RESUMO
MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of HP agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate-by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation; (2) MRI system setup and calibrations; (3) data acquisition and image reconstruction; and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods and Equipment study groups. It further aims to provide a comprehensive reference for future consensus, building as the field continues to advance human studies with this metabolic imaging modality.
Assuntos
Imageamento por Ressonância Magnética , Ácido Pirúvico , Humanos , Ácido Pirúvico/metabolismo , Imageamento por Ressonância Magnética/métodos , Processamento de Imagem Assistida por Computador , Coração , Fígado/diagnóstico por imagem , Fígado/metabolismo , Isótopos de Carbono/metabolismoRESUMO
MRI with hyperpolarized (HP) 13C agents, also known as HP 13C MRI, can measure processes such as localized metabolism that is altered in numerous cancers, liver, heart, kidney diseases, and more. It has been translated into human studies during the past 10 years, with recent rapid growth in studies largely based on increasing availability of hyperpolarized agent preparation methods suitable for use in humans. This paper aims to capture the current successful practices for HP MRI human studies with [1-13C]pyruvate - by far the most commonly used agent, which sits at a key metabolic junction in glycolysis. The paper is divided into four major topic areas: (1) HP 13C-pyruvate preparation, (2) MRI system setup and calibrations, (3) data acquisition and image reconstruction, and (4) data analysis and quantification. In each area, we identified the key components for a successful study, summarized both published studies and current practices, and discuss evidence gaps, strengths, and limitations. This paper is the output of the "HP 13C MRI Consensus Group" as well as the ISMRM Hyperpolarized Media MR and Hyperpolarized Methods & Equipment study groups. It further aims to provide a comprehensive reference for future consensus building as the field continues to advance human studies with this metabolic imaging modality.
RESUMO
A standardized approach to acquiring amyloid PET images increases their value as disease and drug response biomarkers. Most 18F PET amyloid brain scans often are assessed only visually (per regulatory labels), with a binary decision indicating the presence or absence of Alzheimer disease amyloid pathology. Minimizing technical variance allows precise, quantitative SUV ratios (SUVRs) for early detection of ß-amyloid plaques and allows the effectiveness of antiamyloid treatments to be assessed with serial studies. Methods: The Quantitative Imaging Biomarkers Alliance amyloid PET biomarker committee developed and validated a profile to characterize and reduce the variability of SUVRs, increasing statistical power for these assessments. Results: On achieving conformance, sites can justify a claim that brain amyloid burden reflected by the SUVR is measurable to a within-subject coefficient of variation of no more than 1.94% when the same radiopharmaceutical, scanner, acquisition, and analysis protocols are used. Conclusion: This overview explains the claim, requirements, barriers, and potential future developments of the profile to achieve precision in clinical and research amyloid PET imaging.
Assuntos
Doença de Alzheimer , Processamento de Imagem Assistida por Computador , Humanos , Processamento de Imagem Assistida por Computador/métodos , Tomografia por Emissão de Pósitrons/métodos , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Encéfalo/metabolismo , Biomarcadores , Amiloide/metabolismo , Compostos de AnilinaRESUMO
PURPOSE: (99m)Tc-Annexin A5 has been used as a molecular imaging probe for the visualization, characterization and measurement of apoptosis. In an effort to define the quantitative (99m)Tc-annexin A5 uptake criteria that best predict tumor response to treatment, we performed a systematic review and meta-analysis of the results of all clinical imaging trials found in the literature or publicly available databases. METHODS: Included in this review were 17 clinical trials investigating quantitative (99m)Tc-annexin A5 (qAnx5) imaging using different parameters in cancer patients before and after the first course of chemotherapy and/or radiation therapy. Qualitative assessment of the clinical studies for diagnostic accuracy was performed using the QUADAS-2 criteria. Of these studies, five prospective single-center clinical trials (92 patients in total) were included in the meta-analysis after exclusion of one multicenter clinical trial due to heterogeneity. Pooled positive predictive values (PPV) and pooled negative predictive values (NPV) (with 95% CI) were calculated using Meta-Disc software version 1.4. RESULTS: Absolute quantification and/or relative quantification of (99m)Tc-annexin A5 uptake were performed at baseline and after the start of treatment. Various quantitative parameters have been used for the calculation of (99m)Tc-annexin A5 tumor uptake and delta (Δ) tumor changes post-treatment compared to baseline including: tumor-to-background ratio (TBR), ΔTBR, tumor-to-noise ratio, relative tumor ratio (TR), ΔTR, standardized tumor uptake ratio (STU), ΔSTU, maximum count per pixel within the tumor volume (Cmax), Cmax%, absolute ΔU and percentage (ΔU%), maximum ΔU counts, semiquantitative visual scoring, percent injected dose (%ID) and %ID/cm(3). Clinical trials investigating qAnx5 imaging have included patients with lung cancer, lymphoma, breast cancer, head and neck cancer and other less common tumor types. In two phase I/II single-center clinical trials, an increase of ≥25% in uptake following treatment was considered a significant threshold for an apoptotic tumor response (partial response, complete response). In three other phase I/II clinical trials, increases of ≥28%, ≥42% and ≥47% in uptake following treatment were found to be the mean cut-off levels in responders. In a phase II/III multicenter clinical trial, an increase of ≥23% in uptake following treatment was found to be the minimum cut-off level for a tumor response. In one clinical trial, no significant difference in (99m)Tc-annexin A5 uptake in terms of %ID was found in healthy tissues after chemotherapy compared to baseline. In two other clinical trials, intraobserver and interobserver measurements of (99m)Tc-annexin A5 tumor uptake were found to be reproducible (mean difference <5%, kappa = 0.90 and 0.82, respectively) and to be highly correlated with treatment outcome (Spearman r = 0.99, p < 0.0001). The meta-analysis demonstrated a pooled positive PPV of 100% (95% CI 92 - 100%) and a pooled NPV of 70% (95% CI 55 - 82%) for prediction of a tumor response after the first course of chemotherapy and/or radiotherapy in terms of ΔU%. In a symmetric sROC analysis, the AUC was 0.919 and the Q* index was 85.21 %. CONCLUSION: Quantitative (99m)Tc-annexin A5 imaging has been investigated in clinical trials for the assessment of apoptotic tumor responses. This meta-analysis showed a high pooled PPV and a moderate pooled NPV with ΔU cut-off values ranging between 20% and 30%. Standardization of quantification and harmonization of results are required for high-quality clinical research. A standardized uptake value score (SUV, ΔSUV) using quantitative SPECT/CT imaging may be a promising approach to the simple, reproducible and semiquantitative assessment of apoptotic tumor changes.
Assuntos
Anexina A5 , Apoptose , Neoplasias/diagnóstico por imagem , Compostos de Organotecnécio , Tomografia por Emissão de Pósitrons , Compostos Radiofarmacêuticos , Ensaios Clínicos como Assunto , Humanos , Imagem Multimodal , Neoplasias/tratamento farmacológico , Tomografia Computadorizada por Raios XRESUMO
The primary focus of this study was to assess the potential of (99m)Tc-HYNIC-Annexin V for in vivo imaging of apoptosis after systemic chemotherapy and "more localized" radiotherapy in nude mice bearing thymoma tumors and correlating it with TUNEL staining. (99m)Tc-HYNIC-Annexin V was administered intravenously to tumor-bearing mice (n = 25) before and after therapy. Mice were then imaged at 4 hours postinjection, and the animals were subsequently sacrificed. Tumor uptake increased significantly in response to treatment [chemotherapy (n = 8): 1.80 +/- 0.52 %ID/g, p < 0.009; radiotherapy (n = 7): 0.81 +/- 0.07 %ID/g, p < 0.02], compared to the control group (n = 10) (0.57 +/- 0.05 %ID/g). Tumor-to-muscle (T:M) and tumor-to-blood (T:B) ratios were significantly higher in both chemotherapy-treated (p < 0.02 and p < 0.01) and radiotherapy-treated cohorts (p < 0.05 and p < 0.03), compared to the control cohorts at 4 hours postinjection of (99m)Tc-Annxin V. In the post-therapy cohorts, the immunohistochemistry studies indicated a statistically significant correlation between tumor uptake of (99m)Tc-HYNIC-Annexin V and the extent of apoptosis detected by TUNEL-positive staining (r(2) = 0.41). The physiologic localization of the agent was found mainly in the kidneys (34%), liver (11%), and urine (22%). The results suggest that (99m)Tc-HYNIC-Annexin V may be an ideal agent for imaging apoptosis in response to treatment in a thymoma tumor bearing mouse model.
Assuntos
Anexina A5 , Apoptose/efeitos dos fármacos , Apoptose/efeitos da radiação , Compostos de Organotecnécio , Compostos Radiofarmacêuticos , Timoma/diagnóstico por imagem , Neoplasias do Timo/diagnóstico por imagem , Animais , Antineoplásicos Alquilantes/uso terapêutico , Terapia Combinada , Ciclofosfamida/uso terapêutico , Modelos Animais de Doenças , Marcação In Situ das Extremidades Cortadas , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Radiação Ionizante , Cintilografia , Dosagem Radioterapêutica , Timoma/patologia , Timoma/terapia , Neoplasias do Timo/patologia , Neoplasias do Timo/terapia , Distribuição TecidualRESUMO
Molecular imaging tools such as CT, MRI, PET and SPECT, as well as various combinations of these instrument systems, continue to improve and evolve, offering increasingly sensitive and high-resolution images of biological processes in real time. The optimal use of these tools across the continuum of biomedical research and clinical medicine can generate the information that is needed to bridge the gaps that currently exist in drug discovery and development. These gaps negatively affect the promise and potential of translational medicine, in which the knowledge gained from multidisciplinary efforts encompassing genomics, proteomics, biomarker discovery, systems biology and bioinformatics are used to drive R&D, design experiments, predict outcomes, guide patient selection for clinical trials, and define pharmacogenomic parameters for optimizing the safety and efficacy of drug compounds. Thus, molecular imaging tools serve an important role in optimizing the drug discovery and development process.
Assuntos
Pesquisa Biomédica/métodos , Diagnóstico por Imagem/métodos , Desenho de Fármacos , Animais , Ensaios Clínicos como Assunto , HumanosRESUMO
Since its original description in 1972, apoptosis or programmed cell death has been recognized as the major pathway by which the body precisely regulates the number and type of its cells as part of normal embryogenesis, development, and homeostasis. Later it was found that apoptosis was also involved in the pathogenesis of a number of human diseases, cell immunity, and the action of cytotoxotic drugs and radiation therapy in cancer treatment. As such, the imaging of apoptosis with noninvasive techniques such as with radiotracers, including annexin V and lipid proton magnetic resonance spectroscopy, may have a wide range of clinical utility in both the diagnosis and monitoring therapy of a wide range of human disorders. In this chapter we review the basic biochemical and morphologic features of apoptosis and the methods developed thus far to image this complex process in humans.
Assuntos
Apoptose , Diagnóstico por Imagem/métodos , Técnicas de Sonda Molecular , Sondas Moleculares/metabolismo , Animais , Anexina A5/metabolismo , Imagem de Difusão por Ressonância Magnética , Humanos , Lipídeos/química , Espectroscopia de Ressonância Magnética , Pinocitose , Tomografia por Emissão de Pósitrons , Ligação Proteica , Compostos Radiofarmacêuticos/metabolismo , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
OBJECTIVES: The purpose of this study was to evaluate the role of caspase inhibitors on acute resolution of apoptosis in atherosclerotic lesions as evaluated by imaging with annexin A5. BACKGROUND: Extensive apoptosis of macrophages has been reported at the site of plaque rupture in patients dying of acute coronary syndrome. METHODS: Of 31 New Zealand White atherosclerotic rabbits, 6 received broad caspase, 3 received caspase-1, 3 received caspase-3, 3 received caspase-8, and 4 received caspase-9 inhibitors; 12 animals did not receive any caspase inhibitors (treatment control group). Six unmanipulated rabbits were used for comparison (disease control group). Technetium-99m-labeled annexin A5 was used for imaging atherosclerotic lesions; 6 of the 12 uninhibited atherosclerotic rabbits received (99m)Tc-labeled mutant annexin A5 (radiotracer control group). Gamma images were obtained, and quantitative radiotracer uptake was compared with pathologic findings. RESULTS: Atherosclerotic lesions were best visible in untreated atherosclerotic rabbits. Quantitative annexin uptake, defined as the percent of injected dose per g of abdominal aorta tissue, was significantly higher in untreated atherosclerotic animals (mean +/- SD = 0.0515 +/- 0.0099) compared with the normal rabbits (0.0065 +/- 0.0008; p < 0.0001) or atherosclerotic rabbits receiving mutant annexin (0.014 +/- 0.0024; p < 0.0001). Among all caspase inhibitor-treated rabbits, uptake was 39% lower (0.0314 +/- 0.0151) than in untreated atherosclerotic animals (p < 0.01). Uptake was also significantly lower in rabbits receiving broad caspase (0.0206 +/- 0.0058; p < 0.0001) or caspase-1, -3, or -9 (0.0272 +/- 0.0088, p < 0.01; 0.0286 +/- 0.0095, p < 0.01; 0.0300 +/- 0.0021, p < 0.01, respectively) inhibitors. Caspase-8 inhibitor did not affect apoptosis (0.0618 +/- 0.0047; p = NS). Upon histologic characterization, a substantial decrease in macrophage apoptosis was observed in caspase-inhibited animals. CONCLUSIONS: Molecular imaging, using radiolabeled annexin A5, allows the detection of acute resolution of apoptosis as a result of caspase inhibition in experimental atherosclerosis. If proven clinically, this may allow development of novel intervention strategies in acute vascular events.
Assuntos
Anexina A5 , Apoptose/efeitos dos fármacos , Aterosclerose/diagnóstico por imagem , Aterosclerose/tratamento farmacológico , Inibidores de Caspase , Inibidores Enzimáticos , Animais , Aterosclerose/patologia , Masculino , Coelhos , Cintilografia , Compostos Radiofarmacêuticos , Pertecnetato Tc 99m de SódioRESUMO
As positron emission tomography (PET) imaging is becoming more prevalent in clinical practice, it is reasonable to ask if there will be a role for single photon emission computed tomography (SPECT) in the future. This article considers that question, focusing on areas where SPECT can differentiate itself from PET for fundamental reasons: breadth of available radionuclides, simultaneous imaging of multiple agents, cost-effectiveness and adaptability to specific imaging situations. The conclusion is that SPECT will continue to evolve and exist alongside PET and will grow the field of molecular imaging with improved efficiency and patient workflow.
Assuntos
Previsões , Aumento da Imagem/instrumentação , Aumento da Imagem/métodos , Tomografia por Emissão de Pósitrons/instrumentação , Tomografia por Emissão de Pósitrons/tendências , Tomografia Computadorizada de Emissão de Fóton Único/instrumentação , Tomografia Computadorizada de Emissão de Fóton Único/tendências , Estados UnidosRESUMO
BACKGROUND: To determine whether mild to moderate ischemia that is not severe enough to induce myocardial infarction will cause myocardial cell damage or apoptosis, the (99m)Tc-Annexin-V (Tc-A) uptake was studied in groups of rats with various intervals of coronary occlusion and reperfusion times. METHODS AND RESULTS: After left coronary artery occlusion for 15 min (n=23), 10 min (n=23), or 5 min (n=12), Tc-A (80-150 MBq) was injected at 0.5, 1.5, 6, or 24 h after reperfusion. One hour later, to verify the area at risk, (201)Tl (0.74 MBq) was injected just after left coronary artery re-occlusion and the rats were killed 1 min later. Dual tracer autoradiography was performed to assess Tc-A uptake and area at risk. In all 5-min occlusion and reperfusion models, no significant Tc-A uptake was observed in the area at risk. Tc-A uptake ratios in the 15-min and 10-min ischemia models were 4.46+/-3.16 and 2.02+/-0.47 (p=0.078) at 0.5 h after reperfusion, 3.49+/-1.78 and 1.47+/-0.11 (p<0.05) at 1.5 h after reperfusion, 1.60+/-0.43 and 1.34+/-0.23 (p=0.24) at 6 h after reperfusion, 1.50+/-0.33 and 1.28+/-0.33 (p=0.099) at 24 h after reperfusion, respectively. With 15-min ischemia, in 3 of the 5 rats there were a few micro-foci of myocardial cell degeneration and cell infiltration in less than 1% of the ischemic area at 24 h after reperfusion. No significant histological change was observed in rats with 10-min or 5-min ischemia. CONCLUSION: The data indicate that Tc-A binding depends on the severity of ischemia even without a significant amount of histological change or infarction.
Assuntos
Anexina A5/farmacocinética , Compostos de Organotecnécio/farmacocinética , Traumatismo por Reperfusão/metabolismo , Traumatismo por Reperfusão/patologia , Animais , Apoptose , Autorradiografia , Modelos Animais de Doenças , Marcação In Situ das Extremidades Cortadas , Masculino , Miócitos Cardíacos/metabolismo , Miócitos Cardíacos/patologia , Ratos , Ratos Wistar , Índice de Gravidade de Doença , Fatores de TempoRESUMO
To develop a noninvasive direct method for the in vivo tracking of small interfering RNA (siRNA) used in RNA interference, two 18-nucleotide oligoribonucleotides were radiolabeled with technetium-99m ((99m)Tc-RNA). The ability of (99m)Tc-RNA to track delivery was tested in cultured cells and living mice. The cellular delivery of (99m)Tc-RNAs could be quantified by gamma counting and could be visualized by microautoradiography. Radiolabeled RNAs can be efficiently delivered into cells by reaching up to 3x10(5) molecules of small RNAs per cell. Moreover, RNAs were internalized with homogeneous distribution throughout the cytoplasm and nucleus. In tumor-bearing mice, whole-body images and biodistribution studies showed that (99m)Tc-RNAs were delivered to almost all tissues after intravenous injection. The imaging of living animals allowed noninvasive and longitudinal monitoring of the in vivo delivery of these small RNAs. In conclusion, using (99m)Tc radiolabeling, the delivery of small RNAs could be measured quantitatively in cultured cells and could be noninvasively visualized in living animals using a gamma camera. The results of this study could open up a new approach for measuring the in vivo delivery of small RNAs that might further facilitate the development of siRNAs as targeted therapies.
Assuntos
RNA Interferente Pequeno/química , RNA Interferente Pequeno/farmacocinética , Tecnécio/química , Animais , Autorradiografia , Linhagem Celular Tumoral , Células/metabolismo , Cromatografia Líquida de Alta Pressão , Humanos , Marcação por Isótopo , Camundongos , Camundongos Nus , Oligorribonucleotídeos/síntese química , Oligorribonucleotídeos/farmacocinética , Oligorribonucleotídeos Antissenso/síntese química , Oligorribonucleotídeos Antissenso/farmacocinética , RNA Interferente Pequeno/administração & dosagem , Contagem de Cintilação , Frações Subcelulares/metabolismo , Distribuição Tecidual , Transplante HeterólogoRESUMO
UNLABELLED: Labeled annexin V is widely used to detect cell death in vitro and in vivo. Nearly all studies have been done with annexin V derivatized via amine-directed bifunctional agents; it was thought that these molecules retained full bioactivity compared with unmodified protein. We now show that this assumption is incorrect by measuring the affinity of annexin V for cells in vitro by quantitative calcium titration under conditions of low membrane occupancy. METHODS: Annexin V was modified with 4 different amine-directed agents: the N-hydroxysuccinimide esters of hydrazinonicotinic acid, mercaptoacetyltriglycine, and biotin; and with fluorescein isothiocyanate. RESULTS: In all cases, the membrane-binding affinity was decreased by derivatization, even at very low average stoichiometries. A statistical model based on the Poisson distribution accurately predicted the observed heterogeneity of derivatization as a function of average derivatization stoichiometry. This model also showed that multiply derivatized forms, which are the ones most likely to have compromised bioactivity, contributed disproportionately to the binding and imaging signals. The in vitro binding assay correctly predicted in vivo uptake in a mouse liver model of apoptosis for all proteins tested. The annexin V-128 protein, labeled at a single specific site at the N terminus, showed twice as much apoptosis-specific liver uptake as did all forms of annexin V derivatized randomly via amino groups. CONCLUSION: The membrane-binding activity of annexin V is much more sensitive to amine-directed chemical modification than previously realized. New annexin V molecules labeled by site-specific methods will greatly improve sensitivity for detecting cell death in vivo.
Assuntos
Anexina A5/farmacocinética , Apoptose , Marcação por Isótopo/métodos , Fígado/diagnóstico por imagem , Fígado/metabolismo , Modelos Biológicos , Compostos de Organotecnécio/farmacocinética , Animais , Sítios de Ligação , Doença Hepática Induzida por Substâncias e Drogas , Cicloeximida , Hepatopatias/diagnóstico por imagem , Camundongos , Camundongos Endogâmicos BALB C , Ligação Proteica , Cintilografia , Compostos Radiofarmacêuticos/farmacocinéticaRESUMO
The attempt to target the limited copies of messenger RNA (mRNA) in vivo with radiolabeled nucleobase oligomers as antisense probes is challenging. Selecting an antisense molecule with superior properties, enhancing the cellular kinetics, and improving the radiolabeling chemistry would be the reasonable approach to accomplish this goal. The present study reports a method to construct a chimera of phosphorodiamidate morpholino nucleobase oligomer (MORF) covalently conjugated to a peptide containing a cell membrane transduction Tat peptide and an N(2)S(2) chelator for technetium-99m ((99m)Tc) radiolabeling (N(2)S(2)-Tat-MORF). The radiolabeling properties and cellular kinetics of (99m)Tc-N(2)S(2)-Tat-MORF were measured. As hypothesized, the preparation of (99m)Tc-N(2)S(2)-Tat-MORF could be achieved by an instant one-step method with labeling efficiency greater than 95%, and the (99m)Tc-N(2)S(2)-Tat-MORF showed distinct properties in cell culture from those of a control, the same MORF sequence without Tat but with mercaptoacetyltriglycine (MAG(3)) as chelator for (99m)Tc ((99m)Tc-MAG(3)-MORF). (99m)Tc-N(2)S(2)-Tat-MORF achieved maximum accumulation of about 35% within 2 h, while (99m)Tc-MAG(3)-MORF showed lower and steadily increasing accumulations but of less than 1% in 24 h. These preliminary results demonstrated that the proposed chimera has properties for easy labeling, and (99m)Tc-N(2)S(2)-Tat-MORF prepared by this method possesses enhanced cellular kinetics and merits further investigation for in vivo mRNA targeting.
Assuntos
DNA Antissenso/química , Produtos do Gene tat/farmacocinética , Neoplasias Renais/diagnóstico por imagem , Neoplasias Renais/metabolismo , Tecnécio/farmacocinética , Quelantes/química , DNA Antissenso/genética , DNA Antissenso/farmacocinética , Produtos do Gene tat/química , Humanos , Marcação por Isótopo/métodos , Taxa de Depuração Metabólica , Cintilografia , Compostos Radiofarmacêuticos/síntese química , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio/química , Células Tumorais CultivadasRESUMO
PURPOSE: The first aim of the study was to determine whether (99m)Tc-HYNIC-annexin V, a marker of cellular stress and apoptosis, can detect ischemic injury in patients with acute stroke. Secondly, we wished to test radiolabeled annexin's ability to monitor therapy in a rodent model of focal ischemic injury. METHODS: SPECT imaging of patients was performed between 1 and 2 h after intravenous injection of 30 mCi (1,110 MBq) of tracer. Eight MFL4 (anti-FasL) antibody-treated (400 microg i.p. days 0 and 3) and 21 control adult male Sprague-Dawley rats underwent small animal SPECT imaging with 5-10 mCi (185-370 MBq) of tracer, 1 and 6 days after a 2-h intraluminal thread occlusion of the left middle cerebral artery. RESULTS: Two patients with acute stroke had regions of multifocal annexin uptake that correlated with sites of restricted diffusion on MRI. Anti-FasL antibody treatment significantly reduced annexin uptake by 92% with a 60% decrease in the number of caspase-8 staining (apoptotic) neurons on day 1. On day 6, treated animals had an 80% reduction in tracer uptake with a 75% decrease in infarct size as compared with controls. Annexin uptake in controls and treated animals (day 6) linearly correlated with infarct size (r (2)=0.603, p=0.0036) and the number of TUNEL-positive (apoptotic) nuclei (r (2)=0.728, p=0.00084). CONCLUSION: Annexin imaging shows foci of increased uptake at sites of ischemic injury in patients with acute stroke. Annexin imaging can assess the effects of therapy for ischemic cerebral injury in rats, suggesting its potential as a non-invasive indicator of drug efficacy in future clinical trials.
Assuntos
Anexina A5 , Anticorpos Monoclonais/uso terapêutico , Isquemia Encefálica/diagnóstico por imagem , Isquemia Encefálica/tratamento farmacológico , Compostos de Organotecnécio , Acidente Vascular Cerebral/diagnóstico por imagem , Acidente Vascular Cerebral/tratamento farmacológico , Doença Aguda , Animais , Anticorpos Monoclonais Murinos , Isquemia Encefálica/etiologia , Humanos , Masculino , Fármacos Neuroprotetores/uso terapêutico , Prognóstico , Compostos Radiofarmacêuticos , Ratos , Ratos Sprague-Dawley , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Acidente Vascular Cerebral/complicações , Tomografia Computadorizada de Emissão de Fóton Único/métodos , Resultado do TratamentoRESUMO
We conjugated mercaptoacetyltriglycine (MAG(3)) to rh-annexin V to permit radiolabeling with (99m)Tc in an effort to decrease the high kidney and liver accumulation observed for (99m)Tc-labeled Hynic-annexin V. The 36-kDa protein was conjugated at a 5:1 molar ratio with NHS-MAG(3) in HEPES buffer pH 7.8 at room temperature, then quenched with glycine and purified by dialysis. The biopotency of the resulting MAG(3)-annexin was similar to that of Hynic-annexin as determined by a sensitive red blood cell membrane affinity binding assay and a surface plasmon resonance (SPR) assay. The (99m)Tc radiolabeling of MAG(3)-annexin resulted in radiochemical yields of 90% under mildly basic pH conditions. Biodistribution data in normal mice clearly showed a significant decrease in kidney and liver uptake at 1 h postinjection for the (99m)Tc MAG(3)-annexin compared to the (99m)Tc Hynic-annexin (from 24% ID to 4% ID for the liver, and 45% ID to 15% ID for the kidneys, respectively). Autoradiography of the kidneys showed retention of radioactivity in the collecting tubules following administration of both labeled annexins. The (99m)Tc MAG(3)-annexin biodistribution was also characterized by a lower retention of radioactivity in the whole body, but with small intestine accumulation over fivefold higher than observed with (99m)Tc Hynic-annexin. These findings show a definite improvement in renal and hepatic clearance of the MAG(3) radioligand. However, due to the increased radioactivity uptake in the small intestines, the early in vivo detection of ongoing apoptosis in the lower abdomen might be more difficult with (99m)Tc MAG(3)-annexin. Nevertheless, (99m)Tc MAG(3)-annexin may be an attractive alternative to (99m)Tc Hynic-annexin for the in vivo imaging of phosphatidylserine receptors.
Assuntos
Oligopeptídeos/farmacocinética , Compostos Organometálicos/farmacocinética , Tecnécio Tc 99m Mertiatida/farmacocinética , Animais , Quelantes/química , Avaliação Pré-Clínica de Medicamentos , Masculino , Taxa de Depuração Metabólica , Camundongos , Especificidade de Órgãos , Compostos Radiofarmacêuticos/química , Compostos Radiofarmacêuticos/farmacocinética , Tecnécio Tc 99m Mertiatida/química , Distribuição TecidualRESUMO
Apoptosis is a critical factor in AIDS and other viral illnesses, cerebral and myocardial ischemia, autoimmune and neurodegenerative states, organ and bone marrow transplant rejection, and tumor response to chemotherapy and radiation. Improved methods to identify sites of apoptosis are increasing our understanding of the pathophysiology and treatment of these and numerous other human disorders. Here we describe the most used method for labeling annexin V, a protein with a high affinity for apoptotic cells in vitro, with technetium-99m (99mTc) as a radionuclide imaging agent that can localize and non-invasively quantify apoptosis in vivo when coupled with single-photon emission tomography. In this method, annexin V is first attached to the bifunctional chelator molecule hydrazino nicotinate (HYNIC). Once prepared, HYNIC-annexin V can be labeled with 99mTc, a widely available gamma-radiation-emitting radionuclide, for intravenous injection in as little as 30 min without the need for specialized reagents or equipment.
Assuntos
Anexina A5/química , Apoptose , Marcação por Isótopo/métodos , Compostos de Organotecnécio/química , Compostos Radiofarmacêuticos/química , Animais , Anexina A5/análise , Humanos , Masculino , Compostos de Organotecnécio/análise , Traçadores Radioativos , Cintilografia/métodos , Ratos , Ratos Sprague-Dawley , Tecnécio/análise , Tomografia Computadorizada de Emissão de Fóton ÚnicoRESUMO
PURPOSE: (99m)Tc-annexin V (ANX) allows scintigraphic detection of apoptotic cells via specific binding to exposed phosphatidylserine. In myocardial infarction, apoptosis of myocytes is variable and depends especially on the presence or absence of coronary reperfusion. In this study, ANX uptake in non-reperfused experimental myocardial infarcts was compared with uptake of a marker of myocyte necrosis ((111)In-antimyosin antibodies, AM) and an immunohistochemical marker of apoptosis (Apostain). METHODS: The left anterior coronary artery was ligated in 47 Wistar rats, which were then injected with ANX (n=20), AM (n=21) or both (n=6). Myocardial uptake of ANX and AM was determined at 2 h (n=14), 4 h (n=14) and 24 h (n=19) after coronary ligation (CL), by quantitative autoradiography with (n=23) or without (n=24) gamma imaging. Heart-to-lung ratios (HLRs) and infarct-to-remote myocardium activity ratios (INRs) were calculated on the scintigrams and autoradiograms respectively. Cardiac sections were stained with haematoxylin-eosin and Apostain. The above studies were repeated in 12 normal rats. RESULTS: All rats with CL showed increased ANX and AM uptake in cardiac areas on scintigrams 24 h after CL, with HLRs higher than in controls: 3.1+/-0.6 versus 1.5+/-0.3 (p=0.001) for ANX and 1.99+/-0.44 versus 1.01+/-0.05 (p<0.0005) for AM. Autoradiography showed intense ANX and AM uptake in infarcts, with comparable topography and INRs at 2 h, 4 h and 24 h after CL (4.6+/-0.9 versus 5.0+/-1.8 at 24 h), while Apostain staining was very low (0.06+/-0.06% of cells). CONCLUSION: In this model of persistent CL, we observed increased ANX uptake in injured myocardium, comparable in intensity, topography and kinetics to that of AM. There was only minimal Apostain staining in the same areas.
Assuntos
Anexina A5/farmacocinética , Anticorpos Monoclonais/farmacocinética , Apoptose , Infarto do Miocárdio/metabolismo , Infarto do Miocárdio/patologia , Miocárdio/metabolismo , Miosinas/farmacocinética , Compostos de Organotecnécio/farmacocinética , Animais , Coração/diagnóstico por imagem , Radioisótopos de Índio/farmacocinética , Masculino , Taxa de Depuração Metabólica , Infarto do Miocárdio/diagnóstico por imagem , Miocárdio/patologia , Miosinas/imunologia , Cintilografia , Compostos Radiofarmacêuticos/farmacocinética , Ratos , Ratos Wistar , Distribuição TecidualRESUMO
The experimental anticancer drug cyclopentenyl cytosine (CPEC) was associated with cardiotoxicity in a phase I study. The aim of the present study was twofold; first we investigated whether the observed effects could be reproduced in in-vitro and in-vivo rat models. Second, we intended to investigate the underlying mechanism of the possible cardiotoxicity of CPEC. Effects on frequency and contractility were studied on the isolated atria of 18 male Wistar rats. Atria were incubated with 0.1 mmol L(-1) (n = 6) or 1 mmol L(-1) (n = 6) CPEC for 1.5 h and compared with control atria (incubation with buffer solution, n = 6). The cardiac apoptosis-inducing potential was studied in-vivo on 66 rats by 99mTc-Annexin V scintigraphy, followed by postmortem determination of radioactivity in tissues, histological confirmation with the TUNEL assay (late-phase apoptosis), and immunohistochemical staining for cleaved caspase 3 and cytochrome C (early-phase apoptosis). Serum levels of the necrotic cardiomyopathy marker troponin T were also determined. No effect on heart frequency was found in the isolated atria after CPEC treatment. A trend towards a decrease of contraction force was observed. However, the differences were not statistically significant. 99mTc-Annexin V scintigraphy showed no increase in cardiac uptake ratio upon CPEC treatment in the in-vivo rat model, which was confirmed by determination of radioactivity in heart versus blood ratios. At each section a few individual isolated late apoptotic cells (< 5) could be identified by the TUNEL assay in the highest CPEC dose group (90 mg kg(-1)) but not in controls or in rats treated with 60 mg kg(-1) CPEC. Staining for the early apoptosis markers cleaved caspase 3 and cytochrome C did not reveal any significant differences between treated and control rats. Cardiac troponin T levels were not increased after CPEC treatment. CPEC does not affect heart frequency or contraction force in our cardiotoxicity models. Moreover, we did not find an indication of CPEC-induced apoptosis in heart tissue.
Assuntos
Antineoplásicos/toxicidade , Cardiomiopatias/induzido quimicamente , Citidina/análogos & derivados , Coração/efeitos dos fármacos , Animais , Anexina A5/metabolismo , Apoptose/efeitos dos fármacos , Cardiomiopatias/sangue , Cardiomiopatias/patologia , Citidina/toxicidade , Modelos Animais de Doenças , Coração/diagnóstico por imagem , Átrios do Coração/diagnóstico por imagem , Átrios do Coração/efeitos dos fármacos , Átrios do Coração/patologia , Átrios do Coração/fisiopatologia , Marcação In Situ das Extremidades Cortadas , Masculino , Contração Miocárdica/efeitos dos fármacos , Miocárdio/patologia , Compostos de Organotecnécio , Ratos , Ratos Wistar , Tomografia Computadorizada de Emissão de Fóton Único , Troponina T/sangueRESUMO
Pretargeting with bivalent effectors capable of bridging antitumor antibodies has been reported to provide superior results by affinity enhancement. Morpholinos (MORFs) and other DNA analogues used for pretargeting are ideally suited as bivalent effectors since they are easily synthesized and the distance between binding regions, likely to be a determinant of binding, may be adjusted simply by lengthening the chain. The goal of this investigation was to synthesize a bivalent MORF and to determine by surface plasmon resonance (SPR) whether the bivalent MORF exhibited bimolecular binding and whether the MORFs showed improved in vitro hybridization affinity in its bivalent form compared to its monovalent form. An 18 mer amino-derivitized MORF was made bivalent by dimerizing with disuccinimidyl suberate (DSS) in 1-methyl-2-pyrrolidinone (NMP) with N,N-diisopropylethylamine (DIEA) followed by purification by ion exchange chromatography. The in vitro hybridization affinity of bivalent compared to monovalent MORF was then measured by SPR. For these measurements, the complementary biotinylated cDNA was immobilized at coating densities that provided an average spacing of 20-100 angstroms and used to investigate the influence of this spacing on binding of the bivalent MORF with its binding regions separated by 25 A. The yield of bivalent MORF was as high as 45%, and the structure was confirmed by MALDI-TOF mass spectroscopy. When the sensograms obtained by SPR were analyzed using different binding models, the evidence was consistent with bimolecular binding of the bivalent MORF. The dissociation rate constant of the bivalent compared to monovalent MORF was more than 10-fold lower at 2.14 compared to 0.27 x 10(-5) (1/s) (p < 0.05), and since the association rate constants were similar at 8.53 and 5.64 x 10(5) (1/M.s) (p = 0.08), the equilibrium constant for hybridization to the immobilized cDNA of the bivalent compared to the monovalent MORF was almost 20-fold higher at 3.99 compared to 0.21 x 10(10) (1/M) (p < 0.05). In addition, qualitative evidence for bivalent binding of the bivalent MORF was apparent in the lower concentrations necessary to saturate the cDNA. Finally, the stoichiometry interpretation of the binding data provided estimates of the fraction of bivalent MORF binding bimolecularly. Under one set of conditions, this value was 20%. In conclusion, a bivalent MORF was easily synthesized by dimerization of a monovalent MORF. A lower dissociation rate constant and higher equilibrium constant was measured by SPR for the bivalent compared to monovalent MORF in their binding to an immobilized cDNA. These results show that bimolecular binding was occurring in the case of the bivalent MORF and suggest that bivalency may be superior to monovalency in MORF pretargeting applications.
Assuntos
Reagentes de Ligações Cruzadas/química , DNA Antissenso/síntese química , Hibridização de Ácido Nucleico , Ressonância de Plasmônio de Superfície , Aminas , Sítios de Ligação , DNA Complementar/metabolismo , Sistemas de Liberação de Medicamentos , Estrutura Molecular , Morfolinas , MorfolinosRESUMO
OBJECTIVE: The goal of this study was to develop a 99mTc labelled human epidermal growth factor (hEGF) for the in-vivo prediction of cancer cell response to farnesyltransferase inhibitor (FTI) therapy. This is based on the observation that internalization of EGF receptors is inhibited by FTIs. METHODS: We describe the radiolabelling of 99mTc-hEGF using the hydrazinonicotinamide (HYNIC) linker. Binding characteristics of 99mTc-HYNIC-hEGF to the EGF receptor are explored using an in-vitro binding assay. Biodistribution data of the compound in mice and tumour uptake in LoVo tumour bearing athymic mice before and after farnesyltransferase inhibitor therapy are presented. RESULTS: No colloid formation was observed. Binding parameters and LoVo tumour uptake of 99mTc-HYNIC-hEGF did not differ significantly from directly labelled 123I-hEGF values. However, the biodistribution data of the 99mTc-HYNIC-hEGF showed higher uptake in liver and intestines and decreased stomach uptake compared to its 123I analogue. Eight hours after farnesyltransferase inhibitor therapy with R115777, LoVo tumour uptake of 99mTc-HYNIC-hEGF decreased significantly, as shown using planar gamma scintigraphy (the ratio tumour vs. thigh dropped from 2.54+/-0.83 to 0.99+/-0.18). These data confirm the results obtained using 123I-hEGF. CONCLUSION: These data suggest that 99mTc-HYNIC-hEGF is a promising and selective new radiotracer for in-vivo monitoring of the EGF receptor with SPECT. Moreover, 99mTc-HYNIC-hEGF is a possible tool for early therapy response prediction of farnesyltransferase inhibitors.