• View in gallery

    Activity of azithromycin (AZ) against promastigotes of L. (L.) amazonensis (A), L. (V.) braziliensis (B), and L. (L.) chagasi (C) evaluated by the proportional reduction of Alamar blue compared with the drug-free control culture. Amphotericin B (AMB) was used as the reference control drug. Values represent the mean ± SD of three repeated experiments, each one in triplicate. *P ≤ 0.05.

  • View in gallery

    Activity of azithromycin (AZ) against intracellular amastigotes of L. (L.) amazonensis (A), L. (V.) braziliensis (B), and L. (L.) chagasi (C) evaluated by the ration between infection proportions of treated and untreated macrophage cultures: [Macrophage infection ratio = (percentage of infected Mø in the treated culture/ percentage of infected Mø in the drug-free cultures) × 100]. Amphotericin B (AMB) was used as the reference control drug. Values represent the mean ± SD of three repeated experiments, each one in triplicate. *P ≤ 0.05.

  • 1

    Berman JD, 2003. Current treatment approaches to leishmaniasis. Curr Opin Infect Dis 16 :397–401.

  • 2

    Croft SL, Seifert K, Yardley V, 2006. Current scenario of drug development for leishmaniasis. Indian J Med Res 123 :399–410.

  • 3

    Akuffo H, Dietz M, Teklemariam S, Tadesse T, Amare G, Berhan TY, 1990. The use of itraconazole in the treatment of leishmaniasis caused by Leishmania aethiopica.Trans R Soc Trop Med Hyg 84 :532–534.

    • Search Google Scholar
    • Export Citation
  • 4

    Weinrauch L, Livshin R, el-On J, 1987. Ketoconazole in cutaneous leishmaniasis. Br J Dermatol 117 :666–668.

  • 5

    Alrajhi AA, Ibrahim EA, De Vol EB, Khairat M, Faris RM, Maguire JH, 2002. Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major.N Engl J Med 346 :891–895.

    • Search Google Scholar
    • Export Citation
  • 6

    Periti P, Mazzei T, Mini E, Novelli A, 1993. Adverse effects of macrolide antibacterials. Drug Saf 9 :346–364.

  • 7

    Gladue RP, Bright GM, Isaacson RE, Newborg MF, 1989. In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection. Antimicrob Agents Chemother 33 :277–282.

    • Search Google Scholar
    • Export Citation
  • 8

    Foulds G, Madsen P, Cox C, Shepard R, Johnson R, 1991. Concentration of azithromycin in human prostatic tissue. Eur J Clin Microbiol Infect Dis 10 :868–871.

    • Search Google Scholar
    • Export Citation
  • 9

    Scheinfeld NS, Tutrone WD, Torres O, Weinberg JM, 2004. Macrolides in dermatology. Dis Mon 50 :350–368.

  • 10

    Zuckerman JM, 2000. The newer macrolides: azithromycin and clarithromycin. Infect Dis Clin North Am 14 :449–462.

  • 11

    Dunne MW, Singh N, Shukla M, Valecha N, Bhattacharyya PC, Dev V, Patel K, Mohapatra MK, Lakhani J, Benner R, Lele C, Patki K, 2005. A multicenter study of azithromycin, alone and in combination with chloroquine, for the treatment of acute uncomplicated Plasmodium falciparum malaria in India. J Infect Dis 191 :1582–1588.

    • Search Google Scholar
    • Export Citation
  • 12

    Nakornchai S, Konthiang P, 2006. Activity of azithromycin or erythromycin in combination with antimalarial drugs against multidrug-resistant Plasmodium falciparum in vitro.Acta Trop 100 :185–191.

    • Search Google Scholar
    • Export Citation
  • 13

    Miller RS, Wongsrichanalai C, Buathong N, McDaniel P, Walsh DS, Knirsch C, Ohrt C, 2006. Effective treatment of uncomplicated Plasmodium falciparum malaria with azithromycin-quinine combinations: a randomized, dose-ranging study. Am J Trop Med Hyg 74 :401–406.

    • Search Google Scholar
    • Export Citation
  • 14

    Krolewiecki A, Leon S, Scott P, Abraham D, 2002. Activity of azithromycin against Leishmania major in vitro and in vivo.Am J Trop Med Hyg 67 :273–277.

    • Search Google Scholar
    • Export Citation
  • 15

    Tanyuksel M, Bas AL, Araz E, Aybay C, 2003. Determination of intracellular efficacies of azithromycin against Leishmania major infection in human neutrophils in vitro.Cell Biochem Funct 21 :93–96.

    • Search Google Scholar
    • Export Citation
  • 16

    Prata A, Silva-Vergara ML, Costa L, Rocha A, Krolewiecki A, Silva JC, Paula DE, Pimenta EV, Junior FG, Giraldo LE, 2003. Efficacy of azithromycin in the treatment of cutaneous leishmaniasis. Rev Soc Bras Med Trop 36 :65–69.

    • Search Google Scholar
    • Export Citation
  • 17

    Silva-Vergara ML, Silva Ld EA, Maneira FR, da Silva AG, Prata A, 2004. Azithromycin in the treatment of mucosal leishmaniasis. Rev Inst Med Trop Sao Paulo 46 :175–177.

    • Search Google Scholar
    • Export Citation
  • 18

    Krolewiecki AJ, Romero HD, Cajal SP, Abraham D, Mimori T, Matsumoto T, Juarez M, Taranto NJ, 2007. A randomized clinical trial comparing oral azithromycin and meglumine antimoniate for the treatment of American cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis.Am J Trop Med Hyg 77 :640–646.

    • Search Google Scholar
    • Export Citation
  • 19

    Layegh P, Yazdanpanah MJ, Vosugh EM, Pezeshkpoor F, Shakeri MT, Moghiman T, 2007. Efficacy of azithromycin versus systemic meglumine antimoniate (Glucantime) in the treatment of cutaneous leishmaniasis. Am J Trop Med Hyg 77 :99–101.

    • Search Google Scholar
    • Export Citation
  • 20

    Mikus J, Steverding D, 2000. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol Int 48 :265–269.

    • Search Google Scholar
    • Export Citation
  • 21

    Huber W, Koella JC, 1993. A comparison of three methods of estimating EC50 in studies of drug resistance of malaria parasites. Acta Trop 55 :257–261.

    • Search Google Scholar
    • Export Citation
  • 22

    Croft SL, Yardley V, Kendrick H, 2002. Drug sensitivity of Leishmania species: some unresolved problems. Trans R Soc Trop Med Hyg Apr 96 (Suppl1):S127–S129.

    • Search Google Scholar
    • Export Citation
  • 23

    McMahon-Pratt D, Alexander J, 2004. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunol Rev 201 :206–224.

    • Search Google Scholar
    • Export Citation
  • 24

    Cantin L, Chamberland S, 1993. In vitro evaluation of the activities of azithromycin alone and combined with pyrimethamine against Toxoplasma gondii.Antimicrob Agents Chemother 37 :1993–1996.

    • Search Google Scholar
    • Export Citation
  • 25

    Gingras BA, Jensen JB, 1992. Activity of azithromycin (CP-62,993) and erythromycin against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro.Am J Trop Med Hyg 47 :378–382.

    • Search Google Scholar
    • Export Citation
  • 26

    Biswas S, 2001. In-vitro antimalarial activity of azithromycin against chloroquine sensitive and chloroquine resistant Plasmodium falciparum.J Postgrad Med 47 :240–243.

    • Search Google Scholar
    • Export Citation
  • 27

    Orth C, Wllingmyre GD, Lee P, Knirsh C, Milhous W, 2002. Assessment of azithromycin in combination with other anti-malarial drugs Plasmodium falciparum in vitro.Antimicrob Agents Chemother 46 :2518–2524.

    • Search Google Scholar
    • Export Citation
  • 28

    Blais J, Garneau V, Chamberland S, 1993. Inhibition of Toxo-plasma gondii protein synthesis by azithromycin. Antimicrob Agents Chemother 37 :1701–1703.

    • Search Google Scholar
    • Export Citation
  • 29

    Foulds G, Shepard RM, Johnson RB, 1990. The pharmacokinetics of azithromycin in human prostatic tissue. J Antimicrob Chemother 25 (Suppl A):73–82.

    • Search Google Scholar
    • Export Citation

 

 

 

 

Antileishmanial Activity of Azitahromycin Against Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi

View More View Less
  • 1 Laboratory of Clinical Research, Instituto René Rachou, Fundação Oswaldo Cruz-Fiocruz, CEP 30.190-002 Belo Horizonte, Minas Gerais, Brazil

Azithromycin, an azalide antibiotic, is highly concentrated within different phagocytic cells, especially macrophages. The potential antileishmanial activity of azithromycin against three species of Leishmania from the New World was assessed using in vitro models. Azithromycin decreased viability of promastigote cultures of Leishmania (Leishmania) amazonensis, Leishmania (Viannia) braziliensis, and Leishmania (Leishmania) chagasi as determined by the colorimetric Alamar blue assay. In amastigote intracellular cultures, a significant decrease in infected macrophages counts was observed for all three species with IC50 of 20.83 (27 μ mol/L), 2.18 (2.7 μmol/L), and 6.12 (7.8 μmol/L) μg/mL, respectively. Azithromycin showed in vitro activity against L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi and may offer an alternative to current leishmaniasis treatment.

INTRODUCTION

Tegumentary and visceral leishmaniasis are listed among World Health Organization tropical disease priorities because of their significant impact on global public health. Cutaneous leishmaniasis in the Americas are mainly caused by Leishmania (Viannia) braziliensis, Leishmania (Leishmania) amazonensis, and Leishmania (Viannia) guyanensis and produces skin ulcers on exposed parts of the body such as the face, arms, and legs. The lesions vary from mild to severe and may cause serious disability, leaving the patient permanently scarred. Additionally, cutaneous disease caused by the Viannia subgenus may progress to mucosal involvement and infection caused by L. (L.) amazonensis, may result in diffuse cutaneous leishmaniasis, a severe non-responsive form. Visceral leishmaniasis is a severe disease caused by parasites of the Leishmania donovani complex, characterized by long-term fever, hepatosplenomegaly, anemia, leucopenia, and severe weight loss, which is fatal if left untreated. The causative agent of the visceral form in the New World is L. (L.) chagasi.

The classic treatment of different clinical forms of leishmaniasis is the pentavalent antimony in the form of either sti-bogluconate sodium (Pentostam) or meglumine antimoniate (Glucantime; Aventis Pharma Ltda, Suzano, SP-Brazil).1 Pentavalent antimonials remain the first-line drugs against leishmaniasis worldwide, notwithstanding many critical disadvantages concerning parenteral administration, long-term therapy, and potentially severe side effects associated with dosage and toxicity.

In the last decade, new therapeutic options have been introduced for the treatment of visceral and cutaneous leishmaniasis such as aminosidine (paromomycin) for both topical and parenteral administration, lipid-associated amphotericin B, oral miltefosine and sitamaquine.2 Several other compounds have undergone limited evaluation, namely the azoles, itraconazole, ketaconazole, and fluconazole.35 All an-tileishmanial therapeutic formulations impose restrictions because of their adverse side effects, availability, and considerable cost.

Another homogeneous group of antimicrobial drugs include the macrolides, characterized by similar chemical structure, antibacterial spectrum, mechanism of action, and resistance properties, but different pharmacokinetic profiles.6 Among the macrolides, azithromycin presents favorable pharmacokinetic features such as wide tissue distribution and high concentration within cells, including macrophages. Gladue and others7 have shown that azithromycin achieves higher concentrations intracellularly, as high as > 200 times, than those reached extracellularly. Such pharmacokinetic profiles result in high antibiotic concentrations in tissues and body fluids compared with plasma. The concentration of azithromycin exceeds that in the serum 10–100 times, whereas serum/tissue concentration ratios are estimated to reach between 0.5 and 5 in humans.8 Azithromycin has shown to be effective against a wide range of pathogens causing respiratory infections and sexually transmitted diseases,9 as well as enteropathogens.10 Azithromycin has also shown activity against Plasmodium falciparum, isolated or in combination with antimalarial drugs.1113

Two studies have assessed in vitro azithromycin activity against parasites of the genus Leishmania. Krolewiecki and others14 showed both in vitro and in vivo activity against azithromycin in L. (L.) major promastigote and amastigote forms. Tanyuksel and others15 studied the potential leishmanicidal effect in vitro of azithromycin on intracellular amastigote forms of L. (L.) major in peritoneal macrophages from mice. Controversial results have been observed with the clinical use of azithromycin for leishmaniasis treatment. Prata and others,16 in an open non-controlled trial, assessed 20 patients with cutaneous leishmaniasis treated with azithromycin, showing an 85% cure efficacy, and Silva-Vergara and others17 reported the successful treatment of three elderly patients with tegumentary/mucous leishmaniasis through oral administration of azithromycin. However, Krolewiecki and others18 observed that complete re-epithelization without relapse for 12 months after completing therapy was 82.6% for meglumine antimoniate and 45.5% for azithromycin for the treatment of American cutaneous leishmaniasis caused by L. (V.) braziliensis. In another study, azithromycin was determined to be not as effective as glucantime in treatment of Old World cutaneous leishmaniasis when used for 5 days monthly to a maximum of 4 months.19

This study was aimed at determining azithromycin activity against promastigotes and intracellular amastigotes of L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi, three species that are highly prevalent in the Americas.

MATERIALS AND METHODS

Antibiotics.

Azithromycin dihydrate (Zitromax IV; Pfizer, Bedford, OH) was obtained in lyophilized form at 500 mg/vial for intravenous administration. Amphotericin B (Fungizone; Bristol-Myers Squibb, Bedford, OH) was obtained as a sterile powder. The drugs were reconstituted with sterile water and diluted to the appropriate concentration in Schneider Sigma S9895 medium for promastigote cultures or RPMI Sigma R4130 for amastigote-macrophage cultures immediately before assays were performed.

Parasites.

Three species of Leishmania were tested in this study: L. (L.) amazonensis (IFLA/BR/1967/PH8), L. (V.) braziliensis (MHOM/BR/75/M2903), and L. (L.) chagasi (MHOM/BR/70/BH46). Promastigotes forms of L. (V.) braziliensis and L. (L.) chagasi were cultured in Schneider medium supplemented with 10% heat-inactivated fetal bovine serum (FBS; Gibco, Såo Paulo, Brazil) and the antibiotics penicillin (100 U/mL) and streptomycin (100 μg/mL) at 26°C. L. (L.) amazonensis parasites were cultured in biphasic medium Novy, McNeal, and Nicolle (NNN)/liver infusion tryptose (LIT) supplemented with antibiotics, and 10% FBS at 26 °C.

Promastigote assay.

Leishmania (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi amastigotes were isolated from golden hamsters (Mesocricetus auratus) and cultured to log phase at 26°C in Schneider or NNN/LIT supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. Parasites were centrifuged at 1,000g for 10 minutes, and the pellet was re-suspended in Schneider medium with 10% glycerol. Aliquots were kept frozen in liquid nitrogen until use. Cryopreserved promastigotes were thawed at 26°C and washed with Schneider medium or NNN/LIT supplemented with 10% FBS, 100 U/mL penicillin, and 100 μg/mL streptomycin. The pellet was re-suspended in Schneider medium or NNN/LIT with the supplements listed above and incubated at 26°C. To estimate the 50% inhibitory concentration (IC50), the Alamar blue micromethod was used according to Mikus and Steverding20 with modification. Briefly, parasites were grown in axenic cell-free culture in the appropriate medium at 26°C. Promastigotes were diluted in medium to 1 × 106 parasites/mL, and 180 μL of suspension was incubated with varying concentrations of azithromycin, 50 (64 μmol/L), 100 (130 μmol/L), 200 (260 μmol/L), 300 (380 μmol/ L), 500 (640 μmol/L), 750 (957 μmol/L), 1,000 (1.3 mmol/L), 1,250 (1.6 mmol/L), and 1,500 (1.9 mmol/L) μg/mL, and amphotericin B, 0.2 μg/mL (0.2 μmol/L), seeded in triplicate in 96-well flat-bottom microtrays for 72 hours at 26°C. After 64 hours of incubation, 20 μL of Alamar blue was added to each well, and plates were further incubated for 8 hours. The absorbance at 570 and 600 nm was measured simultaneously. The optical density at 570 and 600 nm was read with a 96-well scanner. Three independent experiments in triplicate were performed for determination of the IC50 of each drug. The optical density in the absence of drugs was set as the 100% control.

Amastigote-macrophage assay.

Balb/c mice were injected intraperitoneally with 1.5 mL of 3% thioglycolate medium (Biobrás, Montes Claros, Brazil). After 96 hours, the peritoneal macrophages were harvested by peritoneal lavage using cold RPMI-1640 medium. Cells were counted, centrifuged, and re-suspended at a concentration of 4 × 105/mL in RPMI-1640 medium without supplements. Sterile round glass coverslips (13 mm) were placed in each well of 24-well culture plates. Macrophages were pipetted at a volume of 500 μL/ well and allowed to attach for 2 hours at 37°C in 5% CO2. After 2 hours, the medium was removed from the wells and replaced with 500 μL of warm (37°C) RPMI medium plus 10% FBS and penicillin (50 U/mL) and streptomycin (50 μg/ mL). The following day, a suspension of 4 × 106 amastigotes-like in RPMI was added in a 500-μL volume to each well (for a macrophage/parasite ratio of 1:10). The plates were incubated for 4 hours at 37°C in 5% CO 2, and the medium was aspirated to remove free parasites. Fresh RPMI (1 mL) with or without drugs (azithromycin and amphotericin B) at the appropriate concentration was added in triplicate wells. Plates were incubated for 72 hours at 37°C in 5% CO2. The medium was aspirated, and the coverslips were removed, methanol fixed, air-dried, and glued to microscope slides. After staining with Giemsa, 100 cells on the glass disks were counted along the borderline. Three independent experiments in triplicate for each concentration were performed for determination of azithromycin efficacy. Results are presented as the ratio between infection proportions of treated and untreated macrophage cultures.

Analysis.

IC50s values were calculated by linear regression analysis (MINITAB V. 13.1, PA) or linear interpolation.21 Results are expressed as the mean ± SD, and statistical significance was determined by ANOVA or Kruskal-Wallis; P < 0.05 was considered significant.

RESULTS

Activity of azithromycin on promastigotes in vitro.

Inhibition of promastigotes cultured with azithromycin at 50, 100, 200, 300, 500, 750, 1,000, 1,250, and 1,500 μg/mL for 72 hours was determined by reduction of the dye Alamar blue. Azithromycin was significantly active against L. (L.) amazonensis at concentrations of 1,000 (1.3 mmol/L), 1,250 (1.6 mmol/L), and 1,500 μg/mL (1.9 mmol/L), with values of P < 0.05, P < 0.001, and P < 0.001, respectively, compared with the control without addition of drugs (Figure 1A). The value of IC50 was 789.3 μ g/mL (1.0 mmol/L) as calculated by linear regression (r2 = 0.96). The activity of azithromycin against promastigotes of L. (V.) braziliensis was observed with 750 (957 μmol/L; P < 0.05), 1,000 (1.3 mmol/L; P < 0.01), 1,250 (1.6 mmol/L; P < 0.001), and 1,500 μg/mL (1.9 mmol/L; P < 0.001) compared with control cultures (Figure 1B). The value of IC50 was 458.7 μg/mL (590 μmol/L) as obtained by linear interpolation. In cell-free cultures of promastigotes of L. (L.) chagasi, azithromycin significantly reduced parasite counts after 72 hours at concentrations of 1,000 (1.3 mmol/L; P < 0.01), 1,250 (1.6 mmol/L; P < 0.01), and 1,500 μg/mL (1.9 mmol/L; P < 0.001; Figure 1C). The IC50 value was 1,201 μg/mL (1.5 mmol/L), as determined by linear regression (r2 = 0.79).

Effect of azithromycin on amastigote-macrophage.

Peritoneal macrophages infected with amastigotes of L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi were treated with azithromycin at different concentrations. In the amastigote intracellular assay of L. (L.) amazonensis, the concentrations evaluated were 0.9 (1.1 μmol/L), 1.9 (2.4 μmol/L), 3.8 (4.8 μmol/L), 7.5 (9.5 μmol/L), 15 (19 μmol/L), 30 (38 μmol/ L), and 60 (77 μmol/L) μg/mL. Activity of azithromycin was significant at concentration of 60 μg/mL (P < 0.05; Figure 2A) compared with control (without drugs). An IC50 of 20.83 μg/mL (27 μmol/L) was obtained by linear regression (r2 = 55.4). The activity of azithromycin against intracellular amastigotes of L. (V.) braziliensis was determined at concentrations of 0.1 (0.1 μmol/L), 0.2 (0.2 μmol/L), 1.0 (1.2 μmol/L), 2.5 (3.1 μmol/L), 5.0 (6.3 μmol/L), 10 (13 μmol/L), 25 (32 μmol/L), 50 (64 μmol/L), 75 (96 μmol/L), and 100 (130 μmol/ L) μg/mL. At 25 (P < 0.05), 50 (P < 0.001), 75 (P < 0.01), and 100 μg/mL (P < 0.01) concentrations, significant inhibition was observed compared with the control (Figure 2B). The IC50 value of 2.18 μg/mL (2.7 μmol/L) was determined by linear interpolation. To assess azithromycin activity against intracellular amastigotes of L. (L.) chagasi, concentrations of 2.5 (3.1 μmol/L), 5.0 (6.3 μmol/L), 10 (13 μmol/L), 25 (32 μmol/L), 50 (64 μmol/L), 75 (96 μmol/L), 100 (130 μmol/L), 150 (191 μmol/L), and 175 (223 μmol/L) μg/mL were tested (Figure 2C). At concentrations of 100 and 150 μg/mL, significant inhibition of the number of parasites was observed and an IC50 of 6.12 μg/mL (7.8 μmol/L) was estimated by linear regression (r2 = 62.7).

Table 1 summarizes and presents the comparative inhibitory concentration of azithromycin activity against promastigotes and intracellular amastigotes of three Leishmania spp. evaluated in this study.

DISCUSSION

There is an increasing need for alternative drugs for leishmaniasis treatment that provide effectiveness, safety, low cost, and easy administration. Only two studies have evaluated azithromycin activity in vitro and or in vivo, but both have restricted their experiments to L. (L.) major. As large variation in species-specific drug sensitivity is observed for anti-Leishmania compounds,22 determination of activity against different species is needed to guide the drug development steps and support clinical trial decisions. As appointed by McMahon-Pratt and Alexander,23 the Old World species diverged from the New World species some 40–80 million years ago, and their substantial differences in host–parasite interactions suggest that appropriate general therapeutic intervention strategies may have to be carefully considered.

In this work, we observed the direct action of azithromycin on promastigote forms of the three New World Leishmania species under study and its activity on amastigotes infecting mouse peritoneal macrophages. Krolewiecki and others14 have reported a significant decrease in promastigote forms using azithromycin concentrations of 100 and 1,000 μg/mL. In this study, promastigote growth was reduced at 1,000 μg/mL (1.3 mmol/L) for L. (L.) amazonensis, 750 μg/mL (957 μmol/ L) for L. (V.) braziliensis, and 1,000 μg/mL (1.3 mmol/L) for L. (L.) chagasi. It is not possible to assure that azithromycin is more effective against L. (L.) major than the other species under study caused by protocol differences regarding culture medium and time of drug exposure. These authors have determined azithromycin activity after 6 days of exposure, whereas in this study, parasites were exposed to the drug for 3 days.

The mechanism by which azithromycin reduces the number of parasites remains unclear. There is evidence to suggest that the drug exerts a direct effect on protozoans such as T. gondii24 and Plasmodium2527 by inhibiting protein synthesis.28 Results provided here suggest that azithromycin produces a direct effect on promastigote forms as also shown by Krolewiecki and others.14 Lower azithromycin concentrations were needed to reach an inhibitory effect on intracellular amastigote forms of L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi compared with those necessary to inhibit the growth curve of promastigote cultures. This might be caused by azithromycin ability to reach high tissue concentrations, which may be > 10–100 times compared with serum concentrations. Approximately 37% of a single oral dose of 500 mg is bioavailable and produced a peak serum concentration of 0.4 μg/mL.29

Concerning the leishmanicidal activity, a study by Tanyuksel and others15 suggests an independent phagocytic increasing capacity effect. Krolewiecki and others14 reported that the concentrations of azithromycin able to reduce the amastigote number of L. (L.) major in macrophages (12 μg/mL) were 6.7 times lower than those necessary for inhibition of promastigote growth. In our study, this ratio was 16.6 for L. (L.) amazonensis, being the azithromycin activity against promastigotes observed at the concentration of 1,000 μg/mL (1.3 mmol/L), whereas the concentration of 60 μg/mL (77 μmol/ L) was activity against amastigote forms (Table 1). Concerning L. (V.) braziliensis, the ratio reaches 30 times, because the minimum inhibitory concentration was 750 μg/mL (957 μmol/ L) for promastigotes and 25 μg/mL (32 μmol/L) for intracellular amastigotes. This ratio was 6.6 times for L. (L.) chagasi, because the minimum inhibitory concentration was 1,000 μg/ mL (1.3 mmol/L) for promastigotes and 150 μg/mL (191 μmol/L) for intracellular amastigotes. It is worthy to highlight that the differential response to azithromycin among the three species suggests that some of these New World species are likely to respond better to a standard dose of azithromycin than others, which may have implications for the dose selection in animal and clinical studies.

Our trials have shown a dose-dependent azithromycin effect for the three species studied, which is in accordance with the results provided for L. (L.) major.14 Amphotericin B was used as a control drug for each assay for internal validation caused by its high leishmanicidal activity against different species.

Azithromycin is within the main parameters of an effective medication, comprising a simple oral administration and good tolerance. The drug has been commercialized worldwide and several generic drugs are now available in Brazil. The possibility of an oral treatment is a highly desirable factor, mainly because of difficulties involved in attending health service by most people infected by Leishmania spp.

The results of this study showed the in vitro azithromycin activity against L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi using both models promastigotes and intracellular amastigotes. Azithromycin may be a valuable alternative treatment of the leishmaniasis caused by these species.

Table 1

Comparative IC50 of azithromycin against promastigotes and intra-cellular amastigotes of L. (L.) amazonensis, L. (V.) braziliensis, and L. (L.) chagasi

Values of IC50 (μg/mL)*
Leishmania speciesPromastigoteIntracellular amastigote
* Mean of the results of three separate experiments, each in triplicate.
L. (L.) amazonensis789.3 (1.0 mmol/L)20.83 (27 μmol/L)
L. (V.) braziliensis458.7 (590 μmol/L)2.18 (2.7 μmol/L)
L. (L.) chagasi1201 (1.5 mmol/L)6.12 (7.8 μmol/L)
Figure 1.
Figure 1.

Activity of azithromycin (AZ) against promastigotes of L. (L.) amazonensis (A), L. (V.) braziliensis (B), and L. (L.) chagasi (C) evaluated by the proportional reduction of Alamar blue compared with the drug-free control culture. Amphotericin B (AMB) was used as the reference control drug. Values represent the mean ± SD of three repeated experiments, each one in triplicate. *P ≤ 0.05.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 5; 10.4269/ajtmh.2008.78.745

Figure 2.
Figure 2.

Activity of azithromycin (AZ) against intracellular amastigotes of L. (L.) amazonensis (A), L. (V.) braziliensis (B), and L. (L.) chagasi (C) evaluated by the ration between infection proportions of treated and untreated macrophage cultures: [Macrophage infection ratio = (percentage of infected Mø in the treated culture/ percentage of infected Mø in the drug-free cultures) × 100]. Amphotericin B (AMB) was used as the reference control drug. Values represent the mean ± SD of three repeated experiments, each one in triplicate. *P ≤ 0.05.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 78, 5; 10.4269/ajtmh.2008.78.745

*

Address correspondence to Ana Rabello, Laboratory of Clinical Research, Instituto René Rachou, Fundação Oswaldo Cruz-Fiocruz, Avenida Augusto de Lima 1715, CEP 30.190-002 Belo Horizonte, Minas Gerais, Brazil. E-mail: ana@cpqrr.fiocruz.br

Authors’ addresses: Fernanda de Oliveira-Silva, Eliane de Morais-Teixeira, and Ana Rabello, Laboratory of Clinical Research, Instituto René Rachou, Fundação Oswaldo Cruz-Fiocruz, Avenida Au-gusto de Lima 1715, CEP 30.190-002 Belo Horizonte, Minas Gerais, Brazil, E-mail: ana@cpqrr.fiocruz.br.

Financial support: This work was supported by CNPq (Conselho Na-cional de Desenvolvimento Cientifico e Tecnológico), FAPEMIG (Fundação de Amparo de Pesquisa de Minas Gerais), and FIOCRUZ (Fundação Oswaldo Cruz).

REFERENCES

  • 1

    Berman JD, 2003. Current treatment approaches to leishmaniasis. Curr Opin Infect Dis 16 :397–401.

  • 2

    Croft SL, Seifert K, Yardley V, 2006. Current scenario of drug development for leishmaniasis. Indian J Med Res 123 :399–410.

  • 3

    Akuffo H, Dietz M, Teklemariam S, Tadesse T, Amare G, Berhan TY, 1990. The use of itraconazole in the treatment of leishmaniasis caused by Leishmania aethiopica.Trans R Soc Trop Med Hyg 84 :532–534.

    • Search Google Scholar
    • Export Citation
  • 4

    Weinrauch L, Livshin R, el-On J, 1987. Ketoconazole in cutaneous leishmaniasis. Br J Dermatol 117 :666–668.

  • 5

    Alrajhi AA, Ibrahim EA, De Vol EB, Khairat M, Faris RM, Maguire JH, 2002. Fluconazole for the treatment of cutaneous leishmaniasis caused by Leishmania major.N Engl J Med 346 :891–895.

    • Search Google Scholar
    • Export Citation
  • 6

    Periti P, Mazzei T, Mini E, Novelli A, 1993. Adverse effects of macrolide antibacterials. Drug Saf 9 :346–364.

  • 7

    Gladue RP, Bright GM, Isaacson RE, Newborg MF, 1989. In vitro and in vivo uptake of azithromycin (CP-62,993) by phagocytic cells: possible mechanism of delivery and release at sites of infection. Antimicrob Agents Chemother 33 :277–282.

    • Search Google Scholar
    • Export Citation
  • 8

    Foulds G, Madsen P, Cox C, Shepard R, Johnson R, 1991. Concentration of azithromycin in human prostatic tissue. Eur J Clin Microbiol Infect Dis 10 :868–871.

    • Search Google Scholar
    • Export Citation
  • 9

    Scheinfeld NS, Tutrone WD, Torres O, Weinberg JM, 2004. Macrolides in dermatology. Dis Mon 50 :350–368.

  • 10

    Zuckerman JM, 2000. The newer macrolides: azithromycin and clarithromycin. Infect Dis Clin North Am 14 :449–462.

  • 11

    Dunne MW, Singh N, Shukla M, Valecha N, Bhattacharyya PC, Dev V, Patel K, Mohapatra MK, Lakhani J, Benner R, Lele C, Patki K, 2005. A multicenter study of azithromycin, alone and in combination with chloroquine, for the treatment of acute uncomplicated Plasmodium falciparum malaria in India. J Infect Dis 191 :1582–1588.

    • Search Google Scholar
    • Export Citation
  • 12

    Nakornchai S, Konthiang P, 2006. Activity of azithromycin or erythromycin in combination with antimalarial drugs against multidrug-resistant Plasmodium falciparum in vitro.Acta Trop 100 :185–191.

    • Search Google Scholar
    • Export Citation
  • 13

    Miller RS, Wongsrichanalai C, Buathong N, McDaniel P, Walsh DS, Knirsch C, Ohrt C, 2006. Effective treatment of uncomplicated Plasmodium falciparum malaria with azithromycin-quinine combinations: a randomized, dose-ranging study. Am J Trop Med Hyg 74 :401–406.

    • Search Google Scholar
    • Export Citation
  • 14

    Krolewiecki A, Leon S, Scott P, Abraham D, 2002. Activity of azithromycin against Leishmania major in vitro and in vivo.Am J Trop Med Hyg 67 :273–277.

    • Search Google Scholar
    • Export Citation
  • 15

    Tanyuksel M, Bas AL, Araz E, Aybay C, 2003. Determination of intracellular efficacies of azithromycin against Leishmania major infection in human neutrophils in vitro.Cell Biochem Funct 21 :93–96.

    • Search Google Scholar
    • Export Citation
  • 16

    Prata A, Silva-Vergara ML, Costa L, Rocha A, Krolewiecki A, Silva JC, Paula DE, Pimenta EV, Junior FG, Giraldo LE, 2003. Efficacy of azithromycin in the treatment of cutaneous leishmaniasis. Rev Soc Bras Med Trop 36 :65–69.

    • Search Google Scholar
    • Export Citation
  • 17

    Silva-Vergara ML, Silva Ld EA, Maneira FR, da Silva AG, Prata A, 2004. Azithromycin in the treatment of mucosal leishmaniasis. Rev Inst Med Trop Sao Paulo 46 :175–177.

    • Search Google Scholar
    • Export Citation
  • 18

    Krolewiecki AJ, Romero HD, Cajal SP, Abraham D, Mimori T, Matsumoto T, Juarez M, Taranto NJ, 2007. A randomized clinical trial comparing oral azithromycin and meglumine antimoniate for the treatment of American cutaneous leishmaniasis caused by Leishmania (Viannia) braziliensis.Am J Trop Med Hyg 77 :640–646.

    • Search Google Scholar
    • Export Citation
  • 19

    Layegh P, Yazdanpanah MJ, Vosugh EM, Pezeshkpoor F, Shakeri MT, Moghiman T, 2007. Efficacy of azithromycin versus systemic meglumine antimoniate (Glucantime) in the treatment of cutaneous leishmaniasis. Am J Trop Med Hyg 77 :99–101.

    • Search Google Scholar
    • Export Citation
  • 20

    Mikus J, Steverding D, 2000. A simple colorimetric method to screen drug cytotoxicity against Leishmania using the dye Alamar Blue. Parasitol Int 48 :265–269.

    • Search Google Scholar
    • Export Citation
  • 21

    Huber W, Koella JC, 1993. A comparison of three methods of estimating EC50 in studies of drug resistance of malaria parasites. Acta Trop 55 :257–261.

    • Search Google Scholar
    • Export Citation
  • 22

    Croft SL, Yardley V, Kendrick H, 2002. Drug sensitivity of Leishmania species: some unresolved problems. Trans R Soc Trop Med Hyg Apr 96 (Suppl1):S127–S129.

    • Search Google Scholar
    • Export Citation
  • 23

    McMahon-Pratt D, Alexander J, 2004. Does the Leishmania major paradigm of pathogenesis and protection hold for New World cutaneous leishmaniases or the visceral disease? Immunol Rev 201 :206–224.

    • Search Google Scholar
    • Export Citation
  • 24

    Cantin L, Chamberland S, 1993. In vitro evaluation of the activities of azithromycin alone and combined with pyrimethamine against Toxoplasma gondii.Antimicrob Agents Chemother 37 :1993–1996.

    • Search Google Scholar
    • Export Citation
  • 25

    Gingras BA, Jensen JB, 1992. Activity of azithromycin (CP-62,993) and erythromycin against chloroquine-sensitive and chloroquine-resistant strains of Plasmodium falciparum in vitro.Am J Trop Med Hyg 47 :378–382.

    • Search Google Scholar
    • Export Citation
  • 26

    Biswas S, 2001. In-vitro antimalarial activity of azithromycin against chloroquine sensitive and chloroquine resistant Plasmodium falciparum.J Postgrad Med 47 :240–243.

    • Search Google Scholar
    • Export Citation
  • 27

    Orth C, Wllingmyre GD, Lee P, Knirsh C, Milhous W, 2002. Assessment of azithromycin in combination with other anti-malarial drugs Plasmodium falciparum in vitro.Antimicrob Agents Chemother 46 :2518–2524.

    • Search Google Scholar
    • Export Citation
  • 28

    Blais J, Garneau V, Chamberland S, 1993. Inhibition of Toxo-plasma gondii protein synthesis by azithromycin. Antimicrob Agents Chemother 37 :1701–1703.

    • Search Google Scholar
    • Export Citation
  • 29

    Foulds G, Shepard RM, Johnson RB, 1990. The pharmacokinetics of azithromycin in human prostatic tissue. J Antimicrob Chemother 25 (Suppl A):73–82.

    • Search Google Scholar
    • Export Citation
Save