IHP-102 compound decreases the adhesion of sickle cell disease RBCs to acutely activated endothelial cells in biochip labs endothelial-on-a-chip assay

Abstract Background: The adhesion of red blood cells (RBCs) to the vasculature constitutes a pathophysiological manifestation of Sickle Cell Disease (SCD). The repercussions of the abnormal behavior of RBCs and notable symptomatic cases define a vaso-occlusive episode (VOE). Consequently, anti-adhesive therapies are explored to mitigate the effects of Sickle Cell Disease, particularly VOEs. Crizanlizumab was the first therapy approved by the FDA to diminish adhesion and VOEs in patients with SCD. Regrettably, the efficacy of this therapeutic approach has not matched the initial promising data. Alternative strategies have been investigated to enhance adhesion properties and reduce VOEs in SCD. IHP-102 is a dual complement and P-selectin inhibitor designed for the acute treatment of VOE. In preclinical studies in Townes SS mice, IHP-102 has shown robust inhibition of vaso-occlusion and reduced pain behavior. Here we demonstrate the efficacy of IHP-102 in decreasing the adhesion of human donor SCD RBCs to activated endothelial cells within the BioChip Labs Endothelium-on-a-Chip (EOC) assay. Furthermore, we compare its performance with FDA-approved crizanlizumab. Methods: The EOC utilizes HUVECs (Lonza, Morristown, NJ, USA) maintained at physiological flow for at least 48 hours before the EOC assay. Whole blood EDTA samples from SCD donors with HbSS genotypes were collected at University Hospital, Cleveland, OH, USA, and Emory University, Atlanta, GA, under institutional IRB protocols. To standardize the samples, we isolated RBCs by centrifugation and subsequent washes in Hanks' buffer and resuspended them in basal cell culture medium (EBM; Lonza, Morristown, NJ, USA) at a hematocrit of 20% with 10mM of HEPES. We performed an acute short-term activation, where the (compounds were provided by IHP Therapeutics, San Carlos, CA, USA). Phase-contrast images of the adhered RBCs were acquired with an inverted microscope (DMi8 Leica Microsystems Inc. Deerfield, IL, USA) and quantified based on previously published methods. Paired t-test was used to calculate statistical significance. Initially, we completed a dose response study to compare IHP-102 at concentrations of 100μg/mL to 10μg/mL in 7 samples. Then, we compared IHP-102 at concentrations of 10μg/mL and 1μg/mL in 6 additional samples. The 1μg/mL dose corresponds to IHP-102 blood concentration at effective dose levels, determined from separate PK and pharmacology studies. The ongoing study is comparing the efficacy of IHP-102 treatment with commercially available crizanlizumab. The physiological dose of 1μg/mL of IHP-102 was chosen to be compared to the standard dose of 100μg/mL of crizanlizumab. Results: Acute short-term heme activation induces a high level of RBC adhesion, with a range between 3000 and 50000 adherent RBCs. IHP-102 significantly reduces the adhesion of RBCs in acute activated endothelium at all dose levels tested (p<0.05). These results confirm IHP-102 is effective in reducing RBC adhesion at therapeutically relevant concentrations and that the BioChip Labs EOC assay is a useful tool for monitoring the pharmacodynamic activity of IHP-102. Initial results in ongoing studies comparing IHP-102 (1μg/mL) to crizanlizumab (100μg/mL) show that IHP-102 significantly reduces RBC adhesion while crizanlizumab, even at 100 times higher dose level, does not (n=7 donor samples; p=0.03 vs. p>0.05; IHP-102 vs. crizanlizumab, respectively). Conclusions: IHP-102 inhibits RBC adhesion to acutely activated HUVECs across a diverse dosage spectrum from 100μg/mL down to 1μg/mL. These results further support its potential as an acute treatment for VOE and are a first demonstration of its activity in human SCD donor blood. Our findings indicate that IHP-102 exhibits increased effectiveness when the baseline adhesion level is elevated due to heterogeneity in SCD. Furthermore, IHP-102 demonstrates superior efficacy in comparison to crizanlizumab, even at crizanlizumab concentrations 100 times higher than IHP-102. The BioChip Labs EOC assay represents an innovative and effective methodology for evaluating the efficacy of novel anti-adhesive agents and holds potential for pre-screening and monitoring patients' treatment response to emerging therapies for SCD.