Sachiko Kosaka

Parasiticidal effect of δ‐aminolevulinic acid‐based photodynamic therapy for cutaneous leishmaniasis is indirect and mediated through the killing of the host cells

Abstract:  Several clinical reports have shown promising, but not optimal, results from photodynamic therapy with δ‐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 μm ALA, the PpIX concentration remained at the basal level, whereas after co‐incubation with 0.1 μm 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 for cutaneous leishmaniasis: the effectiveness of topical phenothiaziniums in parasite eradication and Th1 immune response stimulation

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.

Photoinactivation of Mycobacteria In Vitro and in a New Murine Model of Localized Mycobacterium bovis BCG-Induced Granulomatous Infection

ABSTRACT Treatment of tuberculosis is currently hindered by prolonged antibiotic regimens and the emergence of significant drug resistance. Alternatives and adjuncts to standard antimycobacterial agents are needed. We propose that a direct attack utilizing photosensitizers and light-based treatments may be effective in curtailing Mycobacterium tuberculosis in discrete anatomical sites in the most infectious phase of pulmonary tuberculosis. To demonstrate experimental proof of principle, we have applied established photodynamic therapy (PDT) technology to in vitro cultures and an in vivo mouse model using Mycobacterium bovis BCG. We report here in vitro and in vivo PDT efficacy studies and the use of a three-dimensional collagen gel as a delivery vehicle for BCG, subcutaneously inserted, to induce specifically localized granuloma-like lesions in mice. When a benzoporphyrin derivative was utilized as the photosensitive agent, exposure to light killed extracellular and intracellular BCG in significant numbers. Collagen scaffolds containing BCG inserted in situ in BALB/c mice for 3 months mimicked granulomatous lesions and demonstrated a marked cellular infiltration upon histological examination, with evidence of caseating necrosis and fibrous capsule formation. When 105 BCG were present in the in vivo-induced granulomas, a significant reduction in viable mycobacterial cells was demonstrated in PDT-treated granulomas compared to those of controls. We conclude that PDT has potential in the treatment of localized mycobacterial infections, such as pulmonary granulomas and cavities.

Comparative split-face study of 5-aminolevulinic acid photodynamic therapy with intense pulsed light for photorejuvenation of Asian skin.

Photodynamic therapy (PDT) with 5‐aminolevulinic acid (ALA) (ALA‐PDT) using intense pulsed light (IPL) as a light source (IPL‐ALA‐PDT) has been used for photorejuvenation, but it is unclear if this protocol can be applied to darker skin types. We performed this study to assess our IPL‐ALA‐PDT protocol for photorejuvenation in Asian skin. To determine an appropriate dose, ALA ointment (0–20%) was applied to the upper arm of five healthy volunteers and the fluorescence intensity (FI) was measured using a spectrofluorometer. Non‐linear regression analysis of FI 2 h after ALA application with global fitting gave a typical sigmoid dose–response curve with R2 = 0.9705 and saturation after 5% ALA. The entire faces of 16 Japanese women with photodamage were then treated with IPL (500–670 and 870–1400 nm, 23–30 J/cm2) 2 h after application of 5% ALA to one side of the face. Three treatments were delivered at 4‐week intervals with follow‐up visits. Comparative analysis of photorejuvenation showed noticeable improvements on both sides of the face, although the reduction in the photoaging score from baseline did not differ significantly between the two sides in all subjects. Despite this finding, 75% of the patients felt that the IPL‐ALA‐PDT‐treated side of the face showed greater improvement than the IPL‐treated side. However, all IPL‐ALA‐PDT‐treated sides showed adverse effects such as erythema and pain. Therefore, we conclude that the IPL‐ALA‐PDT protocol requires optimization for photorejuvenation in Asians.

Targeting of Sebocytes by Aminolevulinic Acid‐dependent Photosensitization

Abstract Photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX has been developed as a very useful therapeutic modality. Recently, several authors have reported on the efficacy of this procedure for acne. This approach is based on the fact that 5-aminolevulinic acid-induced protoporphyrin IX has strong selectivity for sebaceous glands. We used the immortalized human sebaceous gland cell line SZ95 to investigate cellular mechanisms of photodynamic therapy using 5-aminolevulinic acid-induced protoporphyrin IX. Quantification of induced protoporphyrin IX production showed dependence on the applied 5-aminolevulinic acid dose. When SZ95 sebocytes were differentiated by arachidonic acid treatment, there was no difference between them and the control cells with respect to both the amount of 5-aminolevulinic acid-induced protoporphyrin IX and the phototoxic effects. We altered protoporphyrin IX formation rates by growing cells scattered as single cells in the culture dishes. Single cells produced significantly lower protoporphyrin IX levels than those grown with intercellular contacts. Intracellular localization of protoporphyrin IX was imaged using confocal laser scanning microscopy. The differentiation-specific lipid droplets were virtually excluded from protoporphyrin IX fluorescence. In addition to weak mitochondrial and strong membrane fluorescence, distinctive spots with strong fluorescence were observed. These did not colocalize with fluorescent probes for mitochondria, lysosomes or the Golgi apparati.

Targeting of sebaceous glands by δ‐aminolevulinic acid‐based photodynamic therapy: An in vivo study

Wide application of ALA‐PDT for acne is limited due to relative strong side effects, such as pain and erythema. The objective of this study was to establish a protocol for ALA‐PDT that would provide specific destruction of sebaceous glands at the lowest concentrations and shortest contact times of ALA.

Prospects for the use of differentiation‐modulating agents as adjuvant of photodynamic therapy for proliferative dermatoses

Current interest in photodynamic therapy (PDT) in dermatology stems from its recognized success in dermatological oncology, straightforward approach, easy accessibility and low cost. PDT is a photochemistry‐based modality in which a light‐activated photosensitizer (PS) destroys tissue through oxygen‐dependent and ‐independent mechanisms. Although PDT has been used in dermatology for several decades, its application has still not extended significantly into the routine management of neoplastic and proliferative dermatoses because of continuing issues with the selectivity of the PS for affected tissues. This review analyzes prospects for optimization of PDT for the management of dermatoses with defects in keratinocyte proliferation/differentiation, and discusses the use of differentiating agents that redirect metabolic utilization within cells and lead to high levels of PS accumulation.

Case of necrobiosis lipoidica diabeticorum successfully treated by photodynamic therapy

initiation of dapsone. Engin et al. reported that only four out of 38 patients responded to dapsone at the first month, but finally 36 patients showed complete or partial response up to 3 months. Thus, at least 2–3 months of therapy is required to fully clarify the response to dapsone. Only a short-term use of dapsone may lead to a discouraging result. Histopathologically, perivascular infiltrating cells were mainly composed of neutrophils in the present case. Dapsone appears to reduce a neutrophil adherence function mediated by integrins, and thus inhibits neutrophil migration to extravascular sites. We speculate that wheals disappeared because dapsone inhibited the perivascular recruitment of neutrophils. Importantly, Criado et al. reported that all of three urticaria patients with predominantly neutrophilic infiltrates showed complete response to dapsone during 30–90 days. Taken together, dapsone seems to be a good therapeutic option for chronic idiopathic urticaria especially when histopathology shows neutrophil-rich infiltration. A proper clinical trial is required to obtain a higher evidence level.

Photochemistry-based immune modulation in the treatment of cutaneous leishmaniasis

The destruction of infectious pathogens by photodynamic therapy (PDT) is an emerging modality. We demonstrated the efficacy of PDT for the management of cutaneous leishmaniasis in our previous studies. However, much remains to be done for the improvement of PDT regimens. The modulation of the immune response by photochemistry is an exciting but under-explored area of PDT research. The goal of this study is to understand the mechanisms of the augmentation of the host immune response after PDT of cutaneous leishmaniasis (CL). We found that PDT with phenoxiazine analogues was capable for induction of Th1 immune response due to stimulation of IL- 12 production by dendritic cells. Single PDT treatment facilitated fast healing of the CL lesions due to effective parasite eradication and augmentation of the immune system. Comparative study with different photosensitizers (PS) (porphyrins, pehnoxiazines) demonstrated different immunomodulating properties of PDT depending on chemical class of PS. Knowing the particular profiles and immunomodulating properties of the pertinent PSs allows us to select the optimal PS with regards to both the photodestructive and immunostimulating potential.