Therapeutic Applications of Carbon Monoxide-Releasing Molecules

Review Therapeutic applications of carbon monoxide-releasing molecules Roberto Motterlini†, Brian E Mann & Roberta Foresti 1. Introduction 2. Identification, bioactivity and therapeutic applications of CO-RMs †Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, UK Carbon monoxide (CO), which is formed in mammalian cells through the oxidation of haem by the enzyme haem oxygenase, actively participates in the regulation of key intracellular functions. Emerging evidence reveals that an increased generation of haem oxygenase-derived CO plays a critical role in the resolution of inflammatory processes and alleviation of cardiovascular disorders. The authors have identified a novel class of substances, CO-releasing molecules (CO-RMs), which are capable of exerting a variety of pharmacological activities via the liberation of controlled amounts of CO in biological systems. A wide range of CO carriers containing manganese (CORM-1), ruthenium (CORM-2 and -3), boron (CORM-A1) and iron (CORM-F3) are currently being investigated to tailor therapeutic approaches for the prevention of vascular dysfunction, inflammation, tissue ischaemia and organ rejection. 3. Expert opinion P r o r f o Keywords: bilirubin, carbon monoxide, carbon monoxide-releasing molecules, cardiovascular disease, haem oxygenase-1, inflammation, kidney disease, oxidative stress, vasorelaxation o h Expert Opin. Investig. Drugs (2005) 14(11):xxx-xxx A Ashley Publications www.ashley-pub.com t u 1. Introduction Mammalian haem oxygenase enzymes catalyse the conversion of haem to ferrous iron, carbon monoxide (CO) and biliverdin, which is subsequently reduced to bilirubin by biliverdin reductase [1]. Haem is oxidised at the α-position by haem oxygenase (HO) and participates in the reaction as both a prosthetic group and substrate, whereas O2 and NADPH are required as cofactors [2]. Both the constitutive (HO-2) and inducible (HO-1) isoforms of HO have been characterised, and together they contribute to the control and maintenance of the intracellular haem concentration. Thus, HO-2 is present in normal conditions in organs and tissues, such as brain, liver and endothelium, and its function is associated with neurotransmission and regulation of vascular tone [1,3]. In contrast, stressful insults that cause a threat to cell homeostasis and survival stimulate a high expression of HO-1 in all of the tissues examined, where the protein exerts powerful protective actions (Figure 1) [3-5]. HO-1-mediated protection is intrinsically linked to its enzymatic activity in as much as the fine control of endogenous haem levels leads to at least two important consequences: the risk of oxidative reactions promoted by haem is limited; and haem acts as the substrate for production of the antioxidant couple biliverdin/bilirubin and the signalling molecule CO [1]. Indeed, many of the beneficial effects ascribed to HO-1 are strictly dependent on its metabolic products; for example, studies have shown that pathological insults causing tissue ischaemia/ hypoxia or promoting a redox imbalance induce HO-1 [6,7], which in turn offers cytoprotection and exerts antioxidant activity (Figure 1) [8-11]. Interestingly, biliverdin or bilirubin can achieve similar protective effects whereby cellular and tissue damage imposed by oxidants [9,11,12], ischaemia–reperfusion (I/R) events [10], 10.1517/13543784.14.11.xxx © 2005 Ashley Publications ISSN 1354-3784 1 Therapeutic applications of carbon monoxide-releasing molecules Antioxidant activities Redox imbalance Ischaemia/hypoxia HO-1 ↑ CO ↑ Bilirubin Fe Anti-apoptosis Cytoprotection f o Inflammatory mediators Apoptotic signals Anti-inflammatory action P r o r Figure 1. Schematic diagram on the potential biological activities of the HO-1 pathway. CO: Carbon monoxide; HO: Haem oxyenase. endotoxin challenge [13] or transplantation [14] is prevented or greatly reduced by pretreatment with bile pigments. The role of iron as a potential protective factor has not been thoroughly investigated, and it has been suggested that HO-1 regulates cell viability by modulating iron efflux [15]. However, there is a consistent set of studies that support beneficial effects elicited by CO. The CO that is derived from HO acts as a signalling molecule with antihypertensive [16,17], antiproliferative [18,19] and antiapoptotic properties [20]. In addition, HO-1-mediated anti-inflammatory action [21], which was initially recognised in a model of acute complement-dependent inflammatory response [22], is partially attributable to biliverdin/bilirubin and CO. It appears that bile pigments exert anti-inflammatory effects by reducing the production of nitric oxide (NO) and expression of the adhesion molecule intercellular adhesion molecule-1 (ICAM-1) in vivo [13,23]; recent findings also show that bilirubin blocks vascular cell adhesion molecule-1-dependent leukocytes migration, apparently through suppression of cellular reactive oxygen species production [24]. Likewise, CO gas administered at low concentrations reduces ICAM-1 expression and modulates the production of inflammatory mediators by decreasing pro-inflammatory cytokines such as TNF-α, and increasing crucial anti-inflammatory molecules, such as IL-10, in various experimental models in vitro and in vivo [25,26]. The cellular cascade responsible for the protective effects exerted by CO involves different effectors. Guanylate cyclase is one of the first identified and seems to contribute to the regulation of vascular tone [17], neurotransmission [27] and cell proliferation but also other A 2 t u o h beneficial actions related to organ I/R [14]. Potassium channels are a second pathway utilised by CO to exert biological functions and data in the literature support their involvement in CO-induced vasorelaxation [28,29]. In addition, CO appears to act via mitogen-activated protein kinase (MAPK) pathways (activation of p38) and decreased c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK) phosphorylation, and these effects depend on the cell type and stimulus employed [4,30,31]. It is interesting to note that the targets discovered so far for CO-mediated endogenous functions are in common with those used by NO [32-34]. The similarity between CO and NO as gaseous messengers has been previously emphasised [3]; however, as more investigations shed light on the mechanisms underlying the effects of CO, it is becoming evident that the molecular and biochemical pathways characterising the reactions of the two gases are very different and probably reflect their diverse chemical properties and reactivity. It is through a chemical approach that the authors decided to begin an investigation on the biological, pharmacological and therapeutic activities of CO in physiology and disease. 2. Identification, bioactivity and therapeutic applications of CO-RMs 2.1 Transition metal carbonyl complexes: discovery of bioactive CO-RMs In the late 1990s, the suggestion that CO gas could be used to alleviate pathophysiological conditions characterised by oxidative stress and inflammatory states was rapidly Expert Opin. Investig. Drugs (2005) 14(11) Motterlini, Mann & Foresti emerging [3,35] and incited the authors’ group at Northwick Park, UK to investigate more assiduously on the possibility of exploiting CO as a true therapeutic agent. The concept of delivering low concentrations of CO gas to mitigate disease was quite a remarkable and provocative proposal [25] and was certainly in line with the intriguing notion that endogenous CO derived from HO-1 activity contributes to important intracellular functions that are aimed at increasing the tolerance of organs and tissues to a variety of detrimental effects [16,22,36]. However, the authors’ perception was that the use of gas mixtures in humans could pose some constraints, not least the difficulty of storing and delivering CO in a controlled directed manner. Moreover, it is known that prolonged inhalation of CO may lead to problems related to the systemic effects imposed by this gas on oxygen transport and delivery [37], hence making this approach of limited use in a therapeutic context. From a pharmacological perspective and to facilitate the possible development of novel pharmaceutical agents suitable for therapeutic applications, it was reasoned that this obstacle could be overcome by storing CO in a ‘stable chemical form’, thus permitting the CO groups to be carried and supplied to cells or tissues in a more convenient fashion. It was envisaged that compounds possessing similar features could already have been described in the chemistry literature and, if found, could form the basis for assessing the feasibility of the hypothesis. Indeed, the search led to the encouraging finding of an interesting group of chemicals, transition metal carbonyls, renowned in the organometallic chemistry field for their versatile use in industrial catalysis and purification processes [38]. These complexes contain a transition metal, such as manganese, cobalt or iron, surrounded by a certain number of carbonyl (CO) groups as coordinated ligands. One interesting feature is that some of these compounds are sensitive to light and, under certain conditions, photoexcitation of the metal–carbonyl bond can lead to dissociative loss of CO [39]. After learning from the literature that this was the typical behaviour of manganese decacarbonyl [Mn2(CO)10] [40], the authors tested whether this metal complex has the ability to liberate CO in a biological environment and ultimately promote a pharmacological response that is representative of CO gas. Because the photodissociation of CO from [Mn2(CO)10] has been reported to occur solely in organic solvents and as this metal carbonyl has very limited solubility in water [40], dimethyl sulfoxide (DMSO) was used as the vehicle for the investigation. The initial experiments using in vitro and ex vivo systems provided intriguing results. First, following stimulation with light, Mn2(CO)10 liberated CO in aqueous solutions in a 1:1 ratio as quantified spectrophotometrically by measuring the conversion of deoxymyoglobin to carbonmonoxy myoglobin (MbCO) (see below for details) [41,42]. Second, isolated rat hearts perfused in the presence of Mn2(CO)10 displayed a marked attenuation in coronary vasoconstriction when challenged with an inhibitor of NO synthase (L-nitro-arginine methyl ester [L-NAME]) [41], an effect that could be simulated by activation of the HO-1/ A t u o h CO pathway [16,17]. Finally, and perhaps most importantly, mitigation of coronary vasoconstriction by Mn2(CO)10 was elicited only after stimulation with light to release CO and was absent when perfusion of the metal carbonyl was conducted in the dark [41]. Collectively, these results provided the first evidence that CO liberated from a transition metal carbonyl is directly responsible for the observed pharmacological effect and that similar classes of compounds could effectively deliver CO into cellular and biological environments. The term ‘CO-releasing molecules’ (CO-RMs) was coined to classify bioactive CO carriers and, being the first identified, the acronym of CORM-1 was assigned to Mn2(CO)10 [32], which chemical structure and other properties are reported in Table 1. The encouraging data from the initial studies prompted a search for additional metal carbonyls that, despite their poor solubility in water, could release CO in aqueous solutions through mechanisms that were not restricted to the photodissociation reactions that are distinctive of CORM-1. It was soon realised that ruthenium-based carbonyl complexes could be the ideal candidates for the purpose because the wide range of ligands available for this metal in aqueous solutions enables the modulation of carbonyl binding to the metal centre [43]. From a biological perspective, it is also important to note that compounds containing ruthenium metals are being developed as therapeutic agents in the treatment of cancer and inflammation [43,44]. Tricarbonyldichlororuthenium II dimer ([Ru(CO)3Cl2]2) was selected among a series of commercially available ruthenium-based carbonyls because data from the literature suggested the ability of the metal centre to react reversibly with DMSO, thus leading to the loss of the CO group [45]. The chemical structure of [Ru(CO)3Cl2]2, which was termed CORM-2 in agreement with the nomenclature now used for bioactive CO carriers [32], is reported in Table 1. It should be emphasised that the myoglobin assay used in the experiments is a crucial step for assessing the ability of transition metal carbonyls to liberate CO in aqueous solutions and determining their potential role as bioactive CO-RMs. The absorption spectra of deoxy-myoglobin, oxy-myoglobin and carbonmonoxy myoglobin (MbCO), which is formed after reaction of deoxy-myoglobin with CO gas, are represented in Figure 2A. When CORM-2 is solubilised in DMSO and immediately added to a solution containing deoxymyoglobin, a rapid conversion (< 1 min) to the spectrum typical of MbCO is observed (Figure 2B, purple line). Interestingly, the spectrum of deoxy-myoglobin remains unchanged after addition of Ru(DMSO)4Cl2, a negative control in which the carbonyl groups have been replaced by DMSO (Figure 2B, blue line). Both CORM-2 and its negative control (Ru(DMSO)4Cl2) were then utilised in a series of experiments to demonstrate incontrovertibly the pharmacological action of CO liberated from the transition metal complex. Indeed, CORM-2 was shown to elicit profound vasodilatation in isolated rat aortae, an effect that was mediated by activation of the guanylate cyclase/cGMP pathway, and inhibited by P r o r Expert Opin. Investig. Drugs (2005) 14(11) f o 3 Therapeutic applications of carbon monoxide-releasing molecules Table 1. Chemical structure, properties and CO release profile of CO-RMs. Compound CORM-1* [Mn2(CO)10] CORM-2* [Ru(CO)3Cl2]2 Chemical structure CO CO CO CO CORM-3 [Ru(CO)3Clglycinate] CORM-A1 [Na2H3BCO2] CORM-F3 [C9H5BrFeO5] CO CO CO CO CO Solubility CO release (in PBS, pH = 7.4, 37°C) Year of Refs identification DMSO Ethanol Light dependent Fast (t½ < 1 min) 1 M CO/mole CO-RM 2001 [32,41,42,46] DMSO Ethanol Induced by ligand substitution Fast (t½ ≈ 1 min) 0.75 M CO/mole CO-RM 2002 [32,46] H2O (stable at acidic pH) Induced by ligand substitution/ water-gas shift reaction Fast (t½ ≈ 1 min) ≈ 1 M CO/mole CO-RM 2003 H2O (stable at basic pH) pH-dependent Slow (t½ ≈ 21 min) 1 M CO/mole CO-RM 2004 [50] Induced by metal oxidation Slow (t½ ≈ 55 min) 0.25 M CO/mole CO-RM 2005 ‡ OC OC CO CO Cl Ru Cl CO CO Ru CO CO CO CO OC Ru OC CO O O 2H H B H OH O 2Na+ DMSO Ethanol Br Fe(CO)3 O O t u o h P r o r f o [32,49,53,56] *Some of the recent studies have interchanged CORM-2 for CORM-1 and used their acronyms incorrectly [69,75]. ‡Fairlamb and Motterlini, unpublished observation. CO: Carbon monoxide; CO-RM: CO-releasing molecule; DMSO: Dimethyl sulfoxide; PBS: Phosphate-buffered saline; t½; Half-life. A scavenging CO with myoglobin. The fact that Ru(DMSO)4Cl2 failed to promote vasorelaxation substantiated a direct involvement of CO in the modulation of vessel tone [46]. The vasoactive properties of CORM-2 were confirmed by showing that administration of this metal carbonyl significantly prevented the increase in mean arterial pressure in a rat model of acute hypertension [46]. Thus, the initial results obtained with CORM-1 and -2 clearly identified metal carbonyls as a new class of bioactive agents that could be chemically modified to improve the therapeutic potential of CO and ultimately develop pharmacologically active compounds for the treatment of a variety of disease states [47]. 2.2 The first water-soluble CO-releasing agents Water solubility is of central importance in the process of drug discovery and development from molecular design to pharmaceutical formulation and biopharmacy. As stated in Section 2.1, both CORM-1 and -2 are soluble in only the few organic solvents that are compatible with biological systems (e.g., DMSO and ethanol); this constraint poses no problem 4 for the investigation in animal models but may restrict the use of such compounds as pharmaceuticals. In addition, the ability of these two metal carbonyls to liberate CO once in contact with aqueous solutions is strictly dependent on their intrinsic chemical features as irradiating light in the case of CORM-1 and ligand substitution for CORM-2 are required to favour CO dissociation, which generally occurs at a very fast rate (< 1 min; Table 1). On the other hand, the versatile chemistry of transition metals enables them to be effectively modified by coordinating biological ligands to the metal centre in order to render the molecule more water soluble and eventually less toxic. These criteria motivated the authors’ group to focus on the synthesis and search for novel water-soluble CO carriers. In addition, the possibility of designing or identifying new compounds that are capable of releasing CO with different dissociation rates was also an attractive and promising feature that the group soon began to consider. Among a series of newly synthesised ruthenium carbonyls in which different amino acid groups were coordinated to the metal centre to render the compounds soluble in water, Expert Opin. Investig. Drugs (2005) 14(11) Motterlini, Mann & Foresti A. B. 0.8 0.8 0.6 0.6 Absorbance Absorbance MbCO 0.4 0.2 deoxy-Mb 0.4 0.2 Deoxy-Mb CORM-2 MbCO Oxy-Mb 0.0 500 520 f o Ru(DMSO)4Cl2 0.0 540 560 580 600 500 λ (nm) 520 o r 540 560 580 600 λ (nm) Figure 2. Spectrophotometric detection of CO release from CO-RMs. A. Typical spectra of deoxy-Mb (blue line), MbCO (red line) and oxy-Mb (black line). B. Conversion of deoxy-Mb to MbCO by CORM-2 (see chemical structure in Table 1 for details). Ru(DMSO)4Cl2, which does not contain carbonyl group, was used as a negative control. P r CO: Carbon monoxide; DMSO: Dimethyl sulfoxide; Mb: Myoglobin; MbCO: Carbonmonoxy myoglobin. tricarbonylchloro(glycinato)ruthenium II (Ru[CO]3Cl(glycinate)) was first selected as a potentially promising agent. This compound, which contains glycine and can be obtained in a relatively pure form (> 95%), was termed CORM-3. Second, from the literature it can be seen that the preparation of certain radioimaging compounds containing carbonyl groups can be performed using a boron-based carbonylating agent, potassium boranocarbonate, which acts as a CO source in aqueous solutions [48]. A similar compound (Na2H3BCO2), named CORM-A1, was obtained in a pure form and tested for its ability to liberate CO. The chemical structure of CORM-3 and CORM-A1 are represented in Table 1, and their bioactive properties summarised in Figure 3. As shown, both CORM-3 and -A1 results in the formation of MbCO when added to a solution containing myoglobin 40 µM (Figure 3A). The rate of MbCO formation and, consequently, the rate of CO release, in phosphate buffer solution is much faster in the case of CORM-3 (half-life [t½] ∼ 1 min, at 37°C and pH = 7.4) compared with CORM-A1 (t½ ∼ 21 min at 37°C and pH = 7.4). In addition, CORM-3 and -A1 elicit a significant vasorelaxation when added to an isolated aortic ring precontracted with phenylephrine (Phe). Interestingly, the vasodilatory effect mediated by these two water-soluble CO-RMs appear to reflect the kinetics of CO released from each molecule as CORM-A1 30 µM/kg produced a gradual and sustained relaxation over a period of 30 min, whereas CORM-3 caused a profound relaxation within a few minutes after addition to the organ bath (Figure 3B). Similarly, the effect on mean arterial pressure (MAP) in vivo is in agreement with the different CO-release A t u o h characteristics of the two compounds as CORM-3 produced a rapid decrease in MAP, whereas a gradual hypotensive effect over time was observed in the case of CORM-A1 (Figure 3C). It is important to note that the doses used are unlikely to promote any toxic effect as both CORM-3 and -A1 are relatively safe and do not significantly affect cell viability on smooth muscle cells in vitro at concentrations ≤ 500 µM (Figure 3D). In addition, the lack of effect observed with inactive CO-RMs (iCO-RMs), which had been deliberately depleted of CO and are frequently used as negative controls in the studies, demonstrates that CO is directly responsible for the vasoactive properties mediated by the water-soluble CO-RMs [49,50]. Although to a different extent, both guanylate cyclase and potassium channel activation significantly contribute to the vasorelaxant effect mediated by CORM-3 and -A1, a priori that other mechanisms might also be involved cannot be excluded [49,50]. Thus, the group’s data confirm that the first prototypic water-soluble transition metal carbonyl (CORM-3) and boranocarbonate (CORM-A1) meet the criteria of pharmacologically active CO carriers that can be used to modulate important physiological function both in vitro and in vivo [32,46,49]. 2.3 Cardiovascular effects of CO-RMs The characterisation of water-soluble CO-RMs and the assessment of their vasoactive properties indicate that low concentrations of exogenously applied CO can mimic the activity of endogenous HO-1-derived CO [16,17]; consequently, CO-RMs could be used for the treatment of those pathological states in which the HO-1 system appears Expert Opin. Investig. Drugs (2005) 14(11) 5 Therapeutic applications of carbon monoxide-releasing molecules A. B. 50 CORM-3 (40 µM) CO-RM (100 µM) CORM-A1 (40 µM) 2.5 Tension (g) MbCO (µM) 40 30 20 1.5 1 0.5 10 0.0 0 10 20 C. 100 30 40 Time (min) 50 Phe 60 90 80 70 50 -100 CON (saline) CORM-3 30 µM/kg CORM-A1 30 µM/kg 0 100 200 300 Time (sec) 10 15 D. CO-RM or saline 60 5 400 500 20 25 30 Time (min) 600 o h Cell viability (% of control) 0 Mean arterial pressure (mmHg) CORM-3 CORM-A1 2 120 80 60 40 P r 20 0 f o * 100 0 o r CORM-3 CORM-A1 * 0.1 0.5 Concentration (mM) * 1 Figure 3. Bioactivities of CORM-3 and -A1. A. Rate of MbCO formation after addition of CORM-3 (blue line) or CORM-A1 (red line) 40 µM to a phosphate buffer solution (pH = 7.4, 37°C) containing deoxy-Mb 40 µM. B. Vasodilatory effects of CORM-3 and CORM-A1 in isolated rat aorta precontracted with Phe. Reprinted with permission from Motterlini R, Sawle P, Bains S et al.: CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule. Reprinted with permission from MOTTERLINI R, SAWLE P, BAINS S et al.: CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule. FASEB J. (2005) 19(2):284-286. C. Effect of CORM-3 and -A1 on mean arterial pressure over time in rats. D. Effect of CORM-3 and CORM-A1 on cell viability in rat aortic smooth muscle cells (*p < 0.05 versus 0 µM). t u CO: Carbon monoxide; CO-RM: CO-releasing molecule; CON: Control; Mb: Myoglobin; MbCO: Carbonmonoxy myoglobin; Phe: Phenylephrine. A to play a fundamental and beneficial role. Previous results revealed that HO-1 induction protects cardiomyocytes against the damage inflicted by hypoxia-reoxygenation [11] and significantly ameliorate postischaemic myocardial dysfunction in isolated rat hearts [10]. Transgenic mice overexpressing HO-1 specifically in the heart also showed improved cardiac function following I/R and had reduced inflammatory cell infiltration as well as oxidative tissue damage [51]. Moreover, in a model of mouse-to-rat cardiac transplantation, both HO-1 induction and treatment with CO gas suppressed graft rejection and improved graft survival through mechanisms that involve the inhibition of platelet aggregation, vascular thrombosis and myocardial infarction [36,52]. Based on this preliminary evidence, the group’s initial efforts focused on the possible cardioprotective action of CO-RMs. Specifically, the beneficial effect of CORM-3 on the haemodynamic, biochemical and histological parameters of isolated hearts subjected to I/R was investigated [53] and the data are summarised in Figure 4. It was found that administering CORM-3 10 µM at the beginning of reperfusion following 6 30 min of global ischaemia markedly: improved myocardial perfusion and contractility; reduced the release of myocardial creatine kinase (CK), a specific marker of cardiac tissue injury (Figure 4A); and attenuated infarct size as measured by the tetrazolium red assay, which stains the viable tissue red (Figures 4B and 4C). Interestingly, iCORM-3, a negative control that does not release CO, did not promote any protective effect on the parameters measured and 5-hydroxydecanoic acid (5-HD), an inhibitor of mitochondrial ATP-dependent potassium channels, reversed the cardioprotective action of CORM-3 (Figures 4A, 4B and 4C). CORM-3 also protected cardiomyocytes against cell injury caused by hypoxia-reoxygenation and paraquat, a superoxide anion generator that promotes oxidative stress [46]. Most importantly, using a model of cardiac allograft rejection in mice, the authors found that CORM-3 (but not iCORM-3) considerably prolonged the survival time of transplanted hearts when administered to the recipients for 8 days post-transplant [53]. Several articles followed the original findings to confirm the potential therapeutic effects of CO-RMs against myo- Expert Opin. Investig. Drugs (2005) 14(11) Motterlini, Mann & Foresti A. Creatinine kinase activity 75 CON CORM-3 iCORM-3 5-HD 5-HD + CORM-3 50 25 0 Baseline I/R B. I/R CORM-3 iCORM-3 5-HD C. I/R CORM-3 iCORM-3 5-HD 5-HD + CORM-3 * 10 + 5-HD + CORM-3 + 5-HD + iCORM-3 0 + CORM-3 5 I/R Infarct size (% volume) 15 5-HD + CORM-3 t u o h P r Figure 4. Cardioprotective effects of CORM-3 against myocardial I/R injury. Reprinted with permission from Clark JE, Naughton P, Shurey S et al.: Cardioprotective actions by a watersoluble carbon monoxide-releasing molecule. Circ. Res. (2003) 93(2):e2-e8. A the activation of inducible defensive proteins [58]. A therapeutic role for CO-RMs in other cardiac diseases could also be envisaged as an overexpression of HO-1 inhibits cardiac myocyte hypetrophy induced by endothelin-1 or angiotensin-II [59,60], and a similar antihypertropic effect can be simulated by using CORM-2 [61]. In human pulmonary artery smooth muscle cells, CORM-2 was shown to inhibit proliferation and attenuate the release of the vasoconstrictor endothelin-1, whereas iCORM-2 had no effect [62,63]. A similar antiproliferative action by CORM-2 was observed in Jurkat T cells [64,65] and vascular smooth muscle cells stimulated with platelet-derived growth factor [66]. Recent evidence from collaborators revealed interesting antiproliferative and antiapoptotic actions by CORM-3 in porcine aortic endothelial cells and primate peripheral blood mononuclear cells (PBMCs) [67]. In human microvascular endothelial cells, CORM-2 and -3 appear to affect angiogenesis as they both increase the production of vascular endothelial growth factor (VEGF), cell migration and capillary sprouting [68,69]. After stimulation with light, CORM-1 was reported to cause a concentration-dependent vasodilatation in isolated cerebral arterioles [70], a mechanism that involves activation of guanylate cyclase [71]. The same authors have also demonstrated that CO liberated from CORM-1 by photodissociation activates Ca2+-activated potassium channels in porcine cerebral arteriole smooth muscle cells [72]. Notably, and in agreement with the group’s findings obtained with CORM-3 in isolated rat aorta [49], relaxation of cerebral arterioles by CORM-1 was absent in the presence of inhibitors of NO synthase (NOS) or guanylate cyclase as well as in endothelium-denuded vessels [73], suggesting that NO facilitates the vasodilatory action of CO. In addition, CORM-2 elicited relaxation responses in guinea-pig perfused trachea [74] and rat internal anal sphincter [75], and hyperpolarised the membrane potential of porcine jejunum in a study in which mediators of nonadrenergic non-cholinergic inhibitory neurotransmission was investigated [76]. 5-HD: 5-Hydroxydecanoic acid; CO: Carbon monoxide; CORM: CO-releasing molecule; CON: Control; I/R: Ischaemia–reperfusion. o r f o 2.4 Anti-inflammatory cardial pathophysiology and their ability to protect and preserve vascular function. Guo and colleagues reported that in mice subjected to coronary artery occlusion, an intravenous infusion of CORM-3 3.54 mg/kg before reperfusion significantly reduced infarct size after 24 h; notably, iCORM-3 had no effect [54]. The cardioprotective mechanism of CORM-3 in this model remains to be fully investigated; however, preliminary observations suggest that CORM-3 also promotes delayed protection against myocardial infarction through a mechanism reminiscent of the ischaemic preconditioning phenomenon [55]. Because recently published reports demonstrated that CORM-3 induces HO-1 in vitro and in vivo [56,57], it is tempting to speculate that activation of the HO-1 pathway could contribute to the late-phase preconditioning effect, which is known to be characterised by activities of CO-RMs Beside the emerging evidence on the cardioprotective and vascular effects of CO-RMs, recent progress in the field is corroborating the potential actions of CO carriers in modulating immunosuppresssion and inflammation. In mast cells from guinea-pig and human basophils, CORM-2 markedly decreased the immunological release of histamine and expression of CD203c, suggesting that CO could be used pharmacologically in the treatment of allergic disease [77]. Protection against the immunosuppression induced by solar-simulated ultraviolet (UV) radiation or cis-urocanic acid, an epidermal photoproduct, was observed when CO was delivered to skin topically using lotions containing increasing concentrations of CORM-2; once again, the inactive counterpart (iCORM-2) was ineffective [78]. The production of TNF-α and NO in lipopolysaccharide (LPS)-activated murine J774 macrophages is mediated by NF-κB and can be significantly Expert Opin. Investig. Drugs (2005) 14(11) 7 Therapeutic applications of carbon monoxide-releasing molecules attenuated by induction of the HO-1 pathway. Interestingly, CORM-2 appears to suppress the inflammatory response completely by inhibiting the translocation of NF-κB into the nucleus, indicating a direct contributory effect of CO [79]. A decrease in the expression of inducible NOS (iNOS) and nitrite production stimulated by LPS in RAW264.7 macrophages [80] or smooth muscle cells exposed to IL-1β is also elicited by CORM-2 in a concentration-dependent manner [81]. These data are partially in agreement with the authors’ recent findings showing that both CORM-2 and the water-soluble CORM-3, although they did not affect iNOS expression, markedly reduced nitrite production in LPS-activated RAW264.7 macrophages, suggesting that NOS activity can be inhibited by CO liberated from CO-RMs [69]. To support the feasibility of utilising CO-RMs against the impairment of vascular activity caused by LPS, the authors recently investigated the effect of CORM-3 in a model of septic shock by evaluating the changes in blood pressure induced by endotoxin [82]. In fact, vascular dysfunction and the consequent decrease in blood pressure characteristic of endotoxaemia are usually attributed to an excessive production of NO early after infection [83]. At later times, it appears that HO-1 may also affect vascular tone [84,85] and recent evidence has been reported on the induction of HO-1 in human mesenteric smooth muscle cells of patients with septic shock [86]. The activation of the HO-1 gene and its products, CO and bilirubin may be regarded as a refined response of tissues to counteract the detrimental effects imposed by excessive oxidative [9-11,87-89] and nitrosative stress [3,6,7,90-93] during endotoxaemia. In the experiments, infusion of CORM-3 ∼ 5.3 mg/kg in rats considerably reduced the fall in blood pressure induced by LPS and it was more effective than inducing HO-1 by hemin or treating the endotoxaemic animals with bilirubin [82]. The cellular mechanisms contributing to this effect remain to be fully investigated, but the potential of CORM-3 to modulate the production of inflammatory cytokines in vivo cannot be excluded as: CO gas selectively inhibits the expression of cytokines in LPS-treated mice [25,94]; CORM-3 attenuates the production of NO and TNF-α induced by LPS in murine macrophages [56]; and, in a collaborative study, the group observed that treatment of pigs with CORM-3 prevents the release of TNF-α in isolated PBMCs stimulated with LPS [95,96]. Thus, while the use of CO-RMs to substantiate the cardiovascular function and anti-inflammatory properties of CO in models of disease becomes more assiduous, the authors are gradually learning that these compounds may have promising therapeutic applications in the amelioration of several pathological conditions. In the near future, the authors anticipate that a variety of CO-RMs with different mechanisms of CO release and chemical characteristics will be developed for specific needs. The fact that CORM-F3, an iron-containing carbonyl that slowly liberates CO through oxidation reactions, has been shown to relax blood vessels and inhibit inflammation in vitro (Table 1) is further proof of the versatile A 8 t u o h and resourceful properties of this class of pharmacological agents (Fairlamb and Motterlini, unpublished observations). 2.5 Therapeutic actions of CO-RMs in renal dysfunction The activation of HO-1 and, consequently, the production of CO has been established as a key element in the maintenance of kidney function following injurious events or in conditions affecting the renal system [97]. In fact, using both pharmacological and genetic approaches aimed at overexpressing or inhibiting HO-1 in renal tissue, scientists have demonstrated the critical role of CO in the mitigation of renal vasoconstriction and hypertension [98,99] as well as protection against I/R damage [100], acute renal failure [99], glomerulonephritis [101,102], cisplatin (CP)-induced nephrotoxicity [103] and graft rejection following transplantation [14,104]. Notably, the proteinuria and haematuria that are associated with severe renal tubular injury have been reported as the prime pathological features in the only human case of HO-1 deficiency discovered so far, emphasising the central role of this inducible enzyme in renoprotection [105,106]. The availability and utilisation of CO-RMs in the last 2 years has also confirmed the importance of CO in the mitigation of renal pathophysiology. Arregui and colleagues have shown that CORM-1 elicits an effect on renal haemodynamics and function in vivo. [107]. The authors reported that an intrarenal administration of CORM-1 in Sprague-Dawley rats increases renal blood flow, glomerular filtration rate and urinary cGMP excretion, and that the inhibition of HO activity progressively compromises renal haemodynamics leading to acute renal failure, an effect that was completely reversed by CORM-1. Similarly, Vera and co-workers reported that both CORM-2 and -3 significantly decreased the levels of plasma creatinine and limited renal damage in a mouse model of ischaemia-induced acute renal failure [57]. In line with these findings, the authors have recently examined the possible beneficial effects of CO liberated from CORM-3 and -A1 on the damage inflicted by cold storage and I/R in isolated perfused rabbit kidneys [108]. Kidneys flushed with cold celsior solution supplemented with CO-RMs 50 µM and stored at 4°C for 24 h displayed a significantly higher perfusion flow rate, glomerular filtration rate, and sodium and glucose reabsorption rates at reperfusion compared with control kidneys flushed with Celsior solution alone. The addition of 1H-(1,2,4)oxadiazole(4,3-a)quinoxalin-1-one (ODQ), a guanylate cyclase inhibitor, prevented the increase in perfusion flow rate mediated by CO-RMs. The respiratory control index from kidney mitochondria treated with CO-RMs was also markedly increased. Notably, renal protection was lost when kidneys were flushed with Celsior-containing inactive compounds (iCORMs), which had been deliberately depleted of CO. As nephrotoxicity is one of the main side effects caused by the antineoplastic agent CP, the authors have also investigated the effect of CORM-3 on CP-induced cytotoxicity in renal epithelial cells and explored the potential therapeutic benefits of CO in CP-induced nephrotoxicity in rats [109]. It was found that P r Expert Opin. Investig. Drugs (2005) 14(11) o r f o Motterlini, Mann & Foresti treatment with CORM-3 reduced apoptosis in vitro and resulted in an amelioration of renal dysfunction in vivo as indicated by a reduction of urea and creatinine levels to basal values, decreased number of apoptotic tubular cells and improved histological profile. Predictably, iCORM-3 failed to prevent renal injury suggesting that CO is directly involved in renoprotection. Taken together, these results suggest that CO is dynamically involved in counteracting renal dysfunction in a variety of stress conditions that affect the physiology of the kidney and that CO-RMs can be developed as very effective therapeutic adjuvants in the treatment of renal disease [110]. 3. Expert opinion The development of pharmaceuticals based on transition metal ions is a relatively novel approach that may offer great versatility in the design of more selective and specific drugs. However, this strategy has encountered several obstacles based on the fact that the majority of scientists have the propensity to consider compounds containing metal ions as ‘potentially toxic’ to humans. This inaccurate perception is accentuated by the limited information available on the true toxicological profile of rare transition metals and, as correctly pointed out in previous reports, by the lack of expertise of traditional medicinal chemists and pharmacologists in dealing with metal complexes that could be, after all, biological active [43]. Notwithstanding these drawbacks, the last decade has experienced an increased interest in the synthesis of innovative compounds containing transition metals aimed at being developed for therapeutic and diagnostic applications. For instance, ruthenium, vanadium, gold and technetium have all been used as a core of newly synthesised molecules that could be exploited for the treatment of a diverse range of diseases including cancer, inflammation and diabetes [43]. Specific progresses have been made on ruthenium-based compounds, which possess anticancer activities [111], and with vanadium complexes as effective insulin receptor agonists [112]. The antimetastatic NAMI-A, a ruthenium III complex, has recently completed Phase I trial and it is the first example of a ruthenium metallopharmaceutical to reach clinical testing [111]. The advantage to work with metallo-based compounds in drug development originates from the great adaptability of these complexes as the method of their synthesis is usually very reliable and the ligand substitution to the metal centre as well as their redox potentials can be finely adjusted to specific needs. This is the case for CORM-3, in which the substitution of glycine or the chloride in the trans position of a carbonyl group favours the release of CO; therefore, the rate of CO liberation can be tuned by using ligands with a higher or lower affinity to the metal centre. This chemical feature can be extended to CO-RMs containing a transition metal other than ruthenium; iron-based CO-RMs could be a good alternative (e.g., CORM-F3 in Table 1) as more data are also available on the biological activity of iron metal complexes [113]. It is understood that the promising biochemical and A t u o h pharmacological activities of CORM-3 and other CO-RMs have to be mirrored by good profiles of efficacy and tolerability in vivo. Pharmacokinetic studies as well as data on tissue distribution and elimination of CO-RMs are still awaited. However, the metallopharmaceutical field is rapidly growing and the versatility of metal complexes is such that there is great potential for the use of CO-RMs in biomedicine. In the case of boron, the active centre of CORM-A1, which liberates CO in a slow fashion, there are plenty of reports on the development of boron compounds in the pharmaceutical field; moreover, this non-metallic element is necessary in the human diet and analysis of boron intake, absorption and distribution have been performed showing that boron is relatively safe in humans [114,115]. Thus, based on their ability to liberate CO in aqueous solutions and exert crucial biological activities, CO-RMs have rapidly become a relevant class of compounds that may offer not only a promising solution for a safe and controllable delivery of CO in organisms but also a unique tool to identify intracellular targets responsive to CO [116]. This is exemplified by an elegant study in which CORM-2, and not CO gas, was used to simulate the ability of CO derived from HO-2 to regulate the opening of calcium-sensitive potassium channels present in the carotid body [117]. Activation of different types of potassium channels by both CO gas [28,29] and CO-RMs [49,50,53] have been consistently reported to contribute significantly in the regulation of vessel tone and cardioprotection, but this notion has always been received with great scepticism because it was assumed that potassium channels lack any molecular domain that would validate a direct chemical interaction with CO. Indeed, CO reacts solely and specifically with enzymes and proteins that contain haem or metal centres, making them the most probable ‘molecular switch’ required by cells to transduce the signal elicited by this diatomic gas [32]. Intriguingly enough, calcium-sensitive potassium channels have recently been reported to covalently bind with iron protoporphyrin IX (heam) [118]; moreover, compelling evidence showed that haem is a potent but subtle allosteric regulator of human maxi-K+ channels where the binding of gaseous molecules, including CO, is likely to play an important modulatory function [119]. The fact that haemcontaining transcriptional activators and nuclear factors from bacteria, drosophila and mammals are also emerging as selective CO sensors [120-122] highlights the importance of searching for and identifying new functional and structural proteins with transition metal cores that are sensitive to CO [32]. For instance, the authors have recently demonstrated that CORM-2 appears to modulate the production of reactive oxygen species generated by NAD(P)H oxidase and the mitochondrial respiratory chain in human airway smooth cells, thereby markedly affecting cell proliferation [123]. This is in line with the data showing that addition of CORM-3 and -A1 to cold storage solutions during the preservation of kidneys improves mitochondrial respiration at reperfusion [108]. A direct effect of CO-RMs on the activity P r o r Expert Opin. Investig. Drugs (2005) 14(11) f o 9 Therapeutic applications of carbon monoxide-releasing molecules of cytochromes, oxygen uptake and phosphorylation in isolated mitochondria has also been reported [124]. In addition, in human lung epithelial cells, the authors observed that CORMs inhibit the activity and expression of metalloproteases, which are directly involved in the pathophysiology of emphysema and other diseases related to a proteases/antiproteases imbalance, such as cancer and bacterial infection [125]. 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An excellent study in combination with reference 118 reporting on the covalent binding of haem to potassium channels and their susceptibility to be regulated by gaseous molecules including CO. 120. AONO S: Biochemical and biophysical properties of the CO-sensing transcriptional activator CooA. Acc. Chem. Res. (2003) 36(11):825-831. Expert Opin. Investig. Drugs (2005) 14(11) BOCZKOWSKI J, MOTTERLINI R: Mitochondrial respiratory chain and NAD(P)H oxidase are targets for the antiproliferative effect of carbon monoxide in human airway smooth muscle. J. Biol. Chem. (2005) 280(27):25350-25360. Study reporting that NAD(P)H oxidase and mitochondria, which are both important sources of reactive oxygen species, are targets for CO liberated by CO-RMs. o r • f o 124. SANDOUKA A, BALOGUN E, FORESTI R et al.: Carbon monoxidereleasing molecules (CO-RMs) modulate respiration in isolated mitochondria. Cell. Mol. Biol. (2005) 51(4):425-432. 125. DESMARD M, AMARA M, LANONE S, MOTTERLINI R, BOCZKOWSKI J: Carbon monoxide reduces the expression and activity of matrix metalloproteinases 1 and 2 in alveolar epithelial cells. Cell. Mol. Biol. (2005) 51(4):403-408. Affiliation Roberto Motterlini†1, Brian E Mann2 & Roberta Foresti1 †Author for correspondence 1Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, UK Tel: +44 20 8869 3181; Fax: +44 20 8869 3270; E-mail: r.motterlini@imperial.ac.uk 2Department of Chemistry, University of Sheffield, Sheffield, UK