Synthesis, biophysical properties and pharmacokinetics of ultrahigh molecular weight tense and relaxed state polymerized bovine hemoglobins

Abstract

Hemoglobin-based oxygen carriers (HBOC) are currently being developed as red blood cell (RBC) substitutes for use in transfusion medicine. Despite significant commercial development, late stage clinical results of polymerized hemoglobin (PolyHb) solutions hamper development. We synthesized two types of PolyHbs with ultrahigh molecular weights: tense (T) state PolyHb (M(W)=16.59 MDa and P(50)=41 mmHg) and relaxed (R) state PolyHb (M(W)=26.33 MDa and P(50)=0.66 mmHg). By maintaining Hb in either the T- or R-state during the polymerization reaction, we were able to synthesize ultrahigh molecular weight PolyHbs in distinct quaternary states with no tetrameric Hb, high viscosity, low colloid osmotic pressure and the ability to maintain O(2) dissociation, CO association and NO dioxygenation reactions. The PolyHbs elicited some in vitro RBC aggregation that was less than 6% dextran (500 kDa) but more than 5% human serum albumin. In vitro, T-state PolybHb autoxidized faster than R-state PolybHb as expected from previously reported studies, conversely, when administered to guinea pigs as a 20% exchange transfusion, R-state PolybHb oxidized faster and to a greater extent than T-state PolybHb, suggesting a more complex oxidative processes in vivo. Our findings also demonstrate that T-state PolybHb exhibited a longer circulating half-life, slower clearance and longer systemic exposure time compared to R-state PolybHb.

Figure 1

pO₂ at various stages of the bHb polymerization process for T- and R-state PolybHb solutions. The error bar represents the standard deviation from triplicate reactions

Figure 2

SDS-PAGE of native bHb, T- and R-state PolybHb solutions

Figure 3

Absolute molecular weight distribution of native bHb, T- and R-state PolybHb solutions.

Figure 4

Equilibrium O₂-bHb/PolybHb binding curves of native bHb, T- and R-state PolybHb solutions.

Figure 5

Oxygen affinity (P₅₀) and cooperativity coefficient (n) of native bHb, T- and R-state PolybHb solutions. The error bar represents the standard deviation from triplicate reactions. ✴ p<0.05 with respect to native bHb.

Figure 6

MetHb level of native bHb, T- and R-state PolybHb solutions. The error bar represents the standard deviation from triplicate reactions. ✴ p<0.05 with respect to native bHb.

Figure 7

Rapid kinetics of CO binding with native deoxygenated bHb, T- and R-state PolybHb. Stopped-flow time courses of 15 μM deoxygenated bHb/PolybHb (after mixing) reacting with 250 μM CO solutions for native bHb (○) and T-state PolybHb (□) solutions, or 50 μM CO solution for R-state PolybHb (Δ) were monitored at 437.5 nm in 50 mM phosphate buffer at pH 7.4 and room temperature. The solid lines are from the nonlinear least-square curve fitting of the time courses to the exponential equation. The obtained apparent rate constants were plotted versus CO concentration in the insert for each bHb/PolybHb to generate the second order rate constants of the reaction between deoxygenated bHb/PolybHb and CO (Table 1).

Figure 8

A typical blood smear for aggregation studies obtained from heparinzed hamster blood mixed in a 1:1 volume ratio with test solution. 5% albumin does not produce aggregation (above), however 6% dextran 500 kDa increases aggregation (below).

Figure 9

Pharmacokinetics of T- (panel A) and R- (panel B) state PolybHb: top – photograph of (1) bHb, (2) T- and R-state PolybHb (prior to transfusion), (3) plasma (prior to transfusion), (4) end of transfusion, (5) 0.25 hour, (6) 0.5 hour, (7) 1 hour, (8) 2 hour, (9) 4 hour, (10) 8 hour, (11) 12 hour, (12) 24 hour and (13) 48 hour. Insets show representative visible spectra of plasma from the end of transfusion until 48 hours from 450 to 700 nm. The concentration (heme) versus time (hours) plot shows the plasma concentration ± sem (n=4/time point) of T- and R-state PolybHb as total heme (black), ferrous heme (red) and ferric heme (blue).

Figure 10

Circulating T- and R-state PolybHb polymer distribution over time: (A) Size exclusion chromatography of () bHb, () T-state PolybHb and plasma samples from the end of transfusion to 48 hours (shown as dark grey fading to white). (B) Size exclusion chromatography of () bHb, () R-PolybBv and plasma samples from the end of transfusion to 24 hours (shown as dark grey fading to white).

Figure 11

(A) In vitro autoxidation studies show a significant (p<0.05) attenuation in % R-state PolybHb ferric Hb formation compared to % bHb (*) and % T-state PolybHb (†) ferric Hb formation over 24 hours at 37°C. (B) % ferric Hb in plasma from in vivo studies show a significant (p<0.05) increase in % ferric R-state PolybHb compared to T-state PolybHb (†) within an early time frame following transfusion.