| Background&ObjectiveThe formula of paclitaxel (commodity name:Taxol) is C47H51NO14, molecular weight of853.92, paclitaxel is a kind of white crystal, melting point of213-216℃, extracted from the bark of yew ormosia genera (Taxus) plants, is a pseudoalkaloid with a diterpenoid structure cytotoxic antitumor medicine. Paclitaxel acts as an antineoplastic agent due to its inhibitory effect of cellular growth,hyperstabilising the microtubule by binding to tubulin, blocking the cell replication in the late G2mitotic phase of the cell cycle, but will not affect the production of genetic material in the tumor cells. It has been used to treat a broad range of tumor types, including breast, advanced ovarian, non-small-cell lung and colon.This unique antitumor mechanism of paclitaxel, make it become a hot research all over the world. The commercial formulations of paclitaxel is administered to patients via intravenous injection(i.e. Taxol, Paxene),has been formulated with a mixture of Cremophor EL (polyoxyethylated castor oil) and anhydrous ethanol (1:1, w/w) as solvent. However, Cremophor EL is associated with hypersensitivity reactions and causes leakage of plasticisers from polyvinyl chloride (PVC) infusion bags and polyethylene tubing. In order to minimise these drawbacks,a number of efforts searching and optimal paclitaxel formulation have been performed.Compared to injection preparation, therefore, more safe and convenient paclitaxel oral preparation has become a research hotspot in recent years. Major mechanisms that can explain low oral bioavailability and plasma concentration of paclitaxel,include poor water solubility, extensive metabolism via cytochrome P-450in both liver and small intestine, and efflux from epithelial cell of small intestine by P-gp.In order to overcome these problems and improve the oral bioavailability of anticancer drugs, the implementation of a series of methods. P-glycoprotein inhibitors have been proposed that inhibition of P-gp function and/or expression might be a most popular and useful strategy for developing an oral formulation.P-Glycoprotein (P-gp) is an ATP-dependent plasma membrane glycoprotein about170kDa that belongs to the superfamily of ATP-binding cassette (ABC)transporters. It can actively efflux drugs out of cells, thus reducing the oral bioavailability of drugs. In addition to being expressedin tumor cells, P-gp is also expressed in various normal tissuesincluding liver, kidney, adrenal glands, brain, testis and theintestinal brush border membranes. It has been demonstrated that the intestinal P-gp can be an active secretion system or an absorption barrier by transporting some drugs from the intestinal cells into the lumen. Therefore, intestinal absorption of drugs that are secreted by a P-gp-mediated efflux systemcan be improved by inhibiting the function of P-gp in the intestinal membrane and, as a result, the oral bioavailability of a wide range ofdrugs may be increased. It has been confirmed that many drugs are inhibitors of P-gp, such as verapamil, cyclosporine A, quinidine, some kinds of surfactant and so on.Now there have been three generations of P-gp inhibitors and the other inhibitors.But the development is not yet mature, there are various restrictions, such as verapamil, a recognized P-gp inhibitor, its adverse reaction restricts its clinical use. This subject is to study traditional Chinese medicine extract as P-gp inhibitors, overcome and share the adverse reaction of paclitaxel.We found Euphorbia pekinensis and another inhibitors procyanidins which could inhibit the activity of P-gp.Thereby, I conclude Euphorbia pekinensis and procyanidins which can stimulates oral absorbability of paclitaxel based on the in vitro experiment.To evaluate paclitaxel combinationwith inhibitors after Ussing Chamber experiment by different intestinal segments through the correlation of in vivo. In vivo experiments, we established the LC-MS/MS method to detect drug concentration of paclitaxel in rat plasma, expectations of paclitaxel in vivo absorption through the in vivo environment for testing, to verify the correlation of experiment in vitro and in vivo tests.Further validation of Euphorbia pekinensis, procyanidins can improve oral bioavailability.Content&ResultsFirst, we establish the HPLC method for the determination of paclitaxel in the transdermal solution obtained from the intestinal transport study. There is no need to have Special Handling for the transdermal solution, it can be injected into HPLC after high speed centrifugation on15000r/m. Paclitaxel was analyzed using a Ecosil C18(250mm×4.6mm,5μm) with a flow rate of0.8mL/min at30℃. The mobile phase consisted of methanol-water (70:30, v/v), detection wavelength were272nm in10min.To establish a simple and rapid LC-MS/MS method for the determination of paclitaxel in rat plasma.The100μL of sample plasma was extracted with mixed solvent (ethyl acetate:dichloromethane:acetonitrile=4:1:1) after addition of100ng/mL docetaxel(internal standard), and analyzed by Agilent1260-6460Triple Quadrupole LC-MS/MS system. Detection was performed with multiple reactions monitoring (MRM) using electrospray ionization (ESI) and adding Na ion mode. The precursor/product ion transitions of paclitaxel and docetaxel (internal standard) were monitored at m/z876.29â†'308.1and830.34â†'549.3, respectively. The analysis time was only2min in positive mode; the calibration curve was linear in the concentration range of0.55-550ng/mL. The lowest limit of quantification (LLOQ) reached0.55ng/mL. The extraction recoveries were79.32%,77.54%and78.64%for three QC concentration levels (1.1,110,550ng/mL).The permeability of paclitaxel via different intestinal membranes was evaluated by an in vitro diffusion chamber systemat different concentrations.Data in all experiments were presented as the mean±SD. The groups of different direction and different extents of Papp comparing with factorial design analysis of variance, multiple comparision between the different groups by using SNK test.Differences were considered to be significant when P<0.05.According to the results, the mucosal-to-serosal (M-S) of paclitaxel was increased and its serosal-to-mucosal transport (S-M) transport was decreased with co-administration of P-gp inhibitors across jejunum and ileum transport.However, no action of four groups at test concentration was found on the transport of paclitaxel across colon.The deference in four groups by colon mucosa M-S direction and S-M direction is not significant.Indicates that Euphorbia pekinensis, procyanidins and verapamil can inhibit P-gp,the effect of the permeability of paclitaxel only exists in jejunum and ileum, and have no effect on the colon.Papp between different groups had significant difference (F=15.780, P=0.000), combined with the multiple comparison results, Euphorbia pekinensis group, procyanidins group and control group, there is significant difference (P=0.000,0.001). Euphorbia pekinensis, procyanidins than with verapamil group, there are significant differences (P=0.000). There are significant differences between different sections (F=53.326, P=0.000), combined with the multiple comparison results, there was no significant difference between jejunum and ileum (P=0.142). Colon and also exist significant differences between the two groups (P=0.000). There are significant differences between different direction (F=52.006, P=0.000).Plasma concentrations and pharmacokinetic parametersof paclitaxel in rat were examined by in vivo closed loop method with determination of P-gp inhibitor groups and blank group. Data in all experiments were presented as the mean±SD, using SPSS19.0statistical package software for statistical analysis.Caculated means of Cmax, Tmax, AUC0-720min, F after in vivo experiment. Statistical differences were tested by one-way ANOVA. Differences were considered to be significant when P<0.05.Compare between two groups by SNK method or Dunnett T3method (not neat of variance).It was found that the largest plasma concentration (Cmax), area under curve (AUC) of paclitaxel and biaovailablity after oral administration different P-gp inhibitor sand0.9%sodium chloride(20mL/kg) had statistics difference compaired with blank group when analyzed by one-way ANOVA.The Cmax, Tmax, AUC0-720min of the three P-gp inhibitor groups different from blank group, Euphorbia pekinensis group was higher than that of procyanidins and verapamil group, the results showed that P-gp inhibitors had statistics difference compaired with blank group.Paclitaxel exhibits low(<5%) oral bioavailability. The average absolute bioavailability of blank group (AB%) after oral paclitaxel is2.81%, the average AB%of Euphorbia pekinensis group is7.63%,2.7times that of blank group, the average AB%of verapamil and procyanidins group was about1.5times that of blank group, one of the highest average RB%was Euphorbia pekinensis group(271.75%).The C-max of blank group after oral paclitaxel20mg/kg was95.27ng/mL,smaller than any other P-gp inhibitors groups. The area under the plasma concentration-time curve (AUC) and C-max by the oral paclitaxel(20mg/kg) in P-gp inhibitors groups, increased significantly,1to3times that of blank group,but Euphorbia pekinensis group mostly, procyanidins as same as verapamil group. We believe that P-gp inhibitors can significantly improve the bioavailability of paclitaxel, which inhibits the intestinal mucosa of P-gp influx pump and the metabolism ofsmall intestine P450cell. Obviously, as far as possible to share, paclitaxel oral preparations co-administration P-gp inhibitors can be much convenient than intravenous injection directly.Conclusion&DiscussionIn vitro Ussing Chamber experiment results, which can be concluded that Euphorbia pekinensis, procyanidins and verapamil can inhibit the expression of P-gp,and Euphorbia pekinensis inhibitory effect is more apparent than procyanidins. In clinic, Euphorbia pekinensis and procyanidins have antitumor efficacy, can try to make tumor drug resistance reversal agent, to increase the anticancer curative effect.Also can try in the preparation of drugs, appropriate to combinated with Euphorbia pekinensis or procyanidins, which can improve P-gp substrate drug absorption. From the results of M-S direction and S-M direction, there is significant difference in jejunum and ileum except the colon. Euphorbia pekinensis and verapamil increase drug absorption direction, reduce secrete direction,but the procyanidins group only have effect on the jejunum segment.In vivo experiment results show that the paclitaxel of the average of AUC0-72Omin and Cmax, in the P-gp inhibitor groups was significantly greater than the blank group, and Euphorbia pekinensis group than that of procyanidins and verapamil group, the results showed that co-administrated P-gp inhibitors could actually increase the oral bioavailability of paclitaxel. |
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