Several clinical reports have shown promising, but not optimal, results from photodynamic therapy with delta-aminolevulinic acid-derived protoporphyrin IX, termed ALA-PDT, as a treatment for cutaneous leishmaniasis (CL). Therefore, understanding the basis of the phototoxic response of Leishmania parasites to ALA-PDT may be critical for optimization. We report here both in vitro and in vivo mechanistic studies of ALA-PDT against CL. Following in vitro co-incubation of Leishmania major with 0.1 microM ALA, the PpIX concentration remained at the basal level, whereas after co-incubation with 0.1 microM exogenous PpIX, the PpIX level was 100-fold higher. No differences in ALA-derived PpIX levels were detected between Leishmania-infected and non-infected J774.2 cells, and PDT did not demonstrate any parasiticidal effects on amastigotes. In contrast, in vivo topical ALA-PDT, performed on a murine CL model, resulted in significant reductions of the parasite loads and vigorous tissue destruction. After ALA-PDT, a dramatically decreased percentage of macrophages and increased levels of interleukin-6 were observed in the infected skin. The clinical outcome observed with ALA-PDT is likely the result of unspecific tissue destruction accompanied by depopulation of macrophages rather than direct killing of parasites.

Photodynamic therapy (PDT) is emerging as a therapeutic modality in the clinical management of cutaneous leishmaniasis (CL). The efficacy of PDT against CL has been demonstrated previously with aminolevulinic acid, although the prolonged terms of therapy were less than ideal, and the search for new photosensitizers (PS) is ongoing. However, phenothiaziniums have demonstrated high parasiticidal effects in vitro. The subject of our investigation is the in vivo activity of two PS, 5-ethylamino-9-diethylaminobenzo[a]phenoselenazinium chloride (EtNBSe) and (3,7-Bis(N,N-dibutylamino) phenothiazinium bromide (PPA904). The results of our comparative analysis of the efficacy of these two phenothiazinium analogues demonstrated a high antiparasitic activity of EtNBSe in vitro, and the higher efficacy of PPA904 in a mouse model of CL. The kinetics of photodestruction are different in parasite and mammalian cells, and with both dyes, the macrophages are more susceptible to photodynamic effects than L. major parasites. As the number of parasites in the lesions undergoes a biphasic change, temporarily increasing on days 2-4 and decreasing on days 5-7, more than one treatment is required within an interval of 5 to 7 days. We have also shown that PPA904-PDT can provide an immunomodulating, dose-dependent efflux on IL-12p70 production. This mechanism could be responsible for promoting a more rapid healing in PPA904-PDT treated mice. Our initial data indicate that phenothiaziniums exhibit a high parasiticidal effect in vivo against CL; this finding may be of use in establishing curative PDT regimens for future clinical trials.

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