An evaluation of the CYP2D6 and CYP3A4 inhibition potential of metoprolol metabolites and their contribution to drug–drug and drug–herb interaction by LC‐ESI/MS/MS

The aim of the present study was to evaluate the contribution of metabolites to drug–drug interaction and drug–herb interaction using the inhibition of CYP2D6 and CYP3A4 by metoprolol (MET) and its metabolites. The peak concentrations of unbound plasma concentration of MET, α‐hydroxy metoprolol (HM), O‐desmethyl metoprolol (ODM) and N‐desisopropyl metoprolol (DIM) were 90.37 ± 2.69, 33.32 ± 1.92, 16.93 ± 1.70 and 7.96 ± 0.94 ng/mL, respectively. The metabolites identified, HM and ODM, had a ratio of metabolic area under the concentration–time curve (AUC) to parent AUC of ≥0.25 when either total or unbound concentration of metabolite was considered. In vitro CYP2D6 and CYP3A4 inhibition by MET, HM and ODM study revealed that MET, HM and ODM were not inhibitors of CYP3A4‐catalyzed midazolam metabolism and CYP2D6‐catalyzed dextromethorphan metabolism. However, DIM only met the criteria of >10% of the total drug related material and <25% of the parent using unbound concentrations. If CYP inhibition testing is solely based on metabolite exposure, DIM metabolite would probably not be considered. However, the present study has demonstrated that DIM contributes significantly to in vitro drug–drug interaction. Copyright © 2016 John Wiley & Sons, Ltd.     

Identification of a novel series of alkylitaconic acids in wood cultures of Ceriporiopsis subvermispora by gas chromatography/mass spectrometry.

A novel series of long-chain unsaturated dicarboxylic acids consisting of a long aliphatic chain attached to the C-3 position of itaconic acid has been identified by gas chromatography/mass spectrometry during in vitro decay of eucalypt wood by the white-rot basidiomycete Ceriporiopsis subvermispora. The major compounds were identified as tetradecyl-, 7-hexadecenyl- and hexadecylitaconic acids by their mass fragmentation patterns. Other members of the same compound series, identified as dodecanyl-, tridecanyl-, tetradecenyl-, pentadecanyl-, octadecenyl- and octadecanylitaconic acids, were present in very minor amounts or traces. Whereas hexadecenylitaconic acid has already been reported in cultures of C. subvermispora, to our knowledge this is the first report of the presence of the other alkylitaconic acids in fungal cultures. These new alkylitaconic-type metabolites may constitute a source for peroxidizable lipids involved in lignin degradation during wood decay by C. subvermispora and other white-rot basidiomycetes.     

Characterization of degradation products of Ivabradine by LC‐HR‐MS/MS: a typical case of exhibition of different degradation behaviour in HCl and H2SO4 acid hydrolysis

A validated stability‐indicating HPLC method was established, and comprehensive stress testing of ivabradine, a cardiotonic drug, was carried out as per ICH guidelines. Ivabradine was subjected to acidic, basic and neutral hydrolysis, oxidation, photolysis and thermal stress conditions, and the resulting degradation products were investigated by LC‐PDA and LC‐HR‐MS/MS. The drug was found to degrade in acid and base hydrolysis. An efficient and selective stability assay method was developed on Phenomenex Luna C18 (250 × 4.6 mm, 5.0 µm) column using ammonium formate (10 mM, pH 3.0) and acetonitrile as mobile phase at 30 °C in gradient elution mode. The flow rate was 0.7 ml/min and detection wavelength was 286 nm. A total of five degradation products (I‐1 to I‐5) were identified and characterized by LC‐HR‐MS/MS in combination with accurate mass measurements. The drug exhibited different degradation behaviour in HCl and H₂SO₄ hydrolysis conditions. It is a unique example where two of the five degradation products in HCl hydrolysis were absent in H₂SO₄ acid hydrolysis. The present study provides guidance to revise the stress test for the determination of inherent stability of drugs containing lactam moiety under hydrolytic conditions. Most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation pattern of the drug and its degradation products. In silico toxicity revealed that the degradation products ( I‐2 to I‐5 ) were found to be severe irritants in case of ocular irritancy. The analytical assay method was validated with respect to specificity, linearity, range, precision, accuracy and robustness. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterization of forced degradation products of ketorolac tromethamine using LC/ESI/Q/TOF/MS/MS and in silico toxicity prediction

Ketorolac, a nonsteroidal anti‐inflammatory drug, was subjected to forced degradation studies as per International Conference on Harmonization guidelines. A simple, rapid, precise, and accurate high‐performance liquid chromatography combined with electrospray ionization quadrupole time‐of‐flight tandem mass spectrometry (LC/ESI/Q/TOF/MS/MS) method has been developed for the identification and structural characterization of stressed degradation products of ketorolac. The drug was found to degrade in hydrolytic (acidic, basic, and neutral), photolytic (acidic, basic, and neutral solution), and thermal conditions, whereas the solid form of the drug was found to be stable under photolytic conditions. The method has shown adequate separation of ketorolac tromethamine and its degradation products on a Grace Smart C‐18 (250 mm × 4.6 mm i.d., 5 µm) column using 20 mM ammonium formate (pH = 3.2): acetonitrile as a mobile phase in gradient elution mode at a flow rate of 1.0 ml/min. A total of nine degradation products were identified and characterized by LC/ESI/MS/MS. The most probable mechanisms for the formation of degradation products have been proposed on the basis of a comparison of the fragmentation of the [M + H]⁺ ions of ketorolac and its degradation products. In silico toxicity of the drug and degradation products was investigated by using topkat and derek softwares. The method was validated in terms of specificity, linearity, accuracy, precision, and robustness as per International Conference on Harmonization guidelines. Copyright © 2014 John Wiley & Sons, Ltd.     

In vivo metabolic investigation of cetilistat in normal versus pseudo-germ-free rats using UPLC-QTOFMS/MS and in silico toxicological evaluation of its metabolites

Cetilistat (CET) is a pancreatic lipase inhibitor approved for management of obesity after the serious adverse effects exhibited by its analogue orlistat. Exhaustive literature review reveals lack of comprehensive reports on its biotransformation. With a view to study the same, the present study reports the identification and characterization of metabolites of CET in rats using UPLC–MS/MS. As the small intestine is the site of action for CET, it is important that the role of microbial flora in the metabolism of CET be explored. To achieve this, the metabolic profile of CET was compared between normal and pseudo-germ-free rats. The study involved the administration of a drug suspension to male Sprague–Dawley pseudo-germ-free and normal untreated rats followed by collection of urine, feces, and blood at specific intervals. Sample preparation was performed using liquid–liquid extraction and concentration of samples followed by analysis using LC–MS/MS. Finally, an in silico study was performed on the drug and metabolites to predict their toxicological properties using ADMET Predictor^TM software. Four metabolites of CET were observed in in vivo matrices. As expected, significant changes were observed both qualitatively and quantitatively, implying that formation of metabolites was both CYP enzymes and gut microflora mediated.     

Identification of dual Acetyl-CoA carboxylases 1 and 2 inhibitors by pharmacophore based virtual screening and molecular docking approach

Acetyl-CoA carboxylase (ACC) is a crucial metabolic enzyme that plays a vital role in obesity-induced type 2 diabetes and fatty acid metabolism. To identify dual inhibitors of Acetyl-CoA carboxylase1 and Acetyl-CoA carboxylase2, a pharmacophore modelling approach has been employed. The best HypoGen pharmacophore model for ACC2 inhibitors (Hypo1_ACC2) consists of one hydrogen bond acceptor, one hydrophobic aliphatic and one hydrophobic aromatic feature, whereas the best pharmacophore (Hypo1_ACC1) for ACC1 consists of one additional hydrogen-bond donor (HBD) features. The best pharmacophore hypotheses were validated by various methods such as test set, decoy set and Cat-Scramble methodology. The validated pharmacophore models were used to screen several small-molecule databases, including Specs, NCI, ChemDiv and Natural product databases to identify the potential dual ACC inhibitors. The virtual hits were then subjected to several filters such as estimated $\text{ IC}_{50}$ value, quantitative estimation of drug-likeness and molecular docking analysis. Finally, three novel compounds with diverse scaffolds were selected as potential starting points for the design of novel dual ACC inhibitors.