RESUMO
Microglia are rapidly activated by a wide range of neuropathological insults. Quantifying microglial density in vivo would allow a new, potentially important range of clinic-pathological correlations. Microglia express the 18kDa translocator protein (TSPO) which can be quantified by the positron emission tomography (PET) ligand [(11)C]PK11195, although signal quantification is limited by nonspecific binding. New generation TSPO radioligands with an improved signal-to-noise ratio are now available, but variation in their binding affinity for the TSPO between subjects complicates their use. This review describes the principles of PET imaging, the rationale and challenges in targeting the TSPO as means of quantifying microglial activation in vivo, and disease applications that have been studied with TSPO-PET hitherto.
Assuntos
Química Encefálica/fisiologia , Microglia/metabolismo , Tomografia por Emissão de Pósitrons/métodos , Ensaio Radioligante/métodos , Receptores de GABA/análise , Receptores de GABA/metabolismo , Biomarcadores/metabolismo , Humanos , Microglia/fisiologia , Neuroimagem/métodos , Neuroimagem/tendências , Tomografia por Emissão de Pósitrons/tendências , Ensaio Radioligante/tendênciasRESUMO
Radioligand binding has been used for many years to identify new binding sites, characterize receptors, and identify novel ligands. Although various techniques have been developed to improve the efficiency of preparing the biological source of the receptors and for detecting bound radioligand, the principles of the assays remain the same. This unit reviews theory and provides examples of the parameters that can be calculated from radioligand binding data to characterize ligand-receptor interactions. The important aspects of assay development and validation that allow meaningful interpretation are discussed. The selection of a radioligand, buffer and other assay components is critical to developing a useful binding assay. The nature of the binding interaction can also be probed by varying assay conditions.
Assuntos
Ligantes , Ensaio Radioligante/métodos , Sítio Alostérico/fisiologia , Sítios de Ligação/fisiologia , Ligação Competitiva/fisiologia , Soluções Tampão , Centrifugação/métodos , Simulação por Computador , Filtração/métodos , Humanos , Matemática , Fenômenos de Química Orgânica , Guias de Prática Clínica como Assunto , Ligação Proteica/fisiologia , Ensaio Radioligante/tendências , Estatística como Assunto , TemperaturaRESUMO
Beta-adrenoceptors are predominantly located in the cerebral cortex, nucleus accumbens and striatum. At lower densities, they are also present in amygdala, hippocampus and cerebellum. Beta-2 sites regulate glial proliferation during ontogenic development, after trauma and in neurodegenerative diseases. The densities of beta-1 adrenoceptors are changed by stress, in several mood disorders (depression, excessive hostility, schizophrenia) and during treatment of patients with antidepressants. A technique for beta-adrenoceptor imaging in the human brain is not yet available. Although 24 (ant)agonists have been labeled with either (11)C or (18)F and some of these are successful myocardial imaging agents, only two (S-1'-(18)F-fluorocarazolol and S-1'-(18)F-fluoroethylcarazolol) could actually visualize beta-adrenoceptors within the central nervous system. Unfortunately, these radiopharmaceuticals showed a positive Ames test. They may be mutagenic and cannot be employed for human studies. Screening of more than 150 beta-blockers described in the literature yields only two compounds (exaprolol and L643,717) which can still be radiolabeled and evaluated for beta-adenoceptor imaging. However, other imaging techniques could be examined. Cerebral beta-adrenoceptors might be labeled after temporary opening of the blood-brain barrier (BBB) and simultaneous administration of a hydrophilic ligand such as S-(11)C-CGP12388. Another approach to target beta-adrenoceptor ligands to the CNS is esterification of a myocardial imaging agent (such as (11)C-CGP12177), resulting in a lipophilic prodrug which can cross the BBB and is split by tissue esterases. BBB opening is not feasible in healthy subjects, but the prodrug approach may be successful and deserves to be explored.
Assuntos
Encéfalo/diagnóstico por imagem , Encéfalo/metabolismo , Receptores Adrenérgicos beta/metabolismo , Tomografia Computadorizada de Emissão/métodos , Animais , Humanos , Valor Preditivo dos Testes , Radioisótopos/metabolismo , Ensaio Radioligante/métodos , Ensaio Radioligante/tendências , Tomografia Computadorizada de Emissão/tendênciasRESUMO
By virtue of its ideal nuclear physical characteristics for routine nuclear medicine diagnostics and its ready availability, technetium-99m is of outstanding interest in the development of novel radiopharmaceuticals. The potential for the development of 99mTc-based radioligands for the study the receptor function in the central nervous system (CNS) is also well recognised despite the difficulties to be overcome. A fundamental challenge is the pharmacologically acceptable integration of the transition metal technetium, with its specific coordination chemistry, into the molecular entity of CNS receptor ligands. Conceptually, the ligand molecule can be assembled by three building blocks: a small neutral chelate unit, an organic linker that may also serve as a pharmacological modifier and a receptor-binding region derived from selective receptor antagonists. The recent introduction of novel technetium chelate units, particularly mixed-ligand complexes and low-valency organometallic compounds of technetium, provides an impetus for the further development of CNS receptor ligands. Moreover, progress in receptor pharmacology and the experience gained with positron emission tomography radiotracers have facilitated the design of numerous 99mTc-based CNS receptor ligands. The formidable challenge of developing 99mTc probes as single-photon emission tomography imaging agents targeting CNS receptors can be viewed with optimism given the successful development of [99mTc]TRODAT-1 as a 99mTc complex for imaging dopamine transporters in the brain, although there are a number of receptor-specific imaging agents that have so far resisted all efforts to develop them. This review presents recent advances and discusses the remaining hurdles in the design of 99mTc-based CNS receptor imaging agents.
Assuntos
Sistema Nervoso Central/diagnóstico por imagem , Sistema Nervoso Central/metabolismo , Compostos Radiofarmacêuticos , Tecnécio , Tomografia Computadorizada de Emissão de Fóton Único/tendências , Barreira Hematoencefálica , Desenho de Fármacos , Humanos , Ligantes , Compostos de Organotecnécio/química , Ensaio Radioligante/tendências , Compostos Radiofarmacêuticos/químicaRESUMO
Radioligand binding experiments are easy to perform, and provide useful data in many fields. They can be used to study receptor regulation, discover new drugs by screening for compounds that compete with high affinity for radioligand binding to a particular receptor, investigate receptor localization in different organs or regions using autoradiography, categorize receptor subtypes, and probe mechanisms of receptor signaling, via measurements of agonist binding and its regulation by ions, nucleotides, and other allosteric modulators. This unit reviews the theory of receptor binding and explains how to analyze experimental data. Since binding data are usually best analyzed using nonlinear regression, this unit also explains the principles of curve fitting with nonlinear regression.
Assuntos
Ensaio Radioligante/métodos , Animais , Sítios de Ligação/fisiologia , Humanos , Proteínas de Membrana/metabolismo , Ligação Proteica/fisiologia , Ensaio Radioligante/tendênciasRESUMO
Two oxorhenium and two oxotechnetium [SN(R)S/S] mixed ligand complexes bearing the phenothiazine moiety on the tridentate ligand SN(R)S have been synthesized and characterized. The corresponding complexes at tracer level (99mTc) have also been prepared.
Assuntos
Compostos de Organotecnécio/síntese química , Fenotiazinas/química , Receptores Dopaminérgicos/química , Rênio/química , Diagnóstico por Imagem/métodos , Ligantes , Ensaio Radioligante/tendênciasRESUMO
This overview first discusses issues relating to the selection of radioligand for receptor binding assays, including the isotopic label and considerations pertaining to the pharmacological and chemical profile of the ligand. This is followed by a section on characterization of ligand-binding assays, starting with tissue preparation methods, followed by detection of specific binding, determination of incubation and washing conditions and a discussion of saturation and competition assay formats. Quantification of the assay results can be accomplished by autoradiography or film densitometry. Finally, methods and considerations for analysis of the resulting data are presented.
Assuntos
Ensaio Radioligante/métodos , Ensaio Radioligante/tendências , Receptores de Superfície Celular/química , Receptores de Superfície Celular/metabolismo , Animais , Autorradiografia/métodos , Autorradiografia/tendências , Ligação Competitiva , Humanos , Indicadores e Reagentes/metabolismo , Ligantes , Ligação Proteica , Radioisótopos/metabolismo , Receptores de Superfície Celular/análiseRESUMO
Ligand-binding studies remain a very popular technique among many experimentalists. As far as equilibrium experiments are concerned, saturation and displacement curves are commonly performed for simplicity, convenience or for the sake of tradition. However, alternative protocols, such as 'mixed'-type protocols or multiligand experiments, are also possible. Indeed, there are cases where kinetic experiments, usually considered a 'second-choice' experiment, might have a superior resolving power compared to equilibrium ones. A combination of equilibrium and kinetic experiments might be a powerful solution to overcome limits and shortcomings of each specific technique and is discussed in this issue by G. Enrico Rovati. Thus, a careful choice of the design, a protocol optimization and a computerized analysis of the data can yield a dramatic improvement in the precision of the parameter estimation over more conventional approaches.
Assuntos
Ensaio Radioligante/tendências , Simulação por Computador , Cinética , Ligantes , Modelos BiológicosRESUMO
Immunoassay and other ligand assays have made a major impact on medical research and diagnosis since the first modern (radioisotopically-based) methods emerged. These ubiquitous microanalytic techniques are broadly classifiable as first generation (generally of "competitive" design, e.g., radioimmunoassay), and second generation (generally "noncompetitive," and relying on nonisotopic labels) these (often described as "ultrasensitive") being distinguished by dramatic improvements in sensitivity and performance time. A third generation is now in prospect (based on microarrays of antibody microspots) capable of ultrasensitive determination of hundreds of analytes in a drop of blood. Analogous technology (based on oligonucleotide arrays) is under intensive development for DNA analysis. Array technologies are likely to transform diagnostic medicine in the next decade.
Assuntos
Imunoensaio/tendências , Ensaio Radioligante/tendências , Animais , HumanosRESUMO
Using automatic equipment to perform routine RIA procedures can save time and money, improve precision, and free technologists to work on more-complex, non-routine assays. This article examines some of the instruments available for semiautomated and fully automated RIA testing. The pros and cons of automated testing are explored, and a framework for judging the true value and limitations of systems is presented.