IL-4, IL-10 and IL-13 modulate A beta(1--42)-induced cytokine and chemokine production in primary murine microglia and a human monocyte cell line.

A hallmark of the immunopathology associated with Alzheimer's disease (AD) is the presence of activated microglia surrounding senile plaque deposits of beta-amyloid (A beta) peptides. A beta peptides have been shown to be potent activators of microglia and macrophages, but little is known about endogenous factors that may modulate their responses to amyloid. We investigated whether the 'anti-inflammatory' cytokines IL-4, IL-10 and IL-13 could regulate A beta-induced production of the inflammatory cytokines IL-1 alpha, IL-1 beta, TNF-alpha, IL-6 and the chemokine MCP-1. A beta(1-42) time- and dose-dependently induced the production and secretion of these inflammatory proteins in the human THP-1 monocyte cell line and in primary murine microglia, similar to what was observed for lipopolysaccharide (LPS) stimulated cells. IL-10 was found to suppress all A beta and LPS-induced inflammatory proteins measured (IL-1 alpha, IL-1 beta, IL-6, TNF-alpha and MCP-1) in both cell types with the exception of LPS-induced MCP-1 in THP-1 cells where no change was observed. In contrast to the inhibition observed for IL-10, both IL-4 and IL-13 enhanced MCP-1 secretion. IL-4 and IL-13 reduced IL-6 secretion, but effects on IL-1 alpha, IL-1 beta or TNF-alpha were dependent on cell type and stimulus conditions. Additional experiments using RT-PCR showed that IL-4, IL-10 and IL-13 mRNA is found to be present in human brain tissue. These results show that IL-4, IL-10, and IL-13 differentially regulate microglial responses to A beta and may play a role in the inflammation pathology observed surrounding senile plaques.

Enhancement of beta-amyloid precursor protein transcription and expression by the soluble interleukin-6 receptor/interleukin-6 complex.

We investigated a potential role for the soluble interleukin-6 receptor (sIL-6R) in modulating interleukin-6 (IL-6) function in the central nervous system by assessing IL-6 and sIL-6R effects on beta-amyloid precursor protein (beta-APP) transcription and expression in cells of human neuronal origin. Cells transfected with a luciferase reporter plasmid containing a 3.8 kb DNA fragment of the beta-APP promoter were shown to have inducible promoter activity when treated with phorbol ester or basic fibroblast growth factor, but not when treated with lipopolysaccharide or Il-6. PCR amplification analysis revealed the presence of mRNA encoding the signaling subunit of the Il-6 receptor complex, the gp130 subunit, at levels approximating that found in human cortical tissue. The mRNA encoding the IL-6 receptor, however, was poorly expressed and was detectable only at high amplification cycles. When purified sIL-6R protein was added together with IL-6, there was a rapid induction of promoter activity within 2 h of stimulation followed by elevations in protein levels of both cell-associated and secreted beta-APP. Analysis of mRNA transcripts from human cortical brain tissue and cell cultures derived from fetal human brain demonstrated the presence of an alternatively spliced secreted form of the IL-6 receptor mRNA, suggesting that cells of the central nervous system may themselves be a source of sIL-6R protein. The capacity for sIL-6R to enhance IL-6 function and broaden the IL-6 target cell population in the brain has implications for the regulation of beta-APP expression in disease states such as Alzheimer's disease where elevations in brain IL-6 levels have been reported.

Additive effects of basic fibroblast growth factor and phorbol ester on beta-amyloid precursor protein expression and secretion.

Expression of the beta-amyloid precursor protein (beta-APP), a proteoglycan whose proteolytically derived fragments have been implicated in the neuropathology observed in Alzheimer's disease, is regulated by a variety of stimuli including cytokines, phorbol esters, and growth factors. In this study we report the effects of basic fibroblast growth factor (bFGF) and the protein kinase C activator, phorbol 12-myristate 13-acetate (PMA), on beta-APP expression and secretion in SKNMC human neuroblastoma cells. Treatment of the cells with bFGF for 24 h increased APP promoter activity 200%, cell-associated full-length protein 189%, and secreted amino-terminal fragments 192% compared to basal levels. Treatment of the cells with PMA for 24 h also up-regulated APP expression and secretion with increases of 170, 112, and 161% being observed for promoter activity, cell-associated full-length protein, and secreted amino-terminal fragments, respectively. The effects of bFGF and PMA on the expression and secretion of beta-APP were additive and distinct in that: (a) co-treatment of the cells with maximally stimulating doses of bFGF and PMA had an additive effect on both induced full-length protein expression (242%) and secretion of amino-terminal fragments (311%) compared to basal levels; (b) net levels of full-length protein expression and secretion induced by bFGF and PMA differed significantly from each other; and (c) down-regulation of phorbol ester-stimulated protein kinase C by pre-treatment of the cells for 24 h with 1 microM PMA failed to attenuate bFGF-induced transcription or induced secretion of beta-APP.

Pharmacological activity and safety profile of P10358, a novel, orally active acetylcholinesterase inhibitor for Alzheimer's disease.

1-[(3-Fluoro-4-pyridinyl)amino]-3-methyl-1(H)-indol-5-yl methyl carbamate (P10358) is a potent, reversible acetylcholinesterase inhibitor that produces central cholinergic stimulation after oral and parental administration in rats and mice. P10358 is a 2.5 times more potent acetylcholinesterase inhibitor than THA in vitro (IC50 = 0.10 +/- 0.02 microM vs. IC50 = 0.25 +/- 0.03 microM). It also inhibits butyrylcholinesterase activity as potently as THA (IC50 = 0.08 +/- 0.05 microM vs. IC50 = 0.07 +/- 0.01 microM). Ex vivo, P10358 (0.2 - 20 mg/kg, p.o.) produced dose-dependent inhibition of brain acetylcholinesterase activity. At 10 and 20 mg/ kg, it produced profound and long-lasting hypothermia in mice. P10358 enhanced performance in rats in a step-down passive avoidance task (0.62 and 1.25 mg/kg) and in a social recognition paradigm (0.32, 0.64 and 1.25 mg/kg) in mice. It reversed scopolamine-induced deficits in the Morris Water maze in rats (1.25 and 2.5 mg/kg) and a higher dose elevated striatal homovanillic acid levels. These behavioral and biochemical effects are consistent with central cholinergic stimulation. Hemodynamic studies in the rat demonstrated a 16-fold separation between behaviorally active doses (1.25 mg/kg) and those that elevated arterial pressure (20 mg/kg). Lethality in rats occurred at an oral dose of 80 mg/kg, but not at lower doses. Chemically, P10358 is an N-aminoindole and may not have the hepatotoxic liability associated with aminoacridine structure of tacrine. P10358 had weak affinity (>10 microM) at a variety of aminergic and peptidergic receptors and uptake carriers. These properties suggest that P10358 may be a safe and promising symptomatic treatment for Alzheimer's disease.

Transcriptional inhibition of the beta-amyloid precursor protein by interferon-gamma.

Attenuating beta-amyloid precursor protein (beta-APP) gene expression may have relevance in diseases such as Alzheimer's disease, where beta-APP has been implicated in neuropathological processes. We report here on the transcriptional down-regulation of beta-APP by interferon-gamma (IFN-gamma) in SKNMC human neuroblastoma cells. Treatment of the cells with IFN-gamma resulted in a 85% dose-dependent inhibition of beta-APP promoter activity after 24 h of exposure, with no changes observed at 5 h. For comparison, additional cytokines and signaling agents were also investigated for effects on beta-APP promoter activity. Elevated levels of activity were observed after treatment with phorbol 12-myristate 13-acetate and basic fibroblast growth factor whereas no significant effects were seen after treatment with lipopolysaccharide or interleukin-1 beta. Thus, IFN-gamma was shown here to be a suppressor of beta-APP promoter activity and is the first cytokine reported to possess such down-regulating effects.

Pharmacological evaluation of novel Alzheimer's disease therapeutics: acetylcholinesterase inhibitors related to galanthamine.

Acetylcholinesterase (AChE) inhibitors from several chemical classes have been tested for the symptomatic treatment of Alzheimer's disease; however, the therapeutic success of these compounds has been limited. Recently, another AChE inhibitor, galanthamine hydrobromide (GAL), has shown increased clinical efficacy and safety. Using biochemical, behavioral and pharmacokinetic analyses, this report compares GAL with two of its analogs, 6-O-acetyl-6-O-demethylgalanthamine hydrochloride (P11012) and 6-O-demethyl-6-O[(adamantan-1-yl)-carbonyl]galanthamine hydrochloride (P11149), for their therapeutic potential. P11012 and P11149 were found to be potent, competitive and selective inhibitors of AChE, demonstrating central cholinergic activity, behavioral efficacy and safety. P11012 and P11149, though pharmacokinetic analyses, were shown to act as pro-drugs, yielding significant levels of 6-O-demethylgalanthamine. In vitro, 6-O-demethylgalanthamine was 10- to 20-fold more potent than GAL as an inhibitor of AChE, and it demonstrated greater selectivity for inhibition of AChE vs. butyrylcholinesterase. Like GAL, both P11012 and P11149 showed central cholinergic activity biochemically, by significantly inhibiting rat brain AChE; physiologically, by causing hypothermia; and behaviorally, by attenuating scopolamine-induced deficits in passive avoidance. In addition, GAL, P11012 and P11149 enhanced step-down passive avoidance, another measure of behavioral efficacy. By comparing efficacious doses with primary overt effects, P11012 and P11149 had better oral therapeutic indices than GAL. Oral pharmacokinetic analyses of GAL, P11012 and P11149 revealed differences. Although P11012 and P11149 exhibited similar area under the curve values, 191149 had slower, lower and more sustained concentration maximum levels. P11012 and GAL rapidly reached their concentration maximums, but GAL, in brain had the highest area under the curve and concentration maximum. Because of its composite profile, including duration of action, oral therapeutic index and pharmacokinetics, P11149 is considered the better therapeutic candidate for the treatment of Alzheimer's disease.

Serotonergic activity of HP 184: does spontaneous release have a role?

Examination of HP 184, [N-n-propyl)-N-(3-fluoro-4-pyridinyl) -1H-3-methylindodel-1-amine hydrochloride], in a variety of tests for serotonergic activity revealed some unique properties of this compound. We report here that 100 microM HP 184 enhanced spontaneous release of [3H]serotonin (5-HT) from rat hippocampal slices. This release was independent of the uptake carrier. In vivo assays confirmed that HP 184 (20 mg/kg, i.p.) lacked significant interactions at the norepinephrine (NE) or 5-HT uptake carrier itself. Notably, HP 184 (15 mg/kg, i.p.) reduced drinking behavior in schedule-induced polydipsic (SIP) rats. We previously reported that some selective 5-HT reuptake inhibitors decrease SIP 30-40% after a 14-21 day treatment. In the current study, HP 184 decreased SIP beginning with the first treatment, and this reduction (30%) was maintained for 28 days. We further investigated HP 184 and serotonin metabolite levels. One hour after i.p. administration of 30 mg/kg HP 184, the ratio of whole brain 5-hydroxyindolacetic acid (5-HIAA) to 5-HT was increased, suggesting serotonergic activation. Under these conditions, the brain:plasma ratio of HP 184 was approximately 2:1, with brain concentrations of 1.6 micrograms/gram. We speculate that the spontaneous release effects of HP 184 may be responsible for the behavioral effects observed.

Synthesis and structure-activity relationships of N-propyl-N-(4-pyridinyl)-1H-indol-1-amine (besipirdine) and related analogs as potential therapeutic agents for Alzheimer's disease.

A series of novel N-(4-pyridinyl)-1H-indol-1-amines and other heteroaryl analogs was synthesized and evaluated in tests to determine potential utility for the treatment of Alzheimer's disease. From these compounds, N-propyl-N-(4-pyridinyl)-1H-indol-1-amine (besipirdine, 4c) was selected for clinical development based on in-depth biological evaluation. In addition to cholinomimetic properties based initially on in vitro inhibition of [3H]quinuclidinyl benzilate binding, in vivo reversal of scopolamine-induced behavioral deficits, and subsequently on other results, 4c also displayed enhancement of adrenergic mechanisms as evidenced in vitro by inhibition of [3H] clonidine binding and synaptosomal biogenic amine uptake, and in vivo by reversal of tetrabenazine-induced ptosis. The synthesis, structure-activity relationships for this series, and the biological profile of 4c are reported.

Substituted (pyrroloamino)pyridines: potential agents for the treatment of Alzheimer's disease.

A novel series of substituted (pyrroloamino)pyridines was synthesized, and the compounds were evaluated for cholinomimetic-like properties in vitro (inhibition of [3H]quinuclidinyl benzilate binding) and in vivo (reversal of scopolamine-induced dementia) as potential agents for the treatment of Alzheimer's disease. Compounds displaying significant activity were more broadly evaluated, which revealed the presence of a desirable adrenergic component of activity. The synthesis and structure-activity relationships for this series is presented, along with the biological profiles of selected compounds.

HP 749 enhances calcium-independent release of [3H]norepinephrine from rat cortical slices and synaptosomes.

Previous studies have shown that, at concentrations of 1 microM and 10 microM, HP 749 increased electrically-stimulated release of [3H]norepinephrine (NE) from rat cortical slices. These effects were Ca(2+)-dependent, indicating an effect on release from vesicular stores. At 100 microM, HP 749 had two effects. In addition to enhancing the Ca(2+)-dependent electrically-evoked release, it also induced a rise in the basal efflux (spontaneous release) of [3H]NE, which was observed in both cortical slices and synaptosomes. The spontaneous release effect was (1) not blocked by the reuptake inhibitor nomifensine, (2) not affected by removal of external calcium, (3) not blocked by vesicular depletion with reserpine, and (4) not inhibited by the sodium channel blocker tetrodotoxin (TTX). As would be expected, the spontaneous [3H]NE release induced by the cytoplasmic releaser tyramine and the sodium channel activator veratridine were blocked by nomifensine and TTX, respectively. Notably, however, the Ca(2+)-independent veratridine-induced release was completely blocked by 100 microM HP 749. The mechanism of spontaneous release of [3H]NE caused by 100 microM HP 749 is unresolved at present; however, the data are consistent with this release originating from a cytoplasmic source.

Aminopyridine carbamic acid esters: synthesis and potential as acetylcholinesterase inhibitors and acetylcholine releasers.

4-Amino-3-pyridyl carbamates (2a-c) were synthesized as potential acetylcholinesterase inhibitors and acetylcholine releasers on the basis of the reported activity of the analogous N-(4-amino-3-pyridyl)-N',N'-dimethylurea (1). Although 4-amino-3-pyridyl N,N-dimethylcarbamate (2b) showed good cholinesterase inhibition [concentration that elicited a 50% reduction in the maximal enzyme response (IC50) was 13.4 microM], it had no effect on the stimulated release of [3H]acetylcholine from rat striatal slices. 4-[[(Dimethylamino)methylene]amino]-3-pyridyl N,N-dimethylcarbamate (7a), an intermediate in the synthesis of 2b, demonstrated surprisingly good cholinesterase inhibition (IC50 was 9.4 microM) but showed no activity as a release. A precursor to 7a, N-(3-hydroxy-4-pyridyl)-N',N'-dimethylformamidine (6a), showed some activity in release but was not an esterase inhibitor, whereas the precursor to 6a, 4-amino-3-pyridinol (5a), was a potent releaser. A new synthesis of 5a, based on an ortho-directed lithiation strategy, is also reported.

3-substituted-1,2-benzisoxazoles: novel antipsychotic agents.

A series of 3-substituted-6-fluoro-1,2-benzisoxazoles (II) was synthesized and evaluated for potential antipsychotic activity. Many of the compounds displayed potent antipsychotic-like activity in the apomorphine induced climbing in mice (CMA) or spiroperidol binding assays, and compound 42 (HRP 392, 1-[3-(6-fluoro-1,2-benzisoxazol-3-yl)propyl]-4-(2-methoxyphenyl) piperazine) was selected for more detailed antipsychotic evaluation in a battery of preclinical assays. The results of these studies suggests that 42 is a potential antipsychotic drug with less propensity for EPS than some standard neuroleptics in monkeys. The compound was advanced for toxicological evaluation.