Stability-indicative and conformation-specific enzyme linked immunosorbent assay for analysis of recombinant human gamma interferon.

Recombinant human interferon gamma (rhIFN-γ) is a promising molecule for the treatment of several diseases. A pair of conformation-specific monoclonal antibodies (mAbs) against rhIFN-γ was selected from generated hybridoma cell lines to design a sensitive, stability-indicative, sandwich-type ELISA. The main assay parameters were optimized by the checkerboard method for the highest signal-to-noise ratio: assay buffer composition, coating buffer pH and composition, coating temperature-incubation time parameters, and coating mAb concentration and conjugate dilution. Detection and quantification limits were estimated between 0.019 and 0.078 ng/mL, respectively, and recovery values were from 92.03% to 98.40%. The coefficient of variation of intra-assay precision parameters ranged from 2.32% to 9.21% while the inter-analyst variation was between 4.70% and 10.63%, supporting the method's repeatability. The ELISA was specific for correctly folded and non-aggregated molecular species, as compared to intrinsic Trp fluorescence (chemical denaturation) and optical density at 340 nm (thermal aggregation), respectively. However, the method was not sensitive to the small C-terminal degradation of full-length rhIFN-γ1-144 (losses of 6-12 amino acid residues) as compared to results with mass spectrometry and gel electrophoresis. ELISA showed good correlation with rhIFN-γ antiviral biological activity. This method was applied to the stability evaluation of rhIFN-γ in physiological buffer at low concentrations using polypropylene and glass vials also in the presence of adsorption protectant excipients. Furthermore, ELISA could be adapted to other applications such as quantification of IFN-γ in serum samples, Mycobacterium tuberculosis diagnosis, etc.

Actualización sobre el síndrome de dispersión pigmentaria y el glaucoma pigmentario / Updating of pigmentary dispersion syndrome and pigmentary glaucoma

El síndrome de dispersión pigmentaria se da por la liberación de pigmento del epitelio pigmentado del iris y depósito de gránulos de este en el iris y en las estructuras del segmento anterior, incluyendo el endotelio corneal y la malla trabecular, con posterior aumento de la presión intraocular y glaucoma. El glaucoma pigmentario (GP) es un glaucoma de ángulo abierto, secundario a un síndrome de dispersión pigmentaria, que consiste en el acúmulo de pigmento a nivel de la malla trabecular, aumentando la resistencia a la salida del humor acuoso, ocasionando así un deterioro progresivo e irreversible de las estructuras oculares, llevando a la pérdida visual. El glaucoma pigmentario es el más frecuente de los glaucomas secundarios. Se presenta en pacientes jóvenes durante la tercera o cuarta década de la vida y el diagnóstico se realiza en una etapa de daño glaucomatoso avanzado, por lo que genera un gran impacto social. El propósito de este artículo es revisar la fisiopatología, clínica, tratamiento del GP y proporcionarles recomendaciones según lo estudiado (AU)

Pigmentary dispersion syndrome occurs by the release of pigment and iris pigmented epithelium granules in the iris and the anterior segment structures including corneal endothelium and trabecular meshwork, with a subsequent increase of intraocular pressure and glaucoma. The pigmentary glaucoma is classified as secondary open angle glaucoma. Where the cause is a pigment dispersion syndrome, which consists of the heavy pigmentation at the level of the trabecular meshwork. The pigment increases the resistance of aqueous outflow causing a progressive and irreversible deterioration of the ocular structures contributing to visual loss. Pigmentary glaucoma is the most frequent of secondary glaucoma. It occurs in young patients in the third or fourth decade of life and the diagnosis is made in an advanced stage of glaucomatous damage and a great social impact. The purpose of this article is to revise the pathophysiology, treatment and to express recommendations based on this study (AU)

Stabilization of a recombinant human epidermal growth factor parenteral formulation through freeze-drying.

Development studies were performed to design a pharmaceutical composition that allows the stabilization of a parenteral rhEGF formulation in a lyophilized dosage form. Unannealed and annealed drying protocols were tested for excipients screening. Freeze-dry microscopy was used as criterion for excipients and formulation selection; as well as to define freeze-drying parameters. Excipients screening were evaluated through their effect on freeze-drying recovery and dried product stability at 50 °C by using a comprehensive set of analytical techniques assessing the chemical stability, protein conformation and bioactivity. The highest stability of rhEGF during freeze-drying was achieved by the addition of sucrose or trehalose. After storing the dried product at 50 °C, the highest stability was achieved by the addition of dextran, sucrose, trehalose or raffinose. The selected formulation mixture of sucrose and dextran could prevent protein degradation during the freeze-drying and delivery processes. The degradation rate assessed by RP-HPLC could decrease 100 times at 37 °C and 70 times at 50 °C in dried with respect to aqueous formulation. These results indicate that the freeze-dried formulation represents an appropriate technical solution for stabilizing rhEGF.

Screening for stability and compatibility conditions of recombinant human epidermal growth factor for parenteral formulation: effect of pH, buffers, and excipients.

A successful parenteral formulation can be developed by studying stability and compatibility of biopharmaceuticals as a function of solution composition. Here, we evaluate the influence of pH, buffers, ionic strength, protein concentration and presence of excipients on recombinant human epidermal growth factor (rhEGF) stability. The stability was accessed by reversed-phase high performance liquid chromatography (RP-HPLC), size exclusion chromatography (SEC-HPLC), enzyme-linked immunosorbent assay (ELISA), Far-UV circular dichroism (CD) and light scattering. The overall maximal stability was obtained in pH near to 7.0 in phosphate, Tris and histidine buffers as the results of the different methods revealed. The CD results revealed that this protein is stable in an extensive pH range. Aggregation of rhEGF was minimized at pH values ranged from 6.0 to 8.0 as indicated the SEC-HPLC and light scattering results. Nor the ionic strength neither the rhEGF concentration had significant effect on the reaction rate constants. Most rhEGF-excipient instability occurs among this protein and reducing sugars. Polymers like poly(ethylene glycol) (PEG) and polysorbates increased methionine oxidation. The rhEGF oxidation and deamidation were the most common degradation pathways. This research identified critical solution factors to be considered for the development of a successful rhEGF parenteral formulation.

Síndrome de dispersión pigmentaria y Glaucoma Pigmentario / Pigmentary dispersion syndrome and pigmentary Glaucoma

Se realizó una revisión del síndrome de dispersión pigmentaria y glaucoma pigmentario. Se presentaron los argumentos que proponen diferentes investigadores para apoyar que la presencia del pigmento es la causa del aumento de la presión intraocular, así como las teorías que abogan sobre la lesión de las células endoteliales trabeculares, el factor productor del glaucoma. Se hace referencia a los estudios genéticos con relación a la determinación de la presencia clínica de este síndrome, los posibles mecanismos de aumento de la presión con el ejercicio y el cuadro clínico y la evolución natural de la enfermedad; además de un análisis de los métodos de tratamiento médico y quirúrgico(AU)

A review of the Pigmentary Dispersion Syndrome and Pigmentary Glaucoma was done. The arguments proposed by different researchers to support that the pigment presence is the main cause of intraocular pressure, as well as the theory that accept trabeculars endothelials cells as the factor of the glaucoma field. References to the genetics studies with relation to the determination of the clinical presence of this syndrome are made, along with the possible mechanisms of pressure increase with the practice of blinking, the clinical picture and the natural course of the disease; there is also an analysis of the surgical and medical treatments(AU)

Antioxidant mechanism is involved in the gastroprotective effects of ozonized sunflower oil in ethanol-induced ulcers in rats.

This research was performed in order to determine the potential protective effects of ozonized sunflower oil (OSO) in the injury of rat gastric mucosa induced by absolute ethanol and as well as to elucidate the role of reactive oxygen species (ROS), lipid peroxidation, and some important constituents of antioxidant defense such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in these effects. OSO was administered to rats intragastrically by a cannula and it was applied during four days to animals. The doses of OSO administered daily to each group of rats were 4, 12, and 24 mg/kg, respectively, and one hour after the last treatment, absolute ethanol (1 mL/200 mg body weight) was administered. Our results showed that gastric ulcer index was significantly reduced in rats pretreated with OSO as compared with ethanol-treated controls. However, in rats pretreated with OSO, no significant reduction of TBARS content in gastric mucosa was found as compared to those rats treated with ethanol alone. In contrast, SOD and GSH-Px activities were significantly increased in gastric mucosa of OSO-pretreated rats with respect to those treated with ethanol alone. In summary, our results demonstrate that OSO pretreatment exerts protective effects in ethanol-induced gastric ulcers in rats. Furthermore, these results provide evidence that these protective effects of OSO are mediated at least partially by stimulation of some important antioxidant enzymes such as SOD and GSH-Px, which are scavengers of ROS and therefore prevent gastric injury induced by them.

Antioxidant Mechanism is Involved in the Gastroprotective Effects of Ozonized Sunflower Oil in Ethanol-Induced Ulcers in Rats

This research was performed in order to determine the potential protective effects of ozonized sunflower oil (OSO) in the injury of rat gastric mucosa induced by absolute ethanol and as well as to elucidate the role of reactive oxygen species (ROS), lipid peroxidation, and some important constituents of antioxidant defense such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT) in these effects. OSO was administered to rats intragastrically by a cannula and it was applied during four days to animals. The doses of OSO administered daily to each group of rats were 4, 12, and 24 mg/kg, respectively, and one hour after the last treatment, absolute ethanol (1 mL/200 mg body weight) was administered. Our results showed that gastric ulcer index was significantly reduced in rats pretreated with OSO as compared with ethanol-treated controls. However, in rats pretreated with OSO, no significant reduction of TBARS content in gastric mucosa was found as compared to those rats treated with ethanol alone. In contrast, SOD and GSH-Px activities were significantly increased in gastric mucosa of OSO-pretreated rats with respect to those treated with ethanol alone. In summary, our results demonstrate that OSO pretreatment exerts protective effects in ethanol-induced gastric ulcers in rats. Furthermore, these results provide evidence that these protective effects of OSO are mediated at least partially by stimulation of some important antioxidant enzymes such as SOD and GSH-Px, which are scavengers of ROS and therefore prevent gastric injury induced by them(AU)