Poly-ε -caprolactone microspheres containing interferon alpha as alternative formulations for the treatment of chronic hepatitis C

Interferon-alpha (IFN-alpha) is one of the main drugs used in the treatment of hepatitis C. Use of IFN-alpha has some limitations that result in poor treatment efficacy and low patient compliance. Therefore, the aim of this study was to develop poly-ε-caprolactone (PCL) microspheres containing IFN-alpha as an alternative for the treatment of chronic hepatitis C. Microspheres were prepared using the multiple emulsion followed by solvent evaporation technique. Particle size, surface morphology, drug content and encapsulation efficiency of the microspheres produced were evaluated. The stability of the formulation was assessed after 90 days at -20ºC. An in vitro release study was performed in PBS. In vitro cytotoxicity of the formulation was studied using hepatic cell line. The freeze-dried microspheres had mean particle size, IFN-alpha content, and encapsulation efficiency of 38.52 ± 4.64 µm, 15.52 ± 3.28% and 83.93 ± 5.76%, respectively. There were no significant changes during storage and the structural integrity of the protein was not compromised by the preparation technique. A total of 82% of the IFN-alpha was released after 28 days and the developed microspheres did not present cytotoxicity to the hepatic cell line. In vivo studies are currently underway to evaluate the biological activity of IFN-alpha encapsulated into microspheres.

O interferon alfa (IFN-alfa) é um dos principais fármacos utilizados no tratamento de hepatite C, mas o seu uso apresenta limitações que resultam em baixa eficácia do tratamento e não adesão do paciente. Diante disso, este estudo objetiva o desenvolvimento de microesferas de poli-ε-caprolactona (PCL) contendo IFN-alfa como alternativa ao tratamento de hepatite C crônica. As microesferas foram preparadas pelo método de emulsão múltipla seguido de evaporação do solvente e caracterizadas quanto ao diâmetro médio das partículas, morfologia da superfície, taxa e eficiência de encapsulamento. A estabilidade da formulação foi acompanhada durante 90 dias a -20 ºC. O estudo de liberação in vitro foi realizado em PBS. A citotoxicidade da formulação foi avaliada utilizando linhagem de células hepáticas. As microesferas liofilizadas apresentaram diâmetro médio, taxa de encapsulamento e eficiência de encapsulamento de 38,52 ± 4,64 µm, 15,52 ± 3,28% e 83,93 ± 5,76%, respectivamente. Não foram observadas alterações significativas durante o armazenamento e a integridade estrutural da proteína foi mantida após o preparo. Oitenta e dois por cento de IFN-alfa foram liberados em 28 dias e a formulação desenvolvida não apresentou toxicidade para as células testadas. Estudos in vivo estão em andamento para avaliar a atividade biológica do IFN-alfa encapsulado nas microesferas.

IL-1beta, IL-6 and TNF-alpha in synovial fluid of patients with non-gonococcal septic arthritis.

Interleukin-1 beta (IL-1beta), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) are the main proinflammatory cytokines responsible for the inflammatory process and cartilage destruction of inflammatory arthropathies. The present study sequentially measured the concentrations of these cytokines and their proportions of detectable levels in the synovial fluid (SF) of 23 patients with non-gonococcal (GC) septic arthritis before and after treatment. Persistently high concentrations and proportions of IL-6 and TNF-alpha were found up to day 7 of treatment, while SF IL-1beta concentration declined significantly after day 7 (p = 0.036). SF IL-1beta and TNF-alpha correlated with each other significantly and with SF WBC counts (p < 0.01). Positive correlations between SF IL-1beta concentration and joint effusion (p < 0.01) and between SF TNF-alpha concentration and joint tenderness (p < 0.001) were observed. SF IL-1beta and TNF-alpha were significantly higher in patients with local complications of septic arthritis. In conclusion, high levels of IL-1beta, IL-6 and TNF-alpha were detected in SF of patients with non-GC septic arthritis. Only IL-1beta decreased significantly after day 7 of treatment, but IL-6 and TNF-alpha concentrations were persistently high. SF IL-1beta and TNF-alpha may be useful in predicting the outcome and complications of patients with this disease.

Cuantificación de interferón alfa 2b humano recombinante mediante anticuerpos monoclonales / Quantitation of recombinant human alpha 2b interferon with monoclonal antibodies

Se obtuvieron hibridomas secretores de anticuerpos monoclonales (AcM) contra el IFN alfa-2 humano recombinante (rh*IFN) mediante la hibridación de linfocitos esplénicos de ratones inmunizados y el mieloma P3/x63.Ag8.653. En el tamizaje de las células secretoras de anticuerpos específicos se emplearon sistemas microELISA, con el antígeno unido directamente a la fase sólida, o mediante otro AcM anti rh*IFN. Se seleccionaron nueve clones de hibridomas específicos para rh*IFN, de los cuales se escogió el secretor de AcM denominado CB-IFNA2.4 para los experimentos de desarrollo de un sistema ELISA tipo sandwich. Empleando el AcM CB-IFNA2.3 como anticuerpo de captura y el CB-IFNA2.4 conjugado con peroxidasa, se construyó un sistema ELISA sandwich capaz de detectar 1 ng de antígeno por milimetro. Se realizó un estudio de optimización de los diferentes pasos del ELISA y del reconocimiento del antígeno, cuando este es sometido a distintos tratamientos desnaturalizantes. Este sistema se ha empleado exitosamente en el seguimiento del proceso de producción y purificación del rh*IFN. En experimentos preliminares con ratas inoculadas con esta molecula, se denostró que este ELISA puede detectar su presencia en suero