Radiolabeled octreotide analogs are most successfully being applied today in clinical cancer imaging and treatment. Propagation of this paradigm to other radiopeptide families has been greatly ...hampered by the inherent poor metabolic stability of systemically administered peptide analogs. We hypothesized that the in vivo coadministration of specific enzyme inhibitors would improve peptide bioavailability and hence tumor uptake. Through single coinjection of the neutral endopeptidase inhibitor phosphoramidon (PA), we were able to provoke remarkable rises in the percentages of circulating intact somatostatin, gastrin, and bombesin radiopeptides in mouse models, resulting in a remarkable increase in uptake in tumor xenografts in mice.
The peptide conjugates DOTA-Ala(1)SS14 (DOTA-Ala-Gly-cCys-Lys-Asn-Phe-Phe-Trp-Lys-Thr-Phe-Thr-Ser-Cys-OH), PanSB1 (DOTA-PEG2-dTyr-Gln-Trp-Ala-Val-βAla-His-Phe-Nle-NH2), and DOTA-MG11 (DOTA-dGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) were labeled with (111)In by 20 min of heating at an acidic pH. Metabolic stability was studied with high-performance liquid chromatography analysis of blood samples collected 5 min after the injection of the test radiopeptide alone or with PA into mice. Biodistribution was studied after injection of each (111)In-labeled radiopeptide alone or after coinjection of PA in tumor-bearing severe combined immunodeficient (SCID) mice.
The amount of intact (111)In-DOTA-Ala(1)SS14 detected in the mouse circulation at 5 min after the injection of PA increased impressively-from less than 2% to 86%-whereas the uptake in AR4-2J xenografts rose from less than 1 percentage injected dose per gram of tissue (%ID/g) to 14 %ID/g at 4 h after injection. Likewise, the coadministration of PA resulted in a marked increase in the amount of circulating intact (111)In-PanSB1-from 12% to 80%-at 5 min after injection, and radioligand uptake in human PC-3 xenografts in SCID mice escalated from less than 4 %ID/g to greater than 21 %ID/g at 4 h after injection. In a similar manner, the coadministration of PA resulted in an equally impressive increase in intact (111)In-DOTAMG11 levels in the mouse bloodstream-from less than 5% to 70%-at 5 min after injection, leading to a remarkable increase in radiotracer uptake-from 2 %ID/g to greater than 15 %ID/g-in both AR4-2J tumors and A431(CCKR+) tumors (i.e., tumors induced by A431 cells transfected to stably express the human cholecystokinin subtype 2 receptor) in mice at 4 h after injection. This effect was well visualized by SPECT/CT imaging of AR4-2J tumor-bearing mice at 4 h after injection.
The results of this study clearly demonstrate that the coadministration of key enzyme inhibitors can effectively prolong the survival of radiolabeled peptides in the circulation, securing their safe transit to the target. This strategy clearly provoked an unprecedented increase in radiolabel accumulation in tumor xenografts in mice; this increase might translate into higher diagnostic sensitivity or improved therapeutic efficacy of radiopeptide drugs in cancer patients. Hence, our findings provide exciting new opportunities for the application of biodegradable (radio)peptide drugs of either natural or synthetic origin as well as for the rationale design of analogs that are stable in vivo.
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.
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.
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.
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.
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