Gastrin-releasing peptide receptors (GRPRs) are overexpressed in several human malignancies such as prostate cancer, mammary carcinoma, and lung cancer [1,2,3,4,5,6,7,8,9,10]. Consequently, they have attracted considerable attention as potential biomolecular targets for diagnosis and therapy with radionuclide carriers directed to GRPR-positive cancer lesions [11,12]. Originally, the frog tetradecapeptide bombesin (BBN, Pyr-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2) and its truncated C-terminal octapeptide fragment BBN(7–14) have served as motifs for the development of GRPR-targeting radioligands. However, BBN-like analogs bind with comparable affinity not only to GRPR (BB 2 R), but also to the neuromedin B (NMBR, BB 1 R), another member of the three mammalian bombesin receptor subtypes [1,2]. The above two subtypes are pharmacologically distinguished by their selectivity for different endogenous human homologs of amphibian BBN. Thus, the 27-mer GRP (H-Val-Pro-Leu-Pro-Ala-Gly-Gly-Gly-Thr- Val-Leu-Thr-Lys-Met-Tyr-Pro-Arg-Gly-Asn-His-Trp-Ala-Val-Gly-His-Leu-Met-NH 2), the 14-mer GRP(14–27), and the C-terminal decapeptide GRP(18–27) fragments strongly bind to the GRPR, whereas neuromedin B (NMB, H-Gly-Asn-Leu-Trp-Ala-Thr-Gly-His-Phe-Met-NH 2) exhibits high affinity for the NMBR [13]. The two GRPR and NMBR subtypes are physiologically expressed in the human brain and the gut, especially in stomach, pancreas, and gastrointestinal tract, and they are also implicated in cancer [14,15,16]. It is reasonable to assume, that radiolabeled BBN agonists of poor GRPR selectivity will show increased levels of background radioactivity by virtue of their binding to both GRPR and NMBR populations distributed in the body, especially in the abdomen. Furthermore, additive GRPR- and NMBR-mediated effects in the gastrointestinal tract, such as abdominal smooth muscle contraction and stimulation of gastrointestinal hormone secretion, are to be expected after intravenous injection of BBN-like agonist radioligands [17,18,19,20,21,22].
In contrast to amphibian BBN-like motifs, the respective human homologs have surprisingly remained unexploited as radionuclide carriers for targeting GRPR-positive cancer [13]. Motivated by this gap in the inventory of GRPR-directed radioligands we have expanded our research efforts to native human GRP sequences in order to explore their applicability in GRPR-targeted tumor diagnosis and therapy. First, we introduced a small library of tetraamine derivatized GRP(18–27) analogs labeled with the SPECT radionuclide 99m Tc [23,24]. Compared to previously reported 99m Tc-radiopeptides, which are based on the full-length BBN or its truncated BBN(7–14) octapeptide fragment [25], the 99m Tc-N 4-GRP(18–27) showed high GRPR selectivity and superior in vivo characteristics in tumor-bearing mice, such as faster renal clearance and improved tumor to background ratios. On the other hand, single or double amino acid substitutions in the decapeptide backbone exerted pronounced effects on several biological properties, eventually affecting tumor targeting capabilities and pharmacokinetics. In a following study, a series of differently truncated GRP sequences were coupled to the universal chelator DOTA (1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid) and labeled with 111 In. Receptor affinity, internalization efficiency and tumor uptake of these analogs were favored both by longer peptide chain and by the presence of basic amino acids Lys 13 and Arg 17 in the native GRP sequence [26].
Following this line of research, we herein introduced 99m Tc-N 4-GRP(14–27) and compared its biological profile in PC-3 cells and mice models with 99m Tc-N 4-GRP(18–27) (Figure 1). It should be noted that basic positions Lys 13 and Arg 17 in the native GRP sequence are now occupied by the positively charged N 4+x/[99m Tc(O)2(N 4)]+1-moiety and not by the negatively charged DOTA. This arrangement allows for comparisons with the DOTA-derivatized analogs and further studying the influence of positive/negative charges in 13 and 17 positions of the GRP chain [26]. Next, the selectivity of N 4-GRP(14–27) for each of the three mammalian bombesin receptor subtypes was investigated applying receptor autoradiography in human excised biopsy samples, expressing one of the GRPR, NMBR, and bombesin subtype 3 (BB 3 R) receptors. Finally, the impact of in vivo stability of 99m Tc-N 4-GRP(14–27) and 99m Tc-N 4-GRP(18–27) on tumor targeting and pharmacokinetics was compared in mice. The role of neprilysin (NEP) [27] on the in vivo degradation of the two human GRP-based sequences was monitored by HPLC analysis of blood samples collected without or with coinjection of the NEP-inhibitor phosphoramidon (PA) [28,29], as previously described for BBN-like radioligands [30,31,32,33,34]. The enhancement of radiotracer localization in experimental GRPR-positive PC-3 tumors in mice during transient NEP inhibition induced by PA was assessed.
The bifunctional acyclic tetraamine chelator (6-(carboxy)-1,4,8,11-tetraazaundecane) was covalently coupled by its carboxy functionality at the N-terminal Met 14 of GRP(14–27) or the Gly 18 of GRP(18–27) via an amide bond [24], generating two different length GRP analogs amenable for labeling with the preeminent SPECT radionuclide 99m Tc. Labeling was typically proceeded by a brief incubation with 99m TcO 4− generator eluate, SnCl 2 as reducing agent, and citrate anions as transfer ligand in alkaline pH at ambient temperature at molar activities of 20 to 40 MBq 99m Tc/nmol peptide. Quality control of the radiolabeled products combined HPLC and ITLC analysis. The total radiochemical impurities, comprising 99m TcO 4−, [99m Tc] citrate, and 99m TcO 2 × H 2 O, did not exceed 2%, while a single radiopeptide species was detected by RP-HPLC. In view of labeling yields >98% and >99% radiochemical purity of the resultant 99m Tc-N 4-GRP(14–27) and 99m Tc-N 4-GRP(18–27), the radioligands were used without further purification in all subsequent experiments. Representative radiochromatograms of HPLC analysis of 99m Tc-N 4-GRP(14–27) and 99m Tc-N 4-GRP(18–27) are included in Figure 2a,b, respectively.
The selective affinities of N 4-GRP(14–27) for each of the three bombesin receptor subtypes found in mammals were studied during in vitro competition binding assays against the universal radioligand 125 I-[DTyr 6,_β_ Ala 11,Phe 13,Nle 14]BBN(6–14) [6]. Receptor autoradiography was applied in cryostat sections of well characterized human cancers, preferentially expressing one of the subtypes. As summarized in Table 1, N 4-GRP(14–27) showed high affinity for the GRPR expressed in resected prostate carcinoma specimens (IC 50 = 4.2 ± 1.0 nM, _n_ = 3, vs. IC 50 = 2.4 ± 1.0 nM, _n_ = 3 for N 4-GRP(18–27) [23]), very low affinity for NMBR present in ileal carcinoid biopsy samples (IC 50 = 72 ± 7.6 nM, _n_ = 3, vs. IC 50 = 106 ± 13 nM; _n_ = 2 for N 4-GRP(18–27) [23]), and no affinity for the BB 3 R expressed in bronchial carcinoid samples (IC 50> 1000 nM, _n_ = 3, identical to N 4-GRP(18–27) [23]). Thus, N 4-GRP(14–27) similarly to N 4-GRP(18–27), displayed good selectivity for the GRPR. Hence, the GRP-based analogs turned out to be more GRPR-preferring compared to BBN-based radioligands, like Demobesin 3 (N 4-[Pro 1,Tyr 4]BBN) [25] or [DTyr 6,_β_ Ala 11,Phe 13,Nle 14]BBN(6–14) (Table 1).
As shown in Figure 3a, N 4-GRP(14–27) and N 4-GRP(18–27) as well as the respective GRP(14–27) and GRP(18–27) parent peptide references displaced [125 I-Tyr 4]BBN from GRPR-sites on PC-3 cell membranes in a monophasic and dose-dependent manner. The respective half-maximal inhibitory concentration (IC 50) values differed, yielding the following rank of decreasing receptor affinity: N 4-GRP(14–27) (IC 50 0.32 ± 0.03 nM) > GRP(14–27) (IC 50 0.45 ± 0.02 nM) > N 4-GRP(18–27) (IC 50 0.63 ± 0.06 nM) > GRP(18–27) (IC 50 1.66 ± 0.20 nM). We observe that the longer-chain peptides consistently showed higher binding affinity to GRPR than their shorter chain counterparts. Moreover, coupling of the positively charged acyclic tetraamine unit in the N-terminus of parent GRP(14/18–27) references improved the affinity of resulting analogs to the GRPR, as previously reported for similarly modified peptide analogs [35].
During incubation at 37 °C in PC-3 cells, both 99m Tc-N 4-GRP(14–27) and 99m Tc-N 4-GRP(18–27) were taken up by the cells via a GRPR-mediated process, as demonstrated by the lack of internalization observed in the presence of excess [Tyr 4]BBN. In both cases the bulk of cell-associated radioactivity was found in the cells with 99m Tc-N 4-GRP(14–27) internalizing much faster in PC-3 cells compared to 99m Tc-N 4-GRP(18–27) at all time intervals (Figure 3b). For example, at 1 h, 12.7 ± 0.7% of total added 99m Tc-N 4-GRP(14–27) specifically internalized in the cells vs. 5.0 ± 0.3% of 99m Tc-N 4-GRP(18–27), whereas at 2 h these values increased to 19.5 ± 1.4% and 6.9 ± 1.5%, respectively.
The two 99m Tc-N 4-GRP(14–27) and 99m Tc-N 4-GRP(18–27) radiotracers exhibited distinct resistance to degrading proteases after injection in mice. As revealed by HPLC analysis of blood samples collected at 5 min postinjection (pi), 99m Tc-N 4-GRP(14–27) was found less stable (20.1 ± 4.5% intact, _n_ = 3) than the shorter chain 99m Tc-N 4-GRP(18–27) (31.0 ± 0.9% intact, _n_ = 3). Representative radiochromatograms are shown in Figure 4a,b, respectively.
It should be noted that coinjection of the NEP-inhibitor PA remarkably enhanced the in vivo stability of 99m Tc-N 4-GRP(14–27) (66.5 ± 4.8% intact, _n_ = 3) and 99m Tc-N 4-GRP(18–27) (70.8 ± 5.4% intact, _n_ = 3) in the circulation, revealing NEP as a major degrading protease for both radiotracers in mice. Representative radiochromatograms are included in Figure 4c,d, respectively.
The biodistribution of 99m Tc-N 4-GRP(14–27) and 99m Tc-N 4-GRP(18–27) was studied in severe combined immune deficiency (SCID) mice bearing human PC-3 xenografts expressing the human GRPR. Subcutaneous tumors of suitable size developed in the flanks of mice about four weeks after inoculation of a suspension of prostate adenocarcinoma PC-3 cells and biodistribution was conducted.
Cumulative biodistribution results for 99m Tc-N 4-GRP(14–27) at the 1-, 4-, and 24-h pi intervals are summarized in Table 2, and are expressed as mean % injected dose per gram (%ID/g) values ± sd, _n_ = 4. The radiotracer washed rapidly from the blood and the background tissues predominantly via the kidneys and the urinary system. High uptake was observed in the PC-3 tumor at 1-h pi (10.20 ± 0.72%ID/g) that remained at comparably high levels at 4-h pi (8.41 ± 4.16%ID/g; _p_> 0.05), declining by ~50% at 24-h pi (4.50 ± 0.69%ID/g). Tumor uptake at 4-h pi was significantly lower in the animals treated with excess [Tyr 4]BBN (0.62 ± 0.24%ID/g; _p_< 0.001), suggestive of a GRPR-mediated process. Likewise, 99m Tc-N 4-GRP(14–27) highly localized in the GRPR-rich mouse pancreas via a GRPR-specific process, as demonstrated by the lack of pancreatic uptake during GRPR-blockade by coinjection of excess [Tyr 4]BBN (35.24 ± 4.70%ID/g vs. 0.83 ± 0.24%ID/g in block; _p_< 0.001).
Comparative biodistribution results for 99m Tc-N 4-GRP(14–27) and 99m Tc-N 4-GRP(18–27) at the 4-h pi interval are included in Table 3. Data from additional 4-h pi animal groups coinjected with the NEP-inhibitor PA (300 µg) is also included in the Table. The radiotracers displayed similar tissue distribution patterns. The observed higher tumor and pancreatic uptake of 99m Tc-N 4-GRP(14–27) did not however differ significantly to that of 99m Tc-N 4-GRP(18–27) (_p_> 0.05) [24]. Treatment with PA induced a drastic increase in tumor values for both radiotracers with 99m Tc-N 4-GRP(14–27) showing superior tumor values than the shorter chain 99m Tc-N 4-GRP(18–27) (38.19 ± 4.79%ID/g vs. 28.37 ± 8.05%ID/g, respectively; _p_< 0.01). Pancreatic values increased by >3-fold for both radiotracers as well. Thus, in agreement with previous studies on BBN-based analogs [31,32,33,34], NEP inhibition likewise resulted in significant stabilization of GRP(14/18–27)-based radioligands in peripheral mouse blood and notable improvement of localization in GRPR-expressing lesions in mice.
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
