Characterization of a versatile organometallic pro-drug (CORM) for experimental CO based therapeutics

Abstract

The complex fac-[Mo(CO)(3)(histidinate)]Na has been reported to be an effective CO-Releasing Molecule in vivo, eliciting therapeutic effects in several animal models of disease. The CO releasing profile of this complex in different settings both in vitro and in vivo reveals that the compound can readily liberate all of its three CO equivalents under biological conditions. The compound has low toxicity and cytotoxicity and is not hemolytic. CO release is accompanied by a decrease in arterial blood pressure following administration in vivo. We studied its behavior in solution and upon the interaction with proteins. Reactive oxygen species (ROS) generation upon exposure to air and polyoxomolybdate formation in soaks with lysozyme crystals were observed as processes ensuing from the decomposition of the complex and the release of CO.

Figure 1

Structure of ALF186

Figure 2

Time evolution of UV-Visabsorption spectrum of ALF186 in PBS buffer at pH7.4 at rt under aerobic conditions: a) decay in absorbance measured at 294 nm and 309 nm with ALF186 alone (250 μM). b) Decay in absorbance measured at 308 nm and 386 nm with ALF186 and HSA (250 μM and 50 μM, respectively; HSA spectrum subtracted). The variation of the absorbance with time is given in the insets.

Figure 3

Plot of equivalents of CO released versus time from a solution of pure ALF186 in PBS buffer at pH7.4 (X) and from a solution of a 5:1 molar ratio mixture of ALF186:BSA (○). Experiments done at rt with GC-TCD detection of CO. The solution and headspace volume, as well as the mass and concentration of ALF186 (10mM), was the same in both experiments. The amount of CO₂ released after 4h is only 0.04 equivalents.

Figure 4

Structure of polyoxomolybdate cluster [PMo₁₂O₄₀]³⁻ hydrogen bonded to HEWL. For clarity, only molybdenum (brown) and phosphorus (yellow) atoms are shown; (a) cartoon representation of the crystal packing with the Keggin’s ion surrounded by four protein molecules(b) 2mFo-DFc maps (contoured at 1.5 σ, in blue) and anomalous peaks (contoured at 2.5 σ, in yellow) obtained after model building and refinement. In the inset is a representation of the structure of the Keggin’s ion from Cambridge structural database.

Figure 5

Toxicity of ALF186 in vitro: HepG2, LLC-PK1 and RAW264.7 cell lines. Cells were incubated for 24h (37°C, 5% CO₂) in the presence of ALF186 at concentrations up to 100 μM. The percentage of cell survival was calculated considering 100% survival for control cells.

Figure 6

a) %COHb levels after i.p. administration of 20 mg/kg and 40 mg/kg of ALF186 in Balb/C mice, in PEG300/water (1:4). Average of three mice for each dose b) %COHb levels after oral administration of 500 mg/kg of ALF186 in Balb/C mice, in PEG300/water (1:4). %COHb at each time point is the average of three different mice.

Figure 7

Bottom plot (dashed lines): time evolution of the % COHb levels in mice treated with inhaled CO (250 ppm) and with ALF186 (20 mg/kg, i.p. administration). Top plot (solid lines): time evolution of the mean arterial blood pressure (mmHg) for mice treated with inhaled CO (iCO) as in the bottom plot. Both plots include the control values for mice breathing normal air. These are similar to the controls for i.p. administration of inactivated ALF186 (not shown).