Sepsis involves an unregulated response to bacterial infection, the inflammatory response leading to persistent hypotension, leading to a precipitous fall in arterial blood pressure. The major trigger for the hypotension in sepsis and septic shock is the release of lipopolysaccharide (LPS) from the cell wall of gram-negative bacteria, which generates a major inflammatory response. LPS activates toll-like receptors (TLR4), leading to the release of an array of inflammatory mediators, which most notably activate inducible nitric oxide (NO) synthase and release copious amounts of NO. This is responsible for the hypotension. Despite the development of a large number of newer drugs for treating sepsis, none has emerged as superior to existing treatments. Early vasopressor therapy remains an integral life-saving strategy to treat the hypotension. Noradrenaline remains the vasopressor of choice; however, it has a number of limitations which are discussed. Improvements in vasopressor therapies are required and research from the authors is used to advance the case for using trace amines, such as β-phenylethylamine (PEA). Although usually regarded as sympathomimetic amines, PEA and related amines such as amphetamine and ephedrine exert vasoconstrictive effects via trace amine-associated receptors (TAARs), particularly when administered by infusion. The sympathomimetic and TAAR vasoconstrictor actions are demonstrated on anesthetized rat blood pressure. Unlike noradrenaline, PEA is not a universal vasoconstrictor; it also dilates other vessels, including mesenteric vascular beds. This would provide a superior profile of activity for use in restoring blood pressure in sepsis. The ability of PEA to reverse the vasodilator action of LPS is demonstrated in a simple in vitro blood vessel model. This review therefore opens the possibility of using trace amines for restoring blood pressure and organ perfusion in septic shock.

BACKGROUND:Substantial evidence indicates trace amines can induce vasoconstriction independently of noradrenaline release. However, the mechanism underlying noradrenaline-independent vasoconstrictor responses to trace amines has not yet been established. This study evaluates the role of trace amine-associated receptor 1 (TAAR1) and other biogenic amine receptors in mediating β-phenylethylamine and the TAAR-1 selective agonist RO5256390-induced vasoconstriction. METHODS:Vasoconstrictor responses to β-PEA and the TAAR1-selective agonist, RO5256390 were assessed in vitro in endothelium-denuded aortic rings and third-order mesenteric arteries of male Sprague Dawley rats. RESULTS:β-PEA and RO5256390 induced concentration-dependent vasoconstriction of aortic rings but not third-order mesenteric arteries. Vasoconstrictor responses in aortic rings were insensitive to antagonists of 5-HT. The murine-selective TAAR1 antagonist, EPPTB, had no effect on either β-PEA or RO5256390-induced vasoconstriction. The α1-adrenoceptor antagonist, prazosin, and the α2-adrenoceptor antagonist, yohimbine, induced a shift of the β-PEA concentration response curve too small to be ascribed to antagonism of α1-or α2-adrenoceptors, respectively. The α2-adrenoceptor antagonist atipamezole had no effect on β-PEA or RO5256390-induced vasoconstriction. CONCLUSION:Vasoconstrictor responses to trace amines are not mediated by classical biogenic amine neurotransmitter receptors. Insensitivity of β-PEA vasoconstrictor responses to EPPTB, may be explained by its low affinity for rat rather than murine TAAR1. Therefore, TAAR1 remains the most likely candidate receptor mediating vasoconstrictor responses to trace amines and that prazosin and yohimbine have low affinity for TAAR1.

Abstract Background and Aims For people requiring Kidney Replacement Therapy (KRT) late presentation to a nephrologist (within 90 days of initiating KRT) is associated with adverse outcomes. The ASSIST-CKD kidney function graph surveillance (KFGS) quality improvement project was adopted by the Department of Nephrology in South West (SW) Wales, UK in November 2017. Between the end of 2017 and the end of 2020, late presentation rates reduced from 19.5% [1] to 13.5% [2] across the region, having reduced to 9.7% in 2019 [3]. During KFGS, graphs of eGFR over time are generated for patients <65 years with eGFR ≤50ml/min/1.73 m2 and ≥65 years with eGFR ≤40ml/min/1.73 m2 with significantly deteriorating kidney function. ‘Alerts’ are sent to the GP for review and/or referral to nephrology. Over 100,000 graphs have been reviewed in SW Wales. We present demographic and initial outcome data for patients identified by KFGS. Method An observational retrospective cohort study analysed data of patients identified through KFGS in SW Wales between 1/11/17 and 1/10/21. Data were extracted from ASSIST-CKD and renal (Vital Data) databases using SQL coding. Results Data pertaining to 4811 patients are included for analysis. 44% of patients were male. At initial eGFR the mean age of patients ‘flagged’ was 73 years. The mean [range] initial eGFR was 32ml/min/1.73 m2 [2-50]. The median time [range] from initial eGFR to KFGS alert was 8 days [2-113]. For those with results within 90 days of initial eGFR the mean initial haemoglobin (Hb) was 118 g/L. The mean serum potassium was 4.7 mmol/L. Data for 1144 (24%) patients were included for outcome analysis, where a clear timeline from initial eGFR to KFGS alert, first registration with the nephrology service (registration), and outcome could be established. 3667 patients were excluded from outcome analysis because they were registered before KFGS or were not registered with nephrology, had died or moved out of area after KFGS alert. The median time [range] from alert to registration was 22 days [0-1636]. 20% of patients (n = 232) received CKD education from a specialist nurse. The median time [range] from registration to first contact from a CKD education nurse was 349 days [8-1419]. 34% of patients received IV iron and 31% received an erythropoiesis-stimulating agent (ESA) after alert. The median time [range] from registration to first IV iron and first ESA was 257 [5-1705] and 277 [3-1650] days respectively. 6% (n = 71) of patients required KRT. The late presentation rate was 22.5%. 21% of patients needing haemodialysis (HD) received their first treatment via an arteriovenous (AV) fistula, 1% via AV graft, 59% via non-tunnelled line and 19% via tunnelled line. 44% (7/16) of patients presenting late died within 90 days of starting KRT. 33% (18/55) of patients died within 90 days of starting KRT if they presented to a nephrologist ≥90 days before starting KRT. Conclusion Late presentation rates have reduced in SW Wales since implementing KFGS. The relatively high late presentation rate and low rate of initial definitive access for HD in the present data requires further analysis. eGFR values that prompted alert may denote Acute Kidney Injury (AKI) as opposed to, or as well as CKD progression; increasing the urgency of referral to nephrology. eGFR values pre and post alert will be reviewed to elucidate this. Patients presenting late were more likely to die within 90 days of starting KRT than those presenting early, emphasising the importance of early identification of progressive kidney disease. We aim to compare present data with a cohort referred to nephrology independent of KFGS to inform renal centres of the benefits of KFGS beyond headline late presentation rates, including timely access to specialists. We will consider potentially unwelcome consequences of KFGS, such as inappropriate referral. We cannot draw conclusions of causality between KFGS and outcomes. The GP may not have seen the alert, and there are several reasons why a GP may decide not to refer to nephrology following a KFGS alert.

A high percentage of asthma patients have symptoms that are not well controlled, despite effective drugs being available. One potential reason for this may be that poor inhaler technique limits the dose delivered to the lungs, thereby reducing the therapeutic efficacy. The aim of this study was to assess the prevalence of poor inhaler technique in an asthma patient population and to probe the impact of various demographic parameters on technique quality. This study was conducted at community pharmacies across Wales, UK. Patients diagnosed with asthma and 12 years or older were invited to participate. An aerosol inhalation monitor (AIM, Vitalograph®) was used to measure the quality of patient inhaler technique. A total of 295 AIM assessments were carried out. There were significant differences in the quality of inhaler technique across the different inhaler types (p < 0.001, Chi squared). The best technique was associated with dry-powder inhalers (DPI devices, 58% of 72 having good technique), compared with pressurized metered-dose inhalers (pMDI) or pMDIs with a spacer device (18% of 174 and 47% of 49 AIM assessments, respectively). There were some significant associations between gender, age, and quality of inhaler technique, as determined with adjusted odds ratios. It seems that the majority of asthmatic patients were not using their inhalers appropriately. We recommend that healthcare professionals place more emphasis on assessing and correcting inhaler technique, as poor inhaler technique might be responsible for the observed lack of symptom control in the asthma patient population.

Background and Purposes: Substantial evidence indicates trace amines can induce vasoconstriction independently of noradrenaline release. However, the mechanism underlying noradrenaline-independent vasoconstrictor responses to trace amines has not yet been established. This study evaluates the role of trace amine-associated receptor 1 (TAAR1) and other biogenic amine receptors in mediating trace amine-induced vasoconstriction. Experimental Approach: Vasoconstrictor responses to β-PEA and the TAAR1-selective agonist, RO5256390 were assessed in vitro in endothelium-denuded aortic rings and third-order mesenteric arteries of male Sprague Dawley rats. Key Results: β-PEA and RO5256390 induced concentration-dependent vasoconstriction of aortic rings but not third-order mesenteric arteries. Vasoconstrictor responses in aortic rings were insensitive to antagonists of 5-HT and dopamine. The murine-selective TAAR1 antagonist, EPPTB, had no effect on either β-PEA or RO5256390-induced vasoconstriction. The α1-adrenoceptor antagonist, prazosin, and the α2-adrenoceptor antagonist, yohimbine, induced a small but significant shift of the β-PEA concentration response curve that could not be ascribed to blockade of α1- or α2-adrenoceptors. Conclusion and Implications: Vasoconstrictor responses to trace amines are not mediated by classical biogenic amine neurotransmitter receptors. Although β-PEA vasoconstrictor responses were insensitive to ETTP, it has low affinity for rat TAAR1. Therefore, we propose that TAAR1 remains the most likely candidate receptor mediating vasoconstrictor responses to trace amines and that prazosin and yohimbine have some affinity for TAAR1.

Abstract Background and Purposes: Although trace amines, including β-phenylethylamine (β-PEA), cause vasoconstriction, they also induce endothelial nitric oxide release via an unknown mechanism. This study evaluates the cellular site of action and receptors mediating β-PEA-induced vasodilation. Experimental Approach: In vitro vasodilator responses to β-PEA were assessed using aortic rings and third-order mesenteric arteries of male Sprague-Dawley rats. Key Results: β-PEA-induced concentration-dependent vasodilation of pre-constricted aortic rings and third-order mesenteric arteries were partially sensitive and insensitive to endothelium removal, respectively. In aortic rings vasodilator responses to β-PEA were unaffected by EPPTB, a selective antagonist of murine trace amine associated receptor 1 (TAAR1), or antagonists of β2-adrenoceptors and muscarinic acetylcholine M3 receptors. The inhibitor of uptake-2 transport, decynium-22, abolished β-PEA-induced vasodilation revealing a vasoconstrictor response. Conclusion and Implications: Vasodilator responses to β-PEA do not involve cell surface receptors. We propose that β-PEA utilises uptake-2 transporters to gain access to an intracellular site. Although the identify of the intracellular site is unknown, the mechanism is similar to that previously reported where intracellular TAAR1 medated responses in HEK293 cells. Vasodilatation is the dominant response to trace amines of mesenteric vessels whereas in the aorta, it opposes vasoconstriction.

The role of the community pharmacist has evolved to include the provision of more clinical services for patients. Those people who have stable chronic conditions will be managed in community pharmacies. This qualitative study used semi-structured in-depth interviews to understand the potential of providing additional patient-centred care for patients with stable chronic conditions in community pharmacies and identify potential limitations of this approach. Participants were recruited from Welsh Government, Local Health Boards (LHBS), Community Pharmacy Wales (CPW) and the Royal Pharmaceutical Society Wales (RPSW). The interviews were audio-recorded, transcribed verbatim, and analysed thematically. Eight interviews were conducted. The identified themes were as follows: (1) inconsistency and bureaucracy in commissioning pharmacy services; (2) availability of funding and resources; (3) disagreement and uncertainty about the contribution of the community pharmacy sector; (4) continuity of patient medical information and fragmented care; (5) accessibility, capacity and facilities in community pharmacy; (6) pharmacy education and clinical expertise, and (7) patient acceptability. It was clear that the potential benefit of managing stable chronic diseases in community pharmacies was recognised; however, several limitations expressed by stakeholders of pharmacy services need to be considered prior to moving forward.

Asthma is still an incurable disease, and there is a recognized need for novel small-molecule therapies for people with asthma, especially those poorly controlled by current treatments. We previously demonstrated that calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs), calcilytics, uniquely suppress both airway hyperresponsiveness (AHR) and inflammation in human cells and murine asthma surrogates. Here we assess the feasibility of repurposing four CaSR NAMs, which were originally developed for oral therapy for osteoporosis and previously tested in the clinic as a novel, single, and comprehensive topical antiasthma therapy. We address the hypotheses, using murine asthma surrogates, that topically delivered CaSR NAMs 1) abolish AHR; 2) are unlikely to cause unwanted systemic effects; 3) are suitable for topical application; and 4) inhibit airway inflammation to the same degree as the current standard of care, inhaled corticosteroids, and, furthermore, inhibit airway remodeling. All four CaSR NAMs inhibited poly-L-arginine–induced AHR in naïve mice and suppressed both AHR and airway inflammation in a murine surrogate of acute asthma, confirming class specificity. Repeated exposure to inhaled CaSR NAMs did not alter blood pressure, heart rate, or serum calcium concentrations. Optimal candidates for repurposing were identified based on anti-AHR/inflammatory activities, pharmacokinetics/pharmacodynamics, formulation, and micronization studies. Whereas both inhaled CaSR NAMs and inhaled corticosteroids reduced airways inflammation, only the former prevented goblet cell hyperplasia in a chronic asthma model. We conclude that inhaled CaSR NAMs are likely a single, safe, and effective topical therapy for human asthma, abolishing AHR, suppressing airways inflammation, and abrogating some features of airway remodeling. SIGNIFICANCE STATEMENT Calcium-sensing receptor (CaSR) negative allosteric modulators (NAMs) reduce airway smooth muscle hyperresponsiveness, reverse airway inflammation as efficiently as topical corticosteroids, and suppress airway remodeling in asthma surrogates. CaSR NAMs, which were initially developed for oral therapy of osteoporosis proved inefficacious for this indication despite being safe and well tolerated. Here we show that structurally unrelated CaSR NAMs are suitable for inhaled delivery and represent a one-stop, steroid-free approach to asthma control and prophylaxis.

Research using animal models of asthma is currently dominated by mouse models. This has been driven by the comprehensive knowledge on inflammatory and immune reactions in mice, as well as tools to produce genetically modified mice. Many of the identified therapeutic targets influencing airway hyper-responsiveness and inflammation in mouse models, have however been disappointing when tested clinically in asthma. It is therefore a great need for new animal models that more closely resemble human asthma. The guinea pig has for decades been used in asthma research and a comprehensive table of different protocols for asthma models is presented. The studies have primarily been focused on the pharmacological aspects of the disease, where the guinea pig undoubtedly is superior to mice. Further reasons are the anatomical and physiological similarities between human and guinea pig airways compared with that of the mouse, especially with respect to airway branching, neurophysiology, pulmonary circulation and smooth muscle distribution, as well as mast cell localization and mediator secretion. Lack of reagents and specific molecular tools to study inflammatory and immunological reactions in the guinea pig has however greatly diminished its use in asthma research. The aim in this position paper is to review and summarize what we know about different aspects of the use of guinea pig in vivo models for asthma research. The associated aim is to highlight the unmet needs that have to be addressed in the future.

Background: Structural changes such as sub-epithelial fibrosis occur in inflammatory lung disease and as a natural process in the ageing lung. We have previously shown that activation of the CaSR drives inflammation in in vivo models of inflammatory lung disease, and that negative allosteric modulators of the CaSR, termed calcilytics, reduce these effects. Whether CaSR activation also contributes to extracellular matrix (ECM) remodelling and age-related fibrosis is unknown. Aim: To determine the role of the CaSR in ECM remodelling using in vivo models of allergic and non-allergic asthma and of age-related fibrosis. Methods: Ovalbumin-sensitised/challenged and IL33-sensitised mice as well as 15-month-old mice with targeted CaSR deletion from myofibroblasts and smooth muscle cells (achieved using an sm22a Cre promoter) were used as models of allergic asthma, non-allergic asthma and age-related fibrosis, respectively. Masson’s trichrome staining with semi-quantitative histomorphometry was employed to determine collagen expression beneath the airway epithelium of small and large airways. Results: Calcilytic treatment significantly reduced subepithelial collagen deposition in both large and small airways of ovalbumin-challenged mice and in large, but not small airways of IL33-sensitised mice (p<0.05). Furthermore, collagen expression was significantly reduced in aged mice with targeted CaSR deletion from sm22a-positive cells (p<0.05). Conclusions: CaSR activation drives pulmonary remodelling and fibrosis in animal models of asthma and in ageing mice. In addition to reducing inflammation, calcilytics might prove beneficial at reducing ECM remodelling during inflammatory lung disease.

Introduction: We previously implicated the calcium-sensing receptor (CaSR) in the development of airways inflammation and hyperresponsiveness in human asthma, and that topical CaSR antagonists (calcilytics) reduce both in animal surrogates. Aim: Extend these studies to alarmin-driven airways inflammation by comparing the efficacy of topical (intranasal) calcilytic in a head-to-head comparison in Th2/IgE mediated (OVA) and alarmin (interleukin (IL)-33) driven asthma in a murine surrogate. Methods: IL-33 (naïve mice) or OVA (mice IgE-sensitised to OVA) was delivered once daily intranasally for 6 consecutive days followed by the calcilytic NPSP795 or vehicle control twice daily. On day 7, airways resistance was measured (Flexivent) under anaesthesia then the animals euthanised, after which bronchoalveolar lavage fluid (BALF) was collected, one lung paraffin-embedded for histomorphology and the other homogenised to measure cytokines. Results: Topical calcilytic significantly reduced (1) airways hyperresponsiveness (both stimuli); (2) BALF total and differential eosinophil, neutrophil and lymphocyte counts and lung homogenate concentrations of IL-5, IL-13 and IL-6 (variable according to stimulus); (3) peribronchial inflammatory cellular infiltration in the OVA, but not the IL-33 driven model; (4) airways collagen deposition in the IL-33, but not the OVA driven model. Conclusions: Calcilytics show therapeutic benefit in both IgE/Th2 and alarmin-driven airways inflammation and hyperresponsiveness in animal asthma surrogates in vivo, suggesting that they will be effective against any stimulus potentially exacerbating human asthma by inducing an alarmin response in the airways.

Introduction Exposure to urban particulate matter (UPM) exacerbates the development of asthma and COPD. UPM exposure triggers the release of ‘alarmins’ by the airway epithelium, which causes an inflammatory response such as acceleration of the activation and maturation of dendritic cells (DC). Previously, we have demonstrated that, in surrogate models of allergic asthma, certain environmental stimuli activate the airway calcium-sensing receptor (CaSR), which drives bronchial hyperresponsiveness, inflammation and remodelling. We have also shown that CaSR antagonists, calcilytics, can abrogate these changes. Whether UPM exerts its effects acting at the CaSR is unknown. Aims To test the ability of calcilytics to: Prevent the effects of UPM in recombinant systems expressing the CaSR and in DC; Suppress airways hyperresponsiveness, inflammation and remodelling in a murine model of alarmin–driven (IL–33) asthma. Methods [Ca2+]i responses to UPM were investigated in HEK293 cells stably transfected with human CaSR (HEK-CaSR) or empty vector (HEK-0), ± calcilytic NPS2143. FACS and cytometric bead array were used to evaluate maturation (%CD83) and cytokine release by human monocyte-derived DC following 24h exposure to UPM ± calcilytics. IL-33 was delivered intranasally to naïve mice once daily for 6 consecutive days followed by the calcilytic NPSP795 or vehicle control twice daily from day 2. On day 7, airways resistance was measured (Flexivent) under terminal anaesthesia, after which bronchoalveolar lavage fluid (BALF) analysis was performed, and lungs collected for histomorphology and for measurements of cytokine release. Results UPM increased [Ca2+]i in the HEK-CaSR but not the HEK-0 cells, an effect inhibited by calcilytics. Calcilytics attenuated UPM-induced maturation, and release of the cytokines IL-10 and IL-23p40, but not IL-6 by DC. In vivo, inhaled calcilytics significantly reduced (1) bronchial hyperresponsiveness; (2) BALF inflammatory cell infiltration and lung concentrations of IL-5, IL-13 and IL-6; (3) airways collagen deposition. Conclusions UPM activates the CaSR and induces maturation and activation of DC, an effect inhibited by calcilytics. Furthermore, calcilytics show benefit in alarmin-driven airways inflammation and hyperresponsiveness in an animal asthma surrogate, suggesting that they will be effective against exacerbating stimuli such as UPM.

We have previously shown that inhaled calcium-sensing receptor antagonists, calcilytics, can abrogate airways hyperresponsiveness and inflammation in acute models of ovalbumin (OVA)-induced and triple allergen-sensitised allergic asthma. Here, we examined the efficacy of the calcilytic, NPSP-795, alongside the current standard-of-care, steroid fluticasone propionate (FP), in both short-term and long-term OVA-sensitised/challenged mice. For both experiments, animals were sensitised with two IP injections of OVA and then challenged with two (short-term) or six (long-term) OVA inhalations delivered over 24 hrs or 12 days, respectively. Drug treatments (inhaled calcilytic or the FP) were delivered twice daily over 24 hours (short-term) or six days (long-term). At the end of each experiment, bronchoalveolar lavage fluid (BALF) was collected and total and differential inflammatory cells numbers counted. Long-term inhalation of OVA was associated with a 10-fold greater increase in the mean absolute number of total inflammatory cells in the BALF compared with that measured in the short-term model. BALF cellular infiltration was significantly reduced by both calcilytic and FP in both models. Importantly, the effect of the inhaled calcilytic at the concentration employed was comparable to that of FP in terms of reducing both the total and absolute eosinophil counts. Calcilytics inhibit airways inflammation in both short- and long-term murine asthma models to a degree at least comparable with that of standard-of-care, inhaled corticosteroid. Given their completely distinct mechanisms of action, calcilytics may be a valid alternative therapy for corticosteroid resistant disease.