RESUMO
Chloroquine (CQ) and hydroxychloroquine (HCQ) have shown the ability to inhibit in vitro viral replications of coronaviridae viruses such as SARS-CoV and SARS-CoV-2. However, clinical trial outcomes have been disparate, suggesting that CQ and HCQ antiviral mechanisms are not fully understood. Based on three-dimensional structural similarities between HCQ and the known ACE2 specific inhibitor MLN-4760, we compared their modulation on ACE2 activity. Here we describe, for the first time, in a cell-free in vitro system that HCQ directly and dose-dependently inhibits the activity of recombinant human ACE2, with a potency similar to the MLN-4760. Further analysis suggests that HCQ binds to a noncompetitive site other than the one occupied by MLN-4760. We also determined that the viral spike glycoprotein segment that comprises the RBD segment has no effect on ACE2 activity but unexpectedly was able to partially reverse the inhibition induced by HCQ but not that by MLN-4760. In summary, here we demonstrate the direct inhibitory action of HCQ over the activity of the enzyme ACE2. Then, by determining the activity of ACE2, we reveal that the interaction with the spike protein of SARS-CoV-2 leads to structural changes that at least partially displace the interaction of the said enzyme with HCQ. These results may help to explain why the effectiveness of HCQ in clinical trials has been so variable. Additionally, this knowledge could be used for to develop techniques for the detection of SARS-CoV-2.
Assuntos
Enzima de Conversão de Angiotensina 2 , Antivirais , Tratamento Farmacológico da COVID-19 , Hidroxicloroquina , Enzima de Conversão de Angiotensina 2/antagonistas & inibidores , Enzima de Conversão de Angiotensina 2/química , Enzima de Conversão de Angiotensina 2/metabolismo , Antivirais/química , Antivirais/metabolismo , Antivirais/farmacologia , Humanos , Hidroxicloroquina/química , Hidroxicloroquina/metabolismo , Hidroxicloroquina/farmacologia , Imidazóis/química , Imidazóis/metabolismo , Imidazóis/farmacologia , Leucina/análogos & derivados , Leucina/química , Leucina/metabolismo , Leucina/farmacologia , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , SARS-CoV-2 , Glicoproteína da Espícula de Coronavírus/química , Glicoproteína da Espícula de Coronavírus/metabolismoRESUMO
There has been considerable excitement in the kidney community surrounding the research findings on the genetic contributions to kidney diseases. However, positive outcomes of personalized therapeutic interventions can be circumvented by unpredictable pharmacokinetics of prescribed drugs. Furthermore, unpredictable drug disposition can result in toxicities such as kidney injury. Patient covariates, disease covariates, and pharmacogenetics all contribute to variability in drug disposition. Further treatment personalization and avoidance of drug- and biologic- induced kidney injury will require extensive knowledge and expertise in renal clinical pharmacology. The current review will focus on the pharmacogenetics of drugs and biologics used in the treatment of glomerular kidney diseases and drugs implicated in inducing kidney injury phenotypes.
Assuntos
Injúria Renal Aguda/induzido quimicamente , Azatioprina/metabolismo , Ciclofosfamida/metabolismo , Enzimas/genética , Glomerulonefrite/tratamento farmacológico , Proteínas de Membrana Transportadoras/genética , Farmacogenética , Azatioprina/uso terapêutico , Inibidores de Calcineurina/efeitos adversos , Cisplatino/efeitos adversos , Ciclofosfamida/uso terapêutico , Sistema Enzimático do Citocromo P-450/genética , Humanos , Hidroxicloroquina/metabolismo , Ácido Micofenólico/metabolismo , Polimorfismo Genético , Rituximab/metabolismo , Tacrolimo/metabolismo , Tenofovir/efeitos adversosRESUMO
Hydroxychloroquine has been proposed for HIV treatment; however, little is known about its disposition in the lymphatic system, where replication takes place. Therefore, its distribution in lymphoid tissues (Peyer's patches and popliteal, submandibular, femoral, splenic, and prescapular lymph nodes) was evaluated and compared with that in blood. Results showed a high affinity of hydroxychloroquine for all of these tissues, with higher affinity for the splenic and submandibular lymph nodes, suggesting its potential use as a coadjuvant in HIV therapy.
Assuntos
Fármacos Anti-HIV/metabolismo , Infecções por HIV/tratamento farmacológico , Hidroxicloroquina/metabolismo , Tecido Linfoide/metabolismo , Animais , Fármacos Anti-HIV/sangue , Hidroxicloroquina/sangue , Masculino , CoelhosRESUMO
Hydroxychloroquine (HCQ) is an important chiral drug used, mainly, in the treatment of rheumatoid arthritis, systemic lupus erythematosus and malaria, and whose pharmacokinetic and pharmacodynamic properties look to be stereoselective. Respecting the pharmacokinetic properties, some previous studies indicate that the stereoselectivity could express itself in the processes of metabolism, distribution and excretion and that the stereoselective metabolism looks to be a function of the studied species. So, the in vitro metabolism of HCQ was investigated using hepatic microsomes of rats and mice. The microsomal fraction of livers of Wistar rats and Balb-C mice was separated by ultracentrifugation and 500 μL were incubated for 180 minutes with 10 μL of racemic HCQ 1000 μg mL-1. Two stereospecific analytical methods, high performance liquid chromatography (HPLC) and capillary electrophoresis (CE), were used to separate and quantify the formed metabolites. It was verified that the main formed metabolite is the (-)-(R)-desethyl hydroxychloroquine for both animal species.
A hidroxicloroquina (HCQ) é um importante fármaco quiral usado, principalmente, no tratamento de artrite reumatóide, lupus eritematoso sistêmico e malária e cujas propriedades farmacocinéticas e farmacodinâmicas parecem ser estereosseletivas. Em relação às propriedades farmacocinéticas, alguns estudos prévios indicam que a estereosseletividade pode se expressar nos processos de metabolismo, distribuição e excreção e que o metabolismo estereosseletivo parece ser função da espécie estudada. Sendo assim, o metabolismo in vitro da HCQ foi investigado usando microssomas de fígado de ratos e de camundongos. A fração microssômica de fígados de ratos Wistar e de camundongos Balb-C foi isolada por ultracentrifugação e 500 μL foram incubados por 180 minutos com 10 μL de HCQ racêmica 1000 μg mL-1. Dois métodos analíticos estereoespecíficos, por cromatografia líquida de alta eficiência (HPLC) e eletroforese capilar (CE), foram usados para separar e quantificar os metabólitos formados. Verificou-se que o principal metabólito formado é o (-)-(R)-desetilidroxicloroquina para ambas as espécies de animais.
Assuntos
Animais , Adulto , Camundongos , Ratos , Animais de Laboratório , Modelos Animais de Doenças , Hidroxicloroquina/administração & dosagem , Hidroxicloroquina/metabolismo , Microssomos Hepáticos , Microssomos Hepáticos/metabolismo , Farmacocinética , Efeitos Colaterais e Reações Adversas Relacionados a Medicamentos/metabolismo , Artrite Reumatoide , Cromatografia Líquida de Alta Pressão/métodos , Eletroforese Capilar/métodos , Lúpus Eritematoso Sistêmico/tratamento farmacológico , MaláriaRESUMO
The enantioselective analysis of hydroxychloroquine (HCQ) and its major metabolites was achieved by HPLC and solid-phase microextraction. The chromatographic separation was performed on a Chiralcel OD-H column using hexane/methanol/ethanol (96:2:2, v/v/v) plus 0.2% diethylamine as the mobile phase, at the flow rate of 1.3 mL/min. The main extraction parameters were optimized. The best condition was achieved by the addition of 10% NaCl and 1 mL phosphate buffer 1 mol/L pH 11 to 3 mL human urine. The extraction was conducted for 40 min at 25 degrees C and the desorption time was 3 min using methanol (100%). PDMS-DVB 60 microm fiber was used in this study. The mean recoveries were 9.3, 9.2, and 14.4% for HCQ, desethylhydroxychloroquine (DHCQ), and desethylchloroquine (DCQ), respectively. The method was linear over the range of 50-1000 ng/mL for HCQ enantiomers and over the range of 42-416 ng/mL for DCQ and DHCQ enantiomers. Within-day and between-day precision and accuracy assays for HCQ and its metabolites were lower than 15%. The preliminary 48 h urinary excretion study performed in human urine showed to be stereoselective. The amount of (+)-(S)-enantiomer excreted was higher than its antipode.
Assuntos
Técnicas de Química Analítica/métodos , Cromatografia Líquida de Alta Pressão/métodos , Hidroxicloroquina/metabolismo , Hidroxicloroquina/urina , Extração em Fase Sólida/métodos , Calibragem , Cloroquina/análogos & derivados , Cloroquina/análise , Cromatografia/métodos , Humanos , Concentração de Íons de Hidrogênio , Metanol/química , Modelos Químicos , Reprodutibilidade dos Testes , Estereoisomerismo , Temperatura , Fatores de TempoRESUMO
A rapid, selective, and low-cost chiral capillary electrophoretic method was developed for the simultaneous analysis of hydroxychloroquine (HCQ) and its three chiral metabolites: desethylchloroquine (DCQ), desethylhydroxychloroquine (DHCQ), and bisdesethylchloroquine (BDCQ) in the microsomal fraction of liver homogenates. After liquid-liquid extraction using toluene as extracting solvent, the drug and metabolites were resolved on a fused-silica capillary (50 microm ID, 50 cm total length, and 42 cm effective length), using 100 mmol/L of Tris/phosphate buffer, pH 9.0 containing 1% w/v sulfated-beta-CD and 30 mg/mL hydroxypropyl-beta-CD. Detection was carried out at 220 nm. The extraction procedure was efficient in removing endogenous interferents, and low values (Assuntos
Eletroforese Capilar/métodos
, Hidroxicloroquina/isolamento & purificação
, Hidroxicloroquina/metabolismo
, Microssomos Hepáticos/metabolismo
, Animais
, Eletroforese Capilar/normas
, Humanos
, Hidroxicloroquina/análogos & derivados
, Hidroxicloroquina/química
, Técnicas In Vitro
, Camundongos
, Camundongos Endogâmicos BALB C
, Estereoisomerismo
RESUMO
A one-step chiral method for the quantification of the enantiomers of two hydroxychloroquine (HCQ) metabolites, desethylchloroquine (DCQ) and desethylhydroxychloroquine (DHCQ) by HPLC is described, in addition to its application to the in vitro study of HCQ metabolism in rat liver microsomes. Liquid-liquid extraction was used to extract the enantiomers from microsome samples and the separation was performed on a Chiralpak AD-RH column protected with an RP-8 guard column using hexane:isopropanol (92:8, v/v) plus 0.1% diethylamine as the mobile phase, at a flow rate of 1.0 mL min(-1). The detection was carried out at 343 nm. The method proved to be linear in the range of 50-5000 ng mL(-1) for DCQ enantiomers and 125-2500ngmL(-1) for DHCQ enantiomers, with a quantification limit of 50 and 125 ng mL(-1), respectively. Precision and accuracy, demonstrated by within-day and between-day assays, were lower than 15%. The metabolic study demonstrated that metabolism is stereoselective for HCQ. The major metabolites formed in the incubation of racemic HCQ were (-)-(R)-DCQ and (-)-(R)-DHCQ with R/S ratios of 2.2 and 3.3, respectively.