Topical Treatment of Leishmania tropica Infection Using (â)-Î±

Article pubs.acs.org/jnp

Topical Treatment of Leishmania tropica Infection Using (−)-αBisabolol Ointment in a Hamster Model: Effectiveness and Safety Assessment Victoriano Corpas-López,*,† Gemma Merino-Espinosa,† Margarita López-Viota,‡ Patricia Gijón-Robles,† María Jesús Morillas-Mancilla,† Julian López-Viota,§ Victoriano Díaz-Sáez,† Francisco Morillas-Márquez,† María Concepción Navarro Moll,⊥ and Joaquina Martín-Sánchez*,† †

Departamento de Parasitología, ‡Departamento de Farmacia y Tecnología Farmacéutica, and ⊥Departamento de Farmacología, Facultad de Farmacia, Universidad de Granada, Campus de Cartuja, 18071, Granada, Spain § Vircell Microbiologist, S.L. Avicena 8, 18016, Granada, Spain ABSTRACT: There is currently no reliable treatment for the management of cutaneous leishmaniasis, and intralesional antimonial injections remain the main treatment. The present work aims at evaluating the antileishmanial effectiveness and safety of (−)-α-bisabolol (1) in a novel topical formulation on a cutaneous leishmaniasis model involving Leishmania tropicainfected Syrian hamsters. The topical treatment with 1 reduced lesion thickness to 56% at 2.5%, showing a higher efficacy than the reference control, meglumine antimoniate. Other regimens (ointment at 1% and 5% and oral treatment at 200 mg/kg) reduced the footpad thickness as well. The skin parasite load decreased after the experiment in all treatment groups, particularly in those animals treated with the 2.5% formulation (83.2%). Treatment with (−)-α-bisabolol at different concentrations or through an oral route did not lead to the appearance of toxicity or side effects in healthy hamsters or infected animals. Therefore, topical (−)-α-bisabolol was more effective than meglumine antimoniate in this cutaneous leishmaniasis model without showing toxicity effects on the hamsters. These results are of great interest and might be used for the development of alternatives for the treatment of cutaneous leishmaniasis, either in monotherapy or in combination with other drugs whose skin permeability could be enhanced by this sesquiterpene. field, such as topical treatments that avoid the toxicity caused by systemic administration. Recently, topical paromomycin preparations have been developed and evaluated in clinical trials;6 in addition, some studies have highlighted the efficacy of topical amphotericin B in animal models, and there is an ongoing phase I/II clinical trial evaluating the effectiveness of a 3% amphotericin B cream in Colombia (Anfoleish), sponsored by the Drugs for Neglected Diseases Initiative (DNDi) (https://clinicaltrials.gov/ct2/show/NCT01845727). However, a recent study reported the low efficacy of a topical miltefosine formulation.7 DNDi decided to focus on the development of a treatment for CL that is predominantly caused by L. tropica in the Old World and L. braziliensis species due to their severity and public health importance and due to the fact that CL caused by L. tropica is an anthroponotic disease in many countries. Therefore, treatment of L. tropica cases is essential for controlling transmission.8 In addition, a treatment effective against one of these species is likely to be active against other.

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utaneous leishmaniasis (CL) is a parasitic disease that is endemic in more than 70 countries, with an incidence of 1.2 million cases per year.1 CL is caused by a variety of Leishmania species: mainly L. tropica and L. major in the Old World and L. amazonensis, L. mexicana, and L. braziliensis in the New World. Among these species, L. tropica is the major cause of CL in the Middle East and some areas of North Africa, and it is the only dermotropic anthroponotic species.2 It is also worth mentioning that the rock hyrax has been suggested as a L. tropica reservoir in Israel, where the parasite has been detected in the blood of up to 80% of these animals.3 Recently, the World Health Organization classified 12 countries as “highburden countries” for CL: several countries where L. tropica and L. major are endemic were selected, mainly from North Africa (Morocco, Tunisia, and Algeria), Middle East (Turkey, Syria, Saudi Arabia, and Iran), and also including Afghanistan and Pakistan.4 There is currently no satisfactory treatment for this disease given that the effectiveness of current drugs against CL, mainly antimonials and miltefosine, varies from 55% to 98% depending on the geographical area and the causative species.5 There is an unmet need for a new treatment against CL that is safe, effective, and affordable and can be feasibly administered in the © XXXX American Chemical Society and American Society of Pharmacognosy

Received: August 11, 2016

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DOI: 10.1021/acs.jnatprod.6b00740 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Article

Natural products are a major source of active compounds against parasitic diseases, particularly plant-derived compounds. Compound 1, (−)-α-bisabolol, has been proven effective against Leishmania spp. in vitro,9−12 and in vivo against L. infantum in a murine visceral leishmaniasis model through the oral route without causing toxicity.12 This compound is found in Matricaria chamomilla essential oil, as well as in other plants, at up to 50% concentrations, and it is widely used in cosmetic and dermatologic preparations due to its good organoleptic properties and its anti-inflammatory, anti-irritant, and microbicidal activities.13−15 The toxicity of 1 has been evaluated in several laboratory animal models including dogs, in which it was deemed nontoxic when administered orally [LOAEL (lowest-observed-adverse-effect level) 2 mL/kg].16 This sesquiterpene is effectively absorbed orally17 and shows a good cytochrome P450 inhibitory profile since it is a weak inhibitor of CYP1A2, CYP2C9, and CYP3A4 isoforms and a moderate inhibitor of CYP2D6.18

Figure 1. Evolution of the footpad thickness throughout the treatment and follow-up period. Syrian hamsters (n = 10/group) were inoculated in the foot pads with L. tropica and 7 weeks later, beginning on day 0, 21 consecutive daily treatments with (−)-α-bisabolol, meglumine antimoniate (MA), or vehicle began. All animals were sacrificed 1 week after the final treatment. The figure shows the mean lesion size and standard deviation every week. Control = hamsters treated with ointment lacking (−)-α-bisabolol; MA = hamsters treated with intralesional meglumine antimoniate; Oral = hamsters gavaged with (−)-α-bisabolol at 200 mg/kg; 1% = hamsters treated with a 1% (−)-α-bisabolol ointment; 2.5% = hamsters treated with a 2.5% (−)-αbisabolol ointment; 5% = hamsters treated with a 5% (−)-α-bisabolol ointment.

The mechanism of action of (−)-α-bisabolol is not clear. Some authors have suggested that it may inhibit ergosterol biosynthesis in fungi,19 while others have suggested that it causes mitochondrial damage,10 a mechanism previously reported in cancer cells.20 (−)-α-Bisabolol is a promising compound for the treatment of leishmaniasis. In addition, this sesquiterpene has shown some properties that might be useful in the treatment of CL lesions, such as anti-inflammatory, anti-irritant, curative, and antimicrobial activities. These properties may help prevent subsequent bacterial and fungal infections and the appearance of scars, which are common drawbacks of CL lesions. The aim of this study was to evaluate the safety and effectiveness of this compound when administered orally or applied topically to CL lesions caused by L. tropica in a Syrian hamster model.

Table 1. Parasite Loads in the Different Groups of the Experiments group control oral 1 1% 1 ointment 2.5% 1 ointment 5% 1 ointment intralesional MA

parasite load (parasites/mg)

% parasite load reduction

± ± ± ± ± ±

74 56 83 82 73

7406 1954 3280 1245 1367 2034

980 513a 87a 269a,b 364a,b 328a

a

Denotes a parasite load significantly lower than the control group. Denotes a parasite load significantly lower than the intralesional MA group. b

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Treatment with the 5% (−)-α-bisabolol ointment was also more effective than intralesional antimonial (p = 0.009) but not the 2.5% preparation. The present results constitute an outstanding outcome considering that footpad thickness increased by more than 75% before treatments began on week 7 postinfection and continued decreasing after the end of treatment. Conversely, in untreated hamsters, lesion size increased steadily throughout the 28-day experiment. The anti-inflammatory activity of this natural compound might well have facilitated the lesion size reduction, thus making necessary the quantitation of the parasite load in the footpad. In addition to the decrease in footpad thickness, treatment with (−)-α-bisabolol reduced the parasite load by more than 80% in hamsters treated with the ointment containing 2.5% (−)-α-bisabolol. When administered orally, reduction in lesion thickness was noticeable, but the decrease in parasite load was similar to that induced by the topical treatment. This fact might be due to the greater drug availability achieved by the on-site topical administration. In addition, topical treatment with this sesquiterpene showed a dose− response trend except for the most concentrated ointment [5% (−)-α-bisabolol], which was not more effective than the 2.5%

RESULTS AND DISCUSSION Figure 1 shows the evolution of footpad thickness in all hamster groups from the start of treatment (day 0) to the end of the experiment (day 28). There were no statistically significant differences among groups at day 0 (p > 0.05). At the end of the experiment (day 28), treatment of lesions caused by L. tropica once daily for 3 weeks with 1%, 2.5%, and 5% of (−)-αbisabolol ointment reduced lesion thickness by 49.0%, 56.4%, and 55.2%, respectively (p < 0.01), while oral treatment at 200 mg/kg decreased footpad thickness by 62.5% (p < 0.01). Intralesional meglumine antimoniate (MA) did not lead to a significant thickness reduction (3.2%), whereas hamsters treated with the control ointment underwent a slight increase in footpad thickness (11.0%). Table 1 shows the parasite loads of the different groups of the experiment after euthanasia. The ointments containing (−)-α-bisabolol (1) and the oral treatment were effective at reducing the parasite load (p < 0.01). Treatment with the 2.5% ointment was the most effective at decreasing the parasite load and proved more effective than the reference drug control and the oral treatment with the sesquiterpene (p = 0.001). B



Article

Table 2. Biochemical Tests in Serum and Organ Weight in Healthy and Infected Hamsters after Treatment Administration group uninfected control uninfected olive oil uninfected 1 5% ointment uninfected oral 1 500 mg/kg infected control infected oral 1 200 mg/kg infected ointment 1 1% infected ointment 1 2.5% infected ointment 1 5% meglumine antimoniate

ASTa (U/L) 82.9 83.4 83.4 82.2 77.1 77.9 82.2 87.0 73.0 79.6

± ± ± ± ± ± ± ± ± ±

6.1 4.2 5.9 3.7 0.9 7.0 6.1 3.3 3.0 6.5

ALTb (U/L) 81.9 82.2 82.5 82.6 81.6 81.6 81.9 81.3 81.8 78.8

± ± ± ± ± ± ± ± ± ±

4.3 4.8 3.6 3.7 4.9 4.9 3.0 5.7 2.2 4.1

ALPc (U/L) 14.2 14.4 14.1 14.7 14.4 12.9 13.0 13.3 14.7 13.7

± ± ± ± ± ± ± ± ± ±

1.2 2.0 1.3 1.4 0.9 1.6 3.2 1.3 2.0 2.1

CREATd (mg/dL) 0.18 0.21 0.19 0.19 0.19 0.20 0.22 0.21 0.18 0.19

± ± ± ± ± ± ± ± ± ±

0.1 0.2 0.0 0.1 0.0 0.0 0.1 0.0 0.0 0.0

urea (mg/dL) 35.1 33.2 33.9 33.7 32.5 34.4 33.7 33.6 34.2 34.6

± ± ± ± ± ± ± ± ± ±

1.9 2.8 2.0 1.5 0.8 2.5 1.9 1.7 1.9 0.9

spleen (mg) 182.7 179.4 180.2 181.2 178.8 177.3 175.3 178.4 193.1 192.2

± ± ± ± ± ± ± ± ± ±

20.2 19.8 20.3 23.1 1.2 17.0 12.0 27.7 17.3 23.1

liver (g) 3.5 4.1 3.4 3.5 3.3 3.0 3.2 3.3 3.2 3.3

± ± ± ± ± ± ± ± ± ±

0.6 0.7 0.5 0.3 0.2 0.1 0.2 0.1 0.1 0.1

a AST = mean serum aspartate aminotransferase. bALT = mean serum alanine aminitranferase. cALP = mean serum alkaline phosphatase. dCREAT = mean serum creatinine.

Syrian hamster has been used successfully as a model for mucosal leishmaniasis caused by L. braziliensis or L. panamensis.24,25 Throughout the 28-day experiment, healthy hamsters treated with either (−)-α-bisabolol at 500 mg/kg p.o. or 5% (−)-αbisabolol ointment did not show any signs of toxicity such as diarrhea, stress, pain, footpad edema, or redness. Changes in body weight were similar among the different untreated and treated groups. Spleen and liver weights of treated hamsters were similar to those of the untreated hamsters. Table 2 shows the results of the biochemical functional tests performed. No statistically significant differences were found between the untreated and treated groups (p < 0.05). None of the treatment regimens applied to infected hamsters showed any of the safety issues mentioned above for the healthy animals throughout the 3-week treatment period or the 7-day follow-up period. As shown in Table 2, these treatment regimens did not alter the biochemical values evaluated after the experiment in comparison to the untreated animals. The absence of clinical signs such as erythema and edema at 5% concentration in healthy animals confirms previous results reporting the safety of this natural compound. In addition, neither the oral (500 mg/kg) nor the topical preparation showed signs of toxicity, such as hepato- or splenomegaly, or alterations of biochemical parameters in blood, suggesting that (−)-α-bisabolol is safe in hamsters. These results were also obtained in infected hamsters. Andersen reported that (−)-α-bisabolol is well absorbed topically without causing skin damage or irritation.17 The lipophilic nature of this compound suggested that administration in an oily ointment may enable its absorption through the stratum corneum to reach the parasite. The small size of the sesquiterpene (molecular weight = 222 g/mol) is an advantageous property as well. Furthermore, this natural compound can enhance the absorption of other compounds, as described for 5-fluorouracil.26 Therefore, in addition to the antileishmanial activity shown in the present study, this natural sesquiterpene used in combination may enhance the efficacy of other drugs such as amphotericin B or miltefosine that have not shown an ideal effectiveness in recent clinical trials or in vivo assays using topical formulations. The present study complements previous results reported in a murine L. infantuminduced visceral leishmaniasis model12 that showed high antileishmanial efficacy of this compound in the absence of toxicity when administered orally. In conclusion, topical treatment with (−)-α-bisabolol significantly reduced lesion thickness induced by L. tropica in

preparation. On the other hand, intralesional meglumine antimoniate was significantly more effective than control, but did not reduce lesion thickness. Nevertheless, treatment with this reference drug reduced the parasite load in the footpad, but this reduction was significantly lower than that achieved by the 2.5% (−)-α-bisabolol ointment. The development of an effective topical formulation for the treatment of cutaneous leishmaniasis would represent a significant advance in the therapy of this neglected disease since it would present advantages over systemic treatment, such as lower toxicity and side effects and the ease of administration, which might improve patient compliance. This study has aimed to evaluate the effectiveness and safety of a novel formulation of (−)-α-bisabolol in the treatment of CL due to L. tropica in a hamster model. There is currently no reliable animal model for CL and the most common model, based on Balb/c mice infected with L. major, which leads to the spread of the parasite and the death of the animal, does not recapitulate the clinical outcome in man. Bastien and KillickHendrick evaluated and reviewed the Syrian hamster as a model for L. tropica and found that all hamsters that became infected developed a footpad lesion that increased up to 64 weeks postinfection.21 In our model, lesion thickness increased in control animals until the end of the experiment, but we have assessed the development of the lesion up to 3 months (data not shown). Giemsa-stained imprints were positive for all hamsters. The parasite was isolated in every skin macerate except for two hamsters gavaged with (−)-α-bisabolol that accounted for the lowest parasite loads. However, L. tropica DNA was not detectable by qPCR in the livers or spleens of any hamster studied. In the present study, visceralization was not found in any hamster, in contrast to those authors who confirmed visceral leishmaniasis due to L. tropica in 10% of hamsters.21 This fact might be explained by the longer follow-up period carried out in that study, given that the authors observed visceral leishmaniasis signs after the 12th week postinfection. Viscerotropism has been described for L. tropica in the past in American soldiers returning home after the Gulf War,22 and the geographical origin of the parasite has been suggested as a determinant for this feature.23 The present data support the idea that the golden hamster is a suitable model for cutaneous leishmaniasis caused by L. tropica, leading to a stable and noticeable increase in footpad thickness, high parasite loads, as reported for human CL cases due to this parasite, and the absence of signs of self-healing after 90 days. In addition, the C



Article

DNA was obtained from 10 mg of skin, spleen, or liver using the REAL DNA SSS extraction kit (RBME01). Each DNA extract was rehydrated in a final volume of 50 μL of sterile water. To make sure there was no contamination at this stage, extraction controls were carried out. These consisted of tubes of sterile water to which the whole extraction process was applied simultaneously with the biological samples. One control was used for every group of seven biological samples. The extracted DNA was kept at −20 °C. As an independent procedure DNA was also extracted from L. tropica promastigotes. The parasites were washed and counted with a hemocytometer and adjusted to a final concentration of 1000 parasites/μL to be used as a positive control in the PCR. Various negative controls were used: blank control, extraction controls, and DNA from an uninfected hamster. The evaluation of parasite load in the footpad skin, liver, and spleen was performed by using a TaqMan-based quantitative real-time PCR (qPCR) specific for L. tropica. This qPCR amplifies a genus-specific fragment of the genomic DNA (chromosome 29) using a fluorescent probe specific for L. tropica as described previously.27,28 Each sample was analyzed in triplicate, adding 50 ng of DNA in a final reaction volume of 25 μL. The parasite load was obtained by interpolating the threshold cycle values obtained for each biological sample in a previously constructed calibration curve. This technique has been adjusted to be very reliable at quantifying L. tropica DNA, having an efficiency of 100.3% (slope = −3.315, R2 = 99.1%). Toxicity Evaluation. Hamsters were weighed weekly in order to detect weight loss associated with product administration. They were also investigated for signs such as stress, pain, cutaneous signs (redness and edema in the footpad), or diarrhea. After sacrifice, spleens and livers were weighed to assess spleno- or hepatomegaly. Biochemical functional testsurea, creatinine, alkaline phosphatase, and transaminases serum level testswere carried out by using commercially available kits (Spinreact, Sant Esteve d’en Bas, Spain). Prior to the treatment of infected animals, the subacute toxicity of compound 1 was evaluated. Four groups of noninfected healthy hamsters were treated daily for 28 days with either 5% 1 ointment, 500 mg/kg 1 p.o., olive oil p.o., or a control ointment without 1. Afterward, these animals were euthanized, and all the toxicity tests performed on infected hamsters were carried out as well. Statistical Analysis. A parametric ANOVA test was performed for analyzing differences between footpad thicknesses, parasite loads, animal and organ weights, and serum biochemical data. Data normality was assessed previously. The statistical software package SPSS 20.0 was used.

a Syrian hamster model of CL without causing any toxicity. These results are of great interest given the absence of a reliable topical treatment for this disease, and (−)-α-bisabolol might be useful for the development of novel alternatives for CL treatment, either in monotherapy or in combination with other drugs whose skin permeability could be enhanced by this natural sesquiterpene.

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EXPERIMENTAL SECTION

Chemicals. RPMI-1640 medium, fetal bovine serum (FBS), and (−)-α-bisabolol (≥95%, gas chromatography) were purchased from Sigma-Aldrich (St. Louis, NO, USA). Meglumine antimoniate (Glucantime) was purchased from Sanofi (Paris, France) and was diluted in physiological saline solution up to 50 μL for intralesional use. Cetyl alcohol, lanolin, and white petroleum jelly were supplied by Roig Farma SA (Barcelona, Spain). (−)-α-Bisabolol (1) formulations were prepared as follows: cetyl alcohol was melted by heating to 80 °C and then mixed with lanolin and white petroleum jelly and homogenized until room temperature was reached, when compound 1 was incorporated at up to 1%, 2.5%, and 5% concentration, respectively, in the formulations. For oral administration, 1 was diluted with olive oil to a maximum dose volume of 200 μL. Parasites. L. tropica strain (MHOM/MA/1988/LEM1314) promastigotes were maintained in RPMI-1640 medium supplemented with 10% FBS at 26 °C. Parasites in the stationary phase of growth (day 5 of culture) and freshly isolated from hamster lesions (less than 2 passages) were used for in vivo infections. Animals. Syrian hamsters (80−100 g) were handled in accordance with guidelines for animal experimentation recommended by the Federation for Laboratory Animals Science Associations. They were properly housed under 12 h light cycles and provided with water and food ad libitum. All experiments were performed in accordance with the EU Directive 2010/63/EU for animal experiments and were approved by the Ethics Committee of Animal Experimentation of the University of Granada (CEEA 455-2013). Experimental Infection. Hamsters were inoculated by injection in the left hind footpad with 3 × 107 parasites/50 μL phosphate buffer saline (PBS). A thickness increase is noticeable after 2 weeks, reaching an average 75% increase in the footpad due to the appearance of a nonulcerated lesion in the footpad skin at week 7 postinfection. Then, lesion thickness increases steadily up to 3 months after the infection (data not shown), providing a model of nonhealing cutaneous leishmaniasis in hamsters. The presence of parasites in these lesions has been assessed using parasitological and molecular techniques at different time points. Treatment. Treatment started on week 7 postinfection (day 0), when footpad thickness increased 75% over baseline value. Six experimental groups, each consisting of 10 hamsters (n = 10/group), were designated as follows: control ointment without (−)-α-bisabolol (1), 1% compound 1 ointment, 2.5% 1 ointment, 5% 1 ointment, 200 mg/kg 1 diluted in olive oil for oral administration, and 2 mg SbV/kg intralesional meglumine antimoniate (reference control group). The animals were treated daily for 21 consecutive days (days 0−20). Fifty milligrams of ointment was applied on the footpad lesion, and then the animal was humanely restrained to avoid ointment loss in the cage. The oral administration was performed using an oral gavage. Assessment of Treatment Effectiveness. The efficacy of each treatment was evaluated by measuring the footpad thickness evolution with a caliper every week until the end of the experiment. Animals were sacrificed 7 days after the end of treatment (day 28). Samples of footpad skin, blood, spleen, and liver were collected under sterile conditions. Skin, spleen, and liver samples were divided into two parts for culture and quantitative PCR. Skin imprints were also performed, fixed in pure methanol, and then Giemsa-stained in order to detect amastigotes. Cultures were made with macerates of 20 mg of each tissue, using a combination of EMTM solid phase made with rabbit blood and RPMI supplemented with 20% FBS and 5% human urine as the liquid phase. The cultures were kept for 3 months before being rejected as negative. In vitro subinoculations were performed weekly.

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AUTHOR INFORMATION

Corresponding Authors

*Tel: +34 958 24 20 94. Fax: +34 958 24 38 62. E-mail: [email protected]. *E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was supported by the Project PI14-01024, Ministry of Economy and Competitiveness, Instituto de Salud Carlos III, Madrid, and Feder Funds for Regional Development from the European Union, “One way to make Europe”. The authors wish to thank Dr. Pratlong (University of Montpellier) for kindly donating the Leishmania strain used in this work.

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REFERENCES

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Topical Treatment of Leishmania tropica Infection Using (â)-Î±

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