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Introduction
S-Mephenytoin-4'-hydroxylase (CYP2C19) is a genetically
determined enzyme and its phenotypes can be classified as poor metabolizer
(PM) and extensive metabolizer (EM)[1,2]. When CYP2C19
is the main metabolism enzyme of a drug, the pharmacokinetics of
the drug are different between the PM and Em phenotypes, such as
proton pump inhibitors (PPI). Recently, rabeprazole (RPZ) has been
reported to be metabolized mainly via a non-enzymatic pathway, with
only minor CYP2C19 and CYP3A4 involvement[3-6]. The pharmacokinetics
of RPZ are assumed to be less influenced by the CYP2C19 phenotype.
The incidence of PM for CYP2C19 in the Chinese population is very
high (17.4%)[7]. However, it is not clear whether the
pharmacokinetics and pharmacodynamics of RPZ depend on the CYP2C19
genotype status in Chinese people. Thus, studies examining the effects
of CYP2C19 genetic polymorphism on the metabolism of RPZ
in Chinese people are important. In the present study we observed
the metabolic disposition characteristics and pharmacodynamics of
RPZ after a single dose and after 8 days of repeated doses
with reference to different CYP2C19 genotype groups to provide valuable
data that should be considered when selecting PPI for patients with
acid-related diseases with reference to the CYP2C19 genotype status.
Materials and methods
Subjects and CYP2C19 genotypes Helicobacter pylori
(H pylori) infection was screened using a serological test
(Dot-immunogold kit, Lanbo Bio-Tech Institute, China) and a 13C-urea
breath test. DNA was extracted from each indivi-dual's leucocytes
using a commercially available kit (Promega, Madison, WI, USA).
Genotyping procedures for identifying the CYP2C19 wild-type (CYP2C19*1)
and the two mutated alleles, CYP2C19*2 and CYP2C19*3, were carried
out using the polymerase chain reaction and restriction fragment
length polymorphism method[8].
A total of 20 H pylori-negative healthy volunteers
participated in this study. Seven subjects were classified as homozygous
extensive metabolizers (homEM). Six were heterozygous for exon 5
mutation of CYP2C19 (*1/*2) or heterozygous for the exon 4 mutation
(*1/*3) and were classified as heterozygous extensive metabolizers
(hetEM). The remaining seven subjects were homozygous for the exon
5 mutation (*2/*2) and were classified as the PM group (Table 1).
None of the subjects consumed alcohol or smoking. None of the subjects
had taken any drugs for at least 4 weeks before or during the study.
The Ethics Committee of Anhui Medical University approved the protocol
in advance. Written informed consent was obtained from each subject
before participation in the study.
Study protocol All healthy volunteers were orally treated
with 20 mg RPZ (Pariet, Eisai Company, Tokyo, Japan) for an
8-day period. The medication was taken once daily at 8:00. The 24-h
intragastric pH monitoring and the measurement of serum levels of
RPZ were carried out on day 1 and day 8. Two standard meals (12:00,
18:00), prepared at the hospital, were provided for each subject.
Intragastric pH measurement After overnight fasting, a glass
electrode was inserted transnasally and placed approximately 5 cm
below the cardia. The electrode was calibrated with standard buffers
(pH 1.07 and 7.01) before recording the pH with a Digitrapper pH
(Medtronic, Watford,UK). Intragastric pH recordings started after
the oral dose of RPZ at 8:00 on d 1 and d 8.
Sample collection and concentration assays of rabepra-zole
Blood samples were collected before and 0.5, 1, 1.5, 2, 3, 5, 7,
10, 12, and 24 h after RPZ administration on day 1 and day 8.
After collection, the blood samples were immediately centrifuged
at 4000 r/min for 10 min and 100 µL of 1% diethylamine
solution was added to the 1 mL sample of RPZ plasma. All samples
were stored at -80°C until assayed. Plasma levels of RPZ were
measured using high performance liquid chromatography[9,10]
. The lower detection limit for RPZ was 0.01 mg/L. A
good linearity is obtained from 0.01-0.75 mg/L of
RPZ with r=0.999. The standard curve of RPZ in serum is Y=124950X - 806.05
(n=5). The recoveries of three concentra-tions, 0.05, 0.1,
0.5 mg/L are 75.2%, 84.2%, and
91.0%, respectively. The RSD of intra-day variation of RPZ are 5.1%,
9.2%, and 6.8% and the RSD of inter-day variation are 8.2%, 3.5%,
and 4.2% for the three concentrations, respectively.
Statistical analysis Intragastric pH characters were described
by the median, mean, pH>4 total time and the pH>4 time proportion
of 24 h from the raw pH values. The values for the areas under the
serum concentration-time curves (AUC) from 0 to 24 h for RPZ
were calculated using the 3P87 software. All P values are
two-sided, and P<0.05 indicated statistical significance.
Data were expressed as mean±SD. Statistically significant differences
in the mean AUC values for RPZ and intragastric pH values between
the three different CYP2C19 genotype groups were compared using
a one-way analysis of variance (ANOVA) combined with the least significant
method (LSD). Paired t-tests were used to determine whether
there were differences in the AUC values and intragastric pH values
for RPZ between single and repeated doses. Statistical calculations
were carried out using SPSS 11.0 software (SPSS Inc, Chicago, USA).
Results
Role of the CYP2C19 genotype on the acid-inhibitory efficacy
of rabeprazole Raw data on the mean intragastric pH-time curves
after single and repeated doses of RPZ in the three different genotype
groups are shown in Figure 1. The characteristic values of the 24-h
intragastric pH after single and repeated doses of RPZ in the three
different genotype groups are summarized in Table 2. The median
intragastric pH value of the PM group was the highest, followed
by the hetEM group, and the homEM group had the lowest value.
No significant differences in intragastric pH values were observed
between the three groups after a single dose or after repeated doses
for 8 days of RPZ. In addition, no significant increments in
intragastric pH values from single to repeated doses were observed
in the three different genotype groups.
Role of the CYP2C19 genotype on the kinetic disposition of rabeprazole
The pharmacokinetic parameters are shown in Table 3. The mean
AUC values for RPZ after a single dose differed among the three
different genotype groups, with a relative ratio of 1.0, 1.3, and
1.8 in the homEM, hetEM and PM groups, respectively. The mean AUC
values for RPZ after repeated doses also differed among the three
groups, with a relative ratio of 1.0, 1.1, and 1.7 in the homEM,
hetEM and PM groups, respectively. The mean AUC values for RPZ after
single and repeated doses were significantly different between the
homEM and PM groups, but not between the homEM and hetEM or between
the hetEM and PM groups. No significant increase in the mean AUC
values for RPZ from single to repeated doses was observed in any
of the three different genotype groups. The Cmax
values were significantly different between the homEM and PM groups,
and the hetEM and PM groups after single and repeated doses of RPZ.
Whereas Tmax and T1/2 did not
differ significantly between the three groups on day 1 and
day 8.
Discussion
Proton pump inhibitors, such as omeprazole, lansopra-zole, pantoprazole,
and rabeprazole, have been used widely in the treatment of acid-related
diseases. Recent research has paid more attention to the inhibitory
effects of PPI in relation to the genetic polymorphism of CYP2C19,
a major enzyme for the metabolism of PPI in the liver[11].
These studies have shown that CYP2C19 genetic polymorphism has a
significant influence on acid-inhibitory efficacy and the metabolism
of omeprazole in healthy Chinese Han subjects. However, to date
no studies examining the relationship between the CYP2C19 genotype
and the metabolism of RPZ has been carried out in the Chinese Han
population. in vitro human liver microsomal and in vivo
human pharmacology studies have shown that RPZ is metabolized mainly
via a non-enzymatic reduction to RPZ thioether, and that CYP2C19
and CYP3A4 are partially involved in the metabolism of
RPZ[3-6]. The existence of CYP3A4-related PM has not
been reported in any Chinese population. Therefore, we did take
into account CYP2C19-related genotyping factors in our study.
Rabeprazole has a rapid and powerful onset of pharmacological action[12].
Our study showed that the AUC for RPZ after a single dose exceeded
80% of the AUC after repeated doses and there were no significant
increments from single to repeated doses, which was consistent with
Yasuda et al[9]. In addition, we found that
no significant increment in intragastric pH values was observed
from single to repeated doses. These results suggest that the metabolism
of RPZ after a single dose could attain maximum acid-inhibitory
efficacy. And this appears to be the reason for pH values remaining
elevated for more than 50% of the time, even with very modest exposures,
and when the pharmacokinetic results on day 1 are consistent
with the results on day 8.
Adachi and other researchers[3-6] have found that the
acid-inhibitory efficacy and metabolism of RPZ are not dependent
on CYP2C19 genotype status. However, Horai et al and
Inaba et al[13,14] and Ieiri et al[15]
reported that CYP2C19 genotypic differences affected the metabolism
and kinetics process of RPZ, and influenced gastric pH values and
gastrin level in plasma. In the present study, we found that the
AUC for RPZ differed markedly only between homEM and PM, and the
intragastric pH, the best and most direct pharmacological index
when using PPI, was not significantly different among the different
genotypes after a single or repeated doses of RPZ. As for the discrepancy
between the kinetics and dynamics of RPZ, first we may hypothesize
that the acid-inhibitory effect of RPZ is powerful and rapid, and
that the serum levels of 20 mg RPZ are sufficient for acid-inhibitory
efficacy in Chinese subjects, even in homEM subjects. Second,
no direct and simple relationship between the serum concentration-time
profile of the drug and the pharmacodynamic response has been reported
because of the irreversible blockade of the therapeutic target by
PPI. However, Hussein et al[16] have shown
a clear relationship using the maximum effect (Emax)
model. According to the model, our study suggests a lower half-maximal
effective AUC value (EAUC50) for RPZ than 2 mg·L-1·h.
As shown in Table 3, the mean AUC values of RPZ corresponding
to the maximum acid-inhibitory effect of RPZ in this study may be
greater than this threshold. Therefore, there were no significant
differences for intragastric pH among the three geno-types.
In conclusion, our study focused on investigating the pharmacodynamic
and pharmacokinetic effect of RPZ with reference to different CYP2C19
genotypes. The acid-inhibitory effects of RPZ were independent on
their pharmacokinetic characteristics as well as an individual's
CYP2C19 genotype status. Therefore, RPZ may be a more effective
PPI for treating acid-related disease in relation to CYP2C19 genotype
status.
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