Kazuma Ogawa

Effect of molecular charges on renal uptake of 111In-DTPA-conjugated peptides

The effect of molecular charges on renal accumulation of 111In-DTPA-labeled low molecular weight (LMW) peptides was investigated using 111In-DTPA-octreotide derivatives as models to design radiolabeled peptides that are taken up less by renal cells. The N-terminal D-phenylalanine (Phe) of 111In-DTPA-D-Phe(1)-octreotide was replaced with L-aspartic acid (Asp), L-lysine (Lys), L-methionine (Met) or L-Phe. Cellulose acetate electrophoresis indicated that both 111In-DTPA-L-Phe(1)-octreotide and 111In-DTPA-L-Met(1)-octreotide showed similar net charges, whereas 111In-DTPA-L-alphaLys(1)-octreotide and 111In-DTPA-L-Asp(1)-octreotide had more positive and negative charges, respectively, at pH values similar to those in blood and glomerular filtrate. When injected into mice, significant differences were observed in the renal radioactivity levels. 111In-DTPA-L-alphaLys(1)-octreotide showed the highest radioactivity levels from 10 min to 6 h postinjection, whereas the lowest radioactivity levels were observed with 111In-DTPA-L-Asp(1)-octreotide at all the postinjection intervals. These findings indicated that the replacement of only one amino acid in 111In-DTPA-D-Phe(1)-octreotide significantly altered net molecular charges of the resulting peptides and that the net charges of the 111In-DTPA-octreotide derivatives significantly affected their renal uptake. Thus, an increase of negative charges in peptide molecules may constitute a strategy for designing 111In-DTPA-conjugated LMW peptides with low renal radioactivity levels.

Molecular imaging of active mutant L858R EGF receptor (EGFR) kinase-expressing nonsmall cell lung carcinomas using PET/CT

The importance of the EGF receptor (EGFR) signaling pathway in the development and progression of nonsmall cell lung carcinomas (NSCLC) is widely recognized. Gene sequencing studies revealed that a majority of tumors responding to EGFR kinase inhibitors harbor activating mutations in the EGFR kinase domain. This underscores the need for novel biomarkers and diagnostic imaging approaches to identify patients who may benefit from particular therapeutic agents and approaches with improved efficacy and safety profiles. To this goal, we developed 4-[(3-iodophenyl)amino]-7-{2-[2-{2-(2-[2-{2-([18F]fluoroethoxy)-ethoxy}-ethoxy]-ethoxy)-ethoxy}-ethoxy]-quinazoline-6-yl-acrylamide ([18F]F-PEG6-IPQA), a radiotracer with increased selectivity and irreversible binding to the active mutant L858R EGFR kinase. We show that PET with [18F]F-PEG6-IPQA in tumor-bearing mice discriminates H3255 NSCLC xenografts expressing L858R mutant EGFR from H441 and PC14 xenografts expressing EGFR or H1975 xenografts with L858R/T790M dual mutation in EGFR kinase domain, which confers resistance to EGFR inhibitors (i.e., gefitinib). The T790M mutation precludes the [18F]F-PEG6-IPQA from irreversible binding to EGFR. These results suggest that PET with [18F]F-PEG6-IPQA could be used for the selection of NSCLC patients for individualized therapy with small molecular inhibitors of EGFR kinase that are currently used in the clinic and have a similar structure (i.e., iressa, gefitinib, and erlotinib).

Plasma protein binding of 99mTc-labeled hydrazino nicotinamide derivatized polypeptides and peptides

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare (99m)Tc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, (99m)Tc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of (111)In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the (99m)Tc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of (99m)Tc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this (99m)Tc-labeling method. In mice, [(99m)Tc](HYNIC-IgG)(tricine)(2) and [(99m)Tc](HYNIC-Fab)(tricine)(2) showed persistent localization of radioactivity in tissues when compared with their (125)I-labeled counterparts. [(99m)Tc](HYNIC-IgG)(tricine)(2) eliminated from the blood at a rate similar to that of (125)I-labeled IgG, while [(99m)Tc](HYNIC-Fab)(tricine)(2) showed significantly slower clearance of the radioactivity than (125)I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [(99m)Tc](HYNIC-IgG)(tricine)(2) in murine plasma. However, [(99m)Tc](HYNIC-Fab)(tricine)(2) and [(99m)Tc](HYNIC-RC160)(tricine)(2) demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [(99m)Tc](HYNIC-RC160)(tricine)(2) was stabilized with nicotinic acid (NIC) to generate [(99m)Tc](HYNIC-RC160)(tricine)(NIC). [(99m)Tc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [(99m)Tc](HYNIC-RC160)(tricine)(2). These findings suggested that one of the tricine coligands in (99m)Tc-HYNIC-labeled (poly)peptides would be replaced with plasma proteins to generate higher molecular weight species that exhibit slow blood clearance. In addition, the molecular sizes of parental peptides played an important role in the progression of the exchange reaction of one of the tricine coligands with plasma proteins.

Development of [90Y]DOTA-conjugated bisphosphonate for treatment of painful bone metastases

INTRODUCTION Based on the concept of bifunctional radiopharmaceuticals, we have previously developed (186)Re-complex-conjugated bisphosphonate analogs for palliation of painful bone metastases and have demonstrated the utility of these compounds. By applying a similar concept, we hypothesized that a bone-specific directed (90)Y-labeled radiopharmaceutical could be developed. METHODS In this study, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as the chelating site, and DOTA was conjugated with 4-amino-1-hydroxybutylidene-1,1-bisphosphonate. [(90)Y]DOTA-complex-conjugated bisphosphonate ([(90)Y]DOTA-HBP) was prepared by coordination with (90)Y, and its biodistribution was studied in comparison to [(90)Y]citrate. RESULTS In biodistribution experiments, [(90)Y]DOTA-HBP and [(90)Y]citrate rapidly accumulated and resided in the bone. Although [(90)Y]citrate showed a higher level of accumulation in the bone than [(90)Y]DOTA-HBP, the clearances of [(90)Y]DOTA-HBP from the blood and from almost all soft tissues were much faster than those of [(90)Y]citrate. As a result, the estimated absorbed dose ratios of soft tissues to osteogenic cells (target organ) of [(90)Y]DOTA-HBP were lower than those of [(90)Y]citrate. CONCLUSIONS [(90)Y]DOTA-HBP showed superior biodistribution characteristics as a bone-seeking agent and led to a decrease in the level of unnecessary radiation compared to [(90)Y]citrate. Since the DOTA ligand forms a stable complex not only with (90)Y but also with lutetium ((177)Lu), indium ((111)In), gallium ((67/68)Ga), gadolinium (Gd) and so on, complexes of DOTA-conjugated bisphosphonate with various metals could be useful as agents for palliation of metastatic bone pain, bone scintigraphy and magnetic resonance imaging.

αvβ3 Integrin-targeting radionuclide therapy and imaging with monomeric RGD peptide

The αvβ3 integrin plays a pivotal role in angiogenesis and tumor metastasis. Angiogenic blood vessels overexpress αvβ3 integrin, as in tumor neovascularization, and αvβ3 integrin expression in other microvascular beds and organs is limited. Therefore, αvβ3 integrin is a suitable receptor for tumor‐targeting imaging and therapy. Recently, tetrameric and dimeric RGD peptides have been developed to enhance specificity to αvβ3 integrin. In comparison to the corresponding monomeric peptide, however, these peptides show high levels of accumulation in kidney and liver. The purpose of this study is to evaluate tumor‐targeting properties and the therapeutic potential of 111In‐ and 90Y‐labeled monomeric RGD peptides in BALB/c nude mice with SKOV‐3 human ovarian carcinoma tumors. DOTA‐c(RGDfK) was labeled with 111In or 90Y and purified by HPLC. A biodistribution study and scintigraphic images revealed the specific uptake to αvβ3 integrin and the rapid clearance from normal tissues. These peptides were renally excreted. At 10 min after injection of tracers, 111In‐DOTA‐c(RGDfK) and 90Y‐DOTA‐c(RGDfK) showed high uptake in tumors (7.3 ± 0.6% ID/g and 4.6 ± 0.8% ID/g, respectively) and gradually decreased over time (2.3 ± 0.4% ID/g and 1.5 ± 0.5% ID/g at 24 hr, respectively). High tumor‐to‐blood and ‐muscle ratios were obtained from these peptides. In radionuclide therapeutic study, multiple‐dose administration of 90Y‐DOTA‐c(RGDfK) (3 × 11.1 MBq) suppressed tumor growth in comparison to the control group and a single‐dose administration (11.1 MBq). Monomeric RGD peptides, 111In‐DOTA‐c(RGDfK) and 90Y‐DOTA‐c(RGDfK), could be promising tracers for αvβ3 integrin‐targeting imaging and radiotherapy.

99mTc-HYNIC-derivatized ternary ligand complexes for 99mTc-labeled polypeptides with low in vivo protein binding

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m ((99m)Tc)-labeled polypeptides with tricine as a coligand. However, (99m)Tc-HYNIC-labeled polypeptides show delayed elimination rates of the radioactivity not only from the blood but also from nontarget tissues such as the liver and kidney. In this study, a preformed chelate of tetrafluorophenol (TFP) active ester of [(99m)Tc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare (99m)Tc-labeled polypeptides with higher stability against exchange reactions with proteins in plasma and lysosomes using the Fab fragment of a monoclonal antibody and galactosyl-neoglycoalbumin (NGA) as model polypeptides. When incubated in plasma, [(99m)Tc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [(99m)Tc](HYNIC-Fab)(tricine)(2.) When injected into mice, [(99m)Tc](HYNIC-NGA)(tricine)(BP) was metabolized to [(99m)Tc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [(99m)Tc](HYNIC-NGA)(tricine)(2.) In addition, [(99m)Tc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [(99m)Tc](HYNIC-NGA)(tricine)(2.) Similar results were observed with (99m)Tc-labeled Fab fragments where [(99m)Tc](HYNIC-Fab)(tricine)(BP) exhibited significantly faster elimination rates of the radioactivity not only from the blood but also from the kidney. These findings indicated that conjugation of [(99m)Tc](HYNIC)(tricine)(BP) ternary ligand complex to polypeptides accelerated elimination rates of the radioactivity from the blood and nontarget tissues due to low binding of the [(99m)Tc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [(99m)Tc](HYNIC)(tricine)(BP) complex attractive as a radiolabeling reagent for targeted imaging.

Bone Target Radiotracers for Palliative Therapy of Bone Metastases

The skeleton is one of the most common organs affected by metastatic cancer, and bone metastases often cause severe pain, which significantly affects quality of life. Internal radiotherapy using specifically localized bone-seeking radiopharmaceuticals has proven to be an effective alternative and shows fewer side effects than those associated with other forms of treatment. In this review article, we highlight not only radiopharmaceuticals, which have been approved for the palliation of bone metastases but also boneseeking radiolabeled compounds under investigation in basic research. Specifically, we review the efficacy and prospects of phosphorus- 32, strontium-89 chloride, samarium-153-EDTMP, rhenium-186/188-HEDP, rhenium-186/188-complex conjugated bisphosphonate compounds, yttrium-90-DOTA conjugated bisphosphonate, rhenium-186/188-DMSA, radium-223 chloride, thorium-227-EDTMP, thorium-227-DOTMP, and lead/bismuth-212-DOTMP.

Development of Novel Radiogallium-Labeled Bone Imaging Agents Using Oligo-Aspartic Acid Peptides as Carriers

68Ga (T 1/2 = 68 min, a generator-produced nuclide) has great potential as a radionuclide for clinical positron emission tomography (PET). Because poly-glutamic and poly-aspartic acids have high affinity for hydroxyapatite, to develop new bone targeting 68Ga-labeled bone imaging agents for PET, we used 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a chelating site and conjugated aspartic acid peptides of varying lengths. Subsequently, we compared Ga complexes, Ga-DOTA-(Asp)n (n = 2, 5, 8, 11, or 14) with easy-to-handle 67Ga, with the previously described 67Ga-DOTA complex conjugated bisphosphonate, 67Ga-DOTA-Bn-SCN-HBP. After synthesizing DOTA-(Asp)n by a Fmoc-based solid-phase method, complexes were formed with 67Ga, resulting in 67Ga-DOTA-(Asp)n with a radiochemical purity of over 95% after HPLC purification. In hydroxyapatite binding assays, the binding rate of 67Ga-DOTA-(Asp)n increased with the increase in the length of the conjugated aspartate peptide. Moreover, in biodistribution experiments, 67Ga-DOTA-(Asp)8, 67Ga-DOTA-(Asp)11, and 67Ga-DOTA-(Asp)14 showed high accumulation in bone (10.5±1.5, 15.1±2.6, and 12.8±1.7% ID/g, respectively) but were barely observed in other tissues at 60 min after injection. Although bone accumulation of 67Ga-DOTA-(Asp)n was lower than that of 67Ga-DOTA-Bn-SCN-HBP, blood clearance of 67Ga-DOTA-(Asp)n was more rapid. Accordingly, the bone/blood ratios of 67Ga-DOTA-(Asp)11 and 67Ga-DOTA-(Asp)14 were comparable with those of 67Ga-DOTA-Bn-SCN-HBP. In conclusion, these data provide useful insights into the drug design of 68Ga-PET tracers for the diagnosis of bone disorders, such as bone metastases.

Preparation and evaluation of a radiogallium complex-conjugated bisphosphonate as a bone scintigraphy agent

INTRODUCTION (68)Ga is a radionuclide of great interest as a positron emitter for positron emission tomography (PET). To develop a new bone-imaging agent with radiogallium, 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) was chosen as a chelating site and Ga-DOTA complex-conjugated bisphosphonate, which has a high affinity for bone, was prepared and evaluated. Although we are interested in developing (68)Ga-labeled bone imaging agents for PET, in these initial studies (67)Ga was used because of its longer half-life. METHODS DOTA-conjugated bisphosphonate (DOTA-Bn-SCN-HBP) was synthesized by conjugation of 2-(4-isothiocyanatebenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid to 4-amino-1-hydroxybutylidene-1,1-bisphosphonate (alendronate). (67)Ga-DOTA-Bn-SCN-HBP was prepared by coordination with (67)Ga, and its in vitro and in vivo evaluations were performed. RESULTS (67)Ga-DOTA-Bn-SCN-HBP was prepared with a radiochemical purity of over 95% without purification. (67)Ga-DOTA-Bn-SCN-HBP had great affinity for hydroxyapatite in binding assay. In biodistribution experiments, (67)Ga-DOTA-Bn-SCN-HBP accumulated in bone rapidly but was hardly observed in tissues other than bone. Pretreatment of an excess amount of alendronate inhibited the bone accumulation of (67)Ga-DOTA-Bn-SCN-HBP. CONCLUSIONS (67)Ga-DOTA-Bn-SCN-HBP showed ideal biodistribution characteristics as a bone-imaging agent. These findings should provide useful information on the drug design of bone imaging agents for PET with (68)Ga.

Dynamic Expression of Tenascin-C After Myocardial Ischemia and Reperfusion: Assessment by 125I-Anti–Tenascin-C Antibody Imaging

Tenascin-C, an extracellular matrix glycoprotein, appears only in the early stages of embryonic development. It is not normally expressed in the adult heart but does reappear transiently in distinct areas in association with active tissue remodeling. The aim of this study was to explore serial changes in the expression of tenascin-C after myocardial ischemia and reperfusion, using 125I-labeled anti–tenascin-C antibody (125I-TNC-Ab) in a rat model of acute ischemia and reperfusion. Methods: The left coronary artery was occluded for 20 or 30 min, followed by reperfusion for 1, 3, or 7 d in rats with 20 min of ischemia and for 1, 3, 7, 14, or 28 d in rats with 30 min of ischemia. At the time of the study, 125I-TNC-Ab (1.0–2.5 MBq) was injected. Three to 5 h later, to verify the area at risk, 99mTc-methoxyisobutylisonitrile (100–200 MBq) was injected intravenously just after the left coronary artery reocclusion and the rats were sacrificed 1 min later. Dual-tracer autoradiography was performed to assess 125I-TNC-Ab uptake and the area at risk. Results: In rats with 20 min of ischemia, 125I-TNC-Ab uptake peaked at 3 d after reperfusion, followed by faint uptake after 7 d (uptake ratios at 1, 3, and 7 d after reperfusion were 1.81 ± 0.53, 2.46 ± 0.79, and 1.23 ± 0.17, respectively [P < 0.05 vs. 3 d]). In rats with 30 min of ischemia, uptake was high at 1 and 3 d after reperfusion (2.99 ± 0.90 and 2.71 ± 0.80, respectively), decreased at 7 and 14 d (1.94 ± 0.23 and 2.06 ± 0.37, respectively), and was weak at 28 d (1.47 ± 0.27, P < 0.005 vs. 1 d, P < 0.05 vs. 3 d). Conclusion: These data indicate that 125I-TNC-Ab imaging may be a way to monitor myocardial injury and its repair process after ischemia and reperfusion by visualizing tenascin-C expression.