Anxiolytic-like effects of DAIZAC, a selective high-affinity 5-HT(3) receptor antagonist, in the mouse elevated plus-maze.

Behavioral effects of desamino-3-iodozacopride (DAIZAC) [(S)-5-chloro-3-iodo-2-methoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide], a selective high-affinity 5-HT(3) receptor antagonist (K(D) 0.14 nM), were evaluated in the mouse elevated plus-maze using the anxiolytic benzodiazepine, diazepam, as a positive control. DAIZAC treatment produced a significant dose-related increase in the time spent in the open arm. The increased total time in the open arm resulted from a significant dose-dependent increase in the number of entries into that arm. The minimum dose of DAIZAC associated with a statistically significant increase in entries and time spent in the open arm was 0.05 mg/kg ip, consistent with its high affinity for the 5-HT(3) receptor. DAIZAC did not affect the amount of time spent in the open arm after each entry. Thus, DAIZAC reduced apparent avoidance of the open arm when the animal was in the central compartment, without affecting active avoidance of that arm when the animal was in the exposed condition. The increase in the open-arm entries was accompanied by a corresponding reduction in the number of entries into the closed arm with a consequent reduction in the time spent in the closed arm. The time spent in the closed arm after each entry was not altered by DAIZAC administration. As such, the sole apparent effect of DAIZAC was to alter the choice of arm to enter when the animal was in the central compartment. Diazepam also significantly increased total time in the open arm; however, the increase was not attributable to a single behavioral factor. The anxiolytic-like effects of DAIZAC reached maximum by 20-30 min and returned to baseline levels by 90 min. Ex vivo binding studies found that levels of DAIZAC-like activity assayed in brains of mice 25 min after DAIZAC injection were significantly correlated with the behavioral parameters associated with anxiolysis. These results indicate that DAIZAC produces dose-dependent anxiolytic-like behavioral changes in the mouse elevated plus-maze that are correlated with brain DAIZAC-like activity.

Characterization of (S)-des-4-amino-3-[125I]iodozacopride ([125I]DAIZAC), a selective high-affinity radioligand for 5-hydroxytryptamine3 receptors.

The 5-hydroxytryptamine(HT)3 receptor subtype is present in the central nervous system (CNS) in low abundance, and few selective radiolabeled antagonists with high specific activity are available to study these sites. DAIZAC [desamino-3-iodo-(S)-zacopride; (S)-5-chloro-3-iodo-2-methoxy-N-(1-azobicyclo-[2.2. 2]oct-3-yl)benzamide] is a compound with high affinity and selectivity for the 5-HT3 receptor. Scatchard analysis of specific binding to NCB-20 cell membranes gave a Bmax of 340 +/- 58 fmol/mg protein and a KD of 0.14 +/- 0.03 nM, which is in agreement with the value previously reported in rat brain (KD = 0.15 nM). Nonspecific binding of [125I]DAIZAC in NCB-20 cells was <1% of total binding at the KD for DAIZAC compared with 17% in the rat brain preparation. Unlabeled DAIZAC (10 microM) showed minimal ability to displace binding of radiolabeled ligands selected for their affinities for other CNS receptor and uptake carrier binding sites. The discrimination ratio of DAIZAC for the 5-HT3 receptor over the M1 muscarinic binding site, the non-5-HT3 site at which it was most potent, was >2800. Serotonergic antagonists at every other known CNS serotonergic binding sites (3-30 microM) were ineffective in displacing [125I]DAIZAC binding in rat brain membranes. Similarly, antagonists (3-30 microM) for other nonserotonergic receptors and uptake sites were ineffective in displacing [125I]DAIZAC binding. Autoradiographic studies showed highest specific binding in area postrema and nucleus solitarius, with intermediate levels of binding in entorhinal cortex and hippocampus. DAIZAC inhibited 5-HT3 receptor-mediated inward cation current in NCB-20 cells with an IC50 of 0.24 nM. [125I]DAIZAC is a potent and highly selective ligand for in vitro studies of the 5-HT3 receptor.

Characterization of desamino-5-[125I]iodo-3-methoxy-zacopride ([125I]MIZAC) binding to 5-HT3 receptors in the rat brain.

1. Antagonists at 5-HT3 receptors have shown activity in animal models of mental illness, however, few radiolabeled 5-HT3 ligands are available for preclinical studies. MIZAC, an analogue of the selective 5-HT3 antagonist, zacopride, binds with high affinity (1.3-1.5 nM) to CNS 5-HT3 sites. The authors report here the selectivity of MIZAC for these sites in rat brain homogenates. 2. Ninety-seven percent of total specific binding of [125I]MIZAC (0.1 nM) of was displaced by bemesetron (3 microM), a selective 5-HT3 antagonist. Competition studies using ligands with known affinities for 5-HT3 sites give a high correlation with reported pKi values (r2 0.98). Bemesetron displaceable binding has a regional distribution consistent with that of the 5-HT3 receptor, i.e. highest in cortex and hippocampus, and lowest in striatum and cerebellum. 3. Potent antagonists present at concentrations sufficient to occupy 95% of other 5-HT receptor populations (1A, 1B, 1D, 2A, 2B, 2C, 5A, 5B, 6, and 7) showed minimal ability to displace [125I]MIZAC binding (3 nM). Specificity studies using radioligand binding assays selective for 5-HT4, 5-HT6, and 5-HT7 receptors, and for binding sites of other neurotransmitters indicate a high degree of selectivity of [125I]MIZAC for the 5-HT3 receptor. 4. [125I]MIZAC binds to an apparent low affinity (benzac) site having a unique pharmacology. Low affinity binding was displaceable by benztropine, but not by other muscarinic agents nor inhibitors of dopamine uptake. The regional distribution of the low affinity site differed markedly from that of the high affinity site. The apparent affinity of [125I]MIZAC for the benzac site is two orders of magnitude lower than for the 5-HT3 receptor. Given its high selectivity for 5-HT3 binding sites, [125I]MIZAC appears to be a promising ligand for labeling 5-HT3 receptors in vitro and in vivo.

Synthesis and 5-HT-3 receptor binding of 2- and 3-(halo)alkoxyl substituted (S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chlorobenzamides as potential radioligands.

In an effort to develop selective, high-affinity radioligands for the 5-HT-3 receptor, a series of homologues of 5-chloro-2,3-dimethoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide (2b) was prepared in which individual methoxy groups were replaced by ethoxyl, (2-fluoroethoxyl), allyloxyl, propargyloxyl, or (3-iodoallyl)oxyl groups. Affinities for the 5-HT-3 receptor were determined by displacement of the binding of [125I]MIZAC (2a), a selective 5-HT-3 receptor antagonist radioligand, in rat brain homogenates. The 3-substituted homologues were more potent than the lead compound, 2b. The homologue having the largest 3-substituent, i.e., E-(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-3-(3-iodo-2-propenyl)oxy- 2-methoxybenzamide (3b, THIZAC), had one of the highest affinities, Ki 0.08 nM. The 2-substituted homologues were equipotent with 2b, having Ki 0.2-0.3 nM, regardless of the size of the substituent. The corresponding iodoallyl derivative, E-(S)-N-(1-azabicyclo[2.2.2]oct-3-yl)-5-chloro-2-(3-iodo-2-propenyl)oxy- 3-methoxybenzamide (4, LIZAC), displayed a Ki of 0.29 nM. Saturation binding of [125I]-4 gave a KD of 0.31 +/- 0.04 nM and a Bmax of 2.36 +/- 0.10 fmol/mg of entorhinal cortex. In vivo biodistribution of [125I]-4 in the rat brain showed increased accumulation in hippocampus relative to that in cerebellum. Both the high-affinity ligands [125I]-3b and [125I]-4 are potentially useful radioligands for studying the 5-HT-3 receptor.

Synthesis and radiolabeling of (S)-4-amino-5-iodo-2-methoxy-N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide, the active enantiomer of [125I]iodozacopride, and re-evaluation of its 5-HT3 receptor affinity.

We report an improved synthesis of unlabeled (S)-iodozacopride, the radiolabeling of (S)-[125I]iodozacopride via deschloro-(S)-zacopride, and a re-evaluation of its affinity for the 5-HT3 receptor. Unlabeled (S)-iodozacopride was prepared in seven steps from 4-aminosalicylic acid via alkaline hydrolysis of its 4-acetamide derivative. Catalytic hydrogenation of (S)-iodozacopride gave deschloro-(S)-zacopride, identical to that obtained from (S)-3-amino-quinuclidine and 4-amino-2-methoxybenzoic acid via its corresponding 1-imidazole derivative. Radioiodination to produce (S)-[125I]iodozacopride was accomplished by treatment of deschloro-(S)-zacopride with 5 mCi sodium 125iodide and chloramine-T in hydrochloric acid. Purification of the reaction products using an HPLC system capable of detecting chlorinated side-products revealed a mixture of 2.1 mCi (1.3 nmol) (S)-[125I]iodozacopride and (S)-zacopride (1.5 nmol). Saturation analysis of the binding of the purified (S)-[125I]iodozacopride to whole rat brain homogenates gave an estimated KD of 1.10 +/- 0.07 nM. As anticipated, this is approximately half the KD reported for binding of racemic [125I]iodozacopride, and differs from the previously reported value by an order of magnitude. Analysis of the apparent binding affinity of a 1:1 mixture of (S)-[125I]iodozacopride and (S)-zacopride suggests that the previous result may have been confounded by contamination of the product with unlabeled (S)-zacopride. Competition analysis of the displacement of (S)-[125I]iodozacopride binding by unlabeled (S)-iodozacopride and (S)-zacopride gave Ki values of 0.95 and 0.21 nM, respectively.

Radioreceptor-inactive growth hormone associated with stimulated secretion in normal subjects.

We have previously reported systematic discrepancies between radioreceptor (RRA) and radioimmunoassay (RIA) measurements of growth hormone (hGH) in acromegalic patients. Due to limitations in RRA sensitivity, such comparisons could not be made in normal subjects. RRA methodology has now been adapted to allow detection of hGH at normal circulating levels. Since variations in Na+, K+, Ca++, and Mg++, incubation at 37 C and 4 C, and delayed tracer addition failed to improve assay sensitivity, specimen size was increased to 300 mul and incubation volume to 1.5 ml, while holding the quantity of added receptor constant. Best assay sensitivity, in room temperature incubations in 25 mM Tris for 16 h at pH 7.6 and 10 mM Ca++, was 0.66 +/- 0.30 ng hGH per ml serum. Under these conditions, 200 mug hepatic receptor protein bount 15.8 +/- 0.83% of added 125I-hGH, and 8.72 +/- 0.85% of bound tracer was displaced by 0.25 ng added unlabeled hGH. Nonspecific depression of binding by serum did not impair assay sensitivity with most receptor preparations. The basal hGH measured by RIA (antiserum 68-416) in a group of normal short children was 1.97 ng/ml, similar to the RRA result, 1.89 ng/ml (P = NS). Comparative measurements were also made in selected samples of sufficient volume during the 1 1/2 h following administration of hGH secretagogues (insulin, arginine, L-dopa). In these samples, the RIA value was 9.34 +/- 0.68 and the RRA value 6.29 +/- 0.62 ng/ml (P less than 0.01); the RIA/RRA was 1.77 +/- 0.18. Thus, no significant measurement discrepancy was found in basal samples from normal subjects, in contrast to previous findings in acromegalics. The appearance of such a discrepancy within 90 min after stimulation of hGH might be due to RIA/RRA discordance in secreted molecular subspecies, or might arise from peripheral hGH metabolism.