Characterization of antimalarial activity of artemisinin-based hybrid drugs.

In response to the spread of artemisinin (ART) resistance, ART-based hybrid drugs were developed, and their activity profile was characterized against drug-sensitive and drug-resistant Plasmodium falciparum parasites. Two hybrids were found to display parasite growth reduction, stage-specificity, speed of activity, additivity of activity in drug combinations, and stability in hepatic microsomes of similar levels to those displayed by dihydroartemisinin (DHA). Conversely, the rate of chemical homolysis of the peroxide bonds is slower in hybrids than in DHA. From a mechanistic perspective, heme plays a central role in the chemical homolysis of peroxide, inhibiting heme detoxification and disrupting parasite heme redox homeostasis. The hybrid exhibiting slow homolysis of peroxide bonds was more potent in reducing the viability of ART-resistant parasites in a ring-stage survival assay than the hybrid exhibiting fast homolysis. However, both hybrids showed limited activity against ART-induced quiescent parasites in the quiescent-stage survival assay. Our findings are consistent with previous results showing that slow homolysis of peroxide-containing drugs may retain activity against proliferating ART-resistant parasites. However, our data suggest that this property does not overcome the limited activity of peroxides in killing non-proliferating parasites in a quiescent state.

A polyvalent RNA vaccine reduces the immune imprinting phenotype in mice and induces neutralizing antibodies against omicron SARS-CoV-2.

Immune imprinting is now evident in COVID-19 vaccinated people. This phenomenon may impair the development of effective neutralizing antibodies against variants of concern (VoCs), mainly Omicron and its subvariants. Consequently, the boost doses with bivalent vaccines have not shown a significant gain of function regarding the neutralization of Omicron. The approach to design COVID-19 vaccines must be revised to improve the effectiveness against VoCs. Here, we took advantage of the self-amplifying characteristic of RepRNA and developed a polyvalent formulation composed of mRNA from five VoCs. LION/RepRNA Polyvalent induced neutralizing antibodies in mice previously immunized with LION/RepRNA D614G and reduced the imprinted phenotype associated with low neutralization capacity of Omicron B.1.1.529 pseudoviruses. The polyvalent vaccine can be a strategy to handle the low neutralization of Omicron VoC, despite booster doses with either monovalent or bivalent vaccines.

High-Content Imaging-Based Assay for SARS-CoV-2-Neutralizing Antibodies.

The COVID-19 pandemic and the consequent emergence of new SARS-CoV-2 variants of concern necessitates the determination of populational serum potency against the virus. Here, we standardized and validated an imaging-based method to quantify neutralizing antibodies against lentiviral particles expressing the spike glycoprotein (pseudovirus). This method was found to efficiently quantify viral titers based on ZsGreen-positive cells and detect changes in human serum neutralization capacity induced by vaccination with up to two doses of CoronaVac, Comirnaty, or Covishield vaccines. The imaging-based protocol was also used to quantify serum potency against pseudoviruses expressing spikes from Delta, Omicron BA.1.1.529, and BA.4/5. Our results revealed increases in serum potency after one and two doses of the vaccines evaluated and demonstrated that Delta and Omicron variants escape from antibody neutralization. The method presented herein represents a valuable tool for the screening of antibodies and small molecules capable of blocking viral entry and could be used to evaluate humoral immunity developed by different populations and for vaccine development.

An Overview of the Conventional and Novel Methods Employed for SARS-CoV-2 Neutralizing Antibody Measurement.

SARS-CoV-2 is the etiological agent of the coronavirus disease-19 (COVID-19) and is responsible for the pandemic that started in 2020. The virus enters the host cell through the interaction of its spike glycoprotein with the angiotensin converting enzyme-2 (ACE2) on the host cell's surface. Antibodies present an important role during the infection and pathogenesis due to many reasons, including the neutralization of viruses by binding to different spike epitopes. Therefore, measuring the neutralizing antibody titers in the whole population is important for COVID-19's epidemiology. Different methods are described in the literature, and some have been used to validate the main vaccines used worldwide. In this review, we discuss the main methods used to quantify neutralizing antibody titers, their advantages and limitations, as well as new approaches to determineACE2/spike blockage by antibodies.

Ameliorating the antiparasitic activity of the multifaceted drug ivermectin through a polymer nanocapsule formulation.

Ivermectin (IVM) is a potent antiparasitic widely used in human and veterinary medicine. However, the low oral bioavailability of IVM restricts its therapeutic potential in many parasitic infections, highlighting the need for novel formulation approaches. In this study, poly(ε-caprolactone) (PCL) nanocapsules containing IVM were successfully developed using the nanoprecipitation method. Pumpkin seed oil (PSO) was used as an oily core in the developed nanocapsules. Previously, PSO was chemically analyzed by headspace solid-phase microextraction coupled to gas chromatography/mass spectrometry (HS-SPME/GC-MS). The solubility of IVM in PSO was found to be 4266.5 ± 38.6 µg/mL. In addition, the partition coefficient of IVM in PSO/water presented a logP of 2.44. A number of nanocapsule batches were produced by factorial design resulting in an optimized formulation. Negatively charged nanocapsules measuring around 400 nm demonstrated unimodal size distribution, and presented regular spherical morphology under transmission electron microscopy. High encapsulation efficiency (98-100%) was determined by HPLC. IVM-loaded capsules were found to be stable in nanosuspensions at 4 °C and 25 °C, with no significant variations in particle size observed over a period of 150 days. Nanoencapsulated IVM (0.3 mM) presented reduced toxicity to J774 macrophages and L929 fibroblasts compared to free IVM. Moreover, IVM-loaded nanocapsules also demonstrated enhanced in vitro anthelmintic activity against Strongyloides venezuelensis in comparison to free IVM. Collectively, the present findings demonstrate the promising potential of PCL-PSO nanocapsules to improve the antiparasitic effects exerted by IVM.

A Hybrid of Amodiaquine and Primaquine Linked by Gold(I) Is a Multistage Antimalarial Agent Targeting Heme Detoxification and Thiol Redox Homeostasis.

Hybrid-based drugs linked through a transition metal constitute an emerging concept for Plasmodium intervention. To advance the drug design concept and enhance the therapeutic potential of this class of drugs, we developed a novel hybrid composed of quinolinic ligands amodiaquine (AQ) and primaquine (PQ) linked by gold(I), named [AuAQPQ]PF6. This compound demonstrated potent and efficacious antiplasmodial activity against multiple stages of the Plasmodium life cycle. The source of this activity was thoroughly investigated by comparing parasite susceptibility to the hybrid's components, the annotation of structure-activity relationships and studies of the mechanism of action. The activity of [AuAQPQ]PF6 for the parasite's asexual blood stages was influenced by the presence of AQ, while its activity against gametocytes and pre-erythrocytic parasites was influenced by both quinolinic components. Moreover, the coordination of ligands to gold(I) was found to be essential for the enhancement of potency, as suggested by the observation that a combination of quinolinic ligands does not reproduce the antimalarial potency and efficacy as observed for the metallic hybrid. Our results indicate that this gold(I) hybrid compound presents a dual mechanism of action by inhibiting the beta-hematin formation and enzymatic activity of thioredoxin reductases. Overall, our findings support the potential of transition metals as a dual chemical linker and an antiplasmodial payload for the development of hybrid-based drugs.

Development and in vitro characterization of polymeric nanoparticles containing recombinant adrenomedullin-2 intended for therapeutic angiogenesis.

Cardiovascular diseases (CVD) are the leading cause of death worldwide. Growth factor therapy has emerged as novel therapeutic strategy under investigation for CVD. In this sense, adrenomedullin-2 (ADM-2) has been recently identified as a new angiogenic factor able to regulate the regional blood flow and cardiovascular function. However, the therapeutic value of ADM-2 is limited by its short biological half-life and low plasma stability. Poly (lactic-co-glycolic acid) (PLGA) micro- and nanoparticles have been investigated as growth factor delivery systems for cardiac repair. In this study, we aimed to develop PLGA nanoparticles containing ADM-2 intended for therapeutic angiogenesis. PLGA nanoparticles containing ADM-2 were prepared by a double emulsion modified method, resulting in 300 nm-sized stable particles with zeta potential around - 30 mV. Electron microscopy analysis by SEM and TEM revealed spherical particles with a smooth surface. High encapsulation efficiency was reached (ca.70%), as quantified by ELISA. ADM-2 associated to polymer nanoparticles was also determined by EDS elemental composition analysis, SDS-PAGE and LC-MS/MS for peptide identification. In vitro release assays showed the sustained release of ADM-2 from polymer nanoparticles for 21 days. Cell viability experiments were performed in J774 macrophages and H9c2 cardiomyocyte cells, about which PLGA nanoparticles loaded with ADM-2 did not cause toxicity in the range 0.01-1 mg/ml. Of note, encapsulated ADM-2 significantly induced cell proliferation in EA.hy926 endothelial cells, indicating the ADM-2 bioactivity was preserved after the encapsulation process. Collectively, these results demonstrate the feasibility of using PLGA nanoparticles as delivery systems for the angiogenic peptide ADM-2, which could represent a novel approach for therapeutic angiogenesis in CVD using growth factor therapy.

Avaliação farmacológica de compostos híbridos de ação em múltiplos estágios ou de duração longa para o tratamento da malária / Pharmacological assessment of multistage or long-acting hybrid compounds for the treatment of malaria

INTRODUÇÃO: Nos últimos quatro anos, foram aprovados a vacina Mosquirix™ para a malária falcipária e o medicamento Tafenoquina para a malária vivax, os quais representam avanços importantes no combate à transmissão e no tratamento da malária, respectivamente. Todavia, ainda há inúmeros desafios a serem contornados para combater a malária, tais como o desenvolvimento de novos fármacos com ação antiparasitária frente a múltiplos estágios do ciclo evolutivo do plasmódio e o desenvolvimento de novos fármacos que contornem o tempo de meia-vida plasmático curto das artemisininas, os quais são a primeira linha de tratamento. Uma alternativa para superar tais desafios são os compostos híbridos, os quais podem exercer um papel importante no desenvolvimento de novos fármacos antimaláricos de amplo espectro de ação e com eliminação plasmática mais lenta do que das artemisininas. OBJETIVO: Investigar as bases farmacológicas de compostos híbridos como agentes antiplasmódicos. MATERIAIS E MÉTODOS: Os híbridos foram idealizados, sintetizados e caracterizados quimicamente através de colaborações entre o nosso grupo e outros grupos de pesquisadores. Os ensaios experimentais de potência in vitro foram realizados com o P. falciparum e P. berghei, enquanto os ensaios de eficácia in vivo foram realizados com o P. berghei. RESULTADOS: Os achados experimentais mostraram que a combinação química de grupos farmacofóricos quinolínicos, dando origem aos compostos híbridos MNCS59 (1) e MNCS60 (2), revelou potência, eficácia e espectro de ação mais amplos do que a monoterapia ou terapia combinada com os seus fármacos parentais gametócitos maduros foram os menos suscept. Dentre os diferentes estágios evolutivos do plasmódio, o estágio sanguíneo assexuado foi o mais susceptível ao tratamento com os compostos híbridos, seguido dos esquizontes teciduais; já os gametócitos maduros foram os menos susceptíveis ao tratamento. Coincidentemente, os estágios evolutivos mais susceptíveis ao tratamento com compostos híbridos MNCS59 (1) e MNCS60 (2) são aqueles onde o processo de homeostasia redox e de detoxificação do heme são essenciais ao plasmódio. Os achados experimentais encontrados com a combinação química do grupo farmacofórico endoperóxido existente nas artemisininas, dando origem aos compostos híbridos 163A (3) e LH70 (4), também demonstraram potência, eficácia e espectro de ação mais amplos do que a monoterapia ou terapia combinada com os seus fármacos parentais. Interessantemente, o composto híbrido (4) apresentou potência e eficácia superiores aos demais híbridos e as artemisininas. Estudos subsequentes para explicar o aumento da potência antiparasitária mostraram que o híbrido (4) possui uma estabilidade química e uma ação fenotípica mais duradoura do que as artemisininas. Por apresentar uma ação fenotípica mais duradoura, o composto híbrido (4) foi capaz de reduzir a proliferação de parasitos resistentes as artemisininas, porém não foi tão capaz de reduzir a viabilidade celular de parasitos quiescentes e resistentes as artemisininas. CONCLUSÃO: Com este conjunto de dados, nós concluímos que os compostos híbridos aqui estudados conservam os mecanismos de ação dos endoperóxidos e/ou quinolinas, e também apresentam um potencial terapêutico melhorado e um espectro de ação mais amplo do que a monoterapia ou a terapia combinada, podendo ser considerados como possíveis fármacos para a terapia combinada com as artemininas

Desenvolvimento de nanopartículas para a liberação de adrenomedulina-2 no miocárdio infartado

INTRODUÇÃO: O infarto do miocárdio (IM) representa a manifestação mais significativa da cardiopatia isquêmica em todo o mundo. Atualmente, os tratamentos utilizados são invasivos, de alto custo e difícil acesso pelo sistema público de saúde, além de não apresentarem a capacidade de reparo do coração. Neste sentido, nanopartículas (NPs) à base do copolímero poli (ácido lático-co-glicólico, PLGA) têm sido investigadas como carreadores potenciais de biomoléculas para a regeneração do miocárdio lesionado, devido ao seu baixo potencial imunogênico, adequado perfil de biodegradação e alta biocompatibilidade com o tecido cardíaco. A adrenomedulina-2 (ADM-2), identificada como um novo fator angiogênico, demonstra alto potencial terapêutico em disfunções endoteliais. OBJETIVO: Dessa forma, o objetivo deste trabalho foi produzir nanopartículas poliméricas para a liberação do fator de crescimento ADM-2 no miocárdio infartado. MATERAIS E MÉTODOS: Após a produção de pré-formulações e a padronização da formulação à base de nanopartículas com albumina (BSA) pelo método de emulsão múltipla e extração de solvente, nanopartículas contendo ADM-2 foram preparadas e, em seguida, caracterizadas quanto aos parâmetros físico-químicos e atividade biológica in vitro. RESULTADOS: Em suspensão, as partículas foram caracterizadas quanto ao tamanho, índice de polidispersão (PDI) e potencial zeta por espalhamento de luz dinâmico e mobilidade eletroforética, respectivamente. NPs-ADM-2 apresentaram diâmetro médio de 312,1 nm ± 1,3, PDI de 0,34 ± 0,01 e potencial zeta de -31 mV, sem a presença de agregados. Após 30 dias, houve um leve aumento de tamanho e PDI, mas sem alteração no potencial zeta. NPs-ADM-2 foram avaliadas quanto à morfologia por microscopia eletrônica, apresentando-se dispersas, com forma esférica e superfície lisa. A eficiência de encapsulação foi de 67,28% determinada por ELISA, correspondendo a 134,5 ng de ADM-2 por mg de formulação. NPs-ADM-2 apresentaram compatibilidade in vitro com macrófagos da linhagem J774 e cardiomiócitos da linhagem H9c2, conforme ensaios de citotoxicidade por AlamarBlue®. Com relação à bioatividade, ADM-2 encapsulada foi capaz de estimular a proliferação celular das linhagens endoteliais HUVEC e EA.hy926, indicando que a atividade biológica deste fator de crescimento foi preservada após seu processo de formulação em nanopartículas de PLGA. CONCLUSÃO: Este trabalho é o primeiro estudo de desenvolvimento de uma nanoformulação de ADM-2, com potencial de inovação no âmbito da terapia com fatores de crescimento direcionada ao infarto do miocárdio