Akira Hirose

Characterization of the putative anxiolytic SM-3997 recognition sites in rat brain.

In order to clarify the mechanism of action of the putative nonbenzodiazepine anxiolytic SM-3997 [3a alpha,4 beta,7 beta,7a alpha)-Hexahydro-2-(4-(4-(2-pyrimidinyl)-1- piperazinyl)-butyl)-4,7-methano-1H-isoindole-1,3 (2H)-dione dihydrogen citrate), in vitro binding studies with radiolabeled compound were performed. 3H-SM-3997 bound rapidly, reversibly and in a saturable manner with high affinity to rat brain hippocampal membranes (Kd = 9.4 nM, Bmax = 213 fmol/mg protein). This specific binding was displaced by 5-hydroxytryptamine (5-HT) and related compounds. Especially, 8-OH-DPAT, a 5-HT-1A selective agonist, bound with the highest affinity to these binding sites. 3H-SM-3997 binding, however, was not displaced by a variety of other neurotransmitters, neuropeptides and some other drugs. EDTA and physiological concentration of Na+ inhibited this specific binding, but several divalent cations, Mn2+, Ca2+ and Mg2+, enhanced this binding. GTP decreased the affinity of these binding sites for 3H-SM-3997 without changing the number of binding sites, but GMP and ATP did not influence 3H-SM-3997 binding. Furthermore, 3H-SM-3997 bound with marked regional selectivity to hippocampal membranes. These characteristics and the regional distribution of 3H-SM-3997 binding sites were very similar to those of 3H-8-OH-DPAT binding sites (5-HT-1A receptors). Therefore, these results indicate that SM-3997 binds selectively and with high affinity to 5-HT-1A receptors in rat brain and may be an agonist.

Effects of tandospirone on second messenger systems and neurotransmitter release in the rat brain

1. We studied the effects of tandospirone, a novel serotonin (5-HT)1A receptor-related anxiolytic, on the intracellular second messenger systems and neurotransmitter release. 2. Tandospirone inhibited forskolin-stimulated adenylate cyclase activity in rat hippocampal membranes by activation of 5-HT1A receptors and had high efficacy comparable to 5-HT1A receptor agonists such as 5-HT and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT). 3. Tandospirone suppressed carbachol-stimulated phosphatidyl-inositol metabolism (PI response), which was shown to be a 5-HT1A receptor-mediated event. 4. Tandospirone did not affect the release of 5-HT, norepinephrine (NE), dopamine (DA) and acetylcholine (ACh) from rat brain slice preparations. 5. These findings suggested that tandospirone shows high agonistic efficacy on the postsynaptic 5-HT1A receptors but does not affect the presynaptic autoreceptors located on nerve endings. The modulation of the second messenger system via postsynaptic 5-HT1A receptors might be involved in the anxiolytic efficacy of tandospirone.

Autoradiographic localization and pharmacological characterization of [3H]tandospirone binding sites in the rat brain

The regional distribution and pharmacological properties of [3H]tandospirone binding sites in the rat brain were investigated using quantitative autoradiography. [3H]Tandospirone binding was notably high in the dentate gyrus and CA1 area of the hippocampus, lateral septum, entorhinal cortex, interpeduncular nucleus and dorsal raphe nucleus. The distribution profiles of [3H]tandospirone binding sites significantly correlated with that of serotonin (5-HT)1A receptors identified using [3H]8-OH-DPAT. In competitive binding studies, [3H]tandospirone binding was inhibited by 5-HT, 8-OH-DPAT, pindolol, buspirone and N-(a,a,a-trifluoro-m-tolyl)-piperazine. The potencies of these ligands correlated with their affinities for 5-HT1A receptors. In addition, there was no significant difference in the dissociation constant of [3H]tandospirone binding between the dentate gyrus, CA1 area, dorsal raphe nucleus, lateral septum and entorhinal cortex (about 10 nM) suggesting that [3H]tandospirone binds to 5-HT1A receptors with same affinities in these brain structures. The distribution pattern of binding sites for [3H]tandospirone was also compared with that of benzodiazepine receptors identified using [3H]fludiazepam to find common effector sites for different types of anxiolytics. Some similarities were observed. It is evident in the hippocampal formation that an overlap of intense binding occurred. 5-HT1A receptors in the hippocampus may participate in the anxiolytic effects of tandospirone.

Effects of the putative anxiolytic SM-3997 on central monoaminergic systems

The effects of SM-3997 on central monoaminergic systems were evaluated by ex vivo measurement of monoamines and their metabolite levels in rat brain after intraperitoneal treatment of drugs and by in vitro measurement of monoamine uptake into rat brain slices. The effects of SM-3997 were also compared with those of other new nonbenzodiazepine anxiolytic compounds. SM-3997, buspirone, gepirone and ipsapirone showed no effects on serotonin uptake and dopamine uptake, and a weak inhibition of norepinephrine uptake at the concentration of 100 microM. SM-3997 decreased the serotonin metabolite (5-hydroxyindole-3-acetic acid) level without changing the serotonin level in hippocampus and increased dopamine metabolite (3,4-dihydroxyphenylacetic acid, homovanillic acid) level with no effect on the dopamine level in striatum. SM-3997 also produced an increase in the norepinephrine metabolite (3-methoxy-4-hydroxyphenylglycol) level with a decrease in the norepinephrine levels in hippocampus. Similar effects on serotonin metabolites and norepinephrine metabolites were observed in several other regions. Although the serotonergic effect of SM-3997 was similar to that of buspirone, gepirone and ipsapirone, the dopaminergic effect of SM-3997 was much weaker than that of buspirone.

Benzodiazepines and their metabolites: relationship between binding affinity to the benzodiazepine receptor and pharmacological activity.

Experiments were carried out to study the relationship between binding affinity to the benzodiazepine receptor and pharmacological activity, especially anti-anxiety activity, of clinically useful benzodiazepines. In the in vitro experiments, fludiazepam showed the highest affinity to the benzodiazepine receptor with 4 times more potency than that of diazepam, which paralleled the in vivo activity. Diazepam and nimetazepam also bound with high affinities as expected from their in vivo activities. On the contrary, medazepam and cloxazolam showed extremely low affinities and oxazolam showed no affinity, although they showed moderate in vivo activity. However, their metabolites were found to have both high affinity and in vivo activities. These results strongly suggest that in the case of medazepam, cloxazolam and oxazolam, their metabolites may bind to receptor sites in the brain and then elicit pharmacological action. This conclusion was supported by the fact that a good correlation between the binding affinity and the anti-anxiety activity of the tested compounds was observed.

Neuroleptics-induced changes of tyrosine hydroxylase activity in rat striatum in vitro and in vivo

Abstract The potency of haloperidol and chlorpromazine, but not clozapine, for increasing homovanillic acid and activating tyrosine hydroxylase in the striatum was significantly weakened after the repeated administration in rats. These findings suggest that clozapine could supply enough dopamine to surmount the blockade of dopamine receptors in the striatum even after the repeated administration. This property of clozapine seems to be the cause of low incidence of extrapyramidal side effects in clinical use.