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Background: Most immunotherapies approved for clinical use rely on the use of recombinant proteins and cell-based approaches, rendering their manufacturing expensive and logistics onerous. The identification of novel small molecule immunotherapeutic agents might overcome such limitations., Method: For immunopharmacological screening campaigns, we built an artificial miniature immune system in which dendritic cells (DCs) derived from immature precursors present MHC (major histocompatibility complex) class I-restricted antigen to a T-cell hybridoma that then secretes interleukin-2 (IL-2)., Results: The screening of three drug libraries relevant to known signaling pathways, FDA (Food and Drug Administration)-approved drugs and neuroendocrine factors yielded two major hits, astemizole and ikarugamycin. Mechanistically, ikarugamycin turned out to act on DCs to inhibit hexokinase 2, hence stimulating their antigen presenting potential. In contrast, astemizole acts as a histamine H1 receptor (H1R1) antagonist to activate T cells in a non-specific, DC-independent fashion. Astemizole induced the production of IL-2 and interferon-γ (IFN-γ) by CD4 + and CD8 + T cells both in vitro and in vivo. Both ikarugamycin and astemizole improved the anticancer activity of the immunogenic chemotherapeutic agent oxaliplatin in a T cell-dependent fashion. Of note, astemizole enhanced the CD8 + /Foxp3 + ratio in the tumor immune infiltrate as well as IFN-γ production by local CD8 + T lymphocytes. In patients with cancer, high H1R1 expression correlated with low infiltration by TH1 cells, as well as with signs of T-cell exhaustion. The combination of astemizole and oxaliplatin was able to cure the majority of mice bearing orthotopic non-small cell lung cancers (NSCLC), then inducing a state of protective long-term immune memory. The NSCLC-eradicating effect of astemizole plus oxaliplatin was lost on depletion of either CD4 + or CD8 + T cells, as well as on neutralization of IFN-γ., Conclusions: These findings underscore the potential utility of this screening system for the identification of immunostimulatory drugs with anticancer effects., Competing Interests: Competing interests: OK is a co-founder of Samsara Therapeutics. GK has been holding research contracts with Daiichi Sankyo, Eleor, Kaleido, Lytix Pharma, PharmaMar, Osasuna Therapeutics, Samsara Therapeutics, Sanofi, Tollys, and Vascage. GK has been consulting for Reithera. GK is on the Board of Directors of the Bristol Myers Squibb Foundation France. GK is a scientific co-founder of everImmune, Osasuna Therapeutics, Samsara Therapeutics and Therafast Bio. GK is the inventor of patents covering therapeutic targeting of aging, cancer, cystic fibrosis and metabolic disorders. GK’s wife, Laurence Zitvogel, has held research contracts with 9 Meters Biopharma, Daiichi Sankyo, Pilege, was on the Board of Directors of Transgene, is a co-founder of everImmune, and holds patents covering the treatment of cancer and the therapeutic manipulation of the microbiota. GK’s brother, Romano Kroemer, was an employee of Sanofi and now consults for Boehringer-Ingelheim., (© Author(s) (or their employer(s)) 2023. Re-use permitted under CC BY-NC. No commercial re-use. See rights and permissions. Published by BMJ.)

Iterative medicinal chemistry optimization of an ester-containing astemizole (AST) analogue 1 with an associated metabolic instability liability led to the identification of a highly potent 3-trifluoromethyl-1,2,4-oxadiazole analogue 23 ( Pf NF54 IC 50 = 0.012 μM; Pf K1 IC 50 = 0.040 μM) displaying high microsomal metabolic stability (HLM CL int < 11.6 μL·min -1 ·mg -1 ) and > 1000-fold higher selectivity over hERG compared to AST. In addition to asexual blood stage activity, the compound also shows activity against liver and gametocyte life cycle stages and demonstrates in vivo efficacy in Plasmodium berghei -infected mice at 4 × 50 mg·kg -1 oral dose. Preliminary interrogation of the mode of action using live-cell microscopy and cellular heme speciation revealed that 23 could be affecting multiple processes in the parasitic digestive vacuole, with the possibility of a novel target at play in the organelles associated with it.

The hEag1 (Kv10.1) K + channel is normally found in the brain, but it is ectopically expressed in tumor cells, including osteosarcoma. Based on the pivotal role of ion channels in osteogenesis, we tested whether pharmacological modulation of hEag1 may affect osteogenic differentiation of osteosarcoma cell lines. Using molecular biology (RT-PCR), electrophysiology (patch-clamp) and pharmacology (astemizole sensitivity, IC 50 = 0.135 μM) we demonstrated that SaOS-2 osteosarcoma cells also express hEag1 channels. SaOS-2 cells also express to KCa1.1 K + channels as shown by mRNA expression and paxilline sensitivity of the current. The inhibition of hEag1 (2 μM astemizole) or KCa1.1 (1 mM TEA) alone did not induce Ca 2+ deposition in SaOS-2 cultures, however, these inhibitors, at identical concentrations, increased Ca 2+ deposition evoked by the classical or pathological (inorganic phosphate, Pi) induction pathway without causing cytotoxicity, as reported by three completer assays (LDH release, MTT assay and SRB protein assay). We observed a similar effect of astemizole on Ca 2+ deposition in MG-63 osteosarcoma cultures as well. We propose that the increase in the osteogenic stimuli-induced mineral matrix formation of osteosarcoma cell lines by inhibiting hEag1 may be a useful tool to drive terminal differentiation of osteosarcoma.

The transient receptor potential (TRP) channel TRPV2 is widely expressed in a variety of different cell types and tissues. However, elucidating the exact biological functions of TRPV2 is significantly hampered by the lack of selective pharmacological tools to modulate channel activity in vitro and in vivo. This study aimed to identify new compounds that modify TRPV2 activity via the use of a plate-based calcium imaging approach to screen a drug repurposing library. Three antihistaminic drugs, loratadine, astemizole and clemizole were identified to reduce calcium-influx evoked by the TRPV2 agonist tetrahydrocannabivarin in HEK293 cells expressing murine TRPV2. Using single-cell calcium-microfluorimetry and whole-cell patch clamp recordings, we further confirmed that all three compounds induced a concentration-dependent block of TRPV2-mediated Ca 2+ influx and whole-cell currents, with loratadine being the most potent antagonist of TRPV2. Moreover, this study demonstrated that loratadine was able to block both the human and mouse TRPV2 orthologs, without inhibiting the activity of other closely related members of the TRPV superfamily. Finally, loratadine inhibited TRPV2-dependent responses in a primary culture of mouse endometrial stromal cells and attenuated cell proliferation and migration in in vitro cell proliferation and wound healing assays. Taken together, our study revealed that the antihistaminic drugs loratadine, astemizole and clemizole target TRPV2 in a concentration-dependent manner. The identification of these antihistaminic drugs as blockers of TRPV2 may form a new starting point for the synthesis of more potent and selective TRPV2 antagonists, which could further lead to the unravelling of the physiological role of the channel., (Copyright © 2022. Published by Elsevier B.V.)

Since the beginning of December 2019, a novel Coronavirus severe respiratory disease, caused by Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) which also been termed 2019-new CoV (2019-nCoV), has continued to spread worldwide. As of August 27, 2020, a total of 24,232,429 people have been infected and 826,518 people have died. In our study, we found that astemizole can antagonize ACE2 and inhibit the entry of SARS-COV-2 spike pseudovirus into ACE2-expressed HEK293T cells (ACE2hi cells). We analysied the binding character of astemizole to ACE2 by molecular docking and surface plasmon resonance (SPR) assays and molecule docking, SARS-COV-2 spike pseudotype virus was also taken to investigate the suppression viropexis effect of astemizole. The results showed that astemizole can bind to the ACE2 receptor and inhibit the invasion of SARS-COV-2 Spike pseudoviruses. Thus astemizole represent potential drug candidates that can be re-used in anti-coronavirus therapies., (Copyright © 2021. Published by Elsevier Ltd.)

Disruption of EZH2-embryonic ectoderm development (EED) protein-protein interaction (PPI) is a new promising cancer therapeutic strategy. We have previously reported the discovery of astemizole, a small-molecule inhibitor targeting the EZH2-EED PPI. Herein, we report the cocrystal structure of EED in complex with astemizole at 2.15 Å. The structure elucidates the detailed binding mode of astemizole to EED and provides a structure-guided design for the discovery of a novel EZH2-EED interaction inhibitor, DC-PRC2in-01, with an affinity K d of 4.56 μM. DC-PRC2in-01 destabilizes the PRC2 complex, thereby leading to the degradation of PRC2 core proteins and the decrease of global H3K27me3 levels in cancer cells. The proliferation of PRC2-driven lymphomas cells is effectively inhibited, and the cell cycle is arrested in the G0/G1 phase. Together, these data demonstrate that DC-PRC2in-01 could be an effective chemical probe for investigating the PRC2-related physiology and pathology and providing a promising chemical scaffold for further development.

Background: Anthelminthic treatment options against schistosomiasis are limited. The current treatment relies almost exclusively on a single drug, praziquantel (PZQ). As a consequence, the development of resistance to PZQ and limited activity of PZQ against earlier development stages are respectively a risk and a limitation to achieving the goals of the new WHO roadmap towards elimination. For the discovery of new chemical starting points, the in vitro drug screening on Schistosoma mansoni (S. mansoni) against newly transformed schistosomula (NTS) is still the most predominant approach. The use of only NTS in the initial screening limits sensitivity to potential new compounds which are predominantly active in later developmental stages. Using our recently described highly standardized, straightforward and reliable culture method that generates high rates of juvenile worms, we aimed to repurpose a subset of the National Center for Advancing Translational Sciences (NCATS) Pharmaceutical Collection (340 compounds) to identify new hits with an in vitro worm culture assay., Methodology/principal Findings: Cercariae were mechanically transformed into skin-stage (SkS) schistosomula and continuously cultured for 3-6 weeks to the liver stage (LiS). A commercial source of serum was identified, and decrease of NTS/well along with optimal drug testing conditions was established to test compounds on early and late LiS worms. The library was screened in 96-well format assays using praziquantel (PZQ) as a positive control. Primary screening allowed a 5.9% hit rate and generated two confirmed hits on adult worms; a prophylactic antianginal agent and an antihistaminic drug., Conclusion: With this standardized and reliable in vitro assay, important S. mansoni developmental stages up to LiS worms can be generated and cultured over an extended period. When exposed to a subset of the NCATS Pharmaceutical Collection, 3 compounds yielded a defined anti-schistosomal phenotype on juvenile worms. Translation of activity on perfused adult S. mansoni worms was achieved only for perhexiline (a prophylactic antianginal agent) and astemizole (an antihistaminic drug)., Competing Interests: I have read the journal’s policy and the authors of this manuscript have the following competing interests: TS is an employee of Ares Trading SA, an affiliate of Merck KGaA, Darmstadt, Germany. The other authors have declared that no competing interests exist.

The hERG channel is a voltage-gated potassium channel involved in cardiac repolarization. Off-target hERG inhibition by drugs has become a critical issue in the pharmaceutical industry. The three-dimensional structure of the hERG channel was recently reported at 3.8-Å resolution using cryogenic electron microscopy (cryo-EM). However, the drug inhibition mechanism remains unclear because of the scarce structural information regarding the drug- and potassium-bound hERG channels. In this study, we obtained the cryo-EM density map of potassium-bound hERG channel complexed with astemizole, a well-known hERG inhibitor that increases risk of potentially fatal arrhythmia, at 3.5-Å resolution. The structure suggested that astemizole inhibits potassium conduction by binding directly below the selectivity filter. Furthermore, we propose a possible binding model of astemizole to the hERG channel and provide insights into the unusual sensitivity of hERG to several drugs., Competing Interests: Declaration of interests H.O. and T. Maru are employees of Axcelead Drug Discovery Partners, Inc. R.I. is an employee of Daiichi Sankyo RD Novare Co., Ltd. K.Y. and S.I. are employees of Takeda Pharmaceutical Co., Ltd., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Cytochrome P450 2J2 (CYP2J2) is responsible for the epoxidation of endogenous arachidonic acid, and is involved in the metabolism of exogenous drugs. To date, no crystal structure of CYP2J2 is available, and the proposed structural basis for the substrate recognition and specificity in CYP2J2 varies with the structural models developed using different computational protocols. In this study, we developed a new structural model of CYP2J2, and explored its sensitivity to substrate binding by molecular dynamics simulations of the interactions with chemically similar fluorescent probes. Our results showed that the induced-fit binding of these probes led to the preferred active poses ready for the catalysis by CYP2J2. Divergent conformational dynamics of CYP2J2 due to the binding of each probe were observed. However, a stable hydrophobic clamp composed of residues I127, F310, A311, V380, and I487 was identified to restrict any substrate access to the active site of CYP2J2. Molecular docking of a series of compounds including amiodarone, astemizole, danazol, ebastine, ketoconazole, terfenadine, terfenadone, and arachidonic acid to CYP2J2 confirmed the role of those residues in determining substrate binding and specificity of CYP2J2. In addition to the flexibility of CYP2J2, the present work also identified other factors such as electrostatic potential in the vicinity of the active site, and substrate strain energy and property that have implications for the interpretation of CYP2J2 metabolism.

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 IC 50 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 IC 50 values., Results: All IC 50 values of 21 compounds recorded on the SyncroPatch 384PE correlated well with IC 50 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 IC 50 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., Competing Interests: Declaration of Competing Interest Authors Nina Brinkwirth, Nadine Becker, Alison Obergrussberger, Søren Friis, Takayuki Oka, Atsushi Ohtsuki, Niels Fertig and Sonja Stoelzle-Feix are all employed by Nanion Technologies, the manufacturers of the SyncroPatch 384i, SyncroPatch 384PE and Patchliner used to compile this manuscript., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Current practices in drug development have led to therapeutic compounds being approved for widespread use in humans, only to be later withdrawn due to unanticipated toxicity. These occurrences are largely the result of erroneous data generated by in vivo and in vitro preclinical models that do not accurately recapitulate human physiology. Herein, a human primary cell- and stem cell-derived 3D organoid technology is employed to screen a panel of drugs that were recalled from market by the FDA. The platform is comprised of multiple tissue organoid types that remain viable for at least 28 days, in vitro. For many of these compounds, the 3D organoid system was able to demonstrate toxicity. Furthermore, organoids exposed to non-toxic compounds remained viable at clinically relevant doses. Additional experiments were performed on integrated multi-organoid systems containing liver, cardiac, lung, vascular, testis, colon, and brain. These integrated systems proved to maintain viability and expressed functional biomarkers, long-term. Examples are provided that demonstrate how multi-organoid 'body-on-a-chip' systems may be used to model the interdependent metabolism and downstream effects of drugs across multiple tissues in a single platform. Such 3D in vitro systems represent a more physiologically relevant model for drug screening and will likely reduce the cost and failure rate associated with the approval of new drugs.

Retinoblastoma is the most common pediatric intraocular malignant tumor. Unfortunately, low cure rates and low life expectancy are observed in low-income countries. Thus, alternative therapies are needed for patients who do not respond to current treatments or those with advanced cases of the disease. Ether à-go-go-1 (Eag1) is a voltage-gated potassium channel involved in cancer. Eag1 expression is upregulated by the human papilloma virus (HPV) oncogene E7, suggesting that retinoblastoma protein (pRb) may regulate Eag1 . Astemizole is an antihistamine that is suggested to be repurposed for cancer treatment; it targets proteins implicated in cancer, including histamine receptors, ATP binding cassette transporters, and Eag channels. Here, we investigated Eag1 regulation using pRb and Eag1 expression in human retinoblastoma. The effect of astemizole on the cell proliferation of primary human retinoblastoma cultures was also studied. HeLa cervical cancer cells (HPV-positive and expressing Eag1) were transfected with RB1 . Eag1 mRNA expression was studied using qPCR, and protein expression was assessed using western blotting and immunochemistry. Cell proliferation was evaluated with an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assay. RB1 transfection down-regulated Eag1 mRNA and protein expression. The human retinoblastoma samples displayed heterogeneous Eag1 mRNA and protein expression. Astemizole decreased cell proliferation in primary retinoblastoma cultures. Our results suggest that Eag1 mRNA and protein expression was regulated by pRb in vitro, and that human retinoblastoma tissues had heterogeneous Eag1 mRNA and protein expression. Furthermore, our results propose that the multitarget drug astemizole may have clinical relevance in patients with retinoblastoma, for instance, in those who do not respond to current treatments., Competing Interests: The authors declare no conflict of interest.

Detection of adverse effects of cardiac toxicity at an early stage by in vitro methods is crucial for the preclinical drug screening. Over the years, several kinds of biosensing platforms have been proposed by the scientific society for the detection of cardiac toxicity. However, the proposed tissue platforms have been optimized to measure either mechanophysiology or electrophysiology of the cardiomyocytes but not both. Herein, we demonstrate in detail our successful attempt toward developing a novel "multifunctional microphysiological system" also known as "organs-on-chips" to measure simultaneously the mechanical and electrical characteristics of cardiomyocytes in vitro. The proposed device can rapidly recognize drug-induced cardiovascular toxicity in real time, which is one of the most significant factors for drug discovery and postmarketing surveillance. We confirm that the proposed sensor delivers the direct relationship between the contraction force and cell impedance of cardiomyocytes under the influence of different cardiovascular drugs such as verapamil, astemizole, and lidocaine. The obtained assay results provide a great potential for a deep understanding of the drug effects on the cardiomyocytes in vitro.

Polycomb group proteins are important epigenetic regulators for cell proliferation and differentiation, organ development, as well as initiation and progression of lethal diseases, including cancer. Upregulated Polycomb group proteins, including Enhancer of zeste homolog 2 (EZH2), promote proliferation, migration, invasion and metastasis of cancer cells, as well as self-renewal of cancer stem cells. In our study, we report that EZH2 and embryonic ectoderm development (EED) indicate respective direct interaction with androgen receptor (AR). In the context of AR-positive prostate cancer, EZH2 and EED regulate AR expression levels and AR downstream targets. More importantly, we demonstrate that targeting EZH2 with the small-molecule inhibitor astemizole in cancer significantly represses the EZH2 and AR expression as well as the neoplastic capacities. These results collectively suggest that pharmacologically targeting EZH2 might be a promising strategy for advanced prostate cancer., (© 2019 UICC.)

The objective of this study is to assess the response of telemetered common marmosets to multiple cardiac ion channel inhibitors and to clarify the usefulness of this animal model in evaluating the effects of drug candidates on electrocardiogram (ECG). Six multiple cardiac ion channel inhibitors (sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine) were orally administered to telemetered common marmosets and changes in QTc, PR interval and QRS duration were evaluated. Drugs plasma levels were determined to compare the sensitivity in common marmosets to that in humans. QTc prolongation was observed in the marmosets dosed with sotalol, astemizole, flecainide, quinidine, verapamil and terfenadine. PR prolongation was noted after flecainide and verapamil administration, and QRS widening occurred following treatment with flecainide and quinidine. Drugs plasma levels associated with ECG changes in marmosets were similar to those in humans, except for verapamil-induced QTc prolongation. Verapamil-induced change is suggested due to body temperature decrease. These results indicate that telemetered common marmoset is a useful animal for evaluation of the ECG effects of multiple cardiac ion channel inhibitors and the influence of body temperature change should be considered in the assessment.

Background: Expression and activity of the potassium channel ether-à-go-go-1 (EAG1) are strongly related to carcinogenesis and tumor progression, which can be exploited for therapeutic purposes. EAG1 activity may be reduced by preventing its phosphorylation with epidermal growth factor receptor (EGFR) kinase inhibitors and by astemizole, which blocks the channel pore and downregulates its gene expression., Objective: We aimed to study the potential cooperative antiproliferative effect of the EGFR inhibitor gefitinib and the EAG1-blocker astemizole, in breast cancer cells., Materials and Methods: The cells were characterized by immunocytochemistry. Inhibitory concentrations were determined by non-linear regression analysis using dose-response curves. The nature of the pharmacological effect was evaluated by the combination index equation while cell cycle analysis was studied by flow cy-tometry., Results: Astemizole and gefitinib inhibited cell proliferation in a concentration-dependent manner, with inhibitory concentrations (IC 50) values of 1.72 µM and 0.51 µM, respectively. All combinations resulted in a synergistic antiproliferative effect. The combination of astemizole and gefitinib diminished the percentage of cells in G2/M and S phases, while increased accumulation in G0/G1 of the cell cycle., Conclusions: Astemizole and gefitinib synergistically inhibited proliferation in breast cancer cells expressing both EGFR and EAG1. Our results suggest that the combined treatment increased cell death by targeting the oncogenic activity of EAG1., (Copyright: © 2019 Permanyer.)

In this study, the immunomodulatory effects of astemizole (AST) against lipopolysaccharide (LPS) mediated T cell proliferation and induction of inflammation in RAW macrophages (in vitro), and zebrafish larvae (in vivo) were determined. AST significantly suppressed the phagocytic activity of macrophages (3.303 ± 0.115) and inhibited lysosomal enzyme secretion (13.27 ± 2.52) induced by LPS (100 ng/ml). Moreover, AST subdued the morphological deformities such as yolk sac edema (YSE) and spinal curvature curving (SC) by inhibiting ROS generation in zebrafish larvae 24 h after microinjection of LPS (0.5 mg/ml). AST was also shown to inhibit the production of the major cytokines TNF-α (150.8 ± 0.6), IL-1β (276.5 ± 1.6), and PGE 2 (194.6 ± 0.6) pg/ml in RAW macrophages. It also subdued the ROS induced iNOS and COX-2 generated in response to LPS mediated immune dysfunctions in zebrafish larvae. These results suggested the immunosuppression effect of AST. Furthermore, induction of immune-suppression due to AST resulted in significant down-regulation of innate immunity directed by MAPK (p38, ERK and JNK), which was found to be associated with decreased production of acute inflammatory mediators both in vitro and in vivo. To confirm its activity, splenocytes were prepared using BALB/c mice and a mitogen activated splenocyte proliferation assay was also performed. Our findings suggest that AST has the ability to inhibit T cell proliferation and cytokine secretion both in vitro and in vivo by interfering with MAPK signaling pathway. Taken together, our results showed the potential of AST as a countermeasure to immune dysfunction and suggest its use as immunosuppressant compound in inflammatory disease., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Hepatocellular carcinoma (HCC) accounts for the fifth most common cancer worldwide. Vitamin D and antihistamines have been shown to play an anti-tumor role in various tumors. In the present study, we ought to investigate the synergistic effect of astemizole and Vitamin D in HCC cells. We showed that astemizole enhanced the anti-tumor effect of Vitamin D in HCC both in vitro and in vivo. Astemizole enhanced Vitamin D-induced decrease of cell viability and proliferation, increase of apoptosis, decrease of cell migration and invasion in HCC cells in vitro and decrease of tumor number, mass and incidence in HCC in vivo. Astemizole increased VDR expression both in HCC cells in vitro and in tumor tissues in vivo. Downregulation of VDR significantly inhibited the synergistic effect of Vitamin D and astemizole on HCC cell viability, proliferation, apoptosis, migration and invasion. Bioinformatics analysis identified that miR-125a-5p had a putative binding site in the 3'-UTR of VDR. miR-125a-5p mimics inhibited astemizole-induced increase of VDR and enhancement of the anti-tumor effect of Vitamin D in HCC. Reporter gene assay has confirmed that VDR was regulated by miR-125a-5p. miR-125a-5p inhibitors increased VDR expression and decreased cell viability and proliferation in HCC cells. Moreover, VDR and miR-125a-5p expression in tumor tissues in HCC patients were negatively correlated. We identified that inhibition of miR-125a-5p and subsequent upregulation of VDR was involved in astemizole-induced enhancement of the anti-tumor effect of Vitamin D in HCC. These results highlight the importance of combined treatment of astemizole and Vitamin D and provide novel insights into the role of miR-125a-5p-VDR signaling in HCC., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Voltage-gated ion channels (VGCs) are prime targets for the pharmaceutical industry, but drug profiling on VGCs is challenging, since drug interactions are confined to specific conformational channel states mediated by changes in transmembrane potential. Here we combined various optogenetic tools to develop dynamic, high-throughput drug profiling assays with defined light-step protocols to interrogate VGC states on a millisecond timescale. We show that such light-induced electrophysiology (LiEp) yields high-quality pharmacological data with exceptional screening windows for drugs acting on the major cardiac VGCs, including hNa v 1.5, hK v 1.5 and hERG. LiEp-based screening remained robust when using a variety of optogenetic actuators (ChR2, ChR2(H134R), CatCh, ChR2-EYFP-βArchT) and different types of organic (RH421, Di-4-ANBDQPQ, BeRST1) or genetic voltage sensors (QuasAr1). The tractability of LiEp allows a versatile and precise alternative to state-of-the-art VGC drug screening platforms such as automated electrophysiology or FLIPR readers.

Prostate cancer (PC) is the main cause of cancer mortality in men worldwide. Therefore, novel treatments for PC are needed. Ether à-go-go-1 (Eag1) potassium channels display oncogenic properties, and have been suggested as early tumor markers and therapeutic targets for different cancers. These channels are overexpressed in many human tumors including PC. Astemizole targets several molecules involved in cancer including Eag1 channels, histamine receptors and ABC transporters. Here we studied Eag1 mRNA expression and protein levels in the non-tumorigenic and non-invasive human prostate RWPE-1 cell line, and in the tumorigenic and highly invasive human prostate WPE1-NB26 cell lines. The effect of astemizole on cell proliferation and apoptosis was also studied. The human prostate cell lines RWPE-1 and WPE1-NB26 were cultured following the provider´s instructions. Eag1 mRNA expression and protein levels were studied by real time RT-PCR and immunocytochemistry, respectively. Cell proliferation and apoptosis were studied by a fluorescence AlamarBlue®  assay and flow cytometry, respectively. No difference in Eag1 mRNA expression was observed between the cell lines. However, high Eag1 protein levels were observed in the invasive WPE1-NB26 cells, in contrast to the weak protein expression in RWPE-1 cells. Accordingly, astemizole decreased cell proliferation at nanomolar concentrations only in the invasive WPE1-NB26 cells.  Our results suggest that astemizole may have clinical relevance for prostate cancer treatment in patients with high Eag1 protein levels.

Astemizole is a H 1 -antagonist endowed with antimalarial activity, but has hERG liabilities. Systematic structural modifications of astemizole led to the discovery of analogues that display very potent activity as inhibitors of the growth of the Plasmodium parasite, but show a decreased hERG inhibition, when compared to astemizole. These compounds can be used as starting point for the development of a new class of antimalarials., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Astemizole has gained attention as an antineoplastic drug that targets important ion channels. The present study aimed to investigate the protective effects of astemizole against hydrogen peroxide (H2O2)‑induced oxidative damage to human umbilical vein endothelial cells (HUVECs). HUVECs were pretreated with astemizole (0.5 and 1 µM) for 12 h, then exposed to H2O2 (200 µM) for 12 h. Cell viability was measured using the MTT assay. The levels of malondialdehyde (MDA), superoxide dismutase (SOD), glutathione peroxidase (GSH‑Px), reactive oxygen species (ROS) and apoptotic percentage were determined. Additionally, the protein expression of p53, p21Cip1/Waf1 and p16INK4a was measured by western blot analysis The results demonstrated that astemizole (0.5‑1 µM) was able to significantly restore the viability of HUVECs under oxidative stress and scavenge intracellular ROS induced by H2O2. Astemizole also suppressed the production of lipid peroxides, such as MDA, and restored the activities of endogenous antioxidants, including SOD and GSH‑Px, indicating that cell apoptosis may be inhibited. In addition, astemizole significantly increased p53, p21Cip1/Waf1 and p16INK4a protein expression. In conclusion, astemizole effectively protected endothelial cells against oxidative stress induced by H2O2, a function that may involve ROS/p53/p21Cip1/Waf1/ p16INK4a signaling pathways. The present study therefore served as a preliminary investigation into the ROS‑protective effects of astemizole, and may pave the way for future studies into the development of this compound as a novel therapy for atherosclerosis.

The loss of dopaminergic neurons and the resultant motor impairment are hallmarks of Parkinson's disease. The SH‑SY5Y cell line is a model of dopaminergic neurons, and allows for the study of dopaminergic neuronal injury. Previous studies have revealed changes in Ether à go‑go 1 (Eag1) potassium channel expression during p53-induced SH‑SY5Y apoptosis, and the regulatory involvement of microRNA‑34a (miR‑34a) was demonstrated. In the present study, the involvement of Eag1 and miR‑34a in rotenone‑induced SH‑SY5Y cell injury was investigated. Rotenone is a neurotoxin, which is often used to generate models of Parkinson's disease, since it causes the death of nigrostriatal neurons by inducing intracellular aggregation of alpha synuclein and ubiquitin. In the present study, rotenone resulted in a dose‑dependent decrease in cell viability, as revealed by 3‑(4,5‑dimethylthiazol‑2‑yl)‑2,5‑diphenyltetrazolium bromide (MTT) and trypan blue cell counting assays. In addition, Eag1 was demonstrated to be constitutively expressed by SH‑SY5Y cells, and involved in cell viability. Suppression of Eag1 with astemizole resulted in a dose‑dependent decrease in cell viability, as revealed by MTT assay. Astemizole also enhanced the severity of rotenone‑induced injury in SH‑SY5Y cells. RNA interference against Eag1, using synthetic small interfering RNAs (siRNAs), corroborated this finding, as siRNAs potentiated rotenone‑induced injury. Eag1‑targeted siRNAs (kv10.1‑3 or EAG1hum&#95;287) resulted in a statistically significant 16.4‑23.5% increase in vulnerability to rotenone. An increased number of apoptotic nuclei were observed in cells transfected with EAG1hum&#95;287. Notably, this siRNA intensified rotenone‑induced apoptosis, as revealed by an increase in caspase 3/7 activity. Conversely, a miR‑34a inhibitor was demonstrated to exert neuroprotective effects. The viability of cells exposed to rotenone for 24 or 48 h and treated with miR‑34a inhibitor was restored by 8.4‑8.8%. In conclusion, Eag1 potassium channels and miR‑34a are involved in the response to rotenone-induced injury in SH‑SY5Y cells. The neuroprotective effect of mir‑34a inhibitors merits further investigations in animal models of Parkinson's disease.

Non-small cell lung cancer (NSCLC) is one of the deadliest cancers worldwide. In search for new NSCLC treatment options, we screened a cationic amphiphilic drug (CAD) library for cytotoxicity against NSCLC cells and identified several CAD antihistamines as inducers of lysosomal cell death. We then performed a cohort study on the effect of CAD antihistamine use on mortality of patients diagnosed with non-localized cancer in Denmark between 1995 and 2011. The use of the most commonly prescribed CAD antihistamine, loratadine, was associated with significantly reduced all-cause mortality among patients with non-localized NSCLC or any non-localized cancer when compared with use of non-CAD antihistamines and adjusted for potential confounders. Of the less frequently described CAD antihistamines, astemizole showed a similar significant association with reduced mortality as loratadine among patients with any non-localized cancer, and ebastine use showed a similar tendency. The association between CAD antihistamine use and reduced mortality was stronger among patients with records of concurrent chemotherapy than among those without such records. In line with this, sub-micromolar concentrations of loratadine, astemizole and ebastine sensitized NSCLC cells to chemotherapy and reverted multidrug resistance in NSCLC, breast and prostate cancer cells. Thus, CAD antihistamines may improve the efficacy of cancer chemotherapy., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Apoptosis and autophagy are genetically regulated, evolutionarily conserved processes that can jointly seal cancer cell fates, and numerous death stimuli are capable of activating either pathway. Although crosstalk between apoptosis and autophagy is quite complex and sometimes contradictory, it remains a key factor determining the outcomes of death-related pathologies such as cancer. In the present study, exposure of MCF-7 breast cancer cells to HIS and the H1 receptor antagonist AST both alone and together with HIS (AST-HIS) led to generation of intracellular ROS, which induced massive cellular vacuolization through dilation of the ER and mitochondria. Consequently, apoptosis by Bax translocation, cytochrome c release, and caspase activation were triggered. In addition, AST-HIS caused ER stress-induced autophagy in MCF-7 cells, as evidenced by an increased LC3-II/LC3-I ratio, with surprisingly no changes in Beclin-1 expression. Non-canonical autophagy was induced via p53 phosphorylation, which increased p53-p62 interactions to enhance Beclin-1-independent autophagy as evidenced by immunocytochemistry and immunoprecipitation. In the absence of Beclin-1, enhanced autophagy further activated apoptosis through caspase induction. In conclusion, these findings indicate that AST-HIS-induced apoptosis and autophagy can be regulated by ROS-mediated signaling pathways., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Three new alkaloids (1, 4 and 8), together with nine known analogues (2, 3, 5-7, and 9-12), were isolated from the marine-derived fungus Penicillium expansum Y32. Their structures including the absolute configurations were elucidated by spectroscopic and Mosher's and Marfey's methods, along with quantum electronic circular dichroism (ECD) calculations. Each of the compounds was evaluated for cardiovascular effects in a live zebrafish model. All of the compounds showed a significant mitigative effect on bradycardia caused by astemizole (ASM) in the heart rate experiments. Compounds 4-6 and 8-12 exhibited potent vasculogenetic activity in vasculogenesis experiments. This is the first study to report that these types of compounds show cardiovascular effects in zebrafish. The results suggest that these compounds could be promising candidates for cardiovascular disease lead compounds.

1. QT prolongation is one of the major safety tests used in the development of a new drug. The ICH guidelines for the evaluation of QT prolongation recommend the use of the in vitro hERG assay and the in vivo telemetry test. However, QT intervals change under normal conditions due to circadian rhythm and can affect the results of the tests. In this study, we developed a PK/PD model to describe the QT interval after the administration of astemizole allowing for the normal changes by circadian rhythm. 2. The typical PK parameters of absorption rate constant (ka), volume of distribution (Vc and Vm), metabolism (km), and elimination rate constant (kel and kel-m) were 0.49 h(-1), 4950 L, 20 L, 0.0127 h(-1), 0.0095 h(-1), and 0.95 h(-1), respectively. The final PK/PD model was the biophase model with the modified harmonic model. The typical PK/PD parameters, base QTc interval (QT0), amplitude (T1, T3), period of QTc interval changing (T2, T4), and EC50 were 233 ms, 3.31, 1.5, -9.24 h, 1.85 h, and 0.81 ng/ml, respectively. 3. The PK/PD model to explain the changes of the QT interval that allows normal changes in the circadian rhythm after the administration of astemizole was developed successfully. This final model can be applied to the development of a human model.

Introduction: It has been proposed that proarrhythmia assessment for safety pharmacology testing includes the use of human pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to detect drug-induced changes in cardiac electrophysiology. This study measured the actions of diverse agents on action potentials (AP) and ion currents recorded from hiPSC-CM., Methods: During AP experiments, the hiPSC-CM were paced at 1Hz during a baseline period, and when increasing concentrations of test compound were administered at 4-minute intervals. AP parameters, including duration (APD60 and APD90), resting membrane potential, rate of rise, and amplitude, were measured throughout the entire experiment. Voltage clamp experiments with E-4031 and nifedipine were similarly conducted., Results: E-4031 produced a dose-dependent prolongation of cardiac action potential and blocked the hERG/IKr current with an IC50 of 17nM. At 3nM, dofetilide significantly increased APD90. Astemizole significantly increased APD60 and APD90 at 30nM. Terfenadine significantly increased APD90 at concentrations greater than 10nM. Fexofenadine, a metabolite of terfenadine, did not produce any electrophysiologic changes in cardiac action potentials. Flecainide produced a dose-dependent prolongation of the cardiac action potential at 1 and 3μM. Acute exposure to nifedipine significantly decreased APD60 and APD90 and produced a dose-dependent block of calcium current with an IC50 of 0.039μM. Verapamil first shortened APD60 and APD90 in a dose-dependent manner, until a compensating increase in APD90, presumably via hERG blockade, was observed at 1 and 3μM. Following a chronic exposure (20-24h) to clinically relevant levels of pentamidine, a significant increase in action potential duration was accompanied by early afterdepolarizations (EADs)., Discussion: These experiments show the ability of AP measured from hiPSC-CM to record the interactions of various ion channels via AP recording and avoid the limitations of using several single ion channel assays in a noncardiac tissue., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: Cervical cancer is a major cause of mortality among women in developing countries. Thus, it is necessary to offer novel therapies to treat this malignancy. Astemizole has been suggested as a novel and interesting anticancer agent because it targets several proteins involved in cancer including Eag1 (ether à-go-go-1) potassium channels. Eag1 has been proposed as a tumor marker for different types of cancer. Actually, we previously suggested Eag1 channels as cervical cancer and dysplasia markers. Besides, Eag1 has been proposed as a therapeutic target for different malignancies. However, the effect of astemizole in cervical cancer cells is unknown. Therefore, we investigated the effect of astemizole on the proliferation and apoptosis of cervical cancer cells., Methods: Five cervical cancer cell lines (HeLa, SiHa, CaSki, INBL, and C-33A) were cultured according to manufacturer's instructions. Eag1 protein expression was studied by immunocytochemistry. Cell proliferation was assayed with the MTT method, and apoptosis was investigated by flow cytometry., Results: Eag1 protein expression was observed in different cell lines. Astemizole decreased cell proliferation in up to 40% and increased apoptosis severalfold in all the cell lines studied., Conclusions: Our results suggest astemizole as a potential therapy for cervical cancer.

Some second-generation antipsychotics (SGAs) increase insulin resistance and fat oxidation, but counter intuitively they do not activate lipolysis. This seems unsustainable for meeting energy demands. Here, we measured dose-dependent effects of SGAs on rates of oxygen consumption (VO2), respiratory exchange ratio (RER), and physical activity in C57BL/6J mice. The role of H1-histamine receptors and consequences of blocking fat oxidation were also examined. Olanzapine, risperidone, and clozapine (2.5-10mg/kg) elicited rapid drops in dark-cycle RER (~0.7) within minutes, whereas aripiprazole exerted only modest changes. Higher doses of olanzapine decreased VO2, and this was associated with accumulation of glucose in plasma. Clozapine and risperidone also lowered VO2, in contrast to aripiprazole, whereas all decreased physical activity. Astemizole and terfenadine had no significant effects on RER, VO2, or physical activity. The VO2 and RER effects appear independent of sedation/physical activity or H1-receptors. CPT-1 inhibitors can enhance muscle glucose utilization and prevent fat oxidation. However, after etomoxir (2 × 30 mg/kg), a low dose of olanzapine that did not significantly affect VO2 by itself caused precipitous drops in VO2 and body temperature, leading to death within hours or a moribund state requiring euthanasia. One 30 mg/kg dose of either etomoxir or 2-tetradecylglycidate followed by olanzapine, risperidone, or clozapine, but not aripiprazole, dramatically lowered VO2 and body temperature. Thus, mice treated with some SGAs shift their fuel utilization to mostly fat but are unable to either switch back to glucose or meet their energy demands when either higher doses are used or when fat oxidation is blocked.

Astemizole, a H1R antagonist shows high affinity to the histamine H1 receptor but only a moderate affinity to the histamine H4 receptor. This study aims to modify the astemizole to keep high affinity to the histamine H1 receptor and to increase affinity to the histamine H4 receptor. Therefore, 13 astemizole-derived compounds and astemizole-JNJ7777120-derived hybrid compounds were synthesized and pharmacologically characterized at the histamine H1 and H4 receptors. The new compounds show affinity to the histamine H1 receptor in the pK i range from 5.3 to 8.8, whereas the affinity of these compounds to the histamine H4 receptor was surprisingly rather low (pK i from 4.4 to 5.6). Three representative compounds were docked into the histamine H1 receptor and molecular dynamic studies were performed to explain the binding mode and the experimental results on a molecular level. Furthermore, taking into account the binding mode of compounds with high affinity to the histamine H4 receptor, a H1/H4-pharmacophore hypothesis was developed.

Prion diseases such as Creutzfeldt-Jakob disease (CJD) are incurable and rapidly fatal neurodegenerative diseases. Because prion protein (PrP) is necessary for prion replication but dispensable for the host, we developed the PrP-FRET-enabled high throughput assay (PrP-FEHTA) to screen for compounds that decrease PrP expression. We screened a collection of drugs approved for human use and identified astemizole and tacrolimus, which reduced cell-surface PrP and inhibited prion replication in neuroblastoma cells. Tacrolimus reduced total cellular PrP levels by a nontranscriptional mechanism. Astemizole stimulated autophagy, a hitherto unreported mode of action for this pharmacophore. Astemizole, but not tacrolimus, prolonged the survival time of prion-infected mice. Astemizole is used in humans to treat seasonal allergic rhinitis in a chronic setting. Given the absence of any treatment option for CJD patients and the favorable drug characteristics of astemizole, including its ability to cross the blood-brain barrier, it may be considered as therapy for CJD patients and for prophylactic use in familial prion diseases. Importantly, our results validate PrP-FEHTA as a method to identify antiprion compounds and, more generally, FEHTA as a unique drug discovery platform.

Agents such as sertindole and astemizole affect heart action by inducing long-QT syndrome, suggesting that apart from their neuronal actions through histamine receptors, 5-HT2 serotonin receptors and D2 dopamine receptors they also affect ether-a-go-go channels and particularly ether-a-go-go-related (ERG) potassium (K(+)) channels, comprising the K(v) 11.1, K(v) 11.2 and K(v) 11.3 voltage-gated potassium currents. Changes in ERG K(+) channel expression and activity have been reported and may be linked to schizophrenia [Huffaker, S.J., Chen, J., Nicodemus, K.K., Sambataro, F., Yang, F., Mattay, V., Lipska, B.K., Hyde, T.M., Song, J., Rujescu, D., Giegling, I., Mayilyan, K., Proust, M.J., Soghoyan, A., Caforio, G., Callicott, J.H., Bertolino, A., Meyer-Lindenberg, A., Chang, J., Ji, Y., Egan, M.F., Goldberg, T.E., Kleinman, J.E., Lu, B. & Weinberger DR. (2009). Nat. Med., 15, 509-518; Shepard, P.D., Canavier, C.C. & Levitan, E.S. (2007). Schizophr Bull., 33, 1263-1269]. We have previously shown that histamine H1 blockers augment gamma oscillations (γ) which are thought to be involved in cognition and storage of information. These effects were particularly pronounced for γ induced by acetylcholine. Here we have compared neuronal effects of three agents which interfere with ERG K(+) channels. We found that astemizole and sertindole, but not the K(v) 11 channel blocker E4031, augmented γ induced by acetylcholine in hippocampal slices. Kainate-induced γ were only affected by astemizole. Evoked responses induced by stratum radiatum stimulation in area CA1 revealed that only E4031 augmented stimulus-induced synaptic potentials and neuronal excitability. Our findings suggest that K(v) 11 channels are involved in neuronal excitability without clear effects on γ and that the effect of astemizole is related to actions on H1 receptors., (© 2012 Federation of European Neuroscience Societies and Blackwell Publishing Ltd.)

Ether-à-go-go (EAG) and EAG-related gene (ERG) K(+) channels are close homologues but differ markedly in their gating properties. ERG1 channels are characterized by rapid and extensive C-type inactivation, whereas mammalian EAG1 channels were previously considered noninactivating. Here, we show that human EAG1 channels exhibit an intrinsic voltage-dependent slow inactivation that is markedly enhanced in rate and extent by 1-10 µM 3-nitro-N-(4-phenoxyphenyl) benzamide, or ICA105574 (ICA). This compound was previously reported to have the opposite effect on ERG1 channels, causing an increase in current magnitude by inhibition of C-type inactivation. The voltage dependence of 2 µM ICA-induced inhibition of EAG1 current was half-maximal at -73 mV, 62 mV negative to the half-point for channel activation. This finding suggests that current inhibition by the drug is mediated by enhanced inactivation and not open-channel block, where the voltage half-points for current inhibition and channel activation are predicted to overlap, as we demonstrate for clofilium and astemizole. The mutation Y464A in the S6 segment also induced inactivation of EAG1, with a time course and voltage dependence similar to that caused by 2 µM ICA. Several Markov models were investigated to describe gating effects induced by multiple concentrations of the drug and the Y464A mutation. Models with the smallest fit error required both closed- and open-state inactivation. Unlike typical C-type inactivation, the rate of Y464A- and ICA-induced inactivation was not decreased by external tetraethylammonium or elevated [K(+)](e). EAG1 channel inactivation introduced by Y464A was prevented by additional mutation of a nearby residue located in the S5 segment (F359A) or pore helix (L434A), suggesting a tripartite molecular model where interactions between single residues in S5, S6, and the pore helix modulate inactivation of EAG1 channels.

Background: Calcitriol antiproliferative effects include inhibition of the oncogenic ether-à-go-go-1 potassium channel (Eag1) expression, which is necessary for cell cycle progression and tumorigenesis. Astemizole, a new promising antineoplastic drug, targets Eag1 by blocking ion currents. Herein, we characterized the interaction between calcitriol and astemizole as well as their conjoint antiproliferative action in SUM-229PE, T-47D and primary tumor-derived breast cancer cells., Methodology/principal Findings: Molecular markers were studied by immunocytochemistry, Western blot and real time PCR. Inhibitory concentrations were determined by dose-response curves and metabolic activity assays. At clinically achievable drug concentrations, synergistic antiproliferative interaction was observed between calcitriol and astemizole, as calculated by combination index analysis (CI <1). Astemizole significantly enhanced calcitriol's growth-inhibitory effects (3-11 folds, P<0.01). Mean IC(20) values were 1.82 ± 2.41 nM and 1.62 ± 0.75 µM; for calcitriol (in estrogen receptor negative cells) and astemizole, respectively. Real time PCR showed that both drugs alone downregulated, while simultaneous treatment further reduced Ki-67 and Eag1 gene expression (P<0.05). Astemizole inhibited basal and calcitriol-induced CYP24A1 and CYP3A4 mRNA expression (cytochromes involved in calcitriol and astemizole degradation) in breast and hepatoma cancer cells, respectively, while upregulated vitamin D receptor (VDR) expression., Conclusions/significance: Astemizole synergized calcitriol antiproliferative effects by downregulating CYP24A1, upregulating VDR and targeting Eag1. This study provides insight into the molecular mechanisms involved in astemizole-calcitriol combined antineoplastic effect, offering scientific support to test both compounds in combination in further preclinical and clinical studies of neoplasms expressing VDR and Eag1. VDR-negative tumors might also be sensitized to calcitriol antineoplastic effects by the use of astemizole. Herein we suggest a novel combined adjuvant therapy for the management of VDR/Eag1-expressing breast cancer tumors. Since astemizole improves calcitriol bioavailability and activity, decreased calcitriol dosing is advised for conjoint administration.

Backgrounds: Conventional in vitro approach using human ether-a-go-go related gene (hERG) assay has been considered worldwide as the first screening assay for cardiac repolarization safety. However, it does not always oredict the potential QT prolongation risk or pro-arrhythmic risk correctly. For adaptable preclinical strategiesto evaluate global cardiac safety, an on-chip quasi-in vivo cardiac toxicity assay for lethal arrhythmia (ventricular tachyarrhythmia) measurement using ring-shaped closed circuit microelectrode chip has been developed., Results: The ventricular electrocardiogram (ECG)-like field potential data, which includes both the repolarization and the conductance abnormality, was acquired from the self-convolutied extracellular field potentials (FPs) of a lined-up cardiomyocyte network on a circle-shaped microelectrode in an agarose microchamber. When Astemisol applied to the closed-loop cardiomyocyte network, self-convoluted FP profile of normal beating changed into an early afterdepolarization (EAD) like waveform, and then showed ventricular tachyarrhythmias and ventricular fibrilations (VT/Vf). QT-prolongation-like self-convoluted FP duration prolongation and its fluctuation increase was also observed according to the increase of Astemizole concentration., Conclusions: The results indicate that the convoluted FPs of the quasi-in vivo cell network assay includes both of the repolarization data and the conductance abnormality of cardiomyocyte networks has the strong potential to prediction lethal arrhythmia.

Insulin-like Growth Factor-1 (IGF-1) plays a key role in breast cancer development and cell cycle regulation. It has been demonstrated that IGF-1 stimulates cyclin expression, thus regulating the G1 to S phase transition of the cell cycle. Potassium (K(+)) channels are involved in the G1 phase progression of the cell cycle induced by growth factors. However, mechanisms that allow growth factors to cooperate with K(+) channels in order to modulate the G1 phase progression and cyclin expression remain unknown. Here, we focused on hEag1 K(+) channels which are over-expressed in breast cancer and are involved in the G1 phase progression of breast cancer cells (MCF-7). As expected, IGF-1 increased cyclin D1 and E expression of MCF-7 cells in a cyclic manner, whereas the increase of CDK4 and 2 levels was sustained. IGF-1 stimulated p21(WAF1/Cip1) expression with a kinetic similar to that of cyclin D1, however p27(Kip1) expression was insensitive to IGF-1. Interestingly, astemizole, a blocker of hEag1 channels, but not E4031, a blocker of HERG channels, inhibited the expression of both cyclins after 6-8h of co-stimulation with IGF-1. However, astemizole failed to modulate CDK4, CDK2, p21(WAF1/Cip1) and p27(Kip1) expression. The down-regulation of hEag1 by siRNA provoked a decrease in cyclin expression. This study is the first to demonstrate that K(+) channels such as hEag1 are directly involved in the IGF-1-induced up-regulation of cyclin D1 and E expression in MCF-7 cells. By identifying more specifically the temporal position of the arrest site induced by the inhibition of hEag1 channels, we confirmed that hEag1 activity is predominantly upstream of the arrest site induced by serum-deprivation, prior to the up-regulation of both cyclins D1 and E. This article is part of a Special Issue entitled: 11th European Symposium on Calcium., (2011 Elsevier B.V. All rights reserved.)

Mortality-to-incidence ratio in cancer patients is extremely high, positioning cancer as a major cause of death worldwide. Despite hundreds of clinical trials for anti-cancer drugs that are currently in progress, most clinical trials for novel drug treatments fail to pass Phase I. However, previously developed drugs with novel anti-tumor properties offer a viable and cost-effective alternative to fight cancer. Histamine favors the proliferation of normal and malignant cells. Several anti-histamine drugs, including astemizole, can inhibit tumor cell proliferation. Astemizole has gained enormous interest since it also targets important proteins involved in cancer progression, namely, ether à-go-go 1 (Eag1) and Eag-related gene (Erg) potassium channels. Furthermore, Eag1 is thought to be an important marker and a therapeutic target for several different cancers. Astemizole inhibits Eag1 and Erg channel activity, and in cells expressing the Eag1 channel it decreases tumor cell proliferation in vitro and in vivo. It should be noted that some cardiovascular side effects have been reported for astemizole in a few rare cases. Nevertheless, astemizole stands as a very promising anti-cancer tool because it displays several anti-proliferative mechanisms, may serve as the basis to synthesize new anti-cancer agents, and has been previously administered clinically. In this review we will summarize the main findings relating to histamine and anti-histamines in cancer cell proliferation focusing on astemizole targets (Eag1 and Erg channels), and its anti-cancer effects in vitro and in vivo. We will also describe the side effects of astemizole and discuss proposals to overcome such effects in cancer patients. Finally, we will remark on the relevance of developing novel astemizole-related compounds.

Vorapaxar (SCH 530348), a potent oral thrombin protease-activated receptor 1 antagonist, is being developed as an antiplatelet agent for patients with established vascular disease. The objective of this study was to identify the human liver cytochrome P450 (P450) enzyme(s) responsible for the metabolism of SCH 530348. Human liver microsomes metabolized SCH 530348 to M19, an amine metabolite formed via carbamate cleavage, and M20 (monohydroxy-SCH 530348). Recombinant human CYP3A4 exhibited the most activity (11.5% profiled radioactivity) for the formation of M19, followed by markedly less substrate conversion with CYP1A1 and CYP2C19. Trace levels of M19, a major excreted human metabolite, were detected with CYP1A2, CYP3A5, and CYP4F3A. Formation of M19 by human liver microsomes was inhibited 89% by ketoconazole (IC(50), 0.73 μM), 34% by tranylcypromine, and 89% by anti-CYP3A4 monoclonal antibody. There was a significant correlation between the rate of M19 formation and midazolam 1'-hydroxylation (r = 0.75) or M19 formation and testosterone 6β-hydroxylation (r = 0.92). The results of screening, inhibition, and correlation studies confirmed that CYP3A4 is the major P450 enzyme responsible for M19 formation from SCH 530348. In contrast, formation of M20, a major circulating human metabolite at steady state, was primarily catalyzed by CYP3A4 and CYP2J2. M20 is pharmacologically equipotent to SCH 530348, whereas M19 is an inactive metabolite. Formation of M20 by human liver microsomes was inhibited 89% by ketoconazole, 75% by astemizole (a CYP2J2 inhibitor), and 43% by CYP3A4 monoclonal antibody. These results suggest that CYP3A4 and CYP2J2 are both involved in the formation of M20 metabolite.

Aim of the Study: Poncirus fructus (PF)--also known as the dried, immature fruit of Poncirus trifoliata (L.) Raf. (Rutaceae)--is a natural substance that has long been used for various gastrointestinal disorders in eastern Asia. An aqueous extract of PF (PF-W) has particularly potent gastroprokinetic effects, but its molecular mechanism was not well understood. Identification of the underlying prokinetic mechanism of PF-W was pursued in the present study., Materials and Methods: Changes in in vitro cAMP levels and in vivo intestinal transit rate (ITR) caused by PF-W were measured after pretreatment with GR125487, an antagonist for serotonin receptor subtype 4 (5-HT4R). An [(3)H] astemizole binding assay and electrophysiology experiments were performed to determine if PF-W has any interaction with the human ether-à-go-go related gene (hERG) potassium channel., Results: PF-W induced an increase in intracellular cAMP in 5-HT4R-expressing HEK293T cells, indicating that PF-W does activate 5-HT4R. Moreover, pretreatment with GR125487 successfully blocked the increase, suggesting that the response was 5-HT4R-specific. More importantly, pretreatment of GR125487 in rats inhibited the elevation of ITR by PF-W, indicating that the prokinetic effect of PF-W was indeed exerted via 5-HT4R. On the other hand, both [(3)H]-astemizole binding assay and electrophysiological experiments revealed that PF-W did not interfere at all with the hERG channel., Conclusion: It was found that PF-W exerts its prokinetic activity through a 5-HT4R-mediated pathway, with no interaction with hERG channels. Therefore, PF-W is a good candidate that might be developed as a prokinetic agent with fewer expected cardiac side effects., (Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.)

In vitro hERG blocking potency is measured in drug discovery as part of an integrated cardiovascular risk assessment. Typically, the concentrations producing 50% inhibition are measured in protein-free saline solutions and compared with calculated free therapeutic in vivo Cmax values to estimate a hERG safety multiple. The free/unbound fraction is believed responsible for activity. We tested the validity of this approach with 12 compounds by determining potencies in voltage clamp studies conducted in the absence and presence of 100% dialyzed fetal bovine serum (FBS). Bath drug concentrations in saline solutions were measured to account for loss of compounds due to solubility, stability, and/or adsorption. Protein binding in dialyzed FBS was measured to enable predictions of serum IC50s based on the unbound fraction and the saline IC50. For 11 of 12 compounds, the measured potency in the presence of dialyzed FBS was within 2-fold of the predicted potency. The predicted IC50 in dialyzed FBS for one highly bound compound, amiodarone, was 9-fold higher than the measured serum IC50. These data suggest that for highly bound compounds, direct measurement of IC50s in the presence of 100% serum may provide a more accurate estimate of in vivo potencies than the approach based on calculated serum shifts.

Cryptococcus neoformans is the leading cause of fungal meningitis, a life-threatening infection that occurs predominately in immuocompromised patients. Current drug therapies are limited to amphotericin B, flucytosine and the azoles since the echinocandins have no demonstrated activity against yeast like pathogens. Fluconazole, a drug belonging to the azole class and often the only available antifungal in the developing world, is fungistatic and therefore not effective in clearing cryptococcal infections in immunosuppressed individuals. Here we report that astemizole and a closely related analog (A2) promoted in vitro fungicidal activity of fluconazole against Cryptococcus neoformans var. grubii and Cryptococcus gattii. Astemizole, a second-generation antihistamine drug used as an H1 antagonist, has also been found to have antimalarial activity. Disk diffusion assays and MIC and MFC analysis confirmed that the inhibitory concentrations of these drug combinations were fungicidal. When tested in vivo, astemizole or A2 in combination with fluconazole significantly improved the survival of Galleria mellonella (wax moth caterpillar) that had been previously challenged with C. neoformans but not when caterpillars were challenged with a fluconazole-resistant strain. The findings reported here suggest that fungicidal combinations between azoles and other existing drugs may represent an alternative strategy for improving treatments for fungal infections.

A dual activity, conjugated approach has been taken to form hybrid molecules of two known antimalarial drugs, chloroquine (CQ) and the non-sedating H1 antagonist astemizole. A variety of linkers were investigated to conjugate the two agents into one molecule. Compounds 5-8 possessed improved in vitro activity against a CQ-resistant strain of Plasmodium falciparum, and examples 7 and 8 were active in vivo in mouse models of malaria.

Ion channels represent the third largest class of targets in drug discovery after G-protein coupled receptors and kinases. In spite of this ranking, ion channels continue to be under exploited as drug targets compared with the other two groups for several reasons. First, with 400 ion channel genes and an even greater number of functional channels due to mixing and matching of individual subunits, a systematic collection of ion channel-expressing cell lines for drug discovery and safety screening has not been available. Second, the lack of high-throughput functional assays for ion channels has limited their use as drug targets. Now that automated electrophysiology has come of age and provided the technology to assay ion channels at medium to high throughput, we have addressed the need for a library of ion channel cell lines by constructing the Ion Channel Panel (ChanTest Corp., Cleveland, OH). From 400 ion channel genes, a collection of 82 of the most relevant human ion channels for drug discovery, safety, and human disease has been assembled.Each channel has been stably overexpressed in human embryonic kidney 293 or Chinese hamster ovary cells. Cell lines have been selected and validated on automated electrophysiology systems to facilitate cost-effective screening for safe and selective compounds at earlier stages in the drug development process. The screening and validation processes as well as the relative advantages of different screening platforms are discussed.

Drug-induced QT interval prolongation is a critical issue in development of new chemical entities, so the pharmaceutical industry needs to evaluate risk as early as possible. Common marmosets have been in the limelight in early-stage development due to their small size, which requires only a small amount of test drug. The purpose of this study was to determine the utility of telemetered common marmosets for predicting drug-induced QT interval prolongation. Telemetry transmitters were implanted in common marmosets (male and female), and QT and RR intervals were measured. The QT interval was corrected for the RR interval by applying Bazett's and Fridericia's correction formulas and individual rate correction. Individual correction showed the least slope for the linear regression of corrected QT (QTc) intervals against RR intervals, indicating that it dissociated changes in heart rate most effectively. With the individual correction method, the QT-prolonging drugs (astemizole, dl-sotalol) showed QTc interval prolongations and the non-QT-prolonging drugs (dl-propranolol, nifedipine) did not show QTc interval prolongations. The plasma concentrations of astemizole and dl-sotalol associated with QTc interval prolongations in common marmosets were similar to those in humans, suggesting that the sensitivity of common marmosets would be appropriate for evaluating risk of drug-induced QT interval prolongation. In conclusion, telemetry studies in common marmosets are useful for predicting clinical QT prolonging potential of drugs in early stage development and require only a small amount of test drug.

Pharmacological inhibitors of the human ether-a-go-go (hEAG) potassium channel, astemizole and imipramine, have been used to demonstrate that hEAG plays a role in cancer cell proliferation. Astemizole and imipramine are, however, relatively non-specific ion channel blockers, as astemizole can also block the related potassium channel, human ether-a-go-go-related (hERG). Therefore, we aimed to determine the molecular target of astemizole, in the human mammary carcinoma cell line MCF-7. We initially confirmed the expression of KCNH1 and KCNH2 mRNA and hEAG and hERG channel protein in MCF-7 cells. Using a [3H]-thymidine incorporation assay we determined that astemizole inhibited MCF-7 cell proliferation, whereas the hERG-specific channel blocker E-4031 had no effect. We then determined that E-4031 inhibited the regulatory volume decrease (RVD) observed in these cells following exposure to hypotonic solutions, confirming that functional hERG channels are present and may be important for cell volume regulation in MCF-7 cells. Our results suggest, for the first time, that hERG is involved in cell volume regulation. In addition, the function of hEAG and hERG in MCF-7 cell proliferation can be separated pharmacologically by utilizing the channel inhibitors astemizole and E-4031. The hEAG channel function in MCF-7 cells appears to be involved in the regulation of cell proliferation, whereas hERG is involved in cell volume regulation.

The hERG potassium channel has recently been a matter of extensive studies both at experimental and computational levels, because of its possible involvement in the potentially lethal drug-induced long QT syndrome. In this context, in the absence of an experimentally determined 3D structure, to acquire a detailed description of the channel pore and an understanding of atomic determinants for drug binding is of enormous interest at both academic and industrial levels. To contribute to this aim, we first built the open and closed states of the channel by homology modeling techniques, and then submitted both channel models to ns-time-scale MD simulations in explicit membrane. An in-depth analysis of the dynamical behavior of the channel pore with particular attention to the cavity volume was carried out. Finally, using hERG conformations coming from MD simulations, docking experiments were performed to identify a possible binding mode for the most potent hERG channel blocker so far known, the antihistaminic drug astemizole. We show that the combined use of MD and docking is suitable to identify a possible binding mode of drugs in a fairly good agreement with experiments. Moreover, the exploitation of MD snapshots in the docking experiments allowed us to capture some induced-fit effects related to the side chain conformations of Tyr652 and Phe656, which are residues playing a pivotal role in the hERG drug binding., ((c) 2007 Wiley Periodicals, Inc.)

The present study evaluated the effects of histamine 10(-2) M on longitudinal preparations of rat portal vein. It was observed that histamine 10(-2) M induced relaxation of rat portal vein preparations pre-contracted with phenylephrine 10(-4) M. On the other hand, no pharmacological effects were observed in preparations not pre-contracted. The observed histamine-induced relaxing effect was absent in preparations pre-contracted with KCl (120 mM) or in the presence of depolarizing nutritive solution. However, the histamine-induced relaxation was still present in the endothelium-removed preparations. The histamine-induced relaxation also was not prevented by astemizole (10(-6) M, 10(-5) M and 10(-4) M), cimetidine (10(-5) M, 10(-4) M and 10(-3) M) or thioperamide (10(-6) M, 10(-5) M and 10(-4) M), selective antagonists H(1), H(2) and H(3), respectively. The presence of L-NAME 10(-4) M or L-NAME 10(-4) M plus indomethacin 10(-5) M also did not prevent the histamine-induced relaxation observed in rat portal vein. Thus, the histamine-induced relaxation observed in rat portal vein appears to involve a non-endothelial hyperpolarizing mechanism independent of H(1), H(2) and H(3) receptors.

The H1R antagonist astemizole was identified as a somatostatin 5 (SST5) receptor antagonist by a comparative sequence analysis of the consensus drug binding pocket of GPCRs. Subsequently, a similarity analysis of GPCR affinity profiles of astemizole versus a set of in-house GPCR-biased combinatorial libraries revealed new chemical entry points that led to a second lead series with nanomolar binding affinity.

We disclose the first selective, nonpeptidic, small-molecule somatostatin receptor subtype 5 (SST5R) antagonists that were identified by a chemogenomics approach based on the analysis of the homology of amino acids defining the putative consensus drug binding site of SST5R. With this strategy, opioid, histamine, dopamine, and serotonine receptors were identified as the closest neighbors of SST5R. The H1 antagonist astemizole was chosen as a seed structure and subsequently transformed into a SST5 receptor antagonist with nanomolar binding affinity devoid of the original target activity.