Evaluation of 309 molecules as inducers of CYP3A4, CYP2B6, CYP1A2, OATP1B1, OCT1, MDR1, MRP2, MRP3 and BCRP in cryopreserved human hepatocytes in sandwich culture

Abstract

1. Regulation of hepatic metabolism or transport may lead to increase in drug clearance and compromise efficacy or safety. In this study, cryopreserved human hepatocytes were used to assess the effect of 309 compounds on the activity and mRNA expression (using qPCR techniques) of CYP1A2, CYP2B6 and CYP3A4, as well as mRNA expression of six hepatic transport proteins: OATP1B1 (SCLO1B1), OCT1 (SLC22A1), MDR1 (ABCB1), MRP2 (ABCC2), MRP3 (ABCC3) and BCRP (ABCG2).

2. The results showed that 6% of compounds induced CYP1A2 activity (1.5-fold increase); 30% induced CYP2B6 while 23% induced CYP3A4. qPCR data identified 16, 33 or 32% inducers of CYP1A2, CYP2B6 or CYP3A4, respectively. MRP2 was induced by 27 compounds followed by MDR1 (16)?>?BCRP (9)?>?OCT1 (8)?>?OATP1B1 (5)?>?MRP3 (2).

3. CYP3A4 appeared to be down-regulated (≥2-fold decrease in mRNA expression) by 53 compounds, 10 for CYP2B6, 6 for OCT1, 4 for BCRP, 2 for CYP1A2 and OATP1B1 and 1 for MDR1 and MRP2.

4. Structure–activity relationship analysis showed that CYP2B6 and CYP3A4 inducers are bulky lipophilic molecules with a higher number of heavy atoms and a lower number of hydrogen bond donors. Finally, a strategy for testing CYP inducers in drug discovery is proposed.     

Contribution of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to hepatic uptake of nateglinide, and the prediction of drug-drug interactions via these transporters

Objectives We have investigated the contributions of organic anion transporting polypeptide (OATP) 1B1 and OATP1B3 to the hepatic uptake of nateglinide, and the possibility of drug–drug interactions via these transporters. Methods Uptake studies using transporter‐expressing HEK293 cells and cryopreserved human hepatocytes were performed to examine the contributions of each transporter. Inhibition studies using cryopreserved human hepatocytes were performed to examine the possibility of drug–drug interactions. Key findings The rate of saturable hepatic uptake of nateglinide using human hepatocytes was 47.6%. A certain increase in uptake was observed in the examination using transporter‐expressing HEK293 cells, indicating contributions of OATP1B1 and OATP1B3 to hepatic nateglinide uptake. The 50% inhibitory concentration (IC50) values of nateglinide using cryopreserved human hepatocytes for uptake of estrone 3‐sulfate (substrate of OATP1B1), and cholecystokinin octapeptide (substrate of OATP1B3) were 168 and 17.4 µmol/l, respectively. Moreover, ciclosporin inhibited saturable hepatic uptake of nateglinide with an IC50 value of 6.05 µmol/l. The calculated 1 + I_in,max,u/IC50 values for inhibition of OATP1B1 and OATP1B3 by nateglinide, and the inhibition of saturable uptake of nateglinide by ciclosporin, were all close to 1, indicating a low clinical risk of drug–drug interaction with nateglinide taken up via OATP1B1 and OATP1B3. Conclusions OATP1B1 and OATP1B3 may have contributed to the hepatic uptake of nateglinide, but the possibility of drug–drug interactions appeared to be low.     

Xenobiotic transporters of the human organic anion transporting polypeptides (OATP) family

1.?The organic anion transporting polypeptides (humans OATP; other species Oatp) belong to the SLCO gene superfamily of transporters and are twelve transmembrane domain glycoproteins expressed in various epithelial cells. Some OATPs/Oatps are expressed in a single organ, while others are expressed ubiquitously.

2.?The functionally characterized members mediate sodium-independent transport of a variety of structurally independent, mainly amphipathic organic compounds, including bile salts, hormones and their conjugates, toxins, and various drugs.

3.?This review summarizes the general features and the substrates of the eleven human OATPs. Furthermore, it reviews what is known about the mechanism of their multispecificity, their predicted structure, their role in drug–food interactions, and their role in cancer.

4.?Finally, some open questions are raised that need to be addressed to advance OATP research in the near future.     

Gemfibrozil and its glucuronide inhibit the hepatic uptake of pravastatin mediated by OATP1B1

When pravastatin (40?mg/day) was co-administered with gemfibrozil (600?mg, b.i.d., 3 days) to man, the AUC of pravastatin increased approximately 2-fold. We have clarified that OATP1B1 is a key determinant of the hepatic uptake of pravastatin in humans. Thus, we hypothesized that gemfibrozil and the main plasma metabolites, a glucuronide (gem-glu) and a carboxylic acid metabolite (gem-M3), might inhibit the hepatic uptake of pravastatin and lead to the elevation of the plasma concentration of pravastatin. Gemfibrozil and gem-glu inhibited the uptake of ¹⁴C-pravastatin by human hepatocytes with K_i values of 31.7?µM and 15.7?µM, respectively and also inhibited pravastatin uptake by OATP1B1-expressing Xenopus laevis oocytes with K_i values of 15.1?µM and 7.6?µM. Additionally, we examined the biliary transport of pravastatin and demonstrated that pravastatin was transported by MRP2 using both human canalicular membrane vesicles (hCMVs) and human MRP2-expressing vesicles. However, gemfibrozil, gem-glu and gem-M3 did not affect the biliary transport of pravastatin by MRP2. Considering the plasma concentrations of gemfibrozil and gem-glu in humans, the inhibition of OATP1B1-mediated hepatic uptake of pravastatin by gem-glu would contribute, at least in part, to the elevation of plasma concentration of pravastatin by the concomitant use of gemfibrozil.     

Modulation of transporter activity of OATP1B1 and OATP1B3 by the major active components of Radix Ophiopogonis

Abstract

1. Radix Ophiopogonis is often an integral part of many traditional Chinese formulas, such as Shenmai injection used to treat cardio-cerebrovascular diseases. This study aimed to investigate the influence of the four active components of Radix Ophiopogonis on the transport activity of OATP1B1 and OATP1B3.

2. The uptake of rosuvastatin in OATP1B1-HEK293T cells were stimulated by methylophiopogonanone A (MA) and ophiopogonin D′ (OPD′) with EC₅₀ calculated to be 11.33?±?2.78 and 4.62?±?0.64?μM, respectively. However, there were no remarkable influences on rosuvastatin uptake in the presence of methylophiopogonanone B (MB) or ophiopogonin D (OPD). The uptake of atorvastatin in OATP1B1-HEK293T cells can be increased by MA, MB, OPD and OPD′ with EC₅₀ calculated to be 6.00?±?1.60, 13.64?±?4.07, 10.41?±?1.28 and 3.68?±?0.85?μM, respectively.

3. The uptake of rosuvastatin in OATP1B3-HEK293T cells was scarcely influenced by MA, MB and OPD, but was considerably increased by OPD′ with an EC₅₀ of 14.95?±?1.62?μM. However, the uptake of telmisartan in OATP1B3-HEK293T cells was notably reduced by OPD′ with an IC₅₀ of 4.44?±?1.10?μM, and barely affected by MA, MB and OPD.

4. The four active components of Radix Ophiopogonis affect the transporting activitives of OATP1B1 and OATP1B3 in a substrate-dependent manner.

     

The inhibitory effects of camptothecin (CPT) and its derivatives on the substrate uptakes mediated by human solute carrier transporters (SLCs)

1.?Camptothecin (CPT) and its derivatives are potent candidate compounds in treating cancers. However, their clinical applications are largely restricted by severe toxicities.

2.?The solute carrier transporters (SLCs), particularly the organic anion transporting polypeptides and organic anion/cation transporters (OATs/OCTs) are widely expressed in human key organs and responsible for the cellular influx of many substances including endogenous substrates and many clinically important drugs. Drug–drug interactions through SLCs often result in unsatisfied therapeutic outcomes and/or unexpected toxicities.

3.?This study investigated the inhibitory effects of CPT and its eight derivatives on the cellular uptake of specific substrates mediated by the essential SLCs in over-expressing Human embryonic kidney 293 cells.

4.?Our data revealed that CPT, 10-hydroxycamptothecin (HCPT), 10-methoxycamptothecin (MCPT) and 9-nitrocamptothecin (9NC) significantly inhibit the uptake activity of OAT3. 9NC also inhibited the substrate transport mediated by OAT1. The substrate uptakes of OAT1, OCTN1 and OCTN2 were significantly decreased in the presence of CZ112, while CPT-11 potently down-regulated the transport activity of OCT1 and OCT3.

5.?In summary, our study demonstrated that CPT and its eight derivatives selectively inhibit the substrate uptakes mediated by the essential SLCs. This information contributes to understanding the localized toxicity of CPTs and provides novel molecular targets for the therapeutic optimization of CPTs in the future.     

6种药物对有机阴离子转运多肽OATP1B1及其基因多态性A388G、T521C转运作用的影响

目的 研究6种药物对有机阴离子转运多肽OATP1B1及其基因多态性A388G、T521C转运作用的影响。方法 体外培养稳定高表达OATP1B1和OATP1B1基因多态性A388G、T521C的人胚肾细胞（HEK293）株,高表达空白载体（Mock）的HEK293细胞为空白对照,实时荧光定量PCR（qRT-PCR）法检测各转运体细胞中mRNA表达;放射性标记化合物³Hestrone sulfate作为转运底物、利福平作为阳性抑制剂验证各高表达细胞的转运活性;测定30 μmol·L^-1的达比加群、辛伐他汀、替格瑞洛、卡培他滨、多西他赛、依那普利对各细胞³H-estrone sulfate摄入活性的抑制作用,并依据抑制试验结果,进一步测定辛伐他汀、替格瑞洛、多西他赛对转运体细胞的半数抑制浓度（IC₅₀）。结果 HEK293细胞内导入的各种转运体基因都呈现良好的复制表达;OATP1B1、OATP1B1/A388G、OATP1B1/T521C对底物³H-estrone sulfate（5 μmol·L^-1）的转运活性分别为Mock细胞的39、49和48倍,30 μmol·L^-1利福平添加后,可将细胞的转运活性抑制到50%以下;辛伐他汀、替格瑞洛、多西他赛对OATP1B1的抑制作用较强,30 μmol·L^-1给药的转运活性分别为对照组的（40.09±1.95）%、（33.82±0.61）%、（45.08±0.22）%;辛伐他汀对OATP1B1、OATP1B1/A388G、OATP1B1/T521C的IC₅₀分别为14.2、＞100、＞100 μmol·L^-1,替格瑞洛的IC₅₀分别为19.1、68.4、＞100 μmol·L^-1,多西他赛的IC₅₀分别为17.6、22.9、19.3 μmol·L^-1。结论 OATP1B1基因多态性在一定程度上改变了抑制剂对转运体活性的影响程度。     

Honey flavonoids inhibit hOATP2B1 and hOATP1A2 transporters and hOATP-mediated rosuvastatin cell uptake in vitro

1. Some flavonoids contained in the common diet have been shown to interact with important membrane uptake transporters, including organic anion transporting polypeptides (OATPs). OATP2B1 and OATP1A2 expressed in the apical membrane of human enterocytes may significantly contribute to the intestinal absorption of drugs, e.g. statins. This study is aimed at an evaluation of the inhibitory potency of selected food honey flavonoids (namely galangin, myricetin, pinocembrin, pinobanksin, chrysin and fisetin) toward hOATP2B1 and hOATP1A2 as well as at examining their effect on the cellular uptake of the known OATP substrate rosuvastatin.

2. Cell lines overexpressing the hOATP2B1 or hOATP1A2 transporter were employed as in vitro model to determine the inhibitory potency of the flavonoids toward the OATPs.

3. Chrysin, galangin and pinocembrin were found to inhibit both hOATP2B1 and hOATP1A2 in lower or comparable concentrations as the known flavonoid OATP inhibitor quercetin. Galangin, chrysin and pinocembrin effectively inhibited rosuvastatin uptake by hOATP2B1 with IC₅₀ ～1–10?μM. The inhibition of the hOATP1A2-mediated transport of rosuvastatin by these flavonoids was weaker.

4. The found data indicate that several of the tested natural compounds could potentially affect drug cellular uptake by hOATP2B1 and/or hOATP1A2 at relative low concentrations, a finding which suggests their potential for food–drug interactions.     

Involvement of mitogen-activated protein kinase signaling pathways in microcystin-LR-induced apoptosis after its selective uptake mediated by OATP1B1 and OATP1B3.

The serine/threonine protein phosphatase (PP) 2A inhibitor, microcystin-LR, selectively induces liver damage and promotes hepatocarcinogenesis. It is thought that microcystin-LR affects hepatocellular viability mainly through inhibition of PP2A, partially through PP1, and, in addition, by generation of reactive oxygen species (ROS). However, the molecular basis of the selective liver damage and the balance between cell death and survival remained unclear. We analyzed the cytotoxicity of low doses of microcystin-LR using HEK293 cells stably expressing the human hepatocyte uptake transporters, organic anion transporting polypeptide (OATP)1B1 (HEK293-OATP1B1 cells) and OATP1B3 (HEK293-OATP1B3 cells). HEK293-OATP1B1 (IC(50) 6.6nM) and HEK293-OATP1B3 cells (IC(50) 6.5nM) were equally very sensitive to microcystin-LR. In contrast, control-vector-transfected (HEK293-CV) cells were resistant to microcystin-LR. Using HEK293-OATP1B3 cells, the cytotoxicity was attenuated by substrates and inhibitors of OATP1B3, including bromosulfophthalein, rifampicin, and cyclosporin A. Microcystin-LR was transported into HEK293-OATP1B3 cells with 1.2 microM Km value, and its uptake was inhibited by above substances. Accumulation of microcystin-LR in the HEK293-OATP1B1 and HEK293-OATP1B3 cells was increased in a dose-dependent manner but not in HEK293-CV cells. Cellular serine/threonine PP activity of HEK293-OATP1B3 cells was decreased by microcystin-LR but not in HEK293-CV cells. Apoptotic changes were observed after incubation of the HEK293-OATP1B3 cells with microcystin-LR. We found by FACS analysis that microcystin-LR induced apoptosis but not necrosis in HEK293-OATP1B3 cells. Microcystin-LR activated several mitogen-activated protein kinases (MAPKs) including ERK1/2, JNK, and p38 through inhibition of PP2A. In addition, the cytotoxicity of microcystin-LR was attenuated by the inhibitors of MAPK pathways, including U0126, SP600125, and SB203580. The ROS scavenger N-acetyl-L-cysteine partially attenuated the cytotoxicity of microcystin-LR. Thus, the present study demonstrates that microcystin-LR induces apoptosis through activation of multiple MAPK pathways subsequent to its selective uptake via OATP1B1 and OATP1B3 and followed by inhibition of PP2A, in addition to the ROS generation which might contribute to apoptosis.     

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。     

Palmatine activates AhR and upregulates CYP1A activity in HepG2 cells but not in human hepatocytes

The protoberberine alkaloid palmatine is present in preparations from medicinal plants such as Coptis chinensis and Corydalis yanhusuo. This study examined whether palmatine affects the expression of cytochromes P450 (CYPs) 1A1 and 1A2 in primary cultures of human hepatocytes and human hepatoma HepG2 cells grown as monolayer or spheroids. Gene reporter assays showed that palmatine significantly activated the aryl hydrocarbon receptor (AhR) and increased the activity of CYP1A1 gene promoter in transiently transfected HepG2 cells. In HepG2 monolayer culture, palmatine also significantly increased mRNA and activity levels of CYP1A1, albeit with considerably less potency than 2,3,7,8-tetrachlorodibenzo-p-dioxin, a prototypical CYP1A inducer. On the other hand, CYP1A activity was not significantly elevated by palmatine in HepG2 spheroids. Moreover, palmatine induced mild or negligible changes in CYP1A1 and CYP1A2 mRNA expression without affecting CYP1A activity levels in primary human hepatocytes. It is concluded that palmatine activates the AhR-CYP1A pathway in HepG2 monolayer, while the potential for CYP1A induction is irrelevant in cell systems which are closer to the in vivo situation, i.e. in HepG2 spheroids and primary cultures of human hepatocytes. Possible induction of CYP1A enzymes by palmatine in vivo remains to be investigated.     

The utility of cold-preserved human hepatocytes in studies on cytochrome P450 induction and hepatic drug transport

Abstract

1. Human hepatocytes that had been cold-preserved in SureTran^TM matrix (Abcellute Ltd, Cardiff, UK) were used for studies on cell viability, cytochrome P450 (CYP) 3A4, 2B6 and 1A2 induction and hepatic drug transporters. It has recently been shown that basal CYP activities are maintained in cold-preserved hepatocytes (Palmgren et al., 2012).

2. After 5?d of cold preservation, the viability was still more than 70%, and after 8?d it was around 60%. In hepatocytes that had been cold-preserved for 3?d, the activity of CYP3A4 was induced around 15-fold upon treatment with 8?µM rifampicin for 72?h. For CYP2B6, the activity was induced 4- to 16-fold in hepatocytes that had been cold-preserved for 3?d and thereafter treated with 1?mM phenobarbital for 72?h. The activity of CYP1A2 was low and close to the limit of detection in non-treated cells that had been cold-preserved for up to 3?d, while the activity increased in cells treated with 0.3–25?µM β-naphthoflavone for 72?h. CYP3A4, 2B6 and 1A2 mRNA levels were only determined with hepatocytes from one donor and increased upon treatment with the inducers.

3. Hepatic uptakes of estrone-3-sulfate, taurocholate, ipratropium and rosuvastatin were stable in human hepatocytes that had been cold-preserved for up to 2?d.

4. In summary, cold-preserved human hepatocytes demonstrate retained viability and can advantageously be used for in vitro induction studies and for studies of hepatic uptake transporters.     

OATP and MRP2-mediated hepatic uptake and biliary excretion of eprosartan in rat and human

BackgroundEprosartan is an angiotensin II receptor antagonist, used in the treatment of hypertension and heart failure in clinical patients. The objective of this study was to clarify the mechanism underlying hepatic uptake and biliary excretion of eprosartan in rats and humans.MethodsPerfused rat liver in situ, rat liver slices, isolated rat hepatocytes and human organic anion-transporting polypeptide (OATP)-transfected cells in vitro were used in this study.ResultsExtraction ratio of eprosartan was decreased by rifampicin in perfused rat livers. Uptake of eprosartan in rat liver slices and isolated rat hepatocytes was significantly inhibited by Oatp modulators such as ibuprofen, digoxin, rifampicin and cyclosporine A, but not by tetraethyl ammonium or p-aminohippurate. Uptake of eprosartan in rat hepatocytes indicated a saturable process. Although uptake of eprosartan in OATP1B3-human embryonic kidney cells (HEK) 293 cells was not observed, significant differences in cellular accumulations of eprosartan between vector- and OATP1B1-Madin–Darby canine kidney strain (MDCK) II cells were found in transcellular transport study. Moreover, cumulative biliary excretion rate of eprosartan in the presence of probenecid (Multidrug resistance-associated protein 2 (Mrp2) inhibitor) was significantly decreased in perfused rat livers. Vectorial basal-to-apical transport of eprosartan was also observed in OATP1B1/MRP2 double transfectants.ConclusionsEprosartan was transported by multiple Oatps (at least Oatp1a1 and Oatp1a4)/Mrp2 in rat and OATP1B1/MRP2, at least, in human.     

Organic anion-transporting polypeptide (OATP) 2B1 contributes to the cellular uptake of theaflavin

Organic anion-transporting polypeptide (OATP) 2B1 has been reported in the apical membranes of the human small intestinal epithelium, where it contributes to the intestinal absorption of pharmacologically active drugs. To investigate the potential for OATP2B1-mediated drug–food interactions, the effects of several polyphenolic compounds on OATP2B1-mediated estrone-3-sulfate (E3S) transport were studied by using OATP2B1-expressing HEK293 cells. Our results showed that some compounds, especially theaflavin, were strong inhibitors of OATP2B1-mediated E3S uptake. Theaflavin showed a significantly higher uptake into the OATP2B1-expressing HEK293 cells than the control cells. The concentration dependence of the uptake of theaflavin was determined over a range of concentrations (0.5–100 μM) and the kinetic parameters (K_m and V_max) of theaflavin uptake were found to be 5.12 ± 0.67 μM and 41.6 ± 1.3 pmol/mg protein/min, respectively. The OATP2B1-mediated theaflavin uptake was inhibited by known OATP2B1 substrates such as E3S, bromsulphthalein (BSP), dehydroepiandrosterone-3-sulfate (DHEAS), and fluvastatin. Our results indicate that theaflavin is a novel substrate of OATP2B1. The results of this study might be helpful to predict the potential OATP2B1-mediated drug–theaflavin interactions and to avoid undesirable clinical consequences.     

Orange and apple juice greatly reduce the plasma concentrations of the OATP2B1 substrate aliskiren

AIM

The aim of this study was to investigate the effects of orange juice and apple juice on the pharmacokinetics and pharmacodynamics of aliskiren.

METHODS

In a randomized crossover study, 12 healthy volunteers ingested 200 ml of orange juice, apple juice or water three times daily for 5 days. On day 3, they ingested a single 150-mg dose of aliskiren. Plasma aliskiren concentrations were measured up to 72 h, its excretion into urine up to 12 h and plasma renin activity up to 24 h.

RESULTS

Orange and apple juice reduced aliskiren peak plasma concentrations by 80% (95% CI 63%, 89%, P < 0.001) and 84% (95% CI 72%, 91%, P < 0.001), and the area under the plasma aliskiren concentration–time curve (AUC) by 62% (95% CI 47%, 72%, P < 0.001) and 63% (95% CI 46%, 74%, P < 0.001), respectively, but had no significant effect on its elimination half-life or renal clearance. The decreases in aliskiren AUC by orange and apple juice correlated with aliskiren AUC during the water phase (r = 0.98, P < 0.001). Plasma renin activity was 87% and 67% higher at 24 h after aliskiren during the orange juice and apple juice phases, respectively, than during the water phase (P < 0.05).

CONCLUSIONS

Orange juice and apple juice greatly reduce the plasma concentrations and renin-inhibiting effect of aliskiren, probably by inhibiting its OATP2B1-mediated influx in the small intestine. Concomitant intake of aliskiren with orange or apple juice is best avoided.     

抗癌药细胞色素P450 1B1抑制剂的研究进展

介绍了有关人细胞色素P450 1B1(CYP1B1)抑制剂的研究概况。CYP1B1是一类在导致癌变的雌激素代谢和激活芳香烃化合物致癌性中起重要作用的酶，在正常组织中通常不表达而在许多肿瘤组织中表达活跃，同时对许多抗癌药物的代谢具有重要影响。这表明CYP1B1抑制剂有望成为又一类肿瘤治疗药物。     

Mixed effects of OATP1B1, BCRP and NTCP polymorphisms on the population pharmacokinetics of pravastatin in healthy volunteers

1.?Pravastatin is a 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor used for the treatment of hyperlipidaemia. This study aims to investigate the effects of genetic polymorphisms in OATP1B1, BCRP and NTCP on pravastatin population pharmacokinetics in healthy Chinese volunteers using a non-linear mixed-effect modelling (NONMEM) approach. A two-compartment model with a first-order absorption and elimination described plasma pravastatin concentrations well.

2.?Genetic polymorphisms of rs4149056 (OATP1B1) and rs2306283 (OATP1B1) were found to be associated with a significant (p?<?0.01) decrease in the apparent clearance from the central compartment (CL/F), while rs2296651 (NTCP) increased CL/F to a significant degree (p?<?0.01). The combination of these three polymorphisms reduced the inter-individual variability of CL/F by 78.8%.

3.?There was minimal effect of rs2231137 (BCRP) and rs2231142 (BCRP) on pravastatin pharmacokinetics (0.01?<?p?<?0.05), whereas rs11045819 (OATP1B1), rs1061018 (BCRP) and rs61745930 (NTCP) genotypes do not appear to be associated with pravastatin pharmacokinetics based on the population model (p?>?0.05).

4.?The current data suggest that the combination of rs4149056, rs2306283 and rs2296651 polymorphisms is an important determinant of pravastatin pharmacokinetics.     

有机阴离子转运多肽1B1（OATP1B1）遗传多态性对瑞格列奈药动学的影响

目的：研究有机阴离子转运多肽1B1（OATP1B1）遗传多态性对瑞格列奈药动学的影响。方法：16名健康男性受试者单剂量po 4 mg瑞格列奈片,并考察其SLCO1B1（OATP1B1的编码基因）常见单体型（SLCO1B1＊1a、＊1b、＊5和＊15）,按基因型将其分成＊1b/＊1b、＊1a/＊1a或＊1a/＊1b、＊1a/＊15或＊1b/＊15三组,采用HPLC-MS-MS法测定给药后不同时间瑞格列奈的血药浓度。利用DAS 2.0计算药动学参数和SPSS 12.0评价统计学差异。结果：SLCO1B1＊1b/＊1b、SLCO1B1＊1a/＊1a或＊1a/＊1b和SLCO1B1＊1a/＊15或＊1b/＊15三组受试者的AUC0-8 h分别为（63±20）、（70±26）和（82±24）μg.L-1.h,Cmax分别为（56±16）、（52±17）和（58±34）μg/L,三者之间差异没有统计学意义。结论：OATP1B1遗传多态性对瑞格列奈药动学有影响,但从本试验结果看,三组之间的差异较小,还不足以影响临床用药的安全性和有效性。     

Derivation of CYP3A4 and CYP2B6 degradation rate constants in primary human hepatocytes: A siRNA-silencing-based approach

The first-order degradation rate constant (k_deg) of cytochrome P450 (CYP) enzymes is a known source of uncertainty in the prediction of time-dependent drug–drug interactions (DDIs) in physiologically-based pharmacokinetic (PBPK) modelling. This study aimed to measure CYP k_deg using siRNA to suppress CYP expression in primary human hepatocytes followed by incubation over a time-course and tracking of protein expression and activity to observe degradation. The magnitude of gene knockdown was determined by qPCR and activity was measured by probe substrate metabolite formation and CYP2B6-Glo™ assay. Protein disappearance was determined by Western blotting. During a time-course of 96 and 60 h of incubation, over 60% and 76% mRNA knockdown was observed for CYP3A4 and CYP2B6, respectively. The k_deg of CYP3A4 and CYP2B6 protein was 0.0138 h⁻¹ (±0.0023) and 0.0375 h⁻¹ (±0.025), respectively. The k_deg derived from probe substrate metabolism activity was 0.0171 h⁻¹ (±0.0025) for CYP3A4 and 0.0258 h⁻¹ (±0.0093) for CYP2B6. The CYP3A4 k_deg values derived from protein disappearance and metabolic activity were in relatively good agreement with each other and similar to published values. This novel approach can now be used for other less well-characterised CYPs.