**7. Effects of APLs on the immune system**

includes proteins proposed as likely beta subunits for P4-ATPases [93, 94]. LdMT and LdRos3 proteins are primarily localized to the *Leishmania* plasma membrane and required for the rapid intracellular uptake of miltefosine and additional related choline-bearing lipids. Likewise, in the budding yeast *S. cerevisiae*, members of the two protein families have been found to form stable transporter complexes that function in the translocation of phospholipids from the exoplasmic to the cytoplasmic leaflet of cellular membranes [95, 96]. Despite both LdMT and LdRos3 normally localize to the plasma membrane, they are retained at the endoplasmic reticulum in the absence of the other protein or in the presence of inactivating point mutations in LdMT [94]. Both LdMT and the Cdc50-like protein LdRos3 form a stable complex that plays an essential role in maintaining phospholipid asymmetry in the parasite plasma membrane [97], and constitute part of the same translocation machinery that determines flippase activity, responsible for miltefosine uptake, as well as miltefosine sensitivity in *Leishmania* [94]. Loss of either LdMT or LdRos3 blocks ATP-dependent transport of NBD-labeled phosphatidyletha‐ nolamine and phosphatidylcholine from the outer to the inner plasma membrane leaflet in *L. donovani* promastigotes and results in an increased cell surface exposure of endogenous phosphatidylethanolamine, whereas infectivity was not compromised [97]. A promastigote line, M-mutR, that shows mutations in LdMT, thus leading to defective miltefosine internali‐ zation, is infective to macrophages *in vitro* and in BALB/c mice *in vivo*, and displays a good correlation of *in vitro* resistance between promastigotes and intracellular amastigotes [29]. The fact that M-mutR parasites retain the resistant phenotype *in vivo* indicate that miltefosineresistant *L. donovani* promastigotes transform to miltefosine-resistant amastigotes [29]. It was also observed no cross-resistance to other antileishmanial drugs in M-mutR amastigotes [29]. Some clinical studies have suggested, as indicated above, that miltefosine shows significantly less efficiency against the cutaneous and mucocutaneous leishmaniasis caused by *L. brazilien‐ sis* parasites, mainly due to their inability to internalize the drug because of the low expression levels of the beta subunit LbRos3 [98]. Overexpression of LbRos3 induced increased miltefo‐ sine sensitivity in both *L. braziliensis* promastigotes and intracellular amastigotes, further supporting the notion that miltefosine uptake is a major event in determining miltefosine antileishmanial potency [98]. Miltefosine-resistant promastigotes, displaying cross-resistance to the ether lipid edelfosine, but not to the standard anti-leishmanial drugs, shows no ampli‐ fication of specific genes, including the multidrug resistance P-glycoprotein gene, and resistance has been found to be stable up to 12 weeks in drug-free culture medium [28].

450 Leishmaniasis - Trends in Epidemiology, Diagnosis and Treatment

The evidence gathered so far has shown that reduced miltefosine incorporation has always led to a resistant phenotype. This lower accumulation of miltefosine can be achieved by two processes: a) a decrease in drug uptake, rendered by inactivation of any one of the two proteins responsible for the miltefosine uptake, namely LdMT and its beta subunit LdRos3; b) an increase in drug efflux, mediated by the overexpression of the ABC transporter P-glycoprotein [27]. Thus, in addition to a flaw in the uptake of miltefosine as stated above, an increased efflux of miltefosine has also been implicated in miltefosine resistance. A multidrug resistance (MDR) *L. tropica* line overexpressing a P-glycoprotein-like transporter was found to display significant cross-resistance to the ALP miltefosine and edelfosine, with resistant indices of 9.2- and 7.1 fold, respectively [99]. This resistance was mediated through overexpression of an ABC transporter, namely the *Leishmania* P-glycoprotein-like transporter (*Leishmania* ABCB1 or Even though miltefosine retains its antiparasitic activity against *Leishmania* infection in immunodeficient SCID mice, leading to similar levels of activity in both SCID and BALB/c mouse-*L. donovani* models [52], the immunomodulatory properties of miltefosine have been proposed as an additional factor to its antileishmanial action [104]. Thus, miltefosine's antileishmanial function has been reported to be significantly compromised in interferongamma (IFNγ)-deficient macrophages, suggesting the importance of endogenous IFNγ in the miltefosine-induced antileishmanial functions of macrophages. IFNγ responsiveness is reduced in *L. donovani*-infected macrophages but is significantly restored by miltefosine, as it induces IFNγ, enhances IFNγ receptors, and IFNγ induces STAT-1 phosphorylation but reduces activation of SHP-1, the phosphatase implicated in the downregulation of STAT-1 phosphorylation [104]. *L. donovani*-infected macrophages induced Th2 response, but miltefo‐ sine treatment reversed the response to Th1-type [104].

Miltefosine is able to form stable multilamellar vesicles (MLVs liposomes) to deliver the APL to monocytes/macrophages. Both micellar and liposomal miltefosine have been found to interact with human monocytes and upregulate specific adhesion molecules, including intracellular adhesion molecule-1 and class 1 major histocompatibility complex (MHC-1) antigen in a dose-dependent manner in U937 cells [105], used as a cell line model system to study human monocytes. These actions could be involved in the initial steps of miltefosinemediated recruitment of macrophages [105]. Miltefosine, better in liposomal than in free (micellar) form, has also been reported to induce an increase in tumor necrosis factor (TNF) 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 IFNγ secretion [109].

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].

#### **8. Canine leishmaniasis**

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 observations together with the lack of effect on renal and hepatic parameters and adverse reactions suggests that miltefosine, in combination with allopurinol, might offer a safe and effective alternative treatment option for canine leishmaniasis compared to the reference therapy [119]. A recent study [117], where dogs naturally infected with *L. infantum*/*chagasi* were treated with miltefosine using different therapeutic regimens, has shown that after treatment and during the following 24 months, there was progressive clinical improvement and complete recovery in 50% (7/14) of the treated animals. There was a decrease in the smear positivity of the bone marrow after treatment, and there was also a gradual and constant decrease in positive cultures at the end of the follow-up period. However, the PCR detection of parasite DNA remained positive, and the animals presented a significant increase in parasite load 6 months after treatment. Thus, the fact that the improvement in the clinical symptoms is not followed by total parasitological clearance, raises some doubts about the use of this drug in endemic areas where the dogs are involved in the maintenance of the parasite cycle.
