Introduction

Abstract Introduction Results & Discussion References

The prevalence of simultaneous cocaine and alcohol abuse is very high. The reason for the popularity of this drug combination is that abusers experience more intense and longer lasting euphoric effect after using cocaine and alcohol (1). However, this drug combination has been associated with significant increase in the incidence of medical emergencies, as well as sudden death (2). Alcohol¹ coadministration, has been shown to alter cocaine pharmacokinetics leading to higher plasma concentration in addition to the formation of the pharmacologically active metabolite, cocaethylene (3-6). Although the exact mechanism for the increased toxicity of combined cocaine and alcohol abuse is not known, alteration of cocaine pharmacokinetics and the formation of cocaethylene have been implicated, at least partially, in the increased toxicity of this drug combination (1).

In human studies, administration of cocaine + alcohol resulted in higher cocaine plasma concentration that has been explained by enhanced cocaine absorption and/or inhibition of cocaine elimination (3-5). Also, administration of this drug combination was found to produce a greater increase in heart rate and euphoric effect, compared with administration of cocaine only (3-5). The increase in cocaine effects has been explained by the higher cocaine plasma concentration that can lead to higher cocaine tissue concentration. The objectives of this investigation were to determine if the increase in plasma cocaine concentration after cocaine + alcohol will increase the brain cocaine concentration and to investigate the brain distribution of cocaethylene in the rat. This is the first study that describes the plasma and brain extracellular fluid concentration-time profiles of cocaine and cocaethylene in awake rats after coadministration of cocaine and alcohol.

Materials and Methods

Animal Preparation. Eight male Wistar rats weighing 300-350 g (Simonsen Laboratories, Gilroy, CA) were maintained on a 12-hr light/dark cycle with Purina chow pellets and water ad libitum for at least 7 days before use in experiments. Surgical preparation was done while the animals were fully anesthetized with 40 mg/kg ip sodium pentobarbital, followed by an ip dose of 0.04 mg atropine sulfate, under aseptic condition in two phases. Brain surgery for the placement of the microdialysis guide cannula (CMA 12, BAS; West Lafayette, IN) was performed first, followed 5 days later by the cannulation of the femoral artery and the femoral vein as described previously in detail (7). The only difference in our procedures is that the microdialysis guide cannula was inserted into the nucleus accumbens according to the coordinates of the rat brain atlas (anteroposterior: +1.7 mm from pregma, mediolateral: ±1.3 mm, dorsoventral: 5.3 mm from dura) (8). This position was chosen because good correlation has been reported between cocaine and dopamine concentrations in the nucleus accumbens, and accumbal dopamine transmission plays a significant role in mediating the enhanced locomotor responses to psychostimulants (9).

Animal Recovery. While the rat was still anesthetized after cannulation of the femoral artery and the femoral vein, cannulae were connected to a three-channel swivel. The microdialysis probe (CMA-12; CMA/Microdialysis, Acton, MA) was inserted into the guide cannula, and the probe inlet was connected to the third channel of the swivel. The rat was allowed to recover for at least 2 days before the experiment. During the recovery period, the femoral vein and the femoral artery cannulae were filled with heparinized saline (20 units/ml) to avoid blood clotting. Also, the microdialysis probe was continuously perfused with simulated cerebrospinal fluid (5 mM KCl, 120 mM NaCl, 1.4 mM CaCl₂, 1.2 mM MgCl₂, 5 mM glucose, and 0.2 mM phosphate-buffered saline to adjust the pH to 7.4 and the sodium concentration to 120.7 mM) at rate of 1 µl/min. This recovery period after the insertion of the microdialysis probe was necessary to allow enough time for the tissue surrounding the tip of the probe to heal before the actual microdialysis experiment (10).

Experimental Design. Each rat received cocaine (30 mg/kg, ip) either alone or in combination with alcohol, in a balanced cross-over experimental design allowing 48-hr washout period between treatments. Alcohol was administered as constant rate iv infusion (28 mg/kg/min) via the femoral artery. This alcohol dose was chosen to achieve steady-state alcohol plasma concentrations of ~100-300 mg/dl based on the reported alcohol pharmacokinetic parameters in the rat (6). During cocaine + alcohol treatment, the alcohol iv infusion was started 2 hr before cocaine administration and continued throughout the sampling period. After cocaine administration, eight blood samples each of 0.2 ml were collected over a 4-hr period, through the femoral vein, and the microdialysis probe effluent was collected every 20 min.

Sample Analysis. One plasma sample drawn at the end of the experiment was analyzed for alcohol by fluorescence polarization immunoassay using the Abbott ADx analyzer (Abbott Diagnostics, Chicago, IL). Samples were analyzed for cocaine and cocaethylene by HPLC using slight modification of the method described previously (6). Briefly, after spiking 100 µl of plasma samples with 50 µl of the internal standard solution (8 µg/ml, bupivacaine) and the addition of 0.5 ml of phosphate buffer (pH 6), samples were extracted by shaking with 10 ml of chloroform for 10 min. The chloroformic extract was evaporated under nitrogen, and the residue was reconstituted in the 100 µl mobile phase, then 35 µl was injected into the HPLC (Shimadzu LC-10A; Columbia, MD). The mobile phase consisted of methanol:acetonitrile:50 mM ammonium phosphate (5:7:63), and the flow rate was 0.4 ml/min. Separation was achieved using a Supelcosil ABZ+plus (25 × 0.21 cm, 5 µm) column (Supelco, Bellefonte, PA). The column effluent was monitored at 235 nm, and peak heights were calculated using an electronic integrator. Concentrations of cocaine and cocaethylene in the samples were determined by comparing the peak height ratios in the samples with the peak height ratios obtained from the standard curve.

Pharmacokinetic and Statistical Analysis. The AUC of cocaine and cocaethylene from time 0 to 4 hr after cocaine administration (AUC_0-4) were calculated by the linear trapezoidal rule and extrapolated to time infinity to obtain AUC_0-. The apparent TBC/F was calculated by noncompartmental analysis, and the apparent V_d/F was calculated from TBC/F and the elimination rate constant. Plasma and brain extracellular fluid concentration-time profiles were analyzed by two-way analysis of variance, with repeated measures on both factors using SAS (SAS Institute, Inc., Cary, NC). Treatment (cocaine only or cocaine + alcohol) and time were considered the between subject variability. Multiple comparisons with Bonferroni corrections were conducted to examine differences between treatments at each time point. A difference of p < 0.05 was considered significant.

	Results and Discussion

Abstract Introduction Results & Discussion References

The average alcohol plasma concentration achieved during the iv infusion was 365 ± 36 mg/dl. These alcohol concentrations are in the range of concentrations observed after moderate to heavy alcohol drinking. The average plasma cocaine concentrations were higher after cocaine + alcohol, compared with after cocaine only. Statistical analysis showed that the average plasma cocaine concentrations after cocaine + alcohol were significantly higher at 5 and 15 min, indicating faster rate of cocaine absorption (fig. 1). The average cocaine plasma AUC increased from 1860 ± 210 µg-hr/liter after cocaine only to 2460 ± 389 µg-hr/liter after cocaine + alcohol (p = 0.09, paired t test). This increase in cocaine AUC resulted from a proportional decrease in TBC/F from 6.08 ± 0.59 liters/hr to 5.02 ± 0.72 liters/hr (p = 0.08, paired t test). Also, cocaine maximum plasma concentration significantly increased from 2760 ± 238 µg/liter after cocaine only to 3891 ± 535 µg/liter after cocaine + alcohol (p < 0.05, paired t test). Cocaine elimination half-life was not different after the two treatments (32.1 ± 5.8 min after cocaine only and 30.1 ± 4.4 min after cocaine + alcohol), indicating that the administered alcohol iv infusion did not affect the cocaine elimination rate. These results suggest that the higher cocaine plasma concentration after cocaine + alcohol primarily resulted from faster rate of cocaine absorption. The calculated V_d/F significantly decreased from 4.69 ± 0.46 liters after cocaine only to 3.64 ± 0.52 liters after cocaine + alcohol (p < 0.05, paired t test). This decrease in V_d/F suggests that alcohol coadministration may also affect cocaine tissue distribution or the extent of its absorption after ip administration.

View larger version (12K): [in this window] [in a new window]   Fig. 1.   Cocaine plasma concentration-time profiles. Cocaine plasma concentration-time profiles after administration of () cocaine only and () cocaine + alcohol. Cocaine AUC increased from 1860 ± 210 µg-hr/liter after cocaine only to 2460 ± 389 µg-hr/liter after cocaine + alcohol. The half-life of cocaine was not different after the two treatments. Plasma concentrations are shown as mean ± SEM (N = 8). Asterisks indicate significantly higher concentrations.

The increase in plasma cocaine concentration resulted in proportional increase in brain extracellular fluid cocaine concentration. The average cocaine brain extracellular fluid concentrations were higher after cocaine + alcohol, compared with after cocaine only. The average brain extracellular fluid cocaine concentrations were significantly higher after cocaine + alcohol at 50 and 70 min (fig. 2). Cocaine AUC in the brain extracellular fluid increased from 1990 ± 288 µg-hr/liter when cocaine was administered alone to 2520 ± 327 µg-hr/liter after administration of cocaine + alcohol (p = 0.16, paired t test). This proportional increase in brain extracellular fluid AUC suggests that higher plasma cocaine concentrations will lead to higher brain cocaine concentrations and can result in augmentation of the central nervous system effects of cocaine.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Cocaine brain extracellular concentration-time profiles. Cocaine brain extracellular fluid concentration-time profiles after administration of () cocaine only and () cocaine + alcohol. Cocaine brain AUC increased from 1990 ± 288 µg-hr/liter after cocaine only to 2520 ± 327 µg-hr/liter after cocaine + alcohol. Concentrations are shown as mean ± SEM (N = 8). Asterisks indicate significantly higher concentrations.

There was no evidence that alcohol affected the cocaine brain/plasma distribution ratio. The ratios of cocaine AUC in brain extracellular fluid to that in plasma were not different when cocaine was administered alone and in combination with alcohol (1.20 ± 0.18 after cocaine only and 1.14 ± 0.18 after the combination). These results suggest that the increase in the brain extracellular fluid cocaine concentration resulted from the higher plasma cocaine concentration. Previous investigations reported a significantly higher brain tissue/plasma concentration ratio for cocaine (a ratio of 5:7), compared with the brain extracellular fluid/plasma concentration ratio reported in our study (12, 13). This difference resulted from the fact that microdialysis samples extracellular fluid rather than tissue concentrations of cocaine and, therefore, only the free soluble fraction is sampled.

Cocaethylene was detected only in rats after administration of cocaine + alcohol. Plasma cocaethylene concentrations increased gradually, reaching the maximum value in 15 to 30 min, whereas the maximum cocaethylene concentration in the brain extracellular fluid was achieved in 50-70 min (fig. 3). The average cocaethylene AUC in plasma was 241 ± 24 µg-hr/liter, whereas that in the brain extracellular fluid was 253 ± 48 µg-hr/liter. The brain extracellular fluid to plasma AUC ratio was 1.05 ± 0.15, which is approximately similar to that of cocaine. It has been reported that ip administration of equimolar doses of cocaine and cocaethylene in rats resulted in lower brain cocaethylene concentrations (14), which may seem contradictory to our results. Although the plasma cocaine and cocaethylene concentrations were not determined in this study (14), other investigators have shown that the bioavailability of cocaethylene after ip administration is much lower than that of cocaine (15). This means that, after ip administration, the lower cocaethylene bioavailability resulted in lower plasma and lower brain cocaethylene concentrations, compared with the plasma and brain concentrations of cocaine. Our results showed that the brain extracellular fluid/plasma distribution ratio of cocaine and cocaethylene are similar. This indicates that, after cocaethylene formation, it is distributed into the brain and can contribute to the pharmacological effects produced after administration of cocaine and alcohol.

View larger version (13K): [in this window] [in a new window]   Fig. 3.   Plasma and brain extracellular fluid concentration-time profiles of cocaethylene. Plasma () and brain extracellular fluid () concentration-time profiles of cocaethylene after administration of cocaine + alcohol. Cocaethylene AUC in plasma was 241 ± 24 µg-hr/liter, and in brain extracellular fluid was 253 ± 48 µg-hr/liter. Concentrations are shown as mean ± SEM (N = 8).

In conclusion, coadministration of alcohol with cocaine can cause higher cocaine plasma and brain extracellular fluid concentrations. This increase in cocaine plasma concentrations is primarily due to the effect of alcohol on the rate of cocaine absorption. Although the brain extracellular fluid/plasma distribution ratio was not affected by alcohol coadministration, the increase in plasma cocaine concentrations resulted in a proportional increase in brain extracellular fluid cocaine concentrations. This higher brain extracellular fluid concentration, in addition to the pharmacologically active cocaethylene, are responsible for the increased and longer lasting cocaine effects reported after administration of this drug combination. Because of the clinical importance of this drug interaction, it is important to correlate the changes in cocaine pharmacokinetics with the resulting pharmacological effects and also to determine the contribution of cocaethylene to the observed effects after administration of this drug combination.