DEREK peers at plethora of products in rat urine

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  • Published: Jul 1, 2017
  • Author: Ryan De Vooght-Johnson
  • Channels: Laboratory Informatics / Chemometrics & Informatics
thumbnail image: DEREK peers at plethora of products in rat urine

A detailed understanding of statin metabolism is necessary

Statins, such as fluvastatin are taken by millions of people to prevent cardiovascular disease so it is important to have a thorough understanding of their metabolites.

Statins, such as fluvastatin (sold as Lescol, Vastin and Canef), are taken by millions of people to prevent cardiovascular disease so it is important to have a thorough understanding of their metabolites. For ethical and financial reasons, much drug metabolism work is carried out on rodents, usually rats, rather than humans. However, there may be differences between the metabolites found in rats and those seen in humans.

The Indian scientists examined the in vitro products formed by both rat and human liver microsomes from fluvastatin. Such studies show how compounds are metabolised by the liver. For the in vivo studies, fluvastatin was given to six rats, and samples of blood plasma, urine and faeces were taken.

The Indian researchers used two toxicity software packages: DEREK and TOPKAT. DEREK (deductive estimation of risk from existing knowledge) gives a qualitative assessment of risk based on the toxicity literature concerning the substructures of the molecule, etc. TOPKAT (toxicity prediction by computer assisted technology) gives quantitative toxicity values, expressed as probabilities, based on quantitative structure–toxicity relationships.

Various fluvastatin metabolites seen with microsomes and rats

The samples of urine and plasma were diluted with acetonitrile (three volumes) to precipitate proteins. In the case of faeces, water was added to give a slurry, which was then vortexed and acetonitrile added. Solids were removed by centrifugation, and the resulting solutions were cooled to -30 °C to separate out acetonitrile and water layers. The aqueous layer was extracted using solid-phase extraction (SPE) with a Phenomenex C18-E cartridge. The cartridge was eluted with acetonitrile, the resulting solution being added to the acetonitrile solution previously separated. The samples were evaporated and taken up in 1:1 v/v acetonitrile:water for LC-MS/MS analysis.

UHPLC was carried out with an Agilent 1200 instrument fitted with a Waters Acquity UPLC BEH C18 column. Gradient elution was employed with 0.1% aqueous formic acid and acetonitrile as the two mobile phases; the proportion of the latter was increased from 2 to 90%. The flow rate was 0.3 ml/min. Tandem mass spectroscopy was carried out using an Agilent Q-TOF 6540 instrument (quadrupole time of flight) and Agilent MassHunter software.

The rat urine proved the richest source of metabolites. Fifteen of these, named as M1 to M15, were identified by means of a detailed consideration of their accurate masses and product ions. Six of the metabolites were detected in rat faeces, eight were seen in the rat blood plasma, four were isolated from the human liver microsomes, while three were seen in the rat liver microsomes. Unchanged fluvastatin was also detected in all the tested metabolite mixtures.

Seven metabolites, M1, M2, M7, M9, M11, M13 and M14, were flagged up by the TOPKAT software as possible rodent carcinogens, this being confirmed by the DEREK software for M7 and M14 (DEREK flagged up M11 as a mutagen). Three metabolites, M7, M11 and M14, may therefore be a particular cause for concern, but these three were not seen in previous metabolic studies in humans (Dain et al.). However, M11 was detected from human liver microsomes in the current study.

In silico study raises toxicity questions on fluvastatin metabolites

The use of UHPLC and Q-TOF tandem mass spectrometry was shown to be a powerful method for separating and detecting metabolites. The metabolite profile in rats differed greatly from that seen in previous work in humans (Dain et al.). Further investigation is required to elucidate whether these differences are real or artefacts of the work-up and analytical techniques used. Real carcinogenicity tests may be required if the metabolites that gave carcinogenicity alerts for both in silico packages are present in humans.

Related Links

Journal of Mass Spectrometry, 2016, Early View Paper. Chavan et al., Identification and characterization of fluvastatin metabolites in rats by UHPLC/Q-TOF/MS/MS and in silico toxicological screening of the metabolites.

Drug Metabolism and Disposition, 1993, 21, 567-572. Dain et al.. Biotransformation of fluvastatin sodium in humans.

WIREs Computational Molecular Science, 2016, 6, 147-172. Raies et al.. In silico toxicology: computational methods for the prediction of chemical toxicity.

Article by Ryan De Vooght-Johnson

The views represented in this article are solely those of the author and do not necessarily represent those of John Wiley and Sons, Ltd.

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