Radiolabeled peptides and their expanding role in clinical imaging and targeted cancer therapy.

There is an expanding body of evidence showing that synthetic peptides in combination with radioactive isotopes can be utilized for medical purposes. This area is of particular interest in oncology where applications in diagnosis and therapy are at different stages of development. We review the contributions in this area by the group originally founded by Carlo Pedone in Naples many years ago. We highlight the work of this group in the context of other developments in this area, focusing on three biologically relevant receptor systems: somatostatin, gastrin-releasing peptide, and cholecystokinin-2/gastrin receptors. We focus on key milestones, state of the art, and challenges in this area of research as well as the current and future outlook for expanding clinical applications.

In-vivo inhibition of neutral endopeptidase 1 results in higher absorbed tumor doses of [177Lu]Lu-PP-F11N in humans: the lumed phase 0b study.

BACKGROUND: A new generation of radiolabeled minigastrin analogs delivers low radiation doses to kidneys and are considered relatively stable due to less enzymatic degradation. Nevertheless, relatively low tumor radiation doses in patients indicate limited stability in humans. We aimed at evaluating the effect of sacubitril, an inhibitor of the neutral endopeptidase 1, on the stability and absorbed doses to tumors and organs by the cholecystokinin-2 receptor agonist [177Lu]Lu-PP-F11N in patients. In this prospective phase 0 study eight consecutive patients with advanced medullary thyroid carcinoma and a current somatostatin receptor subtype 2 PET/CT scan were included. Patients received two short infusions of ~ 1 GBq [177Lu]Lu-PP-F11N in an interval of ~ 4 weeks with and without Entresto® pretreatment in an open-label, randomized cross-over order. Entresto® was given at a single oral dose, containing 48.6 mg sacubitril. Adverse events were graded and quantitative SPECT/CT and blood sampling were performed. Absorbed doses to tumors and relevant organs were calculated. RESULTS: Pretreatment with Entresto® showed no additional toxicity and increased the stability of [177Lu]Lu-PP-FF11N in blood significantly (p < 0.001). Median tumor-absorbed doses were 2.6-fold higher after Entresto® pretreatment (0.74 vs. 0.28 Gy/GBq, P = 0.03). At the same time, an increase of absorbed doses to stomach, kidneys and bone marrow was observed, resulting in a tumor-to-organ absorbed dose ratio not significantly different with and without Entresto®. CONCLUSIONS: Premedication with Entresto® results in a relevant stabilization of [177Lu]Lu-PP-FF11N and consecutively increases radiation doses in tumors and organs. Trial registration clinicaltrails.gov, NCT03647657. Registered 20 August 2018.

CUDC-907, a dual PI3K/histone deacetylase inhibitor, increases meta-iodobenzylguanidine uptake (123/131I-mIBG) in vitro and in vivo: a promising candidate for advancing theranostics in neuroendocrine tumors.

BACKGROUND: Neuroblastoma (NB) and pheochromocytoma/paraganglioma (PHEO/PGL) are neuroendocrine tumors. Imaging of these neoplasms is performed by scintigraphy after injection of radiolabeled meta-iodobenzylguanidine (mIBG), a norepinephrine analog taken up by tumoral cells through monoamine transporters. The pharmacological induction of these transporters is a promising approach to improve the imaging and therapy (theranostics) of these tumors. METHODS: Transporters involved in mIBG internalization were identified by using transfected Human Embryonic Kidney (HEK) cells. Histone deacetylase inhibitors (HDACi) and inhibitors of the PI3K/AKT/mTOR pathway were tested in cell lines to study their effect on mIBG internalization. Studies in xenografted mice were performed to assess the effect of the most promising HDACi on 123I-mIBG uptake. RESULTS: Transfected HEK cells demonstrated that the norepinephrine and dopamine transporter (NET and DAT) avidly internalizes mIBG. Sodium-4-phenylbutyrate (an HDACi), CUDC-907 (a dual HDACi and PI3K inhibitor), BGT226 (a PI3K inhibitor) and VS-5584 and rapamycin (two inhibitors of mTOR) increased mIBG internalization in a neuroblastoma cell line (IGR-NB8) by 2.9-, 2.1-, 2.5-, 1.5- and 1.3-fold, respectively, compared with untreated cells. CUDC-907 also increased mIBG internalization in two other NB cell lines and in one PHEO cell line. We demonstrated that mIBG internalization occurs primarily through the NET. In xenografted mice with IGR-NB8 cells, oral treatment with 5 mg/kg of CUDC-907 increased the tumor uptake of 123I-mIBG by 2.3- and 1.9-fold at 4 and 24 h post-injection, respectively, compared to the untreated group. CONCLUSIONS: Upregulation of the NET by CUDC-907 lead to a better internalization of mIBG in vitro and in vivo.

Development of a New Class of CXCR4-Targeting Radioligands Based on the Endogenous Antagonist EPI-X4 for Oncological Applications.

The peptide fragment of human serum albumin that was identified as an inhibitor of C-X-C motif chemokine receptor 4 (CXCR4), termed EPI-X4, was investigated as a scaffold for the development of CXCR4-targeting radio-theragnostics. Derivatives of its truncated version JM#21 (ILRWSRKLPCVS) were conjugated to 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) and tested in Jurkat and Ghost-CXCR4 cells. Ligand-1, -2, -5, -6, -7, -8, and -9 were selected for radiolabeling. Molecular modeling indicated that 177Lu-DOTA incorporation C-terminally did not interfere with the CXCR4 binding. Lipophilicity, in vitro plasma stability, and cellular uptake hinted 177Lu-7 as superior. In Jurkat xenografts, all radioligands showed >90% washout from the body within an hour, with the exception of 177Lu-7 and 177Lu-9. 177Lu-7 demonstrated best CXCR4-tumor targeting. Ex vivo biodistribution and single-photon emission computed tomography (SPECT)/positron emission tomography (PET)/CT imaging of 177Lu-7/68Ga-7 showed the same distribution profile for both radioligands, characterized by very low uptake in all nontargeted organs except the kidneys. The data support the feasibility of CXCR4-targeting with EPI-X4-based radioligands and designate ligand-7 as a lead candidate for further optimization.

Head-to-head comparison of different classes of FAP radioligands designed to increase tumor residence time: monomer, dimer, albumin binders, and small molecules vs peptides.

PURPOSE: Fibroblast activation protein-α (FAP)-targeting radioligands have recently demonstrated high diagnostic potential. However, their therapeutic value is impaired by the short tumor residence time. Several strategies have been tested to overcome this limitation, but a head-to-head comparison has never been done. With the aim to identify strengths and limitations of the suggested strategies, we compared the monomer FAPI-46 versus (a) its dimer (FAPI-46-F1D), (b) two albumin binders conjugates (FAPI-46-Ibu (ibuprofen) and FAPI-46-EB (Evans Blue)), and (c) cyclic peptide FAP-2286. METHODS: 177Lu-labeled ligands were evaluated in vitro in cell lines with low (HT-1080.hFAP) and high (HEK-293.hFAP) humanFAP expression. SPECT/CT imaging and biodistribution studies were conducted in HT-1080.hFAP and HEK-293.hFAP xenografts. The areas under the curve (AUC) of the tumor uptake and tumor-to-critical-organs ratios and the absorbed doses were estimated. RESULTS: Radioligands showed IC50 in the picomolar range. Striking differences were observed in vivo regarding tumor uptake, residence, specificity, and total body distribution. All [177Lu]Lu-FAPI-46-based radioligands showed similar uptake between the two tumor models. [177Lu]Lu-FAP-2286 showed higher uptake in HEK-293.hFAP and the least background. The AUC of the tumor uptake and absorbed dose was higher for [177Lu]Lu-FAPI-46-F1D and the two albumin binder conjugates, [177Lu]Lu-FAPI-46-Ibu and [177Lu]Lu-FAPI-46-EB, in HT1080.hFAP xenografts and for [177Lu]Lu-FAPI-46-EB and [177Lu]Lu-FAP-2286 in HEK293.hFAP xenografts. The tumor-to-critical-organs AUC values and the absorbed doses were in favor of [177Lu]Lu-FAP-2286, but tumor-to-kidneys. CONCLUSION: The study indicated dimerization and cyclic peptide structures as promising strategies for prolonging tumor residence time, sparing healthy tissues. Albumin binding strategy outcome depended on the albumin binding moiety. The peptide showed advantages in terms of tumor-to-background ratios, besides tumor-to-kidneys, but its tumor uptake was FAP expression-dependent.

Peptide Radioligands in Cancer Theranostics: Agonists and Antagonists.

The clinical success of radiolabeled somatostatin analogs in the diagnosis and therapy-"theranostics"-of tumors expressing the somatostatin subtype 2 receptor (SST2R) 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, SST2R-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.

Comparison of the Anti-Tumour Activity of the Somatostatin Receptor (SST) Antagonist [177Lu]Lu-Satoreotide Tetraxetan and the Agonist [177Lu]Lu-DOTA-TATE in Mice Bearing AR42J SST2-Positive Tumours.

Limited experiments have compared the treatment effects of repetitive cycles of radiolabelled somatostatin (SST) analogues. In vitro and in vivo experiments were conducted in an AR42J cancer cell model, comparing the antagonist [177Lu]Lu-satoreotide tetraxetan with the agonist [177Lu]Lu-DOTA-TATE in terms of their binding properties, biodistribution, anti-tumour activity and toxicity. Histopathological and immunohistochemical examinations were performed at different timepoints. In the in vitro assays, [177Lu]Lu-satoreotide tetraxetan recognised twice as many SST2 binding sites as [177Lu]Lu-DOTA-TATE. In mice treated once a week for four consecutive weeks, [177Lu]Lu-satoreotide tetraxetan (15 MBq) revealed a significantly greater median time taken to reach a tumour volume of 850 mm3 (68 days) compared to [177Lu]Lu-DOTA-TATE at 15 MBq (43 days) or 30 MBq (48 days). This was associated with a higher tumour uptake, enhanced DNA damage and no or mild effects on body weight, haematological toxicity, or renal toxicity with [177Lu]Lu-satoreotide tetraxetan (15 MBq). At the end of the study, complete tumour senescence was noted in 20% of animals treated with [177Lu]Lu-satoreotide tetraxetan, in 13% of those treated with [177Lu]Lu-DOTA-TATE at 30 MBq, and in none of those treated with [177Lu]Lu-DOTA-TATE at 15 MBq. In conclusion, repeated administrations of [177Lu]Lu-satoreotide tetraxetan were able to potentiate peptide receptor radionuclide therapy with a higher tumour uptake, longer median survival, and enhanced DNA damage, with a favourable efficacy/safety profile compared to [177Lu]Lu-DOTA-TATE.

Radiolabeled Somatostatin Analogs-A Continuously Evolving Class of Radiopharmaceuticals.

Somatostatin receptors (SSTs) are recognized as favorable molecular targets in neuroendocrine tumors (NETs) and neuroendocrine neoplasms (NENs), with subtype 2 (SST2) being the predominantly and most frequently expressed. PET/CT imaging with 68Ga-labeled SST agonists, e.g., 68Ga-DOTA-TOC (SomaKit TOC®) or 68Ga-DOTA-TATE (NETSPOT®), plays an important role in staging and restaging these tumors and can identify patients who qualify and would potentially benefit from peptide receptor radionuclide therapy (PRRT) with the therapeutic counterparts 177Lu-DOTA-TOC or 177Lu-DOTA-TATE (Lutathera®). This is an important feature of SST targeting, as it allows a personalized treatment approach (theranostic approach). Today, new developments hold promise for enhancing diagnostic accuracy and therapeutic efficacy. Among them, the use of SST2 antagonists, such as JR11 and LM3, has shown certain advantages in improving image sensitivity and tumor radiation dose, and there is evidence that they may find application in other oncological indications beyond NETs and NENs. In addition, PRRT performed with more cytotoxic α-emitters, such as 225Ac, or ß- and Auger electrons, such as 161Tb, presents higher efficacy. It remains to be seen if any of these new developments will overpower the established radiolabeled SST analogs and PRRT with ß--emitters.

Distinct In Vitro Binding Profile of the Somatostatin Receptor Subtype 2 Antagonist [177Lu]Lu-OPS201 Compared to the Agonist [177Lu]Lu-DOTA-TATE.

Treatment of neuroendocrine tumours with the radiolabelled somatostatin receptor subtype 2 (SST2) peptide agonist [177Lu]Lu-DOTA-TATE is effective and well-established. Recent studies suggest improved therapeutic efficacy using the SST2 peptide antagonist [177Lu]Lu-OPS201. However, little is known about the cellular mechanisms that lead to the observed differences. In the present in vitro study, we compared kinetic binding, saturation binding, competition binding, cellular uptake and release of [177Lu]Lu-OPS201 versus [177Lu]Lu-DOTA-TATE using HEK cells stably transfected with the human SST2. While [177Lu]Lu-OPS201 and [177Lu]Lu-DOTA-TATE exhibited comparable affinity (KD, 0.15 ± 0.003 and 0.08 ± 0.02 nM, respectively), [177Lu]Lu-OPS201 recognized four times more binding sites than [177Lu]Lu-DOTA-TATE. Competition assays demonstrated that a high concentration of the agonist displaced only 30% of [177Lu]Lu-OPS201 bound to HEK-SST2 cell membranes; an indication that the antagonist binds to additional sites that are not recognized by the agonist. [177Lu]Lu-OPS201 showed faster association and slower dissociation than [177Lu]Lu-DOTA-TATE. Whereas most of [177Lu]Lu-OPS201 remained at the cell surface, [177Lu]Lu-DOTA-TATE was almost completely internalised inside the cell. The present data identified distinct differences between [177Lu]Lu-OPS201 and [177Lu]Lu-DOTA-TATE regarding the recognition of receptor binding sites (higher for [177Lu]Lu-OPS201) and their kinetics (faster association and slower dissociation of [177Lu]Lu-OPS201) that explain, to a great extent, the improved therapeutic efficacy of [177Lu]Lu-OPS201 compared to [177Lu]Lu-DOTA-TATE.

Radiolabeled Bombesin Analogs.

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 Peptides for Cancer Imaging and Therapy: From Bench-to-Bedside.

Radiolabeled peptides can deliver radiation selectively to tumors via targeting peptide receptors that are overexpressed on the surface of cancer cells. The radiation is used either for detection (imaging) or for destruction (therapy) of these tumors. The Division of Radiopharmaceutical Chemistry at the University Hospital Basel has conducted pioneering work on the development of peptide-based radiopharmaceuticals. Our research covers the entire spectrum of such developments, from bench-to-bedside, and it is illustrated in this article by selective cases.

SPECT Imaging of SST2-Expressing Tumors with 99mTc-Based Somatostatin Receptor Antagonists: The Role of Tetraamine, HYNIC, and Spacers.

[99mTc]Tc-HYNIC-TOC is the most widely used 99mTc-labeled somatostatin receptor (SST) agonist for the SPECT imaging of SST-expressing tumors, such as neuroendocrine tumors. Recently, radiolabeled SST antagonists have shown improved diagnostic efficacy over agonists. 99mTc-labeled SST antagonists are lacking in clinical practice. Surprisingly, when [99mTc]Tc-HYNIC was conjugated to the SST2 antagonist SS01, SST2 imaging was not feasible. This was not the case when [99mTc]Tc-N4 was conjugated to SS01. Here, we assessed the introduction of different spacers (X: ß-Ala, Ahx, Aun and PEG4) among HYNIC and SS01 with the aim of restoring the affinity of HYNIC conjugates. In addition, we used the alternative antagonist JR11 for determining the suitability of HYNIC with 99mTc-labeled SST2 antagonists. We performed a head-to-head comparison of the N4 conjugates of SS01 and JR11. [99mTc]Tc-HYNIC-TOC was used as a reference, and HEK-SST2 cells were used for in vitro and in vivo evaluation. EDDA was used as a co-ligand for all [99mTc]Tc-HYNIC conjugates. The introduction of Ahx restored, to a great extent, the SST2-mediated cellular uptake of the [99mTc]Tc-HYNIC-X conjugates (X: spacer), albeit lower than the corresponding [99mTc]Tc-N4-conjugates. SPECT/CT images showed that all 99mTc-labeled conjugates accumulated in the tumor and kidneys with [99mTc]Tc-HYNIC-PEG4-SS01, [99mTc]Tc-N4-SS01 and [99mTc]Tc-N4-JR11 having notably higher kidney uptake. Biodistribution studies showed similar or better tumor-to-non-tumor ratios for the [99mTc]Tc-HYNIC-Ahx conjugates, compared to the [99mTc]Tc-N4 counterparts. The [99mTc]Tc-HYNIC-Ahx conjugates of SS01 and JR11 were comparable to [99mTc]Tc-HYNIC-TOC as imaging agents. HYNIC is a suitable chelator for the development of 99mTc-labeled SST2 antagonists when a spacer of appropriate length, such as Ahx, is used.

Selection of the First 99mTc-Labelled Somatostatin Receptor Subtype 2 Antagonist for Clinical Translation-Preclinical Assessment of Two Optimized Candidates.

Recently, radiolabelled antagonists targeting somatostatin receptors subtype 2 (SST2) in neuroendocrine neoplasms demonstrated certain superior properties over agonists. Within the ERA-PerMED project "TECANT" two 99mTc-Tetramine (N4)-derivatized SST2 antagonists (TECANT-1 and TECANT-2) were studied for the selection of the best candidate for clinical translation. Receptor-affinity, internalization and dissociation studies were performed in human embryonic kidney-293 (HEK293) cells transfected with the human SST2 (HEK-SST2). Log D, protein binding and stability in human serum were assessed. Biodistribution and SPECT/CT studies were carried out in nude mice bearing HEK-SST2 xenografts, together with dosimetric estimations from mouse-to-man. [99mTc]Tc-TECANT-1 showed higher hydrophilicity and lower protein binding than [99mTc]-TECANT-2, while stability was comparable. Both radiotracers revealed similar binding affinity, while [99mTc]Tc-TECANT-1 had higher cellular uptake (>50%, at 2 h/37 °C) and lower dissociation rate (<30%, at 2 h/37 °C). In vivo, [99mTc]Tc-TECANT-1 showed lower blood values, kidney and muscles uptake, whereas tumour uptake was comparable to [99mTc]Tc-TECANT-2. SPECT/CT imaging confirmed the biodistribution results, providing the best tumour-to-background image contrast for [99mTc]Tc-TECANT-1 at 4 h post-injection (p.i.). The estimated radiation dose amounted to approximately 6 µSv/MBq for both radiotracers. This preclinical study provided the basis of selection of [99mTc]Tc-TECANT-1 for clinical translation of the first 99mTc-based SST2 antagonist.

Evaluation of a New 177Lu-Labeled Somatostatin Analog for the Treatment of Tumors Expressing Somatostatin Receptor Subtypes 2 and 5.

Targeted radionuclide therapy of somatostatin receptor (SST)-expressing tumors is only partially addressed by the established somatostatin analogs having an affinity for the SST subtype 2 (SST2). Aiming to target a broader spectrum of tumors, we evaluated the bis-iodo-substituted somatostatin analog ST8950 ((4-amino-3-iodo)-d-Phe-c[Cys-(3-iodo)-Tyr-d-Trp-Lys-Val-Cys]-Thr-NH2), having subnanomolar affinity for SST2 and SST5, labeled with [177Lu]Lu3+ via the chelator DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid). Human Embryonic Kidney (HEK) cells stably transfected with the human SST2 (HEK-SST2) and SST5 (HEK-SST5) were used for in vitro and in vivo evaluation on a dual SST2- and SST5-expressing xenografted mouse model. natLu-DOTA-ST8950 showed nanomolar affinity for both subtypes (IC50 (95% confidence interval): 0.37 (0.22-0.65) nM for SST2 and 3.4 (2.3-5.2) for SST5). The biodistribution of [177Lu]Lu-DOTA-ST8950 was influenced by the injected mass, with 100 pmol demonstrating lower background activity than 10 pmol. [177Lu]Lu-DOTA-ST8950 reached its maximal uptake on SST2- and SST5-tumors at 1 h p.i. (14.17 ± 1.78 and 1.78 ± 0.35%IA/g, respectively), remaining unchanged 4 h p.i., with a mean residence time of 8.6 and 0.79 h, respectively. Overall, [177Lu]Lu-DOTA-ST8950 targets SST2-, SST5-expressing tumors in vivo to a lower extent, and has an effective dose similar to clinically used radiolabeled somatostatin analogs. Its main drawbacks are the low uptake in SST5-tumors and the persistent kidney uptake.

A new 68Ga-labeled somatostatin analog containing two iodo-amino acids for dual somatostatin receptor subtype 2 and 5 targeting.

BACKGROUND: Somatostatin receptor (SST) targeting, specifically of the subtype 2 (SST2), with radiolabeled somatostatin analogs, is established for imaging and treatment of neuroendocrine tumors. Owing to the concomitant and heterogeneous expression of several subtypes on the same tumor, analogs targeting more subtypes than SST2 potentially target a broader spectrum of tumors and/or increase the uptake of a given tumor. The analog ST8950 ((4-amino-3-iodo)-D-Phe-c[Cys-(3-iodo)-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2), bearing 2 iodo-amino acids, exhibits sub-nanomolar affinity to SST2 and SST5. We report herein the development and preclinical evaluation of DOTA-ST8950 labeled with 68Ga, for imaging SST2- and SST5-expressing tumors. Comparative in vitro and in vivo studies were performed with the de-iodinated DOTA-ST8951 ((4-amino)-D-Phe-c[Cys-Tyr-D-Trp-Lys-Val-Cys]-Thr-NH2) and with the reference compounds DOTA-TATE (SST2 selective) and DOTA-NOC (for SST2 and SST5). RESULTS: Compared with natGa-DOTA-NOC, natGa-DOTA-ST8950 exhibited higher affinity to SST2 and SST5 (IC50 (95%CI), nM = 0.32 (0.20-0.50) and 1.9 (1.1-3.1) vs 0.70 (0.50-0.96) and 3.4 (1.8-6.2), respectively), while natGa-DOTA-ST8951 lost affinity for both subtypes. natGa-DOTA-ST8950 had the same potency for inducing SST2-mediated cAMP accumulation as natGa-DOTA-TATE and slightly better than natGa-DOTA-NOC (EC50, nM = 0.46 (0.23-0.92) vs 0.47 (0.15-1.5) vs 0.59 (0.18-1.9), respectively). [67Ga]Ga-DOTA-ST8950 had a similar internalization rate as [67Ga]Ga-DOTA-NOC in SST2-expressing cells (12.4 ± 1.6% vs 16.6 ± 2.2%, at 4 h, p = 0.0586). In vivo, [68Ga]Ga-DOTA-ST8950 showed high and specific accumulation in SST2- and SST5-expressing tumors, comparable with [68Ga]Ga-DOTA-NOC (26 ± 8 vs 30 ± 8 %IA/g, p = 0.4630 for SST2 and 15 ± 6 vs 12 ± 5 %IA/g, p = 0.3282, for SST5, 1 h p.i.) and accumulation in the SST-positive tissues, the kidneys and the liver. PET/CT images of [68Ga]Ga-DOTA-ST8950, performed in a dual HEK-SST2 and HEK-SST5 tumor xenografted model, clearly visualized both tumors and illustrated high tumor-to-background contrast. CONCLUSIONS: [68Ga]Ga-DOTA-ST8950 reveals its potential for PET imaging SST2- and SST5-expressing tumors. It compares favorably with the clinically used [68Ga]Ga-DOTA-NOC in terms of tumor uptake; however, its uptake in the liver remains a challenge for clinical translation. In addition, this study reveals the essential role of the iodo-substitutions in positions 1 and 3 of [68Ga]Ga-DOTA-ST8950 for maintaining affinity to SST2 and SST5, as the de-iodinated [68Ga]Ga-DOTA-ST8951 lost affinity for both receptor subtypes.

Design and development of the theranostic pair 177 Lu-OPS201/68 Ga-OPS202 for targeting somatostatin receptor expressing tumors.

Radiolabeled somatostatin receptor (sstr) antagonists have shown superiority in different preclinical and clinical settings compared with the well-established and clinically used agonists for targeting sstr-expressing tumors, with regard to pharmacokinetics, tumor uptake, and retention. The theranostic pair 177 Lu-OPS201/68 Ga-OPS202, based on the sstr2 antagonist JR11 (Cpa-c[d-Cys-Aph(Hor)-d-Aph(Cbm)-Lys-Thr-Cys]-d-Tyr-NH2 ), is the most advanced pair of the antagonist family in terms of preclinical development and is currently under clinical evaluation. OPS201 and OPS202 share the same amino acid sequence (JR11) but feature different conjugated chelators needed for radiolabeling, DOTA for OPS201 and NODAGA for OPS202. In this review, the design and development of the peptidic analog, JR11, and the selection of chelators and radiometals that led to 177 Lu-OPS201/68 Ga-OPS202 are discussed. Furthermore, the preclinical evaluation of both radiolabeled analogs from bench to bedside and the clinical trials involving the theranostic pair are presented.

Targeting of the Cholecystokinin-2 Receptor with the Minigastrin Analog 177Lu-DOTA-PP-F11N: Does the Use of Protease Inhibitors Further Improve In Vivo Distribution?

Patients with metastatic medullary thyroid cancer (MTC) have limited systemic treatment options. The use of radiolabeled gastrin analogs targeting the cholecystokinin-2 receptor (CCK2R) is an attractive approach. However, their therapeutic efficacy is presumably decreased by their enzymatic degradation in vivo. We aimed to investigate whether the chemically stabilized analog 177Lu-DOTA-PP-F11N (177Lu-DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Nle-Asp-Phe-NH2) performs better than reference analogs with varying in vivo stability, namely 177Lu-DOTA-MG11 (177Lu-DOTA-dGlu-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2) and 177Lu-DOTA-PP-F11 (177Lu-DOTA-(dGlu)6-Ala-Tyr-Gly-Trp-Met-Asp-Phe-NH2), and whether the use of protease inhibitors further improves CCKR2 targeting. First human data on 177Lu-DOTA-PP-F11N are also reported. Methods: In vitro stability of all analogs was assessed against a panel of extra- and intracellular endoproteases, whereas their in vitro evaluation was performed using the human MTC MZ-CRC-1 and the transfected A431-CCK2R(+) cell lines. Biodistribution without and with the protease inhibitors phosphoramidon and thiorphan was assessed 4 h after injection in MZ-CRC-1 and A431-CCK2R(+) dual xenografts. Autoradiography of 177Lu-DOTA-PP-F11N (without and with phosphoramidon) and NanoSPECT/CT were performed. SPECT/CT images of 177Lu-DOTA-PP-F11N in a metastatic MTC patient were also acquired. Results:natLu-DOTA-PP-F11N is less of a substrate for neprilysins than the other analogs, whereas intracellular cysteine proteases, such as cathepsin-L, might be involved in the degradation of gastrin analogs. The uptake of all radiotracers was higher in MZ-CRC-1 tumors than in A431-CCK2R(+), apparently because of the higher number of binding sites on MZ-CRC-1 cells. 177Lu-DOTA-PP-F11N had the same biodistribution as 177Lu-DOTA-PP-F11; however, uptake in the MZ-CRC-1 tumors was almost double (20.7 ± 1.71 vs. 11.2 ± 2.94 %IA [percentage injected activity]/g, P = 0.0002). Coadministration of phosphoramidon or thiorphan increases 177Lu-DOTA-MG11 uptake significantly in the CCK2R(+) tumors and stomach. Less profound was the effect on 177Lu-DOTA-PP-F11, whereas no influence or even reduction was observed for 177Lu-DOTA-PP-F11N (20.7 ± 1.71 vs. 15.6 ± 3.80 [with phosphoramidon] %IA/g, P < 0.05 in MZ-CRC-1 tumors). The first clinical data show high 177Lu-DOTA-PP-F11N accumulation in tumors, stomach, kidneys, and colon. Conclusion: The performance of 177Lu-DOTA-PP-F11N without protease inhibitors is as good as the performance of 177Lu-DOTA-MG11 in the presence of inhibitors. The human application of single compounds without unessential additives is preferable. Preliminary clinical data spotlight the stomach as a potential dose-limiting organ besides the kidneys.

New Gastrin Releasing Peptide Receptor-Directed [99mTc]Demobesin 1 Mimics: Synthesis and Comparative Evaluation.

We have previously reported on the gastrin releasing peptide receptor (GRPR) antagonist [99mTc]1, ([99mTc]demobesin 1, 99mTc-[N4'-diglycolate-dPhe6,Leu-NHEt13]BBN(6-13)). [99mTc]1 has shown superior biological profile compared to analogous agonist-based 99mTc-radioligands. We herein present a small library of [99mTc]1 mimics generated after structural modifications in (a) the linker ([99mTc]2, [99mTc]3, [99mTc]4), (b) the peptide chain ([99mTc]5, [99mTc]6), and (c) the C-terminus ([99mTc]7 or [99mTc]8). The effects of above modifications on the biological properties of analogs were studied in PC-3 cells and tumor-bearing SCID mice. All analogs showed subnanomolar affinity for the human GRPR, while most receptor-affine 4 and 8 behaved as potent GRPR antagonists in a functional internalization assay. In mice bearing PC-3 tumors, [99mTc]1-[99mTc]6 exhibited GRPR-specific tumor uptake, rapidly clearing from normal tissues. [99mTc]4 displayed the highest tumor uptake (28.8 ± 4.1%ID/g at 1 h pi), which remained high even after 24 h pi (16.3 ± 1.8%ID/g), well surpassing that of [99mTc]1 (5.4 ± 0.7%ID/g at 24 h pi).

Diagnosis of recurrent prostate cancer with PET/CT imaging using the gastrin-releasing peptide receptor antagonist 68Ga-RM2: Preliminary results in patients with negative or inconclusive [18F]Fluoroethylcholine-PET/CT.

PURPOSE/BACKGROUND: [18F]fluoroethylcholine (18FECH) has been shown to be a valuable PET-tracer in recurrent prostate cancer (PCa), but still has limited accuracy. RM2 is a gastrin-releasing peptide receptor (GRPr) antagonist that binds to GRPr on PCa cells. Recent studies suggest that GRPr imaging with PET/CT is a promising technique for staging and restaging of PCa. We explore the value of GRPr-PET using the 68Ga-labeled GRPr antagonist RM2 in a selected population of patients with biochemically recurrent PCa and a negative/inconclusive 18FECH-PET/CT. MATERIAL AND METHODS: In this retrospective study 16 men with biochemical PCa relapse and negative (n = 14) or inconclusive (n = 2) 18FECH-PET/CT underwent whole-body 68Ga-RM2-PET/CT. Mean time from 18FECH-PET/CT to 68Ga-RM2-PET/CT was 6.1 ± 6.8 months. Primary therapies in these patients were radical prostatectomy (n = 13; 81.3%) or radiotherapy (n = 3; 18.7%). 14/16 patients (87.5%) had already undergone salvage therapies because of biochemical relapse prior to 68Ga-RM2-PET/CT imaging. Mean ± SD PSA at 68Ga-RM2-PET/CT was 19.4 ± 53.5 ng/ml (range 1.06-226.4 ng/ml). RESULTS: 68Ga-RM2-PET/CT showed at least one region with focal pathological uptake in 10/16 patients (62.5%), being suggestive of local relapse (n = 4), lymph node metastases (LNM; n = 4), bone metastases (n = 1) and lung metastasis with hilar LNM (n = 1). Seven of ten positive 68Ga-RM2 scans were positively confirmed by surgical resection and histology of the lesions (n = 2), by response to site-directed therapies (n = 2) or by further imaging (n = 3). Patients with a positive 68Ga-RM2-scan showed a significantly higher median PSA (6.8 ng/ml, IQR 10.2 ng/ml) value than those with a negative scan (1.5 ng/ml, IQR 3.1 ng/ml; p = 0.016). Gleason scores or concomitant antihormonal therapy had no apparent impact on the detection of recurrent disease. CONCLUSION: Even in this highly selected population of patients with known biochemical recurrence but negative or inconclusive 18FECH-PET/CT, a 68Ga-RM2-PET/CT was helpful to localize PCa recurrence in the majority of the cases. Thus, 68Ga-RM2-PET/CT deserves further investigation as a promising imaging modality for imaging PCa recurrence.

Biodistribution, Pharmacokinetics, and Dosimetry of 177Lu-, 90Y-, and 111In-Labeled Somatostatin Receptor Antagonist OPS201 in Comparison to the Agonist 177Lu-DOTATATE: The Mass Effect.

Radiolabeled somatostatin receptor (SSTR) antagonists have shown in vivo higher uptake in SSTR-expressing tumors than agonists. In this preclinical study, the SSTR2 antagonist OPS201 (DOTA-JR11; DOTA-[Cpa-c(DCys-Aph(Hor)-DAph(Cbm)-Lys-Thr-Cys)-DTyr-NH2]) labeled with 177Lu, 90Y, and 111In was compared with the SSTR2 agonist 177Lu-DOTATATE. Methods: Biodistribution, pharmacokinetics, SPECT/CT, and dosimetry studies were performed to assess the bioequivalence of all radiotracers. Use of escalated peptide mass and nephroprotective agents were systematically investigated. Results: The tumor residence time was 15.6 h (13.4-17.7) for 177Lu-OPS201 (10 pmol) and 6.4 h (5.4-7.3) for 177Lu-DOTATATE, resulting in a 2.5-times-higher tumor dose for the antagonist than for the agonist (0.854 vs. 0.333 mGy/MBq for a 4-cm tumor). The overall tumor-to-kidney dose ratio was approximately 24% and 32% higher for 177Lu-OPS201 than for 90Y-OPS201 and 177Lu-DOTATATE, respectively. 111In-OPS201 had a biodistribution significantly different from 90Y-OPS201 and is therefore not a surrogate for 90Y-OPS201 dosimetry studies. Importantly, and in contrast to 177Lu-DOTATATE, injection of 10, 200, and 2,000 pmol of 177Lu-OPS201 did not cause any relevant tumor saturation, with tumor uptake 4 h after injection: 23.9, 24.9, and 18.8 percentage of injected activity per gram of tissue (%IA/g), respectively, for the antagonist (P > 0.05), as compared with 17.8, 12.0, and 9.9 %IA/g for the agonist (P < 0.05). Increasing the peptide mass of 177Lu-OPS201 from 10 to 200 pmol drastically decreased the effective dose from 0.0908 to 0.0184 mSv/MBq and decreased the uptake in the liver, bone marrow, and all SSTR2-expressing organs; thus, the therapeutic index improved considerably. Lysine and succinylated gelatine, alone or in combination, significantly reduced the renal dose of 177Lu-OPS201 compared with the control group, by 45%, 25%, and 40%, respectively (P < 0.05). The reduction was similar for 10 and 200 pmol, whereas lysine performed better than succinylated gelatine. Conclusion:177Lu-OPS201 exhibits higher tumor uptake, longer tumor residence time, and improved tumor-to-kidney dose ratio compared with 177Lu-DOTATATE and 90Y-OPS201. Importantly, the mass-escalation study indicates that an optimized antagonist mass might further improve the safety window of peptide receptor radionuclide therapy by reducing the liver and bone marrow doses as well as the effective dose. Clinical studies are warranted to confirm the efficacy and advantageous toxicity profile of 177Lu-OPS201.