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. 2021 Oct 19;22(1):265.
doi: 10.1186/s12931-021-01863-0.

Dual inhibition of αvβ6 and αvβ1 reduces fibrogenesis in lung tissue explants from patients with IPF

Martin L Decaris #  1 , Johanna R Schaub #  1 , Chun Chen #  1 , Jacob Cha  1 , Gail G Lee  1 , Megi Rexhepaj  1 , Steve S Ho  1 , Vikram Rao  1 , Megan M Marlow  1 , Prerna Kotak  1 , Erine H Budi  1 , Lisa Hooi  1 , Jianfeng Wu  1 , Marina Fridlib  1 , Shamra P Martin  1 , Shaoyi Huang  1 , Ming Chen  1 , Manuel Muñoz  1 , Timothy F Hom  1 , Paul J Wolters  2 , Tushar J Desai  3 , Fernando Rock  1 , Katerina Leftheris  1 , David J Morgans  1   4 , Eve-Irene Lepist  1 , Patrick Andre  1   5 , Eric A Lefebvre  1 , Scott M Turner  6
Affiliations

Dual inhibition of αvβ6 and αvβ1 reduces fibrogenesis in lung tissue explants from patients with IPF

Martin L Decaris et al. Respir Res. .

Abstract

Rationale: αv integrins, key regulators of transforming growth factor-β activation and fibrogenesis in in vivo models of pulmonary fibrosis, are expressed on abnormal epithelial cells (αvβ6) and fibroblasts (αvβ1) in fibrotic lungs.

Objectives: We evaluated multiple αv integrin inhibition strategies to assess which most effectively reduced fibrogenesis in explanted lung tissue from patients with idiopathic pulmonary fibrosis.

Methods: Selective αvβ6 and αvβ1, dual αvβ6/αvβ1, and multi-αv integrin inhibitors were characterized for potency, selectivity, and functional activity by ligand binding, cell adhesion, and transforming growth factor-β cell activation assays. Precision-cut lung slices generated from lung explants from patients with idiopathic pulmonary fibrosis or bleomycin-challenged mouse lungs were treated with integrin inhibitors or standard-of-care drugs (nintedanib or pirfenidone) and analyzed for changes in fibrotic gene expression or TGF-β signaling. Bleomycin-challenged mice treated with dual αvβ6/αvβ1 integrin inhibitor, PLN-74809, were assessed for changes in pulmonary collagen deposition and Smad3 phosphorylation.

Measurements and main results: Inhibition of integrins αvβ6 and αvβ1 was additive in reducing type I collagen gene expression in explanted lung tissue slices from patients with idiopathic pulmonary fibrosis. These data were replicated in fibrotic mouse lung tissue, with no added benefit observed from inhibition of additional αv integrins. Antifibrotic efficacy of dual αvβ6/αvβ1 integrin inhibitor PLN-74809 was confirmed in vivo, where dose-dependent inhibition of pulmonary Smad3 phosphorylation and collagen deposition was observed. PLN-74809 also, more potently, reduced collagen gene expression in fibrotic human and mouse lung slices than clinically relevant concentrations of nintedanib or pirfenidone.

Conclusions: In the fibrotic lung, dual inhibition of integrins αvβ6 and αvβ1 offers the optimal approach for blocking fibrogenesis resulting from integrin-mediated activation of transforming growth factor-β.

Keywords: Antifibrotic; PLN-74809; Precision-cut lung slice; Transforming growth factor-β; αv integrin.

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Conflict of interest statement

MLD, JRS, CC, JC, GGL, MR, SSH, VR, MMM, PK, EHB, LH, JW, MF, SPM, SH, MC, MM, TFH, FR, KL, E-IL, EAL, SMT: Employee of Pliant Therapeutics and holds shares in the company. PJW, TJD: Received funding from Pliant Therapeutics for tissue collection/research. DJM, PA: Former employee of Pliant Therapeutics and holds shares in the company.

Figures

Fig. 1
Fig. 1
Effect of αvβ1-selective inhibition (Compound A), αvβ6-selective inhibition (3G9), and dual αvβ6/αvβ1 inhibition (PLN-74809 or Compound A + 3G9) on A COL1A1 mRNA expression and B expression of additional fibrosis-related genes following 7-day culture of PCLSs prepared from lung explants from patients with IPF. Data represent mean (± SD) of 4–5 independent IPF tissues with ≥ 3 slices analyzed per patient tissue. Each symbol within a group represents an individual patient lung, with treatment effects normalized to vehicle. Data in A and B were generated from the same samples. Compound A = 471 nM; 3G9 = 0.5 µg/ml; PLN-74809 = 1.82 µM; TGF-β type I receptor inhibitor (ALK5i [R 268712]) = 1 µM. ALK5i was used as a positive control to confirm TGF-β-driven collagen expression within lung slices. Concentrations selected for integrin inhibitors were ≥ 10 × IC50, determined to inhibit latent TGF-β activation by αvβ1 or αvβ6 in cell-based assays (see Table 1). **P < 0.01 vs DMSO; ****P < 0.0001 vs DMSO. ACTA2: α-smooth muscle actin 2; ALK5i: Activin receptor-like kinase 5 inhibitor; COL1A1: Collagen type I alpha I; COL1A2: Collagen type I alpha II; COL3A1: Collagen type III alpha I; Cpd A: Compound A; CTGF: Connective tissue growth factor; DMSO: Dimethyl sulfoxide; GUSB: Glucuronidase β; IC50: 50% inhibitory concentration; HPRT1: Hypoxanthine phosphoribosyltransferase 1; IPF: Idiopathic pulmonary fibrosis; ITGB6: Integrin subunit β 6; MMP1: Matrix metalloproteinase 1; MMP2: Matrix metalloproteinase 2; MMP7: Matrix metalloproteinase 7; mRNA: Messenger ribonucleic acid; PCLS: Precision-cut lung slice; RPLP0: Ribosomal lateral stalk subunit P0; SD: Standard deviation; SERPINE1: Serpin family E member 1; SNAI1: Snail family transcriptional repressor 1; TGF-β: Transforming growth factor-β; TIMP1: Tissue inhibitor of metalloproteinase 1
Fig. 2
Fig. 2
Effect of selective αvβ6 or αvβ1, dual αvβ6/αvβ1, or multi-αv inhibition on Col1a1 expression in A PCLSs generated from chronic bleomycin-challenged mouse lungs and B PCLSs generated from acute bleomycin-challenged mouse lungs. A Compound A (αvβ1-selective inhibitor) = 471 nM; 3G9 (αvβ6-selective inhibitor) = 0.5 µg/ml; PLN-74809 (dual αvβ6/αvβ1 inhibitor) = 1.82 µM; ALK5i (R 268,712) = 1 µM. Data are mean (± SD) of a single slice from n = 6 mouse lungs. Symbols represent results for individual animals. Culture and treatment were for 7 days. Treatment effects were normalized to average DMSO control. Concentrations selected for integrin inhibitors were ≥ 10 × IC50 determined to inhibit latent TGF-β activation by αvβ1 or αvβ6 (see Table 1). B 3G9 (αvβ6-selective inhibitor) = 1 µg/ml; PLN-74809 (dual αvβ6/αvβ1 inhibitor) = 200 nM; GSK3008348 (multi-αv inhibitor) and CWHM-12 (multi-αv inhibitor) = 1 µM. Data are mean (± SD) of a single slice from n = 5–6 mouse lungs. Culture and treatment were for 3 days. Treatment effects were normalized to DMSO control. **P < 0.01 vs DMSO; ***P < 0.001 vs DMSO; ****P < 0.0001 vs DMSO. ALK5i: Activin receptor-like kinase 5 inhibitor; Col1a1: Collagen type I alpha I; Cpd A: Compound A; DMSO: Dimethyl sulfoxide; IC50: 50% inhibitory concentration; mRNA: Messenger ribonucleic acid; PCLS: Precision-cut lung slice; SD: standard deviation; TGF-β: transforming growth factor-β
Fig. 3
Fig. 3
Mean (± SD) αvβ1 protein levels measured in A lung tissue from healthy subjects vs patients with IPF and B healthy vs fibrotic mouse lung tissue (21 days post-bleomycin challenge). Efficacy of integrin inhibitors of αvβ6 (3G9), αvβ1 (Compound A), or αvβ6/αvβ1 (PLN-74809) at blocking (C) normal human bronchial epithelial cell, D normal human lung fibroblast, and E IPF lung fibroblast adhesion to TGF-β LAP as determined by cell impedance assay. One representative donor cell plotted in (C–E) (mean [± SD] for n = 3 replicate measurements). Pan-αv integrin- and pan-β1 integrin-inhibiting antibodies were also used to demonstrate αvβ1-mediated adhesion of normal and IPF lung fibroblasts to LAP (D, E). Ab: antibody; conc: concentration; Cpd A: Compound A; IPF: idiopathic pulmonary fibrosis; LAP: latency-associated peptide; SD: standard deviation; SMi: small-molecule inhibitor; TGF-β: transforming growth factor-β
Fig. 4
Fig. 4
Pulmonary collagen deposition and Smad3 phosphorylation in bleomycin-challenged mice receiving dual αvβ6/αvβ1 inhibitor. A SHG microscopy images of interstitial collagen deposition (blue) in lung tissue sections collected from sham-challenged or bleomycin-challenged mice (vehicle or 500 mg/kg PLN-74809). B Morphometric analysis of interstitial fibrillar collagen deposition from SHG imaging, and C pSmad3/Smad3 ratio in lung tissue from bleomycin-challenged mice treated with PLN-74809 (100, 250, and 500 mg/kg) or vehicle was compared to sham-challenged mice. PLN-74809 was dosed orally (100–500 mg/kg BID) in mice from 7 to 21 days post-bleomycin-induced lung injury. A Representative images show both fibrotic interstitial fine collagen fibers (blue) and denser normal structural collagens surrounding airways (red). B, C Data presented as box and whisker plot with minimum, 25th, 50th, 75th percentile, and maximum values indicated. *P < 0.05; **P < 0.01; ****P < 0.0001. BID: twice daily; Bleo: bleomycin; PBS: phosphate-buffered saline; pSmad3: phosphorylated Smad3; SHG: second harmonic generation
Fig. 5
Fig. 5
Comparison of plasma PLN-74809 concentrations (red) with pSmad3/Smad3 ratio (blue) in A BAL cells and B lung tissue from bleomycin-challenged mice. C Comparison of plasma PLN-74809 concentrations and pSmad2/Smad2 ratio in BAL cells from healthy mice. D Comparison of plasma PLN-74809 concentrations and pSmad3/Smad3 ratio in lung tissue from healthy mice. Bleomycin-challenged mice received three oral, 250 mg/kg doses BID starting 13 days post-challenge, with lung tissue and BAL cells collected 14 days post-challenge at 2, 4, 8, and 16 h post-dose (n = 2 per group). Healthy mice received continuous infusion of PLN-74809 (1, 3, 10, 30, or 100 mg/kg/day) via osmotic minipump. BAL: Bronchoalveolar lavage; BID: twice daily; conc: concentration; PD: pharmacodynamics; PK: pharmacokinetics; pSmad2: phosphorylated Smad2; pSmad3: phosphorylated Smad3; TGF-β: transforming growth factor-β
Fig. 6
Fig. 6
Effect of dual αvβ6/αvβ1 inhibitor (PLN-74809) and clinical standard-of-care drugs (nintedanib and pirfenidone) on A COL1A1 expression and B fibrosis-related gene expression in PCLSs prepared from explanted lung tissue from patients with IPF, and C Col1a1 expression in PCLSs prepared from chronic bleomycin-challenged mouse lungs. A Data represent mean (± SD) of 5–7 independent IPF tissues with ≥ 3 slices analyzed per patient tissue. Symbols represent results from individual patient tissues. A, B Treatment effects were normalized to DMSO control for each tissue. Culture and treatment were for 7 days. Concentrations used: PLN-74809 = 200 nM; Nin = 75 nM; Pirf = 50 µM; ALK5i (R 268,712) = 1 µM. C Data represent mean (± SD) of a single slice from n = 6 mouse lungs. Symbols represent results for individual slices. Treatment effects were normalized to DMSO control. Culture and treatment were performed for 7 days. *P < 0.05 vs DMSO; **P < 0.01 vs DMSO; ***P < 0.001 vs DMSO; ****P < 0.0001 vs DMSO. ACTA2: α-smooth muscle actin 2; ALK5i: Activin receptor-like kinase 5 inhibitor; IPF: Idiopathic pulmonary fibrosis; COL1A1: Collagen type I alpha I; COL1A2: Collagen type I alpha II; COL3A1: Collagen type III alpha I; CTGF: Connective tissue growth factor; DMSO: dimethyl sulfoxide; GUSB: glucuronidase β; HPRT1: hypoxanthine phosphoribosyltransferase 1; ITGB6: Integrin subunit β 6; MMP1: Matrix metalloproteinase 1; MMP2: Matrix metalloproteinase 2; mRNA: messenger ribonucleic acid; Nin: Nintedanib; PCLS: Precision-cut lung slice; Pirf: pirfenidone; SD: Standard deviation; RPLP0: ribosomal lateral stalk subunit P0; SERPINE1: Serpin family E member 1; SNAI1: Snail family transcriptional repressor 1; TIMP1: tissue inhibitor of metalloproteinase 1
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