Simultaneous transport and metabolism of ethyl nicotinate in hairless rat skin after its topical application: the effect of enzyme distribution in skin
Introduction
Skin is a metabolically active organ, so that one has to pay attention to the first pass metabolism in skin of a topically applied drug. A prodrug or an antedrug may be metabolized into a correspondent active parent drug or a low active compound, respectively, in skin during the membrane transport. Recently bioactive peptides and antisenses have also become candidates for transdermal delivery because they can be delivered through skin by physical penetration-enhancing methods like iontophoresis [1], phonophoresis [2], electroporation [3], and gene gun treatment. The enzymatic barrier of the skin may be a bigger hurdle for the transdermal delivery of such prodrug, antedrug, peptide and antisense than the diffusion barrier. Although a theoretical approach to the drug metabolism in skin can be beneficial in selecting a good drug candidate, it is somewhat difficult because diffusion and metabolism usually take place simultaneously in the viable skin [4]. Metabolic saturation and complex enzyme distribution in skin make a theoretical approach more difficult. Efforts toward a physical and mathematical approach of simultaneous transport and metabolism in skin were made [5], [6], [7]; however, little experimental data were presented on the metabolic saturation and enzyme distribution during drug transport through skin.
In the present study, ethyl nicotinate (EN) was selected as a model drug or prodrug, because the reaction from EN to its metabolite, nicotinic acid (NA), is saturable in viable skin when EN is applied topically at high concentration. The aim of this study was to evaluate the effect of enzyme (esterase) distribution on the simultaneous transport and metabolism of EN in skin.
Section snippets
Physical model for simultaneous transport and metabolism of EN in hairless rat skin
EN is hydrolyzed by general esterases in skin. It is assumed that the esterases are not in the stratum corneum (S.C.) but in the viable epidermis and dermis (V.E.D.). Fig. 1 shows the schematic concentration–distance profiles of EN and NA from the donor to receiver compartment through skin. Two layers, S.C. and V.E.D. are estimated in skin in view of asymmetric enzyme distribution and different diffusivities of penetrants in the layers. The thickness of the two layers is set as Lsc and Lved,
Materials
EN and NA were obtained from Wako (Osaka, Japan) and diisopropylfluorophosphate (DFP), an esterase inhibitor [11], from Sigma (St. Louis, MO, USA). Fluorescein-5-isothiocyanate diacetate was from Funakoshi (Tokyo, Japan). Other chemicals and solvents were of reagent grade and they were used without further purification.
Animals and skin membrane preparations
Male hairless rats (WBN/IL-Ht), 7–8 weeks old, were supplied by Life Science Research Center, Josai University (Sakado, Saitama, Japan) and were used in all of the animal
Simultaneous transport and metabolism of EN through skin
Fig. 2a shows typical in vitro profiles of simultaneous transport and metabolism of EN through excised hairless rat skin after application of EN aqueous solution at a concentration of 145 μmol/ml in the donor compartment of a diffusion cell set. The cumulative amounts of EN and NA in the receiver solution increased linearly with time although a short lag time was found.
The same kind of experiment was done using different concentrations of EN in the donor compartment. Fig. 2b represents the
Discussion
Skin is a metabolically active organ. Some researchers have proposed a theoretical model to describe metabolic kinetics of a drug during its diffusion across skin [6], [7]. A few mathematical solutions were found for the diffusion equation with saturation kinetics like the Michaelis–Menten equation. Unfortunately, few observed data were reported for the metabolic saturation during drug permeation through skin, probably due to low skin permeation rate of a drug. Therefore, even if a mathematical
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