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.