Simultaneous transport and metabolism of ethyl nicotinate in hairless rat skin after its topical application: the effect of enzyme distribution in skin

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Abstract

An in vitro permeation study of ethyl nicotinate (EN) was carried out using excised hairless rat skin, and simultaneous skin transport and metabolism of the drug were kinetically followed. Fairly good steady-state fluxes of EN and its metabolite nicotinic acid (NA) through the skin were obtained after a short lag time for all the concentrations of EN applied. These steady-state fluxes were not proportional to the initial donor concentration of EN: EN and NA curves were concave and convex, respectively, which suggests that metabolic saturation from EN to NA takes place in the viable skin at higher EN application. Further permeation studies of EN or NA were then carried out on full-thickness skin or stripped skin with an esterase inhibitor to measure their permeation parameters, such as partition coefficient of EN from the donor solution to the stratum corneum and diffusion coefficients of EN and NA in the stratum corneum and the viable epidermis and dermis. Separately, enzymatic parameters (Michaelis constant Km and maximum metabolism rate Vmax) were obtained from the production rate of NA from different concentrations of EN in the skin homogenate. The obtained permeation and enzymatic parameters were then introduced to differential equations showing Fick’s second law of diffusion in the stratum corneum and the law with Michaelis–Menten metabolism in the viable epidermis and dermis. The calculated steady-state fluxes of EN and NA by the equations were very close to the obtained data. We then measured the esterase distribution in skin microphotographically using fluorescein-5-isothiocyanate diacetate. A higher enzyme concentration was observed in the epidermal cells and near hair follicles than in the dermis. Simulation studies using the even and the partial enzyme distribution models suggested that no significant difference between the models was observed in the skin permeations of EN and NA, whereas concentration–distance profiles of EN and NA were very different. This finding suggests that the total amount of enzyme in skin which converts EN to NA is a determinant of the metabolic rate of EN in skin. The present approach is a useful tool for analyzing simultaneous transport and metabolism of many drugs, especially those showing Michaelis–Menten type-metabolic saturation 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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