Abstract
Introduction: A feature of scarring in IPF is increased collagen deposition and stiffening by lysyl oxidase (LOX)-family mediated cross-links. LOXL2 is an attractive target for treatment since expression is increased in IPF (Barry-Hamilton et al. Nat Med 2010;16,9:1009-1018) and increased serum LOXL2 is associated with more rapid disease progression (Chien et al. ERJ 2014;43:1430-1438). Compounds A and B are novel LOXL2 selective inhibitors, with 125- and 40-fold selectivity for LOXL2 over LOX.
Aim: To study the effects of compounds A and B on LOX-mediated cross-links in an in-vitro model of fibroblastic foci using IPF-derived human peripheral lung fibroblasts (IHLF).
Method: IHLF were obtained from diagnostic biopsies from confirmed cases of IPF. Cells were seeded onto transwells under conditions appropriate for mature collagen matrix generation ± 1mM BAPN, or compound A or B at 0.001-10nM. After 1 week cultures were treated with TGF-β to induce transformation and contraction of the cultures to spheres. After 6 weeks cultures were harvested, chemically reduced to stabilise bi-valent cross-links, and acid hydrolysed. Pyridinoline (Pyd) cross-links were assessed by ELISA.
Results: Pyd cross-links increased from 24.6 to 108ng per mg total protein (1.5 to 3.1ng Pyd per µg Hyp) from 2 to 6 weeks. Pyd formation was inhibited in a dose-dependent manner by both compound A and B, by 68% and 73% respectively at 1nM, and >90% at 10nM, after 6 weeks of culture.
Conclusions: The LOXL2 selective compounds inhibit formation of Pyd cross-links at concentrations below the cell-free IC50 for LOX in an in-vitro IHLF model of fibroblastic foci.
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