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The effect of repetitive administration of delta-9-tetrahydrocannabinol (delta 9-THC) in mice on behavioral and biochemical tolerance was determined in this study. Mice were injected twice daily with 10 mg/kg delta 9-THC for 6.5 days. On day 8, spontaneous activity was assessed or whole-brain homogenates were prepared for the cannabinoid receptor binding and mRNA studies. Although a twenty-sevenfold tolerance to delta 9-THC was observed in the behavioral assay, there was no significant alteration in receptor binding or mRNA levels.

Title compound, 8, has been synthesized from isoquinolinone, 1 (an improved preparation for which is presented) and separated into its antipodes with D- and L-di-p-toluoyltartaric acids. These antipodes and the racemic precursor have been evaluated (and found active) in two in vivo systems for their effects. The most potent of the three, (+)-8, has an ED50 of 7.13 mumol/kg for inhibition of spontaneous activity and 7.45 mumol/kg for antinociception compared to 4.44 and 4.81 mumol/kg, respectively, for (S)-(-)-nicotine. Compounds (-)-8 and 7 are about one-fourth as potent. Isomer (+)-8 has the 3aR,9bS configuration, the latter corresponding to (S)-(-)-nicotine as determined by X-ray crystallography. However, (+)-8 failed to compete for [3H]-nicotine binding, and its pharmacological effects were not blocked by mecamylamine. These bridged nicotine analogs either are binding to an as-yet-unidentified nicotinic receptor or they represent a novel class of non-nicotinic analgesics.

One aspect of cannabinoid structure-activity relationships (SARs) that has not been thoroughly investigated is the aromatic (A) ring. Although halogenation of the side chain enhances potency, our recent observation that iodination of the A ring also enhanced activity was surprising. The purpose of this investigation was to establish the steric and electrostatic requirements at these sites of the cannabinoid molecule via molecular modeling, while determining pharmacological activity. Molecular modeling was performed using the Tripos molecular mechanics force field and the semiempirical quantum mechanical package AM1. The Ki values for novel cannabinoids were determined in a [3H]CP-55,940 binding assay and ED50 values generated from four different evaluations in a mouse model. The present studies underscore the increase in potency produced by a dimethylheptyl (DMH) side chain. Trifluoro substitutions on the pentyl side chain, or bromination of the DMH side chain, had little effect on the pharmacological activity. Any substitution at the C4 position of the aryl ring resulted in a loss of activity, which appears to be due to steric hindrances. Nitro, but not iodo, substitution at the C2 position essentially produces an inactive analog, and the drastic alteration of the electrostatic potential appears to be responsible. The altered pharmacological profile of the 2-iodo analog seems to be related to an alteration in the highest occupied molecular orbital because there is no alteration in the electron density map compared to delta 8-tetrahydrocannibinol.

The nature of the signaling process activated by neuronal nicotinic receptors has not been fully defined; however, several recent studies have implicated the involvement of calcium ion fluxes in the response to nicotine on a cellular level. Alteration of nicotine-induced antinociception in mice after systemic administration was therefore investigated in the presence of several drugs that increase intracellular calcium. Calcium, (+/-)-BAYK 8644, thapsigargin, glyburide and A23187 administered intrathecally (i.t.) were found to enhance nicotine-induced antinociception by shifting its dose-response curve to the left. Conversely, i.t. administration of agents which decrease intracellular calcium, such as EGTA and alpha-calcitonin gene-related peptide, blocked nicotine-induced antinociception. These findings support a role for spinal intracellular calcium in the pharmacological effects of nicotine. Additionally, blockade of antinociception by nimodipine and nifedipine indicates that a L-type calcium channel is involved in nicotine's effect. However, nicotine did not compete for [3H] nitrendipine binding. Intrathecal administration of mecamylamine, a nicotinic antagonist, resulted in a blockade of antinociception produced by the i.t. injection of thapsigargin, A23187, calcium and (+/-)-BAYK 8644. The mechanism of mecamylamine's antagonism of nicotine is uncertain. However, these results suggest that mecamylamine blocks the effects of drugs which increase intracellular calcium by either a modulation of intracellular calcium-dependent mechanisms or a blockade of calcium channels. Thus, mecamylamine could modulate a calcium signaling process secondary to receptor activation resulting in blockade of antinociception produced by diverse agents.

The roles of sulfhydryl and disulfide groups in the specific binding of synthetic cannabinoid CP-55,940 to the cannabinoid receptor in membrane preparations from the rat cerebral cortex have been examined. Various sulfhydryl blocking reagents including p-chloromercuribenzoic acid (p-CMB), N-ethylmaleimide (NEM), o-iodosobenzoic acid (o-ISB), and methyl methanethiosulfonate (MMTS) inhibited the specific binding of [3H]CP-55,940 to the cannabinoid receptor in a dose-dependent manner. About 80-95% inhibition was obtained at a 0.1 mM concentration of these reagents. Scatchard analysis of saturation experiments indicates that most of these sulfhydryl modifying reagents reduce both the binding affinity (Kd) and capacity (Bmax). On the other hand, DL-dithiothreitol (DTT), a disulfide reducing agent, also irreversibly inhibited the specific binding of [3H]CP-55,940 to the receptor and about 50% inhibition was obtained at a 5 mM concentration. Furthermore, 5 mM DTT was abelt to dissociate 50% of the bound ligand from the ligand-receptor complex. The marked inhibition of [3H]CP-55,940 binding by sulfhydryl reagents suggests that at least one free sulfhydryl group is essential to the binding of the ligand to the receptor. In addition, the inhibition of the binding by DTT implies that besides free sulfhydryl group(s), the integrity of a disulfide bridge is also important for [3H]CP-55,940 binding to the cannabinoid receptor.

Although a receptor exists for cannabinoid drugs, it is uncertain which pharmacological actions this receptor mediates. This structure-activity relationship investigation was initiated to determine which effects might correspond to binding affinity for the cannabinoid receptor, as well as to explore the binding requirements of this site. The ability of nearly 60 cannabinoids to displace [3H]CP-55,940 [(-)-3-[2-hydroxy-4-(1,1-dimethylheptyl) phenyl]-4-[3-hydroxy propyl] cyclohexan-1-ol] was determined before establishing correlations between receptor affinity and in vivo pharmacological potency. Analysis of [3H]CP-55,940 binding indicated a Hill coefficient of 0.97, a Bmax of 499 pM (3.3 pmol/mg of protein) and an apparent Kd of 924 pM. Closer inspection indicated the binding assay exhibited "zone B" characteristics, and use of correction equations indicated a true Kd for CP-55,940 of 675 pM. The structure-activity relationship indicated the importance of side chain structure to high-affinity binding, with the most potent analogs (K1 < 10 nM) possessing either a dimethylheptyl side-chain, a similarly complex branched side chain or a halogen substituent at the 5' position. Comparative analysis of K1 values to in vivo potency in a mouse model indicated a high degree of correlation between parameters for the depression of spontaneous locomotor activity (r = 0.91) and for the production of antinociception (r = 0.90), hypothermia (r = 0.89) and catalepsy (r = 0.85). Similarly high correlations were demonstrated between binding affinity and in vivo potency in both the rat drug discrimination model (r = 0.81) and for psychotomimetic activity in humans (r = 0.88).(ABSTRACT TRUNCATED AT 250 WORDS)

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.

The influence of a calcium agonist (BAYK 8644) and several calcium channel blockers on nicotine-induced antinociception was investigated in mice. The effect of nicotine was sharply increased by BAYK 8644. This potentiation by BAYK 8644 was blocked by pretreating the animals with nifedipine at 2 and 10 mg/kg. The calcium channel antagonists, nifedipine and nimodipine at doses that had no effect on tail-flick time, reduced significantly the antinociception induced by nicotine (1.5 mg/kg, s.c.). However, the effect of verapamil on nicotine was not significant. These results indicate that DHP calcium channels (type L-channel) play a role in some of the pharmacological effects of nicotine, particularly, locomotor activity and antinociception.

Pretreatment with ineffective doses of the D1 antagonist SCH23390 but not the D2 antagonist sulpiride reduced hyperactivity induced by nicotine in mice habituated to the test cage. On the other hand, the D1 and D2 antagonists were ineffective in blocking nicotine-induced hypoactivity in naive mice. Finally, SCH23390 and sulpiride did not block the antinociception induced by nicotine. Our data indicate that the dopamine receptors D1 and D2 are not involved in all the central effects of nicotine in mice, but seems to be a substrate for locomotor activation induced by nicotine under specific experimental conditions.

The binding of [3H]11-OH-delta 9-tetrahydrocannabinol-1, 1-dimethyl-heptyl (THC-DMH), a recently synthesized cannabinoid analog, was characterized in an in vitro brain slice binding assay and compared to that obtained with [3H]CP-55,940, the radiolabeled ligand used originally to characterize cannabinoid binding sites. The binding of both [3H]CP-55,940 and [3H]11-OH-delta 9-THC-DMH exhibited high affinity (Kd of 19 +/- 3 and 29 +/- 9 nM, respectively), and was saturable, reversible and specific. Values of maximal concentration of receptors determined for [3H]11-OH-delta 9-THC-DMH and [3H]CP-55,940 were 4.0 +/- 0.3 and 3.0 +/- 0.5 pmol/mg of protein, respectively. The distribution of [3H]11-OH-delta 9-THC-DMH and [3H]CP-55,940 binding in 30-microns rat brain sections was then compared by autoradiographic analysis. The binding of both ligands was densest in the basal ganglia (substantia nigra pars reticulata, globus pallidus, entopeduncular nucleus and regions of the caudate putamen) and cerebellum (molecular layer). Low levels of binding were observed in discrete brain regions including the brain stem (medulla and pons), thalamic nuclei, hypothalamus, corpus callosum and the deep nuclear layer of the cerebellum. Intermediate levels of binding were seen in layers I and VI of the cortex, and the dentate gyrus and CA pyramidal cell regions of the hippocampus. The ability of selected cannabinoid analogs to compete with [3H]11-OH-delta 9-THC-DMH binding was determined. The Ki's were correlated to the in vivo potencies for producing catalepsy, antinociception, hypothermia and decreasing spontaneous locomotor activity in mice (correlation coefficients > 0.86).(ABSTRACT TRUNCATED AT 250 WORDS)

Six novel aminoalkylindole analogs, related structurally to the dual cyclooxygenase inhibitor and nonopioid analgesic pravadoline, were evaluated in the mouse to determine whether their pharmacological profile of activity was similar to that exhibited by delta 9-tetrahydrocannabinol (delta 9-THC). Analog I (C2-H; C3-methoxy-benzoyl) reduced locomotion, but had no other effects (hypothermia, antinociception or ring-immobility) up to 21 mumol/kg. Analogs II and III (C3-naphthoyl; C2-H and C2-methyl, respectively) possessed all properties exhibited by delta 9-THC with ED50 values ranging from 0.68 to 18 mumol/kg. Analog IV (C2-methyl; C3-anthroyl) was devoid of activity. Stereoselectivity was demonstrated by the fact that (+)-WIN-55,212 (one isomer of a semirigid derivative possessing C2-H and C3-naphthoyl substituents) was moderately potent in all tests (ED50 values ranging from 0.25-23 mumol/kg), but (-)-WIN-55,212 was inactive up to 57 mumol/kg. Active aminoalkylindole compounds were generally least effective in the production of hypothermia. Analogs were also evaluated for their ability to produce delta 9-THC-like discriminative stimulus effects in rats. The ED50 for delta 9-THC as a discriminative stimuli for this model was 1.9 mumol/kg. Analog II and III and (+)-WIN-55,212 produced delta 9-THC-like discriminative effects with ED50 values ranging from 0.33 to 4.3 mumol/kg, whereas analogs I, IV and (-)-WIN-55,212 did not. Although reported to be cannabinoid receptor antagonists in vitro, neither analog I, analog IV nor (-)-WIN-55,212 (at 20 mumol/kg) antagonized the in vivo pharmacological effects of delta 9-THC in the mouse or rat.(ABSTRACT TRUNCATED AT 250 WORDS)

Extensive behavioral and biochemical characterization of cannabinoid-mediated effects on the central nervous system has revealed at least three lines of evidence supporting the role of a putative guanine nucleotide-binding protein-coupled cannabinoid receptor for cannabimimetic effects, (i) stereoselectivity, (ii) inhibition of the adenylate cyclase/cAMP second messenger system, and (iii) radioligand-binding studies with the synthetic cannabinoid [3H]CP-55,940 indicating a high degree of specific binding to brain tissue preparations. Based on recent findings from our laboratory demonstrating that delta 9-tetrahydrocannabinol markedly inhibited forskolin-stimulated cAMP accumulation in mouse spleen cells, the presence of a guanine nucleotide-binding protein-coupled cannabinoid receptor associated with mouse spleen cells and its functional role in immune modulation were investigated. In the present studies, stereoselective immune modulation was observed with the synthetic bicyclic cannabinoid (-)-CP-55,940 versus (+) CP-56,667 and with 11-OH-delta 8-tetrahydrocannabinol-dimethylheptyl, (-)-HU-210 versus (+)-HU-211. In both cases, the (-)-enantiomer demonstrated greater immunoinhibitory potency than the (+)-isomer, as measured by the in vitro sheep red blood cell antibody-forming cell response. Radioligand binding studies produced a saturation isotherm exhibiting approximately 45-65% specific binding to mouse spleen cells. Scatchard analysis demonstrated a single binding site on spleen cells, possessing a Kd of 910 pM and a Bmax of approximately 1000 receptors/spleen cell. RNA polymerase chain reaction of isolated splenic RNA using specific primers for the cannabinoid receptor resulted in the amplification of a 854-kilobase predicted product that hybridized with cannabinoid receptor cDNA, demonstrating the presence of cannabinoid receptor mRNA in mouse spleen. Together, these findings strongly support the role of a cannabinoid receptor in immune modulation by cannabimimetic agents.

Catalepsy is usually defined as a behavioral state in which an animal maintains an unnatural posture for an extended period of time. While numerous laboratory models have been developed for assessing catalepsy, a common problem encountered with most procedures is the difficulty in quantitating immobility. Measurement of catalepsy is still frequently subjective in nature. To eliminate this subjectivity, a computer-based technique was developed for quantitating catalepsy in mice and rats as measured on the elevated ring. The system consisted of a video camera that was focused on either three mice or two rats. Their behavior was recorded during a 5-min session on videotape that was subsequently transmitted to a Macintosh II microcomputer via a Scion Image-Capture 2 board. A modification of the NIH Image 1.17 public domain program allowed the image of the rat to be transformed to a purely black or white image by assigning pixel values of either 0 or 256. The subsequent captured image was preprocessed in an identical manner and each pixel was subtracted from its corresponding pixel in the previous frame. Thus, changes in animal posture between the two frames can be quantitated. One subtraction cycle (acquisition, bilevel processing, and subtraction) was repeated at an average rate of approximately one per second. To quantitate immobility by image analysis, each frame was subtracted from the previous frame during a 5-min session. The resulting data were sorted according to the magnitude of movement (number of changed pixels) and plotted vs. time.(ABSTRACT TRUNCATED AT 250 WORDS)

CP 55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3- hydroxypropyl)cyclohexanol] is a potent bicyclic analog of delta 9-tetrahydrocannabinol (THC) which has been used as a probe for a cannabinoid recognition site in neural tissue. In the present study, CP 55,490 was evaluated for delta 9-THC-like effects in rats and rhesus monkeys trained to discriminate delta 9-THC from vehicle. Rats trained to discriminate delta 9-THC (3.0 mg/kg i.p.) from vehicle were tested with various doses of delta 9-THC and CP 55,940 at both 30 and 90 min postinjection. Catalepsy was measured immediately after these operant tests using an adaptation of the mouse ring-test. In rats, CP 55,940 substituted for delta 9-THC at both 30 and 90 min postinjection at a dose of 0.1 mg/kg that had minimal effects on rates of responding. Doses of delta 9-THC (greater than 3.0 mg/kg) and CP 55,940 (greater than 0.1 mg/kg) that reduced response rates by greater than 50% also produced substantial increases in catalepsy. CP 55,940 and delta 9-THC had a similar time course for discriminative stimulus effects, but CP 55,940 was about 30 times more potent. In monkeys, the training dose of delta 9-THC ranged from 0.04 to 0.16 mg/kg i.m., adjusted individually to minimize response-rate disruption. After training, monkeys were tested with various doses of delta 9-THC and CP 55,940 at 30 min postinjection.(ABSTRACT TRUNCATED AT 250 WORDS)

The 5-hydroxytryptamine (5-HT)2 agonist (+/- )-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane (DOI) and antagonist ketanserin were evaluated for acute and chronic effects on the 5-HT2 receptor-mediated head-twitch response (HTR) in mice. A single dose of DOI resulted in tolerance to the DOI-induced HTR at 24 hr but supersensitivity at 48 hr. This apparent supersensitivity persisted up to 144 hr after the first DOI injection. Chronic once daily DOI administration significantly reduced the HTR frequency (days 2-6), which then returned slowly to control levels by treatment day 13. A 48-hr withdrawal from this chronic regimen produced a similar supersensitivity to that observed after a single DOI injection upon a 48-hr challenge. This effect also persisted up to 144 hr after cessation of chronic treatment. Acute pretreatment with a single injection of ketanserin significantly reduced the DOI-induced HTR frequency when tested 24 and 48 hr, but not 120 hr, after injection of the antagonist. After withdrawal from chronic ketanserin treatment, the DOI-induced HTR was significantly reduced at 24 hr but significantly increased at 48 hr. This enhanced effect subsided when mice were tested with DOI 72 hr after cessation of chronic antagonist treatment. These data suggest that the serotonergic system adapts to chronic exposure of either agonists or antagonists in a fashion distinctly different from that exhibited by other monoamine neurotransmitter systems.

The optical antipodes of N-allyl-N-normetazocine (2; SKF 10047, NANM) were the original compounds used for the classification of the sigma receptor as distinct from other receptors such as the PCP (NMDA), opioid, and dopamine receptors. Later studies showed that (+)-N-(dimethylallyl)-N-normetazocine [(+)-4, (+)-pentazocine] was more potent and selective for the sigma receptor. In order to gain additional structure-activity relationship information, several N-substituted N-normetazocine analogs were prepared and evaluated for their sigma-1 ([3H]-(+)-3-PPP or [3H]-(+)-pentazocine), PCP ([3H]TCP), and mu opioid ([3H]DAMGO) receptor binding affinities. (+)-N-Benzyl-N-normetazocine [(+)-10)] possessed subnanomolar affinities for the sigma site, Ki = 0.67. The analog (+)-10 showed greater than 14,000- and 2400-fold selectivity, respectively, for the sigma receptor relative to the PCP and mu opioid receptors. The N-substituted N-normetazocines were enantioselective for the sigma site. The (+)-N-benzyl analog, (+)-10, showed a 55-fold selectivity relative to (-)-10. Analysis of the data also revealed that (+)-normetazocine [(+)-1] [Ki = 30 nM] possessed the highest affinity for the PCP receptor. However, (+)-metazocine [(+)-5] (Ki = 41 nM) was the most selective compound for the PCP receptor relative to the sigma (51-fold) and mu opioid (greater than 200-fold) sites.

The effects of chronic prenatal methadone exposure (6.3-9.0 mg/kg/day) via osmotic minipumps to pregnant dams on fetal and maternal brain opioid mu-receptors were assessed on gestation day 20 and day 7 postnatally. By using the 3H-DAMGO binding assay, it was shown that chronic methadone treatment (gestation days 7-20) did not affect mu-receptor capacity in both fetal and maternal brains during gestation day 20, nor when tested 7 days after delivery. However, this chronic exposure decreased mu-receptor affinity in both fetal and maternal brain homogenates when determined on day 20 of pregnancy. Scatchard analysis of binding data in both tissues indicated that the methadone-induced increase in KD returned to control values when tested 7 days after delivery. The change in mu-receptor affinity was not due to competition between 3H-DAMGO and residual methadone. Extensive washing of the brain homogenates failed to alter the affinity of the receptor but decreased the concentration of the residual methadone. This decrease in receptor affinity was also observed in extensively washed brain tissue from female adult rats treated acutely with methadone (9.0 mg/kg, IP) or when brain homogenates were exposed to methadone (50 ng/ml) in vitro. Thus, these data suggest that methadone alters mu-receptor affinity by some unknown mechanism.

Marijuana has a long history of abuse yet, as described here by Mary Abood and Billy Martin, there is little evidence that animals will self-administer the primary psychoactive constituent, tetrahydrocannabinol, or that marijuana stimulates brain reward pathways. While marked tolerance develops to marijuana, it has been difficult to demonstrate physical dependence, and until recently the mechanisms by which cannabinoids produced their behavioral effects were poorly defined. The development of new synthetic analogs played a critical role in the characterization and cloning of the cannabinoid receptor. Insight into cannabinoid receptors may lead to a better understanding of marijuana abuse in humans and provide new therapeutic strategies for several disorders.

Cannabinoids have been demonstrated to be effective antinociceptive agents when given intravenously. In order to determine whether spinal antinociception can be achieved while minimizing psychotomimetic properties, the pharmacological activity of delta 9-tetrahydrocannabinol (THC) was evaluated after intrathecal injection in male ICR mice. Although delta 9-THC produced potent antinociception, it also caused hypoactivity, hypothermia, and catalepsy. Intrathecal administration of delta 9-THC in mice which had their spinal cord transected at T12 also produced potent antinociception suggesting a spinal component to the antinociceptive effect. Biodispositional studies of [3H]delta 9-THC demonstrated that brain levels of the drug following intrathecal injection in spinally transected animals were not sufficient to produce the antinociceptive effect. These studies suggest the involvement of a spinal component in the antinociceptive action of the cannabinoids.

Despite the fact that high doses of cocaine are abused chronically, relatively little is known regarding the development of tolerance and/or sensitization under these circumstances. Therefore, male Sprague-Dawley rats were infused continuously i.v. for 10 days, at a rate of 150 mg/kg/day (0.1 mg/kg/min) with cocaine hydrochloride. Body weight, food and water consumption, urine and fecal excretion, as well as blood pressure, heart rate and stereotypic behavior were monitored daily. Blood samples were also drawn daily so that plasma could be analyzed for cocaine and BEG by gas chromatography/mass spectrometry. Severe body weight loss on days 1 through 4 was followed by a gradual return to pre-drug levels. In addition, cocaine's effects on food and water consumption and urine and fecal excretion, which were maximal by day 2, were imperceptible by day 5. Complete tolerance developed rapidly to the remarkable rise in blood pressure noted on the first day. However, tolerance did not develop to the cocaine-induced increase in heart rate. A profound decrease in heart rate was noted in some animals which was interpreted to be cardiotoxicity since these animals subsequently died. On the other hand, sensitization or intensification of behavioral stereotypy occurred during the first 2 days followed by complete tolerance to this effect by day 5. No withdrawal phenomena were noted 24 h after cocaine was abruptly withdrawn. Plasma concentrations of cocaine rose rapidly during the first day and remained elevated at a constant level until day 5. Then, a sharp decline in plasma levels occurred at day 6 which remained depressed for the duration of the infusion.(ABSTRACT TRUNCATED AT 250 WORDS)

The acute and chronic effects of cocaine were evaluated on the 5-hydroxytryptamine (5-HT)-receptor 5-HT2 mediated behavioral function, the head-twitch response (HTR), in mice. In a recent study, we reported that the (+/-)-1-(2,5-dimethoxy-4-iodophenyl)-2-aminopropane HCl (DOI)-induced HTR was dose dependently reduced by cocaine via indirect stimulation of serotonergic 5-HT1A and adrenergic alpha 2 receptors. In the present investigation, the HTR was evoked by the nonselective 5-HT agonist 5-methoxy-N,N-dimethyltryptamine hydrogen oxolate (5-MeO-DMT). Cocaine by itself failed to produce HTR but dose dependently inhibited the 5-MeO-DMT-induced behavior. Cocaine's effects were not due to 5-HT3 antagonism since acute administration of the more potent 5-HT3 antagonist (ICS-205,930) failed to produce or modify the 5-MeO-DMT-induced behavior. During withdrawal from chronic cocaine treatment (5-20 mg/kg), 5-MeO-DMT-induced HTR was enhanced. Depending upon the cocaine dose used, the induced supersensitivity persisted up to 172 h following cessation of cocaine treatment. The mechanisms of cocaine-induced supersensitivity were further investigated using the more selective 5-HT2 agonist DOI. Withdrawal from a low-dose (0.03-1.25 mg/kg) chronic cocaine treatment caused the DOI-induced HTR to increase, whereas withdrawal from a 5- and 10-mg/kg cocaine regimen had no significant effect. The maximal effect persisted up to 36 h following termination of cocaine treatment. Relative to vehicle-exposed controls, withdrawal from cocaine treatment enhanced the inhibitory potency of the 5-HT1A agonist (+-)-8-hydroxy-2-(di-n-propylamino)tetralin HBr (8-OH-DPAT) on DOI-induced HTR.(ABSTRACT TRUNCATED AT 250 WORDS)

Collagen fibres in suspension have been shown to inhibit adenylate cyclase in human platelet preparations. Direct inhibition by collagen fibres was observed when intact platelets were used, although secondary events such as ADP secretion or prostanoid formation were important contributors to the inhibition of adenylate cyclase after treatment of platelets with collagen. The nature of the direct inhibition caused by collagen has been investigated in platelet membrane preparations, with the following results. (1) Collagen fibres inhibit platelet membrane adenylate cyclase in a dose-dependent manner. (2) Inhibition of adenylate cyclase by thrombin, adrenaline or collagen fibres could be abolished in the presence of guanosine 5'-[beta-thio]diphosphate; half-maximal inhibition was obtained at about 100 microM for the inhibitory action of thrombin, and at about 500 microM for that of either adrenaline or collagen. (3) The action of each ligand was blocked to a similar extent by pertussis-toxin treatment of the platelet membranes. Taken together, these results indicate that the action of collagen, like that of thrombin and adrenaline, is G-protein-dependent. (4) inhibition of adenylate cyclase by collagen fibres was additive with that caused by adrenaline, but co-operative with that caused by thrombin, suggesting that inhibitory pathways exists for collagen and adrenaline which are distinct from, but interactive with, that for thrombin. (5) Modification of the collagen fibres by pepsin treatment attenuated the effects of collagen, whereas heat-denaturation of the collagen fibres completely abolished their effects. These data suggest that the effects of collagen are specific, and depend on the detailed structure of the collagen fibres.

Fractional absorption of calcium from bread made either from intrinsically or extrinsically labeled whole-wheat flour was compared in 11 healthy adult women. The intrinsic label was provided by 45Ca injected individually into stems of wheat plants during growth. The extrinsic tag was introduced by adding 45Ca to unlabeled flour via the water used in dough making. The two labeled breads were tested in a randomized crossover design using a standardized breakfast administered after an overnight fast. Approximately 80 g labeled bread was consumed by each subject, providing a total calcium load of 13.3 mg. Fractional absorption from the intrinsically labeled bread averaged 0.812 +/- 0.130 (mean +/- SE) and from the extrinsically labeled bread 0.792 +/- 0.113. The mean difference, within subject, was only 0.025 +/- 0.016 and was not significantly different from zero. Extrinsic labeling of the calcium of whole-wheat flour results in a degree of labeling homogeneity equivalent to that of intrinsic labeling, at least for a leavened bread product.

The cataleptogenic effect of delta 9-THC was compared to its discriminative stimulus effects in rats. The ED50s for the discriminative stimulus and catalepsy were 0.8 and 4.0 mg/kg, respectively, while their time courses were very similar. The ED50 of delta 9-THC for catalepsy in experimentally naive rats was not different from that in rats trained with the drug discrimination procedure, indicating that the cataleptogenic effect was not appreciably attenuated by long-term exposure to low doses of delta 9-THC. Pharmacologically, the catalepsy produced by delta 9-THC more closely resembled that of haloperidol than of morphine, since anticholinergic pretreatment eliminated the delta 9-THC-induced catalepsy while pre-treatment with naloxone had no effect. Although the cataleptogenic effect of delta 9-THC could be pharmacologically manipulated by anticholinergic pre-treatment, its discriminative stimulus effects were not changed in the same animals. These results demonstrate that distinctive mechanisms of action exist for these cannabinoid-induced behaviors.

Objective: To review the potential for anaphylactoid reactions to intravenously administered acetylcysteine when used in the treatment of acetaminophen overdose. This case is unique in that electrocardiographic changes, including ST segment depression and T-wave inversion were associated with the episode and complicated the diagnosis., Data Sources: Reference articles and letters are identified in the text., Data Synthesis: Intravenous administration of acetylcysteine has been used in the treatment of acetaminophen overdose. This route may be considered in some clinical situations where oral therapy is complicated. Anaphylactoid reactions, including cutaneous eruptions, flushing, chest pain, tachycardia, and fever have been reported in up to three percent of patients receiving intravenous acetylcysteine. The nature of these reactions and evidence concerning their etiology suggest a histamine-release phenomenon. Response to intervention with antihistamines and the safety of further acetylcysteine administration are discussed., Conclusions: This case illustrates a variant anaphylactoid reaction to intravenously administered acetylcysteine and emphasizes the need for practitioners to consider the potential for these reactions prior to initiation of therapy and indicates appropriate treatment of these reactions.

Opening of the pyran ring of delta 9-tetrahydrocannabinol (THC) produces cannabidiol, a bicyclic cannabinoid devoid of many pharmacological properties produced by delta 8-THC or delta 9-THC. Interestingly, the bicyclic compound CP-47,497 (VI) has been described as producing many of the pharmacological effects produced by delta 9-THC, and another related bicyclic analog CP-55,940 (XIV) has been used to successfully define a cannabinoid binding site. A series of 16 bicyclic analogs of VI and XIV were evaluated and compared with the pharmacological profile of cannabidiol, delta 8-THC and delta 9-THC. The goals of the studies described herein were to determine whether these bicyclic analogs possess similar pharmacological properties of delta 9-THC, to compare pharmacological activity after s.c. and i.v. administration, and to evaluate the structure-activity relationship of this series of analogs for further insight into cannabinoid mechanism of action. Each analog was evaluated for its ability to produce hypoactivity, hypothermia, antinociception and catalepsy in mice. The ED50 values generated from these assays were averaged to provide an index of activity. The ED50 values for delta 9-THC varied from 1.0 to 1.5 mg/kg, giving an overall index of activity of 1.3. The index for delta 8-THC was 6.0, making this isomer 4-fold less potent. Although several bicyclic analogs (V, VI, VII, VIII, XI, XII, XIV and XVI) proved to be truly cannabimimetic, three (IV, IX and X) were sufficiently unique to be classified as noncannabimimetic. The index of activity of cannabimimetic bicyclic analogs varied from 0.2 to 2.2, although some minor differences between the bicyclics and delta 9-THC exist.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of a 14-day (gestation days 7-20) chronic methadone (6.3-9.0 mg/kg/day) infusion via osmotic minipumps were studied on the induction of physical dependence in both pregnant and nonpregnant female rats. Following continued methadone exposure, an acute injection of naloxone (2.0 mg/kg, SC) produced the following symptoms of withdrawal in both pregnant and nonpregnant methadone-exposed rats: increased frequency of head shakes, teeth-chattering and face-rubbing episodes, as well as the induction of burrowing, diarrhea, facial tremor, squeaking and vaginal sniffing. Increased fetal movement in the maternal abdomen was also observed in the pregnant rats. In the saline-exposed pregnant controls, naloxone failed to induce a significant effect. In addition, brain and plasma methadone levels during the various stages of pregnancy (gestation days 8-20) were determined. The methadone levels in plasma were initially variable (gestation days 8-12) but became more constant (approximately 50 ng/ml) from gestation day 14 to 20. Methadone brain levels also followed a similar pattern, except that the brain methadone content was at least 20-fold greater than plasma concentrations at any given time. Thus, relative to the high brain levels, the present data suggest that acute changes in methadone plasma concentration may not be a good index of pharmacological effect.

The compound 9-beta-hydroxy-hexahydrocannabinol [(-)-9 beta-OH-HHC] was designed to fit a combined theoretical profile of an analgesic cannabinoid (equatorial alcohol at C-9, phenol at C-1 and a C-3 side chain) with reduced psychoactivity (axial C-9 substituent which protrudes into the alpha face). (-)-9 beta-OH-HHC was synthesized by the addition of methyl Grignard to 9-oxo-11-nor-HHC. Its alpha epimer was obtained by the regiospecific epoxide ring opening of 9 alpha, 10 alpha-epoxy-HHC acetate. (-)-9 beta-OH-HHC and (-)-9 alpha-OH-HHC were each evaluated in a battery of tests in mice and were found to be 10-25 times less potent than (-)-trans-delta 9-tetrahydrocannabinol (delta 9-THC) in all tests including the tail flick test for antinociception (analgesia). Molecular mechanics calculations [MMP2(85)] revealed that, in the global minimum energy conformation of (-)-9 beta-OH-HHC, the axial methyl at C-9 protrudes into the alpha face of the molecule, while the axial hydroxyl at C-9 in (-)-9 alpha-OH-HHC protrudes into this same face. These calculations also identified a higher energy carbocyclic ring (twist) conformer of each in which there is no protrusion of a C-9 substituent of the carbocyclic ring into the alpha face. The minimal activity of both compounds is attributed to these higher energy forms.(ABSTRACT TRUNCATED AT 250 WORDS)

-(-)-5'-Bromo-delta 8-THC, (-)-5'-trifluoromethyl-delta 8-THC, (-)-5'-iodo-delta 8-THC, (-)-5'-fluoro-delta 8-THC, (-)-11-fluoro-delta 8-THC and (-)-2-iodo-delta 8-THC were synthesized and evaluated in male ICR mice for their effects on sedation, temperature, catalepsy and antinociception following intravenous injection. The analogs were also tested for relative affinities for cannabinoid binding sites derived from rat cortex membranes, using [3H] CP-55,940 as the tritiated ligand. The results showed that the 5'-bromo, 5'-iodo and 5'-trifluoromethyl analogs were 2-40 times more potent than (-)-delta 8-THC in all biological tests, while the 5'-fluoro and 11-fluoro derivatives were less active. With the 2-iodo analog, a 12-fold separation was observed between antinociception and sedation, pointing to the importance of the side chain orientation in determining cannabinoid activity and to the possible involvement of more than one cannabinoid receptor site. The pharmacological data closely paralleled the data obtained from the binding assay.

The structure-activity relationship studies that have been reported for cannabinoids suggest that 1) the conformation of the C-ring at the C9 position, 2) the A-ring phenolic hydroxyl, and 3) the hydrophobic side chain are important determinants for the production of analgesia, as well as other cannabinoid effects. However, either these previous structure-activity studies described for cannabinoid compounds have not been quantitative in nature or the prediction of the activity of known and unknown compounds based on molecular structure has not been tested in a comprehensive manner. In this study we describe a three-dimensional molecular modeling program using comparative molecular field analysis to derive quantitative structure-activity relationships fitting pharmacological potencies and binding affinities of cannabinoids. The analysis has proven to accurately fit the pharmacological activity of cannabinoid analogs, with cross-validated r2 values of greater than 0.3 and final analysis r2 values of greater than 0.88. Additionally, this study has further characterized the steric and electrostatic properties that account for the variations in their potency. The results from this study indicate that steric repulsion behind the C-ring is associated with decreased predicted binding affinity and pharmacological potency. On the other hand, the steric bulk of a side chain that is extended up to seven carbons contributes to predictions of increased binding affinity and potency. The electrostatic fields of cannabinoid analogs also contribute to the predicted in vitro and in vivo potencies. If the biological activities we have investigated are assumed to be the result of interaction with a single binding site, this method indicates the structural and physicochemical properties necessary for binding to the receptor and producing an effect. By defining cannabinoid binding affinity and behavioral activity pharmacophores, this method can be used for designing cannabinoid agonists and it is capable of predicting the activity of unknowns, thereby serving to facilitate rational drug design.

Numerous cannabinoids have been synthesized that are extremely potent in all of the behavioral assays conducted in our laboratory. An important feature in increasing potency has been the substitution of a dimethylheptyl (DMH) side chain for the pentyl side chain. Our previous studies have shown that (-)-11-OH-delta 8-THC-dimethylheptyl was 80-1150 times more potent than delta 9-THC. Stereospecificity was demonstrated by its (+)-enantiomer which was more than 1400-7500 times less potent. A related series of DMH cannabinoid analogs has recently been synthesized and preliminary evaluations reported here. (-)-11-OH-delta 9-THC-DMH was found to be equipotent with (-)-11-OH-delta 8-THC-DMH. The aldehyde (-)-11-oxo-delta 9-THC-DMH was 15-50 times more potent than delta 9-THC. Surprisingly, (-)-11-carboxy-delta 9-THC-DMH was also active, being slightly more potent than delta 9-THC. In the bicyclic cannabinoid series, the length and bulk of the side chain were found to be equally important. Aminoalkylindoles, which are structurally dissimilar from classical cannabinoids, have been found to exhibit a pharmacological profile similar to delta 9-THC. Though not extremely potent in vivo, they appear to represent an entirely new approach to studying the actions of the cannabinoids. The structural diversity and wide-ranging potencies of the analogs described herein provide the opportunity to develop a pharmacophore for the cannabinoids using molecular modeling techniques.

The primary goal of this research was to synthesize a series of ether analogues of the cannabinoid drug class and to evaluate their agonist and antagonist pharmacological properties in either the mouse or the rat. Agonist and antagonist activity was evaluated in mice using a multiple-evaluation procedure (locomotor activity, tail-flick latency, hypothermia, ring immobility) and activity in rats determined in a discriminative stimulus paradigm. Additionally, novel analogues were evaluated for their ability to bind to the THC receptor site labeled by 3H-CP-55,940. None of the cannabinoid analogues were capable of attenuating the effects of delta 9-THC (3 mg/kg) in either the rat (doses up to 10 mg/kg) or in the mouse (doses up to 30 mg/kg). It also appears that the compounds with minimal in vivo activity are not mixed agonist/antagonists. These data would suggest that the phenolic hydroxyl is important for receptor recognition (binding) and in vivo potency. Additionally, cannabinoid methyl ethers previously considered inactive have been found to produce limited activity. Lastly, data suggest that delta 9,11-THC is more potent than previous reports indicated, and does possess pharmacological activity.

Fractional calcium absorption from wheat products and the influence of co-ingested wheat products on calcium absorption from milk were measured in a series of randomized crossover studies in healthy adult women. The wheat had been intrinsically labeled with 45Ca during growth. In the first study, fractional calcium absorption from leavened whole-wheat bread averaged 0.817 +/- 0.124. By comparison, absorption from milk, ingested at a comparable load in the same women, averaged only 0.589 +/- 0.111. When labeled bread was co-ingested with milk, at the same aggregate load as for bread alone, bread calcium absorption fell to 0.748 +/- 0.103 (P less than 0.05). In a second study, calcium absorption from an extruded cereal prepared from intrinsically labeled wheat bran was compared with milk. Calcium absorption from the cereal (0.223 +/- 0.046) was significantly less than from milk (0.375 +/- 0.072) (P less than 0.001). When the two were co-fed at the same total load, milk calcium absorption fell to 0.258 +/- 0.055 (P less than 0.001). In a third study, the effect of phytate hydrolysis through yeast fermentation and of Maillard browning on calcium absorption was investigated using leavened bread and underbaked and overbaked cookies, each made with intrinsically labeled wheat flour. Calcium absorption from cookies was not affected by the extent of browning and averaged 0.652 +/- 0.087. However, calcium absorption from bread in these same women averaged 0.703 +/- 0.108. This was significantly more than from the cookies (P less than 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

The present study examined whether descending noradrenergic and serotonergic systems mediate the antinociceptive effect of the prototypical cannabinoid, delta-9-tetrahydrocannabinol (delta 9-THC). Rats were administered vehicle or delta 9-THC (10 mg/kg, i.v.) and subsequently given an intrathecal (i.t.) injection of either the alpha 2-noradrenergic antagonist yohimbine, or the non-specific serotonin (5-HT) antagonist, methysergide, through chronically implanted spinal catheters. Whereas yohimbine significantly reversed the cannabinoid-induced elevation of tail-flick latencies, methysergide had no effect. To examine whether yohimbine was indeed blocking the antinociceptive effects of delta 9-THC through a spinal mechanism, it was administered i.t. at either the lumbar or the upper thoracic level of the spinal cord. Antinociception was significantly reduced when yohimbine was administered in the lumbar region; however, administration in the upper thoracic region failed to have an effect. In addition, the effect of i.t. administered yohimbine and methysergide was assessed on two other indices sensitive to cannabinoids, hypothermia and ring immobility. As previously reported, i.v. administration of delta 9-THC led to hypothermia as well as immobility in the ring test which were not blocked by i.t. administration of either monoamine antagonist. To the contrary, methysergide potentiated the hypothermic effect of delta 9-THC. These findings indicate that cannabinoids activate descending noradrenergic neurons resulting in antinociception via the stimulation of spinal alpha 2-adrenoceptors.

The purpose of this study was to investigate whether cannabinoids produce antinociception spinal and supraspinal sites of action. The antinociceptive effect of delta 9-tetrahydrocannabinol (3 or 10 mg/kg), a naturally occurring cannabinoid, and CP-55,940 (0.1, 0.3, or 0.5 mg/kg), a potent synthetic cannabinoid, were assessed in spinally transected and intact rats. Each drug, administered i.v., produced a potent, long-lasting elevation of tail-flick latencies in the intact animals. This antinociception was significantly attenuated by spinal transection. Administration of each cannabinoid (i.t.) to the lumbar region of the spinal cord produced a weak, but long-enduring antinociceptive effect. In contrast, spinal administration of CP-55,940 to the upper thoracic region failed to elevate tail-flick latencies above base-line values. Additionally, i.t. administration of CP-55,940 (30 or 100 micrograms) continued to have a weak antinociceptive effect in spinal rats. In contrast, i.t. administration of CP-56,667, the (+)-enantiomer of CP-55,940, failed to elevate tail-flick latencies above base line at a dose of 1000 micrograms, thus indicating stereoselectivity. Finally, the biodisposition of 3H-delta 9-tetrahydrocannabinol after either i.v. or i.t. administration to spinal and intact rats was also assessed. The levels of radioactivity did not differ between spinal and intact animals in either whole brain, spinal cord, or plasma when the drug was administered i.v. When the drug was administered i.t., however, surgical transection of the spinal cord led to a decreased concentration of labeled substances in the whole brain and plasma. These converging lines of evidence indicate that cannabinoids produce antinociception through multiple mechanisms at the spinal and supraspinal levels of the central nervous system.

Leukotriene B4 is one of a number of agents which stimulate bone resorption by acting on osteoblasts. Some agonists, such as PTH or prostaglandins, are known to activate adenylate cyclase in osteoblasts, whereas others, such as vitamin D3, have no effect on adenylate cyclase. Recent evidence suggests that both classes of agonist may raise the intracellular calcium concentration, although the relative importance for bone resorption of calcium mobilization and adenylate cyclase activity in the osteoblast is not clear. Here it is shown 1) that leukotriene B4 does not activate but may be inhibitory toward adenylate cyclase in intact osteoblasts or membrane preparations, 2) that leukotriene B4 causes an elevation of intracellular calcium levels in osteoblast monolayers, 3) leukotriene B4 rapidly activates phosphatidylinositol bisphosphate breakdown in osteoblast membranes and intact osteoblasts, and 4) that leukotriene B4 stimulates phosphatidylinositol kinase activity concurrently with phosphoinositidase C in intact osteoblasts over a similar timescale. These results suggest that leukotriene B4 may increase the concentration of intracellular calcium in osteoblasts by stimulating phosphoinositide turnover, and support the proposal that calcium signaling rather than activation of adenylate cyclase in osteoblasts may be of overriding importance in the regulation of bone resorption.

1. Incubation of human platelet membranes with guanosine 5'-[beta gamma-imido]triphosphate (p[NH]ppG) causes a time-dependent increase in the activation of adenylate cyclase due to Gs (the stimulatory GTP-binding protein). Forskolin enhances adenylate cyclase activity but does not interfere with the process of activation. The activation follows first-order kinetics in both the presence and the absence of the assay components. 2. ATP in the presence or the absence of an ATP-regenerating system of phosphocreatine and creatine kinase inhibits activation. 3. Hydrolysis of ATP to ADP does not lead to receptor-mediated inhibition of adenylate cyclase acting via Gi (the inhibitory GTP-binding protein). The ADP analogue adenosine 5'-[beta-thio]diphosphate (ADP[S]) does not inhibit the activation process. 4. Phosphocreatine alone inhibits adenylate cyclase activation at concentrations above 1 mM. 5. Inhibition by phosphocreatine is not due to the chelation of free Mg2+ ions. 6. Inhibition by ATP and the other assay components occurs throughout the activation process, decreasing both the rate of activation and the maximum activity obtained. 7. Maximal activation of adenylate cyclase after prolonged incubation with p[NH]ppG slowly reverses in the presence of the assay components. 8. A 10-fold excess of the GDP analogue guanosine 5'-[beta-thio]diphosphate (GDP[S]) over p[NH]ppG inhibits the activation process completely, at all stages of the time course. 9. Preincubations in the presence and absence of ATP, cyclic AMP, phosphocreatine and creatine kinase show equal sensitivity to increasing GDP[S] concentration. These data show that the inhibition observed in the presence of ATP is not due to endogenous or contaminating guanine nucleotides, and suggest that phosphoryl transfer may regulate adenylate cyclase activity.

Despite the great attention given to the pharmacological actions of cocaine in recent years, the mechanisms leading to acute intoxication and related deaths after cocaine smoking have not been fully elucidated. The present study was therefore undertaken to examine the pharmacokinetics and pharmacological effects of cocaine by the inhalation route. Cannulated, male Sprague-Dawley rats were exposed to a constant concentration of cocaine vapor (13.6 +/- 0.4 micrograms cocaine per ml mainstream air), during which time their biodispositional profile and cardiovascular responses were evaluated. The bioavailable doses of cocaine were 0.26 +/- 0.05 and 1.54 +/- 0.46 mg/kg, after 1.5- and 5.0-min exposures, respectively. Peak cocaine plasma concentrations of 95 +/- 26 and 205 +/- 58 ng/ml, for the 1.5- and 5.0-min exposures, respectively, occurred 1 min after the start of exposure. Increasing the duration of exposure significantly increased the bioavailability from 0.29 to 1.03 (P less than .05). Transient changes in heart rate and arterial blood pressure were generally dose-dependent and correlated temporally with peak cocaine plasma concentrations. During exposure, 70% of the animals demonstrated electrophysiological aberrations consistent with atrial arrhythmia and incomplete heart block. These findings suggest that a direct cardiotoxic effect resulted from inhalation of cocaine.

Nine analogues of mecamylamine (2) which differ in the number and substitution pattern of methyl groups, were prepared. In four of these analogues the amine functionality is in an endo orientation. Enantiomers of 2-endo- and 2-exo-N-methylfenchylamine (25 and 26, respectively) were also prepared. The hydrochloride salts of these compounds were tested for nicotinic antagonism relative to mecamylamine in vivo and none was found to be as potent as mecamylamine, although a broad range of activity was observed. In general, methyl substituents at the C1, C2, and C7 positions of the mecamylamine structure do not appear to be significant for antagonistic activity. Methyl substituents at C3, however, appear to be very important for activity. Three sets of enantiomers of N-methylfenchylamine analogues, 28-30, possessing structural features of mecamylamine and nicotine were also prepared. These compounds were inactive as antagonists. Only a small degree of stereoselectivity was elicited in this series, less than that seen with enantiomers of nicotine. Antagonists with the exo N-methylamine functionality are slightly more active than the endo isomers. The extent to which structural modification might change lipophilicities was estimated through calculated partition coefficients; such changes alone appeared insufficient to explain differences in activities of the analogues. Lastly, a tolerance for a tertiary (dimethyl) amine functionality was demonstrated in addition to the lack of tolerance for bulkier substituents at C3 or on the nitrogen.

Cocaine inhibits the 5-HT2-mediated (+/-)-DOI-induced head-twitch response (HTR) in mice in a dose-dependent manner. In order to investigate the possible inhibitory mechanism(s) of cocaine on 5-HT2 receptor function, we studied the effects of the selective adrenergic alpha 2 receptor antagonist yohimbine and the beta-adrenergic/5-HT1 receptor antagonist alprenolol, and the 5-HT3 antagonist ICS 205-930 on the inhibitory action of cocaine on the (+/-)-DOI-induced HTR. Neither yohimbine (0.1 and 0.5 mg/kg) nor alprenolol (10 mg/kg) pretreatment had any significant effect on the (+/-)-DOI-induced HTR. However, both antagonists prevented the inhibitory effects of cocaine on the (+/-)-DOI-induced HTR. The 5-HT3 antagonist ICS 205-930 neither produced HTR nor decreased the (+/-)-DOI-induced HTR frequency. The present results suggest that cocaine inhibits 5-HT2 receptor function by increasing the synaptic concentration of norepinephrine and serotonin via inhibition of their uptake and thus indirectly stimulating the respective inhibitory adrenergic alpha 2 and serotonergic 5-HT1A receptors. Furthermore, cocaine's 5-HT3 antagonist properties appear not to play a role in the inhibition of head-twitch behavior.

In order to establish a functional role for the various populations of serotonin (5-HT) receptors, behavioral studies have been conducted over the past decade with serotonergic agonists and antagonists. And, although there is reason to believe that certain behavioral effects may be mediated via particular populations of 5-HT receptors, evidence now suggests that some serotonin-mediated behaviors may be modulated by the interaction of serotonergic agents at multiple subtypes of 5-HT receptors. The generality of these effects, and the exact mechanism(s) by which they occur, have yet to be elucidated. Nevertheless, over the past year, results from several different laboratories provide a growing recognition of this novel phenomenon.

Computer competition analysis of 3H-DHA (3H-dihydroalprenolol, a nonselective beta-adrenergic radioligand) binding in the presence of unlabeled metoprolol (a beta 1-selective antagonist) indicates the existence of both beta 1- and beta 2-adrenergic receptor subtypes in the rat placenta and confirms previous reports that both beta-adrenoceptors are present in adult rat cortex. In the fetal brain (20th day of gestation), however, only beta 1-receptors were detected. Pregnant rats were chronically exposed to methadone from day 7 to day 20 of gestation via implanted osmotic minipumps (6.3-9.0 mg/kg/day). This treatment schedule did not induce a change in the affinity and density of either beta-receptor subtype in the placental, fetal and maternal brain homogenates. The results are discussed in terms of the reported monoaminergic and opiate receptor functional interactions.

The effects of delta 9-THC and other cannabinoids on cAMP levels in synaptosomes from mouse brains were investigated in order to determine whether cannabinoids produced their behavioral effects through alterations in adenylate cyclase. delta 9-THC (0.01-10 microM) did not significantly alter basal cAMP levels, whereas delta 9-THC and other cannabinoids were able to alter forskolin-stimulated cAMP levels in synaptosomes. In general, three kinds of responses were observed. Some cannabinoids displayed a modest, concentration-dependent decrease in cAMP levels, producing significant inhibition between 1-10 microM. Other cannabinoids, including delta 9-THC and delta 8-THC, appeared to produce a biphasic effect in that inhibition of cAMP was observed only at a single concentration. Finally, some analogs were unable to significantly alter forskolin-stimulated cAMP. There was not a clear relationship between the ability of the cannabinoids to alter cAMP levels in synaptosomes and the behavioral effects observed in mice. However, it was demonstrated that the analogs which are the most potent in producing cannabimimetic effects in mice were the analogs which inhibited cAMP in a concentration-dependent manner. While cannabinoids were able to alter cAMP levels in synaptosomes, the ability to alter cAMP levels does not appear to be absolutely necessary for the production of cannabinoid effects in mice.

Structure-activity relationship studies have suggested that the phenolic hydroxyl group is essential for the pharmacological activity of the cannabinoids. However, it remains to be established whether it is the hydrogen of the phenolic hydroxyl that is important (possibly because this hydrogen can participate in a hydrogen bonding interaction) or whether it is the oxygen of the phenolic hydroxyl that is important (possibly because one of the lone pairs of electrons in this oxygen can serve as a hydrogen bond acceptor). Two new etherified cannabinoids were prepared in which the phenolic hydroxyl oxygen is incorporated into a fourth ring. These new compounds were designed to test the importance both of the phenolic hydroxyl oxygen and of the orientation of its lone pairs of electrons for cannabinoid pharmacological activity. O,2-Propano-delta 8-tetrahydrocannabinol (0,2-Propano-delta 8-THC) was designed to mimic delta 9-THC in its phenol conformation I (C2-C1-O-H = 7 degrees). O,10-Methano-delta 9-tetrahydro-cannabinol (0,10-Methano-delta 9-THC) was designed to mimic delta 9-THC in its phenol conformation II (C2-C1-O-H = 167 degrees). Molecular mechanics calculations revealed that 1) there are two accessible minimum energy conformers for O,2-propano-delta 8-THC, which differ principally in the conformation of the new fourth ring, and 2) there are three accessible minimum energy conformers for O,10-methano-delta 9-THC, the first two of which differ mainly in the conformation of the new fourth ring, whereas the third possesses an alternate pyran ring conformation. Wave functions and molecular electrostatic potential (MEP) maps were calculated for each accessible conformer of O,2-propano-delta 8-THC and of O,10-methano-delta 9-THC. The resultant MEP maps compared well with the corresponding MEP maps generated for delta 9-THC in each of its two minimum energy conformations (two phenolic hydroxyl positions). These results imply that 1) O,2-propano-delta 8-THC should be capable of being recognized at a site that would recognize delta 9-THC in its phenol conformation 1 and 2) O,10-methano-delta 9-THC should be capable of being recognized at a site that would recognize delta 9-THC in its phenol conformation II. Pharmacological evaluation of the analogs revealed that O,10-methano-delta 9-THC was inactive in all mouse tests, as well as the rat drug discrimination model. O,2-Propano-delta 8-THC was similar to delta 8-THC in that it depressed rectal temperature and produced antinociception and ring immobility in mice.(ABSTRACT TRUNCATED AT 250 WORDS)

The oxidative metabolism of CP-55,940 [(-)-cis-3-[2-hydroxy-4-(1,1-dimethylheptyl)phenyl]-trans-4-(3- hydroxypropyl)cyclohexanol] was studied in mouse liver S-9 microsomal preparations. [3H]CP-55,940 was incubated in a microsomal supernatant enriched with the appropriate cofactors for cytochrome P-450 oxidative metabolism. HPLC separation of petroleum ether/diethyl ether (1:1) extracts facilitated the identification of metabolites by GC/MS after derivatization with BSTFA or [2H18]BSTFA. The mass spectral data indicated that five monohydroxylated metabolites had been formed that differed with respect to the position of hydroxylation on the 1',1'-dimethylheptyl side chain. Two additional compounds were detected whose mass spectral data suggested that these metabolites were hydroxylated at two positions on the side chain. Side chain hydroxylation is consistent with the metabolic profile of delta 9-tetrhydrocannabinol (delta 9-THC) and other cannabinoid compounds. It is possible that these side chain-hydroxylated metabolites retain activity, as is the case with similar metabolites formed from delta 9- and delta 8-THC, and thereby contribute to the pharmacological profile seen with this potent synthetic cannabimimetic agent.

delta 9-Tetrahydrocannabinol, the primary psychoactive constituent in marihuana, has been studied extensively for the last 20 years; however, the mechanisms responsible for cannabinoid activity in the central nervous system are not well understood. Although it is thought that lipophilicity plays an important role in the actions of cannabinoids, studies have not been conducted to determine whether a relationship exists between the lipophilicity and behavioral potency of cannabinoid analogs. Two procedures were used to obtain n-octanol/water partition coefficients (Po/w) of naturally occurring and synthetic cannabinoids: reverse-phase high-pressure liquid chromatographic estimation of Po/w and computer calculation of Po/w based on molecular structure. The Po/w value for delta 9-tetrahydrocannabinol obtained in this study, 9.44 x 10(6), is much greater than previously reported values obtained using shake-flask methodology, yet it is in agreement with the computer calculation based on molecular structure or molecular volume. The lipophilicity of the analogs determined in this study ranged from Po/w values of 3.92 x 10(2) to 1.93 x 10(11). All pharmacologically active cannabinoid compounds were extremely lipophilic (log Po/w values greater than 4.5). Several structural alterations were found to exert considerable influence on the lipophilicity of cannabinoid analogs. Increasing the length of the side chain in a homologous series of analogs results in an increase in lipophilicity of approximately 3-fold for each CH2 group added. Introduction of a single hydroxyl group decreased lipophilicity 3- to 40-fold, depending on the site of attachment. The behavioral potency of active analogs was not found to be correlated to lipophilicity. Therefore, data obtained in this study suggest that lipophilicity is a component, but not a primary determinant of pharmacological activity in the cannabinoids.