Efficacy of Apigenin and Miltefosine Combination Therapy against

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Cite This: J. Nat. Prod. 2018, 81, 1910−1913

Efficacy of Apigenin and Miltefosine Combination Therapy against Experimental Cutaneous Leishmaniasis Yago S. S. Emiliano and Elmo E. Almeida-Amaral*

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Laboratório de Bioquímica de Tripanosomatideos, Instituto Oswaldo Cruz (IOC), Fundaçaõ Oswaldo Cruz−FIOCRUZ, Pavilhão Leônidas Deane, 4° andar, sala 405A, Manguinhos, 21045-900, Rio de Janeiro, RJ, Brazil ABSTRACT: Leishmaniasis is a neglected tropical disease caused by several different species of Leishmania. Treatment of leishmaniasis involves a limited drug arsenal that is associated with severe side effects, high costs, and drug resistance. Therefore, combination therapy has emerged as a strategy to improve leishmaniasis treatment. Here, we report the interaction of miltefosine and apigenin in vitro and in vivo. Combination therapy using low doses of these two drugs results in good clinical and parasitological responses.

L

eishmaniasis is a neglected tropical disease that is endemic in 98 countries and affects 12 million people worldwide. Annually, 2 million new cases of this disease are reported, with a mortality rate of 30 000 deaths per year.1 The two clinical forms of this disease are visceral leishmaniasis (VL) and cutaneous leishmaniasis (CL). CL in humans is caused by several different species of Leishmania and has a diverse spectrum of clinical manifestations, including ulcerative skin lesions (localized CL), multiple nonulcerative nodules (diffuse CL), and destructive mucosal inflammation (mucosal leishmaniasis).2 Even after more than 70 years of clinical use, pentavalent antimonials remain the first line of treatment for leishmaniasis.3,4 Pentavalent antimonials have saved thousands of lives; however, these compounds can be administered only by the parenteral route, present severe side effects, and are expensive, and cases of resistance have been reported in some countries.5,6 In northeastern India, miltefosine has replaced antimonials as the first line of treatment for VL. However, an increase in the number of clinical relapses has been reported.7 Therefore, new drugs that are effective and safe and that patients have easy access to are urgently needed. Combination therapy is a strategy for improving the treatment of leishmaniasis. In this study, we investigated the interaction of miltefosine and apigenin (1), a flavonoid with antiparasitic activity, against intracellular amastigotes of L. donovani and promastigotes of L. tropica and L. amazonensis.8−12 The effects of miltefosine and 1 alone have been demonstrated in a murine model of CL using L. amazonensis-infected BALB/c mice.13,14 Compound 1 was effective in the treatment of L. amazonensisinfected BALB/c mice by oral administration, without alterations in serological toxicity markers, and has been proposed as a potential drug candidate for CL chemotherapy.14 © 2018 American Chemical Society and American Society of Pharmacognosy

To determine the effect of the association between miltefosine and 1 on intracellular amastigotes, THP-1-derived macrophages were infected with promastigotes of L. amazonensis. Table 1 shows the EC50, fractional inhibitory concentration (FIC), sum FIC (∑FIC), and mean ∑FIC (x̅∑FIC) values obtained using the modified fixed-ratio isobologram method. Table 1. EC50 and FIC of the Combination of 1 and Miltefosine against Intracellular Amastigotes of Leishmania amazonensisa EC50 combination ratio of 1 with miltefosine

1 (μM)

5:0 4:1 3:2 2:3 1:4 0:5

3.85 2.99 1.43 0.73 0.16

Milt (μM) 0.44 0.73 1.53 1.13 0.75

FIC50 1

Milt

∑FIC50

0.77 0.37 0.18 0.04

0.58 0.96 2.03 1.50

1.35 1.33 2.21 1.54

x̅∑FIC 1.61

a

Milt, miltefosine. FIC, fractional inhibitory concentration at the indicated EC50. ∑FIC, sum of FICs. x̅, mean of ∑FIC.

Received: May 4, 2018 Published: August 10, 2018 1910

DOI: 10.1021/acs.jnatprod.8b00356 J. Nat. Prod. 2018, 81, 1910−1913

Journal of Natural Products

Note

miltefosine (4 and 8 mg/kg) was diluted in phosphate-buffered saline (PBS). Each drug or their combination was incorporated in an oral suspension and administered through an orogastric tube once daily, for a total of seven times per week, until the end of the experiment (day 32), when the animals were euthanized. As shown in Figure 2, compound 1 (2 mg/kg/day; solid squares) and miltefosine (8 mg/kg/day; open circles) separately were able to reduce lesion size (panel A) and parasite load (panel B) as previously demonstrated.13,14 However, the aspartate aminotransferase levels were elevated in mice treated with 8 mg/kg/day miltefosine, indicating possible hepatotoxicity (Table 2).

The effect of the interaction of miltefosine and 1 on intracellular amastigotes exhibited a x̅∑FIC of 1.61, indicating an indifferent interaction.15 Combinations of miltefosine with drugs such as tamoxifen,13 amphotericin B, sodium stibogluconate, sitamaquine, and paromomycin16 have been previously demonstrated to exhibit indifferent interactions (additive interaction), with mean ∑FIC values of >0.5 and ≤4, similar to the value observed in this study. A representative isobologram of the interaction between miltefosine and 1 against intracellular amastigotes is shown in Figure 1.

Table 2. Serological Toxicology Markersa

control compound 1 (2 mg/kg/day) miltefosine (8 mg/ kg/day) compound 1 (1 mg/kg/day) + miltefosine (4 mg/kg/day)

Figure 1. Representative isobologram of the effects of the combination of apigenin (1) and miltefosine against intracellular amastigotes of Leishmania amazonensis in vitro. The isobologram presents the EC50. The x-axis presents the normalized FICs of miltefosine; the y-axis presents the normalized FICs of 1. The bold line corresponds to the predicted position of the experimental points for a simple additive effect. A trend line connects the points corresponding to the FIC values.

CREA (mg/ dL)

AST (U/L)

ALT (U/L)

0.61 ± 0.06 0.63 ± 0.02

161.10 ± 20.99 100.50 ± 6.55

88.41 ± 8.77 43.21 ± 3.36

0.66 ± 0.13

271.10 ± 40,21b,c,d

61.14 ± 3.48

0.69 ± 0.05

115.10 ± 9.17

53.55 ± 2.24

a

Serological toxicology markers in the infected BALB/c mice treated as described above were measured by the Program of Technological Development in Tools for Health-PDTIS-FIOCRUZ. Data are expressed as the mean ± standard error, n = 5. CREA, creatinine; AST, aspartate aminotransferase; ALT, alanine aminotransferase. b Significant differences relative to the control group. cSignificant differences relative to the group treated with compound 1 (2 mg/kg/ day). dSignificant differences relative to the group treated with combined therapy (apigenin 1 mg/kg/day + miltefosine 4 mg/kg/ day).

The in vivo efficacy of the combination of 1 and miltefosine was measured using L. amazonensis-infected BALB/c mice as a murine model of CL. Female BALB/c mice were inoculated with stationary-phase L. amazonensis promastigotes intradermally in the right ear.14,17 The treatment was initiated 7 days following infection. Compound 1 (1 and 2 mg/kg) was diluted in dimethylsulfoxide (DMSO) (1% v/v), and

Therefore, the combined treatment scheme was designed using half the original dose of each drug (1 mg/kg/day

Figure 2. Evaluation of the in vivo efficacy of combined therapy with apigenin (1) and miltefosine in a murine model of cutaneous leishmaniasis. BALB/c mice were infected intradermally with 2 × 106 L. amazonensis promastigotes in the right ear. Panel A: Lesion development in animals treated orally with 1 alone (2 mg/kg/day; solid squares), miltefosine alone (8 mg/kg/day; open circles), or 1 and miltefosine (1 mg/kg/day + 4 mg/kg/day; open squares). The control group was treated with an oral suspension added to DMSO (0.2% v/v) (vehicle for 1; solid circles). All groups were treated by the oral route once per day, seven times per week. The arrow represents the initiation of treatment. Panel B: Parasite burden of L. amazonensis-infected BALB/c mice, untreated or treated with 1 alone (2 mg/kg/day), miltefosine alone (8 mg/kg/day), or a combination (1 mg/kg/day compound 1 + 4 mg/kg/day miltefosine). Ear parasite loads were determined via a limiting dilution assay. Data are expressed as the means ± standard errors, n = 5 ears [* and ** indicate significant differences relative to the control group (p < 0.05 and p < 0.01, respectively). # and ## indicate significant differences relative to the compound 1-treated group (2 mg/kg/day) (p < 0.05 and p < 0.01, respectively)]. ns = no statistical significance. 1911



Note

total number of macrophages) was determined by light microscopy by counting a minimum of 200 cells on each coverslip randomly in duplicate. The EC50 values for each ratio and for each drug were determined by logarithmic regression analysis using GraphPad Prism 6. Determination of FIC Index and Isobologram Construction. The FIC and ∑FICs were calculated as described16,24,25 for all fixedratio solutions. The x̅∑FIC was calculated as the average of the ∑FICs. Isobolograms were built using the FIC value for each drug ratio. x̅∑FIC was used to classify the nature of the interaction. Interactions were considered synergic at an x̅ ∑FIC of ≤0.5, indifferent (additive) at an x̅∑FIC between 0.5 and ≤4, and antagonistic at an x̅∑FIC of >4.15 In Vivo Infection in a Murine Model. BALB/c mice (five animals per group) were maintained under specific pathogen-free conditions. Stationary-phase promastigotes of L. amazonensis (2 × 106 cells in 10 μL of PBS) were inoculated intradermally in the right ear using a 271/2-gauge needle, and treatment was initiated 7 days following infection. Apigenin (1) (1 and 2 mg/kg) was diluted in DMSO (1% v/v), and miltefosine (4 and 8 mg/kg) was diluted in PBS. Compound 1 and miltefosine, separately or combined, were incorporated in an oral suspension and administered through an orogastric tube once daily, for a total of seven times per week, until the end of the experiment (day 32), when the animals were euthanized. Lesion sizes were measured twice per week using a dial caliper.17,26 Parasite Load Quantification. Parasite load was determined using a quantitative limiting dilution assay as described previously.17,26 Briefly, infected ears were excised and weighed before being minced in Schneider’s medium with 10% fetal calf serum. The resulting cell suspension was diluted serially. The number of viable parasites in each ear was estimated from the highest dilution at which promastigotes could be grown after 7 days of incubation at 26 °C. Biochemical Values. Serum levels of toxicological markers (AST, ALT, and CREA) in the infected BALB/c mice treated as described above were measured by the Program for Technological Development in Tools for Health (PDTIS-FIOCRUZ) using dry chemistry technology. Statistical Analysis. All experiments were performed in three independent trials. The data were analyzed using Student’s t test or analysis of variance (ANOVA) followed by Bonferroni’s post-test in GraphPad Prism 6 (GraphPad Software). Results were considered significant at p ≤ 0.05. The data are expressed as means ± standard error.

compound 1 and 4 mg/kg/day miltefosine). Compared with the values in the control group and the group treated with 2 mg/kg/day 1, lesion size (Figure 2A) and parasite load (Figure 2B) in L. amazonensis-infected mice were significantly reduced in the group receiving the combined therapy (1 mg/kg/day compound 1 plus 4 mg/kg/day miltefosine) by 75% (Figure 2A; open squares) and 95% (Figure 2B), respectively, without renal or hepatic toxicity (Table 2). Miltefosine is the first and only oral drug approved for the treatment of leishmaniasis. This drug has been used for the past decade in many countries for the treatment of CL and VL.18 The most common side effects observed during miltefosine treatment are diarrhea, vomiting, and elevated levels of liver enzymes.19 Serum levels of both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) tend to increase during miltefosine treatment,20 necessitating the use of combinatorial therapy using low doses of miltefosine. Taken together, these findings indicate that combined therapy with 1 and miltefosine, using low doses of these two drugs, led to good clinical and parasitological responses. In addition, this treatment could prevent the selection of miltefosine-resistant parasites. However, further studies, such as optimization of the dose ratio of the drugs in this combination, should be conducted.

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EXPERIMENTAL SECTION

Test Compound and Reagents. Apigenin (1) (molecular formula: C15H10O5; molecular weight: 270.24 g/mol; purity ≥96%; lot WE445301/1), Schneider’s Drosophila medium, fetal calf serum, miltefosine, and RPMI 1640 medium were obtained from SigmaAldrich (St Louis, MO, USA). Other reagents were purchased from Merck (São Paulo, Brazil). Deionized distilled water was obtained using a Milli-Q system of resins (Millipore Corp., Bedford, MA, USA) and was used to prepare all solutions. Endotoxin-free sterile disposables were used in all experiments. Apigenin (1) was prepared in DMSO and diluted in culture medium such that the solvent concentration did not exceed 0.2% in the final solution. Ethics Statement. This study was performed in strict accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation (CONCEA). The protocol was approved by the Committee on the Ethics of Animal Experiments of the Instituto Oswaldo Cruz (CEUA-IOC, License Number: L-11/2017). Parasites and Mice. The MHOM/BR/75/LTB0016 strain of L. amazonensis was used throughout this study. The strain was isolated from a human case of cutaneous leishmaniasis in Brazil. L. amazonensis promastigotes were cultivated at 26 °C in Schneider’s Drosophila medium (pH 6.9) supplemented with 10% bovine fetal serum (v/v), 100 μg/mL streptomycin, and 100 U/mL penicillin. Parasite maintenance was promoted by passage every 3 days of culture. Female BALB/c mice (10−12 weeks; provided by the Instituto Ciências e Tecnologia em Biomodelos, ICTB/FIOCRUZ) were used in this study. All animals were bred and maintained at the Fundaçaõ Oswaldo Cruz according to Guide for the Care and Use of Laboratory Animals of the Brazilian National Council of Animal Experimentation (CONCEA). Leishmania−Macrophage Drug Interaction Assay. Stationary-phase L. amazonensis promastigotes were washed with PBS, counted using a Neubauer chamber, and added to THP-1-derived macrophages (THP-1-derived macrophages were obtained as previously described21) at a multiplicity of infection of 3:1 for 3 h at 37 °C in an atmosphere of 5% CO2 in Lab-Tek 16-chamber slides. After 18 h, L. amazonensis-infected THP-1-derived macrophages were incubated with 1 and miltefosine at concentration ratios of 5:0, 4:1, 3:2, 2:3, 1:4, and 0:5, followed by serial dilution (base 2).22,23 The infection index (% of infected macrophages × number of amastigotes/

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AUTHOR INFORMATION

Corresponding Author

*Tel: +55(21)3865-8220. E-mail: elmo@ioc.fiocruz.br. ORCID

Elmo E. Almeida-Amaral: 0000-0003-1363-3176 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors acknowledge the Program of Technological Development in Tools for Health-PDTIS-FIOCRUZ for analyzing the serum toxicological markers. This work was supported by Fundaçaõ Carlos Chagas Filho de Amparo a Pesquisa do Estado do Rio de Janeiro (FAPERJ; E.E.A.A.), ́ Conselho Nacional de Desenvolvimento Cientifico e Tecnológico (CNPq; E.E.A.A.), Programa Estratégio de Apoio a Pesquisa em Saúde (PAPES/FIOCRUZ; E.E.A.A.), and the Fundaçaõ Oswaldo Cruz (FIOCRUZ; E.E.A.A.). E.E.A.A. is the recipient of a research scholarship from Conselho Nacional ́ de Desenvolvimento Cientifico e Tecnológico (CNPq). 1912



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REFERENCES

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Efficacy of Apigenin and Miltefosine Combination Therapy against

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