The present study was designated to study the influence of skin temperature and anaesthesia on the production of porphyrins induced by ALA and ALA-Me in mouse skin.

Porphyrin fluorescence
The fluorescence excitation and emission spectra recorded after topical application of either ALA or ALA-Me in mouse skin are shown in FIG 1. These fluorescence spectra are identical with that observed after administration of pure PpIX in mouse skin (data not shown). Thus we assumed that PpIX was the main fluorescent photosensitizer formed in mouse skin during ALA and ALA-Me application.

Influence of temperature
The present results show that the uptake of ALA and ALA-Me in mouse skin and the subsequent production of PpIX are temperature and anaesthesia dependent processes. The influence of skin temperature on ALA-uptake during 3 h topical application of 20% ALA-cream is shown in FIG. 2. Since the cold skin was kept during 3 h application period the kinetics might represent ALA-penetration, which is significantly decreased in cold skin. Earlier we have found that during short application time (10 min.) ALA-penetration was only slightly dependent on skin temperature. This is not surprising since 3 h is a long period, long enough to allow production of significant amounts of PpIX in control normal skin. It is obviously that both ALA-penetration and PpIX-production decrease in cold skin. ALA was observed to induce a systemic effect in the mice. Significant amounts of PpIX fluorescence was generated in remote sites untreated with ALA-cream, i.e. fluorescence measured on the other flank of a mouse (FIG. 2).

Similar temperature dependence of PpIX fluorescence was found for ALA-Me (FIG. 3). Oppositely to ALA, the methylester derivative did not induce any significant PpIX fluorescence in remote skin sites (data for remote fluorescence are not shown in FIG. 3 since it was equal to a background level). That is in agreement with our previous experience showing PpIX fluorescence all over the mouse after prolonged topical application of ALA-cream, while ALA-Me-induced PpIX fluorescence is localized in a treated spot [13]. Such difference in systemic action of topically applied ALA and ALA-Me was also observed in mice after ice had been removed from treated spot (FIG. 2 and FIG. 3). Furthermore, the influence of various temperatures on the amount of PpIX produced during continuous application of ALA and the corresponding temperature values are shown in FIG. 4. The temperature dependence of ALA-induced PpIX-production in mouse skin is well correlated with our previous observations [7,8].

Influence of anaesthesia
The influence of anaesthesia on the amount of PpIX produced during continuous application of ALA-cream is shown in FIG. 4. As long as mouse skin was kept cold either by anaesthesia or by cooling, significantly less PpIX was found. This is further illustrated in FIG. 5 where the mouse was kept sleeping under continuous anaesthesia and its skin temperature was maintaining at around 22oC. Even after such a short application time (10 min.), enough ALA had penetrated through mouse skin and produced PpIX in a linear manner (FIG. 5), showing that a low metabolic activity strongly reduces rate of PpIX production from ALA and possibly decreases clearance of ALA and/or PpIX from the body.

In conclusion, the conversion of ALA and ALA-Me into PpIX is significantly reduced in cold skin. During topical application of drug, the important physiological parameter such as skin temperature that can be affected by external temperature and anaesthesia should be taken into account.

Acknowledgements ¾ The present work was supported by the Radium Hospital Research Foundation.