Encapsulation of benznidazole in nanostructured lipid carriers and increased trypanocidal activity in a resistant Trypanosoma cruzi strain

Abstract Chagas disease is a neglected parasitic disease caused by Trypanosoma cruzi, whose treatment has remained unsatisfactory for over 50 years, given that it is limited to two drugs. Benznidazole (BZN) is an efficient antichagasic drug used as the first choice, although its poor water-solubility, irregular oral absorption, low efficacy in the chronic phase, and various associated adverse effects are limiting factors for treatment. Incorporating drugs with such characteristics into nanostructured lipid carriers (NLC) is a promising alternative to overcome these limiting obstacles, enhancing drug efficacy and bioavailability while reducing toxicity. Therefore, this study proposed NLC-BZN formulations in different compositions prepared by hot-melt homogenization followed by ultrasound, and the optimized formulation was characterized by FTIR, DRX, DSC, and thermogravimetry. Biological activities included in vitro membrane toxicity (red blood cells), fibroblast cell cytotoxicity, and trypanocidal activity against epimastigotes of the Colombian strain of T. cruzi. The optimized NLC-BZN had a small size (110 nm), negative zeta potential (-18.0 mV), and high encapsulation (1.64% of drug loading), as shown by infrared spectroscopy, X-ray diffraction, and thermal analysis. The NLC-BZN also promoted lower in vitro membrane toxicity (<3% hemolysis), and 50% cytotoxic concentration (CC50) for NLC-BZN in L929 fibroblast cells (110.7 µg/mL) was twice the value as the free BZN (51.3 µg/mL). Our findings showed that the NLC-BZN had higher trypanocidal activity than free BZN against the epimastigotes of the resistant Colombian strain, and this novel NLC-BZN formulation proved to be a promising tool in treating Chagas disease and considered suitable for oral and parenteral administration

Phytochemical screening and evaluation of the toxic potential of ethanolic extract and fractions of Ageratum fastigiatum / Triagem fitoquímica e avaliação do potencial tóxico do extrato etanólico e frações de Ageratum fastigiatum / Cribado fitoquímico y evaluación del potencial tóxico del extracto etanólico y fracciones de Ageratum fastigiatum

SUMMARY Introduction: Ethanolic and hydroalcoholic extracts of Ageratum fastigiatum are used in folk medicine as anti-inflammatory and analgesic agents. Aim: To evaluate toxicity in Artemia salina and in the cells of the connective tissue of mice (L929). Methodology: The extract was partitioned and its hexane, dichloromethane and hydroalcoholic phases were submitted to the same test. The phytochemical screening of the phases of the extract was performed using tests aimed at the detection of secondary metabolites and GC-MS. Regarding A. salina, the hydroalcoholic phase exhibited the highest toxicity (LC50, 1.33 mg/mL) and the crude ethanolic extract was the least toxic (LC50, 4.81 mg/mL). In the assay of L929 cells, the dichloromethane phase was the most toxic (95.48% reduction in cell viability; LC50, 11.39 µg/mL), while the hydroalcoholic phase was the least toxic (cell death percentage, 55.67-19.38 %; LC50, 174.20 µg/mL). Result: The phytochemical study indicated the presence of alkaloids, coumarins, saponins, triterpenes/steroids and tannins. The GC-MS analysis identified the presence of terpenoids and a lycopsamine derivative (a pyrrolizidine alkaloid). Conclusion: These results suggest that the ethanolic extract of A. fastigiatum had constituents (i.e., phenolic compounds) that corroborate its use in folk medicine as an anti-inflammatory agent. However, the toxicity detected and the presence of 3'-acetyl lycopsamine (a chemotaxonomic marker of the genus that is hepatotoxic) indicates that this medicinal plant should be used with caution.

RESUMO Introdução: Os extratos etanólico e hidroalcoólico de Ageratum fastigiatum são usados na medicina popular como agentes antiinflamatório e analgésico. Objetivo: Avalidar quanto à sua toxicidade em Artemia salina e nas células do tecido conjuntivo de camundongos (L929). Metodologia: O extrato foi particionado e suas fases hexânica, diclorometânica e hidroalcoólica foram submetidas ao mesmo teste. A triagem fitoquímica das fases do extrato foi realizada por meio de testes visando a detecção de classes de metabólitos secundários e GC-MS. Resultados: Em relação a A. salina, a fase hidroalcoólica apresentou a maior toxicidade (CL50, 1,33 mg/mL) e o extrato etanólico bruto foi o menos tóxico (CL50_ 4,81 mg/mL). No ensaio de células L929, a fase de diclorometânica foi a mais tóxica (95,48% de redução na viabilidade celular; LC50, 11,39 µg/mL), enquanto a fase hidroalcoólica foi a menos tóxica (porcentagem de morte celular, 55,67-19,38 %; LC50, 174,20 µg/mL). O estudo fitoquímico indicou a provável presença de alcalóides, cumarinas, saponinas, triterpenos/ esteróides e taninos. A análise por GC-MS identificou a presença de terpenóides e um derivado de licopsamina (alcalóide pirrolizidínico). Conclusão: Esses resultados sugerem que o extrato etanólico de A. fastigiatum possui constituintes (ou seja, compostos fenólicos) que corroboram seu uso na medicina popular como agente antiinflamatório. No entanto, a toxicidade detectada e a presença da 3'-acetil licopsamina (um marcador quimiotaxonômico do gênero, que é hepatotóxico) indicam que essa planta medicinal deve ser usada com cautela.

RESUMEN Introducción: Los extractos etanólico e hidroalcohólico de Ageratum fastigiatum se utilizan en la medicina popular como agentes antiinflamatorios y analgésicos. Objetivo: Evaluar la toxicidad en artemia salina y en las células del tejido conectivo de ratones (L929). Metodología: El extracto se fraccionó y se sometieron a la misma prueba sus fases hexano, diclorometano e hidroalcohólica. El tamizaje fitoquímico de las fases del extracto se realizó mediante pruebas dirigidas a la detección de metabolitos secundarios y GC-MS. En cuanto a A. salina, la fase hidroalcohólica presentó la mayor toxicidad (CL50, 1,33 mg/mL) y el extracto etanólico crudo fue el menos tóxico (CL50, 4,81 mg/mL). En el ensayo de células L929, la fase de diclorometano fue la más tóxica (95,48% de reducción de la viabilidad celular; CL50, 11,39 µg/mL), mientras que la fase hidroalcohólica fue la menos tóxica (porcentaje de muerte celular, 55,67-19,38 %; CL50, 174,20 µg/mL). Resultado: El estudio fitoquímico indicó la probable presencia de alcaloides, cumarinas, saponinas, triterpenos/esteroides y taninos. El análisis GC-MS identificó la presencia de terpenoides y un derivado de licopsamina (un alcaloide de pirrolizidina). Conclusión: Estos resultados sugieren que el extracto etanólico de A. fastigiatum tenía constituyentes (es decir, compuestos fenólicos) que corroboran su uso en la medicina popular como agente antiinflamatorio. Sin embargo, la toxicidad detectada y la presencia de 3'-acetil licopsamina (un marcador quimiotaxonómico del género que es hepatotóxico) indica que esta planta medicinal debe utilizarse con precaución.

Nanonization techniques to overcome poor water-solubility with drugs.

INTRODUCTION: Most drugs used in therapy have low water-solubility, a factor that could reduce their dissolution rate and oral bioavailability, representing a challenge in pharmaceutical development. Nanonization of drugs is the reduction of particles to nanoscale, increasing the surface area and consequently the saturation solubility and dissolution rate and resulting in higher bioavailability. AREAS COVERED: This review provides an overview of the consequences of the poor water-solubility and the main strategies applied to increase the solubility of poorly water-soluble drugs. The relationship between the biopharmaceutical classification system and the solubilization process of the drug is also considered. Finally, it includes how drug nanoparticles and nanocarriers, especially lipid-based nanosystems, can overcome these challenges and which of these approaches are already available on the market. EXPERT OPINION: Due to the growing importance of nanomedicines, especially for applications in poorly water-soluble drugs, it is important to clearly establish the specifications and quality criteria for nanonized drugs to ensure the quality and safety of nanoparticles.

Efficacy of Meglumine Antimoniate in a Low Polymerization State Orally Administered in a Murine Model of Visceral Leishmaniasis.

Progress toward the improvement of meglumine antimoniate (MA), commercially known as Glucantime, a highly effective but also toxic antileishmanial drug, has been hindered by the lack of knowledge and control of its chemical composition. Here, MA was manipulated chemically with the aim of achieving an orally effective drug. MA compounds were synthesized from either antimony pentachloride (MA-SbCl5) or potassium hexahydroxyantimonate [MA-KSb(OH)6] and prepared under a low polymerization state. These compounds were compared to Glucantime regarding chemical composition, permeation properties across a cellulose membrane and Caco-2 cell monolayer, and uptake by peritoneal macrophages. MA-SbCl5 and MA-KSb(OH)6 were characterized as less polymerized and more permeative 2:2 Sb-meglumine complexes than Glucantime, which consisted of a mixture of 2:3 and 3:3 Sb-meglumine complexes. The antileishmanial activities and hepatic uptake of all compounds were evaluated after oral administration in BALB/c mice infected with Leishmania infantum chagasi, as a model of visceral leishmaniasis (VL). The synthetic MA compounds given at 300 mg Sb/kg of body weight/12 h for 30 days significantly reduced spleen and liver parasite burdens, in contrast to those for Glucantime at the same dose. The greater activity of synthetic compounds could be attributed to their higher intestinal absorption and accumulation efficiency in the liver. MA-SbCl5 given orally was as efficacious as Glucantime by the parenteral route (80 mg Sb/kg/24 h intraperitoneally). These data taken together suggest that treatment with a less-polymerized form of MA by the oral route may be effective for the treatment of VL.

Glycotriazole-peptides derived from the peptide HSP1: synergistic effect of triazole and saccharide rings on the antifungal activity.

This work proposes a strategy that uses solid-phase peptide synthesis associated with copper(I)-catalyzed azide alkyne cycloaddition reaction to promote the glycosylation of an antimicrobial peptide (HSP1) containing a carboxyamidated C-terminus (HSP1-NH2). Two glycotriazole-peptides, namely [p-Glc-trz-G1]HSP1-NH2 and [p-GlcNAc-trz-G1]HSP1-NH2, were prepared using per-O-acetylated azide derivatives of glucose and N-acetylglucosamine in the presence of copper(II) sulfate pentahydrate (CuSO4·5H2O) and sodium ascorbate as a reducing agent. In order to investigate the synergistic action of the carbohydrate motif linked to the triazole-peptide structure, a triazole derivative [trz-G1]HSP1-NH2 was also prepared. A set of biophysical approaches such as DLS, Zeta Potential, SPR and carboxyfluorescein leakage from phospholipid vesicles confirmed higher membrane disruption and lytic activities as well as stronger peptide-LUVs interactions for the glycotriazole-peptides when compared to HSP1-NH2 and to its triazole derivative, which is in accordance with the performed biological assays: whereas HSP1-NH2 presents relatively low and [trz-G1]HSP1-NH2 just moderate fungicidal activity, the glycotriazole-peptides are significantly more effective antifungal agents. In addition, the glycotriazole-peptides and the triazole derivative present strong inhibition effects on ergosterol biosynthesis in Candida albicans, when compared to HSP1-NH2 alone. In conclusion, the increased fungicidal activity of the glycotriazole-peptides seems to be the result of (A) more pronounced membrane-disruptive properties, which is related to the presence of a saccharide ring, together with (B) the inhibition of ergosterol biosynthesis, which seems to be related to the presence of both the monosaccharide and the triazole rings.

Hepatotoxicity of pentavalent antimonial drug: possible role of residual Sb(III) and protective effect of ascorbic acid.

Pentavalent antimonial drugs such as meglumine antimoniate (Glucantime [Glu; Sanofi-Aventis, São Paulo, Brazil]) produce severe side effects, including cardiotoxicity and hepatotoxicity, during the treatment of leishmaniasis. We evaluated the role of residual Sb(III) in the hepatotoxicity of meglumine antimoniate, as well as the protective effect of the antioxidant ascorbic acid (AA) during antimonial chemotherapy in a murine model of visceral leishmaniasis. BALB/c mice infected with Leishmania infantum were treated intraperitoneally at 80 mg of Sb/kg/day with commercial meglumine antimoniate (Glu) or a synthetic meglumine antimoniate with lower Sb(III) level (MA), in association or not with AA (15 mg/kg/day), for a 20-day period. Control groups received saline or saline plus AA. Livers were evaluated for hepatocytes histological alterations, peroxidase activity, and apoptosis. Increased proportions of swollen and apoptotic hepatocytes were observed in animals treated with Glu compared to animals treated with saline or MA. The peroxidase activity was also enhanced in the liver of animals that received Glu. Cotreatment with AA reduced the extent of histological changes, the apoptotic index, and the peroxidase activity to levels corresponding to the control group. Moreover, the association with AA did not affect the hepatic uptake of Sb and the ability of Glu to reduce the liver and spleen parasite loads in infected mice. In conclusion, our data supports the use of pentavalent antimonials with low residue of Sb(III) and the association of pentavalent antimonials with AA, as effective strategies to reduce side effects in antimonial therapy.

Medicamentos genéricos no Brasil: panorama histórico e legislação / Generic drugs in Brazil: historical overview and legislation

A política de medicamentos genéricos foi implantada no Brasil em 1999 com o objetivo de estimular a concorrência comercial, melhorar a qualidade dos medicamentos e facilitar o acesso da população ao tratamento medicamentoso. O processo de implementação dessa política permitiu a introdução e a discussão de conceitos nunca antes utilizados para o registro de medicamentos no Brasil: biodisponibilidade, bioequivalência, equivalência farmacêutica, medicamentos genéricos, sistema de classificação biofarmacêutica e bioisenção. Este artigo apresenta a definição desses conceitos no contexto das leis brasileiras e oferece uma descrição histórica e cronológica da implementação da política de genéricos no Brasil, listando ainda as resoluções que atualmente estão em vigor. Os resultados contribuem para a compreensão do processo e facilitam a busca e a identificação de ensaios necessários para satisfazer os critérios legais.

The Brazilian generic drugs policy was implemented in 1999 with the aim of stimulating competition in the market, improve the quality of drugs and improve the access of the population to drug treatment. The process of implementing this policy allowed the introduction and discussion of concepts that had never before been used in the context of drug registration in Brazil: bioavailability, bioequivalence, pharmaceutical equivalence, generic drugs, biopharmaceutical classification system, biowaiver. The present article provides definitions for these concepts in the context of Brazilian legislation as well as a historical and chronological description of the implementation of the generic drugs policy in Brazil, including a list of current generic drug legislation. This article contributes to the understanding of the Brazilian generic drugs policy and facilitates the search for information concerning the legal requirements for registration of drugs in Brazil.

Reduced cardiovascular alterations of tartar emetic administered in long-circulating liposomes in rats.

Trivalent antimonial drugs, including tartar emetic (TA), are known to induce important cardiotoxicity observed by electrocardiographic abnormalities. Liposome encapsulation was found to reduce the overall acute toxicity of TA. The present work investigated the cardiovascular parameters alterations of rats submitted to the treatment with free and encapsulated TA in long-circulating liposomes. Liposomes were made using lipids DSPC, DSPE-PEG and cholesterol. The cardiovascular signals, electrocardiogram (ECG) and arterial blood pressure (AP), were recorded from anaesthetized Wistar rats after intravenous (IV) administration of a single specially high dose (17 mg/kg) of TA in liposomes and in free form. The IV administration of TA solution caused significant increase of QT interval of ECG and significant reduction of AP when compared to the control group. These alterations were not observed when liposomes TA were administered and the profile of ECG and AP data was quite similar to the control groups. In conclusion, a liposomal formulation of TA showed a reduced cardiotoxic profile for TA when compared to the free form.

[Generic drugs in Brazil: historical overview and legislation]. / Medicamentos genéricos no Brasil: panorama histórico e legislação.

The Brazilian generic drugs policy was implemented in 1999 with the aim of stimulating competition in the market, improve the quality of drugs and improve the access of the population to drug treatment. The process of implementing this policy allowed the introduction and discussion of concepts that had never before been used in the context of drug registration in Brazil: bioavailability, bioequivalence, pharmaceutical equivalence, generic drugs, biopharmaceutical classification system, biowaiver. The present article provides definitions for these concepts in the context of Brazilian legislation as well as a historical and chronological description of the implementation of the generic drugs policy in Brazil, including a list of current generic drug legislation. This article contributes to the understanding of the Brazilian generic drugs policy and facilitates the search for information concerning the legal requirements for registration of drugs in Brazil.