Antifibrotic Compound Research Articles

Pirfenidone Inhibits Cell Proliferation and Collagen I Production of Primary Human Intestinal Fibroblasts.

Intestinal fibrosis is a common complication of inflammatory bowel disease. So far, there is no safe and effective drug for intestinal fibrosis. Pirfenidone is an anti-fibrotic compound available for the treatment of idiopathic pulmonary fibrosis. Here, we explored the anti-proliferative and anti-fibrotic properties of pirfenidone on primary human intestinal fibroblasts (p-hIFs). p-hIFs were cultured in the absence and presence of pirfenidone. Cell proliferation was measured by a real-time cell analyzer (xCELLigence) and BrdU incorporation. Cell motility was monitored by live cell imaging. Cytotoxicity and cell viability were analyzed by Sytox green, Caspase-3 and Water Soluble Tetrazolium Salt-1 (WST-1) assays. Gene expression of fibrosis markers was determined by quantitative reverse transcription PCR (RT-qPCR). The mammalian target of rapamycin (mTOR) signaling was analyzed by Western blotting and type I collagen protein expression additionally by immunofluorescence microscopy. Pirfenidone dose-dependently inhibited p-hIF proliferation and motility, without inducing cell death. Pirfenidone suppressed mRNA levels of genes that contribute to extracellular matrix production, as well as basal and TGF-β1-induced collagen I protein production, which was associated with inhibition of the rapamycin-sensitive mTOR/p70S6K pathway in p-hIFs. Thus, pirfenidone inhibits the proliferation of intestinal fibroblasts and suppresses collagen I production through the TGF-β1/mTOR/p70S6K signaling pathway, which might be a novel and safe anti-fibrotic strategy to treat intestinal fibrosis.

SUN-155 PROTECTIVE ROLES OF TRIGONELLINE AGAINST OXALATE-INDUCED EPITHELIAL-TO-MESENCHYMAL TRANSITION IN RENAL TUBULAR EPITHELIAL CELLS

Fibrogenesis is a common feature for all types of chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the main processes involving renal fibrosis and its inhibition is thus considered as a preventive/therapeutic strategy for CKD. Trigonelline (TRIG), a plant alkaloid commonly found in herbs, coffee bean, soy bean and other edible food plants, has several beneficial effects on human health and has been proposed to reduce renal fibrosis but with unclear mechanisms. This study thus addressed cellular mechanism underlying the anti-fibrogenic effects of TRIG in renal tubular epithelial cells. MDCK renal tubular cells were pretreated with or without 100 µM TRIG for 2 h followed by treatment with 0.5 mM sodium oxalate for 24 h. The cells were then subjected to cell death assay, cell proliferation assay, morphological study, immunofluorescence staining, gelatin zymogram assay, cell migration assay, and measurement of intracellular reactive oxygen species (ROS). EMT was successfully induced by oxalate treatment as indicated by morphological changes into spindle-shape cells, increased expression of mesenchymal proteins (fibronectin, vimentin and α-smooth muscle actin (α-SMA)), decreased expression of epithelial proteins (E-cadherin and zonula occludens-1 (ZO-1)) and increased activity of a profibrotic factor (matrix metalloproteinase-9 (MMP-9)). Interestingly, these oxalate-induced EMT features could be attenuated by TRIG pretreatment. Moreover, TRIG also prevented oxalate-induced cell migration, ROS overproduction, and down-regulation of Nrf-2 signaling molecule. These data indicated that TRIG could attenuate the effects of oxalate-induced EMT and thus may serve as the anti-fibrotic compound for prevention and/or treatment of CKD.

The antifibrotic effect and mechanism of a novel tyrosine kinase inhibitor, ZSP1603, in preclinical models of pulmonary fibrosis.

This study aimed to investigate the efficacy and molecular mechanisms of ZSP1603 as a novel anti-fibrotic compound. The unilateral left pulmonary fibrosis model was established in the Sprague Dawley (SD) rats. The bilateral pulmonary fibrosis model was established in the C57BL/6J mice. The therapeutic treatment regimen began after the induction of pulmonary fibrosis. The preventive treatment regimen began on the first day of bleomycin administration. Animals were randomly divided into the sham, model, Nintedanib, and ZSP1603 treatment groups. Haematoxylin and eosin (H&E) and Masson's trichrome staining were performed to evaluate pulmonary injury, inflammation, and fibrosis. Cell Counting Kit-8 (CCK-8) assay and Western blot were used to investigate the effects and mechanisms of ZSP1603 on the proliferation of primary human pulmonary fibroblasts (pHPFs). The messenger ribonucleic acid (mRNA) expression of transforming growth factor (TGF)-β1, tissue inhibitor of metalloproteinase 1 (TIMP-1), and collagen 1A1 (COL1A1) in pHPFs was detected by quantitative Real Time-Polymerase Chain Reaction (PCR). ZSP1603 inhibited the proliferation of pHPFs in vitro by blocking the platelet-derived growth factor receptor-β (PDGF-Rβ) and extracellular signal-regulated kinase (ERK) signalling pathway. ZSP1603 also inhibited the differentiation of pHPFs by reducing the expression of TGF-β1, TIMP-1, and COL1A1. ZSP1603 significantly attenuated pulmonary injury, inflammation, and fibrosis in vivo in four independent animal studies of pulmonary fibrosis. ZSP1603 is an effective anti-fibrotic compound with clear mechanisms.

Azithromycin has enhanced effects on lung fibroblasts from idiopathic pulmonary fibrosis (IPF) patients compared to controls

BackgroundIdiopathic pulmonary fibrosis (IPF) is a chronic fatal lung disease without a cure and new drug strategies are urgently needed. Differences in behavior between diseased and healthy cells are well known and drug response can be different between cells isolated from IPF patients and controls. The macrolide Azithromycin (AZT) has anti-inflammatory and immunomodulatory properties. Recently anti-fibrotic effects have been described. However, the anti-fibrotic effects on primary IPF-fibroblasts (FB) directly compared to control-FB are unknown. We hypothesized that IPF-FB react differently to AZT in terms of anti-fibrotic effects.MethodsPrimary normal human lung and IPF-FB were exposed to TGF-β (5 ng/ml), Azithromycin (50 μM) alone or in combination prior to gene expression analysis. Pro-collagen Iα1 secretion was assessed by ELISA and protein expression by western blot (αSMA, Fibronectin, ATP6V1B2, LC3 AB (II/I), p62, Bcl-xL). Microarray analysis was performed to screen involved genes and pathways after Azithromycin treatment in control-FB. Apoptosis and intraluminal lysosomal pH were analyzed by flow cytometry.ResultsAZT significantly reduced collagen secretion in TGF-β treated IPF-FB compared to TGF-β treatment alone, but not in control-FB. Pro-fibrotic gene expression was similarly reduced after AZT treatment in IPF and control-FB. P62 and LC3II/I western blot revealed impaired autophagic flux after AZT in both control and IPF-FB with significant increase of LC3II/I after AZT in control and IPF-FB, indicating enhanced autophagy inhibition. Early apoptosis was significantly higher in TGF-β treated IPF-FB compared to controls after AZT. Microarray analysis of control-FB treated with AZT revealed impaired lysosomal pathways. The ATPase and lysosomal pH regulator ATP6V0D2 was significantly less increased after additional AZT in IPF-FB compared to controls. Lysosomal function was impaired in both IPF and control FB, but pH was significantly more increased in TGF-β treated IPF-FB.ConclusionWe report different treatment responses after AZT with enhanced anti-fibrotic and pro-apoptotic effects in IPF compared to control-FB. Possibly impaired lysosomal function contributes towards these effects. In summary, different baseline cell phenotype and behavior of IPF and control cells contribute to enhanced anti-fibrotic and pro-apoptotic effects in IPF-FB after AZT treatment and strengthen its role as a new potential anti-fibrotic compound, that should further be evaluated in clinical studies.

Anti-fibrotic effects of rosmarinic acid on Tenon's capsule fibroblasts stimulated with TGF-β: therapeutic potential in ocular surgery.

Collagen deposition and myofibroblast differentiation are critical factors related to excessive scarring in ocular surgeries. This study evaluated the anti-fibrotic activity of rosmarinic acid on rabbit Tenon's capsule fibroblasts stimulated with transforming growth factor- β2. Primary cultures of rabbit Tenon's capsule fibroblasts were treated with various concentrations of rosmarinic acid for 12 h, in the presence and absence of transforming growth factor-β2. After 48 h, the proliferation index of rabbit Tenon's capsule fibroblasts and the differentiation of myofibroblasts were investigated through immunofluorescence staining for proliferating cell nuclear antigen and alpha smooth muscle actin. An automated cell counter and colorimetric metabolic activity assay were used to evaluate cell number and viability. Collagen expression and production were determined by quantitative real-time polymerase chain reaction and hydroxyproline assay, respectively. Unstimulated rabbit Tenon's capsule fibroblasts treated with any concentration of rosmarinic acid exhibited diminished collagen expression (p<0.01) but showed no differences in proliferation index. Transforming growth factor-β2 exposure induced myofibroblast differentiation and increased collagen production. Exposure to rosmarinic acid at 1.0 and 3.0 µM concentrations reduced the proliferation index (p<0.02), as well as the collagen expression and hydroxyproline content (p<0.05). Exposure to 3.0 µM rosmarinic acid reduced viability (p=0.035) in unstimulated rabbit Tenon's capsule fibroblasts and cell numbers (p=0.001) in both stimulated and unstimulated rabbit Tenon's capsule fibroblast cultures. Exposure to 1.0 µM rosmarinic acid was noncytotoxic and led to reduced collagen expression and proliferation of stimulated rabbit Tenon's capsule fibroblasts. These findings suggest that rosmarinic acid is a relatively non-injurious anti-fibrotic compound to rabbit Tenon's capsule fibroblasts, with potential application as an adjunctive agent in ocular procedures, particularly in glaucoma surgeries.

194 VCP979 Treatment Attenuates Cardiac Fibroblasts Collagen Synthesis and Sphingolipid Metabolism Regulated by TGFβ

Fibrosis is a major contributor to progressive cardiac dysfunction. However, currently there are no effective therapies that specifically target cardiac fibrosis. TGF-β serves as a key mediator in fibrotic development and recent studies have demonstrated that sphingolipids (SLs) have pro-fibrotic and dihydro-SLs (dhSLs) have anti-fibrotic effects. Together they mediate TGFβ signalling transduction involved in the transformation of fibroblasts to myofibroblasts. The aim of this study is to investigate the underlying mechanisms of the novel antifibrotic compound VCP979. Neonatal rat cardiac fibroblasts (NCF) were isolated from 1 to 2-day old pups using enzymatic digestion. Cells were pre-treated with VCP979 for 2 hours before stimulation with 10 ng/ml TGFβ. Collagen synthesis was determined by 3H-proline incorporation. Sphingolipid levels were determined by HPLC/MS/MS. VCP979 dose-dependently inhibited TGFβ-stimulated NCF collagen synthesis. TGFβ stimulation significantly reduced the level of dhSLs including dihydroceramide (Cer34_0), dihydrosphingosine(dhSph) and dihydrosphingomyelin (SM 33_0; 38_0) and VCP979 (3μM) significantly attenuated this decrease (Figure). This is the first demonstration of VCP979 attenuating the fibrotic process in cardiac fibroblasts through the reversal of sphingolipid metabolism stimulated by TGFβ. Thus, VCP979 may pave the way for a novel approach in reducing cardiac fibrosis.

Clinical Safety and Scar Prevention Study of a Topical Antifibrotic Compound FS2.

Clinical Safety and Scar Prevention Study of a Topical Antifibrotic Compound FS2.

Evaluation of NV556, a Novel Cyclophilin Inhibitor, as a Potential Antifibrotic Compound for Liver Fibrosis.

Hepatic fibrosis can result as a pathological response to nonalcoholic steatohepatitis (NASH). Cirrhosis, the late stage of fibrosis, has been linked to poor survival and an increased risk of developing hepatocellular carcinoma, with limited treatment options available. Therefore, there is an unmet need for novel effective antifibrotic compounds. Cyclophilins are peptidyl-prolyl cis-trans isomerases that facilitate protein folding and conformational changes affecting the function of the targeted proteins. Due to their activity, cyclophilins have been presented as key factors in several stages of the fibrotic process. In this study, we investigated the antifibrotic effects of NV556, a novel potent sanglifehrin-based cyclophilin inhibitor, in vitro and in vivo. NV556 potential antifibrotic effect was evaluated in two well-established animal models of NASH, STAM, and methionine-choline-deficient (MCD) mice, as well as in an in vitro 3D human liver ECM culture of LX2 cells, a human hepatic stellate cell line. We demonstrate that NV556 decreased liver fibrosis in both STAM and MCD in vivo models and decreased collagen production in TGFβ1-activated hepatic stellate cells in vitro. Taken together, these results present NV556 as a potential candidate for the treatment of liver fibrosis.

TGF-β-induced fibrotic stress increases G-quadruplex formation in human fibroblasts.

Scar formation after wound healing is a major medical problem. A better understanding of the dynamic nuclear architecture of the genome during wound healing could provide insights into the underlying pathophysiology and enable novel therapeutic strategies. Here, we demonstrate that TGF-β-induced fibrotic stress increases formation of the dynamic secondary DNA structures called G-quadruplexes in skin fibroblasts, which is coincident with increased expression of collagen 1. This G-quadruplex formation is attenuated by a small molecule inhibitor of intracellular Ca2+ influx and an anti-fibrotic compound. In addition, we identify G-quadruplex-forming sequences in the promoter region of COL1A1, which encodes collagen 1, and confirm their ability to form G-quadruplex structures under physiologically relevant conditions. Our findings reveal a link between G-quadruplexes and scar formation that may lead to novel therapeutic interventions.

Protective roles of trigonelline against oxalate-induced epithelial-to-mesenchymal transition in renal tubular epithelial cells: An in vitro study

Fibrogenesis is a common feature for all types of chronic kidney disease (CKD). Epithelial-to-mesenchymal transition (EMT) of renal tubular epithelial cells is one of the main processes involving renal fibrosis and its inhibition is considered as a preventive/therapeutic strategy for CKD. Trigonelline (TRIG), a plant alkaloid commonly found in herbs, coffee bean, soy bean and other edible food plants, has several beneficial effects on human health and has been proposed to reduce renal fibrosis but with unclear mechanisms. This study thus addressed cellular mechanism underlying the anti-fibrogenic effects of TRIG in renal tubular epithelial cells grown in vitro. EMT was successfully induced by oxalate treatment as indicated by morphological changes into spindle-shape cells, increased expression of mesenchymal proteins (fibronectin, vimentin and α-smooth muscle actin (α-SMA)), decreased expression of epithelial proteins (E-cadherin and zonula occludens-1 (ZO-1)) and increased activity of a profibrotic factor (matrix metalloproteinase-9 (MMP-9)). Interestingly, these oxalate-induced EMT features could be attenuated by TRIG pretreatment. Moreover, TRIG also prevented oxalate-induced cell migration, reactive oxygen species (ROS) overproduction, and down-regulation of Nrf-2 signaling molecule. These data indicated that TRIG could attenuate the effects of oxalate-induced EMT and thus may serve as the anti-fibrotic compound for prevention and/or treatment of CKD.

Abstract 129: The Microbiome Product Urolithin a Abrogates the Tgf-β Egfr Pai-1 Pathway by Inhibiting Egfr Activation and Expression

Introduction: Ellagitanins are highly abundant in various foods including fruits, nuts and wines, and particularly high in commercial muscadine grape-derived supplements. The microbiome metabolizes ellagitanins to urolithins. Urolithin A (UA), the final product of the microbiome processing pathway may contribute to the cardioprotective effects of ellagitanins; however, the cellular signaling mechanisms of UA are unclear. Since the TGFβ-PAI-1 pathway contributes to fibrosis, cellular senescence and progressive kidney injury, we evaluated the effects of UA on TGFβ-induced stimulation of PAI-1 in rat renal proximal tubule cells (rPTCs - NRK52E). We hypothesize that UA may be an endogenous anti-fibrotic compound that attenuates the TGFβ-PAI-1 response. Results: TGFβ (5 ng/ml) treatment markedly increased PAI-1 levels 10-fold [43.9 ± 0.7 vs 3.5 ± 0.3 ng/ml; P&lt;0.001, n=3] in the rPTCs at 24 hrs. The TGFβ receptor (TGFβR) kinase (ALK5) inhibitor SB525334 or UA abolished the increase in PAI-1 to TGFβ [SB: 1.3 ± 0.3; UA: 2.0 ± 0.4 ng/ml; P&lt;0.001, n=3]; UA exhibits an IC50 of 3.1 μM to abrogate the TGFβ-PAI-1 response. Since the TGFβ pathway may transactivate the EGFR, treatment with the EGFR kinase inhibitors AG1478 (AG, 1 μM) and Lapatinib (LAP, 1 μM) also abrogated the PAI-1 response to TGFβ [AG: 3.3 ± 0.4; LAP: 6.2 ± 1.4 ng/ml; P&lt;0.001, n=3]. Addition of EGF alone increased PAI-1 to a similar extent as TGFβ [46.6 ± 2.7 ng/ml; P&lt;0.001, n=3], and treatment with UA or AG abolished the EGF-PAI-1 response [2.4 ± 0.1 and 1.3 ± 0.1 ng/ml, respectively; P&lt;0.001, n=3]. Given the dependence on EGFR signaling for PAI-1, we determined whether UA directly attenuates EGFR stimulation. Indeed, UA treatment inhibited peak EGFR phosphorylation to EGF at 5 mins [10 μM: 67 ± 4%; 50 μM: 71 ± 5%; P&lt;0.05, n=3], and reduced total EGFR expression at 24 hrs [10 μM: 27 ± 11%; 50 μM: 66 ± 7%; P&lt;0.05, n=6]. UA was devoid of cytotoxic effects (LDH release). Conclusion: The present study is the first to establish that UA abrogates the TGFβ-EGFR-PAI-1 pathway via the inhibition of EGFR stimulation and expression. Moreover, the potency of UA to attenuate PAI-1 is comparable to its physiological levels. The microbiome product UA may convey anti-fibrotic actions within the kidney by targeting the EGFR system.

PBI-4050 reduces pulmonary hypertension, lung fibrosis, and right ventricular dysfunction in heart failure.

Heart failure with reduced ejection fraction (HFrEF) causes lung remodelling with myofibroblasts proliferation and fibrosis leading to a restrictive lung syndrome with pulmonary hypertension (PH) and right ventricular (RV) dysfunction. PBI-4050 is a first-in-class anti-fibrotic, anti-inflammatory, and anti-proliferative compound. The present study evaluated the therapeutic impact of PBI-4050 on PH in an HFrEF model. HFrEF was induced after myocardial infarction (MI) in rats. Two weeks later, sham-operated and MI groups received PBI-4050 (200 mg/kg/day by gavage) or saline for 3 weeks. Animals were analysed according to infarct size as large (≥30% left ventricle) or medium MI (<30%). Large MI caused PH and RV hypertrophy (RVH) with a restrictive lung syndrome. PBI-4050 did not adversely affect left ventricular (LV) function but markedly reduced PH and RVH and improved RV dysfunction. PBI-4050 reduced lung remodelling and improved respiratory compliance with decreased lung fibrosis, alveolar wall cellular proliferation and α-smooth muscle actin expression. The increased expression of endothelin-1 (ET-1), transforming growth factor beta (TGF-β), interleukin-6 (IL-6) and of tissue inhibitor of metalloprotease-1 in the lungs from HFrEF were reduced with PBI-4050 therapy. Activation of isolated human lung fibroblasts (HLFs) to a myofibroblastic pro-fibrogenic phenotype was markedly reduced by PBI-4050. The fatty acid receptor GPR84 was increased in HFrEF lungs and in activated HLFs, and reduced by PBI-4050. GPR84 agonists activated fibrogenesis in HLFs and finally, PBI-4050 reduced ERK1/2 phosphorylation. PBI-4050 reduces PH and RVH in HFrEF by decreasing lung fibrosis and remodelling. This novel agent decreases the associated restrictive lung syndrome and recovers RV function. A contributing mechanism involves reducing the activation of lung fibroblasts by IL-6, TGF-β, and ET-1 by antagonism of GPR84 and reduced ERK1/2 phosphorylation. PBI-4050 is a novel promising therapy for targeting lung remodelling in group II PH.

Discovery and evaluation of inhibitor of LARP6 as specific antifibrotic compound

Fibrosis is characterized by excessive production of type I collagen. Biosynthesis of type I collagen in fibrosis is augmented by binding of protein LARP6 to the 5′ stem-loop structure (5′SL), which is found exclusively in type I collagen mRNAs. A high throughput screen was performed to discover inhibitors of LARP6 binding to 5′SL, as potential antifibrotic drugs. The screen yielded one compound (C9) which was able to dissociate LARP6 from 5′ SL RNA in vitro and to inactivate the binding of endogenous LARP6 in cells. Treatment of hepatic stellate cells (liver cells responsible for fibrosis) with nM concentrations of C9 reduced secretion of type I collagen. In precision cut liver slices, as an ex vivo model of hepatic fibrosis, C9 attenuated the profibrotic response at 1 μM. In prophylactic and therapeutic animal models of hepatic fibrosis C9 prevented development of fibrosis or hindered the progression of ongoing fibrosis when administered at 1 mg/kg. Toxicogenetics analysis revealed that only 42 liver genes changed expression after administration of C9 for 4 weeks, suggesting minimal off target effects. Based on these results, C9 represents the first LARP6 inhibitor with significant antifibrotic activity.

Pirfenidone improves survival in IPF: results from a real-life study

BackgroundPirfenidone is an antifibrotic compound approved for the treatment of idiopathic pulmonary fibrosis (IPF). We present our real-world experience in terms of Pirfenidone’s effect on mortality and adverse events profile outside the restrictions of a clinical trial.MethodsThis is a retrospective observational intention to treat study of 82 consecutive IPF patients (UHH cohort).ResultsWe observed a high 3-years survival rate of 73% without excluding patients who discontinued treatment for different reasons. The survival was compared to the survival of an IPF cohort from a tertiary referral center (RBH cohort). After exclusion of severe cases (DLco< 30%), in unadjusted analysis, the survival in the UHH cohort was better than in the RBH cohort (HR:0.32, 95% CI: 0.19–0.53, p < 0.0001). After adjustment for age, gender and FVC, the survival remained higher in the UHH cohort (HR:0.28, 95% CI: 0.16–0.48, p < 0.0001). We observed a similar safety profile compared to previously published data and a lower rate of drug discontinuation due to photosensitivity reactions. Conclusion: Pirfenidone provides a survival benefit in a real-life IPF cohort compared to previously used medications. Counselling patients and proactively managing possible adverse effects can reduce the necessity to discontinue pirfenidone.

Therapeutic administration of inhaled INS1009, a treprostinil prodrug formulation, inhibits bleomycin-induced pulmonary fibrosis in rats

Idiopathic pulmonary fibrosis is a progressive and lethal disease and while there are now two approved drugs (Esbriet® and Ofev®) additional effective treatments are still needed. Recently, prostacyclin analogs such as iloprost and treprostinil (TRE) have been shown to exert some protection against bleomycin-induced pulmonary fibrosis in mice when administered in a prophylactic regimen. In this study, we evaluated the effect of the inhaled treprostinil prodrug hexadecyl-treprostinil (C16TR) formulated in a lipid nanoparticle (INS1009) administered therapeutically in a fibrotic rat model. Male Fischer 344 rats challenged with intra-tracheal saline instillation were then treated with daily inhaled phosphate buffered saline (PBS) while rats challenged with bleomycin sulfate (3.5–4.0 mg/kg) instillation were treated with either daily inhaled PBS, daily inhaled INS1009 (10, 30, or 100 μg/kg), or twice-daily orally with the anti-fibrotic compound pirfenidone (100 mg/kg). Dosing started on day 10 post-bleomycin challenge and continued until day 27 after bleomycin. Lungs were harvested 24 h after the last dose of treatment for evaluation of lung hydroxyproline content and pulmonary histology. Lung hydroxyproline content increased from 421 μg/lung lobe in saline challenged and PBS treated animals to 673 μg/lung lobe in bleomycin challenged and PBS treated rats. Treatment of bleomycin challenged rats with 10, 30, or 100 μg/kg INS1009 dose-dependently reduced lung hydroxyproline content to 563, 501, and 451 μg/lung lobe, respectively, and pirfenidone decreased hydroxyproline content to 522 μg/lung lobe. Histologically, both INS1009 (100 μg/kg) and pirfenidone (100 mg/kg) reduced the severity of subepithelial fibrosis. Single dose pharmacokinetic (PK) studies of inhaled INS1009 in bleomycin challenged rats showed dose-dependent increases in lung C16TR concentration and plasma TRE on day 10 post-bleomycin challenge. Multiple dose PK studies of inhaled INS1009 showed dose-dependent increases only in lung C16TR concentration on day 27 post-bleomycin challenge. We also investigated the effects of TRE on the cytokine transforming growth factor-β1 (TGF-β1)-stimulated collagen gene and protein expressions in cultured human lung fibroblasts, assessed by real-time PCR and Sirius Red staining, respectively. In human fibroblasts, TRE (0.001–10 μM) inhibited TGF-β1 (20 ng/mL)-induced expression of collagen mRNA and protein in a concentration-dependent manner. These results demonstrated that inhaled INS1009, administered in a therapeutic dosing paradigm, dose-dependently (10–100 μg/kg) inhibited bleomycin-induced pulmonary fibrosis in rats. This effect may involve direct actions of TRE in suppressing collagen expression in lung fibroblasts.

Safety and efficacy of nintedanib in idiopathic pulmonary fibrosis: A real-life observational study in Greece

BackgroundNintedanib represents an antifibrotic compound able to slow down disease progression of patients with idiopathic pulmonary fibrosis (IPF). ObjectiveTo investigate the safety and efficacy of nintedanib in patients with IPF in a real-life setting. MethodsThis was a multicentre, retrospective, observational, real-life study for patients with IPF receiving nintedanib between October 2014 and October 2016. ResultsWe identified 94 patients with IPF receiving nintedanib (72 males, mean age±SD: 73.8 ± 7.5, mean%FVC±SD = 68.1 ± 18.3, mean%DLCo±SD = 44.4 ± 14.5). Diarrhea (n = 52, 55.3%) was the most commonly reported adverse event. Twenty patients (21.2%) had to permanently discontinue nintedanib due to severe adverse events. In the 6-months follow-up, median decline in %FVC predicted and %DLCO predicted were 1.36 (95%Cl: 0 to 2.97) and 4.00 (95%Cl: 2.01 to 6.20), respectively, when deaths were censored and excluded from the analysis. At 12 months, mean%FVC±SD and mean%DLCo±SD were 64.5 ± 19.1 and 43.7 ± 15.4, respectively. With regards to mortality, 17 patients (18.1%) died over a study period of 730 days. ConclusionNintedanib demonstrated an acceptable safety and efficacy profile in our real-world observational study. Prospective observational studies in the context of registries that collect well-defined supporting data over time are sorely needed to answer residual questions on drug's performance.

Longitudinal “Real-World” Outcomes of Pirfenidone in Idiopathic Pulmonary Fibrosis in Greece

Pirfenidone is an antifibrotic compound able to slow down disease progression in patients with idiopathic pulmonary fibrosis (IPF). To investigate the safety and efficacy of pirfenidone in patients with IPF in a real-life setting. This was a multicenter, retrospective, real-life, observational study for patients with IPF receiving pirfenidone. We identified 92 patients with IPF receiving pirfenidone. Eighty patients (70 males and 10 females, mean age ± SD: 68.1 + 7.5, mean %FVC ± SD = 74.9 ± 17.2, mean %DLCO ± SD = 48.1 ± 16.9) were included in the analysis. Skin-related (25%) and gastrointestinal (17.5%) adverse events were the most common and led to drug discontinuation in 22.5% of cases. The majority (87%) of patients experienced side effects during the first 6 months of treatment. At 36 months, changes in %FVC and %DLCO were -9.25 ± 16.34 and -9.26 ± 15.26, respectively. At 6, 12, and 24 months after treatment initiation (n = 80, 60, and 26), 18, 15, and 5 patients (22.5, 25, and 19.2%) experienced significant (>10%) and 11, 3, and 3 patients (13.8, 5, and 11.5%) experienced marginal (5-10%) %FVC improvement; and 13, 6, and 1 patient (16.2, 10, and 3.9%) experienced marginal (-5 to -10%) and 20, 21, and 8 patients (25, 35, and 30.8%) experienced significant decline (<-10%) in %FVCpred. Median survival was 851 days, and 41 patients died during the study period. Pirfenidone demonstrated an acceptable safety and therapeutic profile in patients with IPF on a longitudinal basis. Prospective observational registries are urgently needed to provide a real-world view of outcomes of pirfenidone in clinical practice.

Real-Time Bioimpedance Sensing of Antifibrotic Drug Action in Primary Human Cells

Fibrotic diseases are among the most serious health issues with severe burdens due to their chronic nature and a large number of patients suffering from the debilitating effects and long-term sequelae. Collagenase treatment is a nonsurgical option but has limited results. To date, there is no potent noninvasive solution for fibrosis. Part of the reason for this is the lack of appropriate in vitro live cell screening tools to assess the efficacy of new therapeutical agents. Here, we demonstrate the utility of a cell-based electrochemical impedance biosensor platform to screen the efficacy of potential antifibrotic compounds. The platform employs a label-free and noninvasive strategy to detect the progression of fibrosis and the potency of the antifibrotic molecules in real-time. The fundamental principle that governs thisnovel system is that dynamic changes in cell shape and adhesion during fibrosis can be measured accurately by monitoring the changes in the impedance. This is achieved by growing the cells on a transparent interdigitated indium tin oxide (ITO) electrodes. It was demonstrated by monitoring the efficacy of a model antifibrotic compound, PXS64, on cells collected from patients with Dupuytren's contracture. We confirmed the validity of the developed biochemical impedance biosensor as an tool for in vitro screening of antifibrotic compounds and provided quantitative information on subcellular influences of the examined chemical molecules using correlative microscopy analyses that monitor the average cell area, cell morphology, and the amount and directionality of the deposited extracellular matrix protein collagen and measurement of cytosolic Ca2+ changes.

Mogroside IIIE, a Novel Anti-Fibrotic Compound, Reduces Pulmonary Fibrosis through Toll-Like Receptor 4 Pathways.

Idiopathic pulmonary fibrosis is a progressive fibrotic lung disease, and eventually most patients develop respiratory failure with a median survival rate of 2 to 3 years after diagnosis due to the lack of clinically effective therapies. Mogroside IIIE (MGIIIE), a cucurbitane-type compound, was isolated from Siraitia grosvenorii MGIIIE has shown the strongest inhibition of nitric oxide release, a crucial inflammatory factor, from lipopolysaccharide (LPS)-treated RAW264.7 cells compared with other mogrosides. In the pulmonary fibrosis mouse model induced by bleomycin, MGIIIE treatment attenuated pulmonary fibrosis, indicated as a reduction in myeloperoxidase activity, collagen deposition, and pathologic score. MGIIIE also significantly suppressed expression of several important fibrotic markers, e.g., α-smooth muscle actin, collagen I, transforming growth factor-β (TGF-β) signal, and metalloproteinases-9/tissue inhibitor of metalloproteinase-1. Furthermore, MGIIIE blocked tansdifferentiation of lung resident fibroblasts into myofibroblast-like cells induced by TGF-β or LPS and subsequently inhibited collagen production in lung fibroblasts. These data indicate that MGIIIE is a potent inhibitor for pulmonary fibrosis. In vitro and in vivo mechanistic studies have shown that MGIIIE significantly decreased expression of toll-like receptor 4 (TLR4) and its downstream signals of myeloid differentiation factor 88 (MyD88)/mitogen-activated protein kinase (MAPK), an inflammatory signal essential for extracellular matrix (ECM) deposition in pulmonary fibroblasts. Taken together, these results demonstrate that MGIIIE significantly prevents pulmonary fibrosis by inhibiting pulmonary inflammation and ECM deposition through regulating TLR4/MyD88-MAPK signaling. Our study suggests that MGIIIE may have therapeutic potential for treating pulmonary fibrosis in clinical settings.

The effect of non-specific tight junction modulators on the transepithelial transport of poorly permeable drugs across airway epithelial cells

The epithelial barrier in the respiratory system is a major obstacle for drug delivery to the systemic circulation in the lung. Epithelial barrier hinders the transport of large macromolecules or polar drugs. Essential components of this epithelial fence are physical intercellular structures termed tight junctions. Therefore, modulating tight junctions can enhance paracellular transport across epithelial barrier. In this study, the effect of some of non-specific tight junction modulators (TJMs); (Sodium (Na) decanoate, oleic acid and ethyleneglycol-bis-(β-aminoethyl ether)-N, N′-tetraacetic acid (EGTA)) with established effect on intestinal tight junctions was evaluated for its effects on bronchial epithelial cells (Calu-3 cells). It was demonstrated that the effect of TJMs especially Na decanoate resulted in a reversible opening of tight junctions evidenced by the decrease in the transepithelial resistance. It was also demonstrated that this reduction of TEER upon exposing the epithelial cells to the TJMs resulted in a significant increase in Flu-Na (paracellular marker) and PXS25 (anti-fibrotic compound) transepithelial transport through this barrier. In conclusion, among the investigated non-specific TJMs, Na decanoate fulfilled the requirements of an effective, non-toxic and reversible tight junction modulator for Calu-3 lung epithelial cells.