Efflux transport of nicotine,cotinine and trans‐3′‐hydroxycotinine glucuronides by human hepatic transporters

Nicotine is the addiction causing alkaloid in tobacco, and it is used in smoking cessation therapies. Although the metabolic pathways of nicotine are well known and mainly occur in the liver, the transport of nicotine and its metabolites is poorly characterized. The highly hydrophilic nature and urinary excretion of nicotine glucuronide metabolites indicate that hepatic basolateral efflux transporters mediate their excretion. We aimed here to find the transporters responsible for the hepatic excretion of nicotine, cotinine and trans‐3′‐hydroxycotinine (OH‐cotinine) glucuronides. To this end, we tested their transport by multidrug resistance‐associated proteins 1 (MRP1, ABCC1) and MRP3‐6 (ABCC3‐6), which are located on the basolateral membranes of hepatocytes, as well as MRP2 (ABCC2), breast cancer resistance protein (BCRP, ABCG2) and multidrug resistance protein 1 (MDR1, P‐gp, ABCB1) that are expressed in the apical membranes of these cells. ATP‐dependent transport of these glucuronides was evaluated in inside‐out membrane vesicles expressing the transporter of interest. In addition, potential interactions of both the glucuronides and parent compounds with selected transporters were tested by inhibition assays. Considerable ATP‐dependent transport was observed only for OH‐cotinine glucuronide by MRP3. The kinetics of this transport activity was characterized, resulting in an estimated K_m value of 895 µmol/L. No significant transport was found for nicotine or cotinine glucuronides by any of the tested transporters at either 5 or 50 µmol/L substrate concentration. Furthermore, neither nicotine, cotinine nor OH‐cotinine inhibited MRP2‐4, BCRP or MDR1. In this study, we directly examined, for the first time, efflux transport of the three hydrophilic nicotine glucuronide metabolites by the major human hepatic efflux transporters. Despite multiple transporters studied here, our results indicate that an unknown transporter may be responsible for the hepatic excretion of nicotine and cotinine glucuronides.     

Transport Activity of Human MRP3 Expressed in Sf9 Cells: Comparative Studies with Rat MRP3

Purpose. Multidrug resistance-associated protein 3 (MRP3) was initially cloned as a hepatic transporter induced under cholestatic/hyperbilirubinemic conditions. In the present study, transport property of human MRP3 (hMRP3) was compared with that of rat MRP3 (rMRP3). Methods. Adenosine 5 triphosphate (ATP)-dependent uptake of several organic anions into the membrane vesicles isolated from the Sf9 cells expressing hMRP3 and rMRP3 was measured by rapid filtration technique. Results. ATP-dependent uptake of glucuronide conjugates, glutathione conjugates, and [³H]methotrexate (MTX) was stimulated by infection of cDNAs for hMRP3 and rMRP3. The mean (± SE) K_m values for the uptake of 17 estradiol 17-D-glucuronide ([³H]E₂17 G) by hMRP3 and rMRP3 were 42.9 ± 4.3 M and 33.4 ± 2.2 M, respectively. Although the K_i values of glucuronides on the uptake of E₂17G were similar in humans and rats, hMRP3 exhibited higher K_i values toward MTX. In addition, although glycocholate and taurolithocholate 3-sulfate (TLC-S) were transported by both hMRP3 and rMRP3, taurocholate was only transported to a significant degree by rMRP3. Moreover, the inhibitory effect of taurocholate and glycocholate on the transport of E₂17G was much more potent in rMRP3 compared to hMRP3. Conclusion. Collectively, the substrate specificity of hMRP3 resembles that of rMRP3 although differences were observed, particularly in bile acid transport.     

抵抗素在肝脏胰岛素抵抗中的作用及其机制探讨

目的探讨抵抗素在肝脏胰岛素抵抗中的作用及其可能的机制。方法将载有抵抗素基因的重组腺病毒经尾静脉注射构建高抵抗素血症小鼠模型,同时设正常对照组及病毒对照组,取肝脏组织切片行PAS糖原染色半定量观察肝糖代谢变化;以Western blot检测肝腺苷酸活化蛋白激酶(AMPK)的磷酸化,以磷酸化AMPK/总AMPK的比值代表AMPK激活程度;以实时PCR检测肝组织糖异生关键酶葡萄糖6磷酸酶(G6Pase)和磷酸烯醇式丙酮酸羧激酶(PEP-CK)mRNA表达水平的变化。结果重组腺病毒注射d5获得血中抵抗素高表达构建了高抵抗素血症动物模型,糖原染色示高抵抗素血症小鼠肝糖原含量较正常对照及病毒对照组降低(P<0.05);高抵抗素血症组肝AMPK磷酸化水平较正常对照及病毒对照组下降,磷酸化AMPK/总AMPK比值分别为0.78±0.06vs0.93±0.13,0.89±0.05(P<0.05)。高抵抗素血症小鼠G6Pase和PEPCK的mRNA表达升高,G6Pase分别为2.136±0.857vs1.353±0.49,1.250±0.77;PEPCK分别为3.54±0.90vs2.75±0.78,2.63±0.67(P<0.05)。结论抵抗素可能通过抑制肝脏AMPK活性,增加肝糖异生关键酶的表达而影响机体肝糖代谢,降低肝糖储量,参与肝脏胰岛素抵抗的形成。     

奥美拉唑调控肾脏转运体OCT2和P-gp保护顺铂诱导的急性肾损伤

目的：基于肾脏有机阳离子转运体OCT2、多药耐药相关蛋白P-糖蛋白（P-gp）研究奥美拉唑（OME）在大鼠及肾小管上皮细胞中拮抗顺铂诱导急性肾损伤的作用机制。方法：选择雄性SD大鼠随机分为空白组、模型组、药物组（1.8 mg·kg^-1和3.6 mg·kg^-1）以及OME对照组。OME对照组和药物组连续5 d腹腔注射OME,药物组在第5 d腹腔注射顺铂（15 mg·kg^-1）,模型组连续注射5 d生理盐水,第5天腹腔注射顺铂;空白组以上述相同时间注射生理盐水。各组在第7天取血和肾脏,通过HE染色和生化指标BUN、Cr检测观察肾组织损伤程度;MTT法检测肾小管上皮HK-2细胞存活率,Western blot检测OCT2和P-gp蛋白表达。结果：（1）OME能够减轻顺铂对HK-2细胞的损伤程度,提高细胞生存率;（2）与模型组比较,OME降低大鼠死亡率和肾脏肥大指数,改善肾小管空泡变性和炎性浸润,明显降低血清生化指标BUN和Cr水平;（3）Western blot实验结果表明在HK-2细胞和SD大鼠中,OME能够抑制肾脏OCT2的表达,上调外排转运体P-gp的表达。结论：OME能够在大鼠和细胞中有效保护顺铂诱导的急性肾损伤,其保护机制与OME抑制OCT2的表达,上调P-gp的表达有关。     

Faster clearance of omeprazole in mutant Nagase analbuminemic rats: possible roles of increased protein expression of hepatic CYP1A2 and lower plasma protein binding

It is well known that there are various changes in the expression of hepatic and intestinal CYPs in mutant Nagase analbuminemic rats (NARs). It has been reported that the protein expression of hepatic CYP1A2 was increased, whereas that of hepatic CYP3A1 was not altered, and it was also found that the protein expression of the intestinal CYP1A subfamily significantly increased in NARs from our other study. In addition, in this study additional information about CYP changes in NARs was obtained; the protein expression of the hepatic CYP2D subfamily was not altered, but that of the intestinal CYP3A subfamily increased in NARs. Because omeprazole is metabolized via hepatic CYP1A1/2, 2D1, 3A1/2 in rats, it could be expected that the pharmacokinetics of omeprazole would be altered in NARs. After intravenous administration of omeprazole to NARs, the Cl_nr was significantly faster than in the controls (110 versus 46.6 ml/min/kg), and this could be due to an increase in hepatic metabolism caused by a greater hepatic CYP1A2 level in addition to greater free fractions of the drug in NARs. After oral administration of omeprazole to NARs, the AUC was also significantly smaller (80.1% decrease) and F was decreased in NARs. This could be primarily due to increased hepatic and intestinal metabolism caused by greater hepatic CYP1A2 and intestinal CYP1A and 3A levels. In particular, the smaller F could mainly result from greater hepatic and intestinal first‐pass effect in NARs than in the controls. Copyright © 2009 John Wiley & Sons, Ltd.     

反相高效液相色谱法测定奥美拉唑及其代谢产物5′-羟基奥美拉唑和奥美拉唑砜的血药浓度

目的　建立测定奥美拉唑及其代谢产物 5′ 羟基奥美拉唑和奥美拉唑砜的血药浓度的方法 ,用于测定其血药浓度并进行临床药代动力学研究。方法　采用高效液相 -二极管阵列色谱法测定奥美拉唑及其代谢产物 5′ 羟基奥美拉唑和奥美拉唑砜的血药浓度。结果　奥美拉唑的校正标准曲线分别为Y =- 0 0 0 4 4 99+0 0 0 190 9X(r =0 9990 ) ,其5 0、5 0 0、2 0 0 0mg·L-13浓度的血样回收率分别为 90 36 %、10 9 6 2 %、 10 8 91% ;精密度分别 9 86 %、 7 86 %、15 5 2 % ;5′ 羟基奥美拉唑的校正标准曲线分别为Y =-0 0 0 36 5 9+0 0 0 132 8X (r =0 9970 ) ,其 2 0、2 0 0、10 0 0mg·L-13浓度的血样回收率分别为 79 4 2 %、 96 4 9%、95 0 4 % ;精密度分别为 8 95 %、 4 5 2 %、9 73% ;奥美拉唑砜的校正标准曲线分别为Y =0 0 0 92 4 8+0 0 0 176 5X(r =0 9992 ) ,其 2 0、2 0 0、10 0 0mg·L-13浓度的血样回收率分别为 94 4 4 %、 10 5 5 9%、 10 4 2 6 % ;精密度分别为8 72 %、8 5 8%、9 6 0 %。志愿受试者禁食口服 2 0mg奥美拉唑胶囊后 ,奥美拉唑及其代谢产物 5′ 羟基奥美拉唑和奥美拉唑砜的Cmax分别为 14 6 2 2 7、4 0 8 4 332、4 5 4 36 37mg·L-1。结论　此方法可满足测定要求 ,可用于?     

奥美拉唑在葡聚糖硫酸钠诱导的溃疡性结肠炎大鼠肝肾肠微粒体中的代谢研究

目的：研究葡聚糖硫酸钠（DSS）诱导的溃疡性结肠炎（UC）模型大鼠不同病期肝、肾、肠微粒体中奥美拉唑（OME）代谢酶活性的变化。方法：DSS诱导UC急性期（UCA）和恢复期（UCR）大鼠模型。同时制备UCA、UCR以及正常对照（NOR）组大鼠的肝、肾、肠微粒体。将不同浓度的OME与微粒体共孵育,用LC/MS/MS检测样品中5-OH OME的生成量,比较OME在各组微粒体中的代谢活性。结果：连续饮用5% DSS 7 d,UCA和UCR组大鼠表现出明显的结肠炎症状。DSS停药后7 d,UCR组大鼠症状有所好转。UCA组肝微粒体中5-OH OME的生成速率低于NOR组,UCR组的生成速率恢复到正常水平。UCA组的CL_int与NOR组相比降低了45%。OME在3组大鼠肾和肠微粒体中的代谢活性无显著差异。结论：OME在肝微粒中的代谢会受到UC的影响而改变,可能会影响到OME在患者体内的药动学。     

Acetaminophen toxicity up-regulates MRP2 expression in the liver of cats: an old story with new vision

This study was conducted to clarify the role of efflux transporter MRP₂ in acetaminophen-induced hepatotoxicity in cats. Sixteen mixed bred male cats and four liver samples from mixed breed male dogs were used. The cats were assigned into four groups (n?=?4), received saline and 2, 10 and 50?mg/kg doses of acetaminophen orally for 14 days. Unlike the intact dogs, the MRP₂ was not detectable in control cats. MRP₂ at mRNA level was expressed in the liver of cats, which received the medium and high doses. Data suggest that the MRP₂ expression may involve in the acetaminophen-induced hepatotoxicity in cats.     

Characterization of the Cellular Localization,Expression Level,and Function of SNP Variants of MRP2/ABCC2

PURPOSE: The presence of single nucleotide polymorphisms (SNPs) has been reported for multidrug resistance-associated protein 2 (MRP2/ABCC2). The purpose of the current study was to characterize the localization, expression level, and function of MRP2 variants. METHODS: The expression and cellular localization of the wild-type and three kinds of reported SNP variants of MRP2 molecules were analyzed in LLC-PK1 cells after infection with the recombinant Tet-off adenoviruses. Their function was determined by using the isolated membrane vesicles from the infected LLC-PK1 cells. RESULTS: The transport activity for E217betaG, LTC4, and DNP-SG, normalized by the expression level of MRP2, was similar between the wild-type, V417I, and A1450T MRP2s. The transport activity of S789F MRP2 was slightly higher than that of wild-type MRP2. However, the expression level of S789F and A1450T MRP2 proteins was significantly lower compared with the wild-type and V417I MRP2. In addition, although the wild-type and V417I MRP2 were exclusively localized in the apical membrane, S789F and A1450T MRP2 were located in the apical membrane and also in the intracellular compartment. CONCLUSIONS: These results suggest that the most frequently observed V417I substitution may not affect the in vivo function of MRP2, whereas the much less frequently observed S789F and A1450T may be associated with the reduced in vivo function.     

Effects of cytochrome P450 3A (CYP3A) and the drug transporters P-glycoprotein (MDR1/ABCB1) and MRP2 (ABCC2) on the pharmacokinetics of lopinavir

Background and purpose:

Lopinavir is extensively metabolized by cytochrome P450 3A (CYP3A) and is considered to be a substrate for the drug transporters ABCB1 (P-glycoprotein) and ABCC2 (MRP2). Here, we have assessed the individual and combined effects of CYP3A, ABCB1 and ABCC2 on the pharmacokinetics of lopinavir and the relative importance of intestinal and hepatic metabolism. We also evaluated whether ritonavir increases lopinavir oral bioavailability by inhibition of CYP3A, ABCB1 and/or ABCC2.

Experimental approach:

Lopinavir transport was measured in Madin-Darby canine kidney cells expressing ABCB1 or ABCC2. Oral lopinavir kinetics (+/− ritonavir) was studied in mice with genetic deletions of Cyp3a, Abcb1a/b and/or Abcc2, or in transgenic mice expressing human CYP3A4 exclusively in the liver and/or intestine.

Key results:

Lopinavir was transported by ABCB1 but not by ABCC2 in vitro. Lopinavir area under the plasma concentration – time curve (AUC)_oral was increased in Abcb1a/b^−/− mice (approximately ninefold vs. wild-type) but not in Abcc2^−/− mice. Increased lopinavir AUC_oral (>2000-fold) was observed in cytochrome P450 3A knockout (Cyp3a^−/−) mice compared with wild-type mice. No difference in AUC_oral between Cyp3a^−/− and Cyp3a/Abcb1a/b/Abcc2^−/− mice was observed. CYP3A4 activity in intestine or liver, separately, reduced lopinavir AUC_oral (>100-fold), compared with Cyp3a^−/− mice. Ritonavir markedly increased lopinavir AUC_oral in all CYP3A-containing mouse strains.

Conclusions and implications:

CYP3A was the major determinant of lopinavir pharmacokinetics, far more than Abcb1a/b. Both intestinal and hepatic CYP3A activity contributed importantly to low oral bioavailability of lopinavir. Ritonavir increased lopinavir bioavailability primarily by inhibiting CYP3A. Effects of Abcb1a/b were only detectable in the presence of CYP3A, suggesting saturation of Abcb1a/b in the absence of CYP3A activity.     

药物肠道首过效应定量评价及机制研究方法的建立

传统观念认为肝脏是首过效应主要发生器官,但越来越多的研究表明,口服药物在吸收过程即可被代谢,产生肠道首过效应(intestinal first pass effect),甚至对某些药物来说,肠道是其主要代谢器官~([1,2]),但首过效应相关研究中存在的主要问题是肝脏和肠道对药物代谢作用的程度无法予以量化.     

Characterizing the Expression of CYP3A4 and Efflux Transporters (P-gp,MRP1, and MRP2) in CYP3A4-Transfected Caco-2 Cells After Induction with Sodium Butyrate and the Phorbol Ester 12-O-Tetradecanoylphorbol-13-Acetate

Purpose. To examine the changes in expression levels of CYP3A4 and efflux transporters in CYP3A4-transfected Caco-2 (colon carcinoma) cells in the presence of the inducers sodium butyrate (NaB) and 12-O-tetradecanoylphorbol-13-acetate (TPA). To characterize the transport of [³H]-digoxin and the metabolism of midazolam in the cells under different inducing conditions. Methods. CYP3A4-Caco-2 cells were seeded onto cell culture inserts and were grown for 13-14 days. Transport and metabolism studies were performed on cells induced with NaB and/or TPA for 24 h. The expression and localization of P-gp, MRP1, MRP2, and CYP3A4 were examined by Western blot and confocal microscopy. Results. In the presence of both inducers, CYP3A4 protein levels were increased 40-fold over uninduced cells, MRP2 expression was decreased by 90%, and P-gp and MRP1 expression were unchanged. Midazolam 1-OH formation exhibited a rank order correlation with increased CYP3A4 protein, whereas [³H]-digoxin transport (a measure of P-gp activity) was unchanged with induction. P-gp and MRP2 were found on the apical membrane, whereas MRP1 was found peri-nuclear within the cell. CYP3A4 displayed a punctate pattern of expression consistent with endoplasmic reticulum localization and exhibited preferential polarization towards the apical side of the cell. Conclusions. The present study characterized CYP3A4-Caco-2 cell monolayers when induced for 24 h in the presence of both NaB and TPA. These conditions provide intact cells with significant CYP3A4 and P-gp expression suitable for the concurrent study of transport and metabolism.     

Altered expression of MRP2, MRP3 and UGT2B1 in the liver affects the disposition of morphine and its glucuronide conjugate in a rat model of cholestasis

Objectives The aim was to investigate the disposition of morphine and morphine‐3‐glucuronide (M3G) in a rat model of cholestasis induced by bile duct ligation (BDL). Methods Morphine (15 mg/kg) was administered intravenously, and morphine and M3G concentrations in the plasma and urine measured by HPLC. Changes in the mRNA expression of multidrug resistance‐associated protein (MRP)2, MRP3 and UDP‐glucuronosyltransferase (UGT)2B1 in the liver were estimated using RT‐PCR. Key findings Although the plasma morphine concentrations declined exponentially, the elimination was delayed 3 and 5 days after BDL. Plasma M3G concentrations on day 1 after BDL were similar to those in the untreated control group, but were increased 3 and 5 days after BDL. Expression of MRP3 and UGT2B1 mRNA increased after BDL. The urinary excretion of M3G was increased significantly after BDL. Conclusions Enhanced glucuronidation of morphine and transportation of M3G into the blood increased the plasma M3G concentration in the BDL groups. However, M3G disposition 1 day after BDL was similar to that in the untreated control group because urinary excretion of M3G increased.     

酚红外排水平评价肺上皮细胞多药耐药相关蛋白1的功能

目的：以酚红为多药耐药相关蛋白1（MRP1）的底物,丙磺舒为其抑制剂,通过酚红的外排水平建立评价肺上皮细胞MRP1的功能的方法。方法：小鼠静脉注射酚红（250mg/kg）,30min后进行肺泡灌洗,测定灌洗液中酚红的浓度,并以雾化吸入MRP1特异性抑制剂丙磺舒（150mg/kg）作为对照,观察给予抑制剂前后灌洗液中酚红浓度及灌洗液中酚红浓度/血浆中酚红浓度比值的变化,来评价小鼠肺上皮细胞MRP1功能的改变。结果：与未给予抑制剂相比,给予MRP1抑制剂丙磺舒后,血浆中酚红浓度无显著性差异,但与未给予抑制剂相比,给予MRP1抑制剂丙磺舒60min后,其肺泡灌洗液中酚红浓度、灌洗液中酚红浓度/血浆中酚红浓度比值显著性降低。结论：肺泡灌洗液中酚红的外排水平反映小鼠肺上皮细胞MRP1功能的改变。     

Coordinate regulation of UDP-glucuronosyltransferase UGT1A6 induction by 3-methylcholanthrene and multidrug resistance protein MRP2 expression by dexamethasone in primary rat hepatocytes

Concentration-dependent regulation of 3-methylcholanthrene (MC) inducibility of UDP-glucuronosyltransferase UGT1A6 by the synthetic glucocorticoid, dexamethasone (DEX) was studied. Treatment of cultured rat hepatocytes with MC, 0.1, 1, and 10 microM DEX, and MC combined with DEX, resulted in different induction patterns measured in the intact cells compared to that observed in the microsomes prepared from the same cells. DEX treatment in various concentrations caused a concentration-dependent increase in p-nitrophenol (p-NP) conjugation in intact cells (3-, 4-, and 5-fold over control, respectively), and it positively regulated MC induction (4-, 5-, and 6-fold over control, respectively). In contrast, DEX had smaller effect on microsomal p-NP conjugation (115, 200, 220% of control, respectively) and although MC induction was increased significantly by 0.1 microM DEX (520% of control), but higher concentrations of DEX (10 microM) decreased the degree of induction to 410%. Similar results obtained from in vivo experiments showed that at high DEX concentration (100mg/kg), the rate of MC induction (540%) decreased (420%). Permeabilization of the plasma membrane resulted in a 15-fold increase of p-NP conjugation indicating the importance of transport in the rate of overall p-NP elimination, and the induction pattern was similar to that observed in microsomes isolated from cells. Hyper-osmolarity (405 mOsmol/L) led to a 3-fold decrease of p-NP conjugation, the loss of DEX inducibility and reduction of the MRP2 protein level. Our results suggest coordinated regulation of UGT1A6 inducibility and substrate or product transport by DEX.     

Interaction of hesperetin glucuronide conjugates with human BCRP, MRP2 and MRP3 as detected in membrane vesicles of overexpressing baculovirus-infected Sf9 cells

The citrus flavonoid hesperetin (4'-methoxy-3',5,7-trihydroxyflavanone) is the aglycone of hesperidin, the major flavonoid present in sweet oranges. Hesperetin 7-O-glucuronide (H7G) and hesperetin 3'-O-glucuronide (H3'G) are the two most abundant metabolites of hesperetin in vivo. In this study, their interaction with specific ABC transporters, believed to play a role in the disposition and bioavailability of hesperetin, was studied using Sf9 membranes from cells overexpressing human BCRP (ABCG2), MRP2 (ABCC2) and MRP3 (ABCC3). Both H7G and H3'G were tested for their potential to activate and inhibit ATPase activity, and to inhibit vesicular transport by these transporters. Both H7G and H3'G demonstrated interaction with all tested ABC transporters, especially with BCRP and MRP3. An interesting difference between H7G and H3'G was seen with respect to the interaction with BCRP: H7G stimulated the ATPase activity of BCRP up to 76% of the maximal effect generated by the reference activator sulfasalazine, with an EC(50) of 0.45 μM, suggesting that H7G is a high affinity substrate of BCRP, whereas H3'G did not stimulate BCRP ATPase activity. Only moderate inhibition of BCRP ATPase activity at high H3'G concentrations was observed. This study provides information on the potential of hesperetin glucuronide conjugates to act as specific ABC transporter substrates or inhibitors and indicates that regio-specific glucuronidation could affect the disposition of hesperetin.     

The effects of omeprazole on endocrine function in man

Summary We have assessed the effect of omeprazole on various endocrine functions in man. Eight healthy subjects took 60 mg omeprazole or placebo daily for 1 week in a double-blind, randomized, cross-over study. On Day 7 basal concentrations of follicle-stimulating hormone (FSH), luteinising hormone (LH), prolactin, testosterone, thyroid-stimulating hormone (TSH), and serum thyroxine (T₄) and tri-iodothyronine (T₃) were measured, followed by the gonadotrophin response to luteinising hormone releasing hormone (LHRH) and the prolactin and TSH responses to thyrotrophin releasing hormone (TRH).There were no differences in basal or stimulated values between omeprazole and placebo.In a second study, a further 8 subjects were similarly treated, and on Day 7 serial measurements of cortisol and 11-deoxycortisol were made before and for 2.5 h after intravenous adrenocorticotrophin (ACTH).There were no differences in basal values or pattern of response to ACTH for either hormone.Omeprazole in clinical practice is unlikely to cause any significant interference in endocrine function.     

The impact of cytokines on the expression of drug transporters,cytochrome P450 enzymes and chemokine receptors in human PBMC

Background and purpose:

The function of transporters in peripheral blood mononuclear cells (PBMC) has been characterized, but less is known about cytochrome P450 (CYP) enzyme function in these cells. Given that cytokines are dysregulated in many diseases, the purpose of this work was to assess the impact of cytokines on the expression of CYPs, transporters and chemokine receptors in PBMC.

Experimental approach:

Human PBMC were incubated with cytokines for 48 h. ATP-binding cassette (ABC)B1, ABCC1, ABCC2, CYP2B6, CYP3A4, CXCR4 and CCR5 expression were measured by quantitative polymerase chain reaction and flow cytometry at 0, 4, 8, 24 and 48 h. Enzyme activity was assessed using fluorescent probes.

Key results:

We show here functional activity of CYP3A4 and CYP2B6 in PBMC. Furthermore, cytokines had a significant impact on the mRNA and protein expression of all proteins. For example, interleukin-2 (IL-2) had a marked impact on ABCB1 mRNA (% control 4745 ± 11961) and protein (% control 200 ± 57). Increases in drug efflux transporter expression, in response to cytokines, resulted in reduced cellular accumulation of digoxin [decrease of 17% and 26% for IL-2 and interferon-γ (IFNγ) respectively] and saquinavir (decrease of 28% and 30% for IL-2 and IFNγ respectively). The degree to which drug transporter and chemokine receptor expression changed in response to cytokines was positively correlated (e.g. ABCB1 and CXCR4, r² = 0.545).

Conclusions and implications:

These data have important implications for diseases in which cytokines are dysregulated and for which pharmacological intervention targets immune cells.