腫瘍血管新生の可視化を可能にする分子イメージング剤の開発

金沢大学がん研究所VEGFを構成するアミノ酸配列の中で79～93番目の配列を基にして放射性ヨウ素標識を可能にするチロシンを導入したペプチド化合物をドラッグデザインし、化合物のヒト臍帯静脈血管内皮細胞(HUVEC)に対する増殖阻害能をMTSアッセイにより検討してきたが、再現性のある結果が得られなかった。また、[methyl-^3H]thymidine集積実験によりHUVECの増殖能評価を行ってきたが、ペプチド負荷による著しい集積低下は確認されなかった。そこで、HUVECと種々の濃度のペプチドを72時間培養した後に、細胞数を測定した。しかしながら、ペプチド負荷による増殖阻害効果は得られなかった。これまで行ってきた増殖阻害実験では、最大ペプチド濃度を100μMとしてきたが、十分な阻害効果を得るためにはさらに高濃度なペプチドが必要と考えられた。また、各種ペプチドを放射性ヨウ素標識した際に、たくさんのUVピークが観察されたことから、本ペプチドは安定性に問題があることが考えられた。5つのアミノ酸からなる環状RGDペプチドは非常に安定性が高いことから、今後VEGF受容体に対する親和性に影響を及ぼさないアミノ酸を除外し、アミノ酸の少ない環状ペプチドにすることにより、安定性の高い化合物になりうることが予想される。血管新生は、腫瘍イメージングにおける強力なターゲットであることから、本研究で得られた知見を参考に、今後更なるイメージング剤開発を行う。研究課題/領域番号:19659307, 研究期間(年度):2007 – 2009出典：研究課題「腫瘍血管新生の可視化を可能にする分子イメージング剤の開発」課題番号19659307（KAKEN：科学研究費助成事業データベース（国立情報学研究所）） （https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-19659307/）を加工して作

Theragnostic Imaging Using Radiolabeled Antibodies and Tyrosine Kinase Inhibitors

During the past decade, the efficacy of new molecular targeted drugs such as tyrosine kinase inhibitors (TKIs) and monoclonal antibodies has been proven worldwide, and molecular targeted therapies have become the mainstream in cancer therapy. However, clinical use of these new drugs presents unexpected adverse effects or poor therapeutic effects. Therefore, we require diagnostic tools to estimate the target molecule status in cancer tissues and predict therapeutic efficacy and adverse effects. Although immunohistochemical, polymerase chain reaction (PCR) and fluorescence in situ hybridization (FISH) analyses of biopsy samples are conventional and popular for this diagnostic purpose, molecular imaging modalities such as positron emission tomography (PET) and single photon emission computed tomography (SPECT) are also useful for noninvasive estimation of gene and protein expression and drug pharmacokinetics. In this review, we introduce new radiolabeled TKIs, antibodies, and their clinical application in molecular targeted therapy and discuss the issues of these imaging probes

Evaluation of radioiodinated vesamicol analogs for sigma receptor imaging in tumor and radionuclide receptor therapy

金沢大学医薬保健研究域薬学系It has been reported that sigma receptors are highly expressed in a variety of human tumors. In this study, we selected (+)-2-[4-(4-iodophenyl)piperidino] cyclohexanol [(+)-pIV] as a sigma receptor ligand and evaluated the potential of radioiodinated (+)-pIV for tumor imaging and therapy. (+)-[125/131I]pIV was prepared by an iododestannylation reaction under no-carrier-added conditions with radiochemical purity over 99% after HPLC purification. Biodistribution experiments were performed by the intravenous injection of (+)-[125I]pIV into mice bearing human prostate tumors (DU-145). Blocking studies were performed by intravenous injection of (+)-[125I]pIV mixed with an excess amount of unlabeled sigma ligand into DU-145 tumor-bearing mice. For therapeutic study, (+)-[131I]pIV was injected at a dose of 7.4 MBq followed by measurement of the tumor size. In biodistribution experiments, (+)-[125I]pIV showed high uptake and long residence in the tumor. High tumor to blood and muscle ratios were achieved because the radioactivity levels of blood and muscle were low. However, the accumulations of radioactivity in non-target tissues, such as liver and kidney, were high. The radioactivity in the non-target tissues slowly decreased over time. Co-injection of (+)-[125I]pIV with an excess amount of unlabeled sigma ligand resulted in a significant decrease in the tumor/blood ratio, indicating sigma receptor-mediated tumor uptake. In therapeutic study, tumor growth in mice treated with (+)-[131I]pIV was significantly inhibited compared to that of an untreated group. These results indicate that radioiodinated (+)-pIV has a high potential for sigma receptor imaging in tumor and radionuclide receptor therapy. (Cancer Sci 2009). © 2009 Japanese Cancer Association

Reduction of 223Ra retention in the Large Intestine During Targeted Alpha Therapy with 223RaCl2 by Oral BaSO4 Administration in Mice

Background: Targeted alpha therapy with 223RaCl2 is used to treat skeletal metastases of hormone-refractory prostate cancer. The intravenous injection of 223RaCl2 causes gastrointestinal disorders such as nausea, abdominal discomfort, and diarrhea as frequent clinical adverse events caused by radiation. BaSO4 is known to display Ra2+ ion uptake in its structure and is clinically used as a contrast agent for X-ray imaging following oral administration. Here, we investigated the feasibility of a method to reduce 223Ra retention in the large intestine with BaSO4 by biodistribution studies in mice. Methods: 223RaCl2 biodistribution was examined in ddY mice after intravenous administration (10 kBq/mouse).BaSO4 (100 mg/mouse) was orally administered 1 h before 223RaCl2 injection. We also investigated the effect of laxative treatment on BaSO4 activity, since laxatives are clinically used with BaSO4 to avoid impaction in the large intestine. Results: BaSO4 significantly reduced 223Ra retention in the large intestine after 223RaCl2 injection in mice when compared with the control without BaSO4 administration (P < 0.05). Excretion of 223Ra into the feces was significantly increased by BaSO4 administration (P < 0.05). Laxative treatment did not affect BaSO4 activity in reducing 223Ra retention, although no additional effect of laxative treatment to 223Ra excretion was observed in mice. Conclusions. BaSO4 administration was effective in reducing 223Ra retention in the large intestine during 223RaCl2 therapy, and laxative treatment did not attenuate BaSO4 activity. This method could be useful in reducing adverse events caused by radiation exposure to the large intestine during 223RaCl2 therapy

In Vivo Simultaneous Imaging of Vascular Pool and Hypoxia with a HT-29 Tumor Model: the Application of Dual-Isotope SPECT/PET/CT

Investigation of vascularity and hypoxia in tumors is important in understanding cancer biology to developthe therapeutic strategies in cancer treatment. ------------------------------------------------------------------------ *Corresponding author . Recently, an imaging technology with the VECTor SPECT/PET/CT small-animal scanner (MILabs) has been developed to obtain simultaneous images usingtwodifferent tracers labeled with SPECT and PET nuclides, respectively. In this study, we developed amethod to simultaneously visualize vascularity and hypoxia witha human colon carcinoma HT-29tumor-bearing mouse model with 99mTc-labeled human serum albumin (99mTc-HSA) to detect blood pool, and 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM) to detect the over-reduced conditionsunder hypoxia, by applying this SPECT/PET/CT technology.Prior to the in vivo experiments, a phantom study was conducted to confirmquantitativity of the 99mTc/64Cu dual-isotope imaging with the SPECT/PET/CT system,by comparing radioactivities detected by SPECT/PET/CT system and those of standards under the conditions of wide range of radioactivities and various content ratios, in our settings. An in vivoimaging study was conducted with HT-29 tumor-bearing mice. Both 64Cu-ATSM (37 MBq) and 99mTc-HSA (18.5 MBq) were intravenously injected into a mouse (n = 4) at 1 h and 10 min, respectively, before scanning for 20 min; the 99mTc/64Cu dual-isotope SPECT/PET/CT images were then obtained.The phantom study demonstrated that this system has high quantitativity, even when 2 isotopes co-existed and the content ratio was changed over a wide range, indicating the feasibility for in vivo experiments. In vivoSPECT/PET/CT imaging with 64Cu-ATSM and 99mTc-HSA visualized the distribution of each probe and showed that 64Cu-ATSM high-uptake regions barely overlapped with 99mTc-HSA high-uptake regions within HT-29 tumors.We developed a method to simultaneously visualize vascularity and hypoxia within HT-29tumors using in vivodual-isotope SPECT/PET/CT imaging. This methodology would be useful for studies oncancer biology with mouse tumor models anddevelopment of the treatment strategies against cancer. Examination of vascularity and hypoxia within in vivotumors is important in understanding the biology of cancer anddevelopmentof the therapeutic strategies in cancer treatment. For hypervascular tumors, antiangiogenic therapy and antivascular therapy are promising approaches. For antiangiogenic therapy, the anti-vascular endothelial growth factor antibody bevacizumab is now clinically used worldwide [1-4], and for antivascular therapy, a clinical trial withcombrestatin A4 phosphate is conducted[5]. For hypovascular tumor, which is usually associated with hypoxia, intensive treatment is necessary, since tumor hypoxia is reportedly resistant to chemotherapy and radiotherapy [6-8]. In recent years, several therapeutic methods have been proposedto damage to hypoxic regions within tumors, such as intensity modulated radiation therapy with hypoxia positron emission tomography (PET) imaging [9, 10], and carbon-ion radiotherapy, which is able to damage tumor cells even in the absence of oxygen by high linear energy transfer beam [11, 12]. However, considering the difficulty of cancer radical cure at the present moment, more effective drugs and treatment methods for antiangiogenic, antivascular, and antihypoxia therapies need to be developed. In addition, combinations of these therapies would be effective approaches, since they can attacktumor vascularity and hypoxia closely linked each other.However, it is still difficult to observe tumor vascularity and hypoxia both coincidently and concisely in in vivo tumor-bearing mouse model. Recently, a technology of single-photon emission computed tomography/positron emission tomography/computed tomography(SPECT/PET/CT) imaging with the VECTor small-animal scanner, launched from MILabs (Utrecht, Netherlands), has been reportedto obtain truly simultaneous images with twotracers labeled with SPECT and PET nuclides, respectively. Conventionally, dual-isotope imaging studies with SPECT and PET have been performed by obtaining each image independently with 2 separate systems [13, 14]. In contrast, the VECTor system is equipped with a clustered pinhole collimator, which dramatically reduces pinhole-edge penetration of high-energy annihilation ?-photons from PET nuclides and enables it to detect high-energy ?-photons derived from PET nuclides, in a manner similar to SPECT nuclides, and to obtain high-resolution images from positron emitters and single-photon emitters at the same time by separating the images based on the photon energy [15, 16]. Thus, this system has a novel concept to make images of PET nuclides, compared to the typical PET system, which measures the coincidence of annihilation ?-photons. Goorden et al. have reported that this system shows high spatial resolution, with 0.8 mm for PET nuclides and 0.5 mm for SPECT nuclides [15]. Miwa et al. also confirmed its performance in simultaneous detection of 99mTc and 18F using this system [17]. In this study, we developed a methodology to easily observe intratumoralvascularity and hypoxia in a simultaneous manner,by applyingthis SPECT/PET/CT technology. We used 99mTc-labeled human serum albumin (99mTc-HSA) labeled with a SPECT nuclide 99mTc (half-life = 6.0 h; 140 keV ?-ray: 89%) to visualize tumor vascularity by detecting blood pool [18]. The 99mTc-HSAhas been reported to detect tumor blood pool in many types of cancer, including colon cancer, renal cell carcinoma, and liver tumor in both preclinical and clinical studies [19-21]. We also used 64Cu-diacetyl-bis (N4-methylthiosemicarbazone) (64Cu-ATSM), labeled with a PET nuclide 64Cu (half-life = 12.7 h; ?+-decay: 17.4%; ??-decay: 38.5%; and electron capture: 43%) [22], to detect tumor hypoxia. The Cu-ATSM, labeled with Cu radioisotopes, such as 60Cu, 62Cu, and 64Cu, has been developed as an imaging agent targeting hypoxic regions in tumors for use with PET [23-26].Many studies have demonstrated that Cu-ATSM accumulation is associated with hypoxic conditions of tumor in vitro and in vivo[26-29]. The mechanism of radiolabeled Cu-ATSM accumulation has been studied: Cu-ATSM has small molecular sizeand high membrane permeability, and thus rapidly diffuses into cells and is reduced and trapped within cells under highly reduced intracellular conditions such as hypoxia [24, 29-31]. A clinical study with 62Cu-ATSM demonstrated that high levels of hypoxia-inducible factor-1? (HIF-1?) expression were found in Cu-ATSM uptake regions in the tumors of patients with glioma [32]. In this study, we performed simultaneous in vivo imaging using a SPECT/PET/CT with 99mTc-HSA and 64Cu-ATSM for detecting tumor vascularity and hypoxia with a HT-29 tumor-bearing mouse model

血管内皮増殖因子を利用した腫瘍診断及び内部放射線治療の検討

金沢大学医薬保健研究域医学系これまでの研究において、臨床使用が十分可能な高い放射化学的純度、比放射能で血管内皮増殖因子(VEGF_及びVEGF_)の放射性ヨウ素標識を行い、担癌マウスを用いた動態評価を行ってきた。その結果、放射性ヨウ素標識VEGF_がVEGF_よりも腫瘍血管新生イメージング薬剤として有用であることが示唆された。本年度はオートラジオグラフィー法と免疫染色による組織学的な検討から、放射性ヨウ素標識VEGF_の腫瘍血管新生に対する特異性について評価を行った。^I-VEGF_は腫瘍サイズが200mm^3以上の腫瘍では、それ以下の腫瘍に比べ約半分の集積率を示した。オートラジオグラフィーによる検討からサイズの小さい腫瘍では^I-VEGF_は全体的に均一な分布を示していたが、200mm^3以上の腫瘍では腫瘍辺縁でのみに高集積を示した。抗CD34抗体を用いた免疫染色による検討の結果、^I-VEGF_が高集積を示した部位は血管新生が活発に行われていることが確認できた。以上、本研究課題についてまとめる。これまでに、^I-VEGF_による臨床研究が報告されたが、本研究成果から^I-VEGF_よりも^I-VEGF_のほうが血管新生を反映した良好な画像を得られることが期待できる。しかし、^I-VEGF_は血中への放射能が非常に高いため、イメージング薬剤や内部照射治療薬剤としてデメリットである。VEGFレセプターのひとつであるKDRはイメージングや治療ターゲットとしても非常に魅力的であることから、KDRをターゲットにした薬剤開発が今後の研究課題である。今回の研究成果は、血管新生をターゲットにした放射性医薬品開発を行う上で、その方向性を示すことができたことから、非常に有用であったと考える。研究課題/領域番号:15790657, 研究期間(年度):2003 – 2005出典：「血管内皮増殖因子を利用した腫瘍診断及び内部放射線治療の検討」研究成果報告書　課題番号15790657（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/grant/KAKENHI-PROJECT-15790657/)を加工して作

シグナル伝達を指標にした分子イメージングによる癌分子標的薬剤治療効果予測法の開発

金沢大学医薬保健研究域保健学系本研究では、キナゾリンを母体化合物とする種々のヨウ素又は臭素誘導体の合成を行った。これらの化合物はEGFR高発現を示すA431細胞に対し増殖阻害効果を示した。さらに、ヨウ素誘導体であるIPHYやIPYKはEGFR-TKに対して高い親和性と選択性を示し、また、体内分布実験においても腫瘍への高集積を確認した。これらのことから、IPHYとIPYKにEGFR-TKイメージング剤としての有用性が確認された。研究課題/領域番号:18390331, 研究期間(年度):2006 – 2008出典：「シグナル伝達を指標にした分子イメージングによる癌分子標的薬剤治療効果予測法の開発」研究成果報告書　課題番18390331（KAKEN：科学研究費助成事業データベース（国立情報学研究所））（https://kaken.nii.ac.jp/ja/report/KAKENHI-PROJECT-18390331/18390331seika/)を加工して作

Imaging and Therapy Against Hypoxic Tumors with 64Cu-ATSM

In tumors, hypoxia frequently occurs due to poor vascularization and tight packing of cancer cells. Tumor hypoxia is associated with adverse prognosis due to failures in radiotherapy and chemotherapy and to tumor metastasis (Brown 1999). It is thus important to develop methods for diagnosis and therapy of hypoxic tumors. Noninvasive methods to detect hypoxic tumor have been intensively developed (Padhani et al. 2007).Electronic Supplementary Materia

Evaluation of 64Cu-ATSM for an internal radiotherapy agent against CD133+ cancer stem cells with human colon carcinoma: 64Cu-ATSM treatment caused cell apoptosis.

Objectives : Fatty acid synthase (FASN) expression is elevated in several human cancers and this over-expression is associated with poor prognosis. Orlistat, an inhibitor of FASN, is reported to show antitumor effects against FASN-expressing tumors. As variations in FASN expression in individual tumors have been observed, methods to evaluate FASN expression and predict therapeutic effects of orlistat are needed. Here, we examined whether uptake of radiolabeled acetate can act as a surrogate marker for FASN expression and as a predictor of therapeutic effects of orlistat using human prostate carcinoma cells showing various degrees of FASN expression.Methods : FASN expression, radiolabeled acetate uptake and therapeutic effect of orlistat were examined in vitro with human prostate cancer cells, including LNCaP, PC3, 22Rv1 and DU145. In acetate uptake study, cells were incubated in [1-14C]acetate-containing medium and radioactivity was measured. Cell viability after orlistat treatment for 48 h was determined. Tumor-bearing mice were also treated with orlistat (250 mg/kg/day) for 2 weeks to examine therapeutic outcome of orlistat in vivo.Results : LNCaP cells, which express high levels of FASN, showed high acetate uptake, while PC3, 22Rv1 and DU145, which express lower levels of FASN, showed relatively low acetate uptake. There was a significant positive correlation between acetate uptake and FASN expression. % cell viability after orlistat treatment showed significant negative correlations to acetate uptake and FASN expression, respectively. LNCaP tumors (high FASN) showed high acetate uptake and high sensitivity to orlistat treatment, while PC3 and DU145 tumors (low FASN) showed low acetate uptake and less sensitivity, in vivo.Conclusions : Our findings showed that uptake of radiolabeled acetate would be a potential surrogate marker for FASN expression in tumor cells, and is a predictor of the therapeutic effects of orlistat. \nJune 9-13, 2012– SNM Annual Meeting (Miami, Florida, USA) 2250 characters (approx. 300 words) Figure [1-14C]acetate uptake (A), FASN expression (B, C) and therapeutic effect of orlistat (D) in human prostate cancer cells, LNCaP, PC3, 22Rv1 and DU145. There was a significant positive correlation between [1-14C]acetate uptake and FASN expression (E). Percent cell viability after orlistat treatment showed significant negative correlations to [1-14C]acetate uptake (F) and FASN expression (G). Therapeutic effect of orlistat in vivo (H).SNM 2012 Annual Meetin