ALBERT

Description: Key Points The fraction of invariant NKT cells demonstrating activation is increased during painful crises compared with steady state. Regadenoson, an adenosine A2A receptor agonist, decreases the fraction of activated invariant NKT cells during painful crises.

Description: Hydroxyurea and higher hemoglobin F improve the clinical course and survival in sickle cell disease, but their roles in protecting from pulmonary hypertension are not clear. We studied 399 children and adolescents with sickle cell disease at steady state; 38% were being treated with hydroxyurea. Patients on hydroxyurea had higher hemoglobin concentration and lower values for a hemolytic component derived from 4 markers of hemolysis (P ≤ .002) but no difference in tricuspid regurgitation velocity compared with those not receiving hydroxyurea; they also had higher hemoglobin F (P 〈 .001) and erythropoietin (P = .012) levels. Hemoglobin F correlated positively with erythropoietin even after adjustment for hemoglobin concentration (P 〈 .001). Greater hemoglobin F and erythropoietin each independently predicted higher regurgitation velocity in addition to the hemolytic component (P ≤ .023). In conclusion, increase in hemoglobin F in sickle cell disease may be associated with relatively lower tissue oxygen delivery as reflected in higher erythropoietin concentration. Greater levels of erythropoietin or hemoglobin F were independently associated with higher tricuspid regurgitation velocity after adjustment for degree of hemolysis, suggesting an independent relationship of hypoxia with higher systolic pulmonary artery pressure. The hemolysis-lowering and hemoglobin F–augmenting effects of hydroxyurea may exert countervailing influences on pulmonary blood pressure in sickle cell disease.

Description: In adults with sickle cell disease (SCD), an increased tricuspid regurgitation velocity (TRV) by Doppler echocardiography is associated with increased morbidity and mortality. Although sildenafil has been shown to improve exercise capacity in patients with pulmonary arterial hypertension, it has not been evaluated in SCD. We therefore sought to determine whether sildenafil could improve exercise capacity in SCD patients with increased TRV and a low exercise capacity. A TRV ≥ 2.7 m/s and a 6-minute walk distance (6MWD) between 150 and 500 m were required for enrollment in this 16-week, double-blind, placebo-controlled sildenafil trial. After 74 of the screened subjects were randomized, the study was stopped early due to a higher percentage of subjects experiencing serious adverse events in the sildenafil arm (45% of sildenafil, 22% of placebo, P = .022). Subject hospitalization for pain was the predominant cause for this difference: 35% with sildenafil compared with 14% with placebo (P = .029). There was no evidence of a treatment effect on 6MWD (placebo-corrected effect −9 m; 95% confidence interval [95% CI] −56-38; P = .703), TRV (P = .503), or N-terminal pro-brain natriuretic peptide (P = .410). Sildenafil appeared to increase hospitalization rates for pain in patients with SCD. This study is registered at www.clinicaltrials.gov as NCT00492531.

Description: Abstract 1074 Non-Cardiopulmonary Factors Affecting the Six-Minute Walk Distance in Patients with Sickle Cell Disease: Results from the Walk-PHaSST Study. INTRODUCTION: The six-minute walk (6MW) test is frequently used to assess exercise capacity. Patients with sickle cell disease (SCD) can have decreased 6MW distance (6MWD) compared to controls. The 6MWD in conjunction with the TR-jet velocity (TRV) and NT-proBNP have recently been proposed to have a greater predictive value for screening SCD patients suspected of having pulmonary hypertension (PH) than TRV alone. (Parent et al, NEJM, 365; 1, 2011 365 (1):44–53). The American Thoracic Society guidelines recommend caution in controlling for sources of variability in the 6MWD (Am J Respir Crit Care Med 166. 111–117, 2002). Age and height are known confounders of the 6MWD. However, non-cardiopulmonary factors including skeletal-mechanics and pain may also impact the 6MWD. AIM: This study explores whether non-cardiopulmonary factors affect the 6MWD in SCD patients. METHODS: We analyzed data from subjects screened for the walk-PHaSST trial. Walk-PHaSST was a multi-center, placebo-controlled, double-blind, 16-week trial evaluating the safety and efficacy of oral sildenafil for the treatment of Doppler-defined PH (TRV '2.7m/s) in subjects with SCD aged 〉12 years. The primary endpoint in the trial was change in 6MWD. During screening, subjects were evaluated by self-reported medical history, physical examination, blood sampling, echocardiography and 6MWD. Univariate and multivariable linear regression was performed. A two sided p value

Description: Abstract 948 Background: Amino-terminal pro-brain type natriuretic peptide (NT-proBNP) is a widely used clinical laboratory marker of left ventricular stress, although it is also known to be elevated in adults with right ventricular stress due to pulmonary hypertension associated with sickle cell disease (SCD) and other disorders. NT-proBNP is associated with early mortality in adults with SCD. Methods: Using a standard clinical laboratory assay, we measured NT-proBNP in 346 children (median age 12; IQR 7–16 years) with SCD enrolled in the Pulmonary Hypertension and the Hypoxic Response in SCD (PUSH) study, an ongoing, longitudinal and observational multicenter study of children with sickle cell disease. In order to adjust for age, a known confounding factor of NT-proBNP in young children, we compared the characteristics of children with the top quartile of NT-proBNP for each 3-year increment of age with those of children in the remaining three quartiles for that age increment. We examined several factors alone and as part of a logistic regression model. Results: In univariate analyses, high expression of NT-proBNP was associated with lower hemoglobin levels (median 83 vs. 87 gm/L, p=0.0006). The high NT-proBNP group also had a higher hemolytic component, a principal component-derived index of the common properties of the hemolytic markers reticulocyte count, serum lactate dehydrogenase, aspartate aminotransferase and bilirubin (median 0.5 vs. 0.7 relative units, p=0.021). The high NT-proBNP group had a higher prevalence of high left ventricular filling pressure, as indicated by the mitral inflow E wave to tissue Doppler E wave (E/Etdi) above 9.22 (13.1% vs. 2.9%, p9.22 were independently associated with high NT-proBNP levels. Conclusions: After adjustment for age in children with SCD, upper quartile NTproBNP is independently associated with severity of anemia and echocardiographic markers of left ventricular size and diastolic filling pressure. At this age, they have not yet developed the association observed prominently in SCD adults between NT-proBNP and elevated pulmonary arterial pressures. Our data lends support to childhood being considered as a pre-symptomatic phase of pulmonary hypertension; which could be targeted for clinical trials of preventative strategies to prevent adult onset of pulmonary vascular changes associated with higher NT-proBNP and higher TRV. Hydroxyurea therapy is associated in our results from children with SCD with lower NT-proBNP, making it an attractive candidate for a trial in children with SCD to prevent pulmonary hypertension in adulthood. NT-proBNP in childhood SCD remains a marker of left ventricular measures, identifying this additional feature of cardiopulmonary risk. Disclosures: No relevant conflicts of interest to declare.

Description: Despite the high prevalence of PHTN in adults with SCD, the prevalence in the pediatric population with SCD is not known. We hypothesized that elevated pulmonary artery pressures may be found in SCD adolescents with history of pulmonary complications, such as acute chest syndrome (ACS), obstructive sleep apnea (OSA), asthma, and reactive airway disease. Thirty such sickle cell disease adolescents were screened at Howard University or University of Michigan for PHTN with Doppler echocardiography. We defined PHTN as a tricuspid regurgitant jet velocity (TRV) of at least 2.5 m/sec (corresponding to a pulmonary artery systolic pressure greater than 35 mm Hg). PHTN was found in 16 SCD patients (53.3%) and 5 (16.7%) had TRV 〉 3.0 m/sec. Clinical findings according to the presences or absence of PHTN are shown in the table. Potential factors contributing to PHTN in patients with SCD include chronic hemolysis and chronic hypoxia. Our results suggest that PHTN is common among SCD adolescents with a history of pulmonary complications. Consideration should be given to screening such patients for PHTN and exploring treatment options. Further studies are urgently needed to clarify the prevalence and mechanisms of PHTN in adolescents with SCD. Clinical and demographic data of 30 SCD adolescents with pulmonary findings who underwent echocardiography at Howard University Hospital or University of Michigan PHTN (N = 16) No PHTN (N = 14) P Age in years (mean +/− SD) 15.9 +/− 3.2 17.4 +/− 2.3 0.17 Females (no. and %) 5 (31.3) 7 (50) 0.5 Hemoglobin SS Phenotype (no and %) 14 (87.5 11 (78.6) 0.5 Hemoglobin concentration (mean +/− SD) 8.0 +/− 2.1 9.3 +/−1.9 0.11 White blood cells (mean +/− SD) 10.9 +/− 2.9 9.7 +/− 3.7 0.4 Platelet (mean +/− SD) 475 +/− 172 364 +/− 240 0.17 Hemoglobin F percent (mean +/− SD) 5.1 +/− 3.5 6.4 +/− 5.5 0.6 Lactate dehydrogenase (mean +/− SD) 505 +/− 162 264 +/− 50 0.002 Total bilirubin (mean +/− SD) 4.1 +/− 2.6 3.4 +/− 2.6 0.5 Creatinine concentration (mean +/− SD) 0.6 +/− 0.2 0.7 +/− 0.2 0.18 Aspartate transaminase (mean +/− SD) 48 +/− 27 36 +/− 16 0.18 Alanine transaminase (mean +/− SD) 51 +/− 37 39 +/− 20 0.3

Description: Abstract 1651 Background: Pain crises are the most common symptom experienced by individuals with sickle cell anemia (SCA). Frequency of pain crises varies significantly and high rate is a risk factor for higher mortality in adults with SCA. The risk factors for pain crises in children and adolescents with SCA are not completely understood. To determine factors associated with frequent severe pain crises, we analyzed the cohort of children and adolescents with SCA who were enrolled in the prospective study “The Pulmonary Hypertension and the Hypoxic Response in SCD (PUSH)”. All children were evaluated in their steady, non-crisis state. Methods: Family-reported history of number of severe pain crises in the preceding 12 months was recorded prospectively in 365 children and adolescents with SCA. Severe pain crises were defined as painful vaso-occlusive episodes requiring evaluation in Emergency Department (ED) or in-patient hospitalization. Lifetime history of red cell transfusions, echocardiography, and laboratory studies were obtained. Clinical and laboratory characteristics of study subjects who had ≥3 severe pain crises in the preceding year were compared to subjects with 〈 3 severe pain crises. Results: Study subject ranged in age from 3–20 years and 175 (48%) were female. Seventy two children (20 %) had ≥3 severe pain crises in the preceding year (frequent pain crisis group); 293 (80%) children had 〈 3 severe pain crises (infrequent pain crisis group), including 224 (61%) subjects who had no admissions/ ED visits for pain. Associated factors for frequent pain crisis included older age (odds ratio 1.2; 95% confidence interval 1.12–1.35; P 10 lifetime transfusions (42% vs. 22%; p=0.001). Median tricuspid regurgitation jet velocity (TRV) was higher in the frequent pain crisis group (2.41 vs. 2.31; p= 0.001) but the proportion of children with TRV〉2.5 was not different (19.4% vs.11.5% in infrequent crises group; p=0.09). Hydroxyurea use was not different between the groups (51% vs. 40%; p=0.08) nor was fetal hemoglobin (10% vs. 12%; p=0.2). Conclusions: The occurrence of severe pain crisis varies among children and adolescents with SCA with a large number of children experiencing no severe painful episodes. Consistent with the Cooperative Study of Sickle Cell Disease report, the risk of severe pain crisis increases with age. Individuals with α-thalassemia trait are likely to experience more frequent pain crises possibly related to higher hemoglobin concentration and viscosity. However, even after controlling for α-thalassemia trait, children and adolescents who reported more frequent severe pain crises showed evidence of less hemolysis, consistent with a hypothetical model associating the hemolytic subphenotype of SCA with less frequent vasoocclusive pain crises. Further studies are indicated to identify the molecular mechanisms of pain in sickle cell anemia. Disclosures: No relevant conflicts of interest to declare.

Description: Background: Sickle cell disease shares common complications such as vasculopathy and organ dysfunction involving the heart, the lungs, the liver and the kidneys with other hemolytic conditions. We hypothesized that a hemolytic vasculopathy may underlie some of these complications. Distinguishing whether a complication is due to hemolysis or to the degree of anemia has been a challenge. Methods: A prospective, multicenter study of 310 children and adolescents with sickle cell disease in steady state was conducted. The associations of measures of hemolysis and of hemoglobin concentration with disease complications were assessed. Principle component analysis was used to develop a hemolytic index from the measurements of reticulocyte count, lactate dehydrogenase, aspartate aminotransfersae and total bilirubin. In order to determine the independent associations of hemolysis with the clinical manifestations of sickle cell disease, we computed correlation coefficient of the hemolytic index with these manifestations that controlled for hemoglobin concentration. P values were adjusted for multiple comparisons. Results: Hemolysis and hemoglobin had no significant correlation with number of the severe pain episodes, acute chest syndrome and priapism. The hemolytic index correlated with history of stroke (r= 0.19, p=0.026), white blood cell count (r=0.22, p

Description: Background. As a part of a multicenter, observational study in determining the prevalence and risk factors of elevated tricuspid regurgitant jet velocity (TRV) in children, oxygen desaturation correlated with TRV. We further investigated the risk factors and clinical associations of oxygen desaturation at rest and after execrcise in children at steady state. Methods. 310 children and adolescents with sickle cell disease were studied under basal conditions. Pulse oximetry was determined at rest and after a six minute walk test. The relationships of oxygen saturation at rest and desaturation during exercise to the available clinical and laboratory variables were investigated. Results. Among 300 patients with available baseline oxygen saturations, 30 (10%) had saturation 98 percent. Twenty-three (9%) of 244 patients had 〉3 percentage point reduction in oxygen saturation during a six minute walk; the median (interquartile range) baseline saturation was 96 (95– 99) percent among these patients versus 98 (97–100) percent among those with less or no reduction in saturation during the walk (P = 0.0009). Hemoglobin (p3 percentage points during the walk while a hemolytic index (p3 percentage points. Conclusion. Markers of hemolysis, low hemoglobin, PIEDV, LVMI, LVIDD z score, lower TLC, and elevated TRV velocities are associated with ≥3% reduction in oxygen desaturation during six minute walk in children and adolescents with sickle cell disease. A high degree of oxygen desaturation during the six minute walk in sickle cell disease patients might serve as an early biomarker for pulmonary hypertension. Exercise induced changes in oxygen saturation in sickle cell disease children may provide insight into the development of pulmonary hypertension as adults.

Description: Abstract 849FN2 Adenosine 2A receptor (A2AR) agonists decrease pulmonary inflammation and injury in a murine model of sickle cell disease (SCD) by interrupting invariant NKT (iNKT) cell activation that occurs as a result of vaso-occlusion (Clin Immunol 2011). iNKT cells represent a subpopulation of T lymphocytes that rapidly generates pro-inflammatory cytokines upon activation and thereby may play a role in sustaining or propagating vaso-occlusion in human SCD. To evaluate the effects of A2AR activation in human SCD, we are conducting a dose-seeking and safety study of the FDA-approved, highly selective A2AR agonist regadenoson in patients with SCD ≥ 14 years of age. This is a multi-center, phase I clinical trial of infusional regadenoson that utilizes a 3+3 design and is comprised of four stages to determine maximum tolerated dose and safety during a 12 hour infusion in adults at baseline (defined as absence of increased pain, ED or hospital visit in the past 2 weeks) (stage 1), a 24 hour infusion in adults at baseline (stage 2), a 24 hour infusion in adults during a painful vaso-occlusive crisis (VOC) (stage 3), and a 24 hour infusion in children (≥ 14 years) during a painful VOC (stage 4). Three dose levels of infusional regadenoson have been evaluated thus far: 0.24 (level 0), 0.6 (level 1) and 1.44 mcg/kg/hour (level 2). Samples for plasma levels and iNKT cell activation are obtained at 0, 6, 12, 18 and 30 hours after start of infusion. Two methods are used to identify iNKT cells: 1) CD1d tetramers, loaded with the α-GalCer-like glycolipid PBS57 (defined as tetramer+CD3+), and 2) 6B11 antibody to the invariant T cell receptor (defined as CD45+6B11 high). NF-kB activation is measured using phospho-Ser536-p65 antibody (phospho-NF-kB). To date, 15 subjects have received infusional regadenoson. Regadenoson appears to be safe (devoid of cardiovascular or other side effects) and biologically active in reducing iNKT cell activation markers during a 12 hour infusion at dose levels 1 and 2, and we are currently evaluating the safety of a 24 hour infusion at dose level 2. No dose limiting toxicities occurred in the 15 subjects at dose levels 0, 1 or 2. Pharmacokinetic analyses showed mean peak plasma concentrations of regadenoson for dose levels 0, 1 and 2 were 0.37 ng/ml, 1.16 ng/ml and 2.19 ng/ml, respectively. Based on animal models, we suspect that biologically active plasma concentrations are approximately 1 ng/ml. At baseline, iNKT cells were activated in patients with SCD as defined by elevated levels of phospho-NF-kB expression and A2AR expression as assessed by anti-A2AR immunoreactivity. In particular, CD4+ iNKT cells showed increased phospho-NF-kB expression whereas CD4- iNKT cells were not activated. At dose levels 1 and 2, regadenoson decreased iNKT cell activation in patients with SCD. For example, when pre- and post-12 hour regadenoson infusion measurements were compared at dose level 2 (N=5), mean percent of cells expressing phospho-NF-kB in the activation gate was 40% (90% CI 23–58) at baseline and decreased by 53% at the end of infusion to 19% (90% CI 6–33). Similar results were found in measurements of A2ARs. We conclude that infusional regadenoson administered at 0.6 and 1.44 mcg/kg/hour is safe during a 12 hour infusion and decreases activation of iNKT cells. Our next steps are to determine whether infusions of a longer duration (24 and 48 hours) are safe and similarly biologically effective during a VOC. We then intend to conduct a clinical trial to measure the clinical efficacy of regadenoson infusion during VOC and acute chest syndrome episodes. Disclosures: Off Label Use: Regadenoson is an adenosine 2A receptor agonist that is FDA approved for use during myocardial stress imaging. We are examining the safety of regadenoson in patients with sickle cell disease. Nathan:Astellas: Research Funding.