Kenji Fukuzawa

Development of a novel drug delivery system consisting of an antitumor agent tocopheryl succinate

We have developed a novel drug delivery system (DDS) using an antitumor agent, α-tocopheryl succinate (TS). TS has attracted attention as a unique anti-cancer drug for its ability to induce apoptosis in various cancer cells. Furthermore, TS itself readily forms nanovesicles (TS-NVs) and is a prospective tool for use as an antitumor DDS. However, TS-NVs are unstable for encapsulating drugs and passive targeting delivery to tumor tissue via enhanced permeation and retention effect. Therefore, to improve the stability of vesicles, we developed a novel nanovesicle consisting of TS and egg phosphatidylcholine (TS-EPC-NVs). The stability of vesicles of TS-EPC-NVs was significantly higher than that of TS-NVs. As a result, the in vivo antitumor activity of TS-EPC-NVs was more potent than that of TS-NVs. The enhanced antitumor activity of TS-EPC-NVs was found to be due to its effective intratumoral distribution. Moreover, the in vitro anticancer efficiency of TS-EPC-NVs increased seven-fold. We suggest that the improvement is due to homogenous cellular uptake and enhanced cytosolic delivery of the nanovesicles via alteration of intracellular trafficking. Furthermore, TS-EPC-NVs encapsulating siRNA showed significant knockdown efficiency. In summary, TS-EPC-NVs represent a novel and attractive drug delivery system. The system shows antitumor activity of the encapsulated drug and the carrier itself.

Vitamin D3 derivatives increase soluble CD14 release through ERK1/2 activation and decrease IL-8 production in intestinal epithelial cells

Dysfunction of the innate immune system has been reported to cause intestinal inflammation. Vitamin D3 is known to be an important immune system regulator and exerts anti-inflammatory effects. We investigated in vitro effects of vitamin D3 and its derivatives on the innate immune system in HT-29 cells, a line of human colon adenocarcinoma cells. Among the innate immune-related receptors such as Toll-like receptor (TLR) 1, 2, 4, 6, and CD14 examined by flow cytometry, only CD14 was up-regulated by vitamin D3 derivatives. Release of soluble form CD14 (sCD14) was also increased by vitamin D3 derivatives. The 1α,25-dihydroxy-22-oxavitamin D3 (Oxa-D3) induced-sCD14 release was inhibited by U0126 (a specific inhibitor of extracellular signal-regulated kinase; ERK1/2) but not by SB203580 (a specific inhibitor of p38 MAPK), and ERK1/2 phosphorylation was accelerated by Oxa-D3. These results indicate that Oxa-D3 facilitates the release of sCD14 through ERK1/2 activation. IL-8 production stimulated with LPS was diminished by vitamin D3 derivatives. Recombinant sCD14 also lowered the LPS-stimulated IL-8 production, suggesting neutralization of LPS by sCD14. The anti-inflammatory effect of vitamin D3 derivatives was thus associated with diminution of IL-8 production due to increased release of sCD14.

Kinetic study of aroxyl radical-scavenging action of vitamin E in membranes of egg yolk phosphatidylcholine vesicles.

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the reaction rates of vitamin E (α-, β-, γ-, δ-tocopherols, TocH) and tocol with aroxyl radical (ArO·) as model lipid peroxyl radicals in membranes by stopped-flow spectrophotometry. Egg yolk phosphatidylcholine (EYPC) vesicles were used as a membrane model. EYPC vesicles were prepared in the aqueous methanol solution (MeOH:H(2)O=7:3, v/v) that gave the lowest turbidity in samples. The second-order rate constants (k(s)) for α-TocH in MeOH/H(2)O solution with EYPC vesicles were apparently 3.45×10(5)M(-1)s(-1), which was about 8 times higher than that (4.50×10(4)M(-1)s(-1)) in MeOH/H(2)O solution without EYPC vesicles. The corrected k(s) of α-TocH in vesicles, which was calculated assuming that the concentration of α-TocH was 133 times higher in membranes of 10mM EYPC vesicles than in the bulk MeOH/H(2)O solution, was 2.60×10(3)M(-1)s(-1), which was one-seventeenth that in MeOH/H(2)O solution because of the lower mobility of α-TocH in membranes. Similar analyses were performed for other vitamin E analogues. The k(s) of vitamin E in membranes increased in the order of tocol<δ-TocH<γ-TocH∼β-TocH<α-TocH. There was not much difference in the ratios of reaction rates in vesicles and MeOH/H(2)O solution among vitamin E analogues [k(s)(vesicle)/k(s) (MeOH/H(2)O)=7.7, 10.0, 9.5, 7.4, and 5.1 for α-, β-, γ-, δ-TocH, and tocol, respectively], but their reported ratios in solutions of micelles and ethanol were quite different [k(s)(micelle)/k(s)(EtOH)=100, 47, 41, 15, and 6.3 for α-, β-, γ-, δ-TocH, and tocol, respectively]. These results indicate that the reaction sites of vitamin E analogues were similar in vesicle membranes but depended on hydrophobicity in micelle membranes, which increased in the order of tocol<δ-TocH<γ-TocH∼β-TocH<α-TocH.

Involvement of intestinal intraepithelial lymphocytes in turnover of intestinal epithelial cells: Morphological and functional alterations due to daily administration of FK506

To elucidate the interaction of intestinal intraepithelial lymphocytes (IELs) with intestinal epithelial cells (IECs), we investigated alterations of IECs by activating or inactivating IELs. The stimulation of IELs with anti-mouse CD3 monoclonal antibody induced massive apoptosis of IECs. Changes in IECs and IELs from mice that received daily administration of FK506 for 14days were investigated. IELs, particularly TCR-γδ⁺ IELs, were reduced in cell number, and a decrease of cytotoxic activity was observed. Under this condition, loss of apoptotic cells at the tips of villi and delayed turnover of IECs were detected. The expressions of alkaline phosphatase and CD98 amino acid transporters on IECs were decreased. Furthermore, abnormal skeletal organization of villi and weakened binding of IECs to the basement membrane were shown. These results suggest that inactivated IECs, which should be led to apoptosis, remained. It was strongly suggested that IELs participated in IEC turnover.

Down-modulation of toll-like receptor 2 expression on granulocytes and suppression of interleukin-8 production due to in vitro treatment with cellulose acetate beads.

The Adacolumn, which is filled with cellulose acetate beads (CA beads), has been used as a medical device for inflammatory diseases. The CA beads selectively adsorb granulocytes and monocytes and remove them from the peripheral blood. The anti‐inflammatory effects of the Adacolumn are possibly caused by removal of these cells but also due to the functional changes in the processed cells. In this study, we investigated the effects of CA beads treatment on modulation of the expression of innate immunity receptors such as the Toll‐like receptor (TLR) family and production of an inflammatory cytokine, interleukin‐8 (IL‐8). Changes in the expressions of TLR1, 2, 4 and 6 in peripheral leukocytes exposed to CA beads were examined by flow cytometry. TLR2 expression on the surface of granulocytes exposed to CA beads was decreased, but the amount of intracellular TLR2 was increased, possibly by internalization. These changes were not observed in monocytes or lymphocytes. Peptidoglycan (PGN) treatment produced similar changes in TLR2 on granulocytes. We also measured the amounts of IL‐8 in cultured blood treated with lipopolysaccharide (LPS) and PGN, which are known TLR agonists. PGN‐induced IL‐8 production was lower in CA beads‐treated leukocytes than that in non‐treated leukocytes, but LPS did not induce these changes. Based on these findings, we conclude that the down‐modulation of TLR2 and suppression of IL‐8 production on granulocytes by CA beads, may play an important role in the anti‐inflammatory effects of the Adacolumn.

A single exposure of rats to water-immersion restraint stress induces oxidative stress more severely in the thymus than in the spleen

Abstract Objectives We examined whether a single exposure of rats to water-immersion restraint stress (WIRS) induces oxidative stress in the thymus and spleen. Methods Vitamin E, ascorbic acid, reduced glutathione (GSH), and lipid peroxide (LPO) were assayed in the thymus and spleen of rats with and without 6 hours of WIRS. Results In unstressed rats, vitamin E, ascorbic acid, GSH, and LPO levels were higher in the thymus than in the spleen. Thymic ascorbic acid level was lower in stressed rats than in unstressed rats. Splenic ascorbic acid level was similar in both groups. Thymic and splenic GSH levels were lower in stressed rats than in unstressed rats but the reduced amount of GSH was lower in the spleen than in the thymus. Thymic vitamin E level was lower in stressed than in unstressed rats. Splenic vitamin E level was higher in stressed rats than in unstressed rats. Thymic and splenic LPO levels were higher in stressed rats than in unstressed rats but the increased amount of LPO was higher in the thymus than in the spleen. Conclusion It is indicated that a single expose of rats to WIRS induces oxidative stress more severely in the thymus than in the spleen.

Effect of Dietary Vitamin E Supplementation on Liver Oxidative Damage in Rats with Water-Immersion Restraint Stress.

We examined how dietary supplementation of vitamin E protects against liver oxidative damage in rats with water-immersion restraint stress (WIRS). Before WIRS exposure, rats received a normal diet (ND) or vitamin E-supplemented diet (VESD) (500 IU α-tocopherol/kg diet) at a mean dose of 15 g/animal/d for 4 wk. The two diet groups had serum transaminases and lactate dehydrogenase activities and adrenocorticotropic hormone, corticosterone, and glucose levels to a similar extent. VESD-fed rats had higher liver α-tocopherol concentrations and lower liver ascorbic acid, total coenzyme Q9 (CoQ9), reduced CoQ9, reduced CoQ10, and lipid peroxide (LPO) concentrations than ND-fed rats. When the two diet groups were exposed to 6 h of WIRS, the serum liver cell damage index enzyme activities increased more greatly in ND-fed rats than in VESD-fed rats but the serum stress marker levels increased to a similar extent. The WIRS exposure caused no change in liver LPO concentration with the further increase in liver α-tocopherol concentration in VESD-fed rats but increased liver LPO concentration without changing liver α-tocopherol concentration in ND-fed rats. Upon the WIRS exposure, liver reduced glutathione concentration decreased with the further decrease in liver ascorbic acid concentration in VESD-fed rats and those concentrations decreased in ND-fed rats. The WIRS exposure recovered the decreased liver total CoQ9 and reduced CoQ9 concentrations in VESD-fed rats but decreased liver total CoQ9, reduced CoQ9, and reduced CoQ10 concentrations in ND-fed rats. These results indicate that dietary vitamin E supplementation protects against liver oxidative damage without affecting the stress response in rats with WIRS.

A kinetic study of the radical-scavenging action of tocotrienols in the membranes of egg yolk phosphatidylcholine vesicles.

Vitamin E is localized in membranes and functions as an efficient inhibitor of lipid peroxidation in biological systems. In this study, we measured the second-order rate constants (ks) for the reaction of tocotrienol homologues (α-, β-, γ-, and δ-Toc-3Hs) with the aroxyl radical (ArO•) used as a model for lipid peroxyl radicals (LOO•) in the membranes of egg yolk phosphatidylcholine (EYPC) vesicles by stopped-flow spectrophotometry, and compared them to those of tocopherol homologues (α-, β-, γ-, and δ-TocHs). The relative rate constants of Toc-3H homologues to α-Toc-3H in membranes (α/β/γ/δ=100/83.7/63.2/20.2) were not much different to those of TocH homologues to α-TocH (α/β/γ/δ=100/88.4/83.8/17.3). Each ks value of Toc-3H homologues in membranes was 60-80% of that of the corresponding TocH homologues except for the almost identical ks values of δ-homologues, but there was no difference in EtOH solution between each ks value of the corresponding homologues of Toc-3H and TocH. These results indicate that the difference of the alkyl-side chain structure of vitamin E causes a change in the mobility of vitamin E molecules and/or the location of their antioxidant OH-groups in membranes, resulting in lowered radical-trapping rates of Toc-3Hs. By use of the ratio of the kinh value of α-TocH with LOO• (3.20×10(6) M(-1)s(-1)) to the ks value of α-TocH with ArO• (8.05×10(4) M(-1)s(-1)) in chlorobenzene (that is, 39.8), the kinh value for the reaction of α-TocH with LOO• in membrane was estimated to be 1.03×10(5) M(-1)s(-1).