Suppression of heavy drinking and alcohol seeking by a selective ALDH-2 inhibitor

Abstract

Background: Inherited human aldehyde dehydrogenase 2 (ALDH-2) deficiency reduces the risk for alcoholism. Kudzu plants and extracts have been used for 1,000 years in traditional Chinese medicine to treat alcoholism. Kudzu contains daidzin, which inhibits ALDH-2 and suppresses heavy drinking in rodents. Decreased drinking due to ALDH-2 inhibition is attributed to aversive properties of acetaldehyde accumulated during alcohol consumption. However, daidzin can reduce drinking in some rodents without necessarily increasing acetaldehyde. Therefore, a selective ALDH-2 inhibitor might affect other metabolic factors involved in regulating drinking.

Methods: Aldehyde dehydrogenase 2 inhibitors were synthesized based on the co-crystal structure of ALDH-2 and daidzin. We tested the efficacy of a highly selective reversible ALDH-2 inhibitor, CVT-10216, in models of moderate and high alcohol drinking rats. We studied 2-bottle choice and deprivation-induced drinking paradigms in Fawn Hooded (FH) rats, operant self-administration in Long Evans (LE), FH, and inbred P (iP) rats and in cue-induced reinstatement in iP rats. We also assayed blood acetaldehyde levels as well as dopamine (DA) release in the nucleus accumbens (NAc) and tested possible rewarding/aversive effects of the inhibitor in a conditioned place preference (CPP) paradigm.

Results: CVT-10216 increases acetaldehyde after alcohol gavage and inhibits 2-bottle choice alcohol intake in heavy drinking rodents, including deprivation-induced drinking. Moreover, CVT-10216 also prevents operant self-administration and eliminates cue-induced reinstatement of alcohol seeking even when alcohol is not available (i.e., no acetaldehyde). Alcohol stimulates DA release in the NAc, which is thought to contribute to increased drinking and relapse in alcoholism. CVT-10216 prevents alcohol-induced increases in NAc DA without changing basal levels. CVT-10216 does not show rewarding or aversive properties in the CPP paradigm at therapeutic doses.

Conclusion: Our findings suggest that selective reversible ALDH-2 inhibitors may have therapeutic potential to reduce excessive drinking and to suppress relapse in abstinent alcoholics.

Fig. 1.

CVT-10216 inhibits human aldehyde dehydrogenase (ALDH)-2 and ALDH-1 in vitro and increases blood acetaldehyde (AcH) in vivo after alcohol (EtOH) gavage. (A) ALDH activity was assayed in sodium phosphate buffer (50 mM, pH 7.4) containing 1.2 mM NAD⁺, 1 nM ALDH-2, or 10 nM ALDH-1, various concentrations of CVT-10216, and 0.3 mM formaldehyde for ALDH-2 or 0.18 mM acetaldehyde for ALDH-1, substrate concentrations equal to their K_m for ALDH-2 and ALDH-1, respectively. Formaldehyde was used in ALDH-2 assay because ALDH-2 has a low K_m for acetaldehyde (200 nM). Reactions were initiated by adding aldehyde substrate and rates recorded at 25°C in a FluoroMax-2 Fluorimeter with excitation/emission wavelengths of 340/460 nm. IC₅₀ values were determined by fitting concentration-inhibition data to sigmoidal dose–response curves. The IC₅₀ is ~29 nM for ALDH-2 and 1300 nM for ALDH-1 (see Supporting Information Scheme S1 and S2 and Fig. S1 for more details) (B) SD rats with a carotid artery catheter received i.p. CVT-10216 (3.75, 7.5, or 15 mg/kg) 30 minutes before intragastric alcohol (2 g/kg) (n = 3). Timed blood samples obtained after alcohol. Plasma acetaldehyde was measured as a stable DNPH derivative using reverse phase high pressure liquid chromatography with minor modifications.

Fig. 2.

CVT-10216 (i.p.) decreases alcohol intake and seeking in high alcohol drinking rat models. All data are expressed as the mean ± SEM (A) 2-bottle choice alcohol intake (g/kg) in Fawn Hooded (FH) rats (n = 8 to 9). (B) Deprivation-induced alcohol intake (g/kg) in FH rats. Untreated, vehicle, and CVT-10216 rats were alcohol deprived for 5 days. CVT 10216 (15 mg/kg) was administered before providing alcohol on day 6 (n = 6). (C) Alcohol self-administration: alcohol intake (g/kg) and (D) number of alcohol rewards in FH rats (n = 12). (E) Alcohol self-administration: alcohol intake (g/kg) and (F) number of alcohol rewards in iP rats. *p < 0.05, **p < 0.01, ***p < 0.001, compared with vehicle [repeated measure ANOVA (Fisher post hoc) or repeated measures t-test].

Fig. 3.

Cue-induced alcohol-seeking (lever presses) in iP rats. Rats trained to self-administer alcohol received extinction sessions (no cues/fluid), followed by cue-induced reinstatement session under vehicle or CVT-10216 (1.88 and 3.75 mg/kg, n = 10 per dose). *p < 0.05 compared with vehicle; #p < 0.05, compared with extinction [repeated measure ANOVA (Fisher post hoc)].

Fig. 4.

CVT-10216 (i.p.) decreases operant alcohol intake and seeking in Long Evans (LE) rats. All data are expressed as the mean ± SEM (A) Alcohol self-administration (alcohol intake in g/kg) in LE rats (n = 7 to 13). (B) Alcohol seeking (lever presses) in LE rats. Rats were trained as in (A) except no alcohol was available during testing and only 15 mg/kg dose of CVT-10216 was tested (n = 6). *p < 0.05, **p < 0.01, compared with vehicle [repeated measure ANOVA (Fisher post hoc)].

Fig. 5.

(A) CVT-10216 prevents alcohol-activated nucleus accumbens (NAc) dopamine (DA) release in vivo. Long Evans rats with NAc microdialysis probes received i.p. CVT-10216 (7.5 or 15 mg/kg) or vehicle 30 minutes before alcohol (EtOH) (1 g/kg i.p.). Data are the mean ± SEM of NAc DA concentration (% of baseline) (n = 6). (B) CVT-10216 does not affect basal NAc DA levels in vivo. Rats were treated as in (A) except without alcohol administration (n = 4 to 6). **p < 0.01, ***p < 0.001, compared with vehicle [repeated measure ANOVA (Fisher post hoc)].

Fig. 6.

CVT-10216 does not induce conditioned place preference (CPP). Animals treated with CVT-10216 (7.5 and 15 mg/kg) spent a similar amount of time in the nonpreferred side during pre- and postconditioning sessions. Animals treated with cocaine (10 mg/kg) as a positive control showed CPP. Animals in this group spent significantly more time in the nonpreferred side during postconditioning session (n = 8). *p < 0.05 compared with preconditioning time [repeated measure ANOVA (Fisher post hoc)].