Selective effect on punished versus unpunished responding in a conflict test as the criterion for anxiogenic activity.

The punished drinking test has been used successfully for identifying and studying anxiolytic agents. By reducing the level of punishment (i.e., decreasing the intensity of shock), it has also been used as a method for measuring anxiogenic activity. Because anxiogenic behavior is a novel and important concept that is not yet fully established, we have reinvestigated the effects of two putative inverse benzodiazepine agonists and pentylenetetrazol in this conflict test. In a series of experiments, using both our version of the procedure and a replication of a previously published method, we were unable to demonstrate a selective reduction in punished responding over unpunished responding caused by CGS 8216 (3 to 40 mg/kg), FG 7142 (2 to 6 mg/kg), and pentylenetetrazol (10 to 20 mg/kg) as reported previously. A careful comparison of the details of our method and the published procedure failed to reveal the source of this discrepancy. If anxiogenic behavior is to be defined as a selective effect of a drug on punished response, the value of this test will depend on identification of its critical variables.

Molecular determinants of benzodiazepine receptor affinities and anticonvulsant activities.

In vivo convulsant activities profiles and receptor binding studies together with the techniques of theoretical chemistry were used to characterize 15 compounds, from five different chemical families, known to bind to the BDZ receptor. The experimental goals of this study were to determine the affinity of these analogs for this receptor, the effect of gamma-aminobutyric acid on the affinity, and, in a self-consistent manner, the nature of the activity, agonist (anticonvulsant), antagonist, or inverse agonist (proconvulsant, convulsant), elicited by binding to this receptor. To these ends, in vivo studies were made to determine the proconvulsant, convulsant, and anticonvulsant activities and antagonism to anticonvulsant activities of the 15 analogs. Their receptor affinities at 25 degrees were also determined by competitive inhibition of [3H] flunitrazepam and [3H]Ro 15-1788 in the absence and presence of gamma-aminobutyric acid. The goal of the theoretical studies was to identify and calculate molecular properties that modulate these affinities and types of activities and from them to develop a model of receptor recognition and activation that could consistently explain observed behavior and predict new results. Thus, molecular orbital calculations were carried out for all analogs, using semiempirical quantum mechanical methods. In addition to the optimization of structures, a number of electronic properties, such as polarizations, partition coefficients, and proton and electron affinities were computed and examined for their ability to modulate relative affinities and modes of activation of the receptor. From these studies, a model for receptor recognition involving two anchoring hydrogen bond-acceptor sites and for activation involving interaction of the most lipophilic aromatic region of each compound with the receptor was developed, which could systematically account for the three different types of behavior, agonist, antagonist, and inverse agonist, observed for these analogs. Electronic rather than structural properties were found to be the principal modulator of both recognition and activation. A possible mechanism of agonist activation of the receptor involving electron transfer to the agonist, as well as a possible induced conformational change in the receptor, is also suggested by these results. Finally, by complementarity, some steric and electronic characteristics of the receptor binding site could be deduced.

Structure-activity studies of morphine fragments. I. 4-alkyl-4-(m-hydroxy-phenyl)-piperidines.

The 4-(m-OH-phenyl)piperidines are a flexible fragment of the morphine/benzomorphan fused-ring opioids. Analogs in this family were synthesized with varying 4-alkyl substituents increasing in bulk from H through methyl, n-propyl, to t-butyl, each with the three N-substituents methyl, allyl, and phenethyl. These twelve compounds were evaluated for analgetic agonism in mice using two different models for antinociceptive activity, acetic acid writhing and tail-flick, the latter by both subcutaneous and intracerebroventricular routes of administration. Antagonism to morphine analgesia was also measured by the mouse tail-flick procedure. Binding affinities of these new analogs to different opioid receptor subtypes were determined. Energy conformational calculations on these compounds were also carried out using the empirical energy program called MOLMEC, in order to better understand how the 4-R substituents modulate receptor binding affinities and efficacies. The results obtained show that, in general, the compounds studied are mu-selective and vary in agonist potency from weak to morphine-like. Significant differences in rank order of analgetic potencies and their relationship to receptor affinities were obtained from the results of subcutaneous and intracerebroventricular administration. Results of energy-conformational calculations for twelve N-methyl compounds indicate that those with 4-alkyl substituents favor a common, non-morphine-like phenyl axial conformation. The 4-t-butyl compounds are, in fact, the first simple mono-alkyl-substituted 4-phenyl-piperidines predicted to definitely exist in a phenyl axial conformation, as confirmed by X-ray analysis. On the basis of this common phenyl axial conformation, the observed variation in mu receptor affinities and efficacies of the 4-methyl, 4-n-propyl, and 4-t-butyl compounds could be understood and the behavior of 4-ethyl and 4-isopropyl analogs predicted. Two equatorial conformers (rotamers) were found to be the preferred forms of the analogs with 4-R being H or an ester group, or with a 3-methyl group added trans (beta) to the 4-R group. Taking into account the rotational flexibility of these analogs, these two conformers could be used to understand differences in high and low efficacy compounds observed among analogs with preferred phenyl equatorial conformations. None of the analogs exhibit a fused-ring-like N-substituent modulation of efficacy. This result can, perhaps, be understood by their inability in any proposed conformer to totally mimic key receptor interactions of both the phenol-OH and N-substituent portions of the fused compounds.

Novel mu-selective Met-enkephalinamide analogs with antagonist activity. Synthesis, receptor binding, analgesic properties and conformational studies.

Four novel mu-selective peptide antagonists have been synthesized and examined for receptor binding, analgesic agonist and antagonist activity and energy conformational properties. These peptides were designed by analogy to results of molecular modeling of 3-phenyl piperidines which led to incorporating four modified tyrosine residues, m-Tyr, beta-methyl-m-Tyr, N-phenethyl-m-Tyr and alpha, beta-dimethyl-m-Tyr into D-Ala2-Met5-enkephalinamide. Peptides were synthesized by stepwise solution synthesis using an active ester coupling procedure. Receptor binding assays were performed on rat brain homogenates and data were analyzed by a modified version of the program LIGAND. Analgesic agonist and antagonist activity was evaluated by the mouse tail-flick test. Energy-optimized conformations were obtained using a program called Molecule-AIMS. The results demonstrate that relative ratios of in vivo agonist and antagonist potencies in D-Ala2-Met5-enkephalinamides can be modulated by chemical modification of the tyrosine residue. A shift in the phenolic-OH position from para to meta significantly enhances relative antagonist versus agonist activity; addition of a beta-CH3 group to the m-Tyr enhances mu-selectivity and leads to nearly equal agonist/antagonist activity. Energy conformational studies indicate that all analogs with high mu-receptor affinity examined have a common energy accessible B'II 2-3 turn conformation similar to that previously identified for high mu-affinity binding in peptides, lending further support to this candidate conformer. This conformer also has tyrosine side-chain angles which allowed total overlap with the amine and phenolic groups of a known structure of 3-(m-OH phenyl)-piperidine. This structural similarity together with the observation of mixed agonist antagonist activity in both types of opioids confirms the rationale upon which design of these peptides was based.

Intravenous self-administration of ethanol and acetaldehyde by rats.

Fisher strain rats were allowed to self-administer 2, 6, and 18 microM/kg/infusion of ethanol and acetaldehyde into the jugular vein for 22 to 28 days. Thirty-three percent of the rats self-administered 18 microM/kg/infusion of ethanol. Thirty-three, 40, and 33 percent of the rats initiated self-administration of 2, 6, and 18 microM/kg/infusion of acetaldehyde, respectively. It is suggested that acetaldehyde may possess stronger reinforcing effects than ethanol in intravenous self-administration by rats.

Interactions between toluene and alcohol.

Weanling male Fischer-344 rats were exposed by inhalation to air or 2000 ppm toluene for 8 hours each day for 2 weeks. Subgroups had access to water or 6% alcohol as their only fluid sources, respectively. Rats exposed to both toluene and alcohol subsequently showed a marked preference for 6% alcohol in two-bottle choice tests that persisted for up to 20 days for some rats. Rats exposed to toluene without access to alcohol and control rats (exposed to air and water) showed a marked aversion to the alcohol solution, and only 2 of 12 rats forced to drink alcohol without exposure to toluene preferred alcohol in the preference tests. Exposure to both toluene and alcohol also caused greater inhibition of weight gain than exposure to either substance alone, accompanied by greater signs of organ toxicity as indicated by clinical blood chemistries. Exposure to toluene caused marked hearing loss as assessed by a behavioral technique (conditioned avoidance), and there was a trend toward enhancement of this ototoxic effect by forced consumption of alcohol.

Theoretical structure-activity studies of beta-carboline analogs. Requirements for benzodiazepine receptor affinity and antagonist activity.

The techniques of theoretical chemistry have been used to elucidate the molecular properties and modes of receptor binding that modulate receptor affinity and antagonist activity of the beta-carbolines, a class of potent benzodiazepine antagonists. Six analogs were chosen in order to investigate the role of the amine (NH) group, the aromatic nitrogen, and the C3-substituent in determining receptor affinities. Electrostatic potential mapping and characterization of explicit drug-receptor interactions have led to the hypothesis that simultaneous interaction of a model cationic arginine site with the N2 and C3-substituents could play a key role in determining receptor affinities. The electron-withdrawing effects of C3-substituents on the amine nitrogen appear less important, though interactions of these groups with an anionic glutamate or aspartate site could also occur at the receptor. Similarly, stacking interactions with neutral or cationic aromatic residues such as tryptophan or protonated histidine could occur, but do not appear to be determinants of the relative receptor affinity of the beta-carbolines.

Structure-activity studies of beta-carboline analogs.

A number of beta-carboline analogs have been obtained or synthesized, and their in vitro receptor affinities and in vivo antagonist activities determined. The choice of analogs was made in order to explore the importance of the N9 -H, the aromatic nitrogen and the C3-ester moiety for high-receptor affinity and antagonist activity of this class of benzodiazepine antagonist. Among the analogs investigated, we describe the properties of 3-cyano-beta-carboline (1h), the first potent beta-carboline antagonist without a carbonyl at the C3-position. The results obtained indicate: (1) Specific interactions of the C3-substituent with key cationic receptor sites rather than electron-withdrawing properties are important for high-receptor affinity and antagonist activity. (2) Specific in-plane interactions of the aromatic nitrogen with a cationic receptor site, rather than stacking with neutral aromatic residues of the receptor are also important for high affinity and antagonist activity. (3) While the presence of an N9 -H enhances receptor affinity, interaction with an anionic receptor site does not appear essential for antagonist activity.

Replacement of the peptide-backbone amides connecting Tyr-Gly and Gly-Gly in leucine-enkephalin with ketomethylene groups: synthesis and biological activity.

A peptide analogue of Leu-enkephalin was synthesized in which the amide linkages between Tyr-Gly and Gly-Gly were replaced by ketomethylene groups. The resulting analogue, 12, had 1/4000th and 1/2400th the opiate receptor binding activity of Leu-enkephalin when (3H) [D-Ala2,D-Leu5]enkephalin and (3H)naloxone, respectively, were used as tritiated ligands. When tested for analgesia in mice by the tail-flick assay, 12 produced analgesia in 50% of the mice tested at a dose of 24.3 micrograms/mouse (icv), while the ED50 of Leu-enkephalin is 240 micrograms/mouse (icv). At a dose of 40 micrograms/mouse (icv) or higher, 12 caused convulsions in a dose-dependent manner. No analgesia was observed after intravenous (iv) administration of 240 micrograms/mouse of 12.

Assessment of chemicals using a battery of neurobehavioral tests: a comparative study.

Single-dose LD10S and LD50S were determined in male, Fischer-344 rats for acrylamide monomer, arsenic trioxide, chlordecone, lead acetate, methylmercury hydroxide, monosodium salicylate, tetraethyl tin, and triethyl lead chloride. Proportions of the single-dose LD10S were used in a subacute study to estimate the 28-day LD20S for each chemical. Proportions of the 28-day LD20S were used in a subchronic (105 days of dosing) study to determine the effectiveness of a battery of neurobehavioral tests for detecting and characterizing the neurotoxic effects of each chemical. The battery consisted of undifferentiated motor activity, forelimb and hindlimb grip strengths, rotation orientation, thermal sensitivity, startle responsiveness to acoustic and air-puff stimuli, and performance of a multisensory conditioned pole-climb avoidance response task; body weight and rectal temperature were also monitored. The battery of tests was administered on eight occasions, that is, before, at three-week intervals during dosing (PO or IP, five days each week for 15 weeks), and at three and six weeks after dosing. Normative data (controls from each experiment) indicated fair overall stability of the measures over the eight test sessions, but experiment-to-experiment variability in this regard was clearly evident. The inherent statistical sensitivity of the tests varied greatly as estimated by their coefficients of variation, which ranged from 1% (rectal temperature) to over 100% (rotation orientation). Intercorrelations among the various measures were low to moderate indicating relatively little redundancy. The various measures were differentially affected by the eight chemicals: body weight by all eight; rectal temperature by one; undifferentiated motor activity by three; forelimb grip strength by two; hindlimb grip strength by four; rotation orientation by one; thermal sensitivity by one; startle responsiveness by three; and CAR performance by five. A profile analysis using the slopes of the dose-response functions after 15 weeks of dosing indicated clearly different patterns of effect among the eight chemicals. A composite score derived from a multidimensional analysis of the data suggested that the eight chemicals could be ranked from most to least neurotoxic as defined by these tests as acrylamide greater than methylmercury greater than chlordecone greater than tetraethyl tin greater than triethyl lead greater than lead acetate greater than arsenic greater than monosodium salicylate. This ranking and the results in general are in good agreement with what is known about these chemicals from other experiments with animals and from human experience. This battery of tests, or subsets thereof, may therefore have utility in the assessment of the potential neurobehavioral toxicity of various chemicals.

Effects of PAM, proPAM, and DFP on behavior, thermoregulation, and brain AChE in rats.

The effects of pyridine-2 aldoxime methyl iodide (PAM), N-methyl-1,6-dihydro-pyridine-2-carbaldoxime hydrochloride (proPAM), and diisopropyl phosphorofluoridate (DFP) on performance of a conditioned avoidance response (CAR), body temperature, and in vivo acetylcholinesterase (AChE) activity in five brain regions in the rat were examined. Sublethal doses of DFP (1.5 to 2.5 mg/kg, IP) markedly degraded CAR performance. This effect was antagonized by 5 mg/kg, subcutaneously injected (SC) atropine. A 50 mg/kg, SC dose of PAM had no effect on the CAR, but an equal dose of proPAM caused a transient deterioration of performance. Given 10 min or 2 hr after DFP, 50 mg/kg proPAM initially exacerbated the behaviorally toxic effects of DFP. Neither PAM nor proPAM antagonized DFP-induced hypothermia. PAM did not reactivate DFP-inhibited brain AChE, and proPAM reactivated it by only 6 to 12% of control activity.

Analgesics. 1. Synthesis and analgesic properties of N-sec-alkyl- and N-tert-alkylnormorphines.

A series of N-sec- and N-tert-alkylnormorphines was synthesized and evaluated for analgesic potency, antagonist activity, and opiate receptor binding. Computer-assisted conformational analysis profiles were utilized to assist in the selection of compounds for synthesis and correlation of receptor events with in vivo observations. N-tert-Alkylnormorphines 5a-c were devoid of agonist activity; however, some sec-alkyl analogues showed interesting mixed agonist-antagnoist actions. N-sec-Butyl- and N-(alpha-methylally)normorphine were separated into R and S isomers, which exhibited quantitative pharmacological differences. The N-sec-butyl S isomer 10a showed analgesia approximating morphine with nalorphine-like antagonist activity. Preliminary testing indicates only slight evidence for physical dependence with this compound.