The clinical success of radiolabeled somatostatin analogs in the diagnosis and therapy-"theranostics"-of tumors expressing the somatostatin subtype 2 receptor (SST
R) has paved the way for the ...development of a broader panel of peptide radioligands targeting different human tumors. This approach relies on the overexpression of other receptor-targets in different cancer types. In recent years, a shift in paradigm from internalizing agonists to antagonists has occurred. Thus, SST
R-antagonist radioligands were first shown to accumulate more efficiently in tumor lesions and clear faster from the background in animal models and patients. The switch to receptor antagonists was soon adopted in the field of radiolabeled bombesin (BBN). Unlike the stable cyclic octapeptides used in the case of somatostatin, BBN-like peptides are linear, fast to biodegradable and elicit adverse effects in the body. Thus, the advent of BBN-like antagonists provided an elegant way to obtain effective and safe radiotheranostics. Likewise, the pursuit of gastrin and exendin antagonist-based radioligands is advancing with exciting new outcomes on the horizon. In the present review, we discuss these developments with a focus on clinical results, commenting on challenges and opportunities for personalized treatment of cancer patients by means of state-of-the-art antagonist-based radiopharmaceuticals.
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 99mTcTc-Lys7DT1 (DT1, N4-Gly-Arg-Arg-Pro-Tyr-Ile-Leu-OH, N4 = 6-(carboxy)-1,4,8,11-tetraazaundecane) led to the fully stabilized 99mTcTc-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 99mTcTc-DT1 without compromising pharmacokinetics, we now introduce three new 99mTcTc-DT1 mimics, carrying different pendant groups at the ε-amine of Lys7: MPBA (4-(4-methylphenyl)butyric acid)—99mTcTc-DT10; MPBA via a PEG4-linker—99mTcTc-DT11; or a hydrophilic PEG6 chain—99mTcTc-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 99mTcTc-DT10, the longer-chain modified 99mTcTc-DT11 and 99mTcTc-DT12 were significantly stabilized in vivo, resulting in markedly improved tumor uptake compared to 99mTcTc-DT1. 99mTcTc-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 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
TcTc-DT1 (
TcTc-N
-Gly
NT(7-13)) and
TcTc-DT5 (
TcTc-N
-
Ala
,Dab
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
TcTc-DT1 and
TcTc-DT5 in pancreatic cancer models.
The cellular uptake of
TcTc-DT1 and
TcTc-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.
The Entresto + lisinopril combination maximized the metabolic stability of the fast-internalizing
TcTc-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;
< 0.0001).
This study has shown the feasibility of optimizing the uptake of
TcTc-DT1 in pancreatic cancer models with the aid of clinically established NEP/ACE inhibitors, in favor of clinical translation prospects.
The gastrin-releasing peptide receptor (GRPR) is expressed in high numbers in a variety of human tumors, including the frequently occurring prostate and breast cancers, and therefore provides the ...rationale for directing diagnostic or therapeutic radionuclides on cancer lesions after administration of anti-GRPR peptide analogs. This concept has been initially explored with analogs of the frog 14-peptide bombesin, suitably modified at the N-terminus with a number of radiometal chelates. Radiotracers that were selected for clinical testing revealed inherent problems associated with these GRPR agonists, related to low metabolic stability, unfavorable abdominal accumulation, and adverse effects. A shift toward GRPR antagonists soon followed, with safer analogs becoming available, whereby, metabolic stability and background clearance issues were gradually improved. Clinical testing of three main major antagonist types led to promising outcomes, but at the same time brought to light several limitations of this concept, partly related to the variation of GRPR expression levels across cancer types, stages, previous treatments, and other factors. Currently, these parameters are being rigorously addressed by cell biologists, chemists, nuclear medicine physicians, and other discipline practitioners in a common effort to make available more effective and safe state-of-the-art molecular tools to combat GRPR-positive tumors. In the present review, we present the background, current status, and future perspectives of this endeavor.
Radiolabeled somatostatin subtype 2 receptor (SST2R)-antagonists have shown advantageous profiles for cancer theranostics compared with agonists. On the other hand, the newly introduced hybrid ...chelator (6-pentanoic acid)-6-(amino)methyl-1,4-diazepinetriacetate (DATA5m) rapidly binds Ga-68 (t1/2: 67.7 min) at much lower temperature, thus allowing for quick access to “ready-for-injection” 68GaGa-tracers in hospitals. We herein introduce 68GaGa-DATA5m-LM4 for PET/CT imaging of SST2R-positive human tumors. LM4 was obtained by 4Pal3/Tyr3-substitution in the known SST2R antagonist LM3 (H-DPhe-cDCys-Tyr-DAph(Cbm)-Lys-Thr-Cys-DTyr-NH2) and DATA5m was coupled at the N-terminus for labeling with radiogallium (Ga-67/68). 67GaGa-DATA5m-LM4 was evaluated in HEK293-SST2R cells and mice models in a head-to-head comparison with 67GaGa-DOTA-LM3. Clinical grade 68GaGa-DATA5m-LM4 was prepared and injected in a neuroendocrine tumor (NET) patient for PET/CT imaging. DATA5m-LM4 displayed high SST2R binding affinity. 67GaGa-DATA5m-LM4 showed markedly higher uptake in HEK293-SST2R cells versus 67GaGa-DOTA-LM3 and was stable in vivo. In HEK293-SST2R xenograft-bearing mice, it achieved longer tumor retention and less kidney uptake than 67GaGa-DOTA-LM3. 68GaGa-DATA5m-LM4 accurately visualized tumor lesions with high contrast on PET/CT. In short, 68GaGa-DATA5m-LM4 has shown excellent prospects for the PET/CT diagnosis of SST2R-positive tumors, further highlighting the benefits of Ga-68 labeling in a hospital environment via the DATA5m-chelator route.
The overexpression of one or more somatostatin receptors (SST
R) 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 SST
R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala
-Gly
-cCys
-Lys
-Asn
-cCys
-Phe
-DTrp
-Lys
-Thr
-Cys
-Thr
-Ser
-Cys
) and AT6S (DOTA-Ala
-Gly
-cCys
-Lys
-cCys
-Phe
-Phe
-DTrp
-Lys
-Thr
-Phe
-Cys
-Ser
-Cys
), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective
InIn-AT5S and
InIn-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
R and agonistic properties at the SST
R, whereas AT5S lost all affinity to SST
R. Both
InIn-AT5S and
InIn-AT6S remained stable in the peripheral blood of mice, while
InIn-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST
R 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,
InIn-AT6S targeted SST
R-/SST
R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics.
: 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 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 ...99mTcTc-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 (DAsn14DT1) and (ii) DT8 (β-Homoleucine13DT1), modified at the C-terminus, along with (iii) DT9 ((palmitoyl)Lys7DT1), carrying an albumin-binding domain (ABD) at Lys7. The biological profiles of the new 99mTcTc–radioligands were compared with 99mTcTc-DT1, using NTS1R-expressing AsPC-1 cells and mice models without or during NEP/ACE inhibition. The radioligands showed enhanced in vivo stability vs. 99mTcTc-DT1, with 99mTcTc-DT9 displaying full resistance to both peptidases. Furthermore, 99mTcTc-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 99mTcTc-DT1 mimics. To improve the observed suboptimal pharmacokinetics of 99mTcTc-DT9, the replacement of palmitoyl on Lys7 by other ABD groups is currently being pursued.
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.
Gastrin-releasing peptide receptor (GRPR)-antagonists have served as motifs in the development of theranostic radioligands for prostate cancer. Our efforts have been focused on the development of ...radiolabeled RM26 (H-DPhe
-Gln
-Trp
-Ala
-Val
-Gly
-His
-Sta
-Leu
-NH
) analogs, such as
InIn-DOTAGA-PEG2-RM26. We recently showed that its Gly
/Sar
-substituted version,
InIn-AU-RM26-M1, resisted degradation by neprilysin (NEP) while in circulation and achieved higher tumor uptake in mice. We herein introduce the following three new AU-RM26-M1 mimics labeled with In-111, with basic residues in the linker: (i) AU-RM26-M2 (PEG2-Pip), (ii) AU-RM26-M3 (PEG2-Arg), and (iii) AU-RM26-M4 (Arg-Arg-Pip). These analogs were compared in PC-3 cells and animal models vs. AU-RM26-M1 (reference). The new analogs showed high affinity and specificity for the GRPR, exhibiting an uptake and distribution pattern in PC-3 cells typical for a radiolabeled GRPR-antagonist. They showed high stability in peripheral mice blood, except for
InIn-AU-RM26-M3. AU-RM26-M4 achieved the highest tumor uptake and promising background clearance, followed by
InIn-RM26-M2, showing lower background levels. These findings were confirmed for
InIn-AU-RM26-M2 and
InIn-AU-RM26-M4 by micro-SPECT/CT at 4 and 24 h post-injection. Hence, the type of positively charged residues in the linker of AU-RM26-M1 mimics strongly influenced biological behavior. The analogs with Pip next to DPhe
demonstrated the best overall characteristics and warrant further investigation.
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