The Effect of Microbubble-Assisted Ultrasound on Molecular Permeability across Cell Barriers.

The combination of ultrasound and microbubbles (USMB) has been applied to enhance drug permeability across tissue barriers. Most studies focused on only one physicochemical aspect (i.e., molecular weight of the delivered molecule). Using an in vitro epithelial (MDCK II) cell barrier, we examined the effects of USMB on the permeability of five molecules varying in molecular weight (182 Da to 20 kDa) and hydrophilicity (LogD at pH 7.4 from 1.5 to highly hydrophilic). Treatment of cells with USMB at increasing ultrasound pressures did not have a significant effect on the permeability of small molecules (molecular weight 259 to 376 Da), despite their differences in hydrophilicity (LogD at pH 7.4 from -3.2 to 1.5). The largest molecules (molecular weight 4 and 20 kDa) showed the highest increase in the epithelial permeability (3-7-fold). Simultaneously, USMB enhanced intracellular accumulation of the same molecules. In the case of the clinically relevant anti- C-X-C Chemokine Receptor Type 4 (CXCR4) nanobody (molecular weight 15 kDa), USMB enhanced paracellular permeability by two-fold and increased binding to retinoblastoma cells by five-fold. Consequently, USMB is a potential tool to improve the efficacy and safety of the delivery of drugs to organs protected by tissue barriers, such as the eye and the brain.

Evaluation of Cell-Penetrating Peptides as Versatile, Effective Absorption Enhancers: Relation to Molecular Weight and Inherent Epithelial Drug Permeability.

PURPOSE: The poor permeability of new drug candidates across intestinal epithelial membranes complicates their development in oral form. This study investigated the potential of cell-penetrating peptides (CPPs) to improve the intestinal permeation and absorption of low-permeable low-molecular-weight (low-MW) drugs. METHODS: The in vitro epithelial permeation of six different drugs (metformin, risedronate, zanamivir, methotrexate [MTX], tacrolimus, and vincristine [VCR]) across Caco-2 cell monolayers was examined in the presence and absence of L- or D-penetratin, and the correlation between permeation enhancement efficiency and the properties of tested drugs was analyzed. In addition, a rat closed ileal loop absorption study was conducted to determine the in vivo effects of penetratin. RESULTS: MTX and VCR efficiently permeated Caco-2 monolayers in the presence of L- and D-penetratin, suggesting that CPPs enhanced the epithelial permeation of drugs with relatively high molecular weight and resultant limited intrinsic permeability. The in vivo rat closed ileal loop absorption study revealed the stimulatory effect of L- and D-penetratin on the intestinal absorption of MTX and VCR. CONCLUSIONS: CPPs are useful as oral absorption enhancers for low-permeable drugs.

Effects of intestinal luminal contents and the importance of microfold cells on the ability of cell-penetrating peptides to enhance epithelial permeation of insulin.

We previously reported that oral and intestinal absorption of insulin in rats and mice is significantly enhanced in vivo by coadministration with cell-penetrating peptides (CPPs). To evaluate the clinical use of CPPs as absorption enhancers, it is imperative to clarify the mechanisms associated with the permeation-stimulatory effect of CPPs in vitro. The confirmation experiment revealed a discrepancy between in vivo and in vitro effects of CPPs, such as D-octaarginine (D-R8) and L-penetratin, on epithelial permeation of insulin. The present study was designed to determine the factors that work in vivo but are deficient in an in vitro system consisting of Caco-2 cells. The effects of D-R8 and L-penetratin on permeation of insulin through the Caco-2 cell monolayer were partially boosted in fasted-state simulated intestinal fluid (FaSSIF). Contrary to expectation, the effects of CPPs on cellular uptake of insulin and the binding ratio of CPPs to insulin analyzed by surface plasmon resonance in normal buffer and FaSSIF were similar. Also, the effects of CPPs, especially D-R8, on cellular uptake of insulin, were stronger in Caco-2 cell monolayers with microfold cell (M cell)-like properties. These results suggested a key role of intestinal lipids and M cells in the stimulatory effect of CPPs on net epithelial permeation of insulin in vivo.

Development and characterization of a solid dispersion film for the vaginal application of the anti-HIV microbicide UAMC01398.

The purpose of this work was to design and evaluate a vaginal film delivery system for UAMC01398, a novel non-nucleoside reverse transcriptase inhibitor currently under investigation for use as an anti-HIV microbicide. UAMC01398 (1mg) films consisting of hydroxypropylmethylcellulose (HPMC) and polyethylene glycol 400 (PEG400) in different ratios were prepared by solvent evaporation. Based on its flexibility, softness and translucent appearance, the 30% PEG400 and 70% HPMC containing film was selected for further assessment. The vaginal film formulation was fast-dissolving (<10 min in 1 mL of vaginal fluid simulant), stable up to at least one month and safe toward epithelial cells and lactobacilli. Furthermore, formulating UAMC01398 into the film dosage form did not influence its antiviral activity. Powder X-ray diffraction revealed the amorphous nature of the UAMC01398 film, resulting in enhanced compound permeation across the epithelial HEC-1A cell layer, presumably owing to the induction of supersaturation. The in vivo vaginal tissue uptake of UAMC01398 in rabbits, as measured by systemic concentrations, was increased compared to the previously established non-solubilizing gel (significant difference) and sulfobutyl ether-ß-cyclodextrin (5%) containing gel. To conclude, we identified a film formulation suitable for the vaginal delivery of UAMC01398.

Mechanistic study of the uptake/permeation of cell-penetrating peptides across a caco-2 monolayer and their stimulatory effect on epithelial insulin transport.

Our recent studies have demonstrated the potential of cell-penetrating peptides (CPPs) to significantly stimulate the intestinal absorption of therapeutic peptides and proteins. This study examined the mechanisms underlying the intestinal epithelial uptake and permeation of CPPs and their contribution to the enhanced absorption of insulin. Fluorescein-tagged octaarginine (R8) and penetratin were used as the promising CPPs, and in vitro uptake and permeation assays were conducted using Caco-2 cell monolayer. The assay conducted under low temperature conditions revealed that energy-dependent pathways are not involved in d-form arginines (d-R8) uptake or its stimulatory effect on insulin uptake. The Km value (3.82 µM), calculated from the dose dependence of d-R8 uptake, suggested that a part of the d-R8 uptake was saturated at the functional concentration (60 µM d-R8). An analysis based on the binding parameters of insulin and d-R8 also showed an increase in the uptake clearance of the insulin/d-R8 complex, even at a saturated concentration of d-R8, implying that this complex is taken up by Caco-2 cells via pathways that differ from those that take up unbound d-R8. Thus, this study suggests that CPPs such as oligoarginines stimulate the intestinal epithelial transport of peptide and protein drugs via energy-independent unsaturable internalization.