Purification and properties of a human plasma endogenous modulator for the platelet tricyclic binding/serotonin transport complex.

An endogenous modulator for the site labeled by [3H]imipramine which is putatively coupled to the serotonin transporter in human platelets was isolated and purified from plasma. Procedures included sequential chromatography on Cibacron blue-Sepharose 4B, concanavalin A-Sepharose 4B, Mono Q HR 10/10 anion exchange, DuPont GF-250 gel permeation and Mono S HR 5/5 cation exchange columns. The purified modulator is a protein of Mr 45,000 with a very acidic pK (less than 3) and sensitive to various proteinases but heat- and acid-stable. This protein inhibited [3H]imipramine binding to platelet membranes competitively (IC50 approximately 6 microM) and enhanced serotonin uptake in fresh human platelets (EC50 approximately 7 microM). Various physicochemical properties, including chromatographic, electrophoretic and immunological as well as amino acid composition analysis revealed that the isolated protein is most probably the human alpha 1-acid glycoprotein.

A 1-year, placebo-controlled preservation of function survival study of donepezil in AD patients.

OBJECTIVE: To examine the effects of donepezil compared with placebo on the preservation of function in patients with AD over a 1-year period. METHODS: This was a prospective, 54-week, double-blind, placebo-controlled, survival to endpoint study. Patients were required to have at entry: a diagnosis of probable AD (National Institute of Neurological and Communicative Disorders and Stroke criteria); Mini-Mental State Examination score of 12 to 20; Clinical Dementia Rating of 1 or 2; modified Hachinski ischemia score < or =4; and capability of performing 8 of 10 instrumental activities of daily living and 5 of 6 basic activities of daily living. Patients (n = 431) were randomized to placebo or donepezil (5 mg/day for 28 days, 10 mg/day thereafter). Outcome measures were the AD Functional Assessment and Change Scale, the Mini-Mental State Examination, and Clinical Dementia Rating scale. At each visit, investigators determined whether predefined criteria for clinically evident decline in functional status had been met. Patients who met the endpoint criteria were discontinued per protocol. RESULTS: Donepezil extended the median time to clinically evident functional decline by 5 months versus placebo. The probability of patients treated with donepezil remaining in the study with no clinically evident functional loss was 51% at 48 weeks, compared with 35% for placebo. The Kaplan-Meier survival curves for the two treatment groups were different (p = 0.002, log-rank test). CONCLUSIONS: Patients with AD continue to show detectable disease progression over time, but treatment with donepezil for 1 year was associated with a 38% reduction in the risk of functional decline compared with placebo.

Responders to antidepressant drug treatment: a study comparing nefazodone, imipramine, and placebo in patients with major depression.

BACKGROUND: Nefazodone hydrochloride, an antidepressant that acts as a 5-HT2 antagonist and serotonin (5-HT) and norepinephrine uptake inhibitor, was evaluated in a double-blind, imipramine- and placebo-controlled study involving 128 patients with major depression. METHOD: Eligible patients were randomly assigned to receive placebo (2 to 6 capsules/day), imipramine (100 to 300 mg/day), or nefazodone (200 to 600 mg/day) for 8 weeks. The principal efficacy outcome measure assessed was the number of patients who experienced an adequate response during treatment. RESULTS: Based on global improvement (Clinical Global Impressions-Improvement), 67% of nefazodone-treated patients (p < or = .01) and 63% of imipramine-treated patients (p < or = .05) responded during 8 weeks of treatment, compared with 36% of placebo controls. Sixty-two percent of nefazodone-treated, 53% of imipramine-treated, and 26% of placebo-treated patients had 17-item Hamilton Rating Scale for Depression (HAM-D-17) scores < or = 10 on completion of acute treatment. Nefazodone-treated patients had a lower incidence of premature treatment discontinuation and fewer dropouts for adverse events than the imipramine group. CONCLUSION: In a three arm comparison with imipramine and placebo, nefazodone had the greatest number of patients with major depression who responded to therapy. Nefazodone, a new antidepressant with novel pharmacology, is a well-tolerated, efficacious antidepressant.

A placebo-controlled evaluation of single, escalating doses of CL 284,846, a non-benzodiazepine hypnotic.

This report describes the first evaluation in humans of CL 284,846, a non-benzodiazepine compound with a preclinical profile indicative of sedative/hypnotic properties. Healthy, normal male volunteers were assigned randomly to receive single oral doses of 1, 5, 15, 30, or 60 mg of CL 284,846 or placebo on a double-blind basis. Observations were made over the subsequent 25 hours to determine the safety, pharmacokinetic profile, and psychometric effects of the test compound. CL 284,846 was well tolerated in the normal volunteers, causing no significant changes in vital signs, EEG, ECG, hematologic, or clinical chemistry laboratory parameters. Although few adverse events were noted at doses less than 60 mg, at the highest dose (60 mg), all volunteers reported transient neurologically related adverse events (e.g., impaired concentration, difficulty focusing, and impaired coordination), reflecting the central nervous system action of the compound. Although determination of hypnotic efficacy was not an objective in this Phase I study, daytime treatment with 60 mg of CL 284,846 was associated with greater reports of drowsiness and impaired performance on psychomotor tests. However, memory, as assessed by a word recall test, was not affected at any dose of the compound. Pharmacokinetic analyses revealed CL 284,846 to be absorbed and eliminated rapidly (Tmax = 0.9-1.5 hr, T 1/2 = 0.9-1.1 hr), with a dose-proportional AUC (area under cure). Plasma levels of CL 284,859, the primary desethylated metabolite of CL 284,846, were much lower in humans than in other species, indicating that the metabolism of CL 284,846 in humans may differ from that of rodents and dogs. Overall, CL 284,846 was well tolerated, and the authors recommend repeating dose safety and pharmacokinetic studies in healthy volunteers.

Maternal aggression in mice: effects of treatments with PCPA, 5-HTP and 5-HT receptor antagonists.

Drug treatments which influence brain serotonergic systems were administered to lactating female mice during the early postpartum period, and their effects on aggressive behavior, locomotor activity and brain monoamines were examined. P-chlorophenylalanine (200 and 400 mg/kg) and 5-hydroxytryptophan (100 mg/kg) inhibited fighting behavior of postpartum mice toward unfamiliar male intruder mice. These drug-treated postpartum females showed increased latencies to attack male intruders and also reduced frequencies of attack. In addition, postpartum mice treated with the serotonin receptor antagonists, mianserin (2 and 4 mg/kg), methysergide (4 mg/kg) and methiothepin (0.25 and 0.5 mg/kg), displayed significantly less aggressive behavior than control mice, as measured by reduced number of attacks. Whole brain monoamine and monoamine metabolite levels were measured after drug treatments. The behavioral results are discussed in terms of drug-induced changes in brain chemistry and indicate a possible role for serotonin in the mediation of maternal aggressive behavior of mice.

Human platelets possess multiple [3H]imipramine binding sites.

Scatchard analysis of [3H]imipramine binding to human platelets over the concentration range of 0.1-250 nM revealed a biphasic concave upward curve. Computer-assisted analysis indicated the best fit of the data by a two-site model with apparent Kd values of 0.68 and 293 nM and apparent Bmax values of 802 fmol/mg protein and 12.72 pmol/mg protein for the high- and low-affinity components, respectively. These results demonstrate the complex manner in which [3H]imipramine binding to human platelets, suggesting a reason for inconsistencies in Bmax values reported in the literature. Further studies of these low- as well as high-affinity sites may help elucidate the functional role of [3H]imipramine binding sites in affective disorders.

Multiple [3H]imipramine binding sites in brains of male and female Fawn-Hooded and Long-Evans rats.

Comparisons of high- and low-affinity [3H]imipramine binding to whole brain homogenates from adult male and female rats of the Fawn-Hooded and Long-Evans strains were performed. Most strikingly, no significant differences were observed between the two strains in any of the binding parameters, indicating that brain [3H]imipramine binding sites, which may be related to the serotonergic uptake process, appear normal in a strain of rats with serotonin platelet storage pool disease. However, a significant sex difference in high- but not low-affinity whole brain [3H]imipramine Bmax values was observed, with females of both strains having higher densities than males. The observed sex difference in densities of high-affinity [3H]imipramine binding sites necessitates further research into possible sex hormone interactions with this binding site and serotonergic transmitter systems.

Long-term efficacy and safety of donepezil in the treatment of Alzheimer's disease: final analysis of a US multicentre open-label study.

This multicentre, open-label study evaluated the long-term efficacy and safety of donepezil in the treatment of patients with mild to moderately severe Alzheimer's disease (AD). The 133 patients who entered the study had previously completed a 14-week randomized, double-blind, placebo-controlled study with donepezil. In this open-label study, patients were treated initially with 3 mg per day donepezil, which could be increased to 5, 7 and 10 mg per day in a step-wise fashion. Patients attended the clinic for assessments at 3-week intervals for the first 12 weeks, then subsequently at 12-week intervals for up to 240 weeks (254 cumulative weeks). Efficacy was assessed using the Alzheimer's Disease Assessment Scale-cognitive subscale (ADAS-cog) and the Clinical Dementia Rating-Sum of the Boxes scale (CDR-SB), and data were compared with those predicted for historical untreated AD patients. During the first 6-9 months of the study, mean ADAS-cog and CDR-SB scores showed evidence of clinical improvement from baseline. After this time scores gradually deteriorated. Overall the decline was less than that estimated if this cohort of patients had not been treated. The most common adverse events were related to the nervous and digestive systems, and were generally mild and transient, resolving without the need for dose modifications. There was no evidence of hepatotoxicity. In conclusion, these data demonstrate that donepezil is a well-tolerated, realistic symptomatic treatment for AD over a period of up to 4.9 years. An interim report of the first 98 weeks of the study has been published previously.

Two satiety systems revealed by hypothalamic knife cuts in hypophysectomized rats.

When pituitaries are intact, hypothalamic knife cuts produce obesity in adult rats but not in weanlings. Knife cut weaning females do not usually begin to become obese until after they are 7 weeks old. When pituitaries are removed, symmetrical or asymmetrical knife cuts produce obesity promptly in both adults and weanlings. Obesity indices that correct for stunted linear growth reveal that in adults the degree of obesity is independent of the presence or absence of the pituitary. Based on these findings we speculate that there may be two appetite regulating systems, a juvenile one involving the pituitary, and an adult one involving the hypothalamus.

The 5-HT1A receptor probe [3H]8-OH-DPAT labels the 5-HT transporter in human platelets.

The present study characterizes a serotonin (5-HT) binding site on human platelet membranes, using [3H]8-OH-DPAT as the radioligand. [3H]8-OH-DPAT binds specifically and saturably to a site on human platelet membranes with an average KD of 43 nM and Bmax of 1078 fmol/mg protein. Determinations of IC50 values for various serotonergic characterizing agents in platelets for displacement of [3H]8-OH-DPAT were performed. For example, 8-OH-DPAT 5HT1A had an IC50 of 117 nM; TFMPP 5HT1B (2.3 microM0 and PAPP 1A + 5HT2 (9 microM); ipsapirone 5HT1A (21.1 microM) and buspirone 5HT1A (greater than 100 microM); ketanserin 5HT2 (greater than 100 microM); 5-HT uptake inhibitors: paroxetine (13 nM); chlorimipramine (73 nM) and fluoxetine (653 nM). The pharmacological inhibitory profile of the platelet 8-OH-DPAT site is not consistent with profiles reported for brain. 8-OH-DPAT does not inhibit [3H]imipramine binding, however, it does inhibit [3H]5-HT uptake in human platelets near 5-HT's Km value (IC50 = 2-4 microM). These results suggest that the human platelet site labeled by [3H]8-OH-DPAT is pharmacologically different from the neuronal site and probably is a component of the 5-HT transporter.

The serotonergic antidepressant nefazodone inhibits the serotonin transporter: in vivo and ex vivo studies.

Nefazodone HCl (Serzone) is a new antidepressant with a chemical structure unrelated to selective serotonin reuptake inhibitors (SSRIs), tricyclics, tetracyclics, or monoamine oxidase inhibitors (MAOIs). Nefazodone is active in a number of preclinical tests for antidepressant activity and shows clinical efficacy in the treatment of depression with a more favorable side-effect profile than the structurally similar antidepressant trazodone. Previous studies have shown that nefazodone is a potent antagonist of 5-HT2A receptors and binds to the serotonin transporter in vitro and in vivo. Nefazodone also binds to the norepinephrine transporter in vitro and in acute ex vivo studies. To further investigate the ability of nefazodone to modify serotonergic transmission, the ability of systemically administered nefazodone to inhibit the serotonin transporter was assessed by investigating the ability of nefazodone to prevent p-chloroamphetamine- (PCA) induced depletions of cortical 5-HT concentrations. In addition, the ability of acute and subchronic nefazodone administration to inhibit ex vivo [3H]-5-HT uptake was assessed. Acute administration of nefazodone (30, 100, and 150 mg/kg) antagonized PCA-induced depletion of cortical 5-HT concentrations in a dose-dependent manner at 1, 2, and 3 hours post-treatment. This effect was directly correlated with serum nefazodone concentrations. Both 100 mg/kg and 150 mg/kg of nefazodone were equipotent with fluoxetine (10 mg/kg) over the course of the experiment with respect to sparing of 5-HT depletion. Acute administration of nefazodone (100 and 150 mg/kg s.c.) significantly increased the Km for [3H]-5-HT uptake in rat cortical synaptosomes from 60 nmol/L in controls to 230 and 242 nmol/L in nefazodone-treated rats, respectively. Subchronic administration of nefazodone (100 and 150 mg/kg, s.c., b.i.d. x 5.5 days) reduced [3H]-5-HT uptake by 24% and 29%, respectively. Sub-chronic dosing with fluoxetine (5 mg/kg, s.c., b.i.d. x 5.5 days) reduced [3H]-5-HT uptake by 65%. These experiments confirm and extend previous reports concerning the ability of nefazodone to inhibit the 5-HT transporter in vivo.

Effects of dietary tyrosine on L-dopa- and amphetamine-induced changes in locomotor activity and neurochemistry in mice.

Recent findings suggest that intraperitoneal injections of L-tyrosine at high doses (100 mg/kg) alters amphetamine-induced changes in behavior by restoring amphetamine-induced decreases in whole brain norepinephrine (NE). The present study examined the motor effects of L-dihydroxyphenylalanine (L-dopa) and d-amphetamine sulfate in mice after treatment with a basal casein diet supplemented with L-tyrosine. The basal diet supplemented with 1-4% L-tyrosine, or 1-4% L-phenylalanine, produced no changes in motor activity in otherwise untreated mice. Whereas L-dopa (25-100 mg/kg) following inhibition of extracerebral decarboxylase by Ro 4-4602 (25 mg/kg) slightly decreased activity in diet control (casein) animals, this drug treatment enhanced motor activity in a dose-related fashion when L-tyrosine was added to the diet. Increases in motor activity following low doses of amphetamine (0.75-1.5 mg/kg) in casein control mice were antagonized by dietary L-tyrosine, but a higher dose of d-amphetamine (3 mg/kg) interacted with the addition of L-tyrosine producing an increase in motor activity. Neurochemical changes observed in brain concentrations of tyrosine, dopamine (DA), norepinephrine (NE), dihydroxyphenylacetic acid (DOPAC), homovanillic acid (HVA), tryptophan, serotonin (5-HT) and 5-hydroxy-indoleacetic acid (5-HIAA) following drug and diet treatments suggest that 5-HT systems, in addition to catecholamine systems, may be involved in mediating these effects.

Allosteric interaction between the site labeled by [3H]imipramine and the serotonin transporter in human platelets.

The nature of interaction between the site labeled by [3H]imipramine (IMI) and the 5-hydroxytryptamine (5-HT, serotonin) transporter in human platelets was examined. The sulfhydryl characterizing agent N-ethylmaleimide (NEM) differentially affected [3H]5-HT uptake and [3H]IMI binding in human platelet preparations. Concentrations of NEM that completely abolished [3H]5-HT uptake only minimally reduced [3H]IMI binding. Examining the effect of IMI on the kinetics of human platelet [3H]5-HT uptake revealed significant reductions in maximal velocity (Vmax) without altering affinity (Km). IC50 values for selected uptake blockers on [3H]IMI binding and [3H]5-HT uptake were determined. IC50 values of these compounds for uptake and binding revealed that agents such as IMI, chlorpromazine, amitriptyline, and nisoxetine were preferential inhibitors of [3H]IMI binding whereas fluoxetine, CL 216, 303, pyrilamine, and bicifadine were preferential [3H]5-HT uptake blockers. 5-HT was a weak displacer of [3H]IMI binding (IC25 = 3.0 microM) and exhibited a rather low Hill coefficient (nH app = 0.46). Results reported herein support the notion of an allosteric interaction between the [3H]IMI binding site and the 5-HT transporter complex in human platelets.

Characterization and autoradiographic visualization of (+)-[3H]SKF10,047 binding in rat and mouse brain: further evidence for phencyclidine/"sigma opiate" receptor commonality.

The binding specificity of (+)-[3H]N-allylnormetazocine, the dextrorotatory isomer of the prototypical sigma opiate SKF10,047, was determined in rat and mouse brain and the neuroanatomical distribution of its binding sites elucidated by quantitative autoradiography in sections of rat brain. Computer-assisted Scatchard analysis revealed an apparent two-site fit of the binding data in both species and in all rat brain regions examined. In whole rat brain, the Kd values were 3.6 and 153 nM and the maximum binding values were 40 fmol and 1.6 pmol/mg of protein for the apparent high- and low-affinity binding sites, respectively. (+)-SKF10,047, haloperidol and pentazocine were among the most potent inhibitors of 7 nM (+)-[3H]SKF10,047 binding to the higher affinity sites; rank orders of ligand potencies at these sites differ sharply from those that have been reported for the [3H]phencyclidine (PCP) site, or for eliciting PCP-like or SKF10,047-like behaviors. By contrast, rank orders of potency of sigma opiods, PCP derivatives and dioxolanes for displacement of 100 nM (+)-[3H]SKF10,047 from the more numerous lower affinity sites in the presence of 100 nM haloperidol agreed closely with their potencies in the [3H]PCP binding assay as well as their potencies in exerting PCP- or SKF10,047-like behavioral effects. In order to compare directly the anatomical localizations of PCP and (+)-SKF10,047 binding sites, quantitative light microscopy autoradiography utilizing tritium-labeled PCP and (+)-SKF10,047 was carried out in rat brain sections. (+)-[3H]SKF10,047 binding was observed to follow the regional pattern of [3H]PCP binding but also to bind in other regions not associated with PCP receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Donepezil: tolerability and safety in Alzheimer's disease.

The tolerability and safety of donepezil HCI in patients with mild to moderate Alzheimer's disease (AD) were examined in an integrated analysis of phase II/III placebo-controlled trials. Patients with mild to moderately severe AD (n=1,920) were randomised to receive donepezil (n=1,291) or placebo (n=629). Adverse events, physical examinations and clinical laboratory tests were assessed. A high completion rate (79%) was achieved in these trials. Of the 1,291 patients receiving donepezil only, 142 (11%) withdrew because of an adverse event compared with 43 of the 629 (7%) placebo patients. The most common adverse events included nausea, diarrhoea, headache, insomnia, dizziness, rhinitis, vomiting, asthenia/fatigue and anorexia. Donepezil had no clinically significant effect on any laboratory evaluations and was not associated with hepatotoxicity. These results demonstrate that donepezil is well tolerated and has a favourable safety profile at clinically effective, once-daily doses of 5 mg and 10 mg.

A multinational, randomised, 12-week, comparative study of donepezil and rivastigmine in patients with mild to moderate Alzheimer's disease.

This 12-week, multinational study compared the tolerability and cognitive effects of donepezil (up to 10 mg once daily) and rivastigmine (up to 6 mg twice daily) in 111 patients with mild to moderate Alzheimer's disease. Both medications were administered open label according to recommended dosing regimens from the respective product labelling available during the conduct of the study. More patients in the donepezil group (89.3%) completed the study compared with the rivastigmine group (69.1%; p=0.009), and 10.7% of the donepezil group and 21.8% of the rivastigmine group discontinued due to adverse events (AEs); 87.5% of donepezil-treated patients and 47.3% of rivastigmine-treated patients remained on the maximum approved dose of each drug at the last study visit. Both groups showed comparable improvements on the ADAS-cog administered by raters blind to study medication at weeks 4 and 12. Thus, using the recommended dosing schedules, donepezil was better tolerated with fewer discontinuations due to AEs, and both agents improved cognition to a similar extent.