Pharmacological Evaluation of the Long-Term Effects of Xanomeline on the M1 Muscarinic Acetylcholine Receptor

Xanomeline is a unique agonist of muscarinic receptors that possesses functional selectivity at the M1 and M4 receptor subtypes. It also exhibits wash-resistant binding to and activation of the receptor. In the present work we investigated the consequences of this type of binding of xanomeline on the binding characteristics and function of the M1 muscarinic receptor. Pretreatment of CHO cells that stably express the M1 receptor for 1 hr with increasing concentrations of xanomeline followed by washing and waiting for an additional 23 hr in control culture media transformed xanomeline-induced inhibition of [3H]NMS binding from monophasic to biphasic. The high-affinity xanomeline binding site exhibited three orders of magnitude higher affinity than in the case of xanomeline added directly to the binding assay medium containing control cells. These effects were associated with a marked decrease in maximal radioligand binding and attenuation of agonist-induced increase in PI hydrolysis and were qualitatively similar to those caused by continuous incubation of cells with xanomeline for 24 hr. Attenuation of agonist-induced PI hydrolysis by persistently-bound xanomeline developed with a time course that parallels the return of receptor activation by prebound xanomeline towards basal levels. Additional data indicated that blockade of the receptor orthosteric site or the use of a non-functional receptor mutant reversed the long-term effects of xanomeline, but not its persistent binding at an allosteric site. Furthermore, the long-term effects of xanomeline on the receptor are mainly due to receptor down-regulation rather than internalization

Effects of Anacetrapib in Patients with Atherosclerotic Vascular Disease

BACKGROUND: Patients with atherosclerotic vascular disease remain at high risk for cardiovascular events despite effective statin-based treatment of low-density lipoprotein (LDL) cholesterol levels. The inhibition of cholesteryl ester transfer protein (CETP) by anacetrapib reduces LDL cholesterol levels and increases high-density lipoprotein (HDL) cholesterol levels. However, trials of other CETP inhibitors have shown neutral or adverse effects on cardiovascular outcomes. METHODS: We conducted a randomized, double-blind, placebo-controlled trial involving 30,449 adults with atherosclerotic vascular disease who were receiving intensive atorvastatin therapy and who had a mean LDL cholesterol level of 61 mg per deciliter (1.58 mmol per liter), a mean non-HDL cholesterol level of 92 mg per deciliter (2.38 mmol per liter), and a mean HDL cholesterol level of 40 mg per deciliter (1.03 mmol per liter). The patients were assigned to receive either 100 mg of anacetrapib once daily (15,225 patients) or matching placebo (15,224 patients). The primary outcome was the first major coronary event, a composite of coronary death, myocardial infarction, or coronary revascularization. RESULTS: During the median follow-up period of 4.1 years, the primary outcome occurred in significantly fewer patients in the anacetrapib group than in the placebo group (1640 of 15,225 patients [10.8%] vs. 1803 of 15,224 patients [11.8%]; rate ratio, 0.91; 95% confidence interval, 0.85 to 0.97; P=0.004). The relative difference in risk was similar across multiple prespecified subgroups. At the trial midpoint, the mean level of HDL cholesterol was higher by 43 mg per deciliter (1.12 mmol per liter) in the anacetrapib group than in the placebo group (a relative difference of 104%), and the mean level of non-HDL cholesterol was lower by 17 mg per deciliter (0.44 mmol per liter), a relative difference of -18%. There were no significant between-group differences in the risk of death, cancer, or other serious adverse events. CONCLUSIONS: Among patients with atherosclerotic vascular disease who were receiving intensive statin therapy, the use of anacetrapib resulted in a lower incidence of major coronary events than the use of placebo. (Funded by Merck and others; Current Controlled Trials number, ISRCTN48678192 ; ClinicalTrials.gov number, NCT01252953 ; and EudraCT number, 2010-023467-18 .)

Information Sources And Government Research: Ethical Conflicts And Solutions

Governments around the globe have long collected large amounts of information to achieve their goals. The emergence of the Internet has provided new opportunities for information exchange between governments and their citizens. This article presents illustrative examples of this information exchange, identifies associated ethical problems, examines approaches that governments have available to solve ethical dilemmas, and proposes a unified code of government research ethics with particular emphasis on privacy and security of information

Atropine sensitivity of the long-term effects of xanomeline pretreatments on carbachol-stimulated PI hydrolysis in CHO cells stably expressing human M₁ muscarinic acetylcholine receptors.

<p>(A) Presence of atropine during the initial 1 h pretreatment period. Cells were pretreated with 300 nM xanomeline for 1 h in the absence (closed circles) or presence of 10 µM atropine (open circles) followed by washing and incubation in ligand-free media for 23 h. (B) Effects of atropine presence during the 23 h incubation period following xanomeline pretreatment and washing. Cells were pretreated with 300 nM xanomeline for 1 h followed by washing and incubation for 23 h in the absence (closed circles) or presence of 10 µM atropine (open circles). In both figures, control atropine pretreatments were conducted in the absence of xanomeline pretreatments (open squares). Untreated (closed squares) and treated cells were subsequently incubated with increasing concentrations of carbachol for 1 h at 37°C and accumulation of inositol phosphates was measured. Results are expressed as percentages of maximal carbachol elicited PI response in untreated cells (25000±2200 dpm). Values represent the means ± standard error of three to seven experiments conducted in triplicate.</p

Effects of xanomeline pretreatment, followed by washout, on agonist-stimulated PI hydrolysis in CHO cells stably expressing human M₁ muscarinic acetylcholine receptors.

<p>Cells were pretreated with 300 nM xanomeline for 1 h (open circles) or 24 h (open diamonds) followed by washing and agonist-stimulated PI hydrolysis was measured immediately. Alternatively, cells were pretreated for 1 h followed by washing and incubation in agonist-free media for an additional 23 h (closed circles) before measuring agonist-stimulated accumulation of PI hydrolysis. Sham-treated (closed squares) and xanomeline-treated cells were subsequently incubated for 1 hour at 37°C with increasing concentrations of (A) carbachol, (B) oxotremorine, or (C) xanomeline and accumulation of inositol phosphates was measured. Maximal carbachol induced PI response in untreated cells was (A) 24000±1800 dpm, (B) 8300±900 dpm, (C) 19000±1800 dpm. Values represent the means ± standard error of three to eight experiments conducted in triplicate.</p

Effects of xanomeline or carbachol pretreament on activation of PI hydrolysis by carbachol, oxotremorine, or xanomeline in CHO hM₁ cells.

<p>Cells were pretreated with 300 nM xanomeline or 10 µM carbachol for 1 h or 24 h at 37°C followed by washing and immediate use in the functional assay or for 1 h followed by washing and further incubation in the absence of free xanomeline for 23 h. Pretreated or untreated (control) cells were then incubated with increasing concentrations of carbachol, oxotremorine, or xanomeline at 37°C for 1 h and the accumulation of inositol phosphates was determined. Functional parameters were derived from computer-assisted non-linear regression analysis as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015722#s4" target="_blank">Methods</a>, and are presented as mean ± S.E.M. of three to nine individual experiments conducted in triplicate.</p><p>^<i>a</i>Negative logarithm of the midpoint (potency) parameter.</p><p>^<i>b</i>Maximal response. Values are expressed as % maximal response elicited by carbachol in untreated cells (<i>^b1</i> 24000±1800 dpm; <i>^b2</i> 8300±900 dpm; <i>^b3</i>19000±1800 dpm).</p><p>^<i>c</i>Control, naïve cells were incubated with agonist.</p><p>^<i>d</i>Not applicable.</p><p>*ANOVA followed by Dunnett’s post-test detected a significant difference (<i>p</i><0.05) in pEC₅₀ or E_max between the pretreated groups compared with control.</p>†<p>Student’s <i>t</i>-test detected a significant difference (<i>p</i><0.05) in pEC₅₀ or E_max between the pretreated groups compared with control.</p

Effects of various xanomeline treatment conditions on the specific binding of [³H]NMS or [³H]QNB in CHO hM₁ cells.

<p>Data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015722#pone-0015722-g004" target="_blank">Fig. 4</a> were corrected for protein as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015722#s2" target="_blank">results</a>. Parameters derived from nonlinear regression analysis are shown as mean ± S.E.M. of three to four experiments conducted in triplicate. All other details as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015722#pone-0015722-t001" target="_blank">Table 1</a>.</p><p><i>^a</i>Control, naïve cells were incubated simultaneously with xanomeline and the radioligands.</p><p>*Significant difference (<i>p</i><0.05) in pIC₅₀ between the indicated groups and control as determined by one-way ANOVA followed by Dunnett’s post-test.</p

Effects of xanomeline pretreatment, followed by washout, on binding of receptor-saturating concentrations of [³H]NMS and [³H]QNB in CHO cells stably expressing human M₁ muscarinic acetylcholine receptors.

<p>The binding of (A) 2.9 nM [³H]NMS or (B) 1.4 nM [³H]QNB was measured in the presence of increasing concentrations of xanomeline in naïve cells (closed squares), or after pretreating with increasing concentrations of xanomeline for 1 h (open circles) or 24 h (open diamonds) followed by washing and immediate use in the binding assay, or after pretreating with increasing concentrations of agonist for 1 h followed by washing and incubation in agonist-free media for an additional 23 h before use in the binding assay (closed circles). Nonspecific binding was defined by 10 µM atropine. Values represent the means ± standard error of three to four experiments conducted in triplicate.</p

Effects of atropine during pretreatment with 3 µM xanomeline or following washout on [³H]NMS saturation binding parameters and activation of PI hydrolysis by carbachol in CHO hM₁ cells.

<p>Cells were pretreated for 1 h with xanomeline and/or atropine followed by extensive washing and waiting for 23 h in the absence or presence of atropine. Parameters derived from nonlinear regression analysis of data shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015722#pone-0015722-g009" target="_blank">Fig. 9</a> are presented as mean ± S.E.M. of three experiments performed in triplicate. All other details as for <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015722#pone-0015722-t002" target="_blank">Tables 2</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0015722#pone-0015722-t003" target="_blank">3</a>.</p><p><i>^a</i>Expressed as percentage of the maximal response to carbachol in untreated cells (25000±2200 dpm).</p><p><i>^b</i>Control, naïve cells were incubated with radioligand in binding assays, or carbachol in functional assays.</p><p>*ANOVA followed by Tukey’s post-test detected a significant difference (<i>p</i><0.05) between the xanomeline pretreated groups compared with respective control/sham treatment.</p