Search

Increased expression of fetal globin (HBG) has been shown to reduce clinical severity of beta-globin disorders and increase survival in sickle cell disease (SCD), through improved globin chain balance in beta thalassemia and reduced HbS polymerization. Pharmacologic approaches are considered most feasible for a majority of patients. Both proportions of F-cells and quantity of hemoglobin F (HbF)/cell are important for therapeutic effects. PB-04 was identified in a high-throughput screen of US and EU-approved drugs to activate HBG gene transcription, without cytotoxicity, to induce fetal globin expression (gamma globin mRNA, F-cells, HbF/cell, HbF), and to suppress or displace 4 repressors of the fetal globin gene promoter (BCL11A, LSD-1, KLF-1, and HDAC-3) in hemoglobinopathy patients' erythroid progenitors. PB-04 enhanced HbF in progenitors from hydroxyurea (HU)-treated patients with sickle cell disease. In in vivo studies, PB-04 induced fetal globin expression >20-fold with oral dosing in anemic baboons, and by 3.5-fold in mice transgenic for 2 copies of the human β-globin gene locus. This agent has been approved and in broad use for Parkinson's Disease treatment for 5 decades in Europe and Canada, to enhance the PK of an active Parkinson's agent, solely in a combination formulation. Because of its safety with chronic dosing up to 1300 mg/day, PB-04 is therefore of interest for repurposing in the treatment of thalassemia and SCD. This Ph1b trial (NCT004432623) will evaluate safety, tolerability, PK, and preliminary efficacy (fetal globin expression) with at least 3 escalating dose cohorts in up to 24 beta thalassemia intermedia (BTI) patients and 12 patients with sickle cell disease, > 18 years, both genders, with 12 weeks of treatment and 4 weeks of follow-up. Doses to be explored were selected from safe and active human equivalent doses in preclinical toxicology and efficacy studies in 2 species. Primary inclusion criteria include clinical diagnosis of BTI, including HbE beta thalassemia, with at least one beta globin gene mutation, or diagnosis of HbSS or HbSb thalassemia (after dose selection); total Hb levels 6-10 gm/dL. Exclusion criteria include red blood cell (RBC) transfusion within 2 months prior to administration of study medication, receiving regular transfusions, and hepatic or renal function > 3 x institutional ULN. Primary endpoints include occurrence, severity, and duration of adverse events by CTCAE v5.0 criteria and PK parameters. Secondary endpoints include change from baseline in assays of HbF expression, total Hb, markers of hemolysis . Exploratory endpoints include select polymorphisms associated with basal fetal globin expression and in vitro responses to the study drug. Statistical analysis will assess the rate of adverse events, PK parameters by dose, and changes in F-cells, F-reticulocytes, HbF/cell, HbF (% and total), compared to 2 averaged baseline values using the Wilcoxon signed rank test. Changes will be analyzed by mixed effect models during the 12-week dosing and 4-week follow-up period. Currently 3 dose cohorts with BTI are enrolled. This study, if successful, will provide a rational basis for definitive trials of an established safe oral therapeutic, with no overlapping toxicities with other experimental or approved agents, for combined use with HU and other therapeutics which enhance red cell viability in the globin disorders. The expertise and contributions of Jim Cradock of NCATS TRND to this work before his passing are gratefully acknowledged. Disclosures Kuo: Agios: Consultancy, Membership on an entity's Board of Directors or advisory committees; Bioverativ: Membership on an entity's Board of Directors or advisory committees; Celgene: Consultancy; Apellis: Consultancy; Bluebird Bio: Consultancy; Pfizer: Consultancy, Research Funding; Novartis: Consultancy, Honoraria; Alexion: Consultancy, Honoraria. Sheth: CRISPR: Consultancy; Bluebird bio: Consultancy; Bristol Myers Squibb: Consultancy, Research Funding; Chiesi: Consultancy; Imara: Research Funding; Agios: Consultancy; Dispersol: Research Funding. Al-Samkari: Moderna: Consultancy; Amgen: Research Funding; Rigel: Consultancy; Argenx: Consultancy; Novartis: Consultancy; Dova/Sobi: Consultancy, Research Funding; Agios: Consultancy, Research Funding. Pace: Imara Inc.: Consultancy. Kuypers: Forma Therapeutics, Inc.: Research Funding. Nouraie: Phoenicia BioScience Inc.: Consultancy. Perrine: Agios Pharmaceuticals: Consultancy; Emmaus Medical: Consultancy.

The α-globin Constant Spring (CS) mutation (α142 STOP → Gln; TAA → CAA) is the most prevalent non-deletional thalassemia in south East Asia and southern China. DNA diagnosis of Hb H Constant Spring (Hb H CS; 2 α-gene deletions and 1 CS mutation) is often required because it can be missed by electrophoresis. The clinical phenotype of Hb H CS is more severe than classical Hb H disease. We sought to characterize the unique hematological and clinical features of Hb H CS patients, especially compared to those with Hb H disease, who were identified through the Thalassemia Clinical Research Network (TCRN). A total of 836 patients enrolled in the TCRN registry in Canada and the U.S. (2001 to 2005) were screened for this analysis. Genotyping of 836 thalassemia patients identified 106/836 (12.7%) with Hb H and 46/836 (5.5%) with Hb H CS. 2 had other non-deletional mutations. Among Hb H CS patients, 48% were female; mean age was 13±11 years. Among patients who had spleens, splenomegaly was more prevalent in Hb H CS than Hb H patients (16% vs 1%, p=0.001). Among Hb H CS patients who had splenomegaly, average spleen span was 3.67 ± 2.25cm. 13% of the Hb H CS and 2% of the Hb H patients had their spleen removed (p=0.005). Mean Hb level was higher in the splenectomized Hb H CS patients than in the non-splenectomized Hb H CS patients (9.85 ± 2.25 g/dL vs. 8.25 ± 0.76 g/dL, p=0.006). Post-splenectomy portal vein thrombosis was reported in 1 Hb H CS patient. 7.5% (3/40) of non-splenectomized Hb H CS patients had bacteremia or infections requiring intravenous antibiotics. 8.7% (4/46) of Hb H CS patients underwent cholecystectomy. 24% of HbH CS and none of the Hb H patients were placed on regular transfusions (>8/year) and chelation therapy. Mean age of initiation of transfusions was 3.5±1.3 years (range 2–5 years). Mean ferritin level was higher in the non-transfused Hb H CS patients than in the Hb H patients (369.9 ± 409.9 ng/ml vs 175.9 ± 304.2 ng/ml, p=0.01), suggesting increased gastrointestinal iron absorption in Hb H CS. In 5 transfused Hb H CS patients, liver iron concentration was obtained showing elevated levels (27.2 ± 13.9 μgm/gm dry wt). Growth delay was more apparent in the Hb H CS patients (n=19) compared to 20 Hb H patients (Mean height Z score: −1.34 ± 0.98 for Hb H CS vs −0.82 ± 1.15 for Hb H (p=0.16) and mean weight Z score: −1.15 ± 0.88 for Hb H CS vs. −0.83 ± 1.61 for Hb H (p=0.47)). Bone density scans in Hb H CS patients revealed a higher prevalence of low bone mass than that detected in Hb H patients: mean L1-L4 spine Z/T-score of −1.60 ± 0.86 vs. −0.93 ± 0.80 (p=0.02). 40% (4/10) of adult female Hb H CS patients had 1 or more successful pregnancies, some requiring transfusion support through pregnancy. Patients with Hb H CS have a severe phenotype of α-thalassemia. They have moderately severe anemia, which sometimes requires regular transfusions, splenectomy, or both. Patients with Hb H CS commonly have iron overload, growth delay, and reduced bone mass. Early diagnosis in the neonatal period, regular monitoring, and appropriate treatment considerations for initiation of regular transfusions are key.

Pulmonary hypertension (PHT) is associated with high mortality rate and is a leading factor in heart failure and death in thalassemia (Thal) intermedia. However, data on PHT and Thal major is limited. While there are several risk factors for PHT in Thal major, ongoing chronic hemolysis, despite transfusion, supports the development of hemolysis-associated PHT. Recently, we reported in sickle cell disease a novel paradigm whereby hemolysis liberates vasoactive hemoglobin and erythrocyte arginase, resulting in impaired NO bioavailability, endothelial dysfunction, PHT and death. Since arginase activity is also elevated in Thal red cells, we hypothesize that elevated arginase contributes to dysregulated arginine (Arg) metabolism in Thal. Pilot data was analyzed from transfused Thal patients (8 Thal-major, 4 E-beta Thal, 2 Hb H alpha Thal). Echocardiogram were performed in steady state in 10 of the patients; 70% (7/10) demonstrated PHT (tricuspid regurgitant jet velocity ≥ 2.5 m/s). Plasma Arg trended lower in patients with Thal (19.5 to 122mM, median 50 mM) compared to controls. Ornithine (Orn) levels were higher, and Arg/Orn ratio was lower in Thal vs. contrls. Plasma arginase activity was significantly elevated (median 0.83 mmol/cc/hr, range 0.06 – 1.17). Proline, a downstream metabolite of arginase causing pulmonary vascular remodeling was also elevated. Exhaled nitric oxide (NO) levels are significantly elevated in Thal vs. controls (49±41 parts per billion vs.18±8 ppb, p=0.02), suggesting upregulation of NO synthase in the lungs in addition to higher plasma arginase activity. Recent findings indicate diverse origins of PHT share similar pathophysiologic abnormalities, particularly decreased Arg bioavailability and a shift of metabolism towards ornithine-dependent pathways. Hemolysis is most likely a trigger for these pathologic pathyways in hemoglobinopathies. Therapies that maximize Arg and NO bioavailability may benefit patients with thalassemia. Variable NL Control (n=36) Thalassemia (n=14) p* Arginine (μM) 67±18 57±26 (50) 0.15 Ornithine (μM) 62±22 85±68 0.05 Arg/Orn ratio 1.2±0.5 0.79±0.4

A major problem in using murine models for studies of bone marrow allograft rejection in leukemia patients is the narrow margin in which graft rejection can be analyzed. In mice irradiated with greater than 9 Gy total body irradiation (TBI) rejection is minimal, whereas after administration of 8 Gy TBI, which spares a significant number of clonable T cells, a substantial frequency of host stem cells can also be detected. In current murine models, unlike in humans, bone marrow allograft rejection is generally associated with full autologous hematopoietic reconstitution. In the present study, we investigated the effect of the myeloablative drug dimethyl myleran (DMM) on chimerism status following transplantation of T cell-depleted allogenic bone marrow (using C57BL/6 donors and C3H/HeJ recipients, conditioned with 8 Gy TBI). Donor type chimerism 1 to 2 months post-transplant of 1 to 3 x 10(6) bone marrow cells was markedly enhanced by using DMM one day after TBI and prior to transplantation. Conditioning with cyclophosphamide instead of DMM, in combination with 8 Gy TBI, did not enhance engraftment of donor type cells. Artificial reconstitution of T cells, after conditioning with TBI plus DMM, by adding mature thymocytes, or presensitization with irradiated donor type spleen cells 1 week before TBI and DMM, led to strong graft rejection and consequently to severe anemia. The anti-donor responses in these models were proportional to the number of added T cells and to the number of cells used for presensitization, and they could be neutralized by increasing the bone marrow inoculum. These results demonstrate the potential of DMM to facilitate engraftment in unsensitized mice in which the host stem cells may compete with donor type cells; the use of DMM to create models in which mechanisms of immune rejection can be studied without interference due to stem cell competition; and that bone marrow allograft rejection may be overcome by increasing the bone marrow inoculum in these stringent models.