Therapeutic Activity of a Topical Formulation Containing 8-Hydroxyquinoline for Cutaneous Leishmaniasis

Abstract

Cutaneous leishmaniasis exhibits a wide spectrum of clinical manifestations; however, only a limited number of drugs are available and include Glucantime^® and amphotericin B, which induce unacceptable side effects in patients, limiting their use. Thus, there is an urgent demand to develop a treatment for leishmaniasis. Recently, it was demonstrated that 8-hydroxyquinoline (8-HQ) showed significant leishmanicidal effects in vitro and in vivo. Based on that, this work aimed to develop a topical formulation containing 8-HQ and assess its activity in experimental cutaneous leishmaniasis. 8-HQ was formulated using a Beeler base at 1 and 2% and showed an emulsion size with a D₅₀ of 25 and 51.3 µm, respectively, with a shear-thinning rheological behaviour. The creams were able to permeate artificial Strat-M membranes and excised porcine skin without causing any morphological changes in the porcine skin or murine skin tested. In BALB/c mice infected with L. (L.) amazonensis, topical treatment with creams containing 1 or 2% of 8-HQ was found to reduce the parasite burden and lesion size compared to infected controls with comparable efficacy to Glucantime^® (50 mg/kg) administered at the site of the cutaneous lesion. In the histological section of the skin from infected controls, a diffuse inflammatory infiltrate with many heavily infected macrophages that were associated with areas of necrosis was observed. On the other hand, animals treated with both creams showed only moderate inflammatory infiltrate, characterised by few infected macrophages, while tissue necrosis was not observed. These histological characteristics in topically treated animals were associated with an increase in the amount of IFN-γ and a reduction in IL-4 levels. The topical use of 8-HQ was active in decreasing tissue parasitism and should therefore be considered an interesting alternative directed to the treatment of leishmaniasis, considering that this type of treatment is non-invasive, painless, and, importantly, does not require hospitalisation, improving patient compliance by allowing the treatment to be conducted.

Keywords: 8-hydroxyquinoline; L. (L.) amazonensis; cutaneous leishmaniasis; quinolines; topical treatment.

Figure 1

Particle size distribution of Beeler’s base and creams containing 1 or 2% of 8-HQ. Data are expressed as mean ± standard deviation of three independent experiments. * p < 0.05 indicates statistical significance between the indicated groups. The letters “a” and “b” indicate statistical significance among D₅₀ and D₉₀ of cream 2% with Beeler base and cream 1%, respectively.

Figure 2

Viscosity versus applied rpm (A), from 0.5 to 0 rpm. Blank Beeler’s base cream, 1%, and 2% 8-HQ cream. Shear stress versus shear rate (B), from 0.384 to 1.92 (1/s)). Blank Beeler’s base cream with a y = 82.378x + 10.07 and a R² = 0.8257, 1% 8-HQ cream with a y = 97.017x − 3.18 and a R² = 0.9808, 2% 8-HQ cream with a y = 97.404x − 1.9483 and a R² = 0.914.

Figure 3

After the skin permeation test, fragments of porcine ear skin were shaved and fixed in 2% buffered formalin and the histological sections were stained with haematoxylin and eosin. (HE). (A) Cream containing 1% 8-HQ; (B) cream containing 2% 8-HQ. The black bar represents 10 µm.

Figure 4

Histological changes in non-infected BALB/c mice submitted to the treatment. Control mice were not submitted to any topical treatment (A); on the other side, groups of mice were treated for 14 days with blank Beeler’s base (B) or creams containing 1% (C) or 2% of 8-HQ (D) topically or with Glucantime^® (50 mg/kg) by the intralesional route (E). The skin histological section of each group was stained with haematoxylin and eosin (HE) and the main histological changes were compared with the skin histological section collected from untreated BALB/c mice (A). Bars = 20 µm.

Figure 5

BALB/c mice were infected with 10⁶ promastigote forms of L (L.) amazonensis at the base of the tail; at the 5th week post-infection, topical treatment was started with creams containing 1 or 2% of 8-HQ or Glucantime^® (50 mg/kg) by the intralesional route at the site of cutaneous lesion. The animals were treated once a day for 14 days. Lesion development (A) was monitored with a micrometer and at the 7th week PI parasite loads in the skin (B) and lymph nodes (C) were determined using a limiting dilution assay. * p < 0.05 in comparison to the infected group. # p < 0.05 in comparison to the 1% cream.

Figure 6

Histological skin sections of BALB/c mice infected with L. (L.) amazonensis. (A,B) Infected control; (C,D) topical treatment with blank Beeler’s base cream; (E,F) topical treatment with cream 1% of 8-hydroxyquinoline; (G,H) topical treatment with cream 2% of 8-hydroxyquinoline; (I,J) treatment by the intralesional route at the site of cutaneous lesion with 50 mg/kg of Glucantime^®. (A,C,E,G,I) show details of the epidermis and dermis of each group, while (B,D,F,H,J) show details mainly on parasitism, illustrated by the black arrows. Arrowheads indicate areas of necrosis (bars: 40 μm).

Figure 7

The draining lymph nodes of all experimental animals were collected, a single cell suspension was produced, and the supernatants were collected to quantify the amounts of IL-4 (A) and IFN-γ (B) using ELISA. * p < 0.05.