A highly sensitive method for the determination of D-lactic acid and 3-hydroxybutyric acid (3-HB) in rat plasma was developed using high-performance liquid chromatography with octadecylsilica (ODS) connected to a chiral column. At first, (D + L)-lactic acid and 3-HB in the plasma were derivatized with a fluorescent reagent, 4-nitro-7-piperazino-2,1,3-benzoxadiazole (NBD-PZ), separated on the ODS column and determined fluorimetrically at 547 nm with 491 nm of excitation wavelength. During the separation step on the ODS, the peak fraction of (D + L)-lactate derivative was introduced directly to a phenylcarbamoylated beta-cyclodextrin chiral column by changing the flow of the eluent via a six-port valve. Then, D-lactate derivative was separated enantiomerically from the L-lactate derivative, and the enantiomeric ratio was determined from the chromatogram. Intra- and inter-day accuracy values for the determination of D-lactic acid in 10 microL of rat plasma were 97.8-109.2 and 98.4-109.9%, and those for 3-HB were 99.8-108.4 and 99.8-103.8%, respectively. The intra- and inter-day precision values were within 4.6 and 5.1% for D-lactic acid, and 2.7 and 2.4% for 3-HB, respectively. The detection limits for D-lactic acid and 3-HB were approximately 2.0 and 0.04 microM, respectively (signal-to-noise ratio 3). The proposed method was applied to the plasma of diabetic rats induced by intraperitoneal administration of streptozotocin, and the significant increases of both D-lactic acid and 3-HB concentrations were observed in the diabetic rats as compared to the normal rats.

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Inhibitory effects of trehalose liposomes (DMTre) composed of 30mol% l-α-dimyristoylphosphatidylcholine (DMPC) and 70mol% trehalose surfactants on the growth of lymphoblastic leukemia (MOLT-4) cells in vitro and therapeutic effects of DMTre for xenograft mice model of carcinoma in vivo were examined. DMTre inhibited the growth of MOLT-4 cells in a dose-dependent manner due to apoptosis. The activation of caspase-3, -8, and 9 was obtained for MOLT-4 cells after the treatment with DMTre. The clustering of lipid rafts in plasma membranes of MOLT-4 cells was examined with a marker Cholera toxin subunit B conjugates Alexa Fluor (CTB), which binds to the pentasaccharide chains of ganglioside GM1 on the cellular surfaces. The clustering of lipid rafts in plasma membranes of MOLT-4 cells was observed after the treatment with DMTre. Therapeutic effects of DMTre were obtained for xenograft mice model of carcinoma in vivo.

We have produced hybrid liposomes (HL) which can be prepared by sonication of a mixture of vesicular and micellar molecules in a buffer solution. The physical properties of HL such as size, shape, and membrane fluidity can be controlled by changing the constituents and compositional ratio. We have employed HL for chemotherapy and interesting results are as follows; (A) The uniform and stable structure of HL composed of L-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylenedodecyl ether (C₁₂(EO)(n)) with a diameter of 80 nm was revealed. (B) The remarkable inhibitory effects of HL on the growth of various tumor cells were attained in vitro. (C) Induction of apoptosis by HL was obtained and the pathway of apoptosis induced by HL was clarified. (D) A good correlation between the membrane fluidity of HL and inhibitory effects of HL for tumor cells was obtained. (E) Significantly chemotherapeutic effects were obtained using mice model of carcinoma after the treatment with HL without any side effects in vivo. (F) In clinical applications, prolonged survival and remarkable reduction of neoplasm were attained in patients with lymphoma after the treatment with HL without any side effects after the approval of the bioethics committee.

Therapeutic effects of hybrid liposomes (HL) composed of l-α-dimyristoylphosphatidylcholine (DMPC) and polyoxyethylene(25) dodecyl ether (C(12)(EO)(25)) on the metastasis of human colon carcinoma (HCT116) cells were examined in vivo. Remarkably high therapeutic effects were obtained in the xenograft mouse models of colorectal cancer (CRC) liver metastases after treatment with HL-25 on the basis of relative liver weight and histological analysis of the liver tissue sections of mouse models with HE staining, and TUNEL staining for detection of apoptotic cells. The survival effects of HL-25 were obtained using xenograft mouse models of CRC liver metastases. Furthermore, with regard to pharmacokinetics, the accumulation of fluorescent labeled HL-25 was observed in the liver tissue of xenograft mouse models of CRC liver metastases for 24 h after the intravenous injection of fluorescent labeled HL-25. Therapeutic effects of HL without any drugs on the liver metastasis of human CRC were revealed for the first time in vivo.

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Cancer metastasis accounts for the majority of cancer-related deaths. One of the most important strategies for cancer therapy is the control of cancer metastasis. The liver is the most common organ for the metastasis of cancers in the digestive system, and the prognosis for cases with liver metastasis is extremely poor.1-4) Chemotherapy with fluorouracil, irinotecan, and leucovorin has been introduced to improve the postoperative prognosis.5) While the chemotherapy drugs kill tumor cells, they also damage normal cells, causing severe side-effects such as gastrointestinal dysfunction and bone marrow toxicity. Therefore, a chemotherapy that is effective for the metastasis of cancers without any side-effects is required. Docosahexaenoic acid (DHA) is an w-3 polyunsaturated long-chain fatty acid (w-3 PUFAs), whose antitumoral actions have been widely demonstrated in epidemiological and experimental studies. 6) For example, the diets containing fish oil rich in DHA had inhibitory effects against colon tumorigenesis.7-10) Furthermore, it was reported that DHA inhibited the growth of various cancer cells by cytotoxicity of the lipid peroxidation 11) and by apoptotic cell death in vitro. 12)This research suggests that DHA could be an effective drug for cancer chemotherapy. In addition, the relationships between dietary DHA and tumorigenesis have been attracting much attention from many researchers in food and medical science. However, there are few studies on the therapeutic effects of DHA on the growth of tumor cells in vivo. In particular, the therapeutic effects on the mouse models of carcinoma by intravenous treatment of DHA in vivo have not yet been examined due to the poor solubility in water.On the other hand, we have produced hybrid liposomes (HL) which can be prepared by just the sonication of vesicular and micellar molecules in a buffer solution. 13,14) HL are free from any contamination with organic solvents and remain stable for longer periods. The physical properties of these liposomes such as size, membrane fluidity, phase transition temperature, and hydrophobicity can be controlled by changing the constituents and compositional ratios of the HL. In the course of our study for HL, the following interest- 23) In addition, we elucidated the remarkably inhibitory effects on the growth of human colon cancer (HCT116) cells along with apoptosis and differentiation by HL including DHA in vitro. 19) However, therapeutic effects of HL including DHA in vivo have not yet been examined.In this study, we investigated the therapeutic effects of DMPC/DHA hybrid liposomes composed of 50 mol% L-adimyristoylphosphatidylcholine (DMPC) and 50 mol% DHA on the hepatic metastasis mouse models of human colon carcinoma HCT116 cells in vivo. Furthermore, the therapeutic effects on the hepatic metastasis mouse models by intravenous treatment of DMPC/DHA were revealed on the basis of histological analysis of liver tissues. MATERIAL AND METHODSPreparation of DMPC/DHA DMPC/DHA were prepared by sonication of a mixture containing DMPC (N...