Optimizing the use of methods and measurement endpoints in respiratory safety pharmacology.

JPM-06183; No of Pages 6 Journal of Pharmacological and Toxicological Methods xxx (2014) xxx–xxx

Contents lists available at ScienceDirect

Journal of Pharmacological and Toxicological Methods journal homepage: www.elsevier.com/locate/jpharmtox

Review

Optimizing the use of methods and measurement endpoints in respiratory safety pharmacology Dennis J. Murphy ⁎ Department of Safety Pharmacology, GlaxoSmithKline Pharmaceuticals, King of Prussia, PA, USA

a r t i c l e

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Available online xxxx Keywords: Function Methods Nonclinical Pulmonary Respiratory Safety Pharmacology

a b s t r a c t A variety of methods and measurement endpoints are currently available for evaluating respiratory function in animal models. To evaluate drug-induced effects on respiratory function, respiratory safety pharmacology studies generally emphasize the use of conscious animal models and measures of pulmonary ventilation. Respiratory rate, tidal volume and/or a measure of arterial blood gases are the standard measurement parameters. Although these parameters will provide a measure of ventilation, other ventilatory parameters, which can provide mechanistic insight or identify site of action, should also be considered. Such parameters include inspiratory and expiratory times, flows and pauses, and apneic time. Stimulation models involving exercise and exposure to elevated CO2 or reduced O2 should also be considered when enhancing measurement sensitivity or quantifying reductions in ventilatory functional reserve is desired. Although ventilatory measurements are capable of assessing the functional status of the respiratory pumping apparatus, such measurements are generally not capable of assessing the status of the other functional component of the respiratory system, namely, the gas exchange unit or lung. To characterize drug-induced effects on the gas exchange unit, measures of airway patency, lung elastic recoil and gas diffusion capacity need to be considered. The objective of this review is to discuss the value and utility of the methods and measurement endpoints currently available for assessing respiratory function to help optimize the design of respiratory safety pharmacology studies. © 2014 Elsevier Inc. All rights reserved.

Contents 1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Use of ventilatory measurements to detect and characterize respiratory dysfunction . . . . . . . . . . . 3. Use of airway resistance and compliance measurements to identify and characterize respiratory dysfunction 4. Use of arterial blood gas measurements to identify and characterize respiratory dysfunction . . . . . . . . 5. Use of investigative endpoints to provide mechanistic insight . . . . . . . . . . . . . . . . . . . . . . 6. Use of stimulation models to evaluate respiratory dysfunction . . . . . . . . . . . . . . . . . . . . . 7. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1. Introduction Safety pharmacology is a discipline within the nonclinical (preclinical) assessment of drug safety. A recent pharmaceutical industry survey indicates that the current practice of respiratory function assessment within safety pharmacology focuses on the use of conscious rodent models and measures of pulmonary ventilation (Lindgren et al., 2008). ⁎ 709 Swedeland Road, King of Prussia, PA 19406, USA. Tel.: +1 610 270 7410; fax: +1 610 270 7622. E-mail address: [email protected].

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Conscious models help optimize respiratory function measurements by removing the depressant effect most anesthetics and analgesics have on respiratory drive (Chevillard, Megarbane, Risede, & Baud, 2009; Hirshman, McCullough, Cohen, & Weil, 1975; Nussmeier et al., 1991) and the potential alterations of response of airways to bronchoconstrictive agents (Advenier, Boissier, Mallard, & Ruff, 1978; Hirschman & Bergman, 1978). Furthermore, the duration of the experimental measurement time for anesthetized preparations is generally limited to several hours. Respiratory rate, tidal volume and derived minute volume as well as arterial blood gases are the measurement endpoints most commonly used in safety pharmacology studies

http://dx.doi.org/10.1016/j.vascn.2014.03.174 1056-8719/© 2014 Elsevier Inc. All rights reserved.

Please cite this article as: Murphy, D.J., Optimizing the use of methods and measurement endpoints in respiratory safety pharmacology, Journal of Pharmacological and Toxicological Methods (2014), http://dx.doi.org/10.1016/j.vascn.2014.03.174

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D.J. Murphy / Journal of Pharmacological and Toxicological Methods xxx (2014) xxx–xxx

to identify drug-induced changes in ventilation (Lindgren et al., 2008). Although these parameters will provide a measure of ventilation, other parameters, which can provide mechanistic insight, should also be considered. Furthermore, it is important to note that the respiratory system consists of two functional components — the pumping apparatus and the gas exchange unit or lung (see Fig. 1). Although ventilatory measurements are capable of assessing the functional status of the respiratory pumping apparatus, such measurements are generally not capable of assessing the status of the gas exchange unit. To characterize drug-induced effects on the gas exchange unit, measures of airway patency, lung elastic recoil and gas diffusion capacity need to be considered. Such measurement endpoints are not commonly obtained in safety pharmacology studies (Lindgren et al., 2008). Methods for evaluating airway resistance and lung compliance are currently available for use in assessing airway patency and lung elastic recoil, respectively, in animal models. Such methods are available for both anesthetized (Costa, 1985; Diamond & O'Donnell, 1977; Tobin et al., 1987) and conscious (Murphy, Renninger, & Gossett, 1998) rodent models as well as anesthetized (Black, Suki, Madwed, & Jackson, 2001; Van Scott, Aycock, Cozzi, Salleng, & Stallings, 2005) and conscious (Ingram-Ross et al., 2012;

Murphy, Renninger, & Coatney, 2001) non-rodent models. Methods for evaluating lung gas diffusion capacity are also available (Kialouama et al., 2011; Qureshi, 2011). Because the current practice in respiratory safety pharmacology tends to focus on a limited number of respiratory parameters, the objective of this review is to discuss the value and utility of the methods and measurement endpoints currently available for assessing respiratory function to help optimize the design of respiratory safety pharmacology studies. 2. Use of ventilatory measurements to detect and characterize respiratory dysfunction The respiratory system consists basically of two functional components — the pumping apparatus and the gas exchange unit (see Fig. 1). The function of the pumping apparatus is to regulate gas exchange between the environment and the airways to help ensure that sufficient oxygen is supplied to the circulation to meet changing metabolic demands and to remove excess carbon dioxide and other metabolic products. The components of the pump include the respiratory

Evaluation of Respiratory Functions

Pumping Apparatus

Gas Exchange Unit

Function

Function

Regulate gas Exchange between environment and airways

Regulate gas exchange between airways and blood

Components

Components

Respiratory muscles, CNS, chemo/mechano-receptors

Airways, alveoli, vasculature, fibrous network

Core Measurements

Core Measurements

Tidal volume, respiratory rate, minute volume

Airway resistance and lung compliance

Investigative Measurements

Investigative Measurements

Inspiratory and expiratory times and flows ,apneic time and end-inspiratoy and end-expiratory pauses

Gas diffusion capacity–DLCO and PaO2/PaCO2 ratio

Stimulation Models

Stimulation Models

Ventilatory response to elevated CO2 and reduced O2 exposure and graded exercise

Flow-Volume maneuver (PEF, FEV, FEF75, FEF25) Pressure-Volume maneuvers (Cst, IC, FVC, FRC)

Fig. 1. A chart showing the functional and structural components of the respiratory system and the associated models and functional measurement endpoints.



muscles, and the nerves, chemoreceptors and mechanoreceptors that regulate the depth and frequency of the pump. The other functional component is the gas exchange unit or lung. The function of the lung is to ensure that the gas entering the airways is appropriately exchanged with pulmonary arterial blood. To do this, the lung must have patent airways to ensure movement of gases to the alveoli during inspiration and elastic recoil to ensure the removal of gases during expiration. The components of the gas exchange unit include the airways, alveoli, vasculature and elastic fibrous network. Change in the functional status of the pumping apparatus is determined by measuring ventilatory patterns, which should include the standard or core measurement endpoints respiratory rate (frequency) and tidal volume (depth). By monitoring the frequency and depth of the pumping apparatus, the effects of drugs on total pulmonary ventilation (i.e., respiratory stimulation or depression) can be established. Further, inclusion of both tidal volume and respiratory rate is important since the mechanisms controlling the rate and volume are pharmacologically distinct with drugs that are known to affect ventilation generally doing so by selectively affecting either tidal volume or respiratory rate. For example, opioids such as morphine are known to cause respiratory depression in rats by affecting respiratory rate, with no effect on tidal volume (Murphy, Grando, & Joran, 1994), while the respiratory stimulant theophylline increases ventilation in rats by specifically affecting tidal volume with no effect on respiratory rate (Murphy, Joran, & Renninger, 1993). Monitoring ventilatory parameters cannot generally be used to directly assess the status of the gas exchange unit or lung. Studies that have measured drug-induced effects on both ventilatory parameters and airway resistance have demonstrated that mild to moderate (2–3 fold) changes in airway resistance do not produce changes in breathing patterns in either animal models or humans. Studies in our laboratory have demonstrated that 2–3 fold increases in airway resistance using an intravenous infusion of the bronchoconstrictive agent, methacholine, does not cause ventilatory changes in the rat, dog or monkey (unpublished results). Similar findings have been noted in humans (Savoy, Allgower, Courteheuse, & Junod, 1984; Yasukouchi, 1992) and guinea pigs (Wiester, Costa, Tepper, Winsett, & Slade, 2005). Because the absence of a change in ventilatory parameters cannot reliably predict the absence of mild to moderate changes in lung function, the primary use of ventilatory measurements should be to only assess the effects of drugs on the pumping apparatus. 3. Use of airway resistance and compliance measurements to identify and characterize respiratory dysfunction Airway resistance and lung compliance are the standard or core measurement endpoints used to assess airway patency and elastic recoil, respectively, and are obtained by concurrent measurement of both lung airflow and intrapleural pressure changes (Costa, 1985; Diamond & O'Donnell, 1977). Changes in airway resistance and lung compliance are important safety endpoints. An increase in airway resistance can result from obstruction of the airways caused by the constriction of airway smooth muscle (bronchoconstriction), hypertrophy or hyperplasia of cells lining the airways, or hypersecretion of airway mucus. An acute increase in airway resistance can be a lifethreatening event, while a chronic increase can lead to respiratory muscle fatigue and failure (Hunt & Rosenow, 1992). A decrease in lung compliance can result from alterations in the lower respiratory tract, which may involve changes in fibrous network, presence of interstitial or intraalveolar fluid (edema) or inflammatory cells (pneumonitis), pulmonary congestion or surfactant disruption. These effects are characterized histopathologically as diffuse alveolar damage (with edema or hemorrhage), nonspecific interstitial pneumonia (with or without fibrosis) and bronchiolitis obliterans (Erasmus, McAdams, & Rossi, 2002; Flieder & Travis, 2004). These types of changes are associated with the pathological syndromes of acute lung injury (ALI) and acute

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respiratory distress syndrome (ARDS) (Myers, Limper, & Swensen, 2003). Drug treatment is also known to cause pulmonary edema or pneumonitis (Erasmus et al., 2002). Since these effects generally cause a decrease in lung elasticity, they can be detected by measuring lung compliance (Matute-Bello et al., 2011). The use of lung compliance measurement in single dose safety pharmacology studies is usually not considered since lung injury rarely occurs following acute drug treatment or, if injury occurs, it is generally detected by histopathological evaluation in toxicology studies. However, inclusion of lung compliance as an endpoint should be considered when there is concern for acute lung injury following an inhaled dose of drug or when effects, which may not be detected by histopathological evaluation, such as interstitial edema or alterations in alveolar surfactant production, are anticipated. Furthermore, in repeat dose studies where alterations in the fibrous network, surfactant disruption or pulmonary congestion are known to occur with drug treatment (Erasmus et al., 2002), measuring compliance changes could be used to provide an in vivo biomarker for the onset and progression of drug-induced lung injury, as well as providing an understanding of functional consequences. Thus, based on the respiratory system model involving two functional components, the poor reliability of ventilatory change to predict changes in airway resistance, and the known effects of drugs on both ventilatory and lung functions, including core endpoints that assess both the pumping apparatus and the gas exchange unit should be considered for optimizing the ability to detect and quantify drug-induced respiratory dysfunction. 4. Use of arterial blood gas measurements to identify and characterize respiratory dysfunction Changes in the partial pressures arterial blood gases (PaO2 and PaCO2) and the use of hemoglobin oxygen saturation (SaO2) as a surrogate measure for PaO2 are currently accepted methods for assessing drug-induced ventilatory changes in safety pharmacology studies (Anonymous, 2001). To evaluate the value of these endpoints for assessing drug-induced changes in ventilation, two questions need to be addressed: (1) are changes in blood gases the most sensitive measure for detecting ventilatory effects? And (2) is SaO2 an appropriate surrogate measure for arterial blood gases in respiratory safety pharmacology studies? To address the first question, changes in blood gases would not be expected to be a sensitive method for detecting a ventilatory defect. In safety pharmacology studies, healthy, conscious animals are used as the test subjects and, in healthy subjects, homeostatic mechanisms to prevent or minimize blood gas changes during a decrease or increase in lung ventilation are present. To compensate for a decrease in lung ventilation, the lung can minimize the change in blood gases by increasing the gas diffusing capacity of the lung. In general, the lung can increase gas diffusing capacity by approximately three fold (Hsia, 2002). This occurs by increasing lung perfusion, thereby decreasing physiological dead space that normally exists in the lung, and by increasing the number of alveoli that are ventilated. In addition, the blood has a buffering mechanism to minimize changes in plasma PaO2 and PaCO2 (Brown & Clancy, 1965; Ellison, Straumfjord, & Hummel, 1958; Kovtun, Tataurov, Melnikov, & Krivoschekov, 2011). This buffering system includes hemoglobin that binds O2 and minimizes changes in PaO2 and a carbonic anhydrase/bicarbonate system and proteins containing carbamino groups for buffering changes in PaCO2 during periods of reduced ventilation. Studies in healthy conscious humans and animal models have confirmed this functional reserve. A study in humans demonstrated that a 100% increase in minute ventilation produced by removing a resistive load had no effect on end-tidal CO2 (Gallagher, Hoff, & Younes, 1985), while a study in dogs demonstrated that a 96% increase in minute ventilation produced by a respiratory stimulant (albuterol) had no effect on PaO2, PaCO2 or SaO2 (Authier et al., 2008). In conscious rats, reduced levels of activity were associated with up to


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a 40% reduction in minute ventilation with no change in PaO2 or PaCO2 (Polianski, Brun-Pascaud, Jelazko, & Pocidalo, 1984), while a reduction in minute ventilation by 30% using a respiratory depressant (morphine) produced no change in end tidal CO2 (Murphy et al., 1994). SaO2 is typically measured in humans and animals using a methodology called pulse oximetry. This is a non-invasive technology that involves attaching a photoelectric sensor to the surface of the body at a variety of locations including the ear, toe, finger, tail, paw, neck or tongue. The sensitivity of this methodology is limited in that the measurement occurs in a peripheral vascular bed and, being a surface measurement, can be influenced by a variety of factors including body temperature, localized vasoconstriction, room lighting and orientation of probe (Chan, Chan, & Chan, 2013). The sensitivity of this method is also limited by the O2 buffering capacity of hemoglobin. The hemoglobin oxygen dissociation curve is sigmoidal in shape and, in a healthy subject, a decrease in PaO2 of approximately 20% is required before a significant change in hemoglobin oxygen saturation can occur. The lack of sensitivity of SaO2 for detecting blood gas changes was demonstrated in a study using healthy conscious dogs. In that study, minute ventilation was reduced by approximately 68% following the intravenous injection of a respiratory depressant (Remifentanil). The severe hypoventilation was associated with a 21% increase in PaCO2, a 14% decrease in PaO2 and no change in SaO2 (Authier et al., 2008). These findings indicate that a change in blood gases (especially SaO2) is not a sensitive measure of drug-induced ventilatory change in conscious, healthy animals as are used in safety pharmacology studies. Measures of tidal volume, respiratory rate and derived minute volume are more sensitive indicators of ventilatory change and, as such, should be the core measurements used for optimizing the detection of druginduced effects on ventilatory function. 5. Use of investigative endpoints to provide mechanistic insight Core measurement endpoints are those that are considered standard for detecting and quantifying the effects of drugs on respiratory function, while investigative or tier II measurement endpoints are those that further characterize a respiratory effect, provide mechanistic insight or help identify drug targets. Investigative endpoints should be considered, on a case by case basis, in respiratory safety pharmacology studies. Although respiratory rate and tidal volume can detect changes in pulmonary ventilation, the inclusion of additional endpoints such as inspiratory time and flow, expiratory time and flow and apneic time can add value by providing mechanistic insight. For example, a selective increase in inspiratory time or decrease in mean inspiratory flow (tidal volume/inspiratory time) is indicative of a decrease in respiratory drive (Remmers, 1976), while a selective increase in expiratory time or decrease in expiratory flow can be indicative of airway obstruction (Glaab et al., 2002). In addition, the presence of an end-inspiratory or end-expiratory pause has been shown to be indicative of upper airway sensory and lower airway irritant receptor activation, respectively (Ferguson et al., 1986). Such endpoints would be useful for detecting irritation associated with an inhaled drug. Apneic time is the time between breaths and can be used for assessing breathing instability, which is especially important during the sleep state where it is used in clinical studies to identify and grade sleep disordered breathing. Apneic time is generally not considered a core endpoint in respiratory safety pharmacology studies. However, understanding the potential effects of drugs on apneic time may be important since factors that enhance ventilatory instabilities and apneic time in the sleep state have been associated with death in children and adults (Gami, Howard, Olson, & Somers, 2005; Thach, 2005). Sleep apnea is estimated to be present in approximately 2–10% of adults (Leger, Bayon, Laaban, & Philip, 2012) and in 23–62% of the elderly (N65 years of age) (Mayson, Neilan, Awad, & Malhotra, 2012) and has become a major health concern in the respiratory medical community because of its association with the development of systemic hypertension, pulmonary hypertension,

stroke, cardiac arrhythmia, Type II diabetes, cancer and cognitive dysfunction (Benjamin & Lewis, 2008; Kohli, Sarmiento, & Malhotra, 2013). Furthermore, respiratory depressant drugs including phenothiazines and opioids are known to both induce sleep apnea and exacerbate existing sleep apnea (Farney, Walker, Cloward, & Rhondeau, 2003; Kahn, Hasaerts, & Blum, 1985) Thus, when a drug treatment produces evidence of respiratory depression, the inclusion of apneic time and continuous monitoring of ventilation over a 24-hour cycle would be useful for investigating a potential new target in respiratory safety pharmacology. Methods for continuous monitoring respiratory parameters over a complete diurnal cycle (24 h) in animal models are currently available. A method using inductive straps placed around the thorax and abdomen (respiratory inductive plethysmography) is currently available for obtaining ventilatory parameters in the dog (Murphy, Renninger, & Schramek, 2010) and monkey (Ingram-Ross et al., 2012). A method for measuring ventilatory changes using thoracic impedance measurements also exists for dogs (Kearney, Metea, Gleason, Edwards, & Atterson, 2010), while whole body plethysmography has been successfully used in dogs (Talavera et al., 2006), rats (Schierok, Markert, Pairet, & Guth, 2000) and monkeys (Iizuka et al., 2010). Whole body plethysmography is limited in that tidal volume is measured indirectly. In a whole body chamber, tidal volume is estimated from the expansion of inhaled gas, with a calculation that is dependent on the temperature and humidity of the chamber, the animal's body temperature and the rate of breathing since the fraction of heated/humidified exhaled air that is re-inhaled is dependent on breathing rate (Jacky, 1980). Respiratory inductive plethysmography has an advantage over the impedance and plethysmography methods in that it also provides a measure of airway obstruction that can be obtained by analyzing shifts in the timing of the thoracic and abdominal movements (phase angle) (Tobin et al., 1987). Although the measurement of lung compliance can help in the early identification of lower respiratory tract injury, it does not provide a complete measure of the functional consequences. To better characterize the functional consequences of these changes, the impact on blood gas diffusion should be considered. Comparing the relative changes in the partial pressures of arterial O2 (PaO2) and CO2 (PaCO2) is one method for detecting injury that would hinder the diffusion of gases from the alveoli to the pulmonary arterioles as would be caused by interstitial or intra-alveolar accumulation of fluid (edema) or cells (pneumonitis), reduction in alveolar ventilation by small airway or alveolar obstruction (broncholitis obliterans) or the reduction in alveolar blood flow (shunting). A reduction in gas diffusion would be indicated by a selective decrease in PaO2 relative to PaCO2, and is generally quantified by calculating the ratio of PaO2/PaCO2. A reduction in gas diffusion capacity can also be measured as an increase in the ratio of inhaled O2 (FiO2) to PaO2 or a reduction in carbon monoxide diffusion capacity (DLCO) (Kialouama et al., 2011; Qureshi, 2011). Thus, the addition of a measure of blood gas diffusion capacity should be considered when an effect on lung compliance has been detected. 6. Use of stimulation models to evaluate respiratory dysfunction Although a variety of animal models for lung and airway diseases are currently available, these models, in general, do not accurately reflect the etiology or pathophysiology of human diseases. Consequently, such models are of limited value for evaluating drug safety in specific human disease populations. Since a common liability associated with respiratory diseases is the loss of functional reserve, the use of stimulation or stress models that can quantify the loss of functional reserve offers an alternative to disease models. In addition to quantifying the loss of functional reserve, these models can also be of value when a more sensitive measure of effect or an interest in identifying a target site is required. Quantifying the loss of functional reserve could be used when attempting to identify the potential impact or risk of an effect in a sensitive patient population where a loss of functional reserve



has been identified. Stimulation models utilize healthy animals to avoid the complications of induced disease models and use graded stimulation to allow for detection and quantification of the loss of functional reserve. Stimulation models used to detect the loss of ventilatory reserve include the use a graded exercise (running) to assess a decrease in maximal ventilation or gas exchange (Bouitbir et al., 2011; Kittleson, Johnson, & Pion, 1996) and the use of inhaled air mixtures with elevated CO2 or reduced O2 to evaluate the change in maximal ventilatory response or respiratory drive (Bellville & Seed, 1960; Howell, 1993; Schaper, Thompson, & Alarie, 1985; Weil & Zwillich, 1976). By quantifying the loss of functional reserve, the potential impact on morbidity and mortality in human patients with a known loss of functional reserve can be evaluated. Exposure to inhaled air mixtures can also be used to identify a target site. By comparing the ventilatory responses to elevated CO2 and reduced O2 or a low dose of sodium cyanide, a selective drug effect on peripheral or central chemoreceptors can also be determined (Howell & Landrum, 1995; Murphy, Joran, & Grando, 1995). Stimulation models can also be used to evaluate the loss of functional reserve for lung airflow and compliance. One established model in anesthetized animals involves the use of pulmonary forced maneuver procedures, which involve the controlled maximal inflation and deflation of the lung and the simultaneous measurement of flow, volume and transpulmonary pressure (Costa, 1985; Diamond & O'Donnell, 1977). This procedure has also been used in non-human primates (Wegner, Jackson, Berry, & Gillespie, 1984). One maneuver involves inflation of the lung to a maximum volume (inspiratory capacity) and then deflating the lung at a maximal rate by connecting the trachea to a negative pressure reservoir while recording lung airflow and volume. This procedure can be used to calculate flow endpoints such as forced expiratory volume (FEV) at specific expiratory times, peak expiratory flow (PEF), and flow at 25 and 75% of forced vital capacity (FEF25/FEF75). By measuring the change from a maximal or forced (stimulated) value, the loss of functional reserve for lung airflow can be determined. A maneuver involving a slow inflation to inspiratory capacity followed by a slow deflation, and an evaluation of the slope of the expiratory phase of the pressure–volume curve, can also be performed by this procedure to calculate a static or quasi-static lung compliance (Cst) (Costa, 1985; Diamond & O'Donnell, 1977). By measuring compliance under forced (stimulated) volume and pressure conditions, a loss of elastic reserve can be determined. Furthermore, comparing the relative effect of treatment on forced expiratory flows can be used to investigate whether airway obstruction occurred in the larger central airways (PEF or FEF75) or the smaller peripheral airways (FEF25). Since the measure of dynamic lung compliance in spontaneously breathing animals can be influenced by small airway obstruction, measuring static or quasi-static lung compliance can also be used to confirm the presence of a decrease in lung compliance. By measuring changes in inspiratory capacity (IC), forced vital capacity (FVC) and functional residual capacity (FRC) using pressure–volume maneuvers, the effect of drug treatment on gas trapping and loss in the volume capacity for gas exchange can also be investigated. 7. Conclusion A variety of methods and measurement endpoints are available for use in evaluating drug-induced effects on respiratory function in animal models. To optimize the ability of respiratory safety pharmacology studies to detect and characterize drug-induced effects, these studies should include measurement endpoints that are sensitive for detecting and characterizing ventilatory change, include measures that evaluate both the pumping apparatus and gas exchange unit (lung), include investigative measurement endpoints when needed to help identify site of action and/or provide mechanistic insight, and consider the use of respiratory stimulation models when needed to increase sensitivity of detection or to quantify loss of functional reserve.

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