Elevated levels of HbG mRNA, leading to the formation of fetal hemoglobin, is known to ameliorate disease severity in sickle cell and β-thalassemia patients. We have previously shown that the small molecule ACY-957 is a selective inhibitor of HDAC1/2 which induces HbE and HbG in cultured human primary CD34+ cells (Shearstone et al, ASH Annual Meetings 2012-14). In this work, we describe the pharmacokinetics and HbE/HbG induction following once daily oral dosing of ACY-957 in rat and monkey.
To determine the duration of ACY-957 exposure required to induce HbE and HbG in vivo, we first tested ACY-957 in drug washout experiments performed in cultured primary erythroid progenitors. The aminobenzamide class of HDAC inhibitors, such as ACY-957, are known to have slow on rates for HDAC1/2 (Kral et al, Biochemistry 2014; Lauffer et al, J Biol Chem 2013). We found that a 6 h pulse of ACY-957 (1 μM) resulted in undetectable increases in histone H3 lysine 9 acetylation (H3K9ac) and that 4 or 8 h pulses of ACY-957 (1 μM) resulted in undetectable HbE and HbG induction. However, continued exposure resulted in a 2.5-fold and 7-fold increase in H3K9ac after 24 and 48 h of incubation, respectively, leading to a time-dependent increase in HbE and HbG. Based on this data, we hypothesized that in vivo studies would require ACY-957 levels of 1 μM for 24 h in order to observe elevated HbE and HbG. Non-fasted Sprague Dawley rats or cynomolgus monkeys received a single oral dose of 20 mg/kg or 12.5 mg/kg ACY-957, respectively, and pharmacokinetic analysis yielded comparable results with T1/2 = 11.8 and 10.9 h, Cmax = 7.8 and 2.4 μM, and Tmax = 5.3 and 4.0 h, in rat and monkey, respectively. At 24 h post dose, ACY-957 plasma levels in rat and monkey were 1.6 and 0.6 μM, respectively. These findings suggested that the targeted drug exposure could be met with a single daily oral dose of ACY-957.
Since ACY-957 induced HbE in cultured human primary erythroid progenitors, we attempted to measure HbE induction in rat as a surrogate marker for HbG in primate. Rats were dosed with 0, 10 or 30 mg/kg (n=4 per group) by oral gavage, once daily for 6 days, followed by a 13 day washout period. Peripheral blood was sampled every 3 days for isolation of total RNA. Complete blood counts were performed on day 0, 6 and 18. The low and high dose groups showed ACY-957 plasma levels of 1.3 or 5.2 μM, respectively, at 24 h post final dose. No abnormal clinical signs were found during the in-life phase, although a minor, reversible delay in rat weight gain was observed in the high dose group. White blood cells were suppressed by 33% and 68% at day 6 in low and high dose groups, respectively, but recovered to baseline levels by day 18. ACY-957 administration led to a dose-dependent increase in HbE relative to HbB that was detectable at day 3, peaked at day 6, and returned to baseline levels by day 9. Maximum induction of HbE was 2-fold and 5.6-fold for the low and high dosing groups, respectively, relative to animals receiving vehicle only.
Next, monkeys were dosed at 0, 25 or 75 mg/kg (n=3 per group) by oral gavage, once daily for 5 days, followed by a 14 day washout period. Peripheral blood was sampled every 2 to 3 days for isolation of total RNA and analysis of complete blood counts. The low and high dose groups showed ACY-957 plasma levels of 1.9 or 8.0 μM, respectively, at 24 h post final dose. No abnormal clinical signs were found during the in-life phase. White blood cells were suppressed by 25% and 61% at day 5, but recovered to baseline levels by day 9. ACY-957 administration led to a dose-dependent increase in HbE and HbG relative to HbB that was detectable at day 5, peaked at day 7, and returned to baseline levels by day 12. Maximum induction of HbG was 2.2-fold and 7.2-fold for the low and high dosing groups, respectively, relative to animals receiving vehicle only.
These results demonstrate that ACY-957 induces HbE in rat and HbG in monkey to a similar extent. ACY-957 appeared well tolerated in both animals, although a reversible suppression of white blood cells was observed. Together, these findings suggest that optimization of dose and schedule could be performed in rats by monitoring HbE as a surrogate for HbG in primates. The optimized regime could then be validated in cynomolgus monkey. Accordingly, we have initiated experiments that explore the effects of several different ACY-957 dose schedules on HbE induction and white blood cell suppression in rats during a 4 week dosing and 2 week recovery period, which will also be presented.
Shearstone:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Chonkar:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Bhol:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jones:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership. Jarpe:Acetylon Pharmaceuticals, Inc.: Employment, Equity Ownership.
Asterisk with author names denotes non-ASH members.