High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker |
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Authors: | Yapa Udeni Prusakiewicz Jeffery J Wrightstone Ann D Christine Lori J Palandra Joe Groeber Elizabeth Wittwer Arthur J |
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Affiliation: | Pfizer Global Research & Development, Groton, CT 06340, USA. udeni.yapa@pfizer.com |
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Abstract: | Fatty acid amide hydrolase (FAAH) is one of the main enzymes responsible for the degradation of the endocannabinoid anandamide (N-arachidonoylethanolamine, AEA). FAAH inhibitors may be useful in treating many disorders involving inflammation and pain. Although brain FAAH may be the relevant target for inhibition, rat studies show a correlation between blood and brain FAAH inhibition, allowing blood FAAH activity to be used as a target biomarker. Building on experience with a rat leukocyte FAAH activity assay using [3H]AEA, we have developed a human leukocyte assay using stably labeled [2H4]AEA as substrate. The deuterium-labeled ethanolamine reaction product ([2H4]EA) was analyzed by high-performance liquid chromatography–tandem mass spectrometry (HPLC–MS/MS) in the positive electrospray ionization (ESI) mode. The response for [2H4]EA was linear from 10 nM to 10 μM, and the analysis time was less than 6 min/sample. Results using the [2H4]AEA and HPLC–MS/MS method agreed well with those obtained using the [3H]AEA radiometric assay. In addition to using a nonradioactive substrate, the HPLC–MS/MS method had increased sensitivity with lower background. Importantly, the assay preserved partial FAAH inhibition resulting from ex vivo treatment with a time-dependent irreversible inhibitor, suggesting its utility with clinical samples. The assay has been used to profile the successful inhibition of FAAH in recent clinical trials. |
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