Data were analyzed by two-way repeated measures ANOVA with Bonferroni assessments (**p 0.01 compared with corresponding control time point). Effects of Apremilast on Saccharin, Sucrose, and Water Consumption In the 2BC test (continuous 24-h access to saccharin and water) in male mice, apremilast (20 mg/kg) did not alter preference for saccharin or total fluid (saccharin plus water) intake (Fig. 5A,B). mice using the continuous and intermittent 24-h two-bottle choice drinking models. We also studied the effects of apremilast on preference for sucrose or saccharin, spontaneous locomotor activity, and blood ethanol clearance. Finally, apremilast levels in plasma, liver, and brain were measured one or two hours after injection. Results In the continuous and intermittent drinking tests, apremilast (15-50 mg/kg, p.o.) dose dependently reduced ethanol intake and preference in male and female mice. Higher doses of apremilast (30-50 mg/kg) also reduced total fluid intake in these Bay 11-7821 mice. Chronic administration of apremilast (20 mg/kg) produced a stable reduction of ethanol consumption in both drinking tests with no effect on total fluid intake. The drinking effects were reversible after drug treatment was replaced with vehicle administration (saline) for 2-4 days. Six daily apremilast injections did not alter preference for saccharin or sucrose in male or female mice. Apremilast (20 mg/kg) transiently decreased spontaneous locomotor activity and did not alter blood ethanol clearance. The highest levels of apremilast were found in liver followed by plasma and brain. Conclusions Apremilast produces stable reductions in voluntary ethanol consumption and is rapidly distributed to plasma and tissues (including the brain), suggesting that it may be an improved PDE4 inhibitor for medication development and repurposing efforts to treat alcohol abuse. tests, and Student’s analyses showed significant decreases in all drinking parameters at doses of 30-50 mg/kg, while 15 mg/kg of apremilast reduced the amount of ethanol consumed without changing total fluid intake. There was a trend for 15 mg/kg to reduce preference for ethanol, but this did not reach statistical significance. In female mice, apremilast also dose dependently reduced ethanol intake (F4,27 = 15.8, p 0.001), preference for ethanol (F4,27 = 9.4, p 0.001), and total fluid intake (F4,27 = 15.5, p 0.001; Fig. 1D-F). The raw drinking data in female mice are shown in Supplemental Fig. 1D-F. analyses showed that 15-50 mg/kg apremilast significantly decreased the amount of ethanol consumed and preference for ethanol, and 30-50 mg/kg apremilast also reduced total fluid intake. The lowest dose of apremilast tested (5 mg/kg) did not significantly alter ethanol consumption in male or female mice. Based on the dose-response effects, a 20 mg/kg dose was chosen for chronic drug treatment experiments. Open in a separate window Figure 1 Apremilast dose dependently decreases 15% ethanol intake in the continuous 2BC testEffects of apremilast (5-50 mg/kg) on ethanol (EtOH) intake (A), preference for EtOH (B), and total fluid intake (C) in male C57BL/6J mice; n= 7-8 per group. Effects of apremilast (5-50 mg/kg) on EtOH intake (D), preference for EtOH (E), and total fluid intake (F) in female C57BL/6J mice; n= 6-7 per group. Data are presented as the differences between two-day drinking averages after drug injection (days 3, 4 after apremilast) and the first two days of saline (control) injections (days 1, 2). Data were analyzed by one-way ANOVA with Bonferroni tests (*p 0.05, **p 0.01, ***p 0.001 compared with control). Six daily injections of apremilast (20 mg/kg) significantly reduced ethanol consumption (F1,14 = 12.8, p 0.001, effect of treatment; F2,28 = 3.8, p 0.05, effect of time) and preference for ethanol (F1,14 = 9.9, p 0.001, effect of treatment) but did not alter total fluid consumption in male mice (Fig. 2A-C). During the washout phase when injection of apremilast was replaced with saline, mice previously treated with apremilast showed no differences in ethanol consumption or preference compared with control mice. Similar to male mice, 6 daily injections of apremilast (20 mg/kg) in female mice reduced ethanol consumption (F1,16 = 16.4, p 0.001, effect of treatment) and preference for ethanol (F1,16 = 4.6, p 0.05, effect of treatment; F2,32 = 4.4, p 0.05, effect of time) but did not change total fluid consumption (Fig. 2D-F). During the washout phase, female mice previously treated with apremilast showed no differences in ethanol intake or preference compared with control mice. Open in a separate window Figure 2 Apremilast produces stable reduction of 15% ethanol intake in the continuous 2BC testEffects of apremilast (20 mg/kg) on ethanol (EtOH) intake (A), preference for EtOH (B), and total fluid intake (C) Bay 11-7821 in male C57BL/6J mice; n= 8 per group. Effects of apremilast (20 mg/kg) on EtOH intake (D), preference for EtOH (E), and total fluid intake (F) in female.The raw drinking data in female mice are shown in Supplemental Fig. C57BL/6J mice using the continuous and intermittent 24-h two-bottle choice drinking models. We also studied the effects of apremilast on preference for sucrose or saccharin, spontaneous locomotor activity, Bay 11-7821 and blood ethanol clearance. Finally, apremilast levels in plasma, liver, and brain were measured one or two hours after injection. Results In the continuous and intermittent drinking tests, apremilast (15-50 mg/kg, p.o.) dose dependently reduced ethanol intake and preference in male and female mice. Higher doses of apremilast (30-50 mg/kg) also reduced total fluid intake in these mice. Chronic administration of apremilast (20 mg/kg) produced a stable reduction of ethanol consumption in both drinking tests with no effect on total fluid intake. The drinking effects were reversible after drug treatment was replaced with vehicle administration (saline) for 2-4 days. Six daily apremilast Bay 11-7821 injections did not alter preference for saccharin or sucrose in male or female mice. Apremilast (20 mg/kg) transiently decreased spontaneous locomotor activity and did not alter blood ethanol clearance. The highest levels of apremilast were found in liver followed by plasma and brain. Conclusions Apremilast produces stable reductions in voluntary ethanol consumption and is rapidly distributed to plasma and tissues (including the brain), suggesting that it may be an improved PDE4 inhibitor for medication development and repurposing efforts to treat alcohol abuse. tests, and Student’s analyses showed significant decreases in all drinking parameters at doses of 30-50 mg/kg, while 15 mg/kg of apremilast reduced the amount of ethanol consumed without changing total fluid intake. There was a trend for 15 mg/kg to reduce preference for ethanol, but this did not reach statistical significance. In female mice, apremilast also dose dependently reduced ethanol intake (F4,27 = 15.8, p 0.001), preference for ethanol (F4,27 = 9.4, p 0.001), and total fluid intake (F4,27 = 15.5, p 0.001; Fig. 1D-F). The raw drinking data in female mice are shown in Supplemental Fig. 1D-F. analyses showed that 15-50 mg/kg apremilast significantly decreased the amount of ethanol consumed and preference for ethanol, and 30-50 mg/kg apremilast also reduced total fluid intake. The lowest dose of apremilast tested (5 mg/kg) did not significantly alter ethanol consumption in male or female mice. Based on the dose-response effects, a 20 mg/kg dose was chosen for chronic drug treatment experiments. Open in a separate window Figure 1 Apremilast dosage dependently reduces 15% ethanol intake Esm1 in the constant 2BC testEffects of apremilast (5-50 mg/kg) on ethanol (EtOH) intake (A), choice for EtOH (B), and total liquid intake (C) in male C57BL/6J mice; n= 7-8 per group. Ramifications of apremilast (5-50 mg/kg) on EtOH intake (D), choice for EtOH (E), and total liquid intake (F) in feminine C57BL/6J mice; n= 6-7 per group. Data are shown as the variations between two-day taking in averages after medication injection (times 3, 4 after apremilast) as well as the 1st two times of saline (control) shots (times 1, 2). Data had been examined by one-way ANOVA with Bonferroni testing (*p 0.05, **p 0.01, ***p 0.001 weighed against control). Six daily shots of apremilast (20 mg/kg) considerably reduced ethanol usage (F1,14 = 12.8, p 0.001, aftereffect of treatment; F2,28 = 3.8, p 0.05, aftereffect of time) and preference for ethanol (F1,14 = 9.9, p 0.001, aftereffect of treatment) but didn’t alter total liquid consumption in man mice (Fig. 2A-C). Through the washout stage when shot of apremilast was changed with saline, mice previously treated with apremilast demonstrated no variations in ethanol usage or choice weighed against control mice. Just like man mice, 6 daily shots of apremilast (20 mg/kg) in feminine mice decreased ethanol usage (F1,16 = 16.4, p 0.001, aftereffect of treatment) and choice for ethanol (F1,16 = 4.6, p 0.05, aftereffect of treatment; F2,32 = 4.4, p 0.05, aftereffect of time) but didn’t change.
