IHP-Loaded Red Blood Cells Efficacy in Sickle Transgenic Mice

Abstract 2119

Homozygous patients (SS) display the most severe form of sickle cell disease (SCD) with high risk of painful complications during their lifetime including vaso-occlusions (VOC), acute chest syndromes (ACS) and strokes. Even though multiple factors seem to be involved in those sickle related events, hypoxia is the most important triggering factor. Transfusing patients with IHP-RBCs (homologous RBCs loaded with Inositol Hexaphosphate) could potentiate existing transfusion therapy. Indeed, IHP by reducing the oxygen-hemoglobin affinity improves the capacity of transfused RBCs to release more oxygen in the blood stream and to the tissues. In a previous work, we showed that IHP-RBCs were 7-fold more effective than normal RBCs to reduce in vitro sickling of homozygous patient RBCs (Bourgeaux et al, Transfusion, 2010). We hypothesized that in vivo, IHP-RBCs with increased oxygenation properties may reduce the severity of SCD pathophysiology in transgenic mice.

In a first study, BERK transgenic mice which are more representative of SCD in childhood (splenomegaly and RBC sequestration crisis) were used to evaluate the effect of chronic IHP-RBCs transfusions. Mice (n=5-7/group) were subjected to 4 repeated RBC exchanges (every 2 weeks) using IHP-RBCs or control RBCs. One week after the last exchange, mice were challenged with hypoxia and sacrificed for organ and hematological analysis. It was observed an improved survival rate and a prevention of anemia under hypoxic conditions (Hb=7.0±0.6 g/dl vs 5.9±0.4 g/dl for mice receiving control RBCs). On the contrary to control RBCs, IHP-RBCs treatment also corrected impaired brain development observed in female BERK mice with higher brain weight to total body weight ratio (2.12±0.07% versus 1.89±0.08% for untreated mice, p<0.05). IHP-RBCs by modulating cellular blood components and improving the vascular tone also lowered the risk of VOC in BERK mice with decreased proportion of reticulocytes (26±6 % vs 34±4% for treatment with normal RBCs and 42±2 % for untreated mice), reduced levels of soluble thrombospondin-1 involved in adhesion processes (104±22 pg/mL vs 314±56 pg/mL for treatment with normal RBCs and 223±50 pg/mL for untreated mice) as well as endothelin-1, a peptide promoting vaso-constriction.

In a second study, SAD transgenic mice which were shown to be a pertinent model for acute sickle events were used to investigate the efficiency of a simple transfusion of IHP-RBCs. Mice (n=10–12/group) partially exchanged with IHP-RBCs or control RBCs were subjected to an acute hypoxic-reoxygenation stress 24h post-transfusion in order to induce specific lung damages. The therapeutic benefice of IHP-RBCs over classical transfusion to reduce lung injury was evidenced by significantly decreased mRNA levels of molecules involved in intravascular pathology (7-fold and 8-fold reduction in VCAM-1 and Tissue factor mRNA levels as compared to mice receiving control RBCs (p<0.05). AOPP used as specific biomarker of oxidative stress was significantly reduced with IHP-RBCs treatment as compared to untreated mice (2.64±0.27 μmol/L vs 3.87±0.30 μmol/L respectively).

From a safety point of view, one single intravenous administration of IHP-RBCs showed no apparent toxicity in healthy mice and seems to be as safe as classical transfusion. Only moderate hemolysis was observed after transfusion with increased serum bilirubin level but this parameter recovered normal values within 24h.

In conclusion, entrapping IHP into transfused RBCs is a powerful tool to prevent acute or chronic manifestations of SCD in transgenic mice. For therapeutic use in human, IHP-RBCs may potentiate chronic transfusions by reducing the number of RBC units to be transfused but also the frequency of transfusions thus improving patient quality of life. In addition, with the recent improvements in scoring acute manifestations of SCD, IHP-RBCs may also potentiate classical transfusion in the treatment of chest syndromes by acting more rapidly and more efficiently.