Association between Sex Differences and the Pharmacokinetics of Repaglinide among a Malaysian Population

This study was conducted to evaluate the effect of sex differences on the pharmacokinetics of repaglinide in healthy subjects. One hundred twenty one healthy volunteers (61 male and 60 female; aged 18 50 years) were included in the study. Subjects were administered a single 4-mg repaglinide oral dose. Blood samples were taken at 0, 30, 60, 120, 180 and 240 min. Serum repaglinide levels were determined by a high-performance liquid chromatography (HPLC) method. Subjects were also genotyped by polymerase chain reactions-restriction fragment length polymorphisms (PCR-RFLP) for CYP3A4*4, *5 and *18 alleles and by an allele-specific multiplex PCR for CYP2C8*2, *3, *4 and *5 alleles. The pharmacokinetics of repaglinide were comparable between male and female subjects. The mean clearance (CL) of repaglinide was 16.0% lower (p = 0.03), the mean area under the serum concentration-time curve (AUC) was 12.8% higher (p = 0.04) and the peak serum concentration (Cmax) was 13.2% higher (p = 0.03) in females compared to male subjects. The mean rate of elimination (kel) and mean CL of repaglinide were 47.67% (p = 0.03) higher and 29.25% (p = 0.03) higher, respectively, in male subjects having CYP2C8*5 allele compared to female subjects. We also found that the mean half-life (t1/2) of repaglinide was 42.43% higher (p = 0.03), and the mean AUC was 35.83% higher (p = 0.03) in female subjects when compared to the male subjects having CYP2C8*5 allele. Sex differences significantly influence the pharmacokinetics of repaglinide.


Introduction
Repaglinide was the first meglitinide analogue to become available.It is used in type 2 diabetic patients to normalise postprandial hyperglycaemia [1,2].Like the sulfonylureas, repaglinide reduced blood glucose by stimulating insulin release from pancreatic -cells [3].It has a fast onset and short duration of action.Repaglinide is rapidly absorbed from the gastrointestinal tract after oral administration and eliminated in the bile.Only a very small fraction of the administered dose is excreted through the urine.It differs from other antidiabetic agents in its structure, binding profile, duration of action and mode of excretion [3].Cytochrome P4502C8 (CYP2C8) and cytochrome P4503A4 (CYP3A4) are the principal enzymes that participate in its oxidative biotransformation [4].
CYP2C8 is the major human hepatic P450, constitut-ing about 12% of total microsomal CYP content in the liver [5] in which it conducts oxidative metabolism of at least 5% of drugs cleared by phase I metabolism.Drugs for which CYP2C8 contributes significantly to their biotransformation include the anticancer drug paclitaxel [6], the antidiabetic drugs rosiglitazone and troglitazone [7] and repaglinide [4], the antimalarial amodiaquine [6] and the hydroxymethylglutaryl co-enzyme A reductase inhibitors such as cerivastatin and fluvastatin [8].CYP3A4 is involved in the metabolism of more than 60% of all drugs used in humans [5].It is found in human livers, gastrointestinal tracts, kidneys, lungs, brains, endotheliums, placentas, lymphocytes and intestines [9].It plays important roles in the metabolism of drugs used in humans including antidiabetics, antiarrhythmics, antihistamines, synthetic oestrogens, cancer chemotherapeutic drugs, human immunodeficiency virus protease in-hibitors, calcium channel antagonists, immunosuppressants and cholesterol-lowering drugs [10].
Numerous studies have examined the influence of sex differences on drug metabolism.Physiological factors such as differences in body weight and composition, metabolizing enzymes or hormone concentrations may differentially affect the pharmacokinetics and pharmacodynamics of many drugs between women and men [11].Ignoring these sex differences during drug development may lead to side-effects or toxicity as well as inadequate response during drug treatment due to these differences in drug metabolism [11].
Several investigators also have attempted to determine potential sex differences in the metabolic activity of cytochrome P450 (CYP) enzymes including CYP3A and CYP2C.Some studies suggest higher CYP enzymes activities in women than men [12], there are also reports of no sex differences [13] or even lower in activities in women [14].Thus, these findings have been largely inconsistent or inconclusive.
This study was designed to investigate the influence of sex differences on the pharmacokinetics of repaglinide.The secondary objective was to investigate whether sex differences exist in CYP3A4 and CYP2C8 activities by use of population pharmacokinetic modelling methods.

Materials and Methods
The study was conducted following approval by the Research and Ethics Committee, School of Medical Sciences, Universiti Sains Malaysia.All the subjects gave their written informed consent prior to study enrolment.
One hundred twenty one healthy volunteers were recruited, including 61 men and 60 women.All subjects aged 18 to 50 years with a normal body mass index (BMI).These volunteers were students and staff of Universiti Sains Malaysia, Health Campus, Kelantan, Malaysia.They were ascertained to be healthy by a medical history, physical examination and routine laboratory tests.
Subjects were asked to fast overnight before the study.On the day of repaglinide administration, 5 ml of blood were collected in plain vacutainer tubes to determine repaglinide's concentration at 0 min (baseline).Subjects then administered a single 4-mg repaglinide [Novo Nordisk, Denmark] oral dose with 100 ml of water.On each study day subjects were allowed to have a light breakfast, precisely 10 min after repaglinide administration.The breakfast was eaten within 10 min.The breakfast consisted of one sandwich (two pieces of bread with eggs, tomato and margarine) and contained approximately 1550 kJ energy, 70 g carbohydrates, 8 g protein and 6 g fat.Blood samples (5 mL into vacutainer for pharmacokinetic study) were drawn from a cannulated forearm vein at five designated times: 30, 60, 120, 180 and 240 min.Serum were separated within 30 min after blood sampling.
During the day of repaglinide administration, the subjects were under direct medical supervision and blood glucose levels were monitored throughout the day.Additional glucose solution for intravenous use and glucagons for intramuscular use were available in case of severe hypoglycaemia but they were not needed.
The reversed-phase high performance liquid chromatographic (HPLC) methods described by Ruzilawati et al. [15] were used to measure serum repaglinide concentrations.In brief, samples were processed after addition of the internal standard (indomethacin, Sigma-Aldrich, St. Louis, MO, USA).A calibration curve was analysed in the concentration range of 20 -200 ng/ml.Samples were extracted with ethyl acetate and injected into the chromatographic system composed of an automatic sampler (Waters, Mildford, MA, USA).Reversed-phase chromatographic separation was achieved on Purospher  STAR C-18 analytical column (4.8 mm × 150 mm; 5 m particle size).The mobile phase consisted of 60:40 v/v acetonitrile -ammonium formate (pH 2.7; 0.01 M) and was run at 1 ml/min.The retention times of indomethacin and repaglinide were approximately 5.3 and 6.2 min, respectively.
For genetic analysis, a 5 mL ethylenediaminetetraacetic acid (EDTA) blood sample was drawn from each subject and stored at -20˚C until deoxyribonucleic acid (DNA) extraction.DNA was extracted with standard methods (QIAamp DNA Blood Mini Kit, Qiagen, Hilden, Germany).All subjects were genotyped for CYP3A4*4, *5 and *18 alleles by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method as described by Ruzilawati et al. [16].Subjects were also genotyped for CYP2C8*2, *3 and *4 alleles.The method of allele-specific PCR based on a previously published protocol described by Muthiah et al. [17] was used.
The population pharmacokinetic parameters of repaglinide was characterized by the Nonparametric Adaptive Grid (NPAG) programme, a module of USC*PACK ® (version 12.0) [18].
Mann-Whitney tests were used to compare the effect of sex on repaglinide pharmacokinetic parameters using the SPSS package (ver.12, SPSS, Chicago, IL).Data were presented as mean ± SD.A p value of < 0.05 was considered to be statistically significant.

Results
One hundred and twenty one subjects completed the study.Demographic data for the 121 subjects are displayed in Table 1.The comparison between healthy male and female subjects' repaglinide pharmacokinetic is shown in Tables 2-4 summarize CYP3A4 and CYP2C8 genetic polymorphisms by sex in our subjects, respectively.The comparison between the pharmacokinetic parameters of repaglinide after a single oral dose of 4 mg in male and female subjects having CYP2C8*5 allele is shown in Table 5.

Discussion
Gender may be an important variable in the processes of absorption, distribution, metabolism, and excretion.There are several factors that may contribute to sex-related differences in pharmacokinetics.These factors are gastric acid secretion, gastrointestinal blood flow, proportions of muscular and adipose tissue, amount of drug binding    proteins, sex-specific cytochrome P450 isozymes, physiologic and hormonal changes during the menstrual cycle as well as renal blood flow [19].However, there has been little published work evaluating potential sex differences in pharmacokinetics of repaglinide.
The present study indicates that sex difference is significantly influences the pharmacokinetics of repaglinide.We found that the mean clearance (CL) of repaglinide was 16.0% lower (p = 0.03), the mean area under the serum concentration-time curve (AUC) was 12.8% higher (p = 0.04) and the peak serum concentration (C max ) was 13.2% higher (p = 0.03) in female compared to male subjects.
Haidar et al. [20] reported that, on average, female patients had larger AUC for repaglinide when compared to male patients.A higher bioavailability (higher AUC values) has also been reported for females than males by Harris et al. [21].Regarding pharmacokinetic parameters of drugs administered by the oral route, gastrointestinal motility has also been shown to be affected by sex hormones, which is slower in females than in males [22].
Repaglinide is metabolised by oxidation process.Clear-ances of drugs that are metabolised by conjugation or oxidation tend to be slower in women [23,24].In general, clearance of some drugs also depends on the rate of blood flow into the eliminating organ.Women have a smaller liver and a lower liver blood flow.According to Meibohm et al. [19], women have an approximately 10% lower glomerular filtration rate than men when normalized for body surface area.This might explain the observed 16% lower repaglinide clearance in female subjects relative to male subjects.No significant differences were seen in other pharmacokinetic parameters such as rate of elimination (kel), rate of absorbtion (ka), half-life (t 1/2 ), volume of distribution (Vd) and time to reach C max (t max ) between male and female subjects.
Drug concentrations are dependent on the volume of distribution (Vd).The Vd of a drug can affect the amount of drug that give their effects at the site of action.Body composition between women and men are different.This factor might affect the Vd of certain drugs.Women have a higher percentage of adipose tissue than do men.Therefore, as suggested by Greenblatt et al. [25], there should be a much larger Vd in women for lipophilic drugs.This can result in a prolonged half-life of those drugs.However, this did not occur in repaglinide's pharmacokinetics when we compared between males and females.
Differences in pharmacokinetic parameters between males and females could also be explained by differences in sex hormones and the menstrual cycle.However, in this study, the phase of the menstrual cycle was not considered upon enrolment.Therefore, a new study would be necessary to evaluate if menstrual cycle affects repaglinide's pharmacokinetics.
The allele frequencies of the CYP3A4*4 and *5 alleles were 0% respectively for both male and female subjects (Table 3).All five subjects (8.2%) with CYP3A4*18 mutations were found to be male (Table 4).
Although some studies suggested higher CYP3A4 activity in women [26], there are no reports of sex differences in CYP3A4's expression and function [27].One study even reported a lower enzyme activity in women [14].Some drugs that are substrates of CYP3A4 showed higher clearance rates in women than in men, even after correction for physiologic factors such as body weight [21].Some evidence supports the hypothesis that either higher CYP3A4 protein expression in women or female sex steroid modulation of CYP3A4 function could contribute to higher CYP3A4-mediated clearance in women [28].According to Chen et al. [26], women have more CYP3A4.In vitro studies conducted by Wolbold et al. [29] that examined human liver samples detected higher CYP3A4 expression levels in female liver samples.Ta-naka [30] also suggested that drugs that are metabolised by CYP3A4 frequently appear to be eliminated faster by women.To date, however, there are no reports on sex differences in CYP3A4 activity using repaglinide and our study is the first.We did not however find a significant correlation between repaglinide pharmacokinetics and the various genotype groups, perhaps due to the unequal distribution of sexes in the various groups.Furthermore, we were unable to correlate sex and the observed kel and CL values of repaglinide because all of our subjects with the CYP3A4*18 genotype were male.
For CYP2C8, both subjects with CYP2C8*2 and CYP2C8*3 were male subjects.There were five males (8.20%) and six (10.00%) female subjects are having CYP2C8*5 allele.In our study we found that the mean (SD) kel and mean (SD) CL of repaglinide were 47.67% (p = 0.03) higher and 29.25% (p = 0.03) higher, respectively, in male subjects having CYP2C8*5 allele compared to female subjects (Table 5).We also found that the mean (SD) t 1/2 of repaglinide was 42.43% higher (p = 0.03), and the mean (SD) AUC was 35.83% higher (p = 0.03) in female subjects when compared to the male subjects having CYP2C8*5 allele.However, there was no statistically significant change in the mean (SD) Vd, C max or t max of repaglinide.
Potential sex differences in CYP isoenzymes are expected to be most appropriately characterized by quantifying the metabolism of probe drugs that are indicative of a specific CYP activity [31].Sex differences in activity of the CYP enzymes will result in differences in Cl.However there is no prior study on CYP2C8 activity in male and female subjects.There are still large gaps in our knowledge of sex differences in CYP2C8 activity and therefore, significantly more research is needed.
In conclusion, the current data suggest that the pharmacokinetic of repaglinide can be different between women and men.Based on these data, we conclude that gender significantly influences the pharmacokinetics of repaglinide.