Comparison of Cannabinoid Pharmacokinetic Properties in ...

Journal of Analytical Toxicology, Vol. 32, September 2008

Comparison of Cannabinoid Pharmacokinetic Properties in Occasional and Heavy Users Smoking a Marijuana or Placebo Joint Stefan W. Toennes1,*, Johannes G. Ramaekers2, Eef L. Theunissen2, Manfred R. Moeller3, and Gerold F. Kauert1 1Institute

of Forensic Toxicology, Institute for Legal Medicine, Goethe-University of Frankfurt, Germany; of Neuropsychology & Psychopharmacology, Faculty of Psychology, Maastricht University, The Netherlands; and 3Saarland University Hospital, Homburg, Germany

2Department

Abstract Cannabinoid pharmacokinetics in occasional users is well studied, but the interpretation of data from heavy users is difficult. In the present study, blood pharmacokinetic properties were investigated in occasional and heavy users in cannabis and placebo conditions. The results obtained with occasional users were in contrast to those of the heavy users who admitted cannabis use on 4–25 occasions during the previous week. Of the 12 heavy users, 10 exhibited up to 12.3 µg/L ∆9-tetrahydrocannabinol (THC) prior to smoking. During the 8 h after smoking, the distribution and elimination patterns were comparable to those of the occasional users and the concentrations returned to 68–196% (median 110%) of the initial values. However, the maximal concentrations and the areas under the curves were significantly higher with marked interindividual variation. In contrast to the cannabis conditions, the THC concentrations in the placebo phase decreased more slowly (elimination half-life 17.5–43.5 h vs. 1.0–5.9 h) in accordance with a late elimination phase. The elimination half-lives of 11-hydroxy-THC and 11-nor-9-carboxyTHC in the cannabis conditions (medians 3.1 h and 6.2 h, respectively) were longer than those of THC, which was different in the placebo phase (medians 7.2 h and 13.0 h, respectively). From the results, it must be cautioned that cannabinoid blood concentrations from heavy users in a late elimination phase may be difficult to distinguish from concentrations measured in occasional users after acute cannabis use.

Introduction Cannabis is the most widely used illicit drug worldwide. In experimental, placebo-controlled studies, it has been demonstrated that single doses of ∆9-tetrahydrocannabinol (THC) * Author to whom correspondence should be addressed. Dr. Stefan W. Toennes, Institute of Forensic Toxicology, Centre for Legal Medicine, Johann Wolfgang Goethe University, Kennedyallee 104, D-60596 Frankfurt am Main, Germany. E-mail: [email protected].

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cause a dose-dependent reduction in performance at laboratory tasks as well as in driving tests (1). Performance impairment is maximal during the first hour after smoking and sharply declines over 2–4 h after THC use. The forensic proof of impaired driving is usually based on the interpretation of cannabinoid levels determined in a single blood sample obtained some time after the event and the circumstances of the case. Several reviews on the pharmacokinetics of cannabinoids are available (2–6); however, most studies have focused on the acute phase. In occasional users, THC levels declined to values below 1 µg/L within approximately 6 h after smoking (7,8), whereas in heavy users, THC levels exceeding 1 µg/L may be present more than 24 h after the last use (9,10). The aim of the present study was to compare pharmacokinetic properties of cannabinoids in occasional and heavy users during 8 h after smoking a high dose of THC in a placebo-controlled study. Blood samples were obtained during both the cannabis and placebo sessions. The placebo condition of the heavy users was given special attention because residual cannabinoid concentrations due to previous use were expected.

Experimental Chemicals, reference standards, and apparatus

THC (1 mg/mL), 11-hydroxy-THC (THC-OH, 1 mg/mL), 11nor-9-carboxy-THC (THCA, 1 mg/mL), and the deuterated analogues THC-d3, THC-OH-d3, and THCA-d3 (each 0.1 mg/mL) were purchased from Cerilliant (Promochem, Wesel, Germany). The derivatization reagent N-methyl-N-trimethylsilyl trifluoroacetamide (MSTFA) was from Macherey & Nagel (Düren, Germany); all other reagents and organic solvents were of analytical grade and from Merck (Darmstadt, Germany). Gas chromatographic–mass spectrometric (GC–MS) analyses were performed on an Agilent (Waldbronn, Germany)

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Journal of Analytical Toxicology, Vol. 32, September 2008

GC–MS (6890N GC, 7683 series injector, 5973 MSD) with Varian (Darmstadt, Germany) factorFour VF-1MS capillary column (30 m × 0.25-mm i.d., 0.25-µm film thickness), helium carrier gas with a flow rate of 1.0 mL/min. The MS conditions were 280°C transferline temperature and 70 eV ionization energy. Data analysis was performed using Agilent ChemStation software. Study design

The results presented here supplement data from the study by Ramaekers et al. (11) on the influence of cannabis on cognition, impulse control, and psychomotor function in a group of 12 occasional cannabis users (8 males, 4 females) and 12 heavy cannabis users (9 males, 3 females). Specific inclusion criteria were frequent use of cannabis (> 4 times per week) during the previous year for the group of heavy users and weekly use or less for the occasional cannabis users. Urine tests for drugs of abuse were performed at the beginning of the study, where heavy users were required to exhibit a positive result for cannabinoids in contrast to the occasional users. The study was conducted according to a double-blind, placebocontrolled, two-way mixed model design and was approved by the local ethics committee. Written informed consent was obtained from each participant. Groups of occasional and heavy cannabis users received placebo or 500 µg THC per kg body weight (BW), resulting in effective doses of 22.5 to 47.5 mg of THC (median 33 mg). Both groups were in a similar range of age (heavy users 20–31 years, median 22 years, vs. occasional users 20–27 years, median 22 years) and body weight (heavy users 55–84 kg, median 66 kg, vs. occasional users 45–95 kg, median 70 kg). Marijuana cigarettes were prepared for each individual from a stock provided by the Dutch Bureau for Medicinal Cannabis (containing 13% THC), and the dosage was adjusted to each subjects’ body weight. Placebo cigarettes were formally prepared to be similar in weight and size of a marijuana cigarette, but differed in taste, as they contained no active THC. Smoking was performed according to a standard protocol (12) and lasted for approximately 10 min. Blood samples were taken at baseline, 5, 15, 30, 45, and 60 min during the first hour after smoking and then hourly up to 8 h after smoking. The blood samples were immediately centrifuged for serum separation; the serum was stored at –20°C and shipped to the laboratory in Frankfurt/Main. Determination of cannabinoids in serum by GC–MS

The determination of THC, THC-OH, and THCA in serum was performed according to the accredited routine procedure for the determination of cannabinoids in forensic samples. Sample aliquots (1 mL) were diluted with deionized water to 5 mL, and 50 µL of internal standard solution (0.25 ng/µL of THC-d3, THC-OH-d3, and 1 ng/µL of THCA-d3 in methanol) were added. The mixture was vortex mixed and centrifuged for 10 min at 2000 × g. Automated solid-phase extraction was performed using the robot RapidTrace (Caliper LifeSciences, Rüsselsheim, Germany) equipped with 3-mL Bakerbond C18 500-mg cartridges (Baker, Griesheim, Germany). The extraction program consisted of the following steps: conditioning with 6 mL methanol and 3 mL water, application of the sample

at 0.5 mL/min, followed by washing with 1 mL water, 1 mL 0.25 M acetic acid, and 1 mL water, drying for 10 min with compressed air, and elution with 2 mL acetone at 0.5 mL/min. After evaporation to dryness, the residues were dissolved in 40 µL MSTFA and derivatized for 30 min at 60°C. For GC–MS analysis, 1 µL was injected splitless at 250°C with a temperature program from 100°C with an increase of 30°C/min to 310°C, held for 3 min. Quantification of the trimethylsilyl derivatives was performed in the SIM mode using the following fragment ions (m/z, quantifiers underlined): THC-d3 (internal standard) 389, 374, 306, THC 386, 371, 303, THCOH-d3 (internal standard) 374, 462, 477, THC-OH 371, 459, 474, THCA-d3 (internal standard) 476, 374, 491, THCA 473, 371, 488. Relevant validation data on the performance of the method are given in Table I. Samples with cannabinoid concentrations exceeding the upper limit of quantification were reanalyzed after appropriate dilution. Evaluation of the data

The quantitative data were evaluated in the same way as in a previous study (8) with model-independent methods using Microsoft Excel 2003. The highest concentrations observed (Cmax) in this study are probably not the maximal concentrations achieved because the first blood samples after dosing were taken 5 min after smoking. From the concentrationtime curves, it was assumed that one hour after smoking the initial distribution process (β-phase) was negligible. The elimination half-lives (t½ β) were calculated from the result of exponential regression of the data where only samples taken one hour or more after smoking were considered. Concentrations that were lower than the LOQ were omitted. For regression analyses, 89 data sets with 8 points could be used, and in 9 cases, only 6 valid data points were available. The area under Table I. Validation Data for the Assay of THC, THC-OH, and THCA in Serum* Limit of Detection

Linearity (LLOQ–ULOQ)

THC 1 µg/L 2 µg/L 5 µg/L 25 µg/L

0.6 µg/L

1–60 µg/L

THC-OH 1 µg/L 2 µg/L 5 µg/L 25 µg/L

0.3 µg/L

THCA 3 µg/L 6 µg/L 15 µg/L 75 µg/L

0.3 µg/L

Precision (n = 20)

Accuracy (n = 20)

3.0% 2.5% 1.4% 1.8%

3.1% 0.7% 0.8% 1.1%

3.7% 2.8% 3.5% 2.0%

4.6% 2.5% 1.2% 1.4%

4.1% 3.8% 4.3% 3.3%

7.4% 5.2% 3.2% –0.3%

0.3–40 µg/L

3–150 µg/L

* Precision and accuracy data were taken from control charts of the quality control levels monitored during the routinely performed forensic analyses.

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Journal of Analytical Toxicology, Vol. 32, September 2008

the curves (AUC) were estimated using the trapezoidal rule. The data of the occasional cannabis users exhibited a normal distribution, but because the data of the heavy users exhibited a large interindividual variation, the non-parametric MannWhitney test was used to test for significant differences.

Results and Discussion In a previous study, the impact of different THC doses (250 vs. 500 µg/kg BW) on pharmacokinetic properties of THC and its metabolites was examined (8). The main aspect of the present study was to investigate the impact of frequent cannabis use on cannabinoid pharmacokinetics after a dose of 500 µg/kg BW in comparison to a group of occasional users. Pharmacokinetic properties in the cannabis condition of the occasional users

study of Frytak et al. (15) with a 24 h observation time, whereas other authors calculated much higher values (16) from a 72 h sampling time. Pharmacokinetic properties in the cannabis condition of the heavy users

The 12 heavy cannabis users admitted a use of approximately 2 cannabis joints on 4 to 25 (median 7) occasions in the previous week, and all of them exhibited immunochemically positive urine samples before smoking the study dose. In the blood sample prior to the study, THC was present in 10 of 12 samples (range 1.3 to 12.3 µg/L, median 3.2 µg/L). In the first sample after smoking, THC concentrations were maximal with 7.9 to 244.8 µg/L (median 118.8 µg/L). These Cmax values are significantly higher than those observed in occasional users (p < 0.05). Accordingly, the heavy users also exhibited significantly higher THC area AUC values (p < 0.05). Eight hours after smoking, the levels had almost returned to the initial value of each subject (68–196%, median 110%). The elimination half-life of THC was determined in the range of 1.0 to 5.9 h (median 2.7 h), which is significantly longer than that of occasional users (p < 0.01). If the initial THC concentrations are regarded as residual levels, they can be subtracted from each of the following concentrations measured. From these corrected data, the elimination half-life estimates are 0.6 to 3.1 h (median 1.2 h), which is not significantly different from those of the occasional users. The comparison of the uncorrected concentration-time curves of the two groups in a normalized overlay shows a nearly identical course (Figure 1) as previously observed by Ohlsson et al. (17) and Kelly and Jones (14). THC pharmacokinetics is considered to change only slightly in heavy users (6,14,18,19). However, even after correction for the initial THC values, the Cmax and AUC values in the heavy users are significantly higher than those of the occasional users (p < 0.05), which confirms previous reports (17,19) that heavy users smoke more efficiently.

One of the 12 occasional users cancelled his participation after smoking because he did not tolerate the cannabis dose; therefore, only 11 subjects completed the study. The evaluation results are comparable to those obtained with the high dose (500 µg/kg BW) in the previous study (8). Cannabinoids were not detected in blood and urine samples of occasional users at the beginning of the study days as expected. Only 3 of the 11 participants admitted to a single cannabis use in the week before the study. In the first blood sample after smoking (5 min), the THC concentrations were maximal (Cmax) with 11.9 to 86.0 µg/L (median of 53.9 µg/L), which was lower than observed with the same dose in the first study (25.0 to 134.0 µg/L). However, THC concentrations 6 h after smoking were comparable to the previous findings (13,7,8) with very low THC concentrations in only 5 of the 11 subjects (0.7, 0.7, 1.1, 1.5, and 2.4 µg/L). Eight hours after smoking, only one subject exhibited a THC concentration above 1 µg/L (1.2 µg/L). The metabolite THC-OH reached its maximum level (range 2.0 to 16.6 µg/L, median 5.1 µg/L) between 5 and 15 min after smoking as reported previously (7); the metabolite THCA 15 min after smoking (range 10.8–50.8 µg/L, median 27.9 µg/L), which is in the same range as in the first study (7.3 to 48.0 µg/L). THC/THC-OH ratios were in the range of 1.5 to 5.6 (median 2.7) 1 h after smoking and decreased to 0.9 to 4.5 (median 1.7) within the next 2 h, a trend reported previously by Huestis et al. (7). The THC elimination half-life was significantly longer compared to the previous results (1.6 ± 0.2 vs. 1.3 ± 0.2 h, mean ± SD, p < 0.01 in the Student’s t-Test), which can be explained by the longer observation time (8 h vs. 6 h), and which is in accordance with data of Kelly and Jones (14). The elimination halflives of THC-OH (2.4 ± 0.2 vs. 2.0 ± 0.3 h, p < 0.01) and THCA (4.8 ± 0.8 vs. 3.0 ± 0.4 h, p < Figure 1. Overlay of the mean THC concentrations in occasional (n = 11) and heavy users (n = 0.01) were significantly higher than in the 12), each on a separate y-axis to normalize scaling. first study, but also matched results from a

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Journal of Analytical Toxicology, Vol. 32, September 2008

THC-OH values reached their maximum (1.7 to 32.3 µg/L, median 8.4 µg/L) again 15 min after smoking, which seems higher than those of the occasional users and correlates with the higher concentrations of the parent compound. THC/THC-OH ratios matched the values found in occasional users with 1.2 to 6.1 (median 2.7) 1 h after smoking and decreasing to 0.6 to 3.0 (median 1.8) within the next 2 h. The elimination half-life was calculated to be 1.9 to 5.0 h (median 3.1 h), which is longer than that observed in occasional users (p < 0.05). THCA was detected in all blood samples prior to smoking with 3.3 to 241.0 µg/L (median 46.5 µg/L). The maximum levels of THCA were reached within 45 min after smoking cannabis with 9.7 to 310.6 µg/L (median 92.9 µg/L). This was considerably higher in comparison to the values observed in occasional users (p < 0.01). This is due to previous drug use, because after correction for the initial THCA concentrations, the difference was no longer significant. The elimination halflife of 3.4 to 41.9 h (median 6.2 h) was not significantly dif-

ferent from that of the occasional users, which is in accordance with a study by Kelly and Jones (14), but it is much lower than the 5.2 ± 0.8 days (mean ± SD) reported for frequent users (6). However, it has to be mentioned that interindividual variability is very high in this group, which has also been observed in other studies (6,21). The subjects #1 and #2 obviously accumulated high amounts of cannabinoids as a result of their chronic use and are in contrast to the subjects #7, #10, and #12 who exhibited lower (#10) or similar (#7, #12) concentrations than the occasional users (Figure 2). Pharmacokinetic properties in the placebo condition of the heavy users

The blood samples of the occasional users were free of cannabinoids during placebo conditions. The 12 heavy users had admitted use of approximately 2 cannabis joints on 4–7 occasions in the previous week. Accordingly, in 9 of the 12 first blood samples of the placebo phase THC was present in a range of 1.4 to 11.7 µg/L (median 3.2 µg/L) and THC-OH in a range of 0.7 to 11.3 (median 1.7 µg/L). THCA was detectable in all samples in a range of < 3 to 636.5 µg/L (median 37.3 µg/L). THC concentrations at the end of the study day (8 h) were 60.2–95.2% (median 82.5%), THC-OH concentrations 28.9–43.5% (median 42.5%), and THCA concentrations 44.7–96.2% (median 69.1%) of the initial values, respectively. This indicates a slower elimination of THC than of THC-OH or THCA and is also represented by the longer estimate of the elimination halflife of 17.5 to 43.5 h [median 24.1 h, subject #33 was omitted (Table II)], in comparison to 6.1 to 26.9 h (median 7.2 h) for THC-OH and 6.5 to 29.1 h (median 13.0 h) for THCA (Table II). Statements on the time of the last cannabis use prior to the study were not obtained, but as the THC concentration-time courses showed a markedly slower decrease, it can be assumed that the last use was in the previous night (e.g., at least 8 h before the start of the study). THC/THC-OH ratios were 1.0 to 2.6 (median 1.9) in the first blood samples and increased to 2.3 to 5.2 (median 3.2) within the next 8 h. This correlates with the much slower elimination of THC in comparison to that of THC-OH during this later excretion phase than in the first hours after smoking cannabis. Therefore, elevated THC/THC-OH ratios are not necessarily an indication of recent cannabis use. The estimation of the elimination half-life is entirely depending on the study time and on Figure 2. Representative concentration-time plots of THC (■ ▲), and THCA (◆) after ■), THC-OH (▲ the pharmacokinetic model applied. Although smoking cannabis in occasional users (subjects #31 and 33) and heavy users (subjects #1, 5, and the observation time during the placebo con10), and after smoking placebo in heavy users (subjects #1p, 5p, and 10p). Concentration data dition was too short to expect reliable halfare given on a common y-axis except for THCA values in the placebo condition, which are life estimates (c.f. the lower correlation markedly higher in comparison to their corresponding THC or THC-OH values. coefficients in Table II), the THC half-lives in

473

474

6.7 ± 5.1 (0.1 ± 0.1 h)

8.2 (0.3) 15.9 (0.1) 2.0 (0.1) 3.2 (0.1) 2.5 (0.3) 6.0 (0.1) 7.5 (0.3) 3.3 (0.1) 16.6 (0.1) 5.1 (0.3) 3.7 (0.1)

0.4 ± 0.3

0.6 0.9 n.d. n.d. n.d. 0.5 0.7 0.4 0.6 0.5 0.4

< LOQ

< 1.0 < 1.0 n.d. n.d. n.d. n.d. < 1.0 1.2 n.d. n.d. n.d.

C8 h (µg/L)

14 ± 7

17 25 6 9 6 12 19 10 23 13 9

35 ± 14

47 41 17 35 23 35 54 52 41 24 13

AUC0→8 h (µg/L * h)

2.4 ± 0.2

2.4 (0.99) 2.5 (0.98) 2.8 (0.98) 2.5 (1.00) 2.1 (0.98) 2.4 (0.98) 2.5 (0.98) 2.7 (0.99) 2.1 (0.94) 2.1 (0.99) 2.5 (0.97)

1.6 ± 0.2

1.6 (0.99) 1.7 (0.97) 1.6 (0.98) 1.8 (0.98) 1.5 (0.97) 1.3 (0.97) 1.9 (0.98) 2.0 (1.00) 1.7 (0.93) 1.5 (0.99) 1.5 (0.99)

t½ β (h)

1 2 3 4 5 6 7 8 9 10 11 12

1 2 3 4 5 6 7 8 9 10 11 12

Subject

4.8 5.1 n.d. 2.4 n.d. 1.4 n.d. 1.0 1.7 n.d. 0.7 n.d. 2.5 ± 1.8

6.8 12.3 1.9 5.8 1.6 4.5 1.3 2.1 2.9 n.d. 1.9 n.d. 4.1 ± 3.4

C0 h (µg/L)

5.8 10.7 3.7 5.7 1.5 3.0 1.8 2.7 4.3 n.d. 2.3 n.d. 3.5 ± 2.9

C8 h (µg/L)

27.1 (0.1) 4.2 29.4 (0.3) 4.4 32.3 (0.1) 2.1 8.3 (0.1) 2.0 1.7 (0.1) n.d. 11.5 (0.5) 1.7 8.9 (0.1) 0.7 5.3 (0.1) 1.1 8.5 (0.1) 1.5 2.9 (0.1) n.d. 6.8 (0.3) 0.7 5.2 (0.1) n.d. 12.3 ± 10.8 1.5 ± 1.5 (0.1 ± 0.1 h)

228.6 (0.1) 244.8 (0.1) 168.8 (0.1) 122.0 (0.1) 31.1 (0.1) 196.5 (0.1) 87.0 (0.1) 74.9 (0.1) 115.6 (0.1) 7.9 (0.1) 139.7 (0.1) 33.5 (0.1) 120.9 ± 78.1 (0.1 ± 0.0 h)

Cmax (µg/L) (tmax [h])

Heavy Users, Joint/THC Condition

63 77 52 30 6 37 24 21 26 5 18 12 31 ± 23

124 201 96 91 24 114 67 74 118 5 89 23 86 ± 54

AUC0→8 h (µg/L h)

4.0 (0.95) 4.5 (0.88) 1.9 (0.96) 5.0 (0.93) 4.6 (0.95) 2.7 (0.97) 2.2 (0.98) 3.5 (1.00) 3.5 (0.99) 2.5 (0.96) 2.7 (0.97) 2.7 (0.98) 3.3 ± 1.0

3.6 (0.91) 5.9 (0.72) 2.0 (0.91) 5.4 (0.80) 4.3 (0.87) 2.3 (0.95) 2.0 (0.96) 2.6 (0.97) 2.5 (0.94) 1.0 (0.99) 2.2 (0.95) 2.0 (0.96) 3.0 ± 1.5

t½ β (h)

3.6 11.3 n.d. 1.8 0.7 2.4 n.d. 0.7 1.6 n.d. 0.7 n.d. 2.9 ± 3.6

4.5 11.7 1.4 4.5 1.9 4.1 n.d. 1.6 3.2 n.d. 2.0 n.d. 3.5 ± 3.2

C0 h (µg/L)

13

1.6

5 10 4

n.d. 0.7 n.d.

14 ± 17

9 4 11

n.d. n.d. 0.7

0.9 ± 1.6

20 56

1.5 4.8

28 ± 25

12 23

1.4 2.6

3.3 ± 3.1

33 91 10 33 14 24

AUC0→8 h (µg/L h)

3.9 11.1 1.1 3.3 1.8 2.5

C8 h (µg/L)

t½ β (h)

9.5 ± 6.6

26.9 (0.53)

8.9 (0.91) 8.8 (0.94)

7.2 (0.87) 6.8 (0.93) 6.3 (0.94)

6.1 (0.99) 6.3 (0.97)

27.4 ± 9.7

(–64.9 (0.28))

29.1 (0.62) 18.4 (0.70)

20.5 (0.77) 17.5 (0.69) 24.1 (0.42) 43.5 (0.40) 26.1 (0.49) 39.7 (0.32)

Heavy Users, Placebo Condition

* The maximum observed concentration (Cmax) with the corresponding time (tmax) and the last concentration measured 8 h after smoking (C8 h) are given. Detection of analyte below the limit of quantification is indicated (“