A D van Eyk

A comparative light-microscopic, electron-microscopic and chemical study of human vaginal and buccal epithelium.

The scarcity of sizeable specimens of normal oral mucosa for experimental purposes has hampered research on oral epithelium. Because large specimens of viable human vaginal mucosa are readily available and because vaginal and buccal epithelia are microscopically similar, vaginal mucosa has been used successfully to establish a human cyst model in experimental animals. The ultrastructure and distribution of keratin filaments in these epithelia are also similar, as is their permeability to water and a number of chemical substances. Therefore, if vaginal mucosa could be substituted for buccal mucosa it would expedite research on the epithelium of buccal mucosa. To strengthen further the concept that vaginal epithelium could replace buccal epithelium in certain experimental studies, the thickness of these epithelia, their patterns of surface keratinization, the presence or absence of intercellular lipid lamellae and their lipid contents were now compared. Thirty-three specimens of vaginal mucosa from postmenopausal women and 36 of buccal mucosa were investigated. To compare the thickness of the epithelial layers the number of cell layers in sections of 20 vaginal and 20 buccal mucosal specimens were counted in the three thickest and three thinnest regions of each specimen. Surface keratinization was evaluated on sections stained with the Picro-Mallory method. To demonstrate lipid lamellae two vaginal and two buccal mucosa specimens were examined electron microscopically after normal fixation and postfixation in ruthenium tetroxide. Following solvent extraction of 11 vaginal and 14 buccal epithelia, quantitative lipid analyses were performed using thin-layer chromatography. No statistically significant differences were found between the maximum and minimum number of epithelial cell layers. The patterns of surface keratinization and the distribution and appearance of the lipid lamellae in the intercellular spaces were similar. The lipid composition of the two epithelia corresponded, except for the cholesterol esters and glycosylceramides, which were higher in buccal epithelium. Ceramides for vaginal epithelium and triglycerides for buccal epithelium were not determined. Based on structural similarities, a similar lipid composition and earlier findings, it is concluded that vaginal epithelium can be used as a substitute for buccal epithelium in certain in vitro, and possibly for in vivo, studies.

Permeation of sumatriptan through human vaginal and buccal mucosa.

Continued interest in the various routes by which sumatriptan may be administered prompted us to investigate its passage through buccal mucosa. Because human buccal mucosa is scarce, we proposed using the relatively abundant vaginal mucosa, which has been shown to have comparable diffusion rates for a number of widely varying molecules, as a model of buccal mucosa. In addition, by comparing these two tissues with respect to their permeability to sumatriptan, the human vaginal/buccal mucosa model could be further evaluated. Clinically healthy human vaginal and buccal mucosa specimens were used in the permeability studies. Permeability to sumatriptan was determined using a continuous flow‐through diffusion system in the presence and absence of permeation enhancers. No statistically significant differences in permeability could be demonstrated for both mucosae toward sumatriptan. Flux values obtained in the absence and presence of glycodeoxycholate and lauric acid (1:1 molar ratio) to sumatriptan of buccal and vaginal mucosa, respectively, were not significantly different. The results obtained further support the hypothesis of the vaginal/buccal mucosal in vitro permeability model and suggest that this model may be used in conjunction with various absorption enhancers. Further studies on the buccal route of absorption of sumatriptan are thus warranted.

Diffusion of Water across Human Skin in the Presence of Two Barrier Creams

The in vitro permeability of tritiated water through fresh and frozen human skin was evaluated in the presence and absence of two different barrier creams. Treated (10 min) and untreated fresh and frozen human skin disks (4 mm in diameter) were mounted in flow cells of a continuous flow-through diffusion apparatus. Buffer/tritiated water was collected from the acceptor chambers at 2-hour intervals for a total of 20 h and counted in a liquid scintillation counter. The results indicated that both barrier creams lowered the average flux rates of tritiated water through fresh and frozen skin, but no significant differences could be detected between the two preparations. However, different results may be obtained when compounds with molecular weights much higher than water are used.

Transvaginal Diffusion of Synthetic Peptides

Topical microbicide peptides are being developed to combat the transfer of HIV, but little is known about the permeation of these compounds through vaginal epithelium. The object of the present study is to investigate the in vitro permeation of synthetic transport peptides through vaginal mucosa. The permeation kinetics of three FITC (fluorescein isothiocyanate)-labelled peptides MEA-5 (Mw = 2911.4 Da), MDY-19 (Mw = 2409.5 Da) and PCI (Mw = 2325 Da) across human vaginal mucosa was studied by means of a continuous flow-through diffusion system. Permeability studies were conducted at concentrations of 1 mM, 0.75 mM and 0.5 mM in PBS buffer at 37°C and 20°C, respectively, and over a time period of 24 h, using fluorospectrophotometry as detection method. Effects of a surfactant on MDY-19 permeation and de-epithelialisation of the vaginal mucosa were also studied. Statistical tests used included an ANOVA and Duncans multiple range test to establish steady state diffusion kinetics. All three peptides readily penetrate vaginal mucosa. Microbicides may be coupled to MDY-19 and PCI to be transported transmucosally. Although increased size of the peptide/microbicides complex may decrease mucosal permeability, this could possibly be overcome by the addition of a permeation enhancer, e.g. a surfactant. Removal of the vaginal epithelium increased the flux rates of the peptides across the mucosa and may have implications for a more rapid uptake of these and other microbicides in vivo. Concentration- and temperature- dependency of peptide flux rates must be taken into consideration when performing in vitro permeability studies.

Diffusion of two potential anti-HIV microbicides across intact and de-epithelialised, human vaginal mucosa

The lectins derived from Hippeastrum hybrid (Hippeastrum hybrid agglutinin, HHA) and from the stinging nettle Urtica dioica (UDA) have been investigated as anti-HIV microbicides. The present study was conducted to determine their diffusion through intact and de-epithelialised human vaginal epithelium. Both lectins were labelled with fluorescein isothiocyanate groups (FITC) and analysed by SDS-polyacrylamide gel electrophoresis. While UDA appeared to be fairly homogeneous with an average Mw of ∼ 8.5 kDa, HHA was a heterogeneous mixture of compounds with Mws ranging between ∼ 13 and 52 kDa. Fresh human vaginal mucosa was snap-frozen in liquid nitrogen and stored at −85°C. Prior to an experiment, the tissue was defrosted to 20°C in PBS buffer, pH 7.4, and placed in the seven flow cells of a flow-through perfusion apparatus. Either FITC-labelled HHA or UDA was then pipetted into the donor chamber of the flow cell. Samples from each flow cell were collected every 2 hours (1.5 ml/h) over a 24-hour period and analysed by fluorospectrophotometry. Permeability experiments were repeated with vaginal mucosa specimens from which the epithelial layers had been removed by heat-stripping. Both lectins diffused through vaginal mucosa at rates proportionate to their average molecular weights, the flux rates of the smaller UDA being ∼ 5x higher than that of the larger HHA. Removal of the vaginal epithelium increased the flux rates of both HHA and UDA across the mucosa and this may have implications for a more rapid in vivo uptake of these lectins when used as anti-HIV microbicides.

Physicochemical Characteristics of Molecules and Their Diffusion across Human Vaginal Mucosa

The diffusion rate of permeant molecules through mucosal tissue depends on the physicochemical characteristics of the molecules themselves as well as the properties of the tissue. In this study the diffusion kinetics of various molecules was examined through intact as well as de-epithelialised human vaginal mucosa. The molecules studied included tritium-labelled water, 17β-estradiol, reduced-arecoline, vasopressin, oxytocin, bradykinin, tacrolimus, cyclosporin A, dihydro-alprenolol, cimetidine and benzylpenicillin. Freshly harvested human vaginal tissue was frozen in liquid nitrogen and stored at −85°C. A flow-through diffusion apparatus was used for the in vitro permeability studies (24 h, 20°C, 1.5 ml/h). The mean estimated – or mean steady-state flux values for all the molecules studied across intact human vaginal mucosa, were generally found to be lower than those of the corresponding de-epithelialised tissue. Using an F-test and comparing whole curves, statistically significant differences in the diffusion rates of tacrolimus, reduced-arecoline, vasopressin, bradykinin, benzylpenicillin, water and cimetidine were found when comparing intact and de-epithelialised vaginal mucosa. Generally, smaller permeant molecules diffused at a higher rate than larger molecules. The epithelial layer retarded the diffusion rate of molecules carrying charges at physiological pH. Damage to the epithelial layer did not necessarily increase the diffusion rate of all molecules tested and small lipophilic molecules did not necessarily diffuse at higher rates than hydrophilic molecules.