大鼠小肠原位灌流与Caco-2细胞法研究柠檬苦素吸收机制

柠檬苦素广泛存在于柑橘类水果中, 具有抗菌、抗病毒、镇痛、抗炎和抗癌等活性, 但其口服生物利用度较低。本文意在研究柠檬苦素在肠道内的吸收机制, 为其今后的研究奠定基础。实验通过大鼠原位肠灌流和体外Caco-2细胞法进行。大鼠原位肠灌流结果显示, 柠檬苦素可能通过肠道促进扩散机制吸收, 吸收差且在全肠段都有吸收, 没有部位选择性。Caco-2细胞实验结果显示, 维拉帕米和酮康唑能显著提高柠檬苦素的吸收, 而丙磺舒的影响不明显。柠檬苦素吸收较低和生物利用度较差, 可能是P-gp外排以及CYP3A4代谢共同参与的结果。柠檬苦素的肠吸收机制研究将为其剂型设计和临床应用提供重要的参考。     

HPLC考察小檗碱和黄连提取物中小檗碱在Caco-2细胞模型的转运

目的同时考察小檗碱和黄连提取物中小檗碱在Ca-co-2细胞模型的转运。方法以Caco-2细胞单层模型研究药物的双向转运,首先系统考察时间、药物浓度、抑制剂、pH、温度对小檗碱单体吸收转运的影响,然后比较相同浓度的小檗碱单体与黄连提取物中的小檗碱在Caco-2细胞模型的转运差异。运用高效液相色谱法检测药物浓度,计算其表观渗透系数。结果相同浓度的小檗碱从底端(BL)到顶端(AP)的转运量大于顶端(AP)到底端(BL)的转运量,在AP→BP和BP→AP方向,其转运量随时间呈增长趋势。维拉帕米可以明显降低其在BL→AP方向的转运量;在AP→BL方向,其在pH=5的HBSS中的转运量最大;在BL→AP方向的转运对温度有较高的依赖性。在AP→BL和BL→AP方向,提取物中的小檗碱比单一的小檗碱的转运量要低。结论小檗碱在AP→BL方向以被动转运为主,在BL→AP方向以主动转运为主;同时还受到P-gp糖蛋白的调节。提取物中的小檗碱在Caco-2模型中转运的影响因素呈多样化。     

中药肠吸收动力学的研究进展

肠吸收动力学是决定口服药物生物利用度的重要因素之一,在中药口服制剂的开发及评价方面有着重要作用。评价药物肠吸收动力学的方法大致可分为3类：①体外试验法;②在体试验法;③体内试验法。目前中药肠吸收研究常用的实验方法为大鼠原位肠灌注模型、Caco-2细胞模型和血药浓度法;研究热点主要集中在黄酮类、皂苷类及生物碱等单一活性成分,而中药整体或复方的研究甚少。现介绍中药肠吸收动力学的主要研究方法,以及中药肠吸收研究现状,并对存在问题及其原因进行了讨论。     

Mechanism of intestinal transport of an organic cation, tributylmethylammonium in Caco-2 cell monolayers

Many quaternary ammonium salts are incompletely absorbed after their oral administration and may also be actively secreted into the intestine. However, the underlying mechanism(s) that control the transport of these cations across the intestinal epithelium is not well understood. In this study, the mechanism of absorption of quaternary ammonium salts was investigated using Caco-2 cell monolayers, a human colon carcinoma cell line. Tributylmethyl-ammonium (TBuMA) was used as a model quaternary ammonium salts. When TBuMA was administrated at a dose of 13.3 imole/kg via iv and oral routes, the AUC values were 783.7±43.6 and 249.1±28.0 μmole·min/L for iv and oral administration, indicating a lower oral bioavailability of TBuMA (35.6%). The apparent permeability across Caco-2 monolayers from the basal to the apical side was 1.3 times (p<0.05) greater than that from the apical to the basal side, indicating a net secretion of TBuMA in the intestine. This secretion appeared to be responsible for the low oral bioavailability of the compound, probably mediated by p-gp (p-glycoprotein) located in the apical membrane. In addition, the uptake of TBuMA by the apical membrane showed a Na⁺ dependency. Thus, TBuMA appears to absorbed via a Na⁺ dependent carrier and is then secreted via p-gp related carriers.     

Development of an optimized procedure for the preparation of rat intestinal microsomes: comparison of hepatic and intestinal microsomal cytochrome P450 enzyme activities in two rat strains

1. The objective of this study was to characterize cytochrome P450 (CYP) activities in both intestinal and hepatic microsomes from Wistar and Sprague–Dawley rats.

2. Specific probes for measuring CYP activities were selected using rat recombinant CYP.

3. The intestinal microsome preparation was optimized getting a more relevant and reproducible abundance of CYPs to measure CYP activities.

4. Testosterone, propranolol, diclofenac, and midazolam were determined as specific substrates of rat CYP2C11, CYP2D2, CYP2C6, and CYP3A, respectively. Ethoxyresorufin and pentoxyresorufin were not specific substrates of CYP1A2 and CYP2B1, respectively. Hepatic and intestinal microsomes expressed active CYP1A1, CYP1A2, CYP2B1, and CYP3A2. Only liver expressed active CYP2C6, CYP2C11, and CYP2D2. Wistar liver expressed more active CYP1A and CYP3A2, but less active CYP2B1 than Wistar intestine. Sprague–Dawley liver expressed more active CYP2B1 and CYP3A2, but less active CYP1A than Sprague–Dawley intestine.

5. In conclusion, CYP activities were qualitatively equivalent but not quantitatively in both strains.

     

Hepatobiliary excretion and enterohepatic circulation of colchicine in rats

This study investigated the pharmacokinetics of unbound colchicine in rat blood, liver and bile, and its interaction with cyclosporin A (CsA; P-glycoprotein inhibitor) and proadifen (non-specific cytochrome P450 inhibitor) by using a microdialysis and liquid chromatographic system. The pharmacokinetics of colchicine in rat blood showed elimination in a nonlinear manner within the dosage ranges of 1–10 mg/kg. Twenty minutes after administration, colchicine reached maximum concentration in the liver and bile. The liver-to-blood distribution ratios (AUC_liver/AUC_blood) were 1.8 ± 0.6, 1.0 ± 0.2 and 0.8 ± 0.1, and the bile-to-blood distribution ratios (AUC_bile/AUC_blood) were 121.6 ± 24.7, 102.2 ± 13.4 and 116.5 ± 18.4 at dosages of 1, 3 and 10 mg/kg, respectively. The high hepatobiliary excretion of colchicine may lead to increased toxicity in normal tissues and indicates that colchicine undergoes hepatobiliary excretion against the concentration gradient from bile-to-blood. The area under the curse (AUC) of colchicine in the liver increased in the proadifen-treated groups, suggesting that metabolism of colchicine may involve cytochrome P450. CsA pretreatment caused an increase in the AUC of colchicine in the blood, a decreased AUC in the bile, and a profound decline in the bile-to-blood distribution ratio. Furthermore, the acute diarrhea and body weight loss caused by colchicine were delayed by pretreatment with CsA. These results indicate that the hepatobiliary excretion of colchicine was regulated by P-glycoprotein (P-gp) and the related acute diarrhea could be modulated by CsA. By using a paired rats model, the enterohepatic circulation of colchicine was also observed.     

Acetaminophen toxicity in rat and mouse hepatocytes in vitro

Context: Acetaminophen (APAP) hepatotoxicity is often studied in primary cultures of hepatocytes of various species, but there are only few works comparing interspecies differences in susceptibility of hepatocytes to APAP in vitro. Objectives: The aim of our work was to compare hepatotoxicity of APAP in rat and mouse hepatocytes in primary cultures. Materials and methods: Hepatocytes isolated from male Wistar rats and C57Bl/6J mice were exposed to APAP for up to 24?h. We determined lactate dehydrogenase (LDH) activity in culture medium, activity of cellular dehydrogenases (WST-1) and activity of caspases 3 in cell lysate as markers of cell damage/death. We assessed content of intracellular reduced glutathione, production of reactive oxygen species (ROS) and malondialdehyde (MDA). Respiration of digitonin-permeabilized hepatocytes was measured by high resolution respirometry and mitochondrial membrane potential (MMP) was visualized (JC-1). Results: APAP from concentrations of 2.5 and 0.75?mmol/L induced a decrease in viability of rat (p?p?Conclusion: APAP displayed dose-dependent toxicity in hepatocytes of both species. Mouse hepatocytes in primary culture however had approximately three-fold higher susceptibility to the toxic effect of APAP when compared to rat hepatocytes.     

P-糖蛋白对布格呋喃大鼠肠道吸收的影响

本研究采用Caco-2细胞摄取和转运模型、大鼠小肠在体循环灌注、大鼠离体小肠翻转肠小囊模型及P-糖蛋白抑制剂维拉帕米(verapamil)和环孢素(cyclosporine A，CsA)研究P-糖蛋白(P-glycoprotein，P-gp)对布格呋喃(buagafuran)自肠道吸收的影响，UV-HPLC方法测定布格呋喃含量。实验结果表明，布格呋喃可被Caco-2细胞转运和摄取，维拉帕米和环孢素可使布格呋喃由Caco-2细胞绒毛面(apical，A)向基底面(basolateral，B)的转运较对照组增加1.4和1.35倍，基底面向绒毛面的转运则减少为对照组的71%和75%。维拉帕米和环孢素可使低浓度布格呋喃摄取量分别增加4.4和3.4倍。布格呋喃自大鼠小肠吸收较快，灌流90 min后残留量仅为10%。维拉帕米和环孢素可加快布格呋喃吸收，以灌流后30 min最为明显(分别提高12.4%和21.5%)。在大鼠小肠翻转肠小囊内液中布格呋喃浓度可在10 min内下降86%。维拉帕米和环孢素均可使小囊液和小囊匀浆中布格呋喃含量明显升高。以上结果提示，布格呋喃是P-糖蛋白的底物，P-糖蛋白可阻碍布格呋喃在小肠的吸收。肠道P-糖蛋白的外排作用可能是导致布格呋喃生物利用度低的重要原因之一。     

补肾温肺微乳中黄芪甲苷的大鼠在体肠吸收特征研究

目的:研究补肾温肺微乳中黄芪甲苷的大鼠不同肠段的吸收特性,并考察P-糖蛋白(P-gp)对其肠吸收的影响。方法:采用大鼠在体单向肠灌流实验,采用HPLC法测定黄芪甲苷的含量,分别研究肠段不同吸收部位、黄芪甲苷浓度、P-糖蛋白抑制剂盐酸维拉帕米对黄芪甲苷单体及其在补肾温肺微乳中吸收的影响。结果:黄芪甲苷单体在各肠段的吸收速率常数(K_a)和表观吸收系数(P_app)有显著性差异(P<0.05);流速在0.10~0.40 ml·min^-1内和质量浓度在0.1~1.0 mg·ml^-1范围内,十二指肠吸收速率常数和表观吸收系数无显著性差异;补肾温肺微乳中黄芪甲苷的肠灌流试验结果与黄芪甲苷单体相比略小,但无显著性差异;P-糖蛋白抑制剂对黄芪甲苷的肠吸收影响很小。结论:黄芪甲苷和补肾温肺微乳中黄芪甲苷在大鼠不同肠段的吸收具有相似的吸收特性;其在大鼠肠内的吸收不受P-糖蛋白影响。因此,推测黄芪甲苷不是P-gp的底物。     

Inhibition of cytochrome P450s enhances (+)‐usnic acid cytotoxicity in primary cultured rat hepatocytes

(+)‐Usnic acid (UA) is consumed as a dietary supplement to promote weight loss; however, dietary supplements containing UA have been associated with clinical cases of severe liver injury. UA has been shown to be hepatotoxic in rats and is extensively metabolized by hepatic cytochrome P450s (CYPs); therefore, we examined if UA metabolism results in the formation of cytotoxic metabolites or if metabolism is a detoxification process in primary rat hepatocytes. When CYP activity was suppressed by the non‐isoenzyme‐selective inhibitor SKF‐525A (20 μM), or the CYP1A inhibitor alpha‐naphthoflavone (10 μM), or the CYP3A inhibitor ketoconazole (25 μM), the cytotoxicity of UA at 3 ~ 6 μM after 3 ~ 20 h of exposure was significantly increased as measured by lactate dehydrogenase (LDH) leakage. At 2 h after UA exposure, an earlier time point prior to LDH release, these CYP inhibitors potentiated UA‐induced inhibition of cellular respiration as determined by the Clark type oxygen electrode. Cellular adenosine triphosphate (ATP) depletion by UA was also exacerbated by these CYP inhibitors. The CYP2B/2C inhibitor, ticlopidine at 20 μM, showed no effects in parallel experiments. These data demonstrate that UA is bio‐transformed to less toxic metabolites in rat primary hepatocytes, probably mainly by CYP1A and 3A, but not 2B/2C. Published 2013. This article is a U.S. Government work and is in the public domain in the USA.     

小檗碱、巴马汀和药根碱在“三明治”培养大鼠原代肝细胞中的胆汁外排特征

目的研究小檗碱、巴马汀和药根碱在大鼠原代细胞的胆汁外排特征。方法建立"三明治"培养大鼠原代肝细胞(sandwich cultured rat hepatocytes,SCRH)模型,分别加入小檗碱、巴马汀、药根碱,在含Ca²⁺和无Ca²⁺缓冲液中孵育,采用UPLC-MS/MS分别测定3种生物碱的细胞蓄积量,计算胆汁排泄指数和胆汁清除率,评价小檗碱、巴马汀、药根碱在大鼠原代细胞的胆汁外排特征;并考察P-gp、Mrp2抑制剂对3种活性成分在SCRH细胞模型中的外排转运影响。结果随着孵育时间的延长,3种生物碱的细胞蓄积量增加,且含Ca²⁺条件下的细胞蓄积量与无Ca²⁺条件下相比具有明显差异;P-gp抑制剂环孢素A、维拉帕米均能减少小檗碱、巴马汀、药根碱的胆汁排泄,且呈浓度依赖性,Mrp2抑制剂MK571、丙磺舒对3种生物碱的胆汁排泄无明显影响。结论小檗碱、巴马汀、药根碱均经过胆汁外排,P-gp介导了3种生物碱的胆汁外排,Mrp2未参与其胆汁排泄。     

Transport of nanoparticles across an in vitro model of the human intestinal follicle associated epithelium

An in vitro model of the human follicle associated epithelium (FAE) was characterized and the influence of nanoparticle properties on the transcellular transport across the in vitro model was investigated. The model was established by co-culturing Caco-2 and Raji cells, with Caco-2 cells alone as control. The conversion of Caco-2 cells to follicle associated epithelium (FAE) like cells was monitored by following the surface expression of β1-integrins (immunofluorescence) and nanoparticle transport (flow cytometry). The influence of the nanoparticle concentration at the apical side, temperature, size and surface properties of nanoparticles on transport was evaluated, as well as the influence of transport conditions. The conversion of Caco-2 cells into FAE-like cells occurred. The transport was concentration, temperature and size-dependent. Aminated nanoparticles were more efficiently transported than carboxylated nanoparticles, suggesting a role of nanoparticle surface functional groups and hydrophobicity, possibly leading to a different pattern of protein adsorption at their surface. In conclusion, this in vitro model is a promising tool to study the role of M cells in transintestinal nanoparticle transport, as well as to evaluate new drug delivery systems.     

肠道转运Caco-2细胞单层模型的建立及验证评价

目的建立Caco-2细胞单层模型用于药物转运研究。方法按照常规的细胞培养方法,将Caco-2细胞接种到Millicell小室内(接种密度1×10⁶个·mL^-1),培养21 d。定期用细胞电位仪监测跨上皮细胞电阻（TEER）,评价细胞单层的紧密性与完整性;通过荧光黄转运实验检查Caco-2细胞单层模型细胞旁路转运通透性;通过普萘洛尔转运实验验证Caco-2细胞单层模型跨细胞被动转运通透性。结果培养21 d后,TEER值达到（981±123）Ω·cm²,荧光黄和普萘洛尔的表观通透系数分别为0.33×10及16.7×10^-6cm·s^-1。结论本研究建立的Caco-2细胞单层模型紧密、完整,具有良好通透性,可用于药物转运研究。     

Multidrug resistance-associated protein 2 (MRP2) affects hepatobiliary elimination but not the intestinal disposition of tenofovir disoproxil fumarate and its metabolites

The role of multidrug resistance-associated protein 2 (MRP2) on the intestinal disposition and hepatobiliary elimination of tenofovir disoproxil fumarate (DF) and its metabolites [tenofovir (mono)ester and tenofovir] was studied in the Caco-2 system, Ussing chambers and rat in-situ efflux experiments. In the Caco-2 model and Ussing chambers, no statistically significant differences in transport could be observed when the MRP inhibitor probenecid was included. In Ussing chambers, transport was also similar when using intestinal tissue from MRP2-deficient rats. After intravenous administration of tenofovir DF, the excretion of tenofovir [(mono)ester] in bile was significantly decreased in MRP2-deficient rats and in rats treated with probenecid. The area under the blood concentration–time curve was increased in MRP2-deficient rats [1.0?±?0.1 and 0.36?±?0.03?µM.min^?1 for tenofovir and tenofovir (mono)ester, respectively] and rats treated with probenecid (1.42?±?0.04 and 0.36?±?0.02?µM.min^?1) compared with control rats (0.64?±?0.05 and 0.15?±?0.06?µM.min^?1). The appearance of tenofovir [(mono)ester] in intestinal perfusate was similar in control rats upon co-administering probenecid or when using MRP2-deficient rats. In conclusion, MRP2 appeared to have no modulatory effect on the intestinal disposition of tenofovir and tenofovir (mono)ester. However, inhibition (probenecid) or the total absence of MRP2 (MRP2-deficient rats) significantly reduced hepatobiliary elimination, which was accompanied by increased systemic exposure.     

Absorption of fumonisin B1 and aminopentol on an in vitro model of intestinal epithelium; the role of P-glycoprotein.

The aim of the present paper is to evaluate the absorption of fumonisin B1 and its principal metabolite, aminopentol on a human intestinal model, Caco-2 cells, cultured on semi-permeable inserts, that reproduces the two different intestinal compartments: luminal (apical) and serosal (basolateral) side. Following separate exposure in apical and in basolateral compartments, aminopentol passage through the cell layer (in particular from basolateral to apical direction) was shown, while it was not observed for the parent compound. The different aminopentol distribution between the two compartments of the culture system, and its variation in presence of verapamil or probenecid (P-gp and MRP inhibitors respectively), strongly suggests the involvement of P-glycoprotein in the influx/efflux mechanisms of aminopentol in the intestinal cells, reducing its oral bioavailability.     

药物肠吸收实验研究方法进展

介绍各种药物肠吸收模型的实验方法和特点，包括：在体肠回流法、肠襻法、分离肠黏膜法、外翻囊法和Caco-2细胞模型法等。其中，Caco-2单层细胞模型在国外已经作为一种简便、高效的体外模型应用于大量的口服药物的筛选。重点介绍Caco-2细胞模型的方法、特点和应用的进展情况。     

In vitro inhibition of human liver cytochrome P450 (CYP) and UDP-glucuronosyltransferase (UGT) enzymes by rose bengal: system-dependent effects on inhibitory potential

Abstract

1. Rose bengal (4,5,6,7-tetrachloro-2′,4′,5′,7′-tetraiodofluorescein) is being developed for the treatment of cutaneous melanoma and hepatocellular carcinoma. Interestingly, rose bengal can generate singlet oxygen species upon exposure to light.

2. We evaluated rose bengal as an in vitro inhibitor of cytochrome P450 (CYP) or UDP-glucuronosyltransferase (UGT) enzymes in both human liver microsomes (HLM) and cryopreserved human hepatocytes (CHHs) under both yellow light and dark conditions.

3. Rose bengal directly inhibited CYP3A4/5 and UGT1A6 in HLM under yellow light with inhibitor concentration that causes 50% inhibition (IC₅₀) values of 0.072 and 0.035?μM, respectively; whereas much less inhibition was observed in the dark with the IC₅₀ values increasing 43- and 120-fold, respectively. To determine if a more physiologically-relevant test system could be protected from such an effect, rose bengal was evaluated as an inhibitor of CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, 3A4/5 and UGT enzymes in CHH. All IC₅₀ values were similar (64?±?8?μM) and little to no effect of light on inhibitory potential was observed.

4. Given the IC₅₀ values in CHH increased an order of magnitude compared to HLM and the atypical pharmacokinetics of the drug, the risk of rose bengal to cause clinically relevant drug–drug interactions is likely low, particularly when administered to cancer patients on an intermittent schedule.     

Investigation of the influence of glycyrrhizin on the pharmacokinetics of celastrol in rats using LC-MS and its potential mechanism

1.?The aim of this study was to investigate the effects of glycyrrhizin on the pharmacokinetics of celastrol in rats.

2.?Twelve male Sprague–Dawley rats were randomly assigned to two groups: control group and test group. Test group was pretreated with glycyrrhizin at a dose of 100?mg/kg/day for 10 days, and then the two groups were orally administered with celastrol at a dose of 1?mg/kg. The concentration of celastrol was determined using a sensitive and reliable LC-MS method.

3.?The results showed that glycyrrhizin could significantly decrease the plasma concentration (from 64.36?ng/mL to 38.42?ng/mL) and AUC_0?t (from 705.39 to 403.43?μg·h/L) of celastrol in rats. To investigate its potential mechanism, the effects of glycyrrhizin on the transport and metabolic stability of celastrol were investigated using Caco-2 cell monolayer transwell model and rat liver microsome incubation systems. The Caco-2 cell monolayer transwell experiments indicated that glycyrrhizin could increase the efflux ratio of celastrol (4.02 versus 6.51). However, the rat liver microsome incubation experiments showed that glycyrrhizin could significantly increase the intrinsic clearance rate of celastrol from 20.3?±?3.37 to 38.8?±?4.18?μL/min/mg protein.

4.?In conclusion, these results indicated that the herb–drug interaction between glycyrrhizin and celastrol might occur when they were coadministered.