**4. Challenging the target: organometallic compounds**

Although metals have been used in Medicine for centuries, most compounds produced by the pharmaceutical industry are still based on organic molecules. Nevertheless, new perspectives about metal-based drugs and their therapeutic potential against cancer, bacteria, virus, and even trypanosomatid infections have emerged in the last few years. In this context, *Leishmania* infections have been treated with pentavalent antimonials since the 1940s [1], which indicates that new compounds containing other kinds of metals may present anti-*Leishmania* activity, thus opening the possibility to reduce the toxicity based on the metal-drug synergism.

Platinum-derived metals are well known to have antitumor effects due to their ability to bind to DNA molecules. So, since tumor cells and kinetoplastid parasites present similar metabolic pathways [61], the coordination of these metals to organic compounds might be efficient in treating *Leishmania* infections. For example, the (2,2′:6′2″-terpyridine)platinum(II) complexes can inhibit 100% of the growth of *L. donovani* amastigotes at a concentration of 1 μM through the intercalation of terpyridine and platinum(II) into the DNA, probably binding to guanine bases or some enzyme active sites [62]. More recently, cisplatin-derived complexes (*cis*diamminedichloroplatinum(II)), an anticancer drug, were tested on *L*. *infantum* promastigotes and amastigotes, revealing a remarkable anti*-Leishmania* activity with IC50 values of 1.03 μM and 0.10 μM, respectively. Furthermore, the treatment with *cis*-DDP induced loss of mitochondrial transmembrane potential and DNA fragmentation, thus leading to apoptosis-like death [63].

In addition to platinum and its derivatives, other transition metals have been widely studied in terms of antiprotozoal activity. For example, organometallic complexes containing ruthenium(II) and anti-inflammatories were evaluated active against *L. amazonensis* and *L. infantum* promastigotes, presenting IC50 values comparable to the meglumine antimoniate, one of the first-line drugs for *Leishmania* infections [64]. Moreover, the coordination of gold compounds with organic ligands was efficient against *L. amazonensis* and *L. braziliensis* promastigotes while presenting low toxicity to host cells [65]. Thus, the therapeutic mechanism of these organometallic molecules may be related to induction of oxidative damage and alterations in the membrane permeabilization by the inhibition of specific membrane protein channels and zinc-binding proteins. These alterations can lead to parasite cell death by apoptosis-like and necrosis.

Among transition metals, the essential ones, such as zinc and copper, are present in cell structures and involved in many cellular processes, becoming indispensable for host–parasite interactions. Zinc regulates gene transcription processes and cell signaling, while copper is also a critical enzymatic cofactor for organ functioning and multiple metabolic processes [66]. Therefore, the coordination of organic molecules to essential metals regarding new antiprotozoal treatments might increase the drug uptake and contribute to the parasite's elimination. The zinc(II)-dipicolylamine (ZnDPA) coordination complexes were active against *L. major* promastigotes *in vitro* with IC50 values between 12.7 μM and 0.3 μM and minimal mammalian cell toxicity. The compounds also showed *in vivo* activity, with a high affinity for intracellular amastigotes and low toxicity to mice [67]. The effects of a copper dimethoxy bipyridine (CuDMOBP) complex were also investigated against *L. major*; the complex presented significant *in vitro* activity with high selectivity index [68]. Results from quantitative real-time PCR also indicate a significant reduction in cellular expression of IL-10 and TNF-α in macrophages treated with CuDMOBP, probably due to a reduction of the parasite population.

*Use of Cell Biology to Identify Cellular Targets in Drug Development Process... DOI: http://dx.doi.org/10.5772/intechopen.101662*

Metals have also been combined with ergosterol biosynthesis inhibitors, including the azoles family of drugs, which are used to treat fungal infections and present activity against protozoan parasites. For example, a recent study from our group revealed the potent effect of the combination of itraconazole (ITZ) with zinc (Zn) against *L. amazonensis* promastigotes (**Figure 5B**) and intracellular amastigotes [69]. The biological effects were significantly increased when the itraconazole was coupled with zinc, resulting in IC50 values in nanomolar ranges and cell death of parasites in low concentrations [69]. Likewise, the coordination of clotrimazole, ketoconazole (**Figure 5A**), and miconazole to manganese (Mn) provide novel molecules with better activity against *L. major* when compared to the original organic antifungals [70].

Despite Medicine's advances in the past decades, information about organometallic drugs is still lacking. More profound studies must be done to understand the role of metals in host–parasite interactions, thereby better comprising the mechanisms of organometallics drugs against the parasites and mammalian host cells. Nevertheless, the literature available indicates that organometallics are a promising class of drugs for treating leishmaniasis.

#### **Figure 5.**

*Ultrathin sections of* L. amazonensis *promastigotes treated with organometallic compounds. (A) Treatment with 300 nM ketoconazole-ruthenium induced alterations in the mitochondria, such as swelling and loss of the mitochondrial matrix and the formation of large autophagic vacuoles. (B) Treatment with 0.5 μM itraconazole-zinc resulted in mitochondrial alterations and an increase in the secretion of vesicles (arrowhead) at the flagellar pocket. In both panels, the nuclear chromatin appeared altered. F, flagellum; M, mitochondrion; N, nucleus.*

## **5. Therapeutic combination: what do we know?**

There are many strategies to treat leishmaniasis; however, several studies have shown the numerous advantages of therapeutic combination, like observing for other diseases. For example, combining drugs from different chemical classes could reduce the total drug doses or treatment duration. These aspects are important to minimize toxic side effects, submission at treatment, less load on the public health system and reduce cases of drug resistance. Also, the therapeutic combination could improve treatment efficacy for refractory or complicated cases, such as in patients coinfected with HIV. A successful study conducted by the Drugs for Neglected Diseases initiative (DNDi), in partnership with Médicins Sans Frontières (MSF) and other institutions, pointed to evidence of the high efficacy of the combination therapy to treat patients with visceral leishmaniasis (VL) in coinfection with HIV [71]. Although the current WHO guidelines recommend the treatment of HIV/VL coinfection with liposomal amphotericin B (AmBisome®), this work strongly supports a change in the treatment recommendations, from AmBisome monotherapy to combination therapy as the first-line treatment. Moreover, they suggest the combination with miltefosine once this combination therapy has a good safety profile and is highly efficacious [71].

Nowadays, combination therapy is an efficient tool to treat many microbial infections such as AIDS, tuberculosis, malaria, and several other diseases. Recent works have shown that combination therapy for leishmaniasis has progressively been recommended to increase treatment tolerance and efficacy, reduce cost and treatment duration, and limit the growth of drug resistance [72–75]. For the treatment of leishmaniasis, WHO has recommended combination therapy based on many studies showing the efficacy of this therapeutic tool; the combinations include novel synthesized drugs, nanoparticles developed for drug delivery, repositioned drugs, old medicines, and immunomodulatory agents [76–79]. Indeed, several studies have reported the superior efficacies of combination therapies against leishmaniasis. Some of them demonstrated the synergic effects of combinations between amphotericin B with other available medicines, such as meglumine antimoniate, miltefosine, paromomycin, or azithromycin [80–82].

Analysis of drug interactions aimed to show if the interaction between them is classified as synergistic, antagonistic, or indifferent. *In vitro* data are based on an extended ratio and concentration range. However, *in vivo* combinations are more complex and less defined, with the number of doses limited. The synergy between two (or more drugs) occurs when their combined activities are improved over the sum of their separate individual effects. Synergistic drug combinations provide lower concentrations of both compounds, enhancing therapy outcomes by increasing efficacy and reducing side effects. Moreover, synergistic combinations could reintroduce those that have lost activity against drug-resistant strains. The FICI (fractional inhibitory concentration index) value is considered the standard reference parameter to quantify interactions between pairs of drugs. Odds in 2003 [83] established more restrictive criteria to analyze experiments and defined "synergy" as a ≥ 4-fold reduction in the MICs of both compounds in combination when compared to their MICs alone, where the FICI value must be ≤0.5. MIC means the minimum inhibitory concentration, i.e., the lowest drug concentration with no visible cell growth.

*In vitro* studies against *L*. *amazonensis* suggest that combinations between compounds that act in different biosynthetic pathways of the parasite, such as sterol biosynthesis, are promising [32]. Interestingly, a recent study showed that sterol biosynthesis inhibitors and alkylphosphocholine analogs, combined with medicines available to treat other diseases, are efficient against trypanosomatids [32, 36, 78, 84]. Besides, another study showed the *in vivo* efficacy of the combination therapy between miltefosine, an alkylphosphocholine, and amphotericin B or paromomycin [85], a therapeutic alternative to treat antimony-resistant VL cases in India. Furthermore, topical treatment against cutaneous leishmaniasis might be effective when amphotericin B and miltefosine are co-loaded at second-generation ultra-deformable liposomes since *in vivo* studies displayed a significant reduction in the parasitic burden [86]. However, a Brazilian survey against *L. infantum* revealed a decrease of the miltefosine concentration when combined with lopinavir (anti-HIV drug); yet, the synergistic effect was not evidenced [87]. Unexpectedly, the combination of nelfinavir with miltefosine presented better results, with FICI ≤0.5. Thus, this study also concluded that the combination might be helpful to treat patients with visceral leishmaniasis who also are infected with HIV [88].

Combination therapy is a promising strategy to treat several diseases. Therefore, it is urgent to investigate synergistic and other drug combinations to increase novel probabilities of therapeutic protocols to treat leishmaniasis. The discovery and the analysis of drug combinations can be facilitated by the collective use of different

*Use of Cell Biology to Identify Cellular Targets in Drug Development Process... DOI: http://dx.doi.org/10.5772/intechopen.101662*

approaches and methods. Drug combinations have proved to be a successful strategy to shorten the course of therapy and reduce toxicity through lower dosage administration; these strategies should reduce the appearance of new resistant parasites. Thus, recent proposals of combinations have been suggested as state-ofthe-art for the treatment of leishmaniasis. In the short run, combination therapy is an interesting way to improve the treatment for leishmaniasis.

## **6. Where are we going? Nanotechnology**

Recent advances in Nanotechnology have had a profound impact on health sciences, especially Medicine, because of the development of different nanomaterials designed as intracellular carriers to deliver drugs and genes. The development and use of nanocarriers have also been established in the field of Pharmaceutical Sciences by enabling the encapsulation of drugs creating stable and controlled environments, and improving the biocompatibility of these drugs in various biological systems. These nanocarriers were developed to improve the solubility of poorly soluble drugs, control or maintain their release, and protect them from degradation. These characteristics increase drug bioavailability, reduce systemic side effects, and increase drug specificity for biological targets. For these reasons, Nanotechnology is a new field that allows the construction of versatile diagnostic and therapeutic platforms using nanocarriers as molecular machinery for different clinical applications.

The development of nanocarriers began in the 1960s to always seek to improve biocompatibility and reduce the toxicity of nanomaterials. The second generation of nanocarriers was developed around the 1980s and sought to improve the surface of these materials by increasing their stability, stealth, and targeting ability. The third generation of nanocarrier introduced the idea of intelligent nanomedication to enhance the targeting mechanisms and theranostic capabilities of these nanomaterials [89]. The word *nanoparticle* has been widely applied to describe numerous pharmaceutical carriers or imaging systems based on nanoscale materials. Nanoparticles are particulate materials in their solid or dispersed state present in a size scale between 10 nm and 100 nm (ISO/TS 80.004–1:2015). Due to the great diversity of these nanomaterials, the scientific community elaborates a classification based on their characteristics and properties.

Initially, the nanomaterials are divided into two classes: inorganic and organic. However, they are divided into three subgroups, according to some of their characteristics. The first would concentrate single-chain polymer-drug conjugates, polymeric colloids prepared by techniques such as emulsion polymerization, crosslinked nanogel matrices, dendrimers, and carbon nanotubes, where the nanocarrier is a single synthetic molecule with covalent bonds and a relatively large molar mass. The second subgroup of nanocarriers would comprise self-assembly of smaller molecules such as 1) liposomes and polyplexes, the most studied members of this group of nanoparticles; 2) polymersomes and other sets of block copolymers; 3) colloidosomal aggregates of latex particles and sets of proteins or peptides. In this case, the dynamic nature of these types of systems depends on intermolecular forces and biological conditions. Finally, the last subgroup of nanocarriers would include complexes based on fullerenes, silica, colloidal gold, gold nanoshells, quantum dots, and superparamagnetic particles.

Another critical point in developing nanocarriers is the synthesis, which can be rationally divided into two fundamental stages: nucleation and growth. Understanding and manipulating these two steps have created new possibilities allowing researchers to easily control the synthesis of nanoparticles in terms of

size, morphology, and monodispersity. The choice of the synthesis route provides a characteristic set of advantages and disadvantages in nanomaterial production.

In *Leishmania* sp., the use of nanocarriers has been studied since the late 1970s with the development of liposomal amphotericin B [90, 91]. Since its development, liposomal amphotericin B has become more efficient and bioavailable, less toxic, and better tolerated by patients [92, 93]. This formulation also has a broad and rapid biodistribution reaching steady-state plasma concentrations faster with higher total plasma concentration when compared to its deoxycholate form. Furthermore, liposomal amphotericin B is probably inactive because it is fixed to the liposome; thus, the biologically active drug is released only after direct contact with protozoa or fungal cell walls [94]. In 2010, the WHO proposed the administration of liposomal amphotericin B.

The success achieved by the liposomal formulation of amphotericin B is related to its properties as a nanocarrier system, which has numerous advantages. However, despite these advantages, this system has some disadvantages, including its high cost, limiting its use [94]. Thus, the development of new, cheaper, and more efficient nanoformulations is necessary. Furthermore, different nanocarriers have been developed in recent decades, searching for new therapeutic alternatives to treat leishmaniasis, including nanoparticles based on liposomal, lipid, polymeric, and metallic nanomaterials [8, 95–98]. Therefore, choosing the correct nanocarriers is crucial to define properties and characteristics for this proposed new therapeutic approach. Thereby, this enormous diversity of available nanoparticles makes the development of nanocarriers for the treatment of leishmaniasis very promising since each of these carrier systems has advantages and limitations over each other [8].

Some nanoparticles have been generating significant repercussions for presenting theranostic properties, thus allowing them to be used simultaneously for diagnosis and therapy. Superparamagnetic iron oxide nanoparticles (SPIONs) are one example of this type of nanomaterials. SPIONs have excellent biocompatibility, degradability in moderate acid conditions, magnetic properties, and the ability to generate heat when subjected to an alternating current magnetic field [99, 100]. In addition, this type of nanomaterial still has a large surface area presenting great chemical diversity, which can increase the efficacy of the treatment. Finally, these nanomaterials can also be conjugated to specific molecules to facilitate selective and efficient drug delivery to a diseased tissue or organ [101]. The application of this type of nanomaterial for the treatment of leishmaniasis has been studied by different groups and has shown promising results over the past few years (**Figure 6**) [102–105].

#### **Figure 6.**

*Ultrathin sections of* L. amazonensis *promastigotes (A) and amastigotes (B) after treatment with 100 μg/mL SPIONs for 24 h. In both developmental stages, SPIONs were found inside membrane-bound structures (arrowheads). Some alterations were also observed in the treated parasites, such as increased number of lipid bodies (arrows), presence of autophagosome (thin arrows), and secretion of extracellular vesicles (large arrow).*

*Use of Cell Biology to Identify Cellular Targets in Drug Development Process... DOI: http://dx.doi.org/10.5772/intechopen.101662*

In summary, Nanotechnology and the use of nanoparticles inaugurated a new field in health science called Nanomedicine, one of the most promising branches of contemporary Medicine. Thus, the development of new nanomaterials to treat leishmaniasis significantly increases the possibility of finding novel therapeutic alternatives, mainly considering the great diversity of clinical manifestations. An excellent example of this is the use of nanoparticles to develop a topical treatment that can revolutionize the treatment of cutaneous leishmaniasis.
