In Vitro Drug Metabolism Using Liver Microsomes

Knights, Kathleen M.; Stresser, David M.; Miners, John O.; Crespi, Charles L.

doi:10.1002/cpph.9

Cited by 64 publications

(63 citation statements)

References 45 publications

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“…On the other hand, hepatocytes, which contain intact cell membranes and physiological concentrations of enzymes, provide the most physiologically relevant model for predicting hepatic clearance (2,15,18,37,39,48,(55)(56)(57)(58). Some researchers use microsomes in conjunction with hepatocytes to obtain more comprehensive results (7,55,(59)(60)(61).…”

Section: Metabolic Stability and Its Significancementioning

confidence: 99%

Metabolic stability and its role in the discovery of new chemical entities

et al. 2019

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Determination of metabolic profiles of new chemical entities is a key step in the process of drug discovery, since it influences pharmacokinetic characteristics of therapeutic compounds. One of the main challenges of medicinal chemistry is not only to design compounds demonstrating beneficial activity, but also molecules exhibiting favourable pharmacokinetic parameters. Chemical compounds can be divided into those which are metabolized relatively fast and those which undergo slow biotransformation. Rapid biotransformation reduces exposure to the maternal compound and may lead to the generation of active, non-active or toxic metabolites. In contrast, high metabolic stability may promote interactions between drugs and lead to parent compound toxicity. In the present paper, issues of compound metabolic stability will be discussed, with special emphasis on its significance, in vitro metabolic stability testing, dilemmas regarding in vitro-in vivo extrapolation of the results and some aspects relating to different preclinical species used in in vitro metabolic stability assessment of compounds.

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Section: Metabolic Stability and Its Significancementioning

confidence: 99%

Metabolic stability and its role in the discovery of new chemical entities

et al. 2019

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“…Several different kinds of in vitro methods have been established to identify and quantitatively measure the type and extent of CYP inhibition. In such experiments, the CYP enzymes can be introduced into the incubation mixture as individual cDNA-expressed CYP forms or as enzyme mixtures, i.e., tissue fractions such as human liver microsomes (Pelkonen et al, 2005;, Knights et al, 2016.…”

Section: Introductionmentioning

confidence: 99%

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes

et al. 2018

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1. Cytochrome P450 (CYP) enzymes constitute an essential xenobiotic metabolizing system that regulates the elimination of lipophilic compounds from the body. Convenient and affordable assays for CYP enzymes are important for assessing these metabolic pathways. 2. In this study, 10 novel profluorescent coumarin derivatives with various substitutions at carbons 3, 6 and 7 were developed. Molecular modeling indicated that 3phenylcoumarin offers an excellent scaffold for the development of selective substrate compounds for various human CYP forms, as they could be metabolized to fluorescent 7-hydroxycoumarin derivatives. Oxidation of profluorescent coumarin derivatives to fluorescent metabolites by 13 important human liver xenobiotic-metabolizing CYP forms was determined by enzyme kinetic assays. 3. Four of the coumarin derivatives were converted to fluorescent metabolites by CYP1 family enzymes, with 6-methoxy-3-(4-trifluoromethylphenyl)coumarin being oxidized selectively by CYP1A2 in human liver microsomes. Another set of four compounds were metabolized by CYP2A6 and CYP1 enzymes. 7-Methoxy-3-(3methoxyphenyl)coumarin was oxidized efficiently by CYP2C19 and CYP2D6 in a nonselective fashion. 4. The advantages of the novel substrates were 1) an excellent signal-to-background ratio, 2) selectivity for CYP1 forms, and 3) convenient multiwell plate measurement, allowing for precise determination of potential inhibitors of important human hepatic forms CYP1A2, CYP2C19 and CYP2D6.

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“…4,9 As most investigated SCs are prone to extensive biotransformation, parent compounds are rarely detected in urine. [18][19][20][21] Complementary to HLMs, human liver cytosol (HLCYT) contains sulfotransferase (SULT) and can be used to study in vitro sulfation reactions. Since information about biotransformation products of novel SCs is very limited to unavailable, the detection of these illicit drugs in various biological matrices can be challenging.…”

Section: Introductionmentioning

confidence: 99%

“…10,11,[13][14][15][16][17] Pooled human liver microsomes (HLMs) are a suitable source of enzymes for examining in vitro human metabolism as they contain the major drug-metabolizing enzymes: The cytochrome P450 isoenzymes (CYPs) and UDP-glucuronosyltransferases (UGTs). [18][19][20][21] Complementary to HLMs, human liver cytosol (HLCYT) contains sulfotransferase (SULT) and can be used to study in vitro sulfation reactions.…”

Section: Introductionmentioning

confidence: 99%

Suspect and non‐target screening workflows to investigate the in vitro and in vivo metabolism of the synthetic cannabinoid 5Cl‐THJ‐018

Vervliet

Mortelé

Gys

et al. 2018

Drug Testing and Analysis

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The use of synthetic cannabinoids causes similar effects as Δ 9 -tetrahydrocannabinol and long-term (ab)use can lead to health hazards and fatal intoxications. As most investigated synthetic cannabinoids undergo extensive biotransformation, almost no parent compound can be detected in urine, which hampers forensic investigations.Limited information about the biotransformation products of new synthetic cannabinoids makes the detection of these drugs in various biological matrices challenging.This study aimed to identify the main in vitro biotransformation pathways of 5Cl-THJ-018 and to compare these findings with an authentic urine sample of a 5Cl-THJ-018 user. The synthetic cannabinoid was incubated with pooled human liver microsomes and cytosol to simulate phase I and phase II biotransformations. Resulting extracts were analyzed with liquid chromatography coupled to quadrupole time-offlight mass spectrometry (LC-QTOF-MS). Three different data analysis workflows were applied to identify biotransformation products. A suspect screening workflow used an in-house database built from literature data and in silico biotransformation predictions. Two non-target screening workflows used a commercially available software and an open-source software for mass spectrometry data processing. A total of 23 in vitro biotransformation products were identified, with hydroxylation, oxidative dechlorination, and dihydrodiol formation pathways as the main phase I reactions.Additionally, five glucuronidated and three sulfated phase II conjugates were identified. The predominant in vivo pathway was through oxidative dechlorination and in total six metabolites of 5Cl-THJ-018 were identified. Biotransformation products both in vitro and in vivo were successfully identified using complementary suspect and non-target screening workflows. KEYWORDS cytosolic fractions, human liver microsomes, In vitro biotransformation, liquid chromatographymass spectrometry, new psychoactive substances Philippe Vervliet and Olivier Mortelé joint first authors.

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In Vitro Drug Metabolism Using Liver Microsomes

Cited by 64 publications

References 45 publications

Metabolic stability and its role in the discovery of new chemical entities

Metabolic stability and its role in the discovery of new chemical entities

Development of new Coumarin-based profluorescent substrates for human cytochrome P450 enzymes

Suspect and non‐target screening workflows to investigate the in vitro and in vivo metabolism of the synthetic cannabinoid 5Cl‐THJ‐018

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