INTRODUCTION

Considerable experimental data exist to suggest that inflamed tissue and tissue subjected to ischemia-reperfusion are under oxidative stress induced by polymorphonuclear leukocytes (1, 2, 3, 4, 5). Upon activation, neutrophils produce superoxide by the NADPH oxidase-mediated univalent reduction of molecular oxygen (6, 7, 8, 9). Superoxide has been proposed to mediate neutrophil-induced tissue injury in vivo based upon observations that the superoxide dismutase (SOD)¹ enzymes are anti-inflammatory and attenuate ischemia-reperfusion injury (10, 11, 12). The SOD enzymes catalyze the dismutation of superoxide to generate hydrogen peroxide and molecular oxygen with catalytic rate constants as high as 2 × 10⁹ M¹ s¹ (13) (Reaction R1). Since the SOD enzymes are not membrane-permeable, the enzyme is limited in regard to its effectiveness as a probe for elucidating the role of intracellular superoxide in neutrophil-mediated tissue injury. Consequently, there has been considerable interest in the design and synthesis of membrane-permeable, low molecular weight mimetics of the enzyme SOD (14, 15, 16, 17, 18, 19), which could be utilized to probe the role of superoxide in neutrophil-mediated tissue injury as well as in other physiologic and pathologic processes.

With the use of novel Mn(II)-based SOD mimetics, Hardy et al. (20) demonstrated the role of superoxide in human neutrophil-mediated injury to human aortic endothelial cells in vitro. Of critical importance to this study was the accurate measurement of the ability of the SOD mimetics to catalyze the dismutation of superoxide, so that a protective effect could be correlated to the SOD activity of the mimetics. We (21) and others (22, 23, 24) have demonstrated that indirect methods to measure SOD activity of putative SOD mimics, such as the cytochrome c assay, can give false positive or negative results, primarily because these assays do not provide information as to the mechanism of action of the putative SOD mimetics. We have utilized the technique of stopped-flow kinetic analysis to directly monitor the decay of superoxide as a means to quantitate the ability of a putative SOD mimetic to catalyze the dismutation of superoxide (21, 25). By utilizing stopped flow, we have discovered that a Mn(II) dichloro complex of 1,4,7,10,13-pentaazacyclopentadecane (MnPAM) effectively catalyzes the dismutation of superoxide (26).

Herein, we report that the SOD mimetic MnPAM inhibits the tissue injury and the accumulation of neutrophils in an in vivo model of acute colonic inflammation. Structure-activity relationship studies with two novel, carbon-substituted Mn(II)-based SOD mimetics and two Mn(II)-based complexes with little or no detectable SOD activity indicate the importance of superoxide in the mediation of neutrophil infiltration in vivo.

EXPERIMENTAL PROCEDURES

Materials

Potassium superoxide and anhydrous Mn(II) chloride were purchased from Aldrich. Metal-free Hepes free acid and sodium salt were obtained from Calbiochem. Hexadecyltrimethylammonium bromide and o-dianisidine were purchased from Sigma. All other reagents were of the highest quality available.

Synthesis of Mn(II)-based Complexes

MnPAM and the Mn(II) dichloro complexes of 1,4,7,10,13-pentaazacyclohexadecane (MnHEX) and 1,4,7,11,14-pentaazacycloheptadecane (MnHEP) were prepared according to the procedure of Riley and Weiss (26). All reagents were used as received without further purification. Proton nuclear magnetic resonance (¹H NMR) spectra were obtained on a Varian VXR-300 or VXR-400 nuclear magnetic resonance spectrometer. Chemical shifts () are reported relative to tetramethylsilane ( = 0.0). Qualitative and quantitative fast atom bombardment mass spectroscopy was run on a Finnigan MAT90, a Finnigan 4500, and a VG40-250T using m-nitrobenzyl alcohol or m-nitrobenzyl alcohol/lithium chloride as a matrix. Elemental analyses were performed by Atlantic Microlabs (Norcross, GA). Melting points (m.p.) are uncorrected.

Synthesis of Mn(II)-Dichloro(2-methyl-1,4,7,10,13-pentaazacyclopentadecane) (MnMAM)

N,N-Bis(p-toluenesulfonyl)-1,2-diaminopropane

To a stirred solution of p-toluenesulfonyl chloride (270 g, 1.42 mol) and triethylamine (143 g, 1.42 mol) in methylene chloride (1.0 liter) at 0 °C under a dry argon atmosphere was added a solution of 1,2-diaminopropane (50.0 g, 0.675 mol) in methylene chloride (250 ml) over a period of 1 h, maintaining the temperature <10 °C. The mixture was allowed to warm to room temperature and was stirred an additional 19 h. The mixture was poured onto ice (1000 g), and the methylene chloride layer was separated. The methylene chloride layer was washed with 1 N HCl, water, and saturated sodium chloride solution and was dried (anhydrous magnesium sulfate). The solvent was removed in vacuo, and the resulting yellow oil was washed with hexane. The crude product was purified by recrystallization from methylene chloride-hexane to give 241 g (93% yield) of the product as colorless needles: m.p. 105-107 °C; ¹H NMR (chloroform-d)  0.97 (d, J = 6.7 Hz, 3 H), 2.40 (s, 3 H), 2.41 (s, 3 H), 2.91 (m, 2 H), 3.32 (m, 1 H), 5.25 (d, J = 7.6 Hz, 1 H), 5.38 (t, J = 6.3 Hz, 1 H), 7.28 (m, 4 H), 7.73 (m, 4 H).

To a stirred solution of p-toluenesulfonyl chloride (618 g, 3.24 mol) in pyridine (1.5 liters) at 0 °C was added a solution of 1,4,7-triazaheptane (95.5 g, 0.926 mol) in pyridine (150 ml) over a period of 30 min under a dry argon atmosphere, maintaining the temperature 50 °C. The mixture was stirred for 3 h at room temperature. Water (2 liters) was slowly added to the cooled (ice bath) mixture. The heavy white precipitate that formed was filtered and washed thoroughly with water. The pale solid was dissolved in N,N-dimethylformamide (3 liters), and 0.1 N HCl (4 liters) was slowly added at 5 °C. The slurry was filtered, and the pale yellow solid was washed thoroughly with water and dried in vacuo to give 486 g (93% yield) of the product: m.p. 180-181 °C; ¹H NMR (methyl sulfoxide-d₆)  2.39 (s, 3 H), 2.40 (s, 6 H), 2.84 (m, 4 H), 3.04 (t, J = 6.9 Hz, 4 H), 7.40 (d, J = 8.1 Hz, 4 H), 7.59 (d, J = 8.3 Hz, 2 H), 7.67 (m, 6 H).

To a mechanically stirred slurry of 1,4,7-tris(p-toluenesulfonyl)-1,4,7-triazaheptane (486 g, 0.859 mol) in anhydrous ethanol (1.15 liters) heated to reflux under a dry argon atmosphere was added a solution of sodium ethoxide (prepared by dissolving sodium metal (39.5 g, 1.72 mol) in absolute ethanol (1.0 liter)) as rapidly as possible. The clear brown solution that formed rapidly was allowed to cool to room temperature, and ethyl ether (1.0 liter) was added. The crystals were filtered under a dry argon blanket and washed with 3:1 ethanol/ethyl ether and ethyl ether. The crystals were then dried in vacuo to give 509 g (97% yield) of the product as a white powder: ¹H NMR (methyl sulfoxide-d₆)  2.30 (s, 6 H), 2.36 (s, 3 H), 2.63 (t, J = 8.7 Hz, 4 H), 2.89 (t, J = 7.2 Hz, 4 H), 7.11 (d, J = 8.1 Hz, 4 H), 7.28 (d, J = 8.0 Hz, 2 H), 7.46 (m, 6 H).

To a stirred solution of 1,4,7-tris(p-toluenesulfonyl)-1,4,7-triazaheptane-1,7-disodium salt (120 g, 0.197 mol) in anhydrous N,N-dimethylformamide (1.0 liter) was added ethylene carbonate (173 g, 1.97 mol). The resulting mixture was stirred at 60 °C for 24 h. Upon cooling the mixture to room temperature, water (100 ml) was added to quench the reaction, and the solvent was removed in vacuo. The resulting oil was dissolved in chloroform, washed with water and saturated sodium chloride solution, and was then dried (anhydrous magnesium sulfate). The solution was decolorized with activated charcoal, and the solvent was removed in vacuo. The residue was purified by recrystallization from methyl alcohol/water and dried in vacuo to give 124 g (96% yield) of the product as colorless needles: m.p. 110-112 °C; ¹H NMR (chloroform-d)  2.43 (s, 6 H), 2.44 (s, 3 H), 3.24 (t, J = 5.1 Hz, 4 H), 3.39 (m, 8 H), 3.79 (m, 4 H), 7.33 (m, 6 H), 7.72 (m, 6 H).

To a stirred solution of p-toluenesulfonyl chloride (79.6 g, 0.418 mol) and triethylamine (42.3 g, 0.418 mol) in methylene chloride (300 ml) at 0 °C under a dry argon atmosphere was added a solution of 3,6,9-tris(p-toluenesulfonyl)-3,6,9-triazaundecane-1,11-diol (124 g, 0.190 mol) in methylene chloride (300 ml) over a period of 30 min, maintaining the temperature <10 °C. The mixture was allowed to warm to room temperature and was stirred an additional 20 h. The mixture was then poured onto ice (1000 g), and the methylene chloride layer was separated. The methylene chloride layer was washed with 1 N HCl, water, and saturated sodium chloride solution and was dried (anhydrous magnesium sulfate). The solution was decolorized with activated charcoal, and the solvent was removed in vacuo. The resulting oil was washed with hexane. The residue was purified by recrystallization from methylene chloride-hexane to give 143 g (78% yield) of the product as needles: m.p. 158-160 °C; ¹H NMR (chloroform-d)  2.42 (s, 6 H), 2.43 (s, 6 H), 2.46 (s, 3 H), 3.29 (m, 8 H), 3.40 (t, J = 5.3 Hz, 4 H), 4.15 (t, J = 5.5 Hz, 4 H), 7.35 (m, 10 H), 7.74 (m, 10 H).

To a stirred solution of N,N-bis(p-toluenesulfonyl)-1,2-diaminopropane (19.1 g, 50.0 mmol) in anhydrous N,N-dimethylformamide (500 ml) was added sodium hydride (3.00 g; 80% in mineral oil, 0.100 mol) in portions under a dry nitrogen blanket. The resulting mixture was stirred for 30 min under a dry argon atmosphere. The solution was then heated to 100 °C, and a solution of 3,6,9-tris(p-toluenesulfonyl)-3,6,9-triazaundecane-1,11-di-p-toluenesulfonate (48.1 g, 50.0 mmol) in anhydrous N,N-dimethylformamide (250 ml) was added dropwise over a 3-h period, maintaining the temperature at 100 °C. After stirring the solution an additional 1 h at 100 °C, the mixture was concentrated in vacuo to a volume of 300 ml. Water (1.5 liters) was slowly added at room temperature to crystallize the product. The solid was filtered, washed thoroughly with water, and dried in vacuo. The solid was purified by recrystallization from chloroform/methyl alcohol to give 20.6 g (41% yield) of the product as needles: m.p. 255-260 °C; ¹H NMR (chloroform-d)  0.95 (d, J = 6.8 Hz, 3 H), 2.43 (s, 3 H), 2.44 (s, 9 H), 2.46 (s, 3 H), 3.30 (m, 18 H), 4.05 (m, 1 H), 7.33 (m, 10 H), 7.68 (m, 10 H).

A mixture of 2-methyl-1,4,7,10,13-pentakis(p-toluenesulfonyl)-1,4,7,10,13-pentaazacyclopentadecane (20.6 g, 20.6 mmol) and concentrated sulfuric acid (75 ml) was heated at 100 °C with stirring under a dry argon atmosphere for 70 h. To the resulting brown solution, ethanol (100 ml) was added dropwise with stirring at 0 °C, followed by ethyl ether (1 liter). The precipitate was filtered and washed thoroughly with ethyl ether. The solid was then dissolved in water (100 ml), and the resulting solution was washed with ethyl ether. The pH of the solution was adjusted to 10-11 with 10 N NaOH, and the solvent was removed in vacuo. Ethanol (2 × 500 ml) was then added and removed in vacuo. The residue was extracted with hot anhydrous tetrahydrofuran (2 × 500 ml) and filtered. The filtrates were combined, and the solvent was removed in vacuo. The residue was dissolved again in tetrahydrofuran (100 ml) and filtered to remove insoluble impurities. The solvent was removed in vacuo to give a crude solid. The solid was recrystallized from acetonitrile to give 1.30 g (28% yield) of the product as a white crystalline solid: m.p. 73-75 °C; ¹H NMR (chloroform-d)  1.02 (d, J = 6.2 Hz, 3 H), 1.71 (br s, 5 H), 2.36 (m, 1 H), 2.72 (m, 18 H); Anal. Calcd. for C₁₁H₂₇N₅: C, 57.60; H, 11.87; N, 30.53. Found: C, 57.68; H, 11.34; N, 30.48.

A solution of 2-methyl-1,4,7,10,13-pentaazacyclopentadecane (1.2 g, 5.3 mmol) and anhydrous Mn(II) chloride (0.67 g, 5.3 mmol) in anhydrous methanol (60 ml) was refluxed under a dry nitrogen atmosphere for 2 h. A small amount of suspended solid was removed by filtration, and the solvent was removed in vacuo. The residue was recrystallized from ethanol/ethyl ether to give 0.81 g (43% yield) of the product as a white solid: fast atom bombardment mass spectrum (m-nitrobenzyl alcohol), m/z (relative intensity) 354 (M⁺, 3), 319/321 ((M  Cl)⁺, 100/31); Anal. Calcd. for C₁₁H₂₇Cl₂MnN₅: C, 37.19; H, 7.66; Cl, 19.96; N, 19.72. Found: C, 36.73; H, 7.32; Cl, 19.85; N, 19.49.

Synthesis of Mn(II)-Dichloro(2-(2-methylpropyl)-1,4,7,10,13-pentaazacyclopentadecane) (MnBAM)

To a stirred slurry of DL-leucine amide hydrochloride (50.0 g, 0.300 mol) in anhydrous tetrahydrofuran (500 ml) at room temperature under a dry argon atmosphere was added a solution of lithium aluminum hydride (1200 ml; 1.0 M in tetrahydrofuran, 1.20 mol) over a 15-min period. The mixture was refluxed for 8 h and then quenched by the dropwise addition of water (200 ml) while cooling in an ice bath. The mixture was filtered to remove solid, and the filtrate was retained. The solid was washed with tetrahydrofuran (100 ml) and slurried in hot tetrahydrofuran (2 × 1 liter). The tetrahydrofuran filtrates and washings were combined, and the solid was discarded. The tetrahydrofuran solution was acidified to pH 1 with concentrated HCl. The solvent was removed in vacuo to give a pale yellow oil. The oil was dissolved in ethanol, and the solvent was removed in vacuo to give an oil that crystallized. The crude material was purified by recrystallization from methyl alcohol/ethyl ether to give 42.3 g (75% yield) of the product as a white crystalline solid: m.p. 228-230 °C; ¹H NMR (methyl sulfoxide-d₆)  0.89 (d, J = 6.5 Hz, 3 H), 0.90 (d, J = 6.5 Hz, 3 H), 1.51 (t, J = 7.2 Hz, 2 H), 1.75 (septet, J = 6.7 Hz, 1 H), 3.08 (m, 2 H), 3.49 (m, 1 H).

N,N-Bis(p-toluenesulfonyl)-1,2-diamino-4-methylpentane

To a stirred solution of 5 N NaOH (100 ml) was added 1,2-diamino-4-methylpentane dihydrochloride (42.3 g, 0.223 mol). The resulting solution was saturated with sodium chloride, and the amine was extracted with methylene chloride (5 × 200 ml). The extracts were combined, dried (anhydrous magnesium sulfate), and reduced in vacuo to a volume of 200 ml. This solution was then added dropwise over a period of 1 h to a stirred solution of p-toluenesulfonyl chloride (89.4 g, 0.469 mol) and triethylamine (47.5 g, 0.469 mol) in methylene chloride (400 ml) at 0 °C. The mixture was allowed to warm to room temperature and was stirred an additional 19 h. The mixture was then poured onto ice (1000 g), and the methylene chloride layer was separated. The methylene chloride layer was washed with 1 N HCl, water, and saturated NaCl solution and was dried (anhydrous magnesium sulfate). The solvent was removed in vacuo, and the resulting yellow oil was washed with hexane. The crude product was purified by recrystallization from methylene chloride-hexane to give 67.9 g (72% yield) of the product as a white crystalline solid: m.p. 138-140 °C; ¹H NMR (chloroform-d)  0.53 (d, J = 6.5 Hz, 3 H), 0.68 (d, J = 6.5 Hz, 3 H), 1.21 (m, 2 H), 1.34 (septet, J = 6.6 Hz, 1 H), 2.43 (s, 6 H), 2.87 (m, 1 H), 3.02 (m, 1 H), 3.18 (m, 1 H), 4.74 (d, J = 7.5 Hz, 1 H), 5.16 (t, J = 6.3 Hz, 1 H), 7.30 (d, J = 8.8 Hz, 4 H), 7.72 (m, 4 H).

To a stirred solution of N,N-bis(p-toluenesulfonyl)-1,2-diamino-4-methylpentane (21.2 g, 50.0 mmol) in anhydrous N,N-dimethylformamide (500 ml) was added sodium hydride (3.00 g; 80% in mineral oil, 0.100 mol) in portions under a dry nitrogen blanket. The resulting mixture was stirred for 30 min under a dry argon atmosphere. The solution was then heated to 100 °C, and a solution of 3,6,9-tris(p-toluenesulfonyl)-3,6,9-triazaundecane-1,11-di-p-toluenesulfonate (48.1 g, 50.0 mmol) in anhydrous N,N-dimethylformamide (250 ml) was added dropwise over a 3-h period, maintaining the temperature at 100 °C. After stirring the solution an additional 1 h at 100 °C, solvent was removed in vacuo to a volume of 300 ml. A 1:1 mixture of methyl alcohol/water (200 ml) and then water (1.4 liter) were slowly added at room temperature to crystallize the product. The solid was filtered and washed thoroughly with water. The solid was then dissolved in chloroform and dried (anhydrous magnesium sulfate), and the solvent was removed in vacuo to give a solid. The crude product was purified by recrystallization from chloroform/methyl alcohol to give 14.2 g (27% yield) of the product as colorless needles: m.p. 142-145 °C; ¹H NMR (chloroform-d)  0.80 (d, J = 5.9 Hz, 3 H), 0.83 (br s, 3 H), 1.26 (br s, 1 H), 1.37 (br s, 2 H), 2.40 (s, 3 H), 2.44 (s, 3 H), 2.45 (s, 3 H), 2.46 (s, 6 H), 3.17 (br m, 18 H), 4.28 (br s, 1 H), 7.32 (m, 10 H), 7.71 (m, 10 H).

2-(2-Methylpropyl)-1,4,7,10,13-pentakis(p-toluenesulfonyl)-1,4,7,10,13-pentaazacyclopentadecane (14.1 g, 13.5 mmol) and concentrated sulfuric acid (50 ml) were heated at 100 °C with stirring under a dry argon atmosphere for 69 h. To the resulting brown solution, ethanol (70 ml) was added dropwise with stirring at 0 °C, followed by ethyl ether (1 liter). The brown solid was filtered and washed thoroughly with ethyl ether. The solid was then dissolved in water (100 ml) and washed with ethyl ether. The solution was filtered to remove insoluble impurities, and the pH was adjusted to 10-11 with 10 N NaOH. The solvent was removed in vacuo. Ethanol (2 × 250 ml) was added and removed in vacuo. The residue was extracted with hot anhydrous tetrahydrofuran (2 × 500 ml) and filtered at room temperature. The filtrates were combined, the solvent was removed in vacuo, and the residue was dissolved in tetrahydrofuran and filtered. The solvent was removed in vacuo to give the crude product as an oil. The oil was crystallized from cold (20 °C) acetonitrile to give 715 mg (20% yield) of the product as a white crystalline solid: m.p. 70-76 °C; ¹H NMR (chloroform-d)  0.89 (d, J = 6.5 Hz, 3 H), 0.90 (d, J = 6.6 Hz, 3 H), 1.13 (m, 1 H), 1.39 (m, 1 H), 1.64 (septet, J = 6.3 Hz, 1 H), 2.05 (br s, 5 H), 2.34 (m, 1 H), 2.75 (m, 18 H); exact mass (M + Li)⁺. Anal. Calcd.: 278.2896. Found: 278.2919 (C₁₄H₃₃N₅Li).

A solution of 2-(2-methylpropyl)-1,4,7,10,13-pentaazacyclopentadecane (0.66 g, 2.4 mmol) and anhydrous Mn(II) chloride (0.31 g, 2.4 mmol) in anhydrous methyl alcohol (40 ml) was refluxed under a dry nitrogen atmosphere overnight. The solution was filtered, and the solvent was removed in vacuo. The solid was recrystallized from tetrahydrofuran/ethyl ether to give 0.46 g (48% yield) of the product as an off-white solid: fast atom bombardment mass spectrum (m-nitrobenzyl alcohol) m/z (relative intensity) 396 (M⁺, 1), 361/363 ((M  Cl)⁺, 84/86), 272 ((M  MnCl₂ + H)⁺, 100). Anal. Calcd. for C₁₄H₃₃Cl₂MnN₅: C, 42.33; H, 8.37; N, 17.63. Found: C, 41.90; H, 8.22; N, 17.30.

Stopped-flow Kinetic Analysis

The direct technique of stopped-flow kinetic analysis for the measurement of SOD activity has been described in detail by Riley et. al (25). In a stopped-flow spectrometer obtained from Kinetic Instruments Inc. (Ann Arbor, MI), a Me₂SO solution of potassium superoxide (~2 mM) was mixed rapidly with a 60 mM Hepes buffer solution of a Mn(II)-based complex at pH 8.1 and 21 °C to give an initial concentration of superoxide of ~100 µM. The initial concentration of superoxide was always kept at least 10-fold higher than the initial concentration of the Mn(II)-based complex so that only the catalyzed dismutation of superoxide was measured. The decay of superoxide was monitored spectrophotometrically at 245 nm, the absorbance maximum of superoxide. Kinetic analysis of the decay of superoxide established whether the decay was first-order in superoxide (indicating that the Mn(II)-based complex was catalyzing the dismutation of superoxide) or second-order in superoxide (an uncatalyzed self-dismutation of superoxide). An observed rate constant (k_obs) was obtained for the catalyzed decay of superoxide at each concentration of Mn(II)-based complex as described previously (25). The catalytic rate constant (k_cat) was derived from the slope of the plot of k_obs versus the concentration of Mn(II)-based complex (25).

Experimental Animals

Male CD-1 mice (18-30 g) were obtained from Charles River Breeding (Wilmington, MA), acclimatized for 1 week, kept on light/dark cycles of 12 h, and given food and water ad libitum. Procedures were approved by the Searle Institutional Animal Care and Use Committee and conformed to NIH standards for the care and use of laboratory animals.

Dilute Aqueous Acetic Acid-induced Inflammation

The procedure of Krawisz et al. (27), as modified by Fretland et al. (28), was utilized to induce inflammation of the colon in mice. In brief, mice were lightly anesthetized with pentobarbital. A 0.25-ml solution of 4% (v/v) acetic acid in water was instilled intracolonically in the animals via a flexible polyethylene tube. After 24 h, the animals were killed by carbon monoxide anoxia, and the colons were extirpated and frozen at 70 °C until assayed. Mn(II)-based complexes were administered intracolonically (0.2 ml) in mice 30 min prior to dilute aqueous acetic acid instillation.

Myeloperoxidase Activity

Myeloperoxidase activity of isolated colonic tissue samples were measured according to the procedure of Krawisz et al. (27). In brief, colonic tissue samples were weighed, minced, homogenized in 0.5% hexadecyltrimethylammonium bromide in 50 mM phosphate buffer, pH 6.0, and then sonicated. The suspension was centrifuged at 40,000 rpm for 15 min. Myeloperoxidase activity of the supernatant was measured spectrophotometrically by the determination of the decomposition of hydrogen peroxide using o-dianisidine (27). Data are expressed as the mean absorbance at 460 nm ± S.E. at 20 min in units of myeloperoxidase activity per g, wet weight, of tissue. Appropriate vehicle controls were used throughout.

Histological Analysis of Tissue

Colonic tissue samples were assessed histologically following routine procedures for the fixation of tissues with Bouin's fixative, followed by mounting in paraffin, sectioning, and staining with hematoxylin and eosin. All samples were evaluated without prior identification of the experimental group. Five sections/specimen were examined. The tissue sections were selected with a stratified random sampling protocol independent of gross pathology and were thus equivalent from animal to animal.

Statistical Analysis

Statistical evaluation of the myeloperoxidase studies was by a two-sample t test after analysis of variance with significance ascribed when p < 0.05. Correlation data and ED₅₀ were generated by linear regression analysis, and evaluation of histological grading was by inverse mean rank analysis.

RESULTS

The purpose of this study was to utilize synthetic, low molecular weight SOD mimetics and structurally related complexes with little or no detectable SOD activity as molecular probes to evaluate the importance of superoxide in tissue injury and neutrophil infiltration in a model of inflammation in vivo. Herein, we report the use of the Mn(II)-based SOD mimetic MnPAM, two newly synthesized and characterized SOD mimics MnMAM and MnBAM (structures in Fig. 1A), and the Mn(II)-based complexes MnHEX and MnHEP (structures in Fig. 1B), which have little or no detectable SOD activity.

MnPAM and the novel Mn(II)-based complexes MnMAM and MnBAM were synthesized by the reaction of Mn(II) chloride with the appropriate macrocyclic pentaaza ligand in anhydrous methanol under a dry nitrogen atmosphere. The high spin d⁵ Mn(II)-based complexes were white crystalline solids and exhibited a reversible one-electron oxidation in methyl alcohol at an oxidation potential of about 0.7 V (26). Fast atom bombardment mass spectral analysis of the complexes showed that the predominant ion ([M  Cl)⁺, where M equals the molecular weight of the Mn(II)-based complex) arose as a result of the loss of one of the axial chlorides. The complexes were judged analytically pure by elemental analysis.

The SOD activity of the complexes MnPAM, MnMAM, MnBAM, MnHEX, and MnHEP were determined by directly monitoring the decay of superoxide by stopped-flow kinetic analysis in the presence or absence of the complex. In Fig. 2 are shown superimposed stopped-flow traces of the decay of 100 µM superoxide as assessed spectrophotometrically in the presence of 0.0-8.0 µM MnMAM in 60 mM Hepes buffer, pH 8.1, at 21 °C. In the absence of MnMAM, the kinetics of the decay of superoxide were second-order, corresponding to the uncatalyzed, self-dismutation of superoxide (25). With increasing amounts of MnMAM, the decay of superoxide became more rapid and fit a first-order decay at each concentration of MnMAM. These results indicate that MnMAM catalyzed the decomposition of superoxide, since the initial concentration of superoxide was over 10-fold higher than the concentration of MnMAM.

1

1

For each concentration of MnMAM, a first-order rate constant (k_obs) was derived from a plot of ln absorbance versus time (data not shown). The slope of the plot of k_obs versus the concentration of MnMAM (see Fig. 2, inset) gave a k_cat value for the dismutation of superoxide equal to 3.31 × 10⁷ M¹ s¹ at pH 8.1 and 21 °C.

The k_cat values for MnPAM and MnBAM were determined by stopped-flow kinetic analysis to be 2.24 × 10⁷ M¹ s¹ and 1.91 × 10⁷ M¹ s¹, respectively, at pH 8.1 and 21 °C. MnHEX, which has a 16-membered ring pentaaza macrocycle in contrast to the 15-membered ring macrocycle of MnMAM, had very low, but detectable, SOD activity by stopped-flow with a k_cat value of 1.0 × 10⁶ M¹ s¹ (26). MnHEP, which has a 17-membered ring pentaaza macrocycle, had no SOD activity detectable by stopped flow (26). Under these experimental conditions, the limit of detection to measure SOD activity by stopped-flow was a k_cat value of 5 × 10⁵ M¹ s¹, which is equivalent to the self-dismutation of superoxide (25).

The SOD mimic MnPAM was evaluated in vivo for anti-inflammatory activity in an aqueous acetic acid-induced inflammation model (27, 28, 29, 30). In this assay, a solution of 4% (v/v) acetic acid in water was instilled intracolonically in mice. After 24 h, the mice were killed, the colons were isolated, and the colonic tissue was fixed. Histological analysis of the colonic tissue (see Fig. 3B) indicated a large accumulation of neutrophils with massive epithelial destruction, deepithelialized crypts, loss of villar integrity, and bacterial colonization. Colonic tissue isolated from mice that were not treated with 4% (v/v) acetic acid in water showed minimal numbers of neutrophils and an epithelium with normal crypts architecture, intact epithelial cells, and no bacterial colonization (Fig. 3A).

In the mice treated with the SOD mimic, MnPAM was administered intracolonically at a dose of 30 mg/kg 30 min prior to administration of the 4% (v/v) acetic acid in water. After 24 h, the colonic tissue was obtained and fixed for histological analysis. As can be seen from Fig. 3C, the overall architecture of the crypts was preserved with minimal epithelial destruction, minimal neutrophil accumulation, and no apparent bacterial colonization.

The colonic tissue activity of myeloperoxidase, an enzymic marker for neutrophils (27), was utilized to assess the extent of neutrophil infiltration into the colonic tissue in the dilute aqueous acetic acid-induced inflammation assay. In colonic tissue isolated from untreated mice, low levels (0.017 ± 0.003 units/g, wet weight, of tissue) of myeloperoxidase activity were detected, presumably due to the presence of resident granulocytes. When mice were treated intracolonically with 4% (v/v) acetic acid in water and their colons were isolated 24 h after treatment, high levels (up to 0.2 units/g, wet weight, of tissue) of myeloperoxidase activity were detected in the colonic tissue. The histologic appearance of the colonic tissue appeared to correlate with the tissue myeloperoxidase activity and with blinded scoring for severity of inflammation (data not shown). The results are consistent with previous results obtained with this model of acute colonic inflammation (31).

The ability of the SOD mimic MnPAM to attenuate neutrophil infiltration in vivo as assessed by a decrease in colonic tissue myeloperoxidase activity was investigated. As shown in Fig. 4, intracolonic administration of MnPAM over a concentration range of 0-100 mg/kg 30 min prior to instillation of the dilute aqueous acetic acid resulted in a dose-dependent inhibition of colonic tissue myeloperoxidase activity. Doses of 3, 30, and 100 mg/kg MnPAM gave statistically significant reductions in tissue myeloperoxidase activity (Table I). From linear regression analysis of these data, a 50% attenuation of myeloperoxidase activity (i.e. the ED₅₀) by MnPAM would be obtained at a dose of 10 mg/kg.

Table I. Ability of Mn(II) complexes exhibiting or lacking SOD activity to attenuate neutrophil infiltration in mice treated intracolonically with dilute aqueous acetic acid Treatmenta Dose of Mn(II) complex Myeloperoxidase activityb (units/g · wet weight of tissue ± S.E.) kcatc mg/kg M1 s1 × 107 Noned 0 0.017  ± 0.003 4% (v/v) acetic acid in watere 0 0.17  ± 0.022*** MnPAM 1 0.15  ± 0.026 2.24 MnPAM 3 0.092  ± 0.025* MnPAM 30 0.052  ± 0.018* MnPAM 100 0.022  ± 0.007** 4% (v/v) acetic acid in waterf 0 0.22  ± 0.023*** MnMAM 30 0.18  ± 0.047* 3.31 MnBAM 30 0.12  ± 0.021* 1.91 MnHEX 30 0.23  ± 0.027 0.10 MnHEP 30 0.27  ± 0.046 g a  Mn(II) complexes were administered intracolonically to mice at the indicated dose 30 min prior to intracolonic instillation of 0.25 ml of 4% (v/v) acetic acid in water. b  The mice were killed 24 h after treatment with 4% (v/v) acetic acid in water, and the colons were removed. Myeloperoxidase activity was determined on the supernatant of detergent-solubilized colon tissue homogenates. *, p < 0.05 (relative to dilute aqueous acetic acid-treated mice); **, p < 0.01 (relative to dilute aqueous acetic acid-treated mice); ***, p < 0.01 (relative to untreated mice). c  Catalytic rate constants (kcat) for the dismutation of superoxide by the Mn(II) complexes were determined by stopped-flow kinetic analysis at pH 8.1 and 21 °C. d  Mice (n = 12) were untreated (no 4% (v/v) acetic acid in water or Mn(II) complex). e  Mice (n = 12) were treated with 0.25 ml of 4% (v/v) acetic acid in water. This group of animals were compared with mice (n = 6) pretreated with MnPAM (1, 3, 30, or 100 mg/kg) and then treated with 0.25 ml of 4% (v/v) acetic acid in water. f  Mice (n = 12) were treated with 0.25 ml of 4% (v/v) acetic acid in water. This group of animals was compared with mice (n = 6) pretreated with MnMAM, MnBAM, MnHEX, or MnHEP and then treated with 0.25 ml of 4% (v/v) acetic acid in water. g  No detectable SOD activity for this Mn(II) complex was observed (kcat  5 × 105 M1 s1).

To demonstrate that the decrease in tissue myeloperoxidase activity was due to a reduction of neutrophil infiltration into the colonic tissue and not to a direct interaction of MnPAM with the enzyme myeloperoxidase, the ability of MnPAM to inhibit colonic tissue myeloperoxidase activity in vitro was assessed. Colonic tissue was isolated from dilute aqueous acetic acid-treated mice that were not pretreated with MnPAM. The myeloperoxidase activity of the colonic tissue was determined in the presence of 0-1000 µM MnPAM (exogenously added). MnPAM, even at the highest concentration added (1000 µM), did not directly inhibit myeloperoxidase activity in isolated colonic tissue homogenates (data not shown). These results indicate that the MnPAM-dependent reduction of myeloperoxidase activity in colonic tissue isolated from mice treated with dilute aqueous acetic acid is consistent with the inhibition of neutrophil accumulation.

To evaluate whether MnPAM inhibited neutrophil accumulation because of the complex's SOD activity, we assessed the ability of two additional SOD mimetics, MnMAM and MnBAM, and two structurally related Mn(II)-based complexes, MnHEX and MnHEP, which have little or no detectable SOD activity, to attenuate neutrophil infiltration into colonic tissue in dilute aqueous acetic acid-treated mice. The Mn(II)-based complexes were administered intracolonically to mice at a dose of 30 mg/kg 30 min prior to the administration of the 4% (v/v) acetic acid in water. As can be seen from Table I, the Mn(II)-based complexes MnMAM and MnBAM, which have significant SOD activity (k_cat > 1 × 10⁷ M¹ s¹), decreased to a statistically significant extent the myeloperoxidase activity of the isolated colonic tissue. However, the Mn(II) complexes MnHEX and MnHEP, which have little or no detectable SOD activity (k_cat  0.1 × 10⁷ M¹ s¹), did not decrease the myeloperoxidase activity of the colonic tissue homogenates. As with MnPAM, the SOD mimics MnMAM and MnBAM preserved the overall architecture of the crypts and prevented epithelial destruction and bacterial colonization, whereas the Mn(II) complexes MnHEX and MnHEP did not protect the intracolonic epithelium (data not shown). These results are consistent with superoxide acting as a mediator of neutrophil infiltration.

DISCUSSION

The production of reactive oxygen radicals by neutrophils has been implicated as a mechanism of tissue injury in inflammation and ischemia-reperfusion injury (1, 2, 3, 4, 5). A comparison of the anti-inflammatory properties of SOD enzymes demonstrates that the protective effects are quite variable and dependent upon the source of the enzyme (32, 33, 34). Similarly, there are reports that indicate that the SOD enzymes can attenuate ischemia-reperfusion injury (35, 36, 37, 38), whereas other reports have cast doubt on the effectiveness of the enzymes, either by showing a lack of a protective effect (39, 40) or by the observation of a bell-shaped dose response curve (41, 42).

To circumvent the problems associated with the use of SOD enzymes, we have designed and synthesized low molecular weight Mn(II)-based SOD mimetics (26). Other groups have reported the discovery and use of putative SOD mimetics as probes to evaluate superoxide-mediated injury, including manganese complexes of desferal (15, 43), the iron complexes FeTPEN and FeTPAA (17), and the organic nitroxides TEMPO and TEMPOL (44). However, the SOD activities of these putative SOD mimetics were assessed using indirect methodology, such as the cytochrome c assay, which does not provide information on the mechanism by which the compounds were active in the assay (22, 23, 24). By stopped-flow kinetic analysis, a technique that directly measures the ability of a compound to catalyze the dismutation of superoxide (25), we have demonstrated that the manganese complexes of desferal, FeTPEN, TEMPO, and TEMPOL do not effectively catalyze the dismutation of superoxide (k_cat < 0.1 × 10⁷ M¹ s¹) (21).

In this investigation, we report the synthesis and characterization of the novel carbon-substituted Mn(II)-based SOD mimetics MnMAM and MnBAM. The k_cat values for the dismutation of superoxide by the 2-methyl-substituted MnMAM and the 2-(2-methylpropyl)-substituted MnBAM, as determined by stopped-flow kinetic analysis at pH 8.1 and 21 °C, were determined to be 3.31 × 10⁷ M¹ s¹ and 1.91 × 10⁷ M¹ s¹, respectively, and are quite comparable with that of the unsubstituted MnPAM, which has a k_cat value of 2.24 × 10⁷ M¹ s¹. These results indicate that Mn(II)-based complexes can be prepared with substituents on the carbon backbone of the pentaaza macrocycle which retain SOD activity, whereas N-substitution, such as N-methylation, results in a Mn(II)-based complex with no detectable SOD activity (26). The SOD mimetics MnPAM, MnMAM, and MnBAM contain a 15-membered ring pentaaza macrocycle. Mn(II)-based complexes with larger ring sizes, including the 16-membered ring MnHEX and the 17-membered ring MnHEP, have little or no detectable SOD activity (k_cat  0.1 × 10⁷ M¹ s¹) (26).

We have utilized the Mn(II)-based SOD mimics MnPAM, MnMAM, and MnBAM and the structurally related Mn(II)-based complexes MnHEX and MnHEP, which have little or no detectable SOD activity, as probes to assess the role of superoxide as a mediator of inflammation, specifically in regard to tissue injury and the infiltration of neutrophils. All three of the SOD mimetics attenuated the tissue injury (massive epithelial destruction, deepithelialization of the crypts and villi, and bacterial colonization) and neutrophil infiltration (assessed histologically and by the enzymic marker myeloperoxidase) in the colons of mice that were treated with the proinflammatory agent dilute aqueous acetic acid; however, the two complexes MnHEX and MnHEP with little or no detectable SOD activity failed to protect against the tissue injury or to inhibit the neutrophil accumulation. The results are consistent with a role for superoxide in mediating neutrophil infiltration and tissue injury as a result of inflammation.

Of particular interest was the observation that the SOD mimetic MnPAM inhibited neutrophil infiltration in a dose-dependent fashion. These results are in contrast to the bell-shaped dose-response curves observed for the SOD enzymes (34, 41, 42). We attribute the absence of a bell-shaped dose-response curve with the SOD mimetic MnPAM to be due to the compound's inability to react with hydrogen peroxide and generate proinflammatory hydroxyl radicals via Fenton chemistry (45). Therefore, the synthetic, low molecular weight Mn(II)-based superoxide dismutase mimetics may prove useful as therapeutic agents for the treatment of inflammatory conditions that are mediated, or exacerbated, by superoxide.

Superoxide has been proposed to be a phagocyte-produced mediator of inflammation (46, 47). In a previous study, we have shown that the SOD mimic MnPAM protects against human neutrophil-mediated injury to human aortic endothelial cells in vitro without inhibiting the respiratory burst of the neutrophils (20). Endothelial cells can also be a source of superoxide in addition to being a target of oxidative damage (48). In this study, we have discovered that the SOD mimetics may also provide protection against neutrophil-mediated tissue injury in vivo by inhibiting the infiltration of neutrophils. However, the mechanism of action by which superoxide may be involved in the infiltration of neutrophils remains to be established and is currently undergoing active investigation in our laboratory.