Psychopharmacology (1999) 145:153–161

© Springer-Verlag 1999

O R I G I N A L I N V E S T I G AT I O N

Bai-Fang X. Sobel · Anthony L. Riley

The interaction of cocaethylene and cocaine and of cocaethylene and alcohol on schedule-controlled responding in rats

Received: 16 September 1998 / Final version: 16 March 1999

Abstract Rationale: Cocaethylene is a unique metabolite of cocaine, produced only in the presence of alcohol. This metabolite is pharmacologically, physiologically and behaviorally active. Further, it has been reported to interact pharmacokinetically with both cocaine and alcohol, an interaction that may mediate, in part, the interaction of cocaine and alcohol. Although cocaethylene has been shown to interact with both cocaine and alcohol, behavioral assessments of these interactions are limited. Objectives: To examine directly the behavioral interactions between cocaethylene and cocaine and between cocaethylene and alcohol, the present study assessed the effects produced by these combinations on schedule-controlled responding. Methods: Rats were first administered cumulative doses of cocaethylene, cocaine and alcohol to assess their effects alone on responding. Following this, doses of cocaethylene were combined with cumulative doses of cocaine or alcohol. Additionally, doses of cocaine or alcohol were given in combination with cumulative doses of cocaethylene. Results: When administered alone, cocaethylene, cocaine and alcohol produced dose-related decreases in responding. Further, cocaethylene shifted the dose–response functions for both cocaine and alcohol to the left and down, while cocaine and alcohol shifted the dose–response function for cocaethylene to the left and down. An isobolographic analysis revealed that these interactions were additive in nature. Conclusions: The present study suggests behavioral interactions between cocaethylene and cocaine and between cocaethylene and alcohol. The contribution of cocaethylene to the enhanced effects produced by the coadministration of cocaine and alcohol was discussed. Key words Cocaethylene · Cocaine · Alcohol · Interaction · Schedule-controlled responding · Rat B.-F.X. Sobel (✉) · A.L. Riley Psychopharmacology Laboratory, Department of Psychology, American University, Washington, DC 20016, USA e-mail: [email protected] Tel.: +1-202-885-1721, Fax: +1-202-885-1081

Introduction Cocaine is normally metabolized to benzoylecgonine and ecgonine methyl ester by nonspecific hepatic microsomal carboxylesterases (Dean et al. 1991). In the presence of alcohol, however, cocaine is transesterified by a hepatic carboxylesterase into cocaethylene (Dean et al. 1991, 1992; Jatlow et al. 1991; Boyer and Petersen 1992; Baily 1994; Brzezinski et al. 1994). Cocaethylene has been found in the brain, liver, plasma, blood and urine of both human and animals that co-use alcohol and cocaine (Rafla and Epstein 1979; de la Torre et al. 1991; Hearn et al. 1991b; Hime et al. 1991; Bailey 1993, 1996; Watanabe et al. 1997). Depending on the procedures used, the appearance of cocaethylene in the plasma occurs between 2 min and 30 min following the co-administration of cocaine and alcohol (Dean et al. 1992; MaCance-Katz et al. 1993; Bailey 1994). Unlike benzoylecgonine and ecgonine methyl ester, cocaethylene is pharmacologically active (Dean et al. 1991; for detail, see below). Within a number of preparations, cocaethylene has been found to produce similar pharmacological, physiological and behavioral effects as cocaine. For instance, like cocaine, cocaethylene blocks dopamine reuptake into synaptosomes, inhibits binding to the dopamine transporter and results in an increase in extracellular concentrations of dopamine in the nucleus accumbens (Hearn et al. 1991a; Jatlow et al. 1991; Woodward et al. 1991). In addition, cocaethylene produces cardiovascular effects similar to those produced by cocaine, including increased heart rate, cardiac output and blood pressure (Perez-Reyes and Jeffcoat 1992; McCance et al. 1995; Henning and Wilson 1996), as well as depressed myocardial contraction in cardiac myocytes (Crumb and Clarkson 1992; Qiu and Morgan 1993; Welder et al. 1993; Bai et al. 1996). In addition to its cardiotoxicity, cocaethylene also causes hepatic necrosis in mice (Roberts et al. 1992) and blunts the formation of blast cells and interleukin-2 production (Chiappelli et al. 1995). Furthermore, it produces significant restriction on

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brain growth in forebrain, cerebellum and brainstem for neonates (Chen and West 1997). Behaviorally, cocaethylene increases locomotor and rearing activity (Jatlow et al. 1991; Katz et al. 1992), maintains self-administration (Jatlow et al. 1991), produces a conditioned place preference (Schechter 1995) and serves as a discriminative stimulus (Woodward et al. 1991) in a manner similar to that produced by cocaine. In humans, similar subjective effects of cocaethylene and cocaine have been reported (McCance-Katz et al. 1993; McCance et al. 1995). Not only is cocaethylene active (Hearn et al. 1991a, b; Jatlow 1993; McCance-Katz et al. 1993; Hedaya and Pan 1996; Farre et al. 1997; Etkind et al. 1998), but it has also been reported to interact with both cocaine and alcohol. For example, cocaethylene increases alcohol-induced central nervous system depression as measured by the loss of the righting reflex (Wilson et al. 1997). In addition, cocaethylene undergoes an esterase-mediated ethyl-ester exchange with ethanol, resulting in an increase in its elimination half-life (Bourland et al. 1998). Conversely, alcohol affects the bioavailability of cocaethylene via its vasodilatory effects, an interaction that has been offered as an account of the potentiation of cocaethylene-induced lethality by alcohol (Meehan and Schechter 1995). The evidence that cocaethylene interacts with cocaine includes that cocaethylene inhibits cocaine’s metabolism (Parker et al. 1996) and can be transesterified to cocaine by hepatic esterases of the human liver in the presence of methanol (Baily 1994), indicating that cocaethylene can prolong the action and increase the levels of cocaine. Although there have been no direct behavioral assessments of the interaction of cocaethylene and cocaine, studies have showed that there is cross-sensitization between cocaethylene and cocaine, suggesting an interaction between the two compounds (Jatlow 1993; Meehan and Schechter 1996). For example, animals exposed repeatedly to cocaethylene show enhanced locomotor activity and seizures to cocaine, i.e., behavioral sensitization (Jatlow 1993; Meehan and Schechter 1996). It is possible that, via these interactions, cocaethylene contributes to the greater effects often reported when cocaine and alcohol are co-administered. For example, within a number of preparations, the co-administration of cocaine and alcohol yields greater effects than either drug given alone. These preparations include assessments of cardiovascular toxicity (Foltin and Fischman 1989; Perez-Reyes and Jeffcoat 1992; Henning et al. 1994; Pirwitz et al. 1995; Nicolas et al. 1996), hepatoxicity (Boyer and Petersen 1990; Odeleye et al. 1993), immunosuppression (Pirozhkov et al. 1992; Torres 1994), subjective ratings (Farre et al. 1993; Foltin et al. 1993; McCance et al. 1995) and behavioral output (Masur et al. 1989; Misra et al. 1989; Higgins et al. 1992; PecinsThompson and Peris 1993; Lewis and June 1994; Sobel and Riley 1997). In light of this possibility, it is important to assess whether and to what degree cocaethylene interacts with cocaine and alcohol. As indicated, although there are direct assessments of their pharmacological interactions, behavioral work on these interac-

tions has been limited. Thus, the purpose of the present study was to assess directly the behavioral interactions of cocaethylene and cocaine and of cocaethylene and alcohol. Specifically, the effects of cocaethylene and cocaine and of cocaethylene and alcohol (alone and in combination) were assessed with regard to schedule-controlled responding within a cumulative dosing procedure (Thompson and Boren 1977), a procedure useful in the rapid determination of dose–response functions. The present study used an isobolographic analysis to determine the nature of the interactions, i.e., whether they were synergistic (greater than additive), additive or infraadditive (less than additive) (Curry 1977; Levine 1978; for a critical review, see Poch 1993).

Materials and methods Subjects Seventeen experimentally naive, female rats of Long-Evans descent, approximately 120 days old and 260 g in weight at the beginning of the experiment, were studied. They were housed in individual wire-mesh cages and were maintained on a 12-h light/12h dark cycle at an ambient temperature of 23°C. All rats were allowed access to water during the daily experimental sessions and were given supplemental water 2 h following each session to maintain their body weight at 85% of free-feeding levels. They were maintained on ad-libitum access to food for the duration of the study. Apparatus The rats were trained in one of four identical operant chambers (26.5×19.2×16.5 cm). The sides and ceiling of each chamber were made of clear Plexiglas, and the grid floor was constructed of 0.6-cm diameter stainless-steel rods spaced 2 cm apart. A house light was centered on the front wall 2.5 cm from the ceiling. Three equally spaced 1.3-cm diameter holes were positioned 4 cm below the house light. Two stainless-steel rods were mounted on the sides of each hole on the outside of the chambers. Plexiglas blocks were attached to each set of two rods. A stainless-steel tube (blunted 16-gauge needle) was fitted into each Plexiglas block, which was positioned on the rods such that the end of each tube was recessed 6 mm behind each hole in the front wall. This design enabled the subjects to extend their tongues through the holes to lick the tubes. A light was installed in each Plexiglas block and was located 2 mm below each stainless-steel tube. The stainlesssteel tubes were connected via Tygon tubing (S-50-HL) to Teflon solenoid valves (General Valve Corp., Hairfield, N.J.) which, in turn, were attached to water reservoirs (60-cc syringes) via Tygon tubing. The syringes were mounted on a Plexiglas wall which was located outside the chamber 14.5 cm from the front wall. For this experiment, only the center and right tubes were used. Licks on either of these two tubes were detected by a drinkometer (Lafayette Model 55008). The chambers were interfaced via Med Associates interfaces (Model 1080–01) to Apple IIGS microcomputers, which recorded licking responses and delivered contingencies throughout each experimental session. Between each session, the chambers were cleaned and allowed to dry. Drugs Cocaethylene fumarate and cocaine hydrochloride (expressed as salt; generously supplied by the National Institute on Drug Abuse) were prepared in distilled water (0.1–1 mg/ml) and injected in a

155 volume of 1 ml/kg. Alcohol was prepared by diluting 95% alcohol with distilled water and was injected as a 15% (v/v) solution. Procedure Phase I: training Rats were first trained to lick on the right tube. At the outset of training, the house light and right tube light were illuminated for 20 min, during which time each lick on the right tube produced a 0.01-ml drop of water. Licking on the center tube had no programmed consequences. This procedure was repeated daily until rats consumed at least 5 ml over the 20-min session. Following this, rats were then trained to lick on the center tube. During this training period, the house light and center tube light were illuminated at the outset of the session. A single lick on the center tube resulted in the termination of the center tube light and the illumination of the right tube light for 5 s, during which time each lick on the right tube produced a 0.01-ml drop of water. At the end of this 5-s period, the center tube light was again illuminated. As above, a single lick on the center tube resulted in the termination of the center tube light and the illumination of the right tube light for 5 s. As above, each lick on the lighted right tube resulted in the delivery of a 0.01-ml drop of water. These contingencies were in effect for the duration of the 20-min session. Over sessions, the response requirement on the center tube was gradually increased from 1 to the terminal value of 20 (i.e., FR 20). Training at this ratio requirement continued until rats consumed at least 5 ml over the 20-min session for three consecutive days. Following this, an alternating lights-off/lights-on procedure was initiated. Specifically, at the beginning of the session all lights were out for 1 min, and licking on either of the two tubes had no programmed consequences. After 1 min, the house light and center tube light were illuminated for 5 min. Twenty licks on the lighted center tube resulted in the termination of the center tube light and the illumination of the right tube light for 5 s, during which time each lick on the lighted right tube resulted in the delivery of a 0.01-ml drop of water. At the end of this 5-s period, the center tube light was again illuminated. As above, 20 licks on the center tube again resulted in the termination of the center tube light and the illumination of the right tube light for 5 s, during which time each lick on the lighted right tube resulted in the delivery of a 0.01-ml drop of water. These contingencies were in effect for the duration of the 5-min lights-on period. At the end of the 5-min lights-on period, all lights were again out for 1 min and licking had no programmed consequences. At the end of the 1 min lights-off period, the house light and center tube lights were again illuminated and the aforementioned reinforcement contingencies were in effect for the duration of the 5-min lights-on period. This cycle of lights off and lights on was repeated a total of four times within the daily session. Over days, the lights-off periods increased from 1 min to 9 min. The duration of the lights-on periods did not change. Thus, at the end of this phase, each daily session consisted of four alternating cycles of 9-min lights-off and 5-min lights-on periods, totaling 56 min. This daily procedure was in effect until licking over the four lights-on periods was stable (4–6 licks/s) for five consecutive days for individual rats. Phase II: testing During this phase, cocaethylene, cocaine or alcohol (or the combination of cocaethylene with either cocaine or alcohol) was administered intraperitoneally (i.p.) every fourth day to assess its effect on operant licking. On intervening recovery days, subjects received either no injection prior to responding (recovery days 1 and 3) or an injection of the distilled water vehicle at the outset of each lights-off period (recovery day 2). For assessments of cocaethylene, cocaine or alcohol alone, the rats were injected at the outset of each lights-off period with the test drug. The amount injected at the outset of each lights-off period resulted in a 1/8 or 1/4 log increase in the dose of the drug. The spe-

cific cumulative doses of cocaethylene were 5.6, 10, 18 and 24 mg/kg (actual doses for the second, third and fourth injections were 4.4, 8 and 6 mg/kg, respectively), of cocaine were 3.2, 5.6, 10 and 18 mg/kg (actual doses for the second, third and fourth injections were 2.4, 4.4 and 8 mg/kg, respectively) and of alcohol were 0.56, 0.75, 1.0 and 1.3 g/kg (actual doses for the second, third and fourth injections were 0.19, 0.25 and 0.3 g/kg, respectively). After the dose–response function for each of these drugs was determined, cocaethylene was combined with incremental doses of cocaine or alcohol to assess the effects of cocaethylene on the rate-suppressing effects of cocaine or alcohol. Specifically, at the outset of the first lights-off period, rats were injected with a single dose of cocaethylene (5.6, 10 or 18 mg/kg) immediately prior to receiving 3.2 mg/kg cocaine or 0.56 g/kg alcohol. Rats were then injected at the outset of each of the remaining lights-off periods with doses of cocaine cumulating to 5.6, 10 and 18 mg/kg or of alcohol cumulating to 0.75, 1.0 and 1.3 g/kg. Similarly, cocaine and alcohol were combined with incremental 1/4 log doses of cocaethylene to assess the effects of cocaine and alcohol on the rate-suppressing effects of cocaethylene. Specifically, at the outset of the first lights-off period, rats were injected with a single dose of cocaine (3.2 mg/kg or 5.6 mg/kg) or alcohol (0.56 g/kg or 0.75 g/kg) immediately prior to receiving 5.6 mg/kg cocaethylene. Rats were then injected at the outset of the remaining lights-off periods with doses of cocaethylene cumulating to 10, 18 and 24 mg/kg. In administering the drug combinations, cocaethylene was injected on the lower and upper left quadrants and cocaine and alcohol were given on the lower and upper right quadrants. Both during and following assessments of the drug combinations, cumulative doses of cocaethylene, cocaine and alcohol alone were administered to determine the stability of the effects of each of these drugs on scheduled-controlled responding (i.e., whether there were changes in the dose-response functions with repeated drug treatments). Data analysis Lick rates on the center tube over the four lights-on phases following injections of distilled water, cocaethylene, cocaine or alcohol alone were analyzed using one-way repeated-measures analysis of variance (ANOVA) with drug treatment as a factor. Lick rates on the center tube following administration of cocaethylene, cocaine, alcohol, the combination of cocaine and cocaethylene and the combination of alcohol and cocaethylene were compared by means of repeated-measures ANOVA with drug treatment and dose as factors. If there was a significant main effect in the ANOVA statistics, Scheffe F-tests were then used to test for the differences between the cocaethylene and cocaethylene/cocaine or cocaethylene/alcohol combinations, between the cocaine and cocaine/cocaethylene combinations and between the alcohol and alcohol/cocaethylene combinations at specific doses. Effects were considered statistically significant at P≤0.05. An isobolograhic analysis was used to determine the nature of the interactions between cocaethylene and cocaine and between cocaethylene and alcohol (i.e., synergism, additivity or infra-additivity). This analysis employs the x-axis to represent doses of one drug (e.g., cocaethylene) and the y-axis to represent doses of a second drug (e.g., cocaine). An isobologram is depicted by a line connecting dose pairs of drugs that are equieffective with regard to an adequately selected endpoint (Loewe 1957; Gessner and Cabana 1970; Gessner 1974; Curry 1977; Reffenstein and Mah 1984). Ninety-five percent confidence limits are indicated by shading around this line. Points falling along this linear line (and within the 95% confidence intervals) indicate that the drug interaction is additive. Points to the upper right of this line denote an infra-additive interaction, while points to the lower left of the line indicate synergism. In the present study, using SAS probit analysis, the ED50 values for 30% reduction in licking (doses that produced 30% reduction in licking from the baseline levels in 50% of the rats tested) were computed from dose–response curves of cocaethylene, co-

156 caine and alcohol. Isobolograms were constructed by connecting the line between these ED50 values for cocaethylene and cocaine and those for cocaethylene and alcohol. The fixed-dose method was then used to determine the ED50 values of various drug combinations (Gessner 1974). With this method, the dose of one drug is held constant and the dose of the other is varied. The ED50 values of the cocaethylene/cocaine and cocaeathylene/alcohol combinations were calculated using SAS probit analysis on a DELL computer. The values were plotted on the isobologram, and the nature of the interaction was assessed.

Results Cocaine, alcohol and cocaethylene alone Figure 1 depicts the mean rate of licking (licks/s) on the center tube following the distilled water vehicle given at the outset of each lights-off period and following incremental doses of cocaine (top panel), alcohol (middle panel) or cocaethylene (bottom panel). Because there were no significant differences in the dose–response functions for each drug across the multiple dose–response assessments (all P values >0.05), data from these determinations were averaged and these averages are presented in the Fig. 1. Following administration of the vehicle, subjects licked at a constant rate (approximately 4–6.5 licks/s across the session) (F=0.497, P=0.69). These rates were not significantly different from those under non-injection conditions (P values >0.05; data not shown). For all three drugs, there were significant dose effects on licking (F=61.957, P<0.0001 for cocaine; F=55.62, P<0.0001 for alcohol; F=5.76, P=0.015 for cocaethylene). Specifically, licking was significantly decreased following the administration of 10 mg/kg and 18 mg/kg cocaine, 0.75, 1 and 1.3 g/kg alcohol, and 24 mg/kg cocaethylene (all P values <0.05). The effects of cocaethylene on cocaine and alcohol Following separate assessments of the effects of cocaine, alcohol and cocaethylene on lick rates (see above), various doses of cocaethylene were combined with cumulative doses of cocaine or alcohol. Figure 2 illustrates dose–response functions for cocaine (top panel) and alcohol (bottom panel) administered alone and in combination with these doses of cocaethylene. As depicted, cocaine and alcohol produced dose-related decreases in response rate. The dose–response functions for cocaine and alcohol were shifted to the left and down by cocaethylene. In relation to cocaethylene/cocaine combinations, 5.6 mg/kg cocaethylene significantly increased the suppression induced by 10 mg/kg cocaine, whereas 10 mg/kg and 18 mg/kg cocaethylene significantly increased cocaine-induced suppression of licking following administration of 3.2, 5.6 and 10 mg/kg cocaine (all P values <0.05). For the cocaethylene/alcohol combinations, 10 mg/kg cocaethylene significantly enhanced alcoholinduced suppression of licking following 0.56 g/kg alco-

Fig. 1 The mean rate of licking ±SEM (licks/s) on the center tube following the administration of the distilled water vehicle and incremental doses of cocaine (top panel), alcohol (middle panel) and cocaethylene (bottom panel)

hol, and 18 mg/kg cocaethylene significantly increased alcohol-induced suppression of licking following 0.56 g/kg and 0.75 g/kg alcohol (all P values <0.05). The effects of cocaine and alcohol on cocaethylene Figure 3 presents the dose–response functions for cocaethylene administered alone and in combination with cocaine (top panel) and alcohol (bottom panel). As illustrated, cocaethylene alone resulted in dose-related decreases in response rate. The dose–response function for cocaethylene was shifted to the left and down by cocaine and alcohol. With regard to the cocaine/cocaethylene combinations (top panel), 5.6 mg/kg and 10 mg/kg co-

157 Fig. 2 The mean rate of licking ±SEM (licks/s) on the center tube following administration of incremental doses of cocaine alone and in combination with doses of cocaethylene that had little or no effect on operant licking (top right panel) and following administration of incremental doses of alcohol alone and in combination with doses of cocaethylene that had little or no effect on operant licking (bottom right panel). The dose–response curve for cocaethylene (from Fig. 1) is presented on the left side of both the top and bottom panels

caine significantly enhanced suppression of licking following the administration of all four doses of cocaethylene (all P values <0.05). In relation to the alcohol/cocaethylene combinations, 0.56 g/kg and 0.75 g/kg alcohol significantly increased suppression of licking following administration of all four doses of cocaethylene (all P values <0.05, except for 0.56 g/kg alcohol in combination with 5.6 mg/kg and 10 mg/kg cocaethylene). Isobologram Figure 4 depicts the isobologram for the interaction of cocaethylene and cocaine, using the ED50 for 30% reduction in lick rate from the baseline levels as the endpoint. The ED50 value for cocaethylene is depicted on the ordinate and that for cocaine is plotted on the abscissa (there are no 95% confidence limits generated from the probit analysis due to the limitation in our data distribution). The line between the ED50 values for cocaethylene and cocaine (along with its 95% confidence limits) denotes simple additivity. The ED50 values for the various combinations of cocaethylene and cocaine are also plotted. As illustrated, the ED50 values for cocaethylene in combination with 3.2, 5.6 and 10 mg/kg cocaine and for co-

caine in combination with 5.6, 10 and 18 mg/kg cocaethylene did not differ significantly from the additive line (P=0.74), indicating that the interaction was additive. Figure 5 depicts the isobologram for the interaction of cocaethylene and alcohol using the ED50 for 30% reduction in lick rate from the baseline levels as the endpoint. The ED50 value for cocaethylene is depicted on the ordinate, and the ED50 for alcohol is plotted on the abscissa. The line connecting the ED50 values for cocaethylene and alcohol denotes simple additivity. An analysis of the isobologram revealed that the ED50 values for the combination of alcohol and cocaethylene did not differ significantly from the additive line (P=0.42), suggesting that the interaction of these two drugs was additive in nature. In doing the probit analysis to calculate the ED50 values for alcohol and cocaethylene, there were no 95% confidence limits generated from the probit analysis due to the limitation in our data distribution.

Discussion As described, cocaethylene is produced when cocaine and alcohol are concurrently administered (Dean et al. 1991; Baily 1993). Further, cocaethylene is pharmaco-

158 Fig. 3 The mean rate of licking ±SEM (licks/s) on the center tube following administration of incremental doses of cocaethylene alone and in combination with doses of cocaine that had little or no effect on operant licking (top right panel) and with doses of alcohol that had little or no effect on operant licking (bottom right panel). The dose–response curves for cocaine and for alcohol are presented on the left side of the top and bottom panels, respectively

Fig. 4 The isobologram for the interaction of cocaethylene and cocaine on operant licking using the ED50 for 30% reduction in lick rate from the baseline levels as the endpoint. The ED50 values for cocaethylene and cocaine are plotted on the ordinate and abscissa, respectively. The ED50 values for various combinations of cocaethylene and cocaine and their 95% confidence limits (when available) are also plotted

Fig. 5 The isobologram for the interaction of cocaethylene and alcohol on operant licking using the ED50 for 30% reduction in lick rate from the baseline levels as the endpoint. The ED50 values for cocaethylene and alcohol are plotted with their 95% confidence limits on the ordinate and abscissa, respectively. The ED50 values for various combinations of alcohol and cocaethylene and their 95% confidence limits (when available) are also plotted

logically, physiologically and behaviorally active (Dean et al. 1991; Hearn et al. 1991a; Jatlow et al. 1991; Woodward et al. 1991). More importantly, there is evidence that cocaethylene interacts pharmacokinetically with cocaine and alcohol (Bailey 1994; Heith et al. 1995;

Parker et al. 1996; Bourland et al. 1998; Wilson et al. 1997). Behavioral assessments of these interactions, as noted, however, are limited (Jatlow 1993; Meehan and Schechter 1996; Wilson et al. 1997). To examine directly the behavioral interactions between cocaethylene and co-

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caine and between cocaethylene and alcohol, the present study assessed the effects produced by these combinations on schedule-controlled responding. Consistent with other findings, the present experiment demonstrated that cocaethylene shifted the dose–response functions for cocaine and alcohol to the left and down, and cocaine and alcohol shifted the dose–response function for cocaethylene to the left and down, indicating behavioral interactions between cocaethylene and cocaine and between cocaethylene and alcohol. The nature of cocaethylene’s interaction with cocaine and with alcohol, however, is not known. In drug interaction studies, an isobolographic analysis is frequently utilized to assess the nature of drug interactions (Gessner 1974; Reffenstein and Mah 1984; Church et al. 1988; Suhnel 1992). It should be noted that isobolograms are constructed on the basis of dose additivity, which assumes that two compounds work on a common mechanism to produce their effects (Curry 1977; Poch 1993). As such, the combination of doses of two compounds would be equivalent to administering the same drug twice. Employing an isobolographic analysis, the present study revealed that the interactions of cocaethylene and cocaine and of cocaethylene and alcohol were additive in nature. It should be noted, however, that although many researchers view the isobolographic analysis as the method of choice for evaluating possible interaction between biologically active agents (Gessner 1974; Suhnel 1992), not all data are appropriate for such an analysis (Poch 1993). For example, isobolograms can only be constructed when data are appropriately distributed, e.g., when a large sample of subjects or a wide range of doses of drugs is used to allow for a determination of 95% confidence limits of a parameter of effectiveness in 50% of subjects tested (Gessner 1974). Although a relatively large sample of subjects was used in the present experiment, we failed to obtain the 95% confidence intervals for both cocaethylene and cocaine. However, the failure to find the confidence limits can be circumvented by using statistical tests to examine whether a drug combination is truly additive (Church et al. 1988); this was done in the present experiment. In addition to data distribution, the slope of dose–response curves from which isobolograms are constructed and the maximal effects produced by a drug combination should be taken into consideration (Poch 1993). That is, isobolograms should be constructed only when two drugs show the same slope and the same maximum effects. According to Poch (1993), similar characteristics of the dose–response functions and patterns of responding usually indicate a similar underlying mechanism, which was the basis for an isobolographic analysis. Although the steepness in the dose–response curves obtained from the present study was different for cocaine and cocaethylene, these compounds have been reported to share similar pharmacological, physiological and behavioral properties (Hearn et al. 1991a; Jatlow et al. 1991; Woodward et al. 1991), suggesting a common mechanism. Thus, it is possible

that an isobologram may still be constructed despite the difference in the slopes between cocaethylene and cocaine. Although the mechanisms by which cocaethylene interacts with cocaine and with alcohol are not known, that cocaethylene interacts with both cocaine and alcohol may have implications for understanding the aforementioned interaction between cocaine and alcohol. As noted earlier, cocaine and alcohol in combination have been reported to produce greater physiological, pharmacological and behavioral effects than either one alone (Foltin and Fischman 1989; Higgins et al. 1992; Odeleye et al. 1993; Lewis and June 1994; Nicolas et al. 1996; Sobel and Riley 1997). One possible explanation for the greater effects of the combination of cocaine and alcohol is that cocaethylene, a resulting metabolite of the co-administration, contributes to the interaction of cocaine and alcohol. Given that the cocaethylene’s interaction with cocaine and alcohol was additive in nature, it is possible that the dose additivity of cocaethylene and cocaine and/or of cocaethylene and alcohol contributes to the greater effects produced by cocaine and alcohol. For this explanation to be valid, however, the level of cocaethylene produced by exogenous administration has to be comparable with that of cocaethylene produced by coadministration of cocaine and alcohol. Because in some studies the plasma concentrations of cocaethylene following the co-administration of cocaine and alcohol were much lower than those of cocaine (Dean et al. 1992; Perez-Reyes and Jeffcoat 1992; Farre et al. 1993; MaCance-Katz et al. 1993), one might argue that the level of cocaethylene produced by its exogenous administration in the present experiment exceeds those that might be produced by the combination of cocaine and alcohol, thus making it difficult to assess the contribution of cocaethylene as a resulting metabolite of the cocaine and alcohol combinations. However, there are some clinical and forensic cases in which the concentrations of cocaethylene were found to be higher than those of its parent compound, cocaine (Jatlow et al. 1991; Bailey 1993). It is possible, in the present experiment, that some doses of cocaethylene were comparable with those produced by the co-administration of cocaine and alcohol, given that various doses of cocaine and cocaethylene were tested. However, until blood levels of cocaine, alcohol and cocaethylene are directly and concurrently determined when cocaine and alcohol are co-administered, the contribution of cocaethylene to the interaction of cocaine and alcohol remains unknown. In conclusion, the present study has demonstrated that cocaethylene interacts with both cocaine and alcohol. Although the basis for such an interaction remains unknown, it is possible that the effects produced by the combinations of cocaethylene and cocaine and of cocaethylene and alcohol are due to the dose additivity of these combinations. These findings have an implication for understanding the interaction of cocaine and alcohol, i.e., the dose additivity of cocaethylene and cocaine and/or of cocaethylene and alcohol may contribute to the

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greater effects produced by the concurrent use of cocaine and alcohol. Acknowledgements The research was supported in part by a grant from the Mellon Foundation to Anthony L. Riley. Requests for reprints should be sent to Bai-Fang X. Sobel, Psychopharmacology Laboratory, Department of Psychology, American University, Washington, DC 20016.

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