Estudios sobre la eficacia antihelmíntica de oxfendazol : Tioxidazol y Mebendazol en la triquinosis experimental

Tesis doctoral, Univ. Complutense, 1981.Depto. de Microbiología y ParasitologíaFac. de FarmaciaTRUEProQuestpu

Estudios sobre la eficacia antihelmíntica de oxfendazol : Tioxidazol y Mebendazol en la triquinosis experimental

Tesis doctoral, Univ. Complutense, 1981.Depto. de Microbiología y ParasitologíaFac. de FarmaciaTRUEProQuestpu

Transferosomes as nanocarriers for drugs across the skin: Quality by design from lab to industrial scale

Transferosomes, also known as transfersomes, are ultradeformable vesicles for transdermal applications consisting of a lipid bilayer with phospholipids and an edge activator and an ethanol/aqueous core. Depending on the lipophilicity of the active substance, it can be encapsulated within the core or amongst the lipid bilayer. Compared to liposomes, transferosomes are able to reach intact deeper regions of the skin after topical administration delivering higher concentrations of active substances making them a successful drug delivery carrier for transdermal applications. Most transferosomes contain phosphatidylcholine (C18) as it is the most abundant lipid component of the cell membrane, and hence, it is highly tolerated for the skin, decreasing the risk of undesirable effects, such as hypersensitive reactions. The most common edge activators are surfactants such as sodium deoxycholate, Tween® 80 and Span® 80. Their chain length is optimal for intercalation within the C18 phospholipid bilayer. A wide variety of drugs has been successfully encapsulated within transferosomes such as phytocompounds like sinomenine or apigenin for rheumatoid arthritis and leukaemia respectively, small hydrophobic drugs but also macromolecules like insulin. The main factors to develop optimal transferosomal formulations (with high drug loading and nanometric size) are the optimal ratio between the main components as well as the critical process parameters for their manufacture. Application of quality by design (QbD), specifically design of experiments (DoE), is crucial to understand the interplay among all these factors not only during the preparation at lab scale but also in the scale-up process. Clinical trials of a licensed topical ketoprofen transferosomal gel have shown promising results in the alleviation of symptons in orthreothritis with non-severe skin and subcutaneous tissue disorders. However, the product was withdrawn from the market which probably was related to the higher cost of the medicine linked to the expensive manufacturing process required in the production of transferosomes compared to other conventional gel formulations. This example brings out the need for a careful formulation design to exploit the best properties of this drug delivery system as well as the development of manufacturing processes easily scalable at industrial level.Depto. de Farmacia Galénica y Tecnología AlimentariaFac. de FarmaciaInstituto Universitario de Farmacia IndustrialTRUEpu

In vitro effect of new formulations of amphotericin B on amastigote and promastigote forms of Leishmania infantum

The in vitro antileishmanial activities of various new amphotericin B (AMB) formulations were investigated, including microspheres of hydrophilic albumin with three AMB aggregation forms (monomeric, dimeric and multiaggregate) and the polymers of polylactic-co-glycolic acid, Resomer RG502 and RG503 with the multiaggregate AMB form. This in vitro study was performed on the extracellular promastigote form and the intracellular amastigote form of a canine strain of Leishmania infantum (UCM 20) using the infected J774 murine macrophage-like cell line. Albumin-encapsulated forms did not show any toxicity for murine cells and had lower median effective concentration (EC50) values (ca. 0.003 microg/mL) for L. infantum amastigotes than free formulations (0.03 microg/mL). In addition, the aggregation state of AMB had a notable effect on the antileishmanial activity of the drug. Results obtained in vitro point towards interest in monomeric AMB encapsulated in microspheres in the chemotherapeutic control of leishmaniasis.Ministerio de Educación y CienciaDepto. de Bioquímica y Biología MolecularFac. de MedicinaTRUEpu

Identification of proteins interacting with HSP70 mRNAs in Leishmania Braziliensis

HSP70 protein is involved in Leishmania differentiation, apoptosis, antimony-resistance and host-immune response. Therefore, this protein and the regulatory mechanisms of HSP70 gene expression are promising targets for therapeutic intervention against leishmaniasis. The regulation of mRNA expression in trypanosomatids operates mostly through the interaction of trans-acting proteins, and elements located in the untranslated regions of mRNAs. The aim of this work was to identify protein factors interacting specifically with the Leishmania braziliensis HSP70 mRNAs. Thus, the 5' UTR and the two types of 3' UTRs (UTR-I and UTR-II) from L. braziliensis HSP70 genes were used as baits in pull down assays using total protein extracts from parasites cultured at 26 or 35. °C. The captured proteins were resolved by two-dimensional gel electrophoresis (2-DE) and identified by mass spectrometry (MS) analysis. As a result, 52 different proteins were identified based on their binding to the L. braziliensis HSP70-mRNAs. As expected, several of the identified proteins were related to RNA metabolism (27%) and translation process (7%). In addition, five hypothetical conserved proteins having motifs related with RNA interaction were also identified (9.6%). Nevertheless, unexpected proteins, apparently unrelated to the mRNA expression, were also identified. The biological significance of these and others L. braziliensis detected proteins, including the HSP70 itself, is discussed. © 2013 The Authors.Colciencias (Colombia), Research project No. 1203-405-20233; Ministerio de Ciencia y Tecnología (BFU2009-08986); Comunidad Autónoma de Madrid (S2010/BMD-2361); Fondo de Investigaciones Sanitarias (ISCIII-RETIC RD12/0018/0009-FEDER)Peer Reviewe

Enhanced bioavailability and anthelmintic efficacy of mebendazole in redispersible microparticles with low-substituted hydroxypropylcellulose

Antecedentes: el mebendazol (MBZ) es un fármaco extremadamente insoluble y, por lo tanto, poco absorbido y los resultados clínicos variables pueden correlacionarse con las concentraciones sanguíneas. La necesidad de un tratamiento prolongado con dosis altas de este fármaco aumenta el riesgo de efectos adversos. Métodos: En el presente estudio preparamos micropartículas redispersables (RDM) que contienen MBZ, un fármaco oral poco soluble en agua, en diferentes proporciones de hidroxipropilcelulosa poco sustituida (L-HPC). Investigamos las estructuras de micropartículas que emergen espontáneamente tras la dispersión de un RDM en medio acuoso y dilucidamos su influencia en la disolución, y también en su biodisponibilidad oral y eficiencia terapéutica utilizando un modelo murino de infección con el parásito nematodo Trichinella espiralis. Resultados: Se obtuvieron porcentajes elevados de fármaco disuelto con RDM en proporciones 1:2,5 y 1:5 de MBZ:L-HPC. El análisis térmico mostró una amorfización de MBZ en el RDM por la ausencia de un pico de fusión claro de MBZ en las formulaciones. El rápido comportamiento de disolución podría deberse a la disminución de la cristalinidad del fármaco, el rápido tiempo de disolución de portadores como L-HPC, junto con su superior dispersabilidad y excelentes propiedades humectantes. RDM-1:2,5 y RDM-1:5 dieron como resultado un aumento de la concentración plasmática máxima y de los valores de área(s) bajo la curva (AUC)(0-infinito). Asimismo, después de la administración oral de RDM-1:2,5 y RDM-1:5, el AUC(0-infinito) fue 2,67 y 2,97 veces mayor, respectivamente, en comparación con el MBZ puro. La actividad terapéutica, evaluada en el ciclo de vida de Trichinella espiralis, mostró que RDM-1:5 fue el más eficaz para reducir el número de parásitos (4,56 veces) en comparación con el MBZ puro, en estado enquistado. Conclusión: El MBZ: L-HPC RDM podría ser una forma efectiva de mejorar la biodisponibilidad oral y la actividad terapéutica utilizando dosis bajas de MBZ (5 mg/kg), lo que implica un bajo grado de toxicidad para los humanos.Background: Mebendazole (MBZ) is an extremely insoluble and therefore poorly absorbed drug and the variable clinical results may correlate with blood concentrations. The necessity of a prolonged high dose treatment of this drug increases the risk of adverse effects. Methods: In the present study we prepared redispersible microparticles (RDM) containing MBZ, an oral, poorly water-soluble drug, in different proportions of low-substituted hydroxypropylcellulose (L-HPC). We investigated the microparticulate structures that emerge spontaneously upon dispersion of an RDM in aqueous medium and elucidated their influence on dissolution, and also on their oral bioavailability and therapeutic efficiency using a murine model of infection with the nematode parasite Trichinella spiralis. Results: Elevated percentages of dissolved drug were obtained with RDM at 1:2.5 and 1:5 ratios of MBZ: L-HPC. Thermal analysis showed an amorphization of MBZ in the RDM by the absence of a clear MBZ melting peak in formulations. The rapid dissolution behavior could be due to the decreased drug crystallinity, the fast dissolution time of carriers as L-HPC, together with its superior dispersibility and excellent wetting properties. RDM-1:2.5 and RDM-1:5 resulted in increased maximum plasma concentration and area(s) under the curve (AUC)0-∞ values. Likewise, after oral administration of the RDM-1:2.5 and RDM-1:5 the AUC0-∞ were 2.67- and 2.97-fold higher, respectively, compared to those of pure MBZ. Therapeutic activity, assessed on the Trichinella spiralis life cycle, showed that RDM-1:5 was the most effective in reducing the number of parasites (4.56-fold) as compared to pure MBZ, on the encysted stage. Conclusion: The MBZ: L-HPC RDM might be an effective way of improving oral bioavailability and therapeutic activity using low doses of MBZ (5 mg/kg), which implies a low degree of toxicity for humans.Universidad Complutense de MadridDepto. de Farmacia Galénica y Tecnología AlimentariaFac. de FarmaciaTRUEpu

QSAR for RNases and theoretic–experimental study of molecular diversity on peptide mass fingerprints of a new Leishmania infantum protein

The toxicity and low success of current treatments for Leishmaniosis determines the search of new peptide drugs and/or molecular targets in Leishmania pathogen species ( L. infantum and L. major ). For example, Ribonucleases (RNases) are enzymes relevant to several biologic processes; then, theoretical and experimental study of the molecular diversity of Peptide Mass Fingerprints (PMFs) of RNases is useful for drug design. This study introduces a methodology that combines QSAR models, 2D-Electrophoresis (2D-E), MALDI-TOF Mass Spectroscopy (MS), BLAST alignment, and Molecular Dynamics (MD) to explore PMFs of RNases. We illustrate this approach by investigating for the first time the PMFs of a new protein of L. infantum . Here we report and compare new versus old predictive models for RNases based on Topological Indices (TIs) of Markov Pseudo-Folding Lattices. These group of indices called Pseudo-folding Lattice 2D-TIs include: Spectral moments π k (x,y), Mean Electrostatic potentials ξ k (x,y), and Entropy measures θ k (x,y). The accuracy of the models (training/cross-validation) was as follows: ξ k (x,y)-model (96.0%/91.7%)> π k (x,y)-model (84.7/83.3) > θ k (x,y)-model (66.0/66.7). We also carried out a 2D-E analysis of biological samples of L. infantum promastigotes focusing on a 2D-E gel spot of one unknown protein with M <20, 100 and pI 30, but not one >52 (threshold value), the higher value of 42 was for a probable DNA-directed RNA polymerase. However, we determined experimentally the sequence of more than 140 peptides. We used QSAR models to predict RNase scores for these peptides and BLAST alignment to confirm some results. We also calculated 3D-folding TIs based on MD experiments and compared 2D versus 3D-TIs on molecular phylogenetic analysis of the molecular diversity of these peptides. This combined strategy may be of interest in drug development or target identification

Self-assembling, supramolecular chemistry and pharmacology of amphotericin B: Poly-aggregates, oligomers and monomers

Antifungal drugs such as amphotericin B (AmB) interact with lipids and phospholipids located on fungal cell membranes to disrupt them and create pores, leading to cell apoptosis and therefore efficacy. At the same time, the interaction can also take place with cell components from mammalian cells, leading to toxicity. AmB was selected as a model antifungal drug due to the complexity of its supramolecular chemical structure which can self-assemble in three different aggregation states in aqueous media: monomer, oligomer (also known as dimer) and poly-aggregate. The interplay between AmB self-assembly and its efficacy or toxicity against fungal or mammalian cells is not yet fully understood. To the best of our knowledge, this is the first report that investigates the role of excipients in the supramolecular chemistry of AmB and the impact on its biological activity and toxicity. The monomeric state was obtained by complexation with cyclodextrins resulting in the most toxic state, which was attributed to the greater production of highly reactive oxygen species upon disruption of mammalian cell membranes, a less specific mechanism of action compared to the binding to the ergosterol located in fungal cell membranes. The interaction between AmB and sodium deoxycholate resulted in the oligomeric and polyaggregated forms which bound more selectively to the ergosterol of fungal cell membranes. NMR combined with XRD studies elucidated the interaction between drug and excipient to achieve the AmB aggregation states, and ultimately, their diffusivity across membranes. A linear correlation between particle size and the efficacy/toxicity ratio was established allowing to modulate the biological effect of the drug and hence, to improve pharmacological regimens. However, particle size is not the only factor modulating the biological response but also the equilibrium of each state which dictates the fraction of free monomeric form available. Tuning the aggregation state of AmB formulations is a promising strategy to trigger a more selective response against fungal cells and to reduce the toxicity in mammalian cells.European Regional Development FundEuropean Society of Clinical Microbiology an Infection Diseases (ESCMID)Engineering and Physical Sciences Research CouncilDepto. de Farmacia Galénica y Tecnología AlimentariaFac. de FarmaciaInstituto Universitario de Farmacia IndustrialTRUEpu