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Published ahead of print on August 23, 2007, doi:10.1164/rccm.200702-178OC
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American Journal of Respiratory and Critical Care Medicine Vol 176. pp. 1243-1250, (2007)
© 2007 American Thoracic Society
doi: 10.1164/rccm.200702-178OC

Original Article

Role of {alpha}1-Acid Glycoprotein in Therapeutic Antifibrotic Effects of Imatinib with Macrolides in Mice

Momoyo Azuma1, Yasuhiko Nishioka1, Yoshinori Aono1, Mami Inayama1, Hideki Makino1, Jun Kishi1, Masayuki Shono2, Katsuhiro Kinoshita1, Hisanori Uehara3, Fumitaka Ogushi4, Keisuke Izumi3 and Saburo Sone1

1 Department of Internal Medicine and Molecular Therapeutics, 2 Support Center for Advanced Medical Sciences, and 3 Department of Molecular and Environmental Pathology, Institute of Health Biosciences, University of Tokushima Graduate School, Tokushima, Japan; and 4 Clinical Research Center for Allergy and Rheumatology, National Kochi Hospital, Kochi, Japan

Correspondence and requests for reprints should be addressed to Yasuhiko Nishioka, M.D., Ph.D., Department of Internal Medicine and Molecular Therapeutics, Institute of Health Biosciences, University of Tokushima Graduate School, 3-18-15 Kuramoto-cho, Tokushima 770-8503, Japan. E-mail: yasuhiko{at}clin.med.tokushima-u.ac.jp


   ABSTRACT
TOP
 ABSTRACT
AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Rationale: Imatinib is an inhibitor of platelet-derived growth factor receptors. We have reported that treatment with imatinib inhibited bleomycin-induced pulmonary fibrosis in mice. However, late treatment with imatinib had no effect.

Objectives: To clarify why imatinib had no antifibrotic effect when its administration was delayed, we focused on {alpha}1-acid glycoprotein (AGP), because it was reported to bind imatinib and mediate drug resistance.

Methods: The concentration of AGP in serum of mice and patients with idiopathic pulmonary fibrosis was measured by radial immunodiffusion testing. The effects of AGP in vitro were evaluated by assaying the growth of lung fibroblasts. We examined the combined effects of erythromycin (EM) or clarithromycin (CAM) on bleomycin-induced pulmonary fibrosis in mice.

Measurements and Main Results: Addition of AGP abrogated imatinib-mediated inhibition of the growth of fibroblasts. However, treatment with EM or CAM restored the growth-inhibitory effects of imatinib. The elevated level of AGP was detected in serum and lung homogenates in bleomycin-exposed mice and reached a plateau on Day 14. Imatinib alone did not ameliorate pulmonary fibrosis when treatment was started on Day 15, whereas coadministration of imatinib and EM or CAM significantly reduced the fibrogenesis via inhibition of the growth of fibroblasts in vivo. Serum levels of AGP were higher in patients with idiopathic pulmonary fibrosis than in healthy subjects.

Conclusions: AGP is an important regulatory factor modulating the ability of imatinib to prevent pulmonary fibrosis in mice, and combined therapy with imatinib and EM or CAM might be useful for treatment of pulmonary fibrosis.

Key Words: platelet-derived growth factor • erythromycin • clarithromycin • fibroblast



   AT A GLANCE COMMENTARY
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Scientific Knowledge on the Subject
Imatinib inhibited pulmonary fibrosis using a bleomycin model in mice. However, while early treatment (Days 0 to 15) prevented pulmonary fibrosis, late treatment (Days 15 to 28) did not.

What This Study Adds to the Field
Coadministration of imatinib with macrolides reduced pulmonary fibrosis in mice even if started on Day 15.

 
Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal disease of the lungs characterized by the proliferation of fibroblasts and deposition of extracellular matrix (1, 2). Although corticosteroids and other immunosuppressants have been used to treat IPF, the 5-year survival rate of patients with the disease is less than 50% (3, 4). For this reason, novel therapeutic modalities are of great interest.

Imatinib mesylate (Gleevec in the United States and Glivec in Europe) is a potent and specific tyrosine kinase inhibitor acting against bcr-abl, c-kit, and platelet-derived growth factor receptor (PDGFR) (5). Imatinib has been demonstrated to be highly active against chronic myeloid leukemia and gastrointestinal stromal tumors (6–9). Likewise, in some patients with hypereosinophilic syndrome or myeloproliferative diseases having an activated PDGFRA or PDGFRB as a fusion gene, imatinib showed marked therapeutic effects, indicating that it could be an clinically useful PDGFR inhibitor (10, 11). Because PDGF is one of the growth factors playing a role in the pathogenesis of pulmonary fibrosis (12, 13), imatinib has the potential to be useful for the treatment of this disease.

Based on these concepts, we and Daniels and colleagues demonstrated that treatment with imatinib inhibited the development of pulmonary fibrosis using a bleomycin model in mice (14, 15). Furthermore, Abdollahi and coworkers demonstrated the antifibrotic effects of imatinib in murine radiation-induced lung fibrosis (16). Imatinib has also been reported to prevent fibrogenesis in the liver and kidneys (17–20). These results suggest that imatinib might serve as an antifibrotic drug for various fibrotic diseases in humans. In fact, a clinical trial of imatinib in patients with IPF is in progress (21).

However, we found that early treatment (from Days 0 to 15) significantly prevented the development of pulmonary fibrosis in the bleomycin model in mice, whereas late treatment (from Days 15 to 28) did not (15). Neef and colleagues also reported that early (from Days 0 to 21), but not late (from Days 22 to 35) treatment was effective in inhibiting liver fibrosis using a bile duct ligation model (20). These results raise the critical question of whether the antifibrotic effects of imatinib are only prophylactic, not therapeutic.

We therefore examined the mechanisms involved in the failure of imatinib to act in the late phase of pulmonary fibrosis in the bleomycin model. First, we hypothesized that there were some mechanisms of resistance to imatinib in late fibrogenesis, and focused on {alpha}1-acid glycoprotein (AGP), which is a major drug binding protein and was reported to bind imatinib and mediate drug resistance (22, 23). We also examined the effects of 14-membered ring macrolides, including erythromycin (EM) and clarithromycin (CAM), which can compete with imatinib to bind AGP, on the AGP-mediated suppression of imatinib's actions in vitro and in vivo. Some of the results of these studies have been reported previously in the form of an abstract (24).


   METHODS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Detailed methods are described in the online supplement.

Mice and Materials
Eight-week-old female C57BL/6 mice were purchased from Charles River Japan, Inc. (Yokohama, Japan). They were maintained in the animal facility of the University of Tokushima under specific pathogen–free conditions according to the guidelines of our university (15, 25). Imatinib mesylate was kindly provided by Dr. Elisabeth Buchdunger (Novartis, Basel, Switzerland). EM, bovine serum albumin, AGP, and PDGF-BB were purchased from Sigma-Aldrich (St. Louis, MO). CAM was obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Bleomycin was purchased from Nippon Kayaku Co. (Tokyo, Japan).

Bleomycin Treatment
Osmotic minipumps (model 2001; Alza Pharmaceuticals, Palo Alto, CA) containing 200 µl of saline with or without bleomycin (125 mg/kg) were implanted subcutaneously (15, 26, 27). Each experiment was performed in at least three mice per group.

Administration of Imatinib, EM, and CAM
Imatinib powder was dissolved in distilled water (Otsuka Pharmaceutical Co., Tokushima, Japan). EM and CAM were first dissolved in ethanol and then diluted in distilled water. Imatinib (50 mg/kg/d) was injected intraperitoneally, and EM (5 mg/kg/d) or CAM (20 mg/kg/d) was administered subcutaneously from Days 15 to 28 (15).

Collagen Assay
The right lungs were harvested on Day 28. The total amount of collagen in the lungs was determined using the Sircol Collagen Assay kit (Biocolor Ltd., Belfast, Northern Ireland) (15, 27).

Histopathology
The left lungs were fixed in 10% buffered formalin and embedded in paraffin. Sections (3 to 4 µm) were stained with hematoxylin and eosin. For quantitative histologic analysis, a numerical fibrotic scale was used (Ashcroft score) (28). The mean score was considered the fibrotic score. Masson's trichrome staining was also performed.

Fibroblast Isolation
Murine lung fibroblasts were generated according to the method reported by Phan and coworkers (29). These fibroblasts were used at 5 to 10 passages.

Proliferation Assay
Cell proliferation was evaluated by assaying incorporation of 3H-thymidine deoxyribose (TdR) (19). The experiments were performed in triplicate.

Determination of the Concentration of AGP
The concentration of AGP was measured in murine and human serum. The assay was performed using a single radial immunodiffusion plate test (Cardiotech Services, Inc., Louisville, KY) (22). The human study was approved by the ethics committee of the University of Tokushima, and written, informed consent was obtained from all the subjects. IPF was diagnosed according to the American Thoracic Society international consensus statement (3).

Immunoblotting
Immunoblotting for PDGFR and AGP was performed as described previously (15).

Immunostaining
Paraffin-embedded lung sections were stained with rat anti-mouse Ki-67 antigen antibody (Dako, Glostrup, Denmark) using a ready-to-use Vectastain Quick Kit (Vector Laboratories, Burlingame, CA) (15).

Immunofluorescent double-staining was performed with anti-mouse Ki-67 and anti-S100A4/FSP1 antibodies (Lab Vision Corp., Fremont, CA). Fluorescence images were analyzed using a confocal laser scanning microscope (TCS NT; Leica, Heidelberg, Germany) (30).

Statistical Analysis
Comparisons among multiple groups were performed using one-way analysis of variance with Newman-Keuls post hoc correction (GraphPad Prism, version 3.0; GraphPad, San Diego, CA). Correlation coefficients were determined using Pearson's linear regression analysis. The statistical analysis was performed with StatView software (SAS Institute, Inc., Cary NC). Differences were considered statistically significant if P values were less than 0.05.


   RESULTS
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 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The Level of AGP in Serum and Lung Homogenates Is Elevated in Bleomycin-treated Mice
We first examined serum levels of AGP in mice after the administration of bleomycin. The results are shown in Figure 1A. Levels in untreated mice were 90 to 110 µg/ml. After the administration of bleomycin, AGP levels were increased rapidly from Day 3, reaching a plateau of 650 to 1,000 µg/ml on Day 14. They remained high until Day 42. The transient increase in control mice on Day 3 was considered to be due to the procedure of implantation of osmotic minipumps. The elevated level of AGP in serum of mice was confirmed by Western blotting (Figure 1B).


Figure 1
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  		Figure 1. The levels of {alpha}1-acid glycoprotein (AGP) in serum and lung homogenates are elevated in bleomycin-treated mice. Mice were implanted with osmotic minipumps containing saline or bleomycin (125 mg/kg). On the days indicated, three mice per group were killed after blood was harvested from the axillary artery. (A) Elevated level of AGP in serum of mice treated with bleomycin. The level of AGP was determined by radial immunodiffusion assay. Data are presented as the mean ± SD of three mice. Similar results were obtained in two separate experiments. *P < 0.001 versus group treated with saline; {dagger}P < 0.05 versus group treated with saline. (B) Immunoblot analysis for murine AGP. Immunoblotting for AGP with murine serum (1 µl) and lung homogenates (50 µg) was performed as described in the online supplement. Data are representative of three experiments with the different samples from three mice. BLM = bleomycin.

 
On the other hand, the expression of AGP in the lungs was also enhanced in bleomycin-exposed mice from Day 7, and reached a plateau on Day 14 (Figure 1B).

AGP Abrogates the Inhibitory Effects of Imatinib on DNA Synthesis of Lung Fibroblasts in Response to PDGF-BB
Next, we examined whether a high concentration of AGP, comparable to the level in serum of bleomycin-treated mice, affects the biological activities of imatinib in an in vitro assay. As shown in Figure 2, addition of imatinib significantly inhibited the proliferative responses of lung fibroblasts stimulated with PDGF-BB. Furthermore, addition of AGP abrogated the growth-inhibitory effects of imatinib in a dose-dependent manner (Figures 2A–2C). The reversing effects of AGP were dependent on the concentration of imatinib, and at a concentration of more than 200 µg/ml, AGP significantly inhibited the effects of 1 to 3 µM imatinib (Figures 2A–2C). On the other hand, albumin did not affect the growth-inhibitory effects of imatinib (Figure 2D).


Figure 2
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  		Figure 2. {alpha}1-Acid glycoprotein (AGP) abrogates the inhibitory effects of imatinib on PDGF-BB–stimulated DNA synthesis in lung fibroblasts. Primary lung fibroblasts ( 8 x 103 cells/well) were stimulated with PDGF-BB (10 ng/ml). Addition of imatinib (1–3 µM) significantly inhibited the proliferation of lung fibroblasts stimulated with PDGF-BB. AGP (A–C) or albumin (D) was added at various doses (0–1,200 µg/ml). Cells were labeled with 3H-thymidine deoxyribose (3H-TdR) at 1 µCi/well for the final 18 hours and the incorporation of 3H-TdR was measured with a liquid scintillation counter. Data are presented as the mean ± SD of triplicate cultures. Similar results were obtained in three separate experiments. *P < 0.001; {dagger}P < 0.01; or {ddagger}P < 0.05 versus group treated with PDGF-BB and imatinib.

 
Addition of EM or CAM Reverses AGP- mediated Suppression and Restores the Biological Activity of Imatinib In Vitro
Fourteen-membered ring macrolides, including EM and CAM, are known to compete with imatinib to bind AGP (22, 31). Next, we examined whether EM or CAM could reverse the effects of AGP and restore the biological activity of imatinib in an assay of the growth of lung fibroblasts. As shown in Figures 3B and 3C, the addition of either EM or CAM to the culture with AGP (800 µg/ml) and imatinib (1 µM) significantly restored the inhibitory effect of imatinib on the proliferation of lung fibroblasts in a dose-dependent manner. Similar effects of EM or CAM were observed in various doses of AGP (Figure E1 in the online supplement). However, EM or CAM alone did not directly inhibit the uptake of 3H-TdR by fibroblasts stimulated with PDGF-BB (Figure 3A).


Figure 3
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  		Figure 3. Addition of erythromycin (EM) or clarithromycin (CAM) reverses {alpha}1-acid glycoprotein (AGP)–mediated suppression and restores the biological activity of imatinib in vitro. Primary lung fibroblasts were cultured with PDGF-BB (10 ng/ml), imatinib (1 µM), and AGP (800 µg/ml). EM (A, B) or CAM (A, C) was added at various doses (0–25 µM) as indicated. Data are representative of three separate experiments. Cells were pulsed with 3H-thymidine deoxyribose (3H-TdR) (1 µCi/well) for the final 18 hours and the incorporation of 3H-TdR was measured with a liquid scintillation counter. Data are presented as the mean ± SD of triplicate cultures. Similar results were obtained in three separate experiments. *P < 0.001; {dagger}P < 0.01; or {ddagger}P < 0.05 versus the group treated with PDGF-BB, imatinib, and AGP. (D) Immunoblot analysis for tyrosine phosphorylation of PDGFR in primary lung fibroblasts. Primary lung fibroblasts were stimulated with PDGF-BB (10 ng/ml) for 10 minutes. Cell lysates were separated on a 7.5% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred to a nitrocellulose membrane. Immunoblotting was performed with the indicated antibodies. Data are representative of three separate experiments.

 
To clarify the mechanisms involved in the effects of EM or CAM on AGP-mediated suppression of the growth-inhibitory effects of imatinib, we performed immunoblot analysis for tyrosine phosphorylation of PDGFR. As shown in Figure 3D, imatinib inhibited the autophosphorylation of PDGFR induced by PDGF-BB, and addition of AGP restored PDGF-mediated activation of PDGFR. However, treatment with EM or CAM in the presence of AGP and imatinib inhibited the tyrosine phosphorylation of PDGFR, indicating the restoration of the function of imatinib.

Combination of EM or CAM with Imatinib Exerts "Therapeutic" Antifibrotic Effects in Bleomycin-induced Pulmonary Fibrosis in Mice
We further examined the in vivo antifibrotic effects of combined therapy with imatinib and EM or CAM using a bleomycin-induced model of fibrosis in the lungs. In this experiment, bleomycin-treated mice were injected with imatinib and/or EM or CAM from Days 14 to 28 (late treatment). Consistent with previous reports (15, 32), neither imatinib nor the macrolides alone showed any antifibrotic effects with this treatment schedule (Figures 4 and 5). However, combined therapy with imatinib and EM or CAM resulted in a significant reduction in the number of fibrotic lesions in subpleural areas of the lung even when both agents were administered from Days 14 to 28 (late treatment) (Figures 4 and 5). Quantitative histologic analysis demonstrated that the fibrotic score was significantly lower in mice treated with imatinib and EM or CAM than those treated with bleomycin alone (bleomycin, 1.18 ± 0.08, vs. bleomycin + imatinib + EM, 0 ± 0; P < 0.001; bleomycin + imatinib + CAM, 0 ± 0; P < 0.001) (Figure 5A). The amount of collagen in the lung was also significantly smaller in mice treated with imatinib and EM or CAM as opposed to bleomycin alone (bleomycin, 456 ± 272, vs. bleomycin + imatinib + EM, 87 ± 41; P < 0.01; bleomycin + imatinib + CAM, 128 ± 37 µg/ml; P < 0.01) (Figure 5B).


Figure 4
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  		Figure 4. Histologic examination of the antifibrotic effects of the combined use of erythromycin (EM) and imatinib on bleomycin (BLM)-induced pulmonary fibrosis. Mice were implanted with osmotic minipumps containing BLM. Imatinib (50 mg/kg/d) and/or EM (5 mg/kg/d) was intraperitoneally injected from Days 15 to 28. On Day 28, mice were killed and histologic examination was performed using hematoxylin-and-eosin staining (A, C, E, G, I) and Masson's trichrome staining (B, D, F, H, J) (original magnification, x200). (A, F) Phosphate-buffered saline; (B, G) BLM alone; (C, H) BLM + imatinib; (D, I) BLM + EM; (E, J) BLM + imatinib + EM. Data are representative of three separate experiments. Bar = 100 µm.

 

Figure 5
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  		Figure 5. Quantitative examinations of therapeutic antifibrotic effects of imatinib and macrolide antibiotics on bleomycin (BLM)-induced pulmonary fibrosis. Mice were implanted with osmotic minipumps containing saline or BLM. Imatinib (50 mg/kg/d), erythromycin (EM) (5 mg/kg/d), or clarithromycin (CAM) (20 mg/kg/d) was intraperitoneally injected from Days 15 to 28. Mice were killed on Day 28. (A) Evaluation of fibrotic change in the lung using a numerical fibrotic score. The histologic examination of the left lung was performed with hematoxylin-and-eosin staining. The fibrotic score was determined by two pathologists as described in METHODS. Data are presented as the mean ± SD of all fields examined in each group of five mice. (B) Effects of imatinib on the deposition of collagen after treatment with BLM. The amount of collagen in the right lung was measured using a Sircol collagen kit. Data are presented as the mean ± SD in each group of five mice. Data are representative of three separate experiments.

 
Inhibition of Mitogenesis of Pulmonary Fibroblasts by Treatment with Imatinib plus Macrolide in Bleomycin-induced Lung Fibrosis In Vivo
To analyze the mechanism by which combined therapy with imatinib and macrolide attenuated bleomycin-induced lung fibrosis when the treatment was started on Day 14, we examined whether imatinib plus macrolide inhibited the proliferation of lung fibroblasts in vivo on Day 21. Proliferating cells, which were stained with anti–Ki-67 antigen antibody, were detected in the fibrotic areas of bleomycin-treated lungs (Figures 6B–6E). We found a few Ki-67–positive cells in the alveolar wall (Figure 6A), but there was no difference in the number of these cells among the treatment groups (Figure 6F). On the other hand, the proliferating mesenchymal cells in the interalveolar space of the lungs were significantly reduced in mice treated with both imatinib and EM compared with the other groups (Figure 6F).


Figure 6
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  		Figure 6. Coadministration of imatinib and erythromycin (EM) reduces the proliferating mesenchymal cells in the lungs of mice treated with bleomycin (BLM). Mice were implanted with osmotic minipumps containing BLM. Imatinib (50 mg/kg/d) and/or EM (5 mg/kg/d) was administered from Days 15 to 21. On Day 21, mice were killed and immunohistochemical analysis using anti–Ki-67 antigen antibody was performed. (A) Phosphate-buffered saline; (B) BLM alone; (C) BLM + imatinib; (D) BLM + EM; (E) BLM + imatinib + EM (original magnification, x400). Bar = 50 µm; arrows = Ki-67–positive nuclei. (F) Quantitative analysis of Ki-67–positive cells. The Ki-67–positive cells in 20 random fields were counted at x400 original magnification. Data represent three separate experiments, means ± SD.

 
Furthermore, to confirm that the proliferating mesenchymal cells were fibroblasts, we performed immunofluorescent double-staining for a fibroblast-specific marker, S100A4/FSP1, and Ki-67 antigen. The confocal microscopic analysis clearly showed that most Ki-67–positive cells were also positive for S100A4/FSP1 staining (Figure 7).


Figure 7
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  		Figure 7. Confocal immunofluorescence microscope images of tissue slices of the lungs of bleomycin-treated mice with staining for S100A4/FSP1 and Ki-67 antigen. Mice were implanted with osmotic minipumps containing bleomycin. On Day 21, mice were killed and paraffin-embedded lung sections were stained with rat anti–Ki-67 antigen monoclonal antibody (red) and rabbit anti-S100A4/FSP1 polyclonal antibody (green) (A) S100A4/FSP1; (B) Ki-67; (C) S100A4/FSP1 + Ki-67. Bar = 10 µm.

 
Serum Levels of AGP in Patients with IPF
Finally, we measured the levels of AGP in patients with IPF to examine whether AGP-mediated inhibition of the biological activity of imatinib occurs in humans. Serum AGP levels were significantly higher in patients with IPF than in healthy volunteers (1056 ± 555 vs. 455 ± 165 µg/ml, P < 0.001) (Figure 8A). The concentration of AGP was not correlated with that of C-reactive protein (Figure 8B).


Figure 8
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  		Figure 8. Elevated {alpha}1-acid glycoprotein (AGP) concentrations in patients with idiopathic pulmonary fibrosis (IPF). (A) The level of AGP in serum was measured in healthy volunteers (HV) (n = 11) and patients with IPF (n = 25). (B) The correlation between the levels of AGP and C-reactive protein (CRP) in serum was examined.

 

   DISCUSSION
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In the present study, we demonstrated that AGP plays a pivotal role in the antifibrotic effects of imatinib both in vitro and in vivo, and that the coadministration of 14-membered ring macrolides was effective for producing therapeutic, not prophylactic, effects of imatinib in bleomycin-induced pulmonary fibrosis in mice. Furthermore, because the level of AGP was elevated in the serum of patients with IPF, a similar therapeutic effect of such combined treatment might also be predicted in humans.

We and Daniels and colleagues have previously reported that imatinib prevented the development of pulmonary fibrosis in mice using bleomycin- and radiation-induced models (14, 15). However, when the time course of the antifibrotic effects was examined, late treatment with imatinib was found not to be effective in inhibiting pulmonary fibrosis (15), indicating that imatinib might have only a prophylactic, not a therapeutic, effect. These characteristics are of great importance if imatinib is to be used clinically. We therefore examined whether it is more likely that mechanisms of resistance to imatinib exist in the late phase of pulmonary fibrosis, or that imatinib simply does not have therapeutic antifibrotic effects. The present study clearly demonstrated that resistance to imatinib occurred in pulmonary fibrosis, caused by a factor that was identified as AGP. More than 400 µg/ml of AGP significantly reduced the imatinib-mediated suppression of the growth of lung fibroblasts in vitro. In addition, from 700 to 1,000 µg/ml of AGP was detected in the serum of bleomycin-treated mice, indicating the relevance in vivo of the AGP-mediated suppression of imatinib in mice.

Human serum albumin, lipoprotein, and AGP are the most important drug-binding proteins in plasma (33), and AGP might play a major role in binding imatinib because albumin did not affect imatinib-mediated antiproliferative effects in vitro. Next, we examined whether imatinib was likely to have therapeutic antifibrotic effects if the AGP-mediated inhibition was released. To do this, we used 14-membered macrolides, EM and CAM, that compete with imatinib to bind AGP (23). First, the addition of EM or CAM to the culture of lung fibroblasts containing imatinib and AGP clearly reversed the suppressive influence of AGP on the growth-inhibitory effects of imatinib. To abrogate the effects of 800 µg/ml of AGP, more than 1 µM EM or 10 µM CAM was required in vitro. Such concentrations are achievable in mice and humans using standard doses of these macrolides (34, 35). Furthermore, we demonstrated that combined use of EM or CAM and imatinib attenuated the bleomycin-induced pulmonary fibrosis in mice partly via inhibiting the growth of fibroblasts even when both agents were administered from Days 14. It was reported that 14-membered macrolides had antifibrotic effects in a bleomycin-induced model when mice were pretreated, but not post-treated, with the drugs (32, 36). These results strongly suggest that the therapeutic antifibrotic effects of imatinib plus EM or CAM were mainly mediated by the restoration of the activity of imatinib by those macrolides. Although the lack of the data regarding the concentration of imatinib in serum and lung tissues could be a limitation of the present study, a pharmacokinetics study by Gambacorti-Passerini and colleagues showed that the coadministration of imatinib with clindamycin increased the tissue distribution of imatinib, and the free imatinib levels in plasma rose slightly (23). However, we could not completely rule out the possibility that other mechanisms, including antiinflammatory effects, of macrolides play a role in these activities (37).

Finally, we demonstrated that the levels of AGP were higher in patients with IPF than in healthy subjects. The concentration of AGP in 12 of 25 patients with IPF (48%) was higher than 1,000 µg/ml, a level that was demonstrated to reverse the antifibrotic effects of imatinib in vitro. In our study, substantial differences were found in the baseline levels of AGP between mice and humans (<100 µg/ml vs. 400–800 µg/dl) as described previously (22). However, the plasma concentration of imatinib is also higher in humans than in mice (22). Because the effects of imatinib appear to depend on the balance of the concentrations of imatinib and AGP (as shown in Figures 2A–2C), resistance to imatinib caused by AGP might occur in patients with IPF. To clarify these points, the results of ongoing clinical trial might be helpful and further studies examining the changes of AGP levels in different stages and during disease progression in patients with IPF will be needed.

The reason why AGP levels are high in patients with IPF remains unclear. Because AGP is an acute-phase protein synthesized in the liver, it is reasonable that its levels are elevated in patients with inflammatory diseases (33). However, there was no correlation between the levels of AGP and C-reactive protein in patients with IPF at first diagnosis. Furthermore, the expression of AGP in lung homogenates was enhanced in the late-phase fibrosis. Although the precise biological roles of AGP in pulmonary fibrosis have not been fully determined (33), our data together with the previous findings reporting that alveolar macrophages and type II alveolar epithelial cells in fibrotic lungs were able to produce AGP (38–40) may suggest that AGP was preferentially produced in the fibrotic lungs. Further study will be required to clarify the role of AGP in pulmonary fibrosis.

In summary, the present results suggest that combined therapy with imatinib and macrolides or other drugs that compete with imatinib to bind AGP might be useful for treating pulmonary fibrosis, including IPF.


   Acknowledgments
 
The authors thank Ms. Tomoko Oka for technical assistance.


   FOOTNOTES
 
Supported by grants from the Ministry of Health and Welfare and the Ministry of Education, Science, Sports, and Culture of Japan.

This article has an online supplement, which is accessible from this issue's table of contents at www.atsjournals.org

Originally Published in Press as DOI: 10.1164/rccm.200702-178OC on August 23, 2007

Conflict of Interest Statement: None of the authors has a financial relationship with a commercial entity that has an interest in the subject of this manuscript.

Received in original form February 2, 2007; accepted in final form August 23, 2007


   REFERENCES
TOP
 ABSTRACT
 AT A GLANCE COMMENTARY
 METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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