High-performance liquid chromatography-tandem mass spectrometry assay of fatty acid amide hydrolase (FAAH) in blood: FAAH inhibition as clinical biomarker.

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.

An efficient approach for the rapid assessment of oral rat exposures for new chemical entities in drug discovery.

Present study aims to improve efficiency and capacity of in vivo rat pharmacokinetic studies for rapid assessment of systemic exposure (AUC and C(max)) of new chemical entities. Plasma concentration-time profiles in rats from structurally diverse compounds were extracted from the Pfizer database. AUC(0-8) was calculated with 7 data points or a reduced subset of 3 data points. AUC values determined with 7 data points were compared to subset AUC values. A < or = 30% difference in values for 90% of cases was acceptance criteria. In parallel, samples were analyzed individually and pooled at each time point across compounds. For 96% of cases, AUC values estimated using 1, 4, and 8 h were comparable to AUC values obtained from 7 data points suggesting 1, 4, and 8 h sampling should be sufficient to estimate AUC. For C(max), the difference between 1, 4, and 8 h data-point analysis versus 7 data-point analysis is less than 30% for 72% of cases. Concentrations from individual versus pooled sample analysis were found to be equivalent. A rapid rat PK screening paradigm was created by the combination of 1, 4, and 8 h sampling and pooled sample analysis, which improves throughput and cycle time of in vivo PK studies.

Quantification of fumarate and investigation of endogenous and exogenous fumarate stability in rat plasma by LC-MS/MS.

BACKGROUND: Fumaric acid is a commonly used excipient in pharmaceutical products. It is not known if its presence may lead to fluctuation of endogenous fumarate levels. An LC-MS/MS method was developed and validated to quantify fumarate in support of a toxicokinetics study. RESULTS: Stability evaluation showed that endogenous fumarate was stable for 6 h at room temperature, while exogenously added fumaric acid was converted to malate within 1 h due to the presence of fumarase. Citric acid, a fumarase inhibitor, prevented the conversion of added fumaric acid in rat plasma. CONCLUSION: The method was validated in citric acid stabilized rat plasma using a surrogate matrix approach. A discrepancy in stability was observed between endogenous fumarate and exogenously added fumaric acid.