Introduction to the Theme "Pharmacological Individuality: New Insights and Strategies for Personalized and Precise Drug Treatment".

The reviews in Volume 64 of the Annual Review of Pharmacology and Toxicology cover diverse topics. A common theme in many of the reviews is the interindividual variability in the clinical response to drugs. Highlighted areas include emerging developments in pharmacogenomics that can predict the personal risk for drug inefficacy and/or adverse drug reactions. Other reviews focus on the use of circulating biomarkers to define drug metabolism phenotypes and the effect of circadian regulation on drug response. Another emerging technology, digital twins that model individual patients, is used to generate computational simulations of drug effects and identify optimal personalized treatments. Another variable that may affect clinical outcomes, the nocebo response (an adverse reaction to a placebo), complicates clinical trials. These reviews further document that pharmacological individuality is an essential component of the concepts of personalized medicine and precision medicine and will likely have an important impact on patient care.

Ramipril for the Treatment of COVID-19: RAMIC, a Randomized, Double-Blind, Placebo-Controlled Clinical Trial.

INTRODUCTION: Retrospective studies report that angiotensin-converting enzyme inhibitors (ACEIs) may reduce the severity of COVID-19, but prospective data on de novo treatment with ACEIs are limited. The RAMIC trial was a randomized, multicenter, placebo-controlled, double-blind, allocation-concealed clinical trial to examine the efficacy of de novo ramipril versus placebo for the treatment of COVID-19. METHODS: Eligible participants were aged 18 years and older with a confirmed diagnosis of SARS-CoV-2 infection, recruited from urgent care clinics, emergency departments, and hospital inpatient wards at eight sites in the USA. Participants were randomly assigned to daily ramipril 2.5 mg or placebo orally in a 2:1 ratio, using permuted block randomization. Analyses were conducted on an intention-to-treat basis. The primary outcome was a composite of mortality, intensive care unit (ICU) admission, or invasive mechanical ventilation by day 14. RESULTS: Between 27 May 2020 and 19 April 2021, a total of 114 participants (51% female) were randomized to ramipril (n = 79) or placebo (n = 35). The overall mean (± SD) age and BMI were 45 (± 15) years and 33 (± 8) kg/m2. Two participants in the ramipril group required ICU admission and one died, compared with none in the placebo group. There were no significant differences between ramipril and placebo in the primary endpoint (ICU admission, mechanical ventilation, or death) (3% versus 0%, p = 1.00) or adverse events (27% versus 29%, p = 0.82). The study was terminated early because of a low event rate and subsequent Emergency Use Authorization of therapies for COVID-19. CONCLUSION: De novo ramipril was not different compared with placebo in improving or worsening clinical outcomes from COVID-19 but appeared safe in non-critically ill patients with COVID-19. TRIAL REGISTRATION: Clinicaltrials.gov NCT04366050.

Mast cell tolerance in the skin microenvironment to commensal bacteria is controlled by fibroblasts.

Activation and degranulation of mast cells (MCs) is an essential aspect of innate and adaptive immunity. Skin MCs, the most exposed to the external environment, are at risk of quickly degranulating with potentially severe consequences. Here, we define how MCs assume a tolerant phenotype via crosstalk with dermal fibroblasts (dFBs) and how this phenotype reduces unnecessary inflammation when in contact with beneficial commensal bacteria. We explore the interaction of human MCs (HMCs) and dFBs in the human skin microenvironment and test how this interaction controls MC inflammatory response by inhibiting the nuclear factor κB (NF-κB) pathway. We show that the extracellular matrix hyaluronic acid, as the activator of the regulatory zinc finger (de)ubiquitinating enzyme A20/tumor necrosis factor α-induced protein 3 (TNFAIP3), is responsible for the reduced HMC response to commensal bacteria. The role of hyaluronic acid as an anti-inflammatory ligand on MCs opens new avenues for the potential treatment of inflammatory and allergic disorders.

GPCR Signaling Measurement and Drug Profiling with an Automated Live-Cell Microscopy System.

A major limitation of time-lapse microscopy combined with fluorescent biosensors, a powerful tool for quantifying spatiotemporal dynamics of signaling in single living cells, is low-experimental throughput. To overcome this limitation, we created a highly customizable, MATLAB-based platform: flexible automated liquid-handling combined microscope (FALCOscope) that coordinates an OpenTrons liquid handler and a fluorescence microscope to automate drug treatments, fluorescence imaging, and single-cell analysis. To test the feasibility of the FALCOscope, we quantified G protein-coupled receptor (GPCR)-stimulated Protein Kinase A activity and cAMP responses to GPCR agonists and antagonists. We also characterized cAMP dynamics induced by GPR68/OGR1, a proton-sensing GPCR, in response to variable extracellular pH values. GPR68-induced cAMP responses were more transient in acidic than neutral pH values, suggesting a pH-dependence for signal attenuation. Ogerin, a GPR68 positive allosteric modulator, enhanced cAMP response most strongly at pH 7.0 and sustained cAMP response for acidic pH values, thereby demonstrating the capability of the FALCOscope to capture allosteric modulation. At a high concentration, ogerin increased cAMP signaling independent of GPR68, likely via phosphodiesterase inhibition. The FALCOscope system thus enables enhanced throughput single-cell dynamic measurements and is a versatile system for interrogating spatiotemporal regulation of signaling molecules in living cells and for drug profiling and screening.

Guidance for the use and reporting of anaesthetic agents in BJP manuscripts involving work with animals.

Scientists who plan to publish in the British Journal of Pharmacology (BJP) should read this article before undertaking studies utilising anaesthetics in mammalian animals. This editorial identifies certain gaps in the reporting of details on the use of anaesthetics in animal research studies published in the BJP. The editorial also provides guidance, based upon current best practices, for performing in vivo experiments that require anaesthesia. In addition, mechanisms of action and physiological impact of specific anaesthetic agents are discussed. Our goal is to identify best practices and to provide guidance on the information required for manuscripts submitted to the BJP that involve the use of anaesthetic agents in studies with experimental animals.

Introduction to the Theme "Development of New Drugs: Moving from the Bench to Bedside and Improved Patient Care".

Investigations in pharmacology and toxicology range from molecular studies to clinical care. Studies in basic and clinical pharmacology and in preclinical and clinical toxicology are all essential in bringing new knowledge and new drugs into clinical use. The 30 reviews in Volume 63 of the Annual Review of Pharmacology and Toxicology explore topics across this spectrum. Examples include "Zebrafish as a Mainstream Model for In Vivo Systems Pharmacology and Toxicology" and "Artificial Intelligence and Machine Learning for Lead-to-Candidate Decision-Making and Beyond." Other reviews discuss components important for drug discovery and development and the use of pharmaceuticals in a variety of diseases. Air pollution continues to increase globally; accordingly, "Air Pollution-Related Neurotoxicity Across the Life Span" is a timely and forward-thinking review. Volume 63 also explores the use of contemporary technologies such as electronic health records, pharmacogenetics, and new drug delivery systems that help enhance and improve the utility of new therapies.

Planning experiments: Updated guidance on experimental design and analysis and their reporting III.

Scientists who plan to publish in British Journal of Pharmacology (BJP) must read this article before undertaking a study. This editorial provides guidance for the design of experiments. We have published previously two guidance documents on experimental design and analysis (Curtis et al., 2015; Curtis et al., 2018). This update clarifies and simplifies the requirements on design and analysis for BJP manuscripts. This editorial also details updated requirements following an audit and discussion on best practice by the BJP editorial board. Explanations for the requirements are provided in the previous articles. Here, we address new issues that have arisen in the course of handling manuscripts and emphasise three aspects of design that continue to present the greatest challenge to authors: randomisation, blinded analysis and balance of group sizes.

PDE4B Is a Homeostatic Regulator of Cyclic AMP in Dendritic Cells.

Chronic decreases in the second messenger cyclic AMP (cAMP) occur in numerous settings, but how cells compensate for such decreases is unknown. We have used a unique system-murine dendritic cells (DCs) with a DC-selective depletion of the heterotrimeric GTP binding protein Gαs-to address this issue. These mice spontaneously develop Th2-allergic asthma and their DCs have persistently lower cAMP levels. We found that phosphodiesterase 4B (PDE4B) is the primary phosphodiesterase expressed in DCs and that its expression is preferentially decreased in Gαs-depleted DCs. PDE4B expression is dynamic, falling and rising in a protein kinase A-dependent manner with decreased and increased cAMP concentrations, respectively. Treatment of DCs that drive enhanced Th2 immunity with a PDE4B inhibitor ameliorated DC-induced helper T cell response. We conclude that PDE4B is a homeostatic regulator of cellular cAMP concentrations in DCs and may be a target for treating Th2-allergic asthma and other settings with low cellular cAMP concentrations.

Introduction to the Theme "New Insights, Strategies, and Therapeutics for Common Diseases".

The reviews in Volume 62 of the Annual Review of Pharmacology and Toxicology (ARPT) cover a diverse range of topics. A theme that encompasses many of these reviews is their relevance to common diseases and disorders, including type 2 diabetes, heart failure, cancer, tuberculosis, Alzheimer's disease, neurodegenerative disorders, and Down syndrome. Other reviews highlight important aspects of therapeutics, including placebos and patient-centric approaches to drug formulation. The reviews with this thematic focus, as well as other reviews in this volume, emphasize new mechanistic insights, experimental and therapeutic strategies, and novel insights regarding topics in the disciplines of pharmacology and toxicology. As the editors of ARPT, we believe that these reviews help advance those disciplines and, even more importantly, have the potential to improve the health care of the world's population.

Inhaled ß2 Adrenergic Agonists and Other cAMP-Elevating Agents: Therapeutics for Alveolar Injury and Acute Respiratory Disease Syndrome?

Inhaled long-acting ß-adrenergic agonists (LABAs) and short-acting ß-adrenergic agonists are approved for the treatment of obstructive lung disease via actions mediated by ß2 adrenergic receptors (ß2-ARs) that increase cellular cAMP synthesis. This review discusses the potential of ß2-AR agonists, in particular LABAs, for the treatment of acute respiratory distress syndrome (ARDS). We emphasize ARDS induced by pneumonia and focus on the pathobiology of ARDS and actions of LABAs and cAMP on pulmonary and immune cell types. ß2-AR agonists/cAMP have beneficial actions that include protection of epithelial and endothelial cells from injury, restoration of alveolar fluid clearance, and reduction of fibrotic remodeling. ß2-AR agonists/cAMP also exert anti-inflammatory effects on the immune system by actions on several types of immune cells. Early administration is likely critical for optimizing efficacy of LABAs or other cAMP-elevating agents, such as agonists of other Gs-coupled G protein-coupled receptors or cyclic nucleotide phosphodiesterase inhibitors. Clinical studies that target lung injury early, prior to development of ARDS, are thus needed to further assess the use of inhaled LABAs, perhaps combined with inhaled corticosteroids and/or long-acting muscarinic cholinergic antagonists. Such agents may provide a multipronged, repurposing, and efficacious therapeutic approach while minimizing systemic toxicity. SIGNIFICANCE STATEMENT: Acute respiratory distress syndrome (ARDS) after pulmonary alveolar injury (e.g., certain viral infections) is associated with ∼40% mortality and in need of new therapeutic approaches. This review summarizes the pathobiology of ARDS, focusing on contributions of pulmonary and immune cell types and potentially beneficial actions of ß2 adrenergic receptors and cAMP. Early administration of inhaled ß2 adrenergic agonists and perhaps other cAMP-elevating agents after alveolar injury may be a prophylactic approach to prevent development of ARDS.

Detection of GPCR mRNA Expression in Primary Cells Via qPCR, Microarrays, and RNA-Sequencing.

A workflow is described for assaying the expression of G protein-coupled receptors (GPCRs) in cultured cells, using a combination of methods that assess GPCR mRNAs. Beginning from the isolation of cDNA and preparation of mRNA, we provide protocols for designing and testing qPCR primers, assaying mRNA expression using qPCR and high-throughput analysis of GPCR mRNA expression via TaqMan qPCR-based, GPCR-selective arrays. We also provide a workflow for analysis of expression from RNA-sequencing (RNA-seq) assays, which can be queried to yield expression of GPCRs and related genes in samples of interest, as well as to test changes in expression between groups, such as in cells treated with drugs or from healthy and diseased subjects. We place priority on optimized protocols that distinguish signal from noise, as GPCR mRNAs are typically present in low abundance, necessitating techniques that maximize sensitivity while minimizing noise. These methods may also be applicable for assessing the expression of members of families of other low abundance genes via high-throughput analyses of mRNAs, followed by independent confirmation and validation of results via qPCR.

RAMIC: Design of a randomized, double-blind, placebo-controlled trial to evaluate the efficacy of ramipril in patients with COVID-19.

BACKGROUND AND AIMS: Retrospective studies have shown that angiotensin-converting-enzyme (ACE) inhibitors are associated with a reduced risk of complications and mortality in persons with novel coronavirus disease 2019 (COVID-19). Thus, we aimed to examine the efficacy of ramipril, an ACE-inhibitor, in preventing ICU admission, mechanical ventilation and/or mortality while also minimizing the risk of transmission and use of personal protective equipment (PPE). METHODS: RAMIC is a multicenter, randomized, double-blind, allocation-concealed, placebo-controlled trial comparing the efficacy of treatment with ramipril 2.5 mg orally daily compared to placebo for 14 days. The study population includes adult patients with COVID-19 who were admitted to a hospital or assessed in an emergency department or ambulatory clinic. Key exclusion criteria include ICU admission or need for mechanical ventilation at screening, use of an ACE inhibitor or angiotensin-receptor-II blocker within 7 days, glomerular filtration rate < 40 mL/min or a systolic blood pressure (BP) < 100 mmHg or diastolic BP < 65 mmHg. Patients are randomized 2:1 to receive ramipril (2.5 mg) or placebo daily. Informed consent and study visits occur virtually to minimize the risk of SARS-CoV-2 transmission and preserve PPE. The primary composite endpoint of ICU admission, invasive mechanical ventilation and death are adjudicated virtually. CONCLUSIONS: RAMIC is designed to assess the efficacy of treatment with ramipril for 14 days to decrease ICU admission, mechanical ventilator use and mortality in patients with COVID-19 and leverages virtual study visits and endpoint adjudication to mitigate risk of infection and to preserve PPE (ClinicalTrials.gov, NCT04366050).

Introduction to the Theme "Old and New Toxicology: Interfaces with Pharmacology".

The theme of Volume 61 is "Old and New Toxicology: Interfaces with Pharmacology." Old toxicology is exemplified by the authors of the autobiographical articles: B.M. Olivera's work on toxins and venoms from cone snails and P. Taylor's studies of acetylcholinesterase and the nicotinic cholinergic receptor, which serve as sites of action for numerous pesticides and venoms. Other articles in this volume focus on new understanding and new types of toxicology, including (a) arsenic toxicity, which is an ancient poison that, through evolution, has caused most multicellular organisms to express an active arsenic methyltransferase to methylate arsenite, which accelerates the excretion of arsenic from the body; (b) small molecules that react with lipid dicarbonyls, which are now considered the most toxic oxidative stress end products; (c) immune checkpoint inhibitors (ICIs), which have revolutionized cancer therapy but have numerous immune-related adverse events, including cardiovascular complications; (d) autoimmunity caused by the environment; (e) idiosyncratic drug-induced liver disease, which together with the toxicity of ICIs represents new toxicology interfacing with pharmacology; and (f) sex differences in the development of cardiovascular disease, with men more susceptible than women to vascular inflammation that initiates and perpetuates disease. These articles and others in Volume 61 reflect the interface and close integration of pharmacology and toxicology that began long ago but continues today.

Inflammation and thrombosis in COVID-19 pathophysiology: proteinase-activated and purinergic receptors as drivers and candidate therapeutic targets.

Evolving information has identified disease mechanisms and dysregulation of host biology that might be targeted therapeutically in coronavirus disease 2019 (COVID-19). Thrombosis and coagulopathy, associated with pulmonary injury and inflammation, are emerging clinical features of COVID-19. We present a framework for mechanisms of thrombosis in COVID-19 that initially derive from interaction of SARS-CoV-2 with ACE2, resulting in dysregulation of angiotensin signaling and subsequent inflammation and tissue injury. These responses result in increased signaling by thrombin (proteinase-activated) and purinergic receptors, which promote platelet activation and exert pathological effects on other cell types (e.g., endothelial cells, epithelial cells, and fibroblasts), further enhancing inflammation and injury. Inhibitors of thrombin and purinergic receptors may, thus, have therapeutic effects by blunting platelet-mediated thromboinflammation and dysfunction in other cell types. Such inhibitors include agents (e.g., anti-platelet drugs) approved for other indications, and that could be repurposed to treat, and potentially improve the outcome of, COVID-19 patients. COVID-19, caused by the SARS-CoV-2 virus, drives dysregulation of angiotensin signaling, which, in turn, increases thrombin-mediated and purinergic-mediated activation of platelets and increase in inflammation. This thromboinflammation impacts the lungs and can also have systemic effects. Inhibitors of receptors that drive platelet activation or inhibitors of the coagulation cascade provide opportunities to treat COVID-19 thromboinflammation.

Targeting the renin-angiotensin signaling pathway in COVID-19: Unanswered questions, opportunities, and challenges.

The role of the renin-angiotensin signaling (RAS) pathway in COVID-19 has received much attention. A central mechanism for COVID-19 pathophysiology has been proposed: imbalance of angiotensin converting enzymes (ACE)1 and ACE2 (ACE2 being the severe acute respiratory syndrome coronavirus 2 [SARS-CoV-2] virus "receptor") that results in tissue injury from angiotensin II (Ang II)-mediated signaling. This mechanism provides a rationale for multiple therapeutic approaches. In parallel, clinical data from retrospective analysis of COVID-19 cohorts has revealed that ACE inhibitors (ACEIs) or angiotensin receptor blockers (ARBs) may be beneficial in COVID-19. These findings have led to the initiation of clinical trials using approved drugs that target the generation (ACEIs) and actions (ARBs) of Ang II. However, treatment of COVID-19 with ACEIs/ARBs poses several challenges. These include choosing appropriate inclusion and exclusion criteria, dose optimization, risk of adverse effects and drug interactions, and verification of target engagement. Other approaches related to the RAS pathway might be considered, for example, inhalational administration of ACEIs/ARBs (to deliver drugs directly to the lungs) and use of compounds with other actions (e.g., activation of ACE2, agonism of MAS1 receptors, ß-arrestin-based Angiotensin receptor agonists, and administration of soluble ACE2 or ACE2 peptides). Studies with animal models could test such approaches and assess therapeutic benefit. This Perspective highlights questions whose answers could advance RAS-targeting agents as mechanism-driven ways to blunt tissue injury, morbidity, and mortality of COVID-19.