Colloidal carriers have been used for many years mainly as the modified formulation of drug molecules exhibiting low aqueous solubility. The discovery of new therapeutic agents has facilitated a demand for more sophisticated carrier systems which are able to protect agents from inactivation due to chemical or enzymatic degradation, migrate and selectively accumulate at target sites in the body, thus enhancing the performance of the delivered agents. The recent progress in polymer science and nanotechnology certainly lend a strong basis to develop such colloidal carriers with high performance and modulated targetability.

Among these colloidal carriers, polymeric micelles have received significant attention as a promising supramolecular carrier system due to their small size and stability which lead to a prolonged blood circulation with reduced non-specific accumulation in normal tissues and preferential accumulation in solid tumors by the EPR effect (Enhanced Permeability and Retention effect). In addition to these exceptional properties, a high loading capacity of hydrophobic drug in the micelle core establishes polymeric micelle as a unique anticancer drug delivery system.

Previously, cis-dichlorodiammineplatinum(II) (cisplatin, CDDP, figure 1), a widely used anticancer drug, was incorporated into poly(ethylene glycol)-poly(amino acid) block copolymers [poly(aspartic acid) or poly(glutamic acid)] forming polymeric micelles. The physicochemical and biological properties of this micelle were extensively studied indicating that CDDP-loaded micelles are an effective delivery system for CDDP complexes.

CDDP shows acute dose-related side effects (such as nephrotoxicity, ototoxicity, neurotoxicity, nausea, vomiting and mielosupression) and the appearance of intrinsic and acquired resistance. Thus, since the discovery of CDDP in the mid 1960s, enormous efforts had been devoted to developing improved CDDP analogs. Nevertheless, only two of these analogs reached final approval, cis-diammin(cyclobutane-dicarboxylato1,1(2-0)-0,0)platinum(II) (carboplatin) and oxalate 1,2-diaminocyclohexane platinum(II) (oxaliplatin, figure 1). Oxaliplatin is a derivative of the highly active dichloro(1,2-diaminocyclohexane)platinum(II) (DACHPt, figure 1). DACHPt has shown a wide and markedly different spectrum of activity than CDDP, such as lower toxicity than CDDP and no cross-resistance with CDDP in many CDDP resistant cancers. However, the solubility of DACHPt in water is much lower than CDDP (0.25mg/ml for DACHPt vs. 1.2mg/ml for CDDP). Oxaliplatin is a third generation platinum drug approved by the Food and Drug Administration in U.S.A. (FDA) in 2004. Stability, solubility, formulation and safety issues were more promising for oxaliplatin than for other DACH-platinum compounds initially selected for preclinical testing and evaluated in early clinical trials. Oxaliplatin possesses an oxalate group, which is displaced by water and nucleophiles in biological media to activate the drug, and it also has a non-hydrolysable diaminocyclohexane (DACH) ligand, which is maintained in the final active cytotoxic metabolites of the drug.

Even though oxaliplatin shows better relative tolerability compared to other platinum compounds, a small number of side effects (like cumulative peripheral distal neurotoxicity and acute dysesthesias) restrains the range of working doses. Consequently, enormous effort has been dedicated to develop drug delivery systems that increase the blood residence time of oxaliplatin, and target it to solid tumors by taking advantage of the enhanced permeability and retention (EPR) effect. Liposomes and macromolecular carriers (water soluble polymer-drug conjugates) were the first to be attempted However, successful formulations have not been developed yet due to the unfavorable properties of platinum drugs.

The development of polymeric micelles loaded with DACHPt could lead to an oxaliplatin carrier with superior properties, such as prolonged blood circulation or higher tumor accumulation. Thus, in the present study, novel polymeric micelles entrapping DACHPt in their core were prepared through the polymer metal-complex formation between DACHPt and poly(ethylene glycol)-poly(glutamic acid) [PEG-P(Glu)] or poly(ethylene glycol)-poly(aspartic acid) [PEG-P(Asp)] block copolymers in distilled water (Figure 2).

The DACHPt-loaded micelle size ranged from 25 to 50nm with narrow distribution determined by dynamic light scattering measurement. The release of platinum complexes from the micelles cores was measured in phosphate buffer saline (pH 7.4; 150mM NaCl) at 37°C. DACHPt-loaded micelle prepared with PEG-P(Glu) showed a sustained release rate of platinum after an induction period of 12 hours whereas DACHPt-loaded micelles prepared with PEG-P(Asp) released the drug rapidly during the first 12h and reached a plateau of 30% drug released at 48h . In the same conditions, the kinetic stability of DACHPt-loaded micelle prepared with PEG-P(Glu) was found to be very high, keeping the initial size, for 240 hours. Conversely, DACHPt-loaded micelles prepared from PEG-P(Asp) showed much lower kinetic stability, maintaining the initial size for only 36h in phosphate buffered saline. Therefore, PEG-P(Glu) seems to be a promising platform for the construction of DACHPt-loaded micelles since prolonged blood circulation and increased tumor accumulation are strongly associated with micellar stability in the bloodstream.

DACHPt-loaded micelle activity was studied in vitro against human and murine cancer cell lines with the objective of establishing its anticancer potential and also to analyze whether or not the way of supplying the drug affected the drug efficacy.

DACHPt-loaded micelles exhibited considerable in vitro cytotoxicity, lower than oxaliplatin but increasing with exposure time as a result of the release of platinum complexes from the micelle. The in vitro cytotoxity of DACHPt-loaded micelles was also studied on CDDP-resistant human non-small cell lung cancer (PC-9/CDDP) and CDDP-resistant human small lung cell cancer (SBC-3/CDDP). Free CDDP and free oxaliplatin presented a decreased activity against the CDDP-resistant cancer cells. Meanwhile, CDDP-loaded micelles and DACHPt-loaded micelles maintained their cytotoxic activity on SBC-3/CDDP. The intracellular accumulation of platinum after drug exposure was studied by ICP-MS in the same cancer cell lines. The accumulation was much higher for free drugs than micelles in non-resistant cancer cells. However, for SBC-3/CDDP cell free drugs accumulated ten times less. DACHPt-loaded micelles presented similar accumulation level in both resistant and non-resistant cancer cells. Moreover, the DNA of these cancer cells was isolated and the DNA platination level was measured. DACHPt-loaded micelles and oxaliplatin showed similar DNA-bound platinum at equitoxic doses after 24h of incubation. However, after 48h of incubation, the Pt-DNA-adducts were much lower for DACHPt-loaded micelles than for oxaliplatin at equitoxic doses, suggesting that DACHPt-loaded micelles generate different cytotoxic mechanisms or increase the toxicity of the Pt-adducts. The in vitro cytotoxicity was also evaluated against the human tumor cell panel of the Japanese Foundation for Cancer Research. DACHPt-loaded micelles and oxaliplatin showed a similar fingerprint, however, DACHPt-loaded micelles showed higher activity in some tumor cell lines. From these results, DACHPt-loaded micelles have been proven to have potent in vitro anticancer activity, at times higher than free oxaliplatin suggesting that DACHPt-loaded micelles can enhance the efficiency of the active platinum complexes.

In vivo studies were performed to characterize the biological behavior of the micelles as well as to optimize the micelle formulation. Therefore, biodistribution studies of DACHPt-loaded micelles prepared with PEG-P(Glu) 12-40 (PEG-P(Glu) 12-40: MwPEG: 12,000; p(Glu) units: 40) were performed at different times on mice bearing subcutaneous C-26 tumors. This DACHPt-loaded micelles have long circulation in blood and showed 20-fold greater accumulation in tumor even after 48h. Morevover, the biodistribution of DACHPt-loaded micelles prepared with PEG-P(Glu) block copolymer having different p(Glu) lengths [p(Glu): 20, 40, and 70 units] was studied with the aim of optimizing the system's biological performance. The micelles prepared from PEG-b-P(Glu) with 20 units of P(Glu) exhibited the lowest non-specific accumulation in the liver and spleen to critically reduce non-specific accumulation, resulting in higher specificity to solid tumors. In addition, DACHPt-loaded micelles prepared with PEG-P(Glu) 12-20 or 12-40 presented extremely high antitumor activity against a subcutaneous murine colon adenocarcinoma 26 while toxicity was considerably reduced for the PEG-P(Glu) 12-20 formulation. Furthermore, DACHPt-loaded micelles presented extremely high antitumor activity against a series of intractable tumor models: a subcutaneous BxPC3 pancreatic tumor model that is refractive to established therapies and hypovascular, a melanoma lung metastasis model and a HeLa intraperitoneal metastasis. The outstanding results obtained in all the tumor models suggest that DACHPt-loaded micelles could be an outstanding drug delivery system for oxaliplatin in the treatment of solid tumors.

Nevertheless, the activity of most long circulating drug carriers decreases due to their poor cellular uptake, which is often a disadvantage for exerting drug efficacy compared to free drugs that rapidly move into the interior of the cells. Therefore, it is likely that if a macromolecular drug carrier is actively incorporated by cancer cells, the specificity and bioavailability of those drug delivery systems should increase more than a drug delivery system that just takes advantage of the EPR effect to target the tumor site. There are many examples for utilizing the existing endocytosis pathways (Figure 3) for specific delivery of drugs. The conjugation of normally endocytosed ligands with macromolecular carriers frequently improves intracellular accumulation of the prodrug.

Among these ligands, the folic acid has been intensively investigated as a means for tumor-specific delivery of a broad range of experimental therapies including several conceptually new treatments. Herein, folic acid was conjugated on the surface of polymeric micelles incorporating the active complexes of Cisplatin or DACHPt in order to obtain folate-bound polymeric micelles directed to the Folate Binding Protein (FBP) overexpressed on cancer cell surfaces (Figure 3).

Platinum drug-loaded micelles were prepared through metal-polymer complex formation with Acetal-Benzyl-poly(ethylene glycol)-b-poly(glutamic acid) (Ac-Bz-PEG-b-P(Glu)). A hydrazide group was introduced to folic acid to obtain folate-hydrazide. Then, the folate-hydrazide was bounded to the surface of the micelles to obtain folate-conjugated platinum drug-loaded micelles. The folate-conjugated platinum drug-loaded micelles presented narrowly distributed diameters of approximately 40nm. The zetapotential of folate-conjugated platinum drug-loaded micelles at pH 7.4 decreased as the percentage of conjugated folate on the micelle surface increased. The interaction of folate-bound platinum drug-loaded micelles with the folate binding protein (FBP) was determined to be very strong by surface plasmon resonance (SPR) studies. Moreover, folate-conjugated platinum drug-loaded micelles showed enhanced in vitro growth inhibitory activity against the pharyngeal cancer KB cells after short incubation much higher than non-targeted platinum drug-loaded micelles and comparable to that of free drugs. These results suggest that the folate-targeting method for platinum drug-loaded micelles seems to be a very effective way to increase the specificity and activity of platinum drug-loaded micelles to cancer cells.

From the results attained in this study it can be concluded that the DACHPt-loaded micelle offers an exceptional platform for the delivery of the active complexes of oxaliplatin specifically targeting the tumor tissue and enhancing the drug activity. Furthermore, since polymeric micelles can be tuned relatively easily, the future perspectives for this micellar system are enormous.

Figure 1. Platinous anticancer drugs. CDDP and Oxaliplatin have been clinically approved

Figure 2. A. Preparation of DACHPt-loaded micelles from PEG-P(Asp) or PEG-P(Glu) by polymer complex formation. B. In chloride containing media, the chloride ions diffuse into the micelle core and cleave the metal-polymer complex producing the DACHPt release

Figure 3. Folate receptor-mediated endocytosis of folate-drug conjugates. Exogenously added folate-drug conjugates bind specifically to the folate binding protein with high affinity. The plasma membrane invaginates around the conjugate/folate receptor complex to form an intracellular vesicle (endosome). As the lumen of the maturing endosome acidifies up to pH 5.5, the receptor changes conformation and releases the conjugate. Eventually, the fates of the folate ligand, attached drug cargo and the folate receptor are determined during a sorting process within late endosomal elements.

生体適合性に優れたナノスケールの微粒子である高分子ミセルは、高い血中滞留性を示す一方で、腫瘍毛細血管の選択的透過性亢進作用(EPR効果)によって、固形ガンに効果的に集積することが示されている。これらの特性を利用することによって、高分子ミセル内核に薬物を搭載することで新規な制ガン剤送達システムの創製が期待できる。これまでにポリエチレングリコール(PEG)-ポリアミノ酸ブロック共重合体が形成する高分子ミセル内核に、広く臨床応用されている制ガン剤であるシスプラチンを導入し、その物理化学的、生物学的評価から、高分子ミセルの制ガン剤送達システムとしての有用性が確認されている。本研究では、マテリアル工学的観点から第三世代の白金系制ガン剤であるオキサリプラチンの活性形であるDichloro(1,2-Diaminocyclohexane)Platinum(II) (DACHPt)を導入した高分子ミセルを新たに構築し、その新規制ガン剤送達システムとしての機能を検証し、高分子ミセル型薬物キャリアにおける構造-機能相関を明らかにすることを目的としている。以下、各章毎に、本論文の審査結果の概要を述べる。

第1章の序論においては、ガンの標的治療における高分子キャリアの重要性から説き起こし、PEG-ポリアミノ酸ブロック共重合体からなる制ガン剤内包高分子ミセルの有用性を指摘している。さらに、制ガン剤内包高分子ミセルのこれまでのガン標的治療における展開を述べるとともに、白金系制ガン剤を内包する高分子ミセルを創製する意義および新たに第三世代の白金系制ガン剤であるDACHPtを高分子ミセルに内包することの論点を明確に述べている。

第2章では、DACHPtとPEG-poly(glutamic acid)ブロック共重合体 (PEG-PGlu)、もしくはPEG-poly(aspartic acid)ブロック共重合体 (PEG-PAsp)との間の金属錯体形成に基づくDACHPt内包高分子ミセルを調製し、その粒径や生理的塩濃度に調製した緩衝液中における薬物放出挙動などのキャラクタリゼーションを行っている。その結果、両者ともに粒径40 nm程度の極めて単分散性が良好な高分子ミセルを与えることを明らかにしている。また、これらのDACHPt内包高分子ミセルは純水中では長期にわたっての安定性を維持するが、生理的塩濃度下の緩衝液中においては、長期にわたる薬物徐放挙動を示すことが示されている。さらに、内包薬物の徐放挙動に関して、PEG-PAspとPEG-PGluから形成される高分子ミセルについて詳細な比較解析を行っており、後者の方がより長期にわたって一定の薬物徐放が可能であること、ならびに徐放が開始されるまでに十分な誘導期を有することを見出している。このような挙動について、両方のブロック共重合体におけるポリアミノ酸セグメントの疎水性ならびに白金錯体の配位機構の面からの考察を行い、PEG-PGluから形成されるDACHPt内包高分子ミセルの方が薬物キャリアとして優れた性質を有することを結論づけている。

第3章では、マウス大腸ガン由来の細胞株であるC26を用いることによって、DACHPt内包高分子ミセルのin vitro制ガン活性評価を低分子のプロドラッグであるオキサリプラチンと比較しながら検討している。高分子ミセルに内包したDACHPtはオキサリプラチンよりも一桁ほど毒性が低いものの、長期接触によってその活性が上昇していくことが観察され、時間とともに高分子ミセルより薬物が徐放されることを確認している。さらに、ヒト腫瘍細胞パネルを用いて各種ガン細胞に対する活性を網羅的に検討した結果、一部の細胞種においては、オキサリプラチン単独の場合よりも高い細胞毒性を示すことを見いだしている。また、DACHPt内包高分子ミセルは、シスプラチン耐性ガン細胞に対してオキサリプラチンよりも高い活性を有することを見いだしており、白金錯体制ガン剤に対して耐性を獲得したガン細胞についても高い効果を発揮することを示唆している。さらに、これらの結果についての考察を行い、DACHPt内包高分子ミセルが直接、細胞内に取り込まれることによって、従来の低分子制ガン剤とは異なる機序で細胞へ作用する可能性を指摘している。

第4章では、DACHPt内包高分子ミセルのin vivo評価を行っている。DACHPt内包高分子ミセルは、オキサリプラチンに比べて高い血中滞留性を有するとともに、48時間後において約20 倍という優れた腫瘍集積性を示すことを確認している。また、ブロック共重合体の組成との関連においては、PGlu鎖長の短い方が、相対的に高分子ミセル外殻のPEG密度が上昇する結果、ガン以外への一般臓器、特に肝臓への非特異的集積が抑制され、腫瘍への選択的集積性が増大することを見出している。さらに、C26をはじめとする種々の腫瘍モデルに対して、DACHPt内包高分子ミセルが高い制ガン活性を示すことを確認し、その固形ガン治療における有用性を示している。さらに、DACHPt内包高分子ミセルは悪性黒色腫の肺転移モデルに対しても高い増殖抑制効果をもたらすことを見いだしており、本系が原発ガンのみならず転移ガンの治療に対しても高い有用性を示すことを指摘している。

第5章では制ガン剤内包高分子ミセル表面への葉酸コンジュゲートについて述べている。これは、葉酸を高分子ミセル表面に担持させることで、腫瘍細胞に特異的に過剰発現している葉酸受容体との選択的結合を介して、高分子ミセルが細胞内へ取り込まれることを意図した分子設計である。葉酸担持高分子ミセルが葉酸受容体に対して特異的な結合活性を有することを確認した後に、in vitroにおける細胞毒性評価を行い、葉酸担持高分子ミセルが葉酸非担持高分子ミセルよりも優れた効果を示すことを見いだしている。この結果から、高分子ミセル表面に葉酸などのリガンドを導入することで、制ガン剤内包高分子ミセルの腫瘍細胞への特異的選択性の向上が可能であると結論づけている。

以上のように本論文では、高分子ミセルがオキサリプラチン活性型薬物であるDACHPtを送達するキャリアとなり得ることを培養細胞レベルのみならず系統的な動物実験を行うことから明らかとしており、その際には、ブロック共重合体組成の精密制御が不可欠であるというマテリアル工学的見地から重要な結論を導き出している。さらに、固形ガン部位における血管透過性の亢進を利用した受動的ターゲティング(EPR効果)のみならず、高分子ミセル表面にリガンド分子を搭載することで腫瘍に対して特異的に集積し、顕著な制ガン活性を発現し得るという能動的ターゲティングの可能性をも示している。リガンド分子の機能発現が例示する化学的修飾による機能性付与の可能性は、今後の高分子ミセル系薬物送達システムにおける新たな展開を示しており、優れた展開であると考えられる。また、本研究の成果に基づき、DACHPt内包高分子ミセルの臨床開発が始まっていることも、特筆に値する事項である。本論文の内容は、白金錯体系制がん剤による新規治療法を高分子材料設計の観点より提案するものであり、その独創的なアプローチやガン化学療法における有用性から考えて、バイオマテリアルを中心とする医工学研究分野において極めて秀逸であると判定される。

よって本論文は博士(工学)の学位請求論文として合格と認められる。