**4. Discussion**

The aim of the study was to evaluate PPIX formation due to ALA, MAL, and cream sample mixtures from both (M2, M3, M4, M5, and M6) application on normal skin models (porcine and human) and then to show that there is a narrow correlation between both models. In this work we use ALA and MAL on topical application as the precursor of PPIX, since these are the most common drugs applied to clinical topical PDT. The fluorescence measurements were collected after 3 h of cream incubation time since this time is also applied to clinical PDT [19, 22].

In our group [22] the clinical PDT studies on skin cancer are done using 20% ALA and MAL cream application in 3 h of incubation time before light irradiation. During this time, PPIX production is elevated since the previous preparation was performed (curettage).

The fluorescence measurements were done using two techniques: fluorescence spectroscopy and widefield fluorescence imaging. With fluorescence spectroscopy using a 532 nm laser (green light) it is possible to evaluate the skin at greater depths (reaching the dermal papillae) when compared with widefield fluorescence imaging using a 405 nm LED (violet light) bringing images from PPIX on the superficial skin [2, 19].

In the study the choice of animal age had great influence; in agreement with the literature the thickness of porcine skin is similar to human skin at around 2 months after birth [8, 13].

PPIX formation on normal skin is not homogeneous and depends on ALA, MAL, and mixtures from both (M3, M4, M5, and M6) penetration through the skin; evaluations using images by widefield fluorescence imaging can be useful and decrease the variability on experiments. Fluorescence spectroscopy evaluation, despite being collected punctually, which can lead to erroneous measurements and high variability, reveals information about PPIX formation on the deeper skin [2, 19] and is important to understand the replacement mechanism of PPIX from deeper layers up to superficial skin layers.

As shown by Valentine et al. [23], there was no difference after increasing the amount of PPIX using ALA and MAL when analyzed by fluorescence spectroscopy using a laser emission at 405 nm (violet light). Fluorescence emission due to 405 nm illumination allows us to measure the output of PPIX on the superficial skin (stratum corneous and superior epidermis). In our work, this superficial skin analysis was performed using widefield fluorescence imaging.

There are few studies concerning the comparison of ALA and MAL in healthy human skin, but Lesar et al. [10] compared the formation efficiency of PPIX from these precursors in various parts of the human body (arm, forearm, back, and legs) with fluorescence (4–29 h) after topical application. They then observed that there were differences in PPIX production, which applied regardless of where the ALA accumulated more PPIX, but the location (back) where they applied the tape striping difference was only after 24 h.

The kinetics study observed that ALA, M4, and M5 indicated the least time of PPIX production (high PPIX production velocity) at the skin. Both studies, human and porcine skin, showed the same behavior. IF50 values acquired by widefield fluorescence imaging for both models were very close, with the exception of M3, M6, and MAL. Thereby, it is possible to appreciate the similarity of porcine skin with human skin by first performing clinical tests on porcine skin.

However, it is known that *in vitro* and *in vivo* experiments using the same species show less variability than experiments using human volunteers. The authors suggest that human experiments are done using a greater number of volunteers. The measurement of the correlation coefficient proved that porcine and human skin models have the same behavior with respect to the production of PPIX in quantity as well as in speed of PPIX production through optical methods. The correlation coefficient is a measure of how well the predicted values from a forecast model fit with the real data. We suggest that the best correlation is between porcine and human skin by widefield fluorescence imaging, suggesting this optical method as an important tool to develop new clinical topical PDT protocols.
