**9. Conclusions**

release and nitric oxide (NO) generation after *in vitro* co-culture of mouse peritoneal macro‐ phages or U937 cells with lipopolysaccharide (LPS) [106-108]. Miltefosine has also been reported to enhance the immune response of IL-2-stimulated mononuclear cells resulting in granulocyte-macrophage colony-stimulating factor (GM-CSF) and IFNγ gene expression and

However, recent studies on the effects of miltefosine on dendritic cells (DC) in *L. major*infection have challenged the putative role of the immunomodulatory action of miltefosine on its antiparasitic action, and suggest that miltefosine functions independently of the immune system, mostly through direct toxicity against the *Leishmania* parasite [110]. DC are critical for initiation of protective immunity against *Leishmania* through induction of Th1 immunity via interleukin 12 (IL-12), and when co-cultured with miltefosine for 4 days, most of the *in vitro*infected DC were free of parasites. However, miltefosine treatment did not influence DC maturation (upregulation of major histocompatibility complex II [MHC II] or co-stimulatory molecules, e.g., CD40, CD54, and CD86), did not significantly alter cytokine release (IL-12, tumor necrosis factor alpha [TNF-α], or IL-10), antigen presentation, or NO production [110].

Miltefosine is marketed as Milteforan® (Virbac, Carros, France) for the treatment of canine visceral leishmaniasis that is the result of infection with *L. infantum* in the Old World and *L. chagasi* in the New World. These two *Leishmania* species are considered sibling and indistin‐ guishable species, and several genetic studies have shown evidence for the synonym of *L. infantum* and *L. chagasi*, and suggest the introduction of *L. infantum* from Southwest Europe into the New World in recent history [111-114]. Thus, *L. infantum* (Old World) and *L. chagasi* (New World) belong to the same species, and therefore *L. chagasi* has been synonymized with *L. infantum*. Dogs are considered the primary reservoir hosts *of L. infantum/chagasi*, and infection of dogs with *L. infantum/chagasi* involves cells of the lymphatic series resulting in visceralization of infection. The domestic dog seems to be a main reservoir for human visceral leishmaniasis, rendering canine disease control a critical issue. Unfortunately efforts to control leishmaniasis in dogs have been largely unsuccessful so far. Oral administration of miltefosine at a dose of 2 mg/kg body weight once a day for 28 days leads to significant reduction of parasite loads and clinical symptoms, whereas adverse reactions were not serious and observed in less that 12% of the dogs, the most frequent one being vomiting, which was transient, self-limiting, and reversible [115-117]. *Leishmania* DNA quantification by real-time PCR has shown that miltefosine treatment of dogs leads to a drastic and progressive reduction of parasite load in lymph node aspirates, but does not suppress the parasite in lymph nodes [118]. Miró et al. [119] has shown that the treatment of miltefosine-allopurinol combination therapy (2 mg/kg miltefosine orally once daily for 28 days and 10 mg/kg of allopurinol orally twice daily for 7 months) behaved similarly to the current reference combination therapy, namely meglumine antimoniate-allopurinol (50 mg/kg of meglumine antimoniate sub-cutaneously twice daily for 28 days and 10 mg/kg of allopurinol orally twice daily for 7 months), in promoting a significant reduction in total clinical score and parasite load over the 7-month study period. These

IFNγ secretion [109].

452 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

**8. Canine leishmaniasis**

At present the control of protozoan parasite *Leishmania* infections relies primarily on chemo‐ therapy, but the armoury of drugs available for treating *Leishmania* infections is rather limited and includes a few drugs with unknown cellular targets and unclear mode of action. These drugs include pentavalent antimonials, pentamidine, amphotericin B, miltefosine, paromo‐ mycin, fluconazole, allopurinol, and few other drugs at various stages of their development process. The recent inclusion of miltefosine as a new antileishmanial drug has been a break‐ through in the treatment of leishmaniasis, as it constituted the first effective oral drug, thus facilitating medical access and making treatment more accessible to rural and remote areas. Interestingly, miltefosine belongs to a family of lipid compounds collectively known as APLs, some of them showing also interesting and promising antileishmanial activities in addition to their well known antitumor action. Thus, additional APLs or APL-related compounds might be of interest to identify novel drugs to combat *Leishmania* infections both in humans and animals, especially in dogs as major reservoirs for human visceral leishmaniasis (*L. infantum*/ *chagasi*). Identification of the death signaling pathways activated in miltefosine-sensitive parasites will be essential for a better understanding of the molecular mechanisms of action and resistance in these parasites. In this regard, the knowledge acquired for the antitumor action of APLs is being and will be of further aid to unveil the mode of action of miltefosine and putative additional APLs with potent antileishmanial activities. Furthemore, elucidation of the molecular mechanisms underlying miltefosine- and APL-mediated cell death will facilitate the design of new therapeutic strategies against *Leishmania* parasites. The proneness to generate *in vitro* resistance to miltefosine raises some concerns about its putative life span in clinical use, thus favoring research on additional APLs that could improve the current antileishmanial features of miltefosine as well as on combination therapy regimens. APLs have shown themselves as potent antileishmanial drugs, and current evidence warrants further research on these promising lipid drugs that could make a difference in the clinical setting and medical care of leishmaniasis.
