Localization of 99mTc-GRP Analogs in GRPR-Expressing Tumors: Effects of Peptide Length and Neprilysin Inhibition on Biological Responses

The overexpression of gastrin-releasing peptide receptors (GRPRs) in frequently occurring human tumors has provided the opportunity to use bombesin (BBN) analogs as radionuclide carriers to cancer sites for diagnostic and therapeutic purposes. We have been alternatively exploring human GRP motifs of higher GRPR selectivity compared to frog BBN sequences aiming to improve pharmacokinetic profiles. In the present study, we compared two differently truncated human endogenous GRP motifs: GRP(14–27) and GRP(18–27). An acyclic tetraamine was coupled at the N-terminus to allow for stable binding of the SPECT radionuclide 99mTc. Their biological profiles were compared in PC-3 cells and in mice without or with coinjection of phosphoramidon (PA) to induce transient neprilysin (NEP) inhibition in vivo. The two 99mTc-N4-GRP(14/18–27) radioligands displayed similar biological behavior in mice. Coinjection of PA exerted a profound effect on in vivo stability and translated into notably improved radiolabel localization in PC-3 experimental tumors. Hence, this study has shown that promising 99mTc-radiotracers for SPECT imaging may indeed derive from human GRP sequences. Radiotracer bioavailability was found to be of major significance. It could be improved during in situ NEP inhibition resulting in drastically enhanced uptake in GRPR-expressing lesions.


Discussion
A considerable number of radiolabeled analogs of frog BBN have been developed for potential application in the diagnosis and therapy of GRPR-expressing tumors in Homo sapiens [11,12]. This pursuit is based on the overexpression of GRPRs on the surface of malignant cells serving as easily accessible biomolecular targets on cancer lesions [3][4][5][6][7][8][9][10]. Joining this effort, we have previously introduced a series of BBN-like analogs, generated by covalently coupling of an acyclic tetraamine at the N-terminus, Demobesin 3-6 [25]. Like native BBN, these peptide ligands displayed indistinguishable binding affinities for the human bombesin receptor subtypes GRPR and NMBR, but no affinity for the BB 3 R. The respective radiotracers [ 99m Tc] Demobesin 3-6 specifically localized in GRPR-expressing human PC-3 xenografts in mice. Moreover, one of the analogs, [ 99m Tc] Demobesin 4, was able to visualize malignant lesions in a small number of prostate cancer patients with SPECT/CT [36].
We have recently expanded our search toward radioligands based on human endogenous GRP sequences [23,24,26]. The latter are reported for higher GRPR selectivity compared to their frog homolog BBN [13]. This approach has surprisingly remained unexplored up to now, although it offers two major advantages. First, radioactivity levels in abdominal tissues physiologically expressing both GRPR and NMBR subtypes would favorably decrease when GRPR-selective radioligands are used [14][15][16]. Second, injection of GRPR-selective agonists will not activate the NMBR subtype populations in the gut and is hence associated with less adverse effects [17][18][19][20][21][22]. Promising GRPR-selective radioligands based on receptor antagonists have been developed and evaluated in animals and in human, showing excellent tumor targeting and pharmacokinetic profiles [37,38]. Yet, the suitability and efficacy of noninternalizing GRPR antagonists for therapy with low-range beta, alpha, or Auger emitters of high LET has not been established thus far [39,40]. Therefore, the study of radiolabeled GRPR-selective agonists is warranted and may provide an alternative platform for the development of radioligands exhibiting new combinations of biological features particularly suited for cancer theranostics.
In agreement with this observation, the internalization rate of 99m Tc-N 4 -GRP(14-27) in PC-3 cells was clearly superior to that of 99m Tc-N 4 -GRP(18-27) (Figure 3b). A crucial feature for efficient tumor targeting is metabolic stability that will ensure sufficient radioligand supply to tumor sites after injection in the living organism [31]. Recent studies have revealed the significance of in vivo stability assessment of BBN-like radiotracers vs. in vitro determinations in serum or tissue homogenate incubates to more accurately predict the actual protease(s) encountered by the radioligand after entering the circulation. These studies have demonstrated NEP as a major degrading protease of BBN and its radiolabeled analogs [41,42]. In a recently proposed approach, NEP-inhibitors, like PA [28,29], coinjected with the BBN-based radioligand drastically increased metabolic stability leading to notable enhancement of tumor uptake in animal models [31][32][33][34]. Following this rationale, we have tested the in vivo stability of the two 99m Tc-N 4 -GRP(14-27) and 99m Tc-N 4 -GRP (18)(19)(20)(21)(22)(23)(24)(25)(26)(27) radiotracers in peripheral mouse blood collected 5 min pi by radioanalytical HPLC (Figure 4). The longer chain 99m Tc-N 4 -GRP(14-27) displayed somewhat poorer stability (20.1 ± 4.5% intact, n = 3) compared to shorter chain 99m Tc-N 4 -GRP(18-27) (31.0 ± 0.9% intact, n = 3). Thus, the presence of additional amide bonds in the longer peptide chain offered further degradation sites for attacking proteases. It is interesting to note, that coinjection of PA drastically increased the stability of both analogs in the circulation, indicating NEP as a major protease in the catabolism of GRP sequences as well.
The present study has shown that GRP motifs of different chain length may provide new opportunities for the development of promising GRPR-selective radioligands. The latter may prove to be a useful asset in the arsenal of anti-GRPR radioactive drugs, by internalizing in cancer cells while evading binding to and activation of bombesin receptor subtypes other than the GRPR in the gut.
Furthermore, it has shown the impact of in vivo metabolic stability in maximizing tumor localization. Structural modifications in peptide lead-structures to improve the stability of radiopeptides, such as amino acid replacements, cyclization, or changes of cleavable peptide bonds, may deteriorate other important biological features, as for example receptor affinity, cell uptake, tumor targeting, and overall pharmacokinetics. On the other hand, transient in situ inhibition of NEP represents a smart method to accomplish this goal and warrants further efforts for translation in the clinic. Despite challenges related to biosafety, regulatory and financial hurdles, once established in a proof-of-principal study, this strategy is expected to boost the development and clinical application of other NEP-catabolized radioligands, considerably saving costs and resources [31][32][33][34].
Quality control of the radiolabeled products comprised radioanalytical HPLC and instant thin-layer chromatography (ITLC). HPLC analyses were performed on a Waters Chromatograph coupled to a 2998 photodiode array UV detector (Waters, Vienna, Austria) and a Gabi gamma detector (Raytest RSM Analytische Instrumente GmbH, Germany). For analysis, a Waters Symmetry Shield RP-18 cartridge column (5 µm, 3.9 mm × 20 mm) was eluted at a 1.  [34]. Methionine was added to the purified radioligand solution to prevent reoxidation of Met 14 to the corresponding sulfoxide and the resulting stock solution in 0.1% BSA-PBS was kept at −20 • C; aliquots thereof were used in competition binding assays (molar activity of 81.4 GBq/µmol).

Cell Lines and Culture
Human androgen-independent prostate adenocarcinoma PC-3 cells endogenously expressing the human GRPR (LGC Promochem, Teddington, UK) were used in the present study [43]. Cells were cultured in Roswell Park Memorial Institute (RPMI)-1640 medium, supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 U/mL penicillin, and 100 µg/mL streptomycin, and kept in a controlled humidified atmosphere containing 5% CO 2 at 37 • C. Passages were performed weekly using a trypsin/EDTA (0.05%/0.02% w/v) solution. All culture media were purchased from Gibco BRL, Life Technologies and supplements were provided by Biochrom KG Seromed.

Induction of PC-3 Xenografts in SCID Mice
A suspension containing freshly harvested human PC-3 cells (≈150 µL of a ≈1.2 × 10 7 cells) was subcutaneously injected in the flanks of female SCID mice (15 ± 3 g, six weeks of age at the day of arrival, NCSR "Demokritos" Animal House Facility). The animals were kept under aseptic conditions and 4 weeks later developed well-palpable tumors (80-200 mg) at the inoculation sites.

Biodistribution in PC-3 Xenograft-Bearing SCID Mice
For the biodistribution study, animals in groups of 4 received via the tail vein a 100 µL bolus of 99m Tc-N 4 -GRP(14-27) (180-370 kBq, corresponding to 10 pmol total peptide) coinjected either with injection solution (100 µL; control) or PA-solution (300 µg PA dissolved in 100 µL injection solution; 4 h + PA), or with excess [Tyr 4 ]BBN (100 µL injection solution containing 50 µg [Tyr 4 ]BBN for in vivo GRPR-blockade; 4 h block). Animals were euthanized at 1-, 4-, and 24-h pi; in the case of 99m Tc-N 4 -GRP(18-27) two animal groups were included at the 4-h pi interval, namely the control and PA groups described above. Mice were dissected; samples of blood, tumors, and organs of interest were collected, weighed, and measured for radioactivity in the gamma counter. Intestines and stomach were not emptied of their contents. Data was calculated as percent injected dose per gram tissue (%ID/g) with the aid of standard solutions and represent mean values ± sd, n = 4. All animal experiments were performed in compliance with national and European guidelines and approved by national authorities (Prefecture of Athens, EL 25 BIO 021; #1609 and #1610).