Effects of Quercetin on the Pharmacokinetics of Etoposide after Oral or Intravenous Administration of Etoposide in Rats

Materials and Methods

Materials. Etoposide, quercetin and podophyllotoxin (an internal standard for high performance liquid chromatography, HPLC analysis of etoposide) were purchased from Sigma-Aldrich Co. (St. Louis, MO, USA). Injectable etoposide (20 mg/ml) was purchased from Boryung Chemical Co. (Seoul, Republic of Korea). Methanol and tert-butylmethylether were acquired from the Merck Co. (Darmstadt, Germany). The other chemicals were of reagent grade or HPLC grade.

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Figure 1.

Chromatogram of blank rat plasma (A) and plasma spiked with etoposide and the internal standard, podophyllotoxin (B).

Animal experiments. Seven- to 8-week-old male Sprague-Dawley rats weighing 270-300 g were purchased from Dae Han Laboratory Animal Research Co. (Choongbuk, Republic of Korea) and given free access to commercial rat chow (No. 322-7-1, Superfeed Co., Gangwon, Republic of Korea) and tap water ad libitum. The animals were housed, four or five per cage, in laminar flow cages maintained at 22±2°C and 50-60% relative humidity, under a 12:12 h light-dark cycle. The experiments began after acclimation to these conditions for at least one week. The protocols of the animal studies were approved by Animal Care Committee of Chosun University (Gwangju, Republic of Korea).

Drug administration. The rats were divided into four groups (n=6) for each of the oral and i.v. etoposide administration routes (9 mg/kg for oral; 3 mg/kg for intravenous) with 0 (control), 1, 5 or 15 mg/kg of orally administered quercetin. The rats were fasted for at least 24 h prior to beginning the experiments and had free access to tap water. Each animal was lightly anaesthetized with ether, and the femoral artery and vein were cannulated using polyethylene tubing (SP45; i.d., 0.58 mm, o.d., 0.96 mm; Natsume Seisakusho Co. Ltd., Tokyo, Japan) for blood sampling and i.v. administration. Etoposide (20 mg/ml injectable solution diluted in 0.9% NaCl-injectable solution) at a dose of 3 mg/kg was injected (total injection volume, 1.5 ml/kg) over 1 min via the femoral vein 30 min after the administration of 0 (control), 1, 5 or 15 mg/kg of quercetin (dissolved in distilled water, homogenized at 36°C for 30 min; total oral volume, 3.0 ml/kg) through a feeding tube. Blood samples (0.2 ml) were collected into heparinized tubes via the femoral artery at 0 (as a control), 0.017 (at the end of injection), 0.1, 0.25, 0.5, 0.75, 1, 2, 4, 6 and 8 h the after the intravenous injection of etoposide. The blood samples were immediately centrifuged for 5 min at 13,000 rpm and a 0.1 ml aliquot of plasma was stored in a freezer at -40°C until HPLC analysis. Etoposide (the same solution as used for i.v. administration) was administered orally at a dose of 9 mg/kg (total oral volume, 3.0 ml/kg) without or with 1, 5 or 15 mg/kg of orally administered quercetin. Blood samples (0.2 ml) were collected into heparinized tubes via the femoral artery at 0 (as a control), 0.1, 0.25, 0.5, 0.75, 1, 2, 4, 6, 8 and 10 h after the oral administration of etoposide. The other procedures were similar to those in the i.v. studies.

HPLC analysis. The plasma concentrations of etoposide were determined by the HPLC assay reported by Liliemark et al. (22) and Manouilov et al. (23) with slight modification. Briefly, 50 μl of podophyllotoxin (50 ng/ml dissolved in methanol) and 1.2 ml of tert-butylmethylether were mixed with a 0.1 ml aliquot of the plasma sample in a 2.0 ml polypropylene microtube (Axygen Scientific Co., Calif., USA). The resulting mixture was mixed vigorously with a vortex-mixer (Scientific Industries Co., NY, USA) for 1 min and centrifuged at 13,000 rpm for 10 min with a high-speed micro centrifuge (Hitachi Co., Tokyo, Japan). A 1.1 ml aliquot of the upper layer was transferred to another clean microtube and evaporated under nitrogen gas at 38°C in an MG 2100 Eyela dry thermo bath (Rikakikai Co., Tokyo, Japan). The residue was dissolved in 0.2 ml of 50% methanol in deionized water, and a 50 μl aliquot of the solution was injected into the HPLC system.

The HPLC was equipped with a Waters 1515 isocratic HPLC Pump, a Waters 717 plus autosampler and a WatersTM 474 scanning fluorescence detector (Waters Co., Milford, MA, USA). Data were acquired and processed with breeze™ Software (Version 3.2) (Waters Co.). Chromatographic separations were achieved using a Symmetry® C₁₈ column (4.6×150 mm, 5 μm, Waters Co.) and a μBondapak™ C₁₈ HPLC Precolumn (10 μm, Waters Co.). The mobile phase consisted of methanol-deionized water-acetic acid (50:50:0.5, v/v/v) and run at a flow-rate of 1.0 ml/min. Chromatography was performed at 30°C, which was set by a HPLC column temperature controller (Phenomenex Inc., CA, USA). The fluorescence detector was operated at an excitation wavelength of 230 nm with an emission wavelength of 330 nm. Podophyllotoxin and etoposide eluted with retention times of 5.4 and 11.1 min (Figure 1), respectively. The presence of quercetin did not interfere with the detection of etoposide and podophyllotoxin. The detection limit of etoposide in plasma was 10 ng/ml. The intra- and inter-day variation coefficients of etoposide in the plasma samples were below 13.6% .

Pharmacokinetic analysis. The plasma concentration data were analyzed by noncompartmental methods using WinNonlin software version 4.1 (Pharsight Corporation, Mountain View, CA, USA). The elimination rate constant (K_el) was calculated by log-linear regression of etoposide concentration data during the elimination phase and the terminal half-life (t_1/2) was calculated as 0.693/K_el. The peak concentration (C_max) of etoposide in the plasma and the time to reach C_max (t_max) were obtained by visual inspection of the data from the concentration-time curve. The area under the plasma concentration-time curve (AUC) from time zero to the time of last measured concentration (C_last) was calculated by the linear trapezoidal rule. The AUC zero to infinite (AUC_0-∞) was obtained by adding AUC_0-t and the extrapolated area determined by C_last/K_el. The total body clearances of i.v. (CL_t) and oral (CL/F) etoposide were calculated from the quotient of the dose (D) and the AUC_0-∞ of the i.v. and oral routes. The absolute bioavailability (AB) was calculated by AUC_oral/AUC_i.v. × Dose_i.v./Dose_oral × 100. The relative bioavailability (RB) was calculated by AUC_coadmin/AUC_control × 100.

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Figure 2.

Mean plasma concentration-time profiles of etoposide after oral administration of etoposide at a dose of 9 mg/kg to rats in the absence or presence of quercetin at doses of 1, 5 or 15 mg/kg (Mean±S.D., n=6). Bars represent the standard deviation. (●) without quercetin or with (▿) 1 mg/kg, (○) 5 mg/kg and (▾) 15 mg/kg quercetin.

Statistical analysis. The pharmacokinetic parameters were compared using a one-way ANOVA, followed by a posteriori testing with the Dunnett correction. Differences were considered significant at a level of p<0.05. All the data are expressed in terms of mean±S.D.