m-40403 and 3-nitrotyrosine

M40403, [manganese(II)dichloro[(4R,9R,14R,19R)-3,10,13,20,26 pentaazatetracyclo[20.3.1.0.(4,9)0(14,19)]hexacosa-1(26),-22(23),24-triene]], is a low-molecular-weight, synthetic, manganese-containing superoxide dismutase mimetic that removes superoxide anions without interfering with other reactive species known to be involved in inflammatory responses (e.g., nitric oxide, NO and peroxynitrite, ONOO-). As such, M40403 represents an important pharmacological tool to dissect the roles of superoxide anion in acute and chronic inflammation. For this purpose, the pharmacological profile of M40403 was evaluated in a rat model of periodontitis. Periodontitis was induced in rats by placing a 2/0 braided silk around the lower left first molar. On day 8 the gingivomucosal tissue encircling the first molar was removed for biochemical and histological analysis. Ligation significantly increased inducible nitric oxide synthase activity and expression, and gingival tissue revealed increased neutrophil infiltration, lipid peroxidation and positive staining for nitrotyrosine formation and poly (ADP-ribose) polymerase activation. Ligation significantly increased Evans blue extravasation in gingivomucosal tissue and alveolar bone destruction. Intraperitoneal injection of M40403 (10 mg/kg daily for 8 days) significantly decreased all of the above-described markers of inflammation. This suggests compounds that inhibit the generation of superoxide anion, such as M40403 may be potentially useful for the treatment of periodontitis.

Topics: Alveolar Bone Loss; Animals; Capillary Permeability; Ligation; Lipid Peroxidation; Male; Manganese; Neutrophil Infiltration; Organometallic Compounds; Periodontitis; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Tyrosine

Superoxide is increased in the vessel wall of spontaneously hypertensive rats (SHR) where, if "blocked," potentiates endothelium-dependent vasodilation. The purpose of this study was to determine the role of superoxide anion in hypertension and its interaction with nitric oxide (NO). For this purpose we used a low molecular weight synthetic superoxide dismutase mimetic (M40403), known to remove selectively superoxide anion. Baseline mean arterial pressure (MAP) was significantly elevated in the SHR compared with its normal counterpart, Wistar Kyoto (WKY). M40403 at a dose (2 mg x kg(-1) x h(-1)), which had no effect in the WKY, significantly decreased MAP in SHR rats. To determine whether superoxide anion increases MAP by inactivating NO, NO synthesis was blocked with N(G) nitro-arginine methyl ester (L-NAME, 3 mg/kg i.v.), a nonselective nitric oxide synthase inhibitor. L-NAME (3 mg/kg, i.v) blocked the anti-hypertensive effect of M40403 (2 mg/kg over 30 min). When used at a dose that yielded similar increases in MAP, norepinephrine (2.1 microg/kg) failed to alter the anti-hypertensive effects of M40403 in the SHR. To investigate whether the anti-hypertensive effect of M40403 was associated with an improvement of the alterations in vascular reactivity, a separate group of experiments was carried out ex vivo. Endothelium-dependent vasorelaxation to acetylcholine (10 nM-10 microM), an index of endothelial function, was reduced in aortic rings taken from SHR rats when compared with WKY rats. In vivo treatment with M40403 caused an improvement of the degree of the endothelial dysfunction in SHR rats. Furthermore, immunohistochemical analysis for nitrotyrosine (the product formed from the interaction of nitric oxide with superoxide) revealed a positive staining in aorta from SHR rats. The degree of staining for nitrotyrosine was markedly reduced in tissue sections obtained from SHR rats treated with M40403. Our data suggest that overt production of superoxide in SHR couples with nitric oxide, reducing its function and leading to a loss of blood vessel tone and hypertension. Another important effect appears to be at the level of endothelial cellular integrity, where by interacting with nitric oxide, superoxide anion forms peroxynitrite and subsequent endothelial cell dysfunction. By removing superoxide, M40403 restores blood pressure to near-to-normal values.

Topics: Animals; Aorta; Blood Pressure; Culture Techniques; Hypertension; Kinetics; Manganese; Organometallic Compounds; Rats; Rats, Inbred SHR; Rats, Inbred WKY; Superoxides; Tyrosine; Vasodilation

M40403 is a low-molecular-weight, synthetic manganese-containing biscyclohexylpyridine superoxide dismutase mimetic (SODm) that removes superoxide anions (O(2)(-)) without interfering with other reactive species known to be involved in cardiovascular alterations (e.g. nitric oxide [NO] and peroxynitrite [ONOO(-)]). As such, M40403 represents an important pharmacological tool to dissect the roles of O(2)(-) in functional and biochemical cardiovascular alterations induced by perfusion of high glucose concentrations into the heart. Perfusion of a high glucose concentration of glucose into the heart elicited important cardiovascular alterations characterized by QT interval prolongation, increase in coronary perfusion pressure (CPP), lipid peroxidation, decrease in MnSOD activity and DNA damage. All parameters of cardiovascular alteration were attenuated by M40403 (1-10 mg/l). Furthermore, perfusion of a high of glucose concentration induced a significant formation of nitrotyrosine as well as an activation of poly(adenosine diphosphate [ADP]-ribose) synthetase (PARS), as determined by immunohistochemical analysis of heart tissue. The extent of staining for nitrotyrosine and PARS was reduced by M40403. These results clearly indicate that O(2)(-) plays a critical role in the development of the functional and biochemical cardiovascular alterations induced by perfusion of a high concentration of glucose into the heart. Therefore, synthetic enzymes of SOD, such as M40403, offer a novel therapeutic approach for the management of various cardiovascular diseases where these radicals have been postulated to play a role.

Topics: Animals; Antioxidants; Cells, Cultured; DNA Damage; Endothelial Cells; Endothelium, Vascular; Glucose; Heart; Humans; In Vitro Techniques; Lipid Peroxidation; Male; Malondialdehyde; Manganese; Mitochondria; Myocardial Reperfusion Injury; Myocardium; Organometallic Compounds; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Superoxides; Tyrosine

1. Myocardial injury caused by ischaemia and reperfusion comes from multiple pathogenic events, including endothelial damage, neutrophil extravasation into tissue, mast cell activation, and peroxidation of cell membrane lipids. These events are followed by myocardial cell alterations resulting eventually in cell necrosis. An enhanced formation of reactive oxygen species is widely accepted as a stimulus for tissue destruction and cardiac failure. 2. In this study, we have investigated the cardioprotective effects of M40403 in myocardial ischaemia-reperfusion injury. M40403 is a low molecular weight, synthetic manganese containing superoxide dismutase mimetic (SODm) that selectively removes superoxide anion. Ischaemia was induced in rat hearts in vivo by ligating the left anterior descending coronary artery. Thirty minutes after the induction of ischaemia, the ligature was removed and reperfusion allowed to occur for at least 60 min. M40403 (0.1-1 mg kg(-1)) was given intravenously 15 min before ischaemia. 3. The results obtained in this study showed that M40403 significantly reduced the extent of myocardial damage, mast cell degranulation and the incidence of ventricular arrhythmias. Furthermore, M40403 significantly attenuated, in a dose-dependent manner, neutrophil infiltration in the myocardium as well as the associated induction of lipid peroxidation. Calcium overload seen post-reperfusion of the ischaemic myocardium was also reduced by M40403. 4. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in cardiac tissue taken after reperfusion: this was attenuated by M40403. Moreover reperfused cardiac tissue sections showed positive staining for P-selectin and for anti-intercellular adhesion molecule (ICAM-1) in the vascular endothelial cells. M40403 treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in these tissues. No staining for nitrotyrosine, P-selectin or ICAM-1 was found in cardiac tissue taken at the end of the ischaemic period. 5. Overall, M40403 treatment reduced the morphological signs of myocardial cell injury and significantly improved survival. 6. Taken together, these results clearly indicate that M40403 treatment exerts a protective effect against ischaemia-reperfusion-induced myocardial injury, supporting a key role for superoxide anion in reperfusion injuries. This suggests that synthetic enzymes of SOD such as M40403, offer a novel therapeutic approach for the treatment of ischaemic hea

Topics: Animals; Cardiotonic Agents; Immunohistochemistry; Intercellular Adhesion Molecule-1; Male; Manganese; Molecular Mimicry; Myocardial Ischemia; Myocardial Reperfusion Injury; Organometallic Compounds; P-Selectin; Peroxidase; Rats; Rats, Wistar; Superoxide Dismutase; Tyrosine

Gentamicin is an antibiotic effective against Gram-negative infection, whose clinical use is limited by its nephrotoxicity. Oxygen free radicals are considered to be important mediators of gentamicin-mediated nephrotoxicity, but the exact nature of the radical in question is not known with certainty. We have investigated the potential role of superoxide in gentamicin-induced renal toxicity by using M40403, a low molecular weight synthetic manganese containing superoxide dismutase mimetic, which selectively removes superoxide. Administration of gentamicin at 100 mg/kg, s.c. for 5 days to rats induced a marked renal failure, characterised by a significant decrease in creatinine clearance and increased plasma creatinine levels, fractional excretion of sodium, lithium, urine gamma glutamyl transferase (gamma GT) and daily urine volume. A significant increase in kidney myeloperoxidase activity and lipid peroxidation was also observed in gentamicin-treated rats. M40403 (10 mg/kg, i.p. for 5 days) attenuated all these parameters of damage. Immunohistochemical localisation demonstrated nitrotyrosine formation and poly(ADP-ribose) synthetase (PARS) activation in the proximal tubule of gentamicin-treated rats. Renal histology examination confirmed tubular necrosis. M40403 significantly prevented gentamicin-induced nitrotyrosine formation, poly(ADP-ribose) synthetase activation and tubular necrosis. These results confirm our hypothesis that superoxide anions play an important role in gentamicin-mediated nephropathy and support the possible clinical use of low molecular weight synthetic superoxide dismutase mimetics in those conditions that are associated with over production of superoxide.

Topics: Animals; Anti-Bacterial Agents; Enzyme Activation; Free Radical Scavengers; Gentamicins; Kidney; Lipid Peroxidation; Male; Manganese; Molecular Mimicry; Neutrophil Infiltration; Organometallic Compounds; Oxidants; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Renal Circulation; Renal Insufficiency; Superoxide Dismutase; Superoxides; Tyrosine

1. Splanchnic artery occlusion shock (SAO) causes an enhanced formation of reactive oxygen species (ROS), which contribute to the pathophysiology of shock. Here we have investigated the effects of M40401, a new S:,S:-dimethyl substituted biscyclohexylpyridine Mn-based superoxide dismutase mimetic (SODm, k(cat)=1.2x10(+9) M(-1) s(-1) at pH=7.4), in rats subjected to SAO shock. 2. Treatment of rats with M40401 (applied at 0.25, 2.5 or 25 microg kg(-1), 15 min prior to reperfusion), attenuated the mean arterial blood and the migration of polymorphonuclear cells (PMNs) caused by SAO-shock. M40401 also attenuated the ileum injury (histology) as well as the increase in the tissue levels of myeloperoxidase (MPO) and malondialdehyde (MDA) caused by SAO shock in the ileum. 3. Immunohistochemical analysis for nitrotyrosine revealed a positive staining in ileum from SAO-shocked rats. The degree of staining for nitrotyrosine was markedly reduced in tissue sections obtained from SAO-shocked rats which had received M40401. Reperfused ileum tissue sections from SAO-shocked rats showed positive staining for P-selectin and for anti-intercellular adhesion molecule (ICAM-1) in the vascular endothelial cells. M40401 treatment markedly reduced the intensity and degree of P-selectin and ICAM-1 in tissue sections from SAO-shocked rats. M40401 treatment significantly improved survival. 4. Additionally, the very high catalytic activity of this new mimetic (comparable to the native human Cu/Zn SOD enzyme and exceeding the activity of the human Mn SOD enzyme) translates into a very low dose ( approximately microg kg(-1)) required to afford protection in this SAO model of ischemia reperfusion injury. 5. Taken together, our results clearly demonstrate that M40401 treatment exerts a protective effect, and part of this effect may be due to inhibition of the expression of adhesion molecules and peroxynitrite-related pathways with subsequent reduction of neutrophil-mediated cellular injury.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Arterial Occlusive Diseases; Catalysis; Cytokines; Fluorescent Antibody Technique; Ileum; Leukocyte Count; Male; Malondialdehyde; Manganese; Nitrates; Nitrites; Organometallic Compounds; P-Selectin; Peroxidase; Rats; Rats, Sprague-Dawley; Reperfusion Injury; Splanchnic Circulation; Superoxide Dismutase; Superoxides; Tyrosine

1. To further explore the effect of antioxidants in preventing diabetes-induced vascular and neural dysfunction we treated streptozotocin-induced diabetic rats daily with subcutaneous injections of 10 mg kg(-1) of M40403 (n=11) and compared the results obtained from 17 control rats and 14 untreated diabetic rats. M40403 is a manganese(II) complex with a bis(cyclo-hexylpyridine)-substituted macrocyclic ligand that was designed to be a selective functional mimetic of superoxide dismutase. Thus, M40403 provides a useful tool to evaluate the roles of superoxide in disease states. 2. Treatment with M40403 significantly improved diabetes-induced decrease in endoneurial blood flow, acetylcholine-mediated vascular relaxation in arterioles that provide circulation to the region of the sciatic nerve, and motor nerve conduction velocity (P<0.05). M40403 treatment also reduced the appearance of superoxide in the aorta and epineurial vessels and peroxynitrite in epineurial vessels. Treating diabetic rats with M40403 reduced the diabetes-induced increase in thiobarbituric acid reactive substances in serum but did not prevent the decrease in lens glutathione level. Treating diabetic rats with M40403 did not improve sciatic nerve Na(+)/K(+) ATPase activity or the sorbitol, fructose or myo-inositol content of the sciatic nerve. 3. These studies provide additional evidence that diabetes-induced oxidative stress and the generation of superoxide and perhaps peroxynitrite may be partially responsible for the development of diabetic vascular and neural complications.

Topics: Acetylcholine; Animals; Blood Glucose; Blood Vessels; Body Weight; Diabetes Mellitus, Experimental; Dose-Response Relationship, Drug; Fatty Acids, Nonesterified; Fructose; Inositol; Male; Manganese; Neural Conduction; Organometallic Compounds; Rats; Rats, Sprague-Dawley; Regional Blood Flow; Sciatic Nerve; Sodium-Potassium-Exchanging ATPase; Sorbitol; Superoxides; Thiobarbituric Acid Reactive Substances; Triglycerides; Tyrosine; Vasodilation; Vasodilator Agents

Inflammatory bowel disease is characterised by oxidative and nitrosative stress, leukocyte infiltration, and up-regulation of intercellular adhesion molecule 1 (ICAM-1) expression in the colon. The aim of the present study was to examine the effects of M40403, a superoxide dismutase mimetic, in rats subjected to experimental colitis. Colitis was induced in rats by intracolonic instillation of trinitrobenzene sulfonic acid (TNBS). Rats experienced bloody diarrhoea and significant loss of body weight. At 4 days after TNBS administration, the colon damage was characterised by areas of mucosal necrosis. Neutrophil infiltration (indicated by myeloperoxidase activity in the mucosa) was associated with up-regulation of ICAM-1 and expression of P-selectin and high levels of malondialdehyde. Immunohistochemistry for nitrotyrosine and poly (ADP-ribose) synthetase showed an intense staining in the inflamed colon. Treatment with M40403 (5 mg/kg daily i.p.) significantly reduced the appearance of diarrhoea and the loss of body weight. This was associated with a remarkable amelioration of the disruption of the colonic architecture as well as a significant reduction of colonic myeloperoxidase activity and malondialdehyde levels. M40403 also reduced the appearance of nitrotyrosine and poly (ADP-ribose) synthetase immunoreactivity in the colon as well as reduced the up-regulation of ICAM-1 and the expression of P-selectin. The results of this study suggested that administration of a superoxide dismutase mimetic may be beneficial for treatment of inflammatory bowel disease.

Topics: Animals; Body Weight; Colitis; Colon; Cytokines; Enzyme Activation; Free Radical Scavengers; Immunohistochemistry; Intercellular Adhesion Molecule-1; Lipid Peroxidation; Male; Manganese; Organometallic Compounds; P-Selectin; Peroxidase; Poly(ADP-ribose) Polymerases; Rats; Rats, Sprague-Dawley; Superoxide Dismutase; Survival Analysis; Time Factors; Tyrosine

To investigate the effects of M40403, a synthetic mimetic of superoxide dismutase (SOD), on collagen-induced arthritis (CIA) in rats.. CIA was elicited in Lewis rats by intradermal injection of 100 microl of an emulsion of bovine type II collagen (CII) in Freund's incomplete adjuvant at the base of the tail. A second injection was given on day 21.. Immunization induced an erosive arthritis of the hind paws. Macroscopic evidence of CIA first appeared as periarticular erythema and edema in the hind paws by days 24-26 after the first injection, with a 100% incidence by days 27. Severity progressed over a 35-day period. Radiography revealed soft tissue swelling and focal resorption of bone, together with osteophyte formation in the tibiotarsal joint. Histopathologic features included erosion of the articular cartilage at the joint margins and subchondral bone resorption associated with bone-derived multinucleated cell-containing granulomatous lesions. Treatment with M40403 (2-10 mg/kg/day) starting at the onset of arthritis (day 25) ameliorated the clinical signs on days 26-35 and improved the histologic findings in the joint and paw. Immunohistochemical analysis for nitrotyrosine (a marker of peroxynitrite formation) and poly(ADP-ribose) polymerase (PARP; a nuclear enzyme activated by DNA single-strand damage) revealed positive staining in the inflamed joints of CII-treated rats, suggestive of the formation of peroxynitrite and DNA damage, both of which were markedly reduced by M40403 treatment. Radiographic evidence of protection from bone resorption, osteophyte formation, and soft tissue swelling was apparent in the tibiotarsal joints of M40403-treated rats. Arthritic rats treated with M40403 gained weight at the same rate and to the same extent as normal, nonarthritic rats.. This study shows that a low molecular weight mimetic of SOD, M40403, attenuates the degree of chronic inflammation, tissue damage, and bone damage associated with CIA in the rat, and supports the possible use of SOD mimetics as therapeutic agents for the management of chronic diseases such as rheumatoid arthritis.

Topics: Animals; Anti-Inflammatory Agents, Non-Steroidal; Antibody Formation; Arthritis, Experimental; Arthritis, Rheumatoid; Arthrography; Collagen; Collagen Type XI; Disease Models, Animal; Interleukin-1; Joints; Male; Manganese; Molecular Weight; Organometallic Compounds; Proteins; Rats; Rats, Inbred Lew; Superoxide Dismutase; Tumor Necrosis Factor-alpha; Tyrosine; Weight Gain