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INTRODUCTION: C-kit is an important diagnostic and therapeutic target molecule for several malignancies, and c-kit-targeted drugs have been used clinically. Because abundant c-kit expression in tumors is a prerequisite for successful c-kit-targeted therapy, imaging of c-kit expression is expected to play a pivotal role in the therapeutic decision for each patient. We evaluated (64)Cu-labeled Fab of anti-c-kit antibody 12A8 as a positron emission tomography (PET) imaging probe. METHODS: (111)In- or (125)I-Labeled 12A8 Fab was evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution in mice bearing c-kit-expressing and -non-expressing tumors. Next, Fab fragment was labeled with the positron emitter (64)Cu and evaluated by PET. RESULTS: Radiolabeled 12A8 Fab showed specific binding to c-kit-expressing cells with high affinity and internalized into cells after binding to c-kit on cell surface. Although tumor accumulation of [(111)In]Fab was lower than that of [(111)In]IgG, the faster blood clearance of [(111)In]Fab provided higher tumor-to-blood ratio at 6 h postinjection onwards. Blood clearance of (64)Cu-labeled 12A8 Fab was slower than that of [(111)In]Fab, but PET using [(64)Cu]Fab clearly visualized the tumor at 6 h postinjection onwards. CONCLUSION: The (64)Cu-labeled 12A8 Fab could be used for c-kit-specific PET imaging and might help in selecting appropriate patients for c-kit-targeted treatments.

We have devised and estimated a new strategy to prolong the residence time of radiolabeled antibodies in tumor in which an octaarginine peptide (R₈) was used as an anchoring molecule to fix antibodies against CD20 (NuB2; IgG2a) on tumor cells. Conjugation of R₈ with antibodies was performed by maleimide-thiol chemistry using thiol groups generated by reducing the disulfide bonds of the antibody. The R₈-conjugated NuB2 was then reacted with succinimidyl meta-[¹²⁵I]iodobenzoate to prepare [¹²⁵I]SIB-NuB2(I) (0.92 R₈/NuB2) and [¹²⁵I]SIB-NuB2(III) (3.38 R₈/NuB2). Both SIB-NuB2(I) and SIB-NuB2(III) exhibited size-exclusion HPLC elution profiles and immunoreactivity to CD20-positive cells similar to those of NuB2. NuB2(I) also possessed isoelectric focusing (IEF) profile similar to NuB2. However, NuB2(III) registered a broad IEF band toward higher pI. When incubated with CD20-positive cells, [¹²⁵I]SIB-NuB2(I) and [¹²⁵I]SIB-NuB2(III) exhibited 1.4 and 4.0 times higher cell-associated radioactivity than [¹²⁵I]SIB-NuB2. After the cells were washed and reincubated in a fresh medium for 3 h, [¹²⁵I]SIB-NuB2(I) and [¹²⁵I]SIB-NuB2(III) exhibited significantly higher cell-associated radioactivity than [¹²⁵I]SIB-NuB2. In biodistribution studies in normal mice, while both [¹²⁵I]SIB-NuB2(I) and [¹²⁵I]SIB-NuB2 exhibited similar biodistribution profiles, [¹²⁵I]SIB-NuB2(III) showed faster clearance from the blood and higher hepatic radioactivity levels than [¹²⁵I]SIB-NuB2. In SCID mice bearing CD20-positive xenografts, [¹³¹I]SIB-NuB2(I) exhibited significantly higher radioactivity in xenografts than those of [¹²⁵I]SIB-NuB2 with no significant increase being observed in other tissues. The findings indicate that appropriate R₈ modification of antibodies satisfies both specific targeting ability of antibody and strong cell-association property of R₈, which was reflected in the increased radioactivity levels in tumor. These findings supported the applicability of this approach to enhance target-specific accumulation of radiolabeled antibodies.

Background Malignant mesothelioma is a highly aggressive form of cancer. Curative surgery is the only effective therapy for mesothelioma, and therefore early diagnosis is important. However, early diagnosis is difficult using current diagnostic imaging techniques, and a new imaging method for early diagnosis is urgently required. We evaluated the affinity of radiolabeled monoclonal antibodies to the C-terminal fragment of ERC/mesothelin for this purpose. Methods In-111-labeled or I-125-labeled IgG against C-terminal fragment of ERC and its Fab fragment were evaluated in vitro by cell binding, competitive inhibition, and cellular internalization assays, and in vivo by biodistribution in mice bearing ERC-expressing tumors. Next, the Fab fragment was labeled with the positron emitter Cu-64 and evaluated by positron emission tomography (PET). Results Radiolabeled IgG and Fab showed specific binding to ERC-expressing mesothelioma cells with high affinity. Both radiolabeled IgG and Fab internalized into cells after binding to ERC on the cell surface. In-111-labeled IgG accumulated in ERC-expressing tumors and resulted in a moderate tumor-to-blood ratio at 4 days after injection. Furthermore, PET using Cu-64-labeled Fab visualized the tumor at 6 h after injection. Conclusion Cu-64-labeled Fab can be useful for ERC-specific PET imaging, and can thus facilitate improved diagnosis of patients with early-stage mesothelioma. Nucl Med Commun 31:380-388 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.

INTRODUCTION: Gastrointestinal stromal tumor (GIST) is the most common mesenchymal tumor arising from the gastrointestinal tract and highly expresses mutated c-kit. We aimed to develop a specific and sensitive method for detecting GISTs using radiolabeled anti-c-kit monoclonal antibody. METHODS: A mutated c-kit-expressing cell clone was established by transfecting an expressing vector of mutated c-kit gene into HEK293 human embryonic kidney cells. The tumors were developed by inoculating c-kit-expressing cells into nude mice. (125)I- and (111)In-labeled anti-c-kit antibodies (12A8 and 41A11) were evaluated in vitro by cell binding, competitive inhibition and cellular internalization assays, and in vivo by biodistribution and imaging studies in tumor-bearing mice. RESULTS: Both (125)I- and (111)In-labeled antibodies showed specific binding with c-kit-expressing cells with high affinity (dissociation constants = 2.2-7.1x10(9) M(-1)). Internalization assay showed that (125)I-labeled antibodies were rapidly internalized and dehalogenated, with the release of (125)I from the cells, resulting in reduction of cell-associated radioactivity with time. In contrast, (111)In-labeled antibody was internalized but did not result in the reduced radioactivity associated with tumor cells. Reflecting this phenomenon, the in vivo tumor uptake of (125)I-labeled antibody was low on Day 1, further decreasing with time, while tumor uptake of (111)In-labeled antibody was high on Day 1, further increasing with time. The xenografted tumor was clearly visualized by scintigraphy after injection of (111)In-labeled antibody. CONCLUSION: The anti-c-kit monoclonal antibody labeled with a metal radionuclide would be promising for c-kit-targeted imaging of GISTs.

Poly(amidoamine) (PAMAM) dendrimers are highly branched spherical polymers that have a unique surface of primary amine groups and provide a versatile design for targeted delivery of pharmaceuticals and imaging agents. Acetylation or succinylation of surface amine groups of PAMAM dendrimer derivatives is frequently performed to reduce nonspecific uptake. However, since targeting molecules, drugs/imaging agents, and acylating reagents react with the amine groups on dendrimer, such modification may limit the number of targeting molecules and/or drugs or may result in insufficient charge reduction. In this study, a gamma-glutamyl PAMAM dendrimer was designed and synthesized as a new precursor for targeting device. The relationship between surface electrical properties of the PAMAM dendrimer derivatives and pharmacokinetics was also determined. A PAMAM dendrimer (generation 4.0) was modified with a small number of Bolton-Hunter reagent to prepare Phe-P (pI 9.2). The amine residues of Phe-P were gamma-glutamylated to prepare Glu-P (pI 7.1). The alpha-amine residues of Glu-P were then acetylated or succinylated to prepare Ac-Glu-P (pI 5.3) or Suc-Glu-P (pI 3.6). For comparison, Phe-P was acetylated or succinylated to prepare Ac-P (pI 6.0) or Suc-P (pI 5.1). All the PAMAM dendrimer derivatives exhibited similar molecular size (7.2 to 7.8 nm) except for Ac-P (5.1 nm). The biodistribution studies were performed after radioiodination of each PAMAM dendrimer derivative with Na[(125)I]I. When injected intravenously to mice, both [(125)I]Ac-P and [(125)I]Suc-P exhibited prolonged radioactivity levels in the blood and significantly lower hepatic and renal radioactivity levels than those of [(125)I]Phe-P. Both [(125)I]Glu-P and [(125)I]Ac-Glu-P showed residence times in the blood similar to those of [(125)I]Ac-P and [(125)I]Suc-P. However, [(125)I]Glu-P also registered higher radioactivity levels in the kidney. High hepatic and renal radioactivity levels were observed with highly anionic [(125)I]Suc-Glu-P. These results indicate that, while the manipulation of pI between 5 to 6 would be appropriate to enhance blood retention and reduce renal and hepatic uptake, the amount of primary amine residues on dendrimer surface may also play a crucial role in their renal uptake. The findings in this study show that gamma-glutamyl PAMAM dendrimers would constitute versatile precursors to prepare PAMAM dendrimer-based targeting devices due to their neutral molecular charge (pI 7.1) and the presence of a large number of alpha-amine residues available for conjugation of targeting molecules and drugs/imaging agents.

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.

Radiolabeled anti-CD20 antibodies have demonstrated impressive efficacy in the treatment of relapsed non-Hodgkin lymphoma. This encourages the treatment of solid tumor with radiolabeled antibody fragments and peptides. However, both preclinical and clinical studies revealed that persistent localization of radioactivity in the kidney constitutes a major obstacle that compromises therapeutic efficacy. Recent extensive studies show that long residence times of radiolabeled end products from lysosomes are responsible for the renal radioactivity levels. Recent studies have also elucidated the involvement of megalin-cubilin in renal tubular reabsorption of radiolabeled antibody fragments and peptides. In light of these findings, efforts are being made to block tubular reabsorption of radiolabeled antibody fragments and peptides by competitive inhibitors, charge modification, and PEGylation. An interposition of an enzyme-cleavable linkage between antibody fragments and radiolabels would constitute an alternative approach to reduce renal radioactivity levels. Recent findings of these studies will be described.

Left ventricular (LV) remodeling after acute myocardial infarction (MI) causes heart failure, and thus it is important to evaluate cardiac repair as the early stage of LV remodeling. Tenascin-C (TNC), an extracellular matrix glycoprotein, is transiently and abundantly expressed in the heart during the early stage of tissue remodeling after MI. However, it is not expressed in healthy adult heart. This study was undertaken to develop a new noninvasive diagnostic technique to detect cardiac repair after acute MI using 111 In Fab fragment of a monoclonal antibody specific for TNC. 111 In-anti-TNC-Fab was injected intravenously in 13 rats at 1 (D1, n = 3), 3 (D3, n = 5), and 5 (D5, n = 5) days after producing MI and in 5 sham-operated rats (S). We performed autoradiography and dual-isotope single-photon emission computed tomography imaging (SPECT) of 111 In-anti-TNC-Fab and 99mTc methoxyisobutyl isonitrile (MIBI). The radioactivity in the heart was significantly higher in D (D1, 0.45 +/- 0.06% injected-dose/g; D3, 0.64 +/- 0.12; D5, 0.38 +/- 0.07) than S (0.27 +/- 0.06, P < 0.01 versus D1 and D3, P < 0.05 versus D5). By autoradiography, higher radioactivities were observed in the infarcted area than in the noninfarcted area of MI hearts. Dual-isotope SPECT demonstrated the regional myocardial uptake of 111 In-anti-TNC-Fab, which was complementary to the perfusion image. The results of the present study indicated that we can localize the infarcted region in the heart by ex vivo and in vivo imaging methods using 111 In-anti-TNC-Fab, and suggested the potential usefulness of noninvasive detection of cardiac repair.

In a search of coordination molecules suitable to the fac-{(99m)Tc(CO)3}(+) core as a synthon for (99m)Tc-radiopharmaceuticals, nonradioactive rhenium complexes of two macrocyclic triamine compounds with different chelate ring structures, 1,4,7-triazacyclononane (9N3) and 1,5,9-triazacyclododecane (12N3), were synthesized and characterized. (99m)Tc-labeled 9N3 and 12N3 compounds were also prepared using [(99m)Tc(OH 2)3(CO)3](+) and were characterized by both in vitro and in vivo studies. 9N3 produced a single rhenium complex, whereas 12N3 generated two major complexes. The crystallographic data and infrared absorption wavenumber assigned to the C-O stretch suggested that the coordination geometry of 9N3 would be more suitable to fac-{Re(CO)3}(+) than that of 12N3. In contrast, both 9N3 and 12N3 provided a single (99m)Tc-labeled compound. However (99m)Tc-labeled 9N3 exhibited higher stability than (99m)Tc-labeled 12N3 in rat plasma and in the presence of histidine at an elevated temperature. In biodistribution studies, both (99m)Tc-labeled compounds did not show any specific accumulation of radioactivity in any organs except for the excretory organs such as the liver and kidney. These findings showed that 9N3 would constitute a macrocyclic chelating molecule of choice to prepare (99m)Tc radiopharmaceuticals using a fac-{(99m)Tc(CO)3}(+) core.

The development of 99mTc-labeled fatty acid analogues metabolized by beta-oxidation in the myocardium constitutes an unsolved challenge. On the basis of our recent findings that [188Re]tricarbonyl(cyclopentadienylcarbonate)rhenium ([188Re]CpTR-COOH) was recognized as an aromatic compound and was metabolized as such in the body, [99mTc]cyclopentadienyltricarbonyltechnetium ([99mTc]CpTT) was conjugated at the omega-position of pentadecanoic acid to prepare [99mTc]CpTT-PA. When injected into rats, [99mTc]CpTT-PA exhibited the maximum myocardial accumulation and heart-to-blood ratio of 3.85 %ID/g at 1 min and 4.60 at 10 min postinjection, respectively. The metabolic study using isolated Langendorff perfused rat hearts demonstrated that approximately 67% of perfused [99mTc]CpTT-PA was incorporated and [99mTc]CpTT-propionic acid, the metabolite after six cycles of beta-oxidation of [99mTc]CpTT-PA, was detected as the major radiometabolite in the perfusate and myocardium. These findings indicate that [99mTc]CpTT-PA was recognized, transported, and metabolized as a long chain fatty acid analogue for energy production in the myocardium.

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. We have reported that Fab fragments labeled with 3'-[I-131]iodohippuryl N-epsilon-maleoyl-lysine (HML) showed markedly low renal radioactivity levels even shortly after injection, due to a rapid and selective release of m-[I-131]iodohippuric acid by the action of brush border enzymes. To estimate the applicability of the molecular design to metallic radionuclides, [Re-188]tricarbonyl(cyclopentadienylcarbonate)rhenium ([Re-188]CpTR-COOH) was conjugated with N-epsilon-tert-butoxycarbonyl-glycyl-lysine or N-epsilon-maleoyl-glycyl-lysine to prepare [Re-188]CpTR-GK-Boc or [Re-188]CpTR-GK. The cleavage of the glycyl-lysine linkage of the two compounds generates a glycine conjugate of [Re-188]CpTR-COOH ([Re-188]CpTR-Gly), which possesses in vivo behaviors similar to those of m-iodohippuric acid. The hydrolysis rate of the peptide bond in [Re-188]CpTR-GK-Boc was compared with that in 3'-[I-125]iodohippuryl N-epsilon-Boc-lysine ([I-125]HL-Boc) using brush border membrane vesicles (BBMVs) prepared from rat kidneys. [Re-188]CpTR-GK was conjugated to thiolated Fab fragments to prepare [Re-188]CpTR-GK-Fab. The biodistribution of radioactivity after injection of [Re-188]CpTR-GK-Fab was compared with that of [I-125]HML-Fab and [Re-188]CpTR-Fab prepared by conjugating N-hydroxysuccinimidyl ester of [Re-188]CpTR-COOH with antibody fragments. While [Re-188]CpTR-GK-Boc liberated [Re-188]CpTR-Gly in BBMVs, [I-125]HL-Boc liberated m-[I-125]iodohippuric acid at a much faster rate. In addition, although [I-125]HL-Boc was hydrolyzed by both metalloenzymes and nonmetalloenzymes, metalloenzymes were responsible for the cleavage of the peptide linkage in [Re-188]CpTR-GK-Boc. In biodistribution studies, [Re-188]CpTR-GK-Fab exhibited significantly lower renal radioactivity levels than did [Re-188]CpTR-Fab. However, the renal radioactivity levels of [Re-188]CpTR-GK-Fab were slightly higher than those of [I-125]HML-Fab. The analysis of urine samples collected for 6 h postinjection of [Re-188]CpTR-GK-Fab showed that [Re-188]CpTR-Gly was the major radiometabolite. In tumor-bearing mice, [Re-188]CpTR-GK-Fab significantly reduced renal radioactivity levels without impairing the radioactivity levels in tumor. These findings indicate that the molecular design of HML can be applied to metallic radionuclides by using a radiometal chelate of high inertness and by designing a radiometabolite of high urinary excretion when released from antibody fragments following cleavage of a glycyl-lysine linkage. This study also indicates that a change in chemical structure of a radiolabel attached to a glycyl-lysine linkage significantly affected enzymes involved in the hydrolysis reaction. Since there are many kinds of enzymes that cleave a variety of peptide linkages on the renal brush border membrane, selection of a peptide linkage optimal to a radiometal chelate of interest may provide radiolabeled antibody fragments that exhibit renal radioactivity levels similar to those of [I-131]HML-labeled ones. The in vitro system using BBMVs might be useful for selecting an appropriate peptide linkage.

Introduction: The preferable pharmacokinetics of rhenium-186 (Re-186)-monoaminemonoamidedithiol-conjugated or Re-186-mercaptoacetyl-triglycine-conjugated bisphosphonates (Bps) suggested that the molecular design would be applicable to other radionuclides such as Ga-68, (TC)-T-99m, Sm-153 and Lu-177. In this study, a key factor affecting the pharmacokinetics of a chelate-conjugated BP was investigated to estimate the validity and the applicability of molecular design. Methods: Chemically inert and well-characterized tricarbony][Re-186][(cyclopentadienylcarbonyl amino)-acetic acid]rhenium ([Re-186]CpTR-Gly) was conjugated with 3-amino-l-hydroxypropylidene-l, l-bisphosphonate and purified by high-performance liquid chromatography (HPLC) to prepare [Re-186](1-{3-[tricarbonyl(cyclopentadienylcarbonyl amino)-acetylamido]-l-hydroxy-l-phosphono-propyl}-phosphonic acid)rhenium ([Re-186]CpTR-Gly-APD). Plasma stability, plasma protein binding, hydroxyapatite (HA) binding and the pharmacokinetics of [Re-186]CpTR-Gly-APD were compared with those of Re-186 l-hydroxyethylidene-l,l-diphosphonate (HEDP). The effect of HEDP coadministration and preadministration on the pharmacokinetics of [ 116 Re]CpTR-Gly-APD was also determined. Results: The HPLC-purified [186 Re]CpTR-Gly-APD showed higher plasma stability, higher HA binding, higher bone accumulation and lower plasma protein binding than did Re-186-HEDP. However, HA binding of [Re-186]CpTR-Gly-APD decreased to levels slightly higher than that of Re-186-HEDP at similar HEDP concentrations. Bone accumulation of [Re-186]CpTR-Gly-APD also decreased to levels similar to that of Re-186-HEDP when [Re-186]CpTR-Gly-APD was coinjected with HEDP equivalent to that in Re-186-HEDP. In contrast, HEDP pretreatment did not impair bone accumulation of the two Re-186-labeled compounds. However, a delay in blood clearance and an increase in renal radioactivity levels were observed particularly with Re-186-HEDP. Conclusions: Although Re-186-HEDP possessed HA binding and bone accumulation similar to those of [Re-186]CpTR-Gly-APD, the specific activity of Re-186-labeled BPs was found to play a crucial role in bone accumulation and blood clearance. Thus, the molecular design of chelate-conjugated BP would be useful for the development of bone-seeking radiopharmaceuticals with a variety of radionuclides by selecting chelating molecules that provide high specific activities. (c) 2007 Elsevier Inc. All rights reserved.

Renal localization of radiolabeled antibody fragments presents a problem in targeted imaging and radiotherapy. We recently reported that Fab fragments labeled with 3′-[131I]iodohippuryl Nε-maleoyl- L-lysine (HML) demonstrated markedly low renal radioactivity levels from early postinjection in mice. Previous studies suggested that low renal radioactivity levels were attributable to cleavage of the glycyl-lysine sequence in HML by the action of renal brush border enzymes, followed by urinary excretion of the resulting m-iodohippuric acid. In this study, an in vitro system using brush border membrane vesicles (BBMVs) isolated from the rat kidney cortex was developed to estimate renal brush border enzyme(s)-mediated cleavage of the peptide linkage. Low molecular weight HML derivatives, 3′-[125I]iodohippuryl L-lysine (HL), 3′-[125I] iodohippuryl Nε-tert-butoxycarbonyl-L-lysine (HBL), and their D-amino acid counterparts, were synthesized and incubated in BBMVs. Both [125I]HL and [125I]HBL generated m-[125I] iodohippuric acid after incubation in BBMVs at 37 °C while the latter liberated significantly higher amounts of the metabolite. [125I]D-HL and [125I]D-HBL failed to release the metabolite under similar conditions. The liberation of m-[125I]iodohippric acid from [125I]HL was significantly facilitated or completely inhibited by the addition of an activator or an inhibitor for carboxypeptidase M. The release of m-[125I]iodohippuric acid from [125I]HBL increased by the addition of the activator, whereas the inhibitor partially inhibited the release of the metabolite from [125I]-HBL. The BBMV-mediated release of m-[125I]iodohippuric acid from [125I]HBL was not impaired by the addition of inhibitors for neutral endopeptidase or renal dipeptidase. These findings showed that the glycyl-L-lysine sequence in HML would be recognized and cleaved by metalloenzymes and nonmetalloenzymes on the renal brush border even when iodine was incorporated into a benzene ring and the Nε-amine residue of lysine was chemically modified, which supported the hypothesis that low renal radioactivity levels of HML-conjugated Fab fragments would be attributed to the release of m-iodohippuric acid by renal brush border enzymes. This study suggested that this in vitro system using BBMVs would be useful to estimate radiolabeling reagents of antibody fragments or peptides designed to reduce renal radioactivity with a variety of radionuclides. © 2005 American Chemical Society.

医薬品開発において，前臨床試験から臨床試験に進んだ候補薬剤の約40%が脱落している．その多くは，前臨床試験における薬物の体内挙動が臨床のそれと大きく異なることに起因する．最近，欧州医薬品機構により極微量の薬物を用いる臨床試験に関する指針が提示されたのを受けて，本格的な第I相試験を行う前に，放射性核種で標識された極微量の候補薬剤をヒトに投与して，体内動態や代謝を追跡する予試験的な臨床試験が進められている．加速器分析法（accelerator mass spectrometry: AMS）を用いるマスバランス試験や陽電子断層撮像法（positron emission tomography: PET）を用いる画像解析によるヒトにおける予試験的な評価は，候補薬剤の開発期間の短縮や経済的負担の大幅な低減を可能としている．<br>

Tc-99m-labeled mannosyl-neoglycoalbumin (NMA) was prepared and evaluated as a radiopharmaceutical for sentinel lymph node (SLN) identification, since Tc-99m-labeled human serum albumin (HSA) rapidly cleared from injection sites. NMA was conjugated with 6-hydrazinopyridine-3-carboxylic acid (HYNIC) and reacted with [Tc-99m](tricine)(2) to prepare [Tc-99m](HYNIC-NMA)(tricine)(2). After subcutaneous injection of [Tc-99m](HYNIC-NMA)(tricine)(2) from murine foot pad, radioactivity levels in the popliteal and lumbar lymph nodes, the injection site and other tissues were compared with those of [Tc-99m](HYNIC-HSA)(tricine)(2) and Tc-99m-labeled colloidal rhenium sulfate ([Tc-99m]colloid). [(TC)-T-99m](HYNIC-NMA)(tricine)2 demonstrated significantly higher radioactivity levels in the popliteal lymph node, the SLN in this model, than did [Tc-99m](HYNIC-HSA)(tricine)(2) and [Tc-99m]colloid at 0.5, 1, and 6 h post-injection. [Tc-99m](HYNIC-NMA)(tricine)(2) showed a dose-dependent decrease in the popliteal accumulation while the radioactivity levels in the blood, liver and spleen increased with an increase in the molar dose of NMA. [Tc-99m]colloid registered a decrease in the radioactivity levels in the popliteal lymph node, blood, liver, and spleen with dilution. However, the radioactivity levels at the injection site increased with dilution of [ Tc-99m] colloid. Both [Tc-99m](HYNIC-NMA)(tricine)(2) and [Tc-99m](HYNIC-HSA)(tricine)(2) showed the radioactivity levels at the injection site similar each other. These findings indicated that an addition of a macrophage binding function to Tc-99m-labeled HSA provided high and selective accumulation of the radioactivity in the SLN without affecting the elimination rate from the injection site. Such characteristics render [Tc-99m](HYNIC-NMA)(tricine)(2) attractive as a radiopharmaceutical for SLN identification. This study also demonstrated that the number of non-radiolabeled colloidal particles and the molar dose of mannosylated compounds play a crucial role in the SLN accumulation. (C) 2004 Elsevier Inc. All rights reserved.

C-11- and I-123-labeled long chain fatty acid derivatives have been reported as useful radiopharmaceuticals for the estimation of myocardial fatty acid metabolism. We have reported that Tc-99m-labeled N-[[[(2-mercaptoethyl)amino]carbonyl]methyl]-N-(2-mercaptoethyl) -6-aminohexanoic acid ([99mTc]-MAMA-HA), a medium chain fatty acid derivative, is metabolized by β-oxidation in the liver and that the MAMA ligand is useful for attaching to the omega-position of fatty acid derivatives as a chelating group for Tc-99m. On the basis of these findings, we focused on developing a Tc-99m-labeled long chain fatty acid derivative that reflected fatty acid metabolism in the myocardium. In this study, we synthesized a dodecanoic acid derivative, MAMA-DA, and a hexadecanoic acid derivative, MAMA-HDA, and performed radiolabeling and biodistribution studies. [99mTc]MAMA-DA and [99mTC]MAMA-HDA were prepared using a ligand-exchange reaction. Biodistribution studies were carried out in normal mice and rats. Then, a high initial uptake of Tc-99m was observed, followed by a rapid clearance from the heart. The maximum heart/blood ratio was 3.6 at 2 min postinjection of [99mTc]MAMA-HDA. These kinetics were similar to those with postinjection of p-[125I]iodophenylpentadecanoic acid. Metabolite analysis showed [99mTc]MAMA-HDA was metabolized by β-oxidation in the body. In conclusion, [99mTc]MAMA-HDA is a promising compound as a long chain fatty acid analogue for estimating β-oxidation of fatty acid in the heart.

For purpose of reducing renal accumulation of radioactivity of a known radiopharmaceutical agent, i.e.,111In-DTPA (diethylenetriaminepentaacetic acid)-D-Phe1-octreotide, a derivative in which p-carboxy-L-phenylalanine is substituted for D-Phe1was synthesized. Biodistribution study of the resultant compound having carboxy-substituted L-Phe1revealed that the renal accumulation was significantly lower than that of control compound having unsubstituted L-Phe1, demonstrating that the presence of negative charge on the N-terminal amino acid of octreotide is effective to reduce the renal accumulation. This effect can be attributed to the reduction of lipophilicity and also the repulsive force arisen from the negative charge of renal brush border membrane. © 2004 Pharmaceutical Society of Japan.

In vivo metabolism of [(188)Re]tricarbonyl(carboxycyclopentadienyl)rhenium ([(188)Re]CpTR-COOH) and its glycine conjugate ([(188)Re]CpTR-Gly) was investigated to estimate the applicability of cyclopentadienyltricarbonylrhenium (CpTR) compounds to (186/188)Re-labeling reagents for polypeptides and peptides. Both [(188)Re]CpTR derivatives were stable after incubation in a buffered-solution and in murine plasma at 37 degrees C for 6 h. Plasma protein binding was hardly observed with the two derivatives. However, different biodistribution and metabolic fates were observed with the two CpTR derivatives. While more lipophilic [(188)Re]CpTR-COOH was excreted by both hepatobiliary and urinary excretion, the majority of less lipophilic [(188)Re]CpTR-Gly was excreted by urinary excretion. In addition, while [(188)Re]CpTR-Gly was rapidly excreted into urine as its intact structure, [(188)Re]CpTR-COOH was metabolized to more hydrophilic compounds including its glycine conjugate, [(188)Re]CpTR-Gly. Renal excretion of [(188)Re]CpTR-Gly was significantly reduced in probenecid retreated mice. The present studies reinforced that CpTR core remained stable under biological environment. CpTR-COOH was partially recognized as an aromatic acid and was metabolized as such. However, glycine conjugation rendered CpTR-COOH hydrophilic enough to be excreted into urine without further metabolism. These findings suggested that radiolabeling reagents that liberate [(186/188)Re]CpTR-Gly from covalently conjugated (186/188)Re-labeled polypeptides and peptides by the action of renal brush border enzymes would be useful to reduce renal radioactivity levels.

BACKGROUND: Although the identification of inflammatory infiltrates in endomyocardial biopsy specimens is necessary for the definite diagnosis of myocarditis, the biopsy test is invasive and is not sensitive. Therefore, a new diagnostic technique for the early and noninvasive evaluation of myocarditis has been awaited. Expression of tenascin-C (TNC), one of the oligometric extracellular glycoproteins, is induced in various pathological states, including inflammation, suggesting that TNC can be a molecular marker of myocarditis. METHODS AND RESULTS: An 111In anti-TNC monoclonal antibody Fab' fragment was injected intravenously into rats with experimental autoimmune myocarditis (EAM), and the biodistribution of this radiotracer was measured. Rapid clearance of radioactivity from the blood was observed in both EAM and control rats (<1% at 6 hours after injection). Myocardial uptake of the tracer was much higher in EAM rats than in control rats (7.54-, 4.39-, and 3.51-fold at 6, 24, and 48 hours after injection, respectively). By autoradiography, high radioactivities were clearly observed in the regions indicative of inflammation in EAM rats. Single-photon emission CT imaging demonstrated the focal myocardial uptake of 111In anti-TNC Fab' in vivo. CONCLUSIONS: Radiolabeled anti-TNC Fab' may be useful for the noninvasive diagnosis of myocarditis.

The purpose of this study was to develop an indium-111 ((111)ln)-based residualizing label for estimating the pharmacokinetics of proteins. 1,4,7,10-Tetraazacyclododecane-N,N',N",N'"-tetraacetic acid (DOTA), which produced a highly stable and hydrophilic (111)ln chelate, was selected as the chelating site, and the monoreactive DOTA derivative with a tetrafluorophenyl group as the protein binding site (mDOTA) was designed to avoid cross-linkings of proteins. mDOTA was synthesized with an overall yield of 11%. The stability in murine plasma, the radioactivity retention in the catabolic sites of proteins and the radiochemical yields of (111)ln-labelled proteins via mDOTA were investigated using human serum albumin (HSA), galactosyl-neoglycoalbumin (NGA) and cytochrome c (cyt c) as model proteins. (111)ln-labelled HSA via mDOTA was highly stable for 5 days after incubation in murine plasma. Long retention of radioactivity in the catabolic sites was observed after injection of (111)ln DOTA-NGA in mice, due to the slow elimination of the radiometabolite from the lysosome. At a chelator concentration of 42.2 mum, (111)ln-DOTA-cyt c was produced with over 91% radiochemical yield. On the other hand, (111)ln-DOTA-lysine and (111)ln-DOTA were obtained with high radiochemical yields at lower chelator concentrations. These findings indicated that mDOTA would be an appropriate (111)ln-labelling agent for estimating protein pharmacokinetics. These findings also suggested that the introduction of a protein binding site at a position distal from the unmodified DOTA structure would be preferable to preparing (111)ln-DOTA-labelled proteins with higher specific activity.

The effect of molecular charges on renal accumulation of111In-DTPA-labeled low molecular weight (LMW) peptides was investigated using111In-DTPA-octreotide derivatives as models to design radiolabeled peptides that are taken up less by renal cells. The N-terminal D-phenylalanine (Phe) of111In-DTPA-D-Phe1-octreotide was replaced with L-aspartic acid (Asp), L-lysine (Lys), L-methionine (Met) or L-Phe. Cellulose acetate electrophoresis indicated that both111In-DTPA-L-Phe1-octreotide and111In-DTPA-L-Met1-octreotide showed similar net charges, whereas111In-DTPA-L-αLys1-octreotide and111In-DTPA-L-Asp1-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-αLys1-octreotide showed the highest radioactivity levels from 10 min to 6 h postinjection, whereas the lowest radioactivity levels were observed with111In-DTPA-L-Asp1-octreotide at all the postinjection intervals. These findings indicated that the replacement of only one amino acid in111In-DTPA-D-Phe1-octreotide significantly altered net molecular charges of the resulting peptides and that the net charges of the111In-DTPA-octreotide derivatives significantly affected their renal uptake. Thus, an increase of negative charges in peptide molecules may constitute a strategy for designing111In-DTPA-conjugated LMW peptides with low renal radioactivity levels. © 2001 Elsevier Science Inc. All rights reserved.

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. Recently, we reported use of a novel radioiodination reagent, 3′-[131I]iodohippuryl N∈-maleoyl-L-lysine (HML), that liberates m-iodohippuric acid before antibody fragments are incorporated into renal cells. In mice, HML-conjugated Fab demonstrated low renal radioactivity levels from early postinjection times. In this study, renal metabolism of HML-conjugated Fab fragments prepared by different thiolation chemistries and by direct radioiodination were investigated to determine the mechanisms responsible for the low renal radioactivity levels. Fab fragments were thiolated by 2-iminothiolane modification or by reduction of disulfide bonds in the Fab fragments, followed by conjugation with radioiodinated HML to prepare [131I]HML-IT-Fab and [125I]HML-Fab, respectively. In biodistribution studies in mice, both [131I]HML-IT-Fab and [125I] HML-Fab demonstrated significantly lower renal radioactivity levels than those of [125I]Fab. In subcellular distribution studies, [125I]Fab showed migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection. On the other hand, the majority of the radioactivity was detected only in the membrane fraction at the same time points after injection of both [131I]HML-IT-Fab and [125I]HML-Fab. In metabolic studies, while [125I]Fab remained intact at 10 min postinjection, both HML-conjugated Fab fragments generated m-iodohippuric acid as a radiometabolite at the same postinjection time. [131I]HML-IT-Fab registered two radiometabolites (intact [131I]HML-IT-Fab and m-iodohippuric acid), whereas additional radiometabolites were observed with [125I]HML-Fab. This suggested that metabolism of both HML-conjugated Fab fragments would occur in the membrane fractions of the renal cells. The findings of this study reinforced our previous hypothesis that radiochemical design of antibody fragments that liberate radiometabolites that are excreted into the urine by the action of brush border enzymes would constitute a useful strategy to reduce renal radioactivity levels from early postinjection times.

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) is a representative agent used to prepare technetium-99m (99mTc)-labeled polypeptides with tricine as a coligand. However,99mTc-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 [99mTc](HYNIC)(tricine)(benzoylpyridine: BP) ternary complex was synthesized to prepare99mTc-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, [99mTc](HYNIC-Fab)(tricine)(BP) showed significant reduction of the radioactivity in high molecular weight fractions compared with [99mTc](HYNIC-Fab)(tricine)2.When injected into mice, [99mTc](HYNIC-NGA)(tricine)(BP) was metabolized to [99mTc](HYNIC-lysine)(tricine)(BP) in the liver with no radioactivity detected in protein-bound fractions in contrast to the observations with [99mTc](HYNIC-NGA)(tricine)2. In addition, [99mTc](HYNIC-NGA)(tricine)(BP) showed significantly faster elimination rates of the radioactivity from the liver as compared with [99mTc](HYNIC-NGA)(tricine)2. Similar results were observed with99mTc-labeled Fab fragments where [99mTc](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 [99mTc](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 [99mTc](HYNIC)(tricine)(BP) complex with proteins in the blood and in the lysosomes. Such characteristics would render the TFP active ester of [99mTc](HYNIC)(tricine)(BP) complex attractive as a radiolabeling reagent for targeted imaging. © 2001 Elsevier Science Inc.

6-Hydrazinopyridine-3-carboxylic acid (HYNIC) constitutes one of the most attractive reagents to prepare99mTc-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed,99mTc-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of111In-DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the99mTc labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of99mTc-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this99mTc-labeling method. In mice, [99mTc](HYNIC-IgG)(tricine)2and [99mTc](HYNIC-Fab)(tricine)2showed persistent localization of radioactivity in tissues when compared with their125I-labeled counterparts. [99mTc](HYNIC-IgG)(tricine)2eliminated from the blood at a rate similar to that of125I-labeled IgG, while [99mTc](HYNIC-Fab)(tricine)2showed significantly slower clearance of the radioactivity than125I-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [99mTc](HYNIC-IgG)(tricine)2in murine plasma. However, [99mTc](HYNIC-Fab)(tricine)2and [99mTc](HYNIC-RC160)(tricine)2demonstrated significant increases in the radioactivity in higher molecular weight fractions in plasma. Formation of higher molecular weight species was reduced when [99mTc](HYNIC-RC160)(tricine)2was stabilized with nicotinic acid (NIC) to generate [99mTc](HYNIC-RC160)(tricine)(NIC). [99mTc](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [99mTc](HYNIC-RC160)(tricine)2. These findings suggested that one of the tricine coligands in99mTc-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. © 2001 Elsevier Science Inc.

Hydrazino nicotinate (HYNIC) has been shown to produce technetium-99m (99mTc)-labeled proteins and peptides of high stability with high specific activities. However, persistent localization of radioactivity was observed in nontarget tissues such as the liver and kidney after administration of [99mTc]HYNIC-labeled proteins and peptides, which compromises the diagnostic accuracy of the radiopharmaceuticals. Since lysosomes are the principal sites of intracellular catabolism of proteins and peptides,99mTc-HYNIC-labeled galactosyl-neoglycoalbumin (NGA) was prepared using tricine as a co-ligand to investigate the fate of the radiolabel after lysosomal proteolysis in hepatocytes. When injected into mice, over 90% of the injected radioactivity was accumulated in the liver after 10 min injection. At 24 h postinjection, ca. 40% of the injected radioactivity still remained in liver lysosomes. Size-exclusion HPLC analyses of liver homogenates at 24 h postinjection showed a broad radioactivity peak ranging from molecular masses of 0.5-50 kDa. RP-HPLC analyses of liver homogenates suggested the presence of multiple radiolabeled species. However, most of the radioactivity migrated to lower molecular weight fractions on size-exclusion HPLC after treatment of the liver homogenates with sodium triphenylphosphine-3-monosulfonate (TPPMS). The TPPMS-treated liver homogenates showed a major peak at a retention time similar to that of [[99mTc](HYNIC-lysine)(tricine)(TPPMS)] on RP-HPLC. Similar results were obtained with urine and fecal samples. These findings suggested that the chemical bonding between99mTc and HYNIC remains stable in the lysosomes and following excretion from the body. The persistent localization of radioactivity in the liver could be attributed to the slow elimination rate of the final radiometabolite, [[99mTc](HYNIC-lysine)(tricine)2], from lysosomes, and subsequent dissociation of one of the tricine co-ligands in the low pH environment of the lysosomes in the absence of excess co-ligands, followed by binding proteins present in the organelles. The findings in this study also suggested that the development of appropriate co-ligands capable of preserving stable bonding with the Tc center is essential to reduce the residence time of radioactivity in nontarget tissues after administration of [99mTc]HYNIC-labeled proteins and peptides.

The renal uptake of radiolabeled antibody fragments presents a problem in targeted imaging and therapy, We hypothesized that the renal radioactivity levels of radiolabeled antibody fragments could be reduced if radiolabeled compounds of urinary excretion were released from glomerularly filtered antibody fragments before they were incorporated into renal cells by the action of brush border enzymes, present on the lumen of renal tubules, 3'-[I-131]Iodohippuryl N-epsilon-maleoyl-L-lysine ([I-131]HML) was conjugated with a thiolated Fab fragment because the glycyl-lysine sequence in HML is a substrate for a brush border enzyme and meta-iodohippuric acid is released by cleavage of the linkage. Fab fragments were also radiolabeled by direct radioiodination (I-125-Fab) or by conjugation with meta-[I-125]-iodohippuric acid via an amide bond [N-(5-maleimidopentyl) 3'-iodohippuric acid amide ([I-125]MPH-Fab)] or an ester bond [maleimidoethy 3'-iodohippurate ([I-125]MIH-Fab)] by procedures similar to those used for [I-131]HML-Fab. In biodistribution experiments in mice, [I-131]HML-Fab demonstrated markedly low renal radioactivity levels with kidney:blood ratios of radioactivity of 1 from 10 min to 1 h due to rapid release of meta-[I-131]iodohippuric acid. [I-125]MIH-Fab and I-125-Fab reached their peak ratios of 3.8 and 7.3 at 1 h, respectively, and [I-125]MPH-Fab showed the maximum ratio of 16.8 at 6 h, In subcellular distribution studies, both [I-125]MIH-Fab and I-125-Fab showed migration of radioactivity from the membrane to the lysosomal fraction of the renal cells from 10 to 30 min postinjection, whereas the majority of the radioactivity was detected only in the membrane fraction after administration of [I-131]HML-Fab at both time points. In nude mice, [I-131]HML-Fab showed one-quarter of the renal radioactivity of simultaneously administered I-125-Fab without impairing the target radioactivity levels 3 h after injection. These findings indicated that HML is a useful reagent for targeted imaging and therapy using antibody fragments as vehicles, These findings also suggested that the radiochemical design of radiolabeled antibody fragments that liberate radiometabolites of urinary excretion from antibody fragments by the action of brush border enzymes may constitute a new strategy for reducing the renal radioactivity levels of antibody fragments.

The persistent localization of radioactivity in the kidney after administration of In-111-DTPA-D-Phe(1)-octreotide impairs the diagnostic accuracy of this radiopharmaceutical. To better understand the mechanisms responsible for the renal radioactivity levels of In-111-DTPA-D-Phe(1)-octreotide, the renal metabolism of this compound was compared with In-111-DTPA-L-Phe(1)-octreotide, where the N-terminal D-phenylalanine was replaced with L-phenylalanine to facilitate metabolism. DTPA-D-Phe(1)-octreotide and DTPA-L-Phe(1)-octreotide were synthesized by solid-phase methods. Both In-111-DTPA-conjugated octreotide analogues were prepared with radiochemical yields of over 96%, and both remained stable after a 3 h incubation in murine serum at 37 degrees C. When injected into mice, the two In-111-DTPA-conjugated octreotide analogues showed similar radioactivity elimination rates from the blood and accumulation in the kidney with about 60% injected radioactivity being excreted in the urine by 24 h postinjection. Over 85% of the radioactivity in the urine existed as intact peptides for both analogues. Despite the similar renal radioactivity levels, significant differences were observed in the radiolabeled species remaining in the kidney between the two; while In-111-DTPA-L-Phe(1)-octreotide was rapidly metabolized to the final radiometabolite, In-111-DTPA-L-Phe, the metabolic rate of In-111-DTPA-D-Phe(1)-octreotide was so slow that various intermediate radiolabeled species were observed. However, both In-111-DTFA-D-Phe and In-111-DTPA-L-Phe remained in the lysosomal compartment of the renal cells as the final radiometabolites for long periods. These findings indicated that although the metabolic stability of In-111-DTPA-D-Phe(1)-octreotide in the renal cells may be partially involved, the slow elimination rate of the radiometabolite derived from In-111-DTPA-D-Phe(1)-octreotide from the lysosomal compartment of renal cells would be predominantly attributable to the persistent renal radioactivity levels of In-111-DTPA-D-Phe(1)-octreotide.