Advancing Asthma Management with Exhaled Nitric Oxide

R E S O U R C E K I T

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Table of Contents

Summary 2

1 The Burden of Asthma 3

2 Exhaled Nitric Oxide (eNO) 4

3 Insight™ eNO System 6

4 Case Studies 9

5 Exhaled Nitric Oxide Cost Model 12

6 Coding Information for Exhaled Nitric Oxide (eNO)-CPT 95012 12

SummaryExhaled nitric oxide (eNO) is an established marker of airway inflammation that can be safely and accurately measured in people with asthma. This resource kit provides comprehensive information about the benefits and technology of eNO testing, and highlights the medical necessity and the extent of acceptance of eNO testing.

The Asthma and Airway Inflammation DilemmaIt is estimated that 22 million+ people in the U.S. suffer from asthma, making it one of the most common and costly of all diseases. One quarter of all emergency room visits are asthma related and asthma is the one of the leading chronic childhood diseases.

Controlling inflammation of the airways has become the central focus for managing asthma. Strong clinical evidence suggests that asthma management and control can be significantly improved by regularly monitoring airway inflammation. However, current methods used to monitor and manage asthma, such as lung function tests, do not measure airway inflammation.

Physicians have relied largely on correlating symptoms and disease severity to assess their patients. Until now, the degree of airway inflammation has not been measurable in a simple and practical way.

The eNO SolutionExhaled nitric oxide has been established as a reliable marker of airway inflammation in asthma for over 10 years. It has been the central focus of studies, establishing its link to optimization of medication and prediction of asthma exacerbations.

Measurement of eNO in the physician’s office is a much awaited breakthrough in medical technology that provides physicians with a reliable tool to measure airway inflammation as an adjunct to the current diagnostic measures, such as lung function.

Clinical Utility of eNO

Monitoring of eNO and adjusting medication accordingly could significantly improve disease management, resulting in reduction of the severity of symptoms, optimization of drug usage and improvement of compliance in individual patients, and fewer exacerbations. The end result is optimal use of healthcare resources and improved quality of life for asthma patients.

Reimbursement for eNO Testing In 2007, the CPT editorial committee added a specific code to the CPT Coding Book for Nitric Oxide Expired Gas Determination (CPT Code 95012©). With this action, the procedure should be recognized as a standard service provided to patients. Exhaled nitric oxide (eNO) determination is considered a service which is consistent with contemporary medical practice for the evaluation of patients with respiratory complaints.

Apieron and the Insight™ eNO SystemApieron, Inc. is a medical device company based in Menlo Park, California. The company was founded in 2001 to develop the first practical, office-based device for routine measurement of exhaled nitric oxide. In March 2008, the company received FDA 510(k) clearance for the Insight™ eNO System, which is now commercially available. Apieron’s goal is to offer a better way to manage asthma and improve the standard of care for patients who live with the disease.

The Insight system is a highly accurate device, expressly designed for the physician’s office to measure nitric oxide in expired human breath (eNO). It is non-invasive, safe, easy to use, and provides results in less than a minute. Apieron’s unique biosensor utilizes proprietary technology to detect trace amounts of nitric oxide in a single human breath.

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1. The Burden of Asthma

Asthma OverviewAsthma is a chronic disease characterized by inflammation of the airways caused by allergens and other triggers. When airways are inflamed, the inner walls of the airways swell making them irregular. This causes the flow of air to become turbulent.

The events that lead to obstruction of airflow and thus to asthma symptoms are complex and usually involve the following events:

Bronchoconstriction, where the smooth muscle surrounding the •airways tightens in response to a trigger and narrows the airway.

Inflammation, where inner walls of airways swell. •

Mucus formation within the airways that obstructs airflow. •

Typical asthma symptoms include wheezing, coughing, chest tightness (dyspnea) and shortness of breath.

The Role of Inflammation in AsthmaAsthma signs and symptoms evolve from three basic characteristics that underlie the disease and its exacerbations: airway obstruction, airway hyperresponsiveness and airway inflammation. Airway obstruction and hyperresponsiveness represent the classic physiology of asthma, and their contribution to the disease process and symptomatology have been well recognized for some time. Appreciation of the role of airway inflammation in asthma has evolved more recently.

Today asthma experts consider airway inflammation a central feature of asthma pathogenesis and its clinical manifestations. In fact, airway inflammation likely plays a critical role in airway obstruction and hyperresponsiveness. In recent years, clinical and scientific knowledge of asthma has evolved from a model of episodic constriction of bronchial smooth muscle to a model which involves chronic airway inflammation.

Airway inflammation precedes symptoms. Evidence of inflammation is present at the onset of symptoms in newly diagnosed patients with asthma. Accordingly, treatment algorithms for asthma have emphasized treatment of the underlying inflammation, as well as the bronchoconstrictive symptoms. By acquiring a better understanding and appreciation of the inflammatory process, physicians can employ treatments to inhibit specific steps in the process and improve control over asthma and its symptoms.

The Cost of AsthmaAsthma affects 22 million Americans. A principal clinical consequence of both acute and chronic inflammation is the development of asthma exacerbations. Exacerbations of asthma are not only an important clinical marker of inadequately controlled or worsening asthma but are probably the most important outcomes from both a humanistic and health economics viewpoint. Severe asthma exacerbations lead to about 4,000 deaths and nearly 500,000 hospitalizations per year.

Healthcare costs for asthma include outpatient visits to physician offices and hospital outpatient departments, visits to hospital emergency departments (EDs) and hospitalizations. When

considering the prevalence of asthma and the frequency of such visits, as outlined in the statistics below, the costs become monumental.

34.1 million people have been diagnosed with asthma during •their lifetime.1

22.8 million people have asthma.• 1

There were 14.1 million outpatient asthma visits to private •physician offices and hospital outpatient departments.2

Children less than 18 years had 7 million physician office and •outpatient visits.3

Morbidity

Asthma accounts for one quarter of all emergency room visits •in the U.S. each year, with 2 million emergency room visits.4

Each year, asthma accounts for more than 1 million outpatient •visits4 and 500,000 hospitalizations.5

The average length of stay (LOS) for asthma hospitalizations is •3 days.6

Nearly half (41%) of asthma-related hospitalizations are for •children less than 19 years old.5

Respiratory illnesses like asthma are the leading cause of •hospitalization for children. 6

Mortality

There are about 4,000 deaths due to asthma each year, many •of which are avoidable with proper treatment and care.7

Social and Economic Costs

The annual cost of asthma is estimated to be nearly $18 billion. •

Direct costs accounted for nearly $10 billion (hospitalizations •the single largest portion of direct cost) and indirect costs of $8 billion (lost earnings due to illness or death).8

For adults, asthma is one of the leading causes of work •absenteeism and “presenteeism,” resulting in nearly 13 million missed or lost (“less productive”) workdays each year.9

Among children ages 5 to 17, asthma is the leading cause •of school absences from a chronic illness. It accounts for an annual loss of more than 13 million school days per year.3 It is estimated that children with asthma spend a nearly 8 million days per year restricted to bed.8

1. “National Health Interview Survey,” National Center for Health Statistics, CDC, 2006.

2. CDC: http://www.cdc.gov/nchs/fastats/asthma.htm - accessed on Sep 2, 2008.

3. “State of childhood asthma in the United States,” CDC,1980-2005.

4. “National Hospital Ambulatory Medical Care Survey,” CDC, 2001-2004.

5. “National Hospital Discharge Survey,” National Center for Health Statistics, CDC, 2001-2004.

6. “National Health Statistics Survey,” National Center for Health Statistics, CDC, 2006.

7. “National Vital Statistics Reports,” Vol 56, Number 10, CDC, 2005.

8. “The Costs of Asthma,” Asthma and Allergy Foundation 1992 and 1998 Study, 2000 Update.

9. “Asthma Prevalence, Health Care Use and Mortality,” CDC, 2003-2005.

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2. Exhaled Nitric Oxide (eNO)

Current Methods for Assessing Airway InflammationAirway inflammation is shown to be an appropriate target for improving asthma control10, and while it is recognized as playing a key role in the pathophysiology of asthma, current methods to evaluate a patient’s asthma status fall short because they do not directly measure the degree of airway inflammation. Physicians attempt to monitor asthma severity through clinical exam and pulmonary function testing using spirometry or peak flow meters. Unfortunately, these lung function tests do not directly measure airway inflammation. Other markers of inflammation such as bronchoalveolar lavage and induced sputum are invasive and impractical to perform in a physician’s office. Therefore, when prescribing and titrating medication, physicians have had to rely on qualitative measures such as correlation of disease severity and symptoms. Adding to this challenge is the fact that patients with severe asthma may have a compromised perception of airflow obstruction and dyspnea compared to normal individuals11 and, thus, underestimate the severity of their symptoms.

Without a convenient means to accurately and regularly assess inflammation, it is difficult for physicians to manage asthma. Inhaled corticosteroids (ICS) are the mainstay of treatment for chronic asthma, and dosing should be adequate to control asthma symptoms but also be as low as possible to avoid side effects. Under-medication and over-medication are sub-optimal both clinically and economically. If not managed properly, people with asthma could experience permanent and irreversible damage to their airways. Since the dose of medication required is highly variable, both among patients and within individual patients, physicians need an easy, effective and safe method to assist them with titrating medication precisely.

The lack of knowing a patient’s degree of airway inflammation creates a significant gap in asthma management today. With direct markers of inflammation, this gap can be closed to improve the overall treatment and management of asthma. Insight into a patient’s airway inflammation could enable physicians to make earlier interventions with appropriate levels of therapy, thereby preventing emergency room visits and hospitalizations.

Exhaled Nitric Oxide (eNO) as a Biomarker of Airway InflammationNitric oxide (NO), a free radical, is produced by the body as part of the inflammatory response and can be detected as exhaled nitric oxide in expired human breath. Nitric oxide is produced in the airway epithelial cells by a family of NO synthases called inducible nitric oxide synthase. Inducible NO synthase expression is sensitive to steroids and therefore shows a change in response to treatment with anti-inflammatory medication like inhaled steroids. Nitric oxide is a mediator of inflammation and it is an

10. Green RH, Brightling CE, McKenna S, Hargadon B, Parker D, Bradding P, Wardlaw AJ, Pavord ID. Asthma exacerbations and sputum eosinophil counts: A randomised controlled trial. Lancet 2002;360:1715-1721.

11. Kikuchi Y, Okabe S, Tamura G, Hida W, Homma M, Shirato K, Takishima T. Chemosensitivity and perception of dyspnea in patients with a history of near-fatal asthma. N Engl J Med. 1994;330(19):1329–1334.

effective local vasodilator. It causes smooth muscle relaxation thereby matching regional airflow and blood flow. Nitric oxide is also important for ciliary action. Numerous studies over the past decade have validated that exhaled nitric oxide (eNO) can serve as a biomarker of airway inflammation in asthma.

In 1991, NO in exhaled breath was first reported.12 Studies have shown that eNO is correlated with other markers of inflammation. For example, exhaled nitric oxide correlates with eosonophilic airway inflammation measured in induced sputum13 and in bronchoscopy (lavage and biopsy), most commonly seen in asthma but also in any condition where eosonophils are present, such as allergic rhinitis, eosonophilic bronchitis14 and chronic obstructive pulmonary disease (COPD).15

It has also been shown that eNO levels are elevated in steroid-naive asthma,16 and eNO levels fall rapidly with anti-inflammatory medication (e.g. inhaled corticosteroids)17, oral steroids, anti-leukotrienes (e.g. montelukast)18 and anti-IgE (e.g. Xolair).19 Exhaled nitric oxide also correlates with non-specific bronchial reactivity to methacholine (typically assessed in methacholine challenge) in steroid-naïve patients. Increased reactivity is reflected by higher eNO levels and is an indirect marker of airway inflammation.

It is now well established that the concentration of nitric oxide in exhaled breath (eNO) is a reliable indicator of the degree of inflammation in the airways. Exhaled nitric oxide levels increase as inflammation increases and decrease as inflammation decreases.

Clinical Recommendations for eNOClinical recommendations exist for use of eNO measurements in asthma management, and such recommendations are currently being reviewed and updated:

The American Thoracic Society and European Respiratory •Society (ATS/ERS) have published recommendations for the standardized measurement of eNO.20

12. Gustafsson LE, Leone AM, Persson MG, Wiklund NP, Moncada S. Endogenous nitric oxide is present in the exhaled air of rabbits, guinea pigs and humans. Biochem Biophys Res Commun. 1991;181(2):852-857.

13. Jatakanon A, Lim S, Kharitonov SA, Chung KF, Barnes PJ. Correlation between exhaled nitric oxide, sputum eosinophils, and methacholine responsiveness in patients with mild asthma. Thorax 1998;53:91-95.

14. Berlyne GS, Parameswaran K, Kamada D, Efthimiadis A, Hargreave FE. A comparison of exhaled nitric oxide and induced sputum as markers of airway inflammation. J Allergy Clin Immunol 2000;106:638-644.

15. Fabbri LM, Romagnoli M, Corbetta L, et al. Differences in airway inflammation in patients with fixed airflow obstruction due to asthma or chronic obstructive pulmonary disease. Am J Respir Crit Care Med 2003;167:418-424.

16. Alving K, Weitzberg E, and Lundberg JM. Increased amount of nitric oxide in exhaled air of asthmatics. Eur Respir J. 1993;6(9):1368-1370.

17. Yates DH, Kharitonov SA, Robbins RA, Thomas PS, Barnes PJ. Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide. Am J Respir Crit Care Med. 1995;152(3):892-896.

18. Bratton DL, Lanz MJ, Miyazawa N, White CW, Silkoff PE. Exhaled nitric oxide before and after montelukast sodium therapy in school-age children with chronic asthma: A preliminary study. Pediatr Pulmonol 1999;28:402-407.

19. Silkoff PE, Romero FA, Gupta N, Townley RG, Milgrom H. Exhaled nitric oxide in children with asthma receiving xolair (omalizumab), a monoclonal anti-immunoglobulin e antibody. Pediatrics 2004;113:e308-312.

20 Recommendations for standardization procedures for the online and offline measurement of exhaled lower respiratory nitric oxide and nasal nitric oxide in adults and in children. Am J Respir Crit Care Med. 2005;171:913-930.

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Separate recommendations have also been developed for children.

Clinical Utility of eNOExhaled nitric oxide measurement offers new benefits for asthma management.

Predicting steroid response•

An elevated eNO level is highly predictive of a positive response to ICS.21 Smith et al showed that eNO levels are very good indicators of response to steroid in people with undiagnosed respiratory symptoms. Exhaled nitric oxide was shown to be more accurate than spirometry, peak flow meters, bronchodilator response and airway hyper-responsiveness (AHR). This study followed 52 subjects in a single-blind, fixed sequence, placebo-controlled trial of inhaled fluticasone over 4 weeks. Similar results were found in another study 22 (Figure 1.1 below) involving 73 steroid-naïve subjects with uncontrolled asthma which showed about 50% reduction in eNO levels following ICS therapy over 2 weeks. The study also showed that eNO was a significantly better predictor of response to steroid therapy than conventional lung function tests. Additionally, a 35% improvement in asthma symptom scores were seen following the 2-week therapy.

eNO is a more reliable predictor of steroid response than lung function

4-var TAS eNO Rev FEV

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. TAS = Total Asthma Score

eNO = Exhaled Nitric Oxide

FEV1 = Forced Expiratory Volume in 1 Second

Rev = FEV1 Reversibility

4-Var = Regression Model Using All 4 Variables

Figure 1.1

Improving titration of medication•

Smith et al showed that using eNO to guide ICS dose in addition to clinical management compared with clinical management alone significantly reduced the dose of inhaled steroid while showing a numerical reduction in exacerbations.23 In a single-blind, placebo-controlled trial, the researchers randomly assigned 97 patients with asthma who had been regularly receiving treatment with inhaled corticosteroids, to have their corticosteroid dose adjusted, in a stepwise fashion, on the basis of either eNO measurements or an algorithm based on conventional guidelines. After the optimal dose was determined (phase 1), patients were followed up for 12 months (phase 2). They found that a 40% reduction in the dose of ICS can be achieved without loss of asthma control.

21 Smith AD, Cowan JO, Brassett KP, et al. Exhaled nitric oxide: a predictor of steroid response. Am J Respir Crit Care Med. 2005;172(4):453-459.

22 Dreon DM, Berger WE, Hutchins EE, and Parikh BR. Exhaled Nitric Oxide Predicts Use of Controller Medication. J Allergy Clin Immunol 2008;121(2):S157 Scientific poster at AAAAI 2008 (NOT PUBLISHED).

23 Smith AD, Cowan JO, Brassett KP, Herbison GP, Taylor DR. Use of exhaled nitric oxide measurements to guide treatment in chronic asthma. N Engl J Med. 2005.;352(21):2163-2173.

Monitoring patient compliance•

An elevated eNO level is suggestive of an inadequate anti-inflammatory regimen, which may be due to poor compliance or under-medication.24 A study by Beck-Ripp and his colleagues showed that there was a strong correlation between reduction in eNO values and patient compliance (measured as percentage of prescribed medication taken). In this study, the researchers followed 54 patients between the ages of 6 and 16 over 16 weeks. The results showed that there was a 50% reduction in eNO values with higher compliance.

Predicting exacerbations•

An elevated eNO level at a clinic visit has been shown to be associated with an increased risk for an exacerbation in the following two weeks.25 In this study, moderate and severe-persistent asthma patients were evaluated during a routine clinic visit and then noted whether they had an exacerbation within 2 weeks of the initial appointment. Those with an exacerbation had a higher mean eNO (29.67 ppb ± 14.48) compared to those who did not (12.92 ppb ± 5.17), p = 0.002. A nominal logistic regression model to determine those variables that predict asthma exacerbation found that eNO was the only significant predictor, p = 0.03.

In another study26 with 31 subjects (19 adults and 12 children) using the Insight system, it was shown that by regularly monitoring the diurnal changes in eNO levels, it is possible to predict asthma exacerbations up to 1 week in advance. In this study, spirometry measurements showed no indication of an impending exacerbation. During this study, subjects measured and recorded eNO levels and peak flow values twice daily (morning and evening). Once a week they also performed a spirometry measurement at the study site. The subjects also maintained a diary of asthma symptoms. The analysis showed that a change in the week-to-week pattern of diurnal change in eNO values predicted an exacerbation. It was also clear that morning eNO values are better predictors than evening eNO values. See Figure 2.1.

Evening eNO

Morning eNO

Overnight Change

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Morning PEF

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Figure 2.1

24 Beck-Ripp J, Griese M, Arenz S, Koring C, Pasqualoni B, Bufler P. Changes of exhaled nitric oxide during steroid treatment of childhood asthma. Eur Respir J 2002;19(6):1015-1019.

25 Harkins MS, Fiato KL, Iwamoto GK. Exhaled nitric oxide predicts asthma exacerbation. J Asthma. 2004;41(4):471-476.

26 Wolfe JD, Dreon DM, Hutchins EE, and Parikh BR. Relationship of Exhaled Nitric Oxide and Peak Expiratory Flow to Loss of Asthma Control. J Allergy Clin Immunol 2008;121(2):S159 Scientific poster at AAAAI 2008 (NOT PUBLISHED).

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Performing eNO Testing is Simple and StraightforwardExhaled nitric oxide testing is an accurate and reliable measure of airway inflammation that is:

Non-invasive•

Patient-friendly•

Fast and suitable for routine clinical analysis•

The eNO test is simple to administer compared to some of the conventional lung function testing methods or breath analyses. The patient has to be seated and is required to exhale steadily for 11 seconds after taking in a deep breath. The device measures the eNO level and displays a number on the screen. The interpretation is based on this single numerical value rather than on a complex tracing or profile. Historical values of eNO can be added to gain better understanding of the patient’s disease progression.

Important aspects of any system that measures exhaled nitric oxide include:

Detection of trace amounts of the gas in parts per billion (ppb)•

Constant expiratory flow rate due to flow dependence of •exhaled nitric oxide

Exclusion of nasal nitric oxide•

3. Insight™ eNO System

Indications for UseThe intended use of the Apieron Insight™ eNO System is to quantitatively measure exhaled nitric oxide (eNO) in expired human breath as a marker of inflammation in persons with asthma. Measurement of eNO in expired human breath by the Apieron Insight eNO System is a non-invasive, simple and safe method to measure a decrease in eNO in asthma patients that often occurs after treatment with anti-inflammatory pharmacological therapy as an indication of the therapeutic effects in patients with elevated eNO levels. The Apieron Insight eNO System is suitable for use in children 8 to 17 years of age, and in adults 18 years of age and older. As an adjunct to established clinical assessments, such as spirometry and physical examination, eNO measurements give the physician an objective marker to evaluate the patient’s response to anti-inflammatory therapy. The Apieron Insight eNO System can be used by trained operators in a physician’s office or laboratory setting. The Apieron Insight eNO System should not be used in critical care, emergency care or in anesthesiology.

Prescription device: Federal law restricts this device to sale by or on the order of a physician.

A Convenient Way to Measure eNO LevelsThe Insight eNO System was developed to provide a practical and accurate means for measuring eNO levels via a simple breath test. Designed for routine use in physician offices and clinics, the Insight system enables physicians to monitor and manage asthma on a regular basis. The system includes a small desktop monitor with a large color display. It employs an eNO sensor to measure nitric oxide from the breath sample. The patient breathes out

steadily through a disposable breath tube, and the results appear on the display within one minute. Patient data can be stored and maintained in patient cards for trend analysis or downloaded to a printer for patients’ files.

With an accurate measure of their patients’ airway inflammation and customized reports for each patient, physicians can significantly improve the precision of prescribed therapy.

Figure 3.1. Insight Monitor

Insight Sensor TechnologyApieron is the first company to successfully implement a technique utilizing sol-gel technology to make a commercial biosensor for the detection of exhaled nitric oxide. The Insight sensor is a disposable, plastic cartridge that contains a path for exhaled breath to flow through and a sol-gel based biosensor for detecting nitric oxide. The gas flow path contains desiccant to control the environment and condition the gas inside the sensor.

The biosensor consists of a sol-gel matrix (a glass-based three-dimensional matrix) with a porous structure that envelopes protein molecules which are sensitive to nitric oxide. The biosensor matrix changes its light absorption characteristics when exposed to nitric oxide (NO). This optical change can be measured and correlated to the concentration of nitric oxide.

Figure 3.2. Insight Sensor

How Is eNO Measured?The biosensor encapsulates a heme protein that has a high affinity for nitric oxide. The heme protein has a metal active site that is specific to nitric oxide (Figure 3.3). As exhaled nitric

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oxide molecules from the breath sample flow into the sensor, they diffuse into the porous matrix and react with the protein molecules. This results in a change in the optical transmission properties of the matrix in a consistent and measurable way when light of a certain wavelength (412 nm) is passed through the matrix (see Figure 3.4).

The optical signal generated is proportional to the concentration of nitric oxide and enables the Insight system to accurately detect trace amounts of nitric oxide molecules in a single breath sample. Nitric oxide measurement results are reported in parts per billion (ppb).

Optically transparent porous glass matrix

Embedded heme-protein

Figure 3.3. Biosensor

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Figure 3.4. Absorption Spectra (dotted line)

Overcoming Technical ChallengesAlthough the heme protein is highly selective for NO, certain factors will affect the optical absorbance properties of the biosensor. Detrimental factors include exposure to CO2 from the breath sample, changes in humidity and changes in temperature. The Insight system incorporates specific features to counteract these factors:

A carbon dioxide filter to remove CO2 from the breath sample.•

A preconditioned desiccant to control the concentration of •water vapor surrounding the biosensor and to condition the incoming breath sample.

Temperature controllers to control the temperature of the •sensor and gas and to prevent temperature-induced shifts in optical absorbance.

Insight Monitor TechnologyThe Insight monitor consists of three subsystems controlled by a central microprocessor: an eNO measurement system, a pneumatic system and an user interface.

Measurement system—contains the hardware required to •stabilize the Insight sensor to its operating temperature and measure the optical absorbance of the biosensor material when exposed to the breath sample.

Pneumatic system—controls the exhalation flow rate as outlined •in the American Thoracic Society 2005 recommendations.

User-friendly interface—guides the operator through the test •sequence and guides the patient through the breath sampling maneuver.

Accuracy of the SystemA study was conducted on 82 non-randomized asthmatic subjects at a single site comparing eNO measurements taken with both the Insight eNO System and the Aerocrine NIOX® System. The demographic data for this study is presented below. A total of 58 adults (ages ≥18 years) and 20 children (ages <18 years) completed the study resulting in 78 subjects eligible for analysis. Each subject performed two breath maneuvers on each system, and the results ranged from 10 ppb to 197 ppb on the Insight eNO System. The testing was performed by three trained technicians. Results were analyzed to evaluate the performance of the Insight eNO System as it compares to the NIOX System in a clinical setting.

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Figure 4.1. Correlation to reference

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Parameter Analysis Results [95% CI]

Agreement

Ordinary Least Squares Regression R2 = 0.95

Slope 0.95 [0.90 to 1.00]

Intercept 1.3 ppb [-1.9 to 4.5]

Bias Mean Difference -1.14 ppb [-3.08 to 0.80]

Precision Mean Absolute Difference 5.86 ppb

Insight Repeatability Within-Subject Standard Deviation 3.96 ppb (2.95 to 4.98)

Clinical Agreement @ 30 ppb

Positive Percent Agreement 100%

Negative Percent Agreement 93%

Table 4.1. Performance of the Insight eNO System

Agreement: Regression analysis performed on the first measurement from each system demonstrated agreement, with an R2 of 0.95, (y=0.95x+1.3 ppb) between the Insight eNO System and the NIOX System.

Bias: Mean difference between the Insight eNO System and the NIOX System was -1.14 ppb, demonstrating that there is no consistent bias between the two systems in clinical use.

Precision: The mean absolute difference between the Insight eNO System and the NIOX System was 5.86 ppb.

Repeatability: The first and second measurements taken on the Insight eNO System were compared, and the average standard deviation between measurements was 3.96 ppb for the Insight eNO System.

Clinical Agreement: Using a clinically relevant decision point (30 ppb), an analysis was performed that compared the results of measurements taken on the Insight eNO System as they compared to the NIOX System results from the same subject. The 51 subjects identified as elevated by the NIOX System were also identified as elevated by the Insight eNO System (100% positive agreement). Twenty-seven subjects were identified as normal by the NIOX System and 25 of these were identified as normal by the Insight eNO System (93% negative agreement). This demonstrates that both the NIOX System and the Insight eNO System are highly consistent in detecting subjects with both normal and elevated eNO measurements.

InterferenceTwo sets of endogenous interference experiments were conducted.

In the first set of experiments, interference testing was performed on the Insight eNO System for the following common constituents of exhaled breath:

200 ppm H• 2 (hydrogen)

3 ppm CO (carbon monoxide)•

100% Relative Humidity (water vapor)•

0.5% CO• 2 (carbon dioxide)

0 ppb NO (nitric oxide)•

Twenty-four samples containing all of the above potential interferents (balance air) and 24 samples containing only air were prepared and tested on the Insight eNO System. The mean difference in response was less than 0.05 ppb and was not statistically significant between the samples containing potential interferents and those containing only air.

In the second set of experiments seven additional compounds (isoprene, ethanol, acetone, ammonia, acetaldehyde, methanethiol and methane) were tested at physiologically relevant concentrations by comparing the response of the Insight eNO System to a test gas. The test gas contained 0 ppb NO (nitric oxide). A compound was deemed non-interfering if, at the tested concentration, the response was within ±5 ppb NO equivalent when compared to the nitrogen control.

None of the tested compounds exhibited interference greater than ±5 ppb equivalent NO when compared to a nitrogen control.

Clinical interference testing (exogenous compounds):

The influence of mouthwash containing alcohol, alcohol-free mouthwash, toothpaste, breath mints, throat lozenges, carbonated beverage with caffeine and caffeine-free carbonated beverage on eNO was assessed in a clinical study. Twelve healthy adult subjects (9 males and 3 females; mean age 36 ± 11.8 yrs; range 20-62 yrs) participated in the clinical study. The eNO measurements for adults ranged from 5 ppb to 99 ppb. The endpoint was the difference in eNO before as compared to one hour after exposure to each compound. None of the tested exogenous compounds were found to interfere (at ± 5 ppb) with the eNO measurement at one hour post-exposure.

In pediatric subjects, the influence of alcohol-free mouthwash, toothpaste, breath mints, and caffeine-free carbonated beverage on eNO was assessed in a clinical study. Seven healthy children (6 males and 1 female; ages 5-17 yrs) participated in the clinical study. The eNO measurements for children ranged from 3 ppb to 27 ppb. The endpoint was the difference in eNO before as compared to one hour after exposure to each compound. None of the exogenous compounds tested were found to interfere (at ± 5 ppb) with the eNO measurement at one hour post-exposure.

These studies show that the Insight eNO System can accurately and reliably measure exhaled nitric oxide

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Maya is 10 years old and loves to go fishing with her dad on the weekends. She has moderate persistent asthma. She is very compliant and often corrects her dad when it comes to managing her asthma. She gets on very well with her doctor, who has been seeing her for 3 years now.

Maya has been on Pulmicort® DPI 200 mcg QD. Based on her record of asthma control, her doctor wanted

to optimize Maya’s therapy. But he had no objective measure of her airway inflammation to monitor her response. Once he had access to exhaled nitric oxide (eNO) measurement with

the Insight system, he incorporated exhaled nitric oxide (eNO) testing into her treatment plan. When he first measured Maya’s eNO, it was 44 ppb. He felt it was a little high for her age, but he wanted to establish her usual eNO levels over time before changing her medication. Over the next 8 weeks, he found that her eNO values settled at around 30 ppb after initially fluctuating. He increased her Pulmicort dosage to 200 BID. Over the next 8 weeks, Maya’s eNO values decreased to the low 20’s. Her doctor was very pleased with the results but the next time he measured her, she was at 40 ppb. An almost two-fold increase in her eNO bothered him but further investigation with Maya and her father revealed that she had missed taking her medication recently. Her eNO values dropped back down to around 20 ppb once she resumed her treatment. By continuing to monitor Maya’s eNO levels regularly, her doctor was confident that he could fine-tune her medication.

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Benefits of eNO:

Treatment gap revealed by eNO measurement with the Insight™ eNO System

Objective validation for a sound clinical decision•

Insight into airway inflammation not manifesting as symptoms•

Timely identification of suboptimal compliance to medication and therapy•

Patient benefits from the new treatment paradigm

Better titrated dosage enables patient to maintain control •

Maya’s history and medical information

Age:10-year-old female •

Classification:Moderate persistent•

Medication:Pulmicort•

Dosage:200 mcg QD•

FEV1:80% •

eNO:44 ppb•

Other information:•

- Non-allergic rhinitis

- Non-allergic conjunctivitis

- Cold sores

- Occasional headaches

“Let’s go fishing dad!” – Maya, 10, Pensacola FL

4. Case Studies

Case Study 1

Titrate medication and monitor compliance with precise information

10

80

70

60

50

40

30

20

10

0

eNO

in p

pb

Prednisone

Improved compliance

Advair 500 BID

Advair 500 BID to

Flovent 220/2inh BID

Time (months)1 2 3 4 5 6 7

Prednisone & Augmentin

Scott is a talented young racer who loves to drive his all-wheel drive car on wet and slippery dirt roads. He competes in many local and regional events. He has a keen eye for those fast corners and usually leaves the rest of the racers in the dust.

Scott spends a lot of time outdoors and has complained about worsening asthma symptoms with increased activities. His doctor, Jim

Brewster, knows him too well to ask him to reduce his need for speed. Dr. Brewster decided to assess Scott’s airway inflammation via exhaled nitric oxide (eNO) values as an aid to titrating his controller medications. Scott was on Advair® 500 BID. He suspected that Scott was not compliant and Dr. Brewster knew that Scott had some asthma attacks in the past that landed him in emergency care.

Scott’s eNO was 32 ppb at his first visit, but Dr. Brewster planned to measure his eNO values over several visits and look for patterns before making any treatment changes. Consistent with Scott’s irregular compliance, his eNO values varied widely. However, these variations did not coincide with Scott’s symptoms. Most of the time, Scott’s symptoms were normal and he felt fine, but his eNO levels were high. Dr. Brewster then shared the eNO values with Scott and explained the significance, hoping to convince Scott to take his medications seriously and regularly. Scott liked having a number that showed him the status of the inflammation of his airways, and started to improve his compliance which resulted in a reduction and stability of his eNO levels. Subsequently Dr. Brewster changed Scott’s medication from Advair 500 BID (a combination of the 500 mcg of the inhaled steroid fluticasone and salmeterol, a long acting bronchodilator) to fluticasone 220 BID alone. Scott’s eNO values are now in the low 20’s and do not fluctuate like before. Scott has not had any exacerbations since he started monitoring his eNO values, and Dr. Brewster has been able to reduce the daily dose of inhaled steroids by 50% and stop salmeterol.

Benefits of eNO testing

Treatment gap revealed by eNO testing

Simple and compelling evidence to highlight to the patient the need to follow treatment plan and medication•

Confirm suboptimal compliance with medication and treatment plan•

Provide objective validation for clinical treatment decisions•

Patient benefits from the new treatment paradigm

More confidence from a precisely tailored therapy that fits lifestyle requirements•

Greater awareness and motivation to improve compliance•

Scott’s history and medical information

Age:23-year-old male •

Classification:Severe persistent •

Medication:Advair•

Dosage:500 BID•

FEV1:117% •

eNO:32 ppb•

Other information•

- Allergic rhinitis

- Severe asthma attacks

- Hospitalized /intubated in last 18 months

- Occasional headaches

Case Study 2

“I like to win.” – Scott, 23, Fresno CA

Improve patient compliance and confidence

11

QVAR 80 2inh BID

Moved to anew house

Decreased to QVAR 80 1 inh BID

140

120

100

80

60

40

20

0

eNO

in p

pb

Time (months)

1 2 3 4 5 6 7 8

Lynn works in emergency care at the city hospital as a nurse and loves her job. She lives with her husband and two children. Her life is demanding to say the least. She has been living with asthma for almost 14 years now and feels that she can’t quite get it under control. Her health has been a big concern for her whole family. She

has persistent asthma with symptoms most of the time. She has missed many days at work due to her asthma.

While on Advair® 250/50 BID and QVAR® 80mcg BID (i.e. 2 inhaled steroids), her doctor decided to monitor Lynn’s inflammation using eNO testing. Her eNO levels were very high (80-128 ppb where normal is about 20 ppb) and Lynn continued to have almost daily symptoms. Her doctor doubled her QVAR dosage to 160 BID. Over time, this brought her eNO down to the 45-50 ppb range but these levels were still elevated. Around that time, Lynn moved to a new house in Long Island (a surprise family inheritance) and after the move, there was a further drop in eNO to around 20 ppb. Lynn also reported that her symptoms were getting much better. Lynn and her doctor figured that her older house, which had a moldy basement, might have triggered her symptoms. Her doctor then reduced her dosage of QVAR back to just 1 inhalation. He felt confident that he could further drop or even eliminate her dosage of QVAR and significantly reduce her medication by following her eNO levels regularly. Lynn continues to work in ER and is happy to be able to focus more on her work and family.

Benefits of eNO testing

Treatment gap revealed by eNO testing

Despite 2 controller medications, eNO indicated that airway inflammation was uncontrolled•

Exhaled nitric oxide (eNO) showed that airway inflammation was decreasing after a change in environment. •

Exhaled nitric oxide (eNO) allowed the tapering of medication in the better environment. •

Patient benefits from the new treatment paradigm

Titration of therapy to optimize asthma control•

Improved quality of life•

Lynn’s history and medical information

Age: 36-year-old female•

Classification:Severe persistent•

Medication: Advair, QVAR•

Dosage:Advair 250/50 BID and QVAR 80 BID •

FEV1:51%•

eNO: 85 ppb•

Other information:•

- Asthma x 14 years

- Regularly symptomatic

- Allergic Rhinitis

- Ragweed, mold, dander

- Intermittent psoriasis

Case Study 3

“It feels good to be in control.” - Lynn, 36, Long Island NY

Track disease progression and see impact of environment

12

© 2008 Apieron, Inc. Insight™ is a trademark of Apieron, Inc.

All other trademarks are properties of their respective owners. ML00031.A

www.apieron.com

5. Exhaled Nitric Oxide Cost ModelThis cost model provides an insurance payer with the reimbursement costs of eNO patient testing with the Insight system and the expected savings in medical resources resulting from improved asthma control.The cost model:

Estimates the number of patients that are likely to undergo •eNO testing over a three year period.

Estimates total eNO testing reimbursement costs to the •insurance payer.

Estimates the number of asthma exacerbation events likely to •be avoided by patients monitored with eNO.

Estimates cost savings in medical resources (hospitalization, •ER visits, office visits, drug use) due to avoidance of asthma exacerbation events.

Estimates the net cost to the insurance payer of eNO testing •reimbursement minus medical resource savings.

Please contact us at care@apieron.com if you would like to see how the model can be applied to your plan.

6. Coding Information for Exhaled Nitric Oxide (eNO)-CPT 95012In 2007, the CPT editorial committee added a specific code to the CPT Coding Book for Nitric Oxide Expired Gas Determination. With this action, the procedure should be recognized as a standard service provided to patients. The committee felt it appropriate to add this code to the Category I codes instead of placing it in the Category III codes as a temporary or new services code. The Category I codes are considered consistent with contemporary medical practice and are performed by providers across the country. Determination of exhaled nitric oxide (eNO) is considered a service which is consistent with contemporary medical practice for the evaluation of patients with respiratory complaints.

Centers for Medical Services (Medicare) also determined that the service should be recognized as a payable service for all Medicare beneficiaries. In doing this, the RUC committee (Relative Value Update Committee) assigned an RVU value of 0.50. This value does not have a physician work component RVU, only an overhead and malpractice RVU. RVU or relative value unit determines what the reimbursement will be for Medicare as well as many other carriers. Medicare has a conversion factor which may be higher or lower than the third party payers to determine the final reimbursement for the code.

The code assigned for the Nitric Oxide Expired Gas Determination is 95012©. According to the CPT subsection heading for 2008, the interpretation and report for the test is included in the testing code. If a “significant and separately identifiable” evaluation and management service is provided in addition to the testing, then an evaluation and management code may be coded in addition to the testing code. The ‘25 modifier’ would need to be added to the evaluation and management code (E/M) in order for both the test, and the evaluation and management code to be appropriately reimbursed.

Managing asthma is a breath away™