LC/MS/MS evaluation of cocaine and its
metabolites in different brain areas, peripheral
organs and plasma in cocaine self-administering rats
Beata Bystrowska1, Przemys³aw Adamczyk2, Andrzej Moniczewski1, Magdalena Zaniewska2, Kjell Fuxe3, Ma³gorzata Filip1,2
1Department of Toxicology, Collegium Medicum, Jagiellonian University, Medyczna 9, PL 30-688 Kraków, Poland
2Laboratory of Drug Addiction Pharmacology, Department of Pharmacology, Institute of Pharmacology Polish Academy of Sciences, Smêtna 12, PL 31-343 Kraków, Poland
3Department of Neuroscience, Karolinska Institutet, SE-171 77 Stockholm, Sweden
Correspondence: Beata Bystrowska, e-mail: bbystrow@cm-uj.krakow.pl
Abstract:
Background: We employed a cocaine intravenous self-administration model based on positive reinforcement of animals’ instru- mental reactions (i.e., lever pressing) rewarded by a dose of the drug. We also carried out simultaneous characterization of the phar- macokinetics of cocaine and its metabolites in rats during withdrawal; in this part of the experiments, we investigated the cocaine (2 mg/kg, iv)-induced changes in the distribution, rate constant, clearance and t1/2of the parent drug and its metabolites in different structures of the brain and in peripheral tissues.
Methods: By using liquid chromatography-tandem mass spectrometry (LC/MS/MS) we measured the levels of cocaine and its ma- jor metabolites.
Results: Our results demonstrate differences in the levels of cocaine after cocaine self-administration in the rat, with the highest con- centration seen in the striatum and the lowest in the cerebellum. Cocaine metabolites determined in the rat brain remained at very low levels (benzoylecgonine), irrespectively of the brain area, whereas the norcocaine concentration varied from 1.56 µg/g (the nucleus accumbens) to 2.73 µg/g (the striatum).
Conclusion: A tandem LC/MS/MS is a valid method for evaluation of brain and peripheral levels of cocaine and its metabolites. Our results demonstrate brain area-dependent differences in the levels of cocaine after its self-administration in the rat. There were also differences in pharmacokinetic parameters among the brain areas and peripheral tissues following a bolus iv injection of cocaine to rats withdrawn from cocaine; among brain structures the slowest metabolic rate was detected for the striatum.
Key words:
cocaine, norcocaine, benzoylecgonine, metabolism, brain structures, LC/MS/MS, kinetic
Abbreviations: BE – benzoylecgonine, CER – cerebellum, EME – ecgonine methyl ester, FC – frontal cortex, HIP – hip- pocampus, LC/MS/MS – liquid chromatography with tandem mass spectrometry, NAC – nucleus accumbens, NC – norco- caine, PFC – prefrontal cortex, STR – striatum
Introduction
Cocaine is an abused psychostimulant and cocaine ad- diction is a grave socio-medical burden affecting the
whole human population (UNDOC, 2010). Being a lipophilic compound, cocaine easily penetrates the blood-brain barrier and both in the periphery and in the brain its mechanism of action includes interaction with aminergic (dopamine, noradrenaline and sero- tonin) neurotransmitter systems via binding to their transporter sites and reuptake inhibition [3, 11, 33, 38]. Apart from its direct pharmacological actions, cocaine is degraded in the blood, peripheral tissues and brain to many pharmacologically active metabo- lites (Fig. 1). Studies in humans and animals have demonstrated two major metabolic transformations of cocaine: (i) hydrolysis by esterases in blood and tis- sues (accounting for 80–90% of total elimination) and (ii) oxidation by microsomal mixed-function oxidases (i.e., cytochrome P-450 (CYP) enzymes), mainly in the liver [26, 43]. The hydrolytic pathway of cocaine metabolism in the body includes rapid inactivation of the ester bonds to form benzoylecgonine (BE) and ecgonine methyl ester (EME) (Fig. 1). BE is formed by nonenzymatic hydrolysis, while EME formation is catalyzed by esterases localized in the liver and serum [42]. Both BE and EME are bioactive metabolites of cocaine [30, 41], less toxic than the parent drug [46]
and having an approximately five times longer half- life (t1/2) in biological matrices than cocaine [7, 29, 45, 46]. Ten percent of cocaine administered to the body is catalyzed by the liver microsomal reactions via CYP2B1 enzymes in rats [2], and by CYP3A en- zymes in mice and humans [34–36] leading to the for- mation of a pharmacologically active N-demethylated metabolite, norcocaine (NC). NC has a greater tissue af- finity than cocaine [1, 2, 46] but the pharmacologic or toxic effects of cocaine and norcocaine are similar [32, 48]. Like cocaine, lipophilic NC can penetrate to the brain and has been isolated from brain tissue minutes af- ter systemic administration [24, 31] indicating that this metabolite is either formed in or enters the brain.
The aim of the present study was to measure the lev- els of cocaine and its major metabolites (BE and NC) in several brain structures (dorsal striatum (STR), nu- cleus accumbens (NAC), prefrontal cortex (PFC), frontal cortex (FC), hippocampus (HIP) and cerebel- lum (CER)), some peripheral tissues (heart, liver and kidney) and in serum in rats addicted to cocaine. To this end, we employed a cocaine intravenous self- administration model based on positive reinforcement of animals’ instrumental reactions (i.e., lever press- ing) rewarded by a dose of the drug. We also carried out simultaneous characterization of the pharmacoki- netics of cocaine and NC in rats with stabilized co-
caine self-administration and extinction; in this part of experiments we investigated the cocaine (2 mg/kg, iv)-induced changes in the distribution, rate constant, clearance and t1/2of the parent drug and its metabo- lites in different structures of brain and peripheral tis- sues. The cocaine, BE and NC levels were measured by liquid chromatography-tandem mass spectrometry (LC/MS/MS).
Materials and Methods
Animals
Male Wistar rats (280–300 g; n = 26) delivered by a li- censed breeder (Charles River, Germany) were housed individually in standard plastic rodent cages in a col- ony room maintained at 20 ± 1°C and at 40–50% hu- midity under a 12-h light-dark cycle (lights on at 6:00).
Animals had free access to standard animal food and water during the 7-day habituation period. All experi- ments were conducted during the light phase of the light-dark cycle (between 8:00–15:00) and were car- ried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Ani- mals and with approval of the Bioethics Commission as compliant with the Polish Law (21 August 1997).
The animals were experimentally naive.
Drugs
Cocaine hydrochloride (Sigma-Aldrich, USA), dis- solved in sterile 0.9% NaCl, was used. Cocaine was given iv (0.1 ml/infusion or 1 ml/kg).
Cocaine self-administration procedure
Rats (n = 10) were trained to press the lever of stan- dard operant conditioning chambers (Med-Associates, USA) under a fixed ratio 5 schedule of water rein- forcement. Two days following “lever-press” training and free access to water, the rats were chronically im- planted with a silastic catheter in the external right jugular vein, as described previously [17]. Catheters were flushed every day with 0.1 ml of saline solution containing heparin (70 U/ml) and 0.1 ml of solution of cephazolin (10 mg/ml; Biochemie GmbH, Austria).
After a 10-day recovery period, all animals were wa-
Fig. 1. Metabolic pathways of cocaine
ter deprived for 18 h and trained to lever press to fixed ratio 5 schedule of water reinforcement over a 2-h ses- sion. Subjects were then given access to cocaine during 2-h daily sessions performed 6 days/week (mainte- nance). The house light was illuminated throughout each session. Each completion of five presses on the
“active” lever complex (fixed ratio 5 schedule) re- sulted in a 5-s infusion of cocaine (0.5 mg/kg per 0.1 ml) and a 5-s presentation of a stimulus complex (activation of the white stimulus light directly above the “active” lever and the tone generator, 2,000 Hz;
15 dB above ambient noise levels). Following each in- jection, there was a 20-s time-out period during which responding was recorded but had no programmed consequences. Response on the “inactive” lever never resulted in cocaine delivery. Acquisition of the condi- tioned operant response lasted a minimum of 10 days until subjects met the following criteria: minimum re- quirement of 22 reinforcements with an average of 4 days and active lever presses with an average of 4 consecutive days and a standard deviation within those 4 days of < 10% of the average; this criterion was selected based on our prior experiments [14, 15].
After the last session all the animals were decapitated, the brain structures were dissected, and peripheral organs were removed, and frozen on dry ice. The trunk blood was collected to EDTA-filled tubes, samples were centri- fuged at 800 × g for 15 min and plasma was removed.
Next, the tissues were stored at –80°C until analyses.
Pharmacokinetic analyses
After 16 cocaine self-administration sessions (see above), a separate group of animals (n = 16) under- went a 10-day withdrawal (no access to cocaine).
Later on, the animals were randomly assigned to four groups (n = 4/group) that were given a single dose of COC (2 mg/kg) in a bolus iv injection. Rats were de- capitated following either 5, 15, 30 or 60 min, their brain structures were dissected and peripheral organs were removed on dry ice. The tissues were stored at –80°C until analyses.
Measurement of cocaine and its metabolite levels
Chemicals and reagents
LC/MS/MS grade water, acetonitrile, chloroform, for- mic acid, methanol and sodium carbonate (Na2CO3) were obtained from Merck (Germany). Cocaine, BE,
NC and their deuterated derivatives used as internal standards, were provided by Sigma-Aldrich (St.
Louis, USA). A working internal standard solution of COC-d3 and BE-d3 was prepared at a concentration 5 µg/ml in methanol. All standards were stored at 4°C in the dark.
Sample preparation
Frozen tissues were weighed, thawed at room tem- perature and homogenized in mixture of chloroform and methanol (2:1; v/v) in proportion 10 mg tissue per 150 µl of mixture. Next, 0.15 ml of homogenate were mixed with 2 µl of internal standard in methanol and 250 µl of 0.1 M Na2CO3. Chloroform and methanol (2:1; v/v) were added and the samples were shaken for 10 min on an oscillating shaker. The samples were then centrifuged for 10 min at 2,000 × g and the lower organic layer was collected. The organic phase was evaporated to dryness under a stream of nitrogen at 40°C. The residues were reconstituted in 40 µl of ace- tonitrile and subjected to chromatographic analysis.
Figure 2 shows the extracted ion current (XIC) for a sample of brain tissue as an example.
Calibration curves
Calibration curves were constructed for every batch of samples. The homogenized tissue samples were spiked with cocaine, BE and NC to the following con- centration: blank, 0.10, 0.50, 1.00, 1.50, 50.00 µg/g wet tissues. Cocaine-d3 and BE-d3 were used as the internal standard. These samples were prepared according to the procedure described above (sample preparation section). The number of QC’s per batch was six (three concentration’s level in duplicate). They were assessed according to 4-6-20 rule [5, 9, 25].
LC/MS/MS conditions
Separation of COC and metabolites was performed on a reversed-phase, high performance liquid chromatog- raphy (HPLC) system Agilent 1100 series (Agilent Technologies, USA), equipped with a binary pump, an autosampler and degasser. Separation was per- formed using a LiChrospher 60 RP-select B column (125 mm × 4.6 mm ID, 5 µm particle size) in combi- nation with an appropriate guard column (4 mm × 4 mm;
5 µm particle size) (Merck, Germany). The column was thermostated at 30°C. The optimized mobile
phase solvents were: 0.1% formic acid in water (phase A) and 0.1% formic acid in acetonitrile (phase B). Cocaine and its metabolites were separated at a flow rate of 0.7 ml/min with a linear gradient.
Mass spectrometric detection was performed using a tandem quadruple mass spectrometer API 2000 (Applied Biosystems) equipped with an ESI source (electrospray). Mass spectral data were obtained in positive electrospray mode. High purity nitrogen used as a sheath gas, was generated with a nitrogen generator.
All experiments were carried out in the positive ion mode. The main working parameters of the mass spec- trometer were as follows: nitrogen (sheath gas) flow rate 20 l/min, ion spray voltage 5,000, capillary temperature 300°C. The quantization analysis was performed using the MRM mode and tandem LC/MS/MS. The following pairs of ions were monitored with the following values of m/z: 304.1/182.3 for cocaine; 290.2/168.3 for BE and NC; 307.3/185.3 for COC-d3and 293.3/171.3 for BE-d3. Data were analyzed by using the Analyst software 1.4
(Perlan Technologies). Levels of cocaine and its me- tabolites were calculated using the calibration standard curves, constructed by linear regression analysis of peak area versus concentration curves.
Statistical analyses
Data were expressed as the mean (± SEM). For co- caine self-administration, the number of responses on the active and inactive lever (including time out re- sponding) as well as cocaine infusions were analyzed by one- or two-way analysis of variance (ANOVA) for repeated measures, respectively; a post-hoc Dun- nett’s or Duncan’s test was used to analyze differ- ences between daily sessions. Tandem LC/MS/MS data were analyzed by one-way ANOVA; a post-hoc Dunnett’s test was used to analyze differences be- tween group means; p < 0.05 was considered statisti- cally significant for all tests.
Cocaine- (IS)
Cocaine
Norcocaine Benzoylecgonine-
Benzoylecgonine
Cocaine-d (IS)
Cocaine
Norcocaine Benzoylecgonine-
Benzoylecgonine 1.3e4
1.2e4 1.1e4
9000.0 8000.0 1.0e4
7000.0 6000.0
4000.0 3000.0 5000.0
2000.0 1000.0 0.0
Intensity,cps
0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 7.5 8.0 8.5 9.0 9.5 Time, min
d (IS)
Fig. 2. An example of the extracted ion current for a sample of brain tissue; IS – internal standard
Results
Cocaine self-administration
After 16 self-administration sessions rats reached a stable lever responding during the sessions with an acquisition criterion requiring that the rate of active lever presses varied by less than 10%. Following 16 sessions of cocaine self-administration, animals re- ceived approximately 166.8 ± 5.7 mg/kg of the drug throughout the experiment. Before decapitation (the last experimental 2-h session), the animals self- administered about 24–32 injections of cocaine with the daily mean cocaine intake between 12.7–16.3 mg/kg (Tab. 2). Rats responded significantly more frequently on the active lever than on the inactive lever (p <
0.001), independently of self-administration day.
Validation LC/MS/MS analyses
The method was validated for the following parame- ters: linearity, recovery, sensitivity, precision, accu- racy, selectivity and stability, limits of quantification and detection. It showed good linearity (r2³ 0.99) in the range 0.1–50 µg/g. The lower limit of detection was 0.05 µg/g in all tissues. The parameters of valida- tion are presented in Table 1.
Cocaine and metabolite levels
Peripheral organs and plasma
As shown in Table 2, after the last cocaine self- administration session the levels of cocaine reached 0.211–10.45 µg/g tissue. The maximum level of co- caine was recorded in the heart, followed by the kid- ney, while in the serum and liver cocaine concentra- tions were at similar levels and were ca. 40 times lower than in the heart.
Table 2 also shows the mean levels of cocaine me- tabolites in serum and peripheral organs. The BE and NC concentration levels were the highest in the kid- ney, followed by the liver and serum, while in the heart the formation of these metabolites was minimal.
Tab. 2. Concentration of cocaine, benzoylecgonine and norcocaine in peripheral organs and plasma following cocaine self-administration
Tissue COC (iv) self-administration
(mg/kg/injection)
Tissue concentration (µg/g)
BE/COC NC/COC
COC BE NC
total (16 sessions)
last session Serum
166.8 ± 5.7 14.5 ± 1.8
0.2880 ± 0.03279 0.7960 ± 0.1045g,h,i 0.0890 ± 0.01456 2.76r,s 0.31y,z,aa Liver 0.2110 ± 0.05305 1.474 ± 0.2131 0.1370 ± 0.02708j,k 6.99t,u,v 0.65ab,ac,ad Kidney 3.586 ± 0.4677a,b,c 4.532 ± 0.5098 0.3590 ± 0.08563l,m,n 1.26w,x 0.10ae,af,ag Heart 10.47 ± 1.875d,e,f 0.1275 ± 0.01411 0.03711 ± 0.001369o,p,q 0.01 0.003
COC – cocaine, BE – benzoylecgonine, NC – norcocaine. N = 10 rats.ap < 0.0001 vs. COC (serum);bp < 0.0001 vs. COC (liver);cp < 0.0001 vs.COC (heart);dp < 0.0001 vs. COC (serum);ep < 0.0001 vs. COC (liver);fp < 0.0001 vs. COC (kidney);gp < 0.05 vs. BE (liver);hp < 0.0001 vs.BE (kidney);ip < 0.0001 vs. BE (heart);jp < 0.05 vs. NC (kidney);kp < 0.05 vs. NC (heart);lp < 0.05 vs. NC (serum);mp < 0.05 vs. NC (liver);np < 0.05 vs. NC (heart);op < 0.05 vs. NC (serum);pp < 0.05 vs. NC (liver);qp < 0.05 vs. NC (kidney);rp < 0.0001 vs. BE/COC (liver);
sp < 0.0001 vs. BE/COC (heart);tp < 0.0001 vs. BE/COC (heart);up < 0.0001 vs. BE/COC (kidney);vp < 0.0001 vs. BE/COC (serum);wp <
0.05 vs. BE/COC (heart);xp < 0.0001 vs. BE/COC (liver);yp < 0.0001 vs. NC/COC (heart);zp < 0.0001 vs. NC/COC (liver);aap < 0.0001 vs.
NC/COC (kidney);abp < 0.0001 vs. NC/COC (serum);acp < 0.0001 vs. NC/COC (kidney);adp < 0.0001 vs. NC/COC (heart);aep < 0.001 vs.
NC/COC (serum);afp < 0.001 vs. NC/COC (liver);agp < 0.001 vs. NC/COC (heart)
Tab. 1. Parameters of validation for cocaine, benzoylecgonine and norcocaine
Drug Equation of the calibration curve
R2 CV (%)
COC y = 0.3939x – 0.4491 0.9909 1.92–9.77 BE y = 0.182x – 0.2558 0.9975 4.44–7.43 NC y = 0.0247x – 0.0632 0.9936 2.29–11.02
COC – cocaine, BE – benzoylecgonine, NC – norcocaine
As expected, the liver showed the highest meta- bolic activity to form BE (6.99) and NC (0.65).
Among the investigated tissue, the heart was the one with the lowest rate of metabolism amounting to 0.003 (NC/cocaine) and 0.01 (BE/cocaine) (Tab. 2).
Brain areas
The levels of cocaine and its metabolites in cocaine self-administering rats differed in dependence on the rat brain area. As shown in Table 3, cocaine levels ranged from 0.94–1.77 µg/g; the highest concentra- tion was seen in the STR while the lowest in the CER.
Both cocaine metabolites were detected in the rat brain: BE concentrations were at the same (very low) level (0.29–0.34 µg/g) irrespectively of the brain area, whereas the NC concentration varied from 1.56 µg/g (the NAC) to 2.73 µg/g (the STR).
Table 3 presents the mean levels of cocaine me- tabolites in rat brain areas. Two groups of brain areas with similar metabolic rates can been distinguished.
The first group with a faster metabolism included the FC, HIP and CER. In this group the metabolic rates remained similar with respect to BE/cocaine (0.30–0.32) and NC/cocaine (1.89–2.27). The other selected brain areas showed significantly lower meta- bolic rate, and they included the STR, NAC and PFC.
Between these brain areas, the STR showed the slow-
est metabolic activity to form BE (0.16) while the PFC to form NC (1.01).
Pharmacokinetic analyses
Following cocaine self-administration sessions and 10-day withdrawal, rats were given a single dose of COC (2 mg/kg) in a bolus iv injection and were de- capitated following either 5, 15, 30 or 60 min. The ki- netic analyses included cocaine and NC, while BE was not under similar analyses in the present study because its levels due to a long t1/2were equal during the study period (5–60 min).
Figure 3 shows the mean brain cocaine and NC concentration-time profiles for the iv cocaine. The peak cocaine and NC concentration was reached at 5 min following cocaine injection while after 60 min the parent drug and its metabolite were not detected in the brain areas. There were area-dependent differences in the concentration-time profiles for cocaine and NC as well as for their metabolic rates. The slowest metabo- lism was observed in the PFC (5–60 min), NAC, HIP and PFC (5–30 min) while the fastest in the CER.
The pharmacokinetic parameters (clearance, K1.0 and t1/2) calculated with WINNONLIN nonlinear esti- mation program (version 5.3) are presented in Table 3.
Clearance in the selected brain regions ranged from 0.03 to 0.066 g/(min × µg/g)/kg for cocaine and from
Tab. 3. Concentrations of cocaine, benzoylecgonine and norcocaine in brain areas following cocaine self-administration
Brain area
COC (iv) self-administration
(mg/kg/injection)
Tissue concentration (µg/g)
BE/COC NC/COC total
(16 sessions)
last
session Cocaine BE NC
NAC
166.8 ± 5. 7 14.5 ± 1.8
1.296 ± 0.132a,b,c 0.288 ± 0.02 1.560 ± 0.3512g 0.22j,k 1.20q,r,s,t
STR 1.775 ± 0.213 0.291 ± 0.028 2.727 ± 0.8324h 0.16l,m,n 1.54u,v,w,x
PFC 1.675 ± 0.224d,e,f 0.336 ± 0.027 1.695 ± 0.6250i 0.20o,p 1.01y,z,aa
FC 1.069 ± 0.1534 0.3440 ± 0.036615 2.332 ± 0.7368 0.32 2.18
HIP 0.9750 ± 0.2379 0.2910 ± 0.01841 2.217 ± 0.7663 0.30 2.27
CER 0.9400 ± 0.2031 0.2900 ± 0.01880 1.777 ± 0.3534 0.31 1.89
COC – cocaine, BE – benzoylecgonine, NC – norcocaine, CER – cerebellum, FC – frontal cortex, HIP – hippocampus, NAC – nucleus accum- bens, PFC – prefrontal cortex, STR – striatum. N = 10 rats.ap < 0.05 vs. COC (NAC);bp < 0.05 vs. COC (FC);cp < 0.05 vs. COC (CER);dp <
0.05 vs. COC (FCX). ep < 0.05 vs. COC (HIP).fp < 0.05 vs. COC (CER);gp < 0.05 vs. NC (STR);hp < 0.001 vs. NC(PFC);ip < 0.05 vs. NC (FC);jp < 0.05 vs. BE/COC (HIP);kp < 0.05 vs. BE/COC (CER);lp < 0.05 vs. BE/COC (FC);mp < 0.05 vs. BE/COC (HIP);np < 0.05 vs. BE/COC (CER);op < 0.05 vs. BE/COC (HIP);pp < 0.05 vs. BE/COC (CER);qp < 0.05 vs. NC/COC (PFC);rp < 0.001 vs. NC/COC (FC);sp < 0.001 vs.
NC/COC (HIP);tp < 0.001 vs. NC/COC (CER);up < 0.05 vs. NC/COC (PFC);vp < 0.001 vs. NC/COC (FC);wp < 0.001 vs. NC/COC (HIP);xp <
0.001 vs. NC/COC (CER);yp < 0.001 vs. NC/COC (FC);zp < 0.001 vs. NC/COC (HIP);aap < 0.001 vs. NC/COC (CER)
Fig. 3. Cocaine (COC) (a), norcocaine (NC) (b) concentration-time profiles, and metabolic rate NC/COC (c) in rats given a bolus (iv) injection of 2 mg/kg cocaine; n = 4 rats/group for the particular time (5, 15, 30 or 60 min) analyses
0.034 to 0.058 g/(min × µg/g)/kg for NC. The fastest cocaine clearance was observed for in the NAC and CER while NC clearance in the PFC and CER.
The maximum K1.0for cocaine was detected in the FC, HIP and CER (0.11–0.15 µg/min), while the minimum K1.0 values (0.045–0.08 µg/min) in the NAC, STR and PFC. For NC, the highest (0.075 µg/
min) and lowest (0.025 µg/min) K1.0levels were seen in the STR and the PFC, respectively.
There were differences in t1/2values of cocaine and NC between brain structures. For cocaine the longest t1/2(> 12 min) was detected in the STR and NAC, the shortest (ca. 4.5–6 min) in the FC, HIP and CER. For NC, average values of t1/2 were estimated at from 9.18 min (STR) to 27.94 min (PFC).
In peripheral organs, the maximal cocaine and nor- cocaine clearance was observed in the heart (0.119 g/
(min × µg/g)/kg) and the liver (0.056 g/(min × µg/
g)/kg), respectively. The lowest value of cocaine K1.0 was observed in the liver (0.007 µg/min), while the highest in the kidney (0.145 µg/min). For the cocaine metabolite, the K1.0values ranged from 0.045 µg/min (heart) to 0.05 µg/min (liver).
The longest t1/2 for cocaine was seen in the liver (> 104 min) since the drug concentrations were simi- lar at different time points (after 5 min – 0.1810 µg/g, after 15 min – 0.152 µg/g; after 30 min – 0.1284 µg/g;
after 60 min – 0.1272 µg/g). In other peripheral organs t1/2for cocaine varied from 4.7 to 7.8 min.
For NC, the longest t1/2(26.32 min) was found in the kidney and the shortest (7.28 min) in the liver.
The summary of kinetic parameters for cocaine and norcocaine are shown in Table 4.
Discussion
This study documents the levels of cocaine and its metabolites in several brain areas, peripheral organs and plasma of animals undergoing cocaine self- administration. Additionally, it was also of interest to characterize some kinetic parameters and distribution of cocaine and NC in the rat brain following with- drawal from cocaine self-administration and exposure to a bolus injection of cocaine.
To the best of our knowledge, this is the first at- tempt to simultaneously characterize the tissue levels of cocaine and its metabolites during voluntary iv co- caine intake in rats using tandem LC/MS/MS. Numer- ous brain regions have been examined, with particular attention to the structures engaged in the rewarding and/or motivational effects of cocaine (e.g., NAC, STR, PFC, HIP [10, 18, 23, 40]. Our results demon- strated that cocaine self-administration over 16 days
Tab. 4. Summary of kinetic parameters for cocaine and norcocaine
Tissue
CL g/(min ´ µg/g)/kg
K1.0
(1 µg/min)
t1/2
(min)
Cocaine Norcocaine Cocaine Norcocaine Cocaine Norcocaine
NAC 0.061 n.c. 0.056 n.c. 12.376 n.c.
STR 0.030 0.034 0.045 0.075 15.549 9.184
PFC 0.052 0.058 0.080 0.025 8.661 27.948
FC 0.049 0.044 0.116 0.053 5.980 13.045
HIP 0.034 0.045 0.151 0.041 4.583 16.885
CER 0.066 0.053 0.120 0.044 5.756 15.834
Liver 0.076 0.056 0.007 0.095 104.709 7.283
Kidney 0.027 0.031 0.145 0.068 4.794 26.324
Heart 0.119 0.042 0.090 0.045 7.736 15.247
CER – cerebellum, FC – frontal cortex, HIP – hippocampus, NAC – nucleus accumbens, PFC – prefrontal cortex, STR – striatum; CL – clear- ance, K1.0– the elimination rate constant from the central compartment, t1/2– elimination half-life; n.c. – not calculated
resulted in rat brain area-dependent differences in the levels of the drug with the highest cocaine concentra- tions in the STR and PFC (1.68–1.78 µg/g tissue) and the lowest in the HIP and CER (0.94–0.98 µg/g tis- sue). It should be added that the concentrations of co- caine and its metabolites in brain tissues of rats re- ceiving the drug passively (so-called yoked cocaine animals) reached the same levels as in rats self- administered cocaine; in other words the active vs.
passive cocaine groups were not statistically different (data not shown). In other words, the maximal con- centration of cocaine appeared in the rat brain struc- tures importantly implicated in the addiction pro- cesses and contributed to these processes. Till now only two reports described brain cocaine levels in rats trained to self-administer this drug [11, 37] but those authors did not separate particular brain areas. The differences reported in the present study are in line with previous observations on the cocaine brain distri- bution after the drug oral, ip or sc administration. For instance, in male Sprague-Dawley rats found that af- ter acute cocaine (10–20 mg/kg, ip) treatment, the me- dial PFC drug concentration was nearly 1.3- and 2- fold higher than in the STR or limbic areas (the NAC, olfactory tubercle and pyriform cortex), respectively [6]. Comparison between brain samples indicated the statistically significant differences (8–12%) between STR vs. NAC after 10 mg/kg ip [19], however, such regional concentration difference was not always ob- served after ip administration of cocaine by other authors [8, 21, 39].
The other important observation is a marked differ- ence between brain, peripheral organs and serum co- caine concentration. In fact, cocaine levels in the plasma were ca. 3.3–6.2-fold lower as compared to brain areas. Similar higher brain-lower plasma co- caine distribution was also observed after acute drug injection [6, 12, 27] while the plasma concentration following 10–20 mg/kg cocaine, ip, paralleled the range detected in the present study (0.17–0.31 µg/g).
Furthermore, we found the same range of cocaine lev- els in plasma and liver (< 0.3 µg/g tissue) whereas the heart was the organ in which measured cocaine levels were the highest among studied tissues.
The major cocaine metabolites NC and BE were detected by the tandem LC/MS/MS in both brain and peripheral organs. We observed a tissue-dependent metabolite concentration with NC preferentially pres- ent in the brain and BE dominating in peripheral or- gans and plasma. In the brain, the NC levels ranged
from 1.56 µg/g (the NAC) to 2.73 µg/g (the STR) and were 4.6–9.6 times higher than BE levels. In contrast, BE concentrations were the highest in the kidney, fol- lowed by the liver and serum while in the brain small (ca. 0.30 µg/g tissue) and almost the same concentra- tions were measured in its several areas. Interestingly, in the heart the formation of either BE or NC was small or minimal, respectively, as evidenced also by the low metabolic ratio levels (0.003–0.01). The liver was the peripheral organ with the fastest metabolic transformation of cocaine (6.99), followed by plasma (2.76) and the kidney (1.26) while in the brain two groups of areas with different metabolic rate were dis- tinguished. One group included the “faster” me- tabolizer areas (i.e., FC, HIP and CER) while the STR, NAC and PFC showed “slower” metabolism.
This finding means that in the latter brain areas, known as neuroanatomical targets for addictive be- haviors [10, 18, 23, 40], the pharmacological action of the parent drug was longer lasting around the mem- brane targets and cocaine pharmacokinetics might be a contributor to such effects.
It should be added that the tandem LC/MS/MS method permits to quantitatively measure NC plasma levels following iv cocaine as HPLC analyses in simi- lar injection condition do not allow for detection of this metabolite [20, 22, 28, 44, 45].
The second part of the study was focused on the distribution and simultaneous characterization of the pharmacokinetic parameters (clearance, rate constant and t1/2) after a bolus injection of cocaine (2 mg/kg, iv) in rats with stabilized cocaine self-administration and extinction. A simplified kinetic studies showed that cocaine was rapidly distributed in the body; in the brain the peak levels of this drug were achieved within 5 min. The distribution pattern of cocaine in rats after iv treatment was similar to that observed in cocaine human addicts [16, 47] with the preferential cocaine accumulation in the STR. Moreover, in hu- mans a temporal correspondence between the fast up- take of the drug in the brain and the subjective experi- ence of the ‘high’ was reported [47].
Limbic brain areas, including the PFC, FC and HIP as well as the CER showed a relatively uniform co- caine rate constant, clearance and a terminal elimina- tion t1/2of 5.6–8.7 min, while for the NAC and STR t1/2 were 1.5 or 2 times longer, respectively. On the other hand, the latter brain areas showed a slower kinetics, evidenced also by the shorter t1/2value for NC; such metabolic ratios prolonged the duration of cocaine ac-
tion. In peripheral organs, cocaine had the longest t1/2 in the liver while those in the heart and kidney paral- leled pharmacokinetic parameters in the brain areas.
Our study is the first to report plasma levels of NC following a bolus 2 mg/kg injection of cocaine to rats withdrawn from cocaine self-administration. We re- port that the NC peak levels occurred at 5 min, i.e., at the same time as the peak of cocaine. Moreover, t1/2 value for NC was shorter in the STR and liver or longer in the rest of brain areas and plasma peripheral organs than the t1/2 value for cocaine. It should be stressed that NC is a lipophilic drug thus it is able to penetrate to the brain and is isolated from brain tissue minutes after their systemic administration [24, 31].
The concentration of NC in the brain comes from its in situ formation or from the periphery through cross- ing the blood-brain barrier. Apart from the STR, the short time for NC elimination was detected in the liver where a close correlation between NC levels, N-demethylase activity and liver toxicity have been reported in rodents [2, 4].
The BE levels were not a subject of the pharma- cokinetic studies since the experiment stopped at 60 min following cocaine injection while the peak plasma BE levels usually occur at 1–3 h after the psy- chostimulant [13, 49].
To summarize, a tandem LC/MS/MS is a valid method for evaluation of brain and peripheral levels of cocaine and its metabolites. Our results demon- strate brain area-dependent differences in the levels of cocaine after its self-administration in the rat. There were also differences in pharmacokinetic parameters among the brain areas and peripheral tissues follow- ing a bolus iv injection of cocaine to rats withdrawn from cocaine.
Acknowledgments:
This study was supported by the grant no. N N404 273040 (Ministry of Science and Higher Education) and by the statutory activity of the Institute of Pharmacology (Kraków).
References:
1.Benuck M, Reith M, Lajtha A: Presence of the toxic me- tabolite N-hydroxy-norcocaine in brain and liver of the mouse. Biochem Pharmacol, 1988, 37, 1169–1172.
2.Boelsterli UA, Lanzotti A, Goldlin C, Oertle M: Identifi- cation of cytochrome P-450IIB1 as a cocaine-bioactivating
isoform in rat hepatic microsomes and in cultured rat he- patocytes. Drug Metab Dispos, 1992, 20, 96–101.
3.Bossert JB, Ghitza UE, Lu L, Epstein DH, Shaham Y:
Neurobiology of relapse to heroin and cocaine seeking:
an update and clinical implications. Eur J Pharmacol, 2005, 526, 36–50.
4.Boyer CS, Petersen DR: Pharmacokinetic analysis of the metabolism of cocaine to norcocaine and N-hydroxy- norcocaine in mice. Drug Metab Dispos, 1992, 20, 863–868.
5.Bressolle F, Bromet-Petit M, Audran M: Validation of liquid chromatographic and gas chromatographic meth- ods. Applications to pharmakokinetics. J Chromatogr B Biomed Sci Appl, 1996, 686, 3–10.
6.Carey RJ, DePalma G: A simple, rapid HPLC method for the concurrent measurement of cocaine and catechol- amines in brain tissue samples. J Neurosci Methods, 1995, 58, 25–28.
7.Carmonaa GN, Schindlera ChW, Greiga NH, Hollowaya HW, Jufera RA, Conea EJ, Gorelicka DA: Intravenous butyrylcholinesterase administration and plasma and brain levels of cocaine and metabolites in rats. Eur J Pharmacol, 2005, 517, 186–190.
8.Cass W, Zahniser N: Cocaine levels in striatum and nu- cleus accumbens: augmentation following challenge in- jection in rats withdrawn from repeated cocaine admini- stration. Neurosci Lett, 1993, 152, 177–180.
9.Causon R: Validation of chromatographic methods in biomedical analysis. Viewpoint and discussion.
J Chromatogr B Biomed Sci Appl, 1997, 689, 175–180.
10.Di Chiara G: From rats to humans and return: testing addiction hypotheses by combined PET imaging and self-reported measures of psychostimulant effects.
Commentary on Volkow et al. ‘Role of dopamine in drug reinforcement and addiction in humans: results from imaging studies’. Behav Pharmacol, 2002, 13, 371–377.
11.Edwards S, Whisler K, Fuller D, Orsulak P, Self D:
Addiction-related alterations in D1and D2dopamine re- ceptor behavioural responses following chronic cocaine self-administration. Neuropsychopharmacology, 2007, 32, 354–366.
12.Falk J, Ma F, Lau Ch: Chronic oral cocaine self- administration: pharmcokinetics and effects on spontane- ous and discriminative motor functions. J Pharmacol Exp Ther, 1991, 257, 457–465.
13.Festa ED, Rosso SJ, Gazi FM, Niyomchai T, Kemen LM, Lin SN, Foltz R et al.: Sex differences in cocaine- induced behavioral responses, pharmacokinetics, and monoamine levels. Neuropharmacology, 2004, 46, 672–687.
14.Fija³ K, Pachuta A, McCreary AC, Wydra K, Nowak E, Papp M, Bieñkowski P et al.: Effects of serotonin (5-HT)6 receptor ligands on responding for cocaine re- ward and seeking in rats. Pharmacol Rep, 2010, 62, 1005–1014.
15.Filip M: Role of serotonin (5-HT)2receptors in cocaine self-administration and seeking behavior in rats. Pharma- col Rep, 2005, 57, 35–46.
16.Fowler JS, Volkow ND, Wolf AP, Dewey SL, Schlyer DJ, Macgregor RR, Hitzemann R et al.: Mapping co-
caine binding sites in human and baboon brain in vivo.
Synapse, 1989, 4, 371–377.
17.Frankowska M, Go³da A, Wydra K, Gruca P, Papp M, Filip M: Effects of imipramine or GABABreceptor ligands on the immobility, swimming and climbing in the forced swim test in rats following discontinuation of cocaine self-administration. Eur J Pharmacol, 2010, 627, 142–149.
18.Fuchs RA, Eaddy JL, Su Z, Bell GH: Interactions of the basolateral amygdala with the dorsal hippocampus and dorsomedial prefrontal cortex regulate drug context-induced reinstatement of cocaine-seeking in rats.
Eur J Neuroscience, 2007, 26, 487–498.
19.Gulley J, Hoover B, Larson G, Zanisher N: Individual differences in cocaine-induced locomotor activity in rats:
behavioral characteristics, cocaine pharmacokinetics, and the dopamine transporter. Neuropsychopharmacol- ogy, 2003, 28, 2089–2101.
20.Hoja H, Marquet P, Vemeuil B, Lifti H, Penicaut B, Lachatr G: Applications of liquid chromatography – mass spectrometry in analytical toxicology. J Anal Toxicol, 1997, 21, 116–126.
21.Javaid JL, Davis JM: Cocaine disposition in discrete re- gions of rat brain. Biopharm Drug Dispos, 1993, 14, 357–364.
22.Johansen SS, Bhatia HM: Quantitative analysis of co- caine and its metabolites in whole blood and urine by high-performance liquid chromatography coupled with tandem mass spectrometry. J Chromatogr B, 2007, 852, 338–344.
23.Kalivas PW, McFarland K: Brain circuitry and the rein- statement of cocaine-seeking behavior. Psychopharma- cology, 2003, 168, 44–56.
24.Kloss MW, Rosen GM, Rauckman EJ: Biotransforma- tion of norcocaine to norcocaine nitroxide by rat micro- somes. Psychopharmacology, 1984, 84, 221–224.
25.Kringle RO: An assesment of the 4-6-20 rule for accep- tance of analytical runs in bioavailability, bioequivalence and pharmacokinetics studies. Pharm Res, 1994, 11, 24–27.
26.Laizure SC, Mandrell T, Gades NM, Parker RB: Co- caethylene metabolism and interaction with cocaine and ethanol: role of carboxylesterases. Drug Metab Dispos, 2003, 31, 16–20.
27.Ma F, Falk J, Lau Ch: Cocaine pharmacodynamics after intravenous and oral administration in rats: relations to pharmacokinetics. Psychopharmacology, 1999, 144, 323–332.
28.Ma F, Zhang J, Lau CE: Determination of cocaine and its metabolites in serum microsamples by high-performance liquid chromatography and its application to pharma- cokinetics in rats. J Chromatogr B Biomed Sci Appl, 1997, 693, 307–312.
29.Mets B, Diaz J, Soo E, Jamdar S: Cocaine, norcocaine, ecgonine methylester and benzoylecgonine pharmacoki- netics in the rat. Life Sci, 1999, 65, 1317–1328.
30.Misra AL, Nayak PK, Bloch R, Mule SJ: Estimation and disposition of [3H]benzoylecgonine and pharmacological activity of some cocaine metabolites. J Pharm Pharma- col, 1975, 27, 784–786.
31.Misra AL, Nayak PK, Patel MN, Vadlamani NL, Mule SJ: Identification norcocaine as a metabolite of [3H]-cocaine in rat brain. Experentia, 1974, 30, 1312–1314.
32.Mule S, Casella G, Misra A: Intracellular disposition of [3H]-cocaine. [3H]-norcocaine. [3H]-benzoylecgonine and [3H]-benzoylnorcocaine in the brain of rats. Life Sci, 1976, 19, 1585–1596.
33.Nestler EJ: Molecular mechanisms of drug addiction.
Neuropharmacology, 2004, 47, 24–32.
34.Pellinen P, Honkakoski P, Stenback F, Niemitz M, Alhava E, Pelkonen O, Lang MA, Pasanen M:Cocaine N-demethylation and the metabolism- related hepatotox- icity can be prevented by cytochrome P4503A inhibitors.
Eur J Pharmacol, 1994, 270, 35–43.
35.Pellinen P, Kulmala L, Kontilla J, Auriola S, Pasanen M, Juvonen R: Kinetic characterisitcs of norcocaine N-hydroxylation in mouse and human liver microsomes:
involvement of CYP enzymes. Arch Toxicol, 2000, 74, 511–520.
36.Pellinen P, Stenback F, Raunio H, Pelkonen O, Pasanen M: Modification of hepatic cytochrome P450 profile by cocaine-induced hepatotoxicity in DBA/2 mouse.
Eur J Pharmacol, 1994, 292, 57–65.
37.Piazza P, Deroche-Gamonent V, Rouge-Pont F, Le Moal M: Vertical shifts in self-administration dose-response functions predict a drug-vulnerable phe- notype predisposed to addiction. J Neurosci, 2000, 20, 4226–4232.
38.Ritz MC, Cone EJ, Kuhar MJ: Cocaine inhibition of ligand binding at dopamine. norepinephrine and sero- tonin transporters: a structure-activity study. Life Sci, 1990, 46, 635–645.
39.Ruth JA, Ullman EA, Collins AC: An analysis of co- caine effects on locomotor activities and heart rate in four inbred mouse strains. Pharmacol Biochem Behav, 1988, 29, 157–162.
40.Schmidt HD, Andersen SM, Famous KR, Kumaresan V, Pierce RCH: Anatomy and pharmacology of cocaine priming-induced reinstatement of drug seeking. Eur J Pharmacol, 2005, 526, 65–76.
41.Schuelke GS, Konkol RJ, Terry LC, Madden JA:
Effect of cocaine metabolites on behavior: possible neuroendocrine mechanisms. Brain Res Bull, 1996, 39, 43–48.
42.Shuster L, Casey E, Welankiwar SS: Metabolism of co- caine and norcocaine to N-hydroxynorcocaine. Biochem Pharmacol, 1983, 32, 3045–3051.
43.Soloway AH, Patil PN: Potential role of reactive metabo- lites of addictive drugs at the receptor: A novel hypothe- sis. Med Hypotheses, 2011, 77, 889–894.
44.Srinivasan K, Wang P, Eley T, White C, Bartlett M:
Liquid chromatography-tandem mass spectrometry analysis of cocaine and its metabolites from blood, amniotic fluid, placental and fetal tissues: study of the metabolism and distribution of cocaine in pregnant rats.
J Chromatogr B, 2000, 745, 287–303.
45.Sun L, Lau Ch: Simultaneous pharmacokinetics modeling of cocaine and its metabolites, norcocaine and benzoylecgonine, after intravenous and oral admini-
stration in rats. Drug Metab Dispos, 2001, 29, 1183–1189.
46.Toennes S, Thiel M, Walther M, Kauert G: Studies on metabolic pathways of cocaine and its metabolites using microsome preparations from rat organs. Chem Res Toxicol, 2003, 16, 375–381.
47.Volkow ND, Wang GJ, Fowler JS: Imaging studies of cocaine in the human brain and studies of the cocaine addict. Ann NY Acad Sci, 1997, 820, 41–54.
48.Wang Q, Simpao A, Sun L,·Falk JL, Lau CE: Contribu- tion of the active metabolite norcocaine to cocaine’s ef- fects after intravenous and oral administration in rats:
pharmacodynamics. Psychopharmacology, 2001, 153, 341–352.
49.Wansaw M, Lin Shen-Nan, Morrell J: Plasma cocaine levels, metabolites, and locomotor activity after subcuta- neous cocaine injection are stable across the postpartum period in rats. Pharmacol Biochem Behav, 2005, 82, 55–66.
Received: March 20, 2012; in the revised form: June 21, 2012;
accepted: June 28, 2012.
