Potent cyano and carboxamido side-chain analogues of 1', 1'-dimethyl-delta8-tetrahydrocannabinol

The synthesis and pharmacological profile of several cyano (1a-e) and carboxamido (2a-h) side-chain-substituted analogues of 1', 1'-dimethyl-Delta8-THC are described. Commercially available cyano compound 3 was transformed to the resorcinol 6 in a three-step sequence. Condensation of 6 with p-menth-2-ene-1,8-diol formed the THC 7a which, with sodium cyanide/DMSO, gave 1b. Protection of the phenol in 7a as the MOM derivative provided the common intermediate 8 for the synthesis of 1a,c,e. Compound 1d was also synthesized from 7a via the aldehyde 9a. Base hydrolysis of 1b gave the acid 10 which, via its acid chloride and subsequent treatment with the appropriate amine, formed the target compounds 2a-h. The pharmacological profile indicated that the cyano analogues 1a-e had very high CB1 binding affinity (0.36-13 nM) and high in vivo potency as agonists. Two analogues (1a,b) had extremely high potency in the mouse tetrad tests. The dimethylcarboxamido analogue 2a showed a similar profile to 1a,b. The high potency was also retained in analogue 2c. In contrast the sulfonamide analogue 2d was unique as it had greater affinity than Delta9-THC, yet it was practically devoid of agonist effects. This study suggests that the incorporation of a cyano or an amide substituent in the side chain of Delta8-THC-DMH can enhance potency and can also lead to compounds with a unique profile which have high binding affinity and are practically devoid of agonist effects.     

Competitive inhibition of delta8-tetrahydrocannabinol and its active metabolites for cannabinoid receptor binding

In vitro binding characteristics of delta8-tetrahydrocannabinol (delta8-THC) and its metabolites, 11-hydroxy-delta8-THC (11-OH-delta8-THC) and 11-oxo-delta8-THC, as well as an inactive metabolite, delta8-THC-11-oic acid, as a cannabinoid receptor site from bovine cortex were examined using the specific agonist [3H]CP-55940. 11-OH-delta8-THC and 11-oxo-delta8-THC strongly inhibited the specific binding of [3H]CP-55940. The Ki values of 11-OH-delta8-THC and 11-oxo-delta8-THC for the specific binding of [3H]CP-55940 were 52 and 143 nM, respectively, whereas that of delta8-THC-11-oic acid was 917 nM. Scatchard plot analyses indicated that 11-OH-delta8-THC and 11-oxo-delta8-THC caused a significant increase in the apparent KD value without changing the apparent Bmax. These results reveal that active metabolites of delta8-THC also competitively bind to the cannabinoid receptor as agonists.     

Discriminative stimulus effects of delta 9-tetrahydrocannabinol and delta 9-11-tetrahydrocannabinol in rats and rhesus monkeys

Previous reports have suggested that delta 9-11-tetrahydrocannabinol (delta 9-11-THC), an exocyclic analog of delta 9-tetrahydrocannabinol (delta 9-THC), may have weak agonist effects as well as antagonistic properties. The purpose of the present study was to examine the effects of delta 9-11-THC in substitution and antagonism tests in rats and in rhesus monkeys trained to discriminate delta 9-THC from vehicle in two-lever drug-discrimination procedures. The substitution studies showed that delta 9-11-THC generalizes from the training dose of delta 9-THC in rats and in monkeys, although it was less potent in both species. The magnitude of the potency difference was greater in monkeys than in rats. When administered immediately following injection with the training dose of delta 9-THC, delta 9-11-THC failed to block the delta 9-THC cue in rats and showed a lack of dose-responsive inhibition in monkeys. These results suggest that delta 9-11-THC is devoid of antagonistic properties in the drug discrimination paradigm.     

Pharmacokinetics of delta9-tetrahydrocannabinol in dogs

The pharmacokinetics of intravenously administered 14C-delta9-tetrahydrocannabinol and derived radiolabeled metabolites were studied in three dogs at two doses each at 0.1 or 0.5 and 2.0 mg/kg. Two dogs were biliary cannulated; total bile was collected in one and sampled in the other. The time course for the fraction of the dose per milliliter of plasma was best fit by a sum of five exponentials, and there was no dose dependency. No drug was excreted unchanged. The mean apparent volume of distribution of the central compartment referenced to total drug concentration in the plasma was 1.31 +/- 0.07 liters, approximately the plasma volume, due to the high protein binding of 97%. The mean metabolic clearance of drug in the plasma was 124 +/- 3.8 ml/min, half of the hepatic plasma flow, but was 4131 +/- 690 ml/min referenced to unbound drug concentration in the plasma, 16.5 times the hepatic plasma flow, indicating that net metabolism of both bound and unbound drug occurs. Apparent parallel production of several metabolites occurred, but the pharmacokinetics of their appearance were undoubtedly due to their sequential production during liver passage. The apparent half-life of the metabolic process was 6.9 +/- 0.3 min. The terminal half-life of delta9-tetrahydrocannabinol in the pseudo-steady state after equilibration in an apparent overall volume of distribtuion of 2170 +/- 555 liters referenced to total plasma concentration was 8.2 +/- 0.23 days, based on the consistency of all pharmacokinetic data. The best estimate of the terminal half-life, based only on the 7000 min that plasma levels could be monitored with the existing analytical sensitivity, was 1.24 days. However, this value was inconsistent with the metabolite production and excretion of 40-45% of dose in feces, 14-16.5% in urine, and 55% in bile within 5 days when 24% of the dose was unmetabolized and in the tissue at that time. These data were consistent with an enterohepatic recirculation of 10-15% of the metabolites. Intravenously administered radiolabeled metabolites were totally and rapidly eliminated in both bile and urine; 88% of the dose in 300 min with an apparent overall volume of distribution of 6 liters. These facts supported the proposition that the return of delta9-tetrahydrocannabinol from tissue was the rate-determining process of drug elimination after initial fast distribution and metabolism and was inconsistent with the capability of enzyme induction to change the terminal half-life.     

Tolerance to delta9-tetrahydrocannabinol in adapted and nonadapted rabbits

Two groups of New Zealand white rabbits, one which had been adapted to the testing chamber and one which had not been adapted to the testing chamber, were given delta9-tetrahydrocannabinol (delta9-THC; 0.5 mg/kg, IV) daily for 12 days. During vehicle control and on the first and last day of delta9-THC administration, electroencephalograms (EEG's) were recorded from the motor cortex and hippocampus, while standing, sprawling and behavioral activity were recorded concurrently. The results showed that tolerance to the behavioral and EEG effects of delta9-THC occurs in rabbits and that acute and chronic effects produced by delta9-THC are influenced by environmental factors.     

Regulation of delta opioid receptors by delta9-tetrahydrocannabinol in NG108-15 hybrid cells

In this study we employed the neuroblastoma x glioma NG 108-15 cell line as a model for investigating the effects of long-term activation of cannabinoid receptors on delta opioid receptor desensitization, down-regulation and gene expression. Exposure of NG 108-15 cells to (-)-delta9-tetrahydrocannabinol (delta9-THC) reduced opioid receptor binding, evaluated in intact cells, by approximately 40-45% in cells exposed for 24 h to 50 and 100 nM delta9-THC and by approximately 25% in cells exposed to 10 nM delta9-THC. Lower doses of delta9-THC (0.1 and 1 nM) or a shorter exposure time to the cannabinoid (6 h) were not effective. Down-regulation of 6 opioid receptors was not observed in cells exposed for 24 h to pertussis toxin (PTX) and then treated for 24 h with 100 nM delta9-THC. In cells that were exposed for 24 h to the cannabinoid, the ability of delta9-THC and of the delta opioid receptor agonist [D-Ser2, Leu5, Thr6]enkephalin to inhibit forskolin-stimulated cAMP accumulation was significantly attenuated. Prolonged exposure of NG 108-15 cells to 100 nM delta9-THC produced a significant elevation of steady-state levels of delta opioid receptor mRNA. This effect was not observed in cells pretreated with PTX. The selective cannabinoid receptor antagonist SR 141716A blocked the effects elicited by delta9-THC on delta opioid receptor desensitization, down-regulation and gene expression; thus indicating that these are mediated via activation of cannabinoid receptors. These data demonstrate the existence, in NG 108-15 cells, of a complex cross-talk between the cannabinoid and opioid receptors on prolonged exposure to delta9-THC triggered by changes in signaling through Gi and/or G0-coupled receptors.     

Reactive derivatives of phosphorothioate oligonucleotide analogues. II. Properties of phosphorothioate oligodeoxyribonucleotide derivatives containing an aromatic residue of nitrogen mustard gas

STUDY OBJECTIVE: To evaluate current practice in preoperative testing of healthy children undergoing elective surgery that is not expected to result in significant blood loss. DESIGN: Survey of members of the Society for Pediatric Anesthesia. SETTING: Anesthesiologists practicing in North America. POPULATION: A total of 1,200 questionnaires were mailed. INTERVENTIONS: Questionnaires were mailed to all members of the Society for Pediatric Anesthesia. All members were asked to specify which tests were routinely performed and to state why. Specific questions were asked about performing complete blood count (CBC), hemoglobin (Hb), hematocrit (Hct), and urine analysis (UA) in all patients, pregnancy test in adolescents, prothrombin time (PT) and activated partial thrombin time (PTT) prior to tonsillectomy, and sickle cell testing in black and/or Mediterranean children. MEASUREMENTS AND MAIN RESULTS: 685 of 1,200 (57%) questionnaires were returned. No attempt was made to identify and follow-up with nonresponders. Hb testing is routinely performed in 27% to 48% of the children depending on the age of the patient. UA is ordered preoperatively in less than 15% of the children. Pregnancy test was ordered by 43% of the respondents. Hemostatic tests prior to tonsillectomy were conducted by 45% of the anesthesiologists. CONCLUSION: The results indicate the present practice of routine preoperative laboratory testing for children undergoing elective outpatient surgery. In spite of the many studies that indicate no specific benefits of performing routine preoperative testing in healthy children undergoing scheduled surgery, many physicians continue to order these tests in all such children.     

Hydroxypropyl-beta-cyclodextrin and its combination with hydroxypropyl-methylcellulose increases aqueous solubility of delta9-tetrahydrocannabinol

Delta9-tetrahydrocannabinol (THC) is the main psychoactive constituent of Cannabis sativa L. and its therapeutic effects are currently under intensive study. However, THC has a very low aqueous solubility (1-2 microg/mL), which restricts its use as a pharmaceutical. The present study demonstrates that THC forms a drug-cyclodextrin complex in an aqueous solution of hydroxypropyl-beta-cyclodextrin (HP-beta-CD), resulting in a thousand-fold increase in THC solubility. This improvement in solubility can be further increased by adding 0.1% hydroxypropylmethylcellulose to the HP-beta-CD solution. The present results suggest that the use of cyclodextrins might be a simple and useful method to overcome the poor water solubility of THC.     

Reduction of the behavioral effects of delta9-tetrahydrocannabinol by hyperbaric pressure

The temporal order of development of olfactory, hippocampal and thalamocortical connections has been determined by light microscopy. Scalpel lesions were made to interrupt these connections and the resulting terminal degeneration was stained by Eager's method (1970). A post-operative survival time of one to four days was used. Evidence of the development of these connections was first obtained at the following ages: Olfactory mucosa to olfactory bulb: axon fascicles by 16 days of gestation and terminals in glomeruli at birth; Olfactory bulb to prepyriform cortex at birth; Prepyriform to entorhinal cortex at 13 days after birth; Entorhinal cortex to hippocampus (the perforant path) at 9 days; Hippocampal dentate-Ammonic mossy fibres at 9 days; Hippocampal efferent projection to the septum at birth; Subicular projections to the anterior thalamus at birth and to the mammillary body at 6 days; Hippocampal commissural connections at birth; Corticothalamic and thalamocortical connections by 2 days. These results are discussed in relation to the question of how the development of brain connections is programmed.     

A GC-MS study of three major acidic metabolites of delta 1-tetrahydrocannabinol

The major urinary metabolite of delta 1-tetrahydrocannabinol (delta 1-THC) (1), delta 1-THC-7-oic acid (2), has been extensively studied for several purposes, including testing in the workplace for drug abuse. Immunoassays in combination with more specific methods such as gas chromatography-mass spectrometry (GC-MS), are commonly used for verification of positive results in the screening. Two additional and recently synthesized acidic metabolites of 1, 4",5"-bisnor-delta 1-THC-7,3"-dioic acid (3) and 4"-hydroxy-delta 1-THC-7-oic acid (4), were studied to widen the scientific basis in the analysis. Five different derivatives were examined using GC-MS. In addition, a new deuterated internal standard for 2, [2H10]-2, was evaluated. According to our results, suitable derivatives of 2, 3, and 4, according to chromatographic properties, are the methyl ester/silyl ether (procedure a), the methyl ester/trifluoroacetate (procedure b), or the silyl ester/silyl ether (procedure c). The estimated recoveries of [2H5]-3 and [2H6]-4 using liquid-liquid extraction were 24% and 50%, respectively. The properties of [2H10]-2 as internal standard were equivalent to those of [2H9]-2 and, under the conditions used, did not appear to give rise to a significantly higher chromatographic resolution from that of 2. However, [2H10]-2 produces ions at different mass numbers, which makes it useful as a complement to the existing deuterated internal standards of 2.     

Effects of delta9-tetrahydrocannabinol on neurotransmitter accumulation and release mechanisms in rat forebrain synaptosomes

Previous studies involving the end-to-end fusion of the forelimbs of the adult newt have demonstrated that new limbs can regenerate from the transected ends of proximo-distally reversed limb segments. The limb regeneration could only have been initiated by nerve fibers of contralateral origin. The purpose of the present study is to describe histologically the manner in which nerve fibers of contralateral origin regenerate through the junction of fused limbs into the opposite limb. The first sign of nerve regeneration into the opposite limb was observed at eight days post fusion. The nerves crossed over into the opposite, originally denervated limb in a highly dispersed manner. These nerve fibers eventually aggregated, however, either under the skin or within persisting nerve trunks. By 19 days post fusion the nerve fibers had reached the elbow region of the originally denervated limb and by 25 days they were seen at the most proximal extent of the limb. The diameters of the axons seemed smaller than the diameters of regenerating axons observed in non-fused newt forelimbs.     

Covalent sequestration of the nitrogen mustard mechlorethamine by metallothionein

The research reported here demonstrates covalent binding to the metal-binding protein metallothionein (MT) by the therapeutic nitrogen mustard mechlorethamine. The most surprising aspect of this interaction is the selectivity of the alkylating agent for specific residues of MT. A combination of MS and proteolytic and enzymatic methods was used to deduce specific locations of mechlorethamine alkylation. These experiments indicated that alkylation occurs predominantly in the carboxyl domain of MT, with one molecule of mechlorethamine covalently cross-linking two cysteine residues. Electrospray MS revealed the retention of all seven metal ions in the cross-linked MT/mechlorethamine adducts, highlighting the uniqueness of this protein. Computerized docking experiments supported the hypothesis that selective binding precedes selective alkylation, and the structure of the drug indicates the minimal structural requirements for this binding. These results support the idea that MT overexpressed in tumor cells contributes to the inactivation of anticancer drugs.     

Effects of acute administration of delta9-tetrahydrocannabinol on pulmonary hemodynamics of anesthetized dogs

In an attempt to diminish the severity of the acute and late effects of irradiation to the rectum of dogs, oral prednisone was administered to 10 dogs for 1 week prior to, during, and for 1 month following a 3-week fractionated course of 60Co exposures to the pelvis. A control group of 10 dogs received irradiation alone. The dogs were observed clinically, serial rectal biopsies were done during and following the acute reaction, and the rectum was studied following sacrifice. Observations suggest that prednisone has no beneficial effect on the acute inflammatory reaction, and increases the severity of the late tissue damage.     

An acute dose of delta 9-tetrahydrocannabinol affects behavioral and neurochemical indices of mesolimbic dopaminergic activity

Cannabinoid consumption has been reported to affect several neurotransmitter systems and their related behaviors. The present study has been designed to examine cannabinoid effects on certain behaviors, which have been currently located in the limbic forebrain, in parallel to their effects on mesolimbic dopaminergic neurons. To this end, male rats treated with an oral dose of delta 9-tetrahydrocannabinol (THC) or vehicle were used 1 h after treatment for two different behavioral tests or neurochemical analyses of mesolimbic dopaminergic activity. Treatments, behavioral tests and sacrifice were performed in the dark phase of photoperiod because it corresponds to the maximum behavioral expression in the rat. Behavioral tests were a dark-light emergence test, which allows measurements of emotional reactivity, and a socio-sexual approach behavior test, which allows measurements of sexual motivation and also of spontaneous and stereotypic activities. Neurochemical analyses consisted of measurements of dopamine (DA) and L-3,4-dihydroxyphenylacetic acid (DOPAC) contents, tyrosine hydroxylase activity, in vitro DA release and number and affinity of D1 receptors in the limbic forebrain. Results were as follows. THC exposure markedly altered the pattern executed by the animals in both tests. Concretely, THC-exposed animals exhibited a low number of visits to an incentive female in addition to high time spent in the vicinity of an incentive male, both observed in the socio-sexual approach behavior test, and an increased emergence latency to go out of a dark compartment in the dark-light emergence test. However, the fact that THC also decreased spontaneous activity and the frequency of rearing and self-grooming behaviors, in addition to the observations of either low total number of visits to both incentive sexual areas or high escape latency to go out of a light compartment, when the animal is placed in this compartment, also suggest the possible existence of an accompanying motor deficit. These behavioral effects were accompanied by increases in DA and DOPAC contents and in D1 receptor density in the limbic forebrain and to a slight decrease in the pattern of K(+)-evoked DA release in vitro from perifused limbic fragments, with no changes in the remaining neurochemical parameters. Collectively, these results allow us to conclude that acute THC markedly altered the behavioral pattern executed by the animals in a socio-sexual approach behavior test and in a dark-light emergence test, presumably indicating loss of sexual motivation and increased emotionality, although also accompanied by motor deficiencies.(ABSTRACT TRUNCATED AT 400 WORDS)     

delta9-tetrahydrocannabinol and the hippocampus: effects on CA1 field potentials in rats

The effects of delta9-tetrahydrocannabinol (THC) on ortho- and antidromically elicited CA1 field potentials were observed in locally anesthetized rats and in anesthetized with urethane. THC augmented amplitudes of population EPSP's as well as orthodromic and antidromic population spikes from pyramidal cells in locally anesthetized animals. Latencies to peak amplitude of these response were increased. Conditioning-test shock experiments revealed taht THC also depressed recurrent inhibition probably mediated by basket cells. In animals under urethane anesthesia THC enhanced test responses, but failed to augment population response to the conditioning stimulus. It was concluded that THC enhanced postsynaptic excitatory processes but attenuated recurrent inhibition. Urethane anesthesia completely blocked the postsynaptic excitatory effect of THC but had little apparent influence on THC's disinhibitory action.