Tetrahydrobiopterin (6R-BH4): Novel Therapy for Endothelial Dysfunction in Sickle Cell Disease
Authors: Hsu Lewis , Ataga Kenneth I , Gordeuk Victor R. , Swerdlow Paul S. , Kutlar Abdullah , Smith Wally , Johnston J. Martin , Chen Ian A , Greist Anne , Flug Frances , Inoue Susumu , Nwose Olise M. , Sile Saba , Kakkis Emil 
Published: October 2019

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.