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Amiodarone and itraconazole improve the activity of pentavalent antimonial in the treatment of experimental cutaneous leishmaniasis
Accepted Manuscript Title: Amiodarone and itraconazole improve the activity of pentavalent antimonial in the treatment of experimental cutaneous leishmaniasis Author: Laís Anversa, Monique Gomes Salles Tiburcio, Lara Rocha Batista, Marília Beatriz Cuba, Gabriel Antonio Nogueira Nascentes, Tábata Yamasaki Martins, Virgínia Bodelão Richini Pereira, Luciana da Silva Ruiz Menezes, Valdo José Dias da Silva, Luis Eduardo Ramirez PII: DOI: Reference:
S0924-8579(17)30217-0 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.06.007 ANTAGE 5157
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
11-8-2016 17-6-2017
Please cite this article as: Laís Anversa, Monique Gomes Salles Tiburcio, Lara Rocha Batista, Marília Beatriz Cuba, Gabriel Antonio Nogueira Nascentes, Tábata Yamasaki Martins, Virgínia Bodelão Richini Pereira, Luciana da Silva Ruiz Menezes, Valdo José Dias da Silva, Luis Eduardo Ramirez, Amiodarone and itraconazole improve the activity of pentavalent antimonial in the treatment of experimental cutaneous leishmaniasis, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.06.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Amiodarone and itraconazole improve the activity of pentavalent
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antimonial in the treatment of experimental cutaneous leishmaniasis
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Laís Anversaa,*, Monique Gomes Salles Tiburciob, Lara Rocha Batistac,
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Marília Beatriz Cubad, Gabriel Antonio Nogueira Nascentese, Tábata Yamasaki Martinsf,
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Virgínia Bodelão Richini Pereirag, Luciana da Silva Ruiz Menezesh, Valdo José Dias da
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Silvai, Luis Eduardo Ramirezj
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a
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
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Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
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Brazil. [email protected]
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b
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Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
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Brazil. [email protected]
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c
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Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
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Brazil. [email protected]
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d
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Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
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Brazil. [email protected]
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e
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Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
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Brazil. [email protected]
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
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f
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Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
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Brazil. [email protected]
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g
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Lutz. R Rubens Arruda, Qd 6, Centro, 17015-110, Bauru, São Paulo, Brazil.
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[email protected]
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h
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Lutz. R Rubens Arruda, Qd 6, Centro, 17015-110, Bauru, São Paulo, Brazil.
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[email protected]
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i
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Terra, 330, Nossa Senhora da Abadia, 38025-015, Uberaba, Minas Gerais, Brazil.
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[email protected]
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j
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Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais, Brazil.
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[email protected]
Programa de Iniciação Científica do Laboratório de Parasitologia, Universidade Federal do
Núcleo de Ciências Biomédicas, Centro de Laboratório Regional de Bauru, Instituto Adolfo
Núcleo de Ciências Biomédicas, Centro de Laboratório Regional de Bauru, Instituto Adolfo
Laboratório de Fisiologia Humana, Universidade Federal do Triângulo Mineiro. Pç Manoel
Laboratório de Parasitologia, Universidade Federal do Triângulo Mineiro. Av Getúlio
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* Corresponding author
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Centro de Laboratório Regional de Bauru,
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Instituto Adolfo Lutz. R Rubens Arruda,
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Qd 6, 17015-110,
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Bauru (SP),
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Brazil.
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Phone: +55 14 32231175.
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E-mail address: [email protected]
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Highlights
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The therapeutic options for leishmaniasis are still very limited
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Amiodarone and itraconazole improve the activity of glucantime in leishmaniasis
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Abstract
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Leishmaniasis affect millions of people, causing morbidity and mortality, especially in
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developing tropical and subtropical countries. Unfortunately, the possibilities of treatment for
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these infections are still quite limited and most of the available drugs present serious side
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effects. The objective of this paper was to evaluate the therapeutic role of amiodarone and
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itraconazole in the treatment of cutaneous leishmaniasis caused by Leishmania (Leishmania)
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amazonensis. In order to perform this evaluation, hamsters were infected with 1 x 106
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metaciclic promastigotes of the parasite in the hind footpad and, after the onset of the lesions,
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were treated with glucantime, amiodarone, itraconazole, glucantime and amiodarone,
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glucantime and itraconazole or amiodarone and itraconazole. The treatments’ efficacy was
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evaluated per analysis of the size of the cutaneous lesions and by parasitic investigation of the
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infected foot (by histopathological examination and PCR) and possible side effects were
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analyzed taking into account the weight of the animals and some biochemical and metabolic
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parameters (glucose, urea, creatinine, AST, ALT and ALP). The results have shown that, in
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hamsters, amiodarone and itraconazole, either used isolated or in combination, are unable to
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stop the development of cutaneous lesions caused by L. (L.) amazonensis, but improve the
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activity of glucantime in the treatment of these lesions and seem to present no evident side
71
effects. More studies are necessary in order to investigate the clinical potential of these
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combinations, so there can be the possibility of broadening the therapeutic options available,
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especially in resistant cases.
The association of drugs seems to be an alternative to treat leishmaniasis
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Keywords: Leishmania; leishmaniasis; treatment; amiodarone; itraconazole; combination
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therapy.
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1. Introduction
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Leishmaniasis are caused by many different protozoans’ species from the Trypanosomatidae
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family and Leishmania genus, affecting men and different animals. This parasitosis is
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endemic in at least 98 countries in the entire world and more than two million new cases
83
occur, each year, with high morbidity and mortality levels [1]. Among other factors,
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depending on the infecting species and the immune response of the host, the disease presents
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different clinical manifestations, such as: (a) the local cutaneous, characterized by the
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presence of lesions exclusively in the site of the vector insect bite; (b) the mucocutaneous,
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with destructive lesions in the upper respiratory tract; (c) the diffuse cutaneous, with multiple
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non ulcerated nodules, classically unresponsive to available treatments; (d) and the visceral, a
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systemic form affecting especially lymphatic nodes, the spleen, the liver and the bone marrow
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[2].
91 92
Leishmania (Leishmania) amazonensis is found and widely distributed in the Amazon
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rainforest region of Brazil, and has been reported as expanding to the Northeast, the Southeast
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and the Center West of the country, east of Paraguay and other Amazon areas of Bolivia, Peru
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Ecuador, Colombia and Venezuela [3,4]. Commonly, the species of L. (L.) amazonensis
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induce localized lesions, and nevertheless may also cause the diffuse cutaneous and even
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mucocutaneous and visceral forms of the disease [5].
98 99
Conventional therapy for all clinical forms of leishmaniasis include the pentavalent
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antimonials’ (Sb+5) use, such as antimonate of N-methylglucamine (Glucantime®) and
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sodium stibogluconate (Pentostam®) [6]. However, these drugs demand long term parenteral
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administration, present serious toxic side effects, especially on cardiac, renal and hepatic
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systems [7] and have shown gradually decreasing efficacy due to the appearance of resistance
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in many countries [8]. Amphotericin B, pentamidine and paromomycin are alternative drugs,
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also administered by parenteral via, that can present a variety of cure rates and many and
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frequent side effects [9,10]. Miltefosine, on the other hand, was introduced in India, in 2002,
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and represented a major advance in the treatment of visceral leishmaniasis, being the first
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drug possible to be orally administered, but the relatively high cost and the concerns related to
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teratogenicity and the potent development of resistance have limited its use [11]. Therefore,
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alternative treatments are intensely searched in order to achieve better results, with less side
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effects and higher patient adherence.
112 113
Amiodarone, an antiarrhythmic drug class III, commonly used to treat cardiopathies,
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including arrhythmias in the chronic phase of Chagas disease, have been subjected to recent
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studies investigating its use as antimycotic agent and anti trypanosomatid, since this drug has
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excellent pharmacokinetics properties and has relatively low cost [12,13]. It has been shown
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that amiodarone has direct activity against Trypanosoma cruzi and Leishmania (Leishmania)
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mexicana, affecting the viability of the parasitic forms in vitro experiments and reducing the
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parasitemia and the development of lesions, respectively, in mice infected and treated with
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this drug [14,15]. Amiodarone acts in the parasite in many different ways: (a) blocking the
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biosynthesis of membrane steroids; (b) promoting alterations in the potential of the
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mitochondrial membrane; (c) breaking the homeostasis of Ca2+ and inducing a rapid increase
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in the cytoplasmic calcium; (d) increasing the production of oxygen reactive intermediates.
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Even more than that, the efficacy of amiodarone in the infection caused by Leishmania can
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also be favored by its lipid nature (highly soluble), extense tissue distribution and important
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excretion through the skin, factors that assure distribution to infection sites [16].
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Other drugs orally administered, especially azoles - such as ketoconazole, fluconazole and
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itraconazole - are also being applied experimentally in the treatment of leishmaniasis,
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especially cutaneous leishmaniasis. Taking into account that these antifungals inhibit the
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biosynthesis of ergosterol (the major sterol present in the plasmatic membrane of some
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microorganisms, including trypanosomatids) the use of these drugs in the treatment of
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infections caused by Leishmania is coherent by biochemical standards [17,18]. However,
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clinical essays with azoles are still limited and results obtained are ambiguous [19,20].
135 136
Currently, there are great expectations regarding the therapy with a combination of drugs
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designed to reduce dosages and length of the treatment, therefore improving tolerance and
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conformity of drugs already available [21]. Thus, the objective of this paper was to evaluate
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the therapeutic role of amiodarone and itraconazole in the treatment of experimental
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cutaneous leishmaniasis caused by L. (L.) amazonensis, in order to generate new perspective
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in the therapeutic approach of this important parasitic endemic disease.
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2. Materials and methods
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This study (Permit Number: 181/2011) was approved by the Ethics Committee for Animal
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Experimentation of the Universidade Federal do Triângulo Mineiro (Brazil). All animals
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received humane care in compliance with the “Principles of laboratory animal care”
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formulated by the National Society for Medical Research and the “Guide for the care and use
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of laboratory animals” prepared by the National Academy of Sciences (Washington, DC).
150 151
2.1. Parasites
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The strain of L. (L.) amazonensis IFLA/BR/67/PH8 (isolated from phlebotomine
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Lutzomyia flaviscutellata, in Belém, Pará, Brazil, 1976) was kindly donated by Prof. Dr.
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Maria Norma Melo, of the Parasitology Department of the Instituto de Ciências Biológicas of
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the Universidade Federal de Minas Gerais (UFMG) and was kept in the laboratory for
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successive inoculations in hamsters and cultured at 26°C in alfa-MEM medium (Minimum
157
Essential Medium) (Gibco®), supplemented with 10% of fetal bovine serum (SFB) and 100
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µg/ml gentamicin.
159 160
2.2. Drugs
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Glucantime® (N-metilglucamine antimonate) was supplied by the Regional Health
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Management of Uberaba (Lot: 9E1030), amiodarone (C25H30ClI2NO3) was bought from
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Sigma-Aldrich and itraconazole (C35H38Cl2N8O4) was bought from Janssen-Cilag.
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2.3. Experimental animal infection
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A total of 105 Syrian hamsters (Mesocricetus auratus) were used, not isogenic, males aged
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between six and eight weeks, obtained from the biotery of the Laboratory of the Parasitology
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discipline of the Universidade Federal do Triângulo Mineiro (UFTM) and kept in the same
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place, at a controlled temperature of 23ºC ± 1ºC, light-dark cycle with food and water ad
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libitum.
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Hamsters were inoculated by intradermal injection at the right hind footpad, with 100 μl
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phosphate-buffered saline (PBS) containing 1 x 106 L. (L.) amazonensis metacyclic
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promastigote from culture in alpha-MEM medium and quantified in Neubauer chamber (in
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triplicate).
176 177
2.4. Treatments
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Hamsters were randomly divided in seven experimental groups (15 animals/group) and 20
179
days after inoculation, when infection was already well established and lesions were present,
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treatments were started. Each group was treated daily with the drugs (one dose per day and at
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the same time schedule) for 20 consecutive days (TRAT 1), as follows: GLUC Group - 100
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mg Sb+5/kg/day glucantime; AMIO Group - 50 mg/kg/day amiodarone; ITRA Group - 50
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mg/kg/day itraconazole; GLUC+AMIO Group - 100 mg Sb+5/kg/day glucantime and 50
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mg/kg/day amiodarone; GLUC+ITRA Group - 100 mg Sb+5/kg/day glucantime and 50
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mg/kg/day itraconazole; AMIO+ITRA Group - 50 mg/kg/day amiodarone and 50 mg/kg/day
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itraconazole; CONTR Group (control group) - sodium chloride 0.9% (intraperitoneal via) and
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distilled water (oral via). After a 10 day interval, treatments were repeated in the same fashion
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for another 20 consecutive days (TRAT 2).
189 190
Glucantime was administered by intraperitoneal via and amiodarone (diluted in distilled water
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heated to 80°C, in water bath) and itraconazole (diluted in distilled water and submitted to
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ultrasonic bath until the achievement of homogenous suspension) were orally administered by
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gavage.
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The dosages of the drugs used were chosen according to published articles, involving animal
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experimentation [16,22].
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2.5. Evaluation of treatment efficacy
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2.5.1. Measure of the cutaneous lesions
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The size of the infected hamsters’ cutaneous lesions was determined by measuring the
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diameter of both rear feet and by subtracting the measurement of the uninfected foot from that
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of the infected foot. Measures, in millimeters, were performed by direct reading with an
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electronic pachymeter, immediately before the start of the treatment and after, for each 10
204
days during all the research.
205 206
2.5.2. Parasitic investigation
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Animals were subjected to euthanasia (5 animals/group) in three moments: right after TRAT
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1, right after TRAT 2 and 20 days after the end of the treatments, with the objective of
209
identification of possible relapses. After euthanasia, tissue cuts of inoculated foot were
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collected for histopathological and molecular examination and samples of liver and spleen
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were collected for molecular investigation of possible parasitic dissemination.
212 213
(I) Histopathological analysis: after confection and fastening of paraffin blocks, tissue cuts of
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inoculated foot (4 cuts/foot) were stained by hematoxylin and eosin (HE) and analyzed by
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light microscopy to identify presence or absence of Leishmania.
216 217
(II) Molecular analysis - Polymerase Chain Reaction (PCR): DNA extraction was performed
218
by alkaline lise method. For Leishmania DNA specific detection and amplification the
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initiators JW11 (5’-CCTATTTTACACCAACCCCCAGT-3’) and JW12 (5’-
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GGGTAGGGGCGTTCTGCGAAA-3’) were employed, directed to the preserved sequences
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of the kinetoplast minicircle (kDNA), amplifying fragments of 120 bp [23]. PCR was
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performed in a final volume of 40 μL, containing 2 μL DNA, 10 mM Tris-HCl (pH=9,0), 50
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mM KCl, 0,1% Triton X-100, 2 mM MgCl2, 250 µM of each dNTP, 10 mol of each initiator
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(JW11 and JW12) and 0,5 U of Taq polymerase. Amplification consisted in an initial
225
denaturation at 94°C (4 min), 40 denaturation cycles at 94°C (1 min), girdling at 58°C (30 s)
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and extension at 72°C (30 s), followed by a final extension at 72°C (10 min) in a thermal
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cycler MJ Research PTC-100 (Inc. Watertown, MA, USA). Amplicons were seen in
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polyacrylamide gels with molecular weight markers of 100 bp, stained with 0,2% silver
229
nitrate.
230 231
2.6. Evaluation of the side effects of the drugs
232
Animals were weighted in the start of the experiments (before inoculation) and, after, for each
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10 days during the research.
234 235
Before inoculation, immediately before and after the treatments and at the end to the research,
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animals fasted for 13-16 hours and blood samples were collected (by retro orbital via) for
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performance of the following tests: glucose, urea, creatinine, aspartate aminotransferase
238
(AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Serum
239
concentrations were quantified by enzymatic colorimetric tests, with spectrophotometric
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identification in an automatized biochemical analyzer, employing commercial kits (Cobas®,
241
Roche), complying to the instructions of the producer.
242 243
2.7. Statistical analysis
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Statistical analysis were performed employing the Statsoft Statistica 10 software, and
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differences were considered as significant when higher than p<0.05. In comparison analysis
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variables were submitted to Shapiro-Wilk test for data distribution analysis and, in case of
247
independent samples, to Levene test in order to verify homogeneity of variances.
248
Comparisons between independent samples were performed by paired t test or Wilcoxon test,
249
for parametric and non-parametric samples, respectively. In comparisons between
250
independent samples the parametric ANOVA test was performed, followed by the Bonferroni
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test or non-parametric Kruskal-Wallis test, followed by Dunn test. In analysis of categorical
252
data, the chi-square test (χ2) was performed.
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3. Results
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3.1. Evaluation of the efficacy of the treatments
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3.1.1. Measure of cutaneous lesions
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Analysis of the size of the lesions showed at the final of TRAT 1 (40 days) animals treated
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with GLUC, ITRA, GLUC+AMIO, GLUC+ITRA and AMIO+ITRA showed significantly
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smaller lesions, when compared to animals of the CONT group, untreated. At this moment of
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the research, smaller lesions were observed in animals receiving GLUC+AMIO and
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GLUC+ITRA. It is important to emphasize that the lesions presented by the animals treated
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with GLUC+AMIO were significantly smaller than lesions exhibited by hamsters treated with
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GLU, AMIO, ITRA and AMIO+ITRA. As for the lesions presented by the animals receiving
265
GLUC+ITRA, these were significantly smaller than those presented by the animals treated
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with AMIO, ITRA and AMIO+ITRA (Figs. 1 and 2A, Table 1).
267 268
Immediately after TRAT 2 (70 days) only the lesions of animals treated with GLUC+AMIO
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and GLUC+ITRA showed significant differences when compared to CONT animals.
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Hamsters receiving GLUC+AMIO still presented significantly smaller lesions in relation to
271
hamsters treated with AMIO, ITRA and AMIO+ITRA. Animals treated with GLUC+ITRA
272
showed, furthermore, lesions significantly smaller than those treated with AMIO (Figs. 1 and
273
2B, Table 1).
274 275
As well as for all other moments of the research, at the end of the study (90 days) the smaller
276
lesions were observed in animals receiving GLUC+AMIO and GLUC+ITRA, these being
277
significantly smaller to the ones seen in animals treated with AMIO and in CONT animals.
278
Besides, instead of most experimental groups presented cutaneous lesions with smaller
279
average size than those observed in the CONT group, only hamsters treated with
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GLUC+AMIO and GLUC+ITRA presented lesions significantly smaller when compared to
281
CONT hamsters (Figs. 1 and 2C, Table 1).
282 283
Finally, analysis of the evolution of lesion size showed that, in general, in all experimental
284
groups there was a progressive increase of the average size of the lesion during the research,
285
however in animals treated with the association of GLUC+AMIO and GLUC+ITRA, the
286
increase observed was not significant (Figs. 1 and 2, Table 1).
287 288
3.1.2. Parasitic investigation
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(I) Histopathological analysis: in general, the group treated with GLUC+AMIO was the one
290
presenting the higher number of animals (30.8%) with no parasites in tissue cuts of inoculated
291
foot, especially right after the finish of TRAT 1 (Table 2).
292 293
(II) Molecular analysis - PCR: only some of the hamsters treated with GLUC (7.1%),
294
GLUC+AMIO (15.4%) and GLUC+ITRA (7.7%), euthanized right after the end of TRAT 1
295
did not show amplification products corresponding to the Leishmania genus in tissue cuts of
296
the inoculated foot. Additionally, investigation of possible parasitic dissemination showed the
297
presence of the parasite DNA in samples obtained from the liver and the spleen of the
298
majority of the animals, however animals treated with GLUC+AMIO and GLUC+ITRA were
299
those showing the smaller percent of dissemination, either hepatic or splenic (Fig. 3, Table 3).
300 301
3.2. Evaluation of side effects of the drugs
302
Significantly, hamsters treated with AMIO, GLUC+AMIO and AMIO+ITRA lost weight
303
during the treatment periods, presenting, in some of the cases, lower weight when compared
304
to CONT group animals. However, analysis of the evolution of the animals showed that, in
14Page 14 of 28
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general, hamsters gained weight along the research and at the end of the study, as well as in
306
the intervals of the treatments, no significant difference was observed regarding the weight of
307
animals treated or untreated with the drugs (Supplemental material).
308 309
Regarding the biochemical and metabolic parameters, the animals did not present significant
310
alterations in serum concentrations of glucose, urea and creatinine in relation to normal
311
reference values. Nonetheless elevated levels of AST, ALT and ALP were observed during all
312
the study, but no significant increases were seen in hamsters treated with different drugs when
313
compared to CONT group animals (Supplemental material).
314 315
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4. Discussion
317
Leishmaniasis represent an important public health problem in many different countries of the
318
world and the search for new or alternative therapies against the different forms of the disease
319
is still a clinical priority.
320 321
Investigating the action of amiodarone and itraconazole against L. (L.) amazonensis, De
322
Macedo-Silva et al. [27,28] demonstrated in vitro the powerful antiproliferative,
323
ultrastructural and physiological effects of these drugs on the promastigote and amastigote
324
forms of the parasite. Nevertheless our results have shown that infections caused by L. (L.)
325
amazonensis in hamsters are resistant to amiodarone and itraconazole, since, at the end of the
326
study, there were no observable significant differences in lesion size and parasitic cure among
327
the animals treated with these drugs and untreated animals.
328 329
It is common to observe variability and discordance of results when confronting in vitro and
330
in vivo tests, after all, in vitro studies are performed in an environment that is distinct from in
331
vivo environments, in which countless parasite/host interactions occur. The ability of the
332
immune response of the host, the degree of evolution of the disease, the dynamics of the drug
333
concentration and of the infectious agent are examples of factors that may interfere in the
334
treatment [29].
335 336
In addition, data from the literature show that amiodarone and itraconazole are able to hinder
337
the development of lesions in mice BALB/c experimentally infected with L. (L.) mexicana
338
and L. (L.) major, respectively [15,30], and that glucantime presents good efficacy in
339
hamsters infected with L. (V.) panamensis [31]. However, when these data are compared to
340
our findings, we must consider that: (a) BALB/c mice may be more resistant to experimental
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infections by some species of Leishmania, such as L. (V.) brasiliensis, being able to show
342
nodules and swellings that rapidly evolve to cure [32], and that is why we used as model
343
animal the hamster, taking into account that these are more appropriate for researches
344
involving the pathogenesis and therapeutics of leishmaniasis, since they present higher
345
susceptibility to the parasite, with the development of chronic lesions, allowing immunologic
346
and therapeutic follow up for long periods [33]; (b) often, the species of L. (L.) amazonensis
347
are less sensitive to the available drugs, when compared to the other species of Leishmania,
348
such as, for example, L. (L.) mexicana, L. (V.) braziliensis and L. (L.) infantum (syn. Chagasi)
349
[28,34].
350 351
At every moment of our research, smaller cutaneous lesions were observed in hamsters
352
receiving the association of GLUC+AMIO and GLUC+ITRA. Complementary, in general,
353
animals treated with these associations were the only ones that did not present significant
354
increase in the average size of the lesion during the study, presented the higher number of
355
negative parasitic results in the analysis of tissue cuts of inoculated legs and presented the
356
smaller percent of hepatic and splenic dissemination. Together, our data suggest that
357
amiodarone or itraconazole improve the activity of glucantime in the treatment of these
358
lesions.
359 360
Supporting our findings, Serrano-Martín et al. [15] also investigated synergic action in vitro
361
and in vivo, between amiodarone and miltefosine against L. (L.) mexicana and De Morais-
362
Teixeira et al. [35] exalted, in vitro, the synergic activity between glucantime and
363
paromomycin against L. (V.) braziliensis and between paromomycin and miltefosine against
364
L. (L.) amazonensis.
365
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Currently, there are great expectations regarding drug association. Combined treatments are
367
being considered more and more as the first choice therapy, especially in visceral
368
leishmaniasis cases [36]. Clinical phase III essays were finished, in India, in 2010, involving
369
three combination of drugs (miltefosine and paromomycin, miltefosine and liposomal
370
amphotericin B, liposomal amphotericin B and paromomycin) and all of them disclosed
371
efficacy higher than 95% in the treatment of the visceral form [21]. Combination drug therapy
372
has the advantages to reduce doses and the length of the treatment and, therefore, improving
373
the tolerance and adherence of the patients. Also, it is possible that drug association may
374
prevent and/or delay the appearance of resistance [37].
375 376
Taking into account that biochemical analysis of the blood, along with other factors, may
377
issue important information to clear the clinical picture and pathologic alterations and,
378
therefore, broaden experimental results, we analyzed, during all the study, the weight of the
379
animals and some biochemical and metabolic parameters related to possible liver lesions
380
and/or renal impairment. No significant alterations were seen in serum concentrations of
381
glucose, urea and creatinine in relation to normal reference levels found in the literature [24-
382
26], but high levels of AST, ALT and ALP were seen during all the research. Taking into
383
account that animals were not germfree, that the reports of biochemical reference values in
384
hamsters are extremely scarce and often discrepant, due to the different enzymatic methods
385
employed, and present variations related to sex, age, lineage, genotype, diet, handling, the
386
environment, blood collection via and other interfering factors [25,38], we performed
387
comparisons between the different treatment groups and the CONT group and did not find
388
significant increase that could possibly be related to experimental treatment.
389
18Page 18 of 28
390
Finally, it is important to point out that many studies have already described important side
391
effects of the treatment with amiodarone, suggesting caution in its employment [39].
392
However, structural changes in its molecule have enabled the development of new drugs, such
393
as dronedarone, that seem not to present the same serious side effects, providing safer
394
therapeutic options [40].
395 396
19Page 19 of 28
397
5. Conclusions
398
The results indicate that, in hamsters, amiodarone and itraconazole, either used isolated or in
399
combination, are unable to hinder the development of cutaneous lesions caused by L. (L.)
400
amazonensis. However, the results suggest that amiodarone or itraconazole improve the
401
activity of glucantime in the treatment of these lesions and seems not to present evident side
402
effects. More studies are necessary in order to investigate the clinical potential of these
403
combinations, in order to widen therapeutic options, especially in resistant cases.
404
Furthermore, investigation of the use of such drugs in the treatment of leishmaniasis caused
405
by other species of Leishmania would be opportune.
406 407
Declarations
408
Funding: This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de
409
Nível Superior (Capes) (32/2010).
410
Competing Interests: None declared.
411
Ethical Approval: This study (Permit Number: 181/2011) was approved by the Ethics
412
Committee for Animal Experimentation of the Universidade Federal do Triângulo Mineiro
413
(Brazil).
414 415
20Page 20 of 28
416
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520 521
Figure Legends
522
Fig. 1. Evolution of the average size of cutaneous lesions (in millimeters) in hamsters
523
experimentally infected with L. (L.) amazonensis and treated with different drugs.
524
Fig. 2. Images of the cutaneous lesions of hamsters experimentally infected with L. (L.)
525
amazonensis and treated with different drugs. (A) After the end of TRAT 1 (40 days). (B)
526
After the end of TRAT 2 (70 days). (C) At the end of the study (90 days).
527
Fig. 3. Polyacrylamide gel representative of PCR with initiators JW11 and JW12 (amplifying
528
fragments of 120 bp, corresponding to the Leishmania genus) performed at the tissue cuts of
529
hamsters experimentally infected with L. (L.) amazonensis and treated with different drugs.
530
Lane 1, molecular weight marker; lane 2, PCR mixture without DNA (negative control); lane
531
3, L. (L.) amazonensis genomic DNA (positive control); lanes 4 to 11, DNA from tissue cuts
532
of inoculated foot of hamsters treated with GLUC+AMIO, lane 12, DNA from tissue cuts of
533
inoculated foot of hamster untreated. The absence of a 120 bp band indicates the absence of
534
Leishmania in the lesion.
535
25Page 25 of 28
536
Table 1. Size of the cutaneous lesions (in millimeters) of hamsters experimentally infected with L. (L.) amazonensis and treated with different drugs. Size of the lesion (mm)1 20 days
30 days
40 days
50 days
60 days
70 days
80 days
90 days
Value-p (TRAT 1)2
Value-p (TRAT 2)2
Value-p (Final)2
GLUC
1.81±0.50
2.52±0.59 b
2.71±0.79 bc
3.66±1.24 bcd
4.32±1.73 bc
5.31±2.50 bcd
5.41±1.45 abc
5.87±1.06 bc
< 0.001
0.007
0.002
AMIO
1.85±0.39
3.15±0.39 a
3.47±0.76 ab
5.05±1.58 ab
6.77±1.69 ab
8.78±1.77 a
9.12±1.70 a
7.84±0.94 a
< 0.001
0.001
< 0.001
ITRA
1.97±0.44
2.86±0.62 ab
3.25±0.80 b
4.32±1.64 abc
5.24±2.01 abc
5.96±2.51 bc
4.95±2.09 abc
6.34±2.40 abc
< 0.001
0.008
0.014
GLUC+AMIO
1.91±0.40
2.46±0.75 b
1.55±0.73 d
2.11±0.85 d
2.78±1.95 c
3.18±1.66 d
3.42±1.56 c
3.97±1.91 c
0.138
0.086
0.054
GLUC+ITRA
2.18±0.45
2.46±0.66 b
2.08±0.72 cd
2.64±1.36 cd
3.38±1.21 c
4.19±2.07 cd
3.86±3.12 bc
4.02±2.94 c
0.534
0.002
0.253
AMIO+ITRA
1.86±0.35
2.62±0.47 b
2.91±0.82 b
4.25±1.98 abc
4.87±2.75 abc
5.75±2.80 bc
4.47±0.84 bc
6.03±1.03 abc
< 0.001
0.011
0.001
CONT
1.90±0.35
3.41±0.88 a
4.59±1.55 a
6.14±1.29 a
7.27±1.78 a
7.53±2.24 ab
7.77±2.32 ab
7.81±1.67 ab
< 0.001
0.037
0.001
Value-p (Groups ≠)3
0.270
0.001
< 0.001
< 0.001
< 0.001
< 0.001
0.001
0.023
Groups
537 538 539 540 541 542 543 544
1
Values of the lesion size are expressed in mean + standard deviation. Value-p (TRAT 1) represents the result of the comparison between the start (20 days) and the end (40 days) of TRAT 1. Value-p (TRAT 2) represents the result of the comparison between the start (50 days) and the end (70 days) of TRAT 2. Value-p (Final) refers to the result of the comparison between the start (20 days) and the end (90 days) of all the study. These comparisons were performed for each one of the experimental groups employing paired t test or Wilcoxon test. 3 Value-p (Groups ≠) represents the result of the ANOVA test or Kruskal-Wallis in the comparison between the different experimental groups, for each time interval alloted for the study. In variables with p<0.05, means followed by different letters (a, b, c or d) are significantly different from each other according to Bonferroni’s or Dunn’s multiple comparison tests. 2
26
Page 26 of 28
545 546
Table 2. Results of the parasitic investigation by histopathological analysis of tissue cuts of inoculated foot of hamsters experimentally infected with L. (L.) amazonensis and treated with different drugs. Number of negative animals/Total Euthanasia 1
Euthanasia 2
Euthanasia 3
General
Value-p (Euthanasia)1
GLUC
3/4 (75.0%)
1/5 (20.0%)
0/5 (0.0%)
4/14 (28.6%)
0.041
AMIO
2/4 (50.0%)
0/4 (0.0%)
0/5 (0.0%)
2/13 (15.4%)
0.070
ITRA
1/4 (25.0%)
0/5 (0.0%)
0/5 (0.0%)
1/14 (7.1%)
0.260
GLUC+AMIO
4/4 (100.0%)
0/4 (0.0%)
0/5 (0.0%)
4/13 (30.8%)
0.002
GLUC+ITRA
2/3 (66.7%)
0/5 (0.0%)
1/5 (20.0%)
3/13 (23.1%)
0.094
AMIO+ITRA
1/4 (25.0%)
0/4 (0.0%)
1/5 (20.0%)
2/13 (15.4%)
0.579
CONT
0/5 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/15 (0.0%)
-
0.057
0.473
0.506
0.273
Groups
Value-p (Groups ≠)
547 548 549 550 551 552 553
2
1
Value-p (Euthanasia) represents the result of chi-square test (χ2) in the comparison between the different moments in which animals were euthanized (Euthanasia 1: right after the end of TRAT 1; Euthanasia 2: right after the end of TRAT 2; Euthanasia 3: 20 days after the end of TRAT 2). These comparisons were performed for each one of the experimental groups. 2 Value-p (Groups ≠) represents the result of the chi-square test (χ2) in the comparison among the different experimental groups, for each one of the moments in which animals were euthanized and in general.
554
27
Page 27 of 28
555 556
Table 3. Results of the parasitic investigation by molecular analysis (PCR) of tissue cuts of inoculated foot, liver and spleen of hamsters experimentally infected with L. (L.) amazonensis and treated with different drugs. Number of negative animals/Total Euthanasia 3
General
Value-p (Euthanasia)1
Groups Euthanasia 1
Euthanasia 2 Inoculated foot
GLUC
1/4 (25.0%)
0/5 (0.0%)
0/5 (0.0%)
1/14 (7.1%)
0.260
AMIO
0/4 (0.0%)
0/4 (0.0%)
0/5 (0.0%)
0/13 (0.0%)
-
ITRA
0/4 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/14 (0.0%)
-
GLUC+AMIO
2/4 (50.0%)
0/4 (0.0%)
0/5 (0.0%)
2/13 (15.4%)
0.070
GLUC+ITRA
1/3 (33.3%)
0/5 (0.0%)
0/5 (0.0%)
1/13 (7.7%)
0.164
AMIO+ITRA
0/4 (0.0%)
0/4 (0.0%)
0/5 (0.0%)
0/13 (0.0%)
-
0/5 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/15 (0.0%)
-
0.219
-
-
0.309
CONT Value-p (Groups ≠)
2
Liver GLUC
1/4 (25.0%)
0/5 (0.0%)
0/5 (0.0%)
1/14 (7.1%) ab
0.260
AMIO
1/4 (25.0%)
0/4 (0.0%)
0/5 (0.0%)
1/13 (7.7%) ab
0.260
ITRA
0/4 (0.0%)
1/5 (20.0%)
0/5 (0.0%)
1/14 (7.1%) ab
0.379
GLUC+AMIO
3/4 (75.0%)
1/4 (25.0%)
1/5 (20.0%)
5/13 (38.5%) b
0.194
GLUC+ITRA
1/3 (33.3%)
0/5 (0.0%)
1/5 (20.0%)
2/13 (15.4%) ab
0.420
AMIO+ITRA
0/4 (0.0%)
0/4 (0.0%)
0/5 (0.0%)
0/13 (0.0%) a
-
0/5 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/15 (0.0%) a
-
0.099
0.476
0.506
0.022
CONT Value-p (Groups ≠)
2
Spleen
557 558 559 560 561 562 563 564
GLUC
1/4 (25.0%)
0/5 (0.0%)
0/5 (0.0%)
1/14 (7.1%)
0.260
AMIO
1/4 (25.0%)
0/4 (0.0%)
0/5 (0.0%)
1/13 (7.7%)
0.260
ITRA
0/4 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/14 (0.0%)
-
GLUC+AMIO
3/4 (75.0%)
0/4 (0.0%)
0/5 (0.0%)
3/13 (23.1%)
0.012
GLUC+ITRA
1/3 (33.3%)
0/5 (0.0%)
1/5 (20.0%)
2/13 (15.4%)
0.420
AMIO+ITRA
0/4 (0.0%)
0/4 (0.0%)
0/5 (0.0%)
0/13 (0.0%)
-
CONT
0/5 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/15 (0.0%)
-
Value-p (Groups ≠)2
0.099
-
0.404
0.159
1
Value-p (Euthanasia) represents the result of chi-square test (χ2) in the comparison among the different moments of euthanasia (Euthanasia 1: right after the end of TRAT 1; Euthanasia 2: right after the end of TRAT 2; Euthanasia 3: 20 days after the end of TRAT 2). These comparisons were performed for each one of the experimental groups. 2 Value-p (Groups ≠) represents the result of chi-square test (χ2) in the comparison among the different experimental groups for each of the moments of euthanasia and in general. In variables with p<0.05, values followed by different letters (a or b) are significantly different from each other.
28
Page 28 of 28
S0924-8579(17)30217-0 http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.06.007 ANTAGE 5157
To appear in:
International Journal of Antimicrobial Agents
Received date: Accepted date:
11-8-2016 17-6-2017
Please cite this article as: Laís Anversa, Monique Gomes Salles Tiburcio, Lara Rocha Batista, Marília Beatriz Cuba, Gabriel Antonio Nogueira Nascentes, Tábata Yamasaki Martins, Virgínia Bodelão Richini Pereira, Luciana da Silva Ruiz Menezes, Valdo José Dias da Silva, Luis Eduardo Ramirez, Amiodarone and itraconazole improve the activity of pentavalent antimonial in the treatment of experimental cutaneous leishmaniasis, International Journal of Antimicrobial Agents (2017), http://dx.doi.org/doi: 10.1016/j.ijantimicag.2017.06.007. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
1
Amiodarone and itraconazole improve the activity of pentavalent
2
antimonial in the treatment of experimental cutaneous leishmaniasis
3 4
Laís Anversaa,*, Monique Gomes Salles Tiburciob, Lara Rocha Batistac,
5
Marília Beatriz Cubad, Gabriel Antonio Nogueira Nascentese, Tábata Yamasaki Martinsf,
6
Virgínia Bodelão Richini Pereirag, Luciana da Silva Ruiz Menezesh, Valdo José Dias da
7
Silvai, Luis Eduardo Ramirezj
8 9
a
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
10
Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
11
Brazil. [email protected]
12
b
13
Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
14
Brazil. [email protected]
15
c
16
Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
17
Brazil. [email protected]
18
d
19
Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
20
Brazil. [email protected]
21
e
22
Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
23
Brazil. [email protected]
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
Programa de Pós-Graduação em Medicina Tropical e Infectologia, Universidade Federal do
1
Page 1 of 28
24
f
25
Triângulo Mineiro. Av Getúlio Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais,
26
Brazil. [email protected]
27
g
28
Lutz. R Rubens Arruda, Qd 6, Centro, 17015-110, Bauru, São Paulo, Brazil.
29
[email protected]
30
h
31
Lutz. R Rubens Arruda, Qd 6, Centro, 17015-110, Bauru, São Paulo, Brazil.
32
[email protected]
33
i
34
Terra, 330, Nossa Senhora da Abadia, 38025-015, Uberaba, Minas Gerais, Brazil.
35
[email protected]
36
j
37
Guarita S/N, Abadia, 38001-970, Uberaba, Minas Gerais, Brazil.
38
[email protected]
Programa de Iniciação Científica do Laboratório de Parasitologia, Universidade Federal do
Núcleo de Ciências Biomédicas, Centro de Laboratório Regional de Bauru, Instituto Adolfo
Núcleo de Ciências Biomédicas, Centro de Laboratório Regional de Bauru, Instituto Adolfo
Laboratório de Fisiologia Humana, Universidade Federal do Triângulo Mineiro. Pç Manoel
Laboratório de Parasitologia, Universidade Federal do Triângulo Mineiro. Av Getúlio
39 40
* Corresponding author
41
Centro de Laboratório Regional de Bauru,
42
Instituto Adolfo Lutz. R Rubens Arruda,
43
Qd 6, 17015-110,
44
Bauru (SP),
45
Brazil.
46
Phone: +55 14 32231175.
47
E-mail address: [email protected]
48 49 2
Page 2 of 28
50
Highlights
51
The therapeutic options for leishmaniasis are still very limited
52
Amiodarone and itraconazole improve the activity of glucantime in leishmaniasis
53
54
Abstract
55
Leishmaniasis affect millions of people, causing morbidity and mortality, especially in
56
developing tropical and subtropical countries. Unfortunately, the possibilities of treatment for
57
these infections are still quite limited and most of the available drugs present serious side
58
effects. The objective of this paper was to evaluate the therapeutic role of amiodarone and
59
itraconazole in the treatment of cutaneous leishmaniasis caused by Leishmania (Leishmania)
60
amazonensis. In order to perform this evaluation, hamsters were infected with 1 x 106
61
metaciclic promastigotes of the parasite in the hind footpad and, after the onset of the lesions,
62
were treated with glucantime, amiodarone, itraconazole, glucantime and amiodarone,
63
glucantime and itraconazole or amiodarone and itraconazole. The treatments’ efficacy was
64
evaluated per analysis of the size of the cutaneous lesions and by parasitic investigation of the
65
infected foot (by histopathological examination and PCR) and possible side effects were
66
analyzed taking into account the weight of the animals and some biochemical and metabolic
67
parameters (glucose, urea, creatinine, AST, ALT and ALP). The results have shown that, in
68
hamsters, amiodarone and itraconazole, either used isolated or in combination, are unable to
69
stop the development of cutaneous lesions caused by L. (L.) amazonensis, but improve the
70
activity of glucantime in the treatment of these lesions and seem to present no evident side
71
effects. More studies are necessary in order to investigate the clinical potential of these
72
combinations, so there can be the possibility of broadening the therapeutic options available,
73
especially in resistant cases.
The association of drugs seems to be an alternative to treat leishmaniasis
74
3
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75
Keywords: Leishmania; leishmaniasis; treatment; amiodarone; itraconazole; combination
76
therapy.
77 78
4
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79
1. Introduction
80
Leishmaniasis are caused by many different protozoans’ species from the Trypanosomatidae
81
family and Leishmania genus, affecting men and different animals. This parasitosis is
82
endemic in at least 98 countries in the entire world and more than two million new cases
83
occur, each year, with high morbidity and mortality levels [1]. Among other factors,
84
depending on the infecting species and the immune response of the host, the disease presents
85
different clinical manifestations, such as: (a) the local cutaneous, characterized by the
86
presence of lesions exclusively in the site of the vector insect bite; (b) the mucocutaneous,
87
with destructive lesions in the upper respiratory tract; (c) the diffuse cutaneous, with multiple
88
non ulcerated nodules, classically unresponsive to available treatments; (d) and the visceral, a
89
systemic form affecting especially lymphatic nodes, the spleen, the liver and the bone marrow
90
[2].
91 92
Leishmania (Leishmania) amazonensis is found and widely distributed in the Amazon
93
rainforest region of Brazil, and has been reported as expanding to the Northeast, the Southeast
94
and the Center West of the country, east of Paraguay and other Amazon areas of Bolivia, Peru
95
Ecuador, Colombia and Venezuela [3,4]. Commonly, the species of L. (L.) amazonensis
96
induce localized lesions, and nevertheless may also cause the diffuse cutaneous and even
97
mucocutaneous and visceral forms of the disease [5].
98 99
Conventional therapy for all clinical forms of leishmaniasis include the pentavalent
100
antimonials’ (Sb+5) use, such as antimonate of N-methylglucamine (Glucantime®) and
101
sodium stibogluconate (Pentostam®) [6]. However, these drugs demand long term parenteral
102
administration, present serious toxic side effects, especially on cardiac, renal and hepatic
103
systems [7] and have shown gradually decreasing efficacy due to the appearance of resistance
5
Page 5 of 28
104
in many countries [8]. Amphotericin B, pentamidine and paromomycin are alternative drugs,
105
also administered by parenteral via, that can present a variety of cure rates and many and
106
frequent side effects [9,10]. Miltefosine, on the other hand, was introduced in India, in 2002,
107
and represented a major advance in the treatment of visceral leishmaniasis, being the first
108
drug possible to be orally administered, but the relatively high cost and the concerns related to
109
teratogenicity and the potent development of resistance have limited its use [11]. Therefore,
110
alternative treatments are intensely searched in order to achieve better results, with less side
111
effects and higher patient adherence.
112 113
Amiodarone, an antiarrhythmic drug class III, commonly used to treat cardiopathies,
114
including arrhythmias in the chronic phase of Chagas disease, have been subjected to recent
115
studies investigating its use as antimycotic agent and anti trypanosomatid, since this drug has
116
excellent pharmacokinetics properties and has relatively low cost [12,13]. It has been shown
117
that amiodarone has direct activity against Trypanosoma cruzi and Leishmania (Leishmania)
118
mexicana, affecting the viability of the parasitic forms in vitro experiments and reducing the
119
parasitemia and the development of lesions, respectively, in mice infected and treated with
120
this drug [14,15]. Amiodarone acts in the parasite in many different ways: (a) blocking the
121
biosynthesis of membrane steroids; (b) promoting alterations in the potential of the
122
mitochondrial membrane; (c) breaking the homeostasis of Ca2+ and inducing a rapid increase
123
in the cytoplasmic calcium; (d) increasing the production of oxygen reactive intermediates.
124
Even more than that, the efficacy of amiodarone in the infection caused by Leishmania can
125
also be favored by its lipid nature (highly soluble), extense tissue distribution and important
126
excretion through the skin, factors that assure distribution to infection sites [16].
127
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128
Other drugs orally administered, especially azoles - such as ketoconazole, fluconazole and
129
itraconazole - are also being applied experimentally in the treatment of leishmaniasis,
130
especially cutaneous leishmaniasis. Taking into account that these antifungals inhibit the
131
biosynthesis of ergosterol (the major sterol present in the plasmatic membrane of some
132
microorganisms, including trypanosomatids) the use of these drugs in the treatment of
133
infections caused by Leishmania is coherent by biochemical standards [17,18]. However,
134
clinical essays with azoles are still limited and results obtained are ambiguous [19,20].
135 136
Currently, there are great expectations regarding the therapy with a combination of drugs
137
designed to reduce dosages and length of the treatment, therefore improving tolerance and
138
conformity of drugs already available [21]. Thus, the objective of this paper was to evaluate
139
the therapeutic role of amiodarone and itraconazole in the treatment of experimental
140
cutaneous leishmaniasis caused by L. (L.) amazonensis, in order to generate new perspective
141
in the therapeutic approach of this important parasitic endemic disease.
142 143
7
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144
2. Materials and methods
145
This study (Permit Number: 181/2011) was approved by the Ethics Committee for Animal
146
Experimentation of the Universidade Federal do Triângulo Mineiro (Brazil). All animals
147
received humane care in compliance with the “Principles of laboratory animal care”
148
formulated by the National Society for Medical Research and the “Guide for the care and use
149
of laboratory animals” prepared by the National Academy of Sciences (Washington, DC).
150 151
2.1. Parasites
152
The strain of L. (L.) amazonensis IFLA/BR/67/PH8 (isolated from phlebotomine
153
Lutzomyia flaviscutellata, in Belém, Pará, Brazil, 1976) was kindly donated by Prof. Dr.
154
Maria Norma Melo, of the Parasitology Department of the Instituto de Ciências Biológicas of
155
the Universidade Federal de Minas Gerais (UFMG) and was kept in the laboratory for
156
successive inoculations in hamsters and cultured at 26°C in alfa-MEM medium (Minimum
157
Essential Medium) (Gibco®), supplemented with 10% of fetal bovine serum (SFB) and 100
158
µg/ml gentamicin.
159 160
2.2. Drugs
161
Glucantime® (N-metilglucamine antimonate) was supplied by the Regional Health
162
Management of Uberaba (Lot: 9E1030), amiodarone (C25H30ClI2NO3) was bought from
163
Sigma-Aldrich and itraconazole (C35H38Cl2N8O4) was bought from Janssen-Cilag.
164 165
2.3. Experimental animal infection
166
A total of 105 Syrian hamsters (Mesocricetus auratus) were used, not isogenic, males aged
167
between six and eight weeks, obtained from the biotery of the Laboratory of the Parasitology
168
discipline of the Universidade Federal do Triângulo Mineiro (UFTM) and kept in the same
8
Page 8 of 28
169
place, at a controlled temperature of 23ºC ± 1ºC, light-dark cycle with food and water ad
170
libitum.
171 172
Hamsters were inoculated by intradermal injection at the right hind footpad, with 100 μl
173
phosphate-buffered saline (PBS) containing 1 x 106 L. (L.) amazonensis metacyclic
174
promastigote from culture in alpha-MEM medium and quantified in Neubauer chamber (in
175
triplicate).
176 177
2.4. Treatments
178
Hamsters were randomly divided in seven experimental groups (15 animals/group) and 20
179
days after inoculation, when infection was already well established and lesions were present,
180
treatments were started. Each group was treated daily with the drugs (one dose per day and at
181
the same time schedule) for 20 consecutive days (TRAT 1), as follows: GLUC Group - 100
182
mg Sb+5/kg/day glucantime; AMIO Group - 50 mg/kg/day amiodarone; ITRA Group - 50
183
mg/kg/day itraconazole; GLUC+AMIO Group - 100 mg Sb+5/kg/day glucantime and 50
184
mg/kg/day amiodarone; GLUC+ITRA Group - 100 mg Sb+5/kg/day glucantime and 50
185
mg/kg/day itraconazole; AMIO+ITRA Group - 50 mg/kg/day amiodarone and 50 mg/kg/day
186
itraconazole; CONTR Group (control group) - sodium chloride 0.9% (intraperitoneal via) and
187
distilled water (oral via). After a 10 day interval, treatments were repeated in the same fashion
188
for another 20 consecutive days (TRAT 2).
189 190
Glucantime was administered by intraperitoneal via and amiodarone (diluted in distilled water
191
heated to 80°C, in water bath) and itraconazole (diluted in distilled water and submitted to
192
ultrasonic bath until the achievement of homogenous suspension) were orally administered by
193
gavage.
9
Page 9 of 28
194 195
The dosages of the drugs used were chosen according to published articles, involving animal
196
experimentation [16,22].
197 198
2.5. Evaluation of treatment efficacy
199
2.5.1. Measure of the cutaneous lesions
200
The size of the infected hamsters’ cutaneous lesions was determined by measuring the
201
diameter of both rear feet and by subtracting the measurement of the uninfected foot from that
202
of the infected foot. Measures, in millimeters, were performed by direct reading with an
203
electronic pachymeter, immediately before the start of the treatment and after, for each 10
204
days during all the research.
205 206
2.5.2. Parasitic investigation
207
Animals were subjected to euthanasia (5 animals/group) in three moments: right after TRAT
208
1, right after TRAT 2 and 20 days after the end of the treatments, with the objective of
209
identification of possible relapses. After euthanasia, tissue cuts of inoculated foot were
210
collected for histopathological and molecular examination and samples of liver and spleen
211
were collected for molecular investigation of possible parasitic dissemination.
212 213
(I) Histopathological analysis: after confection and fastening of paraffin blocks, tissue cuts of
214
inoculated foot (4 cuts/foot) were stained by hematoxylin and eosin (HE) and analyzed by
215
light microscopy to identify presence or absence of Leishmania.
216 217
(II) Molecular analysis - Polymerase Chain Reaction (PCR): DNA extraction was performed
218
by alkaline lise method. For Leishmania DNA specific detection and amplification the
10Page 10 of 28
219
initiators JW11 (5’-CCTATTTTACACCAACCCCCAGT-3’) and JW12 (5’-
220
GGGTAGGGGCGTTCTGCGAAA-3’) were employed, directed to the preserved sequences
221
of the kinetoplast minicircle (kDNA), amplifying fragments of 120 bp [23]. PCR was
222
performed in a final volume of 40 μL, containing 2 μL DNA, 10 mM Tris-HCl (pH=9,0), 50
223
mM KCl, 0,1% Triton X-100, 2 mM MgCl2, 250 µM of each dNTP, 10 mol of each initiator
224
(JW11 and JW12) and 0,5 U of Taq polymerase. Amplification consisted in an initial
225
denaturation at 94°C (4 min), 40 denaturation cycles at 94°C (1 min), girdling at 58°C (30 s)
226
and extension at 72°C (30 s), followed by a final extension at 72°C (10 min) in a thermal
227
cycler MJ Research PTC-100 (Inc. Watertown, MA, USA). Amplicons were seen in
228
polyacrylamide gels with molecular weight markers of 100 bp, stained with 0,2% silver
229
nitrate.
230 231
2.6. Evaluation of the side effects of the drugs
232
Animals were weighted in the start of the experiments (before inoculation) and, after, for each
233
10 days during the research.
234 235
Before inoculation, immediately before and after the treatments and at the end to the research,
236
animals fasted for 13-16 hours and blood samples were collected (by retro orbital via) for
237
performance of the following tests: glucose, urea, creatinine, aspartate aminotransferase
238
(AST), alanine aminotransferase (ALT) and alkaline phosphatase (ALP). Serum
239
concentrations were quantified by enzymatic colorimetric tests, with spectrophotometric
240
identification in an automatized biochemical analyzer, employing commercial kits (Cobas®,
241
Roche), complying to the instructions of the producer.
242 243
2.7. Statistical analysis
11Page 11 of 28
244
Statistical analysis were performed employing the Statsoft Statistica 10 software, and
245
differences were considered as significant when higher than p<0.05. In comparison analysis
246
variables were submitted to Shapiro-Wilk test for data distribution analysis and, in case of
247
independent samples, to Levene test in order to verify homogeneity of variances.
248
Comparisons between independent samples were performed by paired t test or Wilcoxon test,
249
for parametric and non-parametric samples, respectively. In comparisons between
250
independent samples the parametric ANOVA test was performed, followed by the Bonferroni
251
test or non-parametric Kruskal-Wallis test, followed by Dunn test. In analysis of categorical
252
data, the chi-square test (χ2) was performed.
253 254
12Page 12 of 28
255
3. Results
256
3.1. Evaluation of the efficacy of the treatments
257
3.1.1. Measure of cutaneous lesions
258
Analysis of the size of the lesions showed at the final of TRAT 1 (40 days) animals treated
259
with GLUC, ITRA, GLUC+AMIO, GLUC+ITRA and AMIO+ITRA showed significantly
260
smaller lesions, when compared to animals of the CONT group, untreated. At this moment of
261
the research, smaller lesions were observed in animals receiving GLUC+AMIO and
262
GLUC+ITRA. It is important to emphasize that the lesions presented by the animals treated
263
with GLUC+AMIO were significantly smaller than lesions exhibited by hamsters treated with
264
GLU, AMIO, ITRA and AMIO+ITRA. As for the lesions presented by the animals receiving
265
GLUC+ITRA, these were significantly smaller than those presented by the animals treated
266
with AMIO, ITRA and AMIO+ITRA (Figs. 1 and 2A, Table 1).
267 268
Immediately after TRAT 2 (70 days) only the lesions of animals treated with GLUC+AMIO
269
and GLUC+ITRA showed significant differences when compared to CONT animals.
270
Hamsters receiving GLUC+AMIO still presented significantly smaller lesions in relation to
271
hamsters treated with AMIO, ITRA and AMIO+ITRA. Animals treated with GLUC+ITRA
272
showed, furthermore, lesions significantly smaller than those treated with AMIO (Figs. 1 and
273
2B, Table 1).
274 275
As well as for all other moments of the research, at the end of the study (90 days) the smaller
276
lesions were observed in animals receiving GLUC+AMIO and GLUC+ITRA, these being
277
significantly smaller to the ones seen in animals treated with AMIO and in CONT animals.
278
Besides, instead of most experimental groups presented cutaneous lesions with smaller
279
average size than those observed in the CONT group, only hamsters treated with
13Page 13 of 28
280
GLUC+AMIO and GLUC+ITRA presented lesions significantly smaller when compared to
281
CONT hamsters (Figs. 1 and 2C, Table 1).
282 283
Finally, analysis of the evolution of lesion size showed that, in general, in all experimental
284
groups there was a progressive increase of the average size of the lesion during the research,
285
however in animals treated with the association of GLUC+AMIO and GLUC+ITRA, the
286
increase observed was not significant (Figs. 1 and 2, Table 1).
287 288
3.1.2. Parasitic investigation
289
(I) Histopathological analysis: in general, the group treated with GLUC+AMIO was the one
290
presenting the higher number of animals (30.8%) with no parasites in tissue cuts of inoculated
291
foot, especially right after the finish of TRAT 1 (Table 2).
292 293
(II) Molecular analysis - PCR: only some of the hamsters treated with GLUC (7.1%),
294
GLUC+AMIO (15.4%) and GLUC+ITRA (7.7%), euthanized right after the end of TRAT 1
295
did not show amplification products corresponding to the Leishmania genus in tissue cuts of
296
the inoculated foot. Additionally, investigation of possible parasitic dissemination showed the
297
presence of the parasite DNA in samples obtained from the liver and the spleen of the
298
majority of the animals, however animals treated with GLUC+AMIO and GLUC+ITRA were
299
those showing the smaller percent of dissemination, either hepatic or splenic (Fig. 3, Table 3).
300 301
3.2. Evaluation of side effects of the drugs
302
Significantly, hamsters treated with AMIO, GLUC+AMIO and AMIO+ITRA lost weight
303
during the treatment periods, presenting, in some of the cases, lower weight when compared
304
to CONT group animals. However, analysis of the evolution of the animals showed that, in
14Page 14 of 28
305
general, hamsters gained weight along the research and at the end of the study, as well as in
306
the intervals of the treatments, no significant difference was observed regarding the weight of
307
animals treated or untreated with the drugs (Supplemental material).
308 309
Regarding the biochemical and metabolic parameters, the animals did not present significant
310
alterations in serum concentrations of glucose, urea and creatinine in relation to normal
311
reference values. Nonetheless elevated levels of AST, ALT and ALP were observed during all
312
the study, but no significant increases were seen in hamsters treated with different drugs when
313
compared to CONT group animals (Supplemental material).
314 315
15Page 15 of 28
316
4. Discussion
317
Leishmaniasis represent an important public health problem in many different countries of the
318
world and the search for new or alternative therapies against the different forms of the disease
319
is still a clinical priority.
320 321
Investigating the action of amiodarone and itraconazole against L. (L.) amazonensis, De
322
Macedo-Silva et al. [27,28] demonstrated in vitro the powerful antiproliferative,
323
ultrastructural and physiological effects of these drugs on the promastigote and amastigote
324
forms of the parasite. Nevertheless our results have shown that infections caused by L. (L.)
325
amazonensis in hamsters are resistant to amiodarone and itraconazole, since, at the end of the
326
study, there were no observable significant differences in lesion size and parasitic cure among
327
the animals treated with these drugs and untreated animals.
328 329
It is common to observe variability and discordance of results when confronting in vitro and
330
in vivo tests, after all, in vitro studies are performed in an environment that is distinct from in
331
vivo environments, in which countless parasite/host interactions occur. The ability of the
332
immune response of the host, the degree of evolution of the disease, the dynamics of the drug
333
concentration and of the infectious agent are examples of factors that may interfere in the
334
treatment [29].
335 336
In addition, data from the literature show that amiodarone and itraconazole are able to hinder
337
the development of lesions in mice BALB/c experimentally infected with L. (L.) mexicana
338
and L. (L.) major, respectively [15,30], and that glucantime presents good efficacy in
339
hamsters infected with L. (V.) panamensis [31]. However, when these data are compared to
340
our findings, we must consider that: (a) BALB/c mice may be more resistant to experimental
16Page 16 of 28
341
infections by some species of Leishmania, such as L. (V.) brasiliensis, being able to show
342
nodules and swellings that rapidly evolve to cure [32], and that is why we used as model
343
animal the hamster, taking into account that these are more appropriate for researches
344
involving the pathogenesis and therapeutics of leishmaniasis, since they present higher
345
susceptibility to the parasite, with the development of chronic lesions, allowing immunologic
346
and therapeutic follow up for long periods [33]; (b) often, the species of L. (L.) amazonensis
347
are less sensitive to the available drugs, when compared to the other species of Leishmania,
348
such as, for example, L. (L.) mexicana, L. (V.) braziliensis and L. (L.) infantum (syn. Chagasi)
349
[28,34].
350 351
At every moment of our research, smaller cutaneous lesions were observed in hamsters
352
receiving the association of GLUC+AMIO and GLUC+ITRA. Complementary, in general,
353
animals treated with these associations were the only ones that did not present significant
354
increase in the average size of the lesion during the study, presented the higher number of
355
negative parasitic results in the analysis of tissue cuts of inoculated legs and presented the
356
smaller percent of hepatic and splenic dissemination. Together, our data suggest that
357
amiodarone or itraconazole improve the activity of glucantime in the treatment of these
358
lesions.
359 360
Supporting our findings, Serrano-Martín et al. [15] also investigated synergic action in vitro
361
and in vivo, between amiodarone and miltefosine against L. (L.) mexicana and De Morais-
362
Teixeira et al. [35] exalted, in vitro, the synergic activity between glucantime and
363
paromomycin against L. (V.) braziliensis and between paromomycin and miltefosine against
364
L. (L.) amazonensis.
365
17Page 17 of 28
366
Currently, there are great expectations regarding drug association. Combined treatments are
367
being considered more and more as the first choice therapy, especially in visceral
368
leishmaniasis cases [36]. Clinical phase III essays were finished, in India, in 2010, involving
369
three combination of drugs (miltefosine and paromomycin, miltefosine and liposomal
370
amphotericin B, liposomal amphotericin B and paromomycin) and all of them disclosed
371
efficacy higher than 95% in the treatment of the visceral form [21]. Combination drug therapy
372
has the advantages to reduce doses and the length of the treatment and, therefore, improving
373
the tolerance and adherence of the patients. Also, it is possible that drug association may
374
prevent and/or delay the appearance of resistance [37].
375 376
Taking into account that biochemical analysis of the blood, along with other factors, may
377
issue important information to clear the clinical picture and pathologic alterations and,
378
therefore, broaden experimental results, we analyzed, during all the study, the weight of the
379
animals and some biochemical and metabolic parameters related to possible liver lesions
380
and/or renal impairment. No significant alterations were seen in serum concentrations of
381
glucose, urea and creatinine in relation to normal reference levels found in the literature [24-
382
26], but high levels of AST, ALT and ALP were seen during all the research. Taking into
383
account that animals were not germfree, that the reports of biochemical reference values in
384
hamsters are extremely scarce and often discrepant, due to the different enzymatic methods
385
employed, and present variations related to sex, age, lineage, genotype, diet, handling, the
386
environment, blood collection via and other interfering factors [25,38], we performed
387
comparisons between the different treatment groups and the CONT group and did not find
388
significant increase that could possibly be related to experimental treatment.
389
18Page 18 of 28
390
Finally, it is important to point out that many studies have already described important side
391
effects of the treatment with amiodarone, suggesting caution in its employment [39].
392
However, structural changes in its molecule have enabled the development of new drugs, such
393
as dronedarone, that seem not to present the same serious side effects, providing safer
394
therapeutic options [40].
395 396
19Page 19 of 28
397
5. Conclusions
398
The results indicate that, in hamsters, amiodarone and itraconazole, either used isolated or in
399
combination, are unable to hinder the development of cutaneous lesions caused by L. (L.)
400
amazonensis. However, the results suggest that amiodarone or itraconazole improve the
401
activity of glucantime in the treatment of these lesions and seems not to present evident side
402
effects. More studies are necessary in order to investigate the clinical potential of these
403
combinations, in order to widen therapeutic options, especially in resistant cases.
404
Furthermore, investigation of the use of such drugs in the treatment of leishmaniasis caused
405
by other species of Leishmania would be opportune.
406 407
Declarations
408
Funding: This work was supported by the Coordenação de Aperfeiçoamento de Pessoal de
409
Nível Superior (Capes) (32/2010).
410
Competing Interests: None declared.
411
Ethical Approval: This study (Permit Number: 181/2011) was approved by the Ethics
412
Committee for Animal Experimentation of the Universidade Federal do Triângulo Mineiro
413
(Brazil).
414 415
20Page 20 of 28
416
References
417
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Worldwide and Global Estimates of Its Incidence. PLoS ONE 2012; 7: e35671.
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2. Lainson R, Shaw JJ. New World Leishmaniasis - the neotropical Leishmania species. In:
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Cox FEG, Kreier JP, Wakelin D (org.). Topley and Wilson's Microbiology and Microbial
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Infections. London: Arnold, 2005; 313-49.
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3. Rangel EF, Lainson R. Proven and putative vectores of American cutaneous leishmaniasis
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in Brazil: aspects of their biology and vectorial competence. Mem Inst Oswaldo Cruz
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4. Convit J, Ulrich M, Fernandez CT, Tapia FJ, Cáceres-Ditmar G, Cástes M, et al. The
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clinical and immunological spectrum of American cutaneous leishmaniasis. Trans Roy
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5. Piscopo TV, Azzopardi CM. Leishmaniasis. Postgrad Med J 2006; 82: 649-57.
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6. Croft SL, Yardley V. Chemotherapy of leishmaniasis. Curr Pharm Des 2002; 8: 319-42.
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520 521
Figure Legends
522
Fig. 1. Evolution of the average size of cutaneous lesions (in millimeters) in hamsters
523
experimentally infected with L. (L.) amazonensis and treated with different drugs.
524
Fig. 2. Images of the cutaneous lesions of hamsters experimentally infected with L. (L.)
525
amazonensis and treated with different drugs. (A) After the end of TRAT 1 (40 days). (B)
526
After the end of TRAT 2 (70 days). (C) At the end of the study (90 days).
527
Fig. 3. Polyacrylamide gel representative of PCR with initiators JW11 and JW12 (amplifying
528
fragments of 120 bp, corresponding to the Leishmania genus) performed at the tissue cuts of
529
hamsters experimentally infected with L. (L.) amazonensis and treated with different drugs.
530
Lane 1, molecular weight marker; lane 2, PCR mixture without DNA (negative control); lane
531
3, L. (L.) amazonensis genomic DNA (positive control); lanes 4 to 11, DNA from tissue cuts
532
of inoculated foot of hamsters treated with GLUC+AMIO, lane 12, DNA from tissue cuts of
533
inoculated foot of hamster untreated. The absence of a 120 bp band indicates the absence of
534
Leishmania in the lesion.
535
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536
Table 1. Size of the cutaneous lesions (in millimeters) of hamsters experimentally infected with L. (L.) amazonensis and treated with different drugs. Size of the lesion (mm)1 20 days
30 days
40 days
50 days
60 days
70 days
80 days
90 days
Value-p (TRAT 1)2
Value-p (TRAT 2)2
Value-p (Final)2
GLUC
1.81±0.50
2.52±0.59 b
2.71±0.79 bc
3.66±1.24 bcd
4.32±1.73 bc
5.31±2.50 bcd
5.41±1.45 abc
5.87±1.06 bc
< 0.001
0.007
0.002
AMIO
1.85±0.39
3.15±0.39 a
3.47±0.76 ab
5.05±1.58 ab
6.77±1.69 ab
8.78±1.77 a
9.12±1.70 a
7.84±0.94 a
< 0.001
0.001
< 0.001
ITRA
1.97±0.44
2.86±0.62 ab
3.25±0.80 b
4.32±1.64 abc
5.24±2.01 abc
5.96±2.51 bc
4.95±2.09 abc
6.34±2.40 abc
< 0.001
0.008
0.014
GLUC+AMIO
1.91±0.40
2.46±0.75 b
1.55±0.73 d
2.11±0.85 d
2.78±1.95 c
3.18±1.66 d
3.42±1.56 c
3.97±1.91 c
0.138
0.086
0.054
GLUC+ITRA
2.18±0.45
2.46±0.66 b
2.08±0.72 cd
2.64±1.36 cd
3.38±1.21 c
4.19±2.07 cd
3.86±3.12 bc
4.02±2.94 c
0.534
0.002
0.253
AMIO+ITRA
1.86±0.35
2.62±0.47 b
2.91±0.82 b
4.25±1.98 abc
4.87±2.75 abc
5.75±2.80 bc
4.47±0.84 bc
6.03±1.03 abc
< 0.001
0.011
0.001
CONT
1.90±0.35
3.41±0.88 a
4.59±1.55 a
6.14±1.29 a
7.27±1.78 a
7.53±2.24 ab
7.77±2.32 ab
7.81±1.67 ab
< 0.001
0.037
0.001
Value-p (Groups ≠)3
0.270
0.001
< 0.001
< 0.001
< 0.001
< 0.001
0.001
0.023
Groups
537 538 539 540 541 542 543 544
1
Values of the lesion size are expressed in mean + standard deviation. Value-p (TRAT 1) represents the result of the comparison between the start (20 days) and the end (40 days) of TRAT 1. Value-p (TRAT 2) represents the result of the comparison between the start (50 days) and the end (70 days) of TRAT 2. Value-p (Final) refers to the result of the comparison between the start (20 days) and the end (90 days) of all the study. These comparisons were performed for each one of the experimental groups employing paired t test or Wilcoxon test. 3 Value-p (Groups ≠) represents the result of the ANOVA test or Kruskal-Wallis in the comparison between the different experimental groups, for each time interval alloted for the study. In variables with p<0.05, means followed by different letters (a, b, c or d) are significantly different from each other according to Bonferroni’s or Dunn’s multiple comparison tests. 2
26
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545 546
Table 2. Results of the parasitic investigation by histopathological analysis of tissue cuts of inoculated foot of hamsters experimentally infected with L. (L.) amazonensis and treated with different drugs. Number of negative animals/Total Euthanasia 1
Euthanasia 2
Euthanasia 3
General
Value-p (Euthanasia)1
GLUC
3/4 (75.0%)
1/5 (20.0%)
0/5 (0.0%)
4/14 (28.6%)
0.041
AMIO
2/4 (50.0%)
0/4 (0.0%)
0/5 (0.0%)
2/13 (15.4%)
0.070
ITRA
1/4 (25.0%)
0/5 (0.0%)
0/5 (0.0%)
1/14 (7.1%)
0.260
GLUC+AMIO
4/4 (100.0%)
0/4 (0.0%)
0/5 (0.0%)
4/13 (30.8%)
0.002
GLUC+ITRA
2/3 (66.7%)
0/5 (0.0%)
1/5 (20.0%)
3/13 (23.1%)
0.094
AMIO+ITRA
1/4 (25.0%)
0/4 (0.0%)
1/5 (20.0%)
2/13 (15.4%)
0.579
CONT
0/5 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/15 (0.0%)
-
0.057
0.473
0.506
0.273
Groups
Value-p (Groups ≠)
547 548 549 550 551 552 553
2
1
Value-p (Euthanasia) represents the result of chi-square test (χ2) in the comparison between the different moments in which animals were euthanized (Euthanasia 1: right after the end of TRAT 1; Euthanasia 2: right after the end of TRAT 2; Euthanasia 3: 20 days after the end of TRAT 2). These comparisons were performed for each one of the experimental groups. 2 Value-p (Groups ≠) represents the result of the chi-square test (χ2) in the comparison among the different experimental groups, for each one of the moments in which animals were euthanized and in general.
554
27
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555 556
Table 3. Results of the parasitic investigation by molecular analysis (PCR) of tissue cuts of inoculated foot, liver and spleen of hamsters experimentally infected with L. (L.) amazonensis and treated with different drugs. Number of negative animals/Total Euthanasia 3
General
Value-p (Euthanasia)1
Groups Euthanasia 1
Euthanasia 2 Inoculated foot
GLUC
1/4 (25.0%)
0/5 (0.0%)
0/5 (0.0%)
1/14 (7.1%)
0.260
AMIO
0/4 (0.0%)
0/4 (0.0%)
0/5 (0.0%)
0/13 (0.0%)
-
ITRA
0/4 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/14 (0.0%)
-
GLUC+AMIO
2/4 (50.0%)
0/4 (0.0%)
0/5 (0.0%)
2/13 (15.4%)
0.070
GLUC+ITRA
1/3 (33.3%)
0/5 (0.0%)
0/5 (0.0%)
1/13 (7.7%)
0.164
AMIO+ITRA
0/4 (0.0%)
0/4 (0.0%)
0/5 (0.0%)
0/13 (0.0%)
-
0/5 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/15 (0.0%)
-
0.219
-
-
0.309
CONT Value-p (Groups ≠)
2
Liver GLUC
1/4 (25.0%)
0/5 (0.0%)
0/5 (0.0%)
1/14 (7.1%) ab
0.260
AMIO
1/4 (25.0%)
0/4 (0.0%)
0/5 (0.0%)
1/13 (7.7%) ab
0.260
ITRA
0/4 (0.0%)
1/5 (20.0%)
0/5 (0.0%)
1/14 (7.1%) ab
0.379
GLUC+AMIO
3/4 (75.0%)
1/4 (25.0%)
1/5 (20.0%)
5/13 (38.5%) b
0.194
GLUC+ITRA
1/3 (33.3%)
0/5 (0.0%)
1/5 (20.0%)
2/13 (15.4%) ab
0.420
AMIO+ITRA
0/4 (0.0%)
0/4 (0.0%)
0/5 (0.0%)
0/13 (0.0%) a
-
0/5 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/15 (0.0%) a
-
0.099
0.476
0.506
0.022
CONT Value-p (Groups ≠)
2
Spleen
557 558 559 560 561 562 563 564
GLUC
1/4 (25.0%)
0/5 (0.0%)
0/5 (0.0%)
1/14 (7.1%)
0.260
AMIO
1/4 (25.0%)
0/4 (0.0%)
0/5 (0.0%)
1/13 (7.7%)
0.260
ITRA
0/4 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/14 (0.0%)
-
GLUC+AMIO
3/4 (75.0%)
0/4 (0.0%)
0/5 (0.0%)
3/13 (23.1%)
0.012
GLUC+ITRA
1/3 (33.3%)
0/5 (0.0%)
1/5 (20.0%)
2/13 (15.4%)
0.420
AMIO+ITRA
0/4 (0.0%)
0/4 (0.0%)
0/5 (0.0%)
0/13 (0.0%)
-
CONT
0/5 (0.0%)
0/5 (0.0%)
0/5 (0.0%)
0/15 (0.0%)
-
Value-p (Groups ≠)2
0.099
-
0.404
0.159
1
Value-p (Euthanasia) represents the result of chi-square test (χ2) in the comparison among the different moments of euthanasia (Euthanasia 1: right after the end of TRAT 1; Euthanasia 2: right after the end of TRAT 2; Euthanasia 3: 20 days after the end of TRAT 2). These comparisons were performed for each one of the experimental groups. 2 Value-p (Groups ≠) represents the result of chi-square test (χ2) in the comparison among the different experimental groups for each of the moments of euthanasia and in general. In variables with p<0.05, values followed by different letters (a or b) are significantly different from each other.
28
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