Percutaneous Penetration And Metabolism Of Ketoprofen Isopropyl Ester Through A Tissue Engineering Skin Reconstructed With HaCaT Cells And Its Molecule Mechanism

Transdermal drug delivery system has been paid more attention for its distinct advantages, including avoidance of variable absorption rates and first-pass hepatic metabolism with oral delivery and improved therapeutic activity. Usually animal (rat, pig, et al) or human cadaver skins were chosen for the transcutaneous absorption as the model skin. There are many physiological and histological differences between normal human skin and the model skin. Therefore more and more researchers turned to human skin equivalents for normal skin substitution. Normal human cells isolated from fresh skin specimens were generally used for establishing in vitro skin equivalents. Extrapolation of studies using primary cultures of normal human skin cells may be hampered due to the limitation resource of human skin and the donor-to-donor variability and variation between cells originating from different body regions. The uses of established cell lines will not noly allow unlimited access by passaging, but may also improve the reproducibility of skin models.Skin is the largest organ of our body. For many years, it was considered that skin was a passive barrier between the body and the environment. More and more investigations have shown that skin is an active metabolic organ. Many species of metabolizing enzymes are expressed in skin and take an important position in the bioconversion of xenobiotics. Drug metabolism in skin may directly affect the extent of skin penetration of lipophilic drugs. More hydrophilia drugs metabolized, more lipophilic drugs penetrated through skin. The bioconversion of a lipophilic drug is an important consideration when designing prodrugs enhancing the skin permeability of drugs. Variable metabolizing enzymes are expressed in skin. Modification of drugs with esteratic or amide linkage would improve the liposolubility.In this study, a reconstructed human skin using an immortalized normal human abdominal keratinocyte cell line, HaCaT, was generated. Ketoprofen and ketoprofen isopropyl ester were chosen as the model drugs for investigation the penetration and metabolism of drugs in the reconstructed skin. The ketoprofen isopropyl ester wasstereoselectively hydrolyzed into R-ketoprofen when penetrated through namal human epidermis and the tissue engineering skin. The mechanism of the stereoselective hydrolysis was investigated.Collagen type I was extracted from bovine tendon. Dermal fibroblasts were embedded in collagen. HaCaT cells were seeded on the top of the gel. Effects of variations in culture media on tissues morphology were investigated. After 4 weeks air-liquid interface culture, sections of the cultured skin were stained with hematoxylin and eosin and examined under microscopy. When grown at the air-liquid interface, HaCaT cells initially developed a multilayered epithelium, but showed a parakeratotic stratum corneum. Vitamin C enhanced cell proliferation obviously. Vitamin D3 promoted cell differentiation. And estradiol showed few effects on the tissue engineering skin.To determine the penetration of ketoprofen, ketoprofen isopropyl ester and the ketoprofen enantiomers, reversed high performance liquid chromatographic methods were estabolisned. The calibration curves were linear, the recovery rate, the precision values of intra-day and inter-day were suitable for the analysis.Ketoprofen and ketoprofen isopropyl ester were chosen as model drugs for the permeation and metabolism properties through the tissue engineering skin and normal human foreskin epidermis. Ketoprofen itself could penetrate through the reconstructed skin and the epidermis, with a bit higher permeation rate (174.14 (ig-cm"2-h"') in the former than the later (125.13 jag-cm^-h"1). There was no stereoselective when ketoprofen penetrated through skins. Both through the reconstructed skin and epidermis, ketoprofen isopropyl ester were hydrolyzed to ketoprofen. Only R-ketoprofen was found in the reception fluid when determind with HPLC. When pentrated through the reconstructed skin, the permeation and metabolism rate of ketoprofen isopropyl ester was a bit slowly (1.69 |j.g-cm~2h"') compared with the epidermis (2.49 |ag-cm~2-h"'). These results indicated that, the tissue engineering skin was comparable with the normal skin when ketoprofen or ketoprofen isopropyl ester were chosen as model drugs. This cultured skin could serve as an appropriate model for drug percutaneous metabolism and skin irritation.Two different cell lines, HaCaT cells and SGC-7901 cells, were chosen for the investigation of the stereoselective hydrolysis mechanisem. Ketoprofen isopropyl ester was metabolized to ketoprofen without obviously stereoselective when it was incubated in the culture media of SGC-7901. When it was incubated in the homogenate of SGC-7901, ketoprofen isopropyl ester was metabolized to ketoprofen with the R enantiomer as the main metabolite (90%). The hydrolysis of ketoprofen isopropyl ester was comparable in the culture media of HaCaT cells with DMEM. While in the cell homogenate of HaCaT, the metabolism was stereoselective. R-ketoprofen was the only found metabolite. The kinetics of the hydrolysis of ketoprofen isopropyl ester in the HaCaT cell homogenate was investigated. The Km and Fmax were 255.38 umol and 1.37 jimol'mg'^min"1 respectively. Preliminary inhibitory studies suggested that ketoprofen isopropyl ester were hydrolyzed by a carboxylesterase, because this reaction was inhibited by Triphenyl phosphate. Mercuric chloride (an inhibitor of A-esterase and arylesterase) had little inhibitory effect on the hydrolysis. While galanthamine hydrobromide (a cholinesterase inhibitor) had no inhibitory effect. The total RNA was extracted from HaCaT cell and SGC-7901 cell. RT-PCR indicated that only human carboxylesterase 2 was expressed in both cells, while the human carboxylesterase 1 mRNA were neither detectable.There were some identical metabolism properties of ketoprofen isopropyl ester in HaCaT cell and SGC-7901 cell. First, both cells hydrolyzed ketoprofen isopropyl ester stereoselectively. Second, the stereoselective activity enzyme could not be excreted to the culture media. Third, both cells express the same human carboxylesterase as hCE-2. Therefore we hypothesized that the human carboxylesterase 2 was responsed for the stereoselective hydrolysis.To verifying our hypothesis, hCE-2 was cloned and expressed in COS-7 cells. The total RNA was extracted from HaCaT cell. Fragments containing the cds of hCE-2 were obtained by RT-PCR. The fragments were ligated to pMD-18T vector and were cloned to E.coli TOP10 cells. The plasmid extracted from E.coli TOP10 cells as the template, the cds fragments of hCE-2 were obtained by PCR. After...