Alison B. Lansley

Nonionic oil-in-water microemulsions: the effect of oil type on phase behaviour

The formation of oil-in-water (o/w) microemulsions stabilized by the nonionic surfactants, polyoxyethylene-10-dodecyl ether, polyoxyethylene-10-oleyl ether, N,N-dimethyldodecylamine-N-oxide and N,N-dimethyloleylamine-N-oxide and containing a variety of pharmaceutically acceptable oils, namely ethyl butyrate, ethyl caprylate, ethyl oleate and the triglycerides, soybean oil, Miglyol 812 and tributyrin, has been examined at 298 K. The effect on microemulsion formation of replacing water with phosphate buffered saline (PBS) and complete PBS has been established. In addition, the effect of changing temperature (from 298 to 310 K) on the phase behaviour of microemulsions formulated using PBS as continuous phase has been determined. Although some small differences in phase behaviour were noted when altering the continuous phase, the greatest difference in phase behaviour was observed when changing the experimental temperature, particularly for microemulsions stabilized by polyoxyethylene-10-oleyl ether. Regardless of the temperature and aqueous phase used, however the larger molecular volume oils (soybean oil, Miglyol 812 and ethyl oleate) were solubilized to a lower extent than the smaller molecular volume oils (namely, ethyl butyrate and ethyl caprylate). The only exception to this rule was when polyoxyethylene-10-oleyl ether was used as surfactant, particularly at 298 K, where it was the larger molecular volume oils that were solubilized to the greatest extent. Cloud point/phase inversion temperature experiments suggested that the higher molecular volume oils were incorporated into the microemulsions prepared using the polyoxyethylene-based surfactants in a different way than the smaller molecular volume oils and suggest that the smaller molecular volume oils are acting in much the same way as a cosurfactant in that they interchelate with their hydrophilic group interspersed in the surfactant head group region. As N,N-dimethyldodecylamine-N-oxide does not exhibit a cloud point it was not possible to determine the mode of oil incorporation in microemulsions prepared with this surfactant.

Regulation of airway ciliary activity by Ca2+: simultaneous measurement of beat frequency and intracellular Ca2+.

Airway ciliary activity is influenced by [Ca2+]i, but this mechanism is not fully understood. To investigate this relationship, ciliary activity and [Ca2+]i were measured simultaneously from airway epithelial ciliated cells. Ciliary beat frequency was determined, for each beat cycle, with phase-contrast optics and high-speed video imaging (at 240 images s-1) and correlated with [Ca2+]i determined, at the ciliary base, by fast imaging (30 images s-1) of fura-2 fluorescence. As a mechanically induced intercellular Ca2+ wave propagated through adjacent cells, [Ca2+]i was elevated from a baseline concentration of 45 to 100 nM, to a peak level of up to 650 nM. When the Ca2+ wave reached the ciliary base, the beat frequency rapidly increased, within a few beat cycles, from a basal rate of 6.4 to 11.6 Hz at 20-23 degrees C, and from 17.2 to 26.7 Hz at 37 degrees C. Changes in [Ca2+]i, above 350 nM, had no effect on the maximum beat frequency. We suggest that airway ciliary beat frequency is 1) controlled by a low range of [Ca2+]i acting directly at an axonemal site at the ciliary base and 2) that a maximum frequency is induced by a change in [Ca2+]i of approximately 250-300 nM.

The human bronchial epithelial cell line 16HBE14o- as a model system of the airways for studying drug transport.

The 16HBE14o- cell line, which forms polarised cell layers in vitro, provides a promising opportunity to develop a convenient epithelial cell culture model in which respiratory drug transport can be evaluated in vitro. This study investigated the effect of cell seeding density, collagen substratum and time in culture on the development of barrier properties in this cell line, after which the permeability of the 16HBE14o- cell layers to a series of solutes was studied. Seeding cells at a density of 2.5 x 10(5) cells per cm(2) on a monofibrillar Vitrogen-100 collagen substratum, followed by culture at an air-liquid interface for 6 days resulted in cell layers with a transepithelial electrical resistance (TER) of 247+/-47 omegacm(2) and an apparent permeability coefficient of 2.5 x 10(-6)cms(-1) for mannitol. The permeability of the 16HBE14o- cells to hydrophilic molecules (logP<1.9) was of an order of magnitude greater than that of typical alveolar cell cultures, possibly reflecting barrier properties more representative of the airways. More lipophilic drugs showed higher permeabilities indicating a sigmoidal relationship between permeability and lipophilicity similar to that observed for solute transport across primary cultured epithelial cell layers. These results indicate that under appropriate culture conditions, 16HBE14o- cell layers provide a discriminatory barrier to solute transport.

Light-scattering investigations on dilute nonionic oil-in-water microemulsions

Dilute 3-component nonionic oil-in-water microemulsions formulated with either a polyoxyethylene surfactant (C18∶1E10 or C12E10) or the alkylamine-N-oxide surfactant, DDAO (C12AO), and containing either a triglyceride or an ethyl ester oil have been examined using dynamic and static light-scattering techniques. Analysis of the results showed distinct differences in the tested oils mode of incorporation into the microemulsion droplets, with both the molecular volume of the oil and the hydrophobic chain length of the surfactant being important. For example, microemulsions formulated by C18∶1E10 and containing one of the larger molecular volume oils (that is, either a triglyceride, Miglyol 812, or soybean oil) or the ethyl ester of fatty acid oil, ethyl oleate, exhibited first a decrease and then an increase in hydrodynamic size and surfactant aggregation number, suggesting that the asymmetric C18∶1E10 micelles became spherical upon the addition of a small amount of oil and grew thereafter because of further oil being incorporated into the core of the spherical microemulsion droplet. A similar conclusion of sphericity could not be drawn for microemulsions stabilized by C18∶1E10 and containing one of the oils smaller in molecular volume (namely tributyrin, ethyl butyrate, or ethyl caprylate) where neither the aggregation number nor the hydrodynamic radius changed much upon the addition of oil. This result suggested that these oils were preferentially located in the interfacial surfactant monolayer, behaving in much the same way as a cosurfactant. A different trend of results, however, was seen for microemulsions prepared using C12E10 and C12AO, most likely because these surfactants produced approximately spherical micelles. In this case, the microemulsions containing the oils larger in molecular volume tended to exhibit an increase in surfactant aggregation number and hydrodynamic size, suggesting the growth of spherical micelles, while the smaller oils (in particular ethyl butyrate) caused a significant decrease in surfactant aggregation number incompatible with their being incorporated into the centre of the droplet, suggesting that the oils were being located in the interfacial surfactant monolayer. These results suggest that the various oils are incorporated into the microemulsions in very different ways.

Evaluation of the bioadhesive properties of hyaluronan derivatives: Detachment weight and mucociliary transport rate studies

Hyaluronan of various molecular weights, and microspheres made from several of its esters, were assessed for adhesiveness in vitro by means of detachment weight and mucociliary transport rate. Microspheres made from esters of alginic acid and gellan gum were also evaluated. The results were compared with those obtained from Carbopol 974 which was used as a positive control. Hyaluronan and its autocross-linked esters displayed comparable adhesion to Carbopol in both studies. All microsphere preparations were less adhesive than Carbopol (p < 0.05, Mann-Whitney U-test) when tested for detachment weight (using mucosal epithelium) and mucociliary transport rate. Adhesion to a mucus gel was similar for most preparations. Hyaluronan has been shown to possess excellent adhesion in vitro. Although formulation of hyaluronan into microspheres tends to reduce its inherent adhesive properties, the microspheres formed displayed significantly decreased mucociliary clearance. The inclusion of drug into such a biodegradable and biocompatible dosage form is an attractive prospect for transmucosal delivery.

Mucociliary clearance and drug delivery via the respiratory tract

Disruption of normal mucociliary clearance in the airways is undesirable due to the associated risk of increased respiratory disease. Accordingly, pharmaceutical formulations delivered via the respiratory tract should not cause irreversible damage to the mucociliary clearance apparatus. On the other hand, mucociliary clearance reduces the amount of time a formulation is in contact with its absorbing membrane with important consequences for the bioavailability of the delivered drug. In this review the components of the mucociliary apparatus are described and methods of measuring ciliary beat frequency, mucociliary transport and mucociliary clearance are reviewed. Substances known to alter components of mucociliary clearance are detailed as well as efforts that have been made to overcome the effect of mucociliary clearance on drug absorption.

Mitigation of surfactant erythrocyte toxicity by egg phosphatidylcholine

Polyoxyethylene alkyl ether surfactants have been shown to have excellent penetration enhancing abilities although they are associated with a high level of local toxicity. We have compared the toxicity of a range of polyoxyethylene alkyl ethers (Brij 96, Brij 76, Brij 56, 10 lauryl ether and 9 lauryl ether) to an anionic surfactant (sodium dodecyl sulphate (SDS)), an ampholytic surfactant (lysophosphatidylcholine) and a cationic surfactant (tetradecyltrimethylammonium bromide (TTAB)), in the presence and absence of egg phosphatidylcholine.

L-(6,7-dimethoxy-4-coumaryl) alanine: an intrinsic probe for the labelling of peptides

The asymmetric synthesis, spectral properties and incorporation into solid phase peptide synthesis are described for L-(6,7-dimethoxy-4-coumaryl)alanine (Dca). Dca has great utility as a specific and highly sensitive intrinsic probe for fluorescence labelling and quantitation of peptides and proteins.

Systematic investigations of the influence of molecular structure on the transport of peptides across cultured alveolar cell monolayers

AbstractPurpose. To determine how the structures of peptides influence theiralveolar permeability. Methods. The studies were performed using 14 synthetic ‘model’peptides, labelled with a novel, non-intrusive amino acid fluorophore, andtheir transport studied using rat alveolar cell monolayers cultured onpermeable supports. Results. The passage of the peptides across the epithelial cellmonolayers is shown to be primarily paracellular, with an inverse dependenceon molecular size, and an enhanced flux observed for cationic peptides.The apparent permeability coefficients (Papp) for the peptides(together with those for other organic solutes, taken from the literature) areshown to be well-modelled assuming two populations of ‘pores’ in themonolayers, modelled as cylindrical channels of radii 15 Å and 22nm. The former pores are shown to be numerically equatable withthe monolayer tri-junctional complexes, and the latter are taken asmonolayer defects. Conclusions. The various monolayer Papp values correlatewell with the results from in vivo transport experiments, and the conclusion isdrawn that the pulmonary delivery of peptide drugs is perfectlyexploitable.

Biofilm formation and changes in bacterial cell surface hydrophobicity during growth in a CAPD model system

Peritonitis is a frequent complication of continuous ambulatory peritoneal dialysis (CAPD), with patients suffering recurrent attacks. The microorganisms most frequently implicated in the infection are the skin microflora, in particular, the coagulase‐negative staphylococci such as Staphylococcus epidermidis. These microorganisms gain access to the peritoneal cavity via the in‐dwelling silicone rubber catheter in the abdominal wall and often persist as biofilms on the surface of the catheter. The surface characteristics of S. epidermidis were monitored during growth in a CAPD in‐vitro model together with their ability to adhere to silicone rubber substrata. Fresh dialysis fluid exerted an injurious effect on the cells leading to a decrease in cell numbers but during the simulated dialysis period the cells adapted to the applied stresses. Over a 96‐h period in the model both a clinical isolate and a skin isolate of S. epidermidis adopted a more hydrophobic phenotype. The data presented here show that the bacteria grown in this in‐vivo reflective CAPD model continually adapt to their environment and become more tolerant to the stresses imposed. The adapted cells were seen to colonise silicone rubber substrata.