Phenotyping of CYP450 in human liver microsomes using the cocktail approach

The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-of-flight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs

Inhibition of vascular calcification by inositol phosphates derivatized with ethylene glycol oligomers

Myo-inositol hexakisphosphate (IP6) is a natural product known to inhibit vascular calcification (VC), but with limited potency and low plasma exposure following bolus administration. Here we report the design of a series of inositol phosphate analogs as crystallization inhibitors, among which 4,6-di-O-(methoxy-diethyleneglycol)-myo-inositol-1,2,3,5-tetrakis(phosphate), (OEG <sub>2</sub> ) <sub>2</sub> -IP4, displays increased in vitro activity, as well as more favorable pharmacokinetic and safety profiles than IP6 after subcutaneous injection. (OEG <sub>2</sub> ) <sub>2</sub> -IP4 potently stabilizes calciprotein particle (CPP) growth, consistently demonstrates low micromolar activity in different in vitro models of VC (i.e., human serum, primary cell cultures, and tissue explants), and largely abolishes the development of VC in rodent models, while not causing toxicity related to serum calcium chelation. The data suggest a mechanism of action independent of the etiology of VC, whereby (OEG <sub>2</sub> ) <sub>2</sub> -IP4 disrupts the nucleation and growth of pathological calcification

In vitro assessment of human hepatic cytochrome P450 activities using liquid chromatography-mass spectrometry

Les cytochromes P450 (CYPs) catalysent le métabolisme hépatique de phase I des molécules endogènes, mais aussi des xénobiotiques. Une grande variabilité interindividuelle et intraindividuelle est observée dans le taux d'expression et/ou dans l'activité des différentes CYPs. Caractériser leur activité est donc primordial. Cette thèse a pour but de développer des stratégies de criblage à haut débit pour étudier in vitro les activités des CYPs tout en utilisant les procédures conventionnelles. Ce travail s'est ainsi focalisé sur le développement de différentes approches cocktail pour monitorer simultanément l'activité de plusieurs CYPs en un seul essai. Avec la méthode sélectionnée, des nouveaux défis se sont présentés pour l'analyse par chromatographie liquide couplée à la spectrométrie de masse (LC-MS) étant donné la grande complexité de l'échantillon in vitro. C'est pourquoi, plusieurs stratégies analytiques ont été développées couplant la chromatographie liquide à ultra-haute pression (UHPLC) à la spectrométrie de masse à basse et haute résolution

An extensive cocktail approach for rapid risk assessment of in vitro CYP450 direct reversible inhibition by xenobiotic exposure

Acute exposure to environmental factors strongly affects the metabolic activity of cytochrome P450 (P450). As a consequence, the risk of interaction could be increased, modifying the clinical outcomes of a medication. Because toxic agents cannot be administered to humans for ethical reasons, in vitro approaches are therefore essential to evaluate their impact on P450 activities. In thiswork, an extensive cocktail mixturewas developed and validated for in vitro P450 inhibition studies using human liver microsomes (HLM). The cocktail comprised eleven P450-specific probe substrates to simultaneously assess the activities of the following isoforms: 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, 2J2 and subfamily 3A. The high selectivity and sensitivity of the developed UHPLC-MS/MS methodwere critical for the success of this methodology, whose main advantages are: (i) the use of eleven probe substrates with minimized interactions, (ii) a low HLMconcentration, (iii) fast incubation (5 min) and (iv) the use of metabolic ratios as microsomal P450 activities markers. This cocktail approach was successfully validated by comparing the obtained IC50 values for model inhibitorswith those generatedwith the conventional single probe methods. Accordingly, reliable inhibition values could be generated 10-fold faster using a 10-fold smaller amount of HLM compared to individual assays. This approach was applied to assess the P450 inhibition potential of widespread insecticides, namely, chlorpyrifos, fenitrothion, methylparathion and profenofos. In all cases, P450 2B6 was the most affected with IC50 values in the nanomolar range. For the first time, mixtures of these four insecticides incubated at lowconcentrations showed a cumulative inhibitory in vitro effect on P450 2B6

Coupling ultra-high-pressure liquid chromatography with mass spectrometry for in-vitro drug-metabolism studies

This review emphasizes the benefits of ultra-high-pressure liquid chromatography (UHPLC) in the context of in-vitro drug-metabolism and pharmacokinetics (DMPK) studies. The analysis throughput afforded by UHPLC is particularly useful during the early DMPK stages, when rapid, reliable data must be generated on a large number of new chemical entities (NCEs) to select the most appropriate drug candidates. UHPLC also offers high chromatographic resolution, an important feature for the identification of unknown metabolites that are potentially generated from NCEs by a few dozen biochemical enzymes and combinations of them. Most DMPK studies require selectivity and peak-assignment certainty that is only afforded by mass spectrometry (MS) detectors. We therefore thoroughly discuss the use of UHPLC-MS and UHPLCtandem MS with regard to DMPK, highlighting the coupling of UHPLC with quadrupole-basedMS analyzers, time-of-flight MS analyzers and hybrid analyzers

Phenotyping of CYP450 in human liver microsomes using the cocktail approach

The cocktail approach is an advantageous strategy used to monitor the activities of several cytochromes P450 (CYPs) in a single test to increase the throughput of in vitro phenotyping studies. In this study, a cocktail mixture was developed with eight CYP-specific probe substrates to simultaneously evaluate the activity of the most important CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and the CYP3A subfamily. After cocktail incubation in the presence of human liver microsomes (HLMs), the eight selected substrates and their specific metabolites were analyzed by ultra-high-pressure liquid chromatography and electrospray ionization quadrupole time-offlight mass spectrometry. Qualitative and quantitative data were simultaneously acquired to produce an overview of the extended phase I biotransformation routes for each probe substrate in the HLMs and to generate phenotypic profiles of various HLMs. A comparison of the cocktail strategy with an individual substrate assay for each CYP produced similar results. Moreover, the cocktail was tested on HLMs with different allelic variants and/or in the presence of selective inhibitors. The results were in agreement with the genetic polymorphisms of the CYPs and the expected effect of the alterations. All of these experiments confirmed the reliability of this cocktail assay for phenotyping of the microsomal CYPs

A cocktail approach for assessing the in vitro activity of human cytochrome P450s: An overview of current methodologies

An assessment of cytochrome P450 (CYP) enzyme activity is essential for characterizing the phase I metabolism of biological systems or to evaluate the inhibition/induction properties of xenobiotics. CYPs have generally been investigated individually by single probes, and metabolite formation has been monitored by liquid chromatography-mass spectrometry (LC–MS). To increase the throughput, many probes have been applied to assess multiple CYP activities simultaneously within a single experiment. This strategy is called the cocktail approach, and it has already been reviewed for in vivo applications, but never for in vitro ones. This review focuses for the first time on an in vitro cocktail approach, and it references the most notable articles on this topic. The advantages and limitations of applying cocktails for the in vitro activity assessment of major human CYPs, namely, CYP1A2, CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1 and subfamily CYP3A, are discussed. This article considers the probe reaction selections for each CYP according to regulatory recommendations, probe metabolic properties (i.e., specificity and turnover), probe concentrations and analytical sensitivity, but it also highlights a challenge specific to cocktail design, which is probe-probe interaction. The last part of the review reports some methodologies for incubating these cocktails and discusses some important issues regarding the incubation time,enzyme concentrations and sample preparation

ERE-dependent transcription and cell proliferation: Independency of these two processes mediated by the introduction of a sulfone function into the weak estrogen estrothiazine

The synthetic coumestrol derivative 6,12-dihydro-3-methoxy-1-benzopyrano[3,4-b][1,4]benzothiazin-6-one (estrothiazine, ESTZ) has been identified as a weak estrogen receptor α (ERα) ligand unable to compete with tritiated estradiol. The biological activity of this compound, supported by a methoxy group in position 3, seems mainly to result from its capacity to activate ERα dimerization without any participation of coactivators. In support of this view and referring to conventional estrogens, an ESTZ metabolism study conducted with hepatic human microsomes failed to provide any argument in favour of an estrogenic activity dependent on a metabolic conversion of the compound into hydroxylated metabolites with strong receptor activation ability. Interestingly, we failed to detect any oxidation of the sulfur atom of the compound. In the light of pharmacological literature data concerning sulfonylation, we assessed ERα-mediated activities generated by two sulfonylated ESTZ derivatives in which the methoxy group that plays a key role in its mechanism of action was maintained or removed. Sulfonylated ESTZ, even in its demethoxylated form, induced ERE-mediated transcriptions in MCF-7 breast cancer cells, without affecting the ERα turnover rate. In contrast to ESTZ, this compound failed to enhance the proliferation of ERα-positive breast cancer cells, suggesting that its sulfone function confers upon the receptor a capacity to elicit some of the known characteristics associated with estrogenic responses. Moreover, we demonstrated that this sulfone may contribute to ERα dimerization without any requirement of the methoxy group. Nevertheless, it seems to cooperate with this group, as reflected by a weak ability of the sulfonylated form of ESTZ to compete with tritiated estradiol for ERα-binding. Assessment of the docking of this compound within the ligand-binding domain of the receptor by molecular dynamics provided an explanation for this observation since the sulfone is engulfed in a small hydrophobic pocket involving the residues Leu-346, Leu-349, Ala-350 and Leu-384, also known to recruit coactivators. This work not only reports the sulfone functional group as a pharmacophore for estrogenic activity, but also opens new perspectives for the development of estrogenic molecules with therapeutic purpose and devoid of proliferative side effects.SCOPUS: ar.jinfo:eu-repo/semantics/publishe

Inhibition screening method of microsomal UGTs using the cocktail approach

A rapid method for the simultaneous determination of the in vitro activity of the 10 major human liver UDP-glucuronosyltransferase (UGT) enzymes was developed based on the cocktail approach. Specific substrates were first selected for each UGT: etoposide for UGT1A1, chenodeoxycholic acid for UGT1A3, trifluoperazine for UGT1A4, serotonin for UGT 1A6, isoferulic acid for UGT1A9, codeine for UGT2B4, azidothymidine for UGT2B7, levomedetomidine for UGT2B10, 4-hydroxy-3-methoxymethamphetamine for UGT2B15 and testosterone for UGT2B17. Optimal incubation conditions, including time-based experiments on cocktail metabolism in pooled HLMs that had been performed, were then investigated. A 45- min incubation period was found to be a favorable compromise for all the substrates in the cocktail. Ultra-high pressure liquid chromatography coupled to an electrospray ionization time-of-flight mass spectrometer was used to separate the 10 substrates and their UGT-specific glucuronides in less than 6 min. The ability of the cocktail to highlight the UGT inhibitory potential of xenobiotics was initially proven by using well-known UGT inhibitors (selective and nonselective) and then by relating some of the screening results obtained by using the cocktail approach with morphine glucuronidation (substrate highly glucuronidated by UGT 2B7). All the results were in agreement with both the literature and with the expected effect on morphine glucuronidation

Contribution of various types of liquid chromatography–mass spectrometry instruments to band broadening in fast analysis

When performing fast LC with 50mm narrow-bore columns packed with small particles, the LC instrumentation can give rise to non-negligible band broadening. In the present study, the loss in chromatographic efficiency attributed to nine different mass spectrometers of various brands, ionization source geometries and types of analyzers was assessed. In their standard configurations, the extra-column variance of these UHPLC-MS systems was estimated to vary from 20 to >100μL(2). However, it was demonstrated that these differences arise exclusively from the chromatographic system (i.e., injector, tubing, valves, heater) and from the tubing employed to interface the UHPLC instrument with the MS device. By minimizing the tubing used for each UHPLC system, the extra-column variance was reduced to approximately 17-19μL(2) at 600μL/min, for all types of configurations. To achieve optimal chromatographic performance, it is therefore of prime importance to optimize the UHPLC configuration prior to conducting MS. The tubing located between the UHPLC system and the ionization source entrance was found to be particularly critical, as it contributes to band broadening even in the gradient mode. Using an optimized UHPLC-MS configuration, the loss in efficiency with a 50×2.1mm I.D. column was negligible for k>7. However, the efficiency loss with 1mm I.D. columns remained non-negligible for all current instrumentation, even for solutes with a value of k>20. Indeed, for a mixture of isobaric substrates and metabolites analyzed in gradient mode, the peak widths decreased by approximately 50% between a standard and optimized UHPLC-MS configuration, considering a 50×2.1mm, 1.7μm column. The peak broadening was changed by 230% on a 50×1mm, 1.7μm stationary phase, for the same system configurations