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CORM-A1: a new pharmacologically active carbon monoxide-releasing molecule

Roberto Motterlini^*,1, Philip Sawle^*, Jehad Hammad^*, Sandip Bains^*, Roger Alberto, Roberta Foresti^* and Colin J. Green^*

^* Vascular Biology Unit, Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, UK; and
Department of Chemistry, University of Zurich, Switzerland

¹Correspondence: Department of Surgical Research, Northwick Park Institute for Medical Research, Harrow, Middlesex, HA1 3UJ, UK. E-mail: r.motterlini{at}imperial.ac.uk)

SPECIFIC AIMS

Carbon monoxide (CO) is emerging as an important and versatile mediator of physiological processes in that treatment of animals with exogenous CO gas has beneficial effects in a range of vascular and inflammatory-related disease models. The recent discovery that certain transition metal carbonyls function as CO-releasing molecules (CO-RMs) in biological systems highlighted the potential of exploiting this and similar classes of compounds as a stratagem to deliver CO for therapeutic purposes. We report on the biochemical features of a newly identified water-soluble CO releaser (Na₂[H₃BCO₂], here termed CORM-A1) that, unlike the first prototypic molecule recently described by us (CORM-3), does not contain a transition metal and liberates CO at a much slower rate under physiological conditions. The intrinsic biochemical behavior of CORM-A1 as a slow CO releaser reflects its pharmacological actions in terms of vessel tone and blood pressure regulation.

PRINCIPAL FINDINGS

1. CORM-A1 liberates CO in a pH- and temperature-dependent manner
The spectrophotometric assay that detects the formation of carbonmonoxy myoglobin (MbCO) from deoxy-Mb is a reliable method for assessing the extent and kinetics of CO liberation from CO-RMs. Addition of 60 µM CORM-A1 (Na₂[H₃BCO₂]) to a phosphate buffer solution containing Mb at 37°C and pH = 7.4 resulted in a gradual formation of MbCO over time. The time required to fully saturate Mb with CO liberated from CORM-A1 gradually decreased by lowering the pH to 7.0, 6.5 and 5.5, indicating that the rate of CO release from CORM-A1 is pH dependent. From curves fitted to the spectral data (Fig. 1 A), we can calculate that the half-lives of CORM-A1 at 37°C are 21 min at pH = 7.4, 7.1 min at pH = 7.0, 3.3 min at pH = 6.5, and 2.5 min at pH = 5.5. Predictably, a decomposed CORM-A1 that had been inactivated and does not release CO (iCORM-A1) did not generate any MbCO. Because CO is promptly liberated to Mb at pH = 5.5, we used these conditions to generate a standard curve that clearly indicates that the conversion of the carboxyl group in Na₂[H₃BCO₂] to CO goes to completion as 1 mol of CO is formed per mole of CORM-A1 (Fig. 1B, C ). Therefore, iCORM-A1 is a useful negative control when testing the direct involvement of CO in the biological activities mediated by CORM-A1. We also found that the rate of MbCO formation from CORM-A1 decreases by gradually lowering the temperature of the solutions.

View larger version (25K): [in this window] [in a new window]   Figure 1. The rate of CO release from CORM-A1 is pH dependent. A) The amount of MbCO formed over time at 37°C was measured after addition of CORM-A1 (60 µM) to the Mb solution; half-lives (t1/2) of this compound at different pHs were calculated from the fitted curves. Note that the inactive compound (iCORM-A1) did not generate any MbCO. B, C) Spectra of MbCO formation after addition of different concentrations of CORM-A1 to Mb at pH = 5.5 and relative standard curve.

CORM-A1 is pharmacologically active: effects on vessel tone and blood pressure
CORM-3, a CO releaser containing ruthenium (Ru) as transition metal, has been shown to promote a rapid and significant relaxation in isolated vessels, and this effect has been shown to be mediated by CO. In this study, CORM-A1 (80 µM) caused a profound but gradual dilatory effect over time that was maximal (96%) 33 min after addition of the compound to the organ bath. Precontracted aortic rings were treated with 80 µM iCORM-A1 (the inactive compound) or sodium borohydride (NaBH4), used as an additional negative control to exclude any effect of boron on vessel tone; both compounds failed to produce vasodilatation. The vasodilatation elicited by CORM-A1 was partially inhibited in the presence of a guanylate cyclase blocker (ODQ, 30 µM) but was unaffected by glibenclamide (GLI, 10 µM), an inhibitor of K_ATP potassium channels. It is known that the benzylindazole derivative YC-1 sensitizes guanylate cyclase to activation by CO. When rings were preincubated with YC-1 at 1 µM, the vasoactivity of CORM-A1 (1–20 µM) was markedly intensified at all concentrations tested, further sustaining the direct contribution of guanylate cyclase to the effect elicited by CORM-A1-derived CO (Fig. 2 A). The effect of YC-1 and CORM-A1 was examined on mean arterial pressure (MAP) in vivo (Fig. 2B ). Compounds were injected i.v. as a bolus at a final dose of 30 µmol/kg for CORM-A1 (or iCORM-A1) and 1.2 µmol/kg for YC-1. When the two compounds were used in combination, YC-1 was administered 10 min prior to CORM-A1. CORM-A1 produced a gradual and sustained decrease in MAP; 60 min after injection, MAP decreased by 6.3 ± 1.5 mmHg from the initial baseline value. Administration of YC-1 alone or in combination with the negative control iCORM-A1 produced only a transient decrease in MAP, reaching a maximum of 5.5 ± 1.0 mmHg after 10 min and returning to basal levels 50 min after injection. The combination of CORM-A1 and YC-1 produced a synergistic effect resulting in a rapid and profound hypotension. In fact, MAP significantly decreased by 16.1 ± 5.6 mmHg after 10 min and remained at this level thereafter.

View larger version (22K): [in this window] [in a new window]   Figure 2. Pharmacological activities of CORM-A1. A) Vasodilatory responses to CORM-A1 (1–20 µM) with or without pretreatment with YC-1 (1 µM). *P < 0.05 compared with CORM alone. B) Effect of CORM-A1 and YC-1 on MAP in vivo. CORM-A1 (30 µmol/kg, i.v.) or the inactive compound (iCORM-A1) was injected alone or in combination with YC-1 (1.2 µmol/kg, i.v.) and MAP was measured over time. *P < 0.05 compared with baseline values; P < 0.05 compared with YC-1, CORM-A1, or iCORM-A1 + YC-1. Data represent the mean ± SE of 4–5 independent experiments.

SIGNIFICANCE AND CONCLUSIONS

The discovery of compounds able to carry and deliver CO to tissues and organs is likely to facilitate the development of novel pharmaceutical agents suitable for therapeutic applications in which CO gas is needed. Certain transition metal carbonyls have been shown to exert interesting biological activities insofar as they reproduce many of the pharmacological effects mediated by CO gas and thus can be used as CO-releasing molecules (CO-RMs) for experimental purposes. Our early investigations revealed that manganese and Ru-containing carbonyl complexes (CORM-1 and CORM-2), which are soluble only in organic solvents (e.g., DMSO), promote relaxation of blood vessels and mitigate acute hypertension in vivo. More recently, the first prototype of a water-soluble carbonyl complex that contains the amino acid glycine covalently bound to a Ru metal (CORM-3) was described. This compound promote cardioprotection in both in vitro and in vivo models of myocardial infarction and in cardiac transplantation. In our persistent search for agents that could be safely used as CO-RMs in biological systems, we have identified sodium boranocarbonate (Na₂[H₃BCO₂], or CORM-A1) as an extremely promising water-soluble compound that spontaneously liberates CO in aqueous solutions. CORM-A1 differs from the original CO-RMs as it does not contain a transition metal and releases CO with a slower rate (t_1/2 at physiological pH and temperature is 21 min). A schematic representation summarizing the chemical properties of the CO-RMs identified so far is illustrated in Fig. 3 . In line with its intrinsic chemical properties, CORM-A-1 added to isolated aortic rings precontracted with phenylephrine promoted a gradual and sustained vasorelaxation that was maximal after 33 min. Similarly, the rate of change in MAP observed in vivo after injection of rats with CORM-A1 is in agreement with its behavior as a slow CO releaser. When the compound was depleted of CO and rendered inactive (iCORM-A1), no CO was detected with the Mb assay; consequently, no vasorelaxation could be observed. Sodium borohydride (NaBH4), used as another negative control, was equally ineffective. The experiments to assess changes in MAP and vessel contractility by the combined administration of CORM-A1 and YC-1, a well-known benzylindazole derivative that activates the guanylate cyclase/cGMP pathway, further supported the role of CO as a potential pharmaceutical agent. While YC-1 alone or iCORM-A1 plus YC-1 slightly and only temporarily decreased MAP, the combination of CORM-A1 and YC-1 produced a significant and sustained hypotensive effect in vivo; the presence of YC-1 also markedly amplified the extent of vasodilatation caused by CORM-A1 in isolated aortas.

View larger version (22K): [in this window] [in a new window]   Figure 3. Classes of CO-RMs and their chemical properties. Schematic diagram illustrating the types of bioactive CO-RMs identified so far, the year of their discovery, and their intrinsic chemical properties.

Thus, our attempt to diversify the portfolio of CO-RMs that possess a variety of chemical characteristics (i.e., water-soluble vs. lipid-soluble, slow vs. fast releasers) will help to elucidate the biological function of cellular targets that are responsive to CO and will facilitate the therapeutic delivery of CO in a safe, measurable, and controllable fashion. For instance, irrespective of their solubility in DMSO or aqueous solutions, the metal carbonyls tested so far have been demonstrated to promptly liberate CO upon addition to biological systems, as ligands present in the extracellular or plasma environment (i.e., phosphate, glutathione) appear to accelerate dissociation of CO from the metal center. In the case of the water-soluble CORM-3, the release of CO is further accelerated when this compound is added to a solution containing myoglobin. Therefore, CORM-3 would fall into a category of compounds that release CO very rapidly ("fast releasers") in biological systems, which would be ideal for therapeutic applications where CO acts as a prompt signaling mediator. However, chemicals that release CO with a slow kinetic ("slow releasers"), as with CORM-A1, could be more versatile in the treatment of certain chronic diseases where the continuous effect of CO may be required. CORM-A1 might mimic more closely the natural function of heme oxygenase (HO), which is expected to generate endogenous CO from heme in a sustained manner, particularly in vascular and inflammatory disease states typified by up-regulation of the inducible HO-1.

FOOTNOTES

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