莱柯霉素在Caco-2细胞单层模型中的转运特性研究

doi:10.16431/j.cnki.1671-7236.2023.12.031

摘要/Abstract

摘要： 【目的】研究莱柯霉素在Caco-2细胞单层模型中的转运特性,为进一步深入了解莱柯霉素在体内的吸收过程提供试验依据。【方法】建立Caco-2细胞单层模型,通过测定跨膜电阻(TEER)值、低渗透性药物荧光素钠通透性试验的表观渗透率Papp_(AP-BL)来验证Caco-2细胞单层模型的致密性并进行模型筛选。考察在Caco-2细胞单层模型中莱柯霉素从顶侧(apical side,AP)到基底侧(basolateral side,BL)及基底侧到顶侧的转运特性,分析药物浓度、pH及外排转运蛋白(P-gp、MRP2和BCRP)抑制剂3种因素对莱柯霉素转运的影响。用超高效液相色谱串联质谱(UPLC-MS/MS)法检测药物浓度,计算其表观渗透率和外排率。【结果】连续20 d的跨膜电阻值检测及荧光素钠通透性试验显示,随培养时间增加,Caco-2细胞单层模型的跨膜电阻值逐渐增加,在20 d时达到400 Ω·cm²左右;荧光素钠在0.1~6 μg/mL内线性关系良好(R²=0.991),计算荧光素钠AP侧向BL侧的表现渗透率(Papp_(AP-BL))为(1.57×10^-7±9.92×10^-9)cm/s,小于试验规定的0.5×10^-6cm/s,表明Caco-2细胞单层模型建立成功,可用于莱柯霉素的转运特性研究。莱柯霉素Papp_(AP-BL)值随时间无明显变化且水平较低,而BL侧向AP侧的表现渗透率(Papp_(BL-AP))值随时间递增,在孵育120 min时达到最高水平(2.0350×10^-6 cm/s),表明肠道外排莱柯霉素的能力较强。随莱柯霉素浓度递增,Papp_(BL-AP)值呈上升趋势,具有浓度依赖性。当溶液pH为8.0时,Papp_(BL-AP)值(39.3871×10^-6 cm/s)明显高于pH为6.5和7.4的溶液,表明碱性环境可加速莱柯霉素外排。维拉帕米、丙磺舒和GF120918 3种转运蛋白抑制剂均能显著抑制莱柯霉素由BL侧向AP侧的转运,外排率均＞1.5,可能有外排蛋白参与转运。【结论】莱柯霉素在体外Caco-2细胞单层模型上的转运较差,吸收率低,表现为低渗透性化合物;随着浓度和pH的升高,莱柯霉素外排增强;外排转运蛋白P-gp、MRP2及BCRP可能参与莱柯霉素转运过程。

关键词: Caco-2细胞单层模型; 莱柯霉素; 转运; 表观渗透率

Abstract: 【Objective】 This experiment was aimed to investigate the transport characteristics of lekethromycin across Caco-2 cell monolayer model,and to provide further experimental evidence for further understanding of the absorption process of lekethromycin in vivo.【Method】 A Caco-2 cell monolayer model was established to verify the compactness of the Caco-2 cell monolayer model and to screen the model by measuring the transmembrane resistance (TEER) value and the apparent permeability Papp_(AP-BL) of the low permeability drug fluorescein sodium permeability test.The transport characteristics of lekethromycin from the apical side (AP) to the basolateral side (BL) and from the basolateral side to the apical side in the Caco-2 cell monolayer model were investigated,and the effects of three factors,drug concentration,pH and efflux transporter (P-gp,MRP2 and BCRP) inhibitors on the transportation of lekethromycin in Caco-2 monolayer model were analyzed.The drug concentrations were measured by UPLC-MS/MS,and its apparent permeability and efflux ratio were calculated.【Result】 The resistance value test and sodium fluorescein leakage test for 20 days showed that with the increase of culture time,the transmembrane resistance value of Caco-2 cell monolayer model gradually increased and reached about 400 Ω·cm² at the 20th day.The linear relationship of fluorescein sodium was good within 0.1-6 μg/mL (R²=0.991),and the calculated fluorescein sodium Papp_(AP-BL) was (1.57×10^-7±9.92×10^-9) cm/s,which was less than 0.5×10^-6 cm/s specified in the test.The Caco-2 cell monolayer model was successfully established and could be used to study the transport characteristics of lekethromycin.Lekethromycin Papp_(AP-BL) values did not change appreciably over time and were low,while Papp_(BL-AP) values increased over time,reaching a maximum level of 2.0350×10^-6 cm/s at 120 min of incubation,and intestinal efflux of lekethromycin was strong.Papp_(BL-AP) values increased with increasing concentrations of lekethromycin in a concentration-dependent manner.When the pH of the solution was 8.0,the Papp_(BL-AP) value (39.3871×10^-6 cm/s) was significantly higher than that of the solutions at pH 6.5 and 7.4,and alkaline intestinal fluid accelerated lekethromycin efflux.Verapamil,probenecid,and GF120918 inhibitors all significantly inhibited the lateral transport of lekethromycin from BL to AP,with efflux ratios greater than 1.5,and efflux proteins might be involved in transport.【Conclusion】 Lekethromycin showed poor transport and low absorption rate in the in vitro Caco-2 cell monolayer model,showing low permeability compounds.With the increase of concentration and pH,efflux was enhanced,efflux transporters P-gp,MRP2 and BCRP might be involved in the transport process of lekethromycin.

Key words: Caco-2 cell monolayer model; lekethromycin; transport; apparent permeability

中图分类号:

S859.7

温泽宇, 肖红芝, 孙攀, 刘羽, 杨宇欣, 王悦, 李靖, 张璐, 龚晓会, 周德刚, 曹兴元. 莱柯霉素在Caco-2细胞单层模型中的转运特性研究[J]. 中国畜牧兽医, 2023, 50(12): 5094-5103.

WEN Zeyu, XIAO Hongzhi, SUN Pan, LIU Yu, YANG Yuxin, WANG Yue, LI Jing, ZHANG Lu, GONG Xiaohui, ZHOU Degang, CAO Xingyuan. Transport Characteristics of Lekethromycin Across Caco-2 Cell Monolayer Model[J]. China Animal Husbandry and Veterinary Medicine, 2023, 50(12): 5094-5103.

参考文献

[1] 尹晖,王亦琳,叶妮,等.UPLC-MS/MS法检测禽蛋中10种大环内酯类药物多残留的研究[J].中国兽医杂志,2020,56(8):77-83. YIN H,WANG Y L,YE N,et al.Determination of macrolides residue in poultry eggs by UPLC-MS/MS[J].Chinese Journal of Veterinary Medicine,2020,56(8):77-83.(in Chinese)
[2] 宫晓炜,马文瑛,陈启伟,等.畜禽支原体耐药性及耐药机制研究进展[J].中国畜牧兽医,2017,44(8):2489-2495. GONG X W,MA W Y,CHEN Q W,et al.Research progress on drug resistance and its mechanism of Mycoplasma in livestock and poultry[J].China Animal Husbandry&Veterinary Medicine,2017,44(8):2489-2495.(in Chinese)
[3] 李盛安,何国成,陈楠,等.高效液相色谱-质谱法测定脆肉鲩中大环内酯类药物的残留[J].农业与技术,2022,42(20):112-115. LI S A,HE G C,CHEN N,et al.Determination of macrolide residues in crisped grass carp by HPLC-MS/MS method[J].Agriculture and Technology,2022,42(20):112-115.(in Chinese)
[4] 杜国辉,姜海芳,焦燕,等.超高效液相色谱-串联质谱法测定配合饲料中大环内酯类药物[J].饲料研究,2021,44(16):104-107. DU G H,JIANG H F,JIAO Y,et al.Analysis of macrolides in complete feeds by ultra-performance liquid chromatography-tandem mass spectrometry[J].Feed Research,2021,44(16):104-107.(in Chinese)
[5] JOHNSON A R,CARLSON E E.Structure elucidation of macrolide antibiotics using MSn analysis and deuterium labelling[J].Journal of the American Society for Mass Spectrometry,2019,30(8):1464-1480.
[6] XIAO H, SUN P, QIU J,et al. Determination of lekethromycin, a novel macrolide lactone, in rat plasma by UPLC-MS/MS and its application to a pharmacokinetic study[J].Molecules, 2020, 25(20):4676.
[7] SUN P,XIAO H,QIU J,et al.Plasma protein binding rate and pharmacokinetics of lekethromycin in rats[J].Antibiotics,2022,11(9):1241.
[8] JUNG K H,OH S J,KANG K J,et al.Effects of P-gp and BCRP as brain efflux transporters on the uptake of[18F] FPEB in the murine brain[J].Synapse,2019,73(11):e22123.
[9] CUI J,LIU X,CHOW L.Flavonoids as P-gp inhibitors:A systematic review of SARs[J].Current Medicinal Chemistry,2019,26(25):4799-4831.
[10] AUBETS J,JANSAT J M,SALVA M,et al.No evidence for interactions of dimethylfumarate (DMF) and its main metabolite monomethylfumarate (MMF) with human cytochrome P450(CYP) enzymes and the P-glycoprotein (P-gp) drug transporter[J].Pharmacology Research&Perspectives,2019,7(6):e00540.
[11] ZHANG L,LIANG C,XU P,et al.Characterization of in vitro MRP2 transporter model based on intestinal organoids[J].Regulatory Toxicology and Pharmacology,2019,108:104449.
[12] BI X,YUAN Z,QU B,et al.Piperine enhances the bioavailability of silybin via inhibition of efflux transporters BCRP and MRP2[J].Phytomedicine,2019,54:98-108.
[13] XU P,ZHOU H,LI Y Z,et al.Baicalein enhances the oral bioavailability and hepatoprotective effects of silybin through the inhibition of efflux transporters BCRP and MRP2[J].Frontiers in Pharmacology,2018,9:1115.
[14] LARREGIEU C A,BENET L Z.Drug discovery and regulatory considerations for improving in silico and in vitro predictions that use Caco-2 as a surrogate for human intestinal permeability measurements[J].The AAPS Journal,2013,15:483-497.
[15] 杨海涛,王广基.Caco-2单层细胞模型及其在药学中的应用[J].药学学报,2000,10:797-800. YANG H T,WANG G J.Caco-2 cell monolayers model and its application in pharmacy[J].Acta Pharmaceutica Sinica,2000,10:797-800.(in Chinese)
[16] SONG P,XIAO S,ZHANG Y,et al.Mechanism of the intestinal absorption of six flavonoids from zizyphi spinosi semen across Caco-2 cell monolayer model[J].Current Drug Metabolism,2020,21(8):633-645.
[17] NATOLI M,LEONI B D,D'AGNANO I,et al.Good Caco-2 cell culture practices[J].Toxicology in Vitro,2012,26(8):1243-1246.
[18] SRINIVASAN B,KOLLI A R,ESCH M B,et al.TEER measurement techniques for in vitro barrier model systems[J].Journal of Laboratory Automation,2015,20(2):107-126.
[19] 张宇,班玉娟,王忠元,等.新型DPP-4抑制剂LGT-6在Caco-2细胞单层模型中的转运特性研究[J].化学试剂,2021,43(4):434-440. ZHANG Y,BAN Y J,WANG Z Y,et al.Transport characteristics of novel DPP-4 inhibitor LGT-6 in Caco-2 cell monolayer model[J].Chemical Reagents,2021,43(4):434-440.(in Chinese)
[20] CHEN J,LIN H,HU M.Absorption and metabolism of genistein and its five isoflavone analogs in the human intestinal Caco-2 model[J]. Cancer Chemotherapy and Pharmacology,2005,55:159-169.
[21] DE SOUZA J,BENET L Z,HUANG Y,et al.Comparison of bidirectional lamivudine and zidovudine transport using MDCK,MDCK-MDR1,and Caco-2 cell monolayers[J].Journal of Pharmaceutical Sciences, 2009,98(11):4413-4419.
[22] 胡杰,侯佳,李月婷,等.灯盏细辛提取物中3种活性成分在Caco-2细胞模型吸收机制的研究[J].中国药理学通报,2016,32(3):373-377. HU J,HOU J,LI Y T,et al.In vitro absorption mechanism of Erigeron breviscapus extract in Caco-2 cell monolayer model[J].Chinese Pharmacological Bulletin,2016,32(3):373-377.(in Chinese)
[23] DI L,KERNS E.Drug-like properties:Concepts,Structure Design and Methods from ADME to Toxicity Optimization[M].New York:Academic Press,2015.
[24] 黄静,肖红琴,李莹,等.红禾麻提取物在Caco-2细胞模型中的摄取转运研究[J].中国中药杂志,2022,47(20):5617-5626. HUANG J,XIAO H Q,LI Y,et al.Uptake and transport of laportea bulbifera extract in Caco-2 cell model[J].China Journal of Chinese Materia Medica,2022,47(20):5617-5626.(in Chinese)
[25] 蔡进,张丽,彭金刚,等.树豆酮酸A在Caco-2细胞单层模型的转运机制研究[J].中国药学杂志,2018,53(10):793-798. CAI J,ZHANG L,PENG J G,et al.Transport characteristics of cajanonic acid A across Caco-2 cell monolayer model[J].Chinese Pharmaceutical Journal,2018,53(10):793-798.(in Chinese)
[26] DAI D,TANG J,ROSE R,et al.Identification of variants of CYP3A4 and characterization of their abilities to metabolize testosterone and chlorpyrifos[J]. Journal of Pharmacology and Experimental Therapeutics,2001,299(3):825-831.
[27] YAMAGUCHI S,ZHAO Y L,NADAI M,et al.Involvement of the drug transporters P glycoprotein and multidrug resistance-associated protein MRP2 in telithromycin transport[J]. Antimicrobial Agents and Chemotherapy,2006,50(1):80-87.
[28] SUGIE M,ASAKURA E,ZHAO Y L,et al.Possible involvement of the drug transporters P glycoprotein and multidrug resistance-associated protein MRP2 in disposition of azithromycin[J].Antimicrobial Agents and Chemotherapy,2004,48(3):809-814.
[29] HE X J,ZHAO L M,QIU F,et al.Influence of ABCB1 gene polymorphisms on the pharmacokinetics of azithromycin among healthy Chinese Han ethnic subjects[J].Pharmacological Reports,2009,61(5):843-850.
[30] HE Y Z,WANG H,FANG J H,et al.Research progress on the traditional Chinese medicine-pharmaceutical drug interaction mediated by the ABC transporter family[J].Acta Pharmaceutica Sinica,2021,56(7):1778-1788.
[31] 郭源源,王晖,陈垦.药物肠道吸收机制中转运载体、酶、电位的研究进展[J].中国临床药理学与治疗学,2006,9:966-971. GUO Y Y,WANG H,CHEN K.Mechanism profile of drug intestinal absorption about transporters,enzyme and potential[J].Chinese Journal of Clinical Pharmacology and Therapeutics,2006,9:966-971.(in Chinese)
[32] KATSURA T,INUI K.Intestinal absorption of drugs mediated by drug transporters:Mechanisms and regulation[J].Drug Metabolism and Pharmacokinetics,2003,18(1):1-15.
[33] 王崇,刘克辛.外排型转运体与CYP450酶所介导的药物相互作用[J].药学学报,2014,49(5):590-595. WANG C,LIU K X.The drug-drug interaction mediated by efflux transporters and CYP450 enzymes[J].Acta Pharmaceutica Sinica,2014,49(5):590-595.(in Chinese)
[34] ZUCKERMAN J M.Macrolides and ketolides:Azithromycin,clarithromycin,telithromycin[J].Infectious Disease Clinics,2004,18(3):621-649.
[35] HARDY D J,GUAY D R P,JONES R N.Clarithromycin,a unique macrolide:A pharmacokinetic,microbiological,and clinical overview[J].Diagnostic Microbiology and Infectious Disease,1992,15(1):39-53.
[36] 张萍,毕福林,杨劲.口服药物吸收分数的评价及影响因素综述[J/OL].药学学报:1-19[2023-09-13].http://kns.cnki.net/kcms/detail/11.2163.r.20230808.1114.027.html. ZHANG P,BI F L,YANG J.Review of evaluation and influencing factors of oral drug absorption fraciton[J/OL].Acta Pharmaceutica Sinica:1-19[2023-09-13]. http://kns.cnki.net/kcms/detail/11.2163.r.20230808.1114.027.html.(in Chinese)
[37] 田娜,张彦青,李卫,等.转运体介导的纳米递送系统促进药物口服吸收的研究进展[J/OL].中国实验方剂学杂志:1-13[2023-09-13]. https://doi.org/10.13422/j.cnki.syfjx.20230863. TIAN N,ZHANG Y Q,LI W,et al.Transporter-mediated Nano-delivery system in promoting oral absorption of drugs:A review[J/OL].Chinese Journal of Experimental Traditional Medical Formulae:1-13[2023-09-13]. https://doi.org/10.13422/j.cnki.syfjx.20230863.(in Chinese)