Nanoparticles Formulation Improves the Antifibrogenic Effect of Quercetin on an Adenine-Induced Model of Chronic Kidney Disease.

Renal fibrosis is the final stage of chronic kidney injury characterized by glomerulosclerosis and tubulointerstitial fibrosis with parenchymal destruction. Quercetin belongs to the most studied flavonoids with antioxidant, anti-inflammatory, antifibrogenic, and antitumor activity. It modifies the TGF-ß/Smad signaling pathway, decreasing profibrogenic expression molecules and inducing the expression of antioxidant, anti-inflammatory, and antifibrogenic molecules. However, quercetin exhibits poor water solubility and low absorption and bioavailability. This limitation was solved by developing a nanoparticles formulation that improves the solubility and bioavailability of several bioactive compounds. Therefore, we aimed to investigate the in vivo antifibrogenic effect of a quercetin nanoparticles formulation. Male C57BL/6 mice were induced into chronic renal failure with 50 mg/kg of adenine for four weeks. The animals were randomly grouped and treated with 25, 50, or 100 mg/kg of quercetin, either macroparticles or nanoparticles formulation. We performed biochemical, histological, and molecular analyses to evaluate and compare the effect of macroparticles versus nanoparticles formulation on kidney damage. Here, we demonstrated that smaller doses of nanoparticles exhibited the same beneficial effect as larger doses of macroparticles on preventing kidney damage. This finding translates into less quercetin consumption reaching the desired therapeutic effect.

Gene therapy with urokinase-type plasminogen activator regenerates liver tissue.

BACKGROUND AND OBJECTIVES: Gene therapy using urokinase-type plasminogen activator (uPA) has been shown to induce extracellular matrix degradation, hepatocyte proliferation and liver tissue function restoration in liver cirrhosis models. Physiologically, uPA activates plasminogen conversion to plasmin, which leads, depending on the organ, to thrombolysis or extracellular matrix degradation. The purpose of this study was to compare the regenerative effect of gene therapy with adenoviruses encoding wild-type uPA (huPA), as well as its truncated isoform (ΔhuPA), in healthy and cirrhotic animals. In addition, possible adverse effects on coagulation were assessed. METHODS: 6 x 1011 vp/kg of Ad-huPA or Ad-ΔhuPA were administered via the iliac vein to healthy male Wistar rats or to male Wistar rats with cirrhosis induced by chronic poisoning with carbon tetrachloride (CCl4). The animals were sacrificed at day 2, 4 or 6 post-treatment. Liver fibrosis, proliferating cell nuclear antigen expression, alanine aminotransferase (ALT) and aspartate aminotransferase (AST) serum levels and coagulation markers were evaluated. RESULTS: On day 6 post-treatment, a fibrosis reversal of 48.7-41.5% was achieved. AST and ALT levels did not change in cirrhotic animals treated with ΔhuPA, but showed an elevation in healthy animals. Cell proliferation increased in healthy and cirrhotic animals with both transgene isoforms. No coagulation adverse effects were observed in the ΔhuPA group, and by day 6, they had disappeared in the huPA group. CONCLUSIONS: Gene therapy with Ad-huPA and Ad-ΔhuPA favors cell proliferation in cirrhotic animals, without important side effects.

ANTECEDENTES Y OBJETIVOS: La terapia génica empleando el activador de plasminógeno tipo urocinasa (huPA) ha demostrado que induce la degradación de matriz extracelular, la proliferación de hepatocitos y el restablecimiento de la funcionalidad tisular en modelos de cirrosis hepática. Fisiológicamente, el uPA activa la conversión del plasminógeno en plasmina, lo que conlleva, dependiendo del órgano, una trombólisis o a degradación de la matriz extracelular. El objetivo de este estudio fue comparar el efecto regenerador de la terapia génica con adenovirus codificando la forma silvestre (huPA), así como su isoforma truncada (DhuPA) en animales sanos y cirróticos. Además, se valoraron los posibles efectos adversos sobre la coagulación. MÉTODOS: Se administró 6x1011 pv/kg del Ad-huPA o Ad-ΔhuPA a ratas Wistar macho sanas o con cirrosis por intoxicación crónica con tetracloruro de carbono (CCl4) vía vena ilíaca. El sacrificio fue al día 2, 4 o 6 postratamiento. Se evaluó la fibrosis hepática, la expresión de antígeno nuclear de proliferación celular y niveles séricos de alanina aminotransferasa (ALT) y aspartato aminotransferasa (AST), y marcadores de coagulación. RESULTADOS: Al día 6 postratamiento se alcanzó una reversión de fibrosis del 48.7-41.5%. Los niveles de AST y ALT no cambiaron en animales cirróticos tratados con DhuPA, pero aumentaron en animales sanos comparados con el control sano no tratado. La proliferación aumenta en animales sanos y cirróticos con ambas isoformas del transgén. No se observaron efectos adversos en la coagulación en el grupo DhuPA y para el día 6 habían desaparecido en el grupo huPA. CONCLUSIONES: La terapia génica con Ad-uPA y Ad-ΔhuPA favorece la proliferación celular en animales cirróticos, sin efectos secundarios importantes.

Adenovirus Biodistribution is Modified in Sensitive Animals Compared to Naïve Animals.

Pre-existing immune response against adenovirus could diminish transgene expression efficiency when Ad is employed in humans as gene therapy vector. We previously used Ad-hΔuPA (Recombinant adenovirus expressing human urokinase-type plasminogen activator) as antifibrotic gene therapy in cirrhosis models and demonstrated its effectiveness. As a further clinical approach, transient Cyclosporine A (CsA) immunosuppression was induced in cirrhotic animals to determine whether Ad-hΔuPA administration retained efficacy. Adenovirus sensitization was achieved by systemic administration of non-therapeutic Ad-ßGal (Recombinant adenovirus expressing beta-galactosidase) after 4 weeks of intraperitoneal carbon tetrachloride (CCl4) regimen. Cirrhosis induction continued up to 8 weeks. At the end of CCl4 intoxication, immunosuppression was achieved with three CsA doses (40 mg/kg) as follows: 24 h before administration of Ad-hΔuPA, at the moment of Ad-hΔuPA injection and finally, 24 h after Ad-hΔuPA inoculation. At 2 and 72 h after Ad-hΔuPA injection, animals were sacrificed. Liver, spleen, lung, kidney, heart, brain, and testis were analyzed for Ad-biodistribution and transgene expression. In naïve animals, Ad-hΔuPA genomes prevailed in liver and spleen, while Ad-sensitized rats showed Ad genomes also in their kidney and heart. Cirrhosis and Ad preimmunization status notably diminished transgene liver expression compared to healthy livers. CsA immunosuppression in cirrhotic animals has no effect on Ad-hΔuPA biodistribution, but increments survival.

Treatment with pirfenidone for two years decreases fibrosis, cytokine levels and enhances CB2 gene expression in patients with chronic hepatitis C.

BACKGROUND: The aim of this study was to assess whether two-years treatment with Pirfenidone influences necroinflammation, fibrosis and steatosis, serum levels of TGF-ß1, IL-6, TNF-α and CB1 and CB2 gene expression, in patients with chronic hepatitis C (CHC). METHODS: Twenty-eight patients out of 34 with CHC virus infection were enrolled in the study and received Pirfenidone (1200 mg/day) for 24 months. Six patients dropped out after 12 months of PFD. Liver biopsies and serum samples were obtained at the beginning and end of treatment. Modified HAI was calculated. CB1 and CB2 gene expression was correlated with fibrosis progression alongside with necroinflammation and steatosis. TGF-ß1, IL-6, TNF-α and liver transaminases were measured in serum at two-months intervals. HCV genotype and viral load were also assessed. Quality of life was evaluated by SF36 questionnaires and the prognosis of disease was assessed with Child-Pugh score. The Wilcoxon test matched-pair signed ranks were used to analyze the outcomes. RESULTS: Intention to treat analyses were performed for biochemistry and clinical parameters. At the end of treatment, necroinflammation grading was reduced in an average of 3.2 points in 82% of patients (p < 0.05) and Ishak's fibrosis stage decreased 2-points average in 67% of patients (p < 0.05). Steatosis decreased in 61% of patients. IL-6 and TGF-ß1 serum levels decreased significantly in 93% and 67% of patients (p < 0.05), respectively, while TNF-α diminished in 47% of patients. ALT and AST tended to normalize in 81% of patients; CB2 mRNA levels increased in 86% and CB1 expression diminished in 29% of patients. Both, quality of life and Child-Pugh score improvements were reported in all patients. CONCLUSIONS: Pirfenidone for two years benefits CHC patients and improves inflammation, fibrosis and steatosis in higher number of patients as previously shown for 12-months treatment with PFD. Additionally, PFD improved TGFß1 and IL-6 levels and diminished liver expression of anti-fibrogenic receptor CB2. TRIAL REGISTRATION: ClinicalTrials.gov identifier: NCT02161952. Protocol Registration Date: 06/11/2014.

[Adeno-associated viral vectors: methods for production and purification for gene therapy applications]. / Vectores virales adeno-asociados: métodos de producción, purificación y aplicaciones en terapia génica.

Viral vectors based on adeno-associated virus (AAV) are widely used in gene therapy protocols, because they have characteristics that make them valuable for the treatment of genetic and chronic degenerative diseases. AAV2 serotype had been the best characterized to date. However, the AAV vectors developed from other serotypes is of special interest, since they have organ-specific tropism which increases their potential for transgene delivery to target cells for performing their therapeutic effects. This article summarizes AAV generalities, methods for their production and purification. It also discusses the use of these vectors in vitro, in vivo and their application in gene therapy clinical trials.