Comparative structural analysis of the human and Schistosoma glutathione transferase dimer interface using selective binding of bromosulfophthalein.

The binding channel of Schistosoma glutathione transferase (SGST) has been identified to possess a non-substrate site implicated in enzyme inhibition. This binding channel is formed by the interface of the GST dimer. We produced a comparative characterization of the SGST dimer interface with respect to that of human GST (hGST) analogues using the selective binding of bromosulfophthalein (BSP). First, two SGST and three hGST structures were used as search queries to assemble a data set of 48 empirical GST structures. Sequence alignment to generate a universal residue indexing scheme was then performed, followed by local superposition of the dimer interface. Principal component analysis revealed appreciable variation of the dimer interface, suggesting the potential for selective inhibition of SGST. BSP was found to dock invariably in the dimer interface core pocket, placing it in proximity to the GST catalytic domains, through which it may exert its inhibitory behavior. Binding poses across the GST forms were distinguished with ligand interaction profiling, where SGST complexes showed stabilization of ligand aromatic- and sulfonate moieties, which altogether anchor the ligand and produce a tight association. In comparison, missing aromatic stabilization in the hGST complexes impart large bonding distances, causing mobile poses likely to dissociate. Altogether, this study illustrates the potential for selective inhibition of SGST, rationalizes the selective behavior of the BSP inhibitor, and produces a reliable metric for construction and validation of pharmacophore models of the SGST binding channel.

Liver function from Y to Z: The guidance of William Jakoby.

In the 1960s, my lab was interested in understanding how bilirubin and other organic anions are transferred from the plasma through the liver cell and into the bile. We performed gel filtration of liver supernatants and identified two protein fractions, designated Y and Z, which bound organic anions including bilirubin, and thus we proposed that they were involved in hepatic uptake of organic anions from plasma. Subsequently, the Y and Z proteins responsible for this binding activity were purified, cloned, and sequenced. With Bill Jakoby, we identified Y protein as a member of the glutathione S-transferase (GST) protein family. In separate studies, Z was found to be a member of the fatty acid-binding protein (FABP) family. These proteins have since been shown to have additional surprising roles, but understanding of their full role in physiology and disease has not yet been achieved. In the 1960s, bilirubin metabolism was a "hot" topic. Along with other groups, my lab was studying various forms of inheritable jaundice in an effort to dissect the mechanism of bilirubin's transfer from plasma into the hepatocyte and its role in intracellular metabolism and biliary secretion. These processes were eventually identified and found to be related to the basic mechanisms whereby the liver handles many anionic drugs, metabolites, and hormones. Because the mechanism of hepatic uptake of bilirubin was unknown, A.J. Levi, Z. Gatmaitan, and I took advantage of advances in gel permeation chromatography to study this process. In 1969, we described two hepatic cytoplasmic protein fractions, designated Y and Z, that bound bilirubin and various organic anionic dyes in vivo and in vitro and, based on tissue distribution, abundance, and effects of genetic and pharmacologic models, were proposed to participate in organic anion uptake (Levi et al., 1969) [1]. In the decades since then, the Y and Z proteins have been identified as members of large protein families that were cloned and sequenced. Several surprising functions emerged, whereas others are proposed based on binding properties. Many challenges remain in understanding the full role of these proteins in physiology and disease.

Bromosulfophthalein suppresses inflammatory effects in lipopolysaccharide-stimulated RAW264.7 macrophages.

OBJECTIVE: It has been reported that glutathione (GSH), the most abundant cellular antioxidant, can inhibit production of pro-inflammatory cytokines by activated macrophages. Bromosulfophthalein (BSP) has been recognized as an inhibitor of the efflux of reduced GSH from cells, leading to an increase in the intracellular GSH level. In this study, we evaluated, for the first time, whether BSP possessed anti-inflammatory effects in lipopolysaccharide (LPS)-stimulated macrophages. MATERIALS AND METHODS: RAW 264.7 cells were treated with BSP and the levels of proinflammatory cytokines, GSH, and nitrite were assessed. Gene expression of inducible nitric oxide synthase (iNOS), tumor necrosis factor alpha (TNF α), interleukin-1beta (IL-1ß), and interleukin-6 (IL-6) was analyzed via quantitative RT-PCR. We also examined various inflammatory signaling pathways including Akt/forkhead box protein O1 (FoxO1)/toll-like receptor 4 (TLR4), mitogen-activated protein kinases (MAPKs), and Fas protein by Western blot and flow cytometry analysis. RESULTS: Our study demonstrated that BSP induced an increase in intracellular GSH level in LPS-stimulated macrophages. BSP inhibited production of nitric oxide and proinflammatory cytokines. BSP increased phosphorylation of Akt and nuclear exclusion of FoxO1 and suppressed TLR4 expression. Additionally, BSP decreased MAPKs activation and Fas expression. DISCUSSION AND CONCLUSION: Taken together, these data suggest that BSP can attenuate inflammation through multiple signaling pathways. These findings highlight the potential of BSP as a new anti-inflammatory agent.

OATP1B3-1B7 (LST-3TM12) Is a Drug Transporter That Affects Endoplasmic Reticulum Access and the Metabolism of Ezetimibe.

Drug transporters play a crucial role in pharmacokinetics. One subfamily of transporters with proven clinical relevance are the OATP1B transporters. Recently we identified a new member of the OATP1B family named OATP1B3-1B7 (LST-3TM12). This functional transporter is encoded by SLCO1B3 and SLCO1B7 OATP1B3-1B7 is expressed in hepatocytes and is located in the membrane of the smooth endoplasmic reticulum (SER). One aim of this study was to test whether OATP1B3-1B7 interacts with commercial drugs. First, we screened a selection of OATP1B substrates for inhibition of OATP1B3-1B7-mediated transport of dehydroepiandrosterone sulfate and identified several inhibitors. One such inhibitor was ezetimibe, which not only inhibited OATP1B3-1B7 but is also a substrate, as its cellular content was significantly increased in cells heterologously expressing the transporter. In humans, ezetimibe is extensively metabolized by hepatic and intestinal uridine-5'-diphospho-glucuronosyltransferases (UGTs), the catalytic site of which is located within the SER lumen. After verification of OATP1B3-1B7 expression in the small intestine, we determined in microsomes whether SER access can be modulated by inhibitors of OATP1B3-1B7. We were able to show that these compounds significantly reduced accumulation in small intestinal and hepatic microsomes, which influenced the rate of ezetimibe ß-D-glucuronide formation as determined in microsomes treated with bromsulphthalein. Notably, this molecule not only inhibits the herein reported transporter but also other transport systems. In conclusion, we report that multiple drugs interact with OATP1B3-1B7; for ezetimibe, we were able to show that SER access and metabolism is significantly reduced by bromsulphthalein, which is an inhibitor of OATP1B3-1B7. SIGNIFICANCE STATEMENT: OATP1B3-1B3 (LST-3TM12) is a transporter that has yet to be fully characterized. We provide valuable insight into the interaction potential of this transporter with several marketed drugs. Ezetimibe, which interacted with OATP1B3-1B7, is highly metabolized by uridine-5'-diphospho-glucuronosyltransferases (UGTs), whose catalytic site is located within the smooth endoplasmic reticulum (SER) lumen. Through microsomal assays with ezetimibe and the transport inhibitor bromsulphthalein we investigated the interdependence of SER access and the glucuronidation rate of ezetimibe. These findings led us to the hypothesis that access or exit of drugs to the SER is orchestrated by SER transporters such as OATP1B3-1B7.

Monitorable Mitochondria-Targeting DNAtrain for Image-Guided Synergistic Cancer Therapy.

It is highly desirable to realize real-time monitoring of the drug delivery/release process in cancer treatment. Herein, a monitorable mitochondria-specific DNAtrain (MitoDNAtrs) was developed for image-guided drug delivery and synergistic cancer therapy. In this system, mitochondria-targeting Cy5.5 dye served as the "locomotive" to guide the DNA "vehicle" selectively accumulating in the cancer cells in a detectable manner. More importantly, Cy5.5 showed reactive oxygen species (ROS) generation ability, which made it a promising adjuvant chemotherapy amplifier for cancer theranostics.

Analysis of amino acid residues in the predicted transmembrane pore influencing transport kinetics of the hepatic drug transporter organic anion transporting polypeptide 1B1 (OATP1B1).

The hepatic uptake transporters OATP1B1 (SLCO1B1) and OATP1B3 (SLCO1B3) mediate the uptake of endogenous metabolites and drugs from blood into hepatocytes. Alterations of transport function are accompanied with variations in drug plasma concentrations and the risk of adverse drug effects. Thus, knowledge on amino acids determining substrate recognition or transport kinetics are important to predict alterations in transport kinetics. Therefore, we analyzed the charged amino acids His54 and Tyr169, both located at the extracellular entry of the predicted transmembrane pore of OATP1B1. Based on a computational analysis we established HEK293 cell lines overexpressing the mutant OATP1B1 proteins HEK-OATP1B1p.H54Q, -p.H54A, -p.Y169H and -p.Y169A and analyzed protein expression, localization and transport kinetics of the four OATP1B1 substrates bromosulfophthalein, estradiaol-17ß-glucuronide, taurocholate and pravastatin. Consequences on transport were detected for all mutants and these were different for each amino acid exchange and for each substrate tested. For example, the exchange H54Q resulted in reduced transport for BSP (78% of wildtype OATP1B1 transport at 0.05µM, P<0.01) with reduced affinity to this substrate (Km value increases from 0.76µM to 8.04µM) but in stimulated E217ßG transport (138% compared to wildtype transport at 10µM, P<0.001). Investigating amino acid exchanges located at the extracellular entry of the transport pore of the OATP1B1 protein we demonstrated that these residues are involved in modulating transport kinetics and this participation strongly depends on the substrate and not on the physicochemical character of the investigated amino acid.

Characterization of Organic Anion Transporter 2 (SLC22A7): A Highly Efficient Transporter for Creatinine and Species-Dependent Renal Tubular Expression.

The contribution of organic anion transporter OAT2 (SLC22A7) to the renal tubular secretion of creatinine and its exact localization in the kidney are reportedly controversial. In the present investigation, the transport of creatinine was assessed in human embryonic kidney (HEK) cells that stably expressed human OAT2 (OAT2-HEK) and isolated human renal proximal tubule cells (HRPTCs). The tubular localization of OAT2 in human, monkey, and rat kidney was characterized. The overexpression of OAT2 significantly enhanced the uptake of creatinine in OAT2-HEK cells. Under physiologic conditions (creatinine concentrations of 41.2 and 123.5 µM), the initial rate of OAT2-mediated creatinine transport was approximately 11-, 80-, and 80-fold higher than OCT2, multidrug and toxin extrusion protein (MATE)1, and MATE2K, respectively, resulting in approximately 37-, 1850-, and 80-fold increase of the intrinsic transport clearance when normalized to the transporter protein concentrations. Creatinine intracellular uptake and transcellular transport in HRPTCs were decreased in the presence of 50 µM bromosulfophthalein and 100 µM indomethacin, which inhibited OAT2 more potently than other known creatinine transporters, OCT2 and multidrug and toxin extrusion proteins MATE1 and MATE2K (IC50: 1.3 µM vs. > 100 µM and 2.1 µM vs. > 200 µM for bromosulfophthalein and indomethacin, respectively) Immunohistochemistry analysis showed that OAT2 protein was localized to both basolateral and apical membranes of human and cynomolgus monkey renal proximal tubules, but appeared only on the apical membrane of rat proximal tubules. Collectively, the findings revealed the important role of OAT2 in renal secretion and possible reabsorption of creatinine and suggested a molecular basis for potential species difference in the transporter handling of creatinine.

The potential drug-drug interactions of ginkgolide B mediated by renal transporters.

Ginkgolide B (GB) is a selective and strong antagonist of platelet-activating factor with great benefits in CNS diseases treatment. The renal excretion constitutes the predominant secretory pathway of GB. Here, we investigated the potential role of renal drug transporters in GB urinary excretion. The intravenous administration of GB was conducted at 10 min post-administration of probenecid (potential inhibitor of organic anion transporters/organic anion transporting polypeptides) or bromosulfophthalein (traditional inhibitor of multi-drug resistance proteins) in rats. Pretreated with probenecid, the systemic exposure of GB was significantly elevated from 8.319 ± 1.646 to 14.75 ± 1.328 µg · mL(-1) ∙h but with reduced total clearance from 1.17 ± 0.331 to 0.596 ± 0.0573 L · h(-1) ∙kg(-1) accompanying no changes in plasma elimination half-lives compared with control group. With no pronounced effect on metabolic elimination, the decreased total clearance was closely pertained to the reduced renal excretion, indicating the potential effect of organic anion transporters and/or organic anion transporting polypeptides in renal secretory of GB from blood to urine. However, the possible effect of bromosulfophthalein was restricted within a minor extent, suggesting the mild role of multi-drug resistance protein in GB renal excretion.

Integrated approach of in vivo and in vitro evaluation of the involvement of hepatic uptake organic anion transporters in the drug disposition in rats using rifampicin as an inhibitor.

Cumulative studies describe the importance of drug transporters as one of the key determinants of pharmacokinetics that necessitate investigation and assessment of the involvement of drug transporters in drug discovery and development. The present study investigated an integrated in vivo and in vitro approach to determine the involvement of organic anion transporting polypeptides (Oatps) in the disposition of drugs in rats using rifampicin as an inhibitor. When bromosulfophthalein (BSP) and HMG-CoA reductase inhibitors (statins), which were used as model substrates for Oatps, were administered intravenously (3 and 1 mg/kg, respectively) to rats pretreated with rifampicin orally (30 mg/kg), the total plasma clearance of BSP and statins was attenuated compared with that in control rats, suggesting the involvement of Oatps in the disposition of these drugs in vivo. On the other hand, the pharmacokinetics of midazolam, used as a model substrate of cytochrome P450 3a (Cyp3a), was unchanged between control rats and rifampicin-pretreated rats. The involvement of Oatps in the disposition of statins observed in vivo was further clarified by employing an in vitro hepatic uptake study and media-loss assay in the presence or absence of 100 µM rifampicin. Hepatic intrinsic clearance was reduced in the presence of rifampicin in both the media-loss assay and hepatocyte uptake study. The present study suggests in vivo investigations in rats using rifampicin together with in vitro investigations with a media-loss assay and/or uptake assay using rat hepatocytes can help determine whether a clinical drug-drug interaction study is necessary in drug development.

Identification of multiple binding sites for substrate transport in bovine organic anion transporting polypeptide 1a2.

Organic anion transporting polypeptides (OATP) have been extensively recognized as key determinants of absorption, distribution, metabolism, and excretion of various drugs because of their broad substrate specificity, wide tissue distribution, and the involvement of drug-drug interaction. As the first cloned human OATP, OATP1A2 has been found to transport a wide spectrum of endogenous and exogenous compounds. Bovine Oapt1a2 shared high homology with the human transporter and is considered as its functional ortholog. In the present study, we expressed bovine Oatp1a2 in human embryonic kidney 293 cells and found that, unlike human OATP1A2, the transport of estrone-3-sulfate (E-3-S) exhibited biphasic saturation kinetics. The K(m) values are 0.25 ± 0.08 and 46.6 ± 18.5 µM, and V(max) values were 24.5 ±4.4 and 375 ± 142 pmol/mg protein/min for high- and low-affinity sites, respectively, suggesting the presence of multiple binding sites. Further study on other Oatp1a2 substrates showed that the high affinity component for E-3-S is responsible for the interaction with taurocholate, bromsulphthalein, and rifampicin and is sensitive to proton concentration change, whereas the low affinity binding site is only involved in the binding of the antitumor drug methotrexate and had no response to change of pH.

Substrate-dependent inhibition of organic anion transporting polypeptide 1B1: comparative analysis with prototypical probe substrates estradiol-17ß-glucuronide, estrone-3-sulfate, and sulfobromophthalein.

Organic anion transporting polypeptide (OATP) 1B1 plays an important role in the hepatic uptake of many drugs, and the evaluation of OATP1B1-mediated drug-drug interactions (DDIs) is emphasized in the latest DDI (draft) guidance documents from U.S. and E.U. regulatory agencies. It has been suggested that some OATP1B1 inhibitors show a discrepancy in their inhibitory potential, depending on the substrates used in the cell-based assay. In this study, inhibitory effects of 14 compounds on the OATP1B1-mediated uptake of the prototypical substrates [³H]estradiol-17ß-glucuronide (E2G), [³H]estrone-3-sulfate (E1S), and [³H]sulfobromophthalein (BSP) were studied in OATP1B1-transfected cells. Inhibitory potencies of tested compounds varied depending on the substrates. Ritonavir, gemfibrozil, and erythromycin caused remarkable substrate-dependent inhibition with up to 117-, 14-, and 13-fold difference in their IC50 values, respectively. Also, the clinically relevant OATP inhibitors rifampin and cyclosporin A exhibited up to 12- and 6-fold variation in their IC50 values, respectively. Regardless of the inhibitors tested, the most potent OATP1B1 inhibition was observed when [³H]E2G was used as a substrate. Mutual inhibition studies of OATP1B1 indicated that E2G and E1S competitively inhibited each other, whereas BSP noncompetitively inhibited E2G uptake. In addition, BSP inhibited E1S in a competitive manner, but E1S caused an atypical kinetics on BSP uptake. This study showed substrate-dependent inhibition of OATP1B1 and demonstrated that E2G was the most sensitive in vitro OATP1B1 probe substrate among three substrates tested. This will give us an insight into the assessment of clinically relevant OATP1B1-mediated DDI in vitro with minimum potential of false-negative prediction.

Intestinal ischemia-reperfusion suppresses biliary excretion of hepatic organic anion transporting polypeptides substrate.

PURPOSE: Intestinal ischemia-reperfusion (I/R) causes gut dysfunction and promotes multi-organ failure. The liver and kidney can be affected by multi-organ failure after intestinal I/R. Organic anion transporting polypeptides (OATPs) and organic anion transporters (OATs) are recognized in a broad spectrum from endogenous compounds to xenobiotics, including clinically important drugs. Therefore, it is important for understanding the pharmacokinetics to obtain evidence of alterations in OATPs and OATs expression and transport activities. In the present study, we investigated the expression of rat Oatps and Oats after intestinal I/R. METHODS: We used intestinal ischemia-reperfusion (I/R) model rats. Real-time PCR and Western blotting were used to assess mRNA and protein expression levels. Plasma concentration and biliary excretion of sulfobromophthalein (BSP), which is used as a model compound of organic anion drugs, were measured after intravenous administration in intestinal I/R rats. RESULTS: Although Oat1 and Oat3 mRNA levels were not altered in the kidney, Oatp1a1, Oatp1b2 and Oatp2b1 mRNA levels in the liver were significantly decreased at 1-6 h after intestinal I/R. Moreover, Oatp1a1 and Oatp2b1 protein expression levels were decreased at 1 h after intestinal I/R. Plasma concentration of BSP, which is a typical substrate of Oatps, in intestinal I/R rats reperfused 1 h was increased than that in sham-operated rats. Moreover, the area under the concentration-time curve (AUC0₋90) in intestinal I/R rats reperfused 1 h was significantly increased than that in sham-operated rats. The total clearance (CL(tot)) and the biliary clearance (CL(bile)) in intestinal I/R rats reperfused 1 h were significantly decreased than those in sham-operated rats. CONCLUSIONS: Oatp1a1 and Oatp2b1 expression levels are decreased by intestinal I/R. The decreases in these transporters cause alteration of pharmacokinetics of organic anion compound. The newly found influence of intestinal I/R on the expression and function of Oatps may be a key to perform appropriate drug therapy.

Disposition of ceftriaxone in hepatopathic goats following single-intramuscular dosing.

Hepatopathy sometimes may interfere with metabolism and/or elimination of drugs which undergo major hepatic clearance. Twelve healthy goats were equally divided into two groups (I and II) and hepatopathy was induced by carbontetrachloride in the second group (group II). A single dose of ceftriaxone at 50 mg/kg was administered to each group intramuscularly. Disposition of ceftriaxone in plasma of healthy goats showed a typical absorption-reabsorption phase. However, the reabsorption phase was totally absent in hepatopathic goats and the disposition of ceftriaxone showed only absorption and distribution/elimination phase. The drug persisted in plasma for 6 h in hepatopathic animals, whereas the drug can only be detected up to 2 h in healthy animals indicating longer persistence of ceftriaxone in the former group. Ceftizoxime, the active metabolite of ceftriaxone was available in urine of group I animals, whereas only ceftriaxone was detected in the urine of hepatopathic animals suggesting impairment of metabolism of the parent drug in hepatopathy. Therefore, the reabsorption and metabolism of ceftriaxone in goats should be taken into consideration for drug monitoring.

Molecular dynamics-derived pharmacophores of Schistosoma glutathione transferase in complex with bromosulfophthalein: Screening and analysis of potential inhibitors.

Schistosoma glutathione transferases (GSTs) have been identified as attractive drug targets for the design of novel antischistosomals. Here, we used in silico methods to validate the discriminative inhibitory properties of bromosulfophthalein (BSP) against the 26-kDa GST from S. japonicum (Sj26GST), and the 28-kDa GST from S. haematobium (Sh28GST), versus human GST (hGST) isoforms alpha (hGSTA), mu (hGSTM) and pi (hGSTP). The use of BSP as an archetypal selective inhibitor was harnessed to produce molecular dynamics-derived pharmacophores of the two targets. Pharmacophore-based screening using a large dataset of experimental and approved drug compounds was performed to produce a shortlist of candidates. The top candidate for each target was prioritised via molecular docking, yielding guanosine-3'-monophosphate-5'-diphosphate (G3D) for Sj26GST, and quercetin-3'-O-phosphate (Q3P) for Sh28GST. Comparative molecular dynamics studies of both candidates compared to BSP showed similar characteristics of binding stability and strength, suggesting their potential to emulate the inhibitory effects of BSP.

Substrate- and pH-specific antifolate transport mediated by organic anion-transporting polypeptide 2B1 (OATP2B1-SLCO2B1).

Human organic anion-transporting polypeptide (OATP) 2B1 (OATP-B; SLCO2B1) is expressed in the apical membrane of the small intestine and the hepatocyte basolateral membrane and transports structurally diverse organic anions with a wide spectrum of pH sensitivities. This article describes highly pH-dependent OATP2B1-mediated antifolate transport and compares this property with that of sulfobromophthalein (BSP), a preferred OATP2B1 substrate. At pH 5.5 and low substrate concentrations (~2.5 µM), only [(3)H]pemetrexed influx [in contrast to methotrexate (MTX), folic acid, and reduced folates] could be detected in OATP2B1-transfected HeLa R1-11 cells that lack endogenous folate-specific transporters. Influx was optimal at pH 4.5 to 5.5, falling precipitously with an increase in pH >6.0; BSP influx was independent of pH. Influx of both substrates at low pH was markedly inhibited by the proton ionophore 4-(trifluoromethoxy)phenylhydrazone; BSP influx was also suppressed at pH 7.4. At 300 µM MTX, influx was one-third that of pemetrexed; influx of folic acid, (6S)5-methyltetrahydrofolate, or (6S)5-formyltetrahydrofolate was not detected. There were similar findings in OATP2B1-expressing Xenopus laevis oocytes. The pemetrexed influx K(m) was ~300 µM; the raltitrexed influx K(i) was ~70 µM at pH 5.5. Stable expression of OAPT2B1 in HeLa R1-11 cells resulted in substantial raltitrexed, but modest pemetrexed, growth inhibition consistent with their affinities for this carrier. Hence, OATP2B1 represents a low-affinity transport route for antifolates (relative affinities: raltitrexed > pemetrexed > MTX) at low pH. In contrast, the high affinity of this transporter for BSP relative to antifolates seems to be intrinsic to its binding site and independent of the proton concentration.

Pharmaceutical excipients influence the function of human uptake transporting proteins.

Although pharmaceutical excipients are supposed to be pharmacologically inactive, solubilizing agents like Cremophor EL have been shown to interact with cytochrome P450 (CYP)-dependent drug metabolism as well as efflux transporters such as P-glycoprotein (ABCB1) and multidrug resistance associated protein 2 (ABCC2). However, knowledge about their influence on the function of uptake transporters important in drug disposition is very limited. In this study we investigated the in vitro influence of polyethylene glycol 400 (PEG), hydroxypropyl-ß-cyclodextrin (HPCD), Solutol HS 15 (SOL), and Cremophor EL (CrEL) on the organic anion transporting polypeptides (OATP) 1A2, OATP2B1, OATP1B1, and OATP1B3 and the Na(+)/taurocholate cotransporting polypeptide (NTCP). In stably transfected human embryonic kidney cells we analyzed the competition of the excipients with the uptake of bromosulfophthalein in OATP1B1, OATP1B3, OATP2B1, and NTCP, estrone-3-sulfate (E(3)S) in OATP1A2, OATP1B1, and OATP2B1, estradiol-17ß-glucuronide in OATP1B3, and taurocholate (TA) in OATP1A2 and NTCP cells. SOL and CrEL were the most potent inhibitors of all transporters with the strongest effect on OATP1A2, OATP1B3, and OATP2B1 (IC(50) < 0.01%). HPCD also strongly inhibited all transport proteins but only for substrates containing a sterane-backbone. Finally, PEG seems to be a selective and potent modulator of OATP1A2 with IC(50) values of 0.05% (TA) and 0.14% (E(3)S). In conclusion, frequently used solubilizing agents were shown to interact substantially with intestinal and hepatic uptake transporters which should be considered in drug development. However, the clinical relevance of these findings needs to be evaluated in further in vivo studies.

Involvement of specific transport system on uptake of lactone form of SN-38 in human intestinal epithelial cell line Caco-2.

The aim of this study was to elucidate the characteristics of the transport of lactone and carboxylate forms of SN-38 (SN-38L and SN-38C, respectively), a metabolite of irinotecan hydrochloride (CPT-11), with the human intestinal epithelial cell line, Caco-2. We examined SN-38L and SN-38C uptake from the apical side into Caco-2, and the effects of various compounds on the uptake of SN-38L. SN-38L and SN-38C in the cells were determined by HPLC with a fluorescence detector. When either SN-38L (0.5 µM) or SN-38C (0.5 µM) was added extracellularly at 37°C, the accumulation of SN-38L into the cells was about 10-fold higher than that of SN-38C, suggesting a dominant role of the lactone form in the uptake of SN-38 into Caco-2. The accumulation of SN-38L in Caco-2 increased time-dependently up to 10 min at 37°C, whereas the accumulation markedly decreased at 4°C. The initial uptake rate of SN-38L approached saturation at high concentrations with Michaelis-Menten constant and 'Hill coefficient,' 2.84±1.00 µM and 2.13±1.14, respectively (mean±S.E.). The accumulation of SN-38L was markedly inhibited by baicalin, an active ingredient of a Chinese herbal medicine, Hange-Shashin-To, as well as CPT-11. The type of inhibition by baicalin was competitive. In contrast, concomitant sulfobromophthalein, taurocholate and estrone 3-sulfate significantly increased SN-38L uptake. These results suggest that apical uptake of SN-38 by Caco-2 is dominantly performed as a lactone form through a specific transporter, which is competitively inhibited by baicalin.

Role of melatonin in the oxidative damage prevention at different times of hepatic regeneration.

The process of regenerating liver is the result of a balance between stimulating factors and inhibitors of hepatocyte proliferation. Melatonin and its metabolites have been found to protect tissues against oxidative damage generated by a variety of toxic agents and metabolic processes. Furthermore, studies in liver of rats showed a decrease in the liver mitochondrial hydroxylation of drugs returning to the normal state after the administration of antioxidants. This study was designed to determine, in experimental animals, whether the administration of an antioxidant agent such as melatonin could prevent cells events leading to tissue injury and hepatic dysfunction after partial hepatectomy (PH). Biliary flow (BF), oxidative stress in hepatic tissue and Na⁺/K⁺ ATPase activities in whole plasma membrane were determined. PH decreased the Na⁺/K⁺ ATPase activity. PH significantly reduced the BF (36%) and promoted oxidative stress with an increase of lipoperoxidation and decrease of glutathione peroxidase and catalase activities. Treatment with melatonin prevented the decrease of BF in rats with hepatectomy and normalized the Na⁺/K⁺ ATPase activity. Moreover, melatonin markedly attenuated oxidative stress produced by PH. This may be the results of the higher efficacy of melatonin in scavenging various free radicals and also because of its ability in stimulating the antioxidant enzymes. We suggest that oxidative stress before and during liver regeneration has a crucial role in cholestasis, apoptotic/necrotic hepatocellular damage and the impairment in liver transport function induced by PH and that melatonin could modulate the degree of oxidative stress and through it prevent the alterations in liver function carrier.

Induction of multidrug resistance-associated protein 2 in liver, intestine and kidney of streptozotocin-induced diabetic rats.

Many studies have demonstrated that Mrp2 is highly regulated in some physiopathological situations. The aim of this study was to investigate effects of diabetes mellitus on function and expression of multidrug resistance-associated protein 2 (Mrp2) in rat liver, kidney and intestine. Diabetic rats were induced by an intraperitoneal administration of streptozotocin (65 mg/kg) and randomly divided into diabetic (DM) rats and insulin-treated diabetic rats. Sulfobromophthalein (BSP), a substrate of Mrp2, was used to evaluate Mrp2 function in vivo. Data from excretion experiments demonstrated that compared with normal rats, diabetes markedly enhanced BSP excretion via bile, urine and intestinal perfusate, which contributed to the elevated plasma clearance of BSP after intravenous administration of 45 µmol/kg BSP. Western blot results showed higher levels of hepatic, renal and intestinal Mrp2 protein in DM rats, although no difference was observed in renal Mrp2. Insulin treatment partly reversed these alterations. Induction of Mrp2 by diabetes was in parallel with the increase in bile flow, levels of biliary and plasma total bile acid (TBA), and plasma conjugated bilirubin in DM rats. Diabetes may enhance Mrp2 function and expression in liver, kidney and intestine, which might be due to insulin deficiency, increased TBA and conjugated bilirubin.

Biophysical description of Bromosulfophthalein interaction with the 28-kDa glutathione transferase from Schistosoma japonicum.

Glutathione transferases (GSTs) are major detoxification enzymes vital for the survival and reproduction of schistosomes during infection in humans. Schistosoma encode two GST isoenzymes, the 26- and 28-kDa isoforms, that show different substrate specificities and cellular localisations. Bromosulfophthalein (BSP) has been identified and characterised as a potent 26-kDa Schistosoma japonicum GST (Sj26GST) inhibitor with an anthelmintic potential. This study describes the structure, function, and ligandin properties of the 28-kDa Schistosoma japonicum GST (Sj28GST) towards BSP. Enzyme kinetics show that BSP is a potent enzyme inhibitor, with a specific activity decreases from 60.4 µmol/min/mg to 0.0742 µmol/min/mg and an IC50 in the micromolar range of 0.74 µM. Far-UV circular dichroism confirmed that purified Sj28GST follows a typical GST fold, which is predominantly alpha-helical. Fluorescence spectroscopy suggests that BSP binding occurs at a site distinct from the glutathione-binding site (G-site); however, the binding does not alter the local G-site environment. Isothermal titration calorimetry studies show that the binding of BSP to Sj28GST is exergonic (∆G°= -33 kJ/mol) and enthalpically-driven, with a stoichiometry of one BSP per dimer. The stability of Sj28GST (∆G(H2O) = 4.7 kcal/mol) is notably lower than Sj26GST, owing to differences in the enzyme's dimeric interfaces. We conclude that Sj28GST shares similar biophysical characteristics with Sj26GST based on its kinetic properties and susceptibility to low concentrations of BSP. The study supports the potential benefits of re-purposing BSP as a potential drug or prodrug to mitigate the scourge of schistosomiasis.