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. 2019 Sep 20;44(1):79–87. doi: 10.1007/s12639-019-01162-y

Drug-containing hydrophobic dressings as a topical experimental therapy for cutaneous leishmaniasis

Viviane Pereira 1,2, Neuza Biguinati de Barros 1, Sharon Rose Aragão Macedo 1, Amália dos Santos Ferreira 1, Luiz Alberto Kanis 3, Roberto Nicolete 1,4,✉
PMCID: PMC7046828  PMID: 32174708

Abstract

Cutaneous leishmaniasis (CL), a clinical condition caused mainly by Leishmania amazonensis in Brazil, is characterized by topical, painless ulcers. The current treatment, based on intravenous administration of pentavalent antimonials, presents low adherence by patients and may cause serious adverse effects, leading to the need for searching new therapeutic options. Thus, this study aimed at evaluating a topical administration of “intelligent dressings” as an alternative treatment for CL. BALB/c mice were infected with L. amazonensis promastigotes. Afterward, lesions were treated with hydrophobic dressings incorporated with clinically used drugs. After lesion development, the following analyses were carried out: measurement of lesion diameters, biochemical analyses of serum, evaluation of the recovery of amastigote forms and histological analyses. No significant clinical changes in serum parameters were observed. The group that was treated with dressings impregnated with Glucantime® displayed the lowest number of amastigotes recovered from tissues (parasite load). Conventional treatment with Glucantime® (i.p.) was also able to reduce parasite load. After 6 weeks from the measurement of the lesions mice treated with dressings impregnated with Pentamidine displayed the smallest values. Representative histological aspects of the lesions showed the absence or few amastigotes inside the macrophages when mice were treated with dressings impregnated with Glucantime® and Pentamidine, respectively. The findings presented here indicate that the topical treatments may constitute an alternative treatment option for CL.

Keywords: Cutaneous leishmaniasis, Topical treatment, Hydrophobic “intelligent” bandage aid, Antileishmanial drugs

Introduction

Leishmaniases are neglected diseases, constituting a public health problem, being one of the six endemic parasitic diseases of the highest relevance, owing to its high coefficient of detection and capacity to cause deformities (Chappuis et al. 2007; Anonymous 2015).

The American Cutaneous Leishmaniasis (ACL) present several patterns which may vary according to the phlebotomine vectors involved in their transmissions, susceptibility, the human population involved and level of exposure, as well as the diversity of hosts (de Brito et al. 2012). The disease is known to produce ulcerative, limited cutaneous lesions, and disfiguring lesions in the mucous tissues of nose, mouth and pharynx in the mucocutaneous form (Murback et al. 2011).

Leishmania amazonensis, the main causative agent of ACL in humans, is widely distributed in Brazil. The typical ulcer is painless and is usually present in exposed areas of the skin and are rounded or oval-shaped; measure from a few milimeters to some centimeters; present erythematous infiltrated papule of firm consistency; borders are well limited and elevated, containing a high number of parasites; the bottom of the lesion is red (Andrade et al. 2005).

It is estimated that between 2005 and 2013, 1.7 million cases were reported worldwide, with approximately 11% of these reports occurring in Brazil (WHO 2015). In the year 2013 alone, 18,675 confirmed cases of ACL were reported, with the detection rate in the country being 9.16/100,000 inhabitants. In Brazil, most of the cases occur in the North region, being 4 to 6 times higher than the national average, with a detection rate of 50.1/100,000 habitants (Anonymous 2016).

Teles et al. (2015) stated that the wide variety of etiological agents hinders the control measures against the disease and thus specific strategies are needed according to the epidemiology and transmission pattern of each region in Brazil.

For the treatment of leishmaniasis, the pentavalent antimonials (Sodium stibogluconate, Pentostam® or meglumine antimoniate, Glucantime®) remains as the first line treatment for Cutaneous Leishmaniasis (CL) in most of the countries (Reveiz et al. 2013). The second line drugs, such as Pentamidine and Amphotericin B, also present high toxicity, might not cure, and may lead to resistance (Croft et al. 2003; Lima et al. 2007). Miltefosine (Godinho et al. 2012) and Tamoxifen (Miguel et al. 2008) are also options for alternative chemotherapy, however they have not yet been clinically approved in Brazil.

Recent studies have demonstrated that the topical administration may be promising for the treatment of CL (Carneiro et al. 2012). The advantages of topical treatments include reduced cost; lower systemic toxicity; and adhesion of the treatment by the patient (ease of administration) (Garnier et al. 2007). The application of a topical formulation presenting low toxicity and a higher local concentration of the drug appears as a possibility to overcome the lack of therapeutic options, by the administration of a safer, more efficient regime (Berman 2005).

In the search for the use of intelligent dressings for topical treatment of ACL, Oliveira (2013) has found promising results, with up to 70% reduction in the number of treated ulcers, culminating in clinical cure, according to the author. In a study by Celes et al. (2016), who tested a topical formulation of bacterial cellulose (BC) membranes containing Diethyldithiocarbamate (DETC), infection by Leishmania braziliensis was found to be notably reduced after three weeks of treatment.

Thus, Kanis (2005) proposed the preparation of dressings using a homogeneous monolithic system with a drug release process by diffusion to the surface of the matrix. It was developed a monolithic system that was used in the production of the transdermal system in order to increase the time of constant drug release with controlled diffusion gradient.

In order to better stablish an alternative approach to the treatment of ACL, it is of great relevance to develop an experimental model using clinically antileishmanial drugs that are granted by the Brazilian Ministry of Health (Glucantime®, Amphotericin B, Pentamidine), delivered through flexible Band-Aid®-type dressings, as presented in this study.

Materials and methods

Parasites

Leishmania amazonensis (IFLA/BR/67/PH8) promastigotes were cultured in RPMI 1640 medium + 10% Fetal Bovine Serum (FBS), as recommended by Barros et al. (2013). Cultures were maintained from BALB/c mice previously inoculated with 105 of L. amazonensis promastigotes, subcutaneously, at the dorsum and base of the tail.

Animals

For the experiments, 25 male, 8–10 weeks old BALB/c mice were used, weighing 25-30 g, obtained from a vivarium (FIOCRUZ-RO). The experiments were performed according to the orientations established by the Animal Ethics Committee (CEUA) and approved under no. 2015/12 of Fundação Oswaldo Cruz-RO (FIOCRUZ-RO).

Dressings structure and drug-delivery system

The dressings used in this experimental model were manufactured by incorporating the drugs used in the treatment of leishmaniasis: Glucantime® (2 mg/mL/cm2), Amphotericin B (0.1 mg/mL/cm2) and Pentamidine solution (1 mg/mL/cm2).

For the production of the devices, a system composed of a porous Non Woven Fabric (TNT) base coated with a layer of Polyvinyl Acetate (PVA) (99% hydrolysis) and 10% glycerol was used as plasticizer containing the drugs. The proposed system for drug delivery in dressings is based on monolithic or matrix systems wherein the drug is evenly distributed in a polymer matrix in the form of a solution, suspension or dispersed solid (Kanis 2005).

Experimental treatment of BALB/c mice

After the lesion development (≅ 100 days), the treatment started using dressings and the mice (n = 25) were grouped according to the size of their ulcerative lesions and identified according to the treatment administered to them in five animal groups (Control/no drug—Group 1; Glucantime® i.p.—Group 2; dressings impregnated with: Glucantime®—Group 3; Amphotericin B—Group 4 and Pentamidine—Group 5).

The drug-containing dressings treatment was conducted according to the recommendations of the Brazilian Ministry of Health, which corresponded to each animal group with Glucantime® at 2 mg/mL/cm2, Amphotericin B at 0.1 mg/mL/cm2 and Pentamidine solutions at 1 mg/mL/cm2. Glucantime® i.p. was also administered at 300 mg/kg. The treatment lasted 15 days in both topical and i.p. treatments. In this period, the progress of the ulcerative lesions was evaluated daily, plus 10 days without using the dressings.

Biochemical analyses of the serum

The biochemical parameters of the animals analyzed in this study were those that are requested for patients undergoing treatment according to Brazilian Ministry of Health (Anonymous 2007), for renal function: urea, creatinine; pancreatic function: alkaline phosphatase and hepatic function: transaminases (ALT/AST). Also, it was conducted the analysis of amylase levels.

Blood samples were obtained after the animals were euthanized from the abdominal vena cava. The samples were centrifuged for 5 min in order to obtain a serum sample from each treatment group. For the analysis, an automatic equipment (Trademark Konelab®, model 60i with the kits Wiener Lab®), was used, and the analyses were performed at the Municipal Laboratory of Clinical Analyses of the São Rafael Laboratory, Porto Velho, RO. The biochemical analyses were compared to reference values proposed by Spinelli et al. (2012) and Almeida et al. (2008).

Evaluation of ulcer size

The ulcers were monitored through linear measure of the lesion size. The readings were performed at the beginning of the use of the dressing, at the end, and afterward every 10 days until the animals were euthanized. The assessment of ulcer size was performed by linear measurements of the size or extent of wounds (length and width), showing the tendency of the healing process, as described by Carvalho et al. (2006).

Amastigotes recovered from lesions of the dorsum tissue of infected and treated mice

After the treatment and evaluation of ulcer progression, the animals were euthanized and the ulcer tissues, together with necrotic skin were removed in sterile conditions, with only viable tissues being used. Afterward, part of the tissues was macerated in RPMI medium in order to release the L. amazonensis amastigotes.

As recommended by De Barros et al. (2018) for in vitro culture of promastigote forms, tissue samples (2 mg) of each paw were macerated and diluted 100 × in RPMI medium + 10% FBS. Subsequently, the samples were plated, and the concentrate was centrifuged at 200×g for 10 min at 4 °C. After 7 days in a B.O.D incubator at 25 °C, an aliquot of transformed promastigotes was diluted in erythrosin B (0.04%) and counted in a Neubauer chamber using an optical microscope (400×). Red-colored parasites were considered to be dead, whereas those that were birefringent and mobile were considered to be alive.

Histological analysis

After the animals were euthanized, the tissues were collected and fixed in 10% formaldehyde (pH 7.2) for a period of 24 h. After this period, tissues were stored in buffered formalin (10% v/v) until histological processing and visualization using an optical microscope (400×).

In this experiment, a quali-quantitative analysis was done. It was qualitative in respect to the presence or absence of parasites in the tissue of the ulcer of infected mice (treated and untreated), and quantitative in respect to the features of tissue lesion. These analyses were performed in collaboration with the Laboratory of Parasitology of the Federal University of Acre - UFAC.

Statistical analysis

Statistical analyses were performed after the end of all experiments, using the software GraphPad Prism 5.0, by employing the analysis of variance (ANOVA and the Tukey post-test, with the p value < 0.05.

Results

Serum parameters of the biochemical analysis of hepatic, renal and pancreatic function

The biochemical analysis displayed normal variation for all serum parameters assessed (Table 1), according to track reference values. It is worth to point out that all groups displayed altered AST/ALT levels with no distinction among the treatments and no clinical implications.

Table 1.

Serum biochemical parameters from the infected mice after the treatments

Biochemical parameters Track ref. Group 1 Group 2 Group 3 Group 4 Group 5
Amylase (U/L) 632.80–897.34 484 437 402 452 465
Creatinine (mg/dL) 0.21–0.33 0.3 0.3 0.2 0.3 0.3
ALP (U/L) 191.19–360.21 79 60 62 73 95
SGPT/ALT (U/L) 21.88–32.12 28 35 42 57 48
SGOT/AST(U/L) 18.09–30.31 67 64 86 87 70
Urea (mg/dL) 44.22–54.46 36 39 43 43 40
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Blood samples were collected for the evaluation of renal, pancreatic and hepatic functions. Track references (Almeida et al. 2008; Spinelli et al. 2012) are reported. Representative values (mean) of Control (no drug)—Group 1; Glucantime® (300 mg/kg, i.p.)—Group 2; topically with hydrophobic dressings containing Glucantime® (2 mg/mL/cm2)—Group 3, Amphotericin B (0.1 mg/mL/cm2)—Group 4 and Pentamidine (1 mg/mL/cm2)—Group 5

Parasite load and ulcerative lesion size progress after treatments

In the tissue recovered from the lesions, the parasite load of the Control group was approximately 56 × 105L. amazonensis promastigotes; in the group treated with Glucantime® (i.p.) the parasite load was approximately 23 × 105 parasites. The group treated with Glucantime® impregnated in the dressings displayed the lowest number of parasites recovered (3 × 105 promastigotes). Also, dressings impregnated with Pentamidine displayed low number of promastigotes (approximately 20 × 105) (Fig. 1).

Fig. 1.

Fig. 1

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Quantification of the parasite load of the lesions after treatment. Experimental groups: Control (no drug); Glucantime® (300 mg/kg, i.p.; topically with hydrophobic dressings containing Glucantime® (2 mg/mL/cm2), Amphotericin B (0.1 mg/mL/cm2) and Pentamidine (1 mg/mL/cm2). Data are expressed as mean ± S.D. from two different experiments. **p < 0.01 compared to the control group; # p < 0.05 compared to the Amphotericin B group

The analyses of the lesions revealed significant differences in the progression of lesions area measured in cm2 during the treatment, which were statistically significant (p < 0.05) when compared Control to Pentamidine group; also Glucantime® (i.p.) compared to Pentamidine group and Amphotericin B compared to Pentamidine group (Fig. 2). Especially, Glucantime® (i.p.) and Pentamidine-impregnated dressings were able to greatly reduce the lesion sizes compared to other groups. On the other hand, those dressings impregnated with Amphotericin B were not able to reduce the lesion sizes at the end of the treatment. Visual evolution of the ulcers during each treatment can also be observed in the panels of Fig. 3. Groups treated with the dressings impregnated with Glucantime® and Pentamidine displayed lesions with partial/total wound healing after six weeks.

Fig. 2.

Fig. 2

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Area progression (cm2) of the ulcers during the treatments. L. amazonensis promastigotes were inoculated at the dorsum and base of the tail of Balb/c mice. After lesion development, they were treated for 15 days with Glucantime® (300 mg/kg) (i.p.) and topically with hydrophobic dressings containing Glucantime® (2 mg/mL/cm2), Amphotericin B (0.1 mg/mL/cm2) and Pentamidine (1 mg/mL/cm2). The analyses of the lesions revealed statistically significant difference (p < 0.05); Control compared to Pentamidine group (asterisk); Glucantime® compared to Pentamidine group (right pointing triangle); Amphotericin B compared to Pentamidine group (circlet times operator). Data are expressed as mean ± S.D. from two different experiments

Fig. 3.

Fig. 3

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Visual evolution of the ulcers during the treatments. Mice were inoculated with L. amazonensis promastigotes in the dorsum at the base of the tail and treated 15 days with Glucantime® 300 mg/kg (i.p.) and topical treatment with hydrophobic dressings containing Glucantime® (2 mg/mL/cm2), Amphotericin B (0.1 mg/mL/cm2) and Pentamidine (1 mg/mL/cm2)

Histological analysis of the parasite presence and inflammatory infiltrate

In the quali-quantitative histological analysis, protozoa could be observed in one animal of the group, i.e., in 33.3% of the animals treated with Glucantime® i.p. (300 mg/kg) and in the groups treated with Amphotericin B and Pentamidine. The protozoa were visualized in two microscopic slides for each sample. No parasite was detected in the animal group treated via topical application of Glucantime®, i.e., in 100% of the animals (Fig. 4).

Fig. 4.

Fig. 4

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Representative histological aspects of the lesions on the dorsum of BALB/c mice. Animals were infected with L. amazonensis promastigotes by the intradermal route. Visualization of X400 (10 μm) for the acquisition of images. a Group 1- Control; b Group 2- Glucantime® I.P; d Group 4 Amphotericin; b and e Group 5 Pentamidine: presence of internalized amastigotes in macrophages (indicated by arrows); c Group 3 Glucantime®: Absence of internalized amastigotes

The histological analysis of parts of the ulcer tissues was qualitatively marked by either the presence or absence of Leishmania amastigotes in the intracellular medium of the macrophages in the tissue. Numerous macrophages in the cytoplasm, which were large, pale and vacuolized were found in the dermis of mice of the following groups infected with L. amazonensis: Control (Group 1)—Panel A; Glucantime® i.p. (Group 2)—Panel B; Amphotericin B (Group 4)—Panel D and Pentamidine (Group 5) - Panel E. In the group treated with Glucantime® (Group 3)—Panel C, no parasites were found. In histological sections, it was possible to observe chronic granulomatous inflammation in 85% and chronic necrotizing inflammation in 25% of the analyzed tissues by optical microscopy.

Discussion

The L. amazonensis infection used in this study constitutes a viable model to evaluate the experimental therapy with the dressings evaluated here, since it allowed us to study the interference of antileishmanial drugs with a pattern of therapeutic response closer to that observed in humans.

The clinical parameters for the experimental model proposed through the use of an intelligent dressing, containing a diffusion drug-delivery system, presented significant preliminary results in the analyzed conditions here.

During the treatment period, lesions were healed in treated animals with dressings containing Pentamidine, during the period from 2 to 5 weeks, but they did not remain stable, as there was opening of the cicatricial tissue, developing a new lesion. For the other dressings, a size reduction was obtained in all treatments, that is, 66.6% of the animals, in the third week. However, when the dressings were removed, there was a considerable increase in lesion size in all animals treated with Amphotericin B (group 4).

In the experimental model conducted by Oliveira (2013), using a bioactive dressing, through hydrogel membranes, chitosan and clay, associated with Glucantime®, a 70% reduction of ulcers were obtained in some animals, being validated as a clinical cure only by the presence of the scar tissue. However, scarring of the lesion is not a mandatory and definitive requirement for the animal model, since in literature studies involving the use of Amphotericin B show reduction and disappearance of lesions during treatment, with a posterior recurrence of the lesions after a certain period (Solomon et al. 2011).

In this reasoning, Costa et al. (2009), mention that other criteria besides ulcer healing should be considered, such as biochemical laboratory analysis and histological analysis of tissues. These analyses proposed by the mentioned author were carried out in this experimental model to determine the validation of the dressings used.

In the present study, the histological analysis confirmed the presence of amastigote forms, in nests and internalized in macrophages in the groups treated with Glucantime® (i.p), Amphotericin B and Pentamidine, and also the presence of inflammation that was described and characterized by a granulomatous inflammation in 85% and 25% by necrotizing inflammation in the tissue of the biopsies.

From a histopathological point of view, the tissues affected by ACL are characterized by a lymphoplasmocytic infiltrate with the presence of granulomas and lack or small number of parasites. However, according to Bittencourt (2009), the frequency of parasitism and the presence of granulomatous reaction should also be considered. Silveira et al. (2008) state that ACL is a chronic inflammatory disease, and may or not be accompanied by tissue necrosis and granulomatous reactions caused by macrophages.

In respect to necrotic and inflammatory processes, the parasites also exert an important role in the immune response, contributing to the aggravation of the ulcers, which may also disseminate through the body. Carvalho et al. (2006) found that the non-regression and reappearing of the lesion might be due to the persistence of the inflammatory process induced by IL-10, mediated by IFN-γ and TNF-α. However, because this study tried to validate the intelligent dressings and its action with the drugs in the topical administration, the systemic profile of the immune response exerted by Glucantime® was not evaluated by the quantification of serum cytokines. Here, other analyses were carried out, such as calculating parasite death selectivity after amastigotes recovery from macerated ulcer tissues. The average of dead parasites for all the treatments was calculated and compared to the untreated infected control group. The topical action of Glucantime® dressing revealed the greatest parasite control (lowest number of parasite load) when compared to the intraperitoneal treatment with Glucantime®, followed by the treatments consisting in dressings containing Amphoterin B and also Pentamidine. These results indicate an efficient action of the topical Glucantime® treatment.

It is noteworthy that the ulcers presented between the groups were different and histological analysis did not reveal the presence of parasites in the biopsy specimens of each animal. Considering this phenomenon, which revealed the non-healing of the lesions, one may also suggest the presence of opportunistic infections, since the animals were not isolated in individual cages and the environment of the vivarium does not guarantee the absence of pathogens.

As reported by Silva et al. (2016), Glucantime® administered by the intralesional route, when used for the treatment of ACL, has potential advantages including the use of low total doses of antimonials and the possibility of a more flexible treatment scheme, without the need for daily drug administration. These authors also state that the cure rates described by this intralesional administration of the treatment are similar to those observed by other authors and also resemble the rates obtained with the systemic therapy.

When evaluating pancreatic, hepatic, renal and metabolic changes due to the use of the drugs administered during the treatment of ACL, they should be assessed before, during and after treatment (Bergonso 2007). The biochemical dosages of a blood pool for hepatic, renal and pancreatic function assessment, after the end of the treatment, presented absolute serum values that were compared with reference values proposed by Almeida et al. (2008) and Spinelli et al. (2012), which revealed changes in values for hepatic transaminases for all treatments and even for the control group (without treatment). These transaminases are specific markers of liver damage. However, Mincis and Mincis (2001) point out that there is little correlation between hepatic damage intensity and transaminase levels, but may be related to drug use, corroborating possible adverse reactions of the drugs used in this study. Rath et al. (2003) and Oliveira et al. (2011) reported that the use of Glucantime® causes a reversible elevation of liver enzymes and that hepatotoxicity is also frequent and may occur in as many as 50% of treated cases due to the accumulation of the drug in the liver, which is not observed in the possible adverse reactions caused by Amphotericin B and Pentamidine (Anonymous 2010).

The results of the experimental model achieved in this study corroborate the studies conducted by Alkhawajah et al. (1997), which compared the intramuscular treatment with the intralesional administration of Glucantime® in humans with ACL, with a healing of 73% of the ulcers after 30 days of treatment, and without alterations of hematological and biochemicals parameters. For the other parameters analyzed, they were considered unsatisfactory due to the presence of amastigotes in the tissues and worsening with an increase of the lesion size and inflammatory infiltrate.

Pinheiro (2004) and Kanis (2005) stated that the permeation potential through the use of dressings, membranes and others using diffusion drug release process presents great advantages and may be promising as a topical treatment. In this context, the topical dressing named as “intelligent dressing” was able to release the drug through diffusion, guaranteeing clinical safety by maintaining the renal and pancreatic systems of the animals without major changes, while ensuring its antileishmanial activity by inducing parasite death by an average of 98.21%, also characterized by the total absence of parasites in the tissues of the lesions by histological analysis.

Thus, formulations for topical administration are said to be of great importance as they may offer numerous advantages from application to treatment adherence. In addition, they facilitate the penetration of drugs through the skin barrier, favoring the absorption of topical drugs, constituting an alternative to parenteral administration. The search for new therapeutic alternatives for the treatment of ACL is of high necessity. Improving the experimental model is essential, since a validated treatment based on intelligent dressings may result in a higher adherence of the treatment by the patient, less adverse effects and ease of treatment administration, especially when ACL mostly occurs in locations in which the healthcare is precarious.

The findings observed in this study indicated that the topical therapy may be an alternative for the current treatment of cutaneous leishmaniasis, especially, the topical treatment with Glucantime® and Pentamidine. With the results achieved here, new strategies for pharmacological association between the dressings developed and low doses of the antileishmanial drugs can be further applied in this experimental model. However, this study still presented some limitations, and thus, new experimental assay models are required to further validate the findings observed here.

Acknowledgements

The authors express their gratitude to Fundação Oswaldo Cruz (FIOCRUZ), Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES).

Author contributions

VP, NBB and SRAM conducted all experimental assays and standardization. ASF provided help in some assays. LAK developed the topical hydrophobic bandages and RN supervised the study and mentored the students.

Funding

Dr. Nicolete reports grants from FAPERO, grants from FIOCRUZ during the conduct of the study.

Compliance with ethical standard

Conflict of interest

All authors declare that they have no conflict of interest.

Ethical standard

The study was conducted according to all aspects of ethical standard.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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