药用辅料抑制肠道P-糖蛋白药泵作用研究
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摘要
P-糖蛋白(P-glycoprotein, P-gp)是一种170kDa大小的ATP能量依赖型的外排蛋白,即“药物溢出泵”,能将吸收进入浆膜侧的药物重新泵回粘膜侧,导致药物吸收减少,生物利用度降低。抑制P-糖蛋白的药物溢出泵作用可有效地提高某些口服药物的吸收率,降低肝、肾清除率。许多早期的外排泵抑制剂如维拉帕米、普罗帕酮等,会产生一些不可接受的毒副作用,而限制其临床应用。因此,寻找一些无毒、无药理活性的逆转剂成为国内外药剂学者关注的新焦点。本论文以药用辅料为研究对象,选用塞利洛尔为P-gp探针药物,采用Caco-2细胞单层膜模型、离体小肠外翻模型、在体小肠灌流模型与原位小肠吸收模型考察P-gp药泵作用在塞利洛尔跨膜转运与透膜吸收中发挥的作用,比较不同药用辅料种类及不同浓度对P-gp药泵作用的抑制强度,希望筛选出具有较强P-gp抑制作用的辅料。
Caco-2细胞模型已经作为一种简便、高效的体外模型广泛应用于口服药物吸收机制及候选药物的高通量筛选。本实验采用Caco-2细胞模型,利用测定跨膜电阻值和酚红透过试验检验其完整性。完整性检测合格后可用于探针药物塞利洛尔跨膜转运实验,以维拉帕米为阳性对照,利用HPLC法测定药物跨膜转运量,计算跨膜转运参数表观渗透系数Papp与外排率ER值来评价药用辅料对Caco-2细胞膜上P-gp外排功能的抑制作用。塞利洛尔在Caco-2细胞单层膜AP→BL方向的Papp值远小于BL→AP方向的Papp值,阳性P-gp逆转剂维拉帕米使塞利洛尔AP→BL方向跨膜转运的Papp值增加30%,BL→AP方向的Papp值降低50%,说明P-gp在塞利洛尔跨膜转运过程中发挥重要作用。辅料F-68,Cremophor EL,Cremophor RH40,T-80,PEG200,PEG400,PEG4000,β-CD,HP-β-CD,PVP K30,TritonX 100,S-40可增加塞利洛尔AP→BL方向跨膜转运的Papp值并降低BL→AP方向的Papp值;T-20,T-60,PEG1000,PEG2000,PEG6000,PVP K10随浓度的提高可增加塞利洛尔AP→BL方向跨膜转运的Papp值并降低BL→AP方向的Papp值;Lecithin对塞利洛尔AP→BL方向跨膜转运的Papp值与BL→AP方向的Papp值均无明显影响;SDS具有较大的细胞毒性使塞利洛尔AP→BL方向跨膜转运的Papp值与BL→AP方向的Papp增加异常。进一步通过外排率ER值评价辅料抑制P-gp作用强度,结果表明F-68、Cremophor EL、Cremophor RH40、T-20、T-60、T-80、PEG200、PEG400、β-CD、HP-β-CD、S-40等可显著抑制塞利洛尔细胞膜外排方向转运,在Caco-2细胞模型中具有明显抑制P-gp药泵作用。
大鼠离体外翻小肠吸收与外排模型分别选择大鼠小肠空肠段与结肠段进行研究。以维拉帕米作阳性对照,考察F-68、Cremophor EL、Cremophor RH40、T-60、T-80、PEG200、PEG400、PEG1000、HP-β-CD、S-40十种药用辅料对塞利洛尔从外翻小肠囊的粘膜层向浆膜层(M→S)、浆膜层向粘膜层(S→M)转运情况,评价低中高辅料浓度逆转小肠P-gp药泵作用对塞利洛尔促吸收与抑制外排作用。离体外翻小肠吸收模型实验结果表明F-68、Cremophor RH40、PEG400、T-80具有较强的促吸收作用;Cremophor EL、T-60、PEG200、PEG1000、S-40随浓度提高促吸收作用增强;HP-β-CD对药物M→S吸收影响不大。离体外翻小肠外排模型实验结果表明F-68、Cremophor EL、Cremophor RH40、T-60、T-80、PEG200、PEG400、S-40具有较强的抑制外排作用; PEG1000与HP-β-CD随浓度提高抑制外排作用增强。
大鼠在体小肠灌流模型不仅用于评价药物及其制剂口服吸收情况,还广泛应用于生物膜的转运机制研究。本文选用该模型研究F-68、Cremophor EL等10种药用辅料对在体小肠灌流后塞利洛尔吸收的影响。结果表明F-68、Cremophor EL、Cremophor RH40、T-60、T-80、PEG200、PEG400、S-40可显著提高药物吸收速率常数ka与有效透过系数Peff,由于塞利洛尔为P-gp典型底物,因此推断药物促吸收作用与抑制P-gp外排作用密切相关;但辅料PEG1000与HP-β-CD对塞利洛尔在体肠灌流吸收无明显促进作用。
在Caco-2细胞模型、离体与在体动物模型研究基础上,进一步采用整体动物模型原位小肠吸收实验考察F-68、Cremophor EL等10种药用辅料对塞利洛尔十二指肠处给药后透膜吸收的影响。采用HPLC法测定血药浓度,DAS软件计算药代动力学参数,评价药用辅料对P-gp抑制作用及促进塞利洛尔吸收的影响。从药时曲线结果分析,给予F-68、Cremophor EL、Cremophor RH40、T-80、PEG200、PEG400、S-40后塞利洛尔血药浓度均有不同程度的提高,但给予辅料T-60、PEG1000、HP-β-CD后塞利洛尔药时曲线与阴性对照组相当,远低于维拉帕米阳性对照组水平。从药代动力学参数分析,给予药用辅料后药物半衰期t1/2有所延长,清除率CL降低。同时药时曲线下面积AUC统计分析结果表明: F-68、Cremophor RH40、PEG200、PEG400、S-40高浓度组与Cremophor EL、T-80中高浓度组可明显提高塞利洛尔药时曲线下面积AUC,从而促进药物吸收;同时上述各辅料其它浓度组均可在一定程度上提高药物AUC,但统计学分析无显著性差异。辅料T-60、PEG 1000与HP-β-CD在整体动物模型中对塞利洛尔各参数几乎无影响,提示上述三种辅料不影响药物吸收过程。
选择四种具有抑制P-gp药泵作用的辅料逆转剂F-68、Cremophor EL、T-80、PEG400考察其对大鼠小肠P-gp表达的影响,分别采用BCA蛋白浓度测定试剂盒、运用SDS-聚丙烯酰胺凝胶电泳与凝胶图像分析技术、免疫印迹技术分析逆转剂与P-gp表达之间的关系。单独给予药物塞利洛尔或联合给予塞利洛尔与辅料逆转剂均可促进P-gp表达,但发现辅料逆转剂与维拉帕米可明显诱导外排蛋白的进一步表达。
选取更昔洛韦作为实验药物,考察辅料逆转剂F-68、Cremophor EL、T-80、PEG400对更昔洛韦在Caco-2细胞模型及原位小肠吸收实验模型中跨膜转运与透膜吸收情况,评价辅料逆转剂抑制P-gp作用对提高更昔洛韦口服吸收的影响。在Caco-2细胞模型中维拉帕米与辅料逆转剂可提高更昔洛韦AP→BL方向表观渗透系数Papp,降低BL→AP方向的表观渗透系数Papp,从而使外排率ER值明显下降,统计分析结果表明:维拉帕米组及辅料逆转剂各中高浓度组的外排率ER值与阴性对照组间有显著性差异,辅料逆转剂其它浓度组也均使更昔洛韦外排率降低,但无统计学意义。在原位小肠吸收实验中给予辅料逆转剂F-68、Cremophor EL、T-80、PEG400后更昔洛韦血药浓度均有一定程度的提高,其中Cremophor EL低中浓度组、T-80低浓度组、PEG400低浓度组对药时曲线下面积有显著性影响,其它高浓度组无明显影响。
本文通过考察20种药用辅料在Caco-2细胞模型中抑制P-gp外排作用,筛选出10种辅料进一步进行离体、在体、整体动物实验模型筛选,并考察具有较强P-gp抑制作用的辅料逆转剂对肠道P-gp蛋白表达影响,结果表明非离子表面活性剂等药用辅料具有抑制P-gp药泵外排功能,减少药物外排量,能促进塞利洛尔与更昔洛韦肠道吸收,可提高底物药物口服生物利用度。但辅料逆转剂在抑制P-gp药泵外排功能的同时可促进其蛋白表达。从作用机制角度分析,药物吸收提高的同时蛋白表达量增加,提示抑制P-gp药泵作用机制不是通过抑制蛋白表达而可能通过其它途径如抑制ATP酶活性、竞争性结合位点、P-gp构象改变等实现的。
辅料逆转剂具有毒性低,刺激性小,生物相容性好等特点,可用于水溶性、脂溶性、甚至一些难溶性药物的载体,在药剂学领域将会有更广泛的应用。此外,通过抑制P-gp的外排功能,一些药用辅料具有逆转肿瘤细胞多药耐药的作用,为临床提高抗肿瘤药物的疗效,减少肿瘤细胞对药物的外排,提高其对药物的敏感性,提供了研究方向和试验手段。
P-glycoprotein (P-gp), a 170 kDa ATP dependent membrane transporter, acts as a drug efflux pump. P-gp can exports structurally diverse hydrophobic compounds from serosa to mucosa and thus reduce the absorption and bioavailability of a wide range of drugs which are substrates for this membrane transporter. Inhibiting the drug efflux pump can increase the absorption of some orally drugs and decrease the renal clearance and hepatic-clearance. Many early P-gp modulator agents, such as verapamil and propafenone, often result in unacceptable toxicity that limits their clinical use. Therefore, to develop some P-gp modulators with less toxicity and non-pharmaco-activity is very important and pharmaceutical scientists all over the world have focus on this research field. In this dissertation pharmaceutical excipients were selcted to investigate their inhibitory effect on P-glycoprotein to search for some P-gp modulator agents with non-toxicity and non-pharmaco-activity. In this study celiprolol was used as the P-gp probe drug and Caco-2 monolayer model, everted gut sacs experiment in vitro, intestinal perfusion in situ and intestine absorption model in vivo were employed to study the effect of P-gp efflux pump on transmembrane transport and absorption of celiprolol. Pharmaceutical excipients with strong inhibitory effect on P-gp were sieved by comparing the inhibitory tensity of pharmaceutical adjuvant sorts and concentration.
Caco-2 cell monolayer model has gained enormous popularity as a convenient and high-effective in vitro model for absorption mechanism study of oral drugs and high throughput screening method for drug candidates. The integrity of Caco-2 cell monolayer was dectected by testing the TEER value by Millicell-ERS equipment and phenol permeation experiment. Caco-2 cell monolayer model was then established to perform the bi-directional transport experient of celiprolol with verapamil as positive control. The concentration of celiprolol was determined by HPLC and apparent permeability coefficient(Papp) and efflux ratio (ER) value were calculated according to drug transmembrane transport amount. Then the inhibitory effect of pharmaceutical excipients on P-gp was evaluated with the parameters of Papp and ER. The results showed that Papp of celiprolol transport from apical to basolateral was far lower than that of basolateral to apical direction. With P-gp inhibitor verapamil, Papp of celiprolol transport from apical to basolateral increased by 30% and that from basolateral to apical decreased by 50%, which indicates that efflux protein P-gp plays a vital role in the transepithelial transport of celiprolol. Pharmaceutial adjuvants F-68, Cremophor EL, Cremophor RH40, T-80, PEG200, PEG400, PEG4000,β-CD, HP-β-CD, PVP K30, TritonX 100 and S-40 can also enhance Papp of apical to basolateral direction and reduce Papp of basolateral to apical direction. With the concentration of T-20, T-60, PEG1000, PEG2000, PEG6000 and PVP K10 increased, they would exhibit similar effect. But Lecithin almost has no influence on Papp of both direction transport, while SDS showed great cytotoxicity which result in abnormal alteration in this parameter. Furthermore, ER value was analyzed with SPSS software to elucidate the inhibitory effect strength of pharmaceutical adjuvants on P-gp. The result showed that F-68, Cremophor EL, Cremophor RH40, T-20, T-60, T-80, PEG200, PEG400,β-CD, HP-β-CD and S-40 could significantly inhibit excretion cross Caco-2 monolayers and enhance absorption. In conclusion, these pharmaceutical excipients had an ability to inhibit the P-gp efflux pump obviously in Caco-2 cell monolayer model.
Everted gut sacs in vitro was used for the study of the transport function of P-gp with probe drug celiprolol. Jejunal segment and colon segment of rat intestine were studied in the absorption model and efflux model in vitro, respectively. With verapamil set as positive control, the transport characteristic of celiprolol under the influence of low, medium and high concentration of pharmaceutical adjuvants F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400, PEG1000, HP-β-CD and S-40 was investigated to assess the contribution of P-gp on celiprolol absorptive promotion and efflux suppression. The transport samples from mucosal to serosal side and serosal to mucosal side in the present of various concentrations of pharmacrutical excipients were dectected by HPLC. The results of everted gut sac absorption model processed by SPSS show that F-68, Cremophor RH40, PEG400 and T-80 could significantly promote intestinal absorption while only at the higher concentration Cremophor EL, T-60, PEG200, PEG1000 and S-40 could increase absorptive transport across intestinal mucous membrane. However, there is no significant influence of HP-β-CD on M→S absorption. Furthermore, the results of excretive model in vitro showed that F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400 and S-40 could inhibit the efflux of Celiprolol at the low, middle and high concentration while PEG1000 and HP-β-CD inhibit the P-gp efflux pump at much higher concentration.
Rat intestinal perfusion model in situ was widespreadly used not only for oral application and absorption of drugs and preparations, but also for transport mechanism across biomembrane. This model was established and performed using celiprolol Krebs solution containing phenol to indicate the water ingredient alteration. To study the effect of 10 pharmaceutical excipients on the efflux function of P-gp the concentration of celiprolol in the effluent solution was detected and parameters absorptive rate constant (ka) and effective permeability coefficients (Peff) were calculated. The results processed by SPSS indicated that F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400 and S-40 could increase ka and Peff which showed that these pharmaceutical excipients facilitate absorption process via inhibiting P-gp, but these parameters were not affected by PEG1000 and HP-β-CD.
Intestinal absorption experiment in vivo was set up to detect the concentration of celiprolol by collecting blood plasma samples. This in vivo model was used for detecting whether 10 pharmaceutical excipients such as F-68, Cremophor EL and T-80 had the ability of inhibiting P-gp efflux pump at the low, middle, high concentration. The plasma drug concentration-time data was processed by pharmacokinetic software DAS. The inhibitory effect on P-gp in vivo was investigated by the pharmacokinetics parameters of celiprolol after the administration of different doses of pharmaceutical excipients. From the concentration-time curve, it could be obtained that the drug concentration in plasma raised to a distinct extent with coadministration of F-68, Cremophor EL, Cremophor RH40, T-80, PEG200, PEG400 and S-40 while that in plasma maintain at the same level of negative control with coadministration of T-60, PEG1000 and HP-β-CD. From the pharmacokinetics parameters analyzed by DAS, the half life time (t1/2) of celiprolol prolonged and clearance rate (CL) decreased after giving pharmaceutical excipients. Statistical analysis results of the area under the curve (AUC) showed that high concentration group of F-68, Cremophor RH40, PEG200, PEG400 and S-40 and medium, high concentration group of Cremophor EL and T-80 could increase the absorption of P-gp substrate celiprolol. But these three pharmaceutical adjuvants T-60, PEG1000 and HP-β-CD do not affect the pharmacokinetic process in vivo.
Based on the screening resuluts of different models, four pharmaceutical excipients F-68, Cremophor EL, T-80 and PEG400 were selected to investigate the influence of intestinal P-gp expression. The relationship between P-gp inhibitor and protein expression in intestine was studied by BCA protein assay kit, SDS-PAGE with gel image analysis technique and western blot analysis. In contrast with control group, experimental groups coadministration with F-68, Cremophor EL, T-80 or PEG400 increased the expression of P-gp dramatically. Thus efflux protein P-gp could be induced by its substrate and inhibitors.
To investigate the effect of four pharmaceutic excipients on the absorption of ganciclovir, Caco-2 cell model and intestinal absorption experiment in vivo were exploited to value the extent of absorption promotion. The results in cell model showed that Papp in AP→BL direction increased and that in BL→AP direction decreased, which resulted in that ER reduced significantly. From the statistical analysis results, there exist significant difference between negative control group and verapamil group or medium and high concentration group of pharmaceutical excipients inhibitor F-68, Cremophor EL, T-80 and PEG400 while other groups showed no significant difference. Moreover, intestinal absorption experiment in vivo discovered that only low and medium concentration group of Cremophor EL, low concentration group of T-80 and PEG400 put significant effect on AUC of ganciclovir while other groups showed their enhancement effectiveness with no significant difference.
In conclusion, 20 pharmaceutical excipients were choosed to investigate the inhibitory effect on P-gp in Caco-2 model and then 10 adjuvants were selected for animal research. Furthermore, research on intestinal P-gp expression and ganciclovir transport feature were studied with the presence and absence of F-68, Cremophor EL, T-80 and PEG400. The results indicated that several pharmaceutical exicipients such as nonionic surfactant can reverse the efflux function of P-gp and improve the absorption and bioavailability of P-gp substrate celiprolol and ganciclovir, but these inhibitors also enhance the expression level of P-gp. Thus, excipient inhibitors could ameliorate absorprion and pharmacokinetic characters in vivo of some certain drugs via reversing the intestinal efflux function of P-gp not by inhibiting P-gp expression but probably by inhibiting ATP enzyme activity, competitive binding function site and P-gp conformational change.
Pharmaceutical excipients were used as a vehicle of water-soluble, liposoluble and even indissolvable drugs with low toxicity and satisfactory biocompatibility. Furthermore, it provides direction and method to decrease efflux of cytotoxic drugs from the cancer cells, promote the effect of clinical therapeutic efficacy, recover the sensitivity of the cancer cells to the cytotoxic drugs, and improve the characteristics of pharmacokinetics of some drugs such as increasing the absorption of some orally drugs, decreasing the renal clearance and hepat-clearance.
Caco-2细胞模型已经作为一种简便、高效的体外模型广泛应用于口服药物吸收机制及候选药物的高通量筛选。本实验采用Caco-2细胞模型,利用测定跨膜电阻值和酚红透过试验检验其完整性。完整性检测合格后可用于探针药物塞利洛尔跨膜转运实验,以维拉帕米为阳性对照,利用HPLC法测定药物跨膜转运量,计算跨膜转运参数表观渗透系数Papp与外排率ER值来评价药用辅料对Caco-2细胞膜上P-gp外排功能的抑制作用。塞利洛尔在Caco-2细胞单层膜AP→BL方向的Papp值远小于BL→AP方向的Papp值,阳性P-gp逆转剂维拉帕米使塞利洛尔AP→BL方向跨膜转运的Papp值增加30%,BL→AP方向的Papp值降低50%,说明P-gp在塞利洛尔跨膜转运过程中发挥重要作用。辅料F-68,Cremophor EL,Cremophor RH40,T-80,PEG200,PEG400,PEG4000,β-CD,HP-β-CD,PVP K30,TritonX 100,S-40可增加塞利洛尔AP→BL方向跨膜转运的Papp值并降低BL→AP方向的Papp值;T-20,T-60,PEG1000,PEG2000,PEG6000,PVP K10随浓度的提高可增加塞利洛尔AP→BL方向跨膜转运的Papp值并降低BL→AP方向的Papp值;Lecithin对塞利洛尔AP→BL方向跨膜转运的Papp值与BL→AP方向的Papp值均无明显影响;SDS具有较大的细胞毒性使塞利洛尔AP→BL方向跨膜转运的Papp值与BL→AP方向的Papp增加异常。进一步通过外排率ER值评价辅料抑制P-gp作用强度,结果表明F-68、Cremophor EL、Cremophor RH40、T-20、T-60、T-80、PEG200、PEG400、β-CD、HP-β-CD、S-40等可显著抑制塞利洛尔细胞膜外排方向转运,在Caco-2细胞模型中具有明显抑制P-gp药泵作用。
大鼠离体外翻小肠吸收与外排模型分别选择大鼠小肠空肠段与结肠段进行研究。以维拉帕米作阳性对照,考察F-68、Cremophor EL、Cremophor RH40、T-60、T-80、PEG200、PEG400、PEG1000、HP-β-CD、S-40十种药用辅料对塞利洛尔从外翻小肠囊的粘膜层向浆膜层(M→S)、浆膜层向粘膜层(S→M)转运情况,评价低中高辅料浓度逆转小肠P-gp药泵作用对塞利洛尔促吸收与抑制外排作用。离体外翻小肠吸收模型实验结果表明F-68、Cremophor RH40、PEG400、T-80具有较强的促吸收作用;Cremophor EL、T-60、PEG200、PEG1000、S-40随浓度提高促吸收作用增强;HP-β-CD对药物M→S吸收影响不大。离体外翻小肠外排模型实验结果表明F-68、Cremophor EL、Cremophor RH40、T-60、T-80、PEG200、PEG400、S-40具有较强的抑制外排作用; PEG1000与HP-β-CD随浓度提高抑制外排作用增强。
大鼠在体小肠灌流模型不仅用于评价药物及其制剂口服吸收情况,还广泛应用于生物膜的转运机制研究。本文选用该模型研究F-68、Cremophor EL等10种药用辅料对在体小肠灌流后塞利洛尔吸收的影响。结果表明F-68、Cremophor EL、Cremophor RH40、T-60、T-80、PEG200、PEG400、S-40可显著提高药物吸收速率常数ka与有效透过系数Peff,由于塞利洛尔为P-gp典型底物,因此推断药物促吸收作用与抑制P-gp外排作用密切相关;但辅料PEG1000与HP-β-CD对塞利洛尔在体肠灌流吸收无明显促进作用。
在Caco-2细胞模型、离体与在体动物模型研究基础上,进一步采用整体动物模型原位小肠吸收实验考察F-68、Cremophor EL等10种药用辅料对塞利洛尔十二指肠处给药后透膜吸收的影响。采用HPLC法测定血药浓度,DAS软件计算药代动力学参数,评价药用辅料对P-gp抑制作用及促进塞利洛尔吸收的影响。从药时曲线结果分析,给予F-68、Cremophor EL、Cremophor RH40、T-80、PEG200、PEG400、S-40后塞利洛尔血药浓度均有不同程度的提高,但给予辅料T-60、PEG1000、HP-β-CD后塞利洛尔药时曲线与阴性对照组相当,远低于维拉帕米阳性对照组水平。从药代动力学参数分析,给予药用辅料后药物半衰期t1/2有所延长,清除率CL降低。同时药时曲线下面积AUC统计分析结果表明: F-68、Cremophor RH40、PEG200、PEG400、S-40高浓度组与Cremophor EL、T-80中高浓度组可明显提高塞利洛尔药时曲线下面积AUC,从而促进药物吸收;同时上述各辅料其它浓度组均可在一定程度上提高药物AUC,但统计学分析无显著性差异。辅料T-60、PEG 1000与HP-β-CD在整体动物模型中对塞利洛尔各参数几乎无影响,提示上述三种辅料不影响药物吸收过程。
选择四种具有抑制P-gp药泵作用的辅料逆转剂F-68、Cremophor EL、T-80、PEG400考察其对大鼠小肠P-gp表达的影响,分别采用BCA蛋白浓度测定试剂盒、运用SDS-聚丙烯酰胺凝胶电泳与凝胶图像分析技术、免疫印迹技术分析逆转剂与P-gp表达之间的关系。单独给予药物塞利洛尔或联合给予塞利洛尔与辅料逆转剂均可促进P-gp表达,但发现辅料逆转剂与维拉帕米可明显诱导外排蛋白的进一步表达。
选取更昔洛韦作为实验药物,考察辅料逆转剂F-68、Cremophor EL、T-80、PEG400对更昔洛韦在Caco-2细胞模型及原位小肠吸收实验模型中跨膜转运与透膜吸收情况,评价辅料逆转剂抑制P-gp作用对提高更昔洛韦口服吸收的影响。在Caco-2细胞模型中维拉帕米与辅料逆转剂可提高更昔洛韦AP→BL方向表观渗透系数Papp,降低BL→AP方向的表观渗透系数Papp,从而使外排率ER值明显下降,统计分析结果表明:维拉帕米组及辅料逆转剂各中高浓度组的外排率ER值与阴性对照组间有显著性差异,辅料逆转剂其它浓度组也均使更昔洛韦外排率降低,但无统计学意义。在原位小肠吸收实验中给予辅料逆转剂F-68、Cremophor EL、T-80、PEG400后更昔洛韦血药浓度均有一定程度的提高,其中Cremophor EL低中浓度组、T-80低浓度组、PEG400低浓度组对药时曲线下面积有显著性影响,其它高浓度组无明显影响。
本文通过考察20种药用辅料在Caco-2细胞模型中抑制P-gp外排作用,筛选出10种辅料进一步进行离体、在体、整体动物实验模型筛选,并考察具有较强P-gp抑制作用的辅料逆转剂对肠道P-gp蛋白表达影响,结果表明非离子表面活性剂等药用辅料具有抑制P-gp药泵外排功能,减少药物外排量,能促进塞利洛尔与更昔洛韦肠道吸收,可提高底物药物口服生物利用度。但辅料逆转剂在抑制P-gp药泵外排功能的同时可促进其蛋白表达。从作用机制角度分析,药物吸收提高的同时蛋白表达量增加,提示抑制P-gp药泵作用机制不是通过抑制蛋白表达而可能通过其它途径如抑制ATP酶活性、竞争性结合位点、P-gp构象改变等实现的。
辅料逆转剂具有毒性低,刺激性小,生物相容性好等特点,可用于水溶性、脂溶性、甚至一些难溶性药物的载体,在药剂学领域将会有更广泛的应用。此外,通过抑制P-gp的外排功能,一些药用辅料具有逆转肿瘤细胞多药耐药的作用,为临床提高抗肿瘤药物的疗效,减少肿瘤细胞对药物的外排,提高其对药物的敏感性,提供了研究方向和试验手段。
P-glycoprotein (P-gp), a 170 kDa ATP dependent membrane transporter, acts as a drug efflux pump. P-gp can exports structurally diverse hydrophobic compounds from serosa to mucosa and thus reduce the absorption and bioavailability of a wide range of drugs which are substrates for this membrane transporter. Inhibiting the drug efflux pump can increase the absorption of some orally drugs and decrease the renal clearance and hepatic-clearance. Many early P-gp modulator agents, such as verapamil and propafenone, often result in unacceptable toxicity that limits their clinical use. Therefore, to develop some P-gp modulators with less toxicity and non-pharmaco-activity is very important and pharmaceutical scientists all over the world have focus on this research field. In this dissertation pharmaceutical excipients were selcted to investigate their inhibitory effect on P-glycoprotein to search for some P-gp modulator agents with non-toxicity and non-pharmaco-activity. In this study celiprolol was used as the P-gp probe drug and Caco-2 monolayer model, everted gut sacs experiment in vitro, intestinal perfusion in situ and intestine absorption model in vivo were employed to study the effect of P-gp efflux pump on transmembrane transport and absorption of celiprolol. Pharmaceutical excipients with strong inhibitory effect on P-gp were sieved by comparing the inhibitory tensity of pharmaceutical adjuvant sorts and concentration.
Caco-2 cell monolayer model has gained enormous popularity as a convenient and high-effective in vitro model for absorption mechanism study of oral drugs and high throughput screening method for drug candidates. The integrity of Caco-2 cell monolayer was dectected by testing the TEER value by Millicell-ERS equipment and phenol permeation experiment. Caco-2 cell monolayer model was then established to perform the bi-directional transport experient of celiprolol with verapamil as positive control. The concentration of celiprolol was determined by HPLC and apparent permeability coefficient(Papp) and efflux ratio (ER) value were calculated according to drug transmembrane transport amount. Then the inhibitory effect of pharmaceutical excipients on P-gp was evaluated with the parameters of Papp and ER. The results showed that Papp of celiprolol transport from apical to basolateral was far lower than that of basolateral to apical direction. With P-gp inhibitor verapamil, Papp of celiprolol transport from apical to basolateral increased by 30% and that from basolateral to apical decreased by 50%, which indicates that efflux protein P-gp plays a vital role in the transepithelial transport of celiprolol. Pharmaceutial adjuvants F-68, Cremophor EL, Cremophor RH40, T-80, PEG200, PEG400, PEG4000,β-CD, HP-β-CD, PVP K30, TritonX 100 and S-40 can also enhance Papp of apical to basolateral direction and reduce Papp of basolateral to apical direction. With the concentration of T-20, T-60, PEG1000, PEG2000, PEG6000 and PVP K10 increased, they would exhibit similar effect. But Lecithin almost has no influence on Papp of both direction transport, while SDS showed great cytotoxicity which result in abnormal alteration in this parameter. Furthermore, ER value was analyzed with SPSS software to elucidate the inhibitory effect strength of pharmaceutical adjuvants on P-gp. The result showed that F-68, Cremophor EL, Cremophor RH40, T-20, T-60, T-80, PEG200, PEG400,β-CD, HP-β-CD and S-40 could significantly inhibit excretion cross Caco-2 monolayers and enhance absorption. In conclusion, these pharmaceutical excipients had an ability to inhibit the P-gp efflux pump obviously in Caco-2 cell monolayer model.
Everted gut sacs in vitro was used for the study of the transport function of P-gp with probe drug celiprolol. Jejunal segment and colon segment of rat intestine were studied in the absorption model and efflux model in vitro, respectively. With verapamil set as positive control, the transport characteristic of celiprolol under the influence of low, medium and high concentration of pharmaceutical adjuvants F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400, PEG1000, HP-β-CD and S-40 was investigated to assess the contribution of P-gp on celiprolol absorptive promotion and efflux suppression. The transport samples from mucosal to serosal side and serosal to mucosal side in the present of various concentrations of pharmacrutical excipients were dectected by HPLC. The results of everted gut sac absorption model processed by SPSS show that F-68, Cremophor RH40, PEG400 and T-80 could significantly promote intestinal absorption while only at the higher concentration Cremophor EL, T-60, PEG200, PEG1000 and S-40 could increase absorptive transport across intestinal mucous membrane. However, there is no significant influence of HP-β-CD on M→S absorption. Furthermore, the results of excretive model in vitro showed that F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400 and S-40 could inhibit the efflux of Celiprolol at the low, middle and high concentration while PEG1000 and HP-β-CD inhibit the P-gp efflux pump at much higher concentration.
Rat intestinal perfusion model in situ was widespreadly used not only for oral application and absorption of drugs and preparations, but also for transport mechanism across biomembrane. This model was established and performed using celiprolol Krebs solution containing phenol to indicate the water ingredient alteration. To study the effect of 10 pharmaceutical excipients on the efflux function of P-gp the concentration of celiprolol in the effluent solution was detected and parameters absorptive rate constant (ka) and effective permeability coefficients (Peff) were calculated. The results processed by SPSS indicated that F-68, Cremophor EL, Cremophor RH40, T-60, T-80, PEG200, PEG400 and S-40 could increase ka and Peff which showed that these pharmaceutical excipients facilitate absorption process via inhibiting P-gp, but these parameters were not affected by PEG1000 and HP-β-CD.
Intestinal absorption experiment in vivo was set up to detect the concentration of celiprolol by collecting blood plasma samples. This in vivo model was used for detecting whether 10 pharmaceutical excipients such as F-68, Cremophor EL and T-80 had the ability of inhibiting P-gp efflux pump at the low, middle, high concentration. The plasma drug concentration-time data was processed by pharmacokinetic software DAS. The inhibitory effect on P-gp in vivo was investigated by the pharmacokinetics parameters of celiprolol after the administration of different doses of pharmaceutical excipients. From the concentration-time curve, it could be obtained that the drug concentration in plasma raised to a distinct extent with coadministration of F-68, Cremophor EL, Cremophor RH40, T-80, PEG200, PEG400 and S-40 while that in plasma maintain at the same level of negative control with coadministration of T-60, PEG1000 and HP-β-CD. From the pharmacokinetics parameters analyzed by DAS, the half life time (t1/2) of celiprolol prolonged and clearance rate (CL) decreased after giving pharmaceutical excipients. Statistical analysis results of the area under the curve (AUC) showed that high concentration group of F-68, Cremophor RH40, PEG200, PEG400 and S-40 and medium, high concentration group of Cremophor EL and T-80 could increase the absorption of P-gp substrate celiprolol. But these three pharmaceutical adjuvants T-60, PEG1000 and HP-β-CD do not affect the pharmacokinetic process in vivo.
Based on the screening resuluts of different models, four pharmaceutical excipients F-68, Cremophor EL, T-80 and PEG400 were selected to investigate the influence of intestinal P-gp expression. The relationship between P-gp inhibitor and protein expression in intestine was studied by BCA protein assay kit, SDS-PAGE with gel image analysis technique and western blot analysis. In contrast with control group, experimental groups coadministration with F-68, Cremophor EL, T-80 or PEG400 increased the expression of P-gp dramatically. Thus efflux protein P-gp could be induced by its substrate and inhibitors.
To investigate the effect of four pharmaceutic excipients on the absorption of ganciclovir, Caco-2 cell model and intestinal absorption experiment in vivo were exploited to value the extent of absorption promotion. The results in cell model showed that Papp in AP→BL direction increased and that in BL→AP direction decreased, which resulted in that ER reduced significantly. From the statistical analysis results, there exist significant difference between negative control group and verapamil group or medium and high concentration group of pharmaceutical excipients inhibitor F-68, Cremophor EL, T-80 and PEG400 while other groups showed no significant difference. Moreover, intestinal absorption experiment in vivo discovered that only low and medium concentration group of Cremophor EL, low concentration group of T-80 and PEG400 put significant effect on AUC of ganciclovir while other groups showed their enhancement effectiveness with no significant difference.
In conclusion, 20 pharmaceutical excipients were choosed to investigate the inhibitory effect on P-gp in Caco-2 model and then 10 adjuvants were selected for animal research. Furthermore, research on intestinal P-gp expression and ganciclovir transport feature were studied with the presence and absence of F-68, Cremophor EL, T-80 and PEG400. The results indicated that several pharmaceutical exicipients such as nonionic surfactant can reverse the efflux function of P-gp and improve the absorption and bioavailability of P-gp substrate celiprolol and ganciclovir, but these inhibitors also enhance the expression level of P-gp. Thus, excipient inhibitors could ameliorate absorprion and pharmacokinetic characters in vivo of some certain drugs via reversing the intestinal efflux function of P-gp not by inhibiting P-gp expression but probably by inhibiting ATP enzyme activity, competitive binding function site and P-gp conformational change.
Pharmaceutical excipients were used as a vehicle of water-soluble, liposoluble and even indissolvable drugs with low toxicity and satisfactory biocompatibility. Furthermore, it provides direction and method to decrease efflux of cytotoxic drugs from the cancer cells, promote the effect of clinical therapeutic efficacy, recover the sensitivity of the cancer cells to the cytotoxic drugs, and improve the characteristics of pharmacokinetics of some drugs such as increasing the absorption of some orally drugs, decreasing the renal clearance and hepat-clearance.
引文
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