Discovery of Novel TASK-3 Channel Blockers Using a Pharmacophore-Based Virtual Screening.

TASK-3 is a two-pore domain potassium (K2P) channel highly expressed in the hippocampus, cerebellum, and cortex. TASK-3 has been identified as an oncogenic potassium channel and it is overexpressed in different cancer types. For this reason, the development of new TASK-3 blockers could influence the pharmacological treatment of cancer and several neurological conditions. In the present work, we searched for novel TASK-3 blockers by using a virtual screening protocol that includes pharmacophore modeling, molecular docking, and free energy calculations. With this protocol, 19 potential TASK-3 blockers were identified. These molecules were tested in TASK-3 using patch clamp, and one blocker (DR16) was identified with an IC50 = 56.8 ± 3.9 µM. Using DR16 as a scaffold, we designed DR16.1, a novel TASK-3 inhibitor, with an IC50 = 14.2 ± 3.4 µM. Our finding takes on greater relevance considering that not many inhibitory TASK-3 modulators have been reported in the scientific literature until today. These two novel TASK-3 channel inhibitors (DR16 and DR16.1) are the first compounds found using a pharmacophore-based virtual screening and rational drug design protocol.

Quantitative determination of talatisamine and its pharmacokinetics and bioavailability in mouse plasma by UPLC-MS/MS.

Talatisamine, as the efficacy ingredient of Aconitum, was known as a novel specific blocker for the delayed rectifier K+ channels in rat hippocampal neurons. In this study, a rapid, selective and reproducible UPLC-MS/MS separation method was established and fully validated for the quantitative determination of talatisamine levels in ICR (Institute of Cancer Research) mouse blood. A total of 24 healthy male ICR mice were divided into four groups that was administered talatisamine via intravenous at a dose of 1 mg/kg and oral administration of three doses (2, 4, 8 mg/kg). All blood samples were protein precipitate by using acetonitrile with an internal standard (IS) deltaline. The effective chromatographic separation was carried out through an UPLC BEH C18 analytical column (2.1 mm × 50 mm, 1.7 µm) with an initial mobile phase that consisted of acetonitrile and 10 mmol/L ammonium acetate aqueous solution (containing 0.1% formic acid) with a gradient elution pumped at a flow rate of 0.4 mL/min. Also, an electrospray ionization (ESI) was applied to quantify the talatisamine in the positive ions mode. The method validation demonstrated good linearity over the range of 1-1000 ng/mL (r2 ≥ 0.9993) for talatisamine in mouse blood with a lower limit of quantification (LLOQ) at 1 ng/mL. The accuracy values of the method were within 89.4% to 113.3%, and the matrix effects were between 103.2% and 106.3%. The mean extraction recoveries for talatisamine obtained from four concentrations of QC blood samples were exceeded 71.7%, and the relative standard deviation (RSD) both of intra- and inter-day precision values for replicate quality control samples did not exceed 15% respectively for all analytes during the assay validation. This method was successfully applied to the evaluation of the pharmacokinetic of talatisamine, regardless of intragastric or intravenous administration in mice. Based on the pharmacokinetics data, the bioavailability of talatisamine in mice was >65.0% after oral administration, exhibiting an excellent oral absorption.

Distribution and kinetics of the Kv1.3-blocking peptide HsTX1[R14A] in experimental rats.

The peptide HsTX1[R14A] is a potent and selective blocker of the voltage-gated potassium channel Kv1.3, which is a highly promising target for the treatment of autoimmune diseases and other conditions. In order to assess the biodistribution of this peptide, it was conjugated with NOTA and radiolabelled with copper-64. [64Cu]Cu-NOTA-HsTX1[R14A] was synthesised in high radiochemical purity and yield. The radiotracer was evaluated in vitro and in vivo. The biodistribution and PET studies after intravenous and subcutaneous injections showed similar patterns and kinetics. The hydrophilic peptide was rapidly distributed, showed low accumulation in most of the organs and tissues, and demonstrated high molecular stability in vitro and in vivo. The most prominent accumulation occurred in the epiphyseal plates of trabecular bones. The high stability and bioavailability, low normal-tissue uptake of [64Cu]Cu-NOTA-HsTX1[R14A], and accumulation in regions of up-regulated Kv channels both in vitro and in vivo demonstrate that HsTX1[R14A] represents a valuable lead for conditions treatable by blockade of the voltage-gated potassium channel Kv1.3. The pharmacokinetics shows that both intravenous and subcutaneous applications are viable routes for the delivery of this potent peptide.

Population Pharmacokinetics/Pharmacodynamics of 3,4-Diaminopyridine Free Base in Patients With Lambert-Eaton Myasthenia.

Lambert-Eaton myasthenia (LEM) is a rare autoimmune disorder associated with debilitating muscle weakness. There are limited treatment options and 3,4-diaminopyridine (3,4-DAP) free base is an investigational orphan drug used to treat LEM-related weakness. We performed a population pharmacokinetic/pharmacodynamic (PK/PD) analysis using 3,4-DAP and metabolite concentrations collected from a phase II study in patients with LEM. The Triple Timed Up & Go (3TUG) assessment, which measures lower extremity weakness, was the primary outcome measure. A total of 1,270 PK samples (49 patients) and 1,091 3TUG data points (32 randomized patients) were included in the PK/PD analysis. A two-compartment and one-compartment model for parent and metabolite, respectively, described the PK data well. Body weight and serum creatinine partially explained the variability in clearance for the final PK model. A fractional inhibitory maximum effect (Emax ) model characterized the exposure-response relationship well. The PK/PD model was applied to identify a suggested dosing approach for 3,4-DAP free base.

Prolonged immunomodulation in inflammatory arthritis using the selective Kv1.3 channel blocker HsTX1[R14A] and its PEGylated analog.

Effector memory T lymphocytes (TEM cells) that lack expression of CCR7 are major drivers of inflammation in a number of autoimmune diseases, including multiple sclerosis and rheumatoid arthritis. The Kv1.3 potassium channel is a key regulator of CCR7- TEM cell activation. Blocking Kv1.3 inhibits TEM cell activation and attenuates inflammation in autoimmunity, and as such, Kv1.3 has emerged as a promising target for the treatment of TEM cell-mediated autoimmune diseases. The scorpion venom-derived peptide HsTX1 and its analog HsTX1[R14A] are potent Kv1.3 blockers and HsTX1[R14A] is selective for Kv1.3 over closely-related Kv1 channels. PEGylation of HsTX1[R14A] to create a Kv1.3 blocker with a long circulating half-life reduced its affinity but not its selectivity for Kv1.3, dramatically reduced its adsorption to inert surfaces, and enhanced its circulating half-life in rats. PEG-HsTX1[R14A] is equipotent to HsTX1[R14A] in preferential inhibition of human and rat CCR7- TEM cell proliferation, leaving CCR7+ naïve and central memory T cells able to proliferate. It reduced inflammation in an active delayed-type hypersensitivity model and in the pristane-induced arthritis (PIA) model of rheumatoid arthritis (RA). Importantly, a single subcutaneous dose of PEG-HsTX1[R14A] reduced inflammation in PIA for a longer period of time than the non-PEGylated HsTX1[R14A]. Together, these data indicate that HsTX1[R14A] and PEG-HsTX1[R14A] are effective in a model of RA and are therefore potential therapeutics for TEM cell-mediated autoimmune diseases. PEG-HsTX1[R14A] has the additional advantages of reduced non-specific adsorption to inert surfaces and enhanced circulating half-life.

Measuring kinetics and potency of hERG block for CiPA.

INTRODUCTION: The Comprehensive in vitro Proarrhythmic Assay (CiPA) aims to update current cardiac safety testing to better evaluate arrhythmic risk. A central theme of CiPA is the use of in silico approaches to risk prediction incorporating models of drug binding to hERG. To parameterize these models, accurate in vitro measurement of potency and kinetics of block is required. The Ion Channel Working Group was tasked with: i) selecting a protocol that could measure kinetics of block and was easily implementable on automated platforms for future rollout in industry and ii) acquiring a reference dataset using the standardized protocol. METHODS: Data were acquired using a 'step depolarisation' protocol using manual patch-clamp at ambient temperature. RESULTS: Potency, kinetics and trapping characteristics of hERG block for the CiPA training panel of twelve drugs were measured. Timecourse of block and trapping characteristics could be reliably measured if the time constant for onset of block was between ~500ms and ~15s. Seven drugs, however had time courses of block faster than this cut-off. DISCUSSION: Here we describe the implementation of the standardized protocol for measurement of kinetics and potency of hERG block for CiPA. The results highlight the challenges in identifying a single protocol to measure hERG block over a range of kinetics. The dataset from this study is being used by the In Silico Working Group to develop models of drug binding for risk prediction and is freely available as a 'gold standard' ambient temperature dataset to evaluate variability across high throughput platforms.

An Accelerator Mass Spectrometry-Enabled Microtracer Study to Evaluate the First-Pass Effect on the Absorption of YH4808.

14 C-labeled YH4808, a novel potassium-competitive acid blocker, was intravenously administered as a microtracer at 80 µg (11.8 kBq or 320 nCi) concomitantly with the nonradiolabeled oral drug at 200 mg to determine the absolute bioavailability and to assess the effect of pharmacogenomics on the oral absorption of YH4808. The absolute bioavailability was low and highly variable (mean, 10.1%; range, 2.3-19.3%), and M3 and M8, active metabolites of YH4808, were formed 22.6- and 38.5-fold higher after oral administration than intravenous administration, respectively. The product of the fraction of an oral YH4808 dose entering the gut wall and the fraction of YH4808 passing on to the portal circulation was larger in subjects carrying the variants of the CHST3, SLC15A1, and SULT1B1 genes. A combined LC+AMS is a useful tool to construct a rich and highly informative pharmacokinetic knowledge core in early clinical drug development at a reasonable cost.

A Physiologically Based Pharmacokinetic Model of Amiodarone and its Metabolite Desethylamiodarone in Rats: Pooled Analysis of Published Data.

BACKGROUND AND OBJECTIVE: Amiodarone (AMD) is one of the most effective drugs for rhythm control of atrial fibrillation. The use of AMD is also associated with adverse effects in multiple tissues. Both the parent compound and its major metabolite desethylamiodarone (DEA) contribute to the drug's therapeutic and toxic action. The present study aimed to build a whole-body physiologically based pharmacokinetic (PBPK) model for AMD and DEA in rats. METHODS: Pharmacokinetic data from multiple studies were collected. Some of the data were pooled together to develop the PBPK model; others were used to evaluate the model. Development of the model also involved in vitro to in vivo extrapolation based on in vitro metabolism data. RESULTS: The final model consisted of 11 tissue compartments, including therapeutic target organs and those to which AMD and DEA may be harmful. Model simulations were in good agreement with the observed time courses of the drug-metabolite pair in tissues, under various dosing scenarios. The key pharmacokinetic properties of AMD, such as extensive tissue distribution, substantial storage in the fat tissue, and long half-lives in many tissues, were closely reflected. CONCLUSION: The developed PBPK model can be regarded as the first step towards a PBPK-pharmacodynamic model that can used to mechanistically evaluate and explain the high adverse event rate and potentially to determine which factors are the primary drives for experiencing an adverse event.

Design, synthesis and evaluation of MCH receptor 1 antagonists--Part II: Optimization of pyridazines toward reduced phospholipidosis and hERG inhibition.

Despite recent success there remains a high therapeutic need for the development of drugs targeting diseases associated with the metabolic syndrome. As part of our search for safe and effective MCH-R1 antagonists for the treatment of obesity, a series of 3,6-disubstituted pyridazines was evaluated. During optimization several issues of the initial lead structures had to be resolved, such as selectivity over related GPCRs, inhibition of the hERG channel as well as the potential to induce phospholipidosis. Utilizing property-based design, we could demonstrate that all parameters can significantly be improved by consequently increasing the polarity of the compounds. By this strategy, we succeeded in identifying potent and orally available MCH-R1 antagonists with good selectivity over M1 and 5-HT2A and an improved safety profile with respect to hERG inhibition and phospholipidosis.

Kinetics of drug interaction with the Kv11.1 potassium channel.

The Kv11.1 potassium channel is the molecular target for the majority of drugs implicated in acquired long QT syndrome, the most common cause of drug-induced sudden cardiac death, and a common reason for drug restriction or withdrawal from the market. While the IC50 for block of Kv11.1 is commonly used to estimate the risk of acquired long QT syndrome, this approach is crude, and it is widely accepted that the kinetics of drug interactions with the channel are a critical component in understanding their mechanism of action and risk profiles. In this study we report the first directly measured kinetics of block and unblock of Kv11.1 by a QT prolonging drug: the antipsychotic clozapine. Our data show that clozapine binding to Kv11.1 is complex. There are at least two kinetically distinct components to both block and unblock, while the kinetics of unblock are dependent on the dose or duration of drug application. Based on these observations, we have proposed a model incorporating kinetically distinct binding to the open and inactivated states of Kv11.1 that can describe the observed kinetic features of clozapine block and correctly predict the overall affinity and apparent nonstate-dependent interaction of clozapine with Kv11.1. Mechanistic insights into drug block of Kv11.1 gained though detailed kinetic analyses such as this have a potential role in development of drugs targeted to specific channel states to reduce unwanted side effects, as well as in the design of better high-throughput preclinical tests for assessing the proarrhythmic effects of QT prolonging drugs.

A K⁺ channel blocking peptide from the Cuban scorpion Rhopalurus garridoi.

A proteomic analysis of the venom obtained from the Cuban scorpion Rhopalurus garridoi was performed. Venom was obtained by electrical stimulation, separated by high performance liquid chromatography, and the molecular masses of their 50 protein components were identified by mass spectrometry. A peptide of 3940 Da molecular mass was obtained in pure form and its primary structure determined. It contains 37 amino acid residues, including three disulfide bridges. Electrophysiological experiments showed that this peptide is capable of blocking reversibly K(+)-channels hKv1.1 with a Kd close to 1 µM, but is not effective against hKv1.4, hERG1 and EAG currents, at the same concentration. This is the first protein component ever isolated from this species of scorpion and was assigned the systematic number α-KTx 2.14.

Role of the activation gate in determining the extracellular potassium dependency of block of HERG by trapped drugs.

Drug induced long QT syndrome (diLQTS) results primarily from block of the cardiac potassium channel HERG (human-ether-a-go-go related gene). In some cases long QT syndrome can result in the lethal arrhythmia torsade de pointes, an arrhythmia characterized by a rapid heart rate and severely compromised cardiac output. Many patients requiring medication present with serum potassium abnormalities due to a variety of conditions including gastrointestinal dysfunction, renal and endocrine disorders, diuretic use, and aging. Extracellular potassium influences HERG channel inactivation and can alter block of HERG by some drugs. However, block of HERG by a number of drugs is not sensitive to extracellular potassium. In this study, we show that block of WT HERG by bepridil and terfenadine, two drugs previously shown to be trapped inside the HERG channel after the channel closes, is insensitive to extracellular potassium over the range of 0 mM to 20 mM. We also show that bepridil block of the HERG mutant D540K, a mutant channel that is unable to trap drugs, is dependent on extracellular potassium, correlates with the permeant ion, and is independent of HERG inactivation. These results suggest that the lack of extracellular potassium dependency of block of HERG by some drugs may in part be related to the ability of these drugs to be trapped inside the channel after the channel closes.

Comparative effects of liensinine and neferine on the human ether-a-go-go-related gene potassium channel and pharmacological activity analysis.

Liensinine and neferine, a kind of isoquinoline alkaloid, can antagonize the ventricular arrhythmias. The human ether-a-go-go-related gene (hERG) is involved in repolarization of cardiac action potential. We investigated the effects of liensinine and neferine on the biophysical properties of hERG channel and the underlying structure-activity relationships. The effects of liensinine and neferine were examined on the hERG channels in the stable transfected HEK293 cells using a whole-cell patch clamp technique, western blot analysis and immunofluorescence experiment. The pharmacokinetics and tissue distribution determination of liensinine and neferine in rats were determined by a validated RP-HPLC method. Liensinine and neferine induced decrease of current amplitude in dose-dependent. Liensinine reduced hERG tail current from 70.3±6.3 pA/pF in control group to 56.7±2.8 pA/pF in the 1 µM group, 53.0±2.3 pA/pF (3 µM) and 17.8±0.7 pA/pF (30 µM); the corresponding current densities of neferine-treated cells were 41.9±3.1 pA/pF, 32.3±3.1 pA/pF and 16.2±0.6 pA/pF, respectively. Neferine had binding affinity for the open and inactivated state of hERG channel, liensinine only bound to the open state. The inhibitory effects of liensinine and neferine on hERG current were attenuated in the F656V or Y652A mutant channels. Neferine distributed more quickly than liensinine in rats, which was found to be in higher concentration than liensinine. Both liensinine and neferine had no effect on the generation and expression of hERG channels. In conclusion, neferine is a more potent blocker of hERG channels than liensinine at low concentration (<10 µM), which may be due to higher hydrophobic nature of neferine compared with liensinine. Neferine may be safety even for long-term treatment as an antiarrhythmic drug.

Vernakalant, a mixed sodium and potassium ion channel antagonist that blocks K(v)1.5 channels, for the potential treatment of atrial fibrillation.

Despite being the most common arrhythmia currently treated by cardiologists, safe and effective treatments for atrial fibrillation (AF) remain elusive. To address this issue, Astellas Pharma Inc, Merck & Co Inc and Cardiome Pharma Corp are developing vernakalant (RSD-1235), a drug which dose-dependently inhibits sodium channels and several potassium repolarizing currents. Of particular note, vernakalant inhibits I(Kur) (K(v)1.5), a current that is more predominant in atrial than in ventricular tissue. Consistent with this observation, vernakalant produced increases in atrial refractory period with minimal actions on QTc interval or ventricular refractory period in both humans and animals. Intravenous vernakalant terminated recent-onset AF in several animal models, and also in patients with short-duration AF or AF following cardiac surgery enrolled in phase II and III clinical trials. Vernakalant was well tolerated and adverse reactions were transient and mild. Thus, vernakalant holds considerable promise for the treatment of recent-onset AF; however, given its relatively short half-life, continuous dosing may be required in order to maintain sinus rhythm following conversion from AF. The efficacy and safety of vernakalant for the long-term management of AF remains to be determined. Phase III clinical trials with intravenous vernakalant are ongoing, and phase II clinical trials are also being conducted with an oral formulation intended for chronic use.

Role of estrogen in renal handling of organic cation, tetraethylammonium: in vivo and in vitro studies.

This study examined the effect of estrogen (17beta-estradiol) on renal handling of organic cation, tetraethylammonium (TEA), both in vivo and in vitro. Clearance of TEA in ovariectomized (OVX) mice was increased by 38% above intact animals, which was able to be returned to control level by estrogen supplementation. The mechanism of this effect was examined in isolated mouse renal proximal tubules (mRPT), showing that [(3)H]-TEA uptake was higher in OVX mice than control, and estrogen supplementation returned uptake to normal level. Kinetics analysis of [(3)H]-TEA uptake indicated an increase in numbers of organic cation transporters in OVX mice but no change in substrate affinity. However, mRNA levels determined by quantitative real time polymerase chain reaction of the relevant transporters at basolateral (organic cation transporter (OCT)1, OCT2 and OCT3) and apical (organic cation/carnitine transporter (OCTN)1, OCTN2 and multidrug and toxin extrusion (MATE)1) membranes of OVX mice were not significantly changed, with only MATE2 mRNA of OVX mice being significantly decreased. The realization that estrogen status affects renal clearance of organic cations will be of importance when assessing the susceptibility of an individual to drug-induced toxicity.

Methadone-induced mortality in the treatment of chronic pain: role of QT prolongation.

Methadone is increasingly prescribed for chronic pain, yet the associated mortality appears to be rising disproportionately relative to other opioid analgesics. We review the available evidence on methadone-associated mortality, and explore potential pharmacokinetic and pharmacodynamic explanations for its greater apparent lethality. While methadone shares properties of central nervous system and respiratory depression with other opioids, methadone is unique as a potent blocker of the delayed rectifier potassium ion channel (IKr). This results in QT-prolongation and torsade de pointes (TdP) in susceptible individuals. In some individuals with low serum protein binding of methadone, the extent of blockade is roughly comparable to that of sotalol, a potent QT-prolonging drug. Predicting an individual's propensity for methadone-induced TdP is difficult at present given the inherent limitations of the QT interval as a risk-stratifier combined with the multifactorial nature of the arrhythmia. Consensus recommendations have recently been published to mitigate the risk of TdP until further studies better define the arrhythmia risk factors for methadone. Studies are needed to provide insights into the clinical covariates most likely to result in methadone-associated arrhythmia and to assess the feasibility of current risk mitigation strategies.

Triggering and amplification of insulin secretion by dimethyl alpha-ketoglutarate, a membrane permeable alpha-ketoglutarate analogue.

Cytosolic alpha-ketoglutarate is a potential signalling compound at late steps of stimulus-secretion-coupling in the course of insulin secretion induced by glucose and other fuels. This hypothesis is mainly based on the insulin-releasing effect of the membrane permeable ester dimethyl alpha-ketoglutarate which enters the beta-cell and is cleaved to produce cytosolic monomethyl alpha-ketoglutarate and eventually alpha-ketoglutarate. The present study tested this hypothesis. Insulin release, K(ATP) channel currents, membrane potential, ATP/ADP ratio and fluorescence of NAD(P)H (reduced pyridine nucleotides) were measured in mouse pancreatic islets and beta-cells. At a substimulatory glucose concentration (5 mM), dimethyl alpha-ketoglutarate (15 mM) produced a sustained insulin release, but no change of the islet ATP/ADP ratio and NAD(P)H fluorescence. In the absence of glucose, however, dimethyl alpha-ketoglutarate (15 mM) did not stimulate insulin release although it increased the ATP/ADP ratio and NAD(P)H fluorescence. Insulin secretion induced by a maximally effective concentration of the K(ATP) channel-blocking sulfonylurea glipizide was strongly amplified by dimethyl alpha-ketoglutarate in the presence of 5 mM glucose, but only moderately in the absence of glucose. Dimethyl alpha-ketoglutarate directly inhibited K(ATP) channels in inside-out membrane patches, depolarized the plasma membrane of intact beta-cells and generated action potentials. In conclusion, the stimulation of insulin secretion by extracellularly applied dimethyl alpha-ketoglutarate depends on inhibition of beta-cell K(ATP) channels by direct action of dimethyl alpha-ketoglutarate. The metabolism of alpha-ketoglutarate generated intracellularly by ester cleavage contributes to stimulation of insulin secretion both by indirect K(ATP) channel inhibition (via activation of ATP production) and by an amplifying effect.

Drug-induced long QT syndrome in women: review of current evidence and remaining gaps.

BACKGROUND: Women are at an increased risk of drug-induced long QT syndrome (LQTS). This major cardiac adverse effect may lead to malignant polymorphic ventricular tachycardias, termed torsades de pointes, which may degenerate into ventricular fibrillation and cause sudden death. OBJECTIVE: This article reviews current evidence and remaining gaps in knowledge about drug-induced LQTS in women. METHODS: Using the search terms gender, sex, and sex differences in combination with cardiac electrophysiology, long QT syndrome, HERG, membrane transporters, and cytochromes, we conducted a systematic review of the available literature in the PubMed database. Relevant English- and French-language publications (to October 2007) on sex differences in LQTS were identified. RESULTS: Clinical and experimental studies have reported that gonadal hormones play a role in sex-related differences of QT interval prolongation. Androgens may diminish drug effects on heart repolarization, and estrogens may facilitate arrhythmias. Furthermore, sex-related differences in the density of ion channels may partially explain this phenomenon. However, the magnitude of hormone-dependent differences observed in these studies remains very small compared with the large differences observed in clinical settings. Therefore, many scientists agree that the mechanisms responsible for sex-related differences in the risk of proarrhythmia from drugs remain largely undefined. CONCLUSIONS: Other factors, such as sex-related modulation of drug disposition in situ, may fill the gaps in our understanding of the sex differences observed in drug-induced LQTS. We suggest that mechanisms such as the modulation of the pharmacokinetics of IKr (rapid component of the delayed rectifier potassium current) blockers, via modulation of intra- and extracellular concentrations, may be of major importance. Sex-specific changes in drug transport and metabolism will result in different plasma and intracellular levels acting along a dose-response effect on IKr block. Consequently, important hormone-dependent factors such as metabolic enzymes and membrane transporters need to be investigated in new basic research studies.

Vernakalant: RSD 1235, RSD-1235, RSD1235.

Vernakalant is an atrial-selective antiarrhythmic drug discovered by Cardiome Pharma (formerly Nortran Pharmaceuticals). Vernakalant may have potential in the treatment of atrial arrhythmias, including acute atrial fibrillation and atrial flutter. Vernakalant is a mixed sodium/potassium channel blocker and selectively blocks ion channels in the heart that are known to be active during episodes of atrial fibrillation. An IV formulation of vernakalant is awaiting registration in the US for the acute conversion of atrial fibrillation. Also, an oral formulation of the compound is in phase II clinical development as a chronic-use product for the maintenance of normal heart rhythm following termination of atrial fibrillation. Cardiome is seeking co-development partners for intravenous vernakalant in the treatment of atrial arrhythmia, atrial fibrillation and atrial flutter in Europe and Japan. In October 2003, Cardiome Pharma and Fujisawa Healthcare, the US subsidiary of Fujisawa Pharmaceutical Co., Ltd (now Astellas Pharma), executed a $US68 million strategic partnership agreement for the co-development of vernakalant. On 1 April 2005, Fujisawa merged with Yamanouchi to form Astellas Pharma. The partnership grants Astellas Pharma exclusive commercialisation rights for vernakalant. Under the terms of the agreement, Cardiome and Astellas Pharma will co-develop vernakalant as an intravenous formulation for the treatment of atrial fibrillation and atrial flutter for North American markets. Astellas Pharma will be financially responsible for 75% of all future clinical development costs, with Cardiome responsible for the remaining 25% of costs. Astellas Pharma will be responsible for the development plan, NDA application (and NDA re-submission costs) and registration, along with the commercial manufacturing, marketing and sale of vernakalant. Cardiome will manage the phase III trials ACT 1 and ACT 2 and will also be responsible for the continued manufacturing of clinical supplies of vernakalant. Cardiome will receive royalties on end-user sales of vernakalant reflective of Cardiome's 25% share of development costs and other financial considerations. Product rights to the IV formulation of vernakalant for markets outside of North America and world rights to the oral formulation of vernakalant for chronic atrial fibrillation are not included within the scope of this partnership. Cardiome intends to form future additional alliances for these product opportunities or maintain such opportunities for commercialisation on its own. Cardiome and Astellas amended their agreement for vernakalant in relation to the re-submission of the NDA with the US FDA. Under the terms of the new agreement, Astellas agreed to fund 100% of the costs associated with re-submission, including engagement and external consultants. Astellas also agreed to modify the timing of the $US10 million NDA milestone to the date of resubmission. In February 2005, Cardiome Pharma received a $US6 million milestone payment from its co-development partner, Fujisawa Healthcare Inc. This milestone payment was triggered by the successful completion of ACT 1.A pivotal phase II trial demonstrated in September 2002 that vernakalant rapidly and effectively terminated recent onset atrial fibrillation and the study met both primary and secondary study endpoints. Following discussions with the FDA, Cardiome initiated three separate phase III clinical trials in order to enable Cardiome to apply for marketing approval for vernakalant. In August 2003, Cardiome Pharma commenced patient dosing in its first phase III efficacy study of vernakalant for the acute treatment of atrial fibrillation. This initial study, called ACT 1 (Atrial fibrillation Conversion Trial 1), measured the safety and efficacy of vernakalant in 416 patients with atrial arrhythmias. The placebo-controlled study was carried out in 45 centers in the US, Canada and Scandinavia. The ACT 1 study included two substudies of 60 patients with atrial flutter and 119 patients with longer term atrial fibrillation. The primary efficacy endpoint was acute conversion of atrial arrhythmia to normal heart rhythm. Cardiome commenced its second phase III efficacy study in March 2004, known as ACT 2. The ACT 2 study in post-cardiac surgery (coronary artery bypass graft) patients with atrial fibrillation, evaluated the safety and efficacy of vernakalant (IV) in the termination of atrial arrhythmias in patients after cardiac surgery. Around 210 patients from 25 centres in the US, Canada and Europe were enrolled in this study. The primary efficacy endpoint was acute conversion of atrial arrhythmias to normal heart rhythm. The ACT 2 study is ongoing. The third phase III study, known as ACT 3 (Atrial arrhythmia Conversion Trial 3), was initiated by Cardiome Pharma in July 2004. In September 2005, Cardiome and Astellas reported that ACT 3 had been completed, achieving its primary endpoint, with over half of the 170 patients with recent-onset atrial fibrillation (AF) who received vernakalant intravenously converting to normal heart rhythm, compared with only 4% in the placebo group. The study was being conducted by co-development partner Astellas Pharma and measured the safety and efficacy of intravenous vernakalant in recent onset atrial arrhythmia patients. The placebo-controlled study was being carried out in 276 patients in more than 50 centres throughout the world.ACT 4, a phase III safety study evaluating safety of IV vernakalant in approximately 120 AF patients from 30 centres in the US, Canada and Europe, was initiated in October 2005. Results from this trial are expected to supplement trial results from the pivotal ACT 1 and 3 trials. This study is ongoing. Cardiome Pharma successfully completed phase I studies for its controlled-release oral formulation of vernakalant in 2005. The oral, controlled-release formulation of vernakalant is expected to help prevent or slow the recurrence of atrial fibrillation, and will be used as a follow-on therapy to intravenous vernakalant.

Targeting effector memory T cells with the small molecule Kv1.3 blocker PAP-1 suppresses allergic contact dermatitis.

The voltage-gated potassium channel Kv1.3 has been recently identified as a molecular target that allows for selective pharmacological suppression of effector memory T (T(EM)) cells without affecting the function of naïve and central memory T cells. We here investigated whether PAP-1, a small molecule Kv1.3 blocker (EC50=2 nM), could suppress allergic contact dermatitis (ACD). In a rat model of ACD, we first confirmed that the infiltrating cells in the elicitation phase are indeed CD8+ CD45RC- memory T cells with high Kv1.3 expression. In accordance with its selective effect on T(EM) cells, PAP-1 did not impair sensitization, but potently suppressed oxazolone-induced inflammation by inhibiting the infiltration of CD8+ T cells and reducing the production of the inflammatory cytokines IFN-gamma, IL-2, and IL-17 when administered intraperitoneally or orally during the elicitation phase. PAP-1 was equally effective when applied topically, demonstrating that it effectively penetrates skin. We further show that PAP-1 is not a sensitizer or an irritant and exhibits no toxicity in a 28-day toxicity study. Based on these results we propose that PAP-1 could potentially be developed into a drug for the topical treatment of inflammatory skin diseases such as psoriasis.