Improved Hemolytic Anemia with GBT1118 Is Reno-Protective in Transgenic Sickle Mice

Abstract Intravascular hemolysis releases cell-free hemoglobin and heme into the circulation. When haptoglobin and hemopexin are depleted, as observed in sickle cell anemia, cell-free hemoglobin and heme can cause direct oxidative injury, upregulation of inflammatory and immune response pathways, and depletion of nitric oxide. The kidneys are the primary route for clearance of non-scavenged cell-free hemoglobin and heme and are particularly susceptible to these deleterious pathways (PMID 31455889). In patients with sickle cell anemia, increased markers of hemolysis and hemoglobinuria are risk factors for kidney disease and kidney disease progression (PMID 24329963). Voxelotor is a small molecule allosteric modulator that binds and maintains sickle hemoglobin in the oxygenated state, thereby preventing hemoglobin S polymerization and red blood cell sickling. In a phase 3 study, voxelotor improved the degree of hemolysis, as reflected by a rise in hemoglobin concentration and a reduction in indirect bilirubin and reticulocyte percentage (PMID 31199090). The benefits of reducing hemolytic anemia with voxelotor on kidney function are unknown. We investigated whether GBT1118, an analogue of voxelotor with improved pharmacokinetic properties in transgenic sickle mice, would improve biomarkers of kidney damage and kidney function in sickle cell anemia. Transgenic sickle mice (Hb SS; Townes model, Jackson Laboratory) were treated with either GBT1118 (5 male, 5 female) or control (5 male, 5 female) chow from 12 to 24 weeks of age. Urine was collected using metabolic cages for 24 hours at 3 week intervals and blood was collected at baseline, 6 weeks, and 12 weeks of therapy. We compared markers of hemolysis (hemoglobin, reticulocyte percentage), reactive oxygen species (thibarbituric acid reactive substances [TBARS], kidney injury (kidney injury molecule-1 [KIM-1], nephrin), and kidney function (urine albumin and protein, serum blood urea nitrogen [BUN] and creatinine) at the respective time points using ANOVA and adjusting for gender. Mean and standard error values are provided. Consistent with the clinical results observed in the voxelotor phase 3 study, we observed significant improvements in the hemoglobin concentrations and reticulocyte percentages of Hb SS mice treated with GBT1118 versus control (Figure 1A and 1B). The hemoglobin occupancy of GBT1118 after 6 weeks (33 ± 2%) and 12 weeks (28 ± 2%) of therapy were also similar to what was observed in the phase 3 clinical study. An improvement in hemolysis led to reduced hemoglobinuria (6 weeks: treated = 0.07 ± 0.03 vs. control = 0.72 ± 0.2 ng/day, P = 0.004; 12 weeks: treated = 0.04 ± 0.03 vs. control = 1.06 ± 0.32 ng/day, P = 0.006) and less oxidant damage in the kidneys, assessed by urine TBARS concentration, in the treated versus control mice (6 weeks: treated = 31 ± 6 vs. control = 60 ± 10 nmol/day, P = 0.001; 12 weeks: treated = 29 ± 4 vs. control = 71 ± 9 nmol/day, P < 0.001). At 24 weeks of age, markers of glomerular (nephrin: treated = 3.5 ± 0.7 vs. control = 8.5 ± 1.6 ng/day) and tubular injury (KIM-1: treated = 169.6 ± 19.4 vs. control = 348.9 ± 48.2 pg/day) were significantly improved in the treated versus control Hb SS mice (P< 0.001) (Figure 1C). Albuminuria remained stable in the treated Hb SS mice and was relatively close in value to the degree of albuminuria observed in Hb AA mice (Figure 1D). In contrast, albuminuria progressively increased with older age in the control Hb SS mice. A similar pattern was observed for proteinuria (24 week: Hb SS treated = 2.02 ± 0.17 mg/day, Hb SS control = 3.65 ± 0.44 mg/day, Hb AA = 1.65 ± 0.27 mg/day). At 24 weeks of age, the treated Hb SS mice had serum BUN and creatinine values that were similar to what was observed at 12 weeks of age in the Hb SS mice and to the 24-week old Hb AA mice (Figure 1E, 1F). Furthermore, the values for serum BUN and creatinine were significantly lower in the treated versus control Hb SS mice at 24 weeks of age (P ≤ 0.04). In conclusion, we demonstrate that an improvement in hemolysis results in preserved kidney function in transgenic sickle mice. Our findings highlight the clinical importance of hemolytic anemia in the pathophysiology of sickle cell nephropathy. Our results also provide support for developing strategies to mitigate hemolysis in sickle cell anemia in order to provide a targeted approach to improve kidney disease, a devastating complication associated with high morbidity and mortality, in sickle cell anemia. Figure 1 Figure 1. Disclosures Gordeuk: Modus Therapeutics: Consultancy; Novartis: Research Funding; Incyte: Research Funding; Emmaus: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; CSL Behring: Consultancy. Saraf: Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding.

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Hemoglobinuria Is a Risk Factor For Kidney Disease Progression In Sickle Cell Anemia

Blood ◽

10.1182/blood.v122.21.996.996 ◽

2013 ◽

Vol 122 (21) ◽

pp. 996-996

Author(s):

Santosh L. Saraf ◽

Xu Zhang ◽

Tamir Kanias ◽

James P. Lash ◽

Robert E. Molokie ◽

...

Keyword(s):

Steady State ◽

Kidney Disease ◽

Kidney Function ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Conflicts Of Interest ◽

Red Blood Cell Transfusion ◽

Free Hemoglobin ◽

Arterial Blood

Abstract Chronic kidney disease (CKD) is a frequent complication of sickle cell anemia (SCA) and is a predictor of early mortality. To determine the predictors of deteriorating kidney function in SCA, we followed 164 patients treated at the University of Illinois at Chicago for a median of 32 months (range 3-88 months). Steady-state estimated glomerular filtration (eGFR), albuminuria, and hemoglobinuria assessments were obtained at baseline and during the follow-up period. Steady-state was defined as greater than four weeks from a vaso-occlusive pain episode or a red blood cell transfusion. Hemoglobinuria was defined as positive for blood on dipstick and < 2 red blood cells on microscopy. Fifty-six (34%) of the patients had hemoglobinuria at baseline. We confirmed in a subset of 43 patients that dipstick positive hemoglobinuria (n=17) was associated with higher urine cell-free hemoglobin concentrations determined by ELISA than dipstick negative urine (n=26) (23.1 vs. 11.5 ng/mL, p<0.0001) (Figure 1). Age and mean arterial blood pressures were similar in patients with hemoglobinuria at baseline compared to those without but markers of hemolysis were higher (LDH, indirect bilirubin, AST, and reticulocyte percentage; p<0.0001). Sixty-one percent (95%CI: 48-73%) of patients with hemoglobinuria at baseline had hemoglobinuria at most recent follow up compared to 9% (95%CI: 5-18%) of patients without hemoglobinuria at baseline (p<0.0001). The proportion of patients with CKD progression defined by a 50% reduction in eGFR calculated by the CKD-EPI formula or requirement for hemodialysis or kidney transplant was higher in patients with baseline hemoglobinuria (13%, 7/56) versus without hemoglobinuria (1%, 1/108) (HR 14, 95%CI: 2-113; logrank p=0.001) (Figure 2). Progression of albuminuria category from normoalbuminuria (albuminuria < 30mg/g creatinine) to either microalbuminuria (albuminuria = 30-300 mg/g creatinine) or macroalbuminuria (albuminuria > 300mg/g creatinine) or microalbuminuria to macroalbuminuria was also higher in patients with baseline hemoglobinuria (42%, 11/26) versus without hemoglobinuria (13%, 9/67) (HR 3.1, 95%CI: 1.3-7.7; logrank p=0.004) (Figure 3). In conclusion, hemoglobinuria determined by urinalysis at steady-state is a valid assessment of increased urine cell-free hemoglobin concentration and is fairly consistent on repeat testing at steady-state visits. The presence of hemoglobinuria is significantly associated with a greater risk for progression of CKD and albuminuria. Our findings are consistent with the possibility that cell-free hemoglobin contributes to the progression of kidney disease in SCA. Further research including measures to decrease cell-free hemoglobin exposure to preserve kidney function are warranted.Figure 1Figure 1. Figure 2Figure 2. Figure 3Figure 3. Disclosures: No relevant conflicts of interest to declare.

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Effects of Hydroxyurea and Renin-Angiotensin Blockade on Kidney Function in Sickle Cell Disease

Blood ◽

10.1182/blood-2020-138753 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 21-22

Author(s):

Jin Han ◽

Andrew Srisuwananukorn ◽

Michel Gowhari ◽

Faiz Ahmed Hussain ◽

Franklin Njoku ◽

...

Keyword(s):

Kidney Disease ◽

Kidney Function ◽

Sickle Cell ◽

Ace Inhibitors ◽

Research Funding ◽

Cell Disease ◽

Advisory Committees ◽

Average Change ◽

The Mean ◽

Sickle Cell Nephropathy

Kidney disease is a common complication that leads to increased morbidity and early mortality in patients with sickle cell disease (SCD). Vaso-occlusion, hyperfiltration, hypertension, and cell-free hemoglobin/heme-mediated toxicity may contribute to the pathophysiology of kidney disease. Treatments for SCD-related kidney disease have been adopted from therapies used to treat other SCD-related complications (hydroxyurea [HU]) or diabetic nephropathy (angiotensin converting enzyme [ACE]-inhibitors or angiotensin receptor blockers [ARBs]), although their effects on kidney function are not clear. We evaluated the effects of HU and ACE-inhibitors or ARBs on kidney function in a longitudinal cohort of 439 SCD patients enrolled into a prospective registry between 2011 and 2019. Patients were considered for the analysis if they had 6 months of kidney function values pre-therapy and remained on therapy for 6 months or longer. Changes in the estimated glomerular filtration rate (eGFR) and urine albumin concentration were compared prior to and after starting therapy using a mixed effects model. The effects of HU on kidney function were evaluated in 49 SCD patients. The mean age was 38 years (standard deviation [SD] 11 years), 43% were male, and 80% were Hb SS/Sβ0-thalassemia genotype. The eGFR improved from an average decline of -3.3 mL/min/1.73m2 in the 6 months prior to starting HU to an increase of +9.5 mL/min/1.73m2 during HU therapy (P = 0.0002) (Table). The average change in albuminuria also improved from an increase of +1.2 mg/g creatinine pre-HU therapy to a decline of -1.2 mg/g creatinine during HU therapy, although the difference was not statistically significant (P = 0.17). In 47 SCD patients started on ACE-inhibitors or ARBs, the mean age was 45 years (SD 11 years), 43% were male, and 87% were Hb SS/Sβ0-thalassemia genotype. During ACE-inhibitor or ARB therapy, there was no observed difference in the change in eGFR pre- versus during therapy (P = 0.9) (Table). Albuminuria improved from an average change of -1.0 mg/g creatinine pre-therapy to -1.6 mg/g creatinine during therapy (P = 0.009). Because clinical data are limited, current American Society of Hematology guidelines have conditional recommendations with low levels of certainty for the use of HU and ACE-inhibitors or ARBs to treat sickle cell nephropathy. In a longitudinal cohort of SCD patients, we demonstrate that during 6 months of therapy, there may be a benefit of HU in improving eGFR and of ACE-inhibitors or ARBs in reducing albuminuria. Larger and longer follow up studies of HU, ACE-inhibitors and ARBs as well as new targeted therapies to treat sickle cell nephropathy are urgently needed. Figure Disclosures Gordeuk: Novartis: Consultancy; Ironwood: Research Funding; CSL Behring: Consultancy, Research Funding; Global Blood Therapeutics: Consultancy, Research Funding; Imara: Research Funding. Saraf:Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Other: Advisory Boards, Speakers Bureau; Novartis, Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees; Pfizer, Global Blood Therapeutics, Novartis: Research Funding.

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HMOX1 and Acute Kidney Injury in Sickle Cell Anemia

Blood ◽

10.1182/blood.v130.suppl_1.686.686 ◽

2017 ◽

Vol 130 (Suppl_1) ◽

pp. 686-686

Author(s):

Santosh L. Saraf ◽

Maya Viner ◽

Ariel Rischall ◽

Binal Shah ◽

Xu Zhang ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Acute Kidney Injury ◽

Risk Factor ◽

Kidney Disease ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Kidney Injury ◽

Ckd Progression ◽

Kdigo Guidelines

Abstract Acute kidney injury (AKI) is associated with tubulointerstitial fibrosis and nephron loss and may lead to an increased risk for subsequently developing chronic kidney disease (CKD). In adults with sickle cell anemia (SCA), high rates of CKD have been consistently observed, although the incidence and risk factors for AKI are less clear. We evaluated the incidence of AKI, defined according to Kidney Disease Improving Global Outcomes (KDIGO) guidelines as a rise in serum creatinine by ≥0.3mg/dL within 48 hours or ≥1.5 times baseline within seven days, in 158 of 299 adult SCA patients enrolled in a longitudinal cohort from the University of Illinois at Chicago. These patients were selected based on the availability of genotyping for α-thalassemia, BCL11A rs1427407, APOL1 G1/G2, and the HMOX1 rs743811 and GT-repeat variants. Median values and interquartile range (IQR) are provided. With a median follow up time of 66 months (IQR, 51-74 months), 137 AKI events were observed in 63 (40%) SCA patients. AKI was most commonly observed in the following settings: acute chest syndrome (25%), an uncomplicated vaso-occlusive crisis (VOC)(24%), a VOC with pre-renal azotemia determined by a fractional excretion of sodium <1% or BUN-to-creatinine ratio >20:1 (14%), or a VOC with increased hemolysis, defined as an increase in serum LDH or indirect bilirubin level >1.5 times over the baseline value at the time of enrollment (12%). Compared to individuals who did not develop AKI, SCA adults who developed an AKI event were older (AKI: median and IQR age of 35 (26-46) years, no AKI: 28 (23 - 26) years; P=0.01) and had a lower estimated glomerular filtration rate (eGFR) (AKI: median and IQR eGFR of 123 (88-150) mL/min/1.73m2, no AKI: 141 (118-154) mL/min/1.73m2; P=0.02) by the Kruskal-Wallis test at the time of enrollment. We evaluated the association of a panel of candidate gene variants with the risk of developing an AKI event. These included loci related to the degree of hemolysis (α-thalassemia, BCL11A rs1427407), to chronic kidney disease (APOL1 G1/G2 risk variants), and to heme metabolism (HMOX1) . Using a logistic regression model that adjusted for age and eGFR at the time of enrollment, the risk of an AKI event was associated with older age (10-year OR 2.6, 95%CI 1.4-4.8, P=0.002), HMOX1 rs743811 (OR 3.1, 95%CI 1.1-8.7, P=0.03), and long HMOX1 GT-repeats, defined as >25 repeats (OR 2.5, 95%CI 1.01-6.1, P=0.04). Next, we assessed whether AKI is associated with a more rapid decline in eGFR and with CKD progression, defined as a 50% reduction in eGFR, on longitudinal follow up. Using a mixed effects model that adjusted for age and eGFR at the time of enrollment, the rate of eGFR decline was significantly greater in those with an AKI event (β = -0.51) vs. no AKI event (β = -0.16) (P=0.03). With a median follow up time of 66 months (IQR, 51-74 months), CKD progression was observed in 21% (13/61) of SCA patients with an AKI event versus 9% (8/88) without an AKI event. After adjusting for age and eGFR at the time of enrollment, the severity of an AKI event according to KDIGO guidelines (stage 1 if serum creatinine rises 1.5-1.9 times baseline, stage 2 if the rise is 2.0-2.9 times baseline, and stage 3 if the rise is ≥3 times baseline or ≥4.0 mg/dL or requires renal replacement therapy) was a risk factor for CKD progression (unadjusted HR 1.6, 95%CI 1.1-2.3, P=0.02; age- and eGFR-adjusted HR 1.6, 95%CI 1.1-2.5, P=0.03). In conclusion, AKI is commonly observed in adults with sickle cell anemia and is associated with increasing age and the HMOX1 GT-repeat and rs743811 polymorphisms. Furthermore, AKI may be associated with a steeper decline in kidney function and more severe AKI events may be a risk factor for subsequent CKD progression in SCA. Future studies understanding the mechanisms, consequences of AKI on long-term kidney function, and therapies to prevent AKI in SCA are warranted. Disclosures Gordeuk: Emmaus Life Sciences: Consultancy.

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Manifestations of Reduced Kidney Function Occur at a Higher Estimated Glomerular Filtration Rate in Sickle Cell Anemia

Blood ◽

10.1182/blood-2019-124737 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2268-2268

Author(s):

Santosh L. Saraf ◽

Vimal K. Derebail ◽

Xu Zhang ◽

Mark T Gladwin ◽

Victor R Gordeuk ◽

...

Keyword(s):

Alkaline Phosphatase ◽

Kidney Disease ◽

Kidney Function ◽

Sickle Cell ◽

Research Funding ◽

Estimated Glomerular Filtration Rate ◽

Linear Trend ◽

Kidney Dysfunction ◽

Lower Egfr ◽

Reduced Kidney Function

Kidney disease is a common complication in sickle cell anemia (SCA), which leads to increased morbidity and early mortality. The National Kidney Foundation guidelines use an estimated glomerular filtration rate (eGFR) cutoff of 60 mL/min/1.73m2 to define chronic kidney disease (CKD). However, many SCA patients have an elevated baseline eGFR due to low serum creatinine levels from reduced muscle mass, abnormal tubular secretion of serum creatinine into the urine, and/or high cardiac output from the hemolytic anemia (PMID: 23894560, 20185605). The standard definition of CKD may represent a greater decline from "normal" kidney function in SCA patients compared to the general population. In two independent SCA (Hb SS or Sβ0-thalassemia) cohorts, we investigated eGFR cutoffs for when kidney dysfunction, assessed by altered electrolyte (serum potassium) and acid-base (serum HCO3) balance, osteodystrophy (alkaline phosphatase), increased blood pressure and impaired erythropoiesis (hemoglobin < 9 g/dL and absolute reticulocyte count < 250 x 109/L), were observed. Laboratory and clinical variables were obtained at outpatient visits at the time of enrolment. The eGFR categories were grouped as follows: > 120, 90 - 120, 60 - 89, and < 60 mL/min/1.73m2. We compared linear and categorical variables by eGFR category using the test for linear trend and Cochran's test for linear trend, respectively. Mean values and standard error bars are provided in the figures. We first conducted our analysis in 270 SCA patients treated at the University of Illinois at Chicago (UIC). The median age of the cohort was 31 years (interquartile range (IQR), 23 - 42 years), 59% were female, and 52% were on hydroxyurea. The proportion of SCA patients by eGFR category was as follows: 69% with eGFR > 120 mL/min/1.73m2, 13% with eGFR 90 - 120 mL/min/1.73m2, 9% with eGFR 60 - 89 mL/min/1.73m2, and 9% with eGFR < 60 mL/min/1.73m2. With progressively lower eGFR category, we observed higher serum potassium, alkaline phosphatase, systolic blood pressure, and proportion of patients with ineffective erythropoiesis and lower serum HCO3 (Figure 1A) (P ≤ 0.0002). We repeated our analyses in 456 SCA patients from the multi-center Walk-Treatment of Pulmonary Hypertension and Sickle cell disease with Sildenafil Therapy (Walk-PHaSST) cohort. The median age of the cohort was 34 years (IQR, 24 - 45 years), 52% were female, and 43% were on hydroxyurea. The proportion of SCA patients by eGFR category was as follows: 68.5% with eGFR > 120 mL/min/1.73m2, 15% with eGFR 90 - 120 mL/min/1.73m2, 8.5% with eGFR 60 - 89 mL/min/1.73m2, and 8% with eGFR < 60 mL/min/1.73m2. Manifestations of reduced kidney function were progressively worse with lower eGFR category (Figure 1B) (P ≤ 0.02). We then assessed the association of eGFR with altered kidney function using the test for linear trend in a combined analysis of SCA patients from UIC and Walk-PHaSST as well as in non-SCA African Americans adults from the National Health and Nutrition Examination Survey (NHANES) cohort (n = 1331). The median age of the NHANES cohort was 48 years (IQR, 29 - 62) and 53% were female. The associations between eGFR and kidney dysfunction, based on the beta coefficients, were stronger for serum HCO3, potassium, and alkaline phosphatase in SCA versus non-SCA patients (Figure 1C). The most significant associations between eGFR and kidney dysfunction were observed at an eGFR cutoff of 80 mL/min/1.73m2 for SCA patients, which was higher than the cutoffs observed in non-SCA patients for HCO3 (40 mL/min/1.73m2), potassium (50 mL/min/1.73m2), and alkaline phosphatase (60 mL/min/1.73m2). In conclusion, we demonstrate that kidney dysfunction occurs in SCA patients at eGFR values that are above the standard thresholds currently used to define CKD. Manifestations of kidney dysfunction progressively worsen with lower eGFR category and the differences are most significant at an eGFR < 80 mL/min/1.73m2. Future studies to redefine kidney disease in SCA based on eGFR may help identify high-risk patients for earlier intervention strategies and for the avoidance of potential nephrotoxins, such as nonsteroidal anti-inflammatory drugs and intravenous contrast. Disclosures Saraf: Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding. Derebail:Retrophin: Consultancy; RTI: Honoraria; Novartis: Consultancy. Gladwin:Globin Solutions, Inc: Patents & Royalties: Provisional patents for the use of recombinant neuroglobin and heme-based molecules as antidotes for CO poisoning; United Therapeutics: Patents & Royalties: Co-inventor on an NIH government patent for the use of nitrite salts in cardiovascular diseases ; Bayer Pharmaceuticals: Other: Co-investigator. Gordeuk:Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; CSL Behring: Consultancy, Honoraria, Research Funding; Inctye: Research Funding; Modus Therapeutics: Consultancy, Honoraria; Novartis: Consultancy, Honoraria, Research Funding; Pfizer: Research Funding; Ironwood: Research Funding; Imara: Research Funding. Little:Hemex Health, Inc.: Patents & Royalties; GBT: Research Funding.

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Iron Unloading By Therapeutic Phlebotomy in Previously Transfused Children with Sickle Cell Anemia: The Twitch Experience

Blood ◽

10.1182/blood.v128.22.1018.1018 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1018-1018 ◽

Cited By ~ 1

Author(s):

Banu Aygun ◽

Nicole Mortier ◽

Zora R. Rogers ◽

William Owen ◽

Beng Fuh ◽

...

Keyword(s):

Iron Overload ◽

Board Of Directors ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Research Funding ◽

Chelation Therapy ◽

Venous Access ◽

Study Entry ◽

Iron Loading ◽

Advisory Committees

Abstract Background: TCD With Transfusions Changing to Hydroxyurea (TWiTCH, ClinicalTrials.gov NCT01425307), an NHLBI-sponsored Phase III multicenter trial, compared transfusions to hydroxyurea for maintaining TCD velocities in children with sickle cell anemia who previously received transfusions for abnormal TCD velocities. Iron overload was treated with serial phlebotomy in children randomized to hydroxyurea. At the first scheduled interim analysis, non-inferiority of hydroxyurea was demonstrated and the study was terminated prematurely. Methods: Participants randomized to hydroxyurea received decreasing volumes of monthly transfusions during hydroxyurea dose escalation to maximum tolerated dose (MTD), averaging 6-7 months. During this transfusion overlap period, no chelation therapy was given. After hydroxyurea MTD was reached, transfusions were discontinued and children started monthly phlebotomy if their entry liver iron concentration (LIC) by MRI-R2 (FerriScan®) was ≥2 mg Fe/g dry weight liver (DWL). The prescribed phlebotomy volume was 10 mL/kg (maximum 500 mL) with adjustments for anemia (5 mL/kg for Hb 8.0-8.5 g/dL and held if Hb <8.0 g/dL). Phlebotomy was performed over 30 minutes with immediate equal volume normal saline replacement, typically using peripheral venous access. LIC was assessed at study entry, midpoint (12 months), and exit (24 months/early closure). Ferritin was monitored monthly using a centralized laboratory. Iron loading calculations were based on actual transfusion and phlebotomy volumes. Results: Sixty children (mean age 9.7±3.2 years; range 5.2-19.0 years; 48% male) were randomized to the Hydroxyurea Treatment Arm. The average duration of previous transfusions was 4.5±2.8 years. Almost all (51/60, 85%) had previously received chelation, primarily deferasirox, and 48 (80%) were on chelation therapy at study enrollment. Hydroxyurea MTD was achieved in 57 children (95%), and 54 commenced phlebotomy (two had low iron burden with LIC <2 and one had Hb <8.0 g/dL). A total of 914 phlebotomy procedures were scheduled per protocol for these 54 children and 756 (83%) were fully completed. There were 77 procedures cancelled due to anemia and another 81 procedures cancelled due to planned anesthesia (16), provider preference (14), hydroxyurea-related cytopenia (13), intercurrent illness (11), inadequate iv access (9), family request (5) or other (13). In 94% of phlebotomy procedures that were initiated, the full volume was removed; for the remaining 6% (47 procedures), a reduced volume was removed due to loss of venous access (37), symptoms such as headache or lightheadedness (7), or other reasons (3). A total of 18 Adverse Events (17 Grade 2 and one Grade 3) occurred in 14 participants in association with phlebotomy (2.3% prevalence). The most common complication was light headedness/near-syncope (6) followed by anemia (4), hypotension (3), headache (3), and pain at the venous access site (1). One subject had a syncopal episode followed by transient weakness, which was centrally adjudicated as TIA. An average of 53.6±21.8 mL/kg blood was administered in the hydroxyurea-treated arm, which calculates to an average iron loading of 40.1±16.3 mg Fe/kg, while an average of 112 mL/kg of venous blood was removed by phlebotomy, which calculates to an average iron unloading of 36.1±15.7 mg Fe/kg. For the 54 children who received phlebotomy, the average LIC was 12.0± 9.7 mg/g at study entry, 13.4±10.3 at midpoint reflecting overlap transfusions without chelation, and 9.7±8.9 at study exit reflecting serial phlebotomy, for an average net LIC decrease of 2.3±4.1 mg/g. Average serum ferritin at study entry was 3105±741 ng/mL and 1392±1542 ng/mL at study exit. For 39 children who completed all 24 months of treatment before study closure, the overall average LIC decrease was 3.2±3.8 mg/gram DWL and 10 had final LIC measurements <3 mg Fe/g. Calculated net iron loading was not significantly associated with measured changes in LIC or ferritin. Conclusions: In the TWiTCH trial, phlebotomy was a feasible, safe, well-tolerated, and effective treatment for transfusional iron overload in children with sickle cell anemia. Although initial overlap transfusions without chelation limited the phlebotomy effects, in children who reached hydroxyurea MTD and discontinued chronic transfusions, monthly phlebotomy led to net iron unloading and lower LIC, and significantly reduced iron burden. Disclosures Rogers: Apopharma: Consultancy. Kalfa:Baxter/Baxalta/Shire: Research Funding. Kwiatkowski:Sideris Pharmaceuticals: Consultancy; Luitpold Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Apopharma: Research Funding; Ionis pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; Shire Pharmaceuticals: Consultancy. Wood:World Care Clinical: Consultancy; Biomed Informatics: Consultancy; Biomed Informatics: Consultancy; Celgene: Consultancy; Celgene: Consultancy; AMAG: Consultancy; Apopharma: Consultancy; Apopharma: Consultancy; AMAG: Consultancy; World Care Clinical: Consultancy; Vifor: Consultancy; Vifor: Consultancy; Ionis Pharmaceuticals: Consultancy; Ionis Pharmaceuticals: Consultancy. Ware:Global Blood Therapeutics: Consultancy; Biomedomics: Research Funding; Bayer Pharmaceuticals: Consultancy; Addmedica: Research Funding; Nova Laboratories: Consultancy; Bristol Myers Squibb: Research Funding.

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Outcomes in Children with Severe Vasculopathy from Sickle Cell Anemia after Treatment with Chronic Transfusion, Surgical Revascularization and/or Allogenic Hematopoetic Stem Cell Transplantation

Blood ◽

10.1182/blood-2018-99-118693 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 1098-1098

Author(s):

Courtney W. Johnson ◽

Suvankar Majumdar ◽

Andrew D. Campbell ◽

Suresh Magge ◽

Deepika S. Darbari ◽

...

Keyword(s):

Stem Cell ◽

Board Of Directors ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Research Funding ◽

Moyamoya Syndrome ◽

Surgical Revascularization ◽

Advisory Committees ◽

Time Of Intervention

Abstract Background: Cerebral vasculopathy is a frequent complication of sickle cell anemia (SCA) and is associated with a high risk for stroke. This vasculopathy seen in SCA can be progressive and severe. Sickle cell patients with severe vasculopathy, including Moyamoya syndrome are at increased risk for neurological disabilities and death. While chronic transfusions decrease the risk of stroke in SCA; unfortunately, progression of vasculopathy can occur despite treatment. Limited data exists regarding long term outcomes for this population. We evaluated effectiveness of three treatment approaches at our center, namely chronic transfusions, surgical revascularization plus chronic transfusions and allogenic hematopoietic stem cell transplant (HSCT). Methods: A retrospective chart review was preformed to identify patients with SCA (hemoglobin SS, Sβ0) and severe vasculopathy including Moyamoya syndrome between 1986 to 2017. Severe vasculopathy was defined as having at least one cerebral artery with > 70% stenosis and/or occlusion as seen on MR angiogram (MRA), CT angiogram (CTA) or conventional angiogram (DSA) as determined by a neuroradiologist at our institution. Patients were identified from an institutional stroke database. Patients were included for analysis if they received at least one of the following: chronic transfusions, surgical revascularization (i.e. encephalo-duro-arterio-synagiosis (EDAS) plus chronic transfusions or HSCT. For HSCT, all graft types (bone marrow, peripheral blood stem cells, umbilical cord blood), conditioning regimens and donor types (related, unrelated and haploidentical) were included. Time to event analyses were performed from the time of intervention (transfusion, HSCT, EDAS/chronic transfusions) using overt clinical stroke, new silent infarcts, progression of vasculopathy or new vasculopathy. Survival curves were analyzed using the log-rank (Mantel-Cox) test. Results: Of 35 patients identified, 54% (n =19) underwent chronic transfusions, 23% (n=8) of patients underwent HSCT after being on chronic transfusions, 23% (n=8) underwent EDAS with chronic transfusions and 1 patient underwent each of the above three modalities (Table 1). Median age at time of intervention was similar for all three cohorts (Table 1). Males were overrepresented in all treatment arms (62.5-79% of patients). Average hemoglobin level prior to intervention was also similar: 7.6 g/dL (IQR 7.1-8.3) for the chronic transfusion cohort, 7.3 gm/dL (IQR 6.3-8.2) for the HSCT cohort, and 7.5 gm/dL (IQR 7.2-8) for the EDAS/chronic transfusion cohort. Absolute reticulocyte count was 492.9 K/ul (IQR 358.4-550) for the chronic transfusion group, 389.4 (IQR 174.3-449) for HSCT, and 250.2 (IQR 107.3-393) for EDAS/chronic transfusions (p=0.08). One patient died of overt stroke in the chronic transfusion cohort. The median follow-up times for the transfusion, HSCT and EDAS plus transfusion groups were 4.4, 2.4 and 6 years respectively. Time from date of intervention (transfusion, HSCT, EDAS) to overt clinical or silent stroke was evaluated (Fig 1). Two of the nineteen patients in the chronic transfusion cohort suffered an overt stroke, while one of eight and two of eight had strokes in the post-HSCT and EDAS plus chronic transfusion cohorts respectively. Fourteen of nineteen (74%) in the chronic transfusion cohort had progression of severe vasculopathy after being on transfusions while two of eight (25%) in the HSCT and four of the eight (50%) patients in the EDAS plus chronic transfusion cohorts had progression. The one patient with all three different interventions did not have additional infarction (clinical or silent) or vasculopathy progression during 1.5 years of follow-up. Conclusions: The risk for cerebral infarction and/or vasculopathy progression after initiation of treatment with either chronic transfusion, HSCT or EDAS is still a major concern. Our data suggest HSCT and surgical revascularization with chronic transfusion provide the greatest benefit in reducing stroke risk and HSCT reduces risk for progression of a severe vasculopathy. Additional, large population studies are needed to clarify the risk. Disclosures Majumdar: NIMHD: Research Funding. Campbell:Functional Fluitics: Membership on an entity's Board of Directors or advisory committees; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding.

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Thrombomodulin and Endothelial Dysfunction in Sickle Cell Anemia

Blood ◽

10.1182/blood-2019-123862 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 3558-3558

Author(s):

Maria Armila Ruiz ◽

Binal N. Shah ◽

Jin Han ◽

Rasha Raslan ◽

Victor R Gordeuk ◽

...

Keyword(s):

Endothelial Cell ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Research Funding ◽

Multiorgan Failure ◽

Plasma Concentrations ◽

Organ Damage ◽

Free Hemoglobin ◽

Failure Event

Thrombomodulin (THBD) is a transmembrane protein that regulates endothelial function by 1) binding thrombin and inhibiting its interaction with fibrinogen, 2) augmenting protein C activation, and 3) down-regulating complement activation by inactivating C3a and C5a (PMID 29866818). Reduced THBD function has been implicated in several vasculopathies (PMID 29866818). Oxidative damage increases cleaved non-functional THBD in circulation and increased circulating non-functional THBD predicts the severity of organ damage in thrombotic microangiopathies (PMID 11190905). Vascular endothelial dysfunction is a hallmark feature of sickle cell disease (SCD) that leads to acute and chronic organ damage and may, in part, be mediated by hemolysis (PMID 28248201). Hemolysis leads to release of cell-free hemoglobin which may lead to vasculopathy through consumption of NO, activation of TLR4 pathways, and direct oxidative damage. We investigated whether decreased THBD activity may be implicated in the pathophysiology of SCD vasculopathy in endothelial cells, transgenic sickle mice, and in a prospective cohort of patients with sickle cell anemia. In vitro: We exposed endothelial cells (EA.hy926, ATTC® CRL-2922TM; Manassas, VA) to incremental doses of cell-free hemoglobin. With higher doses of cell-free hemoglobin we observed reduced surface endothelial cell THBD activity at 6 hours of incubation, assessed by cleavage of chromogenic substrate for activated protein C in the presence of thrombin (Figure 1A). In conjunction with the reduced endothelial THBD activity, there were increased concentrations of cleaved THBD in the supernatant by ELISA (R&D Systems, Minneapolis, MN) (Figure 1B). Transgenic sickle mice: At 6 months of age, transgenic sickle mice (Townes model, Jackson Laboratory; Bar Harbor, Maine) had higher plasma concentrations of cleaved non-functional THBD versus hemoglobin AA mice (Figure 2A). Furthermore, staining of the glomerular microvasculature demonstrated decreased endothelial-bound THBD (Figure 2B). Patients with sickle cell anemia: We evaluated whether plasma concentrations of cleaved non-functional THBD are predictive of acute multiorgan failure syndrome (PMID 8109600) in a cohort of 103 SCD patients recruited into a longitudinal kidney cohort study. Clinical and laboratory variables and plasma samples were obtained at the time of enrolment and the patients were monitored prospectively for acute multiorgan failure syndrome. Hemoglobinuria was defined by urine dipstick positive for blood with < 2 red blood cells/high power field. The median age of this cohort was 35 years (interquartile range, 28 - 44 years), 46% were female, 87% had hemoglobin SS genotype, and 47% were on hydroxyurea at the time of enrolment. In cross-sectional analysis, plasma THBD concentrations were greater in patients with hemoglobinuria, a marker of intravascular hemolysis-derived cell-free hemoglobin in circulation exceeding scavenging capability and filtering through the glomerulus, versus without hemoglobinuria (6.1 ± 0.6 µg/mL vs. 3.6 ± 0.4 µg/mL, P = 0.004) (Figure 3A). With a median follow up of 5.5 years (interquartile range, 1.4 - 5.9 years), 18 (17%) SCD patients had a multiorgan failure event. SCD patients with a multiorgan failure event were older (43 vs. 34 years, P = 0.01) but without significant differences in sex, SCD genotype, or hydroxyurea therapy. After adjusting for age, baseline THBD concentrations predicted a greater risk for multi-organ failure syndrome (log-transformed OR 4.0, 95% CI: 1.2 - 13.3; P = 0.01) (Figure 3B). In conclusion, circulating non-functional THBD, a protein that normally functions to reduce vasculopathy when bound to the endothelium, is increased after endothelial cell exposure to incremental doses of hemoglobin in vitro, in hemoglobin SS vs. AA mice, and in SCD patients with versus without hemoglobinuria. Furthermore, circulating THBD is a biomarker that predicted the risk for multi-organ failure syndrome on longitudinal follow up in SCD patients. Future studies investigating the role of THBD in SCD vasculopathy may help improve our understanding for the catastrophic multiorgan failure syndrome and therapies to augment endothelial cell THBD function may guide future intervention practices. Figure 1 Disclosures Gordeuk: Novartis: Consultancy, Honoraria, Research Funding; Emmaus: Consultancy, Honoraria; Global Blood Therapeutics: Consultancy, Honoraria, Research Funding; Modus Therapeutics: Consultancy, Honoraria; Pfizer: Research Funding; Inctye: Research Funding; CSL Behring: Consultancy, Honoraria, Research Funding; Ironwood: Research Funding; Imara: Research Funding. Saraf:Novartis: Consultancy, Membership on an entity's Board of Directors or advisory committees; Pfizer: Research Funding.

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Pharmacokinetics in Children with Sickle Cell Anemia Following Single Dose Versus Chronic Treatment with Hydroxyurea

Blood ◽

10.1182/blood.v128.22.1314.1314 ◽

2016 ◽

Vol 128 (22) ◽

pp. 1314-1314

Author(s):

Joana Marie Mack ◽

Pawel Wiczling ◽

Joseph Moen ◽

Guolian Kang ◽

Robert I. Liem ◽

...

Keyword(s):

Plasma Concentration ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Research Funding ◽

Systemic Exposure ◽

Coefficient Of Determination ◽

Average Dose ◽

Multiple Dosing ◽

Advisory Committees ◽

Post Dose

Abstract Introduction: Hydroxyurea (HU) reduces vaso-occlusive complications, hospitalizations and transfusion requirements in children with sickle cell anemia (SCA; HbSS and HbSβ0thalassemia). Despite linear pharmacokinetics (PK) and apparent dose dependency for HU effect, there remains unexplained large inter-individual variability in drug response. To better understand this variability, we conducted a PK study of HU to assess: 1) effect of multiple dosing on PK of HU, and 2) explore the utility of using a single post-dose HU plasma concentration as a basis for therapeutic drug monitoring. Methods: Data from two prospective trials, "Pharmacokinetics of Liquid Hydroxyurea in Pediatric Patients with Sickle Cell Anemia" (NCT01506544) and "Single-Dose (SD) and Steady-State (SS) Pharmacokinetics of Hydroxyurea in Children and Adolescents with Sickle Cell Disease", were utilized for this analysis. Participants were children (≤18 years) with HgbSS and HbSβ0 thalassemia from 8 medical centers in the U.S. One cohort of patients had never been treated with HU and the second cohort had been treated with HU for at least ≥3 months at a stable dose. All participants received a single oral dose of HU and plasma PK samples were collected pre-dose, then at 15, 30, 45, and 60 minutes, and 1.5, 2, 4, 6, and 8 hours after study drug was administered under direct supervision. HU was quantitated from plasma and urine using a validated HPLC method. PK parameters for HU were determined from each patient using a standard model-independent approach (apparent Cmax observed from plasma concentration vs time data; AUC determined via a log-linear approach). PK parameters were compared using parametric (two-sample t-test) or nonparametric (Wilcoxon Rank Sum test) as appropriate based on normality of distribution. The coefficient of determination was used to determine the most predictive relationship between post-peak HU plasma concentrations and systemic exposure (AUC). The significance limit accepted for all statistical analyses was a = 0.05. Results: A complete plasma HU PK profile was obtained for 59 children. Participants with PK after the first dose (n=7, HUfirst) group received an average dose of 17.9 ±2.6 mg/Kg of HU whereas those with PK after multiple doses (n=52, HUchronic group) received an average dose of 23.8 ±5.1 mg/Kg (p < 0.01). Absorption of HU was rapid in both groups with a time to maximal plasma concentration (Tmax) of 0.9 ±0.58 hours in the HUfirst group and 0.8 ±0.47 hours in the HUchronic group (p=0.68). The mean dose/weight- normalized Cmax in the HUfirst group (2.0 mg/L per 1 mg/kg dose) was 1.4 fold higher than in the HUchronic (1.4 mg/L per 1 mg/kg) (p=0.03). A similar relationship was observed in mean dose/weight-normalized AUCinf, where the HUfirst group was 1.3 fold higher than in the HUchronic group (5.9 vs 4.6 mg/L*hr per 1 mg/kg; p=0.002). Weight-normalized mean apparent oral clearance (Cl/F) was significantly lower in the HUfirst cohort (0.17 L/hr/kg) as compared to the HUchronic (0.23 L/hr/kg) (p<0.001). The mean apparent volume of distribution (Vz/F) for the HUfirst cohort (0.52 L/kg) was not significantly different than that in the HUchronic cohort (0.61 L/kg) (Table 1). As suggested by data in Figure 1a, the apparent mean elimination half-life did not vary between the groups (e.g., 2.1 hr in HUfirst vs. 2.3 hr in HUchronic). Finally, between 45 minutes post-dose and through the last blood sampling point, the 4 hour post-dose concentration most accurately predicted the AUC of HU (r2= 0.78) (Figure 1b). Conclusion: Weight and dose-normalized PK parameters for HU suggest potential differences in the bioavailability of the drug with multiple dosing. This finding may contribute to the known wide variability in HU response. Finally, a single 4 hour post-dose HU plasma concentration adequately predicts systemic exposure (AUC) to HU and thus, could provide an approach to facilitate dose individualization / optimization. Mean hydroxyurea (HU) concentration (μg/mL) per time (hour) profiles in children on chronic HU therapy (HUchronic) and children who are receiving the first dose of drug (HUfirst). Error bars represent standard deviation of the mean.Figure 1A. The coefficient of determination (R2) of the linear correlation between plasma concentration (DV) and observed AUCinf at 4.0 hours in all children (HUtreated and HUfirst) to therapy. Mean hydroxyurea (HU) concentration (μg/mL) per time (hour) profiles in children on chronic HU therapy (HUchronic) and children who are receiving the first dose of drug (HUfirst). Error bars represent standard deviation of the mean.Figure 1A. The coefficient of determination (R2) of the linear correlation between plasma concentration (DV) and observed AUCinf at 4.0 hours in all children (HUtreated and HUfirst) to therapy. Figure 1B Figure 1B. Disclosures Liem: National Institute of Health: Research Funding; Ann & Robert H. Lurie Children's Hospital of Chicago: Research Funding; Ann & Robert H. Lurie Children's Hospital of Chicago: Employment. Estepp:Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees, Research Funding; Global Blood Therapeutics: Membership on an entity's Board of Directors or advisory committees, Research Funding; Pfizer: Research Funding; National Institute of Health: Research Funding.

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An Analysis Of The Pediatric Sub-Group From The Phase 2 Study Of GMI 1070 – A Novel Agent For The Vaso-Occlusive Crisis Of Sickle Cell Anemia

Blood ◽

10.1182/blood.v122.21.2206.2206 ◽

2013 ◽

Vol 122 (21) ◽

pp. 2206-2206

Author(s):

Timothy L. McCavit ◽

Lakshmanan Krishnamurti ◽

Lewis L. Hsu ◽

Charles T. Quinn ◽

Isaac Odame ◽

...

Keyword(s):

Median Time ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Research Funding ◽

Study Drug ◽

Acute Chest Syndrome ◽

Phase 2 ◽

Opioid Use ◽

Advisory Committees ◽

Secondary Outcomes

Abstract Introduction Vaso-occlusive crisis (VOC) is the hallmark of sickle cell anemia (SCA), yet VOC treatment has not improved in decades. Recently the role of adhesive interactions between leukocytes, erythrocytes, and the vascular endothelium has been recognized in VOC. GMI 1070, a pan-selectin inhibitor, was designed to decrease the contribution of leukocyte adhesion to VOC. Herein, we report a sub-analysis of the pediatric patients enrolled in a study of GMI 1070 with the aims of determining the efficacy and toxicities in this subgroup and comparing pediatric to adult patients. Methods This multi-center, randomized, double-blind, placebo-controlled phase 2 trial enrolled afebrile patients ≥ 12 yrs with HbSS or HbSβ0 thalassemia presenting with VOC. Subjects had no organ dysfunction or other acute SCA complications. GMI 1070 was administered IV as a loading dose, then in up to 14 q12 h maintenance doses. Following a pre-specified interim PK analysis including 1 pediatric and 10 adult subjects, the loading and maintenance doses were doubled. Other management was at the discretion of the treating physician. Pain intensity was measured with a 10 cm visual analog scale. The primary outcome was time to resolution of VOC, defined as either a sustained 1.5 cm decrease in the pain score and cessation of IV analgesics; readiness for discharge; or hospital discharge. Secondary outcomes included time to discharge, time to transition from IV to oral analgesics, opioid usage, and safety profile. Median time-to-event was compared between arms using the Kaplan-Meier (KM) method. Analysis of covariance was used to compare the mean hourly opioid use, by hospital day. Results Seven sites enrolled 20 pediatric subjects: GMI 1070 - 13 vs placebo - 7. The median age was 14 years, and 40% were female. Time to 1st dose of study drug was a mean of 15 h from initial medical evaluation. Median length of stay was 105 h. The GMI 1070 arm had a 60.7 h reduction in the median time to resolution of VOC compared to placebo (Fig. 1a). Similarly, the median difference in time to transition to oral opioids and time to discharge were clinically significant between GMI 1070 and placebo at 87.8 h and 96 h, respectively (Fig. 1b & 1c). Mean hourly opioid use was lower with GMI 1070 than placebo in the first 24 h, but the trajectory thereafter did not differ (Fig. 1d). The effect of GMI 1070 on the primary and secondary outcomes was similar for pediatric and adult subjects (Table 1). Differences between pediatric and adult subjects included which opioid was used (pediatrics – 80% morphine vs adults – 80% hydromorphone). Also more pediatric subjects received IV antibiotics (Table 1), particularly in the first 24 hrs of study drug (pediatrics – 35% vs adults – 14%). The proportion of pediatric subjects experiencing a serious adverse event (SAE) was similar between arms (GMI 1070 – 31% vs placebo – 43%). Most SAE's were VOC recurrence; 1 SAE was an episode of acute chest syndrome (ACS) in the GMI 1070 arm (0 in the placebo arm). Of 4 total ACS events (1 SAE, 3 AE), 3 occurred within 24 h of 1st study drug, 1 required red blood cell transfusion and 0 required intensive care. No severe or unusual infections occurred in either arm. Conclusions GMI 1070 is a promising agent for reducing duration of VOC in SCA. Compared to adults, pediatric subjects demonstrated similar efficacy and safety. The ACS cases in the GMI 1070 arm are noteworthy but are not definitively associated with study drug. The strong efficacy signal in adolescents, along with minimal safety concerns, warrants inclusion of younger children in a subsequent phase 3 clinical trial of GMI 1070. Disclosures: McCavit: Pfizer, Inc.: Consultancy; GlycoMimetics, Inc.: Research Funding. Krishnamurti:GlycoMimetics, Inc.: Research Funding. Hsu:GlycoMimetics, Inc.: Research Funding. Quinn:Glycomimetics: Research Funding; Eli Lilly: Research Funding; MAST Therapeutics: Research Funding; American Society of Hematology: Advisory Committees, Advisory Committees Other, Honoraria. Odame:Glycomimetics: Research Funding. Alvarez:Glycomimetics: Research Funding. Driscoll:Glycomimetics: Research Funding. Smith-Whitley:GlycoMimetics, Inc: Research Funding. Rhee:Rho, Inc.: Employment; GlycoMimetics, Inc.: Research Funding. Wun:Emmaus, Inc.: Clinical Adjudication Committee Other; Pfizer, Inc.: Consultancy; GlycoMimetics: Research Funding. Telen:GlycoMimetics, Inc.: Research Funding; Dilaforette, NA: Research Funding; Pfizer, Inc.: Consultancy. Thackray:GlycoMimetics, Inc.: Employment, Equity Ownership.

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Genetic Modifiers Influencing the Development of Albuminuria in Children with Sickle Cell Anemia

Blood ◽

10.1182/blood.v126.23.3393.3393 ◽

2015 ◽

Vol 126 (23) ◽

pp. 3393-3393

Author(s):

Beverly A Schaefer ◽

Jonathan M Flanagan ◽

Banu Aygun ◽

Ofelia A. Alvarez ◽

Stephen C Nelson ◽

...

Keyword(s):

Chronic Kidney Disease ◽

Kidney Disease ◽

Renal Disease ◽

Candidate Genes ◽

Genetic Polymorphisms ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

Genetic Variants ◽

Research Funding ◽

Increased Risk

Abstract Background: Nephropathy in sickle cell anemia (SCA) begins in childhood and portends chronic kidney disease, renal failure, and early mortality among affected adults. Individuals of African descent have disproportionately higher rates of developing non-diabetic renal disease. Several candidate genetic variants have been identified, including some specific to African Americans, which are associated with the development of albuminuria and renal disease. The influence of genetic polymorphisms on albuminuria and elevated glomerular filtration rate (GFR) in children with SCA, both early signs of sickle nephropathy, has not been investigated. Objectives: To determine the influence of selected single nucleotide polymorphisms (SNPs) on the development of albuminuria and elevated GFR in children with SCA; to identify novel genetic variants influencing albuminuria and GFR by whole exome sequencing (WES). Design/Methods: Genomic DNA was collected on children with SCA enrolled in two prospective studies with pre-hydroxyurea renal assessments (n=185): (1) Hydroxyurea Study of Long-Term Effects (HUSTLE, NCT00305175, n=79) with no prior disease-modifying therapy; and (2) Transcranial Doppler (TCD) With Transfusions Changing to Hydroxyurea (TWiTCH, NCT 01425307, n=106) on chronic transfusions for abnormal TCD velocities. Albuminuria was defined as ≥30mg albumin/gm creatinine on the pre-hydroxyurea urine specimen. GFR was measured in HUSTLE using plasma DTPA (technetium 99m-labeled diethylenetriaminepentaacetic acid) clearance, and estimated GFR (eGFR) in TWiTCH based on serum creatinine. DNA samples were genotyped for 8 candidate SNPs previously associated with renal disease, using PCR-based allelic discrimination, bidirectional Sanger sequencing, and analysis of variable number tandem repeats (VNTR). Associations between albuminuria and genetic polymorphisms were tested using an additive model and correlation trend test. Linked WES data from the same patients were analyzed to identify other variants associated with albuminuria and GFR. Results: Albuminuria was present in 13.1% of patients, including 16.3% in HUSTLE and 11.0% in TWiTCH. APOL1 genetic variants were common (G1 allele frequency = 21.9%, G2 allele = 16.0%, Table) and similar to published cohorts. Children with two APOL1 G1 alleles had an increased risk of albuminuria that approached statistical significance (p=0.053). Conversely, the presence of the DARC SNP that confers Duffy antigen expression had a protective effect (p=.038). WES analysis did not identify additional non-synonymous APOL1 variants linked with albuminuria. However, 93 non-synonymous variants were associated with DTPA GFR (p<0.001). Using patients with eGFR as a validation cohort, 7 variants in FUBP1, ZFAND4, CD163, GMFG and HLA-E maintained their association with kidney filtrative function (p<0.05). In particular, two variants in CD163, which is a macrophage scavenger receptor for hemoglobin-haptoglobin complexes, were strongly associated with increased GFR in both patient cohorts. Table 1. Candidate genes associated with microalbuminuria. All SNPs were tested with either an additive or recessive genetic model. *The eNOS VNTR was analyzed by the chi-square method. Gene SNP Location MAF Albuminuria (Additive) Variant frequency Cases Controls APOL1 G1 rs73885319 22q12 21.9% 0.053 32.0% 20.3% APOL1 G2 rs71785313 22q12 16.0% 0.445 10.0% 16.0% DARC rs2814778 1q23 13.6% 0.038 4.2% 15.7% eNOS 4a VNTR 7q35 31.2% 0.333* 31.5% 22.7% eNOS rs1799983 7q35 12.5% 0.663 14.6% 12.2% eNOS rs2070744 7q35 15.6% 0.299 10.4% 16.3% CUBN rs7918972 10p12 16.2% 0.457 12.5% 16.8% CUBN rs1801239 10p12 2.7% 0.199 0.0% 3.1% Conclusion: Genetic polymorphisms associated with chronic kidney disease in African American adults may influence the development of early-onset albuminuria among children with SCA, including an increased risk among children with ≥1 APOL1 G1 alleles and a decreased risk associated with the DARC SNP. Previously published eNOS and CUBN variants had no measureable effects. WES analysis suggests novel genetic variants including CD163 SNPs may influence the development of elevated GFR in children with SCA, and provide candidate genes for future research. Disclosures Off Label Use: Hydroxyurea is FDA approved for the treatment of sickle cell anemia in adults, but has not yet been approved in children. . Nottage:Janssen Pharmaceuticals: Employment. Ware:Biomedomics: Research Funding; Bristol Myers Squibb: Research Funding; Bayer Pharmaceuticals: Consultancy; Eli Lilly: Other: DSMB membership.

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