Pharmaco- and immunotherapeutic strategies for the topical treatment of cutaneous leishmaniasis

  1. Juana Schwartz Mota
Supervised by:
  1. Socorro Espuelas Millán Director
  2. Paul Alain Nguewa Kamsu Director

Defence university: Universidad de Navarra

Year of defence: 2016

Committee:
  1. Juan Manuel Irache Garreta Chair
  2. María del Carmen Sanmartín Grijalba Secretary
  3. Manuel Soto Álvarez Committee member
  4. Enrique Martínez Carretero Committee member
  5. María Manuela Gaspar Committee member

Type: Thesis

Abstract

The lack of effective topical formulations for the treatment of cutaneous leishmaniasis urges the need to develop new therapeutic options. In addition, the fact that skin immune response has a strong influence on treatment response and lesion resolution requires more in depth studies in order to find new treatment options. In our first work, we prepared, optimized and evaluated a chitosan hydrogel containing a new chemically-synthesized diselenide drug solubilized with cyclodextrins. This diselenide drug showed antileishmanial activity against L. major and we decided to formulate it in a hydrogel because L. major leads to ulcer formation, so the more hydrophilic epidermis would have a higher affinity for a hydrophilic formulation. Chitosan was chosen because of its wound healing properties. Franz cells diffusion studies showed that the diselenide drug could accumulate in the skin dermis and that little amounts would reach systemic circulation. In a L. major infection BALB/c model, the formulation did not lead to infection cure, which could have been due to the fact that in mice, hyperkeratosis develops upon Leishmania infection, and this could have hindered the permeation of the drug. Also, because the new drug exhibited high hydrogen bonding ability, it may have been retained in the upper skin layer. In the second study, we prepared, optimized and evaluated lecithin-chitosan nanoparticles containing the natural origin drug β-lapachone. β-lapachone showed antileishmanial activity against L. major that was enhanced when it was encapsulated in the nanoparticles. Franz cell skin diffusion studies confirmed that the nanoformulation containing β-lapachone was able to lead to drug accumulation in the dermis. In L. major-infected BALB/c mice, the formulation did not lead to infection cure but it stopped lesion progression. The reduction of the ulceration area, crust, and necrosis of the lesion, as well as the reduction of the neutrophilic infiltrate could be linked to the down-regulation of IL-1β and COX-2. Finally, our aim was to evaluate which strategy could favor infection control through parasite death and modulation of skin microenvironment: (i) the use of dapsone and curcumine, that are antileishmanial and antiinflammatory drugs (double mechanism of action), or the (ii) combination of the leishmanicidal drug paromomycin with biological molecules that have anti- or proinflammatory effects (anti-TNFα or the TLR3 agonist Poly(I:C), respectively). In vitro studies were carried out using L.major and L. braziliensis strains, as treatment outcome differs between species and L. braziliensis has the potential to cause MCL. Dapsone, curcumine and paromomycin showed antileishmanial activity against both strains. In addition, in a mouse model of Imiquimod®-induced psoriasis, dapsone, curcumine, paromomycin and anti-TNFα showed the ability to penetrate through the skin and anti-inflammatory activity. The efficacy of the formulations was assessed in L. major-infected BALB/c mice. Topical formulations containing dapsone and curcumine were not effective, but both paromomycin plus PIC or anti-TNFα reduced parasite burden in skin, lymph nodes, liver, and spleen, and led to lesion reduction. However, the combination of paromomycin plus anti-TNFα led to reduction of ulceration, necrosis, and crust, and reduced the neutrophilic infiltrate in the infected skin, which could be linked to the reduction in the gene expression of TNFα, IL-1β, IL-17, and CCL3.