Gallic and Ellagic Acids Are Promising Adjuvants to Conventional Amphotericin B for the Treatment of Cutaneous Leishmaniasis

In this study, we demonstrated the potential associative effect of combining conventional amphotericin B (Amph B) with gallic acid (GA) and with ellagic acid (EA) in topical formulations for the treatment of cutaneous leishmaniasis in BALB/c mice. Preliminary stability tests of the formulations and in vitro drug release studies with Amph B, GA, Amph B plus GA, EA, and Amph B plus EA were carried out, as well as assessment of the in vivo treatment of BALB/c mice infected with Leishmania major.

ABSTRACT

In this study, we demonstrated the potential associative effect of combining conventional amphotericin B (Amph B) with gallic acid (GA) and with ellagic acid (EA) in topical formulations for the treatment of cutaneous leishmaniasis in BALB/c mice. Preliminary stability tests of the formulations and in vitro drug release studies with Amph B, GA, Amph B plus GA, EA, and Amph B plus EA were carried out, as well as assessment of the in vivo treatment of BALB/c mice infected with Leishmania major. After 40 days of infection, the animals were divided into 6 groups and treated twice a day for 21 days with a gel containing Amph B, GA, Amph B plus GA, EA, or Amph B plus EA, and the negative-control group was treated with the vehicle. In the animals that received treatment, there was reduction of the lesion size and reduction of the parasitic load. Histopathological analysis of the treatments with GA, EA, and combinations with Amph B showed circumscribed lesions with the presence of fibroblasts, granulation tissue, and collagen deposition, as well as the presence of activated macrophages. The formulations containing GA and EA activated macrophages in all evaluated parameters, resulting in the activation of cells of the innate immune response, which can generate healing and protection. GA and EA produced an associative effect with Amph B, which makes them promising for use with conventional Amph B in the treatment of cutaneous leishmaniasis.

INTRODUCTION

Leishmaniasis is a parasitic disease caused by protozoa of the genus Leishmania. Leishmaniasis is considered a neglected tropical disease (1), and it is estimated that 1.5 million new cases arise each year (2–4). Widely distributed, the infection is transmitted through the blood of female sand flies, which live in forest areas, caves, and rodent dens and also in peridomiciliary areas (5, 6). Among the forms of clinical manifestation (cutaneous, mucocutaneous, and visceral), the cutaneous form is responsible for causing physical deformities in patients, with Leishmania major being one of the main species responsible for this disease (7, 8). Due to the lack of an effective vaccine and limited control measures, chemotherapeutic treatment is still the main way to fight the disease (8).

The drugs used in treatment are limited and costly and have a range of side effects (3, 9, 10). Natural products have been extensively investigated in an attempt to discover new antileishmanial bioactive compounds that possess activity against the parasite as well as the ability to act in synergism with the immune response of the host (11–13). In this regard, the antileishmanial and immunomodulatory potentials of gallic acid (GA) and ellagic acid (EA), derived from the secondary metabolism of plants, were studied previously by our group (11).

Among the various drugs used to treat leishmaniasis, amphotericin B (Amph B) is a polyene antibiotic used in the treatment of leishmaniasis (14, 15). The selective activity of this drug against leishmania parasites is due to its high affinity for 24-substituted sterols, which are predominant in the plasma membrane of these microorganisms, compared to the cholesterol present in the membranes of mammalian cells (15, 16). However, the use of Amph B is limited by its toxic side effects, especially cardio- and nephrotoxicity, in addition to myalgia, arthralgia, anorexia, fever, and urticaria; it also exhibits hepatic, splenic, and renal toxicity, as revealed by abnormal laboratory tests such as urea, creatinine, aspartate aminotransferase, and alanine aminotransferase (ALT) (17–19).

Drug discovery is an expensive process that requires an averages of 10 to 20 years, in addition to an investment of more than $1.0 billion before a new compound with activity against a specific target can be released for clinical use (20). Delivery systems carrying conventional medicines can improve the efficacy of these compounds and, consequently, reduce their adverse effects (21). Concurrently with these systems, combinations of drugs can provide potential synergism and thus better therapeutic success in treating disease, and this represents one of the most promising approaches for the development of new antileishmanial formulations (22, 23).

The aim of the present study was to evaluate the therapeutic potential of topical formulations containing Amph B, GA, and EA and the therapeutic and immunomodulatory effects of the combinations Amph B + GA and Amph B + EA, carried in a poloxamer 407 gel, using BALB/c mice as an experimental model (24, 25).

RESULTS

Preparation and preliminary stability of topical formulations.

Table 1 presents the results of the evaluation of the preliminary stability of the formulations, determined by testing for content (as a percentage), pH, conductivity, and organoleptic characteristics at time zero (T₀) after thermal stress and freeze-thawing.

TABLE 1.

Stability of topical gels containing Amph B, AG, Amph B + AG, AE, and Amph B + AE

Time and formulation	Content (%)	pH	Conductivity (μS/cm)	Organoleptic characteristics
Time 0
Amph B	90.89 ± 2.92	5.02 ± 0.01	142.8 ± 4.75	Yellow, characteristic odor, yellowish-colored gel without precipitate
AG	108.15 ± 0.99	3.33 ± 0.02	146.57 ± 0.58	Yellow, characteristic odor, yellowish-colored gel without precipitate
Amph B + AG	109.49 ± 5.61, 91.48 ± 1.11	3.66 ± 0.01	228.87 ± 0.45	Yellow, characteristic odor, yellowish-colored gel without precipitate
AE	94.01 ± 3.32	3.76 ± 0.03	4,269.67 ± 9.50	Yellow, characteristic odor, yellowish-colored gel without precipitate
Amph B + AE	108.71 ± 5.55, 90.76 ± 2.92	4.60 ± 0.01	2,753.33 ± 15.28	Yellow, characteristic odor, yellowish-colored gel without precipitate
After thermal stress
Amph B	90.31 ± 3.87	5.11 ± 0.01a	254.00 ± 3.46a	Slightly modified
AG	102.77 ± 3.34	3.44 ± 0.01a	149.70 ± 1.11a	Slightly modified
Amph B + AG	107.58 ± 3.48, 90.80 ± 1.67	3.89 ± 0.01a	222.73 ± 0.12a	Slightly modified
AE	96.61 ± 7.89	4.01 ± 0.02a	4,263.33 ± 5.77	Slightly modified
Amph B + AE	99.45 ± 3.69, 91.69 ± 1.07	4.82 ± 0.01a	2,856.67 ± 57.74a	Slightly modified
After freeze-thaw cycle
Amph B	62.78 ± 3.12a	4.97 ± 0.05	214.40 ± 2.61a	Slightly modified
AG	97.14 ± 1.73a	3.48 ± 0.03a	154.20 ± 0.61a	Slightly modified
Amph B + AG	104.02 ± 3.11, 82.05 ± 3.63a	3.87 ± 0.03a	232.57 ± 0.55a	Slightly modified
AE	90.86 ± 2.92	4.03 ± 0.02a	4,316.67 ± 5.77a	Slightly modified
Amph B + AE	107.38 ± 1.66, 79.76 ± 2.00a	4.95 ± 0.04a	2,906.67 ± 5.77a	Slightly modified

^aStatistically significant difference compared with time 0 (P > 0.05; Student’s t test).

With regard to the content at T₀ and after thermal stress, we observed that all the formulations were within the acceptable limit (90% to 110%) for semisolid formulations (26). However, there was a reduction to 62.78% of the content in the formulation of Amph B after the freeze-thaw cycling. The formulations had a pH compatible with that of the skin; however, Amph B and Amph B + EA were within the limit established for formulations designed for prolonged residence on the skin, between 4 and 7 (26, 27).

The determination of the conductivity is an attempt to classify the type of formulation based on the phase distribution, allowing the determination of the conductivity of polar or nonpolar domains. At T₀, an increase in conductivity was observed in the combination formulations Amph B + GA and Amph B + EA. This may be due to the incorporation of acids into the formulation. After thermal stress and freeze-thaw cycling, slight increases in pH and conductivity and a slight modification of the organoleptic characteristics were observed in all the formulations.

In vitro release profiles of topical formulations.

The formulations showed the following accumulated quantities released (liberated): Amph B, 80.77 ± 2.35 μg cm⁻² (2.38%); GA, 2,547.01 ± 125.58 μg cm⁻² (73.80%); EA, 1,511.95 ± 230.08 μg cm⁻² (63.13%); Amph B + GA, 70.18 ± 2.34 μg cm⁻²/1,180.65 ± 136.74 μg cm⁻² (4.05%/68.23%); and Amph B + EA, 94.06 ± 13.98 μg cm⁻²/1,245.69 ± 166.08 μg cm⁻² (5.41%/71.75%) (Fig. 1). GA showed the largest amount released, followed by EA and then Amph B, in 6 h. The combined formulations showed excellent GA and EA release ability and also helped increase the release of Amph B due to its low aqueous solubility. Thus, these formulations have potential for the topical treatment of cutaneous leishmaniasis.

FIG 1.

In vitro release kinetics of gel containing Amph B, GA, Amph B + GA, EA, and Amph B + EA. (A) The single formulations of Amph B, GA, and EA released, respectively, 2.38%, 73.80%, and 63.13%. (B) In combination with GA and EA, Amph B exhibited discernible increases in the quantity released, corresponding to 4.05% and 5.41%, respectively.

Topical antileishmanial activity.

During the 40 days after the BALB/c infection by L. major, the period that preceded the beginning of treatment, all groups showed uniformity in the clinical development of the disease. From this period, a delay in advancement of lesions and/or significant clinical improvement of the disease was observed in animals that received treatment of topical formulations containing Amph B, GA, Amph B + GA, EA, and Amph B + EA for 21 consecutive days. This effect lasted until the end of the experiment, which was 14 days after the end of the 21-day treatment (Fig. 2).

FIG 2.

Clinical evaluation of BALB/c mice infected with L. major and treated with topical formulations. After 40 days of infection, the animals were treated twice a day for 21 days. A reduction in lesions was observed in animals treated with topical formulations containing Amph B, GA, Amph B + GA, EA, and Amph B + EA.

The groups that received treatment with Amph B + GA and Amph B + EA had results similar to those obtained with Amph B, GA, and EA, even with the reduction of the concentration of these drugs in the formulations, which indicates a possible associative effect between these drugs.

When the parasite load of BALB/c mice was evaluated, a significant reduction on a logarithmic scale in the number of parasites recovered from the site of injury was observed in animals that received treatment with topical formulations containing Amph B, GA, Amph B + GA, EA, and Amph B + EA (Fig. 3). The percentages of recovered parasites were 74%, 77.7%, 85.1%, 81.4%, and 85.1%, respectively. It was also observed that these values remained low (51.4%, 68.5%, 82.8%, 77.1%, and 85.7%, respectively) even 14 days after the end of the experiment, suggesting a protective effect of the gallic acid and ellagic acid used in the topical gels, possibly because they activate the immune response. For the Amph B gel, a greater increase in parasite load was observed when the treatment was finished than with the other treatments. The formulations containing combinations of Amph B and the adjuvant GA or EA, even with the reduction of concentration of each active ingredient, resulted in noticeable reductions of the parasite load during the treatment period, thus demonstrating a potential associative effect between Amph B and GA and EA.

FIG 3.

Parasites (log₁₀/ml) recovered from lesions caused by L. major. After 40 days of infection, the animals were treated twice a day for 21 days. A reduction of parasite load was observed in the animals treated with topical formulations containing Amph B, GA, Amph B + GA, EA, and Amph B + EA during and after the treatment.

During ex vivo evaluation, skin fragments removed from the leishmaniotic ulcers were imprinted and the morphology of macrophages was observed. Macrophages were observed to be widely spread, with a fusiform and smoky appearance, from animals treated with GA and EA as well as those treated with Amph B + GA and Amph B + EA, despite reduction of the active principle. These characteristics indicate that these cells were activated and that, once activated by the action of the drugs, they were able to reduce the infection (Fig. 3). In the control and Amph B groups, macrophages were observed with a large amount of unspread amastigotes and large parasitic vacuoles.

Histopathological analysis.

The observed histopathological characteristics of the skin were similar during and after treatment. The animals in the control group showed diffuse lesions covering the deep and superficial dermis, with areas of necrosis, secondary bacterial infection, ulcerations (but without signs of fibrosis or presence of granulation tissue), degeneration of muscle fibers, and macrophages with large parasitic vacuoles filled with leishmaniae in the area of interface dermatitis (Fig. 4A). The animals treated with Amph B gel displayed areas of necrosis, ulcer, interface dermatitis, and the presence of nonactivated macrophages with large parasitic vacuoles filled with leishmaniae and intraepidermal pustules (Fig. 4B). In animals treated with GA gel, the presence of diffuse eosinophils and proliferation of fibroblasts in the granulation tissue were observed, besides a circumscribed area of perivascular dermatitis with spreading, roundish and amoeboid macrophages (Fig. 4C). For the animals treated with Amph B + GA gel, a focal area of acanthosis, panniculitis, and diffuse dermatitis with activated and spreading macrophages intercalated with nodular granulation tissue having numerous neoformed vessels, fibroblast proliferation, and deposition of collagen fibers were observed (Fig. 4D). The animals treated with EA gel demonstrated multifocal lesions in the deep dermis (multifocal dermatitis), with the presence of eosinophils and small shiny eosinophilic debris, as well as diffuse mononuclear inflammatory infiltrate with activated spreading macrophages, present in the immature granulated tissue (Fig. 4E). Finally, the animals that were treated with gel containing Amph B + EA exhibited circumscribed lesions (perivascular dermatitis) interspersed with immature granulation tissue, with the presence of eosinophils as well as activated, hemorrhaging, and spreading macrophages, besides focal calcification of the conjunctive tissue (Fig. 4F).

FIG 4.

Photomicrographs of cutaneous lesions caused by L. major in BALB/c mice after topical treatment with gel containing Amph B, GA, Amph B + GA, EA, and Amph B + EA. (A) A large area of epidermal necrosis, diffuse mixed inflammatory infiltrate throughout the dermis (+), interface dermatitis due to the presence of a large number of amastigotes forms in the papillary dermis (¬), and ulceration and necrosis (*) were observed in the animals of the control group. (B) In the animals that received treatment with topical formulations containing Amph B, an area of necrosis, diffuse inflammatory lesions (+), and intraepidermal pustular ulcerations (*) were observed. (C to F) The animals receiving GA (C), Amph B + GA (D), EA (E), and Amph B + EA (F) showed circumscribed lesions (+), with the presence of activated macrophages, fibroblasts, granulation tissue, and perivascular dermatitis (#) and right focal calcification. Tissues were stained with hematoxylin-eosin. Scale bars, 250 μm and 125 μm.

Activation of macrophages in ex vivo assays.

Peritoneal macrophages were removed from the peritoneal cavities of BALB/c mice during and after the treatment period for assessment of phagocytic capacity, lysosomal activity, and induction of nitric oxide synthesis and quantification of intracellular calcium.

Phagocytic capacity.

For the macrophages of the groups which received topical formulations containing GA, EA, and the combinations Amph B + GA and Amph B + EA, an increase in phagocytic capacity, as determined by zymosan phagocytosis, was observed during the treatment period and lasted for 14 days after treatment (Fig. 5A). Macrophages from animals treated with gel containing Amph B did not show increased phagocytic capacity.

FIG 5.

Activation of murine peritoneal macrophages. Zymosan phagocytosis (A), neutral red (B), nitrite concentration (C), and intracellular calcium (D) were assessed in macrophages obtained from animals treated with topical formulations containing GA, Amph B + GA, EA, and Amph B + EA.

Lysosomal activity.

For the macrophages of the groups treated with topical formulations containing GA, EA, and the combinations Amph B + GA and Amph B + EA, an increased lysosomal activity was observed during the treatment period and lasted for 14 days after the treatment, compared to the control and macrophages removed from normal animals (Fig. 5B). Macrophages from animals treated with Amph B gel did not demonstrate increased lysosomal activity, and those from EA-treated animals showed increased lysosomal activity only after the treatment period.

Nitric oxide synthesis.

The macrophages obtained from BALB/c mice treated with topical formulations containing GA, EA, Amph B + GA, and Amph B + EA exhibited an increase in nitric oxide synthesis, as determined by nitrite concentration (nitric oxide end product), during the treatment period compared to controls and to macrophages removed from normal animals, and this effect lasted for 14 days after the treatment (Fig. 5C). Macrophages from animals treated with Amph B gel did not exhibit increased nitric oxide synthesis.

Quantification of intracellular calcium.

In images captured by confocal microscopy for evaluation of intracellular calcium concentrations by Fluo-3 AM staining, the macrophages from BALB/c mice treated with topical formulations containing GA, EA, Amph B + GA, and Amph B + EA showed increased fluorescence intensity, indicating increased intracellular calcium concentrations. When intracellular calcium concentrations were determined, an increase was observed during the treatment period compared to controls and to macrophages from normal animals, and this increase lasted for 14 days after treatment (Fig. 5D). Macrophages from animals treated with Amph B gel showed no increase in intracellular calcium concentration.

Evaluation of peritoneal macrophages infected with L. major and treated with topical gel.

Macrophages from BALB/c mice treated with topical formulations containing GA, EA, Amph B + GA, and Amph B + EA were infected with promastigote forms of L. major to evaluate the percentages of infected macrophages and the average numbers of surviving amastigotes internalized by macrophages, in order to evaluate the possible resistance of the host organism to possible relapses of the disease. The animals treated with gel containing GA, EA, Amph B + GA, and Amph B + EA showed a reduction in the number of infected macrophages of about 25% compared to the other groups during the treatment and posttreatment periods (Fig. 6A). Concomitantly, a reduction of the number of surviving amastigotes by about 50% was observed, i.e., the number representing half of surviving amastigotes found in the other groups (Fig. 6B).

FIG 6.

Murine peritoneal macrophages infected with promastigotes of L. major and survival index of internalized amastigotes. Significant reductions in the percentages of infected macrophages (A) and surviving amastigotes (B) were observed in the animals treated with topical formulations containing GA, EA, and the combinations Amph B + GA and Amph B + EA.

Toxicity in BALB/c mice infected with L. major and treated with topical gel.

Toxicity was evaluated by assessing biochemical parameters like serum urea, creatinine, and alanine aminotransferase (ALT), as well as hematocrit and total weight evaluation of the animals, during and after topical treatment with gel formulations of Amph B, GA, GA + Amph-B, EA, and EA + Amph B, compared to the control group and to normal animals.

The results show that the treated groups displayed normal levels of serum enzymes, as well as hematocrit and total weight, indicating no apparent toxicity during the treatment period or after treatment. There was a noticeable reduction in the weights of the animals in the group that received treatment with Amph B gel, but this was not statistically significant. The groups that received GA, EA, Amph B + GA, and Amph B + EA exhibited values close to the parameters observed in normal mice. These results are shown in Table 2.

TABLE 2.

Toxicity of topical formulations of Amph B, AG, and AE in BALB/c mice infected with L. major^a

Mouse group	Urea (mg/dl)		Creatinine (mg/dl)		ALT (U/liter)		Hematocrit (%)		Total wt (g)
	Treatment	Posttreatment	Treatment	Posttreatment	Treatment	Posttreatment	Treatment	Posttreatment	Treatment	Posttreatment
Normal	56.7 ± 6.8	54.2 ± 4.4	0.2 ± 0.05	0.2 ± 0.05	67 ± 2.9	67.7 ± 4.6	49.7 ± 1.7	50 ± 3.9	32 ± 2	32.2 ± 1.8
Control	54.5 ± 6.4	46.5 ± 10.2	0.2 ± 0.05	0.2 ± 0.05	69.3 ± 4.9	69.7 ± 4.2	49 ± 2.8	52.2 ± 2.7	29.8 ± 4.7	29.1 ± 3.8
Amph B	60.5 ± 7.7	58.6 ± 7	0.2 ± 0.07	0.2 ± 0.08	76 ± 13.7	69.6 ± 3.5	44 ± 6.3	52 ± 5.4	24 ± 3.8	24.7 ± 1.4
AG	50 ± 4.2	55.6 ± 5.8	0.2 ± 0.01	0.2 ± 0.05	67.5 ± 2.1	66 ± 4.1	47.3 ± 2.8	52 ± 4.2	27 ± 3.9	30.3 ± 4.2
Amph B + AG	57 ± 8.4	55.6 ± 5.8	0.2 ± 0.05	0.2 ± 0.05	74.25 ± 5.6	70 ± 3.6	49 ± 1.4	51 ± 2.6	26.1 ± 3.2	26.4 ± 3.2
AE	52.5 ± 4.9	55 ± 3.4	0.2 ± 0.05	0.2 ± 0.05	60 ± 11.3	60.5 ± 0.7	51 ± 1.4	50 ± 2	27.7 ± 3.7	28 ± 1.7
Amph B + AE	54.5 ± 3.5	58 ± 5.9	0.2 ± 0.05	0.2 ± 0.05	65 ± 5.5	64 ± 9	47.2 ± 5.5	49.5 ± 3.3	27.9 ± 2.5	28.7 ± 4.2

^aIn vivo toxicity was evaluated by comparing biochemical and hematological parameters, as well as total weight, of L. major-infected animals that received treatment and normal (“noninfected”) animals. The results demonstrate that there was no in vivo toxicity in BALB/c mice treated with gels.

DISCUSSION

In a previous study, our group reported the great potential of the antileishmanial and immunomodulator agents gallic acid and ellagic acid and described them as promising molecules in antileishmanial therapy (11). However, because they are hydrophilic molecules, their oral administration is compromised due to the first-pass hepatic metabolism (28). Amphotericin B, the conventional drug of choice for the resolution of the disease, is given by long-term parenteral administration, besides being toxic; in addition, there are already reports of resistance by the parasite (15, 16).

Topical treatments are a promising alternative for the treatment of leishmaniasis, since they offer several advantages over the parenteral route. These advantages are not only limited to the practicality of administration but may also include better therapeutic efficacy and greater safety due to the reduction of the amount of amphotericin B used for the treatment, leading to less toxic side effects (27, 29).

In the preliminary stability tests, our formulations demonstrated changes in the parameters evaluated after thermal stress and the freeze-thaw cycles, which was expected, since poloxamer 407 is composed of two long-chain copolymers that are capable of structural change when subjected to stress. However, it has many advantages, such as the property of being solid at room temperature and liquid at refrigeration temperature. Thus, when in contact with the skin, the gel facilitates the adhesion of the product, acting as an excellent carrier molecule with tensoactive properties and as a promoter of cutaneous permeability (27, 30, 31). In addition, poloxamer gel formulations show good drug release ability, with excellent potential for topical application, including for the topical treatment of cutaneous leishmaniasis, due to its good adhesion to the contact site (30).

In this study, the marked potential of the poloxamer 407-based topical formulations in the treatment of cutaneous leishmaniasis was demonstrated. The single Amph B-, GA-, and EA-based formulations resulted in similar clinical improvement when administered at the same concentrations. Interestingly, these responses were enhanced by administration of Amph B + GA and Amph B + EA combined formulations, compared with Amph B, GA, and EA alone. Furthermore, despite the use of half-concentrations of Amph B, GA, or EA in these combinations, as well as the lower in vitro release of these compounds in combination compared with single formulations, the combinations exhibited higher efficacy in all experimental protocols (27, 32).

In the histopathological analysis, the potential associative effect of Amph B with GA or EA was clear. The presence of activated and eosinophilic macrophages points to a specific cellular response against parasitic agents, and thus, the animals that received this treatment of association demonstrated promising progress in the resolution of the infection and progression to the cicatrization of the wound (27). Studies of conventional drugs and adjuvant molecules, such as Amph B with oleic acid in the same topical delivery type and trivalent antimonial with ascorbic acid in liposomes, also yielded histopathological results that demonstrated progress of the wound toward healing, with the formation of granulomas and activation, which corroborates the results of this study (27, 33).

Because leishmaniasis is an immunopathology, the search for new drugs is carried out by investigating not only the effects of new molecules on the parasite itself but also their ability to act in collaboration with the immune system (25, 32). Macrophages are one of the main targets studied, because they are highly specialized cells that destroy intracellular pathogens when activated (34, 35). The images of the lesions of mice treated with GA, EA, Amph B + GA, and Amph B + EA gels showed the presence of these activated cells, with spreading and hemorrhaging, indicating that these cells were activated and able to resolve the infection (11).

Phagocytic capacity, lysosomal activity, nitric oxide production, and intracellular calcium concentration are examples of functional activities related to the effective activation of macrophages against leishmaniasis (12, 36). Macrophages perform several functions, including phagocytosis, expression of tumor cytotoxicity, cytokine secretion, and antigen presentation. These phagocytes represent an innate line of defense against pathogens and tumor cells, recognizing and destroying them (37, 38). Characteristically, activated macrophages demonstrate increased adhesion and spreading capacity, stimulation of DNA synthesis, increase of intracellular calcium, modification of cytokine secretion, increase of lysosomal enzyme levels, increase of both antimicrobial and antitumoral activity by increased NO and of reactive oxygen species (ROS), and an increase of membrane ruffles, which improve the performance of functions such as locomotion and phagocytosis (39).

Phagocytosis and the lysosomal system are critical for the functions of macrophages, including internalization of pathogens, processing of antigens, and presentation of antigens to specific cells of the adaptive immune system. After endocytosis of the pathogen, the newly created phagosome undergoes sequential fusion events with endosomes and then with lysosomes to produce a phagolysosome (40). The phagolysosome is a compartment full of acid hydrolases, proteins, and ROS (reactive oxygen species), where most of the degradation of the encompassed content occurs. Drugs that act by activating macrophages in this target are able to induce these cells to destroy internalized pathogens in their phagolysosomes, besides helping with upregulation in the presentation of antigen (41, 42).

In antileishmanial activity, an important route that may be involved is the production of NO by macrophages. It has long been considered the most effective mechanism involved in defense against Leishmania species. Within the phagolysosome, NO combines with superoxide anion to produce peroxynitrite, which is highly reactive and antimicrobial (43, 44). The parasite survives within the macrophages by the ability to inhibit inducible nitric oxide synthase (iNOS) expression or activity by inhibiting the production of cytokines involved in the regulation of iNOS, inhibition of NO synthesis by glycosylinositol, surface phospholipids of amastigotes, or stimulation of the production of transforming growth factor β (TGF-β) (35). NO-activating drugs function via activation of macrophages by cytokines, such as gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α), which increase the production of nitric oxide synthase, an enzyme that catalyzes l-arginine to generate NO and citrulline (45). Once inside the phagolysosome, NO combines with superoxide to produce peroxide nitrite, which is highly reactive and acts as a microbicide (43, 45).

The mechanism of action of Amph B is to inhibit the synthesis of ergosterols of the parasite, thus causing the rupture of its plasma membrane (15, 19). When Amph B was combined with these natural immunomodulators, it was also observed to act in association with the immune response of the host, which makes these compounds promising agents for treatment of disease. Our results demonstrate that the activity of the substances tested involves macrophage activation pathways, which possibly potentiated Amph B in association with these molecules, even at a reduced dose.

In settings where leishmaniasis is endemic, there is a high probability that an individual who has already been or is being treated for this disease will be exposed to the infection again, so-called reinfections (5). In this study, it was shown that the immunomodulatory function of the compounds we tested activates macrophages systemically during treatment and also after treatment, suggesting possible generation of immune memory; in addition, a gel vehicle with 20% propylene glycol may have favored penetration of the molecules, since the propylene glycol is a classic promoter of the first generation, and the poloxamer also has promoter characteristics (30, 46). This fact is explained by the reduction in infection and infectivity in the macrophages from the mice treated with the natural compounds and the combination of Amph B with them when they were subjected to infection by promastigotes of L. major, simulating what happens in an area of endemicity.

These compounds also demonstrated safety in terms of hepatic function, renal function, and total weight of the mice. Although there were no statistical differences compared to Amph B, it is known that Amph B is extremely toxic and causes numerous types of discomfort in patients (47). GA and EA have an excellent selectivity index, being more selective in terms of causing toxic effects to the parasite rather than mammalian cells (11). The combinations Amph B + GA and Amph B + EA demonstrated, according to the parameters evaluated in this study, values closer to those in normal animals than to those in animals treated only with Amph B.

This study is the first to describe the effect of conventional Amph B combinations with phenolic compounds in a topical gel, highlighting the excellent immunomodulatory capacity of these natural compounds, which minimize the toxicity of the conventional drug and maximize the therapeutic potential. Therefore, these combinations are promising for the treatment of leishmaniasis, since BALB/c mice were used as an experimental model, and this lineage is highly susceptible to L. major infection (29).

Conclusion.

Topical gels containing GA, EA, Amph B + GA, and Amph B + EA had good drug release capacity, while the gel vehicle with 20% propylene glycol favored the penetration of the molecules, presenting great potential in treating cutaneous leishmaniasis wounds caused by L. major in experimentally infected BALB/c mice. The combinations Amph B + GA and Amph B + EA were shown to be promising in the clinical progress toward a cure by acting systemically on the activation of cellular immune response and relieving the infection, thus preventing possible recurrences. Therefore, GA and EA are promising in the treatment of the disease as monotherapy or in combination with conventional amphotericin B.

MATERIALS AND METHODS

Chemicals.

Schneider's culture medium, RPMI medium, fetal bovine serum (FBS), and the antibiotics penicillin and streptomycin were acquired from Sigma Chemical (Sigma-Aldrich Brazil). Gallic acid (GA) and ellagic acid (EA) were acquired from Alfa Easar (Brazil). Fluo-3 AM was acquired from Sigma-Aldrich (USA). The antibiotic amphotericin B and the anesthetics thiopental and lidocaine were acquired from Cristália (São Paulo, SP, Brazil). Poloxamer 407 and propylene glycol were obtained from ChemSpecs and Dynamics, respectively.

Preparation and preliminary stability of topical formulations.

To obtain the Amph B, GA, GA + Amph B, EA, and EA + Amph B gels, poloxamer 407 was hydrated under refrigeration for 24 h. The drugs were then incorporated by levigation with propylene glycol (approximately 20%). After 24 h of preparation of the formulations, the homogenization and stabilization of the formulations were macroscopically evaluated (30, 48). A concentration of 3% was used for the formulation of Amph B, GA, and EA (27); for the formulations of Amph B + GA and Amph B + EA, the concentration was reduced to 1.5% of each compound in order to evaluate the effect of the combination.

The preliminary study of the formulations’ stability was performed by analyzing the following parameters: organoleptic characteristics, pH, electrical conductivity, and content. In the organoleptic analysis of the formulations, their aspect, color, and homogeneity, as well as instability-related processes such as phase separation, were observed. The parameters were analyzed at the beginning of the studies (T₀) and soon after the thermal stress and freeze-thaw cycles.

The samples for the thermal stress test were subjected to temperatures over the range from 40 to 80°C, with a rate of increase of 10°C/30 min, in a thermostatic water bath. The formulations were evaluated at the end of the 80°C period, after the preparations returned to room temperature (25 ± 2°C). In the freeze-thaw cycle test, samples were subjected to temperatures of −5°C ± 2°C in a freezer for 24 h and then 50°C ± 2°C in an oven for 24 h, completing one cycle. At the end of 12 days (6 cycles), the formulations were analyzed after returning to room temperature (25°C) (26).

Release kinetics of in vitro topical formulations.

The study was conducted using cellulose acetate membranes in Franz-type diffusion cells with a diffusion area of 1.77 cm² and volume of approximately 15 ml. The receptor compartment was filled with phosphate buffer (pH 7.4) mixed with 20% EtOH in a system composed of individual cells (32 ± 0.5°C, 100 rpm). A 200-mg portion of each formulation was applied in the receptor compartment directly on the membrane. Samples of the receiving solution were collected at 0.5, 1, 1.5, 2, 4, and 6 h. The readings for the collected aliquot were performed using UV/visible light with wavelengths of 266.6 nm for GA, 363.4 nm for EA, and 405.4 nm for Amph B. The total volume of the receiving phase was replaced at each sampling for medium replacement and maintenance of sink conditions (26).

Animals and parasites.

Female BALB/c mice (Mus musculus), aged 6 to 8 weeks and weighing between 20 and 25 g, were used. The experiments were approved by the Ethics Committee on Animal Experimentation of Federal University of Piauí (UFPI) (CEEA/UFPI 265/16).

Leishmania major MHOM/IL/80/Friedlin, maintained by successive passages in BALB/c mice, was replicated in cell culture flasks with Schneider's inactivated culture medium and penicillin-streptomycin solution (10,000 IU/ml and 50 μg/ml) and kept in ovens with biochemical oxygen demand (BOD) at 26 ± 1°C (34, 35).

Infection of mice with L. major and treatment with topical formulations.

The animals were infected in the region of the tail base with 1 × 10⁶ of L. major metacyclic promastigotes in 50 μl of sterile saline solution (NaCl, 0.9%). After 40 days, the development of nodules or lesions at the inoculum site was observed. The animals were then divided into six groups (n = 8) according to the treatment to be applied: controls (placebo group, treated with empty poloxamer gel), Amph B (3%), GA (3%), Amph B (1.5%) + GA (1.5%), EA (3%), and Amph B (1.5%) + EA (1.5%).

The animals were treated after this period, twice a day, for 21 days, with 50 μl of the topical formulation for that group, using an automatic micropipette. Euthanasia for collection of biological material and ex vivo trials was performed in two stages. The first was after the end of treatment, and the second was 14 days after the end of treatment.

Clinical evaluation of BALB/c mice infected with L. major and treated with topical formulations.

The weight of the animals was measured during and after treatment. We also performed daily observation of the lesions and weekly measurement of the diameters of the ulcerative lesions or nodes using a pachymeter (Messen). After the two measurements, the mean diameter was calculated. At the end of treatment, the animals were monitored for a period of 14 days, and their lesions were measured weekly to verify possible changes.

Evaluation of parasite load.

Skin fragments from the lesions were weighed and diluted in supplemented Schneider's medium, with 100 mg of tissue in 1,000 μl. They were then crushed and centrifuged at 207 × g at 4°C for 10 min. The supernatant (20 μl) was removed and diluted in 180 μl of supplemented Schneider's medium, and from this, successive serial dilutions were made in 96-well culture plate, in duplicate, conserving the same order of magnitude, starting from a dilution of 10⁻² to a dilution of 10⁻¹⁰. Then, the plates were incubated at 26°C, and after 5 days of culture, the presence or absence of promastigotes forms was observed under an optical microscope, daily for 14 days (25).

Histopathological analysis.

Skin samples from the lesions were fixed in 10% buffered formaldehyde and maintained for a minimum of 48 h. After fixation of the fragments, the samples were taken through the usual histological routine for subsequent analysis by light microscopy, which constitutes the processes of dehydration, clarification, paraffin embedding, packaging, and microtomy. Next, using a manual microtome (Leica RM2235), sections of 4.0 μm were made, adhered to glass slides, subjected to the process of diaphanization (dewaxing) and rehydration in xylol and alcohol, and stained with hematoxylin-eosin (49).

Evaluation of macrophage activation parameters.

(i) Preparation of solutions. A stock solution of neutral red dye (Sigma-Aldrich, St. Louis, MO, USA) was prepared by solubilizing 0.002 g of the dye in 1 ml dimethyl sulfoxide (DMSO). The extraction solution used to evaluate phagocytic capacity and lysosomal activity consists of 96% glacial acetic acid (1% [vol/vol]) and pure ethanol (50% [vol/vol]) dissolved in double-distilled water. Zymosan (Sigma-Aldrich, St. Louis, MO, USA) for evaluation of phagocytic capacity was obtained by diluting 0.3 ml of the stock solution of neutral red and 0.02 g of zymosan in 3 ml of phosphate-buffered saline (PBS), while the fixative used was Baker's formaldehyde-calcium, composed of 4% (vol/vol) formaldehyde, 2% (wt/vol) sodium chloride, and 1% (wt/vol) calcium acetate in distilled water (50).

(ii) Cultivation of macrophages. Peritoneal macrophages from normal animals that had been infected with L. major and treated with the previously described topical formulations were plated, diluted in RPMI medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, to 2 × 10⁵ cells per well in triplicate in 96-well plates, and incubated in an incubator at 37°C and 5% CO₂ for 4 h. After this period, the supernatant was removed and 100 μl of RPMI was added and the plates were incubated again under the same conditions overnight.

(iii) Determination of phagocytic capacity. After cell incubation as described above, 10 μl of stained zymosan solution was added and incubated for 30 min at 37°C. After this procedure, 100 μl of Baker fixative was added to stop the phagocytosis process, and 30 min later, the plates were washed with 0.9% saline to remove the zymosan and neutral red that were not phagocytized by the macrophages. The supernatant was removed, 100 μl of extraction solution was added, and after solubilization on a Kline shaker, the absorbances were read in a Biotek plate reader (ELx800) at 550 nm (50).

(iv) Evaluation of lysosomal activity. After cell incubation as described above, 10 μl of a DMSO–2% neutral red solution was added and incubated for 30 min. After this time, the supernatant was discarded, the wells were washed with 0.9% saline at 37°C, and 100 μl of extraction solution was added to solubilize the neutral red present inside the lysosomal secretory vesicles. After 30 min on a Kline shaker, the absorbances were read at 550 nm. (51).

(v) Quantification of nitrite. After cell incubation as described above, the supernatants were transferred from the cell culture to another 96-well plate to determine nitrite dosages. The standard curve was prepared with sodium nitrite in RPMI medium at concentrations of 1, 5, 10, 25, 50, 75, 100, and 150 μM. At dosages, equal parts of the samples (supernatants) or the solutions prepared to obtain the standard curve were mixed with the same volume of Griess reagents (1% sulfanilamide in 10% [vol/vol] H₃PO₄ in Milli-Q water, added in equal parts to 0.1% naphthylenediamine in Milli-Q water), and the absorbances were read at 550 nm; the result was plotted as the concentrations of nitrite (in micromolar units) (52).

(vi) Quantification of cytosolic calcium. After cell incubation as described above, the calcium probe Fluo-3 AM was added at a concentration of 10 μM, and cells were incubated again for 30 min. Then, the entire supernatant was removed and calcium-containing Hanks balanced salts solution (HBSS) medium was added to it. Then, a microscopic analysis of the mixture was performed using a disk scanning unit (DSU) confocal fluorescence microscope (model IX81; Olympus, Japan) coupled to a high-speed charge-coupled device (CCD) camera (Hamamatsu, Japan). Excitation filters (340/380 nm) were used for the evaluation of the levels of cytoplasmic Ca²⁺ in macrophages, and the emitted fluorescence (525 nm) was quantified with Cell̂R (Olympus, Japan) software.

The cytosolic calcium concentration ([Ca²⁺]i) was estimated as K_d × [(F − F_min)/(F_max − F)], where K_d is the Fluo-3 dissociation constant (450 nM), F is the variable fluorescence intensity, and F_max and F_min are, respectively, the maximum and minimum fluorescence obtained in the presence of ionomycin (5 μM) or EGTA (20 mM) (53). Calcium imaging data were representative of two independent experiments of single-cell analysis carried out in duplicate.

Effect of topical formulations on peritoneal macrophage infection.

Peritoneal macrophages from normal animals that were infected with L. major and treated with the topical formulations described above were plated, diluted in RPMI medium supplemented with 10% heat-inactivated FBS, 100 U/ml penicillin, and 100 μg/ml streptomycin, to 2 × 10⁵ cells per well in triplicate, in a 24-well plate containing 13-mm-diameter circular coverslips, and incubated in an oven at 37°C and 5% CO₂ for 4 h. After this period, the supernatant was removed, 500 μl of RPMI containing 2 × 10⁶ metacyclic promastigotes of L. major was added, and the plates were incubated again under the same conditions overnight. After this period, the medium was aspirated to remove noninternalized parasites, and the plate was washed 2 times with saline solution (0.9% NaCl). Then the coverslips were removed and stained using rapid panoptic stain. For each treatment, the number of infected macrophages and the number of surviving amastigotes inside each cell were determined, traversing the sample fields until 100 macrophages were counted, using optical microscopy at a magnification of ×1,000 (54).

Toxicity evaluation of topical formulations in BALB/c mice infected with L. major. (i) Hematocrit.

Capillary tubes containing whole blood from normal animals that had been infected with L. major and treated with the topical formulations described above were centrifuged at 825 × g for 5 min, and the hematocrit value was quantified on a millimeter scale of percentages using the hematocrit ruler.

(ii) Biochemical parameters of liver and kidney function.

Liver function in serum samples was analyzed through ALT enzyme levels, while nephrotoxicity was evaluated by determining blood urea nitrogen and serum creatinine levels using commercial kits (Labtest Diagnostica). Normal mouse serum was used as the control.

Statistical analysis.

Analysis of variance (ANOVA) and Student’s t test were performed for the evaluation of preliminary stability of formulations. The Kruskal-Wallis test was performed to compare clinical progress between groups. ANOVA and Bonferroni's posttest were used for comparisons between groups in the ex vivo trials, with each study being performed in triplicate. Differences were considered significant when P was <0.05.