Influence of Energy Drinks on Pharmacokinetic Parameters of Sildenafil in Rats

1Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan. 2Faculty of Pharmacy and Medical Sciences, Applied Science University, Amman, Jordan. 3Faculty of Pharmacy and Medical Silences, Al-Ahliyya Amman University, Jordan. 4School of Biomedical Science, Faculty of Health Sciences, University Sultan Zainal Abidin, 21300 Kuala Nerus, Terengganu, Malaysia. *Corresponding author E-mail: eyad782002@yahoo.com

Sildenafil citrate is an oral drug used as a therapy for erectile dysfunction 1  NO activates the enzyme guanylate cyclase, which increases the levels of cyclic guanosine monophosphate (cGMP) 3 .
The production of cGMP leads to relaxation of smooth muscles in the corpus cavernosum and increases blood flow into the penis 4   .
Sildenafil citrate has no direct relaxant effect, but it increases the levels of cGMP by inhibiting phosphodiesterase type 5 (PDE5), which is responsible for degradation of cGMP 5 .Sildenafil is primarily metabolized by the CYP3A4 enzyme, which is the principal enzyme responsible for the oxidative metabolism of the majority of drugs 6 .The interaction between sildenafil and other drugs that are also metabolized by CYP3A4 should be taken into consideration when drugs are prescribed to avoid undesired pharmacological effects [6][7][8] .
RED BULL ® is the best-selling energy drink in the United States of America and worldwide.It contains different ingredients such as sugar, caffeine, taurine, glucuronolactone and B vitamins 9 .Caffeine is probably the most common ingredient in RED BULL ® , as it acts as a stimulant 10 .Taurine is a homocysteine derived amino acid, but not a constituent of proteins 11 .It is present freely in the intracellular fluid and distributed widely in the sarcoplasm of the cardiac tissues, hepatocytes, central nervous system, and retina.Taurine mediates the homeostasis of different physiological functions 12 including osmoregulation, antioxidation, detoxification, neuromodulation, and brain and retinal development 13 .Moreover, a number of studies have documented some pharmacological functions of taurine against congestive heart failure, liver disease, hyperlipidemia 14 .
Several papers reported that Sildenafil pharmacokinetics are altered in response to food-drug (or drug-drug) interactions 5,[15][16][17] .It has been demonstrated the co-administration of sildenafil with potent CYP3A4 inhibitors such as azole antifungal agents, macrolide antibiotics, and protease inhibitors causes a significant increase in plasma sildenafil levels 18,19 .Another example of interaction is Sitaxentan (endothelin receptor antagonist), which when administered with sildenafil, demonstrated a weak, but a statistically significant interaction 20 .The effect of concomitant administration of caffeine with certain drugs used for cardiovascular, CNS, gastrointestinal, infectious, respiratory and skin disorders has been extensively studied as well 21   .whereas, the effect of other ingredients in Red Bull has not been studied for the pharmacokinetic effect on sildenafil.Caffeine has been reported to interact with many medications such as certain selective serotonin reuptake inhibitors (particularly fluvoxamine), antiarrhythmics (mexiletine), antipsychotics (clozapine), psoralens, idrocilamide and phenylpropanolamine, quinolones (enoxacin) and bronchodilators (furafylline and theophylline) 10 .Thus, pharmacokinetic interactions at the CYP1A2 enzyme level may cause toxic effects during concomitant administration of caffeine and certain drugs used for cardiovascular, CNS, gastrointestinal, infectious, respiratory and skin disorders 9 .
Recently, drug-drug and drug-food interactions are being the center of our interest [22][23][24][25][26] .The idea of this research work had arisen from observations of about the concomitant use of sildenafil with RED BULL ® by men seeking for better aphrodisiac effect during sexual intercourse.Both Sildenafil and RED BULL ® claimed to boost up energy.Since RED BULL ® is one of the most commonly used energy drinks, it is easily and widely available for daily use.The current study was conducted to investigate the effect of RED BULL ® ingestion on the pharmacokinetics of Sildenafil in rats plasma using HPLC in a crossover study.

Preclinical study and protocol
Adult female Sprague-Dawley laboratory rats were supplied by the animal house of Applied Science University.The average weight of these rats was about 240g (200g -260), and they were in healthy condition.They were placed in an airconditioned environment (20-25 °C) and were exposed to 12 hours light /12 hours dark cycle daily.
All animals in the experiments were handled in compliance with FELASA guidelines (Federation of European Laboratory Animal Science Association) and the study protocol was approved by the research committee (February 2014) at the Faculty of Pharmacy and Medical Sciences, University of Petra, Amman, Jordan.
10 rats were included in the drug interaction study in a crossover study.The rats were divided into 2 groups (group A and group B), 5 rats each.In the first period of the experiment, group (A) had received sildenafil aqueous solution with a concentration of 0.57 mg/ml and a dose of 2.85/kg (200mg/70kg) alone as a single dose, while group (B) had received sildenafil aqueous solution as a single dose with RED BULL ® (purchased from local market), respectively and spontaneously.Washing out period of 2 weeks was implemented, after which the test items were switched between the 2 groups, Group (A) received sildenafil solution and RED BULL ® while group (B) received sildenafil solution alone.
The dose used is corresponding to 200 mg for human of 70 kg weight, 200 mg is the double dose of sildenafil regular dose.This dose was chosen to clarify any possible interaction, as it found to be used in the previous clinical trial 27 .
The least weight of rats was 200g and the concentration was calculated based on this weight.Rats weighing 200 mg took 1 ml of the solution, and then other rats took more than 1ml according to their weights.
The rats which were aimed to take RED BULL ® in each part of the experiment were supplied with 15 ml, 16 hours before sildenafil solution administration.They received another 5ml of RED BULL ® orally after the administration of sildenafil solution.
A syringe with a specific needle was used for the oral administration of both sildenafil and RED BULL ® solutions.For rats which were aimed to take sildenafil solution alone, drinking water was administrated insufficient amount all the time.
Blood samples were drawn into EDTAcontaining microtubes.The microtubes were immediately centrifuged for 10 minutes at 5,000 rpm and separated plasma was transferred into labeled Eppendorf tubes then stored at -20 °C until analysis.

Preparation of solutions
Preparation of sildenafil solution for oral use 0.081 g of sildenafil citrate (equals to 0.057 g sildenafil) was dissolved in 100 ml of distilled water, vortex to have complete dissolution to get a concentration of 0.57 mg/ml.

Preparation of stock solution of sildenafil
An equivalent weight of 10 mg of sildenafil working standard was dissolved in 10 ml of methanol to get a concentration of 1000 µg/ ml stock solution of sildenafil.

Preparation of stock solution of carbamazepine internal standard
An equivalent weight of 10 mg of carbamazepine working standard was dissolved in 100 ml of Acetonitrile to get a concentration of 100 µg/ml stock solution of carbamazepine.

P re p a r a t i o n o f w o r k i n g s o l u t i o n o f carbamazepine i.s
20 µl were taken from carbamazepine stock solution (100µg/ml) and diluted to 100 ml using ACN which was considered to be an I.S working solution that contains 20 ng/ml of carbamazepine.

Method of extraction
The described procedures were applied for subject samples, calibrator, and quality control samples.
In order to perform the sample extraction, disposable Eppendorf tubes were labeled and placed on a rack, then 100.0 µl aliquots of each test sample (blank, zero, standards, quality control low, quality control medium, quality control high and rat samples) were drawn into the tubes by pipette and mixed with 150.0 µl of internal standard (20 ng/ml carbamazepine in ACN), then vortex of each sample vigorously for 1.0 min, and finally Centrifugation at 14000 rpm for 15 minutes.

Chromatographic conditions and detection conditions were as follow in table I: analytical method validation accuracy and Precision
The intra-day precision and accuracy were evaluated by analyzing six replicates of the QC samples (low, mid, high) and lower limit of quantification (LLOQ) samples on a single day.The inter-day precision and accuracy were determined by analyzing three runs of QC samples and LLOQ samples on three different days.The accuracy (%) was calculated by dividing a measured mean concentration over the nominal analyte concentration.Precision was presented as CV%.The acceptable limits of accuracy and precision should be below 15% except at the LLOQ, for which accuracy and precision should be below 20% 28 .linearity Linearity was determined by a series of six injections to a seven calibration concentration levels for the analyte.Peak areas of the calibration standards were plotted in the Y-axis against the nominal standard concentration, and the linearity of the plotted curve was evaluated through the value of the correlation coefficient (R2) which should be more than 0.98 28 .Stability of the analyte in the rat serum was evaluated using both low and high QC samples, which were analyzed immediately after preparation and after the applied storage conditions.Evaluation included: autosampler stability, freeze-thaw stability "after 3 cycles", and Short-term stability at room temperature "24 h".
The mean concentration should be within ±15% of the nominal concentration 28 .

Validation
The validation of sildenafil was conducted according to EMA guideline 28 .Selected chromatograms for sildenafil validation (blank, zero, LLOQ) are shown in figures (4, 5 and 6).

accuracy and precision
Table II represents inter-day precision and accuracy for quality control samples of sildenafil in three days of validation.All of the Therefore, validation results passed the required criteria in term of linearity and accuracy.

stability
Tables IV shows data for short-term stability indicated by two QC concentrations (low, high) for sildenafil after preparation procedure (auto-sampler stability), T=10°C and for room temperature or processing temperature.Regarding to the stability of plasma samples during freezing and thawing processes; two QC samples were stored and frozen in a freezer at the intended temperature -60°C and there after thawed and processed at room temperature twice for 72 hours.All results were within the accepted range of 85%-l 15%.

Pharmacokinetic data of sildenafil in presence of red Bull ®
The plasma concentrations of sildenafil with or without RED BULL ® were measured in the population rats using a sample size of 10 rats for the drug alone and then repeated for the combination of the drug with RED BULL ® in a crossover study with washout period of 2 weeks.
7 discrete samples were obtained from rats following drug administration to the last time interval of 7.5 hours.
This study was conducted on female rats since the sildenafil bioavailability is more comparable to humans (regardless the gender) and the plasma levels of sildenafil male rats are much lower when compared to its bioavailability in female rats ( 16) (17).
The plasma levels of sildenafil fed rats has reached its maximum level at the first 0.5 hours (C max ) (162.05 ng/ml) (T max ) when administered alone as depicted in figures (3, 7).
Whereas, sildenafil plasma levels were intimately decreased when administered with RED BULL ® as C max was only 44.68 ng/ml at the first 0.5 hours, as shown in figures (3, 8).The sharp decrease in sildenafil levels as a consequent of administration with RED BULL ® reached a proportion of 72% decrease (P<0.001) compared with sildenafil fed rats alone.

Figure 3shows
Mean plasma sildenafil concentration of female rats in a crossover study, sildenafil was administered alone and in combination with RED BULL ® .Each data point represents the mean (n=10).
The pharmacokinetic interaction of Sildenafil and RED BULL ® could happen at any stage of ADME: "Absorption, Distribution, Metabolism, and Excretion." However, the absorption of sildenafil from the intestine happens through a passive mechanism, no carriers are needed so we rule out this is the cause of the difference 29 .The pH of RED BULL ® solutions (with and without Sildenafil) is 3.28, which indicates the minimum effect on stomach pH.In addition, we noted that complete dissolution occurred for Sildenafil in RED BULL®.
However, this cannot rule out the presence of interaction at the absorption level in the stomach as the presence of REDBULL will effect of the gastric emptying time 26 .
By plotting the data on the semilog scale as shown in figure (9), the slope (which represents elimination rate constant) of Sildenafil when administered with RED BULL beverage was steeper, this indicates a higher elimination rate of the drug when combined with RED BULL.
Figure 9: Mean plasma sildenafil concentration in female rats with time after drug administration, compared to its combination with RED BULL ® on a semilog scale.Each data point represents the mean (n=10).
The lower AUC, Cmax is accompanied by an increase in the clearance of the drug and shortening of the half-life.These changes elimination parameters indicate an enhancement in elimination processes of the drug which mainly eliminated by metabolism in the liver.By reviewing the literatures, it has been demonstrated that taurine has an activating effect on the expression level of CYP 3A4 in the presence of CYP3A4 inducer.Taurine enhances the induction of CYP3A4 mRNA in a concentrationdependent manner 30 .Taurine is a potent inducer of CYP3A4 mRNA via pregnane X receptor (PXR), an orphan member of the steroid/retinoid/ thyroid hormone receptor superfamily of ligandactivated transcription factors 31,32 ,.It was recently reported that PXR serves as a functional bile acids receptor, and lithocholic acid, which is a kind of bile acid, induces CYP3A mRNA [33][34][35] .Attention should be paid to drug interactions when taurinecontaining medicinal agents, foods, and drinks are administered together with both sildenafil and drugs metabolized mainly by CYP3A4 35 .
Moreover, in a similar manner of drug interaction, high levels of taurine have lowered the plasma concentrations of acetaminophen and rifampicine by increasing the enzyme activity of CYP3A4 36,37 .Moreover, we tried to find a single clinical report about overdose abuse of sildenafil and REDBULL but up to the date there is no case report about the adverse effect of Sildenafil intake with energy drink.Our findings could explain the reason of why there are no reports on sildenafil interaction with such drinks but even though we don't know anything about the sildenafil clinical effect since it has reached to a sub therapeutic dose.
In addition, we propose that the change in sildenafil plasma levels in female rates is an enzyme induction mechanism due to a significant change in elimination parameters.Whereas Kel has been changed significantly from 0.45 hr-1 for sildenafil alone to 0.56 hr-1 when given in combination with RED BUL beverage.The terminal elimination t1/2 was shortened significantly from 1.51 hr to 1.15 hr in combination, and the clearance increased also highly significantly from to 135 L/hr/kg to 535.6 L/hr/kg in combination.
Also, the MRT-t was shortened.All these changes suggest an enhancement in elimination processes by metabolism of sildenafil by hepatic enzymes.However, the interaction still could have done at any stage of ADME.
Moreover, caffeine has a great impact on the cardiovascular system by causing increased heart rate and blood pressure which results in increased cardiac output 38 .So the blood flow to the liver will increase, and the metabolism of sildenafil in presence of caffeine may increase because of this increase in blood flow.
This interaction between sildenafil and RED BULL ® could be of clinical significance, because the decrease of sildenafil concentrations to subtherapeutic levels may make the effect of sildenafil clinically insignificant.Table V shows Pharmacokinetic data of sildenafil.
The difference between both C max and AUC of sildenafil alone and sildenafil with RED BULL ® was statistically significant (P<0.001).

conclusion
There is a pharmacokinetic interaction between sildenafil and RED BULL ® when administrated concomitantly.Such interaction effects significantly (P<0.001) the C max and AUC of sildenafil.The interaction between sildenafil and RED BULL ® could happen in all stages with high probability to be executed in metabolism stage.
This in vivo trial on rats suggests further investigations, such as assessing the possible interaction between sildenafil and RED BULL ® in humans, checking the pharmacodynamic effect of this combination, testing sildenafil levels in plasma in presence of certain concentrations of caffeine and explaining the exact mechanism of this interaction and the responsible compounds.

Fig. 3 .
Fig. 3. Mean plasma sildenafil concentration in female rats with time after drug administration, compared to its combination with RED BULL ® .Each data point represents the mean (n=10)

Fig. 8 .Fig. 9 .
Fig. 8. Sildenafil with RED BULL ® sample chromatogram after 0.5 hours of administration for one group

acknowledgMent
This research was funded by the deanship of scientific research at University of Petra.The

table 1 .
Chromatographic conditions and detection conditions.

table 2 .
Inter-day precision and accuracy data for sildenafil samples in the three days of validation

table 3 .
Raw data of the calibration curve for sildenafil

table 4 .
Short and long term stability data for Sildenafil

table 5 .
Pharmacokinetic data of sildenafil