: 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.
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.
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.
Minigastrin radiotracers, such as (111)In-DOTAMG0 ((111)In-DOTA-DGlu(1)minigastrin), have been considered for diagnostic imaging and radionuclide therapy of CCK2R-positive human tumors, such as ...medullary thyroid carcinoma. However, the high kidney retention assigned to the pentaGlu(2-6) repeat in the peptide sequence has compromised their clinical applicability. On the other hand, truncated des(Glu)(2-6)-analogs, such as (111)In-DOTAMG11 ((111)In-DOTA-DGlu(10),desGlu(2-6)minigastrin), despite their low renal uptake, show poor bioavailability and tumor targeting. (111)InCP04 ((111)In-DOTA-DGlu(1-6)minigastrin) acquired by Glu(2-6)/DGlu(2-6) substitution showed promising tumor-to-kidney ratios in rodents. In the present study, we compare the biological profiles of (111)InCP04, (111)In-DOTAMG11, and (111)In-DOTAMG0 during in situ neutral endopeptidase (NEP) inhibition, recently shown to improve the bioavailability of several peptide radiotracers. After coinjection of the NEP inhibitor, phosphoramidon (PA), the stability of (111)InCP04 and (111)In-DOTAMG0 in peripheral mouse blood increased, with an exceptional >14-fold improvement monitored for (111)In-DOTAMG11. In line with these findings, PA treatment increased the uptake of (111)InCP04 (8.5 ± 0.4%ID/g to 16.0 ± 2.3%ID/g) and (111)In-DOTAMG0 (11.9 ± 2.2%ID/g to 17.2 ± 0.9%ID/g) in A431-CCK2R(+) tumors at 4 hours postinjection, whereas the respective increase for (111)In-DOTAMG11 was >6-fold (2.5 ± 0.9%ID/g to 15.1 ± 1.7%ID/g). Interestingly, kidney uptake remained lowest for (111)In-DOTAMG11, but unfavorably increased by PA treatment for (111)In-DOTAMG0. Thus, overall, the most favorable in vivo profile was displayed by (111)In-DOTAMG11 during NEP inhibition, highlighting the need to validate this promising concept in the clinic.
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 99mTc-labeled gastrins of varying peptide chain length: 99mTcSG6 (99mTc-N4-Gln1gastrin(1–17)), 99mTcDG2 (99mTc-N4-Gly4,DGlu5gastrin(4–17)) and 99mTcDG4 (99mTc-N4-DGlu10gastrin(10–17)).
Mouse blood samples were collected 5min after injection of each of 99mTcSG6/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). 99mTcSG6 or 99mTcDG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for 99mTcDG2 control and PA animal groups were only included.
Treatment of mice with PA induced significant stabilization of 99mTc-radiotracers in peripheral blood, while treatment with Lis or Lis+PA affected the stability of des(Glu)5 99mTcDG4 only. In line with these findings, PA coinjection led to notable amplification of tumor uptake of radiopeptides compared to controls (P<0.01). Only 99mTcDG4 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 99mTcDG4 (<1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios.
In situ NEP/ACE-inhibition diversely affected the in vivo profile of 99mTc-radioligands based on different-length gastrins. Truncated 99mTcDG4 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.
Abstract 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 three99m Tc-labeled gastrins of varying peptide chain length: 99m TcSG6 (99m Tc-N4 -Gln1 gastrin(1–17)), 99m TcDG2 (99m Tc-N4 -Gly4 ,DGlu5 gastrin(4–17)) and 99m TcDG4 (99m Tc-N4 -DGlu10 gastrin(10–17)). Methods Mouse blood samples were collected 5 min after injection of each of 99m TcSG6/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 4 h postinjection (pi). 99m TcSG6 or 99m TcDG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for 99m TcDG2 control and PA animal groups were only included. Results Treatment of mice with PA induced significant stabilization of99m Tc-radiotracers in peripheral blood, while treatment with Lis or Lis + PA affected the stability of des (Glu)5 99m TcDG4 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 TcDG4 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 TcDG4 (< 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 of99m Tc-radioligands based on different-length gastrins. Truncated 99m TcDG4 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.
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)TcSG6 ((99m)Tc-N4-Gln(1)gastrin(1-17)), (99m)TcDG2 ((99m)Tc-N4-Gly(4),DGlu(5)gastrin(4-17)) and (99m)TcDG4 ((99m)Tc-N4-DGlu(10)gastrin(10-17)).
Mouse blood samples were collected 5min after injection of each of (99m)TcSG6/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)TcSG6 or (99m)TcDG4 was coinjected with either vehicle or the above described NEP/ACE-inhibitor regimens; for (99m)TcDG2 control and PA animal groups were only included.
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)TcDG4 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)TcDG4 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)TcDG4 (<1.9%ID/g) resulting in the most optimal tumor-to-kidney ratios.
In situ NEP/ACE-inhibition diversely affected the in vivo profile of (99m)Tc-radioligands based on different-length gastrins. Truncated (99m)TcDG4 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.
Radiolabeled gastrin analogs represent attractive candidates for diagnosis and therapy of cholecystokinin subtype-2 receptor (CCK2R)-expressing tumors. Radiolabeled des(Glu)5-gastrins show favorably ...low renal accumulation, but localize poorly in CCK2R-positive lesions. We introduce herein three truncated DOTA-DGlu(10)gastrin(10-17) analogs, with oxidation-susceptible Met(15) replaced by: (1), (2), or (3), and study the profile of (111)In1/2/3 during in vivo inhibition of neutral endopeptidase (NEP) in comparison to the non-truncated ((111)In4) reference.
Blood samples collected from mice 5 min postinjection (pi) of (111)In1/2/3/4 without or with phosphoramidon (PA) coinjection were analyzed by RP-HPLC. Biodistribution was conducted in SCID mice bearing A431-CCK2R(+) or AR42J xenografts 4h after administration of (111)In1/2/3/4 without or with PA coinjection.
Firstly, we observed remarkable increases in the amount of radiopeptides detected intact in the blood of PA-treated mice at 5 min pi compared to controls. Secondly, we noted impressive enhancement of (111)In1/2/3 localization in AR42J and A431-CCK2R(+) tumors in mice after PA coinjection. Specifically, the uptake of (111)In1 at 4h pi increased from 2.6 ± 0.3%ID/g to 13.3 ± 3.5%ID/g in the AR42J tumors and from 4.3 ± 0.6%ID/g to 20.4 ± 3.6%ID/g in the A431-CCK2R(+) xenografts, with comparable improvements noted for (111)In2 and (111)In3 as well. Thirdly, renal uptake remained favorably low and unaffected by PA (<2.5%ID/g). Conversely, although the stability and tumor targeting of (111)In4 improved, its high renal uptake (>85%ID/g) increased even further by PA (>140%ID/g).
In situ inhibition of NEP represents a promising new tool to enhance the diagnostic efficacy of biodegradable gastrin radioligands in the visualization of CCK2R-positive lesions in man.
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