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1.
Cell ; 185(25): 4801-4810.e13, 2022 Dec 08.
Artigo em Inglês | MEDLINE | ID: mdl-36417914

RESUMO

Drug-drug interaction of the antiviral sofosbuvir and the antiarrhythmics amiodarone has been reported to cause fatal heartbeat slowing. Sofosbuvir and its analog, MNI-1, were reported to potentiate the inhibition of cardiomyocyte calcium handling by amiodarone, which functions as a multi-channel antagonist, and implicate its inhibitory effect on L-type Cav channels, but the molecular mechanism has remained unclear. Here we present systematic cryo-EM structural analysis of Cav1.1 and Cav1.3 treated with amiodarone or sofosbuvir alone, or sofosbuvir/MNI-1 combined with amiodarone. Whereas amiodarone alone occupies the dihydropyridine binding site, sofosbuvir is not found in the channel when applied on its own. In the presence of amiodarone, sofosbuvir/MNI-1 is anchored in the central cavity of the pore domain through specific interaction with amiodarone and directly obstructs the ion permeation path. Our study reveals the molecular basis for the physical, pharmacodynamic interaction of two drugs on the scaffold of Cav channels.


Assuntos
Amiodarona , Sofosbuvir , Sofosbuvir/efeitos adversos , Amiodarona/farmacologia , Antivirais/farmacologia , Miócitos Cardíacos/metabolismo , Sítios de Ligação , Canais de Cálcio Tipo L/metabolismo , Cálcio/metabolismo
2.
J Pharmacol Exp Ther ; 389(2): 229-242, 2024 Apr 18.
Artigo em Inglês | MEDLINE | ID: mdl-38453526

RESUMO

The drug-drug interaction (DDI) between amiodarone (AMIO) and sofosbuvir (SOF), a direct-acting hepatitis-C NS5B nucleotide polymerase inhibitor, has been associated with severe bradyarrhythmia in patients. Recent cryo-EM data has revealed that this DDI occurs at the α-subunit of L-type Cav channels, with AMIO binding at the fenestration site and SOF [or MSD nucleotide inhibitor #1 (MNI-1): analog of SOF] binding at the central cavity of the conductance pathway. In this study, we investigated the DDI between 21 AMIO analogs, including dronedarone (DRON) and MNI-1 (or SOF) in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and hCav1.2 models. Our findings indicate that among the tested AMIO analogs in hiPSC-CMs at clinically relevant concentrations, only three analogs (AA-9, AA-10, and AA-17) were able to effectively substitute for AMIO in this DDI with 1 µM MNI-1. This highlights the importance of the diethyl amino group of AMIO for interacting with MNI-1. In the hCav1.2 model, desethylamiodarone (AA-12) demonstrated synergy with 90 µM MNI-1, while three other analogs with modifications to the position of the diethyl amino group or removal of iodo groups showed weaker synergy with 90 µM MNI-1. Interestingly, DRON did not exhibit any interaction with 270 µM SOF or 90 µM MNI-1, suggesting that it could safely replace AMIO in patients requiring SOF treatment, other clinically relevant differences considered. Overall, our functional data align with the cryo-EM data, highlighting that this DDI is dependent on the structure of AMIO and cardiomyocyte resting membrane potential. SIGNIFICANCE STATEMENT: Our findings point to specific residues in the AMIO molecule playing a critical role in the DDI between AMIO and MNI-1 (SOF analog), confirming cryo-EM results. Applied at clinically relevant AMIO's concentrations or projected MNI-1's concentrations at the resting potentials mimicking the sinoatrial node, this DDI significantly slowed down or completely inhibited the beating of hiPSC-CMs. Finally, these in vitro results support the safe replacement of AMIO (Cordarone) with DRON (Multaq) for patients requiring SOF treatment, other clinical caveats considered.


Assuntos
Amiodarona , Células-Tronco Pluripotentes Induzidas , Humanos , Amiodarona/farmacologia , Amiodarona/metabolismo , Nucleotídeos/farmacologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Interações Medicamentosas , Relação Estrutura-Atividade
3.
J Pharmacol Exp Ther ; 386(1): 26-34, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37068911

RESUMO

Vericiguat and its metabolite M-1 were assessed for proarrhythmic risk in nonclinical in vitro and in vivo studies. In vitro manual voltage-clamp recordings at room temperature determined the effect of vericiguat on human Ether-a-go-go Related Gene (hERG) K+ channels. Effects of vericiguat and M-1 on hERG K+, Nav1.5, hCav1.2, hKvLQT1/1minK, and hKv4.3 channels were investigated via automated voltage-clamp recordings at ambient temperature. Effects of vericiguat and M-1 on hERG K+ and Nav1.5 channels at pathophysiological conditions were explored via manual voltage-clamp recordings at physiologic temperature. Single oral doses of vericiguat (0.6, 2.0, and 6.0 mg/kg) were assessed for in vivo proarrhythmic risk via administration to conscious telemetered dogs; electrocardiogram (ECG) and hemodynamic parameters were monitored. ECG recordings were included in 4- and 39-week dog toxicity studies. In manual voltage-clamp recordings, vericiguat inhibited hERG K+-mediated tail currents in a concentration-dependent manner (20% threshold inhibitory concentration ∼1.9 µM). In automated voltage-clamp recordings, neither vericiguat nor M-1 were associated with biologically relevant inhibition (>20%) of hNav1.5, hCav1.2, hKvLQT1, and hKv4.3. No clinically relevant observations were made for hNav1.5 and hKvLQT1 under simulated pathophysiological conditions. Vericiguat was associated with expected mode-of-action-related dose-dependent changes in systolic arterial blood pressure (up to -20%) and heart rate (up to +53%). At maximum vericiguat dose, corrected QT (QTc) interval changes from baseline varied slightly (-6 to +1%) depending on correction formula. Toxicity studies confirmed absence of significant QTc interval changes. There was no evidence of an increased proarrhythmic risk from nonclinical studies with vericiguat or M-1. SIGNIFICANCE STATEMENT: There was no evidence of an increased proarrhythmic risk from in vitro and in vivo nonclinical studies with vericiguat or M-1. The integrated risk assessment of these nonclinical data combined with existing clinical data demonstrate administration of vericiguat 10 mg once daily in patients with heart failure with reduced ejection fraction is not associated with a proarrhythmic risk.


Assuntos
Insuficiência Cardíaca , Compostos Heterocíclicos com 2 Anéis , Humanos , Animais , Cães , Guanilil Ciclase Solúvel/metabolismo , Pirimidinas , Vasodilatadores , Canais de Potássio Éter-A-Go-Go
4.
Toxicol Appl Pharmacol ; 430: 115725, 2021 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-34536444

RESUMO

An effective in vitro screening assay to detect seizure liability in preclinical development can contribute to better lead molecule optimization prior to candidate selection, providing higher throughput and overcoming potential brain exposure limitations in animal studies. This study explored effects of 26 positive and 14 negative reference pharmacological agents acting through different mechanisms, including 18 reference agents acting on glutamate signaling pathways, in a brain slice assay (BSA) of adult rat to define the assay's sensitivity, specificity, and limitations. Evoked population spikes (PS) were recorded from CA1 pyramidal neurons of hippocampus (HPC) in the BSA. Endpoints for analysis were PS area and PS number. Most positive references (24/26) elicited a concentration-dependent increase in PS area and/or PS number. The negative references (14/14) had little effect on the PS. Moreover, we studied the effects of 15 reference agents testing positive in the BSA on spontaneous activity in E18 rat HPC neurons monitored with microelectrode arrays (MEA), and compared these effects to the BSA results. From these in vitro studies we conclude that the BSA provides 93% sensitivity and 100% specificity in prediction of drug-induced seizure liability, including detecting seizurogenicity by 3 groups of metabotropic glutamate receptor (mGluR) ligands. The MEA results seemed more variable, both quantitatively and directionally, particularly for endpoints capturing synchronized electrical activity. We discuss these results from the two models, comparing each with published results, and provide potential explanations for differences and future directions.


Assuntos
Convulsivantes/toxicidade , Potenciais Evocados/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Convulsões/induzido quimicamente , Testes de Toxicidade , Animais , Células Cultivadas , Feminino , Idade Gestacional , Ácido Glutâmico/metabolismo , Hipocampo/embriologia , Hipocampo/metabolismo , Hipocampo/fisiopatologia , Técnicas In Vitro , Ligantes , Masculino , Neurônios/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Receptores de Glutamato Metabotrópico/metabolismo , Reprodutibilidade dos Testes , Medição de Risco , Convulsões/metabolismo , Convulsões/fisiopatologia , Transdução de Sinais
5.
Antimicrob Agents Chemother ; 60(8): 4830-9, 2016 08.
Artigo em Inglês | MEDLINE | ID: mdl-27246784

RESUMO

Oxabicyclooctane-linked novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of recently described antibacterial agents with broad-spectrum activity. NBTIs dually inhibit the clinically validated bacterial targets DNA gyrase and topoisomerase IV and have been shown to bind distinctly from known classes of antibacterial agents directed against these targets. Herein we report the molecular, cellular, and in vivo characterization of AM-8722 as a representative N-alkylated-1,5-naphthyridone left-hand-side-substituted NBTI. Consistent with its mode of action, macromolecular labeling studies revealed a specific effect of AM-8722 to dose dependently inhibit bacterial DNA synthesis. AM-8722 displayed greater intrinsic enzymatic potency than levofloxacin versus both DNA gyrase and topoisomerase IV from Staphylococcus aureus and Escherichia coli and displayed selectivity against human topoisomerase II. AM-8722 was rapidly bactericidal and exhibited whole-cell activity versus a range of Gram-negative and Gram-positive organisms, with no whole-cell potency shift due to the presence of DNA or human serum. Frequency-of-resistance studies demonstrated an acceptable rate of resistance emergence in vitro at concentrations 16- to 32-fold the MIC. AM-8722 displayed acceptable pharmacokinetic properties and was shown to be efficacious in mouse models of bacterial septicemia. Overall, AM-8722 is a selective and potent NBTI that displays broad-spectrum antimicrobial activity in vitro and in vivo.


Assuntos
Antibacterianos/farmacologia , Ciclo-Octanos/farmacologia , DNA Girase/metabolismo , DNA Topoisomerase IV/antagonistas & inibidores , DNA Topoisomerases Tipo II/metabolismo , Inibidores da Topoisomerase II/farmacologia , Animais , Linhagem Celular , DNA Bacteriano/genética , Cães , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Infecções por Escherichia coli/tratamento farmacológico , Humanos , Camundongos , Testes de Sensibilidade Microbiana , Ratos , Ratos Wistar , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/genética
6.
Toxicol Appl Pharmacol ; 308: 66-76, 2016 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-27520758

RESUMO

Several clinical cases of severe bradyarrhythmias have been reported upon co-administration of the Hepatitis-C NS5B Nucleotide Polymerase Inhibitor (HCV-NI) direct-acting antiviral agent, sofosbuvir (SOF), and the Class-III anti-arrhythmic amiodarone (AMIO). We model the cardiac drug-drug interaction (DDI) between AMIO and SOF, and between AMIO and a closely-related SOF analog, MNI-1 (Merck Nucleotide Inhibitor #1), in functional assays of human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), to provide mechanistic insights into recently reported clinical cases. AMIO co-applied with SOF or MNI-1 increased beating rate or field potential (FP) rate and decreased impedance (IMP) and Ca(2+) transient amplitudes in hiPSC-CM syncytia. This action resembled that of Ca(2+) channel blockers (CCBs) in the model, but CCBs did not substitute for AMIO in the DDI. AMIO analog dronedarone (DRON) did not substitute for, but competed with AMIO in the DDI. Ryanodine and thapsigargin, decreasing intracellular Ca(2+) stores, and SEA-0400, a Na(+)/Ca(2+) exchanger-1 (NCX1) inhibitor, partially antagonized or suppressed DDI effects. Other agents affecting FP rate only exerted additive or subtractive effects, commensurate with their individual effects. We also describe an interaction between AMIO and MNI-1 on Cav1.2 ion channels in an over-expressing HEK-293 cell line. MNI-1 enhanced Cav1.2 channel inhibition by AMIO, but did not affect inhibition of Cav1.2 by DRON, verapamil, nifedipine, or diltiazem. Our data in hiPSC-CMs indicate that HCV-NI agents such as SOF and MNI-1 interact with key intracellular Ca(2+)-handling mechanisms. Additional study in a Cav1.2 HEK-293 cell-line suggests that HCV-NIs potentiate the inhibitory action of AMIO on L-type Ca(2+) channels.


Assuntos
Amiodarona/farmacologia , Antivirais/farmacologia , Hepacivirus/efeitos dos fármacos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Canais de Cálcio Tipo L , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Miócitos Cardíacos/citologia
7.
Bioorg Med Chem Lett ; 25(17): 3630-5, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26152426

RESUMO

Oxabicyclooctane linked novel bacterial topoisomerase inhibitors (NBTIs) are new class of recently reported broad-spectrum antibacterial agents. They target bacterial DNA gyrase and topoisomerase IV and bind to a site different than quinolones. They show no cross-resistance to known antibiotics and provide opportunity to combat drug-resistant bacteria. A structure activity relationship of the C-2 substituted ether analogs of 1,5-naphthyridine oxabicyclooctane-linked NBTIs are described. Synthesis and antibacterial activities of a total of 63 analogs have been summarized representing alkyl, cyclo alkyl, fluoro alkyl, hydroxy alkyl, amino alkyl, and carboxyl alkyl ethers. All compounds were tested against three key strains each of Gram-positive and Gram-negative bacteria as well as for hERG binding activities. Many key compounds were also tested for the functional hERG activity. Six compounds were evaluated for efficacy in a murine bacteremia model of Staphylococcus aureus infection. Significant tolerance for the ether substitution (including polar groups such as amino and carboxyl) at C-2 was observed for S. aureus activity however the same was not true for Enterococcus faecium and Gram-negative strains. Reduced clogD generally showed reduced hERG activity and improved in vivo efficacy but was generally associated with decreased overall potency. One of the best compounds was hydroxy propyl ether (16), which mainly retained the potency, spectrum and in vivo efficacy of AM8085 associated with the decreased hERG activity and improved physical property.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Naftiridinas/química , Relação Estrutura-Atividade , Animais , Antibacterianos/síntese química , Antibacterianos/farmacocinética , Técnicas de Química Sintética , Ciclo-Octanos/química , DNA Girase/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Canal de Potássio ERG1 , Enterococcus faecium/efeitos dos fármacos , Canais de Potássio Éter-A-Go-Go/metabolismo , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Ratos Sprague-Dawley , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Inibidores da Topoisomerase II/química , Inibidores da Topoisomerase II/farmacologia
8.
Bioorg Med Chem Lett ; 25(17): 3636-43, 2015 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-26141771

RESUMO

Oxabicyclooctane linked 1,5-naphthyridinyl-pyridoxazinones are novel broad-spectrum bacterial topoisomerase inhibitors (NBTIs) targeting bacterial DNA gyrase and topoisomerase IV at a site different than quinolones. Due to lack of cross-resistance to known antibiotics they present excellent opportunity to combat drug-resistant bacteria. A structure activity relationship of the pyridoxazinone moiety is described in this Letter. Chemical synthesis and activities of NBTIs with substitutions at C-3, C-4 and C-7 of the pyridoxazinone moiety with halogens, alkyl groups and methoxy group has been described. In addition, substitutions of the linker NH proton and its transformation into amide analogs of AM-8085 and AM-8191 have been reported. Fluoro, chloro, and methyl groups at C-3 of the pyridoxazinone moiety retained the potency and spectrum. In addition, a C-3 fluoro analog showed 4-fold better oral efficacy (ED50 3.9 mg/kg) as compared to the parent AM-8085 in a murine bacteremia model of infection of Staphylococcus aureus. Even modest polarity (e.g., methoxy) is not tolerated at C-3 of the pyridoxazinone unit. The basicity and NH group of the linker is important for the activity when CH2 is at the linker position-8. However, amides (with linker position-8 ketone) with a position-7 NH or N-methyl group retained potency and spectrum suggesting that neither basicity nor hydrogen-donor properties of the linker amide NH is essential for the activity. This would suggest likely an altered binding mode of the linker position-7,8 amide containing compounds. The amides showed highly improved hERG (functional IC50 >30 µM) profile.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Ciclo-Octanos/química , Avaliação Pré-Clínica de Medicamentos/métodos , Relação Estrutura-Atividade , Inibidores da Topoisomerase/química , Administração Oral , Animais , Antibacterianos/administração & dosagem , Técnicas de Química Sintética , DNA Topoisomerase IV/antagonistas & inibidores , Canal de Potássio ERG1 , Canais de Potássio Éter-A-Go-Go/metabolismo , Compostos Heterocíclicos com 2 Anéis/química , Compostos Heterocíclicos com 2 Anéis/farmacologia , Camundongos , Testes de Sensibilidade Microbiana , Naftiridinas/química , Naftiridinas/farmacologia , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/patogenicidade , Inibidores da Topoisomerase/farmacologia
9.
Bioorg Med Chem Lett ; 25(11): 2409-15, 2015 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-25911300

RESUMO

Bacterial resistance is rapidly growing, necessitating the need to discover new agents. Novel bacterial topoisomerase inhibitors (NBTIs) are new class of broad-spectrum antibacterial agents targeting bacterial DNA gyrase and topoisomerase IV. This class of inhibitors binds to an alternative binding site relative to fluoroquinolones and shows no cross-resistance to quinolones. NBTIs consist of three structural motifs. A structure activity relationship of the left hand motif 1,5-naphthyridine of oxabicyclooctane-linked NBTIs is described. Fifty five compounds were evaluated against a panel of key Gram-positive and Gram-negative strains of bacteria, as well as for hERG activity and five compounds were tested for in vivo efficacy in murine model of Staphylococcus aureus infection. These studies suggest that only a narrow range (activating and deactivating) of substitutions at C-2 and C-7 are tolerated for optimal antibacterial activity and spectrum. An alkoxy (methoxy) and CN at C-2, and a halogen and hydroxyl at C-7, appeared to be preferred in this series. Substitutions on the other three carbons generally have detrimental effect on the activity. No clear hERG activity SAR emerged from these substitutions.


Assuntos
DNA Topoisomerases/metabolismo , Infecções Estafilocócicas/tratamento farmacológico , Staphylococcus aureus/efeitos dos fármacos , Inibidores da Topoisomerase/química , Inibidores da Topoisomerase/farmacologia , Animais , Camundongos , Estrutura Molecular , Infecções Estafilocócicas/microbiologia , Relação Estrutura-Atividade
10.
Bioorg Med Chem Lett ; 25(12): 2473-8, 2015 Jun 15.
Artigo em Inglês | MEDLINE | ID: mdl-25978963

RESUMO

Novel bacterial topoisomerase inhibitors (NBTIs) are a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. (R)-Hydroxy-1,5-naphthyridinone left-hand side (LHS) oxabicyclooctane linked pyridoxazinone right-hand side (RHS) containing NBTIs showed a potent Gram-positive antibacterial profile. SAR around the RHS moiety, including substitutions around pyridooxazinone, pyridodioxane, and phenyl propenoids has been described. A fluoro substituted pyridoxazinone showed an MIC against Staphylococcus aureus of 0.5 µg/mL with reduced functional hERG activity (IC50 333 µM) and good in vivo efficacy [ED90 12 mg/kg, intravenous (iv) and 15 mg/kg, oral (p.o.)]. A pyridodioxane-containing NBTI showed a S. aureus MIC of 0.5 µg/mL, significantly improved hERG IC50 764 µM and strong efficacy of 11 mg/kg (iv) and 5 mg/kg (p.o.). A phenyl propenoid series of compounds showed potent antibacterial activity, but also showed potent hERG binding activity. Many of the compounds in the hydroxy-tricyclic series showed strong activity against Acinetobacter baumannii, but reduced activity against Escherichia coli and Pseudomonas aeruginosa. Bicyclic heterocycles appeared to be the best RHS moiety for the hydroxy-tricyclic oxabicyclooctane linked NBTIs.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Naftiridinas/química , Inibidores da Topoisomerase/química , Inibidores da Topoisomerase/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/síntese química , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , DNA Girase/química , DNA Girase/metabolismo , Escherichia coli/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Oxazóis/química , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos , Relação Estrutura-Atividade , Inibidores da Topoisomerase/síntese química
11.
Bioorg Med Chem Lett ; 25(9): 1831-5, 2015 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-25851938

RESUMO

Novel bacterial topoisomerase inhibitors (NBTIs) represent a new class of broad-spectrum antibacterial agents targeting bacterial Gyrase A and ParC and have potential utility in combating antibiotic resistance. A series of novel oxabicyclooctane-linked NBTIs with new tricyclic-1,5-naphthyridinone left hand side moieties have been described. Compounds with a (R)-hydroxy-1,5-naphthyridinone moiety (7) showed potent antibacterial activity (e.g., Staphylococcus aureus MIC 0.25 µg/mL), acceptable Gram-positive and Gram-negative spectrum with rapidly bactericidal activity. The compound 7 showed intravenous and oral efficacy (ED50) at 3.2 and 27 mg/kg doses, respectively, in a murine model of bacteremia. Most importantly they showed significant attenuation of functional hERG activity (IC50 >170 µM). In general, lower logD attenuated hERG activity but also reduced Gram-negative activity. The co-crystal structure of a hydroxy-tricyclic NBTI bound to a DNA-gyrase complex exhibited a binding mode that show enantiomeric preference for R isomer and explains the activity and SAR. The discovery, synthesis, SAR and X-ray crystal structure of the left-hand-side tricyclic 1,5-naphthyridinone based oxabicyclooctane linked NBTIs are described.


Assuntos
Antibacterianos/farmacologia , Ciclo-Octanos/farmacologia , DNA Topoisomerases Tipo II/metabolismo , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Naftiridinas/farmacologia , Inibidores da Topoisomerase II/farmacologia , Antibacterianos/síntese química , Antibacterianos/química , Ciclo-Octanos/síntese química , Ciclo-Octanos/química , Relação Dose-Resposta a Droga , Bactérias Gram-Negativas/enzimologia , Bactérias Gram-Positivas/enzimologia , Testes de Sensibilidade Microbiana , Modelos Moleculares , Estrutura Molecular , Naftiridinas/síntese química , Naftiridinas/química , Relação Estrutura-Atividade , Inibidores da Topoisomerase II/síntese química , Inibidores da Topoisomerase II/química
12.
Nat Rev Drug Discov ; 2024 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-38773351

RESUMO

Secondary pharmacology screening of investigational small-molecule drugs for potentially adverse off-target activities has become standard practice in pharmaceutical research and development, and regulatory agencies are increasingly requesting data on activity against targets with recognized adverse effect relationships. However, the screening strategies and target panels used by pharmaceutical companies may vary substantially. To help identify commonalities and differences, as well as to highlight opportunities for further optimization of secondary pharmacology assessment, we conducted a broad-ranging survey across 18 companies under the auspices of the DruSafe leadership group of the International Consortium for Innovation and Quality in Pharmaceutical Development. Based on our analysis of this survey and discussions and additional research within the group, we present here an overview of the current state of the art in secondary pharmacology screening. We discuss best practices, including additional safety-associated targets not covered by most current screening panels, and present approaches for interpreting and reporting off-target activities. We also provide an assessment of the safety impact of secondary pharmacology screening, and a perspective on opportunities and challenges in this rapidly developing field.

13.
J Pharmacol Toxicol Methods ; 123: 107297, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37499956

RESUMO

INTRODUCTION: In the framework of the IMI2-NeuroDeRisk consortium, three in vitro electrophysiology assays were compared to improve preclinical prediction of seizure-inducing liabilities. METHODS: Two cell models, primary rat cortical neurons and human induced pluripotent stem cell (hiPSC)-derived glutamatergic neurons co-cultured with hiPSC-derived astrocytes were tested on two different microelectrode array (MEA) platforms, Maestro Pro (Axion Biosystems) and Multiwell-MEA-System (Multi Channel Systems), in three separate laboratories. Pentylenetetrazole (PTZ) and/or picrotoxin (PTX) were included in each plate as positive (n = 3-6 wells) and ≤0.2% DMSO was used as negative controls (n = 3-12 wells). In general, concentrations in a range of 0.1-30 µM were tested, anchored, when possible, on clinically relevant exposures (unbound Cmax) were tested. Activity thresholds for drug-induced changes were set at 20%. To evaluate sensitivity, specificity and predictivity of the cell models, seizurogenic responses were defined as changes in 4 or more endpoints. Concentration dependence trends were also considered. RESULTS: Neuronal activity of 33 compounds categorized as positive tool drugs, seizure-positive or seizure-negative compounds was evaluated. Acute drug effects (<60 min) were compared to baseline recordings. Time points < 15 min exhibited stronger, less variable responses to many of the test agents. For many compounds a reduction and cessation of neuronal activity was detected at higher test concentrations. There was not a single pattern of seizurogenic activity detected, even among tool compounds, likely due to different mechanisms of actions and/or off-target profiles. A post-hoc analysis focusing on changes indicative of neuronal excitation is presented. CONCLUSION: All cell models showed good sensitivity, ranging from 70 to 86%. Specificity ranged from 40 to 70%. Compared to more conventional measurements of evoked activity in hippocampal slices, these plate-based models provide higher throughput and the potential to study subacute responses. Yet, they may be limited by the random, spontaneous nature of their network activity.


Assuntos
Células-Tronco Pluripotentes Induzidas , Ratos , Humanos , Animais , Microeletrodos , Células Cultivadas , Convulsões/induzido quimicamente , Neurônios
14.
APL Bioeng ; 7(4): 046113, 2023 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-38046544

RESUMO

There is critical need for a predictive model of human cardiac physiology in drug development to assess compound effects on human tissues. In vitro two-dimensional monolayer cultures of cardiomyocytes provide biochemical and cellular readouts, and in vivo animal models provide information on systemic cardiovascular response. However, there remains a significant gap in these models due to their incomplete recapitulation of adult human cardiovascular physiology. Recent efforts in developing in vitro models from engineered heart tissues have demonstrated potential for bridging this gap using human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) in three-dimensional tissue structure. Here, we advance this paradigm by implementing FRESH™ 3D bioprinting to build human cardiac tissues in a medium throughput, well-plate format with controlled tissue architecture, tailored cellular composition, and native-like physiological function, specifically in its drug response. We combined hiPSC-CMs, endothelial cells, and fibroblasts in a cellular bioink and FRESH™ 3D bioprinted this mixture in the format of a thin tissue strip stabilized on a tissue fixture. We show that cardiac tissues could be fabricated directly in a 24-well plate format were composed of dense and highly aligned hiPSC-CMs at >600 million cells/mL and, within 14 days, demonstrated reproducible calcium transients and a fast conduction velocity of ∼16 cm/s. Interrogation of these cardiac tissues with the ß-adrenergic receptor agonist isoproterenol showed responses consistent with positive chronotropy and inotropy. Treatment with calcium channel blocker verapamil demonstrated responses expected of hiPSC-CM derived cardiac tissues. These results confirm that FRESH™ 3D bioprinted cardiac tissues represent an in vitro platform that provides data on human physiological response.

15.
Clin Pharmacol Ther ; 109(2): 310-318, 2021 02.
Artigo em Inglês | MEDLINE | ID: mdl-32866317

RESUMO

Defining an appropriate and efficient assessment of drug-induced corrected QT interval (QTc) prolongation (a surrogate marker of torsades de pointes arrhythmia) remains a concern of drug developers and regulators worldwide. In use for over 15 years, the nonclinical International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) S7B and clinical ICH E14 guidances describe three core assays (S7B: in vitro hERG current & in vivo QTc studies; E14: thorough QT study) that are used to assess the potential of drugs to cause delayed ventricular repolarization. Incorporating these assays during nonclinical or human testing of novel compounds has led to a low prevalence of QTc-prolonging drugs in clinical trials and no new drugs having been removed from the marketplace due to unexpected QTc prolongation. Despite this success, nonclinical evaluations of delayed repolarization still minimally influence ICH E14-based strategies for assessing clinical QTc prolongation and defining proarrhythmic risk. In particular, the value of ICH S7B-based "double-negative" nonclinical findings (low risk for hERG block and in vivo QTc prolongation at relevant clinical exposures) is underappreciated. These nonclinical data have additional value in assessing the risk of clinical QTc prolongation when clinical evaluations are limited by heart rate changes, low drug exposures, or high-dose safety considerations. The time has come to meaningfully merge nonclinical and clinical data to enable a more comprehensive, but flexible, clinical risk assessment strategy for QTc monitoring discussed in updated ICH E14 Questions and Answers. Implementing a fully integrated nonclinical/clinical risk assessment for compounds with double-negative nonclinical findings in the context of a low prevalence of clinical QTc prolongation would relieve the burden of unnecessary clinical QTc studies and streamline drug development.


Assuntos
Drogas em Investigação/efeitos adversos , Síndrome do QT Longo/induzido quimicamente , Animais , Arritmias Cardíacas/induzido quimicamente , Desenvolvimento de Medicamentos/métodos , Indústria Farmacêutica/métodos , Eletrocardiografia/métodos , Humanos , Medição de Risco , Torsades de Pointes/induzido quimicamente
16.
Int J Toxicol ; 29(1): 3-19, 2010.
Artigo em Inglês | MEDLINE | ID: mdl-19903873

RESUMO

Vorinostat (SAHA, Zolinza), a histone deacetylase inhibitor, is assessed in nonclinical studies to support its approval for cutaneous T-cell lymphoma. Vorinostat is weakly mutagenic in the Ames assay; is clastogenic in rodent (ie, CHO) cells but not in normal human lymphocytes; and is weakly positive in an in vivo mouse micronucleus assay. No effects are observed on potassium ion currents in the hERG assay up to 300 microM (safety margin approximately 300-fold the approximately 1 microM serum concentration associated with the 400 mg/d maximum recommended human dose. No rat respiratory or central nervous system effects are found at 150 mg/kg (>2-fold maximum recommended human dose). No cardiovascular effects, including effects on QTc interval, are observed after a single oral dose (150 mg/kg) in dogs. Vorinostat is orally dosed daily in rats (controls, 20, 50, or 150 mg/kg/d) and dogs (controls, 60, 80, or 100/125/160 mg/kg/d) for 26 weeks with a 4-week recovery. Rat vorinostat-related adverse findings are decreased food consumption, weight loss, and hematologic changes; a no observed adverse effects level is not established. In dogs, adverse effects are primarily gastrointestinal; the no observed adverse effects level is 60 mg/kg/d (approximately 6-fold maximum recommended human dose). Toxicities are reversible and can be monitored in the clinic.


Assuntos
Inibidores Enzimáticos/toxicidade , Histona Desacetilases , Ácidos Hidroxâmicos/toxicidade , Animais , Células Sanguíneas/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Células CHO , Cricetinae , Cricetulus , DNA/efeitos dos fármacos , Cães , Avaliação Pré-Clínica de Medicamentos , Ingestão de Alimentos/efeitos dos fármacos , Inibidores Enzimáticos/farmacocinética , Mucosa Gástrica/efeitos dos fármacos , Mucosa Gástrica/patologia , Humanos , Ácidos Hidroxâmicos/farmacocinética , Camundongos , Micronúcleos com Defeito Cromossômico/efeitos dos fármacos , Testes para Micronúcleos , Ratos , Salmonella typhimurium/efeitos dos fármacos , Salmonella typhimurium/genética , Úlcera Gástrica/induzido quimicamente , Úlcera Gástrica/patologia , Testes de Toxicidade , Vorinostat , Redução de Peso/efeitos dos fármacos
17.
Toxicol Sci ; 167(2): 573-580, 2019 02 01.
Artigo em Inglês | MEDLINE | ID: mdl-30365015

RESUMO

Calcium channel blockers (CCBs), such as diltiazem, nifedipine, and verapamil, cause tachycardia effects on several commercially available human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs), when tested in culture media provided by suppliers, rather than bradycardia effects, as seen in vivo. We found that in test conditions where Na+ current of hiPSC-CMs was reduced to certain threshold by either specific Na+ channel blocker tetrodotoxin (TTX), or by voltage-dependent inactivation using elevated extracellular potassium concentrations, CCBs produced bradycardia effects on hiPSC-CMs. However, elevated extracellular potassium concentrations or the presence of TTX did not change other pharmacological responses of hiPSC-CMs, including CCBs' effects on contraction intensity and duration; beating rate change by calcium channel opener FPL64176, HCN blocker ivabradine, and ß-adrenergic agonist isoproterenol; and action potential duration prolongation by hERG channel blocker dofetilide. We concluded that action potentials of hiPSC-CMs, with regards to the CCB phenotype, were Na+ current driven. When Na+ channel availability was reduced to a critical level, their action potentials became Ca2+ current driven, and their responses to CCBs correlated well to those seen in vivo. Importantly, the corrected bradycardia effect of calcium channel block with our defined conditions will provide more reliable results in cardiac safety readouts of test compounds that integrate multiple effects including calcium channel inhibition.


Assuntos
Potenciais de Ação/efeitos dos fármacos , Bloqueadores dos Canais de Cálcio/farmacologia , Avaliação Pré-Clínica de Medicamentos , Células-Tronco Pluripotentes Induzidas/efeitos dos fármacos , Miócitos Cardíacos/efeitos dos fármacos , Canais de Cálcio/metabolismo , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Meios de Cultura , Frequência Cardíaca/efeitos dos fármacos , Humanos , Potássio/farmacologia , Bloqueadores dos Canais de Sódio/farmacologia , Tetrodotoxina/farmacologia
18.
JRSM Cardiovasc Dis ; 8: 2048004019854919, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31217965

RESUMO

OBJECTIVE: We investigated if there is IKs, and if there is repolarization reserve by IKs in human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). DESIGN: We used a specific KCNQ1/KCNE1 channel blocker, L-000768673, with an IC50 of 9 nM, and four hERG-specific blockers, astemizole, cisapride, dofetilide, and E-4031 to investigate the issue. RESULTS: L-000768673 concentration-dependently prolonged feature point duration (FPD)-a surrogate signal of action potential duration-from 1 to 30 nM without pacing or paced at 1.2 Hz, resulting from IKs blockade in hiPSC-CMs. At higher concentrations, the effect of L-000768673 on IKs was mitigated by its effect on ICa-L, resulting in shortened FPD, reduced impedance amplitude, and increased beating rate at 1 µM and above, recapitulating the self-limiting properties of L-000768673 on action potentials. All four hERG-specific blockers prolonged FPD as expected. Co-application of L-000768673 at sub-threshold (0.1 and 0.3 nM) and threshold (1 nM) concentrations failed to synergistically enhance the effects of hERG blockers on FPD prolongation, rather it showed additive effects, inconsistent with the repolarization reserve role of IKs in mature human myocytes that enhanced IKr response, implying a difference between hiPSC-CMs used in this study and mature human cardiomyocytes. CONCLUSION: There was IKs current in hiPSC-CMs, and blockade of IKs current caused prolongation of action potential of hiPSC-CMs. However, we could not demonstrate any synergistic effects on action potential duration prolongation of hiPSC-CMs by blocking hERG current and IKs current simultaneously, implying little or no repolarization reserve by IKs current in hiPSC-CMs used in this study.

19.
J Pharmacol Toxicol Methods ; 99: 106598, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31201864

RESUMO

We investigated whether human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) created from diverse origins could have qualitatively (not just quantitatively) different responses to pharmacological reagents. Specifically, we challenged six hiPSC-CM syncytia made from a female Caucasian, a female black non-Hispanic, a female white non-Hispanic, a male Caucasian non-Hispanic, a male Asian Indian, and a male-Asian, respectively, with eight different classes of pharmacological reagents (hERG channel blocker cisapride and dofetilide, calcium channel opener FPL64176, ß-adrenergic agonist Isoproterenol, HCN channel blocker Ivabradine, IKs current blocker L-000768673, sodium channel blocker tetrodotoxin, and calcium channel blocker verapamil). We focused our analysis and comparison on qualitative differences (e.g., yes or no), and, found the following: hiPSC-CMs from female donors were uniformly more sensitive to dofetilide or cisapride, whereas those from male donors of all races were less sensitive to the two typical hERG blockers; isoproterenol had no chronotropic effect at all in one line; and two lines reacted to tetrodotoxin at very low concentrations and were more sensitive to external stimulation. We conclude that not all hiPSC-CMs are suitable for drug testing in terms of cardiac safety assessment, and pre-set acceptance criteria need to be established before any hiPSC-CMs can be used in CiPA-style study to evaluate cardiac liabilities of drug candidates.

20.
Toxicol Sci ; 170(2): 345-356, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31020317

RESUMO

The goal of this research consortium including Janssen, MSD, Ncardia, FNCR/LBR, and Health and Environmental Sciences Institute (HESI) was to evaluate the utility of an additional in vitro assay technology to detect potential drug-induced long QT and torsade de pointes (TdP) risk by monitoring cytosolic free Ca2+ transients in human stem-cell-derived cardiomyocytes (hSC-CMs). The potential proarrhythmic risks of the 28 comprehensive in vitro proarrhythmia assay (CiPA) drugs linked to low, intermediate, and high clinical TdP risk were evaluated in a blinded manner using Ca2+-sensitive fluorescent dye assay recorded from a kinetic plate reader system (Hamamatsu FDSS/µCell and FDSS7000) in 2D cultures of 2 commercially available hSC-CM lines (Cor.4U and CDI iCell Cardiomyocytes) at 3 different test sites. The Ca2+ transient assay, performed at the 3 sites using the 2 different hSC-CMs lines, correctly detected potential drug-induced QT prolongation among the 28 CiPA drugs and detected cellular arrhythmias-like/early afterdepolarization in 7 of 8 high TdP-risk drugs (87.5%), 6 of 11 intermediate TdP-risk drugs (54.5%), and 0 of 9 low/no TdP-risk drugs (0%). The results were comparable among the 3 sites and from 2 hSC-CM cell lines. The Ca2+ transient assay can serve as a user-friendly and higher throughput alternative to complement the microelectrode array and voltage-sensing optical action potential recording assays used in the HESI-CiPA study for in vitro assessment of drug-induced long QT and TdP risk.


Assuntos
Arritmias Cardíacas/induzido quimicamente , Cálcio/metabolismo , Síndrome do QT Longo/induzido quimicamente , Miócitos Cardíacos/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Miócitos Cardíacos/metabolismo , Risco , Células-Tronco/citologia
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