Evaluation and estimation of diuretic activity of the linalyl acetate in the rats.

This study aimed to explore the diuretic activity of linalyl acetate (LA). LA is an essential oil, it is an integral phyto-constituent of various plants. In this study, acute and chronic diuretic activities were explored by measuring the levels of different electrolytes and pH in the urine of experimental rats. Rats were divided into five groups. The control group was given 10 mg/kg normal saline, the treated group was given 10 mg/kg furosemide, and the remaining 3 groups received different doses of LA including 25, 50, and 75 mg/kg through intraperitoneal route, to determine its diuretic potential. Urine volume for acute diuretic activity was measured for 6 hours however for chronic diuretic activity was measured for 6 days. For a comparative study of LA with a control group and treated group with reference drug, diuretic index was used. Moreover, the underlying mechanism of the diuretic activity was also explored by comparing atropine, L-NAME, and indomethacin. The results of each group with 6 rats in each group were obtained by ± standard error of the mean of every group. Analysis of Variance (ANOVA) was used for statistical analysis. Results revealed that the LA 75 mg/kg dose showed comparable results as of furosemide. Moreover, this study revealed the involvement of muscarinic receptors to produce diuresis in comparison with atropine with very little involvement of prostanoids and no effect on NO pathway induced by indomethacin and L-NAME respectively. It is concluded that LA possess anti-diuretic potential. Muscarinic receptors might be involved in producing diuretic effects.

A muscarinic receptor antagonist reverses multiple indices of diabetic peripheral neuropathy: preclinical and clinical studies using oxybutynin.

Preclinical studies indicate that diverse muscarinic receptor antagonists, acting via the M1 sub-type, promote neuritogenesis from sensory neurons in vitro and prevent and/or reverse both structural and functional indices of neuropathy in rodent models of diabetes. We sought to translate this as a potential therapeutic approach against structural and functional indices of diabetic neuropathy using oxybutynin, a muscarinic antagonist approved for clinical use against overactive bladder. Studies were performed using sensory neurons maintained in vitro, rodent models of type 1 or type 2 diabetes and human subjects with type 2 diabetes and confirmed neuropathy. Oxybutynin promoted significant neurite outgrowth in sensory neuron cultures derived from adult normal rats and STZ-diabetic mice, with maximal efficacy in the 1-100 nmol/l range. This was accompanied by a significantly enhanced mitochondrial energetic profile as reflected by increased basal and maximal respiration and spare respiratory capacity. Systemic (3-10 mg/kg/day s.c.) and topical (3% gel daily) oxybutynin reversed paw heat hypoalgesia in the STZ and db/db mouse models of diabetes and reversed paw tactile allodynia in STZ-diabetic rats. Loss of nerve profiles in the skin and cornea of db/db mice was also prevented by daily topical delivery of 3% oxybutynin for 8 weeks. A randomized, double-blind, placebo-controlled interventional trial was performed in subjects with type 2 diabetes and established peripheral neuropathy. Subjects received daily topical treatment with 3% oxybutynin gel or placebo for 6 months. The a priori designated primary endpoint, significant change in intra-epidermal nerve fibre density (IENFD) in skin biopsies taken before and after 20 weeks of treatments, was met by oxybutynin but not placebo. Secondary endpoints showing significant improvement with oxybutynin treatment included scores on clinical neuropathy, pain and quality of life scales. This proof-of-concept study indicates that muscarinic antagonists suitable for long-term use may offer a novel therapeutic opportunity for treatment of diabetic neuropathy. Trial registry number: NCT03050827.

Prevention of MK-801-induced amnestic effect with combined activation of 5-HT1A and muscarinic receptors in mice.

BACKGROUND: Muscarinic or 5-HT1A receptors are crucial in learning and memory processes, and their expression is evident in the brain areas involved in cognition. The administration of the activators of these receptors prevents the development of cognitive dysfunctions in animal models of schizophrenia induced by MK-801 (N-methyl-d-aspartate receptor antagonist) administration. GABAergic dysfunction is considered as one of the most important causes of MK-801-induced spatial learning deficits. METHODS: Novel object recognition (NOR) and Morris water maze (MWM) tests were used to study the anti-amnestic effect of the biased 5-HT1A receptor agonist (F15599) alone or in combinations with VU0357017 (M1 receptor allosteric agonist), VU0152100 (M4 receptor positive allosteric modulator), and VU0238429 (M5 receptor positive allosteric modulator) on MK-801-induced dysfunctions. The compounds were administered for 5 consecutive days. Animals tested with the MWM underwent 5-day training. Western blotting was used to study the expressions of 5-HT1A receptors and the level of GAD65 in the frontal cortices (FCs) and hippocampi of the animals. RESULTS: F15599 prevented the amnestic effect induced by MK-801 in the MWM at a dose of 0.1 mg/kg. The co-administration of the compound with muscarinic receptors activators had no synergistic effect. The additive effect of the combinations was evident in the prevention of declarative memory dysfunctions investigated in NOR. The administration of MK-801 impaired 5-HT1A expression in the hippocampi and decreased GAD65 levels in both the FCs and hippocampi. The administration of muscarinic ligands prevented these MK-801-induced deficits only in the hippocampi of MWM-trained animals. No effects of the compounds were observed in untrained mice. CONCLUSION: Our results indicate that F15599 prevents schizophrenia-related spatial learning deficits in the MWM; however, the activity of the compound is not intensified with muscarinic receptors activators. In contrast, the combined administration of the ligands is effective in the NOR model of declarative memory. The muscarinic receptors activators reversed MK-801-induced 5-HT1A and GAD65 dysfunctions in the hippocampi of MWM-trained mice, but not in untrained mice.

Molecular mechanism of muscarinic acetylcholine receptor M3 interaction with Gq.

Muscarinic acetylcholine receptor M3 (M3) and its downstream effector Gq/11 are critical drug development targets due to their involvement in physiopathological processes. Although the structure of the M3-miniGq complex was recently published, the lack of information on the intracellular loop 3 (ICL3) of M3 and extensive modification of Gαq impedes the elucidation of the molecular mechanism of M3-Gq coupling under more physiological condition. Here, we describe the molecular mechanism underlying the dynamic interactions between full-length wild-type M3 and Gq using hydrogen-deuterium exchange mass spectrometry and NanoLuc Binary Technology-based cell systems. We propose a detailed analysis of M3-Gq coupling through examination of previously well-defined binding interfaces and neglected regions. Our findings suggest potential binding interfaces between M3 and Gq in pre-assembled and functionally active complexes. Furthermore, M3 ICL3 negatively affected M3-Gq coupling, and the Gαq AHD underwent unique conformational changes during M3-Gq coupling.

Antiemetic activity of abietic acid possibly through the 5HT3 and muscarinic receptors interaction pathways.

The present study was designed to evaluate the antiemetic activity of abietic acid (AA) using in vivo and in silico studies. To assess the effect, doses of 50 mg/kg b.w. copper sulfate (CuSO4⋅5H2O) were given orally to 2-day-old chicks. The test compound (AA) was given orally at two doses of 20 and 40 mg/kg b.w. On the other hand, aprepitant (16 mg/kg), domperidone (6 mg/kg), diphenhydramine (10 mg/kg), hyoscine (21 mg/kg), and ondansetron (5 mg/kg) were administered orally as positive controls (PCs). The vehicle was used as a control group. Combination therapies with the referral drugs were also given to three separate groups of animals to see the synergistic and antagonizing activity of the test compound. Molecular docking and visualization of ligand-receptor interaction were performed using different computational tools against various emesis-inducing receptors (D2, D3, 5HT3, H1, and M1-M5). Furthermore, the pharmacokinetics and toxicity properties of the selected ligands were predicted by using the SwissADME and Protox-II online servers. Findings indicated that AA dose-dependently enhances the latency of emetic retching and reduces the number of retching compared to the vehicle group. Among the different treatments, animals treated with AA (40 mg/kg) exhibited the highest latency (98 ± 2.44 s) and reduced the number of retching (11.66 ± 2.52 times) compared to the control groups. Additionally, the molecular docking study indicated that AA exhibits the highest binding affinity (- 10.2 kcal/mol) toward the M4 receptors and an elevated binding affinity toward the receptors 5HT3 (- 8.1 kcal/mol), M1 (- 7.7 kcal/mol), M2 (- 8.7 kcal/mol), and H1 (- 8.5 kcal/mol) than the referral ligands. Taken together, our study suggests that AA has potent antiemetic effects by interacting with the 5TH3 and muscarinic receptor interaction pathways. However, additional extensive pre-clinical and clinical studies are required to evaluate the efficacy and toxicity of AA.

Cardiovascular Events with the Use of Long-Acting Muscarinic Receptor Antagonists: An Analysis of the FAERS Database 2020-2023.

PURPOSE: This study aimed to examine reports of cardiovascular adverse events (CV AEs) observed in the real-world during treatment with aclidinium, tiotropium, glycopyrronium, and umeclidinium alone or in combination with a LABA and, in the context of triple therapy, with the addition of an ICS, and submitted to the food and drug administration adverse event reporting system (FAERS). METHODS: A retrospective disproportionality analysis was conducted utilizing CV AE reports submitted to the FAERS from January 2020 to 30 September 2023. Disproportionality was measured by calculating the reporting odds ratio. RESULTS: Compared with ipratropium, tiotropium was associated with fewer reports of CV AEs. Compared with tiotropium, other LAMAs were more likely to be associated with reports of CV AEs. Combinations of glycopyrronium with indacaterol or formoterol and umeclidinium with vilanterol significantly reduced reports of CV AEs compared with the respective LAMA. The addition of an ICS to these combinations further reduced the risk of CV AE reports. CONCLUSION: Our study suggests that inhaled LAMAs are not free from cardiac AE risks. This risk may be more evident when the newer LAMAs are used, but it is generally significantly reduced when COPD patients are treated with dual bronchodilators or triple therapy. However, these results do not prove that LAMAs cause CV AEs, as FAERS data alone are not indicative of a drug's safety profile. Given the frequency with which COPD and cardiovascular disease co-exist, a large study in the general population could shed light on this very important issue.

Muscarinic control of cardiovascular function in humans: a review of current clinical evidence.

PURPOSE: To review the available evidence on the impact of muscarinic receptor modulation on cardiovascular control in humans. METHODS: In this narrative Review we summarize data on cardiovascular endpoints from clinical trials of novel subtype-selective or quasi-selective muscarinic modulators, mostly PAMs, performed in the last decade. We also review the cardiovascular phenotype in recently described human genetic and autoimmune disorders affecting muscarinic receptors. RESULTS: Recent advancements in the development of compounds that selectively target muscarinic acetylcholine receptors are expanding our knowledge about the physiological function of each muscarinic receptor subtype (M1, M2, M3, M4, M5). Among these novel compounds, positive allosteric modulators (PAMs) have emerged as the preferred therapeutic to regulate muscarinic receptor subtype function. Many muscarinic allosteric and orthosteric modulators (including but not limited to xanomeline-trospium and emraclidine) are now in clinical development and approaching regulatory approval for multiple indications, including the treatment of cognitive and psychiatric symptoms in patients with schizophrenia as well as Alzheimer's disease and other dementias. The results of these clinical trials provide an opportunity to understand the influence of muscarinic modulation on cardiovascular autonomic control in humans. While the results and the impact of each of these therapies on heart rate and blood pressure control have been variable, in part because the clinical trials were not specifically designed to measure cardiovascular endpoints, the emerging data is valuable to elucidate the relative cardiovascular contributions of each muscarinic receptor subtype. CONCLUSION: Understanding the muscarinic control of cardiovascular function is of paramount importance and may contribute to the development of novel therapeutic strategies for treating cardiovascular disease.

Activation of M1 cholinergic receptors in mouse somatosensory cortex enhances information processing and detection behaviour.

To optimise sensory representations based on environmental demands, the activity of cortical neurons is regulated by neuromodulators such as Acetylcholine (ACh). ACh is implicated in cognitive functions including attention, arousal and sleep cycles. However, it is not clear how specific ACh receptors shape the activity of cortical neurons in response to sensory stimuli. Here, we investigate the role of a densely expressed muscarinic ACh receptor M1 in information processing in the mouse primary somatosensory cortex and its influence on the animal's sensitivity to detect vibrotactile stimuli. We show that M1 activation results in faster and more reliable neuronal responses, manifested by a significant reduction in response latencies and the trial-to-trial variability. At the population level, M1 activation reduces the network synchrony, and thus enhances the capacity of cortical neurons in conveying sensory information. Consistent with the neuronal findings, we show that M1 activation significantly improves performances in a vibriotactile detection task.

Muscarinic receptor activation promotes destabilization and updating of object location memories in mice.

The storage of long-term memories is a dynamic process. Reminder cues can destabilize previously consolidated memories, rendering them labile and modifiable. However, memories that are strongly encoded or relatively remote at the time of reactivation can resist destabilization only being rendered labile under conditions that favour memory updating. Using the object location recognition task, here we show in male C57BL/6 mice that novelty-induced destabilization of strongly-encoded memories requires muscarinic acetylcholine receptor (mAChR) activation. Furthermore, we use the objects-in-updated locations task to show that updating of object location memories is mAChR-dependent. Thus, mAChR stimulation appears to be critical for spatial memory destabilization and related memory updating. Enhancing our understanding of the role of ACh in memory updating should inform future research into the underlying causes of behavioural disorders that are characterized by persistent maladaptive memories, such as age-related cognitive inflexibility and post-traumatic stress disorder.

Differential effects of intra-RMTg infusions of pilocarpine or 4-DAMP on regulating depression- and anxiety-like behaviors.

Depression and anxiety are associated with dysfunction of the mesolimbic dopamine system. The rostromedial tegmental nucleus (RMTg) is predominantly composed of GABAergic neurons that exhibit dense projections and strongly inhibit mesolimbic dopaminergic neurons, proposed as a major "brake" for the system. Consequently, the RMTg may be a crucial brain region for regulating these emotions. The central cholinergic system, particularly the muscarinic receptors, plays an important regulatory role in depression and anxiety. M3 muscarinic receptors are distributed on GABAergic neurons in the RMTg, but their involvement in the regulation of depression and anxiety remains uncertain. This study aimed to examine the effects of RMTg M3 muscarinic receptors on regulating depression- and anxiety-like behaviors in adult male Wistar rats, as assessed through the forced swim, tail suspension, and elevated plus maze tests. The results showed that intra-RMTg injections of the M1/M3 muscarinic receptors agonist, pilocarpine (3, 10, and 30 µg/side), or the M3 muscarinic receptors antagonist, 4-DAMP (0.5, 1, and 2 µg/side), did not alter the immobility time in the forced swim and tail suspension tests. Additionally, pilocarpine (30 µg/side) decreased time spent in open arms and increased time in closed arms in the elevated plus maze; while 4-DAMP (1 and 2 µg/side) played the opposite role by increasing time spent in open arms and decreasing time in closed arms. These findings suggest that RMTg M3 muscarinic receptors have differential effects on regulating depression- and anxiety-like behaviors. Enhancing or inhibiting these receptors can produce anxiogenic or anxiolytic effects, but have no impact on depression-like behavior. Therefore, RMTg M3 muscarinic receptors are involved in regulating anxiety and may be a potential therapeutic target for anxiolytic drugs.

M2 muscarinic receptors negatively modulate cell migration in human glioblastoma cells.

Glioblastoma (GB) is a very aggressive human brain tumor. The high growth potential and invasiveness make this tumor surgically and pharmacologically untreatable. Our previous work demonstrated that the activation of the M2 muscarinic acetylcholine receptors (M2 mAChRs) inhibited cell proliferation and survival in GB cell lines and in the cancer stem cells derived from human biopsies. The aim of the present study was to investigate the ability of M2 mAChR to modulate cell migration in two different GB cell lines: U87 and U251. By wound healing assay and single cell migration analysis performed by time-lapse microscopy, we demonstrated the ability of M2 mAChRs to negatively modulate cell migration in U251 but not in the U87 cell line. In order to explain the different effects observed in the two cell lines we have evaluated the possible involvement of the intermediate conductance calcium-activated potassium (IKCa) channel. IKCa channel is present in the GB cells, and it has been demonstrated to modulate cell migration. Using the perforated patch-clamp technique we have found that selective activation of M2 mAChR significantly reduced functional density of the IKCa current in U251 but not in U87 cells. To understand whether the M2 mAChR mediated reduction of ion channel density in the U251 cell line was relevant for the cell migration impairment, we tested the effects of TRAM-34, a selective inhibitor of the IKCa channel, in wound healing assay. We found that it was able to markedly reduce U251 cell migration and significantly decrease the number of invadopodia-like structure formations. These results suggest that only in U251 cells the reduced cell migration M2 mAChR-mediated might involve, at least in part, the IKCa channel.

Assessing the effect of breed, age, and sex on muscarinic receptor distribution in atria and ventricles of turkeys.

Atrial and ventricular myocardium from young (6-wk-old), young adult (3-6-mo-old), and aged (14-15-mo-old) meat-type (B.U.T. Big 6) and wild-type (Cröllwitzer) turkeys were used to study the influence of age and sex on cholinergic muscarinic receptors using [3H]-N-methyl-scopolamine (3H-NMS) binding studies. In both breeds, saturation experiments indicated the presence of regional-, sex-, and age-related differences in the density of cholinergic muscarinic receptors (Bmax), that is, a decrease or increase. Except for right atria, Bmax was decreased in both male and female B.U.T. Big 6 hearts with increasing age. Similarly, a negative correlation between Bmax and age could be seen in female and male atria of Cröllwitzer turkeys, while positive correlation could be seen in right and left ventricles of male, and only right ventricles of female Cröllwitzer turkeys. The affinity of the receptor (KD) was not affected by age, sex and breed. In all cardiac chamber tissues, the M2-subtype was shown to be predominant followed by the M3-subtype and to a lesser extent the M1-subtype. Aspects of this age-dependent remodeling of the heart differ between sexes, resulting in maladaptive changes in older turkeys with a high degree of frailty. These observations may help explain why males and females are susceptible to different cardiovascular diseases as they age and why frail older adults are most often affected by these diseases.

Separation and simultaneous estimation of enantiomers and Diastereomers of muscarinic receptor antagonist Solifenacin using stability-indicating Normal-phase HPLC technique with chiral stationary phase amylose tris-(3,5-dimethylphenylcarbamate).

The R,S-enantiomer impurity and diastereomer impurities (S,S-isomer and R,R-isomer) of the solifenacin (S,R-enantiomer) were effectively separated and quantified simultaneously utilizing normal-phase high-performance liquid chromatography with a chiral stationary phase consisting of amylose tris (3,5-dimethylphenylcarbamate) coated on silica-gel (Chiralpak, AD-H). The enantiomeric and stereo-selective separation was achieved within a run time of 35 minutes using a mobile phase of 'n-hexane, ethanol, and diethylamine' in an isocratic elution mode with a detection wavelength of 220 nm. The validation attributes assessed were accuracy (which showed excellent recoveries between 97.5% and 100.4%) and linearity (which was proven in the range of 0.081-1.275 µg.mL-1 , with a linear regression of 0.999). The stress testing experiments proved that the developed methodology by the HPLC technique has stability-indicating characteristics, as all closely eluting peak pairs were separated well with a resolution of 2.3 and without any interference. The proposed methodology was highly efficient in separating and simultaneously determining the chiral impurities (enantiomers and diastereomers) of the solifenacin in the release and stability sample analyses of drug substances and tablets in pharmaceutical formulations.

Effect of spirocyclopiperazinium salt compound LXM-15 on spinal nerve injury in rats.

BACKGROUND: Currently available therapies for neuropathic pain show limited efficacy. This study aimed to investigate the anti-nociceptive effect of the spirocyclopiperazinium salt compound LXM-15 in spinal nerve ligation (SNL) rats and to explore the potential mechanisms. METHODS: Mechanical allodynia and thermal hyperalgesia tests were used to evaluate the effects of LXM-15 in SNL rats. The expression of CaMKIIα, CREB, JAK2, STAT3, c-fos and TNF-α was detected by western blotting, ELISA or qRT-PCR analysis. Receptor blocking test was performed to explore possible target. RESULTS: Administration of LXM-15 (1, 0.5, 0.25 mg/kg, i.g.) dose-dependently attenuated mechanical allodynia and thermal hyperalgesia in rats subjected to SNL (p < 0.01, p < 0.05), and the effects were completely blocked by peripheral α7 nicotinic or M4 muscarinic receptor antagonist (p > 0.05). LXM-15 significantly decreased the overexpression of phosphorylated CaMKIIα, CREB, JAK2 and STAT3 proteins and the mRNA levels of TNF-α and c-fos (p < 0.01, p < 05). All of the effects could be blocked by α7 or M4 receptor antagonist. Furthermore, LXM-15 reduced the protein expression of TNF-α and c-fos (p < 0.01, p < 0.05). No significant acute toxicity or abnormal hepatorenal function was observed. CONCLUSIONS: This is the first study to report that LXM-15 exerts significant anti-nociceptive effect on SNL rats. This effect may occur by activating peripheral α7 nicotinic and M4 muscarinic receptors, further inhibiting the CaMKIIα/CREB and JAK2/STAT3 signalling pathways, and finally inhibiting the expression of TNF-α and c-fos. SIGNIFICANCE: Existing treatments for neuropathic pain show limited efficacy with severe adverse reactions. This paper is the first to report that LXM-15, a new spirocyclopiperazinium salt compound, exerts a significant anti-nociception in SNL rats without obvious toxicity. The underlying mechanisms include activating peripheral α7 nicotinic and M4 muscarinic receptors, then inhibiting the signalling pathways of CaMKIIα/CREB and JAK2/STAT3 and the expressions of TNF-α and c-fos. This study sheds new light on the development of novel analgesic drugs with fewer side effects.

Targeting the Actions of Muscarinic Receptors on Dopamine Systems: New Strategies for Treating Neuropsychiatric Disorders.

Cholinergic regulation of dopamine (DA) signaling has significant implications for numerous disorders, including schizophrenia, substance use disorders, and mood-related disorders. The activity of midbrain DA neurons and DA release patterns in terminal regions are tightly regulated by cholinergic neurons found in both the striatum and the hindbrain. These cholinergic neurons can modulate DA circuitry by activating numerous receptors, including muscarinic acetylcholine receptor (mAChR) subtypes. This review specifically focuses on the complex role of M2, M4, and M5 mAChR subtypes in regulating DA neuron activity and DA release and the potential clinical implications of targeting these mAChR subtypes.

Population-Based Characterization of the Pharmacokinetics and Food Effect of ANAVEX3-71, a Novel Sigma-1 Receptor and Allosteric M1 Muscarinic Receptor Agonist in Development for Treatment of Frontotemporal Dementia, Schizophrenia, and Alzheimer Disease.

Pharmacokinetic (PK) data from 28 subjects who received 5-200-mg single ascending doses of ANAVEX3-71, formerly AF710B, were analyzed to characterize the PK of ANAVEX3-71 and its M8 metabolite. PK data from 12 subjects who received 160 mg ANAVEX3-71 under fed and fasted conditions were analyzed to characterize the effect of food on the PK of the drug and its M8 metabolite. PK was characterized using the standard 2-stage approach and the nonlinear mixed-effects modeling approach. Dose proportionality was determined using the power model. Two- and 3-compartment linear PK models were tested for the characterization of the PK of ANAVEX3-71 and its M8 metabolite. The PK of ANAVEX3-71 is linear, dose proportional, and time invariant. The drug is rapidly eliminated with a mean (standard deviation) apparent terminal elimination half-life of 3.56 (4.09) hours, while the M8 metabolite was eliminated with a mean (standard deviation) apparent terminal elimination half-life of 6.59 (1.64) hours. The population PK model was used to investigate the effects of covariates on the PK of ANAVEX3-71 and M8. Age, weight, and creatinine clearance were not explanatory of the variability in apparent clearance and apparent volume of the central compartment of ANAVEX3-71. Food had no effect on the PK of ANAVEX3-71 and its M8 metabolite.

Efficacy and safety of the muscarinic receptor agonist KarXT (xanomeline-trospium) in schizophrenia (EMERGENT-2) in the USA: results from a randomised, double-blind, placebo-controlled, flexible-dose phase 3 trial.

BACKGROUND: New treatments with new mechanisms are urgently needed for people with schizophrenia. Xanomeline is a dual M1 and M4-preferring muscarinic receptor agonist that does not block D2 dopamine receptors, unlike all currently approved treatments for schizophrenia. Xanomeline-trospium (KarXT) combines xanomeline with the peripherally restricted muscarinic receptor antagonist trospium chloride with the goal of ameliorating xanomeline-related adverse events associated with peripheral muscarinic receptors. The EMERGENT-2 trial aimed to assess the efficacy and safety of KarXT in people with schizophrenia experiencing acute psychosis. METHODS: EMERGENT-2 was a randomised, double-blind, placebo-controlled, flexible-dose, 5-week, inpatient, phase 3 trial in people with schizophrenia. Participants were adults aged 18-65 years with a diagnosis of schizophrenia who had a recent worsening of psychosis warranting hospital admission, a Positive and Negative Syndrome Scale (PANSS) score of 80 or higher, and a Clinical Global Impression-Severity score of 4 or higher. The participants were recruited from 22 inpatient sites in the USA, and were randomly assigned (1:1) to KarXT or placebo twice per day. Participants randomly assigned to KarXT received 50 mg xanomeline and 20 mg trospium twice per day for the first 2 days and then 100 mg xanomeline and 20 mg trospium twice per day for days 3-7. Beginning on day 8, KarXT dosing was flexible with an optional increase to 125 mg xanomeline and 30 mg trospium twice per day and the option to return to 100 mg xanomeline and 20 mg trospium based on tolerability. The primary endpoint was change from baseline to week 5 in PANSS total score. Efficacy analyses used the modified intention-to-treat population (all randomly assigned participants who received at least one trial medication dose and had at least one post-baseline PANSS assessment). Least squares mean change from baseline, SE, and least squares mean difference between the KarXT and placebo groups at week 5, along with the 95% CI and two-sided p values were calculated for the primary and secondary continuous efficacy endpoints. Safety analyses included all participants receiving at least one trial medication dose and used descriptive statistics. This trial is registered with ClinicalTrials.gov (NCT04659161). FINDINGS: From Dec 16, 2020, to April 13, 2022, of 407 people who were screened, 252 participants meeting enrolment criteria were randomly assigned to the KarXT (n=126) or placebo (n=126). Baseline PANSS total scores were 98·3 (KarXT; n=126) and 97·9 (placebo; n=125). The trial met the primary endpoint with a mean change from baseline to week 5 in PANSS total score that favoured KarXT (-21·2 points, SE 1·7) versus placebo (-11·6 points, 1·6; least squares mean difference -9·6; 95% CI -13·9 to -5·2; p<0·0001, Cohen's d effect size=0·61). All secondary endpoints were also met, and favoured KarXT versus placebo (p<0·05). The most common adverse events with KarXT versus placebo were constipation (27 [21%] vs 13 [10%]), dyspepsia (24 [19%] vs 10 [8%]), headache (17 [14%] vs 15 [12%]), nausea (24 [19%] vs seven [6%]), vomiting (18 [14%] vs one [1%]), hypertension (12 [10%] vs one [1%]), dizziness (11 [9%] vs four [3%]), gastro-oesophageal reflux disease (eight [6%] vs zero [0%]), and diarrhoea (seven [6%] vs four [3%]). Treatment-emergent adverse event rates of extrapyramidal motor symptoms (KarXT, zero [0%] vs placebo, zero [0%]), akathisia (one [1%] vs one [1%]), weight gain (zero [0%] vs one [1%]), and somnolence (six [5%] vs five [4%]) were similar between the KarXT and placebo groups, as were adverse event-related discontinuation rates (nine [7%] vs seven [6%]). INTERPRETATION: In the EMERGENT-2 trial, KarXT was effective in reducing positive and negative symptoms and was generally well tolerated. These results support the potential for KarXT to represent a new class of effective and well tolerated antipsychotic medicines based on activating muscarinic receptors, not the D2 dopamine receptor-blocking mechanism of all current antipsychotic medications. Results from additional trials, including the identical EMERGENT-3 trial and the 52-week, open-label EMERGENT-4 and EMERGENT-5 trials, will provide additional information on the efficacy and safety of KarXT in people with schizophrenia. FUNDING: Karuna Therapeutics.

Precise sensorimotor control impacts reproductive fitness of C. elegans in 3D environments.

The ability of animals to sense and navigate towards relevant cues in complex and elaborate habitats is paramount for their survival and reproductive success. The nematode Caenorhabditis elegans uses a simple and elegant sensorimotor program to track odors in its environments. Whether this allows the worm to effectively navigate a complex environment and increase its evolutionary success has not been tested yet. We designed an assay to test whether C. elegans can track odors in a complex 3D environment. We then used a previously established 3D cultivation system to test whether defect in tracking odors to find food in a complex environment affected their brood size. We found that wild-type worms can accurately migrate toward a variety of odors in 3D. However, mutants of the muscarinic acetylcholine receptor GAR-3 which have a sensorimotor integration defect that results in a subtle navigational defect steering towards attractive odors, display decreased chemotaxis to the odor butanone not seen in the traditional 2D assay. We also show that the decreased ability to locate appetitive stimuli in 3D leads to reduced brood size not observed in the standard 2D culture conditions. Our study shows that mutations in genes previously overlooked in 2D conditions can have a significant impact in the natural habitat, and highlights the importance of considering the evolutionary selective pressures that have shaped the behavior, as well as the underlying genes and neural circuits.

Evaluation of Food Effect on the Pharmacokinetics of Velufenacin, a New Muscarinic Receptor Antagonist, in Healthy Subjects.

Velufenacin (DA-8010) is a new muscarinic receptor antagonist under development for the treatment of overactive bladder. This study aimed to evaluate the effect of food on the pharmacokinetics (PK) and safety of velufenacin in healthy subjects. A randomized, open-label, single-dose, 4-sequence, 4-treatment, 4-period crossover study was conducted. Subjects received a single oral dose of velufenacin 2.5 or 5 mg in a fasted or fed (high-fat meal) state in each period with a 7-day washout. PK parameters including maximum plasma concentration (Cmax ) and area under the concentration-time curve from time 0 to the last measurable point were compared between the fed and fasted states. Twenty-seven subjects completed the study. The mean area under the concentration-time curve from time 0 to the last measurable point of the velufenacin 2.5 and 5 mg doses under the fed condition showed a 1.5- and 1.3-fold increase, respectively, compared to the fasted condition. The corresponding values for Cmax were a 2.3- and 2.0-fold increase, respectively. The time to reach Cmax was comparable regardless of the dose or food intake, showing median values of 4.5-5.0 hours. These results suggest a modest increase of velufenacin absorption by food intake. Velufenacin was generally safe and well tolerated at the 2.5 and 5 mg doses regardless of food.