RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 "Safety of an alkalinizing buffer designed for inhaled medications in humans" Davis, Michael D1 Walsh, Brian K3 Dwyer, Scott T2 Combs, Casey2 Vehse, Nico2 Paget-Brown, Alix4 Pajewski, Thomas5 Hunt, John F 2^, 1Adult Health and Nursing System Virginia Commonwealth University Richmond, VA 23298-0567 Fax/Phone 804-828-3237 2Box 801396 Division of Pediatric Respiratory Medicine University of Virginia Charlottesville, VA 22908 Fax 434-243-5392 Phone 434-243-9377 3Children’s Medical Center of Dallas 1935 Medical District Drive Dallas, TX 75235 4Division of Neonatology University of Virginia Charlottesville, VA 22908 5Department of Anesthesiology University of Virginia Charlottesville, VA 22908 ^Corresponding Author [email protected] Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 Conflicts of Interest: JH is a cofounder of Respiratory Research, Inc, which manufactures exhaled breath condensate collection equipment. JH and the University of Virginia have intellectual property interest in airway pH diagnosis and therapy. Funding Sources: MD is supported by grant K99/R00 NR012016 from the National Institute of Nursing Research. This work was funded in part by The National Institute of Health and the University of Virginia Philip Morris Tobacco Research Fund. Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 "Safety of an alkalinizing buffer designed for inhaled medications in humans" Davis, Michael D1 Walsh, Brian K3 Dwyer, Scott T2 Combs, Casey2 Vehse, Nico2 Paget-Brown, Alix4 Pajewski, Thomas5 Hunt, John F 2^, 1Adult Health and Nursing System Virginia Commonwealth University Richmond, VA 23298-0567 Fax/Phone 804-828-3237 2Box 801396 Division of Pediatric Respiratory Medicine University of Virginia Charlottesville, VA 22908 Fax 434-243-5392 Phone 434-243-9377 3Children’s Medical Center of Dallas 1935 Medical District Drive Dallas, TX 75235 4Division of Neonatology University of Virginia Charlottesville, VA 22908 5Department of Anesthesiology University of Virginia Charlottesville, VA 22908 ^Corresponding Author [email protected] Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 Abstract: BACKGROUND: Airway acidification plays a role in disorders of the pulmonary tract. We hypothesized that the inhalation of alkalinized glycine buffer would measurably alkalinize the airways without compromising lung function or causing adverse events. OBJECTIVE: To evaluate the safety of an inhaled alkaline glycine buffer in both healthy subjects and in subjects with stable obstructive airway disease. METHODS: This work includes two open-label safety studies. The healthy controls were part of a Phase 1 safety study of multiple inhalations of low-dose alkaline glycine buffer; nebulized saline was used as a comparator in 8 of the healthy controls. Subsequently, a Phase 2 study in subjects with stable obstructive airway disease was completed using a single nebulized higher-dose strategy of the alkaline inhalation. We studied 20 non-smoking adults (10 healthy controls and 10 subjects with obstructive airway disease) both at baseline and after inhalation of alkaline buffer. We used spirometry and vital signs as markers of clinical safety. We used changes in exhaled nitric oxide (eNO) and exhaled breath condensate pH (EBC pH) as surrogate markers of airway pH modification. RESULTS: Alkaline glycine inhalation was tolerated by all subjects in both studies with no adverse effects on spirometric parameters or vital signs. Airway alkalinization was confirmed by a median increase in EBC pH of 0.235 pH units (IQR = 0.56-0.03, p = 0.031) in subjects after inhalation of the higher-dose alkaline buffer (2.5 ml of 100 mmol/L glycine).. CONCLUSIONS: Alkalinization of airway lining fluid (ALF) is accomplished with inhalation of alkaline glycine buffer and causes no adverse effects on pulmonary function or vital signs. Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 Introduction: The regulation of airway pH plays a role in the pathogenesis of obstructive lung diseases. Airway acidification, caused by both intrinsic and extrinsic factors, is associated with neutrophilic and eosinophilic inflammation, bronchospasm, bronchial hyperreactivity, ciliary dysfunction, epithelial dysfunction, augmented oxidative damage, abnormal fluid transport, inhibition of transport of cationic drugs such as albuterol, and alteration of cellular death pathways, including inhibition of apoptosis1. Knowledge of the role of airway pH in pulmonary health, along with the development of devices and techniques to measure it, has created interest in treatment of airway pH disturbances. Improved ability to treat or potentially reverse acidic airway pathology by means of therapeutic alteration of airway pH could have an impact in respiratory medicine. The ability to normalize airway pH via inhalation2 may allow introduction of new pulmonary therapeutics. Airway lining fluid (ALF) acidity can be qualitatively determined non-invasively via the collection of exhaled breath condensate (EBC) and the measurement of its pH3. Assays for the measurement of EBC pH have been developed for patients of all ages and sizes, including those receiving mechanical ventilation4-7. EBC pH normally lies within a mildly alkaline range of 7.5-8.27-19. EBC has a minimal buffer capacity, which allows EBC to assess the presence of volatile acids in ALF as indicated by a change in its pH20, 21. Although a normal EBC pH does not exclude airway acidity at some level, a low EBC pH value is highly specific for acidity somewhere within the airway. Using EBC methods, several studies have shown that patients with COPD13, asthma7, 9 , Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 bronchiectasis13, cystic fibrosis9, and chronic cough15 have airway acidification. We incorporated EBC into this study as a non-invasive safety measure to assess for the possibility of excessive alkalinization from our intervention. Airway pH also affects exhaled nitric oxide (eNO) levels by simple chemistry. As the pH of ALF decreases, commonly present nitrite becomes protonated to nitrous acid, which decomposes to nitric oxide 22, which is then in part exhaled. eNO analyzers may be used qualitatively to longitudinally assess the alkalinizing effects of alkaline inhalation therapy through monitoring decreases in eNO levels2, 23. The first aim of the present study was to evaluate the safety of an inhaled aerosol of alkaline glycine buffer in healthy controls; we also investigated the potential of this inhaled, aerosolized buffer to alkalinize the ALF pH in both healthy subjects and those with stable obstructive airway disease. Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 Methods: Protocol: Two open-label safety studies were approved by the University of Virginia Institutional Review Board (UVA IRB) under an Investigational New Drug Application from the United States Food and Drug Administration. Approval and initiation of the second study by the UVA IRB was dependent upon successful completion of the first study and results indicating safety of the intervention. We obtained informed consent from all subjects for both studies. The first study recruited ten healthy volunteers via direct approach from the study team. The study was performed in the General Clinical Research Center (GCRC). No reimbursement was provided. The second study recruited ten subjects with stable obstructive lung disease via referral from their Allergist and was performed in the Allergy/Asthma/Immunology Clinic. Subjects were reimbursed $100.00 for participation. An independent medical safety monitor reviewed the results for each subject daily during the studies. Both studies were executed between 1000 and 1400 hours Eastern Standard Time. For the first study (Study 1), we acquired (in the following order) baseline eNO levels, EBC samples, spirometry (FEV1, FVC, FEV1/FVC, FEF25/75), and vital signs (heart rate, respiratory rate, oxygen saturation and breath sounds) from the subjects. The order these measurements were obtained was kept consistent throughout all points of the study with the exception of EBC collection, which was only collected before the first inhalation and immediately after the third inhalation. All of the subjects were within healthy limits for spirometry (baseline >80% predicted for all observed values) and vital signs, and had not ingested anything except water for 8 hours prior to the study. Exclusion criteria included a > 5 pack-year smoking history, current pregnancy (all Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 female subjects were either abstinent, post-menopausal, or practicing adequate contraception), a history of pulmonary disease (verified through verbal medical history), or acute illness (verified by clinical history or reported by the subject) within five days of study. The Study 1 treatment preparation was an isotonic solution of a sodium chloride diluent mixed with glycine to a concentration of 17.8 mmol/L at pH of 10.5, equating to 44.5 micromoles/dose. The patients received this through a small volume nebulizer in 2.5 mL increments every twenty minutes for a total of three nebulizations and total delivered quantity of 133.5 micromoles of glycine (Figure 1). Each nebulization lasted for ten minutes, with a ten minute break in between, during which vital signs, eNO, and spirometry levels were obtained. A final set of physiologic measurements was performed twenty minutes after the completion of the study. After determining safety in healthy volunteers of serial incremental inhalations of glycine, a second study (Study 2) was conducted in stable subjects with known obstructive lung disease defined as a documented history of asthma or COPD and an FEV1 less than 75% predicted on the day of the study. Exclusion criteria included cigarette smoking in the past six months, any acute illness within five days of the study (verified by clinical history or reported by the subject), or an FEV1 < 50% predicted on the day of the study. Eight of these subjects had a current diagnosis of asthma, one had a current diagnosis of chronic bronchitis, and one had diagnoses of both asthma and chronic bronchitis. The ages of enrolled subjects ranged from 24-62 years old, with a mean of 44 years. This study protocol only varied from the Study 1 protocol by using a single 2.5mL dose of alkaline diluent with 100 mmol/L of glycine (total glycine dose of Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE. RESPIRATORY CARE Paper in Press. Published on December 11, 2012 as DOI: 10.4187/respcare.01753 250 micromoles) instead of the three lower-concentrations nebulizer treatments used in Study 1. Vital signs (heart rate, respiratory rate, oxygen saturation and breath sounds) were obtained prior, during, and after the nebulization. eNO levels, EBC samples, and spirometry were obtained before and after the nebulization. All measurements were obtained in the same order as in Study 1. Instrumentation: eNO levels were measured at an expiratory flow rate of 50ml/s using the Niox Mino (Aerocrine AB, Solna, Sweden). Spirometry was measured in triplicate at each collection point using the SDI SBG spirometer (Queset Medical, Brockton, MA). EBC was collected orally during tidal breathing for 7 minutes at initial temperature of -20 Celsius using the RTube (Respiratory Research, Inc, Austin, TX) without wearing nose clips. All samples underwent gas-standardization with research- grade oxygen for ten minutes at 300 ml/min prior to pH measurement. EBC pH was measured using the Orion 8220BNWP PerpHecT® ROSS® Combination pH Micro Electrode (Thermo Scientific, Waltham, Ma). The probe was calibrated in standard pH 4 and 7 buffers, and then verified in low ionic-strength buffers of the same pH, and then pH of samples was measured immediately after gas- standardization. Nebulizations were administered for ten minutes using the Hudson RCI Micromist nebulizer with a mouthpiece. The nebulizer was powered by 7 liters per minute of room air, a rate at which it averages an output mass median aerodynamic diameter (MMAD) of 2.1 microns, according to manufacturer. The nebulizer cup was Copyright (C) 2012 Daedalus Enterprises Epub ahead of print papers have been peer-reviewed and accepted for publication but are posted before being copy edited and proofread, and as a result, may differ substantially when published in final version in the online and print editions of RESPIRATORY CARE.