ALBERT

Description: Key Points Using genome-wide association study, we found the first replicated genetic association with acute chest syndrome in sickle cell disease patients. The locus identified includes COMMD7, a gene highly expressed in the lung that interacts with NFκB to control inflammatory responses.

Description: Introduction The duration of red blood cell (RBC) storage is currently being examined as a possible determinant of clinical outcomes in transfused patients in large randomized trials in several patient populations, as well as in preclinical models. Biochemical and functional changes coincident with blood storage, referred to as the “storage lesion,” are numerous and complex. Longer storage periods have, for example, been linked to increased tendency of RBCs to adhere to endothelium. How the proadhesive effect of storage influences the transfusion recipient might depend in part on preexisting comorbidities, consistent with a “two-hit” model of transfusion morbidity. We therefore developed a two-hit in vitro model using older stored RBCs and TNF-α pretreatment (activation) of HUVECs in order to better understand mechanisms of post-transfusion complications related to RBC adhesion. Methods Red blood cell (RBC) preparation and labeling: Using an IRB-approved protocol, stored packed RBC segments stored in additive solution (AS)-3 were obtained at 35-42 days storage from the Transfusion Service at Duke Hospital. The stored RBCs were removed aseptically and washed 3 times with PBS, labeled with fluorescent dye PKH26, and allowed to incubate for 3 minutes. BSA (1%) in DPBS was added, and after one minute, the labeled cells were washed three times in PBS with Ca++ and Mg++ while avoiding exposure to light. Finally 5 µL of labeled RBCs was suspended in 3 mL of PBS for adhesion assays. Adhesion assays Human umbilical vein endothelial cells (HUVECs), grown to confluence on glass slides precoated with a 2% gelatin solution in a 5% CO2 incubator at 37.1°C, were placed in a graduated height-flow chamber. The height was measured at 7 different points along the chamber. After 15 minutes of PBS flow (2 mL min-1), the RBC sample was introduced to the chamber slowly at a rate of 1.5 mL min-1. The RBCs were then allowed to incubate without flow for 5 minutes, and the number of adhered cells at each location (height) was recorded. After 5 minutes of fluidic flow with PBS, the number of adherent cells at each location, corresponding to varying shear stresses, was counted. Percent adhesion and shear stress were calculated at each height. TNF-α treatment of HUVECs: Where indicated, TNF-α (20 µL) was added to 20 mL of cell media to yield a final concentration of 10 ng mL-1, and allowed to incubate overnight (16 hours). Results Adhesion of 35-42-days stored RBCs to non-activated HUVECs varied widely, as reported, and at shear stresses of 1, 2, and 5 dynes/cm2 was 42.92%, 10.77%, and 2.157% respectively, with a typical inverse relation to shear stress (p

Description: Introduction: The pan-selectin antagonist rivipansel (GMI-1070) reduced intravascular arrest of red/white blood cell aggregates and improved blood flow and survival in a mouse model of sickle cell disease vaso-occlusive crisis (SCD VOC). In a Phase 1 study of GMI-1070 infusion in SCD adults at steady state (not in VOC), GMI-1070 decreased markers of cellular activation including neutrophil integrins, platelet/neutrophil aggregates, soluble adhesion molecule concentration; and markers of hemostatic activation. Furthermore, in a randomized Phase 2 study of SCD patients, treatment of VOC with GMI-1070 improved clinical outcomes such as time to resolution of crisis, time to discharge, and IV opioid use. Herein we report on the effect of GMI-1070 on biomarkers of cellular and hemostatic cascade activation from this Phase 2 trial. Methods: Patients in VOC enrolled in a prospective, randomized multi-center double-blind Phase 2 trial, ages 12-60 with HbSS or HbSB0thalassemia were treated with GMI-1070 q12h or placebo, in addition to standard treatment per institutional practice, until resolution of VOC. Clinical outcomes and pharmacokinetics have been previously reported (ASH 2013 Abstracts 775, 776, and 2206). Biomarker blood samples were drawn prior to study drug, and on a sparse sampling basis at times starting 30 minutes after initial dose and continuing until 36 hours after the last dose. Analytes measured included: soluble adhesion molecules E-selectin (sEsel), P-selectin, L-selectin, intercellular adhesion molecules 1 and 3, vascular cell adhesion molecule-1; and tissue factor and thrombin-antithrombin complexes by ELISA. At some sites, surface expression of monocyte b2 integrins MAC-1 & LFA-1 and platelet-monocyte aggregates were also measured by flow cytometry. Comparisons were made between the GMI-1070 and placebo groups, and serial expression levels were compared over time. Subgroup analyses were performed by hydroxyurea (HU) use, age group, baseline WBC, and responders' based on clinical outcomes. A mixed effect model was used to test the LS means difference at each time point and ANCOVA model was used to analyze the nadir, peak, and last dose values. Results: ELISA and flow cytometry samples were collected from 70 and 15 subjects, respectively. Soluble E-selectin levels were reduced for the group on GMI-1070 compared to placebo throughout hospitalization, and the differences were statistically significant at some time-points (Figure 1). Baseline sEsel levels were similar; but the peak, nadir, and level at last dose were all lower in the GMI-1070 group (Figure 2). Exploratory subgroup analysis by HU use, age group, response as measured by visual analog scale or opiate use, frequency of VOC in the past, and baseline white blood cell count revealed consistently lower sEsel levels in the GMI-1070 group. Many, but not all, of these differences reached statistical significance. Conclusion: GMI-1070 use during VOC resulted in consistent and significant reductions of sEsel, overall and in sub-groups as compared to placebo. These findings are consistent with the hypothesized effect of GMI-1070 on endothelial activation and/or apoptosis, mediated by inhibition of E-selectin, although an effect on sEsel clearance cannot be excluded. A Phase 3 study is planned to evaluate efficacy and safety of GMI-1070 as treatment for VOC. Soluble E-selectin concentrations may be useful as a biomarker of pharmacodynamic effect. Figure 1: sE-sel was reduced in the GMI-1070 group at all timepoints tested. Comparison to placebo for change from baseline over time is shown. *p

Description: BACKGROUND: We have recently identified a SNP (rs5988) in the gene encoding the factor XIII A chain (F13A) that is highly associated with risk for priapism in SCD. The P value for the comparison of G/G vs. C/G was 0.009, and the odds ratio for having priapism was 2.52 (CI 1.27-5.03) for men with the G/G F13A genotype (encoding FXIIIA 651E) compared to those with the C/G genotype (expressing FXIIIA 651Q/E). We hypothesized that this polymorphism leads to abnormal FXIII-mediated crosslinking and increased heterologous blood cell aggregates. The mechanism whereby heterologous blood cell aggregates form has only been partially elucidated to date. Brittain et al. (2008) showed that fibronectin is detectable within aggregates and plays a critical role in RBC-monocyte interactions, but it is unknown how fibronectin is recruited. We propose that aggregates form via interactions between fibrin(ogen), other plasma proteins (e.g., fibronectin, laminin), and blood cells during blood stasis in the penis during erection. These aggregates might then obstruct vessels, preventing blood egress, and result in SCD-associated priapism. METHODS: We quantitated circulating heterocellular aggregates using flow cytometry and directly labeled antibodies against CD45 (pan-leukocyte), CD235a (glycophorin A), CD41a (platelet GPIIb), and CD14 (monocytes and granulocytes). Heterocellular aggregates were defined as events simultaneously expressing CD45 and CD235a in whole anticoagulated blood. Events were analyzed by forward and side scatter as well as immunostaining characteristics (Canto II flow cytometer, Becton Dickinson, San Jose). Genotypes were ascertained by SNP genotyping using predesigned and custom Taqman SNP Genotyping Assays (ThermoFisher Scientific, Foster City, CA). Mean values for different genotypes were compared using two-tailed t-tests; FXIII activity was compared using 2-way ANOVA. RESULTS: As previously described, heterocellular aggregates occurred more frequently in blood samples from SCD subjects than from HBAA controls. Among SCD subjects, the % WBCs found in aggregates ranged from 14.9% to 88.5% (mean 58.85%). Specifically, lymphocytes and monocytes were found significantly more frequently in aggregates in HbSS compared to HbAA samples (p=0.042 and 0.025, respectively). When analyzed by F13A genotype, we also found that aggregates containing monocytes and lymphocytes were significantly more numerous in individuals with the GG (priapism risk) genotype than with the CC (low-risk) genotype (p = 0.008 and 0.015, respectively). We then tested recombinant (r) FXIIIA isoforms corresponding to the two alleles of F13A for their ability to bind to and crosslink both fibrinogen and fibronectin. Comparison of their ability to bind fibrinogen, fibrin and fibronectin showed no significant differences between the two rFXIII isoforms after activation. However, activated FXIIIA 651E (G allele) crosslinked both fibrinogen and fibronectin significantly more quickly than did activated FXIIIA 651Q (C allele) (p = 0.006 and p = 0.012, respectively), thus suggesting that the G allele might be associated with greater amounts of crosslinked fibrin(ogen) and fibronectin in the circulation to promote aggregate formation. CONCLUSIONS: Our study has demonstrated that the F13A G allele at rs5988 carries a higher risk for SCD-related priapism and is associated with increased involvement of lymphocytes and monocytes in heterocellular aggregates. A possible mechanism is suggested by our observation that rFXIIIA protein encoded by the G allele more rapidly crosslinks fibrinogen and fibronectin than that encoded by the C allele. We theorize that the G genotype may be related to greater fibrin and fibronectin crosslinking, thus promoting the formation of circulating heterocellular aggregates. Disclosures No relevant conflicts of interest to declare.

Description: Abstract 1632 GMI-1070 is a pan-selectin inhibitor that targets E-, P-, and L-selectins and has shown activity in multiple animal models of disease. Sickle cell disease (SCD) is characterized by periodic vaso-occlusive (VOC) episodes in which cell adhesion and aggregation play a crucial role. GMI-1070 has previously been shown to restore blood flow and improve survival in a mouse model of VOC, and safety and PK have been evaluated in normal, healthy volunteers in phase 1. Here we report clinical, safety, and PK results from the first study of GMI-1070 in individuals with SCD. Methods: An open-label phase 1/2 study was performed, enrolling adults with SCD at steady state. GMI-1070 was administered in two IV doses given on the same day: 20 mg/kg in the first dose, followed 10 hours later by 10 mg/kg. Patients were evaluated for safety on days 0, 1, 2, 7 and 28, including adverse events (AEs), routine clinical labs, and clinical exam. Plasma and urine concentrations of GMI-1070 were measured on days 0, 1, and 2, and PK parameters calculated and compared with those from healthy volunteers. Results: Fifteen adults were enrolled at three centers; 13 with HbSS, 2 with HbSB0thal. All were African-American, 9 were male, mean age was 32 years (range 18–50), mean weight was 64.7 kg; 4 were on hydroxyurea. In the past year, 6 had experienced VOC requiring medical care; 2 had ACS; 2 required transfusions; and 1 had an episode of priapism. Five were hospitalized in the past year; 12 were hospitalized in the past 5 years. All subjects received both doses of study drug; all but one were followed for 28 days. The PK in adults with SCD was in good agreement with that in the controls. The elimination half-life of GMI-1070 averaged 7.73 ± 2.45 hours (Figure). Renal clearance averaged 18.0 ± 7.93 mL/min and accounted for essentially all elimination. Physical exam parameters after dosing were unchanged, and all infusions were well tolerated. Four subjects reported headache within 24 hours of dosing, all of which were mild or moderate and resolved within 24 hours. Two subjects experienced VOC not requiring hospitalization, at 2 and 4 weeks after dosing. One subject had worsening anemia requiring transfusion 5 days after dosing. Other adverse events typical of SCD were reported without apparent association with study drug; none were serious adverse events. Routine labs demonstrated no changes from baseline (Hb, reticulocytes, platelets, electrolytes, glucose, ALT, LDH, BUN, Cr, bilirubin, urinalysis) with the exception of white blood cell counts (WBC) and absolute neutrophil counts (ANC). At 24 hours, mean WBC change from baseline was 1.9K/mm3, or 20% (p=0.076, using parametric test with mixed model); mean ANC change was 2.7, or 67% (p=0.019); all returned to baseline by 7 days. One individual had marked leukocytosis 24 hours after dosing (from 10.4 to 28K/mm3), returning to baseline by day 7; no other effects were observed in this subject. Mean C-reactive protein (CRP) increased at 24 and 48 hours, returning to baseline by day 7. Two subjects had marked increases in CRP: one exhibited leukocytosis with dosing and the other had a high baseline WBC count. There was otherwise no apparent correlation between PK, WBC/ANC, hydroxyurea use, or adverse events. In conclusion, GMI-1070, a pan-selectin inhibitor, when administered to adults with SCD at steady state, has a similar safety and PK profile to that in healthy volunteers. However, SCD patients had moderate WBC and ANC increases at 24–48 hours after dosing, which return to baseline without other observed symptomatic adverse events. This study supports further evaluation of GMI-1070 for the treatment of vaso-occlusive crisis. Disclosures: Styles: GlycoMimetics: Consultancy, clinical trial sponsorship. Wun:GlycoMimetics: Consultancy, clinical trial sponsorship. De Castro:GlycoMimetics: clinical trial sponsorship. Telen:GlycoMimetics: Consultancy, clinical trial sponsorship. Kramer:GlycoMimetics: Consultancy. Flanner:GlycoMimetics: Employment, Equity Ownership. Magnani:GlycoMimetics: Employment, Equity Ownership. Thackray:GlycoMimetics: Employment, Equity Ownership. Off Label Use: This drug (GMI-1070) has not been approved for any clinical indication.

Description: The natural history and mechanisms associated with pulmonary hypertension (pHTN) in sickle cell disease (SCD) are incompletely characterized. We investigated the prevalence of pHTN, diagnosed by echocardiography and/or cardiac catheterization, in adults with all types of SCD, to determine whether the frequency of pHTN varied by Hgb diagnosis. We also analyzed which clinical conditions and laboratory findings were associated with pHTN. We screened 125 outpatients with Hgb SS, SC, Sβ0 or Sβ+ thalassemia who presented with symptoms including either shortness of breath, fatigue, or low or decreasing O2 saturation. PHTN was defined by tricuspid regurgitation jet velocity (TRjet) of ≥ 2.5 m/s by echo and was present in 36% (28/77) of SS & Sβ0 and in 25% (12/48) of SC & Sβ+ patients studied. Of patients with pHTN, 16 (57%) of the SS & Sβ0 patients had a peak TR jet 〉3.0 m/sec and 12 (43%) ≥2.5-

Description: Abstract 514 An elevated tricuspid regurgitant jet velocity (TRV) by echocardiography, associated with increased mortality risk, may be reflective of pulmonary and systemic vasculopathy in sickle cell disease (SCD). The epidemiologic associations between an elevated TRV and other sub-phenotypes of SCD, such as leg ulcers, priapism, proteinuria and increased rate of hemolysis, suggest a common pathogenic link across vascular beds. We were interested in identifying genetic modifiers of SCD and hypothesized that an elevated TRV is, in part, genetically determined. Previous candidate gene studies identified single nucleotide polymorphisms (SNPs) in activin A receptor, type II-like 1 (ACVRL1), bone morphogenetic receptor 2 (BMPR2) and bone morphogenetic protein 6 (BMP6) associated with an elevated TRV suggesting a role for the TGF-b pathway in pathogenesis. We performed a genome-wide association study (GWAS) to identify novel genes and pathways that might be associated with an elevated TRV. We first used Illumina 610K arrays in a GWAS on 340 sickle cell disease patients recruited as part of the observational arm of the Pulmonary Hypertension and Sickle Cell Disease with Sildenafil Therapy (Walk-PHaSST) clinical trial (discovery set) and then validated these results using a replication set consisting of 56 patients enrolled in the studies of TRV and SCD at Duke and Boston Universities. All subjects were 17 years or older and had the HbS only phenotype. We analyzed the data using TRV as a continuous variable and we adjusted for potential confounding effects of age and gender. In addition, as there were 9 different clinical centers in the discovery set, we used center-adjusted TRV values for the analysis. We identified 3 SNPs in CUB and sushi multiple domains 1 (CSMD1) (rs12674750, p=9.5 × 10−6, rs7008391, p=1.8×10−5, rs4433172, p=4.1×10−5) and 1 in sorting nexin 31 (SNX31) (rs1609, p=8.2×10−5), also on chromosome 8q, which were also identified in our replication set (p=0.04 for CSMD1 SNPs and 0.01 for SNX31). CSMD1 is a very large gene of more than 2 Mb and the 610 array includes 1710 SNPs in this gene. However, the probability that we observe 3 SNPs replicated in this gene in a sample size of 56 by chance is only 0.0005. Additionally, 2 SNPs in the mediator of innate immunity, lymphocyte antigen 86 (LY86) (rs3827783, p=3.89×10−6, rs9502483, p=1.02×10−5), were identified in our discovery set. We evaluated the top 5 SNPs originally identified in the candidate gene studies and found that 4 were not present on the 610K arrays. The remaining one had a p-value of 0.17 but is in linkage dysequilibrium with another SNP in BMP6 (rs7450930), a rare variant, with a p-value of 2.5 × 10−5. CSMD1 inhibits the classical pathway of complement activation and complement-mediated hemolysis in rats. This is of interest in SCD because of recent data linking cell-free heme and immune modulation. These findings suggest that sickle cell disease patients with an elevated TRV have altered innate immunity and that sickle vasculopathy may be associated with dysregulation of inflammation. Disclosures: No relevant conflicts of interest to declare.