Isobutyramide, an orally bioavailable butyrate analogue, stimulates fetal globin gene expression in vitro and in vivo

Abstract

Butyrate and other short-chain fatty acids stimulate fetal globin gene expression and have potential for ameliorating the beta globin disorders. Butyrate, however, is rapidly metabolized in vivo and reaches only micromolar concentrations in plasma. We report here that a branched-chain derivative of butyrate, isobutyramide, increases gamma globin gene expression in cultured human erythroid progenitors in vitro and stimulates activity from a minimal gamma globin gene promoter linked to a reporter gene in stable and transient expression assays, with slightly less activity in these in vitro assays than butyrate. In vivo, administration of isobutyramide to anaemic adult baboons rapidly stimulates fetal globin synthesis and F-reticulocyte production. Plasma concentrations at millimolar levels are achieved after a single intravenous or oral dose (500-600 mg/kg), and these concentrations are maintained for 9.5-10.5 h. These results indicate that although isobutyramide has slightly less activity than butyrate in vitro in enhancing fetal globin expression at the cellular and molecular level, its prolonged in vivo half-life may provide superior activity as a therapeutic agent for reactivating fetal globin gene expression in vivo.

Short-chain fatty acid derivatives induce fetal globin expression and erythropoiesis in vivo

“…Baboons underwent significant daily phlebotomy to achieve and maintain hemoglobin levels of 6.5 to 7.5 g/dL, a level required for the modulation of fetal globin gene expression in primates at the doses tested, as established in previous reports. 9,24,28,34 The phlebotomy regimen increased endogenous erythropoietin levels from 1.7 to 7 mU/mL before phlebotomy to 10 to 17 mU/mL by the time hemoglobin levels declined to 6.5 to 7.5 g/dL (data not shown). Baboons from the primate breeding colony at the University of Oklahoma typically have low baseline expression of fetal globin, and individual baboons required different amounts of phlebotomy to establish and maintain a constant degree of anemia, ranging from 3 to 7 mL blood withdrawn daily per kilogram body weight.…”

“…34 Oral administration required light sedation with ketamine and fasting before sedation and was accordingly limited in frequency to 3 alternating days per week. Compounds studied in the baboons included 2,2 dimethyl butyric acid, AMHCA, 3-(3,4-dimethoxyphenyl) propionic acid, 3,5 dimethoxy-4-hydrocinanmic acid, cinnamic acid, phenoxyacetic acid, levulinic acid (all purchased from Aldrich), and 2,2 dimethyl hydrocinnamic acid, sodium 2,2 dimethyl butyrate and ␣ methylhydrocinnamate (T. E. Neesby).…”

“…9,24,28,34,40 The SCFADs were, therefore, initially tested on a dose-escalation schedule, beginning with doses in use clinically for arginine butyrate at 500 to 800 mg/kg per dose. Lower doses, between 40 and 200 mg/kg, were tested subsequently if any ␥ globin induction was observed with the higher doses.…”

“…Blood samples were obtained for analysis of concentrations of the test compounds in the plasma and for F-reticulocytes, which were analyzed as previously described. 34,40 Changes in F-reticulocytes were usually observed within 2 to 3 days of administration of a new test compound and diminished within 3 to 5 days of discontinuing administration of the test compound. Because the animal's blood volume was withdrawn approximately every 10 to 20 days through phlebotomy, Hb F levels and F-cell percentages are not considered representative of fetal globin accumulation in previous reports in this model and were not used.…”

“…9 Instead, globin chain protein synthesis in 3 H-leucinelabeled reticulocytes was performed before and 2 to 4 days after the administration of AMHCA and 2,2 dimethyl butyrate in 3 animals, as previously described. 34 Erythropoietin levels were assayed in plasma using a radioimmunoassay kit according to the manufacturer's directions (R&D Systems, Minneapolis, MN). All studies were performed with the approval of the Institutional Animal Care and Use Committees of the University of Oklahoma and of Boston University Schools of Medicine.…”