Effects of 31 recombinant CYP2C19 variants on clomipramine metabolism in vitro

2021 ◽  
pp. 026988112110505
Author(s):  
Tian Lan ◽  
Ya-Qing Ma ◽  
Ya-Min Dang ◽  
Chen-Chen Wang ◽  
Ren-Ai Xu ◽  
...  
Keyword(s):  
High Performance ◽  
Intrinsic Clearance ◽  
Cytochrome P450 Enzyme ◽  
Liquid Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Catalytic Activities ◽  
Enzyme Superfamily ◽  
P450 Enzyme

Background: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31CYP2C19 alleles in the Han Chinese population; studying the effects of CYP2C19 on drug metabolism can help reduce adverse drug reactions and therapeutic failure. Aim: The aim of this study was to assess the catalytic activities of 31 allelic isoforms and their effects on the metabolism of clomipramine in vitro. Methods: The wild-type and 30 CYP2C19 variants were expressed in insect cells, and each variant was characterized using clomipramine as the substrate. Reactions were performed at 37°C with 5–150 μmol/L substrate for 30 min. By using ultra-high-performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of N-desmethyl clomipramine were determined. Results: Among the CYP2C19 variants tested, CYP2C19*29, L16F, and T130M showed extremely increased intrinsic clearance of clomipramine, CYP2C19*3C, and N277K showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 0.65% to 63.28%). In addition, CYP2C19*3 and 35FS could not be detected because they have no detectable enzyme activity. Conclusions: As the first report of 31 CYP2C19 alleles for clomipramine metabolism, our study could provide corresponding reference for clomipramine for further studies in vivo and offer valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.

Effects of CYP2C19 Variants on Fluoxetine Metabolism in vitro

Pharmacology ◽  
2017 ◽  
Vol 100 (1-2) ◽  
pp. 91-97 ◽  
Author(s):  
Ping Fang ◽  
Jia-Yang He ◽  
Ai-Xia Han ◽  
Tian Lan ◽  
Da-Peng Dai ◽  
...  
Keyword(s):  
High Performance ◽  
Insect Cells ◽  
Intrinsic Clearance ◽  
Cytochrome P450 Enzyme ◽  
Liquid Chromatography Mass Spectrometry ◽  
Wild Type ◽  
Catalytic Activities ◽  
Enzyme Superfamily ◽  
P450 Enzyme

Aims: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31 CYP2C19 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of fluoxetine in vitro. Methods: The wild-type and 30 CYP2C19 variants were expressed in insect cells and each variant was characterized using fluoxetine as the substrate. Reactions were performed at 37°C with 20-1,000 µmol/L substrate for 30 min. By using ultra-high performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of norfluoxetine were determined. Results: Among the CYP2C19 variants tested, T130M showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 9.56 to 77.77%). In addition, CYP2C19*3 and *35FS could not be detected because they have no detectable enzyme activity. Conclusion: In China, the assessment of CYP2C19 variants in vitro offers valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.

Assessment of 25CYP2D6alleles found in the Chinese population on propafenone metabolism in vitro

2016 ◽  
Vol 94 (8) ◽  
pp. 895-899 ◽  
Author(s):  
Ying Su ◽  
Bing-qing Liang ◽  
Yan-lin Feng ◽  
Yunyun Zhan ◽  
Ermin Gu ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Spodoptera Frugiperda ◽  
Expression System ◽  
Ultra Performance Liquid Chromatography ◽  
Intrinsic Clearance ◽  
Cytochrome P450 Enzyme ◽  
Signal Collection ◽  
Enzyme Superfamily ◽  
P450 Enzyme

Cytochrome P450 enzyme 2D6 (CYP2D6) is an important member of the cytochrome P450 enzyme superfamily, with more than 100 CYP2D6 allelic variants being previously reported. The aim of this study was to assess the catalytic characteristics of 25 alleles (CYP2D6.1 and 24 CYP2D6 variants) and their effects on the metabolism of propafenone in vitro. Twenty-five CYP2D6 alleles were expressing in 21 Spodoptera frugiperda (Sf) insect cells, and each variant was evaluated using propafenone as the substrate. Reactions were performed at 37 °C with 1–100 μmol/L propafenone for 30 min. After termination, the product 5-OH-propafenone was extracted and used for signal collection by ultra-performance liquid chromatography (UPLC). Compared with wild type CYP2D6.1, the intrinsic clearance (Vmaxand Km) values of all variants were significantly altered. Three variants (CYP2D6.87, CYP2D6.90, CYP2D6.F219S) exhibited markedly increased intrinsic clearance values (129% to 165%), whereas 21 variants exhibited significantly decreased values (16% to 85%) due to increased Kmand (or) decreased Vmaxvalues. These results indicated that the majority of tested alleles had significantly altered catalytic activity towards propafenone hydroxylation in this expression system. Attention should be paid to subjects carrying these rare alleles when treated with propafenone.

scholarly journals Chlorogenic Acid as a Model Compound for Optimization of an In Vitro Gut Microbiome-Metabolism Model

Proceedings ◽  
2019 ◽  
Vol 11 (1) ◽  
pp. 31 ◽  
Author(s):  
Olivier Mortelé ◽  
Elias Iturrospe ◽  
Annelies Breynaert ◽  
Christine Lammens ◽  
Xavier Basil Britto ◽  
...  
Keyword(s):  
Chlorogenic Acid ◽  
Gut Microbiome ◽  
Model Compound ◽  
In Vitro Models ◽  
Cytochrome P450 Enzyme ◽  
Flight Mass Spectrometry ◽  
P450 Enzyme ◽  
Analytical Phase

It has been believed that the metabolism of xenobiotics occurred mainly by the cytochrome P450 enzyme system in the liver. However, recent data clearly suggest a significant role for the gut microbiota in the metabolism of xenobiotic compounds. This microbiotic biotransformation could lead to differences on activation, inactivation and possible toxicity of these compounds. In vitro models are generally used to study the colonic biotransformation as they allow easy dynamic and multiple sampling over time. However, to ensure this accurately mimics communities in vivo, the pre-analytical phase requires optimization. Chlorogenic acid, a polyphenolic compound abundantly present in the human diet, was used as a model compound to optimize a ready-to-use gut microbiome biotransformation platform. Samples of the in vitro gastrointestinal dialysis-model with colon stage were analyzed by liquid chromatography coupled to high resolution time-of-flight mass spectrometry. Complementary screening approaches were also employed to identify the biotransformation products.

Enzymic Cleavage Of In Vivo Formed Maillard-Type Compounds Involved In Haemostasis

1981 ◽  
Author(s):  
L Mester ◽  
L Szabados ◽  
M Mester
Keyword(s):  
Enzyme System ◽  
Basal Membrane ◽  
Enzyme Concentration ◽  
Cytochrome P450 Enzyme ◽  
Microsomal Preparation ◽  
Chemical Hydrolysis ◽  
P450 Enzyme ◽  
Liver Microsomal Preparation

Desoxyfructose derivatives of serotonin (Mester et al.,1975), of haemoglobin (Flückiger and Winterhalter, 1976), of poly-L-lysine (Mester et al., 1975) and of lysine rich histones (Kertesz-Crisba, 1977) are easily formed in vivo by a simple Maillard-type chemical reaction. Some of these compounds interfere with platelet functions (Mester et al.,1976) or contribute to the thickening of the basal membrane of blood vessels (Cerami et al., 1979).While the chemical synthesis of Maillard-type compounds proceeds readily even in vivo, the chemical cleavage of them needs sever conditions which certainly do not exist in vivo (Gottschalk, 1952). However, a slow liberation of serotonin from desoxyfructo-serotonin is observed in vivo, suggesting the existence of an enzyme system for the cleavage of Maillard-type sugar-amine derivatives. In vitro, using a sheep liver microsomal preparation rich in Cytochrome P450 enzyme, the liberation of serotonin is in linear correlation with the enzyme concentration. The cleavage of desoxyfructo-serotonin is activated by NADPH having its optimum at pH=7.4, excluding definitely the occurence of a chemical hydrolysis.Factors interfering with the enzyme system involved in the cleavage of Maillard-type compounds, may also interfere with haemostasis.

scholarly journals Endocannabinoid turnover in GtoPdb v.2021.3

2021 ◽  
Vol 2021 (3) ◽  
Author(s):  
Stephen P.H. Alexander ◽  
Patrick Doherty ◽  
Christopher J. Fowler ◽  
Jürg Gertsch ◽  
Mario Van der Stelt
Keyword(s):  
Cytochrome P450 ◽  
Phospholipase D ◽  
Cytochrome P450 Enzyme ◽  
Facilitated Diffusion ◽  
In Vitro Experiments ◽  
Diacylglycerol Lipase ◽  
P450 Enzyme ◽  
Key Enzyme

The principle endocannabinoids are 2-acylglycerol esters, such as 2-arachidonoylglycerol (2-AG), and N-acylethanolamines, such as anandamide (N-arachidonoylethanolamine, AEA). The glycerol esters and ethanolamides are synthesised and hydrolysed by parallel, independent pathways. Mechanisms for release and re-uptake of endocannabinoids are unclear, although potent and selective inhibitors of facilitated diffusion of endocannabinoids across cell membranes have been developed [28]. FABP5 (Q01469) has been suggested to act as a canonical intracellular endocannabinoid transporter in vivo [17]. For the generation of 2-arachidonoylglycerol, the key enzyme involved is diacylglycerol lipase (DAGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, [70]). A transacylation enzyme which forms N-acylphosphatidylethanolamines has been identified as a cytosolic enzyme, PLA2G4E (Q3MJ16) [62]. In vitro experiments indicate that the endocannabinoids are also substrates for oxidative metabolism via cyclooxygenase, lipoxygenase and cytochrome P450 enzyme activities [5, 23, 72].

scholarly journals Endocannabinoid turnover (version 2019.4) in the IUPHAR/BPS Guide to Pharmacology Database

2019 ◽  
Vol 2019 (4) ◽  
Author(s):  
Stephen P.H. Alexander ◽  
Patrick Doherty ◽  
Christopher J. Fowler ◽  
Jürg Gertsch ◽  
Mario Van der Stelt
Keyword(s):  
Cytochrome P450 ◽  
Phospholipase D ◽  
Cytochrome P450 Enzyme ◽  
Facilitated Diffusion ◽  
In Vitro Experiments ◽  
Diacylglycerol Lipase ◽  
P450 Enzyme ◽  
Key Enzyme

The principle endocannabinoids are 2-acylglycerol esters, such as 2-arachidonoylglycerol (2-AG), and N-acylethanolamines, such as anandamide (N-arachidonoylethanolamine, AEA). The glycerol esters and ethanolamides are synthesised and hydrolysed by parallel, independent pathways. Mechanisms for release and re-uptake of endocannabinoids are unclear, although potent and selective inhibitors of facilitated diffusion of endocannabinoids across cell membranes have been developed [19]. FABP5 (Q01469) has been suggested to act as a canonical intracellular endocannabinoid transporter in vivo [12]. For the generation of 2-arachidonoylglycerol, the key enzyme involved is diacylglycerol lipase (DAGL), whilst several routes for anandamide synthesis have been described, the best characterized of which involves N-acylphosphatidylethanolamine-phospholipase D (NAPE-PLD, [49]). A transacylation enzyme which forms N-acylphosphatidylethanolamines has recently been identified as a cytosolic enzyme, PLA2G4E (Q3MJ16) [43]. In vitro experiments indicate that the endocannabinoids are also substrates for oxidative metabolism via cyclooxygenase, lipoxygenase and cytochrome P450 enzyme activities [4, 16, 51].

scholarly journals Reconstitution of theIn VitroActivity of the Cyclosporine-Specific P450 Hydroxylase from Sebekia benihana and Development of a Heterologous Whole-Cell Biotransformation System

2015 ◽  
Vol 81 (18) ◽  
pp. 6268-6275 ◽  
Author(s):  
Li Ma ◽  
Lei Du ◽  
Hui Chen ◽  
Yue Sun ◽  
Shan Huang ◽  
...  
Keyword(s):  
Redox System ◽  
Cytochrome P450 Enzyme ◽  
Regeneration System ◽  
Nadph Regeneration ◽  
Site Specific ◽  
Whole Cell ◽  
Content Type ◽  
P450 Enzyme

ABSTRACTThe cytochrome P450 enzyme CYP-sb21 fromSebekia benihanais capable of catalyzing the site-specific hydroxylation of the immunosuppressant cyclosporine (CsA), leading to the single product γ-hydroxy-N-methyl-l-Leu4-CsA (CsA-4-OH). Unlike authentic CsA, this hydroxylated CsA shows significantly reduced immunosuppressive activity while it retains a side effect of CsA, the hair growth stimulation effect. Although CYP-sb21 was previously identified to be responsible for CsA-specific hydroxylationin vivo, thein vitroactivity of CYP-sb21 has yet to be established for a deeper understanding of this P450 enzyme and further reaction optimization. In this study, we reconstituted thein vitroactivity of CYP-sb21 by using surrogate redox partner proteins of bacterial and cyanobacterial origins. The highest CsA site-specific hydroxylation activity by CYP-sb21 was observed when it was partnered with the cyanobacterial redox system composed ofseFdx andseFdR fromSynechococcus elongatusPCC 7942. The best bioconversion yields were obtained in the presence of 10% methanol as a cosolvent and an NADPH regeneration system. A heterologous whole-cell biocatalyst usingEscherichia coliwas also constructed, and the permeability problem was solved by usingN-cetyl-N,N,N-trimethylammonium bromide (CTAB). This work provides a useful example for reconstituting a hybrid P450 system and developing it into a promising biocatalyst for industrial application.

scholarly journals Unraveling the Metabolic Routes of Retapamulin: Insights into Drug Development of Pleuromutilins

2018 ◽  
Vol 62 (4) ◽  
Author(s):  
Feifei Sun ◽  
Huiyan Zhang ◽  
Gerard Bryan Gonzales ◽  
Jinhui Zhou ◽  
Yi Li ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Metabolic Pathways ◽  
Enzyme Inhibitors ◽  
Side Chain ◽  
Cytochrome P450 Enzyme ◽  
Cytochrome P450 Enzymes ◽  
Short Term ◽  
P450 Enzyme

ABSTRACT Retapamulin, a semisynthetic pleuromutilin derivative, is exclusively used for the topical short-term medication of impetigo and staphylococcal infections. In the present study, we report that retapamulin is adequately and rapidly metabolized in vitro via various metabolic pathways, such as hydroxylation, including mono-, di-, and trihydroxylation, and demethylation. Like tiamulin and valnemulin, the major metabolic routes of retapamulin were hydroxylation at the 2β and 8α positions of the mutilin moiety. Moreover, in vivo metabolism concurred with the results of the in vitro assays. Additionally, we observed significant interspecies differences in the metabolism of retapamulin. Until now, modifying the side chain was the mainstream method for new drug discovery of the pleuromutilins. This approach, however, could not resolve the low bioavailability and short efficacy of the drugs. Considering the rapid metabolism of the pleuromutilins mediated by cytochrome P450 enzymes, we propose that blocking the active metabolic site (C-2 and C-8 motif) or administering the drug in combination with cytochrome P450 enzyme inhibitors is a promising pathway in the development of novel pleuromutilin drugs with slow metabolism and long efficacy.

scholarly journals The role of cytochrome P450 enzymes in carcinogen activation and detoxication: an in vivo–in vitro paradox

2018 ◽  
Vol 39 (7) ◽  
pp. 851-859 ◽  
Author(s):  
Lindsay Reed ◽  
Volker M Arlt ◽  
David H Phillips
Keyword(s):  
Cytochrome P450 ◽  
Cytochrome P450 Enzyme ◽  
Cytochrome P450 Enzymes ◽  
Predominant Role ◽  
Enzyme Systems ◽  
P450 Enzyme

Cytochrome P450 enzyme systems have been widely used in vitro to determine the pathways of activation of procarcinogens, but paradoxically, these same enzymes can play a more predominant role in carcinogen detoxification in vivo.

Phytochemical, Analytical and Medicinal Studies of Holoptelea integrifolia Roxb. Planch - A Review

2019 ◽  
Vol 5 (4) ◽  
pp. 270-277 ◽  
Author(s):  
Vijay Kumar ◽  
Simranjeet Singh ◽  
Ragini Bhadouria ◽  
Ravindra Singh ◽  
Om Prakash
Keyword(s):  
Liquid Chromatography ◽  
Thin Layer ◽  
Thin Layer Chromatography ◽  
High Performance ◽  
Biologically Active ◽  
Toxicological Studies ◽  
Wide Range ◽  
Layer Chromatography

Holoptelea integrifolia Roxb. Planch (HI) has been used to treat various ailments including obesity, osteoarthritis, arthritis, inflammation, anemia, diabetes etc. To review the major phytochemicals and medicinal properties of HI, exhaustive bibliographic research was designed by means of various scientific search engines and databases. Only 12 phytochemicals have been reported including biologically active compounds like betulin, betulinic acid, epifriedlin, octacosanol, Friedlin, Holoptelin-A and Holoptelin-B. Analytical methods including the Thin Layer Chromatography (TLC), High-Performance Thin Layer Chromatography (HPTLC), High-Performance Liquid Chromatography (HPLC) and Liquid Chromatography With Mass Spectral (LC-MS) analysis have been used to analyze the HI. From medicinal potency point of view, these phytochemicals have a wide range of pharmacological activities such as antioxidant, antibacterial, anti-inflammatory, and anti-tumor. In the current review, it has been noticed that the mechanism of action of HI with biomolecules has not been fully explored. Pharmacology and toxicological studies are very few. This seems a huge literature gap to be fulfilled through the detailed in-vivo and in-vitro studies.

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