Characterization of BBR-SD and BA-SD formulations.

<p>A) DSC analysis of BBR free drug, Physical mixture and BBR-SD formulation. B) DSC analysis of BA free drug, Physical mixture and BA-SD formulation. Scanning electron microscopic analysis of C) BBR-SD and D) BA-SD formulation by using Zeiss FESEM.</p

Combination Approach of YSA Peptide Anchored Docetaxel Stealth Liposomes with Oral Antifibrotic Agent for the Treatment of Lung Cancer

Therapeutic efficacy of nanocarriers can be amplified by active targeting and overcoming the extracellular matrix associated barriers of tumors. The aim of the present study was to investigate the effect of oral antifibrotic agent (telmisartan) on tumor uptake and anticancer efficacy of EphA2 receptor targeted liposomes. Docetaxel loaded PEGylated liposomes (DPL) functionalized with nickel chelated phospholipid were prepared using a modified hydration method. DPL were incubated with various concentrations of histidine tagged EphA2 receptor specific peptide (YSA) to optimize particle size, zeta potential, and percentage YSA binding. Cellular uptake studies using various endocytosis blockers revealed that a caveolae dependent pathway was the major route for internalization of YSA anchored liposomes of docetaxel (YDPL) in A549 lung cancer cell line. Hydrodynamic diameter and zeta potential of optimized YDPL were 157.3 ± 11.8 nm and −3.64 mV, respectively. Orthotopic lung tumor xenograft (A549) bearing athymic nude mice treated with oral telmisartan (5 mg/kg) for 2 days showed significantly (<i>p</i> < 0.05) higher uptake of YDPL in tumor tissues compared to healthy tissue. Average lung tumor weight of the YDPL + telmisartan treated group was 4.8- and 3.8-fold lower than that of the DPL and YDPL treated groups (<i>p</i> < 0.05). Substantially lower expression (<i>p</i> < 0.05) of EphA2 receptor protein, proliferating cell nuclear antigen (PCNA), MMP-9, and collagen 1A level with increased E-cadherin and TIMP-1 levels in immunohistochemistry and Western blot analysis of lung tumor samples of the combination group confirmed antifibrotic effect with enhanced anticancer activity. Active targeting and ECM remodeling synergistically contributed to anticancer efficacy of YDPL in orthotopic lung cancer

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

Pharmacokinetic profile of BA-SD and BBA-SD.

<p>The pharmacokinetic parameters of BA free drug and BA-SD formulations groups after oral administration of 100 mg/kg.</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

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

Western Blot analysis of orthotopic lung tumors lysates.

<p>A) Western blot images of Bcl-2, survivin, cyclin D1, p53, MMP-9, HIF-1α and β-actin expression in control, BBR free drug and BBR-SD formulation treated tumor lysates. B) Densitometric analysis of β-actin relative expression of C) Bcl-2 and C) survivin. Each data point was represented as mean±sem (n = 3–4). ***p<0.001 Vs respective untreated control groups.</p

Anticancer effects of BBR-SD formulations in orthotopic lung cancer model.

<p>A) Lung tumor weights after treatment with BBR free drug and BBR-SD formulations in A549 orthotopic lung tumor models, B) Lung tumor volumes after treatment with BBR free drug and BBR-SD formulations in A549 orthotopic lung tumor models, C) Representative lung tumor images taken from control, BBR free drug and BBR-SD treated (3 weeks daily 100 mg/kg dose) animals. Each data point was represented as mean±sem (n = 6–8). *p<0.05 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

Western blot Densitometric analysis.

<p>A) Densitometric analysis of β-actin relative expression of A) cyclin D1, B) p53, C) MMP-9 and D) HIF-1α. Each data point was represented as mean±sem (n = 3–4). ***p<0.001 Vs respective untreated control groups.</p