Topical treatment with a two‐component gel releasing nitric oxide cures C57BL/6 mice from cutaneous leishmaniasis caused by Leishmania major (2016)

The incidence of the parasitic disease leishmaniasis is 1.5–2 million new cases per year predominantly in (sub)tropical regions of the world.1 Most of the endemic regions belong to developing countries, and the disease is thus a burden especially for the poor and those with coinfections, for example with HIV.1, 2 Due to these findings, there is a strong need for a vaccine and efficient therapeutics against leishmaniasis. Treatment against cutaneous leishmaniasis (CL) is based on systemic or local approaches dependent on the Leishmania (sub)species involved, the type of lesions and the immune status of the patient.3 For systemic treatment, pentavalent antimony, liposomal amphotericin B and miltefosine are most commonly used. Local treatment may be considered in cases with few lesions, in those caused by L. major or L. tropica, and in some cases from the New World that exhibit a low tendency to visceralize and/or induce mucocutaneous disease, and for those who cannot receive systemic treatment, for example during pregnancy.4 Topical treatment modalities include intra-lesional antimony, local excision of single lesions, photodynamic therapy, cryosurgery, thermotherapy and others, but there are limited data about the safety and efficacy of some of these modalities. In prior single-case reports, successful local treatment of CL with topical nitric oxide (NO) donors was reported.5-7 In addition, in a controlled clinical trial a NO-releasing patch revealed cure in ~40% of cases.8 In humans and mice, NO release by activated lesional macrophages (mediated by IFN-γ release from Th1/Tc1 cells) is crucial for intracellular parasite clearance.9,S1–S3 NO induces oligonucleosomal fragmentation of Leishmania DNA, and NO donors have significant leishmanicidal activity in vitro.S4 In addition to this direct antileishmanial effect, NO exerts self-regulating and immunoregulatory effects in adoptive and innate immunity.S5,S6 However, the stability of NO donor formulations is problematic for use in everyday practice. In the presence of oxygen and water, NO is partially oxidized into nitrogen dioxide. Further chemical reactions lead to formation of mainly nitrite and smaller amounts of nitrate ions.S7 To control the rate of NO release, one may admix a sodium nitrite aqueous gel with a second gel containing a reductant like ascorbic acid immediately before use.S8 An acidified nitrite solution containing 50 mmol/L nitrite releases 1.100 mmol NO/min, leading to a transcutaneous penetration of 0.5–1 nmol NO/min × cm2 if used in a cream.S9,S10 In this study, we tested the efficacy of a NO donor gel formulation for treating murine experimental leishmaniasis. We used a two-component gel consisting of ascorbic acid 5% and sodium nitrite 5% (composition described in Table S1).S11 The components are mixed just before application to skin. In this study, we assessed the treatment efficacy of this NO donor gel on the disease course of L. major-infected C57BL/6 mice. This mouse strain is considered to exhibit all main features of human CL.9 First, mice were infected with 2 × 105 infectious-stage parasites into ear dermis (Fig. 1a). To assess the treatment efficacy in mice infected with physiologically relevant low doses of parasite mimicking natural transmission of parasite into human hosts, we also performed infections with as low as 1000 parasites (Fig. 1b). In both settings, starting at the time when lesions became visible, 200 mg of mixed gel was applied twice per week for several weeks. Notably, as soon as treatment was started, progressive ear lesion development stopped and a significant difference between lesions sizes of treated and untreated ears became obvious. Interestingly, in high-dose infections, lesions remained significantly smaller despite end of treatment in week 4, whereas in low-dose infections, lesions' sizes started growing after stopping treatment indicating that parasite replication restarted again. Despite transient hyperaemia and redness, no side effects were obvious. To better mimic the clinical situation in which a patient presents with an already-existing lesion, we also assessed the treatment efficacy of NO donor gel treatment commencing at the peak of the lesions in week 4 (high-dose infection) and week 6 (low-dose infection), respectively. Even when the treatment was initiated for established lesions, the NO donor gel (Fig. 1a,b), but not its inactive single components (Fig. S1), significantly and strongly reduced lesion volumes within 1–2 weeks post-treatment initiation and promoted faster lesion resolution as compared to untreated controls. We next determined parasite loads in CL lesions with and without treatment at different time points. In line with our hypothesis, NO donor gel treatment for >3 weeks induced a strong and clear-cut reduction in lesional parasite loads (Fig. 1c). To our surprise, NO donor gel treatment also led to a reduction of parasite numbers in spleens of infected mice indicating that topical treatment inhibited subsequent parasite visceralization (Fig. 1d). Finally, the cytokine response in supernatants from lymphocytes taken from draining lymph nodes of NO donor gel-treated mice was compared to that of those without treatment. Interestingly, in all treatment groups, we detected elevated levels of IL-12p40 and IFN-γ and unaltered amounts of IL-4 (Fig. 1e). Early treatment with NO donor gel appeared to also influence IL-10 production, whereas treatment of established lesions did not. To assess the dependence of treatment on the ability to induce these proinflammatory cytokines, we assessed the efficacy of topical NO donor gel in the therapeutic setting using IFN-γ−/−, IL-12p40−/− and iNOS−/− C57BL/6 mice (Fig. S2). Of note, all of these strains are known to be highly susceptible to infection despite the genetically resistant background, as these cytokines are essential for the host defense against L. major.9 Interestingly, in IL-12p40 knockout mice, NO donor gel treatment between weeks 4 and 6 induced partial protection, but overall, NO donor gel was unable to prevent progressive disease in all knockout strains studied. Thus, in situations with absent mechanisms normally responsible for parasite elimination, NO donor gel treatment may not be effective enough to induce cure of lesions. In summary, application of NO donor gel prevented the development of full-blown lesions and induced reliably quicker lesion resolution in both a prophylactic and a therapeutic setting. This was paralleled by decreased numbers of viable parasites in both lesions and viscera indicating a direct parasite-eliminating effect of the NO donor gel. Interestingly, the NO donor also shifted the local immune response towards Th1 additionally amplifying anti-Leishmania immunity.S11,S12 This was corroborated by the finding that in IL-12-, IFN-γ- and NO-deficient mice, treatment was ineffective. Thus, it appears that topical NO donor gel application may be highly suitable especially for patients with an (at least partially) competent immune system. Future studies in humans will have to verify the efficacy of this well-tolerated topical treatment against cutaneous leishmaniasis. This work was supported by grants from the DFG to EvS. BL, AG and EvS performed research; AG and EvS designed the study; FB, BL and EvS analysed the data; and FB, AG and EvS wrote the manuscript. The authors have declared no conflicting interests. Figure S1 NO donor gel is only effective when mixing the two inactive components Figure S2 The effect of topical NO donor gel was partially dependent on Th1/Tc1-dependent immune mechanisms Table S1 Two-component-NO-donor gel (1:1 Gel 1 and Gel 2) Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.