CO-MP4, a polyethylene glycol-conjugated haemoglobin derivative and carbon monoxide carrier that reduces myocardial infarct size in rats

Abstract

Background and purpose: MP4 (Hemospan) is a Hb-based oxygen therapeutic agent, based on polyethylene-glycol (PEG) conjugation to Hb, undergoing clinical trials as an oxygen carrier. This study describes the functional interaction between MP4 and carbon monoxide (CO), as a CO delivery agent, and the effects of CO-MP4 on myocardial infarct size following ischaemia and reperfusion in rats.

Experimental approach: Kinetic measurements of CO-MP4 binding were used to evaluate the effects of PEG modification on Hb subunit structure/function and to calculate CO-MP4 equilibrium constants. CO transport by CO-MP4 was shown by ligand (O2/CO) partitioning between MP4 and red blood cell (RBC)-Hb. Pharmacological effects of CO-MP4 were studied on myocardial infarction in rats.

Key results: CO binding kinetics show primary structural/functional effects on beta chains in MP4, with alpha chains maintaining the ability to undergo tertiary conformational transition. CO confers long-term, room-temperature stability and is able to rapidly re-equilibrate between MP4 and RBCs. In a rat model of myocardial infarct, in contrast to oxy-MP4, CO-MP4 reduced infarct size when administered prior to the induction of ischaemia.

Conclusions and implications: MP4 PEGylation chemistry modifies the individual function of Hb subunits, but results in an overall CO equilibrium constant similar to that for unmodified Hb. CO-MP4 is able to deliver CO to the circulation and reduces ischaemia/reperfusion injury in rats, providing the first evidence for this drug as a CO therapeutic agent.

Figures

Figure 1

CO geminate binding to Hb (lower trace) and MP4 (upper trace). Solid and dashed lines show the respective fits. CO, carbon monoxide.

Figure 2

‘R-state' CO association to Hb (a) and MP4 (b), with curves shown at decreasing power from laser photolysis, from bottom to top. (c) shows the normalized CO rebinding time courses following partial photolysis for Hb and MP4; both curves were taken from the middle-power photolysis shown in (a) and (b). The solid lines show fitted curves from Equation (1). Fitted results for the rates are shown in Table 1. CO, carbon monoxide.

Figure 3

‘T-state' normalized time courses of CO association to Hb (a) and MP4 (b). Time courses were measured by mixing the deoxygenated Hbs (6 μM in haem before mixing) with buffer containing CO at 100, 50 or 25 μM before mixing. Only data sampled every 10 ms are shown for clarity. The solid lines show fits of the data using Equation (3). Fitted rates are given in Table 1. CO, carbon monoxide.

Figure 4

‘R-state' CO dissociation from Hb and MP4. The left panel shows the time courses measured by rapid mixing of CO-Hb and CO-MP4 with deoxygenated buffer containing 200 μM NO. Time courses were measured by the change in absorbance at 420 nm and normalized. Only data sampled every 10 s are shown for clarity. The solid lines show the fits using Equation (1), and the rates are given in Table 1. The right panel reports the time courses measured by reaction of CO-Hb and CO-MP4 with MP-11. Experiments in the presence of IHP at a five-fold molar ratio are shown by the closed symbols. Time courses were determined by the change in absorbance at 590 nm and normalized. Solid lines show the fits using Equation (2), and the fitted rates are reported in Table 1. CO, carbon monoxide; IHP, inositol hexaphosphate; NO, nitric oxide.

Figure 5

Size-exclusion chromatography of oxy-Hb (left) and oxy-MP4 (right) at decreasing Hb concentrations in PBS and under dissociating conditions in 0.9 M MgCl2 (bold solid line). Stock concentrations in PBS were 2.6, 0.06 and 0.015 mM (in haem). Final eluted concentrations were approximately 40, 1 and 0.2 μM (in haem), respectively, based on the eluted peak absorbances at 540 nm. Traces were normalized for clarity. Shifts to longer retention times reveal decrease in size due to formation of αβ dimers. Species eluting at the free αβ dimer position in MP4 under dissociating conditions are shown by the arrow. PBS, phosphate buffer saline.

Figure 6

Semi-log plot of met-Hb formation in O2- and CO-liganded MP4 at 37 °C. The slopes of the best fits are 29.0±0.9% and −0.06±0.03% per day for oxy-MP4 and CO-MP4, respectively (r2=0.32 versus 0.98, P=0.04 versus <0.0001). CO, carbon monoxide.

Figure 7

CO re-distribution between MP4 ([Hb] ∼40 g L−1) and red cells ([Hb] ∼160 g L−1). In MP4 → RBC, CO-equilibrated MP4 was mixed 1:1 (v/v) with human blood. In RBC → MP4, CO-equilibrated blood was mixed 1:1 (v/v) with MP4. Percent CO-Hb of the MP4 and blood samples before mixing (time=0) and 30 min after mixing (time=30 min) are displayed. CO, carbon monoxide.

Figure 8

Top: representative digital photographs of heart slices from oxy-MP4 group (left) and CO-MP4 group (right) stained with Evans blue and TTC, followed by overnight immersion in formalin. Middle: myocardial area at risk (percent of total LV). Bottom: infarct size (percent of risk area). *denotes P<0.0125 versus LR group. CO, carbon monoxide; LR=lactated Ringer's solution only; LV, left ventricle; PC=preconditioning; TTC, triphenyl tetrazolium chloride.