Hyo-Jeong Kuh

Pharmacokinetics of doxorubicin after intratumoral injection using a thermosensitive hydrogel in tumor-bearing mice

A novel, thermosensitive hydrogel, poly(organophosphazene), is an injectable drug delivery system that transforms from sol to gel at body temperature. Doxorubicin (DOX) is a cytotoxic drug used for the treatment of several solid tumors. Due to its acute cardiac toxicity, DOX is a good candidate for local chemo-drug delivery system. In this study, we evaluated the pharmacokinetics of DOX (30 mg/kg) when given as an intratumoral injection using poly(organophosphazene) hydrogel in mice with human gastric tumor xenografts. DOX was formulated at 0.6% into a 10% hydrogel, and 40% and 90% of the dose was released in a sustained manner over 5 weeks in vitro and in vivo, respectively. The hydrogel mass was well retained over 7 weeks, and T(1/2beta, tumor) was 1.8-fold longer than that of the solution, but the 2.2-fold lower C(max, tumor), produced a similar AUC(tumor) and antitumor effect. However, solution caused a 2-fold higher systemic exposure (AUC(plasma)), which resulted in significant mortality due to acute cardiac toxicity. These data indicate that hydrogel formulation may have similar efficacy but lower systemic exposure than aqueous solution. In conclusion, poly(organophosphazene) showed adequate characteristics for local intratumoral delivery of DOX, including dose capacity, local retention, and minimal systemic spill-over. The safety and biocompatibility of poly(organophosphazene) should be further evaluated and its application should be extended to other anticancer agents.

Novel application of multicellular layers culture for in situ evaluation of cytotoxicity and penetration of paclitaxel

Limited drug penetration into tumor tissue is one of the major factors causing clinical drug resistance in human solid tumors. The multicellular layers (MCL) of human cancer cells have been successfully used to study tissue pharmacokinetics of anticancer drugs. The purpose of this study was to develop a direct and simple method to evaluate vitality changes in situ within MCL using calcein‐AM. Human colorectal (DLD‐1, HT‐29) and bladder (HT‐1376, J‐82) cancer cells were grown in Transwell inserts to form MCL and subjected to paclitaxel exposure. The drug distribution was evaluated using paclitaxel‐rhodamine. Photonic attenuation and limited penetration of calcein‐AM prevented cellular vitality evaluation on optical sections under confocal microscopy in DLD‐1 MCL. However, direct measurement of the fluorescence intensity on frozen sections of MCL allowed successful vitality assessment in more than 80% depth for HT‐29 and J‐82 MCL and in the upper 40% depth for DLD‐1 and HT‐1376 MCL. The penetration of paclitaxel‐rhodamine was greater in HT‐29 than DLD‐1 and its distribution pattern was correlated to the spatial profile of vitality deterioration in both MCL, suggesting that tissue penetration may be an important determinant of drug effect in tumors. In conclusion, a novel method for vitality evaluation in situ within MCL was developed using calcein‐AM. This method may provide clinically relevant data regarding the spatial pharmacodynamics of anticancer agents within avascular regions of solid tumors. (Cancer Sci 2008; 99: 423–431)

Pharmacokinetics of primaquine and carboxyprimaquine in korean patients with vivax malaria

Primaquine is used for relapses caused by vivax malaria hypnozoites. No studies on the pharmacokinetics of primaquine (PMQ) has been reported in Korean patients. In our study, thirty vivax malaria patients were given 15 mg primaquine daily for 14 days after 3 days of chloroquine treatment. Plasma samples were taken at intervals after each daily dose of PMQ for 3 days. Plasma concentrations of PMQ and carboxyprimaquine (CPMQ), the major metabolite of primaquine, were measured by HPLC. The PMQ concentrations reached a maximum of 0.28±0.18 μg/mL at 1.5 h after the first dose. The maximum concentration of CPMQ was 0.32±0.13 μg/mL at 4 h. Higher drug concentrations with repeated dosing were observed for CPMQ, but not for the parent drug, PMQ. The elimination half-life was 3.76±1.8 h and 15.7±12.2 h, for PMQ and CPMQ, respectively. Large variation in the plasma concentrations of both drugs was observed. Overall, PMQ is absorbed and metabolized rapidly after oral administration. It was noted that the mean peak plasma concentration of PMQ was significantly higher and that of CPMQ was lower in our patients compared to other studies. This suggests a potential difference of inter-ethnic groups, which warrants further investigations.

Penetration and efficacy of VEGF siRNA using polyelectrolyte complex micelles in a human solid tumor model in-vitro

A polyelectrolyte complex(PEC) micelle-based siRNA delivery system has been developed for vascular endothelial growth factor (VEGF), and its antitumor efficacy has been demonstrated using in-vivo animal models. Penetration and distribution through the avascular regions of human solid tumors after extravasation are important issues for antitumor efficacy, especially for macromolecules such as VEGF siRNA PEC micelles. Using an in-vitro solid tumor model, multicellular layers(MCL) culture of human colorectal cancer cells, we evaluated the penetration kinetics and efficacy of VEGF siRNA PEC micelles(PEC-siRNA) in comparison to unmodified siRNA(N-siRNA). The PEC-siRNA showed full penetration (15-17 layers of cells) with a unique punctuated distribution pattern at 48 h following initial accumulation in the top layers and a significant suppression of mRNA and protein expression in a dose-dependent manner after 72 h exposure. Although the initial penetration of N-siRNA was faster than that of PEC-siRNA, N-siRNA showed complete loss of activity due to its instability within 24 h. Our data support the idea that PEC micelle formulation may provide stable penetration tool through the multilayers of cancer cells and ensure the gene silencing effect of VEGF. This study also demonstrated that MCL could serve as a useful in-vitro model to evaluate the dose- and time-dependent profiles of penetration and efficacy of macromolecular delivery systems in human solid tumor avascular regions.

A Long Non-Coding RNA snaR Contributes to 5-Fluorouracil Resistance in Human Colon Cancer Cells

Several types of genetic and epigenetic regulation have been implicated in the development of drug resistance, one significant challenge for cancer therapy. Although changes in the expression of non-coding RNA are also responsible for drug resistance, the specific identities and roles of them remain to be elucidated. Long non-coding RNAs (lncRNAs) are a type of ncRNA (> 200 nt) that influence the regulation of gene expression in various ways. In this study, we aimed to identify differentially expressed lncRNAs in 5-fluorouracil-resistant colon cancer cells. Using two pairs of 5-FU-resistant cells derived from the human colon cancer cell lines SNU-C4 and SNU-C5, we analyzed the expression of 90 lncRNAs by qPCR-based profiling and found that 19 and 23 lncRNAs were differentially expressed in SNU-C4R and SNU-C5R cells, respectively. We confirmed that snaR and BACE1AS were downregulated in resistant cells. To further investigate the effects of snaR on cell growth, cell viability and cell cycle were analyzed after transfection of siRNAs targeting snaR. Down-regulation of snaR decreased cell death after 5-FU treatment, which indicates that snaR loss decreases in vitro sensitivity to 5-FU. Our results provide an important insight into the involvement of lncRNAs in 5-FU resistance in colon cancer cells.

Injectable poly(organophosphazene)-camptothecin conjugate hydrogels: synthesis, characterization, and antitumor activities.

The objective of this study is to develop an effective polymer therapeutics involving camptothecin (CPT) with enhanced efficacy and lessened systemic side-toxicity for cancer treatment. Polymer-CPT conjugates (PCCs), which consisted of CPT-20-glycinate and poly(organophosphazene) bearing carboxylic acid, were synthesized, characterized for physicochemical properties, in vitro degradation and CPT release behaviors from the PCC, and evaluated their anticancer activity. The aqueous solutions of all these PCCs showed a thermo-responsive sol-gel transition behavior for injectable application near room temperature. The CPT incorporated into the hydrogel was proven to be stable in vitro over 15 days. The in vitro cytotoxicity of the PCC was verified to be effective against four kinds of human cancer cell lines. The in vivo anticancer activity study with HT-29 colon cancer cell xenografted mice showed that the intratumorally injected PCC hydrogel inhibited the tumor growth more effectively relative to CPT alone (-29% vs. 130% in tumor size).

Synergistic interaction between gefitinib (Iressa, ZD1839) and paclitaxel against human gastric carcinoma cells.

We have evaluated the antitumor effects of gefitinib (Iressa, ZD1839) in SNU-1 human gastric cancer cells (hMLH1-deficient and epidermal growth factor receptor-overexpressed) when given alone or as a doublet with oxaliplatin (LOHP), 5-fluorouracil (5-FU) or paclitaxel (PTX). The four drugs showed IC50s ranging from 1.81 nM to 13.2 μM. LOHP and PTX induced G2/M arrest, 5-FU increased S phase, and gefitinib increased G1 in a concentration-dependent manner. The analysis using the previously developed cytostatic TPi model showed that 64 and 80% of the overall growth inhibition was attributed to cell cycle arrest in cells exposed to 7.55 μM of LOHP or 10 nM of PTX for 72 h, respectively. PTX+gefitinib showed greatest synergism as determined by combination index analysis and apoptosis induced by PTX was potentiated by the co-administration of gefitinib. LOHP+gefitinib showed a similar, although to a lesser degree, synergistic effect. This study demonstrates the antitumor activity and the significant cell cycle arrest induced by gefitinib in SNU-1 human gastric carcinoma cells, and its synergistic interaction with LOHP and PTX.

Detailed structural analysis on both human MRP5 and mouse mrp5 transcripts

The multidrug-resistant phenotype in tumor cells is attributed in part to anti-cancer drug efflux transporters such as the MRP family. The amino-terminal structure of MRP5 has not been refined. To determine the amino-terminal structure of a major transcript of the MRP5 gene, we performed primer extension analysis to determine a major transcriptional start site of this gene and compared the structure of human MRP5 and that of mouse mrp5. We successfully determined the structures of human MRP5 and mouse mrp5. Estimated amino acid sequences are 1437 and 1436 amino acids for human MRP5 and mouse mrp5 respectively, and were highly conserved (94.1%). We further showed that our previously identified SMRP mRNA was a splicing variant of the MRP5 gene, which was expressed in various human tissues, suggesting that a short form of MRP5 protein encoded by the SMRP mRNA may have a physiological role.

Co-culture of 3D tumor spheroids with fibroblasts as a model for epithelial-mesenchymal transition in vitro.

Epithelial-mesenchymal transition (EMT) acts as a facilitator of metastatic dissemination in the invasive margin of malignant tumors where active tumor-stromal crosstalks take place. Co-cultures of cancer cells with cancer-associated fibroblasts (CAFs) are often used as in vitro models of EMT. We established a tumor-fibroblast proximity co-culture using HT-29 tumor spheroids (TSs) with CCD-18 co fibroblasts. When co-cultured with TSs, CCD-18 co appeared activated, and proliferative activity as well as cell migration increased. Expression of fibronectin increased whereas laminin and type I collagen decreased in TSs co-cultured with fibroblasts compared to TSs alone, closely resembling the margin of in vivo xenograft tissue. Active TGFβ1 in culture media significantly increased in TS co-cultures but not in 2D co-cultures of cancer cells-fibroblasts, indicating that 3D context-associated factors from TSs may be crucial to crosstalks between cancer cells and fibroblasts. We also observed in TSs co-cultured with fibroblasts increased expression of α-SMA, EGFR and CTGF; reduced expression of membranous β-catenin and E-cadherin, together suggesting an EMT-like changes similar to a marginal region of xenograft tissue in vivo. Overall, our in vitro TS-fibroblast proximity co-culture mimics the EMT-state of the invasive margin of in vivo tumors in early metastasis.

Neurotrophic activity of DA-9801, a mixture extract of Dioscorea japonica Thunb. and Dioscorea nipponica Makino, in vitro.

ETHNOPHARMACOLOGICAL RELEVANCE Dioscorea japonica Thunb. has been traditionally used to treat polyuria and diabetes in Korea. AIM OF THE STUDY We previously report the effects of Dioscorea japonica Thunb. extract on glucose control, NGF induction, and neuroprotection in a rodent diabetic model. Since the most potent fraction, DA-9801, was identified from a mixture of Dioscorea japonica Thunb. (DJ) and Dioscorea nipponica Makino (DN) following bioactivity-guided fractionation, here, we investigated the potential mechanism of the extract activity against diabetic peripheral neuropathy (DPN). MATERIALS AND METHODS A 1:3 mixture of DJ and DN was extracted with ethanol (DA-9801) and further fractionated into an ethylacetate-soluble fraction (DA-9801E). Effects of these extracts on neurite outgrowth were measured in PC-12 cells and DRG neurons. Effects on cell viability and TrkA phosphorylation were evaluated in PC-12 cells. NGF induction effect was determined in primary Schwann cells as well as IMS32 cells (immortalized Schwann cells). RESULTS No cytotoxicity was observed in PC-12 cells at the concentration below 500 μg/ml of either DA-9801 or DA-9801E. DA-9801 and DA-9801E at 100 μg/ml and 10 μg/ml, respectively, showed a significant effect on neurite outgrowth in PC-12 cells and DRG neurons in the presence of or absence a low concentration of NGF (2 ng/ml). The Trk-A phosphorylation effect of DA9801 was confirmed in PC-12 cells. An NGF induction effect of these extracts was not detected in either IMS-32 cells, or primary Schwann cells. CONCLUSIONS The NGF agonistic activity of DA-9801 and DA-9801E was demonstrated, which may contribute to their neuroprotective effect against DPN. Studies of the detailed mechanism of these extracts as well as identification of the active components are warranted for the development of an anti-DPN drug from DJ and DN.