The major finding in this report is that the highly selective and reversible ALDH-2 inhibitor CVT-10216 exhibits unequivocal anxiolytic properties in four different rat models of anxiety-like behavior. The social interaction test was used in this study to measure the anxiolytic properties of CVT-10216 against endogenous anxiety-like behavior in the naïve FH rats, alcohol-withdrawal-induced anxiety, stress-induced anxiety and drug-induced anxiety. Locomotor activity was also assayed as a control for nonspecific effects of the treatments on neural function. The usefulness of the social interaction test as an index of anxiety-like behavior is well established (File and Seth, 2003
) and locomotor activity is mostly independent from social interaction behavior (File and Seth, 2003
; Overstreet et al., 2002
). Here we find that CVT-10216 has virtually no effect on locomotor activity when assayed in all of the models tested and in independent tests of locomotor activity. Indeed, changes in locomotor activity were rarely observed when CVT-10216 exhibited clear dose-dependent anxiolytic effects.
Naïve FH rats exhibit endogenous anxiety-like behavior characterized by a reduced amount of social interaction behavior (Kantor et al., 2000
). CVT-10216 significantly and dose-dependently increases the social interaction behavior of the FH rats, without affecting locomotor activity. These findings suggest that the anxiolytic properties of CVT-10216 appear to selectively suppress inherited CNS mechanisms required for anxiety without producing sedation or affecting the control of locomotor activity. Because the development of anxiety is modulated by drugs that influence multiple systems, such as flumazenil, a BZD receptor antagonist, buspirone an 5-HT1A
receptor agonist, CRF1 receptor antagonists and baclofen, a GABA-B receptor agonist (Knapp et al., 2004
; Overstreet et al., 2003a
), it is not possible to suggest which mechanisms underlie the effects of CVT-10216. Kantor et al. (2000)
reported that the FH rat was more sensitive to the anxiogenic effects of the 5-HT2C
agonist mCPP. However, the failure of CVT-10216 to counteract the anxiogenic effects of mCPP in SD rats argues against this mechanism underlying its anxiolytic effects.
In order to identify potential receptor mechanisms underlying the anti-anxiety properties of CVT-10216, we next used an alcohol-withdrawal model of anxiety in SD rats because of the identified role of specific neurotransmitter receptors in response to alcohol withdrawal. We asked whether CVT-10216 would also prevent the development of alcohol-withdrawal-induced anxiety, a well-recognized event that often precipitates relapse drinking (Breese et al., 2005b
). Here we show that CVT-10216 suppresses anxiety-like behavior in rats withdrawn from ethanol. In general, our results suggest that anxiolytic properties of CVT-10216 were greatest after acute treatment, with the 15 mg/kg dose completely normalizing the abnormal behavior produced by repeated withdrawal during 3 cycles of forced alcohol consumption. Moreover, there was also an additional prophylactic effect of CVT-10216 which appeared to prevent the development of anxiogenic behavior during the course of the repeated withdrawals. Thus, CVT-10216 administered during the first two cycles of alcohol withdrawal reduced the degree of anxiety when social interaction was measured after the 3rd cycle of withdrawal 5 days after the last dose of CVT-10216. This finding suggests that the anxiolytic properties of CVT-10216 not only appear to immediately suppress manifestations of anxiety, but also prophylactically prevent the development of neurochemical changes required for the expression of anxiety in the future. Importantly there was no effect of CVT-10216 on locomotor activity in these experiments, an observation that suggests that CVT-10216 targets anxiogenic behavior selectively.
Restraint-induced stress is another model system for producing anxiety-like behavior and searching for anxiolytic agents. CVT-10216 prevented the development of stress-induced anxiety-like behavior in restrained SD rats. A variety of other compounds counteract stress-induced anxiety-like behavior in this model. These compounds include CRF1 receptor antagonists, BZD receptor antagonists, and 5-HT1A
receptor agonists (Breese et al., 2004
). One notable exception to this list is the 5-HT2C
receptor antagonist that does not counteract stress-induced anxiety-like behavior (Breese et al., 2004
) but is effective as an anxiolytic agent in FH rats (Bagdy et al., 2001
; Kantor et al., 2000
) and in alcohol-withdrawal-induced anxiety (Overstreet et al., 2003a
). These observations raised the possibility that CVT-10216 may not target 5-HT2C
mechanisms. Indeed, we find that CVT-10216 is not effective against anxiety produced by mCPP, a 5-HT2C
agonist. Moreover, CVT-10216 also does not affect the suppression of locomotor activity produced by mCPP.
The GABA–BZD receptor system is a well-recognized mechanism that appears to be involved in human anxiety and in rat models of anxiety-like behavior, including those used in this study. Therefore we asked whether CVT-10216 would be therapeutically effective against anxiety-like behavior produced by DMCM, a BZD receptor inverse agonist (Stephens et al., 1984
). These results suggest that the GABA–BZD receptor system is implicated in the anti-anxiety properties of CVT-10216. Consistent with this possibility, flumazenil, a BZD receptor antagonist, is effective in both stress and multiple alcohol-withdrawal protocols (Breese et al., 2004
; Knapp et al., 2007
). Thus, our findings suggest that CVT-10216 has anxiolytic properties that may involve GABA/BZD pathways, even though we have not observed any direct interactions between CVT-10216 and GABA or BZD receptors (unpublished observations, 2007).
Disulfiram, an irreversible ALDH-1 and ALDH-2 inhibitor, does not appear to have anxiolytic properties in humans (Goyer et al., 1984
; Snyder and Keeler, 1981
). This difference may be related, in part, to the fact that disulfiram and its metabolites inhibit many sulfhydryl and metal-containing enzymes while CVT-10216 is a highly selective, reversible inhibitor of ALDH-2 only. Fortunately, unlike the BZD receptor agonists, CVT-10216 does not appear to be an addictive substance (Arolfo et al., 2009
). Studies are underway to determine whether some of the metabolic substrates of ALDH-2 in the brain might play a role in mediating the anxiolytic properties of CVT-10216.
Because the social interaction test was the exclusive test used in these studies, we do not know how general the anxiolytic properties of CVT-10216 are. In particular, anxiety is inferred in the social interaction test from an inhibition of behavior. Whether CVT-10216 will be effective in a model where anxiety is inferred from an increase in behavior, such as the defensive burying test, cannot be answered at this point. It should be emphasized, however, that anxiety tests do not necessarily provide identical information. The FH rats and several other strains with innate low social interaction do not exhibit any differences in the elevated plus maze (Overstreet et al., 1998
; Rezvani et al., 2007
The pattern of effects on anxiety-related behavior resembles those seen for CRF1 receptor antagonists (Breese et al., 2004
; Overstreet et al., 2004
). However, little is known about the effects of CVT-10216 on the HPA axis. It is unlikely that a modulation of this system plays a role in the effects of CVT-10216 because we have previously shown that corticosterone does not modify alcohol-withdrawal-induced anxiety-like behavior (Breese et al., 2004
In summary, CVT-10216 exhibits anxiolytic effects in several well-established rodent model systems. These findings provide a nice complement to the recently established anti-drinking effects of CVT-10216 (Arolfo et al., 2009
). The GABA–BZD system mediates the anxiety-like behavior seen in these models and it is possible that CVT-10216 may be interacting with this system to produce its anxiolytic effects. However, these findings are also consistent with recent reports that a GABA-B receptor agonist appears to have anxiolytic effects in animals (Knapp et al., 2007
), to counteract alcohol-withdrawal signs in humans (Addolorato et al., 2006
), to reduce alcohol drinking in animals and humans (Addolorato et al., 2000
; Flannery et al., 2004
; Colombo et al., 2000
) and to reduce alcohol operant self-administration in alcohol-dependent rats (Walker and Koob, 2007
). Studies are underway to investigate the role of GABA-B receptors in the mechanism of action of CVT-10216.