The Development of Pemetrexed Diacid-Loaded Gelatin-Cloisite 30B (MMT) Nanocomposite for Improved Oral Efficacy Against Cancer: Characterization, In-Vitro and Ex-Vivo Assessment.

AIM: The intention of this investigation was to develop Pemetrexed Diacid (PTX)-loaded gelatine-cloisite 30B (MMT) nanocomposite for the potential oral delivery of PTX and the in vitro, and ex vivo assessment. BACKGROUND: Gelatin/Cloisite 30 B (MMT) nanocomposites were prepared by blending gelatin with MMT in aqueous solution. METHODS: PTX was incorporated into the nanocomposite preparation. The nanocomposites were investigated by Fourier Transmission Infra Red Spectroscopy (FT-IR), Differential Scanning Calorimetry (DSC), Scanning Electron Microscope (SEM) X-Ray Diffraction (XRD) and Confocal Laser Microscopy (CLSM). FT-IR of nanocomposite showed the disappearance of all major peaks which corroborated the formation of nanocomposites. The nanocomposites were found to have a particle size of 121.9 ± 1.85 nm and zeta potential -12.1 ± 0.63 mV. DSC thermogram of drug loaded nanocomposites indicated peak at 117.165 oC and 205.816 oC, which clearly revealed that the drug has been incorporated into the nanocomposite because of cross-linking of cloisite 30 B and gelatin in the presence of glutaraldehyde. RESULTS: SEM images of gelatin show a network like structure which disappears in the nanocomposite. The kinetics of the drug release was studied in order to ascertain the type of release mechanism. The drug release from nanocomposites was in a controlled manner, followed by first-order kinetics and the drug release mechanism was found to be of Fickian type. CONCLUSION: Ex vivo gut permeation studies revealed 4 times enhancement in the permeation of drug present in the nanocomposite as compared to plain drug solution and were further affirmed by CLSM. Thus, gelatin/(MMT) nanocomposite could be promising for the oral delivery of PTX in cancer therapy and future prospects for the industrial pharmacy.

In vitro assessment of iron availability from commercial Young Child Formulae supplemented with prebiotics.

PURPOSE: Iron is essential for development and growth in young children; unfortunately, iron deficiency (ID) is a significant public health problem in this population. Young Child Formulae (YCF), milk-derived products fortified with iron and ascorbic acid (AA, an enhancer of iron absorption) may be good sources of iron to help prevent ID. Furthermore, some YCF are supplemented with prebiotics, non-digestible carbohydrates suggested to enhance iron bioavailability. The aim of our study was to evaluate iron bioavailability of YCF relative to prebiotic and AA concentrations. We hypothesised that YCF with the highest levels of prebiotics and AA would have the most bioavailable iron. METHODS: We used the in vitro digestion/Caco-2 cell model to measure iron bioavailability from 4 commercially available YCF with approximately equal amounts of iron, but varying amounts of: AA and the prebiotics fructo- and galacto-oligosaccharides. Caco-2 cell ferritin formation was used as a surrogate marker for iron bioavailability. RESULTS: The YCF with the highest concentration of prebiotics and AA had the highest iron bioavailability; conversely, the YCF with the lowest concentration of prebiotics and AA had the lowest. After the addition of exogenous prebiotics, so that all tested YCF had equivalent amounts, there was no longer a significant difference between YCF iron bioavailability. CONCLUSION: Our results suggest that ascorbic acid and prebiotics in YCF improve iron bioavailability. Ensuring that iron is delivered in a bioavailable form would improve the nutritional benefits of YCF in relation to ID/IDA amongst young children; therefore, further exploration of our findings in vivo is warranted.

Physicochemical properties and intestinal protective effect of ultra-micro ground insoluble dietary fibre from carrot pomace.

Carrot pomace is an abundant, but underutilized, byproduct from the juice industry. In this study, the insoluble dietary fiber from carrot pomace was treated using an ultra-microgrinding process, and the resulting changes in its physicochemical properties and intestinal protective effect against heavy metal damage were examined. The SEM and fluorescence microscopy results showed that the grinding process could significantly decrease the particle size of carrot insoluble dietary fibre and increase its Brunauer-Emmett-Teller surface area from 0.374 to 1.835 m(2) g(-1). Correspondingly, the water-holding capacity, swelling capacity, and oil-holding capacity increased by 62.09%, 49.25% and 45.45%, respectively. The glucose-, nitrite-, and lead ion-adsorbing abilities also improved significantly compared with the raw samples. In addition, apoptosis assessment by AO/EB revealed that the ground fibre could effectively protect Caco-2 cells from lead ion damage. The MTT assay showed that carrot insoluble dietary fibre has no toxicity for Caco-2 cells at a concentration of 10.0 mg L(-1). The findings of this study highlighted the potential of the ultra-microgrinding process to produce a high added-value fibre ingredient from carrot residues.

A cost-effective system for differentiation of intestinal epithelium from human induced pluripotent stem cells.

The human intestinal epithelium is a useful model for pharmacological studies of absorption, metabolism, drug interactions, and toxicology, as well as for studies of developmental biology. We established a rapid and cost effective system for differentiation of human induced pluripotent stem (iPS) cells into definitive endoderm (DE) cells. In the presence of dimethyl sulfoxide (DMSO), a low concentration of Activin at 6.25 ng/ml is sufficient to give a similar differentiation efficiency with that using Activin at 100 ng/ml at the presence of Wnt activator. In the presence of DMSO, Activin at low concentration triggered hiPS cells to undergo differentiation through G1 arrest, reduce apoptosis, and potentiate activation of downstream targets, such as SMAD2 phosphorylation and SOX17 expression. This increased differentiation into CDX2 + SOX17 + DE cells. The present differentiation procedure therefore permits rapid and efficient derivation of DE cells, capable of differentiating into intestinal epithelium upon BIO and DAPT treatment and of giving rise to functional cells, such as enterocytes.

Isoflavones in food supplements: chemical profile, label accordance and permeability study in Caco-2 cells.

Consumers nowadays are playing an active role in their health-care. A special case is the increasing number of women, who are reluctant to use exogenous hormone therapy for the treatment of menopausal symptoms and are looking for complementary therapies. However, food supplements are not clearly regulated in Europe. The EFSA has only recently begun to address the issues of botanical safety and purity regulation, leading to a variability of content, standardization, dosage, and purity of available products. In this study, isoflavones (puerarin, daidzin, genistin, daidzein, glycitein, genistein, formononetin, prunetin, and biochanin A) from food supplements (n = 15) for menopausal symptoms relief are evaluated and compared with the labelled information. Only four supplements complied with the recommendations made by the EC on the tolerable thresholds. The intestinal bioavailability of these compounds was investigated using Caco-2 cells. The apparent permeability coefficients of the selected isoflavonoids across the Caco-2 cells were affected by the isoflavone concentration and product matrix.

An assessment of the intestinal lumen as a site for intervention in reducing body burdens of organochlorine compounds.

Many individuals maintain a persistent body burden of organochlorine compounds (OCs) as well as other lipophilic compounds, largely as a result of airborne and dietary exposures. Ingested OCs are typically absorbed from the small intestine along with dietary lipids. Once in the body, stored OCs can mobilize from adipose tissue storage sites and, along with circulating OCs, are delivered into the small intestine via hepatic processing and biliary transport. Retained OCs are also transported into both the large and small intestinal lumen via non-biliary mechanisms involving both secretion and desquamation from enterocytes. OCs and some other toxicants can be reabsorbed from the intestine, however, they take part in enterohepatic circulation(EHC). While dietary fat facilitates the absorption of OCs from the small intestine, it has little effect on OCs within the large intestine. Non-absorbable dietary fats and fat absorption inhibitors, however, can reduce the re-absorption of OCs and other lipophiles involved in EHC and may enhance the secretion of these compounds into the large intestine--thereby hastening their elimination. Clinical studies are currently underway to determine the efficacy of using non-absorbable fats and inhibitors of fat absorption in facilitating the elimination of persistent body burdens of OCs and other lipophilic human contaminants.

Surface area assessment of the murine intestinal tract as a prerequisite for oral dose translation from mouse to man.

In many pharmacological and toxicological studies knowledge about the intestinal absorption, which is dependent upon the surface area of absorptive epithelia, is indispensible. Although mice are often used in such preclinical studies, very few quantitative data about their intestinal surface area are available. Especially for locally acting candidate drugs in development, this information is crucial for dose translation towards humans. Therefore, the surface area of the intestinal tract of CD-1 IGS mice was assessed in the present study. The intestinal tracts of 12 mice were collected after euthanasia. From six animals, histological sections from the duodenum, jejunum, ileum, caecum and colon-rectum were made according to common stereological principles. Using these sections, the volumes and surface areas of each intestinal segment were estimated applying stereological counting procedures. In the other six animals, the density and surface area of the microvilli present in each intestinal segment were determined by means of scanning and transmission electron microscopy to assess the increase of the intestinal surface area attributable to the presence of microvilli. The mean total volume and surface area of the intestinal tract were 1.34 cm(3) and 1.41 m(2), respectively. The relative intestinal surface area (intestinal surface area divided by the body surface area) was 119. The relative intestinal surface area of mice is very similar to that of humans. The results of this study are important for the appropriate dose translation of candidate therapeutic compounds in drug development from mouse to humans.

PCR-based assessment of the recovery rate of exfoliated colonocytes or cancer cells from fecal samples depends on the storage conditions after defecation.

BACKGROUND AND AIMS: We recently developed a new methodology for isolating colonocytes from fecal samples. We then applied a DNA-based analysis to the isolated colonocytes to detect colorectal cancer cells originating from any part of the colorectum. The purpose of the present study was to determine how long after defecation and at what temperature the fecal samples should be stored to isolate the colonocytes successfully. METHODS: Fecal samples were collected from 6 patients with colorectal cancer and 6 healthy volunteers soon after defecation at the National Cancer Center Hospital. The fecal samples were stored at 4 degrees C, room temperature or 40 degrees C for 0, 24 or 48 hours. Colonocytes were then isolated from the fecal samples, and the DNA was purified. Finally, PCR for p53, K-ras and APC was conducted to determine whether the corresponding PCR products could be obtained. RESULTS: The colonocyte recovery rate was not reduced, when compared with the data for successful PCR amplification, if the fecal samples were kept at 4 degrees C after defecation and if the colonocytes were isolated within 48 hours after defecation. CONCLUSIONS: The present data provided important clinical knowledge regarding the storage of fecal samples for future mass screening tests.

Development and validation of a reversed-phase HPLC method for the determination of ezetimibe in pharmaceutical dosage forms.

Ezetimibe belongs to a group of selective and very effective 2-azetidione cholesterol absorption inhibitors that act on the level of cholesterol entry into enterocytes. A rapid, specific reversed-phase HPLC method has been developed for assaying ezetimibe in pharmaceutical dosage forms. The assay involved an isocratic elution of ezetimibe in a Kromasil 100 C18 column using a mobile phase composition of water (pH 6.8, 0.05%, w/v 1-heptane sulfonic acid) and acetonitrile (30:70, v/v). The flow rate was 0.5 ml/min and the analyte monitored at 232 nm. The assay method was found to be linear from 0.5 to 50 microg/ml. All the validation parameters were within the acceptance range. The developed method was successfully applied to estimate the amount of ezetimibe in tablets.