Kazuko Horiuchi

The design of a pentavalent 99mTc− dimercaptosuccinate complex as a tumor imaging agent

In our search for a technetium-tumor seeking agent, the chemical characteristic of polynuclear Ga-citrate attracted our attention. On this basis, we selected the ligand dimercaptosuccinic acid (DMS) and appropriate labeling conditions in the design of 99mTc(V)-DMS. Chemical characterization was performed by thin layer chromatography, electrophoresis, Sephadex column chromatography and spectrophotometric studies at the chemical concentration (99Tc). Biodistribution in mice bearing Ehrlich ascites tumor and scintigraphic images in VX-2 tumor-bearing rabbit, indicated the great applicability of 99mTc(V)-DMS as a new tumor imaging agent. Its distinctive characteristic, different from the kidney imaging agent (99mTc-DMSA) is demonstrated and the biological implication of hydrolytic polynucleation of pentavalent technetium, through an anionic specie Tc(V)O3-(4), in the tumor cell is discussed.

Imaging of Soft Tissue Tumors with Tc(v)-99m Dimercaptosuccinic Acid: A New Tumor-seeking Agent

Tumor scintigraphy, using Tc(V)-99m dimercaptosuccinic acid (Tc(V)-DMS) was performed in 58 patients with soft tissue tumors, and the results were compared with that of Ga-67 citrate. Tc(V)-DMS was found to have a sensitivity of 90% for malignant tumors including aggressive fibromatosis compared to that of Ga-67 citrate, which was 56%. However, the specificity of Tc(V)-DMS for these tumors was 71% but with Ga-67 citrate the specificity was 80%. The imaging accuracy in soft tissue tumors with Tc(V)-DMS and Ga-67 citrate was 78% and 71%, respectively. Although the accumulation of Tc(V)-DMS has been detected in some benign soft tissue tumors, the reduced accumulation in inflammatory lesions compared to Ga-67 citrate was recognized, and Tc(V)-DMS could be of great use in the detection of extension or location of malignant soft tissue tumors.

Synthesis and evaluation of a new bifunctional chelating agent for 99mTc labeling proteins: p-carboxyethylphenylglyoxal-di(N-methylthiosemicarbazone)

A new bifunctional chelating agent, p-carboxyethylphenylglyoxal-di(N-methylthiosemicarbazone) (CE-DTS), containing a di(N-methylthiosemicarbazone) as the technetium coordinating site and an aralkyl carboxylate site for the protein conjugation was synthesized. Coupling to human serum albumin (HSA), selected as a model protein was carried out by the phosphorylazide method using diphenylphosphoryl azide (DPPA). The conjugation level of CE-DTS to HSA played a critical role in its biological evaluation. A 99mTc-CE-DTS-HSA with high in vivo stability was obtained when CE-DTS was coupled to HSA at 1:1 molar ratio. This compound showed similar in vivo stability to 131I labeled HSA in mice and rabbits.

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.

In the procurement of a neutral and compact monomeric complex of dithiosemicarbazone (DTS) derivative: 99mTc-KTS.

As a basic structure of a bifunctional radiopharmaceutical (BR), dithiosemicarbazone (DTS) enables the formation of a small conjugated chelating ring with divalent metals (Cu2+, Zn2+, Ni2+), yielding stable and compact complexes. In the development of a neutral and compact monomeric DTS complex of technetium (99mTc), a DTS containing ligand, kethoxal-bis(thiosemicarbazone) (KTS) was selected. A well known monomeric Cu-KTS complex (or 64Cu-KTS) is taken as a model compound, and in vitro (TLC, EP, HPLC) and in vivo (mice biodistribution) studies were compared. Using the stannous chloride method, under conditions to avoid the hydrolytic polynucleation of technetium, a good yield of 99mTc-KTS with characteristics resembling the non-charged, compact and stable Cu-KTS is obtained, in in vitro as well as in vivo studies. The importance of DTS as the constituent of a BR is discussed.

pH sensitive properties of Tc(V)-DMS: analytical and in vitro cellular studies.

Numerous clinical studies with the pentavalent technetium complex of dimercaptosuccinic acid [Tc(V)-DMS] seem to indicate its new role in nuclear oncology. Thus, we questioned what properties of the Tc(V)-DMS molecule associate with its tumoral tissue accumulation. Because studies have reported tumor tissue to be more acidic than normal tissue, acidification might be related to the Tc(V)-DMS localization in tumor tissue. Thus, in the present study, a working hypothesis drew to test the acidification as a plausible factor, and various analytical methods and an in vitro cellular system using Ehrlich ascites tumor cells (EATC) implemented. Analytical methodologies demonstrated the decrease of the overall negative charge of the Tc(V)-DMS molecule, promoted by the acidification of the analytical medium and the sample dilution. In the in vitro cellular experiment, acidification alone showed no effect on the radioactivity accumulation in EATC; nevertheless, if accompanied by a pre-dilution of the Tc(V)-DMS sample added into the cell incubation media, cellular radioactivity accumulation was observed. Thus, acidification as a mediator for the Tc(V)-DMS accumulation in tumoral cells, concurrently with dilution as the promoter of the process, constituted the foundation for discerning the working hypothesis.

67Ga-labeled human fibrinogen: A new promising thrombus imaging agent

Gallium-67 labeled fibrinogen was prepared as a new deep-vein thrombus imaging agent, using deferoxamine (DF) as a bifunctional chelating agent. Excellent physicochemical and biological properties of 67Ga-DF-fibrinogen were obtained. The isotopic clottability was 85.3%. High stability in vivo as well as in vitro was observed. Studies carried out in rabbits with induced thrombi in the femoral vein showed thrombus to blood radioactivity ratio of 11.7, comparable to the value estimated with conventional 131I-fibrinogen 24 h after injection. Remarkable visualization of these thrombi with a scintillation camera provided good evidence for the selective mechanism of this new radiopharmaceutical.

In the procurement of stable 99mTc labeled protein using bifunctional chelating agent.

Recently, technetium-99m (99mTc) labeling of proteins through the use of a bifunctional chelating agent (BCA) has been reported. In previous work, a BCA containing a di(N-methylthiosemicarbazone) (DTS) moiety for 99mTc has offered good potential; however, conjugation with bulky proteins has induced some steric interference in the 99mTc labeling step using the stannous chloride method. In order to minimize this effect, changes in the hydrocarbon chain length (spacer) between the DTS and the protein binding site (carboxyl group), as well as the chemical state of the reducing agent, stannous chloride, were considered of interest. DTS molecules with variable spacer length (n = 0, 2, 4) were synthesized, coupled with human serum albumin and labeled with 99mTc. Also, stannous chloride prepared in different media was evaluated. Validity of the present approach is tested and discussed.

A comparison of the tumor-seeking agent Tc-99m(V) dimercaptosuccinic acid and the renal imaging agent Tc-99m dimercaptosuccinic acid in humans.

Being aware of the ideal nuclear properties of Tc-99m, our interest has been focused on the design of the (+5) oxidation state Tc-99m(V) dimercaptosuccinic acid (Tc(V)-DMSA) as a tumor-seeking agent. Tc-99m(V) DMSA holds a TcO4 3¯ core and, like PO4 3¯, has excellent characteristics for tumor uptake, but has a different distribution than the well-known renal scanning agent, Tc-99m DMSA. The differences in chemical behavior of Tc-99m(V) DMSA and Tc-99m DMSA are discussed. Three cases in which neoplasms were studies with Tc-99m(V) DMSA and Tc-99m DMSA are presented. Tc-99m DMSA and Tc-99m(V) DMSA, having a common ligand and tracer but, with the metal ion core in a different oxidation state, the uptake characteristics are altered markedly.

The development of 99mTc-analog of Cu-DTS as an agent for imaging hypoxia

Works on dithiosemicarbazone (DTS) derivatives radiolabeled with divalent Cu (Cu-62, Cu-64) indicate its potentiality as an ischemic tissue detecting agent. Development of analogous derivatives labeled with the more accessible technetium-99m (Tc) is most desirable. Various synthesized DTS derivatives are radiolabeled with a novel approach, using a macromolecular Sn(II)-complex under an anaerobic condition at pH 3.4-4.5 and stabilization by ascorbate solution at pH 6.7-7.0. Characterization of Tc-DTS derivatives done by various analytical methods (TLC, HPLC, EP, PC) and by in vivo studies in normal mice and in rats myocardial LAD (left anterior descent coronary artery) occlusion model. Among tested DTS, only Tc-ATSE, Tc-ATSM and Tc-ATSM(2) showed distinctive characteristics, with the latter presenting high myocardium uptake in regions of ischemia in LAD rat myocardium model. Potentiality of the Cu-DTS mimetic agent, Tc-ATSM(2) as an ischemia-damaged myocardium agent is discussed.