Desarrollo y evaluación "in vitro e in vivo" de microesferas biodegradables para la vehiculización de cannabinoides. Development and in vitro in vivo evaluation of biodegradable microspheres for cannabinoid vehiculization

Tesis de la Universidad Complutense de Madrid, Facultad de Farmacia, Departamento de Farmacia y Tecnología Farmacéutica, leída el 13-04-2010. La tesis está escrita en inglés y en español.Depto. de Farmacia Galénica y Tecnología AlimentariaFac. de FarmaciaTRUEpu

Exploiting Nanotechnologies and TRPV1 Channels to Investigate the Putative Anandamide Membrane Transporter

Considerable efforts have been made to characterize the pathways regulating the extracellular levels of the endocannabinoid anandamide. However, none of such pathways has been so argued as the existence of a carrier-mediated transport of anandamide across the membrane. Apart from the lack of molecular evidence for such a carrier, the main reasons of this controversy lie in the methodologies currently used to study anandamide cellular uptake. Furthermore, the main evidence in favor of the existence of an "anandamide transporter" relies on synthetic inhibitors of this process, the selectivity of which has been questioned.We used the cytosolic binding site for anandamide on TRPV1 channels as a biosensor to detect anandamide entry into cells, and exploited nanotechnologies to study anandamide membrane transport into intact TRPV1-overexpressing HEK-293 cells. Both fluorescence and digital holographic (DH) quantitative phase microscopy were used to study TRPV1 activation. Poly-epsilon-caprolactone nanoparticles (PCL-NPs) were used to incorporate anandamide, which could thus enter the cell and activate TRPV1 channels bypassing any possible specific protein(s) involved in the uptake process. We reasoned that in the absence of such protein(s), pharmacological tools previously shown to inhibit the "anandamide transporter" would affect in the same way the uptake of anandamide and PCL-NP-anandamide, and hence the activation of TRPV1. However, when masked into PCL-NPs, anandamide cellular uptake became much less sensitive to these agents, although it maintained the same pharmacokinetics and pharmacodynamics as that of "free" anandamide.We found here that several agents previously reported to inhibit anandamide cellular uptake lose their efficacy when anandamide is prevented from interacting directly with plasma membrane proteins, thus arguing in favor of the specificity of such agents for the putative "anandamide transporter", and of the existence of such mechanism

Procedimientos de asistencia odontológica hospitalaria en pacientes discapacitados psíquicos no colaboradores tratados con anestesia general

La literatura existente acerca del manejo odontológico de pacientes discapacitados es escasa. El tratamiento de estos pacientes se realiza frecuentemente bajo anestesia general y en un medio hospitalario. La población de discapacitados psíquicos representa un grupo con una alta incidencia de patologías orales. La falta de diagnostico y las diferencias en el tratamiento contribuyen al empeoramiento de su salud oral y justifica la existencia de unidades especificas para estos pacientes..

Local delivery of cannabinoid-loaded microparticles inhibits tumor growth in a murine xenograft model of glioblastoma multiforme.

Cannabinoids, the active components of marijuana and their derivatives, are currently investigated due to their potential therapeutic application for the management of many different diseases, including cancer. Specifically, Δ(9)-Tetrahydrocannabinol (THC) and Cannabidiol (CBD) - the two major ingredients of marijuana - have been shown to inhibit tumor growth in a number of animal models of cancer, including glioma. Although there are several pharmaceutical preparations that permit the oral administration of THC or its analogue nabilone or the oromucosal delivery of a THC- and CBD-enriched cannabis extract, the systemic administration of cannabinoids has several limitations in part derived from the high lipophilicity exhibited by these compounds. In this work we analyzed CBD- and THC-loaded poly-ε-caprolactone microparticles as an alternative delivery system for long-term cannabinoid administration in a murine xenograft model of glioma. In vitro characterization of THC- and CBD-loaded microparticles showed that this method of microencapsulation facilitates a sustained release of the two cannabinoids for several days. Local administration of THC-, CBD- or a mixture (1:1 w:w) of THC- and CBD-loaded microparticles every 5 days to mice bearing glioma xenografts reduced tumour growth with the same efficacy than a daily local administration of the equivalent amount of those cannabinoids in solution. Moreover, treatment with cannabinoid-loaded microparticles enhanced apoptosis and decreased cell proliferation and angiogenesis in these tumours. Our findings support that THC- and CBD-loaded microparticles could be used as an alternative method of cannabinoid delivery in anticancer therapies

Characterization of cannabinoide-loaded microparticles.

<p>(A) Scanning electron microscopy (500X) of blank, CBD- and THC-loaded PCL MPs. Representative microphotographs of the three types of MPs are shown. (B) Particle size distribution of blank, CBD- and THC-loaded microspheres. Results correspond to microsphere diameter determined by percentage volume distribution. (C) Cannabinoid release profiles of THC and CBD-loaded PCL microspheres. For the in vitro release studies, microspheres were incubated in PBS pH 7.4-Tween®80 0.1% (v/v) and maintained in a shaking incubator at 37°C. At predetermined time intervals supernatants were withdrawn and media was replaced. The concentration of CBD or THC in the release medium was quantified by HPLC. Data correspond to the cumulative amount of each cannabinoid released at the indicated time points, and are expressed as mean percentage of released cannabinoid relative the total amount of cannabinoid loaded into the microspheres ± s.d (n = 3).</p

Cannabinoid-loaded microparticles reduce the growth of U87MG cell-derived tumour xenografts.

<p>(A) Effect of the local administration of placebo MPs, THC-loaded MP (75 mg of MP containing approximately 6.15 mg of THC per administration, one administration every 5 days), CBD-loaded MP (75 mg of MP containing approximately 6.7 mg of CBD per administration, one administration every 5 days), a mixture (1∶1 w:w) of THC- and CBD-loaded MP (37.5 mg of THC-loaded MP and 37.5 mg of CBD-loaded MP per administration, one administration every 5 days), THC (15 mg/kg/day corresponding to 0.5 mg THC per day), CBD (15 mg/kg/day corresponding to 0.5 mg THC per day) or THC + CBD (7.5 mg/kg/day of THC and 7.5 mg/kg/day CBD corresponding to 0.25 mg of THC and 0.25 mg of CBD per day) on the growth of U87MG cell-derived tumor xenografts. No significant differences were found between tumours treated with vehicle in solution or placebo MPs and these data were represented together. For the sake of clarity, comparisons between the effect of THC-loaded MPs and THC in solution (B), CBD-loaded MPs and CBD in solution (C), and THC-loaded MPs + CBD-loaded MPs and THC + CBD in solution (D) on the growth of U87MG cell-derived tumour xenografts are shown. Results are expressed as the mean fold increase ± SEM relative to vehicle treated tumors on the day one of the treatment. (n = 7). Tumours treated with THC-loaded MPs, CBD loaded MPs, a mixture of THC-loaded MPs and CBD-loaded MPs were significantly different (** p<0.01) from vehicle/placebo MPs-treated tumours. Tumours treated with THC in solution, CBD in solution or a mixture of THC and CBD in solution were also significantly different (p<0.01) from vehicle/placebo-treated tumours from day 14 until the end of the treatment (signs of significance are omitted for clarity). No significant differences were found among any of the treatments with cannabinoid-loaded microparticles and any of the treatments with cannabinoids in solution.</p

Cannabinoid-loaded microparticles reduce the weight of U87MG cell-derived tumour xenografts.

<p>(A) Effect of the local administration of placebo MPs, THC-loaded MP (75 mg of MP containing approximately 6.15 mg of THC per administration, one administration every 5 days), CBD-loaded MP (75 mg of MP containing approximately 6.7 mg of CBD per administration, one administration every 5 days), a mixture (1∶1 w:w) of THC- and CBD-loaded MP (37.5 mg of THC-loaded MP and 37.5 mg of CBD-loaded MP per administration, one administration every 5 days), THC (15 mg/kg/day corresponding to 0.5 mg THC per day), CBD (15 mg/kg/day corresponding to 0.5 mg THC per day) or THC + CBD (7.5 mg/kg/day of THC and 7.5 mg/kg/day CBD corresponding to 0.25 mg of THC and 0.25 mg of CBD per day) on tumour weight on the last day of the treatment. (B) Photographs of representative tumors of each experimental condition. (n = 7; ** p<0.01 from vehicle/placebo MPs-treated tumours).</p

Amount of THC or CBD administered to mice and released at the end of the treatment from cannabinoid-loaded microparticles.

*<p>Animals received 75 mg of cannabinoid-loaded MPs every 5 days (corresponding to a total amount of 300 mg of microparticles per animal).</p