Transporter-mediated Uptake Research Articles

Detection of Statin Cytotoxicity Is Increased in Cells Expressing the OATP1B1 Transporter

Cytotoxicity of a compound is determined by the intracellular concentration mediated both by passive permeability and active uptake through drug transporters. However, the major liver uptake transporters were either absent or expressed at significantly lower levels in human liver cell lines than in human liver. When comparing cytotoxicity of five statins, the organic anion transporting polypeptide 1B1 expressing HEK cells showed a significantly higher sensitivity than the wild-type HEK cells. The IC50 shifts ranged from 9- to >100-fold, and the potency shifts collapsed in the presence of rifampicin, the inhibitor for OATPs. The extent of the IC50 shift correlated with the permeability of the statins with high permeable compounds having smaller shifts and low permeable compounds having larger shifts. The changes in statin potency in transporter-transfected cells reflect the active uptake of statins into the cells, and the increased intracellular drug concentration lead to increased toxicity. The data suggested that uptake transporters have a significant impact on the outcomes of a cell-based assay and should be considered during the early stages of compound toxicity screening in drug discovery. For compounds with low permeability that are likely to undergo transporter-mediated uptake, it is important to test them in transporter-competent cell models.

Organic Anion Transporting Polypeptide 1B (OATP1B) Transporters Modulate Hydroxyurea Pharmacokinetics

Abstract 1008 Background:Sickle cell is the most common inherited blood disorder affecting millions worldwide. Hydroxyurea is currently the only FDA approved drug for the treatment of sickle cell anemia (SCA) that alters disease pathophysiology. Known for inducing fetal hemoglobin production, hydroxyurea can decrease hospitalizations, vaso-occlusive pain episodes, and mortality associated with SCA. Although efficacious for many patients, a large degree of inter-patient variability in pharmacokinetic (PK) and pharmacodynamic (PD) properties of hydroxyurea has been noted, but sources of this variation remain largely undefined. Plasma transmembrane transporters have been shown to play an important role in the determination drug PK and PD of various xenobiotics. Our recent in vitro studies have demonstrated that hydroxyurea is a substrate for specific solute carrier (SLC) transporters including members of the organic anion transporting polypeptide 1B (OATP1B) subfamily which consists of OATP1B1, and OATP1B3 in humans, and one mouse orthologue Oatp1b2. These transporters are highly expressed on the sinusoidal membrane of hepatocytes, and function as uptake transporters bringing substrates from the blood into the liver. Limited reports have suggested that Oatp1b2 transcript is also found in kidney and intestine as well. In the present study we evaluated the uptake of the hydroxyurea by different haplotypes of hOATP1B3 transporter in vitro, as well as the influence of the Oatp1b2 transporter on hydroxyurea PK in vivo using a transporter knock-out mouse model. Methods:Transporter-mediated cellular uptake of hydroxyurea was determined in vitro by measuring [14C]-hydroxyurea accumulation in Xenopus laevis oocytes that over express two common haplotypes of OATP1B3. hOATP1B3*1 and the functional variant hOATP1B3*2 that contains the 334T>G and 699G>A SNPs were assessed. In vivo PK Studies were performed using a knockout model mouse model in which mOATP1b2 was eliminated from the DBA/LcaJ1 background. Mice were dose with 50mg/kg of [14C]-hydroxyurea via oral gavage or intravenous (i.v.) injection. Serial blood samples were collected at defined timepoints and concluded with terminal blood and tissue collections at 2 hours post injection. Hydroxyurea levels were determined based on radioactivity in plasma and liver, and kidney homogenate. Accumulation in the urine was also measured over a 3 day period. Results:In vitro kinetic studies did not show any differences between OATP1B3*1 and OATP1B3*2 haplotypes in time-, or concentration-dependent accumulation of hydroxyurea. The inhibitor substrates naringen and rifampin decreased hydroxyurea accumulation by at least 50% in OATP1B3*1 but did not decrease accumulation in OATP1B3*2. These in vitro studies suggest that the genetic mutations associated with these two haplotypes do not have a functional impact on the transport of hydroxyurea, but may have significant implications for potential drug-drug interaction. In vivo, Hydroxyurea PK was altered in the absence of the Oatp1b2 transporter. Plasma levels of hydroxyurea were similar after i.v. injections, but total exposure (AUC) in Oatp1b2-KO mice was decreased by 20% with an AUC of 2257 mg-min/ml compared to 2872 mg-min/ml observed in the wildtype mice (p=0.04). Hydroxyurea levels in the kidneys of Oatp1b2-KO mice were approximately 50% of wildtype with a mean accumulation of 360 pmol/g tissue (p= 0.0025). In addition, 30% less hydroxyurea was excreted in urine of Oatp1b2-KO mice by 72 hours (p=0.023). Accumulation of hydroxyurea in liver of Oatp1b2-KO mice and wildtype mice were 107 and 132 pmol/g tissue respectively, but this difference was not statistically significant. These results suggest that hydroxyurea PK is altered in the absence of Oatp1b2 transporter after oral administration. Conclusions:These studies provide the first in vivo evidence that specific transporters may be key determinants of hydroxyurea PK. Future studies are warranted to investigate the influence of human OATP1B and other transporters that modulate hydroxyurea PK. Identification of such transporters may help to elucidate factors such as genetic variations in the transporters and/or drug-drug interactions that may contribute to PK and PD variability observed in sickle cell patients. Disclosures:No relevant conflicts of interest to declare.

Characterization of Organic Anion Transporting Polypeptide (OATP) Expression and Its Functional Contribution to the Uptake of Substrates in Human Hepatocytes

Since the substrate specificities of OATP1B1, 1B3, and 2B1 are broad and overlapping, the contribution of each isoform to the overall hepatic uptake is of concern when assessing transporter-mediated drug-drug interactions (DDIs) or genetic polymorphism impact in the clinic. Herein, we quantitatively measured OATP proteins in cryopreserved hepatocytes, sandwich-cultured human hepatocytes (SCHH), and the liver, and examined the relationship with functional uptake of OATP substrates in an effort to identify the OATP isoform(s) contributing to the hepatic uptake of pitavastatin. The modulation of OATP expression in SCHH was found to be lot-dependent. However, OATP protein measurements averaged from 5 lots of SCHH were comparable to that of suspended hepatocytes. All three OATP transporters in suspended hepatocytes and SCHH were significantly lower than those in the liver. In SCHH, the uptake of CCK-8 and pravastatin was found to be associated with the expression of OATP1B3 and OATP1B1, respectively. In suspended hepatocytes, OATP1B1 appeared to show a positive trend with respect to the uptake of pitavastatin, which suggests a selective contribution of OATP1B1 to pitavastatin transport and thus an OATP quantitative protein expression-activity relationship. While the passive diffusion of rosuvastatin in SCHH was consistent across hepatocyte lots, the passive diffusion of pitavastatin varied over a broad range (>4-fold) in suspended hepatocytes and was inversely correlated with transporter-mediated uptake, presumably due to cell membrane alterations imparted by cryopreservation. Collectively, SCHH maintains OATP protein expression and membrane integrity and, if feasible when considering research goals, would be considered a superior tool for the characterization of in vitro transport parameters without the complication of membrane leakage.

Comprehensive study on regional human intestinal permeability and prediction of fraction absorbed of drugs using the Ussing chamber technique

The purpose of this study was to evaluate the use of human intestinal tissue in Ussing chamber to predict oral and colonic drug absorption and intestinal metabolism. Data on viability, correlation between apparent permeability coefficients (Papp) and fraction absorbed (fa) after oral and colonic administration, regional permeability, active uptake and efflux of drugs as well as intestinal metabolism were compiled from experiments using 159 human donors. Permeability coefficients for up to 28 drugs were determined using one or several of four intestinal regions: duodenum, jejunum, ileum and colon and 10 drugs were studied bidirectionally. Viability was monitored simultaneously with transport experiments by recording potential difference (PD), short-circuit current (SCC) and the resistance (TER). Intestinal metabolism was studied using testosterone and midazolam as probe substrates.There was a steep sigmoidal correlation between Papp in the Ussing chamber, using jejunal segments, and oral fa in humans, for a set of 25 drugs (R2: 0.85, p<0.01). A clear sigmoidal relationship was also obtained between Papp in colonic segments and fa after colonic administration in humans for a set of 10 drugs (R2: 0.93, p<0.05). Regional permeability data showed a tendency for highly permeable compounds to have higher or similar Papp in colon as in the small intestinal segments, while the colonic regions showed a lower Papp for more polar compounds as well as for d-glucose and l-leucine. Bidirectional transport (mucosa to serosa and serosa to mucosa direction) in jejunum showed well functioning efflux- and uptake asymmetry. Intestinal metabolic extraction during transport across jejunum segments was found for both testosterone and midazolam.In conclusion, viable excised human intestine mounted in the Ussing chamber, is a powerful technique for predicting regional fraction absorbed (fa), transporter-mediated uptake or efflux as well as intestinal metabolism of drug candidates in man. Furthermore, a sigmoidal relationship of Papp vs. fa was obtained when permeability data from the present study were merged with data from 2 other independent laboratories (R2: 0.83, p<0.01). The correlation curve reported can be used by any laboratory for predictions of human permeability and fa. In addition, for the first time a correlation curve between colonic Papp and human colonic fa is reported, which demonstrates the usefulness of this methodology in early assessment of the colonic absorption potential of extended release formulation candidates.

Enantioselective uptake of fexofenadine by Caco-2 cells as model intestinal epithelial cells

Fexofenadine contains a chiral carbon in its chemical structure and is orally administered as a racemic mixture. This study evaluated the selective uptake of fexofenadine enantiomers by Caco-2 cells as a model of intestinal epithelial cells. R(+)-fexofenadine or S(-)-fexofenadine was applied to Caco-2 cells, followed by incubation. After incubation, the amounts of fexofenadine enantiomers in cells were determined. The kinetic parameters for the uptake of fexofenadine enantiomers by Caco-2 cells were estimated using the Michaelis-Menten equation. The transporter-mediated uptake rate of R(+)-fexofenadine was 1.7-fold higher than that of S(-)-fexofenadine. The difference in transporter-mediated R(+)-fexofenadine and S(-)-fexofenadine uptake was completely diminished under ATP-depleted conditions and in the presence of organic anion transporter peptide (OATP) inhibitors. Also, a Dixon plot showed that each fexofenadine enantiomer was competitively inhibited by the other enantiomer. The ratio of R(+)-fexofenadine uptake to S(-)-fexofenadine uptake in the case of a racemic mixture was higher than that in the case of a single enantiomer. This study suggested that the selective absorption of fexofenadine enantiomers by intestinal epithelial cells might have been due to the selective uptake mediated by OATPs and that the difference in intestinal absorption was enhanced with a racemic mixture.

Evaluation of transporter-mediated hepatic uptake in a non-radioactive high-throughput assay: a study of kinetics, species difference and plasma protein effect

1. In this manuscript we describe a non-radioactive, high-throughput method to evaluate hepatic uptake using cryopreserved hepatocytes. We have validated the uptake of pravastatin with different amounts of hepatocytes and the impact of the oil layer used in separation. The time- and concentration-dependent uptake profiles of several anionic and cationic charged drugs were evaluated. The results with our method compare favourably with the literature for pravastatin, atorvastatin and estrone 3-sulfate.2. Two approaches for kinetic determination (temperature difference and fitting the linear and non-saturable passive diffusion rate in the equation, i.e. V = (Vmax × S)/(Km + S) + Pdif × S) have been evaluated. Kinetic studies indicate that the different approaches for determining passive diffusion can affect Km and Vmax, but not the clearance of active uptake (Vmax/Km).3. Using pravastatin as a probe substrate, species differences were observed in the organic anion-transporting polypeptide (OATP) 1B1 and 1B3 activities. Plasma protein significantly reduced the uptake of atorvastatin, but not pravastatin.4. Our data suggests that evaluation of the role of active uptake in hepatic clearance in humans should consider the relative ratio of active uptake to passive diffusion, species differences and plasma protein binding when applying in vitro uptake data.

Microcystins as novel leads against OATP1B3-expressing tumors

Microcystins are cyclic heptapeptides produced by cyanobacteria. They potently inhibit the eukaryotic protein phosphatase families PP1 and PP2A. They are able to display toxicity only after transporter-mediated uptake by the cell. This uptake is mediated by the organic anion transporting polypeptides OATP1B1, OATP1B3, and OATP1A2. Liver cells express OATP1B1 and OATP1B3 transporters, which results in a pronounced liver toxicity of microcystins. However, OATP1B3 is also expressed in a significant percentage of hepatocellular carcinoma cells as well as gastrointestinal, lung, breast and colon tumors. Interestingly, compared to OATP1B1, OATP1B3 is found only in low abundance in liver cells. Thus selectivity that favors OATP1B3 over OATP1B1 should lead to a decreased hepatic clearance and toxicity, and an increased uptake in OATP1B3-expressing tumors, thus creating a therapeutic window for the respective compound.

Neuroprotection by lowering cholesterol: A decrease in membrane cholesterol content reduces transporter-mediated glutamate release from brain nerve terminals

Background: In our earlier work, a reduction of cholesterol content increased the extracellular glutamate level in rat brain nerve terminals (synaptosomes) that was a result of the lack of transporter-mediated glutamate uptake. The aim of this study was to assess transporter-mediated release of glutamate from cholesterol-deficient synaptosomes. In stroke, cerebral hypoxia/ischemia, and traumatic brain injury, the development of neurotoxicity is provoked by enhanced extracellular glutamate, which is released from nerve cells mainly by glutamate transporter reversal — a distinctive feature of these pathological states. Methods: Laser scanning confocal microscopy, spectrofluorimetry, radiolabeled assay, and glutamate dehydrogenase assay. Results: Cholesterol acceptor methyl-β-cyclodextrin (15mM) reduced the cholesterol content in the synaptosomes by one quarter. Transporter-mediated glutamate release from synaptosomes: 1) stimulated by depolarization of the plasma membrane; 2) by means of heteroexchange with competitive transportable inhibitor of glutamate transporters dl-threo-β-hydroxyaspartate; 3) in low [Na+] medium; and 4) during dissipation of the proton gradient of synaptic vesicles by the protonophore cyanide‐p‐trifluoromethoxyphenyl‐hydrazon (FCCP); was decreased under conditions of cholesterol deficiency by ~24, 28, 40, and 17%, respectively. Conclusions: A decrease in the level of membrane cholesterol attenuated transporter-mediated glutamate release from nerve terminals. Therefore, lowering cholesterol may be used in neuroprotection in stroke, ischemia, and traumatic brain injury which are associated with an increase in glutamate uptake reversal. This data may explain the neuroprotective effects of statins in these pathological states and provide one of the mechanisms of their neuroprotective action. However, beside these disorders, lowering cholesterol may cause harmful consequences by decreasing glutamate uptake in nerve terminals.

Absorption, Disposition, and Pharmacokinetics of Saponins from Chinese Medicinal Herbs: What Do We Know and What Do We Need to Know More?

Saponins are a group of amphiphilic glycosides containing one or more sugar chains linked to a nonpolar triterpene or steroid aglycone skeleton, which are believed to be responsible for the pharmacological activities of many Chinese medicinal herbs. The purpose of this paper is to summarize the contemporary knowledge of the absorption, disposition, and pharmacokinetics of some important saponins, including ginsenosides, licorice saponins, dioscorea saponins, astragalosides, and saikosaponins. Poor intestinal absorption of saponins is mainly due to their unfavorable physicochemical traits, such as large molecular mass ( > 500 Da), high hydrogen-bonding capacity ( > 12), and high molecular flexibility ( > 10), that underlie poor membrane permeability. Rapid and extensive biliary excretion is another primary factor that limits the oral bioavailability of most saponins. However, several saponins, including ginsenosides Ra3, Rb1, Rc, and Rd, and dioscin, are excreted slowly into the bile and in turn have significantly long elimination half lives (7-25 h in rats). These longcirculating saponins may be used as pharmacokinetic markers to substantiate systemic exposure to the ingested herb extracts. In addition to biliary excretion for elimination of most saponins unchanged, renal excretion may also be important for certain saponins. Saponins can be hydrolyzed by the colonic microflora. After absorption, the deglycosylated aglycones undergo phase I and/or II metabolism by the host. In line with the poor permeability, saponin concentrations in most rat tissues are lower than the concurrent plasma level and the brain level is usually very low. However, the liver concentrations of many saponins, as well as the kidney levels of certain saponins, can be quite high, which involves transporter-mediated uptake mechanisms. Repeated p.o. ingestion of glycyrrhizin appears to be able to induce CYP3A in rodents and humans, while several deglycosylated products of ginsenosides can moderately inhibit CYP activities in vitro with IC50 values of 10-50 μM. More research is required for elucidation of the absorption, disposition, and pharmacokinetics of multiple saponins to enhance understanding which saponins are most likely to exert pharmacological effects in vivo, as well as influence of complex herb matrix. In addition, research is also needed to characterize the microbiotal deglycosylation and the subsequent aglycone metabolism by the host for a broader range of saponins, as well as the hepatobiliary transporter phenotyping for and the interaction with saponins. Furthermore, in vitro and in vivo studies of saponin-based herb-drug interactions are also warranted.

1006 Combination of Laparoscopy and Enhanced Recovery Program Improves Outcomes After Ileocecal Resection for Crohn's Disease

Activation of sweet taste receptors may enhance glucose uptake several fold in rat intestine. AIM: To explore mechanisms of sweet taste receptor activation in glucose uptake in 3 intestinal cell lines. HYPOTHESIS: The artificial sweetener, acesulfame potassium (AceK), increases glucose uptake via activating sweet taste receptors to induce translocation GLUT2 to the apical membrane through the PLC βII pathway. METHODS: Caco-2, RIE-1, and IEC-6 cells (human, rat, and rat intestinal cell lines) were seeded on a 24-well plate at a density of 4x104 cells/cm2 in growth culture media and left to differentiate for 15 days after confluence. Caco-2 and RIE-1 cells express GLUT2, while IEC-6 cells do not. Cells were starved from glucose for 1 h and pre-incubated with and without 10 mM AceK for 30 min. Glucose uptake was measured by incubating the cells for 1 to 10 min with 0.5-50 mM glucose with and without 10 mM AceK. 14C-D-glucose was used to measure stereospecific, transporter-mediated uptake and 3H-L-glucose to measure passive uptake with or without the inhibitors 10 μM U-73122, a PLC βII inhibitor, 10 μM chelerythrine, a PKC inhibitor, and 2 μM cytochalasin B, a microtubular system disrupter. Glucose uptake was stopped by adding ice-cold PBS; cells were washed with PBS 2 times and solubilized with 0.1 N NaOH. All experiments were done on at least 3 separate occasions in triplicate. RESULTS: In Caco2 and RIE-1 cells, 10 mM AceK increased carrier-mediated glucose uptake by 20-30% at apical glucose concentrations >25 mM (p 25 mM) glucose concentrations during the 5-min incubation; chelerythrine and cytochalasin B had similar effects. No effect was seen in IEC-6 cells. CONCLUSION: The artificial sweetener AceK, a known sweet taste receptor agonist, has no effect on glucose uptake in low ( 25 mM) in our cell culture models when GLUT2 translocation occurs. The role of artificial sweeteners on glucose uptake appears to act in part by effects on the enterocyte itself. (Support: NIH DK39337-MGS)

Neuropeptide Y Y5 receptor antagonism attenuates cocaine-induced effects in mice

Several studies suggest a role for neuropeptide Y (NPY) in addiction to drugs of abuse, including cocaine. However, the NPY receptors mediating addiction-related effects remain to be determined. To explore the potential role of Y5 NPY receptors in cocaine-induced behavioural effects. The Y5 antagonist L-152,804 and Y5-knockout (Y5-KO) mice were tested in two models of cocaine addiction-related behaviour: acute self-administration and cocaine-induced hyperactivity. We also studied effects of Y5 receptor antagonism on cocaine-induced c-fos expression and extracellular dopamine with microdialysis as well as dopamine transporter-mediated uptake of dopamine in vitro. Immunocytochemistry was used to determine whether dopamine neurons express Y5-like immunoreactivity. In self-administration, L-152,804 prominently decreased nose-poking for the peak dose of cocaine and shifted the dose-response curve for cocaine downward. Y5-KO mice also showed modestly attenuated self-administration. Cocaine-induced hyperactivity was attenuated by L-152,804 and in Y5-KO mice. Cocaine failed to increase c-fos expression in the nucleus accumbens and striatum of L-152,804-treated mice, indicating that the Y5 antagonist could act by influencing neural activity in these regions. Accordingly, the cocaine-induced increase in accumbal extracellular dopamine was attenuated by L-152,804 and in Y5-KO mice, suggesting that Y5 antagonism influences cocaine-induced behaviour by regulating dopamine. Consistent with this concept, dopamine neurons in the ventral tegmental area appeared to contain Y5 receptors. In contrast, neither L-152,804 nor NPY influenced dopamine transporter-mediated dopamine uptake. The present data indicate that Y5 antagonism may attenuate cocaine-induced behavioural effects, suggesting that Y5 receptors could be a potential therapeutic target in cocaine addiction.

Identification of drugs and drug metabolites as substrates of multidrug resistance protein 2 (MRP2) using triple‐transfected MDCK‐OATP1B1‐UGT1A1‐MRP2 cells

The coordinate activity of hepatic uptake transporters [e.g. organic anion transporting polypeptide 1B1 (OATP1B1)], drug-metabolizing enzymes [e.g. UDP-glucuronosyltransferase 1A1 (UGT1A1)] and efflux pumps (e.g. MRP2) is a crucial determinant of drug disposition. However, limited data are available on transport of drugs (e.g. ezetimibe, etoposide) and their glucuronidated metabolites by human MRP2 in intact cell systems. Using monolayers of newly established triple-transfected MDCK-OATP1B1-UGT1A1-MRP2 cells as well as MDCK control cells, single- (OATP1B1) and double-transfected (OATP1B1-UGT1A1, OATP1B1-MRP2) MDCK cells, we therefore studied intracellular concentrations and transcellular transport after administration of ezetimibe or etoposide to the basal compartment. Intracellular accumulation of ezetimibe was significantly lower in MDCK-OATP1B1-UGT1A1-MRP2 triple-transfected cells compared with all other cell lines. Considerably higher amounts of ezetimibe glucuronide were found in the apical compartment of MDCK-OATP1B1-UGT1A1-MRP2 monolayers compared with all other cell lines. Using HEK cells, etoposide was identified as a substrate of OATP1B1. Intracellular concentrations of etoposide equivalents (i.e. parent compound plus metabolites) were affected only to a minor extent by the absence or presence of OATP1B1/UGT1A1/MRP2. In contrast, apical accumulation of etoposide equivalents was significantly higher in monolayers of both cell lines expressing MRP2 (MDCK-OATP1B1-MRP2, MDCK-OATP1B1-UGT1A1-MRP2) compared with the single-transfected (OATP1B1) and the control cell line. Ezetimibe glucuronide is a substrate of human MRP2. Moreover, etoposide and possibly also its glucuronide are substrates of MRP2. These data demonstrate the functional interplay between transporter-mediated uptake, phase II metabolism and export by hepatic proteins involved in drug disposition.

Intestinal OATP1A2 inhibition as a potential mechanism for the effect of grapefruit juice on aliskiren pharmacokinetics in healthy subjects

To conduct a mechanistic investigation of the interaction between aliskiren and grapefruit juice in healthy subjects. Twenty-eight subjects received an oral dose of aliskiren 300 mg (highest recommended clinical dose) with 300 mL of either water or grapefruit juice in a two-way crossover design. Safety and pharmacokinetic analyses were performed. In vitro studies were performed in HEK293 cells to investigate the role of organic anion transporting polypeptide (OATP) transporter-mediated uptake of aliskiren. Co-administration of a single dose of aliskiren with grapefruit juice decreased the plasma concentration of aliskiren, with mean decreases in the AUC(inf), AUC(last), and C(max) of 38, 37, and 61%, respectively. The uptake of [¹⁴C]aliskiren into OATP2B1-expressing cells was essentially the same as that into control cells, and the inhibitor combination atorvastatin and rifamycin had no effect on [¹⁴C]aliskiren accumulation in either cell type. The uptake of [¹⁴C]aliskiren and [³H]fexofenadine was linear in OATP1A2-expressing cells and was reduced by naringin, with IC₅₀ values of 75.5 ± 11.6 and 24.2 ± 2.0 μM, respectively. Grapefruit juice decreases exposure of aliskiren partially via inhibition of intestinal OATP1A2.

Microglial zinc uptake via zinc transporters induces ATP release and the activation of microglia

Previously, we demonstrated that extracellular zinc plays a key role in transient global ischemia-induced microglial activation through sequential activation of NADPH oxidase and poly(ADP-ribose) polymerase (PARP)-1. However, it remains unclear how zinc causes the sequential activation of microglia. Here, we examined whether transporter-mediated zinc uptake is necessary for microglial activation. Administration of zinc to microglia activated them through reactive oxygen species (ROS) generation and poly(ADP-ribose) (PAR) formation, which were suppressed by intracellular zinc chelation with 25 μM TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine) or 2 μM BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). The (65)Zn uptake by microglia was temperature- and dose-dependent, and it was blocked by metal cations, but not by L-type calcium channel blockers nifedipine and nimodipine. Expression of Zrt-Irt-like protein (ZIP)1, a plasma membrane-type zinc transporter, was detected in microglia, and nickel, a relatively sensitive substrate/inhibitor of ZIP1, showed cis- and trans-inhibitory effects on the (65)Zn uptake. Exposure of microglia to zinc increased the extracellular ATP concentration, which was suppressed by intracellular zinc chelation and inhibition of hemichannels. mRNA expression of several types of P2 receptors was detected in microglia, and periodate-oxidized ATP, a selective P2×7 receptor antagonist, attenuated the zinc-induced microglial activation via NADPH oxidase and PARP-1. Exogenous ATP and 2'(3')-O-(4-benzoyl-benzoyl) ATP also caused microglial activation through ROS generation and PAR formation. These findings demonstrate that ZIP1-mediated uptake of zinc induces ATP release and autocrine/paracrine activation of P2X(7) receptors, and then activates microglia, suggesting that zinc transporter-mediated uptake of zinc is a trigger for microglial activation via the NADPH oxidase and PARP-1 pathway. © 2011 Wiley-Liss, Inc.

Diglycolic Acid Is the Nephrotoxic Metabolite in Diethylene Glycol Poisoning Inducing Necrosis in Human Proximal Tubule Cells In Vitro

Diethylene glycol (DEG), a solvent and chemical intermediate, can produce an acute toxic syndrome, the hallmark of which is acute renal failure due to cortical tubular degeneration and proximal tubular necrosis. DEG is metabolized to two primary metabolites, 2-hydroxyethoxyacetic acid (2-HEAA) and diglycolic acid (DGA), which are believed to be the proximate toxicants. The precise mechanism of toxicity has yet to be elucidated, so these studies were designed to determine which metabolite was responsible for the proximal tubule cell death. Human proximal tubule (HPT) cells in culture, obtained from normal cortical tissue and passaged 3-6 times, were incubated with increasing concentrations of DEG, 2-HEAA, or DGA separately and in combination for 48 h at pH 6 or 7.4, and various parameters of necrotic and apoptotic cell death were measured. DEG and 2-HEAA did not produce any cell death. DGA produced dose-dependent necrosis at concentrations above 25 mmol/l. DGA did not affect caspase-3 activity and increased annexin V staining only in propidium iodide-stained cells. Hence, DGA induced necrosis, not apoptosis, as corroborated by severe depletion of cellular adenosine triphosphate levels. DGA is structurally similar to citric acid cycle intermediates that are taken up by specific transporters in kidney cells. HPT cells, incubated with N-(p-amylcinnamoyl)anthranilic acid, a sodium dicarboxylate-1 transporter inhibitor showed significantly decreased cell death compared with DGA alone. These studies demonstrate that DGA is the toxic metabolite responsible for DEG-induced proximal tubular necrosis and suggest a possible transporter-mediated uptake of DGA leading to toxic accumulation and cellular dysfunction.

Inhibition of xc- transporter-mediated cystine uptake by sulfasalazine analogs

A series of sulfasalazine analogs were synthesized and tested for their ability to block cystine-glutamate antiporter system xc⁻ using L-[(14)C]cystine as a substrate. Replacement of sulfasalazine's diazo group with an alkyne group led to an equally potent inhibitor, 2-hydroxy-5-((4-(N-pyridin-2-ylsulfamoyl)phenyl)ethynyl)benzoic acid 6. Our SAR studies also revealed that the carboxylate group of sulfasalazine is essential for its inhibitory activity while the phenolic hydroxyl group is dispensable. Truncated analogs lacking an N-pyridin-2-ylsulfamoyl moiety were less potent than sulfasalazine, but may serve as more tractable templates because of their low molecular weight by applying a variety of fragment growing approaches. Given that sulfasalazine is rapidly metabolized through cleavage of the diazo bond, these analogs may possess a more desirable pharmacological profile as system xc- blockers, in particular, for in vivo studies.

Kinetic Characterization of Rat Hepatic Uptake of 16 Actively Transported Drugs

To explore the determinants of hepatic uptake, 16 compounds were investigated with different physicochemical and disposition characteristics, including five statins, three sartans, saquinavir, ritonavir, erythromycin, clarithromycin, nateglinide, repaglinide, fexofenadine, and bosentan. Freshly isolated rat hepatocytes in suspension were used with the oil-spin method to generate kinetic parameters. Clearances, via passive diffusion (P(diff)) and active uptake (CL(active), characterized by maximal uptake rate and K(m)), were estimated from the initial uptake rate data over a 0.01 to 100 μM concentration range. The K(m) values had a range of 15-fold, with 10 of the 16 drugs with K(m) < 10 μM (median 6 μM). Both CL(active) and P(diff) ranged over 100-fold (median 188 and 14 μl/min/10⁶ cells). Assessment of the relative contribution of P(diff) and CL(active) indicated that, at low concentrations (approximately 0.1 μM), the active process contributes >80% to the overall uptake for 13 drugs. Although high P(diff) values were obtained for ritonavir and repaglinide, active process contributed predominantly to uptake; in contrast, high passive permeability dominates over transporter-mediated uptake for saquinavir over the full concentration range. For bosentan and erythromycin, active and passive processes were equally important. Hepatocyte-to-medium unbound concentration ratio was >10 for 9 of the 16 drugs, ranging from 2 to 494 for bosentan and atorvastatin, respectively. Some drugs showed extensive intracellular binding (fraction unbound range 0.01-0.6), which was not correlated with active uptake. LogD₇.₄ correlated significantly with P(diff) and the extent of intracellular binding but not with active uptake. This study provides systematic assessment of the role of active uptake relative to the passive process; implications of the findings are discussed.

Peptide Absorption After Massive Proximal Small Bowel Resection: Mechanisms of Ileal Adaptation

BackgroundProtein absorption occurs as di- and tri-peptides via H+/peptide co-transporter-1 (PepT1). AimThe aim of this study is to identify mechanisms of ileal adaptation after massive proximal enterectomy. HypothesisIleal adaptation in uptake of peptides is mediated through upregulation of PepT1 gene expression. Study DesignRats underwent 70% jejunoileal resection. Total mucosal cellular levels of messenger RNA (mRNA) and protein and transporter-mediated uptake per centimeter of the di-peptide glycyl-sarcosine (Gly-Sar) were compared in remnant ileum 1 and 4 weeks postoperatively to control and to 1-week sham laparotomy rats. Histomorphology, food consumption, and weights of rats were monitored. ResultsAfter 70% resection, although mRNA per cell for PepT1 decreased at 1 week (p = 0.002), expression of mRNA at 4 weeks and protein at 1 and 4 weeks in remnant ileum were unchanged (p > 0.1). Ileal Gly-Sar uptake (Vmax—nanomoles per centimeter per minute, i.e., number of transporters per centimeter) increased at 1 and 4 weeks compared to control and 1-week sham (p < 0.05 each); Km (i.e., transporter function) was unchanged. Villous heights (millimeters) in remnant ileum increased at 1- and 4-week time points over controls (0.45 and 0.57 vs 0.21, resp; p < 0.001). ConclusionsIleal adaptation to proximal resection for peptide absorption occurs through cellular proliferation (hyperplasia) and not through cellular upregulation of PepT1 mRNA or protein per enterocyte.

An alternative and expedient synthesis of radioiodinated 4-iodophenylalanine

Radiolabeled amino acids have been used extensively in oncology both as diagnostic and therapeutic agents. In our pursuit to develop radiopharmaceuticals to target breast cancer, we were interested in determining the uptake of radioiodinated 4-iodophenylalanine, among other labeled amino acids, in breast cancer cells. In this work, we have developed an alternative method for the synthesis of this agent. The novel tin precursor, ( S)- tert-butyl 2-( tert-butoxycarbonylamino)-3-(4-(tributylstannyl)phenyl)propanoate ( 3) was synthesized from the known, corresponding iodo derivative. Initially, the labeled 4-iodophenylalanine was synthesized from the above tin precursor in two steps with radiochemical yields of 91.6±2.7% and 83.7±1.7% ( n=5), for the radioiodination (first) and deprotection (second) step, respectively. Subsequently, it was synthesized in a single step with an average radiochemical yield of 94.8±3.4% ( n=5). After incubation with MCF-7 breast cancer cells for 60 min, an uptake of up to 49.0±0.7% of the input dose was seen; in comparison, the uptake of [ 14C]phenylalanine under the same conditions was 55.9±0.5%. Furthermore, the uptake of both tracers was inhibited to a similar degree in a concentration-dependent manner by both unlabeled phenylalanine and 4-iodophenylalanine. With [ 14C]phenylalanine as the tracer, IC 50 values of 1.45 and 2.50 mM were obtained for Phe and I-Phe, respectively, and these values for [ 125I]I-Phe inhibition were 1.3 and 1.0 mM. In conclusion, an improved and convenient method for the synthesis of no-carrier-added 4-[ ⁎I]phenylalanine was developed and the radiotracer prepared by this route demonstrated an amino acid transporter-mediated uptake in MCF-7 breast cancer cells in vitro that was comparable to that of [ 14C]phenylalanine.

Determination of OATP-, NTCP- and OCT-mediated substrate uptake activities in individual and pooled batches of cryopreserved human hepatocytes

While the utility of cryopreserved human hepatocyte suspensions (CHHS) for in vitro drug metabolism assays has been established, less is known about the effects of cryopreservation on transporter activity in human hepatocytes. In the present study, the activities of NTCP (sodium taurocholate co-transporting polypeptide; SLC10A1), as well as of the hepatic OATP (organic anion transporting polypeptide; SLCO gene family) and OCT (organic cation transporter; SLC22A) isoforms were assessed in 14 individual and four pooled batches of CHHS. For comparative purposes, substrate accumulation rates were also measured in sandwich-cultured human hepatocytes. In CHHS, the mean accumulation clearance of the NTCP substrate taurocholate (1 μM) was 27.5 (±15.0) μl/min/million cells and decreased by 10-fold when extracellular sodium was replaced by choline. The accumulation clearance of digoxin and of the OATP substrates estrone-3-sulfate and estradiol-17β- d-glucuronide (E 2-17β-G; 1 μM) amounted to 9.5 (±4.9), 99 (±67) and 5.2 (±2.6) μl/min/million cells, respectively. Presence of the known OATP inhibitor rifampicin (25 μM) significantly ( p < 0.01) decreased the accumulation of estrone-3-sulfate and E 2-17β-G to 48% and 70% of the control value, respectively, while no significant effect on digoxin accumulation was observed. The mean accumulation clearance of the OCT substrate 1-methyl-4-phenylpyridinium amounted to 19.8 (±10.9) μl/min/million cells. Co-incubation with the OCT1 inhibitor prazosin (3 μM) and the OCT3 inhibitor corticosterone (1 μM) resulted in a significant ( p < 0.01) decrease to 72% and 85% of the accumulation in control conditions, respectively. Experiments in pooled CHHS generally showed accumulation values that were comparable with the mean of the individual batches. A good correlation ( R 2 = 0.93) was observed between estrone-3-sulfate accumulation values and OATP1B3 mRNA levels, as determined in five batches of CHHS. Compared to substrate accumulation measured in sandwich-cultured human hepatocytes, accumulation values in CHHS were comparable (taurocholate and digoxin) to slightly higher (estrone-3-sulfate). Our data indicate that cryopreserved human hepatocyte suspensions are a reliable in vitro model to study transporter-mediated substrate uptake in the liver. Systematic characterization of multiple batches of CHHS for transporter activity supports rational selection of human hepatocytes for specific applications.