Immunogenicity of PvCyRPA, PvCelTOS and Pvs25 chimeric recombinant protein of Plasmodium vivax in murine model.

In the Americas, P. vivax is the predominant causative species of malaria, a debilitating and economically significant disease. Due to the complexity of the malaria parasite life cycle, a vaccine formulation with multiple antigens expressed in various parasite stages may represent an effective approach. Based on this, we previously designed and constructed a chimeric recombinant protein, PvRMC-1, composed by PvCyRPA, PvCelTOS, and Pvs25 epitopes. This chimeric protein was strongly recognized by naturally acquired antibodies from exposed population in the Brazilian Amazon. However, there was no investigation about the induced immune response of PvRMC-1. Therefore, in this work, we evaluated the immunogenicity of this chimeric antigen formulated in three distinct adjuvants: Stimune, AddaVax or Aluminum hydroxide (Al(OH)3) in BALB/c mice. Our results suggested that the chimeric protein PvRMC-1 were capable to generate humoral and cellular responses across all three formulations. Antibodies recognized full-length PvRMC-1 and linear B-cell epitopes from PvCyRPA, PvCelTOS, and Pvs25 individually. Moreover, mice's splenocytes were activated, producing IFN-γ in response to PvCelTOS and PvCyRPA peptide epitopes, affirming T-cell epitopes in the antigen. While aluminum hydroxide showed notable cellular response, Stimune and Addavax induced a more comprehensive immune response, encompassing both cellular and humoral components. Thus, our findings indicate that PvRMC-1 would be a promising multistage vaccine candidate that could advance to further preclinical studies.

In silico bioavailability for BCS class II efavirenz tablets using biorelevant dissolution media for IVIVR and simulation of formulation changes.

OBJECTIVE: This work aims to evaluate the ability of biorelevant dissolution media to simulate the bioavailability of efavirenz tablets, establish an in vitro-in vivo relationship (IVIVR) based on in vivo data using GastroPlus® and simulate formulation changes using DDDPlus™. METHODS: Solubility and drug release profiles were conducted in SLS 0.5% and biorelevant media, such as FaSSIF, FeSSIF, FaSSIF-V2, and FeSSIF-V2. The efavirenz physicochemical properties were used to simulate the plasma concentration profile and compare the simulated pharmacokinetic parameters in fasted and fed states. An IVIVR was developed using Loo-Riegelman as the deconvolution method to estimate drug bioavailability. DDDPlus™ was used to perform virtual trials of formulations to evaluate whether formulations changes and the efavirenz particle size could influence the bioavailability. RESULTS: The drug dissolution displayed higher levels in the biorelevant media that simulated gut-fed state (FeSSIF and FeSSIF-V2). The absorption model successfully predicted the efavirenz pharmacokinetics, and FeSSIF-V2 was chosen as the predictive dissolution media, while an IVIVR was established using the Loo-Riegelman deconvolution method. CONCLUSIONS: The present work provides valuable information about efavirenz solubility and kinetics in the gastrointestinal tract, allowing an IVIVR to support future formulation changes. This understanding is essential for rational science-driven formulation development. At least, this study also showed the validity and applicability of in vitro and in silico tools in the regulatory scenario helping on drug development.

Targeting ROS overgeneration by N-benzyl-2-nitro-1-imidazole-acetamide as a potential therapeutic reposition approach for cancer therapy.

BackgroundWe investigated the role of reactive oxygen species (ROS) in the anticancer mechanism of N-benzyl-2-nitro-1-imidazole-acetamide (BZN), a drug used in Chagas' disease treatment. MethodsBALB/c mice, inoculated with Ehrlich ascites carcinoma (EAC), were treated with BZN or BZN + Nacylcysteine (NAC) or NAC for 9 days. Subsequently, the inhibition of tumor growth and angiogenesis as well as animal survival were evaluated. Apoptosis and the cell cycle were evaluated using fluorescence microscopy and flow cytometry, while oxidative stress was evaluated by measuring TBARS content, DNA damage, calcium influx and ROS generation and antioxidant defenses (CAT, SOD, GPx, GST and GR). Immunoblotting was used to evaluate key death and cell cycle proteins. Results BZN treatment inhibited tumor progression (79%), angiogenesis (2.8-fold) and increased animal survival (29%). Moreover, BZN increased ROS levels (42%), calcium influx (55%), TBARS contents (1.9-fold), SOD (4.4-fold), GPx (17.5-fold) and GST (3-fold) activities and GSH depletion (2.5-fold) also caused DNA fragmentation (7.6-fold), increased cleaved PARP and promoted the trapping of cells in the G1 phase, as corroborated by the reduction in cyclin A and increased CDK2 protein levels. In silico DNA and molecular dynamic simulations showed H-bonds and hydrophobic interactions that were confirmed by circular dichroism. Increased apoptosis (232%), induced by treatment with BZN, was demonstrated by apoptotic cell staining and p53 level. Conclusion The current findings indicate that BZN acts as a tumor growth inhibitor and anti-angiogenic agent by ROS overgeneration, which interact with DNA causing damage and triggering apoptosis.

Mapping Salmonella typhimurium pathways using 13C metabolic flux analysis.

In the last years, Salmonella has been extensively studied not only due to its importance as a pathogen, but also as a host to produce pharmaceutical compounds. However, the full exploitation of Salmonella as a platform for bioproduct delivery has been hampered by the lack of information about its metabolism. Genome-scale metabolic models can be valuable tools to delineate metabolic engineering strategies as long as they closely represent the actual metabolism of the target organism. In the present study, a 13C-MFA approach was applied to map the fluxes at the central carbon pathways of S. typhimurium LT2 growing at glucose-limited chemostat cultures. The experiments were carried out in a 2L bioreactor, using defined medium enriched with 20% 13C-labeled glucose. Metabolic flux distributions in central carbon pathways of S. typhimurium LT2 were estimated using OpenFLUX2 based on the labeling pattern of biomass protein hydrolysates together with biomass composition. The results suggested that pentose phosphate is used to catabolize glucose, with minor fluxes through glycolysis. In silico simulations, using Optflux and pFBA as simulation method, allowed to study the performance of the genome-scale metabolic model. In general, the accuracy of in silico simulations was improved by the superimposition of estimated intracellular fluxes to the existing genome-scale metabolic model, showing a better fitting to the experimental extracellular fluxes, whereas the intracellular fluxes of pentose phosphate and anaplerotic reactions were poorly described.

Drug Release from Inert Spherical Matrix Systems Using Monte Carlo Simulations.

BACKGROUND: Computational approaches for predicting release properties from matrix devices have recently been purposed as an approach to better understand and predict such systems. The objective of this research is to study the behavior of drug delivery from inert spherical matrix systems of different size by means of computer simulation. METHODS: To simulate the matrix medium, a simple cubic lattice was used, which was sectioned to make a spherical macroscopic system. The sites within the system were randomly occupied by drug-particles or excipient-particles in accordance with chosen drug/excipient ratios. Then, the drug was released from the matrix system simulating a diffusion process. RESULTS: When the released fraction was processed until 90% release, the Weibull equation suitably expressed the release profiles. On the basis of the analysis of release equations, it was found that close to the percolation threshold an anomalous released occurs, while in the systems with an initial drug load greater than 0.45, the released was Fickian type. It was also possible to determine the amount of drug trapped in the matrix, which was found to be a function of the initial drug load. The relationship between the two mentioned variables was adequately described by a model that involves the error function. Based on the these results and by means of a non-linear regression to the previous model, it was possible to determine the drug percolation threshold in these matrix devices. CONCLUSION: It was found that the percolation threshold is consistent with the value predicted by the percolation theory.