Controlled Nitric Oxide Releasing Patch Versus Meglumine Antimoniate in the Treatment of Cutaneous Leishmaniasis
Controlled Nitric Oxide Releasing Patch Versus Meglumine Antimoniate in the Treatment of Cutaneous Leishmaniasis
Cutaneous leishmaniasis is a worldwide disease, endemic in 88 countries, that has shown an
 increasing incidence over the last two decades. So far, pentavalent antimony compounds have
 been considered the treatment of choice, with a percentage of cure of about 85%. However, the
 high efficacy of these drugs is counteracted by their many disadvantages and adverse events.
 Previous studies have shown nitric oxide to be a potential alternative treatment when
 administered topically with no serious adverse events. However, due to the unstable nitric
 oxide release, the topical donors needed to be applied frequently, making the adherence to
 the treatment difficult. The electrospinning technique has allowed the production of a
 multilayer transdermal patch that produces a continuous and stable nitric oxide release. The
 main objective of this study is to evaluate this novel nitric oxide topical donor for the
 treatment of cutaneous leishmaniasis.

 A double-blind, randomized, double-masked, placebo-controlled clinical trial, including 620
 patients from endemic areas for leishmaniasis in Colombia was designed to investigate whether
 this patch is as effective as meglumine antimoniate for the treatment of cutaneous
 leishmaniasis but with less adverse events. Subjects with ulcers characteristic of cutaneous
 leishmaniasis will be medically evaluated and laboratory tests and parasitological
 confirmation performed. After checking the inclusion/exclusion criteria, the patients will be
 randomly assigned to one of two groups. During 20 days Group 1 will receive simultaneously
 meglumine antimoniate and placebo of nitric oxide patches while Group 2 will receive placebo
 of meglumine antimoniate and active nitric oxide patches. During the treatment visits, the
 medications will be administered daily and the presence of adverse events assessed. During
 the follow-up, the research group will visit the patients at days 21, 45, 90 and 180. The
 healing process of the ulcer, the health of the participants, recidivisms and/or reinfection
 will also be assessed. The evolution of the ulcers will be photographically registered. In
 the case that the effectiveness of the patches is demonstrated, a novel and safe therapeutic
 alternative for one of the most important public health problems in many countries will be
 available to patients.
BACKGROUND

 Cutaneous Leishmaniasis (CL) is a worldwide disease that is endemic in 88 countries [1] . It
 is estimated that 1.5 million people suffer from CL annually and that more than 350 million
 are at risk of contracting the infection [2-4]. In America, 60,000 new cases of CL are
 reported annually [5], being endemic in 20 of its 22 countries and in 2 islands of the
 Caribbean [2]. Currently, CL has affected more than 500 U.S. Army soldiers serving in Iraq
 [6]. In the Andean region, the incidence of Leishmaniasis has been increasing dramatically
 over the last two decades; reaching more than 14,000 cases per year from 1996-98 [7]. In
 Colombia 6,500 cases have been reported [8]. The increase in the reported cases of CL in
 Colombia has been related to factors such as migration, deforestation, the multiplication of
 illicit plantations, the armed political conflict and the behavioral changes of the vector.
 The main strains of Leishmania in Colombia are L. panamensis, L. brazilensis, L. infantum and
 L. guyanensis, which are distributed throughout the entire national territory, predominantly
 in the rural areas [10]. CL is caused by intracellular protozoan parasites of the genus
 Leishmania [1] and is transmitted to humans through the bite of a small percentage of the
 species of phlebotomus and lutzomyia sandflies classified to date [10]. In the digestive
 system of the sandflies, this dimorphic parasite presents an extracellular flagellated form
 called a promastigote, which upon its release in the host blood, is phagocytized by the
 macrophage, losing its flagella and turning into an amastigote [12]. Dogs, rodents and
 didelphidae are the natural hosts of the parasite while man is an incidental host [11]. This
 zoonosis has suffered an interesting urbanization phenomenon, changing from an eminent rural
 entity affecting mainly men of an active age, to a disease that is affecting all people,
 especially children [8,9]. The characteristic lesions of this disease are ulcers that heal
 spontaneously over a period of three months to a year, depending on the isolate, and that
 leave a flat, atrophic and depigmented scar [13-15]. The CL, especially the one produced by
 L. brazilensis can evolve into mucocutaneous Leishmaniasis (MCL), which has a worse prognosis
 owing to the deforming character of its lesions [16]. The spontaneous cure of these lesions
 allows for the acquisition of partial resistance to reinfection, which could explain the
 higher pathogenicity observed in the children and young adult population [9]. Previous
 studies have shown a higher incidence of CL and a poor response to treatment in the children
 population [17]. The program of epidemiological vigilance in Colombia requires that the
 probable cases of CL (identified by ulcer features and by the patient's origin) be confirmed
 by microscopic direct examination of a secession sample obtained from the ulcer, if these are
 negative, by biopsy of the wound. Once confirmed, the cases must be notified to the Local
 Health Secretary using clinical-epidemiological records. This institution, in charge of the
 epidemiological vigilance, studies the sources of transmission and distributes the medication
 to the people affected. Currently, various aspects are considered when treating CL, among
 which, the risk of developing MCL, the grade, localization, number, size, evolution and
 persistence of the lesions, are the most important [18]. For more than 60 years, the
 pentavalent antimony compounds: sodium stibogluconate, (Pentostan®, produced by
 Glaxo-Wellcome) and meglumine antimoniate, (Glucantime®, produced by Sanofi-Aventis) have
 been considered the treatments of choice for this disease [19]. Studies made in Colombia
 reported a percentage of cure of 85%, using meglumine antimoniate [20,21]. Despite the fact
 that the efficacy of these drugs is high, they present many disadvantages such as parenteral
 administration, and, reversible secondary effects such as nausea, vomiting, muscular and
 abdominal pain, cardiac problems, a rise in the concentration of hepatic aminotransferases,
 and chemical pancreatitis [21,22]. Additionally, the adherence to the treatment is affected
 by its duration (several weeks) and its availability by the restriction in its distribution.
 Therapeutic alternatives of second line have been proposed; amphotericin B and pentamidine
 have been used with excellent results, nevertheless their high cost, little availability, the
 necessity to hospitalize the patients for their administration and the severity of their
 secondary effects have limited their use [23,24]. In the last decade new treatments for CL
 have been developed, using oral agents such as mefloquine, itraconazole, miltefosine,
 paromomycin, ketoconazole, allopurinol and dapsone, however, they have not shown enough
 evidence of their effectiveness [19,21,25,26]. In an effort to develop a topical treatment
 for CL, paromomycin has been used in different preparations. However, rates of curation
 achieved with this medication have not been higher than conventional treatments, even when
 compared with placebo [27,28]. In several studies, in vitro and in vivo, it has been
 demonstrated that nitric oxide (NO) is effective to eliminate various strains of Leishmania
 in its amastigote form [29-35]. The production of NO from the oxidation of L-arginine caused
 by the inducible nitric oxide synthase (iNOS) constitutes one of the most important defense
 mechanisms of the macrophages [36], in which two oxidative forms of defense against
 Leishmania have been identified. During the first phase of infection, in response to the
 phagocytosis process, some promastigotes are eliminated due to the release of the superoxide
 ion, a process which is catalyzed by the NADPH oxidase [29]. Those promastigotes that survive
 this defense mechanism evolve into amastigotes, activating the production of IL-12 in the
 macrophages and promoting the presentation of the antigens of Leishmania [29] to the T
 helpers 1 lymphocytes that enhance the cytotoxic activities of the macrophages toward the
 intracellular parasites via the interferon gamma (INFγ) and the tumor necrosis factor alpha
 (TNFα) by promoting the production of NO catalyzed by iNOS [31-33]. A recent study shows a
 higher activity of iNOS in the macrophages of subjects infected with CL, suggesting a vital
 role of NO in the immunological activity against Leishmania [34]. In Studies with rodents
 resistant to Leishmania infection (C57BL/6), where L. major, L. chagasi or L. donovani were
 inoculated, the application of iNOS inhibitors like NG-monomethyl-L-arginine (L-NMMA) caused
 a higher rate of survival and virulence of the parasites in macrophages [33,35,37,38]. After
 inoculating L. major in mice with the genetic susceptibility to develop infections with
 Leishmania (BALB/C), no activity of iNOS was observed. However, the application of IL-12, was
 able to control the infection by activating iNOS [31]. In humans, several clinical trials
 have been realized with topical treatments containing NO donors [39,40]. In Ecuador, our
 group developed and tested a NO generating topical cream with S-nitroso-N-acetylpenicillamine
 (SNAP), evidencing a beneficial effect in the management of this type of ulcers with no
 reports of any serious adverse event. Nevertheless, due to the unstable nitric oxide release,
 the cream had to be applied frequently (4 times a day) making the adherence to the treatment
 difficult [39]. In Syria, another group used potassium nitrate acidified with salicylic acid
 and ascorbic acid for the topical treatment of L. tropica [40]. In vitro, this NO generating
 mixture destroyed the amastigotes and promastigotes of Leishmania; however, in vivo, the
 study of 40 patients presented inconsistent results, reducing the size of the ulcer in 28% of
 the subjects and healing only 12%. The discrepancy in these results is believed to be due to
 the technique used to obtain the NO. The acidification of nitrite produces an instant blast
 of NO, but its release is not maintained over a long period of time [40]. The difficulty of
 controlling the liberation of NO has created the necessity of looking for new techniques to
 regulate its release. The nanofiber polymers produced by the electrospinning technique have
 been studied in order to guarantee the constant release of pharmaceuticals on the lesion. In
 the electrospinning process, a high voltage is used to create an electrically charged jet of
 polymer solution, which dries and solidifies to leave behind a dry polymer fiber [41]. As
 this jet travels through the air, the solvent evaporates leaving behind a charged fiber that
 can be electrically deflected and collected on a metal screen [42,43]. Fibers with a variety
 of cross sectional shapes and sizes are produced from different polymers. With this
 technique, the encapsulation or entrapment of several pharmaceuticals, enzymes and proteins
 has been successful. In a previous study, nanofiber patches were successfully used as
 releasing vehicles of tetracycline hydrochloride. The release of tetracycline was constant
 for a period of 5 days [41;43]. Using the same model, a multilayer transdermal patch has been
 produced, in which nitrite is bound to an ion exchange resin (DOWEX) and electrospun into a
 polyurethane nanofibers layer. A solution containing Waterlock® superabsorbent and
 polyurethane is electrospun on top of the nitrite-DOWEX layer. The ascorbic acid entrapped in
 the polyurethane solution is electrospun onto a third layer, with another layer of Waterlock®
 superabsorbent and polyurethane as the fourth and final one. Upon hydration, this Nitric
 Oxide Releasing Patch (NOP) produces a stable release of 3.5 µmol of NO during 12 hrs
 [41,44,45]. In a pilot study, developed in Landazuri, Santander, Colombia, a
 placebo-controlled clinical trial was conducted with 35 patients who presented 68 ulcers
 produced by L. panamensis. Using the NOP, a 65% improvement was observed in the treated
 ulcers, with only a 25% improvement in the placebo group (p=0.001). In this pilot study the
 unique adverse event described was pruritus in the area where the patch was applied
 (unpublished data). Taking into account the wide distribution of CL, the changes in its form
 of transmission and the difficulty related with the availability of medication, this study
 proposes to investigate whether the NO donor transdermal patch, produced by electrospinning
 is, at least, as effective as the meglumine antimoniate for the treatment of CL, with less
 adverse events and a lower cost, constituting therefore an effective therapeutic alternative.
 In case that the effectiveness of the NOP is demonstrated in this study, a novel and safe
 therapeutic alternative of easy access and higher adherence for one of the most important
 public health problems in our country will be made available.

 OBJECTIVES

 General Objective

 To evaluate the effectiveness and safety of NOP in the treatment of CL compared with
 meglumine antimoniate (Glucantime®).

 Specific objectives

 1. To evaluate the healing rate of CL ulcers using a NOP compared with the plan of
 treatment with meglumine antimoniate recommended by the health ministry.

 2. To identify adverse events associated with the application of NOP and compare them with
 the ones produced by the treatment with meglumine antimoniate.

 3. To identify and compare the recidivisms that may occur with both NOP and meglumine
 antimoniate.

 4. To advance in the search of a therapeutic alternative for CL in Colombia.

 DESIGN

 Double blind, randomized, double-masked, placebo-controlled clinical trial, comparing nitric
 oxide releasing patches with meglumine antimoniate.

 SAMPLE SIZE

 The sample was calculated according to the arccosine formula using a power of 80% and a type
 1 error of 0.05%. Assigning a successful rate of 85% for meglumine antimoniate and 75% for
 NOP, 558 patients will be needed. After adjustment for a loss rate of 5%, the total of
 patients that must be recruited is 620 (310 patients per treatment group).

 POPULATION

 The population will be composed from two regions of Colombia. The first one is an endemic
 zone in Santander, Colombia, located between the Magdalena Valley and the East Mountain
 Range, which includes the municipalities of Landazuri, El Carmen, San Vicente, El Playon and
 Rionegro. The second region is an endemic zone in North Tolima, which includes the
 municipalities of Chaparral, San Antonio, Libano, Falan, Palocabildo and Mariquita.

 SELECTION OF THE PATIENTS

 Inclusion Criteria

 1. Men and women between 18 and 50 years old

 2. Cutaneous ulcers of more than two weeks of evolution

 3. Positive parasitological diagnosis for CL

 4. Patients that voluntarily agree to participate in the study and sign the informed
 consent.

 5. Disposition to attend all the visits punctually (initial, treatment and follow-up)

 6. Acceptation of not using any other treatment for CL while in the study

 Exclusion Criteria

 1. Pregnant women

 2. Presence of any condition or disease that compromises the patient immunologically (i.e.
 diabetes, cancer, etc.) or, any other, that, based on the judgment of the researcher,
 could alter the course of CL.

 3. Diffuse CL or more than five active lesions.

 4. Mucocutaneous leishmaniasis (no lesion must be located less than 2 cm from the nasal,
 uro-genital, and/or anal mucous membranes or from the edge of the lips).

 5. Visceral leishmaniasis

 6. Complete or incomplete treatment with antimony compounds in the last three months.

 7. Patients with history of hepatic, renal or cardiovascular disease.

 8. Mentally or neurologically disabled patients that are considered not fit to approve
 their participation in the study.

 STUDY DEVELOPMENT

 Logistic Phase

 This phase will last 4 months and will include the following activities:

 1. Training of the personnel that will participate in the study.

 2. Acquisition of the materials required for the development of the project.

 3. Elaboration of flyers, promotional and educative material, procedures manual and case
 report forms (CRFs).

 4. Treatment randomization. The treatment randomization will be realized by the
 epidemiologist of the Cardiovascular Foundation of Colombia. This randomization will be
 done in blocks in order to keep the size of the treatment groups similar, to avoid long
 sequences of the same treatment and to balance when possible some of the bias inherent
 to the simple randomization process. Additionally, this randomization in blocks will
 facilitate the execution of interim analyses.

 Recruitment Phase

 This phase will take 22 months. In the selected municipalities, the health personnel that
 work in hospitals and health centers will receive training regarding the disease and the
 study methodology. Simultaneously an epidemiological focus study will be done with the
 leaders of the community and the health personnel to identify the geographic and demographic
 conditions with the purpose of developing strategies for the recruitment of possible cases of
 leishmaniasis. Subsequently, the subjects that present active ulcers with more than 2 weeks
 of evolution, with or without parasitological confirmation of CL, will be invited by the
 health promoters to attend the screening visit (Table 1).

 Screening Visit

 During this visit a complete clinical history will be elaborated and data about antecedents
 of leishmaniasis obtained (administered treatment, localization, number of lesions, etc). A
 full medical evaluation will be realized based on universally accepted techniques. The
 inclusion/exclusion criteria will be applied and the selected candidates informed about the
 study after which they will sign an informed consent. For those subjects with ulcers of more
 than two weeks of evolution without parasitological diagnosis a direct test will be performed
 by the parasitologist. If the first direct test is negative, it will be repeated up to three
 times; after which, in case of negative results, a biopsy will be performed. In case
 leishmaniasis is not confirmed the patient will be remitted to the original health center.

 Initial Visit

 The included patients will be randomly assigned in one of two groups. A culture in
 Novy-Nicole-McNeal (NNN) medium will be taken for strain identification and a blood sample
 withdrawn from the antecubital vein for hepatic enzymes, creatinine, and pancreatic amylase
 determination. During this visit, a complete medical evaluation will be performed, the ulcer
 will be measured and a picture will be taken. Before taking the picture, a graduated rule
 will be placed next to the ulcer along with a sticker marked with the identification code of
 the participant, and the date of the visit. The first NOP (active or placebo) will be applied
 covering the lesion and the first shot of meglumine antimoniate (active or placebo)
 administered. The patients will receive information on how not to damage the patches and how
 to identify and report adverse events. To help the patients to do so, they will receive a
 diary to register them. The two groups randomly composed will be divided as follows:

 Group 1: During 20 days this group will receive simultaneously intramuscular (IM) meglumine
 antimoniate (Glucantime® 20 mg/kg/day with a maximum dose of 3 ampules per day); and a NOP
 placebo.

 Group 2: During 20 days this group will receive simultaneously placebo of IM meglumine
 antimoniate (5-15cc/day) and an active NOP.

 Treatment Visits

 The patients will visit the health center daily during 20 days to receive both the NOP
 (placebo or active) and meglumine antimoniate (placebo or active). Daily, the subjects and
 their adherence to the treatment will be assessed and the data collected. If any adverse
 event is detected, the patient will be referred immediately to the medical staff who will
 make an evaluation and report it to the adverse event committee that will take the final
 decision in each case.

 Follow-up visits

 This phase will last 10 months. During the follow-up, the patients will be seen by the
 research group in 4 opportunities. The first visit will take place the day after the end of
 the treatment (day 21) in which new blood samples will be taken for biochemical
 determinations. The second, third and fourth visits will be realized on day 45, 90 and 180
 respectively. During these visits the healing process of the ulcer, the presence of
 recidivisms and/or reinfection and the health of the participants will be assessed. The
 evolution of the ulcers will be photographically registered.

 The maximum and minimum diameters of the ulcer will be measured using a graduated ruler, and
 the induration using the ballpoint pen technique. The ulcer and induration areas will be
 calculated separately, and then registered in the CRF. The evaluation of the clinical
 response will be based on the ulcer showing the least improvement.

 1. Complete clinical response: Complete reepithelization of the ulcer and disappearance of
 the induration.

 2. Clinical Improvement: Reduction of more than 50% of the ulcer and the induration areas
 in relation to the last clinical evaluation.

 3. Absence of clinical response: Increase or reduction of less than 50% of the ulcer and
 the induration areas in relation to the last clinical evaluation.

 4. Therapeutic failure:

 1. Increase in the size of the ulcer by more than 50% in relation to the last clinical
 evaluation

 2. Presence of the ulcer three months after the beginning of the treatment.

 3. Reactivation: Appearance of a lesion on the edge or in the center of the scar, with
 positive parasitological diagnosis, after a period of complete reepithelization.

 4. Affection of the mucous membranes: Presence of affections of the mucous membranes
 during the treatment, at the end of it or in the follow-up visits.

 5. Reinfection: Activation of an ulcer in an area different from the original lesion.

 For subjects whose treatment will have been considered a failure the code will be broken and
 they will be remitted to their original health center to look for another therapeutic
 approach.

 Procedures

 Physical Examination

 A complete physical examination will be realized and vital signs will be measured.

 Blood Samples Withdrawn

 Blood samples will be withdrawn from the antecubital vein to perform the following
 biochemical analyses:

 1. Creatinine by spectrophotometry

 2. Alanine transaminase (ALT) by spectrophotometry

 3. Aspartate transaminase (AST) by spectrophotometry

 4. Pancreatic amylase by spectrophotometry

 Sampling Technique for the Direct Test of CL

 A direct test is performed to confirm the presence of cells with amastigotes in a dermis
 scrape[46,47]; the sample must be, in so far as it is possible, free of blood, cellular
 detritus or pus. If the patients present several lesions, the sample must be taken from the
 one with the shortest time of evolution, the biggest induration area and/or the least
 purulent discharge. If the lesion has a scab it must be removed to improve the quality of the
 sample. The sample can be taken from the active edge or from the bottom of the ulcer. When
 the sample is taken from the active edge asepsis must be realized with alcohol at 70% and
 hemostasis then performed using the first and second fingers to make sure that there is no
 blood in the sample. A small incision of about 3mm in length and depth is made with a scalpel
 in the active edge parallel to the edge of the ulcer. With the sharp side of the scalpel, a
 scrape is done in the dermic wall of the incision to obtain tissue. The extracted material is
 extended on two microscope slides. The sample is dried at room temperature, and then fixed
 with methanol and stained with Giemsa, Wright or Field. Using immersion oil the sample is
 observed under a microscope with a 100X lens, the presence of amastigotes of Leishmania
 assessed and their structure verified (nucleus, kinetoplast and cell membrane). If the sample
 is taken from the center of the lesion the same hemostatic technique must be used, the scab
 removed, and the bottom of the ulcer well cleaned using the sharp side of the scalpel to
 prevent the presence of cellular detritus and/or purulent material. This procedure must be
 continued until the granulomas are seen at the bottom. With the same technique used to
 process the samples from the active edge the presence of amastigotes of Leishmania is
 verified.

 Technique for the sampling of cultures

 The sample for the culture may be obtained by suctioning the ulcer active edge or by
 extracting a fragment of tissue which is then macerated in a phosphate-buffered saline
 solution (PBS) with antibiotics (1000 UI of crystalline penicillin per cc), before it is put
 in the culture medium. A tuberculin syringe with a thin needle (26G), containing 0.3 cc of
 PBS solution with antibiotics is used in the suction technique. Previous asepsis of the ulcer
 with alcohol at 70%, a needle is introduced into the dermis and through rotating movements a
 small amount of tissue is macerated by the needle bevel during about a minute, after which it
 is suctioned into the syringe. The sample is deposited in aseptic conditions into a NNN
 culture medium and incubated at 26ºC during 4 weeks [46,47]. Every week a drop is extracted
 from the culture medium and placed between two slides to be observed under a microscope. In
 case that promastigotes are not found, the cultures are rejected as negative [46]. The
 strains are identified by species using the monoclonal antibodies developed by Dr. Diane Mc
 Mahon Pratt [48,49].

 Evaluation and management of adverse events

 During the treatment and the follow-up visits, the patients will be asked about adverse
 events. Each adverse event will be classified by the physician as serious or
 non-serious(Table 2 and 3). A serious adverse event should meet one or more of the following
 criteria:

 1. Death

 2. Life-threatening (i.e., immediate risk of death)

 3. In-patient hospitalization or prolongation of existing hospitalization

 4. Persistent or significant disability/incapacity The presence of a serious adverse event
 that puts the patient's life at risk and/or requires immediate medical or surgical
 procedure will call for the discontinuation of the treatment and the initiation of the
 pertinent medical management of the patients. The investigator will notify the Adverse
 Event Committee (AEC) of the FCV of any serious adverse event within 24 hours of
 learning about it.

 A non serious adverse event will be classified as follows:

 1. Mild: The patients are aware of their symptoms and/or signs, but those are tolerable.
 They do not require medical intervention or specific treatment.

 2. Moderate: Patients present troubles that interfere with their daily activities. They
 require medical intervention or specific treatment.

 3. Severe: The patients are unable to work or to attend their daily activities. They
 require medical intervention or specific treatment.

 The possible relationship between the adverse events and the tested medication will be
 classified by the investigator on the basis of his/her clinical judgment and the following
 definitions:

 1. Definitely related: Event can be fully explained by the administration of the tested
 medication.

 2. Probably related: Event is most likely to be explained by the administration of the
 tested medication rather than other medications or by the patient's clinical state.

 3. Possibly related: Event may be explained by the administration of the tested medication
 or other medications or by the patient's clinical state.

 4. Not related: Event is most likely to be explained by the patient's clinical state or
 other medications, rather than the tested one.

 All the events will be reported to the AEC that, depending on their criteria, will decide for
 the continuity or the withdrawal of the patient from the study and therefore the breaking of
 the code.

 Although the project has been designed to minimize the inherent risks, any adverse event
 related to the study medications will be carefully evaluated by the AEC and the costs
 generated by the required treatment will be covered by the study.

 Data analysis phase

 This phase will last 6 months. After completing all the data entry to the CFR, the results
 will be audited and the detected errors evaluated and corrected by the person in charge. The
 information will be entered in two different databases by two different people and the
 records will be compared to detect any discrepancy. The original CFR will be used to correct
 any mistake in the database.

 For the statistical analysis, Stata 8.0 will be used. The descriptive analysis will be
 composed of medians and proportions according to the nature of the variables, with its
 respective 95% confidence intervals. As a dispersion measurement the standard deviation will
 be calculated. The distribution of the variables will be studied using the Shapiro-Wilk test.
 To detect any difference between the groups, a T-test or a Wilcoxon test will be performed
 according to its distribution. The categorical variables will be compared using the Chi2 test
 or the exact Fisher's test. If required, a model of multiple logistic regressions or a
 covariance analysis will be done.

 Two interim analyses will be performed when 35% and 70% of the calculated sample is
 collected, with the objective of determining the differences in effectiveness and safety
 between the treatments.

 Endpoints

 At the end of the study two endpoints will be evaluated:

 1. Successful Treatment:

 1. Complete reepithelization three months after the beginning of the treatment.

 2. Absence of reactivation and affections of the mucous membranes during the 6 months
 of the study.

 2. Treatment Failure:

 1. Incomplete reepithelization three months after the beginning of the treatment.

 2. Increase in the size of the ulcer by more than 50% in relation to the last clinical
 evaluation

 3. Reactivation and/or affections of the mucous membranes during the 6 months of the
 study.

 Final Report

 At the end of the study the results will be evaluated and discussed and a final report
 presented to COLCIENCIAS, entity that is sponsoring the project. The relevant results will be
 published in both, national and international journals, and presented in congresses and
 scientific meetings.

 ETHICAL ASPECTS

 This study will be conducted in accordance with the Declaration of Helsinki and with the
 Colombian legislation as per the Resolution 8430/93 from the Ministry of Health. Prior to the
 admission of the patients in the study, the objectives and the methodology will be explained
 and the informed consent obtained. The study was approved by the Research Ethic Committee of
 the Cardiovascular Foundation of Colombia (Act# 105/January 28/2005). The right to
 confidentiality of the patients will be maintained in all the phases of the study.

 COMPETING INTERESTS

 Non Financial competing interests

 The authors of this manuscript declare that they have no competing interests, including
 political, personal, religious, ideological, academic, intellectual, or any other competing
 interests.

 Financial competing interests

 We have not received in the past five years reimbursements, fees, funding or salary from any
 organization that may in any way gain or lose from the publication of this manuscript, either
 now or in the future. We don't hold any stocks or shares in an organization that may in any
 way gain or lose financially from the publication of the manuscript, either now or in the
 future. We don't hold or are currently applying for any patents relating to the content of
 the manuscript. We have not received reimbursements, fees, funding or salary from an
 organization that holds or has applied for patents relating to the content of the manuscript.
Inclusion Criteria:

 -  Men and women between 18 and 50 years old

 -  Cutaneous ulcers of more than two weeks of evolution

 -  Positive parasitological diagnosis for CL

 -  Patients that voluntarily agree to participate in the study and sign the informed
 consent.

 -  Disposition to attend all the visits punctually (initial, treatment and follow-up)

 -  Acceptation of not using any other treatment for CL while in the study

 Exclusion Criteria:

 -  Pregnant women

 -  Presence of any condition or disease that compromises the patient immunologically
 (i.e. diabetes, cancer, etc.) or, any other, that, based on the judgment of the
 researcher, could alter the course of CL.

 -  Diffuse CL or more than five active lesions.

 -  Mucocutaneous leishmaniasis (no lesion must be located less than 2 cm from the nasal,
 uro-genital, and/or anal mucous membranes or from the edge of the lips).

 -  Visceral leishmaniasis

 -  Complete or incomplete treatment with antimony compounds in the last three months.

 -  Patients with history of hepatic, renal or cardiovascular disease.

 -  Mentally or neurologically disabled patients that are considered not fit to approve
 their participation in the study.