Digitoxin for Airway Inflammation in Cystic Fibrosis: Preliminary Assessment of Safety, Pharmacokinetics, and Dose Finding

Abstract

Rationale: Cystic fibrosis (CF) lung disease progresses by a combination of airway inflammation, bacterial colonization, and infection. Airway inflammation is predominantly neutrophilic and complicates airway clearance therapies through cellular debris; excessive DNA; excessive and viscous mucus; and high concentrations of neutrophils, IL-8, and related cytokines liberated along the nuclear factor-κB signaling pathway.

Objectives: We conducted a preliminary, single-site, randomized, double-blind, placebo-controlled study to evaluate the effects over 28 days of two dose levels (0.05 mg and 0.1 mg daily) of an older cardiac glycoside, digitoxin, as compared with placebo, on safety, pharmacokinetics, and inflammatory markers in induced sputum obtained from 24 subjects with mild to moderate CF lung disease.

Methods: Patients with CF 18-45 years old with any genotype combination were eligible. The primary objective was to measure the effects of digitoxin on IL-8 and neutrophil counts in induced sputum. Secondary objectives were to measure (1) the pharmacokinetics of digitoxin in sera of patients with stable CF; (2) safety indices, including ECG changes and sputum microbiology; (3) the effect of digitoxin on gene expression in nasal epithelial cells of patients with stable CF; and (4) quality-of-life scores using the Cystic Fibrosis Questionnaire-Revised.

Measurements and main results: It took several weeks to achieve a therapeutic serum level of digitoxin in subjects with CF. No safety concerns emerged during the study. Digitoxin treatment showed a trend toward reduction in sputum free neutrophil elastase and neutrophil counts, but not a reduction in sputum IL-8. Digitoxin treatment did not reach statistical significance for the primary or secondary outcome measures over the 28-day study period. However, the nasal mRNA from the group receiving 0.1 mg of digitoxin daily had a distinct distribution of global gene expression levels as compared with either the 0.05-mg dose or placebo treatment. The mRNAs encoding chemokine/cytokine or cell surface receptors in immune cells were decreased in nasal epithelial cells at the higher dose, leading to pathway-mediated reductions in IL-8, IL-6, lung epithelial inflammation, neutrophil recruitment, and mucus hypersecretion.

Conclusions: At a dose of 0.1 mg daily for 28 days, digitoxin was safe for adults with CF lung disease, but it did not achieve a significant decrease in sputum inflammatory markers. Clinical trial registered with www.clinicaltrials.gov (NCT00782288).

Keywords: cardiac glycoside; inflammation; interleukins; pharmacokinetics; sputum.

Figures

Figure 1.

Consolidated Standards of Reporting Trials (CONSORT) flow diagram of the study.

Figure 2.

Pharmacokinetic data. Data points are means ± SEM.

Figure 3.

Primary and secondary outcome measures. The data in some of these graphs are expressed by box-and-whisker plots. The box indicates the lower quartile (Q1 = 25th percentile), the median (50th percentile), and the upper quartile (Q3 = 75th percentile). The middle 50% of values is given by the interquartile range (IQR = Q3–Q1). The upper whisker on the box plot is drawn to the maximum value in the data set that does not exceed Q3 + 1.5 IQR; thelower whisker is drawn to the minimum value in the data set that does not fall below Q1–1.5 IQR. Thesingle dots are outliers falling above or below these thresholds. Dose assignment groups are placebo, 0.05 mg of digitoxin daily, and 0.1 mg of digitoxin daily. (A) Change in induced sputum neutrophil counts per milliliter over the 28-day treatment period (change in log10 neutrophil count per milliliter, Day 28 minus Day 1), by dose assignment. Units are given in log10 cells × 104/ml. (B) Change in log10 neutrophil count per milliliter in induced sputum over 28 days by Day 1 log10 neutrophil count per milliliter, by dose assignment. The relationship suggests that the higher the baseline induced sputum neutrophil count, the larger the reduction in sputum neutrophils with digitoxin treatment. (C) Change in induced sputum IL-8 over the 28-day treatment period (change in log10 IL-8 in picograms per milliliter, Day 28 minus Day 1), by dose assignment. (D) Change in induced sputum neutrophil elastase over the 28-day treatment period (change in log10 change in neutrophil elastase in micrograms per milliliter, Day 28 minus Day 1), by dose assignment. (E) Change in induced sputum IL-6 over the 28-day treatment period (change in log10 IL-6 in picograms per milliliter, Day 28 minus Day 1), by dose assignment. (F) Change in log10 IL-6 over 28 days by Day 1 log10 IL-6, by dose assignment. The relationship suggests that the higher the baseline induced sputum IL-6, the greater the reduction in sputum IL-6 with digitoxin treatment. (G) Change in induced sputum tumor necrosis factor-α (TNF-α) over the 28-day treatment period (change in log10 TNF-α in picograms per milliliter, Day 28 minus Day 1), by dose assignment. (H) Change in induced sputum IL-1β over the 28-day treatment period (change in log10 IL-1β in picograms per milliliter, Day 28 minus Day 1), by dose assignment.

Figure 4.

Pathway analysis in nasal epithelial cells from participants treated with digitoxin (DIG) or placebo. (A) Principal component analysis (PCA). PCA shows that samples from 0.1-mg digitoxin treatment have distinct distribution of global gene expression levels compared with the samples from 0.05-mg digitoxin or placebo treatment. Red is for 0.1 mg,blue is for 0.05 mg, and greenis for placebo treatment. The source of variation analysis (bar graphs) used to determine noise versus signal level is shown. Mean F-ratio for error was set as 1 for all three groups. Treatment is comparison between pre- and post-treatment. The y-axis is the meanF-ratio for error. Treatment with 0.1-mg digitoxin shows a higher signal level than 0.05-mg digitoxin or placebo. As we expected, different individual patients show large mean F-ratios. (B–E) Ingenuity Pathway Analysis. Green represents downregulation, andred is for upregulation.Orange represents predicted activation, andblue is for predicted inhibition.Unfilled diagram represents undetected molecule from the microarray analysis. The solid line is for direct interaction, and the broken line is for indirect interaction. (B) IL-6 signaling pathway. Digitoxin treatment shows decrease of IL-8 and predicted inhibition of IL-6 via downregulation of receptors for IL-1, tumor necrosis factor (TNF), and predicted inhibition of CCAAT/enhancer binding protein-β (CEBPB). (C) IL-8 signaling pathway. Digitoxin-treated patient samples show suppression of inflammation by reduction in nuclear factor-κB (NF-κB)–mediated induction of Il-8. (D) IL-17F signaling in allergy. Ingenuity Pathway Analysis shows decrease of lung inflammation, mucus production, and neutrophil recruitment to the lung via predicted downregulation of TNF receptor–associated factor 6 (TRAF6). (E) IL-17A signaling in airway epithelial cell shows decrease of chemoattraction, inflammation, and mucus hypersecretion via predicted inhibition of NF-κB. There was unexpected upregulation of defensin β4A (DEFB4A), resulting in neutrophil recruitment.ABCB1 = ATP-binding cassette, subfamily B, member 1; CCL = CC chemokine ligand;COL1A1 = collagen, type I, α1; CXCL = chemokine (C-X-C motif) ligand;CYP19A1 = cytochrome P450, family 19, subfamily A, polypeptide 1;ERK1/2 = extracellular signal-regulated kinase 1/2;HSP27 = heat shock protein 27;IKK = inhibitor of IκB kinase; IRAK1 = IL-1 receptor–associated kinase 1;Jnk = c-Jun N-terminal kinase;Jnkk = Jnk kinase;MAPK = mitogen-activated protein kinase;MAPKAPK2 = mitogen-activated protein kinase-activated protein kinase 2;MAP3K7 = mitogen-activated protein kinase kinase kinase 7;MAP3K7IP1 = mitogen-activated protein kinase kinase kinase 7–interacting protein 1;MKK3/6 = mitogen-activated protein kinase kinase 3/6;MMP-13 = matrix metalloproteinase 13;Muc5AC = mucin 5AC;Mus5B = murine STAT5B;NIK = Nck-interacting kinase;PTGS2 = prostaglandin-endoperoxide synthase 2; RELA = v-rel reticuloendotheliosis viral oncogene homolog A;TM = transmembrane;TNFAIP6 = tumor necrosis factor-α–induced protein 6;TRAF3IP2 = tumor necrosis factor receptor-α–associated factor 3–interacting protein 2.