AR42J Tumor Research Articles

Preclinical Evaluation of 226Ac as a Theranostic Agent: Imaging, Dosimetry, and Therapy.

226Ac (t½ = 29.37 h) has been proposed as a theranostic radioisotope leveraging both its diagnostic γ-emissions and therapeutic α-emissions. 226Ac emits 158 and 230 keV γ-photons ideal for quantitative SPECT imaging and acts as an invivo generator of 4 high-energy α-particles. Because of these nuclear decay properties, 226Ac has potential to act as a standalone theranostic isotope. In this proof-of-concept study, we evaluated a preclinical 226Ac-radiopharmaceutical for its theranostic efficacy and present the first 226Ac-targeted α-therapy study. Methods: 226Ac was produced at TRIUMF and labeled with the chelator-peptide bioconjugate crown-TATE. [226Ac]Ac-crown-TATE was selected to target neuroendocrine tumors in male NRG mice bearing AR42J tumor xenografts for SPECT imaging, biodistribution, and therapy studies. A preclinical SPECT/CT scanner acquired quantitative images reconstructed from both the 158 and the 230 keV emissions. Mice in the biodistribution study were euthanized at 1, 3, 5, 24, and 48 h after injection, and internal radiation dosimetry was derived for the tumor and organs of interest to establish appropriate therapeutic activity levels. Mice in the therapy study were administered 125, 250, or 375 kBq treatments and were monitored for tumor size and body condition. Results: We present quantitative SPECT images of the invivo biodistribution of [226Ac]Ac-crown-TATE, which showed agreement with ex vivo measurements. Biodistribution studies demonstrated high uptake (>30%IA/g at 5 h after injection) and retention in the tumor, with an estimated mean absorbed dose coefficient of 222 mGy/kBq. [226Ac]Ac-crown-TATE treatments significantly extended the median survival from 7 d in the control groups to 16, 24, and 27 d in the 125, 250, and 375 kBq treatment groups, respectively. Survival was prolonged by slowing tumor growth, and no weight loss or toxicities were observed. Conclusion: This study highlights the theranostic potential of 226Ac as a standalone therapeutic isotope in addition to its demonstrated diagnostic capabilities to assess dosimetry in matched 225Ac-radiopharmaceuticals. Future studies will investigate maximum dose and toxicity to further explore the therapeutic potential of 226Ac-radiopharmaceuticals.

PET imaging of [52 Mn]Mn-DOTATATE and [52 Mn]Mn-DOTA-JR11.

Manganese-52 is gaining interest as an isotope for PET imaging due to its desirable decay and chemical properties for radiopharmaceutical development. Somatostatin receptor 2 (SSTR2) is significantly overexpressed by neuroendocrine tumors (NETs) and is an important target for nuclear imaging and therapy. As an agonist, [68Ga]Ga-DOTATATE has demonstrated significant internalization upon interaction with receptor ligands, whereas [68Ga]Ga-DOTA-JR11(as an antagonist) exhibits limited internalization but better pharmacokinetics and increased tumor uptake. The goal of this study was to label both DOTATATE and DOTA-JR11 peptides with 52Mn in high radiochemical yields (RCY) and sufficient specific activity. A comparison of these two compounds was performed in in vitro and in vivo studies in animals with somatostatin receptor-positive xenografts to characterize differences in cell, tumor, and tissue uptake. Radiolabeling of DOTATATE and DOTA-JR11 was carried out by combining varying concentrations of the peptides with [52Mn]MnCl2. In vitro stability of the radiotracers was determined in mouse serum. In vitro cell uptake and internalization assays were performed in SSTR2 + AR42J cells and negative controls. In vivo biodistribution and longitudinal PET imaging was evaluated in mice bearing AR42J tumors. Both [52Mn]Mn-DOTATATE and [52Mn]Mn-DOTA-JR11showed affinity for SSTR2 in AR42J cells. However, the uptake of [52Mn]Mn-DOTATATE was higher (11.95 ± 0.71%/ mg) compared to [52Mn]Mn-DOTA-JR11 (7.31 ± 0.38%/ mg) after 2 h incubation. After 4 h incubation, 53.13 ± 1.83% of the total activity of [52Mn]Mn-DOTATATE was internalized, whereas only 20.85 ± 0.59% of the total activity of [52Mn]Mn-DOTA-JR11 was internalized. The PET images revealed similar biodistribution results, with [52Mn]Mn-DOTATATE showing a significant tumor uptake of 11.16 ± 2.97% ID/g, while [52Mn]Mn-DOTA-JR11 exhibited a lower tumor uptake of 2.11 ± 0.30% ID/g 4 h post-injection. The synthesis of both radiotracers was accomplished with high RCY and purity. The cell uptake and internalization of [52Mn]Mn-DOTATATE showed higher levels compared to [52Mn]Mn-DOTA-JR11. PET images of the radiotracers in AR42J tumor bearing mice demonstrated similar biodistribution in all organs except the tumor, with [52Mn]Mn-DOTATATE showing higher tumor uptake compared to [52Mn]Mn-DOTA-JR11. The variations in properties of these tracers could be used to guide further imaging and treatment studies.

Preclinical evaluation of [225Ac]Ac-crown-TATE – An alpha-emitting radiopharmaceutical for neuroendocrine tumors

BackgroundTargeted alpha therapy (TAT) of somatostatin receptor-2 (SSTR2) positive neuroendocrine tumors (NETs) involving Ac-225 ([225Ac]Ac-DOTA-TATE) has previously demonstrated improved therapeutic efficacy over conventional beta particle-emitting peptide receptor radionuclide therapy agents. DOTA-TATE requires harsh radiolabeling conditions for chelation of [225Ac]Ac3+, which can limit the achievable molar activities and thus therapeutic efficacy of such TAT treatments. Macropa-TATE was recently highlighted as a potential alternative to DOTA-TATE, owing to the mild radiolabeling conditions and high affinity toward [225Ac]Ac3+; however, elevated liver and kidney uptake were noted as a major limitation and a suitable imaging radionuclide is yet to be reported, which will be required for patient dosimetry studies and assessment of therapeutic benefit. Previously, [155Tb]Tb-crown-TATE has shown highly effective imaging of NETs in preclinical SPECT/CT studies, with high tumor uptake and low non-target accumulation; these favourable properties and the versatile coordination behavior of the crown chelator may therefore show promise for combination with Ac-225 for TAT. MethodsCrown-TATE was labeled with Ac-225, and radiochemical yield was analyzed as the function of crown-TATE concentration. LogD7.4 was measured as the indication of hydrophilicity. Free [225Ac]Ac3+ release from [225Ac]Ac-crown-TATE in human serum was studied. Biodistribution studies of [225Ac]Ac-crown-TATE in mice bearing AR42J tumors was evaluated at 1, 4, 24, 48, and 120 h, and the absorbed dose to major organs calculated. Therapy-monitoring studies with AR42J tumor bearing mice were undertaken using 30 kBq and 55 kBq doses of [225Ac]Ac-crown-TATE and compared to controls treated with PBS or crown-TATE. Results[225Ac]Ac-crown-TATE was successfully prepared with high molar activity (640 kBq/nmol), and characterized as a moderately hydrophilic radioligand (LogD7.4 = −1.355 ± 0.135). No release of bound Ac-225 was observed over 9 days in human serum. Biodistribution studies of [225Ac]Ac-crown-TATE showed good initial tumor uptake (11.1 ± 1.7% IA/g at 4 h) which was sustained up to 120 h p.i. (6.92 ± 2.03% IA/g). Dosimetry calculations showed the highest absorbed dose was delivered to the tumors. Therapy monitoring studies demonstrated significant (log-rank test, P < 0.005) improved survival in both treatment groups compared to controls. ConclusionsThis preclinical study demonstrated the therapeutic efficacy of [225Ac]Ac-crown-TATE for treatment of NETs, and highlights the potential of using crown chelator for stable chelation of Ac-225 under mild conditions.

In vivo quantitative SPECT imaging of actinium-226: feasibility and proof-of-concept

Objective. 225Ac radiopharmaceuticals have tremendous potential for targeted alpha therapy, however, 225Ac (t 1/2 = 9.9 d) lacks direct gamma emissions for in vivo imaging. 226Ac (t 1/2 = 29.4 h) is a promising element-equivalent matched diagnostic radionuclide for preclinical evaluation of 225Ac radiopharmaceuticals. 226Ac has two gamma emissions (158 keV and 230 keV) suitable for SPECT imaging. This work is the first feasibility study for in vivo quantitative 226Ac SPECT imaging and validation of activity estimation. Approach. 226Ac was produced at TRIUMF (Vancouver, Canada) with its Isotope Separator and Accelerator (ISAC) facility. [226Ac]Ac3+ was radiolabelled with the bioconjugate crown-TATE developed for therapeutic targeting of neuroendocrine tumours. Mice with AR42J tumour xenografts were injected with either 2 MBq of [226Ac]Ac-crown-TATE or 4 MBq of free [226Ac]Ac3+ activity and were scanned at 1, 2.5, 5, and 24 h post injection in a preclinical microSPECT/CT. Quantitative SPECT images were reconstructed from the 158 keV and 230 keV photopeaks with attenuation, background, and scatter corrections. Image-based 226Ac activity measurements were assessed from volumes of interest within tumours and organs of interest. Imaging data was compared with ex vivo biodistribution measured via gamma counter. Main results. We present, to the best of our knowledge, the first ever in vivo quantitative SPECT images of 226Ac activity distributions. Time-activity curves derived from SPECT images quantify the in vivo biodistribution of [226Ac]Ac-crown-TATE and free [226Ac]Ac3+ activity. Image-based activity measurements in the tumours and organs of interest corresponded well with ex vivo biodistribution measurements. Significance. Here in, we established the feasibility of in vivo 226Ac quantitative SPECT imaging for accurate measurement of actinium biodistribution in a preclinical model. This imaging method could facilitate more efficient development of novel actinium labelled compounds by providing accurate quantitative in vivo pharmacokinetic information essential for estimating toxicities, dosimetry, and therapeutic potency.

Safety, dosimetry, and efficacy of an optimized long-acting somatostatin analog for peptide receptor radionuclide therapy in metastatic neuroendocrine tumors: From preclinical testing to first-in-human study

Peptide receptor radionuclide therapy (PRRT) with radiolabeled SSTR2 agonists is a treatment option that is highly effective in controlling metastatic and progressive neuroendocrine tumors (NETs). Previous studies have shown that an SSTR2 agonist combined with albumin binding moiety Evans blue (denoted as 177Lu-EB-TATE) is characterized by a higher tumor uptake and residence time in preclinical models and in patients with metastatic NETs. This study aimed to enhance the in vivo stability, pharmacokinetics, and pharmacodynamics of 177Lu-EB-TATE by replacing the maleimide-thiol group with a polyethylene glycol chain, resulting in a novel EB conjugated SSTR2-targeting radiopharmaceutical, 177Lu-LNC1010, for PRRT. In preclinical studies, 177Lu-LNC1010 exhibited good stability and SSTR2-binding affinity in AR42J tumor cells and enhanced uptake and prolonged retention in AR42J tumor xenografts. Thereafter, we presented the first-in-human dose escalation study of 177Lu-LNC1010 in patients with advanced/metastatic NETs. 177Lu-LNC1010 was well-tolerated by all patients, with minor adverse effects, and exhibited significant uptake and prolonged retention in tumor lesions, with higher tumor radiation doses than those of 177Lu-EB-TATE. Preliminary PRRT efficacy results showed an 83% disease control rate and a 42% overall response rate after two 177Lu-LNC1010 treatment cycles. These encouraging findings warrant further investigations through multicenter, prospective, and randomized controlled trials.

Functionalization of 68Ga-Radiolabeled Nanodiamonds with Octreotide Does Not Improve Tumor-Targeting Capabilities.

Nanodiamonds (NDs) are emerging as a novel nanoparticle class with growing interest in medical applications. The surface coating of NDs can be modified by attaching binding ligands or imaging probes, turning them into multi-modal targeting agents. In this investigation, we assessed the targeting efficacy of octreotide-functionalized 68Ga-radiolabelled NDs for cancer imaging and compared it with the tumor uptake using [68Ga]Ga-DOTA-TOC. In vivo studies in mice bearing AR42J tumors demonstrated the highest accumulation of the radiolabeled functionalized NDs in the liver and spleen, with relatively low tumor uptake compared to [68Ga]Ga-DOTA-TOC. Our findings suggest that, within the scope of this study, functionalization did not enhance the tumor-targeting capabilities of NDs.

Bis(Disulfide)-Bridged Somatostatin-14 Analogs and Their [111In]In-Radioligands: Synthesis and Preclinical Profile.

The overexpression of one or more somatostatin receptors (SST1-5R) 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 SST2R-prefering radioligands. In pursuit of this goal, we now introduce two bicyclic somatostatin-14 (SS14) analogs, AT5S (DOTA-Ala1-Gly2-c[Cys3-Lys4-Asn5-c[Cys6-Phe7-DTrp8-Lys9-Thr10-Cys11]-Thr12-Ser13-Cys14]) and AT6S (DOTA-Ala1-Gly2-c[Cys3-Lys4-c[Cys5-Phe6-Phe7-DTrp8-Lys9-Thr10-Phe11-Cys12]-Ser13-Cys14]), suitable for labeling with trivalent radiometals and designed to sustain in vivo degradation. Both AT5S and AT6S and the respective [111In]In-AT5S and [111In]In-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 SST1-5R and agonistic properties at the SST2R, whereas AT5S lost all affinity to SST1-5R. Both [111In]In-AT5S and [111In]In-AT6S remained stable in the peripheral blood of mice, while [111In]In-AT6S displayed low, but specific uptake in AR4-2J tumors and higher uptake in HEK293-SST3R 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, [111In]In-AT6S targeted SST2R-/SST3R-positive xenografts in mice. These results call for further research on pansomatostatin-like radioligands for cancer theranostics.

Structural modifications toward improved lead-203/lead-212 peptide-based image-guided alpha-particle radiopharmaceutical therapies for neuroendocrine tumors

PurposeThe lead-203 (203Pb)/lead-212 (212Pb) elementally identical radionuclide pair has gained significant interest in the field of image-guided targeted alpha-particle therapy for cancer. Emerging evidence suggests that 212Pb-labeled peptide-based radiopharmaceuticals targeting somatostatin receptor subtype 2 (SSTR2) may provide improved effectiveness compared to beta-particle-based therapies for neuroendocrine tumors (NETs). This study aims to improve the performance of SSTR2-targeted radionuclide imaging and therapy through structural modifications to Tyr3-octreotide (TOC)-based radiopharmaceuticals.MethodsNew SSTR2-targeted peptides were designed and synthesized with the goal of optimizing the incorporation of Pb isotopes through the use of a modified cyclization technique; the introduction of a Pb-specific chelator (PSC); and the insertion of polyethylene glycol (PEG) linkers. The binding affinity of the peptides and the cellular uptake of 203Pb-labeled peptides were evaluated using pancreatic AR42J (SSTR2+) tumor cells and the biodistribution and imaging of the 203Pb-labeled peptides were assessed in an AR42J tumor xenograft mouse model. A lead peptide was identified (i.e., PSC-PEG2-TOC), which was then further evaluated for efficacy in 212Pb therapy studies.ResultsThe lead radiopeptide drug conjugate (RPDC) — [203Pb]Pb-PSC-PEG2-TOC — significantly improved the tumor-targeting properties, including receptor binding and tumor accumulation and retention as compared to [203Pb]Pb-DOTA0-Tyr3-octreotide (DOTATOC). Additionally, the modified RPDC exhibited faster renal clearance than the DOTATOC counterpart. These advantageous characteristics of [212Pb]Pb-PSC-PEG2-TOC resulted in a dose-dependent therapeutic effect with minimal signs of toxicity in the AR42J xenograft model. Fractionated administrations of 3.7 MBq [212Pb]Pb-PSC-PEG2-TOC over three doses further improved anti-tumor effectiveness, resulting in 80% survival (70% complete response) over 120 days in the mouse model.ConclusionStructural modifications to chelator and linker compositions improved tumor targeting and pharmacokinetics (PK) of 203/212Pb peptide-based radiopharmaceuticals for NET theranostics. These findings suggest that PSC-PEG2-TOC is a promising candidate for Pb-based targeted radionuclide therapy for NETs and other types of cancers that express SSTR2.

Abstract 5044: Antitumor activity comparison of two somatostatin receptor ligands radiolabeled with Lutetium-177 (SSO110 and DOTA-TATE) alone or combined with chemotherapy in mice bearing AR42J SST2-positive tumors

Abstract Only a limited number of studies have compared the therapeutic effects of repetitive cycles of radiolabeled somatostatin (SST) analogues or assessed potential synergy of these analogues with chemotherapy agents. Hence, a comparison was conducted in an in vivo AR42J tumor model with an SST2 antagonist, [177Lu]Lu-satoreotide tetraxetan ([177Lu]LuSSO110), and a SST2 agonist, [177Lu]Lu-DOTA-TATE. The efficacy of treatment combining [177Lu]Lu-satoreotide tetraxetan with capecitabine plus temozolomide was also assessed. Swiss Nude mice were xenografted with AR42J tumor cells. For the evaluation of the tumor response to radiation therapy, animals received one weekly dose of [177Lu]LuSSO110 or [177Lu]Lu-DOTA-TATE for four consecutive weeks. For combined treatments, animals received one weekly dose of [177Lu]LuSSO110 at 20 MBq for four consecutive weeks in association with daily doses of capecitabine and temozolomide (CAPTEM). Weekly treatment for four consecutive weeks with [177Lu]LuSSO110 at 15 MBq revealed a significantly reduced tumor growth, with 68 days to reach a tumor volume of 850 mm3, compared to [177Lu]Lu-DOTA-TATE at 15 MBq or 30 MBq, with, respectively, 43 days and 48 days. This was associated with a 3.5 fold higher tumor uptake of [177Lu]LuSSO110 (15 MBq) compared to [177Lu]Lu-DOTA-TATE, with no or mild effects on body weight, hematological toxicity, or renal toxicity. In AR42J tumor-bearing mice treated once a week for four consecutive weeks, [177Lu]LuSSO110 (15 MBq) associated with CAPTEM regimen did not significantly increase the median time to reach a tumor volume of 1000 mm3 as compared to treatment with [177Lu]Lu-satoreotide alone: 50.7 vs 42.2 days, respectively. The association of the two treatments only led to mild weight losses. Repeated administrations of [177Lu]LuSSO110 were able to potentiate peptide receptor radionuclide therapy with a higher tumor uptake and longer median survival compared to [177Lu]Lu-DOTA-TATE. The The combination of [177Lu]Lu-satoreotide with capecitabine and temozolomide did not show any synergy, but no antagonism was observed either. Citation Format: Olivier Raguin, Marcel Krüger, Peggy Provent, Funda Cay, Elodie Marie dit chatel, Marie Lux, Stéphane Lezmi, Florence Meyer-Losic, Pascale Plas, Ben Pais, Cyril Berthet. Antitumor activity comparison of two somatostatin receptor ligands radiolabeled with Lutetium-177 (SSO110 and DOTA-TATE) alone or combined with chemotherapy in mice bearing AR42J SST2-positive tumors. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 5044.

Evaluation of the Efficacy of a Combined Treatment Using the mTOR-Inhibitor Everolimus and [177Lu]Lu-DOTA-TATE in Nude CD1 Mice with SSTR-Expressing Pancreatic AR42J Xenograft Tumors.

Therapy options for advanced pancreatic neuroendocrine tumors (pNETs) include the mTOR inhibitor everolimus and peptide receptor radionuclide therapy (PRRT) with [177Lu]Lu-DOTA-TATE, however further optimization in the therapeutic landscape is required as response rates are still low. In this study, we investigated the synergistic and potentially enhanced efficacy of a combined treatment with everolimus and [177Lu]Lu-DOTA-TATE in a mouse model. Baseline [68Ga]Ga-DOTA-TATE PET scans were obtained five days after athymic CD1 mice were inoculated with AR42J tumor cells, before separating the animals into four groups. Group 1 received a placebo, group 2 everolimus, group 3 a placebo and PRRT, and group 4 everolimus and PRRT. The treatment response was monitored by manually measuring the tumor volumes (manual tumor volume, MTV) and conducting sequential [68Ga]Ga-DOTA-TATE PET scans at one, two, and four weeks after treatment induction. The biological tumor volume (BTV) was derived from PET scans using threshold-based volume of interest (VOI) measurements. Tracer uptake was measured semi-quantitatively as a tumor to background ratio (TBR). Mice were euthanized due to excessive tumor growth according to the ethics protocol; blood samples were drawn for the preparation of full blood counts and kidneys were obtained for histological analysis. For the histological assessment, a standardized score (renal damage score, RDS) was used. Full blood counts showed significantly increased numbers of neutrophils and lymphocytes in the groups receiving PRRT. All other parameters did not differ relevantly. In the histological analysis, groups receiving PRRT had a significantly higher RDS, whereas everolimus only tended to cause an increase in the RDS. Mice in groups 1 and 2 had to be euthanized due to excessive tumor growth two weeks after the start of the therapy, whereas follow-up in groups 3 and 4 comprised four weeks. PRRT significantly inhibited tumor growth; the administration of everolimus did not induce an additional effect. A good correlation existed between MTV and BTV. PRRT significantly reduced the TBR. [68Ga]Ga-DOTA-TATE PET is suitable for monitoring tumor growth in the applied model. The high efficacy of [177Lu]Lu-DOTA-TATE is not enhanced by the combination with everolimus.

The preparation, biodistribution, and human's absorbed dose evaluation of Radio-Scandium-HYNIC-TOC for somatostatin-receptor-positive neuroendocrine tumors therapy by animal study.

Most of the neuroendocrine tumors (NETs) express Somatostatin receptors (SSTr), which are the main bases for the development of several radiopharmaceuticals for therapy and imaging of these types of tumors. In this study, 46 Scandium nuclide was used to label a peptide compound via hydrazinonicotinyl-Tyr3-Octreotide (HYNIC-TOC) and researched further for somatostatin-receptor NETs treatment. The labeling procedure was conducted at 95°C for 10 min. The compound stability was tested in the environment of human serum at 37°C. The biodistribution of compound was investigated in balb/c normal mice and mice bearing AR4-2J tumor. Absorbed Doses of Human Organs were estimated by extrapolation of the biokinetics data of compound in mice to human's organs and then the absorbed doses were estimated by application of MATLAB and MIRDOSE software. Labeling yield was more than 90% with 555 MBq/mg specific activity. The radio-labeled compound expressed well consistency in human serum. The tumor uptake reached 3.831 ID/g% until 4 h post-injection and increased to 5.564%ID/g until 24 h post-injection. The main achievement of this study was high tumor uptake of 46 Sc-HYNIC-TOC which may be therapeutically valuable for the therapy of NETs. The estimation of the absorbed dose of human from 47 Scandium-HYNIC-TOC showed low absorbed doses in critical organs and the elimination of the radiopharmaceutical was through the gastrointestinal tract.

Combination of terbium-161 with somatostatin receptor antagonists\u2014a potential paradigm shift for the treatment of neuroendocrine neoplasms

PurposeThe β¯-emitting terbium-161 also emits conversion and Auger electrons, which are believed to be effective in killing single cancer cells. Terbium-161 was applied with somatostatin receptor (SSTR) agonists that localize in the cytoplasm (DOTATOC) and cellular nucleus (DOTATOC-NLS) or with a SSTR antagonist that localizes at the cell membrane (DOTA-LM3). The aim was to identify the most favorable peptide/terbium-161 combination for the treatment of neuroendocrine neoplasms (NENs).MethodsThe capability of the 161Tb- and 177Lu-labeled somatostatin (SST) analogues to reduce viability and survival of SSTR-positive AR42J tumor cells was investigated in vitro. The radiopeptides’ tissue distribution profiles were assessed in tumor-bearing mice. The efficacy of terbium-161 compared to lutetium-177 was investigated in therapy studies in mice using DOTATOC or DOTA-LM3, respectively.ResultsIn vitro, [161Tb]Tb-DOTA-LM3 was 102-fold more potent than [177Lu]Lu-DOTA-LM3; however, 161Tb-labeled DOTATOC and DOTATOC-NLS were only 4- to fivefold more effective inhibiting tumor cell viability than their 177Lu-labeled counterparts. This result was confirmed in vivo and demonstrated that [161Tb]Tb-DOTA-LM3 was significantly more effective in delaying tumor growth than [177Lu]Lu-DOTA-LM3, thereby, prolonging survival of the mice. A therapeutic advantage of terbium-161 over lutetium-177 was also manifest when applied with DOTATOC. Since the nuclear localizing sequence (NLS) compromised the in vivo tissue distribution of DOTATOC-NLS, it was not used for therapy.ConclusionThe use of membrane-localizing DOTA-LM3 was beneficial and profited from the short-ranged electrons emitted by terbium-161. Based on these preclinical data, [161Tb]Tb-DOTA-LM3 may outperform the clinically employed [177Lu]Lu-DOTATOC for the treatment of patients with NENs.

Simultaneous Visualization of 161Tb- and 177Lu-Labeled Somatostatin Analogues Using Dual-Isotope SPECT Imaging.

The decay of terbium-161 results in the emission of β¯-particles as well as conversion and Auger electrons, which makes terbium-161 interesting for therapeutic purposes. The aim of this study was to use dual-isotope SPECT imaging in order to demonstrate visually that terbium-161 and lutetium-177 are interchangeable without compromising the pharmacokinetic profile of the radiopharmaceutical. The 161Tb- and 177Lu-labeled somatostatin (SST) analogues DOTATOC (agonist) and DOTA-LM3 (antagonist) were tested in vitro to demonstrate equal properties regarding distribution coefficients and cell uptake into SST receptor-positive AR42J tumor cells. The radiopeptides were further investigated in AR42J tumor-bearing nude mice using the method of dual-isotope (terbium-161/lutetium-177) SPECT/CT imaging to enable the visualization of their distribution profiles in the same animal. Equal pharmacokinetic profiles were demonstrated for either of the two peptides, irrespective of whether it was labeled with terbium-161 or lutetium-177. Moreover, the visualization of the sub-organ distribution confirmed similar behavior of 161Tb- and 177Lu-labeled SST analogues. The data were verified in quantitative biodistribution studies using either type of peptide labeled with terbium-161 or lutetium-177. While the radionuclide did not have an impact on the organ distribution, this study confirmed previous data of a considerably higher tumor uptake of radiolabeled DOTA-LM3 as compared to the radiolabeled DOTATOC.

Enhancing the anti-tumour activity of 177Lu-DOTA-octreotate radionuclide therapy in somatostatin receptor-2 expressing tumour models by targeting PARP

Peptide receptor radionuclide therapy (PRRT) is an important treatment option for patients with somatostatin receptor-2 (SSTR2)-expressing neuroendocrine tumour (NET) though tumour regression occurs in only a minority of patients. Therefore, novel PRRT regimens with improved therapeutic activity are needed. Radiation induced DNA damage repair is an attractive therapeutic target to increase PRRT efficacy and consequently, we have characterised a panel of preclinical models for their SSTR2 expression, in vivo growth properties and response to 177Lu-DOTA-octreotate (LuTate) PRRT to identify models with features suitable for evaluating novel therapeutic combinations. In vitro studies using the SSTR2 expressing AR42J model demonstrate that the combination of LuTate and the small molecule Poly(ADP-ribose) polymerase-1 (PARP) inhibitor, talazoparib led to increased DNA double strand breaks, as assessed by γ-H2AX foci formation, as compared to LuTate alone. Furthermore, using the AR42J tumour model in vivo we demonstrate that the combination of LuTate and talazoparib significantly improved the anti-tumour efficacy of LuTate alone. These findings support the clinical evaluation of the combination of LuTate and PARP inhibition in SSTR2-expressing NET.

Peptide Receptor Radionuclide Therapy with 67Cu-CuSarTATE Is Highly Efficacious Against a Somatostatin-Positive Neuroendocrine Tumor Model.

Peptide receptor radionuclide therapy (PRRT) using radiolabeled octreotate is an effective treatment for somatostatin receptor 2-expressing neuroendocrine tumors. The diagnostic and therapeutic potential of 64Cu and 67Cu, respectively, offers the possibility of using a single somatostatin receptor-targeted peptide conjugate as a theranostic agent. A sarcophagine cage amine ligand, MeCOSar (5-(8-methyl-3,6,10,13,16,19-hexaaza-bicyclo[6.6.6]icosan-1-ylamino)-5-oxopentanoic acid), conjugated to (Tyr3)-octreotate, called 64Cu-CuSarTATE, was demonstrated to be an imaging agent and potential prospective dosimetry tool in 10 patients with neuroendocrine tumors. This study aimed to explore the antitumor efficacy of 67Cu-CuSarTATE in a preclinical model of neuroendocrine tumors and compare it with the standard PRRT agent, 177Lu-LuDOTA-Tyr3-octreotate (177Lu-LuTATE). Methods: The antitumor efficacy of various doses of 67Cu-CuSarTATE in AR42J (rat pancreatic exocrine) tumor-bearing mice was compared with 177Lu-LuTATE. Results: Seven days after a single administration of 67Cu-CuSarTATE (5 MBq), tumor growth was inhibited by 75% compared with vehicle control. Administration of 177Lu-LuTATE (5 MBq) inhibited tumor growth by 89%. Survival was extended from 12 d in the control group to 21 d after treatment with both 67Cu-CuSarTATE and 177Lu-LuTATE. In a second study, the efficacy of fractionated delivery of PRRT was assessed, comparing the efficacy of 30 MBq of 67Cu-CuSarTATE or 177Lu-LuTATE, either as a single intravenous injection or as two 15-MBq fractions 2 wk apart. Treatment of tumors with 2 fractions significantly improved survival over delivery as a single fraction (67Cu-CuSarTATE: 47 vs. 36 d [P = 0.036]; 177Lu-LuTATE: 46 vs. 29 d [P = 0.040]). Conclusion: This study demonstrates that 67Cu-CuSarTATE is well tolerated in BALB/c nude mice and highly efficacious against AR42J tumors in vivo. Administration of 67Cu-CuSarTATE and 177Lu-LuTATE divided into 2 fractions over 2 wk was more efficacious than administration of a single fraction. The antitumor activity of 67Cu-CuSarTATE in the AR42J tumor model demonstrated the suitability of this novel agent for clinical assessment in the treatment of somatostatin receptor 2-expressing neuroendocrine tumors.

Fundamental study of radiogallium-labeled aspartic acid peptides introducing octreotate derivatives.

Somatostatin receptors are highly expressed in neuroendocrine tumors, and many radiolabeled somatostatin analogs for diagnosis and treatment have been developed. To simultaneously detect not only primary cancer but also bone metastases, this study aimed to develop a positron emission tomography probe using generator-produced nuclide Gallium-68 (T1/2 = 68min), in which a carrier for primary cancer, a carrier for bone metastases lesions, and a stable gallium complex are introduced into the one molecule. Based on this strategy, the somatostatin receptor-targeted peptide, [Tyr3]-octreotate (TATE), aspartic acid peptide (Dn) with high binding affinity for hydroxyapatite, and Ga-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA) as a stable gallium complex were selected. The novel complexes, Ga-DOTA-Dn-TATE (n = 0, 2, 5, 8, or 11), were designed, synthesized, and evaluated. The radiogallium complexes were prepared using the easy-to-handle radioisotope 67Ga due to relatively long half-life. The radiogallium complex precursor DOTA-Dn-TATE was synthesized by the Fmoc-based solid-phase method and by the air oxidation method to form the disulfide bond. [67Ga]Ga-DOTA-Dn-TATE was synthesized by reacting DOTA-Dn-TATE and 67Ga. Hydroxyapatite binding assays, in vitro cellular uptake experiments in AR42J tumor cells, in biodistribution experiments in AR42J tumor-bearing mice, were performed using [67Ga]Ga-DOTA-Dn-TATE. The radiochemical purities of [67Ga]Ga-DOTA-Dn-TATE were > 96.0%. In in vitro and in vivo experiments, [67Ga]Ga-DOTA-D11-TATE had a high affinity for hydroxyapatite and highly accumulated in bone. However, the uptake of [67Ga]Ga-DOTA-D11-TATE into somatostatin receptor-positive AR42J cells was lower than that of [67Ga]Ga-DOTA-TATE, and the accumulation of [67Ga]Ga-DOTA-D11-TATE in tumor was significantly low. Ga-DOTA-D11-TATE may not be recognized by somatostatin receptor by the introduction of D11, and the charge adjustment may be important for somatostatin receptor-positive cell uptake.

Chelation, formulation, encapsulation, retention, and in vivo biodistribution of hydrophobic nanoparticles labelled with 57Co-porphyrin: Oleylamine ensures stable chelation of cobalt in nanoparticles that accumulate in tumors

Background and motivationWhile small molecules can be used in cancer diagnosis there is a need for imageable diagnostic NanoParticles (NPs) that act as surrogates for the therapeutic NPs. Many NPs are composed of hydrophobic materials so the challenge is to formulate hydrophobic imaging agents. To develop individualized medical treatments based on NP, a first step should be the selection of patients who are likely responders to the treatment as judged by imaging tumor accumulation of NPs. This requires NPs with the same size and structure as the subsequent therapeutic NPs but labelled with a long-lived radionuclide. Cobalt isotopes are good candidates for NP labelling since 55Co has half-life of 17.5 h and positron energy of 570 keV while 57Co (t1/2 271.6 d) is an isotope suited for preclinical single photon emission tomography (SPECT) to visualize biodistribution and pharmacokinetics of NPs. We used the hydrophobic octaethyl porphyrin (OEP) to chelate cobalt and to encapsulate it inside hydrophobic liquid NPs (LNPs). We hypothesized that at least two additional hydrophobic axial ligands (oleylamine, OA) must be provided to the OEP-Co complex in order to encapsulate and retain Co inside LNP. Results1. Cobalt chelation by OEP and OA. The association constant of cobalt to OEP was 2.49 × 105 M−1 and the formation of the hexacoordinate complex OEP-Co-4OA was measured by spectroscopy. 2. NP formulation and characterization: LNPs were prepared by the fast ethanol injection method and were composed of a liquid core (triolein) surrounded by a lipid monolayer (DSPC:Cholesterol:DSPE-PEG2000). The size of the LNPs loaded with the cobalt complex was 40 ± 5 nm, 3. Encapsulation of OEP-Co-OA: The loading capacity of OEP-Co-OA in LNP was 5 mol%. 4. Retention of OEP-57Co-4OA complex in the LNPs: the positive effect of the OA ligands was demonstrated on the stability of the OEP-57Co-4OA complex, providing a half-life for retention in PBS of 170 h (7 days) while in the absence of the axial OA ligands was only 22 h. 5 Biodistribution Study: the in vivo biodistribution of LNP was studied in AR42J pancreatic tumor-bearing mice. The estimated half-life of LNPs in blood was about 7.2 h. Remarkably, the accumulation of LNPs in the tumor was as high as 9.4% ID/g 24 h after injection with a doubling time for tumor accumulation of 3.22 h. The most important result was that the nanoparticles could indeed accumulate in the AR42J tumors up to levels greater than those of other NPs previously measured in the same tumor model, and at about half the values reported for the molecular agent 57Co-DOTATATE. ConclusionsThe additional hydrophobic chelator OA was indeed needed to obtain a stable octahedral OEP-Co-4OA. Cobalt was actually well-retained inside LNP in the OEP-Co-4OA complex. The method described in the present work for the core-labelling of LNPs with cobalt is now ready for labeling of NPs with 55Co, or indeed other hexadentate radionuclides of interest for preclinical in vivo PET-imaging and radio-therapeutics.

Radiosynthesis and Preclinical Evaluation of 18F-Fluoroglycosylated Octreotate for Somatostatin Receptor Imaging.

Short synthetic octapeptide analogs derived from the native somatostatin peptides SST-14 and SST-28, namely, octreotate (TATE) or octreotide (TOC), bind with high affinity to somatostatin receptors (sstr), mainly to subtypes 2 and 5, which are expressed in high density on neuroendocrine tumors (NET). Therefore, radiolabeled TATE or TOC derivatives represent highly valuable imaging probes for NET diagnosis by positron emission tomography (PET). The aim of our study was the development of an 18F-labeled octreotate analog as an alternative radiotracer for the clinically established 68Ga-DOTATOC and 68Ga-DOTATATE. We applied our previously developed method based on copper(I)-catalyzed azide-alkyne cycloaddition (CuAAC) to the radiosynthesis of 18F-fluoroglycosylated TATE ([18F]FGlc-TATE). [18F]FGlc-TATE was obtained in high yields of 19-22% (non-decay-corrected, referred to [18F]fluoride) and in high specific activities of 32-106 GBq/μmol. [18F]FGlc-TATE showed high affinity to sstr expressed on AR42J cells (IC50 = 4.2 nM) with fast and high internalization, and a beneficial logD7.4 of -1.8. In AR42J tumor bearing nude mice, [18F]FGlc-TATE showed high and specific tumor uptake of 5.6%ID/g at 60 min post-injection, as determined by blocking experiments using octreotide, and fast clearance from other organs, resulting in excellent tumor-to-blood ratios increasing from 9 to 17 from 30 to 60 min post-injection. Small animal PET studies revealed high uptake of [18F]FGlc-TATE in the tumor which could be blocked with octreotide by >99%. Overall, [18F]FGlc-TATE revealed excellent in vitro and in vivo properties and is therefore a viable alternative 18F-labeled radiopeptide for imaging somatostatin receptor-positive tumors by PET.

An assessment tumor targeting ability of 177Lu labeled cyclic CCK analogue peptide by binding with cholecystokinin receptor

The cholecystokinin (CCK) receptor is known as a receptor that is overexpressed in many human tumors. The present study was designed to investigate the targeting ability of cyclic CCK analogue in AR42J pancreatic cells. The CCK analogues, DOTA-K(glucose)-Gly-Trp-Nle-Asp-Phe (DOTA-glucose-CCK) and DOTA-Nle-cyclo(Glu-Trp-Nle-Asp-Phe-Lys-NH2) (DOTA-[Nle]-cCCK), were synthesized and radiolabeled with 177Lu, and competitive binding was evaluated. The binding appearance of synthesized peptide with AR42J cells was evaluated by confocal microscopy. And bio-distribution was performed in AR42J xenografted mice. Synthesized peptides were prepared by a solid phase synthesis method, and their purity was over 98%. DOTA is the chelating agent for 177Lu-labeling, in which the peptides were radiolabeled with 177Lu by a high radiolabeling yield. A competitive displacement of 125I-CCK8 on the AR42J cells revealed that the 50% inhibitory concentration value (IC50) was 12.3 nM of DOTA-glucose-CCK and 1.7 nM of DOTA-[Nle]-cCCK. Radio-labeled peptides were accumulated in AR42J tumor in vivo, and %ID/g of the tumor was 0.4 and 0.9 at 2 h p.i. It was concluded that 177Lu-DOTA-[Nle]-cCCK has higher binding affinity than 177Lu-DOTA-glucose-CCK and can be a potential candidate as a targeting modality for a CCK receptor over-expressing tumors.

Preclinical in vivo application of (152)Tb-DOTANOC: a radiolanthanide for PET imaging.

BackgroundTerbium has attracted the attention of researchers and physicians due to the existence of four medically interesting radionuclides, potentially useful for SPECT and PET imaging, as well as for α- and β−-radionuclide therapy. The aim of this study was to produce 152Tb (T1/2 = 17.5 h, Eβ+av = 1140 keV) and evaluate it in a preclinical setting in order to demonstrate its potential for PET imaging. For this purpose, DOTANOC was used for targeting the somatostatin receptor in AR42J tumor-bearing mice.Methods152Tb was produced by proton-induced spallation of tantalum targets, followed by an online isotope separation process at ISOLDE/CERN. After separation of 152Tb using cation exchange chromatography, it was directly employed for radiolabeling of DOTANOC. PET/CT scans were performed with AR42J tumor-bearing mice at different time points after injection of 152Tb-DOTANOC which was applied at variable molar peptide amounts. 177Lu-DOTANOC was prepared and used in biodistribution and SPECT/CT imaging studies for comparison with the PET results.ResultsAfter purification, 152Tb was obtained at activities up to ~600 MBq. Radiolabeling of DOTANOC was achieved at a specific activity of 10 MBq/nmol with a radiochemical purity >98 %. The PET/CT scans of mice allowed visualization of AR42J tumor xenografts and the kidneys, in which the radiopeptide was accumulated. After injection of large peptide amounts, the tumor uptake was reduced as compared to the result after injection of small peptide amounts. PET images of mice, which received 152Tb-DOTANOC at small peptide amounts, revealed the best tumor-to-kidney ratios. The data obtained with 177Lu-DOTANOC in biodistribution and SPECT/CT imaging studies confirmed the 152Tb-based PET results.ConclusionsProduction of 30-fold higher quantities of 152Tb as compared to the previously performed pilot study was feasible. This allowed, for the first time, labeling of a peptide at a reasonable specific activity and subsequent application for in vivo PET imaging. As a β+-particle-emitting radiolanthanide, 152Tb would be of distinct value for clinical application, as it may allow exact prediction of the tissue distribution of therapeutic radiolanthanides.Electronic supplementary materialThe online version of this article (doi:10.1186/s13550-016-0189-4) contains supplementary material, which is available to authorized users.