Hypothermic action of delta 9-tetrahydrocannabinol, 11-hydroxy-delta 9-tetrahydrocannabinol and 11-hydroxy-delta 8-tetrahydrocannabinol in mice

The hypothermic activity of Δ⁹-tetrahydrocannabinol (Δ⁹-THC), a metabolite, 11-hydroxy-Δ⁹-tetrahydrocannabinol (11-OH-Δ⁹-THC) and 11-hydroxy-Δ⁸-tetrahydrocannabinol (11-OH-Δ⁸-THC) has been determined in male mice maintained at an ambient temperature of 20 ± 1°C. The mean body temperature of mice that received 2, 4, 16 or 32 mg/kg, i. v., of a tetrahydrocannabinol was significantly lower than that of vehicle treated mice (p <0.05) within 2 minutes after drug administration. Dose-response relationships show the intrinsic activity of Δ⁹-THC to be significantly greater than that of 11-OH-Δ⁹-THC or 11-OH-Δ⁸-THC in this system (p <0.05). The data indicate that the hypothermic activity of Δ⁹-THC cannot be explained entirely by metabolism to 11-OH-Δ⁹-THC.     

8 alpha, 9 alpha-Epoxyhexahydrocannabinol formation from delta 8-tetrahydrocannabinol by mouse liver microsomes

8α,9α-Epoxyhexahydrocannabinol ( EHHC ) was formed from Δ⁸-tetrahydrocannabinol ( THC ) by mouse liver microsomes. The reaction required O₂, and partially inhibited by CO. The optimal pH for the epoxide formation was from 7.4 to 8.0. EDTA did not affect the epoxide formation, but SKF 525-A, α-naphthoflavone and CCl₄ caused a significant inhibition. In addition, the rate of epoxidation increased significantly after treatment. with phenobarbital and 3-methylcholanthrene, but decreased after CoCl₂ treatment. 8β,9β-EHHC, a stereoisomer of 8α,9α-EHHC, was not found under all the conditions used in this study. These results indicate that 8α,9α-EHHC formation is mediated by monooxygenase system involving cytochrome P-450.     

Influence of anticonvulsant cannabinoids on posttetanic potentiation at isolated bullfrog ganglia

The effects of anticonvulsant cannabinoids on posttetanic potentiation (PTP) at bullfrog paravertebral ganglia $\text{in vitro}$ were investigated electrophysiologically. Two Δ⁹-tetrahydrocannabinol metabolites, 11-hydroxy-Δ⁹-tetrahydrocannabinol and 8α, 11-dihydroxy-Δ⁹-tetrahydrocannabinol, as well as cannabidiol, markedly depressed PTP. In contrast, Δ⁹-tetrahydrocannabinol had no such effect. Thus, the findings of the present study clearly demonstrate that pharmacological properties of these hydroxylated metabolites of Δ⁹-tetrahydrocannabinol are not identical to those of their parent compound.     

Anticonvulsant properties of delta 9-tetrahydrocannabinol and other cannabinoids

Anticonvulsant doses of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) markedly lower body temperature in mice at an ambient temperature of 22°C, but there is little such effect at 30°C. The anticonvulsant properties of Δ⁹-THC are as follows: The drug abolishes hind-limb extension in a maximal electroshock (MES) test, elevates both the MES (extensor) and 6-Hz-electroshock thresholds, exerts no effect on the 60-Hz-electroshock threshold, and enhances minimal seizures caused by pentylenetetrazol. All anticonvulsant properties studied, with the exception of the 60-Hz-electroshock threshold, were unaffected by the hypothermia resulting at 22°C. Additional experiments with Δ⁹-THC indicated that chronic treatment results in the development of tolerance, as determined by the MES test with rats. The four principal naturally occurring cannabinoids, Δ⁹-THC, Δ⁸-THC, cannabinol and cannabidiol, display anticonvulsant activity, as does the major, primary metabolite of Δ⁹-THC, 11-hydroxy-Δ⁹-THC. Of all agents investigated in mice, the synthetic cannabinoids, dimethylheptylpyran and its isomers, are the most potent anticonvulsants. The results of a study of the relative motor toxicity and anticonvulsant activity of the cannabinoids demonstrate that these properties are at least partially separable among the various agents.     

Further evidence for a role of 2-phenylethylamine in the mode of action of delta 9-tetrahydrocannabinol

In rabbits, Δ⁹-tetrahydrocannabinol (Δ⁹-THC) increased the recovery of labeled 2-phenylethylamine (PEA) from brain following its intraventricular administration. Δ⁹-THC also enhanced the excitatory effect of iontophoretic PEA on cortical unit potentials. Although Δ⁹-THC induced sedation in mice, the subsequent injection of reserpine induced transient excitement. Low doses of PEA, which do not significantly alter the behavior of mice, induced marked excitement in mice pretreated with Δ⁹-THC. In mice treated with pargyline, Δ⁹-THC induced excitement (instead of sedation); this excitement was increased by PEA and reduced by phenylethanolamine. These results suggest that Δ⁹-THC inhibits the disposition of PEA. Since endogenous PEA may be one of the adrenergic ergotropic modulators, it may play a role in the euphoriant effect of marihuana.     

Effects of Δ8- and Δ9-Tetrahydrocannabinol on experimentally induced seizures

Effects of Δ⁸- and Δ⁹-tetrahydrocannabinol (Δ⁸- and Δ⁹-THC) on three experimentally induced seizure models, i.e., audiogenic seizure (AS) test, maximal electroshock seizure (MES) test and pentylenetetrazol (PTZ)-induced seizure test were determined in the audiogenic rat. Both tetrahydrocannabinols possess a dose-related anticonvulsant effect against AS, MES and PTZ-induced maximal seizure. Although anticonvulsant potencies do not significantly differ, Δ⁸THC is three times more neurotoxic than Δ⁹THC. In addition, both THC's are without effect on minimal seizure and lethality induced by PTZ. Furthermore, the low protective indexes (TD50/ED50) determined in this study suggest that Δ⁸ and Δ⁹ THC may have poor therapeutic potentials as antiepileptic drugs.     

Brain microsomal oxidation of delta 8- and delta 9-tetrahydrocannabinol

Brain microsomes of mice, rats, guinea pigs and rabbits catalyzed the oxidation of delta 8- and delta 9-tetrahydrocannabinol to their monohydroxylated metabolites. The most prominent metabolite was the 4'-hydroxylated metabolite on the pentyl side chain of the cannabinoids in all species tested, except that the 5'-hydroxylation of delta 9-tetrahydrocannabinol was most abundant in the guinea pig. These results are quite different from the metabolic profile of the cannabinoids with hepatic microsomes.     

Δ1-Tetrahydrocannabinol and 1α,2α-epoxyhexahydrocannabinol: Mutagenicity investigation in the ames test

1,2-Epoxyhexahydrocannabinol is a metabolite of Δ¹-tetrahydrocannabinol. Because many epoxides are mutagens, we investigated 1,2-epoxyhexahydrocannabinol as well as Δ¹-tetrahydrocannabinol for mutagenicity with Salmonella typhimurium TA1535, TA1537, TA98 and TA100 in the presence and in the absence of S9 mix from liver homogenate of rats treated with Aroclor 1254. Additionally, an epoxide hydratase inhibitor was used in some experiments. Whereas several other epoxides and further positive controls, not requiring activation or activated under the same conditions, respectively, showed strong mutagenicity, no indications of a mutagenic hazard by 1,2-epoxyhexahydrocannabinol or by Δ¹-tetrahydrocannabinol were found.     

A Structural Insight into the Molecular Recognition of a (−)-Δ-Tetrahydrocannabinol and the Development of a Sensitive, One-Step, Homogeneous Immunocomplex-Based Assay for Its Detection

(−)-Δ⁹-Tetrahydrocannabinol (THC) is the main psychoactive compound found in cannabis. In this study, an anti-THC Fab fragment, designed T3, was isolated from a display library cloned from the spleen cells of a mouse immunized with a THC-bovine serum albumin conjugate, and the crystal structures of the T3 Fab in its free form and in complex with THC were determined at 1.9 Å and 2.0 Å resolution, respectively. The THC binding site of the T3 Fab is a narrow cavity: the n-pentyl group of THC protrudes deep into the interface area between the variable domains and the C₁₀ monoterpene moiety of the hapten is partially exposed to solvent. The metabolites of THC, with modifications in the C₁₀ monoterpene moiety, 11-nor-9-carboxy-Δ⁹-tetrahydrocannabinol and 11-hydroxy-Δ⁹-tetrahydrocannabinol, are bound by the T3 Fab with a higher affinity than THC. The crystal structures suggest that Ser52H and Arg53H of the T3 Fab are able to make hydrogen bonds with the metabolites, which leads to an increased binding against these metabolites. By developing a T3 Fab-Δ⁹-THC immunocomplex binding antibody from a naïve antibody phage display library, the specificity of the Δ⁹-THC binding is highly increased, which allows a one-step, homogeneous, fluorescence resonance energy transfer-based sensitive immunoassay, with a detection limit of 20 ng/ml from saliva samples.     

Delta-9-tetrahydrocannabinol and human spermatozoa

Thein vitro effect of δ⁹-tetrahydrocannabinol on adenosine triphosphatase and phosphodiesterase activities as well as on the cyclic-AMP content of human spermatozoa has been studied. At a concentration of 1.0 ▪g, sperm metabolism may be increased as shown by increased cyclic AMP and adenosine-triphosphatase activity while at a higher concentration (10 ▪g tetrahydrocannabinol), it may be reversed.     

CD11cCD8 T cells: Two-faced adaptive immune regulators

Regulatory cells, important controllers of immune homeostasis, carry out a multi-pronged attack by deleting overactive pathogenic immune cells, by supporting anergy, and by blocking effector functions, thereby contributing to the amelioration of disease. CD8⁺ T cells co-expressing CD11c are a new addition to the growing list of regulatory cells. Naïve mice harbor CD11c-expressing CD8⁺ T cells (<3%) that expand further in an antigen-dependent manner. Although activated CD11c⁺CD8⁺ T cells express suppressive cytokines such as IL-10 and TGF-β, their production of IFN-γ is central to their immune suppressive potential. The CD11c⁺CD8⁺ T cells target pathogenic CD4⁺ T cells in a cell-cell contact dependent manner via IDO- and GCN2-dependent mechanisms. Adoptive transfer of activated CD11c⁺CD8⁺ T cells halts the progression of autoimmune rheumatoid arthritis and colitis. However, in certain virus and cancer models the CD11c⁺CD8⁺ T cells assume the role of immune effectors, boosting immune potential. This seemingly dual nature of these cells - exerting regulatory vs. effector activities - makes them an attractive therapeutic target. In this review, we discuss the discovery, origins and developmental requirements of CD11c⁺CD8⁺cells, and the basis of their immuno-suppressive and effector potentials.     

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 chronic administration of delta-9-tetrahydrocannabinol and cannabidiol on rat testicular esterase isozymes

The effects of Δ⁹-tetrahydrocannabinol and cannabidiol on the esterase isozymes of rat testicular tissue were examined using acrylamide gel electrophoretic techniques followed by staining for non-specific esterase activity. Both Δ⁹-tetrahydrocannabinol and cannabidiol caused a specific depression of an esterase isozyme located in the interstitial tissue of the testis.     

Crystal structure of phenylacetic acid degradation protein PaaG from Thermus thermophilus HB8

Microbial degradation of phenylacetic acid proceeds via the hybrid pathway that includes formation of a coenzyme A thioester, ring hydroxylation, non‐oxygenolytic ring opening, and β‐oxidation‐like reactions. A phenylacetic acid degradation protein PaaG is a member of the crotonase superfamily, and is a candidate non‐oxygenolytic ring‐opening enzyme. The crystal structure of PaaG from Thermus thermophilus HB8 was determined at a resolution of 1.85 Å. PaaG consists of three identical subunits related by local three‐fold symmetry. The monomer is comprised of a spiral and a helical domain with a fold characteristic of the crotonase superfamily. A putative active site residue, Asp136, is situated in an active site cavity and surrounded by several hydrophobic and hydrophilic residues. The active site cavity is sufficiently large to accommodate a ring substrate. Two conformations are observed for helix H2 located adjacent to the active site. Helix H2 is kinked at Asn81 in two subunits, whereas it is kinked at Leu77 in the other subunit, and the side chain of Tyr80 is closer to Asp136. This indicates that catalytic reaction of PaaG may proceed with large conformational changes at the active site. Asp136 is the only conserved polar residue in the active site. It is located at the same position as those of 4‐chlorobenzoyl‐CoA dehalogenase and peroxisomal Δ³,Δ²‐enoyl‐CoA isomerase, indicating that PaaG may undergo isomerization or a ring‐opening reaction via a Δ³,Δ²‐enoyl‐CoA isomerase‐like mechanism. Proteins 2009. © 2009 Wiley‐Liss, Inc.     

Endocrine effects of chronic intraventricular administration of delta9-tetrahydrocannabinol to prepuberal and adult male rats.

The daily intraventricular administration of Δ⁹-tetrahydrocannabinol (Δ⁹-THC) in microgram amounts for a week to prepuberal and adult rats had definite endocrine effects. Prostate weights were reduced and plasma and pituitary levels of growth hormone (GH) were increased in prepuberal rats. Pituitary levels of prolactin (PRL) were increased both in prepuberal and in adult animals while pituitary and adrenal weights and plasma corticosterone (B) levels were increased in adult rats. On the other hand, brain weights were significantly reduced by Δ⁹-THC in prepuberal and significantly increased in adult animals. No changes in brain levels of noradrenaline (NA), dopamine (DA) or serotonin (5-HT) were found in treated animals. These results indicate that Δ⁹-THC may modify some endocrine functions when injected directly into the brain in microgram amounts. They show on the other hand that young and adult animals may respond differently to the chronic administration of the psychoactive drug, although the difference may be due to a biphasic effect of different doses.     

Blocking of the effect of delta1 tetrahydrocannabinol (delta1THC) on spontaneous motor activity in the rat by immunization with a protein conjugate of delta1THC

Rats actively immunized with porcine gamma globulin- hemisuccinate-Δ¹-tetrahydrocannabinol (PγG-HS-Δ¹THC) showed higher spontaneous motor activity after intraperitoneal administration of Δ¹THC at a dose of 10 mg. per kg. than did rats immunized with a control antigen, porcine gamma globulin-hemisuccinate-phenol (PγG-HS-Phenol). The capacity to neutralize the effect of Δ¹THC was found to depend on the degree of immunization; thus, the difference in mean spontaneous motor activity after injection of Δ¹THC was significant in rats which had received five injections of the immunogen over a period of 86 days, and not in those which had received only two injections over a period of 34 days.In view of the observations that Δ¹-tetrahydrocannabinol induces a decrease in spontaneous motor activity in rats, the observed neutralization of the effect of δ¹THC in animals receiving multiple injections of protein conjugates of Δ¹THC may be due to the binding of the drug by anti-THC antibodies (which are expected to be produced on active immunization with these conjugates), thus preventing Δ¹THC from reaching drug-receptor sites.     

Delta9-tetrahydrocannabinol: subcortical spike bursts and motor manifestations in a Fischer rat treated orally for 109 days

A total of 12 Fischer rats was prepared surgically for chronic EEG recording from cortical and subcortical sites. Most rats, within 2 to 9 weeks after electrode implantation, developed polyspike activity in cortical and subcortical recordings that were without motor manifestations. Six of these rats, chronically treated po with Δ⁹-tetrahydrocannabinol (Δ⁹-THC) 10 mg/kg exhibited acute EEG changes with more frequent occurrence of EEG desynchronization and polyspike activity. On day 109 one of 6 rats displayed consulsive activity, with jerky movements of the head and paws, characteristics of Δ⁹-THC neurotoxicity. EEG alterations concomitant with motor signs included bursts of spikes of approximately 0.2 sec that occurred in subcortical, but not in cortical, recordings. It is concluded that in the Fischer rat acute and chronic treatment with Δ⁹-THC facilitated the occurrence of surgically-induced “polyspike” activity while chronic treatment caused occasional transient subcortical spike bursts with concomitant motor manifestations.     

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.     

Quantitative measurement of delta 9-tetrahydrocannabinol and two major metabolites in physiological specimens using capillary column gas chromatography negative ion chemical ionization mass spectrometry

delta 9-Tetrahydrocannabinol and two of its metabolites, 11-hydroxy-delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, can be measured in a single 1-ml sample of blood, plasma, or urine by a new assay which combines a relatively rapid extraction procedure with capillary column gas chromatography and negative ion chemical ionization mass spectrometry. Deuterium-labeled analogs of each cannabinoid are added to the physiological specimen as internal standards. Two extracts are obtained from each sample: a neutral fraction containing delta 9-tetrahydrocannabinol and 11-hydroxy-delta 9-tetrahydrocannabinol, and an acid fraction containing 11-nor-9-carboxy-delta 9-tetrahydrocannabinol. The neutral fraction is derivatized by treatment with trifluoroacetic anhydride; the acid fraction is first treated with BF3-methanol followed by reaction with trifluoroacetic anhydride. Under electron-capture chemical ionization conditions the derivatized delta 9-tetrahydrocannabinol and 11-nor-9-carboxy-delta 9-tetrahydrocannabinol give abundant molecular anions ideally suited for selected ion monitoring. The negative ion chemical ionization spectrum of the HO-THC-trifluoroacetate shows no molecular anion. Consequently, quantitation of the hydroxy metabolite is achieved by monitoring a fragment ion formed by loss of CF3CO2 from its molecular anion. The limits of reliable measurement are judged to be 0.1 ng ml-1 for 11-nor-9-carboxy-delta 9-tetrahydrocannabinol, 0.2 ng ml-1 for delta 9-tetrahydrocannabinol and 0.5 ng ml-1 for 11-hydroxy-delta 9-tetrahydrocannabinol. Four examples are given of the application of the assay to the analysis of specimens of medico-legal importance.     

Rapid reversed-phase high-performance liquid chromatographic method for the assay of urinary 11-nor-Δ9-tetrahydrocannabinol-9-carboxylic acid and confirmation of use of cannabis derivatives

The main active cannabis (Marijuana and hashish) derivative Δ⁹-tetrahydrocannabinol is, in vivo, transformed and excreted mainly as 11-nor-Δ⁹-tetrahydrocannabinol-9-carboxylic acid (THC-COOH) and its glucuronide. The method presented here allows the confirmation of the presence of THC-COOH by means of a basic hydrolysis, solid-phase extraction clean-up on reversed-phase (RP) disposable cartridges followed by analysis on a C₈ RP column and UV detection; the mobile phase used was a 55% acetonitrile solution in acid phosphate buffer. Over 600 samples both from drug addicts in therapeutic communities and subjects who were not on any drugs therapy were analysed. This method was precise with a linearity range from 10 to more than 500 ng/ml [the lower limit proposed by the National Institute on Drug Abuse (NIDA) for cannabinoid confirmation method is 15 ng/ml]. The sample preparation is simple and fast, allowing the analysis of large numbers of samples. Perfect correlation was observed between data from the HPLC method and a fluorescence polarization immunoassay screening method. The THC-COOH metabolite was found to constitute 30% of all the cannabinoids excreted in urine of abusers.