リポソーム ト ホタイケイ ノ ソウゴ サヨウ ニ カンスル ケンキュウ

生体内投与後のリポソームの排除機構として，血中での不安定化と単 核食細胞系(MPS)による取り込みという二つの機構が想定されている. 両機構において重要な役割を果たすと考えられているのが，補体系であ る.補体系は20種類以上の血液成分から構成される体液性初期生体防御 機構の一つであり，補体系によるリポソームの認識は， MPS上の補体レ セプターのリガンドであるC3フラグメントの結合と，内封物質の漏出を 促す小孔(MAC)の形成を促進させるため，リポソームの体内動態に大き な影響をおよぼすと考えられる. したがって，補体系がリポソームをどの ように異物として認識するか，そしてその相互作用がどのような要因によって 支配されているかを明らかにすることは，有用なリポソーム製剤を開発する上 で非常に有用な情報を提供すると考えられる. そこで，補体系とリポソームとの相互作用に関して詳細な検討を行った. I　補体系のリポソームの体内動態への寄与の検討　 上述のとおり，補体系によるオプソニン化(C3フラグメント結合)および膜 破壊(MAC形成)がリポソームの動態と密接な関係にあると考えられる.そ こで，リポソーム表面に結合したC3フラグメント量と，血清中でのリポソーム の崩壊度を測定し， リポソームの体内動態とどのような関係にあるか検討した. その結果， C3フラグメント結合量は，リポソームの肝移行性の指標である肝ク リアランスと良好な相関関係にあることが明らかとなった.このことは，肝臓 によるリポソームの取り込みが， C3フラグメント結合量，すなわち補体系との 相互作用によって支配されていることを示すものである.一方，飽和リン脂質 を基剤としたリポソームの血清中での不安定化は補体依存的であったのに対し て，不飽和リン脂質からなるリポソームの不安定化は補体系以外，例えばHDL などのリポタンパクとの相互作用によることが示された. しかし血清中での 補体依存的な崩壊の程度と， in vivoでの不安定化の指標である腎クリアランス との間には一定の相関関係があることが示された.このことから，in vitroで補 体依存的な崩壊を示すリポソームにあっては，in vivoにおいてもその安定性が 補体系に支配されることが明らかとなった. 以上の結果から，補体系との相互 作用，特にC3フラグメントの結合およびMAC形成がリポソームの動態と密接 な関係にあることが明らかとなった.　 E　補体系によるリポソーム認識機構の検討 補体系の活性化経路には古典経路と第二経路の二つの経路があることが知ら れている. 検討の結果， 22%， 33%コレステロール含有リポソームは古典経路 を， 44%コレステロール含有リポソームは第二経路を活性化することが示され， リポソーム組成中のコレステロール含量(ホスファチジルコリン含量)の違い により活性化される経路が異なることが明らかとなった.これまでに，コレス テロール(あるいはホスファチジルコリン)含量の違いがリポソームによる補 体活性化経路を変化させるという報告はなく，本報告が初めてである.そこで， この原因について検討した.その結果，低温下(0℃でリポソーム表面に吸着 する血清因子の寄与により起動されることが明らかとなった.また，興味深い ことに，古典経路に寄与する因子と第二経路に寄与する因子とは別個のもので あり，特に第二経路に寄与する因子はコレステロールに親和性を有する可能性 が示唆された また，このような活性化経路の違いがおよぼす補体系によるオプソニン化お よび膜破壊への影響について検討した. 古典経路を介したオプソニン化は非常 に速やかであり，第二経路を介したオプソニン化は僅かなlag timeの後緩やかに 上昇することが明らかとなった.また古典経路を介した膜破壊は非常に速や かであったのに対して，第二経路を介した場合lag timeの後上昇することが明ら かとなった. したがって，補体活性化の経路の違いは，リポソームと補体系と の相互作用の速度を変化させることが明らかとなった.　 Ⅲ　ヒト補体系との相互作用に関する検討 本来，リポソームはヒトへの適応が志向されており， ヒト補体系との相互作 用について検討することは， ヒトにおけるリポソームの動態を推察する上で有 用な情報を与えると考えられる.そこで，糖修飾リポソーム(Man-Lip)をモ デルリポソームとしてヒト補体系によるリポソームの認識機構について 検討した.その結果， Man-Lipによるヒト補体系の活性化は古典経路を 介したものであり，この活性化には自然抗体由来IgMのMan-Lip表面への 吸着が必須であることが明らかとなった. IgMは，分子量約900KDaの巨 大血液蛋白であり，ヒト補体系による粒子径依存的なMan-Lipの認識に おいて重要な役割を果たす可能性が示唆された. 以上の検討から，生体内投与後のリポソームの動態は，それらを異物として 認識する生体防御機構と密接に関連しており，その中でも特に自然抗体などの 補体活性化因子のリポソームへの結合，そしてその結合に連動した補体系の活 性化としづ基本的な体液性生体防御機構の寄与が大きいことが明らかとなった. このことは， リポソームを用いた薬物送達システムの開発が，単にリポソーム 化による薬物の薬効発現あるいは副作用の軽減を目指すだけでなく，生体防御 機構との相互作用を十分に考慮し，基本的な生体反応に立脚して行われるべき であることを示唆していると思われる.また， リポソームの脂質組成(コレス テロール含量）の変化により補体活性化経路が変化するという新たな知見は， 未だ明らかでない古典経路と第二経路の関係を解明する上で，有用な情報を与 えると考えられる

Lidocaine self-sacrificially improves the skin permeation of the acidic and poorly water-soluble drug etodolac via its transformation into an ionic liquid

Poor transdermal penetration of active pharmaceutical ingredients (APIs) impairs both bioavailability and therapeutic benefits and is a major challenge in the development of transdermal drug delivery systems. Here, we transformed a poorly water-soluble drug, etodolac, into an ionic liquid in order to improve its hydrophobicity, hydrophilicity and skin permeability. The ionic liquid was prepared by mixing etodolac with lidocaine (1:1, mol/mol). Both the free drug and the transformed ionic liquid were characterized by differential scanning colorimetry (DSC), infrared spectroscopy (IR), and saturation concentration measurements. In addition, in vitro skin-permeation testing was carried out via an ionic liquid-containing patch (Etoreat patch). The lidocaine and etodolac in ionic liquid form led to a relatively lower melting point than either lidocaine or etodolac alone, and this improved the lipophilicity/hydrophilicity of etodolac. In vitro skin-permeation testing demonstrated that the Etoreat patch significantly increased the skin permeation of etodolac (9.3-fold) compared with an etodolac alone patch, although an Etoreat patch did not increase the skin permeation of lidocaine, which was consistent with the results when using a lidocaine alone patch. Lidocaine appeared to self-sacrificially improve the skin permeation of etodolac via its transformation into an ionic liquid. The data suggest that ionic liquids composed of approved drugs may substantially expand the formulation preparation method to meet the challenges of drugs which are characterized by poor rates of transdermal absorption

A novel polyethylene glycol (PEG)-drug conjugate of Venetoclax, a Bcl-2 inhibitor, for treatment of acute myeloid leukemia (AML)

Background: Venetoclax (VTX) is an anticancer drug. It is a selective Bcl-2 inhibitor that is clinically used for the treatment of patients with lymphomas and leukemias. Treatment with VTX, however, is accompanied by severe adverse events such as tumor lysis syndrome and neutropenia, because VTX readily binds to serum proteins, which results in poor pharmacokinetics and poor tumor tissue concentration. To avoid such adverse events, VTX is administered using a daily or weekly ramp-up schedule that is cumbersome in clinical situations. Aims: To overcome these shortcomings, we prepared a novel polyethylene glycol (PEG)-drug conjugate of VTX (PEG-VTX) and evaluated its cytotoxic effects on acute myeloid leukemia (AML) both in vitro and in vivo. Methods and results: VTX and 4-armed PEG derivatives were covalently attached through an amide bond linker. In a series of in vitro studies, PEG-VTX selectively induced potent growth inhibition of MV4-11 human AML cells via the inducement of Bcl-2-mediated apoptosis. PEG-VTX had the effect of free VTX, presumably due to the protease-mediated release of VTX from the conjugates. In in vivo studies with AML tumor-xenograft mice models, intravenous PEG-VTX promoted sufficient tumor growth suppression. Compared with a regimen of oral free VTX, the intravenous regimen in those studies used a VTX dosage that was 15–30 times smaller for an OCI-AML-2 xenograft model and a dosing regimen that was less frequent for an MV4-11 xenograft model. The most important development, however, was the absence of weight loss related to severe side effects throughout the treatments. An increase in water solubility and the resultant hydrodynamic size of VTX via PEGylation improved the pharmacokinetics of VTX by avoiding protein interactions and lessening the extravasation from blood. The result was an increase in tumor accumulation and a decrease in the nonspecific distribution of VTX. Conclusion: The results of this study suggest that PEG-VTX could be an alternative therapeutic option for the safe and effective treatment of patients with AML

Anti-PEG antibodies : Properties, formation, testing and role in adverse immune reactions to PEGylated nano-biopharmaceuticals

Conjugation of polyethylene glycols (PEGs) to proteins or drug delivery nanosystems is a widely accepted method to increase the therapeutic index of complex nano-biopharmaceuticals. Nevertheless, these drugs and agents are often immunogenic, triggering the rise of anti-drug antibodies (ADAs). Among these ADAs, anti-PEG IgG and IgM were shown to account for efficacy loss due to accelerated blood clearance of the drug (ABC phenomenon) and hypersensitivity reactions (HSRs) entailing severe allergic symptoms with occasionally fatal anaphylaxis. In addition to recapitulating the basic information on PEG and its applications, this review expands on the physicochemical factors influencing its immunogenicity, the prevalence, features, mechanism of formation and detection of anti-PEG IgG and IgM and the mechanisms by which these antibodies (Abs) induce ABC and HSRs. In particular, we highlight the in vitro, animal and human data attesting to anti-PEG Ab-induced complement (C) activation as common underlying cause of both adverse effects. A main message is that correct measurement of anti-PEG Abs and individual proneness for C activation might predict the rise of adverse immune reactions to PEGylated drugs and thereby increase their efficacy and safety

Use of the Post-Insertion Technique to Insert Peptide Ligands into Pre-Formed Stealth Liposomes with Retention of Binding Activity and Cytotoxicity

Purpose: Simple methods for the large-scale manufacture of ligand-targeted liposomes will be needed if clinical trials are to proceed. We tested a recently developed technology for inserting peptide ligands into preformed Stealth liposomes. Antagonist G-targeted liposomes (PLG) were prepared and loaded with doxorubicin and their cellular association and cytotoxicity were evaluated using the human small cell lung cancer H69 cell line

Systemically Administered RNAi Molecule Sensitizes Malignant Pleural Mesotheliomal Cells to Pemetrexed Therapy

Pemetrexed (PMX) is a key drug for the management of malignant pleural mesothelioma (MPM). However, its therapeutic efficacy is cruelly restricted in many clinical settings by the overexpression of thymidylate synthase (TS) gene. Recently, we emphasized the efficacy of locally administered shRNA designed against TS gene in enhancing the cytotoxic effect of PMX against orthotopically implanted MPM cells in tumor xenograft tumor model. Herein, we explored the efficiency of systemic, rather than local, delivery of TS RNAi molecule in sensitizing MPM cells to the cytotoxic effect of PMX. We here designed a PEG-coated TS shRNA-lipoplex (PEG-coated TS shRNA-lipoplex) for systemic injection. PEG modification efficiently delivered TS shRNA in the lipoplex to tumor tissue following intravenous administration as indicated by a significant suppression of TS expression level in tumor tissue. In addition, the combined treatment of PMX with systemic injection of PEG-coated TS shRNA-lipoplex exerted a potent antitumor activity in a s.c. xenograft tumor model, compared to a single treatment with either PMX or PEG-coated TS shRNA-lipoplex. Metastasis, or the spread, of mesothelioma substantially dedicates the effectiveness of treatment options. The systemic, in addition to local, delivery of tumor targeted anti-TS RNAi system we propose in this study might be an effective option to extend the clinical utility of PMX in treating malignant mesothelioma

S-1 mediated tumor priming enhances intratumor liposomal fate

The efficient delivery of nanocarrier-based cancer therapeutics into tumor tissue is problematic. Structural abnormalities, tumor vasculature heterogeneity, and elevated intratumor pressure impose barriers against the preferential accumulation of nanocarrier-based cancer therapeutics within tumor tissues and, consequently, compromise their therapeutic efficacy. Recently, we have reported that metronomic S-1, orally available tegafur formulation, dosing synergistically augmented the therapeutic efficacy of oxaliplatin (l-OHP)-containing PEGylated liposome without increasing the toxicity in animal model. However, the exact mechanism behind such synergistic effect was not fully elucidated. In this study, therefore, we tried to shed the light on the contributions of metronomic S-1 dosing to the enhanced accumulation and/or spatial distribution of PEGylated liposome within tumor tissue. Tumor priming with metronomic S-1 treatment induced a potent apoptotic response against both angiogenic endothelial cells and tumor cells adjacent to tumor blood vessels, resulting in enhanced tumor blood flow via transient normalization of tumor vasculature, along with alleviation of intratumor pressure. Such a change in the tumor microenvironment imparted by S-1 treatment allows efficient delivery of PEGylated liposome to tumor tissue and permits their deep penetration/distribution into the tumor mass. Such a priming effect of S-1 dosing can be exploited as a promising strategy to enhance the therapeutic efficacy of nanocarrier-based cancer therapeutics suffering from inadequate/heterogeneous delivery to tumor tissues

Metronomic S-1 dosing and thymidylate synthase silencing have synergistic antitumor efficacy in a colorectal cancer xenograft model

Metronomic chemotherapy is currently considered an emerging therapeutic option in clinical oncology. S-1, an oral formulation of Tegafur (TF), a prodrug of 5-fluorouracil (5-FU), is designed to improve the antitumor activity of 5-FU in tandem with reducing its toxicity. Clinically, metronomic S-1 dosing has been approved for the standard first- and second-line treatment of metastatic or advanced stage of colorectal (CRC). However, expression of intratumor thymidylate synthase (TS), a significant gene in cellular proliferation, is associated with poor outcome to 5-FU-based chemotherapeutic regimens. In this study, therefore, we examined the effect of a combination of TS silencing by an RNA interfering molecule, chemically synthesized short hairpin RNA against TS (shTS), and 5-FU on the growth of human colorectal cancer cell (DLD-1) both in vitro and in vivo. The combined treatment of both shTS with 5-FU substantially inhibited cell proliferation in vitro. For in vivo treatments, the combined treatment of metronomic S-1 dosing with intravenously injected polyethylene glycol (PEG)-coated shTS-lipoplex significantly suppressed tumor growth, compared to a single treatment of either S-1 or PEG-coated shTS-lipoplex. In addition, the combined treatment increased the proportion of apoptotic cells in the DLD-1 tumor tissue. Our results suggest that metronomic S-1 dosing combined with TS silencing might represent an emerging therapeutic strategy for the treatment of patients with advanced CRC

FTY720 Reduces Lipid Accumulation by Upregulating ABCA1 through Liver X Receptor and Sphingosine Kinase 2 Signaling in Macrophages

Formation of foam cells as a result of excess lipid accumulation by macrophages is a pathological hallmark of atherosclerosis. Fingolimod (FTY720) is an immunosuppressive agent used in clinical settings for the treatment of multiple sclerosis and has been reported to inhibit atherosclerotic plaque development. However, little is known about the effect of FTY720 on lipid accumulation leading to foam cell formation. In this study, we investigated the effects of FTY720 on lipid accumulation in murine macrophages. FTY720 treatment reduced lipid droplet formation and increased the expression of ATP-binding cassette transporter A1 (ABCA1) in J774 mouse macrophages. FTY720 also enhanced the expression of liver X receptor (LXR) target genes such as FASN, APOE, and ABCG1. In addition, FTY720-induced upregulation of ABCA1 was abolished by knockdown of sphingosine kinase 2 (SphK2) expression. Furthermore, we found that FTY720 treatment induced histone H3 lysine 9 (H3K9) acetylation, which was lost in SphK2-knockdown cells. Taken together, FTY720 induces ABCA1 expression through SphK2-mediated acetylation of H3K9 and suppresses lipid accumulation in macrophages, which provides novel insights into the mechanisms of action of FTY720 on atherosclerosis

Adjuvant Antitumor Immunity Contributes to the Overall Antitumor Effect of Pegylated Liposomal Doxorubicin (Doxil®) in C26 Tumor-Bearing Immunocompetent Mice

Doxorubicin (DXR) has been reported to have direct cytotoxicity against cancer cells and indirect immunotoxicity by modulation of host antitumor immunity. Hence, it may prevent cancer progression by a dual mechanism. Doxil®, a formulation of DXR encapsulated in polyethylene glycol modified (PEGylated) liposomes, is the most widely used of the clinically approved liposomal anticancer drugs. However, the effect of Doxil® on host antitumor immunity is not well understood. In this study, Doxil® efficiently suppressed tumor growth in immunocompetent mice bearing C26 murine colorectal carcinomas, but not in T cell-deficient nude mice, indicating a contribution of T cells to the overall antitumor effect of Doxil®. In immunocompetent mice, Doxil® increased major histocompatibility complex (MHC-1) levels in C26 tumors, which may be an indicator of increased immunogenicity of tumor cells, and potentially amplified tumor immunogenicity by decreasing immunosuppressive cells such as regulatory T cells, tumor-associated microphages and myeloid-derived suppressor cells that collectively suppress T cell-mediated antitumor responses. This suggests that encapsulation of DXR into PEGylated liposomes increased the therapeutic efficacy of DXR though effects on host antitumor immunogenicity in addition to direct cytotoxic effects on tumor cells. This report describes the role of host antitumor immunity in the overall therapeutic effects of Doxil®. Manipulating pharmacokinetics and biodistribution of chemotherapeutic agents with immunomodulatory properties may increase their therapeutic efficacies by amplifying host antitumor immunity in addition to direct cytotoxic effects on tumor cells