Validated analytical study of the effect of Lycopene on the pharmacokinetics of Paracetamol and Chlorzoxazone in rats

Lycopene was reported to influence some cytochrome P450 enzymes activity. The present study investigates the effect of lycopene on the pharmacokinetics of paracetamol and chlorzoxazone. Lycopene (20 mg/kg) was intra-peritoneally administered to two groups of rats for eight consecutive days and two other groups were given vehicle. On the eighth day, chlorzoxazone and paracetamol were separately intravenously administered to a lycopene group and a control group. Blood samples were collected at different time intervals, treated and analyzed using HPLC. The HPLC method used for paracetamol analysis was based on isocratic elution using a mobile phase consisting of water: methanol, (77:23 v/v) at a flow rate 1 mL min−1, Kromasil C18 column, and UV detection at 254 nm using caffeine as internal standard. About chlorzoxazone, separation was carried out using water: acetonitrile (60: 40, v/v) as the mobile phase at a flow rate 1 mL min−1, Inertsil ODS-3 C18 column, UV detection at 283 nm and esomeprazole as internal standard. Statistical analysis of the pharmacokinetic data using student t test showed a significant increase in AUC0–t, AUC0-Inf and t1/2 of paracetamol (P<0.05) and of chlorzoxazone (P<0.05) in the groups pretreated with lycopene (20 mg/kg), significant increase in the volume of distribution of paracetamol (P < 0.05), but no significant difference in that of chlorzoxazone. In other words, paracetamol and chlorzoxazone showed significant decrease (P < 0.05), respectively. These results demonstrate that treatment of rats with Lycopene (20mg/kg, ip) has a significant effect on the metabolic clearance and the pharmacokinetics of both drugs.


INTRODUCTION
Lycopene is a non provitamin A carotenoid, concentrated in tomatoes and other red fruits (Schulzová, Hajšlová, 2007). Great interest to lycopene was recently observed because most studies proved that those who consume more lycopene and accordingly have high concentrations in plasma have lower incidence for chronic illness such as cancer and coronary heart diseases (Gerster, 1997;Rao, Agarwal, 1999). More studies proved the effect of lycopene to decrease bone resorption and thus improve osteoporosis (MacKinnon et al., 2011).
In a previous study, Tomato juice showed a potent inhibitory effect on nifedipine oxidation activity, which was comparable to that on testosterone 6β-hydroxylation activity, and weak inhibitory effect on midazolam 1′-hydroxylation activity ,by one or more mechanismbased and competitive inhibitor(s) of CYP3A4 (Sunaga et al., 2012).
Hence, the carotenoid lycopene is increasingly gaining scientific attention because of its potential health effects. However, little is known about the metabolic interactions between lycopene and clinically used drugs like paracetamol and chlorzoxazone.
Paracetamol (acetaminophen, N-acetyl-paminophenol, PAR) is one of the most popular and widely used drugs for the treatment of pain and fever. Due to its good tolerability profile, PAR is often the analgesic or antipyretic of choice, especially in patients in whom salicylates or other non-steroidal anti-inflammatory drugs are contraindicated (Portolés et al., 2003;Wang et al., 2018).
Cytochrome P450 enzymes, CYP2E1 and CYP1A2, catalyze oxidation of PAR to the reactive metabolite iD N-acetyl-p-benzoquinone imine (NAPQI) (McGill, Jaeschke, 2013). Numerous drugs have been reported to interact with PAR leading to exacerbation of its toxicity, so PAR should be administered carefully to people on isoniazid treatment or consuming excessive amounts of alcohol to prevent hepatotoxicity due to the induction of CYP2E1. PAR toxicity can occur when CYP2E1 metabolism is impaired by drug-drug interactions (Zand et al., 1993).
Chlorzoxazone (5-chloro-2(3H)-benzoxazolone; CZX), a skeletal muscle relaxant used for the treatment of painful muscle spasms is primarily metabolized to 6-hydroxychlorzoxazone (OH-CZX), which is subsequently glucuronidated and excreted in the urine (Kar, 2005;Kramer et al., 2003). Hepatic microsomal cytochrome P450 enzyme (CYP2E1) plays an important role in the formation of OH-CZX from CZX in humans and rats (Rockich, Blouin, 1999;Peter et al., 1990). Previous studies showed that lycopene has the ability to inhibit CYP2E1 in the rat microsomal model (Louisa et al., 2009). So far, there has been no information about the effects of Lycopene on the pharmacokinetics of CZX and PAR. The aim of this study was to determine this influence in rats using bioanalytical HPLC method.

Chemicals and solvents
Lycopene extract was kindly supplied from UG PHARMA company, Badr City, Egypt. Paracetamol, chlorzoxazone, esomeprazole and caffeine were kindly supplied from Memphis Company, Cairo, Egypt. Heparin (Calheparin 5000UI AMP) was purchased from Amoun Pharmaceutical Industries Company, Kaliobeya, Egypt). Methanol for HPLC and ethyl acetate were purchased from Sigma-Aldrich, Munich, Germany. Bi-distilled water was produced in-house (Aquatron Water Still, A4000D, UK).

Lycopene stock solution
Lycopene extract 10% equivalent to 200 mg was dissolved in 10 mL water to obtain a final concentration 20 mg/mL of pure lycopene.

Animal experiment
24 Male Wister rats, 250-450 g, obtained from the Laboratory Animal Center, Faculty of Pharmacy, Cairo University, were housed, six rats per cage, and given free access to food and water in a temperature-controlled room (25˚C) with a 12 h light/dark cycle. The study protocol was reviewed and approved (PC (1687) on 26 th April 2016 by the Institutional Review Board (REC-FOPCU; Research Ethics Committee-Faculty of Pharmacy, Cairo University) in Egypt. All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. The rats were randomLy divided into four groups: (1) Lycopene group of PAR (pretreated with lycopene (20 mg/kg, n=6) (2) Control group of PAR (pretreated with vehicle (water for injection), n=6) (3) Lycopene group of CZX (pretreated with lycopene (20 mg/kg), n=6) (4) Control group of CZX pretreated with vehicle (normal saline), n=6).
All the rats were given vehicle (control group) or lycopene (lycopene group) intraperitoneally on a daily basis for 8 consecutive days. On the eighth day, access to the diet was removed and only water was provided. After treatment with water or lycopene, PAR (25 mg/kg) was given to group (1) and group (2) and CZX (20 mg/kg) was given to group (3) and group (4) by intravenous administration in the tail.
After administration of PAR and CZX, 300 µL blood samples were collected from the vein of the eye at different time intervals (0, 0.25, 0.5, 1, 2, 4, 6 hr). The plasma samples were separated via Eppendorf containing 50µL of diluted heparin (1:10) by centrifugation at 4,000 rpm for 15 min and stored at -20 o C until analysis.

Extraction method of PAR
A liquid-liquid extraction method was used for extraction of PAR from plasma matrix. In an Eppendorf, an aliquot of 20 µL internal standard stock solution (Caffeine 400µg/mL) and 60 µL water were added to 120 µL plasma samples taken from groups (1) and (2) and then 1000 µL of a mixture of methanol and ethyl acetate (1:9) was added. The solution was vortexed for 30 s and the supernatant was separated by centrifugation at 4,000 rpm for 10 min. The organic layer was separated and evaporated to dryness at Page 3 / 11 40 °C under a gentle stream of nitrogen. The residue was reconstituted in 100 µL mixture of methanol and water (77: 23), then 20 µL was injected onto HPLC. The final concentration of IS is 80µg/mL.

Extraction method of CZX
A protein precipitation method was used for extraction of CZX from plasma matrix. An aliquot of 20 µL internal standard stock solution (Esomeprazole, 880µg/mL) and 60 µL water were added to 120 µL plasma samples taken from groups (3) and (4) in Eppendorf and then 200 µL of a mixture of water and acetonitrile (4:6) was added. The solution was vortexed for 30 s and the supernatant was separated by centrifugation at 6,000 rpm for 15 min, then 20 µL was injected onto HPLC. The final concentration of IS is 44µg/mL.

Assay of PAR in rat plasma
PAR plasma concentration was determined using HPLC. Chromatographic separation was achieved on a Kromasil C18 column (150 X 4.6 mm, 5μm) using a mobile phase consisting of water: methanol (77:23, v/v) at a flow rate 1 mL min −1 . The column temperature was 25 °C. UV detector was operated at 254 nm. The injection volume was 20 µL.

Assay of CZX in rat plasma
Chromatographic separation of CZX plasma concentration was achieved on an Inertsil C18 column (250 X 4.6 mm, 5μm) using a mobile phase consisting of water: acetonitrile (60:40, v/v) at a flow rate 1 mL min −1 . The column temperature was 25 °C. UV detector was operated at 283 nm. The injection volume was 20 µL.

Preparation of Calibration standards and quality control samples
Stock solutions of PAR (2.5-200 µg mL -1 ) were diluted in water to get concentrations of calibration curve (0.5-40 µg mL -1 ). Stock solutions of PAR (2.5, 7.5, 100, 150 µg mL -1 ) were diluted in water to get quality control samples (LLOQ = 0.5, Low = 1.5, Med =20, High = 30 µg mL -1 ). Stock solution of caffeine (400 µg mL -1 ) was diluted in distilled water to get concentration (80 µg mL -1 ) (IS). A 100 µL aliquot of rat plasma was spiked with 20 µL of stock solution, 20 µL of Caffeine stock solution (IS) and 60 µL water. To 200 μL of the spiked calibration plasma standards or QC samples, 1000 µL mixture of ethyl acetate and methanol (9:1) was added and the extraction procedure was continued as mentioned in "Extraction method of PAR".
Calibration curves were constructed by plotting peak area ratio of the drug to IS versus the corresponding drug concentration in both PAR and CZX. The constructed calibration curves were found to be linear and precise for PAR and CZX over the linearity range of 0.5-40 µg mL -1 . The regression equations were also computed (Y = 0.0287x + 0.0013, R 2 = 0.9998) for CZX and (Y = 0.0448x + 0.0099, R 2 = 0.998) for PAR.

Selectivity
Selectivity was checked by using 6 randomLy selected drug-free rats' plasma, (processed by the protein precipitation extraction procedure for CZX or liquid-liquid extraction for PAR) which was analyzed to determine the extent to which endogenous plasma components may contribute to the interference at the retention time of analytes and IS. There was no significant interference at the retention times of both CZX, PAR or IS from the six different batches of drug-free rat plasma used for analysis, as shown in Figures 2 and 3.

Carry-over
Carry-over was assessed by injecting blank samples after a high concentration sample (30 µg mL -1 ) of both PAR and CZX. Carry over in the blank sample did not exceed 20% of the lower limit of quantification and 5% for the internal standard.

Accuracy and precision (inter and intra-day)
For the evaluation of precision, the deviation of each concentration level from the nominal concentration should to be within ±15.0%. Similarly, the mean accuracy should not deviate by ± 15.0% of the nominal   Tables I and II.

Stability
To evaluate the stability of both PAR and CZX under different storage conditions, three aliquots of each low and high QC samples were stored in a deep freezer at −80 ± 2 °C for two weeks. After two weeks, the samples were processed along with precision and accuracy batches. Concentrations obtained were compared with nominal concentrations to determine the long-term stability of both PAR and CZX in rat plasma.
The short-term stability was determined by keeping three aliquots of unprocessed QC samples at ambient temperature for 6.0 h. After 6.0 h, the samples  were processed, analyzed and compared with nominal concentrations. The stability of QC samples was also studied after three freeze and thaw cycles. Three aliquots of unprocessed QC samples were stored at −80 ± 2°C and subjected to three freeze and thaw cycles. After the completion of the third cycle, the samples were processed, analyzed and the results were compared with nominal values and the results represented in Tables III  and IV.

Pharmacokinetic analysis
Non-compartmental pharmacokinetic analyses were performed using Excel (Microsoft, Redmond, WA, USA) add-in program, PK solver. The estimated parameters included area under the plasma concentration-time curve (AUC 0-t ), area under the plasma time-concentration curve from time zero to infinity (AUC 0-∞ ), maximum plasma concentration (C max ) and time to maximum plasma   The plasma concentration-time curves after IV administration at PAR (25 mg/kg) and CZX (20 mg/ kg) with either control or lycopene pretreatment (20mg/kg, ip) are shown in Figure 6.
The pharmacokinetic parameters of PAR and CZX indicated that pretreatment of rats with Lycopene at daily dosage of 20mg/kg for eight consecutive days resulted in a (20.374% & 45.997%, P < 0.05) reduction in the total clearance (cL), respectively, compared with the control group. Nevertheless, the difference in volume of distribution (V d ) was not significant between the two groups of CZX, where there was a significant increase (1.694 fold) in Vd of PAR compared to the control group.
The difference in C max and T max did not reach statistical significance in both PAR and CZX, but the AUC 0-t , AUC 0-inf, t1/2 were significantly increased by 1.245,1.295 and 2.09 folds in PAR and 1.631, 1.914 and 2.216 folds in CZX (P < 0.05), respectively, following pretreatment with Lycopene (P < 0.05).

Statistical analysis
The different parameters (AUC, C max , T max , V d , cL and t 1/2 ) of both PAR and CZX between control group FIGURE 4 -Chromatogram of paracetamol in plasma taken from a rat after IV administration and spiked with caffeine (80 µg/mL).

DISCUSSION
To determine the affecting dose of lycopene, many preliminary trials were carried out. Lycopene (10 mg/kg) was given IP to rats for six consecutive days followed by IV administration of PAR (25 mg/kg) or CZX (20mg/kg). Blood samples were collected from each group at different time intervals (0 -6 h). The results of this experiment were compared to that carried out using lycopene (20 mg/kg). Control group was carried out for each drug by pretreating with the vehicle instead of lycopene. Both experiments showed an increase in AUC 0-t , AUC 0-inf and a decrease in cL of both PAR and CZX when pretreated with lycopene but this effect was more significant in the rats pretreated with (20 mg/kg) IP lycopene dose.
This experiment was repeated but with pretreating the rats with lycopene 20 mg/kg for eight consecutive days instead of six then PAR or CZX was IV administered. This showed that the effect of lycopene was more significant when the duration of lycopene administration was increased.
A herbal or food drug interaction occurs when a particular food alters an enzyme activity leading to changes in drug metabolism by this enzyme (Saxena et al., 2008;Schmidt, Dalhoff, 2002).
Recent study showed the inhibitory effect of tomato juice on the metabolism of some substrates of CYP3A4 such as nifedipine, testosterone and midazolam due to the inhibitor effect of lycopene and other flavonoids on CYP3A4 (Sunaga et al., 2012). But this work showed the inhibitory effect of lycopene on pharmacokinetic of paracetamol and chlorzoxazone, which is mainly metabolized by CYP2E, due to the inhibitory effect of lycopene on CYP2E1.
There are three main metabolic pathways for PAR leading to the formation of toxic or non-toxic products and eventually excreted in urine (Veronese, McLean, 1991). In the third pathway, however, the intermediate product N-acetyl-p-benzoquinone imine (NAPQI) is toxic. NAPQI is primarily responsible for the toxic effects of PAR. Production of NAPQI is due primarily to two cytochrome P450 enzymes: CYP2E1 and CYP3A4 (Foye, Lemke, Williams, 2008).
According to experimental results, it can be deduced that pretreatment of rats with lycopene in a dose of 20 mg/kg lead to an increase in the AUC and a decrease in cL of both PAR and CZX and this could be a result of inhibiting CYP2E1. Because both PAR and CZX have low to intermediate hepatic extraction ratio ( (Coleman, 2006;Mehvar, Vuppugalla, 2006), therefore, their hepatic clearance depends mainly on the intrinsic clearance (cL int ) ( (Bryant, Knights, 2014;Davies, Morris, 1993;Lin, Lu, 1997). So, according to equation (1), AUC increases when clearance (or intrinsic clearance in case of low clearance drugs) decreases (Lin, Lu, 1997): When the rats were pretreated with Lycopene, the intrinsic and hepatic clearances decreased and AUC 0-t , AUC 0-inf of both PAR and CZX increased, whereas Lycopene had no significant effect on C max as both were administered intravenously.
The elimination half-life t1/2 increased when the volume of distribution increased, and the total clearance decreased (Lin, Lu, 1997) as shown in Equation (2), and this was consistent with the experimental results that showed an increase of the elimination half-life of both PAR and CZX more than 2 folds: t 1/2 =0.693×Vd/Cl (2) Using FDA rodent-human dose conversion based on 60 kg average human weight (Nair, Jacob, 2016), should consume 193 mg of lycopene per day (20*60/6.2 = 193 mg) to show the difference of the pharmacokinetic parameters for the studied drugs. 6.2 is the ratio that compares the body surface area of rats with humans.

CONCLUSION
In conclusion, the pharmacokinetic behavior of paracetamol and chlorzoxazone was remarkably changed in rats after intra-peritoneal administration of lycopene (20mg/kg), and thus a human being should consume 193 mg of lycopene to show the difference of pharmacokinetic parameters for the studied drugs. Hence, it could be presumed that the increase of AUC and the decrease of cL might be due to the inhibitory effect of lycopene on CYP2E1, the main enzyme in the metabolism of paracetamol and chlorzoxazone. The results of our research might contribute to guide future clinical studies.

NOTES
The authors declare no competing conflict of interest and no financial contributions were received.