Vasorelaxing effects and inhibition of nitric oxide in macrophages by new iron-containing carbon monoxide-releasing molecules (CO-RMs).

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.

Cardioprotective actions by a water-soluble carbon monoxide-releasing molecule.

Carbon monoxide, which is generated in mammals during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator. Transition metal carbonyls have been recently shown to function as carbon monoxide-releasing molecules (CO-RMs) and to elicit distinct pharmacological activities in biological systems. In the present study, we report that a water-soluble form of CO-RM promotes cardioprotection in vitro and in vivo. Specifically, we found that tricarbonylchloro(glycinato)ruthenium(II) (CORM-3) is stable in water at acidic pH but in physiological buffers rapidly liberates CO in solution. Cardiac cells pretreated with CORM-3 (10 to 50 micromol/L) become more resistant to the damage caused by hypoxia-reoxygenation and oxidative stress. In addition, isolated hearts reperfused in the presence of CORM-3 (10 micromol/L) after an ischemic event displayed a significant recovery in myocardial performance and a marked and significant reduction in cardiac muscle damage and infarct size. The cardioprotective effects mediated by CORM-3 in cardiac cells and isolated hearts were totally abolished by 5-hydroxydecanoic acid, an inhibitor of mitochondrial ATP-dependent potassium channels. Predictably, cardioprotection is lost when CORM-3 is replaced by an inactive form (iCORM-3) that is incapable of liberating CO. Using a model of cardiac allograft rejection in mice, we also found that treatment of recipients with CORM-3 but not iCORM-3 considerably prolonged the survival rate of transplanted hearts. These data corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO and highlight the bioactivity and potential therapeutic features of CO-RMs in the mitigation of cardiac dysfunction. The full text of this article is available online at http://www.circresaha.org.

Carbon monoxide-releasing molecules (CO-RMs) attenuate the inflammatory response elicited by lipopolysaccharide in RAW264.7 murine macrophages.

The enzyme heme oxygenase-1 (HO-1) is a cytoprotective and anti-inflammatory protein that degrades heme to produce biliverdin/bilirubin, ferrous iron and carbon monoxide (CO). The anti-inflammatory properties of HO-1 are related to inhibition of adhesion molecule expression and reduction of oxidative stress, while exogenous CO gas treatment decreases the production of inflammatory mediators such as cytokines and nitric oxide (NO). CO-releasing molecules (CO-RMs) are a novel group of substances identified by our group that are capable of modulating physiological functions via the liberation of CO. We aimed in this study to examine the potential anti-inflammatory characteristics of CORM-2 and CORM-3 in an in vitro model of lipopolysaccharide (LPS)-stimulated murine macrophages. Stimulation of RAW264.7 macrophages with LPS resulted in increased expression of inducible NO synthase (iNOS) and production of nitrite. CORM-2 or CORM-3 (10-100 microM) reduced nitrite generation in a concentration-dependent manner but did not affect the protein levels of iNOS. CORM-3 also decreased nitrite levels when added 3 or 6 h after LPS exposure. CORM-2 or CORM-3 did not cause any evident cytotoxicity and produced an increase in HO-1 expression and heme oxygenase activity; this effect was completely prevented by the thiol donor N-acetylcysteine. CORM-3 also considerably reduced the levels of tumor necrosis factor-alpha, another mediator of the inflammatory response. The inhibitory effects of CORM-2 and CORM-3 were not observed when the inactive compounds, which do not release CO, were coincubated with LPS. These results indicate that CO liberated by CORM-2 and CORM-3 significantly suppresses the inflammatory response elicited by LPS in cultured macrophages and suggest that CO carriers can be used as an effective strategy to modulate inflammation.

Metal carbonyls: a new class of pharmaceuticals?

It is now established that NO is a messenger molecule in mammals despite its high toxicity. As NO(+) and CO are isoelectronic, it should not be unexpected that CO could also have a role as a messenger. CO is produced naturally in humans at a rate of between 3 and 6 cm(3) per day, and this rate is increased markedly by certain inflammatory states and pathological conditions associated with red blood cell hemolysis. Over the last 10 years, the interest in the biological effects of CO has greatly increased, and it is now established in the medical literature that CO does have a major role as a signaling molecule in mammals. It is particularly active within the cardiovascular system, for example, in suppressing organ graft rejection and protecting tissues from ischemic injury and apoptosis. Recently it has been shown that metal carbonyls can also function as CO-releasing molecules and provide similar biological activities. This opens the possibility to develop pharmaceutically important metal carbonyls.

Bioactivity and pharmacological actions of carbon monoxide-releasing molecules.

Carbon monoxide (CO) is a resourceful gas as recent advances in the area of cell signaling are revealing an unexpected physiological role for CO in the cardiovascular, immune and nervous systems. Transition metal carbonyls have been lately discovered to function as CO-releasing molecules (CO-RMs) and elicit distinct pharmacological activities in biological systems. Studies currently ongoing in our laboratories are investigating both the chemical and bioactive features of a series of water-soluble CO-RMs and their specific utilization as vasoactive mediators, anti-inflammatory agents and inhibitors of cellular and tissue damage. The data presented in this review corroborate the notion that transition metal carbonyls could be used as carriers to deliver CO in mammals and highlight the bioactivity and potential therapeutic features of CO-RMs in the mitigation of cellular and organ dysfunction.

Vasoactive properties of CORM-3, a novel water-soluble carbon monoxide-releasing molecule.

1 Carbon monoxide (CO), one of the end products of heme catabolism by heme oxygenase, possesses antihypertensive and vasodilatory characteristics. We have recently discovered that certain transition metal carbonyls are capable of releasing CO in biological fluids and modulate physiological functions via the delivery of CO. Because the initial compounds identified were not water soluble, we have synthesized new CO-releasing molecules that are chemically modified to allow solubility in water. The aim of this study was to assess the vasoactive properties of tricarbonylchloro(glycinato)ruthenium(II) (CORM-3) in vitro and in vivo. 2 CORM-3 produced a concentration-dependent relaxation in vessels precontracted with phenylephrine, exerting significant vasodilatation starting at concentrations of 25-50 microm. Inactive CORM-3, which does not release CO, did not affect vascular tone. 3 Blockers of ATP-dependent potassium channels (glibenclamide) or guanylate cyclase activity (ODQ) considerably reduced CORM-3-dependent relaxation, confirming that potassium channels activation and cGMP partly mediate the vasoactive properties of CO. In fact, increased levels of cGMP were detected in aortas following CORM-3 stimulation. 4 The in vitro and in vivo vasorelaxant activities of CORM-3 were further enhanced in the presence of YC-1, a benzylindazole derivative which is known to sensitize guanylate cyclase to activation by CO. Interestingly, inhibiting nitric oxide production or removing the endothelium significantly decreased vasodilatation by CORM-3, suggesting that factors produced by the endothelium influence CORM-3 vascular activities. 5 These results, together with our previous findings on the cardioprotective functions of CORM-3, indicate that this molecule is an excellent prototype of water-soluble CO carriers for studying the pharmacological and biological features of CO.

A re-investigation of [Fe(L-cysteinate)2(CO)2]2-: an example of non-heme CO coordination of possible relevance to CO binding to ion channel receptors.

[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.

Iron indenyl carbonyl compounds: CO-releasing molecules.

New CO-releasing molecules, CO-RMs, based on indenyl iron carbonyls have been identified. Half-lives for carbon monoxide (CO) release, (1)H NMR, (13)C NMR, IR, mass spectra, elemental analysis and biological data have been determined for the compounds. Limited correlations have been made between half-lives and ΔG(‡) for CO release and spectroscopic parameters, ν(CO) and δ((13)CO). X-ray structures have been determined for [Fe(η(5)-C(9)H(7))(CO)(2)L][BF(4)] where L is CO, NCMe, PPh(3), P(OPh)(3), NC(5)H(5) or 1-methylimidazole. Improved preparations of [Fe(η(5)-C(9)H(7))(CO)(2)](2) and [Fe(η(5)-C(9)H(7))(CO)(3)][BF(4)] are reported.

Metal carbonyls as pharmaceuticals? [Ru(CO)3Cl(glycinate)], a CO-releasing molecule with an extensive aqueous solution chemistry.

The pharmacologically active [Ru(CO)(3)Cl(glycinate)] is shown to be in equilibrium with [Ru(CO)(2)(CO(2)H)Cl(glycinate)](-) (isomers) at around pH 3.1 which then at physiological pH reacts with more base to give [Ru(CO)(2)(CO(2))Cl(glycinate)](2-) (isomers) or [Ru(CO)(2)(CO(2)H)(OH)(glycinate)](-) (isomers). The ease with which [Ru(CO)(3)Cl(glycinate)] reacts with hydroxide results in it producing a solution in water with a pH of around 2 to 2.5 depending on concentration and making its solutions more acidic than those of acetic acid at comparable concentrations. Acidification of [Ru(CO)(3)Cl(glycinate)] with HCl gives [Ru(CO)(3)Cl(2)(NH(2)CH(2)CO(2)H)]. The crystal structures of [Ru(CO)(3)Cl(glycinate)] and [Ru(CO)(3)Cl(2)(NH(2)CH(2)CO(2)Me)] are reported.

[(Eta-C5H4R)Fe(CO)2X], X = Cl, Br, I, NO3, CO2Me and [(eta-C5H4R)Fe(CO)3]+, R = (CH2)nCO2Me (n = 0-2), and CO2CH2CH2OH: a new group of CO-releasing molecules.

A new group of CO-releasing molecules, CO-RMs, based on cyclopentadienyl iron carbonyls have been identified. X-Ray structures have been determined for [(eta-C(5)H(4)CO(2)Me)Fe(CO)(2)X], X = Cl, Br, I, NO(3), CO(2)Me, [(eta-C(5)H(4)CO(2)Me)Fe(CO)(2)](2), [(eta-C(5)H(4)CO(2)CH(2)CH(2)OH)Fe(CO)(2)](2) and [(eta-C(5)H(4)CO(2)Me)Fe(CO)(3)][FeCl(4)]. Half-lives for CO release, (1)H, (13)C, and (17)OC NMR and IR spectra have been determined along with some biological data for these compounds, [(eta-C(5)H(4)CO(2)CH(2)CH(2)OH)Fe(CO)(3)](+) and [[eta-C(5)H(4)(CH(2))(n)CO(2)Me]Fe(CO)(3)](+), n = 1, 2. More specifically, cytotoxicity assays and inhibition of nitrite formation in stimulated RAW264.7 macrophages are reported for most of the compounds analyzed. [(eta-C(5)H(5))Fe(CO)(2)X], X = Cl, Br, I, were also examined for comparison. Correlations between the half-lives for CO release and spectroscopic parameters are found within each group of compounds, but not between the groups.

Protection against cisplatin-induced nephrotoxicity by a carbon monoxide-releasing molecule.

Nephrotoxicity is one of the main side effects caused by cisplatin (CP), a widely used antineoplastic agent. Here, we examined the effect of a novel water-soluble carbon monoxide-releasing molecule (CORM-3) on CP-mediated cytotoxicity in renal epithelial cells and explored the potential therapeutic benefits of carbon monoxide in CP-induced nephrotoxicity in vivo. Exposure of LLC-PK(1) cells to CP (50 microM) caused significant apoptosis as evidenced by caspase-3 activation and an increased number of floating cells. Treatment with CORM-3 (1-50 microM) resulted in a remarkable and concentration-dependent decrease in CP-induced caspase-3 activity and cell detachment. This effect involved activation of the cGMP pathway as 1H-oxadiazole [4, 3-a] quinoxaline-1-ore (ODQ), a guanylate cyclase inhibitor, completely abolished the protection elicited by CORM-3. Using a rat model of CP-induced renal failure, we found that treatment with CP (7.5 mg/kg) caused a significant elevation in plasma urea (6.6-fold) and creatinine (3.1-fold) levels, which was accompanied by severe morphological changes and marked apoptosis in tubules at the corticomedullary junction. A daily administration of CORM-3 (10 mg/kg ip), starting 1 day before CP treatment and continuing for 3 days thereafter, resulted in amelioration of renal function as shown by reduction of urea and creatinine levels to basal values, a decreased number of apoptotic tubular cells, and an improved histological profile. A negative control (iCORM-3) that is incapable of liberating CO failed to prevent renal dysfunction mediated by CP, indicating that CO is directly involved in renoprotection. Our data demonstrate that CORM-3 can be used as an effective therapeutic adjuvant in the treatment of CP-induced nephrotoxicity.

Administration of a CO-releasing molecule induces late preconditioning against myocardial infarction.

Mounting evidence suggests that carbon monoxide (CO) exerts powerful cytoprotective actions. CO-releasing molecules (CORMs) offer an effective means of delivering CO to tissues in vivo. The goal of the present study was to determine whether a water-soluble CORM, tricarbonylchloro(glycinato)ruthenium(II) (CORM-3), induces delayed protection against myocardial infarction 24 h later and to explore the duration of this protection. Mice received a 60-min i.v. infusion of CORM-3 or inactive CORM-3 (which does not release CO) and then, 24, 72, or 120 h later, underwent a 30-min coronary occlusion followed by 24 h of reperfusion. Pretreatment with CORM-3 24 h prior to coronary occlusion markedly reduced infarct size (24.8% +/- 2.9% of the risk region vs. 43.8% +/- 4.4% with inactive CORM-3). The infarct-sparing effect of CORM-3 was still evident 72 h after administration of the CO donor (20.4% +/- 3.7% of the risk region vs. 41.9% +/- 2.5% with inactive CORM-3) but was no longer apparent at 120 h. Both at 24 and 72 h, the protective effects of CORM-3 were equivalent to those afforded by the late phase of ischemic preconditioning (PC; 27.0% +/- 2.9% and 30.3% +/- 3.9% of the risk region, respectively). We conclude that the novel CO-releasing compound, CORM-3, induces delayed protection against myocardial infarction which is similar to that afforded by the late phase of ischemic PC, and that this salubrious effect is sustained for 72 h. To our knowledge, this is the first report that exposure to CO causes the heart to shift to a preconditioned phenotype. In addition, this study provides the first evidence that the cardioprotective actions of ischemic PC persist for 72 h in the mouse.