**2. Leishmaniasis — Background and treatment strategies**

There are two main forms of leishmaniasis, visceral (VL) and tegumentary (TL) leishmaniasis, which are also respectively called Kala Azar and Bauru ulcer. The later, received its name because of the original high prevalence in Bauru, a city in the countryside of the State of São

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Paulo, in Brazil. The tegumentary leishmaniasis is characterized by skin lesions (cutaneous-CL) and mucocutaneous lesions (such as, nasal and mouth regions) [2].

Leishmaniasis is a common zoonosis, with domestic (dogs and cats) and wild (rodents, marsupials, edentulous and wild canids) reservoirs. It is transmitted to humans by sand flies, which comprise the genus Lutzomyia and Phlebotomus. Details of the etiology and patho‐ physiology of the disease are out of the scope of this chapter and we suggest that the reader consult reviews that focus on these subjects [3].

The current scenario of leishmaniasis treatment is not promising. Therapeutic approaches include systemic administrations of anti-parasitic medications, which often present serious side effects. Few drugs are available in the clinic, mainly antimonials and amphotericin, and the frequency of resistance development is rising. Therefore, there is an urgent need to establish new and more effective treatments for both VL and TL. The treatment of TL (the focus of this chapter) urges new drugs and new therapeutic forms, that allows for more effective and conveniently administered treatments [4].

One of the promising approaches, and the one discussed in here, is photodynamic therapy (PDT). The main expectation of this approach is that it treats lesions in a localized manner, without damaging healthy tissues [5]. The few reports that are available in the literature have validated this hypothesis. In addition, no sign of systemic toxicity is reported in PDT, elimi‐ nating one of the major health issues related to existing TL treatments.[6] These points will be further discussed in this chapter.

The use of light as a therapeutic modality has gained strong impulse recently due to the development of efficient and affordable light sources. Consequently, photo-activated drugs (PhotoSensitizers-PS) play key roles in the present clinical portfolio, and more importantly, are the major lead in the development of new drugs to treat a variety of diseases such as cancer, microbial infections and tropical diseases. However, increasing the efficiency of PDT photo‐ sensitizers remains challenging [7-9].

The use of PDT in veterinary is much less common even considering the benefits that such strategies could bring in the treatment of high-value reproducing animals, as well as, in the treatment of animals that are reservoirs of human diseases [10].

In terms of developing effective treatments against leishmaniasis in endemic areas, it is important to think of comprehensive strategies that could cause a quick decrease in the pool of infected patients (Figure 1). It is also important to emphasize that leishmaniasis is a neglected tropical disease and, therefore, it is highly relevant to consider low-cost strategies that would serve as an alternative for public medicine in poor countries [9]. Developing efficient clinical protocols that would cure/control the disease would not only favor the patient itself, but also, would decrease the chance of this infection being transmitted to others by the vectors or by blood transfusion. In the next sections, we will explain how PDT can be helpful in the treatment of patients, as well as, of all the possible reservoirs and transmitting vectors that would favor the parasite infection cycle (Figure 1). Some of this potential has been attained and some are still in the step of hypothesis testing.

**Figure 1**: Schematic representation of a comprehensive strategy to control leishmania disease in endemic areas by using PDT. Besides treating patients and animals; killing vectors and disinfecting blood, should be considered in a PDT strategy to control leishmaniasis. The star **Figure 1.** Schematic representation of a comprehensive strategy to control leishmania disease in endemic areas by using PDT. Besides treating patients and animals; killing vectors and disinfecting blood, should be considered in a PDT strategy to control leishmaniasis. The star represents the multi-target characteristic of the PDT strategy.

#### **3. Mechanisms in Photodynamic Therapy 3. Mechanisms in photodynamic therapy**

represents the multi-target characteristic of the PDT strategy.

Paulo, in Brazil. The tegumentary leishmaniasis is characterized by skin lesions (cutaneous-

Leishmaniasis is a common zoonosis, with domestic (dogs and cats) and wild (rodents, marsupials, edentulous and wild canids) reservoirs. It is transmitted to humans by sand flies, which comprise the genus Lutzomyia and Phlebotomus. Details of the etiology and patho‐ physiology of the disease are out of the scope of this chapter and we suggest that the reader

The current scenario of leishmaniasis treatment is not promising. Therapeutic approaches include systemic administrations of anti-parasitic medications, which often present serious side effects. Few drugs are available in the clinic, mainly antimonials and amphotericin, and the frequency of resistance development is rising. Therefore, there is an urgent need to establish new and more effective treatments for both VL and TL. The treatment of TL (the focus of this chapter) urges new drugs and new therapeutic forms, that allows for more effective and

One of the promising approaches, and the one discussed in here, is photodynamic therapy (PDT). The main expectation of this approach is that it treats lesions in a localized manner, without damaging healthy tissues [5]. The few reports that are available in the literature have validated this hypothesis. In addition, no sign of systemic toxicity is reported in PDT, elimi‐ nating one of the major health issues related to existing TL treatments.[6] These points will be

The use of light as a therapeutic modality has gained strong impulse recently due to the development of efficient and affordable light sources. Consequently, photo-activated drugs (PhotoSensitizers-PS) play key roles in the present clinical portfolio, and more importantly, are the major lead in the development of new drugs to treat a variety of diseases such as cancer, microbial infections and tropical diseases. However, increasing the efficiency of PDT photo‐

The use of PDT in veterinary is much less common even considering the benefits that such strategies could bring in the treatment of high-value reproducing animals, as well as, in the

In terms of developing effective treatments against leishmaniasis in endemic areas, it is important to think of comprehensive strategies that could cause a quick decrease in the pool of infected patients (Figure 1). It is also important to emphasize that leishmaniasis is a neglected tropical disease and, therefore, it is highly relevant to consider low-cost strategies that would serve as an alternative for public medicine in poor countries [9]. Developing efficient clinical protocols that would cure/control the disease would not only favor the patient itself, but also, would decrease the chance of this infection being transmitted to others by the vectors or by blood transfusion. In the next sections, we will explain how PDT can be helpful in the treatment of patients, as well as, of all the possible reservoirs and transmitting vectors that would favor the parasite infection cycle (Figure 1). Some of this potential has been attained and some are

CL) and mucocutaneous lesions (such as, nasal and mouth regions) [2].

consult reviews that focus on these subjects [3].

394 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

conveniently administered treatments [4].

further discussed in this chapter.

sensitizers remains challenging [7-9].

still in the step of hypothesis testing.

treatment of animals that are reservoirs of human diseases [10].

infecting microorganisms by light-induced reactions, generically called photosensitization reactions. Photosensitization occurs when PS absorb light and transfer its energy to neighboring molecules, such that light converts into chemical reactivity[11-13] After the end of a photo-cycle, PS returns to the ground state and may absorb another photon. The photophysical step that allows the formation of an efficient PS is the intersystem crossing (ICS), that converts singlet into triplet species, which are long lived and highly reactive PDT is a clinical modality based on the damage caused in biological tissues or in infecting microorganisms by light-induced reactions, generically called photosensitization reactions. Photosensitization occurs when PS absorb light and transfer its energy to neighboring molecules, such that light converts into chemical reactivity [11-13]. After the end of a photocycle, PS returns to the ground state and may absorb another photon. The photophysical step that allows the formation of an efficient PS is the intersystem crossing (ICS), that converts singlet into triplet species, which are long lived and highly reactive (Figure 2) [13].

PDT is a clinical modality based on the damage caused in biological tissues or in

(Figure 2)[13]. The Photooxidation of biomolecules is responsible for changes in their structure and function. It can occur by two main mechanisms: electron transfer reaction (excited states are stronger oxidizing and reducing species than their respective ground states) catalyzing the formation of various radical species, including the highly reactive hydroxyl radical. These The photooxidation of biomolecules is responsible for changes in their structure and function. It can occur by two main mechanisms: electron transfer reaction (excited states are stronger oxidizing and reducing species than their respective ground states) catalyzing the formation of various radical species, including the highly reactive hydroxyl radical. These reactions are classified as type I. The photooxidation can also occur through energy transfer with molecular oxygen, catalyzing the formation of singlet oxygen, a mechanism called type II (Figure 2) [14].

reactions are classified as type I. The photooxidation can also occur through energy transfer with molecular oxygen, catalyzing the formation of singlet oxygen, a mechanism called type II (Figure 2) [14]. It is considered that type II mechanism is the most relevant effector of photooxidation, because type I reactions usually lead to PS degradation [15]. However, in biological systems, there usually is shifts between these two mechanisms (type I versus type II), for several reasons, with other biomolecules and PS aggregation [17-21].

including local concentrations of oxygen and of reducing species, interaction of PS with other biomolecules and PS aggregation [17-21]. Free radicals and singlet oxygen have different reactivity towards biological targets, but both can react with them [14,22]. Singlet oxygen mainly reacts by addition to double

It is considered that type II mechanism is the most relevant effector of photooxidation,

because type I reactions usually lead to PS degradation [15]. However, in biological systems, there usually is shifts between these two mechanisms (type I versus type II), for several

Free radicals and singlet oxygen have different reactivity towards biological targets, but both can react with them [14,22]. Singlet oxygen mainly reacts by addition to double bonds (Figure 2). The efficiency of photo-induced cell killing seems to depend more on the amount of PS that is located in the intracellular environment and on the specific intracellular location than on the *in-vitro* photophysical efficiency of the PS [23-28]. bonds (Figure 2). The efficiency of photo-induced cell killing seems to depend more on the amount of PS that is located in the intracellular environment and on the specific intracellular location than on the *in-vitro* photophysical efficiency of the PS [23-28].

**Figure 2**: Top scheme. Main mechanisms of photooxidation. PS, <sup>1</sup> PS, <sup>3</sup> PS: photosensitizer ground state, singlet and triplet species, respectively. O2 and 1O2 correspond to oxygen in the ground state and the singlet excited state, respectively. *hv* represents light absorption at a specific wavelength and ICS is intersystem crossing between the singlet and the triplet states. Bottom scheme: Reaction of singlet oxygen with a double bond forming a hydroperoxide, **Figure 2.** Top scheme. Main mechanisms of photooxidation. PS, 1PS, 3PS: photosensitizer ground state, singlet and triplet species, respectively. O2 and 1O2 correspond to oxygen in the ground state and the singlet excited state, respec‐ tively. *hv* represents light absorption at a specific wavelength and ICS is intersystem crossing between the singlet and the triplet states. Bottom scheme: Reaction of singlet oxygen with a double bond forming a hydroperoxide, which is the main reaction of singlet oxygen with lipid double bonds.

which is the main reaction of singlet oxygen with lipid double bonds. PDT combines three components to kill cells (eukaryotic and prokaryotic) and noncellular organisms such as virus: PS, light and oxygen. PS is applied either topically or systemically and it must incorporate in the biological tissue to be treated, which is exposed to light in the presence of oxygen. The PS needs to absorb efficiently the incident light and form triplet species [14]. There are hundreds of PS molecules that have been synthesized and tested. In Figure 3 we present the chemical structures of few that are worth commenting in this chapter, because they either have been involved on treatments of leishmania or have the potential to be. Methylene Blue (MB) and Crystal Violet (CV) are positively charged and low-cost photosensitizers that enter cells and react mainly by type II and type I mechanisms, PDT combines three components to kill cells (eukaryotic and prokaryotic) and non-cellular organisms such as virus: PS, light and oxygen. PS is applied either topically or systemically and it must incorporate in the biological tissue to be treated, which is exposed to light in the presence of oxygen. The PS needs to absorb efficiently the incident light and form triplet species [14]. There are hundreds of PS molecules that have been synthesized and tested. In Figure 3 we present the chemical structures of few that are worth commenting in this chapter, because they either have been involved on treatments of leishmania or have the potential to be. Methylene Blue (MB) and Crystal Violet (CV) are positively charged and low-cost photosen‐ sitizers that enter cells and react mainly by type II and type I mechanisms, respectively. MB has been used to treat several diseases including leishmania [27], while CV should be tested since it has a great potential as a positively dye that mainly accumulates in mitochondria [28]. Riboflavin (RF, vitamin B2), is a natural PS that absorbs in the 400-500 nm region and has been used for blood disinfection as well as in test-tube leishmania killing assays [29]. Hypericin is another natural PS that is extract from St. John's wort and has been used in several PDT studies [30]. ALA is the first compound in the porphyrin synthesis pathway. Protoporphyrin IX is

formed intracellularly after the treatment with ALA and/or methyl ALA and is the most used PS in leishmaniasis treatment [31-35]. Chlorophyll is the main pigment of photosynthesis and their derivatives hold promising potential as low-cost PS [36]. methyl ALA and is the most used PS in leishmaniasis treatment [31-35]. Chlorophyll is the main pigment of photosynthesis and their derivatives hold promising potential as low-cost PS [36].

respectively. MB has been used to treat several diseases including leishmania[27], while CV should be tested since it has a great potential as a positively dye that mainly accumulates in mitochondria[28]. Riboflavin (RF, vitamin B2), is a natural PS that absorbs in the 400-500 nm region and has been used for blood disinfection as well as in test-tube leishmania killing assays [29]. Hypericin is another natural PS that is extract from St. John's wort and has been

including local concentrations of oxygen and of reducing species, interaction of PS with other

It is considered that type II mechanism is the most relevant effector of photooxidation,

Free radicals and singlet oxygen have different reactivity towards biological targets,

Type I **FREE RADICALS**

PS, <sup>3</sup>

PS: photosensitizer

but both can react with them [14,22]. Singlet oxygen mainly reacts by addition to double bonds (Figure 2). The efficiency of photo-induced cell killing seems to depend more on the amount of PS that is located in the intracellular environment and on the specific intracellular

because type I reactions usually lead to PS degradation [15]. However, in biological systems, there usually is shifts between these two mechanisms (type I versus type II), for several reasons, including local concentrations of oxygen and of reducing species, interaction of PS

Free radicals and singlet oxygen have different reactivity towards biological targets, but both can react with them [14,22]. Singlet oxygen mainly reacts by addition to double bonds (Figure 2). The efficiency of photo-induced cell killing seems to depend more on the amount of PS that is located in the intracellular environment and on the specific intracellular location than on the

location than on the *in-vitro* photophysical efficiency of the PS [23-28].

e-

O2

Type II

PS

**Figure 2**: Top scheme. Main mechanisms of photooxidation. PS, <sup>1</sup>

which is the main reaction of singlet oxygen with lipid double bonds.

ground state, singlet and triplet species, respectively. O2 and 1O2 correspond to oxygen in the ground state and the singlet excited state, respectively. *hv* represents light absorption at a specific wavelength and ICS is intersystem crossing between the singlet and the triplet states. Bottom scheme: Reaction of singlet oxygen with a double bond forming a hydroperoxide,

**Figure 2.** Top scheme. Main mechanisms of photooxidation. PS, 1PS, 3PS: photosensitizer ground state, singlet and triplet species, respectively. O2 and 1O2 correspond to oxygen in the ground state and the singlet excited state, respec‐ tively. *hv* represents light absorption at a specific wavelength and ICS is intersystem crossing between the singlet and the triplet states. Bottom scheme: Reaction of singlet oxygen with a double bond forming a hydroperoxide, which is

PDT combines three components to kill cells (eukaryotic and prokaryotic) and non-cellular organisms such as virus: PS, light and oxygen. PS is applied either topically or systemically and it must incorporate in the biological tissue to be treated, which is exposed to light in the presence of oxygen. The PS needs to absorb efficiently the incident light and form triplet species [14]. There are hundreds of PS molecules that have been synthesized and tested. In Figure 3 we present the chemical structures of few that are worth commenting in this chapter, because they either have been involved on treatments of leishmania or have the potential to be. Methylene Blue (MB) and Crystal Violet (CV) are positively charged and low-cost photosen‐ sitizers that enter cells and react mainly by type II and type I mechanisms, respectively. MB has been used to treat several diseases including leishmania [27], while CV should be tested since it has a great potential as a positively dye that mainly accumulates in mitochondria [28]. Riboflavin (RF, vitamin B2), is a natural PS that absorbs in the 400-500 nm region and has been used for blood disinfection as well as in test-tube leishmania killing assays [29]. Hypericin is another natural PS that is extract from St. John's wort and has been used in several PDT studies [30]. ALA is the first compound in the porphyrin synthesis pathway. Protoporphyrin IX is

 PDT combines three components to kill cells (eukaryotic and prokaryotic) and noncellular organisms such as virus: PS, light and oxygen. PS is applied either topically or systemically and it must incorporate in the biological tissue to be treated, which is exposed to light in the presence of oxygen. The PS needs to absorb efficiently the incident light and form triplet species [14]. There are hundreds of PS molecules that have been synthesized and tested. In Figure 3 we present the chemical structures of few that are worth commenting in this chapter, because they either have been involved on treatments of leishmania or have the potential to be. Methylene Blue (MB) and Crystal Violet (CV) are positively charged and low-cost photosensitizers that enter cells and react mainly by type II and type I mechanisms,

biomolecules and PS aggregation [17-21].

396 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

*in-vitro* photophysical efficiency of the PS [23-28].

ICS

PS <sup>3</sup>

*hν*

PS

1

1 O2

the main reaction of singlet oxygen with lipid double bonds.

with other biomolecules and PS aggregation [17-21].

**Figure 3**. Molecular structure of relevant photosensitizers in PDT: (A) methylene blue; (B) crystal violet; (C) Riboflavin, (D) Hypericin; (E) ALA, Methl ALA and Protoporphyrin IX; **Figure 3.** Molecular structure of relevant photosensitizers in PDT: (A) methylene blue; (B) crystal violet; (C) Riboflavin, (D) Hypericin; (E) ALA, Methl ALA and Protoporphyrin IX; (F) chlorophyll.

(F) chlorophyll. The ability of PDT to act as an anti-microbial treatment, i.e., to treat fungi, bacteria and virus infections, is well described in the scientific literature [37-39]. Many research groups have developed experiments that prove the effectiveness of this therapy for a large number of diseases, including certain parasitic diseases [40]. *In vitro* studies of photoinduced inactivation of parasites have been used to unravel important aspects of the therapy including, the action mechanisms, light dosimetry, structural-activity relationships, PS uptake and localization. PDT has been used in the treatment of human and experimental murine leishmaniasis of the Old and New Word. Despite the small number of cases related, literature highlights the ability of PDT to deliver better results compared to traditional treatments, emphasizing its better effectiveness in leading to amastigote-free lesions in a shorter time periods, in addition to its excellent esthetic results.
