In vitro to in vivo extrapolation and physiologically based modeling of cytochrome P450 mediated metabolism in beagle dog gut wall and liver.

The beagle dog is a widely used in vivo model to guide clinical formulation development and to explore the potential for food effects. However, the results in dogs are often not directly translatable to humans. Consequently, a physiologically based modeling strategy has been proposed, using the dog as a validation step to verify model assumptions before making predictions in humans. One current weakness in this strategy is the lack of validated tools to incorporate gut wall metabolism into the dog model. In this study, in vitro to in vivo extrapolation factors for CYP2B11 and CYP3A12 mediated metabolism were established based on tissue enzyme abundance data reported earlier. Thereafter, physiologically based modeling of intestinal absorption in beagle dog was conducted in GastroPlus using V(max) and K(m) determined in recombinant enzymes as inputs for metabolic turnover. The predicted fraction of absorbed dose escaping the gut wall metabolism (F(g)) of all five reference compounds studied (domperidone, felodipine, nitrendipine, quinidine, and sildenafil) were within a two-fold range of the value estimated from in vivo data at single dose levels. However, further in vivo studies and analysis of the dose-dependent pharmacokinetics of felodipine and nitrendipine showed that more work is required for robust forecasting of nonlinearities. In conclusion, this study demonstrates an approach for prediction of the gut wall extraction of CYP substrates in the beagle dog, thus enhancing the value of dog studies as a component in a strategy for the prediction of human pharmacokinetics.

Liposome fluidity alters interactions between the ganglioside GM1 and cholera toxin B subunit.

Cholera toxin is a complex protein with a biologically active protein (A subunit) and a cell targeting portion (B subunit). The B subunit is responsible for specific cell binding and entry of the A subunit. One way to limit potential toxicity of the toxin after exposure is to introduce cellular decoys to bind the toxin before it can enter cells. In this study the ganglioside GM1, a natural ligand for cholera toxin, was incorporated into liposomes and the interaction between fluorescent B subunit and the liposome determined. Liposome membrane fluidity was determined to play a major role in the binding between liposomes and the cholera toxin B subunit. Liposomes with lower fluidity demonstrated greater binding with the B subunit. The findings from this study could have important implications on formulation strategies for liposome decoys of toxins.

Effect of lyophilization and freeze-thawing on the stability of siRNA-liposome complexes.

The purpose of this research was to describe the application of lyophilization in the delivery of siRNA using cationic lipids by addressing the long-term formulation/stability issues associated with cationic lipids and to understand the mechanism of lyoprotection. siRNA liposomes complexes were formed in different potential cyro/lyoprotectants and subjected to either lyophilization or freeze thaw cycles. siRNA, liposomes and/or lipoplexes were tested for activity, SYBR Green I binding, cellular uptake and particle size. The lipoplexes when lyophilized in the presence of sugars as lyoprotectants could be lyophilized and reconstituted without loss of transfection efficacy but in ionic solutions they lost 65-75% of their functionality. The mechanism of this loss of activity was further investigated. The lyophilization process did not alter siRNA's intrinsic biological activity as was evident by the ability of lyophilized siRNA to retain functionality and SYBR green I binding ability. While the lipoplex size dramatically increased ( approximately 50-70 times) after lyophilization in the absence of non-ionic lyoprotectants. This increase in size correlated to the decrease in cellular accumulation of siRNA and a decrease in activity. In conclusion, siRNAs can be applied in cationic lipid lyophilized formulations and these complexes represent a potential method of increasing the stability of pre-formed complex.

Evaluation of two cationic delivery systems for siRNA.

Small interference RNA (siRNA) is an important research tool, and also has the potential for clinical application. RNA interference (RNAi) approaches allow degradation of selective mRNA coding for pathogenic or disease-related proteins. RNAi pathway can be taken advantage of by the delivery of chemically synthesized siRNA. To fully attain its potential a sufficient siRNA must be delivered to the cell's cytoplasm. Cellular delivery of polyanions such as siRNA, while a challenging problem, may be addressed by the use of cationic macromolecules, the two major classes being lipids and polymers. In this study we compared two model cationic vectors liposomes (lipoplexes) and polyethelyenimine (PEI) (polyplexes). Complexes of the cationic macromolecules and siRNA did not differ in terms of their cellular uptake as determined by flow cytometry. However, it was demonstrated that the lipoplexes decomplexed more easily than the polyplexes. Differences in the biological activity of the siRNA were observed using commercially available siTOX siRNA. Lipoplexes resulted in dose-dependent siRNA activity; to 76.4 +/- 5.9% cell death was seen 48 hours posttransfection using 80 nmol siTOX. In summary, the selection of delivery vector can have a profound impact on biological activity of siRNA molecules. siRNA decomplexation from the cationic vector might be an important factor in the future development of new vectors.

Antimicrobial activities of human beta-defensins against Bacillus species.

Natural defences in the human body function to protect us from numerous environmental toxins and exposure to potential harmful biological agents. An important frontline defence is antimicrobial peptides. These peptides occur at environmental interfaces and serve to limit bacterial invasion. There has been little work comparing specific peptides as potential antimicrobial compounds. In this study, we evaluated the antimicrobial activity of peptides from the human beta-defensin (HBD) family against four species of Bacillus, chosen as models for Bacillus anthracis, a potential bioweapon. The impact of peptide concentration, sequence and protein binding was evaluated on their biological activity. The results indicated that HBD-3 was the most biologically active against Bacillus subtilis and Bacillus licheniformis, whilst HBD-2 was found to be most active against Bacillus cereus and Bacillus thuringiensis. Moreover, the antimicrobial activity of the peptides was directly related to peptide concentration and indirectly related to albumin concentration (i.e. protein binding).

A gene delivery approach for antimicrobials: expression of defensins.

INTRODUCTION: Peptide antibiotics as new therapeutic agents are becoming a popular option to investigate due to their broad bacterial target selectivity and limited resistance problems. Although attractive, these new drug candidates have several limitations including low potency and delivery issues which face all peptides/proteins. METHODS: In this study, we designed a plasmid expression system for human beta defensin 3. This sequence was cloned from a human epithelial lung cell into a CMV driven expression cassette. This expression plasmid was then evaluated for its ability to produce human-beta defensin 3 with the use of the non-viral transfection agent, polyethylenimine (PEI). RESULTS: The results indicate the expression cassette was transcriptionally active in HEK 293 cells, as measured by RT-PCR and that a beta defensin peptide was produced by the cells as confirmed by Western blot. The biological activity of the peptide was confirmed against both gram negative E. coli and gram positive Bacillus species using in vitro screening. CONCLUSION: Both the cultured media as well as the transfected cell lysate demonstrated biological activity demonstrating the peptide is also secreted.

Sulfhydryl based cationic surfactants and the impact of polyanions on disulfide bond formation: implications for gene transfer vectors.

Compacting plasmid DNA (pDNA) into a small size is a fundamental necessity for the efficient in vivo transfer of nucleic acids to somatic cells. An approach for accomplishing this is to condense pDNA using cationic detergents with sulfhydryl groups, near their critical micelle concentration. In this study, a model surfactant was used to study how the rate of disulfide bond formation relates to environmental factors. It was shown that the thiol detergent had the ability to form a disulfide bond when oxidized and the presence of polyanions was significantly increased. The addition of a reducing agent disrupted the disulfide bonds initially, but this was followed by disulfide bond reformation in a short time period.

Applications of a 7-day Caco-2 cell model in drug discovery and development.

Oral delivery is the preferred route of administration and therefore good absorption after oral dosing is a prerequisite for a compound to be successful in the clinic. The prediction of oral bioavailability from in vitro permeability assays is thus a valuable tool during drug discovery and development. Caco-2 cell monolayers mimic the human intestinal epithelium in many aspects. These monolayers form tight junctions between cells and have been widely used as a model of human intestinal absorption. Caco-2 cells also express a variety of transporter proteins although the transformed nature of the cells results in unpredictable differentiation markers, transport properties and enzyme expression. Thus various modifications of the Caco-2 assay are used in laboratories across the globe. The purpose of this paper is to provide an overview of a time and resource saving 7-day Caco-2 assay protocol. We also discuss the impact of various experimental conditions on permeability measurements and its applications during lead optimization in early discovery and for clinical candidate characterization, specifically for prediction of absorption in human, at a later stage in drug development.

Using ovality to predict nonmutagenic, orally efficacious pyridazine amides as cell specific spleen tyrosine kinase inhibitors.

Inhibition of spleen tyrosine kinase has attracted much attention as a mechanism for the treatment of cancers and autoimmune diseases such as asthma, rheumatoid arthritis, and systemic lupus erythematous. We report the structure-guided optimization of pyridazine amide spleen tyrosine kinase inhibitors. Early representatives of this scaffold were highly potent and selective but mutagenic in an Ames assay. An approach that led to the successful identification of nonmutagenic examples, as well as further optimization to compounds with reduced cardiovascular liabilities is described. Select pharmacokinetic and in vivo efficacy data are presented.

Liposomal siRNA delivery.

With the recent discovery of small interfering RNA (siRNA), to silence the expression of genes in vitro and in vivo, there has been a need to deliver these molecules to the cell nucleus. Forming a lipid/nucleic acid complex has become a solution and is explored here. Certain methods and ideas are used, such as: the positive/negative electrostatic interaction with a cationic lipid and an anionic RNA molecule, the size of the lipid vesicle aiding the uptake target tissues, targeted lipoplexes which can increase efficiency, and the protection of the siRNA molecule from the natural defenses of the immune system. Many lipid formulations exist and can be experimented with to achieve varying results depending on the application.

Polyethylenimine strategies for plasmid delivery to brain-derived cells.

The introduction of effective transfection reagents has had a dramatic impact on basic scientific studies over the past decade and is methodically becoming a clinical relevant agent. An area where these agents have had little impact to date is in transfection of neuronal cells either in vivo or in vitro. The poor results, obtained with these cells, likely arise from the innate properties of the cell itself such as its post-mitotic state and its fragility to the transfection agent. In this report, we investigated the transfection efficiency of branched and linear form of polyethylenimine (PEI) for a commonly used tissue culture cell line, the human CF bronchial epithelial cell line IB3-1, rat brain-derived glial, and neuronal cell lines. In addition, the effect of reaction conditions, such as ratio of PEI/plasmid, polymer molecular weight, and shape, was addressed on the transfection effects. The results indicate that branched PEI is more effective for the brain-derived cells. It is also shown that PEI 25 is more effective for the glial cells and PEI 50-100 is more effective for the neuronal cells under the evaluation conditions.