The use of azythromycin and N-methyl glucamine for the treatment of cutaneous Leishmaniasis caused by Leishmania (Leishmania) amazonensis in C57BL6 mice

Abstracts

FUNDAMENTOS: O tratamento de primeira escolha da leishmaniose tegumentar americana é a N-metil-glucamina que tem alta toxicidade, exige administração parenteral e nem sempre cura. A azitromicina mostrou ação in vitro e resultado contraditório na doença humana. OBJETIVO: Verificar se a associação N-metil-glucamina+azitromicina é mais eficaz do que N-metil-glucamina no tratamento da leishmaniose experimental. MÉTODOS: 25 camundongos inoculados com a cepa C57BL/6 de L. (L.) amazonensis foram divididos em dois grupos. Um foi tratado com 400mgSbV/kg/dia de N-metil-glucamina associado a 200mg/kg/dia de azitromicina durante 20 dias, e o outro com N-metil-glucamina, na mesma dose, durante o mesmo tempo. Foi feita avaliação clínica e parasitológica com análise estatística. RESULTADO: Na avaliação clínica, pesquisa de amastigotas e das culturas, não houve diferença estatística. Verificou-se, entretanto, diferença significante no resultado das culturas realizadas através de diluição limitante, que desfavoreceu a associação NMG+ azitromicina. CONCLUSÃO: A associação N-metil-glucamina e azitromicina não demonstrou mais eficácia do que o N-metil-glucamina em uso isolado.

Azitromicina; Leishmaniose cutânea; Leishmaniose cutânea

CLINICAL, EPIDEMIOLOGICAL, LABORATORY AND THERAPEUTIC INVESTIGATION

The use of azythromycin and N-methyl glucamine for the treatment of cutaneous Leishmaniasis caused by Leishmania (Leishmania) amazonensis in C57BL6 mice

Raimunda Nonata Ribeiro Sampaio^I; Íris Campos Lucas^II; Arnoldo Velloso da Costa Filho^III

^IAssociate Professor, Universidade de Brasília (UnB), Head of the Dermatology Unit, Hospital Universitário de Brasília (UnB) – Brasilia (DF), Brazil

IIScientific Initiation Student, UnB-CNPq, in 2005/2006. Laboratory of Dermatomycology, Universidade de Brasília (UnB) – Brasilia-DF, Brazil

IIIScientific Initiation Student, UnB-CNPq, in 2005/2006. Laboratory of Dermatomycology, Universidade de Brasília (UnB) – Brasilia-DF, Brazil

Mailing Address

ABSTRACT

BACKGROUND: The first choice treatment for cutaneous Leishmaniasis is N-methyl glucamine: it has high toxicity, requires parenteral administration and cure is not always reached. Azythromycin showed in vitro action and controversial results in humans with the disease.

OBJECTIVE: To verify if the association of N-methyl-glucamine - azythromycin is more effective than N-methyl-glucamine alone for the treatment of experimental Leishmaniasis.

METHODS: Twenty-five C57BL/6 mice were inoculated with L. (L.) amazonensis strain and divided into two groups. One group was treated with 400mgSbV/kg/day of N-methyl glucamine and 200mg/kg/day of azythromycin for 20 days and the other group received the same dose of N-methyl glucamine alone during the same period of time. Clinical and parasitological evaluations were submitted to statistical analyses.

RESULTS: There was no statistical difference in clinical analysis, in amastigotes investigation and in cultures. There were significant differences in cultures using limiting dilution, which showed lower efficacy of the association N-methyl glucamine -azythromycin.

CONCLUSION: N-methyl glucamine-azythromycin association was not more effective than N-methyl glucamine alone.

Keywords: Azythromycin; Leishmaniasis, cutaneous; Leishmaniasis, cutaneous/therapy

INTRODUCTION

The epidemiological importance of leishmaniasis in public health demands the development and reduction of price of drugs used to treat it. Several factors hinder the control of this disease: lack of vaccination, wide variety of leishmanias and phlebotomes, limited knowledge and prevention of the disease by the population, among others.

The first line medication to treat leishmaniasis in Brazil and in other non-English speaking countries is antimonial N-methyl-glucamine pentavalent (NMG), a drug that has toxicity ^1-3, which requires parenteral administration and may not be effective in treatment^{2, 4}. In this case, other drugs such as classical amphotericin B and pentamidine, which are also toxic to kidney-heart-liver-pancreas and injectable, may be used.

Azythromycin is a macrolide antibiotic used for over 40 years in many infectious conditions, especially those that affect the respiratory tract and in sexually transmitted diseases, which has potential activity against leishmaniasis ^5-7. The macrolide class acts by inhibiting protein synthesis, dissociating the peptide from the ribosome ⁶. Its characteristics are to get concentrated into the tissues, especially in macrophages, with levels 100 to 200 times over those in serum, becoming an attractive option to manage infections caused by intracellular microorganisms. Other advantages are the possibility of administering it orally or by injection, good oral availability and long half-life, relative safety to be used in pregnant women and children, and low toxicity profile. Many different protozoa such as Plamodium falciparum, Plasmodium vivax and Toxoplasma gondii have shown susceptibility to azythromycin in in vitro and in vivo experiments at different levels ⁶.

Azythromycin has shown action against L. major in vitro and in vivo, even though there has been no understanding of its action mechanism. Using it to treat patients, it showed action against leishmaniasis with therapeutic response slower than for antimonial agents 5. In in vitro experiments, azythromycin did not contribute to the phagocytosis of L. major, but it increased the intracellular death rate of amastigotes 8. Another possibility is that azythromycin has an immunomodulating effect, reducing the number of amastigotes and activating the neutrophils, preventing the production of mediators and proinflammatory cytokines ^{5, 9}. In vitro, it has also shown efficacy against L. (L.) amazonensis, L. (V.) brazililensis) and L. (L.) chagasi in the concentration 150µg/ml. ¹⁰

In a clinical study carried out in Manaus, in which species L. (V.) guyanensis is predominant, there was low efficacy of the drug given to 26 patients with cutaneous leishmaniasis (CL) for 20 days, who also reported adverse events such as diarrhea in 60%, abdominal pain in 28%, and headache and nausea in 12% of the cases ¹¹. Another clinical study analyzed efficacy of azythromycin in three elderly patients with mucosa leishmaniasis (ML) associated with chronic cardiopathy. The drug was administered orally in a single daily dose of 500 mg for 10 days, in three series separated by one month interval. There was good outcome and no adverse events. However, two patients had recurrence after six months and were cured with a new cycle of azythromycin ¹².

The objective of the present study was to assess whether the association between NMG and azythromycin was more effective than the current first line treatment (NMG in isolation) in experimental CL caused by L.(L. ) amazonensis.

MATERIAL AND METHOD

The study used 25 isogenic, male mice of C57BL/6 strain coming from the Institute of Chemistry of State of Goias – Iquego. The inoculated parasites were L. (L.) amazonensis isolated from culture of PH8 strains cryopreserved at Laboratory of Dermatomycology, UnB.

Before treatment, diameter of the animal paws was measured and documented. The paws were inoculated on the right plantar pad with 3. 106 metacyclic promastigotes of L.(L.) amazonensis and they were examined periodically to detect the onset of cutaneous lesions. Next, the animals were randomly divided into two groups, one treated with NMG dose of 400mg SbV/kg/day IM for 20 days (control group) and the other with the same dose NMG associated with azythromycin 200mg/Kg/Day PO (using gavage) for 20 days.

To measure mice paws, we used precision pachymeter marked at millimeter decimals.

After treatment, the animals were assessed using clinical and parasitological criteria. According to clinical criteria, inoculated paws were measured immediately before and 20 days after treatment.

Parasitological assessment was made by counting leishmanias in swab cultures of McNeal, Novy and Nicolle (NNN) and culture in limiting dilution method.

To make the swab, exudate from the inoculated paw was aspirated. It was placed over slides, fixed and stained by Giemsa, submitted to optical microscope reading.

To perform the traditional culture, the aspirated material was placed on a culture tube with NNN medium, maintained in the hot oven at 24º C, and the daily reading was made under inverted light microscopy until one month was completed.

To make limiting dilution, one paw from each mice group was used. Under laminar flow hood, they were dissected and smashed and the content was submitted to successive 10-time dilutions and cultured in Elisa plaques of 96 wells containing RPMI medium enriched with fetal bovine serum. A plaque was used for each group and they were maintained in hot oven BOD at 24º C temperature for 30 days. Numeric data were obtained from counting the number of wells in which there was growth of leishmanias promastigotes, checking the reading under inverted light microscopy, and the mean of the values was calculated for the two groups by Elida® software. ¹³

The statistical tests used were t Student and chi-square.

RESULTS

Before treatment, the measures of mice paws were compared between the two groups to avoid the bias of having pre-experiment differences (p=0.11).

The mean diameter of the mice paws in the group NMG + azythromycin was greater than for group NMG (36.0 X 31.75 mm) after treatment. There was no statistically significant difference according to t Student test (p= 0.23) (Table 1).

In the parasitological assessment of traditional cultures made after treatment there was one positive culture and three negative ones in the NMG + azythromycin group, and two positive and two negative cultures in the NMG group. There was no statistically significant difference between the two groups (Fisher test, calculated by Statcalc module of software Epi Info 6.04 [p= 0.23]).

Similarly, the difference between the two groups concerning presence of amastigotes in the swab was calculated and the results were two positive swabs and two negative swabs in NMG group, and two positive and three negative results in the group with association NMG + azythromycin (p=0.23), according to Fisher test and the software described above.

The estimated mean number of leishmanias present on the paw of each group of mice, through limiting dilution culture, showed differences between the groups (NMG: 528 leishmanias and association NMG+AZYTHR: 12,700 leishmanias) (Table 2) (p=0.007), by chi-square test, used by software SPSS.

DISCUSSION

During the project execution, mortality of mice was high, which interfered in the small sampling of results for NNN medium culture and number of amastigotes in swab. Concerning the measurement of mice paws, culture and amastigote number, there was no statistically significant difference between the groups NMG + Azythromycin and NMG.

The results of limiting dilution showed major difference between the groups (NMG: 528 leishmanias and association NMG + Azythromycin: 12,700 leishmanias), with statistically significant difference unfavorable to the group NMG + azythromycin. The test consisted on the most careful assessment of the study. There was difference between the results, suggesting that the association of the drugs NMG + azythromycin had much lower action over leishmanias than isolated NMG.

It was concluded that the association of drugs was not more efficient than NMG to treat experimental CL.

Literature data about the efficacy of azythromycin in American Tegumentary Leishmaniasis (ATL) are controversial. As we have seen, in vitro studies ^{5, 10} in mice ⁵ and clinical trials ^{7, 12} have shown that azythromycin may be effective to manage different species of leishmanias. However, a clinical trial carried out in Manaus with azythromycin in 26 patients had low cure rate and many adverse events ¹³. The differences between experimental and actual leishmaniasis in humans, as well as the variety of species that cause ATL, may have resulted in such result discrepancy.

The action mechanisms for NMG and azythromycin over leishmanias are unknown, which hinders the hypothesis of a synergistic action between them. These results make us believe that there was no maximization of NMG on the treatment of experimental CL when it was associated with azythromycin. It would be recommendable to have confirmation of these results and completion of the study about the synergism between the drugs to treat ATL, given that the association of drugs represents a new hope in face of the threat of having increased therapeutic resistance.

ACKNOWLEDGEMENT

We would like to thank Mr. Tercio Rodrigues Pereira, from Laboratory of Dermatomycology, for his technical support, and Fundação de Empreendimentos Científicos e Tecnológicos – Finatec, for its partial financial support. In addition, we would like to thank Dr Jorgeth O. C. da Motta and Juliana S. F. e Silva.

REFERENCES

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*

Trabalho realizado no Laboratório de Dermatomicologia da Universidade de Brasília (UnB) – Brasília (DF), Brasil.

Conflito de interesse: Nenhum Suporte financeiro: Apoio financeiro parcial da Fundação de Empreendimentos Científicos e Tecnológicos – Finatec.

Publication Dates

• Publication in this collection
  03 June 2009
• Date of issue
  Apr 2009