Development of Mechanistic Mathematical Models for Gene-mediated Drug-drug Interactions

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Abstract

The glucocorticoid receptor (GR) is a member of the nuclear hormone receptors family and has been shown to exert significant effects on the induction of cytochrome P450 (CYP) enzymes responsible for the metabolism of many xenobiotics. CYP3A4/5 and CYP2C9 are important CYP enzymes which metabolise more that 60% of drugs. Induction or inhibition of the enzymatic activity and the levels of these enzymes can have significant effects on drug metabolism. Understanding the role of GR and other nuclear receptors, pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), in the mechanisms effecting CYP3A4/5 and CYP2C9 levels and activity can aid in the development of in vitro and in vivo models which have become a target for scientists in the clinic and the industry. The commonly prescribed synthetic glucocorticoid (GC) drug, dexamethasone (Dex), can induce GR, PXR and CAR and was used in this study to analyse its effects on the CYP enzymes studied. The hypothesis of this project was that changes in CYP3A4/5 and CYP2C9 gene expression affect drug metabolism and changes in gene expression of these CYP enzymes was under GR, PXR and CAR control, thus affecting the concentration and therapeutic activity of drugs metabolized by these enzymes during chronic use of GCs in conditions such as rheumatoid arthritis and asthma. This study aimed to measure mRNA, protein, ROS and enzymatic activity levels in human HepG2 hepatocytes treated with Dex for 120 h and analyze the results for various time points to produce a mathematical model. Our study has shown that changes in mRNA, protein and enzymatic activity levels of CYP3A4/5 and CYP2C9 in HepG2 cells were induced by Dex at sub-micromolar (0.1 µM) and supra-micromolar (1.5 mM) concentrations. The induction of CYP3A4/5 and CYP2C9 enzymes during 120 h treatment with Dex may be affected by the NRs studied; GR, phosphorylated GR, PXR and CAR protein levels were also shown to be induced by Dex. The efflux transporter, P-gp’s protein levels were also induced by 0.1 µM Dex, highlighting the importance of considering bioavailability of other drugs co-administered with Dex. The results of some of these laboratory experiments have been used to produce mechanistic mathematical models by MATLAB software with reference to previous studies in rats concentrating on the effects of steroids on GR. The models developed were not effective at the lower Dex concentration of 0.1 µM but were better modelled at the higher Dex concentration of 1.5 mM. The basic mechanistic models developed using HepG2 cells in this study can be utilised to design and conduct drug-drug interaction (DDI) analyses of the induction of CYP3A4/5 and CYP2C9 in other human liver cells and starting pre-clinical studies in animals to aid in drug development.

Layman's Abstract

The glucocorticoid receptor (GR) is a member of the nuclear hormone receptors family and has been shown to exert significant effects on the induction of cytochrome P450 (CYP) enzymes responsible for the metabolism of many xenobiotics. CYP3A4/5 and CYP2C9 are important CYP enzymes which metabolise more that 60% of drugs. Induction or inhibition of the enzymatic activity and the levels of these enzymes can have significant effects on drug metabolism. Understanding the role of GR and other nuclear receptors, pregnane X receptor (PXR) and the constitutive androstane receptor (CAR), in the mechanisms effecting CYP3A4/5 and CYP2C9 levels and activity can aid in the development of in vitro and in vivo models which have become a target for scientists in the clinic and the industry. The commonly prescribed synthetic glucocorticoid (GC) drug, dexamethasone (Dex), can induce GR, PXR and CAR and was used in this study to analyse its effects on the CYP enzymes studied. The hypothesis of this project was that changes in CYP3A4/5 and CYP2C9 gene expression affect drug metabolism and changes in gene expression of these CYP enzymes was under GR, PXR and CAR control, thus affecting the concentration and therapeutic activity of drugs metabolized by these enzymes during chronic use of GCs in conditions such as rheumatoid arthritis and asthma. This study aimed to measure mRNA, protein, ROS and enzymatic activity levels in human HepG2 hepatocytes treated with Dex for 120 h and analyze the results for various time points to produce a mathematical model. Our study has shown that changes in mRNA, protein and enzymatic activity levels of CYP3A4/5 and CYP2C9 in HepG2 cells were induced by Dex at sub-micromolar (0.1 µM) and supra-micromolar (1.5 mM) concentrations. The induction of CYP3A4/5 and CYP2C9 enzymes during 120 h treatment with Dex may be affected by the NRs studied; GR, phosphorylated GR, PXR and CAR protein levels were also shown to be induced by Dex. The efflux transporter, P-gp’s protein levels were also induced by 0.1 µM Dex, highlighting the importance of considering bioavailability of other drugs co-administered with Dex. The results of some of these laboratory experiments have been used to produce mechanistic mathematical models by MATLAB software with reference to previous studies in rats concentrating on the effects of steroids on GR. The models developed were not effective at the lower Dex concentration of 0.1 µM but were better modelled at the higher Dex concentration of 1.5 mM. The basic mechanistic models developed using HepG2 cells in this study can be utilised to design and conduct drug-drug interaction (DDI) analyses of the induction of CYP3A4/5 and CYP2C9 in other human liver cells and starting pre-clinical studies in animals to aid in drug development.

Keyword(s)

CYP450; Dexamethasone

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