A pilot study of the effect of atorvastatin on endothelial function and albuminuria in sickle cell disease

Abstract Dysregulated nitric oxide (NO) homeostasis, a consequence of hemolysis, is a central feature of endothelial dysfunction (ED) in Sickle Cell disease (SCD). In addition to ED, scavenging of NO by free heme leads to increased cell adhesion and inflammation. Vascular inflammation and the production of superoxide may decrease BH4, an essential cofactor for NO production, thus creating an acquired BH4 deficiency. Restoring BH4 levels could potentially improve ED thereby favorably impacting complications of SCD. We assessed the safety and efficacy of 6R-BH4 on endothelial function in a Phase 2a, open-label, dose escalation study in SCD subjects using a non-invasive, operatorindependent technique of peripheral arterial tonometry (Endo-PAT; Itamar, Israel). Endo-PAT (PAT) scores were quantitatively determined as the ratio between the arterial pulse wave amplitude following a 5 min arterial occlusion in the forearm to the pre-occlusion value. A value of ≤1.67 represents an impaired response or endothelial dysfunction. Only patients with HbSS and HbSC disease and at least 15 years of age were enrolled. Patients were excluded if they: were on chronic hypertransfusion; had sickle cell crisis within 30 days of screening; had a history of bone marrow or stem cell transplant or were on hydroxyurea (HU) therapy during the 3 months prior to screening. Thirty-two African-American subjects, mean age 29 years (41% male) were sequentially treated for 4 weeks each with 6R-BH4 at 2.5, 5, 10, and 20 mg/kg/day at 12 US sites. Nine subjects discontinued therapy for various reasons including loss of follow up and pregnancy. Twenty-seven subjects had baseline PAT scores and the number of subjects with PAT scores varied at each treatment dose. There were no deaths and only one subject had a drug related adverse effect resulting in discontinuation. Overall, 6R-BH4 is safe and well-tolerated in subjects with SCD. The mean PAT scores for all participants at baseline was 1.58 ± 0.43 (mean ± SD). Mean PAT scores at baseline were 1.33 ± 0.17 in 18 patients with abnormal PAT scores and 2.09 ± 0.31 (p=<0.001) in 9 patients with normal PAT scores. Mean PAT score for all subjects demonstrated significant improvement at 5mg/kg/day and 10mg/kg/day (dose, N, mean change +/− SD, mean % change and p value) (5 mg/kg/day, N=24, 1.79 ± 0.64, 22.4%, p= 0.042; 10 mg/kg/day, N=24, 1.95 ± 0.46, 28.2%, p=0.003). Eighteen of the 27 (67%) subjects who had abnormal PAT scores at baseline had statistically significant dose-dependent improvements over the 16 weeks of therapy with 6R-BH4 (2.5 mg/kg, N=15, 1.63 ± 0.37, 24.7%, p=0.012; 5mg/kg, N=14, 1.69 ± 0.56, 31.2%, p=0.025; 10mg/kg, N=15, 1.84 ± 0.47, 39.9%, p<0.001; 20mg/kg, N=15, 2.01 ± 0.76, 56.6%, p=0.005). Consistent with the mechanism of action of 6R-BH4 subjects with normal Endo-PAT scores at baseline demonstrated no improvement with therapy. HbSS subjects appear to have more ED based on PAT scores compared with HbSC subjects, although the difference was not statistically significant 1.52 ± 0.45 vs 1.67 ± 0.39. More importantly, both HbSS and HbSC subjects demonstrated an improvement in mean change in endothelial function with increasing doses of 6R-BH4 with corresponding % mean changes from baseline being 48.8% and 15.5% respectively following 16 weeks of treatment. The majority of subjects in the study (17/27; 63%) were prescribed folic acid supplement by their physicians at baseline and throughout the study. Post hoc analysis demonstrated no difference in baseline PAT scores between subjects on folic acid supplementation and those not on it (1.60 ± 0.47 vs 1.55 ± 0.37). However, patients on folic acid demonstrated a better dose response to treatment with 6R-BH4 compared to patients not receiving folic acid (2.5 mg/kg: 1.72 ± 0.38 vs 1.69 ± 0.41; 5mg/kg: 1.93 ± 0.74 vs 1.56 ± 0.38; 10mg/kg: 1.89 ± 0.51 vs 2.06 ± 0.34; 20 mg/kg: 2.09 ± 0.73 vs 1.62 ± 0.34) In summary, 6R-BH4 is safe, well-tolerated and demonstrates a dose-dependent improvement in endothelial function in subjects with SCD. Best results were achieved in those with baseline endothelial dysfunction. Improvement in ED occurs regardless of genotype. Finally, patients receiving folic acid showed a better response to 6R-BH4 than those not receiving this supplement. These data provide further support for the development of 6R-BH4 as a treatment for sickle cell disease. 6R-BH4 is a potentially new effective modulator of NO for SCD patients who have ED.

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Low Ascorbate Levels in Transfused Patients Are Associated with Correlates of Vascular Damage in Sickle Cell Disease.

Blood ◽

10.1182/blood.v114.22.2570.2570 ◽

2009 ◽

Vol 114 (22) ◽

pp. 2570-2570

Author(s):

Susan Claster ◽

Susan Carson ◽

Thomas C Hofstra ◽

Thomas Coates ◽

John C Wood

Keyword(s):

Nitric Oxide ◽

Sickle Cell Disease ◽

Endothelial Function ◽

Sickle Cell ◽

Transferrin Saturation ◽

Comprehensive Treatment ◽

Cell Disease ◽

Free Hemoglobin ◽

Convenience Sample ◽

Hs Crp

Abstract Abstract 2570 Poster Board II-547 Introduction: Ascorbate is vital for endothelial homeostasis because it is responsible for regeneration of tetrahydrobiopterin (BH4) from dihydrobiopterin (BH2). BH4 is essential for proper electron transport from oxygen to arginine in the synthesis of nitric oxide (NO) by endothelial nitric oxide synthase (eNOS). When BH4 is deficient, eNOS becomes uncoupled from arginine and synthesizes superoxide, producing a vasoconstrictive rather than a vasodilatory phenotype. Low BH4/BH2 ratios are correlated with decreased flow-mediated dilation, a known marker of nitric oxide-dependent endothelial function. Dietary supplementation with ascorbate has been show to acutely improve NO-mediated vasodilation in smokers and patients with coronary artery disease. Since patients with sickle cell disease (SCD) are known to have low levels of NO and impaired vascular reactivity, we examined the correlates of ascorbate deficiency in our chronically transfused SCD patients. Methods: We recently studied micronutrient levels in convenience sample of our chronically transfused SCD patients. In that study, 56.7% of SCD patients had ascorbate levels below the lower limit of normal (0.2–1.9 mg/dl). We screened the following parameters as predictors of abnormal ascorbate: LDH, cell-free Hb, total bilirubin, liver iron concentration (LIC), pancreas iron, cardiac iron, ferritin, transferrin saturation, insulin, glucose, brain natriuretic peptide (BNP) and high sensitivity C reactive peptide( hs-CRP). A total of 28 patients (21 females, 7 males), ranging in age from 1.4 to 31.4 years old, who had low ascorbate levels at the time of micronutrient measurements were studied. All patients had chelating drugs held for 24 hours prior to being evaluated. Results: None of the above predictors demonstrated significant linear relationships with ascorbate levels. However, low or undetectable ascorbate levels were observed with extremes of a number of parameters including LIC > 30 mg/g, BNP > 40 pg/ml, LDH > 1500 U/Lcell-free hemoglobin > 30 mg/dl,hs-CRP > 7mg/L,and homocysteine > 10 μM/L. LDH, cell-free hemoglobin and hs-CRP exhibited the best specificity for prediction of ascorbate levels and were the only variables to achieve statistical significance by Fischer's Exact test (Table 1). More importantly, there was sufficient independence among these predictors that having and extreme value for one of the above parameters identified 14/15 patients with low ascorbate, with only 1/13 false positives. Discussion: These observations suggest that low ascorbate levels in SCD patients result from a combination of chronic inflammation, hemolysis, and hyperhomocysteinemia. Since ascorbate is vital for proper endothelial function, these observations are consistent with known associations of inflammation and hemolysis with sickle vasculopathy. Although hyperhomocysteinemia is a known vascular stressor in other diseases, its contribution in SCD is less clear because treatment with pyridoxine and folate, which reduce homocysteine levels, do not affect other markers of SCD vascular dysfunction. Taken together, these data suggest that comprehensive treatment of sickle vasculopathy may require multiple interventions, including decreasing hemolysis and inflammation as well as B vitamin supplementation. Ascorbate replacement alone is unlikely to be effective without correction of upstream stressors, similar to its use in other complex vascular disorders. Disclosures: No relevant conflicts of interest to declare.

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