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      A cocktail probe approach to evaluate the effect of hormones on the expression and activity of CYP enzymes in human hepatocytes with conditions simulating late stage of pregnancy

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          Abstract

          Purpose

          Pregnancy-mediated physiological and biochemical changes contribute to alterations in the pharmacokinetics of certain drugs. There is a paucity of data on the systematic evaluation of the underlying mechanisms. The objective of the current study was to examine the impact of changes in circulating and tissue hormonal concentration during the late stage of pregnancy on the activity and expression of hepatic cytochrome P450 (CYP) enzymes using a cocktail probe approach.

          Methods

          Freshly isolated primary human hepatocytes were incubated with third trimester physiologic (plasma) and projected liver (ten-fold higher) concentrations of female hormones: progesterone (2 µM), estradiol (0.3 µM), estriol (0.8 µM), estrone (0.2 µM), 17α-hydroxyprogesterone (0.1 µM), and human growth hormone (0.005 µM). The metabolic activity of the hepatocytes was assessed using a cocktail of isozyme-specific P450 probe substrates (CYP1A2 (phenacetin), CYP2C9 (diclofenac), CYP2C19 (S-mephenytoin), CYP2D6 (dextromethorphan), and CYP3A4 (testosterone)). A validated LC–MS/MS assay was used to measure the corresponding metabolite concentrations. CYP450 protein and mRNA levels were measured using western blot and qRT-PCR, respectively.

          Results

          Female hormones at projected third-semester hepatic concentrations significantly enhanced mRNA and protein expression and increased the metabolic activity of CYP3A4. The expression and activity of other CYP450 enzymes studied were not affected by mixtures of female hormones at concentrations used.

          Conclusion

          The increased activity of CYP3A4 is consistent with the clinically observed increase in clearance of CYP3A4 substrates during pregnancy. Overall expression and activity of CYP450 isozymes are differentially regulated during pregnancy.

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          Most cited references37

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          Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method.

          The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data. Copyright 2001 Elsevier Science (USA).
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            Measurement of protein using bicinchoninic acid

            Bicinchoninic acid, sodium salt, is a stable, water-soluble compound capable of forming an intense purple complex with cuprous ion (Cu1+) in an alkaline environment. This reagent forms the basis of an analytical method capable of monitoring cuprous ion produced in the reaction of protein with alkaline Cu2+ (biuret reaction). The color produced from this reaction is stable and increases in a proportional fashion over a broad range of increasing protein concentrations. When compared to the method of Lowry et al., the results reported here demonstrate a greater tolerance of the bicinchoninate reagent toward such commonly encountered interferences as nonionic detergents and simple buffer salts. The stability of the reagent and resulting chromophore also allows for a simplified, one-step analysis and an enhanced flexibility in protocol selection. This new method maintains the high sensitivity and low protein-to-protein variation associated with the Lowry technique.
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              Pregnancy-induced changes in pharmacokinetics: a mechanistic-based approach.

              Observational studies have documented that women take a variety of medications during pregnancy. It is well known that pregnancy can induce changes in the plasma concentrations of some drugs. The use of mechanistic-based approaches to drug interactions has significantly increased our ability to predict clinically significant drug interactions and improve clinical care. This same method can also be used to improve our understanding regarding the effect of pregnancy on pharmacokinetics of drugs. Limited studies suggest bioavailability of drugs is not altered during pregnancy. Increased plasma volume and protein binding changes can alter the apparent volume of distribution (Vd) of drugs. Through changes in Vd and clearance, pregnancy can cause increases or decreases in the terminal elimination half-life of drugs. Depending on whether a drug is excreted unchanged by the kidneys or which metabolic isoenzyme is involved in the metabolism of a drug can determine whether or not a change in dosage is needed during pregnancy. The renal excretion of unchanged drugs is increased during pregnancy. The metabolism of drugs catalysed by select cytochrome P450 (CYP) isoenzymes (i.e. CYP3A4, CYP2D6 and CYP2C9) and uridine diphosphate glucuronosyltransferase (UGT) isoenzymes (i.e. UGT1A4 and UGT2B7) are increased during pregnancy. Dosages of drugs predominantly metabolised by these isoenzymes or excreted by the kidneys unchanged may need to be increased during pregnancy in order to avoid loss of efficacy. In contrast, CYP1A2 and CYP2C19 activity is decreased during pregnancy, suggesting that dosage reductions may be needed to minimise potential toxicity of their substrates. There are limitations to the available data. This analysis is based primarily on observational studies, many including small numbers of women. For some isoenzymes, the effect of pregnancy on only one drug has been evaluated. The full-time course of pharmacokinetic changes during pregnancy is often not studied. The effect of pregnancy on transport proteins is unknown. Drugs eliminated by non-CYP or non-UGT pathways or multiple pathways will need to be evaluated individually. In conclusion, by evaluating the pharmacokinetic data of a variety of drugs during pregnancy and using a mechanistic-based approach, we can start to predict the effect of pregnancy for a large number of clinically used drugs. However, because of the limitations, more clinical, evidence-based studies are needed to fully elucidate the effects of pregnancy on the pharmacokinetics of drugs.
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                Author and article information

                Contributors
                rv@pitt.edu
                Journal
                Eur J Clin Pharmacol
                Eur J Clin Pharmacol
                European Journal of Clinical Pharmacology
                Springer Berlin Heidelberg (Berlin/Heidelberg )
                0031-6970
                1432-1041
                15 April 2023
                : 1-13
                Affiliations
                [1 ]GRID grid.21925.3d, ISNI 0000 0004 1936 9000, Department of Pharmaceutical Sciences, , University of Pittsburgh School of Pharmacy, ; Pittsburgh, PA 15261 USA
                [2 ]GRID grid.440757.5, ISNI 0000 0004 0411 0012, Clinical Pharmacy Department, College of Pharmacy, , Najran University, ; Najran, Saudi Arabia
                [3 ]GRID grid.21925.3d, ISNI 0000 0004 1936 9000, Department of Pharmacy and Therapeutics, , University of Pittsburgh School of Pharmacy, ; Pittsburgh, PA 15261 USA
                [4 ]GRID grid.411487.f, ISNI 0000 0004 0455 1723, Department of Obstetrics and Gynecology, , UPMC Magee Women’s Hospital, ; Pittsburgh, PA 15213 USA
                Article
                3489
                10.1007/s00228-023-03489-1
                10105140
                37060457
                cc2d407f-aacc-4fb4-b1c5-f6c98662c47f
                © The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature 2023, Springer Nature or its licensor (e.g. a society or other partner) holds exclusive rights to this article under a publishing agreement with the author(s) or other rightsholder(s); author self-archiving of the accepted manuscript version of this article is solely governed by the terms of such publishing agreement and applicable law.

                This article is made available via the PMC Open Access Subset for unrestricted research re-use and secondary analysis in any form or by any means with acknowledgement of the original source. These permissions are granted for the duration of the World Health Organization (WHO) declaration of COVID-19 as a global pandemic.

                History
                : 12 July 2022
                : 28 March 2023
                Funding
                Funded by: FundRef http://dx.doi.org/10.13039/100009633, Eunice Kennedy Shriver National Institute of Child Health and Human Development;
                Award ID: HD-047905-2
                Award ID: HD-047905-2
                Award Recipient :
                Categories
                Research

                Pharmacology & Pharmaceutical medicine
                pregnancy,female hormones,primary human hepatocytes,cocktail probes,cytochrome p450,liquid chromatography-tandem mass spectrometry (lc–ms/ms),quantitative real-time polymerase chain reaction (qrt-pcr)

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