Although effective agents are available to prevent painful vaso-occlusive episodes of sickle cell disease (SCD), there are no disease-modifying therapies for ongoing painful vaso-occlusive episodes; ...treatment remains supportive. A previous phase 3 trial of poloxamer 188 reported shortened duration of painful vaso-occlusive episodes in SCD, particularly in children and participants treated with hydroxyurea.
To reassess the efficacy of poloxamer 188 for vaso-occlusive episodes.
Phase 3, randomized, double-blind, placebo-controlled, multicenter, international trial conducted from May 2013 to February 2016 that included 66 hospitals in 12 countries and 60 cities; 388 individuals with SCD (hemoglobin SS, SC, S-β0 thalassemia, or S-β+ thalassemia disease) aged 4 to 65 years with acute moderate to severe pain typical of painful vaso-occlusive episodes requiring hospitalization were included.
A 1-hour 100-mg/kg loading dose of poloxamer 188 intravenously followed by a 12-hour to 48-hour 30-mg/kg/h continuous infusion (n = 194) or placebo (n = 194).
Time in hours from randomization to the last dose of parenteral opioids among all participants and among those younger than 16 years as a separate subgroup.
Of 437 participants assessed for eligibility, 388 were randomized (mean age, 15.2 years; 176 45.4% female), the primary outcome was available for 384 (99.0%), 15-day follow-up contacts were available for 357 (92.0%), and 30-day follow-up contacts were available for 368 (94.8%). There was no significant difference between the groups for the mean time to last dose of parenteral opioids (81.8 h for the poloxamer 188 group vs 77.8 h for the placebo group; difference, 4.0 h 95% CI, -7.8 to 15.7; geometric mean ratio, 1.2 95% CI, 1.0-1.5; P = .09). Based on a significant interaction of age and treatment (P = .01), there was a treatment difference in time from randomization to last administration of parenteral opioids for participants younger than 16 years (88.7 h in the poloxamer 188 group vs 71.9 h in the placebo group; difference, 16.8 h 95% CI, 1.7-32.0; geometric mean ratio, 1.4 95% CI, 1.1-1.8; P = .008). Adverse events that were more common in the poloxamer 188 group than the placebo group included hyperbilirubinemia (12.7% vs 5.2%); those more common in the placebo group included hypoxia (12.0% vs 5.3%).
Among children and adults with SCD, poloxamer 188 did not significantly shorten time to last dose of parenteral opioids during vaso-occlusive episodes. These findings do not support the use of poloxamer 188 for vaso-occlusive episodes.
ClinicalTrials.gov Identifier: NCT01737814.
Decades of research have established that the most effective treatment for sickle cell disease (SCD) is increased fetal hemoglobin (HbF). Identification of a drug specific for inducing γ-globin ...expression in pediatric and adult patients, with minimal off-target effects, continues to be an elusive goal. One hurdle has been an assay amenable to a high-throughput screen (HTS) of chemicals that displays a robust γ-globin off-on switch to identify potential lead compounds. Assay systems developed in our labs to understand the mechanisms underlying the γ- to β-globin gene expression switch during development has allowed us to generate a cell-based assay that was adapted for a HTS of 121,035 compounds. Using chemical inducer of dimerization (CID)-dependent bone marrow cells (BMCs) derived from human γ-globin promoter-firefly luciferase β-globin promoter-Renilla luciferase β-globin yeast artificial chromosome (γ-luc β-luc β-YAC) transgenic mice, we were able to identify 232 lead chemical compounds that induced γ-globin 2-fold or higher, with minimal or no β-globin induction, minimal cytotoxicity and that did not directly influence the luciferase enzyme. Secondary assays in CID-dependent wild-type β-YAC BMCs and human primary erythroid progenitor cells confirmed the induction profiles of seven of the 232 hits that were cherry-picked for further analysis.
In response to sodium butyrate and trichostatin A treatment in erythroid cells, p38 mitogen activated protein kinase (MAPK) mediates fetal hemoglobin (HbF) induction by activating cAMP response ...element binding protein 1 (CREB1). To expand on this observation, we completed studies to determine the role of p38 MAPK in steady-state γ-globin regulation. We propose that p38 signaling regulates Gγ-globin transcription during erythroid maturation through its downstream effector CREB1 which binds the Gγ-globin cAMP response element (G-CRE). We demonstrated that a loss of p38 or CREB1 function by siRNA knockdown resulted in target gene silencing. Moreover, gain of p38 or CREB1 function augments γ-globin transcription. These regulatory effects were conserved under physiological conditions tested in primary erythroid cells. When the G-CRE was mutated in a stable chromatin environment Gγ-globin promoter activity was nearly abolished. Furthermore, introduction of mutations in the G-CRE abolished Gγ-globin activation via p38 MAPK/CREB1 signaling. Chromatin immunoprecipitation assays (ChIP) demonstrated that CREB1 and its binding partner CREB binding protein (CBP) co-localize at the G-CRE region. These data support the role of p38 MAPK/CREB1 signaling in Gγ-globin gene transcription under steady-state conditions.
Increased expression of developmentally silenced fetal globin (HBG) reduces the clinical severity of β-hemoglobinopathies. Benserazide has a relatively benign safety profile having been approved for ...50 years in Europe and Canada for Parkinson's disease treatment. Benserazide was shown to activate HBG gene transcription in a high throughput screen, and subsequent studies confirmed fetal hemoglobin (HbF) induction in erythroid progenitors from hemoglobinopathy patients, transgenic mice containing the entire human β-globin gene (β-YAC) and anemic baboons. The goal of this study is to evaluate efficacies and plasma exposure profiles of benserazide racemate and its enantiomers to select the chemical form for clinical development. Intermittent treatment with all forms of benserazide in β-YAC mice significantly increased proportions of red blood cells expressing HbF and HbF protein per cell with similar pharmacokinetic profiles and with no cytopenia. These data contribute to the regulatory justification for development of the benserazide racemate. Additionally, dose ranges and frequencies required for HbF induction using racemic benserazide were explored. Orally administered escalating doses of benserazide in an anemic baboon induced γ-globin mRNA up to 13-fold and establish an intermittent dose regimen for clinical studies as a therapeutic candidate for potential treatment of β-hemoglobinopathies.
•Fetal hemoglobin induction for individuals living with β-hemoglobinopathies are most feasible via pharmacologic approaches.•Racemate (R,S)-Benserazide and its enantiomers induce fetal globin expression in vivo in β-YAC transgenic mice.•A clinical trial of (R,S)-Benserazide in patients with hemoglobin disorders was initiated based on the findings.
Therapeutic strategies aimed at reactivating
gene transcription and fetal hemoglobin (HbF) synthesis remain the most effective strategy to ameliorate the clinical symptoms of sickle cell disease ...(SCD). We previously identified microRNA29B (MIR29B) as a novel HbF inducer
targeting enzymes involved in DNA methylation. We provided further evidence that the introduction of MIR29B into KU812 leukemia cells significantly reduced MYB protein expression. Therefore, the aim of this study was to determine the extent to which MIR29B mediates HbF induction
targeting
in KU812 leukemia cells and human primary erythroid progenitors and to investigate the role of MIR29B in HbF induction
in the humanized Townes SCD mouse model.
Human KU812 were cultured and normal CD34 cells (
= 3) were differentiated using a two-phase erythropoiesis culturing system and transfected with MIR29B (50 and 100 nM) mimic or Scrambled (Scr) control
. A luciferase reporter plasmid overexpressing MYB was transfected into KU812 cells. Luciferase activity was quantified after 48 h. Gene expression was determined by quantitative real-time PCR.
studies were conducted using Townes SCD mice (6 per group) treated with MIR29B (2, 3, and 4 mg/kg/day) or Scr control by 28-day continuous infusion using subcutaneous mini osmotic pumps. Blood samples were collected and processed for complete blood count (CBC) with differential and reticulocytes at weeks 0, 2, and 4. Flow cytometry was used to measure the percentage of HbF-positive cells.
analysis predicted complementary base-pairing between MIR29B and the 3'-untranslated region (UTR) of
. Overexpression of MIR29B significantly reduced
mRNA and protein expression in KU812 cells and erythroid progenitors. Using a luciferase reporter vector that contained the full-length
3'-UTR, we observed a significant reduction in luciferase activity among KU812 cells that co-expressed MIR29B and the full-length
3'-UTR as compared to cells that only expressed
3'-UTR. We confirmed the inhibitory effect of a plasmid engineered to overexpress
on
activation and HbF induction in both KU812 cells and human primary erythroid progenitors. Co-expression of MIR29B and
in both cell types further demonstrated the inhibitory effect of MIR29B on
expression, resulting in
reactivation by real-time PCR, Western blot, and flow cytometry analysis. Finally, we confirmed the ability of MIR29B to reduce sickling and induce HbF by decreasing expression of
and
gene expression in the humanized Townes sickle cell mouse model.
Our findings support the ability of MIR29B to induce HbF
in Townes sickle cell mice. This is the first study to provide evidence of the ability of MIR29B to modulate
transcription by
gene silencing
. Our research highlights a novel MIR-based epigenetic approach to induce HbF supporting the discovery of new drugs to expand treatment options for SCD.
The fetal and adult globin genes in the human β-globin cluster on chromosome 11 are sequentially expressed to achieve normal hemoglobin switching during human development. The pharmacological ...induction of fetal γ-globin (HBG) to replace abnormal adult sickle βS-globin is a successful strategy to treat sickle cell disease; however the molecular mechanism of γ-gene silencing after birth is not fully understood. Therefore, we performed global gene expression profiling using primary erythroid progenitors grown from human peripheral blood mononuclear cells to characterize gene expression patterns during the γ-globin to β-globin (γ/β) switch observed throughout in vitro erythroid differentiation.
We confirmed erythroid maturation in our culture system using cell morphologic features defined by Giemsa staining and the γ/β-globin switch by reverse transcription-quantitative PCR (RT-qPCR) analysis. We observed maximal γ-globin expression at day 7 with a switch to a predominance of β-globin expression by day 28 and the γ/β-globin switch occurred around day 21. Expression patterns for transcription factors including GATA1, GATA2, KLF1 and NFE2 confirmed our system produced the expected pattern of expression based on the known function of these factors in globin gene regulation. Subsequent gene expression profiling was performed with RNA isolated from progenitors harvested at day 7, 14, 21, and 28 in culture. Three major gene profiles were generated by Principal Component Analysis (PCA). For profile-1 genes, where expression decreased from day 7 to day 28, we identified 2,102 genes down-regulated > 1.5-fold. Ingenuity pathway analysis (IPA) for profile-1 genes demonstrated involvement of the Cdc42, phospholipase C, NF-Kβ, Interleukin-4, and p38 mitogen activated protein kinase (MAPK) signaling pathways. Transcription factors known to be involved in γ-and β-globin regulation were identified.The same approach was used to generate profile-2 genes where expression was up-regulated over 28 days in culture. IPA for the 2,437 genes with > 1.5-fold induction identified the mitotic roles of polo-like kinase, aryl hydrocarbon receptor, cell cycle control, and ATM (Ataxia Telangiectasia Mutated Protein) signaling pathways; transcription factors identified included KLF1, GATA1 and NFE2 among others. Finally, profile-3 was generated from 1,579 genes with maximal expression at day 21, around the time of the γ/β-globin switch. IPA identified associations with cell cycle control, ATM, and aryl hydrocarbon receptor signaling pathways.
The transcriptome analysis completed with erythroid progenitors grown in vitro identified groups of genes with distinct expression profiles, which function in metabolic pathways associated with cell survival, hematopoiesis, blood cells activation, and inflammatory responses. This study represents the first report of a transcriptome analysis in human primary erythroid progenitors to identify transcription factors involved in hemoglobin switching. Our results also demonstrate that the in vitro liquid culture system is an excellent model to define mechanisms of global gene expression and the DNA-binding protein and signaling pathways involved in globin gene regulation.
Sickle cell disease (SCD) is a group of inherited blood disorders that have in common a mutation in the sixth codon of the β-globin (HBB) gene on chromosome 11. However, people with the same genetic ...mutation display a wide range of clinical phenotypes. Fetal hemoglobin (HbF) expression is an important genetic modifier of SCD complications leading to milder symptoms and improved long-term survival. Therefore, we performed a genome-wide association study (GWAS) using a case-control experimental design in 244 African Americans with SCD to discover genetic factors associated with HbF expression. The case group consisted of subjects with HbF≥8.6% (133 samples) and control group subjects with HbF≤£3.1% (111 samples). Our GWAS results replicated SNPs previously identified in an erythroid-specific enhancer region located in the second intron of the BCL11A gene associated with HbF expression. In addition, we identified SNPs in the SPARC, GJC1, EFTUD2 and JAZF1 genes as novel candidates associated with HbF levels. To gain insights into mechanisms of globin gene regulation in the HBB locus, linkage disequilibrium (LD) and haplotype analyses were conducted. We observed strong LD in the low HbF group in contrast to a loss of LD and greater number of haplotypes in the high HbF group. A search of known HBB locus regulatory elements identified SNPs 5′ of δ-globin located in an HbF silencing region. In particular, SNP rs4910736 created a binding site for a known transcription repressor GFi1 which is a candidate protein for further investigation. Another HbF-associated SNP, rs2855122 in the cAMP response element upstream of Gγ-globin, was analyzed for functional relevance. Studies performed with siRNA-mediated CREB binding protein (CBP) knockdown in primary erythroid cells demonstrated γ-globin activation and HbF induction, supporting a repressor role for CBP. This study identifies possible molecular determinants of HbF production.
Individuals living with sickle cell disease experience high levels of morbidity that result in frequent utilization of the emergency department. The objective of this study was to provide updated ...national estimates of emergency department utilization associated with sickle cell disease in the U.S.
Data from the National Hospital Ambulatory Medical Care Survey for the years 1999-2020 were analyzed. Complex survey analysis was utilized to produce national estimates overall and by patient age groups.
On average, approximately 222,612 emergency department visits occurred annually among individuals with sickle cell disease, a nearly 13% increase from prior estimates. The annual volume of emergency department visits steadily increased over time, and pain remains the most common patient-cited reason for visiting the emergency department. Patient-reported pain levels for individuals with sickle cell disease were high, with 64% of visits associated with severe pain and 21% associated with moderate pain. Public insurance sources continue to cover most visits, with Medicaid paying for 60% of visits and Medicare paying for 12% of visits. The average time spent in the emergency department increased from previous estimates by about an hour, rising to approximately 6 hours. The average wait time to see a provider was 53 minutes.
Utilization of the emergency department by individuals living with sickle cell disease remains high, especially for pain. With more than half of patients with sickle cell disease reporting severe pain levels, emergency department staff should be prepared to assess and treat sickle cell disease-related pain following evidence-based guidelines and recommendations. The findings of this study can help improve care in this population.
Although mentoring is critical for career advancement, underrepresented minority (URM) faculty often lack access to mentoring opportunities. We sought to evaluate the impact of peer mentoring on ...career development success of URM early career faculty in the National Heart Lung and Blood Institute-sponsored, Programs to Increase Diversity Among Individuals Engaged in Health-Related Research-Functional and Translational Genomics of Blood Disorders (PRIDE-FTG). The outcome of peer mentoring was evaluated using the Mentoring Competency Assessment (MCA), a brief open-ended qualitative survey, and a semi-structured exit interview. Surveys were completed at baseline (Time 1), 6 months, and at the end of PRIDE-FTG participation (Time 2). The following results were obtained. Between Time 1 and Time 2, mentees' self-assessment scores increased for the MCA (p < 0.01) with significant increases in effective communication (p < 0.001), aligning expectations (p < 0.05), assessing understanding (p < 0.01), and addressing diversity (p < 0.002). Mentees rated their peer mentors higher in the MCA with significant differences noted for promoting development (p < 0.027). These data suggest that PRIDE-FTG peer mentoring approaches successfully improved MCA competencies among URM junior faculty participants with faculty ranking peer mentors higher than themselves. Among URM faculty, peer mentoring initiatives should be investigated as a key strategy to support early career scholar development.
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