Preclinical evaluation of new GRPR-antagonists with improved metabolic stability for radiotheranostic use in oncology.

BACKGROUND: The gastrin-releasing peptide receptor (GRPR) has been extensively studied as a biomolecular target for peptide-based radiotheranostics. However, the lack of metabolic stability and the rapid clearance of peptide radioligands, including radiolabeled GRPR-antagonists, often impede clinical application. Aiming at circumventing these drawbacks, we have designed three new GRPR-antagonist radioligands using [99mTc]Tc-DB15 ([99mTc]Tc-N4-AMA-DIG-DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt; AMA: p-aminomethylaniline; DIG: diglycolate) as a motif, due to its high GRPR-affinity and stability to neprilysin (NEP). The new analogues carry the DOTAGA-chelator (1,4,7,10-tetraazacyclododecane-1-glutaric acid-4,7,10-triacetic acid) through different linkers at the N-terminus to allow for labeling with the theranostic radionuclide pair In-111/Lu-177. After labeling with In-111 the following radioligands were evaluated: (i) [111In]In-AU-SAR-M1 ([111In]In-DOTAGA-AMA-DIG-DPhe-Gln-Trp-Ala-Val-Sar-His-Leu-NHEt), (ii) [111In]In-AU-SAR-M2 ([111In]In-[DOTAGA-Arg]AU-SAR-M1) and (iii) [111In]In-AU-SAR-M3 ([111In]In-[DOTAGA-DArg]AU-SAR-M1). RESULTS: These radioligands were compared in a series of in vitro assays using prostate adenocarcinoma PC-3 cells and in murine models. They all displayed high and GRPR-specific uptake in PC-3 cells. Analysis of mice blood collected 5 min post-injection (pi) revealed similar or even higher metabolic stability of the new radioligands compared with [99mTc]Tc-DB15. The stability could be further increased when the mice were treated with Entresto® to in situ induce NEP-inhibition. In PC-3 xenograft-bearing mice, [111In]In-AU-SAR-M1 displayed the most favourable biodistribution profile, combining a good tumor retention with the highest tumor-to-organ ratios, with the kidneys as the dose-limiting organ. CONCLUSIONS: These findings strongly point at AU-SAR-M1 as a promising radiotherapeutic candidate when labeled with Lu-177, or other medically appealing therapeutic radiometals, especially when combined with in situ NEP-inhibition. To this goal further investigations are currently pursued.

New Insights into the Hypotensins from Tityus serrulatus Venom: Pro-Inflammatory and Vasopeptidases Modulation Activities.

The Tityus serrulatus scorpion is considered the most dangerous of the Brazilian fauna due to the severe clinical manifestations in injured victims. Despite being abundant components of the venom, few linear peptides have been characterized so far, such as hypotensins. In vivo studies have demonstrated that hypotensin I (TsHpt-I) exerts hypotensive activity, with an angiotensin-converting enzyme (ACE)-independent mechanism of action. Since experiments have not yet been carried out to analyze the direct interaction of hypotensins with ACE, and to deepen the knowledge about these peptides, hypotensins I and II (TsHpt-II) were studied regarding their modulatory action over the activities of ACE and neprilysin (NEP), which are the peptidases involved in blood pressure control. Aiming to search for indications of possible pro-inflammatory action, hypotensins were also analyzed for their role in murine macrophage viability, the release of interleukins and phagocytic activity. TsHpt-I and -II were used in kinetic studies with the metallopeptidases ACE and NEP, and both hypotensins were able to increase the activity of ACE. TsHpt-I presented itself as an inhibitor of NEP, whereas TsHpt-II showed weak inhibition of the enzyme. The mechanism of inhibition of TsHpt-I in relation to NEP was defined as non-competitive, with an inhibition constant (Ki) of 4.35 µM. Concerning the analysis of cell viability and modulation of interleukin levels and phagocytic activity, BALB/c mice's naïve macrophages were used, and an increase in TNF production in the presence of TsHpt-I and -II was observed, as well as an increase in IL-6 production in the presence of TsHpt-II only. Both hypotensins were able to increase the phagocytic activity of murine macrophages in vitro. The difference between TsHpt-I and -II is the residue at position 15, with a glutamine in TsHpt-I and a glutamic acid in TsHpt-II. Despite this, kinetic analyzes and cell assays indicated different actions of TsHpt-I and -II. Taken together, these results suggest a new mechanism for the hypotensive effects of TsHpt-I and -II. Furthermore, the release of some interleukins also suggests a role for these peptides in the venom inflammatory response. Even though these molecules have been well studied, the present results suggest a new mechanism for the hypotensive effects of TsHpt-I.

In Vivo Stabilized SB3, an Attractive GRPR Antagonist, for Pre- and Intra-Operative Imaging for Prostate Cancer.

PURPOSE: The gastrin-releasing peptide receptor (GRPR), overexpressed on various tumor types, is an attractive target for receptor-mediated imaging and therapy. Another interesting approach would be the use of GRPR radioligands for pre-operative imaging and subsequent radio-guided surgery, with the goal to improve surgical outcome. GRPR radioligands were successfully implemented in clinical studies, especially Sarabesin 3 (SB3) is an appealing GRPR antagonist with high receptor affinity. Gallium-68 labeled SB3 has good in vivo stability, after labeling with Indium-111; however, the molecule shows poor in vivo stability, which negatively impacts tumor-targeting capacity. A novel approach to increase in vivo stability of radiopeptides is by co-administration of the neutral endopeptidase (NEP) inhibitor, phosphoramidon (PA). We studied in vivo stability and biodistribution of [111In]SB3 without/with (-/+) PA in mice. Furthermore, SPECT/MRI on a novel, state-of-the-art platform was performed. PROCEDURES: GRPR affinity of SB3 was determined on PC295 xenograft sections using [125I]Tyr4-bombesin with tracer only or with increasing concentrations of SB3. For in vivo stability, mice were injected with 200/2000 pmol [111In]SB3 -/+ 300 µg PA. Blood was collected and analyzed. Biodistribution and SPECT/MRI studies were performed at 1, 4, and 24 h postinjection (p.i.) of 2.5 MBq/200 pmol or 25 MBq/200 pmol [111In]SB3 -/+ 300 µg PA in PC-3-xenografted mice. RESULTS: SB3 showed high affinity for GRPR (IC50 3.5 nM). Co-administration of PA resulted in twice higher intact peptide in vivo vs [111In]SB3 alone. Biodistribution studies at 1, 4, and 24 h p.i. show higher tumor uptake values with PA co-administration (19.7 ± 3.5 vs 10.2 ± 1.5, 17.6 ± 5.1 vs 8.3 ± 1.1, 6.5 ± 3.3 vs 3.1 ± 1.9 % ID/g tissue (P < 0.0001)). Tumor imaging with SPECT/MRI clearly improved after co-injection of PA. CONCLUSIONS: Co-administration of PA increased in vivo tumor targeting capacity of [111In]SB3, making this an attractive combination for GRPR-targeted tumor imaging.

Amide-to-triazole switch vs. in vivo NEP-inhibition approaches to promote radiopeptide targeting of GRPR-positive tumors.

INTRODUCTION: Radiolabeled bombesin (BBN)-analogs have been proposed for diagnosis and therapy of gastrin-releasing peptide receptor (GRPR)-expressing tumors, such as prostate, breast and lung cancer. Metabolic stability represents a crucial factor for the success of this approach by ensuring sufficient delivery of circulating radioligand to tumor sites. The amide-to-triazole switch on the backbone of DOTA-PEG4-[Nle14]BBN(7-14) (1) was reported to improve the in vitro stability of resulting 177Lu-radioligands. On the other hand, in-situ inhibition of neutral endopeptidase (NEP) by coinjection of phosphoramidon (PA) was shown to significantly improve the in vivo stability and tumor uptake of biodegradable radiopeptides. We herein compare the impact of the two methods on the bioavailability and localization of 177Lu-DOTA-PEG4-[Nle14]BBN(7-14) analogs in GRPR-positive tumors in mice. METHODS: The 1,4-disubstituted [1-3]-triazole was used to replace one (2: Gly11-His12; 3: Ala9-Val10) or two (4: Ala9-Val10 and Gly11-His12) peptide bonds in 1 (reference) and all compounds were labeled with 177Lu. Each of [177Lu]1-[177Lu]4 was injected without (control) or with PA in healthy mice. Blood samples collected 5min post-injection (pi) were analyzed by HPLC. Biodistribution of [177Lu]1-[177Lu]4 was conducted in SCID mice bearing human prostate adenocarcinoma PC-3 xenografts at 4h pi. Groups of 4 animals were injected with radioligand, alone (controls), or with coinjection of PA, or of a mixture of PA and excess and [Tyr4]BBN to determine GRPR-specificity of uptake (Block). RESULTS: The in vivo stability of the radioligands was: [177Lu]1 (25% intact), [177Lu]2 (45% intact), [177Lu]3 (30% intact) and [177Lu]4 (40% intact). By PA-coinjection these values notably increased to 90%-93%. Moreover, treatment with PA induced an impressive and GRPR-specific uptake of all radioligands in the PC-3 xenografts at 4h pi: [177Lu]1: 4.7±0.4 to 24.8±4.9%ID/g; [177Lu]2: 8.3±1.2 to 26.0±1.1%ID/g; [177Lu]3: 6.6±0.4 to 21.3±4.4%ID/g; and [177Lu]4: 4.8±1.6 to 13.7±3.8%ID/g. CONCLUSIONS: This study has shown that amide-to-triazole substitutions in 177Lu-DOTA-PEG4-[Nle14]BBN(7-14) induced minor effects on bioavailability and tumor uptake in mice models, whereas in-situ NEP-inhibition(s) by PA impressively improved in vivo profiles.

(99m)Tc-labeled gastrins of varying peptide chain length: Distinct impact of NEP/ACE-inhibition on stability and tumor uptake in mice.

INTRODUCTION: In situ inhibition of neutral endopeptidase (NEP) has been recently shown to impressively increase the bioavailability and tumor uptake of biodegradable gastrin radioligands. Furthermore, angiotensin converting enzyme (ACE) has been previously shown to cleave gastrin analogs in vitro. In the present study, we have assessed the effects induced by single or dual NEP/ACE-inhibition on the pharmacokinetic profile of three (99m)Tc-labeled gastrins of varying peptide chain length: [(99m)Tc]SG6 ([(99m)Tc-N4-Gln(1)]gastrin(1-17)), [(99m)Tc]DG2 ([(99m)Tc-N4-Gly(4),DGlu(5)]gastrin(4-17)) and [(99m)Tc]DG4 ([(99m)Tc-N4-DGlu(10)]gastrin(10-17)). METHODS: Mouse blood samples were collected 5min after injection of each of [(99m)Tc]SG6/DG2/DG4 together with: a) vehicle, b) the NEP-inhibitor phosphoramidon (PA), c) the ACE-inhibitor lisinopril (Lis), or d) PA plus Lis and were analyzed by RP-HPLC for radiometabolite detection. Biodistribution was studied in SCID mice bearing A431-CCK2R(+/-) xenografts at 4h postinjection (pi). [(99m)Tc]SG6 or [(99m)Tc]DG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for [(99m)Tc]DG2 control and PA animal groups were only included. RESULTS: Treatment of mice with PA induced significant stabilization of (99m)Tc-radiotracers in peripheral blood, while treatment with Lis or Lis+PA affected the stability of des(Glu)5 [(99m)Tc]DG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (P<0.01). Only [(99m)Tc]DG4 profited by single Lis (2.06±0.39%ID/g vs 0.99±0.13%ID/g in controls) or combined Lis+PA coinjection (8.91±1.61%ID/g vs 4.89±1.33%ID/g in PA-group). Furthermore, kidney uptake remained favourably low and unaffected by PA and/or Lis coinjection only in the case of [(99m)Tc]DG4 (<1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios. CONCLUSIONS: In situ NEP/ACE-inhibition diversely affected the in vivo profile of (99m)Tc-radioligands based on different-length gastrins. Truncated [(99m)Tc]DG4 exhibited overall the most attractive profile during combined NEP/ACE-inhibition in mouse models, providing new opportunities for CCK2R-expressing tumor imaging in man with SPECT.

Impact of clinically tested NEP/ACE inhibitors on tumor uptake of [(111)In-DOTA]MG11-first estimates for clinical translation.

BACKGROUND: We have recently shown that treatment of mice with the neutral endopeptidase (NEP) inhibitor phosphoramidon (PA) improves the bioavailability and tumor uptake of biodegradable radiopeptides. For the truncated gastrin radiotracer [(111)In-DOTA]MG11 ([(DOTA)DGlu(10)]gastrin(10-17)), this method led to impressively high tumor-to-kidney ratios. Translation of this concept in the clinic requires the use of certified NEP inhibitors, such as thiorphan (TO) and its orally administered prodrug racecadotril (Race). Besides NEP, angiotensin-converting enzyme (ACE) has also been implicated in the catabolism of gastrin analogs. In the present study, we first compared the effects induced by NEP inhibition (using PA, TO, or Race) and/or by ACE inhibition (using lisinopril, Lis) on the biodistribution profile of [(111)In-DOTA]MG11 in mice. In addition, we compared the efficacy of PA and TO at different administered doses to enhance tumor uptake. METHODS: [(111)In-DOTA]MG11 was coinjected with (a) vehicle, (b) PA (300 µg), (c) TO (150 µg), (d) Lis (100 µg), (e) PA (300 µg) plus Lis (100 µg), or (f) 30-40 min after intraperitoneal (ip) injection of Race (3 mg) in SCID mice bearing AR42J xenografts. In addition, [(111)In-DOTA]MG11 was coinjected with vehicle, or with progressively increasing amounts of PA (3, 30, or 300 µg) or TO (1.5, 15, and 150 µg) in SCID mice bearing twin A431-CCK2R(+/-) tumors. In all above cases, biodistribution was conducted at 4 h postinjection (pi). RESULTS: During NEP inhibition, the uptake of [(111)In-DOTA]MG11 in the AR42J tumors impressively increased from 1.8 ± 1.0 % ID/g (controls) to 15.3 ± 4.7 % ID/g (PA) and 12.3 ± 3.6 % ID/g (TO), while with Race tumor values reached 6.8 ± 2.8 % ID/g. Conversely, Lis had no effect on tumor uptake and no additive effect when coinjected with PA. During the dose dependence study in mice, PA turned out to be more efficacious in enhancing tumor uptake of [(111)In-DOTA]MG11 in the CCK2R-positive tumors compared to equimolar amounts of TO. In all cases, renal accumulation remained low, resulting in notable increases of tumor-to-kidney ratios. CONCLUSIONS: This study has confirmed NEP as the predominant degrading enzyme of [(111)In-DOTA]MG11 and ruled out the involvement of ACE in the in vivo catabolism of the radiotracer. NEP inhibition with the clinically tested NEP inhibitors TO and Race resulted in significant enhancement of tumor-to-kidney ratios vs. CONTROLS: However, compared with PA, TO and its prodrug Race induced less potent increases of tumor uptake, highlighting the significance of inhibitor type, administration route, and dose for implementing a first proof-of-principle study in human.

Endothelial permeability in vitro and in vivo: protective actions of ANP and omapatrilat in experimental atherosclerosis.

Increased arterial endothelial cell permeability (ECP) is considered an initial step in atherosclerosis. Atrial natriuretic peptide (ANP) which is rapidly degraded by neprilysin (NEP) may reduce injury-induced endothelial cell leakiness. Omapatrilat represents a first in class of pharmacological agents which inhibits both NEP and angiotensin converting enzyme (ACE). We hypothesized that ANP prevents thrombin-induced increases of ECP in human aortic ECs (HAECs) and that omapatrilat would reduce aortic leakiness and atherogenesis and enhance ANP mediated vasorelaxation of isolated aortas. Thrombin induced ECP determined by I(125) albumin flux was assessed in HAECs with and without ANP pretreatment. Next we examined the effects of chronic oral administration of omapatrilat (12 mg/kg/day, n=13) or placebo (n=13) for 8 weeks on aortic leakiness, atherogenesis and ANP-mediated vasorelaxation in isolated aortas in a rabbit model of atherosclerosis produced by high cholesterol diet. In HAECs, thrombin-induced increases in ECP were prevented by ANP. Omapatrilat reduced the area of increased aortic leakiness determined by Evans-blue dye and area of atheroma formation assessed by Oil-Red staining compared to placebo. In isolated arterial rings, omapatrilat enhanced vasorelaxation to ANP compared to placebo with and without the endothelium. ANP prevents thrombin-induced increases in ECP in HAECs. Chronic oral administration of omapatrilat reduces aortic leakiness and atheroma formation with enhanced endothelial independent vasorelaxation to ANP. These studies support the therapeutic potential of dual inhibition of NEP and ACE in the prevention of increased arterial ECP and atherogenesis which may be linked to the ANP/cGMP system.