Hepatocyte-based flow analytical bioreactor for online xenobiotics metabolism bioprediction.

The research for new in vitro screening tools for predictive metabolic profiling of drug candidates is of major interest in the pharmaceutical field. The main motivation is to avoid late rejection in drug development and to deliver safer drugs to the market. Thanks to the superparamagnetic properties of iron oxide nanoparticles, a flow bioreactor has been developed which is able to perform xenobiotic metabolism studies. The selected cell line (HepaRG) maintained its metabolic competencies once iron oxide nanoparticles were internalized. Based on magnetically trapped cells in a homemade immobilization chamber, through which a flow of circulating phase was injected to transport nutrients and/or the studied xenobiotic, off-line and online (when coupled to a high-performance liquid chromatography chain) metabolic assays were developed using diclofenac as a reference compound. The diclofenac demonstrated a similar metabolization profile chromatogram, both with the newly developed setup and with the control situation. Highly versatile, this pioneering and innovative instrumental design paves the way for a new approach in predictive metabolism studies.

Study of nucleic acid-ligand interactions by capillary electrophoretic techniques: A review.

The understanding of nucleic acids-ligand (proteins, nucleic acids or various xenobiotics) interactions is of fundamental value, representing the basis of complex mechanisms that govern life. The development of improved therapeutic strategies, as well as the much expected breakthroughs in case of currently untreatable diseases often relies on the elucidation of such biomolecular interactions. Capillary electrophoresis (CE) is becoming an indispensable analytical tool in this field of study due to its high versatility, ease of method development, high separation efficiency, but most importantly due to its low sample and buffer volume requirements. Most often the availability of the compounds of interest is severely limited either by the complexity of the purification procedures or by the cost of their synthesis. Several reviews covering the investigation of protein-protein and protein-xenobiotics interactions by CE have been published in the recent literature; however none of them promotes the use of these techniques in the study of nucleic acid interactions. Therefore, various CE techniques applicable for such interaction studies are discussed in detail in the present review. The paper points out the particular features of these techniques with respect the estimation of the binding parameters, in analytical signal acquisition and data processing, as well as their current shortcomings and limitations.

Ultra high performance liquid chromatography method for the determination of pentamidine and analog in rat biological fluids.

Pentamidine isethionate (PTMD) is an antiprotozoal agent used in different parasitic diseases as Human African Trypanosomiasis or Pneumocystis pneumonia. Given its side effects, numerous analogs are still under development worldwide. PTMD has been recently described having a potential activity in myotonic dystrophy (type 1). Here we present an UPLC method coupled to fluo or PDA detection for PTMD and one analog determination in rat plasma or urine. The chromatographic separation was achieved on a Acquity UPLC® HSS T3 analytical column using a mobile phase combining formic acid 0.1% (v/v) and acetonitrile (ACN) at a constant flow rate of 0.4 mL/min. Preliminary, an innovative µSPE (solid phase extraction) procedure using Oasis® WCX sorbent was processed and gave satisfying and reproducible results in terms of extraction yields. Additionally, the methods were successfully validated using the accuracy profiles approach (ß=95% and acceptance limits=15%) over the ranges 2.88-287.52 ng/mL and from 143.76 ng/mL to 1.72 µg/mL in rat plasma and urine for PTMD and for EBAB, from 4.23 to 423.39 ng/mL and from 211.69 ng/mL to 2.54 µg/mL for plasma and urine, respectively. The validated protocols were applied to a pharmacokinetic (PK) study on rats and permitted to point out some relevant PK parameters on PTMD and its studied analog.

Three optimized and validated (using accuracy profiles) LC methods for the determination of pentamidine and new analogs in rat plasma.

Three novel LC-UV methods for the determination of pentamidine (PTMD) and two of its new analogs in rat plasma are described. The chromatographic conditions (wavelength, acetonitrile percentage in the mobile phase, internal standard) were optimized to have an efficient selectivity. A pre-step of extraction was simultaneously developed for each compound. For PTMD, a solid phase extraction (SPE) with Oasis(®) HLB cartridges was selected, while for the analogs we used protein precipitation with acetonitrile. SPE for PTMD gave excellent results in terms of extraction yield (99.7 ± 2.8) whereas the recoveries for the analogs were not so high but were reproducible as well (64.6 ± 2.6 and 36.8 ± 1.6 for analog 1 and 2, respectively). By means of a recent strategy based on accuracy profiles (ß-expectation tolerance interval), the methods were successfully validated. ß was fixed at 95% and the acceptability limits at ± 15% as recommended by the FDA. The method was successfully validated for PTMD (29.6-586.54 ng/mL), analog 1 (74.23-742.3 ng/mL) and analog 2 (178.12-890.6 ng/mL). The first concentration level tested was considered as the LLOQ (lower limit of quantification) for PTMD and analog 1 whereas for analog 2, the LLOQ was not the first level tested and was raised to 178.12 ng/mL.