A combination of nanosystems for the delivery of cancer chemoimmunotherapeutic combinations: 1-Methyltryptophan nanocrystals and paclitaxel nanoparticles.

IDO is an enzyme that tumors use to create a state of immunosupression. 1-d-methyltryptophan (1-MT) is an IDO pathway inhibitor. After being successfully evaluated in preclinical studies, current clinical trials are actually analyzing its efficacy as monotherapy or in combination with multiple chemotherapeutic agents such as paclitaxel. 1-MT very poor solubility in water and many other solvents precludes its ease parenteral administration. It is currently administered by oral route because high daily doses were well-tolerated and effectively inhibited the IDO activity although only 25% of dose was recovered in plasma. The present work describes the preparation and characterization of 1-MT nanocrystals in order to enhance its solubility, dissolution rate, biodisponibility as well as facilitate its administration by parenteral route. A bottom-down approach of nanoprecipitation with an antisolvent was used for the fabrication of the nanocrystals and the choice of stabilizers was critical for reducing the size. Thermal analysis and x-ray diffraction indicated modifications in the drug crystalline state by the process. Through the reduction size and crystalline state modifications the dissolution characteristics of raw material were significantly increased. In a Lewis Lung cancer mice model, the nanocrystals strategy facilitated the sc administration and its antitumoral activity was similar to that of i.v. paclitaxel. The best efficacy was achieved when sc 1-MT nanocrystals were administered in combination with oral paclitaxel loaded in poly(anhydride) nanoparticles. Take together, 1-MT nanocrystals delivery performs a nanotechnological strategy suitable to modify the current route and schedule for its administration.

[Mucopenetrating nanoparticles: vehicles for the oral administration of paclitaxel]. / Nanoparticules mucopénétrantes: véhicules pour l'administration orale du paclitaxel.

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.

Comparison of liposomal drug formulations for transdermal iontophoretic drug delivery.

This study was aimed to evaluate the in vitro transdermal direct/pulsed current iontophoretic delivery of an amphiphilic model compound from various lipid vesicle-encapsulated formulations compared to free-drug formulation. Conventional, pegylated, ultradeformable liposomes (transfersomes) and ethosomes loaded with a negatively charged drug diclofenac sodium (DS) were prepared and characterized. All the liposomes possessed an average size of ≈100-150nm and negative zeta potential. No changes in colloidal stability were detected after 8h incubation of any vesicle formulation under constant or pulsed iontophoretic current. DS was released from all the liposome formulations with a similar, limited rate (≈50% in 24h), leading therefore to significantly lower transdermal fluxes across full-thickness porcine skin compared to the respective free drug formulation. From the tested lipid vesicle formulations, the transfersomes resulted in the highest passive flux and the ethosomes in the highest iontophoretic flux under direct constant current treatment. Higher negative surface charge of the vesicle led to better transport efficiency due to the higher mobility of the drug carrier under electric field. Pulsed current iontophoresis had no advantage over constant current treatment in combination with any type of lipid vesicular nanocarriers, in contrast to what has been described earlier with drug-loaded polymeric nanocarriers.

Albumin nanoparticles for the intravitreal delivery of anticytomegaloviral drugs.

Albumin nanoparticles (NP) were proved to be effective and safe carriers for delivering anticytomegaloviral compounds in the vitreous. NP improved the antiviral activity of both ganciclovir and the phosphodiester oligonucleotide analog to formivirsen. NP appeared to be fusogenic carriers able to target the nucleus of cells. In addition, these drug carriers were well tolerated when administered by the intravitreal route and did not induce autoimmune reactions.

Determination of oligonucleotide ISIS 2922 in nanoparticulate delivery systems by capillary zone electrophoresis.

ISIS 2922 is an antisense oligonucleotide with antiviral activity against cytomegalovirus. However, its rapid degradation in biological fluids and its low capacity for diffusion across cell membranes limit its therapeutical use. One possibility to overcome these drawbacks consists of using nanoparticles as drug carriers. The aim of this study was to develop an analytical method for determining the amount of ISIS 2922 loaded into albumin nanoparticles. For this purpose, capillary zone electrophoresis (CZE) was performed on a fused-silica capillary filled with borate buffer (12.5 mM, pH 9.5). Paracetamol was used as an internal standard and a diode-array detection system was set at 270 nm. Under these conditions, the limit of quantitation of ISIS 2922 was 1.27 microg and the precision and accuracy of the method did not exceed 7%. Moreover, the use of paracetamol as internal standard and the quantification by means of a 'corrected area' procedure enabled us to reduce the peak variability and accurately determine the amount of oligonucleotide loaded in the albumin nanoparticles. In summary, this assay is a selective and sensitive CZE method for the accurate quantitation of ISIS 2922 oligonucleotide in albumin nanoparticles.

Bovine serum albumin modified the intracellular distribution and improved the antiviral activity of an oligonucleotide.

In the present work, we describe the ability of bovine serum albumin (BSA) to improve the cytoplasmatic delivery of a phosphodiester oligonucleotide (PO), whose phosphorotioate analogue is ISIS 2922 (Vitravene). The changes in intensity and lambda(max) in fluorescence spectroscopy and the increase in fluorescence anisotropy upon complex formation between the oligonucleotide and the protein (PO-BSA) were used to determine the dissociation constant, Km=6.3 x 10(-8) M. The stoichiometry of the complex is mainly 1:1, although 2 PO per BSA have been detected at high PO/BSA ratios. The complexation did not protect PO against enzymatic degradation by snake venom phosphodiesterase (0.1 mg/mL). Fluorescence microscopy revealed that PO-BSA complexes showed a decreased uptake and modified pattern of intracellular distribution with a rapid nuclear accumulation (rather than vesicular localization in the cytoplasm observed for free oligonucleotide). The effect was only observed over a certain threshold of BSA concentration (higher than found in media supplemented with 10% serum). By this way, the interaction with albumin increased the antiviral activity of the oligonucleotide, tested by a plaque reduction assay in MRC-5 fibroblasts infected with human cytomegalovirus (strain RC 256) at a MOI of 0.0035. At 10 microM PO concentration, free PO decreased virus plaques to 85% of the control, while PO-BSA complexes reduced to 60%, in the same order than ISIS 2922. The phosphorotioate analogue complexed by BSA (ISIS 2922-BSA) showed the highest activity with 45% of the control plaques.

Albumin nanoparticles as carriers for a phosphodiester oligonucleotide.

The goal of this study was the evaluation of albumin nanoparticles as drug delivery systems for antisense oligonucleotides. Bovine serum albumin (BSA) nanoparticles were prepared by a coacervation process. A phosphodiester oligonucleotide was either incorporated into the matrix of the particles by incubation with the albumin prior the coacervation process or adsorbed onto the pre-formed nanoparticles. Incorporated and/or adsorbed oligonucleotide was estimated by capillary electrophoresis and fluorescence spectroscopy. The adsorbed amount of oligonucleotide was dramatically dependent on the pH of the medium. Desorption of the oligonucleotide was also affected by the pH and ionic strength of the medium. This indicated that electrostatic forces play a major role in the interaction between the oligonucleotide and the nanoparticles. When the oligonucleotide was incubated with the albumin prior to nanoparticle formation, the profile of release confirmed that a fraction was incorporated into the matrix and its release was controlled by the albumin degradation. The hybridisation capability of the oligonucleotide in both nanoparticle formulations was retained. However, only the oligonucleotide incorporated into the nanoparticle matrix was protected against enzymatic degradation.

[Controlled liberation of active principles by means of new galenic formulations]. / Liberación controlada de principios activos mediante el empleo de formulaciones galénicas.

Drugs inside a conventional galenic form are distributed between specific biological targets and other anatomical tissues. With the aim to obtain a more rational and a better therapeutic, one of the most promising possibilities by using the concept of vectorization: association of an active principle to an appropriate vector with the object to increase its action efficiency and efficacy. By this means, they do not just increase the affinity of the drug to the target but also active principle gets protected from a potentially hostile environment (hydrolytic enzymes, acid pH, etc.). The success in the extension of the applications of the vectorización depends more and more of an appropriate design, for what the fundamental objective of this revision will be the one of presenting the general characteristics and some of the current applications in these new galenic forms.

Innovative lead compounds and formulation strategies as newer kinetoplastid therapies.

The protozoan diseases leishmaniasis, human African trypanosomiasis (HAT) and Chagas disease (CD) are responsible for substantial global morbidity and mortality in tropical and subtropical regions. Environmental changes, drug resistance and immunosuppression are contributing to the emergence and spread of these diseases. In the absence of safe and efficient vaccines, chemotherapy, together with vector control, remains the most important measure to control kinetoplastid diseases. Nevertheless, the current chemotherapeutic treatments are clearly inadequate because of their toxic effects, generation of resistances as well as route and schedules of administration not adapted to the field-conditions. This review overlooks the strategies that can be addressed to meet immediately the patient needs such as the reconsideration of current regimens of administration and the rational combination of drugs in use. In the medium-long term, due to new methodologies of medicinal-chemistry, the screening from natural products and the identification of new therapeutic targets, new lead compounds have great chance to advance through the drug development pipeline to clinic. Modern pharmaceutical formulation strategies and nanomedicines also merit a place in view of the benefits of a single dose of liposomal Amphotericin B (AmBisome®) against visceral leishmaniasis. BBB-targeted nanodevices could be suited for selective delivery of drugs against HAT encephalitic phase. Bioadhesive nanoparticles can be proposed to enhance the bioavailability of drugs after oral administration by reason of improving the drug solubility, and permeability across the intestinal epithelia.

Increased oral bioavailability of paclitaxel by its encapsulation through complex formation with cyclodextrins in poly(anhydride) nanoparticles.

The aim of this work was to study the oral bioavailability in rats of paclitaxel (PTX) when encapsulated as a complex with cyclodextrins in poly(anhydride) nanoparticles (NP). For this purpose three different cyclodextrins were selected: beta-cyclodextrin (CD), 2-hydroxypropyl-beta-cyclodextrin (HPCD) and 6-monodeoxy-6-monoamino-beta-cyclodextrin (NHCD). A single dose of 10mg paclitaxel per kg body weight as PTX-cyclodextrin nanoparticles was used. Plasma curves were characterised by a plateau of paclitaxel concentration close to the C(max) from T(max) till 24h post-administration. For PTX-CD NP and PTX-HPCD NP, these sustained levels of the anticancer drug were found to be between 27 and 33-fold higher than the reported value of drug activity whereas the relative oral bioavailability of paclitaxel was calculated to be higher than 80%. These facts would be directly related with a synergistic effect obtained by the combination of the bioadhesive properties of poly(anhydride) nanoparticles and the inhibitory effect of cyclodextrins on the activity of P-glycoprotein and cythocrome P450.

Combined hydroxypropyl-beta-cyclodextrin and poly(anhydride) nanoparticles improve the oral permeability of paclitaxel.

The aim of this work was to study the effect of the combination between 2-hydroxypropyl-beta-cyclodextrin (HPCD) and bioadhesive nanoparticles on the encapsulation and intestinal permeability of paclitaxel (PTX). In this context, a solid inclusion complex between PTX and HPCD was prepared by an evaporation method. Then, the complex was incorporated in poly(anhydride) nanoparticles by a solvent displacement method. The resulting nanoparticles, PTX-HPCD NP, displayed a size of about 300 nm and a drug loading of about 170 microg/mg (500-fold higher than in the absence of HPCD). The effect of these nanoparticles on the permeability of intestinal epithelium was investigated using the Ussing chamber technique. The apparent permeability (P(app)) of PTX was found to be 12-fold higher when formulated as PTX-HPCD NP than when formulated as Taxol (control). Furthermore, when interaction between nanoparticles and the mucosa was avoided, the permeability of PTX significantly decreased. In summary, the association between PTX-HPCD and poly(anhydride) nanoparticles would induce a positive effect over the intestinal permeability of paclitaxel, being the bioadhesion a mandatory condition in this phenomena.

Gamma interferon loaded onto albumin nanoparticles: in vitro and in vivo activities against Brucella abortus.

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.

Intradermal immunization with ovalbumin-loaded poly-epsilon-caprolactone microparticles conferred protection in ovalbumin-sensitized allergic mice.

BACKGROUND: Although immunotherapy has been reported as the only treatment able to revert the T-helper type 2 (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 these 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-epsilon-caprolactone (PCL) microparticles as adjuvants in immunotherapy using ovalbumin (OVA) as an 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 by immunizing BALB/c mice intradermically. Also, OVA-sensitized mice were treated with OVA PCL and OVA adsorbed to aluminium hydroxide (OVA-Alum). Fifteen days after therapy, animals were challenged with OVA and different signs of anaphylactic shock were evaluated. RESULTS: One single shot by an intradermal route with OVA PCL resulted in a Th2-type immune response. In OVA-sensitized mice, treatment with OVA PCL 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.

In vitro reversion of amphotericin B resistance in Leishmania donovani by poloxamer 188.

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.

Fluconazole encapsulation in PLGA microspheres by spray-drying.

Fluconazole-loaded PLGA microspheres were prepared by the spray-drying process. The influence of some process parameters on the physical characteristics of the microspheres was evaluated. Neither type nor polymer concentration influenced significantly the mean diameter of the microspheres, their size distribution and encapsulation efficiency of the drug. However, the drug loading greatly affected their size and the physical state in which fluconazole can exist in the matrix of the carriers, and, thus, affected the release rate of the drug. Results obtained by differential thermal analysis and X-ray powder diffraction revealed that at low nominal drug loading, fluconazole was incorporated in an amorphous state or in a molecular dispersion in the matrix of the microspheres and at high nominal drug loading part of the drug was in a crystalline form. Release profiles of fluconazole from the microspheres displayed a biphasic shape. The duration and extent of each phase were affected mainly by polymer nature, drug loading and physical state in which fluconazole existed in the polymeric matrix.

Synthetic particulate antigen delivery systems for vaccination

La encapsulación de un antígeno (proteína, péptido o DNA)en partículas sintéticas constituye un punto de partida razonablepara el diseño de vacunas subcelulares efectivas. Al dotarlo delas dimensiones de una partícula, se favorece su visualización porcélulas presentadoras de antígeno (APC), como las células dendríticas(DC). Utilizando distintos materiales y procedimientosde fabricación, la tecnología farmacéutica puede construir partículascon un amplio rango de tamaños y variadas propiedadesde superficie. Es nuestro objetivo con esta revisión analizar cómoestas propiedades físicas afectan a la intensidad y al tipo de respuestainmunitaria que producen. Así, partículas de distinto tamañopodrían poner en juego distintas APC y subtipos de DC. Susuperficie puede ser recubierta con ligandos que promueven laendocitosis receptor-específica y algún inmunoestimulante intrínsecoco-encapsulado junto con el antígeno dentro de la mismapartícula. El mecanismo de captura antigénica y el tipo de señalde activación pueden ser decisivos para potenciar y modular larespuesta inmunitaria al tipo deseado, Th2/humoral o Th1/citotoxica.Adicionalmente, las partículas que son de naturaleza poliméricapueden liberar lentamente el antígeno encapsulado, deforma continua o pulsátil, evitando la necesidad de administracionesrepetidas normalmente requerida para las vacunas tradicionales.Las partículas sintéticas para la encapsulación de antígenosconstituyen por tanto, una buena herramienta que puede hacerposible el diseño de vacunas seguras y efectivas con una sola administración,algunos de los atributos de una vacuna ideal. Existenciertos problemas tecnológicos, pero no parecen insalvables

Synthetic particles as carriers for antigens display a greatpotential and versatility for the design of effective vaccines. Supportedby multiple raw materials and preparation procedures,they can be engineered over a wide range of sizes, variable surfacenature and patterns of antigen release. It is our goal in thisreview to evaluate how these physical characteristics decide thetype of immune response the particles will develop, aiming at amore rational design of effective and safer synthetic subunit vaccines.So, as a function of their size, they will be uptaken by differentantigen presenting cell (APC) or dendritic cell (DC) subsets;certain ligands can be attached to the particle surfaces to promotereceptor-specific endocitosis and any immunopotentiatorco-encapsulated with the antigen in the same particle. The mechanismof antigen uptake and the signal for activating DC can bedecisive to increase and skew a Th1/cytotoxic or Th2/humoralresponse. Also, the antigen release rate has influence on the generationof immunological memory and an adequate profile of antigenrelease from these particles can mimic boosters of the traditionalvaccines.The current tendencies are oriented towards the developmentof new vaccines containing perfectly characterized antigens, establishedand safe, and with composition and preparation methodsrigorously controlled. The use of synthetic particles as vectorsmay be considered as adjuvants that accomplish with these requisites,being able to induce a response even at the mucosal level.There are technological handicaps faced by developers of syntheticparticles based vaccines but do not appear to be insurmountable

Synthetic particulate antigen deliverysystems for vaccination

La encapsulación de un antígeno (proteína, péptido o DNA)en partículas sintéticas constituye un punto de partida razonable para el diseño de vacunas subcelulares efectivas. Al dotarlo de las dimensiones de una partícula, se favorece su visualización por células presentadoras de antígeno (APC), como las células dendríticas(DC). Utilizando distintos materiales y procedimientos de fabricación, la tecnología farmacéutica puede construir partículas con un amplio rango de tamaños y variadas propiedades de superficie. Es nuestro objetivo con esta revisión analizar cómo estas propiedades físicas afectan a la intensidad y al tipo de respuesta inmunitaria que producen. Así, partículas de distinto tamaño podrían poner en juego distintas APC y subtipos de DC. Susuperficie puede ser recubierta con ligandos que promueven la endocitosis receptor-específica y algún inmunoestimulante intrínsecoco-encapsulado junto con el antígeno dentro de la misma partícula. El mecanismo de captura antigénica y el tipo de señal de activación pueden ser decisivos para potenciar y modular la respuesta inmunitaria al tipo deseado, Th2/humoral o Th1/citotoxica. Adicionalmente, las partículas que son de naturaleza polimérica pueden liberar lentamente el antígeno encapsulado, de forma continua o pulsátil, evitando la necesidad de administraciones repetidas normalmente requerida para las vacunas tradicionales.Las partículas sintéticas para la encapsulación de antígenos constituyen por tanto, una buena herramienta que puede hacer posible el diseño de vacunas seguras y efectivas con una sola administración,algunos de los atributos de una vacuna ideal. Existen ciertos problemas tecnológicos, pero no parecen insalvables (AU)

Synthetic particles as carriers for antigens display a greatpotential and versatility for the design of effective vaccines. Supported by multiple raw materials and preparation procedures,they can be engineered over a wide range of sizes, variable surfacenature and patterns of antigen release. It is our goal in this review to evaluate how these physical characteristics decide the type of immune response the particles will develop, aiming at amore rational design of effective and safer synthetic subunit vaccines.So, as a function of their size, they will be uptaken by different antigen presenting cell (APC) or dendritic cell (DC) subsets;certain ligands can be attached to the particle surfaces to promotereceptor-specific endocitosis and any immunopotentiatorco-encapsulated with the antigen in the same particle. The mechanism of antigen uptake and the signal for activating DC can be decisive to increase and skew a Th1/cytotoxic or Th2/humoral response. Also, the antigen release rate has influence on the generation of immunological memory and an adequate profile of antigen release from these particles can mimic boosters of the traditionalvaccines.The current tendencies are oriented towards the development of new vaccines containing perfectly characterized antigens, establishedand safe, and with composition and preparation methods rigorously controlled. The use of synthetic particles as vectors may be considered as adjuvants that accomplish with these requisites, being able to induce a response even at the mucosal level.There are technological handicaps faced by developers of syntheticparticles based vaccines but do not appear to be insurmountable (AU)

Liberación controlada de principios activos mediante el empleo de formulaciones galénicas / Controlled liberation of active principles by means of new galenic formulations

Los principios activos incluidos en una forma galénica convencional se distribuyen indistintamente entre dianas biológicas específicas y otros tejidos anatómicos. Con el fin de obtener una terapéutica más racional y mejor adaptada, una de las posibilidades más prometedoras es la que utiliza el concepto de vectorización: asociación del principio activo a un vector apropiado, con objeto de aumentar la eficacia y la especificidad de acción del mismo. De esta manera, no solo aumenta la afinidad del fármaco por la diana, sino que además queda protegido de un ambiente potencialmente hostil (enzimas hidrolíticas, pH ácido, etc.). El éxito en la extensión de las aplicaciones de la vectorización depende cada vez más de un diseño adecuado, por lo que el objetivo fundamental de esta revisión será la de presentar las características generales y algunas de las actuales aplicaciones de estas nuevas formas farmacéuticas (AU)