HISTOLOGICAL AND BIOCHEMICAL STUDY OF THE EFFECT OF FEXOFENADINE ON SALIVARY GLAND IN RATS.

Fexofenadine is a newly introduced oral non-sedating agent used for allergic diseases. We sought to investigate the effects of the use of fexofenadine on the salivary gland of adult male albino rats. 30 adult male albino rats were classified randomly into 3 groups, as follows: Group A (control group) which consisted of 10 healthy rats. Group B (treated group) which consisted of 10 rats received FEX 5mg/kg/day, and Group C (treated group) which consisted of 10 rats received FEX 10mg/kg/day. Blood samples were obtained to assess serum levels of Thioredoxin reductase (TRX) and malondialdehyde (MDA). Salivary glands were removed and prepared for histological examination. This study showed that significantly (p<0.05) higher TRX and MDA levels were observed in group B and group C, compared to group A. The histological examination for salivary tissues revealed degenerative changes in serous cells of acini were present with deep pyknotic nuclei. Vacuolar cytoplasmic degeneration is also seen in other certain cells. Blood congestion was present in the intralobular blood vessels, particularly around the striated ducts. The glandular secretion duct contained mucus and serous secretion and the wall of the duct was surrounded by many WBCs with macrophage. Fexofenadine hydrochloride use induces remarkable histopathological changes with dose-dependent response and remarkably linked to elevation of oxidative stress markers.

Intracellular Binding of Terfenadine Competes with Its Access to Pancreatic ß-cell ATP-Sensitive K+ Channels and Human ether-à-go-go-Related Gene Channels.

Most blockers of both hERG (human ether-à-go-go-related gene) channels and pancreatic ß-cell ATP-sensitive K+ (KATP) channels access their binding sites from the cytoplasmic side of the plasma membrane. It is unknown whether binding to intracellular components competes with binding of these substances to K+ channels. The whole-cell configuration of the patch-clamp technique, a laser-scanning confocal microscope, and fluorescence correlation spectroscopy (FCS) were used to study hERG channels expressed in HEK (human embryonic kidney) 293 cells and KATP channels from the clonal insulinoma cell line RINm5F. When applied via the pipette solution in the whole-cell configuration, terfenadine blocked both hERG and KATP currents with much lower potency than after application via the bath solution, which was not due to P-glycoprotein-mediated efflux of terfenadine. Such a difference was not observed with dofetilide and tolbutamide. 37-68% of hERG/EGFP (enhanced green-fluorescent protein) fusion proteins expressed in HEK 293 cells were slowly diffusible as determined by laser-scanning microscopy in the whole-cell configuration and by FCS in intact cells. Bath application of a green-fluorescent sulphonylurea derivative (Bodipy-glibenclamide) induced a diffuse fluorescence in the cytosol of RINm5F cells under whole-cell patch-clamp conditions. These observations demonstrate the presence of intracellular binding sites for hERG and KATP channel blockers not dialyzable by the patch-pipette solution. Intracellular binding of terfenadine was not influenced by a mutated hERG (Y652A) channel. In conclusion, substances with high lipophilicity are not freely diffusible inside the cell but steep concentration gradients might exist within the cell and in the sub-membrane space.

hERG block potencies for 5 positive control drugs obtained per ICH E14/S7B Q&As best practices: Impact of recording temperature and drug loss.

According to the ICH S7B guideline, drug candidates are screened for hERG block prior to first-in-human testing to predict the likelihood of delayed repolarization associated with a rare, but life-threatening, ventricular tachyarrhythmia. The new ICH E14 Q&As guideline allows hERG results to be used in later clinical development for decision-making (Q&As 5.1 and 6.1). To pursue this path, the hERG assay should be conducted following the new ICH S7B Q&A 2.1 guideline, which calls for best practice considerations of the recording temperature, voltage protocol, stimulation frequency, recording/data quality, and concentration verification. This study investigated hERG block by cisapride, dofetilide, terfenadine, sotalol, and E-4031 - positive controls commonly used to demonstrate assay sensitivity - using the manual whole cell patch clamp method and an action potential-like voltage protocol presented at 0.2 Hz. Recordings were conducted at room and near physiological temperature. Drug concentrations were measured using samples collected during real patch clamp experiments and satellite experiments. Results showed temperature effects for E-4031, terfenadine, and sotalol, but not cisapride and dofetilide. Cisapride and terfenadine showed substantial concentration losses, largely due to nonspecific binding to the perfusion apparatus. Using concentrations measured from the real and satellite experiments to assess block potencies yielded comparable results, indicating that satellite sample collection may be viable for drugs with nonspecific binding concerns only. In summary, this study provides block potencies for 5 hERG positive controls, and serves as a case study for hERG assays conducted, and results illustrated in accordance with the new ICH E14/S7B Q&As.

High throughput measurement of hERG drug block kinetics using the CiPA dynamic protocol.

The Comprehensive in vitro Proarrhythmic Assay (CiPA) has promoted use of in silico models of drug effects on cardiac repolarization to improve proarrhythmic risk prediction. These models contain a pharmacodynamic component describing drug binding to hERG channels that required in vitro data for kinetics of block, in addition to potency, to constrain them. To date, development and validation has been undertaken using data from manual patch-clamp. The application of this approach at scale requires the development of a high-throughput, automated patch-clamp (APC) implementation. Here, we present a comprehensive analysis of the implementation of the Milnes, or CiPA dynamic protocol, on an APC platform, including quality control and data analysis. Kinetics and potency of block were assessed for bepridil, cisapride, terfenadine and verapamil with data retention/QC pass rate of 21.8% overall, or as high as 50.4% when only appropriate sweep lengths were considered for drugs with faster kinetics. The variability in IC50 and kinetics between manual and APC was comparable to that seen between sites/platforms in previous APC studies of potency. Whilst the experimental success is less than observed in screens of potency alone, it is still significantly greater than manual patch. With the modifications to protocol design, including sweep length, number of repetitions, and leak correction recommended in this study, this protocol can be applied on APC to acquire data comparable to manual patch clamp.

Molecular dynamics of hERG channel: insights into understanding the binding of small molecules for detuning cardiotoxicity.

Evaluation of cardiotoxicity potential of new chemical entities (NCEs) has lately become one of the stringent filters in the drug discovery and development process. Cardiotoxicity is caused mainly by the inhibition of human ether-a-go-go related gene (hERG) channel protein. Inhibition of the hERG channel leads to a life-threatening condition known as cardiac arrhythmia. Knowledge of the structural behaviour of the hERG would aid greatly in the design of new drug molecules that do not interact with the protein and add to the safety index. In this study, a computational model for the active-state of hERG was developed. This model was equilibrated by performing the molecular dynamics simulations for 100 ns followed by clustering and selection of a representative structure based on the largest populated cluster. To study the changes in the protein structure on inhibition, three inhibitory ligands, namely, dofetilide, cisapride and terfenadine were docked, followed by molecular dynamics simulations of 200 ns for the apo and each ligand-bound structure. It was observed that docking and simulation studies of the hERG model exhibited noticeable conformational changes in the protein upon ligand-binding. A significant change in the kink of the S6-transmembrane helix was observed. Inter-chain distances between the crucial residues Y652 and F656 (present below the ion-selectivity filter), their side-chain orientation and hydrogen bonding indicated a probable collapse of the pore. These changes may infer the initiation in transition of hERG from an open to an inactive state. Hence, these findings would help in designing compounds devoid of hERG inhibition with reduced cardiotoxicity.Communicated by Ramaswamy H. Sarma.

Intestinal Absorption of Alogliptin Is Mediated by a Fruit-Juice-Sensitive Transporter.

Alogliptin (ALG), an inhibitor of dipeptidylpeptidase-4, is used in the management of type 2 diabetes mellitus, and has a high absorption rate (>60-71%), despite its low lipophilicity (logP=-1.4). Here, we aimed to clarify the mechanism of its intestinal absorption. ALG uptake into Caco-2 cells was time-, temperature-, and concentration-dependent, but was not saturated at concentrations up to 10 mmol/L. The uptake was significantly inhibited by the organic anion transporting polypeptide (OATP) substrate fexofenadine and by the OATP inhibitor 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), but was not inhibited by organic cation transporter (OCT)/organic cation/carnitine transporter (OCTN) or peptide transporter 1 (PEPT1) substrates. Grapefruit, orange, and apple juices and their constituents, which are known to strongly inhibit intestinal OATPs, significantly inhibited ALG uptake into Caco-2 cells. The pH dependence was bell-shaped, indicating the involvement of a pH-sensitive transporter. However, ALG uptake by HEK293 cells overexpressing OATP2B1, a key intestinal OATP transporter of amphiphilic drugs, was not different from that of mock cells. In a rat in vivo study, apple juice reduced systemic exposure to orally administered ALG without changing the terminal half-life. These observations suggest that intestinal absorption of ALG is carrier-mediated, and involves a fruit-juice-sensitive transporter other than OATP2B1.

Inhibitory effect of terfenadine on Kir2.1 and Kir2.3 channels.

Terfenadine is a second-generation H1-antihistamine that despite potentially can produce severe side effects it has recently gained attention due to its anticancer properties. Lately, the subfamily 2 of inward rectifier potassium channels (Kir2) has been implicated in the progression of some tumoral processes. Hence, we characterized the effects of terfenadine on Kir2.x channels expressed in HEK-293 cells. Terfenadine inhibited Kir2.3 channels with a strikingly greater potency (IC50 = 1.06 ± 0.11 µmol L-1) compared to Kir2.1 channels (IC50 = 27.8 ± 4.8 µmol L-1). The Kir2.3(I213L) mutant, possessing a larger affinity for phosphatidylinositol 4,5-bisphosphate (PIP2) than the wild-type Kir2.3, was less sensitive to terfenadine inhibition (IC50 = 13.0 ± 2.9 µmol L-1). Additionally, the PIP2 intracellular application had largely reduced the inhibition of Kir2.1 channels by terfenadine. Our data support that Kir2.x channels are targets of terfena-dine by affecting their interaction with PIP2, which could be regarded as a mechanism of the antitumor properties of terfenadine.

Reliable identification of cardiac liability in drug discovery using automated patch clamp: Benchmarking best practices and calibration standards for improved proarrhythmic assessment.

INTRODUCTION: Screening compounds for activity on the hERG channel using patch clamp is a crucial part of safety testing. Automated patch clamp (APC) is becoming widely accepted as an alternative to manual patch clamp in order to increase throughput whilst maintaining data quality. In order to standardize APC experiments, we have investigated the effects on IC50 values under different conditions using several devices across multiple sites. METHODS: APC instruments SyncroPatch 384i, SyncroPatch 384PE and Patchliner, were used to record hERG expressed in HEK or CHO cells. Up to 27 CiPA compounds were used to investigate effects of voltage protocol, incubation time, labware and time between compound preparation and experiment on IC50 values. RESULTS: All IC50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC50 values from the literature (Kramer et al., 2013) regardless of voltage protocol or labware, when compounds were used immediately after preparation, but potency of astemizole decreased if prepared in Teflon or polypropylene (PP) compound plates 2-3 h prior to experiments. Slow acting compounds such as dofetilide, astemizole, and terfenadine required extended incubation times of at least 6 min to reach steady state and therefore, stable IC50 values. DISCUSSION: Assessing the influence of different experimental conditions on hERG assay reliability, we conclude that either the step-ramp protocol recommended by CiPA or a standard 2-s step-pulse protocol can be used to record hERG; a minimum incubation time of 5 min should be used and although glass, Teflon, PP or polystyrene (PS) compound plates can be used for experiments, caution should be taken if using Teflon, PS or PP vessels as some adsorption can occur if experiments are not performed immediately after preparation. Our recommendations are not limited to the APC devices described in this report, but could also be extended to other APC devices.

Potential synergistic effects of novel hematopoietic prostaglandin D synthase inhibitor TAS-205 and different types of anti-allergic medicine on nasal obstruction in a Guinea pig model of experimental allergic rhinitis.

Nasal obstruction is one of the most bothersome symptoms of allergic rhinitis (AR) affecting sleep-related quality of life in AR patients. Although several treatments were tested to control nasal obstruction, some patients with moderate to severe AR do not respond to current treatments, including the combined administration of different types of anti-allergic medicine. Thus, new options for AR treatment are needed. This study aimed to evaluate the effects of combined treatment with a novel inhibitor of hematopoietic prostaglandin D synthase (HPGDS), TAS-205, and different types of anti-allergic medicine on nasal obstruction in AR. Firstly, we demonstrated that TAS-205 selectively inhibited prostaglandin D2 (PGD2) synthesis in an enzymatic assay in a cell-based assay and in vivo models of AR. Moreover, treatment with TAS-205 alone suppressed eosinophil infiltration into the nasal cavity and late phase nasal obstruction. The combined administration of TAS-205 with montelukast, a cysteinyl leukotriene receptor 1 antagonist, showed significant additive inhibitory effects on eosinophil infiltration and late phase nasal obstruction compared to treatment with each agent alone. In contrast, concomitant treatment with TAS-205 and fexofenadine, a histamine H1 blocker, showed inhibitory effects on late phase and early phase nasal obstruction, although the magnitude of the inhibitory effects upon combined administration was comparable to that of each single treatment. These results suggest that combined treatment with an HPGDS inhibitor and different types of anti-allergic medicine may be a promising strategy to control nasal obstruction in AR patients.

Investigating the utility of adult zebrafish ex vivo whole hearts to pharmacologically screen hERG channel activator compounds.

There is significant interest in the potential utility of small-molecule activator compounds to mitigate cardiac arrhythmia caused by loss of function of hERG1a voltage-gated potassium channels. Zebrafish (Danio rerio) have been proposed as a cost-effective, high-throughput drug-screening model to identify compounds that cause hERG1a dysfunction. However, there are no reports on the effects of hERG1a activator compounds in zebrafish and consequently on the utility of the model to screen for potential gain-of-function therapeutics. Here, we examined the effects of hERG1a blocker and types 1 and 2 activator compounds on isolated zkcnh6a (zERG3) channels in the Xenopus oocyte expression system as well as action potentials recorded from ex vivo adult zebrafish whole hearts using optical mapping. Our functional data from isolated zkcnh6a channels show that under the conditions tested, these channels are blocked by hERG1a channel blockers (dofetilide and terfenadine), and activated by type 1 (RPR260243) and type 2 (NS1643, PD-118057) hERG1a activators with higher affinity than hKCNH2a channels (except NS1643), with differences accounted for by different biophysical properties in the two channels. In ex vivo zebrafish whole hearts, two of the three hERG1a activators examined caused abbreviation of the action potential duration (APD), whereas hERG1a blockers caused APD prolongation. These data represent, to our knowledge, the first pharmacological characterization of isolated zkcnh6a channels and the first assessment of hERG enhancing therapeutics in zebrafish. Our findings lead us to suggest that the zebrafish ex vivo whole heart model serves as a valuable tool in the screening of hKCNH2a blocker and activator compounds.

Fexofenadine inhibits TNF signaling through targeting to cytosolic phospholipase A2 and is therapeutic against inflammatory arthritis.

OBJECTIVE: Tumour necrosis factor alpha (TNF-α) signalling plays a central role in the pathogenesis of various autoimmune diseases, particularly inflammatory arthritis. This study aimed to repurpose clinically approved drugs as potential inhibitors of TNF-α signalling in treatment of inflammatory arthritis. METHODS: In vitro and in vivo screening of an Food and Drug Administration (FDA)-approved drug library; in vitro and in vivo assays for examining the blockade of TNF actions by fexofenadine: assays for defining the anti-inflammatory activity of fexofenadine using TNF-α transgenic (TNF-tg) mice and collagen-induced arthritis in DBA/1 mice. Identification and characterisation of the binding of fexofenadine to cytosolic phospholipase A2 (cPLA2) using drug affinity responsive target stability assay, proteomics, cellular thermal shift assay, information field dynamics and molecular dynamics; various assays for examining fexofenadine inhibition of cPLA2 as well as the dependence of fexofenadine's anti-TNF activity on cPLA2. RESULTS: Serial screenings of a library composed of FDA-approved drugs led to the identification of fexofenadine as an inhibitor of TNF-α signalling. Fexofenadine potently inhibited TNF/nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) signalling in vitro and in vivo, and ameliorated disease symptoms in inflammatory arthritis models. cPLA2 was isolated as a novel target of fexofenadine. Fexofenadine blocked TNF-stimulated cPLA2 activity and arachidonic acid production through binding to catalytic domain 2 of cPLA2 and inhibition of its phosphorylation on Ser-505. Further, deletion of cPLA2 abolished fexofenadine's anti-TNF activity. CONCLUSION: Collectively, these findings not only provide new insights into the understanding of fexofenadine action and underlying mechanisms but also provide new therapeutic interventions for various TNF-α and cPLA2-associated pathologies and conditions, particularly inflammatory rheumatic diseases.

ERG Channels Regulate Excitability in Stellate and Bushy Cells of Mice Ventral Cochlear Nucleus.

ERG (ether-a-go-go-related gene) channels are the members of the voltage-dependent potassium channel family, which have three subtypes, as ERG1 (Kv 11.1), ERG2 (Kv 11.2), and ERG3 (Kv11.3). There is no information on ERG channels in the cochlear nucleus (CN) neurons, which is the first relay station of the auditory pathway. As occur in some of congenital long QT Syndromes (LQTS), mutation of the KCNQ11 genes for ERG channel has been reported to be accompanied by hearing loss. For that reason, we aimed to study biophysical properties and physiological importance, and contribution of ERG K+ currents to the formation of action potentials in the stellate and bushy neurons of the ventral cochlear nucleus (VCN). A total of 70 mice at 14-17 days old were used for this study. Electrophysiological characterization of ERG channels was performed using patch-clamp technique in the CN slices. In current clamp, ERG channel blockers, terfenadine (10 µM) and E-4031 (10 µM), were applied in both cell types. The activation, inactivation, and deactivation kinetics of the ERG channels were determined by voltage clamp. In conclusion, the findings obtained in the present study suggest that stellate and bushy neurons express ERG channels and ERG channels appear to contribute to setting action potential (AP) frequency, threshold for AP induction, and, possibly, resting membrane potentials in this cells.

Histamine receptor 1 inhibition enhances antitumor therapeutic responses through extracellular signal-regulated kinase (ERK) activation in breast cancer.

Histamine receptor 1 (HRH1) belongs to the rhodopsin-like G-protein-coupled receptor family. Its activation by histamine triggers cell proliferation, embryonic development, and tumor growth. We recently established that HRH1 is up-regulated in basal and human epidermal growth factor receptor 2 (HER2)-enriched human breast tumors and that its expression correlates with a worse prognosis. Nevertheless, the functional role of HRH1 in basal and HER2-targeted therapy-resistant breast cancer (BC) progression has not yet been addressed. Using terfenadine, a selective chemical inhibitor of HRH1, we showed that the inhibition of HRH1 activity in basal BC cells leads to sub-G0 cell accumulation, suppresses proliferation, promotes cell motility and triggers the activation of extracellular signal-regulated kinase (ERK) signaling, initiating the mitochondrial apoptotic pathway. Furthermore, HER2-targeted therapy-resistant cells express higher levels of HRH1 and are more sensitive to terfenadine treatment. Moreover, in vivo experiments showed that terfenadine therapy reduced the tumor growth of basal and trastuzumab-resistant BC cells. In conclusion, our results suggest that targeting HRH1 is a promising new clinical approach to consider that could enhance the effectiveness of current therapeutic treatment in patients with basal and BC tumors resistant to HER2-targeted therapies.

Curcumin as an In Vivo Selective Intestinal Breast Cancer Resistance Protein Inhibitor in Cynomolgus Monkeys.

To estimate the clinical impact of pharmacokinetic modulation via breast cancer resistance protein (BCRP), in vivo approaches in nonclinical settings are desired in drug development. Clinical observation has identified curcumin as a promising candidate for in vivo selective BCRP inhibition, in addition to several well known inhibitors, such as lapatinib and pantoprazole. This study aimed to confirm the inhibitory efficacy of curcumin on gastrointestinal BCRP function in cynomolgus monkeys and to perform comparisons with lapatinib and pantoprazole. Oral area under the plasma concentration-time curve (AUC) and bioavailability of well known BCRP (sulfasalazine and rosuvastatin), P-glycoprotein (fexofenadine, aliskiren, and talinolol), and CYP3A (midazolam) substrates were investigated in the presence and absence of inhibitors. Oral exposures of sulfasalazine and rosuvastatin were markedly elevated by curcumin with minimal changes in systemic clearance, whereas pharmacokinetic alterations after fexofenadine, aliskiren, and talinolol oral exposure were limited. Curcumin increased oral midazolam exposure without affecting systemic clearance, presumably owing to partial inhibition of intestinal CYP3A. Lapatinib increased the oral AUC for sulfasalazine to a greater extent than curcumin did, whereas pantoprazole had a smaller effect. However, lapatinib also exerted significant effects on fexofenadine, failed to selectively discriminate between BCRP and P-glycoprotein inhibition, and had an effect on oral midazolam exposure comparable with that of curcumin. Thus, pharmacokinetic evaluation in monkeys demonstrated that pretreatment with curcumin as an in vivo selective BCRP inhibitor was more appropriate than pretreatment with lapatinib and pantoprazole for the assessment of the impact of BCRP on gastrointestinal absorption in nonrodent models.

Terfenadine combined with epirubicin impedes the chemo-resistant human non-small cell lung cancer both in vitro and in vivo through EMT and Notch reversal.

The acquired resistance of non-small cell lung cancer (NSCLC) to taxanes eventually leads to the recurrence and metastasis of tumours. Thus, the development of therapeutic strategies based on the mechanisms by which cells acquire resistance to prolong their survival rate in chemotherapy drug treatment failure patients are warranted. In this study, we found that the resistant cells acquired increased migratory and invasive capabilities, and this transformation was correlated with epithelial-mesenchymal transition (EMT) and Notch pathway deregulation in the resistant cells. Finally, we reported for the first time that terfenadine augmented the effect of epirubicin (EPI) better than Taxol and cisplatin (DDP) by inhibiting migration, invasion, and the EMT phenotype, and the combination therapy also reversed Notch signalling pathway and enhanced the accumulation of fluorescent P-gp substrate rhodamine 123 (Rh123). Similar activities of terfenadine on EPI were observed in xenografts. All of our results confirmed that terfenadine combined with EPI synergistically inhibits the growth and metastatic processes of resistant cells both in vitro and in vivo. Therefore, terfenadine or its derivatives are a promising approach for the clinical challenge of resistance in patients with advanced NSCLC.

Mechanism of As2O3-Induced Action Potential Prolongation and Using hiPS-CMs to Evaluate the Rescue Efficacy of Drugs With Different Rescue Mechanism.

Arsenic trioxide (As2O3) has been verified as a breakthrough in the management of acute promyelocytic leukemia in recent decades. However, cardiotoxicity, especially long QT syndrome (LQTS) has become the most important issue during As2O3 treatment. The characterized mechanisms behind this adverse effect are inhibition of cardiac hERG channel trafficking and increase of cardiac calcium currents. In our study, we found a new pathway underlying As2O3-induced cardiotoxicity that As2O3 accelerates lysosomal degradation of hERG on plasma membrane after using brefeldin A (BFA) to block protein trafficking. Then we explored pharmacological rescue strategies on As2O3-induced LQTS, and found that 4 therapeutic agents exert rescue efficacy via 3 different pathways: fexofenadine and astemizole facilitate hERG trafficking via promotion of channel-chaperone formation after As2O3 incubation; ranolazine slows hERG degradation in the presence of As2O3; and resveratrol shows significant attenuation on calcium current increase triggered by As2O3. Moreover, we used human-induced pluripotent stem cell derived cardiomyocytes (hiPS-CMs) to evaluate the rescue effects of the above agents on As2O3-induced prolongation of action potential duration (APD) and demonstrated that fexofenadine and resveratrol significantly ameliorate the prolonged APD. These observations suggested that pharmacological chaperone like fexofenadine and resveratrol might have the potential to protect against the cardiotoxicity of As2O3.

Stimulating Effect of Terfenadine on Erythrocyte Cell Membrane Scrambling.

BACKGROUND/AIMS: The antihistaminic drug Terfenadine may trigger apoptosis of tumor cells, an effect unrelated to its effect on histamine receptors. Similar to apoptosis of nucleated cells, erythrocytes may enter eryptosis, the suicidal death of erythrocytes characterized by cell shrinkage and cell membrane scrambling with phosphatidylserine translocation to the erythrocyte surface. Signaling triggering eryptosis include increase of cytosolic Ca2+ activity ([Ca2+]i), oxidative stress, and ceramide. The present study explored, whether Terfenadine is capable to trigger eryptosis. METHODS: Flow cytometry was employed to estimate phosphatidylserine abundance at the erythrocyte surface from annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, abundance of reactive oxygen species (ROS) from 2',7'-dichlorodihydrofluorescein (DCF) diacetate dependent fluorescence, and ceramide abundance at the human erythrocyte surface utilizing specific antibodies. Hemolysis was quantified from haemoglobin concentration in the supernatant. RESULTS: A 48 hours exposure of human erythrocytes to Terfenadine (≥ 5 µM) significantly increased the percentage of annexin-V-binding cells and triggered hemolysis without significantly modifying the average forward scatter. Terfenadine (7.5 µM) significantly increased Fluo3-fluorescence, but did not significantly modify DCF fluorescence or ceramide abundance. The effect of Terfenadine on annexin-V-binding was significantly blunted but not abolished by removal of extracellular Ca2+. Exposure of human erythrocytes to Ca2+ ionophore ionomycin (1 µM, 15 min) triggered annexin-V-binding, an effect augmented by Terfenadine pretreatment (10 µM, 48 hours). CONCLUSIONS: Terfenadine triggers phospholipid scrambling of the human erythrocyte cell membrane, an effect in part due to entry of extracellular Ca2+ and in part due to sensitizing human erythrocyte cell membrane scrambling to Ca2+.

Replacement of cardiotoxic aminopiperidine linker with piperazine moiety reduces cardiotoxicity? Mycobacterium tuberculosis novel bacterial topoisomerase inhibitors.

Recently numerous non-fluoroquinolone-based bacterial type II topoisomerase inhibitors from both the GyrA and GyrB classes have been reported as antibacterial agents. Inhibitors of the GyrA class include aminopiperidine-based novel bacterial type II topoisomerase inhibitors (NBTIs). However, inhibition of the cardiac ion channel remains a serious liability for the aminopiperidine based NBTIs. In this paper we replaced central aminopiperidine linker with piperazine moiety and tested for its biological activity. We developed a series of twenty four compounds with a piperazine linker 1-(2-(piperazin-1-yl)ethyl)-1,5-naphthyridin-2(1H)-one, by following a multistep protocol. Among them compound 4-(2-(7-methoxy-2-oxo-1,5-naphthyridin-1(2H)-yl)ethyl)-N-(4-nitrophenyl)piperazine-1-carboxamide (11) was the most promising inhibitor with Mycobacterium tuberculosis (MTB) DNA gyrase enzyme supercoiling IC50 of 0.29±0.22µM, with a good MTB MIC of 3.45µM. These kind of compounds retains good potency and showed reduced cardiotoxicity compared to aminopiperidines.

Mechanism and pharmacological rescue of berberine-induced hERG channel deficiency.

Berberine (BBR), an isoquinoline alkaloid mainly isolated from plants of Berberidaceae family, is extensively used to treat gastrointestinal infections in clinics. It has been reported that BBR can block human ether-a-go-go-related gene (hERG) potassium channel and inhibit its membrane expression. The hERG channel plays crucial role in cardiac repolarization and is the target of diverse proarrhythmic drugs. Dysfunction of hERG channel can cause long QT syndrome. However, the regulatory mechanisms of BBR effects on hERG at cell membrane level remain unknown. This study was designed to investigate in detail how BBR decreased hERG expression on cell surface and further explore its pharmacological rescue strategies. In this study, BBR decreases caveolin-1 expression in a concentration-dependent manner in human embryonic kidney 293 (HEK293) cells stably expressing hERG channel. Knocking down the basal expression of caveolin-1 alleviates BBR-induced hERG reduction. In addition, we found that aromatic tyrosine (Tyr652) and phenylalanine (Phe656) in S6 domain mediate the long-term effect of BBR on hERG by using mutation techniques. Considering both our previous and present work, we propose that BBR reduces hERG membrane stability with multiple mechanisms. Furthermore, we found that fexofenadine and resveratrol shorten action potential duration prolongated by BBR, thus having the potential effects of alleviating the cardiotoxicity of BBR.

Impact of fexofenadine, osthole and histamine on peripheral blood mononuclear cell proliferation and cytokine secretion.

This paper compares results of peripheral blood mononuclear cell (PBMC) incubation with fexofenadine (FXF) and osthole. FXF is a third-generation antihistamine drug and osthole is assumed a natural antihistamine alternative. To our best knowledge, this is the first comparative study on FXF, osthole and histamine cytokine secretion and cytotoxicity in PBMC in vitro cultures using cell proliferation ELISA BrdU. The cultures were treated 12, 42, 48 and 72h with FXF and osthole at 150, 300 and 450ng/ml concentrations and histamine at 50, 100 and 200ng/ml. Our study results confirm that FXF, osthole and histamine exert no cytotoxic effect on PBMCs and that IL-6, IL-10 and TNF-α cytokine secretion following osthole cell stimulation was similar to that by FXF stimulation.This confirms our hypothesis that osthole is a natural histamine antagonist, and can therefore be beneficially applied in antihistamine treatment.