利用人源Caco-2细胞单层模型研究罂粟碱、N-甲基四氢罂粟碱和头花千金藤碱在人肠道的吸收

研究罂粟碱(papaverine，PAP)、N-甲基四氢罂粟碱(laudanosine，LAU)和头花千金藤碱(cepharanthine，CEP)在人小肠的吸收。利用人源结肠腺癌细胞系Caco-2细胞单层模型研究PAP、LAU和CEP由绒毛面(AP侧)到基底面(BL侧)、BL侧到AP侧两个方向的转运过程。应用HPLC-UV对上述3个生物碱进行定量分析，计算转运参数和表观渗透系数，并与易吸收性对照药普萘洛尔和难吸收性对照药阿替洛尔进行比较。PAP、LAU和CEP由AP侧到BL侧的表观渗透系数(P_app)分别为(3.524±0.223)×10^-5、(2.821±0.050)×10^-5和(6.524±0.052)×10^-5 cm·s^-1；由BL侧到AP侧的P_app分别为(5.095±0.508)×10^-5、(2.646±0.146)×10^-5和(5.495±0.036)×10^-5 cm·s^-1，与在Caco-2细胞单层模型上呈良好吸收的阳性对照药普萘洛尔基本一致。PAP、LAU和CEP的P_{app A→B}/P_{app B→A}分别为0.69、1.07和1.19；PAP外流是摄取的1.45倍。PAP、LAU和CEP可以通过小肠上皮细胞被动吸收进入体内，属于良好吸收的药物。在三者的吸收转运过程中，油/水分配系数起着关键性的作用。PAP在Caco-2细胞单层模型中的转运可能存在外流机制。     

普朗尼克抑制P-糖蛋白药泵的作用

采用Caco-2细胞和动物模型，以维拉帕米为阳性对照，考察普朗尼克对塞利洛尔在Caco-2单层膜与肠道黏膜吸收的影响。用高效液相色谱法检测药物浓度，计算表观透过系数、吸收速率常数与有效透过系数等参数,评价普朗尼克对P-糖蛋白药泵的抑制作用。结果显示，塞利洛尔Caco-2细胞膜转运基底端(BL)到顶端(AP)的透过系数P_app大于AP到BL的P_app，分别为(2.10±0.13)×10^-6和(0.333±0.018)×10^-6 cm·s^-1，且双向转运受到抑制剂维拉帕米和普朗尼克的影响。大鼠在体肠灌流实验中塞利洛尔在十二指肠段、空肠、回肠与结肠段的吸收速率常数k_a分别为(0.09±0.03)，(0.14±0.04)，(0.11±0.03)与(0.05±0.02) h^-1；合用维拉帕米后各肠段吸收速率常数k_a分别为(0.14±0.03)，(0.24±0.02)，(0.25±0.03)和(0.23±0.02) h^-1；合用普朗尼克后各肠段吸收速率常数k_a分别为(0.13±0.02)，(0.22±0.02)，(0.22±0.03)和(0.20±0.03) h^-1。可见，普朗尼克通过抑制P-gp外排作用，促进塞利洛尔Caco-2细胞膜和大鼠肠道黏膜的吸收。     

三七皂苷的口服吸收机制

目的研究三七总皂苷(PNS)的口服吸收机制。方法采用Caco-2细胞和动物等模型研究PNS中人参皂苷Rb_l(Rb_l)和人参皂苷Rg_l(Rg_l)的胃肠道内稳定性、肠道黏膜吸收机制及吸收过程中胃、肠及肝对药物的影响。结果Rb_l和Rg_l在胃液酸性环境下易被破坏，而在近中性环境内基本保持稳定。Rb_l和Rg_l在大肠内容物中易降解，尤以Rb_l降解较为明显；二者在小肠内容物中则相对稳定。Rb_l和Rg_l在Caco-2细胞层的摄取受温度的影响，而pH的变化及环孢菌素A的加入对二者摄取均无显著性影响。在实验考察的浓度范围内，细胞内Rb_l(或Rg_l)的摄取量随Rb_l(或Rg_l)的浓度的增加而呈线性增加，Rb_l(或Rg_l)单体与总皂苷中的Rb_l(或Rg_l)在Caco-2细胞模型中的吸收特性无明显差异。而Rg_l的细胞摄取量［(1.07±0.16) μg·mg^-1(protein)］(C₀=1 mg·mL^-1)相对Rb_l［(0.77±0.03) μg·mg^-1(protein)］(C₀=1 mg·mL^-1)较高。Caco-2细胞转运实验表明，Rb_l和Rg_l单体的转运透过系数(P_app)分别为(5.9±1.0)×10^{-8cm·s-1和(2.59±0.17)×10-7 cm·s-1(C₀=1 mg·mL-1)，二者转运都不受环孢菌素A影响。PNS溶液灌胃、十二指肠及门静脉给药后测得Rb_l大鼠绝对生物利用度分别为0.71%，2.75%和65.77%；Rg_l分别为3.29%，6.60%和50.56%。结论三七总皂苷(包括Rb_l和Rg_l)的肠道吸收机制为单纯被动扩散，吸收过程不受细胞膜内P-gp和MRP外排载体的调控，PNS中其他成分对Rb_l或Rg_l的吸收特性无明显影响。胃液的酸性环境、大肠菌丛产生的酶及肝脏的首过作用均对其口服吸收产生影响，而肠道黏膜的透过性低是其口服吸收差的主要影响因素。}     

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。     

蛇床子素在兔体内药物代谢动力学

目的研究蛇床子素在兔体内的药物代谢动力学。方法用高效液相色谱法,以丹皮酚为内标,以甲醇-水(80∶20)为流动相,测定兔血液中蛇床子素(iv,10 mg·kg^-1)的含量。采用3P87程序计算药物代谢动力学参数。结果蛇床子素iv药代动力学符合二房室开放模型,T_1/2α=5.81 min,T_1/2β=42.2 min,K21=0.036 0·min^-1,K₁₂=0.045 0·min^-1,K₁₀=0.054 0·min^-1,AUC=235 mg·min·L^-1,CLs=0.043 0 L·min^-1·kg^-1,V_C=0.780 L·kg^-1。结论蛇床子素在兔体内分布及消除较快     

代谢综合征MSG肥胖大鼠胰岛β 细胞功能紊乱机制的初步研究

评价代谢综合征动物模型MSG肥胖大鼠的胰岛β细胞功能，并对其功能紊乱机制进行初步研究。采用高葡萄糖钳夹技术证实肥胖性胰岛素抵抗MSG大鼠存在β细胞功能缺陷，GIR值较正常动物降低33.8%［MSG (23.1±3.2) mg·kg^{-1·min-1， NOR (34.9±8.4) mg·kg-1·min-1， P<0.05］；考察病理与生化指标显示MSG大鼠胰岛β细胞数目减少，胰腺甘油三酯含量、NO含量显著增加，SOD水平、胰腺线粒体中总ATP酶和Na+-K+-ATP酶活性均显著降低。结果提示，肥胖性胰岛素抵抗MSG大鼠具有典型的代谢综合征特征，同时伴有胰岛β细胞功能缺陷。这可能与其肥胖导致的炎症因子增加，机体抗氧化能力及膜结构中ATP酶活性下降有关。}     

常咯啉在实验性心律失常狗的药代动力学-药效动力学分析

用Harris冠脉结扎法诱发的心律失常狗研究常咯啉药代动力学-药效动力学。7只狗按83.33μg·kg^-1·min^-1静脉滴注60min,在给药期间和停药后不同时间记录ECG及测定血药浓度。C-T数据用药代程序计算药代参数;药效数据用药代-药效同步分析模型计算药效动力学参数,K₁₀, T_1/2,Vd,Cl分别为0.0087min^-1,78.03min,40.55ml·kg^-1和0.421ml·kg^-1·min^-1;K_eO和Ce(50)分别为0.0048min^-1和2.01μg·ml^-1.     

HPLC-荧光法测定人血浆中羟基喜树碱的两种构型及其相互间的转化

目的 建立高效液相色谱法(HPLC)测定人血浆中羟基喜树碱两种活性成分盐型羟基喜树碱(C-HCPT)和酯型羟基喜树碱(L-HCPT)含量的方法,并且对两者之间的相互转化进行了初步研究。方法 采用HPLC-荧光法,Lichrospher C₁₈色谱柱,流动相为乙腈-0.075 mol·L^-1醋酸铵缓冲液(32∶68,pH 6.4),流量1.0 ml·min^-1,检测器激发波长为363 nm,发射波长为530 nm,内标法计算药物浓度。结果 人血浆样品中C-HCPT、L-HCPT峰分离良好,内源性杂质不干扰样品峰,最低检测浓度均为5 ng·ml^-1。C-HCPT、L-HCPT浓度范围在15～2000 ng·ml^-1间呈线性相关,回归方程分别为Y₁=9.393×10^-4X₁-9.1×10^-6和Y₂=1.0207×10^-3X₂-3.4×10^-6,相关系数分别为r₁=0.9997和r₁=0.9998,相对回收率分别为96.79%～119.64%和100.23%～112.93%,日内和日间变异均小于10%。两者之间的转化具有一定规律性;C-HCPT标准血样-20℃冻存20 d稳定,L-HCPT标准血样的稳定性有待进一步研究。结论 本方法专属性强，灵敏、可靠，适用于HCPT 类制剂给药后血药浓度的监测和体内的药代动力学特征研究；有关两者相互转化的机制及L-HCPT 稳定性问题尚待进一步深入研究。     

2',4'-二羟基-3'-甲基-3-甲氧基查耳酮上调p21抑制人肝癌HepG2细胞的生长

目的 探讨2'',4''-二羟基-3''-甲基-3-甲氧基查耳酮(C20)对人肝癌HepG2细胞的体外抗肿瘤作用及其潜在的作用机制。方法 通过CCK-8法、集落形成实验、5-乙炔基-2''-脱氧尿苷(EdU)染色法检测C20对人肝癌HepG2细胞增殖的影响;通过彗星实验检测C20(10 μmol·L^-1)对HepG2细胞DNA损伤的影响;通过流式细胞术检测C20(5、10 μmol·L^-1)对HepG2细胞周期阻滞的影响;通过Hoechst染色和流式细胞术检测C20(5、10 μmol·L^-1)对HepG2细胞凋亡的影响。借助Western blotting法检测C20(5、10 μmol·L^-1)处理对HepG2细胞中与凋亡、DNA损伤、细胞周期阻滞相关蛋白表达水平的调控作用。结果 与对照组比较,C20显著抑制HepG2细胞的活力(P<0.001),给药48 h的半数抑制浓度(IC₅₀)为7.937 μmol·L^-1;5 μmol·L^-1 C20能够显著抑制HepG2细胞的集落形成能力(P<0.01);EdU染色结果显示5、10 μmol·L^-1的C20能够抑制人肝癌HepG2细胞的增殖能力;5、10 μmol·L^-1的C20显著诱导HepG2细胞G₂/M期阻滞(P<0.001);5、10 μmol·L^-1的C20显著促进HepG2细胞凋亡(P<0.001),并显著上调Caspas-3、Caspase-9以及PARP的剪切水平(P<0.01);10 μmol·L^-1的C20能够诱导HepG2细胞发生DNA损伤,并且5、10 μmol·L^-1的C20显著上调γH2AX、p21的蛋白水平(P<0.01)。结论 C20能够造成HepG2细胞发生DNA损伤,上调p21蛋白水平,导致细胞G₂/M期阻滞,并进一步诱发凋亡,发挥体外抗肝癌作用。     

五味子醇甲在大鼠肝微粒体内的代谢动力学和性别差异

体外研究五味子醇甲(schizandrin，SZ)在大鼠肝微粒体内的代谢动力学和性别差异。制备正常雌、雄大鼠肝微粒体，与SZ共同温孵，以高效液相色谱法测定SZ及其代谢产物。SZ在雄鼠肝微粒体内代谢反应的最大速率V_max、米氏常数K_m和清除率Cl_int分别为(21.88±2.30) μmol·L^-1·min^-1·mg^-1(protein)，(389.00±46.26) μmol·L^-1和(0.056 3±0.000 7) min·mg^-1(protein)；在雌鼠肝微粒体内代谢反应的最大速率V_max、米氏常数K_m和清除率Clint_{分别为(0.61±0.07) μmol·L^-1·min^-1·mg^-1(protein)，(72.64±13.61) μmol·L^-1和(0.008 4±0.000 8) min·mg^-1(protein)，雌、雄鼠肝微粒体内SZ的主要代谢物不同，分别为7,8-顺二羟基五味子醇甲(M1)和7,8-顺二羟基-2-去甲基五味子醇甲(M2b)。酮康唑、奎尼丁和奥芬得林对SZ的在雌、雄大鼠肝微粒体内代谢均有不同程度的抑制作用，西咪替丁对其在雄鼠肝微粒体内的代谢也有一定的抑制作用。SZ在雌、雄大鼠肝微粒体中代谢动力学及代谢产物存在明显的性别差异，这种差异可能主要是由CYP3A和CYP2C11在大鼠肝微粒体内的性别差异引起的。}     

Transport characteristics of 9-nitrocamptothecin in the human intestinal cell line Caco-2 and everted gut sacs

The intestinal absorptive characteristics and the efflux mechanisms of 9-nitrocamptothecin (9-NC), a novel water-insoluble camptothecin (CPT) derivative, were investigated. The Caco-2 cells and the everted gut sacs were used as models of the intestinal mucosa to assess transepithelial transport of 9-NC. The determination of 9-NC was performed by HPLC. In the Caco-2 cells, the absorptive transport of 9-NC was pH dependent and the transport was enhanced at weakly acidic pH on the apical side. No concentration dependence and saturation were observed for the absorptive transport of 9-NC at concentrations up to 250 microM, while secretory transport were concentration dependent and saturable process (K(m) was 49.8 +/- 1.2 microM, V(max) was 38.28 +/- 0.8 ng/cm(2)/min). In the presence of verapamil (100 microM) and CsA (10 microM), potent inhibitors of P-glyprotein (P-gp)/MRP2 (cMOAT), the P(appBL-AP)/P(appAP-BL) ratio was decreased from 3.4 to 1.4 and 1.3, respectively, and permeation of apical to basolateral was enhanced approximately two-fold. In the everted gut sacs, the absorption of 9-NC was passive diffusion and had no significant difference in different gut regions. Adding verapamil in the everted gut sacs over a concentration ranging from 10 to 100 microM, the absorption of 9-NC was significantly enhanced, especially more markedly in lower small intestine (P < 0.05). Overall, the current study suggests that pH and efflux transporters are capable of mediating the absorption and efflux of 9-NC, and they may play significant roles in limiting the oral absorption of 9-NC.     

Circumventing P-glycoprotein-mediated cellular efflux of quinidine by prodrug derivatization

The objective of this study is to investigate whether transporter-targeted prodrug derivatization of quinidine, a model P-glycoprotein (P-gp) substrate, can circumvent P-gp-mediated efflux. The L-valine ester of quinidine (val-quinidine) was synthesized in our laboratory. Uptake and transport studies were carried out using the MDCKII-MDRI cell line at 37 degrees C for 10 min and 3 h, respectively. [3H]Ritonavir and cyclosporine were also used as model P-gp substrates to delineate the kinetics of translocation of val-quinidine across the MDCKII-MDRI cell monolayer. The rate of uptake of [3H]ritonavir by MDCKII-MDRI cells exhibited a 2-fold increase in the presence of 75 microM quinidine, but 75 microM val-quinidine did not demonstrate any effect on [3H]ritonavir uptake. The rate of transport of quinidine from the basolateral to the apical membrane [(18.3 +/- 1.25) x 10(-6) cm s(-1)] and from the apical to the basolateral membrane [(6.5 +/- 0.66) x 10(-6) cm s(-1)] exhibited a 3-fold difference. However, transport of val-quinidine from the apical to the basolateral membrane [(5.13 +/- 0.49) x 10(-6) cm s(-1)] and from the basolateral to the apical membrane [(6.17 +/- 1.28) x 10(-6) cm s(-1)] did not demonstrate any statistically significant difference. Moreover, cyclosporine, a potent P-gp substrate and/or inhibitor, did not alter the transport kinetics of val-quinidine. The rates of uptake of [3H]Gly-Sar and various amino acid model substrates were reduced in the presence of 200 microM val-quinidine. Results from this study clearly indicate that prodrug derivatization of quinidine into val-quinidine can overcome P-gp-mediated efflux. Val-quinidine once bound to a peptide or amino acid transporter is probably not recognized and cannot be accessed by the P-gp efflux pump. Transporter-targeted prodrug derivatization seems to be a viable strategy for overcoming P-gp-mediated efflux.     

Ketoconazole and the modulation of multidrug resistance-mediated transport in Caco-2 and MDCKII-MDR1 drug transport models

The hypothesis tested was that ketoconazole can modulate P-glycoprotein, thereby altering cellular uptake and apparent permeability (P(app)) of multidrug-resistant substrates, such as cyclosporin A (CSA) and digoxin, across Caco-2, MDCKII-MDR1, and MDCKII wild-type cell transport models. (3)H-CSA/(3)H-digoxin transport experiments were performed with and without co-exposure to ketoconazole, and (3)H-ketoconzole transport experiments were performed with and without co-exposure to dietary flavonoids, epigallocatechin-3-gallate, and xanthohumol. Ketoconazole (3 microM) reduced the P(app) efflux of CSA and digoxin from 5.07 x 10(-6) to 2.91 x 10(-6) cm s(-1) and from 2.60 x 10(-6) to 1.41 x 10(-6) cm s(-1), respectively, in Caco-2 cells. In the MDCKII-MDR1 cells, ketoconazole reduced the P(app) efflux of CSA and increased the P(app) absorption of digoxin. Cellular uptake of ketoconazole in the Caco-2 cells was significantly inhibited by CSA and digoxin, whereas epigallocatechin-3-gallate and xanthohumol exhibited biphasic responses. In conclusion, ketoconazole modulates the P(app) of P-glycoprotein substrates by interacting with MDR1 protein. Epigallocatechin-3-gallate and xanthohumol modulate the transport and uptake of ketoconazole.     

The influence of P-glycoprotein on morphine transport in Caco-2 cells. Comparison with paclitaxel

In vitro monolayer studies using Caco-2 cells were employed here to explore P-glycoprotein mediated transport of morphine. Bi-directional transport studies of 10-75 microM morphine showed efflux to be twofold higher than influx (4 x 10(-6) compared to 2 x 10(-6) cm/s) and cellular accumulation in the efflux direction was eightfold higher. The cyclosporin analogue (PSC-833) equilibrated morphine transport in both directions. Depletion of intracellular glutathione had a greater effect on increasing cellular morphine accumulation than P-glycoprotein inhibitors, suggesting a role for glutathione in morphine transport. P-glycoprotein had a substantially greater effect on paclitaxel accumulation, efflux and bi-directional transport than for morphine. Paclitaxel transport was below detection (<0.1 x 10(-6) cm/s) in the influx direction, yet efflux was very high (18.4 x 10(-6) cm/s) and P-glycoprotein inhibition increased accumulation >100-fold. These results reinforce the substantial role P-glycoprotein has in paclitaxel transport. Conversely, P-glycoprotein regulated morphine transport is weak. Nevertheless, morphine transport rates could be doubled when administered with P-glycoprotein substrates. Therefore, increased analgesia through P-glycoprotein inhibition should be possible.     

金丝桃苷在Caco-2细胞模型中的跨膜转运方式

目的研究金丝桃苷在Caco-2细胞模型中的吸收机制。方法用Caco-2细胞单层模型研究金丝桃苷的双向转运,考察pn、药物质量浓度、方向、温度、抑制剂对金丝桃苷细胞转运的影响。采用HPLC法检测金丝桃苷的含量,计算其表观渗透系数(P_app)。结果金丝桃苷的细胞转运P_app。具有pH依赖性。金丝桃苷肠腔（A）侧→基底（B）侧P_app>B→A,并且随着金丝桃苷质量浓度的增大而减小,具有浓度依赖性。P-gp抑制剂维拉帕米增加金丝桃苷的细胞正向转运P_app,降低了其逆向转运P_app。金丝桃苷较高浓度时,MRPl抑制剂吲哚美辛和ATP抑制剂叠氮化钠显著降低了金丝桃苷的转运量。结论金丝桃苷在Caco-2细胞单层模型中的转运具有pH依赖性和浓度依赖性,是以主动转运为主,被动扩散为辅,同时涉及外排蛋白作用的转运方式。     

Use of Caco-2 cells and LC/MS/MS to screen a peptide combinatorial library for permeable structures

The transepithelial transport of a synthetic peptide combinatorial library containing 375,000 individual peptides was assessed using Caco-2 cell monolayers in order to screen for permeability and deliverability. A series of 150 pools, each containing 2500 tripeptide sequences, were applied to the apical side of Caco-2 monolayers. Basolateral side samples were collected after 4 h and screened by capillary high-pressure liquid chromatography. The majority of pools showed no permeable species, due to low solubility, limited permeability and extensive metabolism. Several pools contained permeable structure, and transport proved reproducible with passage number and time. Permeable structures were identified by liquid chromatography/mass spectrometry/mass spectrometry (LC/MS/MS). To discriminate between isobaric structures, several tripeptides were resynthesized and tested as discrete compounds. For example, 1-2% D-Phe-D-Ala-D-Ser-OH was transported across the Caco-2 cell monolayer with a Papp value of 0.35-0.69 x 10(-6) cm/s, which is comparable with the permeability of amino acids (Leu, Papp = 0.30 x 10(-6) cm/s) and dipeptides (L-Val-L-Val, Papp = 0.18 x 10(-6) cm/s) (Lennernas, H., Palm, K., Fagerholm, U., Artursson, P., 1996. Comparison between active and passive drug transport in human intestinal epithelial (Caco-2) cells in vitro and human jejunum in vivo. Int. J. Pharm. 127, 103-107; Tamura, K., Bhatnagar, P.K., Takata, J.S., Lee, C.P., Smith, P.L., Borchardt, R.T., 1996. Metabolism, uptake, and transepithelial transport of the diastereomers of Val-Val in the human intestinal cell line Caco-2. Pharm. Res. 13, 1213-1218). These studies demonstrate the techniques used to screen combinatorial libraries for permeability across Caco-2 cells and structurally identify the resulting compounds. Such methodology can be of importance in the achievement of structure-permeability relationships, useful in the design of pharmaceutically bioavailable drugs.     

Effects of dietary flavonoids on the transport of cimetidine via P-glycoprotein and cationic transporters in Caco-2 and LLC-PK1 cell models

1. The hypotheses tested were to study cimetidine as a substrate of P-glycoprotein (P-gp) and organic cation transport systems and the modulatory effects of eight flavonoid aglycones and glycosides on these transport systems using Caco-2 and LLC-PK1 cells. 2. Transport and uptake experiments of (20 microM) (3)H-cimetidine were performed with and without co-exposure to quercetin, quercetrin, rutin, naringenin, naringin, genistein, genistin, and xanthohumol. Co-treatment decreased basolateral to apical (B to A) permeability (P(app)) of cimetidine from 2.02 to 1.24 (quercetin), 1.06 (naringenin), 1.24 (genistein), and 0.96 (xanthohumol) x 10(-6) cm s(-1) in Caco-2 cells and from 10.76 to 1.65 (quercetin), 2.05 (naringenin), 2.88 (genistein), and 1.95 (xanthohumol) x 10(-6) cm s(-1) in LLC-PK1 cells. Genistin significantly reduced B to A P(app) of cimetidine to 1.24 x 10(-6) cm s(-1) in Caco-2 cells. Basolateral intracellular uptake rate of cimetidine was enhanced 145-295% when co-treated with flavonoids. Co-treatment with P-glycoprotein and organic cation transporter inhibitors, verapamil and phenoxybenzamine, resulted in reduced B to A permeability and slower basolateral intracellular uptake rate of cimetidine. Intracellular uptake rate of (14)C-tetraethylammonium (TEA) was reduced in the presence of quercetin, naringenin and genistein in LLC-PK1 cells. 3. In conclusion, quercetin, naringenin, genistein, and xanthohumol reduced P-gp-mediated transport and increased the basolateral uptake rate of cimetidine. Quercetin, naringenin, genistein, but not xanthohumol, reduced intracellular uptake rate of TEA in LLC-PK1 cells. These results suggest that flavonoids may have potential to alter the disposition profile of cimetidine and possibly other therapeutics that are mediated by P-gp and/or cation transport systems.     

Apricot extract inhibits the P-gp-mediated efflux of talinolol

Within the framework of developing strategies to enhance the intestinal absorption of P-glycoprotein (P-gp) substrates, the modulatory effect of a standardized apricot extract on P-gp-related efflux carriers was investigated in the Caco-2 system, Ussing chambers and the rat in situ perfusion model using talinolol as a model substrate. Using the Caco-2 system, polarity in transport of talinolol could be observed, the absorptive transport being much lower than the secretory transport (P(app-abs) = 1.08 +/- 0.29 x 10(-6) cm/s and P(app-secr) = 11.74 +/- 0.80 x 10(-6) cm/s). Inclusion of apricot extract (1%) in the apical medium resulted in a statistically significantly diminished polarity (P(app-abs) = 4.88 +/- 0.96 x 10(-6) cm/s and P(app-secr) = 9.39 +/- 0.58 x 10(-6) cm/s, p < 0.05). In addition, the inhibitory effect of apricot extract on P-gp related efflux mechanisms was shown to be concentration (0% approximately 0.1% < 0.3% < 1%) and pH dependent. Experiments performed with the Ussing chambers resulted in similar observations. In the rat in situ perfusion model, inclusion of apricot extract (1%) in the perfusion medium resulted in a threefold increase of the amount of talinolol appearing in the collected blood compared to the reference condition (23.6 +/- 5.53 pmol/cm. min and 7.13 +/- 1.08 pmol/cm. min, respectively; p < 0.05). Coadministration of this standardized apricot extract might be a safe and useful strategy to enhance the intestinal absorption of P-gp substrates. The nature and structure of the compound(s) responsible for this inhibiting effect on P-gp-related efflux carriers remain to be elucidated, as well as the exact mechanism by which apricot extract exerts its inhibitory function.     

Transport of Schisandra chinensis extract and its biologically-active constituents across Caco-2 cell monolayers - an in-vitro model of intestinal transport

We have determined the intestinal transport of Schisandra chinensis extract and its lignans (gomisin A, gomisin N and schisandrin C) in the Caco-2 cell monolayer model. The transport across monolayers was examined for 2 h in absorptive and secretory directions. Quantitation of lignans was performed by HPLC. Out of the three lignans, gomisin A exhibited bi-directional transport, with P(app) values in the range of 25-29 x 10(-6) cm s(-1), indicating a passive diffusion. Gomisin N, mixture and Schisandra extract displayed a higher transport in the secretory direction with efflux ratios in the range of 2.2-5.2. The efflux was decreased in the presence of inhibitors of multidrug resistance protein (MRP) transporter (MK-571) and P-glycoprotein (verapamil) indicating a possible involvement of an efflux pump and MRP in the transport of Schisandra lignans. Poor transport of schisandrin C was observed which could not be quantitated. The permeability of gomisin A in the isolated form was significantly different compared with the mixture or extract.     

Characterization of rPEPT2-mediated Gly-Sar transport parameters in the rat kidney proximal tubule cell line SKPT-0193 cl.2 cultured in basic growth media

The rat proximal kidney tubule cell line SKPT-0193 cl.2 (SKPT) expresses the di-/tripeptide transporter PEPT2 (rPEPT2) and has been used to study PEPT2-mediated transport. Traditionally, SKPT cells have been cultured in growth media supplemented with epidermal growth factor (EGF), apotransferrin, dexamethasone, and insulin. It was recently demonstrated that omission of EGF from the culture media caused a drastic increase in the expression of rPEPT2. The hypothesis was therefore that the SKPT cell line might be able to differentiate and express rPEPT2 in the absence of the four agonists traditionally added. The aim of the study was thus to characterize Gly-Sar transport parameters in SKPT cells cultured in basic growth media (conventional media without added agonists). Morphology was studied using confocal laser scanning microscopy (CLSM) and immunohistochemistry. Monolayer integrity was evaluated using transepithelial electrical resistance (TEER) measurements and [(3)H]-mannitol permeabilities. Di-/tripeptide transporter activity was studied using [(14)C]-glycylsarcosine ([(14)C]-Gly-Sar). SKPT cells grown in basic media for 4 days formed confluent monolayers with a TEER of 5.03 +/- 0.33 kOmega.cm(2) (n = 5). Apical Gly-Sar uptake peaked after 3-6 days in culture. Uptake at day 4 was 5.89 +/- 0.30 pmol.cm(-2).min(-1) (n = 3). Di-/tripeptide uptake displayed an optimum at approximately pH 6. Affinity values for cephalexin, kyotorphin, and delta-aminolevulinic acid were comparable to those obtained in other PEPT2-expressing model systems. It can be concluded that SKPT cells grown in the absence of the agonists traditionally added to the culture media retain all necessary properties for PEPT2-mediated peptide uptake studies. Furthermore, the absence of the agonists might facilitate studies of hormonal regulation of PEPT2 expression and transport activity.