Cannabinoid receptor 2 (Cb2r) mediates cannabinol (CBN) induced developmental defects in zebrafish.

Of the three primary cannabinoids in cannabis: Δ9-Tetrahydrocannabinol (Δ9-THC), cannabidiol (CBD) and cannabinol (CBN), very little is known about the actions of CBN, the primary oxidative metabolite of THC. Our goal was to determine if CBN exposure during gastrulation alters embryonic development, and if so, does it act via the canonical cannabinoid receptors. Zebrafish embryos were exposed to CBN during gastrulation and exhibited dose-dependent malformations, increased mortality, decreased locomotion and a reduction in motor neuron branching. Moreover, larva showed a significant reduction in the response to sound stimuli. CBN exposure altered the development of hair cells associated with otic vesicles and the lateral line. Pharmacological block of Cb2rs with AM 630 or JTE 907 prevented many of the CBN-induced developmental defects, while block of Cb1rs with AM 251 or CP 945598 had little or no effect. Altogether we show that embryonic exposure to CBN results in alterations in embryonic growth, neuronal and hair cell development, physiology and behavior via Cb2r-mediated mechanisms.

Cannabinol inhibits oxytosis/ferroptosis by directly targeting mitochondria independently of cannabinoid receptors.

The oxytosis/ferroptosis regulated cell death pathway recapitulates many features of mitochondrial dysfunction associated with the aging brain and has emerged as a potential key mediator of neurodegeneration. It has thus been proposed that the oxytosis/ferroptosis pathway can be used to identify novel drug candidates for the treatment of age-associated neurodegenerative diseases that act by preserving mitochondrial function. Previously, we identified cannabinol (CBN) as a potent neuroprotector. Here, we demonstrate that not only does CBN protect nerve cells from oxytosis/ferroptosis in a manner that is dependent on mitochondria and it does so independently of cannabinoid receptors. Specifically, CBN directly targets mitochondria and preserves key mitochondrial functions including redox regulation, calcium uptake, membrane potential, bioenergetics, biogenesis, and modulation of fusion/fission dynamics that are disrupted following induction of oxytosis/ferroptosis. These protective effects of CBN are at least partly mediated by the promotion of endogenous antioxidant defenses and the activation of AMP-activated protein kinase (AMPK) signaling. Together, our data highlight the potential of mitochondrially-targeted compounds such as CBN as novel oxytotic/ferroptotic inhibitors to rescue mitochondrial dysfunction as well as opportunities for the discovery and development of future neurotherapeutics.

Activity of THC, CBD, and CBN on Human ACE2 and SARS-CoV1/2 Main Protease to Understand Antiviral Defense Mechanism.

THC, CBD, and CBN were reported as promising candidates against SARS-CoV2 infection, but the mechanism of action of these three cannabinoids is not understood. This study aims to determine the mechanism of action of THC, CBD, and CBN by selecting two essential targets that directly affect the coronavirus infections as viral main proteases and human angiotensin-converting enzyme2. Tested THC and CBD presented a dual-action action against both selected targets. Only CBD acted as a potent viral main protease inhibitor at the IC50 value of 1.86 ± 0.04 µM and exhibited only moderate activity against human angiotensin-converting enzyme2 at the IC50 value of 14.65 ± 0.47 µM. THC acted as a moderate inhibitor against both viral main protease and human angiotensin-converting enzymes2 at the IC50 value of 16.23 ± 1.71 µM and 11.47 ± 3.60 µM, respectively. Here, we discuss cannabinoid-associated antiviral activity mechanisms based on in silico docking studies and in vitro receptor binding studies.

Differential Interactions of Selected Phytocannabinoids with Human CYP2D6 Polymorphisms.

Cytochrome P450 2D6 (CYP2D6) is primarily expressed in the liver and in the central nervous system. It is known to be highly polymorphic in nature. It metabolizes several endogenous substrates such as anandamide (AEA). Concomitantly, it is involved in phase 1 metabolism of several antidepressants, antipsychotics, and other drugs. Research in the field of phytocannabinoids (pCBs) has recently accelerated owing to their legalization and increasing medicinal use for pain and inflammation. The primary component of cannabis is THC, which is well-known for its psychotropic effects. Since CYP2D6 is an important brain and liver P450 and is known to be inhibited by CBD, we investigated the interactions of four important highly prevalent CYP2D6 polymorphisms with selected phytocannabinoids (CBD, THC, CBDV, THCV, CBN, CBG, CBC, ß-carophyllene) that are rapidly gaining popularity. We show that there is differential binding of CYP2D6*17 to pCBs as compared to WT CYP2D6. We also perform a more detailed comparison of WT and *17 CYP2D6, which reveals the possible regulation of AEA metabolism by CBD. Furthermore, we use molecular dynamics to delineate the mechanism of this binding, inhibition, and regulation. Taken together, we have found that the interactions of CYP2D6 with pCBs vary by polymorphism and by specific pCB class.

Inhibition of UDP-Glucuronosyltransferase Enzymes by Major Cannabinoids and Their Metabolites.

The UDP-glucuronosyltransferase (UGT) family of enzymes play a central role in the metabolism and detoxification of a wide range of endogenous and exogenous compounds. UGTs exhibit a high degree of structural similarity and display overlapping substrate specificity, often making estimations of potential drug-drug interactions difficult to fully elucidate. One such interaction yet to be examined may be occurring between UGTs and cannabinoids, as the legalization of recreational and medicinal cannabis and subsequent co-usage of cannabis and therapeutic drugs increases in the United States and internationally. In the present study, the inhibition potential of the major cannabinoids Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN), as well as their major metabolites, was determined in microsomes isolated from HEK293 cells overexpressing individual recombinant UGTs and in microsomes from human liver and kidney specimens. The highest inhibition was seen by CBD against the glucuronidation activity of UGTs 1A9, 2B4, 1A6, and 2B7, with binding-corrected IC50 values of 0.12 ± 0.020 µM, 0.22 ± 0.045 µM, 0.40 ± 0.10 µM, and 0.82 ± 0.15 µM, respectively. Strong inhibition of UGT1A9 was also demonstrated by THC and CBN, with binding-corrected IC50 values of 0.45 ± 0.12 µM and 0.51 ± 0.063 µM, respectively. Strong inhibition of UGT2B7 was also observed for THC and CBN; no or weak inhibition was observed with cannabinoid metabolites. This inhibition of UGT activity suggests that in addition to playing an important role in drug-drug interactions, cannabinoid exposure may have important implications in patients with impaired hepatic or kidney function. SIGNIFICANCE STATEMENT: Major cannabinoids found in the plasma of cannabis users inhibit several UDP-glucuronosyltransferase (UGT) enzymes, including UGT1A6, UGT1A9, UGT2B4, and UGT2B7. This study is the first to show the potential of cannabinoids and their metabolites to inhibit all the major kidney UGTs as well as the two most abundant UGTs present in liver. This study suggests that as all three major kidney UGTs are inhibited by cannabinoids, greater drug-drug interaction effects might be observed from co-use of cannabinods and therapeutics that are cleared renally.

Cannabinoid Metabolites as Inhibitors of Major Hepatic CYP450 Enzymes, with Implications for Cannabis-Drug Interactions.

The legalization of cannabis in many parts of the United States and other countries has led to a need for a more comprehensive understanding of cannabis constituents and their potential for drug-drug interactions. Although (-)-trans-Δ9-tetrahydrocannabinol (THC), cannabidiol (CBD), and cannabinol (CBN) are the most abundant cannabinoids present in cannabis, THC metabolites are found in plasma at higher concentrations and for a longer duration than that of the parent cannabinoids. To understand the potential for drug-drug interactions, the inhibition potential of major cannabinoids and their metabolites on major hepatic cytochrome P450 (P450) enzymes was examined. In vitro assays with P450-overexpressing cell microsomes demonstrated that the major THC metabolites 11-hydroxy-Δ9-tetra-hydrocannabinol and 11-nor-9-carboxy-Δ9-THC-glucuronide competitively inhibited several major P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6 (apparent Ki,u values = 0.086 ± 0.066 µM and 0.90 ± 0.54 µM, 0.057 ± 0.044 µM and 2.1 ± 0.81 µM, 0.15 ± 0.067 µM and 2.3 ± 0.54 µM, respectively). 11-Nor-9-carboxy-Δ9- tetrahydrocannabinol exhibited no inhibitory activity against any CYP450 tested. THC competitively inhibited CYP1A2, CYP2B6, CYP2C9, and CYP2D6; CBD competitively inhibited CYP3A4, CYP2B6, CYP2C9, CYP2D6, and CYP2E1; and CBN competitively inhibited CYP2B6, CYP2C9, and CYP2E1. THC and CBD showed mixed-type inhibition for CYP2C19 and CYP1A2, respectively. These data suggest that cannabinoids and major THC metabolites are able to inhibit the activities of multiple P450 enzymes, and basic static modeling of these data suggest the possibility of pharmacokinetic interactions between these cannabinoids and xenobiotics extensively metabolized by CYP2B6, CYP2C9, and CYP2D6. SIGNIFICANCE STATEMENT: Major cannabinoids and their metabolites found in the plasma of cannabis users inhibit several P450 enzymes, including CYP2B6, CYP2C9, and CYP2D6. This study is the first to show the inhibition potential of the most abundant plasma cannabinoid metabolite, THC-COO-Gluc, and suggests that circulating metabolites of cannabinoids play an essential role in CYP450 enzyme inhibition as well as drug-drug interactions.

Antioxidants help favorably regulate the kinetics of lipid peroxidation, polyunsaturated fatty acids degradation and acidic cannabinoids decarboxylation in hempseed oil.

The seed of the hemp plant (Cannabis sativa L.) has been revered as a nutritional resource in Old World Cultures. This has been confirmed by contemporary science wherein hempseed oil (HSO) was found to exhibit a desirable ratio of omega-6 and omega-3 polyunsaturated fatty acids (PUFAs) considered optimal for human nutrition. HSO also contains gamma-linoleic acid (GLA) and non-psychoactive cannabinoids, which further contribute to its' potential bioactive properties. Herein, we present the kinetics of the thermal stability of these nutraceutical compounds in HSO, in the presence of various antioxidants (e.g. butylated hydroxytoluene, alpha-tocopherol, and ascorbyl palmitate). We focussed on oxidative changes in fatty acid profile and acidic cannabinoid stability when HSO was heated at different temperatures (25 °C to 85 °C) for upto 24 h. The fatty acid composition was evaluated using both GC/MS and 1H-NMR, and the cannabinoids profile of HSO was obtained using both HPLC-UV and HPLC/MS methods. The predicted half-life (DT50) for omega-6 and omega-3 PUFAs in HSO at 25 °C was about 3 and 5 days, respectively; while that at 85 °C was about 7 and 5 hours respectively, with respective activation energies (Ea) being 54.78 ± 2.36 and 45.02 ± 2.87 kJ/mol. Analysis of the conjugated diene hydroperoxides (CDH) and p-Anisidine value (p-AV) revealed that the addition of antioxidants significantly (p < 0.05) limited lipid peroxidation of HSO in samples incubated at 25-85 °C for 24 h. Antioxidants reduced the degradation constant (k) of PUFAs in HSO by upto 79%. This corresponded to a significant (p < 0.05) increase in color stability and pigment retention (chlorophyll a, chlorophyll b and carotenoids) of heated HSO. Regarding the decarboxylation kinetics of cannabidiolic acid (CBDA) in HSO, at both 70 °C and 85 °C, CBDA decarboxylation led to predominantly cannabidiol (CBD) production. The half-life of CBDA decarboxylation (originally 4 days) could be increased to about 17 days using tocopherol as an antioxidant. We propose that determining acidic cannabinoids decarboxylation kinetics is a useful marker to measure the shelf-life of HSO. The results from the study will be useful for researchers looking into the thermal treatment of hempseed oil as a functional food product, and those interested in the decarboxylation kinetics of the acidic cannabinoids.

In Vitro Inhibition of Carboxylesterase 1 by Major Cannabinoids and Selected Metabolites.

The escalating use of medical cannabis and significant recreational use of cannabis in recent years has led to a higher potential for metabolic interactions between cannabis or one or more of its components and concurrently used medications. Although there have been a significant number of in vitro and in vivo assessments of the effects of cannabis on cytochrome P450 and UDP-glucuronosyltransferase enzyme systems, there is limited information regarding the effects of cannabis on the major hepatic esterase, carboxylesterase 1 (CES1). In this study, we investigated the in vitro inhibitory effects of the individual major cannabinoids and metabolites ∆9-tetrahydrocannabinol (THC), cannabidiol (CBD), cannabinol (CBN), 11-nor-THC-carboxylic acid, and 11-hydroxy-THC on CES1 activity. S9 fractions from human embryonic kidney 293 cells stably expressing CES1 were used in the assessment of cannabinoid inhibitory effects. THC, CBD, and CBN each exhibited substantial inhibitory potency, and were further studied to determine their mechanism of inhibition and kinetic parameters. The inhibition of CES1 by THC, CBD, and CBN was reversible and appears to proceed through a mixed competitive-noncompetitive mechanism. The inhibition constant (K i) values for THC, CBD, and CBN inhibition were 0.541, 0.974, and 0.263 µM (0.170, 0.306, and 0.0817 µg/ml), respectively. Inhibition potency was increased when THC, CBD, and CBN were combined. Compared with the potential unbound plasma concentrations attainable clinically, the K i values suggest a potential for clinically significant inhibition of CES1 by THC and CBD. CBN, however, is expected to have a limited impact on CES1. Carefully designed clinical studies are warranted to establish the clinical significance of these in vitro findings.

Development of a Liquid Chromatography-Tandem Mass Spectrometry Method for the Simultaneous Determination of Four Cannabinoids in Umbilical Cord Tissue.

In utero exposure to marijuana may cause various short- and long-term health problems, such as stillbirth, low birth weight and decreased cognitive function. Detection of in utero marijuana exposure with a relatively new specimen type, umbilical cord tissue, can be used to plan treatment and guide social management. In this study, a liquid chromatography-tandem mass spectrometry (LC-MS-MS) assay was developed for the simultaneous identification of four cannabinoids in umbilical cord tissue, including ∆9-tetrahydrocannabinol (THC), 11-nor-9-carboxy-∆9--THC (THC-COOH), 11-hydroxy-∆9-THC (11-OH-THC) and cannabinol (CBN). Within- and between-run imprecision, accuracy, linearity, sensitivity, carryover, recovery, matrix effects and specificity were evaluated using drug-free umbilical cord tissue spiked with non-deuterated and deuterated standards. Calibration curves were reproducible and linear (r > 0.995) for all four analytes in the range of 0.2 ng/g lower limit of quantitation (LLOQ) and 30 ng/g upper limit of quantitation (ULOQ). Total imprecisions (% coefficient of variation) were 7.8% (THC), 13.3% (THC-COOH), 11.8% (11-OH-THC) and 10.6% (CBN) at low QC (n = 15, 0.25 ng/g), and were 7.2% (THC), 10.0% (THC-COOH), 9.5% (11-OH-THC) and 5.8% (CBN) at high QC (n = 15, 4 ng/g), respectively. No interfering substances were identified, and no carryover was observed. The average accuracies (N = 25) were 94-95%. The average recoveries observed for THC, THC-COOH, 11-OH-THC and CBN were 74, 82, 58 and 86%, respectively. By analyzing authentic clinical specimens that had been previously tested for cannabinoids by enzyme-linked immunoassay, positive and negative result agreements were 100 and 53.8%. In summary, the presented method can be used for the assessment of in utero exposure to four common cannabinoids.

Exogenous cannabinoids as substrates, inhibitors, and inducers of human drug metabolizing enzymes: a systematic review.

Exogenous cannabinoids are structurally and pharmacologically diverse compounds that are widely used. The purpose of this systematic review is to summarize the data characterizing the potential for these compounds to act as substrates, inhibitors, or inducers of human drug metabolizing enzymes, with the aim of clarifying the significance of these properties in clinical care and drug interactions. In vitro data were identified that characterize cytochrome P-450 (CYP-450) enzymes as potential significant contributors to the primary metabolism of several exogenous cannabinoids: tetrahydrocannabinol (THC; CYPs 2C9, 3A4); cannabidiol (CBD; CYPs 2C19, 3A4); cannabinol (CBN; CYPs 2C9, 3A4); JWH-018 (CYPs 1A2, 2C9); and AM2201 (CYPs 1A2, 2C9). CYP-450 enzymes may also contribute to the secondary metabolism of THC, and UDP-glucuronosyltransferases have been identified as capable of catalyzing both primary (CBD, CBN) and secondary (THC, JWH-018, JWH-073) cannabinoid metabolism. Clinical pharmacogenetic data further support CYP2C9 as a significant contributor to THC metabolism, and a pharmacokinetic interaction study using ketoconazole with oromucosal cannabis extract further supports CYP3A4 as a significant metabolic pathway for THC and CBD. However, the absence of interaction between CBD from oromucosal cannabis extract with omeprazole suggests a less significant role of CYP2C19 in CBD metabolism. Studies of THC, CBD, and CBN inhibition and induction of major human CYP-450 isoforms generally reflect a low risk of clinically significant drug interactions with most use, but specific human data are lacking. Smoked cannabis herb (marijuana) likely induces CYP1A2 mediated theophylline metabolism, although the role of cannabinoids specifically in eliciting this effect is questionable.

Cytochrome P450 enzymes involved in the metabolism of tetrahydrocannabinols and cannabinol by human hepatic microsomes.

In this study, tetrahydrocannabinols (THCs) were mainly oxidized at the 11-position and allylic sites at the 7alpha-position for Delta(8)-THC and the 8beta-position for Delta(9)-THC by human hepatic microsomes. Cannabinol (CBN) was also mainly metabolized to 11-hydroxy-CBN and 8-hydroxy-CBN by the microsomes. The 11-hydroxylation of three cannabinoids by the microsomes was markedly inhibited by sulfaphenazole, a selective inhibitor of CYP2C enzymes, while the hydroxylations at the 7alpha-(Delta(8)-THC), 8beta-(Delta(9)-THC) and 8-positions (CBN) of the corresponding cannabinoids were highly inhibited by ketoconazole, a selective inhibitor of CYP3A enzymes. Human CYP2C9-Arg expressed in the microsomes of human B lymphoblastoid cells efficiently catalyzed the 11-hydroxylation of Delta(8)-THC (7.60 nmol/min/nmol CYP), Delta(9)-THC (19.2 nmol/min/nmol CYP) and CBN (6.62 nmol/min/nmol CYP). Human CYP3A4 expressed in the cells catalyzed the 7alpha-(5.34 nmol/min/nmol CYP) and 7beta-hydroxylation (1.39 nmol/min/nmol CYP) of Delta(8)-THC, the 8beta-hydroxylation (6.10 nmol/min/nmol CYP) and 9alpha,10alpha-epoxidation (1.71 nmol/min/nmol CYP) of Delta(9)-THC, and the 8-hydroxylation of CBN (1.45 nmol/min/nmol CYP). These results indicate that CYP2C9 and CYP3A4 are major enzymes involved in the 11-hydroxylation and the 8-(or the 7-) hydroxylation, respectively, of the cannabinoids by human hepatic microsomes. In addition, CYP3A4 is a major enzyme responsible for the 7alpha- and 7beta-hydroxylation of Delta(8)-THC, and the 9alpha,10alpha-epoxidation of Delta(9)-THC.

Novel cannabinol probes for CB1 and CB2 cannabinoid receptors.

The observation that the phenolic hydroxyl of THCs was important for binding to the CB1 receptor but not as critical for binding to the CB2 receptor prompted us to extend this finding to the cannabinol (CBN) series. To study the SAR of CBN analogues, CBN derivatives with substitution at the C-1, C-3, and C-9 positions were chosen since these positions have played a key role in the SAR of THCs. CBN-3-(1',1'-dimethylheptyl) analogues were prepared by sulfur dehydrogenation of Delta(8)-THC-3-(1',1'-dimethylheptyl) analogues. 9-Substituted CBN analogues were prepared by the standard sulfur dehydrogenation of 9-substituted Delta(8)-THC analogues (Scheme 1), which in turn were prepared following our previous procedure using selenium dioxide oxidation of the corresponding Delta(8)-THCs followed by sodium chlorite oxidation to give the 9-carboxy-Delta(8)-THC derivatives. 11-Hydroxy-CBN analogues were prepared from the corresponding 9-carbomethoxy-CBN analogues by reduction with LiAlH(4). Deoxy-CBN analogue 14 was prepared from the corresponding Delta(8)-THC analogue 11 by conversion of the phenolic hydroxyl to the phosphate derivative 12, followed by lithium ammonia reduction to provide the deoxy-Delta(8)-THC analogue 13, which in turn was dehydrogenated with sulfur to provide the deoxy-CBN analogue 14 (Scheme 2). The various analogues were assayed for binding both to the brain and the peripheral cannabinoid receptors (CB1 and CB2). We have found that the binding profile differs widely between the CBN and the THC series. Specifically, in the CBN series the removal of the phenolic hydroxyl decreases binding affinity to both the CB1 and CB2 receptors, whereas in the THC series, CB1 affinity is selectively reduced. Thus, in the CBN series, the selectivity of binding observed with the removal of the hydroxy group is decreased severalfold as compared to what occurs in the THC series. Generally, high affinity for the CB2 receptor was found in analogues when the phenolic hydroxyl was present. The 3-(1', 1'-dimethylheptyl) derivatives were found to have much higher affinities than the CBN analogues, which is in complete agreement with previously reported work by Rhee et al.

Cannabinol derivatives: binding to cannabinoid receptors and inhibition of adenylylcyclase.

Several derivatives of cannabinol and the 1,1-dimethylheptyl homolog (DMH) of cannabinol were prepared and assayed for binding to the brain and the peripheral cannabinoid receptors (CB1 and CB2), as well as for activation of CB1- and CB2-mediated inhibition of adenylylcyclase. The DMH derivatives were much more potent than the pentyl (i.e., cannabinol) derivatives. 11-Hydroxycannabinol (4a) was found to bind potently to both CB1 and CB2 (Ki values of 38.0 +/- 7.2 and 26.6 +/- 5.5 nM, respectively) and to inhibit CB1-mediated adenylylcyclase with an EC50 of 58.1 +/- 6.2 nM but to cause only 20% inhibition of CB2-mediated adenylylcyclase at 10 microM. It behaves as a specific, though not potent, CB2 antagonist. 11-Hydroxycannabinol-DMH (4b) is a very potent agonist for both CB1 and CB2 (Ki values of 100 +/- 50 and 200 +/- 40 pM; EC50 of adenylylcyclase inhibition 56.2 +/- 4.2 and 207.5 +/- 27.8 pM, respectively).

Construction of a steric map of the binding pocket for cannabinoids at the cannabinoid receptor.

In order to gain information about the topology of the brain cannabinoid receptor (CB1), a Receptor Steric (RS) Map for cannabinoids at this receptor was calculated. The classical cannabinoids (-)-11-hydroxy-delta-9-tetrahydrocannabinol (K1 = 210 +/- 56 nM), (-)-9-nor-9-beta-hydroxy-hexahydrocannabinol (K1 = 124 +/- 17 nM), nabilone (K1 = 120 +/- 13 nM), and the non-classical cannabinoid, CP-55,244 (K1 = 1.4 +/- .3 nM) were used as template molecules. The RS map was obtained as the union of the van der Waals' volumes of only those accessible conformers identified by MMP2 calculations that were able to clear a region of steric interference at the CB1 receptor previously characterized by us [Reggio, P.H., Panu, A.M. and Miles, S. (1993), J. Med. Chem., 36, 1761-1771]. The utility of the RS Map was explored by screening the accessible conformers of the classical cannabinoid, cannabinol (CBN), (K1 = 3200 +/- 450 nM), for its ability to fit within the RS map. Only the global minimum energy conformer of CBN (53.2% abundance at 298K) was able to fit within the RS map. These results imply that one reason for the reduced affinity of CBN may be that only 53.2% of CBN molecules are shaped properly to fit in the binding pocket for cannabinoids at the CB1 receptor.

In vitro metabolism of cannabinol in rat, mouse, rabbit, guinea pig, hamster, gerbil and cat.

Metabolism of cannabinol (CBN) was studied in hepatic microsomal incubates from mouse, rat, rabbit, guinea pig, cat, hamster and gerbil. Metabolites were extracted with ethyl acetate, concentrated by chromatography on Sephadex LH-20 and identified by GC/MS as TMS derivatives. Six monohydroxy metabolites were identified. These had hydroxy groups at C-11 and at all positions of the pentyl side-chain. Metabolism varied considerably between the species. 11-Hydroxylation was the most prominent route in the majority of species, but in the hamster and cat the major metabolic pathway was 4'-hydroxylation. Metabolites hydroxylated in the pentyl chain were generally more abundant in guinea pig, hamster and cat.

Psychopharmacological effects of cannabis.

The use of cannabis can lead to an acute toxic psychosis, 'flashbacks', depersonalization, derealization and marked cognitive and psychomotor impairment. Further research is needed to establish whether a functional psychosis can be provoked, aggravated or prolonged by cannabis intake. Perhaps of greatest significance among the physical sequelae is the potential to suppress the immune system, impair reproduction, produce respiratory disease and increase the risk of lung cancer.

The effects of pH and temperature on the in vitro bindings of delta-9-tetrahydrocannabinol and other cannabinoids to bovine serum albumin.

Albumin is a major carrier of drugs and fatty acids in biological fluids. These protein-drug complexes serve to solubilize, transport these compounds to sites of action, and have been associated with increased half-life for these compounds. The authors are interested in the pH and temperature effects of the binding of delta-9-tetrahydrocannabinol to albumin. Ultrafiltration techniques were used in the separation of free to bound compounds. Cannabinoids bind to bovine serum albumin rapidly. The cannabinoid binding sites are more sensitive to temperature changes (37-47 degrees C) than changes in pH with 37 degrees C and pH 7.4 resulting in optimal binding. These conditions would result in the greatest viability in the cells, while allowing for the use of a variety of compounds in in vitro studies for the administration of compounds to isolated cells and cell lines.

The pharmacological activity of the fatty acid conjugate 11-palmitoyloxy-delta 9-tetrahydrocannabinol.

A long-retained cannabinoid metabolite has been detected in rat tissue after intravenous administration of delta 9-tetrahydrocannabinol (THC) and has been identified as a fatty acid conjugate of psychoactive 11-hydroxy-delta 9-tetrahydrocannabinol (11-OH-delta 9-THC) and palmitic acid. The objective of these studies was to determine if this compound, 11-palmitoyloxy-delta 9-tetrahydrocannabinol (11-palm-delta 9-THC) is pharmacologically active. Intravenously injected 11-palm-delta 9-THC decreased thermal sensitivity and induced catalepsy in rats, responses similar to those produced by 11-OH-delta 9-THC, but less pronounced and more delayed. To further characterize the response, animals were intracisternally injected with 11-OH-delta 9-THC or 11-palm-delta 9-THC. Catalepsy and decreased thermal sensitivity were seen in the 11-OH-delta 9-THC and 11-palm-delta 9-THC groups, and again, 11-OH-delta 9-THC appeared to be the more potent of the two cannabinoids. In contrast to the intravenous study, 11-palm-delta 9-THC-induced effects were seen soon after treatment and appeared to be fully developed by the first test time (15 min). The intracisternal results suggest that 11-palm-delta 9-THC itself is active; however, since it is known that the fatty acid conjugate is hydrolyzed in vivo to 11-OH-delta 9-THC in the rat, the possibility remains that the effects of 11-palm-delta 9-THC are due to metabolic conversion to 11-OH-delta 9-THC.

Interactions of cannabinoids with bovine serum albumin.

There is no shift of emission maximum (F470nm) of bovine serum albumin (BSA)-1-anilino-8-naphthalene sulphonic acid (ANS) complex in the presence of delta-9-tetrahydrocannabinol (delta-9-THC) alone and cannabidiol (CBD) or cannabinol (CBN) in the presence and absence of delta-9-THC. Delta-9-THC (1.66-13.33 microM) and CBD at higher concentrations (13.33-20.0 microM) produce a concentration-dependent significant quenching of fluorescence of BSA-ANS complex, but CBN (1.66-20.0 microM) as well as CBD at lower concentrations (1.66-6.66 microM) fails to produce any significant change in fluorescence intensity under similar conditions. Furthermore, neither CBD nor CBN is able to affect the delta-9-THC-induced quenching of fluorescence intensity of BSA-ANS complex. These results indicate that the binding of cannabinoids to the ANS binding sites of BSA molecule are in the order delta-9-THC greater than CBD greater than CBN, and CBD or CBN does not have any influence on the binding of delta-9-THC to BSA molecules under these conditions.

The properties of UDP-glucuronyltransferase for cannabinoids in rat liver microsomes.

The glucuronidation of delta 9-tetrahydrocannabinol (delta 9-THC), cannabidiol (CBD) and cannabinol (CBN) in rat liver microsomes was studied. The enzyme activities for the cannabinoids were 26.3 (delta 9-THC), 52.9 (CBD) and 104.8 (CBN) pmol/min/mg protein. The apparent Km values of UDP-glucuronyltransferase for the cannabinoids were 0.29 (delta 9-THC), 0.18 (CBD) and 2.78 (CBN) mM, while Vmax were 40.3 (delta 9-THC), 104.9 (CBD) and 593.3 (CBN) pmol/min/mg protein. Following treatment of rats with 3-methylcholanthrene, the enzyme activities for delta 9-THC, CBD and CBN were increased 132, 43 and 1198%, respectively, whereas the corresponding increases in microsomes from phenobarbital-treated rats were 127, 13 and 97%, respectively. The cannabinoid glucuronidation was activated 2 to 3 folds by the addition of UDP-N-acetylglucosamine, but not activated by the addition of Triton X-100 in vitro. The properties of cannabinoid UDP-glucuronyltransferase were discussed from the above results.