Platinum-based chemotherapy is widely used for the treatment of many malignancies [1]. Oxaliplatin, a diaminocyclohexane-containing platinum, is a member of the family of Pt-containing chemotherapeutic agents that also include cisplatin and carboplatin. Oxaliplatin is the only Pt-containing drug to have been shown to be effective in the treatment of colorectal cancer, and even though discovered 25 year ago, still remains standard of care for this particular disease. The prognosis for these patients, however, is poor because of frequent chemoresistance acquired during treatment [2-3].

The use of polymeric drug delivery systems can have enhanced anticancer effects compared with the therapeutic entities they contain due to more specific targeting of tumor tissues. The increased permeability of the tumor vasculature allows the tumor to accumulate large molecules more efficiently than normal tissues. At the same time, the poor lymphatic drainage of tumors allows high concentrations of polymeric drug to be retained in these tissues. This has been referred to as the enhanced permeability and retention (EPR) effect [4].

Previously, we have successfully prepared polymeric micelles incorporating (1,2-diaminocyclohexane)platinum(II) (DACHPt/m) [5-6], the parent complex of oxaliplatin. This system is at present being evaluated in phase I clinical trials. In preclinical experiments, DACHPt/m resulted in a high and selective accumulation of platinum at tumor tissue, and they showed to be effective in murine colon adenocarcinoma 26 tumors, which are not sensitive to oxaliplatin treatment. Moreover, DACHPt/m showed a high antitumor activity against intraperitoneal HeLa metastases, while oxaliplatin failed to suppress metastatic growth [6]. Although these appear encouraging results, the cellular activity of DACHPt/m remains to be characterized.

Recently, several studies have indicated that macromolecular drugs may have improved cellular pharmacology, or even overcome multidrug resistance (MDR) to mainly natural-product-based drugs which is caused by the overexpression of ATP-dependent efflux pumps such as P-glycoprotein (P-gp). Since macromolecular drugs enter cells by endocytic internalization, these drugs are able to penetrate cells without being recognized by P-gp leading to high intracellular drug concentration that can overcome efflux-pump mediated drug resistance [7]. Unlike the other chemotherapeutic agents, platinum drugs are not affected by P-gp expression [8]. The major mechanisms of platinum drugs are decreased membrane transport, increased cytoplasmic detoxification, increased DNA repair, and increased tolerance to platinum-induced DNA damage [9].

In the present work, the cytotoxicity of DACHPt/m against HT29 human colon cancer cells was studied. It was demonstrated that DACHPt/m has a greater cytotoxic effect compared with oxaliplatin. Moreover, the cell growth inhibition profile of DACHPt/m was examined using a human cancer cell panel. DACHPt/m seemed to be more effective against several cancer cell lines such as melanoma LOXIM VI, colon cancer HT29 and Breast cancer BSY-1 cell lines probably due to different cellular internalization or distribution of DACHPt/m from that of oxaliplatin.

In this study, a new method to assess cellular distribution and processing pathways of DACHPt/m in living cells by time lapse imaging using 2 different fluorescence-labeled DACHPt/m (F-DACHPt/m) was developed (Figure 1). During micelle state, only the dye conjugated to the micelle shell produces fluorescence while the core-conjugated dye remains quenched. In chloride containing media, the DACHPt is released from the micelle core, and as the platinum density at the core decreases, the core-conjugated dye is dequenched. Using this method, I could follow the cellular uptake of the micelles and their intracellular fate by time-lapse and confocal microscopy. The release rate of the platinum drug from the micelles was consistent with the fluorescence dequenching profile of the core-conjugated dye.

Moreover, the release of DACHPt from DACHPt/m under different intracellular conditions mimicking early endosome: 10 mM phosphate buffer, pH6.9, plus 20 mM NaCl, and late endosome: 10 mM phosphate buffer, pH5.5, plus 70 mM NaCl [10], was evaluated by the dialysis method. The release rate of platinum complexes in the environment of late endosomes accelerated up to 24 hours and was found to be faster than the release rate at early endosomal conditions, suggesting that DACHPt/m may mainly release DACHPt after reaching the late endosomal or lysosomal compartments. In addition, the release rates of F-DACHPt/m at endosomal conditions were consistent with those of DACHPt/m while the changes in the fluorescence intensity corresponded to the DACHPt release profiles.

To study the intracellular trafficking of F-DACHPt/m, HT29 cells were incubated with F-DACHPt/m and analyzed by time-lapse microscopic observation. After 6 h incubation, HT29 cells treated with F-DACHPt/m emitted the fluorescence only from the shell-conjugated dye (Bodipy-FL) while the core-conjugated dye remained quenched (Bodipy-TR) indicating that F-DACHPt/m might enter the cells in micelle form. After 24 h incubation, F-DACHPt/m displayed the fluorescence from the core-conjugated dye at the perinuclear areas.

To verify whether or not the F-DACHPt/m is located in the acidic late endosomal and lysosomal compartments, confocal microscopy studies were performed by incubation with LysoTracker. After 6h incubation, F-DACHPt/m accumulation in the late endosomal and lysosomal compartments became clear. After 24 h incubation, the accumulation of F-DACHPt/m in the late endosome and lysosome was significantly increased, and after 55 h, the core fluorescence (Bodipy TR) became evident and showed colocalization with the fluorescence from the shell and LysoTracker at the perinuclear regions.

At the previous study, the profile of DACHPt release was comparable to the fluorescence profile of the core fluorescence. Thus, the specific accumulation of F-DACHPt/m in the late endosome and lysosome, and the preferential release of DACHPt in those environments suggest that DACHPt/m may enter cancer cells by endocytosis and deliver the platinum complexes at the perinuclear region.

Additionally, the total cellular Pt levels and the amount of Pt bound to DNA after drug exposure (10μM) were measured by ICP-MS. In both intracellular Pt accumulation and amount of Pt-DNA adducts, exposure to oxaliplatin and DACHPt/m resulted in a time-dependent increase in Pt levels. For the total Pt levels, the exposure of HT29 cells to oxaliplatin resulted in 2-fold higher Pt accumulation compared to DACHPt/m. In contrast, no significant differences in DNA platination were observed between oxaliplatin and DACHPt/m exposure. Moreover, the ratio between platinum adducts and intracellular accumulation presented a much higher value for micelle being consistent with the hypothesis that DACHPt/m probably enhance platinum delivery to the nucleus. It should be noted that in the late endosome condition at 24 hours only 28% of the drug loaded in the micelles is released, suggesting that the DNA binding efficiency is markedly high, since the drug bound to the polymer might not bind to DNA.

Since DACHPt/m showed differences in cellular uptake, selective release at perinucleus, and efficient platinum delivery to DNA, the possibility that DACHPt/m retain antitumor activity against oxaliplatin resistant cell lines (HT29/ox) was studied. Thus, HT29/ox cells were prepared by exposure to oxaliplatin for 1 hour every other day. The level of resistance to oxaliplatin was evaluated by MTT assay. Thus, HT29/ox exhibited 10-fold resistance to oxaliplatin as compared to the sensitive parental HT29 cells while DACHPt/m overcame acquired resistance to oxaliplatin in HT29/ox cells.

To further confirm the enhancement of in vivo antitumor activity by DACHPt/m, Balb/c nu/nu mice bearing subcutaneous HT29 cells or HT29/ox cells were treated i.v. three times at 2-day intervals with oxaliplatin at doses of 8 mg/kg or DACHPt/m at doses of 4 mg/kg on a Pt basis which is the maximum tolerated value for each drug. The tumor volume in mice treated by DACHPt/m was approximately 10 times smaller than non-treatment group (p<0.01), and approximately 3 times smaller than those treated with oxaliplatin after 3 weeks (Figure 2A). Importantly, a similar high antitumor activity of DACHPt/m was observed against HT29/ox, while the change in tumor size after treatment with 8 mg/kg of oxaliplatin injected by the i.v. route was not different from control (P>0.1) (Figure 2B).

26 genes were selected among those available in the complete NCI database as potentially related to sensitivity or resistance to platinum compounds from the data available in the literature [11-23]. The coefficients of correlation were obtained between the cytotoxicity of two drugs (i.e.,oxaliplatin and DACHPt/m) or relative GI50 (the ratio of GI50 of DACHPt/m and GI50 of oxaliplatin) against a human cancer cell panel, and the level of expression of those markers. Metallothionein (MT1Q) and methionine synthase (MTR) were found positively correlated to GI50 of oxaliplatin, while there was no correlation with DACHPt/m. Thus, it was of interest to establish whether or not MT1Q and MTR expression was also upregulated in oxaliplatin resistant cells. The confirmation of the mRNA expression was determined by quantitative real-time RT-PCR. The expression of MT1Q and MTR protein in HT29 and HT29/ox were determined by Western blotting. Thus, MT1Q and MTR expression were found to be significantly higher in oxaliplatin resistant cells compared to parent cells.

Similar to other platinum drugs, the biological activity of oxaliplatin is based on its ability to form lethal DNA lesions, including interstrand DNA crosslinks and DNA-protein crosslinks. Once inside cells, oxaliplatin is activated by the addition of water molecules to form chemically reactive DACHPt aqua species. This is facilitated by the relatively low chloride concentrations that are found within cells [24]. Since DACHPt aqueous complexes easily react with other organelles and proteins leading to inactivation of drug, and activation of cellular defense mechanisms, it is important for platinum drugs to sneak up into the nucleus to avoid recognition by the cellular detoxification system. On the contrary, DACHPt/m probably enter via endocytosis pathway and might protect their cargo from interaction with high sulphur species, such as methionine and metallothioneins, which reacts with the activated aqua species and effectively export DACHPt form the cells, during the intracellular trafficking (Figure 3), resulting in the enhancement of drug delivery to the nucleus and the avoidance of certain cellular defense mechanisms in cytoplasm.

In summary, it has been proved that DACHPt/m was internalized by endocytosis, accumulated at perinuclear region and efficiently deliver DACHPt to DNA. This unique intracellular behavior of DACHPt/m probably has responsibility for overcoming resistance in HT29/ox. This mechanism might be different from previously described polymeric drug conjugates overcoming certain kinds of multidrug resistance. Thus, this research provides new aspects for cancer therapy using drug delivery systems.

Figure 1 Fluorescence tagged block copolymer micelles, containing dichloro(1,2-diaminocyclohexane)

platinum(II)(DACHPt, the oxaliplatin parent complex, self-assembled through polymer-metal complex formation of DACHPt with Bodipy FL -poly(ethylene glycol)-b-poly(glutamic acid)[PEG-b-P(Glu)]-Bodipy TR in distilled water. During micelle formation, only the shell-conjugated dye produces fluorescence while the core-conjugated dye remains quenched. As DACHPt is released from the micelle in chloride ion containing media, the core-conjugated dye is dequenched and emits fluorescence.

Figure 2. Antitumor activity of DACHPt/m against s.c.HT29 tumor model. Saline(crosses); oxaliplatin at 8 mg/kg(open circles);DACHPt/m at 4mg/kg (filled circles). A. Tumor volume against HT29 model; B. Tumor volume against HT29/ox model. Data are expressed as mean. Error bars show s.e.m.; n=4. *P>0.1, **P<0.05, ***P<0.01

Figure 3. Micelle mechanisms. Scheme depicting the possible reaction pathway of oxaliplatin and DACHPt/m in the cell. Oxaliplatin might enter cells by passive or through the copper transporter CTR1. Once oxaliplatin is in the cytoplasm, most of the activated aqua species are eliminated by detoxification system, while a small fraction binds to DNA. DACHPt/m enter tumor cells by endocytosis, which is accompanied by an increase of acidity and chloride concentration inside the vesicle as it matures into late endosomes. The release of platinum from DACHPt/m is 7 fold higher at late endosome environment than early endosome. Thus, DACHPt/m may release DACHPt at perinucleus resulting in enhanced delivery to the nucleus.

近年、がん化学療法において、薬剤の効果を高めるために様々なナノテクノロジーの開発が試みられており、ナノキャリアはその基盤として多大な注目を集めている。ナノキャリアは固形がんにおいて、その病態生理学的特徴を活かし、選択的に薬剤を運搬することを可能にする。標的部位にナノキャリアが集積された後、薬剤を放出することが高い制がん効果を得るために非常に重要であるものの、細胞内におけるナノキャリアの経路、その機能的影響は十分に解明されていない。したがって、従来型の長期血中滞留性ミセルは内包された薬剤より活性が低く、同程度の抗腫瘍効果を示すために高い濃度を必要とする。そこで、本研究では、ミセルを構成するブロック共重合体の両末端に二種類の異なる蛍光を導入し、そのミセルの安定性と薬剤が放出される時期をin vitro、in vivoにおいてモニターできるシステムを開発した。以下に、その論文内容を示す。

第一章は序論であり、がんの疫学、現在の抗がん剤について説明し、分子標的薬は従来の抗がん剤に比べ、副作用の軽減が期待されているものの、依然として未知の副作用を招く可能性があること、白金錯体制がん剤は広く使用されているものの耐性を獲得しやすいことから新たなアプローチが要求されていること、他の高分子キャリアにおいて、薬剤の細胞内動態(取り込み過程、細胞内局在、細胞内での薬剤の放出パターン)に変化をもたらし、薬理作用に影響を与えるという報告があること、現在、Phase I試験が実施されているダハプラチン内包ミセルは、オキサリプラチンが効果不良のマウス大腸がん、転移がんにも有効であることが示されているが細胞内動態については未解決であることなどの研究背景を説明し、本研究内容である蛍光標識を利用したミセルの細胞内動態の評価法の確立とそれを利用したダハプラチンミセルのin vitroおよびin vivo評価の概要について記述している。

第二章は蛍光ラベルしたミセルの作成方法について解説したのち、蛍光標識したミセルのサイズ、様々な生理的条件下(細胞外、初期エンドソーム、後期エンドソーム)における薬剤の放出および蛍光強度の時間変化を評価している。また、これら物理化学的特性(サイズ、薬剤の放出パターン)は蛍光標識ミセルと非標識ミセル間で同様であったことを述べている。

第三章は共焦点顕微鏡により、蛍光ラベルしたミセルがミセルの形状を維持したままエンドサイトーシスにより取り込まれ、核付近に集積することを示し、in vivoにおいても、ミセル内核に標識された色素の蛍光が徐々に現れてくることを確認している。また、細胞内Pt量、DNAアダクト量の評価から、ミセルはPtのDNAへの送達効率を高めうることを説明している。

第四章はオキサリプラチン耐性がんの作成方法を説明するとともに、ダハプラチンミセルのオキサリプラチン耐性がんに対する制がん活性をin vitroおよびin vivo実験により明らかにしている。また、cell panelとNCI databaseによる解析結果から、metallothionein、methionine synthaseの遺伝子が強く発現している細胞種に対し、オキサリプラチンは低い細胞毒性を示したが、ダハプラチンミセルの細胞毒性はそれらのタンパク質の発現量に影響を受けないことを示し、本研究で作成した耐性がんはこれらのタンパク質の発現が高いことをreal time RT-PCRおよびWestern blottingにより確認している。以上の結果から、ダハプラチンミセルの細胞内動態について、次のように考察している。白金錯体制がん剤は能動輸送あるいはトランスポーターにより細胞内に取り込まれ、metallothioneinおよびmethionine synthaseによって解毒作用を受けるため、DNAアダクト量はわずか5~10%と考えられている。一方、ダハプラチンミセルはミセルの形状を維持したままエンドサイトーシスにより取り込まれ、初期エンドソームにおいてはPtを放出せず、後期エンドソーム(核付近)でPtを放出することによって、上記の解毒機構を回避し、DNAへのPtの送達効率を3倍程度高めることが可能になる。このため、ダハプラチンミセルはある細胞種において、オキサリプラチンより高い制がん作用を示し、耐性がんにも有効であったと考えられるとしている。

第五章は結論であり、蛍光標識したミセルで細胞内動態を確認するシステムの開発により、ダハプラチンミセルのin vitroおよびin vivoにおける挙動が明らかになり、その結果としてダハプラチンミセルがオキサリプラチン耐性の克服など優れた薬効を示したことなど、研究内容全般の総括を行っている。

以上、本論文は高分子ミセル型DDSのin vitroおよびin vivoにおける挙動を明らかにし、その制御によって薬効を高めることが可能であることを示した先駆的な研究内容となっており、ドラッグデリバリーをはじめとする医工融合領域の研究の進展に寄与するところが少なくない。よって本論文は博士(工学)の請求論文として合格であると認められる。