Colin W. Pouton

Formulation of self-emulsifying drug delivery systems

Abstract Self-emulsifying drug delivery systems (SEDDS) are mixtures of oils and surfactants, ideally isotropic, sometimes including cosolvents, which emulsify under conditions of gentle agitation, similar to those which would be encountered in the gastro-intestinal tract. Hydrophobic drugs can often be dissolved in SEDDS allowing them to be encapsulated as unit dosage forms for peroral administration. When such a formulation is released into the lumen of the gut it disperses to form a fine emulsion, so that the drug remains in solution in the gut, avoiding the dissolution step which frequently limits the rate of absorption of hydrophobic drugs from the crystalline state. Generally this can lead to improved bioavailability, and/or a more consistent temporal profile of absorption from the gut. Ultra-low oil-water interfacial tension and/or substantial interfacial disruption are required to achieve self-emulsification. SEDDS are usually formulated with triglyceride oils and ethoxylated nonionic surfactants, usually at surfactant concentrations greater than 25%. In practice, disruption of the oil-water interface is caused by penetration of water into the formulation or diffusion of cosolvents away from the formulation. Both of these phenomena can be studied using equilibrium phase diagrams, which in combination with particle size measurements allow the optimisation of performance of SEDDS. The precise mechanisms of emulsification remain the subject of speculation but there is an empirical link between self-emulsification, liquid crystal formation, oil-water phase-inversion temperature and enhanced solubilization of water by oily formulations, and these phenomena are indicators of the efficiency of emulsification. This article describes strategies used for formulation of SEDDS, methods used for assessment of efficiency of emulsification and practical considerations regarding the use of SEDDS for enhancement of the bioavailability of drugs from the gastro-intestinal tract.

Self-emulsifying drug delivery systems: formulation and biopharmaceutic evaluation of an investigational lipophilic compound.

Self-emulsifying drug delivery systems (SEDDSs) represent a possible alternative to traditional oral formulations of lipophilic compounds. In the present study, a lipophilic compound, WIN 54954, was formulated in a medium chain triglyceride oil/nonionic surfactant mixture which exhibited self-emulsification under conditions of gentle agitation in an aqueous medium. The efficiency of emulsifi-cation was studied using a laser diffraction sizer to determine particle size distributions of the resultant emulsions. An optimized formulation which consisted of 25% (w/w) surfactant, 40% (w/w) oil, and 35% (w/w) WIN 54954 emulsified rapidly with gentle agitation in 0.1 N HCl (37°C), producing dispersions with mean droplet diameters of less than 3 µm. The self-emulsifying preparation was compared to a polyethylene glycol 600 (PEG 600) solution formulation by administering each as prefilled soft gelatin capsules to fasted beagle dogs in a parallel crossover study. Pharmacokinetic parameters were determined and the absolute bioavailability of the drug was calculated by comparison to an i.v. injection. The SEDDS improved the reproducibility of the plasma profile in terms of the maximum plasma concentration (Cmax) and the time to reach the maximum concentration (tmax). There was no significant difference in the absolute bioavailability of WIN 54954 from either the SEDDS or the PEG formulations.

Biosynthetic polyhydroxyalkanoates and their potential in drug delivery

Abstract Polyhydroxyalkanoates (PHAs) are naturally occurring biodegradable polyesters produced as energy storage materials by many bacteria. The most common PHA, poly(3-hydroxybutyrate) (PHB), can be produced in high yield by fermentation of a variety of bacterial strains. PHB is a isotactic semi-crystalline polyester with great potential as a biodegradable commodity; it has useful physico-mechanical properties and appears to be biocompatible. Hydrolytic degradation occurs by surface erosion which makes it an attractive material for controlled release applications. The homopolymer PHB has a relatively high melting point and crystallizes rapidly, making entrapment of drug technically difficult. The related copolymers with 3-hydroxyvalerate, P(HB-HV)s, have similar semi-crystalline properties though their slower rates of crystallization result in matrices with different properties; this merits further investigation. Release of low molecular weight drugs from PHB and P(HB-HV) matrices tends to proceed by penetration of water and pore formation, at least above loadings of approximately 5% drug. Release from such matrices is predominantly independent of polymer erosion; though at lower loadings it is possible to trap drug more effectively. PHB and P(HB-HV) matrices lose mass very slowly when compared to bulk-degrading poly(lactide-glycolide) systems. Therefore the applications of these materials in drug delivery are likely to depend on the formulation of suitable blends with other biocompatible polymers. Porosity, erosion rate and hence drug release rate can be controlled by blending techniques. At a more fundamental level there is considerable potential for design and bioengineering of other PHAs for applications in drug delivery. For example, medium chain PHAs are rubbery materials with low melting points which may be much more suitable as matrices for applications in drug delivery.

NKX2-5eGFP/w hESCs for isolation of human cardiac progenitors and cardiomyocytes

NKX2-5 is expressed in the heart throughout life. We targeted eGFP sequences to the NKX2-5 locus of human embryonic stem cells (hESCs); NKX2-5eGFP/w hESCs facilitate quantification of cardiac differentiation, purification of hESC-derived committed cardiac progenitor cells (hESC-CPCs) and cardiomyocytes (hESC-CMs) and the standardization of differentiation protocols. We used NKX2-5 eGFP+ cells to identify VCAM1 and SIRPA as cell-surface markers expressed in cardiac lineages.

Influence of lipolysis on drug absorption from the gastro-intestinal tract

Abstract Bioavailability of hydrophobic drugs from the gastro-intestinal (GI) tract can be enhanced by formulation in digestible oils. This form of delivery is an effective way of avoiding the slow dissolution step which limits availability from solid dosage forms. Essentially the drug remains in solution during its passage and prior to absorption. Digestion of the oily components of the formulation will have a major influence on the fate of the drug in the gut. In many cases digestion will be advantageous in that the drug may be solubilized within mixed micelles of bile components and the products of triglyceride lipolysis. The solubility of hydrophobic drugs in the presence of such micelles is greatly enhanced. This has the effect of dispersing the drug extremely finely and allows rapid partitioning of the drug into the aqueous continuum for absorption. However, formulation of oils with surfactants may inhibit lipolysis, which suggests that formulation should include an in vitro assessment of the lipolysis of the delivery system. Here we review the solubilization of drugs in bile salt micelles, describe methods which can be used for assessment of lipolysis in vitro, and present preliminary biostudies using formulations optimised for rapid lipolysis. There is a need for more systematic studies on the influence of lipolysis on absorption from the GI tract, but current data suggest that optimisation of lipolysis will be an important strategy in formulation of oily systems.

Self-emulsifying drug delivery systems: assessment of the efficiency of emulsification

Abstract Self-emulsifying formulations have potential uses as vehicles for the administration of lipophilic drugs by the oral route. In this study model oil-surfactant mixtures were allowed to self-emulsify under conditions of gentle agitation. During emulsification the relative intensity of light scattered by the dispersion was monitored continuously which enabled the rates of emulsification to be compared. The particle sizes of resultant emulsions were compared by light microscopy and using a Coulter Nano-Sizer. Efficient self-emulsifying formulations were produced by the oils Miglyol 812 or Miglyol 840 in combination with the surfactant Tween 85. The properties of these systems have been studied over a range of mixture compositions. The finest dispersions were produced rapidly and in reproducible time by a mixture of 30% w/w Tween 85 and 70% w/w Miglyol 812.

Polycation-DNA complexes for gene delivery : a comparison of the biopharmaceutical properties of cationic polypeptides and cationic lipids

DNA plasmids formed particulate complexes with a variety of cationic polyamino acids and cationic lipids, which were used to transfect mammalian cells in culture. Complexation was studied by assaying for exclusion of ethidium using a fluorometric assay, which indicated that complexation with cationic polyamino acids took place with utilisation of the majority of charged functional groups. The particle sizes and zeta potentials of a range of complexes were determined. Generally polyamino acids formed uniform particles 80-120 nm in diameter in water, but their particle size increased on dilution of the particles in electrolytes or cell culture media. The efficiency of transfection was compared using complexes of pRSVlacZ, a reporter construct which expressed beta-galactosidase under the control of the Rous sarcoma virus promoter. Positively charged DNA/polyamino acid complexes were taken up by cells but required an endosomolytic agent, such as chloroquine, to facilitate transfection. Polyornithine complexes resulted in the highest levels of expression, in comparison with other homopolyamino acids (polyornithine>poly-L-lysine=poly-D-lysine>polyarginine). Copolyamino acids of lysine and alanine condensed DNA but were less active in transfection experiments. Copoly(L-Lys, L-Ala 1:1) was inactive even in the presence of chloroquine. In contrast DNA/cationic lipid complexes transfected cells spontaneously, and chloroquine did not improve the extent of expression, rather it usually reduced efficiency. There was little correlation between comparative efficiencies of lipid complexes between cell lines suggesting that the nature of the cell membrane and differences in mechanisms of internalisation were determinants of efficiency. In an effort to explore better cell culture models for gene delivery, monolayers of Caco-2 cells were transfected in filter culture. As the cells differentiated and formed a polarized monolayer, expression of beta-galactosidase was reduced until at day 27 expression was not significantly different from basal activity. The Caco-2 filter culture model merits further attention as a model of gene delivery to epithelial surfaces, such as would be encountered in the lung after inhalation.

Using polymeric precipitation inhibitors to improve the absorption of poorly water-soluble drugs: A mechanistic basis for utility

The inclusion of certain polymers within solid dispersion or lipid-based formulations can maintain drug supersaturation after dispersion and/or digestion of the vehicle, leading to improvements in bioavailability and variability in exposure. This review presents an overview of the fundamental principles that underpin drug precipitation mechanisms, describes the mechanisms by which precipitation may be inhibited, discusses the methods that can be used to identify polymeric precipitation inhibitors (PPIs), and summarizes current literature evidence of the most effective PPIs. Preliminary data from our laboratory is also presented, which describes the precipitation inhibition behavior of 53 polymeric materials using supersaturated solutions of danazol as a model, poorly water-soluble drug. These studies identify a group of PPIs with superior precipitation inhibition qualities, the majority of which are cellulose-based. These new results in combination with previous published data indicate that PPIs represent an appealing new technology with the potential to improve drug absorption for poorly water-soluble drugs. The molecular determinants of polymer utility, however, remain relatively poorly understood, although the cellulose derivates appear, in general, to provide the most benefit. More detailed studies are therefore required to define the parameters that most effectively predict and quantify the drug–polymer relationships that control precipitation inhibition.

Embryonic stem cells as a source of models for drug discovery

Embryonic stem cells (ESCs) will become a source of models for a wide range of adult differentiated cells, providing that reliable protocols for directed differentiation can be established. Stem-cell technology has the potential to revolutionize drug discovery, making models available for primary screens, secondary pharmacology, safety pharmacology, metabolic profiling and toxicity evaluation. Models of differentiated cells that are derived from mouse ESCs are already in use in drug discovery, and are beginning to find uses in high-throughput screens. Before analogous human models can be obtained in adequate numbers, reliable methods for the expansion of human ESC cultures will be needed. For applications in drug discovery, involving either species, protocols for directed differentiation will need to be robust and affordable. Here, we explore current challenges and future opportunities in relation to the use of stem-cell technology in drug discovery, and address the use of both mouse and human models.

Preparation and in Vitro Evaluation of Novel Lipopeptide Transfection Agents for Efficient Gene Delivery

Gene therapy by delivery of nonviral expression vectors is highly desirable, due to their safety, stability, and suitability for production as bulk pharmaceuticals. However, low transfection efficiency remains a limiting factor in application on nonviral gene delivery. Despite recent advances in the field, there are still major obstacles to overcome. In an attempt to construct more efficient nonviral gene delivery vectors, we have designed a series of novel lipopeptide transfection agents, consisting of an alkyl chain, one cysteine, 1 to 4 histidine and 1 to 3 lysine residues. The lipopeptides were designed to facilitate dimerization (by way of the cysteine residues), DNA binding at neutral pH (making use of charged lysine residues), and endosomal escape (by way of weakly basic histidine residues). DNA/lipopeptide complexes were evaluated for their biophysical properties and transfection efficiencies. The number and identity of amino acids incorporated in the lipopeptide construct affected their DNA/lipopeptide complex forming capacity. As the number of lysine residues in the lipopeptide increased, the DNA complexes formed became more stable, had higher zeta potential (particle surface charge), and produced smaller mean particle sizes (typically 110 nm at a charge ratio of 5.0 and 240 nm at a charge ratio of 1.0). The effect of inclusion of histidines in the lipopeptide moiety had the opposite effect on complex formation to lysine, but was necessary for high transfection efficiency. In vitro transfection studies in COS-7 cells revealed that the efficiency of gene delivery of the luciferase encoding plasmid, pCMV-Luc, mediated by all the lipopeptides, was much higher than poly(L-lysine) (PLL), which has no endosomal escape system, and in two cases was slightly higher than that of branched polyethylenimine (PEI). Lipopeptides with at least two lysine residues and at least one histidine residue produced spontaneous transfection complexes with plasmid DNA, indicating that endosomal escape was achieved by incorporation of histidine residues. These low molecular weight peptides can be readily synthesized and purified and offer new insights into the mechanism of action of transfection complexes.