Unexpected scaffold rearrangement product of pirenzepine found in commercial samples.

Pharmacovigilance aims at a better understanding of the molecular events triggered by medications to prevent adverse effects, which despite significant advances in our analytical repertoire plague the use of drugs until today. In this study, we find that clinically prescribed and commercially available pirenzepine may not be the correct compound. Pirenzepine can undergo an unexpected scaffold rearrangement from the pharmaceutical active ingredient (API) to a previously uncharacterized benzimidazole. The rearrangement occurs under highly acidic conditions, which were believed to favour the dihydrochloride formation of pirenzepine. The rearranged products of pirenzepine and the structurally related telenzepine have significantly decreased affinity for the muscarinic acetylcholine receptor, the pharmacological target of these compounds. Fortunately, in situ rearrangement after oral application is no safety issue, as we show that reaction kinetics in gastric acid prevent rearrangement. The research community should consider appropriate measures to perform reliable receiving inspections in the commercial supply of well described and frequently used chemicals, in particular if experiments yield unexpected results.

Effects of Dose, Age, Sex, Body Weight, and Smoking on Plasma Concentrations of Olanzapine and N-desmethyl Olanzapine in Inpatients With Schizophrenia.

PURPOSE: This study aimed to investigate the combined effects of dose, age, sex, body weight, and smoking on plasma concentrations of olanzapine (OLA) and N-desmethyl olanzapine (DMO) in Chinese inpatients with schizophrenia. METHODS: A retrospective study including 185 inpatients was conducted. The steady-state plasma concentrations of OLA (COLA) and DMO (CDMO) were measured using high-performance liquid chromatography-tandem mass spectrometry. The combined effects of dose, age, sex, body weight, and smoking on COLA and CDMO were evaluated. FINDINGS: Multiple linear regression analyses revealed that dose, age, body weight, and smoking had significant effects on COLA and CDMO in inpatients with schizophrenia treated with OLA. The dose was the most important determinant of COLA and CDMO and was positively correlated with both. Furthermore, smokers exhibited a significantly lower COLA and COLA + DMO, whereas higher body weight led to the reduction of COLA, CDMO, and COLA + DMO. Advanced age was associated with lower CDMO. IMPLICATIONS: These results suggest that dose, age, body weight, and smoking have a significant influence on the plasma concentration of OLA and its metabolite DMO. Clinicians should consider the combined effects when prescribing OLA to patients with schizophrenia.

Muscarinic M1, but not M4, receptor antagonism impairs divided attention in male rats.

Divided attention may be more important than ever to comprehend, given ubiquitous distractors in modern living. In humans, concern has been expressed about the negative impact of distraction in education, the home, and the workplace. While acetylcholine supports divided attention, in part via muscarinic receptors, little is known about the specific muscarinic subtypes that may contribute. We designed a novel, high-response rate test of auditory sustained attention, in which rats complete variable-ratio runs on one of two levers, rather than emitting a single response. By doing this, we can present a secondary visual distractor task during some trials, for which a correct nosepoke response is reinforced with a more palatable food pellet. The nonspecific muscarinic antagonist scopolamine impaired performance, and slowed and reduced lever press activity. We then explored antagonists that preferentially block the M1 and M4 subtypes, because these receptors are potential therapeutic targets for cognitive enhancers. Telenzepine, an M1-preferring antagonist, impaired divided attention performance, but not performance of the attention task without distraction. Telenzepine also had fewer nonspecific effects than scopolamine. In contrast, the M4-preferring antagonist tropicamide had no effects. Analysis of overall behavior also indicated that accuracy in the main attention task decreased as a function of engagement with the distractor task. These results implicate the M1 receptor in divided attention.

Segmental hair analysis of olanzapine and N-desmethyl-olanzapine in postmortem hair from mentally ill patients by LC-MS/MS.

Hair analysis is a useful tool for establishing long-term drug intake. Segmental analysis, in particular, where the hair is cut into defined segments, can potentially provide a calendar of patients' drug intake as drugs are incorporated into the growing hair through the bloodstream with an average growth rate of 1 cm per month. Forensic investigations of hair require knowledge of typical concentrations of common pharmaceuticals in hair, which are rarely reported. The aim of this study was to provide values for olanzapine and N-desmethyl-olanzapine concentrations in postmortem hair from chronic olanzapine consumers to contribute to the establishment of a reference interval for this drug. We analyzed postmortem head hair samples from 37 suspected mentally ill patients, who were part of the SURVIVE population, a Danish national autopsy-based study. Each sample was cut into 1 cm segments, and up to six segments, corresponding to up to six months of hair growth prior to death, were analyzed depending on the hair length. The hair extracts were analyzed by liquid chromatography tandem mass spectrometry. Olanzapine and N-desmethyl-olanzapine were added to a published and validated method. The 37 patients were 12 females and 25 males aged 25-81 years. Their hair colors varied from blond to black, with the majority brown, thus no trend could be discerned from the hair colors. Drugs other than olanzapine were found in all cases except one, and illicit drugs were found in the hair samples of 38 % of the cases. We report olanzapine concentrations ranging from 0.005-20.9 ng/mg (median 0.128 ng/mg) and N-desmethyl-olanzapine concentrations from 0.027 to 0.187 ng/mg (median 0.068 ng/mg) for all 141 analyzed segments. Metabolite-to-drug ratios ranged from 0.010 to 3.31 (median 0.590). Dose calculations based on prescription pick-up demonstrated no correlation with the concentrations in hair, but olanzapine concentrations in the proximal hair segment correlated significantly with olanzapine concentrations in postmortem blood. Olanzapine concentrations decreased considerably from the proximal to distal segments, emphasizing the importance of reporting the length of the measured hair when reporting drug concentrations in hair. This study can contribute to the establishment of a reference interval for olanzapine and N-desmethyl-olanzapine concentrations in hair by reporting concentrations in hair from chronic consumers.

Central muscarinic receptor subtypes (M1 and M3) involved in carbacol-induced hypophagia in neonatal broiler chicken.

Aim: Food intake regulated by a complex of physiologic mechanisms in the nervous system. Muscarinergic system has an important role in the central regulation of appetite in mammals, but there is no information for Muscarinic receptors in avian. The purpose of this study was to examine the effects of intracerebroventricular injection of carbachol (cholinergic agonist), Telenzepine (M1 receptor antagonist), AF-DX116 (M2 receptor antagonist), 4-DAMP (M3 receptor antagonist), and PD102807 (M4 receptor antagonist) on feeding behavior in 3-h food-deprived (FD3) neonatal broiler chicken.Materials and Methods: In experiment 1, chicken intracerebroventricular injected with carbachol (125, 250, and 500 nmol). In experiment 2, birds intracerebroventricular injected with telenzepine (125, 250, and 500 nmol). In experiments 3-5, birds intracerebroventricular injected with AF-DX 116 (125, 250, and 500 nmol), 4-DAMP (125, 250, and 500 nmol), and PD102807 (125, 250, and 500 nmol), respectively. In experiment 6, broilers intracerebroventricular injected with carbacol (500 nmol), co-injection of telenzepine (125 nmol)+carbacol (500 nmol), and 4-DAMP (125 nmol)+carbacol (500 nmol). In experiment 7, injection procedure was carbacol (500 nmol), co-injection of AF-DX116 (125 nmol)+carbacol (500 nmol), and PD102807 (125 nmol)+carbacol (500 nmol). Then, food intake measured until 120 min after injection.Results: According to the data, carbachol (250 and 500 nmol) significantly decreased food intake in comparison with control group (P < 0.05). Intracerebroventricular injection of telenzepine (250 and 500 nmol) and 4-DAMP (250 and 500 nmol) significantly increased food intake (P < 0.05). In addition, carbacol-induced hypophagia was significantly attenuated by co-injection of telenzepine + carbacol (P < 0.05). Also, co-injection of 4-DAMP + carbacol decreased the effect of carbacol on food intake (P < 0.05). However, AF-DX116 and PD102807 had no effect on hypophagia induced by carbacol (P > 0.05).Conclusion: These results suggest, hypophagic effect of muscarinergic system is mediated via M1 and M3 receptors in neonatal chicken.

Participation of acetylcholine and its receptors in the contractility of inflamed porcine uterus.

This study analyzed the effect of inflammation on acetylcholine (ACh)-induced muscarinic receptors (MR)2 and MR3 conducted contractility of the porcine uterus. On Day 3 of the estrous cycle, either E.coli suspension (E.coli group) or saline (SAL group) was injected into uterine horns or laparotomy was performed (CON group). Eight days later, infected gilts developed severe acute endometritis. Compared to the period before ACh treatment, ACh (10-5 M) increased the tension in myometrium (MYO) and endometrium/myometrium (ENDO/MYO) of the CON group (P < 0.01) and in ENDO/MYO of the SAL group (P < 0.01), the amplitude in strips of the CON (P < 0.05) and SAL (MYO: P < 0.05, ENDO/MYO: P < 0.001) groups and the frequency in strips of the CON (MYO: P < 0.01, ENDO/MYO: P < 0.001) and SAL (P < 0.01) groups. In the E.coli group, ACh (10-5 M) reduced the amplitude in MYO (P < 0.05) and ENDO/MYO (P < 0.001), increased the frequency in MYO (P < 0.01) and ENDO/MYO (P < 0.001) and did not change (P > 0.05) the tension. ACh (10-5 M) in ENDO/MYO of the E.coli group, reduced the tension compared to the CON group (P < 0.05) and the amplitude compared to other groups (P < 0.001), while increased the frequency in relation to the SAL group (P < 0.05). MR2 antagonist (AF-DX 44 116) and ACh (10-5 M) reduced (by 16.92%, P < 0.01) the tension in MYO of the CON group and increased (P < 0.01) it in the E.coli group compared to the period before antagonist and ACh addition. In MYO of the SAL group, the tension was increased (P < 0.01) in response to MR3 antagonist (4-DAMP) and ACh (10-7, 10-6 M). In the E.coli group, these substances did not change (P > 0.05) the tension, but it was lower (P < 0.001) in MYO (ACh: 10-7 M) and ENDO/MYO (ACh: 10-5 M) than in the SAL group. MR2 or MR3 antagonists and ACh (10-5 M) increased (P < 0.05-0.001) the amplitude in strips of the CON and SAL groups and reduced it in the E.coli group (P < 0.001) compared to the period before antagonists and ACh use. This parameter in the E.coli group was lower (P < 0.001) after using MR2 or MR3 antagonists and ACh (10-6, 10-5 M) than in other groups. Both antagonists and ACh (10-5 M) reduced the frequency in the CON, SAL (P < 0.05) and E.coli (MR2 antagonist: P < 0.01, MR3 antagonist: P < 0.05) groups compared to period before antagonists and ACh addition. Data show that ACh reduces the contractility of the inflamed porcine uterus by MR2 and MR3, which suggests that pharmacological modulation of these receptors can be used to raise the contractility of an inflamed uterus.

Muscarinic M1 and M2 receptor subtypes play opposite roles in LPS-induced septic shock.

BACKGROUND: To compare pharmacologic effects of pirenzepine and AF-DX116, a selective competitive antagonist for M1 and M2 subtype muscarinic cholinergic receptors (mAChRs), respectively, with atropine, a non-selective competitive antagonist for mAChRs, on Lipopolysaccharide (LPS). METHODS: Male C57BL/6 mice were used to establish models of LPS-induced experimental endotoxemia. Mice were intraperitoneally injected 10 min prior to LPS injection with control (saline), atropine, pirenzepine and AF-DX116, respectively. Overall survival time was estimated using Kaplan-Meier plots. Inflammatory cytokine tumor necrosis factor-α (TNF-α) was monitored at various intervals after LPS injection and individual reagent administration. Pathological alternations in lungs and liver were analyzed. RESULTS: Pirenzepine and atropine pretreatment improved survival rate of LPS-induced septic shock; in contrast, AF-DX116 accelerated death from sepsis. Moreover, TNF-α plasma level was decreased in response to pirenzepine or atropine, whereas increased in response to AF-DX116. Pirenzepine and atropine relieved whereas AF-DX116 accelerated LPS-induced pulmonary and hepatic injury. Pirenzepine reduced proportion of M1 subtype of macrophages, while AF-DX116 promoted polarization of macrophages to M1 subtype. Pirenzepine pretreatment reduced while AF-DX116 enhanced expression of SOCS3 at mRNA level. CONCLUSIONS: The administration of pirenzepine and atropine may have beneficial effects on septic shock.

Metabolic and endocrinal effects of N-desmethyl-olanzapine in mice with obesity: Implication for olanzapine-associated metabolic changes.

Clinical use of the antipsychotic drug olanzapine (OLA) is associated with metabolic side effects to variable degrees. N-desmethyl-olanzapine (DMO) is one major metabolite of OLA, but its potential involvement in the metabolic responses remains unclear. Here we examined whether DMO can directly impact the metabolic, endocrinal and inflammatory parameters under conditions of metabolic disturbance. DMO administration (2 mg/kg, i.g.) to high-fat diet induced obesity mice for 4 weeks induced a remarkable loss of body weight and fat mass. DMO improved insulin resistance and energy expenditure in mice, but had no significant effects on dyslipidemia or hepatic steatosis. Moreover, DMO induced morphological changes in the white adipose tissue, accompanied by reduced interleukin-1ß (IL-1ß) production and increased UCP1 expression. These findings demonstrate that DMO is devoid of the metabolic side effects commonly observed for OLA during obesity, which suggests that the N-desmethyl metabolism may function to regulate the metabolic responses to OLA.

Structural insights into the subtype-selective antagonist binding to the M2 muscarinic receptor.

Human muscarinic receptor M2 is one of the five subtypes of muscarinic receptors belonging to the family of G-protein-coupled receptors. Muscarinic receptors are targets for multiple neurodegenerative diseases. The challenge has been designing subtype-selective ligands against one of the five muscarinic receptors. We report high-resolution structures of a thermostabilized mutant M2 receptor bound to a subtype-selective antagonist AF-DX 384 and a nonselective antagonist NMS. The thermostabilizing mutation S110R in M2 was predicted using a theoretical strategy previously developed in our group. Comparison of the crystal structures and pharmacological properties of the M2 receptor shows that the Arg in the S110R mutant mimics the stabilizing role of the sodium cation, which is known to allosterically stabilize inactive state(s) of class A GPCRs. Molecular dynamics simulations reveal that tightening of the ligand-residue contacts in M2 receptors compared to M3 receptors leads to subtype selectivity of AF-DX 384.

Striatal cholinergic interneurons regulate cognitive and affective dysfunction in partially dopamine-depleted mice.

Early non-motor symptoms such as mood disorders and cognitive deficits are increasingly recognised in Parkinson's disease (PD). They may precede the characteristic motor symptomatology caused by dopamine (DA) neuronal loss in the substantia nigra pars compacta (SNc). It is well known that striatal cholinergic interneurons (ChIs) are emerging as key regulators of PD motor symptom, however, their involvement in the cognitive and affective alterations occurring in the premotor phase of PD is poorly understood. We used optogenetic photoinhibition of striatal ChIs in mice with mild nigrostriatal 6-hydroxydopamine (6-OHDA) lesions and assessed their role in anxiety-like behaviour in the elevated plus maze, social memory recognition of a congener and visuospatial object recognition. In transgenic mice specifically expressing halorhodopsin (eNpHR) in cholinergic neurons, striatal ChIs photoinhibition reduced the anxiety-like behaviour and reversed social and spatial short-term memory impairment induced by moderate DA depletion (e.g., 50% loss of tyrosine hydroxylase TH-positive neurons in the SNc). Systemic injection of telenzepine (0.3 mg/kg), a preferential M1 muscarinic cholinergic receptors antagonist, improved anxiety-like behaviour, social memory recognition but not spatial memory deficits. Our results suggest that dysfunction of the striatal cholinergic system may play a role in the short-term cognitive and emotional deficits of partially DA-depleted mice. Blocking cholinergic activity with M1 muscarinic receptor antagonists may represent a possible therapeutic target, although not exclusive, to modulate these early non-motor deficits.

PKC delta activation increases neonatal rat retinal cells survival in vitro: Involvement of neurotrophins and M1 muscarinic receptors.

Protein kinase C (PKC) is a family of serine/threonine kinases related to several phenomena as cell proliferation, differentiation and survival. Our previous data demonstrated that treatment of axotomized neonatal rat retinal cell cultures for 48 h with phorbol 12-myristate 13-acetate (PMA), a PKC activator, increases retinal ganglion cells (RGCs) survival. Moreover, this treatment decreases M1 receptors (M1R) and modulates BDNF levels. The aim of this work was to assess the possible involvement of neurotrophins BDNF and NGF in the modulation of M1R levels induced by PKC activation, and its involvement on RGCs survival. Our results show that PMA (50 ng/mL) treatment, via PKC delta activation, modulates NGF, BDNF and M1R levels. BDNF and NGF mediate the decrease of M1R levels induced by PMA treatment. M1R activation is essential to PMA neuroprotective effect on RGCs as telenzepine (M1R selective antagonist) abolished it. Based on our results we suggest that PKC delta activation modulates neurotrophins levels by a signaling pathway that involves M1R activation and ultimately leading to an increase in RGCs survival in vitro.

Inhibition of basal and amphetamine-stimulated extracellular signal-regulated kinase (ERK) phosphorylation in the rat forebrain by muscarinic acetylcholine M4 receptors.

The mitogen-activated protein kinase (MAPK), especially its extracellular signal-regulated kinase (ERK) subfamily, is a group of kinases enriched in the mammalian brain. While ERK is central to cell signaling and neural activities, the regulation of ERK by transmitters is poorly understood. In this study, the role of acetylcholine in the regulation of ERK was investigated in adult rat striatum in vivo. We focused on muscarinic M1 and M4 receptors, two principal muscarinic acetylcholine (mACh) receptor subtypes in the striatum. A systemic injection of the M1-preferring antagonist telenzepine did not alter ERK phosphorylation in the two subdivisions of the striatum, the caudate putamen and nucleus accumbens. Similarly, telenzepine did not affect ERK phosphorylation in the medial prefrontal cortex (mPFC), hippocampus, and cerebellum. Moreover, telenzepine had no effect on the ERK phosphorylation induced by dopamine stimulation with the psychostimulant amphetamine. In contrast to telenzepine, the M4-preferring antagonist tropicamide consistently increased ERK phosphorylation in the striatum and mPFC. This increase was rapid and transient. Tropicamide and amphetamine when coadministered at subthreshold doses induced a significant increase in ERK phosphorylation. These results demonstrate that mACh receptors exert a subtype-specific modulation of ERK in striatal and mPFC neurons. While the M1 receptor antagonist has no effect on ERK phosphorylation, M4 receptors inhibit constitutive and dopamine-stimulated ERK phosphorylation in these dopamine-innervated brain regions.

Role of rho-kinase (ROCK) in tonic but not phasic contraction in the frog stomach smooth muscle.

AIMS: Rho/Rho-kinase (ROCK) signaling has extensively been shown to take part in mammalian smooth muscle contractions in response to diverse agents yet its role in the contraction of amphibian smooth muscle has not been investigated. Therefore, we aimed to explore any role of this pathway in the contractions of frog stomach smooth. MAIN METHODS: The strips were prepared and suspended in organ baths filled with Ringer solution. Changes in the circular strips of the frog stomach muscle length were recorded isotonically with a force transducer in organ baths. KEY FINDINGS: Carbachol (CCh) exerted both phasic and tonic contractions. In contrast, atropin abolished all types of contractions by CCh. The phasic contractions were suppressed by a Ca2+ channel blocker, nifedipine but not by the ROCK inhibitor, Y-27632. However, the tonic contractions were markedly attenuated by Y-27632. Selective M1 receptor blocker, pirenzepin, selective M3 receptor blocker and DAMP had no effects on CCh-elicited contractions. On the other hand, selective M2 receptor blocker, AF-DX suppressed all types of contractile activity by CCh. SIGNIFICANCE: These data suggest that M2 receptor activation could mainly mediate CCh-induced phasic and tonic contractions, and ROCK seems to be involved in the CCh-induced tonic but not phasic contractions of the frog stomach smooth muscle.

Early changes in M2 muscarinic acetylcholine receptors (mAChRs) induced by sarin intoxication may be linked to long lasting neurological effects.

The effect of sarin on the binding parameters (KD & Bmax) of M2 muscarinic acetylcholine receptor (mAChR) was studied 24h and 1 week post exposure. Male & female Sprague-Daweley rats were poisoned with 1XLD50 sarin (80µg/kg, im) followed by treatment of trimedoxime bromide and atropine (7.5:5mg/kg, im) 1min later. Brains were removed and analyzed for M2 mAChR binding, using [3H]AFDX384, an M2 selective antagonist. A significant increase in KD of M2 mAChR was found in the cortex 24h post poisoning, displaying elevation from 4.65±1.16 to 8.45±1.06nM and 5.24±0.93 to 9.29±1.56nM in male and female rats, respectively. A rise in KD was also noted 1 week following exposure from 5.04±1.20 to 11.75±2.78 and from 5.37±1.02 to 11.66±1.73nM, presenting an added increase of 51 and 40% (compared to 24h) in males and females, respectively. Analysis of M2 receptor density (Bmax) revealed a significant reduction of 68% in males and insignificant reduction of 22% in females, 24h after sarin exposure which was followed by 37% recovery in males and 100% recovery in females, 1 week later. These results indicate that sarin induces a long-term decreased affinity in M2 mAChR (elevated KDs) and a transient effect on the number of this receptor subtype (Bmax). We hypothesize that the reduced affinity of the M2 receptors (negative auto-regulatory receptors) may cause long-term brain deficits by impairing the normal regulation release of ACh into the synaptic cleft.

Application of plasma levels of olanzapine and N-desmethyl-olanzapine to monitor metabolic parameters in patients with schizophrenia.

Metabolic disturbance is a common side effect of olanzapine (OLZ); however, the relationships between plasma OLZ concentration (COLZ) and metabolic disturbance remain unclear. Our previous study revealed that COLZ≧22.77ng/mL was a positive predictor of therapeutic efficacy in patients with schizophrenia. This study aimed to investigate the roles of OLZ or N-desmethyl-olanzapine (DMO) in metabolic outcomes among OLZ-treated patients with schizophrenia. The metabolic syndrome (MS) was diagnosed based on the modified the National Cholesterol Education Program Adult Treatment Panel III criteria for Asians. HPLC-ECD analytical system was applied to determine the COLZ and DMO concentration (CDMO). The absolute drug levels and concentration-to-dose ratios (C/D ratios) were tested for their correlations to metabolic parameters. Total 151 fasting blood samples from patients with schizophrenia were collected. DMO C/D ratio negatively correlated with weight, body mass index, waist circumference, and C-peptide level. The receiver operator characteristic analysis determined a threshold CDMO>5.63ng/mL and DMO C/D ratio>0.35ng/mL/mg were negative predictors of MS. The COLZ/CDMO ratio>6.03 was identified as positive predictor of MS. Combined with previous study result, we proposed that the optimal OLZ treatment should maintain COLZ/CDMO ratio between 3 and 6 to maximize the clinical efficacy and minimize the metabolic side effects. Our findings suggested that therapeutic drug monitoring on OLZ and DMO is a valuable tool to monitor metabolic side effects in OLZ-treated patients with schizophrenia.

Muscarinic receptor binding changes in postmortem Parkinson's disease.

Parkinson's disease (PD) is a devastating disorder, affecting approximately 2% of people aged 60 and above. It is marked by progressive neurodegeneration that has long been known to impact dopaminergic cells and circuits, but more recently the acetylcholine system has also been implicated in the complex aetiology and symptomatology of the disease. While broad changes in cholinergic markers have been described, insight into the contribution of specific acetylcholine receptors is less clear. To address this important unknown, in this study we performed [3H] pirenzepine, [3H] 4DAMP, and [3H] AF-DX 384 in situ radioligand binding on postmortem tissues from Brodmann's area 6, 9, 46, and the caudate putamen, from PD and matched controls to detect muscarinic M1, M3, and M1/2/4 receptors, respectively. We found no difference in [3H] pirenzepine binding between PD and controls across all regions assessed. [3H] 4DAMP binding was found to be higher in PD CPu and BA9 than in controls. [3H] AF-DX 384 was higher in BA9 of PD compared with controls. In sum, we show selective increase in M3 receptors in cortical and subcortical regions, as well as increased M2/M4 in cortical area BA9, which together support a role for cholinergic dysfunction in PD.

Muscarinic acetylcholine receptors act in synergy to facilitate learning and memory.

Understanding how episodic memories are formed and retrieved is necessary if we are to treat disorders in which they malfunction. Muscarinic acetylcholine receptors (mAChR) in the hippocampus and cortex underlie memory formation, but there is conflicting evidence regarding their role in memory retrieval. Additionally, there is no consensus on which mAChR subtypes are critical for memory processing. Using pharmacological and genetic approaches, we found that (1) encoding and retrieval of contextual memory requires mAChR in the dorsal hippocampus (DH) and retrosplenial cortex (RSC), (2) memory formation requires hippocampal M3 and cooperative activity of RSC M1 and M3, and (3) memory retrieval is more impaired by inactivation of multiple M1-M4 mAChR in DH or RSC than inactivation of individual receptor subtypes. Contrary to the view that acetylcholine supports learning but is detrimental to memory retrieval, we found that coactivation of multiple mAChR is required for retrieval of both recently and remotely acquired context memories. Manipulations with higher receptor specificity were generally less potent than manipulations targeting multiple receptor subtypes, suggesting that mAChR act in synergy to regulate memory processes. These findings provide unique insight into the development of therapies for amnestic symptoms, suggesting that broadly acting, rather than receptor-specific, mAchR agonists and positive allosteric modulators may be the most effective therapeutic approach.

Dynamic Regulation of Quaternary Organization of the M1 Muscarinic Receptor by Subtype-selective Antagonist Drugs.

Although rhodopsin-like G protein-coupled receptors can exist as both monomers and non-covalently associated dimers/oligomers, the steady-state proportion of each form and whether this is regulated by receptor ligands are unknown. Herein we address these topics for the M1 muscarinic acetylcholine receptor, a key molecular target for novel cognition enhancers, by using spatial intensity distribution analysis. This method can measure fluorescent particle concentration and assess oligomerization states of proteins within defined regions of living cells. Imaging and analysis of the basolateral surface of cells expressing some 50 molecules·µm(-2) human muscarinic M1 receptor identified a ∼75:25 mixture of receptor monomers and dimers/oligomers. Both sustained and shorter term treatment with the selective M1 antagonist pirenzepine resulted in a large shift in the distribution of receptor species to favor the dimeric/oligomeric state. Although sustained treatment with pirenzepine also resulted in marked up-regulation of the receptor, simple mass action effects were not the basis for ligand-induced stabilization of receptor dimers/oligomers. The related antagonist telenzepine also produced stabilization and enrichment of the M1 receptor dimer population, but the receptor subtype non-selective antagonists atropine and N-methylscopolamine did not. In contrast, neither pirenzepine nor telenzepine altered the quaternary organization of the related M3 muscarinic receptor. These data provide unique insights into the selective capacity of receptor ligands to promote and/or stabilize receptor dimers/oligomers and demonstrate that the dynamics of ligand regulation of the quaternary organization of G protein-coupled receptors is markedly more complex than previously appreciated. This may have major implications for receptor function and behavior.

Application of Plasma Levels of Olanzapine and N-Desmethyl-Olanzapine to Monitor Clinical Efficacy in Patients with Schizophrenia.

BACKGROUND: This therapeutic drug monitoring (TDM) study aimed to determine the role of olanzapine (OLZ) and N-desmethyl-OLZ (DMO) levels in the therapeutic efficacy of OLZ in patients with schizophrenia. METHOD: Plasma concentrations of OLZ (COLZ) and DMO (CDMO) in schizophrenic patients 12 hours post-dose were assessed. The correlations of COLZ and CDMO with the various scores of the Positive and Negative Syndrome Scale (PANSS) were evaluated. A receiver operating characteristic curve (ROC) was utilized to identify the threshold COLZ and COLZ/CDMO ratio for maintenance of satisfactory efficacy. RESULTS: A total of 151 samples from patients with schizophrenia were analyzed for individual COLZ and CDMO levels. The mean COLZ and CDMO levels were 37.0 ± 25.6 and 6.9 ± 4.7 ng/mL, respectively, and COLZ was ~50% higher in female or nonsmokers (p<0.01). In all patients, the daily dose of OLZ was positively correlated with COLZ and CDMO. Linear relationships between COLZ and OLZ dose were observed in both nonsmokers and smokers (rs = 0.306, 0.426, p<0.01), although CDMO was only correlated with OLZ dose in smokers (rs = 0.485, p<0.01) and not nonsmokers. In all patients, COLZ was marginally negatively correlated with the total PANSS score. The total PANSS score was significantly negatively correlated with the COLZ/CDMO ratio (p<0.005), except in smokers. The ROC analysis identified a COLZ/CDMO ratio ≥2.99 or COLZ ≥22.77 ng/mL as a predictor of maintenance of an at least mildly ill status (PANSS score ≤58) of schizophrenia in all patients. CONCLUSIONS: A significantly negative correlation between the steady-state COLZ/CDMO ratio and total PANSS score was observed in Taiwanese schizophrenic patients. TDM of both OLZ and DMO levels could assist clinical practice when individualizing OLZ dosage adjustments for patients with schizophrenia.

Muscarinic receptor subtypes differentially control synaptic input and excitability of cerebellum-projecting medial vestibular nucleus neurons.

Neurons in the vestibular nuclei have a vital function in balance maintenance, gaze stabilization, and posture. Although muscarinic acetylcholine receptors (mAChRs) are expressed and involved in regulating vestibular function, it remains unclear how individual mAChR subtypes regulate vestibular neuronal activity. In this study, we determined which specific subtypes of mAChRs control synaptic input and excitability of medial vestibular nucleus (MVN) neurons that project to the cerebellum. Cerebellum-projecting MVN neurons were labeled by a fluorescent retrograde tracer and then identified in rat brainstem slices. Quantitative PCR analysis suggested that M2 and M3 were the possible major mAChR subtypes expressed in the MVN. The mAChR agonist oxotremorine-M significantly reduced the amplitude of glutamatergic excitatory post-synaptic currents evoked by stimulation of vestibular primary afferents, and this effect was abolished by the M2-preferring antagonist AF-DX 116. However, oxotremorine-M had no effect on GABA-mediated spontaneous inhibitory post-synaptic currents of labeled MVN neurons. Furthermore, oxotremorine-M significantly increased the firing activity of labeled MVN neurons, and this effect was blocked by the M3-preferring antagonist J104129 in most neurons tested. In addition, AF-DX 116 reduced the onset latency and prolonged the excitatory effect of oxotremorine-M on the firing activity of labeled MVN neurons. Our findings suggest that M3 is the predominant post-synaptic mAChR involved in muscarinic excitation of cerebellum-projecting MVN neurons. Pre-synaptic M2 mAChR regulates excitatory glutamatergic input from vestibular primary afferents, which in turn influences the excitability of cerebellum-projecting MVN neurons. This new information has important therapeutic implications for treating vestibular disorders with mAChR subtype-selective agents. Medial vestibular nucleus (MVN) neurons projecting to the cerebellum are involved in balance control. We found that activation of pre-synaptic M2 muscarinic receptors inhibit glutamatergic input from vestibular primary afferents, whereas stimulation of post-synaptic M3 muscarinic receptors increases the firing activity of cerebellum-projecting MVN neurons. This new information advances our understanding of the cholinergic mechanism regulating the vestibular system.