One Step Closer to Clinical Translation: Enhanced Tumor Targeting of [99mTc]Tc-DB4 and [111In]In-SG4 in Mice Treated with Entresto

Background: Peptide radioligands may serve as radionuclide carriers to tumor sites overexpressing their cognate receptor for diagnostic or therapeutic purposes. Treatment of mice with the neprilysin (NEP)-inhibitor phosphoramidon was previously shown to improve the metabolic stability and tumor uptake of biodegradable radiopeptides. Aiming to clinical translation of this methodology, we herein investigated the impact of the approved pill Entresto, releasing the potent NEP-inhibitor LBQ657 in vivo, on the stability and tumor uptake of two radiopeptides. Methods: The metabolic stability of [99mTc]Tc-DB4 (DB4, N4-Pro-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH2) and [111In]In-SG4 (SG4, DOTA-DGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2) was tested in LBQ657/Entresto-treated mice vs. untreated controls. The uptake in gastrin-releasing peptide receptor (GRPR)-, or cholecystokinin subtype 2 receptor (CCK2R)-positive tumors respectively, was compared between LBQ657/Entresto-treated mice and untreated controls. Results: LBQ657/Entresto treatment induced marked stabilization of [99mTc] Tc-DB4 and [111In]In-SG4 in peripheral mice blood, resulting in equally enhanced tumor uptake at 4 h post-injection. Accordingly, the [99mTc]Tc-DB4 uptake of 7.13 ± 1.76%IA/g in PC-3 tumors increased to 16.17 ± 0.71/17.50 ± 3.70%IA/g (LBQ657/Entresto) and the [111In]In-SG4 uptake of 3.07 ± 0.87%IA/g in A431-CCK2R(+) tumors to 8.11 ± 1.45/9.61 ± 1.70%IA/g. Findings were visualized by SPECT/CT. Conclusions: This study has shown the efficacy of Entresto to notably improve the profile of [99mTc]Tc-DB4 and [111In]In-SG4 in mice, paving the way for clinical translation of this approach.


Introduction
Latest advances in medicine have been aiming at personalized management of patients. Toward this goal, modern anti-cancer drugs are being designed to specifically interact with tumor-associated biomolecular targets, sparing healthy tissues devoid of target expression. For example, peptide analogs may serve as radionuclide carriers to tumor-located receptors, allowing for 3 of 14 Pharmaceutics 2020, 12, x FOR PEER REVIEW 3 of 14

Quality Control
Reversed-phase high-performance liquid chromatography (RP-HPLC) was performed on a Waters Chromatograph based on a 600E multi-solvent delivery system coupled to a Waters 2998 photodiode array detector (Waters, Vienna, Austria) and a Gabi gamma-detector (Raytest, RSM Analytische Instrumente GmbH, Straubenhardt, Germany). Data processing and chromatography were controlled by the Empower Software (Waters, Milford, MA, USA). For quality control, aliquots of the radiolabeling solution were loaded on a Symmetry Shield RP18 cartridge column (5 µm, 3.9 × 20 mm, Waters, Eschborn, Germany), eluted for 40 min with 0.1% trifluoroacetic acid (TFA) in H 2 O/MeCN applying a linear gradient starting from 0% MeCN and a 2% increase per min at 1 mL/min flow rate (system 1 Human androgen-independent prostate adenocarcinoma PC-3 cells endogenously expressing the GRPR [30] were obtained from LGC Standards GmbH (Wesel, Germany), whereas the human epidermoid carcinoma A431 cell line transfected to stably express the human CCK 2 R (A431-CCK 2 R(+)) [31,33] or devoid of CCK 2 R expression (A431-CCK 2 R(-)) was a gift from Prof. O. Boerman (Department of Nuclear Medicine, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands) and Prof. L. Aloj (Istituto di Biostrutture e Bioimmagini, Consiglio Nazionale delle Ricerche, Naples, Italy). All culture reagents were obtained from Gibco BRL, Life Technologies (Grand Island, NY, USA) or from Biochrom KG Seromed (Berlin, Germany).

Animal Studies
European guidelines were applied in all animal studies in supervised and licensed facilities (EL 25 BIO 021). The study protocols were approved by the Department of Agriculture and Veterinary Service of the Prefecture of Athens (revised protocol number 1609 approved on 24 April 2019 for the stability studies and revised protocol number 1610 approved on 24 April 2019 for biodistribution and imaging studies). For stability experiments, 20 healthy male Swiss albino mice (30 ± 5 g, NCSR "Demokritos" Animal House Facility) were used, divided in groups of 3 or 4 (vide infra). For biodistribution studies with [ 99m Tc]Tc-DB4, 16 female severe combined immune deficiency (SCID) mice (14.3 ± 1.3 g, six weeks of age at the day of arrival, NCSR "Demokritos" Animal House Facility) were employed, divided in 4 groups of 4. Biodistribution and imaging studies with [ 111 In]In-SG4 were conducted in 17 male SCID mice (17.2 ± 3.0 g, six weeks of age at the day of arrival) from the same provider. 12 animals divided in 3 groups of 4 were used in biodistribution studies and 5 in SPECT/CT imaging.

In Vivo Stability Tests
A 100 µL bolus of [ 99m Tc]Tc-DB4 (50-60 MBq, 3 nmol total peptide in vehicle: saline/EtOH 9/1 v/v) or [ 111 In]In-SG4 (11-22 MBq, 3 nmol of total peptide in saline/EtOH 9/1 v/v) was injected in the tail vein of mice together with vehicle (100 µL; control; [ 99m Tc]Tc-DB4, n = 3 or [ 111 In]In-SG4, n = 4) or with LBQ657 solution (100 µL injection solution containing 10 µg LBQ657, n = 3 for both analogs). In a third set of animals per radioligand, mice received, by gavage, a single Entresto dose (12 mg/200 mL per animal, prepared as described in Section 2.1.1.) 30 min prior to injection of either radiotracer together with vehicle (100 µL; Entresto-group; [ 99m Tc]Tc-DB4, n = 4 or [ 111 In]In-SG4, n = 3). Mice were euthanized 5 min after radioligand injection (5 min post-injection (pi)) and a prechilled syringe was used to withdraw blood (0.5-1 mL) directly from the heart. Blood was transferred in a pre-chilled EDTA-containing Eppendorf tube on ice and subsequently centrifuged for 10 min at 2000 g/4 • C. The plasma was collected and an equal volume of ice-cold MeCN was added. The mixture was centrifuged for 10 min at 15,000 g/4 • C. The supernatant was collected and a N 2 -flux at 40 • C was applied to reduce the volume to 0.05-0.1 mL. After diluting with normal saline (0.4 mL), the solution was passed through a 0.22 µm Millex GV filter (Millipore, Milford, USA). Filtrate aliquots were then analyzed by radio-HPLC. A Symmetry Shield RP18 (5 µm, 3.9 × 20 mm) column served as stationary phase, eluted at a flow rate of 1 mL/min by 0.1% TFA in H 2 O (A) and MeCN (B) combinations as mobile phase. In case of [ 99m Tc]Tc-DB4, the following linear gradient system was applied: 0%B at 0 min, 10%B at 10 min and reaching 25%B at 40 min (system 2a); for [ 111 In]In-SG4, the gradient started with 0%B increasing linearly with 1%/min to 40%B (system 2b). The t R of the intact radiopeptide was determined by co-injection with the respective [ 99m Tc]Tc-DB4 (t R = 31.5 min)/[ 111 In]In-SG4 (t R = 30.0 min) reference in the HPLC.

Biodistribution of [ 99m Tc]Tc-DB4 and [ 111 In]In-SG4 in Tumor-Bearing Mice
For the biodistribution study, animals in groups of 4 received a 100 µL bolus of [ 99m Tc]Tc-DB4 via the tail vein (200 kBq, corresponding to 10 pmol total peptide in saline/EtOH 9/1 v/v-GRPR-tumor-bearing mice) or [ 111 In]In-SG4 (50 kBq, 10 pmol total peptide-twin A431-CCK 2 R(+)/A431-CCK 2 R(−) tumor-bearing mice) co-injected either with injection solution (100 µL; control) or with LBQ657 solution (100 µL injection solution containing 10 µg LBQ657). In additional sets of animals, mice received, by gavage, a single Entresto dose (12 mg/200 mL per animal, prepared as described in Section 2.1.1) 30 min prior to injection of the respective radiotracer together with vehicle (100 µL; Entresto group), or with a [Tyr 4 ]BBN solution (100 µg in 100 µL vehicle; block group). Mice were euthanized at 4 h pi and dissected. Samples of blood, tumors and organs of interest were collected, weighed and measured for radioactivity in the γ-counter (an automated well-type gamma counter with a NaI(Tl) 3 ' crystal, Canberra Packard Auto-Gamma 5000 series instrument). Intestines and stomach were not emptied of their contents; exceptionally, in the case of [ 111 In]In-SG4-injected animals, stomachs were emptied of their contents. Data was calculated as percent injected activity per gram tissue (%IA/g) with the aid of standard solutions and represented as mean values ± standard deviation (SD), n = 4.

Statistical Analysis
The two-way analysis of variance (ANOVA) with multiple comparisons was used for statistical analysis of biological results, applying Tukey's post hoc analysis (GraphPad Prism Software 6, San Diego, CA, USA). p-values below 0.05 were considered to be statistically significant.

SPECT/CT Imaging of [ 111 In]In-SG4 in A431-CCK 2 R(+) Tumor-Bearing Mice
Five mice bearing A431-CCK 2 R(+) xenografts on their shoulders were used for SPECT/CT imaging. Three out of five mice received Entresto by gavage 30 min prior to radiotracer injection. All five animals were injected with a bolus containing [ 111 In]In-SG4 (100 µL, 5 MBq of HPLC isolated preparation) via the tail vein and were euthanized 4 h later. The y-CUBE/x-CUBE systems (Molecubes, Belgium) were employed for tomographic SPECT/CT imaging [34]. The SPECT system was based on monolithic NaI detectors attached to SiPMs, with a 0.6 mm intrinsic resolution. The CT system was based on a structured CMOS detector of CsI with pixels of 75 µm and operated between 35 and 80 kVp, 10-500 µA tube current, with a 33 µm fixed focal spot size. For SPECT scans, a 60 min duration protocol was applied, based on the injected activity. A CT scan followed after each SPECT scan, according to a General Purpose protocol under 50 kVp for co-registration purposes. The MLEM reconstruction method was applied for the reconstruction of SPECT images with a 250 µm voxel size and 500 iterations. The ISRA reconstruction method with a 100 µm voxel size was applied for the reconstruction of CT images. Images were exported and post-processed on VivoQuant software, version 4.0 (Invicro, Boston, MA, USA). A smoothing median filter (0.6 mm, spherical window) was applied to the images and for consistency purposes, the bladder was removed. Both radiotracers were catabolized to a great extent 5 min after their entry in mice circulation. As revealed by HPLC analysis of blood samples collected at 5 min pi (Figure 2a), only 26.9% ± 3.7% [ 99m Tc]Tc-DB4 was found intact in peripheral mice blood in this period (Figure 2b). Co-injection of LBQ657 led to notable stabilization of the radiotracer (74.3% ± 8.6% intact; p < 0.0001), whereas a similar stabilization effect was observed in mice treated with Entresto 30 min prior to radioligand injection (71.5% ± 1.8% intact; p < 0.0001). A summary of these results in numerical values is included in Table 1.

Peptides and Radioligands
Labeling of DB4 with technetium-99m typically proceeded by brief peptide-conjugate incubation with [ 99m Tc]TcO4 -generator eluate, SnCl2 as reducing agent and citrate anions as transfer ligand in alkaline pH at ambient temperature at molecular activities of 18-

Stability of [ 99m Tc]Tc-DB4 and [ 111 In]In-SG4 in Mice
Both radiotracers were catabolized to a great extent 5 min after their entry in mice circulation. As revealed by HPLC analysis of blood samples collected at 5 min pi (Figure 2a), only 26.9% ± 3.7% [ 99m Tc]Tc-DB4 was found intact in peripheral mice blood in this period (Figure 2b). Co-injection of LBQ657 led to notable stabilization of the radiotracer (74.3% ± 8.6% intact; p < 0.0001), whereas a similar stabilization effect was observed in mice treated with Entresto 30 min prior to radioligand injection (71.5% ± 1.8% intact; p < 0.0001). A summary of these results in numerical values is included in Table 1. Numerical average values ± standard deviation (SD) are summarized in Table 1. Likewise, HPLC analysis of blood samples collected at 5 min pi of [ 111 In]In-SG4 (Figure 3a) revealed only 11.5% ± 3.2% intact radiotracer (Figure 3b).

Biodistribution of [ 99m Tc]Tc-DB4 SCID Mice Bearing PC-3 Xenografts
The biodistribution of [ 99m Tc]Tc-DB4 at 4 h pi was studied in SCID mice bearing subcutaneous PC-3 tumors expressing the human GRPR, without or during treatment with LBQ657 or Entresto. Biodistribution data, expressed as mean %IA/g ± SD, n = 4, is summarized in Table 2. The radiotracer has washed out from the blood and the body of mice predominantly via the kidneys and the urinary system, with some degree of intestinal uptake. High uptake is seen in the GRPR-rich pancreas (27.71 ± 6.36%IA/g) as well as in the implanted tumors (7.13 ± 1.76%IA/g). Numerical average values ± SD are summarized in Table 1.

Biodistribution of [ 99m Tc]Tc-DB4 SCID Mice Bearing PC-3 Xenografts
The biodistribution of [ 99m Tc]Tc-DB4 at 4 h pi was studied in SCID mice bearing subcutaneous PC-3 tumors expressing the human GRPR, without or during treatment with LBQ657 or Entresto. Biodistribution data, expressed as mean %IA/g ± SD, n = 4, is summarized in Table 2. The radiotracer has washed out from the blood and the body of mice predominantly via the kidneys and the urinary system, with some degree of intestinal uptake. High uptake is seen in the GRPR-rich pancreas (27.71 ± 6.36%IA/g) as well as in the implanted tumors (7.13 ± 1.76%IA/g).
Co-injection of LBQ657 or treatment of mice with Entresto resulted in clear enhancement of tumor uptake (to 16.17 ± 0.71%IA/g; p < 0.0001 and to 17.50 ± 3.70%IA/g; p < 0.0001 respectively, vs. controls). No significant increase was observed in any other organ or tissue, except for the pancreas. In this case, the radioligand stabilization by either LBQ657 or Entresto led to significant increases of pancreatic uptake in the mice (to 56.06 ± 4.24%IA/g; p < 0.0001 and to 60.98 ± 6.41%IA/g; p < 0.0001 respectively, vs. controls). It should be noted that uptake in the tumors as well as in pancreas and in mice intestines was significantly reduced in mice co-injected with excess [Tyr 4 ]BBN 30 min after treatment with Entresto, revealing a GRPR-mediated process (to 0.55 ± 0.04%IA/g; p < 0.0001, to 0.68 ± 0.04%IA/g; p < 0.0001 and to 1.23 ± 0.21%IA/g; p < 0.01 respectively, vs. the Entresto group).

SPECT/CT of [ 111 In]In-SG4 in A431-CCK 2 R(+) Xenograft-Bearing Mice
Mice SPECT/CT images obtained 4 h after injection of [ 111 In]In-SG4 are presented in Figure 5. Significant accumulation was achieved in the CCK 2 R-expressing A431-CCK 2 R(+) xenografts, while much lower radioactivity levels remained in the kidneys. Clear differences in tumor uptake could be observed between controls (Figure 5a

Discussion
A major challenge in a wider application of peptide radioligands in cancer theranostics is linked to their fast catabolism in the biological milieu by peptidases [10,11]. We have previously reported on the prominent role of NEP in the rapid in vivo degradation of radioligands originating from various peptide families, including bombesin and gastrin [12]. NEP is an ectoenzyme with a broad

Discussion
A major challenge in a wider application of peptide radioligands in cancer theranostics is linked to their fast catabolism in the biological milieu by peptidases [10,11]. We have previously reported on the prominent role of NEP in the rapid in vivo degradation of radioligands originating from various peptide families, including bombesin and gastrin [12]. NEP is an ectoenzyme with a broad substrate repertoire anchored on epithelial cell membranes of blood vessels and major organs of the body in high local concentrations [13,14]. A great number of radiopeptide analogs entering the circulation become exposed to its action and get swiftly degraded, with only a fraction of the original number of molecules reaching the tumor-associated receptors intact. As a result, diagnostic contrast and/or therapeutic efficacy are strongly compromised. We were able to successfully interfere with this chain of disadvantageous events by inducing transient NEP-inhibition through co-injection of PA [15,16] in mice. As a result, marked increases in the metabolic stability of various circulating peptide radiotracers could be achieved, directly favoring tumor uptake [12,[17][18][19][20][21]24]. These exciting results, documented at the preclinical level in a multitude of cases, warrant further validation in cancer patients. Clinical proof of this concept may promptly enrich the arsenal of anti-cancer theranostic tools, since the profiles of available radiopeptides can be optimized in situ. In this way, time-/cost-intensive structure-activity relationship studies with the development of new radiotracer libraries may be circumvented.
Toward this goal, we have decided to explore the efficacy of approved NEP-inhibitors that could replace PA in a clinical setting [21,25]. Most interestingly, Entresto was approved and became commercially available recently as an antihypertensive drug [26][27][28]. Entresto pills for oral administration contain a combination of valsartan and sacubitril. The latter is the precursor form of the potent NEP-inhibitor sacubitrilat (LBQ657) [29], which is quickly released in vivo via de-esterification by endogenous esterases (Figure 1c). In the present work, we were interested to study the effects of both Entresto and the active substance LBQ657 on the stability and biodistribution profile of two radiopeptide examples, [ 99m Tc]Tc-DB4 and [ 111 In]In-SG4 (Figure 1a, b, respectively). LBQ657 was intravenously injected in mice together with either radioligand. The pill was orally administered 30 min in advance of radiotracer injection, so as to accomplish similar levels of the active substance in mice plasma and hence similar NEP-inhibition efficacy [32].
As depicted in Figures 2 and 3, neither of the two radiotracers were detected intact in peripheral mice blood in the absence of inhibitors as soon as 5 min pi, revealing a very rapid action of native peptidases. Co-injection of the NEP-inhibitor significantly increased the metabolic stability of both radiopeptides, directly implicating NEP in their swift in vivo degradation. Most interestingly, treatment of mice with the pill induced the same stabilization levels of the radiotracers, confirming the indistinguishable efficacy of Entresto and LBQ657 to inhibit NEP in situ [29,32]. It should be noted that PA was previously shown to stabilize BBN-/MG-based radiotracers, including [ 111 In]In-SG4, with comparable efficacy [12].
We next investigated how the aforementioned radioligand stabilization affected tumor targeting and overall pharmacokinetics of the two radioligands. As shown in Table 2, we could observe a highly significant increase in the uptake of [ 99m Tc]Tc-DB4 in the PC-3 xenografts at 4 h pi in both Entresto-and LBQ657-treated mice compared with untreated controls, with the two treatments being equally effective. It should be noted that in the "block" animal group, whereby mice were treated with Entresto and a high excess of [Tyr 4 ]BBN, tumor uptake was minimized. This finding is suggestive of GRPR-specificity, ruling out GRPR-unrelated interferences of the pill in the observed tumor uptake enhancement. Additional increases of radioactivity levels could be observed in mice pancreas and intestines. Such increases could be attributed to the known physiological expression of GRPR in the healthy organs, as implied by the respective values in the "block" animal group. Again, similar observations were previously reported for most BBN-based radioagonists after treatment of mice with PA [12,[17][18][19]35].
The impact of treating mice with Entresto and LBQ657 on the uptake of [ 111 In]In-SG4 in the A431-CCK 2 R(+) xenografts compared with untreated controls is summarized in Table 3. Again, the stabilization of the radiotracer in peripheral mice blood by either Entresto or LBQ657 led to significant and comparable increases in the CCK 2 R-expressing tumors compared with untreated controls. Furthermore, the observed enhancements were apparent only in the A431-CCK 2 R(+), but not in the A431-CCK 2 R(−) xenografts, suggesting CCK 2 R-specificity. Similar enhancements could be induced previously by transient NEP-inhibition by PA [12,20,21,24]. In view of the fact that background radioactivity levels remained the same between the Entresto and the control animal groups, including the mice CCK 2 R-rich gastric mucosa, we further proceeded to visualize these effects by SPECT/CT. As shown in the representative images of Figure 5 taken 4 h pi of [ 111 In]In-SG4, a striking increase of tumor uptake could be established in the Entresto-treated mice group over the non-treated controls.

Conclusions
This study has shown that Entresto, an approved antihypertensive pill to be taken orally and promptly releasing the potent NEP-inhibitor LBQ657 in vivo [29], can induce comparable stabilization effects with LBQ657 and the non-licensed NEP-inhibitor PA of GRPR-/CCK 2 R-directed radiopeptides. The observed stabilization was shown to drastically improve the uptake of two representative radiopeptides, [ 99m Tc]Tc-DB4 and [ 111 In]In-SG4, in tumor-bearing mice via GRPR-/CCK 2 R-specific pathways, most probably by enhancing the supply of intact radiotracers to tumor-residing receptors. These findings represent an important further step for the proof-of-principle of this approach in cancer patients and may thus contribute in a broader and more effective application of radiopeptides in nuclear oncology.