Synthesis and preliminary binding studies of 4,4-ditritio-(-)-nicotine of high specific activity.

4,4-Ditritio-(-)-nicotine (5) of high specific activity (4.7 Ci/mmol) has been synthesized from (-)-nicotine via the readily prepared 4,4-dibromocotinine (3). Scatchard analysis of the binding of 5 to the crude mitochondrial fraction of whole rat brain revealed a Ka of 4.7 X 10(6) M-1 and 13 fmol of binding sites/mg of protein.

Synthesis and in vitro and in vivo activity of (-)-(1R,5R,9R)- and (+)-(1S,5S,9S)-N-alkenyl-, -N-alkynyl-, and -N-cyanoalkyl-5, 9-dimethyl-2'-hydroxy-6,7-benzomorphan homologues.

Two of the synthesized (-)-(1R,5R,9R)-N-homologues (N-but-3-enyl- and N-but-3-ynyl-5,9-dimethyl-2'-hydroxy-6,7-benzomorphan (9, 13)) were found to be about 20 times more potent than morphine in the mouse tail-flick assay (ED(50) = 0.05 mg/kg), and (-)-(1R,5R, 9R)-N-but-2-ynyl-5,9-dimethyl-2'-hydroxy-6,7-benzomorphan ((-)-(1R, 5R,9R)-N-but-2-ynylnormetazocine, 12) was about as potent as the opioid antagonist N-allylnormetazocine (AD(50) in the tail-flick vs morphine assay = 0.3 mg/kg). All of the homologues examined had higher affinity for the kappa-opioid receptor than the mu-receptor except (-)-N-but-2-ynyl-normetazocine (12), which had a kappa/mu ratio = 7.8 and a delta/mu ratio = 118. The (-)-N-2-cyanoethyl (3), -allyl (8), and -but-3-ynyl (13) analogues had good affinity (<10 nM) for delta-opioid receptors. Two homologues in the (+)-(1S,5S,9S)-normetazocine series, N-pent-4-enyl (24) and N-hex-5-enyl (25), were high-affinity and selective sigma(1)-ligands (K(i) = 2 nM, sigma(2)/sigma(1) = 1250, and 1 nM, sigma(2)/sigma(1) = 750, respectively); in contrast, N-allylnormetazocine (22) had relatively poor affinity at sigma(1), and its sigma(1)/sigma(2) ratio was <100.

Antipodal alpha-N-(methyl through decyl)-N-normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans): in vitro and in vivo properties.

The enantiomeric (-)- and (+)-N-(methyl through decyl) normetazocines (5,9 alpha-dimethyl-2'-hydroxy-6,7-benzomorphans) were synthesized and their in vitro and in vivo activities determined. Increasingly bulky enantiomeric N-alkyl homologs were prepared until their interaction with the sigma 1 receptor decreased and their insolubility became a hindrance to their evaluation in vivo and/or in vitro. The (-)-methyl, -pentyl, -hexyl, and -heptyl homologs were essentially as potent as, or more potent than, morphine in the tail-flick, phenylquinone, and hot-plate assays for antinociceptive activity; the (-)-propyl homolog had narcotic antagonist activity between that of nalorphine and naloxone in the tail-flick vs morphine assay, and it also displayed antagonist properties in the single-dose suppression assay in the rhesus monkey. The antinociceptively potent (-)-heptyl homolog did not substitute for morphine in monkeys but did show morphine-like properties in a primary physical-dependence study in continuously infused rats. All five potent compounds showed high affinity for the mu opioid receptor from both rat and monkey preparations and the kappa opioid receptor (< 0.05 microM), and all except the (-)-methyl homolog interacted reasonably well at the delta receptor (K(i) < 0.1 microM). The (-)-propyl compound was equipotent (K(i) 1.5-2.0 nM) at mu and kappa receptors. The pattern of interaction of the (-)-enantiomeric homologs with mu receptors from rat and monkey preparations was similar, but not identical. The enantioselectivity of the homologs for mu receptors was greater in the rat than in the monkey preparation for all but the N-H and butyl compounds, and the enantioselectivity of the lower homologs (methyl through butyl) for the mu (monkey) receptor was greater than for the kappa or delta receptors. However, bulkier homologs (hexyl through decyl) displayed higher enantioselectivity at kappa or delta receptors than at the mu (monkey) receptor. The (+)-butyl through (+)-octyl homologs were essentially equipotent with, or more potent than, (+)-pentazocine at the sigma receptor. Only the (+)-H and (+)-methyl homologs had high affinity (< 0.05 microM) at PCP binding sites.

Common stereospecificity of opioid and dopamine systems for N-butyrophenone prodine-like compounds.

The two optical isomers of 1-[3-(p-fluorobenzoyl) propyl]-3-methyl-4-phenyl-4-propionoxypiperidine (FPP) were obtained by resolution of (+/-)-r-3-methyl-4-phenyl-c-4-piperidinol followed by N-alkylation and O-propionylation. These, as well as the racemate, were evaluated for their antinociceptive, opioid, and neuroleptic properties using in vivo and in vitro test systems. The results are remarkable in two respects, namely, the dextrorotatory isomer is consistently the most potent on all tests, and it acts on both opioid (mu) and neuroleptic (D2) receptors.

3-Alkyl ethers of clocinnamox: delayed long-term mu-antagonists with variable mu efficacy.

In recent years there has been considerable interest in the relationship between clocinnamox (C-CAM) and its methyl ether methoclocinnamox (MC-CAM). While C-CAM appears to be an insurmountable mu-antagonist, MC-CAM has been shown to be a potent partial agonist at mu-opioid receptors. To further investigate this relationship we prepared other ethers of C-CAM and evaluated these in opioid receptor binding assays and in vivo in mouse antinociceptive assays and in morphine-dependent monkeys. In opioid binding assays, the ethers were generally mu-selective with affinity equivalent to that of C-CAM itself. Although they displayed little or no efficacy in vitro, some of the ethers showed substantial agonist activity in the in vivo antinociceptive tests. Two of the ethers, the propargyl ether 7 and the cyclopropylmethyl ether 5, were chosen for more detailed analysis in vivo. 7 was shown to have significant mu-agonist character and was able to substitute for morphine in morphine-dependent monkeys. Interestingly, when this agonist effect abated, 7 displayed long-lasting mu-antagonism. In contrast, 5 displayed little agonist activity in vivo and was characterized as a potent, long-acting mu antagonist. Although further work is needed to determine whether metabolism is a crucial factor in determining the pharmacological profile of these ethers, it is clear that 3-O-alkylation is a useful means of varying the mu efficacy displayed by this class of acyl-substituted 14-aminomorphinones. MC-CAM itself has generated considerable interest as a potential pharmacotherapy for opiate abuse. These analogues with differing mu efficacy but retaining the long-lasting mu-antagonist effects provide further opportunities for the development of treatment drugs.

Blockade of dynorphin A(1-13) overt psychomotor effects by naloxone.

Each of the dynorphin A(1-13) or dynorphin (dyn) treatment groups receiving naloxone showed a significant overall reduction of overt signs compared with the dyn controls. The data suggested that the overt psychomotor effects of dyn in the rhesus monkey were especially prone to blockade by naloxone, and probably involved opioid mechanisms.

Intracerebellar nicotinic-cholinergic participation in the cerebellar adenosinergic modulation of ethanol-induced motor incoordination in mice.

Many epidemiological studies have suggested a high correlation between the use of tobacco and ethanol, the two most frequently abused psychoactive drugs. Recently, we reported behavioral interactions between (-)-nicotine, (-)-cotinine and ethanol within the CNS. The present report is a confirmation and an extension of that study. Using a 2 g/kg ethanol-induced motor incoordination (EIMI) as the test response, possible behavioral interactions between (-)-nicotine, (-)-cotinine and ethanol and between (-)-nicotine, (-)-cotinine and adenosine agonist + ethanol in the cerebellum were investigated. (-)-Nicotine, 0.625, 1.25 and 5 ng intracerebellarly (ICB) significantly attenuated EIMI in a dose-related manner. Likewise, ICB injection of 1.25, 2.5, and 5 ng (-)-cotinine, a major metabolite of nicotine, significantly attenuated EIMI after the same i.p. dose of ethanol as in case of (-)-nicotine but less markedly compared to (-)-nicotine. No change in normal motor coordination was observed when the highest dose of (-)-nicotine or (-)-cotinine was injected ICB followed by saline control, suggesting selectivity of their behavioral interactions with ethanol. The attenuation of EIMI by (-)-nicotine and (-)-cotinine was blocked by ICB hexamethonium (1 microgram) and trimethaphan (100 ng), the purported nicotinic-cholinergic antagonists. Finally, the ICB injection of adenosine agonists, N6-cyclohexyladenosine (CHA) or 5'-N-ethylcarboxamidoadenosine (NECA), produced marked accentuation of EIMI which was significantly antagonized by ICB (-)-nicotine and (-)-cotinine. The data obtained in the present study suggested, for the first time, a cerebellar adenosinergic-nicotinic cholinergic interaction and modulation of EIMI. The data also suggested participation of cerebellar nicotinic-cholinergic receptors in EIMI.

Nicotine's opioid and anti-opioid interactions: proposed role in smoking behavior.

Nicotine produced antinociception in mice which was antagonized noncompetitively by naloxone. In addition, at significantly lower doses, nicotine noncompetitively antagonized morphine-induced antinociception. A speculative suggestion regarding the opiatergic and anti-opiatergic actions of nicotine is that it significantly promotes and maintains smoking behavior.

Etorphines: mu-opioid receptor-selective antinociception and low physical dependence capacity.

Comparative analgesic studies revealed that dihydroetorphine was more potent than etorphine in the tail-flick and hot-plate tests, respectively and nearly equipotent in the phenylquinone assay. Both compounds were short acting. Studies with selective opioid receptor antagonists beta-funaltrexamine, nor-binaltorphimine and naltrindole revealed that both etorphines were mu-selective agonists. Presumptive evidence for competitive antagonism of these compounds with naloxone was provided by Schild regressions with slopes of near unity. In a suppression test in rhesus monkeys maximally dependent on morphine, dihydroetorphine and etorphine dose-dependently replaced morphine. Drug-naive simians chronically exposed to frequent, intermittent and escalating doses of dihydroetorphine for 42 days showed few withdrawal signs when challenged with large doses of naloxone or were abruptly withdrawn from this drug. The results suggest that these atypical opioids may be useful in the clinical treatment of pain and opiate drug abuse.

Central behavioral interactions between ethanol, (-)-nicotine, and (-)-cotinine in mice.

A high correlation between alcohol use and smoking has long been suggested by epidemiological data. We examined the potential behavioral interactions between ethanol and nicotine using ethanol-induced motor incoordination as the test response in mice. Effect of pretreatment of various doses of (-)-nicotine, (-)-cotinine, a major metabolite of nicotine, and (+)-nicotine administered ICV on ethanol (IP)-induced motor incoordination was investigated. (-)-Nicotine (0.19, 0.38, 0.77 and, 1.54 nmoles ICV) produced significant attenuation of motor incoordination due to ethanol (2 g/kg IP) in a nearly dose-related manner which was blocked by ICV hexamethonium and trimethaphan, both purported nicotinic antagonists. (-)-Cotinine (0.35, 0.70, 1.41 nmole ICV) produced similar attenuation but was les potent than (-)-nicotine. Attenuation by (+)-nicotine (0.19, 0.38, and 0.77 nmoles ICV) was also significant but only at 0.77 nmole dose level. (+)-Nicotine-induced attenuation of motor incoordination by ethanol was antagonized by nicotinic antagonists. Data obtained suggest a central behavioral interaction between ethanol and nicotine at least through the participation of cholinergic nicotinic receptors.

Automated radioimmunoassay of nicotine.

We have developed an automated nonequilibrium procedure for the radioimmunoassay of nicotine. The use of a unique iodinated nicotine derivative in this procedure gave a sensitivity of 10 micrograms/l for nicotine with a between-run precision of 7.4% and within-run precision of 6.0%. Nicotine levels of 60 to 67 micrograms/ml were found in subjects 15 min after smoking one standard cigarette. The technique herein reported is a very rapid, and sensitive radioimmunoassay for nicotine and facilitates the determination of nicotine in smoking subjects during the actual process of smoking.

Comparison of the ability of opioid analgesics to increase endogenous opioid-like activity in the cerebrospinal fluid of rabbits.

We have previously demonstrated that the acute administration of morphine increases the level of endogenous substances, which have antinociceptive activity, in cerebrospinal fluid (CSF). The present study was conducted to determine whether other opioid analgesics exert a similar effect. CSF was withdrawn from the cisterna magna of anesthetized rabbits before and after s.c. injections of meperidine, pentazocine, levorphanol and methadone, and was bioassayed for opioid-like activity in the mouse tail-flick and phenylquinone writhing tests. The opioid-like activity of CSF taken 60 min after meperidine (50 mg/kg) was significantly increased in both bioassays, and the CSF level of meperidine was insufficient to account for this effect. Pentazocine (25-75 mg/kg) also significantly increased opioid-like activity in rabbit CSF, but the effects of methadone (5-10 mg/kg) and levorphanol (20 mg/kg) were less marked. Dextrorphan (20 mg/kg), diazepam (10 mg/kg) and pentobarbital (20 mg/kg) administration did not significantly increase opioid-like activity in CSF. It is concluded that the antinociceptive action of some opioid analgesics in rabbits may be mediated in part by the release of endogenous antinociceptive substances.

Cocaine, ecgonine methyl ester, and benzoylecgonine plasma profiles in rhesus monkeys.

From a public health point of view, cocaine (COC) presents serious clinical problems and deaths from overdose and lifelong addiction patterns, not to mention its involvement in crime, in the United States. This study subjected rhesus monkeys to one intravenous administration of COC (1 mg/kg), which closely imitates the smoking of "crack" COC with regard to dose and effect. We monitored plasma concentrations over time, beginning when the primates were in a state of hyperarousal. Blood was sampled at 1, 6, 12, and 40 min after dosing. Plasma concentrations of COC decreased rapidly with a half life of 15.7 min. Mean COC concentrations in the drug-treated group (n = 7) for the four timepoints were 296, 225, 187, and 80 ng/mL, respectively. Ecgonine methyl ester (EME) concentrations ranged from 57 to 91 ng/mL. When compared with the 1-min COC concentrations, the mean EME concentration was 30.7%. Benzoylecgonine (BZE) ranged from 34 to 42 ng/mL, and the mean concentration was 11.5% of the mean COC concentration at 1 min. EME and BZE concentrations did not vary appreciably over the time course of the study. Plasma norcocaine concentrations were less than the limit of detection of 25 ng/mL. Because a rapid decline in plasma COC concentrations over time was observed along with a very small change in EME and BZE concentrations, we attribute tissue redistribution of COC, particularly to the brain, as significant and metabolism or hydrolysis of COC as minor.

Cocaine-induced rausch: overt behaviour and plasma concentrations in rhesus monkeys.

This study was designed to characterize the cocaine-induced rausch or hyperarousal syndrome in rhesus monkeys. This syndrome mimics the stage observed in human abusers bingeing on cocaine and is considered crucial in the progression from recreational use to compulsive abuse. However, little research has focused on this important aspect of cocaine use. Cocaine was administered i.v. at doses of 0.0, 0.5, 1.0 and 2.0 mg/kg. Plasma concentrations were determined by gas chromatograph mass spectrometry (GC/MS) using deuterated internal standards d3 cocaine and d3 benzoylecgonine (BE). Mean plasma concentrations of cocaine, were on samples collected 1 min after infusion, 46 +/- 31, 88 +/- 15 and 275 +/- 116 mg/microliters in the 0.5, 1.0 and 2.0 mg/kg dose groups, respectively. There were no detectable concentrations of BE in any of the specimens nor was cocaine detected in the saline controls. Analysis of the behavioural data revealed that the 0.5 and 1.0 mg/kg results were intermediate between the results obtained at doses of 0.0 and 2.0 mg/kg and that the 1.0 mg/kg dose produced a higher response than the 0.5 mg/kg dose up to the 12 min. Regarding individual behavioural signs, those designated escape attempts, checking, feinting, restlessness, searching, vocalizing, chewing, crouching and wide-eyed were noted most frequently. The results showed dose-response relationships for both plasma concentrations of cocaine and for the total number of overt behavioural signs. The plasma concentrations were in the range reported for human cocaine abusers.

Plasma concentrations of nicotine in rats during tolerance and chronic exposure studies.

A convenient GC/MS method for the quantitation of nicotine is described. Brief and rapid tolerance to the hypertensive action of nicotine was observed during acute administration. Rats continuously exposed to (+)- or (-)-nicotine for 6 days showed significant dose-related suppression of water intake and body-weight decreases for the initial 4 days; then water consumption slowly returned to control levels, while body weight increased, but failed to reach control levels. During the withdrawal period, water consumption rose to levels significantly higher than that of the tartaric acid and water controls. Body weight during the withdrawal phase continued to increase but remained below those of control animals. Blood concentration of nicotine during acute tolerance was found to be 64.3 +/- 17.8 ng/ml whereas the saline controls showed levels of 0.67 +/- 0.67 ng/ml. Nicotine levels which were not detectable before the administration of nicotine, were elevated and constant during days 1 and 6 of the infusion period (320 +/- 80 ng/ml of plasma) and fell to below levels of detectability 24 hr after the termination of the infusion.

Suppression of opiate withdrawal and cocaine hyperarousal syndromes by buspirone. Possible pharmacotherapeutic applications.

Buspirone (CAS 36505-84-7) was evaluated in three animal models which were designed to study stages of drug abuse most likely associated with compulsive abuse. Buspirone attenuated abrupt withdrawal in rhesus monkeys maximally-dependent on morphine. In addition, it completely blocked the emergence of cocaine-induced stereotyped behavior in rats and attenuated the hyperarousal or rausch syndrome in morphine-dependent and non-dependent rhesus monkeys. Buspirone was active at doses which caused little, if any, impairment in the animals. The results suggest that buspirone may possibly find application in the pharmacotherapy of opioid and cocaine abuse.

In vivo mammalian metabolism of methylamine and methylurea and their metabolic interrelationship.

The metabolism of methylamine has been investigated in the rabbit and rat, and evidence has been presented to show that the metabolism of the compound leads to the formation and excretion of methylurea, a metabolite suspected, but never conclusively shown, to exist by previous investigators. Urinary methylurea, after the administration of methyl-14C-amine, was shown to have 14C activity at both the carbonyl and methyl portions (groups) of the molecule. 14C activity in the carbonyl group of methylurea was derived from the same metabolic CO2 pool that is used in the formation of urinary urea and respiratory carbon dioxide. Administration of methyl-14C-amine led to the formation of respiratory 14CO2, as noted by various investigators, and it was shown that during a 24-hr period the ratio of specific activity (urea/respiratory CO2) approached 1, in agreement with previous data reported by Mackenzie and du Vigneaud [J. Biol. Chem. 172, 353-354 (1948)]. In order to quantitatively investigate some aspects of metabolism of methylurea, a procedure was developed for the convenient synthesis of N-methyl-14C-carbonyl-urea. In a series of experiments, the urinary urea of the animals injected with N-methyl-14C-carbonyl-urea and N-methyl-14C-urea was examined for radioactivity. Only limited amounts of 14C activity were found in the carbonyl group of urea following administration of N-methyl-14C-urea. However, after the administration of N-methyl-14C-carbonyl-urea, urinary urea contained a 200-fold excess of 14C activity.(ABSTRACT TRUNCATED AT 250 WORDS)

The enzymatic systems involved in the mammalian metabolism of methylamine.

The metabolism of methylamine has been investigated in the rat in order to elucidate the role of monoamine oxidase (MAO; EC 1.4.3.4) and intestinal bacteria in the metabolism of the compound. In a series of experiments in which short- and long-acting inhibitors of MAO were administered either alone or in combination prior to methyl-[14C]amine hydrochloride injection, the excretion of radioactivity in the expired air and the urine was examined to indirectly assess the role of MAO in the metabolism of methylamine. The data presented provide indirect evidence to demonstrate that the effect of iproniazid, an inhibitor of methylamine oxidation, is mediated through enzyme systems separate from MAO systems which have been invoked as major contributors to metabolism of methylamine by other investigators. The bacterial oxidation of methylamine in the intestine plays a minor role in the overall metabolism of the compound.

Tissue disposition of [3H]sarin and its metabolites in mice.

The biodisposition and metabolic fate of [3H]sarin was investigated in mice after iv administration of a sublethal dose (80 micrograms/kg). Within 1 min of administration, all tissues contained substantial quantities of radioactivity of which less than 10% represented [3H]sarin. The major portion of radioactivity corresponded to free [3H]isopropyl methylphosphonic acid (IMPA), the pharmacologically inactive hydrolytic product of [3H]sarin. Somewhat lesser quantities were present as bound [3H]IMPA which resulted from phosphorylation of protein. Plasma contained high concentrations of bound [3H]IMPA, consistent with sarin's very reactive nature, which were sustained throughout the time course. Plasma concentrations of free [3H]IMPA diminished rather quickly. The high concentrations of metabolites in kidneys implied that this organ played a major role in the detoxification and excretion of [3H]sarin. Large quantities of free and bound [3H]IMPA were also found in lung which suggested an important site for toxicity. Only trace quantities of [3H]sarin were found in brain after 15 min. The major portion of radioactivity was present as either free and bound [3H]IMPA or as nonextractable material which presumably was [3H]methylphosphonic acid. Examination of the time course of sarin-induced motor hypoactivity and hypothermia revealed an immediate onset of action that lasted for 24 hr. However, substantial quantities of bound [3H]IMPA remained in brain at 24 hr which suggested that only a small portion of phosphorylation in brain accounted for these pharmacological effects.