Young-Joon Park

Effect of the solid-dispersion method on the solubility and crystalline property of tacrolimus.

Three solid dispersions containing poorly water-soluble tacrolimus were prepared with hydroxypropyl-beta-cyclodextrin (HP-beta-CD) and dioctyl sulfosuccinate (DOSS) using a spray-drying technique via the solvent-evaporation method with a methylene chloride/ethanol mixture, the solvent-wetting method with ethanol and the surface-attached method with water, respectively. The solubility and dissolution of the drug in the three solid dispersions were evaluated compared to drug powder. Furthermore, their physicochemical properties were investigated using SEM, DSC and powder X-ray diffraction. The solubility and dissolution of the drug were significantly improved in the order of the tacrolimus-loaded solid dispersion prepared by: solvent-evaporation method>solvent-wetting method>surface-attached method. The solid dispersions prepared by solvent evaporation appeared as an aggregated form with the amorphous form. In particular, the solid dispersion prepared by the solvent-evaporation method improved solubility about 900-fold and dissolution of tacrolimus 15-fold because of its reduced particle size, increased surface area and close contact between the hydrophilic carrier and the drug. In the solvent-wetting method, the drug, which was changed to an amorphous form, was attached onto the surface of undissolved carriers. However, the solid dispersion prepared by the surface-attached method gave an unchanged crystalline form. In this solid dispersion, the carriers were attached to the surface of the undissolved drug, resulting in changing the drug from being hydrophobic to hydrophilic. As the crystal form of drug in this solid dispersion was not converted to the amorphous form unlike other solid dispersions, it gave relatively less solubility and dissolution of the drug than did the others. Thus, in the development of a solid-dispersion system containing poorly water-soluble drugs, the method of preparation plays an important role in the solubility and crystallinity of the drugs.

Physicochemical characterization of tacrolimus-loaded solid dispersion with sodium carboxylmethyl cellulose and sodium lauryl sulfate

To develop a novel tacrolimus-loaded solid dispersion with improved solubility, various solid dispersions were prepared with various ratios of water, sodium lauryl sulfate, citric acid and carboxylmethylcellulose-Na using spray drying technique. The physicochemical properties of solid dispersions were investigated using scanning electron microscopy, differential scanning calorimetery and powder X-ray diffraction. Furthermore, their solubility and dissolution were evaluated compared to drug powder. The solid dispersion at the tacrolimus/CMC-Na/sodium lauryl sulfate/citric acid ratio of 3/24/3/0.2 significantly improved the drug solubility and dissolution compared to powder. The scanning electron microscopy result suggested that carriers might be attached to the surface of drug in this solid dispersion. Unlike traditional solid dispersion systems, the crystal form of drug in this solid dispersion could not be converted to amorphous form, which was confirmed by the analysis of DSC and powder X-ray diffraction. Thus, the solid dispersion system with water, sodium lauryl sulfate, citric acid and CMC-Na should be a potential candidate for delivering a poorly water-soluble tacrolimus with enhanced solubility and no convertible crystalline.

Clotrimazole Ameliorates Intestinal Inflammation and Abnormal Angiogenesis by Inhibiting Interleukin-8 Expression through a Nuclear Factor-κB-Dependent Manner

Increased interleukin (IL)-8 plays an important role not only in activation and recruitment of neutrophils but also in inducing exaggerated angiogenesis at the inflamed site. In the present study, we investigated the fact that clotrimazole (CLT) inhibits intestinal inflammation, and the inhibitory action is mediated through suppression of IL-8 expression. In the trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model, CLT dose-dependently protected from the TNBS-induced weight loss, colon ulceration, and myeloperoxidase activity increase. In the lesion site, CLT also suppressed the TNBS-induced angiogenesis, IL-8 expression, and nuclear factor (NF)-κB activation. In a cellular model of colitis using tumor necrosis factor (TNF)-α-stimulated HT29 colon epithelial cells, treatment with CLT significantly suppressed TNF-α-mediated IL-8 induction and NF-κB transcriptional activity revealed by a luciferase reporter gene assay. Furthermore, cotreatment with CLT and pyrrolidine dithiocarbamate, a NF-κB inhibitor, synergistically reduced the NF-κB transcriptional activity as well as IL-8 expression. In an in vitro angiogenesis assay, CLT suppressed IL-8-induced proliferation, tube formation, and invasion of human umbilical vein endothelial cells. The in vivo angiogenesis assay using chick chorioallantoic membrane also showed that CLT significantly inhibited the IL-8-induced formation of new blood vessels. Taken together, these results suggest that CLT may prevent the progression of intestinal inflammation by not only down-regulating IL-8 expression but also inhibiting the action of IL-8 in both colon epithelial and vascular endothelial cells during pathogenesis of intestinal inflammation.

Anti-inflammatory action of mollugin and its synthetic derivatives in HT-29 human colonic epithelial cells is mediated through inhibition of NF-κB activation

Mollugin is the active compound of Rubia cordifolia, which has been used as a traditional Chinese medicine for the treatment of various inflammatory diseases including arthritis and uteritis. In the present study, we investigated for the first time the inhibitory effects and the mechanisms of action of mollugin (M1) and its synthetic derivatives (M2-M4) on tumor necrosis factor (TNF)-alpha-induced inflammatory responses in HT-29 human colon epithelial cells. Treatment with M1 and its derivatives M2-M4 significantly inhibited TNF-alpha-induced attachment of U937 monocytic cells to HT-29 cells, which mimics the initial phase of colon inflammation. TNF-alpha-induced mRNA induction of the chemokines, monocyte chemoattractant protein (MCP)-1 and interleukin (IL)-8, and the intercellular cell adhesion molecule (ICAM)-1, which are involved in adhesion between leukocytes and epithelial cells, was suppressed by M1-M4, and M1 was the most efficacious. In addition, M1-M4 significantly suppressed TNF-alpha-induced NF-kappaB transcriptional activity. Such NF-kappaB inhibitory activity of M1-M4 (20 microM) correlated with their ability to suppress TNF-alpha-induced chemokine expression and U937 monocytic cell adhesion to HT-29 colonic epithelial cells. Treatment of HT-29 cells with M1 and PDTC, a NF-kappaB inhibitor, synergistically suppressed both TNF-alpha-induced NF-kappaB activation and monocytic cell adhesion to HT-29 cells. These results suggest that M1-M4 inhibit TNF-alpha-induced expression of inflammatory molecules via NF-kappaB, and that M1, a potent NF-kappaB inhibitor, may be a valuable new drug candidate for the treatment of colon inflammation.

Clotrimazole ameliorates intestinal inflammation and abnormal angiogenesis by inhibiting IL-8 expression through a NF-κB-dependent manner

Increased interleukin (IL)-8 plays an important role not only in activation and recruitment of neutrophils but also in inducing exaggerated angiogenesis at the inflamed site. In the present study, we investigated the fact that clotrimazole (CLT) inhibits intestinal inflammation, and the inhibitory action is mediated through suppression of IL-8 expression. In the trinitrobenzene sulfonic acid (TNBS)-induced rat colitis model, CLT dose-dependently protected from the TNBS-induced weight loss, colon ulceration, and myeloperoxidase activity increase. In the lesion site, CLT also suppressed the TNBS-induced angiogenesis, IL-8 expression, and nuclear factor (NF)-κB activation. In a cellular model of colitis using tumor necrosis factor (TNF)-α-stimulated HT29 colon epithelial cells, treatment with CLT significantly suppressed TNF-α-mediated IL-8 induction and NF-κB transcriptional activity revealed by a luciferase reporter gene assay. Furthermore, cotreatment with CLT and pyrrolidine dithiocarbamate, a NF-κB inhibitor, synergistically reduced the NF-κB transcriptional activity as well as IL-8 expression. In an in vitro angiogenesis assay, CLT suppressed IL-8-induced proliferation, tube formation, and invasion of human umbilical vein endothelial cells. The in vivo angiogenesis assay using chick chorioallantoic membrane also showed that CLT significantly inhibited the IL-8-induced formation of new blood vessels. Taken together, these results suggest that CLT may prevent the progression of intestinal inflammation by not only down-regulating IL-8 expression but also inhibiting the action of IL-8 in both colon epithelial and vascular endothelial cells during pathogenesis of intestinal inflammation.

Development of novel ibuprofen-loaded solid dispersion with improved bioavailability using aqueous solution

To develop a novel ibuprofen-loaded solid dispersion with enhanced bioavailability, various ibuprofen-loaded solid dispersions were prepared with water, HPMC and poloxamer. The effect of HPMC and poloxamer on aqueous solubility of ibuprofen was investigated. The dissolution and bioavailability of solid dispersion in rats were then evaluated compared to ibuprofen powder. When the amount of carrier increased with a decreased in HPMC/poloxamer ratio, the aqueous solubility of ibuprofen was elevated. The solid dispersion composed of ibuprofen/HPMC/poloxamer at the weight ratio of 10:3:2 improved the drug solubility approximately 4 fold. It gave significantly higher initial plasma concentration, AUC and Cmax of drug than did ibuprofen powder in rats. The solid dispersion improved the bioavailability of drug about 4-fold compared to ibuprofen powder. Thus, this ibuprofen-loaded solid dispersion with water, HPMC and poloxamer was a more effective oral dosage form for improving the bioavailability of poor water-soluble ibuprofen.

Inhibitory effects of clotrimazole on TNF-α-induced adhesion molecule expression and angiogenesis

Cell adhesion molecules play a pivotal role in chronic inflammation and pathological angiogenesis. In the present study, we investigated the inhibitory effects of clotrimazole (CLT) on tumor necrosis factor (TNF)-α-induced changes in adhesion molecule expression. CLT dose-dependently inhibited monocyte chemoattractant protein-1 (MCP-1), intercellular cell adhesion molecule-1 (ICAM-1), and vascular cell adhesion molecule-1 (VCAM-1) expressions in TNF-α-stimulated HT29 colonic epithelial cells. This inhibitory action of CLT correlated with a significant reduction in TNF-α-induced adhesion of monocytes to HT29 cells, which was comparable to the inhibitory effects of anti-ICAM-1 and VCAM-1 monoclonal antibodies on monocyte-epithelial adhesion. These inhibitory actions of CLT were, at least in part, attributable to the inhibition of redox sensitive NF-κB activation, as CLT inhibited TNF-α-induced ROS generation as well as NF-κB nuclear translocation and activation in HT29 cells. Furthermore, the inhibition of TNF-α-induced monocyte adhesion was also mimicked by the specific NF-κB inhibitor, pyrrolidine dithiocarbamate (PDTC). Inflammatory mediators including TNF-α have known to promote angiogenesis, which in turn further contributes to inflammatory pathology. Therefore, we additionally evaluated whether CLT modulates TNF-α-induced angiogenesis using in vivo chick chorioallantoic membrane (CAM) assay. The CAM assay showed that CLT dose-dependently attenuated TNF-α-induced angiogenesis, and the effect was correlated with decreased inflammation of the CAM tissue. In conclusion, our results suggest that CLT can inhibit TNF-α-triggered expression of adhesion molecules, ICAM-1 and VCAM-1, and angiogenesis during inflammation.

Protective effects of Pogostemon cablin Bentham water extract on inflammatory cytokine expression in TNBS-induced colitis in rats

In inflammatory bowel disease (IBD), colon epithelial cells express a variety of inflammatory mediators, including chemokines, which perpetuate inflammatory response. In the current study, we report that water extract of Pogostemon cablin Bentham aerial parts (PCW), which has traditionally been used for treatment of the common cold and infectious disease, suppressed colon inflammation. Treatment with PCW resulted in effective inhibition of tumor necrosis factor (TNF)-α-induced adhesion of monocytes to HT-29 human colonic epithelial cells. In a trinitrobenzene sulfonic acid (TNBS)-induced rat model of IBD, PCW suppressed clinical signs of colitis, including weight loss, colon tissue myeloperoxidase activity, a marker for inflammatory cell infiltration, and cyclooxygenase-2 expression in a dose-dependent manner. In addition, PCW suppressed TNBS-induced mRNA expression of IL-8, MCP-1, and IL-6 in rat colon. The nuclear level of NF-κB in TNBS-treated rat colon and NF-κB luciferase reporter gene activity in TNF-α-treated HT-29 cells were significantly inhibited by PCW. Taken together, the results of this study suggest that PCW suppressed colon inflammation via suppression of NF-κB-dependent expression of pro-inflammatory cytokines.

Highly efficient gene silencing and bioimaging based on fluorescent carbon dots in vitro and in vivo

Small interfering RNA (siRNA) is an attractive therapeutic candidate for sequence-specific gene silencing to treat incurable diseases using small molecule drugs. However, its efficient intracellular delivery has remained a challenge. Here, we have developed a highly biocompatible fluorescent carbon dot (CD), and demonstrate a functional siRNA delivery system that induces efficient gene knockdown in vitro and in vivo. We found that CD nanoparticles (NPs) enhance the cellular uptake of siRNA, via endocytosis in tumor cells, with low cytotoxicity and unexpected immune responses. Real-time study of fluorescence imaging in live cells shows that CD NPs favorably localize in cytoplasm and successfully release siRNA within 12 h. Moreover, we demonstrate that CD NP-mediated siRNA delivery significantly silences green fluorescence protein (GFP) expression and inhibits tumor growth in a breast cancer cell xenograft mouse model of tumor-specific therapy. We have developed a multifunctional siRNA delivery vehicle enabling simultaneous bioimaging and efficient downregulation of gene expression, that shows excellent potential for gene therapy.

High paclitaxel-loaded and tumor cell-targeting hyaluronan-coated nanoemulsions.

The purpose of this study was to develop hyaluronan-coated nanoemulsions (HNEs) with high solubilizing capacity and tumor cell targeting capability for the poorly soluble paclitaxel. The HNEs were composed of dl-a-tocopheryl acetate, soybean oil, polysorbate 80, and ferric chloride and were coated with hyaluronic acid (HA) as a targeting moiety. The nanoemulsions (NEs) and HNEs with or without paclitaxel (PTX) were prepared using high-pressure homogenization with a microfluidizer and were lyophilized with d-mannitol. The particle diameter and zeta potential of the HNEs were 65±15nm and -39.5±0.33mV, respectively. The concentration of PTX loaded in the NEs was 6mg/mL, which was higher than that in any other nanocarrier. The HNEs were coated with HA on the outer surface of the sphere and the amount of HA was 0.82±0.10% (w/w). The lyophilized formulation was stable at 4°C for 12 months and the reconstituted HNE solution was stable for at least 96h, even though Taxol® can be maintained for only 72h. In the cell affinity studies with SK-OV-3 (cluster of differentiation 44 [CD44]+) and OVCAR-3 (CD44-) cells, the HNEs displayed a 10-fold higher targeting capability than the NEs did. Therefore, the HNEs displayed high drug loading capability, excellent stability, and targeting of tumor cells overexpressing CD44, which suggested they were a potentially effective nanocarrier for carrying poorly soluble paclitaxel and targeting tumors.