RESUMO
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.
Assuntos
Cromatografia Líquida de Alta Pressão , Fumaratos/sangue , Espectrometria de Massas em Tandem , Animais , Radioisótopos de Carbono/química , Cromatografia Líquida de Alta Pressão/normas , Ácido Cítrico/química , Ácido Cítrico/metabolismo , Estabilidade de Medicamentos , Fumarato Hidratase/antagonistas & inibidores , Fumarato Hidratase/metabolismo , Fumaratos/normas , Marcação por Isótopo , Malatos/análise , Malatos/metabolismo , Controle de Qualidade , Ratos , Espectrometria de Massas em Tandem/normas , TemperaturaRESUMO
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.
Assuntos
Amidoidrolases/sangue , Cromatografia Líquida de Alta Pressão/métodos , Leucócitos/enzimologia , Espectrometria de Massas por Ionização por Electrospray/métodos , Espectrometria de Massas em Tandem/métodos , Amidoidrolases/antagonistas & inibidores , Biomarcadores/sangue , HumanosRESUMO
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.