Richard J. Labotka

Hydroxyurea therapy in children severely affected with sickle cell disease

HYPOTHESIS The major clinical manifestations of sickle cell disease (SCD) are hemolytic anemia, predisposition to infection, and recurrent vaso-occlusive episodes resulting in pain, organ dysfunction, or both. There has been no satisfactory treatment for children with recurrent severe painful episodes caused by SCD. Hydroxyurea is an antimetabolite drug shown in adults with SCD to increase fetal hemoglobin levels and reduce the symptoms of SCD. We hypothesized that hydroxyurea therapy in children with severe (defined as > or = 3 vaso-occlusive events per year) SCD could improve hematologic parameters and reduce vaso-occlusive events. OBJECTIVE To assess the safety and efficacy of hydroxyurea for the treatment of severe SCD in children. STUDY DESIGN After obtaining informed consent, we initiated hydroxyurea therapy at a dosage of 10 to 20 mg/kg per day in 15 patients with severe SCD (hemoglobin SS, hemoglobin SS-alpha thalassemia, or hemoglobin S-beta0-thalassemia). Doses were escalated as tolerated. Patients were monitored with bimonthly physical examinations and monthly laboratory measures. To assess the impact of hydroxyurea on clinical symptoms, we recorded the number of inpatient days for each patient during the period of treatment and compared it with the number of inpatient days for the 12- to 24-month period before institution of hydroxyurea therapy, using the subject as his or her own control subject. RESULTS Thirteen patients received hydroxyurea for a median of 24 months (range, 6 to 39 months). The mean dose of hydroxyurea was 21.4 +/- 5.2 mg/kg. Treatment with hydroxyurea induced statistically significant increases in the total hemoglobin concentration, mean corpuscular volume, and percentage of hemoglobin F, and a decrease in the serum concentration of bilirubin. Toxic effects included three episodes of a reversible myelotoxic reaction, two of which required transfusion of packed erythrocytes. For the entire group, hospitalization decreased from a prehydroxyurea median of 3.9 +/- 2.0 days per month to 1.1 +/- 2.1 days per month of therapy (p = 0.09). For those subjects (n = 10) completing at least 1 year of treatment, the decrease in hospitalization from 4.1 +/- 2.2 to 1.0 +/- 1.7 days per month was significant (p = 0.03). CONCLUSIONS In this pilot trial, hydroxyurea treatment of severe SCD in children was associated with improved hematologic parameters, acceptable toxic effects, and a trend to reduced hospitalization. Hydroxyurea appears to be a safe and potentially effective agent for the treatment of severe SCD in children. A prospective, controlled trial to investigate the efficacy of hydroxyurea in children is therefore warranted.

Heinz bodies induce clustering of band 3, glycophorin, and ankyrin in sickle cell erythrocytes.

In earlier model studies we demonstrated that artificially denatured hemoglobin binds to and clusters the protein, band 3, in the plane of the erythrocyte membrane. To determine whether denatured hemoglobin also clusters band 3 in vivo, we have compared the locations of denatured hemoglobin aggregates (Heinz bodies) with band 3 in sickle cells using phase contrast and immunofluorescence microscopy. We report that where Heinz bodies are found associated with the cytoplasmic surface of the membrane, clusters of band 3 are usually colocalized within the membrane. In contrast, normal erythrocyte membranes and regions of sickle cell membranes devoid of Heinz bodies display an uninterrupted staining of band 3. Similarly, ankyrin and glycophorin are periodically seen to aggregate at Heinz body sites, but the degree of colocalization is lower than for band 3. These data demonstrate that the binding of denatured hemoglobin to the membrane forces a redistribution of several major membrane components.

Imaging of the diffusion of single band 3 molecules on normal and mutant erythrocytes

Membrane-spanning proteins may interact with a variety of other integral and peripheral membrane proteins via a diversity of protein-protein interactions. Not surprisingly, defects or mutations in any one of these interacting components can impact the physical and biological properties on the entire complex. Here we use quantum dots to image the diffusion of individual band 3 molecules in the plasma membranes of intact human erythrocytes from healthy volunteers and patients with defects in one of their membrane components, leading to well-known red cell pathologies (hereditary spherocytosis, hereditary elliptocytosis, hereditary hydrocytosis, Southeast Asian ovalocytosis, and hereditary pyropoikilocytosis). After characterizing the motile properties of the major subpopulations of band 3 in intact normal erythrocytes, we demonstrate that the properties of these subpopulations of band 3 change significantly in diseased cells, as evidenced by changes in the microscopic and macroscopic diffusion coefficients of band 3 and in the compartment sizes in which the different band 3 populations can diffuse. Because the above membrane abnormalities largely arise from defects in other membrane components (eg, spectrin, ankyrin), these data suggest that single particle tracking of band 3 might constitute a useful tool for characterizing the general structural integrity of the human erythrocyte membrane.

Refining the value of secretory phospholipase A2 as a predictor of acute chest syndrome in sickle cell disease: results of a feasibility study (PROACTIVE)

Acute chest syndrome (ACS) is defined as fever, respiratory symptoms and a new pulmonary infiltrate in an individual with sickle cell disease (SCD). Nearly half of ACS episodes occur in SCD patients already hospitalized, potentially permitting pre‐emptive therapy in high‐risk patients. Simple transfusion of red blood cells may abort ACS if given to patients hospitalized for pain who develop fever and elevated levels of secretory phospholipase A2 (sPLA2). In a feasibility study (PROACTIVE; ClinicalTrials.gov NCT00951808), patients hospitalized for pain who developed fever and elevated sPLA2 were eligible for randomization to transfusion or observation; all others were enrolled in an observational arm. Of 237 enrolled, only 10 were randomized; one of the four to receive transfusion had delayed treatment. Of 233 subjects receiving standard care, 22 developed ACS. A threshold level of sPLA2 ≥ 48 ng/ml gave optimal sensitivity (73%), specificity (71%) and accuracy (71%), but a positive predictive value of only 24%. The predictive value of sPLA2 was improved in adults and patients with chest or back pain, lower haemoglobin concentration and higher white blood cell counts, and in those receiving less than two‐thirds maintenance fluids. The hurdles identified in PROACTIVE should facilitate design of a larger, definitive, phase 3 randomized controlled trial.

Reproductive Decisions in People With Sickle Cell Disease or Sickle Cell Trait

In the context of an inherited condition such as sickle cell disease (SCD), it is critical to understand how people with SCD or carriers (sickle cell trait [SCT]) face the challenges of making informed reproductive health decisions. The purpose of this analysis was to examine the beliefs, attitudes, and personal feelings of people with sickle cell disease or sickle cell trait related to making informed reproductive health decisions. Three focus groups were conducted with a total of 15 people who had either SCD or SCT. Five themes were identified: health-related issues in sickle cell disease, testing for sickle cell trait, partner choice, sharing sickle cell status with partners, and reproductive options. These findings enhance understanding of the reproductive experiences in people with SCD and SCT and provide the groundwork for developing an educational intervention focused on making informed decisions about becoming a parent.

A Phase 1/2 Trial of HQK-1001, an Oral Fetal Globin Inducer, in Sickle Cell Disease

Therapeutics which reduce the pathology in sickle cell syndromes are needed, particularly noncytotoxic therapeutics. Fetal hemoglobin (HbF, α2γ2) is established as a major regulator of disease severity; increased HbF levels correlate with milder clinical courses and improved survival. Accordingly, sodium dimethylbutyrate (HQK‐1001), an orally‐bioavailable, promoter‐targeted fetal globin gene‐inducing agent, was evaluated in a randomized, blinded, dose‐ranging Phase I/II trial in 24 adult patients with HbSS or S/β thalassemia, to determine safety and tolerability of three escalating dose levels. The study therapeutic was administered once daily for two 6‐week cycles, with a two‐week interim dose holiday. Twenty‐one patients completed the study. Five patients received study drug at 10 or 20 mg/kg doses, seven patients received study drug at 30 mg/kg/dose, and 4 patients received placebo. HQK‐1001 was well‐tolerated with no unexpected drug‐related adverse events; a dose‐limiting toxicity was not identified. Plasma drug levels were sustained above targeted levels for 24 hr. Increases in HbF above baseline were observed particularly with 30 mg/kg/day doses; in five of seven treated patients, a mean absolute increase in HbF of 0.2 g/dl and a mean increase in total hemoglobin (Hgb) of 0.83 g/dl above baseline were observed, whereas no increases occurred in placebo‐treated controls. These findings of favorable PK profiles, tolerability, early rises in HbF, and total Hgb indicate that trials of longer duration appear warranted to more definitively evaluate the therapeutic potential of HQK‐1001 in sickle cell disease. Am. J. Hematol., 2012.

Reproducibility of tricuspid regurgitant jet velocity measurements in children and young adults with sickle cell disease undergoing screening for pulmonary hypertension

The reproducibility of tricuspid regurgitant jet velocity (TRJV) measurements by Doppler echocardiography has not been subjected to systematic evaluation among individuals with sickle cell disease (SCD) undergoing screening for pulmonary hypertension. We examined sources of disagreement associated with peak TRJV in children and young adults with SCD. Peak TRJV was independently measured and interpreted a week apart by separate sonographers and readers, respectively, in 30 subjects (mean age, 15.8 ± 3.3 years) who provided 120 observations. We assessed intra‐/inter‐reader, intra‐/inter‐sonographer, sonographer‐reader, and within subject agreement using Intraclass Correlation Coefficient (ICC) and Cohens kappa (κ). Agreement was examined graphically using Bland‐Altman plots. Although sonographers could estimate and measure peak TRJV in all subjects, readers designated tricuspid regurgitation nonquantifiable in 10–17% of their final interpretations. Intra‐reader agreement was highest (ICC = 0.93 [95% CI 0.86, 0.97], P = 0.0001) and within subject agreement lowest (ICC = 0.36 [95% CI 0.02, 0.64], P = 0.021) for single TRJV measurements. Similarly, intra‐reader agreement was highest (κ = 0.74 [95% CI 0.53, 0.95], P = 0.0001) and within subject lowest (κ = 0.14 [95% CI −0.17, 0.46], P = 0.38) when sonographers and readers categorized TRJV measurements. On Bland‐Altman plots, absolute differences in observations increased with higher mean TRJV readings for intra‐/inter‐reader agreement. Peak TRJV measurements in individual children and young adults with SCD are affected by several sources of disagreement, underscoring the need for methodological improvements that ensure reproducibility of this screening modality for making clinical decisions in this population. Am. J. Hematol., 2010.

Barriers to hematopoietic cell transplantation clinical trial participation of African American and black youth with sickle cell disease and their parents

African Americans and Blacks have low participation rates in clinical trials and reduced access to aggressive medical therapies. Hematopoietic cell transplantation (HCT) is a high-risk but potentially curative therapy for sickle cell disease (SCD), a disorder predominantly seen in African Americans. We conducted focus groups to better understand participation barriers to HCT clinical trials for SCD. Nine focus groups of youth with SCD (n=10) and parents (n=41) were conducted at 3 sites representing the Midwest, South Atlantic, and West South Central US. Main barriers to clinical trial participation included gaps in knowledge about SCD, limited access to SCD/HCT trial information, and mistrust of medical professionals. For education about SCD/HCT trials, participants highly preferred one-on-one interactions with medical professionals and electronic media as a supplement. Providers can engage with sickle cell camps to provide information on SCD/HCT clinical trials to youth and local health fairs for parents/families. Youth reported learning about SCD through computer games; investigators may find this medium useful for clinical trial/HCT education. African Americans affected by SCD face unique barriers to clinical trial participation and have unmet HCT clinical studies education needs. Greater recognition of these barriers will allow targeted interventions in this community to increase their access to HCT.

Sickle Cell Disease: An Opportunity for Palliative Care Across the Life Span

Sickle cell disease is a chronic illness that affects patients physically and emotionally and can do so at an early age. An ecological model of palliative care that involves improved communication among the health care team, patients, and their families can be beneficial. Open and honest communication regarding advance care planning, disease management, relief of pain and other symptoms, and bereavement and grief are all important for the patient, family, and health care team. Given the multiple acute and chronic complications of sickle cell disease, an approach to care that is holistic and comprehensive may help to improve a patients biologic function and the perceived health, functional status, and quality of life of the patient and family.

The temperature dependence of human erythrocyte transport of phosphate, phosphite and hypophosphite

The temperature dependence of the erythrocyte anion transport protein (Band 3 or AE1) mediated influx of three nonspherical substrates, the divalent anions phosphate and phosphite, and the monovalent hypophoshite, were determined. Phase transitions were found in the temperature dependence of the influxes of all three anions. The 95% confidence limits for the transition temperatures were: 34.6-38.1 degrees C, 7.4-9.1 degrees C and 6.7-9.7 degrees C for phosphate, phosphite and hypophosphite, respectively, while the critical influx rates at the transitions were 29-50, 64-102 and 26-58 ions/s per carrier, respectively. That the critical rates rather than the transition temperatures are of similar magnitude indicates that the transitions are related to transport mechanisms rather than to thermal protein conformational changes. These critical rates are two orders of magnitude lower than those reported for the self-exchange of Cl- and Br- (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). The critical rate of monovalent hypophosphite is similar to that of divalent phosphate and phosphite, but not to that of Cl- indicating that this effect is mediated by the structure of the substrate rather than by its charge. The disparity in the rates rc at which phase transitions occur in AE1-mediated transport of spherical and nonspherical anions indicates a difference in the interaction between the two classes of anions and the protein.