Eric P. Krenning

Background/Objectives: The neurotensin subtype-1 receptor (NTS₁R) is overexpressed in a number of human tumors, representing a valid biomolecular target for neurotensin-based radionuclide carriers. The latter are designed to deposit diagnostic–therapeutic radionuclides specifically on cancer lesions. This review summarizes research efforts toward the development, preclinical evaluation and first clinical outcomes of neurotensin-based radioligands in the diagnosis and therapy—radiotheranostics—of NTS₁R-positive cancer. Methods: The preclinical and few clinical studies related to NTS₁R-targeted radiotheranostics are discussed. The crucial issue of in vivo metabolic stability is highlighted. Results: Much effort has been dedicated to the development of effective NTS₁R-directed radioligands for cancer theranostics, most of which are peptide-based. However, clinical success has been sub-optimal thus far, partly as a result of their fast degradation in the biological milieu. Conclusions: The administration of key protease inhibitor regimens, the introduction of branched or otherwise stabilized analogs and the advent of non-peptidic NTS₁R-antagonists are some of the new promising approaches towards achieving clinical breakthroughs in the battle against NTS₁R-expressing cancer.

Background/Objectives: Radiotheranostics of neurotensin subtype 1 receptor (NTS₁R)-expressing tumors, like pancreatic, gastrointestinal, or prostate cancer, has attracted considerable attention in recent years. Still, the fast degradation of neurotensin (NT)-based radioligands, by angiotensin-converting enzyme (ACE), neprilysin (NEP), and other proteases, has considerably compromised their efficacy. The recently introduced [^99mTc]Tc-DT11 (DT11, N₄-Lys(MPBA-PEG4)-Arg-Arg-Pro-Tyr-Ile-Leu-OH; N₄, 6-(carboxy)-1,4,8,11-tetraazaundecane) has displayed promising uptake in NTS₁R-positive tumors in mice and enhanced resistance to both ACE and NEP by virtue of the lateral MPBA-PEG4 (MPBA, 4-(4-methylphenyl)butyric acid; PEG4, 14-amino-3,6,9,12-tetraoxatetradecan-1-oic acid) chain attached to the ε-NH₂ of Lys⁷. We were next interested in investigating whether these qualities could be retained in DT14D, likewise modified at Lys⁷ but carrying the universal chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) via a (βAla)₃ spacer at the α-NH₂ of Lys⁷. This chelator switch enables the labeling of DT14D with a wide range of trivalent radiometals suitable for true theranostic applications, not restricted to the diagnostic imaging of NTS₁R-positive lesions only by single-photon emission computed tomography (SPECT). Methods: DT14D was labeled with Ga-67 (a surrogate for the positron emission tomography radionuclide Ga-68), In-111 (for SPECT), and Lu-177 (applied in radiotherapy). The resulting radioligands were tested in NTS₁R-expressing pancreatic cancer AsPC-1 cells and mice models. Results: [⁶⁷Ga]Ga/[¹¹¹In]In/[¹⁷⁷Lu]Lu-DT14D displayed high affinity for human NTS₁R and internalization in AsPC-1 cells. They remained >70% intact 5 min after entering the mice’s circulation, displaying NTS₁R-specific uptake in AsPC-1 xenografts. Conclusions: Suitably side-chain modified NT analogs show enhanced metabolic stability and hence better prospects for radiotheranostic application in NTS₁R-positive cancer.

Background: The concept of radiotheranostics relies on the overexpression of a biomolecular target on malignant cells to direct diagnostic/therapeutic radionuclide-carriers specifically to cancer lesions. The concomitant expression of more than one target in pathological lesions may be elegantly exploited to improve diagnostic sensitivity and therapeutic efficacy. Toward this goal, we explored a first example of a combined application of [^99mTc]Tc-DT11 (DT11, N₄-Lys(MPBA-PEG4)-Arg-Arg-Pro-Tyr-Ile-Leu-OH; NTS₁R-specific) and [^99mTc]Tc-DB7(DB7, N₄-PEG2-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt; GRPR-specific) in prostate cancer models. Methods: Accordingly, the behavior of [^99mTc]Tc-DT11 was compared with that of the [^99mTc]Tc-DT11+[^99mTc]Tc-DB7 mixture in prostate adenocarcinoma PC-3 cells and xenografts in mice. The impact of stabilizing both radiotracers by Entresto^®, as a source of the potent neprilysin inhibitor sacubitrilat, was also investigated. Results: The PC-3 cell binding of the [^99mTc]Tc-DT11+[^99mTc]Tc-DB7 mixture surpassed that of [^99mTc]Tc-DT11. Likewise, the PC-3 tumor uptake of the [^99mTc]Tc-DT11+[^99mTc]Tc-DB7 mixture at 4 h post-injection was superior (7.70 ± 0.89%IA/g) compared with [^99mTc]Tc-DT11 (4.23 ± 0.58%IA/g; p < 0.0001). Treatment with Entresto^® led to further enhancement of the tumor uptake (to 11.57 ± 1.92%IA/g; p < 0.0001). Conclusions: In conclusion, this first preclinical study on prostate cancer models revealed clear advantages of dual NTS₁R/GRPR targeting, justifying further assessment of this promising concept in other cancer models.

The overexpression of one or more somatostatin receptors (SST_1–5R) in human tumors has provided an opportunity for diagnosis and therapy with somatostatin-like radionuclide carriers. The application of “pansomatostatin” analogs is expected to broaden the clinical indications and upgrade the diagnostic/therapeutic efficacy of currently applied SST₂R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala¹-Gly²-c[Cys³-Lys⁴-Asn⁵-c[Cys⁶-Phe⁷-DTrp⁸-Lys⁹-Thr¹⁰-Cys¹¹]-Thr¹²-Ser¹³-Cys¹⁴]) and AT6S (DOTA-Ala¹-Gly²-c[Cys³-Lys⁴-c[Cys⁵-Phe⁶-Phe⁷-DTrp⁸-Lys⁹-Thr¹⁰-Phe¹¹-Cys¹²]-Ser¹³-Cys¹⁴]), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective [¹¹¹In]In-AT5S and [¹¹¹In]In-AT6S were evaluated in a series of in vitro assays, while radioligand stability and biodistribution were studied in mice. The 8/12-mer bicyclic AT6S showed expanded affinity for all SST_1–5R and agonistic properties at the SST₂R, whereas AT5S lost all affinity to SST_1–5R. Both [¹¹¹In]In-AT5S and [¹¹¹In]In-AT6S remained stable in the peripheral blood of mice, while [¹¹¹In]In-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST₃R tumors in mice. In summary, high radioligand stability was acquired by the two disulfide bridges introduced into the SS14 motif, but only the 8/12-mer ring AT6S retained a pansomatostatin profile. In consequence, [¹¹¹In]In-AT6S targeted SST₂R-/SST₃R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics.

Radiolabeled neurotensin analogs have been developed as candidates for theranostic use against neurotensin subtype 1 receptor (NTS₁R)-expressing cancer. However, their fast degradation by two major peptidases, neprilysin (NEP) and angiotensin-converting enzyme (ACE), has hitherto limited clinical success. We have recently shown that palmitoylation at the ε-amine of Lys⁷ in [^99mTc]Tc-[Lys⁷]DT1 (DT1, N₄-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-OH, N₄ = 6-(carboxy)-1,4,8,11-tetraazaundecane) led to the fully stabilized [^99mTc]Tc-DT9 analog, displaying high uptake in human pancreatic cancer AsPC-1 xenografts but unfavorable pharmacokinetics in mice. Aiming to improve the in vivo stability of [^99mTc]Tc-DT1 without compromising pharmacokinetics, we now introduce three new [^99mTc]Tc-DT1 mimics, carrying different pendant groups at the ε-amine of Lys⁷: MPBA (4-(4-methylphenyl)butyric acid)—[^99mTc]Tc-DT10; MPBA via a PEG4-linker—[^99mTc]Tc-DT11; or a hydrophilic PEG6 chain—[^99mTc]Tc-DT12. The impact of these modifications on receptor affinity and internalization was studied in NTS₁R-positive cells. The effects on stability and AsPC-1 tumor uptake were assessed in mice without or during NEP/ACE inhibition. Unlike [^99mTc]Tc-DT10, the longer-chain modified [^99mTc]Tc-DT11 and [^99mTc]Tc-DT12 were significantly stabilized in vivo, resulting in markedly improved tumor uptake compared to [^99mTc]Tc-DT1. [^99mTc]Tc-DT11 was found to achieve the highest AsPC-1 tumor values and good pharmacokinetics, either without or during NEP inhibition, qualifying for further validation in patients with NTS₁R-positive tumors using SPECT/CT.

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.

(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: 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 overexpression of neurotensin subtype 1 receptors (NTS1Rs) in human tumors may be elegantly exploited for directing neurotensin (NT)-based radionuclide carriers specifically to cancer sites for theranostic purposes. We have recently shown that [^99mTc]Tc–DT1 ([^99mTc]Tc–[N₄–Gly⁷]NT(7–13)) and [^99mTc]Tc–DT5 ([^99mTc]Tc–[N₄–βAla⁷,Dab⁹]NT(7–13)) show notably improved uptake in human colon adenocarcinoma WiDr xenografts in mice treated with neprilysin (NEP) inhibitors and/or angiotensin-converting enzyme (ACE) inhibitors compared with untreated controls. Aiming toward translation of this promising approach in NTS1R-positive pancreatic ductal adenocarcinoma (PDAC) patients, we now report on the impact of registered NEP/ACE inhibitors on the performance of [^99mTc]Tc–DT1 and [^99mTc]Tc–DT5 in pancreatic cancer models. Methods: The cellular uptake of [^99mTc]Tc–DT1 and [^99mTc]Tc–DT5 was tested in a panel of pancreatic cell lines, and their stability was assessed in mice treated or not treated with Entresto, lisinopril, or their combinations. Biodistribution was conducted in severe combined immunodeficiency (SCID) mice bearing pancreatic AsPC-1 xenografts. Results: The Entresto + lisinopril combination maximized the metabolic stability of the fast-internalizing [^99mTc]Tc–DT1 in mice, resulting in notably enhanced tumor uptake (7.05 ± 0.80% injected activity (IA)/g vs. 1.25 ± 0.80% IA/g in non-treated controls at 4 h post-injection; p < 0.0001). Conclusions: This study has shown the feasibility of optimizing the uptake of [^99mTc]Tc–DT1 in pancreatic cancer models with the aid of clinically established NEP/ACE inhibitors, in favor of clinical translation prospects.

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.