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

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

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

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

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

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

AFM Images for NMG formulation.

<p>(a) Topographic (b) Phase mode (c) 3D topography (d) Mean NP radius.</p