ALBERT

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: Ineffective erythropoiesis, the hallmark of β-thalassemia, is a result of α/non-α globin chain imbalance.1 One strategy to redress globin-chain imbalance is to induce γ-globin gene (HBG) expression. Repression of HBG in adult erythroid cells involves DNA methylation and other epigenetic changes. Therefore, the cytosine analog decitabine, which can deplete DNA methyltransferase 1 (DNMT1), can potentially activate HBG. In 5 patients with β-thalassemia intermedia, a dose and schedule of decitabine intended to deplete DNMT1 without causing significant cytotoxicity (0.2 mg/kg subcutaneous 2 times per week for 12 weeks) increased total hemoglobin from 7.88 ± 0.88 g/dL to 9.04 ± 0.77 g/dL (P = .004) and absolute fetal hemoglobin from 3.64 ± 1.13 g/dL to 4.29 ± 1.13 g/dL (P = .003). Significant favorable changes also occurred in indices of hemolysis and red blood cell densitometry. Consistent with a noncytotoxic, differentiation altering mechanism of action, the major side effect was an asymptomatic increase in platelet counts without erythrocyte micronucleus or VDJ recombination assay evidence of genotoxicity. This study was registered at www.clinicaltrials.gov as #NCT00661726.

Description: Background Mitochondrial dysfunction is associated with abnormal cell function, oxidative stress and cell death. A progressive impairment of mitochondrial function and/or increased oxidative damage likely contribute to the pathophysiology of SCD, however little is known about bioenergetics in SCD. While previous work demonstrated mitochondrial dysfunction in the platelets of adult patients with SCD characterized by deficient complex IV and V activity which was associated with hemolysis, no data has yet been described in children with SCD. Objective To initiate investigation into mitochondrial function in children with SCD. Methods Children with a diagnosis of SCD requiring parenteral opioids for vaso-occlusive pain episodes (VOE) were prospectively enrolled within 24 hours of presentation to an urban emergency department (ED). Blood was obtained for biomarkers of hemolytic rate, and estimated pulmonary pressures were noninvasively measured by a point-of-care Doppler echocardiography (echo). Platelet rich plasma was isolated from patient whole blood by differential centrifugation and flash frozen for mitochondrial studies. The activities of Complexes V and IV were measured since these enzymes are the sites of oxygen consumption (Cx IV) and ATP production (CxV). The activity of citrate synthase, a mitochondrial matrix protein, was also determined as an indicator of mitochondrial number. Results 11 children with SCD seen in the ED or admitted for VOE were included in this pilot study. Mean age was 17±3 years (range: 9-21 years), 64% were male, and 45.5% had HbSS, 45.5% HbSC and 9% HbSbThal. Mean Hb was 9.9±2 g/dL, reticulocyte (retic) count was 7.6±5%, and 45.5% were on HU. Mean tricuspid regurgitant jet velocity (TRV) = 2.0±0.4 m/s. Of interest patients on HU had significantly higher citrate synthase activity (Figure 1), although Complex IV and V activities were similar in both groups. Citrate synthase activity inversely correlated with TRV (r=-0.63, p=0.04). Similar to the previously reported adult data, complex IV activity inversely correlated with retic count (R2=0.27), although not reaching statistical significance, likely due to small sample size. No difference in mitochondrial studies were found when HbSS patients were compared to those with HbSC. Conclusions These data suggest mitochondrial dysfunction and potentially decreased mitochondrial number in SCD children with increased hemolytic rate (TRV). Further, the greater citrate synthase activity in platelets of patients with SCD on HU compared to those not treated with HU may be indicative of increased mitochondrial number with HU therapy. These observations have never been previously described, and may suggest a novel mechanism of action for HU warranting further study. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 1023 Introduction. Pulmonary hypertension (PH) is a complication associated with thalassemia syndromes, particularly thalassemia intermedia. There are limited data on the safety and efficacy of selective pulmonary vasodilators in this at-risk population. Methods. We evaluated the safety and efficacy of a 12-week prospective, phase 1/2 pilot scale study of sildenafil (100 mg TID) in ten b-thalassemia patients with elevated TRV on Doppler-echocardiography ≥ 2.5 m/s suggestive of PH risk. Patients were evaluated at baseline and at weeks 2, 4, 8 and 12 of sildenafil therapy, and six minute walk distance (6MWD), biomarkers of hemolysis, coagulation, inflammation and adhesion were assessed. Results. Our study population had an average age of 37±12.3 years, 8/10 were male, and 50% were thalassemia intermedia. Splenectomy prevalence was high (90%), and only 30% of patients were transfused since infancy. The mean pre-transfusion hemoglobin was 10.4±1.5 g/dL. A 12-week open-label trial resulted in a significant decrease in TRV by 13.3% (3.0±0.7 vs. 2.6±0.5m/s, p=0.04, Figure 1), improvements in left ventricular end systolic/diastolic volume (p≤0.02), diffusion capacity for carbon monoxide (DLCO, p=0.003) and a trend towards a reduced Borg Dyspnea Score and improved NYHA Functional class. No significant change in 6MWD was noted, although 6MWD correlated strongly with DLCO (ρ=0.72, p=0.03) suggesting that oxygen diffusion across the alveolar-capillary membrane was an important determinant of exercise capacity. Sildenafil was generally well tolerated, but most patients experienced anticipated transient headaches and visual/color disturbances associated with sildenafil use. One patient withdrew from the study due to worsening dyspnea. No other serious adverse events were reported. A strong direct correlation between total dose of sildenafil (mg) taken and % change in plasma NO metabolite concentration was observed (ρ =0.80, p=0.01). A significant increase in plasma and erythrocyte arginine concentration occurred, without an associated change in plasma arginase activity/concentration, nitric oxide metabolites or vascular endothelial growth factor. However arginase concentration was elevated in this cohort similar to prior reports, and correlated inversely to hemoglobin (ρ=-0.41, p=0.01), and directly to ALT (ρ=0.40, p=0.004), AST (ρ=0.38, p=0.04), left ventricular end systolic volume (ρ=0.77, p=0.001), and end-diastolic volume (ρ=0.79, p=0.001). Conclusion. Our study suggests that sildenafil is safe and may improve cardiopulmonary hemodynamics in patients at risk for PH, however improved exercise capacity as reflected by an improved 6MWD was not observed. The reduction in left ventricular dimensions is promising, and could reflective of either increased inotropy or chronotropy, or decreased systemic afterload. This is also the first report of an influence of sildenafil on diffusion capacity of the lungs in patients with thalassemia and the first description of increased plasma and erythrocyte arginine concentration after sildenafil therapy. Given the association of arginine bioavailability with long-term survival in cardiovascular disease, this is an unexpected effect of sildenafil that warrants further investigation. These data support the need for further clinical trials evaluating the use of sildenafil in thalassemia. Disclosures: Taher: Novartis: Research Funding, Speakers Bureau.

Description: Abstract 87 Introduction: Engagement of selectins by their ligands leads to cellular activation and adhesion and plays a role in thrombus formation. In sickle cell disease (SCD), the selectins underlie vaso-occlusion, which results in vaso-occlusive crisis (VOC). In SCD patients high levels of soluble E-selectin (sEsel) are associated with increased mortality (Kato, BJH 2005). In addition, SCD patients exhibit chronic activation of the coagulation cascade and of leukocytes. Previously, we showed in animal models of SCD VOC that pan-selectin antagonist GMI-1070 reduced arrested RBC/WBC aggregates and improved blood flow and survival. In a Phase 1 study of SCD adults at steady state (not in VOC), GMI-1070 inhibited neutrophil activation and platelet/neutrophil aggregate formation and increased circulating neutrophils. Herein we report on the effect of GMI-1070 on biomarkers of monocyte, endothelial cell, and coagulation cascade activation; and on the effect of hydroxycarbamide (hydroxyurea or HU) on these biomarkers for patients on this trial. Methods: SCD adults at steady state (n=15) received an IV loading dose of GMI-1070 (20 mg/kg) and a second dose ten hours later (10 mg/kg). Safety and PK were reported elsewhere. HU use was noted. Biomarker blood samples were drawn prior to treatment, and at 4, 8, 24 and 48 hrs after the loading dose. Analytes measured included: soluble adhesion molecules sEsel, soluble P-selectin (sPsel) and intracellular adhesion molecule-1 (ICAM-1) by multiplex ELISA; tissue factor and thrombin-antithrombin complexes (TF and TAT) by ELISA; and, surface expression of monocyte b2 integrins MAC-1 & LFA-1; and platelet-monocyte aggregates (PMA) by flow cytometry. Expression levels were compared against pre-treatment, and stratified by HU use. Analysis was by ANOVA F-test from mixed effects model. Data are reported for 11 subjects (biomarkers) and 15 subjects (neutrophils). Results: Soluble adhesion markers were reduced after 8 hrs (sEsel), or 4 and 8 hrs (sPsel; ICAM-1). Tissue factor (TF) was reduced at 4 and 8 hrs. Thrombin-antithrombin complex (TAT) levels and expression of MAC-1 and LFA-1 were reduced at all time points. The percentage of PMA was reduced at 8 hrs. (Table) When HU use was considered (HU–No; HU–Yes), the levels of sEsel, sPsel, ICAM-1, TF, TAT, MAC-1, and neutrophil counts were higher and more variable at baseline in the HU-No group, significantly so for ICAM-1 (p=0.048) and MAC-1 (p=0.001). After GMI-1070, significant reduction from baseline was seen in both groups: in the HU-No group for ICAM-1, TF, PMA, LFA-1, and in the HU-Yes group for sEsel, MAC-1, TF, TAT. Neutrophil counts increased at 24 hours in the HU-No group only (p=0.001). Conclusion: In this small sample of adults with SCD at steady state, selectin inhibition by treatment with GMI-1070 resulted in reduction in soluble adhesion markers, leukocyte activation, PMA, and markers of coagulation activation. This suggests that selectin inhibition affects downstream processes in vivo, continuing after plasma clearance of 〉97% of the drug. This may represent interference in the processes that lead to VOC in SCD. A phase 2 study is underway to further explore the effect of this experimental drug on these markers and in the treatment of VOC. Disclosures: Magnani: GlycoMimetics: Employment, Equity Ownership. Kuypers:GlycoMimetics: Research Funding. Patton:GlycoMimetics: Employment. Larkin:GlycoMimetics: Research Funding. Styles:GlycoMimetics: Research Funding. DeCastro:GlycoMimetics: Research Funding. Telen:GlycoMimetics: Research Funding. Wun:GlycoMimetics: Research Funding. Thackray:GlycoMimetics: Employment, Equity Ownership.

Description: Abstract 2122 Introduction: Pulmonary hypertension (PH) commonly develops in thalassemia syndromes, but is poorly characterized. Since limited data are available we set out to provide a comprehensive description of the cardiopulmonary and biological profile of patients with thalassemia at-risk for PH. Methods: We designed a case-control study of thalassemia patients at-risk for PH, defined by a tricuspid-regurgitant-jet velocity (TRV)≥2.5m/s on Doppler-echocardiography (echo) compared to those with low PH-risk, define by a TRV

Description: Abstract 3167 Introduction The membrane of erythrocytes is composed of a bilayer of phospholipids and cholesterol. It is strengthened by a membraneskeleton consisting of the proteins spectrin, ankyrin, pallidin, band 3 and band 4.1. Hereditary elliptocytosis (HE) is caused by mutations in the spectrin protein, resulting in a typical elliptocytic shape. These cells have a decreased deformability and a shortened lifespan. Most mutations in HE are located in the head to head self association site of the α- and β dimers of spectrin. HE Patients with heterozygous mutations in α spectrin show little or no hemolysis because α spectrin is synthesized in an excess relative to β spectrin. In the heterozygous situation, plenty of normal wild type α spectrine (Wt α) is available for incorporation in to the membraneskeleton. In Hereditary Pyropoikilocytosis (HPP) with its bizarre shapes, a second mutation in the α spectrin is present, in addition to the HE mutation. It is responsible for a defect in the synthesis of α spectrin, resulting in the production of 50% functional α spectrin. This mutation is called LELY (Low Expression LYon). Thus, when the LELY mutation is located in trans on the allele in respect to the HE allele, the expression of the Wt α spectrin protein is reduced. As a consequence, more abnormal HE spectrin will be incorporated in to the membraneskeleton. This enhanced expression of the HE mutation results in an unstable membraneskeleton of the HPP cell. Method The deformability of erythrocytes is measured using the ektacytometer LORRCA Maxsis of Mechatronics (Hoorn, The Netherlands). DNA was isolated from white blood cells from peripheral blood. The HE mutations are found by a PCR of the α spectrin exons where most mutations exists for HE, followed by DNA sequencing using the ABI prism genetic analyzer from Applied Biosystems. The LELY mutation is proven by a PCR of exon 40 of the α spectrin gene followed by RFLP agarose gel electrophoresis.(59 G/A) mutation and the LELY mutation are present. In addition to these Exon 2 and LELY mutations, a third mutation in Exon 6 (103 T/C) is found in the α spectrin of brother E.α spectrin is synthesized and incorporated into the membraneskeleton. The same mutations hold for brother E., but he has HPP. This is attributed to a third additional mutation in Exon 6 of the α spectrin gene. This Exon 6 mutation is located in trans to the Exon 2 mutation that in turn is in cis with the LELY mutation. Like in brother W. the expression of the Exon 2 mutated spectrin is reduced due to the LELY mutation. However relatively more of the Exon 6 mutated α spectrin comes available for the dimerization with β-spectrin resulting in an unstable membrane skeleton, causing the HPP of brother E. Results The B. family is examined for the presence of HE or HPP (figure 1). Brother E. has clinical symptoms, poikilocytes and a typical ektacytometric deformability matching HPP. Brother W. and his daughter N. have no clinical symptoms but elliptocytes and an ektacytometric deformability typical for HE. In all affected individuals the same Exon 2. Conclusion The combination of Exon 2 and LELY mutations normally leads to HPP. Concerning brother W. and his daughter N. this is not the case. They have a mild form of HE. The explanation for this finding is, that the Exon 2- and LELY mutation are located in a cis rather than in a trans position on the α spectrin gene (figure 1). In that case the mutated Exon 2 is less expressed and more Wt. Disclosures: No relevant conflicts of interest to declare.

Description: Abstract 4752 Background. An elevated TRV on Doppler echocardiography (echo) identifies pulmonary hypertension (PH) risk, is common in SCD, and is associated with mortality in adults. An elevated TRV is found in 11–15% of children with SCD, and is associated with decreased exercise capacity and low oxygen saturations. An acute rise in pulmonary presssures can compromise oxygenation and contribute to risk of erythrocyte sickling. Acute TRV elevation in SCD has been recently described in adults with VOE and acute chest syndrome (ACS) however studies in children have not previously been performed. The impact of oxygen administration on TRV during VOE is unknown. Objective. To investigate TRV in children with SCD and VOE together with biomarkers of hemolysis, and the impact of oxygen administration. Methods. Investigational intervention without random assignment; Prospective Descriptive Study. Children 1–21 years with a diagnosis of SCD requiring parenteral narcotics for VOE were enrolled within 24 hours of presentation to an urban emergency department (ED). Estimated pulmonary pressures were noninvasively measured by transthoracic echo. A TRV≥2.5 m/s was used to define PH risk. All measurements were made by a single trained sonographer at bedside. Patients with a TRV〉2.5 m/s were administered oxygen by nasal cannula at 2 LPM for 10 minutes, followed by repeated TRV measurement. Secondary outcome measures included correlation of biomarkers of hemolysis and QTc interval with TRV, as well as the effect of oxygen administration on TRV. Results. 22 events were studied in 19 unique subjects, 3 of whom were enrolled twice. Mean age was 16±5 years (range: 4–21 years), 64% were female, and 64% had Hb SS, 27% Hb SC and 9% Hb SbThal. Pain scores were 6±3, mean Hb was 9.6±1.8 g/dL, 9% of children had a borderline/prolonged QTc, and all but 1 patient was admitted for VOE. Mean length of admission was 5±3 days. Chest radiography done on 5/15 (33.3%) revealed an infiltrate, while 3/5 (60%) patients presenting with fever and VOE ultimately developed ACS. 6/22 (27%) episodes required transfusion. Measurements of TRV during VOE were recorded in all 22 patients (mean: 2.11±0.46 m/s; range: 0.98–2.72 m/s). 3 subjects (2 unique subjects, 10%) had a TRV≥2.5 m/s during VOE suggesting PH-risk. Oxygen was administered to patients meeting criteria of PH-risk and all had a reduction in TRV of 0.26, 0.41 and 0.2 m/s respectively (Figure 1). Echos were reviewed by a blinded cardiologist with a concordance correlation coefficient of 0.9431 between evaluators, reflecting good inter-rater reliability. Correlation of secondary outcome measures revealed a statistically significant relationship between reticulocyte count and TRV (ρ=0.5, p=0.02) only. No associations between TRV and Hb, WBC, cell-free Hb, LDH, arginase concentration, pain scores, ACS or QTc were observed. As previously reported, arginase concentration correlated strongly to cell-free Hb (r=0.60, p=0.04). Conclusions. Our preliminary data suggests that an elevated TRV may be less common in children compared to adults with SCD and VOE based on previous published reports. A 10% prevalence of TRV elevation identified in this study is similar to the reported baseline prevalence in children. The role of oxygen administration during VOE and its impact on TRV requires further study. A reduction of TRV in our small cohort however may prove to be clinically relevant, particularly in the acute ED setting. 9% of children presented with a borderline/prolonged QTc, which may have implications when utilizing medications which prolong QTc. Further data is required before conclusions may be drawn regarding TRV elevation in children with SCD and VOE and enrollment is ongoing. Disclosures: No relevant conflicts of interest to declare.