In vitro and in vivo evaluation of a 99mTc(I)-labeled bombesin analogue for imaging of gastrin releasing peptide receptor-positive tumors1

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Abstract

A new radiolabeled bombesin analogue, [99mTc(I)-PADA-AVA]bombesin (7-14), was synthesized and in vitro and in vivo characterized. High affinity and rapid internalization were obtained in binding assays. A specific binding towards gastrin releasing peptide receptors-positive tissues, pancreas and tumor, was observed in CD-1 nu/nu mice bearing PC-3 prostate adenocarcinoma xenografts. We therefore conclude that [99mTc(I)-PADA-AVA]bombesin (7-14) might have promising characteristics for applications in nuclear medicine, namely for diagnosis of GRP receptor overexpressing tumors.

Introduction

Small neuropeptides have attracted an increasing interest in the area of cancer imaging and therapy in recent years [19], [21]. Various malignant tumor cells express neuropeptide receptors on their surface in much higher concentration than most healthy tissues [13]. Thus, these overexpressed receptors could be used as potential targets for imaging and therapy with radiolabeled neuropeptides [24]. Furthermore, due to their low molecular weight, small peptides are expected to better penetrate into tumor tissue, exhibit a faster clearance from normal tissue and thus lead to better tumor-to-background ratios than high molecular weight compounds like monoclonal antibodies and their constructs. Accordingly, the successful application of radiolabeled somatostatin and vasoactive intestinal peptide analogues for imaging of neuropeptide receptor-positive tumors [18], [36], and the development of further regulatory peptides such as bombesin, cholecystokinin, neurotensin and substance P [5], [17], [28], [29] as potential imaging agents have been promoted intensively.

Bombesin (pGlu-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) is a tetradecapeptide, originally isolated from the skin of the amphibian Bombina orientalis [26], with high affinity to the GRP receptor. Bombesin and its mammalian counterpart, the gastrin releasing peptide (GRP), has been found to produce a wide range of biological responses in peripheral tissues as well as in the central nervous system, for instance stimulation of gastrointestinal hormone release, exocrine secretion and maintenance of circadian rhythms. Bombesin-like peptides may also act as paracrine/autocrine growth stimulators in neoplasms [3], [9]. GRP receptors were found to be overexpressed in a variety of tumors, including breast and prostate cancer [15], [23]. Thus, the interest for an application of radiolabeled bombesin analogues in nuclear medicine has rapidly increased.

Among all radionuclides used in nuclear medicine, technetium-99m is still the most widely applied for diagnostic purposes, mainly because of its favorable γ-ray emission characteristics (t1/2 = 6 h, Eγ = 140 keV, 85%) and its suitable availability. It is commonly used in the oxidation state +V as O=Tc=O+ core, wrapped by tetradentate N- and S-ligands, which provide high thermodynamic stability. Organometallic 99mTc precursors in lower oxidation states have not yet gained much attention, mainly because their synthesis was perceived to be difficult, and they had been presumed to be unstable against hydrolysis and oxidation [37]. We report here on the application of a novel and convenient method to synthesize such a low oxidation state 99mTc compound, [99mTc(OH2)3(CO)3]+ [1], for labeling bombesin analogues. As a reflection of its low-spin d6 electronic configuration, this 99mTc(I)-moiety is kinetically remarkably stable in aqueous solution for a long period of time [12], [37]. This might also provide the basis for future application of the more sensitive homologues 186Re and 188Re as therapeutic radionuclides [20]. PADA, 2-picolylamine-N,N-diacetic acid, was chosen as bifunctional chelating agent, mainly because of the high stability of the corresponding 99mTc(I)-complex and, less important (depending on the degradation of the peptide yielding the free complex) due to its fast clearance from the body through the urinary and the hepatobiliary pathways [1], [30]. The additional introduction of 5-aminovaleric acid (AVA) as spacer was thought to avoid interferences of the chelating moiety with the receptor binding [17]. The resulting bioconjugate fac-[M(I)-PADA-AVA]bombesin (7-14) (M = 99mTc, Re; fac: the three carbonyls, positioned in the corners of one facet of the octahedral complex, are forming together with the metal center a pyramid) is shown in Fig. 1. The objective of the present study is the characterization of the first bombesin analogue labeled with the novel fac-[99mTc(OH)2(CO)3]+. We report here on the synthesis, binding affinity, internalization, chemical and metabolic stability of [99mTc(I)-PADA-AVA]bombesin (7-14) as well as its biodistribution in CD-1 nu/nu mice bearing PC-3 prostate adenocarcinoma xenografts.

Section snippets

Materials

Fmoc-protected amino acids and 4-(2′,4′-dimethoxyphenyl-Fmoc-aminoethyl)-phenoxy resin were purchased from NovaBiochem (Läufelingen, Switzerland), and Fmoc-AVA-OH was purchased from Fluka (Buchs, Switzerland). Bombesin and Neuromedin B were obtained from Bachem (Bubendorf, Switzerland). The [125I-Tyr4]bombesin was purchased from NEN Life Science Products (Zaventem, Belgium). Na[99mTcO4] was eluted from a Mallinckrodt 99Mo/99mTc generator (Petten, The Netherlands) using 0.9% saline. Cell culture

Synthesis and labeling

The ES-MS analysis of PADA-AVA-bombesin (7-14) (calculated 1243.45 Dalton, observed 1245.2 Dalton), and [Re(I)-PADA-AVA]bombesin (7-14) (calculated 1513.68 Dalton, observed 1514.7 Dalton) showed that protonated forms of the compounds were detected. The HPLC result showed both [99mTc(I)-PADA-AVA]bombesin (7-14) and [Re(I)-PADA-AVA]bombesin (7-14) at the same retention time of 21.9 min, while the free peptide was found after 15.9 min.

Chemical stability: challenging

Neither histidine nor cysteine led to significant ligand

Discussion

The 14 amino acid neuropeptide bombesin may function as an autocrine/paracrine growth factor for neoplastic tissues, it is for instance suggested to be involved in the modulation of biological effects in prostate carcinoma [3]. These data are supported by the fact of a massive GRP receptor overexpression in neoplastically transformed prostate and breast tissue [23], [31]. Such data provide a molecular basis for the clinical application of bombesin-like peptides for early tumor diagnosis by GRP

Acknowledgements

We would like to thank René Bugmann and Alain Blanc for their excellent technical assistance. This work has partly been funded by Biomed 2 project Nr. BMH4-CT98–3198 and by the Swiss Cancer Liga grant KFS 559–9-1997.

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    Abbreviations: AVA, 5-Aminovaleric acid; fac, facial; PADA, 2-picolylamine-N,N-diacetic acid; GRP, Gastrin releasing peptide; BB, Bombesin; NMB, Neuromedin B.

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