Modulation On The P-glycoprotein In The Intestine By Concerted Pplication Of Radix Glycyrrhizae And Its Antagonisms As Well As Influence Of Incompatibility Pairs On Drug Intestinal Segment Delivery System

Background&ObjectiveP-Glycoprotein (P-gp) is an ATP-dependent plasma membrane glycoprotein of about170kDa that belongs to the superfamily of ATP-binding cassette (ABC) transporters. It can actively pump drugs out of cells, thus reducing the oral bioavailability of drugs. In addition to being expressed in tumor cells, P-gp is also expressed in various normal tissues including liver, kidney, adrenal glands, brain, testis and the intestinal 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 system can be improved by inhibiting the function of P-gp in the intestinal membrane and, as a result, the oral bioavailability of a wide range of drugs 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. Eighteen incompatible medicaments is one kind of taboo which has been accepted for thousands of years in Chinese medical circles. It has reported that Radix Glycyrrhizae exerted different modulation on Cytochrome P450with or without Euphorbia pekinensis, Euphorbia kansui, and Flos Genkwa. It is interesting that P-gp and Cytochrome P450isozyme have some similar substrates, such as rifampicin, lidocaine, digoxin and cyclosporine, etc. Therefore, these incompatible medicaments maybe can produce different effects on intestinal P-gp, which may be one of reasons why combination of incompatible medicaments could be able to generate toxity. Literature retrieval manifested that, so far, there was no reported about the relationship between intestinal P-gp expression as well as function and the combined oral administration of Eighteen incompatible medicaments, and thus, this study is academicly meaningful.Content&ResultsThe permeability of R123or CF via the intestinal membranes was evaluated by an in vitro diffusion chamber system after the intestinal membranes were isolated from the intestine in rat, after oral administration of saline, verapamil and decoctions. And the concentration of R123or CF in the receptor was determined by the fluorospectrophotometry. The serosal-to-mucosal transport (S-M) of R123was much greater than its mucosal-to-serosal (M-S) transport (about quattuor), indicating that R123gives first palce to secretory transport in the intestinal membranes. CF, transported by a passive diffusion in both absorptive and secretory direction, showed no apparent permeability (Papp) difference between M-S and S-M (ER=1.96). These results were compatible with the published reports, so it is feasible to carry out in vitro diffusion chamber experiment under our laboratory condition.The permeability of R123or CF via Wistar rat intestinal membranes was evaluated by in vitro diffusion chamber system after oral administration of different decoctions and0.9%sodium chloride (20mL·kg-1) and verapamil for1week. In low concentration of Radix Glycyrrhizae group, the absorptive directed transport of R123was no significantly difference compared with control group, and the same situation was found on the secretory transport of R123. Meanwhile, the other three kinds of decoctions can decrease the permeability of secretory directed transport and increase the permeability of absorptive directed transport, and the Papp of R123between the different decoctions of Radix Glycyrrhizae combined with other incompatibility pairs has no statistics difference. Meanwhile, Radix Glycyrrhizae had no effect on transport of CF across the intestinal tissues, though the other groups can decrease the permeability of CF, as compared with control group.A new, rapid and sensitive method was developed for the quantifying of rhodamine123(R123) in rat plasma using liquid chromatography tandem mass spectrometry (LC-MS/MS). Rhodamine6G (R6G) was used as the internal standard (IS). R123and IS were extracted from aliquots of plasma with ethyl acetate and dichloromethane (4:1) as the solvent. Chromatographic separation was performed using a Zorbax Eclipse Plus C18column. The mobile phase was composed of A: ammonium formate-formic acid buffer containing5mM ammonium formate and0.1%formic acid and B:methanol (A:B,5:95, v/v). To quantify R123and IS respectively, multiple reaction monitoring (MRM) transition of m/z345.2→285.2and m/z443.3→415.2were performed. The analysis lasted for4min, in the positive mode, the results of which were presented as a linear calibration curve over the concentration range of1-200ng/ml and sampling volume of2μl. The lowest limit of quantification (LLOQ) reached1ng/ml. The intra and inter-day precision were6.7%and9.2%for the low quality control (QC) sample (2ng/ml), respectively. Intra and inter-day precision of the medium QC sample (100ng/ml) were both less than3.4%. Intra and inter-day precision of high QC sample (150ng/ml) were both less than2.1%, while the intra and inter-day relative errors ranged between-7.4%and9.1%for the above three QC concentration levels. The LC-MS/MS method proved to be simple, accurate, reliable and with a shorter running time and has been successfully applied to an absorption experiment in the rat.Rat plasma concentrations and pharmacokinetic parameters of R123and CF were examined by in situ closed loop method after oral administration different decoctions and0.9%sodium chloride (20mL·kg-1) for1week. For R123, It was found that the largest plasma concentration (Cmax), area under curve (AUC) and bioavailability (F) of Radix Glycyrrhizae group had no statistics difference compaired with control group. However, The other three groups significantly enhanced the intestinal absorption of R123in rats (P<0.01,compared with control group), while the groups administration Radix Glycyrrhizae combined with Euphorbia pekinensis, have been close to the results with administration Euphorbia pekinensis, alone. The pharmacokinetic parameters of R123between the different decoctions of Radix Glycyrrhizae combined with Euphorbia pekinensis, has no statistics difference (P>0.05). For CF, Euphorbia pekinensis,(0.5g·mL-1) didn’t change the absorption of CF in intestinal tract. After oral administration of decoction of Euphorbia pekinensis and combine decoction of the two herbs, P-gp expression levels decreased, whereas decoction of liquorice root group had no significant effect compared with negative control group (p<0.05).Conclusion&DiscussionBased on the results from the above In vitro and In situ experiments, it can deduce that Euphorbia pekinensis can notablely inhibit the P-gp expression in the intestine. So in clinical, Euphorbia pekinensis may can increase the anticancer curative effect by compatibling resists tumour medicine which causes resistance easily, or manufacturing tumour medicine resistance reversing agent; we also can attempt to add Euphorbia pekinensis to medicine appropriately. Some compositions in Euphorbia pekinensis may inhibit P-gp function, and some others strengthen the tight junction between cells in the intestinal membrane to decrease permeability of CF, which explained the reson of these results.Traditional Chinese medicine has a kind of statement:Nine prescriptions contain Radix Glycyrrhizae in ten prescriptions; and for most cases, no Radix Glycyrrhizae, no prescription can be formed. Radix Glycyrrhizae compatibility with other drugs, can enhance the curative effect. Our experiment indicated Radix Glycyrrhizae may slightly inhibit P-gp function in the intestinal membrane, and change permeability of intestinal membrane, thereby increasing intestinal absorption of some drugs. Hence, our result provided a very good direction for explaining Radix Glycyrrhizae mechanism of "Mediate all medicine, strengthen a curative effect", in some degree. In future, we may try to investigate the effects of Radix Glycyrrhizae on all kinds of drug transporter in the intestinal membrane, as to disclose the possible mechanism of Radix Glycyrrhizae on mediating the transport of other drugs via intestinal membrane.Oral administration of decoctions of Radix Glycyrrhizae combined with Euphorbia pekinensis can enhance the inhibitory action to P-gp, compared with that of decoctions of Euphorbia pekinensis alone. However, the effect on P-gp between the different decoctions of Radix Glycyrrhizae combined with Euphorbia pekinensis has no statistics difference. It manifested that the disparity of HPLC for the extracts of Radix Glycyrrhizae with Euphorbia pekinensis at different decoctions, had no influence to intestinal P-gp of this different physic liquor. In conclusion, the reason why combination of Radix Glycyrrhizae and Euphorbia pekinensis can cause toxigenicity is the inhibitory action to P-gp in the intestine, resulting in the absorption enhancement of toxiferous compositions from Euphorbia pekinensis or from Radix Glycyrrhizae, which are p-gp substrates. Thinking from another aspect, it also is possible that Euphorbia pekinensis increase absorption of Radix Glycyrrhizae’s toxiferous ingredient (P-gp substrate).As a result of time and energy, the existence is insufficient of this research. For instance, we only analyzed the effects of Radix Glycyrrhizae and Euphorbia pekinensis on the jejunum intestinal P-gp expression, hadn’t evaluate the other intestinal mucosa; we adopted7d of intragastric administration, but hadn’t make the animal experiment of different intragastric administration time; we only made quantitive analysis, but hadn’t make qualitative analysis, etc. These disappointments hoped to perform in future research.