Abstract
Background:Voriconazoleis(VRC) often used in complex therapeutic environments for the treatment and prevention of invasive fungal infections. The steady-state valley concentration (Cminss) of VRC not only varies between individuals, but also within individuals, which is difficult to fully explain by pharmacogenomic theory. It is necessary to propose a new perspective to explain the variation of voriconazole steady-state valley concentration.
Objectives: Based on the regulation of ADME gene expression by DNA methylation, this study aimed to explore the effect ofCYP2C19 DNA methylation level on the VRC Cminss.
Methods : In this study, 116 concentration points were divided into low concentration group (Cminss<1.0mg/L), standard concentration group (Cminss =1.0-5.5mg/L) and high concentration group (Cminss>5.5mg/L) according to Voriconazole Cmin standard range of 1.0-5.5 mg/L. The effect of CYP2C19 DNA methylation was highlighted by predisposition score matching to exclude other confounding factors.
Results: The CYP2C19 CpG25 methylation level was different between low concentration group and standard concentration group (p=0.047). There was no difference in the CYP2C19 DNA methylation between the high concentration group and the standard concentration group, but there were significant differences in CRP (p<0.001), Alb (p=0.007) and T-BIL (p=0.024) between the high concentration group and the standard concentration group.
Conclusions: The VRC Cminss in the low concentration group may be related to the methylation degree of CYP2C19 CpG25 site, while the VRC Cminss in the high concentration group may be unrelated to the methylation degree of CYP2C19 but related to the levels of CRP, Alb and T-BIL.
Key words: Voriconazole, CYP2C19 , DNA methylation, Cminss, Chinese population, CRP, Alb,T-BIL
Introduction
Invasive fungal infections (IFI) mostly occur in people with low immunity and have high morbidity and mortality1. Voriconazole (VRC) is a second-generation triazole broad-spectrum antifungal drug, which has been recommended as a first-line drug for the treatment and prevention of IFI by many guidelines2,3. However, VRC is often in a complex therapeutic environment4. VRC steady-state valley concentration(Cminss) not only varies between individuals, but also within individuals, which is difficult to be fully explained by pharmacogenomics theories. Previous studies have found that in the case of CYP2C19 and CYP3A4 genotyping, high levels of inflammation may slow down voriconazole metabolism in adult patients, resulting in higher Cminss 5,6. It is necessary to explore the new factors affecting VRC in order to promote the personalized medicine of VRC.
Epigenetic regulation is a reversible, heritable change in gene function that ultimately leads to phenotypic change without changing the DNA sequence. More and more studies have found that epigenetic factor is also important reasons for individual differences in drugs7. DNA methylation is a key epigenetic mechanism8. The DNA methylation level of specific pharmacokinetic gene will affect the its mRNA and protein expression, and affect the disposal and effect of corresponding drugs9. CYP2C19 DNA methylation is associated with its mRNA expression10,11. However, whether this epigenetic regulation is responsible for individual differences in VRC Cminss has not been studied. The DNA methylation level of pharmacokinetic genes may also be influenced by some individual factors (e.g. parental exposure, environmental pollutants exposure, obesity and diet, drug, etc.)12. This study investigated the effect of CYP2C19 DNA methylation on the VRC Cminss by excluding the interference of other factors through propensity score matching (PSM), which is expected to provide a new perspective for the personalized administration of voriconazole.
Materials and methods
Study design
This study was approved by the Ethics Committee of Peking University People’s Hospital (No. 2019PHB064-01). Patients receiving VRC for the prevention or treatment of IFD aged at least 18 years who had at least one VRC trough concentration determined at our institute between June 2019 and May 2022 were included. Patients were excluded if they were pregnant, were using potentially interacting drugs described in the drug package, had prior severe liver dysfunction, had a CYP2C19metabolic phenotype of ultrafast and slow metabolism, and were co-using DNA methyltransferase inhibitors (azacytidine, decitabine). The trough concentration of VRC (Cminss) is defined as 2 doses after the first loading dose was administered, and 5 doses if no loading dose was given. A loading dose was considered as patients treated with two 400 mg of VRC on the first day and followed 200 mg every 12 h daily. Blood samples were collected just prior to the subsequent dosage when the plasma concentration would be at a steady state.
Voriconazole and voriconazole N-oxide assay
Blood samples from patients were centrifuged at 4000 rpm for 10 minutes at 4°C, and separated plasma was stored at -20°C until analysis. The methodology was established with blank plasma of patients and rats. Plasma Voriconazole and voriconazole N -oxide concentrations were measured by HPLC-MS/MS. The linear range of VRC and VNO was 0.01-10.00 μg/ml. The calibration curve showed good linearity, with R2 > 0.99. The intra- and inter- day accuracy values, expressed as percent CV, were all within ± 15 %, and precision values (as percent CV) were all less than 10 % in each calibration curve. Accuracy, method recovery and stability were all met the requirements.
Chromatographic conditions: Column Hypersil Gold C18 (1.9μm, 2.1×50 mm, Thermo Scientific, USA); Mobile phase: deionized water (0.1% formic acid, 10mM ammonium formate, solvent A), Acetonitrile (0.1% formic acid, solvent B); Elution conditions: 0.0-0.5 min, 90% A; 0.5-1.0 min, 90%-5% A; 1.0-3.5 min, 5% A; 3.5-3.6 min, 5%-90% A; 3.6-6.0 min, 90% A; Flow rate: 0.5mL•min-1, Column temperature: 50℃, Automatic sampler temperature: 4℃, Sample size: 2μL.
Mass spectrometry conditions: Mass Ionization was performed using Electrospray Ionization (ESI) and Multiple Reaction Monitoring (MRM) modes. Ion transitions (in m/z) of VRC, VNO, and Voriconazole d3 (internal standard) were 350.2 to 224.2, 366.2 to 224.2 and 353.2 to 224.2, respectively. The declustering voltage and the collision energy were 75V and 25V respectively.