Cocaine-induced seizures account for approximately 10% of cocaine-related emergency room visits to hospitals and are a common manifestation of cocaine toxicity. While there are no widely accepted pharmacotherapies for cocaine addiction, disulfiram has shown recent promise as a treatment for cocaine dependence. Unfortunately, this treatment may be hazardous to some patients, due to its mechanism of action. Disulfiram inhibits the enzyme DBH, which could lead to increases in seizures and cocaine sensitivity. Therefore, we chose to examine how pharmacological DBH inhibition and DBH genotype affects CIS probability and frequency in an animal model. In order to assess this, we pretreated Dbh +/+ and -/- mice with disulfiram or nepicastat, a direct, selective DBH inhibitor, prior to administering a high dose of cocaine. We hypothesized that Dbh -/- mice would be hypersensitive to CIS and that disulfiram would exacerbate CIS in a Dbh genotype-dependent manner. We also assessed changes in serum cocaine levels to determine whether the effects of these drugs could be attributed to changes in cocaine metabolism.
Disulfiram had two distinct effects on CIS; it increased the probability of having a seizure in Dbh +/+ and Dbh -/- mice and increased CIS frequency in Dbh +/+ mice only. Nepicastat did not increase seizure probability, but increased the frequency of CIS in Dbh +/+ mice only. These results indicate that pharmacological DBH inhibition is responsible for increasing the frequency of CIS, while disulfiram's ability to raise the probability of CIS is mediated by a DBH-independent mechanism. Given disulfiram's mechanism of action as a copper chelator, the inhibition of cocaine metabolic enzymes, such as cholinesterase and carboxylesterase, could underlie its effects on CIS probability. However, we did not find this to be the case, as disulfiram did not alter serum cocaine concentrations in wild-type mice. Interestingly, nepicastat actually tended to inhibit CIS in Dbh -/- mice. Nepicastat does not chelate copper and is a direct, potent inhibitor of DBH, and initial screening did not reveal any other high-affinity targets aside from DBH. It appears that the lack of DBH in Dbh -/- mice revealed a secondary anticonvulsant target for nepicastat.
Because pharmacological DBH inhibition increased CIS frequency, Dbh
-/- mice would be expected to demonstrate more frequent CIS. We did not find this to be the case. One possible explanation is that compensatory changes in monoamine neurotransmitters arise following chronic and complete knockout of DBH and NE function. One likely compensatory candidate is the serotonin (5-HT) system. Serotonergic activity modulates seizure activity in response to cocaine and seems to have proconvulsant effects. Selective 5-HT reuptake inhibitors, such as fluoxetine, citalopram, paroxetine and the tricyclic antidepressant imipramine, all facilitate CIS (O'Dell et al., 1999
; Ritz and George, 1997
), whereas 5-HT2
receptor antagonists decrease CIS (O'Dell, et al., 1999
; Ritz and George, 1997
; Schechter and Meehan, 1995
). Since cocaine is known to inhibit 5-HT transporters (SERT), increases in 5-HT following cocaine administration can lead to accumulation of serotonin in synapses, which in turn can increase seizure activity via 5-HT2
receptor activation. The serotonergic raphe nuclei receive dense innervation from brainstem noradrenergic nuclei (Baraban and Aghajanian, 1981
; Marcinkiewicz et al., 1989
; Fritschy and Grzanna, 1990
; Peyron et al., 1996
), and activation of α1
-adrenergic receptors increases tonic excitatory activity in the dorsal raphe nucleus (Baraban and Aghajanian, 1980
; Vandermaelen and Aghajanian, 1983
; Hertel et al., 1998
; Pudovkina et al., 2003
; Judge and Gartside, 2006
). Therefore, Dbh
-/- mice should have lower levels of extracellular 5-HT, because they lack the noradrenergic excitatory drive on the serotonergic system. Indeed, when compared with control mice, Dbh
-/- mice have decreased 5-HT release in the nucleus accumbens following amphetamine treatment (D. Weinshenker and S. Puglisi-Allegra, unpublished observations), as well as in the hippocampus following fluoxetine administration (Cryan et al., 2004
). This decreased concentration of the proconvulsant 5-HT in Dbh
-/- mice may explain their “normal” CIS susceptibility at baseline.
Because disulfiram acts as a copper chelator, this drug is relatively nonspecific and inhibits two cocaine-metabolizing enzymes, cholinesterase and carboxylesterase (Zemaitis and Greene, 1976
; Nousiainen and Törrönen, 1984
; Savolainen et al., 1984
). Disulfiram treatment increased cocaine plasma levels and decreased cocaine clearance in humans following intranasal cocaine administration (McCance-Katz et al., 1998a
; Hameedi et al., 1995
; Baker et al., 2007
). Thus, it is possible that the disulfiram-induced increase in CIS was a direct result of decreased cocaine metabolism. However, disulfiram did not alter peak serum cocaine levels in most cases in our study. The single exception was an interaction between disulfiram treatment and genotype; the higher dose of cocaine (60 mg/kg) significantly increased cocaine plasma levels in Dbh
-/- mice only. The mechanims underlying this synergy is unclear. One possibility is that NE limits the spread of cocaine through the bloodstream via its vasoconstrictive properties, while cholinesterase and carboxylesterase are responsible for its metabolism. Perhaps at low doses of cocaine, either mechanism alone is sufficient to maintain “normal” peak cocaine serum levels, but at high doses, the impairment of both noradrenergic function and cocaine metabolic enzymes results in increased serum levels. It is not clear why disulfiram did not increase peak serum cocaine levels in wild-type mice. The differences between the effects of disulfiram on serum cocaine levels in humans and rodents may be due to different routes of cocaine administration (intranasal in humans vs. intraperitoneal in mice) or to species differences in cocaine metabolism.
Our results show that acute disulfiram administration increases the probability and frequency of CIS, which may present a clinical problem during cocaine addiction treatment with disulfiram pharmacotherapy. It should be noted that, while CIS have not been reported during cocaine dependence clinical trials examining disulfiram efficacy, there have been several reports of individuals without a history of epilepsy developing seizures following treatment with therapeutic doses of disulfiram (Liddon and Satran, 1967
; Price and Silberfarb, 1976a, 1976b; McConchie et al., 1983
; Daniel et al., 1987
). Thus, clinicians should be cautious when considering disulfiram as a cocaine pharmacotherapy, particularly in patients with a history of epilepsy or cocaine overdose. The more selective DBH inhibitor nepicastat may be a safer alternative to disulfiram for treating cocaine dependence, as it does not increase CIS probability and is in fact anticonvulsant in Dbh
-/- mice. DBH activity is genetically controlled and highly variable in humans (Weinshilboum, 1978
; Zabetian et al., 2001). The haplotype associated with low DBH activity in humans is also associated with more cocaine-induced paranoia (Cubells et al., 2000
; Kalayarisi et al., 2007). This increase in one of the aversive properties of cocaine may underlie the effectiveness of DBH inhibition via disulfiram in curbing cocaine intake. Given that the proconvulsant effect of disulfiram on CIS frequency is absent in Dbh
-/- mice, disulfiram pharmacotherapy might perhaps be safer for cocaine addicts with low DBH activity. Preliminary data suggest that disulfiram is most effective for these individuals (R. Schottenfeld and J. Cubells, personal communication), possibly due to their enhanced aversion to cocaine. Our results indicate that they may also be resilient to disulfiram-induced exacerbation of CIS and possibly other toxic effects of cocaine.