2. Universidad Cardenal Herrera-CEU

Leishmaniasis a ects around 12 million people worldwide and is estimated to cause the ninth-largest disease burden. There are three main forms of the disease, visceral (VL), cutaneous (CL), and mucocutaneous (MCL), leading to more than one million new cases every year and several thousand deaths. Current treatments based on chemically synthesized molecules are far from ideal. In this study, we have tested the in vitro and in vivo e cacy of ursolic acid (UA), a multifunctional triterpenoid with well-known antitumoral, antioxidant, and antimicrobial e ects on di erent Leishmania strains. The in vitro antileishmanial activity against the intracellular forms was six and three-fold higher compared to extracellular forms of L. amazonensis and L. infantum, respectively. UA also showed to be a potent antileishmanial drug against both VL and CL manifestations of the disease in experimental models. UA parenterally administered at 5 mg/kg for seven days significantly reduced the parasite burden in liver and spleen not only in murine acute infection but also in a chronic-infection model against L. infantum. In addition, UA ointment (0.2%) topically administered for four weeks diminished (50%) lesion size progression in a chronic infection model of CL caused by L. amazonensis, which was much greater than the e ect of UA formulated as an O/W emulsion. UA played a key role in the immunological response modulating the Th1 response. The exposure of Leishmania-infected macrophages to UA led to a significant di erent production in the cytokine levels depending on the Leishmania strain causing the infection. In conclusion, UA can be a promising therapy against both CL and VL.

A stronger Th1 (cellular) immune response in canine leishmaniosis (CanL) leads to a better prognosis. Dietary nucleotides plus AHCC® have shown beneficial e ects in dogs with clinical leishmaniosis and in clinically healthy Leishmania-infected dogs. The potential leishmanicidal activity of nucleotides and AHCC was assessed by quantifying nitric oxide (NO) production and replication of parasites. Their e ects on lymphocyte proliferation were studied with and without soluble Leishmania infantum antigen (SLA) stimulation. Cytokine level variations were assessed using naïve and L. infantum-infected macrophages/lymphocytes cocultures. Promastigotes and amastigotes proliferation and NO macrophage production were not directly a ected. Lymphocyte proliferation was significantly enhanced by nucleotides, AHCC, and their combinations only after SLA stimulation. Nucleotides and AHCC significantly increased the production of IL-1 , IL-2, IL-5, IL-9, IL-10, and IL-12 by naïve immune cells. In naïve and L. infantum-infected macrophage/lymphocyte cocultures, nucleotides with or without AHCC led to significant increases in IFN- and TNF- . Given that these cytokines are involved in the e ective Th1 immune response against Leishmania parasites, these mechanisms of action could explain the previously reported in vivo clinical e cacy of such combination and further support the use of nucleotides with or without AHCC in the management of CanL patients.

The current therapies of leishmaniasis, the second most widespread neglected tropical disease, have limited e ectiveness and toxic side e ects. In this regard, natural products play an important role in overcoming the current need for new leishmanicidal agents. The present study reports a bioassay-guided fractionation of the ethanolic extract of leaves of Piper pseudoarboreum against four species of Leishmania spp. promastigote forms, which a orded six known alkamides (1–6). Their structures were established on the basis of spectroscopic and spectrometric analysis. Compounds 2 and 3 were identified as the most promising ones, displaying higher potency against Leishmania spp. promastigotes (IC50 values ranging from 1.6 to 3.8 M) and amastigotes of L. amazonensis (IC50 values ranging from 8.2 to 9.1 M) than the reference drug, miltefosine. The e cacy of (E)-piplartine (3) against L. amazonensis infection in an in vivo model for cutaneous leishmaniasis was evidenced by a significant reduction of the lesion size footpad and spleen parasite burden, similar to those of glucantime used as the reference drug. This study reinforces the therapeutic potential of (E)-piplartine as a promising lead compound against neglected infectious diseases caused by Leishmania parasites.

A phytochemical investigation of the ethanolic extract of leaves from Piper pseudoarboreum led to the isolation of 3-chlorosintenpyridone 1, an unprecedented chlorinated piperamide, together with the known compounds 2-12. Their structures were established based on 1D and 2D (COSY, ROESY, HMQC, and HMBC) NMR spectroscopy, in addition to high resolution mass spectrometry. The proposed biosynthetic pathway of compound 1 is discussed. Compounds 1-12 were tested in vitro for their leishmanicidal potential against promastigote stages of Leishmania amazonensis, L braziliensis, L. guyanensis and L. infantum. Two compounds from this series, the alkamide 1 (IC50 3.4-5.2 μM) and the fatty acid 9 (IC50 18.7- 29.6 μM) displayed higher or similar potency to Miltefosine, used as the reference drug.

Leishmaniasis is a neglected tropical disease that currently affects 12 million people, and over 1 billion people are at risk of infection. Current chemotherapeutic approaches used to treat this disease are unsatisfactory, and the limitations of these drugs highlight the necessity to develop treatments with improved efficacy and safety. To inform the rational design and development of more efficient therapies, the present study reports a chemoinformatic approach using the ChEMBL database to retrieve benzimidazole as a target scaffold. Our analysis revealed that a limited number of studies had investigated the antileishmanial effects of benzimidazoles. Among this limited number, L. major was the species most commonly used to evaluate the antileishmanial effects of these compounds, whereas L. amazonensis and L. braziliensis were used least often in the reported studies. The antileishmanial activities of benzimidazole derivatives were notably variable, a fact that may depend on the substitution pattern of the scaffold. In addition, we investigated the effects of a benzimidazole derivative on promastigotes and amastigotes of L. infantum and L. amazonensis using a novel fluorometric method. Significant antileishmanial effects were observed on both species, with L. amazonensis being the most sensitive. To the best of our knowledge, this chemoinformatic analysis represents the first attempt to determine the relevance of benzimidazole scaffolds for antileishmanial drug discovery using the ChEMBL database. The present findings will provide relevant information for future structure–activity relationship studies and for the investigation of benzimidazole-derived drugs as potential treatments for leishmaniasis.

La leishmaniosis es una zoonosis en la que distintas especies animales actúan como reservorio del parásito. El perro es el principal reservorio de la enfermedad sufriendo graves patologías que pueden llegar a ser letales en el animal. La leishmaniosis canina es endémica es todos los países de la Cuenca Mediterránea, aunque en las últimas décadas ha comenzado a extenderse además por la zona norte de Europa. En el momento actual no existe cura farmacológica, ya que con los tratamientos actuales (principalmente Glucantime® y alopurinol) se consigue reducir la carga parasitaria y mejorar los síntomas clínicos de la enfermedad, pero después de un determinado periodo de tiempo, se vuelven a producir recidivas. Por lo tanto, existe una necesidad clínica en el mundo veterinario de desarrollar nuevos fármacos o nuevas formulaciones de fármacos ya comercializados que sean capaces de mejorar la evolución de esta enfermedad en perros. En la leishmaniasis, los parásitos se acumulan preferentemente en los macrófagos del sistema retículo endotelial. En este tipo de enfermedad, es interesante vectorizar el fármaco hacia el lugar donde se localizan los parásitos y por lo tanto, favorecer la opsonización y fagocitosis de los fármacos administrados por parte de los macrófagos, lo cual puede realizarse por medio de cambios en el estado de agregación, tamaño de partícula e hidrofobicidad de las partículas que se vayan a administrar por vía intravenosa. El uso de la anfotericina B se encuentra relegado a un segundo plano en el tratamiento de la leishmaniosis canina, a pesar de ser el tratamiento de elección en casos de leishmaniasis visceral en humanos. Las formulaciones lipídicas comercializadas de anfotericina B (AmBiome®, Amphocil® y Abelcet®) en el momento actual son las que presentan un mejor perfil de seguridad en comparación con la anfotericina B convencional (Fungizona® o anfoterician B dimérica); sin embargo, el coste del tratamiento es bastante elevado, limitando el uso de anfotericina B en el mundo veterinario. Por esta razón, el objetivo de esta tesis se ha centrado en la evaluación farmacocinética y farmacodinámica de una nueva formulación de anfotericina B poliagregada que sea segura y al mismo tiempo coste-efectiva con aplicación a nivel veterinario en el tratamiento de la leishmaniasis canina. En los estudios farmacocinéticos realizados en perros Beagle sanos, se observó que con la formulación poliagregada aunque las concentraciones plasmáticas no fueron tan elevadas como las obtenidas por el AmBisome®, sin embargo, se obtuvo un balance beneficio/riesgo favorable, ya que se pudieron administrar dosis de hasta 10 mg/kg sin que ser observados efectos adversos agudos en los Beagles y además se mantuvieron en el organismo concentraciones eficaces durante periodos de hasta 15 días. Esto facilita establecer un régimen de administración más cómodo y no hace falta una administración diaria de dosis bajas como en el caso de la Fungizona® debido a su nefrotoxicidad. Además, se evaluó el perfil farmacocinético de la formulación poliagregada encapsulada en microsferas de albúmina. Dicha formulación aunque parece que permitió vectorizar mejor la anfotericna B ya que presentó menor riesgo de interacciones con fármacos que se unen a proteínas plasmáticas en un alto porcentaje, sin embargo, presentó un balance beneficio/riesgo menor que la formulación poliagregada sin encapsular, debido al riesgo de producir reacciones anafilácticas. Con la administración de tres dosis de 5 mg/kg cada quince días de anfotericina B poliagregada se mantuvieron concentraciones eficaces en el organismo siendo esta la pauta elegida para comenzar los estudios de eficacia en perros infectados de forma natural. Además, se incluyó otra segunda pauta de tratamiento con dosis ligeramente inferiores, que consistió en 4 mg/kg cada quince días. Con ambas dosis, se consiguió la reducción de los síntomas clínicos (en torno al 40%) al final del tratamiento, siendo las lesiones cutáneas las que mejoraron en mayor medida en la mayoría de los perros objeto de estudio. Con la dosis de 5 mg/kg además, se consiguió una reducción de los títulos de anticuerpos más rápida que con la dosis de 4 mg/Kg. En conclusión, la anfotericina B poliagregada es una formulación sencilla de preparar, coste-efectiva, fácilmente escalable a nivel industrial y que es capaz de mejorar los síntomas de la leishmaniosis canina pudiendo ser de gran aplicación en el ámbito veterinario. / Leishmaniosis is a zoonosis in which different animal species act as reservoir of the parasite. The dog is the main reservoir of the disease suffering serious pathologies that can become lethal in the animal. Canine leishmaniasis is endemic is all the countries of the Mediterranean Basin, although in the last decades it has begun to spread also in the northern part of Europe. At the present time, there is no pharmacological cure, since the current treatments (mainly Glucantime® and allopurinol) reduce the parasitic load and improve the clinical symptoms of the disease, but after a certain period of time, recidives occur. Therefore, there is a clinical need in the veterinary world to develop new drugs or new formulations of already marketed drugs that are capable of improving the evolution of this disease in dogs. In leishmaniasis, the parasites preferentially accumulate in the macrophages of the reticulo-endothelial system. In this type of disease, it is interesting to target the drug to the place where the parasites are located and by means favour of the opsonization and phagocytosis of the drugs by the macrophages, which can be done thorugh changes in the state of aggregation, particle size and hydrophobicity of the particles administered. The use of amphotericin B is relegated in the treatment of canine leishmaniasis despite being the treatment of choice of visceral leishmaniasis in humans. The commercially available lipid formulations of amphotericin B (AmBiome®, Amphocil® and Abelcet®) are those with a better safety profile compared to conventional amphotericin B (Fungizone® or amphoteric B); however, the cost of treatment is quite high, limiting the use of amphotericin B in the veterinary world. For this reason, the aim of this thesis has focused on the pharmacokinetic and pharmacodynamic evaluation of a new formulation of poly-aggregated amphotericin B that is safe and at the same time cost-effective with veterinary application in the treatment of canine leishmaniasis. In the pharmacokinetic studies performed on healthy Beagle dogs, it was observed that with the poly-aggregated formulation although plasma concentrations were not as high as those obtained by AmBisome®, however, showed a favorable benefit / risk balance, since the formulation could be administered up to doses of 10 mg / kg without being observed acute adverse effects in the Beagles and in addition effective concentrations were maintained in the organism for periods of up to 15 days. This makes easier to establish a more convenient regimen of administration and does not require a daily administration of low doses as in the case of Fungizone® due to its nephrotoxicity. In addition, the pharmacokinetic profile of the encapsulated poly-aggregated formulation in albumin microspheres was evaluated. Although this formulation appears to have allowed better targeting of the drug (specially lower risk of interactions with drugs that bind to plasma proteins in a high percentage), it presented a lower benefit / risk balance than the free poly-aggregated formulation due to the risk of producing anaphylactic reactions. The administration of three doses of 5 mg / kg every fifteen days of poly-aggregated amphotericin B led to effective concentrations that were maintained in the organism. For this reason, this was the regime chosen for the pharmacodynamics studies in naturally infected dogs. In addition, a second treatment regimen was included with slightly lower doses, which consisted on 4 mg / kg every fortnight. With both doses, a reduction in the clinical symptoms (around 40%) was achieved at the end of treatment, being cutaneous lesions improved in most of the dogs under study. At the dose of 5 mg / kg, a reduction in the antibody titles was achieved faster than at the dose of 4 mg / kg. In conclusion, the poly-aggregated amphotericin B is a simple, cost-effective formulation that is easily scalable at the industrial level and is capable of improving the symptoms of canine leishmaniasis and may be of great application in the veterinary field.