In previous work we demonstrated that systemic injection of Albu-CocH blocked the reinstatement of cocaine seeking effectively, though acutely (7
). In the present study we extended these findings and used gene transfer of CocH (via adenoviral delivery) to drastically enhance the duration of CocH activity, for at least 6 mo. Elevated enzyme levels at the end of this period suggested that the expression could have lasted significantly longer if the rats had been left to continue. Our data provide evidence that a single delivery of CocH to rats via hdAD vector provides a sustained level of enzyme activity that selectively blocks reinstatement of cocaine-seeking behavior (relapse) for a prolonged period in both male and female rats. Thus, during each of 11 cocaine-primed reinstatement sessions that took place over 6 months of testing, responding by rats treated with CocH vector resembled responding following saline priming sessions, and it was significantly lower than that of controls ( – ). This outcome can be regarded as evidence that CocH vector treatment resulted in lasting reduction in the reinforcing effects of cocaine. Further evidence was a significant reduction in the vector-treated (vs. control) group of reinstatement responding (Fig. S4 in the Supplement
) that typically generalizes to the inactive
lever when there is elevated reinstatement responding on the previously active lever (12
). The present study is the first demonstration of a long-lasting treatment to prevent resumption of cocaine-seeking behavior in “cocaine experienced” animals for which cocaine self-administration access has been terminated.
The lack of sex differences in the present study contrasts with previous findings from our laboratories and others indicating that females outperform males on the maintenance, extinction, and reinstatement of cocaine-seeking behavior (10
). However, such sex differences are not always consistent and depend on several factors such as dose of cocaine, durations of experimental sessions, and degree of challenge in the schedule of reinforcement (11
). For example, previous work indicated that females self-administer greater amounts of cocaine than males at low doses of cocaine (e.g., 0.2 mg/kg), long session lengths (e.g., 6 hr), and more demanding requirements for reinforcement (e.g., progressive-ratio schedule) (11
), whereas the present study used higher doses, shorter sessions, and simple schedules (FR1). Another interesting outcome of these experiments was that male and female rats treated with CocH vector showed a similar decrease in cocaine-primed reinstatement responding, whereas pharmacological treatments are usually more effective against reinstatement in females than males (10
). The failure to detect sex differences here might have been due to a floor effect produced by the CocH vector treatment or the limited number of male and female subjects used in the study. Nevertheless, to make an optimistic interpretation, the present results suggest that treatment with CocH vector would be equally effective in men and women.
Therapeutically efficacious treatments for cocaine relapse should be both pharmacologically and behaviorally specific in their action. In previous work we demonstrated that direct injection of Albu-CocH safely and specifically blocked cocaine-primed reinstatement without affecting amphetamine-primed responding, general locomotor activity, and food-maintained responding (7
). Similarly, in the present study we demonstrated that the ability of CocH vector to block cocaine-primed reinstatement (relapse) was pharmacologically specific and did not reflect a general suppression of behavior motivated by a stimulant reward, as demonstrated by the vigorous amphetamine-primed reinstatement by CocH-vector treated rats either at week 3 () or 24 (). In addition, the lack of an effect of CocH vector on the spontaneous recovery of reinstatement responding () and open field locomotor activity (Fig. S5 in the Supplement
) provides further evidence against a non-specific, debilitating “vector effect” on general behavior. Food and water intake by rats treated with CocH vector and controls were also similar following cocaine injections, indicating that CocH vector was well tolerated and without apparent adverse effects.
In our view, restricted drug access of cocaine to key centers in the brain is the most parsimonious hypothesis to account for the ability of CocH to reduce cocaine-driven behavior. Prior work from our laboratories has demonstrated extremely rapid elimination of cocaine in rats exhibiting plasma cocaine hydrolase activities comparable to those achieved in the present study. In fact, that elimination was so rapid that plasma drug half-life became too short to measure accurately (7
). The rapid elimination of cocaine by viral delivered CocH was reconfirmed in the present study, which indicated that cocaine plasma levels remained near baseline throughout a 2-hr period following a 3.5 mg/kg i.v. injection of drug (Fig. S1 in the Supplement
). Further evidence for effective pharmacological interception of cocaine by gene transfer of CocH comes from a study in progress, where a similar treatment effectively blocked the locomotor response to acute cocaine, 10 mg/kg, i.p.
When the reinstatement responding in vector-treated rats was compared with CocH plasma levels, no simple linear relationship emerged. In particular, there was no indication that CocH enzyme levels were inversely proportional to the extent of reinstatement (relapse) responding. An inverse relationship might have been expected across time in a given animal if, as a result of falling enzyme levels in the later months, cocaine metabolism became too slow to reduce the drug's reward value. Two explanations deserve consideration. First, even the relatively low CocH expression in some rats at later stages may have still been adequate to blunt cocaine's central effects by metabolizing most drug before it entered the brain. Alternatively it is possible that, during protracted, high CocH expression, rats unlearned the association between cocaine intake and cocaine reward. Such an outcome is not implausible, even if the benzoic acid or ecgonine methyl ester produced by accelerated cocaine metabolism had neutral properties. If either metabolite were mildly aversive, however, suppression of responses to cocaine priming might well outlast the high enzyme expression. Other explanations are also possible.
In addition to vector-based therapies, an additional approach to the long-term treatment of cocaine relapse by restricting cocaine's access to the brain involves the use of cocaine vaccines. Cocaine vaccines facilitate the production of antibodies that target blood-born cocaine and block cocaine-induced neurobiological and behavioral effects in rodents (9
). The therapeutic efficacy of cocaine vaccines has been successfully demonstrated in Phase I trials in human cocaine addicts (9
). The mechanisms by which hydrolases and anti-cocaine antibodies deactivate cocaine differ but are almost certainly not mutually exclusive. Whereas BChE-like enzymes such as CocH work at low protein levels to metabolize cocaine in blood plasma, anti-cocaine antibodies, present in higher molar concentrations, rapidly bind and sequester substantial quantities of drug. Recently, in an in vitro
experiment Gao et al. (2010) (17
) demonstrated that CocH relinquished cocaine from saturated anti-cocaine antibodies thereby synergistically enhancing cocaine clearance. One hypothesis is that enzyme-based gene therapies and cocaine vaccines may work in concert as a combination therapy with an additive power to prevent cocaine relapse (17
). A combination therapy approach could be applied to the procedure used in the present study to determine additive treatment effects on cocaine-primed reinstatement.
The present experiments were terminated at 6 months in order to test other relevant behavioral parameters. We anticipate that the favorable effect observed could last considerably longer. This point deserves further study because it may have significant translational impact for human cocaine abuse. It will also be important in future work to examine the effectiveness of hydrolase vector treatment in other animal models of clinical relevance. Among these are paradigms in which animals are allowed to reacquire cocaine self-administration, a model of brief relapse in human addicts under treatment, or in those that provide an opportunity to escalate drug intake. Future work should also test CocH vector treatment in other species (e.g., nonhuman primates) and compare it with other pharmacological approaches to drug abuse or related disorders.