99mTc-N2S2-Tat(49-57)-Lys3-bombesin

Excerpt

^99mTc-N₂S₂-Tat(49-57)-Lys³-bombesin (^99mTc-Tat-BN) is a hybrid peptide synthesized and labeled with ^99mTc by Santos-Cuevas et al. for molecular imaging of tumors expressing gastrin-releasing peptide receptor (GRPR) (1).

Bombesin (BN) is an amphibian neuropeptide consisting of 14 amino acids (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH₂) (2, 3), first isolated from frog skin in 1970 (4). The search for its mammalian counterpart led to the discovery of gastrin-releasing peptide (GRP), which consists of 27 amino acids. GRP and BN share an identical C-terminal region (-Trp-Ala-Val-Gly-His-Leu-Met-NH₂), which is necessary for receptor binding and signal transduction (5, 6). Besides the release of gastrin, GRP and BN-like peptides also produce a wide range of other biological responses in diverse tissues such as secretion of endocrine and exocrine glands, maintenance of circadian rhythm, regulation of satiety, and contraction of smooth muscles (7). They also act as potential growth factors for both normal and cancer cells (3, 5, 6). There are four members of the BN receptor family, including three mammalian receptors: GRPR (BB₂ or BRS2; 384 amino acids), neuromedin B receptor (NMBR, BB₁, or BRS1; 390 amino acids), and BN-like receptor 3 (BB₃, BRS3, or orphan; 399 amino acids) (5, 7, 8); the fourth receptor (BB₄) has only been found in amphibians. GRPR is the only well characterized receptor of this family. GRPR is a glycosylated, 7-transmembrane, G-protein–coupled receptor that, upon binding with its ligands, gives rise to a complex cascade of intracellular reactions. It is normally found in non-neuroendocrine tissues of the breast and pancreas and in neuroendocrine cells of the brain, gastrointestinal tract, lung, and prostate (9). Interestingly, GRPR is overexpressed in prostate cancer as well as tumors of breast, lung, pancreas, ovary, kidney, and gastrointestinal tract. It has been reported that GRPR is expressed at a high density in the intraepithelial neoplasia and primary carcinoma of prostate, whereas normal prostate tissue and, in most cases, benign prostate hyperplasia are predominantly GRPR-negative (10-13).

GRPR has attracted significant interest as a target for tumor detection, tumor staging, and evaluation of tumor response to therapy (5, 6, 8, 11). A large number of BN derivatives have been developed, and they have been labeled with ^99mTc, ¹⁷⁷Lu, ⁶⁷Ga, and ¹¹¹In for single-photon emission computed tomography and with ⁶⁴Cu, ⁶⁸Ga, and ¹⁸F for positron emission tomography. The published BN derivatives can be generally classified as truncated BN (6–14 or 7–14) or full-length BN (1-14) analogs (1, 10, 12-17). The truncated BN analogs seem to be favorable because they are usually more stable than the full-length tetradecapeptides for in vivo applications, and they still bind to the GRPR adequately. However, the full-length peptides offer different labeling methods by attachment of functional groups to the amino acid 1 to 6. In many cases, the amino acids on position 13 (Leu) and 14 (Met) have been replaced by unnatural amino acids (cyclohexylalanine (CyHAla) and norleucine (Nle)), and Lys has been placed on position 3 for attachment of radiolabels with reactive esters. Spacers, chelators, or radiometals have also been widely used for conjugation and for favorable kinetics (18). The BN derivatives can also be divided into agonists and antagonists (19-21). By far, most BN derivatives are agonists. Agonists are internalized into and accumulate within cells, and they have been assumed to exhibit higher uptake by cancer cells than antagonists. However, some reports have shown that uptake of antagonists is much higher than that of agonists because antagonists may have stronger binding for the GRPR than agonists (19, 20).

An optimal BN-like radiotracer needs to meet several requirements: high affinity for GRPR, with rapid and specific tumor uptake; high hydrophilicity, with preferred renal excretion and low hepatobiliary excretion; and high stability but relatively rapid clearance from blood (6). Despite a large number of published derivatives, a valid comparison among them for the feasibility of tumor imaging is difficult because standardization between studies is lacking. Generally speaking, the majority of the radiotracers have relatively high renal and hepatic uptake, resulting in low tumor/liver and tumor/kidney ratios. In an effort to increase the tumor cell uptake and consequently improve the image contrast of tumors and their metastases, Santos-Cuevas et al. developed a hybrid peptide radiotracer (^99mTc-Tat-BN) and compared it with the GRPR agonist ^99mTc-EDDA/HYNIC-[Lys³]-BN for the in vitro and in vivo uptake kinetics in GRPR-expressing cancer cells and tumor xenografts (1, 22). ^99mTc-Tat-BN was constructed by linking the HIV Tat-derived peptide (Tat(49-57)) with the GRPR agonist Lys³-BN peptide. Tat(49-57) is a small basic peptide with a sequence of H-Arg-Lys-Lys-Arg-Arg-Gln-Arg-Arg-Arg-NH₂, and it has been successfully used for delivering a large variety of cargoes into cells because of its cell-penetrating properties (11).