Towards the optimization of tumor targeting radiolabeled peptides for molecular imaging and therapy

Radiopharmaceuticals based on regulatory peptides have become an indispensable tool in nuclear medicine for the diagnosis (molecular imaging) and radionuclide therapy of cancer. The specificity of these radiopeptides towards G-protein-coupled receptors (GPCR), which are overexpressed by various cancer cells and their favorable pharmacological properties make them ideal vectors for the targeted delivery of radioactivity to tumors and metastases. However, there are still challenges to be met in order to develop peptide-based radiopharmaceuticals with ideal properties in terms of imaging quality and therapeutic efficacy where therapeutic radionuclides are employed.
A potential drawback of several radiolabeled peptides under investigation is represented by a rapid washout of radioactivity after receptor-mediated internalization into tumor cells. In certain cases, the washout of radioactivity from cells occurs at a rate comparable to that required for accumulation in cancerous tissues. This not only renders the initial efforts of targeted delivery in part futile but also results in an imaging quality and therapeutic efficiency lower than achievable. To address this issue, novel strategies are needed to improve the cellular retention of the radioactivity. A possible approach may include the employment of multi-targeting radioconjugates made of different moieties specific for extra- and intracellular targets. Towards this goal, we investigated the combination of tumor targeting peptides with an additional moiety specific for an intracellular target and radiolabeled the conjugate with the 99mTc-tricarbonyl core as a reporter probe for single-photon emission computed tomography (SPECT). We envisioned that enabling interactions of radioconjugates with intracellular targets after receptor-mediated uptake by endocytosis would result in the trapping of radioactivity in tumors.
Specifically, we combined a modified binding sequence of the peptide bombesin, [Nle14]BBS(7-14), for extracellular targeting of the tumor-associated gastrin releasing peptide receptor (GRP-r) with a triphenylphosphonium group for intracellular targeting of the organelle mitochondria or with the peptide shepherdin, an inhibitor of the cytosolic chaperon heat-shock protein 90 (Hsp90). The conjugates were assembled by the "Click-to-Chelate" approach, an efficient synthetic strategy for the preparation of bifunctional 99mTc-labeled radiopharmaceuticals. The radioconjugates were evaluated in vitro using GRP-r-overexpressing PC-3 cells. Our investigations revealed that the additional moiety for intracellular targeting did not impact the tumor-targeting capability of the bombesin-derived conjugates but neither did it result in an improved cellular retention of the radioactivity. Drawing from our experience and considering recent literature data, we conclude that endosomal entrapment or lysosomal degradation of the bifunctional radiopeptide conjugates is likely to impede with intracellular interactions and thus, responsible for the observed unaltered cellular efflux of radioactivity. Future studies will be directed towards the combination of bifunctional radiopeptide conjugates with drug delivery systems designed to facilitate endosomal escape.
A different approach for the optimization of peptidic radiotracers includes the improvement of their metabolic stability since most of them exhibit a very short biological half-life due to rapid degradation by endogenous peptidases. Enhancement of the stability of radiopeptides results in a prolonged circulation time in the blood and, as a consequence, an improved tumor uptake in vivo. A number of different strategies have been reported for the stabilization of regulatory peptides, however, with varying degree of success in providing peptidomimetics with retained affinity to the corresponding GPCR. In an effort to probe a novel peptide backbone modification methodology, the use of 1,4-disubstituted 1,2,3-triazoles as metabolically stable trans amide bond isosters was investigated. The systematic replacement of amide bonds within the binding sequence of the tumor-affine peptide bombesin, [Nle14]BBS(7-14), by triazoles provided a series of 177Lu-labeled peptidomimetics with both retained affinity towards GRP-r and an increased stability in blood serum. In vivo evaluation of a lead compound in xenografted mice showed that the enhanced stability of the radiopeptidomimetic resulted in a doubling of the uptake of radioactivity in tumors. The described amide-to-triazole substitution methodology is currently being applied to other tumor targeting peptides of medicinal interest.
The specificity and affinity of radiopeptides towards different receptor subtypes is another aspect to consider for optimizations. Inhomogeneous expression of receptor subtypes by tumors may influence the efficiency of a radiotracer. For example, intratumoral administration of radiolabeled substance P (SP) led to significant differences in the clinical response of patients suffering from gliomas despite proven expression of its target, the neurokinin-1 receptor (NK1R). In an effort to identify factors that may be responsible for the varying therapeutic outcome observed, several SP conjugates were evaluated in vitro using four established glioma cell lines differing in their level of RNA expression of the full length and truncated receptor isoforms. Cell binding and internalization of SP-conjugates were only observed with cell lines exhibiting high expression of RNA of the full-length NK1R. Pre-therapeutic screening for NK1R isoforms may therefore be advisable for the selection of glioma patients for NK1R-targeted radionuclide therapy.