ABSTRACT
The oral efficacy and safety of a leishmanicidal nitrochalcone (CH8) were studied in BALB/c mouse infections with Leishmania amazonensis and Leishmania infantum. Although 10-fold-higher doses of CH8 were needed to produce the same antiparasitic effect as that seen with the reference drug miltefosine, the latter was nephrotoxic, whereas CH8 restored disease toxicity markers to normal. This study shows the therapeutic potential of an orally active and hepato-/nephroprotective chalcone against cutaneous and visceral leishmaniases.
TEXT
Cutaneous (CL) and visceral (VL) leishmaniases are vector-borne neglected tropical diseases caused by protozoan parasites of the genus Leishmania that multiply inside phagocytic cells. Together, they affect nearly 12 million people worldwide (1). While CL is characterized by chronic skin lesions that can progress into disfiguring diffuse and mucosal disease forms, VL is a potentially fatal disease affecting macrophage-rich internal organs, such as spleen, liver, and bone marrow. Despite their distinct clinical manifestations, CL and VL infections are commonly treated with parenteral injections of pentavalent antimony compounds (meglumine antimoniate and sodium stibogluconate) or pentamidine and amphotericin B formulations in the case of drug resistance or intolerance. Major limitations associated with these injections are systemic toxicity, high drug and hospital costs, and low patient compliance (2). The only oral drug with recognized efficacy against leishmaniasis, particularly VL, in the Indian subcontinent is miltefosine, an alkyl phospholipid compound originally intended for cancer. Miltefosine drawbacks are gastrointestinal toxicity, teratogenesis, and decreased liver and kidney functions (3). Recent treatment failures due to emerging resistance have prompted combination therapy (4). Miltefosine efficacy against human disease forms is controversial in Brazil (5), and recent approval for treatment in dogs may promote the spread of resistance. Thus, potential toxicity and emerging drug resistance justify the search for novel oral treatments.
Chalcones are a class of biologically active flavonoids, with >330 compounds described with antileishmanial activity in the last decades (6). Pioneer studies in Leishmania major-infected mice and Leishmania donovani-infected hamsters demonstrated the intraperitoneal efficacy of licochalcone A against CL and VL (7). Later, the intralesional subcutaneous activity of the natural oxygenated chalcone DMC (2′,6′-dihydroxy-4′-methoxychalcone) against murine CL caused by Leishmania amazonensis was established (8). Due to its low extraction yield, a DMC analogue, 3-nitro-2′-hydro-4′,6′-dimethoxychalcone (CH8) (Fig. 1) was synthesized and shown to be more selective (selectivity index >143) than DMC (9). Intralesional subcutaneous treatment of CL with CH8 was improved after being loaded in biodegradable microparticles for sustained release (10). A recent biodistribution study in mice given an oral gavage with 12.5 mg/kg of 99mTc-radiolabeled CH8 demonstrated drug presence in blood and liver (30% and 40% of absorbed drug, respectively) and detectable amounts in tissues, such as spleen, bone marrow (femur), lymph nodes, and feet, 2 h after administration (11). Evidence that CH8 is orally absorbed and distributed to VL and CL target tissues prompted us to explore its efficacy and safety in comparison with miltefosine.
Chalcone CH8 structure.
All the experiments were carried out in female BALB/c mice from our animal house facilities (Federal University of Rio de Janeiro, Brazil), used at 8 weeks of age and 25 g body weight. Animals were kept under controlled temperature, lighting, bedding, and feeding conditions and manipulated according to the Guide for the Care and Use of Laboratory Animals (NIH). The experiments were approved by the Committee on the Ethics of Animal Use of the Federal University of Rio de Janeiro under codes CEUA17 and IBCCF118.
The CH8 (>97% purity) used here was synthesized as previously described (8). To evaluate efficacy in CL infection, mice were intradermally infected in the right ear pinna with 2 × 106 L. amazonensis promastigotes (WHOM/BR/75/Josefa) transfected with green fluorescent protein (L. amazonensis-GFP) (12). Ten days after infection, when lesions became detectable, animals were treated daily for 30 days by intragastric gavage with 100 μl of CH8 (40, 120, and 240 mg/kg) in propylene glycol vehicle, previously shown to be inert. Controls received miltefosine (>98% purity; Sigma-Aldrich) at 12 mg/kg (13) or were left untreated. For clinical follow-up, ear thicknesses were periodically measured with a caliper gauge. For parasite burden, on day 42 of infection, the individual ear tissue homogenates were assayed for specific fluorescence intensity by using a florescence plate reader (485/528 nm, FLx800; Bio-Tek Instruments, Winooski, VT) as previously described (14). To evaluate treatment safety, serum samples were individually collected for measurement of aspartate transaminase (AST), alanine transaminase (ALT), and creatinine as parameters of liver and kidney toxicity using colorimetric commercial kits (Doles, Brazil) adapted for microvolumes (15). Positive controls were serum samples from mice treated intraperitoneally with 1% of carbon tetrachloride in 200 μl of soybean oil (CCl4; Sigma-Aldrich) (16). Data were analyzed using two-way or one-way analysis of variance and Dunnett's test as posttest with GraphPad Prism 6 software and considered different when the P value was ≤0.05. Treatment of CL infection was effective with CH8 doses as low as 40 mg/kg, which, after 33 days of treatment, reduced lesion size by 54% in relation to untreated animals (Fig. 2A). Lesion decrease was accompanied by a 48% reduction in parasite burden (Fig. 2B). With 120 mg/kg of CH8, 74% and 68% reductions were achieved in lesion size and parasite burden, respectively; a similar effect was observed with 12 mg/kg of miltefosine. Doubling CH8 doses to 240 mg/kg produced a 92% reduction in ear parasitism, although the corresponding lesion sizes were not further diminished, possibly due to a scarring effect. The doses required for oral CH8 efficacy are higher than those used with miltefosine because of its low oral bioavailability, typical of chalcone scaffolds (17); however, absorbed drug concentrations in the liver and blood highlight its potentiality in VL treatment (11).
Effect of oral CH8 in CL. Mice were infected in the ear pinna with L. amazonensis-GFP. On days 10 to 43, they were treated daily with the indicated oral doses of CH8 or miltefosine (Miltef). Controls were left untreated. (A) Lesion growth as monitored by the increase in ear thickness measured with a dial caliper on the indicated days. (B) On day 43 of infection (23 oral doses), the parasite loads in the infected ears were quantified by fluorimetry. Fluorimetric values were corrected for uninfected ear background values, which were 4.63 ± 0.16 arbitrary fluorescence units (FU): 100% (untreated), 9.52 ± 1.61 FU. Values are means ± SEM (n = 5). *, P < 0.05 in relation to untreated animals; #, P < 0.05 in relation to miltefosine treatment; &, P < 0.05 in relation to treatment with CH8 40 mg/kg.
To assess CH8 efficacy in VL infection, mice were infected with 2 × 107 Leishmania infantum promastigotes (NCL strain) in the tail vein. Treatment with daily oral gavage with CH8 (150 mg/kg in 100 μl of propylene glycol) was started on day 1 of infection and followed until day 29, totaling 28 doses. Controls were given miltefosine (2 and 12 mg/kg) or were left untreated. On day 30, the parasite loads were quantified in the spleen by limiting dilution assay (18) and in the liver by Leishman-Donovan units according to Stauber (19). Figure 3 shows that a 28-day treatment with CH8 150 mg/kg was effective in the VL model, inducing significant (P < 0.01) reductions in parasite burden in spleen (82%) and liver (74%) compared with no treatment. These reductions were comparable to those achieved with miltefosine 2 mg/kg (90% and 82%, respectively). The CH8 dose used for VL was slightly higher than that used for CL for optimization. For miltefosine, only results using lower doses (2 mg/kg) are displayed because 12-mg/kg doses were lethal to the mice with VL. After only 12 doses, animals were visibly ill, and the death rate was 60%, so the experiment was aborted (data not shown). The differential miltefosine toxicity seen in CL- and VL-infected animals possibly reflected the debilitated organ functions in VL.
Effect of oral CH8 in LV. Mice were infected in the tail vein with L. infantum. On days 1 to 29 of infection, they were treated daily by the oral route with CH8 150 mg/kg or miltefosine 2 mg/kg. Controls were left untreated. On day 30 of infection, the numbers of parasites were quantified in individual spleens (A) by limiting dilution analysis (100% in vehicle, 2.9 × 107 amastigotes/spleen) and in individual livers (B) by Leishman-Donovan units (100% in vehicle, 6.7 × 102 amastigotes/1,000 host cell nuclei). Values are means ± SEM (n = 5). *, P < 0.05 compared with untreated animals.
To address CH8 safety, ALT, AST, and creatinine serum levels were evaluated in CL and VL models (Fig. 4). Unlike CL infection, which alone did not affect any of the toxicity parameters (Fig. 4A to C), VL infection alone raised all of them (Fig. 4D to F), which was compatible with findings of previous studies (20). As seen in Fig. 4A to C, CH8 was safe in CL models even when administered at the highest dose (240 mg/kg). Interestingly, when used in VL models at the therapeutic dose (150 mg/kg), CH8 restored AST and creatinine levels to normal (Fig. 4E). On the other hand, miltefosine 12 mg/kg in CL and 2 mg/kg in VL significantly raised creatinine levels (30% and 73%, respectively) compared with levels in untreated animals. These findings are compatible with those of previous studies showing miltefosine changes in kidney biochemical and morphological parameters (21) and support the assumption that the VL model used here was more sensitive to the toxic effects of miltefosine than the CL model.
Safety of oral CH8 treatment in both CL and VL. Mice were infected with L. amazonensis for CL (A to C) or L. infantum for VL (D and E), as described in Figs. 2 and 3, respectively. After receiving 23 (A to C) or 29 (D and E) daily oral doses of CH8 and miltefosine as indicated, serum samples were individually assayed for ALT (A and D), AST (B and E), and creatinine (C and F). The values were corrected considering the CCl4 group (positive controls) as 100% of toxicity. CCl4 values for ALT, AST, and creatinine were 61.00 U/ml, 157.50 U/ml, and 773.25 μg/ml, respectively. Values are means ± SEM (n = 5). *, P < 0.05 compared with respective untreated animals; #, P < 0.05; &, P < 0.05 compared with normal (untreated uninfected) animals.
The observed liver and kidney protective effect of CH8 may reflect its antioxidative properties (9), pointing to an interesting potential for combination therapy with other effective but hepato-/nephrotoxic drugs, such as miltefosine. Unraveling the CH8 mechanism of action is important to predict drug interaction. Previous studies with the DMC natural analogue indicated alterations in parasite mitochondria and interference with membrane sterol biosynthesis (8), although these do not seem to be the primary parasite targets. More recently, the use of fluorescent molecular probes has helped to more directly identify the unique parasite tryparedoxin peroxidase (TXNPx) enzyme as the main CH8 target (data not shown). This target does not seem to be the same as that of miltefosine, which appears to act mainly on the parasite lipid biosynthesis, Ca+2 homeostasis, and apoptosis-like death (22). Future studies in vitro and in vivo will provide further information on the feasibility of combination therapy with CH8 and miltefosine. In sole therapy, an innovative strategy that may increase CH8 bioavailability and efficacy is entrapment in nanostructured systems, such as solid lipid nanocapsules, which were shown to favor intestinal absorption of other flavonoids (15, 23).
In conclusion, this study demonstrates in mice the broad spectrum and therapeutic potential of CH8 for oral treatment of LC and LV infection. In addition, it shows the protective effect of CH8 against liver and kidney damage caused by VL infection, which is of relevance in combination therapies with other effective but toxic drugs.
FOOTNOTES
- Received 20 April 2018.
- Returned for modification 8 June 2018.
- Accepted 5 July 2018.
- Accepted manuscript posted online 16 July 2018.
REFERENCES
- Copyright © 2018 American Society for Microbiology.
