Pain Frequency and Health Care Utilization Patterns in Women with Sickle Cell Disease Experiencing Menstruation-Associated Pain Crises.

Background: Pain crises in sickle cell disease (SCD) lead to high rates of health care utilization. Historically, women have reported higher pain burdens than men, with recent studies showing a temporal association between pain crisis and menstruation. However, health care utilization patterns of SCD women with menstruation-associated pain crises have not been reported. We studied the frequency, severity, and health care utilization of menstruation-associated pain crises in SCD women. Materials and Methods: A multinational, cross-sectional cohort study of the SCD phenotype was executed using a validated questionnaire and medical chart review from the Consortium for the Advancement of Sickle Cell Research (CASiRe) cohort. Total number of pain crises, emergency room/day hospital visits, and hospitalizations were collected from a subcohort of 178 SCD women within the past 6 months and previous year. Results: Thirty-nine percent of women reported menstruation-associated pain crises in their lifetime. These women were significantly more likely to be hospitalized compared with those who did not (mean 1.70 vs. 0.67, p = 0.0005). Women reporting menstruation-associated pain crises in the past 6 months also experienced increased hospitalizations compared with those who did not (mean 1.71 vs. 0.75, p = 0.0016). Forty percent of women reported at least four menstruation-associated pain crises in the past 6 months. Conclusions: Nearly 40% of SCD women have menstruation-associated pain crises. Menstruation-associated pain crises are associated with high pain burden and increased rates of hospitalization. Strategies are needed to address health care disparities within gynecologic care in SCD.

Combinatorial targeting of epigenome-modifying enzymes with decitabine and RN-1 synergistically increases HbF.

Increased fetal hemoglobin (HbF) levels reduce the symptoms of sickle cell disease (SCD) and increase the lifespan of patients. Because curative strategies for bone marrow transplantation and gene therapy technologies remain unavailable to a large number of patients, the development of a safe and effective pharmacological therapy that increases HbF offers the greatest potential for disease intervention. Although hydroxyurea increases HbF, a substantial proportion of patients fail to demonstrate an adequate response. Pharmacological inhibitors of DNA methyltransferase (DNMT1) and lysine-specific demethylase 1A (LSD1), 2 epigenome-modifying enzymes associated with the multiprotein corepressor complex recruited to the repressed γ-globin gene, are powerful in vivo inducers of HbF. The hematological side effects of these inhibitors limit feasible clinical exposures. We evaluated whether administering these drugs in combination could reduce the dose and/or time of exposure to any single agent to minimize adverse effects, while achieving additive or synergistic increases in HbF. The DNMT1 inhibitor decitabine (0.5 mg/kg per day) and the LSD1 inhibitor RN-1 (0.25 mg/kg per day) administered in combination 2 days per week produced synergistic increases in F-cells, F-reticulocytes, and γ-globin messenger RNA in healthy baboons. Large increases in HbF and F-cells were observed in healthy, nonanemic, and anemic (phlebotomized) baboons. Combinatorial therapy targeting epigenome-modifying enzymes could thus be a useful strategy for producing larger increases in HbF to modify the clinical course of SCD.

Reticulocyte mitochondrial retention increases reactive oxygen species and oxygen consumption in mouse models of sickle cell disease and phlebotomy-induced anemia.

Sickle cell disease (SCD) is caused by a mutation of the ß-globin gene that results in the production of hemoglobin S (HbS). People with SCD experience anemia, severe acute pain episodes, persistent chronic pain, multiorgan damage, and a reduced life span. The pathophysiology of SCD caused by the polymerization of HbS on deoxygenation results in red cell deformability and the generation of reactive oxygen species (ROS). These 2 factors lead to red cell fragility and hemolysis. Reticulocytosis is an independent predictor of disease morbidity and mortality in SCD. We previously established that humans and mice with SCD exhibit abnormal mitochondrial retention in erythrocytes increasing ROS-associated hemolysis. Here, we investigated the hypothesis that mitochondrial retention and increased ROS are a consequence of stress erythropoiesis. Our results show clearly that stress erythropoiesis in phlebotomized, anemic AA mice results in mitochondrial retention and increased ROS in reticulocytes. We observed that elevated mitochondrial retention in reticulocytes also alters oxygen consumption and potentially contributes to increased HbS polymerization and red blood cell hemolysis. Therefore, these events occurring due to stress erythropoiesis contribute significantly to the pathology of SCD and suggest new therapeutic targets.

Pain Burden in the CASiRe International Cohort of Sickle Cell Patients: United States and Ghana.

OBJECTIVES: Sickle Cell Disease (SCD) is a genetic blood disorder affecting over 1 million people globally. The aim of this analysis is to explore the pain burden of patients with SCD in two countries: the United States and Ghana. METHODS: The Consortium for the Advancement of Sickle Cell Research (CASiRe) was created to better understand the clinical severity of patients with SCD worldwide. Data regarding gender, SCD genotype, prior medical diagnoses, and validated pain burden measures were analyzed from the CASiRe database. The Sickle Cell Pain Burden Interview (SCPBI) was used to assess pain burden, the impact of pain on physical, emotional, and social function. RESULTS: Most subjects identified as Black/African American (n = 298, 97.0%). Patient ages ranged from 6 to 73 years. 35.9% resided in the United States, 64.1% resided in Ghana, 40.9% were men, and 58.7% were women. The mean SCPBI score for US SCD patients was 6.53(±5.89) vs 4.04(±5.10) for Ghanaian patients, P <0.001. Pain burden was higher in US men vs Ghanaian men (6.74(±5.68) vs 3.54(±4.46), P = .003) and in US women vs Ghanaian women (6.37 ± 6.06 vs 4.44(±5.54), P = .032). Pain burden was higher in US patients than Ghanaian patients for both the Hb SC/SBeta+ genotype (5.40(±5.29) vs 2.82(±4.86), P = .054) and Hb SS/SBeta0 genotype (6.79(±6.01) vs 4.49(±5.13), P = .003). Pain burden was significantly higher in SCD patients with comorbid conditions independent of geographic origin including stroke, cholecystectomy, gallstones, depression, and headache. DISCUSSION: US patients with SCD have a higher pain burden than Ghanaian patients. Further studies should investigate underlying contributors to pain burden in these populations and further explore the etiology of geographic differences in pain.

Global geographic differences in healthcare utilization for sickle cell disease pain crises in the CASiRe cohort.

BACKGROUND: Sickle cell disease (SCD) is characterized by frequent, unpredictable pain episodes and other vaso-occlusive crises (VOCs) leading to significant healthcare utilization. VOC frequency is often an endpoint in clinical trials investigating novel therapies for this devastating disease. PROCEDURE: The Consortium for the Advancement of Sickle Cell Research (CASiRe) is an international collaboration investigating clinical severity in SCD using a validated questionnaire and medical chart review standardized across four countries (United States, United Kingdom, Italy and Ghana). RESULTS: This study, focused on pain crisis incidence and healthcare utilization, included 868 patients, equally represented according to age and gender. HgbSS was the most common genotype. Patients from Ghana used the Emergency Room/Day Hospital for pain more frequently (annualized mean 2.01) than patients from other regions (annualized mean 1.56 U.S.; 1.09 U.K.; 0.02 Italy), while U.K. patients were hospitalized for pain more often (annualized mean: U.K. 2.98) than patients in other regions (annualized mean 1.98 U.S.; 1.18 Ghana; Italy 0.54). Italy's hospitalization rate for pain (annualized mean: 0.57) was nearly 20 times greater than its emergency room/day hospital only visits for pain (annualized mean: 0.03). When categorized by genotype and age, similar results were seen. CONCLUSIONS: Geographic differences in pain crisis frequency and healthcare utilization may correlate with variable organization of healthcare systems among countries and should be considered regarding trial design, endpoints, and analysis of results when investigating novel agents for clinical benefit.

An Analysis of Racial and Ethnic Backgrounds Within the CASiRe International Cohort of Sickle Cell Disease Patients: Implications for Disease Phenotype and Clinical Research.

Millions are affected by sickle cell disease (SCD) worldwide with the greatest burden in sub-Saharan Africa. While its origin lies historically within the malaria belt, ongoing changes in migration patterns have shifted the burden of disease resulting in a global public health concern. We created the Consortium for the Advancement of Sickle Cell Research (CASiRe) to understand the different phenotypes of SCD across 4 countries (USA, UK, Italy, and Ghana). Here, we report the multi-generational ethnic and racial background of 877 SCD patients recruited in Ghana (n = 365, 41.6%), the USA (n = 254, 29%), Italy (n = 81, 9.2%), and the UK (n = 177, 20.2%). West Africa (including Benin Gulf) (N = 556, 63.4%) was the most common geographic region of origin, followed by North America (N = 184, 21%), Caribbean (N = 51, 5.8%), Europe (N = 27, 3.1%), Central Africa (N = 24, 2.7%), and West Africa (excluding Benin Gulf) (N = 21, 2.4%). SCD patients in Europe were primarily West African (73%), European (10%), Caribbean (8%), and Central African (8%). In the USA, patients were largely African American (71%), Caribbean (13%), or West African (10%). Most subjects identified themselves as Black or African American; the European cohort had the largest group of Caucasian SCD patients (8%), including 21% of the Italian patients. This is the first report of a comprehensive analysis of ethnicity within an international, transcontinental group of SCD patients. The diverse ethnic backgrounds observed in our cohort raises the possibility that genetic and environmental heterogeneity within each SCD population subgroup can affect the clinical phenotype and research outcomes.

A study of the geographic distribution and associated risk factors of leg ulcers within an international cohort of sickle cell disease patients: the CASiRe group analysis.

Vasculopathy is a hallmark of sickle cell disease ultimately resulting in chronic end organ damage. Leg ulcer is one of its sequelae, occurring in ~ 5-10% of adult sickle cell patients. The majority of leg ulcer publications to date have emanated from single center cohort studies. As such, there are limited studies on the geographic distribution of leg ulcers and associated risk factors worldwide. The Consortium for the Advancement of Sickle Cell Research (CASiRe) was formed to improve the understanding of the different phenotypes of sickle cell disease patients living in different geographic locations around the world (USA, UK, Italy, Ghana). This cross-sectional cohort sub-study of 659 sickle cell patients aimed to determine the geographic distribution and risk factors associated with leg ulcers. The prevalence of leg ulcers was 10.3% and was associated with older age, SS genotype, male gender, and Ghanaian origin. In fact, the highest prevalence (18.6%) was observed in Ghana. Albuminuria, proteinuria, increased markers of hemolysis (lower hemoglobin, higher total bilirubin), lower oxygen saturation, and lower body mass index were also associated with leg ulceration. Overall, our study identified a predominance of leg ulcers within male hemoglobin SS patients living in sub-Saharan Africa with renal dysfunction and increased hemolysis.

Oral administration of the LSD1 inhibitor ORY-3001 increases fetal hemoglobin in sickle cell mice and baboons.

Increased levels of fetal hemoglobin (HbF) lessen the severity of symptoms and increase the life span of patients with sickle cell disease (SCD). More effective strategies to increase HbF are needed because the current standard of care, hydroxyurea, is not effective in a significant proportion of patients. Treatment of the millions of patients projected worldwide would best be accomplished with an orally administered drug therapy that increased HbF. LSD1 is a component of corepressor complexes that repress γ-globin gene expression and are a therapeutic target for HbF reactivation. We have shown that subcutaneous administration of RN-1, a pharmacological LSD1 inhibitor, increased γ-globin expression in SCD mice and baboons, which are widely acknowledged as the best animal model in which to test the activity of HbF-inducing drugs. The objective of this investigation was to test the effect of oral administration of a new LSD1 inhibitor, ORY-3001. Oral administration of ORY-3001 to SCD mice (n = 3 groups) increased γ-globin expression, Fetal Hemoglobin (HbF)-containing (F) cells, and F reticulocytes (retics). In normal baboons (n = 7 experiments) treated with ORY-3001, increased F retics, γ-globin chain synthesis, and γ-globin mRNA were observed. Experiments in anemic baboons (n = 2) showed that ORY-3001 increased F retics (PA8695, predose = 24%, postdose = 66.8%; PA8698: predose = 13%, postdose = 93.6%), γ-globin chain synthesis (PA8695: predose = 0.07 γ/γ+ß, postdose = 0.20 γ/γ+ß; PA8698: predose = 0.02 γ/γ+ß, postdose = 0.44 γ/γ+ß), and γ-globin mRNA (PA8695: predose = 0.06 γ/γ+ß, postdose = 0.18 γ/γ+ß; PA8698: predose = 0.03 γ/γ+ß, postdose = 0.33 γ/γ+ß). We conclude that oral administration of ORY-3001 increases F retics, γ-globin chain synthesis, and γ-globin mRNA in baboons and SCD mice, supporting further efforts toward the development of this drug for SCD therapy.

Potential role of LSD1 inhibitors in the treatment of sickle cell disease: a review of preclinical animal model data.

Sickle cell disease (SCD) is caused by a mutation of the ß-globin gene (Ingram VM. Nature 180: 326-328, 1957), which triggers the polymerization of deoxygenated sickle hemoglobin (HbS). Approximately 100,000 SCD patients in the United States and millions worldwide (Piel FB, et al. PLoS Med 10: e1001484, 2013) suffer from chronic hemolytic anemia, painful crises, multisystem organ damage, and reduced life expectancy (Rees DC, et al. Lancet 376: 2018-2031, 2010; Serjeant GR. Cold Spring Harb Perspect Med 3: a011783, 2013). Hematopoietic stem cell transplantation can be curative, but the majority of patients do not have a suitable donor (Talano JA, Cairo MS. Eur J Haematol 94: 391-399, 2015). Advanced gene-editing technologies also offer the possibility of a cure (Goodman MA, Malik P. Ther Adv Hematol 7: 302-315, 2016; Lettre G, Bauer DE. Lancet 387: 2554-2564, 2016), but the likelihood that these strategies can be mobilized to treat the large numbers of patients residing in developing countries is remote. A pharmacological treatment to increase fetal hemoglobin (HbF) as a therapy for SCD has been a long-sought goal, because increased levels of HbF (α2γ2) inhibit the polymerization of HbS (Poillin WN, et al. Proc Natl Acad Sci USA 90: 5039-5043, 1993; Sunshine HR, et al. J Mol Biol 133: 435-467, 1979) and are associated with reduced symptoms and increased lifespan of SCD patients (Platt OS, et al. N Engl J Med 330: 1639-1644, 1994; Platt OS, et al. N Engl J Med 325: 11-16, 1991). Only two drugs, hydroxyurea and l-glutamine, are approved by the US Food and Drug Administration for treatment of SCD. Hydroxyurea is ineffective at HbF induction in ~50% of patients (Charache S, et al. N Engl J Med 332: 1317-1322, 1995). While polymerization of HbS has been traditionally considered the driving force in the hemolysis of SCD, the excessive reactive oxygen species generated from red blood cells, with further amplification by intravascular hemolysis, also are a major contributor to SCD pathology. This review highlights a new class of drugs, lysine-specific demethylase (LSD1) inhibitors, that induce HbF and reduce reactive oxygen species.

Evolving treatment paradigms in sickle cell disease.

Sickle cell disease (SCD) is an inheritable hemoglobinopathy characterized by polymerization of hemoglobin S in red blood cells resulting in chronic hemolytic anemia, vaso-occlusive painful crisis, and multiorgan damage. In SCD, an increased reactive oxygen species (ROS) generation occurs both inside the red blood cells and inside the vascular lumen, which augment hemolysis and cellular adhesion. This review discusses the evolving body of literature on the role of ROS in the pathophysiology of SCD as well as some emerging therapeutic approaches to SCD with a focus on the reduction of ROS.

Pharmacological inhibition of LSD1 and mTOR reduces mitochondrial retention and associated ROS levels in the red blood cells of sickle cell disease.

Sickle cell disease (SCD), an inherited blood disorder caused by a point mutation that renders hemoglobin susceptible to polymerization when deoxygenated, affects millions of people worldwide. Manifestations of SCD include chronic hemolytic anemia, inflammation, painful vaso-occlusive crises, multisystem organ damage, and reduced life expectancy. Part of SCD pathophysiology is the excessive formation of intracellular reactive oxygen species (ROS) in SCD red blood cells (RBCs), which accelerates their hemolysis. Normal RBC precursors eliminate their mitochondria during the terminal differentiation process. Strikingly, we observed an increased percentage of RBCs retaining mitochondria in SCD patient blood samples compared with healthy individuals. In addition, using an experimental SCD mouse model, we demonstrate that excessive levels of ROS in SCD are associated with this abnormal mitochondrial retention. Interestingly, the LSD1 inhibitor, RN-1, and the mitophagy-inducing agent mammalian target of rapamycin (mTOR) inhibitor, sirolimus, increased RBC lifespan and reduced ROS accumulation in parallel with reducing mitochondria-retaining RBCs in the SCD mouse model. Furthermore, gene expression analysis of SCD mice treated with RN-1 showed increased expression of mitophagy genes. Our findings suggest that reduction of mitochondria-retaining RBCs may provide a new therapeutic approach to preventing excessive ROS in SCD.

The LSD1 inhibitor RN-1 recapitulates the fetal pattern of hemoglobin synthesis in baboons (P. anubis).

Increased fetal hemoglobin levels lessen the severity of symptoms and increase the lifespan of patients with sickle cell disease. Hydroxyurea, the only drug currently approved for the treatment of sickle cell disease, is not effective in a large proportion of patients and therefore new pharmacological agents that increase fetal hemoglobin levels have long been sought. Recent studies identifying LSD-1 as a repressor of γ-globin expression led to experiments demonstrating that the LSD-1 inhibitor RN-1 increased γ-globin expression in the sickle cell mouse model. Because the arrangement and developmental stage-specific expression pattern of the ß-like globin genes is highly conserved between man and baboon, the baboon model remains the best predictor of activity of fetal hemoglobin-inducing agents in man. In this report, we demonstrate that RN-1 increases γ-globin synthesis, fetal hemoglobin, and F cells to high levels in both anemic and non-anemic baboons with activity comparable to decitabine, the most potent fetal hemoglobin-inducing agent known. RN-1 not only restores high levels of fetal hemoglobin but causes the individual 5' Iγ- and 3' Vγ-globin chains to be synthesized in the ratio characteristic of fetal development. Increased fetal hemoglobin was associated with increased levels of acetylated Histone H3, H3K4Me2, H3K4Me3, and RNA polymerase II at the γ-globin gene, and diminished γ-globin promoter DNA methylation. RN-1 is likely to induce clinically relevant levels of fetal hemoglobin in patients with sickle cell disease, although careful titration of the dose may be required to minimize myelotoxicity.

The LSD1 inhibitor RN-1 induces fetal hemoglobin synthesis and reduces disease pathology in sickle cell mice.

Inhibition of lysine-specific demethylase 1 (LSD1) has been shown to induce fetal hemoglobin (HbF) levels in cultured human erythroid cells in vitro. Here we report the in vivo effects of LSD1 inactivation by a selective and more potent inhibitor, RN-1, in a sickle cell disease (SCD) mouse model. Compared with untreated animals, RN-1 administration leads to induced HbF synthesis and to increased frequencies of HbF-positive cells and mature erythrocytes, as well as fewer reticulocytes and sickle cells, in the peripheral blood of treated SCD mice. In keeping with these observations, histologic analyses of the liver and spleen of treated SCD mice verified that they do not exhibit the necrotic lesions that are usually associated with SCD. These data indicate that RN-1 can effectively induce HbF levels in red blood cells and reduce disease pathology in SCD mice, and may therefore offer new therapeutic possibilities for treating SCD.

Adverse Reactions to Pneumococcal Vaccine in Pediatric and Adolescent Patients with Sickle Cell Disease.

STUDY OBJECTIVE: To review five cases of severe adverse reactions after vaccination with the 23-valent pneumococcal polysaccharide vaccine (PPSV23) in pediatric and adolescent patients with sickle cell disease (SCD), and to evaluate the prevalence of adverse reactions to PPSV23 in patients with SCD by analyzing data from the Vaccine Adverse Event Reporting System (VAERS). DESIGN: Case series and retrospective analysis of data from the VAERS. DATA SOURCES: Medical records from a tertiary care hospital and the VAERS database. MEASUREMENTS AND MAIN RESULTS: Five cases of severe adverse reactions after vaccination with PPSV23 in pediatric and adolescent patients with SCD (aged 2-22 years) were reviewed. The adverse reactions occurred within 24 hours after immunization, and all five patients required medical attention. Analysis of data from the VAERS found that PPSV23 was the most commonly reported vaccine causing adverse events in patients with SCD, accounting for 62% of all vaccine-induced adverse events. This rate is significantly higher than the rate of adverse events related to PPSV23 in patients with human immunodeficiency virus (HIV) or asthma (62% vs 16%, p<0.0001). The reported number of adverse reactions in pediatric patients (< 18 years old) with SCD was 4 times higher than that reported in adult patients (18-39 years old) with SCD. CONCLUSION: The risk of developing severe adverse reactions to PPSV23 is greater in patients with SCD than in patients with HIV or asthma, and especially in pediatric and adolescent patients with SCD compared with their adult counterparts. Health care professionals should closely monitor for potential adverse events after PPSV23 vaccination or revaccination in patients with SCD, adhere to the recommended time interval between PCV13 and PPSV23 administration, and possibly consider avoiding simultaneous administration of other vaccines with PPSV23.

Hydroxymethylcytosine and demethylation of the γ-globin gene promoter during erythroid differentiation.

The mechanism responsible for developmental stage-specific regulation of γ-globin gene expression involves DNA methylation. Previous results have shown that the γ-globin promoter is nearly fully demethylated during fetal liver erythroid differentiation and partially demethylated during adult bone marrow erythroid differentiation. The hypothesis that 5-hydroxymethylcytosine (5 hmC), a known intermediate in DNA demethylation pathways, is involved in demethylation of the γ-globin gene promoter during erythroid differentiation was investigated by analyzing levels of 5-methylcytosine (5 mC) and 5 hmC at a CCGG site within the 5' γ-globin gene promoter region in FACS-purified cells from baboon bone marrow and fetal liver enriched for different stages of erythroid differentiation. Our results show that 5 mC and 5 hmC levels at the γ-globin promoter are dynamically modulated during erythroid differentiation with peak levels of 5 hmC preceding and/or coinciding with demethylation. The Tet2 and Tet3 dioxygenases that catalyze formation of 5 hmC are expressed during early stages of erythroid differentiation and Tet3 expression increases as differentiation proceeds. In baboon CD34+ bone marrow-derived erythroid progenitor cell cultures, γ-globin expression was positively correlated with 5 hmC and negatively correlated with 5 mC at the γ-globin promoter. Supplementation of culture media with Vitamin C, a cofactor of the Tet dioxygenases, reduced γ-globin promoter DNA methylation and increased γ-globin expression when added alone and in an additive manner in combination with either DNA methyltransferase or LSD1 inhibitors. These results strongly support the hypothesis that the Tet-mediated 5 hmC pathway is involved in developmental stage-specific regulation of γ-globin expression by mediating demethylation of the γ-globin promoter.

RN-1, a potent and selective lysine-specific demethylase 1 inhibitor, increases γ-globin expression, F reticulocytes, and F cells in a sickle cell disease mouse model.

Increased levels of fetal hemoglobin are associated with decreased symptoms and increased lifespan in patients with sickle cell disease (SCD). Hydroxyurea, the only drug currently approved for SCD, is not effective in a large fraction of patients, and therefore, new agents are urgently needed. Recently it was found that lysine demethylase 1, an enzyme that removes monomethyl and dimethyl residues from the lysine 4 residue of histone H3, is a repressor of γ-globin gene expression. In this article, we have compared the ability of tranylcypromine (TCP) and a more potent TCP derivative, RN-1, to increase γ-globin expression in cultured baboon erythroid progenitor cells and in the SCD mouse model. The results indicate that the ability of RN-1 to induce F cells and γ-globin mRNA in SCD mice is similar to that of decitabine, the most powerful fetal hemoglobin-inducing drug known, and greater than that of either TCP or hydroxyurea. We conclude that RN-1 and other lysine demethylase 1 inhibitors may be promising new γ-globin-inducing agents for the treatment of SCD that warrant further studies in other preclinical models, such as nonhuman primates.