Yasuyuki Sadzuka

Enhanced antitumor activity of different double arms polyethyleneglycol-modified liposomal doxorubicin.

The effect of polyethyleneglycol (PEG)-modified liposome as a drug carrier has been demonstrated clinically. We designed and synthesized a novel PEG-lipid, 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-PEG (different double arms PEG, DDA-PEG) which had two PEG chains of 500 and 2000 in one molecule to develop more useful PEG-modified liposome. DDA-PEG-modified liposomal doxorubicin (DDA-LDOX(7.5)) had the biggest fixed aqueous layer thickness (FALT) compared with other PEG-lipid-modified liposomes even if the added amount was a few. It was thought that FALT was the indication of blood circulation time. In DOX uptake in tumor cells, DDA-LDOX(7.5) group increased the DOX concentration in tumor cells because it had contact ability with tumor cells. Hence, DDA-LDOX(7.5) which has long circulation time in the bloodstream and contact ability with tumor cells, also had a strong antitumor effect on mice bearing M5076 ovarian sarcoma cells which were DOX low sensitive cells according to the expression of multidrug resistance protein. Furthermore, this liposome maintained a high DOX concentration in a tumor for a long time. These results indicated that the useful antitumor effect of DDA-LDOX(7.5) against M5076 ovarian sarcoma cells is a promising DDS carrier for therapies against drug resistant tumors.

Critical micelle concentration and particle size determine adjuvanticity of cyclic lipopeptides

Cyclic lipopeptides such as surfactin and polymyxin have potent mucosal adjuvant properties. Cyclic lipopeptides are tensioactive compounds, but the relationship between adjuvanticity and surface activity is unknown. Here, we show that the critical micelle concentration (cmc) of surfactant and particle size of the surfactant‐protein complex are important determinants of cyclic lipopeptide adjuvanticity. We found that the diameter of cyclic lipopeptide‐ovalbumin (OVA) complex particles was significantly larger than that in the solutions of OVA alone at cyclic lipopeptide concentrations above the cmc. OVA‐specific antibody titres in mice immunized intranasally with OVA and a cyclic lipopeptide at concentrations above its cmc were significantly higher than those in mice immunized with OVA plus the same dose of the cyclic lipopeptide but administered with formulations in which cyclic lipopeptide concentration was below the cmc. Thus, the concentration of the cyclic lipopeptide in the formulation at immunization, but not its overall dose, was critical for its adjuvanticity. Furthermore, two types of aggregates, the cyclic lipopeptide simplex micelles and the cyclic lipopeptide‐OVA complex micelles, were found in formulations with SF concentrations above its cmc. Degranulation of mast cells exposed to SF simplex micelles was more pronounced when SF concentration was above the cmc. In conclusion, our study showed that surface activity properties, such as the cmc and the size of surfactant‐protein complex, contribute to the adjuvanticity of cyclic lipopeptides. Our study proposes a novel idea that cmc is a key parameter for tensioactive adjuvants.

The effects of vitamin B6 compounds on cell proliferation and melanogenesis in B16F10 melanoma cells

B16F10 murine melanoma cells are frequently used for the study of cancer and melanogenesis. The cells are usually cultured in Dulbeccos Modified Eagle Medium, with the addition of 20 µM pyridoxal (PL) or pyridoxine (PN) for vitamin B6. The difference between these vitamin B6 compounds is thought not to affect cell proliferation, whereas their influence on other physiological effects is poorly understood. In the present study, the effects of PL and PN on cell proliferation and melanogenesis in B16F10 cells were compared. At 500 µM PL significantly suppressed cell growth but the growth inhibitory effect of PN was weak. Although neither of the vitamin B6 compounds affected cell growth at 20 µM, melanogenesis was suppressed by 20 µM PL compared with the effect of PN. In addition, the expression levels of tyrosinase, which is the rate-limiting enzyme, correlated with the melanin content. The results of the present study indicate that PL may be more useful for melanoma therapy and suppression of skin pigmentation than PN. The results also signify the importance of medium selection for cell culture.

Dual-effect liposomes with increased antitumor effects against 67-kDa laminin receptor-overexpressing tumor cells

This study sought 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 of PEG. Doxorubicin (DOX)-loaded EGCG-PEG-modified liposome (EPL) significantly decreased tumor size in mice bearing high 67LR-high-expressing tumors. Caspase-3 activity, which indicates induction of apoptosis, was also elevated only in the EPL group. The importance of PEG for the antitumor effects of EGCG was noted, as soluble EGCG did not accumulate at a sufficient concentration to exert an apoptotic effect. Moreover, EPL significantly increased caspase-8 activity, suggesting that EPL-induced apoptosis occurred due to caspase-8 activity induced following the binding of EGCG to 67LR as a cell-death ligand. In conclusion, EPL appear to have superior antitumor activity against high 67LR-expressing tumor cells, as the liposomes had dual effects, namely antitumor effects due to the loaded DOX and apoptosis induced by the bound EGCG.

Abstract 2678: Antitumor effect against 67 kDa laminin receptor expressed on high-grade tumor cells

[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 (5x10 5 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.

Abstract 5529: The advanced strategy for novel EGCG derivative modified liposome with high targeting ability to tumor

Purpose: The aim of this study is increase of antitumor effect and decrease of adverse effect by liposomes which have high affinity with 67 kDa laminin receptor (67LR). It was reported that 67LR is expression specifically on tumor cell membrane. When (-)-epigallocatechin-3-gallate (EGCG) as one of the green tea polyphenol are connected with 67LR, apoptosis is induced on tumor cells. On the other hand, liposome is often used for cancer chemotherapy. Then we have studied about EGCG modified liposome containing antitumor agent as drug delivery system. In this study, important strategies are 3 points. The first point is targets to 67LR on tumor cell by EGCG modification around liposomal membrane. The second point is to induce apoptosis mediated by 67LR. Last point is to increase antitumor effect by antitumor agent loaded into liposome. Based on these strategies, EGCG modified liposome was evaluated in vivo distribution. Methods: Three derivatives were synthesized for EGCG modification on liposomal membrane. The liposome with each EGCG derivative were prepared and assessed by measuring particle sizes and zeta potentials. EGCG was only modified liposome (EL) and both EGCG and polyethyleneglycol (PEG) were modified liposome (EPL). On P388 leukemia cells, cytotoxicity was evaluated by WST-8 assay and shown IC50. In the distribution study, C57BL/6 mice were injected (i.v.) each liposome, and collected blood and removed organs with definite time after injection. Results and Discussion: Synthesized EGCG derivatives with di-palmitoyl base were inserted directly into liposomal membrane. Two structures of other derivatives obtained PEG or is able to connect with PEG. In all liposomes, the particle sizes were from 100 to 200 nm and zeta potentials were negative charge. The cytotoxicity of EL was stronger than EGCG unmodified liposome. Moreover, 50% inhibitory concentration of EPL was smaller than EL, namely the cytotoxicity of EPL was strong. It was suggested that this effect was mediated with 67LR needed gallate base in the structure of EGCG because EPL showed strong effect compared with EL. The EGCG derivative in EL had di-palmitoyl base at gallate base, and its bind was not free. In mice experiment, EL rapidly disappeared from the blood circulation after injection. This concentration was one sixteenth of common PEG modified liposome at 15 min after injection. On the other hand, accumulation levels into liver and spleen as reticuloendothelial system (RES) reached high level immediately after injection. This phenomenon indicated that disappearance of EL depends on RES trap. EPL which had EGCG-PEG was significantly improved blood circulating time. In conclusion, it was expected that EGCG-PEG modified liposome (EPL) maintains long circulation time in blood, and increases targeted ability to tumor cells and antitumor activity. Citation Format: Ikumi Sugiyama, Kunihiro Kaihatsu, Nobuo Kato, Yasuyuki Sadzuka. The advanced strategy for novel EGCG derivative modified liposome with high targeting ability to tumor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5529. doi:10.1158/1538-7445.AM2015-5529

Abstract 5408: Liposomal doxorubicin with modified EGCG increased antitumor activity by topical targeting efficiency into tumor

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Purpose: The aim of this study is increase of antitumor activity using novel liposome with (-)-epigallocatechin-3-gallate (EGCG) as one of the green tea components. Liposome has been known as a one of useful drug carriers for delivering drug into targeting site. Moreover, some ligand is able to modify around liposomal membrane by a simple method. On the other hand, it was reported that surface of high grade tumor cell expresses 67kDa laminin receptor (67LR) which intermediates of cytostatic effect of EGCG, and EGCG is able to induce apoptosis selectively. Our strategy was to prepare liposomal doxorubicin with EGCG as carrier ligand for getting antitumor activity by both apoptosis and doxorubicin at tumor site. In this study, we tried to prepare EGCG-modified liposome and evaluated the antitumor activity in vitro. Methods: The liposomal doxorubicin was prepared by L-α-distearoylphosphatidylcholine, cholesterol, L-α-distearoylphosphatidyl-DL-glycerol and doxorubicin (100:100:60:18 μmol). Doxorubicin included EGCG-modified liposome (EL-DOX) was prepared to add EGCG-dipalmitate. Amount of EGCG modification on liposomal membrane was determined using DPPH assay. On P388 leukemia cells and M5076 ovarian sarcoma cells, cytotoxicity was evaluated by WST-8 assay and shown IC50. DOX uptake into tumor cells was evaluated in vitro. The concentration of doxorubicin into tumor cells was determined with a fluorescence spectrophotometer at an excitation wavelength of 500 nm and an emission wavelength of 550 nm. Results and Discussion: Particle sizes of EL-DOX were 147 nm on average, zeta potential was -33.1 mV and encapsulated ratio of doxorubicin was 96.5 %. These data of sizes and zeta potentials were better for intravenous administration of liposomes. EGCG ratio on liposomal membrane (71.0 %) was enough to show its ability. In the study of doxorubicin uptake into tumor cells, EL-DOX group was increased amount of doxorubicin for 30 min. The doxorubicin level of EL-DOX was significantly higher compared with that of unmodified liposomal doxorubicin (p<0.05). At cytotoxicity, EL-DOX was stronger than unmodified liposomal doxorubicin. It was provided that IC50 of EL-DOX was also 125 times higher than EGCG solution which was reported to have been antitumor activity. Furthermore, co-incubation of EGCG solution and liposomal doxorubicin was not shown the enhancement effect both doxorubicin uptake into cells and the cytotoxicity. It was suggested that superior effect were shown in modification of EGCG around liposomal membrane, not mixture of EGCG and liposome. In conclusion, EL-DOX exhibited strong antitumor activity efficiently. Citation Format: Ikumi Sugiyama, Kunihiro Kaihatsu, Nobuo Kato, Yasuyuki Sadzuka. Liposomal doxorubicin with modified EGCG increased antitumor activity by topical targeting efficiency into tumor. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5408. doi:10.1158/1538-7445.AM2014-5408

Abstract 4343: Therapeutic efficacy of different double arms polyethyleneglycol-modified liposome containing doxorubicin on pulmonary metastasis.

[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, BDF 1 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 (R 2 =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

Abstract 4515: Improvement of tissue distribution by the treatment of novel different double-arms polyethyleneglycol modified liposome encapsulating doxorubicin.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC [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

Abstract 1961: Evaluated mechanism of dual targeting using liposomal doxorubicin-modified novel PEG by imaging analysis

[Purpose] Liposome is one of the useful drug carriers and it may be applying to a lot of diseases from now on. One of the most reasons which liposomes were using as medicine was improvement of circulation time in bloodstream by modifying polyethyleneglycol (PEG)-lipid. We synthesized novel PEG-lipid with two different PEG lengths of 2000 and 500 (different double arms-PEG; DDA-PEG) for developing more superior passive targeting liposome. In our previous study, doxorubicin (DOX) containing liposome which was modified DDA-PEG (DDA-PEG modified liposomal DOX; DDA-LDOX) had dual effect for primary and hepatic metastasis cancer. In this study, we observed tissue distribution at primary tumor using the near infrared fluorescence and analyzed hepatic metastasis by histological observation for explain this dual effect. Moreover, this effect for pulmonary metastasis was also evaluated. [Methods] Liposome (DSPC/cholesterol/DSPG-Na/DDA-PEG = 100:100:60:7.5 μmol) was prepared according to the method of Bangham. For observing tissue distribution of liposome at primary tumor model, liposome was contained indocyanine green (ICG) as fluorescence probe. This liposome was injected in tail vein and sequentially observed using fluorescent imaging device (Clairvivo OPT, SHIMADZU). In histological observation at hepatic metastasis murine model, M5076 ovarian sarcoma cells were inoculated in tail vein of mice. These mice were injected liposome at a dose of 2.5 mg/kg (i.v.) as DOX on 6, 9 and 12 d after tumor inoculation. At 14 d, this liver was removed and examined by microscope after HE stain. At assessment of pulmonary metastasis, B16F10 melanoma cells were implanted in tail vein and treated similarly to liver metastasis.[Results and discussion] In fluorescent imaging of primary tumor, DDA-PEG modified liposome assembled into tumor at 48 h after injection. This analysis supported our previous finding that DDA-LDOX significantly decreased tumor size. Furthermore, it was proved that DDA-PEG modified liposome or encapsulated drug remained in tumor site for a long time. In histological examination for hepatic metastasis, the tumor area in the liver was 5% by DDA-LDOX injection (normal group: 90%). More lymphocytes around tumor thrombus were positive for CD45. It was suggested that macrophage or lymphocytes, especially T cell, were concerned in this therapy. Pulmonary metastasis was depressed by DDA-LDOX compared with control or DOX containing PEG unmodified liposome group. In DDA-LDOX group, DOX concentration in lung was 2.6 times higher than that in PEG unmodified liposome group. It is known that metastasis is the most severe factor in cancer chemotherapy. Antitumor agent containing DDA-PEG modified liposome may have not only superior effect for primary tumor but also hepatic or pulmonary metastasis. Hence, it was suggested that DDA-PEG modified liposome was useful drug carrier for passive targeting. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1961. doi:1538-7445.AM2012-1961