COMPOSITION, PHARMACEUTICAL COMPOSITION, USE OF A STABLE TOPICAL COMPOSITION COMPRISING A NANOEMULSION AND OF AT LEAST ONE ANTILEISHMANIAL COMPOUND, AND METHOD FOR THE TREATMENT OF CUTANEOUS LEISHMANIASIS

Abstract

The drugs available for the treatment of cutaneous leishmaniasis have unsatisfactory efficacy, frequent and serious adverse effects, and require long treatment regimens. Thus, the search for new treatment alternatives for cutaneous leishmaniasis is considered a priority by the World Health Organization. Parenteral administration of pentavalent antimonials for the treatment of all forms of leishmaniasis, including cutaneous leishmaniasis, has several limitations. The therapy is long, requires repeated doses, and adverse reactions are frequent. Topical treatment is an attractive alternative for cutaneous leishmaniasis, offering significant advantages over systemic therapy: fewer adverse effects, ease of administration, and lower costs. The present inventors aimed to provide a fixed-dose topical composition containing at least one antileishmanial compound, providing adequate absorption of the active ingredient. Another objective of the present invention is to provide a topical, fixed-dose formulation containing a combination of antileishmanial compounds that has sufficient efficacy and safety to be used in the treatment of cutaneous leishmaniasis.

Claims

1. A composition characterized by the fact that it comprises: (a) a stable topical composition comprising a nanoemulsion, and (b) at least one antileishmanial compound incorporated into said nanoemulsion.

2. A composition, according to claim 1, characterized by the fact that the antileishmanial compound is selected from tamoxifen, meglumine antimoniate, sodium stibogluconate, amphotericin B, pentamidine isethionate, miltefosine, paromomycin, imiquimod, and buparvaquone, or combinations thereof.

3. A composition according to claim 1 or 2, characterized by the fact that the antileishmanial compound is selected from tamoxifen, meglumine antimoniate, paromomycin, and amphotericin B, their salts, or combinations thereof.

4. A composition according to any of the claims 1 to 3, characterized by the fact that the antileishmanial compound is paromomycin.

5. A composition according to any one of the claims 1 to 3, characterized by the fact that the antileishmanial compound is a combination of paromomycin and amphotericin B.

6. A composition according to any one of the claims 1 to 3, characterized by the fact that the antileishmanial compound is a combination of meglumine antimoniate and amphotericin B.

7. A composition according to any one of the claims 1 to 3, characterized by the fact that the antileishmanial compound is a combination of two or three selected from tamoxifen, meglumine antimoniate, and amphotericin B.

8. A composition according to any one of the claims 1 to 7, characterized by the fact that the nanoemulsion comprises at least one non-ionic emulsifier, at least one amphoteric surfactant, at least one emollient, at least one humectant, and at least one moisturizer.

9. A composition according to any one of the claims 1 to 8, characterized by the fact that at least one antileishmanial compound is incorporated into the oil globules of said nanoemulsion in the presence of one or more oxygen carriers and optionally one or more oily vehicles, permeation promoters, and moisturizers.

10. A pharmaceutical composition characterized by the fact that it comprises a composition, as defined in any one of the claims 1 to 9, and at least one adjuvant and/or excipient.

11. A pharmaceutical composition according to claim 10, characterized by the fact that it is in the form of a suspension, emulsion, lotion, spray, unguent, cream, gel, plaster, film, ointment, or adhesive.

12. Use of a stable topical composition comprising a nanoemulsion and at least one antileishmanial compound characterized by the fact that it is for the manufacture of a drug for the treatment of cutaneous leishmaniasis.

13. Use, according to claim 12, characterized by the fact that the antileishmanial compound is selected from tamoxifen, meglumine antimoniate, sodium stibogluconate, amphotericin B, pentamidine isethionate, miltefosine, paromomycin, imiquimod, and buparvaquone, or combinations thereof.

14. Use, according to claim 12 or 13, characterized by the fact that the antileishmanial compound is selected from tamoxifen, meglumine antimoniate, paromomycin, and amphotericin B, their salts, or combinations thereof.

15. Use, according to any one of the claims 12 to 14, characterized by the fact that the antileishmanial compound is paromomycin.

16. Use, according to any one of the claims 12 to 14, characterized by the fact that the antileishmanial compound is a combination of paromomycin and amphotericin B.

17. Use, according to any one of the claims 12 to 14, characterized by the fact that the antileishmanial compound is a combination of meglumine antimoniate and amphotericin B.

18. Use, according to any one of the claims 1 to 3, characterized by the fact that the antileishmanial compound is a combination of two or three selected from tamoxifen, meglumine antimoniate, and amphotericin B.

19. Use, according to any one of the claims 12 to 18, characterized by the fact that the nanoemulsion comprises at least one non-ionic emulsifier, at least one amphoteric surfactant, at least one emollient, at least one humectant, and at least one moisturizer.

20. Use, according to any one of the claims 12 to 19, characterized by the fact that at least one antileishmanial compound is incorporated into the oil globules of said nanoemulsion in the presence of one or more oxygen carriers and optionally one or more oily vehicle, permeation promoters, and moisturizers.

21. Method for treating cutaneous leishmaniasis characterized by the fact that it comprises administering a composition as defined in any one of the claims 1 to 9 or the pharmaceutical composition as defined in claim 10 or 11 to a patient in need thereof.

Description

BRIEF DESCRIPTION OF THE IMAGES

[0068] FIG. 1Average lesion size (mm) of male hamsters (Mesocricetus auratus) (n=6) infected at the base of the tail with 200 L suspension of L. (V.) braziliensis amastigotes and treated with spray nanoemulsions of paromomycin, amphotericin B, and combination of paromomycin+amphotericin. The lesions were measured every seven days, and the animals were euthanized thirty-three days after the start of treatment (D33). The vertical bars represent the standard deviation of the average of the largest diameter of the lesions in each group.

[0069] FIG. 2Viable parasites in the lesion (A) and spleen (B) of male hamsters (Mesocricetus auratus) (n=6) infected at the base of the tail with 200 L suspension of L. (V.) braziliensis amastigotes and treated with spray nanoemulsions of paromomycin, amphotericin B and combination of paromomycin+amphotericin. The parasites were determined using the limiting dilution technique on the day the animals were euthanized, D33 (33 days from the start of treatment). The horizontal bars represent the average number of viable parasites in the lesions or spleens of each group. *There was bacterial contamination on the lesion parasite culture plates from three animals in the PA 10%+AnfB 3% group.

[0070] FIG. 3Average size of the largest diameter of lesions (mm) of male hamsters (Mesocricetus auratus) (n=4) infected at the base of the tail with 1105/200 L metacyclic promastigotes of L. (V.) braziliensis and treated with combinations of spray nanoemulsions of amphotericin B 6%+meglumine antimoniate 12%, amphotericin B 6%+tamoxifen 0.5%; amphotericin B 6%+meglumine antimoniate 12%+tamoxifen 0.5% and the isolated formulations amphotericin B 6%, meglumine antimoniate 12% and tamoxifen 0.5%.

DETAILED DESCRIPTION OF THE INVENTION

[0071] Unless defined differently, all technical and scientific terms used herein have the same meaning as understood by someone with expertise in the subject matter to which the invention pertains. Conventional molecular biology and immunology techniques are well known to an expert in the field. The narrative report also provides definitions of terms to assist in the interpretation of what is described here and the claims. Unless otherwise indicated, all figures expressing quantities, percentages, proportions, and other numerical values used in the descriptive report and in claims are to be understood as being modified in all cases by the term about. Thus, unless otherwise stated, the numerical parameters shown in the descriptive report and in the claims are approximations that may vary depending on the properties to be obtained.

[0072] The present invention comprises a topical composition containing at least one antileishmanial compound.

[0073] Among the antileishmanial compounds according to the present invention, we can highlight pentavalent antimonial compounds, such as meglumine antimonate and sodium stibogluconate, amphotericin B, pentamidine isethionate, miltefosine, paromomycin, imiquimod, and buparvaquone. Preferably, the present invention is about a combined fixed-dose topical formulation of paromomycin and amphotericin B.

[0074] According to the present invention, the antileishmanial compounds are incorporated into the formulation through carriers, such as microcarriers and nanocarriers.

[0075] When applied to intact skin, microcarriers and nanocarriers can increase dermal or transdermal penetration of drugs, depending on the composition and size of the vesicles. Different mechanisms of action for micro and nanocarriers as drug administration systems into the skin have been suggested: enhanced penetration by the individual particle components; adsorption of the vesicle and/or merging with the stratum corneum (SC); intact penetration of the particle into and through intact skin; and follicular penetration.

[0076] The use of micro and nanocarriers in the topical treatment of cutaneous leishmaniasis, when compared to conventional formulations, may be beneficial because, when such particles are applied to skin with a normal or compromised barrier, they can dramatically increase drug penetration.

[0077] Among the preferred micro and nanocarriers, according to the present invention, we can highlight microemulsions and nanoemulsions.

[0078] According to the present invention, microemulsions are aqueous dispersions of particles, averaging in size between 1 nm and 1,000 m, composed of a lipid core surrounded by monolayers of surfactants and/or co-surfactants. Nanoemulsions, which are a subgroup of microemulsions, are colloidal systems that include micelles, liposomes, virosomes, nanosuspensions, and other polymeric solutions, with an average size between 1 nm and 10,000 nm.

[0079] Preferably, the antileishmanial drugs according to the present invention shall be incorporated into the nanoemulsion, hereinafter referred to as Biolipid B2. Biolipid B2, developed by Evidence Group, was first described in Brazilian patent PI 1002486-7, as well as its preparation process and methods for incorporating active ingredients in the composition described. Biolipid B2 is a stable, biocompatible nanoemulsion capable of delivering drugs for transdermal administration.

[0080] According to the present invention, when antileishmanial drugs are incorporated into Biolipid B2, the latter can be used directly as a final product or additionally mixed with other adjuvants and excipients to form other compositions suitable for topical administration.

[0081] Some examples of these other formulations suitable for topical administration include suspensions, emulsions, lotion sprays, unguents, creams, gels, plasters, films, ointments, and adhesive-incorporated compositions, all of which are known in the technique of topical formulations and preparations. Lotion, cream, ointment, and spray formulations are preferred according to the present invention.

[0082] Among the adjuvants and excipients that can be used for the preparation of nanoemulsions, Biolipid B2, or the compositions according to the present invention, can be highlighted: [0083] the permeation enhancers, such as dibutyl adipate, isopropyl myristate, dimethyl sulfoxide, diethylene glycol monoethyl ether, propylene glycol dicaprylocaprate, isopropyl myristate, sodium lauryl sulfate, polyoxyethylene sorbitan monooleate, and sorbitan monolaurate; [0084] the oxygen carriers selected from perfluorocarbons, preferably perfluorotrialkylamines, such as perfluorohexane, perfluorodimethylcyclohexane, octofluorooctane, and perfluorodecalin; [0085] fatty alcohols such as those having about 4 to 30 carbon atoms, such as stearyl alcohol, cetyl alcohol, cetostearyl alcohol, and myristyl alcohol; [0086] emollients such as dibutyl adipate, diisobutyl adipate, diisopropyl adipate, dimethicone, fatty acid triglyceride esters such as caprylic/capric triglycerides, hydroxylated lanolin, isopropyl myristate, mineral oil, soy sterol, cetyl stearate and petrolatum, linolenic acid, linoleic acid and oleic acid; [0087] emulsifiers, such as steareth-2, steareth-21, glyceryl monostearate SE (a mixture of glyceryl stearate and PEG-100 stearate) and laureth-4, cetearyl, sorbitan, and ceteareth from mixtures of fatty acid esters resulting from the saponification of vegetable oil, selected from coconut oil, palm oil, olive oil, soybean oil, sunflower seed oil, or animal oil; [0088] humectants such as glycerin, propylene glycol, sorbitol, lactose, mannitol, sodium pyrrolidone carboxylic acid, panthenol, hyaluronic acid, and chondroitin; [0089] the amphoteric surfactants such as saponins, lecithin, and soy proteins; and [0090] the moisturizers such as trehalose, maltose, and sucrose; [0091] as well as any combinations or mixtures thereof, and other similar and equivalent compounds.

[0092] Other additives may also be incorporated into the compositions of the present invention, such as ultraviolet absorbers or sunscreens, antioxidants, preservatives, and others, for improved stability during use and storage. Non-limiting examples of appropriate antioxidants and preservatives include, but are not limited to, butylated hydroxytoluene, butylated hydroxyanisole (BHA), sorbic acid, benzoic acid, benzyl alcohol, imidazolidinyl urea, diazolidinyl urea, methylparaben, propylparaben, potassium sorbate, and mixtures or combinations thereof.

[0093] It should also be understood that the compositions of the present invention may include other components commonly used in conventional topical cosmetic formulations, such as suspending agents, thickening agents, film formers, preservatives, and fragrance oil. The thickening agents are preferably those that are compatible with the composition, such as bentones, xanthan gum, silica, and ethyl cellulose. Dyes, fragrances, and other cosmetic additives may also be present. The exemplified and specifically listed cosmetic components may be freely substituted with other conventional and well-known components to obtain the desired texture and lubricity of the compositions, on the condition that the substitutes do not react adversely with any component of the composition and do not interfere with the homogeneity of the composition.

[0094] The present invention is also described by the non-limiting example below, which is merely illustrative. Various modifications and variations of the embodiments are evident to the expert in the subject without straying from the spirit and scope of the invention.

[0095] Numerous variations affecting the scope of protection of the present application are allowed. Thus, it is reinforced that the present invention is not limited to the particular configurations/embodiments described above.

EXAMPLES

Example 1

Objective

[0096] Evaluate nanoemulsions in spray presentation of paromomycin, amphotericin B, and the combination of paromomycin+amphotericin for the treatment of skin lesions caused by Leishmania (Viannia) braziliensis in experimentally infected hamsters. This model is on the spectrum of susceptibility to infection, difficult therapeutic responses, and a tendency to reactivation with parasite persistence, even when leishmanicides with good action in humans are used.

Development

Parasites

[0097] The study was conducted with the reference strain Leishmania (V.) braziliensis MHOM/BR/75/M2903, and with the reference strain Leishmania (Leishmania) major (MHOWIL/80/Friendlin), characterized and deposited in the strain bank of the Leishmania Collection of the Reference Center for Leishmania Typing at the Instituto Oswaldo Cruz.

Animals

[0098] The animals used in this study were kept in the vivarium of the Instituto Ren Rachou (IRR) and the procedures performed were previously approved by the Ethics Committee on Animal Use of the Fundao Oswaldo CruzPermit LW06/13.

Formulations Evaluated

[0099] Samples containing antileishmanial drugs incorporated into Biolipid B2 nanoemulsions, produced according to the preparation process described in patent PI 1002486-7.

TABLE-US-00001 Samples Paromomycin Sulfate 10% P/V + Amphotericin B 3% P/V PH: 6.7 Paromomycin Sulfate 10% P/V PH: 6.7 Amphotericin B 6% P/V PH: 6.7

Infection and Treatment of the AnimalsL. (V.) Braziliensis

[0100] Twenty-eight male hamsters (Mesocricetus auratus) of approximately 148.7 g of body mass were infected by subcutaneous injection at the base of the tail with 200 L of a suspension of amastigotes of the L. (V.) braziliensis M2903 strain. After 52 days of evolution (Apr. 11, 2018start of treatment), the average size (largest diameter) of the skin lesions was measured with a digital caliper (12.5 mm2.5 mm). For each treatment, the animals were grouped into seven groups of four animals each. Grouping was performed in such a way that the average lesion sizes between the groups were similar.

Treatment Groups:

[0101] 1PA 10%+AnfB 3%: Paromomycin Sulfate 10% P/V+Amphotericin B 3% P/VpH: 6.7, administered twice a day, for 30 consecutive days; [0102] 2AnfB 6%: Amphorericin B 6% P/VpH: 6.7, administered twice a day, for 30 consecutive days; [0103] 3PA 10%: Paromomycin Sulfate 10% P/VpH: 6.7, administered twice a day, for 30 consecutive days;

[0104] 4Control: No treatment control: infected and untreated animals.

Evaluation of Clinical Efficacy

[0105] The effectiveness of the treatment was evaluated by weekly measurement of the size of the lesions. Before, during, and after treatment, the average lesion size (mm) was determined by measuring the largest lesion diameter using a digital caliper. Measurement was performed at D1the day treatment started, D77 days after treatment started, D1414 days after treatment started, D2121 days after treatment started, D2828 days after treatment started, and D333 days after treatment ended (the day the animals were euthanized). The percentage reduction in average lesion size calculated by the difference in lesion size at the start of treatment (D1) and three days after the end of treatment (D33) was determined. The percentage of animals that had complete healing of the lesion was determined at D33.

Evaluation of Parasitological Efficacy

[0106] To evaluate the effect of treatment in reducing or eliminating the parasite load of infected and treated animals, three days after the end of treatment (D33), the lesion and spleen were removed, ground in a tissue homogenizer, centrifuged, and the contents of the final pellet were distributed on culture plates in a 10 serial dilution. After seven days of culture, the plate wells were read under an inverted microscope and viable parasites were determined.

[0107] Evaluation of Treatment Toxicity

[0108] Every seven days (D1, D7, D14, D21, D28, and D33three days after the end of treatment), the animals were weighed to evaluate the possible toxic effects of the treatment. General physical aspects such as piloerection, behavioral changes, diarrhea, and others were also observed for this purpose.

Data Analysis

[0109] The data were processed using GraphPad Prism 5 software. To compare parasite loads, animal weights, and lesion sizes between groups, an one-way analysis of variance followed by Tukey's test was applied. The difference was considered significant when the p-value was less than 0.05. Parasite load data were log10+1 transformed and evaluated for normality using the Kolmogorov-Smirnov test.

Results

[0110] Clinical Efficacy: The reduction in lesion size over time for each treatment regimen was evaluated. A statistically significant difference (*p<0.05) compared to the untreated control group was observed for the group of animals treated with the combination PA 10%+AnfB 3%, starting on D21 (21 days after the start of treatment). For the other groups, despite the evidence of reduction in the average size of the lesions, there was no statistically significant difference at any of the times evaluated (p>0.05) (FIG. 1).

[0111] The percentage of lesion size reduction was calculated on the day of euthanasia (D33) compared to the initial treatment time (D1) and the percentage of complete healing of the animals (TABLE 1). A 94.2% reduction in lesion size is observed for the PA 10%+AnfB 3% group. This group also showed 83.3% (5/6) complete healing of the lesion. The AnfB 6% and PA 10% groups showed a reduction percentage of 16.6% and 50%, respectively. Lesion healing of the animals in the 10% PA group was 50%, and there was no complete healing for the animals in the 6% AnfB group. There was no reduction in the size of the animals' lesions and complete healing of the lesion for the untreated control group (TABLE 1).

TABLE-US-00002 TABLE 1 Clinical efficacy of paromomycin, amphotericin B, and paromomycin + amphotericin B combination spray nanoemulsions in hamsters experimentally infected with L. (V.) braziliensis Clinical Efficacy % reduction in % of animals with complete Group lesion size.sup.1 healing of the lesion.sup.2 PA10% + AnfB 3% 94.2 83.3 (5/6) AnfB 6% 16.6 0.0 (0/6) PA 10% 50.0 50.0 (3/6) Control 0.0 0.0 (0/6) * The average size of lesions was calculated by the largest diameter (mm). The percentages of average lesion size reduction and complete healing were obtained by considering the ratio between the average lesion values at time D 1 (start of treatment) and D 33 (day of euthanasia).

Parasitological Efficacy

[0112] Regarding the evaluation of the parasite load, there was a statistically significant difference in the lesion (A) for the group of animals treated with the combination of paromomycin+amphotericin (PA 10%+AnfB 3%) compared to the untreated control. In the groups that received paromomycin (PA 10%) and amphotericin B (AnfB 6%) alone, no statistically significant difference was observed. In the spleen, a statistically significant difference was observed for the 6% AnfB group compared to the untreated control (*p<0.05) (FIG. 2).

[0113] Drug toxicity was evaluated through body mass during and after treatment. The animals did not lose weight at any time during the evaluation, nor did they show signs that could indicate toxicity (raised fur, aggressiveness, diarrhea, or others).

Conclusions

[0114] Paromomycin 10% spray nanoemulsion showed moderate efficacy in L. (V.) braziliensis infection leading to reduction of lesion size by 50%. Amphotericin B 6% nanoemulsion showed moderate efficacy in reducing the average size and viable parasites in the lesion and was also able to significantly reduce the viable parasite load in the spleen of the animals, a finding not observed with PA 10% and well with the combination of PA 10% and AnfB 3%.

[0115] The combination nanoemulsion of Paromomycin 10%+Amphorericin B 3% was significantly effective in reducing the average lesion size and viable parasite load in the animals' lesions.

Example 2

Objective

[0116] To evaluate spray-presentation nanoemulsions of amphotericin B, meglumine antimonate, and tamoxifen and combinations of amphotericin B+meglumine antimoniate, amphotericin B+tamoxifen, and amphotericin B+meglumine antimoniate+tamoxifen for the treatment of skin lesions caused by Leishmania (Viannia) braziliensis in experimentally infected hamsters.

Development

Parasites

[0117] The study was conducted with the reference strain Leishmania (V.) braziliensis MHOM/BR/75/M2903.

Animals

[0118] In this study, the experimental infection model was used in golden hamsters (Mesocricetus auratus). The animals used were kept in the vivarium of the Instituto Ren Rachou (IRR) and the procedures performed were previously approved by the Ethics Committee on Animal Use of the Fundao Oswaldo CruzPermit LW04/20.

Evaluated Formulations

[0119] Samples containing antileishmanial drugs incorporated into Biolipid B2 nanoemulsions, produced according to the preparation process described in patent PI 1002486-7.

TABLE-US-00003 Samples Formula 1- Amphotericin B 6% + Meglumine antimoniate 12% Extremely absorbable biolipid Formula 2- Amphotericin B 6% + Tamoxifen 0.5% Extremely absorbable biolipid Formula 3- Amphotericin B 6% + Meglumine antimoniate 12% + tamoxifen 0.5% Extremely absorbable biolipid Formula 4- Amphotericin B 6%. Extremely absorbable biolipid Formula 5- Meglumine antimoniate 12%. Extremely absorbable biolipid Formula 6- Tamoxifen 0.5%. Extremely absorbable biolipid
Infection and Treatment of the AnimalsL. (V.) braziliensis

[0120] Twenty-eight male hamsters (Mesocricetus auratus) of approximately 135.5 g of body mass were infected by subcutaneous injection at the base of the tail with 110.sup.5/200 L metacyclic promastigotes of the L. (V.) braziliensis M2903 strain. After 50 days of evolution (Sep. 16, 2021start of treatment), the average size (largest diameter) of the skin lesions was measured with a digital caliper (13.3 mm2.9 mm). For each treatment, the animals were grouped into seven groups of five or four animals each. Grouping was performed in such a way that the average lesion sizes between the groups were similar.

Treatment Groups:

[0121] F1Amphotericin B 6%+Meglumine antimoniate 12%, administered twice a day, topically (spray) for 49 consecutive days; [0122] F2Amphotericin B 6%+Tamoxifen 0.5%, administered twice a day, topically (spray) for 49 consecutive days; [0123] F3Amphotericin B 6%+Meglumine antimoniate 12%+tamoxifen 0.5%, administered twice a day, topically (spray) for 49 consecutive days; [0124] F4Amphotericin B 6%, administered twice a day, topically (spray) for 49 consecutive days; [0125] F5Meglumine antimoniate 12%, administered twice a day, topically (spray) for 49 consecutive days; [0126] F6Tamoxifen 0.5%, administered twice a day, topically (spray) for 49 consecutive days; [0127] F7Control: infected and untreated animals.

Evaluation Of Clinical Efficacy

[0128] The effectiveness of the treatment was evaluated by weekly measurement of the size of the lesions using a digital caliper (Digimess). Before, during, and after treatment, the average lesion size (mm) was determined by measuring the largest lesion diameter. Measurement was performed at D1the day treatment started, D77 days after treatment started, D1414 days after treatment started, D2121 days after treatment started, and D2828 days after treatment started, D3535 days after treatment started, D42forty-two days after treatment started, and D49forty-nine days after treatment started. The percentage reduction in average lesion size was calculated by the difference in lesion size at the start of treatment (D1) and 49 days after the start of treatment (D49). The percentage of animals that showed complete healing of the lesion was determined at D49. The magnitude of the change in lesion size between the start of treatment and the end of observation was also determined by the ratio of the largest ulcer diameter at D49 to the largest lesion diameter on the first day of treatment (D1), as well as the proportion of animals with complete epithelialization at D49.

Evaluation of Treatment Toxicity

[0129] Every seven days (D1, D7, D14, D21, D28, D35, D42, and D49), the animals were weighed to evaluate the possible toxic effects of the treatment. General physical aspects such as pilo-erection, behavioral changes, diarrhea, and others were also observed for this purpose.

Data Analysis

[0130] The database for this study was built using Microsoft Office Excel 2007 spreadsheets, exported to GraphPad Prism 5 for Windows (GraphPad Software, San Diego, California, USA).

[0131] The analysis strategy consisted of comparing the groups with the different treatments in relation to the parameter of clinical evolution of the animal's skin lesion. When evaluating the clinical response, in addition to the graph of the evolution of lesion measurements (largest ulcer diameter), the magnitude of ulcer reduction, calculated by the ratio of the largest lesion diameter on D49 in relation to the largest lesion diameter on D0 (start of treatment), and the number of animals in each group that showed complete epithelialization of the ulcer were also evaluated.

[0132] Comparisons were performed by parametric and nonparametric hypothesis tests, and the distribution of the continuous variables (weight and lesion size) was assessed for normality by the Kolmogov-Sminrnov (KS) and D'Agostino & Pearson tests. For continuous variables with normal distribution, comparison of averages was done using Tukey's multiple comparisons test for comparison of more than two groups. Analysis of continuous variables with non-normal distributions was performed by comparing averages using the Kruskal-Wallis test for three or more groups. For all comparisons, the significance level considered was 5%.

Results

[0133] At D1 and D7, the groups showed no statistically significant difference between them (p>0.5). At D14, the group amphotericin B 6%+meglumine antimoniate 12%+tamoxifen 0.5% showed a statistically significant difference compared to the untreated control, tamoxifen 0.5% group, and amphotericin B 6% (p<0.05).

[0134] At D21, the group amphotericin B 6%+meglumine antimoniate 12% started to show a statistically significant difference compared to the untreated control and tamoxifen 0.5% group (p<0.05).

[0135] At D28, significant statistical differences were observed between the amphotericin B 6%+meglumine antimoniate 12% groups compared to the control group and tamoxifen 0.5% (p<0.05). At the same time, the group amphotericin B 6%+meglumine antimoniate 12% +tamoxifen 0.5% showed a statistically significant difference compared to the animals treated with tamoxifen 0.5% (p<0.5).

[0136] At D35, the statistical differences observed at D28 for the amphotericin B 6% +meglumine antimoniate 12% group remain , and also between this group and the amphotericin B 6% treatment (p<0.5). The combined formulation of amphotericin B 6%+meglumine antimoniate 12%+tamoxifen 0.5% showed a statistically significant difference at D35 compared to the tamoxifen 0.5% treated group (p<0.5).

[0137] At D42 and D49, the statistically significant differences observed at D35 for the group of animals treated with the combination amphotericin B 6%+meglumine antimoniate 12% remain (p<0.5) and at D49, the triple combination of amphotericin B 6%+meglumine antimoniate 12%+tamoxifen 0.5% showed a statistically significant difference compared to the tamoxifen 0.5% group and also the untreated control group (P<0.5).

[0138] Lesions were measured every seven days at the beginning (1), seven (7), 14, 21, 28, 35, 42, and 49 days after treatment. The vertical bars in FIG. 3 represent the standard deviation of the average of the largest diameter of the lesions in each group. At D49, a statistically significant difference (p<0.05) was observed for the group of animals treated with the combined formulation of meglumine antimoniate 12% and amphotericin B 6% compared to the untreated control and the isolated formulations of tamoxifen 0.5% and amphotericin B 6% and between the group amphotericin B 6%+meglumine antimoniate 12%+tamoxifen 0.5% and the untreated control and tamoxifen 0.5% (P<0.05).

[0139] The percentage reduction in lesion size was calculated from the measurements taken on day 49 (D49) of treatment compared to the measurements taken on the first day of treatment (D1) (TABLE 2). At D49, a reduction in lesion size of 65.9% is observed for the amphotericin B+meglumine antimoniate group; 52.6% for the amphotericin B+meglumine antimoniate+tamoxifen group; and 25.3% for the group treated with meglumine antimoniate alone. Complete lesion healing for these groups was 40%, 20%, and 0%, respectively. There was no reduction in the size of the animals' lesions and complete healing of the lesion for the untreated control group and the other treatment groups (TABLE 2).

[0140] The calculation of lesion magnitude (TABLE 2) confirmed the reduction in lesion size for treatments with the formulas amphotericin B 6%+meglumine antimoniate 12%; amphotericin B 6% +meglumine antimoniate 12%, +tamoxifen 0.5% and meglumine antimoniate 12%.

TABLE-US-00004 TABLE 2 Clinical efficacy of spray nanoemulsions of leishmanicidal drugs amphotericin, meglumine antimoniate, and tamoxifen combined and isolated produced by Evidence in hamsters experimentally infected with L. (V.) braziliensis. Magnitude of Clinical Efficacy lesion % of animals variation: % reduction in with complete D 49/D 1* Group lesion size.sup.1 complete lesion (average SD) AnfB 6% + 65.9 40.0 (2/5) 0.40 0.5 AM 12% AnfB 6% + 0.0 0.0 (0/5) 1.22 0.3 Tamoxi 0.5% AnfB 6% + 52.6 20.0 (1/5) 0.46 0.3 AM 12% + Tamoxi 0.5% AnfB 6% 0.0 0.0 (0/5) 1.13 0.3 AM 12% 25.3 0.0 (0/5) 0.75 0.2 Tamoxifen 0.5%. 0.0 0.0 (0/4) 0.98 0.1 Control 0.0 0.0 (0/4) 1.04 0.1 The average size of the lesions was calculated by the largest diameter (mm). .sup.1The percentages of reduction in the average size of the lesions were obtained by considering the ratio between the average values of the lesions at D 1 (the start of treatment) and D 49 (49 days after the start of treatment). *calculated as the ratio of the average of the largest lesion diameter at D 49 (49 days after the start of treatment) to the average of the largest lesion diameter at D 1 (the start of treatment). AnfB 6% = Amphotericin B 6%; AM 12% = meglumine antimoniate 12% and Tamoxi 0.5% = tamoxifen 0.5%.

Final Considerations

[0141] Drug toxicity was evaluated by measuring body mass during and after treatment. The animals did not lose weight at any time during the evaluation, nor did they show signs that could indicate toxicity (raised fur, aggressiveness, diarrhea, or others). No animals died by the end of the experiment (D49).

[0142] The nanoemulsions containing the leishmanicidal drugs: a) amphotericin B 6%+meglumine antimoniate 12%; and b) amphotericin B 6%+meglumine antimoniate 12%+tamoxifen 0.5%, were effective in reducing the size of experimentally induced cutaneous leishmaniasis lesions in an animal model, compared to the control group of untreated animals;

Final Conclusion

[0143] The nanoemulsion containing the leishmanicidal drugs amphotericin B 6%+meglumine antimoniate 12% is sufficient and shows superior therapeutic capacity than that obtained with the drugs alone, as demonstrated by the significant reduction in the size of experimentally induced cutaneous leishmaniasis lesions in an animal model, compared to the untreated control group.

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