Paclitaxel (PTX) is one of the most frequently used anticancer agent for treating refractory ovarian cancer, metastatic breast cancer and non-small cell lung cancer. However, its oral administration is impeded by very low bioavailability (<5%) due to the Pglycopprotein (P-gp) efflux pump effect. This study investigated
Paclitaxel (PTX) is an anticancer agent, which has a significant activity against a wide variety of tumors, including refractory ovarian cancer, metastatic breast cancer, and non-small cell lung cancer (Wang
The currently marketed PTX (Taxol [Bristol-Myers Squibb, New York, USA] 6 mg/mL PTX in a 1:1 Cremophor EL [Sigma-Aldrich, Sigma-Aldrich, St. Louis, MO, USA]/ethanol mixture) is dissolved in a 0.9% NaCl solution for clinical use and administered IV infusion (Colin
Other limitation would be the presence P-glycoprotein (Pgp) efflux pump preventing from the absorption of PTX in the intestine (Sparreboom
Above all, it is considered for P-gp to be clinically more significant than the other transporters, therefore, is viewed as a therapeutic target for MDR cancer cells to respond to anticancer drugs (Min
Paclitaxel and 4-Hydroxybenzoic acid n-hexyl ester (internal standard) were purchased from Samyang Genex (Daejeon, Korea) and Tokyo Kassei Kogyo (Tokyo, Japan), respectively. Antibiotic-antimycotic agent, 0.25% trypsin-1 mM EDTA and RPMI 1640 medium were supplied by GIBCO (Rockville, MD, USA). Fetal bovine serum and Tris, N-(2-hydroxyethyl) peperazine-N′-2-ethanesulfonic acid (HEPES) were obtained from HyClone (Logan, UT, USA) and USB (Cleveland, OH, USA), respectively. Daunomycin was supplied by EMD Chemicals (San Diego, CA, USA). Diethyl ether and Phosphoric acid were purchased from Samchun pure chemical (Pyeongtaek, Korea) and Showa Chemical (Tokyo, Japan), respectively. Heparin sodium injection was obtained from Hanlim Pharm (Yongin, Korea). All other agents were purchased from Sigma-Aldrich (St. Louis, MO, USA). Human breast cancer cell line (MCF-7) and P-gp overexpressed human breast cancer cell line (MCF-7/ADR) were kindly gifted by Dr. Marilyn E. Morris (State University of New York at Buffalo, USA).
To determine the effect of adamantyl derivatives on P-gp function, cytotoxicity assay was carried out in MCF-7/ADR cells (P-gp overexpressed human breast cancer cell line). The tumor cells were grown in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mM L-glutamine, 10 mM HEPES, 24 mM NaHCO3, and 1% antibiotic-antimycotic agent, and maintained at 37°C in a humidified 5% CO2 atmosphere. MCF-7/ADR cells were seeded in 96-well plates at 5,500 cells per well and incubated for 24 h. Daunomycin (DNM) was diluted with cell culture media and treated on the plates to achieve the final concentrations of 0, 0.05, 0.1, 0.5, 1, 5, 10, 25, 50 and 100 μM with or without 5 μM of adamantly derivatives, AC-603 and AC-786 or Verapamil (VER). VER is one of the first generation of P-gp inhibitors. DNM containing 0.4% DMSO and DNM with 5 μM VER were used as negative and positive control, respectively. Each DNM concentration was assayed in three wells and in three independent experiments. After 2 h incubation, cells were washed and replaced with 200 μL of fresh culture media and then incubated for additional 72 h. After 72 h incubation, 50% TCA was treated on the wells to affix cells on the bottom of plates and then removed. Sulforhodamine B (SRB, 0.4%) was used to dye cell membrane and the cells were washed with 1% acetic acid. Dyed cells were treated with 10 mM Tris-base and dissolved on the orbital shaker for about 80 minutes at 163–167 rpm. The cell cytotoxicity was assessed using ELISA reader supplied by Molecular Devices. After placing plates on the ELISA reader, the light intensity of remained cells was measured at a wavelength of 515 nm. IC50 values were calculated with Table curve Windows program.
Healthy male Sprague-Dawley rats (6–7 weeks old), weighing 240–295 g, were purchased from Orient Bio Inc (Seongnam, Korea). All animal procedures for animal studies were approved by Institutional Animal Care and Use Committee (IACUC) at Ewha Womans University, Seoul, Korea. They were kept in an environmentally controlled breeding room for at least five days before starting the experiments.
The rats were given surgical procedure for blood sampling one day before the experiments. The common carotid artery of the rat was surgically cannulated using polyethylene tubing (PE-60) for blood sampling. After surgery, rats were housed individually to allow them to recover. Before the experiments, rats were fasted overnight with free access to water. The rats were assigned randomly to eight groups. PTX was dissolved in Cremophor® EL and anhydrous ethanol (1/1, v/v), followed by dilution of the drug solution with isotonic saline (1/2, v/v) before use. AC-603, AC-786 and VER were also dissolved using Taxol® formulation. Two groups of rats were intravenously (IV) or orally (PO) administered with PTX, 2 mg/kg or 25 mg/kg, respectively, as control. To the other six groups, 25 mg/kg of PTX was orally co-administered with VER (0.5, 5 mg/kg), AC-603 (0.5, 5 mg/kg), or AC-786 (0.5, 5 mg/kg). For the IV injection group, blood samples (0.2 mL) were taken from the common carotid artery at 0, 0.033, 0.083, 0.25, 0.5, 1, 2, 3, 4, 6, 10 and 24 h after the IV injection. For the oral administration groups, 0.2 mL of blood samples were taken at 0, 0.25, 0.5, 1, 2, 3, 4, 6, 8, 10 and 24 h after the oral administration. The same volume of heparinized 0.9% NaCl solution (100 IU/mL, 0.2 mL) was administered to keep the blood volume and to prevent blood clotting. Plasma samples (100 μL) were obtained after centrifugation for 15 min at 13,000 rpm immediately after collection and stored at −20°C until used for HPLC analysis.
The analysis was carried out on an Agilent HP1100 series system. The chromatographic separation was performed on a Capcell-pak C18 MG120 column (3.0×250 mm, 5 μm, Shiseido, Tokyo, Japan). The mobile phase was composed of acetonitrile and 0.1% phosphoric acid (1/1, v/v) at a flow rate of 0.5 mL/min. The UV-detector wavelength was set at 227 nm. A volume of 5 μL of n-hexyl 4-hydroxybenzoate (internal standard, IS) was added to 50 μL of each plasma sample and then 45 μL of acetonitrile were added to precipitate the plasma protein. The mixture was vortexed for 2 min, followed by centrifugation at 13,000 rpm for 15 min to remove all the precipitated material. The auto-sampler was programmed to inject 40 μL of the sample solution into the HPLC system. An analytical method validation for PTX was performed according to the FDA guidance for Industry (U. S. Department of Health and Human Services, Food and Drug Administration, Center for Drug Evaluation and Research and Center for Veterinary Medicine, 2001).
The pharmacokinetic parameters of PTX after a single intravenous or oral administration to rats were investigated by non-compartmental analysis using WinNonlin® Professional version 5.2 software (Pharsight Corporation, Mountain View, CA, USA). The pharmacokinetic parameters estimated from the plasma concentration-time profiles were as follows: the area under the plasma concentration-time curve from 0 h to the last sampling time (AUClast), the area under the plasma concentration-time curve from 0 h to infinity (AUCinf), elimination half-life (t1/2), elimination rate constant (Ke), apparent volume of distribution (Vd), total clearance (Clt), apparent volume of distribution following oral administration (Vd/F) and oral clearance (Clt/F). The maximum plasma concentration (Cmax) and the time required to reach Cmax (Tmax) were measured directly from the concentration-time data.
The absolute bioavailability (AB, %) of PTX was calculated by the following equation:
The relative bioavailability (RB, %) of PTX was calculated by the following equation:
, where AUCoral control is the AUC obtained from the oral administration of PTX alone, and AUCco-admin is the AUC obtained from the oral co-administration of PTX with VER, AC-603, or AC-786.
All the means are presented with their standard deviation (mean ± SD). All data acquired from this study used Graph-Pad Instat 3.0 statistics program (GraphPad Software, Inc., La Jolla, CA, USA) for Windows to calculate the descriptive statistics. To determine any significant difference between the control and PTX treated with compounds for each variable, one-way ANOVA was executed and SNK (Student-Newman-Keuls) was performed for post verification. The
The peaks of PTX and IS were well separated from endogenous interfering peaks in rat plasma, confirming the specificity of the method. The retention times of PTX and IS were approximately 13.7 and 23.4 min, respectively. The analytical method exhibited excellent linearity over the concentration range of 0.02–10.0 μg/mL with a high correlation coefficient (r=0.9999).
The intra- and inter-day precision and accuracy acceptance criteria for each QC sample (0.1, 0.5 and 5 μg/mL) was ≤15% and all the values successfully met the criteria. The LLOQ (the signal-to-noise ratio >5) was determined to be 0.02 μg/mL. All the values for precision and accuracy of LLOQ were within the acceptable range (± 20%). The mean relative extraction recoveries of PTX were in the range of 85–115% at the concentrations of 0.1, 1 and 5 μg/mL. The mean relative recovery of IS was 96.1 ± 0.10% at 5 μg/mL which was used for the analysis. As expected, the efficiency of PTX and IS extraction from rat plasma was coherent, regardless of concentration.
The mean plasma concentration-time curves for PTX obtained after oral co-administration of PTX (25 mg/kg) with two different doses of VER (0.5 mg/kg and 5 mg/kg, Fig. 2), AC-603 (0.5 mg/kg and 5 mg/kg, Fig. 3) and AC-786 (0.5 mg/kg and 5 mg/kg, Fig. 4) are shown in each Figure. In addition, all of them were compared with the mean plasma concentration-time curve obtained after oral administration of PTX (25 mg/kg) alone (control). All the relevant pharmacokinetic parameters for PTX were summarized in Table 2. The data were used to calculate the absolute bioavailability (AB, %) of PTX after oral administration in the absence or presence of compounds (Table 2).
After oral co-administration of PTX with each compound including a positive control, VER, the mean area under the plasma concentration-time curve (AUClast) were significantly increased as compared with control group (
The main purpose of this study was to improve oral bioavailability of PTX by intestinal P-gp inhibition using adamantyl derivatives (Fig. 1). In previous study, AC-603 and AC-786 were found to be two most potent P-gp inhibitors among twenty adamantyl derivatives. The dose-dependency of P-gp inhibitory function was not studied
The pharmacokinetic studies were carried out using two administration routes (IV and PO) to compare the effects of compounds (VER, AC-603 and AC-786) on PTX bioavailability (Fig. 2
In conclusion, adamantyl derivatives, AC-603 and AC-786, significantly improved oral absorption of PTX, resulting in increased bioavailability via the inhibition of intestinal P-gp. These results suggest that the co-administration of AC-603 and AC-786 could provide a therapeutic benefit on the oral delivery of P-gp substrate drugs.
This work was supported by the Research Grant funded by Ewha Womans University.