CYP3A和P-gP对双环醇在大鼠小肠吸收的影响

目的：研究CYP3A和P-gp对双环醇在小肠吸收特性的影响。方法：建立大鼠小肠single-pass灌流模型，收集灌流后不同时间点灌流液和肠系膜静脉血液，应用LC-MS联用法测定灌注液和血浆中双环醇含量，通过公式Plumen=（Q/2πr）LN（c1／co）和Pblood=（△MB／△t）／（2πrl〈C〉）计算双环醇渗透系数，从而反映双环醇的代谢变化。同时，观察CYP3A特异性抑制剂醋竹桃霉素（TAO20uM），CYP3A和P-gp联合抑制剂环孢素A（CsA 10uM）对双环醇自肠道吸收的影响。结果：双环醇在10-l00uM剂量范围内，随着灌流液中药物浓度的增加，肠系膜静脉血中双环醇浓度也相应增加，l00uM时呈现饱和现象。分别加入选择性抑制剂TAO和CsA后，肠系膜静脉血中的双环醇（50uM）的累积量分别为对照组的12以及13倍，静脉血中双环醇的渗透系数（Pblood）分别是对照组的13以及17倍，代谢分别减少33％，36％。结论：CYP3A和P-gp特异性抑制剂可明显减少双环醇在大鼠肠道的首过效应，促进双环醇的肠道吸收，提示CYP3A和P-gp在肠道的联合作用是导致双环醇口服生物利用度低的重要因素。与P-gp相比，CYP3A对双环醇肠道首过效应的影响更为显著。     

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。     

布格呋喃与大鼠肝脏药物代谢酶的相互作用

目的：研究参与布格呋喃代谢的CYP450同工酶类型和体外代谢的酶促动力学特征,并观察口服布格呋喃对大鼠肝脏药物代谢酶的影响。方法：采用比色／动态荧法光及UV—HPLC法检测大鼠口服布格呋喃（8mg·kg^-1·d^-1,连续3日）后肝脏CYP450同工酶（CYP1A2、2C6、2C11、2D2、2E1和3A2）和Ⅱ相酶-谷胱甘肽巯基转移酶（GST）、尿苷二磷酸葡萄糖醛酸转移酶（UDPGT）和谷胱甘肽还原酶（GR）活性。应用肝微粒体温孵法测定布格呋喃体外代谢速率（Vmax和米氏常数Km）。比较布格呋喃在正常及高诱导大鼠肝微粒体中代谢速率的差异以及CYP1A2、2C6、2C11、2D2、2E1和3A2的选择l生抑制剂（呋拉茶碱、磺基苯吡唑、奥美拉唑、奎尼丁、戒酒硫和酮康唑）对布格呋喃代谢的抑制程度,鉴定参与布格呋喃代谢的CYP450同工酶类型。结果：布格呋喃在乙醇、地塞米松和3-甲基胆葸诱导大鼠肝微粒体中的Km、Vmax分别为正常组的11倍、6倍和1.3倍;应用CYP3A2、2E1和IA2的选择性抑制剂酮康唑、戒酒硫和呋拉茶碱可不同程度地抑制布格呋喃的代谢,使代谢速率下降为对照组的34％、47％和78％。     

在体单向肠灌流模型研究小檗碱及其在复方配伍环境中的大鼠肠吸收特性

本实验主要考察药物浓度、肠段、pH、P-糖蛋白 (P-glycoprotein, P-gp) 及Na⁺依赖型葡萄糖转运体 (Na⁺-dependent glucose transporter, SGLT1) 对小檗碱肠吸收的影响, 并探索复方配伍环境中小檗碱的肠吸收情况。实验以酚红为标示物, 采用大鼠在体单向肠灌流模型, 运用高效液相色谱法考察小檗碱单体 (36.70、46.17和92.33 μg·mL⁻¹)、黄连解毒汤模拟体系 (主要指标成分单体混合物: 小檗碱 + 黄芩苷 + 栀子苷, 其中小檗碱浓度为92.33 μg·mL⁻¹) 及黄连解毒汤全方 (小檗碱浓度为92.33 μg·mL⁻¹) 中小檗碱在大鼠各肠段的吸收变化情况。结果表明: 小檗碱在一定浓度范围内 (20～100 μg·mL⁻¹), 部分大鼠小肠吸收速率参数 (K_a) 无显著性差异, 提示药物的吸收机制可能为被动扩散, 其转运过程可能受SGLT1和P-gp的影响; 单体混合物和全方中小檗碱在各肠段的K_a和有效渗透系数 (P_eff) 值均有显著性增加 (P < 0.05)。说明小檗碱在整个肠段均有吸收, 具有广泛的吸收窗, 其中结肠段吸收较好; 配伍对小檗碱的大鼠肠吸收特性有一定影响, 较单体而言, 单体混合物和全方灌流时小檗碱在小肠中吸收更好。     

在体单向灌流法研究阿哌沙班肾衰大鼠肠吸收特性

目的 研究阿哌沙班在肾衰大鼠体内的肠吸收特性,并考察P糖蛋白(P-glycoprotein,P-gp)抑制剂对阿哌沙班吸收行为的影响。方法 选择肾衰大鼠在体单向灌流法进行肠吸收实验,建立大鼠阿哌沙班肠灌流液HPLC分析方法,以考察大鼠在体肠吸收影响因素。结果 阿哌沙班在各肠段的吸收速率常数（K_a）存在显著性差异（P<0.05）,但表观吸收系数（P_app）未见明显差异（P>0.05）;大鼠回肠段的K_a和P_app值随药物浓度的增加而降低;加入 P-gp抑制剂盐酸维拉帕米（0.1 mmol/L）后,阿哌沙班在空肠和回肠段的K_a和P_app值均明显增加。结论 阿哌沙班在各肠段均有吸收;P-gp抑制剂对阿哌沙班在空肠和回肠段的吸收均有明显的促进作用,表明阿哌沙班为P-gp底物,推测其吸收机制为主动转运。     

柚皮素、橙皮素的大鼠在体肠吸收特性研究

目的 考察柚皮素、橙皮素在大鼠肠道的吸收特性,并考察P-糖蛋白(P-gp)对这2种成分吸收的影响。方法 采用大鼠在体肠灌流模型,以超高效液相色谱(UPLC)测定灌流液中柚皮素、橙皮素的含量,计算有效渗透系数(P_eff*)及10 cm肠段吸收百分比。结果 20 μmol·L^-1柚皮素在十二指肠、空肠、回肠、结肠4个肠段的P_eff*值分别为2.77±0.43,2.39±0.30,1.90±0.53,3.15±0.30;20 μmol·L^-1橙皮素分别为2.51±0.18,2.29±0.12,1.99±0.14,3.38±0.20。当两者加了P-gp抑制剂维拉帕米后,柚皮素、橙皮素在4个肠段的吸收均显著增加(P<0.05)。结论 柚皮素、橙皮素在大鼠肠道内吸收较好,但两者均可被肠黏膜上的P-gp外排。     

大鼠肠道对左旋延胡索乙素及其消旋体的吸收差异研究

考察延胡索乙素(THP)的吸收机制，并研究其消旋体与左旋延胡索乙素(l-THP)在大鼠肠道的吸收差异。应用单向灌流模型，采用HPLC法测定THP及l-THP在灌流液中的浓度变化。灌流液中THP质量浓度为8，16，32 μg·mL^-1时，THP吸收速率常数和有效吸收系数均无统计差异(P>0.05)，各肠段的吸收速率常数和有效吸收系数也无统计差异(P>0.05)； l-THP和THP在大鼠肠道吸收存在显著性差异(P<0.05)； 在肠道灌流液中加入P-糖蛋白(P-gp)抑制剂维拉帕米后，THP吸收显著增加，而l-THP吸收几乎不变。THP在肠黏膜的转运为被动扩散过程，无特殊吸收窗口；THP消旋体与l-THP的吸收差异可能与P-gp与右旋THP的选择性结合有关。     

甘草次酸在人细胞色素CYP450中体外代谢研究(英)

甘草根是中医临床常用解毒草药, 其活性成分甘草次酸主要是通过肝脏代谢。本文研究了人肝微粒体以及人源性CYP450s对甘草次酸的体外代谢影响, 以及甘草次酸对几种CYP450酶活性的影响。实验结果表明, 甘草次酸体外主要代谢酶为CYP3A4。体外药代动力学参数K_m, V_max和CL_int分别为18.6 μmol·L⁻¹, 4.4 nmol·mg⁻¹(protein)·min⁻¹和0.237 mL·mg⁻¹(protein)·min⁻¹。体外抑制试验显示, 50 μmol·L⁻¹甘草次酸可以抑制CYP2C19、CYP2C9、CYP3A4酶的活性, 其抑制率可高达50%以上。     

环孢素A对银杏内酯B大鼠体内药动学的影响

研究环孢素A (cyclosporine A, CyA) 对大鼠静脉注射中药成分银杏内酯B (ginkgolide B, GB) 药动学的影响, 并进行作用机制研究。实验建立了测定大鼠血浆、脑组织和尿液中GB浓度的LC-MS方法, 研究了CyA对GB在大鼠体内血药浓度、脑分布和肾排泄的影响。结果表明静脉注射CyA (10及20 mg·kg⁻¹) 可以显著增加GB的AUC_{0−360 min} (P < 0.01), 降低其CL (P < 0.001); 静脉注射CYP3A抑制剂伊曲康唑 (itraconazole, ICZ) 则对GB的药动学行为无影响。脑分布结果显示, 高、中、低剂量的CyA均可增加GB在脑中的浓度, 此作用具有浓度和时间依赖性, 在5 min时不同剂量的CyA均可显著增加GB在脑中的分布 (P < 0.001), 但在20及60 min时仅高剂量CyA组可显著增加GB脑中的浓度 (P < 0.01及P < 0.001)。不同P-gp抑制剂均可减少GB的肾排泄, 其中CyA (20 mg·kg⁻¹) 组、维拉帕米 (verapamil, VER) 组及地高辛 (digoxin, DGX) 组GB 8 h累积排泄百分率仅分别为对照组的34.8% (P < 0.001)、59.4% (P < 0.001) 及79.7% (P < 0.05); 而ICZ组则无此作用。大鼠静脉给予P-gp抑制剂CyA可以显著提高GB的血药浓度, 减少肾脏对GB排泄且能增加GB在脑中的分布。     

磷脂对甘草酸二铵小肠吸收的影响

研究磷脂对甘草酸二铵及甘草次酸小肠吸收的影响。建立大鼠原位单向肠灌流及伴有肠系膜插管的肠灌流模型，采用高效液相色谱法检测灌流液及血浆中的药物浓度，计算表观通透系数P_app和吸收速率常数K_a。建立大鼠灌胃后肝门静脉取血模型，测定门静脉血浆中药物浓度，以对灌流结果进一步验证。甘草酸二铵和甘草次酸均能跨肠壁吸收，其P_app分别为0.36和5.73 cm·min^-1，加入磷脂后，P_app分别提高到0.68和7.98 cm·min^-1。磷脂使甘草酸二铵本身的吸收略有增加，但统计学检验差异不显著，但对其肠道菌群代谢物甘草次酸的吸收有显著促进作用。     

Intestinal permeability of mitragynine in rats using in situ absorption model

The intestinal permeability of mitragynine was investigated in situ using a single pass intestinal perfusion (SPIP) absorption model, in small intestine of rat using mitragynine in the absence/presence of the permeability markers, P-gp and/or CYP3A4 inhibitors. Mitragynine demonstrated high intestinal permeability (P_eff of 1.11 × 10^?4 cm/s) that is in the range of highly permeable drugs such as propranolol (P_eff of 1.27 × 10^?4 cm/s) indicating that it readily crosses the intestine. The addition of azithromycin (P-glycoprotein inhibitor) and ciprofloxacin (CYP3A4 inhibitor) or combination of both has no effect on intestinal permeability of mitragynine across the rat small intestine.     

Factors influencing regional differences in intestinal absorption of UK-343,664 in rat: possible role in dose-dependent pharmacokinetics

The objective of this study was to evaluate potential contributions of intestinal export and metabolism to the oral dose-dependent pharmacokinetics of the human cGMP-specific phosphodiesterase type 5 inhibitor, UK-343,664. Differences between jejunal and ileal handling of this CYP3A and P-gp substrate were investigated. CYP3A and P-gp display differing activities in the upper and lower mammalian small intestine and their impact on variable drug absorption can be mechanistically assessed for individual compounds with in situ perfusion of rat's small intestine. Isolated segments of rat jejunum and ileum were perfused with UK-343,664 solution and measurements were made as a function of drug concentration for dose dependence and in the presence of CYP3A and P-gp inhibitors. Intestinal permeability and metabolism were measured by total drug disappearance and major metabolite, UK-347,334 (N-desethyl metabolite), appearance in the intestinal lumen. Intestinal tissue and mesenteric blood measurements of drug and metabolite were also determined. The effective permeability (P(eff)) of UK-343,664 and metabolite formation (F(met)) increased as a function of concentration. Regional differences in P(eff) and F(met) were observed with low-intestinal metabolism of UK-343,664 in both regions (<10%). P-gp inhibition caused significant increase in P(eff) and F(met) in jejunum and ileum while ketoconazole, a P-gp and CYP3A inhibitor, has only limited effect on metabolism. In conclusion, UK-343,664 absorption is mainly regulated by P-gp in jejunum and ileum while CYP3A intestinal metabolism has minimal effect. This role of P-gp could explain the dose-dependent pharmacokinetics of UK-343,664 and its unusual behavior of t(max) as a function of dose.     

Unmasking the dynamic interplay between efflux transporters and metabolic enzymes

Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the bioavailability of many CYP3A4 substrates. We have demonstrated that the interplay between P-gp and CYP3A4 at the apical intestinal membrane can increase the opportunity for drug metabolism by determining bidirectional extraction ratios across CYP3A4-transfected Caco-2 cells for two dual P-gp/CYP3A4 substrates, K77 (an experimental cysteine protease inhibitor) and sirolimus, as well as two negative control, CYP3A4 only substrates, midazolam and felodipine. Studies were carried out under control conditions, with a P-gp inhibitor (GG918) and with a dual inhibitor (cyclosporine). Measurement of intracellular concentration changes is an important component in calculating the extraction ratios. We hypothesize that the inverse orientation of P-gp and CYP3A4 in the liver will result in an opposite interactive effect in that organ. In vivo rat intestinal perfusion studies with K77 and rat liver perfusion studies with tacrolimus under control conditions and with inhibitors of CYP3A4 (troleandomycin), P-gp (GG918) and both CYP3A4/P-gp (cyclosporine) lend support to our hypotheses. These results serve as a template for predicting enzyme-transporter (both absorptive and efflux) interactions in the intestine and the liver.     

Disposition of everolimus in mdr1a−/1b− mice and after a pre-treatment of lapatinib in Swiss mice

The aim of this study was to document the in vivo transport of everolimus (inhibitor of mTOR) by P-glycoprotein (P-gp), and to investigate the influence of lapatinib (inhibitor of P-gp) on everolimus disposition.Pharmacokinetics of everolimus (0.25 mg/kg) has been investigated after oral administration in mdr1a−/1b− mice compared to the wild type. Also, everolimus pharmacokinetics was characterized after oral administration on Swiss mice either alone or after 2 days of pre-treatment of lapatinib (200 mg/kg). The influence of lapatinib pre-treatment on intestinal P-gp expression was investigated by Western blot analysis. The non-compartimental analysis was performed using Winonlin^® professional version 4.1 software (Pharsight, Mountain View, CA). The areas under the plasma concentration-time curve (AUC) were compared using Bailer's method.A significant 1.3-fold increase of everolimus AUC observed in mdr1a−/1b− mice suggested that everolimus is transported in vivo by intestinal P-gp in mice. In addition, a 2.6-fold significant increase of everolimus AUC with lapatinib pre-treatment as compared with the everolimus alone group was noticed. The elimination half-life was comparable (t_1/2 = 5.3 h vs. t_1/2 = 4 h). A 38.5% significant decrease of P-gp expression was observed in duodenum segment in lapatinib pre-treated group as compared with control group.In conclusion, lapatinib enhanced everolimus absorption by decreasing intestinal P-gp expression. An inhibition of CYP 450 could not be excluded. These results confirm the necessity of a therapeutic monitoring of everolimus combined with an inhibitor of the P-gp and CYP 450 like lapatinib in a future anti-tumor treatment.     

Transporter-enzyme interactions: implications for predicting drug-drug interactions from in vitro data

As discussed in earlier articles, predictions of in vivo drug-drug interactions from in vitro studies is a subject of high interest with obvious therapeutic as well as economic benefits. Up until now little attention has been given to the potential interplay between metabolic enzymes and transporters that could confound the in vivo-in vitro relationships. Drug efflux by intestinal P-glycoprotein (P-gp) is known to decrease the bioavailability of many CYP3A4 substrates. We have demonstrated that the interplay between P-gp and CYP3A4 at the apical intestinal membrane can increase the opportunity for drug metabolism by determining bidirectional extraction ratios across CYP3A4 transfected Caco-2 cells for two dual P-gp/CYP3A4 substrates, K77 (an experimental cysteine protease inhibitor) and sirolimus, as well as two negative control, CYP3A4 only substrates, midazolam and felodipine. Studies were carried out under control conditions, with a P-gp inhibitor (GG918) and with a dual inhibitor (cyclosporine). Measurement of intracellular concentration changes is an important component in calculating the extraction ratios. We hypothesize that the inverse orientation of P-gp and CYP3A4 in the liver will result in an opposite interactive effect in that organ. In vivo rat intestinal perfusion studies with K77 and rat liver perfusion studies with tacrolimus under control conditions and with inhibitors of CYP3A4 (troleandomycin), P-gp (GG918) and both CYP3A4/P-gp (cyclosporine) lend support to our hypotheses. These results serve as a template for predicting enzyme- transporter (both absorptive and efflux) interactions in the intestine and the liver.     

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。     

Pharmacokinetic interaction between oral lovastatin and verapamil in healthy subjects: role of P-glycoprotein inhibition by lovastatin

Background  

Lovastatin is an inhibitor of P-glycoprotein (P-gp) and is metabolized by the cytochrome P450 (CYP) 3A4 isoenzyme. Verapamil is a substrate of both P-gp and CYP3A4. It is therefore likely that lovastatin can alter the absorption and metabolism of verapamil.