Assessment of Substrate-Dependent Ligand Interactions at the Organic Cation Transporter OCT2 Using Six Model Substrates.

Organic cation transporter (OCT) 2 mediates the entry step for organic cation secretion by renal proximal tubule cells and is a site of unwanted drug-drug interactions (DDIs). But reliance on decision tree-based predictions of DDIs at OCT2 that depend on IC50 values can be suspect because they can be influenced by choice of transported substrate; for example, IC50 values for the inhibition of metformin versus MPP transport can vary by 5- to 10-fold. However, it is not clear whether the substrate dependence of a ligand interaction is common among OCT2 substrates. To address this question, we screened the inhibitory effectiveness of 20 µM concentrations of several hundred compounds against OCT2-mediated uptake of six structurally distinct substrates: MPP, metformin, N,N,N-trimethyl-2-[methyl(7-nitrobenzo[c][1,2,5]oxadiazol-4-yl)amino]ethanaminium (NBD-MTMA), TEA, cimetidine, and 4-4-dimethylaminostyryl-N-methylpyridinium (ASP). Of these, MPP transport was least sensitive to inhibition. IC50 values for 20 structurally diverse compounds confirmed this profile, with IC50 values for MPP averaging 6-fold larger than those for the other substrates. Bayesian machine-learning models of ligand-induced inhibition displayed generally good statistics after cross-validation and external testing. Applying our ASP model to a previously published large-scale screening study for inhibition of OCT2-mediated ASP transport resulted in comparable statistics, with approximately 75% of "active" inhibitors predicted correctly. The differential sensitivity of MPP transport to inhibition suggests that multiple ligands can interact simultaneously with OCT2 and supports the recommendation that MPP not be used as a test substrate for OCT2 screening. Instead, metformin appears to be a comparatively representative OCT2 substrate for both in vitro and in vivo (clinical) use.

The Effect of H2 Receptor Antagonist in Acid Inhibition and Its Clinical Efficacy.

The first histamine H2 receptor antagonists (H2RAs) were developed in the early 1970s. They played a dominant role in treating peptic ulcer disease and gastroesophageal reflux disease (GERD). H2RAs block the production of acid by H+, K+-ATPase at the parietal cells and produce gastric luminal anacidity for varying periods. H2RAs are highly selective, and they do not affect H1 receptors. Moreover, they are not anticholinergic agents. Sequential development of H2RAs, proton pump inhibitors (PPIs), and discovery of Helicobacter pylori infection changed the paradigm of peptic ulcer disease with marked decrease of morbidity and mortality. PPIs are known to be the most effective drugs that are currently available for suppressing gastric acid secretion. Many studies have shown its superiority over H2RAs as a treatment for acid-related disorders, such as peptic ulcer disease, GERD, and Zollinger-Ellison syndrome. However, other studies have reported that PPIs may not be able to render stomach achlorhydric and have identified a phenomenon of increasing gastric acidity at night in individuals receiving a PPI twice daily. These nocturnal acid breakthrough episodes can be eliminated with an addition of H2RAs at night. The effectiveness of nighttime dose of H2RA suggests a major role of histamine in nocturnal acid secretion. H2RAs reduce secretion of gastric acid, and each H2RA also has specific effects. For instance, nizitidine alleviates not only symptoms of GERD, but also provokes gastric emptying, resulting in clinical symptom improvement of functional dyspepsia. The aim of this paper was to review the characteristics and role of H2RAs and assess the future strategy and treatment of upper gastrointestinal disease, including acid related disorders.

Design of PEG-grafted-PLA nanoparticles as oral permeability enhancer for P-gp substrate drug model Famotidine.

Bioavailability of oral drugs can be limited by an intestinal excretion process mediated by P-glycoprotein (P-gp). Polyethylene glycol (PEG) is a known P-gp inhibitor. Dispersion of Famotidine (a P-gp substrate) within PEGylated nanoparticles (NPs) was used to improve its oral bioavailability. In this work, we evaluated the potential impact of NPs prepared from a grafted copolymer of polylactic acid and PEG on P-gp function by studying in vitro permeability of Famotidine across Caco-2 cells. Copolymers of PEG grafted on polylactic acid (PLA) backbone (PLA-g-PEG) were synthesised with 1 mol% and 5 mol% PEG vs. lactic acid monomer using PEG 750 and 2000 Da. The polymers were used to prepare Famotidine-loaded NPs and tested in vitro on Caco-2 cells. Significant decrease in basolateral-to-apical transport of Famotidine was observed when Famotidine was encapsulated in NPs prepared from PLA-g-PEG5%. NPs prepared from PLA-g-PEG5% are promising to improve oral bioavailability of P-gp substrates.

Pharmacologic Issues in Liver Disease.

This article describes key concepts for drug dosing considerations in liver disease. Included in this article is a review of pharmacokinetic changes that are known to occur in patients with liver disease that can aide in drug dosing. Although limited, the currently available pharmacokinetic data for medications that are commonly used in the critical care setting are also included.

Indication of acid suppression therapy and predictors for the prophylactic use of protonpump inhibitors vs. histamine-2 receptor antagonists in a Malaysian tertiary hospital / Indicación del tratamiento de supresión ácida y predictors del uso profiláctico de inhibidores de la bomba de protones vs. Antagonista de receptores de histamina-2 en un hospital terciario malayo

Background: Proton-pump inhibitors (PPI) and histamine-2 receptor antagonists (H2RA) are common acid suppressants used in gastrointestinal disorders. The trend of usage in Malaysia has changed from predominantly H2RA to PPI from 2007 to 2008, 3.46 versus 2.87 and 2.99 versus 3.24 DDD (Defined Daily Dose)/1000 population/day respectively. This raises concerns as PPI overutilization amounts to higher cost expenditure and are associated with various untoward consequences such as Clostridium difficile-associated diarrhea, pneumonia, and osteoporosis. Objectives: To evaluate the indication of acid suppression therapy (AST) and to look for predictors associated with the prophylactic use of PPI as compared to H2RA. Methods: Data collection was conducted via a standardized surveillance form over a 2-month period in the general medical wards of Sarawak General Hospital. All patients who received at least one dose of PPI or H2RA in any dosage form were included in the study. Appropriateness of prophylaxis was determined using current available guidelines. Selected risk factors were analysed using simple logistic regression to look for predictors associated with the choice of PPI in prophylactic AST. Results: Out of 212 cases in the present cohort, about three quarters (75.5%, n=160) of acid suppressants were given as prophylaxis. Over half of these did not have appropriate indications for prophylactic AST (58.1%, n=93). Among all cases given prophylactic AST, 75.0% (n=120) of them were given PPI. Renal insufficiency was identified as the only predictor associated with the use of prophylactic PPI in preference to H2RA (OR=2.86, 95%CI 1.21:6.72, p=0.011). Conclusion: Inappropriate prophylactic AST is a major concern and may even be underestimated due to the lack of appropriate guidelines. More data is required to guide the selection between PPI and H2RA, specifically the more cost-effective use of H2RA in patients with lower gastrointestinal risk or in whom PPI has no clear advantage (AU)

Antecedentes: Los inhibidores de la bomba de protones (PPI) y los antagonistas de los receptores de histamina-2 (H2RA) son antiácidos comunes que se usan en desordenes gastrointestinales. La tendencia de uso en Malasia ha cambiado de la predominancia de H2RA a PPI entre 2007 y 2008 de 3,46 vs. 2,87 y 2,99 vs. 3,24 DDD (Dosis Diarias Definida)/1000 habitantes/día, respectivamente. Esto preocupa ya que la sobreutilización de PPI produce mayores costes y están asociados a varias consecuencias indeseadas como diarrea asociada a Clostridium difficile, neumonía, y osteoporosis. Objetivos: Evaluar la indicación del tratamiento de supresión ácida (AST) y buscar predictores asociados con el uso de PPI comparados con H2RA. Métodos: Se recogieron datos mediante un formulario de vigilancia estandarizado durante 2 meses en los servicios de medicina heneral del Hospital General de Sarawak. Todos los pacientes que recibieron, al menos una dosis de PPI o de H2RA en cualquier forma fueron incluidos en el estudio. La adecuación del tratamiento fue determinada usando las guías disponibles actualmente. Se analizaron los factores de riesgo usando una regresión logística simple para buscar predictores asociados con la elección de PPI en AST profiláctica. Resultados: De los 200 casos presentes en la cohorte, cerca de tres cuartos (75,5; n=160) de los antiácidos fueron usado como profilácticos. Más de la mitad de ellos no tenían una indicación apropiada para AST profiláctica (58,1%, n=93). Entre todos los casos con AST profiláctica, el 75,0% (n=120) eran PPI. La insuficiencia renal fue el factor asociado con el uso de profilaxis con PPI en preferencia a los H2RA (OR=2,86; 95%CI 1,21:6,72, p=0,011). Conclusión: La AST profiláctica inadecuada es una gran problema y podría estar subestimada debido a la ausencia de guías apropiadas. Se requieren más datos para guiar la selección entre PPI y H2RA, específicamente el uso más coste-efectivo de los H2RA en pacientes con riesgo gastrointestinal bajo, o en los que no hay una clara ventaja de los PPI (AU)

Effects of antihistamines on the function of human α7-nicotinic acetylcholine receptors.

Effects of the histamine H1 receptor (H1R) antagonists (antihistamines), promethazine (PMZ), orphenadrine (ORP), chlorpheniramine (CLP), pyrilamine (PYR), diphenhydramine (DPH), citerizine (CTZ), and triprolidine (TRP) on the functional properties of the cloned α7 subunit of the human nicotinic acetylcholine receptor expressed in Xenopus oocytes were investigated. Antihistamines inhibited the α7-nicotinic acetylcholine receptor in the order PYR>CLP>TRP>PMZ>ORP≥DPH≥CTZ. Among the antihistamines, PYR showed the highest reversible inhibition of acetylcholine (100 µM)-induced responses with IC50 of 6.2 µM. PYR-induced inhibition was independent of the membrane potential and could not be reversed by increasing the concentration of acetylcholine. Specific binding of [¹²5I] α-bungarotoxin, a selective antagonist for α7-nicotinic acetylcholine receptor, was not changed in the presence of PYR suggesting a non-competitive inhibition of nicotinic receptors. In line with functional experiments, docking studies indicated that PYR can potentially bind allosterically with the α7 transmembrane domain. Our results indicate that the H2-H4 receptor antagonists tested in this study (10 µM) showed negligible inhibition of α7-nicotinic acetylcholine receptors. On the other hand, H1 receptor antagonists inhibited the function of human α7-nicotinic acetylcholine receptor, with varying potencies. These results emphasize the importance of α7-nicotinic acetylcholine receptor for future pharmacological/toxicological profiling.

Signal transduction mechanism of biased ligands at histamine H2 receptors.

7TMRs (seven-transmembrane receptors) exist as conformational collections in which different conformations would lead to differential downstream behaviours such as receptor phosphorylation, G-protein activation and receptor internalization. In this context, a ligand may cause differential activation of some, but not all, of the signalling events, which are associated to a particular receptor, and it would lead to biased agonism. The aim of the present study was to investigate whether H2R (histamine H2 receptor) ligands, described as inverse agonists because of their negative efficacy at modulating adenylate cyclase, could display some positive efficacy concerning receptor desensitization, internalization or even signalling through an adenylate-cyclase-independent pathway. Our present findings indicate that treatment with H2R inverse agonists leads to receptor internalization in HEK (human embryonic kidney)-293T transfected cells, by a mechanism mediated by arrestin and dynamin, but independent of GRK2 (G-protein-coupled receptor kinase 2)-mediated phosphorylation. On the other hand, we prove that two of the H2R inverse agonists tested, ranitidine and tiotidine, also induce receptor desensitization. Finally, we show that these ligands are able to display positive efficacy towards the ERK1/2 (extracellular-signal-regulated kinase 1/2) pathway by a mechanism that involves Gßγ and PI3K (phosphoinositide 3-kinase)-mediated signalling in both transfected HEK-293T cells and human gastric adenocarcinoma cells. These results point to the aspect of pluridimensional efficacy at H2R as a phenomenon that could be extended to naïve cells, and challenge previous classification of pharmacologically relevant histaminergic ligands.

Optimization and evaluation of gastroretentive ranitidine HCl microspheres by using design expert software.

The current study involves the development and optimization of their drug entrapment and ex vivo bioadhesion of multiunit chitosan based floating system containing Ranitidine HCl by ionotropic gelation method for gastroretentive delivery. Chitosan being cationic, non-toxic, biocompatible, biodegradable and bioadhesive is frequently used as a material for drug delivery systems and used to transport a drug to an acidic environment where it enhances the transport of polar drugs across epithelial surfaces. The effect of various process variables like drug polymer ratio, concentration of sodium tripolyphosphate and stirring speed on various physiochemical properties like drug entrapment efficiency, particle size and bioadhesion was optimized using central composite design and analyzed using response surface methodology. The observed responses were coincided well with the predicted values given by the optimization technique. The optimized microspheres showed drug entrapment efficiency of 74.73%, particle size 707.26 µm and bioadhesion 71.68% in simulated gastric fluid (pH 1.2) after 8 h with floating lag time 40s. The average size of all the dried microspheres ranged from 608.24 to 720.80 µm. The drug entrapment efficiency of microspheres ranged from 41.67% to 87.58% and bioadhesion ranged from 62% to 86%. Accelerated stability study was performed on optimized formulation as per ICH guidelines and no significant change was found in drug content on storage.

Homology modeling and antagonist binding site study of the human histamine H2 receptor.

On the basis of the high resolution crystal structures of bovine rhodopsin, human beta2-adrenergic receptor and human A(2a) adenosine receptor, three-dimensional structure of the human histamine H2 receptor (HHR2) was developed by homology modeling. Results of the evaluations suggest that a high quality homology model for HHR2 has been obtained. Three antagonists, cimetidine, ranitidine and nizatidine, were applied to binding site study with this model through molecular docking, molecular dynamics simulations and the molecular mechanics-Poisson-Boltzmann surface area calculations. One aspartic acid, Asp98 in transmembrane domain 7 (TM3), has been identified as major contributors to ligand binding by H-bond interactions. Asn159 in TM4 and Asp186 in TM5 are of great importance in stabilizing HHR2- antagonist complexes. Two hydrophobic sites especially two residues, Val99 in TM3 and Phe254 in TM6, were identified to be essential for their strong hydrophobic interactions with antagonists. The findings reported here are in agreement with available experimental mutagenesis data. This study should be very helpful for a better understanding of the action mode of the antagonist and for guiding further drug design and mutagenesis studies.

Properties of gastroretentive sustained release tablets prepared by combination of melt/sublimation actions of L-menthol and penetration of molten polymers into tablets.

A novel floating sustained release tablet having a cavity in the center was developed by utilizing the physicochemical properties of L-menthol and the penetration of molten hydrophobic polymer into tablets. A dry-coated tablet containing famotidine as a model drug in outer layer was prepared with a L-menthol core by direct compression. The tablet was placed in an oven at 80°C to remove the L-menthol core from tablet. The resulting tablet was then immersed in the molten hydrophobic polymers at 90°C. The buoyancy and drug release properties of tablets were investigated using United States Pharmacopeia (USP) 32 Apparatus 2 (paddle 100 rpm) and 900 ml of 0.01 N HCl. The L-menthol core in tablets disappeared completely through pathways in the outer layer with no drug outflows when placed in an oven for 90 min, resulting in a formation of a hollow tablet. The hollow tablets floated on the dissolution media for a short time and the drug release was rapid due to the disintegration of tablet. When the hollow tablets were immersed in molten hydrophobic polymers for 1 min, the rapid drug release was drastically retarded due to a formation of wax matrices within the shell of tablets and the tablets floated on the media for at least 6 h. When Lubri wax® was used as a polymer, the tablets showed the slowest sustained release. On the other hand, faster sustained release properties were obtained by using glyceryl monostearate (GMS) due to its low hydrophobic nature. The results obtained in this study suggested that the drug release rate from floating tablets could be controlled by both the choice of hydrophobic polymer and the combined use of hydrophobic polymers.

Computational investigation of interactions between human H2 receptor and its agonists.

Type 2 histamine receptor (H(2)R) is widely distributed in the body. Its main function is modulating the secretion of gastric acid. Most gastric acid-related diseases are closely associated with it. In this study, a combination of pharmacophore modeling, homology modeling, molecular docking and molecular dynamics methods were performed on human H(2)R and its agonists to investigate interaction details between them. At first, a pharmacophore model of H(2)R agonists was developed, which was then validated by QSAR and database searching. Afterwards, a model of the H(2)R was built utilizing homology modeling method. Then, a reference agonist was docked into the receptor model by induced fit docking. The 'induced' model can dramatically improve the recovery ratio from 46.8% to 69.5% among top 10% of the ranked database in the simulated virtual screening. The pharmocophore model and the receptor model matched very well each other, which provided valuable information for future studies. Asp98, Asp186 and Tyr190 played key roles in the binding of H(2)R agonists, and direct interactions were observed between the three residues and agonists. Residue Tyr250 could also form a hydrogen bond with H(2)R agonists. These findings would be very useful for the discovery of novel and potent H(2)R agonists.

Should the concomitant use of erlotinib and acid-reducing agents be avoided? The drug interaction between erlotinib and acid-reducing agents.

CONTEXT: Erlotinib, an epidermal growth factor receptor tyrosine kinase inhibitor, exhibits a drug interaction with proton pump inhibitors (PPIs) and histamine-2 receptor antagonists (H2RAs). The manufacturer recommends avoidance of the combination however, the extent of the drug interaction is not clearly understood. Evidence acquisition. A literature search was performed and the pharmacokinetics and pharmacology of acid-reducing agents were reviewed. RESULTS: Acid-reducing agents reduce the solubility, and subsequent absorption, of erlotinib by raising gastric pH. Our literature search was unable to identify any published studies or case reports that address this issue. Until more information is available, the clinical relevance of this interaction, and whether it actually leads to failure of therapy, is unknown. PPIs would all be expected to exhibit a similar effect on erlotinib. Of the H2RAs, co-administration appears to have a greater impact than administering them separately. Ranitidine, famotidine, and nizatidine should likely have similar effects on erlotinib absorption. Cimetidine has a shorter duration of action, but should be used with caution because of its effects on cytochrome P450 3A4, a pathway also utilized by erlotinib. Antacids are not expected to have a significant effect. CONCLUSIONS: The clinical relevance of this drug interaction is unknown. Until more information is available, decision making regarding this interaction should be on a patient-by-patient basis. The indication for acid-reducing therapy should be reevaluated and stopping therapy or changing therapy can be considered. However, there may be occasions where any benefit of such actions will be exceeded by its risks.

Increased use of acid-suppressing drugs before the occurrence of ischemic events: a potential source of confounding in recent observational studies.

STUDY OBJECTIVE: To determine if the use of acid-suppressing drugs is increased before the occurrence of ischemic events. DESIGN: Population-based, nested case-control analysis. DATA SOURCE: Administrative databases in Saskatchewan, Canada. PATIENTS: Cases were 1612 patients (aged ≥ 40 yrs) who started a first-ever antihypertensive drug between January 1, 1994, and December 31, 2003, and were hospitalized for a first ischemic heart event of either myocardial infarction (1002 patients) or unstable angina (610 patients); five control patients were matched to each case patient by age, sex, and year of first antihypertensive prescription (8060 controls). MEASUREMENTS AND MAIN RESULTS: Within the case and control groups, we calculated exposure to acid-suppressing therapy, defined as proton pump inhibitors (PPIs) or histamine(2)-receptor antagonists (H(2)RAs), within 90 days leading up to the event. Exposure to acid-suppressing therapy was higher among cases than controls (15.3% [246/1612] vs 10.4% [837/8060], adjusted odds ratio [AOR] 1.26, 95% confidence interval [CI] 1.06-1.49, p<0.009). Exposure to each acid suppressant was similarly higher among cases than controls: H(2)RA users (11.7% [188/1612] vs 8.4% [678/8060], AOR 1.21, 95% CI 1.00-1.46, p<0.048) and PPI users (4.0% [64/1612] vs 2.2% [179/8060], AOR 1.32, 95% CI 0.95-1.84, p=0.094). Use of other drugs was also significantly increased during this period. CONCLUSIONS: Use of acid-suppressing drugs increased before the occurrence of ischemic events regardless of the type (PPI or H(2)RA) or whether other drugs, such as clopidogrel, were concurrently administered. In addition, significant increases in overall drug use were observed during this time frame, suggesting that many patients exhibit warning signs before an acute hospitalization. Thus, PPI use before the occurrence of ischemic events may simply be a marker of unmeasured and uncontrolled confounding in observational studies that have implicated a PPI-clopidogrel interaction as a cause of recurrent ischemic events.

Marked increase in proton pump inhibitors use in Australia.

OBJECTIVES: To examine the trends in the prescribing of subsidized proton pump inhibitors (PPIs) and histamine receptor antagonists (H2RAs), in the Australian population from 1995 to 2006 to encourage discussion regarding appropriate clinical use. PPIs and H2RAs are the second highest drug cost to the publicly subsidized Pharmaceutical Benefits Scheme (PBS). DESIGN: Government data on numbers of subsidized scripts, quantity and doses for PPIs and H2RAs were analysed by gender and age, dose and indication. MAIN OUTCOME MEASURE: Drug utilisation as DDD [defined daily dose]/1000 population/day. RESULTS: The use of combined PPIs increased by 1318%. Utilisation increased substantially after the relaxation of the subsidized indications for PPIs in 2001. Omeprazole had the largest market share but was substituted by its S-enantiomer esomeprazole after its introduction in 2002. There was considerable use in the elderly with the peak use being in those aged 80 years and over. The utilisation of H2RAs declined 72% over 12 years. CONCLUSIONS: PPI use has increased substantially, not only due to substitution of H2RAs but to expansion in the overall market. Utilisation does not appear to be commensurate with prevalence of gastro-oesophageal reflux disease (GORD) nor with prescribing guidelines for PPIs, with significant financial costs to patients and PBS. This study encourages clinical discussion regarding quality use of these medicines.

Effects of betahistine at histamine H3 receptors: mixed inverse agonism/agonism in vitro and partial inverse agonism in vivo.

We previously suggested that therapeutic effects of betahistine in vestibular disorders result from its antagonist properties at histamine H(3) receptors (H(3)Rs). However, H(3)Rs exhibit constitutive activity, and most H(3)R antagonists act as inverse agonists. Here, we have investigated the effects of betahistine at recombinant H(3)R isoforms. On inhibition of cAMP formation and [(3)H]arachidonic acid release, betahistine behaved as a nanomolar inverse agonist and a micromolar agonist. Both effects were suppressed by pertussis toxin, were found at all isoforms tested, and were not detected in mock cells, confirming interactions at H(3)Rs. The inverse agonist potency of betahistine and its affinity on [(125)I]iodoproxyfan binding were similar in rat and human. We then investigated the effects of betahistine on histamine neuron activity by measuring tele-methylhistamine (t-MeHA) levels in the brains of mice. Its acute intraperitoneal administration increased t-MeHA levels with an ED(50) of 0.4 mg/kg, indicating inverse agonism. At higher doses, t-MeHA levels gradually returned to basal levels, a profile probably resulting from agonism. After acute oral administration, betahistine increased t-MeHA levels with an ED(50) of 2 mg/kg, a rightward shift probably caused by almost complete first-pass metabolism. In each case, the maximal effect of betahistine was lower than that of ciproxifan, indicating partial inverse agonism. After an oral 8-day treatment, the only effective dose of betahistine was 30 mg/kg, indicating that a tolerance had developed. These data strongly suggest that therapeutic effects of betahistine result from an enhancement of histamine neuron activity induced by inverse agonism at H(3) autoreceptors.

Inhibition of brain [(3)H]cimetidine binding by improgan-like antinociceptive drugs.

[(3)H]cimetidine, a radiolabeled histamine H(2) receptor antagonist, binds with high affinity to an unknown hemoprotein in the brain which is not the histamine H(2) receptor. Improgan, a close chemical congener of cimetidine, is a highly effective pain-relieving drug following CNS administration, yet its mechanism of action remains unknown. To test the hypothesis that the [(3)H]cimetidine-binding site is the improgan antinociceptive target, improgan, cimetidine, and 8 other chemical congeners were studied as potential inhibitors of [(3)H]cimetidine binding in membrane fractions from the rat brain. All compounds produced a concentration-dependent inhibition of [(3)H]cimetidine binding over a 500-fold range of potencies (K(i) values were 14.5 to >8000nM). However, antinociceptive potencies in rats did not significantly correlate with [(3)H]cimetidine-binding affinities (r=0.018, p=0.97, n=10). These results suggest that the [(3)H]cimetidine-binding site is not the analgesic target for improgan-like drugs.

Evaluation of 4',6-diamidino-2-phenylindole as a fluorescent probe substrate for rapid assays of the functionality of human multidrug and toxin extrusion proteins.

Multidrug and toxin extrusion protein 1 (MATE1) and MATE2-K are organic cation/H(+) antiporters that have recently been identified and suggested to be responsible for the brush border secretory transport of many cationic drugs in renal tubules. We here report our finding that 4',6-diamidino-2-phenylindole (DAPI) can be used as a probe substrate for rapid assays of the functionality of the human MATEs, hMATE1, and hMATE2-K, by taking advantage of its fluorescent nature. The specific cellular uptakes of DAPI by cloned hMATE1 and hMATE2-K, which were assessed by fluorescence intensity, were found to be rapid and saturable with the Michaelis constants of 1.13 and 3.16 microM, respectively, indicating that DAPI is a good substrate of both hMATEs. It was found that many organic cations inhibit the specific uptake of DAPI by hMATE1 and hMATE2-K, and the extents of inhibition are in good correlation with those of inhibition of the specific uptake of [(3)H]cimetidine as a typical substrate, indicating comparable performances of both substrates as probes in identifying inhibitors. Thus, DAPI can be an alternative probe substrate that enables fluorometric rapid assays of the functionality of both hMATEs. It was also found that the other major renal organic cation transporters, human organic cation transporter 2 (hOCT2), hOCT3, human novel organic cation transporter 1 (hOCTN1), and hOCTN2, cannot transport DAPI, although hOCT1, which is mainly expressed in the liver, can. Therefore, the DAPI uptake assay can be a method specific to the hMATEs among organic cation transporters in the human kidney.

The H2 receptor antagonist nizatidine is a P-glycoprotein substrate: characterization of its intestinal epithelial cell efflux transport.

The aim of this study was to elucidate the intestinal epithelial cell efflux transport processes that are involved in the intestinal transport of the H(2) receptor antagonist nizatidine. The intestinal epithelial efflux transport mechanisms of nizatidine were investigated and characterized across Caco-2 cell monolayers, in the concentration range 0.05-10 mM in both apical-basolateral (AP-BL) and BL-AP directions, and the transport constants of P-glycoprotein (P-gp) efflux activity were calculated. The concentration-dependent effects of various P-gp (verapamil, quinidine, erythromycin, ketoconazole, and cyclosporine A), multidrug resistant-associated protein 2 (MRP2; MK-571, probenecid, indomethacin, and p-aminohipuric acid), and breast cancer resistance protein (BCRP; Fumitremorgin C) inhibitors on nizatidine bidirectional transport were examined. Nizatidine exhibited 7.7-fold higher BL-AP than AP-BL Caco-2 permeability, indicative of net mucosal secretion. All P-gp inhibitors investigated displayed concentration-dependent inhibition on nizatidine secretion in both directions. The IC(50) of verapamil on nizatidine P-gp secretion was 1.2 x 10(-2) mM. In the absence of inhibitors, nizatidine displayed concentration-dependent secretion, with one saturable (J(max) = 5.7 x 10(-3) nmol cm(-2) s(-1) and K(m) = 2.2 mM) and one nonsaturable component (K(d) = 7 x 10(-4) microL cm(-2) s(-1)). Under complete P-gp inhibition, nizatidine exhibited linear secretory flux, with a slope similar to the nonsaturable component. V(max) and K(m) estimated for nizatidine P-gp-mediated secretion were 4 x 10(-3) nmol cm(-2) s(-1) and 1.2 mM, respectively. No effect was obtained with the MRP2 or the BCRP inhibitors. Being a drug commonly used in pediatrics, adults, and elderly, nizatidine susceptibility to efflux transport by P-gp revealed in this paper may be of significance in its absorption, distribution, and clearance, as well as possible drug-drug interactions.