The state of the art in secondary pharmacology and its impact on the safety of new medicines.

Secondary pharmacology screening of investigational small-molecule drugs for potentially adverse off-target activities has become standard practice in pharmaceutical research and development, and regulatory agencies are increasingly requesting data on activity against targets with recognized adverse effect relationships. However, the screening strategies and target panels used by pharmaceutical companies may vary substantially. To help identify commonalities and differences, as well as to highlight opportunities for further optimization of secondary pharmacology assessment, we conducted a broad-ranging survey across 18 companies under the auspices of the DruSafe leadership group of the International Consortium for Innovation and Quality in Pharmaceutical Development. Based on our analysis of this survey and discussions and additional research within the group, we present here an overview of the current state of the art in secondary pharmacology screening. We discuss best practices, including additional safety-associated targets not covered by most current screening panels, and present approaches for interpreting and reporting off-target activities. We also provide an assessment of the safety impact of secondary pharmacology screening, and a perspective on opportunities and challenges in this rapidly developing field.

Counselling for Chronic Insomnia in Swiss Pharmacies: A Survey Study Based on Case Vignettes.

(1) Introduction: Chronic insomnia (CI) reduces quality of life and may trigger depression and cardiovascular diseases. The European Sleep Research Society recommends cognitive behavioural therapy (CBT-I) as the first-line treatment. Because a recent study in Switzerland demonstrated that this recommendation was inconsistently followed by primary care physicians, we hypothesised that pharmacists also deviate from these guidelines. The aim of this study is to describe current treatment practices for CI recommended by pharmacists in Switzerland, compare them to guidelines and examine their attitudes towards CBT-I. (2) Methods: A structured survey was sent to all the members of the Swiss Pharmacists Association, containing three clinical vignettes describing typical CI pharmacy clients. Treatments had to be prioritised. The prevalence of CI, and the pharmacists' knowledge and interest in CBT-I were assessed. (3) Results: Of 1523 pharmacies, 123 pharmacists (8%) completed the survey. Despite large variations, valerian (96%), relaxation therapy (94%) and other phytotherapies (85%) were most recommended. Although most pharmacists did not know about CBT-I (72%) and only 10% had recommended it, most were very interested (64%) in education. Missing financial compensation hampers the recommendation of CBT-I. (4) Conclusions: Contrary to existing European guidelines, community pharmacists in Switzerland mostly recommended valerian, relaxation therapy and other phytotherapies for treating CI. This might be connected to the client's expectation of pharmacy services, e.g., medication dispensing. While pharmacists recommend sleep hygiene regularly, most did not know of CBT-I as an overarching concept but were willing to learn. Future studies should test the effects of dedicated training about CI and changes in the financial compensation for counselling for CI in pharmacies.

Antidiabetic Medication Utilisation before and during Pregnancy in Switzerland between 2012 and 2019: An Administrative Claim Database from the MAMA Cohort.

Background: The incidence of diabetes mellitus (both pregestational and gestational) is increasing worldwide, and hyperglycemia during pregnancy is associated with adverse pregnancy outcomes. Evidence on the safety and efficacy of metformin during pregnancy has accumulated resulting in an increase in its prescription in many reports. Aims: We aimed to determine the prevalence of antidiabetic drug use (insulins and blood glucose-lowering drugs) before and during pregnancy in Switzerland and the changes therein during pregnancy and over time. Methods: We conducted a descriptive study using Swiss health insurance claims (2012-2019). We established the MAMA cohort by identifying deliveries and estimating the last menstrual period. We identified claims for any antidiabetic medication (ADM), insulins, blood glucose-lowering drugs, and individual substances within each class. We defined three groups of pattern use based on timing of dispensation: (1) dispensation of at least one ADM in the prepregnancy period and in or after trimester 2 (T2) (pregestational diabetes); (2) dispensation for the first time in or after T2 (GDM); and (3) dispensation in the prepregnancy period and no dispensation in or after T2 (discontinuers). Within the pregestational diabetes group, we further defined continuers (dispensation for the same group of ADM) and switchers (different ADM group dispensed in the prepregnancy period and in or after T2). Results: MAMA included 104,098 deliveries with a mean maternal age at delivery of 31.7. Antidiabetic dispensations among pregnancies with pregestational and gestational diabetes increased over time. Insulin was the most dispensed medication for both diseases. Between 2017 and 2019, less than 10% of pregnancies treated for pregestational diabetes continued metformin rather than switching to insulin. Metformin was offered to less than 2% of pregnancies to treat gestational diabetes (2017-2019). Conclusion: Despite its position in the guidelines and the attractive alternative that metformin represents to patients who may encounter barriers with insulin therapy, there was reluctance to prescribe it.

Vision and practice of self-care for community pharmacy in Switzerland.

Switzerland is a federal country with a liberal health system built on private mandatory health insurance where the government has three different roles (health protector, guarantor of the offered care and regulator). Health is mostly considered as a responsibility that lies with the individual person. Swiss health policies do not include the term "self-care", although, the federal policy strategy established for this decade (Health2030) includes objectives and lines of action, some of which could be classified as self-care. Swiss policies do not specify the role of health professionals; therefore, it is up to each canton (the terminology used to describe a state of the Swiss Confederation), organization or enterprise to stipulate it. Regarding pharmacists, 1844 community pharmacies (CPs) take care of nearly 260,000 patients each day. The CPs play an important role in self-care that includes activities such as improving patients' health literacy, screening for different health problems, self-medication education or recommendation related to non-prescription medication. The government understands and emphasizes the importance of CPs' role in primary health care to overcome some of the health care system challenges, part of these actions related to self-care. However, there is scope for expansion regarding the role of the CPs in self-care. Nowadays the services and activities related are driven by health authorities (i.e., pharmacists' autonomous prescribing, vaccination, strategy for the prevention of non-communicable diseases or digitization of electronic patients' record), professional pharmacy associations (i.e., netCare® or screening tests), health foundations (i.e., prevention of addiction) and/or private stakeholders such as chain pharmacies (i.e., screening tests). The possibility of including some of these services related to self-care (even when no medication is supplied) as covered services for the mandatory health insurance is currently politically discussed. Long-term actions that also include remuneration, monitoring and quality assurance, or communication/information to public should be considered to support a broader implementation and the sustainability of CPs' services related to self-care.

Use of high throughput ion channel profiling and statistical modeling to predict off-target arrhythmia risk - One pharma's experience and perspective.

INTRODUCTION: The use of high throughput patch clamp profiling to determine mixed ion channel-mediated arrhythmia risk was assessed using profiling data generated using proprietary internal and clinical reference compounds. We define the reproducibility of the platform and highlight inherent platform issues. The data generated was used to develop predictive models for cardiac arrhythmia risk, specifically Torsades de Pointes (TdP). METHODS: A retrospective analysis was performed using profiling data generated over a 3-year period, including patch clamp data from hERG, Cav1.2, and Nav1.5 (peak/late), together with hERG binding. RESULTS: Assay reproducibility was robust over the 3-year period examined. High throughput hERG patch IC50 values correlated well with GLP-hERG data (Pearson = 0.87). A disconnect between hERG binding and patch was observed for ∼10% compounds and trended with passive cellular permeability. hERG and Cav1.2 potency did not correlate for proprietary compounds, with more potent hERG compounds showing selectivity versus Cav1.2. For clinical compounds where hERG and Cav1.2 activity was more balanced, an analysis of TdP risk versus hERG/Cav1.2 ratio demonstrated low TdP probability when the hERG/Cav1.2 potency ratios were < 1. Modeling of clinical compound data revealed a lack of impact of the Nav1.5 (late) current in predicting TdP. Moreover, models using hERG binding data (ROC AUC = 0.876) showed an improved ability to predict TdP risk versus hERG patch clamp (ROC AUC = 0.787). DISCUSSION: The data highlight the value of high throughput patch clamp data in the prediction of TdP risk, as well as some potential limitations with this approach.

Comprehensive Nonclinical Safety Assessment of Nirmatrelvir Supporting Timely Development of the SARS-COV-2 Antiviral Therapeutic, Paxlovid™.

COVID-19 is a potentially fatal infection caused by the SARS-CoV-2 virus. The SARS-CoV-2 3CL protease (Mpro) is a viral enzyme essential for replication and is the target for nirmatrelvir. Paxlovid (nirmatrelvir co-administered with the pharmacokinetic enhancer ritonavir) showed efficacy in COVID-19 patients at high risk of progressing to hospitalization and/or death. Nonclinical safety studies with nirmatrelvir are essential in informing benefit-risk of Paxlovid and were conducted to support clinical development. In vivo safety pharmacology assessments included a nervous system/pulmonary study in rats and a cardiovascular study in telemetered monkeys. Potential toxicities were assessed in repeat dose studies of up to 1 month in rats and monkeys. Nirmatrelvir administration (1,000 mg/kg, p.o.) to male rats produced transient increases in locomotor activity and respiratory rate but did not affect behavioral endpoints in the functional observational battery. Cardiovascular effects in monkeys were limited to transient increases in blood pressure and decreases in heart rate, observed only at the highest dose tested (75 mg/kg per dose b.i.d; p.o.). Nirmatrelvir did not prolong QTc-interval or induce arrhythmias. There were no adverse findings in repeat dose toxicity studies up to 1 month in rats (up to 1,000 mg/kg daily, p.o.) or monkeys (up to 600 mg/kg daily, p.o.). Nonadverse, reversible clinical pathology findings without clinical or microscopic correlates included prolonged coagulation times at ≥60 mg/kg in rats and increases in transaminases at 600 mg/kg in monkeys. The safety pharmacology and nonclinical toxicity profiles of nirmatrelvir support clinical development and use of Paxlovid for treatment of COVID-19.

Characterization of a high throughput human stem cell cardiomyocyte assay to predict drug-induced changes in clinical electrocardiogram parameters.

Human induced pluripotent stem cell derived cardiomyocytes (hIPSC-CM's) play an increasingly important role in the safety profiling of candidate drugs. For such models to have utility a clear understanding of clinical translation is required. In the present study we examined the ability of our hIPSC-CM model to predict the clinically observed effects of a diverse set of compounds on several electrocardiogram endpoints, including changes in QT and QRS intervals. To achieve this, compounds were profiled in a novel high throughput voltage-sensitive dye platform. Measurements were taken acutely (30 min) and chronically (24 h) to ensure that responses from compounds with slow onset kinetics or that affected surface ion channel expression would be captured. In addition, to avoid issues associated with changes in free drug levels due to protein binding, assays were run in serum free conditions. Changes in hIPSC-CM threshold APD90 values correlated with compound plasma exposures that produced a +10 ms change in clinical QTc (Pearson r2 = 0.80). In addition, randomForest modeling showed high predictivity in defining TdP risk (AUROC value = 0.938). Risk associated with QRS prolongation correlated with an increase in action potential rise-time (AUROC value = 0.982). The in-depth understanding of the clinical translatability of our hIPSC-CM model positions this assay to play a key role in defining cardiac risk early in drug development. Moreover, the ability to perform longer term studies enables the detection of compounds that may not be highlighted by more acute assay formats, such as inhibitors of hERG trafficking.

Time for a Fully Integrated Nonclinical-Clinical Risk Assessment to Streamline QT Prolongation Liability Determinations: A Pharma Industry Perspective.

Defining an appropriate and efficient assessment of drug-induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc-prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14-based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B-based "double-negative" nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high-dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double-negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.

Translation of in vitro cannabinoid 1 receptor agonist activity to in vivo pharmacodynamic endpoints.

INTRODUCTION: Building an understanding of in vivo efficacy based on the evaluation of in vitro affinity or potency is critical in expediting early decision making in drug discovery and can significantly reduce the need for animal studies. The aim of the present study was to understand the translation of in vitro to in vivo endpoints for the cannabinoid receptor 1 (CB1). METHODS: Using a selection of CB1 agonists we describe an evaluation of in vitro to in vivo translation comparing in vitro receptor affinity or functional potency, using both cAMP and ß-arrestin endpoints, to various in vivo CB1 agonist-associated endpoints. RESULTS: We demonstrate that in vitro CB1 agonism significantly correlates with the CB1-induced cue in the drug discrimination model in vivo, but not with other purported CB1 agonist-mediated in vivo endpoints, including hypothermia and sedation. Thus, these data challenge common perceptions regarding CB1 agonist-induced tetrad effects in rodents. DISCUSSION: This work exemplifies how in vitro profiling of receptor affinity or potency can predict in vivo pharmacodynamic effects, using the CB1 as an example system. The translatability of in vitro activity to in vivo efficacy allows for the ability to rapidly contextualize off-target CB1 in vitro findings, allowing clear and rapid definition of the risk posed by such activity without the need for extensive animal studies. This has significant implications in terms of early decision making in drug discovery and reducing the use of animals in research, while also outlining a template for expanding the approach for additional targets.

Late-Stage Lead Diversification Coupled with Quantitative Nuclear Magnetic Resonance Spectroscopy to Identify New Structure-Activity Relationship Vectors at Nanomole-Scale Synthesis: Application to Loratadine, a Human Histamine H1 Receptor Inverse Agonist.

An experimental approach is described for late-stage lead diversification of frontrunner drug candidates using nanomole-scale amounts of lead compounds for structure-activity relationship development. The process utilizes C-H bond activation methods to explore chemical space by transforming candidates into newly functionalized leads. A key to success is the utilization of microcryoprobe nuclear magnetic resonance (NMR) spectroscopy, which permits the use of low amounts of lead compounds (1-5 µmol). The approach delivers multiple analogues from a single lead at nanomole-scale amounts as DMSO-d6 stock solutions with a known structure and concentration for in vitro pharmacology and absorption, distribution, metabolism, and excretion testing. To demonstrate the feasibility of this approach, we have used the antihistamine agent loratadine (1). Twenty-six analogues of loratadine were isolated and fully characterized by NMR. Informative SAR analogues were identified, which display potent affinity for the human histamine H1 receptor and improved metabolic stability.

A practical guide to secondary pharmacology in drug discovery.

Secondary pharmacological profiling is increasingly applied in pharmaceutical drug discovery to address unwanted pharmacological side effects of drug candidates before entering the clinic. Regulators, drug makers and patients share a demand for deep characterization of secondary pharmacology effects of novel drugs and their metabolites. The scope of such profiling has therefore expanded substantially in the past two decades, leading to the implementation of broad in silico profiling methods and focused in vitro off-target screening panels, to identify liabilities, but also opportunities, as early as possible. The pharmaceutical industry applies such panels at all stages of drug discovery routinely up to early development. Nevertheless, target composition, screening technologies, assay formats, interpretation and scheduling of panels can vary significantly between companies in the absence of dedicated guidelines. To contribute towards best practices in secondary pharmacology profiling, this review aims to summarize the state-of-the art in this field. Considerations are discussed with respect to panel design, screening strategy, implementation and interpretation of the data, including regulatory perspectives. The cascaded, or integrated, use of in silico and off-target profiling allows to exploit synergies for comprehensive safety assessment of drug candidates.

Author Correction: Exploring use of unsupervised clustering to associate signaling profiles of GPCR ligands to clinical response.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Exploring use of unsupervised clustering to associate signaling profiles of GPCR ligands to clinical response.

Signaling diversity of G protein-coupled (GPCR) ligands provides novel opportunities to develop more effective, better-tolerated therapeutics. Taking advantage of these opportunities requires identifying which effectors should be specifically activated or avoided so as to promote desired clinical responses and avoid side effects. However, identifying signaling profiles that support desired clinical outcomes remains challenging. This study describes signaling diversity of mu opioid receptor (MOR) ligands in terms of logistic and operational parameters for ten different in vitro readouts. It then uses unsupervised clustering of curve parameters to: classify MOR ligands according to similarities in type and magnitude of response, associate resulting ligand categories with frequency of undesired events reported to the pharmacovigilance program of the Food and Drug Administration and associate signals to side effects. The ability of the classification method to associate specific in vitro signaling profiles to clinically relevant responses was corroborated using ß2-adrenergic receptor ligands.

Simultaneous assessment of compound activity on cardiac Nav1.5 peak and late currents in an automated patch clamp platform.

INTRODUCTION: High throughput in vitro profiling of the cardiac Nav1.5 peak sodium current (INa) is widely used in cardiac safety screening. However, there is no standardized high throughput method to measure late INa. This study assessed the pharmacological and biophysical properties of veratridine and ATX-II, as well as the channel mutation (Nav1.5-∆KPQ) on the late INa. We describe a method for simultaneous measurement of both peak and late INa. METHODS: The planar patch clamp technique (QPatch) was applied to record the peak and late INa. RESULTS: The Nav1.5-∆KPQ mutant produced a small late INa (41.9 ±â€¯5.4 pA) not large enough to enable compound profiling. In contrast in wild type Nav1.5 expressing cells veratridine (100 µM) and ATX-II (100 nM) enhanced concentration-dependent increases in the late INa (maximum responses of 1162.2 ±â€¯258.5 pA and 392.4 ±â€¯71.3 pA, respectively). Veratridine inhibited, whereas, ATX-II had a minimal effect, on the peak INa and preserved the current-voltage curve. Peak and late INa inhibition was characterized for 25 clinical INa blockers in the presence of ATX-II. Compound IC50 values for peak INa generated in the absence or presence of ATX-II correlated. The potency of the late INa block was found to be dependent on whether it was measured at the end of the depolarizing pulse or during the ramp. DISCUSSION: In the presence of ATX-II, both peak and late INa could be assessed simultaneously. Late INa may be best assessed using the maximum response obtained during the ramp of the voltage protocol.

Ligand biological activity predicted by cleaning positive and negative chemical correlations.

Predicting ligand biological activity is a key challenge in drug discovery. Ligand-based statistical approaches are often hampered by noise due to undersampling: The number of molecules known to be active or inactive is vastly less than the number of possible chemical features that might determine binding. We derive a statistical framework inspired by random matrix theory and combine the framework with high-quality negative data to discover important chemical differences between active and inactive molecules by disentangling undersampling noise. Our model outperforms standard benchmarks when tested against a set of challenging retrospective tests. We prospectively apply our model to the human muscarinic acetylcholine receptor M1, finding four experimentally confirmed agonists that are chemically dissimilar to all known ligands. The hit rate of our model is significantly higher than the state of the art. Our model can be interpreted and visualized to offer chemical insights about the molecular motifs that are synergistic or antagonistic to M1 agonism, which we have prospectively experimentally verified.

In vitro secondary pharmacological profiling: An IQ-DruSafe industry survey on current practices.

INTRODUCTION: In 2015, IQ DruSafe conducted a survey of its membership to identify industry practices related to in vitro off target pharmacological profiling of small molecules. METHODS: An anonymous survey of 20 questions was submitted to IQ-DruSafe representatives. Questions were designed to explore screening strategies, methods employed and experience of regulatory interactions related to in vitro secondary pharmacology profiling. RESULTS: The pharmaceutical industry routinely utilizes panels of in vitro assays to detect undesirable off-target interactions of new chemical entities that are deployed at all stages of drug discovery and early development. The formats, approaches and size of panels vary between companies, in particular i) choice of assay technology; ii) test concentration (single vs. multiple concentrations) iii) rationale for targets and panels selection (taking into account organizational experience, primary target, therapeutic area, availability at service providers) iv) threshold level for significant interaction with a target and v) data interpretation. Data are generated during the early phases of drug discovery, principally before in vivo GLP studies (i.e., hit-to-lead, lead optimization, development candidate selection) and used to contextualize in vivo non-clinical and clinical findings. Data were included in regulatory documents, and around half of respondents experienced regulatory questions about the significance of the results. CONCLUSION: While it seems that in vitro secondary pharmacological profiling is generally considered valuable across the industry, particularly as a tool in early phases of drug discovery for small molecules, there is only loose consensus on testing paradigm, the required interpretation and suitable follow up strategies to fully understand potential risk.

Discovery of Potent and Selective Periphery-Restricted Quinazoline Inhibitors of the Cyclic Nucleotide Phosphodiesterase PDE1.

We disclose the discovery and X-ray cocrystal data of potent, selective quinazoline inhibitors of PDE1. Inhibitor ( S)-3 readily attains free plasma concentrations above PDE1 IC50 values and has restricted brain access. The racemic compound 3 inhibits >75% of PDE hydrolytic activity in soluble samples of human myocardium, consistent with heightened PDE1 activity in this tissue. These compounds represent promising new tools to probe the value of PDE1 inhibition in the treatment of cardiovascular disease.

Lead Diversification at the Nanomole Scale Using Liver Microsomes and Quantitative Nuclear Magnetic Resonance Spectroscopy: Application to Phosphodiesterase 2 Inhibitors.

In this report, we describe a method whereby lead molecules can be converted into several new analogues each using liver microsomes. Less than one micromole of substrate is incubated with liver microsomes (mouse, rat, hamster, guinea pig, rabbit, dog, monkey, or human) to produce multiple products which are isolated and analyzed by quantitative cryomicroprobe NMR (qNMR) spectroscopy. The solutions from qNMR analysis were then used as stocks that were diluted into biochemical assays. Nine human phosphodiesterase-2 (PDE2) inhibitors yielded 36 new analogues. Products were tested for PDE2 inhibition, intrinsic clearance in human hepatocytes, and membrane permeability. Two of the products (2c and 4b) were 3-10× more potent than their respective parent compounds and also had improved metabolic stability. Others offered insights into structure-activity relationships. Overall, this process of using liver microsomes at a submicromole scale of substrate is a useful approach to rapid and cost-effective late-stage lead diversification.

Late-Stage Microsomal Oxidation Reduces Drug-Drug Interaction and Identifies Phosphodiesterase 2A Inhibitor PF-06815189.

Late-stage oxidation using liver microsomes was applied to phosphodiesterase 2 inhibitor 1 to reduce its clearance by cytochrome P450 enzymes, introduce renal clearance, and minimize the risk for victim drug-drug interactions. This approach yielded PF-06815189 (2) with improved physicochemical properties and a mixed metabolic profile. This example highlights the importance of C-H diversification methods to drug discovery.

Cardiac sodium channel antagonism - Translation of preclinical in vitro assays to clinical QRS prolongation.

INTRODUCTION: Cardiac sodium channel antagonists have historically been used to treat cardiac arrhythmias by preventing the reentry of the electrical impulse that could occur following myocardial damage. However, clinical studies have highlighted a significant increase in mortality associated with such treatment. Cardiac sodium channel antagonist activity is now seen as an off-target pharmacology that should be mitigated during the drug development process. The aim of this study was to examine the correlation between in vitro/ex vivo assays that are routinely used to measure Nav1.5 activity and determine the translatability of the individual assays to QRS prolongation in the clinic. METHODS: A set of clinical compounds with known Nav1.5 activity was profiled in several in vitro/ex vivo assays (binding, membrane potential, patch clamp and the Langendorff isolated heart). Clinical data comprising compound exposure levels and changes in QRS interval were obtained from the literature. Sensitivity/specificity analysis was performed with respect to the clinical outcome. RESULTS: The in vitro assays showed utility in predicting QRS prolongation in the clinic. Optimal thresholds were defined for each assay (binding: IC20; membrane potential: IC10; patch clamp: IC20) and sensitivity (69-88%) and specificity (53-84%) values were shown to be similar between assay formats. DISCUSSION: The data provide clear statistical insight into the translatability of Nav1.5 antagonism data generated in vitro to potential clinical outcomes. These results improve our ability to understand the liability posed by such activity in novel development compounds at an early stage.