Noninvasive PET imaging of tumor PD-L1 expression with 64Cu-labeled Durvalumab.

Breast cancer (BrCa) ranks as the most prevalent malignant neoplasm affecting women worldwide. The expression of programmed death-ligand 1 (PD-L1) in BrCa has recently emerged as a biomarker for immunotherapy response, but traditional immunohistochemistry (IHC)-based methods are hindered by spatial and temporal heterogeneity. Noninvasive and quantitative PD-L1 imaging using appropriate radiotracers can serve to determine PD-L1 expression in tumors. This study aims to demonstrate the viability of PET imaging with 64Cu-labeled Durvalumab (abbreviated as Durva) to assess PD-L1 expression using a murine xenograft model of breast cancer. Durvalumab, a human IgG1 monoclonal antibody against PD-L1, was assessed for specificity in vitro in two cancer cell lines (MDA-MB-231 triple-negative breast cancer cell line and AsPC-1 pancreatic cancer cell line) with positive and negative PD-L1 expression by flow cytometry. Next, we performed the in vivo evaluation of 64Cu-NOTA-Durva in murine models of human breast cancer by PET imaging and ex vivo biodistribution. Additionally, mice bearing AsPC-1 tumors were employed as a negative control. Tumor uptake was quantified based on a 3D region-of-interest (ROI) analysis of the PET images and ex vivo biodistribution measurements, and the results were compared against conventional IHC testing. The radiotracer uptake was evident in MDA-MB-231 tumors and showed minimal nonspecific binding, corroborating IHC-derived results. The results of the biodistribution showed that the MDA-MB-231 tumor uptake of 64Cu-NOTA-Durva was much higher than 64Cu-NOTA-IgG (a nonspecific radiolabeled IgG). In Conclusion, 64Cu-labeled Durvalumab PET/CT imaging offers a promising, noninvasive approach to evaluate tumor PD-L1 expression.

Development and evaluation of 89Zr-trastuzumab for clinical applications.

Objectives: Due to the suitable physical characteristics of 89Zr as a PET radionuclide and affinity of Trastuzumab monoclonal antibody against HER2, [89Zr]Zr-Trastuzumab was prepared and went through preclinical evaluations for ultimate human applications. Methods: 89Zr was produced by using 89Y(p,n)89Zr reaction at a 30 MeV cyclotron (radionuclide purity>99.9%, specific activity of 17 GBq/µg). p-SCN-Bn-Deferoxamine (DFO); was conjugated to trastuzumab, followed by labeling with 89Zr in oxalate form at optimized condition. Cell binding, internalization and, radioimmuno-activity assays were studied using HER2+ BT474 and HER2- CHO cell lines. Finally, the biodistribution of the radioimmunoconjugate was assessed in normal and HER2+ BT474 tumor-bearing mice using tissue counting and imaging at different intervals after injection. Also, a woman with HER2-positive metastatic breast cancer under treatment with Herceptin underwent both [89Zr]Zr-Trastuzumab and, [18F]FDG PET/CTs. Results: 89Zr was produced with high radionuclidic and radiochemical purities (>99%) and [89Zr]Zr-DFO-Trastuzumab was prepared with radiochemical purity of >98% and specific activity of 9.85 GBq/µmol. The radioimmunoconjugate was stable both in PBS buffer and in human serum for at least 48 h. The radioimmunoactivity assay demonstrated about 70% of [89Zr]Zr-DFO-Trastuzumab is bound to the BT474 cells at the number of 250×106 cells. Cell binding studies showed that about 28% of radioimmunoconjugate is attached to BT474 cells after 90 min. Internalization studies showed that 50% of [89Zr]Zr-Trastuzumab is internalized to BT474 cells only in 6 h. The biodistribution study of the labeled compound in normal mice demonstrated the same pattern of the monoclonal antibodies which is entirely different from the biodistribution of free 89Zr. Biodistribution and imaging studies in tumor-bearing mice showed the significant uptake values of [89Zr]Zr-Trastuzumab in tumor sites. [89Zr]Zr-Trastuzumab PET/CT revealed metastatic lesions documented previously with [18F]FDG PET/CT scan in a woman with breast cancer who was under treatment with Herceptin. Although the [18F]FDG PET/CT scan had better quality images, the valuable and unique advantage of [89Zr]Zr-Trastuzumab PET/CT is delineating HER2+ metastasis, which is essential in diagnosis and HER2-based treatments. Conclusion: The prepared [89Zr]Zr-Trastuzumab has a high potential radio-pharmaceutical for immune-PET imaging of the patients with HER2+ tumors.

Development of 177Lu-Cetuximab-PAMAM dendrimeric nanosystem: a novel theranostic radioimmunoconjugate.

PURPOSE: Epidermal growth factor receptors (EGFRs) are overexpressed in a wide range of tumors and are attractive candidates to target in targeted therapies. This study aimed to introduce a novel radiolabeled compound, 177Lu-cetuximab-PAMAM G4, for the treatment of EGFR-expressing tumors. METHODS: In this study, the cetuximab mAb was bound to PAMAM G4 and labeled with 177Lu via DTPA-CHX chelator. The synthesized nanosystem was confirmed by different analyses such as DLS, FT-IR, TEM, and RT-LC. Cell viability of the radioimmunoconjugate was assessed over the EGFR-expressing cell line of SW480. The biodistribution of 177Lu-Cetuximab-PAMAMG4 was determined in different intervals after injection of the radiolabeled compound in normal and tumoral nude mice via scarification and SPECT images. RESULTS: The average size of PAMAM G4 and PAMAM-Cetuximab-DTPA-CHX nanoparticles were 2 and 70 nm, respectively. 177Lu-Cetuximab-PAMAMG4 was prepared with radiochemical purity of more than 98%. The survival rates of SW480 cells at 24, 48, and 72 h post-treatment with177Lu-Cetuximab-PAMAMG4 (500 nM) were 18%, 15%, and 14%, respectively. The biodistribution studies showed a significant accumulation of 177Lu-Cetuximab-PAMAM in the EGFR-expressing tumor. CONCLUSION: According to the results, this new agent can be considered as an efficient therapeutic complex for tumors expressing EGFR receptors.

An Overview of Radiolabeled RGD Peptides for Theranostic Applications.

Angiogenesis phenomenon, as a highly affecting factor on the growth and spread of cancer cells, depends on specific molecular interactions between components of the extracellular matrix and vascular cells. αv integrin acts as a cell adhesive molecule involved in tumor invasion and angiogenesis. Among the various combinations of integrin subunits expressed on the surface of cells, αvß3 integrin has a particularly interesting expression pattern during angiogenesis. The αvß3 integrin is a vital receptor affecting tumor growth, tumor invasiveness, metastasis, and angiogenesis overexpressed on various human tumors, leading to the development of different theranostics probes and radiopharmaceuticals. The αvß3 integrin can recognize several extracellular matrix molecules in the base of the RGD adhesive sequence. This review provides an overview of the status, trends and future of the most studied αvß3 integrin-binding ligand, RGD tripeptides, labeled with various radioisotopes. An overview of the pre-clinical models for radiolabeled RGD peptides and clinical aspects of the RGD- based radiopharmaceuticals is provided with some new considerations and ways forward.

Human Absorbed Dose Estimation of 111In-DOTA-PR81 as a Novel High Potential Agent for Breast Cancer Imaging.

Purpose: In this study, the human absorbed dose of 111In-DOTA-PR81 as a new radioimmunoconjugate for single-photon emission computed tomography (SPECT) imaging of MUC1 + breast cancer was determined. Materials and Methods: The complex was prepared at optimized conditions in about 1 h and 38°C. The radiochemical purity of the tracer was investigated using the instant thin-layer chromatography method method, showing purity of higher than 96%. After evaluating the stability of the product in human serum and room temperature, the biological distribution of the radiolabeled compound was studied in normal rats and tumor-bearing mice. Finally, the human absorbed dose of the complex was estimated based on animals' data using radiation dose assessment resource and Spark et al. methods. Results: High uptake of the complex in MUC1 + breast tumors compared to other nontarget organs shows that the radioimmunoconjugate is a beneficial agent for SPECT imaging of MUC1 + breast cancer. Human organs absorbed dose estimation of the complex demonstrated the highest amounts of the absorbed dose are in the liver and kidneys with 0.384 and 0.245 mGy/MBq, respectively. Conclusions: 111In-DOTA-PR81 radioimmunoconjugate is a high potential agent for MUC1 + breast cancer SPECT imaging and estimated absorbed dose values could helpfully use for the determination of the maximum injectable dose.

Preparation and preclinical characterization of 111In-DTPA-Anti-MUC1 as a radioimmunoconjugate for diagnosis of breast cancer by single-photon emission computed tomography.

Context: Targeting MUC1 antigens which are overexpressed in 80% of breast cancers can be widely used in the field of radioimmunoscintigraphy (RIS) of breast cancer. Aims: The aim of this study was to develop a new diagnostic labeled compound for breast cancer RIS. Settings and Design: In this study, an efficient indirect labeling method of PR81 with Indium-111 was developed and preliminary preclinical qualifications were reported. Subjects and Methods: 111In-DTPA-PR81 was prepared and its radiochemical purity and stabilities in human serum and in phosphate-buffered saline (PBS) buffer were surveyed. Furthermore, cellular studies including complex reactivity, binding specificity, cell toxicity, etc., were examined. Finally, biodistribution and scintigraphy of the complex were studied in normal and tumoral animals. Statistical Analysis Used: Statistical analyses were performed using SPSS 10.0. Results: 111In-DTPA-PR81 was prepared with a radiochemical purity of >99% at optimized conditions. Stability studies showed the radiochemical purity of >90% in PBS buffer after 96 h, while the stability in human serum showed decrement to 81% after 96 h. Reactivity of the complex with MUC1 was significantly (P < 0.005) higher than bovine serum albumin (BSA) (about 7-8 times), even though BSA concentration was about twice the MUC1. The binding specificity of the complex to the MUC1 antigen was confirmed by means of immunoreactivity assay. Cell toxicity examination showed no significant lethal effect of the radiolabeled compound on the cells. Biodistribution studies of the complex in normal rats were consistent with the biodistribution of antibodies and high accumulation was observed in the tissues expressing MUC1 antigen. The results of 111In-DTPA-PR81 scintigraphy in tumoral female BALB/c mice at 24 and 48 h after injection showed an increasement of the accumulation in the tumor site. Conclusions: 111In-DTPA-PR81 can be considered as a potential agent for imaging of the MUC1 +breast tumors.

Human Dose Assessment of 68Ga-NODAGA-RGD-BBN Heterodimer Peptide based on Animal Data.

Aims: Calculation of the absorbed dose in human organs is one of the first steps for developing new radiopharmaceuticals. The aim of this study is to estimate the human absorbed dose of a newly developed 68Ga-NODAGA-RGD-BBN radiolabeled compound. Materials and Methods: 68Ga-NODAGA-RGD-BBN was prepared by varying different parameters at optimized conditions. The stability of the radiolabeled peptide in phosphate-buffered saline (PBS) and in human serum was evaluated for 120 min. Afterward, the biodistribution of the complex was assessed in normal and tumor-bearing mice, at least for 120 min postinjection. Finally, the human absorbed dose of 68Ga-NODAGA-RGD-BBN was estimated based on mice data using Radiation Dose Assessment Resource and Spark method. Results: 68Ga-NODAGA-RGD-BBN was produced with radiochemical purity of more than 98% (high-performance liquid chromatography/ radio thin layer chromatography (RTLC)) with high stability in PBS buffer and in human serum at least for 2 h. The complex demonstrated high uptake in gastrin-releasing peptide receptor-expressing tumors compared to other nontarget organs. Furthermore, the dose assessment for the complex showed that the kidneys receive the highest absorbed dose in comparison with other organs. Conclusion: The result of this study showed that 68Ga-NODAGA-RGD-BBN is an effective and radiolabeled ligand for tumor detection, however more studies are still needed.

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.

Preparation, quality control, and biodistribution assessment of [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors.

In this study, [111 In]In-DOTA-PR81 was developed, and its preliminary preclinical qualifications were assessed for single photon emission computed tomography (SPECT) imaging of breast cancer. DOTA-NHS-ester was practiced and successively purified by molecular filtration. The chelate:mAb ratio was determined by spectrophotometry. DOTA-PR81 was radiolabeled with In-111 and its radiochemical yield, in vitro stability, in vitro internalization, and immunoreactivity tests were performed. SPECT imaging and tissue counting were applied to evaluate the tissue distribution of [111 In]In-DOTA-hIgG and [111 In]In-DOTA-PR81 in BALB/c mice bearing breast tumors. The radiochemical yield of [111 In]In-DOTA-PR81 complex was >95.0 ± 0.5% (ITLC), and the specific activity was 170 ± 44 MBq/mg. Conjugation reaction resulted in the average number of chelators attached to a mAb (c/a) of 3.4 ± 0.3:1. The radioimmunoconjugate showed immunoreactivity towards MCF7 cell line and MUC1 antigen while its significant in vitro and in vivo stability were investigated over 48 h, respectively (93.0 ± 1.2% in phosphate-buffered saline (PBS) and 84.0 ± 1.3% in human serum). The peak concentration of internalized activity of [111 In]In-DOTA-PR81 was between 4 to 6 h. In comparison with control probes, the complex was accumulated with high specificity and sensitivity at the tumor site. Achieved results indicated that [111 In]In-DOTA-PR81 could be contemplated as an appropriate radiotracer for prognostic imaging of antigens in oncology.

Development of 186/188Re-Chitosan as an Effective Therapeutic Agent for Rheumatoid Arthritis.

BACKGROUND: Rheumatoid arthritis (RA) is an inflammatory chronic disease characterized by inflammation, pain, swelling and disability, and radiosynovectomy is one of the disease treatment lines. In this study, the possibility of providing rhenium-186/rhenium-188 chitosan radiopharmaceuticals, optimization of conditions for their production and bio-distribution are reported. OBJECTIVE: In order to build perrhenic acid for labeling, natural rhenium was exposed to radiation. Radionuclidic and radiochemical purities of (186/188Re)-NaReO4 were examined by gamma spectroscopy and paper chromatography methods, respectively. METHODS: Labeling of chitosan with rhenium was done in different acidic situations. The radiochemical purity 186/188Re-chitosan was applied by radio thin layer chromatography (RTLC). Lastly, the bio-distribution of the radiolabeled chitosan was studied in various organs after intra articular injection of the complex to lab rats. Gamma spectrometry confirmed the high rhenium radionuclidic purity. Chromatography results showed that perrhenic acid was produced with purity greater than 97% and rhenium chitosan labeling was done over 98% in pH = 3. Dissection results showed a high bio-distribution of 186/188Re-chitosan after injection into the joint with no leakage to surrounding organs. CONCLUSION: According to the results, there is a possibility of labeling rhenium with chitosan in very high radiochemical purity. Regarding the high retention of these radiopharmaceuticals in joints with no leakage to surrounding organs, 186/188Re-chitosan can be applied as new radiosynovectomy drugs for rheumatoid arthritis treatment.

IAEA contribution to the development of 64Cu radiopharmaceuticals for theranostic applications.

Copper-64 is a very attractive radioisotope with unique nuclear properties that allow using it as both a diagnostic and therapeutic agent, thus providing an almost ideal example of a theranostic radionuclide. A characteristic of Cu-64 stems from the intrinsic biological nature of copper ions that play a fundamental role in a large number of cellular processes. Cu-64 is a radionuclide that reflects the natural biochemical pathways of Cu-64 ions, therefore, can be exploited for the detection and therapy of certain malignancies and metabolic diseases. Beside these applications of Cu-64 ions, this radionuclide can be also used for radiolabelling bifunctional chelators carrying a variety of pharmacophores for targeting different biological substrates. These include peptide-based substrates and immunoconjugates as well as small-molecule bioactive moieties. Fueled by the growing interest of Member States (MS) belonging to the International Atomic Energy Agency (IAEA) community, a dedicated Coordinated Research Project (CRP) was initiated in 2016, which recruited thirteen participating MS from four continents. Research activities and collaborations between the participating countries allowed for collection of an impressive series of results, particularly on the production, preclinical evaluation and, in a few cases, clinical evaluation of various 64Cu-radiopharmaceuticals that may have potential impact on future development of the field. Since this CRP was finalized at the beginning of 2020, this short review summarizes outcomes, outputs and results of this project with the purpose to propagate to other MS and to the whole scientific community, some of the most recent achievements on this novel class of theranostic 64Cu-pharmaceuticals.

Investigations of proton and deuteron induced nuclear reactions on natural and enriched Titanium, Calcium and Vanadium targets, with special reference to the production of 47Sc.

47Sc could be used in SPECT imaging and also suitable for targeted therapy of small tumors. The excitation functions for the production of 47Sc and accompanying impurities via proton and deuteron bombardment of Calcium, Titanium and Vanadium targets were evaluated by three nuclear codes, ALICE, TALYS and EMPIRE. Therefrom, integral yields of 47Sc and also 46gSc as a main impurity were calculated. The various production routes of 47Sc were compared together. The results consistency with available experimental data was checked for each reaction. Based on the results, the 46Ca(d,n)47Sc reaction can leads to the high purity 47Sc with the moderate yield.

Internalization capabilities of gold-198 nanoparticles: Comparative evaluation of effects of chitosan agent on cellular uptake into MCF-7.

In this study, internalization of positively charged chitosan-coated nanoparticles (198AuNPs@chitosan) on MCF-7 cells was investigated by γ-ray spectroscopy and then statistically compared to that of 198Au and negatively charged citrate-stabilized nanoparticles (198AuNPs). Sub-50 nm 198AuNPs@chitosan had a higher internalization compared to 198Au and 198AuNPs (p < 0.05). More cellular uptake of 198AuNP@chitosan means a higher dose of radioactivity to the tumor cells which, in turn, more effective treatment of the cancer.

The effect of 111In radionuclide distance and auger electron energy on direct induction of DNA double-strand breaks: a Monte Carlo study using Geant4 toolkit.

PURPOSE: In this study, the effect of 111In position and Auger electron energy on direct induction of DSBs was investigated. MATERIALS AND METHODS: The Geant4-DNA simulation toolkit was applied using a simple B-DNA form extracted from PDBlib library. First, the simulation was performed for electrons with energies of 111In and equal emission probabilities to find the most effective electron energies. Then, 111In Auger electrons' actual spectrum was considered and their contribution in DSB induction analysed. RESULTS: The results showed that the most effective electron energy is 183 eV, but due to the higher emission probability of 350 eV electrons, most of the DSBs were induced by the latter electrons. Also, it was observed that most of the DSBs are induced by electrons emitted within 4 nm of the central axis of the DNA and were mainly due to breaks with <4 base pairs distance in opposing strands. Whilst, when 111In atoms are very close to the DNA, 1.3 DSBs have been obtained per decay of 111In atoms. CONCLUSIONS: The results show that the most effective Auger electrons are the 350 eV electrons from 111In atoms with <4 nm distance from the central axis of the DNA which induce ∼1.3 DSBs per decay when bound to the DNA. This value seems reasonable when compared with the reported experimental data.

Evaluation of the Possible Utilization of 68Ga-DOTATOC in Diagnosis of Adenocarcinoma Breast Cancer.

OBJECTIVES: Studies have indicated advantageous properties of [DOTA-DPhe1, Tyr3] octreotide (DOTATOC) in tumor models and labeling with gallium. Breast cancer is the second leading cause of cancer mortality in women, and most of these cancers are often an adenocarcinoma. Due to the importance of target to non-target ratios in the efficacy of diagnosis, the pharmacokinetic of 68Ga-DOTATOC in an adenocarcinoma breast cancer animal model was studied in this research, and the optimized time for imaging was determined. METHODS: 68Ga was obtained from 68Ge/68Ga generator. The complex was prepared at optimized conditions. Radiochemical purity of the complex was checked using both HPLC and ITLC methods. Biodistribution of the complex was studied in BALB/c mice bearing adenocarcinoma breast cancer. Also, PET/CT imaging was performed up to 120 min post injection. RESULTS: The complex was produced with radiochemical purity of greater than 98% and specific activity of about 40 GBq/mM at optimized conditions. Biodistribution of the complex was studied in BALB/c mice bearing adenocarcinoma breast cancer indicated fast blood clearance and significant uptake in the tumor. Significant tumor: blood and tumor:muscle uptake ratios were observed even at early times post-injection. PET/CT images were also confirmed the considerable accumulation of the tracer in the tumor. CONCLUSION: Generally, the results proved the possible application of the radiolabelled complex for the detection of the adenocarcinoma breast cancer and according to the pharmacokenitic data, the suitable time for imaging was determined as at least 30 min after injection.

Radioiodination and preclinical evaluation of 4-benzyl-1-(3-[125I]-iodobenzylsulfonyl)piperidine as a breast tumor imaging tracer in mouse.

OBJECTIVE: 4-Benzyl-1-(3-iodobenzylsulfonyl)piperidine, 4-B-IBSP, has shown high-binding affinity to both sigma (σ) receptors in our previous work. In current study, radiolabeling and preclinical evaluation of 4-benzyl-1-(3-[125I]-iodobenzylsulfonyl)piperidine, 4-B-[125I]IBSP, in human ductal breast carcinoma (T47D) cells and in breast adenocarcinoma-bearing BALB/c mice are described. METHODS: Radioiodination of this new σ ligand was performed by a palladium-catalyzed stannylation approach followed by oxidative iododestannylation reaction using Iodo-Gen. Competition-binding assays for binding of 4-B-[125I]IBSP to guinea pig brain membranes and to T47D cells were performed with known σ ligands. The selectivity and binding characteristics (B max and K d) were analyzed. In vitro stability and in vivo blood metabolism studies were also evaluated. Moreover, biodistribution studies were performed in normal and into the tumor-bearing mice at interval time points post-injection (p.i.). Both in vitro and in vivo blockade experiments were done in the presence of the σ receptors blocking agents. RESULTS: Radioiodinated ligand was obtained in high yield and high specific activity. The σ inhibition constants (K i, nM) for 4-(3-iodobenzyl)-1-(benzylsulfonyl)piperazine (4-IBBSPz), (+)-pentazocine, haloperidol, DTG, and 4-B-IBSP were 1.37 ± 0.19, 3.90 ± 0.77, 2.69 ± 0.33, 30.62 ± 2.01, and 0.61 ± 0.05, respectively. 4-B-[125I]IBSP bound to σ receptor sites preferably to very high-affinity binding sites on T47D cells. The radioligand showed acceptable in vitro and in vivo stabilities in the blood pool. However, in vivo biodistribution studies in normal Swiss albino mice revealed fast clearance of 4-B-[125I]IBSP from blood and the other normal organs. Biodistribution experiments of 4-B-[125I]IBSP in breast adenocarcinoma tumor-bearing BALB/c mice showed a relatively high tumor uptake at 30 min p.i. (4.13 ± 0.95) that reaches to 1.57 ± 0.24 even after 240 min p.i. A pre-injection of 4-B-IBSP and haloperidol with 4-B-[125I]IBSP resulted in 36-57% decrease in activity in the tumor, liver, and brain at 60 min p.i. CONCLUSIONS: The high affinity of 4-B-[125I]IBSP to σ receptor-binding sites, its relatively high uptake, and preferential retention in the tumor as well as an increasing trend observed in the tumor to blood and in the tumor to muscle ratios suggests that an iodine-123 labeled counterpart, 4-B-[123I]IBSP, would be a promising σ radioligand for pursuing further studies to assess its potential for breast tumors imaging with SPECT.

Development of a (68)Ga-peptide tracer for PET GnRH1-imaging.

OBJECTIVE: Total synthesis, quality control and preclinical evaluation of [(68)Ga]-DOTA-triptorelin ([(68)Ga]-DOTA-TRP) is reported as a possible PET radiotracer for GnRH receptor imaging. METHODS: DOTA-TRP was totally synthesized in two steps and after characterization went through radiolabelling optimization studies followed by tracer stability. The biodistribution of the tracer in normal male rats and 4T1 tumour-bearing mice was performed in 120 min after i.v. injection. RESULTS: The peptide and the conjugates were synthesized with >95 % chemical purity. [(68)Ga]-DOTA-TRP complex was prepared in high radiochemical purity (>99 %, ITLC, HPLC) and specific activity of 1400-2100 MBq/nM at 95 °C using 40-60 µg of the peptide in 5-7 min followed by solid phase purification. The IC50 [nM] DOTA-TRP was comparable to the intact peptide, 0.11 ± 0.01 and 0.22 ± 0.05, respectively. The biodistribution of the tracer demonstrated kidney, stomach, and testes significant uptake, all in accordance with GnRH receptor ligands. Significant tumour uptake was observed in 4T1 tumour-bearing female mice 30-120 min post-injection with tumour:blood and tumour:muscle ratios of 28 and >50 in 60 min, respectively. Kidney is rapidly washed from the tracer. [(68)Ga]-DOTA-TRP can be proposed as a possible tracer for GnRH-R imaging studies.

Development of [64Cu]-DOTA-PR81 radioimmunoconjugate for MUC-1 positive PET imaging.

OBJECTIVE: Breast cancer radioimmunoscintigraphy targeting MUC1 expression is a growing field of work in nuclear medicine research. PR81 is a monoclonal antibody that binds with high affinity to MUC1, which is over expressed on breast tumors. In this study, we report production, quality control and preclinical qualifications of a copper-64 labeled PR81 for PET imaging of breast cancer. METHODS: PR81 was conjugated with DOTA-NHS-ester and purified by molecular filtration followed by chelate:mAb ratio determination by spectrophotometric method. DOTA-PR81 was labeled with (64)Cu followed by radiochemical purity, in vitro stability, in vitro internalization and immunoreactivity determination. The tissue biodistribution of the (64)Cu-DOTA-PR81 and (64)Cu-DOTA-hIgG was evaluated in BALB/c mice with breast carcinoma tumors using tissue counting and imaging. RESULTS: The radiochemical purity of radioimmunoconjugate was >95±1.9% (ITLC) (specific activity; 4.6 µCi/µg). The average number of chelators per antibody was 3.4±0.3:1. The (64)Cu-DOTA-PR81 showed immunoreactivity towards MUC1 antigen and MCF7 cell line with significant in vitro stability (>89% in PBS and 78±0.5% in human serum) over 48 h. Maximum internalized activity of radiolabeled PR81 in 4-8 h was 81.5%. The biodistribution and scintigraphy studies showed the accumulation of the complex at the site of tumors with high sensitivity and specificity compared to control probes. CONCLUSION: The results showed that (64)Cu-DOTA-PR81 may be considered as a potential PET tracer for diagnosis and follow-up of MUC1 expression in oncology.

Preclinical evaluation of [¹¹¹ In]-DOTA-trastuzumab for clinical trials.

CONTEXT: Herceptin and its fragments have been radiolabeled and used in the imaging of human epidermal growth factor receptor 2 (HER2)/neu-positive tumors and development of diagnostic kits is of great importance in radiopharmacy. AIMS: In this study, ¹¹¹ In-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid-trastuzumab (¹¹¹ In-DOTA-trastuzumab) was successively prepared and evaluated for ultimate use in the HER2 antigen imaging in oncology. SETTINGS AND DESIGN: The conjugate was prepared, labeled and evaluated using in vitro (radioimmunoassay [RIA], enzyme-linked immunosorbent assay (ELISA), stability, binding, internalization)/in vivo (bio-distribution, single-photon emission computed tomography [SPECT]) experiments. MATERIALS AND METHODS: ¹¹¹ In-DOTA-trastuzumab was prepared followed by determination of radiochemical purity (RCP), integrity of protein, immunoreactivity of radiolabeled antibody with HER2/neu antigen (by SkBr3 cell line binding and RIA methods) were determined followed by stability tests, internalization studies and the tissue bio-distribution determination in wild-type rats as well as SPECT imaging in SkBr3-bearing mice. STATISTICAL ANALYSIS USED: All values were expressed as mean ± standard deviation (mean ± SD) and the data were compared using Student's t-test. Statistical significance was defined as P < 0.05. RESULTS: ¹¹¹ In-DOTA-trastuzumab was prepared (RCP >95 ± 0.5%, S.A. 5.3 µCi/µg) with the average number of chelators per antibody of 6:1 showing significant immune-reactivity retention using ELISA. In vitro stability was >90% in phosphate buffered saline and 80 ± 0.5% in serum over 48 h. Cell binding was significant (>0.79). In vitro internalization reached up to %12-13 in 10 h. Significant tumor uptake was observed. CONCLUSIONS: In vitro and in vivo/SPECT imaging in SkBr3-bearing mice demonstrated that ¹¹¹ In-DOTA-trastuzumab is a potential compound for molecular imaging of SPECT for diagnosis and follow-up of HER2 expression in oncology.

Preclinical evaluation of holmium-166 labeled anti-VEGF-A(Bevacizumab).

Radiolabeled antiangiogenic monoclonal antibodies are potential agents for targeted therapy in specific types of malignancies. In this study, (166)Ho-DOTA-Bevacizumab was used in biodistribution studies using single-photon emission computed tomography (SPECT) to acquire dosimetric aspects of the radiolabeled antibody in mice. The liver toxicity of the radiolabeled antibody was also determined using serum glutamic pyruvic transaminase, serum glutamic oxaloacetic transaminase and alkaline phosphatase assay 2-7 days post-injection. The SPECT biodistribution demonstrated a similar pattern as the other radiolabeled anti-vascular endothelial growth factor A (VEGF-A) immunoconjugates. (166)Ho-DOTA-Bevacizumab was revealed as a potential compound for therapy/imaging of VEGF-A expression in oncology.