A chloroquinoline derivate presents effective in vitro and in vivo antileishmanial activity against Leishmania species that cause tegumentary and visceral leishmaniasis

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Highlights

  • A new chloroquinoline derivative, AM1009, was tested against Leishmania parasites.

  • The molecule was in vitro effective against axenic amastigotes and promastigotes.

  • AM1009 caused low toxicity in two types of mammalian cells.

  • The mechanism of action was based on apoptosis/necrosis of the parasites.

  • In vivo experiments showed effective action to treat against L. amazonensis infection.

Abstract

The identification of new therapeutics to treat leishmaniasis is desirable, since available drugs are toxic and present high cost and/or poor availability. Therefore, the discovery of safer, more effective and selective pharmaceutical options is of utmost importance. Efforts towards the development of new candidates based on molecule analogs with known biological functions have been an interesting and cost-effective strategy. In this context, quinoline derivatives have proven to be effective biological activities against distinct diseases. In the present study, a new chloroquinoline derivate, AM1009, was in vitro tested against two Leishmania species that cause leishmaniasis. The present study analyzed the necessary inhibitory concentration to preclude 50% of the Leishmania promastigotes and axenic amastigotes (EC50 value), as well as the inhibitory concentrations to preclude 50% of the murine macrophages and human red blood cells (CC50 and RBC50 values, respectively). In addition, the treatment of infected macrophages and the inhibition of infection using pre-treated parasites were also investigated, as was the mechanism of action of the molecule in L. amazonensis. To investigate the in vivo therapeutic effect, BALB/c mice were infected with L. amazonensis and later treated with AM1009. Parasitological and immunological parameters were also evaluated. Clioquinol, a known antileishmanial quinoline derivate, and amphotericin B (AmpB), were used as molecule and drug controls, respectively. Results in both in vitro and in vivo experiments showed a better and more selective action of AM1009 to kill the in vitro parasites, as well as in treating infected mice, when compared to results obtained using clioquinol or AmpB. AM1009-treated animals presented significantly lower average lesion diameter and parasite burden in the infected tissue and organs evaluated in this study, as well as a more polarized antileishmanial Th1 immune response and low renal and hepatic toxicity. This result suggests that AM1009 should be considered a possible therapeutic target to be evaluated in future studies for treatment against leishmaniasis.

Introduction

Leishmaniases are diseases caused by protozoan parasites of the Leishmania genus, which present an annual estimated incidence of 1.5 to 2.0 million new cases, which range between 1.0 and 1.5 million cases of tegumentary leishmaniasis (TL), coupled with approximately 0.5 million new cases of visceral leishmaniasis (VL). Epidemiological data have proven that this disease complex is endemic in 98 countries, mainly in subtropical and tropical regions in the world [1,2]. TL can lead to self-healing cutaneous lesions, even causing mutilation and morbidity in patients. The disease is usually caused by Leishmania braziliensis, L. amazonensis, L. guyanensis, L. mexicana, and L. lainsoni species in the Americas, whereas VL, which is considered a fatal disease if acute and left untreated, can be caused by L. donovani and L. infantum species [3,4].

Treatment against leishmaniasis has been based on the use of pentavalent antimonials, although other drugs, such as free and liposomal amphotericin B (AmpB), miltefosine, paramomycin, and pentamidine have been also used [5]. However, problems related to their toxicity, high cost, and/or development of resistant strains have limited the efficacy of these therapeutics [6,7]. The prolonged administration of these compounds can cause adverse effects, such as renal, hepatic, and cardiac toxicity. These can also show variable efficacy according to the parasite strain, immune status of the hosts, and the emergence of drug resistance, all of which have been observed in the subcontinent of India and other regions [8,9]. Consequently, since the search to identify new antileishmanial targets is hampered due to the high investment necessary to develop new products, as well as by the lack of a profitable drug market [10], alternative means through which to identify new antileishmanial agents, such as plant derivates or synthetic products, could help to solve this relevant problem and make it possible to identify new therapeutics to be applied against leishmaniasis [11,12].

The interest in natural and/or synthetic molecules has increased in recent decades, and new agents have been evaluated in experimental trials [[13], [14], [15], [16]], although most have been tested in in vitro studies, and few in vivo treatment experiments have been developed using mammalian models [[17], [18], [19]]. Distinct molecule classes, such as flavonoids, terpenoids, quinolines, among others, have shown variable degrees of antileishmanial activity [[20], [21], [22], [23]]. In this aspect, quinoline derivatives have been applied as an important drug class, showing potent antitumoral, antiprotozoal, antimicrobial, and anti-inflammatory activity, among others, and did not cause significant toxicity in the hosts [[24], [25], [26], [27]].

Research groups have demonstrated the selective antileishmanial activity of the quinoline-based compounds against Leishmania models [[28], [29], [30]]. One of these molecules, clioquinol (5-chloro-7-iodoquinolin-8-ol), demonstrated high efficacy against L. amazonensis and L. infantum promastigotes and amastigotes, without causing toxicity in murine macrophages. It was also effective in the treatment of infected macrophages and in the inhibition of the infection of these cells using pre-treated parasites [31]. In another study, clioquinol was incorporated to a Poloxamer 407-based delivery system, and the formulation proved to be effective in the treatment of L. amazonensis-infected BALB/c mice [32].

In this context, in the present study, a new chloroquinoline derivate, AM1009 [N1-(7-chloroquinolin-4-yl)-N3-cyclohexylpropane-1,3-diamine], was in vitro tested against two Leishmania species that cause VL and TL around the world. The 50% Leishmania inhibitory concentration (EC50) and the 50% macrophage inhibitory concentration (CC50) in murine macrophages and in human red blood cells (RBC50) were investigated. Additionally, the treatment of infected macrophages and the inhibition of infection using pre-treated parasites were also evaluated, as were the mechanism of action in L. amazonensis and the in vivo therapeutic efficacy in treating Leishmania-chronically infected BALB/c mice. As a molecule control, clioquinol was used in the in vitro and in vivo experiments, as was AmpB, which was used as a drug control. Our purpose is to identify new candidates to be tested in future studies for treatment against leishmaniasis, which can present higher activity and selectivity when compared to previously described antileishmanial agents.

Section snippets

Synthesis of AM1009

To perform the synthesis of AM1009, 220 mg (0.668 mmol) N-(3-bromopropyl)-7-chloroquinolin-4-amine was added to a round bottom flask containing 464 mg (4.68 mmol) of cyclohexanamine, 462 mg (3.34 mmol) of potassium carbonate, and 1 mL of dimethylformamide PA. The flask was shaken for 20 h at 120 °C, and the reaction was completed by thin layer chromatography. After, the compound was mixed using distilled water and dichloromethane PA, and AM1009 was purified through a filtration using silica,

Antileishmanial activity, cytotoxicity, and selectivity index

The in vitro antileishmanial activity of AM1009 was evaluated against L. amazonensis and L. infantum promastigotes and axenic amastigotes. As molecule and drug control, clioquinol and AmpB were used, respectively. The EC50 values for AM1009, clioquinol, and AmpB against L. amazonensis promastigotes were 2.41 ± 0.28, 7.90 ± 0.65, and 0.13 ± 0.04 μM, respectively, and of 1.03 ± 0.22, 2.27 ± 0.44, and 0.34 ± 0.11 μM against the axenic amastigotes, respectively. Regarding the L. infantum species,

Discussion

Advances in the biochemical research applied in in vitro and/or in vivo parasitological and immunological studies to kill Leishmania parasites have been developed in order to identify new and non-toxic and cost-effective antileishmanial products to control this neglected disease in the world [38]. However, current therapeutics present problems, such as side effects, including arthralgia, myalgia, fever, weakness, besides renal, hepatic and cardiac toxicity, together with high cost and/or poor

Declaration of Competing Interest

The authors confirm that they have no conflicts of interest in relation to this work.

Acknowledgments

The authors would like thank to CAPES, CNPq, and FAPEMIG for the scholarships. This work was supported by grants from CNPq (APQ-408408/2016-2 and APQ-408675/2018-7).

References (56)

  • E.S. Coimbra et al.

    Quinoline derivatives: synthesis, leishmanicidal activity and involvement of mitochondrial oxidative stress as mechanism of action

    Chem. Biol. Interact.

    (2016)
  • G.F. Zhang et al.

    4-quinolone derivatives and their activities against gram positive pathogens

    Eur. J. Med. Chem.

    (2018)
  • K. Balaraman et al.

    In vitro and in vivo antileishmanial properties of a 2-n-propylquinoline hydroxypropyl β-cyclodextrin formulation and pharmacokinetics via intravenous route

    Biomed. Pharmacother.

    (2015)
  • C. Hernández-Chinea et al.

    In vitro activity of synthetic tetrahydroindeno[2,1-c]quinolines on Leishmania mexicana

    Parasitol. Int.

    (2015)
  • T.G. Soyer et al.

    Evaluation of the in vitro and in vivo antileishmanial activity of a chloroquinolin derivative against Leishmania species capable of causing tegumentary and visceral leishmaniasis

    Exp. Parasitol.

    (2019)
  • G.S.V. Tavares et al.

    A Pluronic® F127-based polymeric micelle system containing an antileishmanial molecule is immunotherapeutic and effective in the treatment against Leishmania amazonensis infection

    Parasitol. Int.

    (2019)
  • S.L. Calixto et al.

    Novel organic salts based on quinoline derivatives: The in vitro activity trigger apoptosis inhibiting autophagy in Leishmania spp.

    Chem. Biol. Interact.

    (2018)
  • P.H.F. Stroppa et al.

    Effect of 1,2,3-Triazole salts, non-classical bioisosteres of miltefosine, on Leishmania amazonensis

    Bioorg. Med. Chem.

    (2017)
  • D.V.C. Mendonça et al.

    In vivo antileishmanial efficacy of a naphthoquinone derivate incorporated into a Pluronic® F127-based polymeric micelle system against Leishmania amazonensis infection

    Biomed. Pharmacother.

    (2019)
  • H. Kato et al.

    The evaluation of frequency of nephrotoxicity caused by liposomal amphotericin B

    J. Infect. Chemother.

    (2018)
  • L.M.R. Antinarelli et al.

    Antileishmanial activity of a 4-hydrazinoquinoline derivative: induction of autophagy and apoptosis-related processes and effectiveness in experimental cutaneous leishmaniasis

    Exp. Parasitol.

    (2018)
  • J.C. Coa et al.

    Synthesis, leishmanicidal, trypanocidal and cytotoxic activity of quinoline-hydrazone hybrids

    Eur. J. Med. Chem.

    (2015)
  • V.S. Gopinath et al.

    Design, synthesis, ADME characterization and antileishmanial evaluation of novel substituted quinoline analogs

    Bioorg. Med. Chem. Lett.

    (2014)
  • World Health Organization, Leishmaniasis
  • P.D. Ready

    Epidemiology of visceral leishmaniasis

    Clin. Epidemiol.

    (2014)
  • O.P. Singh et al.

    Current challenges in treatment options for visceral leishmaniasis in India: a public health perspective

    Infect. Dis.Poverty

    (2016)
  • R.J. Faleiro et al.

    Combined immune therapy for the treatment of visceral leishmaniasis

    PLoS Negl. Trop. Dis.

    (2016)
  • N. Didwania et al.

    Alternative to chemotherapy-the unmet demand against leishmaniasis

    Front. Immunol.

    (2017)
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    These authors contributed equally to this work.

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