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.

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.

Effects of chronic intrahippocampal infusion of lipopolysaccharide in the rat.

Astrogliosis and microglial activation are associated with many neurodegenerative disorders including multiple sclerosis, its animal model experimental allergic encephalomyelitis, and Alzheimer's disease. To address the hypothesis that chronic astroglial or microglial activation could be contributing factors to neuronal death or injury, the immunostimulant lipopolysaccharide was infused into the hippocampus for 16 days using Alzet mini-osmotic pumps attached to a cannula. Placement of the cannula and infusion of vehicle for 16 days caused a hippocampal lesion with a volume of 0.5 +/- 0.1 mm3. Infusion of lipopolysaccharide at the dose of 2.0 micrograms/day produced a lesion of 4.9 +/- 1.3 mm3 (P < 0.01, Newman-Keuls), whereas, a lower dose of 0.2 microgram/day caused a lesion of 1.3 +/- 0.3 mm3 (P < 0.05). The lesion was defined as a focal necrotic reaction with fibrin deposits outlining an area at an early stage of encapsulation. No apparent neuronal loss was observed by Cresyl Violet staining outside the encapsulated necrotic area. There was a pronounced astrogliosis and an increase in activated macrophages throughout the lipopolysaccharide-infused hippocampus as determined by glial fibrillary acidic protein and ED-1 immunohistochemistry, respectively. Choline acetyltransferase and glutamic acid decarboxylase enzyme activities, used as functional measures of neuronal viability for cholinergic and GABAergic neurons, respectively, were unaffected in the hippocampus following a 16 day infusion of lipopolysaccharide at the doses of 0.2, 0.6 and 2.0 micrograms/day. In addition, unilateral infusion of lipopolysaccharide into the hippocampus did not affect 24 h locomotion when tested on day 13, body temperature or weight gain. Under the experimental conditions employed in the present study, chronic infusion of lipopolysaccharide into the hippocampus resulted in a dose-dependent focal necrotic lesion at the site of infusion. In tissue surrounding the encapsulated lesion, neurons were present among the reactive astrocytes and increased number of macrophages suggesting that astrocytes and macrophages can be activated without causing neuronal loss.

The pharmacological profile of iloperidone, a novel atypical antipsychotic agent.

Iloperidone (1-[4-[3-[4-(6-fluoro-1,2-benzisoxazol-3-yl)-1- piperidinyl]propoxy]-3-methoxyphenyl]ethanone) demonstrated a potent antipsychotic profile in several in vitro and in vivo animal models. Iloperidone displaced ligand binding at D2 dopamine receptors (IC50 = 0.11 microM) and displayed a high affinity for serotonin (5-HT2) receptors (IC50 = 0.011 microM) and alpha-1 receptors (IC50 = 0.00037 microM). In vivo, iloperidone antagonized apomorphine-induced climbing behavior in mice at low doses with good oral bioavailability, prevented 5-HT-induced head twitch in rats at low doses, and inhibited self-stimulation behavior in rats, pole climb avoidance in rats and continuous Sidman avoidance responding in monkeys. The latter assay also demonstrated a good duration of action. Iloperidone was substantially less active in models of extrapyramidal side effect (EPS) liability, such as preventing apomorphine-induced stereotypy and causing catalepsy in rats. In single dopamine neuron sampling studies, iloperidone demonstrated clozapine-like effects on the number of active midbrain dopamine neurons. Based on the significant increase in the open arm time seen after iloperidone treatment in the elevated plus maze assay and increased interaction score in social interaction, iloperidone may also have favorable effects in the clinic on anxiety and, possibly, negative symptoms. Clinical trials are under way of the use of iloperidone for the treatment of schizophrenia.

Synthesis and evaluation of 5-amino-5,6,7,8-tetrahydroquinolinones as potential agents for the treatment of Alzheimer's disease.

A series of 5-amino-5,6,7,8-tetrahydroquinolinones was designed and synthesized as acetylcholinesterase inhibitors. The compounds are related to hyperzine A, a naturally occurring cholinesterase inhibitor. They inhibit acetylcholinesterase in vitro, and many are active in vivo in reversing a scopolamine-induced impairment of 24 h memory in a passive avoidance paradigm. Although these compounds were designed as partial structures of huperzine A, it is unlikely that they bind to the enzyme in a similar fashion, since they lack the unsaturated three-carbon bridge of huperzine A and both the quinolinone nitrogen and the amino group must be substituted in order to obtain good enzyme affinity.

Velnacrine maleate improves delayed matching performance by aged monkeys.

Velnacrine maleate is a novel, orally active acetylcholinesterase inhibitor of the acridine class with a longer duration of action than physostigmine. Velnacrine has shown efficacy in the treatment of Alzheimer's disease and in improving both normal and experimentally impaired mnemonic function in animals and humans. To characterize this action further, the present study evaluated velnacrine for its ability to ameliorate the decline in short-term memory associated with aging in non-human primates at two time points after velnacrine administration: (1) 30 min and (2) 24 h. Initially, doses of 1, 2, 4, and 6 mg/kg, PO (free base corrected) were administered once to each of six aged (25-40 years), memory-impaired macaques that had been trained to perform a delayed matching-to-sample (DMTS) paradigm. The dose associated with the greatest improvement in session performance was administered three more times to the same individual. Four of the six monkeys showed improved DMTS performance during the repeated best dose phase (phase 2). Almost all of the improvement occurred during long-delay trials. Compared to placebo trials, velnacrine induced a significant improvement of long delay DMTS (58.0-66.7%, 13.4% of the placebo value). Long delay DMTS remained significantly improved during the test session conducted 24 h following velnacrine administration. Pharmacokinetic analysis following administration of 4 or 6 mg/kg velnacrine to three aged monkeys revealed peak plasma concentrations ranging from 27 to 166 ng/ml, 30-60 min after dosing. Six hours after dosing velnacrine plasma levels decreased to 5.1-11.8 ng/ml; and 24 h after dosing velnacrine plasma levels were less than the limit of quantitation (5 ng/ml).(ABSTRACT TRUNCATED AT 250 WORDS)

Synthesis, resolution, and SAR of (+/-)-2-amino-N-methyl-alpha-(3-methyl-2-thienyl)benzeneethanamine++ + and related analogs as noncompetitive NMDA antagonists with neuroprotective properties.

The preparation and structure-activity relationships of a series of 2-amino-alpha-thienylbenzeneethanamines are described. From this work, (+/-)-2-amino-N-methyl-alpha-(3-methyl-2-thienyl)-benzeneethanamine++ + (3a) and the homologous N-ethyl analog 3b emerged as novel noncompetitive NMDA antagonists with neuroprotective properties. Optical resolution of 3a and X-ray crystallography of (+)3a were performed. The racemate and enantiomers were evaluated for neuroprotective properties in models of ischemia-induced hippocampal damage (gerbil) and cerebral focal ischemia (rat). Pretreatment with 3a, (+)3a, or (-)3a significantly reduced ischemia-induced CA1 hippocampal damage. Posttreatment with 3a afforded a lower degree of neuroprotection. A highly significant reduction in infarct volume was observed with 3a in the cerebral focal ischemia model, with only weak positive effects being displayed by (+)3a. Dose-limiting side effects were associated with all three compounds in this model. In summary, the results demonstrate the utility of noncompetitive NMDA antagonists as neuroprotective agents for ischemia-induced neurodegeneration.

Intracerebral beta-amyloid(25-35) produces tissue damage: is it neurotoxic?

beta-Amyloid (1-40) and (25-35) have been reported to be toxic to primary cultured neurons. beta-Amyloid (1-40) was also reported to induce neurodegeneration following intracerebral injection. We attempted to replicate and extend these findings by injecting both the full length amyloid peptide and the 25-35 fragment. beta 1-40 (3 nmol in 1 microliter) or beta 25-35 (20 nmol in 2 microliters) in a vehicle of 10% DMSO (3 and 10 mM concentration, respectively) induced tissue loss and neurodegeneration. We also attempted to prevent the amyloid-induced damage by coinjecting 200 nmol of Substance P. There was no obvious reduction in the size of the lesions. Other studies, however, have reported antagonism of amyloid toxicity with tachykinin agonists. Since beta-amyloid does not appear to bind to tachykinin receptors, there is some question as to the site of the putative interaction of these peptides and, therefore, the mechanism by which beta-amyloid induces tissue damage. Our own results and published cell culture toxicity studies suggest that aggregation of the peptide and physical displacement of tissue may be responsible for both the neuronal and tissue loss, although this hypothesis is not consistent with other published findings.

Memory modulation with peripherally acting cholinergic drugs.

Physostigmine (PHYSO), in doses as low as 0.003 mg/kg IP, antagonized scopolamine (SCOP, 3 mg/kg) induced amnesia of step-through passive avoidance in mice. The peripherally acting acetylcholinesterase (AChE) inhibitor neostigmine (NEO) was also found to reliably, though less strongly, antagonize the SCOP induced amnesia at a dose of 0.03 mg/kg. The NEO antagonism of the SCOP amnesia could be reversed with SCOP (0.3, 1, and 3 mg/kg) and mecamylamine (MECA, 1, 3, and 10 mg/kg), muscarinic and nicotinic antagonists, respectively, which are active both peripherally and centrally, as well as with M-SCOP (0.3 and 1 mg/kg) and hexamethonium (HEX, 1 and 3 mg/kg), muscarinic and nicotinic antagonists, respectively, which are active only in the periphery. In contrast to the ability of these four compounds to attenuate the SCOP amnesia, only the centrally acting compounds SCOP (3 mg/kg) and MECA (10 mg/kg) induced an amnesia when administered alone. These findings suggest that the induction of amnesia of passive avoidance involves central cholinergic systems, whereas the NEO, and possibly PHYSO, reversal of the SCOP induced amnesia is mediated peripherally by both muscarinic and nicotinic receptors. It is hypothesized that the release of adrenal catecholamines, the influence of which on memory processes is well known, and secondarily glucose, may be responsible for the NEO antagonism of the SCOP amnesia.

Preclinical anticonvulsant and neuroprotective profile of 8319, a non-competitive NMDA antagonist.

8319, ((+-)-2-Amino-N-ethyl-alpha-(3-methyl-2-thienyl)benzeneethanamine 2HCl), is a novel compound with the profile of a non-competitive NMDA antagonist. The compound displaced [3H] TCP with high affinity (IC50 = 43 nM), but was inactive at the NMDA, benzodiazepine and GABA sites; in vivo, 8319 showed good efficacy as an anticonvulsant and potential neuroprotective agent. It blocked seizures induced by NMDLA, supramaximal electroshock, pentylenetetrazol (PTZ), picrotoxin, and thiosemicarbazide with ED50's of 1-20 mg/kg ip. As a neuroprotective agent, 8319 (30-100 mg/kg sc) prevented the death of dorsal hippocampal pyramidal cells induced by direct injection of 20 nmol NMDA. At 15 mg/kg ip, the compound was also effective against hippocampal neuronal necrosis induced via bilateral occlusion of the carotid arteries in gerbils. In summary, 8319 is a noncompetitive NMDA antagonist with good anticonvulsant activity and may possess neuroprotective properties useful in the treatment of brain ischemia.

Preclinical anxiolytic profiles of 7189 and 8319, novel non-competitive NMDA antagonists.

Antagonists at excitatory amino acid receptors, especially the N-methyl-d-aspartate (NMDA) subtype, have been shown to possess anticonvulsant and anxiolytic properties (Clineschmidt et al., 1982; Croucher et al., 1982; Bennett and Amrick, 1986). 7189 and 8319, two closely related benzeneethanamines, are potential novel anxiolytic agents which bind with high affinity to the NMDA receptor at the non-competitive site and are relatively non-toxic (LD50's-160 mg/kg, ip). 7189 and 8319 showed anxiolytic effects in schedule controlled conflict assays as well as in the social interaction (SI) and elevated plus maze (EPM) procedures in rats. Following intraperitoneal administration of 7189 at 20 to 60 mg/kg, conflict responding was increased from 2- to 7-fold in the modified Cook and Davidson and Geller conflict paradigms. 8319, at 2.5 to 5 mg/kg, produced a two fold increase in conflict responding. In the non-schedule controlled procedures, 7189 at 20 mg/kg increased SI time by 23% while in the EPM at 10 to 20 mg/kg, open arm exploration time increased by 41 to 77%. Likewise, 8319 at 2.5 and 5 mg/kg increased open arm exploration and SI time by 50 and 37%, respectively. In summary, 7189 and 8319 were efficacious in four behavioral procedures predictive of potential anxiolytic agents. Although these compounds have not been submitted for clinical evaluation, they may represent a new class of beneficial compounds for the treatment of anxiety.

Scopolamine amnesia of passive avoidance: a deficit of information acquisition.

Despite its increasing use as an animal model of memory deficit in human dementia, relatively few studies have attempted to assess the memory processes involved in the anticholinergic-induced impairment of passive avoidance retention. In the present experiments, the influence of scopolamine administered prior to or immediately following training on 24-h retention of step-through passive avoidance was studied in NMRI mice. In low doses (0.3-3.0 mg/kg ip) pretraining administration (-5 min) of scopolamine induced a very strong amnesia. Post-training scopolamine induced a significant effect only at the highest dose tested (30 mg/kg). In a retention test of longer than normal duration (600 vs 180 s), which resulted in a more favorable comparison value in the control group, an intermediate post-training dose (10 mg/kg) induced a small effect which approached significance; a finding which may account for conflicting reports in the literature concerning the ability of scopolamine to induce a post-training deficit. The pretraining effect does not appear to have been solely the result of state-dependent learning; scopolamine (3 mg/kg) administered before both the training and test sessions induced a deficit of approximately the same magnitude as that found when administered before training or before testing only. The results indicate that scopolamine can induce a small post-trial effect, presumably through an influence on consolidation processes. The much larger effect of pretrial scopolamine, however, indicates a primary influence on processes related to information acquisition. Together with findings from the literature, the present experiments suggest that scopolamine-induced amnesia partially, but not completely, models the memory deficits of human dementia.

Shuttlebox Sidman avoidance in rhesus monkeys: day of week and amphetamine effects.

Due to its stability and sensitivity, the Sidman avoidance schedule has been often used to characterize the psychotropic effects of drugs. In the present study, the effects of d-amphetamine (0.125, 0.25, and 0.5 mg/kg IM) on shuttlebox Sidman avoidance by rhesus monkeys were investigated. Amphetamine resulted in increased avoidance rates as shown by both bin and mean inter-response time (IRT) analyses. These results demonstrate the potential usefulness of this combination of task, species, and apparatus for investigating the effects of psychotropic substances. In addition, analysis of baseline data indicated a small but significant day of week effect with more efficient performance at the end of the week. The implications of this finding for conducting drug studies involving repeated measurements (i.e., cross-over designs) are discussed.

Reversal of scopolamine-induced amnesia of passive avoidance by pre- and post-training naloxone.

In a series of five experiments, the modulating role of naloxone on a scopolamine-induced retention deficit in a passive avoidance paradigm was investigated in mice. Scopolamine, but not methyl scopolamine (1 and 3 mg/kg), induced an amnesia as measured by latency and duration parameters. Naloxone (0.3, 1, 3, and 10 mg/kg) injected prior to training attenuated the retention deficit with a peak of activity at 3 mg/kg. The effect of naloxone could be antagonized with morphine (1, 3, and 10 mg/kg), demonstrating the opioid specificity of the naloxone effect. Post-training administration of naloxone (3 mg/kg) as a single or as a split dose also attenuated the scopolamine-induced amnesia. Control experiments indicated that neither an increase in pain sensitivity (pre-training naloxone) nor an induced aversive state (post-training naloxone) appear to be responsible for the influence of naloxone on the scopolamine-induced retention deficit. These results extend previous findings implicating a cholinergic-opioid interaction in memory processes. A possible mechanism for this interaction involving the septo-hippocampal cholinergic pathway is discussed.