Using automated patch clamp electrophysiology platforms in pain-related ion channel research: insights from industry and academia

Automated patch clamp (APC) technology was first developed at the turn of the millennium. The increased throughput it afforded promised a new paradigm in ion channel recordings: it offered the potential to overcome the time-consuming, low-throughput bottleneck arising from manual patch clamp (MPC) investigations. This has relevance to the fast-paced development of novel therapies for chronic pain. This review highlights the advances in technology, using select examples, that have facilitated APC usage in both industry and academia. It covers both first generation and the latest developments in second-generation platforms. In addition, it also provides an overview of the pain research field and how APC platforms have furthered our understanding of ion channel research and the development of pharmacological tools and therapeutics. APC platforms have much to offer the ion channel research community and this review highlights areas of 'best practice' for both academia and industry. The impact of APC platforms and the prospects for chronic pain ion channel research and improved therapeutics will be evaluated

Comprehensive in vitro Proarrhythmia Assay (CiPA): Pending issues for successful validation and implementation

International audienceIntroduction: The Comprehensive in vitro Proarrhythmia Assay (CiPA) is a nonclinical Safety Pharmacology paradigm for discovering electrophysiological mechanisms that are likely to confer proarrhythmic liability to drug candidates intended for human use.Topics covered: Key talks delivered at the ‘CiPA on my mind’ session, held during the 2015 Annual Meeting of the Safety Pharmacology Society (SPS), are summarized. Issues and potential solutions relating to crucial constituents [e.g., biological materials (ion channels and pluripotent stem cell-derived cardiomyocytes), study platforms, drug solutions, and data analysis] of CiPA core assays are critically examined.Discussion: In order to advance the CiPA paradigm from the current testing and validation stages to a research and regulatory drug development strategy, systematic guidance by CiPA stakeholders is necessary to expedite solutions to pending and newly arising issues. Once a study protocol is proved to yield robust and reproducible results within and across laboratories, it can be implemented as qualified regulatory procedure

Voltage-Gated Sodium Channel Drug Discovery Technologies and Challenges

Voltage-gated sodium (Nav) channels represent an important class of drug target for pain and many other pathology conditions. Despite the recent advances in channelopathies and structure-function studies, the discovery of Nav channel therapeutics is still facing a major challenge from the limitation of assay technologies. This chapter will focus on advancement and challenge of Nav drug discovery technologies including nonelectrophysiological assays, extracellular electrophysiological assays, and the newly evolved high-throughput automated patch clamp (APC) technologies

A Comprehensive Evaluation of Sdox, a Promising H2S-Releasing Doxorubicin for the Treatment of Chemoresistant Tumors

Sdox is a hydrogen sulfide (H2S)-releasing doxorubicin effective in P-glycoprotein-overexpressing/doxorubicin-resistant tumor models and not cytotoxic, as the parental drug, in H9c2 cardiomyocytes. The aim of this study was the assessment of Sdox drug-like features and its absorption, distribution, metabolism, and excretion (ADME)/toxicity properties, by a multi- and transdisciplinary in silico, in vitro, and in vivo approach. Doxorubicin was used as the reference compound. The in silico profiling suggested that Sdox possesses higher lipophilicity and lower solubility compared to doxorubicin, and the off-targets prediction revealed relevant differences between Dox and Sdox towards several cancer targets, suggesting different toxicological profiles. In vitro data showed that Sdox is a substrate with lower affinity for P-glycoprotein, less hepatotoxic, and causes less oxidative damage than doxorubicin. Both anthracyclines inhibited CYP3A4, but not hERG currents. Unlike doxorubicin, the percentage of zebrafish live embryos at 72 hpf was not affected by Sdox treatment. In conclusion, these findings demonstrate that Sdox displays a more favorable drug-like ADME/toxicity profile than doxorubicin, different selectivity towards cancer targets, along with a greater preclinical efficacy in resistant tumors. Therefore, Sdox represents a prototype of innovative anthracyclines, worthy of further investigations in clinical settings

Allosteric modulation and ligand binding kinetics at the Kv11.1 channel

Kv11.1-induced cardiotoxicity has emerged as an unanticipated adverse effect of many pharmacological agents and has become a major obstacle in drug development over the past decades. In this thesis, allosteric modulation of the Kv11.1 channel has been extensively explored, and negative allosteric modulators were shown to relieve the proarrhythmic effects of structurally and therapeutically diverse Kv11.1 blockers. The most potent modulators may be developed as a new class of antiarrhythmic medications in the future. On the other hand, kinetic binding parameters of a wide range of Kv11.1 blockers at the channel have been thoroughly investigated in this thesis. Association and dissociation rates or residence times are strongly suggested to be integrated with equilibrium affinity values into the future paradigms for a better and more comprehensive evaluation of Kv11.1 liability of drug candidates. The __kon-koff-KD__ kinetic map provides a first and promising classification of Kv11.1 blockers, which could be beneficial and indicative for drug researchers to design compounds with less Kv11.1-mediated cardiac side effects in the early stage of drug development. Hopefully, all findings in this thesis have brought new insights into Kv11.1-induced cardiac arrhythmias, and will offer opportunities for restoring or preventing this kind of arrhythmias in the near future.Chinese Scholarship CouncilUBL - phd migration 201

Early ADMET profiling of anti-inflammatory alkaloids using validated LC-MS/MS methods

Natural products provide an important and unique source of new lead compounds for drug discovery. Approximately 50% of all new chemical entities are inspired by nature. In the search of novel anti-inflammatory compounds in the ancient medicinal plant Isatis tinctoria, tryptanthrin, indirubin, and (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one (indolinone) were identified as pharmacologically active constituents. They inhibit, at low µM to nM concentrations, cyclo-oxygenase-2  (COX-2), 5-lipoxygenase (5-LOX) catalyzed leukotriene synthesis, cyclin-dependent kinase (CDK), glycogensynthase kinase-3b (GSK), and mast cell degranulation. While the molecular modes of action of these alkaloids are not yet fully understood, their unique pharmacological profiles, structural drug-like properties, and low cytotoxicity render these molecules promising anti-inflammatory leads. To further evaluate the potential of these alkaloids as novel anti-inflammatory and anti-allergic leads, an assessment of their ADMET properties was warranted. For exact quantification of the compounds, LC-MS/MS methods were developed and validated according to current regulatory guidelines. To get a first prognostic picture for the in vivo performance of our compounds, a pilot PK study was performed in male Sprague Dawley rats after intravenous administration at a concentration of 2 mg/kg b.w.. Tryptanthrin and indirubin showed a half-life (t1/2) of around 40 min, while indolinone was quickly eliminated (t1/2 = 4 min). As most of the drugs are preferentially administered orally, the gastrointestinal tract (GIT) represents the major site of drug absorption. Human colon carcinoma cells (Caco-2 cells) serve as the method of choice to predict human drug absorption across the intestinal wall in vitro. To study the permeability of the three compounds across the epithelial monolayer, the alkaloids were screened at concentrations of 5-10 µM in the Caco-2 assay. As efflux transporters can greatly impact the in vivo absorption and, thus, the bioavailability of a drug candidate, the compounds were tested for possible P-glycoprotein (P-gp) interaction. Therefore, the alkaloids were co-incubated with the P-gp inhibitor verapamil (50 µM). Active efflux was assessed by calculating the efflux ratio (ER) from bidirectional assays. Due to high lipophilicity of indirubin, the compound precipitated in the transporter buffer and was thus excluded for further investigations in aqueous solutions. Tryptanthrin displayed a high permeability (Papp > 32.0 x 10-6 cm/s) across the cell monolayer. The efflux ratio below 2 (< 1.12) and the unchanged Papp values in presence of the P-glycoprotein (P-gp) inhibitor verapamil indicated that tryptanthrin was not involved in P-gp mediated efflux. In the Caco-2 assay, the recovery of indolinone was low, pointing to possibly extensive phase II metabolism. Further investigation by a high-resolution mass spectrometry (HR-MS) system revealed the formation of two sulfate and two glucuronide conjugates for indolinone. Another well-known biological barrier in the human body is the blood-brain barrier (BBB). To evaluate the BBB permeation potential of tryptanthrin and indolinone, the compounds were tested in three cell-based human and animal BBB models. Data obtained with the human and animal BBB models showed good correlation and were indicative of a high BBB permeation potential of tryptanthrin and indolinone. Furthermore, active-mediated efflux was evaluated by calculating the ER from bidirectional assays. The ERs below 2 suggested that both compounds were not involved in active-mediated efflux. Besides P-gp, another critical anti-target in drug development is the human ether-a-go-go (hERG) potassium channel. In the late 1990s, an increasing number of non-cardiovascular drugs have been withdrawn from the market due to cardiotoxic side-effects linked to hERG blocking. Since then, regulatory agencies insist on acquiring experimental hERG data of drug candidates before moving into clinical trials. Possible cardiotoxic liability of the compounds was assessed in vitro, by measurement of an inhibitory effect on hERG tail currents in stably transfected HEK 293 cells using the patch-clamp technique. Slight hERG inhibition was found for tryptanthrin (IC50 of 22 µM) and indolinone (IC50 of 25 µM). Data obtained from the in vitro assays were corroborated by in silico predictions. For tryptanthrin and indolinone, all criteria for high human oral absorption and passive BBB penetration were met. In addition, the slight hERG inhibition found for tryptanthrin and indolinone in vitro could be confirmed by in silico predictions