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.

Background: The GRPR-antagonist ⁶⁸Ga-SB3 visualized prostate cancer lesions in animal models and in patients. Switching radiometal from ⁶⁸Ga to ¹¹¹In impaired tumor targeting in mice, but coinjection of the neprilysin (NEP)-inhibitor phosphoramidon (PA) stabilized ¹¹¹In-SB3 in circulation and remarkably increased tumor uptake. We herein report on the biological profile of ¹¹¹In-SB4: ¹¹¹In-[dAla¹¹]SB3. Methods: The biological responses of ¹¹¹In-SB3/SB4 were compared in PC-3 cells and animal models. Results: Gly¹¹/dAla¹¹-replacement deteriorated GRPR-affinity (SB4 IC₅₀: 10.7 ± 0.9 nM vs. SB3 IC₅₀: 4.6 ± 0.3 nM) and uptake in PC-3 cells (¹¹¹In-SB4: 1.3 ± 0.4% vs. ¹¹¹In-SB3 16.2 ± 0.8% at 1 h). ¹¹¹In-SB4 was more stable than ¹¹¹In-SB3, but PA-coinjection stabilized both radiotracers in peripheral mice blood. Unmodified ¹¹¹In-SB3 showed higher uptake in PC-3 xenografts (8.8 ± 3.0%ID/g) vs. ¹¹¹In-SB4 (3.1 ± 1.1%ID/g) at 4 h pi. PA-coinjection improved tumor uptake, with ¹¹¹In-SB3 still showing superior tumor targeting (38.3 ± 7.9%ID/g vs. 7.4 ± 0.3%ID/g for ¹¹¹In-SB4). Conclusions: Replacement of Gly¹¹ by dAla¹¹ improved in vivo stability, however, at the cost of GRPR-affinity and cell uptake, eventually translating into inferior tumor uptake of ¹¹¹In-SB4 vs. unmodified ¹¹¹In-SB3. On the other hand, in-situ NEP-inhibition turned out to be a more efficient and direct strategy to optimize the in vivo profile of ¹¹¹In-SB3, and potentially other peptide radiotracers.

The neurotensin subtype 1 receptor (NTS₁R) is overexpressed in a number of human tumors, thereby representing a valid target for cancer theranostics with radiolabeled neurotensin (NT) analogs like [^99mTc]Tc-DT1 (DT1, N₄-Gly⁷-NT(8-13)). Thus far, the fast degradation of intravenously injected NT–radioligands by neprilysin (NEP) and angiotensin-converting enzyme (ACE) has compromised their clinical applicability. Aiming at metabolic stability enhancements, we herein introduce (i) DT7 ([DAsn¹⁴]DT1) and (ii) DT8 ([β-Homoleucine¹³]DT1), modified at the C-terminus, along with (iii) DT9 ([(palmitoyl)Lys⁷]DT1), carrying an albumin-binding domain (ABD) at Lys⁷. The biological profiles of the new [^99mTc]Tc–radioligands were compared with [^99mTc]Tc-DT1, using NTS₁R-expressing AsPC-1 cells and mice models without or during NEP/ACE inhibition. The radioligands showed enhanced in vivo stability vs. [^99mTc]Tc-DT1, with [^99mTc]Tc-DT9 displaying full resistance to both peptidases. Furthermore, [^99mTc]Tc-DT9 achieved the highest cell internalization and tumor uptake even without NEP/ACE-inhibition but with unfavorably high background radioactivity levels. Hence, unlike C-terminal modification, the introduction of a pendant ABD group in the linker turned out to be the most promising strategy toward metabolic stability, cell uptake, and tumor accumulation of [^99mTc]Tc-DT1 mimics. To improve the observed suboptimal pharmacokinetics of [^99mTc]Tc-DT9, the replacement of palmitoyl on Lys⁷ by other ABD groups is currently being pursued.

Neurotensin subtype 1 receptors (NTS1R) represent attractive molecular targets for directing radiolabeled neurotensin (NT) analogs to tumor lesions for diagnostic and therapeutic purposes. This approach has been largely undermined by the rapid in vivo degradation of linear NT-based radioligands. Herein, we aim to increase the tumor targeting of three ^99mTc-labeled NT analogs by the in-situ inhibition of two key proteases involved in their catabolism. DT1 ([N₄-Gly⁷]NT(7-13)), DT5 ([N₄-βAla⁷,Dab⁹]NT(7-13)), and DT6 ([N₄-βAla⁷,Dab⁹,Tle¹²]]NT(7-13)) were labeled with ^99mTc. Their profiles were investigated in NTS1R-positive colon adenocarcinoma WiDr cells and mice treated or not with the neprilysin (NEP)-inhibitor phosphoramidon (PA) and/or the angiotensin converting enzyme (ACE)-inhibitor lisinopril (Lis). Structural modifications led to the partial stabilization of ^99mTc-DT6 in peripheral mice blood (55.1 ± 3.9% intact), whereas ^99mTc-DT1 and ^99mTc-DT5 were totally degraded within 5 min. Coinjection of PA and/or Lis significantly stabilized all three analogs, leading to a remarkable enhancement of tumor uptake for ^99mTc-DT1 and ^99mTc-DT5, but was less effective in the case of poorly internalizing ^99mTc-DT6. In conclusion, NEP and/or ACE inhibition represents a powerful tool to improve tumor targeting and the overall pharmacokinetics of NT-based radioligands, and warrants further validation in the field of NTS1R-targeted tumor imaging and therapy.

(1) Background: Theranostic approaches in the management of cholecystokinin subtype 2 receptor (CCK₂R)-positive tumors include radiolabeled gastrin and CCK motifs. Moving toward antagonist-based CCK₂R-radioligands instead, we herein present three analogs of the nonpeptidic CCK₂R-antagonist Z360, GAS1/2/3. Each was conjugated to a different chelator (DOTA, NODAGA or DOTAGA) for labeling with medically relevant trivalent radiometals (e.g., Ga-68, In-111, Lu-177) for potential use as anti-CCK₂R cancer agents; (2) Methods: The in vitro properties of the thee analogs were compared in stably transfected HEK293-CCK₂R cells. Biodistribution profiles were compared in SCID mice bearing twin HEK293-CCK₂R and wtHEK293 tumors; (3) Results: The GAS1/2/3 analogs displayed high CCK₂R-affinity (lower nM-range). The radioligands were fairly stable in vivo and selectively targeted the HEK293-CCK₂R, but not the CCK₂R-negative wtHEK293 tumors in mice. Their overall pharmacokinetic profile was found strongly dependent on the radiometal-chelate. Results could be visualized by SPECT/CT for the [¹¹¹In]In-analogs; (4) Conclusions: The present study highlighted the high impact of the radiometal-chelate on the end-pharmacokinetics of a new series of Z360-based radioligands, revealing candidates with promising properties for clinical translation. It also provided the impetus for the development of a new class of nonpeptidic radioligands for CCK₂R-targeted theranostics of human cancer.

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.