Synthesis, radiolabelling and biological characteristics of a bombesin peptide analog as a tumor imaging agent.

BACKGROUND: Several human cancers, including small cell lung, prostate, breast, gastric, colon and pancreatic cancers, express receptors for bombesin-like peptides. Bombesin (BN) peptides that bind specifically to these receptors are useful for detection of bombesin receptor-expressing cancers in vivo. A new 99mTc-labelled-BN peptide for targeting bombesin receptor-expressing cancers was prepared and characterized. MATERIALS AND METHODS: MAG3-coupled BN peptide (MAG3-BN) was prepared by solid-phase synthesis and radiolabelled with 99mTc by an exchange method. In vitro cell binding assays were conducted on human breast cancer cell lines, MDA-MB-231 and MCF-7. In vivo biodistribution studies were performed in normal and nude mice bearing bombesin receptor-positive tumors. RESULTS: Radiolabelling of MAG3-BN with 99mTc produces a single radioactive species (> 95%). In vitro cell-binding indicated the affinity and specificity of 99mTc-MAG3-BN towards bombesin receptors. In vivo biodistribution in mice demonstrated that 99mTc-MAG3-BN cleared rapidly from the blood and most non-targeted tissues and was excreted mainly via the kidneys. Uptake in bombesin receptor-positive tissues and in the tumor was low to moderate. CONCLUSION: 99mTc-MAG3-BN displays good radiolabelling together with certain favorable biological characteristics and might be a useful peptide radiopharmaceutical in the detection of bombesin receptor-expressing cancers in vivo.

Synthesis and evaluation of 99mTc/99Tc-MAG3-biotin conjugates for antibody pretargeting strategies.

Four 99mTc-MAG3-biotin conjugates were synthesized to determine their potential use in antibody pretargeting strategies for radioimmunoscintigraphy (RIS). To use these 99mTc-MAG3-biotin conjugates as model compounds for 186Re-MAG3-biotin conjugates for radioimmunotherapy (RIT), nanomolar amounts of 99Tc were added as carrier to 99mTc. The biotin derivatives used for the preparation of the conjugates-biocytin, biotin hydrazide, biotinyl-piperazine, and biotinyl-diaminosuccinic acid-differed at the site that is regarded to be susceptible to hydrolysis by biotinidase present in human plasma. All four conjugates were produced with high radiochemical purity, were stable in PBS, and demonstrated full binding capacity to streptavidin. The 99mTc/99Tc-MAG3-labeled biotinyl-piperazine and biotinyl-diaminosuccinic acid conjugates were stable in mouse as well as human plasma, whereas the corresponding biocytin and biotin hydrazide conjugates were rapidly degraded. The biodistribution in nude mice at 30 min after injection was similar for all conjugates, and a rapid blood clearance and high intestinal excretion were both observed. It is concluded that the metabolic routing of a conjugate containing biotin and MAG3 is dominated by these two moieties. For this reason, MAG3-biotin conjugates do not seem suited for pretargeted RIT, for which quantitative and fast renal excretion is a prerequisite to minimize radiation toxicity. However, in a pretargeted RIS approach the 99mTc-MAG3-biotin conjugates might have potential.

Characteristics and biological behaviour of 99mTc-labelled hydroxyacetyltriglycine, a potential alternative to 99mTc-MAG3.

Substitution of the oxidation-sensitive thiol function of mercaptoacetyltriglycine (MAG3) by a hydroxyl group yields a tetraligand (hydroxyacetyltriglycine or HAG3) which is almost insensitive to oxidation and has the advantage over MAG3 that it can be stored safely without protection of the alcohol function. We found that deprotected HAG3 could be directly labelled at alkaline pH (pH>/=11.5) and room temperature in high yield (>95%). Results of electrophoresis experiments suggested a comparable structure for 99mTc-HAG3 and 99mTc-MAG3, namely binding of an oxotechnetium(V)core via three deprotonated amides and a deprotonated hydroxyl group. Biodistribution studies in mice at 10 min and 30 min p.i. showed a slightly higher urinary excretion, a faster renal transit and a significantly lower hepatobiliary handling for 99mTc-HAG3 than for 99mTc-MAG3. In a baboon, the 1-h plasma clearance of 99mTc-HAG3 was clearly higher than that of 99mTc-MAG3. Its plasma protein binding was in the same order as that of Hippuran and much lower than that of 99mTc-MAG3. Evaluation in a human volunteer confirmed the favourable biological characteristics of 99mTc-HAG3, namely a rapid renal excretion, a high 1-h plasma clearance and a negligible hepatobiliary handling. The results indicate that 99mTc-HAG3 may be an easy-to-prepare and practical substitute for 99mTc-MAG3 with improved renal excretion characteristics.

Investigation of the labelling characteristics of 99mTc-mercaptoacetyltriglycine.

S-Benzyl-, S-benzamidomethyl- and S-benzoylmercaptoacetyltriglycine were synthesized and compared in exchange labelling experiments for the preparation of 99mTc-MAG3. The rate of exchange from 99mTc-tartrate to 99mTc-MAG3 starting from the respective precursors was determined in different conditions. Labelling proceeded most rapidly starting from the S-benzoyl protected precursor but efficient labelling was also accomplished using the more stable S-benzamidomethyl- and S-benzylmercaptoacetyltriglycine. 99mTc-MAG3 was also prepared by direct labelling of unprotected mercaptoacetyltriglycine at alkaline pH. Radiochemical purity in these conditions is mainly dependent on the pH during labelling.

Evaluation of technetium-99m-triamide-mercaptide complexes designed to identify properties favoring renal tubular transport.

UNLABELLED: To aid in the design of an improved 99mTc-labeled renal agent, several new [99mTcO(MAG3)]2- analogs were synthesized to determine the effects of varying the position and chemical form of the terminal charged group on renal clearance. METHODS: Clearance, extraction efficiency and plasma protein binding were measured in six Sprague-Dawley rats per complex for ortho, meta and para isomers of [99mTcO(MAG2-ABA)]2-, with MAG2- = mercaptoacetylglycylglycyl- and ABA = aminobenzoate; [99mTcO(MAG2-pASA)]2-, with pASA = p-aminosalicylate; [99mTcO(MAG2-AMS]2-, with AMS = aminomethylsulfonate; and [99mTcO(MAG2-AMP]3-, with AMP = aminomethylphosphonate. For agents with relatively poor clearances, hepatobiliary excretion was evaluated by using a camera-based method. RESULTS: The clearances of the ortho, meta and para isomers of [99mTcO(MAG2-ABA)]2- were 17%, 20% and 59% of those of OIH, respectively. The clearances of [99mTcO(MAG2-pASA)]2-, [99mTcO(MAG2-AMS)]2- and [99mTcO(MAG2-AMP)]3- were 32%, 46% and 39% those of OIH, respectively. CONCLUSION: Optimal tubular transport appears to require a terminal anionic group; a planar carboxylate is preferred over nonplanar -SO3- or -PO3(2-) substituents, suggesting that the smaller size and/or planar shape of the carboxylate group are probably more important than the total charge or charge distribution. Optimal transport also appears to depend on the oxo-carboxylate conformation (syn or anti) and the oxo-carboxylate distance, although these relationships can be modulated by steric interactions. These structure-distribution relationships are important factors to consider in the future design of renal radiopharmaceuticals.