As noted above, perhaps the most direct approach to the pharmacokinetic treatment of cocaine abuse and toxicity is the use of BChE. BChE is an enzyme naturally found in humans that metabolizes cocaine to the inactive metabolite EME. Therefore, the administration of additional BChE may be able to speed the metabolism of cocaine sufficiently to decrease the amount of cocaine in the body and to reduce its entry into the brain. As a result, added BChE may potentially reduce the behavioral and toxic effects of cocaine.
The use of added BChE had been tested for safety prior to its application in cocaine use when it was proposed as a treatment for chemical weapons exposure. BChE was able to protect against the nerve agents soman and sarin. In animals treated with these agents, BChE protected against both the toxic and behavioral effects of these agents [43
]. Furthermore, BChE had a relatively long duration of action of over 50 h [29
] and appeared to be safe when given alone, at least when the form given was derived from that species. In addition, there have also been methods developed for the rapid and large-scale production of BChE necessary for the use of the compound in treatment [47
The enzyme kinetics of BChE in metabolizing cocaine compare favorably to those of the catalytic antibodies (). The Km
value for BChE is below the standard that Landry et al.
suggested would be necessary for a compound to be useful in treatment [28
]. While the Kcat
fails to reach the standard Landry et al.
set, it is clearly improved over the catalytic antibodies and, thus, the catalytic efficiency (Kcat
) is clearly improved. Mattes et al.
used a human form of BChE to treat rats subsequently treated with a high dose of cocaine (179 mg/kg, intraperitoneally [ip.]) [49
]. They found that BChE could reduce plasma and brain concentrations of cocaine by over 80%. Similarly in cats, Mattes and colleagues showed that pretreatment with BChE prior to cocaine could alter cocaine metabolism, with increased levels of EME observed along with decreased levels of the active metabolite norcocaine [49
]. Browne et al.
] showed that human plasma spiked with BChE accelerated the metabolism of cocaine and Carmona et al.
] showed that in rats treated with horse serum-derived BChE, the cocaine half-life was significantly reduced. Also using horse serum-derived BChE, Koetzner and Woods showed that cocaine plasma and brain levels in mice were decreased following treatment with the enzyme [53
Butyrylcholinesterase and derivatives.
Treatment with BChE has also been shown to be effective against cocaine’s toxic effects. Rats pretreated with a human form of BChE survived a high dose (179 mg/kg ip.) cocaine treatment, while rats not treated with BChE did not [49
]. Lynch et al.
] was able to show treatment with BChE post cocaine was also able to protect rats against cocaine’s cardiovascular and lethal effects, an effect that would be necessary if BChE was to be used in an emergency room setting. In cats, BChE pretreatment antagonized cocaine’s pressor effect. Hoffman et al.
showed that purified human BChE given to mice was able to protect against the lethal effect of cocaine [24
Since BChE can speed cocaine metabolism and protect against the toxic effects of cocaine, it is reasonable to assume that BChE may also be able to reverse some of the behavioral effects of cocaine. Mattes et al.
gave rats human BChE prior to treatment with a locomotor-activating dose of cocaine and found that BChE reduced activity compared with rats treated with cocaine alone [49
]. Likewise, Carmona et al.
] and Koetzner and Woods [53
] showed that horse serum-derived BChE could antagonize the locomotor activating effects of cocaine. shows the results from Carmona and co-workers illustrating this effect. Rats were treated with BChE (5000 units) or saline and placed in an activity monitor for 30 min (−30 to −10). The rats initially show high levels of activity, but quickly habituate to the chamber as shown by the decrease in activity over the course of the 30 min habituation period. BChE did not affect habituation. Rats were then treated with 17-mg/kg cocaine or saline ip. Those rats given cocaine following saline show an increase in distance traveled. BChE clearly antagonized this effect, although some increase in activity above saline treated rats was observed. Those animals treated with BChE before saline treatment were comparable to saline–saline-treated animals, showing that BChE itself did not affect activity. Interestingly, there have been no published results investigating the effects of BChE on cocaine self-administration. While not published, our lab treated rats with horse serum-derived BChE prior to cocaine self-administration on a fixed-ratio schedule [GN Carmona, CW Schindler, Unpublished Data]. In the few rats tested, no effect of BChE was observed. This result suggests that the kinetic efficiency of BChE against cocaine may not be sufficient to alter an effect like cocaine self-administration that is dependent on the rapid uptake of cocaine into the brain. As a result, a number of investigators have investigated alterations of BChE in an attempt to increase its ability to metabolize cocaine.
Effects of 5000 IU horse serum-derived butyrylcholinesterase administered intravenously on cocaine-induced locomotor activity
lists mutations of human BChE according to their amino acid substitutions. By noting that BChE was able to metabolize (+)-cocaine more efficiently than (−)-cocaine, investigators were able to focus on mutations to improve the ability of BChE to bind (−)-cocaine. Even a single amino acid substitution was able to increase the catalytic efficiency of BChE. The A328W mutant of BChE had a Kcat
over three-times that of wild-type BChE and a relatively long half-life of 16 h [55
]. In mice treated with A328W prior to cocaine, the locomotor-activating effects of cocaine were attenuated. Additional mutations of BChE proved to be even more efficient. The double mutant A199S/A328W had a Kcat
for cocaine of 173 min−1
], clearly exceeding the standards suggested by Landry et al.
]. The double mutant A328W/Y332A [57
] also clearly exceeds that standard. This mutant, and some other BChE mutants, are sometimes referred to as CocE, which should not be confused with the bacterial CocE described in the next section. The addition of this mutant to plasma or pretreatment with A328W/Y332A accelerated cocaine metabolism and decreased the cocaine half-life in rats [57
]. Gao and Brimijoin showed that pretreatment with this mutant was able to blunt the cardiovascular effects of cocaine. They determined the half-life of the A328W/Y332A mutant to be 10 h [59
]. Treatment with this mutant was also able to completely block the locomotor-activating effect of cocaine [57
The triple mutant A199S/F227A/A328W [56
] and the quadruple mutant F227A/S287G/A328W/Y332M [60
] further improved on the catalytic efficiency of BChE for cocaine (). The quadruple mutant is also referred to as AME359 [61
] and was shown by Gao et al.
] to decrease cocaine plasma levels and to antagonize the cardiovascular effects of cocaine. Another quadruple mutant of BChE (A199S/F227A/A328W/Y332G) has proven to be even more efficient in catalyzing cocaine [60
]. This mutant is often referred to as CocH and is typically fused to the C terminus of human serum albumin (Albu-CocH) to increase the peripheral circulation half-life for use in vivo
. This compound has been studied extensively in animals.
When rats and squirrel monkeys are pre-treated with Albu-CocH, cocaine plasma levels are reduced and EME levels are increased [64
]. Albu-CocH is also able to reduce cocaine brain levels [66
]. This reduction in cocaine plasma levels is associated with a reduction in the toxic effects of cocaine. When rats were pretreated with Albu-CocH, cocaine-induced seizures were reduced [66
], as shown in . In this study, rats were treated with 10-mg/kg Albu-CocH and 10 min later were given cocaine. Seizure activity was then monitored. Pretreatment with Albu-CocH produced a substantial and significant shift to the right in the dose effect function for seizures. This effect of Albu-CocH was also seen when the mutant was delivered after, rather than before, cocaine administration, an important finding given that individuals would necessarily need to be treated after cocaine administration in an emergency room setting. Albu-CocH was also able to blunt the hypertensive effect of cocaine when administered in rats prior to cocaine [66
]. These results suggest that Albu-CocH might be useful in emergency room settings to treat cocaine toxicity.
Pretreatment with 10-mg/kg of quadruple mutant of human butyrylcholinesterase (A199S/F227A/A328W/Y332G) shifts the dose–effect function for cocaine-induced seizures to the right in rats
To be potentially useful as a treatment for cocaine addiction, Albu-CocH should also be able to reduce some of the abuse-related behaviors associated with cocaine administration. Albu-CocH has been shown to antagonize cocaine self-administration in rats responding on a progressive ratio schedule [67
] and in squirrel monkeys responding on a fixed-ratio schedule [65
]. In both rats and squirrel monkeys, Albu-CocH was also able to decrease cocaine-induced reinstatement of cocaine self-administration that had undergone extinction [65
]. Finally, in squirrel monkeys, Albu-CocH is able to antagonize the discriminative stimulus effects of cocaine [65
]. In the effects on squirrel monkey self-administration, Albu-CocH had a duration of action of over 24 h. After 24 h these effects were diminished. In monkeys, some evidence of immunogenicity was also observed. However, since Albu-CocH is derived from human BChE these effects might be less likely in humans.
A mutant with five amino acid substitutions (A199S/F227A/S287G/A328W/E441D) has also shown favorable enzyme kinetics against cocaine [56
]. This compound has been shown to be effective against cocaine-induced seizures and to protect against the lethal effects of cocaine [68
Research on BChE mutants has also focused on extending the presence of these mutants in the body. One technique that has been used successfully is virally mediated gene transfer. Gao et al.
used this technique with the A328W/Y332A mutant [62
]. They found that cocaine-hydrolyzing activity was increased 3000-fold and half-life of the mutant in plasma was 33 h. When this technique was applied to the AME359 mutant, cocaine plasma-hydrolyzing activity was increased 50,000-fold. Cocaine was cleared so rapidly from the body that it was reduced by over 95% at the first-time sample (4 min). This treatment also antagonized the pressor effects of cocaine. In a subsequent study, Gao and Brimijoin injected these vectors directly into the nucleus accumbens and showed a dramatic increase in BChE activity as a result of those injections [69
]. Gao and Brimijoin showed that when treated with the gene-transferred quadruple mutant intravenously, plasma BChE activity was substantially enhanced for up to 7 days and the ability of cocaine to induce c-Fos expression in the caudate nucleus was reduced for up to 7 days [70
]. These results show that gene transfer techniques can be applied to the BChE mutants to substantially increase their duration of action, making them viable candidates for the treatment of cocaine abuse. A similar approach has been applied to BChE itself, but this compound has only been tested against the toxicity of chemical nerve agents or organophosphate poisons [71