Metabolism of Tetrahydrocannabinol in Frequent and Infrequent Marijuana Users

Transcription

1 Metabolism of Tetrahydrocannabinol in Frequent and Infrequent Marijuana Users Peggy Kelly* School of Public Health, University of California-Berkeley, Biomedical and Environmental Health Sciences, Forensic Science Group, Berkeley, California Reese T. Jones Drug Dependence Research Center, Langley Porter Psychiatric Institute, University of California-San Francisco, San Francisco, Cafifomia Abstract J ~0-tetrahydrocannablnol (THC), 11-nor-9-carboxy-Ag-tetra - hydrocannabinol (THC-COOH), and its O-ester glucuronide were measured in plasma by GC/MS and in urine by GC/MS and enzyme Immunoassay after frequent and Infrequent marijuana users were given 5 mg THC intravenously. Plasma THC concentrations were detectable 4-5 h after infusion using solid-phase columns for drug extraction and gas chromatography/mass spectrometry (GC/MS) for detection and quantification. THC-COOH and its O-ester glucuronide were analyzed for 12 days. Concentrations were higher in the plasma and urine of frequent marijuana users. The ratio of THC-COOH to its O-ester glucuronide in plasma was greater than 2 in both frequent and infrequent users 2 to 30 min postinfusion. Ratios for the subsequent 12 days were less than 2. A ratio of less than 1 for total THC-COOH/THC occurred only before 45 min for both frequent and infrequent users. Physiological, psychological, and pharmacokinetic data revealed few differences between frequent and infrequent marijuana users. Introduction Current methods used to detect cannabinoids in plasma or urine can indicate that a person has ingested marijuana but not when the marijuana was ingested. The time of marijuana ingestion is often important information because the psychoactive components of marijuana alter psychomotor and psychological performance most reliably up to 1-2 h after smoking or intravenous infusion. Effects disappear rapidly to virtually no measurable effect by 4 h in most published studies (1). Cannabinoids in plasma and urine samples are measurable long after effects are unmeasurable; for example, metabolites appear in urine as long as 72 days after marijuana ingestion (2). Using sensitive assays and deuterium-labeled THC, elimination half-life of THC in plasma is estimated to be about 4 days on average and days in some research subjects after smoking one cigarette (3). Our study examined the disposition of Ag-tetrahydro - cannabinol (THC) and its major metabolites after intravenous administration of 5 mg of THC to marijuana users who varied in 9 Address correspondence to Dr. Peggy Kelly. Syva Company MS E Yerba Buena Rd.. P.O. Box San Jose. CA frequency of marijuana use. A novel extraction method was devised to detect two major metabolites of THC, ll-nor-9-carboxy-a9-tetrahydrocannabinol (THC-COOH) and its O-ester glucuronide. THC-COOH should be in a higher concentration relative to the THC-COOH glucuronide in plasma shortly after ingestion because the oxidation to form a carboxylic acid precedes the glucuronide conjugation. The first goal of our study was to determine if metabolites of THC could be used to estimate the time of THC dosing. Other researchers have attempted to determine the time of marijuana ingestion after smoking (4) or oral ingestion (5). THC concentrations in plasma samples were the best indication that marijuana had been smoked within the previous six hours. These studies examined the ratio of metabolites/thc and the time since ingestion after smoking or oral ingestion of marijuana. However, this strategy has not been tested with THC intravenous administration. The second goal was to discern if tolerance to THC was evident in the more frequent marijuana users in physiological, psychological, and pharmacokinetic parameters. Pharmacokinetic, physiological, and psychological changes resulting from THC and subsequent tolerance to THC have been noted by many researchers (6). Tolerance development to THC has occurred for heart rate (7,8), blood pressure (8), and skin temperature (7,9). Pharmacokinetic differences between frequent and infrequent users due to tolerance have also been reported (8,10). Methods Human Studies Eight males between 24 and 45 years old were recruited with ads in local newspapers. All were of mesomorphic body type. Mean (+SD) body weight was kg (range = kg). Two populations of marijuana users were selected: four frequent users who smoked marijuana almost daily for at least two years and four infrequent users who smoked marijuana no more than two to three times per month but had done so for at least two years. None had used other illicit drugs regularly or recently. Candidates were in good health as judged by routine clinical laboratory tests, history, and physical examination. Subjects gave informed consent. Infrequent marijuana users were asked to refrain from smoking marijuana at least one week prior to the experiment. Frequent users were asked not to smoke marijuana for 228 Reproduction (photocopying) of editorial content of this journal is prohibited without publisher's permission.

2 12 h before the experiment. The protocol was approved by the UCSF Committee on Human Research. Heart rate, systolic and diastolic blood pressure, skin temperature, and EKG were monitored for at least 20 min prior to the infusion or until stable. An indwelling catheter was inserted into a forearm vein for THC infusion. Another venous catheter in the opposite forearm allowed for blood sampling over the next 10 h. After the subject relaxed and blood pressure and heart rate readings were within normal, quiet, resting range, 5 mg of THC in a 95% ethanol solution was injected by pump into the injection port of a rapidly flowing intravenous normal saline infusion. The THC was delivered as 2.5 ml of 2 mg/ml THC over 2 min at a rate of 1.25 ml/min. The subjects remained at the Drug Dependence Laboratory for at least 10 h after injection. Blood pressure, heart rate, fingertip skin temperature, and "high" ratings, as well as subjective mood, were recorded frequently for the first 10 h. "High" ratings were reported by subjects on a 0 to 100 scale where 0 meant not intoxicated and 100 was the most intoxicated they had ever been after any Cannabis preparation. Blood samples were collected at 2, 5, 20, 30, 45, and 90 min and 3, 4, 5, 8, and 10 h into Vacutainer r tubes containing sodium citrate. Blood was collected daily for the following 12 days via venipuncture. Solid components were separated immediately from plasma and the plasma was frozen at -20~ in silanized glass vials (11). Urine was collected as 24 h specimens with sodium bisulfite (2.5 g/l) in glass silanized jars to ensure a urine ph of at least 3. Urine volume and ph were recorded on every sample. An aliquot was frozen in silanized glass vials. Creatinine measurements were made on each specimen. Materials 0.5% w/v (-)-trans-a9-tetrahydrocannabinol in alcohol, USP, batch # (E800802X) (National Institute on Drug Abuse, NIDA), was used for the intravenous dose. Standards were made with (-)-trans-a9-tetrahydrocannabinol and 9-carboxy-ll-nor-A9-tetrahydrocannabinol. The internal standards were (-)-trans A9-tetrahydrocannabinol -5'-2H3 and 9-carboxy- 11-nor-A9-THC-5'-2H3. These ethanolic standards obtained from NIDA were diluted with methanol to give stock solutions of 100 p.g/ml and stored in the dark at -20~ Working standards were prepared by dilution of the 100-1ag/mL stocks to 10-pg/mL concentrations with methanol. Chemicals used for derivatizations were pentafluoropropionic anhydride (Pierce Chemical Co.), trifluoroacetic anhydride (Aldrich Chemical Co.), and 1,1,1,3,3,3-hexafluoro-2-propanol (Aldrich). Reagents and solvents used in compound elution and derivatization were acetonitrile (HPLC grade), methanol (HPLC grade, American Burdick and Jackson), ethyl acetate (HPLC grade), hexane (HPLC grade), phosphoric acid, hydrochloric acid, glacial acetic acid, and sodium bisulfate (reagent grade, Fisher Scientific Co.). Utak from Utak Laboratories, Inc., and NBS controls from the National Institute of Standards and Technology were used for quality control. Plasma THC and metabollte analysis To extract THC or THC-COOH from plasma samples, 1.0 ml plasma, controls, and calibrators ranging from ng/ml were pipetted to silanized mm glass culture tubes. Every sample except the blank plasma was spiked with I0 lal of a 10-pg/mL working stock of deuterated THC for quantitation of THC samples or 10 IJL of a 10-1ag/mL deuterated THC-COOH for quantitation of THC-COOH samples (12). To determine total THC-COOH, 25 lal of 10N NaOH was added to hydrolyze the THC-COOH glucuronide. The sample was heated in a water bath at 60~ for 20 min at a ph of Another set of plasma samples was extracted to quantify unconjugated THC-COOH by omitting the pre-treatment steps. Conjugated THC-COOH was determined by subtracting the unconjugated THC-COOH from the total THC-COOH. To extract THC-COOH from prepared samples, 1.0 ml of acetonitrile added to the tube and vortexed for 30 s. Tubes were centrifuged for 10 rain at 3000 rpm with a Beckman Model TJ-6 centrifuge. The supernatant was decanted into a clean silanized mm tube containing 9 ml of 0.05M H3PO 4 (ph 2.5). After vortexing this mixture, 150 ~L of glacial acetic acid was added to adjust the ph to 3-4 and vortexed again. THC or THC-COOH was extracted from this mixture by using solid-phase extraction columns (Narc-1 by J.T. Baker). The columns were initially washed with 3 ml of methanol pulled through by vacuum suction (Analytichem International Vacelut manifold). This was followed by 3 ml of 0.05M H3PO 4 (ph 2.5) and the sample, which was aspirated at 5 ml/min. When all the solution passed through, the column was dried by vacuum at 20 mm Hg for 3 min. The column was then washed with 1 ml of acetonitrile-0.1n HC1 (2:3) and then dried for 10 min under a vacuum of 20 mm Hg. The vacuum was turned off and 500 ~tl of hexane was added on the column and drawn through slowly. The column was dried for 10 min under vacuum of 20 mm Hg. The sample was finally eluted with two 750-1JL aliquots of ethyl acetate-hexane (1:1). The elutant was dried in the vials at room temperature with air that had passed over a desiccant. For the derivatization of THC, 100 ILL of trifluoroacetic anhydride and 100 ILL of chloroform were added to the vial. The mixture was capped and heated at 70~ for 15 min. The heat was provided by a Pierce Reactitherm heating block. The derivatized sample mixture was cooled and dried by filtered air. For the derivatization of THC-COOH, 100 tal hexane, 50 pl of pentafluoropropionic anhydride, and 50 ~L of hexafluoro-2-propanol were added to the react-vial and the mixture was capped, vortexed, and heated at 100~ for 10 min. The mixture was cooled and evaporated with dried air at room temperature. Each vial was reconstituted with 10 I.LL of ethyl acetate. Derivatized THC and THC-COOH were then stored in the dark in a -20~ freezer overnight. The samples were injected onto the GC/MS the next day. The standard curve for THC and THC-COOH was linear from 1.0 to 200 ng/ml. The detection limit for THC and THC-COOH in plasma was 1 ng/ml. A 10-ng/mL plasma THC Utak control had a mean and standard deviation of ng/ml (n = 20) and a 100-ng/mL plasma THC Utak control had a mean and standard deviation of ng/ml (n = 20). A 20-ng/mL plasma THC-COOH Utak control had a mean and standard deviation of ng/ml (n = 20) and a 100 ng/ml plasma THC-COOH spiked control had a mean and standard deviation of ng/ml (n = 20). Chromatographic conditions A 5970A Hewlett-Packard mass selective detector with a 5880A gas chromatograph and a 9133 computer was used for GC/MS analysis. The column was a DB-5 capillary column: 30-m crossed-linked phenylmethyl silicone with 0.25 mm i.d. and film thickness of 0.25 wn (J&W Scientific). The temperature program for the THC quantification started with an initial temperature of 200~ held for 2 min, then increased by 20~ to 280~ and held for 4 min. The injector 229

3 temperature was 300~ and the GC/MS transfer line was 250~ All injections were in the splitless mode with a purge delay time of 0.4 min. Column flow was 1 mldmin helium. Ions monitored for THC-TFAA identification were 229, 297, 339, 395, and 410. Under these conditions, the derivatized THC and the derivatized intemal standard coelute between 4 and 6 min, depending on the column length. The ion ratio of the THC derivative ion at 410 m/z (THC-TFAA) and the internal standard 413 (THC-2H3 - TFAA) was used for quantification. Table I. THC Plasma Concentrations (ng/ml) for Frequent and Infrequent Marijuana Users Before and After Infusion of 5 mg THC Time Mean SD Mean SO Pre min min min rain min rain h h h h The temperature program for the THC-COOH quantification started with an initial temperature of 150~ held for 1 min, then increased by 30~ to 300~ and held for 2 min. The injector temperature was 215~ and the GC/MS transfer line was 250~ All injections were in the splitless mode with a purge delay time of 0.4 min. The column flow was I mldmin helium. Ions to monitor for THC-COOH/PFPA-HFIP identification were 477, 489, and 640. Under these conditions, derivatized THC-COOH and the coeluting deuterated THC-COOH eluted between 4 and 6 min, depending on column length. The ion ratio of 640 (THC-COOH- PFPA/HFIP) and the internal standard 643 ion (THC-COOH-2H3 PFPA/HFIP) was used for quantification. Urine metabollte analysis To extract THC-COOH from urine, 4-mL urine samples, controls and calibrators ranging ng/ml were pipetted to silanized 16- x 125-ram glass culture tubes. Every sample, except the urine blank, was spiked with 40 ill of 10 l.tg/ml working deuterated THC-COOH standard (12). To determine total THC- COOH, 100 ILL of 1 ON NaOH was added to one set of tubes to hydrolyze the THC-COOH glucuronide. The contents was vortexed and heated in a 60~ water bath for 20 min. The tubes were cooled to room temperature and 600 I.tL of glacial acetic acid was added to bring the ph to 3-4. Another set of urine samples was extracted to quantify any free THC-COOH present by omitting the hydrolysis procedure. Extraction columns (Narc-1, J.T. Baker) were used to extract THC-COOH from the urine matrix. The columns were condi- Table II. THC-COOH and THC-COOH O-Ester Glucuronlde Plasma Concentrations (ng/ml) for Infrequent Marijuana Users Before and After IV InJectlon of 5 mg THC Subject 5 Subject 6 Subject 7 Subject 8 Time Free Total Conj. Free Total Conj. Free Total Conj. Free Total Conj. Minutes * * * * 33.9 * * * * * * * Days Values not reported because samples not taken or sample lost during extraction. Free = THC-COOH ng/ml which is not conjugated with a glucuronide; Total = Free + Conjugated; Conj. =THC-COOH ng/ml which is conjugated with a glucuronide. 230

4 Table III. THC-COOH and THC-COOH O-Ester Glucuronlde Plasma Concentrations (ng/ml) for Frequent Marijuana Users Before and After Iv InJectlon of 5 mg THC Sublect I Sublect 2 Subject 3 Sublect 4 Time Free Total Conj. Free Total Conj. Free Total Conj. Free Total Conj. Minutes ,5 35, , , , , , , , , , , , , ,6 82, , , Days , , , , ,9 19, ,4 20, , ,1 20, ,1 18, ,4 20,6 4,6 12, , , , , ,7 3,9 13,9 10, , , , , , , , Free = THC-COOH ng/ml which I$ not conjugated with a glucuronlde; Total = Free + Conjugated; Conj. = THC-COOH ng/ml which is conjugated with 9 glucuronlde. SO ~11 9 I 9 9 n I I z 9, -,., -,., -,., SO TIME (HR.) Figure 1. Ratio of THC-COOH to THC-COOH glucuronide plotted vs days postinfusion for infrequent and frequent marijuana users after infusion of 5 mg THC intravenously, [] = Subject #1, 9 = Subject #2, 9 = Subject #3, 9 = Subject #4, 9 = Subject #5, [] = Subject #6, 9 = Subject #7, and 9 = Subject #8, tioned by adding 3 ml of methanol pulled through the column by a vacuum. This was repeated with 3 ml of distilled water and again with 3 ml of 0. IN HCI. One milliliter of 0. IN HCI was layered on top of the packing material in the tube so that the material remained wet. From this point, the procedure for extraction and derivatization of THC-COOH from urine was identical to that of the THC-COOH extracted from the plasma. The standard curve for THC-COOH was linear from 1.0 to 200 ng/ml. The detection limit was 1 ng/ml for THC-COOH. A 20-ng/mL THC-COOH NBS control had a mean and standard deviation of ng/ml (n = 20) and a 100-ng/mL THC- COOH spiked control had a mean and standard deviation of ng/ml (n = 20). EMIT end TDx cannablnold analysis Total amount of cannabinoids in urine was assayed with the semiquantitative EMIT d.a.u. TM cannabinoid assay (Syva Co.) and the TDx fluorescence polarization immunoassay (Abbott Laboratories) using the calibrator kits and procedures supplied by the manufacturers (cutoff levels of 20 ng/ml for EMIT and 25 ng/ml for TDx). PharmacoklneUc analysis Noncompartmental analysis (14,15) was used to determine the volume of distribution (beta and steady state), elimination halflife, and metabolic clearance of THC in the subjects. Additionally, elimination half-life of the plasma THC metabolites and elimination half-life and renal clearance (8) of urinary O-ester THC- COOH glucuronide were estimated using the GC/MS results. Statistical analysis The physiological, chemical, and pharmacokinetic parameters, for the four frequent users and the four infrequent users, were compared with the Mann-Whitney nonparametric test using 0.01 as the level of significance (13). Heart rate, systolic and diastolic blood pressure, and skin temperature were measured as the peak value minus the predosing values for each individual. 231

5 Results and Discussion THC and metabolite plasma concentrations The plasma concentrations of THC for infrequent and frequent marijuana users after infusion of 5 mg of THC intravenously are given in Table I. The time course of plasma THC concentrations following intravenous infusion of 5 mg of THC to marijuana users agrees well with published data (16-18). The time course of the THC levels for the frequent and infrequent marijuana users did not differ significantly. THC rapidly disappeared from plasma, probably because the THC is rapidly redistributed to tissue and then released from these tissues slowly (8). The plasma concentrations of THC-COOH and its O-ester glucuronide were measured in blood for 12 days after THC infusion for the infrequent marijuana users (Table II) and for the frequent marijuana users (Table III). Before and after the infusion of 5 mg of THC, the frequent marijuana users had plasma and urine levels of free THC-COOH and conjugated THC- COOH higher than the infrequent users. This is not surprising because frequent users had measurable amounts of cannabinoids in their plasma and urine before the THC was given. These cannabinoid levels would be additive with the metabolites derived from the THC given intravenously. Metabolites appeared in urine for up to 12 days for some subjects. Previous studies measured plasma metabolites and noted metabolite ratios after dosing volunteers with THC. Wall and Taylor (19) found that after 8 mg THC was given by smoking, the ratio of free THC-COOH to conjugated THC-COOH at the time of maximal THC-COOH concentration in plasma varied from 2.3 to After oral ingestion of Cannabis resin containing 20 mg of THC, the glucuronide was 2.8 times greater than the free THC-COOH at the peak plasma level of THC- COOH occurring 4 h after ingestion (Law et al.) (5). Additionally, at the 1-h post-ingestion time point THC-COOH exceeded the O-ester THC-COOH glucuronide. A study by the same group also detected free THC-COOH and its conjugated glucuronide in random forensic samples (20). The plasma samples had a ratio of free THC-COOH/conjugated THC-COOH of , indicating that the glucuronide form was in higher concentration than the free acid. After a frequent or infrequent marijuana user was infused with 5 mg of THC, we found that a ratio of the metabolite THC-COOH to its O-ester glucuronide greater than 2 occurred only 2 to 30 min postinfusion. After 30 minutes all samples had a ratio of less than 2 (Figure 1). McBurney et al. (4) had subjects smoke a cigarette spiked with 10 mg of THC. For plasma metabolites, the best correlation was log [total THC-COOH/THC] vs. log [time postsmoking], but the authors concluded that the ratio variability was too great to be useful to determine time of ingestion. A similar analysis was done in our study. The correlation coefficient for the [total THC- COOH/THC] vs. [time post-infusion] for frequent and infrequent marijuana users had the highest correlation, r = 0.8 (Figures 2 and 3), when compared with plots of either [free or conjugated THC-COOH/THC] vs. [time post-infusion]. Other studies have attempted to determine the time of marijuana intake by examining the ratio of metabolites to THC. Law et al. (5) gave four subjects oral (20 mg) THC. THC, THC- COOH, and its O-ester glucuronide were detected in blood and THC-COOH and its O-ester glucuronide were detected in urine. The total metabolite/thc ratio in plasma was relatively low and constant during the first 4 h after consumption and rose rapidly thereafter. They concluded that a total metabolite/thc ratio of less than 20 indicates recent oral consumption of marijuana. In our study, a ratio less than 1.0 for total THC- COOI-I/THC occurred only before 45 min for frequent and infrequent marijuana users after the 5 mg THC infusion. Urinary THC metabolites Creatinine correction of the urine samples O-ester THC- COOH glucuronide metabolite indicated that all subjects followed the protocol for refraining from marijuana ingestion in the 12-day period after THC administration. Table IV shows the urine levels of THC metabolites as measured by GC/MS. Many studies have measured urinary cannabinoid metabolites. Most conclude that conjugated THC-COOH is the major metabolite (5,19,21) in urine with a small amount of the free acid present. To preserve the conjugated THC-COOH, the ph of urine should be less than 4. Earlier studies that did not correct for the ph erroneously measured high levels of free acid metabolite. In our study, the ph of the urine was adjusted to 3 upon voiding. The majority of the THC-COOH was in the conjugated THC-COOH glucuronide form with only a few ng/ml of free acid present in the frequent users' urine during the first few days after THC infusion. The occurrence of low levels of free acid in the urine of frequent users has been noted previously (22). Table V shows the EMIT data and Table VI shows the TDx data for frequent and infrequent marijuana users. It is of interest to compare the two immunoassays because they both use immuno-specificity to detect cannabinoids. For the frequent users, 20 y x R.081 y o03slx R.085 I ~ n l o. 9,;o 2;.... Time (mln) Figure 2. Regression of total THC-COOH/IHC vs. time postinfusion after infrequent marijuana users were intravenously dosed with 5 mg THC. 0 -,.,., Time (Bin) Figure 3. Regression of total THC-COOH/FHC vs. time postinfusion after frequent marijuana users were intravenously dosed with 5 mg THC. 232

6 differences occurred on the presample and at Day 1 of the study with the TDx indicating a larger amount of cannabinoids present compared to the EMIT (p = 0.02). For the infrequent users, differences between the two immunoassays occurred at Day 1 (p = 0.04) as well. The ng/ml values are calculated from calibration curves generated from calibrators supplied by the manufacturers. Using the 20-ng/mL cutoff recommended by EMIT and 25- ng/ml cutoff for TDx manufacturers, the infrequent users would on the average test positive to Day 2 or 3 and the frequent users tested positive on the average until between Day 9 to Day 12. The pattern of a few negative urines followed by a positive without any additional Cannabis ingestion has been noted (2). Chronic marijuana users have positive tests on the EMIT for cannabinoids in urine (using a 20-ng/mL cutoff) for as many as 46 consecutive days from cessation of marijuana use and can take as many as 77 days to drop below 20 ng/ml for 10 consecutive days (2,23). Pharmaeoklnetlcs Table VII lists the pharmacokinetic parameters for THC and metabolites. The area under the THC plasma concentration/time curve for the frequent users in the present study was (ng/ml)*min. For the infrequent marijuana users, it was (ng/ml)*min. Lindgren found the AUC to be smaller for frequent users compared with infrequent marijuana users after 5 mg THC intravenously (16). This difference was not evident in our study. The actual amount of marijuana exposure of one group of frequent marijuana smokers may indeed be lower compared to another group of frequent marijuana smokers because we are relying on the hearsay of the subjects. This is a plausible explanation for differences in studies that accounts for different pharmacokinetic responses. The THC volume of distribution (beta) for the frequent marijuana users was 96 L and for the infrequent users was 97 L. The volume of distribution (steady state) was 75 L for frequent users and 74 L for infrequent users, consistent with the drug distributing into deep tissue compartments. The terminal half-life for THC in plasma of frequent users was 117 min and for infrequent users it was 93 min. There were no significant differences between the frequent and infrequent marijuana users. Table IV. Urinary Concentrations (ng/ml) of THC Metabolltes as Measured by GC/MS for Frequent and Infrequent Marijuana Users After IV Injection of 5 mg THC Day Mean SD Mean SD Conjugated COOH-THC Pre Free COOH- THC Pre The volume of distribution (steady state) for THC has been reported in previous studies to be about 700 L (24). Most investigators have estimated the terminal half-life to be h (25). A possible explanation for the difference in values is that previous studies used radiolabeled THC so that the method of detection involved extraction of the drug with a solvent to separate the THC. All of the radioactivity in this solvent extract was considered to be THC; however, other cannabinoid metabolites could have also been present and, if also radioactively labeled, would have been considered THC. It is possible in these earlier studies that the amount of the THC fraction could have been overestimated, thus overestimating the volume of distribution and the terminal half-life. Of course, the sensitivity and specificity of the analytic method can greatly influence the calculation of pharmacokinetic parameters. For example, a recent study using deuterated THC (added to marijuana placebo cigarettes) and a GC/MS assay with a detection limit of 20 pg THC/mL estimated elimination half-life to be 4.3 days (3). In the current study, the mean plasma THC-COOH metabolite half-life was 5.2 days for the frequent users and 6.2 days for the infrequent users. The conjugated THC-COOH mean half-life was 6.8 days for the frequent users and 3.7 days for the infrequent users based on data collected for 12 days. The differences were not statistically significant. Previous radiolabeled THC pharmacokinetic studies assessed total metabolite half-life to be about 2 days using data collected over 4 days (8). It is possible that these times are shorter because they included some quickly eliminated metabolites. We conclude that the free and conjugated THC-COOH metabolites have a half-life of 4-6 days in the plasma when measurement is conducted for 12 days. A longer period of sampling would be needed for a truly valid determination of a substance with so long a half-life. Frequent users had a mean half-life of 1.9 days for the conjugated urine THC-COOH and the infrequent users had a mean half-life of 2.0 days, showing a slow elimination of the metabolite. A previous study using enzyme immunoassay reported total cannabinoid urinary half-lives of days (range days) (26). In the current study, metabolic clearance for the frequent users was ml/min and for the infrequent users ml/min. In the literature, plasma clearance for THC is high, ranging from ml/min (25), consistent with a Table V. Urinary Concentrations (ng/ml) of THC Metabolites as Measured by EMIT for Frequent and Infrequent Marijuana Users after IV Injection of THC Subj. Subj. Subj. Subj. Subj. Subj. Subj. Subj. Day Pre 66 <20 <20 66 <20 <20 <20 < < 20 < < <20 <20 < < <20 <20 <20 < < < 20 < 20 <20 < < <20 <20 <20 < <20 <20 <20 <20 <20 <20 < <20 <20 30 <20 <20 <20 < <20 <20 <20 <20 <20 <20 < <20 <20 <20 <20 <20 <20 < <20 <20 <20 <20 <20 <20 < <20 <20 44 <20 <20 <20 <20 233

7 rapid disappearance of THC from plasma. This study's assessment of metabolic clearance is consistent with values previously reported. The renal clearance of the THC-COOH glucuronide for both the frequent (mean = ml/min) and the infrequent (mean = ml/min) users started out higher and progressively decreased over the next 12 days to a lower rate for both the frequent users ( ml/min) and the infrequent users ( ml/min). There was no statistically significant difference between the frequent and infrequent users' renal clearance. Psychological and physiological results Tolerance is a form of adaptation where continued or repeated exposure to a drug results in decreased effects. Cannabis tolerance occurs during sustained use. Tolerance has been shown to occur with heart rate (8), blood pressure (8), skin temperature (9), and degree of psychological "high" (2,9,27). In the present study, the frequent marijuana users smoked at least once a day, and the infrequent marijuana users smoked marijuana no more than 2-3 times per month to 3-I 1 times per year. Despite this great dif- Table Vl. Urinary Concentrations (ng/ml) of THC Metabolites Measured by TDx for Frequent and Infrequent Marijuana Users after IV Injection of 5 mg THC Subj. Subj. Subj. Subj. Subj. Subj. Subj. Subj. Day Pre < 25 < 25 < 25 < < 25 < 28 < < < 25 < 25 < 25 < < 25 < 25 < 25 < 25 < < 25 < 25 < 25 < 25 < < 25 < 25 < 25 < 25 < 25 8 < < 25 < 25 < 25 < 25 < < < 25 < 25 < 25 < 25 < < 25 < 25 < 25 < 25 < 25 < 25 < <25 <25 <25 <25 <25 <25 <25 <25 12 <25 <25 <25 <34 <25 <25 <25 <25 ference in pattern of marijuana use, there was not a significant difference in the self-reported "high" rating after intravenous THC (Table VIII). The frequent users' heart rate increase, skin temperature, and diastolic blood pressure decrease were not statistically different when compared to the infrequent marijuana users. The systolic blood pressure increase induced by THC was significantly greater for the infrequent marijuana users at the systolic blood peak (Table VIII). Because the only parameter manifesting a tolerance to THC was systolic blood pressure, it is possible that the frequent users had not used Cannabis in doses large enough to display consistent tolerance. Another explanation is that an intravenous dose of THC produces a transient peak brain drug level that is sufficient to overcome the tolerance. Conclusions THC is rapidly distributed and transformed into free and conjugated carboxy-a9-tetrahydrocannabinol within 2 rain post 5 mg THC infusion. Plasma conjugated THC-COOH was detected in Table VII. Noncompartmental Pharmacokinetic Results for Frequent and Infrequent Marijuana Users After IV Infusion of 5 mg THC Mean SD Mean SD THC in Plasma Area under the curve (ng/ml)*min Plasma clearance (L/min) Volume of distribution (L) Volume of distribution SS (L) Elimination half-life (min) THC-COOH in plasma Elimination half-life (day) THC-COOH glucuronide plasma Elimination half-life (day) THC-COOH glucuronide urine Elimination half-life (day) Table VIII. Self-Reported "High" Ratings and Systolic Blood Pressure Readings for Frequent and Infrequent Marijuana Users after IV Injection of 5 mg THC High ratings Systolic blood High ratings Systolic blood (% high) pressure (% high) pressure Time Mean SD Mean SD Mean SD Mean SD Pre min min min min min min hr hr hr hr

8 frequent and infrequent marijuana users for as long as 12 days. Over 12 days, the plasma free THC-COOH was detected in all frequent users and some infrequent users. Conjugated plasma THC-COOH was still detectable by GC/MS for 75% of the frequent users and 25% of the infrequent users at Day 12. The plasma and urine metabolite levels were higher for the frequent users when compared with the infrequent users. Frequent users were distinguishable from infrequent users in that infrequent users' urine samples had only conjugated O-ester THC- COOH glucuronide present after 5 mg THC infusion whereas urine samples from the frequent users contained small quantities of unconjugated THC-COOH during the first few days after the infusion of 5 mg THC. Two different methods for the determination of recent THC exposure were explored. The first was looking at the ratio of the plasma free THC-COOH to the glucuronidated THC-COOH. A ratio of the plasma metabolite THC-COOH/O-ester THC-COOH glucuronide greater than 2 occurs 2 to 30 min post-thc infusion. All other time points for 12 days thereafter exhibit a ratio of less than 2, Secondly, we looked at the ratio of the total THC-COOH to THC levels in the plasma samples. A ratio of l or less for total THC-COOH/THC occurred only before the 45 min postinfusion sample for the infrequent and frequent marijuana users alike. Pharmacokinetic parameters after the intravenous THC revealed no significant differences between the frequent and infrequent marijuana users on AUC, volume of distribution, elimination half-lives of parent THC and metabolites in plasma and urine, and metabolic and renal clearances. Infrequent marijuana users had higher peak systolic blood pressure compared with frequent marijuana users after a 5-mg THC infusion, but tolerance was not evident in heart rate, diastolic blood pressure, skin temperature, and degree of psychological "high." Acknowledgments Peggy Kelly's thesis advisor, Dr. John Thornton, as well as Dr. M.A. Peat and Dr. R.C. Baselt gave help and encouragement. Peggy Kelly's work was funded by the National Institute of Justice. We thank UCLA Clinical Laboratories for the EMIT and TDx assay results. We thank Kaye Welch for her unending patience and support. This work was also supported in part by grants No. DA01696 and DA00053 from NIDA. References 1. L.E. Hollister, H.K. Gillespie, A. Ohlsson, J.E. Lindgren, A. Wahlen, and S. Agurell. Do plasma concentrations of delta-9-thc reflect the degree of intoxication? J. Olin. PharmacoL 21: 171s-77s (1981). 2. G.M. Ellis, MA. Mann, B.A. Judson, N.T. Schramm, and A. Tashchian. Excretion patterns of cannabinoid metabolites after last use in a group of chronic users. Olin. PharmacoL Ther. 38: (1985). 3. E. Johansson, M.M. Halldin, S. Agurell, LE. Hollister, and H.K. Gillespie. Terminal elimination plasma half-life of A-l-tetrahydrocannabinol (t~-i- THC) in heavy users of marijuana. Eur. J. Olin. PharmacoL 37: (1989). 4. L.J. McBumey, B.A. Bobbie, and L.A. Sepp. GO/MS and EMIT analyses for delta-9-tetrahydrocannabinol metabolites in plasma and urine of human subjects. J. Anal ToxicoL 10:56-64 (1986). 5. B. Law, P.A. Mason, A.C. Moffat, R.I. Gleadle, and L.J. King. Forensic as- pects of the metabolism and excretion of cannabinoids following oral ingestion of cannabis resin. J. Pharm. Pharmacol. 36: (1984). 6. L.E. Hollister. Health aspects of cannabis. Pharm. Flev. 38:1-20 (1986). 7. R.T. Jones, N. Benowitz, and J. Bachman. Clinical studies of cannabis tolerance and dependence. Ann. N.Y. Acad. Sci. 282: (1976), 8. A.C. Hunt and R.T. Jones, Tolerance and disposition of THC in Man. J. PharmacoL Exp. Ther, 215:35-44 (1980). 9. J.W. Belleville, J.C. Gasser, and T. Miyake. Tolerance to the respiratory effects of marijuana in man. J. PharmacoL Exp. Ther. 197: (1976). 10. M.E. Wall, B.M. Sadler, D. Brine, H. Taylor, and M. Perez-Reyes. Metabolism, disposition and kinetics of t~-9-thc in men and woman. Olin. Pharmacol. Ther. 34: (1983). 11. D.C. Fenimore, CM. Davis, J.H, Whitford, and C.A. Harrington. Vapor phase silylation of laboratory glassware. Anal Chem. 58: (1976). 12. R.L. Foltz, A.F. Fentiman, and R.B. Foltz, Eds., Delta-9-THC and two metabolites, 11-hydroxy-t~-9-THC and 11-nor-9-carboxy-tL-9-THC. In GO~MS Assays for Abused Drugs in Body Fluids, NIDA Research Monograph #32, 1980, pp T.L. Hodge and E.L. Lehman. The efficiency of some nonparametric competitors of the t-test. Ann. Math. Statist. 27: (1956). 14. L. Shargel and A.B.C, Yu, Eds. Applied Biopharmaceutics and Pharmacokinetics. Appleton Century Crofts, Norwalk, CT, L.Z. Benet and R.L. Galeazzi. Noncompartmental determination of the steady state volume of distribution. J. Pharm. SoL 68: (1979). 16. J.E. Lindgren, A. Ohlsson, S. Agurell, L. Hollister, and H. Gillespie. Clinical effects and plasma levels of ~-9-THC in heavy and light cannabis users. Psychopharmacology 74: (1981). 17. A. Ohlsson, J.E. Lindgren, A. Wahlen, S. Agurell, L.E. Hollister, and H.K. Gillespie. Plasma tl-9-thc concentrations and clinical effects of marijuana after oral and intravenous administration and smoking. Olin. PharmacoL Ther. 28: (1980). 18. A. Ohlsson, J.E. Lindgren, A. Wahlen, S. Agurell, L.E. Hollister, and H.K, Gillespie. Single dose kinetics of deuterium labelled,5-1-thc in heavy and light cannabis users. Biomed. Mass. Spec. 9:6-10 (1982). 19. M.E. Wall and H.L. Taylor. Conjugation of acidic metabolites of A-8 and A- 9-THC in man. In Marihuana '84. Proceedings of the Oxford Symposium on Cannabis, D.J. Harvey, Ed., IRL Press, Oxford, 1985, pp B. Law. Confirmation of cannabis use by the analysis of A-9-THC metabolites in blood and urine by combined HPLC and RIA. J. Anal ToxicoL 8:19-22 (1984). 21. M.M. Halldin, M. Widman, S. Agurell, L.E. Hollister, and S.L. Kanter. Acidic metabolites of A-l-tetrahydrocannabinol excreted in the urine of man. In The Cannabinoids: Chemical, Pharmacologic and Therapeutic Aspects, S. Agurell, Ed., Academic Press, New York, 1984, pp M.E. Alburges and M.A. Peat. Profiles of delta-9-tetrahydrocannabinol metabolites in urine of marijuana users: Preliminary observations by high performance liquid chromatography-radioimmunoassay. J. Forens. SoL 31: (1986). 23. E. Johansson and M.M. Halldin. Urinary excretion half-life of t~-l-tetrahydrocannabinol-7-oic acid in heavy users after smoking. J. Anal ToxicoL 13: (1989). 24. S. Agurell, M. Halldin, J.E. Lindgren, A. Ohlsson, M. Widman, H. Gillespie, and L. Hollister. Pharmacokinetics and metabolism of t~-i-thc and other cannabinoids with emphasis on man. Pharm. Rev. 38:21-43 (1986). 25. S. Agurell, H. Gillespie, M. Halldin, L.E. Hollister, E. Johansson, J. E. Lindgren, A. Ohlsson, M. Szirmai, and M. Widman. A review of recent studies on the pharmacokinetics and metabolism of 4-1 -THC, cannabidiol and cannabinol in man. In Marihuana '84, Proceedings of the Oxford Symposium on Cannabis, D.J. Harvey, Ed., IRL Press, Oxford, 1985, pp J.S. Cridland, D. Rottanburg, and A.H. Robins. Apparent half-life of excretion of cannabinoids in man. Human Toxicol. 2: (1983). 27. M. Fink, J. Volavka, C. Panayiotopoulos, and C. Stefanos. Quantitative EEG studies of marijuana, A-9-THC and hashish in man. In Pharmacology of Marijuana, M. Braude and S. Szara, Eds., Raven Press, New York, 1976, pp Manuscript received May 19, 1989; revision received February 3,