Medications introduced into the systematic circulation must be transported across biological barriers such as skin, gastrointestinal, or bronchial epithelia, which can alter their kinetic and metabolic profiles. It is, therefore, important to understand diffusion kinetics across barrier membranes when choosing a dosing regime that will elicit the greatest cellular response. An in vitro system that combines membrane transport studies with a downstream cell culture chamber has been developed. The system has been tested with skin and a small intestine model (Caco-2 cell monolayers) as barriers, the peroxovanadium compound [VO(O2)2 1, 10 phenanthroline] bpV(phen), as the test chemical, Hep-G2 (liver) as the test cells, and glucose consumption as the test assay. Peroxovanadium has insulin mimetic properties and has been previously demonstrated to effectively lower blood glucose levels in diabetic rats when administered transdermally. A dose of 10 mM bpV(phen) placed on the skin epidermis with a continuous iontophoretic current of 0.5 mA/cm2 for 4.5 h led to a net 22% increase in glucose consumption by Hep-G2 cells. The same dose of bpV(phen) passively diffusing across a Caco-2 cell monolayer led to an increase in glucose consumption by Hep-G2 cells of 23%. This system is highly versatile and can be used to study many other processes, involving a variety of biological membranes, cell types, chemicals and assays, making it a valuable research tool.

The potential for using antisense compounds as therapeutic agents has generated great enthusiasm. Strategies for delivery of these compounds are, therefore, of great interest. Transdermal iontophoresis has been used successfully as an enhancement technique for the transdermal delivery of these compounds in vitro. The effectiveness of using percutaneous penetration as a means to deliver therapeutic levels of these compounds in vivo, however, remains to be demonstrated. The purpose of this work was to demonstrate the ability of iontophoretically delivered compounds to alter enzyme levels in the intact rat. A C5 propyne-modified phosphorothioate oligonucleotide (PS-ODN) targeted to the cytochrome p450-3A2 (CYP3A2) mRNA translational start site and the reverse sequence, used as a control, were synthesized. A patch containing either an oligonucleotide or a buffer control was placed on the animal's back, and an iontophoretic current of 0.5 mA/cm2 was applied for 3.5 hours. Twenty-four hours later, CYP3A2 levels were measured noninvasively using the midazolam-induced sleeping rat model. Liver and small intestinal microsomes were made after completion of sleep studies and assayed for CYP3A2, CYP1A1/2, CYP2B1/2, and CYP2E1. Midozolam-treated animals with antisense to CYP3A2 slept significantly longer than did the controls (p < 0.05). CYP3A2 levels were significantly lower in liver microsomes from antisense-treated animals than in either buffer control (p < 0.001) or reverse sequence animals (p < 0.05). The reverse sequence was also significantly different from the buffer control (p < 0.01), indicating a nonspecific effect of the PS background. Nontarget cytochrome levels were not altered by treatment. There were no significant differences in small intestine CYP3A2 levels between treatment groups. These data demonstrate that transdermally delivered PS-ODN can reach concentrations sufficient to induce changes in specific target enzymes in vivo. Further studies are warranted to investigate potential uses for these molecules.

Topical application of antisense PMO in rats is a feasible delivery strategy for gene targets in liver and underlying skin.

The peroxovanadium compound VO(O2)2 1,10 phenanthroline (bpV(phen)) is capable of lowering blood glucose levels. It is not available in oral form, but it is effective when delivered transdermally. Iontophoresis can significantly reduce the lag time of this response in vivo when compared with passive penetration. To better mimic in vivo insulin release, we explored the effects of various iontophoretic current durations on dermal penetration of bpV(phen). Iontophoretic transport was not related to total applied charge, as steady-state flux was equivalent for current durations ranging from 15 minutes to 9 hours. We hypothesized that the unexpectedly large transport after just 15 minutes of current was caused by an increase in passive penetration of bpV(phen) induced by iontophoresis. Iontophoretic pretreatment with the chelating agent 1,10 phenanthroline increased passive penetration of bpV(phen), whereas neither the nonchelating isomer 1,7 phenanthroline nor the less potent chelator EDTA were effective. The use of 1,10 phenanthroline as a penetration enhancer for other chemicals was examined with the amino acids alanine and leucine. Fifteen minutes of 1,10 phenanthroline iontophoresis enhances alanine transport 11.4-fold over passive, whereas the 1,7 phenanthroline increased transport by a factor of 4.6 and the iontophoretic control of ethanol by 1.9. Surprisingly, phenanthroline did not enhance 3H leucine penetration. The reasons for this selectivity are not clear and warrant further investigation. Overall, the data suggest that chelating agents, specifically 1,10 phenanthroline, may be used as penetration enhancers for the delivery of certain compounds.

Oligonucleotides have been extensively studied for their potential as therapeutic agents. Phosphorothioate oligonucleotides have been demonstrated to be particularly useful due to their stability against nucleases, their ability to be internalized by many cell types, and the ease with which they hybridize with target mRNA. These compounds have previously been delivered across the skin with the aid of iontophoresis. During transdermal delivery, the first viable cells exposed to the oligonucleotides are the keratinocytes. The purpose of this study was to determine the relationship between internalization of these compounds by keratinocytes and their transport across the skin. The in vitro uptake of 15 different fluorescently labeled phosphorothioate oligonucleotides into human keratinocytes was quantitatively measured with a fluorometer. Photomicrographs of keratinocytes indicate diffuse cytoplasmic and nuclear localization. The ability of these molecules to enter cells was linearly related to size. Cellular uptake data were inversely correlated with previously reported steady-state transport levels of oligonucleotides that had been transdermally delivered by iontophoresis across hairless mouse skin. Oligonucleotides that readily entered keratinocytes had a decreased ability to penetrate skin under iontophoretic conditions. The results indicate that oligonucleotide sequences may be designed for treating skin diseases (high uptake, low transport) or systemic disorders (low uptake, high transport).