Comparison in the in vitro inhibitory effects of major phytocannabinoids and polycyclic aromatic hydrocarbons contained in marijuana smoke on cytochrome P450 2C9 activity

Inhibitory effects of Δ9-tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, and polycyclic aromatic hydrocarbons (PAHs) contained in marijuana smoke on catalytic activity of human cytochrome P450 (CYP) 2C9 were investigated. These phytocannabinoids concentration-dependently inhibited S-warfarin 7-hydroxylase and diclofenac 4'-hydroxylase activities of human liver microsomes (HLMs) and recombinant CYP2C9 (rCYP2C9). In contrast, none of the twelve PAHs including benz[a]anthracene and benzo[a]pyrene exerted substantial inhibition (IC50 > 10 μM). The inhibitory potentials of Δ9-THC (Ki = 0.937-1.50 μM) and CBN (Ki = 0.882-1.29 μM) were almost equivalent regardless of the enzyme sources used, whereas the inhibitory potency of CBD (Ki > = 0.954-9.88 μM) varied depending on the enzyme sources and substrates used. Δ9-THC inhibited both S-warfarin 7-hydroxylase and diclofenac 4'-hydroxylase activities of HLMs and rCYP2C9 in a mixed manner. CBD and CBN competitively inhibited the activities of HLMs and rCYP2C9, with the only notable difference being that CBD and CBN exhibited mixed-type inhibitions against diclofenac 4'-hydroxylation and S-warfarin 7-hydroxylation, respectively, by rCYP2C9. None of Δ9-THC, CBD, and CBN exerted metabolism-dependent inhibition. These results indicated that the three major phytocannabinoids but not PAHs contained in marijuana smoke potently inhibited CYP2C9 activity and that these cannabinoids can be characterized as direct inhibitors for CYP2C9.     

Cannabidiol and delta 9-tetrahydrocannabinol metabolism. In vitro comparison of mouse and rat liver crude microsome preparations.

The in vitro biotransformations of cannabidiol (CBD) and Δ⁹-tetrahydrocannabinol (Δ-THC) by hepatic microsomal preparations from mouse and rat were compared and it was found that both cannabinoids are metabolized approximately two to three times faster by a mouse preparation than by a similar preparation from the rat. In both species, however, CBD was metabolized more slowly than Δ⁹-THC. The metabolite patterns resulting from the biotransformations of radioactive CBD and Δ⁹-THC were examined by thin-layer chromatography and were found to be qualitatively identical in both the mouse and rat. In contrast, studies of the rate of cannabinoid metabolism in the presence of excess substrate revealed that CBD metabolism was only linear for about 10 min, whereas Δ⁹-THC metabolism was linear for about 60 min. This difference was noted in both species. The evidence suggests that CBD metabolism is inhibited by its own metabolites. Furthermore, the results of other experiments indicate that CBD metabolites can also inhibit Δ⁹-THC and aminopyrine metabolism. The established ability of CBD to inhibit hepatic microsomal drug metabolism in vivo may also be the consequence of the formation of inhibitory metabolites.     

Interaction of cannabinoids with pentobarbital in rats

The effect of cannabidiol (CBD), cannabigerol (CBG), cannabinol (CBN), Δ⁸-tetrahydrocannabinol (Δ⁸-THC), and Δ⁹-tetrahydrocannabinol (Δ⁹-THC) on the in vivo metabolism of [¹⁴C]pentobarbital (¹⁴C-P) was investigated in rats. The cannabinoids were administered ip (20 mg/kg) 30 min prior to either oral or iv treatment with ¹⁴C-P. When ¹⁴C-P was given po, the ¹⁴C blood concentrations were initially depressed and later elevated by CBG and CBD, increased by Δ⁸-THC and Δ⁹-THC and unaffected by CBN. When ¹⁴C-P was injected iv, the blood ¹⁴C values were elevated by CBD and unchanged by CBG, Δ⁸-THC or Δ⁹-THC. Urinary excretion of total ¹⁴C and the metabolites of P was decreased by CBD, CBG and Δ⁸-THC during the first 6 hr following treatment. The effect of CBD on the blood concentration and urinary excretion of ¹⁴C was dose-related. In rats treated with CBD + P, the liver and serum concentrations of P metabolites were significantly lower and the liver, serum and brain concentrations of unmetabolized P were significantly higher than in P-treated rats. Pentobarbital induction and sleeping times were potentiated by CBD and Δ⁹-THC and antagonized by CBG. It was concluded that CBD delayed P metabolism, CBG decreased the rate of P absorption and excretion. Δ⁸-THC and Δ⁹-THC decreased elimination of P and CBN had little effect on P absorption, metabolism or excretion. The effect of CBD + P on sleeping time was correlated with brain P concentrations.     

Characterization of major phytocannabinoids, cannabidiol and cannabinol, as isoform-selective and potent inhibitors of human CYP1 enzymes

Inhibitory effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), cannabidiol (CBD), and cannabinol (CBN), the three major constituents in marijuana, on catalytic activities of human cytochrome P450 (CYP) 1 enzymes were investigated. These cannabinoids inhibited 7-ethoxyresorufin O-deethylase activity of recombinant CYP1A1, CYP1A2, and CYP1B1 in a competitive manner. CBD most potently inhibited the CYP1A1 activity; the apparent K_i value (0.155 μM) was at least one-seventeenth of the values for other CYP1 isoforms. On the other hand, CBN more effectively decreased the activity of CYP1A2 and CYP1B1 (K_i = 0.0790 and 0.148 μM, respectively) compared with CYP1A1 (K_i = 0.541 μM). Δ⁹-THC less potently inhibited the CYP1 activity than CBD and CBN, and showed low selectivity against the CYP1 inhibition (K_i = 2.47-7.54 μM). The preincubation of CBD resulted in a time- and concentration-dependent decrease in catalytic activity of all the recombinant CYP1 enzymes and human liver microsomes. Similarly, the preincubation of Δ⁹-THC or CBN caused a time- and concentration-dependent inhibition of recombinant CYP1A1. The inactivation of CYP1A1 by CBD indicated the highest k_inact/K_I value (540 l/mmol/min) among the CYP1 enzyme sources tested. The inactivation of recombinant CYP1A1 and human liver microsomes by CBD required NADPH, was not influenced by dialysis and by glutathione, N-acetylcysteine, and superoxide dismutase as trapping agents. These results indicated that CBD and CBN showed CYP1 isoform-selective direct inhibition and that CBD was characterized as a potent mechanism-based inhibitor of human CYP1 enzymes, especially CYP1A1.     

Effects of cannabinoids on bone marrow activity in adult mice

Adult mice were chronically treated with cannabinoids at behavioural doses and acutely treated with a unique injection of the same cannabinoids at high dosage. In acute experiments delta-9-tetrahydrocannabinol (Δ⁹-THC) depressed the number of bone marrow promyelocytes and of blood granulocytes, and enhanced the number of erythroblasts, while the action of cannabidiol (CBD) was opposite, and the one of cannabinol (CBN) was not clearly interpretable. In chronic experiments, after treatment with Δ⁹-THC a reduced proportion of erythroblasts and an increased proportion of myelopoietic cells were noted, associated with a reduction of the number of blood granulocytes and with an increased number of blood lymphocytes. CBD and CBN had initially the same depressive effect on erythropoiesis and a stimulating effect on myelopoiesis, followed by a tendency to normalization.     

Identification of in vivo liver metabolites of Δ1 -tetrahydrocannabinol,cannabidiol, and cannabinol produced by the guinea-pig

The metabolites of Δ¹ -trahydrocannabinol (Δ¹ -THC), cannabidiol (CBD), and cannabinol (CBN) produced in vivo by the guinea-pig have been studied by combined gas-liquid chromatography-mass spectrometry. 45 metabolites of Δ¹ -THC were identified of which 12 have not been reported before. Several other metabolites were detected but not identified. The major metabolic routes involved allylic and aliphatic hydroxylations, oxidations to ketones and acids, oxidative degradation of the side-chain presumably by the β-oxidation pathway, and formation of glucuronide conjugates. Di- and tri-substituted metabolites were abundant. The metabolism differed considerably from that observed in mouse and rat in that 1″- and 6β-hydroxylation and oxidative degradation of the side-chain were major metabolic pathways. 1″-Hydroxy-Δ¹ -THC was found as a pair of diasteroisomers. Similar metabolic pathways were observed with CBD; twenty metabolites were identified of which two were new. Only 6 metabolites of CBN were identified. These were mainly mono-substituted in the same positions as were observed with Δ¹ -THC and CBD.     

Cannabinoid-induced enhancement and depression of cat monosynaptic reflexes

A study of the effects of Δ-tetrahydrocannabinol (Δ⁹-THC) and cannabidiol (CBD) on the spinal monosynaptic pathway in unanesthetized spinal cats was undertaken in order to elucidate potential mechanisms of the convulsant and anticonvulsant actions of these cannabinoids. To this end, the effects of the drugs on pre- and posttetanic monosynaptic responses were compared with those of phenytoin (PHT). The data indicated that small doses of Δ⁹-THC enhanced both pre- and posttetanic potentials, a result which suggests that excitatory effects on synaptic transmission may contribute to the convulsant action of the drug. Relatively large doses of Δ⁹-THC, on the other hand, and all doses of CBD depressed the reflex, as did PHT; such depression may account, at least partially, for the anticonvulsant effects of the cannabinoids. The present study provides direct evidence that the cannabinoids produce both excitatory and depressant effects on central synaptic transmission; such results are consistent with the well-documented effects of the cannabinoids on CNS excitability.     

Differential inhibition of mitochondrial monoamine oxidase from brain by hashish components

Monoamine oxidase (MAO) of porcine brain mitochondria was differentially affected by hashish components: with benzylamine as a substrate, Δ¹-tetrahydrocannabinol (Δ¹-THC) inhibited MAO activity markedly, while cannabidiol (CBD) was essentially innocuous at the same concentrations. When added concomitantly, CBD obviated the inhibitory effect of Δ¹-THC. An extract of hashish was over 10-fold more inhibitory toward MAO than Δ¹-THC on weight basis. A prior incubation of the mitochondrial preparation with the cannabis compounds was required to express the inhibitory effect. Liver mitochondrial MAO was not affected by either Δ¹-THC, CBD or hashish extract, despite a prolonged preincubation period, thus demonstrating tissue selectivity with respect to the cannabinoid effect.     

Immunochemical approach using monoclonal antibody against Δ(9)-tetrahydrocannabinolic acid (THCA) to discern cannabis plants and to investigate new drug candidates

A monoclonal antibody (MAb-4A4) against Δ9- tetrahydrocannabinolic acid (THCA) showing extensive cross-reactivity against various cannabinoids was prepared. Using this antibody, a competitive enzyme-linked immunoassay (ELISA) was developed to detect Δ9-THCA in the range of 1 to 100 mg/ml. Various cannabinoids including Δ9-THC (Δ9-tetrahydrocannabinolic acid), Δ8-THCA (Δ8-tetrahydrocannabinolic acid), Δ8-THC (Δ8-tetrahydrocannabinol), CBD (cannabidiol), and CBN (cannabinol) were recognized by MAb-4A4, and their cross-reactivities were 55-1600% compared with Δ9-THCA (100%). This novel characteristic of this MAb enabled detection of marijuana residues in biological samples by detection of residual cannabinoids. The ELISA using MAb-4A4 was found to be applicable even for withered samples which contained only trace amounts of Δ9-THCA and Δ9-THC. In addition, this method using MAb-4A4 could be useful in forensic analysis since the MAb-4A4 also shows cross-reactivities against cannabinoid metabolites in body fluids. As well as forensic applications using this MAb, an investigation of new drug candidates focusing on cannabinoid metabolites arising from biotransformation in plant tissue was performed using immunochemical screening. The resulting new drug candidates were cannabinoid glycosides biotransformed by Pinellia ternata whose bioactivity is as yet unidentified. Our results indicate the utility of the application of ELISA using MAb-4A4 for further experiments involving marijuana and cannabinoids not only in the forensic field but also in the context of drug discovery.     

Detectability of cannabinoids in the serum samples of cannabis users: Indicators of recent cannabis use? A follow-up study

Forensic toxicologists are frequently required to predict the time of last cannabis consumption. Several studies suggested the utility of minor cannabinoids as indicators of recent cannabis use. Because several factors influence blood cannabinoid concentrations, the interpretation of serum cannabinoid concentrations remains challenging. To assess the informative value of serum cannabinoid levels in cannabis users (in total N = 117 patients, including 56 patients who stated an exact time of last cannabis use within 24 h before blood sampling), the detectability of cannabinoids, namely, delta-9-tetrahydrocannabinol (delta-9-THC), 11-hydroxy-delta-9-THC, 11-nor-9-carboxy-delta-9-THC, cannabichromene (CBC), cannabidiol (CBD), cannabinol (CBN), cannabidivarin, tetrahydrocannabivarin, cannabigerol (CBG), cannabicyclol, delta-8-THC, tetrahydrocannabinolic acid A, cannabichromenic acid, cannabidiolic acid (CBDA), cannabigerolic acid, cannabicyclolic acid (CBLA), 11-nor-9-carboxy-THCV (THCVCOOH), and 11-nor-CBN-9-COOH, was investigated. Excluding CBDA and CBLA, all investigated cannabinoids were detected in at least one analyzed sample. The interval between cannabis consumption and sample collection (reported by the patients) was not correlated with cannabinoid concentrations. Minor cannabinoids tended to be more easily detected in samples obtained shortly after consumption. However, some samples tested positive for minor cannabinoids despite an interval of several hours or even days between consumption and sampling (according to patients' statements). For instance, CBC, CBG, THCVCOOH, CBD, and CBN in certain cases could be detected more than 24 h after the last consumption of cannabis. Thus, findings of minor cannabinoids should always be interpreted with caution.     

Effect of cannabis on pentobarbital-induced sleeping time and pentobarbital metabolism in the rat

The effects of two cannabis extracts with different cannabinoid compositions, as well of as pure Δ¹-tetrahydrocannabinol (THC), cannabinol (CBN) and cannabidiol (CBD), on pentobarbital metabolism were studied in the rat. Extract I, with high proportions of CBN and CBD relative to THC, when given by gavage 21·5, 40 or 63 hr before pentobarbital (30 mg/kg, i.p.), prolonged the sleeping time by 53, 42 and 21 per cent respectively. This effect was paralleled by decreases in the rate of disappearance of [¹⁴C]pentobarbital from the blood, and of pentobarbital metabolism by liver microsomal preparations in vitro. Extract II, with low relative proportions of CBN and CBD, did not have any significant effect on penobarbital metabolism or sleeping time. CBD alone, in the same dose as that given in Extract I, had very similar effects, while a dose of CBD equivalent to that given in Extract II had no effect. THC, CBN and CBD added to normal rat liver microsomes in vitro inhibited pentobarbital metabolism competitively, CBD being a much more potent inhibitor than THC and CBN. The CBD content may, therefore, be a significant factor in interactions between marijuana and other drugs.     

Effects of synthetic progestagens on drug metabolism in rat liver microsomes

The inhibitory action of four progestagens on oxidative drug metabolism was studied. p-Nitroanisole demethylation and aniline hydroxylation in phenobarbital stimulated rat liver musomes were inhibited by norethisterone acetate, d-norgestrel, lynestrenol and allylestrenol. Inhibitor constants were between 19 μM and 49 μM for p-nitroanisole demethylation and between 45 μM and 112 μM for aniline hydroxylation. The four steroids tested exhibited type I difference spectra in musomes under aerobic conditions. The spectral dissociation constants were in the same order of magnitude as the inhibitor constants.     

The effect of cannabinoids (Δ9-THC and Δ8-THC) on hepatic microsomal metabolism of testosterone in vitro

The effects of Δ⁹-THC and Δ⁸-THC on testosterone metabolism by rat liver microsomal enzymes were studied in vitro. Δ⁹-THC (25 μM) inhibit the 5α-reduction of testosterone while Δ⁸-THC has no effect at double the concentration. Both Δ⁹-THC and Δ⁸-THC inhibit the hydroxylation of testosterone. This inhibition is dose dependent over the dose range (25–100 μM) tested. At the same molar concentration, Δ⁸-THC inhibits testosterone hydroxylation to a greater extent than Δ⁹-THC. The kinetic data suggest that the observed inhibition on 5α-reduction and total hydroxylation by the tetrahydrocannabinoids is of the competitive type.     

Human skin permeation of Delta8-tetrahydrocannabinol, cannabidiol and cannabinol

The purpose of this study was to quantify the in-vitro human skin transdermal flux of Delta8-tetrahydrocannabinol (Delta8-THC), cannabidiol (CBD) and cannabinol (CBN). These cannabinoids are of interest because they are likely candidates for transdermal combination therapy. Differential thermal analysis and in-vitro diffusion studies with human tissue were completed for the compounds. Heats of fusion, melting points and relative thermodynamic activities were determined for the crystalline compounds, CBD and CBN. Flux, permeability, tissue concentration and lag times were measured in the diffusion experiments. CBN had a lower heat of fusion and corresponding higher calculated relative thermodynamic activity than CBD. Ethanol concentrations of 30 to 33% significantly increased the transdermal flux of Delta8-THC and CBD. Tissue concentrations of Delta8-THC were significantly higher than for CBN. Lag times for CBD were significantly smaller than for CBN. The permeabilities of CBD and CBN were 10-fold higher than for Delta8-THC. Combinations of these cannabinoids with ethanol will be further studied in transdermal patch formulations in vitro and in vivo, as significant flux levels of all the drugs were obtained. CBD, the most polar of the three drugs, and other more polar cannabinoids will also be the focus of future drug design studies for improved transdermal delivery rates.     

Specific inhibition of an acyltransferase by delta9-tetrahydrocannabinol

Increased phospholipid turnover in mouse lymphocytes during mitogen-induced blastogenesis can be prevented by low levels of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) invitro. Acyl CoA: lysophosphati-dylcholine acyltransferase, a lymphocyte plasma membrane enzyme, is inhibited by Δ⁹-THC with a K_i of 0.35 μM. Other cannabinoids with much lower psychoactivity inhibit the enzyme similarly only if present at more than 100 times the concentration needed for Δ⁹-THC inhibition. For cannabinoids other than Δ⁹-THC and for a variety of membrane-active lipids, the enzyme inhibition at higher concentrations of lipid correlates well with their anesthetic activity as measured by their ability to protect erythrocytes against hemolysis. However, the inhibition by Δ⁹-THC is much greater than that predicted by its anti-hemolytic activity, which indicates stereospecific requirements for inhibition of the enzyme. The inhibition of acyltransferase by Δ⁹-THC is not restricted to the lymphocyte enzyme and can also be demonstrated in mouse brain synaptosomes, suggesting a possible mechanism for psychoactive specificity.     

Effect of cannabinoids on neurotransmitter uptake,atpase activity and morphology of mouse brain synaptosomes

Δ¹-Tetrahydrocannabinol (Δ¹-THC) and cannabidiol (CBD), a psychoactive and a nonpsychoactive constituent of marijuana respectively, inhibit the uptake of ³H-labelled norepinephrine (NE), dopamine (DA), γ-aminobutyric acid (GABA) and serotonin (5-HT), by mouse brain synaptosomes. CBD is more effective than Δ¹-THC in the inhibition of neurotransmitter uptake. At 5 × 10^?5 M both CBD and Δ¹-THC inhibit uptake by 60–100%. The one exception to the above is the differential effect of Δ¹-THC and CBD on 5-HT uptake. At 10^?6 M of Δ¹-THC the uptake is twice that of the control value and at 5 × 10^?5 M uptake is still equal to control value. At the former concentration CBD has no effect on 5-HT uptake whereas at the latter concentration a 50 per cent inhibition is observed. Both Δ¹-THC and CBD inhibit Na⁺-K⁺-ATPase and Mg-ATPase activities; at 5 × 10^?5 M inhibition amounts to 40 per cent. Electron microscopy reveals that synaptosomal preparations are highly damaged at 5 × 10^?5 M. Thus inhibition of uptake could stem from either failure of ATPase activity, from disruption of synaptosomes, or from both.     

Cannabidiol interferes with the effects of delta 9 - tetrahydrocannabinol in man

Based on previous observations that cannabidiol (CBD) blocks some effects of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) in laboratory animals, the present work was carried out to study possible interaction between CBD and Δ⁹-THC in human beings. In a double blind procedure, 40 healthy male volunteers were assigned to 1 of 8 experimental groups, receiving per oral route, placebe, 30 mg Δ⁹-THC, 15 30 or 60 mg of CBD, and mixtures of 30 mg of Δ⁹-THC plus either 15, 30 or 60 mg of CBD respectively. Pulse rate, time production tasks and psychological logical reactions were measured at several time intervals after drug ingestion. 30 mg Δ⁹-THC alone increased pulse rate, disturbed time tasks and induced strong psychological reactions in the subjects. 15–60 mg of CBD alone provoked no effects. On the other hand, CBD was efficient in blocking most of the effects of Δ⁹-THC when both drugs were given together. CBD also decreased the anxiety component of Δ⁹-THC effects, in such a way that the subjects reported more pleasurable effects.     

Cannabinoid effects on plasma corticosterone and uptake of 3H-corticosterone by mouse brain

The effects of three cannabinoids, 11-hyddroxy-Δ⁹-tetrahydracannabinol (11-OH-Δ⁹-THC), Δ⁹-THC and cannabinol (CBN), ranging in behavioral activity from high to low, were studied on two aspects of pituitary-adrenal function. Plasma corticosterone levels were used as an index of adrenocorticotropic hormone (ACTH) release. All three cannabinoids elicited an increase in plasma corticosterone levels in a mannger similr to their behavioral potency. These cannabinoids also elicited an increase in the concentration of ³H-corticosterone taken up by the brains of adrenalectomized mice in a mannger similar to their potency in elevating plasma corticosterone levels. The significance and possible underlying mechanism of the apparent correlation resulting between these effects and the behavioral effects of cannabionoids are discussed.     

Antidepressant-like effect of Δ-tetrahydrocannabinol and other cannabinoids isolated from Cannabis sativa L.

The antidepressant action of cannabis as well as the interaction between antidepressants and the endocannabinoid system has been reported. This study was conducted to assess the antidepressant-like activity of ?⁹-THC and other cannabinoids. Cannabinoids were initially evaluated in the mouse tetrad assay to determine doses that do not induce hypothermia or catalepsy. The automated mouse forced swim (FST) and tail suspension (TST) tests were used to determine antidepressant action. At doses lacking hypothermic and cataleptic effects (1.25, 2.5, and 5 mg/kg, i.p.), both ?⁹-THC and ?⁸-THC showed a U-shaped dose response with only ?⁹-THC showing significant antidepressant-like effects at 2.5 mg/kg (p < 0.05) in the FST. The cannabinoids cannabigerol (CBG) and cannabinol (CBN) did not produce antidepressant-like actions up to 80 mg/kg in the mouse FST, while cannabichromene (CBC) and cannabidiol (CBD) exhibited significant effect at 20 and 200 mg/kg, respectively (p < 0.01). The antidepressant-like action of ?⁹-THC and CBC was further confirmed in the TST. ?⁹-THC exhibited the same U-shaped dose response with significant antidepressant-like action at 2.5 mg/kg (p < 0.05) while CBC resulted in a significant dose-dependent decrease in immobility at 40 and 80 mg/kg doses (p < 0.01). Results of this study show that ?⁹-THC and other cannabinoids exert antidepressant-like actions, and thus may contribute to the overall mood-elevating properties of cannabis.     

Anticonvulsant activity of delta-8- and delta-9-tetrahydrocannabinol in rats

Intraperitoneal injections of Δ⁸-tetrahydrocannabinol (THC) and Δ⁹-THC, two major psychoactive constituents of cannabis, produced dose-related protection against tonic extension induced by electroshock in rats. The cannabinoids provided protection against clonic convulsions induced by pentylenetetrazol (Metrazol) only at very high and sometimes lethal doses, and the protection was quantal rather than dose-related. The two isomers of the THC were equipotent in terms of behavioral toxicity and protection against tonic convulsions. However, the significance of the drugs' anticonvulsant activity must be qualified by the observatin that protection was provided by either drug only at doses producing marked toxic behavioral reactions.