Facultad de Ciencias de la Salud

Leishmaniasis is a neglected disease presenting cutaneous, mucosal and visceral forms and affecting an estimated 12 million mostly low-income people. Treatment of cutaneous leishmaniasis (CL) is recommended to expedite healing, reduce risk of scarring, prevent parasite dissemination to other mucocutaneous (common with New World species) or visceral forms and reduce the chance of relapse, but remains an unmet need. Available treatments are painful, prolonged (>20 days) and require hospitalisation, which increases the cost of therapy. Here we present the development of optimised topical self-nanoemulsifying drug delivery systems (SNEDDS) loaded with buparvaquone (BPQ, a hydroxynapthoquinone from the open Malaria Box) for the treatment of CL from New World species. The administration of topical BPQ-SNEDDS gels for 7 days resulted in a reduction of parasite load of 99.989 ± 0.019 % similar to the decrease achieved with intralesionally administered Glucantime® (99.873 ± 0.204 %) in a L. amazonensis BALB/c model. In vivo efficacy was supported by ex vivo permeability and in vivo tape stripping studies. BPQ-SNEDDS and their hydrogels demonstrated linear flux across non-infected CD-1 mouse skin ex vivo of 182.4 ± 63.0 μg cm-2 h-1 and 57.6 ± 10.8 μg cm-2 h-1 respectively localising BPQ within the skin in clinically effective concentrations (227.0 ± 45.9 μg and 103.8 ± 33.8 μg) respectively. These levels are therapeutic as BPQ-SNEDDS and their gels showed nanomolar in vitro efficacy against L. amazonensis and L. braziliensis amastigotes with excellent selectivity index toward parasites versus murine macrophages. In vivo tape stripping experiments indicated localisation of BPQ within the stratum corneum and dermis. Histology studies confirmed the reduction of parasitism and indicated healing in animals treated with BPQ-SNEDDS hydrogels. These results highlight the potential clinical capability of nano-enabled BPQ hydrogels towards a non-invasive treatment for CL.

Cutaneous fungal and parasitic diseases remain challenging to treat, as available therapies are unable to permeate the skin barrier. Thus, treatment options rely on systemic therapy, which fail to produce high drug local concentrations but can lead to significant systemic toxicity. Amphotericin B (AmB) is highly efficacious in the treatment of both fungal and parasitic diseases such as cutaneous leishmaniasis, but is only reserved for parenteral administration in patients with severe pathophysiology. Here, we have designed and optimised AmB-transfersomes [93.5 % encapsulation efficiency, size of 150 nm, and good colloidal stability (-35.02 mV)] that can remain physicochemically stable (>90 % drug content) at room temperature and 4 °C over 6 months when lyophilised and stored under desiccated conditions. AmBtransfersomes possessed good permeability across mouse skin (4.91 ± 0.41 μg/cm2/h) and 10-fold higher permeability across synthetic Strat-M® membranes. In vivo studies after a single topical application in mice showed permeability and accumulation within the dermis (>25 μg AmB /g skin at 6 h post-administration) indicating the delivery of therapeutic amounts of AmB for mycoses and cutaneous leishmaniasis, while a single daily administration in Leishmania (Leishmania) amazonensis infected mice over 10 days resulted in excellent efficacy (98 % reduction in Leishmania parasites). Combining the application of AmB-transfersomes with metallic microneedles in vivo increased levels in the SC and dermis but is unlikely to elicit transdermal levels. In conclusion, AmB-transfersomes are promising and stable topical nanomedicines that can be readily translated for parasitic and fungal infectious diseases.

The diversity and frequency of enteric parasites in dog populations in the Castellón province (Eastern Spain) was assessed by means of a prospective cross sectional epidemiological survey. A total of 263 canine faecal samples were collected between July 2014 and July 2016. Detection of intestinal parasites was conducted by routine coprological methods. In addition, identification of Giardia duodenalis and Cryptosporidium spp. was carried out by direct immunofluorescence microscopy, whereas the presence of Strongyloides spp. was assessed by real-time PCR in a selected number of specimens. Based on conventional and/or immunofluorescence microscopy examination, 65.8% (95% CI: 59.7%‒71.5%) of the investigated dogs were found infected by at least one gastrointestinal parasite. Giardia duodenalis (35.4%) and members of the family Ancylostomatidae (27.0%) were the most prevalent protozoan and helminth parasites found, respectively. Other pathogens potentially infective to human included Toxocara canis (8.0%), Cryptosporidium spp. (6.8%), and Strongyloides spp. (1.1%). Frequency of occurrence of helminthic, but not protozoan, enteroparasites was geographical origin-dependent (P = 0.02), with dogs living in coastal areas presenting higher infection rates than those living in inland regions. Similarly, rural dogs were significantly more infected than urban dogs (P < 0.001). Our results revealed that zoonotic agents were common in dogs from the Castellón province. Animals from rural areas and sheltered dogs were particularly at risk of these infections.

The detection of multi-drug resistant bacteria is a growing problem, however, the role of domesticated animals in the propagation of antimicrobial resistance has barely been studied. The aim of this study was to identify ESBL-producing Escherichia coli strains in domestic animal feces in order to assess their antimicrobial resistance profile and carry out molecular characterization of the ß-lactamases. A total of 325 samples were collected from 8 animal species. Of these, 34 bacterial isolates were identified as E. coli. The antibiotic resistance profile of the E. coli strains was as follows: 100% resistant to amoxicillin, aztreonam, and cephalosporines; 58.8% resistant to nalidixic acid, ciprofloxacin and trimethoprim/sulphamethoxazole; 41.2% resistant to gentamicin and tobramycin; 11.8% resistant and 32.4% intermediate to cefoxitin, 97.1% sensible and 2.9% intermediate to amoxicillin/clavulanate; and 100% sensible to ertapenem, minocycline, imipenem, meropenem, amikacin, nitrofurantoin, phosphomycin and colistin. All 34 E. coli strains met criteria for ESBL production. In total, 46 ß-lactamase genes were detected: 43.5% blaTEM, 30.4% blaCTX-M (23.9% blaCTX-M-1 and 6.5% blaCTX-M-9) and 26.1% blaSHV (17.4% blaSHV-5 and 8.7% blaSHV-12). All the ß-lactamases were found in dogs except for 4 blaSHV found in falcons. No pAmpC genes were found. The high prevalence of ESBL-producing E. coli strains in animals could become a zoonotic transmission vector.

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.