M1 Receptor Agonist Research Articles

Melatonin receptor agonists in neurodegenerative diseases and psychiatric disorders

Neurodegenerative and psychiatric diseases remain a therapeutic challenge. Mental illnesses such as depression and bipolar affective disorder lead to disruption of the diurnal rhythm of melatonin secretion and disruption of the sleep-wake axis. The use of new melatonergic drugs has received special attention in recent years. Selective melatoninergic M1 and M2 receptor agonists (tasimelteon, ramelteon) show a more favorable metabolic profile, better regulation of circadian rhythms, and effects on total sleep time and sleep quality compared to the already well-studied melatonin. In addition, these drugs show no addictive potential. Agomelatine, through its action on M1 and M2 receptors, as well as serotonergic 5-HT2C and 5-HT2B receptors, exhibits antidepressant, normothymic, and neuroprotective effects, providing primary therapy or adjunctive treatment.

A golden age of muscarinic acetylcholine receptor modulation in neurological diseases.

Over the past 40 years, the muscarinic acetylcholine receptor family, particularly the M1-receptor and M4-receptor subtypes, have emerged as validated targets for the symptomatic treatment of neurological diseases such as schizophrenia and Alzheimer disease. However, despite considerable effort and investment, no drugs have yet gained clinical approval. This is largely attributable to cholinergic adverse effects that have halted the majority of programmes and resulted in a waning of interest in these G-protein-coupled receptor targets. Recently, this trend has been reversed. Driven by advances in structure-based drug design and an appreciation of the optimal pharmacological properties necessary to deliver clinical efficacy while minimizing adverse effects, a new generation of M1-receptor and M4-receptor orthosteric agonists and positive allosteric modulators are now entering the clinic. These agents offer the prospect of novel therapeutic solutions for 'hard to treat' neurological diseases, heralding a new era of muscarinic drug discovery.

The Secure Therapeutic Effects of Recently Developed Antipsychotic Drugs and Updated Neural Networks in Schizophrenia

Introduction:: Schizophrenia and schizoaffective disorder are treated in most cases with antipsychotic drugs of the second generation. These drugs block dopaminergic and serotonergic receptors, i.e., D2 and 5-HT2A receptors, and cause different adverse effects, for example, movement disturbances of the extrapyramidal system and adverse effects of vital parameters and of the heart. These drugs treat positive symptoms in schizophrenia and, to a lesser extent, negative symptoms. This review presents the development of newer antipsychotic drugs. Methods/Material:: References were taken from PubMed after using the following keywords: schizophrenia, schizoaffective disorder, antipsychotic drug, neurotransmitter and neuropeptide. Among these newer antipsychotic drugs are cariprazine, brexipiprazole and lumateperone, which exert a partial agonistic effect at D2 and 5-HT2A receptors, pimavanserin, a 5-HT2A receptor antagonist which treats negative symptoms in schizophrenia as add-on therapy, olanzapine combined with samidorphan, which reduces weight gain, and M4 or M1 receptor agonists, for example, xanomeline with an antipsychotic effect combined with trospium, an anticholinergic drug. Neural networks were updated in order to deduce the antipsychotic mechanism of action of newer antipsychotic drugs, especially xanomeline. Results:: The newer antipsychotic drugs cariprazine, brexipiprazole and lumateperone show antipsychotic, antimanic and anti-depressive effects, however, the efficacy on psychotic symptoms in long-term treatment has not yet been examined. Pimavanserin reduces negative symptoms in schizophrenia as an additional pharmacotherapy to treat this disorder. Olanzapine combined with samidorphan exerts good antipsychotic effects and reduces weight gain. The new antipsychotic drug xanomeline, the antipsychotic effect of which is quite different from the antidopaminergic effect, treats positive and negative symptoms in schizophrenia. Its mechanism of action was deduced from the neural networks presented. The long-term efficacy should still be examined. Conclusion:: This review is focused on newer antipsychotic drugs. The long-term efficacy of cariprazine, brexipiprazole and lumateperone in the treatment of schizophrenia should be examined further. Neural networks in the brain areas involved in schizophrenia should be examined and updated furthermore. Newer antipsychotic drugs ,for example, xanomeline, an M4 or M1 receptor agonist, which has been combined with trospium, an anticholinergic drug, the mechanism of action of which can be derived from the neural network suggested in this review.

New, not anti-dopaminergic antipsychotic drugs in the treatment of schizophrenia, M4 or M1 receptor agonists and TAAR1 agonists

New, not anti-dopaminergic antipsychotic drugs in the treatment of schizophrenia, M4 or M1 receptor agonists and TAAR1 agonists

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

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. 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). 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%]). 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. Karuna Therapeutics.

Early treatment with an M1 and sigma-1 receptor agonist prevents cognitive decline in a transgenic rat model displaying Alzheimer-like amyloid pathology

The application of the selective allosteric M1 muscarinic and sigma-1 receptor agonist, AF710B (aka ANAVEX3-71), has shown to attenuate Alzheimer’s disease-like hallmarks in McGill-R-Thy1-APP transgenic rats when administered at advanced pathological stages. It remains unknown whether preventive treatment strategies applying this compound may be equally effective. We tested whether daily oral administration of AF710B (10 µg/kg) in 7-month-old, preplaque, McGill-R-Thy1-APP rats for 7 months, followed by a 4-week washout period, could prevent Alzheimer’s disease-like pathological hallmarks. Long-term AF710B treatment prevented the cognitive impairment of McGill-R-Thy1-APP rats. The effect was accompanied by a reduction in the number of amyloid plaques in the hippocampus and the levels of Aβ42 and Aβ40 peptides in the cerebral cortex. AF710B treatment also reduced microglia and astrocyte recruitment toward CA1 hippocampal Aβ-burdened neurons compared to vehicle-treated McGill-R-Thy1-APP rats, also altering the inflammatory cytokines profile. Lastly, AF710B treatment rescued the conversion of brain-derived neurotrophic factor precursor to its mature and biologically active form. Overall, these results suggest preventive and disease-modifying properties of the compound.

PS-B03-5: HIGH-DOSES OF LOSARTAN RATHER ENHANCE CATECHOLAMINE SECRETION FROM THE PERFUSED ADRENAL GLAND

Objective: Previously, we have found that Losartan (5∼50 μM), an AT1 receptor blocker, dose-dependently inhibits secretion of catecholamines (CA) evoked by cholinergic and angiotensin II (Ang II) receptor stimulation from the perfused rat adrenal medulla. The aim of the present study was to examine the characteristic effects of high-dose losartan on the CA secretion from the isolated perfused rat adrenal gland, and also to establish its mechanism of action. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: The perfusion of high-dose losartan (150 ∼ 600 μM) into an adrenal vein for 90 min resulted in great increases in CA secretions evoked by acetylcholine in a dose-dependent fashion. Also, losartan (300 μM) enhanced the CA release evoked by Ang II, high K+ and DMPP (a selective nicotinic receptor agonist), McN-A-343 (a selective muscarinic M1 receptor agonist). Also, in the presence of losartan (300 μM), the CA secretory responses to veratridine (a selective Na+ channel activator), Bay-K-8644 (a Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly potentiated, respectively. The facilitatory CA-releasing effect of Ach evoked by high-dose losartan (300 μM) was time-dependently depressed by pretreatment with nifedipine (Ca2+ channel blocker), while not by pretreatment with sodium nitroprusside (an NO donor). High-dose olmesartan (150 ∼ 600 μM), one of ARBs, also potentiated Ach-evoked CA release in time-dependent fashion. However, in the simultaneous presence of losartan (300 μM) and CGP42112 (an AT2 receptor agonist), the facilitatory responses of losartan on the CA secretion evoked by ACh were rather significantly reduced in comparison with that of losartan-treatment alone. Conclusion: Conclusively, these results demonstrate that high-dose losartan can potentiate the adrenomedullary CA secretion in a calcium-dependent fashion. It seems that this facilitatory effect of high-dose losartan may be mediated by activation of the Ca2+ influx through L-type Ca2+ channels into adrenomedullary cells without inhibition of NO synthesis, which seems to be relevant to AT2 receptor blockade. It also suggests that high-dose ARBs may be a risk factor in causing hypertensive response.

S-13-4: INHIBITORY IMPACTS OF CHLOROGENIC ACID ON ADRENAL CATECHOLAMINE SECRETION

(Objective) Chlorogenic acid (CGA) is one of the most available phenolic acid compounds in foods, such as coffee and tea. Thus, the present study was aimed to determine the characteristics of CGA on secretion of catecholamines (CA) in the perfused model of the rat adrenal medulla, and also to validify its mechanism of action. (Design and Methods) The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. (Results) CGA (30∼300 μM), administered into an adrenal vein for 90 min, reduced ACh-induced CA secretion in a dose- and time-dependent manner. CGA also time-dependently depressed the CA secretion induced by McN-A-343 (a selective muscarinic M1 receptor agonist), DMPP (a selective neuronal nicotinic receptor agonist), and angiotensin II. CGA itself failed to influence spontaneous CA secretion. Also, During the perfusion of CGA for 90 min, the CA secretory responses induced by high K+ (56 mM, a direct membrane depolarizer), veratridine (a voltage-dependent Na+ channel activator, cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, Bay-K-8644 (an L-type dihydropyridine Ca2+ channel activator were suppressed. However, in the simultaneous presence of CGA and L-NAME (an inhibitor of NO synthase), the CA secretory responses induced by ACh, Ang II, Bay-K-8644 and veratridine was restored nearly to the control level compared to those of the inhibitory effects of CGA-treatment alone. Virtually, the adrenal NO release was significantly enhanced by CGA-treatment. Also, in the coexistence of CGA and olmesartan, ACh-induced CA secretion was markedly reduced compared to that of olmesartan-treatment alone. (Conclusion) Taken together, we present the first evidence that CGA suppresses the CA secretion induced by activation of cholinergic receptors as well as AT1-receptors. This CGA-induced inhibitory effect seems to be exerted by reducing both the influx of Na+ and Ca2+ through their ionic channels into the adrenomedullary chromaffin cells as well as by suppressing the Ca2+ release from the cytoplasmic calcium store, at least through the elevated NO release by the activation of NO synthase, which is associated to the blockade of neuronal cholinergic receptors and AT1-receptors. Based on these results, the ingestion of CGA may be helpful to alleviate or prevent the cardiovascular diseases, via reduction of adrenal CA secretion and consequent decreased CA level in the circulation. Co-administration of CGA and Olmesartan (ARB) may be clinically beneficial for treatment of cardiovascular diseases, including hypertension and angina pectoris.

PS-B03-6: COMPARISON OF SUPPRESSIVE EFFECTS ON CATECHOLAMINE SECRETION BETWEEN OLMESARTAN AND ENALAPRIL

Objective: The present study was designed to compare olmesartan, an angiotensin II receptor blocker (ARB) with enalapril, an angiotensin converting enzyme inhibitor (ACEI) in the suppressive effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and Methods: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both olmesartan (50 μM) and enalapril (50 μM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 μM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 μM), high K+ (a direct membrane-depolarizer, 56 μM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 μM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 μM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 μM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 μM) were significantly reduced. Based on the same concentration (50 μM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan &gt; enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusion: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

P599. The Muscarinic Agonist Xanomeline Demonstrates Standalone Activity and Augments Clinical Antipsychotics in Rodent Behavioral Models of Psychosis

Evidence indicates that muscarinic acetylcholine receptor agonists exhibit antipsychotic properties. Xanomeline, a centrally active M1 and M4 receptor agonist, is devoid of any direct dopamine receptor affinity or activity. An important question is whether xanomeline can also augment the effects of existing antipsychotics. Two rodent behavioral models were used to test combinations of xanomeline and either risperidone or aripiprazole. The conditioned avoidance response (CAR) and psychostimulant-induced locomotor activity (LMA) assays are commonly used behavioral tests in rodents that have good predictive validity for detecting antipsychotic activity.

Understanding Why Muscarinic Receptor Agonists Have Antipsychotic Properties

All current antipsychotics have direct dopamine (DA) D2 receptor activity, which is associated with problems such as dysphoria, EPS, or prolactin elevation. Muscarinic receptor agonists have shown antipsychotic-like activity across preclinical models and clinical trials. This poster reviews preclinical evidence as to how muscarinic receptor agonists, such as the M1/M4 preferring agonist xanomeline, might have clinically relevant antipsychotic effects. Highlight the novel mechanisms through which muscarinic receptor agonists are associated with antipsychotic effects without having any direct dopaminergic D2 receptor activity. The muscarinic receptor family is composed of 5 G protein-coupled receptors (GPCRs). One of the leading hypotheses explaining the antipsychotic activity of muscarinic receptor agonists is preclinical studies of muscarinic receptor modulation of those DA circuits associated with psychosis. Both M1 and M4 receptors are expressed in DA neural circuits implicated in psychosis, and provide unique regulation of these circuits. Xanomeline has both functional M1 and M4 receptor agonist activity, and shows robust antipsychotic-like effects in several animal models that require the presence of functional M1 and M4 receptors. M4 receptors are autoreceptors on cholinergic neurons that regulate DA circuits in two locations: ventral tegmental area (VTA) and nucleus accumbens (NAc). Cholinergic-rich neurons from the hindbrain at the VTA, where they release acetylcholine (ACh) into DA-rich synaptic spaces. M4 autoreceptors are present on these neurons, and their activation reduces ACh release and lowers ambient synaptic ACh concentrations, leading to reduced DA cell firing. Cholinergic interneurons residing in the NAc also express M4 autoreceptors. These ACh interneurons regulate ACh release with M4 activation also turning off ACh release. Therefore, M4 receptors serve as DA regulators at VTA and NAc, both key sites for dopamine's role in psychotic processes. M1 receptors regulate DA circuits in a different, "top down" manner. M1 receptors are found on cortical inhibitory interneurons. When activated, inhibitory drive onto excitatory output neurons is enhanced, which leads to reduced excitatory tone to VTA DA neurons. Over the last 25years, a growing body of evidence has shown potential for muscarinic receptor agonists to become a new class of medicines with potent antipsychotic activity. Preclinical data at micro-and macro-circuit levels suggest that the M1 and M4 receptor subtypes are most relevant in the regulation of DA circuits. The antipsychotic effects of muscarinic agonists effects may arise from influencing these key muscarinic receptor subtypes that are integral to the regulation of DA neural circuits. In summary, there has been great progress in understanding the potential for muscarinic receptor agonists for the treatment of psychosis. Karuna Therapeutics.

Reduced expression of the psychiatric risk gene DLG2 (PSD93) impairs hippocampal synaptic integration and plasticity

Copy number variants indicating loss of function in the DLG2 gene have been associated with markedly increased risk for schizophrenia, autism spectrum disorder, and intellectual disability. DLG2 encodes the postsynaptic scaffolding protein DLG2 (PSD93) that interacts with NMDA receptors, potassium channels, and cytoskeletal regulators but the net impact of these interactions on synaptic plasticity, likely underpinning cognitive impairments associated with these conditions, remains unclear. Here, hippocampal CA1 neuronal excitability and synaptic function were investigated in a novel clinically relevant heterozygous Dlg2+/− rat model using ex vivo patch-clamp electrophysiology, pharmacology, and computational modelling. Dlg2+/− rats had reduced supra-linear dendritic integration of synaptic inputs resulting in impaired associative long-term potentiation. This impairment was not caused by a change in synaptic input since NMDA receptor-mediated synaptic currents were, conversely, increased and AMPA receptor-mediated currents were unaffected. Instead, the impairment in associative long-term potentiation resulted from an increase in potassium channel function leading to a decrease in input resistance, which reduced supra-linear dendritic integration. Enhancement of dendritic excitability by blockade of potassium channels or activation of muscarinic M1 receptors with selective allosteric agonist 77-LH-28-1 reduced the threshold for dendritic integration and 77-LH-28-1 rescued the associative long-term potentiation impairment in the Dlg2+/− rats. These findings demonstrate a biological phenotype that can be reversed by compound classes used clinically, such as muscarinic M1 receptor agonists, and is therefore a potential target for therapeutic intervention.

Muscarinic signaling regulates voltage-gated potassium channel KCNQ2 phosphorylation in the nucleus accumbens via protein kinase C for aversive learning.

The nucleus accumbens (NAc) plays critical roles in emotional behaviors, including aversive learning. Aversive stimuli such as an electric foot shock increase acetylcholine (ACh) in the NAc, and muscarinic signaling appears to increase neuronal excitability and aversive learning. Muscarinic signaling inhibits the voltage-dependent potassium KCNQ current which regulates neuronal excitability, but the regulatory mechanism has not been fully elucidated. Phosphorylation of KCNQ2 at threonine 217 (T217) and its inhibitory effect on channel activity were predicted. However, whether and how muscarinic signaling phosphorylates KCNQ2 in vivo remains unclear. Here, we found that PKC directly phosphorylated KCNQ2 at T217 in vitro. Carbachol and a muscarinic M1 receptor (M1R) agonist facilitated KCNQ2 phosphorylation at T217 in NAc/striatum slices in a PKC-dependent manner. Systemic administration of the cholinesterase inhibitor donepezil, which is commonly used to treat dementia, and electric foot shock to mice induced the phosphorylation of KCNQ2 at T217 in the NAc, whereas phosphorylation was suppressed by an M1R antagonist. Conditional deletion of Kcnq2 in the NAc enhanced electric foot shock induced aversive learning. Our findings indicate that muscarinic signaling induces the phosphorylation of KCNQ2 at T217 via PKC activation for aversive learning.

From structure to clinic: Design of a muscarinic M1 receptor agonist with the potential to treat Alzheimer’s disease

Current therapies for Alzheimer's disease seek to correct for defective cholinergic transmission by preventing the breakdown of acetylcholine through inhibition of acetylcholinesterase, these however have limited clinical efficacy. An alternative approach is to directly activate cholinergic receptors responsible for learning and memory. The M1-muscarinic acetylcholine (M1) receptor is the target of choice but has been hampered by adverse effects. Here we aimed to design the drug properties needed for a well-tolerated M1-agonist with the potential to alleviate cognitive loss by taking a stepwise translational approach from atomic structure, cell/tissue-based assays, evaluation in preclinical species, clinical safety testing, and finally establishing activity in memory centers in humans. Through this approach, we rationally designed the optimal properties, including selectivity and partial agonism, into HTL9936-a potential candidate for the treatment of memory loss in Alzheimer's disease. More broadly, this demonstrates a strategy for targeting difficult GPCR targets from structure to clinic.

PET Imaging of brain muscarinic receptors with 18F-Fluorobenzyl-Dexetimide: A first in human study

M1 and M4 muscarinic receptor (mAChR) agonists are under development for the treatment of schizophrenia, Alzheimer's and Parkinson's disease. We performed first-in-human PET imaging of mAChR with 18F-Fluorobenzyl-Dexetimide (FDEX) in 10 healthy participants (29.4±4.3yrs). Four underwent dynamic brain scanning for 240 min, and then six underwent static brain scans at 120 and 160-min post injection of 250 MBq of FDEX. Gjedde-Patlak graphical analysis was applied to determine the influx constant (Ki). Regional tissue ratios (SUVR) were calculated using the cerebellar cortex as the reference region. No adverse events were observed. The tracer showed good brain entry (∼4.2% ID at 5 min) but irreversible distribution kinetics over four hours in regions of high mAChR. Binding was consistent with the distribution of mAChR receptors with striatum > cortex > hippocampus >> thalamus >>> cerebellum with low variance in regional binding between subjects. Ki was 0.42±0.04 in the putamen, 0.27±0.01 in frontal cortex, 0.25±0.02 in the hippocampus and 0.10±0.01 in the thalamus. SUVR at 120 and 240 min. were highly correlated with these Ki values with R2 of 0.91 and 0.99 respectively. FDEX yields high quality brain images with uptake in the known distribution of mAChR with remarkably little variance between normal subjects.

Safety and Pharmacokinetics of HTL0018318, a Novel M1 Receptor Agonist, Given in Combination with Donepezil at Steady State: A Randomized Trial in Healthy Elderly Subjects.

IntroductionHTL0018318 is a selective muscarinic M1 receptor partial agonist under development for the symptomatic treatment of dementias, including Alzheimer’s disease. Clinically, HTL0018318 would likely be used alone or in conjunction with cholinesterase inhibitors (e.g. donepezil).ObjectiveWe investigated the safety, tolerability, and pharmacokinetics of HTL0018318 given alone and in combination with donepezil.MethodsThis was a randomized, double-blind, placebo-controlled trial in 42 (to deliver 36 with combination treatment) healthy elderly subjects investigating the effects of oral HTL0018318 15 and 25 mg given alone and combined with donepezil 10 mg at steady state on adverse events (AEs), vital signs, saliva production, sleep quality, pulmonary function, subjective feelings, and pharmacokinetics.ResultsAEs were reported by lower percentages of subjects after HTL0018318 alone than after donepezil alone. There was no increase in the percentage of subjects reporting AEs after co-administration than after donepezil alone. Supine systolic blood pressure was 1.6 mmHg (95% confidence interval [CI] −3.1 to −0.1) lower after HTL0018318 alone than after combination treatment. This was comparable with results from placebo alone: 1.7 mmHg (95% CI −3.2 to 0.2) lower than with combination treatment. Supine pulse rate was 3.3 bpm (95% CI 1.5–5.1) higher after HTL0018318 alone than with co-administration. HTL0018318 and donepezil did not meaningfully affect each other’s pharmacokinetics.ConclusionHTL0018318 was well tolerated when given alone and in combination with donepezil. HTL0018318 and donepezil do not demonstrate pharmacokinetic or pharmacodynamic interactions, indicating that HTL0018318 can be safely administered in combination with donepezil.Clinical trial registrationNetherlands Trial register identifier NL5915, registered on 28 October 2016.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40268-021-00352-5.

Cholinergic regulation of cardiac pacemaker activity by L-type Cav1.3 channels

Abstract Funding Acknowledgements Type of funding sources: Foundation. Main funding source(s): Fondation Recherche Médicale Introduction The cholinergic regulation of heart rate (HR) is mediated by acetylcholine (ACh)-dependent activation of M2-receptors (M2R). Activated M2R promote release of the βγ-subunit of G-proteins to directly gate GIRK1/4 channels (underlying the cardiac IKACh current), while αi-subunits inhibit adenylate cyclase (AC) activity. AC inhibition reduces the intracellular concentration of cAMP, decreasing the activity of ion channels involved in pacemaking, including "funny" f-(HCN4) and L-type Cav1.3 calcium channels. Purpose To determine the role of L-type Cav1.3 channels in cholinergic regulation of heart rate. Methods We recorded the frequency of activation and position of pacemaker leading site in ex vivo sinus nodes and the HR of isolated Langendorff perfused hearts of mice at baseline or during ACh perfusion. We used control wild type (WT) mice, and five genetically modified mouse models: Cav1.3 knockout (KO, ablated Cav1.3-mediated L-type current), GIRK4KO (ablated IKACh current), HCN4-CNBD (selective deletion of cAMP-dependent regulation of HCN4), GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO. We performed in vivo telemetric recordings of heart rate (HR) in WT and GIRK4KO/Cav1.3KO animals. Results Data from optical mapping experiments showed that, under basal conditions, perfusion of 3 μM ACh significantly reduced the frequency of action potentials in WT (44%), HCN4-CNBD (38%), Cav1.3KO (65%) and GIRK4KO (8%) isolated mouse sinus node tissues. ACh application did not significantly affect the frequency of action potentials recorded in tissue from GIRK4KO/HCN4-CNBD and GIRK4KO/Cav1.3KO animals. Furthermore, in all sinus nodes tested, regardless of genotype, ACh shifted the pacemaker leading site from its normal position by at least 0.7 mm. Upon stimulation of the β-adrenergic pathway by Isoproterenol, to reproduce conditions of accentuated antagonism, 3µM ACh reduced HR in isolated hearts from WT (43.8%), HCN4-CNBD (38.7%), Cav1.3KO (25,4%), GIRK4KO (16.9%) and GIRK4KO/HCN4-CNBD (16.4%) mice. No significant HR reduction was recorded in hearts from GIRK4KO/Cav1.3KO animals. In vivo data indicate that HR reduction induced by combined injection of Hexamethonium ( a Nicotinic acetylcholine receptor blocker) with Carbamoylcholine (CCH, M2 receptor agonist) or with 2-Chloro-N6-Cyclopentyladenosine (CCPA, A1 receptor agonist) is higher in WT than in GIRK4KO/Cav1.3KO animals (68% vs 48% CCH, and 79% vs 62% CCPA, respectively). Conclusion Our data indicate that L-type Cav1.3 channels are involved in cholinergic regulation of heart rate in mice. In addition, when the intracellular concentration of cAMP is elevated (i.e. under conditions of accentuated antagonism), cholinergic regulation of sinus node pacemaking is reliant on Cav1.3 and KACh channels.

Safety, pharmacokinetics and exploratory pro-cognitive effects of HTL0018318, a selective M1 receptor agonist, in healthy younger adult and elderly subjects: a multiple ascending dose study

BackgroundThe cholinergic system and M1 receptor remain an important target for symptomatic treatment of cognitive dysfunction. The selective M1 receptor partial agonist HTL0018318 is under development for the symptomatic treatment of Dementia’s including Alzheimer’s disease (AD) and dementia with Lewy bodies (DLB). We investigated the safety, tolerability, pharmacokinetics and exploratory pharmacodynamics of multiple doses of HTL0018318 in healthy younger adults and elderly subjects.MethodsThis randomised, double blind, placebo-controlled study was performed, investigating oral doses of 15–35 mg/day HTL0018318 or placebo in 7 cohorts of healthy younger adult (n = 36; 3 cohorts) and elderly (n = 50; 4 cohorts) subjects. Safety, tolerability and pharmacokinetic measurements were performed. Pharmacodynamics were assessed using a battery of neurocognitive tasks and electrophysiological biomarkers of synaptic and cognitive functions.ResultsHTL0018318 was generally well-tolerated in multiple doses up to 35 mg/day and were associated with mild or moderate cholinergic adverse events. There were modest increases in blood pressure and pulse rate when compared to placebo-treated subjects, with tendency for the blood pressure increase to attenuate with repeated dosing. There were no clinically significant observations or changes in blood and urine laboratory measures of safety or abnormalities in the ECGs and 24-h Holter assessments. HTL0018318 plasma exposure was dose-proportional over the range 15–35 mg. Maximum plasma concentrations were achieved after 1–2 h. The apparent terminal half-life of HTL0018318 was 16.1 h (± 4.61) in younger adult subjects and 14.3 h (± 2.78) in elderly subjects at steady state. HTL0018318 over the 10 days of treatment had significant effects on tests of short-term (working) memory (n-back) and learning (Milner maze) with moderate to large effect sizes.ConclusionMultiple doses of HTL0018138 showed well-characterised pharmacokinetics and were safe and generally well-tolerated in the dose range studied. Pro-cognitive effects on short-term memory and learning were demonstrated across the dose range. These data provide encouraging data in support of the development of HTL0018138 for cognitive dysfunction in AD and DLB.Trial registrationNetherlands Trial Register identifier NTR5781. Registered on 22 March 2016.

COMPARISON OF INHIBITORY EFFECTS EVOKED BY OLMESARTAN AND ENALAPRIL ON CATECHOLAMINE SECRETION

Objective: The present study was designed to compare enalapril, an angiotensin converting enzyme inhibitor (ACEI) with olmesartan an angiotensin II receptor blocker (ARB) in the inhibitory effects on the secretion of catecholamines (CA) from the perfused model of the rat adrenal gland. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: Both enalapril (50 microM) and olmesartan (50 microM) during perfusion into an adrenal vein for 90 min inhibited the CA secretory responses evoked by ACh (5.32 microM), DMPP (a selective neuronal nicotinic Nn receptor agonist, 100 microM), high K+ (a direct membrane-depolarizer, 56 mM), and McN-A-343 (a selective muscarinic M1 receptor agonist, 100 microM) in a time-dependent manner. Also, in the presence of enalapril or olmesartan, the secretory responses of CA evoked by veratridine (an activator of voltage-dependent Na+ channels, 100 microM), Bay-K-8644 (a L-type dihydropyridine Ca2+ channel activator, 10 microM), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor, 10 microM) were significantly reduced. Based on the same concentration (50 microM) of enalapril and olmesartan, for the CA release evoked by Ach, high K+, DMPP, McN-A-343, angiotensin II, veratridine, Bay-K-8644 and cyclopiazonic acid, the following rank order of inhibitory potency was obtained: olmesartan&gt; enalapril. In the simultaneous presence of enalapril and olmesartan, Ach-evoked CA secretory responses were more strongly inhibited in comparison with that of enalapril- or olmesartan-treated alone. Conclusions: Collectively, these results demonstrate that both enalapril and olmesartan inhibit the CA secretory responses evoked by activation of both cholinergic and angiotensin II receptors stimulation as well as by direct membrane depolarization in the perfused model of the isolated rat adrenal medulla. When these two drugs were used in combination, their effects were enhanced, which may also be of clinical benefit. Based on concentration used in this study, it seems that the inhibitory effect of olmesartan on the CA secretion is more potent than that of enalapril.

HIGH-DOSES OF LOSARTAN RATHER ACCELERATE THE SECRETION OF ADRENAL CATECHOLAMINES

Objective: Previously, we have found that Losartan (5∼50 microM), an AT1 receptor blocker, dose-dependently inhibits secretion of catecholamines (CA) evoked by cholinergic and angiotensin II (Ang II) receptor stimulation from the perfused rat adrenal medulla. The aim of the present study was to examine the characteristic effects of high-dose losartan on the CA secretion from the isolated perfused rat adrenal gland, and also to establish its mechanism of action. Design and method: The adrenal gland was isolated and perfused with Krebs-bicarbonate. CA was measured directly by using the fluorospectrophotometer. Results: The perfusion of high-dose losartan (150 ∼ 600 microM) into an adrenal vein for 90 min resulted in great increases in CA secretions evoked by acetylcholine in a dose-dependent fashion. Also, losartan (300 microM) enhanced the CA release evoked by Ang II, high K+ and DMPP (a selective nicotinic receptor agonist), McN-A-343 (a selective muscarinic M1 receptor agonist). Also, in the presence of losartan (300 microM), the CA secretory responses to veratridine (a selective Na+ channel activator), Bay-K-8644 (a Ca2+ channel activator), and cyclopiazonic acid (a cytoplasmic Ca2+-ATPase inhibitor) were significantly potentiated, respectively. The facilitatory CA-releasing effect of Ach evoked by high-dose losartan (300 microM) was time-dependently depressed by pretreatment with nifedipine (Ca2+ channel blocker), while not by pretreatment with sodium nitroprusside (an NO donor). High-dose olmesartan (150 ∼ 600 microM), one of ARBs, also potentiated Ach-evoked CA release in time-dependent fashion. However, in the simultaneous presence of losartan (300 microM) and CGP42112 (an AT2 receptor agonist), the facilitatory responses of losartan on the CA secretion evoked by ACh were rather significantly reduced in comparison with that of losartan-treatment alone. Conclusions: Conclusively, these results demonstrate that high-dose losartan can potentiate the adrenomedullary CA secretion in a calcium-dependent fashion. It seems that this facilitatory effect of high-dose losartan may be mediated by activation of the Ca2+ influx through L-type Ca2+ channels into adrenomedullary cells without inhibition of NO synthesis, which seems to be relevant to AT2 receptor blockade. It also suggests that high-dose ARBs may be a risk factor in causing hypertensive response.