Paediatric drug development:reformulation, in vitro, genomic and in vivo evaluation

Angiotensin converting enzyme (ACE) inhibitors lisinopril and ramipril were selected from EMA/480197/2010 and the potassium-sparing diuretic spironolactone was selected from the NHS specials list for November 2011 drug tariff with the view to produce oral liquid formulations providing dosage forms targeting paediatrics. Lisinopril, ramipril and spironolactone were chosen for their interaction with transporter proteins in the small intestine. Formulation limitations such as poor solubility or pH sensitivity needed consideration. Lisinopril was formulated without extensive development as drug and excipients were water soluble. Ramipril and spironolactone are both insoluble in water and strategies combating this were employed. Ramipril was successfully solubilised using low concentrations of acetic acid in a co-solvent system and also via complexation with hydroxypropyl-β-cyclodextrin. A ramipril suspension was produced to take formulation development in a third direction. Spironolactone dosages were too high for solubilisation techniques to be effective so suspensions were developed. A buffer controlled pH for the sensitive drug whilst a precisely balanced surfactant and suspending agent mix provided excellent physical stability. Characterisation, stability profiling and permeability assessment were performed following formulation development. The formulation process highlighted current shortcomings in techniques for taste assessment of pharmaceutical preparations resulting in early stage research into a novel in vitro cell based assay. The formulations developed in the initial phase of the research were used as model formulations investigating microarray application in an in vitro-in vivo correlation for carrier mediated drug absorption. Caco-2 cells were assessed following transport studies for changes in genetic expression of the ATP-binding cassette and solute carrier transporter superfamilies. Findings of which were compared to in vitro and in vivo permeability findings. It was not possible to ascertain a correlation between in vivo drug absorption and the expression of individual genes or even gene families, however there was a correlation (R2 = 0.9934) between the total number of genes with significantly changed expression levels and the predicted human absorption.

Genomic evaluation during permeability of indomethacin and its solid dispersion

Drug resistance was first identified in cancer cells that express proteins known as multidrug resistance proteins that extrude the therapeutic agents out of the cells resulting in alteration of pharmacokinetics, tissue distribution, and pharmacodynamics of drugs. To this end studies were carried out to investigate the role of pharmacological inhibitors and pharmaceutical excipients with a primary focus on P-glycoprotein (P-gp). The aim of this study was to investigate holistic changes in transporter gene expression during permeability upon formulation of indomethacin as solid dispersion. Initial characterization studies of solid dispersion of indomethacin showed that the drug was dispersed within the carrier in amorphous form. Analysis of permeability data across Caco-2 monolayers revealed that drug absorption increased by 4-fold when reformulated as solid dispersion. The last phase of the work involved investigation of gene expression changes of transporter genes during permeability. The results showed that there were significant differences in the expression of both ATP-binding cassette (ABC) transporter genes as well as solute carrier transporter (SLC) genes suggesting that the inclusion of polyethylene glycol as well as changes in molecular form of drug from crystalline to amorphous have a significant bearing on the expression of transporter network genes resulting in differences in drug permeability. © 2011 Informa UK, Ltd.

Drug Transporter Gene Expression in Human Colorectal Tissue and Cell Lines : Modulation with Antiretrovirals for Microbicide Optimization

This work was supported by the European Union's Seventh Programme for research, technological development and demonstration under grant agreement No 305316 as part of the MOTIF (Microbicides Formulation Through Innovative Formulation for Vaginal and Rectal Delivery) project.

Drug Transporter Gene Expression in Human Colorectal Tissue and Cell Lines : Modulation with Antiretrovirals for Microbicide Optimization

This work was supported by the European Union's Seventh Programme for research, technological development and demonstration under grant agreement No 305316 as part of the MOTIF (Microbicides Formulation Through Innovative Formulation for Vaginal and Rectal Delivery) project.

Le rôle du récepteur scavenger B type I, SR-BI, dans l'absorption et le métabolisme du cholestérol au niveau intestinal

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Engineering polymeric micelles for solubilization of poorly-water soluble drugs : a novel approach for oral drug delivery

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Le rôle du récepteur scavenger B type I, SR-BI, dans l'absorption et le métabolisme du cholestérol au niveau intestinal

Mémoire numérisé par la Direction des bibliothèques de l'Université de Montréal.

Engineering polymeric micelles for solubilization of poorly-water soluble drugs : a novel approach for oral drug delivery

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Nanotechnology in food industry II: risk assessment and legislation

Currently, there is increasing use of nanomaterials in the food industry thanks to the many advantages offered and make the products that contain them more competitive in the market. Their physicochemical properties often differ from those of bulk materials, which require specialized risk assessment. This should cover the risks to the health of workers and consumers as well as possible environmental risks. The risk assessment methods must go updating due to more widespread use of nanomaterials, especially now that are making their way down to consumer products. Today there is no specific legislation for nanomaterials, but there are several european dispositions and regulations that include them. This review gives an overview of the risk assessment and the existing current legislation regarding the use of nanotechnology in the food industry.

An in vitro investigation on the cytotoxic and nuclear receptor transcriptional activity of the mycotoxins fumonisin B1 and beauvericin

Fumonisin B1 (FB1) and beauvericin (BEA) are secondary metabolites of filamentous fungi, which under appropriate temperature and humidity conditions may develop on various foods and feeds. To date few studies have been performed to evaluate the toxicological and endocrine disrupting effects of FB1 and BEA. The present study makes use of various in vitro bioassays including; oestrogen, androgen, progestagen and glucocorticoid reporter gene assays (RGAs) for the study of nuclear receptor transcriptional activity, the thiazolyl blue tetrazolium bromide (MTT) assay to monitor cytotoxicity and high content analysis (HCA) for the detection of pre-lethal toxicity in the RGA and Caco-2 human colon adenocarcinoma cells. At the receptor level, 0.001-10μM BEA or FB1 did not induce any agonist responses in the RGAs. However at non-cytotoxic concentrations, an antagonistic effect was exhibited by FB1 on the androgen nuclear receptor transcriptional activity at 10μM and BEA on the progestagen and glucocorticoid receptors at 1μM. MTT analysis showed no decrease in cell viability at any concentration of FB1, whereas BEA showed a significant decrease in viability at 10μM. HCA analysis confirmed that the reduction in the progestagen receptor transcriptional activity at 1μM BEA was not due to pre-lethal toxicity. In addition, BEA (10μM) induced significant toxicity in both the TM-Luc (progestagen responsive) and Caco-2 cells.

Free fatty acid receptor 1 (GPR40) agonists containing spirocyclic periphery inspired by LY2881835

The free fatty acid receptor 1 (FFA1), a G protein-coupled receptor (GPCR) naturally activated by long-chain fatty acids is a novel target for the treatment of metabolic diseases. The basic amine spirocyclic periphery of Eli Lilly's drug candidate LY2881835 for treatment of type 2 diabetes mellitus (which reached phase I clinical trials) inspired a series of novel FFA1 agonists. These were designed to incorporate the 3-[4-(benzyloxy)phenyl]propanoic acid pharmacophore core decorated with a range of spirocyclic motifs. The latter were prepared via the Prins cyclization and subsequent modification of the 4-hydroxytetrahydropyran moiety in the Prins product. Here, we synthesize 19 compounds and test for FFA1 activity. Within this pilot set, a nanomolar potency (EC50=55nM) was reached. Four lead compounds (EC50 range 55-410nM) were characterized for aqueous solubility, metabolic stability, plasma protein binding and Caco-2 permeability. While some instability in the presence of mouse liver microsomes was noted, mouse pharmacokinetic profile of the compound having the best overall ADME properties was evaluated to reveal acceptable bioavailability (F=10.3%) and plasma levels achieved on oral administration.

Assessment of genotoxicity of aflatoxin M1 and B1 contaminated milks after in vitro human digestion

Introduction - Milk is considered a complete food from the nutritional point of view. Milk can be exposed to various types of contamination, such as mycotoxins. These metabolites are naturally occurring toxic compounds produced by fungi. Several studies on milk samples have reported the presence of aflatoxin B1 (AFB1) and M1 (AFM1), due to the high incidence in samples intended for human consumption, carcinogenicity proven AFB1 and resistance of the contaminants to the process of digestion, making those available for intestinal absorption. Considering these aspects, the objective of this study was to evaluate the genotoxicity of milk samples contaminated by AFB1 and AFM1 before and after the action of lactic acid bacteria using Caco-2 intestinal human cells.

Nanostructured lipid carriers as vehicles for the delivery of poorly water-soluble drugs

186 p.

Novel Anti-Campylobacter Compounds Identified Using High Throughput Screening of a Pre-selected Enriched Small Molecules Library

Campylobacter is a leading cause of foodborne bacterial gastroenteritis worldwide and infections can be fatal. The emergence of antibiotic-resistant Campylobacter spp. necessitates the development of new antimicrobials. We identified novel anti-Campylobacter small molecule inhibitors using a high throughput growth inhibition assay. To expedite screening, we made use of a “bioactive” library of 4,182 compounds that we have previously shown to be active against diverse microbes. Screening for growth inhibition of Campylobacter jejuni, identified 781 compounds that were either bactericidal or bacteriostatic at a concentration of 200 µM. Seventy nine of the bactericidal compounds were prioritized for secondary screening based on their physico-chemical properties. Based on the minimum inhibitory concentration against a diverse range of C. jejuni and a lack of effect on gut microbes, we selected 12 compounds. No resistance was observed to any of these 12 lead compounds when C. jejuni was cultured with lethal or sub-lethal concentrations suggesting that C. jejuni is less likely to develop resistance to these compounds. Top 12 compounds also possessed low cytotoxicity to human intestinal epithelial cells (Caco-2 cells) and no hemolytic activity against sheep red blood cells. Next, these 12 compounds were evaluated for ability to clear C. jejuni in vitro. A total of 10 compounds had an anti-C. jejuni effect in Caco-2 cells with some effective even at 25 µM concentrations. These novel 12 compounds belong to five established antimicrobial chemical classes; piperazines, aryl amines, piperidines, sulfonamide and pyridazinone. Exploitation of analogues of these chemical classes may provide Campylobacter specific drugs that can be applied in both human and animal medicine.

Caraterização in vitro dos efeitos tóxicos da patulina na integridade do epitélio intestinal e potenciais efeitos protetores da cisteína

A mucosa intestinal é a primeira barreira biológica encontrada pelas micotoxinas presentes nos alimentos, sendo a patulina, uma micotoxina produzida por fungos do género Penicillium spp., uma preocupação particular atendendo a que a exposição humana a esta micotoxina pode conduzir a efeitos imunológicos, neurológicos e gastrointestinais. Considerando estes efeitos para a saúde, o presente estudo tem como objetivos a avaliação do efeito tóxico da exposição intestinal a patulina, bem como a determinação do potencial efeito protetor da coadministração de patulina e cisteína na membrana intestinal, utilizando para o efeito células Caco-2. A integridade da membrana intestinal foi determinada pela medição da resistência elétrica transepitelial (TEER). Os resultados evidenciaram um decréscimo acentuado nos valores de TEER após 24 horas de exposição celular a 95 μM de patulina. Para as concentrações mais reduzidas verificou-se uma redução máxima inferior a 25% após 24 horas de exposição. A coadministração de patulina (95 μM) e cisteína (40 μM) revelou um decréscimo nos valores de TEER. O tratamento com cisteína em concentrações superiores ( 400 μM) revelou efeito protetor da membrana intestinal, tendo em conta os valores de TEER. Estes resultados contribuem para uma avaliação do risco mais precisa associada à exposição a contaminantes alimentares.