Stereoselective enhancement by (R)-HA-966 of the binding of [3H]CPP to the NMDA receptor complex.

The enantiomers of the strychnine-insensitive glycine antagonist, HA-966 (1-hydroxy-3-amino-pyrrolidone-2), stereoselectively enhance binding of the N-methyl-D-aspartate (NMDA) competitive antagonist, [3H]CPP (3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid) to rat brain synaptosomal membranes. The enhancement by the more potent (R)-HA-966 is competitively inhibited by the glycine antagonist 7-chlorokynurenic acid and noncompetitively by the polyamine spermine. Thus, (R)-HA-966, apparently at the glycine site, enhances the binding of antagonist to the NMDA receptor, possibly through a mechanism partially in common with that of spermine.

A novel potent non-benzodiazepine anxioselective agent with reduced dependence liability: ICI 198, 256.

ICI 198,256, a member of the cinnoline series, was shown to be a potent anxiolytic agent in several species of animals. In addition, ICI 198,256 exhibited potent activity as an antagonist of both metrazole and bicuculline-induced convulsions. The salient features of ICI 198,256 are that it possesses minimal sedative liability, lower ethanol interaction and possibly lower dependence liability than benzodiazepines (e.g., diazepam). Neurochemically, this structurally novel anxiolytic compound is potent and selective for the Type 1 (cerebellar) BZ receptors in vivo as well as ex vivo, and in addition shows an agonist BZ-like profile in a variety of systems. Thus, ICI 198,256 may offer several significant advantages in the treatment of anxiety in humans than existing benzodiazepines.

Synthesis and structure-activity relationships of a series of anxioselective pyrazolopyridine ester and amide anxiolytic agents.

A series of 1-substituted 4-amino-1H-pyrazolo[3,4-b]pyridine-5-carboxylic acid esters and amides were synthesized and screened for anxiolytic activity in the shock-induced suppression of drinking (SSD) test. The compounds were also tested for their ability to displace [3H]flunitrazepam (FLU) from brain benzodiazepine (BZ) binding sites. Many compounds were active in these screens and, additionally, demonstrated a selectivity for the type 1 BZ (BZ1) receptor over the type 2 BZ (BZ2) receptor as indicated by Hill coefficients significantly less than unity and by analysis of [3H]FLU binding results from different brain regions. Based on the results of structure-activity studies of these compounds, a hypothesis was proposed to explain the structural features necessary for optimal interaction with brain BZ receptors. A detailed pharmacological evaluation of one of the most potent behaviorally active compounds (27) demonstrated it to be BZ1 selective; also, in comparison to diazepam, 27 showed minimal sedative and alcohol interactive properties at therapeutically effective doses.

HA-966 acts at a modulatory glycine site to inhibit N-methyl-D-aspartate-evoked neurotransmitter release.

The role of endogenous glycine in supporting N-methyl-D-aspartate (NMDA)-evoked neurotransmitter release was investigated. HA-966 (1-hydroxy-3-aminopyrrolidone-2) inhibited NMDA-evoked release of [3H]norepinephrine from rat hippocampal brain slices, but was much less effective in inhibiting [3H]norepinephrine release evoked by kainic acid (KA). Glycine (1 mM) reversed the HA-966 (1 mM) antagonism of NMDA-evoked release of [3H]norepinephrine. Strychnine (10 microM) had no effect on the ability of glycine to reverse HA-966 antagonism of NMDA-evoked neurotransmitter release. Other amino acids were also capable of reversing the HA-966 antagonism of NMDA-evoked [3H]norepinephrine release with a rank order of potency: D-serine greater than or equal to glycine much greater than L-serine approximately beta-alanine. These same compounds inhibited strychnine-insensitive [3H]glycine binding to rat cortical membrane fragments with a rank order of potency: glycine greater than D-serine much greater than L-serine greater than or equal to beta-alanine. In addition, HA-966 inhibited [3H]glycine binding (IC50 = 8.5 microM). The results suggest that HA-966 antagonism of NMDA-evoked neurotransmitter release is due to the inhibition of endogenous glycine acting at a strychnine-insensitive modulatory glycine site associated with the NMDA receptor/ionophore complex.

Preclinical studies with pyrazolopyridine non-benzodiazepine anxiolytics: ICI 190,622.

Tracazolate is a pyrazolopyridine anxiolytic that enhances the binding of [3H]-flunitrazepam [( 3H]FLU) to brain tissue. The discovery that a metabolite of tracazolate, desbutyltracazolate, was a weak inhibitor of [3H]FLU binding led to the synthesis of a series of potent anxiolytics. From this series, ICI 190,622 emerged as a viable drug candidate, being a potent anxiolytic in rats and monkeys. This anxiolytic agent appears to produce only minimal sedation. Furthermore, ICI 190,622 appears less likely to potentiate the actions of ethanol than diazepam. ICI 190,622 is also a potent anticonvulsant (anti-metrazol ED50 = 1.1 mg/kg, PO) in rodents. Neurochemically, ICI 190,622 is similar to the benzodiazepine anxiolytics. In vitro, ICI 190,622 competitively inhibited [3H]FLU binding in cerebral cortex with an IC50 of 81 nM and was 4.3-fold more potent in the cerebellum (IC50 = 19 nM). This suggests a selectivity for the Type 1 benzodiazepine binding site. In contrast, diazepam showed similar affinities in both regions (cerebral cortex = 7 nM and cerebellum = 9 nM). Following oral administration, ICI 190,622 displaced [3H]FLU binding from cerebellar membranes more potently than diazepam (ED50 = 3 and 6 mg/kg, respectively, 1 hour after administration). Thus, ICI 190,622 should be an effective anxiolytic with significant advantages over benzodiazepines.

Enhancement of GABA binding by the benzodiazepine partial agonist CGS9896.

Compounds have been reported that act on the benzodiazepine receptor as full agonists (diazepam and CL218872), full antagonists (CGS8216 and RO15-1788), on partial agonists (CGS9896). We examined the effect of these compounds on [3H]GABA binding to membrane fragments from rat brain. Incubations were performed at 37 degrees C in a buffer containing EGTA to reduce free calcium ion levels. Centrifugation was then used to separate bound from free [3H]GABA. Diazepam caused a 20-45% enhancement of [3H]GABA binding and this effect was inhibited by 5 mM CaCl2. The magnitude of the enhancement of [3H]GABA by CL218872 was similar to that of diazepam. In contrast, the benzodiazepine antagonists, RO15-1788 and CGS8216 caused little enhancement of [3H]GABA binding. Finally, the partial agonist CGS9896 was distinguishable from both the benzodiazepine antagonists and full agonists by an intermediate level of enhancement of [3H]GABA binding. The extent of enhancement of [3H]GABA binding appears to be predictive of the pharmacological efficacy of compounds acting at the benzodiazepine receptor.

Enhancement of benzodiazepine and GABA binding by the novel anxiolytic, tracazolate.

Tracazolate (ICI 136,753) 4-butylamine-1-ethyl-6-methyl-1H-pyrazolo[3,4]pyridine-5-carboxylic acid ethyl ester is a non-benzodiazepine with anxiolytic-like activity in animal models. In contrast to the benzodiazepines, it enhances [3H]flunitrazepam binding in rat synaptic membrane fragments. The enhancement is potential by chloride ion and is due to an increase in affinity of the receptor. The enhancement of benzodiazepine binding by gamma-aminobutyric acid (GABA) is additive with that of tracazolate; however, the GABA antagonist bicuculline blocks the enhancement by both compounds. Tracazolate enhances [3H]GABA binding to frozen and thawed Triton X-100-treated membrane fragments. The enhancement is due to an increase in the number of sites and potentiated by chloride. Benzodiazepines also enhanced GABA binding but the effect was due to an apparent change in affinity and not potentiated by chloride. The rank order to chlorodiazepoxide, diazepam and flunitrazepam for enhancement of GABA binding and displacement of [3H]flunitrazepam binding were the same. The enhancement of [3H]GABA binding by flunitrazepam and tracazolate were additive. Possible interactions between these various receptors are discussed.

Sodium-dependent, high-affinity taurine transport into rat brain synaptosomes.

Taurine uptake into rat brain synaptosomal fractions appears to occur by two saturable transport processes and by bulk diffusion. The transport requires the presence of sodium ions. The dependence of the transport on temperature and cellular respiration implies that the uptake is an active process. The active process is specific for taurine and closely related amino acids. Brain regions differ in their ability to transport taurine. Uptake is not due to mitochondrial contamination of the synaptosomal fractions. However, glial contamination might partly contribute to the uptake. Kainic acid lesions of rat corpus striatum and cerebellum reduce taurine uptake implying that the uptake is, at least partly, into neurons.