Search

Serine proteases are among the important groups of enzymes having significant roles in cell biology. Trypsin is a representative member of the serine superfamily of enzymes, produced by acinar cells of pancreas. It is a validated drug target for various ailments including pancreatitis and colorectal cancer. Premature activation of trypsin is involved in the lysis of pancreatic tissues, which causes pancreatitis. It is also reported to be involved in colorectal carcinoma by activating other proteases, such as matrix metalloproteinase (MMPs). The development of novel trypsin inhibitors with good pharmacokinetic properties could play important roles in pharmaceutical sciences. This study reports the crystal structures of bovine pancreatic trypsin with four molecules; cimetidine, famotidine, pimagedine, and guanidine. These compounds possess binding affinity towards the active site (S1) of trypsin. The structures of all four complexes provided insight of the binding of four different ligands, as well as the dynamics of the active site towards the bind with different size ligands. This study might be helpful in designing of new potent inhibitors of trypsin and trypsin like serine proteases., Competing Interests: Declaration of competing interest The authors have no conflicts of interest to declare., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Prolonged inflammation after spinal cord injury is detrimental to recovery. To find pharmacological modulators of the inflammation response, we designed a rapid drug screening paradigm in larval zebrafish followed by testing of hit compounds in a mouse spinal cord injury model. Methods: We used reduced il-1β linked green fluorescent protein (GFP) reporter gene expression as a read-out for reduced inflammation in a screen of 1081 compounds in larval zebrafish. Hit drugs were tested in a moderate contusion model in mice for cytokine regulation, and improved tissue preservation and locomotor recovery. Results: Three compounds robustly reduced il-1β expression in zebrafish. Cimetidine, an over-the-counter H2 receptor antagonist, also reduced the number of pro-inflammatory neutrophils and rescued recovery after injury in a zebrafish mutant with prolonged inflammation. Cimetidine action on il-1β expression levels was abolished by somatic mutation of H2 receptor hrh2b , suggesting specific action. In mice, systemic treatment with Cimetidine led to significantly improved recovery of locomotor behavior as compared to controls, accompanied by decreased neuronal tissue loss and a shift towards a pro-regenerative profile of cytokine gene expression. Conclusion: Our screen revealed H2 receptor signaling as a promising target for future therapeutic interventions in spinal cord injury. This work highlights the usefulness of the zebrafish model for rapid screening of drug libraries to identify therapeutics to treat mammalian spinal cord injury., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

Dendritic cells (DCs) are powerful antigen presentation cells and the initiator of adaptive immune response. Cimetidine, a widely used drug for gastric ulcers treatment, has significant immunomodulatory ability. However, the effects of cimetidine on DC-mediated T cell activation need to be further explored. In this study, we constructed the in vitro and in vivo model of cimetidine exposure, and our data showed that cimetidine stimulated the maturity of immature DCs, and further enhanced its T cell priming capacity. In vivo, the number of rat splenic CD103 + DC were not altered after cimetidine exposure, but the expression of surface markers CD54, CD11c, and MHC-II of which were up-regulated. Importantly, cimetidine interfered with DC-mediated T cell polarization, which was reflected in the up-regulation of Th1 and Th17 cells and the down-regulation of Th2 and Treg cells in vitro and in vivo. These results indicate that cimetidine can induce DC activation and promote DC mediated pro-inflammatory T cell response while weaken immunosuppressive T cell response., (Copyright © 2022 Elsevier Ltd. All rights reserved.)

Purpose: MDCK cells are commonly used to assess drug permeability, but the existence of various strains merits a comparative functional study. Since metformin absorption is largely mediated by transporters and paracellular diffusion, we used it to functionally compare MDCK-wt and MDCK-II., Methods: Uptake, bidirectional transport and efflux experiments were performed using different buffers, pH, and a panel of transporter inhibitors. Relative contributions to total transport in both strains were estimated., Results: Metformin uptake into MDCK-wt was linear but saturable in MDCK-II. Uptake into MDCK-wt or -II was promoted at pH 5.4 or 8.4, respectively. Quinidine and cimetidine similarly inhibited uptake in both strains. Lopinavir (PMAT specific) at pH 5.4 or pyrimethamine (MATE specific) at pH 8.4 differentially inhibited MDCK-wt or -II, respectively. Transport at pH 7.4 was absorptive regardless of strains, but secretory (MDCK-II) or absorptive (MDCK-wt) at pH 5.4. Efflux was largely basolateral in both strains. While paracellular permeability was similar between strains, total transport was dominated by transporters in MDCK-II or paracellular diffusion in MDCK-wt., Conclusions: Metformin transport revealed functional differences between MDCK strains. Apical uptake was governed by MATE in MDCK-II or PMAT in MDCK-wt, such that metformin transport was either secretory or absorptive, respectively.

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 IC 50 values can be suspect because they can be influenced by choice of transported substrate; for example, IC 50 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 µ M 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. IC 50 values for 20 structurally diverse compounds confirmed this profile, with IC 50 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., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

We synthesized [ 11 C]sulpiride as a positron emission tomography probe for investigating the drug distribution in the human body. [ 11 C]Sulpiride was injected to healthy male subjects in either tracer dose of [ 11 C]sulpiride (approximately 222 MBq) or with therapeutic dose of sulpiride (500 mg, peroral) 3 h before the injection in a crossover fashion. Whole-body positron emission tomography imaging demonstrated that [ 11 C]sulpiride accumulated exceedingly in the bladder, followed by liver, gall bladder, and kidney, respectively, at 30 min after the injection, whereas scarcely in the brain. Oral dose of sulpiride decreased the hepatic accumulation of the radioactivity by 60%. From in vitro experiments, we found that sulpiride is a substrate of hOCT1 (K m 2.6 μM), hOCT2 (K m 68 μM), hMATE1 (K m 40 μM), and hMATE2-K (K m 60 μM). Moreover, the uptake of sulpiride by human hepatocytes was diminished by tetraethylammonium, and saturable with K m of 18 μM. Oct1/2 double knockout mice and wild-type mice received Mate1 inhibitors (pyrimethamine/cimetidine) manifested reduced renal clearance of sulpiride, accompanied with its accumulation in the plasma. In conclusion, we found that sulpiride is a substrate of OCT1, OCT2, MATE1, and MATE2-K, and this suggests that [ 11 C]sulpiride would be a useful radioligand to investigate the organic cation transporters in humans., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The objective of this work was to optimize a gel formulation of cimetidine to maximize its transdermal delivery across microporated skin. Specifically, the effect of extent of ionization in formulation on permeation of cimetidine across microporated skin was studied. Cimetidine was formulated into a gel using propylene glycol, water, and carbopol 980NF. Three strengths of gels (0.1% w/w, 0.5% w/w, and 0.8% w/w) were made and Tris base was used to adjust the pH of formulations to pH 5, pH 6.8, and pH 7.5. In vitro permeation testing was performed on vertical Franz cells with dermatomed porcine ear skin. Permeation studies suggested that pH 5 gels showed highest permeation through microchannels. This trend was more prominent with an increase in drug loading. The total amount of cimetidine delivered from 0.8% w/w gel at pH 5 at 24 h was 28.20 ± 4.63 μg, which was significantly higher than that from pH 6.8 (16.89 ± 3.56 μg) and pH 7.5 (12.03 ± 1.66 μg) gels. Cimetidine permeation across microporated skin was found to be pH dependent, with lower pH/highest ionization resulting in greatest permeation. The effect of ionization contributing to faster release was more pronounced when drug concentration was increased., (Copyright © 2017 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

The slow molecular mobility in the amorphous solid state of 3 active pharmaceutical drugs (cimetidine, nizatidine, and famotidine) has been studied using differential scanning calorimetry and the 2 dielectric-related techniques of dielectric relaxation spectroscopy and thermally stimulated depolarization currents. The glass-forming ability, the glass stability, and the tendency for crystallization from the equilibrium melt were investigated by differential scanning calorimetry, which also provided the characterization of the main relaxation of the 3 glass formers. The chemical instability of famotidine at the melting temperature and above it prevented the preparation of the amorphous for dielectric studies. In contrast, for cimetidine and nizatidine, the dielectric study yielded the main kinetic features of the α relaxation and of the secondary relaxations. According to the obtained results, nizatidine displays the higher fragility index of the 3 studied glass-forming drugs. The thermally stimulated depolarization current technique has proved useful to identify the Johari-Goldstein relaxation and to measure τ βJG in the amorphous solid state, that is, in a frequency range which is not easily accessible by dielectric relaxation spectroscopy., (Copyright © 2016 American Pharmacists Association®. Published by Elsevier Inc. All rights reserved.)

Renal transporter-mediated drug-drug interactions (DDIs) are of significant clinical concern, as they can adversely impact drug disposition, efficacy, and toxicity. Emerging evidence suggests that human renal organic cation transporter 2 (hOCT2) and multidrug and toxin extrusion proteins 1 and 2-K (hMATE1/2-K) exhibit substrate-dependent inhibition, but their impact on renal drug secretion and intracellular accumulation is unknown. Using metformin and atenolol as the probe substrates, we found that the classic inhibitors (e.g., cimetidine) of renal organic cation secretion were approximately 10-fold more potent for hOCT2 when atenolol was used, suggesting that atenolol is a more sensitive in vitro substrate for hOCT2 than metformin. In contrast, inhibition of hMATE1/2-K was influenced much less by the choice of substrate. Cimetidine is a much more potent inhibitor for hMATE1/2-K when metformin is the substrate but acts as an equally potent inhibitor of hOCT2 and hMATE1/2-K when atenolol is the substrate. Using hOCT2/hMATE1 double-transfected Madin-Darby canine kidney cells, we evaluated the impact of substrate-dependent inhibition on hOCT2/hMATE1-mediated transepithelial flux and intracellular drug accumulation. At clinically relevant concentrations, cimetidine dose dependently inhibited basal-to-apical flux of atenolol and metformin but impacted their intracellular accumulation differently, indicating that substrate-dependent inhibition may shift the major substrate-inhibitor interaction site between apical and basolateral transporters. Cimetidine is effective only when applied to the basal compartment. Our findings revealed the complex and dynamic nature of substrate-dependent inhibition of renal organic cation drug transporters and highlighted the importance of considering substrate-dependent inhibition in predicting transporter-mediated renal drug interaction, accumulation, and toxicity., (Copyright © 2016 by The American Society for Pharmacology and Experimental Therapeutics.)

Foxp3-expressing Treg cells have been well documented to provide immune regulation by promoting immune tolerance and suppressing immune over-reaction. Cimetidine (CIM), used to inhibit stomach acid secretion, has been reported to promote immune responses and suppress Treg cell function in several studies. However, the underlying mechanism is unknown. To investigate CIM effects on the suppressive function of Treg and Foxp3, here we used CIM to stimulate human CD4 + CD25 + Treg cells and Jurkat T cells and evaluated changes of Foxp3 expression and stability. Our data showed that CIM leads to a reduction of Foxp3 via E3 ligase Stub1-mediated proteosomal degradation, which is dependent on an activated PI3K-AKT-mTOR pathway. Thus, CIM affects the suppressive function of Treg cells by destabilizing their Foxp3 expression.

The objective was to assess the impact of larger than conventional amounts of 14 commonly used excipients on Biopharmaceutics Classification System (BCS) class 3 drug absorption in humans. Cimetidine and acyclovir were used as model class 3 drugs across three separate four-way crossover bioequivalence (BE) studies (n = 24 each) in healthy human volunteers, denoted as study 1A, 1B, and 2. In study 1A and 1B, three capsule formulations of each drug were manufactured, collectively involving 14 common excipients. Capsule formulations that incorporated hydroxypropyl methylcellulose (HPMC) or magnesium stearate exhibited lower absorption. The cimetidine commercial solution contained sorbitol and also resulted in lower absorption. Hence, in study 2, two capsule formulations with lower amounts of HPMC and magnesium stearate, the sorbitol-containing commercial solution, and a sorbitol-free solution were assessed for BE. Overall, 12 common excipients were found in large amounts to not impact BCS class 3 drug absorption in humans, such that these excipients need not be qualitatively the same nor quantitatively very similar to reference, but rather simply be not more than the quantities studied here. Meanwhile, for each HPMC and microcrystalline cellulose, BCS class 3 biowaivers require these two excipients to be qualitatively the same and quantitatively very similar to the reference., (Copyright © 2016. Published by Elsevier Inc.)

Catalase is responsible for converting hydrogen peroxide (H2O2) into water and oxygen in cells. This enzyme has high affinity for hydrogen peroxide and can protect the cells from oxidative stress damage. Catalase is a tetramer protein and each monomer contains a heme group. Cimetidine is a histamine H2 receptor blocker which inhibits acid release from stomach and is used for gasterointestinal diseases. In this research, effect of cimetidine on the activity of liver catalase was studied and the kinetic parameters of this enzyme and its conformational changes were investigated. Cell free extract of mouse liver was used for the catalase assay. The activity of the catalase was detected in the absence and presence of cimetidine by monitoring hydrogen peroxide reduction absorbance at 240 nm. The purified enzyme was used for conformational studies by Fluorescence spectrophotometry. The data showed that cimetidine could inhibit the enzyme in a non-competitive manner. Ki and IC50 values of the drug were determined to be about 0.75 and 0.85 uM, respectively. The Arrhenius plot showed that activation energy was 6.68 and 4.77 kJ/mol in the presence and absence of the drug, respectively. Fluorescence spectrophotometry revealed that the binding of cimetidine to the purified enzyme induced hyperchromicity and red shift which determined the conformational change on the enzyme. Cimetidine could non-competitively inhibit the liver catalase with high affinity. Binding of cimetidine to the enzyme induced conformational alteration in the enzyme.

Large individual variations in drug efficacy and safety could be explained in part by pharmacokinetics regulated by drug transporters and drug-metabolizing enzymes. However, expression and/or function of these proteins often fluctuate in pathological conditions, causing individual pharmacokinetic variability. To achieve a personalized pharmacotherapy after liver transplantation, our group has been investigating the pharmacokinetics of drugs and factors causing its variation based on molecular biological analysis using rats with liver ischemia-reperfusion (I/R) injury as a model for injuries immediately after liver transplantation. The first finding is that the oral bioavailability of cyclosporine A (CsA), which is an immunosuppressant, was decreased by increased first-pass metabolism due to elevated CYP3A and P-glycoprotein (P-gp) specifically in the upper small intestine after liver I/R. Expression of CYP3A in the small intestine was also elevated through transcriptional regulation by endogenous bile acids, whereas expression and function of intestinal P-gp were increased by post-transcriptional regulation via microRNA-145. Next, the pharmacokinetics of a cationic drug, cimetidine, which is eliminated from the kidney, and the expressional variation of drug transporters in the kidney after liver I/R were examined. Liver I/R decreased tubular secretion of cimetidine, mainly because of decreased expression of rat organic cation transporter 2 in the kidney. These findings provide useful information on the etiology of liver I/R injury and appropriate use of immunosuppressants and drugs eliminated from the kidney after liver transplantation.

Biosolids contain a variety of pharmaceuticals and personal care products (PPCPs). Studies have observed the uptake of PPCPs into plants grown in biosolids-amended soils. This study examined the ability of Dynamic Plant Uptake (DPU) model and Biosolids-amended Soil Level IV (BASL4) model to predict the concentration of eight PPCPs in the tissue of plants grown in biosolids-amended soil under a number of exposure scenarios. Concentrations in edible tissue predicted by the models were compared to concentrations reported in the literature by calculating estimated human daily intake values for both sets of data and comparing them to an acceptable daily intake value. The equilibrium partitioning (EqP) portion of BASL4 overpredicted the concentrations of triclosan, triclocarban, and miconazole in root and shoot tissue by two to three orders of magnitude, while the dynamic carrot root (DCR) portion overpredicted by a single order of magnitude. DPU predicted concentrations of triclosan, triclocarban, miconazole, carbamazepine, and diphenhydramine in plant tissues that were within an order of magnitude of concentrations reported in the literature. The study also found that more empirical data are needed on the uptake of cimetidine, fluoxetine, and gemfibrozil, and other ionizable PPCPs, to confirm the utility of both models. All hazard quotient values calculated from literature data were below 1, with 95.7% of hazard quotient values being below 0.1, indicating that consumption of the chosen PPCPs in plant tissue poses de minimus risk to human health.

The aim of the present study was to develop a method for water flux reabsorption measurement in Doluisio's Perfusion Technique based on the use of phenol red as a non-absorbable marker and to validate it by comparison with gravimetric procedure. The compounds selected for the study were metoprolol, atenolol, cimetidine and cefadroxil in order to include low, intermediate and high permeability drugs absorbed by passive diffusion and by carrier mediated mechanism. The intestinal permeabilities (Peff) of the drugs were obtained in male and female Wistar rats and calculated using both methods of water flux correction. The absorption rate coefficients of all the assayed compounds did not show statistically significant differences between male and female rats consequently all the individual values were combined to compare between reabsorption methods. The absorption rate coefficients and permeability values did not show statistically significant differences between the two strategies of concentration correction. The apparent zero order water absorption coefficients were also similar in both correction procedures. In conclusion gravimetric and phenol red method for water reabsorption correction are accurate and interchangeable for permeability estimation in closed loop perfusion method., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Catalase is an important enzyme for the degradation of hydrogen peroxide in cells. Bacteria have potent catalase to deal with H2O2 in their medium culture. Any chemicals that inhibit catalase activity can be harmful for cells. Histamine H2 antagonist drugs such as cimetidine and ranitidine are used for the treatment of gastrointestinal tract disorders. The present results showed that cimetidine could inhibit the catalase activity of Pseudomonas aeruginosa in a competitive inhibition. The determination of IC50 value and Ki (6.5 μM) of cimetidine demonstrated that the enzyme binds to the drug with high affinity. Binding of the drug to the enzyme was pH-dependent and no binding was observed at basic pH (>9) and acidic pH (<6). Moreover, the imidazole ring and cyanoguanidine group of cimetidine may play an important role in inhibition by binding to Fe in heme group and glutamic acid 51 residue on the enzyme, respectively. Ranitidine had no effect on the catalase activity., (© 2014 S. Karger AG, Basel.)

Kidney plays a critical role in the elimination of xenobiotics. Drug-drug interactions (DDIs) via inhibition of renal organic anion (OAT) and organic cation (OCT) transporters have been observed in the clinic. This study examined the quantitative predictability of renal transporter-mediated clinical DDIs based on basic and mechanistic models. In vitro transport and clinical pharmacokinetics parameters were used to quantitatively predict DDIs of victim drugs when coadministrated with OAT or OCT inhibitors, probenecid and cimetidine, respectively. The predicted changes in renal clearance (CLr) and area under the plasma concentration-time curve (AUC) were comparable to that observed in clinical studies. With probenecid, basic modeling predicted 61% cases within 25% and 94% cases within 50% of the observed CLr changes in clinic. With cimetidine, basic modeling predicted 61% cases within 25% and 92% cases within 50% of the observed CLr changes in clinic. Additionally, the mechanistic model predicted 54% cases within 25% and 92% cases within 50% of the observed AUC changes with probenecid. Notably, the magnitude of AUC changes attributable to the renal DDIs is generally less than 2-fold, unlike the DDIs associated with inhibition of CYPs and/or hepatic uptake transporters. The models were further used to evaluate the renal DDIs of Pfizer clinical candidates/drugs, and the overall predictability demonstrates their utility in the drug discovery and development settings.

Improgan, a non-opioid, antinociceptive drug, activates descending analgesic circuits following brain administration, but the improgan receptor remains unidentified. Since biotinylation of drugs can enhance drug potency or facilitate discovery of new drug targets, a biotinylated congener of improgan (CC44) and several related compounds were synthesized and tested for antinociceptive activity. In rats and mice, intracerebroventricular (i.c.v.) administration of CC44 produced dose-dependent reductions in thermal nociceptive (tail flick and hot plate) responses, with 5-fold greater potency than improgan. CC44 also robustly attenuated mechanical (tail pinch) nociception in normal rats and mechanical allodynia in a spinal nerve ligation model of neuropathic pain. Similar to the effects of improgan, CC44 antinociception was reversed by the GABAA agonist muscimol (consistent with activation of analgesic circuits), and was resistant to the opioid antagonist naltrexone (implying a non-opioid mechanism). Also like improgan, CC44 produced thermal antinociception when microinjected into the rostral ventromedial medulla (RVM). Unlike improgan, CC44 (i.c.v.) produced antinociception which was resistant to antagonism by the cannabinoid CB1 antagonist/inverse agonist rimonabant. CC44 was inactive in mice following systemic administration, indicating that CC44 does not penetrate the brain. Preliminary findings with other CC44 congeners suggest that the heteroaromatic nucleus (imidazole), but not the biotin moiety, is required for CC44's antinociceptive activity. These findings demonstrate that CC44 is a potent analgesic compound with many improgan-like characteristics. Since powerful techniques are available to characterize and identify the binding partners for biotin-containing ligands, CC44 may be useful in searching for new receptors for analgesic drugs., (© 2013 Elsevier B.V. All rights reserved.)

Cimetidine has been studied as an additive in cancer chemotherapy. It is claimed to reduce the side effects of Cisplatin. This study focuses on possible interactions between Cisplatin and cimetidine on the molecular level. Due to the fact that cimetidine is metabolized in the liver, interactions between its metabolites and Cisplatin are also investigated. By means of LC/ESI-MS, Cisplatin-cimetidine adducts were detected. In a second step, the metabolism of cimetidine was simulated by electrochemical oxidation. These results were compared with microsomal incubations of cimetidine using rat and human liver cell microsomes. Because the two methods showed a correlation, the electrochemical approach was further used to investigate Cisplatin's interactions with metabolites of cimetidine. However, notable interactions that might take place in the human body could neither be observed for pure cimetidine nor for its metabolites. Finally, the impact of cimetidine on Cisplatin-protein interactions were studied using the model protein β-lactoglobulin A. In the presence of cimetidine, the affinity of Cisplatin towards the model protein appears to be increased., (Copyright © 2013 Elsevier B.V. All rights reserved.)

OCT2 is the entry step for organic cation (OC) secretion by renal proximal tubules. Although many drugs inhibit OCT2 activity, neither the mechanistic basis of their inhibition nor their transport status is generally known. Using representatives of several structural classes of OCT2-inhibitory ligands described recently (Kido Y, Matsson P, Giacomini KM. J Med Chem 54: 4548-4558, 2011), we determined the kinetic basis of their inhibition of 1-methyl-4-phenylpyridinium (MPP) transport into Chinese hamster ovary cells that stably expressed hOCT2. The "cluster II" inhibitors (which contain known OCT2 substrates) metformin and cimetidine interacted competitively with MPP. However, other cluster II compounds, including tetraethylammonium (TEA), diphenidol and phenyltoloxamine, were mixed-type inhibitors of MPP transport (i.e., decreasing J(max) and increasing K(t)). A cluster III (neutral steroid) representative, adrenosterone, and a cluster I (large, flexible cation) representative, carvedilol, displayed noncompetitive inhibitory profiles. Competitive counterflow (CCF) was used to determine whether the inhibitory ligands served as substrates of hOCT2. Carvedilol (cluster I) and adrenosterone (cluster III) did not support CCF, consistent with the prediction that members of these structural classes are likely to be nontransported inhibitors of OCT2. The cluster II representatives MPP, metformin, cimetidine, and TEA all supported CCF, consistent with independent assessments of their OCT2-mediated transport. However, the other cluster II representatives, diphenidol and phenyltoloxamine, failed to support CCF, suggesting that neither compound is transported by OCT2. An independent assessment of diphenidol transport (using liquid chromatography with tandem mass spectroscopy) confirmed this observation. The results underscore the caution required for development of predictive models of ligand interaction with multidrug transporters.

Cimetidine, an H₂ receptor antagonist, has been used to investigate the tubular secretion of organic cations in human kidney. We report a systematic comprehensive analysis of the inhibition potency of cimetidine for the influx and efflux transporters of organic cations [human organic cation transporter 1 (hOCT1) and hOCT2 and human multidrug and toxin extrusion 1 (hMATE1) and hMATE2-K, respectively]. Inhibition constants (K(i)) of cimetidine were determined by using five substrates [tetraethylammonium (TEA), metformin, 1-methyl-4-phenylpyridinium, 4-(4-(dimethylamino)styryl)-N-methylpyridinium, and m-iodobenzylguanidine]. They were 95 to 146 μM for hOCT2, providing at most 10% inhibition based on its clinically reported plasma unbound concentrations (3.6-7.8 μM). In contrast, cimetidine is a potent inhibitor of MATE1 and MATE2-K with K(i) values (μM) of 1.1 to 3.8 and 2.1 to 6.9, respectively. The same tendency was observed for mouse Oct1 (mOct1), mOct2, and mouse Mate1. Cimetidine showed a negligible effect on the uptake of metformin by mouse kidney slices at 20 μM. Cimetidine was administered to mice by a constant infusion to achieve a plasma unbound concentration of 21.6 μM to examine its effect on the renal disposition of Mate1 probes (metformin, TEA, and cephalexin) in vivo. The kidney- and liver-to-plasma ratios of metformin both were increased 2.4-fold by cimetidine, whereas the renal clearance was not changed. Cimetidine also increased the kidney-to-plasma ratio of TEA and cephalexin 8.0- and 3.3-fold compared with a control and decreased the renal clearance from 49 to 23 and 11 to 6.6 ml/min/kg, respectively. These results suggest that the inhibition of MATEs, but not OCT2, is a likely mechanism underlying the drug-drug interactions with cimetidine in renal elimination.

The objective of this study was to investigate the effect of sebum on drug transport across the human stratum corneum (SC) in vivo for two model compounds, 4-cyanophenol (CP) and cimetidine (CM), of different lipophilicity and molecular size by utilizing noninvasive tape-stripping techniques, in conjunction with an unsteady-state diffusion model for data analysis. The results demonstrated that the SC permeability of the relatively hydrophilic CM on the forehead may be as much as four times the permeability on the forearm. The administration of sebum supplementation to the forearm increased the SC permeability of CM more than threefold, but did not have the same effect with regard to CP. Removal of sebum from the forehead demonstrated a small but significant effect (-22%) on the SC permeability of CM. The presence of sebum on the forehead or forearm increased the diffusion of both molecules, but the effect on partition varied between sites and drugs. The change in the SC permeability of the relatively hydrophilic drug using sebum treatment may be attributable to the altered barrier function of the SC due to the disordering structures of the intercellular lipid molecules., (Copyright © 2012 S. Karger AG, Basel.)

CYP3A4, an important drug-metabolizing enzyme, is known to have genetic variants. We have previously reported that CYP3A4 variants such as CYP3A4.2, 7, 16, and 18 show different enzymatic kinetics from CYP3A4.1 (wild type). In this study, we quantitatively investigated the inhibition kinetics of two typical inhibitors, itraconazole (ITCZ) and cimetidine (CMD), on CYP3A4 variants and evaluated whether the genetic variation leads to interindividual differences in the extent of CYP3A4-mediated drug interactions. The inhibitory profiles of ITCZ and CMD on the metabolism of testosterone (TST) were analyzed by using recombinant CYP3A4 variants. The genetic variation of CYP3A4 significantly affected the inhibition profiles of the two inhibitors. In CYP3A4.7, the K(i) value for ITCZ was 2.4-fold higher than that for the wild-type enzyme, whereas the K(i) value for CMD was 0.64-fold lower. In CYP3A4.16, the K(i) value for ITCZ was 0.54-fold lower than that for wild-type CYP3A4, whereas the K(i) value for CMD was 3.2-fold higher. The influence of other genetic variations also differed between the two inhibitors. Docking simulations could explain the changes in the K(i) values, based on the accessibility of TST and inhibitors to the heme moiety of the CYP3A4 molecule. In conclusion, the inhibitory effects of an inhibitor differ among CYP3A4 variants, suggesting that the genetic variation of CYP3A4 may contribute, at least in part, to interindividual differences in drug interactions mediated by CYP3A4 inhibition, and the pattern of the influences of genetic variation differs among inhibitors as well as substrates.

The Biopharmaceutical Classification System (BCS) guidance issued by the FDA allows waivers for in vivo bioavailability and bioequivalence studies for immediate-release (IR) solid oral dosage forms only for BCS class I drugs. However, a number of drugs within BCS class III have been proposed to be eligible for biowaivers. The World Health Organization (WHO) has shortened the requisite dissolution time of BCS class III drugs on their Essential Medicine List (EML) from 30 to 15 min for extended biowaivers; however, the impact of the shorter dissolution time on AUC(0-inf) and C(max) is unknown. The objectives of this investigation were to assess the ability of gastrointestinal simulation software to predict the oral absorption of the BCS class I drugs propranolol and metoprolol and the BCS class III drugs cimetidine, atenolol, and amoxicillin, and to perform in silico bioequivalence studies to assess the feasibility of extending biowaivers to BCS class III drugs. The drug absorption from the gastrointestinal tract was predicted using physicochemical and pharmacokinetic properties of test drugs provided by GastroPlus (version 6.0). Virtual trials with a 200 mL dose volume at different drug release rates (T(85%) = 15 to 180 min) were performed to predict the oral absorption (C(max) and AUC(0-inf)) of the above drugs. Both BCS class I drugs satisfied bioequivalence with regard to the release rates up to 120 min. The results with BCS class III drugs demonstrated bioequivalence using the prolonged release rate, T(85%) = 45 or 60 min, indicating that the dissolution standard for bioequivalence is dependent on the intestinal membrane permeability and permeability profile throughout the gastrointestinal tract. The results of GastroPlus simulations indicate that the dissolution rate of BCS class III drugs could be prolonged to the point where dissolution, rather than permeability, would control the overall absorption. For BCS class III drugs with intestinal absorption patterns similar to those of cimetidine, atenolol or amoxicillin, the dissolution criteria for extension of biowaivers to BCS class III drugs warrants further investigation.

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.

This study was performed to quantify the fraction of excreted creatinine not attributable to creatinine filtration for accurately determining the glomerular filtration rate in mice. To measure this we compared creatinine filtration with the simultaneous measurement of inulin clearance using both single-bolus fluorescein isothiocyanate (FITC)-inulin elimination kinetics and standard FITC-inulin infusion. During anesthesia, creatinine filtration was found to be systematically higher than inulin clearance in both male and female C57BL/6J mice. The secretion fraction was significantly less in female mice. Administration of either cimetidine or para-aminohippuric acid, competitors of organic cation and anion transport respectively, significantly reduced the secretion fraction in male and female mice and both significantly increased the plasma creatinine level. Creatinine secretion in both genders was not mediated by the organic cation transporters OCT1 or OCT 2 since secretion fraction levels were identical in FVB wild-type and OCT1/2 knockout mice. Thus, secretion accounts for about 50 and 35% of excreted creatinine in male and female mice, respectively. Increasing plasma creatinine threefold by infusion further increased the secretion fraction. Renal organic anion transporter 1 mRNA expression was higher in male than in female mice, reflecting the gender difference in creatinine secretion. Hence we show that there is a major secretory contribution to creatinine excretion mediated through the organic anion transport system. This feature adds to problems associated with measuring endogenous creatinine filtration in mice.

An in-line screening and an off-line chiral CE method were developed to determine the stereoselectivity of flavin-containing monooxygenase (FMO) isoforms using cimetidine (CIM) as a substrate. The S-oxygenation of CIM was investigated using achiral chemical oxidants and (human supersomes) enzymatic metabolism procedures. In the off-line setup, the chiral selector sulfobutylether-beta-CD was chosen to separate the CIM S-oxide (CSO) metabolites. The electrophoretic migration order of CSO was confirmed to be (+) before (-) through the use of single enantiomers obtained by preparative chromatography. For the electrophoretically mediated microanalysis method, the in-line enzymatic reaction was performed in 100 mM phosphate reaction buffer (pH 8.3), whereas 50 mM phosphate buffer with 30 mM chiral selector (pH 2.5) was used as a BGE. During the screening of FMO isoenzymes by the electrophoretically mediated microanalysis method, formation of the new chiral center on the CIM sulfur was found to be stereoselective. FMO1 produces more (-)-CSO-enantiomer, while FMO3 generates mainly (+)-CSO-enantiomer. On the other hand, FMO5 shows no activity. The kinetic constants of FMO1 and FMO3 were measured by the off-line method. A K(m)=4.31 mM for the formation of the (+)-CSO-enantiomer and a K(m)=4.56 mM for the (-)-CSO-enantiomer are reported for the first time for FMO1.

In this paper we evaluate a modified approach to the traditional single-pass intestinal perfusion (SPIP) rat model in investigating segmental-dependent permeability along the intestine following oral drug administration. Whereas in the traditional model one single segment of the intestine is perfused, we have simultaneously perfused three individual segments of each rat intestine: proximal jejunum, mid-small intestine and distal ileum, enabling to obtain tripled data from each rat compared to the traditional model. Three drugs, with different permeabilities, were utilized to evaluate the model: metoprolol, propranolol and cimetidine. Data was evaluated in comparison to the traditional method. Metoprolol and propranolol showed similar P(eff) values in the modified model in all segments. Segmental-dependent permeability was obtained for cimetidine, with lower P(eff) in the distal parts. Similar P(eff) values for all drugs were obtained in the traditional method, illustrating that the modified model is as accurate as the traditional, throughout a wide range of permeability characteristics, whether the permeability is constant or segment-dependent along the intestine. Three-fold higher statistical power to detect segmental-dependency was obtained in the modified approach, as each subject serves as his own control. In conclusion, the Triple SPIP model can reduce the number of animals utilized in segmental-dependent permeability research without compromising the quality of the data obtained.

Purpose: Human multidrug and toxin extrusion protein 1 (hMATE1) and hMATE2-K are organic cation/H+ antiporters that have recently been identified and suggested to be involved in the renal brush border secretion of various organic cations. Information about functional characteristics of them has been accumulating, but still insufficient to fully understand their functions and respective roles. The present study was conducted to help clarify them., Methods: The cDNA of hMATE1 was isolated from human brain cDNA by RT-PCR and hMATE2-K cDNA was from human kidney cDNA. HEK293 cells were stably transfected with hMATE1 and hMATE2-K, and the cellular uptakes of [3H]cimetidine and [14C]tetraethylammonium (TEA) were evaluated., Results: It was first found that both hMATE1 and hMATE2-K can transport cimetidine with high affinities, indicated by small Michaelis constants of 8.00 mM and 18.18 mM, respectively. These were much smaller than those for TEA (366 mM and 375 mM, respectively, for hMATE1 and hMATE2-K). Subsequent investigation using cimetidine as a probe substrate into the profiles of inhibition of the two hMATEs by various compounds indicated that they are similar in principle but different to some extent in substrate recognition, reflecting the modest differences in amino acid sequences between them. In fact, cimetidine transport by hMATE1 was correlated to that by hMATE2-K, which is 65% similar to hMATE1, but not as good as to that by rat MATE1, which is 86% similar., Conclusions: Cimetidine was demonstrated to be a high affinity substrate of both hMATEs. Subsequent evaluation of the inhibition of hMATEs by various compounds indicated no major difference in function or role between hMATE1 and hMATE2-K.

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 microM) (3)H-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 (P(app)) of cimetidine from 2.02 to 1.24 (quercetin), 1.06 (naringenin), 1.24 (genistein), and 0.96 (xanthohumol) x 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) x 10(-6) cm s(-1) in LLC-PK1 cells. Genistin significantly reduced B to A P(app) of cimetidine to 1.24 x 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 (14)C-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.

To clarify the transcellular transport of organic cations via basolateral and apical transporters, we established double-transfected Madin-Darby canine kidney (MDCK) cells expressing both human organic cation transporter hOCT1 and hMATE1 (MDCK-hOCT1/hMATE1), and hOCT2 and hMATE1 (MDCK-hOCT2/hMATE1) as models of human hepatocytes and renal epithelial cells, respectively. Using the specific antibodies, hOCT1 and hMATE1 or hOCT2 and hMATE1 were found to be localized in the basolateral and apical membranes of MDCK-hOCT1/hMATE1 or MDCK-hOCT2/hMATE1 cells, respectively. A representative substrate, [14C]tetraethylammonium, was transported unidirectionally from the basolateral to apical side in these double transfectants. The optimal pH was showed to be 6.5 for the transcellular transport of [14C]tetraethylammonium, when the pH of the incubation medium on the apical side was varied from 5.5 to 8.5. The basolateral-to-apical transport also decreased in the presence of 10 mM 1-methyl-4-phenylpyridinium or 1 mM levofloxacin on the basolateral side of both double transfectants. In MDCK-hOCT2/hMATE1 cell monolayers, but not in MDCK-hOCT1/hMATE1 cell monolayers, the accumulation of [14C]tetraethylammonium was decreased in the presence of 10 mM 1-methyl-4-phenylpyridinium, but significantly increased in the presence of 1 mM levofloxacin. The uptake of [14C]tetraethylammonium, [3H]1-methyl-4-phenylpyridinium, [14C]metformin and [3H]cimetidine, but not of [14C]procainamide and [3H]quinidine, by HEK293 cells was stimulated by expression of the hOCT1, hOCT2 or hMATE1 compared to control cells. However, transcellular transport of [14C]procainamide and [3H]quinidine was clearly observed in both double-transfectants. These cells could be useful for examining the routes by which compounds are eliminated, or predicting transporter-mediated drug interaction.

[(3)H]Cimetidine (3HCIM) specifically binds to an unidentified site in the rat brain. Because recently described ligands for this site have pharmacological activity, 3HCIM binding was characterized. 3HCIM binding was saturable, heat-labile, and distinct from the histamine H(2) receptor. To test the hypothesis that 3HCIM binds to a cytochrome P450 (P450), the effects of nonselective and isoform-selective P450 inhibitors were studied. The heme inhibitor KCN and the nonselective P450 inhibitor metyrapone both produced complete, concentration-dependent inhibition of 3HCIM binding (K(i) = 1.3 mM and 11.9 muM, respectively). Binding was largely unaffected by inhibitors of CYP1A2, 2B6, 2C8, 2C9, 2D6, 2E1, and 19A1 but was eliminated by inhibitors of CYP2C19 (tranylcypromine) and CYP3A4 (ketoconazole). Synthesis and testing of CC11 [4(5)-(benzylthiomethyl)-1H-imidazole] and CC12 [4(5)-((4-iodobenzyl)-thiomethyl)-1H-imidazole] confirmed both drugs to be high-affinity inhibitors of 3HCIM binding. On recombinant human P450s, CC12 was a potent inhibitor of CYP2B6 (IC(50) = 11.7 nM), CYP2C19 (51.4 nM), and CYP19A1 (140.7 nM) and had a range of activities (100-494 nM) on nine other isoforms. Although the 3HCIM binding site pharmacologically resembles some P450s, eight recombinant human P450s and three recombinant rat P450s did not exhibit 3HCIM binding. Inhibition by KCN and metyrapone suggests that 3HCIM binds to a heme-containing brain protein (possibly a P450). However, results with selective P450 inhibitors, recombinant P450 isoforms, and a P450 antibody did not identify a 3HCIM-binding P450 isoform. Finally, CC12 is a new, potent inhibitor of CYP2B6 and CYP2C19 that may be a valuable tool for P450 research.

The aim of the study was to develop a novel oral delivery system for the efflux pump substrate acyclovir (ACY) utilizing thiolated chitosan as excipient which is capable of inhibiting P-glycoprotein (P-gp). Three chitosan-4-thiobutylamidine (Chito-TBA) conjugates with increasing molecular mass (Chito-9.4kDa-TBA, Chito-150kDa-TBA and Chito-600kDa-TBA) were synthesized and permeation studies on rat intestinal mucosa and Caco-2 monolayers were performed. Additionally, tablets comprising the conjugates and ACY were tested towards their drug release behaviour. The efflux ratio (secretory P(app)/absorptive P(app)) of ACY across Caco-2 monolayers was determined to be 2.5 and in presence of 100microM verapamil 1.1 which indicates ACY as P-gp substrate. In comparison to buffer only, the transport of ACY in presence of 0.5% (m/v) unmodified chitosan, 0.5% (m/v) Chito-150kDa-TBA and 0.5% (m/v) Chito-150kDa-TBA with 0.5% (m/v) reduced glutathione (GSH), was 1.3-, 1.6- and 2.1-fold improved, respectively. Transport studies across Caco-2 monolayers showed that P-gp inhibition is dependent on the average molecular mass of thiolated chitosan showing following rank order: 0.5% (m/v) Chito-150kDa-TBA/GSH>0.5% (m/v) Chito-9.4kDa-TBA/GSH>0.5% (m/v) Chito-600kDa-TBA/GSH. The higher the molecular mass of Chito-TBA was, the more sustained was the release of ACY. Chito-150kDa-TBA/GSH might be an appropriate sustained release drug delivery system for ACY, which is able to enhance ACY transport due to efflux pump inhibition.

The aim of this study was to investigate the transport behavior and efflux of berberine through the primary culture cortical neurons. High-performance liquid chromatography coupled with an UV-vis detector at 347 nm was applied. The mobile phase was 0.05 m potassium dihydrogen phosphate solution (containing 0.5% triethylamine, pH 3.0)-acetonitrile (73:27, v/v). Neurons were incubated with Coptidis rhizoma extract 6.5 microg/mL (containing 1.91 microg/mL berberine) and verapamil, KCN or cimetidine for 2 h, and then lysed in methanol to extract intracellular berberine. A 20 microL aliquot of sample was injected into the HPLC system to determine berberine concentration. The results showed that metabolic inhibitor KCN and P-glycoprotein inhibitor verapamil could increase berberine concentration within the neurons, indicating that efflux of berberine was energy-dependent and P-glycoprotein was likely to be involved. Moreover, the organic cation transporter inhibitor cimetidine could decrease berberine concentration within the neurons, suggesting that the organic cation transporter might be involved in the berberine transport process., (Copyright (c) 2007 John Wiley & Sons, Ltd.)

This study examined the contribution made by organic cation transporters (hOCT/rOct) to the saturable component of the renal uptake of 1-methyl-4-phenylpyridinium, tetraethylammonium (TEA), cimetidine and metformin into rOct2-expressing HEK293 cells and rat kidney slices. All the test compounds accumulated in the rat kidney slices in a carrier-mediated manner. The Michaelis- Menten constant (K(m)) values for saturable uptake of TEA, cimetidine and metformin into rat kidney slices were relatively comparable with those for the rOct2-expressing HEK293 cells. In addition, the relative uptake activity values of TEA, cimetidine and metformin in rat kidney slices were similar to those in rOct2-expressing HEK293 cells. This suggests that the saturable components involved in the renal uptake of TEA, cimetidine and metformin are mediated mainly by rOct2. The saturable uptake profile of cationic compounds into rat kidney can be evaluated in both cDNA-expressing cells and rat kidney slices, as well as the transporter expression pattern. This approach can also be used to estimate the saturable uptake mechanism of cationic compounds into the human kidney when human kidney slices and hOCT2-expressing cells are used.

A simple, accurate and sensitive spectrophotometric method has been developed and validated for determination of H(2)-receptor antagonists: cimetidine, famotidine, nizatidine and ranitidine hydrochloride. The method was based on the oxidation of these drugs with cerium(IV) in presence of perchloric acid and subsequent measurement of the excess Ce(IV) by its reaction with p-dimethylaminobenzaldehyde to give a red colored product (lambda(max) at 464nm). The decrease in the absorption intensity of the colored product (DeltaA), due to the presence of the drug was correlated with its concentration in the sample solution. Different variables affecting the reaction were carefully studied and optimized. Under the optimum conditions, linear relationships with good correlation coefficients (0.9990-0.9994) were found between DeltaA values and the concentrations of the drugs in a concentration range of 1-20microgml(-1). The assay limits of detection and quantitation were 0.18-0.60 and 0.54-1.53microgml(-1), respectively. The method was validated, in terms of accuracy, precision, ruggedness and robustness; the results were satisfactory. The proposed method was successfully applied to the determination of the investigated drugs in pure and pharmaceutical dosage forms (recovery was 98.3-102.6+/-0.57-1.90%) without interference from the common excipients. The results obtained by the proposed method were comparable with those obtained by the official methods.

The aim of this study was to investigate whether diabetes mellitus (DM) affected breast cancer resistance protein (Bcrp) function and expression in rat brain. 5-week and 8-week diabetic rats were induced by streptozocin (STZ). Bcrp expression and function in brain cortex were assessed by western blot and measuring the brain-to-plasma concentration ratios of two typical substrates prazosin and cimetidine, respectively. The diabetic rats were treated with three different agents insulin, aminoguanidine (AG) and metformin (MET). It was found that the brain-to-plasma ratios of prazosin and cimetidine in diabetic rats were significantly higher than those of control rats, which were dependent on duration of diabetes. Lower levels of Bcrp were found in brain cortex of diabetic rats, which were in parallel with increase of brain-to-plasma ratios. Insulin treatment may attenuate the impairment of Bcrp expression and function induced by diabetes. Aminoguanidine and metformin treatment did not prevent the impairment of Bcrp function and expression in brain cortex of diabetic rats. All results gave a conclusion that STZ-induced DM may induce the impairment of function and expression of Bcrp in brain cortex, and lower levels of insulin may mainly contribute to Bcrp dysfunction in brain.

The aim of the study was to investigate the influence of sodium ozagrel on CYP2D6 (cytochromeP450 2D6) activity. The studies were performed with rat urine and liver microsomes and chemical inhibitors. The metabolism of dextromethorphan (dextrophan/dextromethorphan, dextrophan is a metabolite of dextromethorphan) and phenacetin (paracetamol/phenacetin, paracetamol is a metabolites of phenacetin) was used as probe to measure CYP2D6 and CYP1A2 (cytochromeP450 1A2) activity, respectively, determined by high-performance liquid chromatography (HPLC). The results showed that the metabolism of dextrophan/dextromethorphan in the sodium ozagrel-treated group (37 mg/kg) was higher than that of the control (P<0.05/6) in both in vivo and in vitro studies (r=0.9811). The rate of dextromethorphan metabolism was inhibited by sodium ozagrel and cimetidine in rat liver microsomes prepared from sodium ozagrel-treated rats and control rats group (sodium ozagrel IC(50)=26.5 microM, cimetidine IC(50)=86.3 microM in sodium ozagrel-treated group; sodium ozagrel IC(50)=13.9 microM, cimetidine IC(50)=24.8 microM in control group). The inhibitory effect of sodium ozagrel on CYP2D6 activity was noncompetitive with dextromethorphan with a K(i) of 324.94 microM. Kinetic parameters of the reactions were established by using Lineweaver-Burk with K(m)=0.67 mM and V(max)=2.13 pm/min/mg protein for the sodium ozagrel-treated group and K(m)=0.29 mM, and V(max)=0.91 pm/min/mg protein for the control group, respectively. The expression of CYP2D6 protein in the treated group was higher than that of the control group, as determined by Western blotting. The activity and expression of CYP1A2 did not show obvious differences in the control group and sodium ozagrel treated group. In conclusion, sodium ozagrel metabolism in rats is mediated primarily through CYP2D6, and sodium ozagrel can induce CYP2D6 activity.

The contribution of organic cation transporters to the saturable component in the hepatic uptake of 1-methyl-4-phenylpyridinium (MPP), tetraethylammonium (TEA), cimetidine, and metformin was examined by the use of human/rat organic cation transporter (hOCT1/rOct1)-expressing cells and human/rat hepatocytes. Transfection of rOct1 resulted in a considerable increase in the uptake of metformin, whereas that of hOCT1 resulted in only a slight increase. All test compounds (MPP, TEA, cimetidine, and metformin) accumulated in human and rat hepatocytes in a carrier-mediated manner. The Km values for the uptake of MPP, TEA, cimetidine, and metformin into human and rat hepatocytes were comparable with those into hOCT1 and rOct1-expressing cells, respectively. In addition, the relative uptake activities, which were obtained by normalizing the intrinsic uptake clearances of TEA, cimetidine, and metformin against those values of MPP in human and rat hepatocytes, were similar with the uptake activities in hOCT1 and rOct1, respectively. These results suggest that the saturable component in the hepatic uptake of these cationic compounds may be mediated mainly by hOCT1/rOct1; therefore, it is meaningful to evaluate the saturable uptake profile of cationic compounds by the liver using both hOCT1/rOct1-expressing cells and human/rat hepatocytes.

An electroneutral organic cation (OC)/proton exchanger in the apical membrane of proximal tubules mediates the final step of renal OC excretion. Two members of the multidrug and toxin extrusion family, MATE1 and MATE2-K, were recently identified in human and rodent kidney and proposed to be the molecular basis of renal OC/H(+) exchange. To take advantage of the comparative value of the large database on the kinetic and selectivity characteristics of OC/H(+) exchange that exists for rabbit kidney, we cloned rbMATE1 and rbMATE2-K. The rabbit homologs have 75% (MATE1) and 74% (MATE2-K) amino acid identity to their human counterparts (and 51% identity with each other). rbMATE1 and rbMATE2-K exhibited H(+) gradient-dependent uptake and efflux of tetraethylammonium (TEA) when expressed in Chinese hamster ovary cells. Both transporters displayed similar affinities for selected compounds [IC(50) values within 2-fold for TEA, 1-methyl-4-phenylpyridinium, and quinidine] and very different affinities for others (IC(50) values differing by 8- to 80-fold for choline and cimetidine, respectively). These results indicate that rbMATE1 and rbMATE2-K are multispecific OC/H(+) exchangers with similar, but distinct, functional characteristics. Overall, the selectivity of MATE1 and MATE2-K correlated closely with that observed in rabbit renal brush-border membrane vesicles.

Improgan, a chemical congener of cimetidine, is a highly effective non-opioid analgesic when injected into the CNS. Despite extensive characterization, neither the improgan receptor, nor a pharmacological antagonist of improgan has been previously described. Presently, the specific binding of [(3)H]cimetidine (3HCIM) in brain fractions was used to discover 4(5)-((4-iodobenzyl)thiomethyl)-1H-imidazole, which behaved in vivo as the first improgan antagonist. The synthesis and pharmacological properties of this drug (named CC12) are described herein. In rats, CC12 (50-500nmol, i.c.v.) produced dose-dependent inhibition of improgan (200-400nmol) antinociception on the tail flick and hot plate tests. When given alone to rats, CC12 had no effects on nociceptive latencies, or on other observable behavioral or motor functions. Maximal inhibitory effects of CC12 (500nmol) were fully surmounted with a large i.c.v. dose of improgan (800nmol), demonstrating competitive antagonism. In mice, CC12 (200-400nmol, i.c.v.) behaved as a partial agonist, producing incomplete improgan antagonism, but also limited antinociception when given alone. Radioligand binding, receptor autoradiography, and electrophysiology experiments showed that CC12's antagonist properties are not explained by activity at 25 sites relevant to analgesia, including known receptors for cannabinoids, opioids or histamine. The use of CC12 as an improgan antagonist will facilitate the characterization of improgan analgesia. Furthermore, because CC12 was also found presently to inhibit opioid and cannabinoid antinociception, it is suggested that this drug modifies a biochemical mechanism shared by several classes of analgesics. Elucidation of this mechanism will enhance understanding of the biochemistry of pain relief.

Objective: To investigate potential interactions between lidocaine (lignocaine) metabolism and premedication drugs, i.e. psychotropic and antianxiety agents (diazepam, midazolam), hypnotics (pentobarbital, thiamylal), depolarizing neuromuscular blocking agents (vecuronium, pancuronium and suxamethonium), an antihypertensive agent (clonidine) and an H2-receptor blocking agent (cimetidine) using human liver microsomes in vitro., Methods: The interaction effects between lidocaine and premedication were examined using human liver microsomal preparations and monitored for enzyme activity. The lidocaine and its main metabolite (monoethylglycinexylide) were measured by HPLC/UV., Results: Lidocaine metabolism was non-competitively inhibited by midazolam (Ki = 77.6 microM). Thiamylal was a competitive inhibitor of lidocaine metabolism (Ki = 885 microM). Cimethidine, pancuronium and vecuronium weakly inhibited lidocaine metabolism in a concentration-depend manner over the therapeutic range in human liver microsomes. On the contrary, suxamethonium, pentobarbital and clonidine did not inhibit lidocaine metabolism over the therapeutic range in human liver microsomes., Conclusion: These results show that the interactions between lidocaine and midazolam and thiamylal are of potential toxicological and clinical significance.

We compared the characteristics of several cloned rabbit organic electrolyte (OE) transporters expressed in cultured cells with their behavior in intact rabbit renal proximal tubules (RPT) to determine the contribution of each to basolateral uptake of the weak acid ochratoxin A (OTA) and the weak base cimetidine (CIM). The activity of organic anion transporters OAT1 and OAT3 proved to be distinguishable because OAT1 had a high affinity for PAH (K(t) of 20 microM) and did not support estrone sulfate (ES) transport, whereas OAT3 had a high affinity for ES (K(t) of 4.5 microM) and a weak interaction with PAH (IC(50) > 1 mM). In contrast, both transporters robustly accumulated OTA. Intact RPT also accumulated OTA, with OAT1 and OAT3 each responsible for approximately 50%: ES and PAH each reduced uptake by approximately 50%, and the combination of the two eliminated mediated OTA uptake. The weak base CIM was transported by OAT3 (K(t) of 80 microM) and OCT2 (K(t) of 2 microM); OCT1 had a comparatively low affinity for CIM, and CIM uptake by OAT1 was equivocal. Intact RPT accumulated CIM, with TEA and ES reducing CIM uptake by 20 and 75%, respectively, suggesting that OAT3 plays a quantitatively more significant role in CIM uptake in the early proximal tubule than OCT1/2. In single S2 segments of RPT, ES and TEA each blocked approximately 50% of CIM uptake. Thus the fractional contribution of different OE transporters to renal secretion is influenced by their affinity for substrate and relative expression level in RPT.

Histamine H2 receptor antagonist famotidine and cimetidine are commonly used for treatment of gastrointestinal ulcer diseases. Inasmuch as these drugs are mainly secreted by renal tubules, dosages have been adjusted according to renal function. Although many studies have been performed on the molecular mechanisms of renal handling of cimetidine, little is known about that of famotidine. In this study, to examine the recognition and transport of famotidine by human organic anion transporters (OATs; hOAT1, hOAT3) and human organic cation transporter (OCT; hOCT2), the uptake studies using Xenopus laevis oocytes were performed in comparison with cimetidine. The half-maximal inhibitory concentrations of famotidine for [3H]estrone sulfate transport by hOAT3 and [14C]tetraethylammonium transport by hOCT2 (300 microM and 1.8 mM, respectively) were higher than those of cimetidine (53 and 67 microM, respectively). While cimetidine inhibited p-[14C]aminohippurate transport by hOAT1 in a concentration dependent manner, famotidine did not affect it at 5 mM. In addition, hOAT3 mediated famotidine uptake, but hOAT1 and hOCT2 did not show famotidine transport. These results indicate that there are marked differences between famotidine and cimetidine in the recognition and transport by organic ion transporters and that hOAT3 contributes to the renal tubular secretion of famotidine. Present findings should be useful information to understand the renal handling of famotidine and cimetidine.

The choroid plexus (CP) acts as a site for the elimination of xenobiotic organic compounds from the cerebrospinal fluid (CSF). The purpose of the present study is to investigate the role of rat organic anion transporter 3 (rOat3; Slc22a8) in the uptake of H(2)-receptor antagonists (cimetidine, ranitidine, and famotidine) by the isolated rat CP. Saturable uptake of cimetidine and ranitidine was observed in rOat3-LLC with K(m) values of 80 and 120 microM, respectively, whereas famotidine was found to be a poor substrate. The steady-state concentration of the H(2)-receptor antagonists in the CSF was significantly increased by simultaneously administered probenecid, although it did not affect their brain and plasma concentrations. Saturable uptake of cimetidine and ranitidine was observed in the isolated rat CP with K(m) values of 93 and 170 microM, respectively, whereas 50% of the uptake of famotidine remained at the highest concentration examined (1 mM). The K(i) value of ranitidine for the uptake of cimetidine by the isolated CP (50 microM) was similar to its own K(m) value, suggesting that they share the same transporter for their uptake. The inhibition potency of organic anions such as benzylpenicillin, estradiol 17beta-glucuronide, p-aminohippurate, and estrone sulfate for the uptake of cimetidine by the isolated rat CP was similar to that for benzylpenicillin, the uptake of which has been hypothesized to be mediated by rOat3, whereas a minimal effect by tetraethylammonium excludes involvement of organic cation transporter(s). These results suggest that rOat3 is the most likely candidate transporter involved in regulating the CSF concentration of H(2)-receptor antagonists at the CP.

An extract of toxin(s) from the Australian paralysis tick, Ixodes holocyclus, produced positive inotropic responses in rat left ventricular papillary muscles and positive contractile responses in rat thoracic aortic rings. There was no measurable chronotropic response in rat right atria, but positive inotropic concentrations in papillary muscles produced arrhythmias in right atria. Positive inotropic responses were attenuated by verapamil, but unaffected by metoprolol, cimetidine, pyrilamine, tetrodotoxin and pinacidil. Microelectrode studies on isolated left ventricular papillary muscles demonstrated that the extract prolonged action potential duration at 20, 50 and 90% of repolarisation and delayed ventricular papillary muscle relaxation. Cardiovascular tissues isolated from rats with experimentally induced tick paralysis showed no myocardial damage as identified by histological and ultrastructural examination. The basal rate and force of contraction of isolated cardiac tissues were lower from tick-paralysed than normal rats. Concentration-response curves to dobutamine and calcium chloride were similar between tissues from tick-paralysed and normal rats. Thus, the Australian paralysis tick, I. holocyclus, produces one or more toxins with direct cardiovascular effects which mimic the effects produced by direct blockade of cardiac and vascular K+ channels.

Antitumor effect of cyclophosphamide on LS and P388 tumors is realized via apoptosis and on HA-1 and Krebs-2 tumors resistant to apoptosis via necrosis of tumor cells. Phenobarbital induction of cyclophosphamide-metabolizing enzymes decreases and cimetidine inhibition potentiates the effect of cyclophosphamide on LS and P388 cells and does not modulate the effect on HA-1 and Krebs-2 cells. Presumably, apoptosis and necrosis of tumor cell are induced by different cyclophosphamide metabolites.

Background: Production of N-alpha-methyl-histamine (NAMH), a histamine H(3) receptor (H3R) agonist, is reportedly promoted in Helicobacter pylori infected human gastric mucosa. NAMH was suggested to act directly on histamine H(2) receptors (H2Rs) in animals to stimulate acid secretion and to be a H2R agonist. As H2Rs and H3Rs play different roles in gastric acid secretion, it is very important to verify that NAMH is a H2R agonist., Aims: To determine whether NAMH is a H2R agonist, as well as a H3R agonist., Methods: We used a Chinese hamster ovary (CHO) cell line expressing human H2Rs (CHO-H2R) and control CHO cells. Expression of human H2Rs was confirmed by tiotidine binding. cAMP production in CHO-H2R and control cells in response to histamine or NAMH was measured. cAMP production in response to 10(-7) M NAMH was also measured in the presence or absence of the H2R antagonist famotidine and the H3R antagonist thioperamide., Results: NAMH dose dependently stimulated cAMP productions in CHO-H2R cells. This production was inhibited by famotidine but not by thioperamide. Control CHO cells were unresponsive to either histamine or NAMH. In addition, the effect of NAMH, in terms of cAMP production in CHO-H2R cells, was more potent than that of histamine-that is, with a lower EC(50) concentration and higher maximal cAMP production. Both NAMH and histamine, but not R-alpha-methyl-histamine, effectively inhibited [(3)H] tiotidine binding to CHO-H2R cells., Conclusions: NAMH, which is produced in the gastric mucosa by H pylori, is a potent H2R agonist as well as a H3R agonist.