Background

[PubMed]

4-(2´-Methoxyphenyl)-1-[2´-(N-2´´-1,3 pyrimidino)-p-[¹⁸F]fluorobenzamido]ethylpiperazine ([¹⁸F]FPWAY) is a radioligand developed for positron emission tomography (PET) imaging of serotonin-1A (5-HT_1A) receptors in the central nervous system (1, 2). It is a selective 5-HT_1A antagonist labeled with ¹⁸F, a positron emitter with a physical t_½ of 109.7 min (3, 4).

The serotonin (5-hydroxytryptamine (5-HT)) neurotransmission system consists mainly of neurons in the brainstem, with nerve tracts extending from these neurons to many areas of the brain and spinal cord. During firing, the neurons release 5-HT, a neurotransmitter that is involved in the modulation of various important physiologic functions and behavior, such as thermoregulation, cardiovascular function, aggressive and sexual behavior, mood, appetite, and the sleep–wake cycle. The effects of 5-HT are mediated by as many as seven classes of receptor populations (5-HT₁ to 5-HT₇), many of which also contain several subtypes. There are five receptor subtypes within the G protein-coupled 5-HT₁ receptor family, with the 5-HT_1A subtype located primarily in the limbic forebrain (the hippocampus, entorhinal cortex and septum). 5-HT_1A receptors appear to function both as presynaptic (somatodendritic) autoreceptors in the raphe nuclei and as postsynaptic receptors in the terminal fields. This receptor subtype is involved in the modulation of emotion and the function of the hypothalamus, and is implicated in the pathogenesis of anxiety, depression, hallucinogenic behavior, motion sickness, dementia, schizophrenia, and eating disorders. A radioligand that can be used to assess the in vivo densities of 5-HT_1A receptors and their changes may facilitate investigation of the relationship of these receptors to various neuropsychiatric diseases and aid in the design of novel drugs for their treatment.

Many psychiatric drugs modulate serotonergic transmission or specifically target the 5-HT_1A receptors. Various compounds have been radiolabeled for visualization and quantification of these receptors. WAY 100635 was developed as a highly selective, silent antagonist (possessing no intrinsic agonist activity) of 5HT_1A receptors at both pre- and postsynaptic sites. WAY 100635 radiolabeled with ¹¹C at the carbonyl position is an effective radioligand but it is rapidly cleared and metabolized. The short t_½ of ¹¹C also presents some challenges in clinical application of the radiotracer. A radioligand with slower metabolism and labeled with a longer-lived radioisotope would be a better agent for quantitative PET studies. A number of fluorinated derivatives of WAY 100635 have been developed. In 2000, Lang et al. (5) synthesized [¹⁸F]FPWAY with labeling at the carboxamide moiety to minimize metabolites that cross the blood-brain barrier (BBB). [¹⁸F]FPWAY appears to be an intermediate-affinity antagonist of the 5-HT_1A receptors and may be more sensitive to transient changes in endogenous 5-HT_1A levels than high-affinity analogs (6). In this WAY 100635 analog, the pyridine ring is replaced by pyrimidine and the cyclohexane ring is replaced by fluorobenzyl. With ¹⁸F in the 4 position of the benzamide, [¹⁸F]FPWAY is a very different compound from another WAY 100635 analog with a similar abbreviation, [¹⁸F]6FPWAY, which was synthesized by Marchais et al. (7). [¹⁸F]6FPWAY contains a N-pyridine with ¹⁸F in the 6 position of the pyridine moiety.

Synthesis

[PubMed]

Lang et al. (5, 8) first reported the synthesis of FPWAY based on FBWAY (or p-MPPF). FPWAY was synthesized as an FBWAY analog (FBWAY 1,3 N) with a 1,3 pyrimidine substituent. FBWAY was prepared by reacting WAY 100634 with the corresponding acid chloride in methylene chloride. Radiolabeling involved labeling the 4-trimethylamino precursor with ¹⁸F to produce the radiolabeled 4-fluorobenzoyl chloride. The product was purified by high-performance liquid chromatography (HPLC). The radiochemical yield ranged between 10 and 20%, and the total synthesis time was between 80 and 90 min. The specific activity was 37,000 GBq (1,000 Ci)/mmol at the end of bombardment (EOB).

Ma et al. (9) described a similar synthesis of FPWAY from {2-[4-(2-methoxyphenyl)-1-piperazinyl]-ethyl}-N-(2-pyrimidinyl)amine which was prepared from 2-[4-(2-methoxyphenyl)-1-piperazinyl]ethylamine (obtained commercially) by reaction with 2-chloropyrimidine in ethanol and sodium carbonate overnight. The yield was 79%. The final compound was reacted with 4-fluorobenzoyl chloride in triethylamine and 1,2-dichloroethane overnight to give a final yield of 96%. This precursor was reacted with [¹⁸F]− Kryptofix in dimethyl sulfoxide and heated in a microwave oven for 2 min. The product was purified by reverse-phase HPLC and a C-18 Sep-Pak to give a final yield of 25-35% EOB and radiochemical purity of 99.0%.

In Vitro Studies: Testing in Cells and Tissues

[PubMed]

In vitro affinity of [¹⁸F]FPWAY was studied by Lang et al. (5), and the inhibition constant, K_i, was determined to be <1 nM against [¹⁸H]8-hydroxy-2-dipropylaminotetralin ([³H]8-OH-DPAT) binding to a cloned cell line containing 5-HT_1A receptors. This was relatively weak when compared with other high affinity WAY analogs (MeFWAY and Trans-FCWAY). The antagonist inhibition concentration (IC₅₀)was determined to be 340 nM based on the compound’s ability to inhibit relaxation induced by 0.3 µM 8-OH-DPAT. On the basis of the compound’s ability to relax guinea pig field-stimulated ileum, the authors concluded that [¹⁸F]FPWAY showed no agonist activity.

In vitro metabolism studies of FPWAY and [¹⁸F]FPWAY were performed by Ma et al. (9) in cryopreserved hepatocytes of rat and human liver tissue. Metabolites were identified by liquid chromatography−tandem mass spectrometry. In rat hepatocytes, aromatic-ring oxidation was the major metabolism pathway for FPWAY, but there was also amide hydrolysis. Amide hydrolysis products were the major metabolites of FPWAY in human hepatocytes, and the metabolic rate appeared to be higher than for other fluorinated analogs of WAY 100635 (FBWAY and MeFBWAY). The study suggested that the pyrimidyl for pyridyl substitution in FPWAY made the amide more labile to metabolic hydrolysis. In a [¹⁸F]FPWAY imaging study using monkey blood, it was shown that pure [¹⁸F]FPWAY could be readily extracted by an organic phase (hexane−etyhl acetate 4:1) from its metabolites in a basic aqueous phase (125 mM KCl-NaOH, pH 12.5). This simplified the determination of the input function.

Animal Studies

Human Studies

[PubMed]

No publication is currently available.

NIH Support

NIH Intramural Support. NIH Contract NO1MH2003.

References

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