Photodynamic Therapy Using Methylene Blue to Treat Cutaneous Leishmaniasis

Introduction

T egumentary leishmaniasis is caused by 13 dermotropic species of protozoa from the genus Leishmania, most of which are prevalent in the new world. ¹ Leishmania braziliensis is the most prevalent species and causes muco-cutaneous leishmaniasis. ^2,3 Similar drugs are used to treat cutaneous leishmaniasis (CL) around the world. Antimonials are the most common, although other drugs have also been used. The current treatments for leishmaniasis (pentavalent antimony compounds, amphotericin B, and pentamidine) are problematic, because of the route of intravenous administration, toxicity, and prolonged treatment duration. Physical treatment has been used as a therapeutic option to reduce the side effects and improve the cosmetic outcome of CL. ^4,5 Topical treatment has been used with some success; however, the efficacy of the treatment remains questionable. ⁶ Photodynamic therapy (PDT) using porphyrin precursors as sensitizers has been used to treat CL. Positively charged photosensitizers are extremely effective against leishmania parasites, ^7,8 and porphyrins have been used successfully. ⁹ Aminolevulinic acid (ALA) has been used as a photosensitizer of PDT to treat CL, and the results have been encouraging. ^{10 –17} However, ALA is cost-prohibitive and often cannot be incorporated into the treatment routine. ⁷ Methylene blue (MB) has been used under illumination or in the dark to treat a wide range of medical conditions, ¹⁸ and it exhibits antimicrobial activity against Plasmodium falciparum and Trypanosoma cruzi. ¹⁹ In combination with PDT, MB has been used as a treatment for colon cancer, ²⁰ melanoma, ²¹ HIV, hepatitis, ²² and mycobacterium keratitis. ²³

Based on the clinical success of PDT in the treatment of CL ^17,24 and the unique characteristics of MB in PDT, we performed a series of in vitro studies and evaluated a clinical case to determine the efficacy of PDT and MB in the treatment of CL. We hypothesized that the use of PDT with MB and a non-coherent light source such as infrared would be an effective treatment for CL.

Methods

Results

In vitro study

The dose-dependent response of L. amazonensis to MB with and without light stimulation clearly shows that cell toxicity increases with the increase in MB concentration (5, 10, 20, 30, 40, 50, 70, 80, and 100 μM) and with light stimulation (Fig. 1). The IC₅₀ calculated in the absence of illumination was 100 μM. This effect is highly amplified by light absorption (IC₅₀ 20 μM), causing an increase in toxicity in the parasites. It is important to note that 50 and 100 μM of MB and illumination lead to a partial killing of the parasites (Fig. 1). Based on the effect of MB/PDT in Leishmania promastigotes, we decided to observe the effect of MB/PDT in vivo using a human clinical case.

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FIG. 1.

Dose response curve for L. amazonensis exposed to methylene blue (MB) alone and MB+light (PDT). The Student's t-test (p<0.05) indicates a significant decrease in parasite survival with the addition of PDT to 50 or 100 μM of MB. N=6 for each data point.

Clinical case

A male patient had three CL lesions localized in the left posterior flank, one lesion in the left ear and another in the left cheek, confirmed by Giemsa stain. The efficiency of PDT with MB was evaluated by comparing the results of two large lesions. The experimental lesion was treated with PDT and by low dose SbV, and the control lesion was treated with PA alone. The evolution of the lesions was followed by taking pictures and measuring the percentage of area occupied by open, unhealed wounds of 227 mm² (base) as well as the total ulcer surface area of 227 mm². As illustrated in Fig. 2, the size of the open wound was considerably reduced for the lesion treated with PDT. The diameters of the open wound regions treated with PA and PA +MB/PDT are plotted in Fig. 3 as a function of the number of days after starting treatment. One week after the second PDT dose (14 days), the lesion treated with PDT no longer contained an open wound. In the case of the lesion treated only with PA, the open wound persisted until 28 days after the initiation of treatment (Fig. 3).

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FIG. 2.

Pictures of the leishmaniasis lesions for the control (low dose SbV only) and experimental (low dose SbV+MB/PDT) lesions. Photographs were taken during weeks 0–4, during which PDT treatments 1–4 were given. Below each image is the area (mm²) of the open wound (base) versus the area of the lesion (ulcer).

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FIG. 3.

Percent of open lesions versus days of treatment. The graph indicates that the time to wound closure decreases when the lesion is treated with PDT/MB versus treatment with MB alone.

Discussion

We began our work with several in vitro experiments to test the parasiticidal effect of MB against L. amazonensis promastigotes with and without light. As anticipated, MB exhibited an increased parasiticidal effect that was proportional to its concentration, not only in the absence of light excitation, but also with light exposure.

One major difference between this study and prior PDT studies for leishmaniasis has been the addition of SbV to the experimental regimen. Prior studies, which were performed in the Middle East, dealt with forms of CLs that are at a lower risk for converting to the mucocutaneous disease than the leishmaniasis found in Brazil. As a result, the clinical cure of cutaneous lesions was the standard end point. However, in Brazil, because of the probability of the cutaneous disease converting into a mucocutaneous disease (2–8%), decades after healing the cutaneous lesions ⁵ systemic treatment is still required. Therefore, because of ethical and clinical concerns, a low dose of SbV therapy was added to the experimental treatment regimen. Although this makes it more difficult to clearly determine the efficacy of PDT as a treatment for CL, a comparison with low-dose SbV therapy alone will allow us to distinguish any added effect of the combined therapy. In addition, by combining both a systemic and a local therapy, we can minimize the side effects and costs associated with using SbV to treat CL. Concurrently with our in vitro studies, an initial clinical trial was performed on an individual affected by CL. By using the patient as his own control, we were able to compare the effect of the addition of MB/PDT to the healing process of his cutaneous lesions. Our analysis of the healing process revealed that although a low-dose of SbV did result in cutaneous lesion closure, the addition of MB/PDT accelerated the process, resulting in faster wound closure. It is important to mention that this protocol is inexpensive, allowing it to be used in the public health systems of underserved areas.

Future work should be aimed at understanding if the infected host cell has an effect on parasite susceptibility to the treatment and localization of MB. Because the Leishmania amastigote is more metabolically active, it is unclear if this form or if an infected host cell environment will confer increased resistance to treatment. It will also be important to elucidate if parasite viability and host cell viability are mutually exclusive in MB/PDT. Prior studies using ALA in Leishmania have shown that because of the biochemical properties of ALA/PpIX, parasite death occurs through host cell death. ²⁶ However, MB does not require an enzymatic conversion to become active, and therefore we anticipate that the cell viability will depend upon the type of cell, host or parasite.

Conclusions

Our data show that MB causes damage, decreasing parasite viability fivefold compared with the dark control and decreasing the time necessary to heal a leishmaniasis lesion. These results show that the low-cost PDT protocol using MB and illumination with a non-coherent light source has the potential to be useful in the treatment of leishmaniasis and can be a practical solution for treating this disease in underserved populations. Further studies are needed to test the efficiency of this protocol in a statistically significant population of patients.