Tetrahydrocannabinol (THC): Metabolism and subjective effects

C-14-labeled delta-9-tetrahydrocannabinol (delta-9-THC) was administered in spiked cigarettes to nine experienced marijuana smokers. Blood samples obtained by repeated venipuncture showed that the subjects' subjective estimates of being “high” appeared to parallel the blood concentration of THC metabolites at least as closely as the blood concentration of THC itself. After 30 minutes, subjective effects declined less rapidly than either THC or its metabolites.Substantial inter-individual consistency in THC concentrations was found, suggesting that administration of THC in cigarettes under standardized smoking conditions can produce reliable blood concentrations of THC.A second session was run with the same subjects, this time omitting venipuncture, and using unlabeled THC. Significant differences between the effects of initial doses of THC under stress and no-stress conditions appeared in the detailed subjective effects inventories provided by subjects.     

Inhaled marijuana smoke disrupts mitochondrial energetics in pulmonary epithelial cells in vivo

Habitual marijuana smoking is associated with inflammation and atypia of airway epithelium accompanied by symptoms of chronic bronchitis. We hypothesized that Delta(9)-tetrahydrocannabinol (THC), the primary psychoactive component of marijuana, might contribute to these findings by impairing cellular energetics and mitochondrial function. To test this hypothesis, we examined particulate smoke extracts from marijuana cigarettes, tobacco cigarettes, and placebo marijuana (0% THC) cigarettes for their effects on the mitochondrial function of A549 cells in vitro. Only extracts prepared from marijuana cigarettes altered mitochondrial staining by the potentiometric probe JC-1. With the use of a cross-flow, nose-only inhalation system, rats were then exposed for 20 min to whole marijuana smoke and examined for its effects on airway epithelial cells. Inhalation of marijuana smoke produced lung tissue concentrations of THC that were 8-10 times higher than those measured in blood (75 +/- 38 ng/g wet wt tissue vs. 9.2 +/- 2.0 ng/ml), suggesting high local exposure. Intratracheal infusion of JC-1 immediately following marijuana smoke exposure revealed a diffuse decrease in lung cell JC-1 red fluorescence compared with tissue from unexposed or placebo smoke-exposed rats. Exposure to marijuana smoke in vivo also decreased JC-1 red fluorescence (54% decrease, P < 0.01) and ATP levels (75% decrease, P < 0.01) in single-cell preparations of tracheal epithelial cells. These results suggest that inhalation of marijuana smoke has deleterious effects on airway epithelial cell energetics that may contribute to the adverse pulmonary consequences of marijuana smoking.     

Low platelet monoamine oxidase activity and chronic marijuana use

Mean platelet monoamine oxidase (MAO) activity in 26 consecutively-studied male marijuana smokers was significantly lower than in a comparable group of non-marijuana smoking males. In addition, the level of current marijuana use reported by the subjects was significantly and inversely correlated with MAO activity. No acute reduction in MAO activity was found in response to smoking a marijuana cigarette containing 15 mg of delta-9-THC. Significant $\text{in vitro}$ inhibition of MAO activity by THC was detected only at THC concentrations above 10^?5M, approximately 100 times the peak plasma concentrations seen $\text{in vivo}$ following smoking.     

Acquisition of tolerance to Δ-9-THC as measured by the response of a cellular function

The development of tolerance to delta-9-tetrahydrocannabinol (Δ-9-THC) was investigated by measuring respiration in brain tissue after acute or chronic administration. Mice were given either single or seven daily repeated intraperitoneal injections of 50 mg/Kg of delta-9-tetrahydrocannabinol (Δ-9-THC) or control vehicle. The final injection for all drug treated animals included radiolabeled ³H-Δ-9-THC. The mice were sacrificed at 1 hour, 2 hours, 4 hours, 24 hours, and 7 days after the final injection. Δ-9-THC depressed respiration, but after repeated injections was significantly less effective in this regard, indicating acquisition of tolerance to Δ-9-THC. Because the concentration of radiolabeled cannabinoids in brain tissue from each group is not appreciably different, a cellular as opposed to distributional mode of tolerance is suggested.     

Time course and dose-response effects of orally administered delta-9-THC on interval schedule performance of the rat

Rats that had been trained on fixed or variable interval schedules of food reinforcement were given oral administrations of 4, 8, 16, and 32 mg/kg of delta-9-tetrahydrocannabinol at various times prior to experimental sessions. The two lower doses either had no effect or increased response rates up to 8 hours post-administration. The two higher doses suppressed responding, with a maximum effect about 24 hours post-administration. Responding recovered to baseline levels about 30 hours after drug administration.     

Plasma concentrations of delta-9-tetrahydrocannabinol in dams and fetuses following acute or multiple prenatal dosing in rats

Delta-9-tetrahydrocannabinol (THC) was administered by gastric intubation to pregnant rats to study the effects of dose-level and dosing regimen on plasma concentration in dams and fetuses. Two multiple-dose groups were administered either 15 or 50 mg/kg of delta-9-THC once daily during the last two weeks of gestation. Two acute groups were administered the same dose as above but only once on the last day of gestation. Sixty min after receiving the last dose all dams and their fetuses were sacrificed by decapitation, blood collected, centrifuged and plasma removed. Quantitative measurement of delta-9-THC in plasma was carried out using GS/MS. Among the dams, plasma concentrations covaried with dose and multiple dosing produced higher concentrations than acute, especially at the high dose. Among the fetuses, plasma concentrations were approximately 10% of those found in the dams. The fetuses from the high, multiple-dose dams similarly yielded significantly higher concentrations. These findings are discussed with respect to other studies of the placental transfer of delta-9-THC and effects of postnatal developmental.     

Mode of action of delta-9-tetrahydrocannabinol on hypothalamo-pituitary function in adult female rats.

Administration of delta-9-tetrahydrocannabinol (delta 9-THC) to pro-oestrous rats (5 mg/kg and 10 mg/kg, i.p. for 10 days) decreased the hypothalamic LH-RH content. Serum prolactin levels were reduced but serum LH and FSH and pituitary hormone content were similar to values in dioestrous rats. It is suggested that delta 9-THC acts primarily on the hypothalamus.     

Influence of different barbiturate anesthetics on delta-9-tetrahydrocannabinol effects on spinal monosynaptic reflexes

Two barbiturates, pentobarbital and methohexital, were used as general anesthetics to evaluate their interactions with the effects of delta-9-tetrahydrocannabinol (delta-9-THC) on spinal monosynaptic reflexes in cats with transected spinal cords and ischemically destroyed brains. In animals initially anesthetized with pentobarbital, delta-9-THC over a wide dosage range produced only an enhancement of the reflex, whereas in methohexital-treated animals only depression was elicited. Because delta-9-THC is known to produce both excitatory and depressant effects in conscious animals, the results of the present study demonstrate that the choice of anesthetic may determine which effects manifest themselves. Therefore, if anesthesia is used in the investigation of any cannabinoid, the possibility of such interactions must be considered when interpreting the results.     

The anticonvulsant activity of cannabidiol and cannabinol

The anticonvulsant activity of delta-9-tetrahydrocannabinol was compared with that of two other naturally occurring cannabinoids, cannabidiol and cannabinol, in a maximal electroshock test in mice. The drugs were administered as an emulsion of sesame seed oil, Tween 80 and saline to mice i.p. The results indicate that all three cannabinoids are effective anticonvulsants. The time for peak effect is about 2 hr. In terms of relative potencies, cannabidiol and delta-9-THC are similar but both of them are more active than cannabinol.     

Delta-9-tetrahydrocannabinol stimulates ABP secretion from rat Sertoli cells in vitro

The effect of delta-9-tetrahydrocannabinol (THC) on rat Sertoli cell function was investigated. THC significantly increased ABP secretion by 1.5- to 2.1-fold but did not consistently enhance the stimulation of ABP induced by FSH, testosterone or dibutyryl cyclic AMP. ABP was measured by steady-state polyacrylamide gel electrophoresis, DEAE Bio-Gel and immunoassay; all three methods gave similar results. The minimal concentration of THC that stimulated ABP was 10 ng/ml; maximal stimulation was observed with 100-200 ng/ml. This effect was specific since THC did not affect gamma glutamyl transpeptidase activity or the secretion of plasminogen activator, lactate and transferrin. This observation that THC affects ABP secretion specifically is the first report of any differential effect of a drug on Sertoli cell secretion.     

Seasonal fluctuations in cannabinoid content of Kansas Marijuana

Marijuana (Cannabis sativa L.) was sampled at nine progressive growth stages in Riley County, Kansas, and analyzed for four major cannabinoids: cannabidiol (CBD), della-8-tetrahydrocannabinol (delta-8-THC), delta-9-tetrahydrocannabinol (delta-9-THC), and cannabinol (CBN). Seasonal fluctuation in cannabinoids were related to stage of plant development. Cannabinoids were lowest in seedlings, highest prior to flowering and at an intermediate level thereafter until physiological maturity. Cannabinoids were highest in flowers and progressively lower in leaves, petioles, stems, seeds, and roots. Cannabinoid content of male and female flowers was not significantly different. Cannabidiol occurred in the highest concentrations (0.01 to 0.94% of dry matter) in all plant parts; delta-9-THC, the next highest (0.0001 to 0.06%) in the study over time. Cannabidiol content of leaf tissue of plants sampled from ten locations at flowering, ranged from 0.12 to 1.7%; delta-9-THC, from 0.01 to 0.49%. Some variation was attributed to environmental factors. Results indicate transformation of CBD to delta-9-THC to CBN. Environmental stress apparently increased delta-9-THC concentration, and bivalent ions: Mg, Mn, and Fe of leaf tissue could have regulated enzyme systems responsible for cannabinoid synthesis.     

Involvement of reduced acetylcholine release in Delta9-tetrahydrocannabinol-induced impairment of spatial memory in the 8-arm radial maze

To clarify the mechanism by which Delta9-tetrahydrocannabinol, a major psychoactive component of marijuana, impairs spatial memory in the 8-arm radial maze in rats via the cholinergic system, we used two acetylcholinesterase inhibitors, physostigmine and tetrahydroaminoacridine. Moreover, we examined the effect of Delta9-tetrahydrocannabinol on acetylcholine release in the frontal cortex and dorsal and ventral hippocampus using in vivo microdialysis. Physostigmine (0.01-0.05 mg/kg, i.p.) and tetrahydroaminoacridine (1-5 mg/kg, p.o.) improved the impairment of spatial memory induced by Delta9-tetrahydrocannabinol (6 mg/kg, i.p.) in the 8-arm radial maze. Delta9-tetrahydrocannabinol (6 mg/kg, i.p.) produced a significant decrease in acetylcholine release in the dorsal hippocampus as assessed by microdialysis. Moreover, tetrahydroaminoacridine at a dose of 1 mg/kg, which improved the impairment of spatial memory, reversed the decrease in acetylcholine release induced by Delta9-tetrahydrocannabinol in the dorsal hippocampus during 60-120 min after the Delta9-tetrahydrocannabinol injection. These findings suggest that inhibition of the cholinergic pathway by reduced acetylcholine release is one of the means by which Delta9-tetrahydrocannabinol impairs spatial memory in the 8-arm radial maze.     

Testicular steroidogenesis in the baboon Papio anubis.

We recently reported that the baboon testis converts pregnenolone to testosterone through the delta-4 pathway. The present studies were to determine the metabolism of intermediates of the delta-4 and delta-5 pathway by the baboon testis. Fragments (50 mg) were incubated for 3 hr with 10 muCi of the following tritium-labelled substrates: pregnenolone, progesterone, 17-hydroxypregnenolone, 17-hydroxyprogesterone, dehydroepiandrosterone, androstenedione, or testosterone. Pregnenolone was converted to testosterone primarily through the delta-4 pathway, with accumulation of progesterone, 17-hydroxyprogesterone and 20alpha-dihydroprogesterone as predominant intermediates. Similar results were obtained in progesterone incubations. 17-hydroxyprogesterone was not efficiently metabolized by the fragments, while 17-hydroxypregnenolone and dehydroepiandrosterone were efficiently converted into testosterone and androstenedione. Androstenedione was metabolized primarily to testosterone, while testosterone was not a suitable substrate. Some 5alpha-androstanediol was identified in each incubate. These results suggest that although testosterone is formed from pregnenolone through the delta-4 pathway, the delta-5 intermediates are more suitable substrates for testosterone synthesis in the baboon testis.     

Marihuana, tetrahydrocannabinol and T-cell function.

Conflicting reports exhist on the effect of marihuana smoking on thymus derived lymphocytes (T-cells). Marihuana smoking has been reported to both reduce the formation of T-rosettes and affect mitogenic stimulation of T-cells in man. This present study was conducted to evaluate the effects of marihuana smoking on T-cells during a 24-hour period after smoking. Chronic marihuana smokers, who had abstained from smoking for one month, smoked “street” marihuana, marihuana cigarettes with a known quantity of delta-9-tetrahydrocannabinol (THC), or placebo marihuana cigarettes. In two of three subjects who used “street” marihuana, T-cell rosette formation was reduced 24 hours after smoking. Response to phytohemagglutinin (PHA) stimulation was reduced in one of the above subjects. In a second group of subjects, rosette formation was decreased in five of six subjects 3 to 6 hours after smoking marihuana cigarettes containing 10 mg of THC. However, values in all but one individual returned to control levels within 24 hours. A 50% reduction in PHA stimulation was also observed in this subject 6 hours after smoking. PHA stimulation was not affected following placebo. In the sixth subject, a stimulatory effect on rosette formation was observed following both the use of the THC-containing and placebo marihuana cigarettes. The results of these studies indicate that while marihuana smoking affects certain $\text{in}$$\text{vitro}$ tests of T-cell function, the effects are variable, transitory, and may be associated with factors other than THC.     

Cannabis reinforcement and dependence: role of the cannabinoid CB1 receptor.

Awareness of cannabis dependence as a clinically relevant issue has grown in recent years. Clinical and laboratory studies demonstrate that chronic marijuana smokers can experience withdrawal symptoms upon cessation of marijuana smoking and have difficulty abstaining from marijuana use. This paper will review data implicating the cannabinoid CB1 receptor in regulating the behavioral effects of Delta(9)-tetrahydrocannobinol (THC), the primary psychoactive component of cannabis, across a range of species. The behavioral effects that will be discussed include those that directly contribute to the maintenance of chronic marijuana smoking, such as reward, subjective effects, and the positive and negative reinforcing effects of marijuana, THC and synthetic cannabinoids. The role of the CB1 receptor in the development of marijuana dependence and expression of withdrawal will also be discussed. Lastly, treatment options that may alleviate withdrawal symptoms and promote marijuana abstinence will be considered.     

Relationship between the subcellular distribution of delta-9-tetrahydrocannabinol and its metabolites in rat brain and the duration of the effect on motor activity.

Female rats were injected intraperitoneally with 10 mg/kg of unlabelled delta-9-tetrahydrocannabinol (Δ⁹-THC) and their locomotor activity was recorded every 15 minutes for 12 hours. The maximum depressant effect was observed between the first and fourth hour and had completely disappeared by the eighth hour of treatment. In parallel experiments rats were injected with 10 mg/kg of ³H-delta-9-THC and decapitated either one, four or twelve hours later. The concentrations of unchanged delta-9-THC and metabolites in brain subcellular fractions were determined using thin layer chromatographic methods. There were no substantial differences in the relative specific activities of delta-9-THC or 11-OH-delta-9-THC between all fractions except cytosol, indicating no preferential site of accumulation. However, when the synaptosomal fraction was osmotically shocked, the concentration of delta-9-THC in nerve-ending membranes was markedly higher than that in vesicles or soluble fraction. Our results $\text{in vivo}$ showed a marked decline, over twelve hours, in the relative specific activities of delta-9-THC and 11-OH-delta-9-THC with a concomitant increase in the concentration of highly polar, non-extractable metabolites in all subfractions. It is suggested that the diminution of the depressant effect on motor activity may be related to the formation of highly polar, pharmacologically inactive metabolites of delta-9-THC and/or 11-OH-delta-9-THC inside the brain which do not easily migrate out of the cells.     

Effects of delta-9-tetrahydrocannabinol on human immune function and host defense

This review examines evidence that delta(9)-tetrahydrocannabinol (THC) can regulate and suppress human immune responses. Leukocytes express both cannabinoid receptor type 1 (CB1) and cannabinoid receptor type 2 (CB2), and levels of mRNA encoding for them are increased in peripheral blood leukocytes obtained from marijuana smokers, suggesting cannabinoid receptor activation in vivo. Exposure of human T-cells to THC suppresses their proliferation, inhibits the release of interferon-gamma, and skews the balance of T-helper cytokines towards a type 2 response. The majority of these effects are CB2 receptor-dependent. Consistent with an impact of THC on cell-mediated immunity, alveolar macrophages (AMs) recovered from the lungs of marijuana smokers are suppressed in their ability to release pro-inflammatory cytokines and nitric oxide (NO), and kill bacteria. Macrophage function is restored by treatment with interferon-gamma, a type 1 cytokine. Habitual exposure to THC appears capable of impacting on human cell-mediated immunity and host defense.     

Delta 9-tetrahydrocannabinol disrupts mitochondrial function and cell energetics

We have observed rapid and extensive depletion of cellular energy stores by Delta(9)-tetrahydrocannabinol (THC) in the pulmonary transformed cell line A549. ATP levels declined dose dependently with an IC(50) of 7.5 microg/ml of THC after 24-h exposure. Cell death was observed only at concentrations >10 microg/ml. Studies using JC-1, a fluorescent probe for mitochondrial membrane potential, revealed diminished mitochondrial function at THC concentrations as low as 0.5 microg/ml. At concentrations of 2.5 or 10 microg/ml of THC, a decrease in mitochondrial membrane potential was observed as early as 1 h after THC exposure. Mitochondrial function remained diminished for at least 30 h after THC exposure. Flow cytometry studies on cells exposed to particulate smoke extracts indicate that JC-1 red fluorescence was fivefold lower in cells exposed to marijuana smoke extract relative to cells exposed to tobacco smoke extract. Comparison with a variety of mitochondrial inhibitors demonstrates that THC produced effects similar to that of carbonyl cyanide p-trifluoromethoxyphenylhydrazone, suggesting uncoupling of electron transport. Loss of red JC-1 fluorescence by THC was suppressed by cyclosporin A, suggesting mediation by the mitochondrial permeability transition pore. This disruption of mitochondrial function was sustained for at least 24 h after removal of THC by extensive washing. These results suggest that exposure of the bronchopulmonary epithelium to THC may have important health and physiological consequences.     

Determination of amino acids in different regions of the rat brain. Application to the acute effects of tetrahydrocannabinol (THC) and trimethyltin (TMT)

A modified HPLC method is described for the determination of amino acids [aspartic acid, glutamic acid, glutamine, glycine, taurine, and gamma-aminobutyric acid (GABA)] in brain tissue utilizing precolumn derivatization with o-phthalaldehyde (OPA)-tert-butyl-thiol and electrochemical detection. A simple extraction procedure was employed and DL-homoserine used as internal standard. A neurotoxin previously shown to affect brain amino acids (trimethyltin, TMT) and a psychoactive compound hypothesized to act on these neurochemicals (delta-9-tetrahydrocannabinol, THC) were administered to adult male rats and amino acids were measured. Results revealed a gradient of distribution of most amino acids, with lowest levels posteriorly in the brain stem and increasing to the highest values in anterior cortical regions. TMT increased glutamine significantly in all brain regions examined, but increased glycine and decreased taurine only in the frontal cortex and hippocampus. No significant changes in any amino acid were found in hippocampus after THC treatment. The results establish the validity and usefulness of this HPLC method for detecting neurotoxicity-related changes in brain amino acid metabolism.