Inhibition Of Cation Transport Research Articles

Facilitated Transport of EGFR Inhibitors Plays an Important Role in Their Cellular Uptake.

Epidermal growth factor receptor (EGFR) is a transmembrane protein commonly targeted by tyrosine kinase inhibitors (TKIs) as a front-line therapy for patients with many cancers including nonsmall cell lung cancer (NSCLC). Effective treatment requires efficient intracellular drug uptake and target binding. However, despite the recent success in the development of new TKI drugs, the mechanisms of uptake for many TKIs are still poorly understood due to the difficulty in imaging and measuring nonfluorescent drug molecules at a subcellular resolution. It has previously been shown that weakly basic TKI drugs are sequestered in lysosomes. Leveraging this property, we apply hyperspectral stimulated Raman scattering imaging to directly visualize and quantify two Food and Drug Administration-approved EGFR inhibitor drugs (lapatinib and afatinib) inside living cells and the changes in their cellular uptake upon the addition of organic cation transporter inhibitors. These single-cell quantitative measurements provide new insight into the role of membrane transporters in the uptake of TKI drugs in living cells.

The Influence of Kidneys Ion Transport Inhibitors on the Pharmacokinetic and Tumor Uptake Behaviors of a HER2-targeted Small Size Radiolabeled Peptide.

HER2 over-expression plays a crucial role in the cancer treatment protocol. This study evaluates the effectiveness of organic anion and cation transport inhibitors and substrate on the tumor uptake of 99mTc- HYNIC-(Ser)3-LTVPWY radiotracer in SKOV-3 tumor-bearing nude mice. Before the injection of the radiolabeled peptide, SKOV-3 tumor-bearing nude mice were treated with furosemide, cimetidine, para-amino hippuric acid, and saline. The inhibition effects of the organic anion and cation transport inhibitors were compared with the control group. In both treatment and control groups, the tumor and renal accumulation of radiopeptide in mice bearing SKOV-3 tumors were assessed in biodistribution and SPECT imaging studies. The biodistribution and imaging results suggested that all treated groups showed a higher tumor and higher normal tissue radioactivity compared to the control group. According to the tumor imaging study, the furosemidetreated group had slightly better tumor uptake and a higher tumor to muscle uptake ratio than other treatment groups. Administration of furosemide (an OAT inhibitor) increased radioactivity accumulation in the kidneys and blood and improved tumor radioactivity uptake. PAH (an anion transporter substrate) and cimetidine (an OCT inhibitor) have a minor effect on the accumulation of radioactivity in the kidneys and the acquired images.

Influence of Cation Transporters (OCTs and MATEs) on the Renal and Hepatobiliary Disposition of [11C]Metoclopramide in Mice

PurposeTo investigate the role of cation transporters (OCTs, MATEs) in the renal and hepatic disposition of the radiolabeled antiemetic drug [11C]metoclopramide in mice with PET.MethodsPET was performed in wild-type mice after administration of an intravenous microdose (<1 μg) of [11C]metoclopramide without and with co-administration of either unlabeled metoclopramide (5 or 10 mg/kg) or the prototypical cation transporter inhibitors cimetidine (150 mg/kg) or sulpiride (25 mg/kg). [11C]Metoclopramide PET was also performed in wild-type and Slc22a1/2(−/−) mice. Radiolabeled metabolites were measured at 15 min after radiotracer injection and PET data were corrected for radiolabeled metabolites.Results[11C]Metoclopramide was highly metabolized and [11C]metoclopramide-derived radioactivity was excreted into the urine. The different investigated treatments decreased (~2.5-fold) the uptake of [11C]metoclopramide from plasma into the kidney and liver, inhibited metabolism and decreased (up to 3.8-fold) urinary excretion, which resulted in increased plasma concentrations of [11C]metoclopramide. Kidney and liver uptake were moderately (~1.3-fold) reduced in Slc22a1/2(−/−) mice.ConclusionsOur results suggest a contribution of OCT1/2 to the kidney and liver uptake and of MATEs to the urinary excretion of [11C]metoclopramide in mice. Cation transporters may contribute, next to variability in the activity of metabolizing enzymes, to variability in metoclopramide pharmacokinetics and side effects.

Absorption and Disposition of Coproporphyrin I (CPI) in Cynomolgus Monkeys and Mice: Pharmacokinetic Evidence to Support the Use of CPI to Inform the Potential for Organic Anion-Transporting Polypeptide Inhibition.

Despite a recent expansion in the recognition of the potential utility of coproporphyrin (CP) as an endogenous biomarker of organic anion-transporting polypeptide (OATP) 1B activity, there have been few detailed studies of CP's pharmacokinetic behavior and an overall poor understanding of its pharmacokinetic fate from tissues and excretion. Here, we describe the pharmacokinetics of octadeuterium-labeled coproporphyrin I (CPI-d8) in cynomolgus monkeys following oral and intravenous administration. CPI-d8 has a half-life and bioavailability of 7.6 hours and 3.2%, respectively. Cynomolgus monkeys received oral cyclosporin A (CsA) at 4, 20, and 100 mg/kg which yielded maximum blood concentrations (C max) and area under the plasma concentration-time curve (AUC) values of 0.19, 2.5, and 3.8 µM, and 2.7, 10.5, and 26.6 µM·h, respectively. The apparent CsA-dose dependent increase in the AUC ratio of CPI-d8 (1.8, 6.2, and 10.5), CPI (1.1, 1.4, and 4.4), and CPIII (1.1, 1.8, and 4.6) at 4, 20, and 100 mg, respectively. In contrast, the plasma concentrations of CPI and CPIII were generally not affected by intravenous administration of the renal organic anion and cation transporter inhibitors (probenecid and pyrimethamine, respectively). In addition, tritium-labeled coproporphyrin I ([3H]CPI) showed specific and rapid distribution to the liver, intestine, and kidney after an intravenous dose in mice using quantitative whole-body autoradiography. Rifampin markedly reduced the liver and intestinal uptake of [3H]CPI while increasing the kidney uptake. Taken together, these results suggest that hepatic OATP considerably affects the disposition of CPI in animal models, indicating CPI is a sensitive and selective endogenous biomarker of OATP inhibition. SIGNIFICANCE STATEMENT: This study demonstrated that coproporphyrin I (CPI) has favorable oral absorption, distribution, and elimination profiles in monkeys and mice as an endogenous biomarker. It also demonstrated its sensitivity and selectivity as a probe of organic anion-transporting polypeptide (OATP) 1B activity. The study reports, for the first time, in vivo pharmacokinetics, tissue distribution, sensitivity, and selectivity of CPI as an OATP1B endogenous biomarker in animals. The data provide preclinical support for exploration of its utility as a sensitive and selective circulating OATP biomarker in humans.

Evidence for PMAT- and OCT-like biogenic amine transporters in a probiotic strain of Lactobacillus: Implications for interkingdom communication within the microbiota-gut-brain axis.

The ability of prokaryotic microbes to produce and respond to neurochemicals that are more often associated with eukaryotic systems is increasingly recognized through the concept of microbial endocrinology. Most studies have described the phenomena of neurochemical production by bacteria, but there remains an incomplete understanding of the mechanisms by which microbe- or host-derived neuroactive substances can be recognized by bacteria. Based on the evolutionary origins of eukaryotic solute carrier transporters, we hypothesized that bacteria may possess an analogous uptake function for neuroactive biogenic amines. Using specific fluorescence-based assays, Lactobacillus salivarius biofilms appear to express both plasma membrane monoamine transporter (PMAT)- and organic cation transporter (OCT)-like uptake of transporter-specific fluorophores. This phenomenon is not distributed throughout the genus Lactobacillus as L. rhamnosus biofilms did not take up these fluorophores. PMAT probe uptake into L. salivarius biofilms was attenuated by the protonophore CCCP, the cation transport inhibitor decynium-22, and the natural substrates norepinephrine, serotonin and fluoxetine. These results provide the first evidence, to our knowledge, for the existence of PMAT- and OCT-like uptake systems in a bacterium. They also suggest the existence of a hitherto unrecognized mechanism by which a probiotic bacterium may interact with host signals and may provide a means to examine microbial endocrinology-based interactions in health and disease that are part of the larger microbiota-gut-brain axis.

The in vitro renal cell toxicity of some unconventional anticancer phenanthroline-based platinum(II) complexes

The cytotoxicity of platinum(II) complexes coordinated to a chiral diamine, 1S,2S-diaminocyclohexane or 1R,2R-diaminocyclohexane and 1,10-phenanthroline or 3,4,7,8-tetramethyl-1,10-phenanthroline has been investigated in the renal proximal tubule HK-2 cell line. All platinum(II) complexes exhibited lower cytotoxicity in HK-2 cells (IC50 1.7–25μM) than in A2780 ovarian cancer cells or cisplatin-resistant A2780cisR cells (IC50 0.2–2.1μM) (at 48h). PHENSS ([Pt(1,10-phenanthroline)(1S,2S-dach)]2+) induced apoptosis and necrosis in ovarian cancer cells at concentrations that are relatively cytostatic to renal cells. Cisplatin was similarly or more cytotoxic to renal cells than ovarian cancer cells. Similar trends were reflected with shorter term exposure (1.5h). PHENSS demonstrated a comparatively cytostatic mode of action in renal cell cultures than cisplatin, as demonstrated by lower toxicity at higher concentrations (90μM). PHENSS induced an elongated renal cell morphology, cytoskeletal stress fibre thickening, and increased β-galactosidase activity, but no detectable change in reactive oxygen species generation or cell cycle distribution. In contrast, cisplatin treatment was associated with increased oxidative stress, cellular enlargement, G2/M arrest and apoptosis. The cytotoxicity of all platinum(II) complexes in both renal and ovarian cell lines were reduced in the presence of organic cation transporter (OCT) inhibitors cimetidine, disopyramide and amantadine. PHENSS and analogues demonstrated low level genotoxicity in an in vitro micronuclei assay compared to cisplatin or etoposide. These findings highlight PHENSS and other phen-based unconventional platinum(II) complexes as promising anticancer agents with alternative modes of action that induce lower kidney cell toxicity and genotoxicity, while demonstrating greater cisplatin-resistant ovarian cancer cell toxicity.

The cytotoxicity of some phenanthroline-based antimicrobial copper(II) and ruthenium(II) complexes

The in vitro cytotoxic properties of antimicrobial copper(II) complexes with 3,4,7,8-tetramethyl-1,10-phenanthroline (TMP) or 4,7-dipyridyl-1,10-phenanthroline (DIP) ligands and ruthenium(II) complexes coordinated with TMP or 2,9-dimethyl-1,10-phenanthroline ligands were investigated. Both copper(II) complexes were found to have similar inhibitory concentrations (IC50~2–2.5μM). Their cytotoxicity was found to be necrotic, associated with cytoplasmic vacuolisation, rounding, detachment and lack of apoptosis-associated DNA fragmentation, in comparison to the apoptotic effects of cisplatin which demonstrate adherent cell enlargement or detachment, membrane blebbing and condensation. Antimicrobial ruthenium(II) complexes demonstrated a lower renal cytotoxicity than copper(II) complexes or cisplatin (IC50>60μM). [Cu(DIP)(dach)](ClO4)2 and [Cu(TMP)(dach)](ClO4)2 (where dach=1,2-diaminocyclohexane) induced dihydroethidium-sensitive ROS and the cytotoxicity of both TMP and DIP coordinated copper(II) complexes was mitigated by catalase, highlighting a role of H2O2 generation in their mode of action. The cytotoxicity of either copper(II) complex was not affected by coincubation with organic cation transporter (OCT) inhibitors cimetidine or disopyramide, in contrast to cisplatin, suggesting a non-OCT dependent mode of uptake for the copper(II) complexes in human cells. Coincubation with copper sulfate reduced the cytotoxicity of [Cu(TMP)(dach)](ClO4)2 (3-6×). The TMP complex induced a greater degree of G2/M accumulation and micronuclei generation than the DIP complex, possibly attributable to its greater DNA binding affinity. These results highlight the potentially low genotoxicity of copper(II) complexes coordinated with TMP or DIP and polypyridyl ruthenium(II) complexes as potential antimicrobial agents.

An Exploration of Charge Compensating Ion Channels across the Phagocytic Vacuole of Neutrophils

Neutrophils phagocytosing bacteria and fungi exhibit a burst of non-mitochondrial respiration that is required to kill and digest the engulfed microbes. This respiration is accomplished by the movement of electrons across the wall of the phagocytic vacuole by the neutrophil NADPH oxidase, NOX2. In this study, we have attempted to identify the non-proton ion channels or transporters involved in charge compensation by examining the effect of inhibitors on vacuolar pH and cross-sectional area, and on oxygen consumption. The chloride channel inhibitors 4-[(2-Butyl-6,7-dichloro-2-cyclopentyl-2,3-dihydro-1-oxo-1H-inden-5-yl)oxy]butanoic acid (DCPIB) and flufenamic acid (FFA) were the most effective inhibitors of alkalinisation in human neutrophil vacuoles, suggesting an efflux of chloride from the vacuole. The proton channel inhibitor, zinc (Zn2+), combined with DCPIB caused more vacuolar swelling than either compound alone, suggesting the conductance of osmotically active cations into the vacuole. Support for cation influx was provided by the broad-spectrum cation transport inhibitors anandamide and quinidine which inhibited vacuolar alkalinisation and swelling when applied with zinc. Oxygen consumption was generally unaffected by these anion or cation inhibitors alone, but when combined with Zn2+ it was dramatically reduced, suggesting that multiple channels in combination can compensate the charge. In an attempt to identify specific channels, we tested neutrophils from knock-out mouse models including CLIC1, ClC3, ClC4, ClC7, KCC3, KCNQ1, KCNE3, KCNJ15, TRPC1/3/5/6, TRPA1/TRPV1, TRPM2, and TRPV2, and double knockouts of CLIC1, ClC3, KCC3, TRPM2, and KCNQ1 with HVCN1, and humans with channelopathies involving BEST1, ClC7, CFTR, and MCOLN1. No gross abnormalities in vacuolar pH or area were found in any of these cells suggesting that we had not tested the correct channel, or that there is redundancy in the system. The respiratory burst was suppressed in the KCC3-/- and enhanced in the CLIC1-/- cells, but was normal in all others, including ClC3-/-. These results suggest charge compensation by a chloride conductance out of the vacuole and by cation/s into it. The identity of these channels remains to be established.

The influence of oral cimetidine administration on creatinine clearance in chilren with chronic renal failure: A preliminary study

Background Serum creatinine and creatinine clearance are usedto assess glomerular filtration rate but have a major disadvantagesince a variable amount of creatinine is secreted in the proximaltubule. This may cause an unpredictable overestimation of GFR.Tubular creatinine secretion can be blocked by cimetidine throughcompetitive inhibition of cation transport in the proximal tubularluminal membrane.Objective Cimetidine administration might improve the reliabilityof creatinine as a marker of glomerular filtration.Methods A preliminary study with a one-group pretest-posttestdesign in 11 children with chronic renal failure. Serum cystatin Clevel as reference value was compared with creatinine clearancemeasured before and after oral ingestion of cimetidine. The doseof cimetidine was adjusted with the GFR using Schwartz formula.Statistical evaluation was done with the Wilcoxon signed rankstest.Result The mean creatinine clearance before cimetidine adminis-tration was 27.4 (SD 14.6) ml/minute/1.73 m 2 BSA, and decreasedafter cimetidine to 21.1 (SD 13,1) ml/minute/1.73 m 2 BSA (p=0.015).Conclusion Oral cimetidine was effective in inhibiting creatininetubular secretion. This study could not prove that cimetidine im-proves the accuracy of creatinine clearance

Transport of six tyrosine kinase inhibitors: active or passive?

&lt;p class="ADMETabstracttext"&gt;Transport of erlotinib, gefitinib, sorafenib, sunitinib, dasatinib and crizotinib can be active or passive, which was studied by measuring uptake at low (4 °C; passive) and normal temperature (37 °C; active and passive) and by the use of specific organic cation transporter (OCT) inhibitors. Intracellular accumulation was determined using Caco-2 as monolayers, while for gut permeation we used differentiated Caco-2 as model for intestinal epithelium in the Transwell system. Sorafenib and crizotinib uptake are likely to be dependent on passive transport. Gefitinib, dasatinib and sunitinib uptake seem to be active. Erlotinib’s transport also seems to be active. This study suggests that hOCTs might be involved in the apical to basolateral transport of gefitinib and crizotinib. Overall it can be concluded that the accumulation and transport of these six TKIs are very different, despite the fact that they are all tyrosine kinase inhibitors.&lt;/p&gt;

Cynomolgus Monkey as a Clinically Relevant Model to Study Transport Involving Renal Organic Cation Transporters: In Vitro and In Vivo Evaluation.

Organic cation transporter (OCT) 2, multidrug and toxin extrusion protein (MATE) 1, and MATE2K mediate the renal secretion of various cationic drugs and can serve as the loci of drug-drug interactions (DDI). To support the evaluation of cynomolgus monkey as a surrogate model for studying human organic cation transporters, monkey genes were cloned and shown to have a high degree of amino acid sequence identity versus their human counterparts (93.7, 94.7, and 95.4% for OCT2, MATE1, and MATE2K, respectively). Subsequently, the three transporters were individually stably expressed in human embryonic kidney (HEK) 293 cells and their properties (substrate selectivity, time course, pH dependence, and kinetics) were found to be comparable to the corresponding human form. For example, six known human cation transporter inhibitors, including pyrimethamine (PYR), showed generally similar IC50 values against the monkey transporters (within sixfold). Consistent with the in vitro inhibition of metformin (MFM) transport by PYR (IC50 for cynomolgus OCT2, MATE1, and MATE2K; 1.2 ± 0.38, 0.17 ± 0.04, and 0.25 ± 0.04 µM, respectively), intravenous pretreatment of monkeys with PYR (0.5 mg/kg) decreased the clearance (54 ± 9%) and increased in the area under the plasma concentration-time curve of MFM (AUC ratio versus control = 2.23; 90% confidence interval of 1.57 to 3.17). These findings suggest that the cynomolgus monkey may have some utility in support of in vitro-in vivo extrapolations (IVIVEs) involving the inhibition of renal OCT2 and MATEs. In turn, cynomolgus monkey-enabled IVIVEs may inform human DDI risk assessment.

High Risk of Embryo-Fetal Toxicity: Placental Transfer of T-2 Toxin and Its Major Metabolite HT-2 Toxin in BeWo Cells

Though T-2 toxin is the most harmful mycotoxin to the fetuses, it remains unclear whether T-2 toxin and its major metabolite, HT-2 toxin, could pass the placenta into the fetus and which kind of placental transport is involved in the passage. To illustrate their placenta transfer mechanism, the uptake and efflux of T-2 and HT-2 toxins across apical membranes of placenta with BeWo cells as a model were studied at different temperatures, pHs, and in the presence of transporter inhibitors with a developed liquid chromatography-tandem mass spectrometry to determine the amount of toxins in both fetal and maternal sites. Higher unidirectional transport of T-2 toxin was observed in the apical-to-basolateral direction than basolateral-to-apical one, whereas HT-2 toxin exhibited similar transport rate from the 2 directions. The main ATP-binding cassette transporters had no effect on the efflux of 2 toxins. Initial uptake of T-2 toxin was sodium dependent and saturable, and the apical uptake was temperature dependent and enhanced under acidic condition. The apical uptake of T-2 toxin was inhibited by metabolic inhibitors and the organic anion and organic cation transporter inhibitors. These results suggested that an active transport mechanism was responsible for the uptake of T-2 toxin, whereas passive diffusion was the principal mechanism for HT-2 toxin transport in the placenta. Taken together, these data characterized the placental transfer of T-2 and HT-2 toxins. The present study offered new ways of reducing the risks of T-2 and HT-2 toxins to both mother and fetuses.

Organic Cation Transporters in Human Nasal Primary Culture: Expression and Functional Activity

The majority of drugs cross epithelial cells by either passive diffusion or via carrier-mediated drug transporters. The aim of this study was to investigate the transport characteristics, protein expression and localization of organic cation transporters in human nasal epithelium. The expression, localization and transport characteristics of the transporters were investigated using permeation, PCR and immunohistochemistry. The uptake of 4-(4-(dimethylamino)styryl)-N-methylpyridinium iodide followed Michaelis-Menten kinetics. Its intracellular accumulation of the compound was inhibited by organic cation transporters (OCTs) and carnitine/organic cation transporter (OCTNs) inhibitors. Detected OCT1-3, OCTN1 and OCTN2 gene transcripts correlated with immunohistological staining for OCT1-3, OCTN1 and OCTN2 antibodies. Except for OCTN1, the antibodies were generally localized on the apical side of the epithelial cells. Based on the immunohistochemical and uptake/transport studies, we conclude that the human nasal epithelium expresses OCT1-3, OCTN1 and OCTN2 transporters mainly on the apical side of the nasal cells.

New mechanistic explanation for the localization of ulcers in the rat duodenum: Role of iron and selective uptake of cysteamine

Cysteamine, a coenzyme A metabolite, induces duodenal ulcers in rodents. Our recent studies showed that ulcer formation was aggravated by iron overload and diminished in iron deficiency. We hypothesized that cysteamine is selectively taken up in the duodenal mucosa, where iron absorption primarily occurs, and is transported by a carrier-mediated process. Here we report that cysteamine administration in rats leads to cysteamine accumulation in the proximal duodenum, where the highest concentration of iron in the gastrointestinal tract is found. In vitro, iron loading of intestinal epithelial cells (IEC-6) accelerated reactive oxygen species (ROS) production and increased [14C]cysteamine uptake. [14C]Cysteamine uptake by isolated gastrointestinal mucosal cells and by IEC-6 was pH-dependent and inhibited by unlabeled cysteamine. The uptake of [14C]cysteamine by IEC-6 was Na+-independent, saturable, inhibited by structural analogs, H2-histamine receptor antagonists, and organic cation transporter (OCT) inhibitors. OCT1 mRNA was markedly expressed in the rat duodenum and in IEC-6, and transfection of IEC-6 with OCT1 siRNA decreased OCT1 mRNA expression and inhibited [14C]cysteamine uptake. Cysteamine-induced duodenal ulcers were decreased in OCT1/2 knockout mice. These studies provide new insights into the mechanism of cysteamine absorption and demonstrate that intracellular iron plays a critical role in cysteamine uptake and in experimental duodenal ulcerogenesis.

Src Family Kinase Inhibitor Saracatinib (AZD0530) Impairs Oxaliplatin Uptake in Colorectal Cancer Cells and Blocks Organic Cation Transporters

Elevated Src family kinase (SFK) activity is associated with tumor invasion and metastasis. The SFK inhibitor saracatinib (AZD0530) is currently in phase II trials in patients including those with colorectal cancer (CRC), where links between SFK activity and poor prognosis are particularly striking. Saracatinib is likely to be used clinically in combination regimens, specifically with 5-fluorouracil (5-FU) and oxaliplatin, in CRC. The aim of this study was to determine the effect of saracatinib on oxaliplatin and 5-FU efficacy in CRC cells. Saracatinib did not modulate 5-FU efficacy but antagonized oxaliplatin in a schedule-specific manner through reduced oxaliplatin uptake via an SFK-independent mechanism. Saracatinib resembles the pharmacophore of known organic cation transporter (OCT) inhibitors and reduced oxaliplatin efficacy maximally in cells overexpressing OCT2. These data suggest that oxaliplatin uptake in CRC is attenuated by saracatinib via inhibition of OCT2, a potential consideration for the clinical development of this SFK inhibitor.

No pharmacokinetic interaction between paliperidone extended‐release tablets and trimethoprim in healthy subjects

The effect of trimethoprim, a potent organic cation transport inhibitor, on the pharmacokinetics (PK) of paliperidone extended-release tablets (paliperidone ER), an organic cation mainly eliminated via renal excretion, was assessed. Open-label, two-period, randomized, crossover study in 30 healthy males. Single dose of paliperidone ER 6 mg was administered either alone on day 1 or day 5 during an 8-day treatment period of trimethoprim 200 mg twice daily. Serial blood and urine samples were collected for PK and plasma protein binding of paliperidone and its enantiomers. The 90% confidence interval (CI) of ratios with/without trimethoprim for PK parameters of paliperidone and its enantiomers calculated. Creatinine clearance decreased from 119 to 102 mL min(-1) with trimethoprim. Addition of trimethoprim increased unbound fraction of paliperidone by 16%, renal clearance by 13%, AUC(infinity) by 9%, and t((1/2)) by 19%. The 90% CIs for ratios with/without trimethoprim were within the 80-125% range for C(max), AUC(last), and renal clearance. For AUC(infinity), 90% CI was 79.37-101.51, marginally below the lower bound of the acceptance range. Paliperidone did not affect steady-state plasma concentrations of trimethoprim. No clinically important drug interactions are expected when paliperidone ER is administered with organic cation transport inhibitors.

Involvement of Human Organic Cation Transporter 1 in the Hepatic Uptake of 1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium Bromide (YM155 Monobromide), a Novel, Small Molecule Survivin Suppressant

1-(2-Methoxyethyl)-2-methyl-4,9-dioxo-3-(pyrazin-2-ylmethyl)-4,9-dihydro-1H-naphtho[2,3-d]imidazolium bromide (YM155 monobromide), which is a hydrophilic and cationic compound, exhibits antitumor activity in experimental human hormone refractory prostate carcinoma models. Urinary excretion was 18.3 to 28.6% of the dose in the clinical phase I study, and nonrenal elimination may be explained by the biliary excretion of YM155 in its unchanged form. Because the penetration through the sinusoidal membrane of the hepatocytes is the first step and an important part of biliary excretion, we evaluated the uptake of [(14)C]YM155 into human cryopreserved hepatocytes. YM155 was taken up into hepatocytes in a temperature- and concentration-dependent manner. The saturable uptake component was much higher than the nonsaturable passive diffusion component. In vitro hepatic uptake clearance was consistent with the in vivo hepatic intrinsic clearance calculated using clinical study data. Hepatic uptake of YM155 was inhibited by organic cation transporter (OCT) inhibitors, and the IC(50) values for YM155 uptake were comparable to those reported for human OCT1-mediated transport. The interaction of YM155 with candidate transporter, OCT1, was also characterized using S2 cells stably expressing human OCT1 (OCT1-S2) cells. In OCT1-expressing S2 cells, YM155 inhibited the OCT1-mediated uptake of a typical OCT1 substrate, [(14)C]tetraethylammonium. In addition, YM155 was taken up into OCT1-S2 cells These results indicated that OCT1 was the predominant transporter for the hepatic uptake of YM155, and the transporter-mediated uptake clearance observed in vitro may account for the in vivo intrinsic hepatic clearance.

Renal excretion of emtricitabine I: Effects of organic anion, organic cation, and nucleoside transport inhibitors on emtricitabine excretion

The excretion of emtricitabine (FTC) was characterized using isolated perfused rat kidney (IPK) model. Studies were performed to assess the dose‐linearity of FTC excretion, to evaluate the effect of inhibitors of organic anion (probenecid, PBC), organic cation (tetraethylammonium, TEA; cimetidine, CMD) and nucleoside (uridine, URD) transport systems on FTC excretion, and to determine the potential interaction between FTC and trimethoprim (TMP). FTC excretion was studied over a range of doses (80–1600 µg), targeting concentrations encompassing the therapeutic range of FTC (1–20 µg/mL). FTC (2 µg/mL) was also coperfused with PBC (500 µM), TEA (500 µM), CMD (2 mM), URD (500 µM), and TMP (13.7 µM). FTC dose‐linearity studies revealed that excretion parameters were not significantly different among dosing groups. Of the transport inhibitors tested, FTC XR decreased more than twofold in the presence of CMD (0.32 ± 0.099). PBC, TEA, and URD had no observed effect on FTC excretion. TMP coadministration significantly inhibited FTC excretion (XR = 0.43 ± 0.052). The results suggest that FTC renal transport is likely mediated by a CMD‐sensitive organic cation transporter (OCT) in the kidney. TMP may inhibit the renal excretion of FTC when the two compounds are coadministered in vivo. © 2008 Wiley‐Liss, Inc. and the American Pharmacists Association J Pharm Sci 97:5401–5410, 2008

Effects of dietary flavonoids on the transport of cimetidine via P-glycoprotein and cationic transporters in Caco-2 and LLC-PK1 cell models

1. The hypotheses tested were to study cimetidine as a substrate of P-glycoprotein (P-gp) and organic cation transport systems and the modulatory effects of eight flavonoid aglycones and glycosides on these transport systems using Caco-2 and LLC-PK1 cells.2. Transport and uptake experiments of (20 µM) 3H-cimetidine were performed with and without co-exposure to quercetin, quercetrin, rutin, naringenin, naringin, genistein, genistin, and xanthohumol. Co-treatment decreased basolateral to apical (B to A) permeability (Papp) of cimetidine from 2.02 to 1.24 (quercetin), 1.06 (naringenin), 1.24 (genistein), and 0.96 (xanthohumol) × 10−6 cm s−1 in Caco-2 cells and from 10.76 to 1.65 (quercetin), 2.05 (naringenin), 2.88 (genistein), and 1.95 (xanthohumol) × 10−6 cm s−1 in LLC-PK1 cells. Genistin significantly reduced B to A Papp of cimetidine to 1.24 × 10−6 cm s−1 in Caco-2 cells. Basolateral intracellular uptake rate of cimetidine was enhanced 145–295% when co-treated with flavonoids. Co-treatment with P-glycoprotein and organic cation transporter inhibitors, verapamil and phenoxybenzamine, resulted in reduced B to A permeability and slower basolateral intracellular uptake rate of cimetidine. Intracellular uptake rate of 14C-tetraethylammonium (TEA) was reduced in the presence of quercetin, naringenin and genistein in LLC-PK1 cells.3. In conclusion, quercetin, naringenin, genistein, and xanthohumol reduced P-gp-mediated transport and increased the basolateral uptake rate of cimetidine. Quercetin, naringenin, genistein, but not xanthohumol, reduced intracellular uptake rate of TEA in LLC-PK1 cells. These results suggest that flavonoids may have potential to alter the disposition profile of cimetidine and possibly other therapeutics that are mediated by P-gp and/or cation transport systems.

Mechanism of Cellular Uptake of a Ruthenium Polypyridyl Complex

Transition metal complexes provide a promising avenue for the design of therapeutic and diagnostic agents, but the limited understanding of their cellular uptake is a roadblock to their effective application. Here, we examine the mechanism of cellular entry of a luminescent ruthenium(II) polypyridyl complex, Ru(DIP) 2dppz (2+) (where DIP = 4,7-diphenyl-1,10-phenanthroline and dppz = dipyridophenazine), into HeLa cells, with the extent of uptake measured by flow cytometry. No diminution of cellular uptake is observed under metabolic inhibition with deoxyglucose and oligomycin, indicating an energy-independent mode of entry. The presence of organic cation transporter inhibitors also does not significantly alter uptake. However, the cellular internalization of Ru(DIP) 2dppz (2+) is sensitive to the membrane potential. Uptake decreases when cells are depolarized with high potassium buffer and increases when cells are hyperpolarized with valinomycin. These results support passive diffusion of Ru(DIP) 2dppz (2+) into the cell.