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.     

Hydrocinchonine,cinchonine, and quinidine potentiate paclitaxel‐induced cytotoxicity and apoptosis via multidrug resistance reversal in MES‐SA/DX5 uterine sarcoma cells

Multidrug resistance (MDR) is one of important issues to cause the chemotherapy failure against cancers including gynecological malignancies. Despite some MDR reversal evidences of natural compounds including quinidine and cinchonine, there are no reports on MDR reversal activity of hydrocinchonine with its analogues quinidine and cinchonine especially in uterine sarcoma cells. Thus, in the current study, we comparatively investigated the potent efficacy of hydrocinchonine and its analogues quinidine and cinchonine as MDR‐reversal agents for combined therapy with antitumor agent paclitaxel (TAX). Hydrocinchonine, cinchonine, and quinidine significantly increased the cytotoxicity of TAX in P‐glycoprotein (gp)‐positive MES‐SA/DX5, but not in the P‐gp‐negative MES‐SA cells at nontoxic concentrations by 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐‐diphenyltetrazolium bromide (MTT) assay. Rhodamine assay also revealed that hydrocinchonine, cinchonine, and quinidine effectively enhanced the accumulation of a P‐gp substrate, rhodamine in TAX‐treated MES‐SA/DX5 cells compared with TAX‐treated control. In addition, hydrocinchonine, cinchonine, and quinidine effectively cleaved poly (ADP‐ribose) polymerase (PARP), activated caspase‐3, and downregulated P‐gp expression as well as increased sub‐G1 apoptotic portion in TAX‐treated MES‐SA/DX5 cells. Taken together, hydrocinchonine exerted MDR reversal activity and synergistic apoptotic effect with TAX in MES‐SA/DX5 cells almost comparable with quinidine and cinchonine as a potent MDR‐reversal and combined therapy agent with TAX. © 2010 Wiley Periodicals, Inc. Environ Toxicol, 2011.     

异鼠李素和阿魏酸在大鼠原代肝细胞的肝摄取性质

目的建立大鼠原代肝细胞摄取模型,评价异鼠李素和阿魏酸的肝细胞摄取性质。方法应用RT-qPCR技术测定0,0.5,2和4h悬浮培养原代大鼠肝细胞上摄取转运体有机阴离子转运多肽(Oatp)1和Oatp2,有机阴离子转运体(Oat)2和有机阳离子转运体(Oct)1的mRNA表达。应用已知底物非索非那定、普伐他汀、甲氨蝶呤和阿昔洛韦评价转运体的功能。将已知底物或受试药物在4℃和37℃分别与肝细胞孵育不同时间,LC-MS/MS定量测定细胞摄取量,考察温度和时间对底物摄取的影响。由浓度依赖的摄取实验,计算得到Km、Vmax和主动摄取率等参数。在肝细胞模型上,将转运体的阳性抑制剂与异鼠李素10μmol·L-1和阿魏酸50μmol·L-1共孵育2min,观察抑制剂对受试药转运的影响。结果悬浮培养大鼠原代肝细胞上表达有转运体Oatp1,Oatp2,Oat2和Oct1,并能介导已知底物的主动摄取。转运体的mRNA表达水平随着时间的延长而快速下降,4h的表达水平约为0h的10%。转运体对已知底物摄取活性也随时间呈下降趋势,但程度显著低于表达水平,4h的活性约为0h的28.7%～71.4%。在经验证的肝细胞模型上,异鼠李素的4℃和37℃摄取无显著差异,主动转运率为14.6%。阿魏酸在37℃的摄取量显著高于4℃,主动转运率为84.1%;Oct阳性抑制剂奎尼丁100μmol·L-1能显著抑制阿魏酸的肝摄取,抑制率为64.9%。结论成功建立了大鼠原代肝细胞摄取模型,并用底物进行了验证。异鼠李素通过被动扩散进入肝细胞,阿魏酸的肝细胞摄取则以主动转运为主。     

Predominant Contribution of Rat Organic Anion Transporting Polypeptide-2 (Oatp2) to Hepatic Uptake of β-Lactam Antibiotics

Purpose To identify the rat hepatic basolateral transporters involved in the hepatic uptake of β-lactam antibiotics using nafcillin as a model β-lactam antibiotic that undergoes extensive biliary excretion. Materials and Methods Uptake by isolated rat hepatocytes and Xenopus laevis oocytes expressing organic anion transporting peptides (Oatp1, 2, and 4) and organic anion transporter (OAT2) was evaluated. Results Nafcillin uptake by isolated rat hepatocytes was saturable with the K _m of 210 μM and was significantly inhibited by anionic compounds (estrone-3-sulfate and sulfobromophthalein), but not by cationic compounds (tetraethylammonium and 1-methyl-4-phenylpyridinium). In an in vitro uptake study by Xenopus oocytes expressing hepatic basolateral membrane transporters, nafcillin was transported by multiple Oatps with K _m values of 4120 μM (Oatp1/Oatp1a1), 198 μM (Oatp2/Oatp1a4), and 1,570 μM (Oatp4/Oatp1b2), though it was not transported by hOAT2. Comparison of affinity and analysis by the relative activity factor method indicated that Oatp2 is the predominant contributor to the hepatic uptake of nafcillin. Cefadroxil, cefazolin, cefmetazole, cefoperazone, cefsulodin, and cephalexin, though not cefotaxime or ceftriaxone, were also substrates of Oatp2. Conclusion These findings suggest that Oatp2 plays a key role in the hepatic uptake of nafcillin and most other β-lactam antibiotics in rats.     

2‐Hydroxypropyl‐β‐cyclodextrin‐modified SLN of paclitaxel for overcoming p‐glycoprotein function in multidrug‐resistant breast cancer cells

Objectives This study aimed to evaluate the potential of solid lipid nanoparticles (SLNs) of paclitaxel (PTX) modified with a 2‐hydroxypropyl‐β‐cyclodextrin system to enhance cellular accumulation of PTX into p‐glycoprotein (p‐gp)‐expressing cells. Methods The PTX‐loaded‐SLNs consisted of lipid (stearic acid) and surfactants (lecithin and poloxamer 188) and were then modified with 2‐hydroxypropyl‐β‐cyclodextrin by a sonication method. Key findings In terms of cytotoxicity, PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin showed higher cytotoxicity than other formulations. In particular, the cellular uptake of PTX from PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin was about 5.8‐ and 1.5‐fold higher than that from PTX solution and unmodified PTX‐loaded SLNs in MCF‐7/ADR cells, respectively. After a 4‐h incubation, clear fluorescence images inside cells were observed over time. When PTX‐loaded SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin were incubated with MCF‐7/ADR cells for 4 h, cellular uptake of PTX increased 1.7‐fold versus that of PTX in the presence of verapamil. Conclusions These results suggest that optimized SLNs modified with 2‐hydroxypropyl‐β‐cyclodextrin may have potential as an oral drug delivery system for PTX.     

Multiple transport systems mediate the hepatic uptake and biliary excretion of the metabolically stable opioid peptide [D-penicillamine2,5]enkephalin.

Rapid and extensive biliary excretion of [D-penicillamine2,5]enkephalin (DPDPE) in rats as the unchanged peptide suggests that multiple transport proteins may be involved in the hepatobiliary disposition of this zwitterionic peptide. Although DPDPE is a P-glycoprotein substrate, the role of other transport proteins in the hepatic clearance of DPDPE has not been established. Furthermore, the ability of various experimental approaches to quantitate the contribution of a specific hepatic uptake or excretion process when multiple transport systems are involved has not been addressed. 3H-DPDPE uptake in suspended Wistar rat hepatocytes was primarily (>95%) due to temperature-dependent transport mechanisms; similar results were obtained in suspended hepatocytes from Mrp2-deficient (TR-) rats. Pharmacokinetic modeling revealed that saturable and linear processes were involved in 3H-DPDPE uptake in hepatocytes. The use of transport modulators suggested that hepatic uptake of 3H-DPDPE was mediated by Oatp1a1, Oatp1a4, and likely Oatp1b2. Accumulation of 3H-DPDPE in sandwich-cultured (SC) hepatocytes was rapid; uptake of 3H-DPDPE in SC rat hepatocytes from control and TR- rats was similar. However, the biliary excretion index and biliary clearance decreased by 83 and 85%, respectively, in TR- SC rat hepatocytes, indicating that DPDPE is an Mrp2 substrate. Rate constants for uptake and excretion of 3H-DPDPE in SC rat hepatocytes were determined by pharmacokinetic modeling; data were consistent with basolateral excretion of 3H-DPDPE from the hepatocyte. These results demonstrate the complexities of hepatobiliary disposition when multiple transport mechanisms are involved for a given substrate and emphasize the necessity of multi-experimental approaches for the comprehensive resolution of these processes.     

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.     

Sodium fluorescein is a probe substrate for hepatic drug transport mediated by OATP1B1 and OATP1B3

The aim of this study was to characterize the in vitro hepatic uptake kinetics of sodium fluorescein (NaFluo) and identify the transporters involved. NaFluo exhibited saturable uptake kinetics in suspended rat and human hepatocytes as reflected by K_m values of 22.5 and 14.1 µM, and V_max values of 98.3 and 5.8 pmol/(million cells ? min), respectively. Coincubation with known inhibitors (e.g. rifampicin) of organic anion transporting polypeptide (OATP/Oatp; SLCO gene family) significantly decreased NaFluo uptake in hepatocytes. In contrast, neither inhibitors/substrates of the organic cation transporter or organic anion transporter family nor depletion of extracellular sodium resulted in significant inhibition of NaFluo uptake. To explore the contribution of individual uptake transporters, NaFluo uptake was determined in Chinese hamster ovary cells transfected with OATP1B1, OATP1B3, and OATP2B1. Transporter‐mediated uptake of NaFluo was observed in OATP1B1‐ and OATP1B3‐transfected cells (K_m = 4.2 and 10.9 µM; V_max = 30.9 and 135 [pmol/(mg protein ? min)], respectively). NaFluo can be used as a probe substrate to study Oatp/OATP1B‐mediated drug interactions in fluorescence‐based in vitro transport assays of rat and human liver. Labeling of drugs or bile salts with a fluorescein moiety can be expected to result in fluorescent conjugates with substantially altered hepatic uptake characteristics as compared with the unconjugated compounds. © 2011 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 100:5018–5030, 2011     

Kinetic analysis of saturable hepatic uptake of digoxin and its inhibition by rifampicin

Although the organic anion transporter Oatp2 plays a critical role in determining the hepatic clearance of some drugs, little quantitative information exists about its functional characteristics in relation to inhibition of sinusoidal drug uptake. We investigated the uptake kinetics of the Oatp2 substrate digoxin in the isolated perfused rat liver. In the single-pass perfused liver three consecutive digoxin doses of 15, 30 and 45 micorg were administered in the presence or absence of rifampicin (100 micorM), an inhibitor of Oatp2. Digoxin was determined in the outflow samples by HPLC and all data were analyzed by simultaneous nonlinear regression assuming a Michaelis-Menten uptake mechanism. Hepatocellular uptake of digoxin was concentration-dependent with a Michaelis constant (K(M)) of 577.8 ng/ml. Rifampicin significantly reduced uptake (K(M) increased 2.5-fold) without affecting other parameters.     

Enhanced oral bioavailability and anti‐tumour effect of paclitaxel by 20(s)‐ginsenoside Rg3 in vivo

The purpose of this study was to investigate the effect of paclitaxel in combination with 20(s)‐ginsenoside Rg3 on its anti‐tumour effect in nude mice. In the Caco‐2 transport assay, the apparent permeability from the apical side to the basal side (P_app) (A‐B) and P_app (B‐A) of paclitaxel were measured when co‐incubated with different concentrations of 20(s)‐ginsenoside Rg3. The results indicated that the penetration of paclitaxel through the Caco‐2 monolayer from the apical side to the basal side was facilitated by 20(s)‐ginsenoside Rg3 in a concentration‐dependent manner. Meanwhile, 20(s)‐ginsenoside Rg3 inhibited P‐glycoprotein (P‐gp), and the maximum inhibition was achieved at 80 µ m (p < 0.05). The pharmacokinetic parameters of paclitaxel after oral co‐administration of paclitaxel (40 mg/kg) with various doses of 20(s)‐ginsenoside Rg3 in rats were investigated by an in vivo pharmacokinetic experiment. The results showed that the AUC of paclitaxel co‐administered with 20(s)‐ginsenoside Rg3 was significantly higher (p < 0.001 at 10 mg/kg) compared with the control. The relative bioavailability (RB) % of paclitaxel with 20(s)‐ginsenoside Rg3 was 3.4‐fold (10 mg/kg) higher than that of the control. The effect of paclitaxel orally co‐administered with 20(s)‐ginsenoside Rg3 against human tumour MCF‐7 xenografts in nude mice was also evaluated. Paclitaxel (20 mg/kg) co‐administered with 20(s)‐ginsenoside Rg3 (10 mg/kg) exhibited an effective anti‐tumour activity with the relative tumor growth rate (T/C) values of 39.36% (p <0.05). The results showed that 20(s)‐ginsenoside Rg3 enhanced the oral bioavailability of paclitaxel in rats and improved the anti‐tumour activity in nude mice, indicating that oral co‐administration of paclitaxel with 20(s)‐ginsenoside Rg3 could provide an effective strategy in addition to the established i.v. route. Copyright © 2012 John Wiley & Sons, Ltd.     

5‐Fluorouracil treatment alters the expression of intestinal transporters in rats

5‐Fluorouracil (5‐FU), an anticancer drug, causes severe gastrointestinal damage, which may affect the absorption of orally administered drugs including the substrates of intestinal uptake and efflux transporters. This study aimed to investigate quantitatively the effect of 5‐FU‐induced intestinal damage on the expression of intestinal transporters: P‐glycoprotein (P‐gp), breast cancer resistance protein (BCRP) and peptide transporter 1 (PEPT1) in rats. The rats were treated with 5‐FU (30 mg/kg/day, p.o. ) for 5 days to induce intestinal damage, and then the upper, middle and lower intestinal segments were removed. The mRNA and protein expression levels of these transporters in each segment were determined using quantitative real‐time PCR and Western blotting, respectively. In the 5‐FU‐treated rats, the protein levels of P‐gp and Bcrp in the upper segment were significantly increased to 15‐ and 2.6‐fold of the control, respectively, while those in other segments were unaffected. Pept1 expression was increased by 5‐FU in almost all segments. A remarkable increase in P‐gp expression was shown, the uptake of digoxin, a P‐gp substrate, in each intestinal segment was measured using a rat everted sac. As a result, the uptake of digoxin in the upper segments of 5‐FU‐treated rats was decreased compared with that of the control. In conclusion, 5‐FU‐induced intestinal damage was shown to alter the expression of these transporters, especially in the upper intestinal segment, while the characteristics of the influence varied among the transporters. The 5‐FU‐induced intestinal damage may affect transporter‐mediated drug absorption of orally administered drugs in the clinical setting.     

PXR activation impairs hepatic glucose metabolism partly via inhibiting the HNF4α–GLUT2 pathway

Drug-induced hyperglycemia/diabetes is a global issue. Some drugs induce hyperglycemia by activating the pregnane X receptor (PXR), but the mechanism is unclear. Here, we report that PXR activation induces hyperglycemia by impairing hepatic glucose metabolism due to inhibition of the hepatocyte nuclear factor 4-alpha (HNF4α)‒glucose transporter 2 (GLUT2) pathway. The PXR agonists atorvastatin and rifampicin significantly downregulated GLUT2 and HNF4α expression, and impaired glucose uptake and utilization in HepG2 cells. Overexpression of PXR downregulated GLUT2 and HNF4α expression, while silencing PXR upregulated HNF4α and GLUT2 expression. Silencing HNF4α decreased GLUT2 expression, while overexpressing HNF4α increased GLUT2 expression and glucose uptake. Silencing PXR or overexpressing HNF4α reversed the atorvastatin-induced decrease in GLUT2 expression and glucose uptake. In human primary hepatocytes, atorvastatin downregulated GLUT2 and HNF4α mRNA expression, which could be attenuated by silencing PXR. Silencing HNF4α downregulated GLUT2 mRNA expression. These findings were reproduced with mouse primary hepatocytes. Hnf4α plasmid increased Slc2a2 promoter activity. Hnf4α silencing or pregnenolone-16α-carbonitrile (PCN) suppressed the Slc2a2 promoter activity by decreasing HNF4α recruitment to the Slc2a2 promoter. Liver-specific Hnf4α deletion and PCN impaired glucose tolerance and hepatic glucose uptake, and decreased the expression of hepatic HNF4α and GLUT2. In conclusion, PXR activation impaired hepatic glucose metabolism partly by inhibiting the HNF4α‒GLUT2 pathway. These results highlight the molecular mechanisms by which PXR activators induce hyperglycemia/diabetes.Key words: Pregnane X receptor, Hepatocyte nuclear factor 4-alpha, Glucose transporter 2, Hepatic glucose uptake, Diabetes, Drug-induced hyperglycemia     

Comparison of uptake transporter functions in hepatocytes in different species to determine the optimal model for evaluating drug transporter activities in humans

1. A thorough understanding of species-dependent differences in hepatic uptake transporters is critical for predicting human pharmacokinetics (PKs) from preclinical data.

2. In this study, the activities of organic anion transporting polypeptide (OATP/Oatp), organic cation transporter 1 (OCT1/Oct1), and sodium-taurocholate cotransporting polypeptide (NTCP/Ntcp) in cultured rat, dog, monkey and human hepatocytes were compared.

3. The activities of hepatic uptake transporters were evaluated with respect to culture duration, substrate and species-dependent differences in hepatocytes. Longer culture duration reduced hepatic uptake transporter activities across species except for Oatp and Ntcp in rats. Comparable apparent Michaelis–Menten constant (K_m,app) values in hepatocytes were observed across species for atorvastatin, estradiol-17β-glucuronide and metformin. The K_m,app values for rosuvastatin and taurocholate were significantly different across species. Rat hepatocytes exhibited the highest Oatp percentage of uptake transporter-mediated permeation clearance (PS_inf,act) while no difference in %PS_inf,act of probe substrates were observed across species. The in vitro hepatocyte inhibition data in rats, monkeys and humans provided reasonable predictions of in vivo drug–drug interaction (DDIs) between atorvastatin/rosuvastatin and rifampin.

4. These findings suggested that using human hepatocytes with a short culture time is the most robust preclinical model for predicting DDIs for compounds exhibiting active hepatic uptake in humans.

     

Age‐Related Alteration in Hepatic Disposition of the Neurotoxin 1‐Methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine and Pesticides

Abstract: Idiopathic Parkinson's disease may be caused by environmental neurotoxins such as pesticides, however the major risk factor is old age. We postulated that the high incidence of Parkinson's disease in older people is secondary to age‐related impairment of the hepatic detoxification of xenobiotics. Previously, we have shown that there are significant differences between the hepatic disposition of the neurotoxin 1‐methyl‐4‐phenyl‐1,2,3,6‐tetrahydropyridine (MPTP) and pesticides. Here, we investigated whether there are age‐related differences in the hepatic disposition of MPTP and pesticides, putatively associated with the pathogenesis of Parkinson's disease. We measured the hepatic disposition of paraquat, dichlorodiphenyltrichloroethane (DDT), malathion and MPTP using the multiple indicator dilution technique in the perfused livers of Fischer F344 rats aged 3 and 18 months. The recoveries of MPTP, DDT and malathion were increased from the livers of the older rats (by 258%, 253% and 134% compared with young rats, respectively). The hepatic transport of DDT and malathion into hepatocytes was reduced with age suggesting that part of the impaired uptake of neurotoxins may be secondary to an age‐related barrier to influx. Ageing may increase risk of Parkinson's disease by altering hepatic detoxification and increasing systemic bioavailability of neurotoxins.     

Effect of Cryopreservation on Enzyme and Transporter Activities in Suspended and Sandwich Cultured Rat Hepatocytes

Freshly-isolated rat hepatocytes are commonly used as tools for hepatic drug disposition. From an ethical point of view, it is important to maximize the use of isolated hepatocytes by cryopreservation. The present study compared overall hepatocyte functionality as well as activity of the organic anion transporting polypeptide (Oatp), multidrug resistance-associated protein 2 (Mrp2), and UDP-glucuronosyltransferase 1 (Ugt1), in in vitro models established with cryopreserved and freshly-isolated hepatocytes. A similar culture time-dependent decline in cellular functionality, as assessed by urea production, was observed in sandwich-cultured hepatocytes (SCH) obtained from freshly-isolated and cryopreserved cells. Concentration-dependent uptake kinetics of the Oatp substrate sodium fluorescein in suspended hepatocytes (SH) or SCH were not significantly affected by cryopreservation. Mrp2-mediated biliary excretion of 5 (and 6)-carboxy-2′,7′-dichlorofluorescein by SCH was assessed with semi-quantitative fluorescence imaging: biliary excretion index values increased between day 3 and day 4, but did not differ significantly between cryopreserved and freshly-isolated hepatocytes. Finally, telmisartan disposition was evaluated in SCH to simultaneously explore Oatp, Ugt1, and Mrp2 activity. In order to distinguish between the susceptibilities of the individual disposition pathways to cryopreservation, a mechanistic cellular disposition model was developed. Basolateral and canalicular efflux as well as glucuronidation of telmisartan were affected by cryopreservation. In contrast, the disposition parameters of telmisartan-glucuronide were not impacted by cryopreservation. Overall, the relative contribution of the rate-determining processes (uptake, metabolism, efflux) remained unaltered between cryopreserved and freshly-isolated hepatocytes, indicating that cryopreserved hepatocytes are a suitable alternative for freshly-isolated hepatocytes when studying these cellular disposition pathways.     

The influence of the choice of digestion enzyme used to prepare rat hepatocytes on xenobiotic uptake and efflux

Isolated rat hepatocytes are widely used to assess the metabolism and toxicity of xenobiotics. The choice of digestion enzyme used to prepare the cells has been shown previously to influence their metabolic capability. This study investigates the effect of the digestion enzyme (collagenase II, collagenase A/trypsin inhibitor, or collagenase plus dispase) on the uptake of xenobiotics into, and efflux from, hepatocytes. The choice of digestion enzymes used in this study does not affect uptake of either pravastatin (an organic anion probe substrate for Oatp transporter) or metformin (an organic cation probe substrate for Oct transporter). With regard to efflux transporters, hepatocyte differentiation was better maintained when cells were isolated using collagenase II alone.     

Induction of cytochrome P450 3A by paclitaxel in mice: pivotal role of the nuclear xenobiotic receptor, pregnane X receptor.

Paclitaxel, a taxane anti-microtubule agent, is known to induce CYP3A in rat and human hepatocytes. Recent studies suggest that a member of the nuclear receptor family, pregnane X Receptor (PXR), is a key regulator of the expression of CYP3A in different species. We investigated the role of PXR activation, in vitro and in vivo, in mediating Cyp3a induction by paclitaxel. Pregnenolone 16 alpha-carbonitrile (PCN), an antiglucocorticoid, was employed as a positive control for mouse PXR (mPXR) activation in vitro, and Cyp3a induction in vivo. In cell based reporter gene assays paclitaxel and PCN activated mPXR with an EC(50) of 5.6 and 0.27 microM, respectively. Employing PXR wild-type and transgenic mice lacking functional PXR (-/-), we evaluated the expression and activity of CYP3A following treatment with paclitaxel and PCN. Paclitaxel significantly induced CYP3A11 mRNA and immunoreactive CYP3A protein in PXR wild-type mice. Consistent with kinetics of CYP3A induction, the V(max) of testosterone 6 beta-hydroxylation in microsomal fraction increased 15- and 30-fold in paclitaxel- and PCN-treated mice, respectively. The Cyp3a induction response was completely abolished in paclitaxel- and PCN-treated PXR-null mice. This suggests that paclitaxel-mediated CYP3A induction in vivo requires an intact PXR-signaling mechanism. Our study validates the use of PXR activation assays in screening newer taxanes for potential drug interactions that may be related to PXR-target gene induction.     

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.     

Synthesis,radiosynthesis and in vivo evaluation of [123I]‐4‐(2‐(bis(4‐fluorophenyl)methoxy)ethyl)‐1‐(4‐iodobenzyl)piperidine as a selective tracer for imaging the dopamine transporter

Dopamine transporter (DAT) neuroimaging is a useful tool in Parkinson's disease diagnosis, staging and follow‐up providing information on the integrity of the dopaminergic neurotransmitter system in vivo. 4‐(2‐(Bis(4‐fluorophenyl)methoxy)ethyl)‐1‐(4‐iodobenzyl)piperidine (7) has nanomolar affinity for DAT and better selectivity over the other monoamine transporters compared with the existing SPECT radioligands for DAT. The aim of this study was to synthesize and evaluate [¹²³I]‐7 as an in vivo tracer for DAT. The tributylstannyl precursor was synthesized with an overall yield of 25%. [¹²³I]‐7 was synthesized by electrophilic destannylation with a yield of 40±10%. Radiochemical purity appeared to be >98%, whereas specific activity was at least 667 GBq/µmol. Biodistribution studies in mice showed brain uptake of 0.96±0.53%ID/g at 30 s post injection (p.i.) and 0.26±0.02%ID/g at 3 h p.i. High blood activity was observed at all time points. Pretreatment with Cyclosporin A raised brain uptake indicating that [¹²³I]‐7 is transported by P‐glycoprotein (P‐gp) pumps. In rats, regional brain distribution of [¹²³I]‐7 was not in agreement with DAT distribution. These results indicate that [¹²³I]‐7 is not suitable for mapping DAT in vivo but could be a useful tracer for the P‐gp transporter. Copyright © 2009 John Wiley & Sons, Ltd.     

Species differences in metabolism of a new antiepileptic drug candidate,DSP‐0565 [2‐(2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetamide]

The metabolism and pharmacokinetics of DSP‐0565 [2‐(2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetamide], an antiepileptic drug candidate, was investigated in rats, dogs, and humans. In human hepatocytes, [¹⁴C]DSP‐0565 was primarily metabolized via amide bond hydrolysis to (2′‐fluoro[1,1′‐biphenyl]‐2‐yl)acetic acid (M8), while in rat and dog hepatocytes, it was primarily metabolized via both hydrolysis to M8 and hydroxylation at the benzene ring or the benzyl site to oxidized metabolites. After single oral administration of [¹⁴C]DSP‐0565 to rats and dogs, the major radioactivity fraction was recovered in the urine (71–72% of dose) with a much smaller fraction recovered in feces (23–25% of dose). As primary metabolites in their excreta, M8, oxidized metabolites, and glucuronide of DSP‐0565 were detected. The contribution of metabolic pathways was estimated from metabolite profiles in their excreta: the major metabolic pathway was oxidation (57–62%) and the next highest was the hydrolysis pathway (23–33%). These results suggest that there are marked species differences in the metabolic pathways of DSP‐0565 between humans and animals. Finally, DSP‐0565 human oral clearance (CL/F) was predicted using in vitro–in vivo extrapolation (IVIVE) with/without animal scaling factors (SF, in vivo intrinsic clearance/in vitro intrinsic clearance). The SF improved the underestimation of IVIVE (fold error = 0.22), but the prediction was overestimated (fold error = 2.4–3.3). In contrast, the use of SF for hydrolysis pathway was the most accurate for the prediction (fold error = 1.0–1.4). Our findings suggest that understanding of species differences in metabolic pathways between humans and animals is important for predicting human metabolic clearance when using animal SF.