Comparative evaluation of the new GRPR-antagonist 111 In-SB9 and 111 In-AMBA in prostate cancer models: Implications of in vivo stability.

Gastrin-releasing peptide receptors (GRPRs) are overexpressed in prostate cancer, representing attractive targets for diagnosis and therapy with bombesin (BBN)-like radioligands. GRPR-antagonists have lately attracted much attention owing to inherent biosafety and favorable pharmacokinetics. We herein present the GRPR-antagonist SB9 structurally resembling the known BBN-based agonist AMBA (SB9 = [Leu13 NHEt-desMet14 ]AMBA). The profiles of 111 In-SB9 and 111 In-AMBA were directly compared in PC-3 cells and tumor-bearing mice. SB9 and AMBA displayed high GRPR affinities. 111 In-AMBA strongly internalized in PC-3 cells, while 111 In-SB9 remained bound on the cell surface showing a typical GRPR-radioantagonist profile. 111 In-SB9 was more stable than 111 In-AMBA, but coinjection of the neprilysin (NEP) inhibitor phosphoramidon (PA) stabilized both in vivo. The radioligands displayed high tumor uptake (20.23 ± 3.41 %ID/g and 18.53 ± 1.54 %ID/g, respectively, at 4 hours pi), but 111 In-SB9 washed faster from background. PA coinjection led to significant increase of tumor uptake, combined with better clearance for 111 In-SB9. In short, this study has revealed superior pharmacokinetics and higher stability for the GRPR-antagonist 111 In-SB9 vs the corresponding agonist 111 In-AMBA consolidating previous evidence that GRPR antagonists are preferable to agonists for tumor imaging and therapy. It has also demonstrated that further pharmacokinetic improvements were feasible by in situ metabolic radioligand stabilization using PA.

Antitumor efficacy of Auger electron emitter 111In delivered by modular nanotransporter into the nuclei of cells with folate receptor overexpression.

A new modular nanotransporter (MNT) for the delivery of anticancer agents into the nuclei of cells with folate receptor overexpression was created. An effective method for acceding labeling of this MNT with Auger electron emitter 111In has been developed. A significant therapeutic effect was observed after a single intratumoral injection of the new 111In-labeled MNT to mice grafted with human cervical carcinoma characterized by folate receptor overexpression.

Predictive patient-specific dosimetry and individualized dosing of pretargeted radioimmunotherapy in patients with advanced colorectal cancer.

PURPOSE: Pretargeted radioimmunotherapy (PRIT) with bispecific antibodies (bsMAb) and a radiolabeled peptide reduces the radiation dose to normal tissues. Here we report the accuracy of an (111)In-labeled pretherapy test dose for personalized dosing of (177)Lu-labeled IMP288 following pretargeting with the anti-CEA × anti-hapten bsMAb, TF2, in patients with metastatic colorectal cancer (CRC). METHODS: In 20 patients bone marrow absorbed doses (BMD) and doses to the kidneys were predicted based on blood samples and scintigrams acquired after (111)In-IMP288 injection for individualized dosing of PRIT with (177)Lu-IMP288. Different dose schedules were studied, varying the interval between the bsMAb and peptide administration (5 days vs. 1 day), increasing the bsMAb dose (75 mg vs. 150 mg), and lowering the peptide dose (100 µg vs. 25 µg). RESULTS: TF2 and (111)In/(177)Lu-IMP288 clearance was highly variable. A strong correlation was observed between peptide residence times and individual TF2 blood concentrations at the time of peptide injection (Spearman's ρ = 0.94, P < 0.0001). PRIT with 7.4 GBq (177)Lu-IMP288 resulted in low radiation doses to normal tissues (BMD <0.5 Gy, kidney dose <3 Gy). Predicted (177)Lu-IMP288 BMD were in good agreement with the actual measured doses (mean ± SD difference -0.0026 ± 0.028 mGy/MBq). Hematological toxicity was mild in most patients, with only two (10 %) having grade 3-4 thrombocytopenia. A correlation was found between platelet toxicity and BMD (Spearman's ρ = 0.58, P = 0.008). No nonhematological toxicity was observed. CONCLUSION: These results show that individual high activity doses in PRIT in patients with CEA-expressing CRC could be safely administered by predicting the radiation dose to red marrow and kidneys, based on dosimetric analysis of a test dose of TF2 and (111)In-IMP288.

[Additional external radiotherapy to multiple metastases originating from thymic neuroendocrine tumor following peptide receptor radionuclide therapy: a case report].

We describe the case of a 60-year-old man suffering from an advanced thymic neuroendocrine tumor with left supraclavicular lymph node and multiple bone metastases. The patient initially underwent systemic therapy with somatostatin analogues. Thereafter, peptide receptor radionuclide therapy (PRRT) was considered because the lesions had remained stable despite the pharmacological therapy. PRRT was performed 10 months after the initial treatment in a European hospital. Eighteen months after the treatment, cranial nerve palsy arising from skull base metastases and Horner's syndrome induced by left supraclavicular lymph node metastases became exacerbated. Therefore, a course of external radiotherapy was performed with palliative intent in our hospital. During the radiotherapy planning, the biodistribution of 111In-octreotide was examined to determine whether the absorbed dose of the previous PRRT was acceptable. As a result, external radiotherapy was performed, and an acute radiation reaction was observed; the severity of the reaction was typical of reactions to neck radio-therapy. The treatment course of the present case was considered to be instructive because PRRT cannot be performed in Japan at present.

H(4)octapa-trastuzumab: versatile acyclic chelate system for 111In and 177Lu imaging and therapy.

A bifunctional derivative of the versatile acyclic chelator H4octapa, p-SCN-Bn-H4octapa, has been synthesized for the first time. The chelator was conjugated to the HER2/neu-targeting antibody trastuzumab and labeled in high radiochemical purity and specific activity with the radioisotopes (111)In and (177)Lu. The in vivo behavior of the resulting radioimmunoconjugates was investigated in mice bearing ovarian cancer xenografts and compared to analogous radioimmunoconjugates employing the ubiquitous chelator 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA). The H4octapa-trastuzumab conjugates displayed faster radiolabeling kinetics with more reproducible yields under milder conditions (15 min, RT, ~94-95%) than those based on DOTA-trastuzumab (60 min, 37 °C, ~50-88%). Further, antibody integrity was better preserved in the (111)In- and (177)Lu-octapa-trastuzumab constructs, with immunoreactive fractions of 0.99 for each compared to 0.93-0.95 for (111)In- and (177)Lu-DOTA-trastuzumab. These results translated to improved in vivo biodistribution profiles and SPECT imaging results for (111)In- and (177)Lu-octapa-trastuzumab compared to (111)In- and (177)Lu-DOTA-trastuzumab, with increased tumor uptake and higher tumor-to-tissue activity ratios.

Simultaneous dual-isotope SPECT/CT with (99m)Tc- and (111)In-labelled albumin microspheres in treatment planning for SIRT.

OBJECTIVES: To investigate simultaneous dual-isotope SPECT/CT with two differently radioisotope-labelled albumin-microsphere fractions for treatment planning of hepatic radioembolisation. METHODS: In addition to (99m)Technetium-labelled albumin microspheres (commercially available), we performed labelling with (111)Indium. Binding stability of (111)Indium-labelled microspheres was tested in vitro and in vivo in mice. Simultaneous dual-isotope SPECT/CT imaging was validated in an anthropomorphic torso phantom; subsequently, dual-isotope SPECT/CT was performed under in-vivo conditions in pigs (n = 3) that underwent transarterial injection of (99m)Technetium- and (111)Indium-labelled microspheres in the liver (right and left hepatic artery, respectively), in both kidneys and in the gluteal musculature. In total, n = 18 transarterial injections were performed. RESULTS: In-vitro testing and in-vivo studies in mice documented high binding stability for both (99m)Technetium-labelled and (111)Indium-labelled microsphere fractions. In phantom studies, simultaneous dual-isotope SPECT/CT enabled reliable separation of both isotopes. In pigs, the identified deposition of both isotopes could be accurately matched with intended injection targets (100 %, 18/18 intended injection sites). Furthermore, an incidental deposition of (99m)Technetium-labelled microspheres in the stomach could be correlated to the test injection into a right hepatic artery. CONCLUSION: Simultaneous dual-isotope SPECT/CT after transarterial injection with (99m)Technetium- and (111)Indium-labelled microspheres is feasible. Thus, it may offer additional, valuable information compared to single (99m)Technetium-labelled albumin examinations. KEY POINTS: • Simultaneous dual-isotope SPECT/CT with (111) In- and (99m) Tc-labelled albumin microspheres is feasible. • Differentiation of two microsphere fractions after transarterial injection is possible. • The origin of an extra-hepatic microsphere deposition can be correlated to the corresponding artery. • This technique could reduce the setup time for selective internal radiation treatment.

Development of Novel 111In/225Ac-Labeled Agent Targeting PSMA for Highly Efficient Cancer Radiotheranostics.

Prostate-specific membrane antigen (PSMA) is a promising target for metastatic castration-resistant prostate cancer. We previously reported the effectiveness of PSMA-DA1 as a PSMA-targeting radiotheranostic agent containing an albumin binder moiety. To further enhance tumor uptake, we newly designed PSMA-NAT-DA1 (PNT-DA1) by the introduction of a lipophilic linker into PSMA-DA1. The PSMA affinity of [111In]In-PNT-DA1 was increased (Kd = 8.20 nM) compared with that of [111In]In-PSMA-DA1 (Kd = 89.4 nM). [111In]In-PNT-DA1 showed markedly high tumor accumulation (131.6% injected dose/g at 48 h post-injection), and [111In]In-PNT-DA1 enabled the visualization of the tumor clearly at 24 h post-injection with SPECT/CT imaging. The administration of [225Ac]Ac-PNT-DA1 (2.5 kBq) led to shrinkage of the tumor without marked toxicity, and the antitumor effects of [225Ac]Ac-PNT-DA1 were superior to those of [225Ac]Ac-PSMA-DA1 and [225Ac]Ac-PSMA-617, which is the current gold standard for PSMA-targeting 225Ac-endoradiotherapy. These results suggest that the combination of [111In]In-PNT-DA1 and [225Ac]Ac-PNT-DA1 comprises a promising method of PSMA-targeting radiotheranostics.

¹¹¹In-Bn-DTPA-nimotuzumab with/without modification with nuclear translocation sequence (NLS) peptides: an Auger electron-emitting radioimmunotherapeutic agent for EGFR-positive and trastuzumab (Herceptin)-resistant breast cancer.

Increased expression of epidermal growth factor receptors (EGFR) in breast cancer (BC) is often associated with trastuzumab (Herceptin)-resistant forms of the disease and represents an attractive target for novel therapies. Nimotuzumab is a humanized IgG(1) monoclonal antibody that is in clinical trials for treatment of EGFR-overexpressing malignancies. We show here that nimotuzumab derivatized with benzylisothiocyanate diethylenetriaminepentaacetic acid for labelling with the subcellular range Auger electron-emitter, (111)In and modified with nuclear translocation sequence (NLS) peptides ((111)In-NLS-Bn-DTPA-nimotuzumab) was bound, internalized and transported to the nucleus of EGFR-positive BC cells. Emission of Auger electrons in close proximity to the nucleus caused multiple DNA double-strand breaks which diminished the clonogenic survival (CS) of MDA-MB-468 cells that have high EGFR density (2.4 × 10(6) receptors/cell) to less than 3 %. (111)In-Bn-DTPA-nimotuzumab without NLS peptide modification was sevenfold less effective for killing MDA-MB-468 cells. (111)In-Bn-DTPA-nimotuzumab with/without NLS peptide modification were equivalently cytotoxic to MDA-MB-231 and TrR1 BC cells that have moderate EGFR density (5.4 × 10(5) or 4.2 × 10(5) receptors/cell, respectively) reducing their CS by twofold. MDA-MB-231 cells have intrinsic trastuzumab resistance due to low HER2 density, whereas TrR1 cells have acquired resistance despite HER2 overexpression. Biodistribution and microSPECT/CT imaging revealed that (111)In-NLS-Bn-DTPA-nimotuzumab exhibited more rapid elimination from the blood and lower tumour uptake than (111)In-Bn-DTPA-nimotuzumab. Tumour uptake of the radioimmunoconjugates in mice with MDA-MB-468 xenografts was high (8-16 % injected dose/g) and was blocked by administration of an excess of unlabelled nimotuzumab, demonstrating EGFR specificity. We conclude that (111)In-Bn-DTPA-nimotuzumab with/without NLS peptide modification are promising Auger electron-emitting radioimmunotherapeutic agents for EGFR-positive BC, but (111)In-Bn-DTPA-nimotuzumab may be preferred due to its higher tumour uptake in vivo.

Feasibility of bremsstrahlung dosimetry for direct dose estimation in patients undergoing treatment with 90Y-ibritumomab tiuxetan.

PURPOSE: Radioimmunotherapy with (90)Y-ibritumomab tiuxetan has been used successfully used in the treatment of CD20-positive non-Hodgkin's lymphoma (NHL). Pretherapy imaging with (111)In-ibritumomab tiuxetan has been used in provisional dosimetry studies. Posttherapy imaging of (90)Y-ibritumomab tiuxetan for clinical use is appealing as it would simplify the data acquisition process and allow measurements of actual doses absorbed during treatment. METHODS: The study included 29 patients with non-Hodgkin's lymphoma, of whom 16 (group I) received a pretherapy (111)In-ibritumomab tiuxetan diagnostic study and (90)Y-ibritumomab tiuxetan treatment 1 week later, and 13 (group II) received only (90)Y-ibritumomab tiuxetan treatment. Planar imaging and blood sampling were performed in all patients. The doses absorbed by organs at risk were calculated using a whole-body average attenuation correction factor (relative dosimetry approach) and, in the case of the (111)In-ibritumomab tiuxetan image sets, also using organ-specific attenuation correction factors (absolute dosimetry method). Red marrow absorbed doses were based on gamma counting of blood samples. RESULTS: The estimated red marrow absorbed doses from (111)In and (90)Y data were equivalent. In all cases, the doses absorbed by organs at risk were found to be within prescribed limits. The relative dosimetry approach applied to both the (90)Y and (111)In data significantly underestimated the doses relative to those obtained with the (111)In absolute dosimetry method which is generally accepted as the reference method (MIRD 16). In the case of (111)In, the relative dosimetry approach values were highly correlated (R(2) = 0.61) with the reference method values. Relative dosimetry estimates may be adjusted multiplying by a correction factor of 2.8. The (90)Y-ibritumomab tiuxetan relative dosimetry data correlated poorly with the reference method values (R (2) = 0.02). CONCLUSION: Based on patient-specific dosimetry, the administered activity may be increased by an average factor of 2.4, indicating that most patients could be undertreated. The relative dosimetry approach based on planar imaging largely underestimates doses relative to reference values. Dosimetry based on planar bremsstrahlung imaging is not a dependable alternative to (111)In dosimetry.

Comprehensive evaluation of a somatostatin-based radiolabelled antagonist for diagnostic imaging and radionuclide therapy.

PURPOSE: Targeting of tumours positive for somatostatin receptors (sst) with radiolabelled peptides is of interest for tumour localization, staging, therapy follow-up and targeted radionuclide therapy. The peptides used clinically are exclusively agonists, but recently we have shown that the radiolabelled somatostatin-based antagonist (111)In-DOTA-sst2-ANT may be preferable to agonists. However, a comprehensive study of this radiolabelled antagonist to determine its significance was lacking. The present report describes the evaluation of this novel antagonist labelled with (111)In and (177)Lu in three different tumour models. METHODS: Radiopeptide binding, internalization and dissociation studies were performed using cells expressing HEK293-rsst(2). Biodistribution studies were performed in HEK293-rsst(2), HEK293-hsst(2) and HEK293-rsst(3) xenografted mice. RESULTS: Saturation binding analysis confirmed earlier IC(50) data for (111/nat)In-DOTA-sst2-ANT and showed similar affinity of (177/nat)Lu-DOTA-sst2-ANT for the sst(2). Only low internalization was found in cell culture (6.68 ± 0.06 % at 4 h), which was not unexpected for an antagonist, and this could be further reduced by the addition of sucrose. No internalization was observed in HEK293 cells not expressing sst. Both results indicate that the internalization was specific. (111)In-DOTA-sst2-ANT and (177)Lu-DOTA-sst2-ANT were shown to target tumour xenografts expressing the rat and the human sst(2) receptor with no differences in their uptake or pharmacokinetics. The uptake in rsst(2) and hsst(2) was high (about 30 %IA/g 4 h after injection) and surprisingly long-lasting (about 20-23 %IA/g 24 h after injection). Kidney uptake was blocked by approximately 50 % by lysine or Gelofusine. CONCLUSION: These results indicate that radiolabelled somatostatin-based antagonists may be superior to corresponding agonists. The long tumour retention time of (177)Lu-DOTA-sst2-ANT indicates that this new class of compounds is of relevance not only in diagnostic imaging but also in targeted radionuclide therapy of sst-positive tumours.

Assessment of Cell Cytotoxicity and Comet Assay on HER2/neu Positive Cell Line Due to 111In Auger Electrons as DNA-Targeting Radioimmunoconjugate.

BACKGROUND: Breast cancer Auger electron therapy is a growing field of study in radioimmunotherapy and oncology research. Trastuzumab, a high affinity-binding monoclonal antibody against HER2/neu is which is over-expressed in breast tumors, is used in radiopharmaceutical development. OBJECTIVES: In this work, the lethal effects of 111In3+, 111In-DTPA-trastuzumab and 111In-trastuzumab coupled-nuclear localizing sequence peptide (111In-DTPA-NLS-trastuzumab) on malignant cells were studied in vitro. METHODS: DTPA-NLS-trastuzumab was prepared using sulfosuccinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (sulfo-SMCC) conjugation with NLS peptide in the first step, followed by conjugation with diethylenetriaminepentaacetic acid (DTPA). Both DTPA-trastuzumab and DTPA- NLS-trastuzumab were labeled with 111In followed by purification and quality control techniques. Sk-Br-3 (a HER2/neu+ cell line), was used in the cell viability assessment assay for 111In, 111In-DTPA-trastuzumab and 111In-DTPA-NLS-trastuzumab (3.7 MBq) at 37 ºC. The cytotoxicity of the three species was studied using MTT and comet assay was utilized DNA damage detection. RESULTS: A significant radiochemical purity for 111In-DTPA-NLS-trastuzumab (99.36% ± 0.30%, ITLC) at the DTPA:antibody ratio of 6.90 ± 0.34:1, was obtained. Significant cell viability difference was found for 111In-DTPA-NLS-trastuzumab compared to the other treatments at two-time points. In addition, comet assay demonstrated significant DNA damage at 144 h using 111In-DTPA- NLS-trastuzumab. CONCLUSION: The results of cell viability and cell death using MTT assay and comet assay, respectively, demonstrate the NLS-peptide effectively facilitates 111In-trastuzumab transport into the HER2/neu positive cancer cell nuclei to impose the radiotherapeutic effects of Auger electrons on DNA leading to cell death.

Adjuvant radioimmunotherapy improves survival of rats after resection of colorectal liver metastases.

OBJECTIVE: The aim of this study was to test the hypothesis that adjuvant radioimmunotherapy (RIT) prevents recurrent liver metastases and/or results in improved survival after tumorectomy in an experimental model. BACKGROUND: Although partial hepatectomy can improve 5-year survival of patients with colorectal liver metastases up to 58%, recurrent tumor growth in the liver occurs frequently. Radioimmunotherapy using radiolabeled monoclonal antibodies directed against tumor-associated antigens is considered most suited for treating minimal residual disease and could therefore serve as an adjuvant after surgery. METHODS: Liver metastases were induced in male Wag/Rij rats by a mini-laparotomy with intrahepatic injection of 0.3 × 106 CC531 tumor cells. The biodistribution of the radiolabeled monoclonal antibody MG1, directed against a 80-kDa cell surface antigen on CC531 tumors, in this model was determined at 1, 3, and 7 days after intravenous administration. The therapeutic efficacy of 177Lu-MG1 was compared with that of a sham antibody (UPC10), labeled with the same activity dose of Lu-177, and saline only. Radioimmunotherapy was administered either at the day of the tumorectomy (day 14 after tumor cell inoculation) or 7 days later. Primary endpoint was survival. RESULTS: Radiolabeled MG1 preferentially accumulated in tumor lesions in the liver reaching a maximum 3 days postinjection (8.7 ± 0.6% injected dose per gram). Both the administration of 177Lu-MG1 and 177Lu-UPC10 resulted in a transient decrease in body weight. No other signs of clinical discomfort were registered. The survival curves of the group that received 177Lu-UPC10 and the group that received saline only did not differ (P=0.886). Administration of RIT immediately after surgery improved survival compared to administration of the control antibody (hazard ratio [HR], 1.54; P = 0.051), which was even more pronounced when survival was adjusted for the weight of the resected tumor (HR, 1.71; P = 0.027). A therapeutic efficacy of delayed treatment seemed likely (HR, 2.34; P = 0.055). Survival after early administration did not differ from delayed administration (HR, 1.16; P = 0.763). CONCLUSION: This study provides proof of principle that RIT can be an effective adjuvant treatment modality after surgical treatment of colorectal liver metastases.

Preclinical animal research on therapy dosimetry with dual isotopes.

Preclinical research into radionuclide therapies based on radiation dosimetry will enable the use of any LET-equivalent radionuclide. Radiation dose and dose rate have significant influence on dose effects in the tumour depending on its radiation sensitivity, possibilities for repair of sublethal damage, and repopulation during or after the therapy. Models for radiation response of preclinical tumour models after peptide receptor radionuclide therapy based on the linear quadratic model are presented. The accuracy of the radiation dose is very important for observation of dose-effects. Uncertainties in the radiation dose estimation arise from incomplete assay of the kinetics, low accuracy in volume measurements and absorbed dose S-values for stylized models instead of the actual animal geometry. Normal dose uncertainties in the order of 20% might easily make the difference between seeing a dose-effect or missing it altogether. This is true for the theoretical case of a homogeneous tumour type behaving in vivo in the same way as its cells do in vitro. Heterogeneity of tumours induces variations in clonogenic cell density, radiation sensitivity, repopulation capacity and repair kinetics. The influence of these aspects are analysed within the linear quadratic model for tumour response to radionuclide therapy. Preclinical tumour models tend to be less heterogenic than the clinical conditions they should represent. The results of various preclinical radionuclide therapy experiments for peptide receptor radionuclide therapy are compared to the outcome of theoretical models and the influence of increased heterogeneity is analysed when the results of preclinical research is transferred to the clinic. When the radiation dose and radiobiology of the tumour response is known well enough it may be possible to leave the current phenomenological approach in preclinical radionuclide therapy and start basing these experiments on radiation dose. Then the use of a gamma ray-emitting radionuclides for a chemically comparable beta-particle-emitting paired isotope for therapy evaluation would be feasible.

Two 9°Y-labeled multimeric RGD peptides RGD4 and 3PRGD2 for integrin targeted radionuclide therapy.

We have recently developed a series of new Arg-Gly-Asp (RGD) dimeric peptides for specific targeting of integrin α(v)ß3 with enhanced tumor uptake and improved pharmacokinetics. In this study, we investigated 9°Y-labeled RGD tetramer (RGD4) and the new type of RGD dimer (3PRGD2), for the radionuclide therapy of integrin α(v)ß3-positive tumors. Biodistribution and gamma imaging studies of ¹¹¹In labeled RGD4 and 3PRGD2 were performed. Groups of nude mice were used to determine maximum tolerated dose (MTD) of 9°Y-DOTA-RGD4 and 9°Y-DOTA-3PRGD2. The radionuclide therapeutic efficacy of 9°Y-DOTA-RGD4 and 9°Y-DOTA-3PRGD2 was evaluated in U87MG tumor-bearing nude mice. The U87MG tumor uptake of ¹¹¹In-DOTA-3PRGD2 was slightly lower than that of the ¹¹¹In-DOTA-RGD4 (e.g., 6.13 ± 0.82%ID/g vs 6.43 ± 1.6%ID/g at 4 h postinjection), but the uptake of ¹¹¹In-DOTA-3PRGD2 in normal organs, such as liver and kidneys, was much lower than that of ¹¹¹In-DOTA-RGD4, which resulted in much higher tumor-to-nontumor ratios and lower toxicity. The MTD of 9°Y-DOTA-RGD4 in nude mice is less than 44.4 MBq, while the MTD of 9°Y-DOTA-3PRGD2 in mice is more than 55.5 MBq. 9°Y-DOTA-3PRGD2 administration exhibited a similar tumor inhibition effect as compared with 9°Y-DOTA-RGD4 at the same dose. The tumor vasculature in the 9°Y-DOTA-3PRGD2 treatment group was much less than the control groups. Radionuclide therapy studies exhibited that both 9°Y-DOTA-RGD4 and 9°Y-DOTA-3PRGD2 caused significant tumor growth delay in the U87MG tumor model. Compared to 9°Y-DOTA-RGD4, the low accumulation of 9°Y-DOTA-3PRGD2 in normal organs led to lower toxicity and higher MTD in nude mice, which would make it more suitable for high dose or multiple-dose regimens, in order to achieve maximum therapeutic efficacy.

[Somatostatin receptor endoradiotherapy of neuroendocrine tumors: experience in Hungarian patients]. / Neuroendokrin daganatok szomatosztatinreceptor-endoradioterápiája: hazai betegeken szerzett tapasztalatok.

Beside conventional therapies for the treatment of neuroendocrine tumors, a new therapeutical approach, peptide receptor radionuclide therapy has been developed recently. There are two important features which make this therapy feasible: somatostatin receptors are strongly over-expressed in most neuroendocrine tumors resulting in a high tumor-to-background ratio and internalization of the somatostatin-receptor complex in neuroendocrine cells. Due to these features, neuroendocrine tumors can be treated with radiolabelled somatostatin analogues. For peptide receptor radionuclide therapy, somatostatin analogues are conjugated to a chelator that can bind a radionuclide. The most frequently used radionuclides for neuroendocrine tumor treatment are the ß-emitter Yttrium-90 (9°Y) and the ß+γ emitter Lutetium-177 (¹77Lu). Candidates for somatostatin receptor endoradiotherapy are patients with progressive, metastatic, somatostatin-receptor positive neuroendocrine tumors. Many patients have been successively treated with this approach: according to international results major remission can be achieved in 25% of the cases. Although this therapy is still unavailable in Hungary, Hungarian patients can be treated with somatostatin receptor endoradiotherapy with financial support from the National Health Fund in a co-operation with the University of Basel since 2005. During the past 5 years, 51 Hungarian patients have been treated with this therapy. This review briefly summarizes the theoretical background, indications, effectiveness and side effects of somatostatin receptor endoradiotherapy and the authors present the first data obtained from Hungarian patients.

Multifunctional block copolymer micelles for the delivery of 111In to EGFR-positive breast cancer cells for targeted Auger electron radiotherapy.

Block copolymer micelles (BCMs) can improve the payload delivery of therapeutic agents to tumors. Our aim was to construct hEGF-modified BCMs for the delivery of 111In to tumor cells for Auger electron-emission radiotherapy of EGFR-positive breast cancer (BC). Multifunctional nanosized BCMs were prepared from MePEG(2500)-b-PCL(1200) and 111In-DTPA-PEG(3000)-b-PCL(1600) with or without hEGF-PEG(2900)-b-PCL(1400) (111In-hEGF-BCMs or 111In-BCMs). The resulting BCMs were analyzed by dynamic light scattering and transmission electron microscopy. Cellular uptake and nuclear importation were assessed in MDA-MB-468, MDA-MB-231 and MCF-7 BC cells with decreasing EGFR density. In vitro antiproliferative effects were evaluated using the WST-1 assay after 48 h with 111In-hEGF-BCMs, and the clonogenic assay was used to determine the survival fraction (SF) after a 21 h exposure. Results were compared with 111In-DTPA-hEGF, an established Auger electron-emitting radiotherapeutic that is currently in clinical development. Cell uptake and nuclear importation of 111In-hEGF-BCMs decreased in the following order: MDA-MB-468 > MDA-MB-231 > MCF-7. Cellular uptake of 111In-hEGF-BCMs was less than 111In-DTPA-hEGF (P < 0.05) but was 4-fold higher than for 111In-BCMs (P < 0.001). There was a significant growth inhibition of MDA-MB-468 cells by 111In-hEGF-BCMs (6-fold inhibition, P < 0.05) while the growth of MDA-MB-231 and MCF-7 were not significantly inhibited. The SF of MDA-MB-468 cells was decreased to 2.6% while that for MCF-7 cells was 132.7%. 111In-DTPA-hEGF reduced the SF of MDA-MB-468 cells to 0.4%. Nontargeted 111In-BCMs had minimal effect on the SF of BC cells. Therefore, the 111In-hEGF-BCMs were bound, internalized and transported to the nuclei of EGFR-positive BC cells, where the Auger electron emissions were lethal. The 111In-hEGF-BCMs are a promising delivery system for targeted radiotherapy of BC.

Development of anti-CD30 radioimmunoconstructs (RICs) for treatment of Hodgkin's lymphoma. Studies with cell lines and animal studies.

OBJECTIVES: Comparison of the binding affinity to a CD30-positive Hodgkin lymphoma (HL) cell line and biodistribution in HL bearing mice of new anti-CD30 radioimmunoconjugates (RICs) of varying structure and labelling nuclides. METHODS: The antibodies Ki-4 and 5F11 were radioiodinated by the chloramine T method or labelled with (111)In via p-NCS-Benzyl-DOTA. In addition, the Ki-4-dimer was investigated in the iodinated form. The RICs were analyzed for retained immunoreactivity by immunochromatography. In-vitro binding studies were performed on CD30-positive L540 cell lines. For in-vivo biodistribution studies, SCID mice bearing human HL xenografts were injected with the various radioimmunoconjugates. After 24 h, activities in the organs and tumour were measured for all 5 RICs. Tumour-free animals were studied in the same way with (131)I- Ki-4 24 h p. i. The three RICs with the highest tumour/background ratios 24 h p.i. ((131)I-Ki-4, (131)I-5F11, (111)In-bz-DOTA-Ki-4) were analysed further at 48 h and 72 h. RESULTS: All the RICs were successfully labelled with high specific activities (28-47 TBq/mmol) and sufficient radiochemical yields (>80%). Scatchard plot analysis proved high tumour affinity (KD = 20-220 nmol/l). In-vivo tumour accumulation in % of injected dose per g tissue (%ID/g) lay between 2.6 ((131)I-5F11) and 12.3 % ID/g ((131)I-Ki-4) with permanently high background in blood. Tumour/blood-ratios of all RICs were below one at all time points. CONCLUSIONS: In-vitro tumour cell affinities of all RICs were promising. However, in-vivo biokinetics tested in the mouse model did not meet expectations. This highlights the importance of developing and testing further new anti-CD30 conjugates.

Effectiveness and normal tissue toxicity of Auger electron (AE) radioimmunotherapy (RIT) with [111In]In-Bn-DTPA-nimotuzumab in mice with triple-negative or trastuzumab-resistant human breast cancer xenografts that overexpress EGFR.

INTRODUCTION: Our objective was to evaluate the effectiveness and normal tissue toxicity of nimotuzumab labeled with the Auger electron (AE)-emitter, 111In ([111In]In-Bn-DTPA-nimotuzumab) for radioimmunotherapy (RIT) of human triple-negative breast cancer (TNBC) or trastuzumab-resistant HER2-positive BC tumors overexpressing epidermal growth factor receptors (EGFR) in athymic mice. METHODS: Normal tissue toxicity was studied in non-tumor-bearing Balb/c mice i.v. administered 9.0 or 28.6 MBq (3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab, unlabeled nimotuzumab (3 mg/kg) or normal saline. A complete blood cell count (CBC) and serum alanine aminotransferase (ALT) and creatinine (Cr) were measured at 14 days. Body weight was monitored. RIT studies were performed in CD-1 athymic mice engrafted s.c. with MDA-MB-468 human TNBC tumors or TrR1 HER2-positive but trastuzumab-resistant BC tumors. Mice were i.v. administered two amounts (15.5 MBq; 3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab separated by 14 days. Control mice received unlabeled Bn-DTPA-nimotuzumab (3 mg/kg) or anti-HER2 [111In]In-Bn-DTPA-trastuzumab or normal saline. Tumor growth and body weight were measured for 6 weeks. A tumor growth index (TGI) and body weight index (BWI) were calculated to compare the tumor size and body weight post-treatment with the pre-treatment values. A tumor doubling ratio (TDR) was calculated for each treatment group compared to control mice receiving normal saline. RESULTS: There was no loss of body weight or decreased red blood cells (RBC) or platelets (PLT) or increased serum ALT or Cr in Balb/c mice administered 9.0 or 28.6 MBq (3 mg/kg) of [111In]In-Bn-DTPA-nimotuzumab compared to mice treated with unlabeled Bn-DTPA-nimotuzumab (3 mg/kg) or normal saline. There was a significant decrease in white blood cell (WBC) counts in Balb/c mice receiving 28.6 MBq but not 9.0 MBq of [111In]In-Bn-DTPA-nimotuzumab. Based on these results, an administered amount of 15.5 MBq (3 mg/kg) was selected for RIT studies. Administration of two amounts (15.5 MBq; 3 mg/kg) separated by 14 days to CD-1 athymic mice with s.c. MDA-MB-468 xenografts strongly inhibited tumor growth. The TDR for mice treated with [111In]In-Bn-DTPA-nimotuzumab was 2.15 compared to control mice receiving normal saline. In contrast, treatment with unlabeled Bn-DTPA-nimotuzumab or [111In]In-Bn-DTPA-trastuzumab had no significant effect on tumor growth (TDR = 0.96 and 1.08, respectively). RIT with [111In]In-Bn-DTPA-nimotuzumab also strongly inhibited the growth of TrR1 tumors in athymic mice (TDR = 2.13) compared to unlabeled Bn-DTPA-nimotuzumab (TDR = 0.91). There were no losses in body weight over 6 weeks in tumor bearing mice receiving [111In]In-Bn-DTPA-nimotuzumab, unlabeled Bn-DTPA-nimotuzumab, [111In]In-Bn-DTPA-trastuzumab or normal saline. CONCLUSIONS: [111In]In-Bn-DTPA-nimotuzumab was effective for treatment of TNBC or trastuzumab-resistant HER2-positive human BC tumors in mice that overexpress EGFR at administered amounts that caused no decrease in body weight or normal tissue toxicity in non-tumor-bearing Balb/c mice. ADVANCES IN KNOWLEDGE AND IMPLICATIONS FOR PATIENT CARE: Our results suggest that Auger electron RIT with [111In]In-Bn-DTPA-nimotuzumab may provide a novel therapeutic option for patients with TNBC or trastuzumab-resistant HER2-positive BC that overexpresses EGFR. The low normal tissue toxicity of this approach may allow combination with other targeted therapies such as antibody-drug conjugates (ADCs).

Succinylated Gelatin Improves the Theranostic Potential of Radiolabeled Exendin-4 in Insulinoma Patients.

Being highly expressed in insulinomas, the glucagonlike peptide-1 receptor (GLP-1R) is a potential target for diagnosis, localization, and treatment with the radiolabeled GLP-1R agonist exendin. Tracer accumulation in the kidneys, however, hampers accurate diagnostic visualization of pancreatic tissue and prohibits the therapeutic application of radiolabeled exendin for ß-cell-derived tumors. Therefore, we evaluated the ability of succinylated gelatin (Gelofusine) to reduce the renal accumulation of radiolabeled exendin in humans, and we performed dosimetric calculations to estimate the maximum absorbed insulinoma dose that could be achieved if exendin were to be used for peptide receptor radionuclide therapy. Methods: Ten healthy volunteers received 50 MBq of 111In-exendin-4, in combination with Gelofusine or saline, in a crossover design. SPECT/CT images were obtained after 24 h. The procedure was repeated 3 wk later. Uptake of 111In-exendin was determined by drawing regions of interest around the kidneys and in the pancreas. Planar scintigraphic 111In-exendin images of 5 insulinoma patients were used for dosimetry studies estimating the maximum insulinoma absorbed dose that could be achieved without causing radiotoxicity to other organs. Results: Gelofusine reduced the renal accumulation of 111In-exendin-4 by 18.1%, whereas the pancreatic uptake remained unchanged. In 3 of 10 subjects, the kidney uptake was reduced to such an extent that the pancreatic tail could be better discriminated from the kidney signal. Dosimetric estimations suggested that the insulinoma absorbed dose ranges from 30.3 to 127.8 Gy. This dose could be further increased to maximally 156.1 Gy if Gelofusine was used. Conclusion: We have shown that Gelofusine can reduce the renal accumulation of 111In-exendin-4 in humans. This reduction not only allows more accurate qualitative and quantitative analyses of radiolabeled exendin uptake in the tail region of the pancreas but also potentiates the safe delivery of a higher radiation dose to GLP-1R-positive tumors for therapy.

Drug-resistant AML cells and primary AML specimens are killed by 111In-anti-CD33 monoclonal antibodies modified with nuclear localizing peptide sequences.

UNLABELLED: Multidrug resistance (MDR) is a major challenge to the successful treatment of acute myeloid leukemia (AML). Our purpose was to determine whether (111)In-HuM195 anti-CD33 antibodies modified with peptides harboring nuclear localizing sequences (NLS) could kill drug-resistant AML cell lines and primary AML patient specimens expressing MDR transporters through the emission of Auger electrons. METHODS: HuM195, M195, and irrelevant mouse IgG (mIgG) were conjugated to 10+/-3 NLS peptides and then labeled with (111)In by diethylenetriaminepentaacetic acid substitution to a specific activity of 1-8 MBq/microg. The binding affinity of HuM195 and M195 was determined for HL-60 and mitoxantrone-resistant HL-60-MX-1 cells. Nuclear localization of (111)In-NLS-HuM195, (111)In-NLS-M195, (111)In-HuM195, and (111)In-M195 was measured by subcellular fractionation. The antiproliferative effects of (111)In-NLS-HuM195, (111)In-NLS-M195, (111)In-HuM195, and (111)In-M195 (2.5-250 kBq/well) on HL-60 and HL-60-MX-1 were studied using the WST-1 assay. Clonogenic survival of HL-60 and HL-60-MX-1 leukemic cells and 10 primary AML specimens with MDR phenotype (assessed by flow cytometry) was determined after exposure for 3 h at 37 degrees C to 2.5-250 mBq/cell of (111)In-NLS-HuM195, (111)In-HuM195, or (111)In-NLS-mIgG. Clonogenic survival versus the amount of radioactivity incubated with the cells (mBq/cell) was plotted, and the mean lethal amount of radioactivity and the lower asymptote of the curve (plateau) were determined. RESULTS: The (111)In-labeled anti-CD33 monoclonal antibodies HuM195 and M195 modified with NLS were efficiently routed to the nucleus of HL-60 cells and their mitoxantrone-resistant clone after CD33-mediated internalization. The following are the principal findings of our study: (111)In-NLS-HuM195 was more effective at killing HL-60 and HL-60-MX-1 cells than was (111)In-HuM195, a strong correlation between the specific activity of the (111)In-labeled radioimmunoconjugates and their cytotoxicity toward AML cells existed, and leukemic cells from patients were killed by (111)In-NLS-M195 or (111)In-M195, but the cytotoxic response among specimens was heterogeneous. CONCLUSION: NLS conjugation enhanced the nuclear uptake and cytotoxicity of (111)In-HuM195 and (111)In-M195 toward drug-resistant AML cell lines as well as patient specimens expressing a diversity of MDR phenotypes, including Pgp-170, BCRP1, or MRP1 transporters. Targeted Auger electron radioimmunotherapy using (111)In-labeled anti-CD33 monoclonal antibodies modified with NLS may be able to overcome MDR and provide a means of treating chemotherapy-resistant myeloid leukemias in patients.