Hiromichi Akizawa

In vitro and in vivo evaluations of a radioiodinated thymidine phosphorylase inhibitor as a tumor diagnostic agent for angiogenic enzyme imaging

INTRODUCTION The expression of thymidine phosphorylase (TP) is closely associated with angiogenesis, tumor invasiveness and activation of antitumor agents. We evaluated radioiodinated 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil ([(125)I]IIMU) having high TP-inhibitory potency as the new radiotracer for SPECT targeting of TP expression in tumors. METHODS The characteristics of the radioiodinated TP inhibitor IIMU were determined by evaluating the uptake by tumor cells in vitro and by biodistribution studies in vivo. The distribution of the radiotracer and the extent of TP-specific uptake by tumors were evaluated by a counting method in tumor-bearing mice. RESULTS The in vitro uptake of radiolabeled IIMU by A431 cells along with high TP expressions was attributed to the binding of the radiotracer to its target enzyme, i.e., TP. In vivo distribution of the radiotracer in A431 tumor-bearing mice revealed tumor/blood and tumor/muscle activity uptake ratios of 36 and 106, respectively, at 3 h after the radiotracer injection. On using low TP-expressing tumors and TP blocking studies as controls, minor TP-specific accumulation of the radiotracer was detected in these studies. CONCLUSION According to the binding of radioiodinated IIMU to the angiogenic enzyme TP, it can be concluded that radioiodinated IIMU might be suitable as a SPECT tracer for tumor imaging.

Design, synthesis and biological evaluation of negatively charged 111In-DTPA-octreotide derivatives

Our previous studies indicated that (111)In-diethylenetriaminepentaacetic acid ((111)In-DTPA)-octreotide derivatives with an additional negative charge by replacing N-terminal d-phenylalanine (d-Phe) with an acidic amino acid such as l-aspartic acid (Asp) or its derivative exhibited low renal radioactivity levels when compared with (111)In-DTPA-D-Phe(1)-octreotide. On the basis of the findings, we designed, synthesized and evaluated two Asp-modified (111)In-DTPA-conjugated octreotide derivatives, (111)In-DTPA-Asp(1)-octreotide and (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide. While (111)In-DTPA-Asp(1)-octreotide showed negligible AR42J cell uptake, (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide exhibited AR42J cell uptake similar to that of (111)In-DTPA-D-Phe(1)-octreotide. When administered to AR42J tumor-bearing mice, (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide exhibited renal radioactivity levels significantly lower than did (111)In-DTPA-D-Phe(1)-octreotide at 1 and 3 h post-injection. No significant differences were observed in tumor accumulation between (111)In-DTPA-Asp(0)-D-Phe(1)-octreotide and (111)In-DTPA-D-Phe(1)-octreotide after 1 and 3h injection. The findings in this study suggested that an interposition of an Asp at an appropriate position in (111)In-DTPA-D-Phe(1)-octreotide would constitute a useful strategy to develop (111)In-DTPA-D-Phe(1)-octreotide derivatives of low renal radioactivity levels while preserving tumor accumulation.

The Thymidine Phosphorylase Imaging Agent 123I-IIMU Predicts the Efficacy of Capecitabine

Recently, companion diagnostics with nuclear medicine techniques have been anticipated as more suitable means than biopsy for predicting treatment efficacy. The anticancer effect of capecitabine, an orally administered chemotherapeutic agent activated by thymidine phosphorylase (TP), is positively associated with tumor TP expression levels. This study aimed to assess whether TP imaging using a radiolabeled uracil derivative, 123I-5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (123I-IIMU), could predict the efficacy of capecitabine treatment. Methods: Sensitivity to doxifluridine, a metabolite of capecitabine and direct substrate for TP, was assessed by water-soluble tetrazolium salt assays in vitro for 3 human colon cancer cell lines with different TP expression profiles. The intracellular uptake and retention of 123I-IIMU were evaluated. Mice inoculated with each cell line were treated with capecitabine for 2 wk, and tumor growth was compared. In vivo distribution studies and SPECT/CT imaging of 123I-IIMU were performed in inoculated mice. Results: In vitro experiments showed a positive relation between TP expression levels and doxifluridine sensitivity. In vitro studies revealed that intracellular uptake and retention of 123I-IIMU were dependent on TP expression levels. In vivo experiments in inoculated mice showed that 123I-IIMU accumulation in tumor tissue was in line with TP expression levels and susceptibility to capecitabine treatment. Moreover, SPECT/CT imaging of 123I-IIMU in tumor-inoculated mice showed that 123I-IIMU reflects TP expression levels in tumor tissues. Conclusion: 123I-IIMU could be used as an in vivo companion diagnostic for predicting the efficacy of capecitabine treatment.

Radiolabeled uracil derivative as a novel SPECT probe for thymidine phosphorylase : suppressed accumulation into tumor cells by target gene knockdown

ObjectivesWe have developed a radiolabeled uracil derivative, 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil (IIMU) as a novel single photon emission computed tomography probe for thymidine phosphorylase (TP). This radioiodinated IIMU has a high affinity for TP and highly accumulates in the TP-expressing tumor cell line A431 (human epidermoid carcinoma). To evaluate the specificity of the cellular uptake of IIMU to TP expression, we examined the effects of TP knockdown on the uptake of 125I-labeled IIMU (125I-IIMU) in the tumor cells. MethodsTP-specific small interfering RNA (siRNA), glyceraldehyde-3-phosphate dehydrogenase (GAPDH)-specific siRNA (positive control), and negative control siRNA were transfected into A431 cells, respectively. Target-mRNA and protein expression levels of TP and GAPDH were examined 48 and 72 h after transfection, respectively. The cellular uptake level of 125I-IIMU was also evaluated 72 h after transfection. The results were compared after normalization with the corresponding negative controls. ResultsAfter TP-specific and GAPDH-specific siRNA transfection, the expression levels of TP and GAPDH mRNA decreased significantly to 41 and 29%, respectively, compared with the negative control (P<0.001 for both). The expression levels of TP and GAPDH protein also significantly decreased to 34 and 30%, respectively (P<0.001 for both). After TP-specific siRNA transfection, the cellular uptake level of 125I-IIMU decreased significantly to 66% (P<0.001). In contrast, GAPDH siRNA transfection did not significantly affect the cellular uptake level of 125I-IIMU. ConclusionsiRNA-mediated TP knockdown significantly decreased the cellular uptake level of 125I-IIMU. This finding indicates that the uptake of IIMU in tumor cells is TP specific and directly corresponds to TP expression levels.

New 〔11C〕phosgene based synthesis of 〔11C〕pyrimidines for positron emission tomography

Thymine, 5-FU, and uracil were successfully synthesized through a procedure involving a cyclocondensation of triphosgene with newly developed α-substituted β-aminoacrylamides intermediates (la, X= Me; 1b, X= F; 1c, X= H). The radioligands [2- 11 C]thymine and [2- 11 C]5-fluorouracil were synthesized in high radiochemical yields in 16-17 minutes from the end of bombardment by applying the cyclocondensation method with [ 11 C]COCl 2 .

Redesign of negatively charged 111In-DTPA-octreotide derivative to reduce renal radioactivity ☆

INTRODUCTION Radiolabeled octreotide derivatives have been studied as diagnostic and therapeutic agents for somatostatin receptor-positive tumors. To prevent unnecessary radiation exposure during their clinical application, the present study aimed to develop radiolabeled peptides which could reduce radioactivity levels in the kidney at both early and late post-injection time points by introducing a negative charge with an acidic amino acid such as L-aspartic acid (Asp) at a suitable position in 111In-DTPA-conjugated octreotide derivatives. METHODS Biodistribution of the radioactivity was evaluated in normal mice after administration of a novel radiolabeled peptide by a counting method. The radiolabeled species remaining in the kidney were identified by comparing their HPLC data with those obtained by alternative synthesis. RESULTS The designed and synthesized radiolabeled peptide 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide exhibited significantly lower renal radioactivity levels than those of the known 111In-DTPA-d-Phe1-octreotide at 3 and 24h post-injection. The radiolabeled species in the kidney at 24h after the injection of new octreotide derivative represented 111In-DTPA-d-Phe-OH and 111In-DTPA-d-Phe-Asp-OH as the metabolites. Their radiometabolites and intact 111In-DTPA-conjugated octreotide derivative were observed in urine within 24h post-injection. CONCLUSION The present study provided a new example of an 111In-DTPA-conjugated octreotide derivative having the characteristics of both reduced renal uptake and shortened residence time of radioactivity in the kidney. It is considered that this kinetic control was achieved by introducing a negative charge on the octreotide derivative thereby suppressing the reabsorption in the renal tubules and affording the radiometabolites with appropriate lipophilicity.

Versatile Photorearrangement of Photocycloadducts from 5-Fluoro-1,3-dimethyluracil and Naphthalene

__ Direct UV-irradiation of 5-fluoro-1,3-dimethyluracil (5-FDMU) and naphthalene (1) with a 500 W high-pressure mercury lamp in a degassed Pyrex tube (> 300 nm) predominantly afforded benzopyrimidobarrelene derivative (2) through 1,4-addition, while irradiation in the presence of piperylene in singlet excited states preferentially afforded naphthocyclobutapyrimidine derivative (3) via 1,2-addition. Upon 254 nm light-irradiation of 2 gave rise to the formation of benzopyrimidosemibullvalene (4) in fair yields. The reaction pathway for the formation of 4 is reasonably explained in the terms of di--methane rearrangement. Adduct 3 was newly converted to the corresponding barrelene derivative (2) by long-wave-length irradiation in the presence of a triplet sensitizer. INTRODUCTION In recent years, significant attention has been paid to the photocycloaddition of naphthalenes with alkenes as a useful procedure for constructing certain unique ring systems by way of 1,2-, 1,2 1,4-, 1,2 1,3-, 3 1,8-, 4 and [4 + 4] additions.1,5 We previously reported that UV-irradiation of 5-fluoro-1,3-dimethyluracil (5-FDMU) and naphthalene (1) with a 500 W high-pressure mercury lamp in a degassed Pyrex tube (> 300 nm) predominantly afforded the 1,4-adduct, namely benzopyrimidobarrelene derivative (2) in high yield.6 A time course study of the photoreaction of an acetonirile solution of 5-FDMU and 1 under the same conditions showed that the 1,2-cycloadduct, naphthocyclobutapyrimidine derivative (3) is formed as the major product at the initial stage, which however, is quite labile to the UV-light used, to revert rapidly back to 5-FDMU and 1, while the 1,4-adduct (2) was produced competitively with 3, though less effectively but insensitive to the light, to accumulate in the reaction mixture as the irradiation time is prolonged.7 The addition of the triplet quencher piperylene to the reaction mixture of 1 and 5-FDMU dramatically changed the mode of cycloaddition, resulting in the preferential formation of 3 through 1,2-addition.8 Thus, 1,4and 1,2adducts of the pyrimidine ring and naphthalene have been synthesized mode-selectively in fair yields upon long-wave-length irradiation by a high-pressure mercury lamp with a Pyrex filter (> 300 nm). Furthermore, we have obtained 1,3-cycloadducts, semibullvalene derivatives (4), by the UV-irradiation with a low-pressure mercury lamp (= 254 nm). The semibullvalenes were found to be secondary products derived from initially formed 1,4-adducts under UV-irradiation, although the precise reaction mechanism remained unclear. 9 Thus, we have hitherto demonstrated a mode selective synthesis of three types of cycloadducts, 1,2-, 1,3-, albeit formally, and 1,4-adducts, by controlling the reaction conditions appropriately. Although versatile photorearrangements of 1,4-adducts and 1,2-adducts of aromatic hydrocarbons with alkenes have extensively been explored,10-12 little is known about the photochemical behavior of heteroaromatic-fused barrelenes except pyrazinobarrelenes and benzoquinoxalinobarrelenes.13 Hence, our attention was focused to elucidate an aspect of the reciprocal valence isomerization of the adducts of 5-FDMU with naphthalene. In the present paper we describe the feature of the mutual conversion of the cycloadducts between 5-FDMU and 1. RESULTS AND DISCUSSION Photoisomerization of benzopyrimidobarrelene (2) First, we examined photochemical behavior of 1,4-adduct (2). Upon UV-irradiation with a high-pressure

Intracellular reactions affecting 2-amino-4-([11C]methylthio)butyric acid ([11C]methionine) response to carbon ion radiotherapy in C10 glioma cells

PURPOSE The response of 2-amino-4-([(14)C]methylthio)butyric acid ([(14)C]Met) uptake and [(125)I]3-iodo-alpha-methyl-l-tyrosine ([(125)I]IMT) uptake to radiotherapy of C10 glioma cells was compared to elucidate the intracellular reactions that affect the response of 2-amino-4-([(11)C]methylthio)butyric acid ([(11)C]Met) uptake to radiotherapy. METHODS After irradiation of cultured (3 Gy) or xenografted C10 glioma cells (25 Gy) using a carbon ion beam, the accumulation of [(14)C]Met and [(125)I]IMT in the tumors was investigated. The radiometabolites in xenografted tumors after radiotherapy were analyzed by size-exclusion HPLC. RESULTS [(14)C]Met provided earlier responses to the carbon ion beam irradiation than [(125)I]IMT in both cultured and xenografted tumors. While [(125)I]IMT remained intact in xenografted tumor before and after irradiation, the radioactivity derived from [(14)C]Met was observed both in high molecular fractions and intact fractions, and the former decreased after irradiation. CONCLUSION The earlier response of [(11)C]Met uptake to tumor radiotherapy could be attributable to the decline in the intracellular energy-dependent reactions of tumors due to radiotherapy.

111In-DTPA-d-Phe−1-Asp0-d-Phe1-octreotide exhibits higher tumor accumulation and lower renal radioactivity than 111In-DTPA-d-Phe1-octreotide

INTRODUCTION 111In-DTPA-d-Phe1-octreotide scintigraphy is an important method of detecting neuroendocrine tumors. We previously reported that a new derivative of 111In-DTPA-d-Phe1-octreotide, 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide, accomplished the reduction of prolonged renal accumulation of radioactivity. The aim of this study was to evaluate the tumor accumulation of 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide in vitro and in vivo by comparing it with 111In-DTPA-d-Phe1-octreotide. METHODS The tumor accumulation of this octreotide derivative was determined by measuring its uptake using cultured AR42J cells in vitro and biodistribution studies in vivo. The distribution of the radiotracer and the extent of somatostatin receptor-specific uptake in the tumor were estimated by a counting method using AR42J tumor-bearing mice. The radioactive metabolite species in the tumor and kidney were identified by HPLC analyses at 3 and 24h post-injection of the 111In-DTPA-conjugated peptide. RESULTS In both cases, in vitro and in vivo, the tumor radioactivity levels of 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide were approximately 2-4 times higher than those of 111In-DTPA-d-Phe1-octreotide. On in vitro cellular uptake inhibition and radioreceptor assay, 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide exhibited a binding affinity to somatostatin receptor highly similar to that of 111In-DTPA-d-Phe1-octreotide. As the additional cellular uptake of 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide was significantly lower at low temperature than at 37°C, it was considered that a cellular uptake pathway is involved in energy-dependent endocytotic processes. In the radiometabolite analysis of 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide, 111In-DTPA-d-Phe-Asp-OH was a major metabolite in the tumor at 24h post-injection. CONCLUSION 111In-DTPA-d-Phe-1-Asp0-d-Phe1-octreotide exhibited higher tumor accumulation and persistence of tumor radioactivity than 111In-DTPA-d-Phe1-octreotide. We reasoned that this higher tumor accumulation would not be based on the receptor affinity but on a receptor-mediated endocytotic process involved in temperature-dependent cellular uptake. The present study demonstrated the great potential of the pharmaceutical development of a new radiolabeled peptide with high tumor accumulation and low renal radioactivity by the chemical modification of 111In-DTPA-d-Phe1-octreotide.

Preclinical Evaluation of a Thymidine Phosphorylase Imaging Probe, [123I]IIMU, for Translational Research

The expression of thymidine phosphorylase (TP) is closely associated with angiogenesis, tumor invasiveness, and activation of antitumor agents. We developed a radiolabeled uracil derivative, I-123-labeled 5-iodo-6-[(2-iminoimidazolidinyl)methyl]uracil ([123I]IIMU), as a novel SPECT probe for TP. A clinical study to verify the safety of [123I]IIMU injection was approved by the Institutional Review Board of Hokkaido University Hospital for Clinical Research, and first-in-human (FIH) clinical studies of healthy adults were started.