A viable <i>in vitro</i> excised human skin model was developed to accurately assess cutaneous delivery and metabolism of two ester type compounds; tetracaine (TC) and methyl salicylate (MS). This model could maintain the viability of fresh skin in diffusion cells for 24 hours. Skin viability was assessed using two methods; oxygen consumption measurement and confocal laser scanning microscopy. Two fluorescent probes, calcein AM and ethidium homodimer-1, were used as live and dead markers, respectively. General morphology and localization of nonspecific esterase activity in the skin samples from diffusion cell were checked histologically. Cutaneous delivery and metabolism of MS was evaluated with this viable skin model and compared to human skin homogenate model. A sensitive high performance liquid chromatography (HPLC) assay using reversed phase ion pair was developed/refined to simultaneously analyze TC and its metabolite (4-BABA). Several factors affecting this HPLC system were identified. The limit of detection for TC and 4-BABA was 0.3 ng and 0.5 ng, respectively. The limit of quantitation for TC and 4-BABA was 10 ng and 5 ng, respectively. Linearity was in the range of 10-120 ng for TC and 5-60 ng for 4-BABA. MS was hydrolyzed to salicylic acid (SA) during absorption through fall thickness human breast skin in diffusion cells. The extent of MS hydrolysis was significantly higher in viable skin than in non viable. The extent of absorption of SA through viable and non viable skins was similar. In human skin homogenate, MS was hydrolyzed at the rate of 72.31 nmol/h/[mu]g protein while the hydrolysis in phosphate buffered saline was very low. TC hydrolysis in human skin homogenate was not extensive due to substrate inhibition. From the kinetic study of TC hydrolysis in human skin homogenate, Km was in the 11-28 [mu]M range and Vmax was in the 2.0-2.8 [mu]mol/h/[mu]g protein range. Temperature over 60°C substantially reduced esterase activity in both models therefore caution must be taken during preparation and handling of tissue samples to preserve esterase activity. The viable <i>in vitro</i> excised skin model will provide more accurate quantitation of skin metabolism and absorption of xenobiotics.
| Identifer | oai:union.ndltd.org:USASK/oai:usask.ca:etd-10212004-001942 |
| Date | 01 January 2000 |
| Creators | Asavapichayont, Panida |
| Contributors | Foldvari, Marianna |
| Publisher | University of Saskatchewan |
| Source Sets | University of Saskatchewan Library |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://library.usask.ca/theses/available/etd-10212004-001942 |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Saskatchewan or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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