Decreasing undesirable absorbed radiation to the intestine after administration of radium-223 dichloride for treatment of bone metastases.

[223Ra]RaCl2 is the first alpha-particle emitting radiopharmaceutical to be used for castration-resistant prostate cancer patients with bone metastases because of its excellent therapeutic effects. [223Ra]RaCl2 is excreted via the intestine into feces, and some is absorbed from the intestine into the blood, which may be undesirable in terms of the exposure to radiation. Recently, we showed that a complex of myo-inositol-hexakisphosphate (InsP6) with zinc is a useful decorporation agent against radiostrontium. In this study, we hypothesized that Zn-InsP6 could bind to not only strontium but also to radium, and could inhibit the absorption of radium from the intestine. In in vitro binding experiments, Zn-InsP6 showed a high binding affinity for radium. In in vivo biodistribution experiments by intravenous injection of [223Ra]RaCl2 after treatment of Zn-InsP6, mice treated with Zn-InsP6 showed significantly lower bone accumulation of radioactivity (34.82 ± 1.83%Dose/g) than the mice in the non-treatment control group (40.30 ± 2.78%Dose/g) at 48 h postinjection. These results indicate that Zn-InsP6 bound radium in the intestine and inhibited the absorption of radium into the blood. Therefore, the insoluble Zn-InsP6 complex has high potential to decrease the side effects of [223Ra]RaCl2.

A Novel Anionic-phosphate-platinum Complex Effectively Targets a Cisplatinum-resistant Osteosarcoma in a Patient-derived Orthotopic Xenograft Mouse Model.

BACKGROUND/AIM: We have previously developed a novel bone-targeting platinum compound, 3Pt, and showed that it has strong inhibitory activity against osteosarcoma cells and orthotopic cell-line xenograft mouse models. In the present report, we compared the efficacy of 3Pt to cisplatinum (CDDP) in a CDDP-resistant relapsed osteosarcoma patient-derived orthotopic xenograft (PDOX) mouse model. PATIENTS AND METHODS: The tumor of a patient with osteosarcoma of the distal femur was treated with CDDP-based chemotherapy followed by surgery. The surgical specimen was used to establish a PDOX model. An osteosarcoma cell line was also established from the original patient tumor. Osteosarcoma cell viability was assessed with the WST-8 assay and the IC50 values were calculated. The PDOX models were randomized into three groups: untreated control, CDDP-treated group, and 3Pt-treated group. Tumor size and body weight were measured twice a week. RESULTS: 3Pt had a strong concentration-dependent cytocidal effect in vitro. The IC50 value of 3Pt was significantly lower than that of CDDP. On day 14 of the treatment, 3Pt caused a significantly greater tumor growth inhibition compared to the untreated control and CDDP-treated mice. CONCLUSION: 3Pt is a promising clinical candidate for the treatment of recalcitrant osteosarcoma.

Inorganic and Metal-Organic Nanocomposites for Cascade-Responsive Imaging and Photochemical Synergistic Effects.

Porphyrins coordinated with platinum(II) chemotherapeutic drugs are attractive for the development of photosensitizers for photodynamic therapy (PDT) of cancer. In this paper, inorganic and metal-organic nanocomposites were synthesized with cascade-responsive imaging and photochemical synergistic effects. After endo/lysosomal escape, the outer metal-organic frameworks were degraded, leading to the release of an excellent photosensitizer (tetrapyridylporphyrin, PtTPyP). Subsequently, doxorubicin (DOX), inserted in the adenosine triphosphate (ATP) aptamer-functionalized gold nanoparticles, was released under the stimulation of endogenous ATP, synergistically enhancing cancer treatment. Fluorescence imaging allowed tracking of PtTPyP and DOX for real-time detection and on-demand therapy. This strategy endowed the nanocomposites with stability, responsiveness, effectiveness, and ease of synthesis, namely, sTREE strategy. Accordingly, our demonstration provided a promising and smart nanocarrier for imaging and drug delivery.

Bone invasion-targeted chemotherapy with a novel anionic platinum complex (3Pt) for oral squamous cell carcinoma.

Cisplatin (CDDP) is an important drug for chemotherapy in patients with head and neck squamous cell carcinoma. Nephrotoxicity and lack of an effect on bone invasion are limitations of CDDP. To increase its antitumor effect on bone invasion and reduce toxicity problems, anionic Pt complex (3Pt) has been developed. The present study aimed to characterize the basis of the cytotoxicity of the novel platinum complex 3Pt in comparison with that of CDDP for oral squamous cell carcinoma. The ionic platinum complex was prepared to increase solubility and avoid platinum nephrotoxicity. Furthermore, 3Pt was designed to target bone hydroxyapatite and has germinal bisphosphonate moieties for drug delivery. In vitro antitumor activity was assayed in two oral squamous cell carcinoma cell lines. To investigate the antitumor and nephrotoxic effects of 3Pt, nude mice with OSC-19 were given 3Pt and CDDP. The in vitro growth-inhibitory effect of 3Pt was significantly less than that of CDDP. However, both 3Pt and CDDP showed equivalent antitumor effects in vivo. Mice injected with CDDP developed renal cell apoptosis; however, those injected with 3Pt were almost free of renal cell injury. In addition to similar in vivo antitumor effects, 3Pt decreased the volume of bone resorption compared to that with CDDP in a bone invasion model using OSC-19. In conclusion, considering the potential advantages in terms of noticeable antitumor activity on bone invasion and reduced nephrotoxicity, 3Pt represents a significant improvement in the development of bone-targeting platinum drugs.

Syntheses and evaluation of a homologous series of aza-vesamicol as improved radioiodine-labeled probes for sigma-1 receptor imaging.

Sigma-1 receptor imaging probes for determining the expression levels are desirable for diagnoses of various diseases and companion diagnoses of therapeutic agents targeting the sigma-1 receptor. In this study, we aimed to develop probes with higher affinity for the sigma-1 receptor. For this purpose, we synthesized and evaluated compounds, namely, vesamicol derivatives, in which alkyl chains of varying chain length were introduced between a piperazine ring and a benzene ring. The binding affinity of the vesamicol derivatives for the sigma-1 receptor tended to increase depending on the length of the alkyl chain between the benzene ring and the piperazine ring. The sigma-1 receptor of 2-(4-(3-phenylpropyl)piperazin-1-yl)cyclohexan-1-ol (5) (Ki = 5.8 nM) exhibited the highest binding affinity; therefore, we introduced radioiodine into the benzene ring in 5. The radioiodine labeled probe [125I]2-(4-(3-(4-iodophenyl)propyl)piperazin-1-yl)cyclohexan-1-ol ([125I]10) showed high accumulation in the sigma-1 receptor expressing DU-145 cells both in vitro and in vivo. Co-injection of [125I]10 with an excess level of a sigma receptor ligand, haloperidol, resulted in a significant decrease in the tumor accumulation in vitro and in vivo, indicating sigma receptor-mediated tumor uptake. These results provide useful information for developing sigma-1 receptor imaging probes.

Development of radiolabeled bis(zinc(II)-dipicolylamine) complexes for cell death imaging.

PURPOSE: Although it has been traditionally surmised that phosphatidylserine (PS) externalization is a hallmark of apoptosis, most other non-apoptotic modes of cell death, such as necrosis, are also associated with PS externalization. Bis(zinc-dipicolylamine) (ZnDPA) complexes have been reported to exhibit affinity for PS. The present study aimed to develop novel radiolabeled ZnDPA derivatives for cell death imaging in tumor after treatment with anticancer drugs. METHODS: [125I]IB-EG2-ZnDPA and [99mTc]Tc-MAG3-EG2-ZnDPA were designed and prepared. The stabilities of these radiotracers were determined in 0.1 M phosphate buffer (pH 7.4) or murine plasma at 37 °C, and their 1-octanol/water partition coefficients (logP) were measured. The uptake of radioactivity in cancer cells, which were preincubated in a normal medium or in a medium containing 5-FU, was measured after incubation with radiotracers. Accumulation of [99mTc]Tc-MAG3-EG2-ZnDPA in the tumor was evaluated in tumor-bearing mice treated with or without 5-FU, and then TUNEL staining was performed to detect dead cells in the tumor tissue sections. RESULTS: The radiochemical purities of [125I]IB-EG2-ZnDPA and [99mTc]Tc-MAG3-EG2-ZnDPA exceeded 95%. Although [125I]IB-EG2-ZnDPA gradually decomposing with time, more than 90% of [99mTc]Tc-MAG3-EG2-ZnDPA remained in its intact form in phosphate buffer through 6 h of incubation. Neither [125I]IB-EG2-ZnDPA nor [99mTc]Tc-MAG3-EG2-ZnDPA decomposed so much after 6-h incubation in murine plasma. [125I]IB-EG2-ZnDPA could not specifically recognize PS on the cell surface because of its high lipophilicity. Conversely, [99mTc]Tc-MAG3-EG2-ZnDPA accumulated in cancer cells after treatment with an anticancer drug both in vitro and in vivo, and its accumulation was correlated with the number of TUNEL-positive cells. However, the biodistribution of [99mTc]Tc-MAG3-EG2-ZnDPA was not suitable for imaging because of its low accumulation in tumor and high uptake in abdomen organs. CONCLUSION: [99mTc]Tc-MAG3-EG2-ZnDPA could be useful for the early detection of treatment effects after chemotherapy. Since the signal-to-noise ratio is not enough for single photon emission computed tomography imaging, further modification is needed to improve its biodistribution and affinity for PS.

Fundamental study of radiogallium-labeled aspartic acid peptides introducing octreotate derivatives.

OBJECTIVE: Somatostatin receptors are highly expressed in neuroendocrine tumors, and many radiolabeled somatostatin analogs for diagnosis and treatment have been developed. To simultaneously detect not only primary cancer but also bone metastases, this study aimed to develop a positron emission tomography probe using generator-produced nuclide Gallium-68 (T1/2 = 68 min), in which a carrier for primary cancer, a carrier for bone metastases lesions, and a stable gallium complex are introduced into the one molecule. Based on this strategy, the somatostatin receptor-targeted peptide, [Tyr3]-octreotate (TATE), aspartic acid peptide (Dn) with high binding affinity for hydroxyapatite, and Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a stable gallium complex were selected. The novel complexes, Ga-DOTA-Dn-TATE (n = 0, 2, 5, 8, or 11), were designed, synthesized, and evaluated. The radiogallium complexes were prepared using the easy-to-handle radioisotope 67Ga due to relatively long half-life. METHODS: The radiogallium complex precursor DOTA-Dn-TATE was synthesized by the Fmoc-based solid-phase method and by the air oxidation method to form the disulfide bond. [67Ga]Ga-DOTA-Dn-TATE was synthesized by reacting DOTA-Dn-TATE and 67Ga. Hydroxyapatite binding assays, in vitro cellular uptake experiments in AR42J tumor cells, in biodistribution experiments in AR42J tumor-bearing mice, were performed using [67Ga]Ga-DOTA-Dn-TATE. RESULTS: The radiochemical purities of [67Ga]Ga-DOTA-Dn-TATE were > 96.0%. In in vitro and in vivo experiments, [67Ga]Ga-DOTA-D11-TATE had a high affinity for hydroxyapatite and highly accumulated in bone. However, the uptake of [67Ga]Ga-DOTA-D11-TATE into somatostatin receptor-positive AR42J cells was lower than that of [67Ga]Ga-DOTA-TATE, and the accumulation of [67Ga]Ga-DOTA-D11-TATE in tumor was significantly low. CONCLUSION: Ga-DOTA-D11-TATE may not be recognized by somatostatin receptor by the introduction of D11, and the charge adjustment may be important for somatostatin receptor-positive cell uptake.

Design, synthesis, and biological evaluation of radioiodinated benzo[d]imidazole-quinoline derivatives for platelet-derived growth factor receptor ß (PDGFRß) imaging.

Several malignant tumors and fibrotic diseases are associated with PDGFRß overexpression and excessive signaling, making this receptor attractive for molecular targeting and imaging approaches. A series of benzo[d]imidazole-quinoline derivatives were designed and synthesized to develop radioiodinated compounds as PDGFRß-specific imaging probes. The structure activity relationship (SAR) evaluation of the designed compounds was performed. Among them, 2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (5a) and 4-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (5d) exhibited a relatively high PDGFRß-TK inhibitory potency, whereas iodinated 5a derivative 5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]-8-(piperazin-1-yl)quinoline (8) exhibited a superior inhibitory potency as PDGFRß inhibitor than iodinated 5d derivative 4-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}morpholine (11). Furthermore, [125I]8 and [125I]11 were synthesized and evaluated for PDGFRß radioligand ability, both in vitro and in vivo. Cellular uptake experiments showed that [125I]8 had a higher uptake in BxPC3-luc cells as PDGFRß-positive cells than [125I]11. Incubation of [125I]8 after pretreatment of PDGFRß ligands significantly reduced the uptake of [125I]8. In biodistribution experiments using tumor-bearing mice, [125I]8 accumulation in the tumor 1 h postinjection was higher than that of the benzo[d]imidazol-quinoline derivative [125I]IIQP, used in our previous research. These results indicate that [125I]8 could be a promising PDGFRß imaging agent. Although its clinical application requires further structural modifications, the results obtained in this research may be useful for the development of PDGFRß-specific radioligands.

Radiobrominated benzimidazole-quinoline derivatives as Platelet-derived growth factor receptor beta (PDGFRß) imaging probes.

Platelet-derived growth factor receptor beta (PDGFRß) affects in numerous human cancers and has been recognized as a promising molecular target for cancer therapies. The overexpression of PDGFRß could be a biomarker for cancer diagnosis. Radiolabeled ligands having high affinity for the molecular target could be useful tools for the imaging of overexpressed receptors in tumors. In this study, we aimed to develop radiobrominated PDGFRß ligands and evaluate their effectiveness as PDGFRß imaging probes. The radiolabeled ligands were designed by modification of 1-{2-[5-(2-methoxyethoxy)-1H- benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine (1), which shows selective inhibition profile toward PDGFRß. The bromine atom was introduced directly into C-5 of the quinoline group of 1, or indirectly by the conjugation of 1 with the 3-bromo benzoyl group. [77Br]1-{5-Bromo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinoline-8-yl}piperidin-4-amine ([77Br]2) and [77Br]-N-3-bromobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}-piperidin-4-amine ([77Br]3) were prepared using a bromodestannylation reaction. In a cellular uptake study, [77Br]2 and [77Br]3 more highly accumulatd in BxPC3-luc cells (PDGFRß-positive) than in MCF7 cells (PDGFRß-negative), and their accumulation was significantly reduced by pretreatment with inhibitors. In biodistribution experiments, [77Br]2 accumulation was higher than [77Br]3 accumulation at 1 h postinjection. These findings suggest that [76Br]2 is more promising for positron emission tomography (PET) imaging of PDGFRß than [76Br]3.

Comparison of Radioiodine- or Radiobromine-Labeled RGD Peptides between Direct and Indirect Labeling Methods.

Radiolabeled cyclic peptides containing the (Arg-Gly-Asp) RGD sequence for use in positron emission tomography (PET) imaging, single-photon emission computed tomography (SPECT) imaging, and targeted radionuclide therapy of cancer have been reported. In this study, RGD was used as a model carrier peptide for diagnosis and therapy of cancer. To evaluate the characteristics of radiohalogen-labeled peptides, several kinds of labeled RGD peptides [125I-c(RGDyK), 77Br-c(RGDyK), [125I]SIB-c(RGDfK), [77Br]SBrB-c(RGDfK), [125I]SIB-EG2-c(RGDfK), and [77Br]SBrB-EG2-c(RGDfK)] were designed, prepared, and evaluated. In these initial studies, 77Br (t1/2=57.0 h) and 125I (t1/2=59.4 d) were used because of their longer half-lives. Precursor peptides were synthesized using a standard 9-fluorenylmethyloxycarbonyl (Fmoc)-based solid-phase methodology. Radiolabeled peptides were prepared by chloramine-T method or conjugation of RGD peptides with [125I]N-succinimidyl 3-iodobenzoate ([125I]SIB) or [77Br]N-succinimidyl 3-bromobenzoate ([77Br]SBrB). Measurement of the partition coefficients, integrin binding assay, and biodistribution experiments in tumor-bearing mice were performed. 125I and 77Br labeling were successfully performed using similar methods, and in vitro characteristics and biodistributions were similar between the 125I-labeled and corresponding 77Br-labeled peptides. [125I]SIB- and [77Br]SBrB-conjugated RGD peptides showed higher partition coefficients, lower tumor uptakes, and higher intestinal uptake than 125I-c(RGDyK) and 77Br-c(RGDyK). [125I]SIB-EG2-c(RGDfK) and [77Br]SBrB-EG2-c(RGDfK), which possess an ethylene glycol linker, decreased lipophilicity and uptake in intestine compared with [125I]SIB-c(RGDfK) and [77Br]SBrB-c(RGDfK), which possess no linker. However, the improvement in biodistribution of [125I]SIB-EG2-c(RGDfK) and [77Br]SBrB-EG2-c(RGDfK)] was insufficient. In conclusion, directly radiohalogenated c(RGDyK) peptides are potentially more useful for tumor imaging and therapy than indirectly radiohalogenated ones.

Development of a novel radiobromine-labeled sigma-1 receptor imaging probe.

INTRODUCTION: Sigma-1 receptor is a target for tumor imaging. In a previous study, we synthesized a vesamicol analog, (+)-2-[4-(4-bromophenyl)piperidino]cyclohexanol [(+)-pBrV], with a high affinity for sigma-1 receptor, and synthesized radiobrominated (+)-pBrV. This radiobrominated (+)-pBrV showed high tumor uptake in tumor-bearing mice; however, radioactivity accumulation in normal tissues, such as the liver, was high. We assumed that the accumulation of (+)-pBrV in the non-target tissues was partially derived from its high lipophilicity; therefore, we synthesized and evaluated (+)-4-[1-(2-hydroxycyclohexyl)piperidine-4-yl]-2-bromophenol [(+)-BrV-OH], which is a more hydrophilic compound. Although we aimed to develop a PET tracer using 76Br, in these initial studies, we used 77Br because of its longer half-life. METHODS: (+)-[77Br]BrV-OH was synthesized using the chloramine-T method with a radiochemical purity of 95%. Lipophilicity and affinity for sigma-1 receptor of (+)-[77Br]BrV-OH were determined, and biodistribution experiments were performed. We also performed an in vivo blocking study by co-injecting excess amounts of the sigma-1 receptor ligand, SA4503, into mice. RESULTS: The lipophilicity and affinity for sigma-1 receptor of (+)-[77Br]BrV-OH were lower than those of (+)-[77Br]pBrV. (+)-[77Br]BrV-OH also showed high tumor uptake in biodistribution experiments in DU-145 tumor-bearing mice,. Although (+)-[77Br]pBrV was retained in most tissues, (+)-[77Br]BrV-OH was cleared from these tissues. In blocking studies, the co-injection of SA4503 significantly decreased the tumor uptake of (+)-[77Br]BrV-OH. CONCLUSION: These results indicate that (+)-[76Br]BrV-OH has potential as a PET probe for sigma-1 receptor imaging.

Asymmetric structure of cis-[N-(9-anthracenylmethyl)-1,2-ethanediamine]dipyridineplatinum(II) dinitrate.

Platinum antitumour agents, containing aromatic rings, which are used for targeting DNA in effective therapies for the treatment of cancer. We have synthesized the title metallocomplex with an aromatic ligand and determined its crystal structure. In many cases, complexes of platinum and other metals have a symmetrical structure. In contrast, the platinum(II) complex with pyridine and N-(9-anthracenylmethyl)-1,2-ethanediamine as ligands (systematic name: cis-{N-[(anthracen-9-yl)methyl]ethane-1,2-diamine-κ2N,N'}bis(pyridine-κN)platinum(II) dinitrate), [Pt(C5H5N)2(C17H18N2)](NO3)2, is asymmetric. Of the two pyridine ligands, only one is π-stacked with anthracene, resulting in an asymmetric structure. Moreover, the angle of orientation of each pyridine ligand is variable. Further examination of the packing motif confirms an intermolecular edge-to-face interaction.

A novel anionic-phosphate-platinum complex effectively targets an undifferentiated pleomorphic sarcoma better than cisplatinum and doxorubicin in a patient-derived orthotopic xenograft (PDOX).

A patient high-grade undifferentiated pleomorphic soft-tissue sarcoma (UPS) from a striated muscle was previously orthotopically implanted in the right biceps femoris muscle of nude mice to establish a patient-derived orthotopic xenograft (PDOX) nude-mouse model. In the present study, two weeks after orthotopic transplantation of the UPS, mice were treated intraperitoneally with cisplatinum (CDDP), doxorubicin (DOX) or a novel anionic-phosphate-platinum compound 3Pt. Treatments were repeated weekly for a total of 3 times. Six weeks after transplantation, all mice were sacrificed and evaluated. After two weeks treatment, tumor sizes were as follows: control (G1): 2208.3 mm3; CDDP (G2): 841.8±3 mm3, p=0.0001; DOX (G3): 693.1±3 mm3, p=6.56E-7; 3Pt (G4): 333.7±1 mm3, p=4.8E-8. 3Pt showed significantly more efficacy compared to other therapy drugs tested: CDDP (p=0.0002), DOX (p=0.001). There were no animal deaths in any of the four groups. The present results suggest 3Pt is a promising new candidate for UPS since it was demonstrated to be effective in a PDOX model.

Synthesis and evaluation of radioiodinated 1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}piperidin-4-amine derivatives for platelet-derived growth factor receptor ß (PDGFRß) imaging.

Platelet-derived growth factor receptor ß (PDGFRß) is a transmembrane tyrosine kinase receptor and it is upregulated in various malignant tumors. Radiolabeled PDGFRß inhibitors can be a convenient tool for the imaging of tumors overexpressing PDGFRß. In this study, [125I]-1-{5-iodo-2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinoline-8-yl}piperidin-4-amine ([125I]IIQP) and [125I]-N-3-iodobenzoyl-1-{2-[5-(2-methoxyethoxy)-1H-benzo[d]imidazol-1-yl]quinolin-8-yl}-piperidin-4-amine ([125I]IB-IQP) were designed and synthesized, and their potential as PDGFRß imaging agents was evaluated. In cellular uptake experiments, [125I]IIQP and [125I]IB-IQP showed higher uptake by PDGFRß-positive cells than by PDGFRß-negative cells, and the uptake in PDGFRß-positive cells was inhibited by co-culture with PDGFRß ligands. The biodistribution of both radiotracers in normal mice exhibited hepatobiliary excretion as the main route. In mice inoculated with BxPC3-luc (PDGFRß-positive), the tumor uptake of radioactivity at 1h after the injection of [125I]IIQP was significantly higher than that after the injection of [125I]IB-IQP. These results indicated that [125I]IIQP can be a suitable PDGFRß imaging agent. However, further modification of its structure will be required to obtain a more appropriate PDGFRß-targeted imaging agent with a higher signal/noise ratio.

Water-soluble metalloporphyrinates with excellent photo-induced anticancer activity resulting from high tumor accumulation.

To develop a water-soluble and tumor-targeted photosensitizer for photodynamic therapy (PDT), a porphyrin framework containing the metal ion gallium(III) was combined with platinum(II)-based groups to produce two new pentacationic metalloporphyrinates, Ga-4cisPtTPyP (5,10,15,20-tetrakis{cis-diammine-chloro-platinum(II)}(4-pyridyl)-porphyrinato gallium(III) hydroxide tetranitrate) and Ga-4transPtTPyP (5,10,15,20-tetrakis{trans-diammine-chloro-platinum(II)} (4-pyridyl)-porphyrinato gallium(III) hydroxide tetranitrate). Both complexes exhibited high singlet oxygen quantum yields (Φ∆) and remarkable photocytotoxicity with appreciable phototoxic indexes (PIs). In particular, Ga-4cisPtTPyP showed a low IC50 value (Colon 26: 0.12µM; Sarcoma 180: 0.08µM) under illumination and its PI up to 1000. With outstanding tumor accumulation (tumor/muscle ratio>9), Ga-4cisPtTPyP almost completely inhibited tumor growth over two weeks in an in vivo PDT assay. These results imply that Ga-4cisPtTPyP could be a promising anticancer agent for use in PDT.

Preparation and evaluation of an astatine-211-labeled sigma receptor ligand for alpha radionuclide therapy.

INTRODUCTION: Sigma receptors are overexpressed in a variety of human tumors, making them potential targets for radionuclide receptor therapy. We have previously synthesized and evaluated (131)I-labeled (+)-2-[4-(4-iodophenyl)piperidino]cyclohexanol [(+)-[(131)I]pIV], which has a high affinity for sigma receptors. Therefore, (+)-[(131)I]pIV significantly inhibited tumor cell proliferation in tumor-bearing mice. In the present study, we report the synthesis and the in vitro and in vivo characterization of (+)-[(211)At]pAtV, an (211)At-labeled sigma receptor ligand, that has potential use in alpha-radionuclide receptor therapy. METHODS: The radiolabeled sigma receptor ligand (+)-[(211)At]pAtV was prepared using a standard halogenation reaction generating a 91% radiochemical yield with 98% purity after HPLC purification. The partition coefficient of (+)-[(211)At]pAtV was measured. Cellular uptake experiments and in vivo biodistribution experiments were performed using a mixed solution of (+)-[(211)At]pAtV and (+)-[(125)I]pIV; the human prostate cancer cell line DU-145, which expresses high levels of the sigma receptors, and DU-145 tumor-bearing mice. RESULTS: The lipophilicity of (+)-[(211)At]pAtV was similar to that of (+)-[(125)I]pIV. DU-145 cellular uptake and the biodistribution patterns in DU-145 tumor-bearing mice at 1h post-injection were also similar between (+)-[(211)At]pAtV and (+)-[(125)I]pIV. Namely, (+)-[(211)At]pAtV demonstrated high uptake and retention in tumor via binding to sigma receptors. CONCLUSION: These results indicate that (+)-[(211)At]pAtV could function as an new agent for alpha-radionuclide receptor therapy.

Radiogallium Complex-Conjugated Bifunctional Peptides for Detecting Primary Cancer and Bone Metastases Simultaneously.

(68)Ga (T(1/2) = 68 min, a generator-produced nuclide) is an interesting radionuclide for clinical positron emission tomography (PET). Recently, it was reported that radiogallium-labeled 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-conjugated (Asp)n peptide [Ga-DOTA-(Asp)n] has great potential for bone metastases imaging. In the current study, a compound containing an aspartic acid peptide linker (D11) as a carrier to bone metastases, an RGD peptide [c(RGDfK) peptide] as a carrier to the primary cancer, and Ga-DOTA as a stable radiometal complex for imaging in one molecule, Ga-DOTA-D11-c(RGDfK), was designed, prepared, and evaluated to detect both the primary cancer and bone metastases simultaneously using (67)Ga, which is easy to handle. After DOTA-D11-c(RGDfK) was synthesized using Fmoc-based solid-phase methodology, (67)Ga-DOTA-D11-c(RGDfK) was prepared by complexing DOTA-D11-c(RGDfK) with (67)Ga. Hydroxyapatite binding assays, integrin binding assays, biodistribution experiments, and single photon emission tomography (SPECT) imaging using tumor-bearing mice were performed using (67)Ga-DOTA-D11-c(RGDfK). (67)Ga-DOTA-D11-c(RGDfK) was prepared with a radiochemical purity of >97%. In vitro, (67)Ga-DOTA-D11-c(RGDfK) had a high affinity for hydroxyapatite and αvß3 integrin. In vivo, (67)Ga-DOTA-D11-c(RGDfK) exhibited high uptake in bone and tumor. The accumulation of (67)Ga-DOTA-D11-c(RGDfK) in tumor decreased when it was co-injected with c(RGDfK) peptide. (68)Ga-DOTA-D11-c(RGDfK) has great potential as a PET tracer for the diagnosis of both the primary cancer and bone metastases simultaneously.

Effectiveness of two novel anionic and cationic platinum complexes in the treatment of osteosarcoma.

AIM: This study aimed to characterize the cellular basis of the platinum cytotoxicity of two novel platinum complexes, 3Pt and 1Pt, in comparison with that of cisplatin. 3Pt comprises anionic phosphate moieties, while 1Pt comprises neutral aromatic ligands. METHODS: We compared the cytotoxic potency of 3Pt and 1Pt with that of cisplatin in osteosarcoma cell lines and an orthotopic mouse model. RESULTS: The cytotoxic potency of 3Pt was markedly higher than that of cisplatin in all cell lines. Both novel platinum complexes showed a complete lack of cross resistance in cisplatin-resistant cells. Caffeine enhanced the cytotoxic potency of these novel platinum complexes, as observed for cisplatin. Apoptosis after drug administration was observed by DNA ladder formation and an annexin V/PI assay. DNA double-strand breaks were confirmed by phosphorylation of histone H2AX. In vivo, the antitumor activity of 3Pt and 1Pt was superior and similar, respectively, to that of cisplatin. Both novel platinum complexes exerted strong antitumor effects on osteosarcoma in vitro and in vivo. CONCLUSIONS: 3Pt may be an effective drug for the treatment of bone cancer because the PO3 moiety has a high affinity to bone, as exhibited by bisphosphonates, and is expected to decrease the incidence of side effects at extraskeletal sites and overcome drug resistance. Cationic 1Pt may also be an effective antitumor drug because of its unique chemical structure and properties. Further investigations to detail the antitumor effects of these ionic Pt complexes on osteosarcoma are warranted.

[Development of new peptide-based chelating agents for site-specific radiolabeling with 64Cu].

Radiolabeled monoclonal antibodies (mAbs) are very useful molecular probes for nuclear imaging technique due to their high-target specificity and high-stability in the bloodstream. MAbs are generally labeled by indirect method using the combination of relatively long-lived metallic radionuclides (including (64)Cu, (89)Zr, and (111)In) and bifunctional chelating agents which are capable of binding both antibodies and radio metals. The indirect radiolabeling method has some advantages such as high labeling efficiency and long-term retention ability within their target cells. However, this conventional labeling method can potentially lead to low-target affinity of the mAb probes, because of the non-site-specific introduction of the bifunctional chelators into the active site of the mAbs. To overcome the shortcoming, we proposed a new direct labeling method utilizing fusion proteins comprising mAbs linked to metal binding peptides at the N- or C-terminus. In this study, we synthesized new peptide derivatives possessing an N-terminal tripeptide sequence (Xaa-Yaa-His) called the amino terminal Cu(2+)- and Ni(2+)-binding (ATCUN) motif as (64)Cu binding peptides for the proposed labeling method. Moreover, we studied the stability constants of Cu(2+)-ATCUN peptide complexes by pH titration. From these studies, we found that a low basicity of the N-terminal amine in the peptide resulted in a high stability constant of the complex. This finding may provide valuable guidelines in designing the ATCUN peptide with high-binding affinity toward (64)Cu.

Development and evaluation of a novel (99m)tc-labeled annexin A5 for early detection of response to chemotherapy.

(99m)Tc-HYNIC-annexin A5 can be considered as a benchmark in the field of apoptosis imaging. However, (99m)Tc-HYNIC-annexin A5 has characteristics of high uptake and long retention in non-target tissues such as kidney and liver. To minimize this problem, we developed a novel (99m)Tc-labeled annexin A5 using a bis(hydroxamamide) derivative [C3(BHam)2] as a bifunctional chelating agent, and evaluated its usefulness as an imaging agent for detecting apoptosis. The amino group of C3(BHam)2 was converted to a maleimide group, and was coupled to thiol groups of annexin A5 pretreated with 2-iminothiolane. (99m)Tc labeling was performed by a ligand exchange reaction with (99m)Tc-glucoheptonate. Biodistribution experiments for both (99m)Tc-C3(BHam)2-annexin A5 and (99m)Tc-HYNIC-annexin A5 were performed in normal mice. In addition, in tumor-bearing mice, the relationship between the therapeutic effects of chemotherapy (5-FU) and the tumor accumulation of (99m)Tc-C3(BHam)2-annexin A5 just after the first treatment of 5-FU was evaluated. (99m)Tc-C3(BHam)2-annexin A5 was prepared with a radiochemical purity of over 95%. In biodistribution experiments, (99m)Tc-C3(BHam)2-annexin A5 had a much lower kidney accumulation of radioactivity than (99m)Tc-HYNIC-annexin A5. In the organs for metabolism, such as liver and kidney, radioactivity after the injection of (99m)Tc-HYNIC-annexin A5 was residual for a long time. On the other hand, radioactivity after the injection of (99m)Tc-C3(BHam)2-annexin A5 gradually decreased. In therapeutic experiments, tumor growth in the mice treated with 5-FU was significantly inhibited. Accumulation of (99m)Tc-C3(BHam)2-annexin A5 in tumors significantly increased after 5-FU treatment. The accumulation of radioactivity in tumor correlated positively with the counts of TUNEL-positive cells. These findings suggest that (99m)Tc-C3(BHam)2-annexin A5 may contribute to the efficient detection of apoptotic tumor response after chemotherapy.