Randomized Trial of Liposomal Amikacin for Inhalation in Nontuberculous Mycobacterial Lung

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1 ONLINE DATA SUPPLEMENT Randomized Trial of Liposomal Amikacin for Inhalation in Nontuberculous Mycobacterial Lung Disease Kenneth N. Olivier, David E. Griffith, Gina Eagle, John P. McGinnis II, Liza Micioni, Keith Liu, Charles L. Daley, Kevin L. Winthrop, Stephen Ruoss, Doreen J. Addrizzo-Harris, Patrick A. Flume, Daniel Dorgan, Matthias Salathe, Barbara A. Brown-Elliott, Renu Gupta, Richard J. Wallace, Jr.

2 METHODS Study Design and Treatment This phase 2, randomized, double-blind, placebo-controlled trial followed by an open-label extension study was conducted at 19 sites in North America (Figure E1). Patients were stratified based on the presence or absence of cystic fibrosis and on the predominance of Mycobacterium avium complex versus Mycobacterium abscessus at screening. Patients were randomly assigned in a 1:1 ratio to receive liposomal amikacin for inhalation (LAI) at a dose of 590 mg or placebo (empty liposomes) once daily via a customized investigational eflow technology nebulizer (PARI Pharma GmbH) added to their ongoing stable multi-drug regimen for the initial 84 days of the study. Eligible patients could consent to receive open-label treatment with daily inhalation of LAI at a dose of 590 mg with their continuing background regimen for an additional 84 days. Patients completed follow-up visits 28 days and 1 year (optional) after the last dose of study treatment. Approximately 90 patients were planned for randomization. Patient Eligibility Each site s Institutional Review Board approved the protocol and patient informed consent was obtained. Eligible adults met criteria for pulmonary nontuberculous mycobacterial disease as defined by the ATS/IDSA (1), had received ongoing ATS/IDSA guidelines-based multi-drug treatment (1) for at least 6 months prior to screening, and had persistently positive cultures for M. avium complex or M. abscessus. E2

3 Key exclusion criteria included forced expiratory volume in 1 second (FEV 1 ) of less than 30% predicted, clinically significant cardiac, pulmonary, hepatic, or renal disease, systemic immune deficiency, and malignancy. Efficacy Measurements Microbiology At least two predose expectorated or induced sputum specimens were required at study visits; the heaviest growth for a single visit was reported. Patients were stratified based on the predominance of M. avium complex or M. abscessus as determined by the quantity of the species present on screening cultures. Sputum specimens were processed centrally, with standardized decontamination, fluorochrome microscopy, broth culture (2, 3) (VersaTREK, Thermo Fisher Scientific, Inc., Waltham, MA), and solid media culture with Middlebrook 7H10 agar biplate with and without antibiotics. A positive culture was defined as growth in broth and/or agar. M. avium complex isolates were identified to the complex level using AccuProbe (Hologic-GenProbe, San Diego, CA) and subspecies within the M. abscessus group were identified by polymerase chain reaction (PCR) restriction fragment length analysis (4). Isolates were banked. A semi-quantitative scale (SQS) was used to assess relative mycobacterial growth (Table E1). A negative sputum culture was defined at a single time point, and culture conversion was defined as three consecutive negative culture results after first dose of study drug, with first negative indicating time of conversion. A microbiologic recurrence was defined as one or more positive cultures after culture conversion. Cultures positive for M. avium complex occurring after culture E3

4 conversion were molecularly compared with pretreatment isolates using partial 16S ribosomal RNA gene sequencing and variable number tandem repeat (VNTR) to determine if the isolates belonged to the same M. avium complex species and genotype (relapse) or if their sequencing or VNTR differed (new infection) (5 7). In vitro macrolide and amikacin resistance was assessed by broth microdilution susceptibility testing and by molecular identification of mutations. For amikacin, isolates were sequenced for a mutation at position 1408 in the rrs (16S rrna) gene if the amikacin minimum inhibitory concentration was 64 µg/ml or greater (8). Functional and quality-of-life assessment The 6-minute walk test distance walked at baseline was compared with distances achieved at the end of the double-blind and open-label phases as a measure of change in functional capacity (9). At each visit, quality of life was evaluated using the following patient-reported outcome instruments: Global Rating of Health in all patients, St. George s Respiratory Questionnaire and Quality of Life Bronchiectasis NTM Module in non cystic fibrosis patients, and Cystic Fibrosis Questionnaire Revised and Cystic Fibrosis Respiratory Symptoms Diary in patients with cystic fibrosis. Change from baseline in QoL-B-NTM for non-cf subjects was analyzed using both the stratified Wilcoxon rank sum (van Elteren) test, adjusting for the randomization strata and the analysis of co-variance (ANCOVA) model. The ANCOVA model includes effects for treatment, the randomization strata, and the baseline value as a covariate. Safety E4

5 At each visit, treatment-emergent adverse events were evaluated based on their severity, seriousness and relatedness to study treatment. Safety assessment included change from baseline in vital signs, physical examination, clinical laboratory tests, pulmonary function tests (PFTs), electrocardiography and audiology testing. Infective exacerbations of bronchiectasis or cystic fibrosis were defined by Fuchs criteria using the Respiratory Symptom Severity Questionnaire administered at each patient encounter (10, 11). Endpoints and Statistical Analyses Efficacy and safety analyses were performed using the modified intent-to-treat population defined as all randomized patients who received at least one 1 dose of study drug. The primary efficacy end point was change from baseline to day 84 in the semi-quantitative scale (SQS) (see Table E1) for mycobacterial growth for the LAI versus placebo arm. Change on the SQS ranged from 6 steps (improvement) to +6 steps (worsening); death on/before day 84 was considered failure and was represented by a +7 step worsening. Other reasons for missing data were handled via missing value equals failure as follows: If a post-baseline value is missing and the baseline value is non-missing, then the change value is imputed as 7 (baseline value), where the 7 represents the highest of steps 1 through 7, i.e., 4+ (e.g., if the baseline value were 3+ [step 6], then the change value would be 7 6 = 1). If the baseline value is missing and a post-baseline value is non-missing, then the change value is imputed as (post-baseline value) 1, where the 1 represents the lowest of steps 1 through 7, i.e., culture-negative (e.g., if a post-baseline value were 1+ [step 4], then the change value would be 4 1 = 3). E5

6 If both the baseline and post-baseline values are missing, then the change value is imputed as 6 (7 for post-baseline 1 for baseline). If death occurs, then the convention described above applies, imputing a change value of 7. The conventions above maximize the worst-change value to be used in the analysis. Primary efficacy analysis utilized a stratified Wilcoxon rank sum test, adjusting for randomization strata, to compare treatment arms at a two-sided significance level of Key secondary microbiologic end points included the proportion of patients in each group with sputum cultures negative for nontuberculous mycobacteria at day 84, assessed using a stratified Cochrane Mantel Haenszel test, and time to negative sputum culture for nontuberculous mycobacteria summarized using the Kaplan Meier estimate. Because treatment regimens and response rates for M. avium complex and M. abscessus infections differ considerably, culture conversion to negative was assessed separately in evaluable patients during both the double-blind and open-label periods. Change from baseline in distance walked on the 6-minute walk test was compared between treatment groups using the analysis of covariance model. Distance walked in the 6-minute walk test was also compared between patients who achieved culture conversion by end of study and those who did not. The primary analysis for proportion of patients with negative cultures and the 6MWT as defined in the SAP utilized missing values excluded. In an attempt to account for all randomized patients, the manuscript presents data on these endpoints using LOCF as preferred sensitivity analysis. E6

7 Changes in quality-of-life measures were summarized at each visit by treatment arm, and between-group comparisons were assessed using a stratified Wilcoxon rank sum test of treatment arm. E7

8 REFERENCES 1. Griffith DE, Aksamit T, Brown-Elliott BA, Catanzaro A, Daley C, Gordin F, Holland SM, Horsburgh R, Huitt G, Iademarco MF, Iseman M, Olivier K, Ruoss S, von Reyn CF, Wallace RJ Jr, Winthrop K; on behalf of the ATS Mycobacterial Diseases Subcommittee. An official ATS/IDSA statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases. Am J Respir Crit Care Med 2007;175: Wallace RJ Jr, Brown BA, Griffith DE, Girard WM, Murphy DT, Onyi GO, Steingrube VA, Mazurek GH. Initial clarithromycin monotherapy for Mycobacterium avium intracellulare complex lung disease. Am J Respir Crit Care Med 1994;149(5): Wallace RJ Jr, Brown BA, Griffith DE, Girard WM, Murphy DT. Clarithromycin regimens for pulmonary Mycobacterium avium complex: the first 50 patients. Am J Respir Crit Care Med 1996;153(6 Pt 1): Cousins D, Francis B, Dawson D. Multiplex PCR provides a low-cost alternative to DNA probe methods for rapid identification of Mycobacterium avium and Mycobacterium intracellulare. J Clin Microbiol 1996;34: Hall LK, Doerr KA, Wohlfiel SL, Roberts GD. Evaluation of the MicroSeq system for identification of mycobacteria by 16S ribosomal DNA sequencing and its integration into a routine clinical mycobacteriology laboratory. J Clin Microbiol 2003;41: Iakhiaeva E, Howard ST, Brown-Elliott BA, McNulty S, Newman KL, Falkinham JO III, Williams M, Kwait R, Lande L, Vasireddy R, Turenne C, Wallace RJ Jr. Variable number tandem repeat (VNTR) of respiratory and household water biofilm isolates of Mycobacterium E8

9 avium subspecies "hominissuis with establishment of a PCR database. J Clin Microbiol 2016 Jan 6. Pii: JCM [Epub ahead of print] 7. Iakhiaeva E, McNulty S, Brown-Elliott BA, Falkinham JO III, Williams MD, Vasireddy R, Wilson RW, Turenne C, Wallace RJ Jr. Mycobacterial interspersed repetitive-unit-variablenumber tandem-repeat (MIRU-VNTR) genotyping of Mycobacterium intracellulare for strain comparison with establishment of a PCR-based database. J Clin Microbiol 2013; 51: Brown-Elliott BA, Iakhiaeva E, Griffith DE, Woods GL, Stoudt JE, Wolfe CR, Turenne CY, Wallace RJ Jr. In vitro activity of amikacin against isolates of Mycobacterium avium complex with proposed MIC breakpoints and finding of a 16S rrna gene mutation in treated isolates. J Clin Microbiol 2013;51: ATS Committee on Proficiency Standards for Clinical Pulmonary Function Laboratories. ATS Statement: guidelines for the six-minute walk test. Am J Respir Crit Care Med 2002;166: Fuchs HJ, Borowitz DS, Christiansen DH, Morris EM, Nash M, Ramsey BW, Rosenstein BJ, Smith AI, Wohl ME; for the Pulmozyme Study Group. Effect of aerosolized recombinant human DNase on exacerbations of respiratory symptoms and on pulmonary function in patients with cystic fibrosis. N Engl J Med 1994;331: Lymp J, Hilliard K, Rosenfeld M, Koker P, Hamilton A, Konstan M. Pulmonary exacerbations in a phase 2 clinical trial of BIIL284BS in CF: development and implementation of a respiratory and systemic symptoms questionnaire (RSSQ). Pediatr Pulmonol 2009 (Suppl. 32):288. E9

10 FIGURE LEGENDS Figure E1. Study design and treatment. CF = cystic fibrosis; LAI = liposomal amikacin for inhalation; MAC = Mycobacterium avium complex; Mabs = Mycobacterium abscessus. *2007 American Thoracic Society and Infectious Diseases Society of America (ATS/IDSA) criteria with evidence of nodular bronchiectasis and/or fibrocavitary disease by chest computed tomography. At least two documented positive cultures in the previous 2 years, of which at least one was obtained during the 6 months prior to screening. Receiving ATS/IDSA guidelinesbased treatment for at least 6 months prior to screening with persistently positive cultures. Continuing on ATS/IDSA guidelines-based therapy. Figure E2. Patient disposition. LAI = liposomal amikacin for inhalation; mitt = modified intent-to-treat; SOC = standard of care; SUSAR = suspected unexpected serious adverse reactions. Figure E3.Treatment-emergent adverse events (>5% frequency) during the double-blind phase (A) and open-label phase (B).*Adverse events during the double-blind phase are those with onset from the date of first dose of double-blind study drug, and adverse events during the open-label phase are those with onset from the date of first dose of open-label study drug, All patients in the open-label phase received LAI. LAI = liposomal amikacin for inhalation. E10

11 Table E1. Semi-quantitative Mycobacterial Culture Reporting Method* Step* Solid Media CFUs Present Liquid Medium Result Categorical Result 1 0 Negative Culture-negative (confirmed with no growth in liquid medium) 2 0 Positive Growth in liquid medium only (liquid positive) (manual count on agar) Positive Agar-positive Positive 1+ 5 > Positive 2+ 6 > Positive 3+ 7 >500 Positive 4+ * Movement from row to row within the table above constitutes a step. CFUs = colony-forming units. E11

12 Table E2. Concomitant Antibiotic Combinations Taken by 57 Patients With Mycobacterium avium Complex During the Double-Blind Phase LAI (n = 29) Placebo (n = 28) Macrolide monotherapy or with FQ antibiotics containing macrolide + both RIF and EMB Macrolide + either RIF or EMB and other antibiotics EMB + RIF without a macrolide (+/ FQ) 0 5 EMB + Clofazimine + Moxifloxacin 0 1 Definition of abbreviations: EMB = ethambutol, FQ = fluoroquinolones, LAI = liposomal amikacin for inhalation, RIF = rifampicin/rifabutin. E12

13 Table E3. Concomitant Individual Antibiotics * Taken by 57 Patients with Mycobacterium avium Complex During the Double-Blind Phase LAI (n = 29) Placebo (n = 28) Macrolide (azithromycin or clarithromycin) Ethambutol Rifampicin/rifabutin Fluoroquinolone (levofloxacin, moxifloxacin, ciprofloxacin) Clofazimine 2 3 Amikacin 2 0 Cephalosporin (cephalexin, cefepime, ceftazidime, ceftriaxone, cefdinir) Other antibiotics (clindamycin, vancomycin, trimethoprim/sulfamethoxazole, nitrofurantoin, doxycycline, ampicillin, amoxicillin/clavulanate, aztreonam) Meropenem/imipenem 1 1 Definition of abbreviations: LAI = liposomal amikacin for inhalation. * Range of # antibiotic classes / subject = 1-9. Individual antibiotics were counted once in the event of multiple entries/patient. Each class may contain one or more antibiotic per patient. Each patient may be receiving more than one antibiotic. E13

14 Table E4. Concomitant Individual Antibiotics * Taken by 32 Patients With Mycobacterium abscessus During the Double-Blind Phase LAI (n = 15) Placebo (n = 17) Macrolide (Azithromycin or Clarithromycin) Fluoroquinolones (Moxifloxacin, Levofloxacin, 9 5 Ciprofloxacin) Cefoxitin 2 2 Imipenem/Meropenem 4 5 Ethambutol 1 3 Rifampicin 3 2 Tigecycline 1 5 Clofazimine 5 8 Linezolid 3 2 Other antibiotics (Aztreonam, Cefuroxime, Clindamycin, 7 3 Doxycycline, Piperacillin/Tazobactam, Tobramycin, Trimethoprim/sulfamethoxazole) Definition of abbreviations: LAI = liposomal amikacin for inhalation. * Range of # antibiotic classes/subject was 2-7. E14

15 Individual antibiotics were counted once in the event of multiple entries per patient. Each class may contain one or more antibiotics per patient. Each patient may be receiving more than one antibiotic. Table E5. Summary Statistics of the Amikacin Serum Exposure (µg/ml) Estimates on Day 1 and at Steady-State Parameter Mean (CV%) Median Min Max C max, day (82.9%) AUC 24, day (70.5%) C max, steady-state 2.01 (74.2%) AUC 24, steady-state 21.3 (70%) Definition of abbreviations: AUC 24 = 24-hour area under the curve; C max = maximum serum concentration; CV% coefficient of variation percent. E15

16 Randomized 90 Patients (1:1) Stratified: CF vs. Non-CF MAC vs. Mabs Screening period* day 42 to day 2 R 12 Weeks Once-Daily Dosing, Double-Blind Phase Background therapy + LAI once daily by eflow day 1 Background therapy + placebo once daily by eflow day 1 Figure E1. Primary Endpoint: Efficacy Change from baseline on semi-quantitative scale for mycobacterial culture at day Weeks Once-Daily Dosing, Open-Label Phase LAI once daily by eflow day 85 day Weeks 28-day follow-up No inhaled antibiotics Secondary endpoints included: Proportion of subjects with negative sputum culture for NTM at day 84 Tertiary endpoints included: Change in distance walked in the 6-minute walk test at day Months 12-month followup Standard of care E16

17 Figure E2. Screened (n = 136) 46 screen failures Randomized (n = 90) mitt (n = 89) 9 discontinued study drug 1 death (unrelated) 1 SUSAR Randomized double-blind phase LAI + SOC (n = 44) Completed Study Drug Dosing (n = 35) Placebo + SOC (n = 45) Completed Study Drug Dosing (n = 45) Open-label phase Open-label LAI (n = 78) Completed Study Drug Dosing (n = 59) 2 declined open label 19 discontinued study drug 1 death (unrelated) 1 SUSAR E17

18 A Dysphonia Infective exacerbation of Cough Oropharyngeal pain Fatigue Chest discomfort Hemoptysis Nausea Infective pulmonary Pyrexia Wheezing Abdominal discomfort Dyspnea Ear pain Headache Insomnia Laryngitis Nasal congestion Nasopharyngitis Pneumonia Diarrhea Double-Blind Phase Figure E3. LAI (n = 44) B Infective exacerbation of Hemoptysis Infective pulmonary exacerbation Dysphonia Cough Nausea Oropharyngeal pain Pyrexia Urinary tract infection Ear discomfort Headache Aphonia Abdominal pain Diarrhea Laryngitis Upper respiratory tract infection Dyspnea Oral candidiasis Open-Label Phase* LAI (n = 35) 0% 10% 20% 30% 40% 50% Percentage of Patients 0% 5% 10% 15% 20% 25% 30% 35% 40% 45% 50% Percentage of Patients E18