Biol. Pharm. Bull. 30(3) 532—536 (2007)

routes of administration have been well documented, including avoidance of variable absorption rates and first-pass hepatic metabolism with oral delivery and improved therapeutic activity. In the past, skin penetration studies usually focused only on the physicochemical factors affecting the transport of drugs across the skin. However, recently, many researchers have also been considering the concurrent epidermal metabolism. Various aspects of the metabolism of xenobiotics in the skin have been reviewed, and it has been demonstrated that skin contains various enzymes including esterase capable of hydrolyzing different esters. Ester prodrugs for carboxylic acidor hydroxyl-containing drugs are widely used for the enhancement of percutaneous absorption and have been noted to show stereoselective hydrolysis in various tissue preparations; surprisingly, the molecular mechanism of stereoselective hydrolysis in skin has been overlooked. A great deal of the current knowledge on the metabolic clearance of drugs in the skin is based on studies with homogenates of excised tissues, including excised animal skin or human skin. Inherent drawbacks of these models are the doubtful comparability of animal skin versus human skin, the restricted access to human skin, and the potentially high variability of enzyme activity of human skin samples along with the often poorly defined cellular viability of excised tissues. It has been proposed that using cultured HaCaT keratinocytes as a readily available and reproducible in vitro metabolism model may circumvent some of these disadvantages. The transformed human HaCaT keratinocyte is a well-established cell model of dermal toxicity and metabolism studies in vitro, because it is easily cultured and has the characteristics of basal epidermal keratinocytes. Carboxylesterase is a group of serine esterases found in numerous animal species and a variety of mammalian tissues. These enzymes hydrolyze many different endogenous and xenobiotic compounds and play a role in the metabolism of numerous drugs. Some researchers have reported the purification and partial characterization of two distinct human carboxylesterases designated human carboxylesterase-1 (hCE-1) and human carboxylesterase-2 (hCE-2). These enzymes are expressed in human liver and are both members of the 60-kDalton serine esterase superfamily, but they differ substantially. Sequence homology between the two enzymes is only 48%. hCE-1 is an 180 kDalton trimer with an isoelectric point of 5.8, whereas hCE-2 is a monomer with an isoelectric point of 4.9. Substantial differences in substrate specificity also exist between the two isoforms. Ketoprofen is a nonsteroidal antiinflammatory drug (NSAID) and is used through oral or suppository routes for the treatment of pain and inflammation. Given orally, gastrointestinal side effects are most frequently seen. Therefore percutaneous administration of ketoprofen has been studied to minimize gastrointestinal and other possible systemic side effects due to high plasma peak levels after oral administration. Ketoprofen has unsuitable physiochemical properties, and thus various strategies to aid skin penetration have been studied. The prodrug approach represents an alternative method of enhancing the skin permeability of ketoprofen. A successful dermal prodrug of ketoprofen should exhibit optimum lipophilicity and should be stable against chemical degradation prior to hydrolysis within the skin by enzymes. Previously, we reported that ketoprofen ethyl ester (KPE) was hydrolyzed to ketoprofen during in vitro penetration and this hydrolysis markedly affected the stereoselective cutaneous penetration. Thus, in the present study, the stereoselective hydrolysis of the prodrug in HaCaT keratinocytes was studied to evaluate the hydrolyzing activity of epidermal carboxylesterase, and RT-PCR was used for studying the expression of hCE-1 and hCE-2 with the expression in L02 hepatocytes as a control.