[99mTc]Tc-DB1 Mimics with Different-Length PEG Spacers: Preclinical Comparison in GRPR-Positive Models

Background: The frequent overexpression of gastrin-releasing peptide receptors (GRPRs) in human cancers provides the rationale for delivering clinically useful radionuclides to tumor sites using peptide carriers. Radiolabeled GRPR antagonists, besides being safer for human use, have often shown higher tumor uptake and faster background clearance than agonists. We herein compared the biological profiles of the GRPR-antagonist-based radiotracers [99mTc]Tc-[N4-PEGx-DPhe6,Leu-NHEt13]BBN(6-13) (N4: 6-(carboxy)-1,4,8,11-tetraazaundecane; PEG: polyethyleneglycol): (i) [99mTc]Tc-DB7 (x = 2), (ii) [99mTc]Tc-DB13 (x = 3), and (iii) [99mTc]Tc-DB14 (x = 4), in GRPR-positive cells and animal models. The impact of in situ neprilysin (NEP)-inhibition on in vivo stability and tumor uptake was also assessed by treatment of mice with phosphoramidon (PA). Methods: The GRPR affinity of DB7/DB13/DB14 was determined in PC-3 cell membranes, and cell binding of the respective [99mTc]Tc-radioligands was assessed in PC-3 cells. Each of [99mTc]Tc-DB7, [99mTc]Tc-DB13, and [99mTc]Tc-DB14 was injected into mice without or with PA coinjection and 5 min blood samples were analyzed by HPLC. Biodistribution was conducted at 4 h postinjection (pi) in severe combined immunodeficiency disease (SCID) mice bearing PC-3 xenografts without or with PA coinjection. Results: DB7, -13, and -14 displayed single-digit nanomolar affinities for GRPR. The uptake rates of [99mTc]Tc-DB7, [99mTc]Tc-DB13, and [99mTc]Tc-DB14 in PC-3 cells was comparable and consistent with a radioantagonist profile. The radiotracers were found to be ≈70% intact in mouse blood and >94% intact after coinjection of PA. Treatment of mice with PA enhanced tumor uptake. Conclusions: The present study showed that increase of PEG-spacer length in the [99mTc]Tc-DB7–[99mTc]Tc-DB13–[99mTc]Tc-DB14 series had little effect on GRPR affinity, specific uptake in PC-3 cells, in vivo stability, or tumor uptake. A significant change in in vivo stability and tumor uptake was observed only after treatment of mice with PA, without compromising the favorably low background radioactivity levels.


Introduction
The gastrin-releasing peptide receptor (GRPR) has attracted much attention in nuclear oncology owing to its high-density expression in frequently occurring human cancers, such as prostate cancer, mammary carcinoma, and others [1][2][3][4][5][6][7]. This finding can be elegantly exploited to direct diagnostic and therapeutic radionuclides to tumor sites by means of suitably designed peptide carriers that specifically interact with the GRPR on tumor cells [8][9][10][11]. Diagnostic imaging with gamma emitters (e.g., 99m Tc, 111 In) for single-photon-emission computed tomography (SPECT) or positron emitters (e.g., 68 Ga, 64 Cu) for positron-emission tomography (PET) will allow for initial diagnosis, assessment of disease spread and progression, and selection of patients eligible for subsequent radionuclide therapy. Molecular imaging is likewise essential for dosimetry, therapy planning, and follow-up, enabling a patient-tailored, "theranostic" approach. Therapy per se is conducted with the respective peptide analog carrying a suitable particle emitter (beta, alpha, or Auger electron emitter).
Several analogs of the frog 14 peptide bombesin (BBN; Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH 2 ) and its C-terminal BBN (6)(7)(8)(9)(10)(11)(12)(13)(14) fragment, showing high GRPR affinity, have been derivatized with the appropriate chelator for stable binding of the selected medically relevant radiometal and have been evaluated in animal models and in humans [8,11]. It should be noted that such analogs internalize into target cells and display agonistic profiles at the GRPR. Agonism at the GRPR, however, translates into adverse effects elicited in patients after intravenous injection of BBN analogs and the GRPR activation that follows [12][13][14]. Such effects intensify at the higher peptide doses administered during radionuclide therapy, thereby restricting the broader clinical use of GRPR agonists. A subsequent shift of paradigm from GRPR radioagonists to antagonists revealed unexpected benefits in their use beyond the anticipated inherent biosafety. The clearance GRPR radioantagonists, in contrast to agonists, turned out to be much faster from physiological tissues than from tumor sites [15]. The basis for this clinically appealing feature has not been elucidated yet, although it has been observed for other receptor radioantagonists as well [16].
Molecules 2020, 24, x FOR PEER REVIEW 2 of 13 mammary carcinoma, and others [1][2][3][4][5][6][7]. This finding can be elegantly exploited to direct diagnostic and therapeutic radionuclides to tumor sites by means of suitably designed peptide carriers that specifically interact with the GRPR on tumor cells [8][9][10][11]. Diagnostic imaging with gamma emitters (e.g., 99m Tc, 111 In) for single-photon-emission computed tomography (SPECT) or positron emitters (e.g., 68 Ga, 64 Cu) for positron-emission tomography (PET) will allow for initial diagnosis, assessment of disease spread and progression, and selection of patients eligible for subsequent radionuclide therapy. Molecular imaging is likewise essential for dosimetry, therapy planning, and follow-up, enabling a patient-tailored, "theranostic" approach. Therapy per se is conducted with the respective peptide analog carrying a suitable particle emitter (beta, alpha, or Auger electron emitter). Several analogs of the frog 14 peptide bombesin (BBN; Pyr-Gln-Arg-Leu-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Met-NH2) and its C-terminal BBN (6)(7)(8)(9)(10)(11)(12)(13)(14) fragment, showing high GRPR affinity, have been derivatized with the appropriate chelator for stable binding of the selected medically relevant radiometal and have been evaluated in animal models and in humans [8,11]. It should be noted that such analogs internalize into target cells and display agonistic profiles at the GRPR. Agonism at the GRPR, however, translates into adverse effects elicited in patients after intravenous injection of BBN analogs and the GRPR activation that follows [12][13][14]. Such effects intensify at the higher peptide doses administered during radionuclide therapy, thereby restricting the broader clinical use of GRPR agonists. A subsequent shift of paradigm from GRPR radioagonists to antagonists revealed unexpected benefits in their use beyond the anticipated inherent biosafety. The clearance GRPR radioantagonists, in contrast to agonists, turned out to be much faster from physiological tissues than from tumor sites [15]. The basis for this clinically appealing feature has not been elucidated yet, although it has been observed for other receptor radioantagonists as well [16].

Radiolabeling and Quality Control
Radiolabeling of DB7, DB13, and DB14 with [ 99m Tc]Tc was accomplished by 30 min incubation at room temperature in alkaline aqueous medium containing citrate anions and SnCl 2 as reductant. Quality control of the radiolabeled products ( Figure 1) [25,26]. The latter was accomplished by coinjection of the NEP-inhibitor phosphoramidon (PA) [27] together with each radiotracer. This methodology was previously shown to enhance the metabolic stability of BBN and other peptide radioligands in peripheral blood and to improve the supply of the intact radiopeptide form to tumor sites. As a result, notably improved tumor targeting was observed in mice and recently also in patients [28][29][30][31][32][33][34][35].

Radiolabeling and Quality Control
Radiolabeling of DB7, DB13, and DB14 with [ 99m Tc]Tc was accomplished by 30 min incubation at room temperature in alkaline aqueous medium containing citrate anions and SnCl2 as reductant. Quality control of the radiolabeled products ( Figure 1)
All three radiotracers showed a fast blood and body clearance via the kidneys in displaying low background radioactivity uptake even in the GRPR-rich pancreas [ All animals were injected with 180-230 kBq/10 pmol peptide; 1 animals co-injected wit [Tyr 4 ]BBN for in vivo GRPR-blockade; 2 animals co-injected with 300 μg PA for in situ inhi NEP. ]BBN for in vivo GRPR-blockade; 2 animals co-injected with 300 μg PA for in situ inhi NEP.
All three radiotracers showed a fast blood and body clearance via the kidneys in displaying low background radioactivity uptake even in the GRPR-rich pancreas [  All three radiotracers showed a fast blood and body clearance via the kidneys into the urine, displaying low background radioactivity uptake even in the GRPR-rich pancreas [38]   All three radiotracers showed a fast blood and body clearance via the kidneys into the urine, displaying low background radioactivity uptake even in the GRPR-rich pancreas [38]   All three radiotracers showed a fast blood and body clearance via the kidneys int displaying low background radioactivity uptake even in the GRPR-rich pancreas [  All three radiotracers showed a fast blood and body clearance via the kidneys i displaying low background radioactivity uptake even in the GRPR-rich pancreas [ pancreas for all three tracers). Uptake in the experimental PC-3 tumor was comp compounds in control mice, [ 99m Tc]Tc-DB7: 4.49 ± 1.20%IA/g, [ 99m Tc]Tc-DB13: 4.14 ± 0. [ 99m Tc]Tc-DB14 3.71 ± 1.04%IA/g (p > 0.05). This uptake was reduced in the block anim [ 99m Tc]Tc-DB7 (p < 0.0001), [ 99m Tc]Tc-DB13 (p < 0.0001), and [ 99m Tc]Tc-DB14 (p < 0.01), indi specificity.
Molecules 2020, 24, x FOR PEER REVIEW 5 of 13 activity per gram tissue (%IA/g) ± SD, can be found in   All animals were injected with 180-230 kBq/10 pmol peptide; 1 animals co-injected with 50 μg [Tyr 4 ]BBN for in vivo GRPR-blockade; 2 animals co-injected with 300 μg PA for in situ inhibition of NEP.
Molecules 2020, 24, x FOR PEER REVIEW activity per gram tissue (%IA/g) ± SD, can be found in   All three radiotracers showed a fast blood and body clearance via the kidneys displaying low background radioactivity uptake even in the GRPR-rich pancreas pancreas for all three tracers  All three radiotracers showed a fast blood and body clearance via the kidneys into the urine, displaying low background radioactivity uptake even in the GRPR-rich pancreas [38]  activity per gram tissue (%IA/g) ± SD, can be found in ]BBN for in vivo GRPR-blockade; 2 animals co-injected with 300 μg PA for in situ inhibition of NEP. ]BBN for in vivo GRPR-blockade; 2 animals co-injected with 300 μg PA for in situ inhibition of NEP.
All three radiotracers showed a fast blood and body clearance via the kidneys into the urine, displaying low background radioactivity uptake even in the GRPR-rich pancreas [38]   All three radiotracers showed a fast blood and body clearance via the kidneys displaying low background radioactivity uptake even in the GRPR-rich pancreas pancreas for all three tracers ]BBN for in vivo GRPR-blockade; 2 animals co-injected with 300 μg PA for in situ in NEP. ]BBN for in vivo GRPR-blockade; 2 animals co-injected with 300 μg PA for in situ in NEP.
All three radiotracers showed a fast blood and body clearance via the kidneys displaying low background radioactivity uptake even in the GRPR-rich pancreas pancreas for all three tracers  [25,26]. Therefore, its action is overlooked during in vitro incubation assays of radioligands in plasma or serum. We previously demonstrated that coinjection of NEP inhibitors, such as PA [27], with BBNlike radiopeptides, both agonists and antagonists, improves their metabolic stability in circulation [28,[30][31][32][33][34]. As a result, an appreciably higher amount of intact radiopeptide eventually reaches tumor sites. Accordingly, tumor uptake is markedly enhanced, with clear benefits to be gained both for imaging and therapy. Following this rationale, we decided to study the effects of in situ inhibition of NEP on the in vivo stability of [ 99m Tc]Tc-DB7, [ 99m Tc]Tc-DB13, and [ 99m Tc]Tc-DB14 coinjected with PA in the present work. It is interesting to note that once again, significant enhancement of metabolic stability was documented for all three analogs (Figure 4b). In order to assess how the above properties translate in terms of pharmacokinetics, biodistribution profiles of [ 99m Tc]Tc-DB7, [ 99m Tc]Tc-DB13, and [ 99m Tc]Tc-DB14 were compared in mice bearing GRPRpositive experimental tumors at 4 h pi, without or with PA coinjection. Firstly, we observed declining, but not statistically significant lower, uptake in the PC-3 tumors with increasing length of the PEG chain from 2 to 4 ( Figure 5), as a combined result of (i) the equivalent GRPR affinities of DB7, DB13, and DB14, (ii) the slightly declining PC-3 cell uptake capabilities of [ 99m Tc]Tc-DB7, [ 99m Tc]Tc-DB13, and [ 99m Tc]Tc-DB13, and (iii) their similar metabolic stability. Secondly, overall pharmacokinetics turned out to be very comparable for all three radioantagonists, characterized by favorably low background radioactivity levels. Of particular advantage are the low radioactivity values displayed by the three radioligands in the GRPR-rich pancreas, as well as in the kidneys. Thirdly, treatment of mice with PA led to significant increase of tumor uptake compared with controls (Tables 1, 2, and 3) without provoking any unfavorable rise of background activity, thereby further enhancing tumor to background contrast. Interestingly, the enhanced tumor uptake induced by PA via in situ stabilization Tc]Tc-DB14 (gray bars) for kidneys (Ki), pancreas (Pa), and PC-3 tumors (Tu) at 4 h pi in female SCID mice bearing subcutaneous PC-3 xenografts; results are expressed as %IA/g mean ± SD, n = 4, without (checkered bars) or with coinjection (solid bars) of PA. Only statistical differences in organ/tumor uptake across compounds are included in the diagram; statistical differences between PA-treated and nontreated mice are shown in Table 1, Table 2, and Table 3.

Discussion
Radiotracers based on GRPR antagonists have lately attracted much attention in nuclear medicine, largely because of their higher inherent safety for human use compared with agonists [20]. After intravenous injection to patients, antagonists seek and bind but do not activate the GRPR, and hence they do not elicit adverse effects. Furthermore, GRPR radioantagonists often display attractive pharmacokinetic profiles in preclinical models and in patients, as a combined result of rapid clearance from physiological tissues and good retention in tumor sites. In fact, a general concern in the application of GRPR radioligands for cancer theranostics has been the high-density expression of GRPR not only on tumors, but also in physiological tissues, especially in the pancreas [38]. Previous studies with both GRPR radioagonists and antagonists have demonstrated the significant impact of linkers, introduced between the metal chelate and the peptide part, on pharmacokinetics [23,[39][40][41][42].
In the present study, we were interested to find out whether the elongation of the PEG 2 chain would further improve the biological properties of resulting analogs. For this purpose, [ 99m Tc]Tc-DB13 (PEG 3 ) and [ 99m Tc]Tc-DB14 (PEG 4 ) were newly synthetized and compared to [ 99m Tc]Tc-DB7 ( Figure 1). As revealed by competitive binding assays in PC-3 cell membranes, the elongation of PEG 2 , to PEG 3 and PEG 4 had no apparent impact on the GRPR affinity of the respective DB7, DB13, and DB14 ( Figure 2). On the other hand, the resultant [ 99m Tc]Tc radiotracers showed modest, but statistically significant, decline of uptake in PC-3 cells in vitro as the length of the PEG linker increased from PEG 2 to PEG 4 (Figure 3).
In a subsequent step, we investigated the effect of PEG-chain length on the metabolic stability of the three [ 99m Tc]Tc radiotracers in mouse blood collected 5 min pi. This study did not reveal any statistically significant differences across radiotracers (Figure 4a). NEP has been shown to be a major protease in the rapid in vivo degradation of BBN and its analogs [28,36,37]. NEP is actually an ectoenzyme abundantly present on the epithelial cells of several tissues of the body, including vasculature walls, kidneys, lungs, and intestines, but found only in minute amounts in the blood solute [25,26]. Therefore, its action is overlooked during in vitro incubation assays of radioligands in plasma or serum. We previously demonstrated that coinjection of NEP inhibitors, such as PA [27], with BBN-like radiopeptides, both agonists and antagonists, improves their metabolic stability in circulation [28,[30][31][32][33][34]. As a result, an appreciably higher amount of intact radiopeptide eventually reaches tumor sites. Accordingly, tumor uptake is markedly enhanced, with clear benefits to be gained both for imaging and therapy. Following this rationale, we decided to study the effects of in situ inhibition of NEP on the in vivo stability of [ 99m Tc]Tc-DB7, [ 99m Tc]Tc-DB13, and [ 99m Tc]Tc-DB14 coinjected with PA in the present work. It is interesting to note that once again, significant enhancement of metabolic stability was documented for all three analogs (Figure 4b).
In order to assess how the above properties translate in terms of pharmacokinetics, biodistribution profiles of [ 99m Tc]Tc-DB7, [ 99m Tc]Tc-DB13, and [ 99m Tc]Tc-DB14 were compared in mice bearing GRPR-positive experimental tumors at 4 h pi, without or with PA coinjection. Firstly, we observed declining, but not statistically significant lower, uptake in the PC-3 tumors with increasing length of the PEG chain from 2 to 4 ( Figure 5), as a combined result of (i) the equivalent GRPR affinities of DB7, DB13, and DB14, (ii) the slightly declining PC-3 cell uptake capabilities of [ 99m Tc]Tc-DB7, [ 99m Tc]Tc-DB13, and [ 99m Tc]Tc-DB13, and (iii) their similar metabolic stability. Secondly, overall pharmacokinetics turned out to be very comparable for all three radioantagonists, characterized by favorably low background radioactivity levels. Of particular advantage are the low radioactivity values displayed by the three radioligands in the GRPR-rich pancreas, as well as in the kidneys. Thirdly, treatment of mice with PA led to significant increase of tumor uptake compared with controls (Table 1, Table 2, and Table 3) without provoking any unfavorable rise of background activity, thereby further enhancing tumor to background contrast. Interestingly, the enhanced tumor uptake induced by PA via in situ stabilization of analogs in mouse circulation ended up being reflected in statistically significant differences between the PEG 2/3 and PEG 4 members of the series ( Figure 5).