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In vitro metabolism, reaction phenotyping, enzyme kinetics, CYP inhibition and induction potential of ataluren.

ABSTRACT: Ataluren promotes ribosomal readthrough of premature termination codons in mRNA which result from nonsense mutations. In vitro studies were performed to characterize the metabolism and enzyme kinetics of ataluren and its interaction potential with CYP enzymes. Incubation of [¹⁴ C]-ataluren with human liver microsomes indicated that the major metabolic pathway for ataluren is via direct glucuronidation and that the drug is not metabolized via cytochrome P450 (CYP). Glucuronidation was also observed in the incubation in human intestinal and kidney microsomes, but not in human pulmonary microsomes. UGT1A9 was found to be the major uridine diphosphate glucuronosyltransferase (UGT) responsible for ataluren glucuronidation in the liver and kidney microsomes. Enzyme kinetic analysis of the formation of ataluren acyl glucuronide, performed in human liver, kidney, and intestinal microsomes and recombinant human UGT1A9, found that increasing bovine serum albumin (BSA) levels enhanced the glucuronidation Michaelis-Menten constant (K_m ) and ataluren protein binding but had a minimal effect on maximum velocity (V_max ) of glucuronidation. Due to the decreased unbound Michaelis-Menten constant (K_m,u ), the ataluren unbound intrinsic clearance (CL_int,u ) increased for all experimental systems and BSA concentrations. Human kidney microsomes were about 3.7-fold more active than human liver microsomes, in terms of CL_int,u /mg protein, indicating that the kidney is also a key organ for the metabolism and disposition of ataluren in humans. Ataluren showed no or little potential to inhibit or induce most of the CYP enzymes.

SUBMITTER: Kong R

PROVIDER: S-EPMC7083565 | biostudies-literature | 2020 Apr

REPOSITORIES: biostudies-literature

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In vitro metabolism, reaction phenotyping, enzyme kinetics, CYP inhibition and induction potential of ataluren.

Kong Ronald R Ma Jiyuan J Hwang Seongwoo S Moon Young-Choon YC Welch Ellen M EM Weetall Marla M Colacino Joseph M JM Almstead Neil N Babiak John J Goodwin Elizabeth E

Pharmacology research & perspectives 20200401 2

Ataluren promotes ribosomal readthrough of premature termination codons in mRNA which result from nonsense mutations. In vitro studies were performed to characterize the metabolism and enzyme kinetics of ataluren and its interaction potential with CYP enzymes. Incubation of [<sup>14</sup> C]-ataluren with human liver microsomes indicated that the major metabolic pathway for ataluren is via direct glucuronidation and that the drug is not metabolized via cytochrome P450 (CYP). Glucuronidation was ...[more]

PMID: 32196986

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Project description:Chemicals often require metabolic activation to become genotoxic. Established test guidelines recommend the use of the rat liver S9 fraction or microsomes to introduce metabolic competence to in vitro cell-based bioassays, but the use of animal-derived components in cell culture raises ethical concerns and may lead to quality issues and reproducibility problems. The aim of the present study was to compare the metabolic activation of cyclophosphamide (CPA) and benzo[a]pyrene (BaP) by induced rat liver microsomes and an abiotic cytochrome P450 (CYP) enzyme based on a biomimetic porphyrine catalyst. For the detection of genotoxic effects, the chemicals were tested in a reporter gene assay targeting the activation of the cellular tumor protein p53. Both chemicals were metabolized by the abiotic CYP enzyme and the microsomes. CPA showed no activation of p53 and low cytotoxicity without metabolic activation, but strong activation of p53 and increased cytotoxicity upon incubation with liver microsomes or abiotic CYP enzyme. The effect concentration causing a 1.5-fold induction of p53 activation was very similar with both metabolization systems (within a factor of 1.5), indicating that genotoxic metabolites were formed at comparable concentrations. BaP also showed low cytotoxicity and no p53 activation without metabolic activation. The activation of p53 was detected for BaP upon incubation with active and inactive microsomes at similar concentrations, indicating experimental artifacts caused by the microsomes or NADPH. The activation of BaP with the abiotic CYP enzyme increased the cytotoxicity of BaP by a factor of 8, but no activation of p53 was detected. The results indicate that abiotic CYP enzymes may present an alternative to rat liver S9 fraction or microsomes for the metabolic activation of test chemicals, which are completely free of animal-derived components. However, an amendment of existing test guidelines would require testing of more chemicals and genotoxicity end points.

Dataset Information

In vitro metabolism, reaction phenotyping, enzyme kinetics, CYP inhibition and induction potential of ataluren.

Publications

In vitro metabolism, reaction phenotyping, enzyme kinetics, CYP inhibition and induction potential of ataluren.

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