Highly respirable dry powder inhalable formulation of voriconazole with enhanced pulmonary bioavailability

<p><b>Objective</b>: To develop and characterize a highly respirable dry powder inhalable formulation of voriconazole (VRZ).</p> <p><b>Methods</b>: Powders were prepared by spray drying aqueous/alcohol solutions. Formulations were characterized in terms of particle size, morphology, thermal, moisture responses and aerosolization performance. Optimized powder was deposited onto an air-interface Calu-3 model to assess their uptake across Calu-3 lung epithelia. Optimized formulation was evaluated for stability (drug content and aerosol performance) for 3 months. Additionally, Calu-3 cell viability, lung bioavailability and tissue distribution of optimized formulation were evaluated.</p> <p><b>Results</b>: Particle size and aerosol performance of dry powder containing 80% w/w VRZ and 20% w/w leucine was appropriate for inhalation therapy. Optimized formulation showed irregular morphology, crystalline nature, low moisture sensitivity and was stable for 3 months at room temperature. Leucine did not alter the transport kinetics of VRZ, as evaluated by air-interface Calu-3 model. Formulation was non-cytotoxic to pulmonary epithelial cells. Moreover, lung bioavailability and tissue distribution studies in murine model clearly showed that VRZ dry powder inhalable formulation has potential to enhance therapeutic efficacy at the pulmonary infection site whilst minimizing systemic exposure and related toxicity.</p> <p><b>Conclusion</b>: This study supports the potential of inhaled dry powder VRZ for the treatment of fungal infections.</p

Honokiol modulates Bax/Bcl-2 and Bax/Bcl-xL ratio in human pancreatic cancer cells.

<p>(A) MiaPaCa and Panc1 cells were treated with either honokiol (20, 40 or 60 µM) or DMSO (control) for 24 h. Immunoblotting was performed for Bcl-xl, Bcl-2 and Bax proteins followed by densitometry of immunoreactive bands. Normalized densitometric values are indicated at the top of the bands. (B) Bar diagram summarizing the effects of honokiol treatment on Bax/Bcl-2 ratio (upper panel) and Bax/Bcl-xL ratio (lower panel). Data suggest that honokiol induces apoptosis by upregulating pro-apoptotic Bax and downregulating anti-apoptotic Bcl-2 and Bcl-xL proteins.</p

Honokiol induces apoptosis in human pancreatic cancer cells.

<p>MiaPaCa and Panc1 cells were grown in 6-well plates (1×10⁶ cells /well) and allowed to attain 70–80% confluence. Cells were treated with either honokiol (20, 40 or 60 µM) or DMSO (control) for 24 h and subsequently stained with 7-AAD and PE Annexin V followed by flow cytometry. The lower left quadrants of each panels show the viable cells (negative for both, PE Annexin V and 7-AAD). The upper right quadrants contain necrotic or late apoptotic cells (positive for both, PE Annexin V and 7-AAD). The lower right quadrants represent the early apoptotic cells (PE Annexin V positive and 7-AAD negative). Data show a dose-dependent increase in the number of apoptotic cells in both MiaPaCa and Panc1 cells after treatment with honokiol as compared to control cells, indicating apoptotis inducing potential of honokiol.</p

Honokiol causes G₁ phase cell cycle arrest in human pancreatic cancer cells.

<p>MiaPaCa and Panc1cells (1×10⁶ cells/well) were synchronized by culturing in serum free media for 72 h, followed by incubation in serum-containing media for 24 h and subsequent treatment with either honokiol (20, 40 or 60 µM) or DMSO (control) for 24 h. Distribution of cells in different phases of cell cycle was analyzed by propidium iodide (PI) staining followed by flow cytometry. Enhanced accumulation of MiaPaCa and Panc1 cells in the G₁ phase of the cell cycle was observed after treatment with honokiol in a dose-dependent manner (as indicated by flow histograms) with a concomitant decrease in S-phase cells.</p

Honokiol suppresses growth of human pancreatic cancer cells.

<p>(<b>A</b>) MiaPaCa and Panc1 cells were seeded in 6 well plate (1×10⁵ cells/well) and allowed to attain 70–80% confluence prior to honokiol (10–60 µM) treatment for 48 h. Following treatment, significant change in cell morphology was observed of both the cell types as examined under phase-contrast microscope. Cells became round, shrunken and detached from cell surface in a dose-dependent manner. Representative micrographs are from one of the random fields of view (magnification 200X) of cells treated with 20, 40 or 60 µM honokiol. (<b>B</b>) MiaPaCa and Panc1 cells were grown in 96 well microtitre plates (1×10⁴ cells /well) and treated with honokiol (10–60 µM) at 70–80% confluence. Percent viability of cells was measured by WST-1 assay after 24, 48 and 72 h. An OD value of control cells (treated with an equal volume of DMSO, final concentration, <0.1%) was taken as 100% viability. Honokiol inhibited cell viability in a dose- and time- dependent manner for both the cell types suggesting anti-tumor effect of honokiol. Data are expressed as mean± SD; (n = 3).</p

Honokiol attenuates constitutive NF-κB activation by inhibiting nuclear translocation of NF-κB/p65 in human pancreatic cancer cells.

<p>(A) MiaPaCa and Panc1cells (0.5×10⁶ cells/well) were seeded in 12-well plate. Next day at 60% confluence, cells were co-transfected with NF-κB luciferase reporter and TK-Renilla luciferase (control) plasmids. Twenty-four hours post-transfection, cells were treated with honokiol (20, 40, or 60 µM) for next 24 h. Protein lysates were made and luciferase (Fire-fly; test and Renilla, transfection efficiency control) activity assessed using a dual-luciferase assay system. Data is presented as normalized fold-change in luciferase activity (mean± SD; n = 3, * p<0.05). (B) Total, nuclear and cytoplasmic extracts were prepared from cells treated with honokiol (20, 40, or 60 µM) for 6 h and expression of NF-κB/p65, p-IκB-α (S32/36) and IκB-α was determined by Western blot analysis. β-actin was used as a loading control. Intensities of the immunoreactive bands were quantified by densitometry. Normalized densitometry values are indicated at the top of the bands indicating a decreased localization of NF-κB/p65 in nucleus with a concomitant increase in cytoplasm. In contrast, expression of p-IκB-α was decreased leading to increased levels of IκB-α. Altogether, these data clearly suggest that honokiol inhibits NF-κB activity through stabilization of IκB-α.</p

miR-4723 directly targets Abl kinases in prostate cancer cells.

<p>(A) Profiling of apoptotic genes and, (B) Cell cycle related genes after overexpression of miR-4723 in PC3 cells showing downregulation of Abl1 by miR-4723. (C) Relative mRNA expression of Abl1 gene as assessed by RT-PCR. (D) Schematic representation of the Abl1 and Abl2 3′-UTRs showing the relative positions of putative miR-4723 binding sites. (E) Immunoblots of endogenous Abl1, Abl2 and other miR-4723 targets in PC3 cells transfected as indicated. GAPDH was used a loading control. (F) Abl1 3′ UTR construct encompassing miR-4723 binding sites or the control construct was cotransfected into PC3 cells with miR-4723 or miR-CON and assayed for relative luciferase activity (*p<.05).</p

miR-4723 reexpression induces apoptosis in prostate cancer cells.

<p>(A) Apoptosis assay in PC3 cells after miR-CON (left panels) or miR-4723 (right panels) transfection for 72 hrs as assessed by ANNEXIN V-FITC/7-AAD staining. (B) Representation of average apoptotic fractions (early+late apoptotic) in each group (*p<.05).</p

Clinical significance of miR-4723 expression in prostate cancer.

<p>(A) ROC curve analysis showing the ability of miR-4723 expression to discriminate between malignant and non-malignant prostate tissue samples. (B) Kaplan-Meier survival curves for prostate cancer patients, stratified based on miR-4723 expression (low and high). p value based on log rank test (*p<.05).</p

Expression of Abl kinase is inversely correlated with miR-4723 expression in prostate cancer.

<p>We examined the correlation between miR-4723 and Abl expression in a subset of our clinical cohort by performing immunohistochemical staining for Abl in PCa tissues (n = 12). Relative expression levels of Abl (as scored by IHC) and miR-4723 (as determined by RT-PCR) for the samples analysed are represented in the bar graph.</p