欧前胡素阻断Noxs-ROS通路抑制MKN-45及HT-29细胞增殖活性

目的 探讨欧前胡素对人胃癌细胞(MKN-45)和人结肠癌细胞(HT-29)中烟酰胺腺嘌呤二核苷酸磷酸氧化酶(Noxs)-活性氧(ROS)氧化应激通路的作用及对肿瘤细胞增殖活性的影响。方法 MKN-45和HT-29细胞经过不同浓度(3、10、30、100 μmol/L)的欧前胡素及阳性对照二苯基氯化碘盐(DPI)处理后,使用CCK8法检测细胞增殖活性,使用DHE荧光探针检测细胞内ROS水平,使用Western blotting检测细胞内Nox1、Nox2、Nox3、Nox4、Nox5家族蛋白表达。结果 CCK8法结果显示,欧前胡素可浓度相关性地抑制MKN-45和HT-29细胞增殖活性;DHE荧光探针法结果显示,欧前胡素干预可使MKN-45和HT-29细胞荧光强度明显减弱,ROS水平降低;Western blotting结果显示,给予欧前胡素可使MKN-45细胞Nox1、Nox2、Nox3、Nox4、Nox5蛋白表达水平显著降低(P<0.05、0.01、0.001),使HT-29细胞Nox1、Nox2蛋白表达水平显著降低(P<0.05、0.01)。结论 欧前胡素可通过抑制MKN-45和HT-29细胞中Noxs-ROS通路,诱导细胞增殖活性下降,发挥抗肿瘤作用。     

小檗碱对有机阳离子药物转运体抑制作用研究

目的 研究小檗碱对人有机阳离子转运蛋白（OCTs）—OCT1、OCT2、OCT3、OCTN1和OCTN2的抑制作用。方法 应用由转染试剂Lipo 3 000介导的动物细胞转基因方法、经筛选得到各药物转运体过表达细胞株S2-OCT1、S2-OCT2、S2-OCT3、S2-OCTN1和S2-OCTN2;通过检测OCTs介导的放射性探针底物的跨膜转运,建立OCTs体外评价模型;以野生型（WT）细胞为对照组,应用各转运体抑制剂验证其活性;应用上述方法观察小檗碱对各转运体的抑制作用,并计算小檗碱对各药物转运体活性的半数抑制浓度（IC₅₀）。结果 各转运体细胞组与各自WT细胞株比较,转运活性均提高了5倍多,加入抑制剂后,转运活性均明显下降;小檗碱对OCT1、OCT2、OCT3和OCTN1抑制作用较强,对OCTN2的抑制作用相对较弱,IC₅₀分别为7.63、6.80、2.25、4.66和210.34 μmol/L。结论 小檗碱对这5种有机阳离子转运体均有抑制作用,其中对OCT1、OCT2、OCT3、OCTN1的抑制作用较强,发生由其介导的DDI的可能性较大,对OCTN2的抑制作用相对较弱。     

HPLC法同时测定祛喘胶囊中木兰脂素、欧前胡素、异欧前胡素的含量

摘 要 目的： 建立祛喘胶囊中对木兰脂素、欧前胡素和异欧前胡素的HPLC测定方法。方法: 采用日本资生堂CAPCELL PAK C₁₈ 色谱柱（150 mm×4.6 mm,5 μm）,以乙腈-水为流动相进行梯度洗脱,柱温为30℃,流速为1.0 ml·min^-1,检测波长在0～25 min为278 nm,25～70 min为248 nm,进样量为10 μl。结果: 木兰脂素在6.126～55.134 μg·mL^-1范围内线性关系良好（r=0.999 7）,平均回收率为101.5%,RSD为1.4%;欧前胡素在9.862～88.758 μg·mL^-1范围内线性关系良好（r=0.999 8）,平均回收率为99.6%,RSD为1.2%;异欧前胡素在4.830～43.470μg·mL^-1范围内线性关系良好（r=0.999 8）,平均回收率为100.2%,RSD为1.7%。结论:本法操作简便、结果准确,可用于祛喘胶囊的质量控制。     

羌活醇及异欧前胡素对脂多糖诱导大鼠成纤维样滑膜细胞增殖抑制的影响

目的 探讨羌活醇及异欧前胡素对大鼠成纤维样滑膜细胞增殖抑制的影响。方法 复制经脂多糖(LPS)诱导的大鼠成纤维样滑膜细胞(CIA-FLS)模型,分别给予不同浓度的羌活醇(25、50、100、200、400 μg·mL^-1)、异欧前胡素(25、50、100、200、400 μg·mL^-1)及阳性药泼尼松龙（400 μg·mL^-1）,另设空白对照组,观察给药前后成纤维样滑膜细胞形态差异;采用噻唑蓝(MTT)法测定不同给药时间、不同给药浓度下成纤维样滑膜细胞的抑制率,计算半数抑制浓度（IC₅₀）;硝酸还原酶法测定羌活醇和异欧前胡素对大鼠成纤维化滑膜细胞中一氧化氮（NO）水平的影响;ELISA测定肿瘤坏死因子a (TNF-a)表达的影响。结果 造模细胞较正常细胞增殖力强,细胞粗大,纤维样,呈层叠状生长;羌活醇组给药后细胞增殖力减弱,生长良好;异欧前胡素给药后细胞增殖力锐减,生长状况不佳。给药12 h,羌活醇未测出半数抑制浓度值,异欧前胡素半数抑制浓度值为160 μg·mL^-1;给药36 h,羌活醇、异欧前胡素半数抑制浓度值分别为190、120 μg·mL^-1;给药60 h,分别为80、45 μg·mL^-1,异欧前胡素的增殖抑制能力强于羌活醇（P＜0.01）。给药羌活醇与异欧前胡素对一氧化氮总体含量和细胞因子肿瘤坏死因子a含量均有显著性降低（P＜0.01）,且异欧前胡素的降低更显著。结论 羌活醇及异欧前胡素对大鼠成纤维样滑膜细胞均有增殖抑制作用(P＜0.05),均使一氧化氮含量和细胞因子TNF-a的含量显著减少（P＜0.01）,二者都有很好的增殖抑制能力,且异欧前胡素的抑制效果优于羌活醇。     

HPLC法同时测定玉真散中天麻素、升麻素苷、5-O-甲基维斯阿米醇苷、欧前胡素、异欧前胡素的含量

摘 要 目的：建立HPLC法同时测定玉真散中天麻素、升麻素苷、5-O-甲基维斯阿米醇苷、欧前胡素、异欧前胡素含量。方法: 色谱柱为Phenomenex Gemini C18柱（250 mm×4.6 mm,5 μm）,柱温为25℃,流速为1.0 ml·min^-1 ,流动相为甲醇 水梯度洗脱,检测波长为230 nm,进样量为5 μl。结果: 天麻素在2.68~214.00μg·ml^-1范围内线性关系良好（r=0.999 9）,平均回收率为100.2％,RSD为0.9％（n=6）;升麻素苷在5.22~418.00 μg·ml^-1范围内线性关系良好（r=0.999 7）,平均回收率为99.9％,RSD为0.9％（n=6）;5-O-甲基维斯阿米醇苷在4.57~365.80 μg·ml^-1范围内线性关系良好（r=0.999 5）,平均回收率为99.7％,RSD为1.0％（n=6）;欧前胡素在5.22~417.20μg·ml^-1范围内线性关系良好（r=0.999 6）,平均回收率为99.0％,RSD为0.9％（n=6）;异欧前胡素在5.29~423.20μg·ml^-1范围内线性关系良好（r=0.999 8）,平均回收率为100.2％,RSD为0.8％（n=6）。结论: 该方法简单、准确,可同时测定5种成分的含量,可用于玉真散的质量控制。     

一测多评法测定元胡止痛胶囊中欧前胡素、异欧前胡素和延胡索乙素的含量

目的:建立以一测多评法测定元胡止痛胶囊中欧前胡素、异欧前胡素、延胡索乙素含量的方法,并验证此方法在该制剂中应用的可行性和技术适应性。方法:以欧前胡素对照品为内参物,在一定线性范围内,采用高效液相色谱法建立其与异欧前胡素、延胡索乙素的相对校正因子,并用该校正因子进行异欧前胡素、延胡索乙素的含量计算,实现一测多评;同时采用外标法测定元胡止痛胶囊中这3种成分的含量,并比较2种方法测定结果。结果:8批药品中3种成分含量的2种方法测定结果无显著性差异(RSD<5%)。结论:一测多评法控制元胡止痛胶囊的质量快速、准确、可行,且成本低廉。     

五味子乙素通过ROS介导内质网应激诱导人乳腺癌MDA-MB-231细胞凋亡的研究

目的 研究五味子乙素对人乳腺癌MDA-MB-231细胞凋亡的影响及其作用机制。方法 用细胞计数试剂(CCK-8)检测不同浓度五味子乙素对MDA-MB-231细胞存活率的影响;五味子乙素（10、20、40 μmol/L）作用 MDA-MB-231 细胞 24 h,分别用Annexin V-FITC/PI检测细胞凋亡情况;用DCFA-DA荧光探针检测细胞内活性氧（ROS）水平;用Western blot法检测细胞凋亡及内质网应激相关蛋白（Bcl-2、Bax、CHOP、GPR78、PERK、p-PERK、p-eIF2α、eIF2）的表达。结果 与空白组比较,随着五味子乙素浓度增大,细胞存活率明显降低,其IC₅₀为19.16 μmol/L;与对照组比较,五味子乙素（10、20、40 μmol/L）均能抑制细胞克隆形成(P<0.05),且呈剂量依赖;五味子乙素（10、20、40 μmol/L）均可诱导细胞凋亡（P<0.05）,使抗凋亡蛋白BCL-2的表达显著降低,促凋亡蛋白Bax的表达显著升高(P<0.05);五味子乙素（10、20、40 μmol/L）显著升高细胞内ROS水平(P<0.05),且呈剂量依赖;五味子乙素（10、20、40 μmol/L）能够激发内质网应激,使内质网应激相关蛋白CHOP、GPR78、p-eIF2α表达增多(P<0.05),且呈剂量依赖。结论 五味子乙素可能通过ROS介导内质网应激诱导MDA-MB-231细胞凋亡。     

基于网络药理学及实验验证补骨脂乙素治疗2型糖尿病的作用机制

目的 通过网络药理学与实验验证相结合的方法探究补骨脂乙素治疗2型糖尿病的作用机制。方法 使用ECTM、SwissTargetPrediction、PubChem数据库预测补骨脂乙素的作用靶点,使用GeneCards数据库预测2型糖尿病的潜在靶点。使用Venn数据库进行靶点交互分析,通过STRING数据库计算蛋白相互网络关系(PPI),通过Cytoscape 3.9.1插件MCODE和Cytohubba筛选核心靶点。通过DAVID数据库进行基因本体(GO)和京都基因与基因组百科全书(KEGG)途径的富集分析。建立胰岛素抵抗的HepG2细胞模型,通过2-NBDG检测细胞对葡萄糖摄取的能力,通过RT-PCR和Western blotting检测网络药理学预测的磷脂肌醇-3-激酶(PI3K)/蛋白激酶B(Akt)信号通路相关mRNA及蛋白表达。结果 补骨脂乙素和2型糖尿病共同作用的基因92个,PPI网络(交互得分 ≥ 0.150)包含补骨脂乙素和2型糖尿病共同目标的86个节点和379个"边"。在MCODE和Cytohubba的筛选结果交叉后,获得了35个共同靶点。聚类分析表明,补骨脂乙素作用于2型糖尿病涉及PI3K/Akt信号通路等多种途径及细胞氧化还原反应等多种细胞进程。补骨脂乙素显著增强了胰岛素抵抗细胞对葡萄糖的摄取能力(P<0.05、0.01)。与模型组相比,补骨脂乙素处理后显著增加了胰岛素受体(INS-R)、胰岛素受体底物1(IRS1)、葡萄糖转运蛋白2(GLUT2)、PI3K、Akt mRNA的表达(P<0.01、0.001)。与模型组相比,二甲双胍组和补骨脂乙素组的INS-R、IRS1、GLUT2、Akt、PI3K、p-Akt、p-PI3K的蛋白表达显著上调(P<0.05、0.01、0.001)。结论 补骨脂乙素可能通过PI3K/Akt信号通路增加胰岛素抵抗细胞对葡萄糖的摄取能力,发挥其抗2型糖尿病作用。     

HPLC变换波长法测定伤风停胶囊中橙皮苷、甘草酸和欧前胡素含量

摘 要 目的：建立采用HPLC法测定伤风停胶囊中橙皮苷、甘草酸和欧前胡素含量的方法。 方法: 色谱柱为Thermo Hypersil Gold C18柱（250 mm×4.6 mm,5 μm）,流动相为乙腈（A） 0.1%磷酸（B）,梯度洗脱,波长为284（陈皮苷）,252（甘草酸）,300 nm（欧前胡素）,流速为1.0 ml·min-1,柱温为35℃。结果: 橙皮苷、甘草酸和欧前胡素分别在7.61～114.23,6.14～92.16 ,0.87～13.08 μg·ml-1范围内呈良好的线性关系（r≥0.999 8）;平均加样回收率分别为101.18%,102.70%和98.92%,RSD分别为0.88%,1.24%和1.29%（n=6）。 结论: 该方法准确、稳定、重现性好,可用于伤风停胶囊的质量控制。     

消喘膏的体外透皮吸收研究

摘 要 目的：研究消喘膏中芥子碱硫氰酸盐、延胡索乙素和细辛脂素成分的体外经皮渗透性,为其临床应用提供参考。方法: 采用Franz扩散池法,用LC-MS/MS法测定膏中芥子碱硫氰酸盐、延胡索乙素和细辛脂素成分在一段时间内对大鼠离体皮肤的透皮吸收量。结果: 不同给药量对各成分的累积渗透量影响不大。模型拟合结果显示芥子碱硫氰酸盐透皮行为符合Higuchi方程,延胡索乙素和细辛脂素符合零级方程,延胡索乙素和细辛脂素的透皮速率分别为0.362×10^-1,0.330×10^-2 μg·cm^-2·h^-1。结论:消喘膏具有经皮渗透性能,为其透皮吸收给药途径提供了依据。     

Organic anion transporting polypeptides and organic cation transporter 1 contribute to the cellular uptake of the flavonoid quercetin

Flavonoids such as quercetin and kaempferol mediate several health protective effects, e.g., anticancer effects. They are inhibitors of organic anion transporting polypeptides (OATP) and organic cation transporters (e.g., OCT2). However, little is known whether such transporters contribute to the cellular uptake of flavonoids. Therefore, we investigated the cellular uptake of kaempferol and quercetin using HEK293 cell lines stably expressing different human OATPs or OCT1. Kaempferol was not a substrate of any of the investigated transporters (OATP1A2, OATP1B1, OATP1B3, OATP2A1, OATP2B1, OATP3A1, OATP4A1, OATP5A1, and OCT1). Quercetin showed a significantly higher uptake into the HEK293-OATP1A2, HEK293-OATP2A1, HEK293-OATP2B1, and HEK293-OCT1 cells compared to control cells. The OATP1A2-, OATP2B1-, and OCT1-mediated quercetin uptake was inhibited by known inhibitors such as naringin, cyclosporin A, and quinidine, respectively. The cellular accumulation of quercetin into HEK293-OATP2A1 cells was not inhibited by prostaglandin E₂ and diclofenac. The ionophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (FCCP) reduced the net uptake of quercetin by increasing the uptake in the HEK293-control cells and causing no significant change in the HEK293-OATP2B1 cells indicating that quercetin follows the FCCP-driven proton flux through the plasma membrane. In addition to passive diffusion, the SLC transporters OATP1A2, OATP2B1, and OCT1 contribute to cellular accumulation of quercetin.     

Organic anion-transporting polypeptides contribute to the hepatic uptake of berberine

Abstract

1.?The purpose of this study was to investigate the mechanism of hepatic uptake of berberine. Berberine accumulation in hepatocytes was found to be highly dependent on active uptake, which could not be explained by liver organic cation transporter (OCT) alone.

2.?Our studies indicated that berberine uptake was significantly suppressed by rifampicin, cyclosporine A and glycyrrhizic acid, which act as specific inhibitors of different Oatp isoforms (Oatp1a1, Oatp1a4 and Oatp1b2) in rat hepatocytes. The combination of OCT and OATP inhibitors further reduced berberine accumulation in both rat and human hepatocytes. The uptake of berberine could be increased in human HEK293-OATP1B3 but not in OATP1B1-transfected HEK 293 cells.

3.?Rifampicin could reduce the berberine liver extraction ratio (ER) and double its concentration in the effluent in isolated rat livers. Further in vivo study indicated that berberine plasma exposure could be significantly increased by co-administration of the OATP inhibitor rifampicin or the substrate rosuvastatin.

4.?In conclusion, this study demonstrated that both OCT and OATP contribute to the accumulation of berberine in the liver. OATPs may have important roles in berberine liver disposition and potential clinically relevant drug--drug interactions.     

Hydrophilic anti-migraine triptans are substrates for OATP1A2, a transporter expressed at human blood-brain barrier

1. OATP1A2 is expressed in the luminal membrane of human blood-brain barrier (BBB). The human tissue with the highest OATP1A2 mRNA expression is the brain.

2. We have established a robust BacMam2-OATP1A2 transduced HEK293 system. Among the 36 central nervous system (CNS) marketed drugs tested, hydrophilic triptans, 5-HT_1B/1D receptor agonists for the treatment of migraine attacks, were identified as OATP1A2 substrates. Kinetics (K_m and V_max) were determined for six marketed triptans.

3. Structure-activity relationship (SAR) obtained from 18 triptan structural analogs revealed that the positively charged basic amine atom was essential for efficient OATP1A2-mediated triptan uptake and uptake rate was in the order of tertiary > secondary > primary.

4. Preliminary quantitative SAR analysis of the triptan analogs demonstrated positive correlation between OATP1A2-mediated uptake rate and van der Waals volume (vdw_vol).

5. OATP1A2 was specifically expressed on the apical side of MDCKII monolayer after BacMam2-OATP1A2 transduction and can facilitate transport of triptans across the MDCKII monolayer from apical to basolateral side. Involvement of OATP1A2 for brain penetration of triptans in human requires further investigation.

     

Hydrophilic anti-migraine triptans are substrates for OATP1A2, a transporter expressed at human blood-brain barrier

OATP1A2 is expressed in the luminal membrane of human blood-brain barrier (BBB). The human tissue with the highest OATP1A2 mRNA expression is the brain. We have established a robust BacMam2-OATP1A2 transduced HEK293 system. Among the 36 central nervous system (CNS) marketed drugs tested, hydrophilic triptans, 5-HT(1B/1D) receptor agonists for the treatment of migraine attacks, were identified as OATP1A2 substrates. Kinetics (K(m) and V(max)) were determined for six marketed triptans. Structure-activity relationship (SAR) obtained from 18 triptan structural analogs revealed that the positively charged basic amine atom was essential for efficient OATP1A2-mediated triptan uptake and uptake rate was in the order of tertiary > secondary > primary. Preliminary quantitative SAR analysis of the triptan analogs demonstrated positive correlation between OATP1A2-mediated uptake rate and van der Waals volume (vdw_vol). OATP1A2 was specifically expressed on the apical side of MDCKII monolayer after BacMam2-OATP1A2 transduction and can facilitate transport of triptans across the MDCKII monolayer from apical to basolateral side. Involvement of OATP1A2 for brain penetration of triptans in human requires further investigation.     

Characteristics of Choline Transport Across the Blood-Brain Barrier in Mice: Correlation with In Vitro Data

Purpose. We examined the functional properties of choline transport across the blood-brain barrier (BBB) in mice. We compared the kinetic parameters and transport properties with those found in our in vitro uptake experiments using mouse brain capillary endothelial cells (MBEC4). Methods. The permeability coefficient-surface area product (PS) values of [³H]choline at the BBB were estimated by means of anin situ brain perfusion technique in mice.Results. [³H]Choline uptake was well described by a two-component model: a saturable component and a nonsaturable linear component. The [³H]choline uptake was independent of pH and Na⁺, but was significantly decreased by the replacement of Na⁺ with K⁺. Various basic drugs, including substrates and inhibitors of the organic cation transporter, significantly inhibited the [³H]choline uptake. These in situ (in vivo) results corresponded well to the in vitro results and suggest that the choline transporter at the BBB is a member of the organic cation transporter (OCT) family. Conclusion. The choline transport mechanism at the BBB is retained in MBEC4.     

In vitro assessment of the roles of drug transporters in the disposition and drug–drug interaction potential of olaparib

1.?In vitro assessments were conducted to examine interactions between olaparib (a potent oral inhibitor of poly[ADP-ribose] polymerase) and drug transporters.

2.?Olaparib showed inhibition of the hepatic drug uptake transporters OATP1B1 (IC₅₀ values of 20.3?μM and 27.1?μM) and OCT1 (IC₅₀ 37.9?μM), but limited inhibition of OATP1B3 (25% at 100?μM); inhibition of the renal uptake transporters OCT2 (IC₅₀ 19.9?μM) and OAT3 (IC₅₀ 18.4?μM), but limited inhibition of OAT1 (13.5% at 100?μM); inhibition of the renal efflux transporters MATE1 and MATE2K (IC₅₀s 5.50?μM and 47.1?μM, respectively); inhibition of the efflux transporter MDR1 (IC₅₀ 76.0?μM), but limited inhibition of BCRP (47% at 100?μM) and no inhibition of MRP2. At clinically relevant exposures, olaparib has the potential to cause pharmacokinetic interactions via inhibition of OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K in the liver and kidney, as well as MDR1 in the liver and GI tract. Olaparib was found to be a substrate of MDR1 but not of several other transporters.

3.?Our assessments indicate that olaparib is a substrate of MDR1 and may cause clinically meaningful inhibition of MDR1, OCT1, OCT2, OATP1B1, OAT3, MATE1 and MATE2K.     

齐墩果酸对OATP1B1介导药物转运功能的关联性影响

目的 探讨齐墩果酸对有机阴离子转运多肽1B1（organic anion transporting polypetide1B1,OATP1B1）转运功能的关联性影响。方法 利用稳定表达人OATP1B1的人胚胎肾293（hunan embyonic kindney293,HEK293）细胞株,以氟伐他汀为底物进行OATP1B1摄取反应,观察齐墩果酸对OATP1B1摄取功能的影响。结果 齐墩果酸对OATP1B1摄取氟伐他汀的功能有竞争性抑制作用,抑制常数Ki值为（20.3±2.1）mmol·L^-1。结论 齐墩果酸对肝脏药物转运体OATP1B1转运抑制作用可诱导中西药相互作用。     

Characterization of Calu-3 cell monolayers as a model of bronchial epithelial transport: organic cation interaction studies

Background: To fully exploit organic cation transporters for targeted drug delivery in the lung, the use of a readily available and well-characterized tissue culture model and cheap easily detectable substrates is indispensable.

Objectives: To investigate the suitability of Calu-3 as tissue model for characterizing organic cation permeation across the bronchial cells using a fluorescent dye, 4-(4-(Dimethylamino)styryl)-N-methylpyridinium iodide (4-DI-1-ASP).

Methods: Substrate uptake, inhibition, and transport were performed to establish active transport mechanism. Organic cation transporter expression was determined with quantitative polymerase chain reaction (qPCR), immune-histochemistry, and fluorescent microscopy.

Results: 4-Di-1-ASP uptake in Calu-3 cells was concentration (K_m = 2.7?±?0.3?mM, V_max = 4.6?±?2.6 nmol/µg protein/30?min), temperature (uptake at 37°C>>4°C), and pH dependent (higher uptake at pH ≥ 7). L-carnitine, verapamil, and corticosterone significantly inhibited its uptake with IC₅₀ of 28.2, 0.81, and 0.12?mM, respectively. Transport of the dye across the cells was polarized (AP→BL transport was 2.5-fold > BL→AP), saturable (Km = 43.9?±?3.2) (µM; Vmax =0.0228± nmol/cm²/sec) and reduced 3-fold by metabolic inhibition. The expression pattern of the organic cation transporters (OCT) and carnitine/organic cation transporter (OCTN) isoforms was: OCT1<<OCT3 <OCTN1<OCTN2; OCT2 was not detected.

Conclusions: Based on qPCR, immunohistochemistry, uptake and transport data, the Calu-3 cells can be used as a model for not only studying strategies for optimizing the effect of inhaled organic cations, but also for cross-validating newly-developed respiratory cell lines.     

Organic anion transporting polypeptide (OATP)-mediated transport of coproporphyrins I and III

1.?Organic anion-transporting polypeptides (OATPs) 1B1 and 1B3 are polyspecific transporters that mediate the transport of organic acids into hepatocytes. Inactivating mutations of both OATP1B1 and OATP1B3 alleles lead to Rotor syndrome, a disease characterized by coproporphyrinuria, an elevated urinary excretion of coproporphyrins I and III. It was hypothesized that transport of coproporphyrins I and III was mediated by OATP1B1 and OATP1B3.

2.?This hypothesis was tested using cells transfected with OATP1B1 and OATP1B3. OATP1B-mediated transport of coproporphyrin was time-dependent and concentration-dependent. OATP1B1-mediated transport of coproporphyrins I and III (K_m?=?0.13 and 0.22?µM, respectively), as did OATP1B3 (K_m?=?3.25 and 4.61?µM, respectively). The OATP1B-mediated transport of each coproporphyrin was inhibited by rifampicin.

3.?The specificity of coproporphyrin transport was also investigated where OATP2B1 demonstrated meaningful transport of coproporphyrin III (K_m?=?0.31?µM), while OCT1, OCT2, OAT1, OAT3 and NTCP were negative for coproporphyrin transport.

4.?The identification of coproporphyrins as OATP substrates in vitro more clearly defines the role of OATPs in the hepatic disposition and renal excretion of coproporphyrins I and III and provides compelling evidence for future in vivo exploration of coproporphyrins as biomarkers of OATP activity.     

In vitro evaluation of potential drug interactions mediated by cytochrome P450 and transporters for luseogliflozin,an SGLT2 inhibitor

1.?We evaluated potential in vitro drug interactions of luseogliflozin, a sodium-glucose cotransporter 2 (SGLT2) inhibitor, mediated by CYP inhibition, CYP induction and drug transporters using human liver microsomes, primary hepatocytes and recombinant cells-expressing efflux or uptake transporters, respectively.

2.?Human CYP inhibition studies indicated that luseogliflozin was a weak inhibitor for CYP2C19 with an IC₅₀ value of 58.3?μM, whereas it was not an inhibitor of the other eight major isoforms that were tested. The exposure of primary hepatocytes to luseogliflozin for 72?hrs weakly induced CYP3A4 at a concentration of 10?μM, whereas it did not induce CYP1A2 or CYP2B6 at concentrations of 0.1–10?μM.

3.?An in vitro transport study suggested that luseogliflozin is a substrate for human P-glycoprotein (P-gp), but not for breast cancer resistance protein (BCRP), organic anion transporting polypeptide (OATP) 1B1 and OATP1B3, organic anion transporter (OAT) 1 and OAT3, or organic cation transporter (OCT) 2. Luseogliflozin weakly inhibited OATP1B3 with an IC₅₀ value of 93.1?μM, but those for other transporters are greater than 100?μM.

4.?Based on the therapeutic plasma concentration of the drug, clinically relevant drug interactions are unlikely to occur between luseogliflozin and coadministered drugs mediated by CYPs and/or transporters.