Three-Dimensional Oral Mucosal Equivalents as Models for Transmucosal Drug Permeation Studies.

Oral transmucosal administration, where drugs are absorbed directly through the non-keratinized, lining mucosa of the mouth, represents a solution to drug delivery with several advantages. Oral mucosal equivalents (OME) developed as 3D in vitro models are of great interest since they express the correct cell differentiation and tissue architecture, simulating the in vivo conditions better than monolayer cultures or animal tissues. The aim of this work was to develop OME to be used as a membrane for drug permeation studies. We developed both full-thickness (i.e., connective plus epithelial tissue) and split-thickness (i.e., only epithelial tissue) OME using non-tumor-derived human keratinocytes OKF6 TERT-2 obtained from the floor of the mouth. All the OME developed here presented similar transepithelial electrical resistance (TEER) values, comparable to the commercial EpiOral™. Using eletriptan hydrobromide as a model drug, we found that the full-thickness OME had similar drug flux to EpiOral™ (28.8 vs. 29.6 µg/cm2/h), suggesting that the model had the same permeation barrier properties. Furthermore, full-thickness OME showed an increase in ceramide content together with a decrease in phospholipids in comparison to the monolayer culture, indicating that lipid differentiation occurred due to the tissue-engineering protocols. The split-thickness mucosal model resulted in 4-5 cell layers with basal cells still undergoing mitosis. The optimum period at the air-liquid interface for this model was twenty-one days; after longer times, signs of apoptosis appeared. Following the 3R principles, we found that the addition of Ca2+, retinoic acid, linoleic acid, epidermal growth factor and bovine pituitary extract was important but not sufficient to fully replace the fetal bovine serum. Finally, the OME models presented here offer a longer shelf-life than the pre-existing models, which paves the way for the further investigation of broader pharmaceutical applications (i.e., long-term drug exposure, effect on the keratinocytes' differentiation and inflammatory conditions, etc.).

New concepts for transdermal delivery of oxygen based on catalase biochemical reactions studied by oxygen electrode amperometry.

The development of formulation concepts for improved skin tissue oxygenation, including methods for measuring oxygen (O2) transport across biological barriers, are important research topics with respect to all processes that are affected by the O2 concentration, such as radiation therapy in oncology treatments, wound healing, and the general health status of skin. In this work we approach this topic by a novel strategy based on the antioxidative enzyme catalase, which is naturally present in the skin organ where it enables conversion of the reactive oxygen species hydrogen peroxide (H2O2) into O2. We introduce various applications of the skin covered oxygen electrode (SCOE) as an in-vitro tool for studies of catalase activity and function. The SCOE is constructed by placing an excised skin membrane directly on an O2 electrode and the methodology is based on measurements of the electrical current generated by reduction of O2 as a function of time (i.e. chronoamperometry). The results confirm that a high amount of native catalase is present in the skin organ, even in the outermost stratum corneum (SC) barrier, and we conclude that excised pig skin (irrespective of freeze-thaw treatment) represents a valid model for ex vivo human skin for studying catalase function by the SCOE setup. The activity of native catalase in skin is sufficient to generate considerable amounts of O2 by conversion from H2O2 and proof-of-concept is presented for catalase-based transdermal O2 delivery from topical formulations containing H2O2. In addition, we show that this concept can be further improved by topical application of external catalase on the skin surface, which enables transdermal O2 delivery from 50 times lower concentrations of H2O2. These important results are promising for development of novel topical or transdermal formulations containing low and safe concentrations of H2O2 for skin tissue oxygenation. Further, our results indicate that the O2 production by catalase, derived from topically applied S. epidermidis (a simple model for skin microbiota) is relatively low as compared to the O2 produced by the catalase naturally present in skin. Still, the catalase activity derived from S. epidermidis is measurable. Taken together, this work illustrates the benefits and versatility of the SCOE as an in vitro skin research tool and introduces new and promising strategies for transdermal oxygen delivery, with simultaneous detoxification of H2O2, based on native or topically applied catalase.

The Effect of UVB Irradiation and Oxidative Stress on the Skin Barrier-A New Method to Evaluate Sun Protection Factor Based on Electrical Impedance Spectroscopy.

Sunlight is vital for several biochemical processes of the skin organ. However, acute or chronic exposure to ultraviolet radiation (UVR) has several harmful effects on the skin structure and function, especially in the case of the failing function of antioxidative enzymes, which may lead to substantial tissue damage due to the increased presence of reactive oxygen species (ROS). The aim of this work was to investigate the combined effect of ultraviolet B (UVB) irradiation and oxidative stress on the skin barrier integrity. For this, we employed electrical impedance spectroscopy (EIS) to characterize changes of the electrical properties of excised pig skin membranes after various exposure conditions of UVB irradiation, oxidative stress, and the inhibition of antioxidative enzymatic processes. The oxidative stress was regulated by adding hydrogen peroxide (H2O2) as a source of ROS, while sodium azide (NaN3) was used as an inhibitor of the antioxidative enzyme catalase, which is naturally present throughout the epidermis. By screening for the combined effect of UVB and oxidative stress on the skin membrane electrical properties, we developed a new protocol for evaluating these parameters in a simple in vitro setup. Strikingly, the results show that exposure to extreme UVB irradiation does not affect the skin membrane resistance, implying that the skin barrier remains macroscopically intact. Likewise, exposure to only oxidative stress conditions, without UVB irradiation, does not affect the skin membrane resistance. In contrast to these observations, the combination of UVB irradiation and oxidative stress conditions results in a drastic decrease of the skin membrane resistance, indicating that the integrity of the skin barrier is compromised. Further, the skin membrane effective capacitance remained more or less unaffected by UVB exposure, irrespective of simultaneous exposure of oxidative stress. The EIS results were concluded to be associated with clear signs of macroscopic tissue damage of the epidermis as visualized with microscopy after exposure to UVB irradiation under oxidative stress conditions. Finally, the novel methodology was tested by performing an assessment of cosmetic sunscreen formulations with varying sun protection factor (SPF), with an overall successful outcome, showing good correlation between SPF value and protection capacity in terms of skin resistance change. The results from this study allow for the development of new skin sensors based on EIS for the detection of skin tissue damage from exposure to UVB irradiation and oxidative stress and provide a new, more comprehensive methodology, taking into account both the influence of UVB irradiation and oxidative stress, for in vitro determination of SPF in cosmetic formulations.

In vitro intestinal permeability studies, pharmacokinetics and tissue distribution of 6-methylcoumarin after oral and intraperitoneal administration in Wistar rats

ABSTRACT 6-Methylcoumarin (6MC) is a semisynthetic coumarin with important in vitro and in vivo anti-inflammatory activity. In order to continue the pre-clinical characterization of this molecule, in vitro intestinal permeability, plasma profile and tissue distribution after oral administration in rats were studied. The permeability of 6MC was evaluated by the Caco-2 cellular model in both the apical-basal (A-B) and basal-apical (B-A) directions. The pharmacokinetics and biodistribution were evaluated in rats after oral and intraperitoneal administration at doses of 200 mg/kg. Transport experiments with Caco-2 cells showed that 6MC presented high permeability at all concentrations evaluated. This finding suggested that 6MC could be transported across the gut wall by passive diffusion. The plasma concentration-time curve showed that the maximum concentration (Cmax) was 17.13 ± 2.90 µg/mL at maximum time (Tmax) of 30 min for the oral route and Cmax 26.18 ± 2.47 µg/mL at 6.0 min for the intraperitoneal administration, with elimination constant of (Ke ) 0.0070 min-1 and a short life half time of (T1/2 ) lower that 120 min. The distribution study showed that 6MC has high accumulation in the liver, and widespread distribution in all the organs evaluated.

Biodistribution study of free and microencapsulated 6-methylcoumarin in Wistar rats by HPLC.

A sensitive, specific and reproducible HPLC method has been developed and validated for the quantitative determination of 6-methylcoumarin (6MC) in plasma and other tissues in Wistar rats. A C18 column was used with UV detection at 321 nm and a gradient system consisting of methanol-deionized water was used as mobile phase. The retention time for 6MC was 14.921 min and no interfering peaks were observed for any of the matrices. Linear relationships (r(2) > 0.997) were obtained between the peak height ratios and the corresponding biological sample concentrations over the range 0.4-12.8 µg/mL. Precision and accuracy were evaluated; the coefficient of variation and the relative error for all of the organs were <2 and 7%, respectively. The limit of quantitation was 0.20 µg/mL for the heart and 0.30 µg/mL for the other tissues evaluated. This HPLC method was successfully used in the determination of 6MC in the biodistribution study after administration of 200 mg/kg of both 6MC-free and 6MC-loaded polymeric microparticles. In this study, extensive 6MC was found, in both free and microencapsulated forms, in all the organs tested. The 6MC-free showed a range of between 1.7 and 11.5 µg/g, while the microencapsulated 6MC showed concentrations of between 6.35 and 17.7 µg/g, suggesting that 6MC improved absorption rate.

Metástasis óseas: en adenocarcinoma de mama / Osseo's metastases: in adenocarcinoma of the breast

La metástasis ósea sigue siendo una de las principales complicaciones del cáncer de mama durante el seguimiento de las pacientes. El objetivo de esta serie fue determinar la prevalencia de las metástasis óseas en pacientes diagnosticadas con adenocarcinoma de mama en nuestra institución, su número y localización, y el tratamiento empleado para su manejo y la sobrevida global de este grupo. Se realizó un estudio retrospectivo en pacientes con diagnóstico de adenocarcinoma de mama y metástasis óseas durante diez años, en el lapso comprendido entre 1996-2005, mediante la revisión de los expedientes clínicos de 1610 pacientes femeninas. Se encontraron 200 casos con metástasis óseas, diagnosticadas en 90 por ciento mediante gammagrama óseo. Las edades oscilaron entre 41-50 años. La enfermedad metastásica ósea fue múltiple en el 55 por ciento de los casos. A todas las pacientes se les aplicó radioterapia. La localización predominante de la metástasis fue en columna vertebral en un 33 por ciento. La sobrevida global fue de un año. La metástasis ósea en cáncer de mama es una expresión clínica frecuente con el subsiguiente mal pronóstico, corroborado al comparar nuestros resultados con los reportados por la literatura.

Bone metastasis still represents one of the main complications of breast cancer during the follow up of these patients. The objective of the current series was to determine the prevalence of bone metastasis in patients diagnosed of breast adenocarcinoma at our center, the location and number of lesions, treatment selected and the overall patient survival. A retrospective study of a group of female patients with a diagnosis of breast adenocarcinoma and bone metastasis was carried out during a ten year period, between the years 1996 and 2005. Medical charts of 1610 women were analyzed. We found 200 cases with bone metastases, 90 per cent were diagnosed by bone scans. The ages of these patients ranged between 41 and 50 years. Metastatic bone disease was multiple in 55 per cent of cases. All the patients received radiotherapy treatment. The main location of the bone lesions was the spine, accounting for 33 per cent of all cases. Overall survival was one year in this group. Bone metastasis is a frequent clinical finding in the setting of breast adenocarcinoma. Prognosis is dismal and it is corroborated when we compare our results to those reported in the literature.

Statistical strategy for anisotropic adventitia modelling in IVUS.

Vessel plaque assessment by analysis of intravascular ultrasound sequences is a useful tool for cardiac disease diagnosis and intervention. Manual detection of luminal (inner) and media-adventitia (external) vessel borders is the main activity of physicians in the process of lumen narrowing (plaque) quantification. Difficult definition of vessel border descriptors, as well as, shades, artifacts, and blurred signal response due to ultrasound physical properties trouble automated adventitia segmentation. In order to efficiently approach such a complex problem, we propose blending advanced anisotropic filtering operators and statistical classification techniques into a vessel border modelling strategy. Our systematic statistical analysis shows that the reported adventitia detection achieves an accuracy in the range of interobserver variability regardless of plaque nature, vessel geometry, and incomplete vessel borders.

Tratamiento actual de los sarcomas del utero / Treatment of uterine sarcomas

Entre 1969 y 1993 fueron atendidos en nuestro centro 36 pacientes con el diagnóstico de Sarcoma Uterino. De estos, sólo fueron evaluables 32 pacientes. La tasa de sobrevida global a los cincos años, fue del 18,75 por ciento. La tasa de sobrevida por estadios fue para el Estadio I: 50,00 por ciento, Estadio II: 26,27 por ciento, Estadio III: 14,29 por ciento, y para el Estadio IV: 0 por ciento. Sólo se realizo el diagnóstico de la enfermedad, en forma pre-operatoria, en el 31,35 por ciento de los pacientes. El tratamiento inicial fue quirúrgico en el 84,38 por ciento de los pacientes, y consistió en la histerectomía total con oforosalpingectomía bilateral con el resección parcial o total de epiplón mayor. En algunos casos de cirugía fue más extensa. La radioterapia postoperatotia disminuyó la tasa de recidiva pélvica del 34,76 por ciento a 0 por ciento. La quimioterapia postoperatoria no disminuyó la incidencia de metástasis a distancia, las cuales ocurrieron en el 66,67 por ciento de las pacientes. Los factores pronósticos más importantes fueron el estadio de la enfermedad, el volumen tumoral, el estado menstrual de la paciente y el número de mitosis

Gastropatía por Helicobacter Pylori en pacientes pediátricos / Gastropaty by helicobacter pylori in pediatric patients

Se estudiaron veintinueve (29) casos de pacientes pediátricos, que fueron llevados al Servicio de Gastroenterología del Hospital General del Oeste por presentar diversos signos y síntomas del aparato digestivo, siendo el predominante dolor abdominal recurrente. A todos se les realizó estudio clínico, endoscopia del tracto digestivo superior, prueba de la ureasa y biopsia gástrica. Sus edades estaban comprendidas entre los cinco meses y los catorce años. Dieciséis pacientes fueron del sexo femenino y trece del masculino. En dieciséis se hizo el diagnóstico endoscópico de gastritis y la prueba de la ureasa resultó positiva en doce. Desde el punto de vista histopatológico en doce (12) casos se encontró evidencia inequívoca de helicobacter pylori, observándose en la mayoría de ellos una tríada histopatológica constituida por: 1) gastritis crónica superficial activa con plasmocitos e hiperplasia regenerativa típica; 2) hiperplasia vellosa, y 3) dilatación de criptas y/o glándulas. De los doce casos con presencia inequívoca de Helicobacter pylori, nueve presentaron la prueba de la ureasa positiva