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Bosutinib has been evaluated for treatment of chronic-phase chronic myeloid leukemia (CP-CML) in several clinical studies, including in Japan. This open-label, single-arm, phase 2 study evaluated the efficacy and safety of bosutinib at a starting dose of 400 mg once daily in Japanese patients (n = 60) with newly diagnosed CP-CML. The minimum follow-up period was 3 years and median duration of treatment was 35.9 months. At study completion, 60% of patients were still on treatment. Cumulative rates of major molecular response (MMR), molecular response4 (MR4), and MR4.5 at any time were 70.0%, 53.3%, and 48.3%, respectively. No patient who achieved MMR or MR4 had a confirmed loss of response. No patient experienced on-treatment transformation to accelerated/blast phase or died within 28 days of the last bosutinib dose. Any-grade treatment-emergent adverse events (TEAEs) occurred in 100% (grade ≥ 3: 81.7%) of patients. The most common TEAEs were diarrhea (86.7%), increased alanine aminotransferase (55.0%), and increased aspartate aminotransferase (46.7%). No new safety signals emerged during the follow-up period. Bosutinib continues to demonstrate a favorable benefit/risk profile and is an important treatment option for Japanese patients with newly diagnosed CP-CML. Optimal management of TEAEs during initial treatment with bosutinib should be prioritized.Trial Registration: ClinicalTrials.gov ID: NCT03128411.

Background The phase 3 JAVELIN Bladder 100 trial showed significantly prolonged overall survival (OS) with avelumab as first-line (1L) maintenance therapy + best supportive care (BSC) vs BSC alone in patients with advanced urothelial carcinoma (UC) that had not progressed with 1L platinum-containing chemotherapy. Efficacy and safety were assessed in patients enrolled in Japan. Methods Patients with locally advanced or metastatic UC that had not progressed with 4–6 cycles of 1L platinum-containing chemotherapy were randomized to avelumab (10 mg/kg intravenously every 2 weeks) + BSC or BSC alone. The primary endpoint was OS, and secondary endpoints included progression-free survival (PFS) and safety. Results In Japanese patients (n = 73) randomized to avelumab + BSC (n = 36) or BSC alone (n = 37), median OS was 24.7 months (95% CI, 18.2-not estimable) vs 18.7 months (95% CI, 12.8–33.0), respectively (HR, 0.81 [95% CI, 0.41–1.58]), and median PFS was 5.6 months (95% CI, 1.9–9.4) vs 1.9 months (95% CI, 1.9–3.8), respectively (HR, 0.63 [95% CI, 0.36–1.11]). In the avelumab + BSC and BSC-alone arms, grade ≥ 3 treatment-emergent adverse events (AEs) occurred in 50.0% vs 8.1%, including grade ≥ 3 treatment-related AEs in 13.9% vs 0%, respectively. Efficacy and safety results in Japanese patients were generally consistent with findings in the overall trial population. Conclusion Avelumab 1L maintenance treatment showed a favorable benefit-risk balance in Japanese patients, supporting avelumab 1L maintenance as a new standard of care in Japanese patients with advanced UC that has not progressed with 1L platinum-containing chemotherapy. Trial registration Clinicaltrials.gov NCT02603432.

Introduction : Bosutinib is approved in Japan for patients with newly diagnosed Philadelphia chromosome-positive (Ph+) chronic phase (CP) chronic myeloid leukemia (CML) and Ph+ CML resistant/intolerant to prior therapy. In the primary report of a phase 2 study of first-line bosutinib in Japanese patients with CP CML (NCT03128411), the major molecular response (MMR) rate at 12 months was 55.0% and adverse events (AEs) were manageable and consistent with the known safety profile of bosutinib. Here we report the final 3-year efficacy and safety results of this phase 2 study. Methods : Japanese patients with newly diagnosed CP CML received bosutinib 400 mg once daily (QD). Dose escalation to a maximum of bosutinib 600 mg QD was permitted for unsatisfactory response. The bosutinib dose could be reduced to 300 mg QD for toxicity; following sponsor approval, dose reduction to bosutinib 200 mg QD was permitted for 4 weeks maximum. Long-term secondary endpoints of the study included duration of response, event-free survival, and overall survival (OS). This final analysis was based on ≥3 years of follow-up. Results: A total of 60 patients were treated with bosutinib. Median age was 55 years (range 20-83), 60.0% of patients were male, and 46.7%, 41.7%, and 11.7% had low-, intermediate-, and high-risk Sokal scores, respectively. Median duration of follow-up was 39.2 months (range 13.2-45.1), and median duration of treatment was 35.9 months (range 0.3-44.2). At study completion, 36 (60.0%) patients were still on treatment. The most common reason for treatment discontinuation was AEs (35.0%). Median dose intensity was 357.4 mg/day (range 95.3-548.1), with 400 mg QD being the most commonly utilized dose (>50% of ongoing patients across the treatment period). Dose reductions or interruptions due to AEs occurred in 36 (60.0%) and 50 (83.3%) patients, respectively; dose escalations due to insufficient response occurred in 10 (16.7%) patients. Cumulative rates of MMR, MR 4, and MR 4.5 at any time were 70.0%, 53.3%, and 48.3%, respectively (Table 1). Among patients who achieved MMR or MR 4, none had a confirmed loss of response. Cumulative rates of MMR at any time in patients with low-, intermediate-, and high-risk Sokal scores were 57.1%, 88.0%, and 57.1%, respectively. In patients with dose reductions to 300 (n=36) and 200 (n=9) mg QD, 63.9% (n=23) and 33.3% (n=3) newly achieved MMR or maintained a previously attained MMR after dose reduction. No patient progressed to accelerated/blast phase while on treatment. The cumulative incidence of progression or death adjusting for competing risk of treatment discontinuation at 3 years (90% CI) was 1.7% (0.2-6.4). Two (3.3%) patients died on study, 1 due to disease progression and 1 due to an AE considered unrelated to treatment. The 3-year Kaplan-Meier OS estimate (90% CI) was 96.7% (89.7-98.9). No evaluable patient had an emergent mutation while on treatment or at treatment completion. Any grade treatment-emergent AEs (TEAEs) occurred in 100% of patients and grade ≥3 TEAEs in 81.7% of patients. The most common TEAEs are shown in Table 2. There were no deaths on treatment. The most common (≥10%) TEAEs leading to dose reduction were alanine aminotransferase (ALT) increased (21.7%) and aspartate aminotransferase (AST) increased (13.3%), and the most common (≥10%) TEAEs leading to dose interruption were ALT increased (28.3%), AST increased (16.7%), diarrhea (11.7%), and liver disorder (10.0%). TEAEs leading to treatment discontinuation in ≥2% of patients were ALT increased (10.0%), AST increased (8.3%), lipase increased (3.3%), drug eruption (3.3%), and erythema multiforme (3.3%). Conclusions: After ≥3 years of follow-up, bosutinib continued to show clinical benefit, with approximately half of patients achieving deep molecular responses. The overall efficacy results were consistent with the global BFORE trial of first-line bosutinib. No new safety signals emerged with this longer follow-up. Bosutinib continues to demonstrate a favorable risk/benefit profile and is an important treatment option in Japanese patients with newly diagnosed CP CML. Figure 1 Figure 1. Disclosures Ono: Celgene: Honoraria, Research Funding; Kyowa Kirin Co., Ltd.: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Novartis Pharma KK: Honoraria; Bristol-Myers Squibb Company: Honoraria; Pfizer Japan Inc.: Honoraria; Otsuka Pharmaceutical Co., Ltd.: Honoraria; ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; Takeda Pharmaceutical Company Limited.: Honoraria; Astellas Pharma Inc.: Honoraria; Eisai Co., Ltd.: Honoraria; Janssen Pharmaceutical K.K: Honoraria; DAIICHI SANKYO COMPANY, LIMITED.: Honoraria; Mundipharma K.K.: Honoraria; TAIHO PHARMACEUTICAL CO., LTD.: Research Funding; Merck Sharp & Dohme: Honoraria, Research Funding. Hino: TEIJIN PHARMA LIMITED.: Research Funding; SEKISUI MEDICAL CO., LTD.: Research Funding; Sumitomo Dainippon Pharma Co., Ltd.: Research Funding; Abbott: Research Funding; Asahi Kasei Corporation:: Research Funding; ARKRAY: Research Funding; JCR Pharmaceuticals Co., Ltd.: Research Funding; Janssen Pharmaceutical: Honoraria; Bristol-Myers Squibb Comapany: Honoraria; Pfizer Japan Inc.: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; NIPPON SHINYAKU CO.,LTD.: Honoraria; Japan Blood Products Organization: Honoraria, Research Funding; Chugai Pharmaceutical Co., Ltd.: Honoraria; Takeda Pharmaceutical Company Limited.: Honoraria, Research Funding; Celgene Corporation: Honoraria, Research Funding; Sanofi: Honoraria; Kyowa Kirin Co., Ltd: Honoraria, Research Funding; ONO PHARMACEUTICAL CO., LTD.: Honoraria, Research Funding; Otsuka Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Eisai Co., Ltd: Honoraria, Research Funding; AstraZeneca: Honoraria; Astellas Pharma Inc.: Honoraria; MSD: Honoraria, Research Funding; Meiji Seika Pharma Co., Ltd.: Honoraria; CSL Behring: Honoraria; TAIHO PHARMACEUTICAL CO., LTD.: Research Funding; DAIICHI SANKYO COMPANY, LIMITED.: Research Funding; TOSOH CORPORATION: Research Funding. Matsumura: MSD: Research Funding; Nippon Shinyaku: Research Funding; Novartis: Research Funding, Speakers Bureau; Ono: Research Funding; Otsuka: Consultancy, Research Funding, Speakers Bureau; Pfizer: Research Funding, Speakers Bureau; Shionogi: Research Funding; Taiho: Research Funding; Takeda: Research Funding; Sumitomo Dainippon: Research Funding; Nihon Pharmaceutical: Research Funding; Daiichi Sankyo: Research Funding, Speakers Bureau; Japan Blood Products Organization: Research Funding; Mundipharma: Research Funding; AYUMI Pharmaceutical: Research Funding; Mitsubishi Tanabe: Research Funding; Kyowa Kirin: Research Funding; Eisai: Research Funding; Chugai: Research Funding; Addvie: Research Funding; Eli Lilly Japan: Research Funding; Amgen: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau; Janssen: Speakers Bureau; Astellas: Speakers Bureau; Asahi Kasei: Research Funding. Fujisawa: Novartis: Honoraria, Research Funding; Pfizer: Honoraria, Research Funding; Otsuka: Honoraria, Research Funding; Bristol Myers Squibb: Honoraria, Research Funding. Ishizawa: Otsuka: Research Funding; Novartis: Honoraria, Research Funding, Speakers Bureau; Sanofi: Research Funding; SymBio: Honoraria, Research Funding; Kyowa Kirin: Consultancy; Pfizer: Research Funding; Bristol Myers Squibb: Speakers Bureau; IQVIA: Research Funding; Eisai: Honoraria; Chugai: Honoraria; Ono: Honoraria; Celgene: Honoraria; Takeda: Honoraria; Bayer: Research Funding; AbbVie: Research Funding. Sakaida: Kyowa Kirin: Research Funding; Ono: Research Funding; Chugai: Research Funding; Bristol Myers Squibb: Research Funding. Sekiguchi: Ono: Research Funding; A2 Healthcare: Research Funding; Astellas: Research Funding; Janssen: Research Funding; Merck Sharp & Dohme: Research Funding; Otsuka: Research Funding; Pfizer: Research Funding; PPD-SNBL: Research Funding; Sumitomo Dainippon: Research Funding; Daiichi Sankyo: Research Funding; Bristol Myers Squibb: Research Funding. Ono: Pfizer: Current Employment, Current equity holder in publicly-traded company. Aizawa: Pfizer: Current Employment. Tanetsugu: Pfizer: Current Employment. Koide: Pfizer: Current Employment. Takahashi: Chugai: Research Funding; Eizai: Research Funding; Asahikasei: Research Funding; Novartis Pharmaceuticals: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Pfizer: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Otsuka Pharmaceutical: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding, Speakers Bureau; Ono: Research Funding; Kyowahakko-Kirin: Research Funding; Toyamakagaku: Research Funding.

This long-term follow-up of a completed phase 1/2 study assessed the safety and efficacy of bosutinib in Japanese Philadelphia chromosome-positive, chronic phase (CP) or advanced phase (ADV) chronic myeloid leukemia patients who were resistant/refractory or intolerant to prior tyrosine kinase inhibitor treatment. This analysis included 63 patients with a median bosutinib follow-up of 132 weeks (range 3‒372). In the CP second-line (2L) cohort, the cumulative major cytogenetic response (MCyR) and major molecular response (MMR) rates throughout the study were 73 and 53%, respectively. In the CP third-line (3L) cohort, the cumulative MCyR and MMR rates throughout the study were 70 and 40%, respectively. Of the eight ADV patients, MCyR was attained or maintained by 50% of patients, and complete hematologic response was attained or maintained by 25% of patients. Progression-free survival rate and overall survival rate at 96 weeks were, respectively, 91 and 98% in CP2L, 88 and 100% in CP3L, and 33 and 50% in ADV patients. The most common adverse events (50%) reported were diarrhea (95%), nasopharyngitis (57%), and rash (57%). Bosutinib demonstrated durable efficacy and a manageable tolerability profile over long-term use in Japanese patients.ClinicalTrials.gov: NCT00811070.

Fluorescence probes that can detect pH are useful tools for biological research and clinical diagnosis. Here we report pH-activatable near-infrared fluorescence probes, based on hydroxymethyl germanium-rhodamine (HMGeR), that are suitable for a range of biological applications. The pKa, the ratio of the fluorescent form in an acidic environment, and the absorption/emission wavelengths can all be conveniently optimized. The most promising probe, 2-HM IGeR, offers significant advantages over currently available near-infrared pH probes, notably high quantum efficiency, appropriate pKa value for biological applications, and high photostability. Further, our molecular design strategy allows easy conjugation of the probes to biomolecules without loss of functionality. We illustrate the value of this strategy by developing probe-Herceptin® and probe-avidin conjugates to visualize pH change in cellular vesicles during endocytosis, and to visualize tumors in a mouse model, respectively. We believe 2-HM IGeR is currently among the best-in-class pH-activatable near-infrared probes for biological and medical research. Near-infrared fluorescence probes that respond to pH are effective tools to analyse pH-driven biological processes but share common limitations. Here the authors report a germanium-rhodamine based near-infrared probe with appropriate pKa value for biological applications and improved photostabillity and quantum yield.

425 Background: A randomized phase III trial (JAVELIN Bladder 100; NCT02603432) to investigate avelumab (anti–PD-L1) as 1L maintenance therapy in patients with advanced UC met its primary objective, demonstrating significantly prolonged overall survival (OS) with Ave + BSC vs BSC alone in all randomized patients and in patients with PD-L1+ tumors. We report efficacy and safety in Japanese patients enrolled in this study. Methods: Eligible patients with unresectable locally advanced or metastatic UC that had not progressed with 4-6 cycles of gemcitabine with either cisplatin or carboplatin were randomized 1:1 to receive maintenance Ave (10 mg/kg IV every 2 weeks) + BSC or BSC alone, stratified by best response to 1L chemotherapy (complete/partial response vs stable disease) and by visceral vs nonvisceral disease when initiating 1L chemotherapy. The primary endpoint was OS, assessed from randomization in all randomized patients and in patients with PD-L1+ tumors (Ventana SP263 assay). Secondary endpoints included progression-free survival (PFS) per blinded independent central review and safety. Results: Japanese patients (n=73) were randomized to receive Ave + BSC (n=36) or BSC alone (n=37); 52.8% vs 62.2% had PD-L1+ tumors, respectively. Median OS (95% CI) was 24.7 months (18.2-not estimable [NE]) with Ave + BSC vs 18.7 months (12.8-33.0) with BSC alone (HR, 0.81 [95% CI; 0.409-1.585]) in all randomized patients and 18.6 months (9.4-NE) with Ave + BSC vs 19.4 months (11.7-33.0) with BSC alone (HR, 1.00 [95% CI, 0.413-2.412]) in patients with PD-L1+ tumors. Median PFS (95% CI) was 5.6 months (1.9-9.4) with Ave + BSC vs 1.9 months (1.9-3.8) with BSC alone (HR, 0.63 [95% CI, 0.358-1.113]) in all randomized patients and 5.6 months (1.8-11.2) with Ave + BSC vs 1.9 months (1.9-3.8) with BSC alone (HR, 0.62 [95% CI, 0.298-1.301]) in patients with PD-L1+ tumors. The most common treatment-emergent adverse events (all grade; grade ≥3) in the Ave + BSC arm were pyrexia (10 [27.8%]; 0), nasopharyngitis (7 [19.4%]; 0), and anemia (7 [19.4%]; 4 [11.1%]). Conclusions: Ave 1L maintenance + BSC was efficacious and tolerable in Japanese patients with advanced UC, and results were generally consistent with those in the overall population. Clinical trial information: NCT02603432.

Background: Bosutinib, a Src/Abl tyrosine kinase inhibitor, is approved at a starting dose of 500 mg once daily (QD) in many countries, including Japan, for patients with Philadelphia chromosome-positive (Ph+) chronic phase (CP), accelerated phase (AP), or blast phase (BP) chronic myeloid leukemia (CML) after prior therapy. The indication for bosutinib was expanded to patients with newly diagnosed CP CML, at a starting dose of 400 mg QD, in 2017 by the US Food and Drug Administration and in 2018 by the European Medicines Agency. Approval of first-line bosutinib for CP CML was based on data from the global phase 3 BFORE trial, which demonstrated a significantly higher major molecular response (MMR) rate at Month 12 with bosutinib vs imatinib in patients with Ph+ CP CML and e13a2/e14a2 transcripts (primary endpoint; 47.2% vs 36.9%; 2-sided P=0.02). We conducted a phase 2 study to evaluate the efficacy, safety, and pharmacokinetics (PK) of bosutinib in Japanese patients with newly diagnosed CP CML. Methods: In this open-label, single-arm study (NCT03128411), Japanese patients ≥20 years of age with a molecular diagnosis of CP CML within 6 months, Eastern Cooperative Oncology Group performance status 0 or 1, adequate renal and hepatic function, and no prior treatment for CML (hydroxyurea within 6 months permitted) received bosutinib at a starting dose of 400 mg QD. The primary endpoint was MMR at Month 12 in the modified as-treated population, which included patients who were Ph+ and had e13a2/e14a2 transcripts. A total of 60 patients was required in the modified as-treated population for the study to have >82% power to reject the null hypothesis (25% MMR rate at Month 12) and accept the alternative hypothesis (40% MMR rate at Month 12) with a 1-sided ∝-level of 5%. Secondary endpoints included MMR and complete cytogenetic response (CCyR) by Month 12, event-free survival (EFS), overall survival, safety, and PK. Results: In all, 60 Japanese patients with CP CML were treated with bosutinib; all patients were Ph+ and had e13a2/e14a2 transcripts and were included in the modified as-treated population analyzed for efficacy. Median age was 55 years (range 20-83), 60.0% of patients were male, and 45.0%, 43.3%, and 11.7% had low-, intermediate-, and high-risk Sokal scores, respectively. Median duration of follow-up was 16.6 months (range 11.1-21.9), and median duration of bosutinib treatment was 15.3 months (range 0.3-21.9). After 12 months of follow-up, 42 (70.0%) patients remained on bosutinib; 17 (28.3%) discontinued due to adverse events (AEs) and 1 (1.7%) due to physician decision. Median dose intensity was 354.7 mg/day (range 95.3-494.1). The MMR rate at Month 12 was 55.0% (2-sided 90% confidence interval [CI] 44.4-65.6); the test of the null hypothesis was rejected (1-sided P The most common treatment-emergent AEs (TEAEs) were diarrhea (86.7%), increased alanine aminotransferase (ALT; 55.0%) and increased aspartate aminotransferase (AST; 46.7%; Table). Grade 3/4 TEAEs reported in ≥10% of patients were increased ALT (33.3%), increased AST (18.3%), diarrhea (15.0%), increased lipase (15.0%), and neutropenia (11.7%). The incidence of cardiac, vascular, and hypertension TEAEs was low (5.0%, 1.7%, and 1.7%, respectively). The average bosutinib plasma trough concentration (mean ± standard deviation of concentrations on Days 28, 56, and 84) was 82.7 ± 48.0 ng/mL, ~1.12-fold higher than that observed in bosutinib-treated patients in the global BFORE trial. Conclusions: The primary objective of this phase 2 study was met, and the MMR rate at Month 12 in Japanese patients with newly diagnosed CP CML was similar to that reported in the bosutinib arm of the multinational BFORE trial. The safety profile of bosutinib was consistent with previous studies. These data suggest bosutinib is an effective first-line treatment option for Japanese patients with newly diagnosed CP CML. Disclosures Takahashi: Novartis Pharmaceuticals: Research Funding, Speakers Bureau; Eisai Pharmaceuticals: Research Funding; Chug Pharmaceuticals: Research Funding; Pfizer: Research Funding, Speakers Bureau; Ono Pharmaceutical: Research Funding; Astellas Pharma: Research Funding; Asahi Kasei Pharma: Research Funding; Otsuka Pharmaceutical: Research Funding, Speakers Bureau; Kyowa Hakko Kirin: Research Funding; Bristol-Myers Squibb: Speakers Bureau. Matsumura:Novartis: Speakers Bureau; Otsuka Pharmaceutical: Consultancy, Research Funding; Pfizer: Research Funding, Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. Ishizawa:Otsuka Pharmaceutical: Research Funding; Pfizer: Research Funding; Novartis: Speakers Bureau; Bristol-Myers Squibb: Speakers Bureau. ONO:Otsuka Pharmaceutical Co., Ltd.: Honoraria; Pfizer: Honoraria; Bristol-Myers Squibb: Honoraria; Novartis: Honoraria; Chugai Pharmaceutical Co.,Ltd.: Research Funding; Kyowa Hakko Kirin: Research Funding; ONO Pharmaceutical Co., Ltd.: Honoraria, Research Funding; Merck Sharp & Dohme: Honoraria, Research Funding; Celgene: Honoraria, Research Funding; Takeda Pharmaceutical Co., Ltd.: Honoraria. Sekiguchi:Pfizer: Research Funding. Tanetsugu:Pfizer R&D Japan G.K.: Employment. Fukuhara:Pfizer R&D Japan G.K.: Employment. Ohkura:Pfizer R&D Japan G.K.: Employment. Koide:Pfizer R&D Japan G.K.: Employment. Hino:Alexion: Honoraria; Astellas Pharma Inc: Honoraria, Research Funding; Astellas Amgen BioPharma: Honoraria; Bristol-Myers Squibb: Honoraria; Celgene: Honoraria; Chugai Pharmaceutical Co., Ltd: Honoraria, Research Funding; Daichi-Sankyo: Honoraria, Research Funding; Eisai: Research Funding; Janssen: Honoraria; Japan Blood Products Organization: Honoraria, Research Funding; Kyowa-Hakko Kirin Co.,Ltd: Honoraria, Membership on an entity's Board of Directors or advisory committees, Other: Consulting fee, Research Funding; Mochica Pharmaceutical Co., Ltd: Honoraria; MSD: Honoraria, Research Funding; Mundipharma: Honoraria; Nihon Pharmaceutical Co., Ltd: Research Funding; Nippon Shinyaku: Honoraria; Novartis: Honoraria, Membership on an entity's Board of Directors or advisory committees; Ono Pharmaceutical: Honoraria, Other: Consulting fee, Research Funding; Otsuka Pharmaceutical: Honoraria, Research Funding; Pfizer Japan Inc: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding; Sanofi: Honoraria; Shire Japan KK: Honoraria; Sumitomo Dainippon Parma: Honoraria, Research Funding; Taiho Pharama: Research Funding; Takeda Pharmaceutical Co., Ltd: Honoraria, Research Funding; Teijin: Research Funding; Abbott: Research Funding. OffLabel Disclosure: Bosutinib is approved for newly diagnosed patients with chronic phase chronic myeloid leukemia by the US Food and Drug Administration and by the European Medicines Agency, but it is not yet approved for this patient population in Japan, where this study takes place.

The development of sophisticated fluorescent probes has contributed to the elucidation of the molecular mechanisms of many complex biological phenomena. In particular, fluorescence imaging of the calcium ion (Ca) has become an essential technique for the investigation of signaling pathways involving Ca as a second messenger. For example, changes in the intracellular Ca concentration have been found to be related to physiological responses in obesity, as well as immune responses and pathological responses in Alzheimer s disease. Because Ca signaling is involved in so many biological phenomena, it is expected that the simultaneous visualization of Ca and other biomolecules, that is, multicolor imaging, would be particularly informative. For this purpose, we require a fluorescent probe for Ca that operates in a different color window from that of probes for other molecules. Fluorescent Ca sensors can be categorized into twomain classes: those based on genetically encoded fluorescent proteins and those based on fluorescent small organic molecules. Although both types of sensors have certain advantages and drawbacks, small-molecule-based probes have the particular advantage that their AM ester form (cell-permeable acetoxymethyl ester derivative) can be readily bulk loaded into live cells with no need for transfection. Most currently used small-molecular fluorescent probes for Ca are fluorescein-based, such as Fluo-3, Fluo-4, Calcium Green-1, and Oregon Green 488 BAPTA-1, and emit green fluorescence (ca. 527 nm). There are also some redemitting fluorescent probes for Ca, such as Rhod-2 (ca. 576 nm), which is based on the rhodamine scaffold. These red-emitting fluorescent probes for Ca, including Rhod-2, are also widely used for biological studies; however, the cationic nature of the rhodamine scaffold generally causes Rhod-2 AM to localize into mitochondria. Although this behavior is useful for monitoring the Ca dynamics of mitochondria, the visualization of cytoplasmic Ca is much more important for research on Ca signaling. The influx of Ca into the cytoplasm from the extracellular environment and/or from intracellular stores (including the endoplasmic reticulum) triggers numerous cellular responses mediated by the interaction of Ca with various Ca-binding proteins, such as calmodulin and troponin C. Fura Red is a representative near-infrared fluorescent probe for Ca that is often used in biological research. However, it has extremely low fluorescence quantum efficiency (Ffl 0.013). Accordingly, the fluorescence signal is very small unless a high concentration of Fura Red or a high-powered laser is used. However, the use of a high dye concentration has a buffering effect on Ca, whereas the use of a high laser power causes rapid photobleaching of the dye and phototoxicity to the cells. Thus, a novel fluorescent probe for cytoplasmic Ca with strong emission in the long-wavelength region would be extremely useful, especially for multicolor imaging. In the present study, we designed and synthesized a novel and practical red-fluorescence-emitting probe suitable for monitoring cytoplasmic Ca and confirmed its usefulness for the visualization of stimulus-induced Ca oscillation in HeLa cells. As a fluorophore that emits in the red region, we chose TokyoMagenta (TM). The absorption and fluorescence wavelengths of this fluorescein analogue are 90 nm longer than those of fluorescein. TM was also expected to retain the advantages of the fluorescein scaffold, including cytoplasmic localization. For the development of the red fluorescent probe, we chose a combination of 2-Me-substituted TM as the fluorescent moiety and 1,2-bis(o-aminophenoxy)ethaneN,N,N’,N’-tetraacetic acid (BAPTA) as a specific chelator for Ca, and synthesized CaTM-1 (Figure 1; see also Scheme S1 in the Supporting Information). The fluorescence-activation ratio of CaTM-1 in the presence/absence of Ca is 5.6:1 (Figure 2a,b, Table 1). To further improve this ratio, we decided on the strategy of decreasing the energy of the highest occupied molecular orbital (HOMO) of the fluorophore to obtain a high level of [*] T. Egawa, K. Hirabayashi, Dr. Y. Koide, C. Kobayashi, Dr. N. Takahashi, Dr. T. Terai, Dr. T. Ueno, Dr. T. Komatsu, Dr. Y. Ikegaya, Prof. N. Matsuki, Prof. T. Nagano, Dr. K. Hanaoka Graduate School of Pharmaceutical Sciences The University of Tokyo 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033 (Japan) E-mail: khanaoka@mol.f.u-tokyo.ac.jp

Stereocomplex poly (lactic acid) films were prepared by casting chloroform, acetonitrile or tetrahydrofuran solution of a mixture, mix-poly(lactic acid) (PLA), of poly (L-lactic acid) and poly (D-lactic acid). Time dependence of cloud point (Figure 1), as well as the morphology of the cast films (Figure 2) was markedly different in these three cast-solvents. The cast-solvent dependence of the morphology was studied by assuming that structure of stereocomplex is constructed through two processes.

Background and Objectives: Antimicrobial photodynamic therapy (APDT) employs a non-toxic photosensitizer (PS) and visible light, which in the presence of oxygen produce reactive oxygen species (ROS), such as singlet oxygen (^(1)O_2, produced via Type II mechanism) and hydroxyl radical (HO., produced via Type I mechanism). This study examined the relative contributions of ^(1)O_2 and HO. to APDT killing of Gram-positive and Gram-negative bacteria. Study Design/Materials and Methods: Fluorescence probes, 3′-(p-hydroxyphenyl)-fluorescein (HPF) and singlet oxygen sensor green reagent (SOSG) were used to determine HO. and ^(1)O_2 produced by illumination of two PS: tris-cationic-buckminsterfullerene (BB6) and a conjugate between polyethylenimine and chlorin(e6) (PEI–ce6). Dimethylthiourea is a HO. scavenger, while sodium azide (NaN_3) is a quencher of ^(1)O_2. Both APDT and killing by Fenton reaction (chemical generation of HO.) were carried out on Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis) and Gram-negative bacteria (Escherichia coli, Proteus mirabilis, and Pseudomonas aeruginosa). Results: Conjugate PEI-ce6 mainly produced ^(1)O_2 (quenched by NaN_3), while BB6 produced HO. in addition to ^(1)O_2 when NaN_3 potentiated probe activation. NaN_3 also potentiated HPF activation by Fenton reagent. All bacteria were killed by Fenton reagent but Gram-positive bacteria needed a higher concentration than Gram-negatives. NaN3 potentiated Fenton-mediated killing of all bacteria. The ratio of APDT killing between Gram-positive and Gram-negative bacteria was 2 or 4:1 for BB6 and 25:1 for conjugate PEI-ce6. There was a NaN_3 dose-dependent inhibition of APDT killing using both PEI-ce6 and BB6 against Gram-negative bacteria while NaN_3 almost failed to inhibit killing of Gram-positive bacteria. Conclusion: Azidyl radicals may be formed from NaN_3 and HO.. It may be that Gram-negative bacteria are more susceptible to HO. while Gram-positive bacteria are more susceptible to ^(1)O_2. The differences in NaN_3 inhibition may reflect differences in the extent of PS binding to bacteria (microenvironment) or differences in penetration of NaN_3 into cell walls of bacteria.

Optical imaging is emerging as an important tool to visualize tumors. However, there are many potential choices among the available fluorophores. Optical imaging probes that emit in the visible range can image superficial tumors with high quantum yields, however, if deeper imaging is needed then near infrared (NIR) fluorophores are necessary. Most commercially available NIR fluorophores are cyanine based and are prone to non-specific binding and relatively limited photostability. Silica-containing rhodamine (SiR) fluorophores represent a new class of NIR fluorophores, which permit photoactivation via H-dimer formation as well as demonstrate improved photostability. This permits higher tumor-to-background ratios (TBRs) to be achieved over longer periods of time. Here, we compared an avidin conjugated with SiR700 (Av-SiR700) to similar compounds based on cyanine dyes (Av-Cy5.5 and Av-Alexa Fluor 680) in a mouse tumor model of ovarian cancer metastasis. We found that the Av-SiR700 probe demonstrated superior quenching enabling activation after binding-internalization to the target cell. As a result, Av-SiR700 had higher TBRs compared to Av-Cy5.5, and better biostability compared to Av-Alexa Fluor 680.

The absorption and emission wavelengths of group 14 pyronines and rhodamines, which contain silicon, germanium, or tin at the 10 position of the xanthene chromophore, showed large bathochromic shifts compared to the original rhodamines, owing to stabilization of the LUMO energy levels by σ*-π* conjugation between group 14 atom-C (methyl) σ* orbitals and a π* orbital of the fluorophore. These group 14 pyronines and rhodamines retain the advantages of the original rhodamines, including high quantum efficiency in aqueous media (Φ(fl) = 0.3-0.45), tolerance to photobleaching, and high water solubility. Group 14 rhodamines have higher values of reduction potential than other NIR light-emitting original rhodamines, and therefore, we speculated their NIR fluorescence could be controlled through the photoinduced electron transfer (PeT) mechanism. Indeed, we found that the fluorescence quantum yield (Φ(fl)) of Si-rhodamine (SiR) and Ge-rhodamine (GeR) could be made nearly equal to zero, and the threshold level for fluorescence on/off switching lies at around 1.3-1.5 V for the SiRs. This is about 0.1 V lower than in the case of TokyoGreens, in which the fluorophore is well established to be effective for PeT-based probes. That is to say, the fluorescence of SiR and GeR can be drastically activated by more than 100-fold through a PeT strategy. To confirm the validity of this strategy for developing NIR fluorescence probes, we employed this approach to design two kinds of novel fluorescence probes emitting in the far-red to NIR region, i.e., a series of pH-sensors for use in acidic environments and a Zn(2+) sensor. We synthesized these probes and confirmed that they work well.

To improve optical imaging of Ca(2+) and to make available a distinct color window for multicolor imaging, we designed and synthesized CaSiR-1, a far-red to near-infrared fluorescence probe for Ca(2+), using Si-rhodamine (SiR) as the fluorophore and the well-known Ca(2+) chelator BAPTA. This wavelength region is advantageous, affording higher tissue penetration, lower background autofluorescence, and lower phototoxicity in comparison with the UV to visible range. CaSiR-1 has a high fluorescence off/on ratio of over 1000. We demonstrate its usefulness for multicolor fluorescence imaging of action potentials (visualized as increases in intracellular Ca(2+)) in brain slices loaded with sulforhodamine 101 (red color; specific for astrocytes) that were prepared from transgenic mice in which some neurons expressed green fluorescent protein.

We have developed a series of novel near-infrared (NIR) wavelength-excitable fluorescent dyes, SiR-NIRs, by modifying the Si–rhodamine scaffold to obtain emission in the range suitable for in vivo imaging. Among them, SiR680 and SiR700 showed sufficiently high quantum efficiency in aqueous media. Both antibody-bound and free dye exhibited high tolerance to photobleaching in aqueous solution. Subcutaneous xenograft tumors were successfully visualized in a mouse tumor model using SiR700-labeled anti-tenascin-C (TN-C) antibody, SiR700-RCB1. SiR-NIRs are expected to be useful as labeling agents for in vivo imaging studies including multicolor imaging, and also as scaffolds for NIR fluorescence probes.

The new Si-substituted fluorescence probe (Ia) possesses a high off/on activation ratio with a large red shift and a strong fluorescence intensity.

A far-red to near-infrared (NIR) fluorescence probe, MMSiR, based on Si-rhodamine, was designed and synthesized for sensitive and selective detection of HOCl in real time. MMSiR and its oxidized product SMSiR have excellent properties, including pH-independence of fluorescence, high resistance to autoxidation and photobleaching, and good tissue penetration of far-red to NIR fluorescence emission. The value of MMSiR was confirmed by real-time imaging of phagocytosis using a fluorescence microscope. wsMMSiR, a more hydrophilic derivative of MMSiR, permitted effective in vivo imaging of HOCl generation in a mouse peritonitis model. This probe is expected to be a useful tool for investigating the wide range of biological functions of HOCl.

We present a design strategy for fluorescence probes with a high off/on activation ratio in the red wavelength region, based on a novel fluorescein analogue in which the O atom at the 10 position of the xanthene chromophore is replaced with a Si atom. To demonstrate the usefulness of this strategy, we designed and synthesized a red-fluorescent probe for β-galactosidase, and showed that it works in live HEK293 cells.

We designed and synthesized fluorescent probes for the sensitive and selective detection of highly reactive oxygen species (hROS) in mitochondria of living cells and confirmed their usefulness in vitro. MitoAR and MitoHR are highly selective for hROS (•OH, ONOO-, OCl-) over other ROS. They have exellent properties, including tolerance to autoxidation and photobleaching by laser irradiation during fluorescence microscopy and should be useful tools for biological and pathological applications.

A novel design strategy for controlling the fluorescence and photosensitizing ability of thiazole orange (TO) has been developed. The validity of this approach was demonstrated by the synthesis of a beta-galactosidase-activatable photosensitizer, PhoTO-Gal, in which fluorescence is simultaneously activated. PhoTO-Gal was demonstrated to kill HEK293 lacZ(+) cells, which express beta-galactosidase, but not HEK293 lacZ(-) cells, under light illumination. Such activatable photosensitizers should allow more refined PDT without the side effect of prolonged light sensitivity and should also be useful as tools for reporter enzyme expression-specific cell ablation.

We have designed and synthesized a reversible near-infrared (NIR) fluorescence probe, 2-Me TeR, for reactive oxygen species (ROS), utilizing the redox properties of the tellurium (Te) atom. 2-Me TeR is oxidized to fluorescent 2-Me TeOR by various ROS, while the generated 2-Me TeOR is quickly reduced in the presence of glutathione to regenerate 2-Me TeR. This redox-induced reversible NIR-fluorescence response of 2-Me TeR allowed us to detect the endogenous production of ROS and subsequent homeostatic recovery of the intracellular reductive environment in hydrogen peroxide-stimulated HL-60 cells. This probe is expected to be useful for monitoring the dynamics of ROS production continuously in vivo.