P-糖蛋白对番荔枝酰胺衍生物FLZ在Caco-2细胞模型中跨膜转运的影响

目的考察转运蛋白P-糖蛋白(P-glycoprotein,P-gp)对番荔枝酰胺衍生物FLZ体外跨血脑屏障(Blood-brain barrier,BBB)转运的影响。方法采用体外培养的Caco-2细胞建立体外BBB模型,研究1,5,10μM FLZ跨膜转运特性。并探讨加入P-gp抑制剂5μM zosuquidar后FLZ跨膜转运的表观渗透系数和外排率的变化。结果 FLZ在Caco-2细胞模型上显示出了极性转运特性,Papp(B~A)>Papp(A~B),并呈现出良好的剂量依赖关系。同时FLZ在Caco-2细胞的跨膜转运中也呈现外排现象,1,5,10μM FLZ的外排率ER值分别为2.56,3.67和5.06。P-gp抑制剂zosuquidar可以显著降低FLZ外排,增加转运。10μM FLZ的外排率由5.06降低为1.94,下降了2.6倍。结论 FLZ具有P-gp的底物特性,P-gp参与了FLZ在BBB跨膜转运中的外排。     

芦丁在Caco-2细胞模型中吸收和外排机制的研究

利用Caco-2细胞模型研究芦丁在小肠上皮的摄取、跨膜转运及外排动力学机制,评价孵育时间、芦丁浓度、p-糖蛋白抑制剂环孢素A和多药耐药相关蛋白抑制剂维拉帕米对芦丁的细胞摄取与转运的影响.结果表明,药物摄取量与孵育时间、药物浓度呈正相关,环孢素A和维拉帕米对芦丁的细胞摄取量无显著影响(P＞0.05).不同浓度药物从基底侧(basolateral,BL)到肠腔侧(Apical,AP)的表观渗透系数Papp,BL-AP与AP到BL的Papp,AP-BL比值均在0.5～1.5.试验结果提示芦丁是以被动扩散为主要转运方式被小肠上皮细胞摄取和转运,且不受外排蛋白外排作用的影响.     

药物外排泵和转运蛋白对口服药物吸收影响的研究进展

人体肠道的药物外排泵和转运蛋白对口服药物的吸收有较大影响,常见的药物外排泵有P-糖蛋白和多药耐药相关蛋白,会降低相关底物吸收;其它的一些转运蛋白如有机离子转运蛋白家族、H+/单羧酸共转运蛋白和肽转运蛋白可促进相关底物的吸收.作者对药物外排泵和转运蛋白近年来的相关研究进行了综述,并指出了药物外排泵和转运蛋白今后可能的研究方向.     

坎地沙坦与坎地沙坦酯在Caco-2细胞模型中的转运特性

目的 研究坎地沙坦(Cand)与坎地沙坦酯(Cil)在Caco-2细胞中的跨膜转运特征.方法 采用Caco-2细胞单层膜模型来考察药物的浓度、介质pH与P-gp抑制剂维拉帕米对Cand与Cil跨膜转运的影响,并比较两者双向跨膜转运的差异.结果 两者的吸收转运具有pH依赖性,分泌转运具有浓度依赖性,其分泌(BL-AP)方向转运均快于吸收(AP-BL)方向转运,且Cil的AP-BL方向转运比Cand快;在P-gp抑制剂维拉帕米存在下,两者的转运外排率显著下降(P<0.01).结论 Cil容易通过Caco-2细胞的单层膜,且外排蛋白参与了Cand与Cil的跨膜转运.     

8-异丙胺亚甲基橙皮素(IPHP)在Caco-2细胞模型上跨膜转运的研究

目的利用Caco-2细胞模型研究8-异丙胺亚甲基橙皮素(IPHP)在小肠吸收转运的机制。方法在Caco-2细胞模型上进行IPHP的跨膜转运实验,探讨药物浓度、p H、温度、P-gp抑制剂维拉帕米、MRP2抑制剂MK-571和丙磺舒对IPHP在体外细胞模型上跨膜转运的影响。结果 IPHP在Caco-2细胞模型上的转运具有一定的浓度依赖性,IPHP不同浓度从A侧到B侧的渗透系数Papp(AP-BL)(×10-5)分别为:(2.21±0.200)、(3.56±0.306)、(3.81±0.179)、(4.23±0.229)、(4.17±0.262)cm·s-1,B侧到A侧的渗透系数Papp(BL-AP)(×10-5)分别为:(3.57±0.209)、(4.51±0.113)、(4.97±0.229)、(5.24±0.550)、(5.07±0.557)cm·s-1,外排率分别为:1.61、1.26、1.3、1.23、1.21。温度和p H对其转运均有影响,而P-gp抑制剂对于IPHP的转运没有明显的影响,MRP2抑制剂在一定程度上增加了IPHP的转运量(P<0.05)。结论 IPHP在Caco-2细胞模型上的转运方式主要是被动扩散,且其转运不受P-gp外排蛋白影响,而外排蛋白MRP2可能参与了IPHP的外排转运。     

P-糖蛋白抑制剂对蝙蝠葛碱跨膜转运的影响

目的 研究Caco-2细胞模型中P-糖蛋白抑制剂对蝙蝠葛碱跨膜转运的影响.方法 采用HPLC法测定转运液中蝙蝠葛碱的含量;采用Caco-2细胞模型双向转运实验,考察不同浓度维拉帕米、环孢素A和醋酸地塞米松3种p-糖蛋白抑制剂对蝙蝠葛碱跨膜转运的影响.结果 加入3种P-糖蛋白抑制剂后,蝙蝠葛碱的Papp(A-B)均有一定程度增加,而Papp(B-A)均有显著降低(P＜0.05),即均抑制了蝙蝠葛碱的外排转运.结论 外排转运体P-糖蛋白对蝙蝠葛碱有外排作用,蝙蝠葛碱可能为P-糖蛋白底物.     

鲍曼不动杆菌抗生素主动外排泵转运系统与外排泵抑制剂

鲍曼不动杆菌(Acinetobacter baumannii)是医院内感染的重要病原菌,其耐药率呈现上升的趋势。该菌存在多种耐药机制,其中药物主动外排转运系统或外排泵介导的抗菌药物主动外排与多重耐药(multi-dmg resistance,MDR)密切相关,是近年来研究细菌多重耐药机制的重点。外排泵抑制剂(efflux pump inhibitors,EPIs)的研发有助于克服细菌主动外排机制导致的多重耐药性,为逆转细菌的多重耐药提供了一条新思路。本文就近年来鲍曼不动杆菌的药物主动外排转运系统与外排泵抑制剂的研究进展进行综述。     

细菌多药外排系统及其研究方法的进展

位于细菌细胞膜上的多药外排泵可将多种结构无关药物排出，本文综述了细菌主要外排泵包括初级和次级转运系统的分子生物学特征及外排蛋白结构与药物识别和转运上的关系；同时总结了对外排蛋白进行结构与功能关系研究的主要的方法，包括定点诱变、螺旋空间排列和结晶等。     

细菌RND外排泵的结构与作用机制研究进展

摘要：细菌RND(resistance-nodulation-cell division)外排泵是一类由内膜转运蛋白、周质融合蛋白、外膜外排蛋白组成的蛋 白复合体,能够将抗菌药物和多种小分子化合物如毒素、染料、洗涤剂、脂质、群感信号分子等排出细菌细胞外。通过影响 RND外排泵的组装和功能,可逆转多重耐药性的发生与防止耐药性的发展。细菌具有多种外排泵,本文以RND外排泵为代表, 介绍与其结构以及外排药物的转运和调控机制的最新进展。     

ABC转运蛋白与肿瘤多药耐药

目的 :介绍ABC转运蛋白与肿瘤多药耐药的研究进展 ,阐述ABC转运蛋白高表达是多药耐药的重要机制之一。方法 :检索国内外大量文献资料进行汇总、综述。结果 :ABC转运蛋白是具有ATP结合区的单向底物外排泵。P -糖蛋白高表达是肿瘤细胞产生多药耐药的经典路径 ,多药耐药相关蛋白高表达能导致非P -糖蛋白介导的多药耐药。结论 :开发多药耐药蛋白抑制剂有广阔的应用价值 ,但注重其药理作用的同时还应对其潜在的毒副作用保持高度警惕     

Polymeric and low molecular mass efflux pump inhibitors for oral drug delivery

Transmembrane located transporter proteins can be responsible for the low bioavailability of orally administered drugs. Drug delivery systems which can overcome this barrier caused by efflux pumps are therefore highly on demand. Within the current review, intestinal located efflux transporters, methods to identify efflux pump substrates and inhibitors as well as strategies to minimize efflux pump mediated transport of drugs are discussed. Methods include in silico screening, transport and accumulation studies and monitoring of the ATPase activity. An emphasis has been placed on efflux pump inhibitors including low molecular mass inhibitors such as cyclosporine, PSC833 or KR30031 and polymeric inhibitors such as myrj, thiomers and cremophor EL. Also formulation approaches to circumvent intestinal segments with high efflux pump expression are briefly addressed.     

Efflux transporters and their clinical relevance

It is increasingly recognized that efflux transporters play an important role, not only in chemo protection e.g. multi-drug resistance, but also in the absorption, distribution, and elimination of drugs. The modulation of drug transporters through inhibition or induction can lead to significant drug-drug interactions by affecting intestinal absorption, renal secretion, and biliary excretion, thereby changing the systemic or target tissue exposure of the drug. Few clinically significant drug interactions that affect efficacy and safety are due to a single mechanism and there is considerable overlap of substrates, inhibitors, and inducers of efflux transporters and drug metabolizing enzymes, such as CYP3A. As well, genetic polymorphisms of efflux transporters have been correlated with human disease and variability of drug exposure. Accordingly, this review will discuss drug interactions and suitable probe substrates, as well as, the clinical relevance of the variability and modulation of efflux transporters and the exploitation of substrates as diagnostic tools. An update is given on inhibitors, which clinically reverse drug resistance and minimize the risk of metabolic interactions.     

介导肿瘤多药耐药的ATP结合盒转运体的研究进展

多药耐药（MDR）是阻碍肿瘤化疗成功的一大障碍,其机制之一就是耐药的肿瘤细胞高表达三磷酸腺苷（ATP）结合盒（ABC）转运体。依据此机制提出克服肿瘤细胞耐药的策略即开发外排转运体抑制剂,以期逆转MDR。最近的研究发现肿瘤干细胞也可能是通过表达外排转运体天然耐药,这就提供了一个新的抗癌药物作用靶点。对介导肿瘤细胞多药耐药的ABC转运体及其抑制剂的开发作一综述。     

介导肿瘤多药耐药的ATP结合盒转运体的研究进展

多药耐药（MDR）是阻碍肿瘤化疗成功的一大障碍,其机制之一就是耐药的肿瘤细胞高表达三磷酸腺苷（ATP）结合盒（ABC）转运体。依据此机制提出克服肿瘤细胞耐药的策略即开发外排转运体抑制剂,以期逆转MDR。最近的研究发现肿瘤干细胞也可能是通过表达外排转运体天然耐药,这就提供了一个新的抗癌药物作用靶点。对介导肿瘤细胞多药耐药的ABC转运体及其抑制剂的开发作一综述。     

Human Primary Cell-Based Organotypic Microtissues for Modeling Small Intestinal Drug Absorption

Purpose

The study evaluates the use of new in vitro primary human cell-based organotypic small intestinal (SMI) microtissues for predicting intestinal drug absorption and drug-drug interaction.

Methods

The SMI microtissues were reconstructed using human intestinal fibroblasts and enterocytes cultured on a permeable support. To evaluate the suitability of the intestinal microtissues to model drug absorption, the permeability coefficients across the microtissues were determined for a panel of 11 benchmark drugs with known human absorption and Caco-2 permeability data. Drug-drug interactions were examined using efflux transporter substrates and inhibitors.

Results

The 3D–intestinal microtissues recapitulate the structural features and physiological barrier properties of the human small intestine. The microtissues also expressed drug transporters and metabolizing enzymes found on the intestinal wall. Functionally, the SMI microtissues were able to discriminate between low and high permeability drugs and correlated better with human absorption data (r²?=?0.91) compared to Caco-2 cells (r²?=?0.71). Finally, the functionality of efflux transporters was confirmed using efflux substrates and inhibitors which resulted in efflux ratios of >2.0 fold and by a decrease in efflux ratios following the addition of inhibitors.

Conclusion

The SMI microtissues appear to be a useful pre-clinical tool for predicting drug bioavailability of orally administered drugs.

     

Application and limitation of inhibitors in drug-transporter interactions studies

The objective of the present study was to investigate the reliability of transporter inhibitors in the elucidation of drug-transporter interactions when multiple transporters are present in a test system. The bidirectional permeabilities of digoxin, estrone-3-sulfate (E3S), and sulfasalazine, substrates of P-gp, BCRP/MRP2 and unspecified efflux transporters, respectively, were examined in Caco-2 and MDR-MDCK cells in the absence and presence of transporter inhibitors: CsA (P-gp), FTC (BCRP) and MK571 (MRP). Digoxin showed significant efflux ratios (ER) in both Caco-2 (ER=17) and MDR-MDCK (ER=120), whereas E3S and sulfasalazine only showed significant efflux in Caco-2 (ER=15 and 88, respectively) but not in MDR-MDCK cells (ER=1.1 and 1.3, respectively). CsA at 10 microM showed complete inhibition of digoxin efflux, partial inhibition of E3S efflux and no effect on sulfasalazine efflux. FTC and MK571 had different inhibitory effects on the efflux of these compounds. The present study shows evidence of the functional expression of multiple efflux transporter systems in Caco-2 cells. Although the use of Caco-2 cells and selected inhibitors of efflux transporters can provide useful mechanistic information on drug-drug interactions involving efflux transporters, the potential cross-reaction of inhibitors with multiple transporters makes it difficult to discern the role of individual transporters in drug transport or drug-drug interactions.     

P-Glycoprotein- and cytochrome P-450-mediated herbal drug interactions

P-Glycoprotein (P-gp), the most extensively studied ATP-binding cassette transporter, functions as a biological barrier by extruding toxic substances and xenobiotics out of cells. Drug efflux pumps such as P-gp play a functional role in determining the pharmacokinetics of drugs administered by oral and parenteral routes. Determining the activity of drug efflux transport proteins has important implications in the identification of substrates and/or inhibitors. The significant role of the small intestine in reducing the oral bioavailability of drugs is due to metabolic enzymes and efflux transporters. The role of cytochrome P-450 3A (CYP3A) and P-gp in intestinal drug disposition has been highlighted. This review examines the structure, localisation and functional role of P-gp, the mechanism of drug efflux and drug-herb interactions.     

Role of intestinal efflux transporters in the intestinal absorption of methotrexate in rats

The role of intestinal efflux transporters such as P-glycoprotein (P-gp), breast cancer resistance protein (BCRP) and multidrug resistance-associated proteins (MRPs) in intestinal absorption of methotrexate was examined in rats. In everted intestine, the mucosal efflux of methotrexate after application to serosal side was higher in jejunum than ileum, and the efflux in jejunum was suppressed by pantoprazole, a BCRP inhibitor, and probenecid, an MRP inhibitor, but not by verapamil, a P-gp inhibitor. The mucosal methotrexate efflux in ileum was suppressed by pantoprazole, but not by other inhibitors. On the other hand, the serosal efflux of methotrexate after application to mucosal side was greater in ileum than jejunum, and was suppressed by probenecid. In in-vivo rat studies, the intestinal absorption of methotrexate was significantly higher when methotrexate was administered to ileum than jejunum. Pantoprazole increased methotrexate absorption from jejunum and ileum. Probenecid increased the absorption of methotrexate from jejunum but decreased the absorption from ileum, as evaluated by peak plasma methotrexate levels. In conclusion, BCRP and MRPs are involved in the regional difference in absorption of methotrexate along the intestine, depending on their expression sites.     

Effects of amino acid chirality and the chemical linker on the cell permeation characteristics of cyclic prodrugs of opioid peptides

Previously, our laboratory showed that the oxymethyl-modified coumarinic acid (OMCA) cyclic prodrug of the opioid peptide DADLE ([D-Ala2,D-Leu5]-Enk, H-Tyr-D-Ala-Gly-Phe-D-Leu-OH) exhibited low permeation across both the intestinal mucosa and the blood-brain barrier (BBB). This low cell permeation arose from its strong substrate activity for efflux transporters in these biological barriers. In an attempt to determine whether the chirality of the amino acid asymmetric centers could influence the solution structure of the cyclic prodrugs and thus their substrate activities for efflux transporters, we synthesized cyclic prodrugs of the opioid peptides H-Tyr-Ala-Gly-Phe-D-Leu-OH ([Ala2,D-Leu5]-Enk), H-Tyr-D-Ala-Gly-Phe-Leu-OH ([D-Ala2,Leu5]-Enk), and H-Tyr-Ala-Gly-Phe-Leu-OH ([Ala2,Leu5]-Enk). In an attempt to determine whether the chemical linker (OMCA) bestowed efflux substrate activity on the cyclic prodrugs, we synthesized capped linear derivatives (acetylated on the N-terminal and amidated on the C-terminal end) of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk. The solution conformations of the cyclic prodrugs were determined by molecular dynamics simulations using two-dimensional NMR data. The physicochemical properties (molecular surface area, polar surface area, and cLogP) were estimated computationally using Sybyl. Cell permeation characteristics were assessed using Caco-2 cells in the presence and absence of known inhibitors of efflux transporters. Despite apparent differences in their solution conformations and their physicochemical properties, the cyclic prodrugs of DADLE, [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk all exhibited strong substrate activity for efflux transporters in Caco-2 cells. In contrast, the capped linear derivatives of [Ala2,D-Leu5]-Enk, [D-Ala2,Leu5]-Enk, and [Ala2,Leu5]-Enk exhibited very poor substrate activity for efflux transporters in Caco-2 cells. Therefore, the substrate activities of the cyclic prodrugs for efflux transporters in Caco-2 cells and in the intestinal mucosa and the BBB in vivo are most likely due to the chemical linker used to prepare these molecules and/or its effect on solution structures of the prodrugs.     

Role of Drug Efflux and Uptake Transporters in Atazanavir Intestinal Permeability and Drug-Drug Interactions

Purpose

To investigate the role of membrane-associated drug transporters in regulating the intestinal absorption of the HIV-1 protease inhibitor, atazanavir, and assess the potential contribution of these transporters in clinical interactions of atazanavir with other protease inhibitors and tenofovir disoproxil fumarate (TDF).

Methods

Intestinal permeability of atazanavir was investigated in vitro, using the Caco-2 cell line system grown on Transwell inserts, and in situ, by single-pass perfusion of rat intestinal segments, jejunum and ileum, in the absence or presence of standard transporter inhibitors or antiretroviral drugs.

Results

Atazanavir accumulation by Caco-2 cells was susceptible to inhibition by P-glycoprotein and organic anion transporting polypeptide (OATP) family inhibitors and several antiretroviral drugs (protease inhibitors, TDF). The secretory flux of atazanavir (basolateral-to-apical P_app) was 11.7-fold higher than its absorptive flux. This efflux ratio was reduced to 1.5–1.7 in the presence of P-glycoprotein inhibitors or ritonavir. P-glycoprotein inhibition also resulted in 1.5–2.5-fold increase in atazanavir absorption in situ. Co-administration of TDF, however, reduced atazanavir intestinal permeability by 13–49%, similar to the effect observed clinically.

Conclusions

Drug transporters such as P-glycoprotein and OATPs regulate intestinal permeability of atazanavir and may contribute to its poor oral bioavailability and drug-drug interactions with other protease inhibitors and TDF.