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Interface peptides that mediate protein-protein interactions (PPI) are a class of important lead compounds for designing PPI inhibitors. However, Their potential as precursors for radiotracers has never been exploited. Here we reported that the interface peptides from programmed death-ligand 1 (PD-L1) can be used in positron emission tomography (PET) imaging of the programmed cell death 1 (PD-1) with high accuracy and sensitivity. Moreover, the performance differentiation between murine PD-L1 derived interface peptide (mPep-1) and human PD-L1 derived interface peptide (hPep-1) as PET tracers for PD-1 unveiled an unprecedented role of a non-critical residue in target binding, highlighting of the significance of PET imaging as a companion diagnostics in drug development. Collectively, this study not only provided a first-of-its-kind peptide-based PET tracer for PD-1 but also conveyed a unique paradigm for developing imaging agents for highly challenging protein targets, which could be used to identify other protein biomarkers involved in the PPI networks.

Mutations that increase leucine-rich repeat kinase 2 (LRRK2) activity in the brain are associated with Parkinson's disease. Here, we synthesized a novel compound 4-(6-fluoro-4-(5-isopropoxy-1H-indazol-3-yl)pyridin-2-yl)morpholine (FIPM) and labeled it with fluorine-18 (18F), to develop a positron emission tomography (PET) tracer for in vivo visualization of LRRK2 in the brain. FIPM showed high in vitro binding affinity for LRRK2 (IC50 = 8.0 nM). [18F]FIPM was prepared in 5% radiochemical yield (n = 5), by inserting 18F into a pyridine ring, followed by removal of the protecting group. After HPLC separation and formulation, [18F]FIPM was acquired with >97% radiochemical purity and 103–300 GBq μmol−1 of molar activity at the end of radiosynthesis. Biodistribution and small-animal PET studies in mice indicated a low in vivo specific binding of [18F]FIPM. While [18F]FIPM presented limited potential as an in vivo PET tracer for LRRK2, we suggested that it can be used as a lead compound for developing new radiotracers with improved in vivo brain properties.

Vascular endothelial growth factor receptors (VEGFRs) are well recognized as significant biomarkers of tumor angiogenesis. Herein, we have developed a first-of-its-kind peptide-based VEGFR positron emission tomography (PET) tracer. The novel [64Cu]VEGF125–136 peptide possessed satisfactory radio-characteristics and showed good specificity for the visualization of VEGFR in various mouse models, in which the tumor-specific radioactivity uptake was highly correlated to the VEGFR expression level. Moreover, the tracer showed high tumor uptake (ca. 5.89 %ID/g at 20 min postinjection in B16F10 mice) and excellent pharmacokinetics, achieving the maximum imaging quality within 1 h after injection. These features convey [64Cu]VEGF125–136 as a promising, clinically translatable PET tracer for the imaging of tumor angiogenesis.

We studied the simple separation of radiosilvers from proton- or deuteron-irradiated natural palladium (natPd) targets, and successfully separated radiorhodium, radiosilver and radiopalladium using an anion-exchange resin. The yields of radiosilvers were 105Ag: 1.0 ± 0.32 MBq/μAh; 106mAg: 2.0 ± 0.64 MBq/μAh; 110mAg: 0.019 ± 0.0063 MBq/μAh (n = 4) at the end of bombardment with a total recovery rate of 98 % under the following irradiation conditions (deuteron beam energy: 20 MeV; beam current: 10 μA; irradiation time: 2.25 ± 0.50 h). We also evaluated the recycling of the palladium target.

Targeted radionuclide therapy (TRT) targeting oncoproteins facilitates the delivery of therapeutic radionuclides to tumor tissues with high precision. Herein, we developed two new radiopharmaceuticals, 4-131I-iodo- and 4-211At-astato-N-[4-(6-(isopropylamino)pyridine-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide (131I-IITM and 211At-AITM), targeting the ectopic metabotropic glutamate receptor 1 (mGluR1) in melanomas for TRT studies. Methods: 131I-IITM and 211At-AITM were synthesized by reacting a stannyl precursor with 131I-NaI and 211At in the presence of an oxidizing agent. The therapeutic efficacy and safety of the two radiopharmaceuticals were investigated using mGluR1-expressing B16F10 melanoma cells and melanoma-bearing mice. Results: 131I-IITM and 211At-AITM were obtained with radiochemical purity of >99% and radiochemical yields of 42.7% ± 10.4% and 28.9% ± 9.9%, respectively, based on the total radioactivity of used radionuclides. 131I-IITM and 211At-AITM exhibited maximum uptakes of 4.66% ± 0.70% ID/g and 7.68% ± 0.71% ID/g in the targeted melanomas, respectively, and were rapidly cleared from non-target organs after intravenous injection. Both agents markedly inhibited melanoma growth compared with the controls (61.00% and 95.68%, respectively). In the melanoma model, considerably greater therapeutic efficacy with negligible toxicity was observed using 211At-AITM. Conclusion: The non-toxic radiopharmaceuticals 131I-IITM and 211At-AITM are useful high-precision TRT agents that can be used to target the oncoprotein mGluR1 for melanoma therapy.

Herein, a PD-L1 targeted native peptide was developed for PET imaging. 18F and 64Cu were utilized to label the peptide. To improve the pharmacokinetics and biodistribution of the tracers, the peptide was further pegylated to form star-like tetramers. Consequently, four tracers were synthesized with acceptable radiochemical characteristics and their in vivo pharmacokinetics and PD-L1 imaging capability were systematically evaluated. This proof-of-principle study may provide new possibilities for PD-L1 PET imaging in cancers.

Additional file 1: Figure S1. The system diagram of automated multi-purpose synthesizer developed in-house (Fukumura et al. 2007). Figure S2. The preparative HPLC chromatogram of [18F]FMISO synthesized by 18F-fluorination using 1 and [18F]F-, followed by the removal of the protecting group in [18F]3. The HPLC conditions were as follows: XBridge C18 column (5 μm, 10 mm i.d. × 250 mm length; Waters), with a mixture of ethanol and water (2:98, vol./vol.) as the mobile phase, a flow rate of 5.0 mL/min, and UV detection at 325 nm. Figure S3. The preparative HPLC chromatograms of [18F]PM-PBB3 synthesized by 18F-fluorination using 2 and [18F]F-, followed by the removal of the protecting group in [18F]4. The HPLC conditions were as follows: Capcell Pak C18 column (5 μm, 10 mm i.d. × 250 mm length; Shiseido, Tokyo, Japan), the mixture of acetonitrile, water and triethylamine (40:60:0.1, v/v/v) as the mobile phase, 5.0 mL/min flow rate, and UV detection at 365 nm.

Interface peptides that mediate protein-protein interactions (PPI) are a class of important lead compounds for designing PPI inhibitors. However, their potential as precursors for radiotracers has never been exploited. Here we reported that the interface peptides from PD-L1 can be used in PET imaging of the PD-1 with high accuracy and sensitivity. Moreover, the performance differentiation between murine PD-L1 derived interface peptide (mPep-1) and human PD-L1 derived interface peptide (hPep-1) as PET tracers for PD-1 unveiled an unprecedented role of a non-critical residue in target binding, highlighting the significance of PET imaging as a companion diagnostics in drug development. Collectively, this study not only provided a first-of-its-kind peptide-based PET tracer for PD-1 but also conveyed a unique paradigm for develop ing imaging agents for highly challenging protein targets., 第60回日本核医学会学術総会

我々は多くのがん種に異常発現している癌蛋白質“代謝型グルタミン酸受容体1型（mGluR1）”に着目し、α線の標的アイソトープ治療（TRT)に資する小分子薬剤211At-AITMの開発に成功した。今回は、211At-AITM TRTの実用化を目指し、ヒトメラノーマ、乳がん、大腸がんを対象とするがん種横断的な治療検証を行った。【方法】ヒト由来のメラノーマ細胞A375、A2058、乳がん細胞MDA-MB231、MCF-7、大腸がん細胞DLD1に対して、mGluR1発現量をRT-PCRで確認した上、担がんモデルをそれぞれ樹立し、211At-AITMを静脈投与して、治療効果及び安全性等の評価を行った。【結果】生理食塩水投与のコントロール群に比べ、211At-AITMはmGluR1陰性のA2058、MCF-7担癌モデルに抗腫瘍効果を示さなかったが、mGluR1陽性のA375、MDA-MB231及びDLD1担癌モデルには、顕著な抗腫瘍効果を示した(p＜0.05)。一方、有害事象である体重減少、血液毒性等は認めなかった。【結論】211At-AITM TRTは、癌蛋白質mGluR1を有する複数のがん種に対し安全かつ有効な治療法になりえることが示唆された。, 第60回日本核医学会学術総会

Integrins play a pivotal role in many physiological processes such as neo-angiogenesis, cell adhesion and proliferation, and thus are key proteins in pathological disorders such as cancer metastasis, thrombosis, osteoporosis, and autoimmune diseases. For molecular imaging agents based on small peptidic integrin ligands, such as the αvβ3-integrin ligand c(RGDfK), has been found to greatly improve target affinities and in vivo performance. Kessler et al. described the systematic screening of c(-phg-isoDGR-X-) lead motif aiming to confer to the small lead pentapeptide selectivity toward the Fn-binding integrins α5β1 and αvβ6 (1). The cyclic peptide c(-phg-isoDGR-k-) and c(-phg-isoDGR-K-) displayed an antagonistic activity for α5β1, thus representing the first report of a small-sized, highly active, cyclic peptide targeting α5β1 integrins (2). Copper-64 labeled ligands are useful in PET studies because of the clinically suitable nuclear properties of copper-64 and its availability at high molar activity. This property allows sufficient time for clearance of non-specific radioactivity from background tissues to increase image contrast, which requires long times to circulate throughout the whole body. Moreover, copper-64 labeled ligands are demonstrably effective for radioimmunotherapy. Here, we report radiolabeling and evaluation of copper-64 labeled c(-phg-isoDGR-X(DOTA)-) (X:k or K). Copper-64 for radiolabeling were produced at the National Institute of Radiological Science (3). [64Cu]-c(-phg-isoDGR-k(DOTA)-) (1a) and [64Cu]-c(-phg-isoDGR-K(DOTA)-) (1b) was synthesized by DOTA-containing cyclic peptides with [64Cu]CuCl2 in citric buffer solution (pH 5.5) at r.t. for 30 min. High radiolabeling efficiency of copper-64 for DOTA-containing cyclic peptide yielded 1a and 1b with >99% radiochemical purity. In vivo regional distributions were determined in B16B10-bearing mice. PET sacns for 1a and 1b enabled visualization of α5β1 integlins on B16B10 cells in the mice. (1) Frank, A. O. et al. Angew. Chem., Int. Ed. 2010, 49, 9278−9281. (2) Bochen, A. et al. J. Med. Chem. 2013, 56, 1509−1519. (3) Ohya, T. et al. Nucl. Med. Biol. 2016, 43(11), 685-691., 第57回ペプチド討論会

Objectives: Noninvasive positron emission tomography (PET) imaging of vascular endothelial growth factor receptor 2 (VEGFR-2) expression could be a valuable tool for evaluation of patients with a variety of malignancies, and particularly for monitoring those undergoing antiangiogenic therapies that block VEGF/VEGFR-2 function. VEGF125-136 (QKRKRKKSRYKS) is a 12 amino acid peptide encoded by exon 6 of VEGF-A which was first identified as an effective inhibitor to VEGFR-2. The aim of this work was to develop and evaluate in mice the 64Cu labelled analogue as an in vivo tracer for VEGFR-2 expression in solid tumours. Methods: VEGF125-136 was conjugated with PEG3 and DOTA and then labeled with 64Cu (denoted as [64Cu]VEGF125-136) for small-animal PET of mice bearing B16F10 human melanoma cells, U87MG human glioblastoma cells, and MDA-231 human breast cancer xenografts. Biodistribution studies, autoradiography and immunofluorescence staining were carried out to confirm the noninvasive imaging results. Results: The radiolabeling yield and specific activity of [64Cu]VEGF125-136 were more than 95% and 74.3 ± 3.8 GBq/µmol, respectively. Noninvasive microPET showed that [64Cu]VEGF125-136 had variable uptake in different tumors, with the order: B16F10 > U87MG > MDA-231. The pattern of radiotracer uptake was correlated well with variations in VEGFR-2 expression determined ex vivo by immunohistochemical analysis. Moreover, the tracer showed high tumor uptake (5.89 ± 2.58 %ID/g at 20 min postinjection in B16F10 mice) and excellent pharmacokinetics, achieving the maximum imaging quality within 1 h after injection. Biodistribution studies and autoradiography confirmed the VEGFR-2 specificity of [64Cu]VEGF125-136. Conclusions: We have developed a VEGFR-2-specific PET tracer, [64Cu]VEGF125-136. This tracer may be translated into the clinic for imaging tumor angiogenesis and monitoring antiangiogenic treatment efficacy., SNMMI 2020 Annual Meeting

To introduce the 3‐[18F]fluoro‐2‐hydroxypropyl moiety into positron emission tomography (PET) radiotracers, we performed automated synthesis of (rac)‐, (R)‐, and (S)‐[18F]epifluorohydrin ([18F]1) by nucleophilic displacement of (rac)‐, (R)‐, or (S)‐glycidyl tosylate with 18F− and purification by distillation. The ring‐opening reaction of (R)‐ or (S)‐[18F]1 with phenol precursors gave enantioenriched [18F]fluoroalkylated products without racemisation. We then synthesised (rac)‐, (R)‐, and (S)‐ 2‐{5‐[4‐(3‐[18F]fluoro‐2‐hydroxypropoxy)phenyl]‐2‐oxobenzo[d]oxazol‐3(2H)‐yl}‐N‐methyl‐N‐phenylacetamide ([18F]6) as novel radiotracers for the PET imaging of translocator protein (18 kDa) and showed that (R)‐ and (S)‐[18F]6 had different radioactivity uptake in mouse bone and liver. Thus, (rac)‐, (R)‐, and (S)‐[18F]1 are effective radiolabelling reagents and can be used to develop PET radiotracers by examining the effects of chirality on their in vitro binding affinities and in vivo behaviour.

Vasopressin 1B receptors (V1BRs) are abundantly expressed in the pituitary, and in vivo positron emission tomography (PET) of V1BRs was recently enabled by our development of a specific radioligand, 11C-TASP0434299, derivatized from pyridopyrimidin-4-one. Here, we identified a novel pyridopyrimidin-4-one analog, N-tert-butyl-2-[2-(6-11C-methoxypyridine-2-yl)-6-[3-(morpholin-4-yl)propoxy]-4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl]acetamide (11C-TASP0410699, hereafter referred to as 11C-TASP699), as a potent V1BR radioligand producing a higher image contrast for the target than 11C-TASP0434299. Methods: In vitro properties of TASP699 were assessed by assaying its affinity for human V1BR and its selectivity for off-target molecules. Radioactive uptakes in the pituitary were analyzed using PET in rhesus monkeys after intravenous administration of 11C-TASP699. Serial doses of a selective V1BR antagonist, 2-[2-(3-chloro-4-fluorophenyl)-6-[3-(morpholin-4-yl)propoxy]-4-oxopyrido[2,3-d]pyrimidin-3(4H)-yl]-N-isopropylacetamide hydrochloride (TASP0390325), were administered prior to the radioligand injection. Autoradiographic labeling of monkey pituitary slices with 11C-TASP699 was conducted with or without nonradioactive V1BR antagonists. Results: The half maximal inhibitory concentration (IC50) of TASP699 for human V1BRs (0.165 nM) was lower than that of TASP0434299 (0.526 nM), whereas its IC50 values for off-target molecules exceeded 1 μM. PET imaging in monkeys demonstrated that the peak pituitary uptake of 11C-TASP699 was almost equivalent to that of 11C-TASP0434299 and that pretreatment with TASP0390325 inhibited the retention of 11C-TASP699 in a dose-dependent manner, inducing nearly full occupancy at 0.3 mg/kg. Specific radioligand binding was determined as a specific-to-nondisplaceable uptake ratio at equilibrium using radioactivity retentions at 60 min in baseline and blocking studies. This ratio for 11C-TASP699 was approximately 2.5-fold greater than that of 11C-TASP0434299. A reverse-phase high performance liquid chromatography (HPLC) study identified the parent and polar radiometabolites. Affinities of two predicted metabolite candidates for V1BRs were more than ten times weaker than that of the parent. Intense autoradiographic labeling of the anterior pituitary with 11C-TASP699 was inhibited with TASP0390325 in a concentration-dependent manner. Conclusion:11C-TASP699 yielded PET images of pituitary V1BRs with a higher contrast than 11C-TASP0434299, supporting the applicability of 11C-TASP699 in the assessment of neuropsychiatric diseases and dose findings for test drugs in clinical trials.

これまでに、転移しやすいメラノーマに高発現する癌蛋白質 “代謝型グルタミン酸1型受容体（mGluR1）”に着目し、α線標的アイソトープ治療（TRT)に資する小分子放射性薬剤である211At-AITMの開発に成功した。今回、我々は211At-AITMの実用化を目指し、転移性メラノーマに対する治療検証を行った。癌蛋白質mGluR1が高発現した転移性メラノーマ担癌マウスモデルに211At-AITMあるいは生理食塩水をそれぞれ静脈投与して、治療効果及び安全性等の評価を行った。生理食塩水を投与したコントロール群（6匹）では、移植後約27.5  2.47 日にがんが進行して死亡したのに対し、11At-AITM (2.96 MBq)の単回投与による治療群（12匹）では、有意に全生存期間（61.67  3.17日）が延長した。一方、有害事象である体重減少、血液毒性及び肝、腎機能障害などは認めなかった。α線薬剤211At-AITMは、転移性メラノーマに対し顕著な治療効果を示し、安全かつ高効率なTRT薬になりえることが示唆された。, 第59回日本核医学会学術総会

目的】金属錯体を用いた標識反応の開発で製造困難であったPET薬剤、特に銅錯体を用いた製造方法が数多く報告されている。一方、薬剤に混入する金属不純物の定量はICP-MSを用いた検出方法が用いられる。しかし日常でICP-MSを用いる金属不純物検定は事前準備に大きな負担がかかるため非常に困難である。そこで検定方法としてHPLCポストカラム法を用いた金属不純物測定の検討を行った。【方法】カラムとしてIonPac CS5A analytic column (4.6 × 250mm)、溶離液としてMetPac PDCA溶液、発色液としてMetPac PAR試薬希釈溶液を使用した。PET薬剤として銅錯体を用いて製造した[18F]F-DOPAを用いた。銅溶液は【結果】上述の条件でHPLC測定した結果、銅の保持時間は5.8分であった。検量線は銅の濃度が10 ppbから 200 ppbの間でよい直線性を示した(R2>0.98)。 [18F]F-DOPA注射剤中の銅濃度は30 ppb±10 ppbであり、ICH-Q3Dガイドラインで示された銅混入量下限値濃度よりも大幅に低いことを確認した。, 第59回日本核医学会学術総会

【背景・目的】67Cuはその崩壊形式(β- 100%, 61.9 h)から、内照射療法に適した核種として期待されている。しかしながら、67Cuの製造は多量(数g～)の68Znをターゲットとするため、分離にはイオン交換樹脂とキレート樹脂を組み合わせて使用せねばならず、その結果、多くの手間と時間（1日）を必要とする。今回、我々は共沈を利用した簡便な分離法（共沈法）を開発した。【方法】natZnO(3.5 g)を60MeVのプロトンビームで7時間照射した。ターゲットを塩酸で溶解後、H2SとAgNO3を共沈剤として加え、溶液をフィルターろ過した。その後、酸溶液でフィルターから67Cuを回収した。同様の操作を2回行った後、グラスフィルターでAgを除去した。また、従来のイオン交換樹脂を用いた分離法も行い、両者の品質を比較した。【結果】共沈法の製品は、従来の分離法の製品と同等の品質（不純物金属濃度、放射化学純度）を示した。分離時間は3時間以下であった。【結論】共沈を用いた簡便な分離精製法の開発に成功した。, 第59回日本核医学会学術総会

【目的】[64Cu]Cu-ATSMは脳腫瘍の治療に効果が期待されている日本発の治療用放射性薬剤である。今回、将来の承認申請を見据えた本薬剤の規格値の設定および品質検査法の確立を行い、治験薬の規格に採用したので実績を報告する。【方法】治療用[64Cu]Cu-ATSM注射液の品質検査項目として、製造ごとに液量、放射能、性状、pH、放射性異核種、放射化学的異物、[64Cu]Cu-ATSM特異性、エンドトキシン、無菌試験、1年に1回以上、アセトンとジメチルスルホキシドの濃度測定を行うこととした。【結果】Radio-TLCによる放射化学的異物の測定においては、放射能検出器の定量限界と定量上限から、10%の放射化学的異物を定量可能な検体の放射能濃度を300～500 kBq/μLに設定し、希釈法による検査法を確立し、分析法バリデーションを行った上で採用した。また、他の検査も日本薬局方に準拠した方法で行うことで将来の承認申請に対応できる治療用[64Cu]Cu-ATSM注射液の品質検査法及び規格値を確立できた。さらに、本規格試験を3ロット試験製造および治験薬に適用し、すべての製造ロットについて適合した。, 第59回日本核医学会学術総会

Copper-67 (67Cu), a feasible radionuclide for diagnosis and radiotherapy, is commercially generated from a bulk zinc (Zn) target using the 68Zn(p, 2p)67Cu and 68Zn(γ, p)67Cu nuclear reactions. Be-cause it uses a large amount of zinc, the separation is complex—requiring a combination of three ion exchange columns—and is time-consuming (about 1 day). We developed a quick and easy separation method referred to as “double coprecipitation” using H2S gas and silver nitrate as co-precipitation agents in place of ion exchange columns. We compared this method with a conventional separation method using three ion exchange columns (AG50W-X8, AG1-X8, and Chelex-100) for a natural zinc (natZn) target irradiated by a proton beam. The product quality and the recovery rate with the new method were competitive with the conventional method, and the total operation time was reduced from 1 day to < 3 h.

Background and Objectives: Designing small-molecule radiopharmaceuticals targeting an oncoprotein, such as the ectopic metabotropic glutamate receptor 1 (mGluR1) in melanoma [1], is an attractive strategy to increases our ability to deliver therapeutics to tumor tissues with high precision in targeted radionuclide therapy (TRT). Herein, we focused on 4-fluoro-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-methylbenzamide (FITM), a small-molecule compound with high affinity and specificity for the oncoprotein mGluR1[2,3], as the target carrier, with an -emitter (211At) to design and synthesize a novel TRT radiopharmaceutical: 4-211At-astato-N-[4-(6-(isopropylamino)pyridine-4-yl)-1,3-thiazol-2-yl]-N-methylbenzamide (211At-AITM), and investigated its therapeutic efficacy and safety in mGluR1-expressing B16F10 melanomas. Methods: Radiosynthesis of 211At-AITM was carried out by reacting the stannyl precursor with 211At (Fig. 1A). The binding specificity of 211At-AITM to mGluR1-expressing melanoma cells was assessed with and without the parent compound containing no radionuclide, FITM. Therapeutic effect and safety assessment were performed with a single injection of 211At-AITM with conservative doses (0–2.96 MBq) to B16F10-bearing C57BL/6J mice. Results: 211At-AITM were stably obtained with radiochemical purity >99% and radiochemical yields of 28.9 ± 9.9% (n = 20), based on the radioactivity of the cyclotron-produced 211At. The radiopharmaceuticals 211At-AITM bound to B16F10 cells with 12.87 %ICD/mg, and were specifically displaced by the non-radiolabeled FITM. TRT with 211At-AITM markedly inhibited melanoma growth compared to the 0 MBq (saline) treated controls. Dose-dependent tumor inhibition was observed in melanoma mice treated with 0.11, 1.11, 1.85 or 2.96 MBq 211At-AITM. A single 0.11 MBq dose of 211At-AITM resulted in approximately 32.24% tumor reduction, but this was not statistically significant; other reductions based on the concentration administered include 73.48% at 1.11 MBq; 87.38% at 1.85 MBq; 95.68% at 2.96 MBq compared to the 0 MBq group at 19 days post-administration (Fig. 1B). Throughout the examination period, no weight loss, hepatotoxicity or nephrotoxicity were observed in melanoma mice injected with 0–2.96 MBq 211At-AITM (Fig. 1C). Conclusions: Our results demonstrated the 211At labeled small-molecule radiopharmaceutical 211At-AITM possess high therapeutic efficiency and minimal health risks, thus could be attractive for clinical development as high-precision TRT weapons directed at the oncoprotein mGluR1 for melanoma therapy. References: 1.Pollock PM, Cohen-Solal K, Sood R, et al. Melanoma mouse model implicates metabotropic glutamate signaling in melanocytic neoplasia. Nat Genet. 2003; 34:108-112. 2.Xie L, Yui J, Fujinaga M, et al. Molecular imaging of ectopic metabotropic glutamate 1 receptor in melanoma with a positron emission tomography radioprobe 18 F-FITM. Int J Cancer. 2014;135:1852-9. 3.Fujinaga M1, Xie L, Yamasaki T, et al. Synthesis and evaluation of 4-halogeno-N-[4-[6-(isopropylamino)pyrimidin-4-yl]-1,3-thiazol-2-yl]-N-[11C]methylbenzamide for imaging of metabotropic glutamate 1 receptor in melanoma. J Med Chem. 2015;58:1513-23., WMIC2019でのポスター発表

Integrins play a pivotal role in many physiological processes such as neo-angiogenesis, cell adhesion and proliferation, and thus are key proteins in pathological disorders such as cancer metastasis, thrombosis, osteoporosis, and autoimmune diseases. For molecular imaging agents based on small peptidic integrin ligands, such as the αvβ3-integrin ligand c(RGDfK), has been found to greatly improve target affinities and in vivo performance. Kessler et al. described the systematic screening of c(-phgisoDGR-X-) lead motif aiming to confer to the small lead pentapeptide selectivity toward the Fn-binding integrins α5β1 and αvβ6 (1). The cyclic peptide c(-phg-isoDGR-k-) displayed an antagonistic activity for α5β1 (IC50: 8.7 nM), thus representing the first report of a small-sized, highly active, cyclic peptide selectively targeting α5β1 integrins (2). Cu-64 labeled ligands are useful in PET studies because of the clinically suitable nuclear properties of Cu-64 and its availability at high molar activity. This property allows sufficient time for clearance of non-specific radioactivity from background tissues to increase image contrast, which requires long times to circulate throughout the whole body. Moreover, Cu-64 labeled ligands are demonstrably effective for radioimmunotherapy. Meanwhile, Cu-67 is one of the most promising radioactive metals for radiotherapy. Here, we report radiolabeling and evaluation of c(-phg-isoDGR-k(DOTA)-) using radioactive cooper. (1) Frank, A. O. et al. Angew. Chem., Int. Ed. 2010, 49, 9278−9281. (2) Bochen, A. et al. J. Med. Chem. 2013, 56, 1509−1519., 14th International Comference on Radiopharmaceutical Therapy

Objectives In the last few years new emerging metal-mediated radiolabeling methods have facilitated access to probes which had been so far inaccessible or difficult to produce using conventional labeling procedures. Particularly, procedures for copper-mediated 18F-fluorination discovered by Gouverneur et al. [1] and further developed by inventors and others [2]. On the other hands, a guideline called ICH-Q3D regulates the permission limit of elemental impurities in conventional drugs. Hence, we should consider and prepare to reflect such regulation into daily radio-pharmaceutical productions. The most common detection technique for metal (elements) impurity is inductively coupled plasma mass spectrometry (ICP-MS) detection. Although ICP-MS is a suitable instrument to analyze trace amount of metallic impurities with high sensitivity, it is cumbersome for routine assessment. We report here the metal impurity in PET probe measured by using the HPLC post-column method. Methods Production of [18F]DOPA by copper-mediated method was performed using homemade automation single reactor module. The residual amount of copper in the final solution were analyzed using a metal-free JASCO Extrema HPLC system (Tokyo, Japan) including two pumps, auto sampler, column-oven, and a UV–VIS detector. One pump was used to deliver MetPac PDCA eluent through the analytical column, and the other one was used to deliver PAR diluent. The sample solution was separated through an IonPac CS5A analytic column (4.6 × 250mm), and the eluent mixed with the dye PAR was analyzed by the UV–VIS detector at 520 nm of copper. Flow rates for MetPac PDCA eluent and PAR diluent solution were 1.2 mL/min and 0.6 mL/min, respectively. Calibration curves for copper ranging from 10 to 2000 μg/L were made from the TraceCERT® ICP-MS standard at an injection volume of 50 μL. Results The concentration of copper was measured with a good linearity in the range of 10 to 2000 μg/mL (R2>0.98), repeatability on three injections at the same day in less than 5% relative standard deviation (% RSD). We enabled detecting the residual amount of copper in [18F]DOPA using the HPLC post-column system, and calculate the residual amount of copper in [18F]DOPA (0.03 ppm±0.01ppm). This is significantly below any level of concern according to the ICH Guideline of Elemental Impurities (Q3D). We will report the residual amount of copper in [11C]product using copper-mediated cyanation. Conclusions The post-column evaluation provided considerable sensitivity for metal impurity within a short time (10 min), suggesting that this method is suitable for daily QC because of the simplicity and prompt measurement. Therefore, we used the data obtained by the post-column method only for estimating metal impurity of PET probe in a daily QC protocol. Acknowledgements This work was supported by JSPS KAKENHI Grant Number 18K07281. Reference [1] Tredwell, M., et al., Angew. Chem. 2014, 126, 1-6. [2] Zarrad, F., et al., Molecules 2017, 22, 2231; doi:10.3390/molecules22122231., ISRS2019

Objectives: Immune checkpoint blockade has emerged as a promising cancer treatment paradigm. Unfortunately, there are still a large number of patients and malignancies that do not respond to this therapy. A major barrier to validating biomarkers for the prediction and monitoring of responders to clinical checkpoint blockade has been the lack of imaging tools to accurately assess dynamic immune checkpoint expression. The programmed cell death ligand 1 (PD-L1) is expressed in many cancers, and is an important contributor to the maintenance of immunosuppressive tumor microenvironment. PD-L1 is a prominent target for cancer immunotherapy [1, 2]. An Octapeptide (PWZ-1), was recently screened with high binding affinity for PD-L1 (KD ≈ 10 nM). Here we examine the feasibility of developing the octapeptide as a radiotracer for noninvasive detection of PD-L1 expression in tumors by PET. Methods: PWZ-1 peptide was conjugated with DOTA as a chelator for radioisotope labelling. The binding affinity of PWZ-1-NOTA was evaluated by surface plasmon resonance. PWZ-1 was subjected to radiolabeling with copper-64 and the radiolabeling conditions were optimized. The resulting [64Cu]PWZ-1 was assessed for stability in saline and in mouse serum. The in vitro specificity of the radiolabeled peptides for two PD-L1(+) cell lines MDA-MB-231 and B16F10 was tested. Next, we performed small animal PET imaging in tumor-bearing mice with the radiolabeled peptide which was injected via tail vein. The ex vivo biodistribution of [64Cu]PWZ-1 in mice was also conducted. Results: Surface plasmon resonance showed the DOTA-conjugated PWZ-1 with a similar binding affinity with PWZ-1. Synthesis of [64Cu]PWZ-1 provided radiochemical purity >99% after purification and formulation. Biodistribution in mice demonstrated 5-10 percentage of injected dose per gram (%ID/g) in MDAMB231and B16F10 tumors, respectively, at 1 h postinjection, with high binding specificity noted with coinjection of excess unlabelled PWZ-1. PET imaging showed high uptake contrast in all tumor models tested. Conclusion: We demonstrated the specificity of [64Cu]PWZ-1 to detect PD-L1 expression in multiple xenograft models with variable PD-L1 expressions and described its biodistribution and pharmacokinetics. In addition, the radiolabeling and purification conditions tested for preparation of [64Cu]PWZ-1 could be easily modified to facilitate a kit preparation of the radiotracer for PD-L1 imaging. Such 64Cu-labeled PD-L1 imaging agents with short biological half-life and fit within the routine clinical work flow enables therapy monitoring and stratification of patients in the field of immuno-oncology. References: [1] L. M. Roy, et al. Proc. Natl. Sci. USA. 2015, 112, E6506-14. [2] C. Truillet, et al. Bioconjugate. Chem. 2018, 29, 96-103., ISRS2019

Objectives Silver has many attractive radionuclides such as Ag-111 (T1/2 7.45 d / β-) promising candidate for radiotherapy as beta emitter, Ag-105 (T1/2 41.3 d /EC) and Ag-106m (T1/2 8.28 d /EC) for analytical chemistry, Ag-110m (T1/2 250 d /IT, β-) for industrial application, i.e. labeling for silver nanoparticles. Several production routes of these radionuclides from palladium targets using accelerator have been proposed, while few reports of separation have been reported. In this study, we evaluated a separation method for silver nuclides from bulk palladium target with cyclotron (proton or deuteron beam), and recycle method of the target. Methods A 100 mg of natural palladium powder (palladium black) was used as a target of this production study. Irradiation of vertical beam was conducted by NIRS AVF-930 cyclotron with deuteron particles of 20 MeV at 3 – 10 µA for 1 – 3.5 h for production of silver radionuclides or proton of 60 MeV at 3 – 5 µA for 1 – 2 h for research of separation method. Irradiated target was dissolved by conc-HCl with bubbling of chloride gas for 7 – 9 min. After the evaporation of the solution, it was redissolved in 8ml of 1N-HCl and loaded on anion exchange resin (Dowex 1×8, 100-200 mesh, Cl- form, 4g). A byproduct radioactive rhodium was removed by gradual 1 – 2 N HCl. Then, 5N-HCl was loaded to elute the sliver radionuclides, of which the fraction was evaporated to obtain as final product. Conc. HCl was finally loaded to recover the palladium. In the recovery process, the palladium fraction was evaporated and redissolved in 10 ml of 0.1N-HCl. Then, 20 wt% of NaBH4 in ultrapure water (UPW) was added for reducing palladium from chloride form. The reduced palladium was washed four times with UPW by decantation and centrifugation, and was completely dried (recovered palladium). We used HPGe detector (GX1020 Canberra, Meriden, CT) to identify the interested nuclides such as silver, rhodium and palladium on every process. Results The rhodium and palladium radionuclides (Rh-99, 100, 101m, 102m, Pd-100, 101, 103) were found in the irradiated target as radio-byproduct. We successfully separate the sliver nuclides (Ag-105, 106m, 110m) from the target (Fig. 1), where the recovery of silver was > 95 %. The radio elemental purity of any silver nuclides in the sliver fraction was > 99 %, in which both palladium and rhodium radionuclides were not detected. No residue was found in the evaporation of silver fraction (product). The recovery rate of the palladium was about 74 %. Conclusions The separation method presented here has a potential to prepare silver nuclides from a bulk palladium target. An evaluation of the chemical purity and the reducing rate of the palladium are underway. Acknowledgements We would like to thank our cyclotron staff for their operation of NIRS cyclotron and good support. This work was supported by JSPS KAKENHI Grant Number JP17K10386, ISRS2019

Objectives: Photothermal therapy (PTT) employs the heat generated from the absorbed optical energy by light-absorbing agents accumulated in the tumor to ablate tumor cells. Photodynamic therapy (PDT) is a clinically approved, minimally invasive technique that can exert a procedure involves administration of a photosensitizing agent followed by irradiation at a wavelength corresponding to an absorbance band of the sensitizer. In the presence of oxygen, a series of events lead to direct tumor cell death, damage to the microvasculature and induction of a local inflammatory reaction. Black phosphorus quantum dots (BPQDs), a type of newly developed nanomaterial material, have attracted tremendous research interest owing to their distinctive physical/chemical properties [1]. BP has an excellent photothermal effect under near-infrared (NIR) irradiation, which gives a high photothermal conversion efficiency as high as 28%. BPs possess excellent biocompatibility and biodegradability properties under physiological conditions [2]. Here, we aim to develop a theranostic platform based on the radioisotope-loading BPQDs. In order to maximize the tumor targeting effect, two peptides were conjugated to the surface of QDs, which can sequentially recognize the tumor infiltrated vessels and tumor cell membrane receptor, thereby achieve maximum accumulation in the tumor site. Methods: Chelator-free labeling of BP with copper-64 and nanocomplex forming: 64Cu produced in house was diluted with HEPES buffer and mixed with BPQDs. The reaction was conducted at room temperature for 10 min with constant shaking. The resulting BPQD@64Cu was easily collected by centrifugation. This nanocomplex was resuspended in buffer and the beta hairpin peptide was added. After incubation, peptide-modified BP@64Cu nanocomplex as a final product was obtained by filtration. Surface peptide ligands conjugation: Two peptide ligands, VEGF 125-136 binds to vascular endothelial growth factor (VEGF) and cyclo-RGDyC binds to integrin av3 respectively, were conjugated to poly(oxyethylene) via a 3+2 yne-azide click reaction and a maleimide-thiol reaction, respectively. The peptides-modified PEG was complexed with BPQD@64Cu for 2 hours with constant stirring. The final BPQD@64Cu@PEG-VEGF/RGD was formulated by centrifugation. In vitro cellular uptake and in vivo PET imaging: The cellular uptake mechanism was investigated by fluorescent imaging and radioactivity experiment. The blocking assay was also performed. The release kinetics of 64Cu and peptide from the nanocomplex was studied at different pH values. The in vitro cell viability, the PPT efficiency, and PDT effect were carefully evaluated. PET imaging was performed in B6F10 bearing Balb/c nude mice. Ex vivo biodistribution in the major organs of tumor mice was also performed. Finally, the anti-cancer therapy by PTT and PDT was conducted. Results: 64Cu ions bound tightly to the BPQDs. The BPQD@64Cu@PEG-VEGF/RGD nanocomplex showed effective internalization by the cancer cells and can be blocked by either VEGF or RGD peptides, demonstrating the peptide ligand-mediated active targeting effects. PET imaging results indicated that the 64Cu-nanocomplex was efficiently accumulated in the tumor site. Moreover, this nanocomplex showed significant tumor growth inhibition in the mice model. Conclusion: The BPQD@64Cu@PEG-VEGF/RGD nanocomplex is a promising theranostic platform for PET imaging-guided cancer PTT/PDT therapy. Further preclinical biosafety study of the nanoparticles will be studied. References: [1] K. Hu et al. Small, 2018, 14, 1801701. [2] K Hu et al. J. Mater. Chem. B. 2018, 5, 5433-5440., ISRS2019

We have successfully achieved the preparation of cyclo[Arg-Xaa-Asp-D-Tyr-Lys([1-11C]Tpi)](Xaa=Gly or Ala) using carbon-11 formaldehyde. This labeling technique could be used to increase the overall utility of 11C-labeled oligopeptides as PET probes because this method allows for the incorporation of carbon-11 into a cyclic bond. Furthermore, our study provide that cyclo[Arg-Gly-Asp-D-Tyr-Lys([1-11C]Tpi)] enabled accumulation in pancreatic cancer cell line BxPC3.