RESUMO
Cutaneous leishmaniasis (CL) is classified as a neglected tropical disease by the World Health Organization. As the standard drugs for the treatment of this disease suffer from severe unwanted effects, new effective and safe therapeutic options are required. In our previous work, Arnica tincture showed promising antileishmanial effects in vitro and in vivo. For the potential treatment of human CL patients with Arnica tincture, data on the pharmacokinetic properties of the bioactive, antileishmanial compounds (the sesquiterpene lactone (STL) helenalin and its derivatives) are needed. Therefore, we studied the in vivo absorption of the bioactive compounds after the dermal application of Arnica tincture in rats. Moreover, we analyzed the blood plasma, urine, and feces of the animals by ultra-high-performance liquid chromatography coupled to high-resolution mass spectrometry (UHPLC-HRMS). Although the majority (84%) of the applied STLs (1.0 mg) were absorbed, the concentrations in the plasma, urine, and feces were below the limit of detection (0.3 ng/mL) in the samples for UHPLC-HRMS analysis. This result may be explained by extensive metabolism and slow permeation accompanied by the accumulation of STLs in the skin, as described in our previous work. Accordingly, the plasma concentration of STLs after the topical application of Arnica tincture was very far from a dose where toxicity could be expected. Additionally, tests for corrosive or irritant activity as well as acute and repeated-dose dermal toxicity did not show any positive results after the administration of the amounts of Arnica tincture that would be needed for the treatment of CL. Consequently, in the treatment of CL patients with Arnica tincture, no toxic effects are expected, other than the known sensitization potential of the STLs.
RESUMO
Arnica tincture is a traditional herbal medicine used to treat blunt injuries, e.g., bruises and squeezes. In addition, a potential new use in the treatment of cutaneous leishmaniasis is currently under investigation. Therefore, detailed information about the dermal absorption of the tincture and especially its bioactive constituents, sesquiterpene lactones (STLs) of the helenalin- and 11α,13-dihydrohelenalin type, is mandatory. Consequently, this article reports on dermal absorption studies of Arnica tincture using diffusion cells and porcine skin as well as two human skin samples with different permeability. The amounts of STLs on the skin surfaces, in skin extracts and in the receptor fluids were quantified by ultra-high-performance liquid chromatography with high-resolution mass spectrometry (UHPLC-HRMS). It was found that Arnica STLs permeated into the receptor fluid already 4 h after the application, but the amount was rather low. Within 48 h, a maximum of 8.4%, 14.6% and 36.4% of STLs permeated through porcine skin, human skin A (trans-epidermal water loss (TEWL) = 11.518 g·m-2·h-1) and the more permeable human skin B (TEWL = 17.271 g·m-2·h-1), respectively. The majority of STLs was absorbed (penetrated into the skin; 97.6%, 97.8% and 99.3%) after 48 h but a huge portion could not be extracted from skin and is expected to be irreversibly bound to skin proteins. To better visualize the analytes in different skin layers, a fluorescence-labeled STL, helenalin 3,4-dimethoxycinnamate, was synthesized. Fluorescence microscopic images depict an accumulation of the fluorescent derivative in the epidermis. For the treatment of local, cutaneous complaints, an enrichment of the bioactive substances in the skin may be considered beneficial.
RESUMO
Leishmaniasis may occur in three different clinical forms, namely, visceral, mucocutaneous and cutaneous, which are caused by different species of trypanosomatid protozoans of the genus Leishmania. Pentavalent antimonials are the leading treatment for cutaneous leishmaniasis despite the hepatic, renal, and cardiac toxicity. In addition, the response of some Leishmania species to pentavalent antimonials is increasingly poorer, and therefore new and more potent therapeutic alternatives are needed. Arnica montana L., Asteraceae, is a traditional medicinal plant of Europe and preparations of its flowers are commonly used externally to treat disorders of the musculoskeletal system as well as superficial inflammatory conditions. Previous studies have shown that Arnica tincture (AT), an ethanolic extract prepared from the flowerheads of Arnica montana as well as isolated Arnica sesquiterpene lactones (STLs) have antileishmanial activity in vitro against L. donovani and L. infantum, as well as in vivo against L. braziliensis. In this work, we studied the in vitro cytotoxicity and antileishmanial activity of AT and STLs against both L. braziliensis and L. tropica. The in vivo therapeutic effect of AT was studied in hamsters with cutaneous Leishmaniasis (CL) caused by experimental infection with L. braziliensis and L. tropica. Furthermore, various semisolid Arnica preparations were also evaluated against L. braziliensis. The STLs and the AT possess a very high in vitro activity against both Leishmania species with median effective concentrations (EC50) ranging from 1.9 to 5.9 µg/mL. The AT was not cytotoxic for human tissue macrophages, skin fibroblasts, and hepatic cells. The therapeutic response of hamsters infected with L. braziliensis to the topical treatment with AT was 87.5% at a dose of 19.2 µg STL/2× day/60 d, 72.7% at doses of 19.2 µg STL/1× d/60 d and 67% at a dose of 38.4 µg STL/2× d/60 d. In turn, the therapeutic response in hamsters infected with L. tropica was 100% when treated at a dose of 19.2 µg STL/2× day/60 d and 71% at a dose of 38.4 µg STL/2× d/60 d. On the other hand, the effectiveness of treatment with glucantime administered intralesionally at a dose of 200 mg/every three days for 30 days was 62.5% for L. braziliensis and 37.5% for L. tropica infection. These results are promising and encourage the implementation of clinical trials with AT in CL patients as a first step to using AT as a drug against CL.