RESUMO
Toxoplasma gondii is an obligate intracellular parasite in the phylum Apicomplexa that causes toxoplasmosis in humans and animals worldwide. Despite its prevalence, there is currently no effective vaccine or treatment for chronic infection. Although there are therapies against the acute stage, prolonged use is toxic and poorly tolerated. This study aims to explore the potential of repurposing topotecan and 10-hydroxycamptothecin (HCPT) as drugs producing double strand breaks (DSBs) in T. gondii. DSBs are mainly repaired by Homologous Recombination Repair (HRR) and Non-Homologous End Joining (NHEJ). Two T. gondii strains, RHΔHXGPRT and RHΔKU80, were used to compare the drug's effects on parasites. RHΔHXGPRT parasites may use both HRR and NHEJ pathways but RHΔKU80 lacks the KU80 protein needed for NHEJ, leaving only the HRR pathway. Here we demonstrate that topotecan and HCPT, both topoisomerase I venoms, affected parasite replication in a concentration-dependent manner. Moreover, variations in fluorescence intensity measurements for the H2A.X phosphorylation mark (γH2A.X), an indicator of DNA damage, were observed in intracellular parasites under drug treatment conditions. Interestingly, intracellular replicative parasites without drug treatment show a strong positive staining for γH2A.X, suggesting inherent DNA damage. Extracellular (non-replicating) parasites did not exhibit γH2A.X staining, indicating that the basal level of DNA damage is likely to be associated with replicative stress. A high rate of DNA replication stress possibly prompted the evolution of an efficient repair machinery in the parasite, making it an attractive target. Our findings show that topoisomerase 1 venoms are effective antiparasitics blocking T. gondii replication.
Assuntos
Parasitos , Toxoplasma , Humanos , Animais , Toxoplasma/genética , Topotecan/farmacologia , Topotecan/metabolismo , Reparo do DNA , Dano ao DNARESUMO
OBJECTIVES: This work aimed to evaluate semisolid formulations containing topotecan (TPT) loaded nanostructured lipid carriers (NLC) for topical treatment of skin cancers, as TPT is effective against a variety of tumours. A formulation which increases TPT skin permeation would be extremely desirable. METHODS: TPT-NLC were prepared and incorporated in hydrogels with hydroxyethyl cellulose and chitosan (TPT-NLC-HEC and TPT-NLC-Ch, respectively). Control formulations were obtained by dispersing TPT in HEC and Ch hydrogels (TPT-HEC and TPT-Ch). KEY FINDINGS: TPT-NLC-HEC and TPT-NLC-Ch showed to maintain the drug and nanoparticle dispersions stable for up to 30 days. When nanoparticles were incorporated into gels, TPT release was significantly decreased (P < 0.05). Still, TPT-NLC-HEC increased 2.37 times permeation compared with TPT-HEC (11.9 and 5.0 µg/cm2 , respectively). Cell culture experiments with B16F10 melanoma demonstrated that nanoencapsulation significantly increased TPT cytotoxicity (P < 0.05). TPT-NLC was more toxic than free TPT, with IC50 value of 5.74 µg/ml, whereas free TPT had an IC50 > 20 µg/ml. As skin permeated values of TPT from developed formulation (TPT-NLC) were superior to melanoma IC50, it can be extrapolated that chemotherapeutic permeated amounts may be sufficient for a therapeutic effect. CONCLUSIONS: TPT-NLC-HEC may be a valuable tool for the topical treatment of skin cancers.
Assuntos
Portadores de Fármacos/administração & dosagem , Melanoma Experimental/tratamento farmacológico , Nanopartículas/administração & dosagem , Absorção Cutânea/fisiologia , Neoplasias Cutâneas/tratamento farmacológico , Topotecan/administração & dosagem , Administração Tópica , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/fisiologia , Relação Dose-Resposta a Droga , Portadores de Fármacos/metabolismo , Hidrogéis/administração & dosagem , Hidrogéis/metabolismo , Lipídeos/administração & dosagem , Melanoma Experimental/metabolismo , Camundongos , Nanopartículas/metabolismo , Técnicas de Cultura de Órgãos , Absorção Cutânea/efeitos dos fármacos , Neoplasias Cutâneas/metabolismo , Suínos , Inibidores da Topoisomerase I/administração & dosagem , Inibidores da Topoisomerase I/metabolismo , Topotecan/metabolismo , Resultado do TratamentoRESUMO
Topotecan is an important cytotoxic drug that has gained broad acceptance in clinical use for the treatment of refractory ovarian and small-cell lung cancer. The lactone active form of topotecan can be hydrolyzed in vivo, decreasing the drug's therapeutic efficacy. Lipid encapsulation may promote in vivo stabilization by removing topotecan from aqueous media. Earlier reports of topotecan lipid nanoencapsulation have focused on liposomal encapsulation; however, the higher stability and cost-effectiveness of solid lipid nanoparticles (SLN) highlight the potential of these nanoparticles as an advantageous carrier for topotecan. The initial motivation for this work was to develop, for the first time, solid lipid nanoparticles and nanostructured lipid carriers (NLC) with a high drug loading for topotecan. A microemulsion technique was employed to prepare SLNs and NLCs and produced homogeneous, small size, negatively charged lipid nanoparticles with high entrapment efficiency and satisfactory drug loading. However, low recovery of topotecan was observed when the microemulsion temperature was high and in order to obtain high quality nanoparticles, and precise control of the microemulsion temperature is critical. Nanoencapsulation sustained topotecan release and improved its chemical stability and cytotoxicity. Surprisingly, there were no significant differences between the NLCs and SLNs, and both are potential carriers for topotecan delivery.