甘草与反药配伍对P-糖蛋白影响的相关研究及配伍禁忌对口服药物肠道吸收机制的探讨
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摘要
背景和目的
P-糖蛋白(P-glycoprotein, P-gp)是属于ABC (ATP-binding cassette)转运体超家族的能量依赖性膜蛋白,分子量为170kDa,可发挥外排泵作用,将细胞内的化合物逆浓度梯度转运至胞外,从而降低细胞内的药物浓度。P-gp除在肿瘤细胞中高度表达外,在正常机体组织和器官如肝脏、肾脏、胎盘、大脑、睾丸及肠刷状缘膜等部位均有高水平的表达。存在于肠上皮细胞刷状缘膜中的P-gp能将药物从浆膜侧泵回至粘膜侧而进入肠腔排出,导致药物透膜吸收减少,血药浓度降低。因此,受肠粘膜P-gp调控的经肠道吸收药物可以通过抑制肠粘膜P-gp的活性而增加吸收,提高生物利用度。现已证实,许多药物均为肠粘膜P-gp的抑制剂,例如维拉帕米、环胞菌素A、奎尼丁、一些表面活性剂等。
中药十八反是中医界沿袭数千年的用药禁忌。有研究表明,甘草与反药合用前后对细胞色素P450具有不同的调控作用。令人感兴趣的是P-gp的底物特异性很大程度上与细胞色素P450同工酶(CYP3A1-3)相似,二者有很多重叠的底物,如利福平、利多卡因、地高辛以及环孢菌素等。那么,这些反药对合用前后也有可能对肠粘膜P-gp产生不同的调节作用,这可能是反药合用引起临床上产生毒性的原因。文献检索表明,目前国内外尚未见由探讨肠粘膜P-gp的表达同中药配伍合理性之间的研究报告,因此探讨中药反药对合用后毒性增加是否同P-gp表达的改变等研究具有较高的学术价值。
本课题选择十八反药对中的甘草,甘遂,海藻,京大戟作为模型药进行研究。参考2010年版《中国药典》对所选中药材进行质量控制后,中药材按传统方法进行煎煮,制备甘草水提液,反药水提液及甘草反药合煎水提液,甘草反药合并水提液。选择典型的P-gp抑制剂维拉帕米为阳性对照药物,以生理盐水为阴性对照药物。将中药水提液,维拉帕米,生理盐水分别对Wistar大鼠灌胃造模,利用体外扩散池法(Ussing chamber), Real-time PCR法,在体In situ实验法,分析甘草与反药合用前后对大鼠肠粘膜P-gp表达的影响。实验中,将P-gp典型底物药物罗丹明123(R123)和旁细胞途径药物荧光素钠(CF)分别作为转运方式的标志物进行检测。Ussing chamber体外实验中,R123和CF都具有可见光范围的荧光,故应用荧光分光光度计进行检测,检测灵敏度高。实验成功建立了荧光分光光度计对肠粘膜透过液中R123和CF检测的方法学,选取了灌胃的最佳浓度及能够反应肠道吸收机制的最佳肠段。In situ体内实验中,应用LC-MS/MS首次建立了大鼠血浆中检测R123的方法学,期望通过对R123及CF的检测能间接地说明甘草反药合用前后对P-gp调控的规律性。基因水平实验中,选择mdr1a为目的基因,各个组织中表达量相对恒定的β-actin为内参基因分析造模大鼠不同区段肠道的P-gp表达情况,阐明甘草反药合用引起毒性增加的可能作用机制。
内容和结果
中药根据不同产地、不同炮制方法,质量存在很大差别,因此要首先对实验中所用的甘草,甘遂,海藻,京大戟进行质量控制。参考2010年版《中国药典》含量测定的方法,分别选择甘草酸铵,甘草苷,丹酚酸B(首次从京大戟中分离得到),大戟二烯醇为对照品。选择Inertsil ODS-SP C18柱(4.6×150mm,5μm)为色谱柱；柱温：40℃；流动相：A为乙腈,B为0.05%磷酸水溶液。其中甘草酸铵,甘草苷用于甘草水提液的含量测定：波长为237nm；采取梯度洗脱。大戟二烯醇用于甘遂的含量测定：流动相：A为乙腈,B为0.05%磷酸水溶液；A：B=95：5；波长为237nm,流速：1ml/min。大戟二烯醇,丹酚酸B用于京大戟含量测定：波长为210nm；采用梯度系统。甘草酸铵标准品,甘草苷标准品,丹酚酸B标准品,大戟二烯醇标准品(A用于甘遂水提液检测,B用于京大戟水提液检测)在各自的色谱条件下,重现性良好,各自保留时间分别约为：43.09+0.17mmin,25.02±0.10min,40.09±0.07mmin,13.46±0.18min(A),74.09±0.14min(B)。含量测定中,甘草水提液中所含甘草苷的量为0.6%,甘草酸的量为2.2%,甘遂水提液中所含大戟二烯醇的量为0.2%,均大于药典规定的含量。实验首次对京大戟进行含量测定,京大戟水提液中含大戟二烯醇的量为0.1%,丹酚酸B的量为2.0%。
应用荧光分光光度计,建立R123及CF检测方法学。R123的激发波长为485nm,发射波长为535nm,CF的激发波长为490nm,发射波长为520nm。R123在(10-200)μg·I-1,荧光强度对浓度进行回归,回归方程为Y=0.2237X+2.5658(r2=0.9993,),回收率和日内精密度分别为：99.9%和0.9%。CF在(200-2000)μg·1-1,荧光强度对浓度进行回归,回归方程为Y=0.6386X+0.0679(r2=1),回收率和日内精密度分别为：99.8%和2.3%。
应用LC-MS/MS,建立大鼠血浆中R123的检测方法学。0.1ml血浆样品用乙酸乙酯-二氯甲烷提取。用罗丹明6G作为内标。利用C18柱分离R123及R6G。检测方式为正离子电离,多离子反应监测(MRM),用于定量分析的离子为R123m/z：345-285,R6Gm/z：443-415。样品运行时间为4min,标准曲线的浓度范围为：1-200ng/ml,最低检测限为1ng/ml。低浓度质控样品的日内精密度及日间精密度分别为6.96%和9.2%,中间浓度质控样品的日内精密度及日间精密度分别为3.33%和3.02%,高浓度质控样品的日内精密度及日间精密度分别为1.41%和2.09%,三种浓度的质控样品在日间精密度及日内精密度中的误差均在0.12%-3.24%之间。
使用体外Ussing chamber实验评价R123、CF经不同区段肠粘膜的经时吸收方向和分泌方向的累计透过率和表观渗透系数(Papp)及泵出比ER。计量资料统计结果用均数±标准差(x±s)表示。药物不同浓度不同区段的Papp比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检验；各组累计透过率的比较用重复测量数据的方差分析,不同组间多重比较采用LSD法检验；各组间不同方向,不同区段的Papp及ER的均数比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检验。显著性标准为P<0.05。
生理盐水组,R123在空肠回肠结肠中的分泌方向转运大于吸收方向转运,泵出比ER分别为空肠ER=5.69+4.13；回肠ER=3.51±2.20；结肠ER=2.02±0.84。表明其在小肠中的转运是以分泌为主,说明泵出系统的存在。维拉帕米组,R123在空肠回肠结肠中的吸收方向转运大于分泌方向,泵出比分别为空肠：ER=0.42+0.33,回肠ER=0.41±0.25,结肠ER=I.23±1.16。维拉帕米作为P-gp的典型抑制剂,表明其口服后,可显著抑制P-gp的表达,使R123在小肠中的转运是以吸收为主。
使用体外Ussing chamber实验考察不同浓度(0.25g/ml,0.5g/ml,1g/ml)中药水提液灌胃大鼠1周后,R123经空肠粘膜M-S方向和S-M方向的影响。计量资料统计结果用均数±标准差(x±s)表示。各组标准曲线浓度与吸光度的效应关系用线性回归分析(Linear Regression)。各组中分泌方向累计透过率与吸收方向累计透过率的比较用重复测量数据的方差分析；各组间不同方向,不同区段的Papp及ER的均数比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检测。以P<0.05认为差异有统计学意义。。
3种浓度的甘草水提液灌胃后,R123在吸收方向与分泌方向中的透过与生理盐水组(0.28±0.15和1.16±0.46)比较,没有显著性差异。3种浓度的甘遂水提液,海藻水提液,京大戟水提液灌胃后,R123经空肠粘膜M-S方向的透过按浓度增高而增加。但是1g/ml灌胃时,R123分泌方向的透过均高于0.25g/m1,0.5g/ml的透过。提示浓度过高可能影响了P-gp转运的饱和性。故选择0.5g/ml作为中药灌胃浓度。
将大鼠灌胃各种中药液,1周后用体外Ussing chamber法评价各种药液对R123经各区段肠粘膜透过性的影响。研究空肠吸收时,实验发现甘草液可以降低R123经空肠粘膜吸收分泌双方向的转运,甘遂使R123吸收增加,分泌减小ER显著降低,甘遂合煎与合并液吸收分泌之间无统计学差异,均可以使R123分泌显著降低,吸收显著增加,ER显著降低；海藻使分泌吸收双方向透过增加,但ER有降低趋势,海藻合煎与合并液吸收分泌之间无统计学差异,均可以使双方向透过增加,但是ER显著降低京大戟可以使分泌降低,吸收增加,ER显著降低,其合煎液与合并液之间无统计学差异,但是与京大戟相比,分泌显著降低,吸收增加。研究回肠方向时,甘草降低R123吸收分泌双方向的透过。除海藻外,甘遂与京大戟均时R123吸收方向透过增加,分泌方向减小,ER显著降低。甘草与甘遂,海藻,京大戟合用时,合煎与合并组之间均无统计学差异。除海藻与甘草合用时使双方向透过显著增加,且ER与空白组无显著性差异外,甘草与甘遂合并及甘草与京大戟合并均可使分泌方向降低,吸收方向R123增加,ER与空白组对比,差异具有统计学意义。研究结肠时,药物合用后各自之间没有统计学差异。除京大戟可使R123分泌方向转运降低外,其余药物组的吸收及分泌方向均为升高趋势。
将大鼠灌胃各种中药液,1周后用体外Ussing chamber法评价各种药液对CF经各区段肠粘膜透过性的影响。除了回肠中,京大戟使CF在吸收分泌方向透过均增加外,其余药物组在空肠及回肠中均使CF的透过显著降低。空肠与回肠各组相比趋势相同,提示药物对空肠与回肠的影响相似。各自药物在结肠实验时,均使CF的透过显著降低。
基因水平实验中,选择mdr1a为目的基因,各个组织中表达量相对恒定的β-actin为内参基因分析造模大鼠不同区段肠道的P-gp表达情况,阐明甘草反药合用引起毒性增加的可能作用机制。实验结果用x±s表示,所有实验数据均采用SPSS13.0统计软件进行析因设计资料的方差分析,不同组间多重比较采用LSD法检测；以P<0.05认为差异有统计学意义。。考察发现,空白组空肠,回肠,结肠mdr1a表达依次升高,而维拉帕米组空肠,回肠,结肠ndr1a表达,依次降低。空肠,回肠组海藻及其与甘草合并组对mdr1a表达与空白组相比没有统计学差异。甘遂具有降低mdr1a表达的趋势,但是与空白组相比,没有统计学差异。京大戟可以显著降低mdr1a (?)勺表达,与空白组相比,差异具有统计学差异。甘遂与甘草合并,京大戟与甘草合并,均可以降低mdr1a的表达。各自药物对结肠(?)dr1a的影响均无显著性差异。
根据体外结果,应用小肠作为主要部位,在体In situ实验测定大鼠灌胃甘草水提液,京大戟水提液,甘草京大戟合并药液后,R123和CF在血浆中药物浓度的变化,计算各种药动学参数。实验资料结果用均数±标准差(x±s)表示,采用SPSS13.0统计软件包软件进行统计分析。CF各组浓度与峰面积的效应关系用线性回归分析(Linear Regression)。R123及CF药物在肠道内吸收后Cmax、Tmax、AUC0-240min、F的均数比较各自均采用单向方差分析(One-Way ANOVA),如果有显著性差异,则组间两两比较采用LSD法(方差齐性时)或Dunnett T3法(方差不齐时)；LDH及总蛋白含量测定的均数比较采用采用单向方差分析(One-Way ANOVA),如果有显著性差异,则组间两两比较采用LSD法(方差齐性时)或Dunnett T3法(方差不齐时)。以P<0.05认为差异有统计学意义。。
实验发现甘草组R123最高血浆浓度Cmax、曲线下面积AUC0-240min (?)口生物利用度F与对照组比较无统计学差异,而京大戟、合并组Cmax、AUC0-240min和F均大于对照组(P<0.01),并且合合并组AUC0-240和F与京大戟组相比,增加了将近一倍,差异有统计学意义(P<0.01),；大鼠灌胃京大戟后,CF的各药动学参数与对照组比较无统计学差异。
结论和讨论
结合体内外实验结果,可以推断甘遂和京大戟可以抑制P-gp的表达,且京大戟的抑制作用更加明显,甘遂与京大戟在与甘草分别合并使用后对P-gp表达的抑制加强,这可能是其产生配伍禁忌的原因之一。海藻和甘草单用及合用对P-gp的表达的影响没有统计学意义,提示二者配伍禁忌可能不是由P-gp影响造成的。那么在临床上,甘遂与京大戟有可能配伍一些易产生耐药性的抗肿瘤药物,或者制成肿瘤药物耐药性逆转剂,以增加抗癌疗效；也可以尝试在药物的制备中,适当加入甘遂或京大戟,改善一些P-gp底物药物的吸收。而甘遂对CF的作用可能是甘遂一方面抑制其吸收,另一方面抑制分泌,吸收和分泌量都减少,从而导致其最终生物利用度与对照组无差异,这结果与甘遂是P-gp的抑制剂并不矛盾,京大戟使CF吸收分泌双方向均增加,且ER与空白组无统计学差异,提示可能是由于打开了肠黏膜紧密连接的原因。
中医素有“十方九草,无草不成方”之说,甘草配伍其他药物可以起到增强药物疗效的作用。实验表明甘草对P-gp没有影响,甘草与反药合用后可能直接或间接对P-gp产生调控作用,改变了胃肠粘膜通透性,从而引起不同药物吸收的增加。这为将来对甘草“调和诸药、增强疗效”的作用机制研究,提供了一个很好的方向,可以试图从甘草对肠粘膜中各种转运蛋白的影响方面进行研究,科学地认识甘草对不同转运方式的药物经胃肠道粘膜渗透和吸收影响的可能机制,从而为临床广泛应用甘草配伍其他药物提高疗效提供更多的依据。
甘草反药合用后与单用反药相比,对P-gp抑制作用增强,但是合煎与合并两种炮制方法对P-gp的影响无差异。我们可以推断甘草与反药配伍有毒,可能是甘草对反药有协同作用,使反药中某些抑制P-gp表达的化学成分作用加强,从而使反药中有毒成分的分泌减少,吸收增加而导致其产生毒性作用；从另一方面考虑,反药与甘草合用产生毒性,也有可能是反药使甘草中一些有毒的物质(P-gp底物)吸收增加,这还有待于未来的进一步实验。
Background&Objective
P-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&Results
The 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&Discussion
Based 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.
P-糖蛋白(P-glycoprotein, P-gp)是属于ABC (ATP-binding cassette)转运体超家族的能量依赖性膜蛋白,分子量为170kDa,可发挥外排泵作用,将细胞内的化合物逆浓度梯度转运至胞外,从而降低细胞内的药物浓度。P-gp除在肿瘤细胞中高度表达外,在正常机体组织和器官如肝脏、肾脏、胎盘、大脑、睾丸及肠刷状缘膜等部位均有高水平的表达。存在于肠上皮细胞刷状缘膜中的P-gp能将药物从浆膜侧泵回至粘膜侧而进入肠腔排出,导致药物透膜吸收减少,血药浓度降低。因此,受肠粘膜P-gp调控的经肠道吸收药物可以通过抑制肠粘膜P-gp的活性而增加吸收,提高生物利用度。现已证实,许多药物均为肠粘膜P-gp的抑制剂,例如维拉帕米、环胞菌素A、奎尼丁、一些表面活性剂等。
中药十八反是中医界沿袭数千年的用药禁忌。有研究表明,甘草与反药合用前后对细胞色素P450具有不同的调控作用。令人感兴趣的是P-gp的底物特异性很大程度上与细胞色素P450同工酶(CYP3A1-3)相似,二者有很多重叠的底物,如利福平、利多卡因、地高辛以及环孢菌素等。那么,这些反药对合用前后也有可能对肠粘膜P-gp产生不同的调节作用,这可能是反药合用引起临床上产生毒性的原因。文献检索表明,目前国内外尚未见由探讨肠粘膜P-gp的表达同中药配伍合理性之间的研究报告,因此探讨中药反药对合用后毒性增加是否同P-gp表达的改变等研究具有较高的学术价值。
本课题选择十八反药对中的甘草,甘遂,海藻,京大戟作为模型药进行研究。参考2010年版《中国药典》对所选中药材进行质量控制后,中药材按传统方法进行煎煮,制备甘草水提液,反药水提液及甘草反药合煎水提液,甘草反药合并水提液。选择典型的P-gp抑制剂维拉帕米为阳性对照药物,以生理盐水为阴性对照药物。将中药水提液,维拉帕米,生理盐水分别对Wistar大鼠灌胃造模,利用体外扩散池法(Ussing chamber), Real-time PCR法,在体In situ实验法,分析甘草与反药合用前后对大鼠肠粘膜P-gp表达的影响。实验中,将P-gp典型底物药物罗丹明123(R123)和旁细胞途径药物荧光素钠(CF)分别作为转运方式的标志物进行检测。Ussing chamber体外实验中,R123和CF都具有可见光范围的荧光,故应用荧光分光光度计进行检测,检测灵敏度高。实验成功建立了荧光分光光度计对肠粘膜透过液中R123和CF检测的方法学,选取了灌胃的最佳浓度及能够反应肠道吸收机制的最佳肠段。In situ体内实验中,应用LC-MS/MS首次建立了大鼠血浆中检测R123的方法学,期望通过对R123及CF的检测能间接地说明甘草反药合用前后对P-gp调控的规律性。基因水平实验中,选择mdr1a为目的基因,各个组织中表达量相对恒定的β-actin为内参基因分析造模大鼠不同区段肠道的P-gp表达情况,阐明甘草反药合用引起毒性增加的可能作用机制。
内容和结果
中药根据不同产地、不同炮制方法,质量存在很大差别,因此要首先对实验中所用的甘草,甘遂,海藻,京大戟进行质量控制。参考2010年版《中国药典》含量测定的方法,分别选择甘草酸铵,甘草苷,丹酚酸B(首次从京大戟中分离得到),大戟二烯醇为对照品。选择Inertsil ODS-SP C18柱(4.6×150mm,5μm)为色谱柱；柱温：40℃；流动相：A为乙腈,B为0.05%磷酸水溶液。其中甘草酸铵,甘草苷用于甘草水提液的含量测定：波长为237nm；采取梯度洗脱。大戟二烯醇用于甘遂的含量测定：流动相：A为乙腈,B为0.05%磷酸水溶液；A：B=95：5；波长为237nm,流速：1ml/min。大戟二烯醇,丹酚酸B用于京大戟含量测定：波长为210nm；采用梯度系统。甘草酸铵标准品,甘草苷标准品,丹酚酸B标准品,大戟二烯醇标准品(A用于甘遂水提液检测,B用于京大戟水提液检测)在各自的色谱条件下,重现性良好,各自保留时间分别约为：43.09+0.17mmin,25.02±0.10min,40.09±0.07mmin,13.46±0.18min(A),74.09±0.14min(B)。含量测定中,甘草水提液中所含甘草苷的量为0.6%,甘草酸的量为2.2%,甘遂水提液中所含大戟二烯醇的量为0.2%,均大于药典规定的含量。实验首次对京大戟进行含量测定,京大戟水提液中含大戟二烯醇的量为0.1%,丹酚酸B的量为2.0%。
应用荧光分光光度计,建立R123及CF检测方法学。R123的激发波长为485nm,发射波长为535nm,CF的激发波长为490nm,发射波长为520nm。R123在(10-200)μg·I-1,荧光强度对浓度进行回归,回归方程为Y=0.2237X+2.5658(r2=0.9993,),回收率和日内精密度分别为：99.9%和0.9%。CF在(200-2000)μg·1-1,荧光强度对浓度进行回归,回归方程为Y=0.6386X+0.0679(r2=1),回收率和日内精密度分别为：99.8%和2.3%。
应用LC-MS/MS,建立大鼠血浆中R123的检测方法学。0.1ml血浆样品用乙酸乙酯-二氯甲烷提取。用罗丹明6G作为内标。利用C18柱分离R123及R6G。检测方式为正离子电离,多离子反应监测(MRM),用于定量分析的离子为R123m/z：345-285,R6Gm/z：443-415。样品运行时间为4min,标准曲线的浓度范围为：1-200ng/ml,最低检测限为1ng/ml。低浓度质控样品的日内精密度及日间精密度分别为6.96%和9.2%,中间浓度质控样品的日内精密度及日间精密度分别为3.33%和3.02%,高浓度质控样品的日内精密度及日间精密度分别为1.41%和2.09%,三种浓度的质控样品在日间精密度及日内精密度中的误差均在0.12%-3.24%之间。
使用体外Ussing chamber实验评价R123、CF经不同区段肠粘膜的经时吸收方向和分泌方向的累计透过率和表观渗透系数(Papp)及泵出比ER。计量资料统计结果用均数±标准差(x±s)表示。药物不同浓度不同区段的Papp比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检验；各组累计透过率的比较用重复测量数据的方差分析,不同组间多重比较采用LSD法检验；各组间不同方向,不同区段的Papp及ER的均数比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检验。显著性标准为P<0.05。
生理盐水组,R123在空肠回肠结肠中的分泌方向转运大于吸收方向转运,泵出比ER分别为空肠ER=5.69+4.13；回肠ER=3.51±2.20；结肠ER=2.02±0.84。表明其在小肠中的转运是以分泌为主,说明泵出系统的存在。维拉帕米组,R123在空肠回肠结肠中的吸收方向转运大于分泌方向,泵出比分别为空肠：ER=0.42+0.33,回肠ER=0.41±0.25,结肠ER=I.23±1.16。维拉帕米作为P-gp的典型抑制剂,表明其口服后,可显著抑制P-gp的表达,使R123在小肠中的转运是以吸收为主。
使用体外Ussing chamber实验考察不同浓度(0.25g/ml,0.5g/ml,1g/ml)中药水提液灌胃大鼠1周后,R123经空肠粘膜M-S方向和S-M方向的影响。计量资料统计结果用均数±标准差(x±s)表示。各组标准曲线浓度与吸光度的效应关系用线性回归分析(Linear Regression)。各组中分泌方向累计透过率与吸收方向累计透过率的比较用重复测量数据的方差分析；各组间不同方向,不同区段的Papp及ER的均数比较用析因设计资料的方差分析,不同组间多重比较采用SNK法检测。以P<0.05认为差异有统计学意义。。
3种浓度的甘草水提液灌胃后,R123在吸收方向与分泌方向中的透过与生理盐水组(0.28±0.15和1.16±0.46)比较,没有显著性差异。3种浓度的甘遂水提液,海藻水提液,京大戟水提液灌胃后,R123经空肠粘膜M-S方向的透过按浓度增高而增加。但是1g/ml灌胃时,R123分泌方向的透过均高于0.25g/m1,0.5g/ml的透过。提示浓度过高可能影响了P-gp转运的饱和性。故选择0.5g/ml作为中药灌胃浓度。
将大鼠灌胃各种中药液,1周后用体外Ussing chamber法评价各种药液对R123经各区段肠粘膜透过性的影响。研究空肠吸收时,实验发现甘草液可以降低R123经空肠粘膜吸收分泌双方向的转运,甘遂使R123吸收增加,分泌减小ER显著降低,甘遂合煎与合并液吸收分泌之间无统计学差异,均可以使R123分泌显著降低,吸收显著增加,ER显著降低；海藻使分泌吸收双方向透过增加,但ER有降低趋势,海藻合煎与合并液吸收分泌之间无统计学差异,均可以使双方向透过增加,但是ER显著降低京大戟可以使分泌降低,吸收增加,ER显著降低,其合煎液与合并液之间无统计学差异,但是与京大戟相比,分泌显著降低,吸收增加。研究回肠方向时,甘草降低R123吸收分泌双方向的透过。除海藻外,甘遂与京大戟均时R123吸收方向透过增加,分泌方向减小,ER显著降低。甘草与甘遂,海藻,京大戟合用时,合煎与合并组之间均无统计学差异。除海藻与甘草合用时使双方向透过显著增加,且ER与空白组无显著性差异外,甘草与甘遂合并及甘草与京大戟合并均可使分泌方向降低,吸收方向R123增加,ER与空白组对比,差异具有统计学意义。研究结肠时,药物合用后各自之间没有统计学差异。除京大戟可使R123分泌方向转运降低外,其余药物组的吸收及分泌方向均为升高趋势。
将大鼠灌胃各种中药液,1周后用体外Ussing chamber法评价各种药液对CF经各区段肠粘膜透过性的影响。除了回肠中,京大戟使CF在吸收分泌方向透过均增加外,其余药物组在空肠及回肠中均使CF的透过显著降低。空肠与回肠各组相比趋势相同,提示药物对空肠与回肠的影响相似。各自药物在结肠实验时,均使CF的透过显著降低。
基因水平实验中,选择mdr1a为目的基因,各个组织中表达量相对恒定的β-actin为内参基因分析造模大鼠不同区段肠道的P-gp表达情况,阐明甘草反药合用引起毒性增加的可能作用机制。实验结果用x±s表示,所有实验数据均采用SPSS13.0统计软件进行析因设计资料的方差分析,不同组间多重比较采用LSD法检测；以P<0.05认为差异有统计学意义。。考察发现,空白组空肠,回肠,结肠mdr1a表达依次升高,而维拉帕米组空肠,回肠,结肠ndr1a表达,依次降低。空肠,回肠组海藻及其与甘草合并组对mdr1a表达与空白组相比没有统计学差异。甘遂具有降低mdr1a表达的趋势,但是与空白组相比,没有统计学差异。京大戟可以显著降低mdr1a (?)勺表达,与空白组相比,差异具有统计学差异。甘遂与甘草合并,京大戟与甘草合并,均可以降低mdr1a的表达。各自药物对结肠(?)dr1a的影响均无显著性差异。
根据体外结果,应用小肠作为主要部位,在体In situ实验测定大鼠灌胃甘草水提液,京大戟水提液,甘草京大戟合并药液后,R123和CF在血浆中药物浓度的变化,计算各种药动学参数。实验资料结果用均数±标准差(x±s)表示,采用SPSS13.0统计软件包软件进行统计分析。CF各组浓度与峰面积的效应关系用线性回归分析(Linear Regression)。R123及CF药物在肠道内吸收后Cmax、Tmax、AUC0-240min、F的均数比较各自均采用单向方差分析(One-Way ANOVA),如果有显著性差异,则组间两两比较采用LSD法(方差齐性时)或Dunnett T3法(方差不齐时)；LDH及总蛋白含量测定的均数比较采用采用单向方差分析(One-Way ANOVA),如果有显著性差异,则组间两两比较采用LSD法(方差齐性时)或Dunnett T3法(方差不齐时)。以P<0.05认为差异有统计学意义。。
实验发现甘草组R123最高血浆浓度Cmax、曲线下面积AUC0-240min (?)口生物利用度F与对照组比较无统计学差异,而京大戟、合并组Cmax、AUC0-240min和F均大于对照组(P<0.01),并且合合并组AUC0-240和F与京大戟组相比,增加了将近一倍,差异有统计学意义(P<0.01),；大鼠灌胃京大戟后,CF的各药动学参数与对照组比较无统计学差异。
结论和讨论
结合体内外实验结果,可以推断甘遂和京大戟可以抑制P-gp的表达,且京大戟的抑制作用更加明显,甘遂与京大戟在与甘草分别合并使用后对P-gp表达的抑制加强,这可能是其产生配伍禁忌的原因之一。海藻和甘草单用及合用对P-gp的表达的影响没有统计学意义,提示二者配伍禁忌可能不是由P-gp影响造成的。那么在临床上,甘遂与京大戟有可能配伍一些易产生耐药性的抗肿瘤药物,或者制成肿瘤药物耐药性逆转剂,以增加抗癌疗效；也可以尝试在药物的制备中,适当加入甘遂或京大戟,改善一些P-gp底物药物的吸收。而甘遂对CF的作用可能是甘遂一方面抑制其吸收,另一方面抑制分泌,吸收和分泌量都减少,从而导致其最终生物利用度与对照组无差异,这结果与甘遂是P-gp的抑制剂并不矛盾,京大戟使CF吸收分泌双方向均增加,且ER与空白组无统计学差异,提示可能是由于打开了肠黏膜紧密连接的原因。
中医素有“十方九草,无草不成方”之说,甘草配伍其他药物可以起到增强药物疗效的作用。实验表明甘草对P-gp没有影响,甘草与反药合用后可能直接或间接对P-gp产生调控作用,改变了胃肠粘膜通透性,从而引起不同药物吸收的增加。这为将来对甘草“调和诸药、增强疗效”的作用机制研究,提供了一个很好的方向,可以试图从甘草对肠粘膜中各种转运蛋白的影响方面进行研究,科学地认识甘草对不同转运方式的药物经胃肠道粘膜渗透和吸收影响的可能机制,从而为临床广泛应用甘草配伍其他药物提高疗效提供更多的依据。
甘草反药合用后与单用反药相比,对P-gp抑制作用增强,但是合煎与合并两种炮制方法对P-gp的影响无差异。我们可以推断甘草与反药配伍有毒,可能是甘草对反药有协同作用,使反药中某些抑制P-gp表达的化学成分作用加强,从而使反药中有毒成分的分泌减少,吸收增加而导致其产生毒性作用；从另一方面考虑,反药与甘草合用产生毒性,也有可能是反药使甘草中一些有毒的物质(P-gp底物)吸收增加,这还有待于未来的进一步实验。
Background&Objective
P-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&Results
The 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&Discussion
Based 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.
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