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The aim of the present study is to synthesize new mannosylated dextran derivative that can be labeled with Tc-99m for potential use in sentinel lymph node detection (SLND). The compound was designed to have a dextran with molecular weight of 10 kDa as a backbone, mannose for binding to mannose receptors of the lymph node and S-derivatized cysteine as a suitable chelator for labeling with [99mTc(H2O)3(CO)3]+precursor. Reaction of allyl bromide with dextran (MW 11800) yielded the intermediate allyl-dextran (1) with about 40% coupling. Addition of cysteine to allyl-dextran resulted in the S-derivatized cysteine, compound DC15 (2). The final product DCM20 (3) was obtained in good yield after in situ hydrolysis and activation of cyanomethyl tetraacetyl-1-thio-d-mannopyranoside and coupling to DC15. All derivatives were purified by ultrafiltration and characterized by NMR. DC15 and DCM20 were quantitatively labeled with 99mTc (>95% radiochemical purity) using the fac-[99mTc(OH2)3(CO)3]+precursor and ligand concentration of 1.5 × 10–6M at neutral pH. Both 99mTc-labeled compounds 99mTc(CO)3–DC15 (6) and 99mTc(CO)3–DCM20 (7) remained stable after 6 h incubation at 37 °C in the presence of excess histidine or cysteine, as well as even after 20-fold dilution and incubation for 24 h at room temperature. The characterization of the compounds 6and 7was performed by comparing their HPLC radiochromatograms with those of their rhenium surrogates Re(CO)3–DC15 (4) and Re(CO)3–DCM20 (5) respectively that were prepared using the precursor [NEt4]2fac-[ReBr3(CO)3] and characterized by IR and NMR spectroscopy. When injected subcutaneously from the foot pad of mice, 99mTc-labeled mannosylated dextran (7) showed accumulation in the popliteal lymph node (SLN in this model) higher than that of non-mannosylated analogue (6) and the 99mTc-phytate serving as standard. Compound 7also exhibited lower radioactivity levels at the injection site compared to 99mTc-phytate. The SPECT/CT studies in mice confirmed that 7accumulated in the popliteal lymph node allowing its clear visualization. The present findings demonstrate that compound 7(99mTc(CO)3-DCM20) is promising and merits further evaluation as a radiopharmaceutical for sentinel lymph node detection.

In vivo imaging of β-amyloid (Aβ) peptide aggregates in the brain may lead to early detection of Alzheimer's disease (AD) and monitoring of the progression and effectiveness of AD treatment. The purpose of this study was to develop novel amyloid imaging agents based on flavone as a core structure. Radioiodinated flavone derivatives were designed and synthesized. The binding affinities of flavone derivatives for Aβ aggregates varied from 13 to 77 nM. When in vitro plaque labeling was carried out using post-mortem AD brain sections, all flavones intensely stained not only amyloid plaques but also cerebrovascular amyloids. In biodistribution studies using normal mice, they displayed high brain uptakes ranging from 3.2 to 4.1% ID/g at 2 min postinjection. The radioactivity washed out from the brain rapidly (0.5−1.9% ID/g at 30 min), which is highly desirable for amyloid imaging agents. The results in the study suggest that these classes of radioiodinated flavones may be useful candidates as potential imaging agents for amyloid plaques.

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 N^ε-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 N^ε-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 N^ε-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.

Rhenium-186-1-hydroxyethylidene-1,1-diphosphonate (¹⁸⁶Re-HEDP) has been used for the palliation of metastatic bone pain. Delayed blood clearance and high gastric uptake of radioactivity have been observed upon injection, due to the instability of ¹⁸⁶Re-HEDP in vivo. In this study, on the basis of the concept of bifunctional radiopharmaceuticals, we designed a stable ¹⁸⁶Re-mercaptoacetylglycylglycylglycine (MAG3) complex-conjugated bisphosphonate, [[[[(4-hydroxy-4,4-diphosphonobutyl)carbamoylmethyl]carbamoylmethyl]carbamoylmethyl]carbamoylmethanethiolate]oxorhenium(V) (¹⁸⁶Re-MAG3-HBP). As a precursor, [1-hydroxy-1-phosphono-4-[2-[2-[2-(2-tritylmercaptoacetylamino)acetylamino]acetylamino]acetylamino]butyl]phosphonic acid (Tr-MAG3-HBP) was synthesized by the conjugation of N-[(tritylmercapto)acetyl]glycylglycylglycine (Tr-MAG3) with the bisphosphonate analogue. After deprotection of the trityl group of Tr-MAG3-HBP, ¹⁸⁶Re-labeling was performed by reacting ¹⁸⁶ReO4^- with SnCl2 in citrate buffer. After purification by HPLC, ¹⁸⁶Re-MAG3-HBP showed a radiochemical purity of over 95%. To compare the stability of ¹⁸⁶Re-MAG3-HBP and ¹⁸⁶Re-HEDP, these ¹⁸⁶Re complexes were incubated in phosphate buffer. No measurable decomposition of ¹⁸⁶Re-MAG3-HBP occurred over a 24-h period, while only approximately 30% of ¹⁸⁶Re-HEDP remained intact 24 h postincubation. In biodistribution experiments, the radioactivity level of ¹⁸⁶Re-MAG3-HBP in bone was significantly higher than that of ¹⁸⁶Re-HEDP. Blood clearance of ¹⁸⁶Re-MAG3-HBP was faster than that of ¹⁸⁶Re-HEDP. In addition, the gastric accumulation of ¹⁸⁶Re-MAG3-HBP radioactivity was lower than that of ¹⁸⁶Re-HEDP. In conclusion, ¹⁸⁶Re-MAG3-HBP is expected to be a useful radiopharmaceutical for the palliation of metastatic bone pain.

The reduction of acetylcholinesterase (AChE) activity in the brain has been measured in dementia disorders such as Alzheimer's disease and dementia with Lewy bodies using ¹¹C-labeled acetylcholine analogues, N-[¹¹C]methylpiperidin-4-yl acetate and propionate, and positron emission tomography (PET). Our aim was to develop an ¹⁸F-labeled acetylcholine analogue useful for brain AChE mapping with PET, since ¹⁸F, with a longer half-life, has advantages over ¹¹C. In a preliminary study, a series of N-[¹⁴C]ethylpiperidin-3-yl or -4-ylmethanol esters (acetyl and propionyl esters) were newly designed and evaluated in vitro regarding the reactivity with and specificity to AChE using purified human enzymes, leading to a novel ¹⁸F-labeled acetylcholine analogue, N-[¹⁸F]fluoroethylpiperidin-4-ylmethyl acetate. In rat experiments, the ¹⁸F-labeled candidate showed desirable properties for PET AChE measurement:  high brain uptake of the authentic ester, high AChE specificity, a moderate hydrolysis rate, and low membrane permeability (metabolic trapping) of the metabolite.

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.

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 ^99mTc-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 ^99mTc−HYNIC-polypeptides. Each of the ternary ligands tested provided ^99mTc−HYNIC-labeled galactosyl-neoalbumin (NGA) and Fab fragments of high stability with high radiochemical purity. Moreover, after administration of each ^99mTc−HYNIC-labeled NGA into normal mice, the respective ternary ligand [^99mTc](HYNIC−lysine)(tricine)(L) complexes were generated as final radiometabolites in the hepatic lysosome. The partition coefficients of [^99mTc](HYNIC−lysine)(tricine)(BP), [^99mTc](HYNIC−lysine)(tricine)(AP), [^99mTc](HYNIC−lysine)(tricine)(NIC), and [^99mTc](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 ^99mTc−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 ^99mTc-labeled polypeptides.

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^ε-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.

External imaging of energy production activity of living cells with ^99mTc-labeled compounds is a challenging task requiring good design of ^99mTc-radiopharmaceuticals. On the basis of our recent findings that ¹¹C- and ¹²³I-labeled medium-chain fatty acids are useful for measuring β-oxidation activity of hepatocytes, we focused on development of ^99mTc-labeled medium-chain fatty acid analogues that reflect β-oxidation activity of the liver. In the present study, monoamine−monoamide dithiol (MAMA) ligand and triamido thiol (MAG) ligand were chosen as chelating groups because of the stability and size of their complexes with ^99mTc and their ease of synthesis. Each ligand was attached to the ω-position of hexanoic acid (MAMA-HA and MAG-HA, respectively). In biodistribution studies, [^99mTc]MAMA-HA showed high initial accumulation in the liver followed by clearance of the radioactivity in the urine. Analysis of the urine revealed [^99mTc]MAMA-BA as the sole radiometabolite. Furthermore, when [^99mTc]MAMA-HA was incubated with living liver slices, generation of [^99mTc]MAMA-BA was observed. However, [^99mTc]MAMA-HA remained intact when the compound was incubated with liver slices in the presence of 2-bromooctanoate, an inhibitor of β-oxidation. The findings in this study indicated that [^99mTc]MAMA-HA was metabolized by β-oxidation after incorporation into the liver. On the other hand, poor hepatic accumulation was observed after administration of [^99mTc]MAG-HA.

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 between ^99mTc 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.

To develop chelating molecules that provide ^99mTc-labeled polypeptides of high in vivo stability and high specific activities under mild reaction conditions, an asymmetrical bis(benzohydroxamamide) compound with an amine group, 4‘-aminomethyl-N,N‘-trimethylenedibenzohydroxamamide [NH2−C3(BHam)2], was designed and synthesized. The amine residue of NH2−C3(BHam)2 was converted to a maleimide group by reaction with N-succinimidyl-6-maleimidohexanoate, and the conjugation product was coupled to thiol groups of a monoclonal antibody against osteogenic sarcoma (OST7, IgG1) pretreated with 2-iminothiolane to prepare C3(BHam)2−OST7. ^99mTc radiolabeling of C3(BHam)2−OST7 was performed by the exchange reaction with [^99mTc]glucoheptonate. [^99mTc]C3(BHam)2−OST7 was further characterized using directly radioiodinated OST7 ([¹²⁵I]OST7) and [¹¹¹In]labeled OST7 with 1-[4-[(5-maleimidopentyl)amidobenzyl]ethylenediamine-N,N,N‘N‘-tetraacetic acid (EMCS−Bz-EDTA) as references. [^99mTc]C3(BHam)2−OST7 was obtained with radiochemical yields of over 94% at protein concentrations as low as 0.2 mg/mL at room temperature for 1 h. [^99mTc]C3(BHam)2−OST7 remained stable after incubation in freshly prepared murine plasma and in the presence of cysteine. Similar binding affinities to tumor cells were observed between [^99mTc]C3(BHam)2−OST7 and [¹²⁵I]OST7. When injected into normal mice, [^99mTc]C3(BHam)2−OST7 exhibited radioactivity levels in the blood similar to [¹¹¹In]-EMCS−Bz-EDTA-OST7 up to 24 h postinjection with significantly faster elimination rate of the radioactivity from the liver. In nude mice bearing osteogenic sarcoma, no significant differences were observed in the radioactivity levels in the blood and the tumor between [^99mTc]C3(BHam)2−OST7 and [¹²⁵I]OST7 at 24 h postinjection. These findings indicated that C3(BHam)2 provided ^99mTc chelate of high stability at low concentrations even when conjugated to an intact antibody. Such characteristics render bis(hydroxamamide) compounds useful as chelating molecules for preparation of ^99mTc-labeled polypeptides.

Previous studies on indium-111 (¹¹¹In) labeling of polypeptides and peptides using cyclic diethylenetriaminepentaacetic dianhydride (cDTPA) as a bifunctional chelating agent (BCA) have indicated that DTPA might be a useful BCA for ¹¹¹In labeling of polypeptides at high specific activities when DTPA can be incorporated without inducing intra- or intermolecular cross-linking. To investigate this hypothesis, a monoreactive DTPA derivative with a maleimide group as the peptide binding site (MDTPA) was designed and synthesized. A monoclonal antibody (OST7, IgG1) was used as a model polypeptide, and conjugation of MDTPA with OST7, ¹¹¹In radiolabeling of MDTPA−OST7, and the stability of ¹¹¹In−MDTPA−OST7 were investigated using cDTPA and benzyl-EDTA derivatives as references. SDS−PAGE analysis demonstrated that while cDTPA induced intramolecular cross-linking, no such undesirable side reactions were observed with MDTPA. MDTPA generated ¹¹¹In-labeled OST7 with high radiochemical yields at higher specific activities than those produced using cDTPA and benzyl-EDTA derivatives as the BCAs. Incubation of each ¹¹¹In-labeled OST7 in human serum indicated that MDTPA generated ¹¹¹In-labeled OST7 of much higher and a little lower stability than those derived from cDTPA and benzyl-EDTA derivatives, respectively. These findings indicated that the low in vivo stability of cDTPA-conjugated antibody reported previously is not attributable to low stability of ¹¹¹In−DTPA but to formation of intramolecular cross-linking during cDTPA conjugation reactions. The present study also indicated that MDTPA and its precursor, the tetra-tert-butyl derivative of DTPA, would be useful BCAs for ¹¹¹In radiolabeling of polypeptides that have rapid blood clearance with high specific activities.

Radiochemical design of polypeptides using metabolizable linkages would be attractive to enhance target-selective localization of radioactivity for diagnostic and therapeutic nuclear medicine. However, while use of ester bonds as the linkage allows selective release of the designed radiometabolite from covalently conjugated polypeptides after lysosomal proteolysis in nontarget tissues, low plasma stability of ester bonds causes a decrease in radioactivity levels of the target. In pursuit of new metabolizable linkages that provide stable attachment of radiolabels with polypeptide in plasma while facilitating rapid and selective release of designed radiometabolites of rapid urinary excretion in lysosomes, a new radioiodination reagent with l-lysine as the metabolizable linkage to liberate m-iodohippuric acid (l-HML) was designed and synthesized. Stabilities of the metabolizable linkage in serum and cleavabilities of the linkage in lysosomal proteolysis in hepatic cells were investigated after conjugation of [¹³¹I]-l-HML with galactosyl-neoglycoalbumin (NGA). For comparison, a radioiodination reagent with an ester bond to release m-iodohippuric acid (MIH) was conjugated with NGA under similar conditions. When incubated in human serum, [¹³¹I]-l-HML-NGA liberated less than 3% of the initial radioactivity after 24 h, whereas [¹²⁵I]MIH-NGA released more than 60% of its radioactivity during the same interval. In biodistribution studies, [¹³¹I]-l-HML-NGA demonstrated radioactivity elimination from murine liver at a rate and excretion route similar to [¹²⁵I]MIH-NGA. Analyses of murine urine after injection of [¹³¹I]-l-HML-NGA indicated a single radioactivity peak at fractions identical to those of m-iodohippuric acid. Biodistribution studies of radioiodinated NGAs with d-lysine or cadaverine as the linkages demonstrated a delayed elimination rate from murine liver with significantly higher radioactivity being excreted in the feces at 24 h postinjection. Thus, l-HML is the first reagent that allows stable attachment of radiolabel with polypeptide in serum while facilitating selective release of a radiometabolite with rapid urinary excretion from covalently conjugated polypeptides after lysosomal proteolysis at a rate similar to that of ester bonds. Thus, l-HML is potentially useful for the radioiodination of polypeptides for diagnostic and therapeutic purposes.

The persistent localization of radioactivity in the kidney after administration of ¹¹¹In-DTPA-d-Phe¹-octreotide impairs the diagnostic accuracy of this radiopharmaceutical. To better understand the mechanisms responsible for the renal radioactivity levels of ¹¹¹In-DTPA-d-Phe¹-octreotide, the renal metabolism of this compound was compared with ¹¹¹In-DTPA-l-Phe¹-octreotide, where the N-terminal d-phenylalanine was replaced with l-phenylalanine to facilitate metabolism. DTPA-d-Phe¹-octreotide and DTPA-l-Phe¹-octreotide were synthesized by solid-phase methods. Both ¹¹¹In-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 °C. When injected into mice, the two ¹¹¹In-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 ¹¹¹In-DTPA-l-Phe¹-octreotide was rapidly metabolized to the final radiometabolite, ¹¹¹In-DTPA-l-Phe, the metabolic rate of ¹¹¹In-DTPA-d-Phe¹-octreotide was so slow that various intermediate radiolabeled species were observed. However, both ¹¹¹In-DTPA-d-Phe and ¹¹¹In-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 ¹¹¹In-DTPA-d-Phe¹-octreotide in the renal cells may be partially involved, the slow elimination rate of the radiometabolite derived from ¹¹¹In-DTPA-d-Phe¹-octreotide from the lysosomal compartment of renal cells would be predominantly attributable to the persistent renal radioactivity levels of ¹¹¹In-DTPA-d-Phe¹-octreotide.

Interposition of a metabolizable linkage has been performed to reduce the hepatic radioactivity levels of radiolabeled antibodies. To estimate the validity of this strategy, a radioiodination reagent (HML) that provides a stable attachment for m-iodohippuric acid with proteins in plasma while facilitating rapid and selective release of the compound after lysosomal proteolysis in the liver was conjugated with a monoclonal antibody (mAb) against osteogenic sarcoma (OST7, IgG1). Radiolabeled OST7 conjugates with a plasma-labile ester bond for releasing m-iodohippuric acid (MIH), plasma-stable amide bonds for releasing radiometabolites of hepatobiliary excretion (MPH), or slow elimination rates from hepatocytes ([¹¹¹In]EMCS-Bz-EDTA) were prepared with similar conjugation chemistry. The four radiolabeled OST7 conjugates were characterized both in vitro and in vivo. All the radiolabeled OST7 conjugates had similar radiochromatograms on size-exclusion HPLC and similar antigen binding affinities. While MIH−OST7 indicated accelerated clearance of radioactivity from the blood due to the release of m-iodohippurate, the rest of the three radiolabeled OST7 conjugates remained stable in serum incubation studies and had similar radioactivity elimination from the blood in vivo. When injected into normal mice, HML−OST7 demonstrated tissue-to-blood ratios of radioactivity similar to those of MIH−OST7 and significantly lower than those of the other two radiolabeled OST7 conjugates. In biodistribution studies in nude mice, both HML−OST7 and MIH−OST7 exhibited tumor-to-liver or tumor-to-intestine ratios of radioactivity higher than those of [¹¹¹In]EMCS-Bz-EDTA−OST7 or MPH−OST7, respectively. HML−OST7, MPH−OST7, and [¹¹¹In]EMCS-Bz-EDTA−OST7 indicated there were no changes in the radioactivity levels in the tumor between 24 and 48 h postinjection, whereas MIH−OST7 significantly decreased the radioactivity levels in the tumor at these time points. HML reduced the radioactivity levels in nontarget tissues without impairing the tumor radioactivity levels delivered by OST7. These findings indicated that the design of a radiolabeled mAb that is stable in plasma and liberates the radiometabolite of rapid urinary excretion constitutes an effective strategy for achieving target-selective radioactivity delivery.

Successful imaging of somatostatin receptor-positive tumors with ¹¹¹In-DTPA-d-Phe¹-octreotide has stimulated development of peptide radiopharmaceuticals using DTPA as the chelating agent. However, use of cyclic DTPA dianhydride (cDTPA) resulted in low synthetic yields of DTPA-peptide by either solution or solid-phase syntheses. This paper reports a novel high-yield synthetic procedure for DTPA-d-Phe¹-octreotide that is applicable to other peptides of interest using a monoreactive DTPA derivative. A monoreactive DTPA that possesses one free terminal carboxylic acid along with four carboxylates protected with tert-butyl ester (mDTPA) was synthesized. Fmoc-Thr(^tBu)-ol, prepared from Fmoc-Thr(^tBu)-OH, was loaded onto 2-chlorotrityl chloride resin. After construction of the peptide chains by Fmoc chemistry, mDTPA was coupled to the α amine group of the peptide on the resin in the presence of 1,3-diisopropylcarbodiimide and 1-hydroxybenzotriazole. Treatment of the mDTPA-peptide−resin with trifluoroacetic acid−thioanisole removed the protecting groups and liberated [Cys(Acm)^2,7]-octreotide-d-Phe¹-DTPA from the resin. Iodine oxidation of the DTPA-peptide, followed by the reversed-phase HPLC purification, produced DTPA-d-Phe¹-octreotide in overall 31.8% yield based on the starting Fmoc-Thr(^tBu)-ol-resin. The final product gave a single peak on analytical HPLC, and amino acid analysis and mass spectrometry confirmed the integrity of the product. ¹¹¹In radiolabeling of the product provided ¹¹¹In-DTPA-d-Phe¹-octreotide with >95% radiochemical yield, as confirmed by analytical reversed-phase HPLC, TLC, and CAE. These findings indicated that use of mDTPA during solid-phase peptide synthesis greatly increased the synthetic yield of DTPA-d-Phe¹-octreotide, due to the absence of nonselective reactions that are unavoidable when cDTPA is used. These results also suggested that mDTPA would be a versatile reagent to introduce DTPA with high yield into peptides of interest.

Reduction of radioactivity levels in nontarget tissues such as the liver and kidney constitutes a problem to be resolved in diagnostic and therapeutic applications of radiolabeled monoclonal antibodies (mAbs). A new radioiodination reagent with an ester bond to liberate m-iodohippuric acid from covalently conjugated proteins, maleimidoethyl 3-(tri-n-butylstannyl)hippurate (MIH), was recently developed. MIH liberated m-iodohippuric acid from galactosylneoglycoalbumin in murine liver, and the radiometabolite was rapidly eliminated from the liver into urine as an intact structure. In this study, intact IgG and Fab fragment of a mAb against osteogenic sarcoma were radioiodinated with MIH to further assess the applicability of MIH to radioimmunoimaging and therapy. For comparison, a mAb radioiodinated with N-succinimidyl iodobenzoate (SIB) and indium-111 (¹¹¹In)-labeled mAbs with diethylenetriaminepentaacetic dianhydride (cDTPA) or 1-[4-[(5-maleimidopentyl)amino]benzyl]ethylenediaminetetraacetic acid (EMCS-Bz-EDTA) were used. Size-exclusion HPLC analysis and cell binding assays indicated the preservation of both structure and antigen binding affinity of radioiodinated MIH−OST7 (IgG). In biodistribution studies in mice, [¹²⁵I]MIH−OST7 (IgG) showed faster systemic clearance of radioactivity after 24 h postinjection than did [¹³¹I]SIB− and [¹¹¹In]EMCS-Bz-EDTA−OST7 (IgG). [¹²⁵I]MIH−OST7 (IgG) also exhibited much lower radioactivity levels in nontarget tissues such as the liver and kidney, with higher radioactivity levels in the blood up to 72 h postinjection when compared with [¹¹¹In]cDTPA−OST7 (IgG). Radioactivity excreted from the mice was found in the urine as m-iodohippuric acid, following administration of [¹²⁵I]MIH−OST7 (IgG). In athymic mice bearing osteogenic sarcoma, [¹³¹I]MIH−OST7 (IgG) indicated higher tumor-to-nontarget ratios of radioactivity at both 24 and 48 h postinjection than [¹²⁵I]SIB−OST7 (IgG). Although both radioiodinated OST7s showed similar radioactivity levels in the target at 24 h postinjection, a small but significant decrease in the target radioactivity level was observed with [¹³¹I]MIH−OST7 (IgG) at 48 h postinjection. In addition, [¹³¹I]MIH−OST7 (Fab) showed very rapid cleavage of the ester bond both in vivo and in vitro. These findings indicated that while MIH may be a useful reagent for radioimmunoimaging using IgG mAb, its application to smaller molecular weight mAbs and radioimmunotherapy would be hindered due to the labile characteristics of the ester bond in plasma. Thus, while the present study reinforced the usefulness of metabolizable linkages for reducing nontarget radioactivity levels, a development of plasma-stable metabolizable linkages is also warranted for radioimmunotherapy and for smaller molecular weight polypeptides.