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.

Identification of novel inhibitors of rat Mrp3.

Multidrug resistance-associated protein (MRP; ABCC gene family) mediated efflux transport plays an important role in the systemic and tissue exposure profiles of many drugs and their metabolites, and also of endogenous compounds like bile acids and bilirubin conjugates. However, potent and isoform-selective inhibitors of the MRP subfamily are currently lacking. Therefore, the purpose of the present work was to identify novel rat Mrp3 inhibitors. Using 5(6)-carboxy-2',7'-dichlorofluorescein diacetate (CDFDA) as a model-(pro)substrate for Mrp3 in an oil-spin assay with primary rat hepatocytes, the extent of inhibition of CDF efflux was determined for 1584 compounds, yielding 59 hits (excluding the reference inhibitor) that were identified as new Mrp3 inhibitors. A naive Bayesian prediction model was constructed in Pipeline Pilot to elucidate physicochemical and structural features of compounds causing Mrp3 inhibition. The final Bayesian model generated common physicochemical properties of Mrp3 inhibitors. For instance, more than half of the hits contain a phenolic structure. The identified compounds have an AlogP between 2 and 4.5, between 5 to 8 hydrogen bond acceptor atoms, a molecular weight between 260 and 400, and 2 or more aromatic rings. Compared to the depleted dataset (i.e. 90% remaining compounds), the Mrp3 hit rate in the enriched set was 7.5-fold higher (i.e. 17.2% versus 2.3%). Several hits from this first screening approach were confirmed in an additional study using Mrp3 transfected inside-out membrane vesicles. In conclusion, several new and potent inhibitors of Mrp3 mediated efflux were identified in an optimized in vitro rat hepatocyte assay and confirmed using Mrp3 transfected inside-out membrane vesicles. A final naive Bayesian model was developed in an iterative way to reveal common physicochemical and structural features for Mrp3 inhibitors. The final Bayesian model will enable in silico screening of larger libraries and in vitro identification of more potent Mrp3 inhibitors.

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.

A sensitive liquid chromatography method for analysis of propofol in small volumes of neonatal blood.

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.

Current insights in the complexities underlying drug-induced cholestasis.

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.

Vesicle- and Hepatocyte-Based Assays for Identification of Drug Candidates Inhibiting BSEP Function.

Transporters play a crucial role in the uptake of endo- and exogenous molecules in hepatocytes and efflux into the bile. The bile salt export pump (BSEP; ABCB11) is of major importance for efflux of bile salts to the bile and BSEP inhibition frequently provokes drug-induced cholestasis. This chapter describes two assays to determine inhibition of BSEP-mediated bile salt excretion. The first assay uses inside-out membrane vesicles, prepared from BSEP-transfected cell lines. The cholestasis potential of compounds can be determined by specifically investigating the ability to inhibit BSEP-mediated uptake of tauro-nor-THCA-24-DBD, a fluorescent bile salt derivative. For the second assay, relative accumulation of tauro-nor-THCA-24-DBD in sandwich-cultured hepatocytes, which represents a more biorelevant in vitro system, is investigated. Through incubation with standard or Ca2+/Mg2+-free buffer, the substrate signal can be determined in the cells and bile or the cells alone, respectively. Performing this assay in the presence and absence of potentially interfering compounds of interest enables exploration of the relative effect of these compounds on biliary excretion of the probe substrate.

Detection of Drug-Induced Cholestasis Potential in Sandwich-Cultured Human Hepatocytes.

Drug-induced cholestasis poses a major hurdle for the pharmaceutical industry as it is one the primary mechanisms of drug-induced liver injury. Hence, detection of drug-induced cholestasis during the early stages of drug development is of utmost importance. The most commonly used in vitro models rely on the extent of inhibition of bile salt export pump-mediated taurocholic acid transport, thereby assuming that drug-induced cholestasis mechanisms are merely restricted to the interaction with this sole hepatic transporter. Sandwich-cultured human hepatocytes represent a more holistic in vitro tool to investigate drug-induced cholestasis as they preserve all relevant disposition pathways and cellular functions involved in bile acid homeostasis. We developed and validated a sandwich-cultured human hepatocytes-based in vitro assay which is able to identify compounds causing cholestasis by altering bile acid disposition. The in vitro cholestatic potential is expressed by calculating a drug-induced cholestasis index value, which reflects the relative residual urea formation of sandwich-cultured human hepatocytes co-incubated with bile acids and test compound as compared to sandwich-cultured human hepatocytes treated with test compound alone. In addition, a safety margin can be calculated to determine the in vivo risk for cholestasis based on the determination of the drug-induced cholestasis index at various concentrations and the peak plasma concentration of the drug candidate. This chapter outlines the various steps involved in performing our sandwich-cultured human hepatocytes-based in vitro assay.