ABSTRACT
OBJECTIVE@#To explore the regulatory mechanism of human hepatocyte apoptosis induced by lysosomal membrane protein Sidt2 knockout.@*METHODS@#The Sidt2 knockout (Sidt2-/-) cell model was constructed in human hepatocyte HL7702 cells using Crispr-Cas9 technology.The protein levels of Sidt2 and key autophagy proteins LC3-II/I and P62 in the cell model were detected using Western blotting, and the formation of autophagosomes was observed with MDC staining.EdU incorporation assay and flow cytometry were performed to observe the effect of Sidt2 knockout on cell proliferation and apoptosis.The effect of chloroquine at the saturating concentration on autophagic flux, proliferation and apoptosis of Sidt2 knockout cells were observed.@*RESULTS@#Sidt2-/- HL7702 cells were successfully constructed.Sidt2 knockout significantly inhibited the proliferation and increased apoptosis of the cells, causing also increased protein expressions of LC3-II/I and P62(P < 0.05) and increased number of autophagosomes.Autophagy of the cells reached a saturated state following treatment with 50 μmol/L chloroquine, and at this concentration, chloroquine significantly increased the expressions of LC3B and P62 in Sidt2-/- HL7702 cells.@*CONCLUSION@#Sidt2 gene knockout causes dysregulation of the autophagy pathway and induces apoptosis of HL7702 cells, and the latter effect is not mediated by inhibiting the autophagy-lysosomal pathway.
Subject(s)
Humans , Lysosome-Associated Membrane Glycoproteins/metabolism , Autophagy , Apoptosis , Hepatocytes , Lysosomes/metabolism , Chloroquine/pharmacology , Nucleotide Transport Proteins/metabolismABSTRACT
Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.
Resumo A resistência do Plasmodium falciparum à cloroquina (CQ) é uma causa significativa de mortalidade e morbidade em todo o mundo. Há uma escassez de dados documentados sobre a prevalência de haplótipos mutantes CQ-resistentes dos genes Pfcrt e Pfmdr1 da guerra endêmica da malária em áreas tribais administradas pelo governo federal do Paquistão. O objetivo deste estudo foi investigar a prevalência de resistência a CQ de P. falciparum nesta área. Isolados clínicos foram coletados entre maio de 2017 e maio de 2018 nas agências do Waziristão do Norte e do Waziristão do Sul da Área de Ensaio Administrada Federalmente. Posteriormente, esfregaços de sangue corados com Giemsa foram examinados para detectar Plasmodium falciparum. A extração do DNA da malária foi feita a partir de amostras de P. falciparum positivas para microscopia, e as infecções por P. falciparum foram confirmadas por nested PCR (visando genes de ácido ribonucleico ribossômico de subunidade pequena de Plasmodium (ssrRNA)). Todas as amostras de P. falciparum confirmadas por PCR foram sequenciadas por pirosequenciamento para descobrir a mutação no gene Pfcrt no códon K76T e em pfmdr1 nos códons N86Y, Y184F, N1042D e D1246Y. De 121 microscopias de casos positivos de P. falciparum, 109 amostras foram positivas para P. falciparum por nested PCR. A mutação Pfcrt K76T foi encontrada em 96% dos isolados, a mutação Pfmdr1 N86Y foi observada em 20% e 11% abrigou a mutação Y184F. Todas as amostras eram do tipo selvagem para o códon N1042D e D1246Y de Pfmdr1. No FATA, Paquistão, a frequência do alelo resistente 76T permaneceu alta apesar da remoção de CQ. No entanto, as descobertas atuais do estudo sugerem a fixação completa do genótipo resistente a CQ de P. falciparum na área de estudo.
Subject(s)
Plasmodium falciparum/genetics , Antimalarials/pharmacology , Pakistan , Membrane Transport Proteins/genetics , Drug Resistance/genetics , Protozoan Proteins/genetics , Chloroquine/pharmacology , Multidrug Resistance-Associated Proteins/genetics , AllelesABSTRACT
Abstract Neospora caninum is an apicomplexan parasite that causes abortion in cattle, resulting in significant economic losses. There is no commercial treatment for neosporosis, and drug repositioning is a fast strategy to test possible candidates against N. caninum. In this article, we describe the effects of atovaquone, chloroquine, quinine, primaquine and tetracycline on N. caninum proliferation. The IC50 concentrations in N. caninum were compared to the current information based on previous studies for Plasmodium and Toxoplasma gondii, correlating to the described mechanisms of action of each tested drug. The inhibitory patterns indicate similarities and differences among N. caninum, Plasmodium and T. gondii. For example, atovaquone demonstrates high antiparasitic activity in all the analyzed models, while chloroquine does not inhibit N. caninum. On the other hand, tetracycline is effective against Plasmodium and N. caninum, despite its low activity in T. gondii models. The repurposing of antimalarial drugs in N. caninum is a fast and inexpensive way to develop novel formulations using well-established compounds.
Resumo Neospora caninum é um parasita Apicomplexa relacionado a abortos no gado bovino, que resultam em impactos econômicos. Não há tratamento comercial para neosporosis e o reposicionamento de drogas indica uma estratégia rápida para testar candidatos anti-N. caninum. Neste artigo, são descritos os efeitos da atovaquona, cloroquina, quinino, primaquine e tetraciclina na proliferação de N. caninum. As concentrações IC50 em N. caninum foram comparadas com a informação disponível, baseada em estudos publicados previamente para Plasmodium e Toxoplasma gondii, incluindo a correlação com os mecanismos de ação descritos para cada droga testada. Os padrões de inibição indicam pontos de similaridades e diferenças entre N. caninum, Plasmodium e T. gondii. Por exemplo, a atovaquona demonstra uma alta atividade antiparasitária em todos os modelos testados, enquanto a cloroquina não inibe N. caninum. Por outro lado, a tetraciclina é efetiva contra Plasmodium e N. caninum, em contraste com a baixa atividade em modelos de T. gondii. O reposicionamento de drogas antimaláricas em N. caninum é uma forma rápida e de baixo custo para o desenvolvimento de novas formulações que usam compostos bem estabelecidos.
Subject(s)
Neospora/drug effects , Antimalarials/pharmacology , Primaquine/pharmacology , Quinine/pharmacology , Tetracyclines/pharmacology , Chloroquine/pharmacology , Atovaquone/pharmacologyABSTRACT
Introdução: Responsável pela doença COVID-19, o novo corona-vírus, denominado SARS-CoV-2, foi descoberto na China. A sua rápida propagação causou espanto e preocupação e ocasionou uma das maiores pandemias já registradas na história. Para o tratamento da infecção, alguns profissionais sugerem o uso da Cloroquina e da Hidroxicloroquina, medicamentos utilizados para o tratamento de diversas doenças há mais de 70 anos. Entretanto, a efetividade des-ses medicamentos no controle da COVID-19 ainda não foi confir-mada. Objetivos: Identificar na literatura científica os riscos e bene-fícios da Cloroquina e da Hidroxicloroquina utilizados como terapia medicamentosa para pacientes com COVID-19. Método: Revisão integrativa da literatura, utilizando três bancos de dados on-line (LILACS, SCIELO e PUBMED) a fim de descrever os efeitos da Cloroquina e da Hidroxicloroquina quando usadas no tratamento da doença, permitindo a avaliação crítica de determinado conhecimen-to sobre o assunto e reunindo múltiplos estudos e suas principais considerações. Resultados: Foram analisados dez estudos científi-cos acerca da temática, focando na relação riscos e benefícios que os medicamentos Cloroquina e Hidroxicloroquina, associados ou não a outros medicamentos, possam apresentar no combate à infec-ção. Os dados in vitro apresentaram resultados promissores quan-to ao uso dos medicamentos, porém não demonstraram eficácia no combate à COVID-19 quando utilizados em pacientes. Considera-ções finais: Conclui-se que a utilização de Cloroquina e Hidroxi-cloroquina no tratamento da COVID-19 mostra-se ineficaz e com riscos para quem faz uso. Nota-se uma concordância com relação à necessidade de cautela no momento, com destaque para os riscos cardíacos para o usuário.
Introduction: Responsible for the disease COVID-19, the new coronavirus, called SARS-CoV-2, was discovered in China. Its rapid spread caused astonishment and concern in the population, and it ended up causing one of the largest pandemics ever recorded in history. Some professionals suggest using Chloroquine and Hydroxychloroquine, medicines that have been used to fight various diseases for more than 70 years, to treat this infection. Their effectiveness in controlling COVID-19 has not been confirmed, though. Objectives: This study aims to collect information in the scientific literature on the risks and benefits of the drugs Chloroquine and Hydroxychloroquine used as drug therapy for patients affected by the disease. Method: An integrative literature review was carried out, on three online databases (LILACS, SCIELO, and PUBMED) to describe the effects of Chloroquine and Hydroxychloroquine when used in the treatment of the disease, allowing the critical evaluation of certain knowledge on the subject, and gathering multiple studies and their main outcomes. Results: Ten scientific studies on the theme were analyzed, focusing on the relationship between risks and benefits that the medicines Chloroquine and Hydroxychloroquine, associated or not with other medicines may present in the fight against the infection. In vitro data showed promising results regarding the use of the drugs, but the medicines did not demonstrate efficiency in combating COVID-19 when used in patients. Final considerations: The use of Chloroquine and Hydroxychloroquine in the treatment of COVID-19 proves to be ineffective and it brings risks for those who use them. There is an agreement regarding the need for caution now, emphasizing heart risk to the user.
Subject(s)
COVID-19/drug therapy , Chloroquine/pharmacology , Hydroxychloroquine/pharmacologyABSTRACT
ABSTRACT Chloroquine (CQ) and its analog hydroxychloroquine (HCQ) were recently included in several clinical trials as potential prophylactic and therapeutic options for SARS-COV-2 infection/covid-19. However, drug effectiveness in preventing, treating, or slowing the progression of the disease is still unknown. Despite some initial promising in vitro results, rigorous pre-clinical animal studies and randomized clinical trials have not been performed yet. On the other hand, while the potential effectiveness of CQ/HCQ is, at best, hypothetical, their side effects are factual and most worrisome, particularly when considering vulnerable groups of patients being treated with these drugs. in this comment, we briefly explain the possible mechanisms of action of CQ/HCQ for treating other diseases, possible actions against covid-19, and their potent side effects, in order to reinforce the necessity of evaluating the benefit-risk balance when widely prescribing these drugs for SARS-COV-2 infection/covid-19. We conclude by strongly recommending against their indiscriminate use.
Subject(s)
Humans , Pneumonia, Viral/drug therapy , Chloroquine/pharmacology , Coronavirus Infections/drug therapy , Betacoronavirus/drug effects , Hydroxychloroquine/pharmacology , Antimalarials/pharmacology , Chloroquine/adverse effects , Chloroquine/pharmacokinetics , Risk Assessment , Pandemics , Contraindications, Drug , SARS-CoV-2 , COVID-19 , Hydroxychloroquine/adverse effects , Hydroxychloroquine/pharmacokinetics , Antimalarials/adverse effects , Antimalarials/pharmacokineticsABSTRACT
Abstract Hydroxychloroquine and chloroquine, also known as antimalarial drugs, are widely used in the treatment of rheumatic diseases and have recently become the focus of attention because of the ongoing COVID-19 pandemic. Rheumatologists have been using antimalarials to manage patients with chronic immune-mediated inflammatory rheumatic diseases for decades. It is an appropriate time to review their immunomodulatory and anti-inflammatory mechanisms impact on disease activity and survival of systemic lupus erythematosus patient, including antiplatelet effect, metabolic and lipid benefits. We also discuss possible adverse effects, adding a practical and comprehensive approach to monitoring rheumatic patients during treatment with these drugs.(AU)
Subject(s)
Humans , Chloroquine/therapeutic use , Rheumatic Diseases/drug therapy , Hydroxychloroquine/therapeutic use , Chloroquine/pharmacology , Hydroxychloroquine/pharmacologyABSTRACT
Resumen Nuevos agentes antimaláricos a partir de plantas son estudiados como alternativas en el tratamiento de la malaria. Los principales antimaláricos como la cloroquina tienen varios mecanismos de acción contra parásitos, uno de ellos es la inhibición de polimerización del grupo hemo, modelo que ha permitido el diseño de nuevos candidatos antimaláricos. En este sentido, el objetivo de este trabajo fue evaluar extractos de plantas de género Piper y Calophyllum sobre la capacidad de inhibición de la β-hematina. Se informa las concentraciones inhibitorias de la formación de β-hematina por parte de 40 extractos de diferente polaridad obtenidos a partir de las especies P. piedecuestanum, C. brasiliense, C. longinforium, y Calophyllum. sp. 19 extractos mostraron un mayor potencial para inhibir la formación de β−hematina con CI50 < 3mg / ml. Estas actividades respaldan principalmente, futuros estudios con el género Calophyllum, en el desarrollo y descubrimiento de nuevas sustancias antiplasmodiales con modos de acción conocido.(AU)
Abstract New antimalarial agents from plants are studied as alternatives in the treatment of malaria. The main antimalarials such as chloroquine have several mechanisms of action against parasites, one of which is the inhibition of polymerization of the heme group, a model that has allowed the design of new antimalarial candidates. In this sense the objective of this work was to evaluate extracts of genus Piper and Calophyllum plants on the inhibition capacity of β-hematin. Inhibitory concentrations of β-hematin are reported from 40 extracts of different polarity obtained from the species P. piedecuestanum, C. brasiliense, C. longinforium, and Calophyllum. sp. 19 extracts showed a greater potential to inhibit β-hematin with IC50 < 3 mg/ml. These activities mainly support future studies with the genus Calophyllum in the development and discovery of new antiplasmodial substances with known modes of action.(AU)
Subject(s)
Chloroquine/pharmacology , Polymerization/drug effects , Heme-Binding Proteins , Malaria/drug therapyABSTRACT
OBJECTIVE@#To determine whether chloroquine (CQ), an often used inhibitor of late autophagy and autophagosome/lyosome fusion, can inhibit proliferation of renal carcinoma cells and investigate its effect on sunitinib (ST)-induced apoptosis.@*METHODS@#Renal carcinoma cell line 786 O and ACHN had been used as cellular model and 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt (MTS) assay was carried out to detect the cell viability in response to CQ or ST treatment. Both transmission electron microscope and immunoblotting had been employed to observe apoptotic and autophagic process. To examine the involvement of autophagy in ST-dependent apoptosis, autophagy had been inhibited either chemically or genetically via utilizing autophagy inhibitor or specific small interference RNA (siRNA) targeted to either Ulk1 (unc-51-like kinase 1) or LC3 (microtubule associated protein 1 light chain 3 fusion protein), two essential autophagic proteins.@*RESULTS@#Both ST and CQ induced cell viability loss, indicating that either of them could inhibit renal cancer cell proliferation. Clone formation experiments confirmed the aforementioned results. Furthermore, the combined ST with CQ synergistically promoted the loss of cell viability. By transmission electron microscopy and immunoblotting, we found that the ST induced both autophagy and caspase-dependent apoptosis. While 3-MA, an early autophagy inhibitor, reduced the ST-induced cleavage of poly (ADP-ribose) polymerase-1 (PARP-1), a substrate of caspase 3/7 and often used marker of caspase-dependent apoptosis, CQ promoted the ST-dependent PARP-1 cleavage, indicating that the early and late autophagy functioned differentially on the ST-activated apoptotic process. Moreover, the knock down of either Ulk1 or LC3 decreased the ST-caused apoptosis.Interestingly, we observed that rapamycin, a specific inhibitor of mTOR (mammalian target of rapamycin) and an inducer of autophagy, also showed to inhibit cell viability and increased the cleavage of PARP-1 in the ST-treated cells, suggesting that autophagy was likely to play a dual role in the regulation of the ST-induced apoptosis.@*CONCLUSION@#ST activates both apoptotic and autophagic process in renal carcinoma cells. Although autophagy precedes the ST-induced apoptosis, however, early and late autophagy functions differentially on the apoptotic process induced by this compound. Additionally, ST can coordinate with the inducer of autophagy to inhibit the cell proliferation. Further research in this direction will let us illuminate to utilize CQ as a potential drug in the treatment of renal carcinoma.
Subject(s)
Animals , Antineoplastic Agents/pharmacology , Antirheumatic Agents/pharmacology , Apoptosis/drug effects , Autophagy/drug effects , Caspases , Cell Line, Tumor , Chloroquine/pharmacology , Kidney Neoplasms/drug therapy , Sunitinib/pharmacologyABSTRACT
Ever increasing multi-drug resistance by Plasmodium falciparum is creating new challenges in malaria chemotherapy. In the absence of licensed vaccines, treatment and prevention of malaria is heavily dependent on drugs. Potency, range of activity, safety, low cost and ease of administration are crucial issues in the design and formulation of antimalarials. We have tested three synthetic ozonides NAC89, LC50 and LCD67 in vitro and in vivo against multidrug resistant Plasmodium. In vitro, LC50 was at least 10 times more efficient inhibiting P. falciparum multidrug resistant Dd2 strain than chloroquine and mefloquine and as efficient as artemisinin (ART), artesunate and dihydroartemisinin. All three ozonides showed high efficacy in clearing parasitaemia in mice, caused by multi-drug resistant Plasmodium chabaudi strains, by subcutaneous administration, demonstrating high efficacy in vivo against ART and artesunate resistant parasites.
Subject(s)
Humans , Animals , Female , Antimalarials/pharmacology , Malaria, Falciparum/parasitology , Plasmodium falciparum/drug effects , Artemisinins/pharmacology , Chloroquine/pharmacology , Disease Models, Animal , Mefloquine/pharmacology , Mice , Parasitemia/drug therapyABSTRACT
Viral hepatitis is an important public health problem in Brazil and around the world. To evaluate vaccination coverage against hepatitis B in adolescents and to identify the associated factors and reasons for non-adherence. A cross-sectional population-based study with sampling by clusters and in two stages, carried out from records of 702 adolescents aged 11 to 19 years old, non-institutionalized, living in an urban area of Campinas, São Paulo, Brazil, in 2008/2009. The data were obtained from the Health Survey in the city of Campinas (ISACamp). The prevalence of vaccination (3 doses) was 72.2%. An independent and negative association with the vaccine was observed for the adolescents who were not born in the municipality. The orientation of a health care provider was positively and significantly associated with vaccination. The main reasons for non-adherence were the lack of orientation and not considering the vaccine necessary. Socioeconomic factors, health behaviors and conditions did not restrict the access to vaccination, but the coverage was below the target established by the Ministry of Health in Brazil. Health education programs, addressing the importance of vaccination to prevent the disease; strategies to actively reach out adolescents that did not complete the schedule; as well as orientation from the health care professional about the benefits of the vaccine to the adolescents, parents and guardians can extend the vaccination coverage.
As hepatites virais constituem importante problema de saúde pública no Brasil e em todo o mundo. Avaliar a cobertura vacinal contra hepatite B em adolescentes e identificar os fatores associados e motivos da não adesão. Estudo transversal de base populacional com amostra por conglomerados e em 2 estágios realizado a partir de 702 registros de adolescentes com idade entre 11 e 19 anos, não institucionalizados, residentes em área urbana no município de Campinas, São Paulo, em 2008/2009. Os dados foram obtidos do Inquérito de Saúde no município de Campinas (ISACamp). A prevalência de vacinação (3 doses) foi de 72,2%. Associação independente e negativa com a vacina foi observada para os adolescentes não naturais do município. A orientação de profissional de saúde esteve positiva e fortemente associada à vacinação. Os principais motivos para a não adesão foram a falta de orientação e não considerar a vacina necessária. Condições socioeconômicas, comportamentos e condições de saúde não restringiram o acesso à vacinação, mas a cobertura esteve abaixo da meta estabelecida pelo Ministério da Saúde. Programas de educação em saúde, abordando a importância da vacinação na prevenção da doença, estratégias para busca ativa aos adolescentes que não completaram o esquema, bem como a orientação do profissional de saúde sobre os benefícios da vacina aos adolescentes, pais e responsáveis podem ampliar as coberturas vacinais.
Subject(s)
Humans , Autophagy/drug effects , Chloroquine/pharmacology , Oncogenes , ras Proteins/metabolism , Cell Line, Tumor , Cell Proliferation/drug effects , Lung Neoplasms/pathology , Phagosomes/drug effects , Phagosomes/metabolism , /metabolismABSTRACT
The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 mul) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.
Subject(s)
Humans , Amino Acid Substitution , Antimalarials/pharmacology , Chloroquine/pharmacology , Drug Resistance , Inhibitory Concentration 50 , Malaria, Vivax/parasitology , Membrane Transport Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Mutation, Missense , Myanmar , Parasitic Sensitivity Tests , Plasmodium vivax/drug effects , Protozoan Proteins/genetics , ThailandABSTRACT
The aim of the study was to explore the possible molecular markers of chloroquine resistance in Plasmodium vivax isolates in Thailand. A total of 30 P. vivax isolates were collected from a malaria endemic area along the Thai-Myanmar border in Mae Sot district of Thailand. Dried blood spot samples were collected for analysis of Pvmdr1 and Pvcrt-o polymorphisms. Blood samples (100 mul) were collected by finger-prick for in vitro chloroquine susceptibility testing by schizont maturation inhibition assay. Based on the cut-off IC50 of 100 nM, 19 (63.3%) isolates were classified as chloroquine resistant P. vivax isolates. Seven non-synonymous mutations and 2 synonymous were identified in Pvmdr1 gene. Y976F and F1076L mutations were detected in 7 (23.3%) and 16 isolates (53.3%), respectively. Analysis of Pvcrt-o gene revealed that all isolates were wild-type. Our results suggest that chloroquine resistance gene is now spreading in this area. Monitoring of chloroquine resistant molecular markers provide a useful tool for future control of P. vivax malaria.
Subject(s)
Humans , Amino Acid Substitution , Antimalarials/pharmacology , Chloroquine/pharmacology , Drug Resistance , Inhibitory Concentration 50 , Malaria, Vivax/parasitology , Membrane Transport Proteins/genetics , Multidrug Resistance-Associated Proteins/genetics , Mutation, Missense , Myanmar , Parasitic Sensitivity Tests , Plasmodium vivax/drug effects , Protozoan Proteins/genetics , ThailandABSTRACT
The molecular basis of Plasmodium vivax chloroquine (CQ) resistance is still unknown. Elucidating the molecular background of parasites that are sensitive or resistant to CQ will help to identify and monitor the spread of resistance. By genotyping a panel of molecular markers, we demonstrate a similar genetic variability between in vitro CQ-resistant and sensitive phenotypes of P. vivax parasites. However, our studies identified two loci (MS8 and MSP1-B10) that could be used to discriminate between both CQ-susceptible phenotypes among P. vivax isolates in vitro. These preliminary data suggest that microsatellites may be used to identify and to monitor the spread of P. vivax-resistance around the world.
Subject(s)
Humans , Chloroquine/pharmacology , DNA, Protozoan/isolation & purification , Drug Resistance/genetics , Genetic Variation , Plasmodium vivax/drug effects , Plasmodium vivax/genetics , Brazil/epidemiology , Endemic Diseases/statistics & numerical data , Genetic Markers , Malaria, Vivax/blood , Malaria, Vivax/epidemiology , Parasitic Sensitivity Tests , Phenotype , Polymerase Chain Reaction , Random AllocationABSTRACT
Se evaluó la frecuencia de mutaciones en los genes pfCRT y DHFR/DHPS del Plasmodium falciparum asociados a la resistencia a cloroquina y sulfadoxina-pirimetamina en 83 cepas provenientes de los distritos Esmeralda y Machala ubicados en las fronteras entre Ecuador-Perú y Ecuador-Colombia durante el año 2002. Se empleó la reacción en cadena de polimerasa (PCR) convencional y sus variantes. El gen pfCRT presentó más de 90% de muestras mutantes en Esmeralda y Machala. Para el gen DHFR, el 90% de las cepas fueron muestras mutantes en Esmeralda, tres fueron mutaciones dobles y una triple; en Machala se encontró 25% de formas mutantes simples y 75% de formas mixtas (formas silvestres/mutantes). En conclusión, la resistencia a cloroquina se ha fijado en las cepas portadoras de la mutación K76T pfCRT, mientras que la impronta genética a la resistencia a pirimetamina está en evolución, principalmente en el distrito de Esmeralda.
The frequency of mutations in pfCRT and DHFR/DHPS genes of Plasmodium falciparum associated with resistance to chloroquine and sulfadoxine-pyrimethamine was evaluated in 83 strains from the districts of Esmeralda and Machala, located on the borders of Ecuador-Peru and Ecuador-Colombia in 2002. Polymerase chain reaction (PCR), conventional and its variants, was used. Mutations in the pfCRT gene were found in more than 90% of the samples from Esmeralda and Machala. For the DHFR gene, 90% of the strains were mutant samples from Esmeralda, 3 were double mutations and 1 was a triple mutation. In Machala, 25% were simple mutant forms and 75% mixed mutant forms (wild forms/mutant). In conclusion, resistance to chloroquine has been fixed in strains carrying K76T pfCRT mutation, whereas genetic imprinting for resistance to pyrimethamine is evolving, particularly in the district of Esmeralda.
Subject(s)
Humans , Alleles , Antimalarials/pharmacology , Chloroquine/pharmacology , Mutation , Plasmodium falciparum/drug effects , Plasmodium falciparum/genetics , Pyrimethamine/pharmacology , Sulfadoxine/pharmacology , Colombia , Drug Combinations , Drug Resistance , Ecuador , PeruABSTRACT
The development and rapid spread of chloroquine resistance (CQR) in Plasmodium falciparum have triggered the identification of several genetic target(s) in the P. falciparum genome. In particular, mutations in the Pfcrt gene, specifically, K76T and mutations in three other amino acids in the region adjoining K76 (residues 72, 74, 75 and 76), are considered to be highly related to CQR. These various mutations form several different haplotypes and Pfcrt gene polymorphisms and the global distribution of the different CQR- Pfcrt haplotypes in endemic and non-endemic regions of P. falciparum malaria have been the subject of extensive study. Despite the fact that the Pfcrt gene is considered to be the primary CQR gene in P. falciparum , several studies have suggested that this may not be the case. Furthermore, there is a poor correlation between the evolutionary implications of the Pfcrt haplotypes and the inferred migration of CQR P. falciparum based on CQR epidemiological surveillance data. The present paper aims to clarify the existing knowledge on the genetic basis of the different CQR- Pfcrt haplotypes that are prevalent in worldwide populations based on the published literature and to analyse the data to generate hypotheses on the genetics and evolution of CQR malaria.
Subject(s)
Humans , Antimalarials/pharmacology , Chloroquine/pharmacology , Drug Resistance/genetics , Haplotypes/genetics , Malaria, Falciparum/parasitology , Membrane Transport Proteins/genetics , Plasmodium falciparum/genetics , Protozoan Proteins/genetics , DNA, Protozoan/genetics , Malaria, Falciparum/drug therapy , Polymorphism, Genetic , Plasmodium falciparum/drug effectsABSTRACT
Biofilms formed by Candida albicans, a human pathogen, are known to be resistant to different antifungal agents. Novel strategies to combat the biofilm associated Candida infections like multiple drug therapy are being explored. In this study, potential of chloroquine to be a partner drug in combination with four antifungal agents, namely fluconazole, voriconazole, amphotericin B, and caspofungin, was explored against biofilms of C. albicans. Activity of various concentrations of chloroquine in combination with a particular antifungal drug was analyzed in a checkerboard format. Growth of biofilm in presence of drugs was analyzed by XTT-assay, in terms of relative metabolic activity compared to that of drug free control. Results obtained by XTT-metabolic assay were confirmed by scanning electron microscopy. The interactions between chloroquine and four antifungal drugs were determined by calculating fractional inhibitory concentration indices. Azole resistance in biofilms was reverted significantly (p < 0.05) in presence of 250 µg/mL of chloroquine, which resulted in inhibition of biofilms at very low concentrations of antifungal drugs. No significant alteration in the sensitivity of biofilms to caspofungin and amphotericin B was evident in combination with chloroquine. This study for the first time indicates that chloroquine potentiates anti-biofilm activity of fluconazole and voriconazole.
Subject(s)
Humans , Antifungal Agents/pharmacology , Biofilms/drug effects , Candida albicans/drug effects , Chloroquine/pharmacology , Amphotericin B/pharmacology , Azoles/pharmacology , Biofilms/growth & development , Candida albicans/physiology , Candida albicans/ultrastructure , Drug Synergism , Echinocandins/pharmacology , Microbial Sensitivity Tests , Microscopy, Electron, ScanningABSTRACT
Introducción. La planta Solanum nudum es ampliamente usada en la medicina tradicional del Pacífico colombiano para tratar las fiebres y la malaria, o paludismo, y se ha convertido en una fuente de nuevas moléculas promisorias. Objetivo. Evaluar el efecto citotóxico y daño genético de extractos estandarizados de S. nudum en diferentes modelos celulares. Materiales y métodos. A 66 extractos estandarizados de S. nudum se les evaluó la actividad anti- Plasmodiumin vitro en dos cepas de Plasmodium falciparum, una sensible (NF54) y otra resistente (FCB2) a la cloroquina, y la citotoxicidad en células U937 y HepG2. Se seleccionaron los extractos que presentaron actividad anti- Plasmodium y baja toxicidad, y se les estimó su efecto hemolítico en eritrocitos sanos O + , el efecto mutagénico en las cepas TA98 y TA100 de Salmonella Typhimurium y el efecto genotóxico en células U937. Resultados. Se seleccionaron cinco extractos como promisorios (28MA1, 29MA1, 51MA1, 55MA1 y 61MA1), los cuales fueron activos en las cepas de P. falciparum con concentración inhibitoria 50 (CI 50 ) entre 9,8 y 54,8 µg/ml. El extracto 29MA1 fue el más selectivo para Plasmodium, con índice de selectividad de 4,4 y 14,5 para las células U937 y HepG2, respectivamente. En ningún extracto se observó efecto hemolítico a 250 µg/ml, no causaron mutaciones en las cepas TA98 y TA100 de S.Typhimurium, ni generaron efectos genotóxicos en células U937. Conclusiones. La utilización de extractos estandarizados de S. nudum contribuye con los trabajos encaminados al desarrollo de una nueva formulación farmacéutica para tratar la malaria a partir de productos naturales.
Introduction. The plant Solanum nudum (Solanaceae) is extensively used for the treatment of malaria-related symptoms in traditional medicine practices in the Colombian Pacific. Recently, it has become a significant source of promising new molecules for developing a pharmaceutical malaria treatment. Objective. This research aimed to evaluate the cytotoxic effect and the genetic damage of standardized extracts of S. nudumon different cells. Materials and methods. Sixty six standardized S. nudum extracts were used, evaluating cytotoxicity in U937 and HepG2 cells and the antiplasmodial activity using both a chloroquine-sensitive (NF54) and a chloroquine-resistant (FCB2) strain. The hemolytic effect on healthy O + erythrocytes, the mutagenic effect on S.Typhimurium TA98 and TA100 strains and the genotoxic effect on U937 cells were evaluated. The extracts that displayed both antiplasmodial activity and low toxicity were selected. Results. Five extracts were selected: 28MA1, 29MA1, 51MA1, 55MA1 and 61MA1. These extracts were active against P. falciparum with IC 50 between 9.8 and 54.8 µg/ml and selectivity indexes were calculated between 0.9 and 4.4, the latter for 29MA1. Also, no hemolytic effects in healthy O + erythrocytes were shown at a concentration of 250 µg/ml, nor did they cause mutations in the TA98 and TA100 strains or generate genotoxic effects in U937cells. Conclusion. The use of standardized extracts of S. nudum could contribute to the body of work aimed at developing a new pharmaceutical treatment for malaria using natural products.
Subject(s)
Humans , Antimalarials/toxicity , Plant Extracts/toxicity , Plasmodium falciparum/drug effects , Solanum/chemistry , Antimalarials/isolation & purification , Antimalarials/pharmacology , Biotransformation , Chloroquine/pharmacology , DNA Damage , DNA, Bacterial/drug effects , Drug Evaluation, Preclinical , Drug Resistance , Erythrocytes/drug effects , Hemolysis/drug effects , /drug effects , Medicine, Traditional , Mutagenicity Tests , Phytotherapy , Plant Extracts/isolation & purification , Plant Extracts/pharmacology , Solvents , Salmonella typhimurium/drug effects , /drug effectsABSTRACT
En el presente trabajo se describe la síntesis y la evaluación de la posible actividad Antimalárica y Antineoplásica de una serie de derivados 7-cloroquinolina-4-sustituidos. La estrategia empleada para la síntesis comienza con las obtención de los intermediarios clave 1-(3 ó 4-(7-cloroquinolin-4-ilamino)fenil)etanona (2 y 3) mediante una sustitución nucleofílica aromática entre la 4,7-dicloroquinolina y la 3 y/o 4-amino acetofenona. Los derivados (E)-1-(3 ó 4-(7-cloroquinolin-4-ilamino)fenil)-3-(fenilsustituido)prop-2-eno-1-ona (4 y 5), se sintetizaron a través de una condensación aldólica de Claisen-Schmidt entre los intermediarios clave y diferentes benzaldehídos sustituidos. Los derivados 7-cloro-N-(3 ó 4-(4,5-dihidro-5-(fenilsustituido)-1H-pirazol-3-il)fenil)quinolin-4-amina (6 y 7) y los 1-(3 ó 4-(7-cloroquinolin-4-ilamino)fenil)-3-(fenilsustituido)propano-1-ona (8 y 9) se diseñaron por modificación molecular de la cetona a,b-insaturada de los compuestos finales 4 y 5, (metodología clásica de la Química Medicinal) para obtener dichos derivados rígidos 6 y 7, mediante la formación de un anillo D2-pirazolina y flexibles 8 y 9, a través de su reducción. La síntesis de los derivados 6 y 7 se realizó mediante una reacción de ciclo-condensación con hidrazina monohidratada y los derivados 8 y 9, se obtuvieron a través de una hidrogenación catalítica. En la evaluación de la actividad Antimalárica in vitro se evidenció que los derivados 4, 5, 6 y 7, mostraron actividades inhibitorias la formación de la b hematina importantes (superior al 70 %), siendo los más activos: 4l, 5g, 5c, 5g y 6e, 6f con valores comparable al de la CQ. En la evaluación Antimalárica in vivo se encontró que el derivado 4e fue el más activo con 26,4 días de sobrevivencia post-infección (230 % de incremento) y una parasitemia de 2,4 % (96 % de reducción). Con respecto a los resultados obtenidos en el efecto de estos derivados sobre la viabilidad y proliferación de las líneas celulares neoplásicas Jurkat E6.1, HL60, MCF-7 y A549, los compuestos 4a, 4g, 4l, 4m y 6e mostraron la mayor actividad inhibitoria del crecimiento de las células leucémicas HL60 después de 24h de tratamiento con valores de CI50 de 1,19 µM, 1,08 µM, 0,59 µM, 0,43 µM y 0,94 µM (hasta 3 y 100 veces más activos que la doxorubicina y que la CQ, respectivamente). En lo referente a la evaluación de la actividad proapoptótica en las líneas celulares neoplásicas Jurkat E6.1, HL60, MCF-7 y A549, se evidenció que los derivados 4, 5 y 6, al igual que los controles, generaron un aumento en el porcentaje de células positivas para la Anexina V/FITC dependiente de la dosis (apoptosis temprana y tardía). Ninguno de estos derivados indujo el proceso de necrosis en estas células.
The present investigation describes the synthesis and evaluation of the Antimalarial and Antineoplastic activity possible a series of derivatives of 7-substituted-4-chloro-quinoline. The strategy employed for the synthesis begins with preparation of the key intermediate 1-(3 or 4-(7-chloroquinolin-4-ylamino) phenyl)ethanone (2and 3) by a nucleophilic aromatic substitution between 4,7-dichloroquinoline and the 3 and/or4-amino acetophenone. The derivatives (E)-1-(3 or 4-(7-chloroquinolin-4-ylamino) phenyl)-3-(substitutedphenyl)prop-2-en-1-one (4and 5), were synthesized a through aldol condensation Claisen-Schmidt among different key intermediates and substituted benzaldehydes. The resulting 7-chloro-N-(3 or 4-(4,5-dihydro-5-(substitutedphenyl)-1H-pyrazol-3-yl)phenyl)quinolin-4-amine (6 and 7) and 7-chloro-4-[(3 or 4-(substituted phenyl)ethylcarbonyl)phenyl]aminoquinoline(8 and 9) were designed for the molecular modification ï¡, ï¢-unsaturated ketone of the final compounds 4and 5 (classic methodology Medicinal Chemistry) for said rigid derivatives 6and 7, through the formation of a ï2-pyrazoline ring flexible and 8and 9, through its reduction. The synthesis of derivatives 6and 7were performed using a cycle-condensation reaction with hydrazine monohydrate and 8and 9derivatives were obtained via a catalytic hydrogenation. In the assessment of antimalarial activity in vitro was demonstrated that derivatives 4, 5, 6and 7showed inhibitory activities ï¢forming the major hematin (above 70%), being more active: 4l, 5g, 5c, 5g, 6eand 6f,with values comparable to that of CQ. In vivoantimalarial evaluation found that the derivative 4ewas most active with survival 26.4 dayspost-infection (230% increase) and a parasitemia of 2.4% (96% reduction). With regard to the results on the effect of these derivatives on the viability and proliferation of neoplastic cell lines Jurkat E6.1, HL60, MCF-7 and A549, compounds 4a, 4g, 4l,4mand 6eshow greater activity growth inhibitory HL60 leukemia cells after 24 h of treatment with IC50values of 1.19µM, 1.08µM, 0.59µM, 0.43µMand 0.94 ïM (to 3 and 100 times more active than doxorubicin and the CQ, respectively). Regarding the evaluation of pro-apoptotic activity on neoplastic cell lines Jurkat E6.1, HL60, MCF-7 and A549, was demonstrated that derivatives 4, 5and 6, like the controls, an increase in generated percentage of cells positive for Annexin V/FITC dose dependent (early andlate apoptosis). None of these derivatives induced necrosis process in these cells.
Subject(s)
Humans , Animals , Male , Mice , Quinolines/chemistry , Chloroquine/chemistry , Antimalarials/chemistry , Antineoplastic Agents/chemistry , Plasmodium berghei/drug effects , Quinolines/chemical synthesis , Quinolines/pharmacology , In Vitro Techniques/methods , Cell Line/drug effects , Cell Survival/drug effects , Chloroquine/chemical synthesis , Chloroquine/pharmacology , Apoptosis/drug effects , Cell Proliferation/drug effects , Antimalarials/chemical synthesis , Antimalarials/pharmacology , Antineoplastic Agents/chemical synthesis , Antineoplastic Agents/pharmacologyABSTRACT
Artemisinin is the active antimalarial compound obtained from the leaves of Artemisia annua L. Artemisinin, and its semi-synthetic derivatives, are the main drugs used to treat multi-drug-resistant Plasmodium falciparum (one of the human malaria parasite species). The in vitro susceptibility of P. falciparum K1 and 3d7 strains and field isolates from the state of Amazonas, Brazil, to A. annua infusions (5 g dry leaves in 1 L of boiling water) and the drug standards chloroquine, quinine and artemisinin were evaluated. The A. annua used was cultivated in three Amazon ecosystems (várzea, terra preta de índio and terra firme) and in the city of Paulínia, state of São Paulo, Brazil. Artemisinin levels in the A. annua leaves used were 0.90-1.13% (m/m). The concentration of artemisinin in the infusions was 40-46 mg/L. Field P. falciparum isolates were resistant to chloroquine and sensitive to quinine and artemisinin. The average 50% inhibition concentration values for A. annua infusions against field isolates were 0.11-0.14 μL/mL (these infusions exhibited artemisinin concentrations of 4.7-5.6 ng/mL) and were active in vitro against P. falciparum due to their artemisinin concentration. No synergistic effect was observed for artemisinin in the infusions.