Arnold S. Lippa

Resolution of two biochemically and pharmacologically distinct benzodiazepine receptors

Brain-specific binding sites have been isolated on synaptosomal membrane fragments which recognize pharmacologically active benzodiazepines (BDZs) and triazolopyridazines (TPZs). While early evidence indicated the existence of a single homogeneous class of BDZ binding sites, more recent biological and pharmacological studies support the notion of BDZ receptor multiplicity. We now propose that two biochemically distinct BDZ receptors exist in brain which are responsible for the mediation of different pharmacological activities. Type I BDZ receptors display a high affinity for both BDZs and TPZs, are not coupled to GABA receptors or to chloride ionophores, and are the sites which mediate anxiolytic actions. Type II BDZ receptors display a high affinity for BDZs, display a low affinity for TPZs, are coupled to GABA receptors and/or chloride ionophores, and are the sites which mediate pharmacological effects other than anxiolytic activity.

A synthetic non-benzodiazepine ligand for benzodiazepine receptors: a probe for investigating neuronal substrates of anxiety.

CL 218,872 is the first non-benzodiazepine to selectively displace brain specific 3H-diazepam binding with a potency comparable to that of the benzodiazepines. Like the benzodiazepines, CL 218,872 increased punished responding in a conflict situation and protected against the convulsions induced by pentylenetetrazole. These three pharmacological properties are highly predictive of anxiolytic activity. Unlike the benzodiazepines, however, CL 218,872 was relatively inactive in tests designed to measure effects on neuronal systems which utilize GABA, glycine and serotonin as transmitters. Furthmore, CL 218,872 was relatively free of the ataxic and depressant side effects commonly associated with the benzodiazepines. Because of this high degree of selectivity, CL 218,872 may represent a new probe for investigating neuronal substrates of anxiety.

Brain cholinergic dysfunction and memory in aged rats

Age related alterations in mnemonic ability and in the functional status of muscarinic receptors were evaluated and compared to biochemical measures of pre and post-synaptic cholinergic functioning. Retention of a single trial passive avoidance task was considerably disturbed as a function of aging. The functional status of muscarinic receptors, as measured by the ability of microiontophoretically applied acetylcholine to stimulate the firing of hippocampal pyramidal cells, was similarly disturbed in aged rats. A small, but significant decrease in muscarinic receptors was detected in the dorsal hippocampi of these same aged rats, while choline acetyltransferase activity did not change. When considered with prior psychopharmacological studies, these data suggest that specific muscarinic receptor impairments may play a critical role in the memory disturbances associated with old age.

Human brain receptor alterations in suicide victims

A comparison was made of human postmortem muscarinic-cholinergic, beta-adrenergic and serotonergic (presynaptic) recognition sites in cortical tissues derived from suicide and homicide (control) victims. An elevation of 47% and 35% in the suicide group compared to controls was observed in receptor ligand binding for 3H-quinuclidinyl benzilate (QNB, muscarinic antagonist) and 3H-imipramine (IMI, a presynaptic serotonin marker), respectively. In contrast, no appreciable differences in 3H-dihydroalprenolol (DHA, beta-adrenergic antagonist) binding were observed between the two groups. Additionally, tissues from both groups of subjects were analyzed for tricyclic antidepressive agent (TAD) content. High performance liquid chromatographic (HPLC) tissue analysis revealed no detectable levels of tricyclic agents with an assay sensitivity of 50 picograms/mg tissue. The results presented herein demonstrate neurotransmitter-receptor alterations in suicide subjects compared to homicide (control) victims. The attendant roles of serotonergic and muscarinic-cholinergic processes in the psychobiology of suicide and depression are addressed.

Antidepressant-like actions of DOV 21,947: a ''triple'' reuptake inhibitor

DOV 21,947 [(+)-1-(3,4-dichlorophenyl)-3-azabicyclo-[3.1.0]hexane hydrochloride] inhibits the reuptake of [3H]serotonin, [3H]norepinephrine, and [3H]dopamine in human embryonic kidney (HEK) 293 cells expressing the corresponding human recombinant transporters (IC(50) values of 12, 23, and 96 nM, respectively). This compound also inhibits [125I]RTI 55 (3beta-(4-iodophenyl)tropane-2beta-carboxylic acid methyl ester) binding to the corresponding transporter proteins in membranes prepared from these cells (K(i) values of 99, 262, and 213 nM, respectively). DOV 21,947 reduces the duration of immobility in the forced swim test (using rats) with an oral minimum effective dose of 5 mg/kg. This antidepressant-like effect manifests in the absence of significant increases in motor activity at doses of up to 20 mg/kg. DOV 21,947 also produces a dose-dependent reduction in immobility in the tail suspension test, with a minimum effective oral dose of 5 mg/kg. The ability of DOV 21,947 to inhibit the reuptake of three biogenic amines closely linked to the etiology of depression may result in a therapeutic profile different from antidepressants that inhibit the reuptake of serotonin and/or norepinephrine.

Age-related alterations in neurotransmitter receptors: An electrophysiological and biochemical analysis

The difficulties involved in understanding the complex phenomena of geriatric cognitive dysfunction clearly call for an interdisciplinary approach. We have initiated multi-laboratory studies in order to assess the utility of the aged Fisher 344 rat as a potential animal model. Prior behavioral experiments have demonstrated that these aged animals suffer from age-related memory impairments conceptually similar to those which occur in humans. Neurophysiological and biochemical studies have focused on the task of assessing hippocampal functioning in this potential animal model. The hippocampus was chosen for these studies because changes in hippocampal function may contribute to the memory impairments associated with old age. The overall firing rate of single hippocampal pyramidal cells was reduced in OLD (26–28 months) rats when compared to YOUNG rats (6–8 months). This effect was due to a selective decrease in the simple spike firing rate. Since burst firing was relatively intact, these results argue against a generalized non-specific decrease in neuronal activity. This interpretation is supported by the observation that microiontophoretic application of glutamate stimulated the firing of hippocampal pyramidal cells to the same extent in both young and old animals. However, microiontophoretic application of GABA inhibited hippocampal pyramidal cell firing to a greater extent in old rats. This effect was not accompanied by any age-related alterations in 3H-muscimol binding in the dorsal hippocampi obtained from these same animals. The pyramidal cell stimulation produced by microiontophoretic application of acetylcholine was reduced in OLD rats. The number of muscarinic receptors (as measured by the binding of 3H-agonists and antagonists) in the dorsal hippocampi was reduced approximately 20% in the aged rats. All of these results have generated the following conclusions: (1) The neuronal activity of hippocampal pyramidal cells is reduced in aged rats, an effect which might contribute to the mnemonic disturbances observed in these animals. (2) Age-related alterations in the functional consequences of GABA receptor activation occurred independent of any change in the binding parameters of 3-muscimol. (3) The decrease in muscarinic binding sites is reminiscent of the decreases observed in aged human brain. Together with the decreased functional response to acetylcholine, these results provide strong evidence for a cholinergic hypothesis of geriatric cognitive dysfunction.

Relationship between benzodiazepine receptors and experimental anxiety in rats

The in vitro and in vivo ability of benzodiazepines to inhibit specific 3H-diazepam binding correlated with their ability to increase punished responding in a conflict situation. Conflict and foot shock, the punishing stimulus used in most conflict procedures, also altered 3H-diazepam binding. These data implicate 3H-diazepam binding sites in mediating at least some of the anxiolytic properties of benzodiazepines and suggest the existence of some endogenous substance which might be involved in the etiology of anxiety.

Differential ontogeny of Type 1 and Type 2 benzodiazepine receptors

Abstract The postnatal development of Type 1 and Type 2 benzodiazepine receptors in rat cerebral cortex was studied using CL 218,872, a novel triazolopyridazine. On postnatal day 1 most 3 H-flunitrazepam binding sites appeared to be Type 2 receptors, which increased rapidly during the first week of life and reached adult levels by 3–4 weeks of age. Type 1 receptors, on the other hand, represented only a small percentage of the binding sites on postnatal day 1 and did not begin to increase in number until approximately 7–16 days of age. These results demonstrate a differential postnatal development of two sub-populations of benzodiazepine receptors.

DOV 216,303, a “Triple” Reuptake Inhibitor: Safety, Tolerability, and Pharmacokinetic Profile

This report describes the first evaluation in humans of DOV 216,303, a putative antidepressive that inhibits the reuptake of norepinephrine, serotonin, and dopamine. Subjects received single oral doses of 5 to 150 mg of DOV 216,303 or placebo. At 150 mg, 4 of 7 subjects reported gastrointestinal disturbances. In the multiple‐dose phase of the evaluation, subjects received total daily doses of 50, 75, or 100 mg of DOV 216,303 or placebo for 10 days. At a total daily dose of 100 mg, gastrointestinal disturbances were reported in 4 of 6 volunteers. In both the single‐ and multiple‐dose evaluations, no significant changes were noted in vital signs, electrocardiogram, hematology, or clinical chemistry. DOV 216,303 was rapidly absorbed (plasma tmax of 0.7–1.2 hours and t1/2 of 3.3–4.4 hours), with dose‐proportional Cmax and AUC values. Furthermore, no remarkable difference was apparent in either the Cmax or AUC of DOV 216,303 following 1 and 10 days of dosing. The present results demonstrate that DOV 216,303 is safe and well tolerated both at single doses of up to 100 mg and multiple doses of up to 100 mg/day for 10 days. Plasma concentrations of DOV 216,303 after doses >10 mg exceed its reported IC50 values for inhibition of biogenic amine reuptake.

The role of GABA in mediating the anticonvulsant properties of benzodiazepines

Abstract While it has been suggested that benzodiazepine (BDZ) actions on brain GABA mechanisms might provide a unitary framework for explaining the diverse pharmacological properties of BDZs, recent evidence has necessitated a reevaluation of this hypothesis. Hofstee analysis of 3H-diazepam and 3H-flunitrazepam binding in the presence of various concentrations of novel triazolopyridazines (TPZ) demonstrates the presence of two binding sites. One site has a high affinity for BDZ and TPZ (Type 1 receptors), while another site has a high affinity for BDZ, but a low affinity for TPZ (Type 2 receptors). Cerebellum is enriched in Type 1 receptors, while hippocampus is enriched in Type 2 receptors. CL 218,872, a representative TPZ, is able to potently antagonize pentylenetetrazole (PTZ)-induced convulsions, but only very weakly antagonize bicuculline or isoniazid-induced convulsions, These data suggest that drug actions on Type 1 receptors may be independent of GABA and responsible for antagonizing PTZ-induced convulsions. Conversely, drug actions on Type 2 receptors are GABA associated and responsible for antagonizing bicuculline- and isoniazid-induced convulsions.