Progressive Transforming Growth Factor [beta]1-induced Lung Fibrosis Is Blocked by an Orally Active ALK5 Kinase Inhibitor

Diseases that involve chronic fibrotic changes to the structure of the lungs, such as toxic interstitial lung diseases (e.g., drug injuries, radiation, environmental damage) or idiopathic pulmonary fibrosis (IPF), are challenging clinical problems. The process of lung fibrosis is not well understood, and there is no proven therapy to prevent or reverse it (1). IPF is one of the most common chronic interstitial lung diseases, with a mortality rate of up to 70% 5 years after diagnosis, and most therapeutic strategies have been based on eliminating or suppressing the inflammatory component without evidence of real efficacy (2). A recent clinical trial of long-term treatment with IFN-?1b in IPF showed some promise through effects on mortality, but no impact on the process of fibrogenesis was detected (3).

Transforming growth factor ß1 (TGF-ß1), among a series of cytokines and chemokines, has been implicated in the initiation and progression of fibrosis (4,5). TGF-ß promotes myofibroblast proliferation, induces the synthesis of extracellular matrix (ECM) proteins, and inhibits ECM degradation by induction of antiproteinases or reduction of metalloproteases (6). We have previously demonstrated that transient (7-10 days) overexpression of active TGF-ß1 by adenoviral vector gene transfection in rat lungs induces a severe and progressive fibrosis out to 60 days (7). Additional studies have shown that blocking TGF-ß in different animal models, such as with the use of soluble type II receptors for TGF-ß (8), may be effective at reducing fibrosis.

The active form of TGF-ß interacts with a series of serine/threonine receptors, which are part of a family of related receptor molecules termed activin receptor-like kinases (ALK) (9). TGF-ß receptor I (ALK5) acts downstream of the TGF-ß type II receptor and interacts with members of Smad, a family of cytoplasmic transducer proteins (10). ALK5 is specific for active TGF-ß and/or activin and phosphorylates Smad2 and Smad3 (11). Smad3 appears to be a crucial element in the TGF-ß signal transduction pathway involved in fibrosis (12-14).

This study tests a potent and selective orally active inhibitor of ALK5 kinase activity (SD-208) in an experimental model of pulmonary fibrosis using adenovirus-mediated gene transfer of active TGF-ß1. Using a combination of histologic examinations, biochemical assays, and global gene expression analyses, we demonstrate for the first time that inhibition of ALK5 kinase activity blocks both the early acute fibrogenic response and the progression of established fibrosis. Genes previously implicated in the process of fibrogenesis, such as ECM, growth factors, and proteinase inhibitors, are coordinately regulated in the induction of progressive fibrosis by TGF-ß1 gene overexpression, and regulation of these genes is significantly inhibited by treatment with SD-208. These data indicate an exciting potential role for ALK5 inhibition in the therapy for pulmonary fibrosis. Some of the results of these studies have been previously reported in the form of abstracts (15, 16).

METHODS

Recombinant Adenovirus

AdTGF-ß1^sup 223/225^ expresses biologically active TGF-ß1, as described previously (7, 17). Control vector (AdDL) has no insert in the E1 region.

Kinase Inhibitor

SD-208, a selective and novel 2,4-disubstituted pteridine derivative, is a potent, orally active TGF-ß receptor I kinase inhibitor synthesized by Scios, Inc. (Fremont, CA).

In Vitro Experiments

Primary rat lung fibroblasts were grown as described previously (18) Kinases were assayed by measuring the incorporation of radiolabeled ATP into the relevant kinase, cofactors protein, or peptide substrate. The concentration of compound required to inhibit kinase activity by 50% was recorded as an IC50 value.

Animal Treatment

Female Sprague-Dawley rats (Charles River Laboratories. Montreal, PQ, Canada) were housed under special pathogen-free conditions and provided food and water ad libitum. Procedures used inhalation anesthesia with isofluorane (MTC Pharmaceuticals, Cambridge, ON, Canada).