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

Abstract

Orally bioactive compounds that induce gamma globin gene expression at tolerable doses are needed for optimal treatment of the beta-hemoglobinopathies. Short-chain fatty acids (SCFAs) of 2 to 6 carbons in length induce gamma globin expression in animal models, and butyrate, phenylbutyrate, and valproate induce gamma globin in human patients. The usefulness of these compounds, however, is limited by requirements for large doses because of their rapid metabolism and their tendency to inhibit cell proliferation, which limits the pool of erythroid progenitors in which gamma globin can be induced. Selected short-chain fatty acid derivatives (SCFADs) were recently found to induce gamma globin and to stimulate the proliferation of hematopoietic cells in vitro. These SCFADs are now evaluated in vivo in nonanemic transgenic mice containing the human beta globin gene locus and in anemic phlebotomized baboons. In mice treated with a SCFAD once daily for 5 days, gamma globin mRNA increased 2-fold, reticulocytes increased 3- to 7-fold, and hematocrit levels increased by 27%. Administration of 3 SCFADs in anemic baboons increased F-reticulocytes 2- to 15-fold over baseline and increased total hemoglobin levels by 1 to 2 g/dL per week despite ongoing significant daily phlebotomy. Pharmacokinetic studies demonstrated 90% oral bioavailability of 2 SCFADs, and targeted plasma levels were maintained for several hours after single oral doses equivalent to 10% to 20% of doses required for butyrate. These findings identify SCFADs that stimulate gamma globin gene expression and erythropoiesis in vivo, activities that are synergistically beneficial for treatment of the beta hemoglobinopathies and useful for the oral treatment of other anemias.

Figure 1. Effects of SCFAD on γ globin mRNA and reticulocytes in transgenic and normal mice

(A) Mean peak γ globin mRNA levels relative to baseline in transgenic mice treated once daily for 5 to 7 days with 500 mg/kg intraperitoneal doses of AMHCA, DMB, PAA, ABA, or SB continuously at 1000 mg/kg per day. Control mice were treated intraperitoneally with the same volume (500 μL) normal saline (NS) as the SCFA derivative agents. Values shown are the means ± SE, designated by the vertical lines above each bar. The horizontal bar above each graph designates the treatment period. (B) Reticulocytes in mice treated with PAA; each curve represents values in one animal. (C) Reticulocytes in mice treated with AMHCA; each curve represents values in one animal. (D) Reticulocytes in mice treated with DMB; each curve represents values in one animal. (E) Reticulocytes in mice treated with ABA; each curve represents values in one animal. (F) Reticulocytes in mice treated with SB; each curve represents values in one animal. (G) Reticulocytes in control mice treated with (NS); each curve represents values in one animal. (H) Mean fold increase in reticulocyte counts in normal and transgenic mice treated with each SCFAD or SB or NS. Values shown are the means ± SE, designated by the vertical lines above each bar.

Figure 2. Hematocrit levels in mice treated with normal saline or α methyl hydrocinnamic acid once daily for 5 days

Hematocrit levels in mice treated with normal saline are shown with open symbols, and those with α methyl hydrocinnamic acid treatment are shown with closed symbols. Each curve represents values in one animal; each symbol represents the same animal’s values while receiving either normal saline (open symbols) or α methylhydrocinnamatic acid (closed symbols).

Figure 3. γ Globin expression in baboons treated with SCFADs

(A) Percentage F-reticulocytes in baboons treated with phenoxyacetic acid, administered intravenously once daily at 900 mg/kg for 2 doses and 1100 mg/kg for 1 dose in baboon 225 (○) and at 500 mg/kg per dose daily for 5 days in baboon 1392 (●). (B) F-reticulocytes in baboons treated with 2, 2 dimethyl butyrate are shown. F-reticulocytes were induced by 2 oral doses of 500 and then of 700 mg/kg, every other day, in a dose-escalation safety study in baboon 1392 (▲); 200 mg/kg per dose intravenously 5 days a week in baboon 1997 (■); 500 mg/kg orally administered every other day over 2 weeks to baboon 894 (●); and 150 mg/kg intravenously once daily for 5 days in baboon 2063 (○). (C) F-reticulocytes in baboons treated with α methyl hydrocinnamic acid administered orally at 500 mg/kg for 2 doses every other day and 700 mg/kg once in a dose-escalation safety study in baboon 994 (●), at 200 mg/kg IV once daily for 5 days to baboon 1593 (▲), at 200 mg/kg intravenously once daily for 5 days to baboon 1997 (□), and at 150 mg/kg intravenously once daily for 5 days in baboon 2063 (■;). (D) Globin chain synthesis in a baboon (1573) that under phlebotomy before (i) and after (ii) treatment with α methyl hydrocinnamic acid. Closed symbols designate ³H cpm, and open symbols designate OD 280. The proportion of γ globin synthesis (γ/γ +β × 100) is shown.

Figure 4. Effects of rhu-erythropoietin (rhu-EPO) or SCFA derivatives on hemoglobin and hematocrit in chronically anemic baboons that underwent phlebotomy

Duration of the ongoing phlebotomy is designated by the horizontal bar above each graph. Compound administration is shown by open horizontal bars. In all figure panels: total hemoglobin levels are shown by ●; hematocrit levels, ○. (A) Phlebotomy of 3.5 mL/kg per day was performed in baboon 497, designated by the closed bar above the graph. Administration of rhu-EPO (300 U/kg 3 times per week) is shown by the open bars. During treatment, hemoglobin and hematocrit levels remained stable, despite the ongoing phlebotomy. When rhu-EPO was withdrawn and phlebotomy continued, hemoglobin levels declined by 1.0 g/dL over 5 days. (B) Effects of α methyl hydrocinnamic acid (50 mg/kg per day intravenously for 5 days a week over 3 weeks) administered to the same animal shown in panel A (baboon 497). The baboon underwent similar phlebotomy (3.5 mL/kg per day; shown by the closed horizontal bar). Treatment with α methyl hydrocinnamic acid (open horizontal bars) induced an increase in total hemoglobin level of 2.5 g/dL and an absolute increase in hematocrit level of 7 percentage points (28% of baseline), despite the ongoing daily phlebotomy. (C) Effects of α methyl hydrocinnamic acid in another baboon 196. Daily phlebotomy alone (13 mL/kg per week) resulted in declines in total levels of hemoglobin and hematocrit. Administration of the SCFAD (200 mg/kg per day intravenously) was begun on day 8, shown by the open bars, followed by an increase in total hemoglobin level of 3 g/dL and in hematocrit level of 8 absolute percentage points (40% above baseline). When the phlebotomy was increased further to 21 mL/kg per week on day 14, hemoglobin and hematocrit levels remained stable. (D) Effects of rhu-EPO administered to baboon 894 with a daily phlebotomy of 7 mL/kg per day, shown by the closed horizontal bar. Despite the administration of rhu-EPO (300 U/kg daily [as shown by the open horizontal bars]), a decline in hemoglobin and hematocrit levels occurred with this substantial phlebotomy. (E) Effects of the SCFAD phenoxyacetic acid in the baboon 894, shown in panel D. Daily phlebotomy is designated by the closed horizontal bar. Four doses of phenoxyacetic acid were administered orally (700–900 mg/kg) on alternate days (designated by the open bars) over 2 weeks. An increase in hemoglobin level of 2.4 g/dL and an increase in hematocrit level of 7 absolute percentage points (30% above baseline) were observed. (F) Effects of sodium 2,2 dimethyl butyrate in baboon 1997 administered intravenously (700 mg/kg) 5 days a week for 6 weeks (open bars). Chronic phlebotomy (6.3 mL/kg per day) is designated by the closed bar. An increase in hemoglobin level of 2.0 g/dL and an increase in hematocrit level of 6 percentage points were observed. When the phlebotomy was increased to 7.4 mL/kg per day on day 20, there was no significant further increase, but hemoglobin and hematocrit levels remained stable.

Figure 5. Pharmacokinetic analyses of sodium α methylhydrocinnamate in baboons

Single oral or intravenous doses were administered, and concentrations of the compound were analyzed in plasma samples collected sequentially between 15 minutes and 8 to 24 hours after the administered dose. (A) Comparison of plasma concentrations of sodium α methylhydrocinnamate after a single intravenous dose (■) and a single oral dose (○) of 113 mg/kg in baboon 1997. The compound was detected in the plasma at, or above, the targeted concentration of 100 μM for 6 to 8 hours. (B) Repeat-dose pharmacokinetics of one oral dose (200 mg/kg) of sodium α methylhydrocinnamate administered on 4 separate days over 1 month to baboon 1997. Concentrations in the plasma after the single oral doses were similar and resulted in plasma concentrations above the targeted level (100 μM) for 6 hours. (C) Plasma levels of sodium α methylhydrocinnamate in baboons after the administration of single doses of 50 mg/kg orally (○), 100 mg/kg orally (●), 200 mg/kg orally (□), and 500 mg/kg orally (●).

Figure 6. Pharmacokinetic analyses of sodium 2,2 dimethyl butyrate in baboons

Single oral or intravenous doses were administered and concentrations of the compound were analyzed in plasma samples collected sequentially between 15 minutes and 8 to 24 hours after the administered dose. (A) Comparison of plasma levels after the administration of 100 mg/kg doses of sodium 2,2 dimethyl butyrate given intravenously (○) and orally (●). By either route of administration, the compound was detected above the targeted concentration of 100 μM for 8 hours. (B) Repeat-dose pharmacokinetic analyses of sodium 2,2 dimethyl butyrate after the administration of 150 mg/kg in baboon 2298 administered orally on 3 separate occasions with 2 to 3 days between each dose. Plasma levels are designated by open circles after the first dose, closed circles after the second dose, and open squares after the third dose. Plasma concentrations remained above the targeted plasma level (100 μM) for more than 12 hours. (C) Plasma concentrations of sodium 2,2 dimethyl butyrate after the administration of single doses in 3 baboons. Plasma levels were detected after single doses of 2,2 dimethyl butyrate at 40 mg/kg intravenously in baboon 1197 (○), 75 mg/kg orally in baboon 5014 (△), 100 mg/kg orally in baboon 5014 (▲), 100 mg/kg orally in baboon 1197 (●), and 100 mg/kg orally in baboon 3397 (■). Levels remained above the targeted concentration (100 μM) for 6 to 8 hours.

Figure 7. Pharmacokinetic analyses of 2 additional SCFADs in baboons

(A) Pharmacokinetic analyses of the SCFAD 3-(3,4 dimethoxyphenyl)-propionic acid in 2 baboons. Plasma levels are shown after single doses of 50 mg/kg intravenously (○), 50 mg/kg orally (●) in baboon 1197, 150 mg/kg intravenously (□) and orally (■) in baboon 1997, and 200 mg/kg orally in baboon 1997 (▲). (B) Pharmacokinetic analyses of the SCFAD 4-(3,4 dimethoxyphenyl) butyric acid after the administration of single low oral doses in 3 baboons. Plasma levels are shown after oral doses of 20 mg/kg in baboon 1997 (□) and baboon 3397 (■), after 40 mg/kg in baboon 3397 (△), and after 50 mg/kg given on 2 separate occasions in baboon 5014 (○ and ●).