Unravelling the Hepatic Elimination Mechanisms of Colistin.

PURPOSE: Colistin is an antibiotic which is increasingly used as a last-resort therapy in critically-ill patients with multidrug resistant Gram-negative infections. The purpose of this study was to evaluate the mechanisms underlying colistin's pharmacokinetic (PK) behavior and to characterize its hepatic metabolism. METHODS: In vitro incubations were performed using colistin sulfate with rat liver microsomes (RLM) and with rat and human hepatocytes (RH and HH) in suspension. The uptake of colistin in RH/HH and thefraction of unbound colistin in HH (fu,hep) was determined. In vitro to in vivo extrapolation (IVIVE) was employed to predict the hepatic clearance (CLh) of colistin. RESULTS: Slow metabolism was detected in RH/HH, with intrinsic clearance (CLint) values of 9.34± 0.50 and 3.25 ± 0.27 mL/min/kg, respectively. Assuming the well-stirred model for hepatic drug elimination, the predicted rat CLh was 3.64± 0.22 mL/min/kg which could explain almost 70% of the reported non-renal in vivo clearance. The predicted human CLh was 91.5 ± 8.83 mL/min, which was within two-fold of the reported plasma clearance in healthy volunteers. When colistin was incubated together with the multidrug resistance-associated protein (MRP/Mrp) inhibitor benzbromarone, the intracellular accumulation of colistin in RH/HH increased significantly. CONCLUSION: These findings indicate the major role of hepatic metabolism in the non-renal clearance of colistin, while MRP/Mrp-mediated efflux is involved in the hepatic disposition of colistin. Our data provide detailed quantitative insights into the hereto unknown mechanisms responsible for non-renal elimination of colistin.

Quantitative determination of colistin A/B and colistin methanesulfonate in biological samples using hydrophilic interaction chromatography tandem mass spectrometry.

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.

Viability, function and morphological integrity of precision-cut liver slices during prolonged incubation: Effects of culture medium.

Precision-cut liver slices (PCLS) are an ex vivo model for metabolism and toxicity studies. However, data on the maintenance of the morphological integrity of the various cell types in the slices during prolonged incubation are lacking. Therefore, our aims were to characterize morphological and functional changes in rat PCLS during five days of incubation in a rich medium, RegeneMed®, and a standard medium, Williams' Medium E. Although cells of all types in the slices remain viable, profound changes in morphology were observed, which were more prominent in RegeneMed®. Slices underwent notable fibrosis, bile duct proliferation and fat deposition. Slice thickness increased, resulting in necrotic areas, while slice diameter decreased, possibly indicating cell migration. An increased proliferation of parenchymal and non-parenchymal cells (NPCs) was observed. Glycogen, albumin and Cyp3a1 were maintained albeit to a different level in two media. In conclusion, both hepatocytes and NPCs remain viable and functional, enabling five-day toxicity studies. Tissue remodeling and formation of a new capsule-like cell lining around the slices are evident after 3­4 days. The differences in effects between media emphasize the importance of media selection and of the recognition of morphological changes in PCLS, when interpreting results from toxicological or pharmacological studies.