Drug-induced cholestasis (DIC) poses a major challenge to the pharmaceutical industry and regulatory agencies. It causes both drug attrition and post-approval withdrawal of drugs. DIC represents itself as an impaired secretion and flow of bile, leading to the pathological hepatic and/or systemic accumulation of bile acids (BAs) and their conjugate bile salts. Due to the high number of mechanisms underlying DIC, predicting a compound's cholestatic potential during early stages of drug development remains elusive. A profound understanding of the different molecular mechanisms of DIC is, therefore, of utmost importance. Although many knowledge gaps and caveats still exist, it is generally accepted that alterations of certain hepatobiliary membrane transporters and changes in hepatocellular morphology may cause DIC. Consequently, liver models, which represent most of these mechanisms, are valuable tools to predict human DIC. Some of these models, such as membrane-based in vitro models, are exceptionally well-suited to investigate specific mechanisms (i.e., transporter inhibition) of DIC, while others, such as liver slices, encompass all relevant biological processes and therefore offer a better representation of the in vivo situation. In the current review, we highlight the principal molecular mechanisms associated with DIC and offer an overview and critical appraisal of the different liver models that are currently being used to predict the cholestatic potential of drugs.
Colistin (polymyxin E) is a polycation antibiotic which is increasingly used (administered as colistin methanesulfonate, CMS) as a salvage therapy in critically ill patients with multidrug resistant Gram‐negative infections. Even though colistin has been used for more than 50 years, its metabolic fate is poorly understood. One of the current challenges for studying the pharmacokinetics (PK) is the precise and accurate determination of colistin in in vitro and in vivo studies. In the present study, we developed and validated a series of sensitive and robust liquid chromatography tandem mass spectrometry (LC–MS/MS) methods for analysing biological samples obtained from in vitro and in vivo disposition assays. After a zinc acetate‐mediated precipitation, hydrophilic–lipophilic‐balanced solid phase extraction (HLB‐SPE) was used for the extraction of colistin. The compounds were retained on a hydrophilic interaction liquid chromatography (HILIC) column and were detected by MS/MS. CMS was quantified by determining the produced amount of colistin during acidic hydrolysis. The developed methods are sensitive with lower limits of quantification varying between 0.009 μg/mL and 0.071 μg/mL for colistin A, and 0.002 μg/mL to 0.013 μg/mL for colistin B. The intra‐ and inter‐day precision and accuracy were within ±15%. Calibration curves of colistin were linear (0.063 μg/mL to 8.00 μg/mL) within clinically relevant concentration ranges. Zinc acetate‐mediated precipitation and the use of a HILIC column were found to be essential. The developed methods are sensitive, accurate, precise, highly efficient and allow monitoring colistin and CMS in biological samples without the need for an internal standard.
What is known and objective: Sampling volumes of blood from neonates is necessarily limited. However, most of the published propofol analysis assays require a relatively large blood sample volume (typically ≥0.5 mL). Therefore, the aim of the present study was to develop and validate a sensitive method requiring a smaller sample volume (0.2 mL) to fulfill clinically relevant research requirements. Methods: Following simple protein precipitation and centrifugation, the supernatant was injected into the HPLC-fluorescence system and separated with a reverse phase column. Propofol and the internal standard (thymol) were detected and quantified using fluorescence at excitation and emission wavelengths of 270 nm and 310 nm, respectively. The method was validated with reference to the Food and Drug Administration (FDA) guidance for industry. Accuracy (CV, %) and precision (RSD, %) were evaluated at three quality control concentration levels (0.05, 0.5 and 5 µg/mL). Results and discussion: Calibration curves were linear in the range of 0.005-20 µg/ mL. Intra-and interday accuracy (−4.4%-13.6%) and precision (0.2%-5.8%) for propofol were below 15%. The calculated LOD (limit of detection) and LLOQ (lower limit of quantification) were 0.0021 µg/mL and 0.0069 µg/mL, respectively. Propofol samples were stable for 4 months at −20°C after the sample preparation. This method was applied for analyzing blood samples from 41 neonates that received propofol, as part of a dose-finding study. The measured median (range) concentration was 0.14 (0.03-1.11) µg/mL, which was in the range of the calibration curve. The calculated median (range) propofol half-life of the gamma elimination phase was 10.4 (4.7-26.7) hours. What is new and conclusion: A minimal volume (0.2 mL) of blood from neonates is required for the determination of propofol with this method. The method can be used to support the quantification of propofol drug concentrations for pharmacokinetic studies in the neonatal population.
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