The inducible stress protein heme oxygenase-1 (HO-1) has been linked to tissue and organ protection against the deleterious actions of many pathological conditions, including endotoxin challenge. Similar protection can be achieved by the main products of heme oxygenase activity, namely bilirubin and carbon monoxide (CO). Since the identification of novel chemical compounds that liberate CO in biological systems (CO-releasing molecules or CO-RMs), our group and others have had access to a convenient and simple pharmacological tool that enables to study the role of CO in physiological functions. This article will review the scientific literature published to date on CO-RMs, with emphasis on the in vitro, ex vivo and in vivo experimental models employed to determine the contribution of CO to cellular mechanisms. In addition, we will report on the effect of heme oxygenase-related substances, such as bilirubin, CORM-3 and hemin, in a model of endotoxin-induced hypotension. Among the three different approaches examined, CORM-3 proved the most effective agent in reducing the fall in blood pressure caused by endotoxin. Furthermore, heme oxygenase-related substances affected the endotoxin-stimulated induction and distribution of hepatic HO-1 and inducible nitric oxide synthase (iNOS). Thus, it emerges that CO-RMs could exert important biological actions in the context of endotoxic-mediated dysfunction.

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 μM) 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‐α, 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. British Journal of Pharmacology (2005) 145, 800–810. doi:10.1038/sj.bjp.0706241

Emerging evidence reveals that heme oxygenase-1 (HO-1) and its product carbon monoxide (CO) can exert diverse biological and cytoprotective effects. Our group has recently identified a new class of compounds (CO-releasing molecules or CO-RMs) that can carry and deliver CO to biological systems and can be used to examine the physiological properties of CO. Here, we evaluated the influence of endogenously-generated CO (via HO-1 induction by hemin) and CO liberated from exogenously supplied CO-RMs on mitochondrial function. Renal mitochondria were isolated either from rats with increased HO-1 or from untreated animals, the latter being exposed to different concentrations of CO-RMs (10-100 microM). We found that mitochondrial oxygen uptake was significantly reduced in kidneys after HO-1 induction and, in a similar fashion, CO-RMs inhibited mitochondrial function in a concentration-dependent manner. Specifically, a marked depression of state 3 was observed resulting in a significant decrease in respiratory control index (RCI) values. When mitochondria were incubated with the inactive forms of CO-RMs, which are devoid of CO, the respiratory parameters remained unchanged. In summary, the results indicate that HO-1 induction and enhanced CO decrease renal oxygen consumption and alter mitochondrial function suggesting that CO could be a physiological regulator of mitochondrial oxidative phosphorylation.

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.

P85 Introduction: Carbon monoxide (CO) administration has been shown to protect from ischemia reperfusion (IR) injury associated with organ transplantation. However, CO administration by gas inhalation, as currently done in experimental transplantation, offers the disadvantage of being complicated and unpractical particularly with regard to its possible future clinical application. Therefore, we have studied the potential therapeutic effects of a recently generated CO-releasing molecule (CO-RM) as a new mode of administering CO for clinical use. Methods: In a preliminary pharmacodynamic study in the pig, tricarbonylchloro(glycinato) ruthenium(II) (CORM-3) was administered as a single dose of 4 mg/kg i.v. into two Large White pigs. PBMC isolated at 2, 6, 12, 24, 48, 72 and 168 hrs after CORM-3 administration, were stimulated in vitro with 1-10 μg/ml LPS and the production of TNF-α, one of the key initiators of apoptosis in IR injury, was evaluated by ELISA. Subsequently, four immunosuppressed Large White pigs were induced with 4 mg/kg of CORM-3 one day before renal transplantation and received the drug at 1 mg/kg or 4 mg/kg every second day until the end of the experiment. In these allograft recipients, the efficacy of CORM-3 on the in vitro production of TNF-α was evaluated on the day of transplantation and at the time of euthanasia. All the routine haematological and biochemical parameters were monitored throughout the experiments. Results: A single dose of 4 mg/kg of CORM-3 was able to significantly reduce TNF-α production by PBMC starting from 24 hrs after administration. This effect was maintained for 48 hrs. 72 hrs after a single dose treatment, the levels of TNF-α were back to the control pre-dose values or even higher. The efficacy of CORM-3 as an inhibitor of TNF-α production has also been observed in allotransplanted pigs where PBMC isolated after 24 hr treatment with CORM-3 were unable to release TNF-α after LPS stimulation. In these animals, CORM-3 administration was not associated with any noticeable side-effects. Conclusion: At the doses used in this study, in vivo administration of CORM-3 in a large animal model is well-tolerated and is able to interfere with TNF-α production by PBMC. As TNF-α is a key initiator of the apoptotic events in IR injury, it is expected that this approach may prove useful for the long term survival of transplanted organs.

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.

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 (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.

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.

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.