Inhibition of discoidin domain receptors by imatinib prevented pancreatic fibrosis demonstrated in experimental chronic pancreatitis model

Abstract Discoidin domain receptors (DDR1 and DDR2) are the collagen receptors of the family tyrosine kinases, which play significant role in the diseases like inflammation, fibrosis and cancer. Chronic pancreatitis (CP) is a fibro-inflammatory disease in which recurrent pancreatic inflammation leads to pancreatic fibrosis. In the present study, we have investigated the role of DDR1 and DDR2 in CP. The induced expression of DDR1 and DDR2 was observed in primary pancreatic stellate cells (PSCs) and cerulein-induced CP. Subsequently, the protective effects of DDR1/DDR2 inhibitor, imatinib (IMT) were investigated. Pharmacological intervention with IMT effectively downregulated DDR1 and DDR2 expression. Further, IMT treatment reduced pancreatic injury, inflammation, extracellular matrix deposition and PSCs activation along with inhibition of TGF-β1/Smad signaling pathway. Taken together, these results suggest that inhibition of DDR1 and DDR2 controls pancreatic inflammation and fibrosis, which could represent an attractive and promising therapeutic strategy for the treatment of CP

An Adaptogen: Withaferin A Ameliorates in Vitro and in Vivo Pulmonary Fibrosis by Modulating the Interplay of Fibrotic, Matricelluar Proteins, and Cytokines

Pulmonary fibrosis (PF) is chronic lung disease with only two FDA approved clinically available drugs, with limited safety profile. Inadequate therapy motivated us to explore the effect of vimentin inhibitor Withaferin A, as an anti-fibrotic agent against TGF-β1-induced in vitro fibrotic events and Bleomycin induced in vivo fibrosis with an emphasis on epithelial to mesenchymal transition (EMT), extracellular matrix deposition (ECM), inflammation, and angiogenesis. In vitro EMT and fibrotic events were induced by TGF-β1 in alveolar epithelial cells and human fetal lung fibroblasts followed by treatment with Withaferin A (0.25, 0.5, and 1 μM concentrations) to explore its anti-fibrotic effects. In vivo potential of Withaferin A (2 and 4 mg/kg) was assessed in murine model of Bleomycin induced PF. All the parameters and molecular studies related to PF were performed at the end of treatment period. Withaferin A treatment reduced the progression of PF by modulating the EMT related cell markers both in vivo and in vitro. Withaferin A ameliorated the expression of inflammatory cytokines including NF-κB p65, IL-1β and TNF-α, as well as attenuated the expression of pro-fibrotic proteins including CTGF, collagen 1A2, collagen 3A1, and fibronectin. Expression of angiogenic factors like VEGF, FAK, p38 MAPK, and PLC-γ1 were also inhibited by Withaferin A. Phosphorylation of Smad 2/3 induced by TGF-β1 and Bleomycin were significantly inhibited. Withaferin A suppressed expression of pro-inflammatory, pro-fibrotic, and pro-angiogenic mediators and also reduced the ECM deposition. In a nutshell, Withaferin A could probably prove as an efficient and potential therapeutic against PF

Amelioration of experimentally induced inflammatory arthritis by intra-articular injection of visnagin

Background: Rheumatoid arthritis (RA) is a chronic inflammatory autoimmune disease characterized by synovial hyperplasia, cartilage destruction and bone erosion. Visnagin (VIS) is a proven anti-inflammatory agent and in this study, we aimed to evaluate the anti-arthritic activity of VIS when administered via intra-articular (I.A.) route of administration. Materials and methods: RAW 264.7 ​cells were stimulated with lipopolysaccharide (LPS) (1 ​μg/mL) and treated with VIS at concentrations of 12.5 and 25 ​μM. Arthritis was induced in Sprague Dawley rats by administering Complete Freund's Adjuvant (CFA) (1 ​mg/mL) through (I.A.) route and treated with VIS via (I.A.) route at doses of 3 and 10 ​mg/kg twice a week for 3 weeks. Protective effects were assessed by arthritic score, behavioral studies for pain evaluation, radiological assessment, histopathological examination and molecular studies. Results: Our results indicated that VIS significantly reduced LPS induced inflammation in RAW 264.7 ​cells. While in arthritic rats, VIS reduced the disease scorings with improvement towards pain. Pathological examination demonstrated that VIS reduced knee joint inflammation and cartilage destruction. Radiographic analysis and molecular studies also supported the protective effects of VIS. Conclusion: The results of the study imply that VIS exerted potential anti-inflammatory and anti-arthritic activity in in vitro and in vivo models of RA

IHC analysis and VEGF levels.

<p>A) Quantification of cleaved caspase 3, CD31 positive MVD and TUNEL positive cells by IHC analysis. For IHC quantification, 10 fields were randomly counted from each tumor sections and 4 slides per group were used. B) VEGF levels in serum and tumor lysates after treatment with BBR free drug and BBR-SD. Each data point was represented as mean±sem (n = 3–4). *p<0.05 and ***p<0.001 Vs respective untreated control groups.</p

Anticancer effects of BA-SD formulations in orthotopic and metastatic lung cancer models.

<p>A) Lung tumor weights and tumor volumes after treatment with BA free drug and BA-SD formulations in A549 orthotopic and metastatic lung tumor models, B) Number of lung tumor nodules in peripheral, medial and central lobes in A549 metastatic models after treatment with BA free drug and BA-SD formulations. C) Quantification of TUNEL positive cells in orthotopic lung tumors D) Representative TUNEL assay images of orthotopic lung tumor images from control, BA free drug and BA-SD treated groups. Each data point was represented as mean±sem (n = 6–8). **p<0.01 and ***p<0.001 Vs respective untreated control groups.</p

Approaches to Improve the Oral Bioavailability and Effects of Novel Anticancer Drugs Berberine and Betulinic Acid

<div><p>Background</p><p>The poor bioavailability of Berberine (BBR) and Betulinic acid (BA) limits the development of these promising anticancer agents for clinical use. In the current study, BBR and BA in spray dried (SD) mucoadhesive microparticle formulations were prepared.</p><p>Methods</p><p>A patented dual channel spray gun technology established in our laboratory was used for both formulations. Gastrointestinal (GI) permeability studies were carried out using Caco-2 cell monolayer grown in in-vitro system. The oral bioavailability and pharmacokinetic profile of SD formulations were studied in Sprague Dawley rats. A549 orthotopic and H1650 metastatic NSCLC models were utilized for the anticancer evaluations.</p><p>Results</p><p>Pharmacokinetic studies demonstrated that BBR and BA SD formulations resulted in 3.46 and 3.90 fold respectively, significant increase in plasma C_max concentrations. AUC levels were increased by 6.98 and 7.41 fold in BBR and BA SD formulations, respectively. Compared to untreated controls groups, 49.8 & 53.4% decrease in the tumor volumes was observed in SD formulation groups of BBR and BA, respectively. Molecular studies done on excised tumor (A549) tissue suggested that BBR in SD form resulted in a significant decrease in the survivin, Bcl-2, cyclin D1, MMP-9, HIF-1α, VEGF and CD31 expressions. Cleaved caspase 3, p53 and TUNEL expressions were increased in SD formulations. The RT-PCR analysis on H1650 tumor tissue suggested that p38, Phospho-JNK, Bax, BAD, cleaved caspase 3&8 mRNA expressions were significantly increased in BA SD formulations. Chronic administration of BBR and BA SD formulations did not show any toxicity.</p><p>Conclusions</p><p>Due to significant increase in oral bioavailability and superior anticancer effects, our results suggest that spray drying is a superior alternative formulation approach for oral delivery of BBR and BA.</p></div

Immunohistochemical (IHC) analysis of orthotopic lung tumor sections.

<p>IHC analysis of tumor sections collected from untreated control, BBR free drug and BBR-SD formulation treated animals. First row of images shows the cleaved caspase-3 expression in different groups. The brown color stained cells indicate the cleaved caspase-3 specific positive cells. Second row shows the CD31 expression, the brown colored cells suggest the MVD positive cells. Third row shows the TUNEL assay, brown color stained cells indicate the apoptotic positive cells.</p

Pharmacokinetic profiles of BBR-SD and BA-SD formulations.

<p>A) Plasma concentration time profile of BBR free drug and BBR-SD formulation in Sprague Dawley rats. B) Plasma concentration time profile of BA free drug and BA-SD formulation in Sprague Dawley rats. In both the studies, rats were administered at the dose of 100 mg/kg, orally. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089919#pone-0089919-g003" target="_blank">Figure 3B</a> also shows the plasma concentration and time profiles of BA upon intravenous administration. Each data point was represented as mean±sem (n = 3–4).</p