Elsevier

Toxicology Letters

Volume 173, Issue 2, 10 September 2007, Pages 118-123
Toxicology Letters

The distribution of esterases in the skin of the minipig

https://doi.org/10.1016/j.toxlet.2007.07.004Get rights and content

Abstract

Skin esterases serve an important pharmacological function as they can be utilised for activation of topically applied ester prodrugs. Understanding the nature of these enzymes, with respect to their role and local activity, is essential to defining the efficacy of ester prodrugs. Minipigs are used as models to study the kinetics of absorption of topically applied drugs. Their skin has structural properties very similar to human skin. However, regional distribution differences in esterase activity from site-to-site could influence cross-species extrapolation. Investigation of the regional site variation of minipig skin esterase activity will facilitate standardization of topically applied drug studies. Furthermore, the characterization of regional skin variation, will aid in translation of minipig results to better predictions of human esterase activity. Here we report the variation in rates of hydrolysis by minipig skin taken from different regional sites, using the esterase-selective substrates: phenyl valerate (carboxylesterase), phenyl acetate (arylesterase) and p-nitrophenyl acetate (general esterase). Skin from ears and back of male minipig showed higher activity than female. Skin from minipig ears and the back showed the highest level of esterase activity and was similar to human breast skin used in vitro absorption studies. These results suggest that skin from the minipig back is an appropriate model for preclinical human skin studies, particularly breast skin. This study supports the use of the minipig, with topical application to the back, as a model for the investigation of pharmacokinetics and metabolism of ester prodrugs.

Introduction

Prodrug esters have been developed to increase the absorption of a drug when topically applied. Many prodrug esters are activated by hydrolysis in the skin, which has been shown to contain esterases. Therefore, the hydrolysis rates in the skin determine, to some extent, the human exposure levels of active drug. Absorption through minipig skin has been found to be a more appropriate model of absorption through human skin than rodent. Rodents have much higher skin esterase activity, and therefore, higher prodrug activation than either the human or minipig skin (Prusakiewicz et al., 2006, Jewell et al., 2007). Esterases are members of the hydrolase family of enzymes, which primarily hydrolyse endogenous and exogenous esters with substrate specificity overlapping with lipases (Williams, 1985, Mentlein et al., 1988). They are ubiquitously expressed in mammalian liver and extra-hepatic tissues including blood, skin, kidney, intestines, testes, brain, central nervous system and lung (Satoh and Hosokawa, 1998). Carboxylesterases with a serine active centre are inhibited by organophosphates, whereas arylesterases have a cysteine at the active centre and are not inhibited by organophosphates.

Regional variations in minipig esterase activity could influence the systemic delivery of topically applied ester prodrugs and confound the correlations to humans. Preclinical human dermal absorption studies have been performed primarily on skin from the female breast (mammoplasty) or abdomen (adominoplasty). In vitro absorption studies have generally been conducted using skin excised from the back of the pig (both domestic pig and minipig) or the isolated pig ear (Dick and Scott, 1992). For technical reasons, compounds are usually applied to the backs of the minipigs during in vivo pharmacokinetic studies. Investigation of the variation in metabolising capacity between different areas of minipig skin is warranted to assure that the back is an adequate model of human trunk skin, the usual site of a transdermal patch. It is also important to compare the skin with the liver, when considering all possible sites of prodrug activation. Two carboxylesterases have been identified in the pig liver and have been classified as CES1 and CES by Satoh and Hosokawa (2006). Human carboxylesterases are classified as CES1, CES2 and CES3. David et al. (1998) reported a pig intestinal carboxylesterase that showed high similarity with that of rat and human intestinal carboxylesterases. Carboxylesterases in pig skin have been shown to differ in isozyme distribution from the liver (Jewell et al., 2007). Esterases are present in endoplasmic reticulum and cytosol in keratinocytes of the skin and hepatocytes of the liver (Clark et al., 1993, McCracken et al., 1993a, McCracken et al., 1993b, Mutch et al., 2007). There are few reports describing cytosolic esterase activity. However, it is important to evaluate the contribution of cytosolic esterase activity to the tissue as a whole and to determine whether they differ from microsomal esterases in their activity and specificity.

In this study phenyl valerate (PV), phenyl acetate (PA) and p-nitrophenyl acetate (NPA) were used as esterase substrates to investigate the distribution of esterase activity in minipig skin microsomal and cytosolic fractions taken from various regional locations. PV is substrate for carboxylesterases, NPA for carboxylesterases and arylesterases and PA for arylesterases. By comparing hydrolysis of a range of substrates by microsomal and cytosolic fractions from skin and liver differences in regional esterase activity and specificity can be deduced.

Section snippets

Reagents

Unless otherwise stated chemical reagents were purchased from Sigma–Aldrich (Poole, Dorset, UK).

Human skin preparation

Human skin was obtained following breast reduction surgery of healthy female individuals. Patients gave informed consent and ethical approval was obtained from University Hospital of North Durham, UK

Minipig tissue procurement

Minipig livers and skin from three male and three female, approximately 11 months old, were provided by Pfizer Inc., Framboise, France. A section of tissue from each of the five minipig liver lobes was

Minipig skin preparation

Visually, minipig neck skin was the thickest, followed closely by back skin and then shoulder skin. These sections had thick fatty connective tissue below the skin surface and had thick course hair. Flank skin was much thinner and ear skin was thinnest. Flank and ear skin had far less fatty tissue associated with it and hair was more sparse and finer.

Minipig protein recovery

Overall, skin protein recovery was less than the liver (Table 1). Microsomal protein recovery from minipig skin was 1.40 ± 0.17 mg/g dermatomed

Discussion

Phenyl valerate was hydrolysed by non-specific carboxylesterases in the skin and inhibited by paraoxon (Jewell et al., 2007) whereas phenylacetate has previously been shown to be a substrate for arylesterases in the liver and skin (McCracken et al., 1993a). There is little information on the nature of arylesterases in minipig skin. The relative importance of arylesterases in skin compared to carboxylesterases in the hydrolysis of ester prodrugs depends on the specificity of the substrate. It

Acknowledgment

This research was supported by a grant from Pfizer Inc., Ann Arbor, MI, USA.

References (13)

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