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.

In Vivo Evaluation of Brain [18F]F-FDG Uptake Pattern Under Different Anaesthesia Protocols.

BACKGROUND/AIM: Since the use of anaesthetics has the drawback of altering radiotracer distribution, preclinical positron emission tomography (PET) imaging findings of anaesthetised animals must be carefully handled. This study aimed at assessing the cerebral [18F]F-FDG uptake pattern in healthy Wistar rats under four different anaesthesia protocols using microPET/magnetic resonance imaging (MRI) examinations. MATERIALS AND METHODS: Post-injection of 15±1.2 MBq of [18F]F-FDG, either while awake or during the isoflurane-induced incubation phase was applied. Prior to microPET/MRI imaging, one group of the rats was subjected to forane-only anaesthesia while the other group was anaesthetised with the co-administration of forane and dexmedetomidine/Dexdor® Results: While as for the whole brain it was the addition of dexmedetomidine/Dexdor® to the anaesthesia protocol that generated the differences between the radiotracer concentrations of the investigated groups, regarding the cortex, the [18F]F-FDG accumulation was rather affected by the way of incubation. To ensure the most consistent and highest uptake, forane-induced anaesthesia coupled with an awake uptake condition seemed to be most suitable method of anaesthetisation for cerebral metabolic assessment. Diminished whole brain and cortical tracer accumulation detected upon dexmedetomidine/Dexdor® administration highlights the significance of the mechanism of action of different anaesthetics on radiotracer pharmacokinetics. CONCLUSION: Overall, the standardization of PET protocols is of utmost importance to avoid the confounding factors derived from anaesthesia.

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.

In Vivo Preclinical Assessment of ß-Amyloid-Affine [11C]C-PIB Accumulation in Aluminium-Induced Alzheimer's Disease-Resembling Hypercholesterinaemic Rat Model.

Aluminum (Al) excess and hypercholesterinaemia are established risks of Alzheimer's disease (AD). The aim of this study was to establish an AD-resembling hypercholesterinaemic animal model-with the involvement of 8 week and 48 week-old Fischer-344 rats-by Al administration for the safe and rapid verification of ß-amyloid-targeted positron emission tomography (PET) radiopharmaceuticals. Measurement of lipid parameters and ß-amyloid-affine [11C]C-Pittsburgh Compound B ([11C]C-PIB) PET examinations were performed. Compared with the control, the significantly elevated cholesterol and LDL levels of the rats receiving the cholesterol-rich diet support the development of hypercholesterinaemia (p ≤ 0.01). In the older cohort, a notably increased age-related radiopharmaceutical accumulation was registered compared to in the young (p ≤ 0.05; p ≤ 0.01). A monotherapy-induced slight elevation of mean standardised uptake values (SUVmean) was statistically not significant; however, adult rats administered a combined diet expressed remarkable SUVmean increment compared to the adult control (SUVmean: from 0.78 ± 0.16 to 1.99 ± 0.28). One and two months after restoration to normal diet, the cerebral [11C]C-PIB accumulation of AD-mimicking animals decreased by half and a third, respectively, to the baseline value. The proposed in vivo Al-induced AD-resembling animal system seems to be adequate for the understanding of AD neuropathology and future drug testing and radiopharmaceutical development.

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 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.

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.

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 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.

Recommendations for selection of additives for stabilization of [18F]FDG.

In this work, two relevant recommendations were established for selection of stabilizers for suppression of radiolysis of [18F]FDG. First, the stabilizing effect could be enhanced using selective scavengers of hydroxyl radicals (OH·). Additionally, the initial radiochemical purity could be retained due to compounds with rate constants of their reactions with OH· (kOH) higher than 109 M-1 s-1. According to these recommendations number of additives among carbohydrates, amino acids, B vitamins and organic salts were successfully applied for stabilization of [18F]FDG.

Cold wall effect eliminating method to determine the contrast recovery coefficient for small animal PET scanners using the NEMA NU-4 image quality phantom.

The contrast recovery coefficients (CRC) were evaluated for five different small animal PET scanners: GE Explore Vista, Genisys4, MiniPET-2, nanoScan PC and Siemens Inveon. The NEMA NU-4 2008 performance test with the suggested image quality phantom (NU4IQ) does not allow the determination of the CRC values for the hot regions in the phantom. This drawback of NU4IQ phantom motivated us to develop a new method for this purpose. The method includes special acquisition and reconstruction protocols using the original phantom, and results in an artificially merged image enabling the evaluation of CRC values. An advantageous feature of this method is that it stops the cold wall effect from distorting the CRC calculation. Our suggested protocol results in a set of CRC values contributing to the characterization of small animal PET scanners. GATE simulations were also performed to validate the new method and verify the evaluated CRC values. We also demonstrated that the numerical values of this parameter depend on the actual object contrast of the hot region(s) and this mainly comes from the spillover effect. This effect was also studied while analysing the background activity level around the hot rods. We revealed that the calculated background mean values depended on the target contrast in a scanner specific manner. Performing the artificially merged imaging procedure and additional simulations using the micro hollow sphere (MHS) phantom geometry, we also proved that the inactive wall around the hot spheres can have a remarkable impact on the calculated CRC. In conclusion, we have shown that the proposed artificial merging procedure and the commonly used NU4IQ phantom prescribed by the NEMA NU-4 can easily deliver reliable CRC data otherwise unavailable for the NU4IQ phantom in the conventional protocol or the MHS phantom.