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[Purpose] Vitamin D3 (VD3) is used to treat osteoporosis and causes rickets, osteomalacia and hypocalcemia as deficiencies. On the other hand, it has been reported that large intake of VD3 reduce carcinogenesis risk in colorectal cancer, and VD3 itself has an anticancer effect. However, overdose of VD3 causes severe hypercalcemia as an adverse reaction. Therefore, it was expected that liposomalization of VD3 could improve this problem. VD3 encapsulated liposome as a drug nanocarrier is possible to accumulate of VD3 in a tumor as a target site without adverse reaction. In this study, the optimal preparation of VD3 encapsulated liposomes, their antitumor effects without adverse reaction were examined. [Methods] Cholecalciferol as VD3 encapsulated liposome by thin film method with different amount of cholesterol (CHO) was prepared. L-α-Distearoylphosphatidylcholine, CHO, L-α-distearoylphosphatidyl-DL-glycerol and 1-monomethoxypolyethyleneglycol-2,3-distearoylglyerol were dissolved using chloroform/methanol (4:1,(v/v)). After empty liposome preparation, VD3 is added to this suspension and incubated to encapsulate VD3 (Basic Method). The other method used ethanol as solvent and VD3 was added with the constituent lipids (Modified method). Cytotoxic effect was examined using tumor cell with overexpression of VD3 receptor. In P388 leukemia cell suspension, cytotoxicity by VD3 liposomes or VD3 solution were determined by the WST-8 assay. P388 leukemia cells with VD3 liposomes or VD3 solution were incubated and the intracellular VD3 concentration was measured. In vivo experiment, P388 leukemia cells bearing BDF1 mice were administered VD3 liposomes at 1st, 4th and 7th day. On the next day after final administration, tumor weight and blood calcium concentration were measured. [Results and Discussion] The particle size and zeta potential of VD3 liposomes were same level in some ratio of CHO in lipid composition. The encapsulated amount of VD3 in liposomes increased with the decrease of CHO amount. This may be due to the similar structure of CHO and VD3. VD3 encapsulated level of liposome by modified method was showed high amount. Because VD3 is lipophilicity, the high encapsulated amount of VD3 may have when the addition of VD3 with the formation of lipid film. VD3 receptor expression of P388 leukemia cells was higher than that of B16F10 melanoma cells by Western blots. In P388 leukemia cells, VD3 solution was not have cytotoxicity whereas VD3 liposome showed significant cytotoxicity. Furthermore, VD3 concentration in P388 leukemia cells was slight in VD3 solution group. In contrast, its concentration in VD3 liposome group quickly elevate and maintain a high concentration. In tumor bearing mice after VD3 liposome treatment, the tumor weight was decreased and the blood calcium concentration had lower level than that of control level. Namely, it was suggested that VD3 liposomes has a superior antitumor effect without the increase of adverse reaction. In conclusion, the optimum method of VD3 liposome preparation and its usefulness in novel cancer treatment were clarified. Citation Format: Yukako Soma, Ikumi Sugiyama, Yasuyuki Sadzuka. Advanced properties of Vitamin D3 nanocarrier in cancer chemotherapy [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2877.

[Purpose] This study aims to evaluate the possible antitumor effect of vitamin D. Vitamin D is a group of lipophilic compounds comprising of vitamin D2 and vitamin D3. Vitamin D3 has been shown to play an important role in humans. It has been previously reported that the onset risk of malignant melanoma and leukemia can be decreased by maintaining high vitamin D3 levels in the body. In addition, the cytotoxic effect of vitamin D on breast cancer cells has also been shown. In this study, the relationship between the cytotoxicity of vitamin D3 and vitamin D3 receptor (VDR) expression on melanoma and leukemia cells was examined. [Method] B16F10 melanoma (B16F10) and P388 leukemia (P388) cells were used in this study. Western blot analysis was performed to determine VDR expression in each cell type. To evaluate cytotoxicity, B16F10 or P388 cells were seeded at 1×105 cells/mL, and calcitriol as vitamin D3 (range 9 ng/mL-90 μg/mL) was added at once. After two days, cytotoxicity was evaluated by colorimetric analysis using Cell Counting Kit-8. Additionally, the cytotoxicity of P388 cells was determined following the combined treatment of vitamin D3 and retinoic acid. [Results and Discussion] Western blot analysis revealed that both B16F10 and P388 cell types, expressed VDR with a higher expression in P388 cells. In P388 cells, there was a significant reduction in cell viability following vitamin D3 treatment, which decreased to 10% at 90 μg/mL. In contrast, the viability of B16F10 cells following vitamin D3 treatment only slightly decreased. Interestingly, these results suggest that the cytotoxicity of vitamin D3 correlates with VDR expression. VDR reportedly forms a heterodimer with the retinoid X receptor. However, the presence of retinoic acid did not alter vitamin D3 cytotoxicity. Therefore, it was considered that cytotoxicity induced by vitamin D3 was not connected with the formation of the VDR-retinoid X receptor heterodimer. In conclusion, there was a higher expression of VDR in P388 cells compared with B16F10 cells. This higher expression correlated with a higher level of vitamin D3 cytotoxicity, suggests an antitumor effect of vitamin D3. This study proposes the possibility of using vitamin D3 as a novel treatment for leukemia and other cancers. Citation Format: Takaya Hoshi, Yuta Abe, Ikumi Sugiyama, Yasuyuki Sadzuka. The connection of vitamin D3 induced cytotoxicity with the vitamin D3 receptor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 367.

[Purpose] Novel nanoparticles have the potential to increase the efficacy of cancer chemotherapy. Recently, the use of two chemotherapeutic drugs load to liposomes has been studied. In particular, cytarabine-daunorubicin (1:5) co-loaded liposome (CPX-351) has high therapeutic efficacy against acute myelocytic leukemia. We previously reported that a combination of theanine (a specific amino acid of green tea) with doxorubicin (DOX) had a stronger antitumor effect than DOX alone. The effect is caused by the competitive inhibition of glutamate transport by theanine as a glutamate derivative via glutamate transporters in tumor cells. Subsequently, decreased glutamate levels in tumor cells result in lower glutathione, which makes efflux of DOX from cells difficult. Thus, it was speculated that specific accumulation of DOX, caused by theanine, increases the therapeutic index. In the present study, we examined the availability of a DOX-theanine co-loaded liposome as drug carrier for cancer chemotherapy. [Methods] DOX-theanine co-loaded liposome (DT-lip) was prepared using the thin-lipid method. DOX and theanine were loaded at the lipid membrane layer and internal water layer, respectively. P388 leukemia cells were used to study the influence of DT-lip on DOX influx and efflux. The cell suspension was incubated with DT-lip at 37°C, and the DOX concentration in tumor cells was determined. In the antitumor effect study in vivo, tumor-bearing mice were intravenously administered DT-lip (DOX 2.5 mg/kg) at 17th, 20th, and 23rd days after tumor cell implant. Tumors and normal tissue were removed 48 h after last administration. Tumor weight and DOX concentration in tissue were measured. [Results and Discussion] DT-lip was prepared with three ratios of DOX to theanine (1:0.5, 1:1, and 1:4, w/w). In the influx study, the DOX concentration in tumor cells was the same for the different ratios. DOX influx into tumor cells in the DT-lip group was enhanced compared to liposomes loaded with DOX only (D-lip). At 30 min, the intracellular DOX concentration in the DT-lip was 1.4 fold higher than that in the D-lip group. Conversely, there was no practical difference in DOX efflux from tumor cells between the D-lip and DT-lip group. In other words, DT-lip had an influence on influx system, and this phenomenon differed from the previous results for theanine′s role in the DOX efflux system. This findings suggest that liposomalization of theanine + DOX changed the theanine connected integrated mechanism in tumor cells. In vivo, the antitumor effect of DT-lip was superior to combined administration of both solutions. The adverse effects of DT-lip treatment on normal tissue did not increase because DOX concentration in tissue was comparable across group. In conclusion, the DT-lip, a liposome co-loaded DOX and theanine, is a novel antitumor formulation for cancer chemotherapy. Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. Evauation of doxorubicin-thanine co-loaded nanoparticles for cancer chemotherapy [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3633.

[Purpose] In chemotherapy for cancer, liposomes as drug delivery system (DDS) carriers are expected to have a useful effect. The aim of this study was, therefore to evaluate the antitumor effects of, and elucidate the mechanisms underlying, (-)-epigallocatechin-3-O-gallate (EGCG) and polyethyleneglycol (PEG)-modified liposomes. EGCG functions as a target ligand of the 67 kDa laminin receptor (67LR), which is expressed on high-grade tumor cells. An EGCG derivative was synthesized for binding to the end site of PEG structure. In this study, the antitumor effects of EGCG-modified liposomes as active-targeting liposome were evaluated. [Methods] Liposome was loaded with doxorubicin (DOX) as an antitumor agent. C57BL/6 mice were subcutaneously inoculated with B16F10 mouse melanoma cells (5x105 cells/animal). Each sample was administered intravenously with 2.5 mg/kg DOX. EGCG solution (EGCG sol.) was also administered intravenously at 11.3 mg/kg, based on the amount of EGCG modification in DOX-loaded EGCG-PEG-modified liposomes (EPL). Caspase-3 was evaluated as 7-amino-4-trifluoromethyl coumarin (Ex: 400 nm, Em: 505 nm), following a reaction with the fluorescent substrate DEVD-AFC. Caspase-8 was determined reacted with DEVD-pNA and detected as chromophore p-nitroanilin (λ: 400nm). [Results and Discussion] EPL significantly decreased tumor size against B16F10 mouse melanoma cells, in mice bearing 67LR-high-expression tumors. The tumor weight of the EPL group was significantly lower than that of the control (p Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. Antitumor effect against 67 kDa laminin receptor expressed on high-grade tumor cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2678.

Purpose: Chemotherapy using antitumor agents plays an important role in clinical cancer therapy. Among the treatments involving antitumor agents, the enhancement of antitumor activity was observed by combined chemotherapy. We have shown previously that some amino acids included theanine, taurine and anserine et al. as food components, increased doxorubicin (DOX) induced antitumor effect in vitro and in vivo. These effects by combined amino acids have depended on the increased DOX concentration in the tumor cells. Furthermore, these suppressions of DOX efflux by theanine and taurine were caused by the inhibitions of glutamate transporter and taurine transporter, respectively. The effect of anserine on DOX influx might connect with dipeptide transporter. Thus, other amino acid and unique peptides may act to DOX transport. In this study, the effects of cyclodipeptide on the transport of DOX and its cytotoxicity on tumor cell were clarified. Methods: Cyclo-leucine-proline (cyclo-Leu-Pro), cyclo-phenylalanine-proline (cyclo-Phe-Pro) and cyclo-glycine-proline (cyclo-Gly-Pro) as cyclodipeptide was used in this study. In transport of DOX, DOX influx or efflux was determined with combined cyclodipeptide in M5076 ovarian sarcoma cells or P388 leukemia cells. DOX concentration in tumor cell was measured using spectrofluorometer (Ex. 470 nm, Em. 580 nm). The cytotoxic study of DOX with combined cyclodipeptide was performed using WST-8. Results and Discussion: Cyclo-Phe-Pro and cyclo-Gly-Pro did not affect DOX influx and efflux in M5076 ovarian sarcoma cells and P388 leukemia cells. By combined cyclo-Leu-Pro, DOX influx into both M5076 ovarian sarcoma cells and P388 leukemia cells changed, compared to that of DOX only group. Into M5076 ovarian sarcoma, cyclo-Leu-Pro had strong effect in increased DOX influx in particular. And cyclo-Leu-Pro showed a tendency to suppress DOX efflux from M5076 ovarian sarcoma. In this studies of influx and efflux, concentration of cyclo-Leu-Pro existed optimum amount for increased DOX concentration in tumor cells. It is expected that cyclo-Leu-Pro induces the increment of DOX concentration in the tumor in vivo. DOX had the cytotoxic effects on M5076 ovarian sarcoma cells or P388 leukemia cells in culture. The combined cyclo-Leu-Pro with DOX increased cytotoxic effect, compared to that in the DOX alone group. In conclusion, cyclo-Leu-Pro has increased effect on DOX influx and inhibited effect on DOX efflux, and DOX induced cytotoxicity. It is expected that the combined cyclo-Leu-Pro will improve cancer chemotherapy by DOX. Moreover, cyclo-Leu-Pro may be help to prevent adverse effect since it will be able to decrease DOX dose. Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. The effects of cyclodipeptide on the transport of doxorubicin and its cytotoxicity on tumor cell [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2139. doi:10.1158/1538-7445.AM2017-2139

[Purpose] Metastasis is responsible for most mortality in cancer therapy. Important things of metastatic treatment are not only prevention but also therapy of metastatic tumor. It was indicated that usability of liposomal doxorubicin (DOX) as drug delivery system formulation which were modified with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine dipolyethyleneglycol(different double arms PEG; DDA-PEG) (DDA-LDOX). DDA-PEG had two different PEG lengths, 2000 and 500, in one molecule. In this study, the effect of DDA-LDOX was examined on pulmonary metastasis compared with 1-monomethoxy-PEG(2000)-2,3-distearoylglycerol (PEG2000-DSG) modified liposomal DOX (2000-LDOX).[Methods] The liposomal DOX was prepared by L-α-distearoylphosphatidylcholine, cholesterol, L-α-distearoylphosphatidyl-DL-glycerol and DOX (100:100:60:18 μmol). Liposomal DOX was added 7.5 μmol DDA-PEG or 15 μmol PEG2000-DSG. In anti-metastasis experiment, BDF1 mice were transplanted B16F10 melanoma cells by intravenous inoculation and each liposome (2.5 mg/kg, DOX) was injected at 20, 23 and 26 day after tumor transplantation. After 48 hr from last administration, the mice were dissected and score of pulmonary metastasis was recorded (0∼10, 0: normal). Removed tissues were determined DOX concentration by fluorescence method. In vitro, it was examined DOX uptake into B16F10 melanoma cells from each liposome. [Results and Discussion] Pulmonary metastasis scores of control and 2000-LDOX were 6.5 and 2.6, respectively. In contrast, that of DDA-LDOX was 1.0. Relative pulmonary weight (%) was correlation with metastatic score (R2=0.954) and supported pulmonary metastasis score. DOX concentration of DDA-LDOX was twice higher than that of 2000-LDOX in the lung, namely, it was proved that DDA-LDOX was easy to accumulate in the lung. Especially, it was suggested that DDA-LDOX accumulated into pulmonary metastatic site because DOX concentrations of heart, spleen and kidney were equal level in other group. In the examination of DOX uptake into tumor cells, DOX level in DDA-LDOX group was significantly higher than that in 2000-LDOX group. The data had supported high accumulation into pulmonary metastasis in vivo. In conclusion, it was suggested that DDA-LDOX was useful drug carrier for suppressed pulmonary metastasis since it could have target ability to pulmonary metastasis. Citation Format: Ikumi Sugiyama, Yasuyuki Sadzuka. Therapeutic efficacy of different double arms polyethyleneglycol-modified liposome containing doxorubicin on pulmonary metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4343. doi:10.1158/1538-7445.AM2013-4343

[Purpose] Polyethyleneglycol (PEG) modification on liposomal membrane increases therapeutic index by improvement of targeting based on prolonged blood stream circulation. In contrast, some accumulation of drug after liposome treatment has also been observed in normal tissues, it is necessary to improve liposomal composition on the decrease of adverse reactions. Particularly, doxorubicin (DOX) encapsulated liposome has some adverse reactions as cardiac toxicity or hand-foot syndrome,etc. To contribute superior liposome formulation, different double arms PEG in a molecule(DDA-PEG) was synthesized and was clarified effectiveness in our previous reports. In this study, we examined physical properties of novel synthesized DDA-PEG, and the tissue distribution of the DOX encapsulated liposome with modification of these DDA-PEGs. [Methods] Two DDA-PEGs with (PEG1000 and PEG500) or (PEG2000 and PEG500) were synthesized. Critical micelle concentration (cmc) in 10 mM Tris-HCl buffer (pH7.4) and 10 mM lactate buffer (pH4.0) was measured by the method of florescent probe using the 8-anilinonaphthalene-1-sulfonate (ANS). Octanol-buffer partitioning value was determined. DOX encapsulated liposomes with the modification of each DDA-PEGs were prepared by DSPC/cholesterol/DSPG-Na/DOX/PEG-lipid =100:100:60:18:7.5 μmol according to the method of Bangham. M5076 ovarian sarcoma cells were transplanted into the backs of BDF1 mice, and each liposome was injected intravenously at a dose of 2.5 mg DOX/kg on 15, 18 and 21 day after tumor inoculation. The mice were sacrificed after 2 day from last administration, and the tumor was removed and weighted. Moreover, DOX concentrations in each tissue were determined. [Results and discussion] The cmc of all PEG-lipids were same level. In PEG(2000, 500)-modified liposome, DOX concentrations in tumors were equal to that in PEG2000-modified liposome. On the other hand, DOX concentration in the heart after PEG(2000, 500) modified liposome and PEG(1000,500)-modified liposome treatments were one second and one sixth compared with DOX solution group, respectively. According to these results, it was expected that novel DDA-PEG-modified liposome was able to reduce the cardiac toxicity which is adverse reaction by DOX. The DOX concentration in the liver was showed a low level by PEG (1000, 500) modified liposome. It was suggested that novel DDA- PEG-modified liposome had the effect of avoiding the uptake into the liver. In conclusion, it was suggested that PEG(1000,500) modified liposome could decrease the DOX level in heart, and had ability of accumulation into tumor and avoidance from reticuloendothelial cells as same as PEG2000-modified liposome as known liposome. Citation Format: Mai Yagi, Ikumi Sugiyama, Yasuyuki Sadzuka. Improvement of tissue distribution by the treatment of novel different double-arms polyethyleneglycol modified liposome encapsulating doxorubicin. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 4515. doi:10.1158/1538-7445.AM2013-4515