From structure to clinic: Design of a muscarinic M1 receptor agonist with potential to treatment of 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.

Oxygenation During Venoarterial Extracorporeal Membrane Oxygenation: Physiology, Current Evidence, and a Pragmatic Approach to Oxygen Titration.

OBJECTIVES: This review aims to: 1) identify the key circuit and patient factors affecting systemic oxygenation, 2) summarize the literature reporting the association between hyperoxia and patient outcomes, and 3) provide a pragmatic approach to oxygen titration, in patients undergoing peripheral venoarterial extracorporeal membrane oxygenation (ECMO). DATA SOURCES: Searches were performed using PubMed, SCOPUS, Medline, and Google Scholar. STUDY SELECTION: All observational and interventional studies investigating the association between hyperoxia, and clinical outcomes were included, as well as guidelines from the Extracorporeal Life Support Organization. DATA EXTRACTION: Data from relevant literature was extracted, summarized, and integrated into a concise narrative review. For ease of reference a summary of relevant studies was also produced. DATA SYNTHESIS: The extracorporeal circuit and the native cardiorespiratory circuit both contribute to systemic oxygenation during venoarterial ECMO. The ECMO circuit's contribution to systemic oxygenation is, in practice, largely determined by the ECMO blood flow, whereas the native component of systemic oxygenation derives from native cardiac output and residual respiratory function. Interactions between ECMO outflow and native cardiac output (as in differential hypoxia), the presence of respiratory support, and physiologic parameters affecting blood oxygen carriage also modulate overall oxygen exposure during venoarterial ECMO. Physiologically those requiring venoarterial ECMO are prone to hyperoxia. Hyperoxia has a variety of definitions, most commonly Pa o2 greater than 150 mm Hg. Severe hypoxia (Pa o2 > 300 mm Hg) is common, seen in 20%. Early severe hyperoxia, as well as cumulative hyperoxia exposure was associated with in-hospital mortality, even after adjustment for disease severity in both venoarterial ECMO and extracorporeal cardiopulmonary resuscitation. A pragmatic approach to oxygenation during peripheral venoarterial ECMO involves targeting a right radial oxygen saturation target of 94-98%, and in selected patients, titration of the fraction of oxygen in the mixture via the air-oxygen blender to target postoxygenator Pa o2 of 150-300 mm Hg. CONCLUSIONS: Hyperoxia results from a range of ECMO circuit and patient-related factors. It is common during peripheral venoarterial ECMO, and its presence is associated with poor outcome. A pragmatic approach that avoids hyperoxia, while also preventing hypoxia has been described for patients receiving peripheral venoarterial ECMO.

Composite outcome measures in high-impact critical care randomised controlled trials: a systematic review.

BACKGROUND: The use of composite outcome measures (COM) in clinical trials is increasing. Whilst their use is associated with benefits, several limitations have been highlighted and there is limited literature exploring their use within critical care. The primary aim of this study was to evaluate the use of COM in high-impact critical care trials, and compare study parameters (including sample size, statistical significance, and consistency of effect estimates) in trials using composite versus non-composite outcomes. METHODS: A systematic review of 16 high-impact journals was conducted. Randomised controlled trials published between 2012 and 2022 reporting a patient important outcome and involving critical care patients, were included. RESULTS: 8271 trials were screened, and 194 included. 39.1% of all trials used a COM and this increased over time. Of those using a COM, only 52.6% explicitly described the outcome as composite. The median number of components was 2 (IQR 2-3). Trials using a COM recruited fewer participants (409 (198.8-851.5) vs 584 (300-1566, p = 0.004), and their use was not associated with increased rates of statistical significance (19.7% vs 17.8%, p = 0.380). Predicted effect sizes were overestimated in all but 6 trials. For studies using a COM the effect estimates were consistent across all components in 43.4% of trials. 93% of COM included components that were not patient important. CONCLUSIONS: COM are increasingly used in critical care trials; however effect estimates are frequently inconsistent across COM components confounding outcome interpretations. The use of COM was associated with smaller sample sizes, and no increased likelihood of statistically significant results. Many of the limitations inherent to the use of COM are relevant to critical care research.

Beta-blockade for the treatment of refractory hypoxaemia during venovenous extracorporeal membrane oxygenation: An in-silico study.

INTRODUCTION: Venovenous extracorporeal membrane oxygenation (VV ECMO) is used for refractory hypoxemia, although despite this, in high cardiac output states, hypoxaemia may persist. The administration of beta-blockers has been suggested as an approach in this scenario, however the physiological consequences of this intervention are not clear. METHODS: We performed an in-silico study using a previously described mathematical model to evaluate the effect of beta-blockade on mixed venous and arterial saturations (Sv¯O2, SaO2), in three different clinical scenarios and considered the potential effects of beta-blockers on, cardiac output, oxygen consumption and recirculation. Additionally we assessed the interaction of beta-blockade with haemoglobin concentration. RESULTS: In scenario 1: simulating a patient with high cardiac output and partial lung shunt Sv¯O2 decreased from increased 53.5% to 44.7% despite SaO2 rising from 74.2% to 79.2%. In scenario 2 simulating a patient with high cardiac output and complete lung shunt Sv¯O2 remained unchanged at 52.2% and SaO2 rose from 71.9% to 85%. In scenario 3 a patient with normal cardiac output and high recirculation Sv¯O2 fell from 50.8% to 25.5% and also fell from 82.4% to to 78.3%. Across the remaining modelling examples the effect on Sv¯O2 varied but oxygen delivery was consistently reduced across all scenarios. CONCLUSION: The administration of beta-blockers for refractory hypoxemia during VV ECMO are unpredictable and may reduce oxygen delivery, although this will vary with patient and circuit features. This study does not support the use of beta-blockers for this indication.

Frequency of and associations with alterations of medical emergency team calling criteria in a teaching hospital emergency department.

BACKGROUND: Medical emergency team (METs), activated by vital sign-based calling criteria respond to deteriorating patients in the hospital setting. Calling criteria may be altered where clinicians feel this is appropriate. Altered calling criteria (ACC) has not previously been evaluated in the emergency department (ED) setting. OBJECTIVES: The objectives of this study were to (i) describe the frequency of ACC in a teaching hospital ED and the number and type of vital signs that were modified and (ii) associations between ACC in the ED and differences in the baseline patient characteristics and adverse outcomes including subsequent MET activations, unplanned intensive care unit (ICU) admissions and death within 72 h of admission. METHODS: Retrospective observational study of patients presenting to an academic, tertiary hospital ED in Melbourne, Australia between January 1st, 2019 and December 31st, 2019. The primary outcome was frequency and nature of ACC in the ED. Secondary outcomes included differences in baseline patient characteristics, frequency of MET activation, unplanned ICU admission, and mortality in the first 72 h of admission between those with and without ACC in the ED. RESULTS: Amongst 14 159 ED admissions, 725 (5.1%) had ACC, most frequently for increased heart or respiratory rate. ACC was associated with older age and increased comorbidity. Such patients had a higher adjusted risk of MET activation (odds ratio [OR]: 3.14, 95% confidence interval [CI]: 2.50-3.91, p = <0.001), unplanned ICU admission (OR: 1.97, 95% CI: 1.17-3.14, p = 0.016), and death (OR: 3.87, 95% CI: 2.08-6.70, p = 0.020) within 72 h. CONCLUSIONS: ACC occurs commonly in the ED, most frequently for elevated heart and respiratory rates and is associated with worse patient outcomes. In some cases, ACC requires consultant involvement, more frequent vital sign monitoring, expeditious inpatient team review, or ICU referral.

Corrigendum: Crystal structure of the GLP-1 receptor bound to a peptide agonist.

This corrects the article DOI: 10.1038/nature22800.

Crystal structure of the GLP-1 receptor bound to a peptide agonist.

Glucagon-like peptide 1 (GLP-1) regulates glucose homeostasis through the control of insulin release from the pancreas. GLP-1 peptide agonists are efficacious drugs for the treatment of diabetes. To gain insight into the molecular mechanism of action of GLP-1 peptides, here we report the crystal structure of the full-length GLP-1 receptor bound to a truncated peptide agonist. The peptide agonist retains an α-helical conformation as it sits deep within the receptor-binding pocket. The arrangement of the transmembrane helices reveals hallmarks of an active conformation similar to that observed in class A receptors. Guided by this structural information, we design peptide agonists with potent in vivo activity in a mouse model of diabetes.

Extra-helical binding site of a glucagon receptor antagonist.

Glucagon is a 29-amino-acid peptide released from the α-cells of the islet of Langerhans, which has a key role in glucose homeostasis. Glucagon action is transduced by the class B G-protein-coupled glucagon receptor (GCGR), which is located on liver, kidney, intestinal smooth muscle, brain, adipose tissue, heart and pancreas cells, and this receptor has been considered an important drug target in the treatment of diabetes. Administration of recently identified small-molecule GCGR antagonists in patients with type 2 diabetes results in a substantial reduction of fasting and postprandial glucose concentrations. Although an X-ray structure of the transmembrane domain of the GCGR has previously been solved, the ligand (NNC0640) was not resolved. Here we report the 2.5 Å structure of human GCGR in complex with the antagonist MK-0893 (ref. 4), which is found to bind to an allosteric site outside the seven transmembrane (7TM) helical bundle in a position between TM6 and TM7 extending into the lipid bilayer. Mutagenesis of key residues identified in the X-ray structure confirms their role in the binding of MK-0893 to the receptor. The unexpected position of the binding site for MK-0893, which is structurally similar to other GCGR antagonists, suggests that glucagon activation of the receptor is prevented by restriction of the outward helical movement of TM6 required for G-protein coupling. Structural knowledge of class B receptors is limited, with only one other ligand-binding site defined--for the corticotropin-releasing hormone receptor 1 (CRF1R)--which was located deep within the 7TM bundle. We describe a completely novel allosteric binding site for class B receptors, providing an opportunity for structure-based drug design for this receptor class and furthering our understanding of the mechanisms of activation of these receptors.

First-in-man study to investigate safety, pharmacokinetics and exploratory pharmacodynamics of HTL0018318, a novel M1 -receptor partial agonist for the treatment of dementias.

AIMS: HTL0018318 is a selective M1 receptor partial agonist currently under development for the symptomatic treatment of cognitive and behavioural symptoms in Alzheimer's disease and other dementias. We investigated safety, tolerability, pharmacokinetics and exploratory pharmacodynamics (PD) of HTL0018318 following single ascending doses. METHODS: This randomized, double-blind, placebo-controlled study in 40 healthy younger adult and 57 healthy elderly subjects, investigated oral doses of 1-35 mg HTL0018318. Pharmacodynamic assessments were performed using a battery of neurocognitive tasks and electrophysiological measurements. Cerebrospinal fluid concentrations of HTL0018318 and food effects on pharmacokinetics of HTL0018318 were investigated in an open label and partial cross-over design in 14 healthy subjects. RESULTS: Pharmacokinetics of HTL0018318 were well-characterized showing dose proportional increases in exposure from 1-35 mg. Single doses of HTL0018318 were associated with mild dose-related adverse events of low incidence in both younger adult and elderly subjects. The most frequently reported cholinergic AEs included hyperhidrosis and increases in blood pressure up to 10.3 mmHg in younger adults (95% CI [4.2-16.3], 35-mg dose) and up to 11.9 mmHg in elderly subjects (95% CI [4.9-18.9], 15-mg dose). There were no statistically significant effects on cognitive function but the study was not powered to detect small to moderate effect sizes of clinical relevance. CONCLUSION: HTL0018318 showed well-characterized pharmacokinetics and following single doses were generally well tolerated in the dose range studied. These provide encouraging data in support of the development for HTL0018318 for Alzheimer's disease and other dementias.

Safety, pharmacokinetics and pharmacodynamics of HTL0009936, a selective muscarinic M1 -acetylcholine receptor agonist: A randomized cross-over trial.

AIMS: HTL0009936 is a selective M1 muscarinic receptor agonist in development for cognitive dysfunction in Alzheimer's disease. Safety, tolerability and pharmacokinetics and exploratory pharmacodynamic effects of HTL0009936 administered by continuous IV infusion at steady state were investigated in elderly subjects with below average cognitive functioning (BACF). METHODS: Part A was a four-treatment open label sequential study in healthy elderly investigating 10-83 mg HTL0009936 (IV) and a 24 mg HTL0009936 single oral dose. Part B was a five-treatment randomized, double-blind, placebo and physostigmine controlled cross-over study with IV HTL0009936 in elderly subjects with BACF. Pharmacodynamic assessments were performed using neurocognitive and electrophysiological tests. RESULTS: Pharmacokinetics of HTL0009936 showed dose-proportional increases in exposure with a mean half-life of 2.4 hours. HTL0009936 was well-tolerated with transient dose-related adverse events (AEs). Small increases in mean systolic blood pressure of 7.12 mmHg (95% CI [3.99-10.24]) and in diastolic of 5.32 mmHg (95% CI [3.18-7.47]) were noted at the highest dose in part B. Overall, there was suggestive, but no definitive, positive or negative pharmacodynamic effects. Statistically significant effects were observed on P300 with HTL0009936 and adaptive tracking with physostigmine. CONCLUSIONS: HTL0009936 showed well-characterized pharmacokinetics and single doses were safe and generally well-tolerated in healthy elderly subjects. Due to physostigmine tolerability issues and subject burden, the study design was changed and some pharmacodynamic assessments (neurocognitive) were performed at suboptimal drug exposures. Therefore no clear conclusions can be made on pharmacodynamic effects of HTL0009936, although an effect on P300 is suggestive of central target engagement.

Exploring the appropriateness of antibiotic prescribing for common respiratory tract infections in UK primary care.

OBJECTIVES: To use illness severity scores to evaluate the appropriateness of antibiotic prescribing in UK general practice. METHODS: We describe variations in practice prescribing rates, taking account of illness severity. We used three scores in three studies to measure severity: 'FeverPAIN' in an adult acute sore throat cohort (n=12 829), the '3C score' in an adult acute lower respiratory tract infection cohort (n=28 883) and the STARWAVe score in an acute cough and respiratory infection children's cohort (n=8394). We calculated median ORs to quantify practice-level variation in prescribing rates, adjusted for illness severity. RESULTS: There was substantial variability in practice prescribing rates (ranges of 0%-97%, 7%-100% and 0%-75% in the three cohorts, respectively). There was evidence that higher prescribing practices saw a higher proportion of unwell patients. At the individual level, patients who were more unwell were more likely to receive a prescription, but prescribing levels for those with low scores were still high. The median OR was 2.5 (95% credible interval=2.2-2.9) in the sore throat data set, 2.9 (95% credible interval=2.6-3.2) in the adult cough data set and 2.1 (95% credible interval=1.8-2.4) in the children's cough data set. CONCLUSIONS: Higher prescribing practices may see more unwell patients with high illness severity scores, but the differences in scores account for a minority of between-practice prescribing variation. There is likely to be scope for further reductions in antibiotic prescribing among patients with low illness severity scores. Further research is needed to explore the additional factors that account for variation in prescribing levels.

Identification of a novel allosteric GLP-1R antagonist HTL26119 using structure- based drug design.

A series of novel allosteric antagonists of the GLP-1 receptor (GLP-1R), exemplified by HTL26119, are described. SBDD approaches were employed to identify HTL26119, exploiting structural understanding of the allosteric binding site of the closely related Glucagon receptor (GCGR) (Jazayeri et al., 2016) and the homology relationships between GCGR and GLP-1R. The region around residue C3476.36b of the GLP-1R receptor represents a key difference from GCGR and was targeted for selectivity for GLP-1R.

Glucagon-Like Peptide-1 and Its Class B G Protein-Coupled Receptors: A Long March to Therapeutic Successes.

The glucagon-like peptide (GLP)-1 receptor (GLP-1R) is a class B G protein-coupled receptor (GPCR) that mediates the action of GLP-1, a peptide hormone secreted from three major tissues in humans, enteroendocrine L cells in the distal intestine, α cells in the pancreas, and the central nervous system, which exerts important actions useful in the management of type 2 diabetes mellitus and obesity, including glucose homeostasis and regulation of gastric motility and food intake. Peptidic analogs of GLP-1 have been successfully developed with enhanced bioavailability and pharmacological activity. Physiologic and biochemical studies with truncated, chimeric, and mutated peptides and GLP-1R variants, together with ligand-bound crystal structures of the extracellular domain and the first three-dimensional structures of the 7-helical transmembrane domain of class B GPCRs, have provided the basis for a two-domain-binding mechanism of GLP-1 with its cognate receptor. Although efforts in discovering therapeutically viable nonpeptidic GLP-1R agonists have been hampered, small-molecule modulators offer complementary chemical tools to peptide analogs to investigate ligand-directed biased cellular signaling of GLP-1R. The integrated pharmacological and structural information of different GLP-1 analogs and homologous receptors give new insights into the molecular determinants of GLP-1R ligand selectivity and functional activity, thereby providing novel opportunities in the design and development of more efficacious agents to treat metabolic disorders.

Metabolic costs imposed by hydrostatic pressure constrain bathymetric range in the lithodid crab Lithodes maja.

The changing climate is shifting the distributions of marine species, yet the potential for shifts in depth distributions is virtually unexplored. Hydrostatic pressure is proposed to contribute to a physiological bottleneck constraining depth range extension in shallow-water taxa. However, bathymetric limitation by hydrostatic pressure remains undemonstrated, and the mechanism limiting hyperbaric tolerance remains hypothetical. Here, we assess the effects of hydrostatic pressure in the lithodid crab Lithodes maja (bathymetric range 4-790 m depth, approximately equivalent to 0.1 to 7.9 MPa hydrostatic pressure). Heart rate decreased with increasing hydrostatic pressure, and was significantly lower at ≥10.0 MPa than at 0.1 MPa. Oxygen consumption increased with increasing hydrostatic pressure to 12.5 MPa, before decreasing as hydrostatic pressure increased to 20.0 MPa; oxygen consumption was significantly higher at 7.5-17.5 MPa than at 0.1 MPa. Increases in expression of genes associated with neurotransmission, metabolism and stress were observed between 7.5 and 12.5 MPa. We suggest that hyperbaric tolerance in Lmaja may be oxygen-limited by hyperbaric effects on heart rate and metabolic rate, but that Lmaja's bathymetric range is limited by metabolic costs imposed by the effects of high hydrostatic pressure. These results advocate including hydrostatic pressure in a complex model of environmental tolerance, where energy limitation constrains biogeographic range, and facilitate the incorporation of hydrostatic pressure into the broader metabolic framework for ecology and evolution. Such an approach is crucial for accurately projecting biogeographic responses to changing climate, and for understanding the ecology and evolution of life at depth.

The Effects of Temperature and Hydrostatic Pressure on Metal Toxicity: Insights into Toxicity in the Deep Sea.

Mineral prospecting in the deep sea is increasing, promoting concern regarding potential ecotoxicological impacts on deep-sea fauna. Technological difficulties in assessing toxicity in deep-sea species has promoted interest in developing shallow-water ecotoxicological proxy species. However, it is unclear how the low temperature and high hydrostatic pressure prevalent in the deep sea affect toxicity, and whether adaptation to deep-sea environmental conditions moderates any effects of these factors. To address these uncertainties we assessed the effects of temperature and hydrostatic pressure on lethal and sublethal (respiration rate, antioxidant enzyme activity) toxicity in acute (96 h) copper and cadmium exposures, using the shallow-water ecophysiological model organism Palaemon varians. Low temperature reduced toxicity in both metals, but reduced cadmium toxicity significantly more. In contrast, elevated hydrostatic pressure increased copper toxicity, but did not affect cadmium toxicity. The synergistic interaction between copper and cadmium was not affected by low temperature, but high hydrostatic pressure significantly enhanced the synergism. Differential environmental effects on toxicity suggest different mechanisms of action for copper and cadmium, and highlight that mechanistic understanding of toxicity is fundamental to predicting environmental effects on toxicity. Although results infer that sensitivity to toxicants differs across biogeographic ranges, shallow-water species may be suitable ecotoxicological proxies for deep-sea species, dependent on adaptation to habitats with similar environmental variability.

Recommendations of the 2006 Human Variome Project meeting.

Lists of variations in genomic DNA and their effects have been kept for some time and have been used in diagnostics and research. Although these lists have been carefully gathered and curated, there has been little standardization and coordination, complicating their use. Given the myriad possible variations in the estimated 24,000 genes in the human genome, it would be useful to have standard criteria for databases of variation. Incomplete collection and ascertainment of variants demonstrates a need for a universally accessible system. These and other problems led to the World Heath Organization-cosponsored meeting on June 20-23, 2006 in Melbourne, Australia, which launched the Human Variome Project. This meeting addressed all areas of human genetics relevant to collection of information on variation and its effects. Members of each of eight sessions (the clinic and phenotype, the diagnostic laboratory, the research laboratory, curation and collection, informatics, relevance to the emerging world, integration and federation and funding and sustainability) developed a number of recommendations that were then organized into a total of 96 recommendations to act as a foundation for future work worldwide. Here we summarize the background of the project, the meeting and its recommendations.