Generalization of the discriminative stimulus properties of x0394; 9 in rats

Rats were trained in a water maze to discriminate between IP injections of 3 mg/kg ⁹- (⁹⁽¹¹⁾-THC) and its vehicle. Both ⁸- and ⁹⁽¹¹⁾ were generalized to the training drug. In contrast to our observations in rhesus monkeys, where ⁹-THC is at least 100 times less potent than ⁹-THC, ⁹⁽¹¹⁾ was found to be only seven times less potent in the rat. Relative potencies, expressed as the dosage at which 50% of the animals gave drug responses (ED₅₀) were 1.8 mg/kg and 12.2 mg/kg for ⁹-THC and ⁹⁽¹¹⁾ respectively. Twenty-four hours after receiving 7×ED₅₀=12 mg/kg ⁹ the tests showed intermediate results when conducted with the training dosage; 4×ED₅₀=50 mg/kg ⁹-THC 48 h prior to the training dosage of 3 mg/kg ⁹-THC completely blocked drug-appropriate responses. Coinjection of ED₅₀ dosages of ⁹- and ⁹⁽¹¹⁾-THC led to 90% drug responses, demonstrating the additivity of the cannabis-like effect of both cannabinoids. Differences in the individual sensitivity of the rats to the tested cannabinoids were observed. Findings are interpreted in terms of the receptor mechanism for cannabis-like activity.This paper is dedicated to the memory of Dr. M. Binder who died on February 15, 1984     

Δ9-THC-induced cognitive deficits in mice are reversed by the GABAA antagonist bicuculline

Rationale The results of recent in vitro studies have underscored the important role that activation of CB₁ receptors has on GABAergic activity in brain areas associated with memory.Objectives The primary purpose of this study was to test the hypothesis that the memory disruptive effects of ⁹-tetrahydrocannabinol (⁹-THC) in vivo are mediated through GABAergic systems. Conversely, we also evaluated whether blocking CB₁ receptor signaling would alter memory deficits elicited by GABA agonists.Methods The GABA_A antagonist bicuculline and GABA_B antagonist CGP 36742 were evaluated for their ability to ameliorate ⁹-THC-induced deficits in a mouse working memory Morris water maze task. Mice were also assessed in a T-maze task, as well as non-cognitive behavioral assays. Additionally, the effects of GABA_A and GABA_B agonists were assessed in either CB₁ (–/–) mice or wild type mice treated with the CB₁ antagonist SR 141716.Results Memory deficits resulting from 10 mg/kg ⁹-THC in the Morris water maze were completely reversed by bicuculline, though unaffected by CGP 36742. Bicuculline also blocked the disruptive effects of ⁹-THC in the T-maze, but failed to alter non-mnemonic effects of ⁹-THC. Although CB₁ (–/–) mice exhibited supersensitivity to muscimol-induced water maze deficits compared with wild type control mice, muscimol elicited virtually identical effects in SR 141716-treated and vehicle-treated wild type mice.Conclusions This is the first demonstration of which we are aware showing that GABA_A receptors may play a necessary role in ⁹-THC-induced memory impairment in whole animals.     

Effects of Δ8-THC,and Δ9-THC on the acquisition of a discriminative positional habit in rats

Using a reversal learning paradigm the dissociative effects of two tetrahydrocannabinols (THC) on the acquisition and reversal of a discriminative positional habit in rats were studied. A T-shaped water maze was used. From these experiments it is concluded that learning under the influence of ⁸-THC (10 and 20 mg/kg), and ⁹-THC (5 mg/kg) is state-dependent (StD) in the rat.Numbering system according to IUPAC rules.Parts of the results were presented at the Symposium on the chemistry and biological activity of cannabis, Stockholm, October 26–28, 1971 and at the Symposium on medical plants of Brazil, Sao Paulo, April 17–20, 1972.     

EEG and behavioral effects of natural,synthetic and biosynthetic cannabinoids

The pharmacological effects of marihuana in man and animals have been attributed to ¹ and ⁶ tetrahydrocannabinol (THC).Recently, one of the metabolites of THC, 7-OH-THC, has been reported to have intoxicating properties. A comparative study was carried out on the EEG and behavioral effects of cannabinol, cannabidiol, ⁶-THC, 7-OH- ⁶-THC and 7-Acetoxy ⁶-THC acetate in six chronically implanted rabbits bearing cortical and subcortical leads. Drugs were dissolved in polyethyleneglycol and administered i.v. once every 7 days in a crossover experimental design. 7-OH- ⁶-THC and 7-Acetoxy- ⁶THC acetate proved to be at least twice as active as ⁶-THC in inducing EEG changes (disruption of theta waves, appearance of spikes and waves, blockade of the arousal reaction) and behavioral alterations (excitation, exophthalmus, mydriasis, corneal arreflexia, ataxia and swaying). Pretreatment with 1 or 2 mg/kg of reserpine, s.c., did not substantially alter the subjects response to THC.Amphetamine 2 mg/kg, i.v., in animals pretreated with 2 or 4 mg/kg ⁶-THC, reverses in part the depression induced by THC.     

Effects of chronic exposure to Δ9-tetrahydrocannabinol on delayed matching-to-sample in chimpanzees

Three groups of four chimpanzees were trained on a 20-sec delayed matching-to-sample task and then were exposed to a 152 day chronic drug regimen. Two of the chimpanzees in each group were drug naive. The other two chimpanzees in each group had experienced 45 doses of ⁹-tetrahydrocannabinol ( ⁹-THC) four months prior to the present experiment. One group of animals served as nondrug controls. A second control group received an oral dose of 1.0 mg ⁹-THC per kilogram of body weight following each matching-to-sample session. The experimental group was given the same dose of ⁹-THC prior to each daily session. The initial administrations of the drug before but not after each session produced a significant decrease in matching-to-sample accuracy. During the course of the chronic drug regimen, animals in the experimental group recovered very slowly from this initial impairment in matching-to-sample performance. The extent to which the experimental animals recovered seemed to depend upon their pre-experimental drug histories. The drug-experienced animals developed complete tolerance within five weeks while the previously drug-naive animals did not so do even after five months exposure to the drug. However, no residual or long-term effects were observed following termination of the chronic drug regimen.The authors thank Dr. Monique C. Braude for her advice and support and Dr. Wolfgang Mueller for gas chromatography analysis. Research funded by National Institute of Mental Health Contract HSM 42-71-15. Synthetic ⁹-THC obtained by approval of the FDA-NIMH Psychotomimetic Agents Advisory Committee. The animals involved in this study were maintained in accordance with Guide for Laboratory Animal Facilities and Care as published by the National Academy of Sciences-National Research Council.     

Immunomodulatory effect and quantitation of a cytotoxic glycosphingolipid from Murdannia loriformis

NMR signal reassignments for a cytotoxic glycosphingolipid compound, 2, -O-D-glucopyranosyl-2-(2-hydroxy-Z-6-enecosamide)sphingosine, isolated from an ethanolic extract of the herb Murdannia loriformis, have been achieved by use of FAB-MS, and 1D and 2D ¹H and ¹³C NMR. The amount of 2 in the herb juice was quantitatively determined by use of a validated HPLC method (RP-18, MeOH–H₂O, UV detection at 210 nm). The immunomodulatory effect of the herb juice and of 2 was proved by means of in vitro cellular immunological assays. Compound 2 at a concentration of 13 nmol L^–1 stimulated PBMC proliferation and increased the CD 3,4:CD 3,8 ratio in T lymphocytes.     

Protein Aggregates Seem to Play a Key Role Among the Parameters Influencing the Antigenicity of Interferon Alpha (IFN-α) in Normal and Transgenic Mice

Purpose. During long-term treatment of various malignant or viral diseases with IFN- up to 20% of patients develop anti-IFN- antibodies for as yet unknown reasons. Methods. To address this issue, a mouse model using Balb/C mice was established and the relevance of several potentially anti-IFN- antibodies inducing factors was studied. Results. The model revealed that both a higher frequency of injections and a higher dosage of IFN- were more immunogenic and that the route of administration affected the antibody response to IFN-. The intrinsic immunostimulatory activity of IFN- itself also enhanced the immune response. IFN- protein aggregates (IFN--IFN- and human serum albumin (HSA)-IFN- aggregates), which were recently identified in all marketed IFN- products, were significantly more immunogenic than IFN- monomers. These aggregates broke the tolerance against human IFN- monomers in human IFN- transgenic mice. Conclusions. Based on these animal studies it is proposed that the immune response to IFN- in humans is most probably elicited by a combination of several factors among which IFN- protein aggregates seem to play a key role.     

ATP and endogenous agonists inhibit evoked [3H]-noradrenaline release in rat iris via A1 and P2y-like purinoceptors

Summary Effects of ATP, adenosine and purinoceptor antagonists on field stimulation-evoked (3 Hz, 2 min) [³H]-noradrenaline overflow were investigated in the rat isolated iris.ATP and adenosine inhibited the evoked overflow of [³H]-noradrenaline. 1,3-Dipropyl-8-cyclopentylxanthine (DPCPX) shifted the concentration-response curve of ATP to the right in a concentration-dependent manner, but with a potency (–log K_B = 7.88) much lower than expected for an A1 adenosine receptor. In the continuous presence of DPCPX, the ATP-induced prejunctional inhibition was unaffected by suramin (100 mol/l) and DIDS (4,4-diisothiocyanatostilbene-2,2-disulfonic acid, 50 mol/l) but was antagonized by the P_2Y-receptor antagonist cibacron blue ( = reactive blue 2;30 and 100 mol/l, –log K_B = 4.7)and ,-methylene-ATP (10 mol/l). Whereas the evoked [³H]-noradrenaline overflow was unaffected by suramin and DIDS, cibacron blue and ,-methylene-ATP caused a small and transient increase. Cibacron blue at 30 mol/l failed to antagonize the inhibition of evoked [³H]-noradrenaline overflow that adenosine produced in the absence of DPCPX. Basal [³H]-noradrenaline overflow was enhanced by cibacron blue, not changed by ,-methylene-ATP and DIDS, and decreased by suramin.The results show that exogenous ATP inhibits sympathetic neurotransmission in the rat iris via A₁ and P_2Y-like purinoceptors. The latter have a low apparent affinity for cibacron blue and probably are blocked by ,-methylene-ATP. Under the present conditions, endogenous purines exert a tonic inhibition not only via A₁- but also via these P_2Y-receptors. Correspondence to: H. Fuder at the above address     

CB<Subscript>1</Subscript> receptor mediation of cannabinoid behavioral effects in male and female rats

Rationale Cannabinoids have been shown to produce greater behavioral effects in female than male rats. Although central nervous system CB₁ receptors are known to mediate cannabinoid-induced behavioral effects in male rats, it is not known whether the same is true for females.Objective To determine if cannabinoid-induced antinociception and catalepsy are similarly mediated by central CB₁ receptors in male and female rats.Methods The ability of SR141716A, a CB₁ receptor selective antagonist, administered ICV (1–1000 g) or IT (1–600 g) to block 10 mg/kg IP ⁹-THC-induced antinociception (paw pressure) and catalepsy (bar test), was compared in male and female rats.Results ⁹-THC alone produced slightly greater antinociception, and significantly greater catalepsy in females than males. When administered ICV, SR141716A partially antagonized ⁹-THC-induced antinociception in both females and males. IT SR141716A also antagonized ⁹-THC-induced antinociception in both sexes; it was slightly more potent in males but equally effective in males and females. SR141716A antagonized ⁹-THC-induced catalepsy in a similar manner in males and females when given ICV or IT.Conclusions These results confirm that ⁹-THC-induced behavioral effects are mediated by central CB₁ receptors in male and female rats.     

α-Adrenoceptor activity of arylalkylimidazoles is improved byα-methylation and impaired byα-hydroxylation

Summary To investigate the effects of hydroxyl and methyl substitution of the alkyl bridge bond on the-adrenoceptor activity of arylalkylimidazole derivatives, the cardiovascular effects of the molecules were studied in anaesthetized and pithed rats. The compounds studied were 4(5)-substituted imidazole derivatives with a methano, ethano or etheno bridge between the imidazole and the 2-, 2,3- or 2,6-methyl substituted phenyl rings. The hypotensive and bradycardic activities of the molecules in the anaesthetized rat were always reduced by-hydroxylation and usually augmented by-methylation of the bridge between the imidazole and phenyl rings. Hydroxylation was associated with a consistent, marked decrease in vasopressor and sympatho-inhibitory activity in the cardiovascular system of the pithed rat, but a methyl moiety as a bulky substituent in the-position of the alkyl bridge did not decrease but even caused an increase in-adrenoceptor activity in this test system. The detrimental effect of-hydroxylation of the compounds at ₁- and ₂-adrenoceptors supports the notion that the interaction of the imidazoles at-adrenoceptor is different from that of the classical, noradrenaline-like phenethylamines. The results also suggest that the alkyl bridge between the phenyl and imidazole rings of the imidazoles may contribute directly to the binding process.     

Stimulus effects of Δ9-THC and its interaction with naltrexone and catecholamine blockers in rats

Rats trained in a T-shaped maze to discriminate the effects of i.p. injections of ⁹-tetrahydrocannabinol (⁹-THC, 4mg/kg) and the effects of the vehicle were tested for antagonism and generalization to the ⁹-THC stimulus by naltrexone (4 mg/kg), haloperidol (0.32 mg/kg), propranolol (20 mg/kg), and phenoxybenzamine (10 mg/kg). None of these drugs blocked the ⁹-THC stimulus, nor were they found to generalize to ⁹-THC.     

The effect of two tetrahydrocannabinols, (Δ9-THC and Δ8-THC) on conditioned avoidance learning in rats and its transfer to normal state conditions

Rats trained in conditioned avoidance responding (CAR) after injections of either 7.5 mg/kg ⁹-THC (tetrahydrocannabinol) or 15 mg/kg ⁸-THC, showed no transfer when tested in the non-drugged state. Furthermore, these doses of the isomeric tetrahydrocannabinols exerted a disruptive effect on previously established CAR in rats, trained under normal conditions.Only the ⁹-THC-group showed an impairment of acquisition which was statistically significant compared to the control group.     

Dependence of the cardiovascular effects of pindolol on the individual sympathetic nervous activity

Summary To investigate the mechanism of the relative beta-antagonist and agonist properties of pindolol, the cardiovascular effects of i.v. pindolol 0.01 mg/kg were studied in 23 subjects with different baseline levels of cardiac beta-adrenergic function. Baseline cardiac beta-adrenergic activity was assessed by the decrease in heart rate following intravenous propranolol 0.2 mg/kg (HR) after parasympathetic blockade with intravenous atropine 0.04 mg/kg. Cardiac beta-adrenergic sensitivity was defined by the positive chronotropic effect of a three minute infusion of isoprenaline 0.005 µg/kg/min. The chronotropic effect of intravenous pindolol was negatively correlated with resting beta-adrenergic activity (R=–0.81, p<0.001). The traditional point of HR, at which pindolol shifted from beta-antagonist to beta-agonist action, was 17 beats/min, i.e. pindolol decreased heart rate in subjects with HR greater than 17 bpm, and increased heart rate in those with HR less than 17 bpm. The chronotropic effect of intravenous pindolol, however, was positively correlated with beta-sensitivity (R=+0.73, p<0.001). Thus subjects with the greatest increment in heart rate with isoprenaline had an increased heart rate with pindolol, while those with the lowest increment in heart rate with isoprenaline had a decreased heart rate with pindolol. Intravenous pindolol decreased cardiac index and increased total peripheral resistance in the group with HR more than 17 bpm, and it had the contrary actions in the group with HR less than 17 bpm. Blood pressure in both groups was not significantly changed by pindolol. Compared to endogenous catecholamines, pindolol was considered to possess higher beta-adrenocepter affinity and weaker beta-adrenoceptor stimulating action. Therefore, pindolol produced a net beta-blocking action in subjects with elevated cardiovascular sympathetic nervous activity and beta-stimulating action in subjects with reduced sympathetic nervous activity.     

Pre- and postganglionic stimulation-induced noradrenaline overflow is markedly facilitated by a prejunctional β2-adrenoceptor-mediated control mechanism in the pithed rat

The aim of the study was to further explore the prejunctional -adrenoceptor-mediated control mechanism of noradrenaline release from sympathetic nerves in response to preganglionic nerve stimulation (PNS) and local nerve stimulation of the portal vein, respectively, in the pithed rat.Baseline values as well as the increments of mean arterial blood pressure (-BP), heart rate (-HR) and plasma noradrenaline levels (-NA) in response to four PNS episodes (0.8 Hz, 3 ms, 75 V for 45 s at 20 min intervals), respectively, were evaluated. Fenoterol administration (0.25 mg/kg, i.v.) reduced significantly the basal blood pressure but did not alter d-BP in response to PNS. Basal heart rate markedly increased after fenoterol without any further change in heart rate induced by PNS.The ₁-selective antagonist CGP 20712A attenuated -BP in response to PNS and prevented the fenoterol-induced increase in basal heart rate. The ₂-selective antagonist ICI 118,551 per se did not change the blood pressure and heart rate values, but antagonized the fenoterol-induced decrease in basal blood pressure.Fenoterol enhanced plasma -NA in response to PNS by 105% in comparison to the corresponding control value. This effect of fenoterol could be blocked by pretreatment with ICI 118,551 but not with CGP 20712A (a selective ₁-adrenoceptor antagonist) which per se did not significantly change plasma -NA.Repeated local stimulation of the portal vein (S1–S 3, 2 Hz, 3 ms, 10 mA, for 120 s at 30 min intervals) increased portal plasma noradrenaline without changing mean blood pressure and heart rate in pithed rats. Fenoterol enhanced the increase in portal-vein plasma noradrenaline evoked by nerve stimulation by 110%. Pretreatment with ICI 118,551 antagonized this effect of fenoterol, but had per se no effect on the portal vein nerve stimulation-evoked increase in portal plasma noradrenaline.It is concluded that the increase in plasma noradrenaline evoked both by pre- and postganglionic nerve stimulation can be markedly enhanced by activation of a facilitatory prejunctional ₂-adrenoceptor control mechanism. Correspondence to: V.I. Tarizzo     

Effect of cannabinoids on the turnover rate of acetylcholine in rat hippocampus,striatum and cortex

Summary The effects of ⁹-tetrahydrocannabinol, (⁹THC) the major psychoactive compound of marijuana, and cannabidiol (CBD), a non-psychoactive component, on the acetylcholine (ACh) concentration and the turnover rate of ACh (TR_ACh) have been studied in various regions of the rat brain. Neither ⁹THC doses from 0.2 to 10 mg/kg nor CBD (10 or 20 mg/kg) alter the ACh concentration in the brain areas examined 30 min, after the intravenous injection. However, ⁹-THC (doses from 0.2 to 10 mg/kg) causes a marked dose-related decrease in the TR_ACh in hippocampus whereas CBD is without effect in this brain region even when 20 mg/kg is given. Furthermore, high doses of ⁹-THC (5 mg/kg) and CBD (20 mg/kg) that produce a significant decrease in the TR_ACh of striatum fail to change the TR_ACh in parietal cortex. The low doses of ⁹-THC required to reduce hippocampal TR_ACh suggest that an action on these cholinergic mechanisms may play a role in the psychotomimetic activity of ⁹-THC.     

Prediction of In Vivo Interaction Between Triazolam and Erythromycin Based on In Vitro Studies Using Human Liver Microsomes and Recombinant Human CYP3A4

Purpose. To quantitatively predict the in vivo interaction betweentriazolam and erythromycin, which involves mechanism-basedinhibition of CYP3A4, from in vitro studies using human liver microsomes(HLM) and recombinant human CYP3A4 (REC). Methods. HLM or REC was preincubated with erythromycin in thepresence of NADPH and then triazolam was added. - and 4-hydroxy(OH) triazolam were quantified after a 3 min incubation and the kineticparameters for enzyme inactivation (k_inact and K _app) were obtained.Drug-drug interaction in vivo was predicted based on aphysiologically-based pharmacokinetic (PBPK) model, using triazolam anderythromycin pharmacokinetic parameters obtained from the literature and kineticparameters for the enzyme inactivation obtained in the in vitro studies. Results. Whichever enzyme was used, triazolam metabolism was notinhibited without preincubation, even if the erythromycin concentrationwas increased. The degree of inhibition depended on preincubationtime and erythromycin concentration. The values obtained for k_inactand K _app were 0.062 min^–1 and 15.9 M (-OH, HLM), 0.055 min^–1and 17.4 M (4-OH, HLM), 0.173 min^–1 and 19.1 M (-OH, REC),and 0.097 min^–1 and 18.9 M (4-OH, REC). Based on the kineticparameters obtained using HLM and REC, the AUCpo of triazolamwas predicted to increase 2.0- and 2.6-fold, respectively, followingoral administration of erythromycin (333 mg t.i.d. for 3 days), whichagreed well with the reported data. Conclusions. In vivo interaction between triazolam and erythromycinwas successfully predicted from in vitro data based on a PBPK modelinvolving a mechanism-based inhibition of CYP3A4.     

Δ 1-tetrahydrocannabinol-induced circling behavior in rats: A possible measure of psychotomimetic activity?

Relatively high dosages of ¹-tetrahydrocannabinol ( ¹-THC) markedly suppressed almost all normally occurring behavioral elements in rats as observed in both a small and a large open field. This effect persisted following repeated treatment and testing for 3 consecutive days. The psychotropically inert but related compound cannabidiol (CBD) did not suppress behavior, but in contrast had a mild activating effect. Both compounds decreased defecation during the test procedure. In addition, ¹-THC, but not CBD, induced a bizarre circling and turning response, that was evident over all 3 test days and occurred even when the rats were deeply sedated. It is suggested that an analysis of this phenomenon may be useful in measuring the psychotominetic action of ¹-THC and similar compounds in rats.     

Electrophysiological and electrochemical analysis of the secretion of ATP and noradrenaline from the sympathetic nerves in rat tail artery: effects of α2-adrenoceptor agonists and antagonists and noradrenaline reuptake blockers

Summary The aim of this study was to investigate whether or not nerve impulses release ATP and noradrenaline in parallel from the sympathetic nerve terminals of the rat tail artery. The extracellularly recorded excitatory junction current (EJC) was used to study, pulse by pulse, the release of ATP. An electrochemical method was used to study online the nerve stimulation-induced rise in the extracellular concentration of endogenous noradrenaline at the probe, a carbon fibre electrode (CF). This parameter, which does not directly represent noradrenaline release, but reflects release minus clearance, has been termed [NA]_CF. The effects of a number of pharmacological agents on the EJCs were examined both at 0.1 and 2 Hz, and the effects on the EJC response to 100 pulses at 2 Hz compared with that on the [NA]_CF response. Clonidine and xylazine were used as ₂-agonists, yohimbine and idazoxan as ₂-antagonists and desipramine and cocaine as blockers of noradrenaline reuptake. Most of these agents had unwanted side effects, especially at higher concentrations. However, clonidine and xylazine depressed at lower concentrations the EJC and [NA]_CF responses to about the same extent; these effects were partially or completely reversed by yohimbine. Yohimbine or idazoxan did not affect the EJCs at 0.1 Hz but enhanced the EJC and [NA]_CF responses to 100 pulses at 2 Hz to the same extent. All effects of desipramine (1 M) seemed explainable as a result of block of noradrenaline reuptake, while cocaine (10 M) in addition exerted an unspecific depressant (probably local anesthetic) effect. Under control conditions, both agents depressed the EJC but dramatically enhanced the [NA]_CF response to 100 pulses at 2 Hz. Addition of yohimbine prevented the depressant effect of desipramine on the EJCs completely and reduced that of cocaine, but increased their effects on the [NA]_CF response. These results are compatible with the view that ATP and noradrenaline are released in parallel from the sympathetic nerve terminals of this tissue. The different, and under some conditions even opposite, effects of desipramine or cocaine on the EJC and [NA]_CF responses are explainable in terms of the known post-secretory effects of these agents. Send offprint requests to M. Msghina at the above address     

Effect of Δ9-THC on the open-field activity of the rat

Using the open-field activity of the hooded rat as a model of overall activity, the dose-response and time-action effects of doses of ⁹-THC which did not adversely affect spontaneous activity or behavior on appetitively motivated tasks were studied. Subjects received two exposures to an open field one week apart. Prior to the first exposure subjects were treated with small doses of Tween 80-water. At 30 min or 3 h prior to the second exposure subjects were treated with Tween or ⁹-THC in doses which ranged from 0.5–5 mg/kg. Results indicated that ⁹-THC affected various indices of open-field activity such as grooming, sniffing and ambulation differently depending on the time after injection. Rearing and defecation were affected similarly by THC independent of post-injection intervals.This research was supported by USPHS Grant No. R01-MH18392-01. The ⁹-THC was supplied by Dr. John A. Scigliano, NIMH, Chevy Chase, Maryland. The authors wish to thank Dr. Patrick Deluca for assistance in the solvent studies.