Targeting Plasminogen Activator Inhibitor-1 with a Novel Small Molecule Inhibitor Attenuates Lung Fibrosis

Abstract

Fibrotic lung diseases are associated with significant morbidity and mortality, and few therapies have been FDA-approved for patients with these conditions. Therefore, developing effective anti-fibrotic treatments represents an unmet clinical need. Plasminogen activator inhibitor 1 (PAI-1) is an attractive therapeutic target as its expression is up-regulated in the context of fibrotic lung disease, and a causal role for PAI-1 in lung fibrogenesis has been established in complementary animal models. Here, we study the efficacy of a novel small molecule PAI-1 inhibitor, MDI-2517, to attenuate lung fibrosis. We observed that MDI-2517 administered during the fibrotic phase of complementary murine models reduces the severity of scarring. Furthermore, we found that MDI-2517 treatment beginning on day 21 after lung injury accelerates fibrosis resolution while in vitro data reveal that this drug reverses myofibroblast differentiation. These results motivate targeting PAI-1 as a therapy for lung fibrosis and highlight MDI-2517 as a promising drug.

Figure 1. Comparison of the IC_50s of MDI-2517 to MDI-2268 and Tiplaxtinin.

Analysis of multiple IC₅₀ titrations of PAI-1 inhibition by MDI-2517, MDI-2268, and Tiplaxtinin. A) in buffer alone, B) in buffer with 50nM purified human vitronectin, C) or in buffer containing 10% human PAI-1-depleated plasma. Data is shown as mean ± SD, n is indicated in each figure by the individual data points, significance by one-way ANOVA.

Figure 2. Comparison of the pharmacokinetics in mice of MDI-2517 and MDI-2268.

Mice were treated with a single dose by oral gavage either MDI-2517 or MDI-2268 at 30 mg/kg. At the given time points (0.5h, 2h, 4h, and 7h), blood samples were collected in heparinized calibrated pipettes and centrifuged immediately at 15,000g for 10 min. The plasma was collected and frozen at −80°C for later analysis of the concentration of each drug by LC-MS. The data in the curves are mean at each time point ± SD and are fit with a single-phase exponential decay. The T_1/2 and area under the curve (AUC) for each drug are shown. N=3 for each time point and each drug.

Figure 3. Dose-response of MDI-2517 in inhibiting the development of lung fibrosis in two complimentary lung injury models.

(A) In the single dose bleomycin lung injury model, bleomycin was administered (2.5 u/kg in 50 μl by the oropharyngeal route) on day 0 to C57BL/6 mice. Beginning on day 11, subsets of mice were treated with daily doses of MDI-2517 (10 mg/kg to 200 mg/kg) or vehicle for 10 days. A group of uninjured C57BL/6 mice were included as a negative control. (B) In the targeted AEC2 injury model, diphtheria toxin (12.5 μg/kg) was administered for 14 days to DTR⁺ mice by intraperitoneal injection. Beginning on day 11, subsets of mice were treated with daily doses of MDI-2517 (60 mg/kg or 100 mg/kg) or vehicle for 10 days. A group of DTR⁻ mice treated with PBS was included as a negative control. (C-D) Lungs were harvested on D21 and analyzed for hydroxyproline content. n = 6–13 in single-dose bleomycin-induced injury model and n = 6–7 in targeted AEC2 injury model. Significant p values are shown from a two-way ANOVA and a Tukey’s multiple comparison test.

Figure 4. Efficacy of MDI-2517 compared to Nintedanib in inhibiting lung fibrosis following targeted AEC2 injury.

(A) Diphtheria toxin (12.5 μg/kg) was administered for 14 days to DTR⁺ mice. Beginning on day 11, subsets of mice were treated with daily doses of MDI-2517 (60 mg/kg qD), twice daily doses of nintedanib (60 mg/kg BID) or vehicle for 10 days. A group of DTR⁻ mice treated with PBS were included as a negative control. (B) Mice were weighed intermittently between day 0 and day 21. Lungs were harvested on D21 and analyzed for (C) hydroxyproline content (n = 7–8/group) or (D) histopathologic changes via picrosirius red staining. Significant p values are shown from a two-way ANOVA and a Tukey’s multiple comparison test.

Figure 5. Efficacy of MDI-2517 compared to Nintedanib in inhibiting lung fibrosis following single-dose bleomycin-induced lung injury.

(A) Bleomycin was administered (2.5 u/kg in 50 μl by the oropharyngeal route) on day 0 to C57BL/6 mice. Beginning on day 11, subsets of mice were treated with daily doses of MDI-2517 (60 mg/kg qD), twice daily doses of nintedanib (60 mg/kg BID) or vehicle for 10 days. A group of uninjured C57BL/6 mice were included as a negative control. (B) Mice were weighed intermittently between day 0 and day 21. (C) Lungs were harvested on D21 and analyzed for hydroxyproline content (n = 5–7 per group) or (D) histopathologic changes via picrosirius red staining. Significant p values are shown froma two-way ANOVA and a Tukey’s multiple comparison test.

Figure 6. MDI-2517 treatment decreases pro-fibrotic plasma biomarkers levels following single-dose bleomycin-induced lung injury.

(A) Bleomycin was administered (2.5 u/kg in 50 μl by the oropharyngeal route) on day 0 to C57BL/6 mice. Beginning on day 11, subsets of mice were treated with daily doses of MDI-2517 (60 mg/kg qD) or vehicle for 5 days (n = 5/group). Blood was collected for plasma preparation on day 16 and analyzed by immunologic assay for: (B) Active PAI-1 in plasma, (C) TGFβ, or (D) matrix metallopeptidase 9 (MMP-9). Data and error bars represent the mean ± SEM and significant p values are shown froma two-tailed Student’s t-test.

Figure 7. MDI-2517 treatment inhibits collagen synthetic pathways in the lung following targeted AEC2 injury.

Diphtheria toxin (12.5 μg/kg) was administered for 14 days to DTR⁺ mice. Groups of PBS-administered DTR⁻ mice treated with were included as a negative control. Beginning on day 11, subsets of injured and control mice were treated with daily doses of MDI-2517 (60 mg/kg qD) or vehicle for 7 days. Lungs were harvested and one lung processed for hydroxyproline (A; n = 6–8/group), and one for bulk RNA seq analysis (n = 3/group). (B) Functional enrichment analyses using hypergeometric tests were conducted using the top 500 up/down-regulated genes. and fibrosis-associated pathways that were enriched with genes up-regulated in the AEC2 injury model were significantly enriched among genes down-regulated post MDI-2517 treatment. (C) Statistically significant (following multiple comparisons correction) genes upregulated by MDI-2517 versus vehicle treatment.

Figure 8. Efficacy of MDI-2517 in reversing lung fibrosis following single-dose bleomycin-induced lung injury.

(A) Bleomycin was administered (2.5 u/kg in 50 μl by the oropharyngeal route) on day 0 to C57Bl/6 mice. Groups of uninjured C57Bl/6 mice were included as negative controls. Beginning on day 21, subsets of mice were treated with daily doses of MDI-2517 (60 mg/kg qD) or vehicle for 21 days. (B) Mice were weighed intermittently between day 0 and day 42. (C) Lungs from injured and control mice were harvested on D21 to establish pre-treatment lung collagen content and on day 42 (to establish post-treatment lung collagen content as measured by hydroxyproline concentration (n = 6–8). Significant p values are shown froma two-way ANOVA and a Tukey’s multiple comparison test.

Figure 9. PAI-1 inhibition with MDI-2517 promotes myofibroblast dedifferentiation.

(A) Schematic detailing “reversal” protocol of human lung myofibroblasts. Readouts of myofibroblast reversal following treatment with MDI-2517 (100 μM): qPCR – 48 h treatment (B), Western blot – 96 h treatment (C), and immunofluorescence microscopy of αSMA stress fibers – 96 h treatment (D) using an anti–αSMA-FITC–conjugated antibody. Nuclei were stained with DAPI. Scale bars: 20 μm (top row) and 5 μm (bottom row). The sample number (n) for experiments (B) and (C) is indicated by the number of data points in each histogram. Data and error bars represent the mean ± SEM, respectively. **P < 0.01 and ****P < 0.0001, by 2-way ANOVA.