Accepted Manuscript Title: Determination of domperidone in human plasma using high performance liquid chromatography with fluorescence detection for clinical application Author: Tsuneaki Yoshizato Kimiko Tsutsumi Tsutomu Kotegawa Hiromitsu Imai Shigeyuki Nakano PII: DOI: Reference:
S1570-0232(14)00304-3 http://dx.doi.org/doi:10.1016/j.jchromb.2014.05.004 CHROMB 18927
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
8-2-2014 17-4-2014 4-5-2014
Please cite this article as: T. Yoshizato, K. Tsutsumi, T. Kotegawa, H. Imai, S. Nakano, Determination of domperidone in human plasma using high performance liquid chromatography with fluorescence detection for clinical application, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of domperidone in human plasma using high performance liquid chromatography with fluorescence detection for clinical application
ip t
Tsuneaki Yoshizato1, Kimiko Tsutsumi1, Tsutomu Kotegawa2, Hiromitsu Imai2,
1
cr
Shigeyuki Nakano1
Department of Pharmaceutical Medicine and Communication, Oita University Faculty
us
of Medicine, Oita, Japan Address: Hasama-machi, Yufu-shi, Oita 879-5593, Japan
Department of Clinical Pharmacology and Therapeutics, Oita University Faculty of
an
2
Medicine, Oita, Japan
M
Address: Hasama-machi, Yufu-shi, Oita 879-5593, Japan E-mail: Tsuneaki Yoshizato; [email protected]
d
Tsutomu Kotegawa; [email protected]
te
Hiromitsu Imai; [email protected]
Ac ce p
Shigeyuki Nakano; [email protected]
Corresponding Author: Kimiko Tsutsumi, PhD Department of Pharmaceutical Medicine and Communication, Oita University Faculty of Medicine
Address: Hasama-machi, Yufu-shi, Oita 879-5593, Japan Fax: +81 97 586 6197, Tel: +81 97 586 6196, E-mail address: [email protected]
Page 1 of 30
Abstract
ip t
A simple and reliable method for the determination of domperidone in human plasma has been developed. Plasma samples (1ml) were pre-purified by a solid-phase extraction with
cr
Bond Elut® C18. The separation was achieved with XBridge™ C18 column (150mm ×
us
4.6mm i.d., 5µm) at 40˚C. The mobile phase was a mixture of acetonitrile and 10mM
an
ammonium acetate buffer (36 : 64, v/v), adjusted to pH 9.4 with 20% ammonium solution
M
at a flow rate of 1.0 ml/min. The peak was detected using fluorescence detector at excitation 282 nm and emission 328 nm. Retention times for domperidone and internal
te
d
standard (propranolol) were 8.3 min and 11.2 min, respectively. The method showed a
Ac ce p
good linearity (r > 0.999), precision (relative standard deviations < 10.6%), and extraction recovery (85.7 to 99.7%) over a concentration of 1–100 ng/ml. The lower limit of quantification (LLOQ) was 1.0 ng/ml. This proposed method was successfully applied to a pharmacokinetic interaction study of domperidone in healthy Japanese volunteers.
Key words: domperidone, itraconazole, HPLC, solid phase extraction, fluorescence detection, MDR1
-1Page 2 of 30
1. Introduction
ip t
Domperidone, a selective dopamine D2 receptor antagonist, is an orally active antiemetic and prokinetic agent [1]. Domperidone is rapidly and almost completely absorbed
cr
after oral administration, but its bioavailability is only about 14 % because of an extensive
us
first-pass metabolism in the intestine and liver [2]. The main route of metabolism of
an
domperidone is hydroxylation and oxidative N-dealkylation by CYP3A [3]. In addition,
M
domperidone is a substrate of MDR1 [4]. Domperidone is widely used in patients with nausea and vomiting caused by a wide variety of common disorders and for prevention of
te
d
GI symptoms induced by drugs (eg. cytotoxic drugs) [5]. Therefore, concomitant use of
Ac ce p
other drugs, such as CYP3A and/or MDR1 inhibitors, might affect the pharmacokinetics and pharmacodynamics of domperidone not only by altering systemic pharmacokinetics, but also enhancing tissue distribution of domperidone in the target organ including the brain. Clinical evidence on interactions between domperidone and other drugs is required for a suitable usage of domperidone in clinical practice. Therefore, a simple and reliable analytical method is required for determination of domperidone in human plasma. Several HPLC methods have been reported for the determination of domperidone in plasma [6-12]. Most of these methods required time-consuming laborious liquid–liquid
-2Page 3 of 30
extraction procedures [6, 8, 10, 12]. Kobilińska et al. previously reported an HPLC using
solid-phase
extraction
(SPE),
which
required
a
post-column
ip t
method
photoderivatization for fluorescence detection [9]. Recently, Xu et al. proposed an LC-
cr
MS/MS method with direct injection after simple protein precipitation [7]. LC-MS/MS
us
method could be the best choice in terms of sensitivity and specificity, but this equipment
an
is not always available in many laboratories. Therefore, the objective of the present work
M
was to develop and validate a simple and reliable HPLC method with fluorescence detection method, which is the commonly used analytical technique, for analysis of
Ac ce p
volunteers.
te
d
domperidone. This developed method was applied to a pharmacokinetic study in healthy
2. Materials and methods 2.1 Reagents
Domeridone and propranolol hydrochloride as an internal standard (IS) were
purchased from Sigma-Aldrich Japan Inc. (Tokyo, Japan). Acetonitrile, methanol, 25% aqueous ammonia, ammonium acetate and sodium acetate were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Acetic acid and triethylamine were obtained
-3Page 4 of 30
from Nacalai Tesque Inc. (Kyoto, Japan). All chemicals and solvents were of analytical-
ip t
reagent grade. Purified water was obtained by Milli-Q water purification system (Japan
cr
Millipore Ltd., Tokyo, Japan).
us
2.2 Stock and working standard solutions
an
Stock solutions containing 100 µg/ml domperidone and propranolol (internal standard,
M
IS) were prepared in methanol. Working standard solutions were prepared by dilution of the stock solution with 10mM ammonium acetic acid buffer (pH 9.4) and stored at 4ºC.
te
d
For preparation of calibration standards and quality control samples, appropriate aliquots
Ac ce p
of the stock solutions were added to drug-free plasma. The final concentrations were 1, 2, 5, 10, 50 and 100 ng/ml for the calibration standards and 1, 10, 100 ng/ml for quality control (QC) samples. These plasma samples were stored at –20ºC.
2.3 Chromatography
The HPLC system consisted of a TOSOH DP-8020 pump (Tosoh Co., Tokyo, Japan), an online GASTORR-702 degassor (FLOM Co., Tokyo, Japan), a TOSOH CO-8010 (Tosoh) column oven, a Waters™ 717plus HPLC autosampler (Waters, Milford, MA,
-4Page 5 of 30
USA), and Waters™ 474 scanning fluorescence detector set at 282 nm excitation and 328
ip t
nm emission wavelengths. A Multi station LC-8020 Model II ver. 02.20 (Tosoh) was used for recording peak areas. Chromatographic separations were performed on an XBridge™
cr
C18 (5 µm, 150 mm × 4.6 mm i.d.; Waters) with a guard column of XBridge™ C18 (5
us
µm, 20 mm × 3.0 mm i.d.; Waters). The column temperature was maintained at 40ºC. The
an
mobile phase consisted of acetonitrile and 10mM ammonium acetate buffer (36 : 64, v/v),
te
2.4 Sample preparation
d
M
adjusted to pH 9.4 with 25% ammonium solution. The flow rate was 1.0 ml/min.
Ac ce p
Bond Elut C18® columns (3 ml, 500 mg packing, Varian, CA, USA) were used for sample preparation. The internal standard (propranolol 5ng) in methanol (50 µl) diluted with 950 µl of 0.2M acetate buffer (pH 4.0) was added to plasma sample (1 ml). This mixture was applied to the column. This column was preconditioned with 2 ml of methanol and 2 ml of water in order, and then acidified with 1.5 ml of 0.2M sodium acetate buffer (pH 4.0). After loading the sample, the column was washed with 2 ml of water, 2 ml of 50% methanol/water, and 1ml of methanol. The columns were dried by suction and the analytes were eluted with 1ml of 50mM triethylamine in methanol (TEA
-5Page 6 of 30
solution). The eluate was dried at 40ºC in vacuum. The residue was dissolved in 100 µl of
ip t
HPLC mobile phase. A 50 µl aliquot was injected into the HPLC system.
cr
2.5 Method validation
us
The method validation assays were carried out according to the US Food and Drug
an
Administration (FDA) bioanalytical method validation guidance [13] on selectivity,
te
2.5.1 Selectivity
d
M
linearity, precision, accuracy and recovery.
Ac ce p
Selectivity of the assay method was assessed by evaluating potential interference from endogenous compounds. Six randomly selected blank plasma samples obtained from healthy subjects were analyzed. Each blank sample was tested for interference using the proposed extraction procedure and chromatographic conditions.
2.5.2 Linearity of calibration curves Calibration curves for domperidone were constructed from the working standard solutions over a range 1–100 ng/ml (i.e., 1, 2, 5, 10, 50, 100 ng/ml). The linearity of each
-6Page 7 of 30
calibration curves was determined by the peak area ratio (y) of domperidone to IS versus
ip t
nominal concentration (x) of domperidone. The calibration curve was constructed by weighted (1/y) least squares linear regression. The lower limit of quantification (LLOQ)
cr
was defined as the followings; 1) the lowest concentration on the calibration curve for
M
an
times response of the analyte compared with blank.
us
which an acceptable accuracy within 20% and precision below 20%, and 2) at least 5
2.5.3 Precision and accuracy
te
d
The precision and accuracy of the method were evaluated by assaying the QC samples
Ac ce p
at three different concentrations (1, 10, 100 ng/ml). The intra-day precision and accuracy were evaluated by assaying five spiked samples in a single day. The inter-day precision and accuracy were examined by assaying each QC sample on three different days. Intraassay and inter-assay precision were expressed as the relative standard deviation (RSD, %) of measured concentrations of the QC samples. Accuracy was expressed as the relative error (RE, %) for each QC level by comparing the nominal concentration and the estimated concentration determined after extraction. The precision criterion for the acceptability of data was that the variation for each concentration level should not exceed
-7Page 8 of 30
15% deviation from the nominal values except for the LLOQ, for which it should not
ip t
exceed 20%. Similarly, for accuracy, the mean value should not deviate by ± 15% from the
cr
nominal concentration except for LLOQ, for which the limit was ± 20%.
us
2.5.4 Extraction recovery
an
The extraction recovery of domperidone was determined by comparing the mean peak
M
areas obtained from five replicates of QC samples at three concentrations (1, 10, 100 ng/ml), with the peak areas for pure compounds of the same concentrations in solvent. The
Ac ce p
te
d
recovery of the IS was evaluated at 100ng/mL.
2.6 Application
The proposed method was applied to a pharmacokinetic interaction study between
domperidone and itraconazole, a commonly-used azole antifungal agent, in healthy male volunteers. The study protocol was approved by the Institutional Review Board of Oita University Hospital and a written informed consent was obtained from all participants. The subjects were selected based on an initial prescreening study for the MDR1 C3435T genotype. The genotype of MDR1 was determined by a polymerase chain reaction-
-8Page 9 of 30
restriction fragment length polymorphism (PCR-RFLP) analysis with the method reported
ip t
by Sakaeda et al. [14]. Five homozygous carriers of the wild type (CC), 5 homozygous carriers of the T allele (TT), and 5 heterozygous subjects (CT) were selected to constitute
cr
three groups. The subjects ranged in age from 21 to 32 years and in body weight from 56
us
to 76 kg. They were good in health as judged by their medical history, physical
an
examinations and clinical laboratories tests. They were asked to refrain from consuming
the study and throughout the study.
M
herbal dietary supplements, all fruit products, and fruit juice two weeks before the start of
te
d
This study was a randomized, double-blind, placebo-controlled, crossover design with
Ac ce p
washout periods of at least two weeks. Subjects (n=15; CC=5, TT=5, CT=5) were administered itraconazole 200mg (Itrizole®; Janssen Pharmaceutical KK., Tokyo, Japan) or placebo once a day in the morning for five days. On day 5, after subjects took itraconazole or placebo with 150 ml of water just after a meal at 08:00h, subjects received a single oral dose of domperidone 20 mg (Nauzelin®; Kyowa Hakko Kirin Co. Ltd., Tokyo, Japan) with 150 ml of water at 09:00h. Blood samples were collected into heparinized tubes at time points as; 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after oral domperidone administration. Samples were centrifuged at 3000 rpm for 10 min and
-9Page 10 of 30
plasma was separated and stored at –20ºC until analyzed.
ip t
Pharmacokinetic parameters of domperidone were determined by a twocompartmental model analysis using WinNonlin® Professional (version 5.2, Pharsight Co.,
cr
Mountain View, CA). The elimination half-life (t1/2) was calculated using the equation
us
0.693/β, where β is the terminal slope of time vs. log concentration. The area under the
an
plasma concentration−time curve from zero to infinity (AUC∞) was calculated as AUCt +
M
Ct/β, where AUCt was the area under the plasma concentration−time curve from time zero
Ac ce p
2.7 Statistical analysis
te
d
to the last measurable concentration and Ct was the last measurable plasma concentration.
The results are expressed as mean ± SD. The log-transformed AUC∞, Cmax, and t1/2
were analyzed by the paired t test. Wilcoxon signed rank test was used for tmax. A value of p
S1570-0232(14)00304-3 http://dx.doi.org/doi:10.1016/j.jchromb.2014.05.004 CHROMB 18927
To appear in:
Journal of Chromatography B
Received date: Revised date: Accepted date:
8-2-2014 17-4-2014 4-5-2014
Please cite this article as: T. Yoshizato, K. Tsutsumi, T. Kotegawa, H. Imai, S. Nakano, Determination of domperidone in human plasma using high performance liquid chromatography with fluorescence detection for clinical application, Journal of Chromatography B (2014), http://dx.doi.org/10.1016/j.jchromb.2014.05.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Determination of domperidone in human plasma using high performance liquid chromatography with fluorescence detection for clinical application
ip t
Tsuneaki Yoshizato1, Kimiko Tsutsumi1, Tsutomu Kotegawa2, Hiromitsu Imai2,
1
cr
Shigeyuki Nakano1
Department of Pharmaceutical Medicine and Communication, Oita University Faculty
us
of Medicine, Oita, Japan Address: Hasama-machi, Yufu-shi, Oita 879-5593, Japan
Department of Clinical Pharmacology and Therapeutics, Oita University Faculty of
an
2
Medicine, Oita, Japan
M
Address: Hasama-machi, Yufu-shi, Oita 879-5593, Japan E-mail: Tsuneaki Yoshizato; [email protected]
d
Tsutomu Kotegawa; [email protected]
te
Hiromitsu Imai; [email protected]
Ac ce p
Shigeyuki Nakano; [email protected]
Corresponding Author: Kimiko Tsutsumi, PhD Department of Pharmaceutical Medicine and Communication, Oita University Faculty of Medicine
Address: Hasama-machi, Yufu-shi, Oita 879-5593, Japan Fax: +81 97 586 6197, Tel: +81 97 586 6196, E-mail address: [email protected]
Page 1 of 30
Abstract
ip t
A simple and reliable method for the determination of domperidone in human plasma has been developed. Plasma samples (1ml) were pre-purified by a solid-phase extraction with
cr
Bond Elut® C18. The separation was achieved with XBridge™ C18 column (150mm ×
us
4.6mm i.d., 5µm) at 40˚C. The mobile phase was a mixture of acetonitrile and 10mM
an
ammonium acetate buffer (36 : 64, v/v), adjusted to pH 9.4 with 20% ammonium solution
M
at a flow rate of 1.0 ml/min. The peak was detected using fluorescence detector at excitation 282 nm and emission 328 nm. Retention times for domperidone and internal
te
d
standard (propranolol) were 8.3 min and 11.2 min, respectively. The method showed a
Ac ce p
good linearity (r > 0.999), precision (relative standard deviations < 10.6%), and extraction recovery (85.7 to 99.7%) over a concentration of 1–100 ng/ml. The lower limit of quantification (LLOQ) was 1.0 ng/ml. This proposed method was successfully applied to a pharmacokinetic interaction study of domperidone in healthy Japanese volunteers.
Key words: domperidone, itraconazole, HPLC, solid phase extraction, fluorescence detection, MDR1
-1Page 2 of 30
1. Introduction
ip t
Domperidone, a selective dopamine D2 receptor antagonist, is an orally active antiemetic and prokinetic agent [1]. Domperidone is rapidly and almost completely absorbed
cr
after oral administration, but its bioavailability is only about 14 % because of an extensive
us
first-pass metabolism in the intestine and liver [2]. The main route of metabolism of
an
domperidone is hydroxylation and oxidative N-dealkylation by CYP3A [3]. In addition,
M
domperidone is a substrate of MDR1 [4]. Domperidone is widely used in patients with nausea and vomiting caused by a wide variety of common disorders and for prevention of
te
d
GI symptoms induced by drugs (eg. cytotoxic drugs) [5]. Therefore, concomitant use of
Ac ce p
other drugs, such as CYP3A and/or MDR1 inhibitors, might affect the pharmacokinetics and pharmacodynamics of domperidone not only by altering systemic pharmacokinetics, but also enhancing tissue distribution of domperidone in the target organ including the brain. Clinical evidence on interactions between domperidone and other drugs is required for a suitable usage of domperidone in clinical practice. Therefore, a simple and reliable analytical method is required for determination of domperidone in human plasma. Several HPLC methods have been reported for the determination of domperidone in plasma [6-12]. Most of these methods required time-consuming laborious liquid–liquid
-2Page 3 of 30
extraction procedures [6, 8, 10, 12]. Kobilińska et al. previously reported an HPLC using
solid-phase
extraction
(SPE),
which
required
a
post-column
ip t
method
photoderivatization for fluorescence detection [9]. Recently, Xu et al. proposed an LC-
cr
MS/MS method with direct injection after simple protein precipitation [7]. LC-MS/MS
us
method could be the best choice in terms of sensitivity and specificity, but this equipment
an
is not always available in many laboratories. Therefore, the objective of the present work
M
was to develop and validate a simple and reliable HPLC method with fluorescence detection method, which is the commonly used analytical technique, for analysis of
Ac ce p
volunteers.
te
d
domperidone. This developed method was applied to a pharmacokinetic study in healthy
2. Materials and methods 2.1 Reagents
Domeridone and propranolol hydrochloride as an internal standard (IS) were
purchased from Sigma-Aldrich Japan Inc. (Tokyo, Japan). Acetonitrile, methanol, 25% aqueous ammonia, ammonium acetate and sodium acetate were obtained from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Acetic acid and triethylamine were obtained
-3Page 4 of 30
from Nacalai Tesque Inc. (Kyoto, Japan). All chemicals and solvents were of analytical-
ip t
reagent grade. Purified water was obtained by Milli-Q water purification system (Japan
cr
Millipore Ltd., Tokyo, Japan).
us
2.2 Stock and working standard solutions
an
Stock solutions containing 100 µg/ml domperidone and propranolol (internal standard,
M
IS) were prepared in methanol. Working standard solutions were prepared by dilution of the stock solution with 10mM ammonium acetic acid buffer (pH 9.4) and stored at 4ºC.
te
d
For preparation of calibration standards and quality control samples, appropriate aliquots
Ac ce p
of the stock solutions were added to drug-free plasma. The final concentrations were 1, 2, 5, 10, 50 and 100 ng/ml for the calibration standards and 1, 10, 100 ng/ml for quality control (QC) samples. These plasma samples were stored at –20ºC.
2.3 Chromatography
The HPLC system consisted of a TOSOH DP-8020 pump (Tosoh Co., Tokyo, Japan), an online GASTORR-702 degassor (FLOM Co., Tokyo, Japan), a TOSOH CO-8010 (Tosoh) column oven, a Waters™ 717plus HPLC autosampler (Waters, Milford, MA,
-4Page 5 of 30
USA), and Waters™ 474 scanning fluorescence detector set at 282 nm excitation and 328
ip t
nm emission wavelengths. A Multi station LC-8020 Model II ver. 02.20 (Tosoh) was used for recording peak areas. Chromatographic separations were performed on an XBridge™
cr
C18 (5 µm, 150 mm × 4.6 mm i.d.; Waters) with a guard column of XBridge™ C18 (5
us
µm, 20 mm × 3.0 mm i.d.; Waters). The column temperature was maintained at 40ºC. The
an
mobile phase consisted of acetonitrile and 10mM ammonium acetate buffer (36 : 64, v/v),
te
2.4 Sample preparation
d
M
adjusted to pH 9.4 with 25% ammonium solution. The flow rate was 1.0 ml/min.
Ac ce p
Bond Elut C18® columns (3 ml, 500 mg packing, Varian, CA, USA) were used for sample preparation. The internal standard (propranolol 5ng) in methanol (50 µl) diluted with 950 µl of 0.2M acetate buffer (pH 4.0) was added to plasma sample (1 ml). This mixture was applied to the column. This column was preconditioned with 2 ml of methanol and 2 ml of water in order, and then acidified with 1.5 ml of 0.2M sodium acetate buffer (pH 4.0). After loading the sample, the column was washed with 2 ml of water, 2 ml of 50% methanol/water, and 1ml of methanol. The columns were dried by suction and the analytes were eluted with 1ml of 50mM triethylamine in methanol (TEA
-5Page 6 of 30
solution). The eluate was dried at 40ºC in vacuum. The residue was dissolved in 100 µl of
ip t
HPLC mobile phase. A 50 µl aliquot was injected into the HPLC system.
cr
2.5 Method validation
us
The method validation assays were carried out according to the US Food and Drug
an
Administration (FDA) bioanalytical method validation guidance [13] on selectivity,
te
2.5.1 Selectivity
d
M
linearity, precision, accuracy and recovery.
Ac ce p
Selectivity of the assay method was assessed by evaluating potential interference from endogenous compounds. Six randomly selected blank plasma samples obtained from healthy subjects were analyzed. Each blank sample was tested for interference using the proposed extraction procedure and chromatographic conditions.
2.5.2 Linearity of calibration curves Calibration curves for domperidone were constructed from the working standard solutions over a range 1–100 ng/ml (i.e., 1, 2, 5, 10, 50, 100 ng/ml). The linearity of each
-6Page 7 of 30
calibration curves was determined by the peak area ratio (y) of domperidone to IS versus
ip t
nominal concentration (x) of domperidone. The calibration curve was constructed by weighted (1/y) least squares linear regression. The lower limit of quantification (LLOQ)
cr
was defined as the followings; 1) the lowest concentration on the calibration curve for
M
an
times response of the analyte compared with blank.
us
which an acceptable accuracy within 20% and precision below 20%, and 2) at least 5
2.5.3 Precision and accuracy
te
d
The precision and accuracy of the method were evaluated by assaying the QC samples
Ac ce p
at three different concentrations (1, 10, 100 ng/ml). The intra-day precision and accuracy were evaluated by assaying five spiked samples in a single day. The inter-day precision and accuracy were examined by assaying each QC sample on three different days. Intraassay and inter-assay precision were expressed as the relative standard deviation (RSD, %) of measured concentrations of the QC samples. Accuracy was expressed as the relative error (RE, %) for each QC level by comparing the nominal concentration and the estimated concentration determined after extraction. The precision criterion for the acceptability of data was that the variation for each concentration level should not exceed
-7Page 8 of 30
15% deviation from the nominal values except for the LLOQ, for which it should not
ip t
exceed 20%. Similarly, for accuracy, the mean value should not deviate by ± 15% from the
cr
nominal concentration except for LLOQ, for which the limit was ± 20%.
us
2.5.4 Extraction recovery
an
The extraction recovery of domperidone was determined by comparing the mean peak
M
areas obtained from five replicates of QC samples at three concentrations (1, 10, 100 ng/ml), with the peak areas for pure compounds of the same concentrations in solvent. The
Ac ce p
te
d
recovery of the IS was evaluated at 100ng/mL.
2.6 Application
The proposed method was applied to a pharmacokinetic interaction study between
domperidone and itraconazole, a commonly-used azole antifungal agent, in healthy male volunteers. The study protocol was approved by the Institutional Review Board of Oita University Hospital and a written informed consent was obtained from all participants. The subjects were selected based on an initial prescreening study for the MDR1 C3435T genotype. The genotype of MDR1 was determined by a polymerase chain reaction-
-8Page 9 of 30
restriction fragment length polymorphism (PCR-RFLP) analysis with the method reported
ip t
by Sakaeda et al. [14]. Five homozygous carriers of the wild type (CC), 5 homozygous carriers of the T allele (TT), and 5 heterozygous subjects (CT) were selected to constitute
cr
three groups. The subjects ranged in age from 21 to 32 years and in body weight from 56
us
to 76 kg. They were good in health as judged by their medical history, physical
an
examinations and clinical laboratories tests. They were asked to refrain from consuming
the study and throughout the study.
M
herbal dietary supplements, all fruit products, and fruit juice two weeks before the start of
te
d
This study was a randomized, double-blind, placebo-controlled, crossover design with
Ac ce p
washout periods of at least two weeks. Subjects (n=15; CC=5, TT=5, CT=5) were administered itraconazole 200mg (Itrizole®; Janssen Pharmaceutical KK., Tokyo, Japan) or placebo once a day in the morning for five days. On day 5, after subjects took itraconazole or placebo with 150 ml of water just after a meal at 08:00h, subjects received a single oral dose of domperidone 20 mg (Nauzelin®; Kyowa Hakko Kirin Co. Ltd., Tokyo, Japan) with 150 ml of water at 09:00h. Blood samples were collected into heparinized tubes at time points as; 0, 0.25, 0.5, 0.75, 1, 1.5, 2, 3, 4, 6, 8, and 12 h after oral domperidone administration. Samples were centrifuged at 3000 rpm for 10 min and
-9Page 10 of 30
plasma was separated and stored at –20ºC until analyzed.
ip t
Pharmacokinetic parameters of domperidone were determined by a twocompartmental model analysis using WinNonlin® Professional (version 5.2, Pharsight Co.,
cr
Mountain View, CA). The elimination half-life (t1/2) was calculated using the equation
us
0.693/β, where β is the terminal slope of time vs. log concentration. The area under the
an
plasma concentration−time curve from zero to infinity (AUC∞) was calculated as AUCt +
M
Ct/β, where AUCt was the area under the plasma concentration−time curve from time zero
Ac ce p
2.7 Statistical analysis
te
d
to the last measurable concentration and Ct was the last measurable plasma concentration.
The results are expressed as mean ± SD. The log-transformed AUC∞, Cmax, and t1/2
were analyzed by the paired t test. Wilcoxon signed rank test was used for tmax. A value of p
