Safety and Efficacy of an Inhaled Epidermal Growth Factor Receptor ...

Safety and Efficacy of an Inhaled Epidermal Growth Factor Receptor Inhibitor (BIBW 2948 BS) in Chronic Obstructive Pulmonary Disease Prescott G. Woodruff1,2*, Michael Wolff3*, Jens M. Hohlfeld5, Norbert Krug5, Mark T. Dransfield6, E. Rand Sutherland7, Gerard J. Criner8, Victor Kim8, Antje Prasse9, Michael C. Nivens4, Kay Tetzlaff4,10, Ralf Heilker3*, and John V. Fahy1,2* 1

Division of Pulmonary and Critical Care Medicine, Department of Medicine, and 2Cardiovascular Research Institute, University of California San Francisco, San Francisco, California; 3Department of Lead Discovery, Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach, Germany; 4 Department of Clinical Research Respiratory, Boehringer Ingelheim Pharmaceuticals Inc., Ridgefield, Connecticut; 5Fraunhofer Institute Toxicology and Experimental Medicine (ITEM), Hannover, Germany; 6UAB Lung and Health Center, Birmingham, Alabama; 7National Jewish Health, Denver, Colorado; 8Temple University School of Medicine, Philadelphia, Pennsylvania; 9Department of Pneumology, University Medical Center Freiburg, Freiburg im Breisgau; and 10Department of Sports Medicine, Medical Clinic and Policlinic, University of Tuebingen, Tuebingen, Germany

Rationale: Epidermal growth factor receptor (EGFR) activation is implicated in mucin hypersecretion in chronic obstructive pulmonary disease (COPD). Objectives: To investigate the safety and efficacy of an inhaled EGFR antagonist (BIBW 2948) in COPD. Methods: Multicenter, double-blind, placebo-controlled trial of 4 weeks of treatment with two doses of BIBW 2948 (15 and 30 mg twice a day) on safety and mucin-related outcomes in 48 patients with COPD. The effect of BIBW 2948 on EGFR activation in airway epithelial cells was assessed using an ex vivo assay. Efficacy measures included the volume of mucin in the airway epithelium (Vs mu,bala) in bronchial biopsies and the expression of mucin genes in bronchial brushings. Measurements and Main Results: Inhaled BIBW 2948 induced a doserelated inhibition of EGFR internalization (reflecting decreased EGFR activation) in epithelial cells from treated subjects. However, BIBW 2948 was associated with a dose-related increase in adverse events, including reversible liver enzyme elevation (n 5 2), and reduction in FEV1. The changes in mucin stores and mucin gene expression were not significantly different in the pooled BIBW 2948 group versus placebo (volume of mucin per surface area of basal lamina 5 0.22 6 7.11 vs. 0.47 6 8.06 mm3/mm2; P 5 0.93). However, in the 30 mg twice a day group, the reduction in epithelial mucin stores was greatest in subjects with the greatest degree of EGFR inhibition (Pearson r 5 0.98; 95% confidence interval, 0.71–0.99). Conclusions: Four-week treatment with BIBW 2948 did not significantly decrease epithelial mucin stores and was poorly tolerated in patients with COPD. Ex vivo analyses suggest that higher doses may be more effective at both EGFR inhibition and decreases in mucin stores but that adverse events should be expected. Clinical trial registered with www.clinicaltrials.gov (NCT00423137). Keywords: chronic obstructive pulmonary disease; chronic bronchitis; epidermal growth factor receptor; mucins

(Received in original form September 21, 2009; accepted in final form December 7, 2009) * Indicates equal contribution. Supported by Boehringer Ingelheim Pharmaceuticals, Inc. Correspondence and requests for reprints should be addressed to John Fahy, M.D., M.Sc., 505 Parnassus Avenue, M1307, Box 0130, San Francisco, CA 94143-0130. E-mail: [email protected] This article has an online supplement, which is accessible from this issue’s table of contents at www.atsjournals.org Am J Respir Crit Care Med Vol 181. pp 438–445, 2010 Originally Published in Press as DOI: 10.1164/rccm.200909-14150OC on December 10, 2009 Internet address: www.atsjournals.org

AT A GLANCE COMMENTARY Scientific Knowledge on the Subject

Basic research implicates epidermal growth factor receptor (EGFR) activation in the pathogenesis of mucin hypersecretion in chronic obstructive pulmonary disease (COPD). What This Study Adds to the Field

Four-week treatment with the EGFR antagonist BIBW 2948 did not significantly decrease epithelial mucin stores and was poorly tolerated in patients with COPD. Ex vivo analyses suggest that higher doses may be more effective at both EGFR inhibition and decreases in mucin stores, but that adverse events should be expected.

Cough and sputum production cause significant morbidity in patients with chronic obstructive pulmonary disease (COPD) (1), and these symptoms correlate with accelerated loss of lung function (2). Mucin hypersecretion contributes to the pathophysiology of excess sputum in COPD, and goblet cells in the airway epithelium are an important source of mucins in the airway (3–5). In previous studies in habitual smokers with and without airflow obstruction, we measured the expression of three gel-forming mucins (MUC2, MUC5AC, and MUC5B) in airway epithelial brushings, and we quantified epithelial mucin stores in sections of airway mucosal biopsies collected during research bronchoscopy (6). We found increased mucin stores in the airway epithelium in habitual smokers and abnormal expression of MUC2, MUC5AC, and MUC5B (6). Notably, we found the highest epithelial mucin stores in smokers with airflow obstruction, suggesting a mechanistic link between epithelial mucin dysregulation and airflow obstruction in COPD. The molecular and cellular pathways that underlie increased mucin stores in the airway epithelium in COPD include activation of the epidermal growth factor receptor (EGFR) pathway. Multiple environmental stimuli of relevance in patients with COPD, including smoke and bacterial products, activate EGFR and promote mucin upregulation in airway epithelial cells in cell and animal model systems (7). It is unknown, however, whether blockade of the EGFR pathway in vivo in patients with COPD will result in improvement in airway mucin stores or in a reduction in symptoms of cough and sputum production. BIBW 2948 is a potent and specific inhibitor of EGFR tyrosine kinase autophosphorylation. EGFR

Woodruff, Wolff, Hohlfeld, et al.: EGFR blockade in COPD

tyrosine kinase inhibitors currently used for cancer treatment are generally well tolerated but exhibit system class toxicity, such as skin rashes and diarrhea (8). Therefore, it is important to weigh potential therapeutic benefits with possible side effects in patients with COPD. BIBW 2948 was designed to have minimal systemic effects, because it can be administered to the lungs by inhalation via a dry powder inhaler and it is rapidly hydrolyzed by plasma esterases to an inactive zwitterion form (BIBW 3056 ZW), which, once bound to the EGFR, does not readily cross the cell membrane. We set out here to determine if inhaled BIBW 2948 could normalize changes in epithelial mucin gene expression and epithelial mucin stores in current smokers with COPD. We measured these outcomes in a randomized placebo-controlled trial in which enrolled subjects underwent bronchoscopy and collection of airway tissue samples before and after 4 weeks of treatment with BIBW 2948. To document effective EGFR blockade by BIBW 2948, we measured the effect of BIBW 2948 on EGFR in epithelial brushings in a subset of patients using an ex vivo EGFR internalization assay. Because EGF binding leads to EGFR dimerization, transphosphorylation, and rapid receptor internalization (9–12), this assay allowed us to monitor BIBW 2948 BS–mediated EGFR inhibition through the measurement of EGF-induced receptor internalization. Some of the results of these studies have been previously reported in the form of an abstract (13).

METHODS Study Design This study was a six-center, randomized, double-blind, placebo-controlled, parallel group study with two treatment arms (Figure 1). The study periods were divided into four phases, including screening, baseline, treatment, and follow-up. During the screening visit (visit 1) patients were evaluated for their eligibility to participate in the study. At the following visit (visit 2), baseline assessments included bronchoscopy, bronchoalveolar lavage (BAL), bronchial epithelial brushings, and bronchial biopsy. One week after the baseline visit, at the first treatment visit (visit 3), patients were randomized to receive either placebo or active treatment for 4 weeks. BIBW 2948 BS was packaged in polyethylene capsules (7.5 mg per capsule) and inhaled as a dry powder using a handheld device (HandiHaler for BIBW 2948; Boehringer Ingelheim Pharma GmbH & Co., Ingelheim/Rhein, Germany). At study initiation, patients were randomized to either placebo or to BIBW 2948 BS 30 mg twice a day (as described below, the dose delivered in the active treatment arm was reduced to 15 mg twice a day after 24 subjects were randomized, in response to hepatic transaminase elevations in 2 subjects). Study drug allocation was done at random by assignment of subsequent medication numbers to each patient in blocks with equal allocation of treatments within each block. Randomization lists were generated using a validated system involving a pseudo-random number generator so that resulting treatment was

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reproducible and not predictable. At visit 5 (end of treatment), assessments included bronchoscopy, BAL, brushings, and biopsy. Two follow-up visits occurred after visit 5. visit 6 (24 to 72 h after visit 5) was for collection of pharmacokinetic (PK) blood samples and spirometry. visit 7 (2 wk after visit 5) was for clinical evaluation and collection of additional blood for PK analyses. The study protocol and consent were reviewed and approved by institutional review boards associated with each participating center, and all subjects signed informed consent forms. All studies were performed in accordance with Good Clinical Practices and the principles expressed in the Declaration of Helsinki.

Prespecified Endpoints The primary efficacy endpoint was the change in volume of mucin per surface area of basal lamina (Vs mu,bala) between visit 2 (baseline) and visit 5 (end of treatment). This outcome was chosen because of our prior data showing increased mucin volume in smokers with airflow obstruction using the same approach (6). Secondary endpoints included: (1) volume of mucin per volume of epithelium (Vv mu,ep), (2) goblet cell size and goblet cell number per surface area of basal lamina, (3) the expression of the gel-forming mucins (MUC2, MUC5AC, and MUC5B) and EGFR in airway epithelial brushings, (4) the degree of EGFR internalization (and hence activation) in an ex vivo EGFR internalization assay performed on epithelial brushings, (5) total and differential cell counts in bronchoalveolar lavage samples, and (6) interleukin-8 (IL-8) and myeloperoxidase (MPO) levels in bronchoalveolar lavage.

Study Population Study subjects were male and female current smokers (>10 cigarettes/d and >10 pack-years), aged 40 to 70 years, with a diagnosis of COPD. Other inclusion criteria included a post-bronchodilator FEV1/ FVC less than 70% and FEV1 greater than or equal to 40% predicted at screening, and symptoms of cough and sputum production on most days during at least 3 months for 2 consecutive years. Exclusion criteria included abnormal liver function tests (serum aspartate aminotransferase [AST] .80 IU/L, alanine aminotransferase [ALT] .80 IU/L, bilirubin .2.0 mg/dl, or creatinine .2.0 mg/dl) and other medical conditions outlined in the online supplement. The use of inhaled corticosteroids was permitted provided there were no changes to the therapeutic plan in the prior 6 weeks (or at any time during the study).

Bronchoscopy Bronchoscopy included collection of epithelial brushings, BAL, and six bronchial biopsies using specific methods previously described (6, 14). Additional detail on the method for making these measurements is provided in the online data supplement.

Gene Expression Analyses Epithelial brushings were performed for analysis of airway epithelial gene expression using real-time polymerase chain reaction as described

Figure 1. Study schematic. This study was a doubleblind, placebo-controlled, parallel group study with two treatment arms. The study periods were divided into screening, baseline, treatment, and follow-up phases. During visit 1 patients were evaluated for study eligibility. During visit 2 subjects underwent bronchoscopy, bronchoalveolar lavage, bronchial epithelial brushings, and bronchial biopsy. During visit 3 patients were randomized to receive either placebo or active treatment for 4 weeks with one treatment phase assessment at visit 4. During visit 5 the patients had follow-up assessment, including bronchoscopy. During visit 6, patients underwent pharmacokinetic measurements. Patients then entered a 2-week follow-up period before the final study visit 7.

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previously (6) and primers and probes for MUC2, MUC5AC, MUC5B, and housekeeping genes as listed in Table E1 in the online supplement. Additional detail is provided in the online supplement.

Stereology to Measure Airway Epithelial Mucin Stores Airway epithelial mucin stores were measured in 3-mm sections of endobronchial biopsy tissue stained with Alcian blue (pH 2.5)/periodic acid Schiff and analyzed by methods of design-based stereology, as described previously (6). Additional detail is provided in the online data supplement.

Measures in BAL Cell count and differentials were performed on cytospin preparations of BAL, and MPO and interleukin-8 (IL-8) levels were measured using commercial immunoassays (MPO EIA kit; Assay Designs, Ann Arbor, MI; and IL-8 Quantikine ELISA Kit; R&D, Minneapolis, MN). Additional detail is provided in the online data supplement.

BIBW 2948 BS Pharmacokinetics Because BIBW 2948 BS is not stable in plasma, the major metabolite, BIBW 3056 ZW, was measured after alkaline hydrolysis (to ensure complete conversion of BIBW 2948 BS to BIBW 3056 ZW) using highperformance liquid chromatography coupled to tandem mass spectrometry. This approach measures the concentration of both BIBW 2948 BS and any BIBW 3056 ZW present in the sample before hydrolysis. The relationship between pharmacokinetics and clinical response was explored by calculating the correlation between the exposure parameters area under the concentration curve from time 0 to 6 hours after study drug administration at steady state (AUC0–6,ss), the maximal concentration after study drug administration at steady state (Cmax,ss), and predose concentration at steady state (Cpre,ss), and the efficacy endpoints. Additional detail is provided in the online data supplement.

EGFR Internalization Assay We used an EGFR internalization assay in which airway epithelial brushing cells are incubated with an Alexa Fluor (AF) 647-conjugated EGF. Cells without EGFR activation display AF647-based red fluorescence predominantly at the cell surface, whereas cells with activated EGFR show red fluorescent granules in a cytoplasmic/perinuclear location, corresponding to internalized EGFR. We established an image analysis algorithm to quantify red fluorescence intensity associated with granules to quantify EGF-triggered EGFR internalization. The assay was optimized in experiments in NCI-H292 cells (data not shown), and additional details of the methods in the epithelial brushings are provided in the online supplement.

Statistical Methods In our primary analysis, we compared the change in mucin stores (Vs mu,bala; final value 2 baseline value) in the pooled group of placebo patients to the change in the pooled group of BIBW-treated patients. In secondary analyses, a four-group comparison of the primary outcome was performed with the 15-mg and 30-mg doses of BIBW 2948 BS and their respective placebo groups considered separately. In additional secondary analyses, the change in each respective secondary outcome was evaluated across the same two group comparisons (pooled placebo versus pooled BIBW-treated patients) and four group

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comparisons (each group considered separately). Two group comparisons were made using the Student t test. Four group comparisons were made using one-way analysis of variance followed by group-wise comparisons (adjusted for multiple testing using the Sidak correction). Correlations between study outcomes were assessed using Pearson correlation. There were two data sets predefined for the study: the intent-to-treat set, which included all treated patients, and the PK set, which included all treated patients with evaluable PK samples. In analyses that depended on change in a specific efficacy parameter, subjects missing the sample at the second bronchoscopy could not be included because of missing data (Figure E1). All tests were two-tailed and a P less than 0.05 was taken as statistically significant. Statistical analyses presented here were performed by the University of California San Francisco investigators using STATA 9.0 (StataCorp, College Station, TX).

RESULTS In total, 101 subjects were enrolled into the study; 53 subjects were screen failures and 48 subjects were randomized (Figure E1). The clinical characteristics of these subjects are shown in Table 1. The groups were well matched with respect to age, lung function, and smoking history; however, there were slightly fewer men in the 15 mg twice a day BIBW group. At study initiation, subjects were randomized to either placebo or to BIBW 2948 BS 30 mg twice a day. After 24 subjects were randomized, 2 subjects developed elevations in their hepatic transaminase levels (see below), which prompted a protocol modification and a reduction in dose to 15 mg twice a day for the remaining subjects. Thirty-nine subjects completed treatment, and 36 had evaluable efficacy data from both bronchoscopies (Fig E1). Additionally, 24 subjects had paired and evaluable data for EGFR internalization studies in airway epithelial brushings, 31 subjects had paired brushing data for measures of mucin gene expression, and 33 subjects had paired baseline and end-of-treatment lavage samples for the IL-8, MPO, and differential cell counts. EGFR Internalization

An important step in early phase clinical trials of novel inhibitor drugs is to document that the drug reaches and inhibits its target. Therefore, we used a novel ex vivo assay in which EGFR activity in airway epithelial brushings is determined in an EGF internalization assay based on fluorometric imaging of AF647conjugated EGF. Using this assay, we found that the images from brushing cells obtained pretreatment displayed AF647 tagged EGF in perinuclear compartments, consistent with internalized receptor (Figures 2A and 2C) and that these images were similar for brushing cells from placebo-treated patients at the end of treatment (Figure 2D). In contrast, brushing cells collected from BIBW 2948 BS–treated patients at the end of treatment showed cell surface EGF localization and decreased perinuclear EGF localization compared with baseline, consistent with a lack of EGFR internalization (Figure 2B). To

TABLE 1. SUBJECT CHARACTERISTICS OF THE INTENT-TO-TREAT POPULATION

Total subjects Age, years % Male FEV1, % predicted* FEV1/FVC, %* Pack-year smoking history

Placebo 15 mg twice a day

Placebo 30 mg twice a day

Placebo Total

BIBW 15 mg twice a day

BIBW 30 mg twice a day

BIBW Total

11 56 6 7 73 63 6 14 60 6 12 44 6 10

12 56 6 7 75 72 6 20 59 6 11 52 6 15

23 56 6 8 74 68 6 18 59 6 11 48 6 13

13 54 6 8 46 69 6 24 55 6 10 57 6 21

12 59 6 8 75 74 6 23 56 6 10 61 6 24

25 56 6 8 60 69 6 20 55 6 10 59 6 22

* Post-bronchodilator spirometry at visit 2.


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Figure 2. Calculation of epidermal growth factor (EGF) internalization in airway epithelial brushing cells from patients with chronic obstructive pulmonary disease. Image details from baseline and end-of-treatment samples of (A, B) a representative patient treated twice a day with 30 mg BIBW 2948 BS and (C, D) a representative placebotreated patient. White arrowheads indicate nuclei. Blue arrowheads indicate internalized EGF receptor. Granular red fluorescent spots in proximity to the blue nuclei are more frequent in A, C, and D than in B.

quantify internalized AF647-tagged EGF we used the Definiens Enterprise Image Intelligence Software (15) to create an image analysis algorithm. The number of red fluorescent spots that displayed both a prespecified intensity gradient versus the locally surrounding red fluorescence background and an overlap with the nuclear area or a perinuclear ring region were defined as ‘‘perinuclear EGF spots per cell.’’ We found that the number of labeled EGF spots per cell nucleus in brushed epithelial cells was lower in the pooled BIBW 2948 BS group than in the pooled placebo group, especially in the 30 mg subgroup (Figure 3). Specifically, treatment with a 30 mg twice a day dose of BIBW 2948 BS caused a fourfold inhibition of EGF-induced receptor internalization (P 5 0.02) compared with a 1.7-fold average inhibition for a 15 mg twice a day dose (P 5 0.08). In addition, EGFR internalization correlated with plasma levels of BIBW 2948 BS in the 30 mg subgroup (r 5 20.79; 95% confidence interval [CI], 0.98–0.39), but not in the 15 mg subgroup (r 5 20.15; 95% CI, 20.97 to 0.95). These findings demonstrate that inhaled BIBW 2948 BS had its intended effect on EGFR in vivo at the highest dose delivered. Although the effect of BIBW 2948 BS on EGFR at the lower dose was not statistically significant, an outlier value (Figure 3) and small sample size render definitive interpretation of the data for this group difficult. Safety Outcomes

Twenty-three subjects received placebo (11 subjects received 15 mg twice a day and 12 received 30 mg twice a day), and 25 subjects received BIBW 2948 BS (13 subjects received 15 mg twice a day and 12 received 30 mg twice a day). Treatment was discontinued because of adverse events in 1 of 23 (4.3%) subjects in the placebo group versus 6 of 25 (24%) subjects in the BIBW 2948 BS group (P 5 0.099 by Fisher exact test). One subject suffered unstable angina early in the active treatment

phase (with small vessel coronary artery disease on catheterization, which was managed medically). Another subject had a myocardial infarction 3 weeks after completion of the study (with a 90% lesion in the left anterior descending artery, treated with stenting). Both subjects were on study drug, but both were also older males and habitual smokers. In addition, because myocardial ischemia or infarction have not been reported side effects of EGFR inhibitors such as erlotinib and gefitinib, these events were judged by the investigators unlikely to be related to study drug. The principal adverse events considered related to study drug were declines in FEV1 and abnormalities in liver function. The mean FEV1 decreased from baseline during treatment in the higher dose group (BIBW 30 mg twice a day) but returned to baseline by visit 7 at the end of the 2-week follow-up period (Figure 4). Specifically, the decline in prebronchodilator FEV1 at visit 5 as compared with visit 2 was clinically and statistically significant (176 ml, P 5 0.006) in the 30 mg twice a day dose group as compared with placebo but small and nonsignificant for the 15 mg twice a day dose group (9 ml, P 5 0.93). A similar pattern was observed for postbronchodilator FEV1 with a statistically significant decline from visit 2 to visit 5 in the 30 mg twice a day group compared with placebo (212 ml, P 5 0.023). Although there was a decline in post-bronchodilator FEV1 in the 15 mg twice a day group, it was smaller and not statistically significant (156 ml, P 5 0.35). Again, there was no difference from baseline in post-bronchodilator FEV1 in either group at visit 7. Liver transaminase levels increased in three subjects; one of these subjects was in the placebo subgroup (1.8-fold increase in AST, and almost threefold increase in ALT), and the two others were in the BIBW 2948 BS subgroups (average fold increase in AST was approximately sixfold; ALT was approximately sevenfold). In all three subjects the abnormal values returned to baseline after study drug was discontinued. The two subjects with transaminase abnormalities in the BIBW 2948 BS subgroup had a history of

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Figure 3. BIBW 2948 BS induced epidermal growth factor receptor (EGFR) inhibition in ex vivo airway epithelial cells from patients after 4 weeks of treatment. One hundred twenty images were taken in four separate microtiter plate wells of both baseline and end-of-treatment samples that had been exposed to Alexa Fluor 647-EGF. As background controls, 30 images were taken in one additional microtiter plate well of both baseline and end-of-treatment samples that had been exposed to medium instead of AF647-EGF. Samples from 24 patients treated with 30 mg/15 mg twice a day BIBW 2948 BS or matching placebos were analyzed. Perinuclear EGF spots per cell at baseline was set to 100% for each patient. The %ctl value as indicated on the y axis is calculated as the perinuclear EGF spots per cell at end of treatment, normalized to this baseline value. %ctl values shown are means 6 SEM (n 5 4, 30 microscopic images per experiment). The figure inset provides the means 6 SEM of the four patient subgroups (i.e., treated with 30 mg/15 mg twice a day BIBW 2948 BS or with matching placebos).

daily alcohol use and one had a previous hospitalization for alcoholism that was unknown to the study investigators at the time of randomization. Efficacy Outcomes

Primary outcome. The primary outcome variable was mucin stores in the airway epithelium, as measured by the volume of mucin in epithelium per surface area of basal lamina (Vs mu,bala) in Alcian blue/periodic acid Schiff stained sections of airway mucosal biopsies. We found no significant difference in the change from baseline in Vs mu,bala in the pooled placebo and pooled treatment groups (Figure 5). However, when we examined Vs mu,bala in relation to EGFR internalization, we

found a correlation between reduction in Vs mu,bala and inhibition of EGFR internalization (Figure 6A), which was driven by a reduction in the 30 mg subgroup (Figure 6C; r 5 0.98; 95% CI, 0.71–0.99) and was not present in the 15 mg group (Figure 6B). In addition, the change in Vs mu,bala correlated with mean plasma area under the curve values for BIBW 2948 at steady state in the 30 mg subgroup (r 5 20.73; 95% CI, 20.95 to 0.10), but not in the 15 mg subgroup (r 5 0.58; 95% CI, 20.35 to 0.92). Secondary outcomes. We found no significant changes in the volume of mucin per volume of epithelium (Vv mu,ep), or goblet cell size or number in bronchial biopsies across any group comparisons (Tables 2 and E2). Similarly, there were no

Figure 4. BIBW 2948 BS treatment at 30 mg twice a day is associated with a decline in FEV1. The mean FEV1 (prebronchodilator) in the two subgroups of subjects treated with BIBW 2948. FEV1 decreased from visit 2 to visit 5 in the 30 mg twice a day dose group (decline of 176 ml, P 5 0.006), but the FEV1 returned to baseline at visit 7 (after the 2-week follow-up period). The change in the 15 mg twice a day dose group from visit 2 to visit 5 was not significant (9 ml, P 5 0.93).


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for increased MPO and IL-8 levels in the BIBW 2948 BS subgroups in the pooled analysis (Table 2). In four group comparisons, there was a trend for increased IL-8 levels in the BIBW 2948 BS 15 mg twice a day group versus its placebo group but not in the higher dose BIBW 2948 BS group (Table E2).

DISCUSSION

Figure 5. The effect of BIBW 2948 BS on airway mucin stores. There was no significant difference in the change from baseline in the volume of mucin per surface area of basal lamina (Vs mu,bala) in the pooled placebo and pooled treatment groups (data presented as box plots with the box denoting the 25–75% interquartile range, the center line denoting the median, the whiskers denoting the range, and the dots denoting outliers; all P . 0.05).

significant differences in the gene expression levels of MUC2, MUC5AC, or MUC5B in epithelial brushings (Tables 2 and E2). We found no differences in neutrophil percentages, MPO levels, or interleukin-8 levels in BAL, although there was a trend

Abnormalities in mucin gene expression in airway epithelial cells and increases in epithelial mucins stores because of goblet cell hypertrophy and hyperplasia are important pathologic abnormalities that contribute to mucus hypersecretion and to airflow obstruction in COPD (6). Activation of the EGF receptor is considered important in the regulation of mucin expression in the airway, and studies in animal models have shown that EGFR inhibition can prevent or reverse cigarette smoke–induced changes in mucins in airway epithelial cells (16– 18). To date, there have been no published clinical studies of the effect of EGFR inhibition on airway mucins in COPD. Here we report that inhaled BIBW 2948 BS, an inhibitor of EGFR phosphorylation, does not significantly affect airway epithelial mucin gene expression or epithelial mucin stores in habitual smokers with COPD when given at either 15 mg or 30 mg twice a day. However, we did find that EGFR internalization in airway epithelial cells was reduced by BIBW 2948 BS when given at a dose of 30 mg twice a day, and that the degree of EGFR inhibition in any given subject correlated with reduction

Figure 6. Correlation between reduction in epidermal growth factor receptor (EGFR) internalization and reduction in mucin stores. (A) Pooled BIBW 2948– treated groups. (B) Group treated with BIBW 2948 at 15 mg twice a day. (C ) Group treated with BIBW 2948 at 30 mg twice a day (r 5 0.98; 95% CI, 0.71– 0.99). Inhibition of EGFR internalization was associated with decreases in airway mucin stores in the 30 mg subgroup.

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TABLE 2. EFFECT OF BIBW 2948 BS VERSUS PLACEBO ON PRINCIPAL STUDY OUTCOMES Pooled Placebo

Vs mu,bala, mm3/mm2 Vv mu,ep, mm3/mm3 Ns gc,bala, no./mm2 Nv gc,ep, no./mm3 Mean goblet cell volume, mm3 Log10 MUC2 transcript copy number Log10 MUC5AC transcript copy number Log10 MUC5B transcript copy number Neutrophil % in BAL, median, IQR IL-8 levels in BAL, ng/ml, median, IQR MPO levels in BAL, ng/ml, median, IQR

Pooled BIBW 2948 BS

Pretreatment

Post-treatment

Pretreatment

Post-treatment

P Value for Change in BIBW vs. Placebo

7.95 6 4.42 0.20 6 0.12 2190 6 1464 55,368 6 33,778 3,626 6 1,506 5.16 6 1.02 7.45 6 0.30 6.15 6 0.81 1.3, 0.4–2.2 0.030, 0.014–0.098 0.008, 0.003–0.019

8.17 6 5.89 0.17 6 0.1 3161 6 2195 67,535 6 43,560 2,645 6 862 5.19 6 0.71 7.44 6 0.31 6.07 6 0.73 1.8, 0.6–3.0 0.035, 0.024–0.055 0.007, 0.004–0.009

6.87 6 3.46 0.18 6 0.1 2546 6 1373 68,538 6 38,467 3,334 6 1,438 4.93 6 0.66 7.36 6 0.39 6.02 6 0.69 0.8, 0.4–3.0 0.029, 0.020–0.063 0.004, 0.002–0.010

7.34 6 7.98 0.14 6 0.12 3127 6 3263 59,775 6 50,105 2,679 6 991 5.19 6 0.85 7.31 6 0.60 6.06 6 0.73 3.0, 1.2–4.8 0.084, 0.055–0.16 0.012, 0.009–0.019

0.92 0.70 0.77 0.26 0.79 0.16 0.22 0.74 0.19 0.068 0.071

Definition of abbreviations: BAL 5 bronchoalveolar lavage; IQR 5 interquartile range; MPO 5 myeloperoxidase; Ns gc,bala 5 goblet cell number per surface area of basal lamina; Nv gc,ep 5 goblet cell number per volume of epithelium; Vs mu,bala 5 volume of mucin per surface area of basal lamina; Vv mu,ep 5 volume of mucin per volume of epithelium.

in their airway epithelial mucin stores. These data suggest that higher doses of BIBW 2948 BS could have a beneficial effect on mucin stores. However, because the 30 mg twice a day dose of BIBW 2948 BS was associated with reversible declines in FEV1 and reversible increases in liver transaminases, our data also suggest that higher doses cannot be given without incurring unacceptable adverse effects. Studies of novel therapeutics can be limited by uncertainty about the dose(s) studied or the accessibility of a target to its inhibitor. In our study we incorporated an ex vivo assay to determine if BIBW 2948 BS had its intended effect on airway epithelial EGF receptors. Specifically, bronchial brushings from patients were assayed before and after treatment to measure the in vivo biological activity of BIBW 2948 BS, as evidenced by inhibition of EGF-induced internalization of EGFR. We found a dose-dependent effect of BIBW 2948 BS on EGFR internalization. Treatment with the 30 mg twice a day dose of BIBW 2948 BS caused a statistically significant 4.0-fold average inhibition of EGF-induced receptor internalization compared with a smaller and nonsignificant 1.7-fold inhibition for the 15 mg twice a day dose; placebo-treated patients exhibited either enhanced EGFR internalization or an internalization rate as calculated for the baseline sample. These data provide reassurance that the inhaled drug reached its intended target and that EGFR signaling blockade can be achieved in some patients with the 30 mg twice a day dose. Our primary outcome variable was a measure of mucin stores in airway epithelial cells, as assessed by quantification of the volume of mucin in epithelial cells per surface area of basal lamina in sections of endobronchial biopsies. We found no significant effect of BIBW 2948 BS on this outcome and no significant effects on related mucin outcomes, including goblet cell number and size and the gene expression of three gelforming mucins (MUC2, MUC5AC, and MUC5B). In addition, BIBW 2948 BS did not decrease markers of neutrophilic inflammation measured in BAL. In exploratory analyses, however, we found that reduction in epithelial mucin stores were greatest in subjects with the highest plasma levels of BIBW 2948 BS and in subjects with the greatest inhibition of EGFR internalization, findings which provide some support for the hypothesis that effective inhibition of the EGF receptor could decrease airway mucin stores in patients with COPD. Alternatively, the molecular pathways that drive mucin production in the human airway may be redundant such that inhibition of EGFR alone is insufficient to decrease airway mucin stores, despite good evidence for the importance of this pathway in animal models (19).

Inhaled treatments with BIBW 2948 BS treatment were not well tolerated in this study population of habitual smokers with COPD, especially at the higher dose of 30 mg twice a day The most important adverse effects were reversible increases in liver transaminases in two patients and a decline in FEV1 of 5 to 10% in subjects treated with BIBW 2948 BS at 30 mg twice a day. Elevations in liver enzymes have been reported from treatment with oral EGFR tyrosine kinase inhibitors, such as gefitinib and erlotinib, in patients with cancer (20). Increases in transaminases were reported to be less common than the more frequently seen dermatologic and gastrointestinal side effects. Interestingly, the occurrence of these side effects from both gefitinib and erlotinib was described as being dose dependent. In our study, 2 out of 12 patients receiving the (higher) 30 mg twice a day dose of BIBW 2948 BS experienced a sudden temporary increase in ALT after 4 weeks of treatment. However, we found no evidence for dermatologic and gastrointestinal side effects that might be considered the first signs of systemic toxicity (20). Although a decline of FEV1 has not been reported with EGFR tyrosine kinase inhibitors, there have been reports of interstitial lung disease developing from treatment (8). Despite those reports, we consider the development of an interstitial lung disease in our subjects unlikely because of the lack of typical symptoms and because the changes in lung function were fully reversible after treatment cessation. The decline in FEV1 that we observed with active treatment also raises the alternative possibility that the mucus hypersecretion associated with chronic bronchitis is physiologic and protective. Other than prior data showing increased mucin stores correlate with increased severity of airflow obstruction (6), we do not have data that directly address this possibility. In summary, in examining the effects of the EGFR inhibitor BIBW 2468 BS in patients with COPD, we found that the doses at which therapeutic effects on mucin stores may occur overlap with the doses at which reversible adverse effects occur. Whether the adverse effects of BIBW 2948 BS are a class effect associated with EGFR inhibition in the airway or are specific to BIBW 2948 BS cannot be determined from our clinical trial and will require further study. Conflict of Interest Statement: P.G.W. received more than $100,001 from Genentech in research grants (ongoing) and more than $100,001 from Boehringer in research grants (completed) and is the co-inventor on patents related to asthma biomarkers held by UCSF and Genentech. M.W. is an employee of Boehringer Ingelheim Pharma GmbH & Co. KG. J.M.H. received $1,001–$5,000 in consultancy fees from Boehringer Ingelheim (unrelated to IND discussed in this article) and more than $100,001 from Boehringer Ingelheim in institutional grants. N.K.’s institution received more than $100,001 from Boehringer Ingelheim in sponsored grants. M.T.D. received $1,001–$5,000 from GlaxoSmithKline

Woodruff, Wolff, Hohlfeld, et al.: EGFR blockade in COPD and $1,001–$5,000 from AstraZeneca in consultancy fees, $5,001–$10,000 from GlaxoSmithKline and $1,001–$5,001 from AstraZeneca, $10,001–$50,000 from Aeris Therapeutics, more than $100,001 from Boehringer Ingelheim, more than $100,001 from GlaxoSmithKline, $50,001–$100,000 from Novartis, and more than $100,001 from Roche in industry-sponsored grants for contracted COPD research. E.R.S. received $1,001–$5,000 from Schering Plough (as a DSMB member), up to $1,000 from Meda, up to $1,000 from GlaxoSmithKline (as a DSMB member), up to $1,000 from Archimedes, Inc (as a consultant), and $1,001–$5,000 from TEVA in consultancy fees, $10,001–$50,000 from Dey and $5,001–$10,000 from GlaxoSmithKline in advisory board fees, and $10,001– $50,000 from Boehringer-Ingelheim and $10,001–$50,000 from Novartis in grants to the institution. G.J.C. received $1,001–$5,000 from Boehringer, $1,001–$5,000 from GlaxoSmithKline, and $1,001–$5,000 from Aeris in industry-sponsored grants for a research study. V.K. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. A.P. does not have a financial relationship with a commercial entity that has an interest in the subject of this manuscript. M.C.N. is a full-time employee of Boehringer Ingelheim Pharma US. K.T. is an employee of Boehringer Ingelheim Pharmaceuticals Inc. R.H. is a full-time employee of Boehringer Ingelheim Pharma GmbH & Co KG. J.V.F. received $1,001–$5,000 from GlaxoSmithKline, $1,001–$5,000 from Oxagen, $1,001–$5,000 from Amira, $1,001–$5,000 from Gilead, and $1,001–$5,000 from Merck as a consultant, $5,001–$10,000 from Cytokinetics as a scientific advisory board member, more than $100,001 from Genentech in industry-sponsored grants for clinical studies, more than $100,001 from Boehringer Ingelheim in industry-sponsored grants for clinical trials, and more than $100,001 from Roche in industry-sponsored grants for clinical studies, and holds a patent application for being named as an inventor on a gene signature for Th2 high asthma. Acknowledgment: The authors thank Margaret Solon and Almut Ellwanger for technical support and data management.

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