Optimizing the Profile of [99mTc]Tc-NT(7-13) Tracers in Pancreatic Cancer Models by Means of Protease Inhibitors

Abstract

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.

Keywords: Entresto; [99mTc]Tc radiotracer; angiotensin_converting enzyme; clinical translation; lisinopril; neprilysin; neurotensin; neurotensin subtype 1 receptor; pancreatic cancer; theranostics.

Figure 1

Representative radiochromatograms of high-performance liquid chromatography (HPLC) analysis of the radiolabeling mixture of (a) [^99mTc]Tc–DT1 and (b) [^99mTc]Tc–DT5 with the respective radioligand structures indicated.

Figure 2

(a) Neurotensin subtype 1 receptor (NTS1R)-specific cell uptake of [^99mTc]Tc–DT1 in AsPC-1 (red column), PANC-1 (green column), MiaCapa-2 (blue column), and Capan-1 (black column) cells during 1 h incubation at 37 °C. (b) Comparison of NTS1R-specific uptake of [^99mTc]Tc–DT1 and [^99mTc]Tc–DT5 in AsPC-1 cells during 1 h incubation at 37 °C; solid bars: internalized fraction; checkered bars: membrane-bound fraction. Results represent average values ± SD (n = 3, in triplicate); non-specific values were obtained in the presence of 1 μM neurotensin (NT), and were subtracted from totals to provide specific values. The study was conducted with cells as confluent monolayers.

Figure 3

Time-dependent, NTS1R-specific cell uptake curves of [^99mTc]Tc–DT1 in AsPC -1 (red lines), PANC-1 (green lines), and MiaCapa-2 (blue lines) cells at 37 °C (solid lines: internalized fraction; dotted lines: membrane-bound fraction). Results represent average values ± SD (n = 3, in triplicate); non-specific values were obtained in the presence of 1 μM NT, and were subtracted from totals to provide specific values. The study was conducted with cells as confluent monolayers.

Figure 4

Representative radiochromatograms of HPLC analysis of mouse blood samples collected 5 min pi with (a) [^99mTc]Tc–DT1 and (b) [^99mTc]Tc–DT5; black line graphs correspond to samples from untreated controls, red line graphs to samples from animals receiving per os Entresto 30 min prior to radioligand injection, blue line graphs to samples from animals with Lis co-injected together with the radioligand, and green line graphs to samples from animals receiving Entresto 30 min prior to radioligand co-injection with Lis (HPLC system 2); percentages of intact radioligand are summarized in Table 1.