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Like fluoxetine, the N-demethylated metabolite norfluoxetine exists in R- and S-enantiomeric forms. S-Norfluoxetine inhibited serotonin (5-HT) uptake and [3H]paroxetine binding to 5-HT uptake sites with a pKi of 7.86 and 8.88 or 14 and 1.3 nM, respectively, whereas R-norfluoxetine was 22 and 20 times, respectively, less potent. R- and S-Norfluoxetine were less potent than the corresponding enantiomers of fluoxetine as inhibitors of norepinephrine uptake and [3H]tomoxetine binding to norepinephrine uptake sites. Ex vivo studies showed that S-norfluoxetine inhibited 5-HT uptake with an ED50 of 3 mg/kg intraperitoneally, 4.7 mg/kg subcutaneously, and 9 mg/kg orally (7.3, 11.4 and 21.9 mumol/kg, respectively), while the ED50 for R-norfluoxetine exceeded 20 mg/kg intraperitoneally (48.6 mumol/kg). Inhibition of 5-HT uptake in cerebral cortex ex vivo and decrease in 5-HIAA levels in hypothalamus persisted for 24 hours after administration of S-norfluoxetine as demonstrated with the administration of fluoxetine. Thus, S-norfluoxetine is the active N-demethylated metabolite responsible for the persistently potent and selective inhibition of 5-HT uptake in vivo.

LY248686 is an inhibitor of serotonin (5-hydroxytryptamine; 5-HT) and norepinephrine (NE) uptake in synaptosomal preparations of hypothalamus and cerebral cortex, and 5-HT uptake in human blood platelets, with inhibitor constants near nanomolar concentrations. Upon administration to rats 1 hour before sacrifice, LY248686 caused dose-dependent and parallel decreases of 5-HT and NE uptake in hypothalamus homogenates ex vivo. LY248686 is a positive enantiomer and was slightly more potent than its negative isomer, LY248685, as an inhibitor of 5-HT uptake. Both isomers were only weak inhibitors of dopamine (DA) uptake in striatal synaptosomes. The inhibitory effects on 5-HT and NE uptake after a single administration of LY248686 followed similar time courses and simultaneously persisted for as long as 6 hours. LY248686 in vivo could effectively antagonize the p-chloroamphetamine-induced decreases of 5-HT uptake and levels of 5-HT and 5-hydroxyindoleacetic acid in cerebral cortex, and block the accumulation of 14C-NE in rat hearts. In food deprived rats, LY248686 suppressed food intake synergistically with 5-hydroxytryptophan, a precursor amino acid of 5-HT. Because of its lack of affinity for receptors of 5-HT, NE, DA, acetylcholine, histamine and naloxone, and its ability to inhibit 5-HT and NE uptake simultaneously, LY248686 has a favorable pharmacological profile as a potential antidepressant drug.

The advent of potent, highly selective 5HT3 receptor antagonists has stimulated considerable interest in 5HT3 receptor mediated physiology and pharmacology. To permit detailed biochemical studies regarding interaction of the indazole class of serotonin (5HT) antagonists with 5HT3 receptors in multiple tissues, we synthesized 1-methyl-N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole- 3-carboxamide (LY278584, compound 9) in high specific activity, tritium-labeled form. This radioligand was selected as a synthetic target because of its potency as a 5HT3-receptor antagonist, its selectivity for this receptor viz a viz other 5HT-receptor subtypes, and the ability to readily incorporate three tritia via the indazole N-CH3 substituent. Alkylation of N-(8-methyl-8-azabicyclo[3.2.1]oct-3-yl)-1H-indazole-3-carboxamide (8) with sodium hydride and tritium-labeled iodomethane, followed by HPLC purification, resulted in [3H]-9 with a radiochemical purity of 99% and a specific activity of 80.5 Ci/mmol. This radioligand bound with high affinity to a single class of saturable recognition sites in membranes isolated from cerebral cortex of rat brain. The Kd was 0.69 nM and the Bmax was 16.9 fmol/mg of protein. The specific binding was excellent, and accounted for 83-93% of total binding at concentrations of 2 nM or less. The potencies of known 5HT3-receptor antagonists as inhibitors of [3H]-9 binding correlated well with their pharmacological receptor affinities as antagonists of 5HT-induced decreases in heart rate and contraction of guinea pig ileum, suggesting the central recognition site for this radioligand may be extremely similar to or identical with peripheral 5HT3 receptors.

A number of cytokines have been found to be potent regulators of bone resorption and to share the properties originally attributed to osteoclast-activating factor. One such activity, differentiation-inducing factor (DIF, D-factor) from mouse spleen cells, shares a number of biological and biochemical properties with the recently characterized and cloned leukemia inhibitory factor (LIF). We have assessed the effects of recombinant LIF on bone resorption and other parameters in neonatal mouse calvaria. Both recombinant murine and human (h) LIFs stimulated 45Ca release from prelabeled calvaria in a dose-dependent manner. The increase in bone resorption was associated with an increase in the number of osteoclasts per mm2 bone. The osteolytic effect of hLIF were blocked by 10(-7) M indomethacin. hLIF also stimulated incorporation of [3H] thymidine into calvaria, but the dose-response relationship was distinct from that for bone resorption, and this effect was not blocked by indomethacin. Similarly, hLIF increased [3H]phenylalanine incorporation into calvaria, and this was also not inhibited by indomethacin. It is concluded that LIF stimulates bone resorption by a mechanism involving prostaglandin production, but that a distinct mechanism is responsible for its stimulation of DNA and protein synthesis. The primary structure of LIF differs from that of other fully characterized, bone-active cytokines, and it, thus, represents a novel factor which may be involved in the normal regulation of bone cell function.

A total of 15 cultures of Leptospira were examined for aminopeptidase activity using 22 aminoacyl-beta-naphthylamde substrates. Activity was demonstrated in each of the cultures. Extracts from serovars of Leptospira interrogans preferentially hydrolysed the same range of substrates. The level of hydrolysis of the preferred substrates for the seven strains of L. interrogans was distinctively higher than that demonstrated for the six Leptospira biflexa strains. Extracts from cultures of Leptospira illini and Leptospira parva sp. nov. exhibited profiles different to those demonstrated for the other 13 leptospiral cultures examined.

Isapirone, a 5-hydroxytryptamine1A receptor agonist, stimulated food intake in non-fasted rats in a dose-dependent manner (1, 3 and 10 mg kg-1 s.c.). Fenfluramine, an antiobesity agent and a 5-HT releaser, at 3 and 10 mg kg-1 s.c. antagonized the isapirone-induced (3 mg kg-1 s.c.) feeding. These results are consistent with the known inhibitory role of 5-HT in the control of food intake in rats.

Binding of [3H]LY278584 a 1-methyl-indazole-carboxamide, to putative 5-HT3 recognition sites in membranes isolated from cerebral cortex of rat brain, is examined. Specific binding of [3H]LY278584 accounts for 83-93% of total binding. The unlabelled LY278584 has 500 times greater affinity for [3H]LY278584 recognition sites than its 2-methyl analogue (LY278989), and their potencies parallel their antagonism of the peripheral 5-HT3 receptors. Moreover, the order of potencies of other known antagonists of 5-HT3 receptors supports the conclusion that [3H]LY278584 binds to putative 5-HT3 receptors in cortical membranes.

R- and S-enantiomers of fluoxetine lowered food intake in meal-fed rats and in 2-deoxyglucose-induced hyperphagic rats. In both feeding paradigms, the S-enantiomer was slightly more potent. The potency of the two enantiomers of fluoxetine in producing anorectic effects paralleled their potency as inhibitors of 5-hydroxytryptamine (5HT) uptake in vivo. Both enantiomers were selective inhibitors of 5HT uptake in vitro and showed only weak affinity for 5HT-1, 5HT-1A and 5HT-2 receptors or for other receptors in rat brain. The anorectic effect of fluoxetine in meal-fed rats was not reversed by either centrally or peripherally acting 5HT-2 receptor antagonists (ritanserin, LY53857, xylamidine, BW 501C67) or a nonspecific 5HT receptor antagonist, metergoline. However, the serotonergic mechanism involved in the anorexic effect of fluoxetine is discussed.

LY227942, (+/-)-N-methyl-3-(1-naphthalenyloxy)-3-(2-thiophene)propanamine ethanedioate, is a new, competitive inhibitor of monoamine uptake in synaptosomal preparations of rat brain. LY227942 inhibits uptake of serotonin (5-hydroxytryptamine, 5HT) and norepinephrine (NE) in cortical synaptosomes and uptake of dopamine (DA) in striatal synaptosomes with inhibitor constants (Ki values) of 8.5, 45 and 300 nM, respectively. Upon administration in vivo, LY227942 lowers 5HT and NE uptake in hypothalamus homogenates to half their respective control activities (ED50) at 0.74 and 1.2 mg/kg s.c., 7 and 12 mg/kg i.p., and 12 and 22 mg/kg p.o., but LY227942 at doses up to 30 mg/kg p.o. does not change DA uptake in striatal homogenates. Lowering of 5HT and NE uptake is demonstrated after 15 min and 6 hr, but has dissipated by 16 hr after oral administration. According to radioligand binding determinations, LY227942 possesses only weak affinity for muscarinic receptors, histamine-1 receptors, adrenergic receptors, dopamine receptors and serotonin receptors. These findings suggest that LY227942 has the pharmacological profile of an antidepressant drug and is useful to study the pharmacological responses of concerted enhancement of serotonergic and noradrenergic neurotransmission.

Fluoxetine and nine other antidepressant drugs which interact with brain receptors for neurotransmitters were studied in vitro using radioligand-binding techniques and transmitter-coupled adenylate cyclase assays. Tricyclic antidepressant drugs (desipramine, imipramine, clomipramine, amitriptyline and doxepin) had marked affinity for alpha-adrenergic, muscarinic cholinergic and histaminergic H1 receptors, and lesser affinity for serotonin and dopamine receptors. Mianserin was relatively similar to some of the tricyclic compounds, whereas trazodone had less affinity for most receptors except serotonin and alpha-adrenergic receptors. Fluoxetine had little affinity for any of these receptors, and the same was true for zimelidine and fluvoxamine, two other selective inhibitors of serotonin uptake. None of the compounds showed much affinity for beta-adrenergic receptors, opiate receptors, gamma-aminobutyric acid receptors, or benzodiazepine receptors. The present findings with fluoxetine are consistent with the virtual absence of anticholinergic or other side effects often observed with tricyclic antidepressant drugs in animal models or during the treatment of depressed patients.

LY163502, a partial ergoline and a trans-levorotatory enantiomer, does not stimulate adenylate cyclase in striatal membranes but inhibits 50% binding of 3H-apomorphine, 3H-pergolide and 3H-spiperone at 10, 13 and 151 nM (IC50), respectively. The racemic mixture (LY137157) is less effective, with 3, 2.7 and 1.4 times higher IC50 values, respectively, whereas the dextrorotatory isomer (LY175877) is inactive. LY163502 inhibits binding of 3H-clonidine with an IC50 value of 2600 nM, but not the binding of 3H-WB4101, 3H-dihydroalprenolol, 3H-serotonin, 3H-quinuclidinyl benzilate and 3H-pyramilamine or the uptake of serotonin, norepinephrine or dopamine, suggesting selective affinity toward dopamine receptors in vitro. Both LY163502 and LY137157 elevate striatal acetylcholine (Ach) levels. The elevation of Ach levels by LY163502 is reversed by dopamine antagonists haloperidol, cis-flupenthixol and metoclopramide. Therefore, the levorotatory enantiomer exhibits pharmacology of a D2 type of dopamine agonist.