Superoxide anions (O₂⁻) - catalytic removal (superoxide dismutase mimetic) [1]

Manganese imisoparsonin is a synthetic manganese small molecule that contains a superoxide dismutase mimetic (SODm) that eliminates superoxide anions without interfering with other active ingredients including nitric oxide (NO) and peroxynitrite (ONOO-) that are known to be involved in inflammatory responses [1].

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M40403 is characterized as a low molecular weight, synthetic manganese-containing superoxide dismutase mimetic (SODm) that selectively removes superoxide anion. It catalytically removes superoxide anion at a high rate without interacting with other reactive species including nitric oxide, peroxynitrite, hydrogen peroxide, oxygen or hypochlorite. This selectivity is a key property that distinguishes it from other SOD mimetics like metalloporphyrins. The study references that this property has not been demonstrated by other claimed SOD mimetic compounds such as the MnIII or FeIII porphyrins or the MnIII (Salen) which also react with other pertinent biological oxidants including nitric oxide and peroxynitrite.

A small chemical superoxide dismutase mimic called imisomersonin has demonstrated effectiveness in animal model illness situations where superoxide anion is believed to be important. The inflammatory response in rats following intrapleural injection of carrageenan is inhibited by manganese imisoparsonin. All inflammatory indicators were reduced by imisopasem manganese, with the exception of NOx, PGE2, and IL-10, which did not decrease [1]. Imisopasem manganese pretreatment led to a considerable restoration of lymphoid and hematopoietic tissue in the large intestine (particularly the small intestine) and decreased cell death. Imisoparsonite has the potential to be a novel radioprotective agent that can effectively lessen tissue damage brought on by traumatic brain injury [2].

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In a rat model of carrageenan-induced pleurisy, M40403 (5-20 mg/kg, i.p.) administered 15 minutes before carrageenan dose-dependently attenuated the inflammatory response [1].
M40403 (5-20 mg/kg) dose-dependently reduced exudate volume and polymorphonuclear cell accumulation in the pleural cavity at 4 hours after carrageenan injection [1].
Lung myeloperoxidase activity (a marker of neutrophil infiltration) and malondialdehyde levels (a marker of lipid peroxidation) were significantly reduced by M40403 (5-20 mg/kg) in a dose-dependent manner [1].
Histological examination of lung tissues showed that M40403 (20 mg/kg) significantly reduced tissue injury, edema, and neutrophil infiltration caused by carrageenan [1].
Immunohistochemical analysis revealed that M40403 (20 mg/kg) reduced the upregulation of adhesion molecules ICAM-1 and P-selectin in lung tissues from carrageenan-treated rats [1].
M40403 (20 mg/kg) reduced the immunostaining for nitrotyrosine and poly(ADP-ribose) synthetase in lung tissues from carrageenan-treated rats [1].
At the highest dose tested (20 mg/kg), M40403 attenuated the release of pro-inflammatory cytokines TNFα, IL-1β, and IL-6 in pleural exudates, but had no effect on IL-10 release [1].
M40403 (5-20 mg/kg) had no effect on nitrite/nitrate or PGE₂ levels in pleural exudates from carrageenan-treated rats [1].
In macrophages isolated from pleural exudates of carrageenan-treated rats, M40403 (5-20 mg/kg) dose-dependently reduced peroxynitrite formation [1].
M40403 (5-20 mg/kg) had no effect on iNOS activity or nitrite/nitrate release from pleural macrophages [1].
M40403 (5-20 mg/kg) dose-dependently attenuated DNA single-strand breaks, restored mitochondrial respiration, and prevented NAD⁺ depletion in pleural macrophages from carrageenan-treated rats [1].

No direct enzyme assay protocols for M40403 were described in the provided literature. The study referenced previous work showing that M40403 catalytically removes superoxide at a high rate without interacting with nitric oxide, peroxynitrite, hydrogen peroxide, oxygen, or hydroxyl radicals [1].
iNOS activity was measured in pleural macrophage and lung homogenates by monitoring the conversion of [³H]-L-arginine to [³H]-L-citrulline. Homogenates were incubated with [³H]-L-arginine, NADPH, calmodulin, tetrahydrobiopterin, and EGTA for 20 minutes at 22°C. Reactions were stopped and applied to Dowex 50W columns, and eluted [³H]-L-citrulline activity was measured by scintillation counting. M40403 treatment did not affect iNOS activity [1].

Macrophage Isolation and Culture: Pleural macrophages were collected from rats 4 hours after carrageenan injection by washing the pleural cavity with Tris-HCl buffer. Cells were cultured in DMEM supplemented with L-glutamine, penicillin, streptomycin, and heparin. After 2 hours of adherence at 37°C, non-adherent cells were removed, and adherent macrophages were used for various assays [1].
Peroxynitrite Measurement: Macrophages were rinsed with PBS and incubated with dihydrorhodamine 123 (5 μM) in PBS for 60 minutes at 37°C. Fluorescence of rhodamine-123 was measured at excitation 500 nm and emission 536 nm. M40403 treatment dose-dependently reduced peroxynitrite formation [1].
Mitochondrial Respiration (MTT Assay): Cells in 96-well plates were incubated with MTT (0.2 mg/ml) for 1 hour at 37°C. Culture medium was removed, cells were solubilized in DMSO, and OD₅₅₀ was measured. M40403 treatment restored mitochondrial respiration in macrophages from carrageenan-treated rats [1].
DNA Single-Strand Breaks: DNA damage was assessed by the alkaline unwinding method. Cells were lysed in alkaline solution, and after controlled denaturation and neutralization, ethidium bromide was added. Fluorescence was measured (excitation 520 nm, emission 590 nm), and the percentage of double-stranded DNA was calculated. M40403 treatment attenuated DNA damage in a dose-dependent manner [1].
NAD⁺ Measurement: Cells were extracted in HClO₄, neutralized with KOH, and centrifuged. Supernatants were assayed for NAD⁺ using a colorimetric method involving enzymatic cycling with alcohol dehydrogenase, which reduces MTT to formazan. The rate of MTT reduction is proportional to NAD⁺ concentration. M40403 treatment prevented NAD⁺ depletion [1].

Carrageenan-Induced Pleurisy Model:** Male Sprague-Dawley rats (300-350 g) were anesthetized with isoflurane. A skin incision was made at the left sixth intercostal space, and the underlying muscle was dissected. Saline (0.2 ml) or 1% λ-carrageenan in saline (0.2 ml) was injected into the pleural cavity. The incision was closed, and animals were allowed to recover. M40403 (5, 10, or 20 mg/kg) or vehicle (26 mM sodium bicarbonate buffer, pH 8.1-8.3) was injected intraperitoneally 15 minutes before carrageenan. At 4 hours after carrageenan injection, animals were euthanized with CO₂ [1].
* **Exudate Collection and Analysis:** The chest was opened, and the pleural cavity was rinsed with 2 ml of saline containing heparin (5 U/ml) and indomethacin (10 μg/ml). Exudate and washing solution were removed by aspiration, and total volume was measured. Exudate volume was calculated by subtracting the 2 ml injected volume. Leukocytes in the exudate were suspended in PBS and counted using a Burker's chamber after Trypan Blue staining. Cytokines (TNFα, IL-1β, IL-6, IL-10) and PGE₂ were measured in exudates by ELISA. Nitrite/nitrate levels were measured using the Griess reaction [1].
* **Tissue Collection and Analysis:** Lung tissues were collected at 4 hours after carrageenan injection. For histology, tissues were fixed in 10% buffered formaldehyde, embedded in paraffin, sectioned (7 μm), and stained with trichromic Van Gieson. For MPO and MDA assays, tissues were homogenized and processed as described. For immunohistochemistry, 7 μm cryostat sections were permeabilized with acetone, rehydrated in PBS, and incubated overnight with primary antibodies against ICAM-1, P-selectin, nitrotyrosine, or PARS. Sections were then incubated with appropriate secondary antibodies and observed under a confocal microscope [1].

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Carrageenan-Induced Pleurisy Model: Male Sprague-Dawley rats (300-350 g) were anesthetized with isoflurane. A skin incision was made at the left sixth intercostal space, and the underlying muscle was dissected. Saline (0.2 ml) or 1% λ-carrageenan in saline (0.2 ml) was injected into the pleural cavity. The incision was closed, and animals were allowed to recover. M40403 (5, 10, or 20 mg/kg) or vehicle (26 mM sodium bicarbonate buffer, pH 8.1-8.3) was injected intraperitoneally 15 minutes before carrageenan. At 4 hours after carrageenan injection, animals were euthanized with CO₂ [1].
Exudate Collection and Analysis: The chest was opened, and the pleural cavity was rinsed with 2 ml of saline containing heparin (5 U/ml) and indomethacin (10 μg/ml). Exudate and washing solution were removed by aspiration, and total volume was measured. Exudate volume was calculated by subtracting the 2 ml injected volume. Leukocytes in the exudate were suspended in PBS and counted using a Burker's chamber after Trypan Blue staining. Cytokines (TNFα, IL-1β, IL-6, IL-10) and PGE₂ were measured in exudates by ELISA. Nitrite/nitrate levels were measured using the Griess reaction [1].
Tissue Collection and Analysis: Lung tissues were collected at 4 hours after carrageenan injection. For histology, tissues were fixed in 10% buffered formaldehyde, embedded in paraffin, sectioned (7 μm), and stained with trichromic Van Gieson. For MPO and MDA assays, tissues were homogenized and processed as described. For immunohistochemistry, 7 μm cryostat sections were permeabilized with acetone, rehydrated in PBS, and incubated overnight with primary antibodies against ICAM-1, P-selectin, nitrotyrosine, or PARS. Sections were then incubated with appropriate secondary antibodies and observed under a confocal microscope [1].

[1]. Pharmacological manipulation of the inflammatory cascade by the superoxide dismutase mimetic,M40403. Br J Pharmacol. 2001 Feb;132(4):815-27.

[2]. Protective effects of M40403, a selective superoxide dismutase mimetic, in myocardial ischaemia and reperfusion injury in vivo. Br J Pharmacol. 2002 Jul;136(6):905-17.

M40403 is a low molecular weight synthetic manganese-based superoxide dismutase mimic (SODm) that selectively scavenges superoxide anions. Imidazole persemam manganese is a manganese-based non-peptide compound that mimics human mitochondrial manganese superoxide dismutase (MnSOD) and possesses potential antioxidant and chemical/radioprotective activities. After administration, imidazole persemam manganese mimics the activity of MnSOD, scavenging reactive oxygen species (ROS), such as superoxide anions, thereby preventing damage to macromolecules such as DNA by oxygen free radicals. This can reduce ROS-mediated lipid peroxidation, prevent apoptosis, and protect normal tissues from toxic damage caused by oxygen free radicals. Drug Indications: For the treatment of pain and potentially for the treatment of various cancers. Mechanism of Action: M40403 is a lead drug candidate in the unique class of superoxide dismutase (SOD) mimics. These stable, low-molecular-weight compounds mimic the function of superoxide dismutase, a naturally occurring enzyme designed to scavenge superoxide free radicals present in various pain and inflammation-related diseases.

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Pharmacodynamics
M40403 treatment has a protective effect against ischemia-reperfusion-induced myocardial injury, suggesting that superoxide anions play a crucial role in reperfusion injury.

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M40403 is a low molecular weight, synthetic manganese-containing superoxide dismutase mimetic that catalytically removes superoxide anions (O₂⁻) without interfering with other reactive species such as nitric oxide, peroxynitrite, hydrogen peroxide, oxygen, or hydroxyl radicals [1].
The compound represents an important pharmacological tool to dissect the roles of superoxide in acute and chronic inflammation [1].
Unlike native SOD enzymes, M40403 is a much smaller molecule (MW 483 vs. 30,000 for native SOD), offering advantages in terms of stability, lack of immunogenicity, and ability to penetrate cells [1].
M40403 is not deactivated by peroxynitrite, unlike native MnSOD which is nitrated and deactivated by peroxynitrite [1].
The anti-inflammatory effects of M40403 are mediated through multiple mechanisms including: reducing peroxynitrite formation, down-regulating adhesion molecules (ICAM-1, P-selectin), inhibiting pro-inflammatory cytokine release (TNFα, IL-1β, IL-6), preventing DNA damage, and inhibiting PARS activation [1].
M40403 does not affect iNOS activity, NOx production, or PGE₂ release, indicating selectivity for superoxide-mediated pathways [1].
The study demonstrates that superoxide plays a critical role in the development of the inflammatory response by altering key components of the inflammatory cascade [1].
The results support the potential use of M40403 and similar SOD mimetics as novel therapeutic approaches for managing various inflammatory diseases [1].

C21H35CL2MNN5

483.3802

482.164

218791-21-0

10195666

White to off-white solid powder

4.77

4

7

0

29

381

4

C1CC[C@@H]2[C@@H](C1)NCCN[C@@H]3CCCC[C@H]3NCC4=CC=CC(=N4)CN2.[Cl-].[Cl-].[Mn+2]

WXEMWBBXVXHEPU-XNPJUPKFSA-L

InChI=1S/C21H35N5.2ClH.Mn/c1-3-10-20-18(8-1)22-12-13-23-19-9-2-4-11-21(19)25-15-17-7-5-6-16(26-17)14-24-20;;;/h5-7,18-25H,1-4,8-15H2;2*1H;/q;;;+2/p-2/t18-,19-,20-,21-;;;/m1.../s1

Manganese, dichloro[(4aR,13aR,17aR,21aR)-1,2,3,4,4a,5,6,12,13,13a,14,15,16,17,17a,18,19,20,21,21a-eicosahydro-11,7-nitrilo-7Hdibenzo[b,h][1,4,7,10]tetraazacycloheptadecine-κN5,κN13,κN18,κN21,κN22]-, (PB-7-11-2344'3')-.

M40403 M 40403 M-40403 CG4419 CG 4419 CG-4419 SC72325 SC-72325 SC 72325

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

H2O : ~25 mg/mL (~52.15 mM)

Solubility in Formulation 1: 8.33 mg/mL (17.38 mM) in PBS (add these co-solvents sequentially from left to right, and one by one), clear solution; with sonication (<60°C).

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 (Please use freshly prepared in vivo formulations for optimal results.)