Effects of Interferon gamma-1b on biomarker expression in

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1 Online Methods Supplement Effects of Interferon gamma-1b on biomarker expression in patients with idiopathic pulmonary fibrosis Robert M Strieter, Karen M Starko, Richard I Enelow, Imre Noth, Vincent G Valentine, and the Other Members of the Idiopathic Pulmonary Fibrosis (IPF) Biomarkers Study Group* *The IPF Biomarkers Study Group? K Brown, A Frost, L Lancaster, P Noble, A Chan, MK Glassberg, M Kallay, P Economou, S Sahn, M Wencel, DA Zisman, W Bradford, M Burdick, MP Keane, and T Ganz.

2 Methods A. Inclusion/exclusion Criteria Inclusion Criteria In the absence of clinical features suggesting infection, neoplasm, sarcoidosis, collagen vascular disease, or exposure to known fibrogenic environmental factors, patients must fulfill all of the following criteria to be eligible for enrollment into the study: 1. Clinical symptoms consistent with idiopathic pulmonary fibrosis (IPF) with onset between 3 months and 42 months prior to screening. 2. Worsening as evidenced by any one of the following within the past year:? 10% decrease in percent predicted FVC, worsening chest X-ray, or worsening dyspnea at rest or on exertion. 3. Age 20 through 79, inclusive. Patients aged must have diagnosis by either open or VATS lung biopsy to be eligible. 4. Diagnosis must be made by high resolution computed tomographic scan (HRCT) showing definite or probable IPF AND either of the following (Figure E1): Open or video-assisted thoracoscopic (VATS) lung biopsy showing definite or probable UIP, OR Non-diagnostic transbronchial biopsy to exclude other conditions (including granulomatous disease, sarcoidosis, hypersensitivity pneumonitis), AND abnormal pulmonary function tests (PFTs) (reduced FVC or decreased DL CO or impaired gas exchange with rest or exercise, i.e., outside the expected normal range for patient), AND 2 of the following: a) Age > 50 years b) Insidious onset of otherwise unexplained dyspnea on exertion c) Bibasilar, inspiratory crackles (dry or Velcro type in quality) 5. Able to take prednisone 10 mg for at least 21 days prior to the first bronchoscopy, and willing to continue the same dose until the end of study treatment. 6. Failure to show improvement* after an adequate course of steroids.** *Improvement means an increase of? 10% in the percent predicted FVC from baseline value (before steroids started) to any point after the steroid administration period and before randomization. If a patient has? 10% improvement and then returns to the baseline value, despite

3 the continuation of the same dose of steroids that was associated with the improvement, the patient was eligible for this study if all other criteria were met. **For patients with a diagnosis of IPF established within the prior year, an adequate course of steroids was a total oral dose of prednisone 1800 mg or its equivalent administered over a period of no less than 1 month and no greater than 3 months. For patients with a diagnosis of IPF established > 1 year prior to treatment, an adequate course of steroids is a total oral dose of prednisone 1800 mg or its equivalent administered within a 6-month period. 7. FVC? 50% and = 90% of predicted value at Baseline. 8. DLco? 25% of predicted value at Screening. 9. PaO 2 > 55 mmhg (PaO 2 > 50 mmhg for patients at altitudes > 4000 feet) at rest after 20 minutes or less (see Section 5.3.1) on room air at Baseline (by arterial blood gas) 10. Able to understand and sign a written informed consent form and comply with the requirements of the study; in addition, to sign and understand any site-specific informed consent for bronchoscopy, bronchoalveolar lavage (BAL), and transbronchial biopsy (TBB).

4 Exclusion Criteria Patients with any of the following were excluded from the study: 1. Inability to tolerate fiberoptic bronchoscopy, BAL, or TBB, in the opinion of the Principal Investigator. 2. Use of Coumadin or other anticoagulant. 3. History of abnormal bleeding. 4. Prolonged prothrombin time (PT) or partial thromboplastin time (PTT). 5. Not a suitable candidate for enrollment or unable to comply with the requirements of this study, in the opinion of the site Principal Investigator. 6. History of clinically significant environmental exposure known to cause pulmonary fibrosis (drugs, asbestos, beryllium, radiation, domestic birds, etc.). 7. Known explanation for interstitial lung disease, other than IPF, including but not limited to radiation, sarcoidosis, hypersensitivity pneumonitis, bronchiolitis obliterans organizing pneumonia (BOOP), and cancer. 8. Diagnosis of any connective tissue disease (scleroderma, systemic lupus erythematosus [SLE], rheumatoid arthritis, etc.) according to American College of Rheumatology criteria. 9. Forced expiratory volume in the first second (FEV 1 )/ forced vital capacity (FVC) ratio < 0.6 at Screening (post-bronchodilator). 10. Patients with a residual volume > 120% of predicted at Screening (prebronchodilator). 11. Evidence of active infection, including bronchitis, sinusitis, urinary tract infection (UTI), and cellulitis. 12. Any condition other than IPF which, in the opinion of the site Principal Investigator (PI), is likely to result in the death of the patient within the next year. 13. History of unstable or deteriorating cardiac or neurologic disease within the last 6 months, including but not limited to: a) Myocardial infarction, coronary artery bypass surgery, or coronary angioplasty

5 b) Congestive heart failure requiring hospitalization c) Uncontrolled arrhythmias d) Transient ischemic attacks (TIAs) 14. Any cardiac or neurologic condition which, in the opinion of the site PI, might be significantly exacerbated by the known flu-like syndrome associated with the administration of IFN-? 1b. 15. History of peripheral vascular disease which, in the opinion of the site PI, might be significantly exacerbated by the known flu-like syndrome associated with the administration of IFN-? 1b. 16. History of CNS disorder which, in the opinion of the site PI, might be significantly exacerbated by the known flu-like syndrome associated with the administration of IFN-? 1b. In addition, patients with the following conditions were excluded: a) History of multiple sclerosis b) Seizures within the past 10 years or taking anti-seizure medication 17. History of severe or poorly controlled diabetes. 18. Pregnancy or lactation. Females of childbearing potential are required to have a negative serum or urine pregnancy test prior to treatment and must agree to practice abstinence or prevent pregnancy by at least a barrier method of birth control for the duration of the study. 19. Any of the following liver function test criteria above specified limits: Total bilirubin? 1.5? ULN; aspartate or alanine aminotransferases (AST, SGOT or ALT, SGPT) > 3? ULN; alkaline phosphatase > 3? ULN; and albumin < 3.0 mg/dl at Screening. 20. Hematology outside of specified limits: WBC < 2,500/mm 3, hematocrit < 30% or > 59%, platelets < 100,000 /mm 3 at Screening. 21. Creatinine > 1.5? ULN at Screening. 22. Prior treatment with IFN-? 1b, beta interferon (Avonex), or other interferons. 23. Investigational therapy for any indication within 28 days prior to treatment.

6 24. Use of azathioprine, colchicine, cyclophosphamide, cyclosporine, D- penicillamine, methotrexate, or N-acetyl cysteine within 6 weeks prior to treatment. 25. Investigational therapy, including Perfenidone, within 6 months prior to treatment. B. ELISA A ½-log standard curve was generated for each of the proteins ranging from 0 to 100 ng/ml Plates were read at 450 nm in an automated microplate reader (Bio-Tek Instruments, Inc., Winooski, VT, USA). The lower threshold limit for detection (i.e., sensitivity of each assay) of each protein over either background (0) or the next preceding absorbance was established by the geometric change in the slope of the standard curve. Absorbance levels below the level of detection were determined to represent nondetectible levels for each protein and were assigned zero for statistical analysis. Because assessment of Type III procollagen protein is by radioimmunoassay, it was not measured. TGF-ß was measured using the Duoset ELISA Development System (R&D Systems) according to manufacturer s specifications. Latent TGF-?? 1 was activated to immunoreactive TGF-? using the activation procedure per manufacturer instructions. The lower limits of assay sensitivity for both BALF and plasma were as follows: 5 pg/ml for IL-13 and MDC/CCL22; 10 pg/ml for ENA- 78/CXCL5, IP-10/CXCL10, and MIP-1?/CCL15; 50 pg/ml for IFN-?, ITAC/CXCL11, TGF-ß, IL-8/CXCL8, VEGF, and PDGF-A; 100 pg/ml for PDGF-B, IL-4, and MIG/CXCL9; 1 ng/ml for Type I procollagen (measured in BALF only); 3.2 ng/ml for?-defensin-2 (measured in BALF only); and 4 ng/ml for neutrophil defensin (measured in BALF only).

7 C. QUANTITATIVE REAL-TIME TAQMAN RT-PCR Total cellular mrna from lung tissue was isolated as previouslydescribed (E1, E2). Total mrna was measured, and 2.0 µg of total mrna was reverse-transcribed into cdna using TaqMan Gene Expression Quantification assays (Applied Biosystems Kit Number ). cdna was amplified and quantified using the TaqMan 7700 Sequence Detection System, and predeveloped assay reagents with specific FAM-labeled probes were used to detect the target human genes listed. A VIC-labeled probe was used to detect human GAPDH (Applied Biosystems). Gene expression was quantitatively analyzed using the comparative C T (?C T ) method, in which C T is the threshold cycle number (i.e., the minimum number of cycles needed to detect the gene) (E1, E2, E3). The arithmetic formula for?c T is the difference in threshold cycles for a target gene (e.g., CXCL11) and the endogenous reference (i.e, the housekeeping gene, GAPDH). The amount of target gene expression normalized to the endogenous reference at baseline relative to the target gene expression normalized to an endogenous reference at 6 months is given by the following relative expression (RE) calculation: (? Ct[pre]? Ct[post]) RE = 2 where pre is at baseline and post is at 6 months (E1, E2). The result of this formula gives a relative value when comparing the expression of the target gene at two time points and this value is designated a multiple (i.e. a fold increase) from baseline to 6 months. D. STATISTICAL ANALYSIS

8 For mrna analyses, log-transformed data were analyzed. Variables with normally distributed data were compared between the groups using the contrast t-test from an analysis of covariance (ANCOVA) model, with a classification effect for treatment and the baseline transcription level as the covariate (removed from the model if the p value for the covariate was >0.10). Non-normally distributed data were compared using the Wilcoxon rank-sum test. Two categorical analyses were performed on TaqMan? data at baseline and 6 months: (a) increase, no change, or decrease in gene presence (treatment comparisons with the Wilcoxon rank sum test) and (b) gene presence or absence (descriptive statistics only). Samples with the gene of interest not detected through the 36 th cycle were considered to have no gene expression, and samples with an inadequate quantity of the housekeeping gene (VIC > 34) were excluded from all analyses. For other variables, changes from baseline were analyzed using the ANCOVA (when data were approximately normal in distribution) or the Wilcoxon rank-sum test (when data were not approximately normal in distribution or if categorical). Binary response data were analyzed using Fisher s exact test. Values were carried forward from date of last visit.

9 References E1. Belperio JA, Keane MP, Burdick MD, Lynch JP III, Xue YY, Li K, Ross DJ, Strieter RM. Critical role for CXCR3 chemokine biology in the pathogenesis of bronchiolitis obliterans syndrome. J Immunol 2002;169: E2. Belperio JA, Keane MP, Burdick MD, Lynch JP III, Zisman DA, Xue YY, Ardehali A, Ross DJ, Strieter RM. Role of CXCL9/CXCR3 chemokine biology during pathogenesis of acute lung allograft rejection. J Immunol 2003;171: E3. Livak KJ, Schmittgen TD. Analysis of relative gene expression data using realtime quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 2001;25:

10 Figure Legend: Figure E1. Inclusion Criteria for diagnosis of IPF.

11 Figure E1 Diagnosis of IPF High resolution computed tomography (HRCT) Showing definite or probable IPF and either A or B A Open or video-assisted thoracoscopic (VATS) lung biopsy showing definite or probable usual interstitial pneumonia (UIP) B Non-diagnostic transbronchial biopsy to exclude other conditions AND Abnormal pulmonary function tests (1 or more of the following):? Reduced FVC or? Impaired gas exchange with rest or exercise or? Decreased DL CO AND Two of the following:? Age > 50 years? Insidious onset of otherwise unexplained dyspnea on exertion? Bibasilar, inspiratory crackles (dry or Velcro type in quality)