Apremilast Alters Behavioral Responses to Ethanol in Mice: I. Reduced Consumption and Preference

Abstract

Background: Phosphodiesterase type 4 (PDE4) inhibitors produce widespread anti-inflammatory effects and reduce ethanol (EtOH) consumption in several rodent models. These drugs are potential treatments for several diseases, including central nervous system disorders, but clinical use is limited by their emetic activity. Apremilast is a selective PDE4 inhibitor with fewer gastrointestinal side effects that is FDA-approved for the treatment of psoriasis.

Methods: We measured the acute and chronic effects of apremilast on EtOH consumption in male and female C57BL/6J mice using the continuous and intermittent 24-hour 2-bottle choice drinking models. We also studied the effects of apremilast on preference for sucrose or saccharin, spontaneous locomotor activity, and blood EtOH clearance. Finally, apremilast levels in plasma, liver, and brain were measured 1 or 2 hours after injection.

Results: In the continuous and intermittent drinking tests, apremilast (15 to 50 mg/kg, p.o.) dose dependently reduced EtOH intake and preference in male and female mice. Higher doses of apremilast (30 to 50 mg/kg) also reduced total fluid intake in these mice. Chronic administration of apremilast (20 mg/kg) produced a stable reduction in EtOH 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 to 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 EtOH clearance. The highest levels of apremilast were found in liver followed by plasma and brain.

Conclusions: Apremilast produced stable reductions in voluntary EtOH consumption and was 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.

Keywords: Apremilast Biodistribution; C57BL/6J Mice; Locomotor Activity; PDE4 Inhibitor; Two-Bottle Choice Ethanol Drinking.

Figure 1. Apremilast dose dependently decreases 15% ethanol intake in the continuous 2BC test

Effects 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 post-hoc tests (*p < 0.05, **p < 0.01, ***p < 0.001 compared with control).

Figure 2. Apremilast produces stable reduction of 15% ethanol intake in the continuous 2BC test

Effects of apremilast (20 mg/kg) on ethanol (EtOH) intake (A), preference for EtOH (B), and total fluid intake (C) 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 C57BL/6J mice; n= 9 per group. Each data point represents the average of two days of drinking. S/S represents two-day drinking averages after saline injections for both groups. S/Drug represents the two-day averages after saline or apremilast injections (6 days total). The second S/S period shows the extinction (4 days total) of effects on drinking in mice previously treated with apremilast. Data were analyzed by two-way repeated measures ANOVA.

Figure 3. Apremilast dose dependently decreases 15% ethanol intake in the intermittent 2BC-EOD test

Effects 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= 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= 11 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 post-hoc tests (*p < 0.05, **p < 0.01, ***p < 0.001 compared with control).

Figure 4. Apremilast produces stable reduction of 15% ethanol intake in the intermittent 2BC-EOD test

Effects of apremilast (20 mg/kg) on ethanol (EtOH) intake (A), preference for EtOH (B), and total fluid intake (C) in male C57BL/6J mice; n= 10 per group. Effects of apremilast (20 mg/kg) on EtOH intake (D), preference for EtOH (E), and total fluid intake (F) in female C57BL/6J mice; n= 10 per group. Each data point represents the average of two days of drinking. S/S represents two-day drinking averages after saline injections for both groups. S/Drug represents the two-day averages after saline or apremilast injections (11 days total). The second S/S period shows the extinction (2 days total) of effects on drinking in mice previously treated with apremilast. Data were analyzed by two-way repeated measures ANOVA with Bonferroni post-hoc tests (**p < 0.01 compared with corresponding control time point).

Figure 5. Apremilast does not alter preference for saccharin or water intake

Effects of apremilast (20 mg/kg) on preference for saccharin (A), total fluid intake (B), and water intake (C) in male C57BL/6J mice; n= 6 per group for the saccharin experiment and n= 5-6 per group for the water experiment. Effects of apremilast (20 mg/kg) on preference for saccharin (D), total fluid intake (E), and water intake (F) in female C57BL/6J mice; n= 6 per group. For 2BC saccharin drinking, data represent the average of two days of drinking. For water intake using one bottle of water, data are presented as daily intake. S/S represents two days of saline injections for both groups. S/Drug represents two days of saline or apremilast injections (6 days total for saccharin experiment and 8 days total for water experiment). Data were analyzed by two-way repeated measures ANOVA with Bonferroni post-hoc tests (*p < 0.05 compared with corresponding control time point).

Figure 6. Apremilast does not alter preference for sucrose

Effects of apremilast (20 mg/kg) on preference for sucrose (A) and total fluid intake (B) in male C57BL/6J mice; n= 6 per group. Effects of apremilast (20 mg/kg) on preference for sucrose (A) and total fluid intake (B) in female C57BL/6J mice; n= 6 per group. Data are presented as two-day drinking averages. S/S represents two days of saline injections for both groups. S/Drug represents two days of saline or apremilast injections (6 days total). Data were analyzed by two-way repeated measures ANOVA with Bonferroni post-hoc tests, but no significant group differences were found.

Figure 7. Acute administration of apremilast (20 mg/kg) decreases locomotor activity in female mice

Spontaneous locomotion recorded every hour (A) and area under the curve (B) in male C57BL/6J mice (n= 16 per group). Spontaneous locomotion (C) and area under the curve (D) in female C57BL/6J mice (n= 16 per group). Spontaneous locomotor data were analyzed by two-way repeated measures ANOVA with Bonferroni post-hoc tests (*p < 0.05, **p< 0.01, ***p<0.001, ****p<0.0001 compared with corresponding control time point). Area under the curve data were analyzed using Student's t-tests (**p < 0.01 compared with saline control).

Figure 8. Chronic administration of apremilast (20 mg/kg) decreases locomotor activity in male mice

Apremilast was given daily for 6 days. Spontaneous locomotion recorded every hour (A) and area under the curve (B) in male C57BL/6J mice (n= 16 per group). Spontaneous locomotion (C) and area under the curve (D) in female C57BL/6J mice (n= 16 per group). Spontaneous locomotor data were analyzed by two-way repeated measures ANOVA with Bonferroni post-hoc tests (*p < 0.05, **p< 0.01, ****p<0.0001 compared with corresponding control time point). Area under the curve data were analyzed using Student's t-tests (**p < 0.01 compared with saline control).

Figure 9. Apremilast does not alter clearance of blood ethanol

Effect of saline or apremilast (20 mg/kg) on blood ethanol concentration (BEC) measured over 4h in male (A) and female (B) C57BL/6J mice after i.p. injection of 4 g/kg ethanol (n= 12 per group).