SKIN STRIPPING TO DETERMINE METABOLISM 371 RADIOACTIVITY (Bq) RA DIDACTIVITY (Bq) 25 RADIOACTIVITY (Bq) 300 0 TIME (MINUTES) RADIOACTIVITY (8q) 3 1 2 5 2'0 TIME (MINUTES) 1 30 O 10 20 30 TIME (MINUTES) TIME (MINUTES) Figure 3. Radiochromatograms of 14C-Cyoctol-derived radioactivity in the dosage (A) and the tape ex- tracts at 1 (B), 23 (C), and 45 hours (D) after removal of the dose. front most likely originated from the tritiated drug. It should be noted in this regard that even though liquid scintillation counters have programs to correct for spillovers when •4C and 3H isotopes are counted simultaneously, such programs may not be able to adequately correct if the amounts of •4C isotopes present are rather small in compar- ison to the amounts of 3H isotopes. Radiochromatograms at 20 and 44 hours showed similar profiles, yet at much lower quantities, due to the rapid disappearance of Azone from the stratum corneum (5). Levels of radioactivity were therefore close to the base- line, and this makes the assessment of the percentual contribution of individual peaks meaningless. Nevertheless, all profiles were basically the same at the three collection times, and it can therefore be concluded that only unchanged Azone is present in the stratum corneum. With Cyoctol, the situation appears to be quite different. At one hour after removal of the dose, some metabolites can be detected in small amounts, but the majority of the radioactivity is still present as the parent compound (see Figures 3A and 3B). Table I shows that, as time goes by, the relative contribution of Cyoctol (peak 4), decreases, whereas that of the metabolites increases. At 45 hours after removal of the dose, only about 35 % of the radioactivity in the stratum corneum is present as unchanged Cyoctol.
372 JOURNAL OF THE SOCIETY OF COSMETIC CHEMISTS Table I Relative Contribution of Individual Compounds as a Percentage of the Total Eluted Amount of •4C-Azone (A n = 3) or •4C-Cyoctol (C n = 4) Derived Radioactivity (mean -+ SD) Relative contribution (%) in tape extracts at hour Test Peak compound number Dosage 1 (A + C) 20 (A) or 23 (C) 44 (A) or 45 (C) Azone Cyoctol 1 95.4 --- 1.8 94.0 & 3.8 N.D. 2 N.D. 2 1 1.4 -+ 0.9 9.7 & 5.0 17.5 -+ 3.9 2 4.0 _+ 2.6 12.2 _+ 4.2 15.7 -+ 1.7 3 4.0 -+ 3.1 8.3 -+ 7.0 5.9 -+ 1.6 4 93.9 -+ 1.4 78.3 -+ 16.8 49.5 -+ 19.7 35.4 -+ 13.4 5 6.6 _+ 7.6 9.2 -+ 5.8 14.2 _+ 10.0 The numbers correspond to those in Figures 2 and 3. N.D. = not determined. The possibility of chemical degradation of Cyoctol during the sample work-up could be excluded by having the parent compound undergoing the same sample processing. No compounds other than Cyoctol could be detected. These findings indicate that only unchanged Azone is present in the tape extracts, while in the case of Cyoctol, both the parent compound and its metabolites can be found. The stratum corneum, however, is a layer of dead cells, assumed to be devoid of metabolic activity, as opposed to the underlying viable epidermis and dermis where skin metabo- lism may take place (1). Bioconversion of Cyoctol, therefore, conceivably occurred in one of these layers. Yet, stripping removes only two thirds of the stratum corneum (7) and cannot have removed part of the viable epidermis. The presence of metabolites in the stratum corneum can be explained by assuming outward migration of the metabo- lites formed in the viable epidermis and/or dermis. Outward migration has been de- scribed for compounds following oral administration (8,9), but recently could be estab- lished following dermal application as well in the case of Cyoctol (6,10). Although the majority of the metabolites formed in the viable epidermis will move inwards into the body, favored by a more aqueous environment and systemic removal, a concentration gradient will also exist towards the stratum corneum. As a result of that, the stripping technique will usually underestimate the extent of metabolism. In the case of Cyoctol, for instance, De Zeeuw et al. showed that this drug was completely metabolized during skin passage to a more nonpolar metabolite, corresponding to peak 5 in Figure ! (10). A good indication as to cutaneous metabolism can be obtained from the metabolic profiles of the ipsi- and contralateral plasma samples (11). However, it is sometimes impossible to apply the latter methodology, for example, when dosing on areas such as the back, abdomen, or forehead. Moreover, the levels of radioactivity in the ipsi- and contralateral plasma samples have to be relatively high to obtain reliable metabolic profiles. This presents severe difficulties with drugs that have low dermal absorption such as Azone (5,12,13). The skin stripping methodology does not have these disadvantages and thus seems to be an interesting, simple, and noninvasive alternative to assess in vivo cutaneous metabo- lism, provided that sufficient outward migration of metabolites occurs. The technique
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