RESUMO
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.
Assuntos
Autorradiografia , Modelos Animais de Doenças , Fluordesoxiglucose F18 , Tomografia por Emissão de Pósitrons , Animais , Ratos , Linhagem Celular Tumoral , Distribuição Tecidual , Leucemia Mieloide Aguda/patologia , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/diagnóstico por imagem , Compostos Radiofarmacêuticos , Masculino , HumanosRESUMO
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.
Assuntos
Cobalto , Fluordesoxiglucose F18 , Neoplasias Ovarianas , Humanos , Feminino , Animais , Camundongos , Hipóxia Tumoral , Xenoenxertos , Linhagem Celular Tumoral , Neoplasias Ovarianas/diagnóstico por imagem , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , Hipóxia/diagnóstico por imagemRESUMO
As malignancies remain one of the major health concerns worldwide, increasing focus has been centered around the application of cyclodextrins (CDs) in cancer imaging and therapy due to their outstanding inclusion forming capability. Albeit the physicochemical properties of CDs were intensively elucidated, the spread of their clinical application is limited by the relative paucity of knowledge about their pharmacokinetic profile, especially biodistribution. Studies applying fluorescently- CDs, or CD-based MRI contrast agents revealed much about pharmacokinetics and diagnostic applications; however, derivatives labelled with positron emitters seem superior molecular probes in the investigation of the route of CDs in biological niche. In vivo imaging based on preclinical tumor-bearing model systems are well-suited to evaluate the whole-body distribution of the two most frequently assessed CDs: randomly methylated ß-cyclodextrin (RAMEB), and hydroxypropyl-ß-cyclodextrin (HPBCD). Exploiting the firm signaling interaction between cancer-related cyclooxygenase-2, prostaglandin E2 (PGE2) and RAS oncoprotein, radioconjugated, PGE2-affine CDs project the establishment of novel imaging probes and therapeutic agents. Currently, we provide an overview of the preclinical studies on CD pharmacokinetics highlighting the significance of the integration of translational discoveries into human patient care.
Assuntos
Ciclodextrinas , Neoplasias , Humanos , Ciclodextrinas/química , Distribuição Tecidual , Dinoprostona , 2-Hidroxipropil-beta-Ciclodextrina/química , Neoplasias/diagnóstico por imagem , Neoplasias/tratamento farmacológicoRESUMO
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.
Assuntos
Neoplasias , beta-Ciclodextrinas , Animais , Radioisótopos de Gálio/metabolismo , Dinoprostona/metabolismo , Compostos Heterocíclicos com 1 Anel/metabolismo , Neoplasias/diagnóstico por imagem , Neoplasias/metabolismoRESUMO
Since NGR-tripeptides (asparagine-glycine-arginine) selectively target neoangiogenesis-associated Aminopeptidase N (APN/CD13) on cancer cells, we aimed to evaluate the in vivo tumour targeting capability of radiolabelled, NGR-containing, ANP/CD13-selective [213Bi]Bi-DOTAGA-cKNGRE in CD13pos. HT1080 fibrosarcoma-bearing severe combined immunodeficient CB17 mice. 10 ± 1 days after cancer cell inoculation, positron emission tomography (PET) was performed applying [68Ga]Ga-DOTAGA-cKNGRE for tumour verification. On the 7th, 8th, 10th and 12th days the treated group of tumourous mice were intraperitoneally administered with 4.68 ± 0.10 MBq [213Bi]Bi-DOTAGA-cKNGRE, while the untreated tumour-bearing animals received 150 µL saline solution. In addition to body weight (BW) and tumour volume measurements, ex vivo biodistribution studies were conducted 30 and 90 min postinjection (pi.). The following quantitative standardised uptake values (SUV) confirmed the detectability of the HT1080 tumours: SUVmean and SUVmax: 0.37 ± 0.09 and 0.86 ± 0.14, respectively. Although no significant difference (p ≤ 0.05) was encountered between the BW of the treated and untreated mice, their tumour volumes measured on the 9th, 10th and 12th days differed significantly (p ≤ 0.01). Relatively higher [213Bi]Bi-DOTAGA-cKNGRE accumulation of the HT1080 neoplasms (%ID/g: 0.80 ± 0.16) compared with the other organs at 90 min time point yields better tumour-to-background ratios. Therefore, the therapeutic application of APN/CD13-affine [213Bi]Bi-DOTAGA- cKNGRE seems to be promising in receptor-positive fibrosarcoma treatment.
RESUMO
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.
Assuntos
Radioisótopos de Gálio , beta-Ciclodextrinas , 2-Hidroxipropil-beta-Ciclodextrina , Acetatos , Linhagem Celular Tumoral , Dinoprostona , Compostos Heterocíclicos com 1 Anel , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos , AnimaisRESUMO
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.
Assuntos
Hipercolesterolemia , Neoplasias Renais , Leucemia , Nefroma Mesoblástico , Animais , Ratos , Fluordesoxiglucose F18 , Ratos Long-Evans , Tomografia por Emissão de Pósitrons , Neoplasias Renais/diagnóstico por imagem , Lipídeos , Compostos Radiofarmacêuticos , Tomografia por Emissão de Pósitrons combinada à Tomografia ComputadorizadaRESUMO
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.
Assuntos
Neoplasias da Próstata , Receptores da Bombesina , Acetatos , Animais , Bombesina , Linhagem Celular Tumoral , Radioisótopos de Gálio , Compostos Heterocíclicos com 1 Anel , Humanos , Masculino , Camundongos , Tomografia por Emissão de Pósitrons/métodos , Neoplasias da Próstata/metabolismo , Receptores da Bombesina/metabolismoRESUMO
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.
Assuntos
Neoplasias , beta-Ciclodextrinas , Animais , Bismuto , Linhagem Celular Tumoral , Dinoprostona/metabolismo , Radioisótopos de Gálio/química , Camundongos , Camundongos SCID , Neoplasias/tratamento farmacológico , Tomografia por Emissão de Pósitrons , Radioisótopos , Receptores de Prostaglandina/metabolismo , Receptores de Prostaglandina/uso terapêutico , Distribuição Tecidual , beta-Ciclodextrinas/químicaRESUMO
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.
Assuntos
Neoplasias Renais , Nefroma Mesoblástico , Animais , Linhagem Celular Tumoral , Fluordesoxiglucose F18 , Radioisótopos de Gálio/química , Integrinas , Neoplasias Renais/diagnóstico por imagem , Oligopeptídeos/química , Oligopeptídeos/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ratos , Ratos Endogâmicos F344 , Ensaio de Cápsula Sub-RenalRESUMO
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.
Assuntos
Radioisótopos de Gálio/química , Melanoma Experimental/irrigação sanguínea , Neovascularização Patológica/diagnóstico por imagem , Peptídeos/química , Tomografia por Emissão de Pósitrons/métodos , Animais , Antígenos CD13/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Radioisótopos de Gálio/farmacocinética , Integrina alfaVbeta3/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Neovascularização Patológica/metabolismo , Distribuição Tecidual , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismoRESUMO
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.
Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas Experimentais , Neovascularização Patológica/diagnóstico por imagem , Tomografia por Emissão de Pósitrons , Animais , Carcinoma Hepatocelular/irrigação sanguínea , Carcinoma Hepatocelular/diagnóstico por imagem , Hipóxia Celular , Linhagem Celular , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/diagnóstico por imagem , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Compostos Radiofarmacêuticos/farmacologia , Ratos , Ratos Endogâmicos F344RESUMO
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.
Assuntos
Dinoprostona/metabolismo , Avaliação Pré-Clínica de Medicamentos , Radioisótopos de Gálio/administração & dosagem , Neoplasias/diagnóstico , Neoplasias/tratamento farmacológico , Compostos Radiofarmacêuticos/metabolismo , beta-Ciclodextrinas/administração & dosagem , Acetatos/administração & dosagem , Acetatos/química , Acetatos/metabolismo , Animais , Linhagem Celular Tumoral , Radioisótopos de Gálio/química , Radioisótopos de Gálio/metabolismo , Compostos Heterocíclicos com 1 Anel/administração & dosagem , Compostos Heterocíclicos com 1 Anel/química , Compostos Heterocíclicos com 1 Anel/metabolismo , Humanos , Masculino , Camundongos , Camundongos SCID , Neoplasias/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Compostos Radiofarmacêuticos/administração & dosagem , Compostos Radiofarmacêuticos/química , Distribuição Tecidual/fisiologia , beta-Ciclodextrinas/química , beta-Ciclodextrinas/metabolismoRESUMO
We have developed a unified, versatile vector set for expression of recombinant proteins, fit for use in any bacterial, yeast, insect or mammalian cell host. The advantage of this system is its versatility at the vector level, achieved by the introduction of a novel expression cassette. This cassette contains a unified multi-cloning site, affinity tags, protease cleavable linkers, an optional secretion signal, and common restriction endonuclease sites at key positions. This way, genes of interest and all elements of the cassette can be switched freely among the vectors, using restriction digestion and ligation without the need of polymerase chain reaction (PCR). This vector set allows rapid protein expression screening of various hosts and affinity tags. The reason behind this approach was that it is difficult to predict which expression host and which affinity tag will lead to functional expression. The new system is based on four optimized and frequently used expression systems (Escherichia coli pET, the yeast Pichia pastoris, pVL and pIEx for Spodoptera frugiperda insect cells and pLEXm based mammalian systems), which were modified as described above. The resulting vector set was named pONE series. We have successfully applied the pONE vector set for expression of the following human proteins: the tumour suppressor RASSF1A and the protein kinases Aurora A and LIMK1. Finally, we used it to express the large multidomain protein, Rho-associated protein kinase 2 (ROCK2, 164 kDa) and demonstrated that the yeast Pichia pastoris reproducibly expresses the large ROCK2 kinase with identical activity to the insect cell produced counterpart. To our knowledge this is among the largest proteins ever expressed in yeast. This demonstrates that the cost-effective yeast system can match and replace the industry-standard insect cell expression system even for large and complex mammalian proteins. These experiments demonstrate the applicability of our pONE vector set.
Assuntos
Clonagem Molecular/métodos , Vetores Genéticos , Proteínas Recombinantes/isolamento & purificação , Transfecção/métodos , Animais , Aurora Quinase A/genética , Aurora Quinase A/isolamento & purificação , Escherichia coli/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Quinases Lim/genética , Quinases Lim/isolamento & purificação , Pichia/genética , Pichia/metabolismo , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/isolamento & purificação , Quinases Associadas a rho/genética , Quinases Associadas a rho/isolamento & purificaçãoRESUMO
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.
Assuntos
Receptor 1 de Folato/isolamento & purificação , Radioisótopos de Gálio/química , Nanopartículas/química , Neoplasias Ovarianas/diagnóstico por imagem , Acetatos/química , Animais , Quelantes/química , Quitosana/síntese química , Quitosana/química , Quitosana/uso terapêutico , Modelos Animais de Doenças , Feminino , Receptor 1 de Folato/química , Ácido Fólico/química , Radioisótopos de Gálio/uso terapêutico , Compostos Heterocíclicos com 1 Anel/química , Humanos , Imageamento por Ressonância Magnética/métodos , Camundongos , Nanopartículas/uso terapêutico , Neoplasias Ovarianas/patologia , Ácido Poliglutâmico/química , Tomografia por Emissão de Pósitrons/métodosRESUMO
Melanoma is a devastating form of skin cancer with high tendency to metastasis. This work addresses the development of new targeted nanoparticles that can be used for single-photon emission computed tomography (SPECT) imaging of melanoma. Melanoma-specific glycoprotein nonmetastatic b (GPNMB) antigen targeted and nontargeted gemini nanoparticles were prepared, characterized, and radiolabeled with 111In. 111In-labeled nanoparticles were composed of gemini surfactant grafted with monoclonal antibody Fab fragment that targeted GPNMB. Specific uptake of GPNMB-Fab was studied in six melanoma cell lines using flow cytometry. In vitro cellular uptake and internalization were studied using flow cytometry, confocal laser scanning microscopy, and radiometric techniques. Specific uptake of anti-GPNMB targeted nanoparticles was observed in GPNMB expressing cells, which was higher than low expressing or control cells. In vitro studies showed that conjugation of GPNMB targeted nanoparticles led to enhanced intracellular uptake of the nanodelivery system, which is critical for drug delivery. In vivo distribution of the nanoparticles was studied by microSPECT/CT imaging and ex vivo biodistribution. Tumor uptake was significantly higher ( p < 0.05) in nontargeted nanoparticles (5.47 ± 0.46%IA/cc) compared to GPNMB targeted nanoparticles (1.87 ± 0.27% ID/cc), which might be attributed to the high spleen uptake of the targeted formulation. These findings demonstrated that the radiolabeled gemini nanoparticles are promising for image-guided radiotherapy of melanoma. Formulation optimization is needed to improved tumor uptake and in vivo intracellular delivery for radiotherapeutic applications.
Assuntos
Calcitriol/análogos & derivados , Proteínas do Olho/metabolismo , Índio/química , Melanoma/tratamento farmacológico , Glicoproteínas de Membrana/metabolismo , Nanopartículas/química , Tensoativos/química , Tensoativos/uso terapêutico , Animais , Calcitriol/química , Calcitriol/uso terapêutico , Linhagem Celular Tumoral , Sistemas de Liberação de Medicamentos , Humanos , Melanoma/metabolismo , Camundongos , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
BACKGROUND: With the advances in radiopharmaceutical research, the development of image-guided therapy has become a major interest. While the development of theranostic nanotherapeutics is frequently associated with cancer chemotherapy, phototherapy and radiotherapy, there is little information available on the in vivo monitoring of gene delivery systems and the application of image-guided approach in gene therapy. The goal of this work was to determine the in vivo behavior of DNA delivery nanosystems - based on cationic gemini surfactants - designed for image-guided gene therapy. We tested the feasibility of monitoring tumor accumulation of gene delivery nanoparticles by positron emission tomography. METHODS: To be able to conjugate radiotracers to the nanoparticles, a deferoxamine-modified gemini surfactant was synthesized, DNA-containing lipoplex nanoparticles were formulated, and radiolabeled with Zirconium-89 (89Zr). The pharmacokinetics and biodistribution of 89Zr labeled surfactant and 89Zr labeled nanoparticles were monitored in mice by microPET/CT imaging and ex vivo gamma counting. RESULTS: Modification of the nanoparticles with deferoxamine did not alter their physicochemical properties. The radiolabeled nanoparticles (labeling efficiency of 95±3%) were stable in PBS and serum. The biological half-life of the 89Zr labeled nanoparticles was significantly higher compared to 89Zr labeled surfactant. As expected, the nanoparticles had significantly higher liver accumulation than the radiolabeled surfactant alone and lower kidney accumulation. Tumor uptake was detected at 2 hours post injection and decreased throughout the 3-day monitoring. CONCLUSION: We propose that radiolabeling DNA delivery lipoplex nanosystems is a promising approach for the design and optimization of image-guided nanomedicines, especially in the context of cancer gene therapy.
Assuntos
Técnicas de Transferência de Genes , Imageamento Tridimensional , Lipídeos/química , Nanopartículas/química , Radioisótopos/química , Zircônio/química , Animais , Sobrevivência Celular , Desferroxamina/química , Regulação da Expressão Gênica , Terapia Genética , Camundongos Nus , Nanopartículas/ultraestrutura , Neoplasias/metabolismo , Neoplasias/patologia , Tomografia por Emissão de Pósitrons , Radioisótopos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Tensoativos/química , Distribuição Tecidual , Zircônio/farmacocinéticaRESUMO
Lysyl oxidase (LOX) enzymes as potential drug targets maintain constant attention in the therapy of fibrosis, cancer and metastasis. In order to measure the inhibitory activity of small molecules on the LOX enzyme family members a fluorometric activity screening method was developed. During assay validation, previously reported non-selective small inhibitor molecules (BAPN, MCP-1, thiram, disulfiram) were investigated on all of the major LOX enzymes. We confirmed that MCP-1, thiram, disulfiram are in fact pan-inhibitors, while BAPN inhibits only LOX-like enzymes (preferably LOX-like-protein-2, LOXL2) in contrast to the previous reports. We measured the LOX inhibitory profile of a small targeted library generated by 2D ligand-based chemoinformatics methods. Ten hits (10.4% hit rate) were identified, and the compounds showed distinct activity profiles. Potential inhibitors were also identified for LOX-like-protein-3 (LOXL3) and LOX-like-protein-4 (LOXL4), that are considered as emerging drug targets in the therapy of melanoma and gastric cancer.
Assuntos
Inibidores Enzimáticos/farmacologia , Proteína-Lisina 6-Oxidase/antagonistas & inibidores , Bibliotecas de Moléculas Pequenas/farmacologia , Aminopropionitrilo/química , Aminopropionitrilo/farmacologia , Dissulfiram/química , Dissulfiram/farmacologia , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/química , Humanos , Ligantes , Estrutura Molecular , Proteína-Lisina 6-Oxidase/metabolismo , Piridinas/química , Piridinas/farmacologia , Bibliotecas de Moléculas Pequenas/síntese química , Bibliotecas de Moléculas Pequenas/química , Relação Estrutura-Atividade , Tionas/química , Tionas/farmacologia , Tiram/química , Tiram/farmacologiaRESUMO
PURPOSE: Alpha melanocyte stimulating hormone (α-MSH) enhances melanogenesis in melanoma malignum by binding to melanocortin-1 receptors (MC1-R). Earlier studies demonstrated that alpha-MSH analog NAPamide molecule specifically binds to MC1-R receptor. Radiolabeled NAPamide is a promising radiotracer for the non-invasive detection of melanin producing melanoma tumors by Positron Emission Tomography (PET). In this present study the MC1-R selectivity of the newly developed Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using melanoma tumors. METHODS: DOTA-NAPamide was labeled with Ga-68 and Sc-44 radionuclides. The MC1-R specificity of Ga-68- and Sc-44-labeled DOTA-NAPamide was investigated in vitro and in vivo using MC1-R positive (B16-F10) and negative (A375) melanoma cell lines. For in vivo imaging studies B16-F10 and A375 tumor-bearing mice were injected with 44Sc/68Ga-DOTA-NAPamide (in blocking studies with α-MSH) and whole body PET/MRI scans were acquired. Radiotracer uptake was expressed in terms of standardized uptake values (SUVs). RESULTS: 44Sc/68Ga-labeled DOTA-NAPamide were produced with high specific activity (approx. 19 GBq/µmol) and with excellent radiochemical purity (99%<). MC1-R positive B16-F10 cells showed significantly (p≤0.01) higher in vitro radiotracer accumulation than that of receptor negative A375 melanoma cells. In animal experiments, also significantly (p≤0.01) higher Ga-68-DOTA-NAPamide (SUVmean: 0.38±0.02), and Sc-44-DOTA-NAPamide (SUVmean: 0.52±0.13) uptake was observed in subcutaneously growing B16-F10 tumors, than in receptor negative A375 tumors, where the SUVmean values of Ga-68-DOTA-NAPamide and Sc-44-DOTA-NAPamide were 0.04±0.01 and 0.07±0.01, respectively. Tumor-to-muscle (T/M SUVmean) ratios were approximately 15-fold higher in B16-F10 tumor-bearing mice, than that of A375 tumors, and this difference was also significant (p≤0.01) using both radiotracers after 60 min incubation time. CONCLUSION: Our newly synthesized 44Sc-labeled DOTA-NAPamide probe showed excellent binding properties to melanocortin-1 receptor (MC1-R) positive melanoma cell and tumors. Due to its high specificity and sensitivity 44Sc-DOTA-NAPamide is a promising radiotracer in molecular imaging of malignant melanoma.
Assuntos
Melanoma Experimental/diagnóstico por imagem , Melanoma Experimental/metabolismo , Fragmentos de Peptídeos/farmacocinética , Compostos Radiofarmacêuticos/farmacocinética , Receptor Tipo 1 de Melanocortina/metabolismo , alfa-MSH/análogos & derivados , Animais , Linhagem Celular Tumoral , Feminino , Radioisótopos de Gálio , Imageamento por Ressonância Magnética , Camundongos Endogâmicos C57BL , Camundongos SCID , Tomografia por Emissão de Pósitrons , Radioisótopos , Escândio , alfa-MSH/farmacocinéticaRESUMO
A new biocompatible, biodegradable, self-assembled chitosan-based nanoparticulate product was successfully synthesized and radiolabeled with technetium-99m, and studied as a potential new SPECT or SPECT/CT imaging agent for diagnosis of folate receptor overexpressing tumors. In the present study we examined the conditions of a preclinical application of this labeled nanosystem in early diagnosis of spontaneously diseased veterinary patient using a human SPECT/CT device. The results confirmed that the nanoparticles accumulated in tumor cells overexpressing folate receptors, contrast agent revealed higher uptake in the tumor for a long time. Preclinical trials verified that the new nanoparticles are able to detect folate-receptor-overexpressing tumors in spontaneously diseased animal models with enhanced contrast.