SV40-transformed human corneal keratocytes: optimisation of serum-free culture conditions.

Aiming at the replacement of animal experiments in eye irritation testing, we have established a multilay ered cornea model comprising the co-culture of all three corneal cell types. It was the objective of this study to optimise serum-free culture conditions to preserve both growth and phenotype of an SV40-immortalised human corneal keratocyte cell line (HCK). Our results revealed that HCK continue to proliferate in both monolayer cultures as well as after seeding in a collagen matrix and resemble primary corneal keratocytes in morphology and functional characteristics under defined serum-free conditions. Furthermore, HCK were shown to transform into activated corneal fibroblast phenotypes in response to serum and TGF(beta)1. In summary, HCK cells mimic their in vivo (primary) precursors, both in sustaining the quiescent keratocyte phenotype (serum-starved conditions) and in responding to growth factor stimulation. Hence, this cell line may provide a useful tool to study the toxicity and wound healing response of corneal keratocytes in vitro.

Characterisation of human corneal epithelial cell cultures maintained under serum-free conditions.

Three-dimensional tissue constructs have been proposed as in vitro screening models for ocular irritancy. Based on our previous studies, in which a full-thickness corneal model based exclusively on SV40-immortalised cell lines was generated, we have currently evaluated the effects of a range of commercially-available cell culture media on several cellular parameters in cultures of a human corneal epithelial (HCE) cell line. This cell line was used in an attempt to establish a rational basis for the development of serum-free culture media for the assembly and long-term tissue culture of full-thickness corneal models. Briefly, we investigated the impact of serum-free culture on the proliferation, morphology, barrier function and cytokine expression of HCE cells. The number of cell layers and the epithelial differentiation were evaluated by histology. Barrier properties were characterised via the determination of transepithelial electrical resistance (TEER), fluorescein permeation, and the expression of the tight junction-related protein, zona occludin 1 (ZO-1). The cytokine expression pattern in response to serum-free culture was measured by using an antibody array system. Our results revealed that both the morphology and the barrier function of the epithelial constructs were comparable to those of human donor corneas, when serum-free media were supplemented with ascorbic acid, calcium, hydrocortisone and retinoic acid. Under these conditions, the artificial epithelium based on serum-free HCE cultures represented a valid model for the natural ocular surface.

Reconstructed epidermis and full-thickness skin for absorption testing: influence of the vehicles used on steroid permeation.

A protocol for percutaneous absorption studies has been validated, based on the use of reconstructed human epidermis (RHE) and aqueous solutions of test substances. However, it is often the case that it is more-complex formulations of drugs or chemicals which will make contact with the skin surface. To investigate whether RHE and the reconstructed full-thickness skin model (FT-model) can be used to predict uptake from formulations, we compared the permeation of hydrocortisone and testosterone when applied in emulsion form and as a solution containing the penetration enhancer, ethanol. Human and pig skin and a non-cornified alveolar model served as references. The results were compared with steroid release from the formulations. The permeation rates of the steroids were ranked as: alveolar model >> RHE > FT-model, pig skin > human skin. In accordance with the rapid hydrocortisone release from the formulations, the permeation rates of this steroid exceeded those of testosterone. Only minor differences were observed when comparing the testosterone formulations, in terms of release and permeation. However, the ranking of the permeation of the hydrocortisone formulations was: solution > w/o emulsion > o/w emulsion, which permitted the elucidation of penetration enhancing effects, which is not possible with drug release studies. Differences in penetration were most obvious with native skin and reconstructed tissues, which exhibited a well-developed penetration barrier. In conclusion, RHE and skin preparations may be useful in the development of topical dermatics, and in the framework of hazard analysis of toxic compounds and their various formulations.

Solubility and transdermal permeation properties of a dehydroepiandrosterone cyclodextrin complex from hydrophilic and lipophilic vehicles.

The permeation ability of a compound is due principally to its concentration in the vehicle and to its aptitude to cross the stratum corneum of the skin. In this work ex-vivo permeation studies on newly developed formulations containing dehydroepiandrosterone (DHEA) were carried out to investigate vehicles that increase drug permeation through the skin. To enhance the solubility of DHEA, its complex form with alpha-cyclodextrin was used. In addition, the two forms (pure drug and complex form) were introduced in hydrophilic (water), lipophilic (paraffin oil), and microemulsion vehicles to evaluate the synergic effect of cyclodextrins and microemulsion vehicles on solubility and permeation. From the results, DHEA solubility is notably conditioned by the type of the vehicle used: the highest solubilities (both for pure and complex drug forms) were obtained with microemulsion, followed by paraffin oil and water. Moreover, in all the studied vehicles, the c-DHEA was more soluble than DHEA. Permeation profile fluxes showed very interesting differences. That reflect the varying drug forms (pure drug and complex form), vehicles used, and drug concentrations in the vehicles. The major flux was obtained in complex of DHEA with alpha-cyclodextrins in the microemulsion vehicle. Therefore, this type of vehicle and drug form would be very useful in the development of a topical formulation containing DHEA.

Skin permeation study of dehydroepiandrosterone (DHEA) compared with its alpha-cyclodextrin complex form.

Several studies have shown that dehydroepiandrosterone (DHEA) has promising uses in a large range of pathologies including age-related illnesses (osteoporosis, atherosclerosis), immune dysfunctions, and cancer. A long-term oral dosage form would be favorable, but this type of medication has not been developed yet because of the important first-pass effect and low and variable bioavailability. A proposed alternative strategy is the preparation of DHEA for transdermal permeation, allowing direct absorption in the systemic circulation. DHEA has shown good permeation properties through the stratum corneum, however its poor water solubility limits its dosage form concentration and represents the major obstacle to the formulation of a reservoir transdermal system. However, alpha-cyclodextrins have been shown to improve the solubility, the dissolution properties, and the partition coefficient of DHEA. This paper deals with the formulation and evaluation of a transdermal gel containing an alpha-cyclodextrin complex with DHEA. In in vitro permeation tests on porcine skin, the formulation containing the complex improved the permeation flux of DHEA. The study also shows that a gel formulation improved drug permeation with respect to simple water suspension.