RESUMEN
PURPOSE: We aimed to fulfill a need for a radioligand that may be simply labeled with carbon-11 for effective positron emission tomography (PET) imaging of brain 5-HT(1A) receptors. METHODS: Racemic RWAY (2,3,4,5,6,7-hexahydro-1-[4-[1-[4-(2-methoxyphenyl)piperazinyl]]-2-phenylbutyryl]-1H-azepine) has high affinity for 5-HT(1A) receptors. The enantiomers of RWAY and O-desmethyl-RWAY, synthesized from commercially available materials, were each labeled with carbon-11 by treating the respective O-desmethyl precursor with [(11)C]iodomethane, and injected into rhesus monkey for measurement of regional brain uptake. The 5-HT(1A) selectivity of (R)-[(11)C]RWAY was checked by administering WAY-100635, before and after radioligand administration. Radiometabolites of (R)-[(11)C]RWAY in blood and urine were analyzed by HPLC with partial elucidation of their structures by LC-MS-MS. RESULTS: (R)-[(11)C]RWAY was a 5-HT(1A) receptor antagonist exhibiting high brain uptake with regional distribution consistent with specific binding to 5-HT(1A) receptors. The similar affinity, (S)-[(11)C]RWAY was a weak partial agonist at 5-HT(1A) receptors exhibiting similar brain peak uptake with much less 5-HT(1A) receptor-specific binding. The maximal ratio in receptor-rich cingulate gyrus to receptor-devoid cerebellum reached 6.4 at 87.5 min after injection of (R)-[(11)C]RWAY. After treatment with WAY-100635 before or after (R)-[(11)C]RWAY administration, radioactivity levels in 5-HT(1A) receptor-rich regions were reduced almost to that in cerebellum. Blood and urine radiometabolites were less lipophilic than parent and were not due to hydrolysis but to ring hydroxylations, oxidation, and dephenylation. CONCLUSION: (R)-[(11)C]RWAY is simply prepared and an effective antagonist for imaging brain 5-HT(1A) receptors. This radioligand resists hydrolysis in vivo, gives less lipophilic radiometabolites, and warrants further PET studies in human subjects.
Asunto(s)
Azepinas/farmacocinética , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Piperazinas/farmacocinética , Tomografía de Emisión de Positrones/métodos , Receptor de Serotonina 5-HT1A/metabolismo , Antagonistas del Receptor de Serotonina 5-HT1 , Animales , Azepinas/química , Evaluación Preclínica de Medicamentos , Marcaje Isotópico/métodos , Macaca mulatta , Masculino , Tasa de Depuración Metabólica , Piperazinas/química , Radiofármacos/síntesis química , Radiofármacos/farmacocinética , Reproducibilidad de los Resultados , Sensibilidad y Especificidad , Distribución TisularRESUMEN
[(11)C](R)-(-)-RWAY has been shown to be a promising radioligand for imaging brain 5-HT(1A) receptors with positron emission tomography in rodents and nonhuman primates. We now report the first use of [(11)C](R)-(-)-RWAY in six healthy human subjects, using kinetic brain imaging and serial arterial measurements of plasma parent radiotracer. At 80 min after radiotracer injection, activity ratios were about three for brain receptor-rich regions compared with cerebellum. However, the washout from brain was unexpectedly slow relative to plasma clearance of the parent radiotracer. This disparity between brain and plasma activity was quantified with distribution volume calculated from increasingly truncated brain imaging data. In both receptor-rich regions and cerebellum, distribution volumes were unstable and increased continuously from 90 to 150 min by about 30%. This increasing distribution volume was unlikely due to the variations or errors of plasma input at later time points, since a similar truncation of plasma time points from 150 to 90 min did not significantly affect the analysis of the brain data. When the metabolites of [(11)C](R)-(-)-RWAY in human and monkey were compared, a moderate lipophilic radiometabolite was present at a significantly higher percentage of total plasma radioactivity in human than in monkey. The relatively slow washout of activity from brain and the temporal instability of distribution volume likely reflect the accumulation of radiometabolite(s) in human brain. Although prior studies in rodents and nonhuman primates showed [(11)C](R)-(-)-RWAY to be a promising radiotracer, we suspect that a species difference in metabolism caused this serious deficiency in humans.
Asunto(s)
Azepinas/farmacocinética , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Piperazinas/farmacocinética , Receptor de Serotonina 5-HT1A/metabolismo , Adulto , Autorradiografía , Azepinas/sangre , Unión Competitiva , Encéfalo/anatomía & histología , Mapeo Encefálico , Radioisótopos de Carbono/sangre , Radioisótopos de Carbono/farmacocinética , Femenino , Humanos , Imagen por Resonancia Magnética/métodos , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Tasa de Depuración Metabólica/fisiología , Persona de Mediana Edad , Piperazinas/sangre , Tomografía de Emisión de Positrones , Factores de Tiempo , Distribución TisularRESUMEN
Limited brain uptake of radioligands with otherwise optimal properties for imaging brain receptors can be improved by blocking the effect of P-glycoprotein (P-gp), an efflux pump at the blood-brain barrier. Using small animal positron emission tomography (PET), we investigated how P-gp and its blockade with Cyclosporin A (CsA) affect rodent brain uptake of [(11)C](R)-(-)-RWAY, a radioligand for brain 5-HT(1A) receptors. Brain uptake of radioactivity was compared in control and CsA-treated rats as well as P-gp knockout and wild type mice. Parent radioligand and radiometabolite in plasma and brain samples were determined at 30 min after injection of radioligand. PET binding potential (BP) was calculated with a reference tissue model. P-gp knockout mice had 2.5- and 2.8-fold greater brain uptake of [(11)C](R)-(-)-RWAY than wild type ones, measured by in vivo PET and ex vivo tissue sampling, respectively. Similarly, CsA increased rat brain uptake 4.9- and 5.3-fold, in vivo and ex vivo. In addition, CsA increased the plasma free fraction of [(11)C](R)-(-)-RWAY in rats by 2.7-fold. Although CsA increased brain uptake in wild type mice by 2.5-fold, it had no effect on plasma free fraction in knockout animals. Three radiometabolites of [(11)C](R)-(-)-RWAY were uniformly distributed in rat brain, suggesting they were inactive. CsA treatment increased brain uptake of [(11)C](R)-(-)-RWAY and only one of its radiometabolites. Regional rat brain BP increased 27-70% in the CsA-treated rats and the P-gp knockout mice. [(11)C](R)-(-)-RWAY is a P-gp substrate in rat and mouse. The effects of CsA in rats are mediated by both P-gp blockade and displacement of the radiotracer from plasma proteins. Studies with wild type and knockout mice showed that CsA affected only P-gp in this species.
Asunto(s)
Azepinas/farmacocinética , Sangre/efectos de los fármacos , Encéfalo/efectos de los fármacos , Ciclosporina/farmacología , Inhibidores Enzimáticos/farmacología , Piperazinas/farmacocinética , Antagonistas del Receptor de Serotonina 5-HT1 , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/deficiencia , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/genética , Sangre/diagnóstico por imagen , Encéfalo/diagnóstico por imagen , Mapeo Encefálico , Relación Dosis-Respuesta a Droga , Ratones , Ratones Noqueados , Tomografía de Emisión de Positrones/métodos , Ratas , Factores de TiempoRESUMEN
[11C](R)-(-)-RWAY ([11C]2, 3, 4, 5, 6, 7-hexahydro-1{4-[1[4-(2-methoxyphenyl)-piperazinyl]]-2-phenylbutyry}-1H-azepine) is a new radioligand for imaging brain 5-HT1A receptors with positron emission tomography. In [11C](R)-(-)-RWAY, the direction of the amide bond is expected to reduce metabolism by hydrolysis while allowing easy 11C-labeling at the methoxy position. The purposes of this study were to evaluate different tracer kinetic models in nonhuman primates to quantify 5-HT1A receptors with [11C](R)-(-)-RWAY and to test for the possible action of P-glycoprotein (P-gp), one of the known efflux pumps at the blood-brain barrier. The brain uptake of radioactivity from [11C](R)-(-)-RWAY into 5-HT1A receptor-rich brain regions was severalfold greater than for its antipode ([11C](S)-(+)-RWAY) and could be displaced by receptor saturating doses of the selective 5-HT1A antagonist, WAY-100635. Pretreatment with tariquidar, a potent inhibitor of P-gp, increased brain uptake of [11C](R)-(-)-RWAY about 1.5-fold and the plasma free fraction about 1.8-fold. Thus, the effect of tariquidar on brain uptake may have been caused by displacement of the radioligand binding to plasma proteins. Mathematical modeling showed that the estimated values of regional binding potential were correlated strongly between two-tissue compartment model and multilinear reference tissue model, and thus, supported the use of the cerebellum as a reference region.
Asunto(s)
Azepinas/farmacología , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Piperazinas/metabolismo , Piperazinas/farmacocinética , Receptor de Serotonina 5-HT1A/análisis , Receptor de Serotonina 5-HT1A/metabolismo , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Animales , Unión Competitiva/efectos de los fármacos , Unión Competitiva/fisiología , Barrera Hematoencefálica/efectos de los fármacos , Barrera Hematoencefálica/metabolismo , Encéfalo/anatomía & histología , Mapeo Encefálico/métodos , Radioisótopos de Carbono , Macaca mulatta , Masculino , Tasa de Depuración Metabólica/efectos de los fármacos , Tasa de Depuración Metabólica/fisiología , Modelos Biológicos , Estructura Molecular , Piperazinas/síntesis química , Piperazinas/farmacología , Tomografía de Emisión de Positrones/métodos , Piridinas/metabolismo , Piridinas/farmacocinética , Quinolinas/farmacología , Ensayo de Unión Radioligante , Serotonina/metabolismo , Antagonistas de la Serotonina/metabolismo , Antagonistas de la Serotonina/farmacocinéticaRESUMEN
PURPOSE: [carbonyl-11C]N-(2-(1-(4-(2-methoxyphenyl)-piperazinyl)ethyl)-N-pyridinyl)cyclohexanecarboxamide ([carbonyl-11C]WAY-100635) is an effective radioligand for imaging brain 5-HT1A receptors with positron emission tomography (PET). However, this radioligand has some drawbacks for deriving relative regional receptor densities, including rapid metabolism, which acts against accurate definition of an arterial input function for compartmental modeling, and very low nonspecific binding in brain, which detracts from the accuracy of modeling by a simplified reference tissue (cerebellum) approach. Here, in a search for a radioligand that overcomes these limitations, we investigated the effects of introducing a single methyl group at either of the carbon atoms alpha to the amide bond in [11C]WAY-100635. PROCEDURES: Ligands with a methyl group on the alpha carbon of the cyclohexyl group (SWAY) or the alpha carbon of the C2H4 linker ((R,S)-JWAY) were synthesized and tested for binding affinity and intrinsic activity at 5-HT1A receptors. SWAY was labeled with carbon-11 (t1/2 = 20.4 minutes; beta+ = 99.8%) in its O-methyl group and (R,S)-JWAY in its carbonyl group. Each radioligand was evaluated by PET experiments in cynomolgus monkey. RESULTS: SWAY and (R,S)-JWAY were found to be high-affinity antagonists at 5-HT1A receptors. After injection of [11C]SWAY into monkey, radioactivity uptake in brain reached a maximum of 3% at 4.5 minutes and decreased to 0.7% at 72 minutes. However, over the time span of the experiment, radioactivity concentrations in 5-HT1A receptor-rich brain regions were only fractionally higher than in cerebellum. Radioactivity represented by parent radioligand in plasma was 39% at 45 minutes. After injection of [11C](R,S)-JWAY alone, radioactivity uptake in brain reached a maximum of 4.8% at 2.5 minutes and decreased to 1.2% at 90 minutes. At this time, radioactivity concentration in 5-HT1A receptor-rich brain regions was markedly greater than in cerebellum. In another PET experiment, the monkey was predosed with WAY-100635 before [11C](R,S)-JWAY injection. At 90 minutes after injection, the ratio of radioactivity in 5-HT1A receptor-rich regions to that in cerebellum was reduced to near unity. Radioactivity represented by parent radioligand in plasma was 12% at 45 minutes. CONCLUSIONS: [11C](R,S)-JWAY, but not [11C]SWAY, gives a sizeable 5-HT1A receptor-selective PET signal in monkey. The presence of a C-methyl group adjacent to the amide bond in SWAY or (R,S)-JWAY fails to counter metabolism.
Asunto(s)
Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Haplorrinos/metabolismo , Piperazinas/química , Piperazinas/farmacocinética , Piridinas/química , Piridinas/farmacocinética , Ensayo de Unión Radioligante , Receptores de Serotonina 5-HT1/metabolismo , Aminación , Animales , Radioisótopos de Carbono/química , Interacciones Hidrofóbicas e Hidrofílicas , Concentración 50 Inhibidora , Ligandos , Lípidos/química , Metilación , Estructura Molecular , Piperazinas/sangre , Piperazinas/síntesis química , Tomografía de Emisión de Positrones , Piridinas/sangre , Piridinas/síntesis química , Radioquímica , Antagonistas del Receptor de Serotonina 5-HT1RESUMEN
PURPOSE: We aimed to evaluate radiofluorination at the pyridinyl-6 position of the selective 5-HT(1A) receptor antagonist, WAY-100635 [N-(2-(1-(4-(2-methoxyphenyl)piperazinyl)ethyl))-N-(2-pyridinyl)cyclohexanecarboxamide)], on 5-HT(1A) receptor radioligand behavior in vivo. PROCEDURES: The pyridinyl-6 [(18)F]fluoro derivative of WAY-100635 ([(18)F]6FPWAY) was obtained by direct nucleophilic substitution with [(18)F]fluoride ion in a bromo precursor. After intravenous injection of [(18)F]6FPWAY into Cynomolgus monkey, the uptake of radioactivity into brain regions was assessed with positron emission tomography (PET) and blood samples analyzed by high performance liquid chromatography (HPLC) for parent radioligand and radioactive metabolites. The experiment was repeated after pretreatment of the monkey with a dose of WAY-100635 that blocks brain 5-HT(1A) receptors. RESULTS: After intravenous injection of [(18)F]6FPWAY into Cynomolgus monkey, the uptake of radioactivity into whole brain reached 4.33% of injected dose at 7.5 min. Uptake was highest in 5-HT(1A) receptor-rich regions. Pretreatment with WAY-100635 reduced uptake in these regions to near the levels in receptor-devoid cerebellum. [(18)F]6FPWAY was rapidly metabolized in vivo, as evidenced by the rapid appearance of radioactive metabolites in plasma. CONCLUSION: [(18)F]6FPWAY is selective and moderately useful for imaging brain 5-HT(1A) receptors in vivo. The pyridinyl-6 position is resistant to defluorination and may be an attractive site for the (18)F-labeling of 6FPWAY analogs that resist hydrolysis.
Asunto(s)
Aminopiridinas/farmacocinética , Piperazinas/farmacocinética , Receptor de Serotonina 5-HT1A/análisis , Antagonistas de la Serotonina/farmacocinética , Aminopiridinas/química , Animales , Encéfalo/diagnóstico por imagen , Encéfalo/metabolismo , Cromatografía Líquida de Alta Presión , Radioisótopos de Flúor , Macaca fascicularis , Estructura Molecular , Piperazinas/química , Piperazinas/farmacología , Piridinas/química , Piridinas/farmacología , Ensayo de Unión Radioligante , Antagonistas del Receptor de Serotonina 5-HT1 , Antagonistas de la Serotonina/química , Tomografía Computarizada de EmisiónRESUMEN
In recent years, considerable effort has been spent on the design, synthesis and pharmacological characterization of radiofluorinated derivatives of the 5-HT(1A) receptor antagonist, WAY-100635, for the in vivo study of these receptors in human brain with PET. (Pyridinyl-6)-fluoro- and (pyridinyl-5)-fluoro-analogues of WAY-100635 (6-fluoro and 5-fluoro-WAY-100635, 5a/6a) were synthesized as well as the corresponding chloro-, bromo- and nitro-derivatives as precursors for labelling (5b-d and 6b-d). Comparative radiolabelling of these precursors with fluorine-18 (positron-emitting isotope, 109.8 min half-life) clearly demonstrated that only ortho-fluorination in this pyridine series, and not meta-fluorination, is of interest for the preparation of a radioligand by nucleophilic heteroaromatic substitution. 6-[(18)F]Fluoro-WAY-100635 ([(18)F]5a) can be efficiently synthesized in one step, either from the corresponding 6-bromo precursor (using conventional heating at 145 degrees C for 10 min) or from the corresponding 6-nitro precursor (using microwave activation at 100 W for 1 min). Typically, 15-25 mCi (0.55-0.92 GBq) of 6-[(18)F]fluoro-WAY-100635 ([(18)F]5a, 1-2 Ci/micromol or 37-72 GBq/micromol) were obtained in 50-70 min starting from a 100 mCi (3.7 GBq) aliquot of a batch of cyclotron-produced [(18)F]fluoride. This (18)F-labelled radioligand is now being evaluated in PET studies.