Mary Kremer

Delivery of bioactive peptides and proteins across oral (Buccal) mucosa

The identification of an increasing array of highly potent, endogenous peptide and protein factors termed cytokines, that can be efficiently synthesized using recombinant DNA technology, offers exciting new approaches for drug therapy. However, the physico-chemical and biological properties of these agents impose limitations in formulation and development of optimum drug delivery systems as well as on the routes of delivery. Oral mucosa, including the lining of the cheek (buccal mucosa), floor of mouth and underside of tongue (sublingual mucosa) and gingival mucosa, has received much attention in the last decade because it offers excellent accessibility, is not easily traumatized and avoids degradation of proteins and peptides that occurs as a result of oral administration, gastrointestinal absorption and first-pass hepatic metabolism. Peptide absorption occurs across oral mucosa by passive diffusion and it is unlikely that there is a carrier-mediated transport mechanism. The principal pathway is probably via the intercellular route where the major permeability barrier is represented by organized array of neutral lipids in the superficial layers of the epithelium. The relative role of aqueous as opposed to the lipid pathway in drug transport is still under investigation; penetration is not necessarily enhanced by simply increasing lipophilicity, for other effects, such as charge and molecular size, also play an important role in absorption of peptide and protein drugs. Depending on the pharmacodynamics of the peptides, various oral mucosal delivery systems can be designed. Delivery of peptide/protein drugs by conventional means such as solutions has some limitations. The possibility of excluding a major part of drug from absorption by involuntary swallowing and the continuous dilution due to salivary flow limits a controlled release. However these limitations can be overcome by adhesive dosage forms such as gels, films, tablets, and patches. They can localize the formulation and improve the contact with the mucosal surface to improve absorption of peptides and proteins. Addition of absorption promoters/permeabilizers in bioadhesive dosage forms will be essential for a successful peptide/protein delivery system.

The Innate Immune System Is Activated by Stimulation of Vaginal Epithelial Cells with Staphylococcus aureus and Toxic Shock Syndrome Toxin 1

ABSTRACT Despite knowledge of the effects of toxic shock syndrome (TSS) toxin 1 (TSST-1) on the adaptive immune system, little is known about stimulation of the innate immune system, particularly epithelial cells. This study investigated the interactions of TSS Staphylococcus aureus and TSST-1 with human vaginal epithelial cells (HVECs) and porcine mucosal surfaces. When cocultured with HVECs for 6 h, TSS S. aureus MN8 proliferated, formed aggregates on the HVEC surfaces, and produced exotoxins. Receptor binding studies showed that 35S-TSST-1 bound to 5 × 104 receptors per HVEC, with saturation at 15 min. Affymetrix Human GeneChip U133A microarray analysis determined S. aureus MNSM (100 bacteria/HVEC) caused at least twofold up- or down-regulation of 410 HVEC genes by 6 h; these data were also confirmed with S. aureus MN8. TSST-1 (100 μg/ml) caused up- or down-regulation of 2,386 HVEC genes by 6 h. In response to S. aureus, the HVEC genes most up-regulated compared to those in controls were those coding for chemokines or cytokines—MIP-3α, 478-fold; GRO-α, 26-fold; GRO-β, 14-fold; and GRO-γ, 30-fold—suggesting activation of innate immunity. TSST-1 also caused up-regulation of chemokine/cytokine genes. Chemokine/cytokine gene up-regulation was confirmed by enzyme-linked immunosorbent assays measuring the corresponding proteins induced by S. aureus and TSST-1. S. aureus MN8, when incubated with porcine vaginal tissue, increased the flux of 35S-TSST-1 across the mucosal surface. This was accompanied by influx of lymphocytes into the upper layers of the tissue. These data suggest innate immune system activation through epithelial cells, reflected in chemokine/cytokine production and influx of lymphocytes, may cause changes in vaginal mucosa permeability, facilitating TSST-1 penetration.

Penetration of toxic shock syndrome toxin-1 across porcine vaginal mucosa ex vivo: permeability characteristics, toxin distribution, and tissue damage.

OBJECTIVE The purpose of this study was to evaluate transvaginal penetration of toxic shock syndrome toxin-1 and its effects on permeability and tissue integrity in vitro with the use of excised porcine vaginal mucosa. STUDY DESIGN Permeability to tritiated water (1 and 10 microg/mL applied toxin) and transmucosal flux of (35)S-methionine-labeled toxic shock syndrome toxin-1 (10 and 20 microg/mL) for up to 12 hours were assessed with the use of a continuous flow perfusion system. The location of labeled toxin that penetrated the mucosal tissue strata was determined. The integrity of toxin-treated, intact, scalpel-incised tissue was evaluated histopathologically. RESULTS Toxic shock syndrome toxin-1 caused a non-dose-dependent increase in mucosal permeability and traversed the intact mucosa at a low rate without disrupting tissue integrity. In incised vaginal mucosa, toxic shock syndrome toxin-1 induced subepithelial separation and atrophy that were analogous to clinically relevant vaginal lesions that were reported in fatal cases of menstrual toxic shock syndrome. CONCLUSION An in vitro model could be used to demonstrate that toxic shock syndrome toxin-1 permeates the vaginal mucosa and distributes throughout the tissue. Histologic evaluation of tissues that were exposed to toxic shock syndrome toxin-1 demonstrated lesions that were similar to those lesions that were reported in cases of menstrual toxic shock syndrome.

Organization of the intercellular spaces of porcine epidermal and palatal stratum corneum: a quantitative study employing ruthenium tetroxide.

Previous studies have demonstrated that the intercellular spaces of the stratum corneum contain multilamellar lipid sheets with variable ultrastructure in addition to desmosomes or desmosomal remnants. The intercellular lamellae are thought to provide a permeability barrier whereas the desmosomes are responsible for cell-cell cohesion. In this study, transmission electron microscopy of RuO4-fixed tissue was used to compare the proportions of the intercellular spaces in epidermal and palatal stratum corneum occupied by desmosomes and by different patterns of lamellae. Desmosomes are more abundant in palatal than in epidermal stratum corneum (46.9 vs 15.0% length of intercellular space). In epidermis the most frequent lamellar arrangements involve 3 (23.5%) or 6 (24.2%) lucent bands with an alternating broad-narrow-broad pattern, whereas the most frequent lamellar arrangements in palatal tissue are 2 (17.2%) or 4 (10.5%) lucent bands of uniform width. Most of the nondesmosomal portion of the intercellular space in palatal stratum corneum was dilated and had elongated lamellae at the periphery and short disorganized lamellae and amorphous electron-dense material in the interior. It is concluded that the multilamellar lipid sheets are less extensive in palatal than in epidermal stratum corneum, which could explain the greater permeability of the palate.

Oral Mucosal Permeability and Stability of Transforming Growth Factor Beta-3 In Vitro

AbstractPurpose. To investigate the permeability and localization of topically applied 125I-TGF-β3 in porcine floor-of-mouth mucosa as a function of concentration and exposure. Methods. The 125I-TGF-β3 diluted in three different vehicles was applied to the tissue samples mounted in perfusion cells maintained at 37°C. Flux and Kp values were calculated from the perfusate collected over a 24 hour period. The quantity of 125I-TGF-β3 present in the tissue was determined by horizontal sectioning and subsequent counting. The stability of 125I-TGF-β3 in saliva and in the tissue was analyzed by SDS polyacrylamide gradient gel electrophoresis. Results. 125I-TGF-β3 was relatively stable in saliva and in the epithelium; approximately 50% of the total counts in the deeper epithelium were resident in the 25kDa TGF-β3 homodimer. A steady-state flux was reached ∼6 hours post application and Kp value was 4.0 ± 0.6 × 10-6 (mean ± sem). Penetration of 125I-TGF-β3 to the basal cell layer was concentration dependent but reached nanomolar concentrations even after extensive surface rinsing, representing over one-thousand fold the IC50 for epithelial cell cycle arrest. Conclusions. The data suggest that topical application of TGF-β3 to the oral mucosa in an appropriate vehicle can provide effective therapeutic delivery to the tissue.

Lipids of hamster cheek pouch epithelium

The hamster cheek pouch is a much used but incompletely understood experimental model. In particular, the cheek pouch epithelial lipids, which are important for permeability barrier function as well as other aspects of epithelial biology, have not been completely characterized. In the present study, the complete lipid class composition has been determined by thin-layer chromatography in conjunction with photodensitometry. The major lipid classes were phospholipids, free sterols, and ceramides. Minor amounts of monohexosylceramides, sterol esters, fatty acids, and triglycerides were also present. Significant amounts of covalently bound ω-hydroxyceramide was also detected. Transmission electron micrographs reveal extensive, largely paired, lipid bilayers in the intercellular spaces of the stratum corneum.