PAULO CESAR COTRIM
Projetos de Pesquisa
Unidades Organizacionais
Departamento de Moléstias Infecciosas e Parasitárias, Faculdade de Medicina - Docente
LIM/48 - Laboratório de Imunologia, Hospital das Clínicas, Faculdade de Medicina
LIM/48 - Laboratório de Imunologia, Hospital das Clínicas, Faculdade de Medicina
12 resultados
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- Hydroxymethylnitrofurazone lymphatic uptake with nanostructured lipid carrier after oral administration in rats(2024) SOUZA, Aline de; SCARIM, Caue Benito; COTRIM, Paulo Cesar; BARBOSA JUNIOR, Fernando; ROCHA, Bruno Alves; CALIXTO, Leandro Augusto; CORREIA, Cristiano Jesus; ARAUJO, Gabriel Lima de Barros; LOBENBERG, Raimar; BOU-CHACRA, Nadia Araci; BREITHAUPT-FALOPPA, Ana CristinaBackground: Leishmaniasis, caused by the protozoan Leishmania sp., infects phagocyte cells present in lymphatic organs. This study demonstrates the influence of nanostructured lipid carrier-loaded hydroxymethylnitrofurazone (NLC-NFOH) on lymphatic uptake using a chylomicron-blocking flow model in rats. Method: Lymphatic uptake of NFOH was assessed 1 h after oral administration of dimethyl sulfoxide with NFOH or NLC-NFOH with and without cycloheximide pretreatment. Result: Dimethyl sulfoxide with NFOH and NLC-NFOH showed NFOH serum concentrations of 0.0316 and 0.0291 mu g/ml, respectively. After chylomicron blocking, NFOH was not detected. Conclusion: Despite log P below 5, NFOH was successfully taken up by the lymphatic system. Long-chain fatty acids and particle size might be main factors in these findings. NLC-NFOH is a promising and convenient platform for treating leishmaniasis via oral administration.
- Biodegradable nanocarriers coated with polymyxin B: Evaluation of leishmanicidal and antibacterial potential(2019) COSTA, Juliana Souza Ribeiro; MEDEIROS, Marilia; YAMASHIRO-KANASHIRO, Edite Harumi; ROCHA, Mussya Cisotto; COTRIM, Paulo Cesar; STEPHANO, Marco Antonio; LANCELLOTTI, Marcelo; TAVARES, Guilherme Diniz; OLIVEIRA-NASCIMENTO, LauraMost treatments of leishmaniasis require hospitalization and present side effects or parasite resistance; innovations in drug formulation/reposition can overcome these barriers and must be pursued to increase therapeutic alternatives. Therefore, we tested polymyxin B (polB) potential to kill Leishmania amazonensis, adsorbed or not in PBCA nanoparticles (PBCAnp), which could augment polB internalization in infected macrophages. PBCAnps were fabricated by anionic polymerization and analyzed by Dynamic Light Scattering (size, potential), Nanoparticle Tracking Analysis (size/concentration), vertical diffusion cell (release rate), drug incorporation (indirect method, protein determination) and in vitro cell viability. Nanoparticles coated with polB (PBCAnp-polB) presented an adequate size of 261.5 25.9 nm, low PDI and of 1.79 +/- 0.17 mV (stable for 45 days, at least). The 50% drug release from PBCAnp-polB was 6-7 times slower than the free polB, which favors a prolonged and desired release profile. Concerning in vitro evaluations, polB alone reduced in vitro amastigote infection of macrophages (10 g/mL) without complete parasite elimination, even at higher concentrations. This behavior limits its future application to adjuvant leishmanicidal therapy or antimicrobial coating of carriers. The nanocarrier PBCAnp also presented leishmanicidal effect and surpassed polB activity; however, no antimicrobial activity was detected. PolB maintained its activity against E. coli, Pseudomonas and Klebsiella, adding antimicrobial properties to the nanoparticles. Thus, this coated drug delivery system, described for the first time, demonstrated antileishmanial and antimicrobial properties. The bactericidal feature helps with concomitant prevention/treatment of secondary infections that worst ulcers induced by cutaneous L. amazonensis, ultimately ending in disfiguring or disabling lesions. Author summary Cutaneous leishmaniasis form disfiguring lesions that leads to depression and social isolation. Both consequences result often in economic and education loss for communities already short on resources. The actual treatment aims to eliminate the protozoa disease-vector. Yet, it presents undesired effects or parasite resistance, besides the need for intravenous infusions and hospitalization. There is a few topical leishmanicidals but none with prolonged action. As highlighted, no ideal therapy exists; we must keep searching for alternatives, which includes new drugs, vehicles or therapies. For drugs, the most cost-effective way comprises testing approved molecules for other diseases, such as the antimicrobial polymyxin B (polB). Repositioning a drug has the added benefit of known safety profile and drawbacks, shortening and narrowing the research path. In turn, carriers can decrease side-effects and multiple dosing. They shield and/or improve drug targeting, which in this case means delivering inside macrophages infected with Leishmania. An example is poly (n-butyl cyanoacrylate) nanoparticles (PBCAnp), shown as an efficient carrier for targeting an extended drug release. Therefore, this work aimed to evaluate leishmanicidal and adjuvant properties of polB, alone and absorbed onto PBCAnp.
- Correlation between glucose uptake and membrane potential in Leishmania parasites isolated from DCL patients with therapeutic failure: a proof of concept(2014) PADRON-NIEVES, Maritza; MACHUCA, Claudia; DIAZ, Emilia; COTRIM, Paulo; RODRIGUEZ, Noris; PONTE-SUCRE, AliciaBesides infection with drug-resistant parasites, therapeutic failure in leishmaniasis may be caused by altered drug pharmacokinetics, re-infection, and host immunologic compromise. Our aim has been to evaluate if relapses that occur in patients suffering from diffuse cutaneous leishmaniasis (DCL) associate with changes in the fitness of infecting organisms. Therefore, in isolates from patients suffering DCL, we correlated glucose uptake and plasma membrane potential and compared the results with those obtained from reference strains. The data demonstrate that Leishmania parasites causing DCL incorporate glucose at an efficient rate, albeit without significant changes in the plasma membrane potential as their corresponding reference strains. The isolate that did not change its accumulation rate of glucose compared to its reference strain expressed a less polarized membrane potential that was insensitive to mitochondrial inhibitors, suggesting a metabolic dysfunction that may result in glycolysis being the main source of ATP. The results constitute a proof of concept that indicates that parasites causing DCL adapted well to drug pressure and expressed an increased fitness. That is, that in Leishmania mexicana and Leishmania amazonensis, parasites isolated from DCL patients, a strong modification of the parasite physiology might occur. As consequences, the parasites adapted well to drug pressure, increased their fitness, and they had an efficient glucose uptake rate albeit not significant changes in membrane potential as their corresponding reference strains. Further validation of the concepts herein established and whether or not the third isolate corresponds with a drug-resistant phenotype need to be demonstrated at the genetic level.
- APPLICABILITY OF kDNA-PCR FOR ROUTINE DIAGNOSIS OF AMERICAN TEGUMENTARY LEISHMANIASIS IN A TERTIARY REFERENCE HOSPITAL(2013) SATOW, Marcela M.; YAMASHIRO-KANASHIRO, Edite H.; ROCHA, Mussya C.; OYAFUSO, Luiza K.; SOLER, Rita C.; COTRIM, Paulo C.; LINDOSO, Jose Angelo L.This study evaluated the applicability of kDNA-PCR as a prospective routine diagnosis method for American tegumentary leishmaniasis (ATL) in patients from the Instituto de Infectologia Emolio Ribas (IIER), a reference center for infectious diseases in Sao Paulo - SP, Brazil. The kDNA-PCR method detected Leishmania DNA in 87.5% (112/128) of the clinically suspected ATL patients, while the traditional methods demonstrated the following percentages of positivity: 62.8% (49/78) for the Montenegro skin test, 61.8% (47/76) for direct investigation, and 19.3% (22/114) for in vitro culture. The molecular method was able to confirm the disease in samples considered negative or inconclusive by traditional laboratory methods, contributing to the final clinical diagnosis and therapy of ATL in this hospital. Thus, we strongly recommend the inclusion of kDNA-PCR amplification as an alternative diagnostic method for ATL, suggesting a new algorithm routine to be followed to help the diagnosis and treatment of ATL in IIER.
- Generation of Luciferase-Expressing Leishmania infantum chagasi and Assessment of Miltefosine Efficacy in Infected Hamsters through Bioimaging(2015) REIMAO, Juliana Q.; OLIVEIRA, Jordana C.; TRINCONI, Cristiana T.; COTRIM, Paulo C.; COELHO, Adriano C.; ULIANA, Silvia R. B.Background The only oral drug available for the treatment of leishmaniasis is miltefosine, described and approved for visceral leishmaniasis in India. Miltefosine is under evaluation for the treatment of cutaneous leishmaniasis in the Americas although its efficacy for the treatment of human visceral leishmaniasis caused by Leishmania infantum chagasi has not been described. Drug efficacy for visceral leishmaniasis is ideally tested in hamsters, an experimental model that mimics human disease. Luciferase has been validated as a quantitative tool for the determination of parasite burden in experimental leishmaniasis. However, there are no reports of luciferase detection in the model of progressive visceral leishmaniasis in hamsters. Therefore, the aims of this study were to generate recombinant Leishmania infantum chagasi expressing the luciferase gene (Lc-LUC), characterize the biological properties of this transgenic line as compared with the wild-type parasites and evaluate miltefosine effectiveness in Lc-LUC infected hamsters. Methodology/Principal Findings A transgenic line containing a luciferase encoding gene integrated into the ribosomal DNA locus was obtained and shown to produce bioluminescence which correlated with the number of parasites. Lc-LUC growth curves and susceptibility to pentavalent antimony and miltefosine in vitro were indistinguishable from the wild-type parasites. The effectiveness of pentavalent antimony was evaluated in Lc-LUC infected hamsters through bioimaging and determination of Leishman Donovan Units. Both methods showed concordant results. Miltefosine was effective in the treatment of Lc-LUC-infected hamsters, as demonstrated by the reduction in parasite burden in a dose-dependent manner and by prolongation of animal survival. Conclusions/Significance Luciferase expressing parasites are a reliable alternative for parasite burden quantification in hamsters with advantages such as the possibility of estimating parasite load before drug treatment and therefore allowing distribution of animals in groups with equivalent mean parasite burden. Miltefosine was effective in vivo in an L. infantum chagasi experimental model of infection.
- Targeting Leishmania amazonensis amastigotes through macrophage internalisation of a hydroxymethylnitrofurazone nanostructured polymeric system(2017) MONTEIRO, Lis Marie; LOBENBERG, Raimar; FERREIRA, Elizabeth Igne; COTRIM, Paulo Cesar; KANASHIRO, Edite; ROCHA, Mussya; CHUNG, Man Chin; BOU-CHACRA, NadiaDextran-coated poly (n-butyl cyanoacrylate) nanoparticles (PBCA-NPs) were prepared and were evaluated for enhanced delivery of a promising anti-Leishmania drug candidate, hydroxymethylnitrofurazone (NFOH), to phagocytic cells. Currently available chemotherapy for leishmaniasis, such as pentavalent antimonials, presents low safety and efficacy. Furthermore, widespread drug resistance in leishmaniasis is rapidly emerging. To overcome these drawbacks, the use of nanosized delivery systems can reduce systemic drug toxicity and increase the drug concentration in infected macrophages, therefore improving treatment of leishmaniasis. PBCA-NPs containing NFOH (PBCA-NFOH-NPs) were prepared by an anionic emulsion polymerisation method. The z-average and polydispersity index (PDI) were determined by photon correlation spectroscopy, the zeta potential by microelectrophoresis and the entrapment efficiency by HPLC. Cytotoxicity was determined using macrophages from BALB/c mice. Efficacy tests were performed using Leishmania amazonensis promastigotes and amastigotes. The z-average of PBCA-NFOH-NPs was 151.5 +/- 61.97 nm, with a PDI of 0.104 +/- 0.01, a zeta potential of -10.1 +/- 6.49 mV and an entrapment efficiency of 64.47 +/- 0.43%. Efficacy in amastigotes revealed IC50 values of 0.33 mu M and 31.2 mu M for the nanostructured and free NFOH, respectively (95-fold increase). The cytotoxicity study indicated low toxicity of the PBCA-NFOH-NPs to macrophages. The selectivity index was 370.6, which is 49-fold higher than free NFOH (7.6). Such findings indicated that improved efficacy could be due to NP internalisation following site-specific drug delivery and reactivation of immune protective reactions by the NP components. Thus, PBCA-NFOH-NPs have the potential to significantly improve the treatment of leishmaniasis, with reduced systemic side effects.
- The role of ABC transporters in drug-resistant Leishmania(2018) COELHO, A. C.; COTRIM, P. C.The ATP-binding cassette (ABC) transporters belong to the largest family of transmembrane proteins found in living organisms. These proteins are present in prokaryotes and eukaryotes and are mainly involved in the transport of a variety of molecules across cellular membranes, whereas others are involved in biological processes unrelated to transport. The genome sequencing of several Leishmania species confirmed the presence of members for all eight different subfamilies of ABC transporters (ABCA to ABCH), according to their specific functional and molecular characteristics. These proteins have recently been characterized in Leishmania; some of them associated with drug resistance, which is a significant field in leishmaniasis chemotherapy, a disease still lacking effective treatment, with increasing daily reports of therapeutic failure. In this chapter, we focus our discussion on the association of these proteins with drug resistance in leishmaniasis and its fundamental role in the pathology and pharmacology of this medically important protozoan parasite that currently infects around 12 million people in the world. © Springer International Publishing AG, part of Springer Nature 2018.
- Isolation, typing, and drug susceptibility of Leishmania (Leishmania) infantum isolates from dogs of the municipality of Embu das Artes, an endemic region for canine leishmaniasis in Brazil(2022) FERREIRA, Bianca A.; MARTINS, Thaynan F. C.; COSER, Elizabeth M.; OLIVEIRA, Viviane da L.; YAMASHIRO-KANASHIRO, Edite H.; ROCHA, Mussya C.; PINTO, Marcelo M.; COTRIM, Paulo C.; COELHO, Adriano C.The parasitic protozoa Leishmania (Leishmania) infantum is the etiological agent of human visceral leishmaniasis and canine leishmaniasis in South America, where Brazil is the most affected country. This zoonotic disease is transmitted by the bite of an infected phlebotomine sand fly and dogs constitute the main domestic reservoir of the parasite. In this study, we screened 2348 dogs of the municipality of Embu das Artes, Brazil, for antibodies against the parasite. Prevalence for canine leishmaniasis seropositivity was 2.81%, as assessed using a Dual-Path Platform rapid test for canine leishmaniasis. Twenty-five seropositive dogs were euthanized for parasite isolation and 14 isolates were successful obtained. Nucleotide sequencing of the internal transcribed spacer confirmed the isolates to be L. (L.) infantum, and very low sequence variability was observed among them. The in vitro susceptibility to miltefosine and paromomycin was assessed and moderate variation in paromomycin susceptibility was found among the isolates in the promastigote and intracellular amastigote stages. On the other hand, in vitro susceptibility to miltefosine of these isolates was homogenous, particularly in the amastigote stage (EC50 values from 0.69 to 2.07 mu M). In addition, the miltefosine sensitivity locus was deleted in all the isolates, which does not corroborate the hypothesis that the absence of this locus is correlated with a low in vitro susceptibility. Our findings confirm that the municipality of Embu das Artes is endemic for canine leishmaniasis and that isolates from this region are susceptible to paromomycin and miltefosine, indicating the potential of these drugs to be clinically evaluated in the treatment of human visceral leishmaniasis in Brazil.
- The role of ABC transporters in drug-resistant leishmania(2013) COELHO, A. C.; COTRIM, P. C.The ATP-binding cassette (ABC) transporters belong to the largest family of transmembrane proteins found in living organisms. These proteins are present in prokaryotes and eukaryotes and are mainly involved in the transport of a variety of molecules across cellular membranes, whereas others are involved in biological processes unrelated to transport. The recent sequencing of the Leishmania species genome confirmed the presence of representatives, also called as members, for all eight different subfamilies (ABCA to ABCH), according to their specific functional and molecular characteristics. These proteins have recently been characterized in Leishmania; some of them associated with drug resistance. This is a significant field in leishmaniasis chemotherapy, a disease still lacking effective treatment and with increasing daily reports of therapeutic failure. In this chapter, we focus our discussion on the association of these proteins with drug resistance in leishmaniasisand its fundamental role in the pathology and pharmacology of this medically important protozoan parasite that currently infects around 12 million people in the world. © Springer-Verlag Wien 2013.
- Buparvaquone Nanostructured Lipid Carrier: Development of an Affordable Delivery System for the Treatment of Leishmaniases(2017) MONTEIRO, Lis Marie; LOBENBERG, Raimar; COTRIM, Paulo Cesar; ARAUJO, Gabriel Lima Barros de; BOU-CHACRA, NadiaBuparvaquone (BPQ), a veterinary drug, was formulated as nanostructured lipid carriers (NLC) for leishmaniases treatment. The formulation design addressed poor water solubility of BPQ and lack of human drug delivery system. The DSC/TG and microscopy methods were used for solid lipids screening. Softisan (R) 154 showed highest BPQ solubility in both methods. The BPQ solubility in liquid lipids using HPLC revealed Miglyol (R) 812 as the best option. Response surface methodology (RSM) was used to identify the optimal Softisan154 : Miglyol 812 ratios (7 : 10 to 2 : 1) and Kolliphor (R) P188 and Tween (R) 80 concentration (> 3.0% w/w) aiming for z-average in the range of 100-300 nm for macrophage delivery. The NLC obtained by high-pressure homogenization showed low z-averages (< 350 nm), polydispersity (< 0.3), and encapsulation efficiency close to 100%. DSC/ TG and microscopy in combination proved to be a powerful tool to select the solid lipid. The relationship among the variables, demonstrated by a linear mathematical model using RSM, allowed generating a design space. This design space showed the limits in which changes in the variables influenced the z-average. Therefore, these drug delivery systems have the potential to improve the availability of affordable medicines due to the low cost of raw materials, using well established, reliable, and feasible scale-up technology.