: 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
TcTc-N
-PEGx-DPhe
,Leu-NHEt
BBN(6-13) (N
: 6-(carboxy)-1,4,8,11-tetraazaundecane; PEG: polyethyleneglycol): (i)
TcTc-DB7 (x = 2), (ii)
TcTc-DB13 (x = 3), and (iii)
TcTc-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).
: The GRPR affinity of DB7/DB13/DB14 was determined in PC-3 cell membranes, and cell binding of the respective
TcTc-radioligands was assessed in PC-3 cells. Each of
TcTc-DB7,
TcTc-DB13, and
TcTc-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.
: DB7, -13, and -14 displayed single-digit nanomolar affinities for GRPR. The uptake rates of
TcTc-DB7,
TcTc-DB13, and
TcTc-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.
: The present study showed that increase of PEG-spacer length in the
TcTc-DB7-
TcTc-DB13-
TcTc-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.
The GRPR-antagonist-based radioligands
Ga/
In/
LuNeoBOMB1 have shown excellent theragnostic profiles in preclinical prostate cancer models, while
GaNeoBOMB1 effectively visualized prostate cancer ...lesions in patients. We were further interested to explore the theragnostic potential of NeoBOMB1 in GRPR-positive mammary carcinoma, by first studying
GaNeoBOMB1 in breast cancer models; Methods: We investigated the profile of
GaNeoBOMB1, a
GaNeoBOMB1 surrogate, in GRPR-expressing T-47D cells and animal models;
: NeoBOMB1 (IC
s of 2.2 ± 0.2 nM) and
GaNeoBOMB1 (IC
s of 2.5 ± 0.2 nM) exhibited high affinity for the GRPR. At 37 °C
GaNeoBOMB1 strongly bound to the T-47D cell-membrane (45.8 ± 0.4% at 2 h), internalizing poorly, as was expected for a radioantagonist.
GaNeoBOMB1 was detected >90% intact in peripheral mouse blood at 30 min pi. In mice bearing T-47D xenografts,
GaNeoBOMB1 specifically localized in the tumor (8.68 ± 2.9% ID/g vs. 0.6 ± 0.1% ID/g during GRPR-blockade at 4 h pi). The unfavorably high pancreatic uptake could be considerably reduced (206.29 ± 17.35% ID/g to 42.46 ± 1.31% ID/g at 4 h pi) by increasing the NeoBOMB1 dose from 10 pmol to 200 pmol, whereas tumor uptake remained unaffected. Notably, tumor values did not decline from 1 to 24 h pi; Conclusions:
GaNeoBOMB1 can successfully target GRPR-positive breast cancer in animals with excellent prospects for clinical translation.
Diagnostic imaging and radionuclide therapy of prostate (PC) and breast cancer (BC) using radiolabeled gastrin-releasing peptide receptor (GRPR)-antagonists represents a promising approach. We herein ...propose the GRPR-antagonist based radiotracer 99mTcTc-DB15 (99mTcTc-N4-AMA-DGA-DPhe6,Sar11,LeuNHEt13BBN(6-13); N4: 6-carboxy-1,4,8,11-tetraazaundecane, AMA: aminomethyl-aniline, DGA: diglycolic acid) as a new diagnostic tool for GRPR-positive tumors applying SPECT/CT. The uptake of 99mTcTc-DB15 was tested in vitro in mammary (T-47D) and prostate cancer (PC-3) cells and in vivo in T-47D or PC-3 xenograft-bearing mice as well as in BC patients. DB15 showed high GRPR-affinity (IC50 = 0.37 ± 0.03 nM) and 99mTcTc-DB15 strongly bound to the cell-membrane of T-47D and PC-3 cells, according to a radiolabeled antagonist profile. In mice, the radiotracer showed high and prolonged GRPR-specific uptake in PC-3 (e.g., 25.56 ± 2.78 %IA/g vs. 0.72 ± 0.12 %IA/g in block; 4 h pi) and T-47D (e.g., 15.82 ± 3.20 %IA/g vs. 3.82 ± 0.30 %IA/g in block; 4 h pi) tumors, while rapidly clearing from background. In patients with advanced BC, the tracer could reveal several bone and soft tissue metastases on SPECT/CT. The attractive pharmacokinetic profile of 99mTcDB15 in mice and its capability to target GRPR-positive BC lesions in patients highlight its prospects for a broader clinical use, an option currently being explored by ongoing clinical studies.
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-Gly7NT(7-13)), DT5 (N4-βAla7,Dab9NT(7-13)), and DT6 (N4-βAla7,Dab9,Tle12NT(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.
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.
The metabolic stability of
TcTc-DB4 (DB4, N
-Pro-Gln-Arg-Tyr-Gly-Asn-Gln-Trp-Ala-Val-Gly-His-Leu-Nle-NH
) and
InIn-SG4 (SG4, DOTA-DGlu-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH
) was tested in LBQ657/Entresto-treated mice vs. untreated controls. The uptake in gastrin-releasing peptide receptor (GRPR)-, or cholecystokinin subtype 2 receptor (CCK
R)-positive tumors respectively, was compared between LBQ657/Entresto-treated mice and untreated controls.
LBQ657/Entresto treatment induced marked stabilization of
Tc Tc-DB4 and
InIn-SG4 in peripheral mice blood, resulting in equally enhanced tumor uptake at 4 h post-injection. Accordingly, the
TcTc-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
InIn-SG4 uptake of 3.07 ± 0.87%IA/g in A431-CCK
R(+) tumors to 8.11 ± 1.45/9.61 ± 1.70%IA/g. Findings were visualized by SPECT/CT.
This study has shown the efficacy of Entresto to notably improve the profile of
TcTc-DB4 and
InIn-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-dAla11SB3. 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 overexpression of gastrin-releasing peptide receptors (GRPRs) in frequently occurring human tumors has provided the opportunity to use bombesin (BBN) analogs as radionuclide carriers to cancer ...sites for diagnostic and therapeutic purposes. We have been alternatively exploring human GRP motifs of higher GRPR selectivity compared to frog BBN sequences aiming to improve pharmacokinetic profiles. In the present study, we compared two differently truncated human endogenous GRP motifs: GRP(14–27) and GRP(18–27). An acyclic tetraamine was coupled at the N-terminus to allow for stable binding of the SPECT radionuclide 99mTc. Their biological profiles were compared in PC-3 cells and in mice without or with coinjection of phosphoramidon (PA) to induce transient neprilysin (NEP) inhibition in vivo. The two 99mTc-N4-GRP(14/18–27) radioligands displayed similar biological behavior in mice. Coinjection of PA exerted a profound effect on in vivo stability and translated into notably improved radiolabel localization in PC-3 experimental tumors. Hence, this study has shown that promising 99mTc-radiotracers for SPECT imaging may indeed derive from human GRP sequences. Radiotracer bioavailability was found to be of major significance. It could be improved during in situ NEP inhibition resulting in drastically enhanced uptake in GRPR-expressing lesions.
We recently introduced the potent gastrin-releasing peptide receptor (GRPR) antagonist
Ga-SB3 (
Ga-DOTA-p-aminomethylaniline-diglycolic acid-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt), showing excellent ...tumor localizing efficacy in animal models and in patients. By replacement of the C-terminal Leu
-Met
-NH
dipeptide of SB3 by Sta
-Leu
-NH
, the novel GRPR antagonist NeoBOMB1 was generated and labeled with different radiometals for theranostic use. We herein report on the biologic profile of resulting
Ga-,
In-, and
Lu-NeoBOMB1 radioligands in GRPR-expressing cells and mouse models. The first evidence of prostate cancer lesion visualization in men using
Ga-NeoBOMB1 and PET/CT is also presented.
NeoBOMB1 was radiolabeled with
Ga,
In, and
Lu according to published protocols. The respective metalated species
Ga-,
In-, and
Lu-NeoBOMB1 were also synthesized and used in competition binding experiments against
I-Tyr
BBN in GRPR-positive PC-3 cell membranes. Internalization of
Ga-,
In-, and
Lu-NeoBOMB1 radioligands was studied in PC-3 cells at 37°C, and their metabolic stability in peripheral mouse blood was determined by high-performance liquid chromatography analysis of blood samples. Biodistribution was performed by injecting a
Ga-,
In-, or
Lu-NeoBOMB1 bolus (74, 74, or 370 kBq, respectively, 100 μL, 10 pmol total peptide ± 40 nmol Tyr
-BBN: for in vivo GRPR blockade) in severe combined immunodeficiency mice bearing PC-3 xenografts. PET/CT images with
Ga-NeoBOMB1 were acquired in prostate cancer patients.
NeoBOMB1 and
Ga-,
In-, and
Lu-NeoBOMB1 bound to GRPR with high affinity (half maximal inhibitory concentration, 1-2 nM).
Ga-,
In-, and
Lu-NeoBOMB1 specifically and strongly bound on the cell membrane of PC-3 cells displaying low internalization, as expected for receptor antagonists. They showed excellent metabolic stability in peripheral mouse blood (>95% intact at 5 min after injection). After injection in mice, all 3 (
Ga-,
In-, and
Lu-NeoBOMB1) showed comparably high and GRPR-specific uptake in the PC-3 xenografts (e.g., 30.6 ± 3.9, 28.6 ± 6.0, and >35 percentage injected dose per gram at 4 h after injection, respectively), clearing from background predominantly via the kidneys. During a translational study in prostate cancer patients,
Ga-NeoBOMB1 rapidly localized in pathologic lesions, achieving high-contrast imaging.
The GRPR antagonist radioligands
Ga-,
In-, and
Lu-NeoBOMB1, independent of the radiometal applied, have shown comparable behavior in prostate cancer models, in favor of future theranostic use in GRPR-positive cancer patients. Such translational prospects were further supported by the successful visualization of prostate cancer lesions in men using
Ga-NeoBOMB1 and PET/CT.
Recent advances in oncology involve the use of diagnostic/therapeutic radionuclide-carrier pairs that target cancer cells, offering exciting opportunities for personalized patient treatment. ...Theranostic gastrin-releasing peptide receptor (GRPR)-directed radiopeptides have been proposed for the management of GRPR-expressing prostate and breast cancers. We have recently introduced the PET tracer
Ga-SB3 (SB3, DOTA- p-aminomethylaniline-diglycolic acid-DPhe-Gln-Trp-Ala-Val-Gly-His-Leu-NHEt), a receptor-radioantagonist that enables the visualization of GRPR-positive lesions in humans. Aiming to fully assess the theranostic potential of SB3, we herein report on the impact of switching
Ga to
In/
Lu-label on the biological properties of resulting radiopeptides. Notably, the bioavailability of
In/
Lu-SB3 in mice drastically deteriorated compared with metabolically robust
Ga-SB3, and as a result led to poorer
In/
Lu-SB3 uptake in GRPR-positive PC-3 xenografts. The peptide cleavage sites were identified by chromatographic comparison of blood samples from mice intravenously receiving
In/
Lu-SB3 with each of newly synthesized
In/
Lu-SB3-fragments. Coinjection of the radioconjugates with the neprilysin (NEP)-inhibitor phosphoramidon led to full stabilization of
In/
Lu-SB3 in peripheral mouse blood and resulted in markedly enhanced radiolabel uptake in the PC-3 tumors. In conclusion, in situ NEP-inhibition led to indistinguishable
Ga/
In/
Lu-SB3 profiles in mice emphasizing the theranostic prospects of SB3 for clinical use.
From a series of radiolabelled cholecystokinin (CCK) and gastrin analogues, (111)In-CP04 ((111)In-DOTA-(DGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) was selected for further translation as a diagnostic ...radiopharmaceutical towards a first-in-man study in patients with medullary thyroid carcinoma (MTC). A freeze-dried kit formulation for multicentre application has been developed. We herein report on biosafety, in vivo stability, biodistribution and dosimetry aspects of (111)In-CP04 in animal models, essential for the regulatory approval of the clinical trial.
Acute and extended single dose toxicity of CP04 was tested in rodents, while the in vivo stability of (111)In-CP04 was assessed by HPLC analysis of mouse blood samples. The biodistribution of (111)In-CP04 prepared from a freeze-dried kit was studied in SCID mice bearing double A431-CCK2R(±) xenografts at 1, 4 and 24h pi. Further 4-h animal groups were either additionally treated with the plasma expander gelofusine or injected with (111)In-CP04 prepared by wet-labelling. Pharmacokinetics in healthy mice included the 30min, 1, 4, 24, 48 and 72h time points pi. Dosimetric calculations were based on extrapolation of mice data to humans adopting two scaling models.
CP04 was well-tolerated by both mice and rats, with an LD50>178.5μg/kg body weight for mice and a NOAEL (no-observed-adverse-effect-level) of 89μg/kg body weight for rats. After labelling, (111)In-CP04 remained >70% intact in peripheral mouse blood at 5min pi. The uptake of (111)In-CP04 prepared from the freeze-dried kit and by wet-labelling were comparable in the A431-CCK2R(+)-xenografts (9.24±1.35%ID/g and 8.49±0.39%ID/g, respectively; P>0.05). Gelofusine-treated mice exhibited significantly reduced kidneys values (1.69±0.15%ID/g vs. 5.55±0.94%ID/g in controls, P<0.001). Dosimetry data revealed very comparable effective tumour doses for the two scaling models applied, of 0.045 and 0.044mSv/MBq.
The present study has provided convincing toxicology, biodistribution and dosimetry data for prompt implementation of the freeze-dried kit formulation without or with gelofusine administration in a multicentre clinical trial in MTC patients.
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