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En esta tesis se investigación sobre la posible explotación del berberine como antileishmanial.

The oral administration of dapsone (DAP) for the treatment of cutaneous leishmaniasis (CL) is effective, although serious hematological side effects limit its use. In this study, we evaluated this drug for the topical treatment of CL. As efficacy depends on potency and skin penetration, we first determined its antileishmanial activity (IC50 = 100 ¿M) and selectivity index in vitro against Leishmania major-infected macrophages. In order to evaluate the skin penetration ex vivo, we compared an O/W cream containing DAP that had been micronized with a pluronic lecithin emulgel, in which the drug was solubilized with diethylene glycol monoethyl ether. For both formulations we obtained similar low flux values that increased when the stratum corneum and the epidermis were removed. In vivo efficacy studies performed on L. major-infected BALB/c mice revealed that treatment not only failed to cure the lesions but made their evolution and appearance worse. High plasma drug levels were detected and were concomitant with anemia and iron accumulation in the spleen. This side effect was correlated with a reduction of parasite burden in this organ. Our results evidenced that DAP in these formulations does not have an adequate safety index for use in the topical therapy of CL.

MALIGNANT PHYLLODES BREAST TUMOR IN A 16-YEAR-OLD GIRL: A CASE REPORT AND REVIEWProblem statement Phyllodes tumor (PT) of the breast is a rare fibroepithelial neoplasm, accounting for 0.3% to 0.9% of all breast tumors (mean age 40 years). Clinical presentation consists in a mobile, non-painful, rapidly enlarging breast lump with great predisposition to recurrence. MethodsWe report a 16-year-old girl followed in our Breast Unit, who had previously undergone 6cm-left breast adenoepithelioma excision. She reconsults 15 months later, due to reappearance of a 3-4cm left retro-areolar lump. The ultrasound revealed a 26x15x21mm solid lump with heterogeneous echo pattern, areas of cystic degeneration and increased vascularity, which led to suspicion of adenoepithelioma recurrence (BIRADS 4a). A biopsy was performed and the anatomopathologic examination showed a fibroepithelial lesion with an increased mitotic activity, classifying the tumor as borderline/malignant PT. A tumorectomy was performed with a final diagnosis of malignant 34x30mm PT with positive resection margins. Finally, a left mastectomy with periareolar access (including recent tumorectomy scar) was done. Immediate breast reconstruction was performed and a 295-cc polyurethane prepectoral implant covered with an acellular dermal matrix was placed .Two surgical drains were left. Anatomopatology study did not show features of malignancy and today she remains disease-free. ResultsHystological characteristics are the basis for diagnosis of PT but mammography and ultrasound are useful for its identification. The recommended treatment is complete surgical excision (mastectomy if necessary) with 1 cm margins. Radiation treatment can be an option in selected patients but its use is controversial. The use of routine hormone therapy or chemotherapy is not encouraged. ConclusionMPT is an extremely rare neoplasm that makes its diagnosis and treatment a challenge due to its clinical presentation, rapid evolution and high recurrences rates.

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.

Docetaxel (DTX) is a semisynthetic taxoid, analogue of paclitaxel, derived from the European yew tree (Taxus bacata sp.) and is one of the most effective drugs in chemotherapy. It has proven to be useful against several types of cancers such as breast, ovarian, prostate, head and neck, gastric and non-small lung cancers. The oral bioavailability of DTX is hampered by their both high lipophilic character and low permeability related to their affinity for the P-glycoprotein and cytochrome P450 enzymatic complex. In order to solve these problems and, thus, enhance the oral absorption of these drugs, one possible solution may be the encapsulation of these drugs into polymeric nanoparticles with both mucus-penetrating properties and inhibitory abilities of the efflux pump activity and cythocrome P450 metabolism. In this way, in a first step, these nanoparticles would be capable to cross the mucus layer that covers the surface of the enterocytes and, thus, conduct the drug till the absorptive membrane. Once there, in a second step, the carriers would release the cargo and, in parallel, disturb the activity of enzymes and pumps involved in the presystemic metabolism of the loaded drug. The pegylation of nanoparticles from the copolymer of methyl vinyl ether and maleic anhydride (Gantrez® AN) yields submicronic carriers with these specific properties. In the present work, our aim was to explore the potential of these pegylated nanoparticles as delivery systems for docetaxel. In summary, pegylated nanoparticles provide an adequate device for the oral delivery of docetaxel. When orally administered, these nanoparticles offered prolonged and sustained plasma levels of the anticancer drug for 3 days. In addition, the relative oral bioavailability was calculated to be up 56% and around 12-times higher than for ¿naked¿ nanoparticles. Interestingly, the elimination and distribution of docetaxel in vivo from pegylated nanoparticles and intravenous Taxotere® was found to be similar, suggesting that these nanoparticles after reaching the surface of the enterocytes would remain within the gut and did not enter into the circulation. Finally, toxicity study revealed a reduced toxicity profile for nanoparticles orally administered compared to Taxotere®.

Paclitaxel is a natural cytotoxic agent widely used in clinics against breast, ovarian and non-small cell lung cancers. It is classified as class IV by the Biopharmaceutical Classification System presenting a very low oral bioavailability limiting its oral administration. Recently, poly(anhydride) nanoparticles combined with cyclodextrins and poly(ethylene glycol) have been described to increase the oral uptake of paclitaxel when orally administered to rats. Poly(anhydride) nanoparticles were prepared by the solvent displacement method with rather homogeneous size distribution and with a yield of 60-70%. The drug loading achieved varied between 4.5-15% (w/w). Poly(anhydride) nanoparticles containing paclitaxel orally administered to C57BL/6J mice displayed sustained drug levels in plasma for at least 24 hours, with longer residence time for pegylated nanoparticles, and a wide organ distribution similar to Taxol®. This resulted in an increase in the oral bioavailability of paclitaxel up to 60-80%. Besides, the developed poly(anhydride) nanoparticles were evaluated in a syngenic tumor model of mouse Lewis lung carcinoma. Mice were treated orally with the different poly(anhydride) nanoparticles or intravenously with the commercially available formulation Taxol®. Results showed that oral paclitaxel loaded in poly(anhydride) nanoparticles was able to slow down tumor growth in this tumor model in C57BL/6J mice. Moreover, the combination of these poly(anhydride) nanoparticles encapsulating paclitaxel with a selective IDO inhibitor, such as 1-methyl-tryptophan, was a more effective strategy than the single chemotherapy based on paclitaxel loaded in poly(anhydride) nanoparticles. In conclusion, these nanocarriers appear to be promising systems for the future development in oral chemotherapy even in combination with immunotherapy treatment.

Paclitaxel is an anticancer drug used as solution for perfusion for the treatment of certain types of cancers. In the last years, a number of strategies have been proposed for the development of an oral formulation of this drug. However, this task is quite complicated due to the poor aqueous solubility of paclitaxel as well as the fact that this compound is substrate of the intestinal P-glycoprotein and the cytochrome P450 enzymatic complex. In this work, we have developed pegylated nanoparticles with mucopenetrating properties in order to conduct paclitaxel onto the surface of the enterocyte. These nanoparticles displayed a size of about 180 nm and a drug loading close to 15% by weight. The pharmacokinetic study in mice has shown that these nanoparticles were capable to offer therapeutic plasma levels of paclitaxel up to 72 hours. In addition, the oral relative bioavailability of paclitaxel when loaded in nanoparticles pegylated with poly(ethylene glycol) 2000 (PEG) was found to be 85%. In a subcutaneous model of tumour in mice, these pegylated nanoparticles administered orally every 3 days have demonstrated a similar efficacy than Taxol® administered intravenously every day during 9 days. All of these results suggested that these pegylated nanoparticles were capable to cross the mucus layer of the gut and, then, reach the surface of the enterocytes. The PEG molecules would facilitate the adhesion of nanoparticles to this epithelial surface, minimise the pre-systemic metabolism of paclitaxel and, thus, promote its absorption.

It seems well established that CpG oligonucleotides Th1 biased adjuvant activity can be improved when closely associated with a variety of antigens in, for example, microparticles. In this context, we prepared 1-μm near non-charged PLGA 502 or PLGA 756 microparticles that loaded with high efficiency an antigen (50% ovalbumin (OVA), approximately) into their matrix and CpG-chitosan complexes (near to 20%) onto their surface maintaining OVA and CpG integrity intact. In the intradermal immunization studies, whereas OVA microencapsulated into PLGA 756 alone induced a strong humoral immune response assisted by a very clear Th1 bias (IgG2a/IgG1=0.875) that was decreased by CpG co-delivery (IgG2a/IgG1=0.55), the co-encapsulation of CpG with OVA in PLGA 502 particles significantly improved the antibody response and isotype shifting (IgG2a/IgG1=0.73) in comparison with mice immunized with OVA loaded PLGA 502 (IgG2a/IgG1=0). This improvement was not correlated with the cellular immune response where the effect of co-encapsulated CpG was rather negative (2030.2 pg/mL and 335.3 pg/mL IFN-g for OVA PLGA 502 for OVA CpG PLGA 502, respectively). These results underscore the critical role of polymer nature and microparticle characteristics to show the benefits of coencapsulating CpG motifs in close proximity with an antigen.

The aim of this study was to evaluate the activity of gamma interferon (IFN-gamma) when it was either adsorbed onto or loaded into albumin nanoparticles. Brucella abortus-infected macrophages and infected BALB/c mice were selected as the models for testing of the therapeutic potentials of these cytokine delivery systems, in view of the well-established role of IFN-gamma-activated macrophages for the control of Brucella sp. infections. Whereas the encapsulation of IFN-gamma inside the matrix of nanoparticles completely abrogated its activity, adsorbed IFN-gamma increased by 0.75 log unit the bactericidal effect induced by RAW macrophages activated with free IFN-gamma, along with a higher level of production of nitric oxide. In infected BALB/c-mice, IFN-gamma adsorbed onto nanoparticles was also more active than free cytokine in reducing the number of bacteria in the spleens, and the effect was mediated by an increased ratio of IFN-gamma-secreting (Th1) to interleukin-4-secreting (Th2) cells. Overall, albumin nanoparticles would be suitable as carriers that target IFN-gamma to macrophages and, thus, potentiate their therapeutic activity.

Background Despite immunotherapy has been reported as the only treatment able to revert the Th2 response, its administration has some disadvantages such as the requirement of multiple doses, possible side effects provoked by conventional adjuvants and the risk of suffering an anaphylactic shock. For that reasons, drug delivery systems appear to be a promising strategy due to its ability to i) transport the allergens, ii) protect them from degradation, iii) decrease the number of administrations and iv) act as immuno-adjuvants. Objective The aim of this work was to evaluate the properties of poly- -caprolactone (PCL) microparticles as adjuvants in immunotherapy using ovalbumin (OVA) as allergen model. For this purpose, the protection capacity of these microparticles (OVA PCL) against OVA allergy was studied in a murine model. Methods The humoral and cellular induced immune response generated by OVA encapsulated into PCL microparticles was studied immunizing BALB/c mice intradermically. Beside, OVAsensitized mice were treated with OVA PCL and OVA adsorbed to aluminium hydroxide (OVAAlum). Fifteen days after therapy, animals were challenged with OVA and different signs of anaphylactic shock were evaluated. Results One single shot by intradermal route with OVA PCL resulted in a Th2-type immune response. In OVA-sensitized mice, treatment with OVA PCL treatment elicited high OVA specific IgG but low levels of IgE. Furthermore, OVA PCL mice group displayed lower levels of serum histamine and higher survival rate in comparison with the positive control group. Conclusion The anaphylactic shock suffered by OVA PCL treated mice was weaker than the one induced in the OVA-Alum group. Hence, the intradermal immunization with OVA PCL microparticles induced hyposensitization in OVA-allergic mice.

A micellar formulation of amphotericin B (AmB) solubilized with poloxamer 188 was evaluated against an AmB Leishmania donovani-resistant line. A concave isobologram showed a synergistic effect of this association against promastigotes. This result was confirmed with amastigotes since the 50% effective concentration of the new formulation was 100 times less than that of the control AmB formulation.