Is imatinib mesylate a promising drug in systemic sclerosis?

Authors


Abstract

A patient with therapy-resistant and progressive systemic sclerosis (SSc) with pulmonary involvement who was treated with imatinib mesylate is described herein. Prior to treatment, pulmonary fibroblasts obtained from the patient were cultured and incubated with imatinib mesylate. Preincubation of the fibroblasts for 16 hours with 2.5 μg/ml imatinib mesylate efficiently abrogated platelet-derived growth factor BB–induced fibroblast proliferation. Furthermore, transforming growth factor β1–induced type I collagen gene transcription was blocked. During treatment, the patient's pulmonary involvement stabilized and her skin tightness improved. To our knowledge, this is the first report of a patient with therapy-refractory SSc responding to treatment with imatinib mesylate.

Systemic sclerosis (SSc) is a debilitating generalized autoimmune disorder characterized by fibrosis and vasculopathy of the peripheral and visceral vasculature and variable degrees of extracellular matrix (ECM) accumulation (mainly collagen) in both the skin and viscera. The disease is associated with specific autoantibodies. Different subsets of this disease are distinguished by specific clinical features and variable involvement of internal organs (1).

Involvement of internal organs, including the gastrointestinal tract, lungs, heart, and kidneys, accounts for increased morbidity and mortality. No putative antifibrotic or immunosuppressive agents have yet been shown to be of unequivocal benefit in controlled clinical trials; consequently, treatment in most patients remains supportive (1).

Due to its extensive proliferation and increased collagen synthesis, the fibroblast is regarded as the key effector cell in fibrosis. A host of mediators have been implicated in the pathogenesis of fibrosis because they fit 3 basic criteria: 1) they stimulate fibroblast proliferation and/or procollagen synthesis, 2) the gene expression and protein production of the mediator is increased in fibrotic tissues, and 3) inhibitors of the mediator function attenuate fibrosis in animal models. Platelet-derived growth factor (PDGF) and transforming growth factor β1 (TGFβ1) fit these criteria, and activation of the PDGF and TGFβ signaling pathways is central in the fibrotic response in SSc (2). Increased PDGF levels have been detected in bronchoalveolar lavage fluid from patients with SSc, and PDGF receptors (PDGFRs) were found to be up-regulated on fibroblasts (3). Recently, stimulatory autoantibodies directed against the PDGFR were detected in serum of patients with SSc (4). Furthermore, fibroblasts from patients with SSc express increased levels of TGFβ receptor type I (TGFβRI) and TGFβRII (2). It was recently shown that TGFβ stimulates c-Abl kinase activity and that inhibition or loss of c-Abl kinase prevents TGFβ-induced expression of ECM genes (5).

Imatinib mesylate (Gleevec; Novartis, Basel, Switzerland) is commonly used in the treatment of chronic myelogenous leukemia in order to block the Abl kinase activity associated with the Bcr-Abl translocation. However, imatinib mesylate is also effective in inhibiting PDGFR kinase (6) and TGFβR signaling and has been shown to prevent bleomycin-induced lung fibrosis in mice (5). Considering this, we anticipated that imatinib mesylate might be a promising therapeutic agent in the treatment of SSc. We present herein the case of a patient with therapy-resistant progressive SSc who was treated with the tyrosine kinase inhibitor imatinib mesylate and showed improvement.

CASE REPORT

The patient, a 69-year-old woman, had had SSc for 4 years. She reported tightening of the perioral skin and of the skin of the distal extremities, which led to impaired range of motion of her hands. Her modified Rodnan skin thickness score (MRSS) (7) was 18. Her disease was complicated by pulmonary fibrosis, for which she was treated with 600 mg intravenous cyclophosphamide twice a month for 6 months, together with low-dose corticosteroids (10 mg/day). Cyclophosphamide was well tolerated, apart from an initial reversible hyponatremia. Despite this treatment, however, her pulmonary fibrosis (as assessed by repeated high-resolution computed tomography [HRCT] scanning) deteriorated, resulting in increased exercise intolerance. HRCT scanning of her lungs showed clear progression of honeycombing. Under treatment, the patient's total lung capacity decreased from 60% to 54% of predicted value. Her diffusing capacity for carbon monoxide (DLCO) remained stable at 36% of predicted value.

Because of progression of the pulmonary changes, bronchoalveolar lavage was performed to rule out alveolitis or infection. During this procedure, transbronchial biopsy specimens were obtained. Fibroblasts were obtained by culturing bronchial biopsy specimens in Dulbecco's modified Eagle's medium (DMEM) containing 10% heat-inactivated fetal calf serum (FCS) and antibiotics.

Fibroblast proliferation assay.

The administration of imatinib mesylate (kindly provided by Novartis) to inhibit PDGF-induced fibroblast proliferation was performed essentially as described previously by our group for the PDGFR-specific tyrosine kinase inhibitor tyrphostin AG1296 (8). Briefly, bronchial fibroblasts were seeded at 6 × 103/well into 96-well plates in 100 μl DMEM supplemented with 0.4% FCS and antibiotics and were allowed to adhere for 24 hours. The medium was then changed to DMEM/0.4% FCS with or without 2.5 μg/ml imatinib mesylate (dissolved in 1 mg/ml phosphate buffered saline) for 16 hours. Subsequently, the medium was removed and replaced by DMEM/0.4% FCS in the presence or absence of 50 ng/ml recombinant human PDGF-BB (R&D Systems, Abingdon, UK) (6 replicates per condition). Proliferation was assessed after 24 hours using a methylene blue colorimetric assay and was expressed as the percentage change in mean absorbance from that of cells exposed to DMEM/0.4% FCS alone (8).

Collagen messenger RNA (mRNA) expression.

Bronchial fibroblasts were seeded at a density of 5 × 105/well into 6-well plates in DMEM/1% FCS and allowed to adhere for 24 hours. Subsequently, the fibroblasts were incubated with DMEM/1% FCS in the presence or absence of imatinib mesylate (2.5 μg/ml) for 16 hours. The medium was then replaced by fresh DMEM/1% FCS with or without 10 ng/ml recombinant human TGFβ1 (R&D Systems) for 6 hours. Subsequently, cells were harvested, and RNA was isolated using RNeasy columns according to the manufacturer's instructions (Qiagen, Hilden, Germany) and reverse transcribed into complementary DNA (9). Type I collagen α1-chain (COL1A1) mRNA levels were determined by real-time quantitative polymerase chain reaction using the following primer–probe combination: forward primer, 5′-ACTGGCCCCCCTGGTCC-3′; reverse primer, 5′-GGGCTCTCCAGCAGCACCTT-3′; probe, 5′-CCGGACCCCCAGGCCCACCT-3′. COL1A1 mRNA expression was defined by calculating the ratio relative to the control gene ABL (9).

Fibroblast proliferation was induced to a significant level after 24 hours of stimulation with PDGF-BB at a concentration of 50 ng/ml. Preincubation of the fibroblasts for 16 hours with 2.5 μg/ml imatinib mesylate efficiently abrogated the PDGF-BB–induced fibroblast proliferation (Figure 1). Furthermore, imatinib mesylate inhibited collagen gene transcription induced by TGFβ1 (10 ng/ml for 6 hours), as assessed by type I collagen mRNA synthesis (Figure 2). Basal proliferation and collagen mRNA synthesis were not influenced by imatinib mesylate. Recently, Distler et al (10) reported that imatinib mesylate inhibited PDGF-BB– and TGFβ-induced collagen production by skin fibroblasts obtained from SSc patients, but they observed no differential effect with control fibroblasts. In the current study, we did not test the effect of imatinib mesylate on bronchial fibroblasts from control patients without SSc, and we therefore cannot conclude that the effect is specific for bronchial fibroblasts from SSc patients. Nevertheless, our data and the results of the study by Distler and colleagues indicate that imatinib mesylate inhibits both collagen gene transcription/production and proliferation induced by the key fibrotic mediators TGFβ and PDGF-BB in fibroblasts obtained from the 2 organs most frequently affected in SSc.

Figure 1.

Imatinib mesylate (IM; 2.5 μg/ml) blocks platelet-derived growth factor (PDGF; 50 ng/ml)–induced proliferation of bronchial fibroblasts obtained from a patient with systemic sclerosis. Values are the mean ± SD of 3 independent proliferation experiments. ∗ = P < 0.05 versus fibroblasts not preincubated with imatinib mesylate.

Figure 2.

Imatinib mesylate (IM; 2.5 μg/ml) blocks expression of type I collagen mRNA induced by stimulation for 6 hours with 10 ng/ml transforming growth factor β1 (TGFβ1) in pulmonary fibroblasts obtained from a patient with systemic sclerosis. Values are the mean and SD of 3 independent measurements from 1 experiment. ∗ = P < 0.05 versus fibroblasts not preincubated with imatinib mesylate.

Based on the above findings, and after informed consent was obtained, the patient was treated with 400 mg imatinib mesylate once daily. Treatment was started 2 months after cessation of cyclophosphamide. Low-dose corticosteroids were continued. After 1 month, the patient reported subjective improvement of skin tightness, with improvement in movement of her hands. After 3 months of treatment, her MRSS (still 18 at the start of imatinib mesylate) was reduced to 12, which remained stable during the following 4 months, although the patient herself noticed some additional improvement that enabled her to go shopping again. Followup HRCT scanning of the lungs showed no further deterioration of the pulmonary fibrosis (Figures 3B and C). Pulmonary function test results initially remained stable and later showed a small increase in DLCO. Diffusion corrected for alveolar ventilation increased from 58% of the reference value when imatinib mesylate was started to 60% and 64% of reference at 4 months and 7 months, respectively, after starting imatinib mesylate treatment. Treatment was well tolerated, although the patient developed some temporary periorbital edema. Currently, she is still being treated with imatinib mesylate.

Figure 3.

A, High-resolution computed tomography (HRCT) image of the lungs obtained 2 weeks before the start of cyclophosphamide treatment and 11 months before the start of imatinib mesylate treatment. B, HRCT image of the lungs obtained 11 months after treatment with cyclophosphamide and at the start of imatinib mesylate treatment, showing progression of honeycombing as compared with that shown in A. Progression is most prominent in the dorsal part of the left lower lobe. C, HRCT image of the lungs obtained 5 months after treatment with imatinib mesylate, showing honeycombing similar to that shown in B.

Based on the in vitro data, we believe that the improvement in the patient's condition was due to treatment with imatinib mesylate. However, there is always the possibility that the 6 months of cyclophosphamide therapy may have had some impact.

DISCUSSION

SSc is a severe, debilitating disorder for which no treatment has yet been shown to be unequivocally beneficial in controlled clinical trials. Therefore, treatment of SSc remains supportive.

In this case report, we describe a patient who had progressive SSc despite treatment with cyclophosphamide and who was then treated with imatinib mesylate, a tyrosine kinase inhibitor. Prior to treatment, pulmonary fibroblasts obtained from the patient were cultured and incubated with imatinib mesylate. Preincubation of the fibroblasts for 16 hours with 2.5 μg/ml imatinib mesylate efficiently abrogated the PDGF-BB–induced fibroblast proliferation. Furthermore, TGFβ1-induced type I collagen gene transcription was blocked. The concentrations of imatinib mesylate used in these experiments can easily be achieved in humans by oral administration of standard doses of the drug. During treatment, the pulmonary involvement stabilized and the skin tightness improved.

The results of the fibroblast culture experiments are consistent with data from the study by Distler et al (10), who reported that imatinib mesylate inhibited PDGF-BB– and TGFβ-induced collagen production by skin fibroblasts obtained from SSc patients. They also showed an effect on bleomycin-induced experimental dermal fibrosis in mice. However, until now, there were no data on the effect of imatinib mesylate in humans with SSc. To our knowledge, this is the first description of a patient with therapy-refractory SSc responding to treatment with imatinib mesylate.

Imatinib mesylate appears to be a selective, and therefore relatively nontoxic, compound for inhibition of pathologically increased production of ECM proteins, although a recent report described 10 patients who developed congestive heart failure while being treated with imatinib mesylate for a different indication (11). In our patient, temporary periorbital edema was the only observed side effect.

The observed in vitro effects, the results seen in our patient, and the fact that imatinib mesylate is well tolerated warrant further clinical research. We are currently conducting an open-label trial in patients with therapy-resistant SSc in order to investigate the efficacy of imatinib mesylate in this incurable disease.

AUTHOR CONTRIBUTIONS

Dr. van Daele had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

Study design. Van Daele, Dik, Thio, van Hal, van Laar, Hooijkaas, van Hagen.

Acquisition of data. Van Daele, Dik, Thio, van Hal, Hooijkaas, van Hagen.

Analysis and interpretation of data. Van Daele, Dik, van Laar, Hooijkaas, van Hagen.

Manuscript preparation. Van Daele, Dik, Thio, van Hal, van Laar, van Hagen.

Statistical analysis. Van Daele, Dik, van Hagen.

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