荒野 泰

An enhancement of the target/nontarget ratio of radioactivity levels enables reliable diagnosis and therapy using polypeptide radiopharmaceuticals in nuclear medicine. In the present study, we investigated the effects of the physicochemical properties of radiometabolites on the radioactivity pharmacokinetics after administration of Tc-99m-labeled polypeptides using 6-hydrazinopyridine-3-carboxylic acid (HYNIC). Four ternary ligands (L) [3-benzoylpyridine (BP), 3-acetylpyridine (AP), 3-nicotinic acid (NIC), pyridine (PY)] with different lipophilicity were selected as coligands for the preparation of Tc-99m-HYNIC-polypeptides. Each of the ternary ligands tested provided Tc-99m-HYNIC-labeled galactosyl-neoalbumin (NGA) and Fab fragments of high stability with high radiochemical purity. Moreover, after administration of each Tc-99m-HYNIC-labeled NGA into normal mice, the respective ternary ligand [Tc-99m](HYNIC-lysine)(tricine)(L) complexes were generated as final radiometabolites in the hepatic lysosome. The partition coefficients of [Tc-99m](HYNIC-lysine)(tricine)(BP), [99mTc](HYNIC-lysine)(tricine)(AP), [99mTc](FrYNIC-lysine)(tricine)(NIC), and [Tc-99m(HYNIC-lysine)(tricine)(PY) were determined to be -2.21, -2.37, -2.93, and -2.73, respectively. Elimination rates of these radiometabolites from the lysosome were enhanced in the order of increasing lipophilicity of the radiometabolites. After injection of the four Tc-99m-HYNIC-labeled Fab fragments into normal mice, blood clearances of radioactivity were similar while radioactivity elimination rates from the kidney were enhanced in the order of increasing lipophilicity of the radiometabolites. The present study indicated that the lipophilicity of the radiometabolites constitutes one important factor affecting their elimination rates from the tissues. Thus, as ternary ligands facilitate alteration of the physicochemical properties of radiometabolites, the use of ternary ligand complexes might be applicable for controlling the pharmacokinetics of Tc-99m-labeled polypeptides.

The present study compares distribution and elimination characteristics of In-111-DTPA-D-Phe(1)-octreotide and In-111-DTPA-L-Phe(1)-octreotide in rats and evaluated the effect of the replacement of the terminal L-phenylalanine by D-phenylalanine on pharmacokinetic profiles of the radiolabelled peptides. Both agents exhibited rapid radioactivity clearance from the blood and most organs and tissues with no systematic and significant differences in activity accumulation. The long-term retention and high radioactivity concentrations for both compounds under study were found in the kidneys and organs with a high density of somatostatin receptors, such as the pancreas and adrenals. The residence times in these organs were longer for In-111-DTPA-D-Phe(1)-octreotide in comparison with In-111-DTPA-L-Phe(1)-octreotide. The major elimination pathway for both radiolabelled peptides was relatively rapid excretion into the urine. Analysis of the renal handling by an employment of the perfused rat kidney showed that both peptides were eliminated mainly by the mechanism of glomerular filtration. Rat liver perfusion experiments confirmed a very low value of bile clearance of radioactivity for both agents under study.

99mTc radiopharmaceuticals play an important role in widespread applications of nuclear medicine. When 99mTc radiopharmaceuticals first came into use, major efforts were directed toward the development of 99mTc radiopharmaceuticals for bone imaging and for the excretory functions of the liver and kidneys. In the past 20 years, a significant advance has been made in technetium chemistry, which provided 99mTc radiopharmaceuticals for assessment of regional cerebral and myocardial blood flow. Recent efforts have been directed toward the design of 99mTc-labeled compounds for estimating receptor or transporter functions. A number of bifunctional chelating agents that provide 99mTc labeled proteins and peptides of high in vivo stability with high radiochemical yields have also been developed. More recently, organometallic technetium and rhenium compounds have been introduced as another class of 99mTc radiopharmaceutical design. In this manuscript, recent progress in 99mTc radiopharmaceuticals is reviewed with the major emphasis laid on key innovations in this field to provide the 99mTc radiopharmaceuticals available today.

The present study compares distribution and elimination characteristics of In-111-DTPA-D-Phe(1)-octreotide and In-111-DTPA-L-Phe(1)-octreotide in rats and evaluated the effect of the replacement of the terminal L-phenylalanine by D-phenylalanine on pharmacokinetic profiles of the radiolabelled peptides. Both agents exhibited rapid radioactivity clearance from the blood and most organs and tissues with no systematic and significant differences in activity accumulation. The long-term retention and high radioactivity concentrations for both compounds under study were found in the kidneys and organs with a high density of somatostatin receptors, such as the pancreas and adrenals. The residence times in these organs were longer for In-111-DTPA-D-Phe(1)-octreotide in comparison with In-111-DTPA-L-Phe(1)-octreotide. The major elimination pathway for both radiolabelled peptides was relatively rapid excretion into the urine. Analysis of the renal handling by an employment of the perfused rat kidney showed that both peptides were eliminated mainly by the mechanism of glomerular filtration. Rat liver perfusion experiments confirmed a very low value of bile clearance of radioactivity for both agents under study.

The effect of molecular charges on renal accumulation of In-111-DTPA-labeled low molecular weight (LMW) peptides was investigated using In-111-DTPA-octreotide derivatives as models to design radiolabeled peptides that are taken up less by renal cells. The N-terminal D-phenylalanine (Phe) of In-111-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 In-111-DTPA-L-Phe(1)-octreotide and In-111-DTPA-L-Met(1)-octreotide showed similar net charges, whereas In-111-DTPA-L-(alpha)Lys(1)-octreotide and In-111-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. In-111-DTPA-L-(alpha)Lys(1)-octreotide showed the highest radioactivity levels from 10 min to 6 h postinjection, whereas the lowest radioactivity levels were observed with In-111- DTPA-L-Asp(1)-octreo tide at all the postinjection intervals. These findings indicated that the replacement of only one amino acid in In-111-DTPA-D-Phe(1)-octreotide significantly altered net molecular charges of the resulting peptides and that the net charges of the In-111-DTPA-octreotide derivatives significantly affected their renal uptake. Thus, an increase of negative charges in peptide molecules may constitute a strategy for designing In-111-DTPA-conjugated LMW peptides with low renal radioactivity levels. (C) 2001 Elsevier Science Inc. All rights reserved.

Metabolic studies of In-111-DTPA-labeled polypeptides and peptides showed that the radiolabeled (poly)peptides generated In-111-DTPA-adducts of amino acid that possess long residence times in the lysosomal compartment of the tissues where (poly)peptides accumulated. However, a recent study suggested that metal-chelate-methionine (Met) might possess in vivo behaviors different from metal-chelate adducts of other amino acids. Tn this study, to elucidate whether some biological characteristics of Met may accelerate the renal elimination rate of In-111-DTPA-adduct of Met into urine, In-111-DTPA-Met(1)-octreotide was synthesized and the renal handling of In-111-DTPA-Met was investigated using In-111-DTPA-L-Phe(1)-octreotide (Phe represents phenylalanine), which was reported previously, as a reference. Both In-111-DTPA-conjugated octreotide analogs were stable against 3-h incubation in murine serum at 37 degreesC. Both In-111-DTPA-octreotide analogs also showed rapid clearance of the radioactivity from the blood and similar accumulation of the radioactivity in the kidney. No significant differences were observed in the renal radioactivity levels from 10 min to 24 h postinjection between the two. Metabolic studies indicated that In-111-DTPA-Met(1)-octreotide and In-111-DTPA-L-Phe(1)-octreotide generated In-111-DTPA-adducts of Met and Phe, respectively, as the final radiometabolites at similar rates. These findings suggested that the long residence times of the radioactivity in tissues after administration of In-111-DTPA-labeled peptides and polypeptides would be attributed to inherent characteristics of In-111-DTPA chelate. (C) 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'-[I-131] iodohippuryl N-is an element of-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 [I-131]HML-IT-Fab and [I-125]HML-Fab, respectively. In biodistribution studies in mice, both [I-131]HML-IT-Fab and [I-125]HML-Fab demonstrated significantly lower renal radioactivity levels than those of [125I]Fab. In subcellular distribution studies, [I-125]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 [I-131]HML-IT-Fab and [I-125]- HML-Fab. In metabolic studies, while [I-125]Fab remained intact at 10 min postinjection, both HML-conjugated Fab fragments generated m-iodohippuric acid as a radiometabolite at the same postinjection time. [I-131]HML-IT-Fab registered two radiometabolites (intact [131I]HML-IT-Fab and m-iodohippuric acid), whereas additional radiometabolites were observed with [I-125]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.

Renal localization of radiolabeled antibody fragments constitutes a problem in targeted imaging and radiotherapy. Recently, we reported use of a novel radioiodination reagent, 3'-[I-131] iodohippuryl N-is an element of-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 [I-131]HML-IT-Fab and [I-125]HML-Fab, respectively. In biodistribution studies in mice, both [I-131]HML-IT-Fab and [I-125]HML-Fab demonstrated significantly lower renal radioactivity levels than those of [125I]Fab. In subcellular distribution studies, [I-125]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 [I-131]HML-IT-Fab and [I-125]- HML-Fab. In metabolic studies, while [I-125]Fab remained intact at 10 min postinjection, both HML-conjugated Fab fragments generated m-iodohippuric acid as a radiometabolite at the same postinjection time. [I-131]HML-IT-Fab registered two radiometabolites (intact [131I]HML-IT-Fab and m-iodohippuric acid), whereas additional radiometabolites were observed with [I-125]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) constitutes one of the most attractive reagents to prepare Tc-99m-labeled polypeptides and peptides of various molecular weights in combination with two tricine molecules as coligands. Indeed, Tc-99m-HYNIC-conjugated IgG showed biodistribution of radioactivity similar to that of In-111 DTPA-conjugated IgG. However, recent studies indicated significant plasma protein binding when the Tc-99m labeling procedure was expanded to low molecular weight peptides. In this study, pharmacokinetics of Tc-99m-HYNIC-conjugated IgG, Fab and RC160 using tricine were compared with their radioiodinated counterparts to evaluate this Tc-99m-labeling method. In mice, [Tc-99m](HYNIC-IgG)(tricine)(2) and [Tc-99m](HYNIC-Fab)(tricine)(2) showed persistent localization of radioactivity in tissues when compared with their I-125-labeled counterparts. [Tc-99m](HYNIC-IgG)(tricine)(2) eliminated from the blood at a rate similar to that of I-125-labeled IgG, while [Tc-99m](HYNIC Fab)(tricine)(2) showed significantly slower clearance of the radioactivity than I-125-labeled Fab. On size-exclusion HPLC analyses, little changes were observed in radiochromatograms after incubation of [Tc-99m](HYNIC-IgG)(tricine)(2) in murine plasma. However, [Tc-99m](HYNIC-Fab)(tricine)(2) and [Tc-99m](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 [Tc-99m](HYNIC-RC160)(tricine)(2) was stabilized with nicotinic acid (NIC) to generate [Tc-99m](HYNIC-RC160)(tricine)(NIC). [(TC)-T-99m](HYNIC-RC160)(tricine)(NIC) also demonstrated significantly faster clearance of the radioactivity from the blood than [Tc-99m](HyNIC-RC160)(tricine)(2). These findings suggested that one of the tricine coligands in Tc-99m-HYNIC-labeled (poly)peptides would he 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. (C) 2001 Elsevier Science Inc. All rights reserved.

To elucidate the cause for the different levels of hepatic radioactivity among mammals after injection of protein radiopharmaceuticals, the metabolism of radiolabeled proteins and the fate of their radiometabolites in the parenchymal cells of rat liver were investigated and compared with those of mice. We used galactosyl-neoglycoalbumin (NGA) as a carrier protein, and NGA was labeled with In-111 via 1-(4-isothiocyanatobenzyl)ethylenediaminetetraacetic acid (SCN-Bz-EDTA) or 1-[p-(5-maleimidopentyl) aminobenzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) and with I-125 via direct iodination. All radiolabeled NGAs exhibited rapid accumulation in liver parenchymal cells after intravenous injection into rats. Radioactivity was eliminated following NGA-I-125 injection at similar rates from rat and mouse liver. In contrast, both In-111-labeled NGAs demonstrated much slower elimination of radioactivity in rat when compared with mouse liver. Analyses of radioactivity in bile and liver indicated that both SCN-Bz-EDTA and EMCS-Bz-EDTA rendered mono amino acid adducts as the final radiometabolites, which were generated in rat liver within 1 h postinjection. Subcellular distribution studies suggested that these radiometabolites were copurified with lysosome in rat liver. Because similar results were observed in mice previously, the difference between rats and mice in radioactivity elimination from liver parenchymal cells would be predominantly attributable to the different efflux rate of the In-111-labeled metabolites from the lysosome between these species. Such differences in the efflux rates of radiometabolites from the lysosome among mammals may also account for the different hepatic radioactivity levels of radiolabeled proteins between animal and clinical studies. NUCL MED BIOL 26;3:281-289, 1999. (C) 1999 Elsevier Science Inc. All rights reserved.

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.

Pharmacokinetic analyses of protein pharmaceuticals are of prime importance for their clinical application. Because many proteins have pharmacological activity at low concentrations, radiolabelling of proteins is widely used to identify the sites and determine the rates of protein catabolism in-vivo due to the high sensitivity of detection of radioactivity. Recently, a metallic radionuclide, In-111, has been used to trace the pharmacokinetics of proteins of interest after conjugation of the proteins with diethylenetriaminepentaacetic acid (DTPA). In this study, galactosyl-neoglycoalbumin (NGA) was reacted with the cyclic dianhydride of DTPA and labelled with In-111 to estimate the validity of this radiolabelling procedure for pharmacokinetic analyses, For comparison, we also evaluated direct radioiodination, because directly-radioiodinated proteins are widely used to assess the pharmacokinetics of proteins of interest. The hepatic radioactivity profile after intravenous injection of [I-131]NGA or [In-111]DTPA-NGA into mice was analysed pharmacokinetically, and the first-order rate constant representing the elimination of the respective radiometabolite from hepatic parenchymal cells was determined. The results indicated that direct radioiodination is inappropriate for pursuing the pharmacokinetics of the proteins, because of rapid elimination of the radioactivity from the sites of protein catabolism. These findings also implied that the [In-111]DTPA label could be used to identify the catabolic sites and determine the rates of catabolism of proteins with relatively short biological half-lives, although characterization of radiolabelled species at the sites of accumulation would be required for accurate determination of the catabolic sites of proteins.