Pharmacokinetics interaction of dapoxetine with different doses of green tea extract in male healthy volunteers using midazolam as CYP3A4 enzyme probe

Article history: Received on: 18/09/2015 Revised on: 16/10/2015 Accepted on: 15/11/2015 Available online: 27/12/2015 Objective: This study aimed to investigate the pharmacokinetics interaction of dapoxetine with different doses of green tea extract in healthy volunteers using midazolam (CYP3A4 probe). Method and materials: Twelve healthy males were included in a random three-way crossover study. Each volunteer received dapoxetine 60 mg and midazolam 7.5 mg concurrently after drinking 250 ml of water, 250 ml of fresh extract of 2 gram of green tea or 250 ml of fresh extract of 4 gram of green tea with one week washout period. Plasma samples were analyzed for dapoxetine and midazolam using HPLC. Results: The co-administration of dapoxetine with 4 gm green tea extract significantly increased dapoxetine AUC∞ (from 3218.74 μg.hr/L to 4207.65 μg.hr/L, P<0.05) and dapoxetine Cmax (from 433.1 μg/L to 601.1 μg/L,P<0.05) with a decrease in CL and t1/2 only after administration of 4 gm green tea extract. There was a significant increase in midazolam AUC∞ (from 41.123 μg.hr/L to 58.55 μg.hr/L, P<0.05) and midazolam Cmax (from 36.07 μg/L to 53.53 μg/L,P<0.05) with a decrease in CL and t1/2only after administration of 4 gm green tea extract. However, the intake of 2 gram green tea extract showed no significant change in either dapoxetine or midazolam AUC or Cmax (p≥0.05). Conclusion: High dose of green tea intake increases dapoxetine bioavailability by the inhibiting CYP3A4 enzyme as indicated by the change in midazolam pharmacokinetic. Taking high dose of green tea with dapoxetine should be avoided. However, normal dose of green tea is safe for dapoxetine co-administration.


INTRODUCTION
Premature ejaculation (PE) is a common male sexual disorder which is associated with substantial personal and interpersonal negative psychological factors. The off-label use of anti-depressant SSRIs including paroxetine, sertraline, fluoxetine, citalopram and fluvoxamine has revolutionized the approach to PE treatment (Salonia et al., 2009). However, the lack of an approved drug and total reliance on off-label treatment represents a substantial unmet treatment need. Dapoxetine was the first safe drug developed for PE (McMahon, 2011). Dapoxetine is a potent SSRI structurally similar to fluoxetine (Sorbera et al., 2004). Dapoxetine binds to 5-HT, norepinephrine (NE) and dopamine (DA) re-uptake transporters and inhibits uptake in the following order of potency: 5-HT> NE >DA (Gengo et al., 2005). Dapoxetine was rapidly absorbed, Dapoxetine has a Tmax of 1.0-2.0 hours and rapidly achieves peak plasma concentration (Cmax) following oral administration. Both plasma concentration and area under the curve (AUC) are dose dependent up to 100 mg (Dresser et al., 2004). Elimination was biphasic, with an initial half-life of approximately 1.4 hours and a terminal half-life of approximately 15 hours (Modi et al., 2006).
Dapoxetine is metabolized extensively in the liver and kidney by multiple enzymes mainly CYP3A4 or CYP2D6. The major product at the end of the metabolic pathway is circulating dapoxetine N-oxide, which is a weak SSRI and contributes no clinical effect. The other products presented less than 3% in the plasma are desmethyl dapoxetine and didesmethydapoxetine, which are equipotent to dapoxetine. Although didesmethyldapoxetine is equipotent to the parent dapoxetine, its substantially lower plasma concentration, compared with dapoxetine, limits its pharmacological activity and it exerts little clinical effect . Dapoxetine 30 and 60 mg were well tolerated with a low incidence of adverse effects (AEs). The most frequently reported AEs were nausea, diarrhea, headache, dizziness, insomnia, somnolence, fatigue, and nasopharyngitis (Montejo et al., 2001). Green tea has been widely used as antioxidant and chemo-preventive agents against vascular risk factors, sexual disorders, and cancer (Mostafa et al., 2013). Green tea is derived from Camellia sinensis plant (Graham, 1992). The major catechins in green tea are epicatechin (EC), epigallocatechin (EGC), epicatechin-3-gallate (ECG) and epigallocatechin-3-gallate (EGCG) which is the most abundant, accounting for 50-80% of the total catechins in green tea (Feng, 2006). Previous study showed that green tea extract inhibited CYP3A4 activity in human liver microsomes (Nishikawa et al., 2004). For this reason, the wide variety of medical uses of green tea suggests that the possibility for potential drug interaction is high. Drug-drug interactions (DDI) of CYP3A4 are of particular importance because of the number of marketed drugs that are cleared by this enzyme. Multiple probe substrates are often used for in CYP3A4 DDI studies, including midazolam, felodipine/ nifedipine, and testosterone (Foti et al., 2010). Patients receiving dapoxetine for the treatment of PE could potentially take grean tea as a beverage for its multiple benefits. Green tea can inhibit CYP 3A4 and Dapoxetine undergo CYP3A4 metabolism and the potential for interactions must be evaluated. So, this study aimed to investigate the pharmacokinetic interaction of dapoxetine and different doses of green tea extract in normal healthy male volunteers using midazolam as CYP3A4 probe

Materials
Dapoxetine was obtained from SEIDICO "South Egypt for Drug Industries Co. (Ismailia, Egypt), midazolam was obtained Amoun pharmaceutical company (cairo, Egypt), and clonazepam was obtained from Sigma chemical Co. (St.Louis, MO, USA). Acetonitrile, methanol, and ammonium dihydrogen phosphate, and phosphoric acids were purchased from (Riedel-De Haen, Germany). All solvents were HPLC grade. Diethyl ether of analytical grade was obtained from Honil Limited (London, UK).

Subjects
Twelve healthy males were included in the study. The average age of the volunteers range (25-45) and the average weight was 78 kg (range 60-93). The study was carried out in the Pharmaceutical Research Center of Faculty of Pharmacy, Tanta University, Egypt, from April 2015 to July 2015. The study protocol was approved by the ethical committee of Tanta University in accordance with the Declaration of Helsinki. Participants signed for a written consent form. All volunteer had vital signs, normal kidney and liver functions free from ischemic heart disease and liver disorders.

Study design
A random crossover single dose study was employed. Participants abstain taking any drug for at least 3 days before and till the end of the study period. They were fast for 8 hours before drug intervention. On the day of the study, each volunteer received Dapoxetine tablet 60 mg ( Joypox ® 60 mg, SEDICO "South Egypt for Drug Industries Co.", Cairo, Egypt), midazolam tablet 7.5 mg ( Mediathetic ® 7.5 mg, Amoun pharmaceutical company, Cairo, Egypt) concurrently after drinking 250 ml of water, after drinking 250 ml of fresh extract of 2 gram of green tea (one Green tea packets 2 grams, Mepaco, Anshas El-raml, Sharqia), or after drinking 250 ml of fresh extract of 4 gram of green( two Green tea packets 2 grams, Mepaco, Anshas El-raml, Sharqia). After one week washout period, each volunteer received the second intervention followed by another one week washout period, then each volunteer received the third intervention as shown in the study flow (Table 1). All volunteers take the same standardized meal 4 hours after dapoxetine intake and no smoking was allowed during blood sampling period.

Sampling
Blood samples were obtained after the insertion of peripheral cannula into the forearm by a skilled certified nurse. Samples were obtained before dapoxetine and midazolam intake (blank) and at 0.25, 0.5, 0.75, 1, 1.25, 1.5, 2, 3, 5, 8, 12,24, and 48 hours after sildenafil and midazolam administration. The samples were collected in clean heparinized tubes. Plasma was obtained by centrifugation (Hettich EAB 12 Centrifugator, Germany) and was stored at -20° until assay. Plasma samples were analyzed for dapoxetine and midazolam using HPLC (Shimadzu HPLC LC2010HT, Shimadzu Corporation, Japan). Tolerability was assessed throughout the study using vital-sign measurements, physical examinations, and monitoring of adverse events (AEs) that were recorded in terms of symptoms and signs, duration, and seriousness.

Determination of dapoxetine and midazolam by HPLC
Plasma samples were analyzed for dapoxetine and midazolam in one run using validated HPLC method developed in the laboratory of pharmaceutical research center of faculty of pharmacy, Tanta University, Egypt. A clean test tube were spiked with 50 µl of the internal standard solution "5 µg/ml clonazepam in methanol" and the methanol was left to evaporate in a water bath adjusted at 50°. To this tube, 0.5 ml of the collected plasma sample was added and the tube contents were vortex mixed for 3 min. The plasma samples already spiked with the internal standard were extracted with 4 ml of ether following mixing by vortex for 3 minutes. After centrifugation for 10 min, the ether layer was transferred to a clean test tube and was evaporated in a water bath at 50°. The residue was dissolved in 150 µl of the mobile phase and 50 µl of the resulting solution was injected into the HPLC.
The mobile phase consisted of acetonitril and 50 mmol ammonium dihydrogen phosphate buffer (PH adjusted at 2.5 with phosphoric acid) at a ratio 30:70. Separation was achieved at ambient temperature using column C8 (C 8 150*4.6) at a flow rate of 1.0 ml/min. The column effluent was monitored by UV detector at 220 nm. The retention time (RT) for dapoxetine was 3.3 and (RT) for midazolam was 8.79 as shown in the following chromatogram ( Figure 1).

Standard Curve for dapoxetine
Blank plasma was spiked with the internal standard and known amounts of dapoxetine to produce standard samples with concentrations in the range of 50μg/L -1000 μg/L. Calibration curves were constructed from the obtained peak area and the concentration of dapoxetinein each standard sample.
The concentrations of dapoxetine in the unknown samples were determined from the calibration curves. The assay was fully validated for linearity, selectivity, precision, accuracy and stability.

Standard Curve for midazolam
Blank plasma was spiked with the internal standard and known amounts of midazolam to produce standard samples with concentrations in the range of 5 μg/L-100 μg/L. Calibration curves were constructed from the obtained peak area and the concentration of midazolam in each standard sample. The concentrations of midazolam in the unknown samples were determined from the calibration curves. The assay was fully validated for linearity, selectivity, precision, accuracy and stability.

Pharmacokinetic and statistical calculations
The plasma concentration-time profiles of dapoxetine, and midazolam of each subject were analyzed by a noncompartmental method using WinNonlin ® software (v 6.1; Pharsight Corporation, Mountain View, CA, USA). Pharmacokinetic values for peak concentration (Cmax) and time to Cmax (Tmax) were taken directly from the observed data. Individual concentration-versus time profiles were plotted, and the terminal elimination rate constant (k) was determined by the loglinear regression of at least three data points judged to be in the terminal phase. The half-life (t 1/2 ) equal to 0.693/k. Area under curve (AUC) was determined by the trapezoidal rule from time zero to the time of the last observed concentration (C last ) plus C last /k.
The total body clearance (CL/F) was obtained as dose/AUC . Statistical comparisons of the estimated pharmacokinetic values were performed using ANOVA with Tukey's test. P-values < 0.05 were considered significant. Retention time for midazolam is 3.328 min, for dapoxetineis 8.797 min, for clonazepam (inyernal standard) 7.16 min.

RESULTS
Vital signs were similar after dapoxetine alone as well as with different doses of green tea extract. Dapoxetine was absorbed rapidly after oral administration reaching a maximum plasma concentration in about 1hr. The mean plasma concentration-time profile after a single dose of dapoxetine 60 mg without, after 2 gm, or after 4 gm green tea were shown in Figure (2). There was a significant increase in dapoxetine AUC∞ (from 3218.74μg.hr/L to 4207.65μg.hr/L, P<0.05) and dapoxetine Cmax (from 433.1μg/L to 601.1μg/L, P<0.05) only after co-administration of 4 gm green tea extract with dapoxetine. However, the intake of 2 gram green tea extract with dapoxetine showed no significant increase in dapoxetine AUC or Cmax (p≥0.05).
Table (2) showed the pharmacokinetic analysis for dapoxetine in the three cases. After 4gm intake of green tea with dapoxetine. The CLtot/F was significantly reduced from 18.64 L/hr to 14.25 L/hr (p<0.05) and the elimination of dapoxetine was also significantly delayed. The elimination rate constant was decreased from 0.046hr -1 to 0.034h-1 (p<0.05) and the elimination half life was prolonged from15.06 h to 20.38 h (p<0.05).
On the other hand, the Tmax showed no significant change after either 2 gm or 4 gm intake of green tea extract (p≥0.05). Regarding midazolam, the mean plasma concentrationtime profile after a single dose of midazolam 7.5 mg without, after 2 gm, or after 4 gm green tea were shown in Figure (3). There was a significant increase in midazolam AUC∞ (from 41.123 μg.hr/L to 58.55 μg.hr/L, P<0.05) and midazolam Cmax (from 36.07 μg/L to 53.53μg/L, P<0.05) only after co-administration of 4 gm green tea extract with midazolam. However, the intake of 2 gram green tea extract showed no significant increase in midazolam AUC or Cmax (p≥0.05).
Table (3) showed the pharmacokinetic analysis for midazolam in the three cases. After 4gm intake of green tea with midazolam. The CLtot/F for midazolam was significantly reduced from 182.37 L/hr to 128.09 L/hr ((p<0.05) and the elimination of midazolam was also significantly delayed. The terminal elimination rate constant was decreased from 0.643hr -1 to 0.389hr -1 (p<0.05) and the terminal elimination half life was prolonged from 1.096hr to1.758hr (p<0.05). On the other hand, the Tmax showed no significant change after either 2 gm or 4 gm intake of green tea extract (p≥0.05). Data are presented as mean ± S.E, except for tmax for which median is shown, n=12. *significantly different from dapoxetine (paired t test, p<0.05). k= elimination rate constant, AUC∞=area under the curve, t½= half-life, tmax= time required to achieve maximum plasma concentration, Cmax=maximum plasma concentration, CLtot= total body clearance and F= bioavailability.

Fig. 2:
The mean plasma concentration-time profile after a single dose of dapoxetine 60 mg without, after 2 gm, or after 4 gm green tea. * significant increase AUC and Cmax after co-administration with 4 gm green tea extract compared to dapoxetine alone (paired t test, P<0.05).

DISCUSSION
Dapoxetine is the first effective and safe treatment for premature ejaculation that represents a major advance in sexual medicine. So, the use of dapoxetine is expected to increase. For this reason, it is very important to ensure the safety of this drug in a variety of conditions (McMahon, 2011).Recently, the use of herbal supplements has increased dramatically as an alternative to chemical drugs, making drug interactions with these supplements a major concern to researchers specially that herbal supplements are not subject to the same regulations as prescription drugs (Wanwimolruk et al., 2009).
In this study, the primary pharmacokinetic parameters were the AUC, Tmax, and C max of dapoxetine and midazolam. The results of current study indicated that co-administration of 4 gm green tea extract (high dose than normal) produced a significant increase in the extent of dapoxetine absorption but not the rate of dapoxetine absorption. This was apparent from the significant increase in dapoxetine AUC without any change in Tmax. However, the co-administration of 2 gm green tea extract (normal green tea dose) showed no change in dapoxetine pharmacokinetics. In addition, the current study showed an increase in half life of dapoxetine. This prolongation of the half life by high dose of green tea resulted from decreasing the total body clearance which is attributed to the decrease in dapoxetine metabolism by inhibiting CYP3A4 (Walsky and Obach, 2004). The inhibition of CYP3A4 was confirmed by the increase in midazolam concentration. Midazolam is the most reliable standard substrate for evaluation of the in vivo inhibition of CYP3A4 (Ohno et al., 2007). The change in midazolam bioavailability after dapoxetine intake indicate the possible inhibition of CYP3A4 by dapoxetine.
The most common form of green tea in the market is packet of 2gm green tea leave powder which is considered the normal dose. In contrast, high dose of green tea may be found in some dietary supplement in the market or from taking two packets of 2 gram green tea simultaneously. So, patients taking this high dose should be warranted about the possible drug interaction with dapoxetine and reducing dapoxetine dose may be recommended or seeking physician for advice. Yang and Pan were the first to report that The effect of green tea on drug metabolism is dose dependant supporting current results and that the of tea catechins from dietary supplements containing large doses may produce more profound effects on drug metabolism while normal doses may be safe on drug metabolism (Yang and Pan, 2012).
The green tea used in this study was tested for content in polyphenols (Catechins represent 90 % of polyphenol) by the manufacturing company which reported that the used green tea is Data are presented as mean ± S.E, except for tmax for which median is shown , n=12. *significantly different from control (p<0.05). k= elimination rate constant, AUC∞=area under the curve, t½= half-life, tmax= time required to achieve maximum plasma concentration, Cmax=maximum plasma concentration, CLtot= total body clearance and F= bioavailability. the Chinese species of green tea and one gram of green tea leaves powder contain 0.3 gram of catchins. In our laboratory, qualitative standardization for the composition of green tea leaves was carried out by comparing their TLC fingerprints with authentic sample of green tea leaves. The amount is automatically adjusted by the manufacturing company so variation in amount is reduced to minimum. In the current study, 60 mg dose of dapoxetine were selected because they are the highest recommended dosage. Generally, these drugs are used on-demand; for this reason, this study was conducted after a single oral administration of dapoxetine (Hatzimouratidis et al., 2010). Limited studies are available for dapoxetine pharmacokinetic interactions. Currently, there are no documented drug-drug interactions associated with dapoxetine except for ketoconazole which is potent CYP3A4 inhibitor. Ketoconazole increased dapoxetine exposure to a greater extent. There was a 23% increase in C max and 88% increase in the AUC of dapoxetine. However, other drug interaction studies on dapoxetine and other PDE-5 inhibitors, including tadalafil, sildenafil or udenafil as well as with ethanol reported no clinically significant pharmacokinetic interactions. Despite that contraindication to dapoxetine include that Dapoxetine should not be used in men receiving CYP3A4 inhibitors such as ritonavir, ketoconazole, and telithromycine Kim et al., 2015) The effects of green tea extract on enzyme metabolism is not completely proven. Many previous studies indicated that green tea extract use caused significant interactions with drugs metabolism by inhibiting CYP3A4 and Tea whether used for leisure or medicinal purposes has the potential to inhibit CYP3A4 (Vischini et al., 2011;Tam et al., 2014).On the other hand, few studies indicated that green tea extract might not change or even increase the CYP3A4 activity.
Regarding supporting studies, Mooiman et al. stated that green tea is a potent inhibitor of CYP3A4-mediated metabolism of midazolam (Mooiman et al., 2014) and the effect on CYP3A4 varied among different brands of green tea, possibly due to variations in their content of the herbal product's active ingredients (Wanwimolruk et al., 2009). Engdal and Nilsen stated that although Agaricus, noni juice, mistletoe and green tea inhibited CYP3A4 metabolism in vitro, clinically relevant systemic or intestinal interactions with CYP3A4 were considered only for the green tea (Engdal and Nilsen, 2009). In addition, Chow et al., revealed that four weeks of green tea catechin intervention did not alter the CYP1A2, CYP12D6, and CYP12C9 activities, but resulted in increasing AUC of buspirone concentration through the reduction in CYP3A4 activity (Chow et al., 2009). A recent study showed that, green tea inhibited CYP3A activity in a noncompetitive manner (Misaka et al., 2012). Moreover, green tea extract alters the pharmacokinetics of simvastatin by 3.3 fold increase, probably by inhibiting intestinal CYP3A (Misaka et al., 2013). In another study, the repeated intake of green tea extract aggravated cyclophosphamide-induced body weight loss and malformations of fetuses by modulating CYP2B and CYP3A mRNA (Park et al., 2009). In the opposite side, Donovan was the first to report that green tea is unlikely to alter the disposition of medications primarily dependent on the CYP2D6 or CYP3A4 pathways of metabolism (Donovan et al., 2004). A recent study showed that green tea extract decreased the bioavailability of quetiapine but the exact mechanism was unknown (Asiri and Iqbal, 2015). Boušová et al. who showed that green tea decreased insulin and leptin levels and Long-term administration of green tea extract caused an increase in the activity and mRNA level of CYP3A4 ortholog in the liver as well as in the small intestine in obese individuals and these unexpected result may be related to obese people only (Boušová et al., 2015). The use of Midazolam enabled us to investigate the possible mechanism for this interaction at high dose. Midazolam is considered the clinical standard probe for CYP3A4 enzyme (Ohno et al., 2007). The increase of midazolam AUC and Cmax at the high dose of green tea indicated that the expected mechanism of this interaction is the inhibition of CYP3A4 enzyme in both the intestine and the liver.
In conclusion, at normal doses of green tea, dapoxetine has no clinically important pharmacokinetic interactions with green tea, and the combinations are well tolerated. But, at high doses of green tea, green tea may cause moderate increase in dapoxetine plasma concentration by inhibiting CYP3A4 enzyme as indicated by the significant increase in midazolam AUC and Cmax. Patients taking high dose of green tea should take smaller doses of dapoxetine or should contact physician first before taking dapoxetine therapy. However, further controlled studies for larger numbers of patients may be required.