agents that are known to generate O2^- (X + XO and PQ ++), aconitase activity was determined as a valid marker of O2^-. Generators of O₂^- they selectively and reversibly inactivated aconitase and this was done avoidably by SOD.

Inactivation of aconitase produced by PQ⁺⁺, NMDA and KA correlate with cell death: Cortical cells were treated with varying concentrations of KA, NMDA and PQ⁺⁺ for 18 h. and the LDH activity in the medium was analyzed. Similar cultures were treated with varying concentrations of KA, NMDA, or PQ⁺⁺ for 6,

ES 2 249 784 T3 or 3 h., Respectively, and aconitase activity was analyzed in cell lysates; Aconitase activity was evaluated during a shorter incubation period because neuron death leads to irreversible inactivation of aconitase activity, presumably due to protein degradation. Accordingly, an earlier time point was selected to measure aconitase activity based on the steady-state approach time of reversible activity (Fig. 34 A and B for NMDA 50 µM and KA 300 µ / M, respectively). .

Treatment with KA produced commensurate decreases in aconitase activity and cell death controlled by the release of LDH (Fig. 35A). Likewise, treatment with PQ⁺⁺ produced a proportional decrease in aconitase activity and the amount of LDH release (Fig. 35 B). At higher concentrations (30 to 1000 juM), NMDA produced a proportional decrease in aconitase activity and also in cell death. However, low concentrations (3 and 10 µ / M) of NMDA produced cell death (release of LDH) but not detectable inactivation of aconitase (Fig. 35 C).

MnTBAP prevents the inactivation of aconitase and cell death caused by PQ, NMDA and KA: To determine if MnTBAP can cause the inactivation of aconitase and cell death caused by NMDA, PQ⁺⁺ and KA, cortical cells were incubated with PQ ++, NMDA and KA in the absence and presence of 200 µ / M MnTBAP. PQ ++, NMDA, and KA produced a 70%, 40%, and 42% decrease in aconitase activity after 3, 1, and 6 h, respectively; 200 μM MnTBAP markedly inhibited PQ-mediated decreases in aconitase activity⁺⁺, NMDA and KA (Fig. 36 A). Parallel effects were shown in cell death measured by LDH release. MnTBAP (200 µM) 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 µM) produced a right and downward shift in the concentration response curve for NMDA, providing complete protection at low NMDA concentrations (Fig. 37).

The possibility that MnTBAP simply prevents the activation of NMDA or AMPA receptors was examined and eliminated.

To consolidate the possibility that MnTBAP decreases the inactivation of aconitase and prevents cell death by its SOD mimetic action, the effects of the structurally related congener, ZnTBAP, with decreased SOD activity (i.e. 10 times less) were examined. EthD-1 was used to quantify cell death in these experiments because preliminary studies showed that ZnTBAP interfered with LDH measurement (MnTBAP was found to have no effect on LDH measurement). Concentration response curves for NMDA and KA demonstrated a strong correlation between cell death measured using LDA release and dead cells quantified by EhtD-1. Concentration response curves revealed that MnTBAP exhibited markedly increased neuroprotective effects compared to ZnTBAP against PQ ++, NMDA and KA toxicities (Fig. 38).

To determine whether the addition of MnTBAP after the initiation of the excitotoxic insult exerted neuroprotective effects, 100 µM NMDA was included for 15 min. at HBSS^- (HBSS free of Ca^-- and mg^-- containing 5.6 mM glucose and enriched with CaCl₂ 2 mM, NaHCO₃ 1 mM, 10 mM HEPES and 5 µM glycine). This acute exposure paradigm produced delayed neuronal death (measured 18 h later) and allowed the determination of the temporal relationship between MnTBAP exposure and neuroprotection. The neuroprotective effects of MnTBAP were evaluated when it was: 1) present 15 min. before and for 15 min. of NMDA application (but not during the 18 h period after medium change) ("pre" condition); 2) present 15 min. before and during a 15 min NMDA application. and for the next 18 h. ("pre + post" condition); 3) 15, 30 or 60 min was added. after a 15 min incubation. with 100 µM NMDA and left in the medium for the next 18 h. The "pre" condition produced a 25% reduction in LDH release measured 18 h. later; Using an identical time application cycle of 100 µM D-APV in the "pre" condition, a complete blockade of LDH release was observed. In contrast, MnTBAP incubated as in the "pre + post" condition produced a 51% reduction in LDH release. The addition of 200 µM MnTBAP 15, 30 or 60 min. after an exposure of 15 min. at 100 µM NMDA produced a 38%, 30% and 25% reduction in LDH release respectively. Instead, the addition of MK801 10 µM 15 min. after NMDA treatment it exerted a modest protective effect (17%) when added 15 min. after the NMDA assault. In order to investigate the possibility that the inactivation of aconitase produced by NMDA results from the formation of peroxynitrite, the effects of inhibitors of nitric oxide synthetase (NOS) on the inactivation and cell death of aconitase produced by NMDA were examined. . The inactivation of the aconitase produced by NMDA and the release of LDH were not altered by the presence of 1 mM of N methyl ester.^G- nitro-L-arginine, N^G-nitro-L-arginine or N^G-monomethyl-L-arginine. However, NOS was expressed in the cortical neuronal preparation used. For this reason, peroxynitrite would not appear to play an etiological role in the inactivation of the aconitase produced by NMDA or in cell death under the conditions used.

Example XII

Learning defects in mice genetically modified with EC-SOD

EC-SOD genetically modified mice were obtained from Dr. Lena Carlsson and Dr. Stephan Marklund, Umea, Sweden. Learning was assessed in genetically modified mice and control mice (controls came from litters (+ / +) carrying normal concentrations of EC-SOD) using a maze.

ES 2 249 784 T3 eight-arm radical in which feed was placed in a well at the end of each arm. The feed was concealed from the mice for a period of eight hours and the mice were then placed in the maze. The number of arms that the mice lowered to retrieve the feed was counted before they lowered any arms during a second period. The wild animals randomly descended between four and five different arms of the maze before starting to repeat.

The results presented in Figure 39 demonstrate that when the experiment is carried out in 24 successive days, the control mice learn the configuration and are able to increase the number of arms that go down to find the feed without error. The EC-SOD genetically modified animals were unable to show any significant learning during the full 24 sessions.

Example XIII

Peroxynitrite antioxidation by mimetics

Experimental protocols

Cell culture: J774 macrophages were cultured in DMEM medium, enriched with L-glutamine (3.5 mmol / l) and 10% fetal calf serum. Cells were grown in 96-well plates (200 µl of medium / well) until confluence. To produce the inducible isoform of nitric oxide synthase (iNOS), fresh culture medium containing LPS from E. coli (011: B4; 10 pg / ml) alone or in combination with murine γ-interferon (γ-IFN, 10 u / ml) in the presence or absence of the inhibitor NOS, a SOD mimetic of the combination of the two compounds for 24 h. In addition, cells were exposed to the NO donor compounds, S-nitroso-N-acetyl-DL-penicillamine (SNAP) (3 mM) and diethylamine: NO NONOate (DNO) (3 mM) for 24 hours or a peroxynitrite ( 1 mM) authentic for 1 h. The nitrite / nitrate concentration in the medium and mitochondrial respiration were then measured as described below.

Measurement of nitrite / nitrate production: Nitrite / nitrate production in the supernatant was measured as an indicator of NO synthesis. First, nitrate in the culture medium was reduced to nitrite by incubation with nitrate reductase (670 mU / ml) and NADPH (160 pM) at room temperature for 2 h. After 2 h, the nitrite concentration in the samples was measured by the griess reaction, adding 100 µl of griess reagent (1% sulfanilamide and 0.1% naphthylethylenedimide in 5% phosphoric acid) to 100 µl of samples of conditioned medium. Optical density at 550 nm (OD_55o) using a Spectramax 250 microplate reader (Molecular Devices, Sunnyvale, CA). Nitrate concentrations were calculated by comparison with OD_55o of sodium nitrate standard solutions prepared in culture medium. All measurements were corrected for MnTBAP interference at this wavelength. MnTBAP (up to 300 pM) did not autooxidize nitrite or nitrate and did not interfere with nitrate reductase activity.

Measurement of mitochondrial respiration: Cellular respiration was assessed by the mitochondrial-dependent reduction of 3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyltetrazolium (MTT) bromide to formazan. Cells were incubated in 96-well plates at 37 ° C with MTT (0.2 mg / ml) for 1 hour. The culture medium was removed by aspiration and the cells were solubilized in DMSO (100 µl). The extent of MTT reduction to formazan with cells was quantified by measuring OD_55o. All measurements were corrected for MnTBAP interference at this wavelength.

Measurement of peroxynitrite oxidation of dihydrorhodamine 123: The peroxynitrite-dependent oxidation of dihydrorhodamine 123 to rhodamine 123 was measured, based on the principles of the method described by Kooy et al., Free Radical Biol. Med. 16: 149 (1994 ). Briefly, peroxynitrite was added at 5 pM in phosphate buffered saline containing 10 pM dihydrorhodamine 123, in the absence or presence of MnTBAP (3 to 100 pM). After a 10 min incubation. at 22 ° C, rhodamine 123 fluorescence was measured using a Perkin-Elmer fluorimeter (model LS50B; Perkin-Elmer, Norwalk, CT) at an excitation wavelength of 500 mn, emission wavelength of 536 nm (slit widths of 2.5 and 3.0 nm, respectively).

Measurement of vascular relaxations produced by NO: New Zealand white rabbits weighing 3 to 4 kg were anesthetized with pentobarbital (30 mg / kg). The descending thoracic aorta was isolated, removed, cleaned, and placed in Krebs buffer (pH 7.5). The vessels were cut into 5 mm rings and hung on stirrups connected to force transducers. The rings were suspended in 20 ml jacketed baths that were kept at 37 ° C and sparged with 95% O2 and 5% CO2. The rings were equilibrated with 2 g resting tension for 1 hr prior to use and concentrated with phenylephrine. Saturated NO solution was prepared by bubbling compressed NO gas through a NaOH trap and then into deionized anaerobic water. Aliquots of the nitric oxide solutions (final concentration: 75 to 300 nM) were added to the rings (in the presence or absence of 100 pM MnTBAP) and the relaxing responses were recorded.

Data analysis: All values ​​are expressed as mean ± standard error of the mean of n observations, where n represents the number of wells studied (12 wells from 2-3 independent experiments). Data series were examined by analysis of variance and individual group means were then compared with Student's t test for unpaired data. A p value less than 0.05 was considered significant.

ES 2 249 784 T3

Results

MnTBAP is not a nitric oxide antioxidant: MnTBAP did not inhibit relaxations in vascular rings in response to authentic NO. Furthermore, MnTBAP, at 300 pM, did not inhibit nitrite / nitrate accumulation in the culture medium in response to the NO donor SNAP compound and produced a slight inhibition in response to DNO. These data, and the finding that NO does not affect spectral changes in the Sorét band of MnTBAP, indicate that NO does not complex with manganese in MnTBAP.

MnTBAP inhibits peroxynitrite-mediated oxidation: Peroxynitrite produced a significant oxidation of hydrorhodamine 123 to rhodamine 123, which was inhibited on a dose-dependent basis by MnTBAP with 50% inhibition at 30 pM (Fig. 40). This suggests that MnTBAP, like cysteine, urate, ascorbate, and alpha-tocopherol, inhibit peroxynitrite oxidations.

MnTBAP inhibits the suppression of mitochondrial respiration by authentic peroxynitrite in J774 cells. Mitochondrial respiration was profoundly inhibited by exposure to 1 mM peroxynitrite in 1 h (Fig. 41A). This effect was prevented in part and as a function of dose by MnTBAP.

MnTBAP inhibits the suppression of mitochondrial respiration by NO donors in J774 cells: Mitochondrial respiration was also inhibited by exposure to the NO donor SNAP compound (Fig. 41B) and DNO for 24 h (Fig. 41C). This effect was prevented in part and as a function of dose by MnTBAP.

Effects of MnTBAP on NO suppression and suppression of mitochondrial respiration in immunostimulated J744 macrophages: Immunostimulation of cells by lipopolysaccharide (LPS; 10 pg / ml) alone, and more markedly, in the presence of interferon gamma ( IFN; 10 u / ml), produced the formation of nitrite / nitrate and a marked inhibition of mitochondrial respiration. Administration of IFN alone did not cause detectable nitrite / nitrate production and only produced mild suppression (&lt;15%) of respiration (n = 12).

MnTBAP produced a dose-dependent inhibition of nitrite / nitrate production in LPS-stimulated cells. However, in cells immunostimulated with the combination of LPS and IFN, MnTBAP produced a less marked inhibition of nitrite and nitrate accumulation (300 pM). For example, at 100 pM and at 300 pM, MnTBAP produced a significant 63 and 86% inhibition of nitrite / nitrate accumulation produced by LPS. When administered in the combined presence of LPS and IFN, MnTBAP 100 pM only produced 25% inhibition of nitrite / nitrate accumulation and a marked inhibition (61%) was observed only at the highest concentration of MnTBAP tested (300 pM ). L-NMA (N^G-methyl-L-arginine) produced an almost complete inhibition of NO production in cells stimulated with LPS or LPS and IFN; and MnTBAP had no additional effect on nitrite / nitrate formation in the presence of L-NMA. The inhibition of nitrite / nitrate accumulation by MnTBAP in macrophages stimulated by LPS decreased by more than 50% when the agent was applied 6h after LPS, while in the case of inhibition observed with L-NMA, the extension of the inhibition observed was similar when the compound was administered together with LPS or 6 h later (n = 6).

MnTBAP produced a restoration of the partial, dose-dependent immunostimulatory suppression of mitochondrial respiration in both LPS-treated cells (less potentially) and LPS- and IFN-treated cells. Inhibition of NOS with L-NMA resulted in restoration of respiration to an extent comparable to that of 300 pM MnTBAP. The combined administration of 300 pM MnTBAP and 3 mM L-NMA produced a further restoration of mitochondrial respiration. In the presence of L-NMA and MnTBAP, respiration was restored to baseline levels in LPS-stimulated cells, but remained below normal in LPS and IFN-stimulated cells.

Example XIV

Synthesis Reaction Schemes

Synthesis of 10303 (Fig. 42A)

1. Methyl 2-methoxy-3-methyl benzoate (1)

To a solution of 2-hydroxy-3-methylbenzoic acid (2.22 g, 14.6 mmol), K₂CO₃ finely ground anhydrous (8.06 g, 58.3 mmol) and magnetically stirred acetone (150 ml) was added (CH₃OR)₂SW₂ (2.9 ml, 30.6 mmol). The solution was stirred at room temperature for 18 hours, then heated under reflux until TLC analysis indicated that the reaction was complete (1 to 2 hours). The reaction mixture was cooled to room temperature, filtered, and the excess K2CO3 cake was washed thoroughly with acetone. The filtrate was evaporated and the residue was redissolved in EtOAc (100 to 125 ml) and triethylamine (~ 5 to 9 ml) was added. The reaction mixture was stirred at room temperature for 30 minutes, transferred to a separatory funnel, and washed successively with H₂O (100 mL), 2N HCl (until slightly acidic), H₂O (100 ml) and brine (100 ml), then filtered (Na₂SW₄) and evaporated under reduced pressure. Chromatography of the residue on silica gel (silica height: 15 cm, diameter: 3 cm, eluent: hexanes / Et₂Or 1: 1) provided 2.50 g (95%) of pure (1).

ES 2 249 784 T3

two. Methyl 2-methoxy-4- (a, a-dibromomethyl) benzoate (2)

A solution of 1 (24.43 g, 135.6 mmol), NBS (54.41 g, 305.7 mmol) and CCl₄ (1 L) magnetically stirred was exposed to a 100 watt lamp for 6 hours. TLC analysis indicated that the reaction mixture was composed of mono-, di-, and tribrominated benzoates, in which dibromide was the major product. The reaction mixture was quenched with H₂O (200 ml, then Na₂S₂OR₃ saturated (500 ml) to destroy Br₂. The mixture was transferred to a separatory funnel, stirred thoroughly, and then separated. The organic layer was dried (Na2SO4), filtered and evaporated under reduced pressure. The crude 'II NMR spectrum indicated that methyl-2-methoxy-4- (α, α-dibromomethyl) benzoate was the main product and was used without further purification.

3. Methyl 4-formyl-2-methoxy benzoate (3)

Crude product 2 was dissolved in acetone / H₂O (200 ml, 83:17) then AgNO was added₃ (47.25 g, 278.2 mmol). The flask was covered with aluminum foil to avoid decomposition of AgNO₃ by the light. The reaction mixture was stirred at room temperature for 2 to 3 hours, then the salts of AgBr from the solution were filtered. The filtrate was diluted with EtOAc (400 ml, transferred to a separatory funnel, then washed with saturated NaHCO3 (300 ml) to extract the acid from the aldehyde. The organic layer was washed successively with H2O (300 ml) and brine (300 ml), dried (Na2SO4), filtered and evaporated under reduced pressure. Chromatography of the residue on silica gel (silica height: 15 cm, diameter: 5 cm, eluent: hexanes / Et₂Or 1: 1) provided 11.92 g (42%) of pure aldehyde (3).

Four. Methyl 4-formyl-2-hydroxy benzoate (4)

To a solution of methyl 4-formyl-2-methoxy benzoate (3) (1.82 g, 9.37 mmol) in CH₂Cl₂ (35 ml) magnetically stirred at 0 ° C and in N₂ was added dropwise, BCl₃ 1M (18.7 ml, 18.7 mmol). The reaction mixture was stirred at 0 ° C for 10 to 30 minutes, then quenched with H₂Or (50 ml). Ether (100 ml) was added to the mixture, then it was transferred to a separatory funnel and decanted. The aqueous layer was extracted with ether (35 ml) the combined organic extracts were washed with brine (100 ml), dried (Na₂SW₄), filtered and evaporated under reduced pressure. Chromatography on silica gel (silica height: 12.5 cm, diameter: 5 cm, eluent: hexanes / Et₂Or 1: 1) provided 1.60 g (95%) of methyl 4-formyl-2-hydroxy benzoate as an oil.

5. 2,2 ', 2 ", 2"' - tetrahydroxy-4,4 ', 4 ", 4"' - (21H, 23H-porphine-5,10,15,20-tetrayl) tetrabenzoate tetramethyl (5 )

In a 1 L round-bottomed, 3-necked flask, covered with aluminum foil, fitted with a reflux condenser, a magnetic stirrer, and an inlet for N₂ Methyl 4-formyl-2-hydroxy benzoate (1.01 g, 5.6 mmol), pyrrole (397 // l, 5.6 mmol) and CH₂Cl₂ anhydrous (560 ml). The reaction mixture was stirred at room temperature for 10 to 30 minutes, then BF was added.₃OEt₂ (69 // 1, 0.56 mmol). The extent of the reaction was followed by uv-vis spectrophotometry. After 1.5 hours at room temperature, tetrachloro-1,4benzoquinone (1.03 g, 4.2 mmol) was added and the reaction mixture was refluxed for 3.5 hours. The reaction mixture was allowed to cool to room temperature, then the solvent was evaporated under reduced pressure. After 2 chromatographic purifications on silica gel, 0.38 g (30%) of (5) was obtained as a violet solid.

General procedure for the chromatographic purification of 5

On a 1 g aldehyde scale, the crude product was combined normally with 2 to 3 g of silica gel to prepare a mixture which was then divided into 2 batches for 2 separate chromatographic purifications on silica gel (silica height : 12 to 15 cm, diameter 5 cm). The first eluent used was hexanes / Et₂Either 1: 1 or 4: 1 hexanes / EtOAc (depending on how porphyrin behaves on TLC) to remove hydroquinone by-products and other non-polar impurities. After removing all non-polar impurities, the eluent was changed to CH₂Cl₂ or CHCl₃to elute porphyrin.

6. Trimethyl2,2 ', 2 ", 2"' -tetrahydroxy-4-benzoic-4 ', 4 ", 4"' (porphine-5,10,15,20-tetrayl) -manganzoic tribenzoate chloride (6)

A solution of 5 (0.38 g, 0.42 mmol) and MnCl2 (0.26 g, 2.1 mmol) in DMF (25 ml) was heated under reflux for 1.5 hours, then air was bubbled in for 1.5 hrs as the solution cooled to room temperature. DMF was evaporated under reduced pressure. The residue was then combined with silica gel (1 g) and CH₂Cl₂ (10 ml) to prepare a suspension. CH was evaporated₂Cl₂ leaving a solid mixture which was loaded dry onto a column packed with silica gel (column height: 12.5 cm, diameter: 5 cm, eluent 3% MeOH / CH₂Cl₂). The above purification gave 124 mg (32%) of pure 6 as a dark green solid. (melting point&gt; 300 ° C; UVVIS Tmax 467 nm (131,000); FAB-MS calculated for C₅iH₃₄MnN₄I heard₂ 949, found 949).

Synthesis of 10204 (Fig. 42B)

7. 4,4 ', 4 ", 4"' - (21H, 23H-porphine-5,10,15,20-tetrayl) tetrakis (benzonitrile) (7)

In a 2-L round-bottomed, 3-necked flask covered with aluminum foil, fitted with a reflux condenser, a magnetic stirrer and an inlet for N₂ 4-formyl benzonitrile (1.13 g, 8.6 mmol), pyrrole was added

ES 2 249 784 T3 (0.6 ml, 8.6 mmol) and CH₂Cl₂ (850 ml). The reaction mixture was stirred at room temperature for 15 minutes, then BF was added._rOEt₂ (105 //.l, 0.85 mmol). The extent of the reaction was followed by uv-vis spectrophotometry. After 2 hours, tetrachloro-1,4-benzoquinone (1.56 g, 6.34 mmol) was added and the reaction mixture heated under reflux for 2 hours. The reaction mixture was evaporated to a volume of 50 to 100 ml, then added to 2.8 g of silica gel. The remainder of the solvent was evaporated under reduced pressure to give a solid mixture for chromatographic purifications. After 3 separate purifications, 700 mg (46%) of pure 7 was obtained as a powdered violet solid.

8. 1, r, 1 ", 1" '- tetra- (1H-tetrazol-5-yl) -4,4', 4 ", 4 '' '- (21H, 23H-porphine-5,10,15,20 -tetrail) -tetrakisbenzene (8)

A solution of 7 (0.30 g, 0.42 mmol), NaN₃ (0.24 g, 3.69 mmol), NH₄Cl (0.18 g, 3.37 mmol) and DMF (25 ml) were heated at 120 ° C for 3 days. More NaN was added₃ (0.17 g, 2.59 mmol) and NH₄Cl (0.11 g, 2.05 mmol) portioned on day 2 to bring the reaction to completion. The DMF was evaporated under reduced pressure, then H₂Or cold (10 ml). The resulting solution was acidified with 6N HCl and then CH₂CL₂(10 ml). The resulting precipitate was collected and dried in vacuo to give 0.37 g (99%) of 8 as a greenish solid which was used without further purification.

9. 1,1 ', 1 ", 1"' - tetra- (1H-tetrazjol-5-yl) -4,4 ', 4 ", 4"' - (porphine-5,10,15,20-tetrayl chloride ) -tetrakisbenzene manganese (III) (9).

A solution of 8 (0.35 g, 0.4 mmol) and MnCl2 (0.25 g, 2 mmol) in DMF (20 ml) was heated under reflux for 4 to 5 hours. The reaction mixture was allowed to cool to room temperature, then the DMF was evaporated under reduced pressure. The crude product was adsorbed on 1.5 g of silica gel with 10 to 15 ml of CH₂Cl₂. The CH₂Cl₂ evaporated leaving a solid which was loaded dry onto a column packed with silica gel suspension (silica height:

12.5 cm, diameter: 5 cm, eluent: 6 CHCl₂/ 3 MeOH / 1 NH₄OH). Chromatographic purification gave 0.19 g (50%) of pure 9 as a dark green solid. (Melting point&gt; 320 ° C; 2_maxUV-VIS 469 nm (42,000); FAB-MS calculated for C₄₈H₂₈MnN₂₀ 939, found 939).

Synthesis of 10305 (Fig. 42C)

10. Methyl 3-methoxy-4-methyl benzoate (10)

To a solution of 3-hydroxy-4-methylbenzoic acid (15.1 g, 99.2 mmol), K₂CO₃ finely ground anhydrous (43.7 g, 317 mmol) and magnetically stirred acetone (250 ml) was added (MeO)₂ SW₂ (21 ml, 222 mmol). The reaction mixture was stirred at room temperature for 24 hours, then the K was filtered₂CO₃ in excess. Acetone was removed by evaporation and the residue was redissolved in EtOAc (250 ml), then Et₃N (15 ml). The solution was stirred at room temperature for 30 minutes, transferred to a separatory funnel and washed successively with H₂O (100 mL), 1N HCl (until slightly acidic), NaHCO₃ (100 ml), H₂O (100 ml) and brine (100 ml), dried (Na₂SW₄), filtered and evaporated under reduced pressure. Chromatography of the residue on silica gel (silica gel height: 25 cm, diameter: 4 cm, eluent: CH₂Cl₂/ hexanes 1: 1) provided 16.84 g (94%) of pure 10.

eleven. Methyl 4-formyl-3-methoxy benzoate (11)

To a magnetically stirred solution of 10 (5.87 g, 32.6 mmol) in CH₃CN / H₂Or 1: 1 (250 ml) CuSO was added consecutively₄oh₂O (8.13 g, 32.6 mmol) and K₂S₂OR₈ (26.4 g, 97.7 mmol). The reaction mixture was refluxed for 50 minutes, then transferred to a separatory funnel and extracted with EtOAc (2 x 100 mL). The combined organic extracts were dried over Na₂SW₄, filtered and evaporated. Purification on silica gel by gravity chromatography (silica gel height: 33 cm, diameter: 4 cm, eluent: 5: 1 hexanes / EtOAc, then 4: 1 hexanes / EtOAc) provided 1.47 g (23 %) of 11 pure.

12. 3,3 ', 3 ", 3"' - tetramethoxy-4,4 ', 4 ", 4"' - (21H, 23H-porphine-5,10,15,20-tetrayl) -tetrabenzoate tetramethyl (12)

In a 2-L round-bottomed, 3-necked flask covered with aluminum foil, fitted with a reflux condenser, a magnetic stirrer and an inlet for N₂ 4-formyl-3-methoxy benzoate (11) (1.23 g, 6.3 mmol), pyrrole (448 //.l, 6.3 mmol) and CH₂Cl₂ (630 ml). The reaction mixture was stirred for 15 minutes, then BF was added._rOEt₂ (Jul 78, 0.63 mmol). The reaction was monitored by uv-vis spectrophotometry. After 3 hours, tetrachloro-1,4-benzoquinone (1.17 g, 4.7 mmol) was added and the reaction mixture heated under reflux for

2.5 hours. The reaction mixture was allowed to cool to room temperature, then the solvent was evaporated in vacuo. The crude product was added on 2.6 g of silica gel with 10 to 15 ml of CH2Cl2. The CH2Cl2 was evaporated and the resulting mixture was divided into 3 equal parts for three independent chromatographic purifications. Each part was separately loaded dry onto a column packed with silica gel (silica gel height: 15 cm, diameter: 5 cm, eluent: hexanes / Et₂Or 1: 1 then CH₂Cl₂). 0.461 g of combined product (30%) of pure 12 were obtained as a mixture of rotational isomers.

ES 2 249 784 T3

13. 3,3 ', 3' ', 3 ”' - tetramethoxy-4,4 ', 4' ', 4' '' - (porphine-5,10,15,20-tetrayl) -tetrabenzoate tetramethyl manganese chloride ( III) (13)

A solution of 12 (0.32 g, 0.33 mmol) and MnCl was heated under reflux for 6.5 hours₂ (0.20 g, 1.56 mmol) in DMF (32 ml). Air was then bubbled into the solution for 1.25 hours as the reaction mixture cooled to room temperature. DMF was evaporated and the crude mixture was adsorbed on 1.5 g of silica gel with 10 to 15 ml of CH₂Cl₂. CH was evaporated₂Cl₂ and the resulting solid mixture was loaded dry onto a column packed with silica gel (silica height: 12.5 cm, diameter: 5 cm, eluent: 5% MeOH in CH2Cl2). Chromatographic purification provided 294 mg (84%) of pure 13 as a green-black solid. (melting point&gt; 300 ° C; 2_maxUV-VIS 467 number (145,000); FABS calculated for C₅₆H₄₄MnN₄OR₁₂ 1019, found 1019).

14. 3,3 ', 3 ", 3"' - tetramethoxy-4,4 ', 4 ", 4"' - (porphine-5,10,15,20-tetrayl) tetrakis (benzoic acid) (14)

A magnetically stirred solution of manganese tetrabenzoate 13 in a 10 ml Claisen box (KOH / MeOH / H₂O) was refluxed for 40 to 60 minutes. The reaction was cooled to room temperature and then to 0 ° C. The cold solution was then acidified with 6N HCl then the MeOH was evaporated under reduced pressure. The solids were filtered into the acidic solution and washed thoroughly with cold water. The solids were collected and dried under vacuum at 80 ° C overnight to provide 96 mg (87%) of pure 14 as a dark green solid (melting point&gt; 300 ° C; 2_maxUV-VIS 467 nm (150,000); FAB-MS calculated for C₅₂H₃₆MnN₄OR₁₂ 963, found 963).

Synthesis of 10109 (Fig. 42D)

fifteen. 4,4 ', 4 ", 4"' - (porphine-5,10,15,20-tetrayl) -tetrakis (benzoyl chloride) manganese (III) chloride (15)

MnTBAP (500 mg, 0.57 mmol) was suspended in anhydrous benzene (125 mL) in a 3 necked round bottom flask equipped with a reflux condenser and an N2 inlet. Thionyl chloride (45 ml, 617 mmol) was added to the mixture and the reaction mixture was refluxed for 22 hours. Benzene and SOCl₂ unreacted were removed by distillation. The residue was washed with anhydrous benzene (50 ml) and rotary evaporated to dryness. This washing and evaporation were repeated twice. The dark green residue was dried under high vacuum to give 0.54 g (100%).

16. 4,4 ', 4 ”, 4”' - (porphine-5,10,15,20-tetrayl) -tetrakis (benzamide) chloride of tetra (N, N-dimethyl) manganese (III) (16)

To compound 15 (340mg, 0.36mmol) in THF (20ml) was added dimethylamine in THF solution (10ml, 2M solution). The reaction mixture was refluxed for 6 hours. TLC analysis (eluent: 1 methanol: 3 chloroform) showed complete conversion of the acid chloride to the product. After cooling to room temperature, deionized water (100 ml) was added to the reaction mixture and the solid was filtered. The crude solid was purified by silica gel column chromatography (eluent methylene chloride: methanol = 4: 1). The combined fractions (Rf = 0.75 in CH₂Cl₂: MeOH = 4: 1) were evaporated and dried under high vacuum to provide the product as a green solid 0.11 g (31%). (two_maxUV-VIS 467 nm (56,100); FAB-MS calculated for C₅₆H₄₉MnN₈OR₄ 952, found 952).

Synthesis of 10402, 10602, 11001, 11002, 11003 and 11103

As long as it refers to compounds 10402, 10602, 11001, 11002, 11003 and 11103, the synthesis can be carried out essentially, as described in Example XIV, sections 5 and 6; The following starting material substitutions are required: 4-hydroxybenzaldehyde for methyl 4-formyl-2-hydroxy benzoate for 10402,4-hydroxy3-nitrobenzaldehyde for methyl 4-formyl-2-hydroxy-benzoate for 10602, indole-3-carboxaldehyde for 4-formyl Methyl -2-hydroxy benzoate in the case of 11001,4-quinolinecarboxaldehyde by methyl 4-formyl-2-hydroxy benzoate in the case of 11002, 3-quinolinecarboxaldehyde by methyl 4-formyl-2-hydroxy benzoate in the case of 11003 and 3-fluoro-4-methoxybenzaldehyde by methyl 4-formyl-2-hydroxy benzoate in the case of 11103.

Synthesis of 10207 and 10208

Acetonitrile is reacted with hydroxylamine to prepare the amide oxime. An excess of oxime of the amide 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.

Synthesis of 10209

Commercially available 4-methylbenzoic acid is converted to its acid chloride by reaction with oxalyl chloride or thionyl chloride. The resulting acid chloride is heated in the presence of cuprous cyanide to provide the α-ketonitrile derivative. The α-ketonitrile is hydrolyzed with anhydrous methanol saturated with anhydrous hydrogen chloride gas to give the α-ketonitrile ester derivative. The aromatic methyl group is effectively oxidized to the oxidation state of the aldehyde by the first reaction with N-bromosuccinimide and the subsequent hydrolysis with silver nitrate. The synthesis of 10209 can be carried out essentially as described in Example XIV, sections 5 and 6;

ES 2 249 784 T3 requiring the substitution of the starting material of methyl 4-formyl-2-hydroxy benzoate by the aldehyde described above.

A person skilled in the art will appreciate from reading this discussion that various changes in shape and detail can be made without departing from the true scope of the invention. In addition to the compounds described herein, the compounds set forth in the following references can also be used as oxidant antioxidants consistent with the invention: US Patent No. 5,227,405; Nagele et al., Biochem. Pharmacol. (UK) 47: 555-562 (1994); Baudry et al., Biochem. Biophys. Res. Comm. 192: 964-968 (1993); Duran et al., Cancer Lett (IRE) 69: 167-172 (1993); Itami et al., Biochem. Biophys. Res. Comm. 197: 536-541 (1993); Kitajima et al., Inorg. Chem. 32: 1879-1880 (1993); Riley et al., Free Radical Biol. &amp; Med. 15: 514 (1993); Weiss et al., J. Biol. Chem. (US) 268: 23049-23054 (1993); Foye, Ann. Pharmacother. 26: 1144-1147 (1992); Haseloff et al., J. Biolumin. Chemilumin. (UK) 7: 171-175 (1992); Pelletier, J., Biochem. Pharmacol. 43: 1061-1066 (1992); Yaping et al., J. Free Radic. BiolMed 13: 533-541 (1992); Schechinger et al., Biol. Met. 1: 112 (1988); Linss et al., Inorg. Chim. Acta 125: 117 (1986); and Weser et al., Adv. Exp. Med. Biol. 264: 51 (1990).

Footnotes to Table IX 'Porphine carbon numbers refer to the carbons (5, 10, 15, 20) of the methine bridge to which each R group is attached or to the carbons (2, 4, 7 , 8, 12, 13, 17 or 18) of the pyrrole, to which P. is attached. P is hydrogen unless otherwise defined.

² SOD activity determined as described by McCord and Fridovich, J.Biol. Chem. 244: 6049 (1969).

³Catalase activity determined as described by Del Rio et al., Anal. Biochem. 80: 409 (1977). Metalloporphyrins were tested at various concentrations in the presence of 1 mM hydrogen peroxide for catalase-like activity. A Clark electrode was used to measure the formation of oxygen in the decomposition of hydrogen peroxide over a period of 2 minutes.

⁴The growth rates of the SOD-null E. coli (strain JI132) cultures were followed by turbidimetry at 700 nm to minimize absorbance interferences from test compounds. The culture medium contained 0.2% glucose, 0.2% casamino acids, 30 mg / l of thiamine, 30 mg / l of pantothenic acid and M9 salts in water, the pH was adjusted to 7.0. Test compounds were subjected to ultrafiltration before adding to the medium (Faulkner et al., J. Biol. Chem. 269: 23471 (1994)).

⁵Human Umbilical Cord Vein Endothelial Cells (HUVEC) (ATCC # CRL-1730) (Moldow et al., Methods in Enzymology 105: 378-385 (1984)) were cultured in Ham's F-12K medium enriched with serum 10% fetal bovine, 0.1 mg heparin and 0.03 mg / ml endothelial cell growth factor in T-75 culture flasks in a humidified atmosphere containing 5% carbon dioxide and 95% air . Cells were plated at ~ 3.5 x 10⁴ cells / well in 24-well plates. The experiments were carried out 48 hours later. The medium was changed to EMEM without serum enrichment but including heparin and endothelial cell growth factor at the beginning of each study. The stock solutions of D-amino acid oxidase and metalloporphyrins were diluted in (EMEM) and passed through a 0.2 ml filter before use. HUVEC cells were labeled with Cr⁵¹ incubating them with EMEM containing radiolabeled sodium chromate for 4 h at an isotope concentration of 20 //.Ci/10^{5 6} cells. Cr⁵¹ not incorporated by repeated washes in EMEM. The trauma was initiated by incubating the endothelial cells for 5 hours with a hydrogen peroxide generation system consisting of D-alanine (1 mM) and 40 mU / ml of D-amino acid oxidase in EMEM. D-amino acid oxidase is found in PMNs (polymorphonuclear leukocytes) and is believed to be part of the oxidative unfolding that is a key part of the PMN-mediated inflammatory response (Robinson et al., J. Cell Biology, 77:59 (1978); Cline et al., Microbiology 62: 756 (1969); De Chatelet J. Reticul. Soc. 24:73 (1978)). The supernatant was collected from each well and at the end of the incubation period, the cells were washed with PBS and then lysed with 0.4N sodium hydroxide. The radioactivity in both supernatants and in the cell lysates was measured separately with a gamma counter. A series of wells on each plate were used to measure basal Cr release.⁵¹from HUVEC incubated in EMEM alone. The release of Cr⁵¹ was expressed as a percentage of radioactivity in the supernatant with respect to the total radioactivity [(Cr⁵¹ in the supernatant + Cr⁵¹ in lysate) x 100].

ES 2 249 784 T3

<img file="ES2249784T3_D0059.tif" />

ES 2 249 784 T3

<td> §</td><td>yes or -i *&gt; XZ) X</td><td> +</td><td> +</td><td> +</td><td> 1</td><td> 1</td><td> 1</td><td colspan="2"> 1</td><td colspan="2"></td><td></td>

<td> 5</td><td>isis //from ilo⁴</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>O = CO ^c</td><td> +</td><td> 1</td><td> +</td><td> 1</td><td> 1</td><td> 1</td><td> 1</td><td></td><td></td><td> □</td><td> 1</td>

<td></td><td>m, Q or Ul © CQ OR OT</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>co &lt;9</td><td></td><td></td><td></td><td></td><td></td><td></td><td>ro</td><td></td><td></td><td>ro</td><td></td>

<td></td><td>«&gt; V</td><td></td><td></td><td></td><td rowspan="2">CO CO</td><td></td><td></td><td> &gt;</td><td></td><td colspan="2"> &gt;</td><td></td>

<td></td><td>JS c</td><td>OO</td><td>in</td><td>σ&gt;</td><td>co</td><td>σ&gt;</td><td></td><td></td><td></td><td>or</td><td></td>

<td>&lt;9 or</td><td>S *</td><td>or</td><td></td><td>or'</td><td>or</td><td></td><td>V</td><td>ro _c</td><td></td><td></td><td>ro c</td><td>OR</td>

<td>AND</td><td>or</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td> *3</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>σ or</td><td>or or</td><td></td><td></td><td rowspan="2">ro &gt; or ro</td><td></td><td></td><td></td><td>ro &gt;</td><td></td><td></td><td></td><td>ro &gt;</td>

<td>m</td><td> 9 ^w 2 ³</td><td>co co</td><td>IT</td><td>co ν '</td><td>00 CM</td><td>h » xr</td><td>or ro</td><td></td><td colspan="2">or CM</td><td>g ro</td>

<td></td><td>^w ro</td><td></td><td></td><td>c</td><td></td><td></td><td></td><td>c</td><td></td><td></td><td></td><td>c</td>

<td></td><td>to.</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>a&gt;</td><td>or</td><td>or</td><td>or</td><td>or</td><td>OR</td><td>or</td><td>or</td><td></td><td></td><td>or</td><td>or</td>

<td> 1</td><td>O om o&gt; Ol 2</td><td>or c</td><td>or 'c</td><td>or 'c</td><td>or 'c</td><td>OR 'c</td><td>or 'c</td><td>or c</td><td></td><td></td><td>or c</td><td>or 'c</td>

<td colspan="2">W = ®</td><td>or 'c</td><td>;OR 'c</td><td>or c</td><td>'OR c</td><td>*or 'c</td><td>or c</td><td>•or 'c</td><td></td><td></td><td>or c</td><td>'OR 'c</td>

<td colspan="2">δ -</td><td>What</td><td>ro</td><td>ro</td><td>ro</td><td>ro</td><td>ro</td><td>ro</td><td></td><td></td><td>ro</td><td>ro</td>

<td colspan="2" rowspan="2">CO f?^K °</td><td>X or or</td><td>X</td><td>X (OR or</td><td>Γ T</td><td>X or F,</td><td>X r \</td><td>° c °</td><td>X OR OR</td><td>X or or</td><td>I r&gt;</td><td>m X or</td>

<td>or</td><td></td><td>W 1</td><td>T</td><td>¹ z °</td><td>* Ύ</td><td>xy</td><td>OR 1</td><td>or 1</td><td></td><td>or 1</td>

<td></td><td></td><td>&lt;D</td><td>Φ</td><td>Φ</td><td>Φ</td><td>φ</td><td>you</td><td>ω</td><td>Φ</td><td>CD</td><td>you</td><td>Φ</td>

<td></td><td>to.</td><td>OR</td><td>or</td><td>or</td><td>or</td><td>or</td><td>or</td><td>OR</td><td>OR</td><td>or</td><td>or</td><td>or</td>

<td></td><td>CM (V X rt</td><td>ro c</td><td>ro c</td><td>ro c</td><td>ro c</td><td>ro c</td><td>ro c</td><td>ro c</td><td>ro c</td><td>_ro c</td><td>ro c</td><td>ro c</td>

<td></td><td></td><td>LU</td><td>UJ</td><td>LU</td><td>IXI</td><td>LU</td><td>UJ</td><td>UJ</td><td>UJ</td><td>LU</td><td>UJ</td><td>UJ</td>

<td></td><td></td><td>I</td><td>or £</td><td>I</td><td>j</td><td>I</td><td>I</td><td> 1</td><td> 1</td><td> |</td><td>l</td><td>ο · ό</td>

<td></td><td></td><td></td><td> $</td><td></td><td></td><td></td><td></td><td>&gt; L</td><td rowspan="2">T</td><td rowspan="2">or</td><td rowspan="2">Φ</td><td>o-o</td>

<td></td><td>r- Q_ X “rt O, O,</td><td>Φ</td><td>or 9</td><td>V</td><td>or</td><td></td><td>T</td><td>or</td><td> 0</td>

<td colspan="2">or</td><td>or</td><td>OR</td><td>or</td><td>or</td><td>or</td><td>or</td><td></td><td></td><td></td><td></td><td>OR</td>

<td colspan="2">w. - n</td><td>CM</td><td>CM</td><td>CM</td><td>CM</td><td>CM</td><td>CM</td><td>or</td><td></td><td></td><td></td><td>CM</td>

<td colspan="2">o Λ c</td><td>go</td><td>go</td><td>go</td><td>go</td><td>go</td><td>go</td><td>CM</td><td rowspan="2">IO T * "</td><td>IO</td><td>or</td><td>go</td>

<td>α AND</td><td>® O .h⁷³ £ t</td><td>or'</td><td>or'</td><td>or</td><td>or</td><td>CD</td><td>τ- ο</td><td>or'</td><td>go</td><td>or</td><td>or</td>

<td> 2</td><td>•H.H (9</td><td>go</td><td>go</td><td>go</td><td>V " go</td><td>go</td><td>V— go</td><td>go</td><td></td><td></td><td></td><td>go</td>

<td></td><td>OR</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>om -</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td></td><td>® -2.§ s&gt; ® 8</td><td>__v £ o</td><td>^ CM</td><td>CO</td><td>CO</td><td>LQ CO</td><td>^ co co</td><td colspan="2">Γ-- oc</td><td colspan="2">oo or ^c</td><td>^ σ&gt; co</td>

<td></td><td>Comp (comp metal</td><td>CM</td><td>to</td><td>• 5 CM g, O</td><td>• s CM V</td><td>&lt;r CM ^, or</td><td>«5 CM</td><td colspan="2">CM «5 OR</td><td colspan="2">CM OR</td><td>«R CM * —- O</td>

ES 2 249 784 T3

<td rowspan="2">1 In vitro 1</td><td>&lt;n o yj xx X</td><td> •</td><td>I</td><td colspan="2"> +</td><td>t</td><td> +</td><td> 1</td><td>ND</td><td> 1</td><td> 1</td><td> +</td><td> 1</td>

<td>Null SOD E. Coli Bioassay⁴</td><td> +</td><td></td><td colspan="2"> +</td><td> 1</td><td> +</td><td> +</td><td>Q Z</td><td> 1</td><td> +</td><td> +</td><td>I</td>

<td rowspan="2">| Biochemistry</td><td>«*&gt; ro Io7 «C fifteen ^ANDor</td><td>IT OO or</td><td>cn cm</td><td colspan="2"> ▼—</td><td>Q. Q.</td><td>CM cm</td><td>cn cn</td><td>OR &lt;0 _c</td><td>ro &gt; ro c</td><td>IT or</td><td>or</td><td>cm</td>

<td>o "o ° 9 w OX «O, 3.</td><td>Xf OO</td><td>r ^ oo</td><td colspan="2">00 r-</td><td>* j Q. Q.</td><td>ro &gt; or ro c</td><td>or to</td><td>or co c</td><td>00 co CM</td><td> 00</td><td>r-</td><td>co cn</td>

<td colspan="2">Side group class</td><td>Φ c (OR ω or cr</td><td>φ c ro or&gt; cr</td><td colspan="2">Φ c ro φ Z3 cr</td><td>Φ C ro φ Z3 cr</td><td>φ «♦ -» c ro φ o cr</td><td>φ c ro Φ 2. 3 cr</td><td>Φ 4 ^ c ro TJ X or U— &lt;c ro</td><td>Φ C ro TJ X or c ro</td><td>two 'c</td><td>or L_ .'you 'c</td><td>or TJ 'or 4-" (0 3 to .c</td>

<td colspan="2">__ ~ r&gt; OO ΛΖ "to</td><td>X OR 1</td><td>ro X OR or 1</td><td>X or 1</td><td>X OR (</td><td>X or 1</td><td>on X or or 1</td><td>CO X or or 1</td><td>X or 1</td><td>X or 1</td><td>CN OR z 1</td><td>CN OR z 1</td><td>X 1</td>

<td colspan="2">__ CM rw * "to</td><td>Φ OR ro c LLJ</td><td>Φ or ro c LLI</td><td>Φ OR ro c LLI</td><td>Φ or ro c LLI</td><td>Φ OR ro c LU</td><td>Φ OR ro c LLI</td><td>Φ OR ro c LLI</td><td>Φ OR ro c LLI</td><td>Φ OR ro c LLI</td><td>Φ OR _TO c LLI</td><td>Φ OR ro c LLI</td><td>Φ OR ro c LU</td>

<td colspan="2"></td><td>X to or V</td><td>X to or</td><td>z</td><td>φτ-</td><td>or</td><td>X &lt;f V 4</td><td>JP or</td><td>Λ £, £ fr</td><td>Φ</td><td>£ or T</td><td>X or φ-</td><td>Φ</td>

<td colspan="2">Numbers) from porphyrin carbon (s)</td><td>or CM to or io '</td><td>or CM IT or IT</td><td>to</td><td>or CM to or</td><td>OR CM to or IT</td><td>or CM IT or IO</td><td>or CM to or v— LO ~</td><td>or CM IT or v- IT</td><td>or CM IT or IT</td><td>or CM io or it</td><td>or CM to or it</td><td>or CM to or' IT</td>

<td colspan="2">Compound (complex metallic)</td><td>Έ or • 5 oo</td><td>^ CM C 2</td><td colspan="2">00 S ^coo or T—</td><td>MC O oo έ °</td><td>^ WHAT CO DO</td><td>^ CO C 2 «5 OO</td><td> ?2 *&gt;</td><td>^ CM^C 2 5 6°</td><td>? δ 5 &lt;°</td><td>__CM c O ¿o</td><td>'c ¡s £ o T "</td>

ES 2 249 784 T3

<td rowspan="2">1 ln vitro I</td><td>CO O ω X</td><td> (</td><td> 1</td><td></td><td> +</td><td colspan="2">ND</td><td colspan="2">ND</td><td>Ι- ο. CL</td>

<td>Null SOD E. Coli Bioassay⁴</td><td> 1</td><td>ND</td><td>ND</td><td>ND</td><td colspan="2">ND</td><td colspan="2">ND</td><td>ND</td>

<td rowspan="2">| Biochemistry</td><td>W (Q &lt;/&gt; t- 2S c ra ·? w^ANDOR</td><td> 3,2</td><td> 0,9</td><td> 3,3</td><td> 1,02</td><td colspan="2">I- inactive</td><td colspan="2"> 4,6</td><td> 2,4</td>

<td>OR or 9 ω O Z&gt;^WS. 3.</td><td> 169</td><td> 404</td><td>22 I</td><td> 106</td><td colspan="2">inactive</td><td colspan="2"> 0,6</td><td>ND</td>

<td colspan="2">Side group class</td><td>cyano</td><td>hetero- cyclic</td><td>hetero- cyclic</td><td>hetero- cyclic</td><td colspan="2">halogenated</td><td colspan="2">halogenated</td><td>halogenated</td>

<td colspan="2">__ &lt;Or &lt;and »</td><td>two OR 1</td><td>^Xi</td><td></td><td></td><td>CO X or 1</td><td></td><td>&lt;*&gt; z: or 1</td><td></td><td>LU</td>

<td colspan="2">© Ί ry * "or</td><td>Link</td><td>Link</td><td>Link</td><td>Link</td><td>Link</td><td></td><td>Link</td><td></td><td>Link</td>

<td colspan="2"> 2.-2-</td><td>Φ</td><td>Link</td><td>Link</td><td>Link</td><td>Φ</td><td>-Br</td><td>Φ</td><td>-Br</td><td>to go i</td>

<td colspan="2">Numbers) from porphyrin carbon (s)</td><td> 5, 10, 15, 20</td><td> 5, 10, 15, 20</td><td> 5, 10, 15, 20</td><td> 5, 10, 15, 20</td><td> 5, 10, 15, 20</td><td> 2, 3, 7, 8, 12, 13, 17, 18</td><td> 5, 10, 15, 20</td><td> 2, 3, 7, 8, 12, 13, 17, 18</td><td> 5, 10, 15, 20</td>

<td colspan="2">Compound (complex metallic)</td><td>(Mn) 10901</td><td>(Mn) 11001</td><td>(Mn) 11002</td><td>(Mn) 11003</td><td colspan="2">11101 (Cu)</td><td colspan="2">11102 (Mn)</td><td>(Mn) 11103</td>