Acute Myelomonoblastic Leukemia (My1/De): A Preclinical Rat Model.

BACKGROUND/AIM: Since acute myeloid leukemias still represent the most aggressive type of adult acute leukemias, the profound understanding of disease pathology is of paramount importance for diagnostic and therapeutic purposes. Hence, this study aimed to explore the real-time disease fate with the establishment of an experimental myelomonoblastic leukemia (My1/De) rat model using preclinical positron emission tomography (PET) and whole-body autoradiography. MATERIALS AND METHODS: In vitro [18F]F-FDG uptake studies were performed to compare the tracer accumulation in the newly cultured My1/De tumor cell line (blasts) with that in healthy control and My1/De bone marrow suspensions. Post transplantation of My1/De cells under the left renal capsule of Long-Evans rats, primary My1/De tumorigenesis, and metastatic propagation were investigated using [18F]F-FDG PET imaging, whole-body autoradiography and phosphorimage analyses. To assess the organ uptake profile of the tumor-carrying animals we accomplished ex vivo biodistribution studies. RESULTS: The tracer accumulation in the My1/De culture cells exceeded that of both the tumorous and the healthy bone marrow suspensions (p<0.01). Based on in vivo imaging, the subrenally transplanted My1/De cells resulted in the development of leukemia in the abdominal organs, and metastasized to the mesenterial and thoracic parathymic lymph nodes (PTLNs). The lymphatic spread of metastasis was further confirmed by the significantly higher %ID/g values of the metastatic PTLNs (4.25±0.28) compared to the control (0.94±0.34). Cytochemical staining of the peripheral blood, autopsy findings, and wright-Giemsa-stained post-mortem histological sections proved the leukemic involvement of the assessed tissues/organs. CONCLUSION: The currently established My1/De model appears to be well-suited for further leukemia-related therapeutic and diagnostic investigations.

Concomitant [18F]F-FAZA and [18F]F-FDG Imaging of Gynecological Cancer Xenografts: Insight into Tumor Hypoxia.

BACKGROUND/AIM: Herein we assessed the feasibility of imaging protocols using both hypoxia-specific [18F]F-FAZA and [18F]F-FDG in bypassing the limitations derived from the non-specific findings of [18F]F-FDG PET imaging of tumor-related hypoxia. MATERIALS AND METHODS: CoCl2-generated hypoxia was induced in multidrug resistant (Pgp+) or sensitive (Pgp-) human ovarian (Pgp- A2780, Pgp+ A2780AD), and cervix carcinoma (Pgp- KB-3-1, Pgp+ KB-V-1) cell lines to establish corresponding tumor-bearing mouse models. Prior to [18F]F-FDG/[18F]F-FAZA-based MiniPET imaging, in vitro [18F]F-FDG uptake measurements and western blotting were used to verify the presence of hypoxia. RESULTS: Elevated GLUT-1, and hexokinase enzyme-II expression driven by CoCl2-induced activation of hypoxia-inducible factor-1α explains enhanced cellular [18F]F-FDG accumulation. No difference was observed in the [18F]F-FAZA accretion of Pgp+ and Pgp- tumors. Tumor-to-muscle ratios for [18F]F-FAZA measured at 110-120 min postinjection (6.2±0.1) provided the best contrasted images for the delineation of PET-oxic and PET-hypoxic intratumor regions. Although all tumors exhibited heterogenous uptake of both radiopharmaceuticals, greater differences for [18F]F-FAZA between the tracer avid and non-accumulating regions indicate its superiority over [18F]F-FDG. Spatial correlation between [18F]F-FGD and [18F]F-FAZA scans confirms that hypoxia mostly occurs in regions with highly active glucose metabolism. CONCLUSION: The addition of [18F]F-FAZA PET to [18F]F-FGD imaging may add clinical value in determining hypoxic sub-regions.

Overview of Prostaglandin E2 (PGE2)-Targeting Radiolabelled Imaging Probes from Preclinical Perspective: Lessons Learned and Road Ahead.

As malignancies still represent one of the major health concerns worldwide, early tumor identification is among the priorities of today's science. Given the strong association between cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2), PGE2 receptors (EPs), and carcinogenesis, target-specific molecules directed towards the components of the COX2/PGE2/EP axis seem to be promising imaging probes in the diagnostics of PGE2pos. neoplasms and in the design of anti-cancer drugs. Featured with outstanding inclusion forming capability, ß-cyclodextrins (CDs) including randomly methylated ß-CD (RAMEB) were reported to complex with PGE2. Therefore, radiolabelled ß-CDs could be valuable vectors in the molecular imaging of PGE2-related tumorigenesis. In vivo preclinical small animal model systems applying positron emission tomography (PET) ensure a well-suited scenario for the assessment of PGE2-affine labelled CD derivatives. Previous translational studies dealt with the evaluation of the tumor-homing capability of Gallium-68 (68Ga) and Bismuth-205/206 (205/206Bi)-appended ß-CD compounds conjugated with chelator NODAGA or DOTAGA: [68Ga]Ga-NODAGA-2-hydroxypropyl-ß-cyclodextrin/HPBCD, [68Ga]Ga-NODAGA-RAMEB, [68Ga]Ga-DOTAGA-RAMEB, and [205/206Bi]Bi-DOTAGA-RAMEB in experimental tumors with different PGE2 expression. These imaging probes project the establishment of tailor-made PET diagnostics of PGE2pos. malignancies. In the present review, we provide a detailed overview of the in vivo investigations of radiolabelled PGE2-directed CDs, highlighting the importance of the integration of translational discoveries into routine clinical usage.

In vivo assessment of tumor targeting potential of 68Ga-labelled randomly methylated beta-cyclodextrin (RAMEB) and 2-hydroxypropyl-ß-cyclodextrin (HPßCD) using positron emission tomography.

Cyclodextrin derivates (CyDs) can form complexes with cyclooxygenase-2 induced tumor promoting prostaglandin E2 (PGE2). Based on our previous observations, 68Ga-labelled CyDs may represent promising radiopharmaceuticals in the positron emission tomography (PET) diagnostics of PGE2 positive tumors. We aimed at evaluating the tumor-targeting potential of 68Ga-NODAGA conjugated randomly methylated beta-cyclodextrin (68Ga-NODAGA-RAMEB) and 2-hydroxypropyl-ß-cyclodextrin (68Ga-NODAGA-HPßCD) using in vivo PET imaging with experimental tumor models. Tumor radiopharmaceutical uptake was assessed applying PET and gamma counter in vivo and ex vivo respectively, following the administration of 18FDG, 68Ga-NODAGA-RAMEB or 68Ga-NODAGA-HPßCD via the lateral tail vein to the subsequent tumor-bearing animals: HT1080, A20, PancTu-1, BxPC3, B16-F10, Ne/De and He/De. All investigated tumors were identifiable with both 68Ga-labelled CyDs; however, in vivo results, in correlation with the ex vivo data, revealed that the PGE2 positive BxPC3, A20, Ne/De and He/De tumors presented the highest accumulation. In case of HT1080, A20, B16-F10 tumors significant differences were encountered between the accumulations of both 68Ga-labelled radiopharmaceuticals of the same tumor. Subcutaneously and the orthotopically transplanted Ne/De tumors differed significantly (p ≤ 0.01) regarding tracer uptake. 68Ga-labelled CyDs may open a novel field in the PET diagnostics of PGE2 positive primary tumors and metastases.

In Vivo Assessments of Mesoblastic Nephroma (Ne/De) and Myelomonoblastic Leukaemia (My1/De) Tumour Development in Hypercholesterolemia Rat Models.

Given the rising prevalence of lipid metabolic disorders and malignant diseases, we aimed to establish an in vivo hypercholesterinaemic tumour-bearing rat model for the induction and assessment of these conditions. A normal standard CRLT/N, 2 (baseline),- or 4 (2 + 2, pretreated)-week-long butter and cholesterol rich (BCR) diet was applied to mesoblastic nephroma (Ne/De) and myelomonoblastic leukaemia (My1/De) tumour-bearing and healthy control Long­Evans and Fischer 344 rats. The beginning of chow administration started in parallel with tumour induction and the 2 weeks of pre-transplantation in the baseline and pretreated groups, respectively. Fourteen days post-inoculation, the measurement of lipid parameters and [18F]F-FDG PET/MRI examinations was executed. The comparable lipid status of baseline healthy and tumorous rats proves that regardless of tumour presence, BCR-based hypercholesterolemia was achieved. A higher tumour mass among pretreated tumorous animals was found when compared to the control groups (p < 0.05, p < 0.01). Further, a visually greater [18F]F-FDG accumulation was observed in pretreated BCR tumorous animals; however, the quantitative data (SUVmean: 9.86 ± 0.98, 9.68 ± 1.24; SUVmax: 19.63 ± 1.20; 17.56 ± 3.21 for Ne/De and My1/De, respectively) were not statistically significantly different from those of the CRLT/N tumorous rats (SUVmean: 8.40 ± 1.42, 7.22 ± 1.06 and SUVmax: 15.99 ± 2.22, 12.46 ± 1.96 for control Ne/De and My1/De, respectively). Our model seems to be appropriate for simultaneously investigating hypercholesterolemia and cancer in the same rat.

PET Probes for Preclinical Imaging of GRPR-Positive Prostate Cancer: Comparative Preclinical Study of [68Ga]Ga-NODAGA-AMBA and [44Sc]Sc-NODAGA-AMBA.

Gastrin-releasing peptide receptors (GRPR) are overexpressed in prostate cancer (PCa). Since bombesin analogue aminobenzoic-acid (AMBA) binds to GRPR with high affinity, scandium-44 conjugated AMBA is a promising radiotracer in the PET diagnostics of GRPR positive tumors. Herein, the GRPR specificity of the newly synthetized [44Sc]Sc-NODAGA-AMBA was investigated in vitro and in vivo applying PCa PC-3 xenograft. After the in-vitro assessment of receptor binding, PC-3 tumor-bearing mice were injected with [44Sc]Sc/[68Ga]Ga-NODAGA-AMBA (in blocking studies with bombesin) and in-vivo PET examinations were performed to determine the radiotracer uptake in standardized uptake values (SUV). 44Sc/68Ga-labelled NODAGA-AMBA was produced with high molar activity (approx. 20 GBq/µmoL) and excellent radiochemical purity. The in-vitro accumulation of [44Sc]Sc-NODAGA-AMBA in PC-3 cells was approximately 25-fold higher than that of the control HaCaT cells. Relatively higher uptake was found in vitro, ex vivo, and in vivo in the same tumor with the 44Sc-labelled probe compared to [68Ga]Ga-NODAGA-AMBA. The GRPR specificity of [44Sc]Sc-NODAGA-AMBA was confirmed by significantly (p ≤ 0.01) decreased %ID and SUV values in PC-3 tumors after bombesin pretreatment. The outstanding binding properties of the novel [44Sc]Sc-NODAGA-AMBA to GRPR outlines its potential to be a valuable radiotracer in the imaging of GRPR-positive PCa.

In vivo investigation of Gallium-68 and Bismuth-205/206 labeled beta cyclodextrin for targeted alpha therapy of prostaglandin E2 receptor-expressing tumors in mice.

Prostaglandin E2 (PGE2) molecule and its receptors play an important role in the development of malignancies and metastases therefore PGE2 may play a crucial role in the diagnosis and a new therapeutic target in the field of radionuclide therapy of PGE2-positive tumors. PGE2 form complexes with RAMEB (randomly-methylated-beta-cyclodextrin) with high affinity therefore the aim of this present study was to synthesize a PGE2-specific DOTAGA-RAMEB, which can be labeled with diagnostic and therapeutic isotopes also and binds to PGE2-positive tumors. DOTAGA-RAMEB was labeled with 68Ga and 205/206Bi radionuclides and their radiochemical purity (RCP%), partition coefficient (logP values), and in vitro and in vivo stability were determined. For the assessment of the biological properties and the PGE2 specificity of [68Ga]Ga-DOTAGA-RAMEB and [205/206Bi]Bi-DOTAGA-RAMEB in vivo PET imaging and ex vivo biodistribution studies were performed using healthy control and PGE2-positive BxPC-3 tumor-bearing CB17 SCID mice. The RCP% of the newly synthesized [68Ga]Ga-DOTAGA-RAMEB and [205/206Bi]Bi-DOTAGA-RAMEB was higher than 98 %. In vivo studies showed that the tumor-to-background ratio of [68Ga]Ga-DOTAGA-RAMEB was 2.5 ± 0.2 as a result BxPC-3 tumors were clearly identified on PET images. Beside this the ex vivo biodistribution studies showed that the accumulation rate of [68Ga]Ga-DOTAGA-RAMEB and [205/206Bi]Bi-DOTAGA-RAMEB was similar in the PGE2-positive BxPC-3 tumors.

In Vivo Imaging of Neo-angiogenesis of Transplanted Metastases in Subrenal Capsule Assay Induced Rat Model.

BACKGROUND/AIM: Changes in the expression of neo-angiogenic molecules in the primary tumor and its metastases may significantly affect the efficacy of therapies. The aim of this study was to evaluate the alterations in aminopeptidase N (APN/CD13) and αvß3 integrin receptor expression in serially transplanted mesoblastic nephroma tumor (Ne/De) metastases using 68Gallium (68Ga)-labeled NOTA-cNGR and NODAGA-RGD radiotracers and preclinical positron emission tomography (PET) imaging. MATERIALS AND METHODS: Primary and metastatic mesoblastic nephroma (Ne/De) tumors were induced by subrenal capsule assay (SRCA) in Fischer-344 rats. In vivo PET imaging experiments were performed 8±1 days after the SRCA surgery using intravenously injected 68Ga-NOTA-c(NGR), 68Ga-NODAGA-RGD, and [18F]FDG radiotracers. RESULTS: Among the examined neo-angiogenic molecules, the expression of αvß3 integrin in the tumors was significantly lower than that of APN/CD13. This observation was confirmed by the PET data analysis, where a 2-6-fold higher APN/CD13-specific 68Ga-NOTA-cNGR accumulation was observed in both primary malignancies and metastases. However, a steadily increased accumulation of [18F]FDG, 68Ga-NODAGA-RGD, and 68Ga-NOTA-cNGR was observed in the tumors growing under the renal capsule and in the metastatic parathymic lymph nodes during serial transplantations. The observed increase in 68Ga- NOTA-cNGR accumulation during serial transplantations correlated well with the western blot analysis, where APN/CD13 protein levels were also elevated in the metastatic parathymic lymph nodes. CONCLUSION: The observed increase in glucose metabolism and the up-regulated expression of αvß3 integrin and APN/CD13 during serial transplantations of metastases may indicate enhanced malignancy.

In Vivo Imaging of Ischemia/Reperfusion-mediated Aminopeptidase N Expression in Surgical Rat Model Using 68Ga-NOTA-c(NGR).

BACKGROUND/AIM: Previous studies have already shown that 68Gallium(68Ga)-labeled NGR-based radiopharmaceuticals specifically bind to the neoangiogenic molecule Aminopeptidase N (APN/CD13). The aim of this study was to evaluate the applicability of 68Ga-NOTA-c(NGR) in the in vivo detection of the temporal changes of APN/CD13 expression in the diabetic retinopathy rat model using positron emission tomography (PET). MATERIALS AND METHODS: Ischemia/reperfusion injury was initiated by surgical ligation of the left bulbus oculi of rats. In vivo PET imaging studies were performed after the surgery using 68Ga-NOTA-c(NGR). RESULTS: Significantly higher 68Ga-NOTA-c(NGR) uptake was observed in the surgically-ligated left bulbus, compared to the bulbus of the non-surgical group at each investigated time point. The western blot and histological analysis confirmed the increased expression of the neo-angiogenic marker APN/CD13. CONCLUSION: 68Ga-NOTA-c(NGR) is a suitable radiotracer for the detection of the temporal changes of the ischemia/reperfusion-mediated expression of APN/CD13 in the surgically induced diabetic retinopathy rat model.

A generic gas chromatography method for determination of residual solvents in PET radiopharmaceuticals.

A novel gas chromatography (GC) method for quantitation of volatile organic compounds (VOCs) in 18F- and 11C-radiopharmaceuticals listed in the European Pharmacopoeia (Ph. Eur.) was proposed. Optimized chromatographic parameters were used for separation of ethanol, acetone, acetonitrile, tetrahydrofuran (THF), dibromomethane (DBM), 2-dimethylaminoethanol (deanol), N,N-dimethylformamide (DMF) and dimethyl sulfoxide (DMSO) which could be detected in radioactive drug samples. The calculated peak resolutions (RS) were higher than 2.0 at ethanol concentration of up to 11 m/m%. Reproducible results could be obtained using base deactivated fused silica wool as packing material of inlet liner. Validation parameters showed excellent linearity (r2 ≥0.9998) in the range from 10 to at least 120% of concentration limit of solvents. The accuracy was determined as recovery of concentrations which ranged from 99.3% to 103.8%. Additionally, the relative standard deviation (RSD) of each solvent for inter-day and intra-day precision were in the range of 0.5-4.2% and 0.4-4.4%, respectively. The limit of quantitation (LOQ) for ethanol, acetone, acetonitrile, THF, DBM, deanol, DMF and DMSO was 0.48, 0.42, 0.43, 0.46, 4.35, 0.73, 0.68 and 0.50 mg/L, respectively. The developed procedure was successively applied for quantitation of ethanol, acetone, acetonitrile and deanol in radioactive drug samples of [11C]methionine, [11C]choline, 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) and O-(2-[18F]fluoroethyl)-L-tyrosine ([18F]FET). The proposed GC method applying flame ionization detection (FID) could be adapted in routine quality control of most frequently used positron emission tomography (PET) radiopharmaceuticals to perform the determination of residual solvents with analysis time of 12 min.

Synthesis of 68Ga-Labeled cNGR-Based Glycopeptides and In Vivo Evaluation by PET Imaging.

Tumor hypoxia induces angiogenesis, which is required for tumor cell survival. The aminopeptidase N receptor (APN/CD13) is an excellent marker of angiogenesis since it is overexpressed in angiogenic blood vessels and in tumor cells. Asparagine-glycine-arginine (NGR) peptide analogs bind selectively to the APN/CD13 recepto, therefore, they are important vector molecules in the development of a PET radiotracer which is capable of detecting APN-rich tumors. To investigate the effect of glycosylation and pegylation on in-vivo efficacy of an NGR-based radiotracer, two 68Ga-labeled radioglycopeptides were synthesized. A lactosamine derivative was applied to glycosylation of the NGR derivative and PEG4 moiety was used for pegylation. The receptor targeting potential and biodistribution of the radiopeptides were evaluated with in vivo PET imaging studies and ex vivo tissue distribution studies using B16-F10 melanoma tumor-bearing mice. According to these studies, all synthesized radiopeptides were capable of detecting APN expression in B16-F10 melanoma tumor. In addition, lower hepatic uptake, higher tumor-to background (T/M) ratio and prolonged circulation time were observed for the novel [68Ga]-10 radiotracer due to pegylation and glycosylation, resulting in more contrasting PET imaging. These in vivo PET imaging results correlated well with the ex vivo tissue distribution data.

In vivo preclinical assessment of novel 68Ga-labelled peptides for imaging of tumor associated angiogenesis using positron emission tomography imaging.

Formation and growth of metastases require a new vascular network. Angiogenesis plays an essential role in the expansion and progression of most malignancies. A high number of molecular pathways regulate angiogenesis, including vascular endothelial growth factor (VEGF), αvß3 integrin, matrix metalloproteinases (MMPs), or aminopeptidase N. The aim of this study is to involve new, easily accessible peptide sequences into the of neo-angiogenesis in malignant processes. Labelling of these peptide ligands with 68Ga enable PET imaging of neo-vascularization.

In Vivo Molecular Imaging of the Efficacy of Aminopeptidase N (APN/CD13) Receptor Inhibitor Treatment on Experimental Tumors Using 68Ga-NODAGA-c(NGR) Peptide.

INTRODUCTION: The aminopeptidase N (APN/CD13) receptor plays an important role in the neoangiogenic process and metastatic tumor cell invasion. Clinical and preclinical studies reported that bestatin and actinonin are cytotoxic to APN/CD13-positive tumors and metastases due to their APN/CD13-specific inhibitor properties. Our previous studies have already shown that 68Ga-labeled NGR peptides bind specifically to APN/CD13 expressing tumor cells. The APN/CD13 specificity of 68Ga-NGR radiopharmaceuticals enables the following of the efficacy of antiangiogenic therapy with APN/CD13-specific inhibitors using positron emission tomography (PET). The aim of this in vivo study was to assess the antitumor effect of bestatin and actinonin treatment in subcutaneous transplanted HT1080 and B16-F10 tumor-bearing animal models using 68Ga-NODAGA-c(NGR). MATERIALS AND METHODS: Three days after the inoculation of HT1080 and B16-F10 cells, mice were treated with intraperitoneal injection of bestatin (15 mg/kg) or actinonin (5 mg/kg) for 7 days. On the 5th and 10th day, in vivo PET scans and ex vivo biodistribution studies were performed 90 min after intravenous injection of 5.5 ± 0.2 MBq68Ga-NODAGA-c(NGR). RESULTS: Control-untreated HT1080 and B16-F10 tumors were clearly visualized by the APN/CD13-specific 68Ga-NODAGA-c(NGR) radiopharmaceutical. The western blot analysis also confirmed the strong APN/CD13 positivity in the investigated tumors. We found significantly (p ≤ 0.05) lower radiopharmaceutical uptake after bestatin treatment and higher radiotracer accumulation in the actinonin-treated HT1080 tumors. In contrast, significantly lower (p ≤ 0.01) 68Ga-NODAGA-c(NGR) accumulation was observed in both bestatin- and actinonin-treated B16-F10 melanoma tumors compared to the untreated-control tumors. Bestatin inhibited tumor growth and 68Ga-NODAGA-c(NGR) uptake in both tumor models. CONCLUSION: The bestatin treatment is suitable for suppressing the neoangiogenic process and APN/CD13 expression of experimental HT1080 and B16-F10 tumors; furthermore, 68Ga-NODAGA-c(NGR) is an applicable radiotracer for the in vivo monitoring of the efficacy of the APN/CD13 inhibition-based anticancer therapies.

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.

In vivo assessment of aminopeptidase N (APN/CD13) specificity of different 68Ga-labelled NGR derivatives using PET/MRI imaging.

Aminopeptidase N (APN/CD13) plays an important role in neoangiogenic process in malignancies. Our previous studies have already shown that 68Ga-labelled NOTA conjugated asparagine-glycine-arginine peptide (c[KNGRE]-NH2) specifically bind to APN/CD13 expressing tumors. The aim of this study was to evaluate and compare the APN/CD13 specificity of newly synthesized 68Ga-labelled NGR derivatives in vivo by PET/MRI imaging using hepatocellular carcinoma (He/De) and mesoblastic nephroma (Ne/De) tumor models. PET/MRI and ex vivo biodistribution studies were performed 11 ± 1 days after subcutaneous injection of tumor cells and 90 min after intravenous injection of 68Ga-NOTA-c(NGR), 68Ga-NODAGA-c(NGR), 68Ga-NODAGA-c(NGR) (MG1) or 68Ga-NODAGA-c(NGR) (MG2). The APN/CD13 selectivity was confirmed by blocking experiments and the APN/CD13 expression was verified by immunohistochemistry. 68Ga-labelled c(NGR) derivatives were produced with high specific activity and radiochemical purity. In control animals, low radiotracer accumulation was found in abdominal and thoracic organs. Using tumor-bearing animals we found that the 68Ga-NOTA-c(NGR), 68Ga-NODAGA-c(NGR), and 68Ga-NODAGA-c(NGR) (MG1) derivatives showed higher uptake in He/De and Ne/De tumors, than that of the accumulation of 68Ga-NODAGA-c(NGR) (MG2). APN/CD13 is a very promising target in PET imaging, however, the selection of the appropriate 68Ga-labelled NGR-based radiopharmaceutical is critical for the precise detection of tumor neo-angiogenesis and for monitoring the efficacy of anticancer therapy.

In Vivo Imaging of Hypoxia and Neoangiogenesis in Experimental Syngeneic Hepatocellular Carcinoma Tumor Model Using Positron Emission Tomography.

INTRODUCTION: Hypoxia-induced α ν ß 3 integrin and aminopeptidase N (APN/CD13) receptor expression play an important role in tumor neoangiogenesis. APN/CD13-specific 68Ga-NOTA-c(NGR), α ν ß 3 integrin-specific 68Ga-NODAGA-[c(RGD)]2, and hypoxia-specific 68Ga-DOTA-nitroimidazole enable the in vivo detection of the neoangiogenic process and the hypoxic regions in the tumor mass using positron emission tomography (PET) imaging. The aim of this study was to evaluate whether 68Ga-NOTA-c(NGR) and 68Ga-DOTA-nitroimidazole allow the in vivo noninvasive detection of the temporal changes of APN/CD13 expression and hypoxia in experimental He/De tumors using positron emission tomography. MATERIALS AND METHODS: 5 × 106 hepatocellular carcinoma (He/De) cells were used for the induction of a subcutaneous tumor model in Fischer-344 rats. He/De tumor-bearing animals were anaesthetized, and 90 min after intravenous injection of 10.2 ± 1.1 MBq 68Ga-NOTA-c(NGR) or 68Ga-NODAGA-[c(RGD)]2 (as angiogenesis tracers) or 68Ga-DOTA-nitroimidazole (for hypoxia imaging), whole-body PET/MRI scans were performed. RESULTS: Hypoxic regions and angiogenic markers (α v ß 3 integrin and APN/CD13) were determined using 68Ga-NOTA-c(NGR), 68Ga-DOTA-nitroimidazole, and 68Ga-NODAGA-[c(RGD)]2 in subcutaneously growing He/De tumors in rats. 68Ga-NOTA-c(NGR) showed the strong APN/CD13 positivity of He/De tumors in vivo, by which observation was confirmed by western blot analysis. By the qualitative analysis of PET images, heterogenous accumulation was found inside He/De tumors using all radiotracers. Significantly (p ≤ 0.01) higher SUVmean and SUVmax values were found in the radiotracer avid regions of the tumors than those of the nonavid areas using hypoxia and angiogenesis-specific radiopharmaceuticals. Furthermore, a strong correlation was found between the presence of angiogenic markers, the appearance of hypoxic regions, and the tumor volume using noninvasive in vivo PET imaging. CONCLUSION: 68Ga-DOTA-nitroimidazole and 68Ga-NOTA-c(NGR) are suitable diagnostic radiotracers for the detection of the temporal changes of hypoxic areas and neoangiogenic molecule (CD13) expression, which vary during tumor growth in a hepatocellular carcinoma model.

In vivo preclinical evaluation of the new 68Ga-labeled beta-cyclodextrin in prostaglandin E2 (PGE2) positive tumor model using positron emission tomography.

The cyclooxygenase-2 (COX-2)/prostaglandin E2 (PGE2) pathway plays an important role in tumor development and formation of metastases. It was earlier reported that cyclodextrin derivatives have a high affinity to form complexes with PGE2. Based on these results radiolabeled cyclodextrins - as new radiopharmaceuticals - may open a new pathway in the in vivo imaging and diagnosis of PGE2 positive tumors. The aims of this study were to synthetize the PGE2 specific 68Ga-labeled NODAGA-randomly methylated beta-cyclodextrin (68Ga-NODAGA-RAMEB) and investigate its tumor-targeting properties. NODAGA-RAMEB was labeled with Gallium-68 (68Ga), and the radiochemical purity (RCP%), partition coefficient (logP values), and in vitro-in vivo stability of 68Ga-NODAGA-RAMEB were determined. After intravenous injection of 68Ga-NODAGA-RAMEB the accumulation in organs and tissues was monitored in vivo by positron emission tomography (PET) and ex vivo by gamma counter in BxPC-3 and PancTu-1 tumor-bearing CB17 SCID mice. The RCP% of the newly synthesized 68Ga-NODAGA-RAMEB was higher than 98%. The molar activity was 15.34 ± 1.93 GBq/µmol. The logP of 68Ga labeled NODAGA-RAMEB was - 3.63 ± 0.04. Biodistribution studies showed high accumulation of 68Ga-NODAGA-RAMEB in PGE2 positive BxPC-3 tumors; approximately 15-20-fold higher radiotracer uptake was observed, than that of the background. 68Ga-labeled RAMEB is a promising radiotracer in PET diagnostics of PGE2 positive tumors.

Synthesis of 68Ga-Labeled Biopolymer-based Nanoparticle Imaging Agents for Positron-emission Tomography.

AIM: The purpose of this study was to develop a folate receptor-targeted 68Ga-labeled agent for the detection of cancer cells in mouse models of ovarian cancer by dual positron-emission tomography (PET) and magnetic resonance imaging (MRI). Moreover, we aimed to develop a controlled biopolymer-based chemistry that enables linking metal-binding (here Ga-68) chelators. MATERIALS AND METHODS: The nanoparticle (NP) agent was created by self-assembling of folic acid-modified polyglutamic acid and chelator-modified chitosan followed by radiolabeling with 68Ga (III) ions (68Ga-NODAGA-FA). The structure of modified biopolymers was characterized by spectroscopy. Particle size and mobility were determined. RESULTS: Significant selective binding of NPs was established in vitro using folate receptor-positive KB and - negative MDA-MB-231 cell lines. In vivo tumor uptake of folate-targeted 68Ga3+-radiolabeled NPs was tested using subcutaneous tumor-bearing CB17 SCID mice models. PET/MR dual modalities showed high tumor uptake with 6.5 tumor-to-muscle ratio and NP localization. CONCLUSION: In vivo results supporting the preliminary in vitro tests demonstrated considerably higher 68Ga-NODAGA-FA nanoparticle accumulation in KB tumors than in MDA-MB-231 tumors, thereby confirming the folate receptor-mediated uptake of this novel potential PET imaging agent.

In Vivo Imaging of Experimental Melanoma Tumors using the Novel Radiotracer 68Ga-NODAGA-Procainamide (PCA).

PURPOSE: The most aggressive form of skin cancer is the malignant melanoma. Because of its high metastatic potential the early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of the disease. Previous studies have already shown that benzamide derivatives, such as procainamide (PCA) specifically bind to melanin pigment. The aim of this study was to synthesize and investigate the melanin specificity of the novel 68Ga-labeled NODAGA-PCA molecule in vitro and in vivo using PET techniques. METHODS: Procainamide (PCA) was conjugated with NODAGA chelator and was labeled with Ga-68 (68Ga-NODAGA-PCA). The melanin specificity of 68Ga-NODAGA-PCA was tested in vitro, ex vivo and in vivo using melanotic B16-F10 and amelanotic Melur melanoma cell lines. By subcutaneous and intravenous injection of melanoma cells tumor-bearing mice were prepared, on which biodistribution studies and small animal PET/CT scans were performed for 68Ga-NODAGA-PCA and 18FDG tracers. RESULTS: 68Ga-NODAGA-PCA was produced with high specific activity (14.9±3.9 GBq/µmol) and with excellent radiochemical purity (98%<), at all cases. In vitro experiments showed that 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than Melur cells. Ex vivo biodistribution and in vivo PET/CT studies using subcutaneous and metastatic tumor models showed significantly (p≤0.01) higher 68Ga-NODAGA-PCA uptake in B16-F10 primary tumors and lung metastases in comparison with amelanotic Melur tumors. In experiments where 18FDG and 68Ga-NODAGA-PCA uptake of B16-F10 tumors was compared, we found that the tumor-to-muscle (T/M) and tumor-to-lung (T/L) ratios were significantly (p≤0.05 and p≤0.01) higher using 68Ga-NODAGA-PCA than the 18FDG accumulation. CONCLUSION: Our novel radiotracer 68Ga-NODAGA-PCA showed specific binding to the melanin producing experimental melanoma tumors. Therefore, 68Ga-NODAGA-PCA is a suitable diagnostic radiotracer for the detection of melanoma tumors and metastases in vivo.

Comparative preclinical evaluation of 68Ga-NODAGA and 68Ga-HBED-CC conjugated procainamide in melanoma imaging.

Malignant melanoma is the most aggressive form of skin cancer. The early detection of primary melanoma tumors and metastases using non-invasive PET imaging determines the outcome of this disease. Previous studies have shown that benzamide derivatives (e.g. procainamide) conjugated with PET radionuclides specifically bind to melanin pigment of melanoma tumors. 68Ga chelating agents can have high influence on physiological properties of 68Ga labeled bioactive molecules, as was experienced during the application of HBED-CC on PSMA ligand. The aim of this study was to assess this concept in the case of the melanin specific procaindamide (PCA) and to compare the melanin specificity of 68Ga-labeled PCA using HBED-CC and NODAGA chelators under in vitro and in vivo conditions. Procainamide (PCA) was conjugated with HBED-CC and NODAGA chelators and was labeled with Ga-68. The melanin specificity of 68Ga-HBED-CC-PCA and 68Ga-NODAGA-PCA was investigated in vitro and in vivo using amelanotic (MELUR and A375) and melanin containing (B16-F10) melanoma cell lines. Tumor-bearing mice were prepared by subcutaneous injection of B16-F10, MELUR and A375 melanoma cells into C57BL/6 and SCID mice. 21±2days after tumor cell inoculation and 90min after intravenous injection of the 68Ga-labelledlabeled radiopharmacons whole body PET/MRI scans were performed. 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA were produced with excellent radiochemical purity (98%). In vitro experiments demonstrated that after 30 and 90min incubation time 68Ga-NODAGA-PCA uptake of B16-F10 cells was significantly (p≤0.01) higher than the 68Ga-HBED-CC-conjugated PCA accumulation in the same cell line. Furthermore, significant difference (p≤0.01 and 0.05) was found between the uptake of melanin negative and positive cell lines using 68Ga-NODAGA-PCA and 68Ga-HBED-CC-PCA. In vivo PET/MRI studies using tumor models revealed significantly (p≤0.01) higher 68Ga-NODAGA-PCA uptake (SUVmean: 0.46±0.05, SUVmax: 1.96±0.25,T/M ratio: 40.7±4.23) in B16-F10 tumors in contrast to 68Ga-HBED-CC-PCA where the SUVmean, SUVmax and T/M ratio were 0.13±0.01, 0.56±0.11 and 11.43±1.24, respectively. Melanin specific PCA conjugated with NODAGA chelator showed higher specific binding properties than conjugated with HBED-CC. The chemical properties of the bifunctional chelators used for 68Ga-labeling of PCA determine the biological behaviour of the probes. Due to the high specificity and sensitivity 68Ga-labeled PCA molecules are promising radiotracers in melanoma imaging.