Boudewijn, Ilse M.

Cox, Claire A.

Frijlink, Henderik W.

Hagedoorn, Paul

Lexmond, Anne J.

Schokker, Siebrig

ten Hacken, Nick H. T.

van den Berge, Maarten

Vonk, Judith M.

Vroegop, Sebastiaan J.

American Journal of Respiratory and Critical Care Medicine

University of Groningen

   University of GroningenBronchial Provocation Testing Can Be Improved by Using Dry Powder Adenosine Instead ofNebulized Adenosine MonophosphateLexmond, Anne J.; Boudewijn, Ilse M.; Hagedoorn, Paul; Schokker, Siebrig; Cox, Claire A.;Vonk, Judith M.; ten Hacken, Nick H. T.; Frijlink, Henderik W.; Vroegop, Sebastiaan J.; vanden Berge, MaartenPublished in:American Journal of Respiratory and Critical Care MedicineDOI:10.1164/rccm.201704-0715LEIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionFinal author's version (accepted by publisher, after peer review)Publication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Lexmond, A. J., Boudewijn, I. M., Hagedoorn, P., Schokker, S., Cox, C. A., Vonk, J. M., ten Hacken, N. H.T., Frijlink, H. W., Vroegop, S. J., & van den Berge, M. (2018). Bronchial Provocation Testing Can BeImproved by Using Dry Powder Adenosine Instead of Nebulized Adenosine Monophosphate. AmericanJournal of Respiratory and Critical Care Medicine, 197(3), 391-394. https://doi.org/10.1164/rccm.201704-0715LECopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Bronchial provocation testing can be improved by using dry powder adenosine instead of nebulized AMP  Anne J. Lexmond1, Ilse M. Boudewijn2,3, Paul Hagedoorn1, Siebrig Schokker4, Claire A. Cox2,3, Judith M. Vonk3,5, Nick H.T. ten Hacken2,3, Henderik W. Frijlink1, Sebastiaan J. Vroegop4, Maarten van den Berge2,3  1University of Groningen, Department of Pharmaceutical Technology and Biopharmacy;  2University of Groningen, University Medical Center Groningen, Department of Pulmonary Diseases;  3University of Groningen, University Medical Center Groningen, Groningen Research Institute for Asthma and COPD;  4Martini Hospital Groningen, Department of Pulmonary Diseases 5University of Groningen, University Medical Center Groningen, Department of Epidemiology  Corresponding author: Dr A.J. Lexmond University of Groningen Department of Pharmaceutical Technology and Biopharmacy (XB21) 9713 AV Groningen, The Netherlands Tel: +3150-3633254; Fax: +3150-3632500; E-mail: a.j.lexmond@rug.nl  Funding: This research was supported by a research grant from TEVA Pharma. TEVA Pharma was in no way involved in study design, writing or reviewing of the manuscript. Page 1 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society To the editor:  Airway hyperresponsiveness (AHR) to adenosine has proven to be a good marker for eosinophilic airway inflammation in asthma and can be used to monitor disease activity and therapeutic effectiveness of inhaled corticosteroids (ICS) (1–3). Adenosine is usually administered by nebulization of adenosine monophosphate (AMP) but the highest feasible concentration of AMP often fails to induce sufficient bronchoconstriction in subjects with asthma (4,5). We studied whether this limitation can be resolved by administering adenosine as dry powder formulation. We have previously demonstrated the feasibility of this new bronchial provocation method in a small proof-of-concept study (6). The aim of the present study was to further validate the dry powder adenosine provocation test in a larger cohort of subjects with asthma.   Data were obtained from subjects recruited for the OLIVIA study (clinical trial number: NCT01741285, www.clinicaltrials.gov). Sixty current or ex-smokers with asthma (34 females, 26 males) with FEV1 ≥50% predicted, who did not use ICS for at least four weeks, underwent provocations with both AMP and dry powder adenosine as baseline measurements on subsequent visits (1–2 weeks apart), in addition to blood sampling, spirometry, body plethysmography, impulse oscillometry (IOS) and multiple breath nitrogen washout (MBNW) measurements. Their mean (±SD) age was 45±12 years and baseline FEV1 89±16 %predicted.   AMP was administered by nebulization of doubling concentrations (0.04–320 mg/mL). Dry powder adenosine was administered with an investigational inhaler in doubling doses (0.04–80 mg) (6,7). We determined the provocative concentration (PC20) of AMP and dose (PD20) of adenosine causing the forced expiratory volume in 1 s (FEV1) to drop with 20% by log-Page 2 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society linear interpolation and assessed which clinical characteristics were predictors of these parameters. Provocation tests were negative if no 20% drop in FEV1 was reached after administration of the highest concentration/dose and values were censored to 640 mg/mL for PC20 AMP and 160 mg for PD20 adenosine for analysis. Calculations were performed with the base-2 logarithm (log2) of PC20 AMP and PD20 adenosine to reflect the use of doubling dose steps and normalize the distribution.  We calculated the agreement between the two tests with Cohen’s kappa and correlation analysis. Correlation analysis was also performed to assess associations between subject baseline characteristics and PC20 AMP/PD20 adenosine. Associations with a p-value <0.20 were considered for multiple linear regression analysis, although per baseline measurement procedure maximally one (the most significant) predictor was included to prevent multicollinearity. Forced entry multiple linear regression analysis was performed to determine which parameters independently predict the airway responses.   Forty subjects reached the predefined 20% drop in FEV1 on both AMP and adenosine. Ten subjects obtained a positive adenosine test (PD20 5.4–39 mg) but negative AMP test (PC20 >320 mg/mL), whereas two subjects had a negative adenosine test (PD20 >80 mg) but positive AMP test (PC20 143 and 148 mg/mL). Seven subjects did not reach a 20% drop in FEV1 on either stimulus. One subject, who had a negative AMP test, experienced severe cough during inhalation of dry powder adenosine, leading to early termination of the test. The total percentage of non-responders was 30% (18 out of 60) for AMP and 15% (9 out of 59) for adenosine. Figure 1A shows PC20 AMP and PD20 adenosine values, clearly illustrating the higher responder rate to adenosine. PC20 AMP and PD20 adenosine were strongly correlated Page 3 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society (rSp = 0.799; Figure 1B), yet had only a moderate agreement (κ = 0.42), mainly due to the larger number of non-responders to AMP.   Baseline variables included in multiple linear regression analysis for PC20 AMP were age, smoking status, blood eosinophils, FEV1, residual volume (RV), and the ventilation heterogeneity of the conductive lung zone (Scond). For PD20 adenosine these were age, blood eosinophils, FEV1, and RV. The models obtained by multiple regression analysis were largely similar for PC20 AMP and PD20 adenosine with predictive powers of 34% and 30% respectively (Table 1). Only age (AMP and adenosine) and FEV1 (adenosine) were found to be independent predictors (p < 0.05). Age and FEV1 were positively associated with both PC20 AMP and PD20 adenosine, whereas blood eosinophils and RV exhibited a trend towards an inverse association.  The present work shows that bronchial provocation with dry powder adenosine is a suitable method for measuring AHR in asthmatic subjects. Moreover, the new test method allowed us to administer higher doses, resulting in fewer false negative test results, while the degree of AHR to dry powder adenosine correlated well with the degree of AHR to nebulized AMP. Despite the greater sensitivity, there were still nine subjects with a negative dry powder adenosine provocation test. Although the order of provocation testing was performed non-randomized with AMP first and dry powder adenosine second one to two weeks later and refractoriness has been shown to occur after AMP provocation (8), we consider any remaining effect one to two weeks later to be unlikely given the findings of Singh et al. (9). Some patients may have developed a component of COPD or asthma-COPD overlap, since this study examined current or former smokers. There was, however, no relationship apparent between measures of airway obstruction at baseline and PD20 adenosine (e.g. only two out of Page 4 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society nine had an FEV1/FVC ratio <70%) or with their smoking status (four current and five former smokers). Therefore, we expect that increasing the top dose, which was now arbitrarily chosen at 80 mg, could further reduce the number of false negatives and thus increase the test’s sensitivity even more. However, it cannot be ruled out that there may actually be subjects with asthma that remain unresponsive to even higher doses inhaled adenosine, which requires further investigation.   The subjects did not appear to react more severely to dry powder adenosine than anticipated from their responsiveness to AMP, indicating that the test is safe to use. Severe cough, a side effect that has been shown to hinder applicability of the mannitol provocation test (10), another indirect measure of AHR, was only reported in one subject. No other side effects were observed.  We previously reported that AHR to AMP is associated with eosinophilic inflammation (1). In the present study, blood eosinophils were included in the prediction models, although their individual contributions were not significant for either PC20 AMP or PD20 adenosine (p = 0.066 and p = 0.11 respectively). This can be explained by the fact that in the present study we investigated eosinophilic inflammation in blood rather than sputum, by the smaller study population (60 vs. 120 patients (1)) and the non-parametric distribution due to the high number of non-responders, especially to AMP. Alternatively, differences in smoking behavior of the subjects may have played a role. Smoking has been shown to blunt eosinophilic inflammation, demonstrated by lower numbers of eosinophils in sputum and blood of smokers and ex-smokers compared to never-smokers (11). Further studies in never-smokers are therefore warranted.  Page 5 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society In conclusion, we have shown that bronchial provocation with dry powder adenosine is a suitable alternative to provocation with nebulized AMP, considering the good agreement between the tests and comparable baseline predictors. Moreover, dry powder adenosine appears to offer an improvement over nebulized AMP, because of its higher sensitivity for less hyperresponsive subjects with asthma.   Acknowledgments  We thank Anne H. de Boer and Prof. Dirkje S. Postma (University of Groningen) for their significant scientific contributions to this work.  References 1. Van den Berge M, Meijer RJ, Kerstjens HAM, de Reus DM, Koeter GH, Kauffman HF, et al. PC(20) adenosine 5’-monophosphate is more closely associated with airway inflammation in asthma than PC(20) methacholine. Am J Respir Crit Care Med. 2002;165(5):1546–50.  2. Prosperini G, Rajakulasingam K, Cacciola RR, Spicuzza L, Rorke S, Holgate ST, et al. Changes in sputum counts and airway hyperresponsiveness after budesonide: monitoring anti-inflammatory response on the basis of surrogate markers of airway inflammation. J Allergy Clin Immunol. 2002;110(6):855–61.  3. Choi SH, Kim DK, Yu J, Yoo Y, Koh YY. Bronchial responsiveness to methacholine and adenosine 5’-monophosphate in young children with asthma: their relationship with blood eosinophils and serum eosinophil cationic protein. Allergy. 2007;62(10):1119–24.  4. Prieto L, Reig I, Rojas R, Ferrer A, Domenech J. The effect of challenge method on sensitivity and reactivity to adenosine 5’-monophosphate in subjects with suspected asthma. Chest. 2006;130(5):1448–53. Page 6 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society 5. Cohen J, Postma DS, Douma WR, Vonk JM, de Boer AH, ten Hacken NHT. Particle size matters: diagnostics and treatment of small airways involvement in asthma. Eur Respir J. 2011;37(3):532–40. 6. Lexmond AJ, van der Wiel E, Hagedoorn P, Bult W, Frijlink HW, ten Hacken NHT, et al. Adenosine dry powder inhalation for bronchial challenge testing, part 2: Proof of concept in asthmatic subjects. Eur J Pharm Biopharm. 2014;88:148–52. 7. Lexmond AJ, Hagedoorn P, van der Wiel E, ten Hacken NHT, Frijlink HW, de Boer AH. Adenosine dry powder inhalation for bronchial challenge testing, part 1: Inhaler and formulation development and in vitro performance testing. Eur J Pharm Biopharm. 2014;86(1):105–14. 8. Daxun Z, Rafferty P, Richards R, Summerell S, Holgate ST. Airway refractoriness to adenosine 5’-monophosphate after repeated inhalation. J Allergy Clin Immunol. 1989;83:152–8. 9. Singh D, Fairwood J, Murdoch R, Weeks A, Russell P, Roy K, et al. The reproducibility of adenosine monophosphate bronchial challenges in mild, steroid-naive asthmatics. Br J Clin Pharmacol. 2008;66(2):261–5. 10. Brannan JD, Anderson SD, Perry CP, Freed-Martens R, Lassig AR, Charlton B. The safety and efficacy of inhaled dry powder mannitol as a bronchial provocation test for airway hyperresponsiveness: a phase 3 comparison study with hypertonic (4.5%) saline. Respir Res. 2005;6:144. 11. Telenga ED, Kerstjens HAM, ten Hacken NHT, Postma DS, van den Berge M. Inflammation and corticosteroid responsiveness in ex-, current- and never-smoking asthmatics. BMC Pulm Med. 2013;13(1):58.   Page 7 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society Figure 1: (A) Comparison of the PC20 AMP and PD20 adenosine. The lines indicate the geometric means, * depicts negative test results, which were censored to PC20 AMP = 640 mg/mL and PD20 adenosine = 160 mg in the analyses. (B) Correlation analysis between PC20 AMP and PD20 adenosine, showing a strongly significant correlation between the two test results (rSp = 0.799, p < 0.001).   Table 1: Baseline predictors for PC20 AMP and PD20 adenosine obtained by multiple linear regression analysis. Dependent variable Baseline predictor B CI 95% p-value R2 log2 PC20  AMP Age (years) 0.111 0.035; 0.187 0.005 0.34 Smoking status -0.028 -1.82; 1.77 0.98 Eos blood (% total) -0.306 -0.686; 0.074 0.11 FEV1 (%pred) 0.047 -0.011; 0.104 0.11 RV (%pred) -0.018 -0.054; 0.017 0.31 Scond (/L) -1.94 -42.1; 38.2 0.92 log2 PD20 Adenosine Age (years) 0.059 0.007; 0.112 0.027 0.30 Eos blood (% total) -0.244 -0.542; 0.055 0.11 FEV1 (%pred) 0.052 0.009; 0.096 0.020 RV (%pred) -0.024 -0.050; 0.002 0.073 PC20: provocative concentration causing a 20% drop in FEV1; PD20: provocative dose causing a 20% drop in FEV1; Eos blood: blood eosinophils as percentage of total leukocytes; FEV1: forced expiratory volume in 1 s; RV: residual volume; Scond: ventilation heterogeneity of the conductive lung zone.  Page 8 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society     Figure 1: (A) Comparison of the PC20 AMP and PD20 adenosine. The lines indicate the geometric means, * depicts negative test results, which were censored to PC20 AMP = 640 mg/mL and PD20 adenosine = 160 mg in the analyses. (B) Correlation analysis between PC20 AMP and PD20 adenosine, showing a strongly significant correlation between the two test results (rSp = 0.799, p < 0.001).   164x92mm (300 x 300 DPI)    Page 9 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society 

English

Bronchial Provocation Testing Can Be Improved by Using Dry Powder Adenosine Instead of Nebulized Adenosine Monophosphate

Proceedings - University of Groningen

   University of GroningenBronchial Provocation Testing Can Be Improved by Using Dry Powder Adenosine Instead ofNebulized Adenosine MonophosphateLexmond, Anne J.; Boudewijn, Ilse M.; Hagedoorn, Paul; Schokker, Siebrig; Cox, Claire A.;Vonk, Judith M.; ten Hacken, Nick H. T.; Frijlink, Henderik W.; Vroegop, Sebastiaan J.; vanden Berge, MaartenPublished in:American Journal of Respiratory and Critical Care MedicineDOI:10.1164/rccm.201704-0715LEIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionFinal author's version (accepted by publisher, after peer review)Publication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Lexmond, A. J., Boudewijn, I. M., Hagedoorn, P., Schokker, S., Cox, C. A., Vonk, J. M., ten Hacken, N. H.T., Frijlink, H. W., Vroegop, S. J., & van den Berge, M. (2018). Bronchial Provocation Testing Can BeImproved by Using Dry Powder Adenosine Instead of Nebulized Adenosine Monophosphate. AmericanJournal of Respiratory and Critical Care Medicine, 197(3), 391-394. Advance online publication.https://doi.org/10.1164/rccm.201704-0715LECopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).The publication may also be distributed here under the terms of Article 25fa of the Dutch Copyright Act, indicated by the “Taverne” license.More information can be found on the University of Groningen website: https://www.rug.nl/library/open-access/self-archiving-pure/taverne-amendment.Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Bronchial provocation testing can be improved by using dry powder adenosine instead of nebulized AMP  Anne J. Lexmond1, Ilse M. Boudewijn2,3, Paul Hagedoorn1, Siebrig Schokker4, Claire A. Cox2,3, Judith M. Vonk3,5, Nick H.T. ten Hacken2,3, Henderik W. Frijlink1, Sebastiaan J. Vroegop4, Maarten van den Berge2,3  1University of Groningen, Department of Pharmaceutical Technology and Biopharmacy;  2University of Groningen, University Medical Center Groningen, Department of Pulmonary Diseases;  3University of Groningen, University Medical Center Groningen, Groningen Research Institute for Asthma and COPD;  4Martini Hospital Groningen, Department of Pulmonary Diseases 5University of Groningen, University Medical Center Groningen, Department of Epidemiology  Corresponding author: Dr A.J. Lexmond University of Groningen Department of Pharmaceutical Technology and Biopharmacy (XB21) 9713 AV Groningen, The Netherlands Tel: +3150-3633254; Fax: +3150-3632500; E-mail: a.j.lexmond@rug.nl  Funding: This research was supported by a research grant from TEVA Pharma. TEVA Pharma was in no way involved in study design, writing or reviewing of the manuscript. Page 1 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society To the editor:  Airway hyperresponsiveness (AHR) to adenosine has proven to be a good marker for eosinophilic airway inflammation in asthma and can be used to monitor disease activity and therapeutic effectiveness of inhaled corticosteroids (ICS) (1–3). Adenosine is usually administered by nebulization of adenosine monophosphate (AMP) but the highest feasible concentration of AMP often fails to induce sufficient bronchoconstriction in subjects with asthma (4,5). We studied whether this limitation can be resolved by administering adenosine as dry powder formulation. We have previously demonstrated the feasibility of this new bronchial provocation method in a small proof-of-concept study (6). The aim of the present study was to further validate the dry powder adenosine provocation test in a larger cohort of subjects with asthma.   Data were obtained from subjects recruited for the OLIVIA study (clinical trial number: NCT01741285, www.clinicaltrials.gov). Sixty current or ex-smokers with asthma (34 females, 26 males) with FEV1 ≥50% predicted, who did not use ICS for at least four weeks, underwent provocations with both AMP and dry powder adenosine as baseline measurements on subsequent visits (1–2 weeks apart), in addition to blood sampling, spirometry, body plethysmography, impulse oscillometry (IOS) and multiple breath nitrogen washout (MBNW) measurements. Their mean (±SD) age was 45±12 years and baseline FEV1 89±16 %predicted.   AMP was administered by nebulization of doubling concentrations (0.04–320 mg/mL). Dry powder adenosine was administered with an investigational inhaler in doubling doses (0.04–80 mg) (6,7). We determined the provocative concentration (PC20) of AMP and dose (PD20) of adenosine causing the forced expiratory volume in 1 s (FEV1) to drop with 20% by log-Page 2 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society linear interpolation and assessed which clinical characteristics were predictors of these parameters. Provocation tests were negative if no 20% drop in FEV1 was reached after administration of the highest concentration/dose and values were censored to 640 mg/mL for PC20 AMP and 160 mg for PD20 adenosine for analysis. Calculations were performed with the base-2 logarithm (log2) of PC20 AMP and PD20 adenosine to reflect the use of doubling dose steps and normalize the distribution.  We calculated the agreement between the two tests with Cohen’s kappa and correlation analysis. Correlation analysis was also performed to assess associations between subject baseline characteristics and PC20 AMP/PD20 adenosine. Associations with a p-value <0.20 were considered for multiple linear regression analysis, although per baseline measurement procedure maximally one (the most significant) predictor was included to prevent multicollinearity. Forced entry multiple linear regression analysis was performed to determine which parameters independently predict the airway responses.   Forty subjects reached the predefined 20% drop in FEV1 on both AMP and adenosine. Ten subjects obtained a positive adenosine test (PD20 5.4–39 mg) but negative AMP test (PC20 >320 mg/mL), whereas two subjects had a negative adenosine test (PD20 >80 mg) but positive AMP test (PC20 143 and 148 mg/mL). Seven subjects did not reach a 20% drop in FEV1 on either stimulus. One subject, who had a negative AMP test, experienced severe cough during inhalation of dry powder adenosine, leading to early termination of the test. The total percentage of non-responders was 30% (18 out of 60) for AMP and 15% (9 out of 59) for adenosine. Figure 1A shows PC20 AMP and PD20 adenosine values, clearly illustrating the higher responder rate to adenosine. PC20 AMP and PD20 adenosine were strongly correlated Page 3 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society (rSp = 0.799; Figure 1B), yet had only a moderate agreement (κ = 0.42), mainly due to the larger number of non-responders to AMP.   Baseline variables included in multiple linear regression analysis for PC20 AMP were age, smoking status, blood eosinophils, FEV1, residual volume (RV), and the ventilation heterogeneity of the conductive lung zone (Scond). For PD20 adenosine these were age, blood eosinophils, FEV1, and RV. The models obtained by multiple regression analysis were largely similar for PC20 AMP and PD20 adenosine with predictive powers of 34% and 30% respectively (Table 1). Only age (AMP and adenosine) and FEV1 (adenosine) were found to be independent predictors (p < 0.05). Age and FEV1 were positively associated with both PC20 AMP and PD20 adenosine, whereas blood eosinophils and RV exhibited a trend towards an inverse association.  The present work shows that bronchial provocation with dry powder adenosine is a suitable method for measuring AHR in asthmatic subjects. Moreover, the new test method allowed us to administer higher doses, resulting in fewer false negative test results, while the degree of AHR to dry powder adenosine correlated well with the degree of AHR to nebulized AMP. Despite the greater sensitivity, there were still nine subjects with a negative dry powder adenosine provocation test. Although the order of provocation testing was performed non-randomized with AMP first and dry powder adenosine second one to two weeks later and refractoriness has been shown to occur after AMP provocation (8), we consider any remaining effect one to two weeks later to be unlikely given the findings of Singh et al. (9). Some patients may have developed a component of COPD or asthma-COPD overlap, since this study examined current or former smokers. There was, however, no relationship apparent between measures of airway obstruction at baseline and PD20 adenosine (e.g. only two out of Page 4 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society nine had an FEV1/FVC ratio <70%) or with their smoking status (four current and five former smokers). Therefore, we expect that increasing the top dose, which was now arbitrarily chosen at 80 mg, could further reduce the number of false negatives and thus increase the test’s sensitivity even more. However, it cannot be ruled out that there may actually be subjects with asthma that remain unresponsive to even higher doses inhaled adenosine, which requires further investigation.   The subjects did not appear to react more severely to dry powder adenosine than anticipated from their responsiveness to AMP, indicating that the test is safe to use. Severe cough, a side effect that has been shown to hinder applicability of the mannitol provocation test (10), another indirect measure of AHR, was only reported in one subject. No other side effects were observed.  We previously reported that AHR to AMP is associated with eosinophilic inflammation (1). In the present study, blood eosinophils were included in the prediction models, although their individual contributions were not significant for either PC20 AMP or PD20 adenosine (p = 0.066 and p = 0.11 respectively). This can be explained by the fact that in the present study we investigated eosinophilic inflammation in blood rather than sputum, by the smaller study population (60 vs. 120 patients (1)) and the non-parametric distribution due to the high number of non-responders, especially to AMP. Alternatively, differences in smoking behavior of the subjects may have played a role. Smoking has been shown to blunt eosinophilic inflammation, demonstrated by lower numbers of eosinophils in sputum and blood of smokers and ex-smokers compared to never-smokers (11). Further studies in never-smokers are therefore warranted.  Page 5 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society In conclusion, we have shown that bronchial provocation with dry powder adenosine is a suitable alternative to provocation with nebulized AMP, considering the good agreement between the tests and comparable baseline predictors. Moreover, dry powder adenosine appears to offer an improvement over nebulized AMP, because of its higher sensitivity for less hyperresponsive subjects with asthma.   Acknowledgments  We thank Anne H. de Boer and Prof. Dirkje S. Postma (University of Groningen) for their significant scientific contributions to this work.  References 1. Van den Berge M, Meijer RJ, Kerstjens HAM, de Reus DM, Koeter GH, Kauffman HF, et al. PC(20) adenosine 5’-monophosphate is more closely associated with airway inflammation in asthma than PC(20) methacholine. Am J Respir Crit Care Med. 2002;165(5):1546–50.  2. Prosperini G, Rajakulasingam K, Cacciola RR, Spicuzza L, Rorke S, Holgate ST, et al. Changes in sputum counts and airway hyperresponsiveness after budesonide: monitoring anti-inflammatory response on the basis of surrogate markers of airway inflammation. J Allergy Clin Immunol. 2002;110(6):855–61.  3. Choi SH, Kim DK, Yu J, Yoo Y, Koh YY. Bronchial responsiveness to methacholine and adenosine 5’-monophosphate in young children with asthma: their relationship with blood eosinophils and serum eosinophil cationic protein. Allergy. 2007;62(10):1119–24.  4. Prieto L, Reig I, Rojas R, Ferrer A, Domenech J. The effect of challenge method on sensitivity and reactivity to adenosine 5’-monophosphate in subjects with suspected asthma. Chest. 2006;130(5):1448–53. Page 6 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society 5. Cohen J, Postma DS, Douma WR, Vonk JM, de Boer AH, ten Hacken NHT. Particle size matters: diagnostics and treatment of small airways involvement in asthma. Eur Respir J. 2011;37(3):532–40. 6. Lexmond AJ, van der Wiel E, Hagedoorn P, Bult W, Frijlink HW, ten Hacken NHT, et al. Adenosine dry powder inhalation for bronchial challenge testing, part 2: Proof of concept in asthmatic subjects. Eur J Pharm Biopharm. 2014;88:148–52. 7. Lexmond AJ, Hagedoorn P, van der Wiel E, ten Hacken NHT, Frijlink HW, de Boer AH. Adenosine dry powder inhalation for bronchial challenge testing, part 1: Inhaler and formulation development and in vitro performance testing. Eur J Pharm Biopharm. 2014;86(1):105–14. 8. Daxun Z, Rafferty P, Richards R, Summerell S, Holgate ST. Airway refractoriness to adenosine 5’-monophosphate after repeated inhalation. J Allergy Clin Immunol. 1989;83:152–8. 9. Singh D, Fairwood J, Murdoch R, Weeks A, Russell P, Roy K, et al. The reproducibility of adenosine monophosphate bronchial challenges in mild, steroid-naive asthmatics. Br J Clin Pharmacol. 2008;66(2):261–5. 10. Brannan JD, Anderson SD, Perry CP, Freed-Martens R, Lassig AR, Charlton B. The safety and efficacy of inhaled dry powder mannitol as a bronchial provocation test for airway hyperresponsiveness: a phase 3 comparison study with hypertonic (4.5%) saline. Respir Res. 2005;6:144. 11. Telenga ED, Kerstjens HAM, ten Hacken NHT, Postma DS, van den Berge M. Inflammation and corticosteroid responsiveness in ex-, current- and never-smoking asthmatics. BMC Pulm Med. 2013;13(1):58.   Page 7 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society Figure 1: (A) Comparison of the PC20 AMP and PD20 adenosine. The lines indicate the geometric means, * depicts negative test results, which were censored to PC20 AMP = 640 mg/mL and PD20 adenosine = 160 mg in the analyses. (B) Correlation analysis between PC20 AMP and PD20 adenosine, showing a strongly significant correlation between the two test results (rSp = 0.799, p < 0.001).   Table 1: Baseline predictors for PC20 AMP and PD20 adenosine obtained by multiple linear regression analysis. Dependent variable Baseline predictor B CI 95% p-value R2 log2 PC20  AMP Age (years) 0.111 0.035; 0.187 0.005 0.34 Smoking status -0.028 -1.82; 1.77 0.98 Eos blood (% total) -0.306 -0.686; 0.074 0.11 FEV1 (%pred) 0.047 -0.011; 0.104 0.11 RV (%pred) -0.018 -0.054; 0.017 0.31 Scond (/L) -1.94 -42.1; 38.2 0.92 log2 PD20 Adenosine Age (years) 0.059 0.007; 0.112 0.027 0.30 Eos blood (% total) -0.244 -0.542; 0.055 0.11 FEV1 (%pred) 0.052 0.009; 0.096 0.020 RV (%pred) -0.024 -0.050; 0.002 0.073 PC20: provocative concentration causing a 20% drop in FEV1; PD20: provocative dose causing a 20% drop in FEV1; Eos blood: blood eosinophils as percentage of total leukocytes; FEV1: forced expiratory volume in 1 s; RV: residual volume; Scond: ventilation heterogeneity of the conductive lung zone.  Page 8 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society     Figure 1: (A) Comparison of the PC20 AMP and PD20 adenosine. The lines indicate the geometric means, * depicts negative test results, which were censored to PC20 AMP = 640 mg/mL and PD20 adenosine = 160 mg in the analyses. (B) Correlation analysis between PC20 AMP and PD20 adenosine, showing a strongly significant correlation between the two test results (rSp = 0.799, p < 0.001).   164x92mm (300 x 300 DPI)    Page 9 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society 

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   University of GroningenBronchial Provocation Testing Can Be Improved by Using Dry Powder Adenosine Instead ofNebulized Adenosine MonophosphateLexmond, Anne J.; Boudewijn, Ilse M.; Hagedoorn, Paul; Schokker, Siebrig; Cox, Claire A.;Vonk, Judith M.; ten Hacken, Nick H. T.; Frijlink, Henderik W.; Vroegop, Sebastiaan J.; vanden Berge, MaartenPublished in:American Journal of Respiratory and Critical Care MedicineDOI:10.1164/rccm.201704-0715LEIMPORTANT NOTE: You are advised to consult the publisher's version (publisher's PDF) if you wish to cite fromit. Please check the document version below.Document VersionFinal author's version (accepted by publisher, after peer review)Publication date:2018Link to publication in University of Groningen/UMCG research databaseCitation for published version (APA):Lexmond, A. J., Boudewijn, I. M., Hagedoorn, P., Schokker, S., Cox, C. A., Vonk, J. M., ... van den Berge,M. (2018). Bronchial Provocation Testing Can Be Improved by Using Dry Powder Adenosine Instead ofNebulized Adenosine Monophosphate. American Journal of Respiratory and Critical Care Medicine, 197(3),391-394. https://doi.org/10.1164/rccm.201704-0715LECopyrightOther than for strictly personal use, it is not permitted to download or to forward/distribute the text or part of it without the consent of theauthor(s) and/or copyright holder(s), unless the work is under an open content license (like Creative Commons).Take-down policyIf you believe that this document breaches copyright please contact us providing details, and we will remove access to the work immediatelyand investigate your claim.Downloaded from the University of Groningen/UMCG research database (Pure): http://www.rug.nl/research/portal. For technical reasons thenumber of authors shown on this cover page is limited to 10 maximum.Download date: 12-11-2019Bronchial provocation testing can be improved by using dry powder adenosine instead of nebulized AMP  Anne J. Lexmond1, Ilse M. Boudewijn2,3, Paul Hagedoorn1, Siebrig Schokker4, Claire A. Cox2,3, Judith M. Vonk3,5, Nick H.T. ten Hacken2,3, Henderik W. Frijlink1, Sebastiaan J. Vroegop4, Maarten van den Berge2,3  1University of Groningen, Department of Pharmaceutical Technology and Biopharmacy;  2University of Groningen, University Medical Center Groningen, Department of Pulmonary Diseases;  3University of Groningen, University Medical Center Groningen, Groningen Research Institute for Asthma and COPD;  4Martini Hospital Groningen, Department of Pulmonary Diseases 5University of Groningen, University Medical Center Groningen, Department of Epidemiology  Corresponding author: Dr A.J. Lexmond University of Groningen Department of Pharmaceutical Technology and Biopharmacy (XB21) 9713 AV Groningen, The Netherlands Tel: +3150-3633254; Fax: +3150-3632500; E-mail: a.j.lexmond@rug.nl  Funding: This research was supported by a research grant from TEVA Pharma. TEVA Pharma was in no way involved in study design, writing or reviewing of the manuscript. Page 1 of 9  AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society To the editor:  Airway hyperresponsiveness (AHR) to adenosine has proven to be a good marker for eosinophilic airway inflammation in asthma and can be used to monitor disease activity and therapeutic effectiveness of inhaled corticosteroids (ICS) (1–3). Adenosine is usually administered by nebulization of adenosine monophosphate (AMP) but the highest feasible concentration of AMP often fails to induce sufficient bronchoconstriction in subjects with asthma (4,5). We studied whether this limitation can be resolved by administering adenosine as dry powder formulation. We have previously demonstrated the feasibility of this new bronchial provocation method in a small proof-of-concept study (6). The aim of the present study was to further validate the dry powder adenosine provocation test in a larger cohort of subjects with asthma.   Data were obtained from subjects recruited for the OLIVIA study (clinical trial number: NCT01741285, www.clinicaltrials.gov). Sixty current or ex-smokers with asthma (34 females, 26 males) with FEV1 ≥50% predicted, who did not use ICS for at least four weeks, underwent provocations with both AMP and dry powder adenosine as baseline measurements on subsequent visits (1–2 weeks apart), in addition to blood sampling, spirometry, body plethysmography, impulse oscillometry (IOS) and multiple breath nitrogen washout (MBNW) measurements. Their mean (±SD) age was 45±12 years and baseline FEV1 89±16 %predicted.   AMP was administered by nebulization of doubling concentrations (0.04–320 mg/mL). Dry powder adenosine was administered with an investigational inhaler in doubling doses (0.04–80 mg) (6,7). We determined the provocative concentration (PC20) of AMP and dose (PD20) of adenosine causing the forced expiratory volume in 1 s (FEV1) to drop with 20% by log-Page 2 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society linear interpolation and assessed which clinical characteristics were predictors of these parameters. Provocation tests were negative if no 20% drop in FEV1 was reached after administration of the highest concentration/dose and values were censored to 640 mg/mL for PC20 AMP and 160 mg for PD20 adenosine for analysis. Calculations were performed with the base-2 logarithm (log2) of PC20 AMP and PD20 adenosine to reflect the use of doubling dose steps and normalize the distribution.  We calculated the agreement between the two tests with Cohen’s kappa and correlation analysis. Correlation analysis was also performed to assess associations between subject baseline characteristics and PC20 AMP/PD20 adenosine. Associations with a p-value <0.20 were considered for multiple linear regression analysis, although per baseline measurement procedure maximally one (the most significant) predictor was included to prevent multicollinearity. Forced entry multiple linear regression analysis was performed to determine which parameters independently predict the airway responses.   Forty subjects reached the predefined 20% drop in FEV1 on both AMP and adenosine. Ten subjects obtained a positive adenosine test (PD20 5.4–39 mg) but negative AMP test (PC20 >320 mg/mL), whereas two subjects had a negative adenosine test (PD20 >80 mg) but positive AMP test (PC20 143 and 148 mg/mL). Seven subjects did not reach a 20% drop in FEV1 on either stimulus. One subject, who had a negative AMP test, experienced severe cough during inhalation of dry powder adenosine, leading to early termination of the test. The total percentage of non-responders was 30% (18 out of 60) for AMP and 15% (9 out of 59) for adenosine. Figure 1A shows PC20 AMP and PD20 adenosine values, clearly illustrating the higher responder rate to adenosine. PC20 AMP and PD20 adenosine were strongly correlated Page 3 of 9  AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society (rSp = 0.799; Figure 1B), yet had only a moderate agreement (κ = 0.42), mainly due to the larger number of non-responders to AMP.   Baseline variables included in multiple linear regression analysis for PC20 AMP were age, smoking status, blood eosinophils, FEV1, residual volume (RV), and the ventilation heterogeneity of the conductive lung zone (Scond). For PD20 adenosine these were age, blood eosinophils, FEV1, and RV. The models obtained by multiple regression analysis were largely similar for PC20 AMP and PD20 adenosine with predictive powers of 34% and 30% respectively (Table 1). Only age (AMP and adenosine) and FEV1 (adenosine) were found to be independent predictors (p < 0.05). Age and FEV1 were positively associated with both PC20 AMP and PD20 adenosine, whereas blood eosinophils and RV exhibited a trend towards an inverse association.  The present work shows that bronchial provocation with dry powder adenosine is a suitable method for measuring AHR in asthmatic subjects. Moreover, the new test method allowed us to administer higher doses, resulting in fewer false negative test results, while the degree of AHR to dry powder adenosine correlated well with the degree of AHR to nebulized AMP. Despite the greater sensitivity, there were still nine subjects with a negative dry powder adenosine provocation test. Although the order of provocation testing was performed non-randomized with AMP first and dry powder adenosine second one to two weeks later and refractoriness has been shown to occur after AMP provocation (8), we consider any remaining effect one to two weeks later to be unlikely given the findings of Singh et al. (9). Some patients may have developed a component of COPD or asthma-COPD overlap, since this study examined current or former smokers. There was, however, no relationship apparent between measures of airway obstruction at baseline and PD20 adenosine (e.g. only two out of Page 4 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society nine had an FEV1/FVC ratio <70%) or with their smoking status (four current and five former smokers). Therefore, we expect that increasing the top dose, which was now arbitrarily chosen at 80 mg, could further reduce the number of false negatives and thus increase the test’s sensitivity even more. However, it cannot be ruled out that there may actually be subjects with asthma that remain unresponsive to even higher doses inhaled adenosine, which requires further investigation.   The subjects did not appear to react more severely to dry powder adenosine than anticipated from their responsiveness to AMP, indicating that the test is safe to use. Severe cough, a side effect that has been shown to hinder applicability of the mannitol provocation test (10), another indirect measure of AHR, was only reported in one subject. No other side effects were observed.  We previously reported that AHR to AMP is associated with eosinophilic inflammation (1). In the present study, blood eosinophils were included in the prediction models, although their individual contributions were not significant for either PC20 AMP or PD20 adenosine (p = 0.066 and p = 0.11 respectively). This can be explained by the fact that in the present study we investigated eosinophilic inflammation in blood rather than sputum, by the smaller study population (60 vs. 120 patients (1)) and the non-parametric distribution due to the high number of non-responders, especially to AMP. Alternatively, differences in smoking behavior of the subjects may have played a role. Smoking has been shown to blunt eosinophilic inflammation, demonstrated by lower numbers of eosinophils in sputum and blood of smokers and ex-smokers compared to never-smokers (11). Further studies in never-smokers are therefore warranted.  Page 5 of 9  AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society In conclusion, we have shown that bronchial provocation with dry powder adenosine is a suitable alternative to provocation with nebulized AMP, considering the good agreement between the tests and comparable baseline predictors. Moreover, dry powder adenosine appears to offer an improvement over nebulized AMP, because of its higher sensitivity for less hyperresponsive subjects with asthma.   Acknowledgments  We thank Anne H. de Boer and Prof. Dirkje S. Postma (University of Groningen) for their significant scientific contributions to this work.  References 1. Van den Berge M, Meijer RJ, Kerstjens HAM, de Reus DM, Koeter GH, Kauffman HF, et al. PC(20) adenosine 5’-monophosphate is more closely associated with airway inflammation in asthma than PC(20) methacholine. Am J Respir Crit Care Med. 2002;165(5):1546–50.  2. Prosperini G, Rajakulasingam K, Cacciola RR, Spicuzza L, Rorke S, Holgate ST, et al. Changes in sputum counts and airway hyperresponsiveness after budesonide: monitoring anti-inflammatory response on the basis of surrogate markers of airway inflammation. J Allergy Clin Immunol. 2002;110(6):855–61.  3. Choi SH, Kim DK, Yu J, Yoo Y, Koh YY. Bronchial responsiveness to methacholine and adenosine 5’-monophosphate in young children with asthma: their relationship with blood eosinophils and serum eosinophil cationic protein. Allergy. 2007;62(10):1119–24.  4. Prieto L, Reig I, Rojas R, Ferrer A, Domenech J. The effect of challenge method on sensitivity and reactivity to adenosine 5’-monophosphate in subjects with suspected asthma. Chest. 2006;130(5):1448–53. Page 6 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society 5. Cohen J, Postma DS, Douma WR, Vonk JM, de Boer AH, ten Hacken NHT. Particle size matters: diagnostics and treatment of small airways involvement in asthma. Eur Respir J. 2011;37(3):532–40. 6. Lexmond AJ, van der Wiel E, Hagedoorn P, Bult W, Frijlink HW, ten Hacken NHT, et al. Adenosine dry powder inhalation for bronchial challenge testing, part 2: Proof of concept in asthmatic subjects. Eur J Pharm Biopharm. 2014;88:148–52. 7. Lexmond AJ, Hagedoorn P, van der Wiel E, ten Hacken NHT, Frijlink HW, de Boer AH. Adenosine dry powder inhalation for bronchial challenge testing, part 1: Inhaler and formulation development and in vitro performance testing. Eur J Pharm Biopharm. 2014;86(1):105–14. 8. Daxun Z, Rafferty P, Richards R, Summerell S, Holgate ST. Airway refractoriness to adenosine 5’-monophosphate after repeated inhalation. J Allergy Clin Immunol. 1989;83:152–8. 9. Singh D, Fairwood J, Murdoch R, Weeks A, Russell P, Roy K, et al. The reproducibility of adenosine monophosphate bronchial challenges in mild, steroid-naive asthmatics. Br J Clin Pharmacol. 2008;66(2):261–5. 10. Brannan JD, Anderson SD, Perry CP, Freed-Martens R, Lassig AR, Charlton B. The safety and efficacy of inhaled dry powder mannitol as a bronchial provocation test for airway hyperresponsiveness: a phase 3 comparison study with hypertonic (4.5%) saline. Respir Res. 2005;6:144. 11. Telenga ED, Kerstjens HAM, ten Hacken NHT, Postma DS, van den Berge M. Inflammation and corticosteroid responsiveness in ex-, current- and never-smoking asthmatics. BMC Pulm Med. 2013;13(1):58.   Page 7 of 9  AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society Figure 1: (A) Comparison of the PC20 AMP and PD20 adenosine. The lines indicate the geometric means, * depicts negative test results, which were censored to PC20 AMP = 640 mg/mL and PD20 adenosine = 160 mg in the analyses. (B) Correlation analysis between PC20 AMP and PD20 adenosine, showing a strongly significant correlation between the two test results (rSp = 0.799, p < 0.001).   Table 1: Baseline predictors for PC20 AMP and PD20 adenosine obtained by multiple linear regression analysis. Dependent variable Baseline predictor B CI 95% p-value R2 log2 PC20  AMP Age (years) 0.111 0.035; 0.187 0.005 0.34 Smoking status -0.028 -1.82; 1.77 0.98 Eos blood (% total) -0.306 -0.686; 0.074 0.11 FEV1 (%pred) 0.047 -0.011; 0.104 0.11 RV (%pred) -0.018 -0.054; 0.017 0.31 Scond (/L) -1.94 -42.1; 38.2 0.92 log2 PD20 Adenosine Age (years) 0.059 0.007; 0.112 0.027 0.30 Eos blood (% total) -0.244 -0.542; 0.055 0.11 FEV1 (%pred) 0.052 0.009; 0.096 0.020 RV (%pred) -0.024 -0.050; 0.002 0.073 PC20: provocative concentration causing a 20% drop in FEV1; PD20: provocative dose causing a 20% drop in FEV1; Eos blood: blood eosinophils as percentage of total leukocytes; FEV1: forced expiratory volume in 1 s; RV: residual volume; Scond: ventilation heterogeneity of the conductive lung zone.  Page 8 of 9 AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society     Figure 1: (A) Comparison of the PC20 AMP and PD20 adenosine. The lines indicate the geometric means, * depicts negative test results, which were censored to PC20 AMP = 640 mg/mL and PD20 adenosine = 160 mg in the analyses. (B) Correlation analysis between PC20 AMP and PD20 adenosine, showing a strongly significant correlation between the two test results (rSp = 0.799, p < 0.001).   164x92mm (300 x 300 DPI)    Page 9 of 9  AJRCCM Articles in Press. Published on 26-June-2017 as 10.1164/rccm.201704-0715LE  Copyright © 2017 by the American Thoracic Society 

Bronchial Provocation Testing Can Be Improved by Using Dry Powder Adenosine Instead of Nebulized Adenosine Monophosphate

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