RESUMO
The rapid development of multidrug-resistant pathogens against conventional antibiotics is a global public health problem. The irrational use of antibiotics has promoted therapeutic limitations against different infections, making research of new molecules that can be applied to treat infections necessary. Antimicrobial peptides (AMPs) are a class of promising antibiotic molecules as they present broad action spectrum, potent activity, and do not easily induce resistance. Several AMPs from scorpion venoms have been described as a potential source for the development of new drugs; however, some limitations to their application are also observed. Here, we describe strategies used in several approaches to optimize scorpion AMPs, addressing their primary sequence, biotechnological potential, and characteristics that should be considered when developing an AMP derived from scorpion venoms. In addition, this review may contribute towards improving the understanding of rationally designing new molecules, targeting functional AMPs that may have a therapeutic application.
RESUMO
Physical barrier membranes have been used to release active substances to treat critical bone defects; however, hydrophilic membranes do not present a prolonged release capacity. In this sense, hydrophobic membranes have been tested. Thus, this study aimed to develop hydrophobic membranes based on mixtures of ureasil-polyether-type materials containing incorporated dexamethasone (DMA) for the application in guided bone regeneration. The physicochemical characterization and biological assays were carried out using small-angle X-ray scattering (SAXS), an in vitro DMA release study, atomic force microscopy (AFM), a hemolysis test, and in vivo bone formation. The swelling degree, SAXS, and release results revealed that the u-PPO400/2000 membrane in the proportion of 70:30 showed swelling (4.69% ± 0.22) similar to the proportions 90:10 and 80:20, and lower than the proportion 60:40 (6.38% ± 0.49); however, an equal release percentage after 134 h was observed between the proportions 70:30 and 60:40. All u-PPO materials presented hemocompatibility (hemolysis ≤2.8%). AFM results showed that the treatments with or without DMA did not present significant differences, revealing a flat/smooth surface, with no pores and/or crystalline precipitates. Finally, in vivo results revealed that for both the commercial hydrophilic membrane and u-PPO400/2000 (70:30) after 60 days, the bone formation volume was 21%. In conclusion, hybrid membranes present unique characteristics for treating critical bone defects, considering the delayed and prolonged release results associated with the physical barrier capacity.
RESUMO
BACKGROUND AND PURPOSE: Inflammation associated with the tumour microenvironment (TME) is critical for cancer development, and immunotherapeutic strategies modulating the immune response in cancer have been crucial. In this study, a methotrexate-loaded (MTX) poly(lactic-co-glycolic acid)-based (PLGA) drug nanocarrier covered with polyethyleneimine (Pei) and hyaluronic acid (HA) was developed and combined with an PD-L1 antibody to investigate anti-cancer and immunomodulatory effects in breast cancer TME. EXPERIMENTAL APPROACH: Naked or HA-coated PeiPLGA-MTX nanoparticles (NPs) were assessed on 4T1 breast cancer cells grown in culture and in a mouse model of orthotopic tumour growth. Tumours were evaluated by qRT-PCR and immunohistochemistry. The cell death profile and cell migration were analysed in vitro in 4T1 cells. Polarization of murine macrophages (RAW cells) was also carried out. KEY RESULTS: Naked or HA-coated PeiPLGA-MTX NPs used alone or combined with PD-L1 antibody modified the tumourigenic course by TME immunomodulation, leading to reduction of primary tumour size and metastases. STAT3 and NF-κB were the major genes downregulated by NPs. In tumor-associated macrophages (TAM) such regulation switched M2 phenotype (CD163) towards M1 (CD68) and reduced levels of IL-10, TGF-ß and CCL22. Moreover, malignant cells showed overexpression of FADD, APAF-1, caspase-3 and E-cadherin, and decreased expression of Bcl-2, MDR-1, survivin, vimentin, CXCR4 and PD-L1 after treatment with NPs. CONCLUSION AND IMPLICATIONS: NPs-mediated STAT3/NF-κB signalling axis suppression disrupted crosstalk between immune and malignant cells, reducing immunosuppression and critical pro-tumour events. These findings provide a promising therapeutic approach capable of guiding the immune TME to suppress the development of breast cancer.
Assuntos
Antígeno B7-H1 , Neoplasias da Mama , Animais , Antígeno B7-H1/metabolismo , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Feminino , Humanos , Imunomodulação , Camundongos , NF-kappa B , Fator de Transcrição STAT3 , Microambiente Tumoral , Macrófagos Associados a TumorRESUMO
Scorpion venom constitutes a rich source of biologically active compounds with high potential for therapeutic and biotechnological applications that can be used as prototypes for the design of new drugs. The aim of this study was to characterize the structural conformation, evaluate the antimicrobial activity, and gain insight into the possible action mechanism underlying it, for two new analog peptides of the scorpion peptide Stigmurin, named StigA25 and StigA31. The amino acid substitutions in the native sequence for lysine residues resulted in peptides with higher positive net charge and hydrophobicity, with an increase in the theoretical helical content. StigA25 and StigA31 showed the capacity to modify their structural conformation according to the environment, and were stable to pH and temperature variation-results similar to the native peptide. Both analog peptides demonstrated broad-spectrum antimicrobial activity in vitro, showing an effect superior to that of the native peptide, being non-hemolytic at the biologically active concentrations. Therefore, this study demonstrates the therapeutic potential of the analog peptides from Stigmurin and the promising approach of rational drug design based on scorpion venom peptide to obtain new anti-infective agents.
Assuntos
Substituição de Aminoácidos , Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Bactérias Gram-Positivas/efeitos dos fármacos , Venenos de Escorpião/genética , Trypanosoma cruzi/efeitos dos fármacos , Animais , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Dicroísmo Circular , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Testes de Sensibilidade Microbiana , Modelos Moleculares , Simulação de Dinâmica Molecular , Estabilidade Proteica , Estrutura Secundária de Proteína , Venenos de Escorpião/química , Tripanossomicidas/química , Tripanossomicidas/farmacologiaRESUMO
This study explored the transition of lamellar-type liquid crystal (LLC) to biocompatible oil-in-water nanoemulsions able to modify benznidazole (BNZ) release and target the drug to cells infected with the T. cruzi parasite. Three cosolvents (2methylpyrrolidone [NMP], polyethylene glycol [POL], and propylene glycol [PRO] were tested to induce the transition of anisotropic LLC systems to isotropic nanoemulsions. Mixtures of soy phosphatidylcholine with sodium oleate stabilized the dispersions of medium chain triglyceride in water. Rheological measurements, polarized microscopy, and small angle X-ray scattering demonstrated that there is a phase transition from LLC to desired nanoemulsions. These small and narrow droplet-sized nanocarriers exhibited some advantages and promising features, such as the enhanced BNZ aqueous solubility and slow drug release rate. In vitro cell biocompatibility of formulations was assessed in the Vero E6 and SiHa cell lines. Drug-loaded nanoemulsions inhibited the epimastigote growth of the T. cruzi parasite (IC50 0.208⯱â¯0.052⯵gâ¯mL-1) and reduced its infective life form trypomastigote (IC50 0.392⯱â¯0.107⯵gâ¯mL-1). The oil-in-water nanoemulsions were demonstrated as promising biocompatible liquid drug delivery systems capable of improving the BNZ trypanocidal activity for the treatment of Chagas disease.
Assuntos
Sistemas de Liberação de Medicamentos , Nitroimidazóis/administração & dosagem , Tripanossomicidas/administração & dosagem , Trypanosoma cruzi/efeitos dos fármacos , Animais , Linhagem Celular , Doença de Chagas/tratamento farmacológico , Química Farmacêutica/métodos , Chlorocebus aethiops , Preparações de Ação Retardada , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Emulsões , Humanos , Concentração Inibidora 50 , Cristais Líquidos , Nanoestruturas , Nitroimidazóis/química , Nitroimidazóis/farmacologia , Transição de Fase , Solubilidade , Solventes/química , Tripanossomicidas/química , Tripanossomicidas/farmacologia , Células VeroRESUMO
Scorpion venom constitutes a rich source of biologically active compounds with high potential for therapeutic and biotechnological applications that can be used as prototypes for the design of new drugs. The aim of this study was to characterize the structural conformation, evaluate the antimicrobial activity, and gain insight into the possible action mechanism underlying it, for two new analog peptides of the scorpion peptide Stigmurin, named StigA25 and StigA31. The amino acid substitutions in the native sequence for lysine residues resulted in peptides with higher positive net charge and hydrophobicity, with an increase in the theoretical helical content. StigA25 and StigA31 showed the capacity to modify their structural conformation according to the environment, and were stable to pH and temperature variationresults similar to the native peptide. Both analog peptides demonstrated broad-spectrum antimicrobial activity in vitro, showing an effect superior to that of the native peptide, being non-hemolytic at the biologically active concentrations. Therefore, this study demonstrates the therapeutic potential of the analog peptides from Stigmurin and the promising approach of rational drug design based on scorpion venom peptide to obtain new anti-infective agents.
RESUMO
Scorpion venom is a rich source of biologically active components and various peptides with high-potential therapeutic use that have been characterized for their antimicrobial and antiproliferative activities. Stigmurin is a peptide identified from the Tityus stigmurus venom gland with high antibacterial and antiproliferative activities and low toxicity. Amino acid substitutions in peptides without a disulfide bridge sequence have been made with the aim of reducing their toxicity and increasing their biological activities. The purpose of this study was to evaluate the structural conformation and structural stability, as well as antimicrobial, antiproliferative, and hemolytic activities of two peptide analogs to Stigmurin, denominated StigA6 and StigA16. In silico analysis revealed the α-helix structure for both analog peptides, which was confirmed by circular dichroism. Data showed that the net charge and hydrophobic moment of the analog peptides were higher than those for Stigmurin, which can explain the increase in antimicrobial activity presented by them. Both analog peptides exhibited activity on cancerous cells similar to the native peptide; however, they were less toxic when tested on the normal cell line. These results reveal a potential biotechnological application of the analog peptides StigA6 and StigA16 as prototypes to new therapeutic agents.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos/farmacologia , Venenos de Escorpião/farmacologia , Células 3T3 , Sequência de Aminoácidos , Animais , Anti-Infecciosos/química , Bactérias/efeitos dos fármacos , Candida/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Eritrócitos/efeitos dos fármacos , Células HeLa , Humanos , Camundongos , Simulação de Dinâmica Molecular , Peptídeos/química , Estrutura Secundária de Proteína , Venenos de Escorpião/química , Trypanosoma cruzi/efeitos dos fármacosRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Jatropha species (Euphorbiaceae) are largely used in traditional medicine to treat different pathologies in Africa, Asia and Latin America. In Northeastern Brazilian folk medicine, several Jatropha species, such as Jatropha gossypiifolia L. and Jatropha mollissima (Pohl) Baill., are indistinctly used to treat snakebites. AIM OF THE STUDY: To compare two of the Brazilian most used Jatropha species for snakebites (J. gossypiifolia and J. mollissima), in relation to their ability to inhibit local edematogenic activity of Bothrops erythromelas snake venom in mice, their in vitro antibacterial activity and phytochemical profile. MATERIAL AND METHODS: Aqueous leaf extracts of J. gossypiifolia (AEJg) and J. mollissima (AEJm) were prepared by decoction. AEJg and AEJm were compared chemically, by thin layer chromatography (TLC) and high-performance liquid chromatography with diode array detection (HPLC-DAD) analysis. They were also pharmacologically compared, using the mouse model of paw edema induced by Bothrops erythromelas snake venom (BeV), and in vitro by broth microdilution and agar dilution antimicrobial tests. RESULTS: Flavonoids were detected as the major compounds in both extracts. However, AEJg and AEJm showed quantitatively different chemical profiles by HPLC-DAD. AEJg presented fewer peaks of flavonoids than AEJm, however, when the intensity of peaks were analyzed, these compounds were at high concentration in AEJg, even using the same concentration of both extracts. Differences were also observed in the biological activity of the two extracts. While no difference was observed when the extracts were administered by oral route (P > 0.05), by the intraperitoneal route AEJg presented anti-edematogenic activity significantly (P < 0.001) higher than AEJm. In antimicrobial assays, only AEJg presented antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and Bacillus cereus. CONCLUSIONS: Although used indistinctly by folk medicine, our results suggested that AEJg is more active than AEJm in relation to its antiedematogenic and antibacterial activities. Significant differences were observed in their phytochemical profiles, especially a higher content of C-glycosylated flavonoids in the most active species, which could justify the different biological effects observed. These findings strengthen the potentiality of J. gossypiifolia species for use as complementary treatment for local effects induced by Bothrops venoms and could be helpful for distinction of the species and control quality assessment of future herbal medicines based on Jatropha plants.
Assuntos
Antibacterianos , Anti-Inflamatórios , Edema/tratamento farmacológico , Jatropha , Extratos Vegetais , Mordeduras de Serpentes/tratamento farmacológico , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Anti-Inflamatórios/química , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/uso terapêutico , Bothrops , Venenos de Crotalídeos , Feminino , Flavonoides/análise , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Negativas/crescimento & desenvolvimento , Bactérias Gram-Positivas/efeitos dos fármacos , Bactérias Gram-Positivas/crescimento & desenvolvimento , Jatropha/química , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Compostos Fitoquímicos/análise , Fitoterapia , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Folhas de Planta/químicaRESUMO
Jatropha species (Euphorbiaceae) are largely used in traditional medicine to treat different pathologies in Africa, Asia and Latin America. In Northeastern Brazilian folk medicine, several Jatropha species, such as Jatropha gossypiifolia L. and Jatropha mollissima (Pohl) Baill., are indistinctly used to treat snakebites. To compare two of the Brazilian most used Jatropha species for snakebites (J. gossypiifolia and J. mollissima), in relation to their ability to inhibit local edematogenic activity of Bothrops erythromelas snake venom in mice, their in vitro antibacterial activity and phytochemical profile. Aqueous leaf extracts of J. gossypiifolia (AEJg) and J. mollissima (AEJm) were prepared by decoction. AEJg and AEJm were compared chemically, by thin layer chromatography (TLC) and high-performance liquid chromatography with diode array detection (HPLC-DAD) analysis. They were also pharmacologically compared, using the mouse model of paw edema induced by Bothrops erythromelas snake venom (BeV), and in vitro by broth microdilution and agar dilution antimicrobial tests. RESULTS: Flavonoids were detected as the major compounds in both extracts. However, AEJg and AEJm showed quantitatively different chemical profiles by HPLC-DAD. AEJg presented fewer peaks of flavonoids than AEJm, however, when the intensity of peaks were analyzed, these compounds were at high concentration in AEJg, even using the same concentration of both extracts. Differences were also observed in the biological activity of the two extracts. While no difference was observed when the extracts were administered by oral route (P > 0.05), by the intraperitoneal route AEJg presented anti-edematogenic activity significantly (P < 0.001) higher than AEJm. In antimicrobial assays, only AEJg presented antibacterial activity against Staphylococcus epidermidis, Staphylococcus aureus and Bacillus cereus. Although used indistinctly by folk medicine, our results suggested that AEJg is more active than AEJm in relation to its antiedematogenic and antibacterial activities. Significant differences were observed in their phytochemical profiles, especially a higher content of C-glycosylated flavonoids in the most active species, which could justify the different biological effects observed. These findings strengthen the potentiality of J. gossypiifolia species for use as complementary treatment for local effects induced by Bothrops venoms and could be helpful for distinction of the species and control quality assessment of future herbal medicines based on Jatropha plants.
RESUMO
In this study, biodegradable and biocompatible gamma irradiated poly-(dl-lactide-co-glycolide) (PLGA) spray-dried microparticles were prepared aiming to improve the efficacy of methotrexate (MTX). The experimental design included three formulations of microparticles containing distinct drug amount (9%, 18%, and 27% w/w) and three distinct gamma irradiation dose (15kGy, 25kGy, and 30kGy). The physicochemical and drug release properties of the microparticles supported their biocompatibility and biological efficacy studies in different cell lines. The irradiation induced slight changes in the spherical shape of the microparticles and the formation of free radicals was dependent on the drug loading. However, the amorphous character, particle size, drug loading, and drug release rate of the microparticles were preserved. The drug release data from all microparticles formulation were evaluated by using four drug kinetic models and by comparison of their similarity factor (f2). The gamma irradiation did not induce changes in the biocompatibility of PLGA microparticles and in the biological activity of the MTX-loaded microparticles. Finally, the spray-dried MTX-loaded PLGA microparticles enhanced the efficacy of the drug in the human cervical cancer cells (SiHa cell line). This study demonstrated the feasibility of the gamma irradiated spray dried PLGA microparticles for prolonged release of MTX, supporting a promising antitumor-drug delivery system for parenteral (subcutaneous) or pulmonary use.
Assuntos
Metotrexato/química , Química Farmacêutica , Sistemas de Liberação de Medicamentos , Raios gama , Humanos , Ácido Láctico , Microesferas , Tamanho da Partícula , Ácido PoliglicólicoRESUMO
Several polymers have been investigated for producing cationic nanocarriers due to their ability to cross biological barriers. Polycations such as copolymers of polymethylmethacrylate are highlighted due to their biocompatibility and low toxicity. The purpose of this study was to produce small and narrow-sized cationic nanoparticles able to overcome cell membranes and improve the biological activity of benznidazole (BNZ) in normal and cancer cells. The effect of composition and procedure parameters of the used emulsification-solvent evaporation method were controlled for this purpose. The experimental approach included particle size, polydispersity index, zeta potential, atomic force microscopy (AFM), attenuated total reflectance Fourier transforms infrared spectroscopy (ATR- FTIR), drug loading efficiency, and physical stability assays. Spherical and stable (over six weeks) sub 150nm cationic nanoparticles were optimized, with the encapsulation efficiency >80%. The used drug/copolymer ratio modulated the slow drug release, which was adjusted by the parabolic diffusion mathematical model. In addition, the ability of the cationic nanoparticles improve the BNZ uptake in the normal kidney cells (HEK 293) and the human colorectal cancer cells (HT 29) demonstrate that this novel BNZ-loaded cationic has great potential as a chemotherapeutic application of benznidazole.
Assuntos
Nanopartículas , Portadores de Fármacos , Liberação Controlada de Fármacos , Células HEK293 , Humanos , Nitroimidazóis , Tamanho da PartículaRESUMO
Anionic Peptides are molecules rich in aspartic acid (Asp) and/or glutamic acid (Glu) residues in the primary structure. This work presents, for the first time, structural characterization and biological activity assays of an anionic peptide from the venom of the scorpion Tityus stigmurus, named TanP. The three-dimensional structure of TanP was obtained by computational modeling and refined by molecular dynamic (MD) simulations. Furthermore, we have performed circular dichroism (CD) analysis to predict TanP secondary structure, and UV-vis spectroscopy to evaluate its chelating activity. CD indicated predominance of random coil conformation in aqueous medium, as well as changes in structure depending on pH and temperature. TanP has chelating activity on copper ions, which modified the peptide's secondary structure. These results were corroborated by MD data. The molar ratio of binding (TanP:copper) depends on the concentration of peptide: at lower TanP concentration, the molar ratio was 1:5 (TanP:Cu2+), whereas in concentrated TanP solution, the molar ratio was 1:3 (TanP:Cu2+). TanP was not cytotoxic to non-neoplastic or cancer cell lines, and showed an ability to inhibit the in vitro release of nitric oxide by LPS-stimulated macrophages. Altogether, the results suggest TanP is a promising peptide for therapeutic application as a chelating agent.
Assuntos
Quelantes/química , Cobre/química , Peptídeos/química , Escorpiões/metabolismo , Sequência de Aminoácidos , Animais , Linhagem Celular , Dicroísmo Circular , Camundongos , Simulação de Dinâmica Molecular , Peptídeos/metabolismo , Estrutura Secundária de Proteína , Venenos de Escorpião/química , Venenos de Escorpião/metabolismo , Alinhamento de SequênciaRESUMO
Bothrops erythromelas is a snake of medical importance responsible for most of the venomous incidents in Northeastern Brazil. However, this species is not included in the pool of venoms that are used in the Brazilian polyvalent bothropic antivenom (BAv) production. Furthermore, it is well known that antivenom therapy has limited efficacy against venom-induced local effects, making the search for complementary alternatives to treat snakebites an important task. Jatropha gossypiifolia is a medicinal plant widely indicated in folk medicine as an antidote for snakebites, whose effectiveness against Bothrops jararaca venom (BjV) has been previously demonstrated in mice. In this context, this study assessed the effectiveness of the aqueous extract (AE) of this plant and of the BAv against local effects induced by B. erythromelas venom (BeV). Inhibition of BeV-induced edematogenic and hemorrhagic local effects was assayed in mice in pre-treatment (treatment prior to BeV injection) and post-treatment (treatment post-envenomation) protocols. Inhibition of proteolytic, phospholipase A2 (PLA2) and hyaluronidase enzymatic activities of BeV were evaluated in vitro. BAv cross-reactivity and estimation of antibody titers against BeV and BjV were assessed by Ouchterlony double diffusion test. The results show that in pre-treatment protocol AE and BAv presented very similar effects (about 70% of inhibition for edematogenic and 40% for hemorrhagic activities). However, BAv poorly inhibited edema and hemorrhage in post-envenomation protocol, whilst, in contrast, AE was significantly active even when used after BeV injection. AE was able to inhibit all the tested enzymatic activities of BeV, while BAv was active only against hyaluronidase activity, which could justify the low effectiveness of BAv against BeV-induced local effects in vivo. Ouchterlony's test showed positive cross-reactivity against BeV, but the antibody titers were slightly higher against BjV. Together, these data indicate that despite the presence of immunological cross-reactivity, Brazilian polyvalent bothropic antivenom presented low inhibitory potential against biological and enzymatic effects of BeV, illustrating the need for new strategies in the production of antivenom with broad neutralizing potential in the treatment of Bothrops spp. envenomation throughout the country. Together, the results highlight the antiophidic potential of J. gossypiifolia, suggesting that it can be considered a potential adjuvant in the treatment of bothropic envenomation local effects.
Assuntos
Antivenenos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Jatropha/química , Extratos Vegetais/farmacologia , Animais , Antivenenos/uso terapêutico , Feminino , Masculino , Medicina Tradicional , Camundongos , Extratos Vegetais/química , Extratos Vegetais/uso terapêuticoRESUMO
Microbial resistance to conventional antibiotics is a public health problem worldwide, motivating the search for new therapeutic alternatives in varied natural sources. Cationic peptides without disulfide bridges from scorpions have been targeted in this context, mainly due to their multifunctional action and the limited ability of microorganisms to develop resistance against them. The present study was focused on Stigmurin and TsAP-2, cationic peptides found in the transcriptome of the venom gland from the scorpion Tityus stigmurus. The aims were: to assess the secondary structure of TsAP-2 and the structural stability of both peptides by circular dichroism; to evaluate their antiproliferative effect, and antimicrobial activities in vitro, ex vivo and in vivo; and to investigate their therapeutic potential in a murine model of polymicrobial sepsis. Stigmurin and TsAP-2 secondary structures responded similarly to environment polarity changes, and were stable to temperature and pH variation. Both peptides showed antiproliferative effect on tumor cells. TsAP-2 showed lower cytotoxicity to normal cells, and had a mitogenic activity on murine macrophages. Stigmurin demonstrated bactericidal and bacteriostatic activity, depending on the microorganism, whereas TsAP-2 had bactericidal action upon different bacterial strains analyzed. Both peptides were able to reduce leukocyte migration, TNF-α levels and microorganism load in the peritoneal cavity after induction of experimental sepsis, decreasing inflammation in the lung and cecum of septic animals. TsAP-2 also reduced the release of nitric oxide in the peritoneal cavity. Taken together, these data suggest that Stigmurin and TsAP-2 are structurally stable molecules and are efficient in the control of the infectious focus in polymicrobial sepsis, with potential use as a prototype for the rational design of novel therapeutic agents.
Assuntos
Venenos de Escorpião/química , Venenos de Escorpião/toxicidade , Sepse/tratamento farmacológico , Toxinas Biológicas/uso terapêutico , Sequência de Aminoácidos , Animais , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Dicroísmo Circular , Citocinas/metabolismo , Feminino , Células Hep G2 , Humanos , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Óxido Nítrico/metabolismo , Estrutura Secundária de Proteína , Sepse/metabolismo , Sepse/patologia , Temperatura , Toxinas Biológicas/químicaRESUMO
The presence of bioactive peptides in animal venoms has been targeted in scientific research for assessing biological activities, as well as mechanisms of action. A recent study by our group observed hypotensive action of TistH (Tityus stigmurus Hypotensin), a peptide deduced from the transcriptome of T. stigmurus venom gland. The present study aims to analyze TistH structure properties and to evaluate its toxicity on normal and tumor cells, its in vitro antimicrobial activity, as well as its inflammatory effect. Circular dichroism analyses of TistH showed a general predominance of α-helix conformation in TFE (20-70%) and structural stability to pH variations. TistH was not cytotoxic to normal cell lines (3T3, RAW and HEK), and also not to cancer cell lines (HeLa, B16, 786-0, SiHa and HepG2). The peptide did not present inflammatory activity up to 6 h after administered subcutaneously to Swiss mice. It was observed that concentrations of 4-1024 µg/mL of TistH produced no inhibition against the bacteria Staphylococcus aureus, Staphylococcus epidermidis e Pseudomonas aeruginosa. The results of antifungal assays showed a moderate activity of TistH against Candida albicans strain LM-108 and the filamentous fungus Trichophyton rubrum LM-640, with growth inhibition at a concentration of 1024 µg/mL. In contrast, the peptide presented a greater activity (MIC 128 µg/mL) against C. albicans LM-106, Candida tropicalis ATCC 13308 and Aspergillus flavus strains LM-247 and LM-26, fungi that cause oral and vaginal infections, candidiasis and respiratory allergies, respectively. The present data contribute to a better understanding of TistH and its possible use as a bioactive compound. This multifunctional peptide is capable of acting as anti-hypertensive, as well as to inhibit the growth of fungal strains, having low toxicity, which suggests its safety for using as a pharmacological agent.
Assuntos
Anti-Infecciosos/farmacologia , Peptídeos/química , Peptídeos/farmacologia , Venenos de Escorpião/química , Animais , Linhagem Celular , Linhagem Celular Tumoral , Dicroísmo Circular , Ensaios de Seleção de Medicamentos Antitumorais , Humanos , Camundongos , Estrutura Secundária de Proteína , TemperaturaRESUMO
Bullfrog oil is a natural product extracted from the Rana catesbeiana Shaw adipose tissue and used in folk medicine for the treatment of several diseases. The aim of this study was to evaluate the extraction process of bullfrog oil, to develop a suitable topical nanoemulsion and to evaluate its efficacy against melanoma cells. The oil samples were obtained by hot and organic solvent extraction processes and were characterized by titration techniques and gas chromatography mass spectrometry (GC-MS). The required hydrophile-lipophile balance and the pseudo-ternary phase diagram (PTPD) were assessed to determine the emulsification ability of the bullfrog oil. The anti-tumoral activity of the samples was assessed by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for normal fibroblast (3T3) and melanoma (B16F10) cell lines. Both extraction methods produced yielded around 60% and the oil was mainly composed of unsaturated compounds (around 60%). The bullfrog oil nanoemulsion obtained from PTPD presented a droplet size of about 390 nm and polydispersity = 0.05 and a zeta potential of about -25 mV. Both the bullfrog oil itself and its topical nanoemulsion did not show cytotoxicity in 3T3 linage. However, these systems showed growth inhibition in B16F10 cells. Finally, the bullfrog oil presented itself as a candidate for the development of pharmaceutical products free from cytotoxicity and effective for antineoplastic therapy.
Assuntos
Antineoplásicos/isolamento & purificação , Produtos Biológicos/uso terapêutico , Melanoma Experimental/tratamento farmacológico , Óleos/uso terapêutico , Rana catesbeiana , Células 3T3 , Animais , Antineoplásicos/uso terapêutico , Antineoplásicos/toxicidade , Pesquisa Biomédica/tendências , Linhagem Celular Tumoral , Ensaios de Seleção de Medicamentos Antitumorais , Emulsões , Células HeLa , Humanos , Camundongos , Óleos/química , Óleos/isolamento & purificação , Óleos/toxicidadeRESUMO
In a recent work by our group involving a transcriptomics approach applied to the venom glands from Tityus stigmurus we identified a new family of peptides called Hypotensins (TSTI0006C) (Almeida et al., 2012). The cluster TSTI0006C was analyzed in the main 25 amino acid residues and named T. stigmurus Hypotensin (TistH), showing a molecular mass of 2.7 kDa, an absence of cysteines and the presence of two C-terminal proline residues, which are a bradykinin-potentiating peptide (BPP) signature. Here, we describe the homology modeling of the three-dimensional structure of TistH. In addition, we evaluated the cardiovascular effects elicited by TistH in normotensive rats. Firstly, TistH showed no cytotoxic effect on horse erythrocyte. Furthermore, in normotensive rats TistH was able to potentiate the hypotensive action of bradykinin (BK) and induced a vasorelaxant effect in mesenteric artery rings by endothelium-dependent release of nitric oxide (NO) and demonstrated independent inhibition of angiotensin converting enzyme (ACE). Our data can contribute to a better understanding of the structural and functional characteristics of TistH and suggest its potential use in cardiovascular diseases.
Assuntos
Bradicinina/farmacologia , Venenos de Escorpião/farmacologia , Escorpiões/metabolismo , Vasodilatadores/farmacologia , Inibidores da Enzima Conversora de Angiotensina/química , Inibidores da Enzima Conversora de Angiotensina/farmacologia , Animais , Anti-Hipertensivos/farmacologia , Pressão Sanguínea/efeitos dos fármacos , Bradicinina/química , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/metabolismo , Clonagem Molecular , Biologia Computacional , Modelos Moleculares , Óxido Nítrico/metabolismo , Peptidil Dipeptidase A/metabolismo , Ratos , Ratos Wistar , Venenos de Escorpião/química , Transcriptoma , Vasodilatadores/químicaRESUMO
The interaction of methotrexate (MTX) with beta-cyclodextrin (ß-CD) in the presence of triethanolamine (TEA) was investigated with the aim to elucidate the mechanism whereby self-assembly cyclodextrin systems work in association with this third component. Solubility diagram studies showed synergic increment of the MTX solubility to be about thirty-fold. Experiments using 2D ROESY and molecular modeling studies revealed the inclusion of aromatic ring III of the drug into ß-CD cavity, in which TEA contributes by intensifying MTX interaction with ß-CD and stabilizes MTX:ß-CD:TEA ternary complex by electrostatic interaction. The maintenance of these interactions in solid phase was also studied in ternary MTX:ß-CD:TEA and comparisons were made with freeze dried binary MTX:ß-CD and physical mixtures. FTIR studies evidenced that MTX-ß-CD interaction remained in solid ternary complexes, which was also supported by thermal (differential scanning calorimetry (DSC), thermogravimetric analysis (TG)/first derivative of TG analysis (DTG) and C,N,H elementary analysis) and structural (X-ray diffraction analysis, (XRD)) studies, mainly regarding the increment of drug stability. The efficient in vitro drug dissolution studies successfully demonstrated the contribution of ternary complexes, which highlights the importance of this possible new raw material for further applications in drug delivery systems.
Assuntos
Etanolaminas/farmacologia , Excipientes/química , Metotrexato/química , beta-Ciclodextrinas/química , Varredura Diferencial de Calorimetria , Cristalografia por Raios X , Portadores de Fármacos , Liofilização , Interações Hidrofóbicas e Hidrofílicas , Metotrexato/administração & dosagem , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Solubilidade , Espectroscopia de Infravermelho com Transformada de Fourier , Eletricidade Estática , Termogravimetria , ÁguaRESUMO
Snakebites are a serious public health problem due their high morbi-mortality. The main available specific treatment is the antivenom serum therapy, which has some disadvantages, such as poor neutralization of local effects, risk of immunological reactions, high cost and difficult access in some regions. In this context, the search for alternative therapies is relevant. Therefore, the aim of this study was to evaluate the antiophidic properties of Jatropha gossypiifolia, a medicinal plant used in folk medicine to treat snakebites. The aqueous leaf extract of the plant was prepared by decoction and phytochemical analysis revealed the presence of sugars, alkaloids, flavonoids, tannins, terpenes and/or steroids and proteins. The extract was able to inhibit enzymatic and biologic activities induced by Bothrops jararaca snake venom in vitro and in vivo. The blood incoagulability was efficiently inhibited by the extract by oral route. The hemorrhagic and edematogenic local effects were also inhibited, the former by up to 56% and the latter by 100%, in animals treated with extract by oral and intraperitoneal routes, respectively. The inhibition of myotoxic action of B. jararaca reached almost 100%. According to enzymatic tests performed, it is possible to suggest that the antiophidic activity may be due an inhibitory action upon snake venom metalloproteinases (SVMPs) and/or serine proteinases (SVSPs), including fibrinogenolytic enzymes, clotting factors activators and thrombin like enzymes (SVTLEs), as well upon catalytically inactive phospholipases A2 (Lys49 PLA2). Anti-inflammatory activity, at least partially, could also be related to the inhibition of local effects. Additionally, protein precipitating and antioxidant activities may also be important features contributing to the activity presented. In conclusion, the results demonstrate the potential antiophidic activity of J. gossypiifolia extract, including its significant action upon local effects, suggesting that it may be used as a new source of bioactive molecules against bothropic venom.
Assuntos
Antídotos/farmacologia , Bothrops , Venenos de Crotalídeos/antagonistas & inibidores , Venenos de Crotalídeos/farmacologia , Euphorbiaceae/química , Extratos Vegetais/farmacologia , Adulto , Animais , Anticoagulantes/química , Anticoagulantes/isolamento & purificação , Anticoagulantes/farmacologia , Antídotos/química , Antídotos/isolamento & purificação , Antioxidantes/química , Antioxidantes/isolamento & purificação , Antioxidantes/farmacologia , Coagulação Sanguínea/efeitos dos fármacos , Bothrops/metabolismo , Células HEK293 , Humanos , Camundongos , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificação , Folhas de Planta/químicaRESUMO
Proteolytic enzymes are important macromolecules in the regulation of biochemical processes in living organisms. Additionally, these versatile biomolecules have numerous applications in the industrial segment. In this study we have characterized a protein-rich fraction of Cnidoscolus urens (L.) Arthur leaves, rich in proteolytic enzymes, and evaluated its effects on the coagulation cascade. Three protein-rich fractions were obtained from the crude extract of C. urens leaves by precipitation with acetone. Fraction F1.0 showed higher proteolytic activity upon azocasein, and thus, was chosen for subsequent tests. The proteolytic activity of F1.0 on fibrinogen was dose-dependent and time-dependent. The extract demonstrated procoagulant activity on citrated plasma and reduced the APTT, not exerting effects on PT. Despite the fibrin(ogen)olytic activity, F1.0 showed no defibrinogenating activity in vivo. The fraction F1.0 did not express hemorrhagic nor hemolytic activities. The proteolytic activity was inhibited by E-64, EDTA and in the presence of metal ions, and increased when pretreated with reducing agents, suggesting that the observed activity was mostly due to cysteine proteases. Several bands with proteolytic activity were detected by zymography with gelatin, albumin and fibrinogen. The optimal enzymatic activity was observed in temperature of 60 °C and pH 5.0, demonstrating the presence of acidic proteases. In conclusion, these results could provide basis for the pharmacological application of C. urens proteases as a new source of bioactive molecules to treat bleeding and thrombotic disorders.