Int. Med J Vol. 6 No 2 December 2007

Resistance to Glucantime in Cutaneous Leishmaniasis: proposing an unexpected solution.

Mohammad Ali Nilforoushzadeh (M.D) ^a, Fariba Jaffary (Ph.D) ^a, Roya Derakhshan (M.D) ^a, Nazli Ansari (M.D) ^a Shahram Moradi (M.D) ^a, Mahnaz Derakhshan (M.D) ^b, Hamid R. Ansarian (M.D) ^c*

^a Skin Diseases and Leishmaniasis Research Center, Isfahan University of Medical Sciences, Isfahan, Iran

^b Department of Applied Chemistry and Biochemistry, Kumamoto University, 2-39-1 Kurokami, Kumamoto, Japan

^c SVB Laboratory, Kumamoto University, 2-39-1 Kurokami, Kumamoto, Japan

ABSTRACT:

Objectives: Pentavalent antimony containing compounds are considered the first line treatment for cutaneous leishmaniasis (CL). However resistance to the present treatments is an important issue. The aim of this survey is first, to evaluate the prevalence of Meglumine antimoniate (Glucantime) resistance in Isfahan province of Iran and Second, to find a solution for this problem.  Materials and Methods Patients from a highly infected strip of Isfahan province, Iran, were followed up and their responses to systemic therapy were recorded. 370 patients were included in the study. Systemic Meglumine antimoniate (20 mg/kg/day elemental Sb^V) was given to each patient for 2-3 weeks. Their response to treatment was classified as complete response, partial response, and no response. Data were stored and processed into graphs, percentages, Chi-Square, and Logistic Regression using SPSS software.  Results: 247 men and 123 women were completely followed. The average and the standard deviation of their ages was 26.7 y (years) and 16 y. After one week 16.2% showed partial response. After two weeks, 0.7% showed complete response, and 64.1% showed partial response. After three weeks, 73% showed partial response and 15.1% showed complete response. After 12 weeks, 56.8% showed complete response, 35.1% showed partial response, and 8.1% of patients didn’t show any response. Discussion and conclusion: The pattern of distribution of drug resistance, which was not affected by host factors, indicates that the role of parasite as the dominant mechanism of poor response. One strategy (inspired from nanotechnology) for overcoming this drug resistance is antimoniate-containing nanofibers.

KEYWORDS: Cutaneous leishmaniasis, Drug resistance, Meglumine antimoniate, Iran, Nanotechnology, Nanofiber

INTRODUCTION:

Acute cutaneous leishmaniasis (ACL) is a domestic disease in Iran and one of its big and important health challenges. It is so prevalent that in some villages up to 70% of whole population has at least one leishmaniasis scar. Leishmania tropica and leishmania major are the principal infecting agents. ^[1] For leishmania major, Rhombomys opimus is the principal reservoir. ^[2] For leishmania tropica, human is the main reservoir. ^[3] Reports of subclinical visceral infection by L. tropica, ^[4,5] despite being rare, underscore its ability to disseminate through circulating blood monocytes. Therefore phlebotomus bites from patients with L. tropica Cutaneous lesions, even from locations far from active lesions may infect it. In rural areas some animals are thought to be the reservoir of leishmania tropica, but the full life cycle is still unknown. ^[6] The main transmitter (vector) for both L. tropica and L. major is Phlebotomus papatasi. ^[2-6]

Preventive measures include both control of rodent’s reproduction and effective treatment of patients. Several treatments have been suggested for acute cutaneous leishmaniasis (ACL) but neither of them can be considered a satisfactory cure. The most important and effective treatment for acute cutaneous leishmaniasis (ACL) has been the pentavalent antimony containing compounds (Meglumine antimoniate and pentostam). ^[2-6]. unfortunately resistance to Meglumine antimoniate has been growing up during 5 decades of its widespread use and now has emerged as a significant health problem. There are increasing reports of resistance to pentavalent antimony containing compounds especially in case of Leishmania tropica in recent years. ^[7-8] In an in-vitro study on Leishmania Viannia it has been shown that the profile of carbohydrates at the surface of cell membrane and reactivity to lecithin were directly correlated to Meglumine antimoniate resistance. ^[9] Reduced cellular immune response and reduced levels of gamma-interferon are other important factors that are associated with poor response to antimony containing compounds. ^{[10, 11]}

Considering Meglumine antimoniate being still the first line treatment for tackling acute cutaneous leishmaniasis (ACL) in Iran, this survey aimed at determination of relative prevalence of Meglumine antimoniate resistance in ACL patients from 150000 inhabitants who were living in a highly infected strip extended from north to northern east and eastern part of Isfahan city, Iran.

METHODS:

In a case-series study 370 patients, from 5 to 75 years old, who were admitted to the clinic of Isfahan Dermatology Research Center between October 23^rd 2002 and February 19^th 2003, and met the criteria of being included in the survey, were included in the research after obtaining their written consent. The criteria for inclusion of patients in our survey were as follows:

1. Having at least one of the indications for systemic treatment with meglumine antimoniate.

1.1.  Having big or multiple acute cutaneous leishmaniasis (ACL) lesions (above 3 cm in diameter or more than five lesions, n ³ 5) not later than 12 weeks after the appearance of the first lesion.

1.2.Those patients with ACL lesions in face or other regions that were important in terms of their beauty or were likely to leave disabling scare.

1.3. Some rare manifestations of ACL like sporotrichoid or lymphangitic acute cutaneous leishmaniasis with satellite lesions indicative of active expanding ulcers.

1.4.Patients with diffuse cutaneous leishmaniasis (DCL) or with lesions that had involved nearby mucosal or chondral tissues

2. Positive direct smear test.

3. Personal agreement to participate in the survey.

Breast-feeding or pregnant mothers and those patients who had some concomitant chronic heart, kidney, or liver disease were excluded. Patients received systemic meglumine antimoniate, 70 mg/kg/day of C₇H₁₇NO₅.H.O₃Sb that equals 20 mg/kg/day of pentavalent antimony (Sb^V), with a maximum limiting dose of 10 ml/day (850 mg/day) through deep intra-muscular injection for a period of two or three weeks depending on their clinical situation. Because the effect of meglumine antimoniate is usually continued for another two weeks after cessation of treatment, patients were not given any medication in the last two weeks of Treatment and then if ACL lesions were not completely cured another period of systemic Meglumine antimoniate therapy was started. Patients were visited weekly during their first three weeks of receiving treatment and finally 3 months after cessation of their treatment course. Patient’s response to treatment (both clinical and parasitologic) was determined as follows.

Complete response: both complete remedy of ACL lesions and negative direct smear test.

Partial response: positive direct smear test despite remedy of ACL lesions, or partial remission (decrease in size or indurations), or recurrence during the follow up period. 

No response: no clinical response to the treatment.

 Data were stored and processed into graphs and percentages, Chi-Square, and Logistic Regression using SPSS software.

RESULTS:

Among 370 acute cutaneous leishmaniasis (ACL) patients 247 (66.8%) were men and 123 (33.2%) were women. The average and the standard deviation of their ages were 26.7 y (years) and 16 y. After 12 weeks of medication only 30 patients (8.1%) fell in the category of no response. (Fig. 1) The mean and standard deviation of the recuperation time was 9.5 ± 4 weeks among men and 9.6 ± 3.9 weeks among women. As a whole the mean and standard deviation of the recuperation time was 9.59 ± 3.9 weeks. Table 1 shows the distribution of response to treatment according to type and location of the lesion. In this study the mean and standard deviation of the number of lesions were 3.28 ± 2.6. The maximum number of lesions in one patient was 20. Four patients (1.1%) had 4 lesions, and 26 patients (7%) had 3 lesions. There was no statistically significant correlation (p=0.79) between multiplicity of ACL lesions and the distribution profile of response to systemic meglumine antimoniate. In age group 5- 19 years old, 9 patients (6%), in 20 – 39 years old, 11 patients (8.1%), and in age group 40-59 years old, 8 patients (12.3%) showed drug resistance. Only 2 patients (6.7%) from 20 patients of 60-79 years old had drug resistance. There was no statistically significant correlation between age and drug resistance (p=0.47). Also no statistically significant prediction was revealed in the profile of drug resistance with logistic regression model with 92% sensitivity based on sex, age group, type of lesion, or location of lesions (Table 2). As it is shown in table 2, none of the regression coefficients are above 0.8 and all confidence intervals for odd ratio embraces unity. These statistical indicators tell that the distribution of drug resistance is not affected by any of the above factors.  The complete response rate was 56.8% after 12 weeks. 8.1% of patients didn’t show any response and 35.1% exhibited only a partial remission. Only prevalence of ACL lesions on trunk was significantly lower among women.  It was 18.2% among men and 7.3% among women with a P-value of P= 0.005, that is probably due to women’s obligation to cover up that part of body according to Islamic codes. Also the total number of client women (123) with ACL was half of that of men (247) that again indicates a protecting effect of Islamic dressing code against ACL. There are a number of theories regarding resistance to meglumine antimoniate ranging from changes in membrane carbohydrates profile of parasite to reduced cellular immune response and gamma-interferon level in host. ^[10] In this study, drug resistance showed no discrimination against host factors like sex, age, lesion type or location; a fact that indicates some biomolecular changes in the parasite as the dominant cause in drug resistance rather than host related factors. We also searched the literature for possible new methods of antimoniate delivery to leishman bodies considering peculiarity of pathophysiological aspects of ACL. As this study shows meglumine antimoniate, in its common route of delivery, is effective in the majority of patients (more than 90%) therefore keeping high concentrations of pentavalent antimony in the local area of lesion is a promising strategy. We realized that this challenge now lies within the capacity of nano-science to be addressed. ^[13] Nano-scientists have been engaged in several researches surrounding particulate drug delivery systems for targeting or for sustained release. ^[14] For targeting, surfactant vesicles are classically loaded with a drug and their surfaces are modified by polymers like PEG (Polyethylenglycol) in a way that they are protected from opsonization and macrophageal uptake. Then appropriate ligands like viral proteins, carbohydrates, glycoproteins; hormones, etc. are attached to their surfaces. ^[15] However there are also many other superstructures like ultra-thin microcapsules, ^[16] macromolecular nanoshells, ^[17] icosahedrals, ^[18] or even fibers ^[19] that can either be loaded or embedded with the drug for special targeting or sustained release.

Figure1 The prevalence distribution of response to treatment

Table 1: The prevalence distribution of response to treatment according to type and location of the lesion

Table 2: Prediction of drug resistance by host criteria with forward binary Logistic Regression model

DISCUSSION:

Although this study looks as a case-series, it can be accepted as a cross-sectional study because almost all suspicious cases of ACL in that region were referred to our clinic for diagnosis and treatment and it was very unlikely for patients to go to other centers for treatment. In this study drug resistance showed no discrimination against host factors like sex, age, lesion type or location; a fact that indicates some biomolecular changes in the parasite as the dominant cause of drug resistance rather than host related factors. As we mentioned earlier nano-science today has much to offer to medical community. ^[13,20-23] The specifications that are supposed to be asked from nano-technologist (for the purpose of tackling ACL by furthering the delivery of antimoniate to leishman bodies) can be nanofibers, which will trap antimoniate in their  superstructure. If a system of Antimoniate-induced nanofibrilar aggregates is injected into CL lesions it is expected that antimoniate will not be washed away from the nanofibers and remain in the local tissue. On the other hand these nanofibers will be treated as small foreign bodies by macrophages, which are main reservoirs of leishman bodies. Hence antimoniate will be delivered to leishman bodies (passive targeting), which are mainly located inside the cytosol of macrophages.

CONCLUSION:

Our survey showed that the pattern of distribution of drug resistance was not affected by host factors, which indicates the role of biomolecular changes in the parasite as the dominant mechanism of poor response. One potential solution for overcoming this drug resistance (antimoniate containing nanofibers) is proposed that is inspired from recent developments in nanotechnology.

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Corresponding author

Hamid Reza Ansarian

SVB Laboratory, Kumamoto University,

2-39-1 Kurokami, Kumamoto, Japan

Tel: +81-342-3662

E-mail: ansarian@chem.kumamoto-u.ac.jp