Abstract: Infarct size is a major determinant of outcomes after acute myocardial infarction (AMI). Carbon monoxide–releasing molecules (CORMs), which deliver nanomolar concentrations of carbon monoxide to tissues, have been shown to reduce infarct size in rodents. We evaluated efficacy and safety of CORM-A1 to reduce infarct size in a clinically relevant porcine model of AMI. We induced AMI in Yorkshire White pigs by inflating a coronary angioplasty balloon to completely occlude the left anterior descending artery for 60 minutes, followed by deflation of the balloon to mimic reperfusion. Fifteen minutes after balloon occlusion, animals were given an infusion of 4.27 mM CORM-A1 (n = 7) or sodium borate control (n = 6) over 60 minutes. Infarct size, cardiac biomarkers, ejection fraction, and hepatic and renal function were compared amongst the groups. Immunohistochemical analyses were performed to compare inflammation, cell proliferation, and apoptosis between the groups. CORM-A1–treated animals had significant reduction in absolute infarct area (158 ± 16 vs. 510 ± 91 mm2, P < 0.001) and infarct area corrected for area at risk (24.8% ± 2.6% vs. 45.2% ± 4.0%, P < 0.0001). Biochemical markers of myocardial injury also tended to be lower and left ventricular function tended to recover better in the CORM-A1 treated group. There was no evidence of hepatic or renal toxicity with the doses used. The cardioprotective effects of CORM-A1 were associated with a significant reduction in cell proliferation and inflammation. CORM-A1 reduces infarct size and improves left ventricular remodeling and function in a porcine model of reperfused MI by a reduction in inflammation. These potential cardioprotective effects of CORMs warrant further translational investigations.

Aims: Carbon monoxide (CO)-releasing molecules (CORMs) are candidates for animal and antimicrobial therapeutics. We aimed to probe the antimicrobial potential of a novel manganese CORM. Results: [Mn(CO)(4)S2CNMe(CH2CO2H)], CORM-401, inhibits growth of Escherichia coli and several antibiotic-resistant clinical pathogens. CORM-401 releases CO that binds oxidases in vivo, but is an ineffective respiratory inhibitor. Extensive CORM accumulation (assayed as intracellular manganese) accompanies antimicrobial activity. CORM-401 stimulates respiration, polarizes the cytoplasmic membrane in an uncoupler-like manner, and elicits loss of intracellular potassium and zinc. Transcriptomics and mathematical modeling of transcription factor activities reveal a multifaceted response characterized by elevated expression of genes encoding potassium uptake, efflux pumps, and envelope stress responses. Regulators implicated in stress responses (CpxR), respiration (Arc, Fnr), methionine biosynthesis (MetJ), and iron homeostasis (Fur) are significantly disturbed. Although CORM-401 reduces bacterial growth in combination with cefotaxime and trimethoprim, fractional inhibition studies reveal no interaction. Innovation: We present the most detailed microbiological analysis yet of a CORM that is not a ruthenium carbonyl. We demonstrate CO-independent striking effects on the bacterial membrane and global transcriptomic responses. Conclusions: CORM-401, contrary to our expectations of a CO delivery vehicle, does not inhibit respiration. It accumulates in the cytoplasm, acts like an uncoupler in disrupting cytoplasmic ion balance, and triggers multiple effects, including osmotic stress and futile respiration.

Carbon monoxide (CO) is generated by heme oxygenase-1 (HO-1) and displays important signaling, anti-apoptotic and anti-inflammatory activities, indicating that pharmacological agents mimicking its action may have therapeutic benefit. This study examined the biochemical and pharmacological properties of CORM-401, a recently described CO-releasing molecule containing manganese as a metal center. We used in vitro approaches, ex-vivo rat aortic rings and the EA.hy926 endothelial cell line in culture to address how CORM-401 releases CO and whether the compound modulates vascular tone and pro-angiogenic activities, respectively. We found that CORM-401 released up to three CO/mole of compound depending on the concentration of the acceptor myoglobin. Oxidants such as H2O2, tert-butyl hydroperoxide or hypochlorous acid increased the CO liberated by CORM-401. CORM-401 also relaxed pre-contracted aortic rings and vasorelaxation was enhanced in combination with H2O2. Consistent with the release of multiple CO molecules, CORM-401-induced vasodilation was three times higher than that elicited by CORM-A1, which exhibits a similar half-life to CORM-401 but liberates only one CO/mole of compound. Furthermore, endothelial cells exposed to CORM-401 accumulated CO intracellularly, accelerated migration in vitro and increased VEGF and IL-8 levels. Studies using pharmacological inhibitors revealed HO-1 and p38 MAP kinase as two independent and parallel mechanisms involved in stimulating migration. We conclude that the ability of CORM-401 to release multiple CO, its sensitivity to oxidants which increase CO release, and its vascular and pro-angiogenic properties highlight new advances in the design of CO-releasing molecules that can be tailored for the treatment of inflammatory and oxidative stress-mediated pathologies.

Introduction Conventional pharmacological treatments for acute myocardial infarction (AMI), a life threatening complication of a sudden coronary occlusion, are limited by their efficiency and side effects. New therapeutic strategies are thus needed to improve outcomes. Carbon monoxide (CO) is cardio-protective at nanomolar concentrations. Carbon monoxide-releasing molecules (CORMs), capable of carrying and releasing controlled quantities of CO in cellular systems, are a promising therapeutic that overcome the limitations of CO gas. CORM-A1 is a water soluble and releases CO slowly through hydrolysis at physiological conditions. We investigated the efficacy and safety of CORM-A1 in reducing infarct size in a clinically relevant porcine model of re-perfused AMI. Methods Male Yorkshire White pigs (25–33 kg) underwent a balloon-induced coronary occlusion at the middle segment of left anterior descending artery beyond the first diagonal branch for 60min. From 15min post-occlusion, sodium borate (control) or CORM-A1 (4.27 mM, each) were infused over a period of 60min. Left ventricular (LV) function and blood pressure were assessed by cardiac catheterization. Cardiac biomarkers, hepatic and renal functions were compared between the groups. Seven days after AMI, animals were culled and in-situ double staining with Evans blue and 2,3,5-triphenyltetrazolium (TTC) performed to measure infarct size. Myocardial inflammation, proliferation and apoptosis were evaluated by immunohistochemistry, immunoblotting, and TUNEL assay. Results CORM-A1 treated pigs had a reduced infarcted area and improved LV function, but no significant change in blood pressure, compared to controls. Infarct size was 35 ± 7% of the area at risk (ischaemic area) in CORM-A1 pigs compared to 90 ± 5% in controls (p < 0.0001, n = 3–8/group). Myocardium from CORM-A1 treated animals had fewer TUNEL positive (30.5 ± 4.7 vs. 46.2 ± 6.6%, p < 0.05, n = 37), Ki67 positive (7.7 ± 2.3 vs. 29.0 ± 4.0%, p < 0.01, n = 3–7) and inflammation positive cells (4.3 ± 1.8 vs. 36.7 ± 7.1%, p < 0.01, n = 3–7) in the infarcted regions, compared to controls. CORM-A1 infused animals also had significantly reduced (2–3-fold) neovascular formation (vWF staining) in the infarcted areas compared to controls (22.0 ± 3.6 vs. 53.3 ± 8.9%, p < 0.01, n = 3–7). A similar pattern was seen in the ischaemic areas. These changes were associated with a down-regulation of HIF-1a expression in the myocardium of CORM-A1 treated animals. Conclusions Our data suggest CORM-A1 as a key modulator of myocardial repair following re-perfused AMI injury. Injury is reduced in CORM-A1 treated animals by reducing inflammation, proliferation and cell death whilst maintaining healthy repair via neovascularisation. This study suggests the development of CORM-A1 as a potential new therapeutic for treatment of patients with AMI and warrants further clinical studies.

Aims: Carbon monoxide-releasing molecules (CORMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically, including applications in antimicrobial therapy. Hemes are generally considered the prime targets of CO and CORMs, so we tested this hypothesis using heme-deficient bacteria, applying cellular, transcriptomic, and biochemical tools. Results: CORM-3 [Ru(CO)(3)Cl(glycinate)] readily penetrated Escherichia coli hemA bacteria and was inhibitory to these and Lactococcus lactis, even though they lack all detectable hemes. Transcriptomic analyses, coupled with mathematical modeling of transcription factor activities, revealed that the response to CORM-3 in hemA bacteria is multifaceted but characterized by markedly elevated expression of iron acquisition and utilization mechanisms, global stress responses, and zinc management processes. Cell membranes are disturbed by CORM-3. Innovation: This work has demonstrated for the first time that CORM-3 (and to a lesser extent its inactivated counterpart) has multiple cellular targets other than hemes. A full understanding of the actions of CORMs is vital to understand their toxic effects. Conclusion: This work has furthered our understanding of the key targets of CORM-3 in bacteria and raises the possibility that the widely reported antimicrobial effects cannot be attributed to classical biochemical targets of CO. This is a vital step in exploiting the potential, already demonstrated, for using optimized CORMs in antimicrobial therapy. Antioxid. Redox Signal. 23, 148-162.

Carbon monoxide-releasing molecules (CO-RMs) uncouple respiration in isolated mitochondria1, which may serve as crucial organelles to transduce the anti-inflammatory properties of CO. Here we investigated in detail how CORM-4012, a manganese-based compound, modulates mitochondrial function in BV2 and primary microglia cells using a Seahorse XF analyzer. CORM-401 caused a concentration- and time-dependent increase in oxygen consumption rate (OCR) up to 100 µM, while higher concentrations were inhibitory. The increase in OCR was associated with a decrease in mitochondrial membrane potential and occurred when CORM-401 was added either before or after oligomycin, indicating an activity independent from ATP turnover, i.e. uncoupling (see Figure). Maximal respiration induced by FCCP was decreased by CORM-401, thus diminishing the spare respiratory capacity of cells. The uncoupling effect of CORM-401 was confirmed in permeabilized cells3 with a concentration-dependent increase in state 4 OCR at the level of complex I and III and a mild stimulation of state 3 OCR of complex II. Interestingly, CORM-401 increased glycolysis and prevented the depression of respiration as well as the decrease in ATP levels elicited by lipopolysaccharide in BV2 cells. These findings suggest that the anti-inflammatory activity of CO may be linked to modulation of mitochondrial bioenergetics and metabolism.

Background: CO-releasing molecules (CO-RMs) are used to study biological interactions with this toxic gas. Results: CORM-3 inhibits the NO detoxification activity of the E. coli flavohemoglobin Hmp in vivo but not in vitro. Conclusion: CO-RMs must be used with regard to their chemistry in biological studies. Significance: CORM-3 is a useful tool to study concerted effects of NO and CO in vivo.CO and NO are small toxic gaseous molecules that play pivotal roles in biology as gasotransmitters. During bacterial infection, NO, produced by the host via the inducible NO synthase, exerts critical antibacterial effects while CO, generated by heme oxygenases, enhances phagocytosis of macrophages. In Escherichia coli, other bacteria and fungi, the flavohemoglobin Hmp is the most important detoxification mechanism converting NO and O-2 to the ion nitrate (NO3-). The protoheme of Hmp binds not only O-2 and NO, but also CO so that this ligand is expected to be an inhibitor of NO detoxification in vivo and in vitro. CORM-3 (Ru(CO)(3)Cl(glycinate)) is a metal carbonyl compound extensively used and recently shown to have potent antibacterial properties. In this study, attenuation of the NO resistance of E. coli by CORM-3 is demonstrated in vivo. However, polarographic measurements showed that CO gas, but not CORM-3, produced inhibition of the NO detoxification activity of Hmp in vitro. Nevertheless, CO release from CORM-3 in the presence of soluble cellular compounds is demonstrated by formation of carboxy-Hmp. We show that the inability of CORM-3 to inhibit the activity of purified Hmp is due to slow release of CO in protein solutions alone i.e. when sodium dithionite, widely used in previous studies of CO release from CORM-3, is excluded. Finally, we measure intracellular CO released from CORM-3 by following the formation of carboxy-Hmp in respiring cells. CORM-3 is a tool to explore the concerted effects of CO and NO in vivo.

Aims: Carbon monoxide (CO) delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas. The metal carbonyl Ru(CO)3Cl(glycinate) (CORM-3) is a novel, potent antimicrobial agent. Here, we established its mode of action. Results: CORM-3 inhibits respiration in several bacterial and yeast pathogens. In anoxic Escherichia coli suspensions, CORM-3 first stimulates, then inhibits respiration, but much higher concentrations of CORM-3 than of a classic protonophore are required for stimulation. Proton translocation measurements (H/O quotients, i.e., H extrusion on pulsing anaerobic cells with O2) show that respiratory stimulation cannot be attributed to true ‘‘uncoupling,’’ that is, dissipation of the protonmotive force, or to direct stimulation of oxidase activity. Our data are consistent with CORM-3 facilitating the electrogenic transmembrane movement of K (or Na), causing a stimulation of respiration and H pumping to compensate for the transient drop in membrane potential (DJ). The effects on respiration are not mimicked by CO gas or control Ru compounds that do not release CO. Inhibition of respiration and loss of bacterial viability elicited by CORM-3 are reversible by white light, unambiguously identifying heme-containing oxidase(s) as target(s). Innovation: This is the most complete study to date of the antimicrobial action of a CO-RM. Noteworthy are the demonstration of respiratory stimulation, electrogenic ion transport, and photosensitive activity, establishing terminal oxidases and ion transport as primary targets. Conclusion: CORM-3 has multifaceted effects: increased membrane permeability, inhibition of terminal oxidases, and perhaps other unidentified mechanisms underlie its effectiveness in tackling microbial pathogenesis. Antioxid. Redox Signal. 19, 497–509.

Carbon monoxide (CO) is a toxic gas that binds to haems, but also plays critical signalling and cytoprotective roles in mammalian systems; despite problems associated with systemic delivery by inhalation of the gas, it may be employed therapeutically. CO delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas; CO-RMs are also attractive candidates as novel antimicrobial agents. Salmonella enterica serovar Typhimurium is an enteropathogen causing gastroenteritis in humans. Recent studies have implicated haem oxygenase-1 (HO-1), the protein that catalyses the degradation of haem into biliverdin, free iron and CO, in the host immune response to Salmonella infection. In several studies, CO administration via CO-RMs elicited many of the protective roles of HO-1 induction and so we investigated the effects of a well-characterized water-soluble CO-RM, Ru(CO)3Cl(glycinate) (CORM-3), on Salmonella. CORM-3 exhibits toxic effects at concentrations significantly lower than those reported to cause toxicity to RAW 264.7 macrophages. We demonstrated here, through oxyhaemoglobin assays, that CORM-3 did not release CO spontaneously in phosphate buffer, buffered minimal medium or very rich medium. CORM-3 was, however, accumulated to high levels intracellularly (as shown by inductively coupled plasma MS) and released CO inside cells. Using growing Salmonella cultures without prior concentration, we showed for the first time that sensitive dual-beam integrating cavity absorption spectrophotometry can detect directly the CO released from CORM-3 binding in real-time to haems of the bacterial electron transport chain. The toxic effects of CO-RMs suggested potential applications as adjuvants to antibiotics in antimicrobial therapy.

AIMS Carbon monoxide (CO)-releasing molecules (CO-RMs) are being developed with the ultimate goal of safely utilizing the therapeutic potential of CO clinically. One such application is antimicrobial activity; therefore, we aimed to characterize and compare the effects of the CO-RM, CORM-3, and its inactivated counterpart, where all labile CO has been removed, at the transcriptomic and cellular level. RESULTS We found that both compounds are able to penetrate the cell, but the inactive form is not inhibitory to bacterial growth under conditions where CORM-3 is. Transcriptomic analyses revealed that the bacterial response to inactivated CORM-3 (iCORM-3) is much lower than to the active compound and that a wide range of processes appear to be affected by CORM-3 and to a lesser extent iCORM-3, including energy metabolism, membrane transport, motility, and the metabolism of many sulfur-containing species, including cysteine and methionine. INNOVATION This work has demonstrated that both CORM-3 and its inactivated counterpart react with cellular functions to yield a complex response at the transcriptomic level. A full understanding of the actions of both compounds is vital to understand the toxic effects of CO-RMs. CONCLUSION This work has furthered our understanding of how CORM-3 behaves at the cellular level and identifies the responses that occur when the host is exposed to the Ru compound as well as those that result from the released CO. This is a vital step in laying the groundwork for future development of optimized CO-RMs for eventual use in antimicrobial therapy.

AIMS:Carbon monoxide (CO) delivered to cells and tissues by CO-releasing molecules (CO-RMs) has beneficial and toxic effects not mimicked by CO gas. The metal carbonyl Ru(CO)3Cl(glycinate) (CORM-3) is a novel, potent antimicrobial agent. Here, we established its mode of action. RESULTS:CORM-3 inhibits respiration in several bacterial and yeast pathogens. In anoxic Escherichia coli suspensions, CORM-3 first stimulates, then inhibits respiration, but much higher concentrations of CORM-3 than of a classic protonophore are required for stimulation. Proton translocation measurements (H(+)/O quotients, i.e., H(+) extrusion on pulsing anaerobic cells with O2) show that respiratory stimulation cannot be attributed to true "uncoupling," that is, dissipation of the protonmotive force, or to direct stimulation of oxidase activity. Our data are consistent with CORM-3 facilitating the electrogenic transmembrane movement of K(+) (or Na(+)), causing a stimulation of respiration and H(+) pumping to compensate for the transient drop in membrane potential (ΔΨ). The effects on respiration are not mimicked by CO gas or control Ru compounds that do not release CO. Inhibition of respiration and loss of bacterial viability elicited by CORM-3 are reversible by white light, unambiguously identifying heme-containing oxidase(s) as target(s). INNOVATION:This is the most complete study to date of the antimicrobial action of a CO-RM. Noteworthy are the demonstration of respiratory stimulation, electrogenic ion transport, and photosensitive activity, establishing terminal oxidases and ion transport as primary targets. CONCLUSION:CORM-3 has multifaceted effects: increased membrane permeability, inhibition of terminal oxidases, and perhaps other unidentified mechanisms underlie its effectiveness in tackling microbial pathogenesis.

Carbon monoxide-releasing molecules (CO-RMs) are a class of organometallo carbonyl complexes capable of delivering controlled quantities of CO gas to cells and tissues thus exerting a broad spectrum of pharmacological effects. Here we report on the chemical synthesis, CO releasing properties, cytotoxicity profile and pharmacological activities of four novel structurally related iron-allyl carbonyls. The major difference among the new CO-RMs tested was that three compounds (CORM-307, CORM-308 and CORM-314) were soluble in dimethylsulfoxide (DMSO), whereas a fourth one (CORM-319) was rendered water-soluble by reacting the iron-carbonyl with hydrogen tetrafluoroborate. We found that despite the fact all compounds liberated CO, CO-RMs soluble in DMSO caused a more pronounced toxic effect both in vascular and inflammatory cells as well as in isolated vessels. More specifically, iron carbonyls soluble in DMSO released CO with a fast kinetic and displayed a marked cytotoxic effect in smooth muscle cells and RAW 247.6 macrophages despite exerting a rapid and pronounced vasorelaxation ex vivo. In contrast, CORM-319 that is soluble in water and liberated CO with a slower rate, preserved smooth muscle cell viability, relaxed aortic tissue and exerted a significant anti-inflammatory effect in macrophages challenged with endotoxin. These data suggest that iron carbonyls can be used as scaffolds for the design and synthesis of pharmacologically active CO-RMs and indicate that increasing water solubility and controlling the rate of CO release are important parameters for limiting their potential toxic effects.

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 (Km 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.

Carbon monoxide-releasing molecules (CO-RMs) emulate the beneficial (e.g., anti-inflammatory) effects of CO in biology. CO release from CO-RMs is routinely determined in the presence of reduced deoxy-myoglobin by measuring the formation of carboxy-myoglobin (Mb-CO). Previous studies have highlighted discrepancies between the apparent CO release rates of some CO-RMs established using this assay versus other experimental data where a slower or more complex mechanism of release is suggested. It has been hypothesized that some CO-RMs require a CO acceptor, believed to be reduced myoglobin in Mb-CO assays, in order to facilitate the release of CO. Here, we show, for the first time, that CO is not liberated from the ruthenium (Ru)-based [Ru(CO)3Cl2]2 (CORM-2) and [Ru(CO)3Cl(glycinate)] (CORM-3) at an appreciable rate in the presence of reduced myoglobin alone. Rather, we confirm that it is the reducing agent sodium dithionite that facilitates release of CO from these CO-RMs. Other sulfite compounds, namely sodium sulfite and potassium metabisulfite, also promote the liberation of CO from CORM-3. We describe an alternative oxy-hemoglobin assay that eliminates dithionite and suggest that the efficacy of CO-RMs results from intracellular interactions with anions that facilitate CO delivery to therapeutic targets.

New CO-releasing molecules, [Fe(CO)(3)X-(S2CNR2)] and [Fe(CO)(3)I(S2COEt)], are reported. [Fe-(CO)(3)X(S2CNR2)] releases the first two carbonyls rapidly to myoglobin (t(1/2) < 1 min) and the third carbonyl more slowly. In the case of [Fe(CO)(3)I(S2COEt)], only 0.4 mol of CO are lost. [Fe(CO)(3)Br(S2CNEt2)] has low toxicity. CO loss is much slower from [Fe(CO)(2)(S2CNR2)(2)] (R-2 = Me-2, Et-2, (CH2CH2)(2)O, (CH2C2H2)(2), (CH2CH2OH)(2); t(1/2) >24 h) and is not observed from [Fe(CO)(2)(S2COEt)(2)]. The mechanism of CO loss was investigated using Gaussian 09 calculations for [Fe(CO)(3)Br(S2CNMe2)] and [Fe(CO)(2)(S2CNMe2)(2)]. The X-ray structures of [Fe(CO)(3)Br(S2CNMe2)] and [Fe(CO)(3)I-(S2CNEt2)](2)I-2 were determined.

Objective— We compared the antithrombotic effects in vivo of 2 chemically different carbon monoxide–releasing molecules (CORM-A1 and CORM-3) on arterial and venous thrombus formation and on hemostatic parameters such as platelet activation, coagulation, and fibrinolysis. The hypotensive response to CORMs and their effects on whole blood gas analysis and blood cell count were also examined. Methods and Results— CORM-A1 (10–30 µmol/kg, i.v.), in a dose-dependent fashion, significantly decreased weight of electrically induced thrombus in rats, whereas CORM-3 inhibited thrombosis only at the highest dose used (30 µmol/kg). CORM-A1 showed a direct and stronger inhibition of platelet aggregation than CORM-3 in healthy rats, both in vitro and in vivo. The antiaggregatory effect of CORM-A1, but not CORM-3, correlated positively with weight of the thrombus. Concentration of active plasminogen activator inhibitor-1 in plasma also decreased in response to CORM-A1, but not to CORM-3. Neither CORM-A1 nor CORM-3 had an effect on plasma concentration of active tissue plasminogen activator. CORM-3, but not CORM-A1, decreased the concentration of fibrinogen, fibrin generation, and prolonged prothrombin time. Similarly, laser-induced venous thrombosis observed intravitally via confocal system in green fluorescent protein mice was significantly decreased by CORMs. Although both CORM-A1 and CORM-3 (30 µmol/kg) decreased platelets accumulation in thrombus, only CORM-A1 (3–30 µmol/kg) inhibited platelet activation to phosphatidylserine on their surface. Conclusion— CORM-3 and CORM-A1 inhibited thrombosis in vivo, however CORM-A1, which slowly releases carbon monoxide, and displayed a relatively weak hypotensive effect had a more pronounced antithrombotic effect associated with a stronger inhibition of platelet aggregation associated with a decrease in active plasminogen activator inhibitor-1 concentration. In contrast, the fast CO releaser CORM-3 that displayed a more pronounced hypotensive effect inhibited thrombosis primarily through a decrease in fibrin generation, but had no direct influence on platelet aggregation and fibrynolysis.

The development of CO-releasing molecules, CO-RMs, for medical applications is reviewed from a personal point of view. The review covers the initial discovery of CO-RMs and then concentrates on developments involving the author. The review finishes with suggestions for areas meriting further investigation.

[Mn(CO)(4){S(2)CNMe(CH(2)CO(2)H)}], 1, is shown to be a CO releasing molecule providing at least three moles CO per mole of compound. The mechanism of CO loss is dissociative and reversible and was investigated using Gaussian 09 calculations. The reversible binding of CO results in a relatively stable solution of the compound, while in the presence of a CO receptor or a ligand to prevent the rebinding of CO, the CO is lost rapidly. The X-ray structure was determined.

[Fe(L-cysteinate)(2)(CO)(2)](2-) is a CO releasing molecule which has low cytotoxicity to RAW264.7 macrophages. It provides an example of CO binding using ligands available to ion channels which use CO as a signalling molecule in the absence of heme. Previous work has shown that this compound consists of five isomers and it was proposed that the two isomers with trans-dicarbonyls are dominant. In this work the isomers are re-assigned and shown to be capable of releasing CO, albeit too slowly to act as a signalling receptor. It is shown that by linking the two L-cysteines together to form [Fe(SCH(2)CH{CO(2)H}NHCH(2))(2)(CO)(2)], only one isomer is isolated.

Carbon monoxide (CO) is a classical respiratory inhibitor, but CO‐releasing molecules (CO‐RMs) have therapeutic value, increasing phagocytosis, and reducing sepsis‐induced lethality. CORM‐3, Ru(CO) 3 Cl(glycinate), a ruthenium‐based carbonyl that liberates CO under physiological conditions, has previously been shown to inhibit bacterial growth and respiration, even at high concentrations of oxygen. Here, we report the effects of CORM‐3 on the microaerophilic foodborne pathogen Campylobacter jejuni . Even at CO‐RM ( i.e. , CO) concentrations that exceed dissolved oxygen levels, CORM‐3 does not inhibit microaerobic growth. This insensitivity is not due to failure of CORM‐3 to penetrate cells, as revealed by assay with extracellular myoglobin and by the ability of CO from externally added CORM‐3 to bind intracellular membrane‐associated respiratory oxidases. Even at almost 200 μ M oxygen, CORM‐3 inhibits formate‐dependent respiration and leads to generation of hydrogen peroxide. This work shows that CO‐RMs have valuable properties as antimicrobial agents; however, growth inhibition does not always accompany inhibition of respiration, even when ambient oxygen concentrations are low. © 2011 IUBMB IUBMB Life, 63(5): 363‐371, 2011