药物与肠道肽转运蛋白PEPT1的相互作用及影响因素

H^＋协同转运载体PEPT1主要存在于小肠上皮细胞的刷状缘膜上，肠道PEPT1对于消化道中蛋白质的降解产物二肽、三肽具有转运吸收的功能，另外肽类似药物如β-内酰胺类抗生素、血管紧张素转化酶抑制剂、非肽药物伐昔洛韦等也经此载体转运吸收。肠道PEPT1对于维持机体的内环境稳定以及药物的胃肠道吸收发挥重要作用。随着对PEPT1基因序列、蛋白结构、功能活性等方面研究的逐渐深入，对于调控PEPT1在膜上表达、影响其功能活性以及与底物亲和力的因素及相关的作用机制有了一定的了解，加之PEPT1广泛的底物专属性，使其成为新药开发中重要的药物传递的靶蛋白。了解药物与肠道肽转运蛋白PEPT1的相互作用及其影响因素，对于了解药物一药物相互作用，提高药物口服吸收的生物利用度，研究抗肿瘤药物的靶向治疗以及个体化给药等方面具有十分重要的意义。     

小肠寡肽转运蛋白及其在提高药物口服吸收中的应用

小肠上皮细胞刷状缘侧的寡肽转运蛋白(PEPT1)以及侧底膜的寡肽转运蛋白是寡肽转运蛋白(PEPT)的2种亚型,它们在寡肽及拟肽类药物(peptidomimetic drug)肠转运中发挥重要作用。通过前体药物(prodrug)设计将一些吸收差的药物修饰成类似二肽或三肽的结构,在PEPT1的介导下吸收,能够提高这些药物的口服生物利用度,这是非常有意义的。     

药物传递靶蛋白寡肽转运蛋白1的研究进展

目的:为相关临床安全用药和新药研发提供依据。方法:根据文献,综述了寡肽转运蛋白1(PEPT1)的组织分布、分子结构与功能、转运机制、底物、与药物相互作用等方面的内容。结果:PEPT1主要在小肠表达,肝和肾中表达较少;其基因编码的蛋白上有多个N-糖基化和蛋白激酶的识别位点,它们可能参与肽转运的调控,且其上的His-57是最关键的组氨酸残基,可能是转运蛋白发挥吸收功能时最关键的结合位点;其对大多数的二肽和三肽有高亲和性,能转运某些特定四肽,但不能转运更长的肽段;其转运机制为主动转运;其底物为二肽、三肽化合物等,但不包括氨基酸和四肽以上的大分子;其主要介导口服给药的肽类及肽类似物相关药物的相互作用。结论:虽然现国内有关PEPT1的研究才刚起步,但由于PEPT1具有底物丰富,能够转运亲脂性的、带电荷的以及不同大小的药物分子的特点,使其将会成为设计前药很好的靶蛋白。     

肽转运载体与药物转运

转运寡肽的载体称为肽转运载体(peptide trans- porter,PEFF),分为2种类型,即PEPT1和PEPT2。PEPT能以二肽和三肽作为生理性底物,与其他营养物的转运载体相比,有更广泛的底物特异性。结构相似的外源性化合物如β内酰胺类抗生素也可被PEPT识别。PEPT既可转运营养物质又可转运药     

病理状态对肠寡肽转运体PEPT1活性的调节

PEPT1是位于小肠刷状缘膜的寡肽转运体,介导蛋白消化产物(如二肽和三肽)及类肽药物(如β-内酰胺类抗生素)的摄取和转运。营养不良及代谢失调(如高蛋白饮食、禁食和糖尿病)均可引起PEPT1基因和蛋白表达发生变化,一些临床常见病(如溃疡性结肠炎、克罗恩病、短肠综合征)也可诱导肠道PEPT1的表达和功能发生改变。检索PubMed数据库及参考互联网文献,并进行分析综述,探讨不同病理状态对PEPT1活性调节的机制,为患者的营养支持及药物治疗方案提供理论依据。     

阿糖胞苷5′-缬氨酸酯前体药物的小肠吸收机制

目的研究阿糖胞苷的5′-缬氨酸酯前体药物在大鼠小肠内的吸收情况。方法运用单向灌流模型研究药物在小肠内的吸收机制,利用高效液相色谱法测定药物和酚红在灌流液中的浓度。结果阿糖胞苷5′-缬氨酸酯前体药物的小肠渗透率是原药阿糖胞苷的10.6倍,在小肠内的吸收存在浓度依赖性,能够被小肠寡肽转运蛋白的专属底物头孢氨苄明显抑制。结论阿糖胞苷5′-缬氨酸酯前体药物是小肠寡肽转运蛋白的底物,在大鼠小肠内的吸收是由小肠寡肽转运蛋白介导的主动转运过程。     

阿糖胞苷5’-缬氨酸酯前体药物的小肠吸收机制

目的 研究阿糖胞苷的5’-缬氨酸酯前药在大鼠小肠内的吸收情况。方法 运用单向灌流模型研究药物在小肠内的吸收机制，利用高效液相色谱法测定药物和酚红在灌流液中的浓度。结果 阿糖胞苷 5’-缬氨酸酯前药的小肠渗透率是母药阿糖胞苷的10.6倍，在小肠内的吸收存在浓度依赖性，能够被小肠寡肽转运蛋白的专属底物头孢氨苄明显抑制。结论 阿糖胞苷 5’-缬氨酸酯前药是小肠寡肽转运蛋白的底物，在大鼠小肠内的吸收是由小肠寡肽转运蛋白介导的主动转运过程。     

寡肽转运体在生理、药物转运的重要作用及临床相关性

寡肽转运体（PEPTs）属于溶质转运体（SLC）大家族,以H⁺梯度为驱动力,包括PEPT1和PEPT2。PEPT1是低亲和力、高容量转运蛋白,主要表达于小肠;而PEPT2是高亲和力、低容量的转运蛋白,主要在肾脏、脑和肺中表达,在生物体中分布较广。PEPTs除重吸收二肽和三肽以及维持脑中神经肽的稳态作用外,还能够吸收和处置许多重要的化合物,如一些氨基头孢菌素、血管紧张素转化酶抑制剂、抗病毒前药等,而且PEPTs也与一些肠道疾病和癌症相关。因此综述了PEPTs在生理、药物转运中的重要作用及临床相关性。     

基于procaspase-3激活的抗肿瘤活性分子SM-1吸收机制研究

目的初步探讨新型抗肿瘤活性分子SM-1的吸收特征,为其成药性评价以及剂型设计提供研究基础。方法分别采用Caco-2细胞模型及大鼠在体肠灌流模型研究SM-1吸收特征,并通过大鼠体内药动学研究综合评价SM-1在体内的吸收程度。结果在10⁴0 mg·L-1时SM-1的吸收以被动扩散为主,其双向转运过程可能与浓度无关。在2540 mg·L-1时SM-1的吸收以被动扩散为主,其双向转运过程可能与浓度无关。在25¹00 mg·L-1内,SM-1的Ka与Peff差异无显著性(P>0.05),说明药物吸收无自身浓度抑制作用,SM-1的小肠吸收表现为被动扩散机制,属于高渗透性化合物。SM-1在十二指肠吸收优于其他肠段(P<0.05),在空肠、回肠、结肠的吸收差异无显著性(P>0.05)。初步药动学研究显示,SM-1在大鼠体内绝对生物利用度为29.3%。结论 SM-1渗透性高,在肠道内吸收良好,且吸收机制主要以被动扩散为主,不受转运蛋白外排作用影响,大鼠体内绝对生物利用度较低。     

几种多肽在大鼠离体小肠中的吸收

在大鼠的离体小肠标本中比较几种多肽、蛋白质和氨基酸的吸收率。主要通过两种方法计算肽在小肠中的吸收率:一是通过测定肽吸收前后的蛋白质含量,另一种是运用HPLC测定肽吸收前后分子质量图谱。结果:小肠能吸收多种肽(白蛋白肽、大豆肽、菜籽肽、花生肽和胶原肽),被吸收的肽的相对分子质量多在1000以内。小肠对肽的吸收率高于对氨基酸和蛋白质的吸收率。小肠各段均具有吸收肽的功能。该研究为肽类生物药物的口服途径吸收提供了实验依据。     

Peptide derivation of poorly absorbable drug allows intestinal absorption via peptide transporter

The purpose of the present study was to examine whether the intestinal absorption of low-permeability drugs could be improved by utilization of the intestinal influx transporter PEPT1. We investigated whether peptide derivatives of poorly absorbable nonamino acid-like drugs might be substrates of PEPT1, using rebamipide (Reb) as a model drug. We synthesized several peptide derivatives of rebamipide and examined their inhibitory effect on the uptake of [(3)H]Gly-Sar by PEPT1-expressing HeLa cells. Some of the peptide derivatives inhibited PEPT1-mediated uptake of [(3)H]Gly-Sar. Next, uptake of the inhibitory peptide derivatives was evaluated in PEPT1-expressing Xenopus oocytes and HeLa cells. Ser(Reb)-Gly exhibited significantly increased uptake by PEPT1-expressing cells in comparison with that by mock cells. The permeability of Ser(Reb)-Gly across a Caco-2 cell monolayer was significantly higher than that of rebamipide itself, and the transport was decreased in the presence of PEPT1 substrates. Further, a rat intestinal perfusion study revealed increased absorption of Ser(Reb)-Gly compared with rebamipide. These results demonstrate that the addition of a dipeptide moiety to a poorly absorbable nonpeptide/nonamino acid-like drug can result in absorption via the intestinal transporter PEPT1, though there is some selectivity as regards the structure of the added peptide moiety.     

Floxuridine Amino Acid Ester Prodrugs: Enhancing Caco-2 Permeability and Resistance to Glycosidic Bond Metabolism

Purpose The aim of this study was to synthesize amino acid ester prodrugs of 5-fluoro-2′-deoxyuridine (floxuridine) to enhance intestinal absorption and resistance to glycosidic bond metabolism.Methods Amino acid ester prodrugs were synthesized and examined for their hydrolytic stability in human plasma, in Caco-2 cell homogenates, and in the presence of thymidine phosphorylase. Glycyl-l-sarcosine uptake inhibition and direct uptake studies with HeLa/PEPT1 cells [HeLa cells overexpressing oligopeptide transporter (PEPT1)] were conducted to determine PEPT1-mediated transport and compared with permeability of the prodrugs across Caco-2 monolayers.Results Isoleucyl prodrugs exhibited the highest chemical and enzymatic stability. The prodrugs enhanced the stability of the glycosidic bond of floxuridine. Thymidine phosphorylase rapidly cleaved floxuridine to 5-fluorouracil, whereas with the prodrugs no detectable glycosidic bond cleavage was observed. The 5′-l-isoleucyl and 5′-l-valyl monoester prodrugs exhibited 8- and 19-fold PEPT1-mediated uptake enhancement in HeLa/PEPT1 cells, respectively. Uptake enhancement in HeLa/PEPT1 cells correlated highly with Caco-2 permeability for all prodrugs tested. Caco-2 permeability of 5′-l-isoleucyl and 5′-l-valyl prodrugs was 8- to 11-fold greater compared with floxuridine.Conclusions Amino acid ester prodrugs such as isoleucyl floxuridine that exhibit enhanced Caco-2 transport and slower rate of enzymatic activation to parent, and that are highly resistant to metabolism by thymidine phosphorylase may improve oral delivery and therapeutic index of floxuridine.     

Development of Novel Lipophilic Derivatives of DADLE (Leucine Enkephalin Analogue): Intestinal Permeability Characteristics of DADLE Derivatives in Rats

Purpose: The objective of this study is to examine the intestinal permeability of novel lipophilic derivatives of DADLE (Tyr- D-Ala-Gly-Phe-D-Leu), an enkephalin analogue, using isolated rat intestinal membranes. Methods: The novel lipophilic derivatives of DADLE were synthesized by chemical modification with various fatty acids at the C terminus. The pharmacological activities of these DADLE derivatives were assessed by a hot plate test. The intestinal permeability of these derivatives was estimated by the in vitro Ussing chamber method. Results: We obtained four different DADLE derivatives including acetyl-DADLE (DADLE-C2), butyryl-DADLE (DADLE-C4), caproyl-DADLE (DADLE-C6), and caprylyl-DADLE (DADLE-C8). All the derivatives of DADLE had at least 75 % of the activity of native DADLE, suggesting that chemical modification of DADLE at the C terminus did not markedly affect its pharmacological activity. These DADLE derivatives were more stable than native DADLE in jejunal and colonic homogenates. A bell-shaped profile was observed between the apparent permeability coefficients (Papp) of DADLE derivatives and lipophilicity. In particular, DADLE-C4 had the greatest permeability characteristics across the intestinal membrane of the acyl derivatives studied in this experiment. The permeability of DADLE-C4 across the jejunal membrane was further improved in the presence of puromycin, amastatin, and sodium glycocholate (NaGC), all at a concentration of 0.5 mM. Conclusions: We suggest that the combination of chemical modification with butyric acid and the application of a protease inhibitor are effective for improving the absorption of DADLE across the intestinal membrane.     

5′-Amino Acid Esters of Antiviral Nucleosides, Acyclovir, and AZT Are Absorbed by the Intestinal PEPT1 Peptide Transporter

Purpose. General use of nucleoside analogues in the treatment of viral infections and cancer is often limited by poor oral absorption. Valacyclovir, a water soluble amino acid ester prodrug of acyclovir has been reported to increase the oral bioavailability of acyclovir but its absorption mechanism is unknown. This study characterized the intestinal absorption mechanism of 5-amino acid ester prodrugs of the antiviral drugs and examined the potential of amino acid esters as an effective strategy for improving oral drug absorption. Methods. Acyclovir (ACV) and Zidovudine (AZT) were selected as the different sugar-modified nucleo-side antiviral agents and synthesized to L-valyl esters of ACV and AZT (L-Val-ACV and L-Val-AZT), D-valyl ester of ACV (D-Val-ACV) and glycyl ester of ACV (Gly-ACV). The intestinal absorption mechanism of these 5-amino acid ester prodrugs was characterized in three different experimental systems; in siturat perfusion model, CHO/hPEPTl cells and Caco-2 cells. Results. Testing 5-amino acid ester prodrugs of acyclovir and AZT, we found that the prodrugs increased the intestinal permeability of the parent nucleoside analogue 3- to 10-fold. The dose- dependent permeation enhancement was selective for the L-amino acid esters. Competitive inhibition studies in rats and in CHO cells transfected with the human peptide transporter, hPEPTl, demonstrated that membrane transport of the prodrugs was mediated predominantly by the PEPT1 H⁺/dipeptide cotransporter even though these prodrugs did not possess a peptide bond. Finally, transport studies in Caco-2 cells confirmed that the 5-amino acid ester prodrugs enhanced the transcellular transport of the parent drug. Conclusions. This study demonstrates that L-amino acid-nucleoside chimeras can serve as prodrugs to enhance intestinal absorption via the PEPT1 transporter, providing a novel strategy for improving oral therapy of nucleoside drugs.     

PEPT1 Enhances the Uptake of Gabapentin via Trans-Stimulation of b<Superscript>0,+</Superscript> Exchange

Purpose The aims of this study were (1) to determine whether amino acid and dipeptide loading can improve the effective permeability of gabapentin and (2) to characterize the underlying mechanism that is responsible for this interaction. Materials and Methods An in situ single-pass rat intestinal perfusion model was used to assess the effective permeability of gabapentin in rat, in the absence and presence of cellular loading by amino acid and dipeptide mixtures. Results Compared to gabapentin alone, cellular loading with amino acid and dipeptide mixtures significantly improved the effective permeability of gabapentin by 46–79% in jejunum and by 67–72% in ileum (p ≤ 0.01). However, coperfusion of glycylsarcosine (i.e., PEPT1 substrate), methionine sulfoximine (i.e., glutamine synthase inhibitor), or lysine and arginine (i.e., b^0,+ substrates) with the amino acid and dipeptide mixtures compromised the intestinal uptake of gabapentin. Conclusions These findings demonstrate, for the first time, a direct relationship between the PEPT1-mediated uptake of a dipeptide and the trans-stimulated uptake of gabapentin (an amino acid-like drug) through the transport system b^0,+. This article is posthumous for David Fleisher.     

Quantitative Evaluation of PEPT1 Contribution to Oral Absorption of Cephalexin in Rats

Purpose  PEPT1 mediates the intestinal absorption of many drugs, but its contribution to oral absorption of drugs is still controversial. The objective of this study is to quantitatively evaluate the contribution of PEPT1 to oral absorption of cephalexin, a typical substrate for PEPT1, in rats. Materials and Methods  The absorbability of cephalexin via PEPT1 or passive diffusion was assessed in five intestinal segments by utilizing glycyl-proline as a competitive inhibitor by in-situ closed loop method. Absorption kinetics of cephalexin after oral administration was predicted by GI-Transit-Absorption model. Results  Absorbability of cephalexin was segment-dependent, and concentration-dependent in all the segments except for the lower ileum. Intrinsic absorption rate constant via PEPT1 ranged from 0.64 to 4.07 h⁻¹. The absorption rate constants via passive diffusion ranged from 0.78 to 1.24 h⁻¹. Plasma concentration–time profile of cephalexin was successfully predicted and the substantial contribution of PEPT1 to the oral absorption was calculated to be from 46% to 60% of total absorption. Simulation study indicated that 83% bioavailability would be expected for cephalexin even though PEPT1 does not function. Conclusions  PEPT1 substantially contributes to oral absorption of cephalexin, around a half of total absorption. However, the function of PEPT1 can be compensated by passive diffusion for cephalexin.     

In Vitro Intestinal Permeability of Factor Xa Inhibitors: Influence of Chemical Structure on Passive Transport and Susceptibility to Efflux

Purpose. To study the in vitro intestinal permeability of a number of newly synthesised factor Xa inhibitors to better understand the poor oral absorption of these compounds. Methods. The bidirectional transport of the fXa inhibitors was studied in the Caco-2 cell model and isolated rat ileal tissue. An attempt was made to characterize efflux mechanisms with the help of commonly used substrates and inhibitors of various transport proteins. In addition, the transport of the fXa inhibitors was studied in MDCK cells transfected with the human MDR1 gene and expressing large amounts of P-glycoprotein (Pgp). Results. The in vitro absorptive permeability was low for all but one of the fXa inhibitors. For compounds with non-substituted amidine, a charge (due to ionisation at neutral pH) may have resulted in poor membrane partitioning. Neutral compounds with substituted amidines were effluxed from the epithelial cells. The significance of the secretion process was illustrated by the results obtained for a neutral analogue showing high absorptive Caco-2 cell permeability that was not obviated by efflux. Transport inhibition studies in Caco-2 and permeability studies in the MDR1-transfected MDCK cells consistently showed that Pgp is not involved in the secretion of fXa inhibitors. Besides efflux, metabolic liability limited the permeation of the neutral lipophilic analogues with a carbamate ester. Conclusions. Poor intestinal permeability may be an important factor in the incomplete oral absorption of the bisbenzimidazole-type fXa inhibitors. Poor permeability may be related to poor membrane partitioning for hydrophilic analogues, whereas susceptibility to efflux transports and gastro-intestinal enzymatic degradation may limit the permeability of some of the neutral less hydrophilic derivatives.     

金莲花汤中葛根素、牡荆素、迷迭香酸和咖啡酸的肠吸收研究

目的 考察金莲花汤中葛根素、牡荆素、迷迭香酸和咖啡酸的肠吸收特性,确定金莲花汤中活性成分。方法 采用HPLC法测定肠吸收样品中咖啡酸、葛根素、牡荆素、迷迭香酸,以大鼠肠外翻吸收实验和Caco-2细胞模型转运实验计算各成分的累积吸收量、吸收速率常数和表观渗透系数。结果 随着金莲花汤的质量浓度增加,咖啡酸、葛根素、牡荆素、迷迭香酸的吸收速率常数呈线性关系增加,符合零级吸收速率,且吸收率均小于1.4%,在肠道中吸收效果普遍较差,其中咖啡酸的吸收较为明显。各成分的表观渗透系数值大小为迷迭香酸>咖啡酸>葛根素>牡荆素。结论 金莲花汤中葛根素、牡荆素、迷迭香酸和咖啡酸均可在肠道中被吸收,吸收方式为被动扩散,咖啡酸和迷迭香酸可能是金莲花汤中发挥药效的活性物质,葛根素和牡荆素可能是前药。     

Cellular Uptake Mechanism of Amino Acid Ester Prodrugs in Caco-2/hPEPT1 Cells Overexpressing a Human Peptide Transporter

Purpose. This study characterized the cellular uptake mechanism and hydrolysis of the amino acid ester prodrugs of nucleoside antiviral drugs in the transiently transfected Caco-2 cells overexpressing a human intestinal peptide transporter, hPEPTl (Caco-2/hPEPTl cells). Methods. Amino acid ester prodrugs of acyclovir and AZT were synthesized and their apical membrane permeability and hydrolysis were evaluated in Caco-2/hPEPTl cells. The cellular uptake mechanism of prodrugs was investigated through the competitive inhibition study in Caco-2/hPEPTl cells. Results. L-Valyl ester of acyclovir (L-Val-ACV) was approximately ten fold more permeable across the apical membrane than acyclovir and four times more permeable than D-valyl ester of acyclovir (D-Val-ACV). Correspondingly, L-valyl ester of AZT (L- Val-AZT) exhibited three fold higher cellular uptake than AZT. Therefore, amino acid ester prodrugs significantly increased the cellular uptake of the parent drugs and exhibited the D,L-stereoselectivity. Furthermore, prodrugs were rapidly hydrolyzed to the parent drugs by the intracellular hydrolysis, following the apical membrane transport. In the inhibition studies, cephalexin and small dipeptides strongly inhibited the cellular uptake of L-Val-ACV while L-valine had no effect, indicating that the peptide transporter is primarily responsible for the apical membrane transport of L-Val-ACV. In addition, the cellular uptake of L-Val-ACV was five times higher in Caco-2/hPEPT 1 cells than the uptake in the untransfected Caco-2 cells, implying the cellular uptake of L-Val-ACV was related to the enhancement of the peptide transport activity in Caco-2/hPEPTl cells. Conclusions. Caco-2/hPEPTl system is an efficient in vitro model for the uptake study of peptidyl derivatives. Amino acid ester prodrugs significantly improved the cellular uptake of the parent drugs via peptide transport mechanism and were rapidly converted to the active parent drugs by the intracellular hydrolysis.