Effects of zaleplon or triazolam with or without ethanol on human performance.

Objectives and background: Given that non-selective gamma-aminobutyric acid (GABA) agonist hypnotics impair performance and potentiate the disruptive effects of ethanol, this study was done to determine the performance-impairing and ethanol-potentiating effects of zaleplon, a new selective GABA agonist hypnotic.Methods: Eighteen healthy men (12) and women (six), 31.5+/-5.6 years old, were studied. Each underwent six treatments of 2 days in duration, presented in a Latin square design with 2-12 recovery days between. The treatments were: placebo-placebo; placebo-ethanol; triazolam-placebo; triazolam-ethanol; zaleplon-placebo; and zaleplon-ethanol; with triazolam (0.25 mg) or placebo administered at 08:30 h, zaleplon (10 mg) or placebo at 09:00 h, and ethanol (0.75 g/kg) or placebo consumed from 09:30 h. Performance tests were completed each day at 10:30, 12:00 and 14:30 h.Results: Breath ethanol concentration (BrEC), tested 0.5, 2.0, 4.5 and 6 h post consumption, did not differ among treatments and peaked at 0.052%, declining to 0.037, 0.009 and 0.001%. Triazolam with and without ethanol impaired digit symbol substitution, symbol copying, simple and complex reaction times and divided attention performance relative to placebo-placebo treatment. It did so consistently at 10:30 and 12:00 h, and less consistently at 14:30 h. Zaleplon without ethanol impaired only digit symbol substitution and divided attention tracking, and only at 10:30 h. Zaleplon with ethanol impaired most measures at 10:30 and 12:00 h, but not at 14:30 h. Zaleplon without ethanol consistently differed from triazolam without ethanol in the extent of performance impairment. Zaleplon with ethanol began to differ from triazolam with ethanol in performance impairment on the 12:00 and 14:30 h test sessions. Ethanol itself impaired most measures at 10:30 h, fewer at 12:00 h and none at 14:30 h. All active drug treatments increased self-rated sleepiness compared with placebo-placebo. Triazolam without ethanol produced greater self-rated sleepiness than zaleplon without ethanol. The addition of ethanol to both drugs generally produced comparable levels of self-rated sleepiness.Conclusions: In an absolute sense, zaleplon produced less performance impairment and a shorter period of ethanol potentiation than triazolam.

Efficacy and safety of zaleplon at peak plasma levels.

Zaleplon is a non-benzodiazepine sleep medication that shows efficacy as a sleep inducer comparable to that of other hypnotics but with significantly fewer residual effects. In addition, evaluations of psychomotor or memory function at zaleplon peak plasma levels show much less impairment than noted with other hypnotics, suggesting an improved benefit-to-risk profile for zaleplon compared with older available agents. Thus, zaleplon can be used to treat symptoms of insomnia when they occur without the concern of next-day psychomotor or memory impairment, whether administered at bedtime or later during the night. Such an approach permits physicians to reformulate their strategies for safe and effective management of sleeplessness.

Nuclear tyrosine phosphorylation: the beginning of a map.

Tyrosine phosphorylation is usually associated with cytoplasmic events. Yet, over the years, many reports have accumulated on tyrosine phosphorylation of individual molecules in the nucleus, and several tyrosine kinases and phosphatases have been found to be at least partially nuclear. The question arises as to whether nuclear tyrosine phosphorylation represents a collection of loose ends of events originating in the cytoplasm or if there may be intranuclear signaling circuits relying on tyrosine phosphorylation to regulate specific processes. The recent discovery of a mechanism causing nuclear tyrosine phosphorylation has prompted us to review the cumulative evidence for nuclear tyrosine phosphorylation pathways and their possible role. While we found that no complex nuclear function has yet been shown to rely upon intranuclear tyrosine phosphorylation in an unambiguous fashion, we found a very high number of compelling observations on individual molecules that suggest underlying networks linking individual events. A systematic proteomics approach to nuclear tyrosine phosphorylation should help chart possible interaction pathways.

Zaleplon improves sleep without producing rebound effects in outpatients with insomnia. Zaleplon Clinical Study Group.

The efficacy and safety of three doses of zaleplon, a novel non-benzodiazepine hypnotic, were compared with those of placebo in outpatients with insomnia in this 4-week study, using zolpidem 10 mg as active comparator. Postsleep questionnaires were used to determine treatment effects on the patient's perception of sleep, as well as any development of pharmacological tolerance during therapy or rebound insomnia or withdrawal symptoms upon discontinuation of therapy. During week 1, sleep latency was significantly shorter with zaleplon 5, 10, and 20 mg compared to placebo. The significant decrease in sleep latency persisted through week 4 with zaleplon 20 mg, and was again evident with zaleplon 10 mg at week 3. Zaleplon 20 mg also had significant effects on sleep duration, number of awakenings, and sleep quality compared to placebo. No pharmacological tolerance developed during treatment with zaleplon and there were no indications of rebound insomnia or withdrawal symptoms after treatment discontinuation. Zolpidem 10 mg had significant effects on sleep latency, sleep duration, and sleep quality compared to placebo. However, a significantly greater incidence of withdrawal symptoms and a suggestion of sleep difficulty after treatment discontinuation (rebound insomnia) for all sleep measures was seen with zolpidem compared to placebo. There was no significant difference in the frequency of adverse events with active treatment compared to placebo. These results show that zaleplon provides effective treatment of insomnia with a favourable safety profile.

Comparison of the effects of zaleplon, zolpidem, and triazolam on memory, learning, and psychomotor performance.

Twenty-four healthy male and female subjects, who participated in this randomized, double-blind, crossover study, received single nighttime doses of zaleplon 10 mg (therapeutic dose), zaleplon 20 mg, zolpidem 10 mg (therapeutic dose), zolpidem 20 mg, triazolam 0.25 mg (positive control), and placebo. Subjective behavioral ratings and psychomotor tests were completed before and 1.25 and 8.25 hours after administration of the study drug. The Immediate and Delayed Word Recall tests and the Digit Span Test were used to assess memory. The Digit-Symbol Substitution Test, Paired Associates Learning Test, and Divided Attention Test were used to assess other cognitive skills. Zaleplon 10 mg did not produce any significant changes in memory or learning compared with placebo. All other active treatments, including zolpidem 10 mg, caused psychomotor impairment at the 1.25-hour test battery. Zolpidem 20 mg (twice the therapeutic dose) produced more psychomotor impairment at the 1.25-hour assessment than did any of the other active treatments, including zaleplon 20 mg. At the 8.25-hour time point, test scores for subjects who received zaleplon 10 mg and 20 mg did not differ from the test scores for those who received placebo. However, cognitive impairment persisted up to the 8.25-hour observation for subjects who were administered triazolam 0.25 mg and zolpidem 20 mg. Adverse events associated with the use of zaleplon were transient and mild-to-moderate in severity. Overall, this study shows that zaleplon is a safe hypnotic that does not affect memory, learning, or psychomotor skills associated with vigilance.

A nuclear tyrosine phosphorylation circuit: c-Jun as an activator and substrate of c-Abl and JNK.

The nuclear function of the c-Abl tyrosine kinase is not well understood. In order to identify nuclear substrates of Abl, we constructed a constitutively active and nuclear form of the protein. We found that active nuclear Abl efficiently phosphorylate c-Jun, a transcription factor not previously known to be tyrosine phosphorylated. After phosphorylation of c-Jun by Abl on Tyr170, both proteins interacted via the SH2 domain of Abl. Surprisingly, elevated levels of c-Jun activated nuclear Abl, resulting in activation of the JNK serine/threonine kinase. This phosphorylation circuit generates nuclear tyrosine phosphorylation and represents a reversal of previously known signalling models.

Chimeric amplicons containing the c-myc gene in HL60 cells.

The major amplicon present in HL60 cells is chimeric in nature being composed of 70 kb of DNA sequence derived from the MYC locus linked to 80 kb of novel DNA sequence derived from a non contiguous region located telomeric to the c-myc gene at 8q24 (Feo et al., 1996). Here we show by fluorescence in situ hybridization (FISH) that these coamplified sequences, MCR (Myc Coamplified Region), are derived from a locus located 3-4 Mb telomeric to the c-myc gene in the q24.2-24.3 region of chromosome 8. Genomic cloning and Southern blot analysis indicate the arrangement of chimeric amplicons are in tandem arrays. Analysis of the DNA sequences at the juncture of the MYC locus and the MCR suggest that these non syntenic regions were joined by nonhomologous recombination events. Visualization of the organization of the amplified DNA by fiber-FISH analysis illustrates we have cloned the complete amplicon. This is the first complete mammalian amplicon to be cloned and have its structure visualized. In addition to the major class of tandemly repeated amplicons, a second class of amplicons was detected by fiber-FISH in which the extent of the MCR component is about twice the size of the MCR component in the major amplicon. These longer amplicons most likely contain inverted repeats of MCR and MYC region sequences. Whether the amplicons contain mixtures of these two types of structures or separate amplicons only contain one type of structure has not yet been resolved. Properties of the MCR sequences responsible for retention in the chimeric HL60 amplicons upon long term passage are discussed.

The amplicons in HL60 cells contain novel cellular sequences linked to MYC locus DNA.

We have previously determined that the amplified DNA present in the HL60 promyelocytic leukaemia cell line contained 70 kb of continuous DNA sequences around the c-myc gene. In the work presented here we have further defined the HL60 amplicon and find it to be of the order of 160 kb and to contain a large region of DNA from chromosome 8q24 that is located at least 260 kb telomeric to the c-myc gene, joined to the 70 kb of DNA from the c-myc gene region. The novel chromosome 8 DNA coamplified sequences are not lost during multiple passage of HL60 cells since the composition of the chimeric amplicon is the same in both early passage HL60 cells containing only double minutes (DMs) and late passage isolates containing homogeneous staining regions (HSRs) at different chromosomal locations. This shows that the HL60 HSRs found in late passage cells are not generated anew but are directly derived from the precursor DMs. The HL60 cells contain two copies of chromosome 8, each with different polymorphic markers. Both these chromosome 8 homologues retain a c-myc gene as well as the region containing the coamplified DNA sequences indicating that the HL60 amplicons were not generated by simple DNA deletion. The constraint to maintain the novel DNA sequences coamplified with c-myc gene DNA suggests that these sequences may play some role in maintaining the growth potential and/or differentiation capacity of the HL60 cells.

Modulation of atrial repolarization by site of pacing in the isolated rabbit heart.

BACKGROUND: Single-site or multisite atrial pacing may reduce the incidence of atrial fibrillation in humans. The therapeutic mechanisms may include synchronization of atrial repolarization (repolarization "memory") and/or decreased dispersion of atrial repolarization. These responses have not been well documented in intact atria. METHODS AND RESULTS: Monophasic action potential recordings were made from six atrial epicardial sites in 39 isolated perfused rabbit heart preparations during 3 hours of continuous right atrial, left atrial, or biatrial pacing. Action potential recordings obtained at times 0, 45, 90, 135, and 180 minutes were computer analyzed for activation time (AT) and 90% action potential duration (APD) at each site. No consistent relationship could be demonstrated between APD and AT at any time during atrial pacing (all P > .05). On average, left atrial APDs were longer than right atrial APDs by up to 6.3 ms at all times, regardless of the site of pacing (P < or = .05). At all times, dispersion of atrial repolarization was minimized by left atrial pacing compared with right atrial pacing (21.6 +/- 9.1 versus 32.4 +/- 15.1 ms, respectively, at time 0; P < .05). Biatrial pacing provided no further reduction in dispersion of repolarization compared with left atrial pacing (all P > .05). CONCLUSIONS: No relationship can be demonstrated between atrial AT and APD in the isolated rabbit heart preparation. This differs from ventricular repolarization "memory," which is demonstrable under the same conditions. Left atrial APD is, on average, longer than right atrial APD, suggesting spatial heterogeneity in repolarization. Dispersion of atrial repolarization is minimized by left atrial pacing in this preparation with no further advantage to biatrial pacing.

The synthesis and biochemical pharmacology of enantiomerically pure methylated oxotremorine derivatives.

Previous pharmacological studies of methylated oxotremorine derivatives bearing substituents at the 3-, 4-, and 5-positions of the pyrrolidinone ring have been conducted using racemic mixtures, and not with optically active compounds. The synthesis and radioligand binding data of optically active, methylated oxotremorine derivatives at the 3- and 4-positions are described. There are significant pharmacological differences between the 3- and 4-position derivatives. The 4-position enantiomers have weak, approximately equal affinity and antagonist-like profiles, whereas the 3-position enantiomers have significantly different affinities and partial agonist-like profiles.

Interleukin-1 beta decreases acetylcholine measured by microdialysis in the hippocampus of freely moving rats.

Interleukin (IL-1) is a cytokine which plays an important role in the modulation of the acute response in host defense. This cytokine is also increased in patients with Alzheimer's disease. In the present experiment systemic injection of IL-1 beta (7.5-50 micrograms/kg) decreased extracellular acetylcholine in the hippocampus. This effect could not be attributed entirely to general malaise since lithium chloride (130 mg/kg) had the opposite effect. Heat-inactivation of the cytokine eliminated the reduction of extracellular ACh. The results give further evidence of a relationship between the immune system and the central nervous system and suggest a possible relationship between IL-1 and cholinergic function or dysfunction in the hippocampus.

Chemical and biochemical studies of 2-propynylpyrrolidine derivatives. Restricted-rotation analogues of N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide (BM-5).

A series of optically pure 2-[substituted-3-aminopropynyl]pyrrolidine derivatives, which are restricted-rotation analogues of the muscarinic agent N-methyl-N-(1-methyl-4-pyrrolidino-2-butynyl)acetamide (BM-5, compound 1), have been prepared from d- and l-proline. The compounds when tested in a series of in vitro muscarinic assays [[3H]CD (cortex), [3H]QNB (cortex), [3H]PZ (cortex), [3H]QNB (heart), [3H]QNB + GppNHp (heart)] were found to have weaker muscarinic properties than compound 1. The decrease in affinity was attributed to the increased size of the molecule resulting from the addition of a methylene group to form the pyrrolidine ring. The use of optically active compounds provided a more detailed examination of the complex pharmacological effects of the flexible muscarinic agent 1. The R enantiomers in the acetamide derivatives 12b, 12d, and 12f had a 5-10-fold greater affinity for the muscarinic receptor than the corresponding S enantiomers. A 5-fold difference or less found in the (R)- and (S)-carbamate derivatives 9, 15, and 16 suggested close overlap of the two enantiomers in the receptor binding domain. The affinity differences found in the enantiomeric acetamido derivatives when compared to those of the carbamate analogues may be the result of limited rotation of the acetamido group.

Quinolinic acid: an endogenous metabolite that produces axon-sparing lesions in rat brain.

A current hypothesis links the neuroexcitatory properties of certain acidic amino acids to their ability to cause selective neuronal lesions. Intracerebral injection of the neuroexcitatory tryptophan metabolite, quinolinic acid, has behavioral, neurochemical, and neuropathological consequences reminiscent of those of exogenous excitotoxins, such as kainic and ibotenic acids. Its qualities as a neurotoxic agent suggest that quinolinic acid should be considered as a possible pathogenic factor in neurodegenerative disorders.

Chronic infusion of endogenous excitatory amino acids into rat striatum and hippocampus.

The effects of chronic intrastriatal and intrahippocampal infusions of endogenous excitatory amino acids were examined on the light microscopic level. Repeated manual injections of either glutamate or aspartate (1.8 mumoles/0.5 mu 1 every 12 hours for 14 days), but not of equimolar GABA, caused neuronal degeneration proximal to the tip of the injection cannula. Nerve cell death was limited to a spherical area with a radius of approximately 0.5 mm. Continuous infusion of these amino acids via osmotic minipumps, at total daily doses identical to those used in the experiments employing manual injections, did not result in lesions in striatum or hippocampus. Attempts using minipump administration of cysteine sulfinate or combinations of glutamate with aspartate, cysteine sulfinate or DL-threo-beta-hydroxyaspartate were equally unsuccessful in producing neuronal cell loss. The efficient re-uptake systems for the endogenous amino acids may suffice to continuously remove large quantities of the infused compounds from vulnerable neuronal sites. It appears, however, that these protective mechanisms can be overcome by extremely high local concentrations of glutamate or aspartate. Possible implications for the pathogenesis of human neurodegenerative disorders are discussed.

Huntington's disease. Glutamate and aspartate metabolism in blood platelets.

The metabolism of two excitatory amino acids, glutamate and aspartate, was determined in human blood platelets because of their hypothetical involvement in the etiology of the heritable neurodegenerative disorder, Huntington's disease (HD). The specific activities of the two acids and their amides, glutamine and asparagine, constitute markers for the efficiency of the enzymatic cascades originating from [3H]acetate and [14C]glucose, respectively. Results demonstrate that both precursors are converted by temperature-dependent processes, thus indicating the presence, in platelets, of all glycolytic and tricarboxylic acid cycle enzymes necessary for the biosynthesis of glutamate and aspartate. No differences between normal and HD platelets were found in any of the specific activities investigated. A genetic defect in any of these enzymes is therefore not likely to underlie HD neuropathology.

Platelet glutamate and aspartate uptake in Huntington's disease.

A possible involvement of amino acid uptake mechanisms in the etiology of the human neurodegenerative disease, Huntington's disease (HD), was investigated. Measurement of glutamate (Glu) and aspartate (Asp) uptake was performed in blood platelets, which have previously been shown to constitute a peripheral model system for central amino acid uptake processes. Analyses of Glu and Asp accumulation at 10(-7) M and kinetic examination of the high affinity site for Glu indicate no significant differences between control and HD platelets. A genetically determined defect in amino acid uptake therefore does not seem to underlie the nerve cell loss observed in HD patients.

The human platelet as a model for the glutamatergic neuron: platelet uptake of L-glutamate.

L-Glutamate uptake into human platelets revealed two components: a high-affinity system (KmH = 3.1 microM), which was sodium-dependent, and a low-affinity site (Km = 88 microM) displaying temperature rather than sodium dependency. These kinetic properties were similar to those found in crude synaptosomal preparations and brain slices. However, Vmax values were far higher in brain (VmaxH = 325 +/- 96, VmaxL = 3759 +/- 1116 pmol/mg wet weight per min) than in platelets (VmaxH = 14 +/- 6, VmaxL = 313 +/- 63 pmol/mg platelet protein per 10 min), indicating a denser population in brain than in platelets of qualitatively similar sites. Pharmacological analysis substantiated the resemblance of nerve endings and platelets: the specific uptake inhibitors threo-3-hydroxy-DL-aspartate and DL-aspartate-beta-hydroxamate as well as D- and L-glutamate and L-aspartate showed similar--though not identical-- IC50 values in both preparations; a spectrum of compounds devoid of inhibitory effects in synaptosomes also did not interfere with glutamate uptake in platelets. Uptake parameters were studied in a population of human volunteers to determine the variability of platelet glutamate uptake. Whole blood could be stored up to 6 h after venipuncture without any appreciable change in experimental values. Percentage of variation between 0.09 and 0.28 for three repetitive (weekly) assays in single subjects indicated that glutamate uptake measurements in human platelets are sufficiently suited for future clinical studies.