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[Display omitted] • CORM-2 attenuates cisplatin-induced hearing loss in young adult mice. • CORM-2 co-treatment decreases platinum accumulation in the inner ear. • CORM-2 protects against cisplatin-induced toxic cellular responses including necroptosis, plasma membrane disruption, and apoptotic cell death. • CORM-2 co-treatment reverses the persistent inflammatory environment created by cisplatin. • CORM-2 co-treatment reduces cochlear capillary leakage (hyperpermeability) and maintains the integrity of blood-labyrinth barrier. Cisplatin is a life-saving anticancer compound used to treat multiple solid malignant tumors, while it causes permanent hearing loss. There is no known cure, and the FDA has not approved any preventative treatment for cisplatin-based ototoxicity. This study investigated whether the carbon monoxide (CO)-releasing tricarbonyldichlororuthenium (II) dimer, CORM-2, reverses cisplatin-induced hearing impairment and reduces cisplatin accumulation in the mouse inner ear. Male 6-week-old BALB/c mice were randomly assigned to one of the following groups: control (saline-treated, i.p.), CORM-2 only (30 mg/kg, i.p., four doses), cisplatin only (20 mg/kg, i.p., one dose), and CORM-2 + cisplatin, to determine whether cisplatin-based hearing impairment was alleviated by CORM-2 treatment. Our results revealed CORM-2 significantly attenuated cisplatin-induced hearing loss in young adult mice. CORM-2 co-treatment significantly decreased platinum accumulation in the inner ear and activated the plasma membrane repair system of the stria vascularis. Moreover, CORM-2 co-treatment significantly decreased cisplatin-induced inflammation, apoptosis, and cochlear necroptosis. Because the stria vascularis is the likely cochlear entry point of cisplatin, we next focused on the microvasculature. Cisplatin induced increased extravasation of a chromatic tracer (fluorescein isothiocyanate [FITC]-dextran, MW 75 kDa) around the cochlear microvessels at 4 days post-treatment; this extravasation was completely inhibited by CORM-2 co-therapy. CORM-2 co-treatment effectively maintained the integrity of stria vascularis components including endothelial cells, pericytes, and perivascular-resident macrophage-type melanocytes. CORM-2 co-therapy substantially protects against cisplatin-induced ototoxicity by reducing platinum accumulation and toxic cellular stress responses. These data indicate that CORM-2 co-treatment may be translated into clinical strategy to reduce cisplatin-induced hearing loss. [ABSTRACT FROM AUTHOR]

Background: Although low-dose carbon monoxide (CO) administration has been shown to have an anti-fibrotic effect in various fibrotic diseases, its effects on peritoneal adhesion (PA), one of the postoperative complications, are not elucidated. In this study, the effect of CO-releasing tricarbonyldichlororuthenium (II) dimer (CORM-2) administration on the formation of PA and the underlying factors of its potential effect were investigated. Methods and results: After the induction of PA, rats were divided into four groups with 8 rats in each group. The rats received either (i) dimethyl sulfoxide:saline solution (1:10) as a vehicle, (ii) 2.5 mg/kg CORM-2, (iii) 5 mg/kg CORM-2, or (iv) inactive (i) CORM (iCORM) intragastrically every day for a duration of 7 days. PA was not induced in rats (n = 8) designated as sham controls. Gross, histological, immunohistochemical and quantitative real-time polymerase chain reaction analyses were performed to evaluate the effectiveness of CORM-2 administration. Gross analysis showed that CORM-2 administration reduced PA formation compared to rats treated with vehicle. Histological and immunohistochemical examinations showed that increased collagen deposition, myofibroblast accumulation, microvessel density, and M1 macrophage count in the peritoneal fibrosis area of vehicle-treated rats decreased following CORM-2 treatments. PCR analyses showed that CORM-2 treatments decreased hypoxia-induced Hif1a, profibrotic Tgfb1, ECM components Col1a1 and Col3a1, collagen degradation suppressor Timp1, fibrinolysis inhibitor Serpine1, and pro-inflammatory Tnf mRNA expressions, while increasing the M2 macrophage marker Arg1 mRNA expression. Conclusions: These results suggested that CORM-2 administration reduces PA formation by affecting adhesiogenic processes such as pro-inflammatory response, fibrinolytic system, angiogenesis and fibrogenesis. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) is a known gaseous bioactive substance found across a wide array of body systems. The administration of low concentrations of CO has been found to exert an anti-inflammatory, anti-apoptotic, anti-hypertensive, and vaso-dilatory effect. To date, however, it has remained unknown whether CO influences atrial natriuretic peptide (ANP) secretion. This study explores the effect of CO on ANP secretion and its associated signaling pathway using isolated beating rat atria. Atrial perfusate was collected for 10 min for use as a control, after which high atrial stretch was induced by increasing the height of the outflow catheter. Carbon monoxide releasing molecule-2 (CORM-2; 10, 50, 100 µM) and hemin (HO-1 inducer; 0.1, 1, 50 µM), but not CORM-3 (10, 50, 100 µM), decreased high stretch-induced ANP secretion. However, zinc porphyrin (HO-1 inhibitor) did not affect ANP secretion. The order of potency for the suppression of ANP secretion was found to be hemin > CORM-2 >> CORM-3. The suppression of ANP secretion by CORM-2 was attenuated by pretreatment with 5-hydroxydecanoic acid, paxilline, and 1H-[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one, but not by diltiazem, wortmannin, LY-294002, or NG-nitro-L-arginine methyl ester. Hypoxic conditions attenuated the suppressive effect of CORM-2 on ANP secretion. In sum, these results suggest that CORM-2 suppresses ANP secretion via mitochondrial KATP channels and large conductance Ca2+-activated K+ channels. [ABSTRACT FROM AUTHOR]

The short release half-life of carbon monoxide (CO) is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). The potential of CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) to enhance the release half-life of CO and alleviate skin inflammation was investigated in the present study. CORM-2-UDLs were prepared by using soy phosphatidylcholine to form lipid bilayers and Tween 80 as an edge activator. The deformability of CORM-2-UDLs was measured and compared with that of conventional liposomes by passing formulations through a filter device at a constant pressure. The release profile of CO from CORM-2-UDLs was evaluated by myoglobin assay. In vitro and in vivo anti-inflammatory effects of CORM-2-UDLs were assessed in lipopolysaccharide-stimulated macrophages and TPA-induced ear edema model, respectively. The deformability of the optimized CORM-2-UDLs was 2.3 times higher than conventional liposomes. CORM-2-UDLs significantly prolonged the release half-life of CO from 30 s in a CORM-2 solution to 21.6 min. CORM-2-UDLs demonstrated in vitro anti-inflammatory activity by decreasing nitrite production and pro-inflammatory cytokine levels. Furthermore, CORM-2-UDLs successfully ameliorated skin inflammation by reducing ear edema, pathological scores, neutrophil accumulation, and inflammatory cytokines expression. The results demonstrate that CORM-2-UDLs could be used as promising therapeutics against acute skin inflammation.

Toxic carbon monoxide in small concentrations has pro-apoptotic, anti-allergic, vasodilator effects, and stimulates angiogenesis. The problem with the therapeutic use of low doses of carbon monoxide is that it is difficult to dose. To control the amount and gradual release of carbon monoxide, non-toxic preparation is used - CO donor based on carbonyl compound of ruthenium (CORM-2). The aim – is to identify the effect of CORM-2 on the level of immunoglobulins in the blood serum and bone marrow of mice under conditions of inducing an immune response. Materials and methods. 3 groups of 15 white laboratory mice each were formed. Induction of the immune response was due to the intraperitoneal administration of xenogenic red blood cells. The first experimental group on the first day of immunization received CORM-2 (20 mg/kg), group No. 2 – on the 5th day after immunization (period of the productive phase). The control group consisted of immunized animals that did not receive CORM-2. The amount of IgA, IgM, and IgG in blood serum was determined by ELISA on the 2nd and 6th day after immunization. At the end of the experiment, bone marrow cell populations were counted. Results. After the injection of CORM-2 during the induction phase of the immune response, it inhibits the production of immunoglobulins. In comparison with the control, the level of IgA and IgG is reduced, but the amount of IgM remains unchanged. In the bone marrow, the number of monocytes, erythroblasts, and normoblasts, as well as lymphoblasts and plasma cells, increased. At the same time, the number of myeloblasts, myelocytes, basophilic normoblasts, and megakaryocytes decreased. The use of CORM-2 during the productive phase caused a decrease in the level of IgM and IgG with a simultaneous increase in the level of IgA. The number of neutrophils, eosinophils, monocytes, polychromophilic and oxyphilic normoblasts, lymphocytes, and plasma cells in the bone marrow increased. However, the number of myeloblasts, promyelocytes, myelocytes, metamyelocytes, basophilic normoblasts, and megakaryocytes decreased. Conclusions. The impact of the CORM-2 on the inductive phase of the immune response leads to inhibition of the production of immunoglobulins. The injection of CORM-2 during the productive phase of the immune response decreased the level of IgM and IgG, but at the same time, an increase in the level of IgA was observed. After the injection of CORM-2, in the bone marrow, the number of monocytes, lymphocytes, and plasma cells increased. The results indicate that CORM-2 is able to modulate the immune response.

Carbon monoxide releasing molecule-2 (CORM-2) exhibited protection against cardiac hypoxia reoxygenation injury (H/R). However, its poor water solubility and short half-life restrict its clinical usage. The present study aims to investigate whether encapsulation of CORM-2 in mesoporous silica (MSN-A-CORM-2) can enhance its anti-H/R effect in cardiac cell lines H9C2 (cardiomyoblast) and 3T3 (fibroblast). Mesoporous silica nanoparticles (MSN) were synthesized by co-operative self-assembly technique through solvothermal strategy and terminated with amine functional groups (MSN-A). The ligand, CORM-2, was linked to the terminal amine group in mesoporous silica. The physicochemical characterization of the compound was made by using SEM, TEM, FTIR, and EDS analysis. Further, we evaluated the biological effect of MSN-A-CORM-2 against H/R in H9C2 and 3T3 cells. The results demonstrated that MSN-A-CORM-2 was less toxic as compared to that of standard CORM-2. The comparable potential of CORM-2 in combating H/R got enhanced upon encapsulation with mesoporous silica. [ABSTRACT FROM AUTHOR]

Purpose: Cardioprotective effect of carbon monoxide, a gasotransmitter against myocardial ischemia–reperfusion injury (I/R), is well established in preclinical studies with male rats. However, its ischemic tolerance in post-menopausal animals has not been examined due to functional perturbations at the cellular level. Methods: The protective role of carbon monoxide releasing molecule-2 (CORM-2) on myocardial I/R was studied in female Wistar rats using the Langendorff apparatus. The animals were randomly divided into normal and ovariectomized (Ovx) female rats and were maintained 2 months post-surgery. Each group was further divided into 4 subgroups (n = 6/subgroup): normal, I/R, CORM-2-control (20 μmol/L), and CORM-2-I/R. The cardiac injury was estimated via myocardial infarct size, lactate dehydrogenase, and creatine kinase levels in coronary effluent and cardiac hemodynamic indices. Mitochondrial functional activity was assessed by measuring mitochondrial electron transport chain enzyme activities, swelling behavior, mitochondrial membrane potential, and oxidative stress. Results: Hemodynamic indices were significantly lower in ovariectomized rat hearts than in normal rat hearts. Sixty minutes of reperfusion of ischemic heart exhibited deteriorated cardiac physiological recovery in both ovariectomized and normal groups, where prominent decline was observed in ovariectomized rat. However, preconditioning the isolated heart with CORM-2 improved hemodynamics parameters significantly in both ovariectomized and normal rat hearts challenged with I/R, but with a limited degree of protection in ovariectomized rat hearts. The protective effect of CORM-2 was further confirmed via a reduction in cardiac injury, preservation of mitochondrial enzymes, and reduction in oxidative stress in all groups. Conclusion: CORM-2 administration significantly attenuated myocardial I/R injury in ovariectomized rat hearts by attenuating I/R-associated mitochondrial perturbations and reducing oxidative stress. [ABSTRACT FROM AUTHOR]

We investigated the metabolism of mouse isolated heart under the influence of tricarbonyldichlorothenium (II)- dimer (CORM-2 and 2,3-4,5-bis-O-isopropylidene-β- D-fructopyranose sulfamate (topiramate) as potential blockers of aquaporine channel (AQP3) of cardiac myocytes. The results were compared with those obtained from the group receiving anti-AQP3 monoclonal antibodies. A decrease in coronary flow was found during the period preceding ischemia (topiramate did not cause this effect). However, at the end of reperfusion, CORM-2 was responsible for its stabilization. This compound did not affect glucose intake (topiramate increased it only at the end of reperfusion), decreased Ca2+ deposition in cardiac muscle (AQP3-IgG antibodies and topiramate had similar effect), decreased creatinine release, AST (especially at the end of reperfusion). The action of CORM-2 increased the amplitude of the R waveform before ischemia and during reperfusion. At the end of reperfusion the R-wave amplitude decreased. The effect of topiramate caused an increase in amplitude only at the beginning of reperfusion. Administration of CORM-2, topiramate and antibodies resulted in prolongation of the interval before and during ischemia. At the same time, the effect of these drugs and antibodies reduced the development of ischemic damage. The results indicate that the released CO from CORM-2 has effects similar to those of anti-AQP3 antibodies. The action of topiramate had signs of calcium channel blocking. [ABSTRACT FROM AUTHOR]

The short release half-life of carbon monoxide (CO) is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). The potential of CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) to enhance the release half-life of CO and alleviate skin inflammation was investigated in the present study. CORM-2-UDLs were prepared by using soy phosphatidylcholine to form lipid bilayers and Tween 80 as an edge activator. The deformability of CORM-2-UDLs was measured and compared with that of conventional liposomes by passing formulations through a filter device at a constant pressure. The release profile of CO from CORM-2-UDLs was evaluated by myoglobin assay. In vitro and in vivo anti-inflammatory effects of CORM-2-UDLs were assessed in lipopolysaccharide-stimulated macrophages and TPA-induced ear edema model, respectively. The deformability of the optimized CORM-2-UDLs was 2.3 times higher than conventional liposomes. CORM-2-UDLs significantly prolonged the release half-life of CO from 30 s in a CORM-2 solution to 21.6 min. CORM-2-UDLs demonstrated in vitro anti-inflammatory activity by decreasing nitrite production and pro-inflammatory cytokine levels. Furthermore, CORM-2-UDLs successfully ameliorated skin inflammation by reducing ear edema, pathological scores, neutrophil accumulation, and inflammatory cytokines expression. The results demonstrate that CORM-2-UDLs could be used as promising therapeutics against acute skin inflammation. The short release half-life of carbon monoxide is a major obstacle to the effective therapeutic use of carbon monoxide-releasing molecule-2 (CORM-2). Herein, CORM-2-entrapped ultradeformable liposomes (CORM-2-UDLs) were designed to prolong carbon monoxide release half-life and enhance skin permeation of CORM-2 for better anti-inflammatory effects in skin inflammation model. Image 1 [ABSTRACT FROM AUTHOR]

During the process of human islet isolation a cascade of stressful events are triggered and negatively influence islet yield, viability, and function, including the production of proinflammatory cytokines and activation of apoptosis. Carbon monoxide-releasing molecule 2 (CORM-2) is a donor of carbon monoxide (CO) and can release CO spontaneously. Accumulating studies suggest that CORM-2 exerts cytoprotective and anti-inflammatory properties. However, the effect of CORM-2 on islet isolation is still unclear. In this study, we found that CORM-2 pretreatment significantly decreased the expression of critical inflammatory genes, including tissue factor, intercellular adhesion molecule-1, chemokine (C-C motif) ligand 2, C-X-C motif chemokine 10, Toll-like receptor 4, interleukin-1β, interleukin-6, and tumor necrosis factor-α (TNF-α). The isolated islets of the CORM-2 pretreatment group showed reduced apoptotic rate, improved viability, and higher glucose-stimulated insulin secretion, and functional gene expression in comparison to control group. Importantly, CORM-2 pretreatment prevented the impairment caused by TNF-α, evidenced by the improved glucose-stimulated index and transplantation outcomes. The present study demonstrated the anti-inflammatory property of CORM-2 during human islet isolation, and we suggest that CORM-2 pretreatment is an appealing treatment to mitigate inflammation-mediated islet dysfunction during isolation and culture ex vivo and to preserve long-term islet survival and function. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological ‘gasotransmitter’, in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (Kd 3 μM; His, Kd 130 μM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 μM; GSSG, Kd 25,000 μM). The toxicity of low, but potent, levels (15 μM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data.

OBJECTIVE: Non-small cell lung cancer (NSCLC) is the most common type of lung cancer, which is the leading cause of cancer-related morbidity and mortality worldwide. The carbon monoxide-releasing molecules (CO-RMs) are transition metal carbonyls with the capacity to release carbon monoxide (CO). The aims of our study were to assess the effects and underlying mechanisms of CO-releasing molecules-2 (CORM-2) on proliferation, migration, invasion and apoptosis in NSCLC cells, and to evaluate its potential application for lung cancer. MATERIALS AND METHODS: NSCLC cells Calu-3 were treated with CORM-2, negative control and blank control. Cell proliferation, migration and invasion were assessed by cell Counting Kit-8 (CCK-8), scratch assay and matrigel invasion chamber experiment, respectively. Apoptosis was measured by flow cytometry. Real-time PCR and Western blot were applied to examine the expression of apoptosis-related molecules on mRNA and protein levels. RESULTS: CORM-2 markedly attenuated proliferation, migration and invasion of Calu-3 cells. CORM-2 treatment also significantly reduced the ratio of B cell lymphoma 2 (Bcl-2)/B cell lymphoma 2 associated X protein (Bax) while increased expression of caspase-3 and cytochrome c. The optimal dose of CORM-2 for Calu-3 cells was 100 µM. CONCLUSIONS: CORM-2 modulates biological functions of NSCLC cells and may provide a novel therapeutic strategy for lung cancer. [ABSTRACT FROM AUTHOR]

Carbon monoxide-releasing molecule (CORM-2) acts as a carbon monoxide (CO) deliverer in a more controlled manner without altering carboxyhemoglobin level and exerts potential function in inhibiting inflammation and/or acute nociception. However, the regulatory mechanism of CORM-2 on spinal nerve ligation (SNL)-induced neuropathic pain is not currently clear. Our study aims to investigate the role of CORM-2 in neuropathic pain and the underlying mechanism. We found that spinal cord astrocytes were dramatically activated on day 7 after SNL. L-α-aminoadipate (L-α-AA), an astroglial toxin, reversed SNL-induced astrocyte activation at sub-toxic dose. Intrathecal administration of CO donor CORM-2 induced antiallodynic and antihyperalgesic effects in neuropathic animals induced by SNL and suppressed SNL-induced spontaneous excitatory postsynaptic current (EPSC) frequency in lamina II neurons of spinal cord slices. CORM-2 administration markedly inhibited SNL-induced connexin 43 (Cx43) expression, hemichannel function, and gap junction function on spinal astrocyte membranes. Moreover, exogenous CORM-2 could attenuate HO-1 expression, while overexpressed heme oxygenase-1 (HO-1) increased intracellular CO production, attenuated Cx43 expression, hemichannel function, and gap junction function on spinal astrocyte membranes. Additionally, Cx43 over-expression markedly reduced CORM-2-induced mechanical threshold and thermal hyperalgesia and elevated CORM-2-induced spontaneous EPSC frequency. In conclusion, CORM-2 attenuated SNL-induced neuropathic pain via suppressing Cx43-hemichannel function, which may contribute to understanding of the pathology of neuropathic pain. [ABSTRACT FROM AUTHOR]

Neuropathic pain is still an extremely important problem in today's medicine because opioids, which are commonly used to reduce pain, have limited efficacy in this type of pathology. Therefore, complementary therapy is needed. Our experiments were performed in rats to evaluate the contribution of the purinergic system, especially P2X4 receptor (P2X4R), in the modulation of glia activation and, consequently, the levels of nociceptive interleukins after chronic constriction injury (CCI) of the right sciatic nerve, a rat model of neuropathic pain. Moreover, we studied how intrathecal (ith.) injection of a P2X4R antagonist Tricarbonyldichlororuthenium (II) dimer (CORM-2) modulates nociceptive transmission and opioid effectiveness in the CCI model. Our results demonstrate that repeated ith. administration of CORM-2 once daily (20 mg/5 ml, 16 and 1 h before CCI and then daily) for eight consecutive days significantly reduced pain-related behavior and activation of both spinal microglia and/or astroglia induced by CCI. Moreover, even a single administration of CORM-2 on day 7 after CCI attenuated mechanical and thermal hypersensitivity as efficiently as morphine and buprenorphine. In addition, using Western blot, we have shown that repeated ith. administration of CORM-2 lowers the CCI-elevated level of MMP-9 and pronociceptive interleukins (IL-1β, IL-18, IL-6) in the dorsal L4-L6 spinal cord and/or DRG. Furthermore, in parallel, CORM-2 upregulates spinal IL-1Ra; however, it does not influence other antinociceptive factors, IL-10 and IL-18BP. Additionally, based on our biochemical results, we hypothesize that p38MAPK, ERK1/2 and PI3K/Akt but not the NLRP3/Caspase-1 pathway are partly involved in the CORM-2 analgesic effects in rat neuropathic pain. Our data provide new evidence that P2X4R may indeed play a significant role in neuropathic pain development by modulating neuroimmune interactions in the spinal cord and DRG, suggesting that its blockade may have potential therapeutic utility. [ABSTRACT FROM AUTHOR]

Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by renal 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)2D3 regulates Ca2+ and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)2D3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)2D3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 µM CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)2D3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)2D3 synthesis.

The aim of this study was to enhance the anti-inflammatory effects of carbon monoxide (CO) via sustained release of CO from carbon monoxide-releasing molecule-2-loaded lipid nanoparticles (CORM-2-NPs). CORM-2-NPs were prepared by hot high pressure homogenization method using trilaurin as a solid lipid core and Tween 20/Span 20/Myrj S40 as surfactant mixture. The physicochemical properties of CORM-2-NPs were characterized and CO release from CORM-2-NPs was assessed by myoglobin assay. In vitro anti-inflammatory effects were evaluated by nitric oxide assay in lipopolysaccharide-stimulated RAW 264.7 macrophages. In vivo anti-inflammatory activity was investigated by measuring paw volumes and histological examination in carrageenan-induced rat paw edema. Spherical CORM-2-NPs were around 100 nm with narrow particle size distribution. The sustained CO release from CORM-2-NPs was observed and the half-life of CO release increased up to 10 times compared with CORM-2 solution. CORM-2-NPs showed enhanced in vitro anti-inflammatory effects by inhibition of nitric oxide production. Edema volume in rat paw was significantly reduced after treatment with CORM-2-NPs. Taken together, CORM-2-NPs have a great potential for CO therapeutics against inflammation via sustained release of CO. [ABSTRACT FROM AUTHOR]

Background: Increased resistance to antimicrobial agents is a characteristic of many bacteria growing in biofilms on for example indwelling urinary catheters or in intracellular bacterial reservoirs. Biofilm-related infections caused by multidrug-resistant bacteria, such as extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae, are a major challenge. The aim of this study was to investigate if a carbon monoxide-releasing molecule (CORM-2) has antibacterial effects against ESBL-producing uropathogenic E. coli (UPEC) in the biofilm mode of growth and following colonization of host bladder epithelial cells. Results: The effect of CORM-2 was examined on bacteria grown within an established biofilm (biofilm formed for 24 h on plastic surface) by a live/dead viability staining assay. CORM-2 (500 μM) exposure for 24 h killed approximately 60 % of the ESBL-producing UPEC isolate. A non-ESBL-producing UPEC isolate and the E. coli K-12 strain TG1 were also sensitive to CORM-2 exposure when grown in biofilms. The antibacterial effect of CORM-2 on planktonic bacteria was reduced and delayed in the stationary growth phase compared to the exponential growth phase. In human bladder epithelial cell colonization experiments, CORM-2 exposure for 4 h significantly reduced the bacterial counts of an ESBL-producing UPEC isolate. Conclusion: This study shows that CORM-2 has antibacterial properties against multidrug-resistant UPEC under biofilmlike conditions and following host cell colonization, which motivate further studies of its therapeutic potential. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO), like other gaseous neuromodulators, has a dual nature as both a toxic gas and a physiologically relevant signaling molecule. In the nervous system, high concentrations of CO can lead to neuronal injury while lower concentrations are found to be neuroprotective. The number of cellular targets affected by physiological concentrations of CO is rapidly growing and includes ion channels in various cell types. The modulation of ion channels by CO in neurons, however, and the effect it has on neural activity are incompletely understood. Here, the well-characterized buccal neurons, B5 and B19, of the freshwater snail, Helisoma trivolvis , were used to investigate the role that CO plays in regulating spontaneous firing activity and neuronal excitability. Neurons were studied in single-cell culture, thereby removing other signals normally present in the intact nervous system and allowing for the optimal characterization of physiological effects of CO. We found that the CO donor molecule, carbon monoxide releasing molecule-2 (CORM-2), hyperpolarized the resting membrane potential of B5 neurons and silenced their spontaneous firing activity. These effects were mediated through the inhibition of a persistent sodium current. CORM-2 also inhibited neuronal excitability. This effect was mediated by the inhibition of voltage-gated calcium channels by CO. The general findings of CO acting as a hyperpolarizing signal and an inhibitor of neuronal excitability extended to B19 neurons. Taken together, these findings suggest that CO is a potent modulator of ion channels with broad implications for the modulation of neural activity in a wide range of neuron-types. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO) is one of the cytoprotective byproducts of heme oxygenase (HO)-1 and exerts anti-inflammatory action in various models. However, the detailed mechanisms underlying CO-induced HO-1 expression in primary human cardiomyocytes remain largely unidentified. We used primary left ventricle myocytes as a model and applied CO releasing molecule (CORM)-2 to investigate the relationship of CO and HO-1 expression. We herein used Western blot, real-time PCR, promoter activity and EIA to investigate the role of HO-1 expression protecting against thrombin-mediated responses. We found that thrombin-induced COX-2 expression, PGE 2 release and cardiomyocyte hypertrophy markers (increase in ANF/BNP, α-actin expression and cell surface area) was attenuated by pretreatment with CORM-2 which was partially reversed by hemoglobin (Hb) or ZnPP (an inhibitor of HO-1 activity), suggesting that HO-1/CO system may be of clinical importance to ameliorate heart failure through inhibition of inflammatory responses. CORM-2-induced HO-1 protein expression, mRNA and promoter was attenuated by pretreatment with the inhibitors of Pyk2 (PF431396), PDGFR (AG1296), PI3K (LY294002), Akt (SH-5), p38 (SB202530), JNK1/2 (SP600125), FoxO1 (AS1842856) and Sp1 (mithramycin A). The involvement of these signaling components was further confirmed by transfection with respective siRNAs, consistent with those of pharmacological inhibitors. These results suggested that CORM-2-induced HO-1 expression is mediated through a Pyk2/PDGFR/PI3K/Akt/FoxO1/Sp1-dependent manner and exerts a cytoprotective effect in human cardiomyocytes. [ABSTRACT FROM AUTHOR]

Introduction: Carbon monoxide (CO) released from CORM-2 has anti-inflammatory function, but the critical molecule mediating the inflammation inhibition has not been elucidated. Previous studies indicate that CORM-2 can activate Nrf2, a key transcription factor regulating host defense against oxidative stress and inflammation-related disorders. In this study we use Nrf2 knockout mice to determine the role of Nrf2 in mediating the CO anti-inflammatory action. Methods: We compared CORM-2's inhibiting effect on pro-inflammatory cytokine expressions (TNF-α, IL-1β and IL-6 and iNOS) in primary peritoneal macrophages, mouse liver and brain tissues from Nrf2 and Nrf2 mice. We further assayed the inflammatory cell infiltration in both liver and brain tissues of the Nrf2 and Nrf2 mice. Finally, we examined CORM's influence on mouse mortality in a mouse sepsis model. Results: Our results showed that CORM-2 dramatically inhibited the expression of pro-inflammatory cytokines in Nrf2 mice, but not in Nrf2 mice. Furthermore CORM-2 substantially decreased LPS-induced mouse mortality of Nrf2 mice, but not of Nrf2 mice. Conclusion: We conclude that Nrf2 is indispensable for CORM-2 inhibition of LPS-induced inflammation. [ABSTRACT FROM AUTHOR]

Folic Acid (FA)-tagged protein nanoemulsions were found to be preferentially internalized on B-cell lymphoma cell line (A20 cell line), which, for the first time, is reported to express folate receptor (FR)-alpha. Carbon monoxide releasing molecule-2 (CORM-2) was incorporated in the oil phase of the initial formulation. FA-functionalized nanoemulsions loaded with CORM-2 exhibited a considerable antitumor effect and an increased survival of BALB/c mice bearing subcutaneous A20 lymphoma tumors. The developed nanoemulsions also demonstrated to be well tolerated by these immunocompetent mice. Thus, the results obtained in this study demonstrate that FA-tagged protein nanoemulsions can be successfully used in cancer therapy, with the important ability to delivery drugs intracellularly. From the Clinical Editor In this research, the authors developed folic acid tagged nanoemulsions containing a carbon monoxide releasing protein molecule for targeted cancer cell treatment. In-vitro and in-vivo experiments showed efficacy against B-cell lymphoma cells. The same nanocarrier platform could be applied to other tumor cells expressing folate receptors on the cell surface. [ABSTRACT FROM AUTHOR]

Abstract: Carbon monoxide (CO) releasing molecules (CO-RMs) have been shown to inhibit growth of commensal Escherichia coli (E. coli). In the present study we examined the effect of CORM-2 on uropathogenic E. coli (UPEC) that produces extended-spectrum β-lactamase (ESBL). Viability experiments showed that CORM-2 inhibited the growth of several different ESBL-producing UPEC isolates and that 500 μM CORM-2 had a bactericidal effect within 4 h. The bactericidal effect of CORM-2 was significantly more pronounced than the effect of the antibiotic nitrofurantoin. CORM-2 demonstrated a low level of cytotoxicity in eukaryotic cells (human bladder epithelial cell line 5637) at the concentrations and time-points where the antibacterial effect was obtained. Real-time RT-PCR studies of different virulence genes showed that the expression of capsule group II kpsMT II and serum resistance traT was reduced and that some genes encoding iron acquisition systems were altered by CORM-2. Our results demonstrate that CORM-2 has a fast bactericidal effect against multiresistant ESBL-producing UPEC isolates, and also identify some putative UPEC virulence factors as targets for CORM-2. CO-RMs may be candidate drugs for further studies in the field of finding new therapeutic approaches for treatment of uropathogenic ESBLproducing E. coli. [Copyright &y& Elsevier]

Background/Aims: Carbon monoxide (CO) interferes with cytochrome-dependent cellular functions and acts as gaseous transmitter. CO is released from CO-releasing molecules (CORM) including tricarbonyl-dichlororuthenium (II) dimer (CORM-2), molecules considered for the treatment of several disorders including vascular dysfunction, inflammation, tissue ischemia and organ rejection. Cytochrome P450-sensitive function include formation of 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) by renal 25-hydroxyvitamin D3 1-alpha-hydroxylase (Cyp27b1). The enzyme is regulated by PTH, FGF23 and klotho. 1,25(OH)2D3 regulates Ca2+ and phosphate transport as well as klotho expression. The present study explored, whether CORM-2 influences 1,25(OH)2D3 formation and klotho expression. Methods: Mice were treated with intravenous CORM-2 (20 mg/kg body weight). Plasma 1,25(OH)2D3 and FGF23 concentrations were determined by ELISA, phosphate, calcium and creatinine concentrations by colorimetric methods, transcript levels by quantitative RT-PCR and protein expression by western blotting. Fgf23 mRNA transcript levels were further determined in rat osteosarcoma UMR106 cells without or with prior treatment for 24 hours with 20 µM CORM-2. Results: CORM-2 injection within 24 hours significantly increased FGF23 plasma levels and decreased 1,25(OH)2D3 plasma levels, renal Cyp27b1 gene expression as well as renal klotho protein abundance and transcript levels. Moreover, treatment of UMR106 cells with CORM-2 significantly increased Fgf23 transcript levels. Conclusion: CO-releasing molecule CORM-2 enhances FGF23 expression and release and decreases klotho expression and 1,25(OH)2D3 synthesis. © 2013 S. Karger AG, Basel [ABSTRACT FROM AUTHOR]

Abstract: Background: CO-releasing molecules (CO-RMs) are potential therapeutic agents, able to deliver CO – a critical gasotransmitter – in biological environments. CO-RMs are also effective antimicrobial agents; although the mechanisms of action are poorly defined, haem-containing terminal oxidases are primary targets. Nevertheless, it is clear from several studies that the effects of CO-RMs on biological systems are frequently not adequately explained by the release of CO: CO-RMs are generally more potent inhibitors than is CO gas and other effects of the molecules are evident. Methods: Because sensitivity to CO-RMs cannot be predicted by sensitivity to CO gas, we assess the differential susceptibilities of strains, each expressing only one of the three terminal oxidases of E. coli — cytochrome bd-I, cytochrome bd-II and cytochrome bo′, to inhibition by CORM-3. We present the first sensitive measurement of the oxygen affinity of cytochrome bd-II (K m 0.24μM) employing globin deoxygenation. Finally, we investigate the way(s) in which thiol compounds abolish the inhibitory effects of CORM-2 and CORM-3 on respiration, growth and viability, a phenomenon that is well documented, but poorly understood. Results: We show that a strain expressing cytochrome bd-I as the sole oxidase is least susceptible to inhibition by CORM-3 in its growth and respiration of both intact cells and membranes. Growth studies show that cytochrome bd-II has similar CORM-3 sensitivity to cytochrome bo′. Cytochromes bo′ and bd-II also have considerably lower affinities for oxygen than bd-I. We show that the ability of N-acetylcysteine to abrogate the toxic effects of CO-RMs is not attributable to its antioxidant effects, or prevention of CO targeting to the oxidases, but may be largely due to the inhibition of CO-RM uptake by bacterial cells. Conclusions: A strain expressing cytochrome bd-I as the sole terminal oxidase is least susceptible to inhibition by CORM-3. N-acetylcysteine is a potent inhibitor of CO-RM uptake by E. coli. General significance: Rational design and exploitation of CO-RMs require a fundamental understanding of their activity. CO and CO-RMs have multifaceted effects on mammalian and microbial cells; here we show that the quinol oxidases of E. coli are differentially sensitive to CORM-3. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins. [Copyright &y& Elsevier]

Abstract: Carbon monoxide (CO) may play an important physiological role in regulation of the vascular tone. CO-releasing molecule (CORM-2) is frequently used as a CO-donor to evaluate (patho)physiological properties of CO and its potential therapeutic applications. The aim of this study was to examine the molecular mechanisms underlying the vasodilatory properties of CORM-2 as this has not yet been extensively explored. Isometric tension recordings were performed using mice and rat isolated aortic ring segments as well as mice femoral artery ring segments. Responses to CO (10μmol/l–300μmol/l) and CORM-2 (30μmol/l–600μmol/l) were evaluated in the presence/absence of activators/inhibitors of different molecular pathways. CO was unable to relax mice blood vessels, whereas it induced concentration-dependent relaxations in rat aorta. The response to CO was inhibited by both the soluble guanylyl cyclase (sGC) inhibitor ODQ (10μmol/l) and potassium (K+) channel blocker tetraethyl-ammonium chloride (3mmol/l). CORM-2 relaxed both mice and rat isolated blood vessels in a concentration-dependent manner, however this response was only partially blocked by ODQ and tetraethyl-ammonium chloride. Interestingly, 4-aminopyridine (3mmol/l) inhibited the CORM-2 induced vasodilatation whereas iberiotoxin (100nmol/l) had no influence. The molecular mechanisms underlying CORM-2 induced relaxation differ from those of CO-induced relaxation. While CO relaxes vessels through activation of sGC and/or calcium-activated K+-channels, CORM-2 exerts its vasodilatory properties only partially through sGC or K+-channels activation. CORM-2 induced vasodilatation seems to involve voltage-dependent rather than calcium-activated K+-channels. [Copyright &y& Elsevier]

Abstract: The carbon monoxide releasing molecule tricarbonyldichlororuthenium (CORM-2) displays protective actions like carbon monoxide. The molecular mechanism underlying this effect remains controversial. We hypothesized that CORM-2 mediates cytoprotection via induction of heat shock proteins through activation of p38 mitogen-activated kinase. Embryonic bovine lung cells were incubated with CORM-2. Apoptosis was induced by staurosporine and analyzed by flow cytometry following annexin-V staining, caspase-3 activity assay, and by Western Blot for caspase-3 cleavage. Heat shock response was assessed by DNA-binding activity of heat shock factor 1 and by reporter gene activity. Cells were transfected with siRNA targeting p38 isoforms. Data were analyzed with ANOVA and post-hoc Holm-Sidak test. CORM-2 inhibited staurosporine-induced apoptosis (% annexin-V positive cells: staurosporine=60±4% vs. CORM-2 10μM=48±4%, CORM-2 25μM=42±5%, CORM-2 50μM=40±4% and CORM-2 100μM=38±2%, mean±S.D., P<0.001; caspase-3 activity: staurosporine=92±15 RFUs vs. CORM-2 50μM=60±14 RFUs, mean±S.D. P<0.001). CORM-2 induced phosphorylation of p38 MAPK, but not of JNK and ERK1/2. CORM-2 induced DNA-binding of heat shock factor 1 and elicited a 4-fold induction of gene activity (P<0.05). Incubation with the Hsp inhibitors KNK437 attenuated and 17-AAG abolished the anti-apoptotic effect of CORM-2 (P<0.001). p38 inhibition and silencing of p38β attenuated the anti-apoptotic effect of CORM-2 (P<0.05), most likely by abolishing CORM-2-induced HSF-1 binding activity. These findings suggest that CORM-2-mediated cytoprotection is caused by induction of the heat shock response and by p38 activation. Furthermore, the p38β isoform activation may represent an upstream mechanism of heat shock response induction. [Copyright &y& Elsevier]

Abstract: Doxorubicin (DXR) has been used in variety of human malignancies for decades. Despite its efficacy in cancer, clinical usage is limited because of its cardiotoxicity, which has been associated with oxidative stress and apoptosis. Carbon monoxide-releasing molecules (CORMs) have been shown to reduce the oxidative damage and apoptosis. The present study investigated the effects of CORM-2, a fast CO-releaser, against DXR-induced cardiotoxicity in mice using biochemical, histopathological and gene expression approaches. CORM-2 (3, 10 and 30mg/kg/day) was administered intraperitoneally (i.p.) for 10days and terminated the study on day 11. DXR (20mg/kg, i.p.) was injected before 72h of termination. Mice treated with DXR showed cardiotoxicity as evidenced by elevation of serum creatine kinase (CK) and lactate dehydrogenase (LDH), tissue malondialdehyde (MDA), caspase-3 and decrease the level of total antioxidant status (TAS) in heart tissues. Pre- and post-treatment with CORM-2 (30mg/kg, i.p.) elicited significant improvement in CK, LDH, MDA, caspase-3 and TAS levels. Histopathological studies showed that cardiac damage with DXR has been reversed with CORM-2+DXR treatment. There was dramatic decrease in hematological count in DXR-treated mice, which has been improved with CORM-2. Furthermore, there was also elevation of mRNA expression of heme oxygenase-1, hypoxia inducible factor-1 alpha, vascular endothelial growth factor and decrease in inducible-nitric oxide synthase expression upon treatment with CORM-2 that might be linked to cardioprotection. These data suggest that CORM-2 treatment provides cardioprotection against acute doxorubicin-induced cardiotoxicity in mice and this effect may be attributed to CORM-2-mediated antioxidant and anti-apoptotic properties. [Copyright &y& Elsevier]

Carbon monoxide (CO) is produced endogenously by heme oxygenase (HO) enzymes. HO-1 is highly expressed in many inflammatory disease states, where it is broadly protective. The protective effects of HO-1 expression can be largely mimicked by the exogenous application of CO and CO-releasing molecules (CORMs). Despite a dearth of pharmacological tools for their study, molecular methodologies have identified P2X4 receptors as a potential anti-nociceptive drug target. P2X4 receptors are up-regulated in animal models of inflammatory pain, and their knock-down reduces pain behaviours. In these same animal models, HO-1 expression is anti-nociceptive, and we therefore investigated whether P2X4 was a target for CO and tricarbonyldichlororuthenium (II) dimer (CORM-2). Using conventional whole-cell and perforated-patch recordings of heterologously expressed human P2X4 receptors, we demonstrate that CORM-2, but not CO gas, is an inhibitor of these channels. We also investigated the role of soluble guanylate cyclase and mitochondria-derived reactive oxygen species using pharmacological inhibitors but found that they were largely unable to affect the ability of CORM-2 to inhibit P2X4 currents. A control breakdown product of CORM-2 was also without effect on P2X4. These results suggest that P2X4 receptors are not a molecular target of endogenous CO production and are, therefore, unlikely to be mediating the anti-nociceptive effects of HO-1 expression in inflammatory pain models. However, these results show that CORM-2 is an effective antagonist at human P2X4 receptors and represents a useful pharmacological tool for the study of these receptors given the current dearth of antagonists. [ABSTRACT FROM AUTHOR]

The effect of CO on the expression of iNOS and COX-2 was investigated by using a CO-releasing molecule (CORM)-2 in LPS-activated RAW 264.7 cells in vitro. Interestingly, CORM-2 significantly inhibited iNOS (NO) but not COX-2 (PGE2) expression. PPAR-γ activators such as troglitazone, GW1929, and 15-deoxy-Δ12, 14- prostaglandin J2 showed preferential inhibitory effect on iNOS over COX-2 expression in LPS-activated macrophages. The same effect was shown in lung tissues (iNOS, COX-2) and serum (NO, PGE2) when administered of CORM-2 in LPS-induced septic mice, indicating that CO derived from CORM-2 differentially regulates iNOS and COX-2 through PPAR-γ activation under inflammation state. [ABSTRACT FROM AUTHOR]

The structural and bonding characteristics of several CO-releasing molecules have been investigated using density functional theories. Based on the nature of the physiological buffer compositions used in experiments, several isodesmic reactions involving chloride (Cl-), hypophosphate (HPO42-), and nitrosyl (NO) ligands are set up to find out whether the ligand-substituted products, which could have direct influence in CO releasing mechanism, are thermodynamically feasible. The isodesmic reactions involving the Cl- and HPO42- ligands are exothermic whereas it is endothermic with the NO ligand. © 2009 Wiley Periodicals, Inc. Int J Quantum Chem, 2009 [ABSTRACT FROM AUTHOR]

Abstract: Carbon monoxide (CO) arising from heme degradation, catalyzed particularly by the stress-inducible heme oxygenase-1 (HO-1), has recently been demonstrated to provide cytoprotection against cell death in macrophages stimulated with bacterial lipopolysaccharide (LPS). In the present study, we determined the effects of CO on the production of reactive oxygen species (ROS) and nitric oxide (NO) by the LPS-stimulated RAW 264.7 macrophages. In addition, effect of CO-exposure on the production of superoxide (O2 −) in the phorbol myristate acetate (PMA)-stimulated PLB-985 neutrophils was determined. Production of ROS by the LPS-stimulated macrophages pretreated with 50μM [Ru(CO)3Cl2]2, a CO-releasing molecule (CORM-2), was abolished and the production of O2 − by the PMA-stimulated neutrophils pretreated with the CORM-2 was decreased markedly. The CORM-2 (50μM) was not cytotoxic to both the unstimulated and LPS-stimulated macrophages when determined by employing mitochondrial reductase function test (MTT assay). In macrophages pretreated with increasing doses of CORM-2, both the LPS-derived upregulations of iNOS (NO production) and HO-1 expression (CO production) were suppressed in a dose-dependent manner. Alternatively, when the macrophages were treated with LPS and CO-donor together, the LPS-derived increase in NO production was decreased. Conversely, when the control and LPS-stimulated macrophages were treated with zinc protoporphyrin IX (ZnPP) to inhibit the HO activity blocking endogenous production of CO (basal and enhanced), macrophages died extensively. Interestingly, production of NO in the LPS-stimulated macrophages increased significantly following the ZnPP treatment. Addition of CORM-2 to the LPS-treated cells that were being treated additionally with ZnPP did not prevent the cell death. However, endogenous overproduction of CO by super-induction of HO-1 (obtained by pretreatment of macrophages with either buthionine sulfoximine or hemin) decreased the LPS-derived iNOS expression without affecting cell survival. Combined, these results indicated that enhanced HO activity is essential for the survival of LPS-stimulated macrophages. Thus, upregulation of HO-1 and overproduction of CO may allow the survival of LPS-stimulated macrophages; first, by eliminating the free heme to prevent Fenton reaction, second, by limiting the availability of free heme required for induction of NO-producing heme enzyme (i.e., iNOS), third, by limiting additional production of O2 − and NO via CO-derived inhibition on the activities of heme enzymes like NADPH oxidase and iNOS, respectively. CO may allow the LPS-activated macrophages to return back to the normal quiet state insensitive to additional stimuli causing oxidative stress. [Copyright &y& Elsevier]

Carbon monoxide (CO) is a known gaseous bioactive substance found across a wide array of body systems. The administration of low concentrations of CO has been found to exert an anti-inflammatory, anti-apoptotic, anti-hypertensive, and vaso-dilatory effect. To date, however, it has remained unknown whether CO influences atrial natriuretic peptide (ANP) secretion. This study explores the effect of CO on ANP secretion and its associated signaling pathway using isolated beating rat atria. Atrial perfusate was collected for 10 min for use as a control, after which high atrial stretch was induced by increasing the height of the outflow catheter. Carbon monoxide releasing molecule-2 (CORM-2; 10, 50, 100 μM) and hemin (HO-1 inducer; 0.1, 1, 50 μM), but not CORM-3 (10, 50, 100 μM), decreased high stretch-induced ANP secretion. However, zinc porphyrin (HO-1 inhibitor) did not affect ANP secretion. The order of potency for the suppression of ANP secretion was found to be hemin > CORM-2 >> CORM-3. The suppression of ANP secretion by CORM-2 was attenuated by pretreatment with 5-hydroxydecanoic acid, paxilline, and 1 H -[1,2,4] oxadiazolo [4,3-a] quinoxalin-1-one, but not by diltiazem, wortmannin, LY-294002, or NG-nitro-L-arginine methyl ester. Hypoxic conditions attenuated the suppressive effect of CORM-2 on ANP secretion. In sum, these results suggest that CORM-2 suppresses ANP secretion via mitochondrial KATP channels and large conductance Ca 2+ -activated K + channels.

OBJECTIVES: Oxidative stress caused by reactive oxygen species (ROS) inhibiting the effects of the body's antioxidant system is the main pathology of many diseases. Heme oxygenase (HO) in the cellular antioxidant system is oxidative sensor protein induced by ROS formation or carbon monoxide (CO) production. CORM-2 (tricarbonyldichlororuthenyum-II), Co releasing molecule induces the expression of heme oxygenase-1 (HO-1). The aim of the study is to determine the effect of CORM-2 on hydrogen peroxide (H2O2) induced neuroblastoma cells (SH-SY5Y) through HO-1 induction. MATERIAL&METHODS: To determine the effective concentration of CORM-2, MTT analyze was performed. The cells were treated with 1 μM, 5 μM, 10 μM, 25 μM and 50 μM concentrations of CORM-2. At the end of the MTT analysis 20 μM and 50 μM concentrations were determined for Western Blot and Real-Time PCR experiments. The cells were pre-treated with the 20UM and 50 UM concentrations of CORM-2 for 3 hours, then they were induced with 300 μM H2O2 for 3 hours. The samples were prepared for Western Blot and Real-Time PCR experiments. RESULTS: CORM-2 molecule significantly affected cell viability in SHSY-5Y cells in the dose range of 10 μM- 50 μM. The expression of HO-1 (0.42 ± 0.02 and 0.82 ± 0.13 control and 20 μM respectively, n = 3) was increased in SH-SY5Y cells which are pretreated with the 20 μM and 50 μM CORM-2 concentrations as a result of Western Blot experiments. CONCLUSIONS: In the presence of oxidative stress, CORM-2 increased HO-1 expression. This may be significant in carcinogenesis and neurodegenerative disorders. [ABSTRACT FROM AUTHOR]

Carbon monoxide (CO)-releasing molecules (CORMs) are used to deliver CO, a biological 'gasotransmitter', in biological chemistry and biomedicine. CORMs kill bacteria in culture and in animal models, but are reportedly benign towards mammalian cells. CORM-2 (tricarbonyldichlororuthenium(II) dimer, Ru2Cl4(CO)6), the first widely used and commercially available CORM, displays numerous pharmacological, biochemical and microbiological activities, generally attributed to CO release. Here, we investigate the basis of its potent antibacterial activity against Escherichia coli and demonstrate, using three globin CO sensors, that CORM-2 releases negligible CO (<0.1 mol CO per mol CORM-2). A strong negative correlation between viability and cellular ruthenium accumulation implies that ruthenium toxicity underlies biocidal activity. Exogenous amino acids and thiols (especially cysteine, glutathione and N-acetyl cysteine) protected bacteria against inhibition of growth by CORM-2. Bacteria treated with 30 μM CORM-2, with added cysteine and histidine, exhibited no significant loss of viability, but were killed in the absence of these amino acids. Their prevention of toxicity correlates with their CORM-2-binding affinities (Cys, Kd 3 μM; His, Kd 130 μM) as determined by 1H-NMR. Glutathione is proposed to be an important intracellular target of CORM-2, with CORM-2 having a much higher affinity for reduced glutathione (GSH) than oxidised glutathione (GSSG) (GSH, Kd 2 μM; GSSG, Kd 25,000 μM). The toxicity of low, but potent, levels (15 μM) of CORM-2 was accompanied by cell lysis, as judged by the release of cytoplasmic ATP pools. The biological effects of CORM-2 and related CORMs, and the design of biological experiments, must be re-examined in the light of these data. [ABSTRACT FROM AUTHOR]

Hypoxia-inducible factor prolyl hydroxylase domain 2 (PHD2) is an important oxygen sensor in animals. By using the CO-releasing molecule-2 (CORM-2) as an in situ CO donor, we demonstrate that CO is an inhibitor of PHD2. This report provides further evidence about the emerging role of CO in oxygen sensing and homeostasis., (© 2021 Wiley-VCH GmbH.)

CO-releasing molecule-2 (CORM-2), as a safe donor for releasing CO, has been widely adopted to disclose the physiopathological roles of CO and used as therapeutic drug replacing gaseous CO. The accurate determination of CORM-2 levels in biological systems is central for understanding its safe use as therapeutic agent and the cellular functions of CO. In this paper, a novel fluorescent probe CPD-NB was synthesized, which provided a safe and convenient way for detecting CORM-2 without the requirement of Pd2+ as an additional substrate. It featured a robust fluorescence response to CORM-2 with desirable sensitivity (the detection limit was as low as 4 nM), and showed high selectivity toward CORM-2 over other biologically relevant species. Moreover, with the low cytotoxicity of probe CPD-NB , the visualizations of CORM-2 changes in live cells and zebrafish were also achieved. Thus, we expected that probe CPD-NB could be as an ideal platform for safe and powerful imaging of CORM-2 in biological systems, and may thus promote the progress of CO-related research. Image 1 • The probe could detect CORM-2 without the assistance of Pd2+. • The probe could detect CORM-2 with ultra-sensitivity (detection limit was about 4 nM). • The probe displayed prominent selectivity towards CORM-2. • The probe was successful achieved in imaging the fluctuations of CORM-2 in live cells and zebrafish. [ABSTRACT FROM AUTHOR]